Gene Therapy and Molecular Immunology Unite to Give New Life to Cancer Immunotherapy
Authors: Dale Ando
Journal: Molecular Therapy (2002)

Since the late nineteenth century, researchers have tried to harness the immune system to treat cancer. Initial strategies used bacterial extracts (Coley's toxin), bacilli (for example, BCG), and modification with virus or xenogenization to render tumors immunogenic [1Lindenmann J Klein P Viral oncolysis: increased immunogenicity of host cell antigen associated with influenza virus.J. Exp. Med. 1967; 126: 93-108Crossref PubMed Scopus (197) Google Scholar, 2Pardoll D Cancer vaccines.Nat. Med. 1998; 4: 525-531Crossref PubMed Scopus (484) Google Scholar]. Demonstrations of the efficacy of allogeneic donor lymphocytes to induce remissions in patients with chronic myelogenous leukemia and induction of long-lasting remissions with interleukin-2 therapy in patients with renal cell carcinoma underscore the potential of manipulating the immune system to treat cancer [3Shimoni A Long-term follow-up of recipients of CD8-depleted donor lymphocyte infusions for the treatment of chronic myelogenous leukemia relapsing after allogeneic progenitor cell transplantation.Biol. Blood Marrow Transplant. 2001; 7: 568-575Abstract Full Text PDF PubMed Scopus (56) Google Scholar, 4Fyfe G Results of treatment of 255 patients with metastatic renal cell carcinoma who receive high-dose recombinant interleukin-2 therapy.J. Clin. Oncol. 1995; 13: 688-696PubMed Google Scholar]. However, the success of immunotherapy in cancer therapy has been limited. One reason for this is that an effective immunotherapy must coordinate a complex set of interactions in a given cancer patient. The arrival of the age of molecular biology, molecular immunology, and genomics is now beginning to give us insight on the level of complexity that confronts us. Important basic categories are the antigen, antigen presentation, immune tolerance, type of anti-tumor immune response, host immunosuppressive factors, and killing or control of the tumor.The development of gene transfer techniques along with molecular characterization of "normal" immune recognition has brought fundamental insights into the nature of tumor/host immune interactions. A paper by Itaya et al. in 1987 [5Itaya T Xenogenization of a mouse lung carcinoma (3LL) by transfection with an allogeneic class I major histocompatibility complex gene (H-2Ld).Cancer Res. 1987; 47: 3136-3140PubMed Google Scholar] described the genetic transfer of an allogeneic murine histocompatiblity complex (MHC) class I into a spontaneously arising Lewis lung carcinoma cell line. The specific addition of an allogeneic MHC class I gene to a non-immunogenic tumor after vaccination into a syngeneic host led not only to an immune response against the foreign allogeneic protein, but also to self proteins in the tumor. This clearly demonstrated the concept of "xenogenization," in which a foreign protein (when expressed by a tumor) could bypass immune tolerance to the tumor and generate anti-tumor responses. This observation was followed by a paper by Fearon and colleagues in 1988 showing that syngeneic tumor cells transfected with a xenogeneic viral antigen hemaglutination antigen (HA) could also dramatically increase the immunogenicity of the tumor [6Fearon E Induction in a murine tumor of immunogenic tumor variants by transfection with a foreign gene.Cancer Res. 1988; 48: 2975-2980PubMed Google Scholar]. Finally, this group went on to demonstrate that transfer of the interleukin-2 (IL-2) gene into tumors dramatically increased tumor antigenicity by bypassing T-cell help, demonstrating that a tumor cell vaccine plus cytokines could also circumvent tolerance [7Fearon E Interleukin-2 production by tumor cells bypasses T helper function in the generation of an antitumor response.Cell. 1990; 60: 397-403Abstract Full Text PDF PubMed Scopus (945) Google Scholar]. These studies revealed that gene transfer into tumors could be used to specifically dissect the components of a vaccine that result in breakdown of tolerance to tumors in animal tumor models, and led to a rapid expansion of our knowledge of the complexity of immune tolerance to tumors and immunotherapeutic strategies.The molecular cloning of cytokine genes initially led to their development as protein therapeutics. Cancer immunotherapy in the 1980s focused on clinical trials using IL-2, interferon-α, -β, and -γ, tumor necrosis factor, GM-CSF, macrophage-CSF, IL-12, and so on to treat patients with cancer. This era of nonspecific immune therapy with pharmacologic doses of cytokines had few successes, most notably the use of IL-2 in treating renal cell carcinoma and interferon-α in hairy cell leukemia [4Fyfe G Results of treatment of 255 patients with metastatic renal cell carcinoma who receive high-dose recombinant interleukin-2 therapy.J. Clin. Oncol. 