WO2006026746A2

WO2006026746A2 - Methods to separate and expand antigen-specific t cells

Info

Publication number: WO2006026746A2
Application number: PCT/US2005/031280
Authority: WO
Inventors: Jongming Li; John A. Barrett
Original assignee: The Government Of United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Priority date: 2004-08-31
Filing date: 2005-08-31
Publication date: 2006-03-09
Also published as: WO2006026746A3

Abstract

Methods are provided for producing a population of purified T cells that are specific for a particular antigen, such as a viral antigen or a tumor-associated antigen. In one example the method includes contacting a layer of antigen presenting cells (APCs) with a preselected target antigen for a time sufficient for the antigen to be presented by an APC. The resulting APCs presenting the antigen are incubated with primed T cells under conditions sufficient to allow binding between APCs and primed T cells that recognize the preselected target antigen. After removing non-adherent cells, a population of purified antigen-specific T cells that recognize the preselected target antigen is produced. This population can be expanded by stimulating the cells to proliferate, thereby producing a population of purified antigen-specific T cells. Purified populations of antigen-specific T cells produced using the methods are disclosed herein. In addition, methods of using the antigen-specific T cells, for example to reconstitute immunity, are disclosed.

Description

METHODS TO SEPARATE AND EXPAND ANTIGEN-SPECIFIC T CELLS

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No. 60/606,197 filed August 31, 2004, herein incorporated by reference in its entirety.

FIELD

This application relates to methods that can be used to select and expand antigen- specific T-cells that recognize a preselected target antigen, to purified populations of antigen- specific T-cells that recognize a preselected target antigen, and to therapeutic uses of antigen- specific T-cells that recognize a preselected target antigen.

BACKGROUND

Stem cell transplantation can be used to treat patients having leukemia or other disorders. Transplanted donor T cells (lymphocytes) exert strong alloimmune graft-versus leukemia (GVL) and other anti-tumor effects. However, these donor cells can also cause potentially lethal graft-versus-host disease (GVHD), requiring post-transplant immunosuppression. As a result of such immunosuppression, patients are more likely to contract a potentially fatal infection, such as a cytomegalovirus (CMV) infection, and are less likely to be cured of their malignant disease.

The transfer of T lymphocytes specific for leukemia cells or micro-organism antigens would be useful because therapeutic immune effects would be enhanced while GVHD reactions would not be induced. Although previous reports have indicated that in vitro cultured T cells that recognize chronic myeloid leukemia (CML) cells (Falkenburg et at, Blood 4:1201-8, 1999) or CMV-specific T cells (Peggs et al., Lancet 362:1375-7, 2003) have anti-leukemic effects or prevent CMV reactivation, respectively, currently available methods for isolating and expanding antigen-specific T cells are costly and inefficient (Einsesle and Hamprecht, Lancet 362:1343-4, 2003). Currently, antigen-specific T cells are selected using HLA tetramers or by magnetic beads binding to activation markers, or by laborious limiting dilution techniques. Therefore, improved methods of selecting antigen-specific T cells are needed. In addition, current methods of expanding antigen-specific T cells are unreliable and poorly reproducible, and can require many days to achieve the needed number of cells. In some cases, T cell specificity is lost.

Therefore, a new method of isolating and expanding antigen-specific T cells is needed that is efficient and inexpensive.

SUMMARY

Disclosed herein are novel methods for selecting and expanding antigen-specific T cells that recognize a preselected target antigen. The target antigen(s) can be selected based on the disorder to be treated. For example, in order to enhance antiviral immunity in a subject, one or more viral target antigens are selected, such as a CMV antigen. In another example, to enhance antitumor immunity in a subject, one or more tumor associated antigens (TAAs) are selected. Although antigen-specific T cells are present in peripheral blood mononuclear cell (PBMC) fractions or isolated lymphocyte fractions obtained from a blood sample, the percentage of T cells in the PBMC fraction or the lymphocyte fraction that specifically recognize a particular target antigen is very low. The disclosed methods permit selection of the few desired antigen- specific T cells present in a blood sample, such as a PBMC or lymphocyte fraction, and subsequent expansion of those selected antigen-specific T cells that recognize a preselected target antigen to increase the purity of the antigen-specific T cells.

It was observed that in the presence of presenting antigen, T cells that recognize the antigen bind to antigen presenting cells (APCs) presenting a target antigen more strongly than do T cells that are not specific for the antigen (and are thus not binding in an antigen-specific manner). This permits selection of antigen-specific T cells by exposing target-specific T cells to primed APCs that have been primed by exposure of the APCs to the target antigen recognized by the target-specific T cell. The target-specific T cells are preferentially bound by the APCs, such that T cells that do not specifically recognize the antigen may be preferentially removed. In a particular example, antigen-specific T cells are selected by exposing APCs to a target peptide antigen (such as a target viral or tumor associated antigen) against which desired T cells are to be targeted, such that the APC presents the antigen in association with a major histocompatability complex (MHC). For example, APCs can be exposed to a sufficient amount of a target antigen to sufficiently occupy MHC molecules on the surface of the APC (for example, at least 25% of the MHC molecules are occupied, such at least 50%, at least 75% or at least 90%) and stimulate preferential binding of target T cells to the APCs presenting the target antigen (as compared to APCs that do not present the target antigen). A population of T cells that has been primed for the target antigen is then incubated with the APCs to preferentially adhere the desired T cells to the APCs. T cells that do not preferentially adhere (or bind) to the APCs are then removed, leaving behind a population of cells enriched with the desired T cells that recognize the target antigen.

In particular embodiments, culture conditions are disclosed that allow antigen-specific T cells to be selectively propagated in vitro (or ex vivo). The disclosed methods are efficient and can be inexpensive relative to currently available methods. Generally, the method uses APCs, such as monocyte APCs, to select and then provide a favorable environment to selectively expand antigen-specific T cells that recognize the target antigen. The ability to grow and administer populations of antigen-specific T cells (such as an enriched population, for example a purified population) provides a cost-effective immunotherapy to enhance immune function of a subject receiving the antigen-specific T cells.

In particular examples, the disclosed methods for producing a population of enriched (for example purified) antigen-specific T cells include contacting or incubating APCs (such as monocytes, dendritic cells, or B lymphocytes) with at least one target antigen under conditions sufficient for the antigen to be presented by MHC molecules associated with APCs, thereby generating a population of target antigen-bound APCs. The resulting population of target antigen-bound APCs is then incubated with primed T cells under conditions sufficient to allow preferential binding between target antigen-specific T cells to target antigen-bound APCs. Non¬ adherent (or non-bound) cells are substantially removed, thereby leaving a population of enriched target antigen-specific T cells. The remaining adherent (bound) target antigen-specific T cells can be stimulated to proliferate (for example by incubation with interleukin-2 (IL-2)). This selection process can be repeated one or more times to further increase the purity of the antigen-specific T cells.

The resulting purified target antigen-specific T cells can be cryo-preserved for later use. The method can further include assessing the cytotoxicity of the purified target antigen-specific T cells, or determining whether the purified target antigen-specific T cells are activated. Purified populations of target antigen-specific T cells generated using the disclosed methods are disclosed herein.

The disclosed methods can be used to enrich and expand any target antigen-specific T cell(s) of interest. This broad applicability permits the use of such cells to treat a variety of disorders that benefit from enhanced immune function. For example, purified target antigen- specific T cells can be administered to a subject following an allogenic organ or stem cell transplant to enhance or restore antiviral immunity, or administered to a subject receiving a stem cell allograft or autograft to enhance GVL or other anti-tumor effects. In particular examples in which the target antigen is associated with a viral or fungal pathogen, purified target antigen-specific T cells are administered to a subject infected with or at risk for infection with a virus or fungus. In some examples in which the target antigen is associated with a tumor, purified target antigen-specific T cells are administered to a subject at risk for tumor relapse. Methods are disclosed for decreasing or avoiding graft-versus-host disease (GVHD) in a subject receiving a stem cell transplant from a donor. The method includes contacting APCs (such as monocytes) from the recipient with lymphocytes from the donor under conditions sufficient for donor T cells that recognize alloantigens of the recipient to bind to the recipient's monocytes. The donor T cells that do not bind to the recipient's monocytes are not reactive with the recipient. The donor T cells that do not bind to the recipient's APCs can be administered to the recipient to decrease or prevent GVHD caused by immune reactions to recipient alloantigens following a stem cell transplant, while providing T cells with useful immune functions to the recipient. The recipient's monocytes can be immobilized on a surface, such as a column, and the donor lymphocytes passed over the column. The desired donor T cells that do not bind to the recipient monocytes will flow-through the column and can be collected for later use.

The foregoing and other objects, features, and advantages of the methods and cells described herein will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the first order kinetics of T cell selection.

FIG. 2 is a graph showing the use of allogenic monocytes to select and expand T cells.

SEQUENCE LISTING SEQ ID NO: 1 is an exemplary BK viral antigen.

SEQ ID NO: 2 is an exemplary JC viral antigen. SEQ ID NO: 3 is an exemplary Epstein-Barr (EBV) viral antigen. SEQ ID NO: 4 is an exemplary cytomegalovirus (CMV) viral antigen.

SEQ ID NO: 5 is an exemplary HPV viral antigen.

SEQ ID NO: 6 is an exemplary Influenza A viral antigen.

SEQ ID NO: 7 is a PRAME tumor-associated antigen (TAA). SEQ ID NO: 8 is a (Wilms tumor 1) WTl) TAA.

SEQ ID NO: 9 is a Survivin TAA.

SEQ E) NO: 10 is an alpha feto protein (AFP) TAA.

SEQ E) NO: 11 is a ELF2M TAA.

SEQ E) NO: 12 is a proteinase 3 and its peptide PRl TAA. SEQ ID NO : 13 is a neutrophil elastase TAA.

SEQ ID NO: 14 is a MAGE TAA.

SEQ ID NO: 15 is a MART TAA.

SEQ E) NO: 16 is a tyrosinase TAA.

SEQ ID NO: 17 is a GPlOO TAA. SEQ E) NO: 18 is a NY-Eso-1 TAA.

SEQ E) NO: 19 is a herceptin TAA.

SEQ E) NO: 20 is a carcino-embryonic antigen (CEA) TAA.

SEQ E) NO: 21 is a prostate specific antigen (PSA) TAA.

SEQ E) NOS: 22 and 23 are exemplary BK viral antigens. SEQ E) NOS: 24-29 are exemplary Blastomyces dermatitidis fungal antigens.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS Abbreviations and Terms The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. The singular forms "a," "an," and "the" refer to one or more than one, unless the context clearly dictates otherwise. For example, the term "comprising a target antigen" includes single or plural target antigens and is considered equivalent to the phrase "comprising at least one target antigen." The term "or" refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise. As used herein, "comprises" means "includes." Thus, "comprising A or B," means "including A, B, or A and B," without excluding additional elements.

Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs.

APC antigen-presenting cell

BKV BK polyoma virus

CMV cytomegalovirus

FCS bovine fetal calf serum

GVHD graft-versus-host disease

IL-2 interleukin-2

INF-γ interferon gamma

MHC major histocompatability complex mL milliliter

MON monocytes

NABS normal AB serum

PBL peripheral blood leukocytes

PBMC peripheral blood mononuclear cell

PBS phosphate-buffered saline, pH 7.0

U units μL microliter

Adherent cells: Cells that are able to stick to a substrate, such as a tissue culture dish treated for growth of adherent cells, or are able to bind to other cells. In some examples, such cells (such as APCs) are not dislodged from the substrate during routine washing or culturing of the cells.

Administration: To provide or give a subject an agent (such as a antigen-specific T cells) by any effective route. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes. Alloantigen: A substance genetically inherited by one individual that is capable of inducing a specific immune response by another individual because the latter individual has not inherited the same substance and sees it as "foreign".

Antigen: A substance capable of inducing a specific immune response. A target antigen is an antigen that is selected for recognition, such as a viral-associated antigen, or a tumor-associated antigen, for example those listed in Table 1. In some examples, the target antigen is an antigen involved in the pathogenesis of disease, such as an infection, tumor associated, or autoimmune antigen.

Antigen-presenting cell (APC): A cell that carries on its surface MHC class I or class II molecules capable of presenting an antigen in the context of the MHC molecule to T cells. APCs include, but are not limited to, monocytes, macrophages, dendritic cells, B cells, and Langerhans cells.

APC layer: A thickness of APCs (such as monocytes) formed on a substrate. In some examples the layer is only a single layer of cells thick; however in some examples cells are stacked on top of one another. Does not require 100% confluence of the cells, and includes APCs that are, for example, at least 50% confluent, at least 80% confluent, at least 90% confluent, or even at least 99% confluent.

Antigen-specific T cell: A CD8 or CD4 lymphocyte that recognizes a particular antigen, such as a target antigen. Generally, antigen-specific T cells specifically bind to a particular antigen, but not other antigens. A target antigen-specific T cell specifically binds to a particular target antigen, such as such as a viral-associated antigen, or a tumor-associated antigen, for example those listed in Table 1.

Anti-microbial agent: A compound (or combination of compounds) that inhibits or eliminates the growth, function or activity of an infectious agent, for example by interfering with its multiplication or proliferation. Examples include, but are not limited to anti-viral compounds (such as AZT and protease inhibitors) and anti-fungal compounds (such as amphotericin B).

Bone marrow transplant (BMT): The transfer of bone marrow containing transplantable hematopoietic stem cells, usually from a donor to a recipient, for example by the intravenous infusion of bone marrow. The marrow can be from a previously harvested and stored self-donation (autologous transplant), from a living donor other than the recipient (allogeneic transplant), or from an identical twin donor (syngeneic transplant). Often used to treat malignancies such as leukemia, lymphoma, myeloma, and selected solid tumors, as well as nonmalignant conditions such as aplastic anemia, immunologic deficiencies, and inborn errors of metabolism.

Cancer: Malignant neoplasm that has undergone characteristic anaplasia with loss of differentiation, increased rate of growth, invasion of surrounding tissue, and is capable of metastasis.

CD3 cells: T cells that express CD3 on their surface. CD3 is a complex of at least five membrane-bound polypeptides in mature T-lymphocytes that are non-covalently associated with one another and with the T-cell receptor. Chemotherapy: In cancer treatment, chemotherapy refers to the administration of one or more compounds to kill or slow the reproduction of rapidly multiplying cells, such as tumor cells. Anti-tumor chemotherapeutic agents include, but are not limited to: 5-fluorouracil (5- FU), azathioprine, cyclophosphamide, antimetabolites (such as Fludarabine), antineoplastics (such as Etoposide, Doxorubicin, methotrexate, and Vincristine), carboplatin, cis-platinum and the taxanes (such as taxol).

Decrease: To reduce the quality, amount, or strength of something. In one example, a therapy decreases the incidence of GVHD, or one or more symptoms associated with GVHD, if the subject better tolerates a stem cell transplant as compared to tolerance in the absence of the therapy. In a particular example, a therapy decreases the incidence of GVHD, or one or more symptoms associated with GVHD if the incidence or symptoms of GVHD are decreased subsequent to the therapy, such as a decrease of at least 10%, at least 20%, at least 50%, or even at least 90%. Such decreases can be measured using the methods disclosed herein.

Enhance: To improve the quality, amount, or strength of something. In one example, a therapy enhances the immune system if the immune system is more effective at fighting infection or tumors, as compared to immune function in the absence of the therapy. In a particular example, a therapy enhances the immune system if the number of lymphocytes increases subsequent to the therapy, such as an increase of at least 10%, at least 20%, at least 50%, or even at least 90%. Such enhancement can be measured using the methods disclosed herein, for example determining the number of lymphocytes before and after the therapy using flow cytometry. Enriched cell population: A cell population that is selectively increased in a particular cell type, for example a cell population that has undergone a process to selectively increase a population of T cell that recognize (or fail to recognize) a target antigen.

