COMPOSITIONS AND METHODS FOR ENHANCED ANTIGEN DELIVERY TO ANTIGEN PRESENTING CELLS IN VIVO

Abstract: The present invention provides compositions and methods for enhancing site-specific in vivo delivery of tumor associated antigens. Thus, in one aspect, this invention provides a method of recruiting antigen presenting cells (APCs) to a predetermined site in a subject. Methods of augmenting transduction of a transgene in vivo are also provided by this invention.

Description: COMPOSITIONS AND METHODS FOR ENHANCED ANTIGEN DELIVERY TO ANTIGEN PRESENTING CELLS IN VIVO CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 60/078,909, filed March 20, 1998, the contents of which are hereby incorporated by reference into the present disclosure. TECHNICAL FIELD This invention is in the field of molecular immunology and medicine. In particular, compositions and methods for enhancing site-specific in vivo delivery of tumor associated antigens are provided. BACKGROUND In spite of numerous advances in medical research, cancer remains the second leading cause of death in the United States. In the industrialized nations, roughly one in five persons will die of cancer. Traditional modes of clinical care, such as surgical resection, radiotherapy and chemotherapy, have a significant failure rate, especially for solid tumors. Failure occurs either because the initial tumor is unresponsive, or because of recurrence due to regrowth at the original site and/or metastases. Cellular immunotherapy is emerging as a technologically and intellectually compelling anti-cancer treatment. The generation of an immune response against tumors has been demonstrated in several animal models and has been inferred from reports of spontaneous tumor regression in patients (Stotter and Lotze (1990) Cancer Cells 2:44-55). Cytotoxic T-lymphocyte (CTL) responses can be directed against antigens specifically presented by tumor cells, both in vivo and in vitro, without the need for prior knowledge of the molecular mechanism by which the tumor arose. In animal models, established tumors can be eradicated by the adoptive transfer of T-cells that are specifically immune to the malignant cells (Beun et al. (1994) Immunol. Today 15:11-15). The use of gene therapy vectors expressing tumor associated antigens (TAA) has been proposed for the treatment of certain cancers. One of the critical factors in cancer gene therapy is the route of administration of the vector encoding the TAA transgene. Currently, common routes of administration in the mouse include intraperitoneal (i.p.), intramuscular (i.m.), intravascular (i.v.), subcutaneous (s.c.) and intradermal (i.d.) (Wan et al. (1997) Human Gene Therapy 8:1355-63). The optimal route of administration appears to be dependent upon the type of tumor cell as well the TAA encoded on the transgene. The current limitation of in vivo administration of TAA-expressing is that they do not suppress growth of actively growing tumors. In other words, when vectors expressing TAAs are administered in vivo prior to the challenge with tumor cells, tumor growth is not suppressed unless the vectors are co-administered with interleukin 2 (IL-2). However, even with the IL-2 supplement, tumor growth is suppressed but not completely inhibited. Antigen presenting cells (APCs), a class of cells which includes dendritic cells, monocytes, macrophages, and B cells, are the presumed target for the delivery of TAAs. The TAAs being investigated are, in fact, antigens recognized by cytotoxic T lymphocytes (CTLs). In order to provoke a CTL response, the TAA needs to be presented to cells of the immune system by APCs. Therefore, the APC appears to be the appropriate target for both gene therapy vectors and recombinant TAA proteins. TAA transgenes have been directly delivered to isolated dendritic cells ex vivo, and the transfected dendritic cells are capable of inhibiting actively growing tumors when readministered to the mouse. Furthermore, tumor growth inhibition is accomplished without IL-2 supplementation. It is desirable, from a clinical standpoint, to deliver the tumor antigen to the patient's APCs in vivo rather than first isolating APCs for transfection and subsequent readministration. At the current time, however, in vivo delivery of the TAA transgene to APC is not as effective in suppressing tumor cell growth as delivery of the TAA transgene to APCs ex vivo. There are a number of possible explanations as to why in vivo transfections of APCs is not as efficacious as ex vivo transfection. First, there may not be many APCs in the region where the gene therapy vector is administered. Second, the number of copies of the gene therapy vector per APC may also be too low for effective transfection. Third, the APCs at the site of administration may be at an inappropriate state of maturity for effective transfection with a TAA transgene. As a result, the TAA may not be presented to the immune system in the manner that produced effective tumor growth inhibition. DISCLOSURE OF THE INVENTION The present invention provides compositions and methods for enhancing in vivo site-specific delivery of tumor associated antigens. Thus, in one aspect, this invention provides a method of recruiting antigen presenting cells to a predetermined site in a subject by administering to the subject an effective amount of an antigen presenting cell (APC) recruitment or proliferation factor to the predetermined site. The APC recruitment or proliferation factor may be a proinflammatory agent, a chemotactic agent, a growth factor or a mitogenic factor, and may be administered as a protein, a peptide or in a gene delivery vehicle. In a preferred embodiment, the APC recruitment or proliferation factor is granulocyte-macrophage colony-stimulating factor (GM-CSF), Sepragel, IL4 or macrophage inflammatory protein 3α. In another embodiment, a growth factor, a cytokine, a co-stimulatory molecule and/or a mitogenic factor can also be administered with the APC recruitment or proliferation factor. In another aspect, the present invention provides a method to enhance the presentation of an antigen into antigen presenting cells (APCs) in vivo, by priming a predetermined site in a subject with an effective amount of an APC recruitment or proliferation factor, and administering an effective amount of the antigen to the site. The antigen is administered as a protein, a recombinant protein, or a peptide. Alternatively, the antigen can be administered in the form of an antigne-encoding gene in a gene delivery vehicle. As such, the invention also encompasses a method to augment transduction of a transgene encoding an antigen into APCs in vivo, by priming a predetermined site in a subject with an effective amount of an APC recruitment or proliferation factor prior to the administration of an effective amount the transgene to the site. In one illustrative aspect, the antigen is a tumor- associated antigen (TAA). In one embodiment, the APC recruitment or proliferation factor is a proinflammatory agent, a chemotactic agent, a growth factor and/or a mitogenic factor. In a preferred embodiment, the APC recruitment or proliferation factor is selected from the group consisting of granulocyte- macrophage colony stimulating factor (GM-CSF), Sepragel, interleukin 4 (IL4) and macrophage inflammatory protein 3 alpha (MIP-3α). In another embodiment, a growth factor, a cytokine, a co-stimulatory molecule and/or a mitogenic factor can be administered with the antigen including TAA. MODES FOR CARRYING OUT THE INVENTION Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains. General Techniques The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual," second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M.J. Gait, ed., 1984); "Animal Cell Culture" (R.I. Freshney, ed., 1987); the series "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (D.M. Weir & CC. Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (J.M. Miller & M.P. Calos, eds., 1987); "Current Protocols in Molecular Biology" (F.M. Ausubel et al., eds., 1987, and periodic updates); "PCR: The Polymerase Chain Reaction," (Mullis et al., eds., 1994); "Current Protocols in Immunology" (J.E. Coligan et al., eds., 1991). Definitions As used herein, certain terms may have the following defined meanings. As used in the specification and claims, the singular form "a," "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof. The terms "cancer," "neoplasm," and "tumor," 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, as used herein, includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from 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 such procedures as CAT scan, magnetic resonance imaging (MRI), X-ray, ultrasound or palpation. Biochemical or immunologic findings alone may be insufficient to meet this definition. Tumor cells often express antigens which are tumor specific. The term "tumor associated antigen" or "TAA" refers to an antigen that is associated with or specific to a tumor. Localized increases in dendritic cell populations can be achieved by inducing either migration or proliferation of these cells. Recombinant adenoviral vectors expressing the melanoma TAAs such as gplOO (Ad2/hugpl00 v2), MARTI (Ad2/MART1), TRPl (Ad2/TRP1) and TRP2 (Ad2/TRP2) were made at Genzyme Corporation. The cationic lipids, GL67 and GL 89) and DNA vaccines expressing gplOO (pCFlhugplOO), TRPl (pCFlTRPl), and TRP2 (pCFlTRP2) were made at Genzyme Corporation. The term "immune effector cells" refers to cells that specifically recognize an antigen present, for example on a neoplastic or tumor cell. For the purposes of this invention, immune effector cells include, but are not limited to, B cells, monocytes, macrophages, NK cells and T cells such as cytotoxic T lymphocytes (CTLs), for example CTL lines, CTL clones, and CTLs from tumor, inflammatory, or other infiltrates. "T-lymphocytes" denotes lymphocytes that are phenotypically CD3+, typically detected using an anti-CD3 monoclonal antibody in combination with a suitable labeling technique. The T-lymphocytes of this invention are also generally positive for CD4, CD8, or both. The term "naϊve" immune effector cells refers to immune effector cells that have not encountered antigen and is intended to by synonymous with unprimed and virgin. "Educated" refers to immune effector cells that have interacted with an antigen such that they differentiate into an antigen-specific cell. An "effective amount" is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. The proteins, peptides and polynucleotides of the present invention may be administered or applied transdermally, orally, subcutaneously, intramuscularly, intravenously, intradermally or parenterally. For purposes of this invention, an effective amount of the protein, peptide or polynucleotide is that amount which provokes an antigen-specific immune response in the