Joseph Baar

Interferon-α gene therapy for cancer : retroviral transduction of fibroblasts and particle-mediated transfection of tumor cells are both effective strategies for gene delivery in murine tumor models

Stable transfection of tumor cells with IFN-α genes has been shown to result in abrogation of tumor establishment and induction of antitumor immunity. However, strategies suitable for the clinical application of IFN-α gene therapy for cancer have not been reported. In this study, we investigated two gene delivery systems capable of mediating the local paracrine production of high levels of biologically active IFN-α in murine tumor models: retroviral transduction of fibroblasts and particle-mediated transfection of tumor cells. In spite of the antiproliferative effects of IFN-α, it was possible to obtain stable retroviral producer cell lines and transduce a variety of murine tumor cells including syngeneic fibroblasts to stably secrete 2000–5000 U (40–100 ng) murine IFN-α/106 cells/24 h. IFN-α transduction of tumor cells abrogated tumorigenicity in establishment models and induced antitumor immunity in several murine tumor model systems. Importantly, IFN-α gene delivery using retrovirally transduced syngeneic fibroblasts was capable of suppressing the establishment of the poorly immunogenic TS/A mouse mammary adenocarcinoma and induced antitumor immunity. Particle-mediated transient transfection of tumor cells using the gene gun led to the production of up to 20000 U IFN-α/106 cells during the first 24 h and proved to be equally effective in suppressing establishment of TS/A adenocarcinoma and inducing antitumor immunity. These results suggest that retroviral transduction of autologous fibroblasts can serve as an effective gene delivery method for IFN-α gene therapy of cancer. Particle-mediated transfection of freshly isolated tumor cells may represent a clinically attractive alternative approach for nonviral gene delivery. Both strategies circumvent the difficulties in routinely establishing primary tumor cell lines from the vast majority of human cancers.

Phase I trial of BAY 50-4798, an interleukin-2-specific agonist in advanced melanoma and renal cancer

Purpose: BAY 50-4798 is an analogue of interleukin-2 that selectively activates T cells over natural killer cells. This phase I study was designed to determine the maximum tolerated dose (MTD) and safety of BAY 50-4798, screen for tumor response, and assess pharmacokinetics. Experimental Design: Forty-five patients with metastatic melanoma or renal cancer were enrolled, 31 on escalating doses to determine the MTD, with 20 renal cell carcinoma patients treated at MTD to detect antitumor activity. BAY 50-4798 was delivered i.v. every 8 h, days 1 to 5 and 15 to 19, and could be repeated after 9 weeks if tumor was stable or responding. Results: The MTD was defined by and reported in terms of doses received. The doses tested ranged from 1.3 to 26.1 μg/kg, and the MTD was defined as 10.4 μg/kg based on toxicities similar to those of aldesleukin. Two patients achieved partial responses, one with melanoma and one with renal cell carcinoma. Among all 45 patients, 53% and 9% experienced a grade 3 and 4 toxicity, respectively. Among the patients treated at the MTD of 10.4 μg/kg, 71% and 10% experienced a grade 3 and 4 toxicity, respectively. Pharmacokinetics showed dose-dependent peak concentrations (Cmax) and area under the curve with a half-life of ∼2 h and no evidence of accumulation. Lymphocyte subset analysis confirmed the preferential expansion of T-cell subsets over natural killer cells. Conclusions: The antitumor activity of BAY 50-4798 in malignancies that respond to high-dose interleukin-2 was low. BAY 50-4798 might provide advantages over aldesleukin in antigen-specific immunotherapies.

Oncology thinking cap: scaffolded use of a simulation to learn clinical trial design.

Background: Physicians often are called on to participate in and interpret clinical trials, but their training in this area may not provide them with the inquiry skills that are needed. Simulations have the potential to be a promising tool for helping medical students learn the skills involved in clinical trial design. However, simulations may be complex and require additional scaffolding to support learning. Description: The goal of this study was to teach aspects of cancer clinical trial design through the scaffolded use of a simulation, the Oncology Thinking Cap. The software-based scaffolding provided guidance in designing the trial. Subsequently, the simulation allowed students to run the designed trial, which produces detailed patient histories. This feedback then could be used to redesign the trial. Evaluation: Twenty-four 4th-year medical students were asked to design a clinical trial in advance, on paper, to test a new anticancer drug. Student groups then designed and simulated running the clinical trial assisted by the software environment. Instructional effectiveness was measured using a pretest-posttest design that included having students (a) write a group research proposal and (b) individually critique a flawed proposal. At the group level (N = 6 groups), students demonstrated a 34% increase in the number of elements of a clinical trial that they included in their research proposals. At the individual level (N = 24), students improved by 48% in their critiques of flawed proposals. Conclusions: Scaffolding embedded in the simulator is a promising approach to helping students learn about clinical trial design.