Epitope: An antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic (such as those that elicit a specific immune response). An antibody specifically binds a particular antigenic epitope on a peptide. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2- dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology. Vol. 66, Glenn E. Morris, Ed (1996). Fungal-associated antigen (FAAs): A fungal antigen which can stimulate fungal- specific T-cell-defined immune responses. Exemplary FAAs include, but are not limited to, an antigen from Candida albicans, Cryptococcus (such as d25, or the MP98 or MP88 mannoprotein from C. neoformans, or an immunological fragment thereof), Blastomyces (such as B. dermatitidis, for example WI-I or an immunological fragment thereof), and Histoplasma (such as H. capsulation).

Graft-versus-host disease (GVΗD): An incompatibility reaction in a subject (host) of low immunological competence who has been the recipient of immunologically competent lymphoid tissue from a donor who is immunologically different from the recipient. The reaction is the result of the action of the transplanted cells against those host tissues that possess an antigen not found in the donor. Acute and chronic GVΗD are a major cause of morbidity and mortality among hematopoietic stem cell transplant recipients. Symptoms can include skin rash, intestinal problems similar to colitis, and liver dysfunction

Ηaplotyping or tissue typing: A method used to identify the haplotype or tissue types of a subject, for example by determining which ΗLA locus (or loci) is expressed on the lymphocytes of a particular subject. The ΗLA genes are located in the major histocompatibility complex (MΗC), a region on the short arm of chromosome 6, and are involved in cell-cell interaction, immune response, organ transplantation, development of cancer, and susceptibility to disease. There are six genetic loci important in transplantation, designated HLA-A, HLA-B, HLA-C, and HLA-DR, HLA-DP and HLA-DQ. At each locus, there can be any of several different alleles.

A widely used method for haplotyping uses the polymerase chain reaction (PCR) to compare the DNA of the subject, with known segments of the genes encoding MHC antigens. The variability of these regions of the genes determines the tissue type or haplotype of the subject. Serologic methods are also used to detect serologically defined antigens on the surfaces of cells. HLA-A, -B, and -C determinants can be measured by known serologic techniques. Briefly, lymphocytes from the subject (isolated from fresh peripheral blood) are incubated with antisera that recognize all known HLA antigens. The cells are spread in a tray with microscopic wells containing various kinds of antisera. The cells are incubated for 30 minutes, followed by an additional 60-minute complement incubation. If the lymphocytes have on their surfaces antigens recognized by the antibodies in the antiserum, the lymphocytes are lysed. A dye can be added to show changes in the permeability of the cell membrane and cell death. The pattern of cells destroyed by lysis indicates the degree of histologic incompatibility. If, for example, the lymphocytes from a person being tested for HLA-A3 are destroyed in a well containing antisera for HLA-A3, the test is positive for this antigen group.

Immobilized: Bound to a surface, such as a solid surface. A solid surface can be for example polymeric, such as polystyrene or polypropylene. In one example, the solid surface is the interior bottom surface of a flask or a tissue culture plate. In another example, the solid surface is in the form of a bead. A specific, non-limiting example of a bead is Tosylated magnetic beads (Dynal).

Immune response: A change in immunity, for example, a response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one example, the response is specific for a particular antigen (an "antigen-specific response"), such as a target antigen which has been selected for therapeutic purposes as a target of the immune response. In one example, an immune response is a T cell response, such as a CD4⁺ response or a CD8⁺ response. In another example, the response is a B cell response, and results in the production of specific antibodies. In a particular example, an increased or enhanced immune response is an increase in the ability of a subject to fight off a disease, such as a viral infection or tumor. Immune synapse: The region of association between an APC and an antigen-specific T cell. In a specific example, it is the complex formed between an antigen/MHC complex on an APC and the T cell receptor on the antigen-specific T cell.

Immuno-deplete: To decrease the number of lymphocytes, such as CD4⁺ or CD8⁺ cells, in a subject. In particular examples, immunodepletion decreases the number of lymphocytes in a subject by at least 50%, such as at least 75%, at least 80%, at least 90%, at least 95%, at least 99%, or even 100%. Specific immunodepletion refers to immunodepletion of a particular lymphocyte, such as a T cell involved in the mediation of disease (such as GVHD). Immuno-depleting agent: One or more compounds, when administered to a subject, result in a decrease in the number of cells of the immune system (such as lymphocytes) in the subject. Examples include, but are not limited to, chemotherapeutic agents, monoclonal antibodies, radiation, and other therapies disclosed herein.

Immunogenic peptide: A peptide that includes an allele-specific motif or other sequence such that the peptide will bind an MHC molecule and induce a cytotoxic T lymphocyte ("CTL") response, or a B cell response (such as antibody production) against the antigen from which the immunogenic peptide is derived.

Immunogenic peptides can identified using methods known in the art, such as sequence motifs or other methods, for example neural net or polynomial determinations. Typically, algorithms are used to determine the "binding threshold" of peptides to select those with scores that give them a high probability of binding at a certain affinity and will be immunogenic. The algorithms are based either on the effects on MHC binding of a particular amino acid at a particular position, the effects on antibody binding of a particular amino acid at a particular position, or the effects on binding of a particular substitution in a motif-containing peptide. Within the context of an immunogenic peptide, a "conserved residue" is one which appears in a significantly higher frequency than would be expected by random distribution at a particular position in a peptide. In one example, a conserved residue is one where the MHC structure may provide a contact point with the immunogenic peptide.

Immunogenic peptides can also be identified by measuring their binding to a specific MHC protein (such as HLA-A) and by their ability to stimulate CD4 or CD8 when presented in the context of the MHC protein. Imniunologically compromised: A subject who has reduced immune function as compared to the same subject previously, or to a population in general. A phenotypically- immunodeficient subject is a subject which is genetically capable of generating an immune response, yet has been phenotypically altered such that no response is seen. Examples of phenotvpically-immunodefϊcient subjects are those who are irradiated, treated with chemotherapy, or are immunosuppressed with an immunosuppressant drug (such as cyclosporine) to suppress tissue rejection following an organ transplant.

Infection: Invasion and multiplication of pathogens in a subject, which can cause local cellular injury due to competitive metabolism, toxins, intracellular replication, or antigen- antibody response.

Infectious disease: Any disease caused by an infectious pathogen. Examples of infectious pathogens include, but are not limited to: viruses and fungi. In a particular example, it is a disease caused by at least one type of infectious pathogen. In another example, it is a disease caused by at least two different types of infectious pathogens. Infectious diseases can affect any body system, be acute (short-acting) or chronic (long-acting), occur with or without fever, strike any age group, and overlap each other.

Viral diseases commonly occur after transplants due to re-activation of viruses already present in the recipient. Particular examples of viral infections include, but are not limited to, cytomegalovirus (CMV) pneumonia, enteritis and retinitis; Epstein-Barr virus (EBV) lymphoproliferative disease; chicken pox/shingles (caused by Varicella zoster virus, VZV); HSV-I and -2 mucositis; HSV-6 encephalitis, BK-virus hemorrhagic cystitis; viral influenza; pneumonia from respiratory syncytial virus (RSV); AIDS (caused by HFV); and hepatitis A, B or C.

Examples of fungal infections that occur after transplants include but are not limited to: aspergillosis; thrush (caused by Candida albicans); cryptococcosis (caused by Cryptococcus); and histoplasmosis.

Interferon-gamma (IFN-γ): A protein produced by T lymphocytes in response to specific antigen or mitogenic stimulation.

Includes naturally occurring IFN-γ peptides and nucleic acid molecules and IFN-γ fragments and variants that retain full or partial IFN-γ biological activity. Sequences for IFN-γ are publicly available (for example, exemplary IFN-γ mRNA sequences are available from GenBank Accession Nos: BC070256; AF506749; and J00219, and exemplary IFN-γ protein sequences are available from GenBank Accession Nos: CAA00226; AAA72254; and 0809316A).

Interleukin (IL)-2: A growth factor for all subpopulations of T- lymphocytes. It is an antigen-unspecific proliferation factor for T-cells that induces cell cycle progression in resting cells, and allows clonal expansion of activated T-lymphocytes.

Includes naturally occurring IL-2 peptides and nucleic acid molecules and IL-2 fragments and variants that retain full or partial IL-2 biological activity. Sequences for IL-2 are publicly available (for example, exemplary IL-2 mRNA sequences are available from GenBank Accession Nos: BC066254; BC066257; E00978; and NM_053836, and exemplary DL-2 protein sequences are available from GenBank Accession Nos: AAD14263; AAG53575; and AAK52904).

Isolated: An "isolated" biological component (such as a portion of hematological material, such as blood components) has been substantially separated or purified away from other biological components of the organism in which the component naturally occurs. An isolated cell is one which has been substantially separated or purified away from other biological components of the organism in which the cell naturally occurs. For example, an isolated antigen-specific T cell population is a population of T cells that recognize a target antigen and which are substantially separated or purified away from other blood cells, such as other T cells. Leukemia: A group of bone marrow diseases involving an uncontrolled increase in white blood cells (leukocytes). Particular examples include, but are not limited to: hairy cell leukemia, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia, and acute nonlymphocytic leukemia (AML).

Leukocyte: Cells in the blood, also termed "white cells," that are involved in defending a subject against infective organisms and foreign substances. Leukocytes are produced in the bone marrow. There are five main types, subdivided between two main groups: polymorphomnuclear leukocytes (neutrophils, eosinophils, basophils) and mononuclear leukocytes (monocytes and lymphocytes). Generally, when a subject has an infection, the production of leukocytes increases. Lymphocyte: A type of white blood cell involved in the immune defenses of the body.

There are two main types of lymphocytes: B-cells and T-cells.

Lymphoproliferation: An increase in the production of lymphocytes. Malignant: Cells which have the properties of anaplasia invasion and metastasis.

Monocyte: A large white blood cell in the blood that ingests microbes or other cells and foreign particles and proteins. When a monocyte passes out of the bloodstream and enters tissues, it develops into a macrophage. Neoplasm: Abnormal growth of cells.

Non-adherent cells: Cells that do not stick to a substrate, such as a tissue culture dish or other cells. Such cells usually "float" in the tissue culture medium. In some examples, such cells can be easily dislodged from a substrate during routine washing or culturing of the cells.

Normal Cell: Non-tumor cell, non-malignant, uninfected cell. Peripheral blood stem cell transplant: A method of transplanting bone marrow stem cells by mobilization of bone marrow stem cells into the peripheral blood for the purposes described above in Bone Marrow Transplantation.

Primed T cells: T cells that have been exposed to an antigen and therefore can respond efficiently to recall the same antigen, and can include memory T cells. In some examples, the T cells are present in a population of PBMCs or lymphocytes, wherein the PBMCs or lymphocytes are exposed to the antigen, for example in the form of a viral lysate or peptide. Exposure can occur in vivo or ex vivo.

Purified: The term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified protein, nucleic acid molecule, or cell is one in which the protein, nucleic acid molecule, or cell is more pure than the protein, nucleic acid molecule, or cell in its natural environment, such as within a cell or within an organism.

In particular examples, purified populations of cells refers to populations of cells that are at least 30% pure, such as at least 40% pure, at least 50% pure, at least 70% pure, at least 75% pure, at least 80% pure, at least 90% pure, at least 95% pure, at least 97% pure, at least 98% pure, or at least 99% pure. In one example, a substantially purified population of T cells specific for a target antigen (antigen-specific T cells) is composed of about 95% of those antigen-specific T cells, that is, the population of cells includes less than about 5% of other T cells that recognize other non-target antigens. The purity of a target antigen-specific T cell population can be measured based on cell surface characteristics (for example, as measured by fluorescence activated cell sorting) or by ability to stimulate a particular immune response (for example, as measured by an ELISA assay), as compared to a control. Reconstituting immunity: Increasing the number of lymphocytes, for example in an immuno-depleted subject, such that the immune system of the subject is enhanced relative to the immune system during immuno-depletion.

Specifically binds: To selectively bind with a single binding affinity for a particular antigen/epitope with which it immunoreacts. Examples include antigens and T cells that selectively immunoreact with a target antigen. In a particular example of specific binding, a T cell receptor on a target antigen-specific T cell specifically recognizes and reacts with a target antigen presented on an APC, such as an MHC complex, wherein the binding is a non-random binding reaction between the T cell receptor and a target antigenic determinant. In a specific example, the desired binding specificity of a target antigen-specific T cell is determined from the reference point of the ability of the T cell receptor on the target antigen-specific T cell to bind to an APC presenting the target antigen, but not an unrelated antigen, and therefore distinguish between two different antigens.

Stem Cell: A pluripotent cell that gives rise to progeny in all defined hematolymphoid lineages. Stem cells transferred into a recipient are capable of fully reconstituting lifelong bone marrow function and immunity.

Stem cell transplant: The process of transplanting hematopoietic stem cells derived from the bone marrow or from the peripheral blood of a donor into a recipient for the purposes described in Bone Marrow Transplant. In some examples, the donor and the recipient are the same subject. In another example, the donor and the recipient are different subjects.

Stimulate proliferation: To increase the growth or reproduction of cells, for example to increase the number of antigen-specific T cells.

Subject: Includes any organism having a vascular system and hematopoietic cells in the wild-type organism. In one example, the subject is a mammalian subject, such as a human or veterinary subject.

T Cell: A white blood cell involved in the immune response. T cells include, but are not limited to, CD4⁺ T cells and CD8⁺ T cells. A CD4⁺ T lymphocyte is an immune cell that carries a marker on its surface known as "cluster of differentiation 4" (CD4). These cells, also known as helper T cells, help orchestrate the immune response, including antibody responses as well as killer T cell responses. CD8⁺ T cells carry the "cluster of differentiation 8" (CD8) marker. Therapeutically effective amount: An amount sufficient to produce a desired therapeutic result, for example an amount of purified target antigen-specific T cells sufficient to increase an immune response against the target antigen in a subject to whom the cells are administered. In particular examples, it is an amount effective to increase an immune response in a subject by at least 10%, for example at least 20%, at least 30%, at least 40%, at least 50%, or even at least 75%.

In one example, the therapeutically effective amount includes a quantity of purified target antigen-specific T cells sufficient to improve signs or symptoms a disease such as cancer, complications from a transplant (such as an infection), for example by increasing an immune response. In a particular example, it is an amount of purified target antigen-specific T cells sufficient to increase an anti-tumor immune response, such as a graft-versus-leukemia (GVL) or graft-versus-tumor (GVT) response. In another particular example, it is an amount of donor cells depleted of donor T cells that recognize alloantigens of the recipient sufficient to decrease the effects or severity of GVHD, for example after allogeneic stem cell transplantation. A therapeutically effective amount of target antigen-specific T cells can be administered in a single dose, or in several doses, for example every two weeks, during a course of treatment. However, the effective amount of purified target antigen-specific T cells can depend on the subject being treated, the severity and type of the condition being treated, and the manner of administration. In one example, a therapeutically effective amount of purified target antigen- specific T cells varies from about 10⁵ cells per kg body weight to about 10⁹ cells per kg body weight, for example at least 10⁶ cells per kg body weight.