1995; 13: 688-696PubMed Google Scholar, 8Weiss K Safety profile of interferon-a therapy.Semin. Oncol. 1998; 25: 9-13PubMed Google Scholar]. Response rates were low and associated toxicity was significant. It was clear that infusion of large amounts of cytokines or stem cell factors did not always result in consistent anti-tumor effects.In the early 1990s, gene transfer studies in animals using cytokines and growth factors as paracrine adjuvants for induction of anti-tumor responses demonstrated a clear improvement in efficacy in animal models. This has resulted in tremendous enthusiasm and a renaissance in the evaluation of these cytokines as well as other molecules involved in costimulation of the immune response for cancer therapy. In 1991 Dranoff and colleagues were the first to comparatively evaluate a series of cytokines in animal tumor models, and found that GM-CSF demonstrated comparative superiority in the induction of anti-tumor responses and immune protection in animals [9Dranoff G Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity.Proc. Natl. Acad. Sci. USA. 1993; 90: 3539-3543Crossref PubMed Scopus (2644) Google Scholar]. The discovery that GM-CSF is a major growth and activation factor for dendritic cells that are potent antigen presentation cells for immune responses suggested a mechanism for GM-CSF as a vaccine adjuvant [10Mach N Dranoff G Cytokine-secreting tumor cell vaccines.Curr. Opin. Immunol. 2000; 12: 571-575Crossref PubMed Scopus (115) Google Scholar, 11Borrello I Pardoll D GM-CSF-based cellular vaccines: a review of the clinical experience.Cytokine Growth Factor Rev. 2002; 13: 185-193Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar]. Many animal studies have shown a positive role for T-cell cytokines, interferons, and costimulatory molecules as "molecular adjuvants" for tumor vaccines. In addition, Woo and colleagues showed that addition of the thymidine kinase suicide gene and cytokine activation in a tumor vaccine increased antitumor immune responses [12Chen S Combination suicide and cytokine gene therapy for hepatic metastases of colon carcinoma: sustained antitumor immunity prolongs animal survival.Cancer Res. 1996; 56: 3758-3762PubMed Google Scholar]. As a result of all this research, we are now confronted with a large array of novel gene therapy vaccine strategies: xenogenization, cytokine adjuvants, suicide genes, and so forth.What immunologic framework could account for this sudden increase in effective tumor vaccine strategies in animal models? In 1996 Matzinger and colleagues proposed a "danger" tolerance model in which cell death associated with inflammation or danger signals from the tissue was the key to induction of immune responses, and that cell death associated with a quiet apoptosis of the tissue led to tolerance. This could explain the efficacy of these multiple cytokines as adjuvants and, in combination, the additive effect of induction of cell death with suicide genes [13Fuchs E Matzinger P Is cancer dangerous to the immune system?.Semin. Immunol. 1996; 8: 271-280Crossref PubMed Scopus (258) Google Scholar]. Thus tumor growth and death without inflammation led to immune tolerance in a physiologic fashion. What we needed to do was vaccinate against tumor antigens in the context of an inflammatory/danger immune milieu. Still, substantial questions remain: What is the mechanism of immune tolerance to tumors? What is the best way to reverse immune tolerance? What cytokines are optimal for danger signals? What immune cells should be targeted?? How do we model human tolerance to cancer in animals?Armed with new preclinical efficacy models and hypotheses of tolerance induction by tumors, the next step was to initiate human clinical trials to investigate these ideas. We are now in that stage. Human clinical trials evaluating various theoretical strategies are in progress. For example, the concept of xenogenization is being evaluated in clinical trials using HLA-B7 allogeneic melanoma [14Hersh E Stopeck A Advances in the biological therapy and gene therapy of malignant disease.Clin. Cancer Res. 1997; 3: 2623-2629PubMed Google Scholar, 15Nabel