DNA Vaccines Targeting Dendritic Cells for the Immunotherapy of Cancer

Cytotoxic T lymphocytes (CTL) play a crucial role in the host’s immune response to cancer. The adoptive transfer of tumor-specific CTL can mediate the regression of established tumors in experimental animal models1 as well as in some patients with melanoma2. Recently, a number of genes encoding tumor-associated antigens (TAA) and their peptide products, which are recognized by cytotoxic T lymphocytes in the context of major histocompatibility complex (MHC) class I molecules, have been identified for both murine and human tumors3,4. These insights now need to be translated into the development and application of novel immunotherapies designed to elicit tumor antigen-specific T cells. Dendritic cells (DC) are believed to be critical for the induction of primary, cell-mediated immune responses5,6. Using freshly isolated, as well as, cultured DC pulsed with peptides constituting relevant CTL-defined epitopes, we and others have been able to induce protective and therapeutic antitumor immune responses in mouse tumor models7–13. Furthermore, autologous cultured human DC pulsed with synthetic melanoma peptides were able to stimulate antigen-specific CTL capable of lysing HLA-matched allogeneic melanoma cells that naturally express these epitopes in vitro 14–16. As an alternative to synthetic peptides, that may restrict the immune response to defined tumor-associated epitopes with known MHC restriction, the use of plasmid DNA or recombinant viruses encoding tumor-associated antigens has recently been investigated for the immunotherapy of cancer17–22. Direct inoculation of naked DNA into the skin or muscle of animals results in both humoral and cellular immune responses.

Potentiation of E7 antisense RNA-induced antitumor immunity by co-delivery of IL-12 gene in HPV16 DNA-positive mouse tumor

Down-regulation of oncogene expression by antisense-based gene therapy has been extensively studied, and in some cases, therapeutic effects have been demonstrated. We have previously shown that down-regulation of HPV16 E6 and E7 gene expression inhibited HPV DNA-positive C3 mouse tumor growth. Although not all of the tumor cells were transfected by pU6E7AS plasmid, complete tumor regression was achieved if the tumor size was small at the start of therapy in a syngeneic host. This suggests that some other antitumor mechanisms may be involved in addition to the direct down-regulation of HPV16 E7 oncogene expression by the antisense effect of E7AS. In the current study, we demonstrated that E7AS induces tumor cell apoptosis. More importantly, a strong antitumor immune response was elicited in the pU6E7AS-treated and tumor-regressed mice. There was no tumor growth after rechallenging the tumor-regressed mice with 1 million C3 cells. This E7AS-induced antitumor immune response was augmented by co-delivery of mIL-12 cytokine gene. The combination therapy strategy resulted in complete regression of 26 of 28 (93%) tumors. Only 12 of 31 (38%) tumors from the group treated with pU6E7AS alone and 14 of 28 (50%) tumors from the group treated with pCMVmIL-12 alone had completely regressed. Complete regression was also demonstrated in tumors located 1 cm from the treated tumors, which indicates that a systemic antitumor effect was induced by E7AS and mIL-12. Immunohistochemistry demonstrated that a significant amount of CD4+ and CD8+ cells infiltrated into tumors treated with pU6E7AS, pCMVmIL-12 and pU6E7AS+pCMVmIL-12. These data indicate that host immunity is an important factor for antisense-based gene therapy approach which can be further enhanced by combination with cytokine gene therapy.

Pilot trial of a type I - polarized autologous dendritic cell vaccine incorporating tumor blood vessel antigen-derived peptides in patients with metastatic breast cancer

Cancer vaccines based on tumor-associated antigens are rarely curative in advanced cancer. This limitation relates to the heterogeneity of cancer due to defects in antigen presentation and altered immunophenotypes. Therefore, another method to promote anti-tumor immunity is to prime T cells against tumor-associated stromal cells. We have reported [1] that IL-12 gene therapy of established HLA-A2neg B16 melanomas in HLA-A2+ transgenic mice resulted in CD8+ T cell-mediated immunity against the host HLA-A2+ stromal cells within the tumor microenvironment (TME). We have also shown [2] that vaccines based on a subset of tumor blood vessel antigen (TBVA)-derived peptides (DLK1, EphA2, HBB, NRP1, PDGFRβ, RGS5 or TEM1) prevented HLA-A2neg MC38 tumor establishment and promoted the regression of tumors in HLA-A2+ mice by CD8+ T cell targeting of HLA-A2+ pericytes and vascular endothelial cells in the TME. Based on this pre-clinical data, we propose to undertake a Susan G. Komen -funded (IIR13261822) clinical trial of chemo-immunotherapy using the immunomodulatory drug gemcitabine with a dendritic cell vaccine pulsed with six HLA-A2-presented TBVA-derived peptides (DLK1310-318, EphA2883-891, HBB31-39, NRP1433-441, RGS55-13 and TEM1691-700) in 30 HLA-A2+ patients with metastatic breast cancer. The specific aims of this study are to determine vaccine-induced generation of TBVA-Tc1 immunity and clinical response.