The amount of cells effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro assays can be employed to identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each subject's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

The methods disclosed herein have equal application in medical and veterinary settings. Therefore, the general term "subject being treated" is understood to include all animals (such as humans, apes, dogs, cats, horses, and cows) that require an enhanced immune response, for example subjects having cancer, receiving a transplant, or having at least one infectious disease. Tissue culture vessel: A chamber for growing and expanding cells, such as lymphocytes and monocytes. In some examples, the chamber is treated with an agent to permit growth of adherent cells. Exemplary vessels include, but are not limited to, tissue culture plates/dishes, flasks, or tubes. Transplantation: The transfer of a tissue, cells, or an organ, or a portion thereof, from one subject to another subject, from one subject to another part of the same subject, or from one subject to the same part of the same subject. In one example, transplantation of antigen-specific T cells, such as a purified population of antigen-specific T cells, into a subject involves removal of blood from the subject, selection and expansion of the target antigen-specific T cells ex vivo, and introduction of the purified target antigen-specific T cells into the same or a different subject.

An allogeneic transplant is transplantation from one individual to another, wherein the individuals have genes at one or more loci that are not identical in sequence in the two individuals. An allogeneic transplant can occur between two individuals of the same species, who differ genetically, or between individuals of two different species. An autologous transplant is transplantation of a tissue, cells, or a portion thereof from one location to another in the same individual, or the removal of a tissue such as bone marrow derived stem cells, storage of the cells at low temperature, and reinfusion into the same individual at a later time. A syngeneic or congenic transplant is the transfer of a tissue or a portion thereof from one individual to another, wherein the two individuals are genetically identical.

Treating a disease: "Treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition, such a sign or symptom related to immune suppression, such as decreasing symptoms associated with an infection, halting the progression of a tumor, reducing the size of a tumor, or even elimination of a tumor. Treatment can also induce remission or cure of a condition, such as an infection, GVHD, or a tumor. In particular examples, treatment includes preventing a disease, for example by inhibiting the full development of a disease, such as preventing development of an infection or GVHD in a subject who received a transplant. Prevention of a disease does not require a total absence of disease. For example, a decrease of at least 50% can be sufficient. Tumor: A neoplasm. Includes solid and hematological (or liquid) tumors.

Examples of hematological tumors include, but are not limited to: leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelogenous leukemia, and chronic lymphocytic leukemia), myelodysplastic syndrome, and myelodysplasia, polycythemia vera, lymphoma, (such as Hodgkin's disease, all forms of non-Hodgkin's lymphoma), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease.

Examples of solid tumors, such as sarcomas and carcinomas, include, but are not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, bladder carcinoma, melanoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, meningioma, neuroblastoma and retinoblastoma).

Tumor-associated antigens (TAAs): A tumor antigen which can stimulate tumor- specific T-cell-defmed immune responses. Exemplary TAAs include, but are not limited to, RAGE- 1 , tyrosinase, MAGE- 1 , MAGE-2, NY-ESO- 1 , Melan-A/MART- 1 , glycoprotein (gp) 75, gplOO, beta-catenin, PRAME, MUM-I, WT-I, CEA, and PR-I. Additional TAAs are known in the art (for example see Novellino et ah, Cancer Immunol. Immunother. 54(3): 187- 207, 2005) and includes TAAs not yet identified.

Under conditions sufficient for: A phrase that is used to describe any environment that permits the desired activity. In one example, includes culturing cells in a vessel, for example in tissue culture media, sufficient to allow the desired activity. In particular examples, the desired activity is presentation of a target antigen by an APC. In other particular examples, the desired activity is binding between an APC presenting target antigen and a primed T cell. Viral-associated antigen (VAAs): A viral antigen which can stimulate viral-specific T-cell-defined immune responses. Exemplary VAAs include, but are not limited to, an antigen from BK virus, JC virus, Epstein-Barr virus (EBV), cytomegalovirus (CMV), adenovirus, respiratory syncytial virus (RSV), herpes simplex virus 6 (HSV-6), parainfluenza 3, or influenza B.

Viral lysate: The material generated when cells containing virions are lysed, for example when cells containing virions are lysed in a buffer that includes a detergent such as Triton X-100 or sodium dodecyl sulfate (SDS).

Methods for Purifying and Expanding Antigen-Specific T-cells

Methods are provided for producing an enriched population of antigen-specific T cells that recognize a preselected target antigen. Generally, APCs (such as monocytes) present antigens to T cells that induce an MHC restricted response based on whether the antigens are presented in a class I (CD8) or class II (CD4) restricted fashion. The typical T cell response is activation and proliferation.

The target antigen-specific T cells can be enriched (for example purified) and expanded from a donor sample (allogeneic), or from the subject who will receive the cells (autologous). In some examples, the method includes exposing primed target-specific T cells to APCs presenting target antigen under conditions sufficient for binding of the T cells to APCs that present the target antigen. Non-bound cells are removed, thereby leaving a population of enriched (such as purified) antigen-specific T cells that specifically recognize the preselected target antigen. The target antigen is preselected based on the subject to be treated. For example, if the subject is in need of increased antiviral or antifungal immunity, such as a subject who has received an organ or stem cell transplant, one or more target viral or fungal associated antigens are selected. Exemplary target antigens from viruses include antigens from EBV, CMV, HSV, BK, JC, and HFV, as well as those shown in Table 1. Exemplary target antigens from fungi include antigens from Candida albicans, Cryptococcus, Blastomyces, and Histoplasma, as well as those shown in Table 1. In another example, the subject is in need of increased antitumor immunity, such as a subject who has received a stem cell transplant, one or more target tumor- associated antigens are selected. Exemplary target antigens from tumors include WTl, PSA, PRAME, and others listed in Tables 1 and 2. In some examples, the preselected target antigen includes both a target viral associated antigen and a target tumor associated antigen, both a target fungal associated antigen and a target tumor associated antigen, or a target viral associated antigen, a target fungal associated antigen, and a target tumor associated antigen. In particular examples, the method includes exposing T cells primed for the target antigen to APCs in the presence of one or more target antigens that increases binding of the primed T cells to APCs that present the target antigen. T cells that do not specifically bind to the APCs presenting the target antigen are removed, thereby leaving an enriched population of T cells that specifically recognize one or more preselected target antigens. The resulting enriched population of T cells that specifically recognize one or more preselected target antigens can be formulated into a therapeutic dose for administration to a subject in need of the cells. Such a therapeutic dose can include a pharmaceutical carrier (for example buffered saline) or other therapeutic agents (for example anti-microbial or anti-tumor agents). In another example, the removed T cells are formulated into a therapeutic dose for administration to a subject in need of such cells

The method can further include generating the APCs that present the target antigen. For example, APCs can be incubated with a sufficient amount of one or more different target peptide antigens, under conditions sufficient for the target peptide(s) to be presented on the surface of the APCs. This generates a population of APCs that present the target antigen on

MHC molecules on the surface of the APC. The disclosed methods are not limited to particular methods of presenting the target antigen on the surface of an APC. In some examples, the target antigen binds directly to MHC molecules on the surface of the APCs. For example, immunogenic peptides, such as epitope sequences, can be used to bind MHCs directly without the need for cellular processing. In one example, an immunogenic peptide is about 8-15 contiguous amino acids of a full-length target antigen, such as 8-12, 8-10 or 8-9 contiguous amino acids. Such immunogenic fragments can be incubated with APCs under conditions sufficient for the peptide to bind to MHCs on the APC surface. However, target antigens can also be taken up by endocytosis (or other mechanisms) into APCs, where the target antigen is then complexed with the MHC class I or class II molecules and presented on the APC surface. For example, a full-length target antigen amino acid sequence can be incubated with APCs under conditions sufficient for the peptide to be internalized and processed by the cell, such that the appropriate peptide fragment is complexed with MHC molecules and presented on the surface of the APC. Target antigens can also come from within the APC either naturally or due to the insertion of a gene containing the DNA sequence encoding the target protein antigen, such as CMV pp65 or WT-I . The population of APCs that present a sufficient density of the desired target antigen(s) are incubated with primed T cells (such as primed lymphocytes or primed PBMCs) under conditions sufficient to allow binding between the APCs presenting target antigen and the primed T cells that can specifically immunoreact with the target antigen (antigen-specific T cells). A sufficient number of APCs expressing a sufficient density of target antigen in combination with MHC to stimulate enhance binding of a target T cell to the APC are used. In particular examples, at least 50% of the APCs are presenting the desired target antigen on MHC molecules on the APC surface, such as at least 75% of the APCs, at least 90% of the APCs, at least 95% of the APCs, or at least 99% of the APCs. Primed T cells that specifically bind the target antigen are selectively retained in association with the APCs which permits selective removal of T cells that do not bind to the target antigen. Such binding can occur irrespective of the method used to join the target antigen to the MHC molecule of the APC. In particular examples this binding forms an immune synapse between the MHC/peptide complex on the APCs and T cell receptors on the antigen-specific T cells. After a sufficient amount of binding of the target-antigen specific T cells to the APCs, non-adherent cells are removed (for example by substantially removing the non-adherent cells, such as removal of at least 50% of the non-adherent cells, at least 90%, or at least 98% of the non-adherent cells). This generates a population of enriched (such as purified) antigen-specific T cells that are specific for the preselected target antigen. The method can further include stimulating proliferation of the remaining adherent antigen-specific T cells. In some examples, the resulting population of T cells that are specific for the preselected target antigen is at least 30% pure, such as at least 40% pure, or even at least 50% pure.

In some examples, the selection process is repeated one or more times to further increase the purity of the target antigen-specific T cells. For example, a population of target antigen-specific T cells that is partially purified (such as at least 30% pure) can be incubated with APCs presenting the target antigen under conditions sufficient to allow binding between the APCs and the partially purified population of target antigen-specific T cells. Primed T cells that specifically bind the target antigen are selectively retained in association with the APCs which permits selective removal of T cells that do not bind to the target antigen. Non-adherent cells are removed to increase the purity of the adherent target antigen-specific T cells. If desired, the remaining adherent cells can be stimulated to induce T cell proliferation (for example with IL-2). In some examples, this second round of selection results in a substantially purified population of T cells that are specific for the target antigen, such as a population of target antigen-specific T cells that is at least 80% pure, such as at least 90% pure, at least 95% pure, or even at least 97% pure.

In one example, the method includes further performing an assay to demonstrate that the expanded population of T cells is specific for the target antigen. For example, labeled CD3 and labeled interferon-gamma (INF-γ) can be used to determine the percent of target antigen- specific T cells that are present, wherein target antigen-specific T cells are both CD3 and INF-γ positive. In some examples, the method further includes using labeled CD4 and labeled CD8 to determine the percent of target antigen-specific T cells that are present within a particular CD population, wherein target antigen-specific T cells are those that are INF-γ positive and CD8 or CD4 positive. Flow cytometry can be used to conduct such assays. In some examples, the resulting population of target antigenic T cells is at least 80% pure relative to the total population of CD3 positive cells, such as at least 85% pure, at least 90% pure, at least 93% pure, or even at least 95% pure relative to the total population of CD 3 positive cells. In specific examples, the resulting population of target antigenic T cells is at least 80% pure relative to the total population of CD4 or CD8 positive cells, such as at least 85% pure, at least 90% pure, at least 95% pure, or even at least 98% pure relative to the total population of CD4 or CD8 positive cells.

In another example, the method further includes determining the cytotoxicity of the antigen-specific T cells. Methods for determining cytotoxicity are known in the art, for example a ⁵¹Cr-release assay (for example see Walker et al. Nature 328:345-8, 1987; Qin et al. Acta Pharmacol. Sin. 23(6):534-8, 2002; all herein incorporated by reference).

Isolation of monocytes and lymphocytes Monocytes and lymphocytes can be obtained using any method known in the art.

Generally, these populations are isolated from blood drawn from a subject, for example using apheresis (for example leukapheresis) or venous puncture. In one example, blood is obtained from a donor subject, such as an HLA-matched donor or the same subject who is to receive the antigen-specific T cells (recipient subject). In one example, an HLA-matched donor is one that matches at 5/6 or 6/6 of the HLA loci (such as the A, B, and DR loci). In particular examples, the HLA-matched donor is a first degree relative. Monocytes can be isolated from blood obtained from the subject using methods known in the art. In one example, monocytes are obtained by elutriation of monocytes. In another example, monocytes are obtained from peripheral blood mononuclear cells (PBMCs) using a kit to deplete nonmonocytic cells (for example from Miltenyi Biotec, Auburn, CA) or by positive selection using anti-CD14 magnetic beads as recommended by the manufacturer (Miltenyi Biotec). In another example, PBMCs are prepared by centrifugation over a Ficoll-Paque (Pharmacia, Uppsala, Sweden) density gradient and the monocytes separated from lymphocytes by counterflow centrifugation (for example using the J6-MC elutriator system; Beckman Instruments, Palo Alto, CA) or centrifugation on a continuous Percoll (Pharmacia, Piscataway, NJ) density gradient.

Similarly, lymphocytes can be isolated from blood obtained from the subject using methods known in the art. In one example, lymphocytes are collected by elutriation of the lymphocytes. B cells can also be depleted. In another example, PBMCs are prepared by centrifugation over a Ficoll-Paque density gradient and the lymphocytes separated from monocytes as described above.

In some examples, a monocyte/lymphocyte population (a leukocyte pack or peripheral blood leukocytes (PBL)) is isolated from a subject. PBLs can be obtained by incubation of citrated blood in a medium that lyses erythrocytes, and removal of the lysed cells, thereby generating a PBL population. In one example, blood is incubated in NH₄Cl buffer (0.15 M NH₄Cl, 10 mM NaHCO₃ [pH 7.4]) for 5 minutes at 4°C (this can be repeated three times), followed by a wash in Ca²⁺-Mg²⁺-free phosphate-buffered saline (PBS-A) supplemented with 0.035% (wt/vol) EDTA and centrifugation to remove the lysed erythrocytes. However, this method is exemplary, and other methods known to those of skill in the art can also be utilized. The resultant monocyte, lymphocyte, or monocyte/lymphocyte product can be cryopreserved prior to use, using standard methods (for example using a combination of

Pentastarch and DMSO). In some examples, cells are cryopreserved in aliquots of 5 to 200 x 10⁶ cells/vial, such as 6-10 x 10⁶ monocytes/vial, such as 50-200 x 10⁶ lymphocytes/vial, such as 10-50 x 10⁶ PBL/vial. To qualify for cryopreservation, the cell culture ideally contains predominately monocyte, lymphocyte, or monocyte/lymphocyte cells by flow cytometry. Sterility of the population need not be determined at this stage of the target antigen-specific T cells generation procedure; such a determination can occur after the final co-culture of cells. Methods for obtaining other APC populations, such as dendritic and B lymphoblastoid cells, are known in the art. For example, the Blood Dendritic Cell Isolation Kit II (Miltenyi Biotec Inc., Auburn, CA) can be used to obtain dendritic cells from blood according to the manufacturer's instructions or by culture from blood cells using the method of Wong et al. (Cytotherapy, 4: 65-76, 2002, herein incorporated by reference). B lymphoblastoid cells can be cultured from peripheral blood, for example using the method of Tosato {Current Protocols in Immunology, Ed Coligan et al, Wiley, 1994, 7.22.1, herein incorporated by reference).

Priming of T cells T cells, such as those present in a population of PBMCs or lymphocytes, can be incubated with one or more target antigens, to generate a T cell population that is primed for the one or more target antigens. As discussed above, a target antigen can pre-selected based on the condition to be treated.

T cells can be primed using any method known in the art. In particular examples, PBMCs, lymphocytes, or other population of cells containing T cells obtained from a subject, are incubated in the presence of a purified target peptide antigen. In some examples, the preselected target antigen is a viral- or tumor-associated antigen, such as one or more of the target antigens listed in Table 1. The target antigen can be in a purified form, such as a chemically synthesized peptide. In other examples, the target antigen is present in a non- purified form, such as in a crude lysate, for example a viral lysate. In particular examples, the target antigen is presented by and APC, and the T cells incubated with the APCs.

