Multispecific t cell receptors

Abstract

CD8 + T cells comprising multispecific T-cell receptors and methods for making the same are provided.SOLUTION: A method of generating CD8 + T-cells comprising multispecific T-cell receptors (TCRs), the method comprising: (a) administering to a subject a recombinant cytomegalovirus (CMV) vector comprising a first heterologous antigen-encoding nucleotide sequence; (b) identifying a first CD8 + TCR from a first set of the CD8 + T-cells; (c) administering to the subject a second heterologous antigen-encoding nucleotide sequence; (d) isolating one or more CD8 + T-cells from a second set of the CD8 + T-cells; (e) identifying a second CD8 + TCR from the second set of CD8 + T cells; (f) transfecting a third set of CD8 + T cells with an expression vector; and (g) selecting one or more of the third CD8 + TCRs having the highest avidity for a particular peptide of interest.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 948,691, filed December 16, 2019, which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under grants P01 AI094417, U19 AI128741, R01 AI117802, and R01 AI140888 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention.

[0003] Reference to an electronically submitted sequence listing The contents of the electronically submitted sequence listing in an ASCII text file (Name: 4153_014PC01_Seqlisting_ST25, Size: 12,548 bytes, and Creation Date: November 30, 2020) are incorporated herein by reference in their entirety. [Background technology]

[0004] Conventionally restricted T cell receptors (TCRs) recognize specific peptides or epitopes within a given protein, or antigens, presented by specific alleles of the major histocompatibility complex (MHC) class I or class II. For example, the mouse T cell receptor OT-1 is specific for the mouse MHC-I molecule Kb, which presents the peptide SIINFEKL derived from the antigen ovalbumin.

[0005] Traditionally restricted TCRs are currently in clinical development for the treatment of cancer and chronic infectious diseases. This is typically achieved by first cloning a TCR specific for a desired antigen presented by a common MHC allele. The TCR is then introduced into autologous T cells (i.e., T cells derived from a given patient). Upon in vitro expansion of these cells, such "TCR T cells" are reintroduced into the patient for treatment (similar to T cells expressing chimeric antigen receptors, or CARs). This approach has several drawbacks: a) autologous TCR T cells must be generated anew for each new patient's treatment; b) TCRs can only be used in humans expressing the correct MHC alleles; and c) because TCRs are highly specific for a given peptide, mutations in the peptide sequence can lead to escape from TCR recognition. Regarding a), many efforts have been made in industry and academia to generate "off-the-shelf" xenogeneic T cells, i.e., T cell lines that will not be rejected when given to another person. However, there are currently no solutions for b) and c).

[0006] It has occasionally been observed that a single TCR can recognize more than one peptide presented by the same or different MHC molecules, however, until now, there has been no method that can specifically generate a single TCR with multiple (unrelated) specificities. Summary of the Invention

[0007] The present invention provides a method for generating CD8+ T cells comprising a multispecific TCR, comprising: (a) administering to the subject a recombinant cytomegalovirus (CMV) vector comprising a nucleic acid sequence encoding a first heterologous antigen in an amount effective to generate a first set of CD8+ T cells that recognize a first MHC / xenoantigen-derived peptide complex, wherein the CMV vector does not express active UL128, UL130, UL146, and UL147 proteins 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 / xenoantigen-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 recognize a second MHC / xenoantigen-derived peptide complex; (d) isolating one or more CD8+ T cells from the second set of CD8+ T cells; and (e) isolating one or more CD8+ T cells from the second set of CD8+ T cells. (f) transfecting a third set of CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding the third CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the third CD8+ TCR, and the third CD8+ TCR comprises CDR3α and CDR3β of the second CD8+ TCR, thereby generating one or more CD8+ T cells that recognize the first MHC / xenoantigen-derived peptide complex and the second MHC / xenoantigen-derived peptide complex; and (g) selecting one or more of the third CD8+ TCRs that have the highest avidity for the particular peptide of interest.

[0008] In one embodiment, the recombinant CMV vector does not express active UL18. In one embodiment, the recombinant CMV vector expresses an active UL40 protein or an orthologue thereof, and an active US28 protein or an orthologue thereof.

[0009] In one embodiment, the first MHC / xenogenous antigen-derived peptide complex is an MHC-II / xenogenous antigen-derived peptide complex, an MHC-E / xenogenous antigen-derived peptide complex, or an MHC-I / xenogenous antigen-derived peptide complex. In one embodiment, the second MHC / xenogenous antigen-derived peptide complex is an MHC-II / xenogenous antigen-derived peptide complex or an MHC-E / xenogenous antigen-derived peptide complex.

[0010] In one embodiment, the subject is a human or non-human primate. In one embodiment, the recombinant CMV vector is a recombinant human CMV vector or a recombinant rhesus CMV vector.

[0011] In one embodiment, the first and / or second heterologous antigen comprises a tumor antigen, a pathogen-specific antigen, a tissue-specific antigen, or a host autoantigen. In one embodiment, the tumor antigen is associated with a cancer selected from the group consisting of prostate cancer, kidney cancer, lung cancer, pancreatic cancer, mesothelioma, breast cancer, acute myeloid leukemia, chronic myeloid 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. In one embodiment, the pathogen-specific antigen is associated with 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 parasite, Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), human T-lymphotropic virus type 1 (HTLV1), Merkel virus (MCV), cytomegalovirus, and Mycobacterium tuberculosis.

[0012] In one embodiment, the first CD8+ TCR recognizes a specific MHC-II subtope or supertope. In one embodiment, the first CD8+ TCR recognizes a specific MHC-E subtope or supertope. In one embodiment, the first CD8+ TCR recognizes a specific MHC-I subtope or supertope.

[0013] In one embodiment, the first CD8+ TCR is identified by DNA or RNA sequencing. In another embodiment, the first CD8+ TCR is identified by single cell sequencing.

[0014] In one embodiment, the first heterologous antigen and the second heterologous antigen are the same. In one embodiment, the first heterologous antigen and the second heterologous antigen are different.

[0015] In one embodiment, one or more isolated CD8+ T cells from the second set of CD8+ T cells express CD69 and TNFα.

[0016] In one embodiment, the second CD8+ TCR recognizes one or more specific MHC-II supertopes. In one embodiment, 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.

[0017] In one embodiment, the second CD8+ TCR recognizes an MHC-II supertope and an MHC-E supertope. In one embodiment, the second CD8+ TCR recognizes an MHC-I supertope and an MHC-E supertope. In one embodiment, the second CD8+ TCR recognizes an MHC-I supertope and an MHC-II supertope.

[0018] In one embodiment, the second CD8+ TCR recognizes one or more specific MHC-II subtopes. In one embodiment, the second CD8+ TCR recognizes one or more specific MHC-E subtopes. In one embodiment, the second CD8+ TCR recognizes one or more specific MHC-I subtopes.

[0019] In one embodiment, the second CD8+ TCR recognizes an MHC-II subtope and an MHC-E subtope. In one embodiment, the second CD8+ TCR recognizes an MHC-II subtope and an MHC-I subtope. In one embodiment, the second CD8+ TCR recognizes an MHC-E subtope and an MHC-I subtope.

[0020] In one embodiment, the second CD8+ TCR recognizes an MHC-II subtope or supertope and an MHC-E subtope or supertope. In one embodiment, the second CD8+ TCR recognizes an MHC-II subtope or supertope and an MHC-I subtope or supertope. In one embodiment, the second CD8+ TCR recognizes an MHC-E subtope or supertope and an MHC-I subtope or supertope.

[0021] In one embodiment, the second CD8+ TCR recognizes a specific MHC-II supertope and MHC-II subtope. In one embodiment, the second CD8+ TCR recognizes a specific MHC-E supertope and MHC-E subtope. In one embodiment, the second CD8+ TCR recognizes a specific MHC-I supertope and MHC-I subtope.

[0022] In one embodiment, the second CD8+ TCR recognizes two or more MHC-II supertopes from the same antigen. In one embodiment, the second CD8+ TCR recognizes two or more MHC-E supertopes from the same antigen. In one embodiment, the second CD8+ TCR recognizes two or more MHC-I supertopes from the same antigen.

[0023] In one embodiment, the second CD8+ TCR recognizes two or more MHC-II subtopes from the same antigen. In one embodiment, the second CD8+ TCR recognizes two or more MHC-E subtopes from the same antigen. In one embodiment, the second CD8+ TCR recognizes two or more MHC-I subtopes from the same antigen.

[0024] In one embodiment, 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.

[0025] In one embodiment, the second CD8+ TCR recognizes two or more MHC-II supertopes from two or more antigens. In one embodiment, the second CD8+ TCR recognizes two or more MHC-E supertopes from two or more antigens. In one embodiment, the second CD8+ TCR recognizes two or more MHC-I supertopes from two or more antigens.

[0026] In one embodiment, the second CD8+ TCR recognizes two or more MHC-II subtopes from two or more antigens. In one embodiment, the second CD8+ TCR recognizes two or more MHC-E subtopes from two or more antigens. In one embodiment, the second CD8+ TCR recognizes two or more MHC-I subtopes from two or more antigens.

[0027] In one embodiment, 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.

[0028] In one embodiment, 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.

[0029] In one embodiment, 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.

[0030] In one embodiment, the third CD8+ TCR recognizes a specific MHC-II supertope and MHC-II subtope. In one embodiment, the third CD8+ TCR recognizes a specific MHC-E supertope and MHC-E subtope. In one embodiment, the third CD8+ TCR recognizes a specific MHC-I supertope and MHC-I subtope.

[0031] In one embodiment, the third CD8+ TCR recognizes two or more MHC-II supertopes from one antigen. In one embodiment, the third CD8+ TCR recognizes two or more MHC-E supertopes from one antigen. In one embodiment, the third CD8+ TCR recognizes two or more MHC-I supertopes from one antigen.

[0032] In one embodiment, the third CD8+ TCR recognizes two or more MHC-II subtopes from one antigen. In one embodiment, the third CD8+ TCR recognizes two or more MHC-E subtopes from one antigen. In one embodiment, the third CD8+ TCR recognizes two or more MHC-I subtopes from one antigen.

[0033] In one embodiment, the third CD8+ TCR recognizes one or more MHC-II supertopes and one or more MHC-II subtopes from an antigen. In one embodiment, the third CD8+ TCR recognizes one or more MHC-E supertopes and one or more MHC-E subtopes from an antigen. In one embodiment, the third CD8+ TCR recognizes one or more MHC-I supertopes and one or more MHC-I subtopes from an antigen.

[0034] In one embodiment, the third CD8+ TCR recognizes two or more MHC-II supertopes from two or more antigens. In one embodiment, the third CD8+ TCR recognizes two or more MHC-E supertopes from two or more antigens. In one embodiment, the third CD8+ TCR recognizes two or more MHC-E supertopes from two or more antigens.

[0035] In one embodiment, the third CD8+ TCR recognizes two or more MHC-II subtopes from two or more antigens. In one embodiment, the third CD8+ TCR recognizes two or more MHC-E subtopes from two or more antigens. In one embodiment, the third CD8+ TCR recognizes two or more MHC-I subtopes from two or more antigens.

[0036] In one embodiment, the third CD8+ TCR recognizes a specific MHC-E subtope or supertope and an MHC-II subtope or supertope. In one embodiment, the third CD8+ TCR recognizes a specific MHC-E subtope or supertope and an MHC-I subtope or supertope. In one embodiment, the third CD8+ TCR recognizes a specific MHC-II subtope or supertope and an MHC-I subtope or supertope.

[0037] In one embodiment, the third CD8+ TCR recognizes two or more MHC-II subtopes from the same antigen. In one embodiment, the third CD8+ TCR recognizes two or more MHC-E subtopes from the same antigen. In one embodiment, the third CD8+ TCR recognizes two or more MHC-I subtopes from the same antigen.

[0038] In one embodiment, 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.

[0039] In one embodiment, the nucleic acid sequence encoding the third CD8+ TCR is identical to the nucleic acid sequence encoding the second CD8+ TCR.

[0040] In one embodiment, one or more CD8+ T cells are isolated from a second subject and transfected with a nucleic acid sequence encoding a 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 / xenogenous antigen-derived peptide complex and the second MHC / xenogenous antigen-derived peptide complex.

[0041] In one embodiment, the first MHC-xenogenous antigen-derived peptide complex is an MHC-II / xenogenous antigen-derived peptide complex, an MHC-E / xenogenous antigen-derived peptide complex, or an MHC-I / xenogenous antigen-derived peptide complex. In one embodiment, the second MHC-xenogenous antigen-derived peptide complex is an MHC-II / xenogenous antigen-derived peptide complex, an MHC-E / xenogenous antigen-derived peptide complex, or an MHC-I / xenogenous antigen-derived peptide complex.

[0042] In one embodiment, the transfected CD8+ T cells are administered to a second subject to treat or prevent cancer. In another embodiment, the cancer is selected from the group consisting of prostate cancer, kidney cancer, lung cancer, pancreatic cancer, mesothelioma, breast cancer, acute myeloid leukemia, chronic myeloid 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.

[0043] In one embodiment, the transfected CD8+ T cells are administered to a second subject to treat a pathogen infection.

[0044] In another embodiment, the pathogen 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 parasite, Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), human T-lymphotropic virus type 1 (HTLV1), Merkel virus (MCV), cytomegalovirus, and Mycobacterium tuberculosis.

[0045] In one embodiment, the first subject is a non-human primate and the second subject is a human, and the transfected CD8+ T cells comprise a chimeric non-human primate-human CD8+ TCR comprising a non-human primate CDR3α and CDR3β of a second CD8+ TCR.

[0046] In one embodiment, the third CD8+ TCR comprises the non-human primate CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the second CD8+ TCR. In one embodiment, the third CD8+ TCR comprises the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the second CD8+ TCR.

[0047] In one embodiment, the first subject is a non-human primate, the second subject is a human, and the second CD8+ TCR is a chimeric non-human primate-human CD8+ TCR comprising the non-human primate CDR3α and CDR3β of the first CD8+ TCR.

[0048] In one embodiment, the third CD8+ TCR is a chimeric CD8+ TCR.

[0049] In one embodiment, administering the recombinant CMV vector to the first subject comprises intravenous, intramuscular, intraperitoneal, or oral administration.

[0050] In one embodiment, CD8+ T cells comprising a multispecific TCR are generated by the method.

[0051] In one embodiment, the CD8+ T cells are administered to a subject in need thereof to treat or prevent cancer. In another embodiment, the cancer is selected from the group consisting of prostate cancer, kidney cancer, lung cancer, pancreatic cancer, mesothelioma, breast cancer, acute myeloid leukemia, chronic myeloid 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.

[0052] In one embodiment, CD8+ T cells are administered to a subject in need thereof to treat a pathogenic infection. In another embodiment, 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 parasite, Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), human T-lymphotropic virus type 1 (HTLV1), Merkel virus (MCV), cytomegalovirus, and Mycobacterium tuberculosis.

[0053] In one embodiment, administration of the recombinant CMV vector to the first subject comprises intravenous, intramuscular, intraperitoneal, or oral administration.

