Minal A. Barve

Induction of Immune Responses and Clinical Efficacy in a Phase II Trial of IDM-2101, a 10-Epitope Cytotoxic T-Lymphocyte Vaccine, in Metastatic Non-Small-Cell Lung Cancer

PURPOSE Generation of broad cytotoxic T-lymphocyte responses against multiple epitopes and tumor-associated antigens (TAAs) may provide effective immunotherapy in patients with cancer. We evaluated a single-vial peptide vaccine consisting of nine HLA-A2 supertype-binding epitopes (two native and seven analog epitopes modified for optimal HLA binding or T-cell receptor stimulation) covering five TAAs and the universal helper pan-DR epitope, formulated as a stable emulsion with incomplete Freunds adjuvant (Montanide ISA 51; Seppic SA, Paris, France). The clinical efficacy, safety, and multiepitope immunogenicity of IDM-2101 was evaluated in patients with stage IIIB or IV non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS A total of 63 patients were enrolled who were positive for HLA-A2. End points included survival, safety, and immune response. IDM-2101 (previously EP-2101) was administered every 3 weeks for the first 15 weeks, then every 2 months through year 1, then quarterly through year 2, for a total of 13 doses. Epitope-specific cytotoxic and helper T-lymphocyte immunogenic responses were measured by the interferon gamma enzyme-linked immunosorbent spot assay. RESULTS No significant adverse events were noted. Low-grade erythema and pain at the injection site were the most common adverse effects. One-year survival in the treated patients was 60%, and median survival was 17.3 months. One complete and one partial response were identified. Survival was longer in patients demonstrating an immune response to epitope peptides (P < .001). CONCLUSION IDM-2101 was well tolerated, and evidence of efficacy was suggested.

Phase I Trial of TGF-β2 Antisense GM-CSF Gene-Modified Autologous Tumor Cell (TAG) Vaccine

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.

Summary of bi-shRNAfurin/GM-CSF Augmented Autologous Tumor Cell Immunotherapy (FANG™) in Advanced Cancer of the Liver

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.

Long Term Follow Up: Phase I Trial of "bi-shRNA furin/GMCSF DNA/ Autologous Tumor Cell" Immunotherapy (FANG™) in Advanced Cancer

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.

Follow-up of bi-shRNA furin / GM-CSF Engineered Autologous Tumor Cell (EATC) Immunotherapy Vigil® in patients with advanced melanoma