The amount of target antigen used to prime T cells can be readily determined using methods known in the art. Generally, if the target antigen is used in a purified form, about 1-10 μg/cc of peptide is used. When a viral lysate is used, about 0.1-1.0 cc of lysate, such as about 0.2cc, can be used. When APCs are used, about 10-20 million APCs presenting the target antigen can be used for every 40-60 million T cells (or lymphocytes or PBMCs).

In a specific example, lymphocytes are primed in vitro by incubating them with soluble target antigen or viral lysate for 5-7 days under conditions that permit priming of T cells. Viable T cells are recovered, for example by Ficoll-Hypaque centrifugation, thereby generating primed T cells. If desired, the viable primed T cells can be primed again one or more times, for example by incubation with the target antigen for another 5-7 days under the same conditions as those used for the first priming, and viable T cells recovered. In another example, lymphocytes are primed in vivo by inoculating a subject with the target antigen, for example in the form of a vaccine. In this example, T cells obtained from the subject following immunization are already primed. For example, lymphocytes or PBMC obtained from a subject previously immunized with the preselected target antigen are incubated with APCs as described above, without the need for additional priming.

Culturing of cells

APCs are cultured under conditions that permit an APC to present a target antigen. Sources of APCs include, but are not limited to, monocytes, PBL, and dendritic cells obtained from a subject's blood. If the cells were previously cryopreserved, they are thawed before use. In particular examples, APCs are incubated in a tissue culture vessel until they form a layer in the tissue culture vessel. However, the layer need not be 100% confluent, nor does the layer need to be a single layer thick. For example, a layer of APCs includes cells a population of APCs that are adhering to a tissue culture vessel, such as a population of APCs that are at least 50% confluent, at least 75% confluent, or at least 95% confluent. In some examples, monocytes or PBL are incubated in a tissue culture vessel under conditions sufficient for APCs to adhere to the tissue culture vessel, for example under conditions sufficient to form a monocyte layer. Non-adherent cells (such as dead cells or non-APCs) can be removed.

In some examples, when using a purified monocyte population of cells, approximately 6-10 x 10⁶ cells are used, while when using a PBL population of cells, approximately 40-50 x 10⁶ cells are used. The cells are incubated in a tissue culture vessel (such as a tissue culture dish, flask, or other surface to which APCs can adhere) under conditions sufficient for APCs to adhere to the tissue culture vessel, for example under conditions sufficient to form a monocyte layer in the tissue culture vessel. In some examples, the cells are incubated with DNAse to decrease the presence of cell debris and dead cells in the cell suspension. If a PBL mixture was used, the APCs will adhere to the tissue culture vessel while the lymphocytes will float in the culture medium. The lymphocytes can be removed for later use.

Incubation with target antigen After the APCs have adhered to a substrate, the APCs are incubated with one or more target antigens, under conditions sufficient for the target antigen(s) to be presented on the surface of the APC, wherein the target antigen is associated with an MHC molecule (such as class I or class II) on the APC surface. In specific examples where the target antigen has been inserted into a gene and transfected into the APCs so that the target antigen is presented by the APC, the transfected APCs are allowed to adhere to a substrate and no additional antigen is added. The cell-mediated immune response involves the activity of MHC molecules. In humans, this complex is called the "HLA" ("Human Leukocyte Antigen") complex. In mice, it is referred to as the "H-2" complex. The major histocompatibility complex includes three classes of proteins, MHC class I, MHC class II and MHC class III.

MHC class I molecules are expressed on the surface of nearly all nucleated cells. They present antigen peptides to T_c cells (CD8+). There are three MHC class I gene loci in humans, HLA A, HLA B and HLA C. Each locus is highly polymorphic. Therefore, a human may have up to six different kinds of HLA molecules on the surface of their cells. MHC Class II proteins are expressed primarily on antigen presenting cells such as macrophages, dendritic cells and B cells, where they present processed antigenic peptides to T_H cells. There are three MHC Class II gene loci in humans, HLA DP, HLA DQ and HLA DR. MHC class III proteins are associated with various immune processes, and include soluble serum proteins, components of the complement system and tumor necrosis factors.

MHC class I molecules present epitopes from proteins (such as a target antigen) for presentation to T_c cells. HLA A, HLA B and HLA C molecules bind peptides of about 8 to 12 amino acids in length that have particular anchoring residues. The anchoring residues recognized by an HLA class I molecule depend upon the particular allelic form of the HLA molecule. A CD8+ T cell bears T cell receptors that recognize a specific epitope when presented by a particular HLA molecule on a cell. When a Tc cell that has been stimulated by an antigen presenting cell to become a cytotoxic T lymphocyte contacts a cell that bears such an HLA-peptide complex, the CTL forms a conjugate with the cell and destroys it. Programs are publicly available (for example on the Internet) for the prediction of epitopes that bind MHC.

In particular examples, the presentation of peptides by MHC Class I (MHC I) molecules involves the cleavage of a full-length protein (such as an endogenously produced protein, such as a full-length TAA) into peptides by the proteasome, its processing through the ER and Golgi apparatus, its binding to the cleft in an MHC Class I molecule through the anchor residues of the peptide and ultimate presentation on the cell surface. Depending upon the particular anchor residues, among other things, certain peptides may bind more tightly to a particular HLA molecule than others. Peptides that bind well are referred to as "dominant" epitopes, while those that bind less well are termed "subdominant" or "cryptic" epitopes. Dominant epitopes of either self proteins or foreign proteins evoke strong tolerance or immune responses. Subdominant or cryptic epitopes generate weak responses or no responses at all. However, one skilled in the art will appreciate that antigenic peptides that bind to MHC I molecules can be generated ex vivo (for example instead of being processed from a full-length protein in a cell), and allowed to interact with (such as bind) MHC I molecules on a cell surface.

MHC II antigens are generally derived from pinocytotic or phagocytic mechanisms. However, one skilled in the art will appreciate that antigenic peptides that bind to MHC II molecules can be generated ex vivo (for example instead of being processed from a full-length protein in a cell), and allowed to interact with (such as bind) MHC I molecules on a cell surface. In one example, a target antigen includes a peptide sequence bearing a binding motif for an HLA molecule of the subject. These motifs are well known in the art. For example, HLA- A2 is a common allele in the human population. The binding motif for this molecule includes peptides with 9 or 10 amino acids having leucine or methionine in the second position and valine or leucine in the last positions (for example see SEQ ID NO: 8, 9, 12, 13, and 21). Based on the peptide sequence of a target antigen, one can identify amino acid sequences bearing motifs for any particular HLA molecule. Peptides that include these motifs can be prepared by any method known in the art (such as recombinantly, chemically, etc.). In one example, the target antigen is a self protein (such as a TAA) and the amino acid sequences bearing HLA binding motifs are those that encode subdominant or cryptic epitopes. Those epitopes can be identified by a lower comparative binding affinity for the HLA molecule with respect to other epitopes in the molecule or compared with other molecules that bind to the HLA molecule.

Through the study of single amino acid substituted antigen analogs and the sequencing of endogenously bound, naturally processed peptides, critical residues that correspond to motifs required for specific binding to HLA antigen molecules have been identified (see, for example, Southwood et al., J. Immunol. 160:3363, 1998; Rammensee etal, Immunogenetics 41:178, 1995; Rammensee et al, J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, Curr. Opin. Immunol. 6:13, 1994; Sette and Grey, Curr. Opin. Immunol. 4:79, 1992). Furthermore, x-ray crystallographic analysis of HLA-peptide complexes has revealed pockets within the peptide binding cleft of HLA molecules which accommodate, in an allele- specific mode, residues borne by peptide ligands; these residues in turn determine the HLA binding capacity of the peptides in which they are present. (See, for example, Madden, Annu. Rev. Immunol. 13:587, 1995; Smith et al, Immunity 4:203, 1996; Fremont et al, Immunity 8:305, 1998; Stern et al, Structure 2:245, 1994; Jones, Curr. Opin. Immunol. 9:75, 1997; Brown et al, Nature 364:33, 1993.)

In one example, the target antigen incubated with the APCs is a fusion protein that includes an amino acid sequence from the target antigen (such as 8-50 contiguous amino acids, for example 8-15 or 8-12 contiguous amino acids from the target antigen) fused to an HLA binding motif. Such a fusion protein can be incubated with APCs, and be processed by the APCs into a peptide that can bind to the HLA molecule and that have a target antigen epitope.

In some examples, only a single target antigen is used, but in other embodiments, at least one target antigen is used, such as at least 2 different target antigens, at least 3 different target antigens, at least 4 different target antigens, at least 5 different target antigens, at least 10 different target antigens, at least 15 different target antigens, at least 20 different target antigens, or even at least 50 different target antigens, for example one or more target antigens listed in Table 1.

Table 1: Exemplary target antigens

Any antigenic peptide (such as an immunogenic fragment) from the target antigen can be used to generate a population of T cells specific for that target antigen. Numerous such antigenic peptides are known in the art, such as viral and tumor antigens. This disclosure is not limited to using specific target antigen peptides. Particular examples of antigenic peptides from target antigens, include, but are not limited to, those target antigens that are viral, fungal, and tumor associated, such as those shown in Table 1. Additional antigenic peptides are known in the art (for example see Novellino et al, Cancer Immunol Immunother. 54(3): 187-207, 2005, and Chen et al., Cytotherapy, 4:41-8, 2002, both herein incorporated by reference). Although Table 1 discloses particular fragments of full-length target antigen peptides, one skilled in the art will recognize that other fragments or the full-length protein can also be used in the methods disclosed herein. In one example, a target antigen is an "immunogenic fragment" of a full-length target antigen sequence. An "immunogenic fragment" refers to a portion of a protein which, when presented by a cell in the context of a molecule of the MHC, can in a T-cell activation assay, activate a T-cell against a cell expressing the protein.

Typically, such fragments are 8 to 12 contiguous amino acids of a full length antigen, although longer fragments may of course also be used. In some examples, the immunogenic fragment is one that can specifically bind to an MHC molecule on the surface of an APC, without further processing of the epitope sequence. In particular examples, the immunogenic fragment is 8-50 contiguous amino acids from a full-length target antigen sequence, such as 8-20 amino acids, 8- 15 amino acids, 8-12 amino acids, 8-10 amino acids, or 8, 9, 10, 11, 12, 13, 14, 15 or 20 contiguous amino acids from a full-length target antigen sequence. In some examples, APCs are incubated with the immunogenic fragment under conditions sufficient for the immunogenic fragment to specifically bind to MHC molecules on the APC surface, without the need for intracellular processing. In yet other examples, a target antigen is a full-length target antigen amino acid sequence (such as a full-length FAA, TAA, or VAA, for example a viral lysate or full-length cathepsin G). In some examples, the full-length target antigen is one that is internalized by the APC (such as pinocytosed or endocytosed) and processed intracellularly, such that appropriate immunogenic fragments of the full-length target antigen are presented by an MHC molecule on the surface of the APC.

With knowledge of a target antigen sequence, immunogenic fragment sequences predicted to bind to an MHC can be determined using publicly available programs. For example, an HLA binding motif program on the Internet (Bioinformatics and Molecular Analysis Section-BIMAS) can be used to predict epitopes of any tumor-, viral-, or fungal- associated antigen, using routine methods.

In addition, as yet unidentified target TAA sequences can be identified. For example, TILs from a subject with metastatic cancer are grown and tested for the ability to recognize the autologous cancer in vitro. These TILs are administered to the subject to identify the ones that result in tumor regression. The TILs are used to screen expression libraries for genes that express epitopes recognized by the TILs. Subjects then are immunized with these nucleic acid sequences. Alternatively, lymphocytes are sensitized in vitro against antigens encoded by these genes. Then the sensitized lymphocytes are adoptively transferred into subjects and tested for their ability to cause tumor regression. Rosenberg et al., Immunol. Today 18:175, 1997.

Target antigens (either full-length proteins or an immunogenic fragment thereof) can be produced and purified using standard techniques. For example, epitope or full-length target antigens can be produced recombinantly or chemically synthesized by standard methods. A substantially pure peptide preparation will yield a single major band on a non-reducing polyacrylamide gel. In other examples, the target antigen includes a crude viral lysate.

The optimal length of incubation and amount of target antigen used can depend on the particular antigen used. However, the length of incubation and amount of target antigen are sufficient to present a density of MHC/target antigen on the APC surface to achieve enhanced binding of target T cells to the APCs as compared to non-target T cells. In one example in which the APC is a monocyte, the peptide antigen is incubated with APCs for at least 0.5 hour, such as at least 1 hour, at least 2 hours, or even at least 3 hours, such as 1-3 hours, at 37°C. In particular examples, the amount of antigen added to the APCs is 0.1-100 μM, such as 0.1-10 μM, 10-40 μM, 20-40 μM, such as at least 0.1 μM, at least 1 μM, at least 10 μM, at least 20 μM, at least 40 μM, or at least 50 μM. Particular amounts of antigens and periods of incubation can be varied, and optimal ranges readily determined for each antigen using methods known in the art. For example, various amounts of antigen (such as 0.1 - 10 μM) can be incubated with APCs for various amounts of time (such as 10-120 minutes), and the presence of antigen on the surface of the APC determined, for example by flow cytometry.

Incubation with primed T cells

Once the APCs are presenting the target antigen, excess antigen peptide can be removed, for example by washing the APCs with medium or other isotonic solution. Primed T cells, such as a primed lymphocyte or PBMC population, previously exposed to the target antigen are then incubated with the APCs presenting the target antigen. The optimal amount of primed T cells added can vary depending on the amount of APCs used. In some examples, a T cell:APC ratio of at least 6:1 is used, such as at least 8:1, at least 10:1, at least 12:1, at least 15:1, at least 16:1, at least 20:1, or even at least 50:1.

The primed T cells are incubated with the APCs under conditions sufficient to allow binding between the primed T cells and the APCs presenting target antigen, for example binding between a T cell receptor on the T cell and an MHC/target antigen complex on the APC. In some examples, this binding results in the formation of an immune synapse between the T cells that are specific for the target antigen (target antigen-specific T cells) and the APCs. The optimal length of incubation can depend on the particular antigen used. Methods for determining the optimal incubation time can be determined using methods known in the art, such as using electron or confocal microscopy to determine the amount of time it takes to form an immunological synapse with the APC of interest.

In some examples, the incubation time is between 3 and 120 minutes, such as 3-60 minutes, 3-30 minutes, or 3-10 minutes. In particular examples, if the antigen-specific T cell is a CD4 cell, the incubation time is at least 3 minutes. In other examples, if the target antigen- specific T cell is a CD8 cell, the incubation time is at least 6 minutes. Sήmulaήon of T cells

After binding of the primed T cells that recognize the target antigen and APCs presenting the target antigen, for example by the formation of immune synapses, non-adherent cells (those that did not bind to the adherent APCs, for example those that did not form immune synapses) can be removed, for example by washing the cells. The remaining adherent cells are enriched for antigen-specific T cells that specifically recognize the preselected target antigen.

Any method that substantially removes non-adherent cells can be used. In one example, the cell culture medium is aspirated (thereby substantially removing non-adherent cells), and isotonic liquid added to the tissue culture vessel, thereby suspending the non- adherent cells in the isotonic liquid that were not removed during aspiration. If desired, the tissue culture vessel can be gently agitated under conditions that promote suspension of non¬ adherent cells into the isotonic liquid, but do not significantly dislodge adherent cells. The isotonic liquid can be aspirated, thereby further enriching the adherent population of cells. This process of adding and removing isotonic liquid can be repeated one or more times as desired to achieve the desired enrichment of adherent cells. In particular examples, cells are washed 1, 2, or 3 times. Ideally, isotonic liquids do not lyse or otherwise disrupt the adherent cells. Examples of isotonic liquids are known in the art and include culture medium (which may or may not include serum, for example RPMI or DMSO alone) and phosphate buffered saline (PBS). To increase the number of isolated target antigen-specific T cells, proliferation the cells can be stimulated, for example by incubation in the presence of a cytokine, such as IL-2, IL-7, and IL-12. In particular examples, the partially purified target antigen-specific T cells are incubated in the presence of feeder layers of irradiated cells including CD4+ T cells. The cytokine stimulated or irradiated cells can be added immediately after the non-adherent cells are removed, or a time thereafter, such as at least 1 hour later, at least 6 hours later, at least 12 hours later, or at least 24 hours later.

