The premise of cancer immunotherapy is based on the assumption that tumor-associated antigens (TAAs) exist and that the host immune system can recognize expression of these antigens by tumor cells. A significant advantage of immunotherapy is thus the ability to recruit the immune system to attack disseminated tumor cells and thereby eliminate both local and metastatic disease. In recent years, the identification of human TAAs and elucidation of the mechanisms responsible for the induction of antitumor immunity have provided the requisite background for bringing to bear the potential of immunotherapy in the treatment of cancer. Importantly, the marriage of immunotherapy and gene therapy has provided an opportunity to induce immunity against specific TAAs and to selectively activate the immune response in the tumor microenvironment. Immunomodulatory genes include those encoding cytokines, costimulatory molecules, and TAAs.

Genetic immunotherapy has been developed to induce the immune recognition of TAAs as "foreign" antigens. In ex vivo approaches, autologous tumor cells transfected in culture to express an immunomodulatory gene encoding a cytokine or costimulatory molecule are reimplanted into the patient. In in vivo therapy, the gene is delivered directly into the tumor or, with certain vaccines, into normal tissues. The identification of specific TAAs is not required when tumor cells are used as the source of antigen; however, in this setting, immunity can be induced to epitopes from both TAAs and normal antigens. Alternatively, vaccination with a gene encoding a TAA, while requiring TAAs that are known, induces antitumor immunity that is antigen specific. Although initial studies focused on the delivery of single immunomodulatory genes, experience supports the combination of genes encoding cytokine, costimulatory molecules, and TAAs to maximally reverse immunologic unresponsiveness or tolerance to tumors.

The identification and cloning of cytokine genes has enabled the characterization of the pleiotropic effects of cytokines on the immune system and inflammatory cells. The systemic administration of IL-2 is associated with tumor regressions in subsets of patients with renal cell carcinomas and melanomas; however, systemic delivery of cytokines, such as IL-2, that nonspecifically activate the immune system, is limited by substantial toxicity. As with the delivery of genes encoding prodrug-converting enzymes, cytokine-based gene therapy can restrict expression of the cytokine to the tumor microenvironment. The continuous production of the cytokine by transduced tumor cells also overcomes the disadvantages of circulating peak and trough cytokine levels associated with systemic administration. Of note, cytokine gene immunotherapy functions in the activation of antitumor immunity and is dependent on the expression of TAAs.

Cytokine genes studied in the induction of antitumor immunity include IL-1, IL-2, IL-4, IL-6, IL-12, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF), IFN-a, and IFN-g. The adoptive transfer approach involves transduction of cytokine genes into tumor explants and then lethal irradiation of the tumor cells. For example, prostate cancer tissue is excised, transduced in culture with a retrovirus expressing GM-CSF, irradiated, and then used to vaccinate the patient. ¹⁰⁰ Production of GM-CSF recruits dendritic cells to the immunization site for presentation of tumor antigens to CD4+ and CD8+ T cells. Malignant melanomas have also been targeted by this approach using IL-2 as the cytokine. ¹⁰¹

Direct in vivo delivery of genes into tumors represents an alternative approach. An IFN-g encoding plasmid directly injected into tumors as naked DNA or in a cationic liposome has resulted in the production of IFN-g for 7 days. ²⁰ IFN-g activates T cells, natural killer (NK) cells, and macrophages and induces major histocompatibility complex (MHC) class I and II expression. Gene gun mediated intratumoral delivery has been used for plasmids expressing IFN-g, IL-6, TNF, and IL-2. ²¹ In addition, intramuscular injection of plasmid DNA encoding IFN-a is associated with reductions in tumor growth and in the development of metastases by a CD8+ T-cell-dependent mechanism. ¹⁰² IFN-a activates the immune system, decreases tumor cell proliferation, decreases angiogenesis, induces a T-helper pathway, and upregulates MHC class I expression. A variation on this approach is to add autologous irradiated tumor cells (as a source of antigen) to fibroblasts transfected to express cytokine encoding plasmids. This strategy is advantageous when tumor explants are difficult to culture and transfect ex vivo and has been employed in murine tumor models and Phase I clinical trials. ^{22, 103}

The effective activation of T cells is dependent on at least two signals. The first, mediated by MHC molecules, involves antigen-specific interactions with the T-cell receptor (TCR). The second involves costimulation provided by the interaction of B7 molecules (B7-1, B7-2) with CD28 or CTLA4 on the T cell surface. The antigen-TCR interaction can select antigen-specific cytolytic T cells (CTL); however, costimulation is needed for appropriate signaling and clonal expansion of a CTL population. Although most cells express MHC class I molecules for antigen presentation, costimulatory molecules are predominantly found on professional antigen-presenting cells (APCs), such as dendritic cells, Langerhan cells, B cells, monocytes, and macrophages. The lack of costimulatory molecules on tumor cells results in MHC class I presentation of tumor antigens in the absence of costimulation and thereby T cell anergy. Thus, one mechanism for generating antitumor CTL is through the delivery of costimulatory genes to tumor cells such that antigens are presented in the context for CTL activation.