[0054] The present invention provides a method for generating CD8+ T cells comprising a multispecific T cell receptor (TCR), comprising: (a) administering to a subject a recombinant cytomegalovirus (CMV) vector comprising a nucleic acid sequence encoding a first heterologous antigen in an amount effective to generate a first set of CD8+ T cells that recognize a first MHC-E / xenoantigen-derived peptide complex, wherein the CMV vector does not express active UL128, UL130, UL146, and UL147 proteins or their orthologs; (b) identifying a first CD8+ TCR from the first set of CD8+ T cells, wherein the first CD8+ TCR recognizes a first MHC-E / xenoantigen-derived peptide complex; (c) administering to the subject a second xenoantigen in an amount effective to generate a second set of CD8+ T cells that recognizes a second MHC-E / xenoantigen-derived peptide complex; and (d) identifying a first CD8+ TCR from the second set of CD8+ T cells that recognizes a second MHC-E / xenoantigen-derived peptide complex. (e) identifying a second CD8+ TCR from the second set of CD8+ T cells, wherein the second CD8+ TCR recognizes the first MHC-E / xenoantigen-derived peptide complex and the second MHC-E / xenoantigen-derived peptide complex; and (f) transfecting a third set of CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a third CD8+ TCR and a nucleic acid sequence encoding the third CD8+ TCR. and (g) transfecting one or more of the third CD8+ TCRs with the highest avidity for a particular peptide of interest, wherein the third CD8+ TCR comprises the CDR3α and CDR3β of the second CD8+ TCR, thereby generating one or more CD8+ T cells that recognize the first MHC-E / xenoantigen-derived peptide complex and the second MHC-E / xenoantigen-derived peptide complex; and (g) selecting one or more of the third CD8+ TCRs with the highest avidity for a particular peptide of interest.

[0055] In one embodiment, the subject is a human or non-human primate. In one embodiment, the recombinant CMV vector is a recombinant human CMV vector or a recombinant rhesus CMV vector.

[0056] In one embodiment, the first heterologous antigen comprises a tumor antigen, a pathogen-specific antigen, a tissue-specific antigen, or a host autoantigen. In one embodiment, the tumor antigen is associated with a cancer selected from the group consisting of prostate cancer, kidney cancer, lung cancer, pancreatic cancer, mesothelioma, breast cancer, acute myeloid leukemia, chronic myeloid 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. In one embodiment, the pathogen-specific antigen is associated with 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 parasite, Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), human T-lymphotropic virus type 1 (HTLV1), Merkel virus (MCV), cytomegalovirus, and Mycobacterium tuberculosis.

[0057] In one embodiment, the MRE contains a target site for a microRNA expressed in endothelial cells. In another embodiment, the MRE is specific for a 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.

[0058] In one embodiment, the first CD8+ TCR recognizes a specific MHC-E subtope or supertope.

[0059] In one embodiment, the first CD8+ TCR is identified by DNA or RNA sequencing. In one embodiment, the first CD8+ TCR is identified by single cell sequencing.

[0060] In one embodiment, the second heterologous antigen comprises a tumor antigen, a pathogen-specific antigen, a tissue-specific antigen, or a host autoantigen. In one embodiment, the tumor antigen is associated with a cancer selected from the group consisting of prostate cancer, kidney cancer, lung cancer, pancreatic cancer, mesothelioma, breast cancer, acute myeloid leukemia, chronic myeloid 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. In one embodiment, the pathogen-specific antigen is associated with 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 parasite, Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), human T-lymphotropic virus type 1 (HTLV1), Merkel virus (MCV), cytomegalovirus, and Mycobacterium tuberculosis.

[0061] In one embodiment, the first heterologous antigen and the second heterologous antigen are the same. In one embodiment, the first heterologous antigen and the second heterologous antigen are different.

[0062] In one embodiment, one or more isolated CD8+ T cells from the second set of CD8+ T cells express CD69 and TNFα.

[0063] In one embodiment, the second CD8+ TCR is identified by DNA or RNA sequencing. In one embodiment, the second CD8+ TCR is identified by single cell sequencing.

[0064] In one embodiment, 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-E subtopes. In one embodiment, the second CD8+ TCR recognizes a specific MHC-E supertope and MHC-E subtope.

[0065] In one embodiment, the second CD8+ TCR recognizes two or more MHC-E supertopes from the same antigen. In one embodiment, the second CD8+ TCR recognizes two or more MHC-E subtopes from the same antigen.

[0066] 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 two or more MHC-E supertopes from two or more antigens. In one embodiment, the second CD8+ TCR recognizes two or more MHC-E subtopes from two or more 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.

[0067] 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-E subtopes. In one embodiment, the third CD8+ TCR recognizes a specific MHC-E supertope and MHC-E subtope.

[0068] In one embodiment, the third CD8+ TCR recognizes two or more MHC-E supertopes from one antigen. In one embodiment, the third CD8+ TCR recognizes two or more MHC-E subtopes from one antigen.

[0069] In one embodiment, the third CD8+ TCR recognizes one or more MHC-E supertopes and one or more MHC-E subtopes from one antigen, hi one embodiment, the third CD8+ TCR recognizes two or more MHC-E supertopes from two or more antigens.

[0070] In one embodiment, the third CD8+ TCR recognizes two or more MHC-E subtopes from two or more antigens, hi one embodiment, the third CD8+ TCR recognizes a particular MHC-E supertope and MHC-E subtope.

[0071] In one embodiment, the third CD8+ TCR recognizes two or more MHC-E subtopes from the same antigen, hi one embodiment, the third CD8+ TCR recognizes one or more MHC-E supertopes and one or more MHC-E subtopes from different antigens.

[0072] In one embodiment, the nucleic acid sequence encoding the third CD8+ TCR is identical to the nucleic acid sequence encoding the second CD8+ TCR.

[0073] In one embodiment, one or more CD8+ T cells are isolated from a second subject and transfected with a nucleic acid sequence encoding a 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 / xenogenous antigen-derived peptide complex and the second MHC-E / xenogenous antigen-derived peptide complex.

[0074] In one embodiment, the transfected CD8+ T cells are administered to a second subject to treat or prevent cancer. In another embodiment, the cancer is selected from the group consisting of prostate cancer, kidney cancer, lung cancer, pancreatic cancer, mesothelioma, breast cancer, acute myeloid leukemia, chronic myeloid 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.

[0075] In one embodiment, the transfected CD8+ T cells are administered to a second subject to treat a pathogen infection. In another embodiment, the pathogen 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 parasite, Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), human T-lymphotropic virus type 1 (HTLV1), Merkel virus (MCV), cytomegalovirus, and Mycobacterium tuberculosis.

[0076] In one embodiment, the first subject is a non-human primate and the second subject is a human, and the transfected CD8+ T cells comprise a chimeric non-human primate-human CD8+ TCR comprising a non-human primate CDR3α and CDR3β of a second CD8+ TCR.

[0077] In one embodiment, the third CD8+ TCR comprises the non-human primate CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the second CD8+ TCR. In one embodiment, the third CD8+ TCR comprises the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the second CD8+ TCR.

[0078] In one embodiment, the first subject is a non-human primate, the second subject is a human, and the second CD8+ TCR is a chimeric non-human primate-human CD8+ TCR comprising the non-human primate CDR3α and CDR3β of the first CD8+ TCR.

[0079] In one embodiment, the third CD8+ TCR is a chimeric CD8+ TCR.

[0080] In one embodiment, administering the recombinant CMV vector to the first subject comprises intravenous, intramuscular, intraperitoneal, or oral administration.

[0081] In one embodiment, CD8+ T cells comprising a multispecific TCR are generated by the method.

[0082] In one embodiment, the CD8+ T cells are administered to a subject in need thereof to treat or prevent cancer. In one embodiment, the cancer is selected from the group consisting of prostate cancer, kidney cancer, lung cancer, pancreatic cancer, mesothelioma, breast cancer, acute myeloid leukemia, chronic myeloid 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.

[0083] In one embodiment, the CD8+ T cells are administered to a subject in need thereof to treat a pathogenic infection, in one embodiment, 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 parasite, Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), human T-lymphotropic virus type 1 (HTLV1), Merkel virus (MCV), cytomegalovirus, and Mycobacterium tuberculosis.

[0084] In one embodiment, administering the recombinant CMV vector to the first subject comprises intravenous, intramuscular, intraperitoneal, or oral administration. [Brief explanation of the drawings]

[0085] [Figure 1A-1B] An overview of the primary study cohort is shown. Figure 1A is a timeline showing vaccination dates and sampling windows used in this study. Figure 1B shows 15 overlapping SIVgag peptides recognized by rhesus macaques (RM) using intracellular cytokine staining (ICS). Each T cell-targeting peptide "box" is colored based on MHC restriction, as determined by differential blocking analysis. Green = MHC-E, red = MHC-1a, blue = MHC-II, purple = variable. MHC-E and MHC-II restricted supertopes are labeled. [Figures 2A-2F] Figure 2A shows the TCR clonotype hierarchy of MHC-E supertope responses. Figure 2A shows peripheral blood mononuclear cells (PBMCs) from a RhCMV68-1 / SIVgag-vaccinated RM (Rh-1) stimulated with EK9 peptide in the presence of the secretion inhibitor brefeldin A. Intracellular cytokine (TNF-α vs. IFN-γ) analysis (ICS) was performed to identify EK9-specific CD8+ T cells (left). In parallel, the same PBMCs were stimulated with EK9 in the absence of brefeldin A but in the presence of the TNF-α cleavage inhibitor TAPI-0. Responder cells were identified by activation-induced upregulation of surface CD69 and surface-trapped TNF-α (STTS analysis, right). Figure 2B is a bar graph showing the clonotype hierarchy at each time point based on CDR3 alpha and / or beta sequences. In many cases, a given TCR α / β pair clone was found within the responding fraction after both EK9 and RL9 stimulation (asterisk). FIG. 2F shows a representative ICS experiment in which these transductants were cultured with BLCL pulsed with no peptide, negative control peptide, Gag RL9, or Gag EK9. [Figure 3A-3B]Figure 3A shows SIVgag recognition by TCR transducers. Figure 3A shows the results of a flow cytometry experiment showing target cells 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 expressing both SIV Gag and cleaved NGFR, providing the surface marker (NGFR-T2A-Gag). Figure 3B shows an ICS assay using target cells and the indicated MHC-E-TCR CD8+ T cell transducers. CD8+ T cell transducers expressing the Mamu-A*01-restricted CM9-specific TCR were used as positive controls. Untransduced CD8+ T cells or CD8+ T cell transducers expressing an (irrelevant) MR1-restricted TCR were used as negative controls. [Figures 4A-4D] 1 shows a pie chart depicting the complete clonotype hierarchy for SIV infection recognition. [Figure 5] Analysis of epitope cross-reactivity using TCR transducins. Representative ICS using CD8+ T cell transducins expressing Rh-1 MHC-E-TCR4 versus TCR6-1. These transducins were cultured in RM BLCL pulsed with SIVgag MHC-E-optimized supertope and subtope peptides against Rh-1, as indicated. Responses were measured using IFN-γ and TNF-α staining. [Figures 6A-6B] Responses of CD8+ T cells expressing TCRs recognizing MHC-E-presented SIVgag peptides to MHC-II-presented SIVgag peptides and to peptides derived from unrelated TB antigens are shown. PBMCs from Rh-4 were stimulated with either the MHC-II supertope peptides Gag211-222 (53) or Gag290-301 (73) (Figure 6A) or a pool of overlapping 15-mer peptides from the TB protein Ag85B (Figure 6B). Activated cells were sorted based on sCD69 and stTNF-α, and TCRs were characterized by scRNA-seq. [Figure 7A-7J]Cross-reactivity of MHC-E-restricted TCRs with MHC-Ia-presented CMV IE peptides is shown. Figure 7A shows a flow cytometry experiment analyzing the responses of four RMs to AN10 and VY9 tetramers. Figures 7B-7E are graphs showing the clonotype hierarchy of each peptide-specific response identified by both approaches in each RM (note the concordance of TCR identification by both approaches). PBMCs from each RM were pulsed with the indicated CMV peptide (AN10 or VY9) followed by STTS, or reactive CD8+ T cells were sorted and analyzed by scRNAseq and stained with AN10 or VY9 tetramers. Figure 7F shows ICS analysis of CD8+ transducins expressing TCR2 (top) or TCR4 (bottom) cultured with (Mamu-A*02+ and MHC-E+) BLCL pulsed with the indicated peptides. Figures 7G-7J are pie charts showing clonotype hierarchy from the SIV-infected cell recognition assay, which are identical to Figure 4 except that TCR clones are shaded based on whether they cross-react with AN10 / VY9. [Figure 8A-8B] Figure 8A shows validation of MHC-Ia restriction by VY9 blocking. Figure 8A shows TCR-expressing CD8+ transduced cells were cultured with Mamu-A*02+ and MHC-E+ BLCLs pulsed with the indicated peptides (top row). In parallel, BLCLs were preincubated with the strong MHC-E-binding VL9 peptide before pulsing with the epitope peptide to assess MHC-E restriction of individual responses (bottom row). Figure 8B shows ICS assays from BLCLs preincubated with peptides with various affinities for Mamu-A*02 (CM9 = non-A*02 binder, GY9 = weak A*02 binder, YY9 = strong A*02 binder). After pulsing the pre-treated cells with AN10 peptide, these BLCLs were then used as APCs in ICS assays using TCR14-expressing CD8+ T cell transduced cells. [Figure 9] ICS demonstrating specificity analysis of dual TCR expressing clonotypes are shown. [Figures 10A-10B]Figure 10 shows functional avidity analysis of MHC-Ia and MHC-E restricted responses mediated by the same TCR. Mamu-A1*002 BLCLs were pulsed with 10-fold diluted EK9 or VY9 peptides (starting at 200 μM). BLCLs were washed and incubated with TCR2 CD8+ T cell transducants in three separate experiments. Figure 10A shows representative flow cytometry data from one experiment. Figure 10B is a graph showing results from all experiments. [Figures 11A-11G] Figure 11A shows the transcriptional response of MHC-E-restricted SIVgag-reactive CD8+ T cells with and without MHC-Ia-IE epitope cross-reactivity. Figure 11A shows a tSNE plot of scRNA-seq for purified CD8+ T cells incubated with BLCL pulsed with EK9 and RL9. Cells were clustered based on their transcriptional profiles. Colors indicate the results of unsupervised clustering. Dots indicate cells expressing previously identified TCR pairs and previously identified as MHC-E-restricted (Figure 4). Cells with these MHC-E / SIVgag-specific clones are significantly enriched within the same cluster (shown in red boxes). Figure 11B is a heatmap of the scRNA-seq data. Figure 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, NFKBID, IRF8, CD83, CD82, PLEK, and RGCC]. Figure 11D shows gating of total CD69+ cells. Figure 11E shows a tSNE plot of scRNA-seq of purified CD8+ T cells incubated with BLCL pulsed with EK9 and RL9. Figure 11F shows the activation scores in the tSNE plot. Figure 11G is a graph showing the activation scores of CD8+ T cells expressing each indicated TCR in response to each indicated antigen stimulation. The dotted blue line indicates the threshold at which cells are considered activated. DETAILED DESCRIPTION OF THE INVENTION

[0086] I. Terminology Unless otherwise specified, terminology is used according to conventional usage.

[0087] All publications, patents, patent applications, internet sites, and accession numbers / database sequences (including both polynucleotide and polypeptide sequences) cited herein are hereby incorporated by reference in their entirety for all purposes as if each individual publication, patent, patent application, internet site, or accession number / database sequence was specifically and individually indicated to be incorporated by reference.

[0088] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. In order to facilitate review of the various embodiments of the disclosure, explanations of specific terms are provided below.

[0089] Antigen: As used herein, the terms "antigen" or "immunogen" are used interchangeably to refer to a substance, typically a protein, that can induce an immune response in a subject. The term also refers to a protein that is immunologically active, in the sense that when administered to a subject (either directly or by administering to the subject a nucleotide sequence or vector encoding the protein), the protein is capable of eliciting a humoral and / or cellular immune response directed against the protein.

[0090] Antigen-specific T cells: CD8 that recognize specific antigens + or CD4 + Lymphocytes. Generally, antigen-specific T cells specifically bind to a particular antigen presented by an MHC molecule, but not to other antigens presented by the same MHC.