subject. The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably to refer to polymeric forms of nucleotides of any length. The polynucleotides may contain deoxyribonucleotides, ribonucleotides, and/or their analogs. Nucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The term "polynucleotide" includes single-, double-stranded and triple helical molecules. "Oligonucleotide" refers to polynucleotides of between about 5 and about 100 nucleotides of single- or double-stranded DNA. Oligonucleotides are also known as oligomers or oligos and may be isolated from genes, or chemically synthesized by methods known in the art. A "primer" refers to an oligonucleotide, usually single-stranded, that provides a 3'-hydroxyl end for the initiation of enzyme-mediated nucleic acid synthesis. The following are non-limiting embodiments of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A nucleic acid molecule may also comprise modified nucleic acid molecules, such as methylated nucleic acid molecules and nucleic acid molecule analogs. Analogs of purines and pyrimidines are known in the art, and include, but are not limited to, aziridinycytosine, 4-acetylcytosine, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl- aminomethyluracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1- methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, pseudouracil, 5-pentylnyluracil and 2,6-diaminopurine. The use of uracil as a substitute for thymine in a deoxyribonucleic acid is also considered an analogous form of pyrimidine. By way of example only and not to limit this invention, the polynucleotides encode a peptide, a ribozyme or an antisense sequence. The terms "protein," "oligopeptide," "polypeptide" and "peptide" are used interchangeably to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modification known in the art. A "subject" is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. The term "predetermined site" refers to the region of the subject into which it is intended that the methods described herein will be practices. Preferably, the predetermined site is a region that is naturally rich in antigen presenting cells such as skin. By "priming" is meant any treatment or preparation causing a desired result. For purposes of the this invention, priming a site means preparing that site for administration of transgene by first recruiting antigen presenting cells to that site. The terms "antigen presentation cells" or "APCs" includes both intact, whole cells as well as other molecules which are capable of inducing the presentation of one or more antigens, preferably with class I MHC molecules. Examples of suitable APCs are discussed in detail below and include, but are not limited to, whole cells such as macrophages, dendritic cells and B cells. Dendritic cells (DCs) are potent antigen-presenting cells (APCs). It has been shown that DCs provide all the signals required for T cell activation and proliferation. These signals can be categorized into two types. The first type, which gives specificity to the immune response, is mediated through interaction between the T-cell receptor/CD3 ("TCR/CD3") complex and an antigenic peptide presented by a major histocompatibility complex ("MHC") class I or II protein on the surface of APCs. This interaction is necessary, but not sufficient, for T cell activation to occur. In fact, without the second type of signal, the first type of signals can result in T cell anergy. The second type of signal, called a costimulatory signal, is neither antigen-specific nor MHC-restricted, and can lead to a full proliferation response of T cells and induction of T cell effector functions in the presence of the first type of signals. DCs are minor constituents of various immune organs such as spleen, thymus, lymph node, epidermis, and peripheral blood. For instance, DCs represent merely about 1% of crude spleen (Steinman et al. (1979) J. Exp. Med. 149:1) or epidermal cell suspensions (Schuler et al. (1985) J. Exp. Med. 161:526; and Romani et al. (1989) J. Invest. Dermatol. 93:600), and 0.1-1% of mononuclear cells in peripheral blood (Freudenthal et al. (1990) Proc. Natl. Acad. Sci. USA 87:7698). Methods for generating dendritic cells from peripheral blood or bone marrow progenitors have been described (Inaba et al. (1992) J. Exp. Med. 175:1157; Inaba et al. (1992) J. Exp. Med. 176:1693-1702; Romani et al. (1994) J. Exp. Med. 180:83-93; Sallusto et al. (1994) J. Exp. Med. 179:1109-1118; Bender et al. (1996) J. Imm. Methods 196:121-135; and Romani et al. (1996; J. Imm. Methods 196:137-151). "Co-stimulatory molecules" are molecules involved in the interaction between receptor-ligand pairs expressed on the surface of antigen presenting cells and T cells. "Co-stimulatory activity" was originally defined as an activity provided by bone-marrow-derived accessory cells such as macrophages and dendritic cells, the so called "professional" APCs. Several molecules have been shown to enhance co-stimulatory activity. These are heat stable antigen (HSA) (Liu Y. et al. (1992) J. Exp. Med. 175:437), chondroitin sulfate-modified MHC invariant chain (Ii-CS) (Naujokas M.F. et al. (1993) Cell 74:257), intracellular adhesion molecule 1 (ICAM-1) (Van Seventer G.A. (1990) J. Immunol. 144:4579), B7-1 and B7-2/B70 (Schwartz R.H. (1992) Cell 71:1065) and B7's counter-receptor CD28 or CTLA-4 on T cells (Freeman et al. (1993) Science 262:909; Young et al. (1992) J. Clin. Invest 90: 229; and Nabavi et al. (1992) Nature 360:266). Other important co-stimulatory molecules are CD40, CD54, CD80, CD86. As used herein, the term "co-stimulatory molecule" encompasses any single molecule or combination of molecules which, when acting together with a peptide/MHC complex bound by a TCR on the surface of a T cell, provides a co-stimulatory effect which achieves activation of the T cell that binds the peptide. The term thus encompasses B7, or other co-stimulatory molecule(s) on an antigen-presenting matrix such as an APC, fragments thereof (alone, complexed with another molecule(s), or as part of a fusion protein) which, together with peptide/MHC complex, binds to a cognate ligand and results in activation of the T cell when the TCR on the surface of the T cell specifically binds the peptide. Co-stimulatory molecules are commercially available from a variety of sources, including, for example, Beckman Coulter. It is intended, although not always explicitly stated, that molecules having similar biological activity as wild-type or purified co-stimulatory molecules (e.g., recombinantly produced or muteins thereof) are intended to be used within the spirit and scope of the invention. The terms "antigen presenting cell recruitment or proliferation factors" or "APC recruitment or proliferation factors" includes both intact, whole cells as well as other molecules which are capable of recruiting antigen presenting cells. APC recruitment or proliferation factors include "proinflammatory agents," "chemotactic agents," "growth factors" and "mitogenic agents." Numerous mediators of dendritic cell migration and proliferation have been described in both in vitro and in vivo models. Examples of suitable APC factors include molecules such as interleukin 4 (IL4), granulocyte macrophage colony stimulating factor (GM-CSF), Sepragel and macrophage inflammatory protein 3 alpha (MIP3α). Schering-Plough, Genzyme, Immunex. Other APC recruitment or proliferation factors include cytokines such as IL-2, stem cell factor (SCF), IL-3, IL-6, IL-12, G-CSF, GM-CSF, IL-lα, IL-11, MlP-lα, LIF, c-kit ligand, TPO, and flt3 ligand. Cytokines are commercially available from several vendors such as, for example, Genentech (South San Francisco, CA), Amgen (Thousand Oaks, CA) and Immunex (Seattle, WA). It is intended, although not always explicitly stated, that 10 molecules having similar biological activity as wild-type or purified APC recruitment or proliferation factors (e.g., recombinantly produced) are intended to be used within the spirit and scope of the invention. In vitro, dendritic cell chemotaxis is induced by macrophage-derived chemokine (MDC) (Godiska et al. (1997) J. Exp. Med. 185:1595:1604), formyl peptides, C5a, and chemokines such as monocyte chemotactic protein (MCP)-3 and RANTES (Morelli et al. (1996) Immunology 89:126-134; Sozzani et al. (1995) J. Immunol. 155:3292-3295). However, human dendritic cell chemotaxis is not induced by IL-8, IL-10, MCP-1, and MCP-2 in vitro (Sozzani et al. (1995) supra). The proliferation and maturation of cultured dendritic cells is promoted by granulocyte macrophage-colony stimulating factor (GM-CSF) GM-CSF (Caux et al. (1992) Nature 360:258-261; Witmer-Pack et al. (1987) J. Exp. Med. 166:1484-1498). In vivo, the number of Langerhans cells, a type of dendritic cell, has been increased in response to GM-CSF in the skin (Kaplan et al. (1992) J. Exp. Med. 175:1717); O'Sullivan et al. (1997) Exp. Dermatol. 6:236-242) and in the lungs (Tazi et al. (1993) J. Clin. Invest. 91:566-576). In addition, Langerhans cell recruitment or proliferation to the skin has been induced using splenopentin (a pentapeptide hormone) (Gruner et al. (1990) Arch. Dermatol. Res. 281:526-529). Recombinant murine GM-CSF (mGM-CSF) (Genzyme Corporation, Cambridge, MA) is a glycosylated polypeptide (23-29kD) (Burgess et al. (1977) J. Biol. Chem. 252:1998-2003) of 142 amino acids (Gough et al. (1985) Nature 309:763-767). Human GM-CSF (hGM-CSF) is a glycoprotein of 22kD (Gasson et al. (1984) Science 226:1339-1342) of 144 amino acids (Wong et al. (1985) Cancer Cells 3:235-242). The homology between murine and human GM-CSF is 60% and there is no species cross reactivity. Recombinant prolactin (PRL) (Genzyme Corporation, Cambridge, MA) is a protein with a molecular weight of about 25 kD consisting of 205 amino acids. Sepragel (Genzyme Corporation, Cambridge, MA) is a cross-linked hyaluronic acid gel. Recombinant adenoviral vectors expressing mGM-CSF (Ad2/cmvGMCSFF9ix) and human prolactin (Ad2/EV/PRL) were made at Genzyme (Framingham, MA). 11 A "gene delivery vehicle" is defined as any molecule that can carry inserted polynucleotides into a host cell. A "transgene" is the term given to the polynucleotide carried by the gene delivery vehicle. The term "transduction" refers to the transfer of polynucleotides into a host cell. Examples of gene delivery vehicles are liposomes, viruses, such as baculovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression. A "viral vector" is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro. Examples of viral vectors include retroviral vectors, adeno virus vectors, adeno-associated virus vectors and the like. In aspects where gene transfer is mediated by a retroviral vector, a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof, and a therapeutic gene. As used herein, "retroviral mediated gene transfer" or "retroviral transduction" carries the same meaning and refers to the process by which a gene or nucleic acid sequences are stably transferred into the host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome. The virus can enter the host cell via its normal mechanism of infection or be modified such that it binds to a different host cell surface receptor or ligand to enter the cell. Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus. In aspects where gene transfer is mediated by a DNA viral vector, such as a adenovirus (Ad) or adeno-associated virus (AAV), a vector construct refers to the polynucleotide comprising the viral genome or part thereof, and a therapeutic gene. Adenoviruses (Ads) are a relatively well characterized, homogenous group of viruses, including over 50 serotypes. (see, e.g., WO 95/27071) Ads are easy to 12 grow and do not require integration into the host cell genome. Recombinant Ad- derived vectors, particularly those that reduce the potential for recombination and generation of wild-type virus, have also been constructed, (see, e.g. WO 95/00655; WO 95/11984). Wild-type AAV has high infectivity and specificity integrating into the host cells genome. (Hermonat and Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470; Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996). Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, WI). In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5' and/or 3' untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation. Alternatively, consensus ribosome binding sites can be inserted immediately 5 ' of the start codon to enhance expression. Among these are several non-viral vectors, including DNA/liposome complexes, and targeted viral protein DNA complexes. To enhance delivery to a cell, the nucleic acid or proteins of this invention can be conjugated to antibodies or binding fragments thereof which bind cell surface antigens, e.g., TCR, CD3 or CD4. Liposomes that also comprise a targeting antibody or fragment thereof can be used in the methods of this invention. This invention also provides the targeting complexes for use in the methods disclosed herein. Polynucleotides are inserted into vector genomes using methods well known in the art. For example, insert and vector DNA can be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined together with a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the termini of 13 restricted polynucleotide. These synthetic linkers contain nucleic acid sequences that correspond to a particular restriction site in the vector DNA. Additionally, an oligonucleotide containing a termination codon and an appropriate restriction site can be ligated for insertion into a vector containing, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColEl for proper episomal replication; versatile multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Other means are well known and available in the art. As used herein, "expression" refers to the process by which polynucleotides are transcribed into mRNA and translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA, if an appropriate eukaryotic host is selected. Regulatory elements required for expression include promoter sequences to bind RNA polymerase and transcription initiation sequences for ribosome binding. For example, a bacterial expression vector includes a promoter such as the lac promoter and for transcription initiation the Shine-Dalgarno sequence and the start codon AUG (Sambrook et al. (1989) supra ). Similarly, an eukaryotic expression vector includes a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome. Such vectors can be obtained commercially or assembled by the sequences described in methods well known in the art, for example, the methods described below for constructing vectors in general. "Host cell" is intended to include any individual cell or cell culture which can be or have been recipients for vectors or the incorporation of exogenous nucleic acid molecules, polynucleotides and/or proteins. It also is intended to 14 include progeny of a single cell, and the progeny may not necessarily be completely identical (in morphology or in genomic or total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. The cells may be prokaryotic or eukaryotic, and include but are not limited to bacterial cells, yeast cells, animal cells, and mammalian cells, e.g., murine, rat, simian or human. An "antibody" is an immunoglobulin molecule capable of binding an antigen. As used herein, the term encompasses not only intact immunoglobulin molecules, but also anti-idiotypic antibodies, mutants, fragments, fusion proteins, humanized proteins and modifications of the immunoglobulin molecule that comprise an antigen recognition site of the required specificity. A "composition" is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant. A "pharmaceutical composition" is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo. As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin, REMINGTON'S PHARM. SCI., 15th Ed. (Mack Publ. Co., Easton (1975)). As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but not excluding others. "Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, 15 preservatives, and the like. "Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention. This invention provides a method to enhance the recruitment or increase the number of APCs to a predetermined site or location in a subject by administering to the subject an effective amount of an APC recruitment or proliferation factor and under conditions which favor the recruitment or increase in the number of APC to the site of administration. In one aspect, the factor is formulated for slow release (i.e., in a sponge or liposome) so as to create a sustained localized concentration of the APC recruitment or proliferation factor. The recruitment or proliferation factor is, in some embodiments, GM-CSF or Supragel. The recruitment or proliferation factor also includes, but is not limited to, biological equivalents of these protein such as amino acid sequences having conservative amino acid substitutions and fusion proteins. This may include yet unidentified proteins which may be assayed in the animal model presented below. The biological activity of an equivalent protein can be assayed by using the animal model detailed below. The recruitment or proliferation factor of this invention also can be administered as a polynucleotide or gene coding for the factor. These polynucleotides can be delivered using conventional gene therapy delivery vehicles, vectors and methods as provided below. Either of these means of delivering the recruitment or proliferation factor of this invention can be further modified by the co-administration of a growth factor, a cytokine, a co-stimulatory molecule or a mitogenic molecule, prior to, subsequently to or concurrently with the recruitment or proliferation factor. Alternatively, a host cell can be transduced with gene(s) encoding these molecules which is then administered to the subject. In one aspect of the invention, the host cell containing the gene(s) coding for the ctyokine and/or co-stimulatory molecule is a professional antigen- 16 presenting cell such as a dendritic cell which includes, but is not limited to, a pulsed dendritic cell, a dendritic cell hybrid or an antigen-presenting foster cell. Presentation of the antigens such as tumor associated antigens by the APCs elicits a strong immune response resulting in destruction of tumor cells by antigen-specific immune effector cells such as cytotoxic T lymphocyte cells (CTLs). Thus, in one aspect, this is achieved by recruiting antigen presenting cells to a predetermined site in a subject, by administering an effective amount of an APC recruitment or proliferation factor. The induction of the CTL response is one method to assay for a positive response to the therapy and a means to confirm the biological activity of new factors useful in the methods of this invention. The presence of a large number of T-cells in a tumor has been correlated with a prognostically favorable outcome in some cases (Whiteside and Parmiani (1994) Cancer Immunol. Immunother. 39:15-21). It has been shown that implantation of polyurethane sponges containing irradiated tumor cells can efficiently trap anti- tumor CTLs (4-times greater than lymph fluid, 50-times greater than spleen or peripheral blood). Woolley et al. (1995) Immmunology 84:55-63. Following activation with T-cell cytokines in the presence of their appropriately presented recognition antigen, TILs proliferate in culture and acquire potent anti-tumor cytolytic properties. Weidmann et al. (1994) Cancer Immunol. Immunother. 39:1- 14. Assays to determine T cell response are well known in the art and any method that will compare T cell number prior to, and subsequent to therapy can be utilized. In addition, the induction of co-stimulatory molecules by the polynucleotide could also stimulate anergic or low affinity self-reactive CTL clones. Methods to assay for CTL clones include: standard 51 Cr release assay as described in Kawakami et al. (1988) J. Exp. Med. 168:2183-91. Briefly, cytotoxic T cells are added to target cells previously loaded with 51 Cr and one measures the release of 51 Cr from the lysed target cells. Cytokine release assay can be used as described in Kawakami et al. (1994) Proc. Natl. Acad. Sci. USA 91:3515-19. Briefly, cytotoxic T cells are added to target cells and one measures the amount of IFNγ released by ELIS A. To measure the relative proportion of immune effector 17 cells within a mixed population that recognize a particular target, the Enzyme- Linked immunoSPOT (ELISPOT) assay is employed as described in Czerkinsky et al. (1988) J. Immunol. Methods 110:29-36. Briefly, 96 well nitrocellulose- bottomed plates are coated with an anti-cytokine antibody, generally anti- interferon-γ. Target cells and immune effector cells such as cytotoxic T cells (CTLs) are added to wells. Cytokine released from the CTLs is captured by the anti-interferon-γ antibody and quantitated using a standard ELISA format. In another aspect, the present invention provides a method to enhance presentation of an antigen into antigen presenting cells (APCs) in vivo, by priming a predetermined site in a subject with an effective amount of an APC recruitment or proliferation factor, and administering an effective amount of the antigen to the site. The antigen is administered as a protein, a recombinant protein, or a peptide. This invention f rther provides a method to augment transduction of transgenes into APCs in vivo, by priming a predetermined site in a subject with an effective amount of an APC recruitment or proliferation factor and administering an effective amount of the transgene, which may be a tumor associated antigen. The antigen of this vaccine may be an altered antigen or heterologous (i.e., allogeneic or a homolog from a isolated species, e.g., a murine antigen administered to a human patient). It may be a previously characterized tumor-associated antigens such as gpl 00 (Kawakami et al. (1997) Intern. Rev. Immunol. 