WO2017156365A1

WO2017156365A1 - Methods of generating antigen-specific t cells for adoptive immunotherapy

Info

Publication number: WO2017156365A1
Application number: PCT/US2017/021730
Authority: WO
Inventors: Quenther KOEHNE
Original assignee: Memorial Sloan Kettering Cancer Center
Priority date: 2016-03-11
Filing date: 2017-03-10
Publication date: 2017-09-14
Also published as: AR107857A1

Abstract

Provided herein are methods of generating antigen-specific T cells for therapeutic administration to a human patient having or suspected of having a pathogen or cancer using a CD34 fraction of an apheresis collection from G-CSF mobilized donors. Also disclosed are methods of treating a human patient using antigen-specific T cells generated by such methods, and methods of assessing antigen-specific T cells for suitability for therapeutic administration to a human patient.

Description

METHODS OF GENERATING ANTIGEN-SPECIFIC T CELLS FOR ADOPTIVE

IMMUNOTHERAPY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No.

62/307,240, filed March 11, 2016, which is incorporated by reference herein in its entirety.

1. FIELD

[0002] Provided herein are methods of generating antigen-specific T cells for therapeutic administration to a human patient having or suspected of having a pathogen or cancer using a CD34^" fraction of an apheresis collection from G-CSF mobilized donors. Also disclosed are methods of treating a human patient using antigen-specific T cells generated by such methods, and methods of assessing antigen-specific T cells for suitability for therapeutic administration to a human patient.

2. BACKGROUND

[0003] Antigen-specific T cells can be used in adoptive immunotherapy to treat infections and cancer, such as cytomegalovirus (CMV) infections, EBV-associated lymphoproliferative disorder (EBV-LPD), and WT1 (Wilms tumor l)-positive leukemia (see, e.g., Koehne et al., 2015, Biol Blood Marrow Transplant 21 : 1663-1678; O'Reilly et al., 2012, Seminars in

Immunology 22: 162-172; and Doubrovina et al., 2012, Blood 119:2644-2656). Antigen-specific T cells are usually generated from peripheral blood mononuclear cells (PBMCs) collected before mobilization with granulocyte colony-stimulating factor (G-CSF) from peripheral blood stem cell transplant (PBSCT) donors (Clancy et al., 2013, Biol Blood Marrow Transplant 19:725- 734).

[0004] PBSCT donors are administered G-CSF so as to increase the number of circulating hematopoietic stem cells (i.e., the donors are G-CSF mobilized) so that peripheral blood of the donors can be used for hematopoietic reconstitution (via PBSCT). G-CSF mobilization has been shown to impair T cell function (Bunse et al, 2013, PloS One 8:e77925). For this reason, PBSCT donors need to donate blood for a second time, to generate antigen-specific T cells, either before the G-CSF mobilization or after the G-CSF mobilization at a time (e.g., 6 weeks) when they are no longer G-CSF mobilized, thereby increasing the costs, and increasing the medical risk and pain to the donors associated with blood donation.

[0005] One report suggested that PBMCs exposed to G-CSF can be used to generate CMV- specific T cells that retain functionality, but the method takes an unfractionated portion of the apheresis collection to generate CMV-specific T cells, thereby reducing the number of CD34⁺ cells available for hematopoietic reconstitution of the transplant recipient (Clancy et al., 2013, Biol Blood Marrow Transplant 19:725-734). The method thereby also has the disadvantage that it uses only apheresis collections that have a threshold number of CD34+ cells. Id.

[0006] Therefore, there is a need for methods of generating antigen-specific T cells without incurring the cost, medical risk, and pain associated with additional blood draws, and at the same time without increasing risk of failure of hematopoietic reconstitution to transplant recipients.

[0007] Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

3. SUMMARY OF THE INVENTION

[0008] The present invention provides methods of generating antigen-specific T cells for therapeutic administration to a human patient having or suspected of having a pathogen or cancer using a CD34^" fraction of an apheresis collection from G-CSF mobilized donors. Also disclosed are methods of treating a human patient using antigen-specific T cells generated by such methods, and methods of assessing antigen-specific T cells for suitability for therapeutic administration to a human patient.

[0009] In one aspect, provided herein are methods of generating a population of cells comprising antigen-specific T cells for therapeutic administration to a human patient having or suspected of having a pathogen or cancer, comprising ex vivo sensitizing T cells derived from a CD34^" cell population to one or more antigens of the pathogen or cancer, wherein the CD34^" cell population is the product of a method comprising separating CD34⁺ cells from CD34^" cells in an apheresis collection that comprises T cells from a human donor who is G-CSF mobilized, thereby producing the CD34^" cell population. In various embodiments of the aspect, the methods further comprise, prior to the ex vivo sensitizing step, a step of separating CD34⁺ cells from CD34^" cells in the apheresis collection, to produce the CD34^" cell population. In specific embodiments, the apheresis collection is a leukapheresis collection. [0010] In certain embodiments, the separating step comprises sorting the apheresis collection using an anti-CD34 antibody. In specific embodiments, the anti-CD34 antibody is coupled to magnetic beads, and the sorting of the apheresis collection using the anti-CD34 antibody is performed by magnetic separation.

[0011] In certain embodiments, the methods further comprise, prior to the separating step, a step of administering G-CSF to the human donor to render the human donor G-CSF mobilized. In specific embodiments, the step of administering G-CSF comprises administering G-CSF to the human donor for 5 to 6 consecutive days. In a specific embodiment, the step of administering G- CSF comprises administering G-CSF to the human donor once daily at 10 mcg/kg per dose for multiple consecutive days. In a specific embodiment, the methods further comprise, prior to the separating step and during or after the step of administering G-CSF, a step of subjecting blood from the human donor to apheresis to produce the apheresis collection. In specific embodiments, the subjecting step comprises subjecting blood from the human donor to apheresis daily on the last two days of G-CSF administration.

[0012] In certain embodiments wherein the methods further comprise, prior to the separating step, a step of administering G-CSF to the human donor to render the human donor G-CSF mobilized, the methods further comprise, prior to the separating step and during or after the step of administering G-CSF, a step of subjecting blood from the human donor to apheresis to produce the apheresis collection.

[0013] In certain embodiments, the methods further comprise, between the separating step and the ex vivo sensitizing step, a step of isolating peripheral blood mononuclear cells (PBMCs) from the CD34^" cell population. In specific embodiments, the ex vivo sensitizing step comprises co-culturing the isolated PBMCs with one or more immunogenic peptides or proteins derived from the one or more antigens. In specific embodiments, the ex vivo sensitizing step comprises co-culturing the isolated PBMCs with antigen presenting cells that present the one or more antigens.

[0014] In certain embodiments, the methods further comprise, after the step of isolating PBMCs, a step of enriching T cells from the PBMCs. In specific embodiments, the step of enriching T cells from the PBMCs comprises sorting the PBMCs using an anti-CD3 antibody. In specific embodiments, the ex vivo sensitizing step comprises co-culturing the enriched T cells with one or more immunogenic peptides or proteins derived from the one or more antigens. In specific embodiments, the ex vivo sensitizing step comprises co-culturing the enriched T cells with antigen presenting cells that present the one or more antigens.

[0015] The antigen presenting cells used in the ex vivo sensitizing step can be any antigen presenting cells suitable for presenting the one or more antigens of the pathogen or cancer, such as dendritic cells, cytokine-activated monocytes, PBMCs, Epstein-Barr virus-transformed B- lymphoblastoid cell line cells (EBV-BLCL cells), or artificial antigen presenting cells. In a specific embodiment, the antigen presenting cells used in the ex vivo sensitizing step are dendritic cells. In another specific embodiment, the antigen presenting cells used in the ex vivo sensitizing step are EBV-BLCL cells.

[0016] In some embodiments, the antigen presenting cells are loaded with one or more immunogenic peptides or proteins derived from the one or more antigens. In other embodiments, the antigen presenting cells are genetically engineered to express one or more immunogenic peptides or proteins derived from the one or more antigens.

[0017] In some embodiments, the one or more immunogenic peptides or proteins are a pool of overlapping peptides derived from the one or more antigens. In specific embodiments, the pool of overlapping peptides is a pool of overlapping pentadecapeptides. In other embodiments, the one or more immunogenic peptides or proteins are one or more proteins derived from the one or more antigens.

[0018] In specific embodiments, the human donor is allogeneic to the human patient.

[0019] In a specific embodiment, the methods further comprise, after the separating step, recovering the separated CD34⁺ cells and using the separated CD34⁺ cells in a peripheral blood stem cell transplantation (PBSCT). In some embodiment, the human patient is the recipient of the separated CD34⁺ cells in the PBSCT, and the human patient has or is suspected of having the pathogen or cancer subsequent to the human patient having undergone the PBSCT. In other embodiments, the human patient is not the recipient of the separated CD34⁺ cells in the PBSCT.

[0020] In another aspect, provided herein are methods of treating a human patient having or suspected of having a pathogen or cancer, comprising: (i) generating a population of cells comprising antigen-specific T cells for therapeutic administration to the human patient according to a method described above; and (ii) administering the population of cells comprising antigen- specific T cells to the human patient.

[0021] In specific embodiments, the administering step is by bolus intravenous infusion. [0022] In certain embodiments, the administering step comprises administering at least about 1 x 10⁵ cells of the population of cells per kg per dose per week to the human patient. In specific embodiments, the administering step comprises administering about 1 x 10⁶ to about 5 x 10⁶ cells of the population of cells per kg per dose per week to the human patient.