[0091] Administration: As used herein, the term "administration" means giving or administering an agent, such as a composition comprising an effective amount of a CMV vector containing an exogenous antigen, to a subject by any effective route. Exemplary routes of administration include, but are not limited to, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, nasal, vaginal, and inhalation routes.

[0092] Avidity: As used herein, the term "avidity" refers to the strength of the affinity of multiple individual non-covalent interactions, such as antigen-antibody interactions. Avidity is thus a measure of the overall strength of the antigen-antibody complex.

[0093] Effective amount: As used herein, the term "effective amount" refers to an amount of an agent sufficient to reduce or eliminate signs or symptoms of a condition or disease or to generate a desired response, such as inducing an immune response to an antigen, e.g., a CMV vector containing a heterologous antigen, or the amount of transfected CD8+ T cells that recognize an MHC-E / heterologous antigen-derived peptide complex, an MHC-II / heterologous antigen-derived peptide complex, or an MHC-I / heterologous antigen-derived peptide complex. In some examples, an "effective amount" treats (including prevents) one or more symptoms and / or underlying causes of any of the disorders or diseases. An effective amount can be a therapeutically effective amount, including an amount that prevents one or more signs or symptoms of a particular disease or condition, such as one or more signs or symptoms associated with an infection or cancer, from developing.

[0094] Epitope: As used herein, the term "epitope" refers to a molecular structure that may entirely constitute a specific binding partner or may be part of a specific binding partner for a binding domain or T-cell receptor domain polypeptide of the present invention. Chemically, an epitope may be composed of carbohydrates, peptides, fatty acids, organic, biochemical, or inorganic substances, or derivatives thereof, and any combination thereof. When the epitope is a polypeptide, the epitope typically contains at least three amino acids within the peptide, preferably 8-50 amino acids, and more preferably about 10-20 amino acids. There is no critical upper limit to the length of the peptide and it can include almost the entire polypeptide sequence. Epitopes can be either linear or conformational. A linear epitope is composed of a single segment of the primary sequence of a polypeptide chain. Linear epitopes can be contiguous or overlapping. A conformational epitope is composed of amino acids that are brought together by folding of the polypeptide to form a tertiary structure, and the amino acids are not necessarily adjacent to each other in the linear sequence. Specifically, an epitope is at least a portion of a diagnostically relevant molecule, i.e., the absence or presence of the epitope in a sample correlates qualitatively or quantitatively with either a disease or the health status of a patient, or with the process status or environmental and food status in manufacturing. An epitope may also be at least a portion of a therapeutically relevant molecule, i.e., a molecule that can be targeted by a specific binding domain that alters the course of a disease.

[0095] Heterologous antigen: As used herein, the term "heterologous antigen" refers to any protein or fragment thereof that is not derived from CMV. The heterologous antigen may be a pathogen-specific antigen, a tumor virus antigen, a tumor antigen, a host self-antigen, or any other antigen.

[0096] Immunogenic peptide: A peptide that contains allele-specific motifs or other sequences, such as N-terminal repeats, such that the peptide binds to an MHC molecule and induces a cytotoxic T lymphocyte ("CTL") response or a B cell response (e.g., antibody production) against the antigen from which the immunogenic peptide is derived.

[0097] In one embodiment, immunogenic peptides are identified using sequence motifs or other methods known in the art, such as neural nets or polynomial determination. Typically, an algorithm is used to determine a peptide's "binding threshold" to select peptides that have a score that confers a high probability of binding with a particular affinity and are immunogenic. The algorithm is based on either the effect of a particular amino acid at a particular position on MHC binding, the effect of a particular amino acid at a particular position on antibody binding, or the effect of a particular substitution in a motif-containing peptide on binding. Within the context of immunogenic peptides, a "conserved residue" is a residue that appears at a particular position in a peptide with a frequency significantly higher than expected by random distribution. In one embodiment, a conserved residue is a residue that may provide a contact point for the MHC structure with the immunogenic peptide.

[0098] Mutation: As used herein, the term "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 containing a mutation. Furthermore, a cell or organism having a mutation may be referred to as a mutant. Some types of coding sequence mutations include point mutations (individual nucleotide or amino acid differences), silent mutations (nucleotide differences that do not result in an amino acid change), deletions (differences resulting in the loss of one or more nucleotides or amino acids, up to the deletion of the entire coding sequence of a gene), and frameshift mutations (differences resulting in a change in the amino acid sequence due to the deletion of a number of nucleotides that is not divisible by three). Mutations resulting in amino acid differences may also be referred to as amino acid substitution mutations. Amino acid substitution mutations can be described by the amino acid change relative to the wild-type at a specific position in the amino acid sequence.

[0099] Nucleotide sequence or nucleic acid sequence: The terms "nucleotide sequence" and "nucleic acid sequence" refer to a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequence, including, but not limited to, messenger RNA (mRNA), a DNA / RNA hybrid, or a synthetic nucleic acid. A nucleic acid can be single-stranded or partially or completely double-stranded (duplex). A duplex nucleic acid can be homoduplex or heteroduplex.

[0100] Operably linked: As used herein, the term "operably linked" means that a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is positioned in such a manner that it affects the second nucleic acid sequence. Operably linked DNA sequences can be contiguous or operably spaced apart.

[0101] Promoter: As used herein, the term "promoter" can refer to any of a number of nucleic acid control sequences that direct transcription of a nucleic acid. Typically, eukaryotic promoters include a necessary nucleic acid sequence 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 recognized by one or more transcription factors. Expression by a promoter can be further regulated by enhancer or repressor elements. Numerous examples of promoters are available and well known to those skilled in the art. A nucleic acid containing a promoter operably linked to a nucleic acid sequence encoding a specific polypeptide can be referred to as an expression vector.

[0102] Recombinant: As used herein, the term "recombinant" with respect to a nucleic acid or polypeptide refers to one having a sequence that is not naturally occurring or that is created by the artificial combination of two or more separated sequence segments, such as a CMV vector containing a heterologous antigen. This artificial combination is often achieved by chemical synthesis or, more commonly, by the artificial manipulation of isolated nucleic acid segments, e.g., genetic engineering techniques. A recombinant polypeptide can also refer to a polypeptide made using a recombinant nucleic acid, including a recombinant nucleic acid (e.g., a nucleic acid encoding a polypeptide that forms a CMV vector containing a heterologous antigen) that is transcribed into a host organism that is not the natural source of the polypeptide.

[0103] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" is conventional. Remington's Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, PA, 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the compositions disclosed herein. Generally, the nature of the carrier will depend on the particular mode of administration used. For example, parenteral formulations usually contain an injectable fluid containing a pharmaceutically and physiologically acceptable fluid such as water, physiological saline, balanced salt solution, aqueous dextrose, glycerol, or the like as a vehicle. For solid compositions (such as powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions to be administered may contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate.

[0104] Polynucleotide: As used herein, the term "polynucleotide" refers to a polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). Polynucleotides consist of the four bases adenine, cytosine, guanine, and thymine / uracil (uracil is used in RNA). A coding sequence derived from a nucleic acid indicates the sequence of the protein encoded by the nucleic acid.

[0105] 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 polymers can be linear or branched, can comprise modified amino acids or amino acid analogs, and can be interrupted by chemical moieties other than amino acids. The term also encompasses amino acid polymers that are modified naturally or by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as labeling or conjugation with a biologically active moiety.

[0106] Protein orthologs are typically characterized by having greater than 75% sequence identity across the full length of the amino acid sequence of a particular protein using ALIGN with default parameters. Proteins with even greater similarity to the reference sequence will exhibit increasing percentages of sequence identity, such as at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, or at least 98% sequence identity, as assessed by this method. Furthermore, sequence identity can be compared across the full length of a particular domain of the peptides of the present disclosure.

[0107] Sequence identity / similarity: As used herein, 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 can be measured in terms of the percentage identity, with the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of the percentage identity or similarity (taking into account conservative amino acid substitutions), with 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 perform the same or similar functions to each other (e.g., proteins that perform the same function in different species or mutant forms of proteins that do not change the function or size of the protein) can be referred to as "homologs."

[0108] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in Smith & Waterman, Adv Appl Math 2, 482 (1981), Needleman & Wunsch, J Mol Biol 48, 443 (1970), Pearson & Lipman, Proc Natl Acad Sci USA 85, 2444 (1988), Higgins & Sharp, Gene 73, 237-244 (1988), Higgins & Sharp, CABIOS 5, 151-153 (1989), Corpet et al, Nuc Acids Res 16, 10881-10890 (1988), Huang et al, Computer App Biosci 8, 155-165 (1992), and Pearson et al, Meth Mol Bio 24, 307-331 (1994). Additionally, Altschul et al., J Mol Biol 215, 403-410 (1990) provide a detailed discussion of sequence alignment methods and homology calculations.

[0109] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., (1990), supra) is available from several sources, including the National Center for Biotechnology Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894) and the Internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Additional information can be found at the NCBI website.

[0110] BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. If the two compared sequences are homologous, the specified output file will present the homologous regions as aligned sequences. If the two compared sequences are not homologous, the specified output file will not present aligned sequences.

[0111] Upon alignment, the number of matches is determined by counting the number of positions where identical nucleotides or amino acid residues are present in both sequences. The percent sequence identity is determined by dividing the number of matches by either the length of the sequence shown in the specified sequence or the articulated length (e.g., 100 consecutive nucleotides or amino acid residues from the sequence shown in the specified sequence), and then multiplying the resulting value by 100. For example, a nucleic acid sequence with 1166 matches is 75.0 percent identical to a test sequence having 1154 nucleotides (1166÷1554*100=75.0). The percent sequence identity value is rounded down to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, and 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded down to 75.2. Length values ​​are always integers. In another example, a target sequence comprising a 20 nucleotide region that aligns with 20 consecutive nucleotides from a specified sequence contains a region having 75 percent sequence identity with the specified sequence (i.e., 15÷20×100=75), as follows:

[0112] For comparison of amino acid sequences greater than approximately 30 amino acids, the Blast2 sequence function is used, with the default BLOSUM62 matrix set to default parameters (gap existence cost of 11 and per residue gap cost of 1). Homologs are typically characterized by at least 70% sequence identity, counted over the full length alignment with the amino acid sequence, using gapped blastp with databases such as NCBI Basic Blast2.0, the nr database, the swissprot database, and proprietary sequence databases. Queries searched with the blastn program are filtered with DUST (Hancock & Armstrong, Comput Appl Biosci 10, 67-70 (1994)). Other programs use SEG. Additionally, manual alignments may be performed. Proteins with even greater similarity will exhibit increasing percentages of identity, such as at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the protein when assessed by this method.

[0113] When aligning short peptides (fewer than approximately 30 amino acids), alignments are performed using the Blast2 sequence function with the PAM30 matrix set to default parameters (open gap penalty of 9, extension gap penalty of 1). Proteins with even greater similarity to the reference sequence will exhibit increasing percentages of identity, such as at least approximately 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the protein when assessed by this method. When comparing less than the entire sequence for sequence identity, homologs typically have at least 75% sequence identity over a short window of 10–20 amino acids, and may have at least 85%, 90%, 95%, or 98% sequence identity, depending on the identity to the reference sequence. Methods for determining sequence identity over such short windows are described on the NCBI website.

[0114] One indicator that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions, as described above. Nevertheless, nucleic acid sequences that do not show a high degree of identity may encode the same or similar (conserved) amino acid sequences due to the degeneracy of the genetic code. This degeneracy can be used to alter the nucleic acid sequence to generate multiple nucleic acid molecules that all encode substantially the same protein. Such homologous nucleic acid sequences can, for example, have at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity with the nucleic acid encoding the protein.

[0115] Specific Binding: As used herein, the terms "specifically bind" or "specific binding" refer to the definitive binding reaction of a cognate ligand of interest in a heterogeneous population of molecules. Thus, under specified conditions (e.g., immunoassay conditions), a particular T-cell receptor domain polypeptide binds to its particular "target" but does not bind in significant amounts to other molecules present in the sample.

[0116] Subject: As used herein, the term "subject" refers to living multi-cellular vertebrate organisms, a category that includes both human and non-human mammals.

[0117] Subtope: As used herein, the term "subtope" refers to a subdominant epitope or peptide recognized by T cells.

[0118] Supertope: As used herein, the term "supertope" or "supertope peptide" refers to an epitope or peptide that is recognized by T cells in greater than about 90% of a population, regardless of MHC haplotype, i.e., regardless of the presence or absence of a given MHC-I, MHC-II, or MHC-E allele.

[0119] Treatment: As used herein, the term "treatment" refers to an intervention that ameliorates the signs or symptoms of a disease or pathological condition. As used herein, the terms "treatment," "treat," and "treating" with respect to a disease, pathological condition, or symptom also refer to any observable beneficial effect of treatment. A beneficial effect may be evidenced, for example, by a delay in the onset of clinical symptoms of a disease in a susceptible subject, a reduction in the severity of some or all clinical symptoms of a disease, a delay in the progression of a disease, a reduction in the number of disease recurrences, an improvement in the overall health or well-being of the subject, or other parameters well known in the art that are specific to a particular disease. A preventative treatment is a treatment administered to a subject who does not show signs of a disease or who shows only early signs, with the aim of reducing the risk of developing a pathological condition. A therapeutic treatment is a treatment administered to a subject after the onset of signs and symptoms of a disease.

[0120] Vaccine: An immunogenic composition that can be administered to a mammal, such as a human, to confer immunity, such as active immunity, against a disease or other pathological condition. Vaccines can be used prophylactically or therapeutically. Thus, vaccines can be used to 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, or to limit the progression of a disease or condition (such as a tumor or pathological infection), or to limit the recurrence of a disease or condition (such as a tumor). In certain embodiments, the vaccine is a replication-deficient CMV that expresses a heterologous antigen, such as a tumor-associated antigen from a lung, prostate, ovarian, breast, colon, cervical, liver, kidney, bone tumor, or melanoma.

[0121] Vector: A nucleic acid molecule of a specific sequence can be incorporated into a vector, and then the vector is introduced into a host cell, thereby generating a transformed host cell.A vector can contain a nucleic acid sequence that allows it to replicate in a host cell, such as a replication origin.A vector can also contain one or more selectable marker genes and other gene elements known in the art, including a promoter element that directs nucleic acid expression.The vector can be a viral vector, such as a CMV vector.Viral vectors can be constructed from attenuated viruses, including wild-type viruses or replication-defective viruses.

[0122] T cell receptor: As used herein, the term "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 peptide antigens presented by HLA molecules.

[0123] Multispecific T cell receptor: As used herein, the term "multispecific T cell receptor" refers to a T cell receptor that can bind 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.

[0124] II. Multispecific T cell receptor (TCR) The present invention is directed to TCRs with multiple specificities for unrelated peptides. T cells bearing these TCRs can be used to treat patients.

[0125] The present invention is also directed to a method of generating CD8+ T cells comprising a multispecific T cell receptor (TCR), the method comprising: administering to a subject a recombinant CMV vector comprising a nucleic acid sequence encoding a first heterologous antigen in an amount effective to generate a first set of CD8+ T cells that recognize a first MHC / xenogenous antigen-derived peptide complex, wherein the CMV vector does not express active UL128, UL130, UL146, and UL147 proteins 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 / xenogenous 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 a second MHC / xenogenous antigen-derived peptide complex; and isolating one or more CD8+ T cells from the second set of CD8+ T cells. identifying a second CD8+ TCR from the second set of CD8+ T cells, wherein the second CD8+ TCR recognizes the first MHC / xenoantigen-derived peptide complex and the second MHC / xenoantigen-derived peptide complex; transfecting a third set of CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding the third CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the third CD8+ TCR, and the third CD8+ TCR comprises CDR3α and CDR3β of the second CD8+ TCR, thereby generating one or more CD8+ T cells that recognize the first MHC / xenoantigen-derived peptide complex and the second MHC / xenoantigen-derived peptide complex; and selecting one or more of the third CD8+ TCRs that have the highest avidity for the particular peptide of interest.

