Therapies for advanced hepatocellular carcinoma (HCC) are limited. We carried out a phase I trial of a novel autologous whole-cell tumor cell immunotherapy (FANG™), which incorporates a dual granulocyte macrophage colony-stimulating factor (GM-CSF) expressive/bifunctional small hairpin RNA interference (bi-shRNAi) vector. The bi-shRNAi DNA targets furin, which is a proconvertase of transforming growth factors beta (TGFβ) 1 and 2. Safety, mechanism, immunoeffectiveness, and suggested benefit were previously shown [Senzer et al.: Mol Ther 2012;20:679-689; Senzer et al.: J Vaccines Vaccin 2013;4:209]. We now provide further follow-up of a subset of 8 HCC patients. FANG manufacturing was successful in 7 of 8 attempts (one failure due to insufficient cell yield). Median GM-CSF expression was 144 pg/106 cells, TGFβ1 knockdown was 100%, and TGFβ2 knockdown was 93% of the vector-transported cells. Five patients were vaccinated (1 or 2.5 × 107 cells/intradermal injection, 6-11 vaccinations). No FANG toxicity was observed. Three of these patients demonstrated evidence of an immune response to the autologous tumor cell sample. Long-term follow-up demonstrated survival of 319, 729, 784, 931+, and 1,043+ days of the FANG-treated patients. In conclusion, evidence supports further assessment of the FANG immunotherapy in HCC.

Current treatments for lung cancer are far from optimal. Several immunotherapeutic strategies involving vaccines incorporating different tumor-associated antigens to induce immune responses against tumors are being tested in clinical trials internationally. Although small, benefits have indeed been observed from the early studies of these vaccines, and the future is looking brighter for lung cancer patients as a handful of these immunotherapies reach Phase III trials. In addition, optimizing the induced immune response by these vaccines has become a priority, and a number of techniques are being considered, including addition of adjuvants and combining vaccines, which affect synergy based on their mechanism of action. This review is an update on the current vaccines in production, the benefits observed from their most recent studies, and the upcoming plans for improvements in these immunotherapies.

TAG vaccine is a novel 'triad vaccine' that involves transfection of autologous tumor with a dual plasmid, TGFβ2 antisense gene and GM-CSF gene. Patients with advanced cancer who failed standard therapy were treated. IFN-γ ELISPOT analysis (Enzyme-Linked Immunospot Assay for Interferon Gamma) using TAG autologous vaccine target cells was performed prior to vaccination and at week 12 after the third vaccination. The purpose of this assessment was to correlate the IFN-γ ELISPOT immune response with long-term survival of advanced cancer patients who received TAG vaccination. Twenty-three of 28 patients received ⩾3 TAG vaccinations (two patients withdrew consent and three had disease progression prior to the third vaccination). Eleven patients demonstrated a positive ELISPOT response (>10 spots and ⩾2 × baseline) at week 12 and 12 patients did not (P=0.002). Median survival from time of treatment between ELISPOT-positive and -negative groups was significantly different (550 vs 159 days, P=0.036), as was median survival from the time of procurement (627 vs 257 days, respectively, P=0.043). In conclusion, the IFN-γ ELISPOT assay may provide an effective measure of immune response following treatment with 'triad vaccines', but additional patient numbers and/or other immune modulatory parameters are necessary for future testing.

Study Background: Previously, we demonstrated safety and correlated induced immune response with survival in a Phase I study of FANG immunotherapy in advanced cancer patients. We now report long term follow-up (FU) of Phase I treated patients including assessment of relationships of dose, γIFN-ELISPOT response, and patient demographics to safety and survival. Methods: Safety, γIFN-ELISPOT response, and survival have been followed through 3+ years in advanced cancer patients who received ≥ 2-12 intradermal monthly injections of 1×107 or 2.5×107 cells/injection. Clinical and serological assessments were performed monthly, radiographic evaluations bimonthly, and γ-IFN-ELISPOT at baseline, and start of Cycle 2, 4, 6, 9, 12 then sequentially at FU. Results: Previously, we reported results on 45 patients with successful FANG construction followed for 1 year (28 treated (designated FANG); 17 not treated based on availability of other alternative treatments or failed manufacturing (designated No FANG)). We now report FU results through year 3 on those patients and an additional 29 patients (7 FANG, 22 No FANG) subsequently entered into Phase I study (total N=35 FANG; total N=39 No FANG). The median survival of the current expanded Phase I trial population is 562 days vs. 122 days (p=0.00001). This is similar to the originally published data from two years earlier. The γ-IFN-ELISPOT reaction was positive in 14 of the current FANG treated patients and negative in 12 FANG treated patients at Month 3 or less post first injection. Survival correlated with γ-IFN-ELISPOT reaction; median 836 days vs. 440 days with positive and negative ELISPOT respectively, (p=0.04). No long term adverse toxicity has been seen and there was no significant correlation of immune response or survival with either dose or demographics. Conclusions: Treatment with FANG vaccine continues to show long term safety and evidence of benefit in patients with many types of advanced cancer thereby justifying further efficacy testing.

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.

Purpose: On the basis of the hypothesis that the combined expression of immunostimulatory granulocyte macrophage colony stimulating factor (GM-CSF) and antitumor suppressor TGF-β2 antisense (AS) transgenes can break tolerance and stimulate immune responses to cancer-associated antigens, we constructed an expression plasmid [the tumor-associated glycoprotein (TAG) plasmid] that coexpresses GM-CSF and TGF-β2 AS nucleotide sequences and which was incorporated into an autologous whole-cell vaccine. Experimental Design: Patients undergoing resection were enrolled. Freshly harvested autologous tumor cells were mechanically and enzymatically disaggregated, then electroporated with the TAG vector. The resulting vaccine was irradiated, then aliquoted and cryopreserved until the time of injection. Patients received a minimum of 5 to a maximum of 12 monthly intradermal injections. Immune function was monitored at baseline and at months 3 and 6. Results: Vaccine manufacturing efficiency was 84% (32/38). Twenty-three patients received at least 1 vaccination. There were no grade 3 or 4 toxicities, and grade 1 and 2 events were local in nature. Seventeen of 21 patients had stable disease (SD) at month 2 or later as their best response, and 1 patient with stage IVa malignant melanoma achieved a complete response (CR) following 11 vaccinations and remains without evidence of disease 2 years following initiation of therapy. Six of 13 patients displayed a positive enzyme-linked immunospot (ELISPOT) response to autologous TAG vaccine at week 12 including 3 patients with prolonged SD or CR. The 3 other patients survived through week 24, as compared with none of the 7 ELISPOT-negative patients. Conclusions: On the basis of safety and clinical and immunologic results, further evaluation of bifunctional vaccines is warranted. Clin Cancer Res; 17(1); 183–92. ©2011 AACR.