Immune gene therapy for kidney cancer: the search for a magic trigger
Authors: Hyung L Kim, Arie S Belldegrun, Robert A Figlin
Journal: Molecular Therapy (2003)

It is estimated that approximately 30,000 new cases of renal cell carcinoma (RCC) will be diagnosed in 2002 [1Jemal A. et al.Cancer statistics, 2002.CA Cancer J. Clin. 2002; 52: 23-47Crossref PubMed Scopus (2919) Google Scholar]. Approximately 20–30% of patients with RCC present with metastatic disease and their overall median survival is 6–8 months [2Kirkali Z. Tuzel E. Mungan M.U. Recent advances in kidney cancer and metastatic disease.BJU Int. 2001; 88: 818-824Crossref PubMed Scopus (59) Google Scholar]. One of the most intriguing features of RCC is the dramatically favorable course of the disease in a small subset of patients [3Vogelzang N.J. Stadler W.M. Kidney cancer.Lancet. 1998; 352: 1691-1696Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar]. Oliver et al. observed 73 patients with metastatic renal cell carcinoma without treatment [4Oliver R.T. et al.A phase 2 study of surveillance in patients with metastatic renal cell carcinoma and assessment of response of such patients to therapy on progression.Mol. Biother. 1988; 1: 14-20PubMed Google Scholar]. In this series, 4% had complete and spontaneous regression of disease and 3% had partial regression of disease. Furthermore, 5% had stable disease with no evidence of progression for more than 12 months. There are also numerous cases of patients who recur with metastatic RCC over 10 years after undergoing a nephrectomy for apparently localized disease. It is presumed that microscopic foci of metastatic disease present at the time of nephrectomy had been held "in check" by the immune system. Although patients with such favorable outcomes may represent a small minority, the question is what accounts for the difference in prognosis, and can medical intervention consistently reproduce favorable outcomes? It is possible that nonimmune mechanisms may be involved; however, several observations underscore the importance of the immune system in determining the course of disease. The most obvious observation is the response of RCC to generalized stimulation of the immune system with cytokines. RCC is one of the most immunosensitive tumors, and interleukin 2 (IL-2) and interferon-α represent the standard of care for the treatment of metastatic disease. IL-2, for example, does not have any direct cytotoxic effects; however, it induces the immune system to produce a cellular cytotoxic response that is specific for RCC. Further evidence for the role of the immune system comes from multiple reports of a direct relationship between a favorable response to immunotherapy and the development of autoimmune thyroiditis [5Franzke A. et al.Autoimmunity resulting from cytokine treatment predicts long-term survival in patients with metastatic renal cell cancer.J. Clin. Oncol. 1999; 17: 529-533PubMed Google Scholar]. The development of autoimmunity may be the by-product of successful immunotherapy. The systemic administration of IL-2, either alone or in combination with interferon-α, can produce objective response rates ranging from approximately 15 to 20% and is associated with significant toxicity [6Bukowski R.M. Cytokine combinations therapeutic use in patients with advanced renal cell carcinoma.Semin. Oncol. 2000; 27: 204-212PubMed Google Scholar, 7Fisher R.I. et al.High-dose aldesleukin in renal cell carcinoma long-term survival update.Cancer J. Sci. Am. 1997; 3: S70-72PubMed Google Scholar, 8Figlin R.A. Renal cell carcinoma management of advanced disease.J. Urol. 1999; 161: 381-386Abstract Full Text Full Text PDF PubMed Scopus (231) ]. To date, attempts to stimulate the immune system more selectively and more efficiently have not produced better results than those seen with systemic cytokine therapy. However, new insights about the immune system and the biology of RCC have allowed us to envision more clearly better therapies that can be implemented with the current repertoire of genetic techniques. One of the major obstacles to developing an effective RCC tumor vaccine has been the lack