[0126] Rhesus cytomegalovirus (RhCMV) vectors lacking functional expression of the RhCMV homologs of human CMV UL128, UL130, UL146, and UL147, while expressing homologs of UL40 and US28, efficiently elicit broadly targeted Mamu E-restricted CD8+ T cell responses in rhesus macaques to virtually any protein expressed by this vector, including both RhCMV proteins and exogenous protein inserts (the latter including bacterial, viral, and self-proteins).

[0127] 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 CMV vector. In some embodiments, the recombinant CMV does not express active UL128, UL130, UL146, and UL147 proteins due to the presence of mutations in the nucleic acid sequences encoding UL128, UL130, UL146, and UL147, or their homologs or orthologs (homologous genes in CMV that infect other species). In some embodiments, the recombinant CMV does not express active UL128, UL130, UL146, UL147, and UL18 proteins due to the presence of mutations in the nucleic acid sequences encoding UL128, UL130, UL146, UL147, and UL18, or their homologs or orthologs (homologous genes in CMV that infect other species). Mutation can be any mutation that results in the lack of expression of active UL128, UL130, UL146, UL147 or US18 protein.Such mutation can include point mutation, frameshift mutation, deletion of less than the entire protein-coding sequence (truncating mutation), or deletion of the entire nucleic acid sequence encoding protein, or any other mutation.Exemplary vectors are described in U.S. Patent Nos. 9,783,823 and 9,862,972, and U.S. Patent Application Publication No. 2018 / 0298404 (incorporated herein by reference).

[0128] In some embodiments, the recombinant CMV vector does not express active UL128, UL130, UL146, and UL147 proteins, or their homologs, or orthologues, but expresses active UL40 and US28 proteins, or their homologs, or orthologues. In some embodiments, the recombinant CMV vector does not express active UL128, UL130, UL146, UL147, and UL18 proteins, or their homologs, or orthologues, but expresses active UL40 and US28 proteins, or their homologs, or orthologues.

[0129] In some embodiments, the first MHC / xenogenous antigen-derived peptide complex is an MHC-II / xenogenous antigen-derived peptide complex, an MHC-E / xenogenous antigen-derived peptide complex, or an MHC-I / xenogenous antigen-derived peptide complex. In some embodiments, the second MHC / xenogenous antigen-derived peptide complex is an MHC-II / xenogenous antigen-derived peptide complex, an MHC-E / xenogenous antigen-derived peptide complex, or an MHC-I / xenogenous antigen-derived peptide complex.

[0130] Human or animal CMV vectors, when used as expression vectors, are essentially non-pathogenic in a selected subject, such as a human, hi some embodiments, CMV vectors are modified to render them non-pathogenic (incapable of host-to-host spread) in a selected subject.

[0131] The heterologous antigen can be any protein or fragment thereof not derived from CMV, including a tumor antigen, a pathogen-specific antigen, a model antigen (such as lysozyme, keyhole limpet hemocyanin (KLH), or ovalbumin), a tissue-specific antigen, a host self-antigen, or any other antigen.

[0132] The pathogen-specific antigen can be derived from any human or animal pathogen. The pathogen can be a viral pathogen and the antigen can be a protein derived from the viral pathogen. Viruses include, but are not limited to, retroviruses, polioviruses, adenoviruses, coxsackieviruses, hepatitis A virus, polioviruses, rhinoviruses, herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus, Epstein-Barr virus, Kaposi's sarcoma virus, 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-1), Merkel cell polyomavirus (MCV), cytomegalovirus, and parvovirus B19.

[0133] The pathogen may be a bacterial pathogen and the antigen may be a protein derived from the bacterial pathogen.Pathogenic bacteria include 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 perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Vibrio cholera, and Yersinia pestis are mentioned, but not limited to these.

[0134] The pathogen may be a parasite and the antigen may be a protein derived from the parasitic pathogen. The parasite may be a protozoan or protozoan causing diseases such as, but not limited to, Acanthamoeba, Babesiosis, Balantidiosis, Blastocystosis, Coccidia, Dinulamoeyonia, Amoebiasis, Giardiasis, Isosporosis, Leishmaniasis, Primary Amebic Meningoencephalitis (PAM), Malaria, Rhinosporidiosis, Toxoplasmosis - Parasitic Pneumonia, Trichomoniasis, Sleeping Sickness, and Chagas Disease. The parasite may be Hookworm / hookworm, Anisakiasis, Ascaris - Parasitic Pneumonia, Ascaris - Baylisascariasis, Tapeworm - Infection, Clonorchiasis, Dioctophyme renalis infection, Diphyllobothriasis - tapeworm, Dracunculiasis - dracunculiasis, Echinococcosis - tapeworm, Pinworm - pinworm disease, Clonorchis - fascioliasis, Fascioliasis - intestinal fluke, Gnathostomiasis, Hymenococcosis, Loa filariasis, Calabar swelling, Mansonellosis, Filariasis, Paragonimiasis - intestinal fluke, River blindness, Chinese liver fluke, Paragonimiasis, Lung fluke, Schistosomiasis - bilharzia, bilharziosis or snail The parasite may be a helminth organism or worm, or a disease caused by a helminth organism, such as, but not limited to, Staphylococcus aureus (all types), intestinal schistosomiasis, urinary schistosomiasis, schistosomiasis due to Schistosoma japonicum, Asian intestinal schistosomiasis, sparganosis, strongyloidiasis--a parasitic pneumonia, Beef tapeworm, Pork tapeworm, toxocariasis, trichinosis, Swimmer's itch, whipworm, and elephantiasis (lymphatic filariasis). The parasite may be an organism or a disease caused by an organism, such as, but not limited to, parasitic worm, Harzan's syndrome, myiasis, Chigoe flea, Human botfly, and Candirus.The parasite may be an ectoparasite or a disease caused by an ectoparasite, such as, but not limited to, bed bugs, head lice - pediculosis, body lice - pediculosis, pubic lice - pediculosis, Demodex - demodicosis, scabies, screwworm, and Cochliomyia.

[0135] The antigen may be a protein derived from the cancer. A tumor antigen can be any protein that is relatively restricted to tumor cells and induces an immune response. However, many tumor antigens are host (self) proteins and therefore typically are not considered antigenic by the host immune system. A tumor antigen can also be aberrantly expressed by cancer cells. A tumor antigen can also be a germline / testis antigen expressed in cancer cells, a lineage differentiation antigen not expressed in adult tissues, or an antigen overexpressed in cancer cells. Cancers include acute lymphoblastic leukemia; acute myeloid leukemia; adrenocortical carcinoma; AIDS-related cancer; 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; brain stem glioma; brain tumor; brain tumor, cerebellar astrocytoma; brain tumor, cerebral astrocytoma / malignant glioma; brain tumor, ependymoma; brain tumor, medulloblastoma; brain tumor, supratentorial primitive neuroectodermal tumor ;Brain tumors, visual pathway and hypothalamic glioma;Breast cancer;Bronchial adenoma / carcinoid;Burkitt lymphoma;Carcinoid tumor, pediatric;Carcinoid tumor, gastrointestinal;Carcinoma of unknown primary;Central nervous system lymphoma, primary;Cerebellar astrocytoma, pediatric;Cerebral astrocytoma / malignant glioma, pediatric;Cervical cancer;Childhood cancer;Chronic lymphocytic leukemia;Chronic myeloid leukemia;Chronic myeloproliferative disorder;Colon cancer;Cutaneous T-cell lymphoma;Desmoplastic small round cell tumor;Endometrial Cancer; Ependymoma; Esophageal cancer; Ewing's sarcoma in the Ewing's tumor family; Extracranial germ cell tumors, children; Extragonadal germ cell tumors; Extrahepatic bile duct cancer; Eye cancer, intraocular melanoma; Eye cancer, retinoblastoma; Gallbladder cancer; Gastric (stomach) cancer; Gastrointestinal carcinoid tumors; Gastrointestinal stromal tumors (GIST); Germ cell tumors: extracranial, extragonadal, or ovarian; Gestational trophoblastic tumors; Glioma of the brainstem; Glioma, childhood cerebral astrocytoma ;Glioma, pediatric visual pathway and hypothalamus;Gastric carcinoid;Hairy cell leukemia;Head and neck cancer;Heart cancer;Hepatocellular (liver) cancer;Hodgkin's lymphoma;Hypopharyngeal cancer;Hypothalamus and visual pathway glioma, pediatric;Intraocular melanoma;Pancreatic islet cell carcinoma (endocrine pancreas);Kaposi's sarcoma;Kidney cancer (renal cell carcinoma);Laryngeal cancer;Leukemia;Leukemia, acute lymphoblastic (also called acute lymphocytic leukemia);Leukemia, acute myeloid (also called acute myeloid leukemia);Leukemia, chronic lymphocytic leukemia (also called chronic lymphocytic leukemia); Leukemia, chronic myelogenous (also called chronic myeloid leukemia); Leukemia, hairy cell; Lip and oral cavity cancer; Liver cancer (primary); Lung cancer, non-small cell; Lung cancer, small cell; Lymphoma; Lymphoma, AIDS-related; Lymphoma, Burkitt; Lymphoma, cutaneous T-cell; Lymphoma, Hodgkin; Lymphoma, non-Hodgkin (old classification of all lymphomas except Hodgkin); Lymphoma, primary central nervous system; Marcus Whittle, fatal disease; Macroglobulinemia, warts Ludenstrom's disease; Malignant fibrous histiocytoma / osteosarcoma of bone; Medulloblastoma, childhood; Melanoma; Melanoma, intraocular (eye); Merkel cell carcinoma; Mesothelioma, adult malignant; Mesothelioma, childhood; Metastatic squamous cell carcinoma of the neck of unknown primary; Oral cancer; Multiple endocrine neoplasia syndrome, childhood; Multiple myeloma / plasma cell neoplasm; Mycosis fungoides; Myelodysplastic syndrome; Myelodysplastic / myeloproliferative disorders; Myeloid leukemia, chronic; Myeloid leukemia, adult acute; Myeloid leukemia, childhood acute; Myeloma, multiple (cancer of the bone marrow); Myeloproliferative disorders, chronic; Nasal cavity and paranasal sinus cancer; Nasopharyngeal carcinoma; Neuroblastoma; Non-Hodgkin's lymphoma; Non-small cell lung cancer Cell lung cancer; Oral cavity cancer; Oropharyngeal cancer; Osteosarcoma / Malignant fibrous histiocytoma of bone; Ovarian cancer; Ovarian epithelial cancer (superficial epithelial and stromal tumors); Ovarian germ cell tumors; Ovarian low malignant potential tumors; Pancreatic cancer; Pancreatic islet cell cancer; Paranasal sinus and nasal cavity cancer; Parathyroid cancer; Penile cancer; Pharyngeal cancer; Pheochromocytoma; Pineal astrocytoma; Pineal germinomas; Pineoblastoma and supratentorial primitive neuroectodermal tumors, children; Pituitary adenoma; Plasma cell neoplasm / multiple myeloma; Pleuropulmonary blastoma; Primary central nervous system lymphoma; Prostate cancer; Rectal cancer; Renal cell carcinoma (kidney cancer); Renal pelvis and ureter, transitional cell carcinoma; Retinal blastoma Tumor;Rhabdomyosarcoma, childhood;Salivary gland cancer;Sarcoma, Ewing family of tumors;Sarcoma, Kaposi;Sarcoma, soft tissue;Sarcoma, uterine;Sézary syndrome;Skin cancer (non-melanoma);Skin cancer (melanoma);Skin cancer, Merkel cell;Small cell lung cancer;Small intestine cancer;Soft tissue sarcoma;Squamous cell carcinoma - see Skin cancer (non-melanoma);Squamous cell carcinoma of the neck of unknown primary, metastatic;Gastric cancer;Supratentorial primitive neuroectodermal tumor, childhood;T-cell lymphoma, skin (mycosis fungoides and Sézary syndrome);Testicular cancer;Throat cancer;Thymoma, childhood;Thymoma and thymic carcinoma;Thyroid cancer;Thyroid cancer, childhood;These include, but are not limited to, transitional cell carcinoma of the renal pelvis and ureter; trophoblastic tumor, pregnancy; cancer of unknown primary site, adult; cancer of unknown primary site, pediatric; ureter and renal pelvis, transitional cell carcinoma; urethral cancer; uterine cancer, endometrium; uterine sarcoma; vaginal cancer; visual pathway and hypothalamic glioma, pediatric; vulvar cancer; Waldenstrom's macroglobulinemia; and Wilms' tumor (kidney cancer).

[0136] In some embodiments, the first heterologous antigen and the second heterologous antigen are the same. In some embodiments, the first heterologous antigen and the second heterologous antigen are different.

[0137] In some embodiments, the first CD8+ TCR recognizes a specific MHC-II, MHC-E, or MHC-I subtope or supertope. 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.

[0138] In some embodiments, one or more isolated CD8+ T cells from the second set of CD8+ T cells express CD69 and TNFα.

[0139] In some embodiments, the second CD8+ TCR recognizes one or more specific MHC-II, MHC-E, and / or MHC-I supertopes. In further examples, the second CD8+ TCR recognizes an MHC-II and an MHC-E supertope, an MHC-II and an MHC-I supertope, or an MHC-I and an MHC-E supertope.

[0140] In some embodiments, the second CD8+ TCR recognizes one or more specific MHC-II, MHC-E, and / or MHC-I subtopes. In further examples, the second CD8+ TCR recognizes an MHC-II and an MHC-E subtope, an MHC-II and an MHC-I subtope, or an MHC-I and an MHC-E subtope.

[0141] In some embodiments, the second CD8+ TCR recognizes an MHC-II subtope or supertope and an MHC-E subtope or supertope, an MHC-II subtope or supertope and an MHC-I subtope or supertope, or an MHC-I subtope or supertope and an MHC-E subtope or supertope.

[0142] In some embodiments, the second CD8+ TCR recognizes a particular MHC-II supertope and MHC-II subtope, MHC-E supertope and MHC-E subtope, or MHC-I supertope and MHC-I subtope. In some embodiments, the second CD8+ TCR recognizes two or more MHC-II supertopes from the same antigen, two or more MHC-E supertopes from the same antigen, or two or more MHC-I supertopes from the same antigen. In some embodiments, the second CD8+ TCR recognizes two or more MHC-II subtopes from the same antigen, two or more MHC-E subtopes from the same antigen, or two or more MHC-I subtopes from the same antigen.

[0143] In some embodiments, 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, the second CD8+ TCR recognizes two or more MHC-II supertopes from two or more antigens, two or more MHC-E supertopes from two or more antigens, or two or more MHC-I supertopes from two or more antigens. In some embodiments, the second CD8+ TCR recognizes two or more MHC-II subtopes from two or more antigens, two or more MHC-E subtopes from two or more antigens, or two or more MHC-I subtopes from two or more antigens.

[0144] In some embodiments, 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.

[0145] In some embodiments, the third CD8+ TCR recognizes one or more specific MHC-II supertopes, MHC-E supertopes, or MHC-I supertopes. In some embodiments, 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 a specific MHC-II supertope and MHC-II subtope, a specific MHC-E supertope and MHC-E subtope, or a specific MHC-I supertope and MHC-I subtope.