[0023] In a specific embodiment, the administering step comprises administering 2, 3, 4, 5, or 6 doses of the population of cells to the human patient, and a washout period of at least one week between two consecutive doses, wherein no dose of the population of cells is administered during the washout period. In specific embodiments, the washout period is about 1, 2, 3, or 4 weeks.

[0024] In another aspect, provided herein are methods of assessing a population of cells comprising antigen-specific T cells ex vivo sensitized to one or more antigens of a pathogen or cancer, for suitability for therapeutic administration to a human patient having the pathogen or cancer, comprising determining whether the antigen-specific T cells exhibit a predominant effector memory phenotype, wherein the predominant effector memory phenotype is CD62L^" CCR7^"CD45RA^"CD28⁺CD27^+/"CD57^+/", and wherein determining that the antigen-specific T cells do not exhibit a predominant effector memory phenotype indicates that the population of cells is not suitable for therapeutic administration to the human patients.

[0025] In specific embodiments, the methods of assessing a population of cells comprising antigen-specific T cells further comprise determining whether CD137 is upregulated in the antigen-specific T cells, wherein determining that CD137 is not upregulated indicates that the population of cells is not suitable for therapeutic administration to the human patient.

[0026] In various aspects, the human patient has or is suspected of having a pathogen. The pathogen can be a virus, bacterium, fungus, helminth, or protist. In certain embodiments, the pathogen is a virus.

[0027] In some embodiments, the virus is cytomegalovirus (CMV). In specific

embodiments, the human patient has or is suspected of having a CMV infection. In specific embodiments, the human patient has a CMV infection. In particular embodiments, the one or more antigens of CMV in the methods described in this disclosure is CMV pp65, CMV IE1, or a combination thereof.

[0028] In some embodiments, the virus is Epstein-Barr virus (EBV). In certain

embodiments, the one or more antigens of EBV in the methods described in this disclosure is EBNAl, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMPl, LMP2, or a combination thereof. In specific embodiments, the human patient has or is suspected of having an EB V-positive lymphoproliferative disorder (EBV-LPD). In specific embodiments, the human patient has an EBV-LPD. In a specific embodiment, the EBV-LPD is an EB V-positive lymphoma. In embodiments wherein the human patient has or is suspected of having an EBV-LPD, the one or more antigens of EBV is EBNAl, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMPl, LMP2, or a combination thereof. In specific embodiments, the human patient has or is suspected of having an EB V-positive nasopharyngeal carcinoma. In specific embodiments, the human patient has an EB V-positive nasopharyngeal carcinoma. In such specific embodiment wherein the human patient has or is suspected of having an EB V-positive nasopharyngeal carcinoma, the one or more antigens of EBV is EBNAl, LMPl, LMP2, or a combination thereof.

[0029] In some embodiments, the virus is polyoma BK virus (BKV), John Cunningham virus (JCV), herpesvirus, adenovirus (ADV), human immunodeficiency virus (HIV), influenza virus, ebola virus, poxvirus, rhabdovirus, or paramyxovirus.

[0030] In various aspects, the human patient has or is suspected of having a cancer. In some embodiments, the human patient has a cancer.

[0031] In some embodiments, the cancer is a blood cancer. In other embodiments, the cancer is a cancer of the breast, lung, ovary, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostate, thyroid, brain, or skin.

[0032] In certain embodiments, the one or more antigens of the cancer is WT1 (Wilms tumor 1). In specific embodiments, the cancer is multiple myeloma or plasma cell leukemia.

4. DETAILED DESCRIPTION

[0033] The present invention provides methods of generating antigen-specific T cells for therapeutic administration to a human patient having or suspected of having a pathogen or cancer. In T-cell depleted peripheral blood stem cell transplantation (TCD-PBSCT) using adult peripheral blood, T-cell depletion is usually carried out by selecting the CD34⁺ fraction of the apheresis collection from a G-CSF mobilized transplant donor for use in TCD-PBSCT (O'Reilly et al., 2010, Seminars in Immunology 22: 162-172). While the CD34⁺ cells are used for transplantation, the CD34^" cells are normally discarded. In contrast, according to the present invention, the normally discarded CD34^" fraction of the apheresis collection from a G-CSF mobilized donor is used to generate functional antigen-specific T cells suitable for therapeutic administration to a human patient to provide an immune response against an undesirable pathogen or a cancer in the patient or suspected of being in the patient. By employing the CD34^" fraction of the apheresis collection, donors do not need to undergo additional blood collection prior to the G-CSF mobilization or when they are no longer G-CSF mobilized, thereby avoiding the costs, medical risk, and pain associated with additional blood collections. In addition, since the methods according to the invention do not reduce the number of CD34⁺ cells, they also avoid the risk associated with possibly insufficient CD34⁺ cell content to provide hematopoietic reconstitution of transplant recipients, and can be performed independent of any threshold CD34⁺ cell count in the apheresis collection used to generate the T cells for therapeutic use.

4.1. Methods of Generating Antigen-Specific T Cells for Adoptive Immunotherapy

[0034] In one aspect, provided herein are methods of generating a population of cells comprising antigen-specific T cells for therapeutic administration to a human patient having or suspected of having a pathogen or cancer, comprising ex vivo sensitizing T cells derived from a CD34^" cell population to one or more antigens of the pathogen or cancer, wherein the CD34^" cell population is the product of a method comprising separating CD34⁺ cells from CD34^" cells in an apheresis collection that comprises T cells from a human donor who is G-CSF mobilized, thereby producing the CD34^" cell population. In various embodiments of the aspect, the methods further comprise, prior to the ex vivo sensitizing step, a step of separating CD34⁺ cells from CD34^" cells in the apheresis collection, to produce the CD34^" cell population. In certain embodiments, the separating step also comprises collecting the CD34^" cell population;

alternatively, in certain embodiments, the methods further comprise, after the separating step, a step of collecting the CD34^" cell population. In specific embodiments, the apheresis collection is a leukapheresis collection.

[0035] In certain embodiments, the separating step comprises sorting the apheresis collection using an anti-CD34 antibody. In specific embodiments, the sorting of the apheresis collection using an anti-CD34 antibody is performed by Fluorescence Activated Cell Sorting (FACS). In specific embodiments, the anti-CD34 antibody is coupled to magnetic beads, and the sorting of the apheresis collection using the anti-CD34 antibody is performed by magnetic separation. In a specific embodiment, the sorting of the apheresis collection using the anti-CD34 antibody is performed by a CliniMACS® system, e.g., the CliniMACS® Cell Selection System. In a specific embodiment, the sorting of the apheresis collection using the anti-CD34 antibody is performed using the CliniMACS® CD34 Reagent System.

[0036] In certain embodiments, the methods further comprise, prior to the separating step, a step of administering G-CSF to the human donor to render the human donor G-CSF mobilized. In specific embodiments, the step of administering G-CSF is by subcutaneous administration. In specific embodiments, the step of administering G-CSF comprises administering G-CSF to the human donor for 3 to 8 consecutive days. In specific embodiments, the step of administering G- CSF comprises administering G-CSF to the human donor for 4 to 6 consecutive days. In specific embodiments, the step of administering G-CSF comprises administering G-CSF to the human donor for 5 to 6 consecutive days. In a specific embodiment, the step of administering G-CSF comprises administering G-CSF to the human donor for 4 consecutive days. In another specific embodiment, the step of administering G-CSF comprises administering G-CSF to the human donor for 5 consecutive days. In another specific embodiment, the step of administering G-CSF comprises administering G-CSF to the human donor for 6 consecutive days.

[0037] In specific embodiments, the step of administering G-CSF comprises administering G-CSF to the human donor at 5-20 mcg/kg per day. In specific embodiments, the step of administering G-CSF comprises administering G-CSF to the human donor at 10 mcg/kg per day. In specific embodiments, the step of administering G-CSF comprises administering G-CSF to the human donor daily. In specific embodiments, the step of administering G-CSF comprises administering G-CSF to the human donor twice daily. In a specific embodiment, the step of administering G-CSF comprises administering G-CSF to the human donor once daily at 5-20 mcg/kg per dose for multiple consecutive days. In another specific embodiment, the step of administering G-CSF comprises administering G-CSF to the human donor once daily at 10 mcg/kg per dose for multiple consecutive days.

[0038] In certain embodiments, the methods further comprise, prior to the separating step and during or after the step of administering G-CSF, a step of subjecting blood from the human donor to apheresis (e.g., leukapheresis) to produce the apheresis collection. When the subjecting step is after the step of administering G-CSF, in specific embodiments, the subjecting step is within 6 weeks of the step of administering G-CSF. When the subjecting step is after the step of administering G-CSF, in specific embodiments, the subjecting step is within 1 month of the step of administering G-CSF. When the subjecting step is after the step of administering G-CSF, in specific embodiments, the subjecting step is within three weeks of the step of administering G- CSF. When the subjecting step is after the step of administering G-CSF, in specific

embodiments, the subjecting step is within two weeks of the step of administering G-CSF.