of tumor-specific antigens [9Neumann E. et al.Heterogeneous expression of the tumor-associated antigens RAGE-1, PRAME, and glycoprotein 75 in human renal cell carcinoma candidates for T-cell-based immunotherapies?.Cancer Res. 1998; 58: 4090-4095PubMed Google Scholar]. However, carbonic anhydrase IX (CA9) has recently been identified as a potential target for immunotherapy. CA9 represents the only characterized protein that is widely and specifically expressed in RCC. CA9 is present in over 80% of primary and metastatic tumors, and it is present in 95–100% of the clear cell variant, which is the most common histologic type [10Bui, M. H., et al. (2002). Carbonic anhydrase IX is an independent predictor of survival in advanced renal clear cell carcinoma: implications for prognosis and therapy. Clin. Cancer Res. (in press)Google Scholar, 11Oosterwijk E. et al.Monoclonal antibody G 250 recognizes a determinant present in renal-cell carcinoma and absent from normal kidney.Int. J. Cancer. 1986; 38: 489-494Crossref PubMed Scopus (323) Google Scholar]. In RCC, the overexpression of CA9 is the direct consequence of a mutation in the von Hippel-Lindau gene, which normally functions to degrade and suppress CA9. Patients that are able to generate tumor-specific cytotoxic T cells have a better prognosis [12Uchida A. Fukata H. Role of NK cell cytotoxic factor against fresh human tumors.Nat. Immun. 1993; 12: 267-278PubMed Google Scholar]. This suggests that an effective tumor vaccine needs to recruit cytotoxic T cells specific for RCC, and antigen presentation is the crucial first step. Attempts to use radiated tumor cells modified ex vivo to produce GM-CSF or to inject directly a plasmid vector containing the IL-2 gene into tumors produced only modest results, as tumor cells are not well suited to presenting antigens [13Dummer R. GVAX (Cell Genesys).Curr. Opin. Invest. Drugs. 2001; 2: 844-848PubMed Google Scholar, 14Hoffman D.M. Figlin R.A. Intratumoral interleukin 2 for renal-cell carcinoma by direct gene transfer of a plasmid DNA/DMRIE/DOPE lipid complex.World J. Urol. 2000; 18: 152-156Crossref PubMed Scopus (32) Google Scholar]. Successful antigen presentation requires the processing and presenting of the antigen in the context of MHC and costimulatory molecules (B7-1/B7-2) [15Irving B.A. Chan A.C. Weiss A. Functional characterization of a signal transducing motif present in the T cell antigen receptor zeta chain.J. Exp. Med. 1993; 177: 1093-1103Crossref PubMed Scopus (251) Google Scholar, 16Chan A.C. Irving B.A. Weiss A. New insights into T-cell antigen receptor structure and signal transduction.Curr. Opin. Immunol. 1992; 4: 246-251Crossref PubMed Scopus (40) Google Scholar]. Dendritic cells (DC) are the primary APC for stimulating T-cell-mediated immune responses [17Banchereau J. Steinman R.M. Dendritic cells and the control of immunity.Nature. 1998; 392: 245-252Crossref PubMed Scopus (12177) Google Scholar], and thus, an attractive strategy is to manipulate DC ex vivo to express tumor-specific antigens and to use this product as a vaccine to induce tumor-specific immunity [18Geiger J.D. et al.Vaccination of pediatric solid tumor patients with tumor lysate-pulsed dendritic cells can expand specific T cells and mediate tumor regression.Cancer Res. 2001; 61: 8513-8519PubMed Google Scholar]. DC can be efficiently harvested from peripheral blood and expanded. Currently a clinical trial is under way at UCLA to test a RCC vaccine using DC derived from peripheral blood and loaded with autologous tumor lysate [19Mulders P. et al.Presentation of renal tumor antigens by human dendritic cells activates tumor-infiltrating lymphocytes against autologous tumor implications for live kidney cancer vaccines.Clin. Cancer Res. 1999; 5: 445-454PubMed Google Scholar]. Another potential stumbling block to developing a RCC vaccine may be the existence of an immunosuppressive microenvironment in the tumor. The concentration of mature DC in a RCC tumor is 40-fold higher compared to the peripheral blood; however, the failure to generate an effective anti-tumor response suggests that tumor-associated factors may be suppressing the immune system [20Thurnher M. et al.Human renal-cell carcinoma tissue contains dendritic cells.Int. J. Cancer. 