[0146] In some embodiments, the third CD8+ TCR recognizes two or more MHC-II supertopes from an antigen, two or more MHC-E supertopes from an antigen, or two or more MHC-I supertopes from an antigen. In some embodiments, the third CD8+ TCR recognizes two or more MHC-II subtopes from an antigen, two or more MHC-E subtopes from an antigen, or two or more MHC-I subtopes from an antigen.

[0147] In some embodiments, the third CD8+ TCR recognizes one or more MHC-II supertopes and one or more MHC-II subtopes from an antigen, one or more MHC-E supertopes and one or more MHC-E subtopes from an antigen, or one or more MHC-I supertopes and one or more MHC-I subtopes from an antigen. In some embodiments, the third CD8+ TCR recognizes a specific MHC-E subtope or supertope and an MHC-II subtope or supertope, a specific MHC-E subtope or supertope and an MHC-I subtope or supertope, or a specific MHC-II subtope or supertope and an MHC-I subtope or supertope.

[0148] In some embodiments, the third CD8+ TCR recognizes two or more MHC-II subtopes from the same antigen, the third CD8+ TCR recognizes two or more MHC-E subtopes from the same antigen, or the third CD8+ TCR recognizes two or more MHC-I subtopes from the same antigen. In some embodiments, 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.

[0149] In some embodiments, the nucleic acid sequence encoding the third CD8+ TCR is identical to the nucleic acid sequence encoding the second CD8+ TCR.

[0150] In some embodiments, the method includes isolating one or more CD8+ T cells from the second subject and transfecting the one or more CD8+ T cells with a nucleic acid sequence encoding a 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 / xenogenous antigen-derived peptide complex and the second MHC / xenogenous antigen-derived peptide complex.

[0151] In some embodiments, the first MHC-xenogenous antigen-derived peptide complex is an MHC-II / xenogenous antigen-derived peptide complex, an MHC-E / xenogenous antigen-derived peptide complex, or an MHC-I / xenogenous antigen-derived peptide complex. In some embodiments, the second MHC-xenogenous antigen-derived peptide complex is an MHC-II / xenogenous antigen-derived peptide complex, an MHC-E / xenogenous antigen-derived peptide complex, or an MHC-I / xenogenous antigen-derived peptide complex.

[0152] In certain embodiments, CD8+ T cells comprising multispecific TCRs can be used to prevent or treat disease. The administration route of the T cell population and the amount administered to a human patient can be determined based on the condition of the human patient and the knowledge of the physician. In some embodiments, the administration route is intravenous, intramuscular, intraperitoneal, or oral administration. Generally, administration is intravenous.

[0153] In some embodiments, the CD8+ T cells are administered to treat or prevent cancer. In further examples, the cancer is prostate cancer, kidney cancer, lung cancer, pancreatic cancer, mesothelioma, breast cancer, acute myeloid leukemia, chronic myeloid 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.

[0154] In some embodiments, the CD8+ T cells are administered to treat or prevent a pathogen infection. In further examples, the pathogen infection is human immunodeficiency virus, herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasite, Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), human T-lymphotropic virus type 1 (HTLV1), Merkel virus (MCV), cytomegalovirus, and Mycobacterium tuberculosis.

[0155] In certain embodiments, the administering is by infusion of the population of CD8+ T cells. In some embodiments, the infusion is a bolus intravenous infusion. In certain embodiments, the administering is at least about 1 x 10 of the CD8+ T cell population per kg per dose per week. 5 In certain embodiments, the administering comprises administering at least about 1 x 10 T cells per kg per dose per week of the CD8+ T cell population to the human patient. 6 The method includes administering the T cells to a human patient.

[0156] In certain embodiments, the method of treatment comprises administering at least two doses of the population of CD8+ T cells to the human patient. In certain embodiments, the method of treatment comprises administering 2, 3, 4, 5, or 6 doses of the population of T cells to the human patient.

[0157] In some embodiments, the first subject is a non-human primate and the second subject is a human, and the transfected CD8+ T cells comprise a chimeric non-human primate-human CD8+ TCR comprising the non-human primate CDR3α and CDR3β of the second CD8+ TCR. In some embodiments, the third CD8+ TCR comprises the non-human primate CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the second CD8+ TCR. In some embodiments, the third CD8+ TCR comprises the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the second CD8+ TCR. In some embodiments, the first subject is a non-human primate, the second subject is a human, and the second CD8+ TCR is a chimeric non-human primate-human CD8+ TCR comprising the non-human primate CDR3α and CDR3β of the first CD8+ TCR. In some embodiments, the third CD8+ TCR is a chimeric CD8+ TCR.

[0158] Also disclosed is a method for generating CD8+ T cells comprising a multispecific T cell receptor (TCR), the method comprising: administering to a subject a recombinant CMV vector comprising a nucleic acid sequence encoding a first heterologous antigen in an amount effective to generate a first set of CD8+ T cells that recognize a first MHC-E / xenogenous antigen-derived peptide complex, wherein the CMV vector does not express active UL128, UL130, UL146, and UL147 proteins or orthologues thereof, and 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 / xenogenous 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 a second MHC-E / xenogenous antigen-derived peptide complex; and identifying one or more CD8+ TCRs from the second set of CD8+ T cells that recognize the second MHC-E / xenogenous antigen-derived peptide complex. and identifying a second CD8+ TCR from the second set of CD8+ T cells, wherein the second CD8+ TCR recognizes the first MHC-E / xenoantigen-derived peptide complex and the second MHC-E / xenoantigen-derived peptide complex; transfecting a third set of CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding the third CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the third CD8+ TCR, and the third CD8+ TCR comprises CDR3α and CDR3β of the second CD8+ TCR, thereby generating one or more CD8+ T cells that recognize the first MHC-E / xenoantigen-derived peptide complex and the second MHC-E / xenoantigen-derived peptide complex; and selecting one or more of the third CD8+ TCRs that have the highest avidity for the particular peptide of interest.

[0159] In some embodiments, the first heterologous antigen and the second heterologous antigen are the same. In some embodiments, the first heterologous antigen and the second heterologous antigen are different. In some embodiments, the subject is a human or a non-human primate. In some embodiments, the recombinant CMV vector is a recombinant human CMV vector or a recombinant rhesus CMV vector.

[0160] In some embodiments, the first heterologous antigen comprises a tumor antigen, a pathogen-specific antigen, a tissue-specific antigen, or a host autoantigen. In some embodiments, the tumor antigen is associated with a cancer selected from the group consisting of prostate cancer, kidney cancer, lung cancer, pancreatic cancer, mesothelioma, breast cancer, acute myeloid leukemia, chronic myeloid 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. In some embodiments, the pathogen-specific antigen is associated with 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 parasite, Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), human T-lymphotropic virus type 1 (HTLV1), Merkel virus (MCV), cytomegalovirus, and Mycobacterium tuberculosis.

[0161] In some embodiments, the MRE comprises a target site for a microRNA expressed in endothelial cells, hi further embodiments, the MRE is specific for a 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.

[0162] In some embodiments, the first CD8+ TCR recognizes a specific MHC-E subtope or supertope. 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.

[0163] In some embodiments, one or more isolated CD8+ T cells from the second set of CD8+ T cells express CD69 and TNFα.

[0164] In some embodiments, the second CD8+ TCR recognizes one or more specific MHC-E supertopes. In some embodiments, the second CD8+ TCR recognizes one or more specific MHC-E subtopes. In some embodiments, the second CD8+ TCR recognizes a specific MHC-E supertope and MHC-E subtope.

[0165] In some embodiments, the second CD8+ TCR recognizes two or more MHC-E supertopes from the same antigen. In some embodiments, the second CD8+ TCR recognizes two or more MHC-E subtopes from the same antigen.

[0166] In some embodiments, the second CD8+ TCR recognizes one or more MHC-E supertopes and one or more MHC-E subtopes from the same antigen, hi some embodiments, the second CD8+ TCR recognizes two or more MHC-E subtopes from two or more antigens.

[0167] In some embodiments, the second CD8+ TCR recognizes one or more MHC-E supertopes and one or more MHC-E subtopes from different antigens.

[0168] In some embodiments, the third CD8+ TCR recognizes one or more specific MHC-E supertopes. In some embodiments, the third CD8+ TCR recognizes one or more specific MHC-E subtopes. In some embodiments, the third CD8+ TCR recognizes a specific MHC-E supertope and MHC-E subtope.

[0169] In some embodiments, the third CD8+ TCR recognizes two or more MHC-E supertopes from an antigen. In some embodiments, the third CD8+ TCR recognizes two or more MHC-E subtopes from an antigen.

[0170] In some embodiments, the third CD8+ TCR recognizes one or more MHC-E supertopes and one or more MHC-E subtopes from an antigen.

[0171] In some embodiments, the third CD8+ TCR recognizes two or more MHC-E subtopes from the same antigen, hi some embodiments, the third CD8+ TCR recognizes one or more MHC-E supertopes and one or more MHC-E subtopes from different antigens.

[0172] In some embodiments, the nucleic acid sequence encoding the third CD8+ TCR is identical to the nucleic acid sequence encoding the second CD8+ TCR.

[0173] In some embodiments, the method includes isolating one or more CD8+ T cells from the second subject and transfecting the one or more CD8+ T cells with a nucleic acid sequence encoding a 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 / xenogenous antigen-derived peptide complex and the second MHC-E / xenogenous antigen-derived peptide complex.

[0174] In certain embodiments, CD8+ T cells comprising multispecific TCRs can be used to prevent or treat disease. The administration route of the T cell population and the amount administered to a human patient can be determined based on the condition of the human patient and the knowledge of the physician. In some embodiments, the administration route is intravenous, intramuscular, intraperitoneal, or oral administration. Generally, administration is intravenous.

[0175] In some embodiments, the CD8+ T cells are administered to treat or prevent cancer. In further examples, the cancer is prostate cancer, kidney cancer, lung cancer, pancreatic cancer, mesothelioma, breast cancer, acute myeloid leukemia, chronic myeloid 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.

[0176] In some embodiments, the CD8+ T cells are administered to treat or prevent a pathogen infection. In further examples, the pathogen infection is human immunodeficiency virus, herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasite, Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), human T-lymphotropic virus type 1 (HTLV1), Merkel virus (MCV), cytomegalovirus, and Mycobacterium tuberculosis.

[0177] In certain embodiments, the administering is by infusion of the population of CD8+ T cells. In some embodiments, the infusion is a bolus intravenous infusion. In certain embodiments, the administering is at least about 1 x 10 of the CD8+ T cell population per kg per dose per week. 5 In certain embodiments, the administering comprises administering at least about 1 x 10 T cells per kg per dose per week of the CD8+ T cell population to the human patient.6 The method includes administering the T cells to a human patient.

[0178] In certain embodiments, the method of treatment comprises administering at least two doses of the population of CD8+ T cells to the human patient. In certain embodiments, the method of treatment comprises administering 2, 3, 4, 5, or 6 doses of the population of T cells to the human patient.

[0179] In some embodiments, the first subject is a non-human primate and the second subject is a human, and the transfected CD8+ T cells comprise a chimeric non-human primate-human CD8+ TCR comprising the non-human primate CDR3α and CDR3β of the second CD8+ TCR. In some embodiments, the third CD8+ TCR comprises the non-human primate CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the second CD8+ TCR. In some embodiments, the third CD8+ TCR comprises the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the second CD8+ TCR. In some embodiments, the first subject is a non-human primate, the second subject is a human, and the second CD8+ TCR is a chimeric non-human primate-human CD8+ TCR comprising the non-human primate CDR3α and CDR3β of the first CD8+ TCR. In some embodiments, the third CD8+ TCR is a chimeric CD8+ TCR.

[0180] The multispecific TCRs disclosed herein can be used in a method of inducing an immune response in a subject, comprising administering to the subject a composition comprising CD8+ T cells comprising the multispecific TCR and a pharmaceutically acceptable carrier or diluent. For purposes herein, 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. Of course, a subset of "animal" is "mammal," which for purposes herein includes all mammals except humans.

[0181] With regard to antigens for use in vaccines or immune compositions, see also Stedman's Medical Dictionary (24th ed., 1982, e.g., the definition of vaccine, for a list of antigens used in vaccine formulations). Any such antigens of interest or epitopes derived from those antigens may be used. With regard to tumor antigens, one skilled in the art can select tumor antigens and their encoding DNA without undue experimentation from knowledge of the amino acids and corresponding DNA sequences of peptides or polypeptides, as well as the properties of particular amino acids (e.g., size, charge, etc.) and the codon dictionary.

[0182] A wide variety of suitable host cells may be used to express the multispecific TCRs of the present 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). For example, various 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. Additionally, plants and animals may be used as hosts for expression of the T cell receptors of the present invention. Expression and transfection vectors or cassettes may be selected according to the host to be used.

[0183] Of course, cell-free or cell-free protein expression systems can also be used. In vitro transcription / translation protein expression platforms that produce sufficient amounts of protein offer many of the advantages of cell-free protein expression, eliminating the need for laborious upstream and downstream steps (e.g., host cell transformation, culturing, or lysis) typically associated with cell-based expression systems.

[0184] Immune responses to tumor antigens are generally generated as follows: T cells recognize proteins only when they are cleaved into smaller peptides and presented in complexes called "major histocompatibility complexes (MHC)" located on the surface of other cells. There are two classes of MHC complexes, class I and class II, and each class consists of many different alleles. Different species and individual subjects have different types of MHC complex alleles. They are said to have different MHC species. One type of MHC class I molecule is called MHC-E (HLA-E in humans, Mamu-E in RM, and Qa-lb in mice). Unlike other MHC-I molecules, MHC-E is highly conserved within and between mammalian species.

[0185] Pharmaceutical and other compositions comprising the disclosed multispecific TCRs are further disclosed. Such pharmaceutical and other compositions can be formulated for use in any administration procedure known in the art. Such pharmaceutical compositions can be administered via parenteral routes (intradermal, intramuscular, subcutaneous, intravenous, or other). Administration can also be via mucosal routes such as oral, nasal, or genital.

[0186] The pharmaceutical compositions of the present disclosure may be prepared according to 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 account factors such as the breed or species, age, sex, weight, and condition of the particular patient, as well as the route of administration. The compositions may be administered alone or in combination with other immunological, antigenic, or therapeutic compositions, either simultaneously or sequentially.

[0187] The CMV vectors of the present disclosure can be administered in vivo to generate immunogenic responses, including CD8+ immune responses, including immune responses characterized by a high proportion of CD8+ T cell responses restricted by MHC-E, MHC-II, or MHC-I (or their homologs or orthologs). For example, in some instances, it may be desirable to use the CMV vectors of the present disclosure for preclinical testing of immunogenic compositions and vaccines using RhCMV in laboratory animals such as rhesus monkeys. In other instances, it may be desirable to use the CMV vectors of the present disclosure for actual clinical use of immunogenic compositions using HCMV in human subjects, such as in clinical trials.

[0188] For such in vivo applications, the CMV vectors of the present disclosure are administered as a component of an immunogenic composition further comprising a pharmaceutically acceptable carrier. In some embodiments, the immunogenic compositions of the present disclosure are useful for stimulating an immune response against heterologous antigens, including tumor antigens, tumor virus antigens, or host self-antigens, 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 present disclosure are particularly useful for providing genetic vaccines, i.e., for delivering nucleic acids encoding the antigens of the present disclosure to a subject, such as a human, so that the antigen is then expressed in the subject and induces an immune response.