14: 173- 192); MUC-1 (Henderson et al. (1996) Cancer Res. 56:3763-3770); MART-1 (Kawakami et al. (1994) Proc. Natl. Acad. Sci. USA 91:3515-3519; Kawakami et al. (1997) Intern. Rev. Immunol. 14:173-192; and Ribas et al. (1997) Cancer Res. 57:2865-2869); HER-2/neu (U.S. Patent No. 5,550,214), MAGE (PCT/US92/04354); HPV 16, 18E6 and E7 (Ressing et al. ( 1996) Cancer Res. 56(l):582-588; Rstifo (1996) Curr. Op. Immunol. 8:658-663; Stern (1996) Adv. Cancer Res. 69:175-211; Tindle et al. (1995) Clin. Exp. Immunol. 101:265-271; and van Driel et al. (1996) Annals of Medicine 28:471-477); CEA (U.S. Patent No. 5,274,087); PSA (Lundwall A. (1989) Biochem. Biophys. Research Comm. 161:1151-59); prostate membrane specific antigen (PSMA) (Israeli et al. ( 1993) 18 Cancer Research 53:227-30); tyrosinase (U.S. Patent Nos. 5,530,096 and 4,898,814; Brichard et al. (1993) J. Exp. Med. 178:489-49); tyrosinase related proteins 1 or 2 (TRP-1 and TRP-2); NY-ESO-1 (Chen et al. (1997) Proc. Natl. Acad. Sci. USA 94:1914-18); or the GA733 antigen (U.S. Patent No. 5,185,254). The coding and amino acid sequences of these antigens are available in the art. For example, human and murine MUC1 coding sequences are provided under Genbank Accession No. M35093 and M64928. Biologically equivalent proteins of known antigens also are useful in the methods described herein. These proteins are encoded by polynucleotides that hybridize under stringent conditions to the sequences disclosed in the references described above or known in the art. Alternatively, the proteins are encoded by polynucleotides that are at least 80%, or more preferably, at least 90% or most preferably, at least 95%, identical to the disclosed sequences using as determined using sequence alignment programs and default parameters. "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme. Examples of stringent hybridization conditions include: incubation temperatures of about 25°C to about 37°C; hybridization buffer concentrations of about 6 X SSC to about 10 X SSC; formamide concentrations of about 0% to about 25%; and wash solutions of about 6 X SSC. Examples of moderate hybridization conditions include: incubation temperatures of about 40°C to about 50°C; buffer concentrations of about 9 X SSC to about 2 X SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5 X SSC 19 to about 2 X SSC. Examples of high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about 1 X SSC to about 0.1 X SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about 1 X SSC, 0.1 X SSC, or deionized water. In general, hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 80%, 85%, 90%, or 95%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F.M. Ausubel et al., eds., 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters. In particular, preferred programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non- redundant, GenBank + EMBL + DDB J + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following Internet address: http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST. It also may be a yet undetermined antigen identified by the methods described herein or well known to those of skill in the art. The antigen can be administered as a protein, a recombinant protein, a peptide, or, alternatively, a polynucleotide in a gene delivery vehicle. Furthermore, the invention provides a method for cloning the cDNA and genomic DNA encoding novel recruitment or proliferation factors identified by 20 this invention, by generating degenerate oligonucleotides probes or primers based on the sequence of the epitope. Compositions comprising the nucleic acid and a carrier, such as a pharmaceutically acceptable carrier, a solid support or a detectable label, are further provided by this method as well as methods for detecting the sequences in a sample using methods such as Northern analysis, Southern analysis and PCR. Further provided by this invention are therapeutic and diagnostic oligopeptide sequences determined according to the foregoing methods. Compositions comprising the oligopeptide sequence and a carrier, such as a pharmaceutically acceptable carrier, a solid support or a detectable label, are further provided by this method as well as methods for detecting the oligopeptide sequence in a sample using methods such as Western analysis and ELISA. Harlow and Lane (1988), supra. Materials and Methods Identification of Tumor Associated Antigens Any conventional method, e.g., subtractive library, comparative Northern and/or Western blot analysis of normal and tumor cells, Serial Analysis of Gene Expression (U.S. Patent No. 5,695,937) and Solid PHase Epitope REcovery ("SPHERE," described in PCT WO 97/35035), can be used to identify putative antigens for use in the subject invention. Expression cloning also can be used. This methodology, as described in Kawakami et al. (1994) Proc. Natl. Acad. Sci. USA 91:3515-19, can be used to identify a novel tumor-associated antigen. Briefly, a library of cDNAs corresponding to mRNAs derived from tumor cells is cloned into an expression vector and introduced into target cells which are subsequently incubated with cytotoxic T cells. One identifies pools of cDNAs that are able to stimulate the CTL and through a process of sequential dilution and re-testing of less complex pools of cDNAs one is able to derive unique cDNA sequences that are able to stimulate the CTL and thus encode the cognate tumor antigen. 21 SAGE analysis involves identifying nucleotide sequences expressed in the antigen-expressing cells. Briefly, SAGE analysis begins with providing complementary deoxyribonucleic acid (cDNA) from (1) the antigen-expressing population and (2) cells not expressing that antigen. Both cDNAs can be linked to primer sites. Sequence tags are then created, for example, using the appropriate primers to amplify the DNA. By measuring the differences in these tags between the two cell types, sequences which are aberrantly expressed in the antigen- expressing cell population can be identified. Alternatively, muteins of the antigen as well as allogeneic and antigens from a different species, of previously characterized antigens are useful in the subject invention. MARTI and gplOO are melanocyte differentiation antigens specifically recognized by HLA-A2 restricted tumor-infiltrating lymphocytes (TILs) derived from patients with melanoma, and appear to be involved in tumor regression (Kawakami, Y. et al. (1994) Proc. Natl. Acad. Sci. USA 91:6458-62; Kawakami, Y. et al. (1994) Proc. Natl. Acad. Sci. USA 91:91:3515-9). Recently, the mouse homolog of human MART-1 has been isolated. The full-length open reading frame of the mouse MARTI consists of 342 bp, encoding a protein of 113 amino acid residues with a predicted molecular weight of ~13 kDa. Alignment of human and murine MARTI amino acid sequences showed 68.6% identity. The murine homologue of gplOO has also been identified. The open reading frame consists of 1,878 bp, predicting a protein of 626 amino acid residues which exhibits 75.5% identity to human gplOO. SPHERE, described in PCT WO 97/35035, is a method that will identify wild-type or native antigens as well as provide altered antigens. SPHERE is an empirical screening method for the identification of MHC Class I-restricted CTL epitopes that utilizes peptide libraries synthesized on a solid support (e.g., plastic beads) where each bead contains approximately 200 picomoles of a unique peptide that can be released in a controlled manner. The synthetic peptide library is tailored to a particular HLA restriction by fixing anchor residues that confer high-affinity binding to a particular HLA allele (e.g. , HLA-A2) but contain a 22 variable TCR epitope repertoire by randomizing the remaining positions. Roughly speaking, 50 96-well plates with 10,000 beads per well will accommodate a library with a complexity of approximately 5 X 10 7 . In order to minimize both the number of CTL cells required per screen and the amount of manual manipulations, the eluted peptides can be further pooled to yield wells with any desired complexity. Based on experiments with soluble libraries, it should be possible to screen 10 7 peptides in 96-well plates (10,000 peptides per well) with as few as 2 X 10 6 CTL cells. After cleaving a percentage of the peptides from the beads and incubating them with 51 Cr-labeled APCs (e.g., T2 cells) and the CTL line(s), peptide pools containing reactive species can be determined by measuring 51 Cr-release according to standard methods known in the art. Alternatively, cytokine production (e.g., interferon-γ) or proliferation (e.g., incorporation of 3 H-thymidine) assays may be used. After identifying reactive 10,000-peptide mixtures, the beads corresponding to those mixtures are separated into smaller pools and distributed to new 96-well plates (e.g. , 100 beads per well). An additional percentage of peptide is released from each pool and reassayed for activity by one of the methods listed above. Upon identification of reactive 100- peptide pools, the beads corresponding those peptide mixtures are redistributed at 1 bead per well of a new 96-well plate. Once again, an additional percentage of peptide is released and assayed for reactivity in order to isolate the single beads containing the reactive library peptides. The sequence of the peptides on individual beads can be determined by sequencing residual peptide bound to the beads by, for example, N-terminal Edman degradation or other analytical techniques known to those of skill in the art. In vitro confirmation of the immunogenicity of a putative antigen of this invention can be confirmed using the method described below, which assays for the production of CTLs. Isolation, Culturing and Expansion of APCs, Including Dendritic Cells Bender et al. (1996) J. Immunol. Meth. 196:121-135 describes a method for generating sizable numbers of mature dendritic cells from nonproliferating 23 progenitors in human blood. The procedure requires 1% human plasma in the place of 10% fetal calf serum and involves two steps. The first step or "priming" phase is a 6-7 day culture of T cell depleted mononuclear cells in medium supplemented with GM-CSF and IL-4. The second step or "differentiation" phase requires the exposure to macrophage conditioned medium. Romani et al. (1996) J. Immunol. Methods 196:137-151 describes a method to generate human dendritic cells from hematopoietic precursor cells in peripheral blood. A 3 day maturation culture is added to the initial 6-7 day culture in the presence of GM-CSF and IL-4. Human plasma, rather than fetal calf serum and media approved for clinical use are optimal, additional conditions for use in this method. The following describe additional methods. In one aspect, a large numbers of precommitted APCs already circulating in the blood are isolated. Previous techniques for isolating committed APCs from human peripheral blood have involved combinations of physical procedures such as metrizamide gradients and adherence/nonadherence steps (Freudenthal et al. (1990) PNAS USA 87:7698- 7702); Percoll gradient separations (Mehta-Damani et al. (1994) J. Immunol. 