WO2009042773A1 - Adoptively transferred tumor-specific t cells stimulated ex vivo using herpes simplex virus amplicons - Google Patents

Abstract

Compositions in the treatment of cancer provide tumor specific T lymphocytes which have been administered to patients. The tumor specific T lymphocytes are contacted with tumor cells expressing a co-stimulatory T cell antigen.

Description

ADOPTIVELY TRANSFERRED TUMOR-SPECIFIC T CELLS STIMULATED EX VIVO USING HERPES SIMPLEX VIRUS AMPLICONS

CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims the priority of U.S. provisional patent application No.

60/974,953 entitled filed September 25, 2007, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under ROl CA87978 and ROl CA

742273 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION Embodiments of the invention relate to the generation of antigen specific lymphocytes for adoptive cell therapy in the prevention and treatment of cancer.

BACKGROUND

4-1BB (CD137, ILA, TNFRSF9) is a type I transmembrane glycoprotein belonging to the TNF receptor superfamily. 4- IBB expression is observed in a range of myeloid and lymphoid cells, including CD4⁺ and CD8⁺ T cells, intraepithelial lymphocytes, natural killer cells, monocytes, and dendritic cells (DCs). In contrast to CD28 expression on naive T cells, 4- IBB is induced on T cells following activation. CD28 appears to relay an initial costimulatory signal followed by 4- IBB signaling which serves to further shape the T cell response.

4- IBB ligation induces cytokine secretion, especially IFN-γ, enhances proliferation and survival of T cells in vitro and in vivo, and plays a role in the generation and expansion of effector and memory CTLs. Administration of agonistic anti-4-lBB monoclonal antibody (mAb) enhanced anti-tumor responses in the poorly immunogenic Ag 104 A sarcoma model and improved anti-tumor effects seen with adoptive transfer of CD8⁺ T cells in several tumor models. 4-lBB-mediated anti-tumor effects have been ascribed to the prevention of programmed cell death leading to the accumulation of anti-tumor effector cells. Studies of cellular therapy and vaccination for immunotherapy of cancer are just beginning. The present invention satisfies the need for improving anti-cancer immunotherapy.

SUMMARY

This Summary is provided to present a summary of the invention to briefly indicate the nature and substance of the invention. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims.

In a preferred embodiment, a method of treating a patient suffering from cancer comprising: obtaining a patient sample comprising tumor tissue or tumor cells; administering a composition comprising a lymphocyte co-stimulatory antigen to the tumor cells; and, expressing the co-stimulatory antigen; obtaining lymphocytes and culturing the lymphocytes with the tumor cells expressing the co-stimulatory antigen; administering the lymphocytes to the patient; and, treating the patient suffering from cancer. In another preferred embodiment, the lymphocytes are preferably T lymphocytes comprising autologous, heterologous, syngeneic, allogeneic or xenogeneic T lymphocytes.

In another preferred embodiment, the composition comprising a lymphocyte co- stimulatory antigen comprises a vector expressing the co-stimulatory antigen, polynucleotide or polypeptide. In another preferred embodiment, the co-stimulatory antigen comprises a CD4⁺ or a

CD8⁺ T lymphocyte specific co-stimulatory antigen.

In yet another embodiment, the vector comprises a viral, recombinant or plasmid vector. The viral vector preferably is a herpes viral vector, an adenoviral vector, an adeno- associated viral vector, a retroviral vector, a lentiviral vector, a herpes viral vector, polyoma viral vector or hepatitis B viral vector.

In another preferred embodiment, the vector is a replication deficient herpes simplex virus (HSV) amplicon expressing the 4- IBBL (CD 137L) co-stimulatory antigen. In other embodiments, the co-stimulatory antigen is CD137 (4-1BB), CD28, CD3, OX40, mutants, variants, analogues, homologues, ligands or fragments thereof. In another preferred embodiment, culturing the lymphocytes with the tumor cells expressing the co-stimulatory antigen generates tumor specific T lymphocytes.

In another preferred embodiment, administering of the tumor specific T-lymphocytes to the patient generates tumor specific effector memory T lymphocytes in vivo. In another preferred embodiment, wherein the administering of the tumor specific T lymphocytes to a patient further comprises surgery and/or administering chemokines or cytokines.

In another preferred embodiment, the method of treating a patient suffering from cancer further comprises administration of one or more therapeutic agents. Preferably, the one or more therapeutic agents are co-administered, prior to or after administration of the tumor specific T lymphocytes.

In another preferred embodiment, the one or more therapeutic agents comprise chemotherapy, chemokines, radionuclides, cytokines, anti-angiogenic agents or radiotherapy. In another preferred embodiment, the method of treating a patient with cancer further comprises administering to the patient lymphocytes with specificity for one or more tumor antigens.

In another preferred embodiment, a method of treating cancer comprises obtaining a tumor sample and lymphocytes from a patient; transducing tumor cells from the sample with a vector comprising a polynucleotide sequence encoding a T-lymphocyte co-stimulatory antigen, mutants, variants, homologues and fragments thereof; culturing the tumor cells expressing the antigen with T lymphocytes obtained from the patient; re-infusing the T lymphocytes into the patient; and, treating cancer.

In another preferred embodiment, the vector is a replication deficient herpes simplex virus (HSV) amplicon expressing the 4-1BBL (CD137L) co-stimulatory antigen.

In another preferred embodiment, culturing the lymphocytes with the tumor cells expressing the co-stimulatory antigen generates tumor specific CD8⁺ T lymphocytes.

In another preferred embodiment, administering of the tumor specific T-lymphocytes to the patient generates tumor specific effector memory T lymphocytes in vivo. In another preferred embodiment, the administration of the tumor specific T lymphocytes to a patient further comprises administering of chemokines or cytokines.

In another preferred embodiment, the method of treating a patient suffering from cancer further comprises surgery and/or administration of one or more therapeutic agents.

In another preferred embodiment, the one or more therapeutic agents are co- administered, prior to or after administration of the tumor specific T lymphocytes. Preferably, the one or more therapeutic agents comprise chemotherapy, chemokines, radionuclides, cytokines, anti-angiogenic agents or radiotherapy. In another preferred embodiment, the method of treating a patient with cancer further comprises administering to the patient lymphocytes with specificity for one or more tumor antigens.

In another preferred embodiment, a method of preventing cancer in a patient at risk of developing cancer comprising: obtaining a sample from a patient; administering a composition expressing one or more tumor antigens and a T lymphocyte co-stimulatory antigen; expanding the tumor specific T lymphocytes ex vivo; administering to the patient the tumor specific T lymphocytes; and, preventing cancer in a patient at risk for developing cancer. In another preferred embodiment, the sample comprises isolated cells.

In another preferred embodiment, the isolated cells comprise at least one of stem cells, antigen presenting cells, fibroblasts, kidney cells, lung cells, hepatocytes, myoblasts, myotube cells, neuron cells and oocytes. In another preferred embodiment, an expression vector comprises a polynucleotide sequence encoding at least one of CD137, CD137L, 4-1BB, 4- IBBL, variants, mutants, homologues and fragments thereof.

In another preferred embodiment, the vector is a herpes simplex virus amplicon. Preferably, the polynucleotide sequence comprises CD137L or 4-1BBL, variants, mutants, alleles, homologues and fragments thereof. Other co-stimulatory antigens are also within the scope of embodiments of the invention., e.g. CD28, CD3,; any adhesion molecules and ligands thereof, cytokines and ligands thereof.

In another preferred embodiment, an isolated cell expresses at least one of CD 137, CD137L, 4-1BB, 4-1BBL, variants, mutants, fragments, homologues and fragments thereof.

In another preferred embodiment, the cell comprises at least one of tumor cells, stem cells, antigen presenting cells, fibroblasts, kidney cells, lung cells, hepatocytes, myoblasts, myotube cells, neuron cells and oocytes.

Other aspects are described infra.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1 A-ID show a HSV amplicon vector encoding 4- IBBL and proliferation of

CD8⁺ OT-I T cells in response to HSV amplicon-transduced tumor. Figure IA is a schematic representation showing the HSV.4-1BBL amplicon plasmid DNA, which is packaged into helper virus-free viral stocks using an HSV genome-containing bacterial artificial chromosome (BAC), having its cognate HSV packaging elements (pac sequences) deleted. Vhs, or the viral host shutdown gene, is expressed from a separate plasmid (pBS-vhs) in order to enhance viral titers. Baby hamster kidney (BHK) cell monolayers are transfected with all three vectors, and the HSV amplicon viral particles are later harvested, concentrated, and titered for use. Figure IB is a graph showing LLC/OVA and LLC tumors transduced with HSV amplicons encoding either human B7.1 (HSV.B7.1) or murine 4-1BBL (HSV.4- IBBL) as T cell costimulatory ligands or β-galactosidase (HSV.LacZ) as an HSV control at MOI=I . After 2 days, the tumor cells were treated with 0.4mg/ml mitomycin C and stained for B7.1 and 4-1BBL expression. Solid line indicates staining of untransduced tumor, and dashed line indicates staining of HSV.LacZ-transduced tumor. Figure 1C is a graph showing purified OT-I T cells (10⁵) added to mitomycin-treated tumor cells (2 x 10⁴) and incubated at 37°C. Thymidine was added 20 h before the plate was harvested following 3 days of co- culture. Thymidine uptake of tumor cells alone was minimal. SD of triplicates is shown. Data represents six separate experiments. Figure ID is a graph showing a 10⁵ total CD8⁺ T cells (OT-I and/or normal CD8⁺ splenocytes) incubated with or without HSV.4- IBBL- transduced LLC/OVA (5 x 10⁴) for 3 or 5 days. Thymidine was added 19 h before harvest. SD of triplicates is shown. Data represents two separate experiments.

Figures 2A-2B show the phenotype of OT-I /GFP cells prior to adoptive transfer and experimental outline. Figure 2A is FACS scan showing CD8⁺ OT-l/GFP T cells added to mitomycin C-treated LLC/OVA tumor cells. Three days later OT-l/GFP T cells were stained for surface activation markers and analyzed by flow cytometry. Filled histogram = OT- l/GFP + HSV.4-lBBL-transduced LLC/OVA; Bold solid line= OT-l/GFP + HSV.B7.1- transduced LLC/OVA; Dashed line = OT-l/GFP + HSV.LacZ transduced LLC/OVA; Dotted line = Naive OT-l/GFP. Data is representative of 3 separate experiments. Figure 2B is a schematic illustration showing LLC/OVA tumor cells which were transduced with HSV.4- IBBL or HSV.B7.1 at an MOI of 1 and cultured for one day. Tumor cells were then washed and treated with mitomycin C. CD8⁺ OT-l/GFP cells were purified from spleen and co- cultured with transduced tumor for three days. Stimulated OT-l/GFP cells were separated from tumor cells using anti-CD8antibody-magnetic beads. OT-l/GFP cells were administered i.v. into C57BL/6 mice bearing LLC/OVA tumor. LLC/OVA tumor was palpable following 2-4 days of s.c. injection. Peripheral blood, spleen, TDLN, and the tumor bed were examined for the presence of OT-l/GFP cells. The spleen was analyzed for cytolytic activity, and the phenotype of OT-l/GFP cells were characterized in the spleen, TDLN, and tumor bed. Figures 3A-3D show the presence of OT-l/GFP cells in the peripheral blood, spleen, and tumor bed following adoptive transfer of expanded OT-l/GFP cells. Figure 3 A: Blood was collected 6 days post transfer of OT-l/GFP cells. Percentage of OT-l/GFP cells in blood was assessed using flow cytometry and used to calculate cell number from the volume of blood collected. N=5, 6, and 6 for the tumor bearing group. N=3 for each of the non-tumor bearing groups. The average is shown as a horizontal bar ± SD. p values are statistical results from a Student's t-Test. Figure 3B: splenocytes from mice were collected on day 17 post OT-l/GFP adoptive transfer. They were stained with anti-CD8-Cy-Chrome and analyzed by flow cytometry. Numbers indicate percentage of GFP⁺ cells in the CD8⁺ gate. Figure 3C: Frozen spleen harvested 32 days following adoptive transfer of OT-l/GFP cells were sectioned, fixed by formaldehyde vapor, and counterstained with Hoechst 33342 to show nuclei. GFP⁺ areas indicate infiltration by adoptively transferred CD8⁺ OT-l/GFP cells. Images were taken at 2Ox magnification. Upper left: GFP⁺ spleen (positive control); upper right: transfer of naϊve OT-l/GFP cells; lower left: transfer of HSV.4-lBBL-expanded OT- 1/GFP cells; lower right: transfer of HSV.B7.1 -expanded OT-l/GFP cells. Data represents at least two separate experiments. Figure 3D: tumors from mice were collected on day 6 post OT-l/GFP adoptive transfer. They were stained with anti-CD8-Cy-Chrome and analyzed by flow cytometry.

