Eli Gilboa, Ph.D.

Dodson Professor of Microbiology and Immunology,
Director, Dodson Interdisciplinary Immunotherapy Institute,
Co-leader, Tumor Immunology Research Program, UM/Sylvester Cancer Center

University of Miami, Miller School of Medicine
Room 211
Papanicolaou Cancer Research Building
1550 NW 10th Avenue
Miami, FL 33136

Tel: 305-243-1767 (direct)
305-243-1762 (assistant)
Fax: 305-243-4409

Research Interests: Therapeutic vaccination in the setting of cancer and HIV infection

A multi-pronged approach to cancer immunotherapy

Cancer is a complex disease and formidable challenge, and no “magic bullet” is in sight. The “holy grail” in cancer therapy must, therefore, be a combination approach consisting of multiple & complementary treatments; each treatment providing an incremental benefit and in combination exerting an additive or synergistic impact. Surgery and radiation therapy can reduce, but rarely eliminate, the cancer, necessitating the use of adjuvant therapies to prevent metastasis and recurrence of disease. Chemotherapy, the most frequently used intervention in this setting, is highly effective in some instances but is often limited due to dose-related toxicities. Specific active immunotherapy, or “cancer vaccination” - to induce an immune response against the progressing cancer - offers an alternative or complementary modality. Recent progress and insights from the field of immunology, the identification and isolation of tumor rejection antigens, and vaccination-induced regression of pre-existing tumors in murine studies, have combined to revive the interest in this modality (1).

Conceptually, therapeutic vaccination-immunizing patients with a pre-existing antigenic load-is not unlike prophylactic vaccination, just harder, and will require a potent vaccination strategy. Inducing immunity, namely activating a high proportion of tumor-specific T cells, is only the first step. To engender long lasting protective immunity in the cancer patient it is also important to ensure that the immune response is sustained, to overcome a state of immune suppression in the tumor-bearing patient, and to limit or circumvent the propensity of tumor cells to escape immune elimination (1). Our program, depicted in Figure 1, is developing a multi-pronged approach to cancer immunotherapy addressing the various requisites needed to engender a potent, effective and sustained antitumor response in the cancer patient.

1. Inducing antitumor immunity – activating tumor-specific T cells.

Immunizing with dendritic cells (DC) loaded with tumor antigens is a powerful method of stimulating CD4 + and CD8 + T cell responses and inducing antitumor immunity. Several years ago we introduced the concept of loading DC with antigen by transfection with the corresponding mRNA (2). Generating mRNA encoding tumor antigens is a broadly applicable, simple, and cost effective and comparative studies performed by us and other laboratories have shown that mRNA transfected DC are remarkably effective in stimulating T cell immunity in vitro and in vivo in mice (see for example (3) & reviewed in(4)). A striking feature and hallmark of the early experience was that the majority of vaccinated patients responded immunologically to the vaccine (5, 6). Hints of clinical responses were also seen in early clinical trials (7). A recent clinical trial evaluating the benefits of removing regulatory T cells in renal cancer patients vaccinated with tumor RNA transfected DC has shown that the patient cohort treated with an IL-2-diphteria toxin fusion (ONTAK ®) to deplete regulatory T cells exhibited a statistically significant 3-5 fold increase in the induction of tumor-specific CD8 T cell responses ((8)).

mRNA transfected DC vaccination is arguably a promising approach and could serve as a foundation for an effective treatment, yet in its present form the clinical benefit to the patients will be minimal at best. The challenge and one focus of our program is how to improve this vaccination protocol. This is illustrated with two examples. Dendritic cells maturation is a key and limiting step in the process of generating DC for clinical applications which is accomplished by culturing the DC with a cocktail of biological agents. Current protocols are woefully suboptimal, and the DC generated in such manner exhibit reduced immunopotency. We are developing a strategy that circumvents the need to ex vivo mature DC by injecting immature, antigen-loaded (mRNA transfected), DC into adjuvant pretreated site. The purpose of adjuvant treatment is to induce a local inflammatory reaction conducive for the in situ maturation of the injected DC. Studies in mice have provided preclinical proof-of-concept (9) and a clinical trial in patients with prostate cancer is currently ongoing. In situ DC maturation offers a simpler and potentially superior method to generate immunostimulatory DC for clinical immunotherapy.