153:996-1003); and fluorescence activated cell sorting techniques (Thomas R. et al. (1993) J. Immunol. 151:6840-52). The APC can be precommitted or mature dendritic cells, which can be isolated from the white blood cell fraction of a mammal, such as a murine, simian or a human (See, e.g., WO 96/23060). The white blood cell fraction can be from the peripheral blood of the mammal. This method includes the following steps: (a) providing a white blood cell fraction obtained from a mammalian source by methods known in the art such as leukophoresis; (b) separating the white blood cell fraction of step (a) into four or more subtractions by countercurrent centrifugal elutriation; (c) stimulating conversion of monocytes in one or more fractions from step (b) to dendritic cells by contacting the cells with calcium ionophore; (d) identifying the dendritic cell-enriched fraction from step (c); and (e) collecting the enriched fraction of step (d), preferably at about 4°C. One way 24 to identify the dendritic cell-enriched fraction is by fluorescence-activated cell sorting (FACS). The white blood cell fraction can be treated with calcium ionophore in the presence of other cytokines, such as rhIL-12, rhGM-CSF, or rhIL-4. The cells of the white blood cell fraction can be washed in buffer and suspended in Ca ^ /Mg^ free media prior to the separating step. The white blood cell fraction can be obtained by leukapheresis. The dendritic cells can be identified by the presence of at least one of the following markers: HLA-DR, HLA-DQ, or B7.2, and the simultaneous absence of the following markers: CD3, CD 14, CD 16, 56, 57, and CD 19, 20. Monoclonal antibodies specific to these cell surface markers are commercially available. More specifically, the method requires collecting an enriched collection of white cells and platelets from leukapheresis that is then further fractionated by countercurrent centrifugal elutriation (CCE) (Abrahamsen, T.G. et al. (1991) J. Clin. Apheresis. 6:48-53). Cell samples are placed in a special elutriation rotor. The rotor is then spun at a constant speed of, for example, 3000 rpm. Once the rotor has reached the desired speed, pressurized air is used to control the flow rate of cells. Cells in the elutriator are subjected to simultaneous centrifugation and a washout stream of buffer that is constantly increasing in flow rate. This results in fractional cell separations based largely but not exclusively on differences in cell size. Quality control of APC and more specifically DC collection and confirmation of their successful activation in culture is dependent upon a simultaneous multi-color FACS analysis technique which monitors both monocytes and the dendritic cell subpopulation as well as possible contaminant T lymphocytes. It is based upon the fact that DCs do not express the following markers: CD3 (T cell); CD 14 (monocyte); CD 16, 56, 57 (NK LAK cells); CD 19, 20 (B cells). At the same time, DCs do express large quantities of HLA-DR, significant HLA-DQ and B7.2 (but little or no B7.1) at the time they are circulating in the blood (in addition they express Leu M7 and M9, myeloid markers which are also expressed by monocytes and neutrophils). 25 When combined with a third color reagent for analysis of dead cells, propridium iodide (PI), it is possible to make positive identification of all cell subpopulations (see Table 1): TABLE 1 FACS analysis of fresh peripheral cell subpopulations Color #1 Color #2 Color #3 Cocktail HLA-DR PI 3/14/16/19/20/56/57 Live Dendritic cells Negative Positive Negative Live Monocytes Positive Positive Negative Live Neutrophils Negative Negative Negative Dead Cells Variable Variable Positive Additional markers can be substituted for additional analysis: Color #1 : CD3 alone, CD 14 alone, etc.; Leu M7 or Leu M9; anti-Class I, etc. Color #2: HLA-Dq, B7.1, B7.2, CD25 (IL2r), ICAM, LFA-3, etc. The goal of FACS analysis at the time of collection is to confirm that the DCs are enriched in the expected fractions, to monitor neutrophil contamination, and to make sure that appropriate markers are expressed. This rapid bulk collection of enriched DCs from human peripheral blood, suitable for clinical applications, is absolutely dependent on the analytic FACS technique described above for quality control. If need be, mature DCs can be immediately separated from monocytes at this point by fluorescent sorting for "cocktail negative" cells. It may not be necessary to routinely separate DCs from monocytes because, as will be detailed below, the monocytes themselves are still capable of differentiating into DCs or functional DC-like cells in culture. Once collected, the DC rich/monocyte APC fractions (usually 150 through 190) can be pooled and cryopreserved for future use, or immediately placed in short term culture. 26 Alternatively, others have reported that a method for upregulating (activating) dendritic cells and converting monocytes to an activated dendritic cell phenotype. This method involves the addition of calcium ionophore to the culture media convert monocytes into activated dendritic cells. Adding the calcium ionophore A23187, for example, at the beginning of a 24-48 hour culture period resulted in uniform activation and dendritic cell phenotypic conversion of the pooled "monocyte plus DC" fractions: characteristically, the activated population becomes uniformly CD 14 (Leu M3) negative, and upregulates HLA-DR, HLA- DQ, ICAM-1, B7.1, and B7.2. Furthermore this activated bulk population functions as well on a small numbers basis as a further purified. Specific combination(s) of cytokines have been used successfully to amplify (or partially substitute) for the activation/conversion achieved with calcium ionophore: these cytokines include but are not limited to rhGM-CSF, rhIL-2, and rhIL-4. Each cytokine when given alone is inadequate for optimal upregulation. In one embodiment, the APCs and cells expressing one or more antigens are autologous. In another embodiment, the APCs and cells expressing the antigen are allogeneic, i.e., derived from a different subject. Administration of APC recruitment or proliferation Factor(s) In one embodiment, an APC-recruitment or proliferation factor is administered to mice by subcutaneous injection. The APC-recruitment or proliferation factors is administered either as a protein or as a gene therapy vector that is capable of expressing the gene for the APC-enhancing factor. The effective amount can be readily determined, for instance by administering subcutaneous injections at different dosages and evaluating the subsequent inflammatory response at different times after administration. The APC recruitment or proliferation factors useful in the present invention include, but are not limited to, GM-CSF, PRL, Sepragel, Ad2/cmvGMCSFF9ix, and Ad2/EV/PRL. In addition, it may be necessary to 27 complex the protein form of some of the CEFs to a bead for slow-release into the tissue (Meyer et al. (1994) Biochem. Biophys. Res. Commun. 199:433-438; Tanaka et al. (1991) Cancer Res. 51:3710-3714). The slow release of chemotactic factors establishes a concentration gradient that would facilitate dendritic cell migration towards the APC recruitment or proliferation factor. An in vitro assay to determine the recruitment efficacy of known or novel APC recruitment factors is the chemotactic assay for cultured dendritic cells as described by Godiska et al. (1997) J. Exp. Med. 185:1595-1604; Morelli et al. (1996) Immunology 89:126-134; and Sozzani et al. (1995) J. Immunol. 155:3292- 3295. The inflammatory response at the site of APC recruitment or proliferation factor administration can be evaluated by any other known method, for example, in histology sections or in samples taken by needle aspiration. Alternatively, enhanced numbers of dendritic cells, Langerhans cells, and other antigen presenting cells can also be evaluated using either immunohistochemistry or FACS analysis (Kaplan et al. (1992), supra and Tazi et al. (1993) J. Clin. Invest. 91:566-576). Alternative routes of administration for gene therapy vectors expressing APC recruitment or proliferation factors are also encompassed by the present invention, and are discussed in detail below. By way of example only, topical administration of recombinant adeno viral vectors to debrided skin (Tang et al. (1997) Nature 388:729-730) and gene gun delivery of adenoviral or plasmid vectors (Mahvi et al. (1997) Immunol. Cell. Biol. 75:456-460) can be employed. The subsequent inflammatory response will be evaluated and compared with the response from the subcutaneous administration. Administration of Tumor- Associated Antigens (TAA) Tumor-associated antigens (TAA) can be administered as whole molecules or, preferably, using gene delivery vehicles carrying a polynucleotide encoding 28 the TAA. TAAs may also be administered to APC recruitment or proliferation factor-primed subcutaneous sites in the form of peptides or recombinant proteins. Typically, gene delivery vehicles expressing a TAA will be administered after treatment with the APC recruitment or proliferation factor. In one aspect, the TAA is administered in a recombinant adenoviral vector or cationic lipid:DNA vectors by subcutaneous injection. The TAAs include, but are not limited to, Ad2gpl00; GL67:pCFlgpl00; Ad2TRP2; and GL67:pCFlTRP2. The successful transfer of the TAA transgene to APCs, such as dendritic cells or Langerhans cells, at the site of vector administration may be evaluated by any suitable method, for example, using a combination of fluorescence in situ hybridization (FISH) (Gussoni et al. (1996) Nature Biotechnol. 14:1012-1016); fluorescently labeled lipids or plasmid DNA (Bebok et al. (1996) J. Pharmacol. Exp. Therapeutics 279:1462-1469 and Matsui et al. (1997) J. Biol. Chem. 272:1117-1126); and immunofluorescence. Transfection of these cell types will also be analyzed by measuring the induction of a specific CTL response against the TAAs (Chen et al. (1993) J. Immunol. 151:244-255). Optimization of transfection conditions will include changes in dose and time after APC recruitment or proliferation factor administration. Presentation of Antigen to the APC Peptide fragments from antigens must first be bound to peptide binding receptors ((MHC) class I and class II molecules) that display the antigenic peptides on the surface of the APCs. Palmer E. and Cresswell (1998) Annu. Rev. Immunol. 