622 FANGâ„¢ Cytokine Expression Analyses Update Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy S240 CANCER IMMUNOTHERAPY III 620 Summary of[.]
CANCER-IMMUNOTHERAPY III 620 Summary of bi-shRNAfurin/GM-CSF Augmented Autologous Tumor Cell Vaccine (FANG™) in Advanced Cancer of the Liver Minal Barve,1,3 Phillip B Maples,2 Douglas Orr,5 Joseph Kuhn,6 Mitchell Magee,7 Jeffrey Lamont,5 Cynthia Bedell,1 Gladice Wallraven,2 Beena O Pappen,2 Alyssa Roth,1 Staci Horvath,1 Derek Nemunaitis,1 Padmasini Kumar,2 Neil Senzer,1,2 John Nemunaitis.1,2,3,4 Mary Crowley Cancer Research Centers, Dallas, TX; 2Gradalis, Inc., Dallas, TX; 3Texas Oncology, P.A., Dallas, TX; 4Medical City Dallas Hospital, Dallas, TX; 5Baylor Medical Center, Dallas, TX; 6WLS Surgical Associates, P.A., Dallas, TX; 7Cardiovascular Specialty Associates of North Texas, P.A., Dallas, TX There are limited systemic options available for the management of advanced hepatocellular cancer (HCC) In a Phase I trial of a novel autologous whole-cell tumor cell vaccine (FANG™) incorporating a plasmid with an expressive GM-CSF transgene and a bifunctional shRNA interference moiety targeting furin, we demonstrated safety; confirmed mechanism; verified immunoeffectiveness via tumor-specific gIFN ELISPOT conversion and ELISPOT-survival correlation; and suggested an overall survival benefit [Senzer et al Mol Ther 2012; Senzer et al Vaccines and Vaccination in press] FANG™ vaccine, combining 1) knockdown of the proconvertase furin which consequently limits tumor expression of TGFβ1 and TGFβ2 (both potent inhibitors of immune response), 2) expression of GM-CSF and 3) provision of the full patient-specific tumor antigenic matrix, enables a broadened dimensional approach to stimulation of the afferent arm of the immune response We now provide further follow up of a subset of hepatocellular carcinoma (HCC) patients Vaccine manufacturing was successful in 7/8 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 x 107 cells/ intradermal injection, 6-11 vaccinations) No vaccine toxicity or unique HCC-related toxicities were observed Three of these patients demonstrated evidence of an immune response per gIFN ELISPOT assay Long-term follow up demonstrates survivals of 319, 729, 784, 931+ and 1043+ days of the FANG treated patients In conclusion, the data support Phase II trial assessment of FANG vaccine in HCC 621 Phase I Study of FANG™ Vaccine in Advanced Ewing’s Sarcoma M Ghisoli,1,2 C Lenarsky,1 N Senzer,2,4 M Magee,3 J Lanoue,5 E Mendeloff,6 C Bedell,2 P Kumar,4 D D Rao,4 G Wallraven,4 B O Pappen,4 P B Maples,4 J Nemunaitis.1,2,3,4 Texas Oncology, PA, Dallas; 2Mary Crowley Cancer Research Centers, Dallas; 3Medical City Dallas Hospital, Dallas; Gradalis, Inc, Dallas; 5Pediatric Surgical Associates, Dallas, TX; 6Congenital Heart Surgery Center, Medical City Children’s Hospital, Dallas Ewing’s Sarcoma, uniquely characterized by expression of EWSFLI1 fusion protein, is the second most common malignant bone tumor among children and adolescents Patients and physicians continue to be frustrated by the lack of therapeutic gains in meaningful improvement in overall survival for patients after failure of conventional chemotherapy The FANG™ vaccine contains a plasmid encoding both GMCSF and an RNA interference (RNAi) moiety, bifunctional shRNAfurin (bi-shRNAfurin) that targets and down regulates the proprotein convertase Furin resulting in knockdown of both TGFb1 and b2 The bi-shRNA technology is designed to maximize target inhibition via a process involving amalgamated mRNA cleavage, degradation and p-body sequestration GMCSF enhances dendritic cell functionality and expands the cytotoxic T-cell population This S240 “triad” concept (enhanced effector cell