When the subjecting step is after the step of administering G-CSF, in specific embodiments, the subjecting step is within one week of the step of administering G-CSF. When the subjecting step is after the step of administering G-CSF, in specific embodiments, the subjecting step is multiple days (e.g., 3-7 days) after the step of administering G-CSF. When the subjecting step is after the step of administering G-CSF, in specific embodiments, the subjecting step is two days after the step of administering G-CSF. When the subjecting step is after the step of administering G-CSF, in specific embodiments, the subjecting step is one day after the step of administering G-CSF. When the subjecting step is after the step of administering G-CSF, in preferred embodiments, the subjecting step is on the same day as the step of administering G-CSF. In specific

embodiments, the subjecting step comprises subjecting blood from the human donor to apheresis (e.g., leukapheresis) daily on the last two days of G-CSF administration. In specific

embodiments, the subjecting step comprises subjecting blood from the human donor to apheresis (e.g., leukapheresis) daily on the last day of G-CSF administration. In specific embodiments, the subjecting step comprises subjecting blood from the human donor to apheresis (e.g.,

leukapheresis) daily on the last three days of G-CSF administration. In specific embodiments, the subjecting step comprises subjecting blood from the human donor to apheresis (e.g., leukapheresis) daily on the last day of G-CSF administration and on the day after.

[0039] In a preferred embodiment, the methods comprise administering G-CSF to the human door (e.g., once daily at 10 mcg/kg per dose) for 4 to 6 consecutive days, and subjecting blood from the human donor to apheresis (e.g., leukapheresis) on the last day, or the last two days, or the next to last day, of G-CSF administration to produce the apheresis collection. In a specific embodiment, the methods comprise administering G-CSF to the human donor (e.g., once daily at 10 mcg/kg per dose) for 4 consecutive days, and subjecting blood from the human donor to apheresis (e.g., leukapheresis) daily on the last two days of G-CSF administration to produce the apheresis collection. In another specific embodiment, the methods comprise administering G- CSF to the human donor (e.g., once daily at 10 mcg/kg per dose) for 5 consecutive days, and subjecting blood from the human donor to apheresis (e.g., leukapheresis) daily on the last two days of G-CSF administration to produce the apheresis collection. In another specific embodiment, the methods comprise administering G-CSF to the human donor (e.g., once daily at 10 mcg/kg per dose) for 6 consecutive days, and subjecting blood from the human donor to apheresis (e.g., leukapheresis) daily on the last two days of G-CSF administration to produce the apheresis collection.

[0040] Apheresis can be performed by any method known in the art. In some embodiments, apheresis is performed by centrifugation, for example, by continuous flow centrifugation or intermittent flow centrifugation. In other embodiments, apheresis is performed by filtration. In specific embodiments, apheresis is performed by using an automated apheresis machine.

[0041] The ex vivo sensitizing step can be performed by any method known in the art to stimulate T cells to be antigen-specific ex vivo, such as the method described in the example in Section 5 herein, or a method as described in Koehne et al., 2000, Blood 96: 109-117; Trivedi et al., 2005, Blood 105:2793-2801; Haque et al., 2007, Blood 110: 1123-1131; Hasan et al., 2009, J Immunol 183 : 2837-2850; Feuchtinger et al., 2010, Blood 116:4360-4367; Doubrovina et al., 2012, Blood 120: 1633-1646; Leen et al., 2013, Blood 121 :5113-5123; Papadopoulou et al., 2014, Sci Transl Med 6:242ra83; Sukdolak et al., 2013, Biol Blood Marrow Transplant 19: 1480- 1492; or Koehne et al., 2015, Biol Blood Marrow Transplant 21 : 1663-1678.

[0042] In certain embodiments, the methods further comprise, between the separating step and the ex vivo sensitizing step, a step of isolating peripheral blood mononuclear cells (PBMCs) from the CD34^" cell population. PBMCs can be isolated from the CD34^" cell population by any method known in the art to isolated PBMCs from a blood sample, such as by Ficoll-Hypaque centrifugation as described in Koehne et al., 2000, Blood 96: 109-117; Trivedi et al., 2005, Blood 105:2793-2801; and the example in Section 5 herein. In specific embodiments, the ex vivo sensitizing step comprises co-culturing the isolated PBMCs with one or more immunogenic peptides or proteins derived from the one or more antigens. In specific embodiments, the ex vivo sensitizing step comprises co-culturing the isolated PBMCs with antigen presenting cells that present the one or more antigens.

[0043] In certain embodiments, the methods further comprise, after the step of isolating PBMCs, a step of enriching T cells from the PBMCs. T cells can be enriched from the PBMCs by any method known in the art to enrich T cells from a blood sample or PBMCs. Non-limiting exemplary methods for enriching T cells can be found in Koehne et al., 2000, Blood 96: 109-117; Trivedi et al., 2005, Blood 105:2793-2801; Hasan et al., 2009, J Immunol 183 : 2837-2850; and Koehne et al., 2015, Biol Blood Marrow Transplant 21 : 1663-1678. In specific embodiments, the step of enriching T cells from the PBMCs comprises sorting the PBMCs using an anti-CD3 antibody. In specific embodiments, the step of enriching T cells from the PBMCs comprises depleting of adherent monocytes and natural killer cells from the PBMCs. In specific embodiments, the ex vivo sensitizing step comprises co-culturing the enriched T cells with one or more immunogenic peptides or proteins derived from the one or more antigens. In specific embodiments, the ex vivo sensitizing step comprises co-culturing the enriched T cells with antigen presenting cells that present the one or more antigens.

[0044] The antigen presenting cells used in the ex vivo sensitizing step can be any antigen presenting cells suitable for presenting the one or more antigens of the pathogen or cancer, such as dendritic cells, cytokine-activated monocytes, PBMCs, Epstein-Barr virus-transformed B- lymphoblastoid cell line cells (EBV-BLCL cells), or artificial antigen presenting cells. In a specific embodiment, the antigen presenting cells used in the ex vivo sensitizing step are dendritic cells. In another specific embodiment, the antigen presenting cells used in the ex vivo sensitizing step are EBV-BLCL cells. In a specific embodiment, the antigen presenting cells are derived from the human donor. The antigen presenting cells can be obtained by any method known in the art, such as the method(s) described in Koehne et al., 2000, Blood 96: 109-117; Koehne et al., 2002, Blood 99: 1730-1740; Trivedi et al., 2005, Blood 105:2793-2801; O'Reilly et al., 2007, Immunol Res 38:237-250; Hasan et al., 2009, J Immunol 183 : 2837-2850; Barker et al., 2010, Blood 116:5045-5049; O' Reilly et al., 2011, Best Practice & Research Clinical Haematology 24:381-391; Doubrovina et al., 2012, Blood 120: 1633-1646; or Koehne et al., 2015, Biol Blood Marrow Transplant 21 : 1663-1678.

[0045] In some embodiments, the antigen presenting cells are loaded with one or more immunogenic peptides or proteins derived from the one or more antigens. Non-limiting exemplary methods for loading antigen presenting cells with peptide(s) derived from antigen(s) can be found in Trivedi et al., 2005, Blood 105:2793-2801; and Hasan et al., 2009, J Immunol 183 : 2837-2850. In other embodiments, the antigen presenting cells are genetically engineered to express one or more immunogenic peptides or proteins derived from the one or more antigens. Any appropriate method known in the art for introducing nucleic acid vehicles into cells to express proteins, such as transduction or transformation, can be used to genetically engineer the antigen presenting calls to express the one or more immunogenic peptides or proteins derived from the one or more antigens.

[0046] In some embodiments, the one or more immunogenic peptides or proteins are a pool of overlapping peptides derived from the one or more antigens. In specific embodiments, the pool of overlapping peptides is a pool of overlapping pentadecapeptides. In other embodiments, the one or more immunogenic peptides or proteins are one or more proteins derived from the one or more antigens.

[0047] Preferably, the human donor is seropositive for the one or more antigens of the pathogen or cancer. Preferably, the human donor is an adult.

[0048] In specific embodiments, the human donor is allogeneic to the human patient; thus the population of cells is allogeneic to the human patient.

[0049] In a preferred embodiment, the human donor is a donor for a peripheral blood stem cell transplantation (PBSCT). In a specific embodiment, the methods further comprise, after the separating step, recovering the separated CD34⁺ cells and using the separated CD34⁺ cells in the PBSCT. In some embodiment, the human patient is the recipient of the separated CD34⁺ cells in the PBSCT, and the human patient has or is suspected of having the pathogen or cancer subsequent to the human patient having undergone the PBSCT. In other embodiments, the human patient is not the recipient of the separated CD34⁺ cells in the PBSCT.

[0050] In a specific embodiment, the apheresis collection that is employed in a method of the invention, to generate antigen-specific T cells, is selected for use independent of CD34⁺ cell count in the apheresis collection. In another specific embodiment, the apheresis collection has equal to or less than 2.5 x 10⁶ CD34⁺ cells per kg of the PBSCT recipient's weight. In another specific embodiment, the apheresis collection has equal to or less than 2 x 10⁶ CD34⁺ cells per kg of the PBSCT recipient's weight. In another specific embodiment, the apheresis collection has equal to or less than 1.5 x 10⁶ CD34⁺ cells per kg of the PBSCT recipient's weight. In another specific embodiment, the apheresis collection has equal to or less than 1 x 10⁶ CD34⁺ cells per kg of the PBSCT recipient's weight. In another specific embodiment, the apheresis collection has equal to or less than 5 x 10⁵ CD34⁺ cells per kg of the PBSCT recipient's weight. In another specific embodiment, the apheresis collection has equal to or less than 1 x 10⁵ CD34⁺ cells per kg of the PBSCT recipient's weight. In another specific embodiment, the apheresis collection has equal to or less than 5 x 10⁴ CD34⁺ cells per kg of the PBSCT recipient's weight. In another specific embodiment, the apheresis collection has equal to or less than 1 x 10⁴ CD34⁺ cells per kg of the PBSCT recipient's weight. In another specific embodiment, the apheresis collection has equal to or less than 2.5 x 10⁴ CD34⁺ cells per kg of the PBSCT recipient's weight. In another specific embodiment, the apheresis collection has equal to or less than 1 x 10⁴ CD34⁺ cells per kg of the PBSCT recipient's weight.