Recombinant vaccinia virus has been used to transduce weakly immunogenic syngeneic murine tumor cells in vitro with genes encoding B7-1 or B7-2. ¹⁰⁴ The finding that tumor growth is inhibited following implantation of the transduced tumor cells into immunocompetent mice indicated that costimulation by B7 molecules is sufficient to induce antitumor CTL activity. By contrast, mice immunosuppressed by irradiation failed to reject the B7-transduced tumor cells. Importantly, mice that rejected the transduced tumor also rejected a subsequent challenge with parental nontransduced tumor cells. Other studies demonstrate that B7 costimulation is needed to induce tumor-specific CTL in naïve mice, but is not required for tumor rejection upon rechallenge. ¹⁰⁵ These findings suggest that transduction of B7 genes into one tumor site could confer rejection of B7-negative tumors at other sites. Transduction of multiple myeloma cells with a recombinant AAV expressing B7-1 or B7-2 also induces specific antitumor CTL activity as measured by T-cell proliferation, production of IL-2 and IFN-g, and lysis of target cells. ¹⁰⁶

CD40 ligand (CD40L) is selectively expressed on CD4+ T helper cells and stimulates APCs through binding to the CD40 receptor. As the interaction of CD40L with CD40 stimulates antigen-specific T-cell responses, transduction of tumor cells with the CD40L gene can confer more efficient presentation of TAA and enhanced antitumor immunity. For example, intratumoral delivery of an adenovirus expressing CD40L is associated with CD8+ T-cell-mediated antitumor immunity and inhibition of tumor growth in murine models. ¹⁰⁷

Activation of T cells is dependent on multiple factors. Thus, delivery of a single immunomodulatory gene will probably be insufficient to activate effective antitumor immunity. As certain cytokines are involved in expansion of T cell clones, gene therapy strategies delivering both a cytokine and costimulatory gene could prove synergistic in inducing antitumor immunity. In this context, adenoviral-mediated transduction of both IL-2 and B7-1 genes has resulted in a greater than additive antitumor effect in a breast cancer model. ¹⁰⁸

Gene therapy with recombinant vectors that express a TAA has been developed as vaccines for the induction of active specific immunotherapy. Genes that express TAA can be distinguished as (1) endogenous, nonmutated genes that are often overexpressed in tumors; (2) endogenous genes that are mutated and thereby express an altered protein; and (3) exogenous genes. Examples of nonmutated genes associated with tumors include melanoma/melanocyte differentiation antigens (MART-1/MelanA, gp100, tyrosinase, TRP-1, and TRP-2), testicular cancer/testes antigens (MAGE, BAGE, GAGE, and NY-ESO-1), carcinoembryonic antigen (CEA), PSA, and DF3/MUC1. ^{26, 109} TAAs expressed by endogenous mutated genes include p53, cdk4, caspase 8, and b-catenin.¹¹⁰¹¹² TAAs from exogenous sources are often derived from viral transformation as exemplified in human papillomavirus-positive cervical cancer.

Gene therapy based tumor vaccination induces immunity against specific TAAs and is distinguished from the nonspecific immune stimulation associated with transfer of cytokine or costimulatory genes. One method of vaccination involves delivery of the TAA-encoding gene directly into the patient by viral or nonviral systems. Another approach is accomplished through in vitro transfection or transduction of cells, generally APCs, with the TAA gene and reintroduction of these cells to the patient. Direct delivery of the TAA gene at subcutaneous or intradermal sites is used more widely than the ex vivo approach. Expression of the TAA in the epidermis is associated with processing of the TAA by Langerhans cells and thereby with presentation of TAA peptides to T cells.

Vaccination with genes encoding TAAs has been accomplished by transfection and viral transduction. For example, immunization of mice with naked DNA encoding a TAA was achieved by direct intramuscular injection. ¹¹³ The immunized mice are protected against challenge with tumor cells expressing the TAA. In clinical trials, recombinant vaccinia virus expressing CEA has been administered intradermally. ¹¹⁴ Peripheral blood lymphocytes from certain vaccinated patients responded to stimulation with CEA in vitro. ^{114, 115} Genes encoding the melanoma/melanocyte differentiation antigens MART-1 and gp100 have been used to vaccinate patients with metastatic melanoma. ¹¹⁶ A long-term complete response was achieved in one patient vaccinated with an adenovirus expressing MART-1. Another Phase I trial of a recombinant PSA expressing vaccinia virus vaccine (PROSTVAC) in men with advanced prostate cancer has demonstrated induction of anti-PSA immunity and stabilization of the disease course in certain patients. ¹¹⁷

Multiple genes encoding TAAs can be constructed in a single plasmid or the genes can be coadministered in separate plasmids. In this context, a potential disadvantage of immunization against a single TAA is that expression of the antigen can be downregulated in tumors. Thus, immunization against multiple TAAs could decrease the potential for the development of immunologic resistance by the tumor cell. In this strategy, multiple TAAs for a tumor must be known; however, at present, there are few well-characterized TAAs for most tumors. Another approach has been to codeliver the B7-1 gene with the TAA gene to maximize the antitumor immune response. Recombinant vaccinia viruses encoding B7-1 or CEA have been administered as a mixture at a ratio of 1:3 to further stimulate the anti-CEA response. ¹¹⁸ Similar findings have been obtained following vaccination with mixtures of vaccinia viruses expressing B7-1 and MUC1. ¹¹⁹ More advanced vaccinia and fowlpox vector systems have been developed to express B7-1, intercellular adhesion molecule (ICAM), and lymphocyte function-associated antigen (LFA)-3 in a single virus for coadministration with vectors expressing TAAs. ¹²⁰