16:323 and Germain R.N. (1996) Immunol. Rev. 151:5. T lymphocytes produce an antigen receptor that they use to monitor the surface of APCs for the presence of foreign peptides. The antigen receptors on T H cells recognize antigenic peptides bound to MHC class II molecules whereas the receptors on CTLs react with antigens displayed on class I molecules. For a general review of the methods for presentation of exogenous antigen by APC, see Raychaudhuri and Rock (1998) Nature Biotechnology 16:1025. 29 For purposes of immunization, antigens can be delivered to antigen- presenting cells as protein/peptide or in the form of polynucleotides encoding the protein/peptide ex vivo or in vivo. The methods described below focus primarily on DCs which are the most potent, preferred APCs. Several different techniques have been described to produce genetically modified APCs. These include: (1) the introduction into the APCs of polynucleotides that express antigen or fragments thereof; (2) infection of APCs with recombinant vectors to induce endogenous expression of antigen; and (3) introduction of tumor antigen into the DC cytosol using liposomes. (See Boczkowski D. et al. (1996) J. Exp. Med. 184:465; Rouse et al. (1994) J. Virol. 68:5685; and Nair et al. (1992) J. Exp. Med. 175:609). For the purpose of this invention, any method which allows for the introduction and expression of the heterologous, altered or non-self antigen and presentation by the MHC on the surface of the APC is within the scope of this invention. Antigen Pulsing Pulsing is accomplished in vitro/ex vivo by exposing APCs to antigenic protein or peptide(s). The protein or peptide(s) are added to APCs at a concentration of 1-10 μm for approximately 3 hours. Paglia et al. (1996) J. Exp. Med. 183:317, has shown that APC incubated with whole protein in vitro were recognized by MHC class I-restricted CTLs, and that immunization of animals with these APCs led to the development of antigen-specific CTLs in vivo. Protein/peptide antigen can also be delivered to APC in vivo and presented by the APC. Antigen is preferably delivered with adjuvant via the intravenous, subcutaneous, intranasal, intramuscular or intraperitoneal route of delivery. Grant E.P. and Rock K.L. (1992) J. Immunol. 148:13; Norbury, C C et al. (1995) Immunity 3:783; and Reise-Sousa C. and Germain R.N. (1995) J. Exp. Med. 182:841. 30 Antigen Painting Another method which can be used is termed "painting". It has been demonstrated that glycosyl-phosphotidylinositol (GPI)-modified proteins possess the ability to reincorporate themselves back into cell membranes after purification. Hirose et al. (1995) Methods Enzymol. 250:582; Medof et al. (1984) J. Exp. Med. 160:1558; Medof (1996) FASEB J. 10:574; and Huang et al. (1994) Immunity 1:607, have exploited this property in order to create APCs of specific composition for the presentation of antigen to CTLs. Expression vectors for β2- microglobulin and the HLA-A2.1 allele were first devised. The proteins were expressed in Schneider S2 Drosophila melanogaster cells, known to support GPI- modification. After purification, the proteins could be incubated together with a purified antigenic peptide which resulted in a trimolecular complex capable of efficiently inserting itself into the membranes of autologous cells. In essence, these protein mixtures were used to "paint" the APC surface, conferring the ability to stimulate a CTL clone that was specific for the antigenic peptide. Cell coating was shown to occur rapidly and to be protein concentration dependent. This method of generating APCs bypasses the need for gene transfer into the APC and permits control of antigenic peptide densities at the cell surfaces. Foster Antigen Presenting Cells Foster APCs are derived from the human cell line 174xCEM.T2, referred to as T2, which contains a mutation in its antigen processing pathway that restricts the association of endogenous peptides with cell surface MHC class I molecules (Zweerink et al. (1993) J. Immunol. 150:1763). This is due to a large homozygous deletion in the MHC class II region encompassing the genes TAP1 , TAP2, LMP1, and LMP2, which are required for antigen presentation to MHC class 1 -restricted CD8 + CTLs. In effect, only "empty" MHC class I molecules are presented on the surface of these cells. Exogenous peptide added to the culture medium binds to these MHC molecules provided that the peptide contains the allele-specific binding motif. These T2 cells are referred to herein as "foster" 31 APCs. They can be used in conjunction with this invention to present the heterologous, altered or control antigen. Transduction of T2 cells with specific recombinant MHC alleles allows for redirection of the MHC restriction profile. Libraries tailored to the recombinant allele will be preferentially presented by them because the anchor residues will prevent efficient binding to the endogenous allele. High level expression of MHC molecules makes the APC more visible to the CTLs. Expressing the MHC allele of interest in T2 cells using a powerful transcriptional promoter (e.g., the CMV promoter) results in a more reactive APC (most likely due to a higher concentration of reactive MHC-peptide complexes on the cell surface). Preparation of Hybrid Cells Utilizing Dendritic Cells Hybrid cells typically retain the phenotypic characteristics of the APCs. Thus, hybrids made with dendritic cells will express the same MHC class II proteins and other cell surface markers. Moreover, the hybrids will express those antigens expressed on the cells from which they are derived. A population of APCs are collected and isolated. Preferably, the ratio of APCs:antigen-expressing cells is between about 1:100 and about 1000:1. For example, in one aspect, the fraction enriched for antigen-expressing cells is then fused to APCs, preferably dendritic cells. Fusion between the APCs and antigen- expressing cells can be carried out with any suitable method, for example using polyethylene glycol (PEG) or Sendia virus. In a preferred embodiment, the hybrid cells are created using the procedure described by Gong et al.(1997) Nat. Med. 3(5):558-561. Typically, unfused cells will die off after a few days in culture. Therefore, the fused cells can be separated from the parent cells simply by allowing the culture to grow for several days. In this embodiment, the hybrid cells both survive more and, additionally, are only lightly adherent to tissue culture surfaces. The parent cells are strongly adherent to the containers. Therefore, after about 5 32 to 10 days in culture, the hybrid cells can be gently dislodged and transferred to new containers, while the unfused cells remained attached. Alternatively, it has been shown that fused cells lack functional hypoxanthine-guaninephosphoribosyl transferase ("HGPRT") enzyme and are, therefore, resistant to treatment with the compound HAT. Accordingly, to select these cells HAT can be added to the culture media. However, unlike conventional HAT selection, hybrid cell cultures should not be exposed to the compound for more than 12 days. Gene Delivery Vehicles In general, introduction of the transgene or polynucleotide encoding the APC recruitment or proliferation factor or antigen is accomplished in vivo, ex vivo or in vitro by introducing a vector containing a polynucleotide or transgene encoding a heterologous or an altered antigen or APC recruitment or proliferation factor. A variety of different gene transfer vectors, including viral as well as non- viral systems can be used. Viral vectors useful in the genetic modifications of this invention include, but are not limited to adenovirus, adeno-associated virus vectors, retroviral vectors and adeno-retroviral chimeric vectors. Construction of Recombinant Adenoviral Vectors or Adeno- Associated Virus Vectors Adenovirus and adeno-associated virus vectors useful in the genetic modifications of this invention may be produced according to methods already taught in the art. (see, e.g., Karlsson et al. (1986) EMBO 5:2377; Carter (1992) Curr. Op. Biotechnol. 3:533-539; Muzcyzka (1992) Current Top. Microbiol. Immunol. 158:97-129; GENE TARGETING: A PRACTICAL APPROACH (1992) ed. A. L. Joyner, Oxford University Press, NY). Several different approaches are feasible. Preferred is the helper-independent replication deficient human adenovirus system. 33 The recombinant adenoviral vectors based on the human adenovirus 5 (Virology 163:614-617, 1988) are missing essential early genes from the adenoviral genome (usually El A/El B), and are therefore unable to replicate unless grown in permissive cell lines that provide the missing gene products in trans. In place of the missing adenoviral genomic sequences, a transgene of interest can be cloned and expressed in cells infected with the replication deficient adenovirus. Although adeno virus-based gene transfer does not result in integration of the transgene into the host genome (less than 0.1% adeno virus- mediated transfections result in transgene incorporation into host DNA), and therefore is not stable, adenoviral vectors can be propagated in high titer and transfect non-replicating cells. Human 293 cells, which are human embryonic kidney cells transformed with adenovirus El A/El B genes, typify useful permissive cell lines and are commercially available from the ATCC However, other cell lines which allow replication-deficient adenoviral vectors to propagate therein can be used, including HeLa cells. Additional references describing adenovirus vectors and other viral vectors which could be used in the methods of the present invention include the following: Horwitz M.S. Adenoviridae and Their Replication, in Fields B. et al. (eds.) VIROLOGY, Vol. 2, Raven Press New York, pp. 1679-1721, 1990); Graham, F. et al., pp. 109-128 in METHODS IN MOLECULAR BIOLOGY, Vol. 7: GENE TRANSFER AND EXPRESSION PROTOCOLS, Murray, E. (ed.), Humana Press, Clifton, N.J. (1991); Miller, N. et al. (1995) FASEB Journal 9:190-199 Schreier, H. (1994) Pharmaceutica Acta Helvetiae 68:145-159; Schneider and French (1993) Circulation 88:1937-1942; Curiel, D.T. et al. (1992) Human Gene Therapy 3: 147-154; Graham, F.L. et al., WO 95/00655; Falck-Pedersen, E.S. WO 95/16772; Denefle, P. et al. WO 95/23867; Haddada, H. et al. WO 94/26914; Perricaudet, M. et al. WO 95/02697; and Zhang, W. et al. WO 95/25071. A variety of adenovirus plasmids are also available from commercial sources, including, e.g., Microbix Biosystems of Toronto, Ontario, Canada (see, e.g., Microbix Product Information Sheet: Plasmids for Adenovirus Vector Construction, 1996). See also, the papers 34 by Vile et al. (1997) Nature Biotech. 15:840-841; Feng et al. (1997) Nature Biotech. 15:866-870, describing the construction and use of adeno-retroviral chimeric vectors that can be employed for genetic modifications. Additional references describing AAV vectors which could be used in the methods of the present invention include the following: Carter B., HANDBOOK OF PARVOVIRUSES, Vol. I, pp. 169-228, 1990; Berns, VIROLOGY, pp. 1743-1764 (Raven Press 1990); Carter, B. (1992) Curr. Opin. Biotechnol. 