[0189] Immunization schedules (or regimens) are well known for animals (including humans) and can be readily determined for a particular subject and immunogenic composition. Thus, the immunogen may be administered to a subject one or more times. Preferably, there is a set time interval between separate administrations of the immunogenic composition. This interval varies from subject to subject, but typically ranges from 10 days to several weeks, [often 2, 4, 6, or 8 weeks]. In humans, the interval is typically 2 to 6 weeks. In particularly advantageous embodiments of the present disclosure, the intervals are longer, advantageously about 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, or 70 weeks. The immunization regime typically involves one to six administrations of the immunogenic composition, but may be as few as one, two, or four. The method of inducing an immune response may also include administering an adjuvant with the immunogen. In some cases, the initial immunization protocol may be supplemented with booster immunizations annually, twice a year, or at other longer intervals (5-10 years). The methods of the present invention also include various prime-boost regimens. In these regimens, 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., including a protein or expression vector), route, and formulation of the immunogen may also vary. For example, if an expression vector is used in the priming and boosting steps, it may be of the same or different types (e.g., DNA or bacterial or viral expression vectors). One useful prime-boost regimen provides two priming immunizations four weeks apart, followed by two boosting immunizations at weeks four and eight after the final priming immunization.It should also be readily apparent to one skilled in the art that several permutations and combinations may be encompassed to provide priming and boosting regimens using the DNA, bacterial, and viral expression vectors of the present disclosure. CMV vectors may be used repeatedly, expressing different antigens derived from different pathogens. [Example]

[0190] Example 1: TCR clonotype hierarchy of MHC-E supertope responses On average, after vaccination with an RhCMV vector expressing the UL40 and US28 homologs but lacking functional expression of the RhCMV homologs of human CMV UL128, UL130, UL146, and UL147, approximately one Mamu E-restricted CD8+ T cell epitope (typically a 9-mer) can be identified for every 30–40 amino acid protein length. Responses to some of these epitopes are common to all monkeys, and these epitopes are referred to as supertopes. For example, four animals vaccinated with strain 68-1 RhCMV expressing the antigen SIVgag elicited T cell responses to the MHC-E supertopes gag69 and gag120 (Figures 1A and 1B) (strain 68-1 naturally acquired by genetic recombination, as described above).

[0191] Characterization of the TCRs responsible for SIV-specific CD8+ T cell responses from four rhesus macaques (RMs) vaccinated with the long-term 68-1 RhCMV / SIVgag vector, which developed long-term, well-characterized, unconventionally restricted SIVgag-specific CD8+ T cell responses elicited and maintained by 68-1 RhCMV / SIVgag vaccination in these animals over the past 15 years, is shown in Figures 1A and 1B. These RMs were also used to test the 68-1 RhCMV / TB vector expressing ESAT-6 and Ag85B TB antigens approximately 4 years after RhCMV / SIVgag vaccination.

[0192] Using surface-captured TNF staining (STTS, Figure 2A), viable (non-fixed) epitope-reactive CD8+ T cells could be sorted from these RMs suitable for single-cell (sc) transcriptome analysis, including sequencing of all expressed TCR chains and global analysis of each cell's transcriptome. 276~284 RL9 (Gag69) and Gag 482~499 Characterization of the TCR clonotype structure of 68-1 RhCMV / SIV vector-induced CD8+ T cell responses to EK9 (Gag120) was performed by analyzing sorted CD8+ T cells responding to these epitopes by both CD69 upregulation and surface-acquired TNF expression (this double-positive criterion was used for maximum specificity; however, it is noteworthy that not all responding clonotypes express detectable stTNF, so responding clonotype+ cells may also be in the CD69+ / stTNF- fraction. See Figure 2A for a comparison of parallel ICS and STTS assays). STTS was used longitudinally over a 3-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 RNA sequencing, allowing identification of their complete TCRα / β hierarchy. Remarkably, when Gag supertope-responsive CD8+ T cells were analyzed for TCR expression, each of the four study RMs displayed a stable, highly oligoclonal clonotype hierarchy for both Gag276-284RL9 and Gag482-499EK9, and unexpectedly, many clonotypes were common across these two supertope-specific responses (despite the fact that the two supertope-optimal 9-mer peptides have essentially no sequence homology (Figures 2B-2E)).

[0193] Example 2: Some MHC-E-restricted CD8+ TCRs recognize sequence-unrelated supertopes and endogenously processed antigens Each of the major TCR alpha / beta chain pairs from all four RMs was cloned for specificity analysis using transduction of primary control (SIV Ag naive) RM CD8+ T cells. As shown in Figure 2F, each scRNAseq-identified TCR was specifically expressed in Gag 276~284 RL9, Gag 482~499 EK9, or both, confirming the specificity revealed by scRNAseq and clearly demonstrating that individual TCRs can have dual specificity for these two MHC-E-restricted supertopes.

[0194] Furthermore, these TCRs were able to specifically recognize SIVgag-transfected SIVmac239-infected CD4+ T cells and B lymphoblastoid cell lines (BLCLs) (Figures 3A-3B), demonstrating that SIVgag epitope peptides can be effectively processed and surface-expressed in the context of MHC-E in non-RhCMV-infected cells.

[0195] Example 4: Broad epitope specificity of MHC-E-restricted CD8+ T cell responses is mediated by a small number of TCRs As shown in Figure 1A, the SIVgag-specific CD8+ T cells maintained in these four RMs recognize at least 9–16 different MHC-E-restricted epitopes and 23–27 MHC-II-restricted epitopes. To identify the full range of TCR clonotypes in these RMs that can recognize naturally processed SIVgag in SIV-infected cells, CD8+ T cells from each study RM were stimulated with autologous SIV-infected CD4+ T cells, responder cells were identified by STTS, responder cells were sorted based on sCD69 and stTNF, and then responder cells were analyzed by scRNAseq (shown in Figure 2).

[0196] To determine the clonal hierarchy for SIV infection recognition, purified CD8+ T cells from each study RM were incubated with autologous SIV-infected CD4+ T cells. Activated cells were sorted based on sCD69 and s-tTNF-α staining, followed by single-cell RNA-seq. Pie charts show the relative frequency of each clone. Additionally, clones identified in greater than 5% of responder cells across at least two separate supertope peptide stimulations are also named, although clones present in less than 5% of responder cells in this experiment. Clone names, alpha / beta CDR3 sequences, and V / J segment usage are listed in Tables 1–4. [Table 1] [Table 2] [Table 3] [Table 4]

[0197] Notably, several clones (TCR1-1 / 2, TCR6-1 / 2, and TCR13-1 / 2, highlighted in gray) encoding two alpha chains and one beta were identified. While there are no completely shared clonotypes between RMs, there is one shared alpha chain between Rh-3 and Rh-1 (Figures 4A and 4B, TCR1-1 and TCR4, red).

[0198] Notably, the TCR hierarchy of CD8+ T cells responding to SIV-infected cells was highly oligoclonal and composed almost entirely (90%+) of TCRs previously identified by MHC-E-restricted supertope reactivity (Figures 4A-4D). With the exception of one TCR alpha chain shared between Rh-3 and Rh-1, these TCRs were distinct in each RM. Of note, two Rh-3 clonotypes and one Rh-1 clonotype expressed two TCR alpha chains, raising the possibility that these cells might express two distinct TCRs, where the beta chain could pair with either alpha chain.

[0199] Example 5: MHC-E-restricted TCRs that recognize multiple unrelated peptides These data raise the question of where TCRs recognizing non-supertope epitopes (termed "subtopes") lie in these SIV-infected cell recognition assays: are these subtopes unprocessed or presented on MHC-E in SIV-infected cells (e.g., suggesting that only supertopes are properly processed / presented (a highly unlikely possibility)), or are supertope-responsive TCRs, which have already been shown to be cross-reactive across supertopes in many cases, also cross-react with subtopes? Indeed, as shown in Figure 5, the latter possibility is likely.

[0200] MHC-E-TCR CD8+ T cell transfectants from the four overall RM study cohorts were tested against a panel of MHC-E-restricted optimal peptides recognized in one of the study RMs, as shown in Figure 5. For an epitope to be considered positive, it must have a stimulatory response of >0.3% above background in three or more independent assays. The overall response pattern for each TCR is shown in Tables 5-8 (Note: Not all target MHC-E-presented peptides are triggered in all assays). ND: No data (analysis pending). [Table 5] [Table 6] [Table 7] [Table 8]

[0201] Of 17 supertope-reactive TCRs tested for reactivity to 10 of the most common subtopes, 12 of these TCRs showed cross-reactivity with at least one and up to all 10 of these subtopes (five other TCRs only showed reactivity to one or both supertopes, but cross-reactivity with other untested subtopes cannot be excluded). These data clearly demonstrate that the MHC-E-restricted CD8+ T cell responses elicited and maintained by the 68-1 RhCMV / SIVgag vector primarily use polyspecific TCRs and suggest that the majority of TCR clonotypes comprising the vaccine-induced responses in these RMs have the potential to recognize multiple distinct MHC-E-presented epitopes on the surface of SIV-infected target cells.

[0202] Example 6: Generation of MHC-E-restricted TCRs that recognize more than one antigen As shown in Figure 1A, all RMs in this study cohort were prevaccinated with the 68-1 RhCMV / TB vector expressing the ESAT-6 / Ag85B polyprotein. ScRNA analysis of CD8+ T cell responses to the Ag85B peptide mixture reveals that at least one TCR previously characterized as SIVgag-specific (TCR9) also responds to epitopes within this heterologous Ag (Figure 6A; TCR6 from Rh33034 appears to have similar cross-reactivity). Three of the five dominant clonotypes previously identified by their MHC-E-restricted SIVgag reactivity also respond to one or both MHC-II-restricted SIVgag supertope peptides, and one of these TCRs (TCR9) also responds to the TB Ag85B epitope (Figure 6B).

[0203] Thus, by a mechanism yet to be characterized, RMs vaccinated with 68-1 RhCMV vectors develop Ag-targeted responses via highly cross-reactive TCRs, with cross-reactivity involving not only MHC-E-presented epitopes within the specific Ag insert but also MHC-E-restricted epitopes within heterologous inserts expressed by 68-1 RhCMV-based vaccines administered at different times.

[0204] Example 7: Several MHC-E-restricted SIVgag-specific TCRs are derived from MHC-Ia-restricted RhCMV-IE1-specific TCRs All four of the study RMs were naturally infected with RhCMV during their first year of life and, like all RMs with natural RhCMV infection, expressed classically MHC-Ia-restricted responses to RhCMV Ags, almost certainly including substantial responses to the RhCMV immediate-early-1 (IE-1) protein (a highly expressed viral protein frequently targeted by T cells). All four study RMs typically expressed responses to two highly immunodominant IE-1 epitopes: IE 1313-323 AN10 and IE 1291-299Expressing the VY9-restricting Mamu-A*02 allele, analysis with Mamu-A*02 / AN10 and Mamu-A*02 / VY9 tetramers revealed that all four RMs displayed robust CD8 + T cell responses to both epitopes ( Fig. 7A ).

[0205] We isolated the CD8+ T cells that comprised these responses by sorting based on both Mamu-A*02 / AN10 / Mamu-A*02 / VY9 tetramer binding and sCD69 and stTNF upregulation in response to peptide stimulation with STTS, and analyzed the sorted cells by scRNAseq as described above. Remarkably and quite surprisingly, several TCRs identified by this analysis turned out to be the same TCRs previously shown to be driven by MHC-E-restricted SIVgag supertopes (Figures 7B-7E). Interestingly, as expected for conventional MHC-Ia-restricted CD8+ T cell responses, the TCRs recognizing AN10 and VY9 were distinct, yet both recognized unconventionally restricted SIVgag supertopes / subtopes.

[0206] Functional analysis using TCR transfectants confirmed specific triggering of the relevant TCR by both Mamu-A*02-restricted epitopes and one or both MHC-E-restricted SIVgag supertopes (Fig. 7F).

[0207] Next, we verified MHC-Ia restriction by VY9 blocking. Note that VL9 preincubation blocked binding and TCR2-mediated recognition of the SIVgagEK9 supertope, but not VY9 binding / recognition. Blocking analysis confirmed that the dual reactivity of these transduced CD8+ T cells was distinct with respect to the MHC molecule used for epitope presentation. Recognition of the SIVgag supertope by TCR transducers was blocked by preincubation with the strongly MHC-E-binding VL9 peptide, whereas recognition of the Mamu-A*02-restricted IE-1 epitope by the same transducers was not. Conversely, TCR transducer recognition of the Mamu-A02 IE-1 epitope could be blocked by the Mamu-A*02-binding peptide in proportion to their binding strength (Figures 8A and 8B). Remarkably, TCRs with both conventional IE-1-specific and unconventional SIVgag-specific reactivity accounted for the majority (but not all) of the TCRs involved in SIV-infected cell recognition in these four 68-1 RhCMV / SIVgag-vaccinated RMs ( Figures 7G–7J ).

[0208] Example 8: Dual MHC specificity of CD8+ T cells can result from the expression of two TCR subunits As shown in Figures 4A-4D, three of the SIVgag supertope-reactive T cell clones identified in the analysis of the four study RMs expressed two distinct TCR alpha chains and one TCR beta chain, potentially forming two distinct TCRs. To examine the specificity of each pair, transducants for each pair (TCR6-1 and TCR6-2) were generated separately. The sequences of TCR6-1 and TCR6-2 are shown in Tables 9 and 10. [Table 9] [Table 10]

[0209] Interestingly, both TCRs bind to Gag482~499 Although they recognize EK9 and are broadly (though not identically) cross-reactive with multiple SIVgag subtopes (Fig. 5), only one of these pairs (TCR6.2) recognizes the Mamu-A*02 epitope (VY9) (Fig. 9). These data suggest that conventional VY9 specificity is primarily TCR alpha chain dependent, whereas MHC-E SIVgag specificity is primarily TCR beta chain mediated, and offer unique insights into possible mechanisms by which some of these aberrant cross-reactivities may arise.

[0210] Example 9: Functional avidity of dual MHC-specific TCRs When a given TCR with Mamu-A*02 epitope and MHC-E supertope / subtope multispecificity was analyzed side by side, responses to the Mamu-A*02 epitope were often larger (more responding cells) and stronger (more cytokine production) than optimal MHC-E supertope responses (see Figures 7B-7E, 8A; the TCR6.2 response in Figure 10, bottom panel, is an exception). This is not surprising, given that the initial recruitment of these TCR clonotypes in the memory compartment almost certainly occurred during the original wild-type RhCMV infection, but it was important to compare these functional differences in more detail.

[0211] As a first step toward this goal, we performed a parallel epitope dilution analysis with TCR2 CD8+ T cell transducers. TCR2 was chosen because it is one of the most consistently potent TCRs in terms of its response to MHC-E supertopes and also recognizes the IE-1 VY9 epitope. We hypothesized that if triggering by an MHC-E supertope was impaired by either weak / unstable binding of the supertope peptide within the MHC-E peptide-binding groove or by low TCR avidity for the supertope-MHC-E complex, supertope-mediated triggering would begin similarly (or perhaps less efficiently) as triggering by the conventional VY9 epitope at high epitope doses, but would then fall off more rapidly with epitope dilution, such that demonstrable triggering by the conventional epitope extended to much lower peptide doses than for the non-conventional epitope. Interestingly, this was not observed (Figures 10A and 10B). Even at the highest peptide dose, responses to the MHC-E supertope elicited fewer TCR2-transduced cells than the conventional VY9 epitope, but the response of this smaller population declined at roughly the same rate as the larger population elicited by the conventional epitope. This finding suggests that, at least for this TCR, transduced cells can respond to MHC-E supertopes similarly to conventional epitopes if they have the "right" environment, but not all transduced cells have the correct epigenetic landscape to respond. TCR transduction was performed on peripheral blood CD8+ T cells from control animals, and the activation required for transduction converted all transduced cells to a memory phenotype. However, it is important to remember that the origin of these cells is diverse, and therefore there is likely heterogeneity in the epigenetic landscape of transduced cells. The factors that determine a cell's ability to be elicited by an MHC-E supertope remain to be determined at this point in the project. In addition, scRNA was used to determine whether MHC-E supertope non-responsive transfectants were indeed responsive but had a distinct activation response that did not include TNF-α or γ-IFN production.