The amount of cytokine added is sufficient to stimulate production and proliferation of T cells, and can be determined using routine methods. In some examples, the amount of IL-2, IL-7, or IL-12 added is about 0.1-100 ILVmL, such as at least 1 ILVmL, at least 10 ILVmL, or at least 20 ILVmL. Determining purity and activity of target antigen-specific T cells

During stimulation of proliferation of target antigen-specific T cells, the cells can be counted to determine the cell number. When the desired number of cells is achieved, purity is determined. In some examples, cells are incubated in the presence of a cytokine, such as IL-2, for 7-14 days, such as 7-10 days, such as at least 7 days.

Purity of the population of antigen-specific T cells can be determined using routine methods. In one example, purity is determined using markers present on the surface of target antigen-specific T cells. Antigen-specific T cells are positive for the CD3 marker, along with the CD4 or CD8 marker, and IFN-γ (which is specific for activated T cells). For example, fluorescence activated cell sorting (FACS) can be used to identify (and sort if desired) populations of cells that are positive for CD3, CD4/CD8, and IFN-γ by using differently colored anti-CD3, anti-CD4, anti-CD8 and anti-IFN-γ. Briefly, stimulated T target antigen-specific cells are incubated in the presence of anti-CD3, anti-CD4, anti-CD8 and anti-IFN-γ (each having a different flourophore attached), for a time sufficient for the antibody to bind to the cells. After removing unbound antibody, cells are analyzed by FACS using routine methods.

In one example, the population of purified antigen-specific T cells that specifically recognize the preselected target antigen is at least 30% pure relative to all CD3 cells present, such as at least 40% pure. In a particular example, the population of purified antigen-specific T cells produced is at least 30% pure relative to all CD3 cells present, such as at least 40% pure. In another example, the population of purified antigen-specific T cells produced is at least 50% pure relative to all CD3 cells present, such as at least 60% pure.

Re-selection of antigen-specific T cells The target antigen-specific T cells can be subjected to one or more rounds of selection to increase the purity of the target antigen-specific T cells. For example, the purified target antigen-specific T cells generated above are incubated with APCs presenting the target antigen under conditions sufficient to allow binding between the APCs and the purified target antigen- specific T cells, for example under conditions sufficient to allow the formation of immune synapses to form between the APCs and the purified target antigen-specific T cells. Non¬ adherent cells are removed, further purifying the population of target antigen-specific T cells. The resulting target antigen-specific T cells can be stimulated to proliferate, for example with IL-2.

The resulting antigen-specific T cells that specifically immunoreact with the target antigen are more pure than with only one round of selection. In one example, the population of purified antigen-specific T cells produced is at least 90% pure relative to all CD3 cells present, such as at least 95% pure or at least 98% pure. In a particular example, the population of purified antigen-specific T cells produced is at least 95% pure relative to all CD4 cells present, such as at least 98% pure. In another example, the population of purified antigen-specific T cells produced is at least 90% pure relative to all CD3 cells present, such as at least 93% pure.

Formation of therapeutic compositions

The present disclosure also provides therapeutic compositions that include either the enriched (such as purified) antigen-specific T cells, or the non-specific T cells (such as the non¬ adherent T cells). In particular examples, the resulting enriched population of target antigen- specific T cells (or non-specific T cells) are placed in a therapeutic dose form for administration to a subject in need of them. In one example, such a therapeutic dose is administered to the subject in need of them, for example as disclosed herein.

Purified target antigen-specific T cells Also comprehended by this disclosure are antigen-specific T cells produced by the disclosed method that specifically recognize the preselected target antigen. In one example, the population of purified antigen-specific T cells produced is at least 30% pure relative to all CD3 cells present, such as at least 40% pure, at least 50% pure, at least 80% pure, or even at least 90% pure. In a particular example, the population of purified antigen-specific T cells produced is at least 30% pure relative to all CD3 cells present, such as at least 40% pure, at least 50% pure, at least 80% pure, at least 90% pure, at least 95% pure, or even at least 98% pure. In another example, the population of purified antigen-specific T cells produced is at least 50% pure relative to all CD3 cells present, such as at least 60% pure, at least 75% pure, at least 80% pure, at least 90% pure, or even at least 93% pure. Expanded and selected target antigen-specific T cells can be tested for mycoplasma, sterility, endotoxin and quality controlled for function and purity prior cryopreservation or prior to infusion into the recipient. Methods for Treatment by Transplanting Purified/Expanded Target Antigen-Specific T-cells

Methods are disclosed for increasing the immune response, such as enhancing the immune system in a subject. Administration of the purified antigen-specific T cells disclosed above will increase the ability of a subject to overcome pathological conditions, such as an infectious disease or a tumor, by targeting an immune response against a pathogen (such as a virus or fungus) or neoplasm. Therefore, by purifying and generating a purified population of selected antigen-specific T cells from a subject ex vivo and introducing a therapeutic amount of these cells into the same subject, or into another subject (allogenic transplant), the immune system of the recipient subject will be enhanced by providing exogenous T cells that specifically recognize and direct an immune response against the pathogen or neoplasm, thus treating the infection or tumor. In some examples, the donor and recipient are tissue-typed prior to administration of purified antigen-specific T cells into the recipient.

Providing antiviral immunity

Methods of providing antiviral immunity to a subject are disclosed. Antiviral immunity can be provided to a subject by administration of target antigen-specific T cells that recognize a target viral-associated antigen. Such administration to a recipient will enhance the recipient's immune response to the infection by providing T cells that are targeted to, recognize, and immunoreact with a preselected viral antigen.

Infections in immune deficient people are a common problem in allograft stem cell recipients and in permanently immunosuppressed organ transplant recipients. The resulting T cell deficiency infections in these subjects are usually from reactivation of viruses already present in the recipient. For example, once acquired, most herpes group viruses (such as CMV, EBV, VZV, HSV) are dormant, and kept suppressed by T cells. However, when patients are immunosuppressed by conditioning regimens, dormant viruses can be reactivated. For example, CMV reactivation, Epstein Barr virus (EBV) reactivation which causes a tumor in B cells (EBV lymphoproliferative disease), and BK virus reactivation which causes hemorrhagic cystitis, can occur following immunosuppression. In addition, HIV infection and congenital immune deficiency are other examples of T cell immune deficiency. These viral infections and reactivations can complicate allostem cell transplants and organ transplants. In one example target antigen-specific T cells that recognize a target viral antigen are administered to a subject who has had, or will receive, an allogeneic stem cell transplant or a solid organ transplant, such as kidney, liver, heart, or lung. For example, a therapeutic amount of target antigen-specific T cells can be administered that recognize one or more preselected target viral antigens, for example at least one of the target antigens listed in Table 1, such as a CMV antigen, or a CMV, EBV, and BKV antigen. In particular examples where the recipient receives an allogeneic stem cell transplant, the target antigen-specific T cells are purified from the donor. In other examples where the recipient receives a solid organ transplant, target antigen-specific T cells are purified from the recipient (autologous T cells). For example, a blood sample containing T cells can be obtained from the recipient prior to receiving the transplant.

Administration of a therapeutic amount of such cells can be used prophylactically to prevent reactivation of the virus in the recipient, or to treat an infection caused by reactivation of the virus. Such target antigen-specific T cells can kill cells containing the infectious agent or assist other immune cells in fighting the infection.

Similar methods can be used to enhance antifungal immunity in a subject, except that a preselected fungal antigen (such as those listed in Table 1) is used instead of a viral-associated antigen.

Providing antitumor immunity

Methods of providing antitumor immunity to a subject are disclosed. Antitumor immunity can be provided to a subject by administration of target antigen-specific T cells that recognize a target tumor-associated antigen. Such administration to a recipient will enhance the recipient's immune response to the tumor by providing T cells that are targeted to, recognize, and immunoreact with a preselected tumor antigen. The preselected tumor antigen is chosen based on the recipient's tumor. For example, if the recipient has a breast tumor, a breast tumor- associated antigen is selected, and if the recipient has a prostate tumor, a prostate tumor- associated antigen is selected, and so forth. Shown below in Table 2 are tumors and respective tumor associated antigens that can be used to generate purified target antigen-specific T cells that can be administered to a subject having that particular tumor. However, one skilled in the art will recognize that other tumors can be treated using other tumor antigens. Table 2: Exemplary tumors and their tumor antigens

In one example target antigen-specific T cells that recognize a target tumor-associated antigen are administered m a therapeutically effective amount to a subject who has had, or will receive, a stem cell allograft or autograft, or who has been vaccinated with the target tumor antigen For example, a therapeutic amount of target antigen-specific T cells can be administered that recognize one or more preselected target tumor-associated antigens, for example at least one of the target antigens listed in Tables 1 or 2.

In particular examples where the recipient has a tumor and has or will receive a stem cell allograft, donor target tumor antigen-specific T cells are administered in a therapeutically effective amount after the stem cell allograft to prevent, decrease, or delay tumor recurrence, or to treat a malignant relapse In some examples where the recipient receives a stem cell autograft, lymphocytes containing target antigen-specific T cells are collected prior to receiving the autograft. Target antigen-specific T cells that recognize a preselected tumor antigen are selected and purified from the lymphocytes using the methods disclosed herein. The purified target antigen-specific T cells are re-introduced back into the subject after debulking. In yet another example, the recipient is vaccinated with the preselected tumor antigen, purified preselected target antigen-specific T cells purified from the recipient and then re-introduced into the recipient to increase the recipient's immune system against the tumor. Administration of a therapeutic amount of target tumor antigen-specific T cells can be used prophylactically to prevent recurrence of the tumor in the recipient, or to treat a relapse of the tumor. Such target antigen-specific T cells can kill cells containing the tumor-associated antigen or assist other immune cells in fighting the tumor.

In a specific example, a recipient has a tumor and has or will receive a stem cell allograft to reconstitute immunity. Following bone marrow irradiation or administration of a cytotoxic drug that has ablated or otherwise compromised bone marrow function, at least two types of donor target antigen-specific T cells are administered in a therapeutically effective amount; antigen-specific T cells that specifically recognize a viral-associated antigen (or a fungal-associated antigen) and antigen-specific T cells that specifically recognize a rumor- associated antigen. Such administration can be used to induce an anti-tumor effect (such as a GVL effect) and an anti-viral effect (such as an anti -viral effect).

Immunodepleting the immune system

In examples where T cells are transplanted from one individual into a recipient, the recipient's immune is depleted or ablated by any method known in the art. Examples of immunodepleting methods include, but are not limited to, the use of chemotherapy, radiotherapy and antilymphocyte antibodies such as Campath, ATG, ALG, OKT3. Such treatment is termed a conditioning regimen and is used to prepare the recipient to take (and not reject) the transplant of lymphocytes and marrow stem cells and to debulk the malignant disease if the recipient is being treated for a malignant disease. In one example, the recipient's immune system is depleted or ablated by the administration of total body irradiation and cyclophosphamide. In another example, fludarabine and other chemotherapy such as busulfan cyclophosphamide or melfalan is administered to deplete T cells and to debulk the malignant disease.

In examples where the donor and recipient are the same subject, a population of purified antigen-specific T cells from the recipient is generated prior to administration of immune-depleting agents (such as radiation or chemotherapy to debulk the malignant disease), and the purified antigen-specific T cells administered subsequent to the administration of immune-depleting agents.

Administration of purified antigen-specific T cells

The disclosed antigen-specific T cells are administered to a recipient in a therapeutic amount. In allogeneic stem cell transplant examples, the recipient receives the antigen-specific T cells following depletion or ablation of their immune system. In autologous stem cell transplant and organ transplant examples, the recipient is given a reinfusion of their own antigen-specific T cells obtained prior to the transplant.

Purified antigen-specific T cells that specifically recognize a preselected target antigen are prepared by the methods disclosed herein. The cells can be tested for mycoplasma, sterility, endotoxin and quality controlled for function and purity prior to infusion into the recipient. If the antigen-specific T cells were cryopreserved, they are thawed prior to administration to the recipient.

A therapeutically effective amount of antigen-specific T cells are administered to the subject. Specific, non-limiting examples of a therapeutically effective amount of purified antigen-specific T cells include purified antigen-specific T cells administered at a dose of about I X lO⁵ cells per kilogram of subject to about 1 X 10⁹ cells per kilogram of subject, such as from about 1 X 10⁶ cells per kilogram to about 1 X 10⁸ cells per kilogram, such as from about 5 X 10⁶ cells per kilogram to about 75 X 10⁶ cells per kilogram, such as at about 25 X 10⁶ cells per kilogram, or at about 50 X 10⁶ cells per kilogram.

Purified antigen-specific T cells can be administered in single or multiple doses as determined by a clinician. For example, the cells can be administered at intervals of approximately 2 weeks depending on the response desired and the response obtained. In some examples, once the desired response is obtained, no further antigen-specific T cells are administered. However, if the recipient displays one or more symptoms associated with infection or the presence or growth of a tumor, a therapeutically effective amount of antigen- specific T cells can be administered at that time.

The purified antigen-specific T cells disclosed herein can be administered with a pharmaceutically acceptable carrier, such as saline. In some examples, other therapeutic agents are administered with the antigen-specific T cells. Other therapeutic agents can be administered before, during, or after administration of the antigen-specific T cells, depending on the desired effect. Exemplary therapeutic agents include, but are not limited to, anti-microbial agents, immune stimulants such as interferon-alpha, or peptide vaccines of the same antigen used to stimulate T cells in vitro. In a particular example, compositions containing purified antigen- specific T cells also include one or more therapeutic agents. Screening for response

After administration of the antigen-specific T cells, the response in the recipient can be monitored as determined appropriate by the clinician.