[0051] In another specific embodiment, the apheresis collection has less than 10⁹

mononuclear cells per kg of the PBSCT recipient's weight. In another specific embodiment, the apheresis collection has less than 5 x 10⁸ mononuclear cells per kg of the PBSCT recipient's weight. In another specific embodiment, the apheresis collection has less than 1 x 10⁸ mononuclear cells per kg of the PBSCT recipient's weight. In another specific embodiment, the apheresis collection has less than 5 x 10⁷ mononuclear cells per kg of the PBSCT recipient's weight. In another specific embodiment, the apheresis collection has less than 1 x 10⁷ mononuclear cells per kg of the PBSCT recipient's weight.

[0052] In certain embodiments, the methods further comprise, after the ex vivo sensitizing step, cryopreserving a cell population comprising the ex vivo sensitized T cells for storage. In specific embodiments, the methods further comprise, after the ex vivo sensitizing step, cryopreserving a cell population comprising the ex vivo sensitized T cells for storage, storing the cell population, thawing the cell population, and optionally expanding the ex vivo sensitized T cells of the cell population in vitro to generate the population of cells for therapeutic

administration. The methods can further comprise a step of administering the thawed cell population, or thawed and expanded cell population, to the human patient.

[0053] In certain embodiments, the methods further comprise, after the ex vivo sensitizing step, expanding the sensitized T cells in vitro to generate the population of cells for therapeutic administration, wherein the sensitized T cells have not been cryopreserved for storage.

4.2. Methods of Treating Patients Using the Generated Antigen-Specific T Cells

[0054] In another aspect, provided herein are methods of treating a human patient having or suspected of having a pathogen or cancer, comprising: (i) generating a population of cells comprising antigen-specific T cells for therapeutic administration to the human patient according to a method described in Section 4.1; and (ii) administering the population of cells comprising antigen-specific T cells to the human patient.

4.2.1. Administration and Dosage

[0055] The route of administration of the population of cells and the amount to be

administered to the human patient can be determined based on the condition of the human patient and the knowledge of the physician. Generally, the administration is intravenous. In certain embodiments, the administering step is by infusion of the population of cells. In specific embodiments, the infusion is bolus intravenous infusion.

[0056] In certain embodiments, the administering step comprises administering at least about 1 x 10⁵ cells of the population of cells per kg per dose per week to the human patient. In specific embodiments, the administering step comprises administering about 1 x 10⁶ to about 1 x 10⁷ cells of the population of cells per kg per dose per week to the human patient. In specific

embodiments, the administering step comprises administering about 1 x 10⁶ to about 5 x 10⁶ cells of the population of cells per kg per dose per week to the human patient. In specific

embodiments, the administering step comprises administering about 1 x 10⁶ to about 2 x 10⁶ cells of the population of cells per kg per dose per week to the human patient. In a specific embodiment, the administering step comprises administering about 1 x 10⁶ cells of the population of cells per kg per dose per week to the human patient. In a specific embodiment, the administering step comprises administering about 2 x 10⁶ cells of the population of cells per kg per dose per week to the human patient. In a specific embodiment, the administering step comprises administering about 3 x 10⁶ cells of the population of cells per kg per dose per week to the human patient. In a specific embodiment, the administering step comprises administering about 5 x 10⁶ cells of the population of cells per kg per dose per week to the human patient.

[0057] In certain embodiments, the administering step comprises administering at least 2 doses of the population of cells to the human patient. In specific embodiments, the administering step comprises administering 2, 3, 4, 5, or 6 doses of the population of cells to the human patient. In a specific embodiment, the administering step comprises administering 2 doses of the population of cells to the human patient. In another specific embodiment, the administering step comprises administering 3 doses of the population of cells to the human patient. In another specific embodiment, the administering step comprises administering 4 doses of the population of cells to the human patient [0058] In certain embodiments, the administering step comprises a washout period between two consecutive doses, wherein no dose of the population of cells is administered during the washout period. In specific embodiments, the washout period is at least about 1 week (e.g., about 1-6 weeks). In specific embodiments, the washout period is about 1, 2, 3, or 4 weeks. In a specific embodiment, the washout period is about 2 weeks. In another specific embodiment, the washout period is about 3 weeks. In another specific embodiment, the washout period is about 4 weeks.

[0059] In a specific embodiment, the administering step comprises administering 2, 3, 4, 5, or 6 doses of the population of cells to the human patient, and a washout period of at least one week between two consecutive doses, wherein no dose of the population of cells is administered during the washout period. In specific embodiments, the washout period is about 1, 2, 3, or 4 weeks. In a specific embodiment, the washout period is about 2 weeks. In another specific embodiment, the washout period is about 3 weeks. In another specific embodiment, the washout period is about 4 weeks.

[0060] In specific embodiments, the administering step comprises administering at least two cycles (e.g., 2, 3, 4, 5, or 6 cycles) of one dose per week of the population of cells for at least two consecutive weeks (e.g., 2, 3, 4, 5, or 6 consecutive weeks), each cycle separated by a washout period during which no dose of the population of cells is administered. In a specific

embodiment, the at least two consecutive weeks are 2 consecutive weeks. In another specific embodiment, the at least two consecutive weeks are 3 consecutive weeks. In another specific embodiment, the at least two consecutive weeks are 4 consecutive weeks. In specific embodiments, the washout period is at least about 1 week (e.g., about 1-6 weeks). In specific embodiments, the washout period is about 1, 2, 3, or 4 weeks. In a specific embodiment, the washout period is about 2 weeks. In another specific embodiment, the washout period is about 3 weeks. In another specific embodiment, the washout period is about 4 weeks. Preferably, an additional cycle is administered only when the previous cycle has not exhibited toxicity (for example, no grade 3-5 serious adverse events, graded according to NCI CTCAE 4.0).

[0061] In certain embodiments, a first dosage regimen described herein is carried out for a first period of time, followed by a second and different dosage regimen described herein that is carried out for a second period of time, wherein the first period of time and the second period of time are optionally separated by a washout period. In specific embodiments, the washout period is at least about 1 week (e.g., about 1-6 weeks). In specific embodiments, the washout period is about 1, 2, 3, or 4 weeks. In a specific embodiment, the washout period is about 2 weeks. In another specific embodiment, the washout period is about 3 weeks. In another specific embodiment, the washout period is about 4 weeks. Preferably, the second dosage regimen is carried out only when the first dosage regimen has not exhibited toxicity (for example, no grade 3-5 serious adverse events, graded according to NCI CTCAE 4.0).

[0062] The term "about" shall be construed so as to allow normal variation.

4.2.2. Serial Treatment with Different Cell Populations

[0063] In certain embodiments, the methods of treating a human patient having or suspected of having a pathogen or cancer as described above further comprise, after administering to the human patient a first population of cells comprising antigen-specific T cells generated according to a method described in Section 4.1, administering to the human patient a second population of cells comprising antigen-specific T cells, wherein the second population of cells is restricted by a different HLA allele (different from that of the first population of cells) shared with at least some, optionally all, of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous, and the antigen-specific T cells in the second population of cells are sensitized to an antigen of the pathogen or cancer. In a specific embodiment, the methods of treating a human patient having or suspected of having a pathogen or cancer comprise administering a first cycle of one dose per week of the first population of cells comprising antigen-specific T cells, generated according to a method described in Section 4.1, for at least two consecutive weeks (e.g., 2, 3, 4, 5, or 6 consecutive weeks) followed by a washout period during which no dose of the first or second population of cells is administered, followed by a second cycle of one dose per week of the second population of cells comprising antigen-specific T cells for at least two consecutive weeks (e.g., 2, 3, 4, 5, or 6 consecutive weeks). In specific embodiments, the washout period is at least about 1 week (e.g., about 1-6 weeks). In specific embodiments, the washout period is about 1, 2, 3, or 4 weeks. In a specific embodiment, the washout period is about 2 weeks. In another specific embodiment, the washout period is about 3 weeks. In certain embodiments, the human patient has no response, an incomplete response, or a suboptimal response (i.e., the human patient may still have a substantial benefit from continuing treatment, but has reduced chances of optimal long-term outcomes) after administering the first population of cells comprising antigen-specific T cells generated according to a method described in Section 4.1 and prior to administering the second population of cells comprising antigen-specific T cells.