G Immune response in human melanoma after transfer of an allogeneic class I major histocompatibility complex gene with DNA-liposome complexes.Proc. Natl. Acad. Sci. USA. 1996; 93: 15388-15393Crossref PubMed Scopus (226) Google Scholar]. There are many trials underway using cytokines as tumor vaccine adjuvants [16Soiffer R Vaccination with irradiated autologous melanoma cells engineered to secrete human granulocyte-macrophage colony-stimulating factor generates potent antitumor immunity in patients with metastatic melanoma.Proc. Natl. Acad. Sci. USA. 1998; 95: 13141-13146Crossref PubMed Scopus (499) Google Scholar, 17Wollenberg B Gene therapy-phase I trial for primary untreated head and neck squamous cell cancer (HNSCC) UICC stage II-IV with a single intratumoral injection of hIL-2 plasmids formulated in DOTMA/Chol.Hum. Gene Ther. 1999; 10: 141-147Crossref PubMed Scopus (27) Google Scholar, 18Fujii S Induction of melanoma-associated antigen systemic immunity upon intratumoral delivery of interferon-γ retroviral vector in melanoma patients.Cancer Gene Ther. 2000; 7: 1220-1230Crossref PubMed Scopus (39) Google Scholar, 19Nemunaitis J Phase I trial of retroviral vector-mediated interferon (IFN)-γ gene transfer into autologous tumor cells in patients with metastatic melanoma.Cancer Gene Ther. 1998; 5: 292-300PubMed Google Scholar]. Immune cell targets for these cytokine vaccines include dendritic cells with GM-SCF, lymphocytes with IL-2, and NK and T cells with IL-12. Vectors used have included plasmids, retrovirus, foulpox, and adenovirus. These human studies will provide critical information on what specific molecular strategies will bypass tolerance and increase tumor recognition by the human immune system.Progress in the area of tumor antigen presentation and induction of tumor immune responses using gene therapy addresses the proximal limb of the immune system. Researchers have also actively pursued additional strategies of activating immune cells ex vivo. Tumor infiltrating lymphocytes (TIL) from patients with melanoma can be cultured and expanded ex vivo, and can specifically lyse autologous tumors. Some patients with malignant melanoma have had significant tumor reduction after treatment with autologous ex vivo expanded TIL and IL-2. Steven Rosenberg and colleagues at the NCI and NIH were the first to use retroviral-mediated gene transfer into TIL in 1990 [20Rosenberg S Gene transfer into humans—immunotherapy of patients with advanced melanoma using tumor-infiltrating lymphocytes modified by retroviral gene transduction.N. Engl. J. Med. 1990; 323: 570-578Crossref PubMed Scopus (1072) Google Scholar]. This study was the first gene transfer study performed in humans and the first use of retroviral vectors produced from retroviral packaging cell lines. Significantly, this marking study of TIL to understand the pharmacology and biodistribution of these cells in metastatic melanoma patients was considered to be an appropriate risk-benefit ratio for the first gene therapy trial in humans. The rationale for this was that the TIL were transfected and expanded ex vivo, and testing of the product could be carried out before infusion of the TIL cells into the patients, so that some level of assessment for replication-competent retrovirus (RCR) would be available before infusion of patients. Five patients were treated with neor gene transduced TIL. Circulating gene-marked TIL were present from 20 to 60 days in the peripheral blood, one tumor biopsy was positive at day 64, and there were no adverse events.RCR is a significant issue that was confronted for the first time in this treatment protocol. Extensive molecular engineering of the retroviral producer cell lines involving splitting of the viral genome to minimize the risk of recombination of the vector [21Miller A.D. Retrovirus packaging cells.Hum. Gene Ther. 1990; 1: 5-14Crossref PubMed Scopus (315) Google Scholar]. The vector portion of the retrovirus and packaging and envelope functions were separated in these early packaging cell lines. Animal safety studies were carried out to analyze the safety and oncogenic potential of the murine retroviruses [22Cornetta K Amphotropic murine leukemia retrovirus is not an acute pathogen for primates.Hum. Gene Ther. 1990; 1: 15-30Crossref PubMed Scopus (152) Google Scholar]. Sensitive assays for molecular detection and culture or replication-competent retrovirus were developed. This trial was publicly reviewed by the NIH Recombinant DNA Advisory Committee and the