Phase I trial of "bi-shRNAi(furin)/GMCSF DNA/autologous tumor cell" vaccine (FANG) in advanced cancer

Abstract

We performed a phase I trial of FANG vaccine, an autologous tumor-based product incorporating a plasmid encoding granulocyte-macrophage colony-stimulating factor (GMCSF) and a novel bifunctional short hairpin RNAi (bi-shRNAi) targeting furin convertase, thereby downregulating endogenous immunosuppressive transforming growth factors (TGF) β1 and β2. Patients with advanced cancer received up to 12 monthly intradermal injections of FANG vaccine (1 × 10(7) or 2.5 × 10(7) cells/ml injection). GMCSF, TGFβ1, TGFβ2, and furin proteins were quantified by enzyme-linked immunosorbent assay (ELISA). Safety and response were monitored. Vaccine manufacturing was successful in 42 of 46 patients of whom 27 received ≥1 vaccine. There were no treatment-related serious adverse events. Most common grade 1, 2 adverse events included local induration (n = 14) and local erythema (n = 11) at injection site. Post-transfection mean product expression GMCSF increased from 7.3 to 1,108 pg/10(6) cells/ml. Mean TGFβ1 and β2 effective target knockdown was 93.5 and 92.5% from baseline, respectively. Positive enzyme-linked immunospot (ELISPOT) response at month 4 was demonstrated in 9 of 18 patients serially assessed and correlated with survival duration from time of treatment (P = 0.025). Neither dose-adverse event nor dose-response relationship was noted. In conclusion, FANG vaccine was safe and elicited an immune response correlating with prolonged survival. Phase II assessment is justified.

Figure 1

Vaccine transgene expression. (a) Results of GMCSF gene expression, (b) TGFβ1 knockdown, (c) TGFβ2 knockdown of the 14-day post-vaccine manufacturing quality control assay. The pre-transfection (pre) data are from a tumor cell aliquot removed on day 1 of manufacturing just prior to electroporation. The post-transfection (post) data are from a tumor cell aliquot removed on day 2 of manufacturing just following irradiation. Linear regression was performed on all data sets except for nonlinear regression on the post GMCSF data. The number of patients assayed was 42. Failed vaccines did not have protein data collected. GMCSF, granulocyte-macrophage colony-stimulating factor; TGF, transforming growth factor.

Figure 2

Survival comparison of FANG and No FANG patients. (a) Survival comparison of patients who received <4 FANG (red) vaccinations versus ≥4 vaccinations (blue) (n = 27). (b) Survival comparison of patients receiving FANG (blue) with those not receiving FANG (red) (n = 45). (c) Survival comparison of patients receiving ≥4 vaccinations (blue) with those not receiving FANG (red) (n = 36).

Figure 3

IFN-γ (ELISPOT) in FANG vaccine–treated patient's peripheral blood mononuclear cells in response to non-transfected autologous tumor cells (n = 18). Blue lines indicate nine patients (003, 020, 021, 022, 028, 034, 035, 036, 044) achieving ≥10 IFN-γ producing lymphocytes (positive response) at month 4. Red lines indicate nine patients (008, 018, 019, 024, 031, 032, 039, 041, 042) not achieving positive ELISPOT response at month 4. Green circled dot indicates end of treatment time. ELISPOT, enzyme-linked immunospot.

Figure 4

Survival comparison of patients achieving positive ELISPOT response at month 4 (blue) versus those not achieving positive ELISPOT response at month 4 (red). Survival comparison of patients achieving and not achieving positive ELISPOT response from (a) time of procurement and (b) time of first vaccination (n = 18). ELISPOT, enzyme-linked immunospot.