[0064] In certain embodiments, the methods of treating a human patient having or suspected of having a pathogen or cancer as described above further comprise, before administering to the human patient a first population of cells comprising antigen-specific T cells generated according to a method described in Section 4.1, administering to the human patient a second population of cells comprising antigen-specific T cells, wherein the second population of cells is restricted by a different HLA allele (different from that of the first population of cells) shared with at least some, optionally all, of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous, and the antigen-specific T cells in the second population of cells are sensitized to an antigen of the pathogen or cancer. In a specific embodiment, the methods of treating a human patient having or suspected of having a pathogen or cancer comprise administering a first cycle of one dose per week of the second population of cells comprising antigen-specific T cells for at least two consecutive weeks (e.g., 2, 3, 4, 5, or 6 consecutive weeks) followed by a washout period during which no dose of the first or second population of cells is administered, followed by a second cycle of one dose per week of the first population of cells comprising antigen-specific T cells, generated according to a method described in Section 4.1, for at least two consecutive weeks (e.g., 2, 3, 4, 5, or 6 consecutive weeks). In specific embodiments, the washout period is at least about 1 week (e.g., about 1-6 weeks). In specific embodiments, the washout period is about 1, 2, 3, or 4 weeks. In a specific embodiment, the washout period is about 2 weeks. In another specific embodiment, the washout period is about 3 weeks. In certain embodiments, the human patient has no response, an incomplete response, or a suboptimal response (i.e., the human patient may still have a substantial benefit from continuing treatment, but has reduced chances of optimal long-term outcomes) after administering the second population of cells comprising antigen-specific T cells and prior to administering the first population of cells comprising antigen-specific T cells generated according to a method described in Section 4.1.

[0065] The second population of cells comprising antigen-specific T cells can be generated by a method as described in Section 4.1 or any method as described in Koehne et al., 2000, Blood 96: 109-117; Trivedi et al., 2005, Blood 105:2793-2801; Haque et al., 2007, Blood 110: 1123-1131; Hasan et al., 2009, J Immunol 183 : 2837-2850; Feuchtinger et al., 2010, Blood 116:4360-4367; Doubrovina et al., 2012, Blood 120: 1633-1646; Leen et al., 2013, Blood 121 :5113-5123; Papadopoulou et al., 2014, Sci Transl Med 6:242ra83; Sukdolak et al., 2013, Biol Blood Marrow Transplant 19: 1480-1492; or Koehne et al., 2015, Biol Blood Marrow Transplant 21 : 1663-1678.

[0066] The second population of cells comprising antigen-specific T cells can be

administered by any route and any dosage/administration regimen as described in Section 4.2.1.

[0067] In specific embodiments, two populations of cells comprising antigen-specific T cells that are each restricted by a different HLA allele shared with at least some, optionally all, of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous, are administered serially, wherein at least one of the two populations of cells is generated by a method as described in Section 4.1. In specific

embodiments, three populations of cells comprising antigen-specific T cells that are each restricted by a different HLA allele shared with at least some, optionally all, of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous, are administered serially, wherein at least one of the three populations of cells is generated by a method as described in Section 4.1. In specific embodiments, four populations of cells comprising antigen-specific T cells that are each restricted by a different HLA allele shared with at least some, optionally all, of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous, are administered serially, wherein at least one of the four populations of cells is generated by a method as described in Section 4.1. In specific embodiments, more than four populations of cells comprising antigen-specific T cells that are each restricted by a different HLA allele shared with at least some, optionally all, of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous, are administered serially, wherein at least one of the more than four populations of cells is generated by a method as described in Section 4.1.

4.3. Methods of Assessing Antigen-Specific T Cells for Suitability for Adoptive

Immunotherapy

[0068] In another aspect, provided herein are methods of assessing a population of cells comprising antigen-specific T cells ex vivo sensitized to one or more antigens of a pathogen or cancer, for suitability for therapeutic administration to a human patient having the pathogen or cancer, comprising determining whether the antigen-specific T cells exhibit a predominant effector memory phenotype, wherein the predominant effector memory phenotype is CD62L^" CCR7^"CD45RA^"CD28⁺CD27^+/"CD57^+/", and wherein determining that the antigen-specific T cells do not exhibit a predominant effector memory phenotype indicates that the population of cells is not suitable for therapeutic administration to the human patients. In a specific embodiment, the step of determining whether the antigen-specific T cells exhibit a predominant effector memory phenotype upon the ex vivo sensitizing is performed by FACS.

[0069] In specific embodiments, the methods of assessing a population of cells comprising antigen-specific T cells further comprise determining whether CD137 is upregulated in the antigen-specific T cells, wherein determining that CD137 is not upregulated indicates that the population of cells is not suitable for therapeutic administration to the human patient. In a specific embodiment, the step of determining whether CD137 is upregulated in the antigen- specific T cells upon the ex vivo sensitizing is performed by FACS.

[0070] The ex vivo sensitizing can be performed as described in Section 4.1.

4.4. Characterization of the Population of Cells Comprising Antigen-Specific T Cells

[0071] To select a population of cells comprising antigen-specific T cells for therapeutic administration to a particular human patient, the population of cells comprising antigen-specific T cells as described in this disclosure preferably (1) exhibits substantial cytotoxicity toward fully or partially ULA-matched (relative to the human donor) antigen presenting cells that are loaded with or genetically engineered to express a peptide(s) or protein(s) derived from the one or more antigens of the pathogen or cancer in or suspected of being in the particular human patient; (2) lacks substantial alloreactivity; and/or (3) is restricted by an ULA allele shared with at least some, optionally all, of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous in the human patient, or shares at least 2 ULA alleles with at least some, optionally all, of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous. Thus, preferably, cytotoxicity, alloreactivity, information as to which ULA allele(s) the population of cells is restricted, and/or the ULA assignment of the population of cells are measured by a method known in the art before administration (for example, as described in Koehne et al., 2000, Blood 96: 109-117; Trivedi et al., 2005, Blood 105:2793-2801; Haque et al., 2007, Blood 110: 1123- 1131; Hasan et al., 2009, J Immunol 183 : 2837-2850; Feuchtinger et al., 2010, Blood 116:4360- 4367; Doubrovina et al., 2012, Blood 120: 1633-1646; Leen et al., 2013, Blood 121 :5113-5123; Papadopoulou et al., 2014, Sci Transl Med 6:242ra83; Sukdolak et al., 2013, Biol Blood Marrow Transplant 19: 1480-1492; or Koehne et al., 2015, Biol Blood Marrow Transplant 21 : 1663- 1678).

4.4.1. Cytotoxicity

[0072] The cytotoxicity of a population of cells toward fully or partially HLA-matched (relative to the human donor) antigen presenting cells can be determined by any assay known in the art to measure T cell mediated cytotoxicity. In a specific embodiment, the cytotoxicity is determined by a standard ⁵¹Cr release assay as described in the example in Section 5 herein or as described in Trivedi et al., 2005, Blood 105:2793-2801 or Hasan et al., 2009, J Immunol 183 : 2837-2850.

[0073] In certain embodiments of the methods described in this disclosure, the population of cells comprising antigen-specific T cells exhibits substantial cytotoxicity in vitro toward {e.g., exhibits substantial lysis of) fully or partially HLA matched antigen presenting cells that are loaded with or genetically engineered to express a peptide(s) or protein(s) derived from the one or more antigens of the pathogen or cancer in or suspected of being in the particular human patient. Preferably, the fully or partially HLA-matched antigen presenting cells are fully HLA- matched antigen presenting cells {e.g., antigen presenting cells derived from the human donor). In specific embodiments, the population of cells exhibits lysis of greater than or equal to 20%, 25%, 30%), 35%), or 40% of the fully or partially HLA-matched antigen presenting cells that are loaded with or genetically engineered to express a peptide(s) or protein(s) derived from the one or more antigens of the pathogen or cancer in or suspected of being in the particular human patient. In a specific embodiment, the population of cells exhibits lysis of greater than or equal to 20%) of the fully or partially HLA-matched antigen presenting cells that are loaded with or genetically engineered to express a peptide(s) or protein(s) derived from the one or more antigens of the pathogen or cancer in or suspected of being in the particular human patient.

[0074] Antigen presenting cells that can be used in the cytotoxicity assay include, but are not limited to, dendritic cells, phytohemagglutinin (PHA)-lymphoblasts, macrophages, B-cells that generate antibodies, EBV-BLCL cells, and artificial antigen presenting cells (AAPCs). In a specific embodiment, the antigen presenting cells used in the cytotoxicity assay are dendritic cells.

[0075] In specific embodiments, the fully or partially HLA-matched antigen presenting cells used in the cytotoxicity assay are loaded with a pool of peptides derived from the one or more antigens of the pathogen or cancer in or suspected of being in the particular human patient. The pool of peptides, can be, for example, a pool of overlapping peptides (e.g., pentadecapeptides) spanning the sequence of the one or more antigens of the pathogen or cancer in or suspected of being in the particular human patient.

4.4.2. Alloreactivity

[0076] Alloreactivity can be measured using a cytotoxicity assay known in the art to to measure T cell mediated cytotoxicity, such as a standard ⁵¹Cr release assay, as described in Section 4.4.1, but with antigen presenting cells that are not loaded with or genetically engineered to express a peptide(s) or protein(s) derived from the one or more antigens of the pathogen or cancer in or suspected of being in the particular human patient, and/or HLA-mismatched (relative to the human donor) antigen presenting cells. A population of cells comprising antigen- specific T cells that lacks substantial alloreactivity results generally in the absence of graft- versus-host disease (GvHD) when administered to a human patient.