Figures 4A-4B show BrdU incorporation by adoptively transferred OT-I . Figure 4 A: Thy 1.1+ mice were pulsed with BrdU on days 7 and 10 and sacrificed on day 12 to determine in vivo proliferation of OT-I CD8⁺ cells and their distribution in the spleen, TDLNs, and non- TDLN. Numbers indicate percentage of BrdU⁺ cells in a CD8⁺Thyl.2⁺ gate. Figure 4B: The presence of proliferating OT-I cells in the tumor was assessed by pulsing mice on days 3 and 6 with 1 mg of BrdU and harvesting the tumor on day 8. Numbers indicate percentage in the lymphocyte gate. Data is representative of two experiments.

Figures 5A-5D are graphs showing cytolytic activity, tumor volumes, and tumor eradication in mice following adoptive transfer of OT-l/GFP T cells. CD8⁺ OT-l/GFP T cells were activated in vitro with transduced LLC/OVA at an E:T ratio of 3:2 for 3 days. Figures 5 A and 5B: Activated OT-l/GFP or naϊve OT-l/GFP (2 x 10⁶) cells were administered into the tail vein of mice injected with tumor s.c. 4 days previously. Figure 5 A: Tumors were measured at the time points indicated. Error bars indicate SEM. p values were determined from regression analysis. O/G HSV.4-1BBL= 10 mice; O/G HSV.B7.1 = 5 mice; naϊve O/G = 5 mice; No O/G = 3 mice. Figure 5B: At 22 days following tumor injection, splenocytes were harvested and cultured with recombinant human IL-2 (10 U/ml) and mitomycin C-treated LLC/OVA cells at an E:T ratio of 10:1 for 6 days. LLC/OVA cells were used as targets in an 8-h ⁵¹Cr release assay. Percent lysis with SD is shown. Data represent two separate experiments. Figures 5C and 5D: Activated OT-l/GFP or naive OT-I (4 x 10⁶) were administered into the tail vein of mice injected with LLC/OVA tumor (1 x 10⁶) s. c. 2 days previously. Tumors were measured every 2-4 days, and mice were sacrificed when tumor diameter reached 15mm. Data is representative of three separate experiments.

Figures 6A-6C show CD44, Ly-6C, and CD62L expression of OT-l/GFP in splenocytes and tumor. Splenocytes from mice were collected on day 6 (Figure 6A) or day 17 (Figure 6C) following OT-l/GFP adoptive transfer. Tumor was also analyzed on day 6 (Figure 6B). Cells were stained with anti-CD8-Cy-Chrome and biotinylated-anti-Ly-6C followed by streptavidin-PE (Figures 6A, 6B, 6C), anti-CD44-PE (Figure 6C), or anti- CD62L-PE (Figure 6C) and analyzed by flow cytometry. Numbers indicate percentages in the CD8⁺ gate. Data represent at least three separate experiments.

Figures 7A-7B are graphs showing the proliferation of tumor-specific OT-I cells is not enhanced in the presence of anti-CD28 or anti-CTLA-4 antibodies. OT-I cells (10⁵) were stimulated with either HSV.4-lBBL-transduced (Figure 7A) or HSV.B7.1 -transduced (Figure 7B) LLC/OVA (6.6 x 10⁴) in the presence of insoluble (i) or soluble (s) anti-CD28 or anti- CTLA-4 antibodies. Numbers in parentheses indicate concentrations of antibodies in μg/ml. Hamster IgG2 and hamster IgGl antibodies were added as isotype controls for anti-CD28 and anti-CTLA-4 antibodies, respectively. Thymidine was added 25 h before harvest on Day 3. SD of triplicates is shown. Data represents two separate experiments.

DETAILED DESCRIPTION

The present invention is described with reference to the attached figures, wherein like reference numerals are used throughout the figures to designate similar or equivalent elements. The figures are not drawn to scale and they are provided merely to illustrate the instant invention. Several aspects of the invention are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One having ordinary skill in the relevant art, however, will readily recognize that the invention can be practiced without one or more of the specific details or with other methods. The present invention is not limited by the illustrated ordering of acts or events, as some acts may occur in different orders and/or concurrently with other acts or events. Furthermore, not all illustrated acts or events are required to implement a methodology in accordance with the present invention.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

Definitions

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including", "includes", "having", "has", "with", or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

The term "tumor" or "cancer" is defined as one or more tumor cells capable of forming an invasive mass whose normal growth control mechanisms are disrupted (typically by accumulated genetic mutations), thereby providing the potential for uncontrolled proliferation generally results in progressive displacement or destruction of normal tissues. As used herein, the terms "cancer," "neoplasm," and "tumor," are used interchangeably and in either the singular or plural form, refer to cells that have undergone a malignant transformation that makes them pathological to the host organism. Primary cancer cells (that is, cells obtained from near the site of malignant transformation) can be readily distinguished from non-cancerous cells by well-established techniques, particularly histological examination. The definition of a cancer cell, includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, in vitro cultures and cell lines derived from transformed or cancer cells. When referring to a type of cancer that normally manifests as a solid tumor, a "clinically detectable" tumor is one that is detectable on the basis of tumor mass; e.g., by procedures such as CAT scan, MR imaging, X-ray, ultrasound or palpation, and/or which is detectable because of the expression of one or more cancer-specific antigens in a sample obtainable from a patient. Included within this definition are malignant cells of the hematopoietic system which do not form solid tumors such as leukemias, lymphomas and myelomas.

The term "malignant tumor" is defined as those tumors formed by tumor cells that can develop the property of dissemination beyond their original site of occurrence. "Activation", as used herein, refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions. The term "activated T cells" refers to, among other things, T cells that are undergoing cell division. As used herein, the term "antigen presentation" is meant the biological mechanism by which macrophages, dendritic cells, B cells and other types of antigen presenting cells process internal or external antigens into sub-fragments of those molecules and present them complexed with class I or class II major histocompatibility complex or CDl molecules on the surface of the cell. This process leads to growth stimulation of other types of cells of the immune system (such as CD4⁺, CD8⁺, B and NK cells), which are able to specifically recognize those complexes and mediate an immune response against those antigens or cells displaying those antigens.

The term "antigen" or "Ag" as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

"An antigen presenting cell" (APC) is a cell that is capable of activating T cells, and includes, but is not limited to, monocytes/macrophages, B cells and dendritic cells (DCs). The term "dendritic cell" or "DC" refers to any member of a diverse population of morphologically similar cell types found in lymphoid or non-lymphoid tissues. These cells are characterized by their distinctive morphology, high levels of surface MHC-class II expression. DCs can be isolated from a number of tissue sources. DCs have a high capacity for sensitizing MHC-restricted T cells and are very effective at presenting antigens to T cells in situ. The antigens may be self-antigens that are expressed during T cell development and tolerance, and foreign antigens that are present during normal immune processes.

The term "expression vector" as used herein refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules, siRNA, ribozymes, and the like. Expression vectors can contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operatively linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well.

The term "non-replicating" or "replication-impaired" or "replication deficient" as used herein means not capable of replication in the majority of normal mammalian cells or normal human cells. Viruses which are non-replicating or replication-impaired may have become so naturally (i.e. they may be isolated as such from nature) or artificially e.g. by breeding in vitro or by genetic manipulation, for example deletion of a gene which is critical for replication. Replication of a virus is generally measured in two ways: 1) DNA synthesis and 2) viral titer.

By "encoding" or "encoded", "encodes", with respect to a specified nucleic acid, is meant comprising the information for translation into the specified protein. A nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid, or may lack such intervening non-translated sequences (e.g., as in cDNA). The information by which a protein is encoded is specified by the use of codons. Typically, the amino acid sequence is encoded by the nucleic acid using the "universal" genetic code. As used herein, "heterologous" in reference to a nucleic acid is a nucleic acid that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous structural gene is from a species different from that from which the structural gene was derived, or, if from the same species, one or both are substantially modified from their original form. A heterologous protein may originate from a foreign species or, if from the same species, is substantially modified from its original form by deliberate human intervention.

By "host cell" is meant a cell which contains a vector and supports the replication and/or expression of a vector. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells.

The term "introduced" or "administered" in the context of inserting a nucleic acid into a cell, means "transfection" or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

A cell has been "transformed" or "transfected" by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA.

As used herein, "nucleic acid" includes reference to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids). As used herein, "polynucleotide" includes reference to a deoxyribopolynucleotide, ribopolynucleotide, or analogs thereof that have the essential nature of a natural ribonucleotide in that they hybridize, under stringent hybridization conditions, to substantially the same nucleotide sequence as naturally occurring nucleotides and/or allow translation into the same amino acid(s) as the naturally occurring nucleotide(s). A polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof. Thus, DNAs or RNAs with backbones modified for stability or for other reasons as "polynucleotides" as that term is intended herein.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids. The terms "polypeptide", "peptide" and "protein" are also inclusive of modifications including, but not limited to, phosphorylation, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.

As used herein, the term "autologous" is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual. The term "xenogeneic cell" refers to a cell that derives from a different animal species than the animal species that becomes the recipient animal host in a transplantation or vaccination procedure.

The term "allogeneic cell" refers to a cell that is of the same animal species but genetically different in one or more genetic loci as the animal that becomes the "recipient host". This usually applies to cells transplanted from one animal to another non-identical animal of the same species.

The term "syngeneic cell" refers to a cell which is of the same animal species and has the same genetic composition for most genotypic and phenotypic markers as the animal who becomes the recipient host of that cell line in a transplantation or vaccination procedure. This usually applies to cells transplanted from identical twins or may be applied to cells transplanted between highly inbred animals.

"Sample" is used herein in its broadest sense. A sample comprising polynucleotides, polypeptides, peptides, antibodies and the like may comprise a bodily fluid; a soluble fraction of a cell preparation, or media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA, polypeptides, or peptides in solution or bound to a substrate; a cell; a tissue; a tissue print; a fingerprint, skin or hair; and the like.

The terms "patient" or "individual" are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred. In some cases, the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; and primates.

"Diagnostic" or "diagnosed" means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives"). Diseased individuals not detected by the assay are "false negatives." Subjects who are not diseased and who test negative in the assay, are termed "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.

"Treatment" is an intervention performed with the intention of preventing the development or altering the pathology or symptoms of a disorder. Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. In tumor (e.g., cancer) treatment, a therapeutic agent may directly decrease the pathology of tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents, e.g., radiation and/or chemotherapy. As used herein, "ameliorated" or "treatment" refers to a symptom which is approaches a normalized value (for example a value obtained in a healthy patient or individual), e.g., is less than 50% different from a normalized value, preferably is less than about 25% different from a normalized value, more preferably, is less than 10% different from a normalized value, and still more preferably, is not significantly different from a normalized value as determined using routine statistical tests. For example the term "treat" or "treating" with respect to tumor cells refers to stopping the progression of said cells, slowing down growth, inducing regression, or amelioration of symptoms associated with the presence of said cells.

The "treatment of cancer", refers to one or more of the following effects: (1) inhibition, to some extent, of tumor growth, including, (i) slowing down and (ii) complete growth arrest; (2) reduction in the number of tumor cells; (3) maintaining tumor size; (4) reduction in tumor size; (5) inhibition, including (i) reduction, (ii) slowing down or (iii) complete prevention, of tumor cell infiltration into peripheral organs; (6) inhibition, including (i) reduction, (ii) slowing down or (iii) complete prevention, of metastasis; (7) enhancement of anti-tumor immune response, which may result in (i) maintaining tumor size, (ii) reducing tumor size, (iii) slowing the growth of a tumor, (iv) reducing, slowing or preventing invasion and/or (8) relief, to some extent, of the severity or number of one or more symptoms associated with the disorder.