[0212] Example 10: TCR ex vivo functional analysis To explore differences in triggering efficiency between conventional and nonconventional recognition by cells naturally bearing these TCRs, we used scRNAseq to analyze cells harvested ex vivo from the study RM. While the focus of scRNAseq use to date has been single-cell determination of TCR expression, available data includes whole transcriptomes. Ag-activated CD8+ T cells were primarily sorted prior to scRNAseq analysis (stTNF+ / sCD69+). This was primarily done to enrich for Ag-responsive cells of interest to reduce costs, and the transcriptomes of Ag-responsive cells also provide clear evidence of TCR-mediated activation, readily recognizable by the clustering of responding cells (with their associated TCR) in the tSNE plot (Figure 11A) and by differential gene expression of putatively activated "clusters" from other clusters identified in the tSNE plot, including canonical TCR-triggered genes (Figure 11B). We used differentially upregulated genes associated with TCR-mediated activation to generate a composite activation score that provides a quantitative assessment of cellular responses to a given Ag (Figure 11C). We studied isolated total CD69+ cells to enrich for mostly activated cells (Figures 11D-11F, left panels). Within this mostly activated subset, we then determined the activation scores of CD8+ T cells expressing relevant TCRs within this RM (see Figures 4A-4D, TCRs 4, 5, 6, and 12) against three different stimuli: i) the IE-1 peptide VY9 + AN10, ii) the MHC-E SIVgag supertope peptide RL9 + EK9, and iii) SIV-infected cells (Figures 11D-11G).

[0213] For each assay, total CD69+ cells, enriched for activated cells but containing background and scored for activation as described above, were sorted (Figure 11D). Again, cells expressing cross-reactive TCR clusters were activated and evaluated separately (Figures 11E and 11F). The activation scores of CD8+ T cell responses expressing each indicated TCR to each indicated Ag stimulus were assessed separately (Figure 11G). It is shown that TCR4 and TCR6 respond to AN10 or VY9, whereas TCR5 and TCR12 do not. Of the two TCRs that react with the MHC-E SIVgag supertope and MHC-Ia epitope, the activation distribution suggests, at least for TCR4, more efficient activation by the MHC-Ia epitope (rightward shift), compensated for by the multiple MHC-E SIVgag epitopes presented by SIV-infected cells.

[0214] As noted above (Figure 9), TCR6+ cells express two TCRs that share a common beta chain; therefore, the responses of these cells likely reflect a complex of both TCRs. As expected from the block analysis (Figures 7A-7J), only TCR4 and TCR6 respond to IE-1 VY9+AN10, while TCR5 and TCR12 are either completely absent within the CD69+ gate or, if present, exhibit subzero activation scores. In contrast, the TCR6 response to VY9 / AN10 is robust and unimodal, despite the fact that only one of the two TCRs in this clonotype responds to one of these peptides (VY9). The TCR6 response to supertope peptides and to SIV-infected cells is overall slightly but discernibly left-shifted relative to the IE-1 VY9 response, appearing bimodal, suggesting some cells are fully activated and others have less induction of activation gene sets. While TCR4 responses to IE peptides are slightly weaker than TCR6 responses, a key difference with this TCR is its trimodal response to MHC-E supertopes, which includes strong, weak, and no responses to these peptides, along with a robust response to SIV-infected cells. Notably, TCR4 recognizes four MHC-E subtopes (Figure 5, often more potent than the supertope), and SIV-infected cells likely present these additional target epitopes, almost certainly resulting in a stronger and more uniform response to multivalent stimulation. Conversely, TCR5 is one of the supertope-only TCRs (e.g., no known recognized subtopes; Figure 5), and its response to optimal supertope peptides in native cells is clearly stronger than in SIV-infected cells. Although TCR12 has not yet been tested for subtope reactivity, this TCR exhibits the same supertope > SIV-infected cell triggering pattern as TCR5, suggesting that it may also have lower cross-reactivity with subtopes.

[0215] Overall, these data suggest that, in the context of their native (RhCMV vector-induced) CD8+ T cells, MHC-E SIVgag-reactive TCRs are heterogeneous but clearly capable of inducing "intact" transcriptionally defined activation responses (similar to those elicited by MHC-Ia epitopes) in the majority of cells bearing SIVgag supertope peptides or SIV-infected cells, or both, suggesting that these native (CMV "reared") CD8+ T cells, in contrast to CD8+ T cell TCR transducers, possess the epigenetic landscape necessary to adequately respond to these non-conventional epitopes.

Claims

1. 1. A method for generating CD8+ T cells comprising a multispecific T cell receptor (TCR), comprising: (a) administering to a subject a recombinant cytomegalovirus (CMV) vector comprising a nucleic acid sequence encoding 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 active UL128, UL130, UL146, and UL147 proteins 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 / foreign antigen-derived peptide complex; and (c) administering to the subject a second heterologous antigen in an amount effective to generate a second set of CD8+ T cells that recognize a second MHC / heterologous antigen-derived peptide complex; (d) isolating one or more CD8+ T cells from the second set of CD8+ T cells; and (e) identifying a second CD8+ TCR from the second set of CD8+ T cells, wherein the second CD8+ TCR recognizes the first MHC / xenoantigen-derived peptide complex and the second MHC / xenoantigen-derived peptide complex; (f) transfecting a third set of CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a third CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the third CD8+ TCR, wherein the third CD8+ TCR comprises CDR3α and CDR3β of the second CD8+ TCR, thereby generating one or more CD8+ T cells that recognize the first MHC / xenoantigen-derived peptide complex and the second MHC / xenoantigen-derived peptide complex; (g) selecting one or more of said third CD8+ TCRs that have the highest avidity for a particular peptide of interest.

2. The method of claim 1 , wherein the recombinant CMV vector does not express an active UL18 protein.

3. 3. The method of claim 1 or 2, wherein the recombinant CMV vector expresses an active UL40 protein or an orthologue thereof, and an active US28 protein or an orthologue thereof.

4. The method according to any one of claims 1 to 3, wherein the first MHC / xenogenous antigen-derived peptide complex is an MHC-II / xenogenous antigen-derived peptide complex, an MHC-E / xenogenous antigen-derived peptide complex, or an MHC-I / xenogenous antigen-derived peptide complex.

5. The method according to any one of claims 1 to 4, wherein the second MHC / xenogenous antigen-derived peptide complex is an MHC-II / xenogenous antigen-derived peptide complex or an MHC-E / xenogenous antigen-derived peptide complex.

6. The method of any one of claims 1 to 5, wherein the subject is a human or non-human primate.

7. The method of any one of claims 1 to 6, wherein the recombinant CMV vector is a recombinant human CMV vector or a recombinant rhesus CMV vector.

8. The method of any one of claims 1 to 7, wherein the first and / or second heterologous antigen comprises a tumor antigen, a pathogen-specific antigen, a tissue-specific antigen, or a host self-antigen.

9. 9. The method of claim 8, wherein the tumor antigen is associated with a cancer selected from the group consisting of prostate cancer, kidney cancer, lung cancer, pancreatic cancer, mesothelioma, breast cancer, acute myeloid leukemia, chronic myeloid 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.

10. 9. The method of claim 8, wherein the pathogen-specific antigen is associated with 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 parasite, Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), human T-lymphotropic virus type 1 (HTLV1), Merkel virus (MCV), cytomegalovirus, and Mycobacterium tuberculosis.

11. The method of any one of claims 1 to 10, wherein the first CD8+ TCR recognizes a specific MHC-II subtope or supertope.

12. The method of any one of claims 1 to 10, wherein the first CD8+ TCR recognizes a specific MHC-E subtope or supertope.

13. The method of any one of claims 1 to 10, wherein the first CD8+ TCR recognizes a specific MHC-I subtope or supertope.

14. 14. The method of any one of claims 1 to 13, wherein the first CD8+ TCR is identified by DNA or RNA sequencing.

15. 15. The method of claim 14, wherein the first CD8+ TCR is identified by single-cell sequencing.

16. The method of any one of claims 1 to 15, wherein the first heterologous antigen and the second heterologous antigen are the same.

17. The method of any one of claims 1 to 15, wherein the first heterologous antigen and the second heterologous antigen are different.

18. 18. The method of any one of claims 1 to 17, wherein the one or more isolated CD8+ T cells from the second set of CD8+ T cells express CD69 and TNFα.

19. 19. The method of any one of claims 1 to 18, wherein the second CD8+ TCR recognizes one or more specific MHC-II supertopes.

20. 19. The method of any one of claims 1 to 18, wherein the second CD8+ TCR recognizes one or more specific MHC-E supertopes.

21. 19. The method of any one of claims 1 to 18, wherein the second CD8+ TCR recognizes one or more specific MHC-I supertopes.

22. 21. The method of claim 19 or 20, wherein the second CD8+ TCR recognizes an MHC-II supertope and an MHC-E supertope.

23. 22. The method of claim 20 or 21, wherein the second CD8+ TCR recognizes an MHC-I supertope and an MHC-E supertope.

24. 22. The method of claim 19 or 21, wherein the second CD8+ TCR recognizes an MHC-I supertope and an MHC-II supertope.

25. 19. The method of any one of claims 1 to 18, wherein the second CD8+ TCR recognizes one or more specific MHC-II subtopes.

26. The method of any one of claims 1 to 18, wherein the second CD8+ TCR recognizes one or more specific MHC-E subtopes.

27. The method of any one of claims 1 to 18, wherein the second CD8+ TCR recognizes one or more specific MHC-I subtopes.

28. 27. The method of claim 25 or 26, wherein the second CD8+ TCR recognizes an MHC-II subtope and an MHC-E subtope.

29. 28. The method of claim 26 or 27, wherein the second CD8+ TCR recognizes an MHC-II subtope and an MHC-I subtope.

30. 28. The method of claim 25 or 27, wherein the second CD8+ TCR recognizes an MHC-E subtope and an MHC-I subtope.

31. 27. The method of any one of claims 19-20 and 25-26, wherein the second CD8+ TCR recognizes an MHC-II subtope or supertope and an MHC-E subtope or supertope.

32. 28. The method of any one of claims 19, 21, 25, and 27, wherein the second CD8+ TCR recognizes an MHC-II subtope or supertope and an MHC-I subtope or supertope.

33. 28. The method of any one of claims 20-21 and 26-27, wherein the second CD8+ TCR recognizes an MHC-E subtope or supertope and an MHC-I subtope or supertope.

34. 26. The method of claim 19 or 25, wherein the second CD8+ TCR recognizes a specific MHC-II supertope and MHC-II subtope.

35. 27. The method of claim 20 or 26, wherein the second CD8+ TCR recognizes a specific MHC-E supertope and MHC-E subtope.

36. 28. The method of claim 21 or 27, wherein the second CD8+ TCR recognizes a specific MHC-I supertope and MHC-I subtope.

37. 20. The method of claim 19, wherein the second CD8+ TCR recognizes two or more MHC-II supertopes from the same antigen.

38. 21. The method of claim 20, wherein the second CD8+ TCR recognizes two or more MHC-E supertopes from the same antigen.

39. 22. The method of claim 21, wherein the second CD8+ TCR recognizes two or more MHC-I supertopes from the same antigen.

40. 26. The method of claim 25, wherein the second CD8+ TCR recognizes two or more MHC-II subtopes from the same antigen.

41. 27. The method of claim 26, wherein the second CD8+ TCR recognizes two or more MHC-E subtopes from the same antigen.

42. 28. The method of claim 27, wherein the second CD8+ TCR recognizes two or more MHC-I subtopes from the same antigen.

43. 26. The method of claim 19 or 25, wherein the second CD8+ TCR recognizes one or more MHC-II supertopes and one or more MHC-II subtopes from the same antigen.

44. 27. The method of claim 20 or 26, wherein the second CD8+ TCR recognizes one or more MHC-E supertopes and one or more MHC-E subtopes from the same antigen.

45. 28. The method of claim 21 or 27, wherein the second CD8+ TCR recognizes one or more MHC-I supertopes and one or more MHC-I subtopes from the same antigen.

46. 20. The method of claim 19, wherein the second CD8+ TCR recognizes two or more MHC-II supertopes from two or more antigens.

47. 21. The method of claim 20, wherein the second CD8+ TCR recognizes two or more MHC-E supertopes from two or more antigens.

48. 22. The method of claim 21, wherein the second CD8+ TCR recognizes two or more MHC-I supertopes from two or more antigens.

49. 26. The method of claim 25, wherein the second CD8+ TCR recognizes two or more MHC-II subtopes from two or more antigens.

50. 27. The method of claim 26, wherein the second CD8+ TCR recognizes two or more MHC-E subtopes from two or more antigens.

51. 28. The method of claim 27, wherein the second CD8+ TCR recognizes two or more MHC-I subtopes from two or more antigens.

52. 26. The method of claim 19 or 25, wherein the second CD8+ TCR recognizes one or more MHC-II supertopes and one or more MHC-II subtopes from different antigens.

53. 27. The method of claim 20 or 26, wherein the second CD8+ TCR recognizes one or more MHC-E supertopes and one or more MHC-E subtopes from a different antigen.

54. 28. The method of claim 21 or 27, wherein the second CD8+ TCR recognizes one or more MHC-I supertopes and one or more MHC-I subtopes from different antigens.

55. 19. The method of any one of claims 1 to 18, wherein the third CD8+ TCR recognizes one or more specific MHC-II supertopes.

56. 19. The method of any one of claims 1 to 18, wherein the third CD8+ TCR recognizes one or more specific MHC-E supertopes.

57. 19. The method of any one of claims 1 to 18, wherein the third CD8+ TCR recognizes one or more specific MHC-I supertopes.

58. The method of any one of claims 1 to 18, wherein the third CD8+ TCR recognizes one or more specific MHC-II subtopes.

59. The method of any one of claims 1 to 18, wherein the third CD8+ TCR recognizes one or more specific MHC-E subtopes.

60. The method of any one of claims 1 to 18, wherein the third CD8+ TCR recognizes one or more specific MHC-I subtopes.

61. 59. The method of claim 55 or 58, wherein the third CD8+ TCR recognizes a specific MHC-II supertope and MHC-II subtope.

62. 60. The method of claim 56 or 59, wherein the third CD8+ TCR recognizes a specific MHC-E supertope and MHC-E subtope.

63. 61. The method of claim 57 or 60, wherein the third CD8+ TCR recognizes a specific MHC-I supertope and MHC-I subtope.

64. 56. The method of claim 55, wherein the third CD8+ TCR recognizes two or more MHC-II supertopes from one antigen.

65. 57. The method of claim 56, wherein the third CD8+ TCR recognizes two or more MHC-E supertopes from one antigen.

66. 58. The method of claim 57, wherein the third CD8+ TCR recognizes two or more MHC-I supertopes from one antigen.

67. 59. The method of claim 58, wherein the third CD8+ TCR recognizes two or more MHC-II subtopes from one antigen.

68. 60. The method of claim 59, wherein the third CD8+ TCR recognizes two or more MHC-E subtopes from one antigen.

69. 61. The method of claim 60, wherein the third CD8+ TCR recognizes two or more MHC-I subtopes from a single antigen.

70. 59. The method of claim 55 or 58, wherein the third CD8+ TCR recognizes one or more MHC-II supertopes and one or more MHC-II subtopes from an antigen.

71. 60. The method of claim 56 or 59, wherein the third CD8+ TCR recognizes one or more MHC-E supertopes and one or more MHC-E subtopes from an antigen.