[0077] In certain embodiments of the methods described in this disclosure, the population of cells comprising antigen-specific T cells lacks substantial cytotoxicity in vitro toward antigen presenting cells that are not loaded with or genetically engineered to express a peptide(s) or protein(s) derived from the one or more antigens of the pathogen or cancer in or suspected of being in the particular human patient. In preferred embodiments, such antigen-presenting cells are fully or partially HLA-matched antigen presenting cells (relative to the human donor) {e.g., antigen presenting cells derived from the human donor). In specific embodiments, the population of cells lyses less than or equal to 15%, 10%, 5%, 2%, or 1% of antigen presenting cells that are not loaded with or genetically engineered to express a peptide(s) or protein(s) derived from the one or more antigens of the pathogen or cancer in or suspected of being in the particular human patient. In a specific embodiment, the population of cells lyses less than or equal to 15% of antigen presenting cells that are not loaded with or genetically engineered to express a peptide(s) or protein(s) derived from the one or more antigens of the pathogen or cancer in or suspected of being in the particular human patient.

[0078] In certain embodiments of the methods described in this disclosure, the population of cells comprising antigen-specific T cells lacks substantial cytotoxicity in vitro toward HLA- mismatched (relative to the human donor) antigen presenting cells. In some embodiments, such antigen-presenting cells are loaded with or genetically engineered to express a peptide(s) or protein(s) derived from the one or more antigens of the pathogen or cancer in or suspected of being in the particular human patient. In other embodiments, such antigen-presenting cells are not loaded with or genetically engineered to express a peptide(s) or protein(s) derived from the one or more antigens of the pathogen or cancer in or suspected of being in the particular human patient. In specific embodiments, the population of cells lyses less than or equal to 15%, 10%, 5%), 2%), or 1%) of HLA-mismatched (relative to the human donor) antigen presenting cells. In a specific embodiment, the population of cells lyses less than or equal to 15% of HLA-mismatched (relative to the human donor) antigen presenting cells.

[0079] In certain embodiments of the methods described in this disclosure, the population of cells comprising antigen-specific T cells lacks substantial cytotoxicity in vitro toward antigen presenting cells that are not loaded with or genetically engineered to express a peptide(s) or protein(s) derived from the one or more antigens of the pathogen or cancer in or suspected of being in the particular human patient, as described above, and lacks substantial cytotoxicity in vitro toward HLA-mismatched antigen presenting cells as described above.

[0080] Antigen presenting cells that can be used in the alloreactivity assay include, but are not limited to, dendritic cells, phytohemagglutinin (PHA)-lymphoblasts, macrophages, B-cells that generate antibodies, EBV-BLCL cells, and artificial antigen presenting cells (AAPCs). In a specific embodiment, the antigen presenting cells used in the alloreactivity assay are dendritic cells.

4.4.3. HLA Type

[0081] The HLA assignment {i.e., the HLA loci type) of the population of cells and/or the human donor can be ascertained {i.e., typed) by any method known in the art. Non-limiting exemplary methods for ascertaining the HLA assignment can be found in ASHI Laboratory Manual, Edition 4.2 (2003), American Society for Histocompatibility and Immunogenetics; ASHI Laboratory Manual, Supplements 1 (2006) and 2 (2007), American Society for Histocompatibility and Immunogenetics; Hurley, "DNA-based typing of HLA for

transplantation." in Leffell et al., eds., 1997, Handbook of Human Immunology, Boca Raton: CRC Press; Dunn, 201 1, Int J Immunogenet 38:463-473; Erlich, 2012, Tissue Antigens, 80: 1-1 1 ; Bontadini, 2012, Methods, 56:471-476; and Lange et al., 2014, BMC Genomics 15 : 63. In specific embodiments, at least 4 HLA loci (preferably HLA-A, HLA-B, HLA-C, and HLA-DR) are typed. In a specific embodiment, 4 HLA loci (preferably HLA-A, HLA-B, HLA-C, and HLA-DR) are typed. In another specific embodiment, 6 HLA loci are typed. In another specific embodiment, 8 HLA loci are typed.

[0082] In general, high-resolution typing is preferable for HLA typing. The high-resolution typing can be performed by any method known in the art, for example, as described in ASHI Laboratory Manual, Edition 4.2 (2003), American Society for Histocompatibility and

Immunogenetics; ASHI Laboratory Manual, Supplements 1 (2006) and 2 (2007), American Society for Histocompatibility and Immunogenetics; Flomenberg et al., Blood, 104: 1923-1930; Kogler et al., 2005, Bone Marrow Transplant, 36: 1033-1041 ; Lee et al., 2007, Blood 1 10:4576- 4583; Erlich, 2012, Tissue Antigens, 80: 1-1 1 ; Lank et al., 2012, BMC Genomics 13 :378; or Gabriel et al., 2014, Tissue Antigens, 83 :65-75.

[0083] In specific embodiments, the HLA assignment of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous in the human patient is ascertained by typing the origin of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous (e.g., the human patient or a transplant donor for the human patient, as the case may be). The origin of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous can be determined by any method known in the art, for example, by analyzing variable tandem repeats (VTRs) (which is a method that uses unique DNA signature of small DNA sequences of different people to distinguish between the recipient and the donor of a transplant), or by looking for the presence or absence of chromosome Y if the donor and the recipient of a transplant are of different sexes (which is done by cytogenetics or by FISH

(fluorescence in situ hybridization)).

[0084] The HLA allele by which the population of cells comprising antigen-specific T cells is restricted can be determined by any method known in the art, for example, as described in Trivedi et al., 2005, Blood 105 :2793-2801 ; Barker et al., 2010, Blood 1 16:5045-5049; Hasan et al., 2009, J Immunol, 183 :2837-2850; or Doubrovina et al., 2012, Blood 120: 1633-1646.

[0085] In some embodiments of the methods described in this disclosure, the population of cells comprising antigen-specific T cells is restricted by an HLA allele shared with at least some, optionally all, of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous in the human patient. In other embodiments of the methods described in this disclosure, the population of cells comprising antigen-specific T cells shares at least 2 HLA alleles (for example, at least 2 out of 8 HLA alleles, such as two HLA-A alleles, two HLA-B alleles, two HLA-C alleles, and two HLA-DR alleles) with at least some, optionally all, of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous in the human patient. In other embodiments of the methods described in this disclosure, the population of cells comprising antigen-specific T cells is restricted by an HLA allele shared with at least some, optionally all, of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous in the human patient, and shares at least 2 HLA alleles (for example, at least 2 out of 8 HLA alleles, such as two HLA-A alleles, two HLA-B alleles, two HLA-C alleles, and two HLA- DR alleles) with at least some, optionally all, of the cells harboring or suspected of harboring the pathogen, or the cells that are cancerous or suspected of being cancerous in the human patient.

4.5. Patients

[0086] In various aspects, the human patient has or is suspected of having a pathogen. In some embodiments, the human patient has the pathogen. In a specific embodiment, the human patient has a disorder (e.g., cancer) associated with the pathogen (e.g., resulting from infection with the pathogen). In other embodiments, the human patient is suspected of having the pathogen. In a specific embodiment, the human patient is seropositive for the pathogen, and has symptoms of an infection by the pathogen. In another specific embodiment, the human patient is seropositive for the pathogen, and has symptoms of a disorder (e.g., cancer) associated with the pathogen (e.g., resulting from infection with the pathogen). The pathogen can be a virus, bacterium, fungus, helminth, or protist. In certain embodiments, the pathogen is a virus.

[0087] In some embodiments, the virus is cytomegalovirus (CMV). In specific

embodiments, the human patient has or is suspected of having a CMV infection. In specific embodiments, the human patient has or is suspected of having a CMV infection subsequent to the human patient having undergone an HSCT. In specific embodiments, the human patient has a CMV infection. In a specific embodiment, the human patient has or is suspected of having CMV viremia. In another specific embodiment, the human patient has CMV viremia. In another specific embodiment, the human patient has or is suspected of having CMV retinitis, CMV pneumonia, CMV hepatitis, CMV colitis, CMV encephalitis, CMV meningoencephalitis, CMV- positive meningoma, or CMV-positive glioblastoma multiforme. In another specific

embodiment, the human patient has CMV retinitis, CMV pneumonia, CMV hepatitis, CMV colitis, CMV encephalitis, CMV meningoencephalitis, CMV-positive meningoma, or CMV- positive glioblastoma multiforme. In particular embodiments, the one or more antigens of CMV in the methods described in this disclosure is CMV pp65, CMV IE1, or a combination thereof. In a particular embodiment, the one or more antigens of CMV in the methods described in this disclosure is CMV pp65.

[0088] In some embodiments, the virus is Epstein-Barr virus (EBV). In certain

embodiments, the one or more antigens of EBV in the methods described in this disclosure is EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, or a combination thereof. In specific embodiments, the human patient has or is suspected of having an EBV-positive lymphoproliferative disorder (EBV-LPD) (for example, an EBV-positive post-transplant lymphoproliferative disorder). In specific embodiments, the human patient has an EBV-LPD (for example, an EBV-positive post-transplant lymphoproliferative disorder). The EBV-LPD can be, but is not limited to, B-cell hyperplasia, B-cell lymphoma (for example, diffuse large B- cell lymphoma), T-cell lymphoma, polymorphic or monomorphic EBV-LPD, EBV-positive Hodgkin's lymphoma, Burkitt lymphoma, autoimmune lymphoproliferative syndrome, or mixed PTLD (post-transplant lymphoproliferative disorder). In a specific embodiment, the EBV-LPD is an EBV-positive lymphoma (for example, and EBV-positive B-cell lymphoma). In a specific embodiment, the EBV-LPD is present in the central nervous system of the human patient. In a further specific embodiment, the EBV-LPD is present in the brain of the human patient. In embodiments wherein the human patient has or is suspected of having an EBV-LPD, the one or more antigens of EBV is EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, or a combination thereof. In specific embodiments, the human patient has or is suspected of having an EBV-positive nasopharyngeal carcinoma. In specific embodiments, the human patient has an EBV-positive nasopharyngeal carcinoma. In such specific embodiment wherein the human patient has or is suspected of having an EBV-positive nasopharyngeal carcinoma, the one or more antigens of EBV is EBNA1, LMP1, LMP2, or a combination thereof.