Over the last decade, management of melanoma has dramatically evolved from chemotherapy through targeted molecular therapy (BRAF V600E signaling) and, currently, immunotherapy (checkpoint inhibitors, immunogenic oncolytic viruses). Response, time to progression and survival has improved for many melanoma patients undergoing targeted therapy, but insensitive population subsets, adaptive resistance and toxic side effectslimit therapeutic benefit. Previous studies have shown a correlation between Vigil engineered autologous tumor cell (EATC) immunotherapy induced circulating activated T-cells responsive against autologous tumor cells and survival prolongation. We now assess the safety and response to Vigil (1 x 107 cells/ intradermal injection monthly x 4-12) in 12 patients with advanced metastatic melanoma in comparison with 12 who underwent similar standard of careautologous tumor harvest but received other treatment regimens,not Vigil. None of the patients experienced≥ Grade 3 treatment-related toxicity. Two Grade 2 adverse events (AE) (fatigue, irritability) and local regionalGrade 1 AE (injection site erythema, induration, rash, skin hypopigmentation)in 19 of 63 injections were observed. IFN-γ ELISPOT analysis (PBMC) showed the induction of T-cell activation from 0-1 at baseline to 78 spots/106 cells post first cycle of Vigil. Median survival of Vigil treated patients was 20 months compared to 7 months (KaplanMeier analysis, log rank p=0.00009). In conclusion, preliminary evidence of safety and activity of Vigil supportsfurther clinical evaluation in advanced melanoma. Correspondence to: John Nemunaitis, M.D., Mary Crowley Cancer Research Centers, 12222 Merit Drive, Suite 1500, Dallas, Texas 75251, USA, Tel: 214-6581964, Fax: 214-658-1992, E-mail: jnemunaitis@marycrowley.org Received: September 15, 2016; Accepted: September 25, 2016; Published: September 29, 2016 Introduction MAGE-A3. MAGE-A1, NY-ESO-1 and SSX-2), a high tumor mutation burden (TMB) leading to an increased number of tumor-specific epitopes, and clinicallya reproducible response rate to immunotherapies [1-4] particularly to the recently FDA approved immune checkpoint inhibitors. One of these inhibitors is ipilimumab (Yervoy; a human monoclonal antibody (hMAb) CTLA-4 inhibitor), which was FDA approved in 2011 for patients with advanced, unresectable Stage III and IV melanoma [5]. Results show improvement in recurrence-free survival (RFS) as compared to placebo in the EORTC trial 18071 (HR 0.75, 95% CI 0.64 – 0.90), [6]. Pembrolizumab (Keytruda), ahMAbPD-1 inhibitor, subsequently demonstrated response rates of 36% [7] and has proven to be superior to chemotherapy and single agent ipilimumab in patients with advanced melanoma [8-10] as has nivolumab (Optivo) [11]. However, >60% of melanoma patients do not achieve an optimal response to a single agent checkpoint inhibitor and subsets of patients (i.e. PD-L1-; low TMB) predictively respond less favorably. Although the combination of mechanistically different immune checkpoint inhibitors elicits higher response rates, in a randomized trial of nivolumab alone,ipilimumab alone, or the combinationof the two in treatment-naïve patients with unresectable stage III or IV melanoma,the combination achieved an ORR of 57.6% (compared to 43.7% with nivolumaband 19% with ipilimumab) with a durable response of 11.5 months,but with 55% treatment-related Grade 3 or higher toxicities. Furthermore, in 36.4% of patients the combination leads to treatment-related discontinuation[9]. Although these data confirm the effectiveness of immunotherapy in advanced melanoma, they alsohighlight the need for further development of novel and/or combinatory immunotherapies with increased, predictable effectiveness at a lower risk of toxicity. Talimogenelaherparepvec(T-VEC), a genetically-modified, immuneenhanced H. simplex type I virus, is systemically effective in advanced melanoma [12] but the FDA indication is limited to Stages IIIb, IIIc or IVM1a disease that are unresectable based on regional efficacy shown in Phase III testing [13,14]. Vigil is a DNA engineered autologous tumor cell (EATC) immunotherapy. It contains a dual vector; a bi-shRNA targeting furinthe pro-protein convertase that activates the immunosuppressive TGF-beta 1 and 2 and the gene encoding hGM-CSF. A phase I clinical Barve M (2016) Follow-up of bi-shRNAfurin /GM-CSF Engineered Autologous Tumor Cell (EATC) Immunotherapy Vigil in patients with advanced melanoma Volume 1(3): 81-86 Biomed Genet Genomics, 2016 doi: 10.15761/BGG.1000116 trial of Vigil in patients with heavily pretreated advanced solid tumors showeda significant survival benefit which correlated with induction of an immune response measured by the interferon gamma (IFN-γ) ELISPOT assay. We now update the results of Vigil clinical activity in patients with advanced melanoma. Materials and methods The method and mechanism of construction and manufacturing of Vigil (formerly known as FANG) has previously been described [15,16]. The Vigil vector encodes for GM-CSF expressive cDNA and the bi-sh RNAfurin in autologous tumor cells. Following protocol-specific informed consent, tumor tissue is harvested, placed in sterile media and delivered to the Gradalis, Inc. manufacturing facility (Carrollton, TX, USA). Vigil is manufactured over 2 conservative days. Subsequent manufacturing, following FDA discussion, now utilizes Gentamicin in the sterile media in order to reduce contamination risk. First, autologous tumor cells are dissociated into a single-cell suspension, followed by electroporation(which allows cell transfection with the plasmid), and overnight incubation. Then the cells are irradiated, placed into the final vials, cryopreserved, and undergo release testing. Following product release by Quality Assurance compliance, patients are registered for treatment every 4 weeks with 1.0 x 107cells/injectionof Vigil.

Case Report: Immune-mediated Complete Response in a Patient With Recurrent Advanced Ewing Sarcoma (EWS) After Vigil Immunotherapy.

Ewing sarcoma is a highly resistant disease with a <10% chance of survival at 5 years after failure of frontline chemotherapy. This is a case report of an Ewing sarcoma patient with metastatic disease recurrence <2 years after standard chemotherapy/radiation who achieved a durable and sustained complete response after 2 series of treatments with Vigil (GMCSF/bi-shRNA furin DNA autologous tumor immunotherapy) serially manufactured from first and second recurrences with ELISPOT assay correlation. Results support justification of further testing of Vigil with ELISPOT assay as a biomarker to assess level of immune response and correlation with disease control.