72. 61. The method of any one of claims 57 to 60, wherein the third CD8+ TCR recognizes one or more MHC-I supertopes and one or more MHC-I subtopes from an antigen.

73. 56. The method of claim 55, wherein the third CD8+ TCR recognizes two or more MHC-II supertopes from two or more antigens.

74. 57. The method of claim 56, wherein the third CD8+ TCR recognizes two or more MHC-E supertopes from two or more antigens.

75. 58. The method of claim 57, wherein the third CD8+ TCR recognizes two or more MHC-I supertopes from two or more antigens.

76. 59. The method of claim 58, wherein the third CD8+ TCR recognizes two or more MHC-II subtopes from two or more antigens.

77. 60. The method of claim 59, wherein the third CD8+ TCR recognizes two or more MHC-E subtopes from two or more antigens.

78. 61. The method of claim 60, wherein the third CD8+ TCR recognizes two or more MHC-I subtopes from two or more antigens.

79. 60. The method of any one of claims 55-56 and 58-59, wherein the third CD8+ TCR recognizes a specific MHC-E subtope or supertope and an MHC-II subtope or supertope.

80. 61. The method of any one of claims 56-57 and 59-60, wherein the third CD8+ TCR recognizes a specific MHC-E subtope or supertope and an MHC-I subtope or supertope.

81. 61. The method of any one of claims 55, 57-58, and 60, wherein the third CD8+ TCR recognizes a specific MHC-II subtope or supertope and an MHC-I subtope or supertope.

82. 59. The method of claim 58, wherein the third CD8+ TCR recognizes two or more MHC-II subtopes from the same antigen.

83. 60. The method of claim 59, wherein the third CD8+ TCR recognizes two or more MHC-E subtopes from the same antigen.

84. 61. The method of claim 60, wherein the third CD8+ TCR recognizes two or more MHC-I subtopes from the same antigen.

85. 59. The method of claim 55 or 58, wherein the third CD8+ TCR recognizes one or more MHC-II supertopes and one or more MHC-II subtopes from different antigens.

86. 60. The method of claim 56 or 59, wherein the third CD8+ TCR recognizes one or more MHC-E supertopes and one or more MHC-E subtopes from different antigens.

87. 61. The method of claim 57 or 60, wherein the third CD8+ TCR recognizes one or more MHC-I supertopes and one or more MHC-I subtopes from different antigens.

88. 88. The method of any one of claims 1 to 87, wherein the nucleic acid sequence encoding the third CD8+ TCR is identical to the nucleic acid sequence encoding the second CD8+ TCR.

89. 89. The method of any one of claims 1-88, further comprising 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 / xenoantigen-derived peptide complex and the second MHC / xenoantigen-derived peptide complex.

90. 90. The method of claim 89, wherein the first MHC-xenoantigen-derived peptide complex is an MHC-II / xenoantigen-derived peptide complex, an MHC-E / xenoantigen-derived peptide complex, or an MHC-I / xenoantigen-derived peptide complex.

91. 91. The method of claim 89 or 90, wherein the second MHC-heterologous antigen-derived peptide complex is an MHC-II / heterologous antigen-derived peptide complex, an MHC-E / heterologous antigen-derived peptide complex, or an MHC-I / heterologous antigen-derived peptide complex.

92. 92. The method of any one of claims 89-91, further comprising administering the transfected CD8+ T cells to the second subject to treat or prevent cancer.

93. 93. The method of claim 92, wherein the cancer is selected from the group consisting of prostate cancer, kidney cancer, lung cancer, pancreatic cancer, mesothelioma, breast cancer, acute myeloid leukemia, chronic myeloid 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.

94. 92. The method of any one of claims 89-91, further comprising administering the transfected CD8+ T cells to the second subject to treat a pathogen infection.

95. 93. The method of claim 92, wherein the pathogen 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 parasite, Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), human T-lymphotropic virus type 1 (HTLV1), Merkel virus (MCV), cytomegalovirus, and Mycobacterium tuberculosis.

96. 96. The method of any one of claims 89-95, wherein the first subject is a non-human primate and the second subject is a human, and the transfected CD8+ T cells comprise a chimeric non-human primate-human CD8+ TCR comprising the non-human primate CDR3α and CDR3β of the second CD8+ TCR.

97. 97. The method of any one of claims 89-96, wherein the third CD8+ TCR comprises the non-human primate CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the second CD8+ TCR.

98. 98. The method of any one of claims 89-97, wherein the third CD8+ TCR comprises the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the second CD8+ TCR.

99. 99. The method of any one of claims 89-98, wherein the first subject is a non-human primate, the second subject is a human, and the second CD8+ TCR is a chimeric non-human primate-human CD8+ TCR comprising the non-human primate CDR3α and CDR3β of the first CD8+ TCR.

100. 100. The method of any one of claims 1 to 99, wherein the third CD8+ TCR is a chimeric CD8+ TCR.

101. 101. The method of any one of claims 1-100, wherein administering the recombinant CMV vector to the first subject comprises intravenous, intramuscular, intraperitoneal, or oral administration.

102. A CD8+ T cell comprising said multispecific TCR produced by the method of any one of claims 1 to 101.

103. 103. A method of treating or preventing cancer in a subject in need thereof, comprising administering to said subject the CD8+ T cells of claim 102.

104. 104. The method of claim 103, wherein the cancer is selected from the group consisting of prostate cancer, kidney cancer, lung cancer, pancreatic cancer, mesothelioma, breast cancer, acute myeloid leukemia, chronic myeloid 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.

105. 103. A method of treating a pathogen infection in a subject in need thereof, comprising administering to said subject the CD8+ T cells of claim 102.

106. 106. The method of claim 105, wherein the pathogen 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 parasite, Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), human T-lymphotropic virus type 1 (HTLV1), Merkel virus (MCV), cytomegalovirus, and Mycobacterium tuberculosis.

107. 107. The method of any one of claims 102-106, wherein administering the recombinant CMV vector to the first subject comprises intravenous, intramuscular, intraperitoneal, or oral administration.

108. 1. A method for generating CD8+ T cells comprising a multispecific T cell receptor (TCR), comprising: (a) administering to a subject a recombinant cytomegalovirus (CMV) vector comprising a nucleic acid sequence encoding 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 active UL128, UL130, UL146, and UL147 proteins or orthologues thereof, and 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 / foreign antigen-derived peptide complex; and (c) administering to the subject a second heterologous antigen in an amount effective to generate a second set of CD8+ T cells that recognize a second MHC-E / heterologous antigen-derived peptide complex; (d) isolating one or more CD8+ T cells from the second set of CD8+ T cells; and (e) identifying a second CD8+ TCR from the second set of CD8+ T cells, wherein the second CD8+ TCR recognizes the first MHC-E / xenoantigen-derived peptide complex and the second MHC-E / xenoantigen-derived peptide complex; (f) transfecting a third set of CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a third CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the third CD8+ TCR, wherein the third CD8+ TCR comprises CDR3α and CDR3β of the second CD8+ TCR, thereby generating one or more CD8+ T cells that recognize the first MHC-E / xenoantigen-derived peptide complex and the second MHC-E / xenoantigen-derived peptide complex; (g) selecting one or more of said third CD8+ TCRs that have the highest avidity for a particular peptide of interest.

109. 109. The method of claim 108, wherein the subject is a human or non-human primate.

110. 110. The method of claim 108 or 109, wherein the recombinant CMV vector is a recombinant human CMV vector or a recombinant rhesus CMV vector.

111. 111. The method of any one of claims 108 to 110, wherein the first heterologous antigen comprises a tumor antigen, a pathogen-specific antigen, a tissue-specific antigen, or a host self-antigen.

112. 112. The method of claim 111, wherein the tumor antigen is associated with a cancer selected from the group consisting of prostate cancer, kidney cancer, lung cancer, pancreatic cancer, mesothelioma, breast cancer, acute myeloid leukemia, chronic myeloid 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.

113. 112. The method of claim 111, wherein the pathogen-specific antigen is associated with 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 parasite, Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), human T-lymphotropic virus type 1 (HTLV1), Merkel virus (MCV), cytomegalovirus, and Mycobacterium tuberculosis.

114. 109. The method of claim 108, wherein the MRE comprises target sites for microRNAs expressed in endothelial cells.

115. 115. The method of claim 114, wherein 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.

116. 116. The method of any one of claims 108 to 115, wherein the first CD8+ TCR recognizes a specific MHC-E subtope or supertope.

117. 117. The method of any one of claims 108-116, wherein the first CD8+ TCR is identified by DNA or RNA sequencing.

118. 118. The method of claim 117, wherein the first CD8+ TCR is identified by single-cell sequencing.

119. 119. The method of any one of claims 108 to 118, wherein the second heterologous antigen comprises a tumor antigen, a pathogen-specific antigen, a tissue-specific antigen, or a host self-antigen.

120. 120. The method of claim 119, wherein the tumor antigen is associated with a cancer selected from the group consisting of prostate cancer, kidney cancer, lung cancer, pancreatic cancer, mesothelioma, breast cancer, acute myeloid leukemia, chronic myeloid 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.

121. 120. The method of claim 119, wherein the pathogen-specific antigen is associated with 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 parasite, Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), human T-lymphotropic virus type 1 (HTLV1), Merkel virus (MCV), cytomegalovirus, and Mycobacterium tuberculosis.

122. 122. The method of any one of claims 108 to 121, wherein the first heterologous antigen and the second heterologous antigen are the same.

123. 122. The method of any one of claims 108 to 121, wherein the first heterologous antigen and the second heterologous antigen are different.

124. 124. The method of any one of claims 108-123, wherein the one or more isolated CD8+ T cells from the second set of CD8+ T cells express CD69 and TNFα.

125. 125. The method of any one of claims 108 to 124, wherein the second CD8+ TCR is identified by DNA or RNA sequencing.

126. 126. The method of claim 125, wherein the second CD8+ TCR is identified by single-cell sequencing.

127. 127. The method of any one of claims 108 to 126, wherein the second CD8+ TCR recognizes one or more specific MHC-E supertopes.

128. The method of any one of claims 108 to 126, wherein the second CD8+ TCR recognizes one or more specific MHC-E subtopes.

129. The method of claim 127 or 128, wherein the second CD8+ TCR recognizes a specific MHC-E supertope and MHC-E subtope.

130. 128. The method of any one of claims 108 to 127, wherein the second CD8+ TCR recognizes two or more MHC-E supertopes from the same antigen.

131. 129. The method of any one of claims 108-126 and 128, wherein the second CD8+ TCR recognizes two or more MHC-E subtopes from the same antigen.

132. 130. The method of claim 129, wherein the second CD8+ TCR recognizes one or more MHC-E supertopes and one or more MHC-E subtopes from the same antigen.

133. 128. The method of any one of claims 108 to 127, wherein the second CD8+ TCR recognizes two or more MHC-E supertopes from two or more antigens.

134. 129. The method of any one of claims 108-126 and 128, wherein the second CD8+ TCR recognizes two or more MHC-E subtopes from two or more antigens.

135. 129. The method of any one of claims 108 to 128, wherein the second CD8+ TCR recognizes one or more MHC-E supertopes and one or more MHC-E subtopes from different antigens.

136. 128. The method of any one of claims 108 to 127, wherein the third CD8+ TCR recognizes one or more specific MHC-E supertopes.

137. 129. The method of any one of claims 108-126 and 128, wherein the third CD8+ TCR recognizes one or more specific MHC-E subtopes.

138. The method of claim 136 or 137, wherein the third CD8+ TCR recognizes a specific MHC-E supertope and MHC-E subtope.

139. 137. The method of claim 136, wherein the third CD8+ TCR recognizes two or more MHC-E supertopes from one antigen.

140. 138. The method of claim 137, wherein the third CD8+ TCR recognizes two or more MHC-E subtopes from one antigen.

141. 141. The method of claim 139 or 140, wherein the third CD8+ TCR recognizes one or more MHC-E supertopes and one or more MHC-E subtopes from an antigen.

142. 137. The method of claim 136, wherein the third CD8+ TCR recognizes two or more MHC-E supertopes from two or more antigens.

143. 138. The method of claim 137, wherein the third CD8+ TCR recognizes two or more MHC-E subtopes from two or more antigens.

144. 137. The method of claim 136, wherein the third CD8+ TCR recognizes two or more MHC-E supertopes from the same antigen.

145. 138. The method of claim 137, wherein the third CD8+ TCR recognizes two or more MHC-E subtopes from the same antigen.

146. 144. The method of claim 142 or 143, wherein the third CD8+ TCR recognizes one or more MHC-E supertopes and one or more MHC-E subtopes from different antigens.

147. 147. The method of any one of claims 108 to 146, wherein the nucleic acid sequence encoding the third CD8+ TCR is identical to the nucleic acid sequence encoding the second CD8+ TCR.

148. 148. The method of any one of claims 108-147, further comprising 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 / xenoantigen-derived peptide complex and the second MHC-E / xenoantigen-derived peptide complex.

149. 149. The method of claim 148, further comprising administering the transfected CD8+ T cells to the second subject to treat or prevent cancer.

150. 150. The method of claim 149, wherein the cancer is selected from the group consisting of prostate cancer, kidney cancer, lung cancer, pancreatic cancer, mesothelioma, breast cancer, acute myeloid leukemia, chronic myeloid 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.

151. 149. The method of claim 148, further comprising administering the transfected CD8+ T cells to the second subject to treat a pathogen infection.

152. 150. The method of claim 149, wherein the pathogen 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 parasite, Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), human T-lymphotropic virus type 1 (HTLV1), Merkel virus (MCV), cytomegalovirus, and Mycobacterium tuberculosis.

153. 153. The method of any one of claims 148-152, wherein the first subject is a non-human primate and the second subject is a human, and the transfected CD8+ T cells comprise a chimeric non-human primate-human CD8+ TCR comprising the non-human primate CDR3α and CDR3β of the second CD8+ TCR.

154. 154. The method of any one of claims 148-153, wherein the third CD8+ TCR comprises the non-human primate CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the second CD8+ TCR.

155. 155. The method of any one of claims 148-154, wherein the third CD8+ TCR comprises the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the second CD8+ TCR.

156. 156. The method of any one of claims 148-155, wherein the first subject is a non-human primate, the second subject is a human, and the second CD8+ TCR is a chimeric non-human primate-human CD8+ TCR comprising the non-human primate CDR3α and CDR3β of the first CD8+ TCR.

157. The method of any one of claims 148 to 156, wherein the third CD8+ TCR is a chimeric CD8+ TCR.

158. 158. The method of any one of claims 108-157, wherein administering the recombinant CMV vector to the first subject comprises intravenous, intramuscular, intraperitoneal, or oral administration.

159. A CD8+ T cell comprising said multispecific TCR produced by the method of any one of claims 108 to 158.

160. 160. A method of treating or preventing cancer in a subject in need thereof, comprising administering to said subject the CD8+ T cells of claim 159.

161. 161. The method of claim 160, wherein the cancer is selected from the group consisting of prostate cancer, kidney cancer, lung cancer, pancreatic cancer, mesothelioma, breast cancer, acute myeloid leukemia, chronic myeloid 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.

162. 160. A method of treating a pathogen infection in a subject in need thereof, comprising administering to said subject the CD8+ T cells of claim 159.

163. 161. The method of claim 160, wherein the pathogen 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 parasite, Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), human T-lymphotropic virus type 1 (HTLV1), Merkel virus (MCV), cytomegalovirus, and Mycobacterium tuberculosis.

164. 164. The method of any one of claims 160-163, wherein administering the recombinant CMV vector to the first subject comprises intravenous, intramuscular, intraperitoneal, or oral administration.

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