John J. Nemunaitis

Phase I Study of a Systemically Delivered p53 Nanoparticle in Advanced Solid Tumors

Selective delivery of therapeutic molecules to primary and metastatic tumors is optimal for effective cancer therapy. A liposomal nanodelivery complex (scL) for systemic, tumor-targeting delivery of anticancer therapeutics has been developed. scL employs an anti-transferrin receptor (TfR), scFv as the targeting molecule. Loss of p53 suppressor function, through mutations or inactivation of the p53 pathway, is present in most human cancers. Rather than being transiently permissive for tumor initiation, persistence of p53 dysfunction is a continuing requirement for maintaining tumor growth. Herein, we report results of a first-in-man Phase I clinical trial of restoration of the normal human tumor suppressor gene p53 using the scL nanocomplex (SGT-53). Minimal side effects were observed in this trial in patients with advanced solid tumors. Furthermore, the majority of patients demonstrated stable disease. One patient with adenoid cystic carcinoma had his status changed from unresectable to resectable after one treatment cycle. More significantly, we observed an accumulation of the transgene in metastatic tumors, but not in normal skin tissue, in a dose-related manner. These results show not only that systemically delivered SGT-53 is well tolerated and exhibits anticancer activity, but also supply evidence of targeted tumor delivery of SGT-53 to metastatic lesions.

Vaccines in cancer: GVAX®, a GM-CSF gene vaccine

GVAX® is a granulocyte–macrophage colony-stimulating factor (GM-CSF) gene-transfected tumor cell vaccine. Original work with GM-CSF as a recombinant DNA protein (Leukine) involved proliferative stimulation of macrophages and neutrophils for the purpose of reducing hematopoietic toxicity related to dose-intensive chemotherapy. Following US Food and Drug Administration approval of Leukine several years ago, extensive preclinical results have demonstrated an immunostimulatory effect related to GM-CSF gene when transfected into tumor cells and used as a vaccine (GVAX). Tumor regression and prolonged survival was demonstrated in animal models. Toxicology with GVAX indicated no adverse effects, which enabled further testing in cancer patients. A small number of responses were demonstrated in Phase I trials in immunosensitive cancer patients (renal cell carcinoma and melanoma). However, a series of dramatic complete and durable responses in advanced non-small cell lung cancer patients, demonstrated in recent clinical trials, have generated interest in further development of this vaccine in nontraditional cancer disease types. The rationale of GVAX development and a summary of clinical results are reviewed.

Phase 2 trial of erlotinib with or without PF-3512676 (CPG 7909, a Toll-like receptor 9 agonist) in patients with advanced recurrent EGFR-positive non-small cell lung cancer

This phase 2 study assessed PF-3512676 plus erlotinib in patients with epidermal growth factor receptor-positive advanced non-small cell lung cancer after prior chemotherapy failure. Patients were randomized 1:1 to PF-3512676 (0.20 mg/kg injected subcutaneously once weekly) plus erlotinib (150 mg daily) or erlotinib alone. The primary objective was to estimate progression-free survival (PFS). Patients received PF-3512676 plus erlotinib (n = 18) or erlotinib alone (n = 21). The study was halted because an unplanned interim analysis indicated that large improvement in PFS with addition of PF-3512676 would be unlikely. In the PF-3512676-plus-erlotinib and erlotinib-alone arms, median PFS was 1.6 and 1.7 mo (hazard ratio, 1.00; 95% confidence interval, 0.5–2.0; P = 0.9335), respectively. Salient grade ≥ 3 adverse events in PF-3512676-plus-erlotinib and erlotinib-alone arms were diarrhea (5/0), dyspnea (5/6), fatigue (4/1), other flu-like symptoms (2/0), anemia (2/1), and lymphocytopenia (based on laboratory values, 1/4). Adding PF-3512676 to erlotinib did not show potential for increased progression-free survival over erlotinib alone in patients with advanced recurrent epidermal growth factor receptor-positive non-small cell lung cancer.

Multifunctional vaccines in cancer: the ‘triad’ approach

Are we perhaps unlocking a mystery of cancer management that has eluded us for hundreds of years? Namely, the mystery of how we influence the immune system to control cancer? Tantalizing spontaneous tumor regression has been demonstrated since recorded medical history in 0.1% of melanoma and in rare renal cell cancer patients following primary surgery. Over the last 50 years, in an attempt to duplicate success with infectious disease and antigen awareness, ‘single’ modality vaccines have been tested (whole-cell vaccines, tumor lysates, activated effector cells, various protein small molecules, cytokines, antibody-directed vaccines, and DNAor RNA-based vaccines). However, they have met with limited success and have not approached a level of efficacy (survival advantage) necessary for US FDA product approval. Why is this? There are many reasons, but one which is frequently mentioned is that our animal models are not effective and have minimal correlation to the human response. Another is that we have not identified a surrogate marker or assay consistently correlating immune activity with survival. Nevertheless, our knowledge has improved. For example, there have been many advances in molecular biology that have led to identification of new antigens, cytokines and molecular mechanisms, thereby clarifying our understanding of how immunotherapeutic approaches may impact control over cancer. Dendritic cells (DCs), for instance, engage in cell-mediated immunity and play a central role in the induction of antitumor immunity in tumor-bearing hosts via antigenic cross-presentation [1–4]. DCs efficiently display antigens on MHC class II and stimulate proliferation and activation of CD4 and CD8 T cells. CD4 cells augment the activity of natural killer cells and macrophages, in addition to amplifying antigen-specific immunity via local secretion of cytokines [5,6]. Through this process DCs have been identified as a pivotal component to engage for success of immune-based vaccine technologies.

Current vaccine updates for lung cancer.

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.

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.

Tp53 gene therapy for cancer treatment and prevention

Abnormalities in the tumor suppressor TP53 are among the most common mechanisms of cancer pathogenesis (Lane and Levine 2010) and formed the rationale for TP53 gene therapy to restore normal p53 function in cancer treatment. Several important principles were elucidated in preclinical tumor models which predicted the outcomes of subsequent clinical trials including synergistic antitumor activity for combined TP53 gene therapy plus DNA damaging chemotherapy and radiation (Zhang and Roth 1994; Gjerset and Sobol 1997; Nielsen and Maneval 1998).