activation + inhibition of innate immunosuppression + antigenic matrix exposure) promotes immune de-tolerization and enhances the afferent arm of the immune response Based on our initial encouraging results in adult cancer in which safety, confirmation of component mechanism and apparent survival advantage was demonstrated [Senzer et al Mol Ther 2012], a Phase I study was initiated in patients (≥12 y.o.) with advanced/relapsed Ewing’s sarcoma who failed standard treatment options Briefly, the vaccine is cGMP manufactured at Gradalis, Inc following autologous tumor tissue harvest Vaccine is produced at a dose of 1xcells/injection It is administered intradermally once a month at a minimum of to a maximum of 12 doses ELISPOT analysis of cytotoxic T cell function to autologous tumor antigens is monitored at baseline, Months 2, 4, 6, 9, 12, and 18 GMSCF production is also measured To date, 11 pediatric patients have entered the screening process: with relapsed disease and with progressive disease Vaccine manufacturing was successful in of the 11 harvested patients (2 patients had insufficient viable cells and had contaminated tissue) Five patients have received vaccine; three of whom experienced progressive disease Two patients received and doses, respectively, and both have stable disease (494 and 99 days from enrollment) A total of 16 doses have been given There have been no treatmentrelated serious adverse effects, one patient had a grade treatmentrelated AE limited to skin erythema at the injection site Three of evaluable patients showed ELISPOT activation (see Table) High levels of knockdown were achieved for TGFb1 (88–100%, mean 98%), TGFb2 (84–100%, mean 95%) and Furin (74–98%, mean 89%) This limited initial data shows the safety and feasibility of this study and possible clinical benefit Updated results will be presented ELISPOT rxn’s (+ > 10) Time Pt 062 Pt 081 Pt 083 Base 0 Mo - 143 101 Mo - 108 106 Mo 82 Mo 105 Mo 12 206 Mo 14 186 Mo 17 136 622 FANG™ Cytokine Expression Analyses Update Padmasini Kumar,1 Connor Phalon,1 Beena O Pappen,1 Gladice Wallraven,1 Yang Yu,1 Fabienne Norvell,1 Himabindu Menakuru,1 Chris M Jay,1 Zhaohui Wang,1 Donald D Rao,1 John Nemunaitis,1,2,3,4 Neil Senzer,1,2 Phillip B Maples.1 Gradalis, Inc., Dallas, TX; 2Mary Crowley Cancer Research Centers, Dallas, TX; 3Medical City Dallas Hospital, Dallas, TX; Texas Oncology, P.A., Dallas, TX We have developed an autologous whole cell tumor vaccine, FANG™, incorporating a plasmid encoding GMCSF and a novel bifunctional short hairpin RNA (bi-shRNA) targeting FURIN FURIN, also known as furin/PACE/SPC1, is an enzyme, which belongs to the subtilisin-like proprotein convertase family The proprotein convertases processes latent precursor proteins into their biologically active products One of the furin substrates is the family of transforming growth factor beta (TGFβ) precursors which have been shown to play a significant role in tumor progression, metastasis, and tumor induced immunosuppression Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy CANCER-IMMUNOTHERAPY III An aliquot of cells from before and after transfection with FANG™ plasmid were placed in culture and incubated at 37ºC Cells and supernatants were collected on Days 0, 1, 2, 3, 4, 7, 10 and 14 GMCSF, TGFβ1, TGFβ2 (R&D Systems) and Furin (USCN Life Sciences) proteins were quantified by enzyme-linked immunosorbent assay (ELISA) kits We demonstrated earlier an average increase (n=85) in the expression of GMCSF from 7.3 to 1,108 pg/106cells/ ml at Day Mean TGFβ1 and β2 effective target knockdown was 93.5 and 92.5% from baseline, respectively (Senzer et.al Mol Ther 2011) Furin knockdown was 85.09% The percentage knockdown of TGFβ1 and TGFβ2 were significant with p