The mRNA/DC vaccination approach has been optimized to induce a potent CD8 + CTL response. Yet, accumulating evidence suggests that CD4 + T cells also have a key role in tumor immunity. To address this limitation we are developing strategies to redirect the endogenously expressed antigen into the endosomal class II presentation pathway by appending appropriate targeting signal sequences (10, 11), targeting to autophagosomes or fusing the antigen to enodosomal products.

2. Enhancing persistence of immunity-Aptamer based therapeutics.

The potency of an antitumor immune response is not only a function of how many T cells are activated but also how long they persist. This is perhaps more important in the setting of a chronic disease like cancer where a weak but sustained immune response could be more beneficial than a strong yet transient response. We are developing several approaches to address this important issue. One approach is to co-transfect DC with mRNA encoding ligands to costimulatory molecules such as OX40, 4-1BB or CD40 (12). mRNA transfection of DC avoids the non-specific activation of autoreactive T cells and provides simple and cost effective reagents for preclinical and clinical studies.

A second set of approaches is to use agonistic and antagonistic agents to enhance costimulation, i.e., 4-1BB or OX-40, or block the function of coinhibitory molecules such as CTLA-4 or PD-1. In lieu of using antibodies or soluble ligands, we are developing short oligonuclotide-based aptamers that bind to their respective targets with exquisite specificity and avidity and can either inhibit or activate their function (13). Aptamers represent a new platform technology for drug discovery and offer distinct advantages over protein-based biologicals for human therapy, including manipulating the immune system. In a recent study, Giangrande and her colleagues have shown that aptamers can be used to target siRNA in vivo to selectively kill tumor cells (14). We are developing aptamer-based strategies to potentiate the function of vaccine-induced T cells by targeting siRNA directed to inhibitory products such as TGF b receptor or cAMP cylase fused to aptamers specific to activated T cells such as 4-1BB or OX-40.

3. Countering tumor-induced immune suppression.

There is accumulating evidence suggesting that immune suppression, rather than inherent lack of immunogenicity, plays a dominant role in promoting the continued growth of tumors in the cancer patient. Two strategies targeting immune suppression are currently explored in our program. Aptamer technology is used to block the activity of immune suppressive molecules elaborated by tumor cells such as B7H1 (PD-L1), and decoy receptor-3 (DcR3). A second approach is to remove tumor suppressive regulatory T cells (Treg). As an alternative to targeting CD25, the low affinity IL-2 receptor which is also upregulated on conventional T cells, we are exploring an approach which targets the intracellular Treg-specific foxp3 product using CTL induced by vaccination. Targeting foxp3 with CTL offers important advantages over antibody or targeting of CD25 and early murine studies suggest that the approach has merit (15). Ongoing and future studies will target additional suppressive cells and pathways including myeloid derived suppressive cells, TGF b, Cox-2,, STAT3, the cAMP pathway and more. The goal of these studies is to develop a combination therapy targeting several suppressive pathway to maximize its therapeutic impact.

4. Overcoming immune escape-immunological targeting the tumor stroma.

The well documented genetic instability of tumor cells is responsible for the propensity of tumor cells to evade the immune system via a host of genetic and epigenetic mechanisms. Emergence of treatment-resistant variants will manifest itself in the face of increasingly effective immunization protocols and, not unlike what is seen with chemotherapy, could limit, if not defeat, much of the promise of this treatment modality. Immunological targeting the tumor stroma could go a long way to address this concern. Since stromal cells, unlike tumor cells, are diploid, genetically stable and show limited proliferative capacity, targeting the stroma could substantially reduce the incidence of immune evasion. Stromal products also represent the ultimate form of “universal” antigens that can be targeted in every cancer patients and offer a broad range of candidates to choose from. We and other groups have shown that immunization of mice with DC transfected with mRNA encoding angiogenesis-associated products, VEGR-2, Tie-2 or VEGF, inhibits tumor growth in the absence of significant autoimmune pathology (16). More recently, we have extended this concept to fibroblast infiltrating the tumors by targeting a product, fibroblast activation protein (FAP) which is upregulated on tumor resident fibroblast (17, 18). The purpose of this line of study is to identify a set of 4-6 broadly expressed stromal & tumor antigens that can stimulate potent immunity in the majority of cancer patients with reduced risk of immune escape.