By the term "effective amount", as used herein, is meant an amount that when administered to a mammal, causes a detectable level of T cell response compared to the T cell response detected in the absence of the compound. According to embodiments herein, an effective amount of administration of T cells would result in the "treatment of cancer" as defined above. T cell response can be readily assessed by a plethora of art-recognized methods. The skilled artisan would understand that the amount of the compound or composition administered herein varies and can be readily determined based on a number of factors such as the disease or condition being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, the particular compound being administered, and the like.

As used herein, the term "safe and effective amount" refers to the quantity of a component which is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention. By "therapeutically effective amount" is meant an amount of a compound of the present invention effective to yield the desired therapeutic response. For example, an amount effective to delay the growth of or to cause a cancer, either a sarcoma or lymphoma, or to shrink the cancer or prevent metastasis. The specific safe and effective amount or therapeutically effective amount will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of mammal or animal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives.

"Cells of the immune system" or "immune cells", is meant to include any cells of the immune system that may be assayed, including, but not limited to, B lymphocytes, also called B cells, T lymphocytes, also called T cells, natural killer (NK) cells, natural killer T (NK) cells, lymphokine-activated killer (LAK) cells, monocytes, macrophages, neutrophils, granulocytes, mast cells, platelets, Langerhans cells, stem cells, dendritic cells, peripheral blood mononuclear cells, tumor-infiltrating (TIL) cells, gene modified immune cells including hybridomas, drug modified immune cells, and derivatives, precursors or progenitors of the above cell types. "Immune effector cells" refers to cells capable of binding an antigen and which mediate an immune response selective for the antigen. These cells include, but are not limited to, T cells (T lymphocytes), B cells (B lymphocytes), monocytes, macrophages, natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), for example CTL lines, CTL clones, and CTLs from tumor, inflammatory, or other infiltrates.

"Immune related molecules" refers to any molecule identified in any immune cell, whether in a resting ("non-stimulated") or activated state, and includes any receptor, ligand, cell surface molecules, nucleic acid molecules, polypeptides, variants and fragments thereof. "T cells" or "T lymphocytes" are a subset of lymphocytes originating in the thymus and having heterodimeric receptors associated with proteins of the CD3 complex (e.g., a rearranged T cell receptor, the heterodimeric protein on the T cell surfaces responsible for antigen/MHC specificity of the cells). T cell responses may be detected by assays for their effects on other cells (e.g., target cell killing, activation of other immune cells, such as B-cells) or for the cytokines they produce. "CD4" is a cell surface protein important for recognition by the T cell receptor of antigenic peptides bound to MHC class II molecules on the surface of an APC. Upon activation, naϊve CD4 T cells differentiate into one of at least two cell types, ThI cells and Th2 cells, each type being characterized by the cytokines it produces. "ThI cells" are primarily involved in activating macrophages with respect to cellular immunity and the inflammatory response, whereas "Th2 cells" or "helper T cells" are primarily involved in stimulating B cells to produce antibodies (humoral immunity). CD4 is the receptor for the human immunodeficiency virus (HIV). Effector molecules for ThI cells include, but are not limited to, IFN-γ, GM-CSF, TNF-α, CD40 ligand, Fas ligand, IL-3, TNF-β, and IL-2. Effector molecules for Th2 cells include, but are not limited to, IL-4, IL-5, CD40 ligand, IL-3, GS-CSF, IL-IO, TGF-β, and eotaxin. Activation of the ThI type cytokine response can suppress the Th2 type cytokine response, and reciprocally, activation of the Th2 type cytokine response can suppress the ThI type response.

As used herein, a "pharmaceutically acceptable" component is one that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.

Lymphocyte compositions Exemplary candidates for treatment according to embodiments of the invention include, but are not limited to: (i) non-human animals suffering from neoplasms characterized by a tumor-specific or cell-type specific surface protein; (ii) humans suffering from neoplasms characterized by a tumor-specific or cell-type specific surface protein; or (iii) humans or non-human animals in need of eradication of a particular cell population.

The basic rationale for immune therapy against tumors is the induction of an effective immune response against tumor-associated antigens (TAA), which in turn results in immune- mediated destruction of proliferating tumor cells expressing these antigens. For an immune response to be effective against TAAs comprising protein, these antigens must first be endocytosed by antigen presenting cells (APC) such as macrophages, dendritic cells and B cells. Within APCs, TAAs are degraded in the lysosomal compartment and the resulting peptides are expressed on the surface of the macrophage cell membrane mostly in association with MHC (Major Histocompatibility Complex) Class II molecules but also in association with MHC class I molecules. This expression mediates recognition by specific CD4⁺ helper T cells and subsequent activation of these cells to effect the immune response (Stevenson, 1991, FASEB J. 5:2250; Lanzavecchia, 1993, Science 260:937; Pardoll, 1993, Immunol. Today 14:310).

Instead of a general prophylactic approach, immune treatment of an individual with recurring, or a diagnosed tumor would provide a more directly targeted method. Most tumor cells have unique expression profiles of TAA, but in many cases these TAA are unknown or very difficult to determine or isolate for individual tumors. The use of whole cells alleviates this difficulty, as it provides a whole repertoire of TAA without the need to isolate or characterize those antigens.

T cells isolated from tumor-infiltrating lymphocytes, tumor-draining lymph nodes (TDLNs), or peripheral blood which contain tumor-specific cells, can be expanded ex vivo and transferred into the host. In a preferred embodiment, efficacy of T cell adoptive transfer, is improved through optimization of in vitro expansion, improved characterization of effector populations, and/or enhancing the function and survival of transferred CTLs in order to facilitate establishment of immunologic memory. Adequate activation resulting in priming of naive T-cells depends on two signals derived from professional APCs (antigen-presenting cells) like dendritic cells. The first signal is antigen-specific and normally mediated by stimulation of the clonotypic T-cell antigen receptor (TCR) that is induced by processed antigen presented in the context of MHC class-I or MHC class-II molecules. However, this primary stimulus is insufficient to induce priming responses of naive T-cells, and the second signal is required which is provided by an interaction of specific T-cell surface molecules binding to co-stimulatory ligand molecules on antigen presenting cells (APCs), further supporting the proliferation of primed T-cells. The term "T-cell co-stimulatory ligand" therefore denotes in the light of the present invention molecules, which are able to support priming of naive T-cells in combination with the primary stimulus and include, but are not limited to, members of the B7 family of proteins, including B7-1 (CD80) and B7-2 (CD86), 4- IBB ligand (CD137L), CD40 ligand, OX40 ligand.

The term "activated T cell," as used herein, refers to a T cell that expresses antigens indicative of T-cell activation (that is, T cell activation markers). Examples of T cell activation markers include, but are not limited to, CD25, CD26, CD30, CD38, CD69, CD70, CD71, ICOS, OX-40 and 4-1BB. The expression of activation markers can be measured by techniques known to those of skill in the art, including, for example, western blot analysis, northern blot analysis, RT-PCR, immunofluorescence assays, and fluorescence activated cell sorter (FACS) analysis.

Immune systems are classified into two general systems, the "innate" or "primary" immune system and the "acquired/adaptive" or "secondary" immune system. It is thought that the innate immune system initially keeps the infection under control, allowing time for the adaptive immune system to develop an appropriate response. The various components of the innate immune system trigger and augment the components of the adaptive immune system, including antigen-specific B and T lymphocytes (Kos, Immunol Res. 1998, 17:303; Romagnani, Immunol. Today. 1992, 13: 379; Banchereau and Steinman, Nature. 1988, 392:245). A "primary immune response" refers to an innate immune response that is not affected by prior contact with the antigen. The main protective mechanisms of primary immunity are the skin (protects against attachment of potential environmental invaders), mucous (traps bacteria and other foreign material), gastric acid (destroys swallowed invaders), antimicrobial substances such as interferon (IFN) (inhibits viral replication) and complement proteins (promotes bacterial destruction), fever (intensifies action of interferons, inhibits microbial growth, and enhances tissue repair), natural killer (NK) cells (destroy microbes and certain tumor cells, and attack certain virus infected cells), and the inflammatory response (mobilizes leukocytes such as macrophages and dendritic cells to phagocytose invaders).

Some cells of the innate immune system, including macrophages and dendritic cells (DC), function as part of the adaptive immune system as well by taking up foreign antigens through pattern recognition receptors, combining peptide fragments of these antigens with major histocompatibility complex (MHC) class I and class II molecules, and stimulating naive CD8⁺ and CD4⁺ T cells respectively (Banchereau and Steinman, supra; Holmskov et al, Immunol. Today. 1994, 15:67; Ulevitch and Tobias Annu. Rev. Immunol. 1995, 13:437). Professional antigen-presenting cells (APCs) communicate with these T cells, leading to the differentiation of naive CD4+ T cells into T-helper 1 (ThI) or T-helper 2 (Th2) lymphocytes that mediate cellular and humoral immunity, respectively (Trinchieri Annu. Rev. Immunol.

1995, 13:251; Howard and O'Garra, Immunol. Today. 1992, 13:198; Abbas et al, Nature.

1996, 383:787; Okamura et al, Adv. Immunol. 1998, 70:281; Mosmann and Sad, Immunol. Today. 1996, 17:138; O'Garra Immunity. 1998, 8:275).

A "secondary immune response" or "adaptive immune response" may be active or passive, and may be humoral (antibody based) or cellular that is established during the life of an animal, is specific for an inducing antigen, and is marked by an enhanced immune response on repeated encounters with said antigen. A key feature of the T lymphocytes of the adaptive immune system is their ability to detect minute concentrations of pathogen-derived peptides presented by MHC molecules on the cell surface.

In adaptive immunity, adaptive T and B cell immune responses work together with innate immune responses. The basis of the adaptive immune response is that of clonal recognition and response. An antigen selects the clones of cell which recognize it, and the first element of a specific immune response must be rapid proliferation of the specific lymphocytes. This is followed by further differentiation of the responding cells as the effector phase of the immune response develops. These effector T lymphocytes, in the context of the embodiments, are the tumor specific T lymphocytes.

The phrase "T cell response" means an immunological response involving T cells. The T cells that are "activated" divide to produce antigen specific memory T cells or antigen specific cytotoxic T cells. The cytotoxic T cells bind to and destroy cells recognized as containing the antigen. The memory T cells are activated by the antigen and thus provide a response to an antigen already encountered. This overall response to the antigen is the antigen specific T cell response, e.g. tumor specific. In a preferred embodiment, a method of treating a patient suffering from cancer comprises obtaining a patient sample comprising tumor tissue or tumor cells; administering or introducing a composition comprising a lymphocyte co-stimulatory antigen to the tumor cells; such that the co-stimulatory antigen is expressed; obtaining lymphocytes and culturing the lymphocytes with the tumor cells expressing the co-stimulatory antigen and expanding tumor specific lymphocytes ex vivo prior to administering the lymphocytes to the patient. Preferably, the co-stimulatory antigen is CD137 ligand or 4-1BBL, analogs, mutants, alleles, fragments and chimeric fusion peptides thereof.

In a preferred embodiment, tumor specific T lymphocytes are autologous T lymphocytes. In other embodiments, the T lymphocytes are obtained form other sources, e.g. donor derived.

In another preferred embodiment, the tumor cells are autologous tumor cells. In other embodiments, the tumor cells are donor derived or cell lines.

Embodiments of the invention comprise the use of gene transfer technique to engineer tumor cells to express co-stimulatory antigens, such as for example, CD 137 ligand (human), 4- IBB ligand (mouse) in vitro. The tumor cells used for the pharmaceutical composition of the invention may be autologous, or in another embodiment may be allogeneic or syngeneic. To the extent that universal, or overlapping epitopes or TAA exist between different cancers, the pharmaceutical compositions may be quite widely applicable. The invention also need not be limited to a cancer cell and can include any type of cell. For example, generation of tumor specific T lymphocytes could be achieved by introducing both a co-stimulatory antigen and a tumor antigen to the cells. Immune cell activity that may be measured include, but is not limited to, (1) cell proliferation by measuring the DNA replication; (2) enhanced cytokine production, including specific measurements for cytokines, such as IFN-γ, GM-CSF, or TNF- α; (3) cell mediated target killing or lysis; (4) cell differentiation; (5) immunoglobulin production; (6) phenotypic changes; (7) production of chemotactic factors or chemotaxis, meaning the ability to respond to a chemotactin with chemotaxis; (8) immunosuppression, by inhibition of the activity of some other immune cell type; and, (9) apoptosis, which refers to fragmentation of activated immune cells under certain circumstances, as an indication of abnormal activation.