For example, viral infections can be monitored using methods known in the art. In one example, viral persistence is determined using a sample obtained from the recipient, such as blood or other body fluid. Antibody titers, cultures, and PCR are examples of techniques for following response to therapies. Using methods known in the art, whether viral nucleic acids are present in the subject can be determined, and whether virions are present in the subject can be determined using standard viral culture methods. In addition, the recipient can be monitored for a decrease or loss of symptoms and signs of viral infection, such as fever. In one example, administration of the antigen-specific T cells decreases the presence of virions or viral nucleic acids by at least 10%, for example at least 25%, at least 50%, at least 80%, at least 90%, at least 95% or even at least 99% as compared to an amount present before the administration of the cells. Infection by other pathogens, such as fungi, can be monitored using methods known in the art. For example, the presence of pathogens in the recipient can be determined using a sample obtained from the recipient, such as blood or other body fluid. Using methods known in the art, the sample can be cultured for the presence of particular pathogens. In addition, the recipient can be monitored for a decrease or loss of symptoms and signs of infection, such as fever, the presence of pulmonary infiltrates on an x-ray, or a reduction of infection induced leukocytosis. In one example, therapeutic administration of the antigen-specific T cells that target the preselected fungus decreases the presence of a fungus or fungal nucleic acids by at least 10%, for example at least 25%, at least 50%, at least 80%, at least 90%, at least 95% or even at least 99% as compared to an amount present before the administration of the cells. The response of a malignant disease to the antigen-specific T cells can be determined by methods known in the art. For example, for solid tumors, the tumor can be analyzed using CT, MRI or PET scans to monitor and measure the reduction in size or number of tumors. Tumor markers (such as serum carcinoembryonic antigen (CEA) or alpha-fetal protein (AFP)) can also be followed to assess response to therapy. For liquid tumors, blood and bone marrow can be examined to determine the response. In addition, PCR can be used to monitor for the presence of the appropriate tumor marker, such as WTl expression and BCR/ABL gene quantitation for chronic myelogenous leukemia. In one example, administration of the antigen- specific T cells decreases the size of a tumor, or the number of tumor cells, by at least 10%, for example at least 25%, at least 50%, at least 80%, at least 90%, at least 95% or even at least 99% as compared to the tumor size or cell counts before the administration of the antigen-specific T cells. In addition, the presence of increased numbers of antigen-specific CD4 and CD8 T cells in the recipient can be determined using methods known in the art, such as tetramer analysis, flow cytometric intracellular cytokine staining, ELISPOT and RT-PCR for cytokine production following antigenic stimulation. In one example, administration of the antigen-specific T cells increases the number of antigen-specific CD4 and CD8 T cells by at least 10%, for example at least 25%, at least 50%, at least 80%, at least 90%, at least 95% or even at least 99% as compared an amount of antigen-specific CD4 and CD8 T cells before administration of the cells. In addition, assays can be performed to determine the lymphocyte cytotoxicity of virally- infected or malignant cell targets.

Selective Depletion of Matched or Mis-Matched Donor PBMCs

Methods are disclosed for decreasing graft-versus-host disease (GVHD) in a recipient receiving a stem cell transplant from an allogenic donor. The method includes exposing APCs from the recipient (such as monocytes) to lymphocytes from the donor (such as those present in a PBMC population) for a time sufficient for donor T cells that recognize antigens on recipient APCs to bind to the APCs. In one example, donor T cells are mismatched, for example at 1/6, 2/6, 3/6, 4/6, or 5/6 of the HLA loci (such as the A, B, and DR loci). In such an example, donor T cells that are HLA-mismatched to the recipient are selectively removed from a population of donor cells by exposing the donor cells to the APCs of the recipient under conditions in which the HLA-reacting cells adhere to the APCs. In another example the donor and the recipient are matched at 6/6 of the HLA loci and donor T cells recognizing minor histocompatibility antigens of the recipient are selectively removed.

The donor T cells that do not bind to APCs from the recipient are collected. Regardless of whether HLA antigens are matched or mismatched between the donor and recipient, donor T cells that are reactive with the recipient antigens presented by MHC molecules are removed by the method, thus decreasing the chance of the GVHD reaction of the donor lymphocytes attacking the tissues of the recipients. In addition, donor antigen-specific T cells that recognize antigens on tumor or virus infected cells of the recipient are not removed and are used for therapeutic purposes without risking GVHD because the recipient APC did not present infectious agent antigens or tumor antigens to the donor cells.

The non-binding subset of donor T cells (those that do not bind to recipient APCs) is not reactive with the recipient and can therefore be administered in a therapeutically effective amount to the recipient. This decreases GVHD in the recipient while providing T cells with other useful immune functions to the recipient.

The methods described herein for administration of purified target antigen-specific T cells can be used to intravenously administer the collected donor T cells that are not immunoreactive with the host tissues of the recipient that are not found in the donor. Specific, non-limiting examples of a therapeutically effective amount of donor T cells that are not reactive with the recipient include such cells administered at a dose of about I X lO⁵ cells per kilogram to about I X lO⁹ cells per kilogram. In addition, donor T cells that are not reactive with the recipient can be administered in single or multiple doses.

The recipient APCs can be immobilized on a surface, such as a bead or tissue culture dish. In such examples, the immobilized APCs are exposed to lymphocytes from the donor. This allows for selection or collection of the donor lymphocytes that do not bind to the immobilized APCs.

Disclosure of certain specific examples is not meant to exclude other embodiments. In addition, any treatments described in the specification are not necessarily exclusive of other treatment, but can be combined with other bioactive agents or treatment modalities.

EXAMPLE 1 Selection and Expansion of CMV pp65 antigen-specific T cells This example describes methods used to purify CMV pp65 antigen-specific T cells. In this particular example, monocytes were the particular APCs chosen to select a specific population of target antigen-specific T cells. Similar methods can be used to select and expand any target antigen-specific T cell(s) of interest by substituting another antigen for the CMV antigen. Donor lymphocytes were primed as follows. Lymphocytes (30-60 million) containing

T cells were cultured in the presence of monocytes (10-20 million) and peptide library from CMV pp65 protein (1 μM/cc) (positive CMV serology) for 7 days. If necessary, the lymphocytes were subjected to a second priming, in the presence of TL-2 (20 U/cc).

A monocyte layer of about 6-10 x 10⁶ cells was prepared as follows. Human elutriated monocytes previously obtained and frozen were thawed using 10 mL 10% bovine fetal calf serum (FCS), heat-inactivated and 960 U DNASE per 10 mL cell suspension. The cells were washed once to remove FCS. Alternatively, about 5O x IO⁶ PBL or leukocyte pack was thawed and diluted using 10 mL 10% FCS per 1 mL cell suspension, and washed twice to remove FCS. DNAse (960 U to 2400 U) and 2 mL 10% normal AB serum (NABS), heat-inactivated, were added to the cell pellet. DNAse was provided to decrease clumping of cells. The resuspended cells were dispensed into one well of a 6-well plate and incubated at 37°C for 60- 90 minutes to allow the monocytes to adhere to the plate, and form a monocyte layer. The medium was removed from the cells, and the cells were vigorously washed three times with warm phosphate-buffered saline (PBS) while holding the plate at a 30-45° angle, to remove cells that did not adhere (such as lymphocytes present in the PBL suspension). To the monocyte layer, 2.5 mL of warm 10%NABS/RPMI and peptide

(NLVPMVATV, 0.1-10 μM) (Biosynthesis, MD) or 4 μL of the CMV antigen (250 μg/ml)) was added. The peptide was incubated with the monocyte layer for 1-2.5 hours at 37°C. After removing the medium and washing the monocyte layer three times with 3 mL warm 10% NABS/RPMI, primed lymphocytes (from the same subject as the monocytes were obtained) were added in 2-3 mL 10% NABS to achieve a lymphocyte: APC ratio of at least 6:1. An immunocytochemistry (ICC) IFN-γ assay can be used to determine the optimal time to incubate the lymphocytes.

After 3-30 minutes, the medium and floating cells were removed, and the adherent cells washed three times with 2 mL warm 10% NABS/RPMI, while holding the plate at a 30° angle. For CD4 responders, 3 minutes of incubation is sufficient, while for CD8 responders, 6 minutes is sufficient. If there were a significant amount of floating cells in the center field under the microscope (low field), about 10-15 minutes later the cells were washed again twice with 10% NABS.

As shown in FIG. 1, the selection of antigen-specific T cells occurs with first order kinetics. The Y axis represents the frequency of peptide (CMV pp65: A0201)-specific T cells remaining in the media following selection. The X axis represents selection time (the contacting time between T cells and monocyte layer before the non-adherent cells were removed).

To the remaining adherent cells, recombinant human IL-2 (20 IU/mL) (Roche, Inc.) was added immediately or 24 hours later, and then every 2 days thereafter to stimulate proliferation of the T cells. Cells were incubated in the IL-2 at 37°C (5% CO₂). Media was changed as needed (when yellow). On day 2 or 3, a transfer pipette was used to forcibly agitate the cells, and transferred to a culture flask. The surface of the well was not scraped.

On day 7-10, an immunocytochemistry (ICC) IFN-γ assay was performed to determine the frequency of antigen-specific T cells. At least 10% monocytes were added to act as APCs in the ICC IFN-γ assay. After a first round of selection, a purity of 40-70% of antigen-specific T cells relative to total CD3 cells were obtained. As shown in FIG. 2, T cells from two different donors share the same peptide phenotype.

In order to increase the purity, another round of selection was performed (the resulting antigen-specific T cells were re-incubated with peptide, lymphocytes, and IL-2 as described above). After the second round of selection, a purity of 80-95% of antigen-specific T cells relative to total CD3 cells was obtained.

The CD3 cell counts were also determined using flow cytometry. After starting with about 50 million CD3 cells, by day 7 there were 100 million CD3 cells, with an antigen-specific T cell purity of 0.5% relative to total CD3 cells. After the first selection, at day 14, there were 300 million CD3 cells, with an antigen-specific T cell purity of at least 55% relative to total CD3 cells. After the second selection (by using 30 million T cells from the first selection), at day 21, there were 150 million CD3 cells, with an antigen-specific T cell purity of at least 93% relative to total CD3 cells.

EXAMPLE 2

Identification of CD4 and CD8 BK antigens

This example describes methods used to identify CD4 and CD8 T cell responses to HLA-restricted peptide epitopes of the BK virus. The sequences identified in this example can be used to generate BK-antigen-specific T cells using the methods described herein. PBMC from healthy donors or systemic lupus erythematosus (SLE) patients were cultured for 7 days with BKV-lysate infected DCs or monocytes. A peptide matrix of BKV T- ag (15-mer with 4 amino acid shift) was used to elicit intracellular INF-γ production (ICC) in T cells detected by flow cytometry.

Two HLA class I super motifs were identified: peptide 7 (GNLPLMRKAYLRKCK;

SEQ E) NO: 22) restricted by HLA-B*0702, HLA-B*08 and HLA-DQB 1*0501 and peptide 154 (TFSRMKYNICMGKCI; SEQ E⁾ NO: 23) restricted by HLA-DRBl *0901 plus two or more undetermined HLA class I antigens. Similarly, peptide 15 (TLYKKMEQD VKVAHQ;

SEQ ID NO: 1) was restricted by HLA-DRB 1*0301. INF-γ responses in both normal subjects and SLE patients were similar, ranging from 0.4 - 15% tested in both CD8 and CD4 T lymphocytes after two consecutive weekly stimulations with the original stimulus. Ranges in CD4 T cells were higher than in CD8 lymphocytes. In healthy subjects with HLA-B *07 (n=2), peptide 7 was used to induce proliferative responses expanding the CD8 INF-γ response at least tenfold.

A peptide 7 nonamer (LPLMRKAYL; amino acids 3-11 of SEQ ID NO: 22) was subsequently identified by INF-γ ICC to be responsible for the CD8 T cell expansion. According to SYFPEITHI algorithm, this same sequence is expected to elicit CD8 T cell expansion in HLA-B*08 subjects.

These results indicate that BKV T antigen protein contains peptide epitopes capable of stimulating proliferative responses and INF-γ production in both CD4 and CD8 T cells. These

T antigen peptides can be used to expand donor T cells for adoptive immunotherapy of BKV- induced hemorrhagic cystitis after allogeneic stem cell transplant (SCT).

EXAMPLE 3 Selection and Expansion of BK antigen-specific T cells

This example describes methods used to purify BK antigen-specific T cells. Up to 80% of adults acquire immunity to the BK polyoma virus (BKV) following presumed infection in childhood. BKV reactivation can occur in SLE and in immunosuppressed transplanted patients where it causes hemorrhagic cystitis. Similar methods can be used to select and expand any antigen-specific T cell(s) of interest.

Using the methods described in Example 1, antigen-specific T cells for a BK antigen were generated, using 10 μM/cc of the BK peptide 154 (TFSRMKYNICMGKCI; SEQ E) NO: 23) identified in Example 2. After a first round of selection, a purity of at least 36% of antigen- specific T cells (CD4) relative to total CD3 cells was obtained. In order to increase the purity, another round of selection was performed (the resulting antigen-specific T cells were re-incubated with peptide, monocyte layer, and JL-2 as described in Example 1). After the second round of selection, a purity of 95% of antigen-specific T cells (CD4) relative to total CD3 cells was obtained. The CD4 cell counts were also determined. After starting with about 50 million lymphocytes, by day 14 there were 90 million primed CD3 cells, with an antigen-specific T cell (CD4) purity of 0.4% relative to total CD3 cells. After the first selection, at day 21, there were 200 million CD3 cells, with an antigen-specific T cell (CD4) purity of at least 36% relative to total CD3 cells. After the second selection, at day 28, there were 300 million CD3 cells, with an antigen-specific T cell purity (CD4) of at least 98% relative to total CD3 cells.

EXAMPLE 4 Treatment and Prophylaxis of Viral Infection

This example describes methods that can be used to provide antiviral immunity to a subject using the antigen-specific T cells disclosed herein. One skilled in the art will appreciate that similar methods can be used to provide antifungal immunity, except that fungal preselected target antigens are used instead of antiviral antigens. Such methods can be used in subjects receiving a stem cell or organ transplant in combination with some immunosuppressive therapy, to decrease or prevent reactivation of a dormant infection, to treat an active infection, or to prevent or decrease the likelihood that a subject will contract an infection.

One of the difficulties encountered following an allograft stem cell transplant or organ transplant is infection in the recipient due to immunosuppression. In the case of an allograft stem cell transplant (SCT), the recipient immune system is suppressed or ablated prior to the transplant, for example to treat malignant disease (debulking) or to prevent rejection of the allograft. In one example, the recipient is treated with immunoablative agents such as fludarabine or total body irradiation. Subjects having a congenital disease or aplastic anemia (or other non- malignant condition of the bone marrow) who receive an allograft BMT and donor immune cells (such as lymphocytes) receive immunosuppressive-directed conditioning to immuno-ablate the recipient in order to increase the ability of the subject to accept the donor marrow and donor lymphocytes. In the case of an organ transplant, the recipient immune system is permanently immunosuppressed following the organ transplant, to prevent rejection of the donated organ. Subjects eligible for this approach include, but are not limited to, those with lung failure, renal failure, heart failure, liver failure, pancreatic islet cell failure, and those with resultant diabetes mellitus.

These forms of immune system suppression or ablation can lead to an increased incidence of new infections, and more commonly to reactivation of infections previously present in the recipient. For example, recipients having a suppressed immune system who were previously infected with a herpes virus (such as CMV, EBV, VZV, or HSV) are at risk for reactivation of these dormant viruses. Although such infections and reactivations can occur after autologous transplants, such occurrences are rare. The disclosed methods provide treatment and prophylaxis for such new infections and reactivation. Additional examples of subjects who would benefit from such therapy include, but are not limited to, those having an inherited or acquired immune deficiency disorder such as severe combined immune deficiency disease or AIDS from HTV infection, and those subjects who are refractory to other modalities of treatment, for example those subjects having an infection which was not treatable by other means to control the infection (such as standard anti-microbial therapies).

The antigen-specific T cells of the present disclosure that recognize a preselected target viral antigen (such as those cells generated in Example 1) are used to enhance the immune system towards one or more particular viral antigens. The preselected viral-associated antigen can be chosen based on the subject to be treated. For example, if it is determined that the recipient subject has a dormant herpes virus infection, such as CMV, the preselected viral antigen will be a herpes virus antigen, such as a CMV antigen. If the subject is at risk for reactivation of multiple viruses, multiple antigens for the appropriate viruses can be selected.

Using the methods disclosed herein, antigen-specific T cells that recognize one or more preselected target viral antigens obtained from a donor or recipient subject are purified and expanded ex vivo. The antigen-specific T cells are selected to specifically immunoreact with one or more preselected viral antigens (or fungal antigens). The expanded antigen-specific T cells are introduced at a therapeutically effective dose into the same or another subject to stimulate a subject's immune response to the pathogen. Such methods can be used to reduce one or more signs or symptoms associated with an infection, such as fever, to decrease the severity of an infection, or to decrease the likelihood that the subject will get a new infection, or to prevent or decrease reactivation of a dormant virus in the recipient. In a particular example, the treatment is administered to a subject who is known to have a dormant herpes virus infection, such as CMV.