appendix M document was developed to review human clinical gene therapy experiments [23NIH guidelines for research involving recombinant DNA molecules, http://www4.od.nih.gov/oba/rac/guidelines_02/NIH_Guidelines_Apr_02.htm.Google Scholar]. The issues of informed consent, clinical trial risk benefit, retroviral packaging cell line split genome strategy and risk for RCR, and replication-competent retroviral testing in manufacturing and in patients were extensively discussed in a public setting before the initiation of this trial. The extensive scientific, ethical, and legal review in public that was required for the first gene therapy human clinical protocol set a precedent for how novel clinical gene therapy trials would be evaluated.The gene therapy era of cancer immunotherapy began after this pivotal initial human gene therapy trial showing clinical safety of infusion with ex vivo retroviral gene transduced T cells. Once the safety of gene transfer ex vivo was demonstrated, the next step was a progression to direct vector therapy with retroviral vectors and adenoviral vectors in cancer and non-cancer indications [18Fujii S Induction of melanoma-associated antigen systemic immunity upon intratumoral delivery of interferon-γ retroviral vector in melanoma patients.Cancer Gene Ther. 2000; 7: 1220-1230Crossref PubMed Scopus (39) Google Scholar, 24Merritt J Roth J Logothetis C Clinical evaluation of adenoviral-mediated p53 gene transfer: review of INGN 201 studies.Semin. Oncol. 2001; 28: 105-114Abstract Full Text PDF PubMed Google Scholar]. As of December 2001 there were 314 cancer gene therapy trials out of a total of 500 gene transfer clinical trials performed in the United States in the NIH Recombinant DNA database. Now we can look back at nearly a decade of cancer vaccine basic and clinical trials research using gene therapy in which we have learned more about basic mechanisms and developed the clinical groundwork for gene therapy trials. These basic and clinical studies have lead to novel immunotherapeutic strategies that can now be translated into human clinical trials. This new era of human clinical trials, supported by intensive basic research, will lead to the next level of understanding and manipulating immune responses to tumors in human and, ultimately, to effective therapeutic strategies. Since the late nineteenth century, researchers have tried to harness the immune system to treat cancer. Initial strategies used bacterial extracts (Coley's toxin), bacilli (for example, BCG), and modification with virus or xenogenization to render tumors immunogenic [1Lindenmann J Klein P Viral oncolysis: increased immunogenicity of host cell antigen associated with influenza virus.J. Exp. Med. 1967; 126: 93-108Crossref PubMed Scopus (197) Google Scholar, 2Pardoll D Cancer vaccines.Nat. Med. 1998; 4: 525-531Crossref PubMed Scopus (484) Google Scholar]. Demonstrations of the efficacy of allogeneic donor lymphocytes to induce remissions in patients with chronic myelogenous leukemia and induction of long-lasting remissions with interleukin-2 therapy in patients with renal cell carcinoma underscore the potential of manipulating the immune system to treat cancer [3Shimoni A Long-term follow-up of recipients of CD8-depleted donor lymphocyte infusions for the treatment of chronic myelogenous leukemia relapsing after allogeneic progenitor cell transplantation.Biol. Blood Marrow Transplant. 2001; 7: 568-575Abstract Full Text PDF PubMed Scopus (56) Google Scholar, 4Fyfe G Results of treatment of 255 patients with metastatic renal cell carcinoma who receive high-dose recombinant interleukin-2 therapy.J. Clin. Oncol. 1995; 13: 688-696PubMed Google Scholar]. However, the success of immunotherapy in cancer therapy has been limited. One reason for this is that an effective immunotherapy must coordinate a complex set of interactions in a given cancer patient. The arrival of the age of molecular biology, molecular immunology, and genomics is now beginning to give us insight on the level of complexity that confronts us. Important basic categories are the antigen, antigen presentation, immune tolerance, type of anti-tumor immune response, host immunosuppressive factors, and killing or control of the tumor. The development of gene transfer techniques along with molecular characterization of "normal" immune recognition has brought fundamental insights into the nature of tumor/host immune interactions. A paper by Itaya et al. in 1987 [5Itaya T Xenogenization of a mouse lung carcinoma (3LL) by transfection with an allogeneic class I major histocompatibility complex gene (H-2Ld).Cancer Res. 1987; 47: 3136-3140PubMed Google Scholar] described the genetic transfer of an allogeneic murine histocompatiblity complex (MHC) class I into a spontaneously arising Lewis lung carcinoma cell line. The specific addition of an allogeneic MHC class I gene to a non-immunogenic tumor after vaccination into a syngeneic host led not only to an immune response against the foreign allogeneic protein, but also to self proteins in the tumor. This clearly demonstrated the concept of "xenogenization," in which a foreign protein (when expressed by a tumor) could bypass immune tolerance to the tumor and generate anti-tumor responses. This observation was followed by a paper by Fearon and colleagues in 1988 showing that syngeneic tumor cells transfected with a xenogeneic viral antigen hemaglutination antigen (HA) could also dramatically increase the immunogenicity of the tumor [6Fearon E Induction in a murine tumor of immunogenic tumor variants by transfection with a foreign gene.Cancer Res. 1988; 48: 2975-2980PubMed Google Scholar]. Finally, this group went on to demonstrate that transfer of the interleukin-2 (IL-2) gene into tumors dramatically increased tumor antigenicity by bypassing T-cell help, demonstrating that a tumor cell vaccine plus cytokines could also circumvent tolerance [7Fearon E Interleukin-2 production by tumor cells bypasses T helper function in the generation of an antitumor response.Cell. 1990; 60: 397-403Abstract Full Text PDF PubMed Scopus (945) Google Scholar]. These studies revealed that gene transfer into tumors could be used to specifically dissect the components of a vaccine that result in breakdown of tolerance to tumors in animal tumor models, and led to a rapid expansion of our knowledge of the complexity of immune tolerance to tumors and immunotherapeutic strategies. The molecular cloning of cytokine genes initially led to their development as protein therapeutics. Cancer immunotherapy in the 1980s focused on clinical trials using IL-2, interferon-α, -β, and -γ, tumor necrosis factor, GM-CSF, macrophage-CSF, IL-12, and so on to treat patients with cancer. This era of nonspecific immune therapy with pharmacologic doses of cytokines had few successes, most notably the use of IL-2 in treating renal cell carcinoma and interferon-α in hairy cell leukemia [4Fyfe G Results of treatment of 255 patients with metastatic renal cell carcinoma who receive high-dose recombinant interleukin-2 therapy.J. Clin. Oncol. 1995; 13: 688-696PubMed Google Scholar, 8Weiss K Safety profile of interferon-a therapy.Semin. Oncol. 1998; 25: 9-13PubMed Google Scholar]. Response rates were low and associated toxicity was significant. It was clear that infusion of large amounts of cytokines or stem cell factors did not always result in consistent anti-tumor effects. In the early 1990s, gene transfer studies in animals using cytokines and growth factors as paracrine adjuvants for induction of anti-tumor responses demonstrated a clear improvement in efficacy in animal models. This has resulted in tremendous enthusiasm and a renaissance in the evaluation of these cytokines as well as other molecules involved in costimulation of the immune response for cancer therapy. In 1991 Dranoff and colleagues were the first to comparatively evaluate a series of cytokines in animal tumor models, and found that GM-CSF demonstrated comparative superiority in the induction of anti-tumor responses and immune protection in animals [9Dranoff G Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity.Proc. Natl. Acad. Sci. USA. 1993; 90: 3539-3543Crossref PubMed Scopus (2644) Google Scholar]. The discovery that GM-CSF is a major growth and activation factor for dendritic cells that are potent antigen presentation cells for immune responses suggested a mechanism for GM-CSF as a vaccine adjuvant [10Mach N Dranoff G Cytokine-secreting tumor cell vaccines.Curr. Opin. Immunol. 