3:533-539; MuzyczkaN. (1992) Current Topics in Micro, and Immunol. 158:92-129; Flotte, T.R. et al. (1992) Am. J. Respir. Cell Mol. Biol. 7:349-356; Chatterjee et al. (1995) Ann. NY Acad. Sci. 770:79-90; Flotte, T.R. et al. WO 95/13365; Trempe, J.P. et al. WO 95/13392; Kotin, R. (1994) Human Gene Therapy, 5:793-801; Flotte, T.R. et al. (1995) Gene Therapy 2:357-362; Allen, J.M. WO 96/17947; and Du et al. (1996) Gene Therapy 3:254-261. Construction of Retroviral Vectors Retroviral vectors useful in the methods of this invention are produced recombinantly by procedures already taught in the art. For example, WO 94/29438 describes the construction of retroviral packaging plasmids and packaging cell lines. As is apparent to the skilled artisan, the retroviral vectors useful in the methods of this invention are capable of infecting the cells described herein. The techniques used to construct vectors, and transfix and infect cells are widely practiced in the art. Examples of retroviral vectors are those derived from murine, avian or primate retroviruses. Retroviral vectors based on the Moloney murine leukemia virus (MoMLV) are the most commonly used because of the availability of retroviral variants that efficiently infect human cells. Other suitable vectors include those based on the Gibbon Ape Leukemia Virus (GAL) or HIV. In producing retroviral vector constructs derived from the Moloney murine leukemia virus (MoMLV), in most cases, the viral gag, pol and env sequences are removed from the virus, creating room for insertion of foreign DNA sequences. Genes encoded by the foreign DNA are usually expressed under the control of the 35 strong viral promoter in the LTR. Such a construct can be packed into viral particles efficiently if the gag, pol and env functions are provided in trans by a packaging cell line. Thus, when the vector construct is introduced into the packaging cell, the gag-pol and env proteins produced by the cell, assemble with the vector RNA to produce infectious virions that are secreted into the culture medium. The virus thus produced can infect and integrate into the DNA of the target cell, but does not produce infectious viral particles since it is lacking essential packaging sequences. Most of the packaging cell lines currently in use have been transfected with separate plasmids, each containing one of the necessary coding sequences, so that multiple recombination events are necessary before a replication competent virus can be produced. Alternatively, the packaging cell line harbors an integrated provirus. The provirus has been crippled so that, although it produces all the proteins required to assemble infectious viruses, its own RNA cannot be packaged into virus. Instead, RNA produced from the recombinant virus is packaged. The virus stock released from the packaging cells thus contains only recombinant virus. The range of host cells that may be infected by a retro virus or retroviral vector is determined by the viral envelope protein. The recombinant virus can be used to infect virtually any other cell type recognized by the env protein provided by the packaging cell, resulting in the integration of the viral genome in the transduced cell and the stable production of the foreign gene product. In general, murine ecotropic env of MoMLV allows infection of rodent cells, whereas amphotropic env allows infection of rodent, avian and some primate cells, including human cells. Amphotropic packaging cell lines for use with MoMLV systems are known in the art and commercially available and include, but are not limited to, PA12 and PA317. Miller et al. (1985) Mol. Cell. Biol. 5:431-437; Miller et al. (1986) Mol. Cell. Biol. 6:2895-2902; and Danos et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464. Xenotropic vector systems exist which also allow infection of human cells. 36 The host range of retroviral vectors has been altered by substituting the env protein of the base virus with that of a second virus. The resulting, "pseudotyped" virus has the host range of the virus donating the envelope protein and expressed by the packaging cell line. Recently, the G-glycoprotein from vesicular stomatitis virus (VSV-G) has been substituted for the MoMLV env protein. Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037 and PCT patent application WO 92/14829. Since infection is not dependent on a specific receptor, VSV-G pseudotyped vectors have a broad host range. Usually, the vectors will contain at least two heterologous genes or gene sequences: (i) the therapeutic gene to be transferred; and (ii) a marker gene that enables tracking of infected cells. As used herein, "therapeutic gene" can be an entire gene or only the functionally active fragment of the gene capable of compensating for the deficiency in the patient that arises from the defective endogenous gene. Therapeutic gene also encompasses antisense oligonucleotides or genes useful for antisense suppression and ribozymes for ribozyme-mediated therapy. For example, in the present invention, a therapeutic gene may be one that neutralizes the immunosuppressive factor or counter its effects. Therapeutic genes that encode dominant inhibitory oligonucleotides and peptides as well as genes that encode regulatory proteins and oligonucleotides also are encompassed by this invention. Generally, gene therapy will involve the transfer of a single therapeutic gene although more than one gene may be necessary for the treatment of particular diseases. In one embodiment, the therapeutic gene is a dominant inhibiting mutant of the wild-type immunosuppressive agent. Alternatively, the therapeutic gene could be a wild- type, copy of a defective gene or a functional homologue. More than one gene can be administered per vector or alternatively, more than one gene can be delivered using several compatible vectors. Depending on the genetic defect, the therapeutic gene can include the regulatory and untranslated sequences. For gene therapy in human patients, the therapeutic gene will generally be of human origin although genes from other closely related species 37 that exhibit high homology and biologically identical or equivalent function in humans may be used, if the gene product does not induce an adverse immune reaction in the recipient. The therapeutic gene suitable for use in treatment will vary with the disease. Nucleotide sequences for the therapeutic gene will generally be known in the art or can be obtained from various sequence databases such as GenBank. The therapeutic gene itself will generally be available or can be isolated and cloned using the polymerase chain reaction PCR (Perkin-Elmer) and other standard recombinant techniques. The skilled artisan will readily recognize that any therapeutic gene can be excised as a compatible restriction fragment and placed in a vector in such a manner as to allow proper expression of the therapeutic gene in hematopoietic cells. A marker gene can be included in the vector for the purpose of monitoring successful transduction and for selection of cells into which the DNA has been integrated, as against cells which have not integrated the DNA construct. Various marker genes include, but are not limited to, antibiotic resistance markers, such as resistance to G418 or hygromycin. Less conveniently, negative selection may be used, including, but not limited to, where the marker is the HSV-tk gene, which will make the cells sensitive to agents such as acyclovir and gancyclovir. Alternatively, selections could be accomplished by employment of a stable cell surface marker to select for transgene expressing cells by FACS sorting. The NeoR (neomycin /G418 resistance) gene is commonly used but any convenient marker gene whose sequences are not already present in the recipient cell, can be used. The viral vector can be modified to incorporate chimeric envelope proteins or nonviral membrane proteins into retroviral particles to improve particle stability and expand the host range or to permit cell type-specific targeting during infection. The production of retroviral vectors that have altered host range is taught, for example, in WO 92/14829 and WO 93/14188. Retroviral vectors that can target specific cell types in vivo are also taught, for example, in Kasahara et al. 38 (1994) Science 266:1373-1376. Kasahara, et al. describe the construction of a Moloney leukemia virus (MoMLV) having a chimeric envelope protein consisting of human erythropoietin (EPO) fused with the viral envelope protein. This hybrid virus shows tissue tropism for human red blood progenitor cells that bear the receptor for EPO, and is therefore useful in gene therapy of sickle cell anemia and thalassemia. Retroviral vectors capable of specifically targeting infection of cells are preferred for in vivo gene therapy. The viral constructs can be prepared in a variety of conventional ways. Numerous vectors are now available which provide the desired features, such as long terminal repeats, marker genes, and restriction sites, which may be further modified by techniques known in the art. The constructs may encode a signal peptide sequence to ensure that cell surface or secreted proteins encoded by genes are properly processed post-translationally and expressed on the cell surface if appropriate. Preferably, the foreign gene(s) is under the control of a cell specific promoter. Expression of the transferred gene can be controlled in a variety of ways depending on the purpose of gene transfer and the desired effect. Thus, the introduced gene may be put under the control of a promoter that will cause the gene to be expressed constitutively, only under specific physiologic conditions, or in particular cell types. The retroviral LTR (long terminal repeat) is active in most hematopoietic cells in vivo and will generally be relied upon for transcription of the inserted sequences and their constitutive expression (Ohashi et al. (1992) Proc. Natl. Acad. Sci. USA 89:11332; Correll et al. (1992) Blood 80:331). Other suitable promoters include the human cytomegalovirus (CMV) immediate early promoter and the U3 region promoter of the Moloney Murine Sarcoma Virus (MMSV), Rous Sarcoma Virus (RSV) or Spleen Focus Forming Virus (SFFV). Examples of promoters that may be used to cause expression of the introduced sequence in specific cell types include Granzyme A for expression in T-cells and NK cells, the CD34 promoter for expression in stem and progenitor 39 cells, the CD8 promoter for expression in cytotoxic T-cells, and the CD1 lb promoter for expression in myeloid cells. Inducible promoters may be used for gene expression under certain physiologic conditions. For example, an electrophile response element may be used to induce expression of a chemoresistance gene in response to electrophilic molecules. The therapeutic benefit may be further increased by targeting the gene product to the appropriate cellular location, for example the