The nucleic acid sequence that encodes the co-stimulatory antigen is contained in an appropriate expression vehicle, which transduces the tumor cells. Such expression vehicles include, but are not limited to, eukaryotic vectors, prokaryotic vectors (such as, for example, bacterial vectors), and viral vectors. In a preferred embodiment, the vector encoding the one or more co-stimulatory antigens is a herpes simplex virus (HSV) amplicon. Preferably, the co-stimulation antigen is 4-1BB, 4-1BBL, CD137, CD137L. In a preferred embodiment the co-stimulatory antigen is 4- IBBL or CD137L depending on the species to be treated. Encompassed within the term co-stimulatory antigen is the species homologue, e.g. 4- IBBL for mouse, CD137L for human etc. In other preferred embodiments, the vector encodes for at least one co-stimulatory antigen, however, the vector can encode for more than one co-stimulatory antigen molecule, e.g. increase surface expression. In some embodiments, the vector encodes for different types of co-stimulatory antigens in addition to the CD137L, e.g. CD28. Tumors which may be treated in accordance with the present invention include malignant and non-malignant tumors. Malignant (including primary and metastatic) tumors which may be treated include, but are not limited to, those occurring in the adrenal glands; bladder; bone; breast; cervix; endocrine glands (including thyroid glands, the pituitary gland, and the pancreas); colon; rectum; heart; hematopoietic tissue; kidney; liver; lung; muscle; nervous system; brain; eye; oral cavity; pharynx; larynx; ovaries; penis; prostate; skin (including melanoma); testicles; thymus; and uterus. Examples of such tumors include apudoma, choristoma, branchioma, malignant carcinoid syndrome, carcinoid heart disease, carcinoma (e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, Krebs 2, Merkel cell, mucinous, non-small cell lung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, and transitional cell), plasmacytoma, melanoma, chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma, giant cell tumors, histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcoma, Ewing's sarcoma, synovioma, adenofibroma, adenolymphoma, carcinosarcoma, chordoma, mesenchymoma, mesonephroma, myosarcoma, ameloblastoma, cementoma, odontoma, teratoma, thymoma, trophoblastic tumor, adenocarcinoma, adenoma, cholangioma, cholesteatoma, cylindroma, cystadenocarcinoma, cystadenoma, granulosa cell tumor, gynandroblastoma, hepatoma, hidradenoma, islet cell tumor, Leydig cell tumor, papilloma, Sertoli cell tumor, theca cell tumor, leiomyoma, leiomyosarcoma, myoblastoma, myoma, myosarcoma, rhabdomyoma, rhabdomyosarcoma, ependymoma, ganglioneuroma, glioma, medulloblastoma, meningioma, neurilemmoma, neuroblastoma, neuroepithelioma, neurofibroma, neuroma, paraganglioma, paraganglioma nonchromaffin, angiokeratoma, angiolymphoid hyperplasia with eosinophilia, angioma sclerosing, angiomatosis, glomangioma, hemangioendothelioma, hemangioma, hemangiopericytoma, hemangiosarcoma, lymphangioma, lymphangiomyoma, lymphangiosarcoma, pinealoma, carcinosarcoma, chondrosarcoma, cystosarcoma phyllodes, fibrosarcoma, hemangiosarcoma, leiomyosarcoma, leukosarcoma, liposarcoma, lymphangiosarcoma, myosarcoma, myxosarcoma, ovarian carcinoma, rhabdomyosarcoma, sarcoma (e.g., Ewing's experimental, Kaposi's, and mast-cell), neoplasms and for other such cells. Vectors Expressing Co-Stimulatory Antigens

Specific examples of viral vector systems that have been utilized include: retroviruses; adenoviruses ; adenoviral/retroviral chimeras; adeno-associated viruses; herpes simplex virus 1 or 2; parvovirus; reticuloendotheliosis virus. Other viruses that can be used as vectors for gene delivery include poliovirus, papillomavirus, vaccinia virus, lentivirus, as well as hybrid or chimeric vectors incorporating favorable aspects of two or more viruses.

Viral vectors based on herpes simplex virus (HSV), and especially HSV-I, have shown promise as potent gene delivery vehicles for several reasons: the virus has a very large genome and thus can accommodate large amounts of foreign DNA (greater than 50 kb), the virus can persist long-term in cells, and can efficiently infect many different cell types, including post-mitotic neural cells (Breakefield, X.O., et al., "Herpes Simplex Virus Vectors for Tumor Therapy," in The Internet Book of Gene Therapy: Cancer Gene Therapeutics, R. E. Sobol and K. J. Scanlon, eds., Appleton and Lange, Stamford, Conn., pp. 41-56 (1995); Glorioso, J. C, et al., "Herpes Simplex Virus as a Gene-Delivery Vector for the Central Nervous System," in Viral Vectors: Gene Therapy and Neuroscience Applications, M. G. Kaplitt and A. D. Loewy, eds., Academic Press, New York, pp. 1-23 (1995)).

Two types of HSV vector systems comprise: recombinant and amplicon. Recombinant HSV-I vectors (Wolfe, J. H. et al, Nat. Genet. 1:379-384 (1992)) are created by inserting genes of interest directly into the 152 kb viral genome, thereby mutating one or more of the approximately 80 viral genes, and usually concomitantly reducing cytotoxicity. HSV-I amplicons are bacterial plasmids containing only about 1% of the 152 kb HSV-I genome. Typically, they are packaged into infectious HSV-I particles ("virions") using HSV-I helper virus functions. HSV-I amplicons contain: (i) a transgene cassette with a gene(s) of interest; (ii) sequences that allow plasmid propagation in E. coli, such as the origin of DNA replication co /El and the ampicillin resistance gene; and (iii) non-coding elements of the HSV-I genome, in particular an origin of DNA replication (ori) and a DNA cleavage/packaging signal (pac), to support replication and subsequent packaging of the amplicon DNA into virions in the presence of helper functions (Spaete, R. R. and Frenkel, N., Cell 30:295-304 (1982)). HSV amplicon vectors are one of the most versatile, most efficient, and least toxic, and have the largest transgene capacity of the currently available virus vectors. HSV-I amplicon vectors can support some gene expression for up to one year in non- dividing cells (During, M. J., et al., Science 266:1399-1403 (1994)).

In a preferred embodiment, the vector encoding the co-stimulatory antigen(s) is a herpes simplex virus amplicon. Herpes simplex virus I (HSV) amplicons, replication-defective viral particles, were used for gene transfer of 4- IBBL because of their broad cellular tropism, large transgene capacity, and ability to induce high levels of gene expression. By triggering an innate response, HSV amplicons facilitate a more vigorous adaptive response. Strong activation of several toll-like receptors (TLRs), induction of cytokines, and NKG2D-ligand expression were noted following transduction with HSV amplicons in macrophage cell lines and human chronic lymphocytic leukemia (CLL). Since HSV amplicons can readily transduce primary tumor cells, it was reasoned that HSV amplicons encoding 4- IBBL could facilitate direct antigen presentation by tumor cells in order to expand tumor-specific effectors for adoptive transfer. It was hypothesized that HSV.4-lBBL-expanded CD8+ T cells would show desirable effector properties, including in vivo expansion and therapeutic efficacy, as well as confer a memory response.

HSV.4-1BBL amplicons were used to transduce tumor for purposes of activating and expanding tumor-specific CD8+ OT-I cells in vitro, and studied the behavior of adoptively transferred ex-vivo expanded cells in LLC/OVA tumor-bearing mice. The results demonstrated that HSV.4-1BBL can induce significant expansion of CTLs in vitro and in vivo, and that the adoptive transfer of expanded T cells results in reduction of tumor growth in vivo as well as the persistence of CD44^hlLy-6C^hlCD62L^neg tumor-specific T cells with memory characteristics. In another embodiment, the expression vector encoding the co-stimulatory antigen is a viral vector. Viral vectors which may be employed include, but are not limited to, retroviral vectors, adenovirus vectors, Herpes virus vectors, and adeno-associated virus vectors, or DNA conjugates. Traditionally, the viral vector is a retroviral or adenoviral vector. Examples of retroviral vectors which may be employed include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus.

Retroviral vectors are useful as agents to mediate retroviral-mediated gene transfer into eukaryotic cells. Retroviral vectors are generally constructed such that the majority of sequences coding for the structural genes of the virus are deleted and replaced by the gene(s) of interest. Most often, the structural genes (i.e., gag,pol, and env), are removed from the retroviral backbone using genetic engineering techniques known in the art. These new genes have been incorporated into the proviral backbone in several general ways. The most straightforward constructions are ones in which the structural genes of the retrovirus are replaced by a single gene which then is transcribed under the control of the viral regulatory sequences within the long terminal repeat (LTR). Retroviral vectors have also been constructed which can introduce more than one gene into target cells. Usually, in such vectors one gene is under the regulatory control of the viral LTR, while the second gene is expressed either off a spliced message or is under the regulation of its own, internal promoter.

In Moloney murine leukemia virus (MoMuLV), 5' to the authentic gag start, an open reading frame exists which permits expression of another glycosylated protein (pPrδOgag). Moloney murine sarcoma virus (MoMuSV) has alterations in this 5' region, including a frameshift and loss of glycosylation sites, which obviate potential expression of the amino terminus of pPrδOgag. Therefore, the vector LNL6 was made, which incorporated both the altered ATG of LNL-XHC and the 5' portion of MoMuSV. The 5' structure of the LN vector series thus eliminates the possibility of expression of retroviral reading frames, with the subsequent production of viral antigens in genetically transduced target cells. In a final alteration to reduce overlap with packaging-defective helper virus, extra env sequences immediately preceding the 3' LTR in the LN vector were eliminated (Miller, et al., Biotechniques, 7:980-990, 1989). The vector includes one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and β-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, TK promoters, and B 19 parvovirus promoters.

In another embodiment the invention comprises an inducible promoter. One such promoter is the tetracycline-controlled transactivator (tTA)-responsive promoter {tet system), a prokaryotic inducible promoter system which has been adapted for use in mammalian cells. The tet system was organized within a retroviral vector so that high levels of constitutively- produced tTA mRNA function not only for production of tTA protein but also the decreased basal expression of the response unit by antisense inhibition. See, Paulus, W. et al., "Self- Contained, Tetracycline-Regulated Retroviral Vector System for Gene Delivery to Mammalian Cells", J of Virology, January. 1996, Vol. 70, No. 1, pp. 62-67 ^'. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.

Introduction of the vector to the tumor cells include any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO₄ precipitation.

Tumor Antigens

In another preferred embodiment, the T lymphocytes are cultured with one or more tumor cells expressing different tumor antigens. These cells are transduced with a composition, e.g. HSV amplicon encoding the co-stimulatory antigen, e.g. CD137L. Each of these cells are patient or donor derived tumor cells, cells transduced with a vector expressing a tumor antigen, cell lines and the like. Many tumor antigens are well known in the art. See for example, Van den Eynde BJ, van der Bruggen P. Curr Opin Immunol 1997; 9: 684-93; Houghton AN, Gold JS, Blachere NE. Curr Opin Immunol 2001; 13: 134-140; van der Bruggen P, Zhang Y, Chaux P, Stroobant V, Panichelli C, Schultz ES, Chapiro J, Van den

Eynde BJ, Brasseur F, Boon T. Immunol Rev 2002; 188: 51-64, which are herein incorporated by reference in their entirety.