[0089] In some embodiments, the virus is polyoma BK virus (BKV), John Cunningham virus (JCV), herpesvirus, adenovirus (ADV), human immunodeficiency virus (HIV), influenza virus, ebola virus, poxvirus, rhabdovirus, or paramyxovirus. In particular embodiments, the virus is BKV. In particular embodiments, the virus is JCV. In particular embodiments, the virus is ADV. In particular embodiments, the virus is human herpesvirus-6 (HHV-6) or human herpesvirus-8 (HHV-8).

[0090] In certain embodiments wherein the human patient has a disorder (e.g., cancer) associated with the pathogen (e.g., resulting from infection with the pathogen), the disorder is not responsive to a therapy for the disorder previously administered to the human patient, such as chemotherapy (e.g., combination chemotherapy), radiation therapy, or a combination thereof).

[0091] In certain embodiments wherein the pathogen is a virus and the human patient has an infection associated with the virus, the infection is not responsive to a previous antiviral (small molecule) drug therapy.

[0092] In various aspects, the human patient has or is suspected of having a cancer. In some embodiments, the human patient has a cancer. In other embodiments, the human patient is suspected of having a cancer. In a specific embodiment, the human patient who is suspected of having the cancer is seropositive for one or more antigens of the cancer, and has symptoms normally associated with the cancer. In specific embodiments, the cancer is not associated with a pathogen. In specific embodiments, the cancer is associated with a pathogen.

[0093] An antigen of a cancer, as described herein, can be a cancer-specific or cancer- associated antigen, and thus can be a peptide or protein whose expression is higher in the cancer tissue or cancer cells than in non-cancerous tissues or non-cancerous cells, or a peptide or protein which is uniquely expressed in the cancer tissue or cancer cells relative to non-cancerous tissues or non-cancerous cells.

[0094] In some embodiments, the cancer is a blood cancer. A blood cancer that can be treated using a population of cells comprising antigen-specific T cells described in this disclosure can be, but is not limited to: acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, hairy cell leukemia, T-cell prolymphocytic leukemia, Large granular lymphocytic leukemia, adult T-cell leukemia, plasma cell leukemia, Hodgkin lymphoma, Non-Hodgkin lymphoma, or multiple myeloma.

[0095] In other embodiments, the cancer is a solid tumor cancer. The solid tumor cancer can be a sarcoma, a carcinoma, a lymphoma, a germ cell tumor, a blastoma, or a combination thereof. A solid tumor cancer that can be treated using a population of cells comprising antigen- specific T cells described in this disclosure can be, but is not limited to: a cancer of the breast, lung, ovary, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostate, thyroid, brain, or skin.

[0096] In certain embodiments, the one or more antigens of the cancer is WT1 (Wilms tumor 1). In specific embodiments, the cancer is multiple myeloma or plasma cell leukemia. In a specific embodiment, the cancer is relapsed/refractory multiple myeloma (RRMM), which can be, for example, primary refractory multiple myeloma, relapsed multiple myeloma, or relapsed and refractory multiple myeloma. In another specific embodiment, the cancer is primary plasma cell leukemia. In another specific embodiment, the cancer is secondary plasma cell leukemia.

[0097] In certain embodiments wherein the human patient has a cancer, the cancer is not responsive to an anti-cancer therapy previously administered to the human patient, such as chemotherapy (e.g., combination chemotherapy), radiation therapy, or a combination thereof.

[0098] In certain embodiments, the human patient has or is suspected of having the pathogen or cancer subsequent to the human patient having undergone a hematopoietic stem cell transplantation (HSCT), such as a peripheral blood stem cell transplantation, a bone marrow transplantation, or a cord blood transplantation. In some embodiments, the human donor is the donor of the HSCT. The human donor can be a related donor or unrelated donor of the HSCT. In a specific embodiment, the human patient has or is suspected of having the pathogen or cancer subsequent to the human patient having undergone a PBSCT, and the human donor is the donor of the PBSCT. In other embodiments, the human donor is a third-party donor that is different from the donor of the HSCT.

[0099] In certain embodiments, the human patient has not been the recipient of an HSCT.

[00100] In a specific embodiment, the human patient is an adult (at least age 16). In another specific embodiment, the human patient is an adolescent (age 12-15). In another specific embodiment, the patient is a child (under age 12). 5. EXAMPLE

[00101] This following non-limiting example demonstrates that the CD34^" fraction of an apheresis collection from a G-CSF mobilized donor, normally discarded, is a suitable source to generate antigen-specific T cells for adoptive immunotherapy.

5.1. Material and Methods:

[00102] G-GSF Mobilization

[00103] Beginning 6 days before the day of hematopoietic stem cell transplant, each normal human donor received 10 mcg/kg of G-CSF, administered subcutaneously daily for 6 days. On the fifth and sixth days of this course of G-CSF, the donor underwent daily leukapheresis designed to provide a minimum of 10⁹ mononuclear cells/kg of the transplant recipient's weight.

[00104] Isolation of CD34⁺ hematopoietic progenitor cells for PBSCT and CD34^' cells for generation of antigen-specific T cells with the CliniMACS® System

[00105] Aliquots of the apheresis product were collected. The apheresis product was prepped for the CliniMACS® Cell Selection System. The mechanism of action of the CliniMACS Cell Selection System is based on magnetic-activated cell sorting, which can select or remove specific cell types depending on the cell-specific immunomagnetic label used. The apheresis product was first co-incubated with the CliniMACS® CD34 reagent (antibody-coated paramagnetic particles). Prior to and during incubation of the anti-CD34 beads with the G-CSF mobilized apheresis collection, intravenous gammaglobulin was added to the incubation fluid at a concentration of 1.5 mg/ml. After magnetic labeling and washing, the cells were passed through a high-gradient magnetic separation column in the CliniMACS® clinical cell selection device. Magnetically labeled CD34⁺ cells were retained in the magnetized column, and CD34^" cells flowed through as the effluent fraction. The CD34⁺ cells retained in the column were eluted by removing the magnetic field from the column, then washing the cells through the column and collecting them. The final CD34⁺ cell enriched product was concentrated by centrifugation and tested before final release for administration for PBSCT as per SOPs (Standard Operating Procedures) from the MSKCC Cytotherapy Lab Manual.

[00106] Before infusion, the CD34⁺ cells were washed in normal saline for intravenous infusion containing 1% human serum albumin, and suspended in a volume of 25-50 ml for intravenous administration. Aliquots of the product were taken for in-process and final product testing were performed as per SOPs from the Cytotherapy Lab Manual.

[00107] Generation of CMV CTLs

[00108] For the generation of CMV CTLs (cytotoxic T lymphocytes), PBMCs were isolated after Ficoll-Hypaque centrifugation with 10 ml taken from 8 separate unrelated donor CD34^" apheresis collections. lxlO⁶ cells/mL of unmodified and cryopreserved PBMCs were stimulated with 0.5xl0⁵/mL 6000 CGy irradiated donor-derived dendritic cells (DCs) or 0.5xl0⁵/mL irradiated donor-derived Epstein-Barr virus-transformed B lymphocyte cell lines (BLCLs), both pulsed with the pool of overlapping pentadecapeptides of CMVpp65. Cultures were weekly restimulated and IL-2 was added to the culture at day 8 and 2-3 times weekly thereafter. After 28 days, T cells were harvested and assessed by flow cytometry, MHC tetramer analyses and cytotoxicity assays.

5.2. Results

[00109] CMV CTLs were able to be generated and expanded from all 8 donor-derived CD34^" specimens. In side-by-side comparison using CMVpp65 loaded BLCLs and DCs, CMV-specific T cell were expanded from 8/8 donors for the BLCL group, but only 5/8 in the DC group, which may reflect the previously described impairment of DC function after G-CSF mobilization. While cultures from the BLCL sensitized T cells were predominantly CD8⁺ (95%) and were specific for CMV (64%) as well as EBV (24%) antigens, the DC sensitized cultures consisted of CD8⁺ (68%) and CD4⁺ (32%) T cells with CMV specificity of 42% and 18%, respectively. CMV and EBV specific CD8⁺ T cells were multifunctional expressing high levels of CD 107a, T Fa and IFN-γ. CMV-specific CD4+ T cells also produced IL-2 and up-regulated CD 154, suggesting their potential to sustain T and B cell expansion. Degranulation observed by flow cytometry correlated with high levels of cytotoxicity, assessed by the standard 4h ⁵ Chromium assay, against antigen loaded DCs. No alloreactivity, NK cell expansion or

CD4⁺CD25⁺CD127^lowFOXP3⁺ Tregs were present at the end of any cultures. In the donors expressing HLA-A*0201 and B*0702, for which tetramers to NLV and TPR sequences of pp65- protein were available, high proportions of responses were confirmed to be directed to these epitopes. [00110] Further characterization of the differentiation status of CMV CTLs, identified in cultures by up-regulation of CD 137 upon peptide stimulation, revealed a predominant effector memory phenotype (CD62L^"CCR7^"CD45RA^"CD28⁺CD27^+/"CD57^+/"). Of the check-point inhibitors, expanded cells expressed high levels of PD-1 but no relevant expression of the other markers (LAG-3, TIM-3 or CTLA-4).