nucleus, by attaching the appropriate localizing sequences. The vector construct is introduced into a packaging cell line which will generate infectious virions. Packaging cell lines capable of generating high titers of replication-defective recombinant viruses are known in the art, see for example, WO 94/29438. Viral particles are harvested from the cell supernatant and purified for in vivo infection using methods known in the art such as by filtration of supernatants 48 hours post transfection. The viral titer is determined by infection of a constant number of appropriate cells (depending on the retrovirus) with titrations of viral supernatants. The transduction efficiency can be assayed 48 hours later by a variety of methods, including Southern blotting. After viral transduction, the presence of the viral vector in the transduced cells or their progeny can be verified such as by PCR. PCR can be performed to detect the marker gene or other virally transduced sequences. Generally, periodic blood samples are taken and PCR conveniently performed using it NeoR probes if the NeoR gene is used as marker. The presence of virally transduced sequences in bone marrow cells or mature hematopoietic cells is evidence of successful reconstitution by the transduced cells. PCR techniques and reagents are well known in the art, See, generally, PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS. Innis, Gelfand, Sninsky & White, eds. (Academic Press, Inc., San Diego, CA 1990) and commercially available (Perkin-Elmer). Non- viral vectors, such as plasmid vectors useful in the genetic modifications of this invention, can be produced according to methods taught in the art. References describing the construction of non- viral vectors include the 40 following: Ledley, F.D. (1995) Human Gene Therapy 6:1129-1144, Miller, N. et al. (1995) FASEB 9:190-199; Chonn, A. et al. (1995) Curr. Opin. Biotech. 6:698- 708, Schofield, J.P. et al. (1995) British Med. Bull. 51:56-71; Brigham, K.L. et al. (1993) J. Liposome Res. 3:31-49, Brigham, K.L. WO 91/06309; Feigner, PL. et al. WO 91/17424; Solodin et al. (1995) Biochem. 34:13537-13544; WO 93/19768; Debs et al. WO 93/25673; Feigner, P . et al. U.S. Patent 5,264,618; Epand, R.M. et al. U.S. Patent 5,283,185; Gebeyehu et al. U.S. Patent 5,334,761; Feigner, P . et al. U.S. Patent 5,459,127; Overell, R.W. et al. WO 95/28494; Jessee WO 95/02698; Haces and Ciccarone WO 95/17373; and Lin et al. WO 96/01840. Vaccines The agents identified herein as effective for their intended purpose can be administered to subjects or individuals susceptible to or at risk of developing a disease, such as cancer. When the agent is administered to a subject such as a mouse, a rat or a human patient, the agent can be added to a pharmaceutically acceptable carrier and systemically or topically administered to the subject. To determine patients that can be beneficially treated, a tumor regression can be assayed. Therapeutic amounts can be empirically determined and will vary with the pathology being treated, the subject being treated and the efficacy and toxicity of the therapy. When delivered to an animal, the method is useful to further confirm efficacy of the agent. As an example, an adenoviral vector encoding a tumor antigen can be employed in vivo for direct immunization as described in Zhai et al. (1996) J. Immunol. 156:700-10. C57BL/6 mice are injected subcutaneously at two sites (1.5 x 10 9 IU per site) with an adenoviral vector encoding an antigen presenting cell recruitment or proliferation factor such as IL4. Two days later an adenoviral vector encoding a tumor antigen such as gplOO is injected into these two pre-treated site (1.5 x 10 9 IU per site). Two weeks later, animals are challenged with a lethal dose (2 x 10 4 ) of murine B16F10 melanoma tumor cells by subcutaneous injection. Animals are scored for survival and tumor measurements are taken. 41 Administration in vivo can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents can be found below. The agents and compositions of the present invention can be used in the manufacture of medicaments and for the treatment of humans and other animals by administration in accordance with conventional procedures, such as an active ingredient in pharmaceutical compositions. More particularly, an agent of the present invention also referred to herein as the active ingredient, may be administered for therapy by any suitable route including oral, rectal, nasal, topical (including transdermal, aerosol, buccal and sublingual), vaginal, parental (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary. It will also be appreciated that the preferred route will vary with the condition and age of the recipient, and the disease being treated. 42 Example Enhancement of antigen delivery to antigen presenting cells is tested in murine tumor models where TAAs have been identified. For example, B16-F10 melanoma tumor growth in the C57BL/6 mouse strain can be used since the TAAs MARTI and gplOO have been identified and offer some protective effect against tumor growth (Zhai et al. (1997) J. Immunother. 20:15-25). An alternative model is the K1735 melanoma cells from the C3H murine strain, where a specific CTL response against tumor antigens has been shown (Chen et al. (1993), supra). In another scenario, an APC recruitment or proliferation factor such as IL- 4 is injected subcutaneously in the form of a recombinant protein formulated in either cationic lipids, or in a sponge, to allow for sustained slow release of the APC recruitment or proliferation factor at a localized site. At a time point deemed to be optimal for APC recruitment or proliferation (1 to 3 days post injection), an adenoviral vector encoding an antigen such as the melanoma antigen gpl 00 is injected into the pretreated site to maximize the likelihood that the injected viral particles will contact, and cause gene transfer to APCs. The genetically modified APCs will present tumor antigen derived peptides to the immune system, thereby provoking a potent anti-tumor cell immune response. It is to be understood that while the invention has been described in conjunction with the above embodiments, that the foregoing description and the following examples are intended to illustrate and not limit the scope of the invention. For example, any of the above-noted compositions and/or methods can be combined with known therapies or compositions. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains. 43

Claims: CLAIMS 1. A method of recruiting antigen presenting cells to a predetermined site in a subject, comprising administering to the subject an effective amount of an antigen presenting cell (APC) recruitment or proliferation factor to the predetermined site. 2. The method according to claim 1 , wherein the APC recruitment or proliferation factor is administered as a protein or peptide. 3. The method according to claim 2, wherein the protein or polypeptide is formulated for slow release. 4. The method according to claim 1, wherein the APC recruitment or proliferation factor is administered in a gene delivery vehicle. 5. The method according to claim 1 or 4, wherein the APC recruitment or proliferation factor is selected from the group consisting of a proinflammatory agent, a chemotactic agent, a growth factor and a mitogenic factor. 6. The method according to claim 5, wherein the APC recruitment or proliferation factor is selected from the group consisting of granulocyte- macrophage colony stimulating factor (GM-CSF), Sepragel, interleukin 4 (IL4) and macrophage inflammatory protein 3 alpha (MIP-3α). 7. The method according to claim 1 or 4, further comprising administering a compound selected from the group consisting of a growth factor, a cytokine, a co-stimulatory molecule and a mitogenic factor. 44 8. The method according to claim 7, wherein the compound is administered in a gene delivery vehicle. 9. A method of enhancing presentation of an antigen into antigen presenting cells (APCs) in vivo comprising priming a predetermined site in a subject with an effective amount of an APC recruitment or proliferation factor and administering an effective amount of the transgene at the site. 10. The method according to claim 9, wherein the antigen is administered as a protein, recombinant protein or peptide. 11. The method according to claim 10, wherein the protein or polypeptide is formulated for slow release. 12. The method according to claim 9, wherein the antigen is administered as a polynucleotide encoding the antigen. 13. The method according to claim 9, wherein the antigen is a tumor associated antigen. 14. The method according to claim 12, wherein the tumor associated antigen is selected from the group consisting of gplOO, MUC-1, MART-1, HER- 2, CEA, PSA, prostate specific membrane antigen (PSMA), tyrosinase, tyrosinase related proteins 1 or 2 (TRP-1 and TRP-2), NY-ESO-1, and GA733 antigen. 15. The method according to claim 9 or 12, wherein the APC recruitment or proliferation factor is selected from the group consisting of a proinflammatory agent, a chemotactic agent, a growth factor and a mitogenic factor. 45 16. The method according to claim 9 or 12, wherein the APC recruitment or proliferation factor is selected from the group consisting of granulocyte-macrophage colony stimulating factor (GM-CSF), Sepragel, interleukin 4 (IL4) and macrophage inflammatory protein 3 alpha (MIP-3α). 17. The method according to claim 9 or 12, further comprising administering a compound selected from the group consisting of a growth factor, a cytokine, a co-stimulatory molecule and a mitogenic factor. 18. A method of identifying an APC recruitment or proliferation factor, comprising: (a) identifying a set of unique DNA sequence tags corresponding to mRNAs expressed in a sample cell that secretes an APC recruitment or proliferation factor; (b) identifying a set of sequence tags corresponding to mR A transcripts expressed in a control cell that does not secrete an APC recruitment or proliferation factor; (c) comparing the sequence tags for the sample cell with those of the control cell to identify transcripts that are differentially expressed in the sample cell; (d) using the sequence tags corresponding to the differentially expressed transcripts identified in part (c) to clone the cDNAs from which the tags were derived and in so doing identify candidate cDNAs that may encode an APC recruitment or proliferation factor; and (e) expressing the candidate cDNAs identified in (d) to determine which specific cDNAs encode APC recruitment or proliferation factors. 46

Inventors: PERRICONE MICHAEL A, ROBERTS BRUCE L, PERRICONE, MICHAEL, A., ROBERTS, BRUCE, L.
Applicants: GENZYME CORP, PERRICONE MICHAEL A, ROBERTS BRUCE L, GENZYME CORPORATION, PERRICONE, MICHAEL, A., ROBERTS, BRUCE, L.
Application year: 1999
Main classification: A61K48
IPC codes: A61K31, A61K38, A61K39, A61K48, A61P35, C12Q1