Non-limiting examples of tumor antigens, include, tumor antigens resulting from mutations, such as: alpha-actinin-4 (lung carcinoma); BCR-ABL fusion protein (b3a2) (chronic myeloid leukemia); CASP-8 (head and neck squamous cell carcinoma); beta-catenin (melanoma); Cdc27 (melanoma); CDK4 (melanoma); dek-can fusion protein (myeloid leukemia); Elongation factor 2 (lung squamous carcinoa); ETV6-AML1 fusion protein (acute lymphoblastic leukemia); LDLR-fucosyltransferaseAS fusion protein (melanoma); overexpression of HLA- A2^d (renal cell carcinoma); hsp70-2 (renal cell carcinoma); KIAAO205 (bladder tumor); MART2 (melanoma); MUM- 1 f (melanoma); MUM-2

(melanoma); MUM-3 (melanoma); neo-PAP (melanoma); Myosin class I (melanoma); 0S-9g (melanoma); pml-RARalpha fusion protein (promyelocytic leukemia); PTPRK (melanoma); K-ras (pancreatic adenocarcinoma); N-ras (melanoma). Examples of differentiation tumor antigens include, but not limited to: CEA (gut carcinoma); gplOO / Pmell7 (melanoma); Kallikrein 4 (prostate); mammaglobin-A (breast cancer); Melan-A / MART-I (melanoma); PSA (prostate carcinoma); TRP-I / gp75 (melanoma); TRP-2 (melanoma); tyrosinase (melanoma). Over or under-expressed tumor antigens include but are not limited to: CPSF (ubiquitous); EphA3 ; G250 / MN / CAIX (stomach, liver, pancreas); HER-2/neu; Intestinal carboxyl esterase (liver, intestine, kidney); alpha-foetoprotein (liver ); M-CSF (liver, kidney); MUCl (glandular epithelia); p53 (ubiquitous); PRAME (testis, ovary, endometrium, adrenals); PSMA (prostate, CNS, liver); RAGE-I (retina); RU2AS (testis, kidney, bladder); survivin (ubiquitous); Telomerase (testis, thymus, bone marrow, lymph nodes); WTl (testis, ovary, bone marrow, spleen); CAl 25 (ovarian). The compositions provided herein may be used alone or in combination with conventional therapeutic regimens such as surgery, irradiation, chemotherapy and/or bone marrow transplantation (autologous, syngeneic, allogeneic or unrelated). The chemotherapeutic agents may be administered under a metronomic regimen. As used herein, "metronomic" therapy refers to the administration of continuous low-doses of a therapeutic agent. Therapeutic agents can include, for example, chemotherapeutic agents such as, cyclophosphamide (CTX, 25 mg/kg/day,/?.o.), taxanes (paclitaxel or docetaxel), busulfan, cisplatin, cyclophosphamide, methotrexate, daunorubicin, doxorubicin, melphalan, cladribine, vincristine, vinblastine, and chlorambucil.

As discussed in greater detail below, binding agents and T cells as provided herein may be used for purging of autologous stem cells. Such purging may be beneficial prior to, for example, bone marrow transplantation or transfusion of blood or components thereof. Binding agents, T cells, antigen presenting cells (APC) and compositions provided herein may further be used for expanding and stimulating (or priming) autologous, allogeneic, syngeneic or unrelated tumor-specific T-cells in vitro and/or in vivo. Such tumor-specific T cells may be used, for example, within donor lymphocyte infusions.

Routes and frequency of administration, as well as dosage, will vary from individual to individual, and may be readily established using standard techniques. In general, the pharmaceutical compositions and vaccines may be administered by injection (e.g., intracutaneous, intramuscular, intravenous or subcutaneous. In some tumors, pharmaceutical compositions or vaccines may be administered locally (by, for example, rectocoloscopy, gastroscopy, videoendoscopy, angiography or other methods known in the art). Preferably, between 1 and 10 doses may be administered over a 52 week period. Preferably, 6 doses are administered, at intervals of 1 month, and booster vaccinations may be given periodically thereafter. Alternate protocols may be appropriate for individual patients. A suitable dose is an amount of a compound that, when administered as described above, is capable of promoting an anti-tumor immune response that is at least 10-50% above the basal (i.e., untreated) level. Such response can be monitored by measuring the anti-tumor antibodies in a patient or by vaccine-dependent generation of cytolytic effector cells capable of killing the patient's tumor cells in vitro. Such vaccines should also be capable of causing an immune response that leads to an improved clinical outcome (e.g., more frequent complete or partial remissions, or longer disease-free and/or overall survival) in vaccinated patients as compared to non-vaccinated patients.

In general, an appropriate dosage and treatment regimen provides the active compound(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit. Such a response can be monitored by establishing an improved clinical outcome (e.g., more frequent complete or partial remissions, or longer disease-free and/or overall survival) in treated patients as compared to non-treated patients. Increases in preexisting immune responses to the tumor antigen generally correlate with an improved clinical outcome. Such immune responses may generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which may be performed using samples obtained from a patient before and after treatment.

Within further aspects, methods for inhibiting the development of cancer associated with tumor antigen expression involve the administration of autologous T cells that have been activated in response to a tumor antigen, as described above. Such T cells may be CD4⁺ and/or CD8⁺ lymphocytes, and may be proliferated as described above.

In preferred embodiments, the tumor specific t lymphocytes are cytotoxic (CD8⁺) T lymphocytes. The CD8⁺ T lymphocytes can be separated from other cells by any known means, e.g. FACS scan, magnetic separation and the like. The T cells may be administered to the individual in an amount effective to inhibit the development of the disease and any metastasized cells or growths. Typically, about 1 x l O⁹ to 1 x 10¹¹ T cells/M² are administered intravenously, intracavitary or in the bed of a resected tumor. It will be evident to those skilled in the art that the number of cells and the frequency of administration will be dependent upon the response of the patient. Within certain preferred embodiments, T cells are stimulated prior to an autologous bone marrow transplantation. Such stimulation may take place in vivo or in vitro. For in vitro stimulation, bone marrow and/or peripheral blood (or a fraction of bone marrow or peripheral blood) obtained from a patient, stimulation of lymphocytes is carried preferably as described, and are contacted with the tumor cells expressing the co-stimulatory antigen and/or an APC that expresses a tumor antigen under conditions and for a time sufficient to permit the stimulation of T cells as described above. Bone marrow, peripheral blood stem cells and/or tumor-specific T cells may then be administered to a patient using standard techniques. Within related embodiments, T cells of a related or unrelated donor may be stimulated prior to a syngeneic or allogeneic (related or unrelated) bone marrow transplantation. Such stimulation may take place in vivo or in vitro. Preferably, the lymphocytes are activated in vitro. For in vitro stimulation, bone marrow and/or peripheral blood (or a fraction of bone marrow or peripheral blood) obtained from a related or unrelated donor the tumor cells are transduced with a co-stimulatory antigen and/or APC that expresses a tumor antigen and the co-stimulatory antigen, e.g. CD137, CD137L, 4-1BB, 4-1BBL, variants, alleles, mutants and fragments thereof, under conditions and for a time sufficient to permit the stimulation of T cells as described above. The tumor specific T lymphocytes and, if desired, bone marrow, peripheral blood stem cells may then be administered to a patient using standard techniques. Within other embodiments, tumor-specific T cells as described herein may be used to remove cells expressing tumor antigen from autologous bone marrow, peripheral blood or a fraction of bone marrow or peripheral blood (e.g., CD34⁺ enriched peripheral blood (PB) prior to administration to a patient). Such methods may be performed by contacting bone marrow or PB with such T cells under conditions and for a time sufficient to permit the reduction of tumor antigen expressing cells to less than 10%, preferably less than 5% and more preferably less than 1%, of the total number of myeloid or lymphatic cells in the bone marrow or peripheral blood. The extent to which such cells have been removed may be readily determined by standard methods such as, for example, qualitative and quantitative PCR analysis, morphology, immunohistochemistry and FACS analysis. Bone marrow or PB (or a fraction thereof) may then be administered to a patient using standard techniques.

Isolation and Expansion of Antigen-Specific T Cells

Tumor peptide and co-stimulatory antigens of the present invention can be used to take advantage of adoptive immunotherapy around the reinfusion of T cells specific for an antigen/epitope into a patient in need thereof. Preferred methods have been described in the examples which follow, however, any method can be used wherein the tumor antigen, Major Histocompatibility Complex (MHC; HLA- Human Lymphocyte Antigen) and co-stimulatory T cell antigen are present. For example, a peptide/MHC/co-stimulatory antigens can be conjugated to a physical support (i.e. a streptavidin bead) and therefore provide the opportunity to isolate antigen-specific T cells which when expanded in vitro using conventional methods (i.e. using an APC expressing a tumor specific antigen to T cells) and those disclosed herein can be used for adoptive immunotherapy. These methods can be used in conjunction with each other. Alternatively, the co-stimulatory ligands can be used to sort antigen-specific T cells using a flow cytometry-based cell sorter.

In another aspect of the invention, the peptide/MHC/co-stimulatory antigen can be used to isolate an antigen-specific T cell from a T cell population isolated from a blood sample. A T cell population can be obtained using any method known in the art. Preferably, cells from the circulating blood of an individual are obtained by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis or leukapheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In one embodiment of the invention, the cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. A s those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated "flow-through" centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca⁺²ZMg⁺² free PBS. Alternatively, the undesirable components of the apheresis sample may be removed and the cells are directly resuspended in culture media. In another embodiment, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and by centrifugation through a PERCOLL™ gradient. A specific subpopulation of T cells, such as CD28⁺, CD4⁺, CD8⁺, CD45RA⁺, and CD45RO⁺ T cells, can be further isolated by positive or negative selection techniques. For example, CD3⁺, CD28⁺ T cells can be positively selected using CD3/CD28 conjugated magnetic beads. In one aspect of the present invention, enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. A preferred method is cell sorting and/or selection via negative magnetic immunoadherence.

Pharmaceutical Preparations

According to the invention the tumor specific T lymphocytes are used as either prophylactic or therapeutic treatments of tumors or potential development of such. Thus the invention also includes pharmaceutical preparations for humans and animals involving these cells. Those skilled in the medical arts will readily appreciate that the doses and schedules of pharmaceutical composition will vary depending on the age, health, sex, size and weight of the human and animal. These parameters can be determined for each system by well- established procedures and analysis e.g., in phase I, II and III clinical trials and by review of the examples provided herein. For administration, the lymphocytes cells can be combined with a pharmaceutically acceptable carrier such as a suitable liquid vehicle or excipient and an optional auxiliary additive or additives. The liquid vehicles and excipients are conventional and are commercially available. Illustrative thereof are distilled water, physiological saline, aqueous solutions of dextrose and the like. Suitable formulations for parenteral, subcutaneous, intradermal, intramuscular, oral or intraperitoneal administration, include aqueous solutions of active compounds in water- soluble or water-dispersible form. In addition, suspensions of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, include for example, sodium carboxymethyl cellulose, sorbitol and/or dextran, optionally the suspension may also contain stabilizers. Also, cells can be mixed with immune adjuvants well known in the art such as Freund's complete adjuvant, inorganic salts such as zinc chloride, calcium phosphate, aluminum hydroxide, aluminum phosphate, saponins, polymers, lipids or lipid fractions (Lipid A, monophosphoryl lipid A), modified oligonucleotides, etc. In addition to administration with conventional carriers, active ingredients may be administered by a variety of specialized delivery drug techniques which are known to those of skill in the art.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following examples are offered by way of illustration, not by way of limitation. While specific examples have been provided, the above description is illustrative and not restrictive. Any one or more of the features of the previously described embodiments can be combined in any manner with one or more features of any other embodiments in the present invention. Furthermore, many variations of the invention will become apparent to those skilled in the art upon review of the specification.

All publications and patent documents cited in this application are incorporated by reference in pertinent part for all purposes to the same extent as if each individual publication or patent document were so individually denoted. By their citation of various references in this document, Applicants do not admit any particular reference is "prior art" to their invention.

EXAMPLES The following non-limiting Examples serve to illustrate selected embodiments of the invention. It will be appreciated that variations in proportions and alternatives in elements of the components shown will be apparent to those skilled in the art and are within the scope of embodiments of the present invention.

Embodiments of the invention may be practiced without the theoretical aspects presented. Moreover, the theoretical aspects are presented with the understanding that Applicants do not seek to be bound by the theory presented.

While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. Numerous changes to the disclosed embodiments can be made in accordance with the disclosure herein without departing from the spirit or scope of the invention. Thus, the breadth and scope of the present invention should not be limited by any of the above described embodiments.