5. New challenges - Therapeutic vaccination in the setting of HIV infection.

The cellular arm of the immune response is pivotal in containing spread of HIV in the infected patient. Like for cancer patients, a pre-existing antigenic load and the immune compromised nature of this patient population will require a highly efficient vaccination protocol to induce potent CD8 +, as well as CD4 +, T cell responses to limit HIV replication. mRNA transfected DC offer a powerful approach to induce potent CD4 + and CD8 + T cell responses under such demanding circumstances. Yet, the extent of genetic instability exhibited by this virus, even compared to tumor cells, threatens to significantly limit, if not defeat, the purpose of vaccination. Thus dominant epitopes present in the chosen prototypic HIV antigen(s) used in the vaccine may not be represented in all patients, and more importantly, in face of an effective CTL response variants which lost the dominant epitopes will emerge. One approach to limit the impact of HIV variability and genetic instability, investigated by several groups, is to identify and vaccinate against conserved epitopes that are not, or less likely, to undergo mutations & selection. The RNA technology offers an alternative and complementary approach - to immunize patients against HIV antigens isolated in “real time”, namely using antigens isolated from patients immediately (within 24-48 h) prior to vaccination. The approach, exploiting a unique feature of the mRNA technology, is to isolate and amplify HIV mRNA from a drop of the patient’ blood which is then transfected onto DC and used to immunize the patient. HIV infected patients can be, therefore, immunized against their own HIV quasispecies using a procedure that was optimized to stimulate CD4 + and CD +8 T cell immunity, and which can be easily repeated when circumstances warrant, namely when the emergence of resistant variants are suspected. Similar rationale and strategy can be also developed for HCV infected patients. Recently, Kavanagh et al. have demonstrated that DC transfected with specific and PCR-amplified mRNA from patient’ HIV isolates can stimulate CD4+ and CD8+ T cell responses in vitro(19). A phase I clinical trial in HIV infected patients is currently in preparation.

References

1. Gilboa, E. 2004. The promise of cancer vaccines. Nat Rev Cancer 4:401-411.

2. Boczkowski, D., S.K. Nair, D. Snyder, and E. Gilboa. 1996. Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo. J Exp Med 184:465-472.

3. Weissman, D., H. Ni, D. Scales, A. Dude, J. Capodici, K. McGibney, A. Abdool, S.N. Isaacs, G. Cannon, and K. Kariko. 2000. HIV gag mRNA transfection of dendritic cells (DC) delivers encoded antigen to MHC class I and II molecules, causes DC maturation, and induces a potent human in vitro primary immune response. J Immunol 165:4710-4717.

4. Gilboa, E., and J. Vieweg. 2004. Cancer immunotherapy with mRNA-transfected dendritic cells. Immunol Rev 199:251-263.

5. Heiser, A., D. Coleman, J. Dannull, D. Yancey, M.A. Maurice, C.D. Lallas, P. Dahm, D. Niedzwiecki, E. Gilboa, and J. Vieweg. 2002. Autologous dendritic cells transfected with prostate-specific antigen RNA stimulate CTL responses against metastatic prostate tumors. J Clin Invest 109:409-417.

6. Su, Z., J. Dannull, A. Heiser, D. Yancey, S. Pruitt, J. Madden, D. Coleman, D. Niedzwiecki, E. Gilboa, and J. Vieweg. 2003. Immunological and clinical responses in metastatic renal cancer patients vaccinated with tumor RNA-transfected dendritic cells. Cancer Res 63:2127-2133.