5.3. Conclusion

[00111] This study demonstrates that a CD34^" product, normally discarded, obtained from donors of peripheral blood stem cell transplantations is a suitable source of highly functional memory T cells allowing manufacturing of CMV CTLs for cellular therapies.

6. Incorporation by reference

[00112] All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[00113] Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

What is claimed is:

1. A method of generating a population of cells comprising antigen-specific T cells for therapeutic administration to a human patient having or suspected of having a pathogen or cancer, comprising ex vivo sensitizing T cells derived from a CD34^" cell population to one or more antigens of the pathogen or cancer, wherein the CD34^" cell population is the product of a method comprising separating CD34⁺ cells from CD34^" cells in an apheresis collection that comprises T cells from a human donor who is G-CSF mobilized, thereby producing the CD34^" cell population.

2. The method of claim 1, which further comprises, prior to the ex vivo sensitizing step, a step of separating CD34⁺ cells from CD34^" cells in the apheresis collection, to produce the CD34^" cell population.

3. The method of claim 1 or 2, wherein the apheresis collection is a leukapheresis

collection.

4. The method of any of claims 1-3, wherein the separating step comprises sorting the apheresis collection using an anti-CD34 antibody.

5. The method of claim 4, wherein the anti-CD34 antibody is coupled to magnetic beads, and the sorting of the apheresis collection using the anti-CD34 antibody is performed by magnetic separation.

6. The method of any of claims 1-5, which further comprises, prior to the separating step, a step of administering G-CSF to the human donor to render the human donor G-CSF mobilized.

7. The method of claim 6, wherein the step of administering G-CSF comprises

administering G-CSF to the human donor for 5 to 6 consecutive days.

8. The method of claim 6 or 7, wherein the step of administering G-CSF comprises

administering G-CSF to the human donor once daily at 10 mcg/kg per dose for multiple consecutive days.

9. The method of claim 6, which further comprises, prior to the separating step and during or after the step of administering G-CSF, a step of subjecting blood from the human donor to apheresis to produce the apheresis collection.

10. The method of claim 7 or 8, which further comprises, prior to the separating step and during or after the step of administering G-CSF, a step of subjecting blood from the human donor to apheresis to produce the apheresis collection.

11. The method of claim 10, wherein the subjecting step comprises subjecting blood from the human donor to apheresis daily on the last two days of G-CSF administration.

12. The method of any of claims 1-11, which further comprises, between the separating step and the ex vivo sensitizing step, a step of isolating peripheral blood mononuclear cells (PBMCs) from the CD34^" cell population.

13. The method of claim 12, wherein the ex vivo sensitizing step comprises co-culturing the isolated PBMCs with one or more immunogenic peptides or proteins derived from the one or more antigens.

14. The method of claim 12, wherein the ex vivo sensitizing step comprises co-culturing the isolated PBMCs with antigen presenting cells that present the one or more antigens.

15. The method of claim 12, which further comprises, after the step of isolating PBMCs, a step of enriching T cells from the PBMCs.

16. The method of claim 15, wherein the step of enriching T cells from the PBMCs

comprises sorting the PBMCs using an anti-CD3 antibody.

17. The method of claim 15 or 16, wherein the ex vivo sensitizing step comprises co- culturing the enriched T cells with one or more immunogenic peptides or proteins derived from the one or more antigens.

18. The method of claim 15 or 16, wherein the ex vivo sensitizing step comprises co- culturing the enriched T cells with antigen presenting cells that present the one or more antigens.

19. The method of claim 14 or 18, wherein the antigen presenting cells are dendritic cells, cytokine-activated monocytes, PBMCs, Epstein-Barr virus-transformed B- lymphoblastoid cell line cells (EBV-BLCL cells), or artificial antigen presenting cells.

20. The method of claim 14 or 18, wherein the antigen presenting cells are dendritic cells.

21. The method of claim 14 or 18, wherein the antigen presenting cells are EBV-BLCL cells.

22. The method of any of claims 14 and 18-21, where the antigen presenting cells are loaded with one or more immunogenic peptides or proteins derived from the one or more antigens.

23. The method of any of claims 14 and 18-21, where the antigen presenting cells are genetically engineered to express one or more immunogenic peptides or proteins derived from the one or more antigens.

24. The method of any of claims 13, 17, and 22-23, wherein the one or more immunogenic peptides or proteins are a pool of overlapping peptides derived from the one or more antigens.

25. The method of claim 24, wherein the pool of overlapping peptides is a pool of

overlapping pentadecapeptides.

26. The method of any of claims 13, 17, and 22-23, wherein the one or more immunogenic peptides or proteins are one or more proteins derived from the one or more antigens.

27. The method of any of claims 1-26, wherein the human donor is allogeneic to the human patient.

28. The method of any of claims 1-27, which further comprises, after the separating step, recovering the separated CD34⁺ cells and using the separated CD34⁺ cells in a peripheral blood stem cell transplantation (PBSCT).

29. The method of claim 28, wherein the human patient is the recipient of the separated

CD34⁺ cells in the PBSCT, and the human patient has or is suspected of having the pathogen or cancer subsequent to the human patient having undergone the PBSCT.

30. The method of claim 28, wherein the human patient is not the recipient of the separated CD34⁺ cells in the PBSCT.

31. A method of treating a human patient having or suspected of having a pathogen or

cancer, comprising: (i) generating a population of cells comprising antigen-specific T cells for therapeutic administration to the human patient according to the method of any of claims 1-30; and (ii) administering the population of cells comprising antigen-specific T cells to the human patient.

32. The method of claim 31, wherein the administering step is by bolus intravenous infusion.

33. The method of claim 31 or 32, wherein the administering step comprises administering at least about 1 x 10⁵ cells of the population of cells per kg per dose per week to the human patient.

34. The method of claim 31 or 32, wherein the administering step comprises administering about 1 x 10⁶ to about 5 x 10⁶ cells of the population of cells per kg per dose per week to the human patient.

35. The method of any of claims 31-34, wherein the administering step comprises

administering 2, 3, 4, 5, or 6 doses of the population of cells to the human patient, and a washout period of at least one week between two consecutive doses, wherein no dose of the population of cells is administered during the washout period.

36. The method of claim 35, wherein the washout period is about 1, 2, 3, or 4 weeks.

37. A method of assessing a population of cells comprising antigen-specific T cells ex vivo sensitized to one or more antigens of a pathogen or cancer, for suitability for therapeutic administration to a human patient having the pathogen or cancer, comprising determining whether the antigen-specific T cells exhibit a predominant effector memory phenotype, wherein the predominant effector memory phenotype is CD62L^"CCR7^"CD45RA^" CD28⁺CD27^+/"CD57^+/", and wherein determining that the antigen-specific T cells do not exhibit a predominant effector memory phenotype indicates that the population of cells is not suitable for therapeutic administration to the human patients.

38. The method of claim 37, which further comprises determining whether CD137 is

upregulated in the antigen-specific T cells, wherein determining that CD137 is not upregulated indicates that the population of cells is not suitable for therapeutic administration to the human patient.

39. The method of any of claims 1-38, wherein the human patient has or is suspected of having a pathogen.

40. The method of claim 39, wherein the pathogen is a virus, bacterium, fungus, helminth or protist.

41. The method of claim 39, wherein the pathogen is a virus.

42. The method of claim 41, wherein the virus is cytomegalovirus (CMV).

43. The method of claim 42, wherein the human patient has or is suspected of having a CMV infection.

44. The method of claim 42, wherein the human patient has a CMV infection.

45. The method of any of claims 42-44, wherein the one or more antigens of the pathogen is CMV pp65, CMV IE1, or a combination thereof.

46. The method of claim 41, wherein the virus is Epstein-Barr virus (EBV).

47. The method of claim 46, wherein the human patient has or is suspected of having an EBV-associated lymphoproliferative disorder (EBV-LPD).

48. The method of claim 46, wherein the human patient has an EBV-LPD.

49. The method of claim 47 or 48, wherein the EBV-LPD is an EBV-positive lymphoma.

50. The method of any of claims 46-49, wherein the one or more antigens of the pathogen is EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2, or a combination thereof.

51. The method of claim 46, wherein the human patient has or is suspected of having an EBV-positive nasopharyngeal carcinoma.

52. The method of claim 46, wherein the human patient has an EBV-positive nasopharyngeal carcinoma.

53. The method of claim 51 or 52, wherein the one or more antigens of the pathogen is

EBNA1, LMP1, LMP2, or a combination thereof.

54. The method of claim 41, wherein the virus is BKV, JCV, herpesvirus, adenovirus, human immunodeficiency virus, influenza virus, ebola virus, poxvirus, rhabdovirus, or paramyxovirus.

55. The method of any one of claims 1-38, wherein the human patient has or is suspected of having a cancer.

56. The method of claim 55, wherein the cancer is a blood cancer.

57. The method of claim 55, wherein the cancer is a cancer of the breast, lung, ovary,

stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostate, thyroid, brain, or skin.

58. The method of any of claims 55-57, wherein the one or more antigens of the cancer is WT1 (Wilms tumor 1).

59. The method of claim 58, wherein the cancer is multiple myeloma or plasma cell

leukemia.

60. The method of any of claims 55-59, wherein the human patient has the cancer.