Materials and Methods Animals and cells

C57BL/6 and Thy 1.1 (C57BL/6.PL-Thyla/Cy) mice were obtained from The Jackson Laboratory. Thy 1.1 mice, a C57BL/6J congenic strain, carry the T cell-specific Thy l^a {Thy 1.1) allele. OT-I mice express a transgenic TCR that is specific for OVA257-264 (SIINFEKL, SEQ ID NO: 1) peptide bound to H-2K^b. GFP⁺ mice (Ikawa M, et al. FEBS Lett 1998; 430: 83-7) were bred with OT-I mice to generate OT-I /GFP T cells expressing green fluorescent protein. Mice were maintained in pathogen- free facilities at the University of Miami, and procedures were performed in agreement with the Institutional Animal Care and Use Committee, per National Institutes of Health guidelines.

Lewis lung carcinoma (LLC) cells stably transfected with ovalbumin (LLC/OVA) (Strbo N, et al. Immunity 2003; 18: 381-90) were grown in IMDM plus 10% FBS, penicillin (50 units/ml), streptomycin (50 μg/ml)(I-10), and lmg/ml Geneticin.

Antibodies Anti-mouse CD3ε-PE, anti-mouse CD8a-PE or -Cy-Chrome (PE-Cy5), anti-mouse CD4-FITC, anti-mouse Vα2-FITC, and anti-mouse Vβ5.1,5.2-PE (BD Pharmingen, San Diego, CA) mAbs were used to stain for T cells. Prior to staining, splenocytes were treated with anti-mouse CD16/CD32 (Fc-γIII/II receptor, BD Pharmingen) to block Fc-mediated binding. Antibodies used to assess activation and/or differentiation states of T cells include: anti-CD44-PE (eBioscience, San Diego, CA), anti-CD62L-PE (BD Pharmingen), anti-human granzyme B-PE (Caltag, Burlingame, CA), anti-mouse CD25-biotin (7D4) or anti-mouse Ly- 6C-biotin (BD Pharmingen) followed by streptavidin-PE (Sigma, St Louis, MO), anti-mouse 4- IBB (BD Pharmingen) followed by anti-rat IgGl-biotin (BD Pharmingen) and streptavidin- PE, and anti-CD107a (1D4B) (BD Pharmingen) followed by anti-rat IgG (H+L)-PE (Caltag). B7.1 expression was detected with anti-human CD80-FITC (BD Pharmingen) and 4-1BBL expression with antimurine 4- IBBL (BD Pharmingen), anti-rat IgG (H+L)-biotin (Caltag), and streptavidin-PE (Sigma).

Purification ofCD8⁺ OT-I cells

Splenocytes from OT-I mice were purified by positive column selection using MACS anti-CD8a (Ly-2) MicroBeads (Miltenyi Biotec, Auburn, CA) as described (Strbo N, et al. supra). Purified cells were >97% CD8⁺Vα2⁺Vβ5.1,5.2+ as assessed by flow cytometry.

HSV-amplicon-vector construction and helper virus-free packaging

The cDNA of murine 4- IBBL with a Kpnl site 5' and Nhel site 3' was amplified by

RT-PCR from RNA of C57BL/6 spleen stimulated with LPS (15μg/ml) for 24 hours using the 5' primer: 5 '-GGTACCGCCATGGACCAGCACACACTTG-S ' (SEQ ID NO: 2) and the

3' primer: 5'- GCTAGCTTCCCATGGGTTGTCGGGTTTCAC-3' (SEQ ID NO: 3). The cDNA was inserted into pCR-Script Amp SK(+) (Stratagene, La Jolla, CA)

(415.pCRScript) and again amplified by PCR, to introduce a 5' BamKl site and stop codon plus EcoKI 3' using the 5' primer: 5 '-TCGGATCCGTAATGGACCAGCACACACTTG-S '

(SEQ ID NO: 4) and the 3' primer: 5'-

GAGAATTCTCATTCCCATGGGTTGTCGGGTTTCAC-3' (SEQ ID NO: 5). The complete murine 4- IBBL cDNA was then cloned into the BamHI and EcoBΛ sites in the polylinker region of the HSV-I amplicon vector pHSVPrPUC (Geller A.I. et al. Science 1988;

241: 1667-9). The cDNA of human B7.1 was cloned into HSVPrPUC as previously described (Kutubuddin M, et al. Blood 1999; 93: 643-54). Helper-virus-free replication-defective packaging of HSV-I viral amplicons was performed as described previously (Stavropoulos TA, et al. J Virol 1998; 72: 7137-43; Bowers WJ, et al. Gene Ther 2001; 8:111-20). Amplicons containing the gene for Escherichia coli β-galactosidase (HSV. LacZ) were prepared using the same vector system.

OT-l/GFP in vitro expansion for in vivo administration

LLC/OVA cells were resuspended at 106 cells/1 OOμl I- 10 media, transduced with either HSV.4-1BBL (MOI=I) or HSV.B7.1 (MOI=I), and incubated at 37°C for Ih before being transferred to a six-well plate. One day later, transduced LLC/OVA were resuspended at 5 x 10⁶ cells/ml, treated with 0.4mg/ml mitomycin C for 20min at 37oC in PBS, and washed three times in RPMI 1640 plus 10% FBS, penicillin, streptomycin, and 50μM 2-ME (R-IO). Freshly isolated OT-l/GFP cells were then plated with mitomycin C-treated LLC/OVA at a 3:2 ratio in 24-well plates. Each well contained 2.4 x 10⁶ OT-l/GFP cells plus 1.6 x 10⁶ tumor cells in 2ml R-IO. R-IO media (0.5-lml) was added to each well after 2 days. Cells were harvested on the third day and separated from tumor using anti-CD8a magnetic beads before adoptive transfer.

Intracellular staining and flow cytometry

Cells were stained on the surface with fluorochrome-conjugated anti-CD4 and anti- CD8a antibodies in PBS at 4°C for 20min. Cells were then washed with PBS, fixed using

Cytofix/Cytoperm buffer (BD Pharmingen) for 20min at 4°C. 0 .1% saponin/1% FBS in PBS was used to wash, stain with fluorochrome-conjugated antibodies, and wash cells again.

Cells were analyzed using an LSR flow cytometer and CellQuest software (BD Biosciences,

San Jose, CA).

Blood collection and preparation for flow cytometry

Sodium heparin from a 10ml Vacutainer blood collection tube (Becton Dickinson,

Franklin Lakes, NJ) was resuspended in PBS (5ml) and aliquoted into microfuge tubes (100 μl/tube). Blood was collected from the tail and lysed with ACK buffer three times. Cells were resuspended in PBS for antibody staining.

Detection OfGFP⁺ cells in frozen tissue sections

Spleens were frozen in O. C. T. compound (Sakura Finetek, Torrance, CA) with dry ice and stored at -80⁰C until sectioning. Tissues were sectioned 6μm thick and adhered onto Superfrost plus glass slides (VWR, West Chester, PA). Slides were kept cold to prevent diffusion of GFP and exposed in a closed-lid container to 37% formaldehyde vapor at -20⁰C for 24h. Tissues were outlined with an ImmEdge pen (Vector Laboratories, Burlingame, CA), washed with PBS, and counterstained with lμg/ml Hoechst 33342 (Sigma) for 15min at 37°C. After washing, slides were mounted with Prolong Gold anti-fade reagent (Molecular Probes/Invitrogen, Eugene, OR). Sections were viewed using a Leica DMIRB Inverted Microscope (Bannockburn, IL) and images captured with MetaMorph Imaging System (Molecular Devices Corporation, Downingtown, PA).

Tumor measurements and statistics

Statistical analyses were done using Microsoft Excel, StatView, and SAS® 9.1. Average cell counts were compared by Student's t-test. Tumor burden was calculated as the volume of a sphere with radius based on the average of two diameters, Dl and D2, measured by caliper [Volume = 4/3π((Di+D2)/4)³]. Where possible, t-tests were used to compare the average tumor volume in treatment groups at the end of the experiment. For experiments involving animal sacrifice, tumor growth was compared across groups by fitting a log-linear regression model, and differences in tumor- free mice across groups were compared by Fisher's exact test.

CTL assay

Splenocytes were incubated with mitomycin C-treated LLC/OVA at a 10:1 ratio for 6 days with recombinant mIL-2 (10-20 U/ml) in R- 10 media and then plated in 96-well round- bottom plates at the indicated effector :target ratios. LLC/OVA and LLC targets were labeled with ⁵¹Cr (150 μl/10⁶ cells) and plated at 5 x 10⁴ cells/well. Plates were incubated at 37°C for 8h. Supernatant was collected and added to Ready Safe Liquid scintillation cocktail for aqueous samples (Beckman Coulter, Fullerton, CA). Samples were counted on a LS 6500 multi-purpose scintillation counter (Beckman Coulter). Percent lysis = (Sample counts- Spontaneous counts)/(Maximum counts-Spontaneous counts)* 100.

In vivo BrdU labeling

Three and six days following adoptive transfer of OT-I, Thy 1.I⁺ mice were injected intraperitoneal^ with 100 μl BrdU (lmg) (APC BrdU Flow kit, BD Pharmingen). On Day 8, OT-I numbers and incorporation of BrdU were assessed in the spleen, tumor, draining and nondraining lymph nodes by staining with anti-CD8-PE, anti-Thyl.2-FITC, and anti-BRDU- APC per manufacturer's instructions.

Example 1: 4-1 BBL in adoptive immunotherapy Tumors transduced by HSV A-IBBL express murine 4- IBBL and can induce proliferation ofCD8⁺ OT-I T cells: Murine 4- IBBL cDNA was cloned into the HSV amplicon vector pHS VPrPUC and packaged into amplicons (HSV.4- IBBL) using a helper virus-free packaging method (Figure IA). Packaged virions contain only amplicon genomes, without the propagation of helper virus (Figure IA). HSV.B7.1 and HSV.LacZ amplicons, which encode for human B7.1 and bacterial β-galactosidase, respectively, were also packaged using the helper-free method. Both mouse and human B7.1 can stimulate T cell CD28 receptors of either species.

The ability of the amplicons to transduce LLC/OVA or the parental LLC tumor cell line was tested. LLC and LLC/OVA cells transduced with HSV.B7.1 or HSV.4-1BBL showed high levels of expression of B7.1 or 4-1BBL, respectively, by day 2 as shown by flow cytometric analysis (Figure IB). HSV.LacZ transduction did not induce either B7.1 or 4-1BBL expression (Figure IB). HSV.B7.1 transduction did not induce 4-1BBL expression or vice versa. In order to more accurately follow the effects of 4- IBBL on antigen-specific T cells, the adoptive transfer of CD8⁺ OT-I T cells was utilized in the in vivo mouse experiments. OT-l/GFP cells were derived from TCR-transgenic OT-I mice that were backcrossed into GFP⁺ mice to facilitate monitoring once adoptively transferred. HSV.B7.1- or HSV.4-lBBLtransduced LLC/OVA tumor cells were used to stimulate CD8⁺ OT-I T cells for 3-5 days (Figure 1C). At day 3, markedly increased proliferation was seen for OT-I cells stimulated with either HSV.B7.1- or HSV.4-lBBL-transduced tumors, compared to untransduced tumor (Figure 1C). HSV.LacZ-transduced LLC/OVA did not augment proliferation of OT-I in vitro compared to untransduced LLC/OVA. OT-I stimulated with HSV.4-lBBL-transduced LLC/OVA continued to proliferate vigorously at day 5. OT-I cells which were cultured with parental LLC with or without costimulatory ligands did not proliferate, signifying an absolute requirement for signal one for T cell activation. OT-I cells cultured with HSV.4-lBBL-transduced LLC/OVA did not show increased proliferation on days 3 (Figures 7A, 7B) or 5 with the addition of soluble (2μg) or plate-bound (0.2μg) anti- CD28 antibody. Soluble anti-CD28 antibody instead markedly inhibited proliferation elicited by HSV.B7.1 -transduced LLC/OVA, but not that elicited by HSV.4-lBBLtransduced tumor, indicating soluble anti-CD28 antibody blocked interaction between CD28 on T cells and B7.1 on the tumor cells (Figures 7 A, 7B). Addition of soluble anti-CTLA-4 antibody (12μg) to the co-cultures of OT-I cells with HSV.B7.1- or HSV.4-lBBL-transduced LLC/OVA did not further augment proliferation (Figures 7A, 7B). Titration of OT-I with HSV.4- IBBL- transduced LLC/OVA showed that tumor-specific OT-I cells (10⁵) could expand following serial dilution with normal CD8⁺ splenocytes (FigurelD). Dilutions of up to 1:32 (3,000 OT- 1 cells/well) are shown for days 3 and 5 (FigurelD). On day 5, OT-I cells continued to proliferate at a dilution of 1:32 even when the wells with higher number of OT-I have markedly decreased their proliferation, due possibly to limited nutrients or lack of intact tumor cells. Therefore, tumor-specific cells can be stimulated to proliferate using this system even when present at lower numbers.