7. Su, Z., J. Dannull, B.K. Yang, P. Dahm, D. Coleman, D. Yancey, S. Sichi, D. Niedzwiecki, D. Boczkowski, E. Gilboa, and J. Vieweg. 2005. Telomerase mRNA-transfected dendritic cells stimulate antigen-specific CD8+ and CD4+ T cell responses in patients with metastatic prostate cancer. J Immunol 174:3798-3807.

8. Dannull, J., Z. Su, D. Rizzieri, B.K. Yang, D. Coleman, D. Yancey, A. Zhang, P. Dahm, N. Chao, E. Gilboa, and J. Vieweg. 2005. Enhancement of vaccine-mediated antitumor immunity in cancer patients after depletion of regulatory T cells. J Clin Invest 115:3623-3633.

9. Nair, S., C. McLaughlin, A. Weizer, Z. Su, D. Boczkowski, J. Dannull, J. Vieweg, and E. Gilboa. 2003. Injection of immature dendritic cells into adjuvant-treated skin obviates the need for ex vivo maturation. J Immunol 171:6275-6282.

10. Nair, S.K., D. Boczkowski, M. Morse, R.I. Cumming, H.K. Lyerly, and E. Gilboa. 1998. Induction of primary carcinoembryonic antigen (CEA)-specific cytotoxic T lymphocytes in vitro using human dendritic cells transfected with RNA. Nat Biotechnol 16:364-369.

11. Su, Z., J. Vieweg, A.Z. Weizer, P. Dahm, D. Yancey, V. Turaga, J. Higgins, D. Boczkowski, E. Gilboa, and J. Dannull. 2002. Enhanced induction of telomerase-specific CD4(+) T cells using dendritic cells transfected with RNA encoding a chimeric gene product. Cancer Res 62:5041-5048.

12. Dannull, J., S. Nair, Z. Su, D. Boczkowski, C. DeBeck, B. Yang, E. Gilboa, and J. Vieweg. 2005. Enhancing the immunostimulatory function of dendritic cells by transfection with mRNA encoding OX40 ligand. Blood 105:3206-3213.

13. Santulli-Marotto, S., S.K. Nair, C. Rusconi, B. Sullenger, and E. Gilboa. 2003. Multivalent RNA aptamers that inhibit CTLA-4 and enhance tumor immunity. Cancer Res 63:7483-7489.

14. McNamara, J.O., 2nd, E.R. Andrechek, Y. Wang, K.D. Viles, R.E. Rempel, E. Gilboa, B.A. Sullenger, and P.H. Giangrande. 2006. Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras. Nat Biotechnol 24:1005-1015.

15. Nair, S., D. Boczkowski, M. Fassnacht, D. Pisetsky, and E. Gilboa. 2007. Vaccination against the forkhead family transcription factor Foxp3 enhances tumor immunity. Cancer Res 67:371-380.

16. Nair, S., D. Boczkowski, B. Moeller, M. Dewhirst, J. Vieweg, and E. Gilboa. 2003. Synergy between tumor immunotherapy and antiangiogenic therapy. Blood 102:964-971.

17. Fassnacht, M., J. Lee, C. Milazzo, D. Boczkowski, Z. Su, S. Nair, and E. Gilboa. 2005. Induction of CD4(+) and CD8(+) T-cell responses to the human stromal antigen, fibroblast activation protein: implication for cancer immunotherapy. Clin Cancer Res 11:5566-5571.

18. Lee, J., M. Fassnacht, S. Nair, D. Boczkowski, and E. Gilboa. 2005. Tumor immunotherapy targeting fibroblast activation protein, a product expressed in tumor-associated fibroblasts. Cancer Res 65:11156-11163.

19. Kavanagh, D.G., D.E. Kaufmann, S. Sunderji, N. Frahm, S. Le Gall, D. Boczkowski, E.S. Rosenberg, D.R. Stone, M.N. Johnston, B.S. Wagner, M.T. Zaman, C. Brander, E. Gilboa, B.D. Walker, and N. Bhardwaj. 2006. Expansion of HIV-specific CD4+ and CD8+ T cells by dendritic cells transfected with mRNA encoding cytoplasm- or lysosome-targeted Nef. Blood 107:1963-1969.