2000; 12: 571-575Crossref PubMed Scopus (115) Google Scholar, 11Borrello I Pardoll D GM-CSF-based cellular vaccines: a review of the clinical experience.Cytokine Growth Factor Rev. 2002; 13: 185-193Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar]. Many animal studies have shown a positive role for T-cell cytokines, interferons, and costimulatory molecules as "molecular adjuvants" for tumor vaccines. In addition, Woo and colleagues showed that addition of the thymidine kinase suicide gene and cytokine activation in a tumor vaccine increased antitumor immune responses [12Chen S Combination suicide and cytokine gene therapy for hepatic metastases of colon carcinoma: sustained antitumor immunity prolongs animal survival.Cancer Res. 1996; 56: 3758-3762PubMed Google Scholar]. As a result of all this research, we are now confronted with a large array of novel gene therapy vaccine strategies: xenogenization, cytokine adjuvants, suicide genes, and so forth. What immunologic framework could account for this sudden increase in effective tumor vaccine strategies in animal models? In 1996 Matzinger and colleagues proposed a "danger" tolerance model in which cell death associated with inflammation or danger signals from the tissue was the key to induction of immune responses, and that cell death associated with a quiet apoptosis of the tissue led to tolerance. This could explain the efficacy of these multiple cytokines as adjuvants and, in combination, the additive effect of induction of cell death with suicide genes [13Fuchs E Matzinger P Is cancer dangerous to the immune system?.Semin. Immunol. 1996; 8: 271-280Crossref PubMed Scopus (258) Google Scholar]. Thus tumor growth and death without inflammation led to immune tolerance in a physiologic fashion. What we needed to do was vaccinate against tumor antigens in the context of an inflammatory/danger immune milieu. Still, substantial questions remain: What is the mechanism of immune tolerance to tumors? What is the best way to reverse immune tolerance? What cytokines are optimal for danger signals? What immune cells should be targeted?? How do we model human tolerance to cancer in animals? Armed with new preclinical efficacy models and hypotheses of tolerance induction by tumors, the next step was to initiate human clinical trials to investigate these ideas. We are now in that stage. Human clinical trials evaluating various theoretical strategies are in progress. For example, the concept of xenogenization is being evaluated in clinical trials using HLA-B7 allogeneic melanoma [14Hersh E Stopeck A Advances in the biological therapy and gene therapy of malignant disease.Clin. Cancer Res. 1997; 3: 2623-2629PubMed Google Scholar, 15Nabel G Immune response in human melanoma after transfer of an allogeneic class I major histocompatibility complex gene with DNA-liposome complexes.Proc. Natl. Acad. Sci. USA. 1996; 93: 15388-15393Crossref PubMed Scopus (226) Google Scholar]. There are many trials underway using cytokines as tumor vaccine adjuvants [16Soiffer R Vaccination with irradiated autologous melanoma cells engineered to secrete human granulocyte-macrophage colony-stimulating factor generates potent antitumor immunity in patients with metastatic melanoma.Proc. Natl. Acad. Sci. USA. 1998; 95: 13141-13146Crossref PubMed Scopus (499) Google Scholar, 17Wollenberg B Gene therapy-phase I trial for primary untreated head and neck squamous cell cancer (HNSCC) UICC stage II-IV with a single intratumoral injection of hIL-2 plasmids formulated in DOTMA/Chol.Hum. Gene Ther. 1999; 10: 141-147Crossref PubMed Scopus (27) Google Scholar, 18Fujii S Induction of melanoma-associated antigen systemic immunity upon intratumoral delivery of interferon-γ retroviral vector in melanoma patients.Cancer Gene Ther. 2000; 7: 1220-1230Crossref PubMed Scopus (39) Google Scholar, 19Nemunaitis J Phase I trial of retroviral vector-mediated interferon (IFN)-γ gene transfer into autologous tumor cells in patients with metastatic melanoma.Cancer Gene Ther. 1998; 5: 292-300PubMed Google Scholar]. Immune cell targets for these cytokine vaccines include dendritic cells with GM-SCF, lymphocytes with IL-2, and NK and T cells with IL-12. Vectors used have included plasmids, retrovirus, foulpox, and adenovirus. These human studies will provide critical information on what specific molecular strategies will bypass tolerance and increase tumor recognition by the human immune system. Progress in the area of tumor antigen presentation and induction of tumor immune responses using gene therapy addresses the proximal limb of the immune system. Researchers have also actively pursued additional strategies of activating immune cells ex vivo. Tumor infiltrating lymphocytes (TIL) from patients with melanoma can be cultured and expanded ex vivo, and can specifically lyse autologous tumors. Some patients with malignant melanoma have had significant tumor reduction after treatment with autologous ex vivo expanded TIL and IL-2. Steven Rosenberg and colleagues at the NCI and NIH were