OT-I cells costimulated ex vivo with 4-1BBL display an effector phenotype.

Expanded OT-l/GFP cells were characterized following three days of co-incubation with transduced LLC/OVA (Figure 2A). CD44 is expressed on activated T cells and functions in lymphocyte homing and adhesion. CD25 (IL-2Rα) is a component of the high- affinity IL-2 receptor upregulated on effector T cells. Granzyme B is a serine protease stored in the granules of CTLs along with perform. CD 107a (LAMP-I) is a widely expressed intracellular antigen that appears on CD8⁺ CTLs following activation-induced degranulation. OT-l/GFP stimulated ex vivo with either HSV.4-1BBL- or HSV.B7.1 -transduced LLC/OVA expressed high levels of CD44, intracellular granzyme B, and CD 107a and modestly increased levels of CD25, indicating that they were activated and capable of cytotoxic activity (Figure 2A). HSV.LacZ stimulated OT-l/GFP expressed CD44 and CD 107a at lower levels than those stimulated with HSV.4-1BBL or HSV.B7.1. 4-1BB was expressed on OT-I cells stimulated for 3 days with HSV.4-lBBL-transduced LLC/OVA, but was not detected on naive OT-I cells (Figure 2A). Naϊve OT-l/GFP did not express any of the aforementioned activation markers.

Ly-6C is a marker for previously activated T cells and memory CD8⁺ T cells. Expression of Ly-6C was highest in HSV.4-lBBL-stimulated OT-l/GFP, compared to HSV.B7.1- and HSV.LacZ-stimulated OT-l/GFP and naϊve OT-l/GFP (Figure 2A). Since Ly-6C can be upregulated on T cells by type I interferon secretion by HSV amplicon- transduced tumors, supernatant from co-cultures of OT-I and LLC/OVA transduced with HSV.4-1BBL or HSV.LacZ was collected on days 1-3. IFN-α was not detected by ELISA at a detection threshold of 12.5 pg/ml. More likely, IFN-γ produced by 4-lBBL-stimulated T cells induced Ly-6C expression. T cells costimulated with 4-1 BBL ex vivo expand in vivo in response to tumor.

Ex vz^'vø-stimulated OT-I /GFP cells were adoptively transferred into LLC/OVA tumor-bearing mice, and the extent of expansion and anti-tumor response were measured (Figure 2B). Mice were bled at several time points following transfer of OT-l/GFP cells to detect expansion (Figure 3A). Six days following transfer, the number of OT-l/GFP in the peripheral blood was significantly greater in the LLC/OVA tumor-bearing group receiving HSV.4-lBBL-stimulated OT-l/GFP cells compared to tumor-bearing groups receiving naive (p=0.007) or HSV.B7.1 -stimulated OT-l/GFP (Figure 3A). Greater numbers of OT-l/GFP cells were observed following transfer of 4-lBBL-stimulated OT-l/GFP cells into non- tumor-bearing mice than naive or B7.1 -stimulated OT-l/GFP cells, indicating continued proliferation of 4-lBBL-stimulated OT-l/GFP cells in vivo in the absence of tumor. The number of 4-lBBL-stimulated OT-l/GFP cells was significantly greater in the tumor-bearing mice compared to non-tumor-bearing mice (p=0.017), suggesting that 4-lBBL-stimulated OT-l/GFP cells can respond in vivo to tumor-specific antigen (Figure 3A).

The total numbers and percentages of tumor-specific OT-l/GFP cells in relation to the CD8⁺ population were determined in the spleen of tumor-bearing mice 17 days post-transfer. Representative mice are shown in Figure 3B. The percentage Of GFP⁺ cells in the spleen was the greatest in the tumor-bearing group given OT-l/GFP cells stimulated with HSV.4- BBLtransduced tumor (19.8% of CD8⁺ cells) vs. HSV.B7.1 -transduced tumor (0.1%) or naive OT-l/GFP cells (1.1%) (Figure 3B). Substantial numbers of OT-l/GFP cells were also detected in the spleen of non-tumor-bearing mice 17 days after transfer of 4-lBBL-stimulated OT-l/GFP cells (7% of CD8+ cells), evidencing that tumor-specific 4-lBBL-stimulated T cells can also persist in vivo in the absence of antigen (Figure 3B). In addition, there were greater absolute numbers of OT-l/GFP cells present in the spleen for the 4-lBBL-stimulated OT-l/GFP group (0.9 x 10⁶ ± 0.4 x 10⁶ cells/spleen) vs. B7.1 -stimulated (0.9 x 10⁵ ± 0.7x105) or naϊve OT-l/GFP groups (0.1 x 10⁶ ± 0.3 x 10⁵).

Spleens were sectioned and examined for GFP+ cells 32 days following adoptive transfer of OT-l/GFP. Spleens from mice given HSV.4-lBBL-stimulated OT-l/GFP cells showed increased infiltration with GFP+ cells (Figure 3C). Analysis by flow cytometry indicated that 9.4% of the CD8⁺ splenocytes in the HSV.4-lBBL-stimulated OT-l/GFP group were GFP⁺, compared to 0.1% and 0.2% GFP⁺ in the naϊve OT-l/GFP and HSV.B7.1- stimulated OT-l/GFP groups, respectively. These results indicate that tumor-specific T cells stimulated ex vivo with HSV.4-lBBL-transduced tumor could expand and persist in vivo. The tumor bed was also analyzed for the presence of OT-l/GFP cells. Six days post- transfer, flow cytometry analysis of dispersed tumor showed that the percentage of GFP⁺ cells was 7-8-fold greater in mice which received the 4-lBBL-stimulated OT-l/GFP cells compared to those receiving naive cells (Figure 3D). Later time points could not be examined due to tumor regression in the HSV.4-lBBL-stimulated OT-l/GFP group.

BrdU incorporation by adoptively transferred cells

To determine whether adoptively transferred OT-I divided in vivo, the BrdU uptake of transferred cells was characterized in separate experiments. Thyl.2⁺ OT-I cells were transferred into Thyl .I⁺ mice, which were pulsed with BrdU on day 7 and day 10. Mice were sacrificed on day 12 to determine in vivo proliferation of OT-I CD8+ cells and their distribution in the spleen, TDLNs, and non-TDLNs (Figure 4A). Significantly higher percentages of OT-I cells which had incorporated BrdU were present in the TDLNs in the A- IBBL group compared to the naive OT-I group (Figure 4A). The presence of proliferating OT-I cells in the tumor bed was assessed by pulsing mice on days 3 and 6 with 1 mg of BrdU and harvesting the tumor on day 8. Overall, more Thyl.2⁺ OT-I cells were present in the 4-lBBL-stimulated group as compared to the naive OT-I or B7.1 -stimulated group, and these cells had incorporated BrdU (Figure 4B).

CD8+ T cells expanded in vitro with 4-1 BBL possess cytolytic activity and markedly decrease tumor growth.

CTL activity from splenocytes of mice that received 2 x 10⁶ OT-l/GFP cells by adoptive transfer, was measured. LLC/OVA tumor-bearing mice treated with HSV.4- IBBL- activated OT-l/GFP cells harbored lower tumor burden at day 14 than untreated mice (P <

0.0001) or mice treated with an identical number of HSV.B7.1 -stimulated OT-l/GFP (P <

0.0001) or naive OT-l/GFP cells (P = 0.0003) (Figure 5A). Naive OT-l/GFP treatment did not reduce tumor burden significantly when compared to the untreated group (P >0.05).

Administration of HSV. B7.1 -activated OT-l/GFP cells did not have major inhibitory effects on tumor size when compared to no treatment or to naive OT-l/GFP transfer (P > 0.05)

(Figure 5A).

Splenocytes from the HSV.4-lBBL-activated OT-l/GFP group demonstrated substantially higher CTL activity against LLC/OVA compared to splenocytes from the

HSV.B7.1 -stimulated OT-l/GFP, naive OT-l/GFP, and untreated groups (Figure 5B). These results suggest that T cells expanded with HSV.4-lBBL-transduced tumor cells may have more favorable effector characteristics than those obtained through HSV.B7.1 -mediated stimulation.

In a second experiment, 4 x 10⁶ cells OT-I /GFP cells, either naϊve or stimulated with HSV.4-lBBL-transduced tumor, were transferred into LLC/OVA tumor-bearing mice to determine effects on tumor volume (Figure 5C). Transfer of HSV.4-lBBL-stimulated OT- 1/GFP resulted in a statistically significant decrease in tumor growth compared to no treatment (jXO.OOl) or to naϊve OT-l/GFP transfer (jXO.OOl) (Figure 5C). The estimated rate of growth for the HSV.4-1BBL OT-l/GFP group from the regression model was significantly lower than that for the naϊve OT-l/GFP group (0.013 vs.0.130, /Kθ.001). By Day 30, 88.9% (8/9) mice in the HSV.4-1BBL OT-l/GFP group were tumor-free, compared to 0% (0/7) tumor-free in the no treatment group and 12.5% (1/8) tumor-free in the naϊve OT- l/GFP group. Analysis using Fisher's exact test show that these differences between the HSV.4-1BBL OT-l/GFP group and the no treatment group (p=0.00\) or the naϊve OT-l/GFP group were statistically significant (Figure 5D).

4-lBBL-stimulated CD8+ OT-I cells display memory phenotypic characteristics.

The phenotype of OT-l/GFP cells in each group was characterized post-adoptive transfer (Figure 6). On day 6, GFP+ cells in the spleen (Figure 6A) and tumor bed (Figure 6B) were analyzed for Ly-6C. The HSV.4-lBBL-stimulated OT-l/GFP group had a greater percentage in the lymphocyte gate of Ly-6C⁺GFP⁺ cells present in the spleen and tumor bed than the naϊve OT-l/GFP group. On day 17, OT-1/GFP⁺ cells in tumor-bearing and non- tumor-bearing mice were studied for levels of CD44, Ly-6C, and CD62L (L-selectin) (Figure 6C). Significantly higher levels of CD44⁺ OT-l/GFP were observed in the splenocytes of mice which had received HSV.4-lBBL-stimulated OT-l/GFP (12.7% in tumor-bearing mice, 3.8% in non-tumor-bearing mice) than were seen for either HSV.B7.1 -stimulated (0.1% in tumor-bearing and non-tumor-bearing mice) or naϊve OT-l/GFP transfer (0.4% in tumor- bearing mice, 0.1% in non-tumor-bearing mice) (Figure 6C). HSV.4-lBBL-stimulated OT- l/GFP cells continued to demonstrate higher levels of Ly-6C expression on day 17 in comparison to naϊve or HSV.B7.1 -stimulated OT-l/GFP transferred cells (Figure 6C).

Further analysis showed that OT-l/GFP cells persisting in the spleen were CD62L negative (Fig. 6C) as well as CD25 negative and CD 127 (IL-7Rα) negative. Similar characteristics, indicating the generation of effector memory cells, were observed in OT-l/GFP cells present in the spleen at Day 32. This evidences that upon adoptive transfer of 4-lBBL-stimulated T cells, the T cells persist at high levels and exhibit phenotypic attributes of effector memory cells.

