Results of a randomized phase I gene therapy clinical trial of nononcolytic fowlpox viruses encoding T cell costimulatory molecules

Abstract

Oncolytic viruses have shown promise as gene delivery vehicles in the treatment of cancer; however, their efficacy may be inhibited by the induction of anti-viral antibody titers. Fowlpox virus is a nonreplicating and nononcolytic vector that has been associated with lesser humoral but greater cell-mediated immunity in animal tumor models. To test whether fowlpox virus gene therapy is safe and can elicit immune responses in patients with cancer, we conducted a randomized phase I clinical trial of two recombinant fowlpox viruses encoding human B7.1 or a triad of costimulatory molecules (B7.1, ICAM-1, and LFA-3; TRICOM). Twelve patients (10 with melanoma and 2 with colon adenocarcinoma) enrolled in the trial and were randomized to rF-B7.1 or rF-TRICOM administered in a dose escalation manner (~3.7×10(7) or ~3.7×10(8) plaque-forming units) by intralesional injection every 4 weeks. The therapy was well tolerated, with only four patients experiencing grade 1 fever or injection site pain, and there were no serious adverse events. All patients developed anti-viral antibody titers after vector delivery, and posttreatment anti-carcinoembryonic antigen antibody titers were detected in the two patients with colon cancer. All patients developed CD8(+) T cell responses against fowlpox virus, but few responses against defined tumor-associated antigens were observed. This is the first clinical trial of direct (intratumoral) gene therapy with a nononcolytic fowlpox virus. Treatment was well tolerated in patients with metastatic cancer; all subjects exhibited anti-viral antibody responses, but limited tumor-specific T cell responses were detected. Nononcolytic fowlpox viruses are safe and induce limited T cell responses in patients with cancer. Further development may include prime-boost strategies using oncolytic viruses for initial priming.

FIG. 1.

Antibody responses to rF-B7.1 and rF-TRICOM vaccines. (A) Patient sera before (pre) and after (post) treatment were tested for fowlpox virus antibody by standard ELISA, and resulting antibody titers are shown (n=12 patients). (B and C) ELISA (graph) and immunoblotting results (rectangles at top) from the sera of two patients with colon cancer (patients 1 and 5, respectively) tested for anti-CEA antibody before (pre) and after one (Post 1), two (Post 2), or three (Post 3) rF treatments.

FIG. 2.

Characterization of fowlpox virus-specific HLA-A2 epitopes. Recognition of fowlpox virus HLA-A2-restricted peptides by cytotoxic T lymphocyte (CTL) lines was tested with peptide-pulsed HLA-A2 melanoma cells by LDH assay. (A) Absorbances at 500 nm in response to a fowlpox virus epitope recognized by patient CTLs (ILAPFNFKV), conducted at various ratios of effectors (patient CD8⁺ CTLs) to targets (patient PBMCs loaded with HLA-A2-restricted peptides). (B) Resultant absorbances at 500 nm in response to one of the fowlpox virus epitopes (MLMETSFYI) that did not result in CTL responses. These results shown are representative of three other peptides that did not result in peptide recognition.

FIG. 3.

Inhibition of IFN-γ secretion after HLA-ABC blockade. IFN-γ secretion was measured in postvaccination PBMCs from patients (n=7) treated with fowlpox virus lysate and either control IgG or HLA-ABC blocking antibody. Percent suppression by HLA-ABC blocking antibody was calculated as follows: difference in the number of prevaccination and postvaccination IFN-γ-secreting cells divided by the number of prevaccination IFN-γ-secreting cells.