the first to use retroviral-mediated gene transfer into TIL in 1990 [20Rosenberg S Gene transfer into humans—immunotherapy of patients with advanced melanoma using tumor-infiltrating lymphocytes modified by retroviral gene transduction.N. Engl. J. Med. 1990; 323: 570-578Crossref PubMed Scopus (1072) Google Scholar]. This study was the first gene transfer study performed in humans and the first use of retroviral vectors produced from retroviral packaging cell lines. Significantly, this marking study of TIL to understand the pharmacology and biodistribution of these cells in metastatic melanoma patients was considered to be an appropriate risk-benefit ratio for the first gene therapy trial in humans. The rationale for this was that the TIL were transfected and expanded ex vivo, and testing of the product could be carried out before infusion of the TIL cells into the patients, so that some level of assessment for replication-competent retrovirus (RCR) would be available before infusion of patients. Five patients were treated with neor gene transduced TIL. Circulating gene-marked TIL were present from 20 to 60 days in the peripheral blood, one tumor biopsy was positive at day 64, and there were no adverse events. RCR is a significant issue that was confronted for the first time in this treatment protocol. Extensive molecular engineering of the retroviral producer cell lines involving splitting of the viral genome to minimize the risk of recombination of the vector [21Miller A.D. Retrovirus packaging cells.Hum. Gene Ther. 1990; 1: 5-14Crossref PubMed Scopus (315) Google Scholar]. The vector portion of the retrovirus and packaging and envelope functions were separated in these early packaging cell lines. Animal safety studies were carried out to analyze the safety and oncogenic potential of the murine retroviruses [22Cornetta K Amphotropic murine leukemia retrovirus is not an acute pathogen for primates.Hum. Gene Ther. 1990; 1: 15-30Crossref PubMed Scopus (152) Google Scholar]. Sensitive assays for molecular detection and culture or replication-competent retrovirus were developed. This trial was publicly reviewed by the NIH Recombinant DNA Advisory Committee and the appendix M document was developed to review human clinical gene therapy experiments [23NIH guidelines for research involving recombinant DNA molecules, http://www4.od.nih.gov/oba/rac/guidelines_02/NIH_Guidelines_Apr_02.htm.Google Scholar]. The issues of informed consent, clinical trial risk benefit, retroviral packaging cell line split genome strategy and risk for RCR, and replication-competent retroviral testing in manufacturing and in patients were extensively discussed in a public setting before the initiation of this trial. The extensive scientific, ethical, and legal review in public that was required for the first gene therapy human clinical protocol set a precedent for how novel clinical gene therapy trials would be evaluated. The gene therapy era of cancer immunotherapy began after this pivotal initial human gene therapy trial showing clinical safety of infusion with ex vivo retroviral gene transduced T cells. Once the safety of gene transfer ex vivo was demonstrated, the next step was a progression to direct vector therapy with retroviral vectors and adenoviral vectors in cancer and non-cancer indications [18Fujii S Induction of melanoma-associated antigen systemic immunity upon intratumoral delivery of interferon-γ retroviral vector in melanoma patients.Cancer Gene Ther. 2000; 7: 1220-1230Crossref PubMed Scopus (39) Google Scholar, 24Merritt J Roth J Logothetis C Clinical evaluation of adenoviral-mediated p53 gene transfer: review of INGN 201 studies.Semin. Oncol. 2001; 28: 105-114Abstract Full Text PDF PubMed Google Scholar]. As of December 2001 there were 314 cancer gene therapy trials out of a total of 500 gene transfer clinical trials performed in the United States in the NIH Recombinant DNA database. Now we can look back at nearly a decade of cancer vaccine basic and clinical trials research using gene therapy in which we have learned more about basic mechanisms and developed the clinical groundwork for gene therapy trials. These basic and clinical studies have lead to novel immunotherapeutic strategies that can now be translated into human clinical trials. This new era of human clinical trials, supported by intensive basic research, will lead to the next level of understanding and manipulating immune responses to tumors in human and, ultimately, to effective therapeutic strategies.