To further protect a subject from infections that can result from receiving chemotherapy or other immune-depleting therapy, one or more prophylactic compounds can be administered prior, during, or after, to the start of the therapy to enhance the immune system. Prophylactic compounds can be administered separately, or in combination, depending on the requirements of the subject. In addition, the dosage regimens for the prophylaxis are known to those skilled in the art, and can be found in Mandell (Principles and Practice of Infectious Disease; 5th Edition, Copyright 2000 by Churchill Livingstone, Inc.) Exemplary prophylactic compounds include antimicrobial agents, such as anti viral, antibiotics, or anti-fungal compounds, hi a particular example, at the initiation of pre-transplant induction chemotherapy until administration of immunosuppressive agents is terminated, subjects receive: trimethoprim 160 mg/sulfamethoxazole 800 mg for PCP prophylaxis (if a subject is allergic to sulfonamide antibiotics, aerosolized pentamadine (300 mg) is administered); fluconazole (oral or i.v.) for fungal and bacterial prophylaxis, and acyclovir for HSV prophylaxis or ganciclovir for CMV prophylaxis.

Collection of APCs and Lymphocytes

Blood is collected from a subject, such as an HLA-matched donor, and a purified population of antigen-specific T cells generated, for example using the method disclosed in

EXAMPLES 1 and 2. The subject need not receive any particular treatment prior to harvesting the blood. However, if the donor and recipient are the same subject, ideally APCs (such as monocytes) and lymphocytes are obtained prior to immuno-suppression of the subject. Briefly, the subject undergoes a 2 to 5 liter apheresis procedure, or a 15 to 25 liter large volume whole blood apheresis via a 2-armed approach or via a temporary central venous catheter in the femoral position. The apheresis product is subjected to counterflow centrifugal elutriation. Red blood cells are removed from the apheresis product, for example using ACK lysis buffer (Biofluids, Inc., Rockville, MD). The lymphocyte fraction and the monocyte fraction of the elutriation are collected. In some examples, the lymphocyte fraction is depleted of B cells, for example by incubation with an anti-B cell antibody.

The resultant lymphocytes and APCs are cryopreserved using standard methods (for example using a combination of Pentastarch and DMSO) in aliquots of 1 to 200 x 10⁶ cells/vial. To qualify for cryopreservation, the cell culture should contain predominately lymphocytes or APCs (such as monocytes) as determined by flow cytometry. Sterility of the population need not be tested at this stage; such testing can occur after the final co-culture of antigen-specific T- cells.

Expansion and purification of viral antigen-specific T Cells

T cells that recognize a target viral antigen can be purified from a donor, or from the recipient. In the example of a recipient who receives an allogenic stem cell transplant from a donor, the antigen-specific T cells are purified from a donor sample. In the example of a recipient who receives an allogenic organ transplant from a donor, antigen-specific T cells are purified from the recipient, and re-introduced into the recipient following the transplant. In this example, a sample containing T cells (such as a blood sample) is obtained from the subject prior to the organ transplant.

About 6 million APCs are provided (for example monocytes), for example in a layer or in multiple discrete groups, such in a tissue culture vessel. In a particular example, monocytes are provided in a layer that is 100% confluent.

One or more viral-associated peptide antigens, such as one or more of those listed in Table 1, are added to the APCs, for example 1-100 μM of peptide. In one example at least one of the viral antigens listed in Table 1 is used, such as at least 2, at least 3, or at least 4 of the antigens listed in Table 1. The peptide is incubated with the APCs under conditions that permit the peptide to be presented on the surface of APCs, for example by binding to an MHC molecule on the APC surface, or by being endocytosed and subsequently presented with MHC in a complex on the APC surface. In some examples, the incubation is at least 30 minutes, such as at least 120 minutes. A primed T cell population is incubated with the APCs presenting the target antigen at a ratio of at least 6 T cells to 1 APC under conditions that permit binding between the primed T cells and the APCs, for example incubation for 3-120 minutes. -The primed T cell population can include PBMCs or lymphocytes that have been primed, for example with the preselected target antigen (such as by exposing the cells to APCs presenting the preselected viral antigen) or a viral lysate. Binding between the primed T cells and the APCs can result in the formation of immune synapses between a T cell receptor that is specific for the targeted antigen, and the MHC/target antigen on the APC. Unbound cells are removed, for example by washing the cells. The remaining enriched adherent antigen-specific T cells can be stimulated to increase the number of cells, for example with 1-100 IU/mL IL-2.

After about 7-10 days, the purified antigen-specific T cells can be tested for purity and cytotoxicity. If the desired purity and activity is achieved, the purified antigen-specific T cells are administered to the recipient. If the desired purity and activity are not achieved, the purified antigen-specific T cells are re-selected to further increase purity, using the methods described herein.

Administration of target antigen-specific T cells Purified antigen-specific T cells that recognize one or more preselected target viral antigens are administered by any appropriate route, which is typically intravenously. Examples of other routes of administration are intrathecal and intravitreal administration to sites of infection. If the cells were previously cryopreserved, the cells are thawed and diluted in saline solution to a volume of approximately 125 to 250 ml for intravenous infusion. In a particular example, the dose of purified antigen-specific T cells administered to a subject is in the range of 1 x 10⁵ purified antigen-specific T cells/kg subject to 1 x 10⁹ cells/kg subject, such as 1 x 10^δ - 1 x 10⁸ cells/kg. The cells can be administered in at least one pharmaceutically acceptable carrier, such as a saline solution. In addition, the purified antigen-specific T cells can be administered concurrently (or separately) with other therapeutic agents, such as anti-microbial agents, for example anti-viral agents and anti-fungal agents.

In some examples, a recipient is immuno-suppressed prior to administration of the purified antigen-specific T cells. Examples of such recipients include those who are immuno- suppressed prior to receiving an allogeneic SCT, and those who are immuno-suppressed after to receiving an organ transplant. The recipient can receive a stem cell or organ transplant prior to, or at the same time as, the purified antigen-specific T cells.

The determination of whether a purified target antigen-specific T cell infusion was safe is based on the presence or absence of grade 4-5 infusional toxicity attributable to the cells that occurs in the first 14 days post-transplant (Grade 4 toxicity is considered "life-threatening" whereas Grade 5 toxicity is death) and the presence or absence of acute GVHD which is a severe level of acute GVHD (grade II or IV) that occurs within the first 3 months post- transplant. In one example, if no infusional toxicity or grade III-IV GVHD attributable to the antigen-specific T cells is observed in an initial three subjects receiving a particular dose of cells, then it is determined that that dose level has acceptable toxicity, and accrual to a higher dose level can commence. If grade 4 or 5 toxicity or grade III-IV GVHD attributable to the cells is observed in any of the initial three subjects, the number of cells per dose is decreased. The antigen-specific T cells disclosed herein can be administered to a subject one or more times as necessary for a particular subject. Although one infusion may be sufficient, several infusions can be performed to increase the benefit, as some infections are chronic and difficult to treat. If multiple infusions are performed, they can be separated by a period of about two-four weeks.

During such treatment, the patient can be monitored, for example by performing tests about once or twice during each 2-4 week treatment cycle. Tests can include measurement of pathogen load (such as viral load), measurement of immune recovery panels such as T cell counts and T cell diversity and competence using methods known to those skilled in the art. In addition, the recipient can be monitored for a decrease or loss of symptoms and signs of viral infection, such as fever. The presence of increased numbers of antigen-specific CD4 and CD8 T cells in the recipient can also determined using methods known in the art, such as tetramer analysis, flow cytometric intracellular cytokine staining, ELISPOT and RT-PCR for cytokine production following antigenic stimulation.

EXAMPLE 5

Treatment and Prophylaxis of Tumors

This example describes methods that can be used to decrease the incidence of tumor relapse, and to treat a tumor relapse in a subject, using antigen-specific T cells that recognize a preselected tumor antigen. Such cells can be generated using the methods disclosed.

The antigen-specific T cells of the present disclosure can be used to enhance the immune system in a subject towards one or more preselected tumor associated antigens (TAAs), such as at least one of those listed in Table 1 or 2. Administration of target antigen-specific T cells to a subject can improve the subject's immune response to a tumor, such as a cancer, thereby decreasing the risk that the subject will have a tumor relapse, or treating a recurring tumor. Allogenic SCT

Tumor antigen-specific T cells purified from a donor can be administered to a recipient after the recipient has received a SCT from the donor, to decrease or prevent tumor recurrence, or to treat a malignant relapse. For cancer patients, the development of malignant disease relapse after a SCT is a very poor prognostic sign. To decrease the incidence of relapse after transplantation, the administration of additional immune cells, such as purified tumor antigen- specific T cells, at or before the time of relapse can result in tumor regressions or continued remission, respectively.

In such examples, the recipient is immunodepleted or ablated using methods known in the art, such as those described herein. After this treatment, the recipient receives an autologous SCT. For example, peripheral blood stem cells (PBMCs) obtained from a donor can be cryopreserved, and then thawed and administered intravenously immediately to the recipient. The target dose of the PBMCs is > 4 x 10⁶ CD34⁺ cells per kg and from 40 to 400 x 10⁶ T cells/kg (containing both CD4⁺ and CD8⁺ subsets). However, if apheresis of PBMCs yielded fewer cells per kg, this level of CD34⁺ cell dose is utilized.

Within 24 hours after the SCT, the recipient is administered ex vivo generated purified tumor antigen-specific T cells, using the methods disclosed herein. In some examples, the recipient is administered ex vivo generated purified tumor antigen-specific T cells and the SCT at essentially the same time. This method results in enhanced anti-tumor activity, which can be used to decrease the recurrence of a tumor post-transplant. However, the purified tumor antigen-specific T cells can be administered at a later time, such as at any initial sign of tumor recurrence.

Autologous SCT Tumor antigen-specific T cells purified from the recipient can be administered back to the recipient following (or at the same time as) receiving a stem cell autograft, hi this example a T cell population, such as lymphocytes, are collected from the subject prior to immune suppression for debulking, rumor antigen-specific T cells selected and expanded and then reinfused into the subject after debulking, for example using the methods described above. The tumor antigen-specific T cells can be cryopreserved prior to reintroduction into the subject. In addition to administration of tumor antigen-specific T cells to restore immune function, other autologus hematopoietic stem cells are transplanted to restore marrow function. Other subjects that would benefit from enhanced antitumor activity

The immune system of a subject receiving vaccine therapy or monoclonal antibody therapy to treat or prevent a tumor can be further enhanced. Examples of monoclonal antibody therapies include, but are not limited to: Rituxan and Herceptin. Rituxan is a monoclonal antibody to CD20, which is present on B cell malignancies such as lymphoma. Herceptin is a monoclonal antibody to her2-neu, which is often over-expressed on breast cancer cells.

For example, tumor antigen-specific T cells purified from the subject receiving vaccine therapy or monoclonal antibody therapy are administered back to the subject following immunization with the same preselected tumor antigen recognized by the tumor antigen-specific T cells. Such tumor antigen-specific T cells can be purified and expanded from the subject prior to, or following administration of the vaccine or antibody, and can be cryopreserved for later use. Purified tumor antigen-specific T cells can be administered to the subject prior to, during, or after the vaccine or antibody modalities. Administration of tumor antigen-specific T cells, before, concurrently, or after vaccination enhances reactivity to the tumor antigens.

Administration of tumor antigen-specific T cells, before, concurrently, or after administering a monoclonal antibody therapy enhances the therapy by augmenting the cellular aspect of the immune system.

Additional examples of subjects who would benefit from such therapy include, but are not limited to, those subjects who are refractory to other modalities of treatment, for example those subjects having an tumor which was not treatable by other means to control the tumor (such as standard anti-cancer therapies).

Administration of T cells Using the methods disclosed herein, tumor antigen-specific T cells obtained from a subject are purified and expanded ex vivo. The expanded tumor antigen-specific T cells are introduced at a therapeutically effective dose into the same or another subject to enhance the recipient's specific immune response to the tumor of interest. Such methods can be used to reduce one or more symptoms associated with a tumor or tumor relapse, such as the size of a tumor, the number of tumors, the volume of a tumor, and so forth, or can be used to eliminate residual tumor after debulking treatments or to prevent or decrease the occurrence or recurrence of a tumor. Using the methods disclosed in the EXAMPLES above, such as Example 4, tumor antigen-specific T cells are generated. The only difference is that TAAs are used instead of viral antigens. The TAA chosen is based on the tumor to be treated (or prevented); for example if the subject has a breast cancer, a breast TAA is chosen. One skilled in the art will understand how to preselect one or more TAAs depending on the tumor to be treated in the recipient. Other particular examples of TAAs and their associated cancers are listed in Table 2. In one example, at least 1 of the TAAs in Table 1 or 2 is used, such as at least 2, at least 3 or at least 4 of the TAAs in Table 1 or 2.

Tumor antigen-specific T cells can be administered alone or in the presence of a pharmaceutical carrier (such as saline), with one or more lymphocyte stimulators such as IL-2, IL-12, EL-7, or interferon-alpha, or with other therapies. For example, tumor antigen-specific T cells can be used to treat a subject having a tumor, alone or in combination with another therapy, such as chemotherapy, radiation therapy, or an anti-tumor vaccine therapy.

Monitoring

Although one infusion may be sufficient, several infusions can be performed to increase the benefit, as some tumors are typically chronic and difficult to treat. If multiple infusions are performed, they can be separated by a period of about 2-4 weeks. During such treatment, the patient can be monitored, for example by performing tests about once or twice during each 2-4 week treatment cycle.

Tests can include CT, MRI or PET scans to monitor and measure the reduction in size or number of tumors. For liquid tumors, blood and bone marrow can be examined to determine the response. PCR can be used to monitor for the presence of the appropriate tumor marker, such as WTl expression and BCR/ABL gene quantitation for CML. In addition, the presence of increased numbers of antigen-specific CD4 and CD8 T cells in the recipient can be determined using methods known in the art, such as tetramer analysis, flow cytometric intracellular cytokine staining, ELISPOT and RT-PCR for cytokine production following antigenic stimulation. In addition, assays can be performed to determine the lymphocyte cytotoxicity of malignant cell targets. EXAMPLE 6 Enhancing Tumor and Viral immunity

This example describes methods that can be used to increase both tumor and viral immunity in a subject, for example using the methods described in Examples 4 and 5. Preselected target antigen-specific T cells that recognize tumor antigens, and preselected target antigen-specific T cells that recognize viral antigens are generated, and administered to a subject in a therapeutically effective amount to enhance an immune response to a tumor and a virus, for example following an allogenic stem cell transplant. As discussed herein, the choice of tumor and viral antigen can depend on the subject to be treated. For example, a subject having acute myelogenous leukemia can be administered antigen-specific T cells that recognize a WTl, PRAME, PRl, proteinase 3, elastase, or cathepsin G antigens, or combinations thereof, as well as administered antigen-specific T cells that recognize a viral antigen, such as CMV. The recipient is immunodepleted or ablated using methods known in the art, such as those described herein. After this treatment, the recipient receives an autologous SCT and administration of the target antigen-specific T cells that recognize preselected viral antigens and tumor antigens. Administration of the target antigen-specific T cells can be at the same time as the SCT, or before or after the SCT. Such methods can be used to decrease the incidence of tumor relapse, to treat a tumor relapse in a subject, as well as to decrease viral reactivation and treat viral infection.