1996; 68: 1-7Crossref PubMed Scopus (119) Google Scholar]. In an analysis of nephrectomy specimens, less than 10% of leukocytes in the tumor were DC, and activated DC represented 0.15% of the total leukocyte count [21Troy A.J. et al.Minimal recruitment and activation of dendritic cells within renal cell carcinoma.Clin. Cancer Res. 1998; 4: 585-593PubMed Google Scholar]. Taken together, these findings imply that DC are not effectively recruited to the tumor and the tumor does not active the DC. The inefficient stimulation of DC provides a sound rationale for engineering DC to express tumor-specific antigens, activating DC ex vivo, and reinfusing the product as a tumor vaccine. In animal studies, DC-based tumor vaccines were more effective when combined with systemic cytokine therapy [22Shimizu K. et al.Systemic administration of interleukin 2 enhances the therapeutic efficacy of dendritic cell-based tumor vaccines.Proc. Natl. Acad. Sci. USA. 1999; 96: 2268-2273Crossref PubMed Scopus (142) Google Scholar]. This is not surprising as the cytokine provides the stimulus for perpetuation of the immune response. However, with systemic cytokine therapy, you lose the primary advantage of reduced toxicity. Therefore, an alternative is to engineer the DC ex vivo to express both the tumor-specific antigen and an immunomodulatory cytokine such as GM-CSF. This will result in high local cytokine concentrations in the tumor and lower systemic concentrations. We have recently constructed a fusion protein that combines CA9 and GM-CSF, and we are in the process of characterizing the fusion protein [23Tso C.L. et al.Induction of G250-targeted and T-cell-mediated antitumor activity against renal cell carcinoma using a chimeric fusion protein consisting of G250 and granulocyte/monocyte-colony stimulating factor.Cancer Res. 2001; 61: 7925-7933PubMed Google Scholar]. Our goal is to develop a DC vaccine using DC engineered ex vivo to express the CA9 and GM-CSF fusion protein. Although a variety of cytokines can be used, GM-CSF is an attractive cytokine for use in a tumor vaccine for a number of reasons. GM-CSF is a growth factor that causes proliferation and maturation of myeloid progenitor cells. It enhances the cytotoxic potential of macrophages and granulocytes against tumor cells and also acts as a chemotactic agent. Immunization of animals with GM-CSF gene-modified tumor cells can induce protective immunity against subsequent tumor challenge [24Soiffer R. et al.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, 25Kielian T. et al.Granulocyte/macrophage-colony-stimulating factor released by adenovirally transduced CT26 cells leads to the local expression of macrophage inflammatory protein 1alpha and accumulation of dendritic cells at vaccination sites in vivo.Cancer Immunol. Immunother. 1999; 48: 123-131Crossref PubMed Scopus (37) Google Scholar]. Vaccination of patients with irradiated autologous RCC or melanoma cells engineered to secrete human GM-CSF can also induce potent anti-tumor immunity in patients. More importantly, GM-CSF is an essential growth and differentiation factor for DC [24Soiffer R. et al.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]. In every disease, we are looking for the "magic bullet." In RCC the ultimate goal is to activate the immune system specifically against malignant cells and avoid toxicity. Toward this end, ongoing research efforts are directed at identifying new antigenic targets for RCC, improving techniques to present antigens to the immune system, and characterizing and overcoming the immunosuppressive microenvironment of the tumor. What is fascinating about renal cell carcinoma is that there are examples of the immune system truly functioning as the magic bullet. Spontaneous regression of metastatic tumor in patients who have received no treatment is a well-recognized phenomenon in RCC. If genetic and molecular technology is a "gun" capable of firing the magic bullet, then the search continues for the "magic trigger." discussion 386–387