To determine whether this strategy could be used in a non-transgenic setting, GFP⁺ mice were inoculated with LLC/OVA s.c. for 8 days and then harvested spleens and TDLNs. CD8⁺ T cells in the spleen or lymphocytes from TDLNs were stimulated ex vivo with untransduced LLC/OVA or LLC/OVA transduced with HSV.4-1BBL. Increased Ly-6C expression was observed in vitro on the CD8⁺ splenocytes and to a lesser degree on the CD8⁺ TDLN cells following five days of cultivation with HSV.4-lBBL-transduced LLC/OVA compared to co-culture with untransduced tumor. Although 4-lBBL-transduced LLC/OVA induced proliferation and modest expansion in vivo, levels were ~20-fold lower than found with OT-I, preventing adequate harvest for adoptive transfer. Adoptive transfer of 0.5x106 co-cultured T cells showed modest effects on tumor growth. This may be due to low precursor frequency and reduced expansion. Therefore, further optimization of the ability to generate tumor-specific T cells and/or modification of transferred dose and dose schedules would improve tumor control.

Discussion

The overall purpose of these studies was to understand the potential utility of 4- IBB as a means of expanding and activating tumor-specific CD8⁺ T cells for adoptive immunotherapy. 4- IBB stimulation has been used to sustain nascent responses and prevent activation- induced cell death in T cells. 4- IBB stimulation triggers TRAF2 signaling and NF-κB activation (Saoulli K, et al. J Exp Med 1998; 187: 1849-62; Lee HW, et al. J Immunol 2002; 169: 4882-8), which may in turn induce Bcl-xL and BfI-I, two pro-survival members of the Bcl-2 family. Without wishing to be bound by theory, it was reasoned that cells expanded using 4- IBB costimulation would have favorable effector characteristics and persist in vivo leading to improved tumor control.

HSV.4-1BBL amplicons were used to transduce LLC/OVA tumors in vitro for purposes of ex-vivo expansion of tumor-specific OT-I T cells. OT-I cells responded to co- culture with HSV.4-lBBL-transduced LLC/OVA by proliferating and expressing activation markers, namely CD44, CD25, Ly-6C, CD107a, 4-1BB, and intracellular granzyme B, indicating priming and differentiation into cytolytic effectors.

Adoptive transfer of HSV.4-lBBL-stimulated OT-l/GFP T cells significantly conferred greater protection against LLC/OVA growth compared to naive or HSV.B7.1- stimulated OT-l/GFP cells. Mice treated with HSV.4-lBBL-expanded OT-l/GFP cells exhibited greater CTL activity and showed higher percentages of tumor-specific OT-I /GFP+ cells in the spleen and tumor bed. Following HSV.4-1BBL stimulation, CD8⁺ OT-I T cells expanded in vivo, incorporated BrdU, and expressed high levels of CD44 and the CD8⁺ memory marker Ly-6C. Ly-6C regulates homing of CD8⁺ T cells to lymph nodes and perhaps augments the homing of transferred HSV.4-lBBL-stimulated OT-l/GFP cells to secondary lymphoid organs, where they can be found on days 6, 17, and 32 post-transfer. Most transferred OT-l/GFP cells were CD62L negative even after 20 days post-tumor eradication in the host, indicating, in combination with Ly-6C expression, an effector memory phenotype. Perhaps, longer observations can demonstrate conversion of tumor- specific cells to CD44^hlLy-6C^hlCD62L^hl central memory cells. In these studies, we show T cell costimulation with 4- IBBL expressed on tumor cells is useful in facilitating expansion of tumor-specific T cells in vivo as well as in vitro.

Our studies indicated that it was possible to specifically generate tumor-reactive clones, which can persist in vivo, by using HSV.4-lBBL-transduced tumor cells in the absence of additional CD28 costimulation. Whether the low levels of B7.1 present on

LLC/OVA tumor are adequate for CD28 stimulation to work in combination with 4- IBBL costimulation is not known.

Maus et al. have shown ex vivo expansion of human polyclonal and MHC tetramer- sorted antigen-specific CTL using artificial antigen presenting cells, specifically K562 erythromyeloid cell lines stably transfected to express 4- IBBL and the Fcγ receptor CD32 to bind anti-CD3 and anti-CD28 antibodies on the surface (Nat Biotechnol 2002; 20: 143-8). In contrast to the work by Maus et al., the highly efficient HSV amplicon system provides a means by which to selectively expand tumor-specific effector populations using autologous tumor from patients without need for presorting for tumor-reactive T cells. In addition to 4- IBB costimulation, HSV.4-1BBL amplicon transduction of tumor may provide other stimuli that facilitate the generation of effector cells. HSV amplicons can impart a strong innate response to transduced cells, including macrophage cell lines and human chronic lymphocytic leukemia cells, resulting in cytokine secretion and NKG2D-L expression by the transduced cells. HSV possesses at least three molecular components capable of activating the innate immune system: 1) dsRNA generated through self- hybridization of viral genes transcribed from complementary DNA strands; 2) envelope glycoproteins recognized by TLR2, and 3) unmethylated CpG motifs encoded in the viral genome that activate TLR9. Due to the fact that HSV amplicon DNA is concatamerized, CpG effects on TLR9 may be quite potent. The enhanced capacity of transduced tumors to stimulate an innate immune response may lead to an improved adaptive response.

Future work would include whether HSV.B7.1 will further augment effects seen with HSV.4-lBBLstimulation. HSV.B7.1 -stimulated OT-I did not expand in vivo and inhibit tumor growth. Since B7.1 serves as a ligand for both CD28 and CTLA-4, B7.1 may have also bound to CTLA-4 expressed on activated OT-I cells, inhibiting expansion and survival. Greater proliferation in the presence of soluble anti-CTLA-4 antibody was not observed (Figures 7A, 7B).

In summary, these studies evidence that costimulation with 4- IBBL can be employed to enhance expansion and cytolytic activity of tumor-specific CD8⁺ T cells for the generation of tumor-specific immunity. Adoptive transfer of TCR-transgenic OT-I /GFP T cells was utilized in order to more accurately follow the effects of 4- IBBL on T cells that were specific for defined tumor-related antigens in the in vivo mouse experiments, since antigen-specific CD8⁺ T cells are normally present in low numbers. The vigorous cytolytic effector function as well as the increased expansion and persistence seen using HSV amplicon-transduced tumor evidences this method should be explored further and may be potentially applicable in the human setting. Since HSV vectors are theoretically safe and a highly efficient means of gene transfer, the laboratory is pursuing pre-clinical development of these vectors for potential human use in CLL. Further optimization would allow for efficient expansion of relatively rare precursor anti-tumor T cells.

Although the invention has been illustrated and described with respect to one or more implementations, equivalent alterations and modifications will occur to others skilled in the art upon the reading and understanding of this specification and the annexed drawings. In addition, while a particular feature of the invention may have been disclosed with respect to only one of several implementations, such feature may be combined with one or more other features of the other implementations as may be desired and advantageous for any given or particular application.

The Abstract of the Disclosure is provided to allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the following claims.

Claims

What is claimed:

1. A method of treating a patient suffering from cancer comprising: obtaining a patient sample comprising tumor tissue or tumor cells; administering a composition comprising a lymphocyte co-stimulatory antigen to the tumor cells; and, expressing the co-stimulatory antigen; obtaining lymphocytes and culturing the lymphocytes with the tumor cells expressing the co-stimulatory antigen; administering the lymphocytes to the patient; and, treating the patient suffering from cancer.

2. The method of claim 1, wherein the lymphocytes are T lymphocytes comprising autologous, heterologous, syngeneic, allogeneic or xenogeneic T lymphocytes.

3. The method of claim 1, wherein the composition comprising a lymphocyte co- stimulatory antigen comprises a vector expressing the co-stimulatory antigen, polynucleotide or polypeptide.

4. The method of claim 1, wherein the co-stimulatory antigen comprises a CD4⁺ or a CD8⁺ T lymphocyte specific co-stimulatory antigen.

5. The method of claim 3, wherein the vector comprises a viral, recombinant or plasmid vector.

6. The method of claim 5, wherein the viral vector is a herpes viral vector, an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, a herpes viral vector, polyoma viral vector or hepatitis B viral vector.

7. The method of claim 3, wherein the vector is a replication deficient herpes simplex virus (HSV) amplicon expressing the 4- IBBL (CD 137L) co-stimulatory antigen.

8. The method of claim 6, wherein the co-stimulatory antigen is CD137 (4-1BB), mutants, variants, analogues, homologues, ligands or fragments thereof.

9. The method of claim 1 , wherein culturing the lymphocytes with the tumor cells expressing the co-stimulatory antigen generates tumor specific T lymphocytes.

10. The method of claim 9, wherein the administering of the tumor specific T- lymphocytes to the patient generates tumor specific effector memory T lymphocytes in vivo.

11. The method of claim 1 , wherein the administering of the tumor specific T lymphocytes to a patient further comprises surgery and/or administering chemokines or cytokines.

12. The method of claim 1, wherein the method of treating a patient suffering from cancer further comprises administration of one or more therapeutic agents.

13. The method of claim 10, wherein the one or more therapeutic agents are coadministered, prior to or after administration of the tumor specific T lymphocytes.

14. The method of claim 11, wherein the one or more therapeutic agents comprise chemotherapy, chemokines, radionuclides, cytokines, anti-angiogenic agents or radiotherapy.

15. The method of claim 1, wherein the method of treating a patient with cancer further comprises administering to the patient lymphocytes with specificity for one or more tumor antigens.

16. A method of treating cancer comprising: obtaining a tumor sample and lymphocytes from a patient; transducing tumor cells from the sample with a vector comprising a polynucleotide sequence encoding a T-lymphocyte co-stimulatory antigen, mutants, variants, homologues and fragments thereof; culturing the tumor cells expressing the antigen with T lymphocytes obtained from the patient; re-infusing the T lymphocytes into the patient; and, treating cancer.

17. The method of claim 16, wherein the vector is a replication deficient herpes simplex virus (HSV) amplicon expressing the 4- IBBL (CD 137L) co-stimulatory antigen.

18. The method of claim 17, wherein culturing the lymphocytes with the tumor cells expressing the co-stimulatory antigen generates tumor specific CD8⁺ T lymphocytes.

19. The method of claim 16, wherein the administering of the tumor specific T- lymphocytes to the patient generates tumor specific effector memory T lymphocytes in vivo.

20. The method of claim 16, wherein the administering of the tumor specific T lymphocytes to a patient further comprises administering of chemokines or cytokines.

21. The method of claim 16, wherein the method of treating a patient suffering from cancer further comprises surgery and/or administration of one or more therapeutic agents.

22. The method of claim 21, wherein the one or more therapeutic agents are coadministered, prior to or after administration of the tumor specific T lymphocytes.

23. The method of claim 21, wherein the one or more therapeutic agents comprise chemotherapy, chemokines, radionuclides, cytokines, anti-angiogenic agents or radiotherapy.

24. The method of claim 1 , wherein the method of treating a patient with cancer further comprises administering to the patient lymphocytes with specificity for one or more tumor antigens.

25. A method of preventing cancer in a patient at risk of developing cancer comprising: obtaining a sample from a patient; administering a composition expressing one or more tumor antigens and a T lymphocyte co-stimulatory antigen; expanding the tumor specific T lymphocytes ex vivo; administering to the patient the tumor specific T lymphocytes; and, preventing cancer in a patient at risk for developing cancer.

26. The method of claim 25, wherein the sample comprises isolated cells.

27. The method of claim 26, wherein the isolated cells comprise at least one of stem cells, antigen presenting cells, fibroblasts, kidney cells, lung cells, hepatocytes, myoblasts, myotube cells, neuron cells and oocytes.

28. An expression vector comprising a polynucleotide sequence encoding at least one of CD137, CD137L, 4-1BB, 4-1BBL, variants, mutants, homologues and fragments thereof.

29. The expression vector of claim 28, wherein the vector is a herpes simplex virus amplicon.

30. The expression vector of claim 28, wherein the polynucleotide sequence comprises CD137L or 4-1BBL, variants, mutants, alleles, homologues and fragments thereof.

31. An isolated cell expressing at least one of CD137, CD137L, 4-1BB, 4-1BBL, variants, mutants, fragments, homologues and fragments thereof.

32. The isolated cell of claim 31 , wherein the cell comprises at least one of tumor cells, stem cells, antigen presenting cells, fibroblasts, kidney cells, lung cells, hepatocytes, myoblasts, myotube cells, neuron cells and oocytes.