EXAMPLE 7 Decreasing GVHD This example describes methods that can be used to decrease graft-versus-host disease

(GVHD) in a subject receiving an allogenic SCT, using the antigen-specific T cells disclosed herein.

One complication that can occur following an allogenic stem cell is GVHD, which occurs when donor T cells recognize recipient antigens that are not present in the donor. As a result of this mismatch, recipient antigens are as foreign to the donor T cells, resulting in GVHD. Although mismatches can occur at HLA antigens, in most cases the donor and the patient are completely (6/6) HLA matched. However, problems are still encountered due to mismatch of minor histocompatibility antigens, peptides presented by recipient MHC molecules to the donor. Minor histocompatibility antigen mismatches can cause lethal GVHD. The disclosed methods can decrease the incidence of GVHD by reducing the presence of donor T cells that immunoreact with recipient antigens in a donor SCT graft. In addition, the methods can be used to extend allogenic SCT to recipients who are not completely HLA matched (such as donor-recipient pairs that are HLA-mis-matched at 1/6, 2/6, 3/6, 4/6, 5/6, or 6/6 of the A, B, and DR loci).

The method includes depleting the donor stem cell graft of alloreactive T cells to decrease or prevent GVHD in a recipient. Such methods can be used if the donor and recipient are HLA matched or mismatched. Depletion does not require 100% depletion, and includes reductions of alloreactive T cells. For example, donor T cells present in the stem cell graft that are alloreactive with the recipient are depleted if the number of such cells is reduced by at least 50%, such as at least 75%, at least 90%, at least 95%, at least 99%, or at least 99.9% as compared to an amount present prior to the depletion. Recipient APCs (such as monocytes) are isolated using methods known in the art and described herein. The recipient APCs are incubated with donor lymphocytes (such as those present in a PBMC population) for a time sufficient for donor T cells that are mismatched with the recipient to bind to monocytes from the subject. In one example, donor T cells are HLA- mis-matched at 1/6, 2/6, 3/6, 4/6, 5/6 or 6/6 of the A, B, and DR loci. In another example, donor T cells are matched at 6/6 of the HLA loci. Even in examples of HLA matched donor- recipient pairs (donor and recipient are matched at 6/6 of the HLA loci), recipient minor histocompatibility antigens that differ from the donor can cause the alloresponse in the recipient, resulting in GVHD. Donor T cells that alloreact with the recipient's APC, including those are mismatched for minor histocompatibility antigens and at HLA loci, will bind to the recipient's APCs. The mismatched donor T cells bound to the recipient's APCs can be selectively separated from the remaining lymphocytes that are matched or non-reactive with the recipient and not bound to the recipient's APCs.

The recipient APCs can be immobilized, for example attached to a bead, column, or tissue culture dish. In such examples, the immobilized APCs are exposed to lymphocytes from the donor. The donor lymphocytes that do not bind to the immobilized APCs will then pass over the substrate to which the APCs are immobilized, thereby facilitating their collection. The donor T cells that do not bind to APCs from the recipient subject are administered at a therapeutically effective amount into the recipient. This decreases GVHD in the recipient while providing T cells with other useful immune functions including antitumor and antiviral activities to the recipient. In some examples, such cells are cryopreserved and thawed prior to administration.

The subset of donor T cells that are not reactive with the recipient are administered in a therapeutically effective amount, for example as described in Example 4 for antigen-specific T cells. If the cells were previously cryopreserved, the cells are thawed and diluted in saline solution to a volume of approximately 125 to 250 ml for intravenous infusion. In a particular example, the therapeutically effective dose of donor T cells that are not reactive with recipient APCs is in the range of 1 x 10⁵ cells/kg to 1 x 10⁹ cells/kg, such as 1 x 10⁶ - 1 x 10⁸ cells/kg. The determination of whether a donor T cell infusion was safe is based on the presence or absence of hyperacute GVHD as described in Example 4.

The cells can be administered in at least one pharmaceutically acceptable carrier, such as a saline solution. In addition, the donor T cells that are not reactive with recipient APCs can be administered concurrently (or separately) with other therapeutic agents.

The donor T cells that are not reactive with recipient APCs can be administered to a subject one or more times as necessary for a particular subject. Although one infusion may be sufficient, several infusions can be performed to increase the benefit. If multiple infusions are performed, they can be separated by a period of about two-four weeks. During such treatment, the subject is monitored, for example by performing tests to evaluate GVHD symptoms. In these examples, a recipient is immuno-suppressed or immuno-ablated prior to administration of the donor T cells that are not reactive with recipient APCs. The recipient can receive the SCT prior to, or at the same time as, the purified antigen-specific T cells. The methods disclosed in this example can be combined with the methods described in the examples above to enhance antitumor and antiviral responses, such that the donor stem cell graft is depleted of alloreactive T cells, and also contains purified populations of one or more target antigen-specific T cells that specifically recognize viral or tumor antigens. EXAMPLE 8 Pharmaceutical Compositions and Modes of Administration

Various delivery systems for administering the antigen-specific T cells and the donor T cells that are not alloreactive with recipient APCs disclosed herein are known, and include, but are not limited to, intravenous routes. The present disclosure provides pharmaceutical compositions which include a therapeutically effective amount of purified antigen-specific T cells, or donor T cells that are not reactive with recipient APCs, alone or with a pharmaceutically acceptable carrier. Furthermore, the pharmaceutical compositions or methods of treatment can be administered in combination with other therapeutic treatments, such as chemotherapeutic agents, or antimicrobial agents, or vaccines.

The pharmaceutically acceptable carriers useful herein are conventional. Remington 's Pharmaceutical Sciences, by Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the cells herein disclosed. In general, the nature of the carrier will depend on the mode of administration being employed. For instance, parenteral formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, serum, plasma, serum substitutes, pharmacologically approved tissue culture medium supplemented with autologous serum or blood group AB serum from a blood bank, combinations thereof, or the like, as a vehicle. The carrier and composition can be sterile, and the formulation suits the mode of administration.

hi view of the many possible embodiments to which the principles of our disclosure may be applied, it should be recognized that the illustrated embodiments are only particular examples of the disclosure and should not be taken as a limitation on the scope of the disclosure. Rather, the scope of the disclosure is in accord with the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. A method of producing a population of purified antigen-specific T cells that specifically recognize at least one preselected target antigen, compπsing exposing a population of primed target specific T cells to antigen presenting cells (APCs) presenting the at least one target antigen under conditions sufficient to allow binding between the APCs presenting the at least one target antigen and an antigen-specific pnmed T cell in the population of pnmed T cells that recognizes the at least one target antigen; and removing non-bound cells, thereby leaving a population of enriched antigen-specific T cells that specifically recognize the preselected at least one target antigen.

2. The method of claim 1, further compπsing contacting APCs with at least one preselected target antigen under conditions sufficient for the target antigen to be presented by the APCs, thereby generating APCs presenting the target antigen.

3 The method of claim 1 , further compπsing stimulating proliferation of the enriched antigen-specific T cells that specifically recognize the preselected target antigen.

4. The method of claim 1, further compπsing: contacting the ennched antigen-specific T cells that specifically recognize the preselected target antigen with APCs presenting the target antigen under conditions sufficient to allow binding between APCs presenting the target antigen and an antigen-specific T cell that specifically recognizes the preselected target antigen; and removing non-bound cells, thereby producing a population of substantially purified antigen-specific T cells that specifically recognize the preselected target antigen.

5. The method of claim 1, further compπsing: incubating the ennched antigen-specific T cells that specifically recognize the preselected target antigen with a labeled mterferon-gamma (INF-γ); and determining whether the ennched antigen-specific T cells that specifically recognize the preselected target antigen are activated, wherein detection of binding of the INF-γ to the puπfied antigen-specific T cells indicates that the ennched antigen-specific T cells that specifically recognize the preselected target antigen are activated.

6. The method of claim 1, further comprising cryo-preserving the enriched antigen- specific T cells that specifically recognize the preselected target antigen.

7. The method of claim 1 , wherein the APCs presenting the target antigen are a population of monocytes present in a layer.

8. The method of claim 7, wherein the population of monocytes present in a layer is formed in a tissue culture vessel.

9. The method of claim 1, wherein the at least one preselected target antigen is a viral- associated target antigen.

10. The method of claim 9, wherein the viral-associated target antigen is an antigen from BK virus, JC virus, Epstein-Barr virus (EBV), cytomegalovirus (CMV), adenovirus, respiratory syncytial virus (RSV), herpes simplex virus 6 (HS V-6), parainfluenza 3, or influenza B.

11. The method of claim 1, wherein the at least one preselected target antigen is a tumor-associated target antigen.

12. The method of claim 11, wherein the tumor-associated target antigen is PRAME, WTl, Survivin, cyclin D, cyclin E, proteinase 3 and its peptide PR, neutrophil elastase, cathepsin G, MAGE, MART, tyrosinase, GPlOO, NY-Eso-1, herceptin, carcino-embryonic antigen (CEA), or prostate specific antigen (PSA).

13. The method of claim 1, wherein the at least one preselected target antigen is a fungal-associated target antigen.

14. The method of claim 1, wherein the population of primed target specific T cells comprises a population of peripheral blood mononuclear cells (PBMCs) previously incubated with the at least one target antigen.

15. The method of claim 14, wherein the at least one target antigen comprises a viral lysate.

16. The method of claim 1, wherein binding between APCs presenting the target antigen and the T cell in the population of primed target specific T cells that recognizes the target antigen comprises formation of an immune synapse between the APCs and a T cell receptor on the T cell that recognizes the at least one target antigen.

17. The method of claim 1, wherein exposing the APCs presenting the target antigen with a population of primed target specific T cells comprises adding primed target antigen specific T cells to the APCs presenting the target antigen at a ratio of at least 6:1.

18. The method of claim 1, wherein removing non-bound cells comprises removing culture medium and washing the adherent cells.

19. The method of claim 2, wherein the APCs are contacted with at least two preselected target antigens.

20. The method of claim 1, wherein the enriched antigen-specific T cells that specifically recognize the preselected target antigen are at least 30% pure relative to a total population of CD3 positive cells present.

21. The method of claim 1, further comprising determining cytotoxicity of the purified antigen-specific T cells.

22. The method of claim 3, wherein the enriched antigen-specific T cells that specifically recognize the preselected target antigen are stimulated to proliferate for at least 7 days.

23. The method of claim 3, wherein stimulating proliferation comprises incubating the bound antigen specific T cells with interleukin-2 (IL-2) for a time sufficient to stimulate proliferation.

24. The method of claim 4, wherein the substantially purified antigen-specific T cells that specifically recognize the preselected target antigen are at least 80% pure relative to a total population of CD3 positive cells present.

25. The method of claim 1, further comprising placing the population of enriched antigen-specific T cells that specifically recognize the preselected target antigen into a therapeutic dose form for administration to a subject in need of them.

26. The method of claim 1, further comprising placing the non-bound cells into a therapeutic dose form for administration to a subject in need of them.

27. The method of claim 1, further comprising administering the population of enriched antigen-specific T cells that specifically recognize the preselected target antigen to a subject in need of them.

28. An antigen-specific T cell produced by the method of claim 1.

29. A method of producing a population of purified antigen-specific T cells that specifically recognize a preselected target antigen, comprising: generating a layer of APCs; contacting the APCs with at least one preselected target antigen under conditions sufficient for the antigen to be presented by the APCs, thereby generating a population of APCs presenting the antigen; contacting the population of APCs presenting the target antigen with a population of primed T cells under conditions sufficient to allow formation of an immune synapse between APCs presenting the target antigen and a primed T cell in the population of primed T cells that recognizes the target antigen; removing non- bound cells, thereby leaving a population of enriched antigen-specific T cells that specifically recognize the preselected target antigen; and stimulating proliferation of the population of purified antigen-specific T cells that specifically recognize the preselected target antigen, thereby producing a population of purified antigen-specific T cells that specifically recognize the preselected target antigen.

30. A method of enhancing an immune system in a subject, comprising: administering to the subject a composition comprising a population of the purified antigen-specific T cells of claim 28, wherein administration of the population of purified antigen-specific T cells enhances the immune system of the subject.

31. The method of claim 30, wherein the preselected antigen is a viral-associated antigen, and the immune system is enhanced against the viral-associated antigen.

32. The method of claim 30, wherein the preselected antigen is a tumor-associated antigen, and the immune system is enhanced against the tumor-associated antigen.

33. The method of claim 30, wherein the population of purified antigen-specific T cells are cryopreserved and thawed prior to administering the antigen-specific T cells to the subject.

34. The method of claim 30, wherein the population of purified antigen-specific T cells are administered at a dose of about 1 X 10⁵ to about 2 x 10⁹ purified antigen-specific T cells per kilogram of subject.

35. The method of claim 34, wherein the population of purified antigen-specific T cells are administered at a dose of about 1 X 10⁶ to about 1 x 10⁸ purified antigen-specific T cells per kilogram of subject.

36. The method of claim 30, wherein the composition further comprises a pharmaceutically acceptable carrier.

37. The method of claim 31 or 32, wherein the subject previously received an autologous stem cell transplant the population of purified antigen-specific T cells that specifically recognize a preselected target antigen are obtained from a donor subject.

38. The method of claim 32, wherein the subject previously received an organ transplant and the population of purified antigen-specific T cells that specifically recognize a preselected target antigen are obtained from the subject.

39. The method of claim 37, wherein the subject is a risk for developing a tumor recurrence or has a tumor relapse and the composition is administered to treat or prevent the tumor.

40. A method of treating a subject having a CMV infection or preventing the infection due to reactivation of CMV, comprising: producing a population of purified antigen-specific T cells that specifically recognize a preselected target antigen using the method of claim 10, wherein the preselected target antigen is a CMV antigen; and administering the population of purified antigen-specific T cells that specifically recognize a preselected target antigen to the subject, wherein administration of the purified antigen-specific T cells treats or prevents CMV infection in the subject.

41. A method of enhancing a subject's immune system against a tumor, comprising: producing a population of purified antigen-specific T cells from the subject that specifically recognize a preselected target antigen using the method of claim 11 ; administering a conditioning regimen or other therapy to the subject to treat the tumor; and administering the population of purified antigen-specific T cells to the subject, wherein administration of the antigen-specific T cells enhances the subject's immune system against the tumor.

42. A method of enhancing an immune system against a tumor in a subject, comprising: producing a population of purified antigen-specific T cells from a donor subject that specifically recognize a preselected target antigen using the method of claim 11; administering an immunosuppressive conditioning regimen to the subject; administering stem cells from the donor to the subject; and administering the population of purified antigen-specific T cells to the subject, wherein administration of the antigen-specific T cells enhances the immune system of the subject against the tumor in the subject.

43. A method of treating or preventing recurrence of a tumor in a subject, comprising: administering to the subject a composition comprising a population of purified antigen- specific T cells produced by the method of claim 11, wherein administration of the population of purified antigen-specific T cells treats or prevents recurrence of the tumor in the subject.

44. A method of decreasing graft-versus-host disease (GVHD) in a transplant recipient, comprising: contacting APCs from the transplant recipient with lymphocytes from a transplant donor under conditions sufficient for transplant donor T cells that recognize alloantigens of the transplant recipient to bind to the APCs from the transplant recipient; collecting transplant donor T cells that do not bind to APCs from the transplant recipient, wherein the transplant donor T cells that do not bind to APCs from the transplant recipient are not reactive with the transplant recipient; and administering the transplant donor T cells that do not bind to APCs from the transplant recipient, thereby decreasing GVHD in the transplant recipient.

45. The method of claim 44, wherein the APCs are immobilized on a surface, and contacting APCs with lymphocytes comprises contacting the lymphocytes with the immobilized surface.

46. The method of claim 44, further comprising administration of the antigen-specific T cell of claim 28.