Claire Coeshott

Whole recombinant yeast vaccine activates dendritic cells and elicits protective cell-mediated immunity

There is currently a need for vaccines that stimulate cell-mediated immunity—particularly that mediated by CD8+ cytotoxic T lymphocytes (CTLs)—against viral and tumor antigens. The optimal induction of cell-mediated immunity requires the presentation of antigens by specialized cells of the immune system called dendritic cells (DCs). DCs are unique in their ability to process exogenous antigens via the major histocompatibility complex (MHC) class I pathway as well as in their ability to activate naive, antigen-specific CD8+ and CD4+ T cells. Vaccine strategies that target or activate DCs in order to elicit potent CTL-mediated immunity are the subject of intense research. We report here that whole recombinant Saccharomyces cerevisiae yeast expressing tumor or HIV-1 antigens potently induced antigen-specific, CTL responses, including those mediating tumor protection, in vaccinated animals. Interactions between yeast and DCs led to DC maturation, IL-12 production and the efficient priming of MHC class I- and class II-restricted, antigen-specific T-cell responses. Yeast exerted a strong adjuvant effect, augmenting DC presentation of exogenous whole-protein antigen to MHC class I- and class II-restricted T cells. Recombinant yeast represent a novel vaccine strategy for the induction of broad-based cellular immune responses.

Safety, tolerability and immunogenicity of GS-4774, a hepatitis B virus-specific therapeutic vaccine, in healthy subjects: a randomized study.

BACKGROUND GS-4774 is a recombinant, heat-killed, yeast-based immunotherapy engineered to express hepatitis B virus (HBV)-specific antigens. GS-4774 is being developed as a therapeutic vaccine for chronic HBV infection. The aim of this study was to assess the safety, tolerability and immunogenicity of GS-4774 in healthy subjects. DESIGN This was a randomized, open-label, dose-ascending study. Subjects were allocated to one of three dose groups (n=20 per group) to receive 10, 40 or 80 yeast units (YU; 1YU=10(7) yeast) of GS-4774 in two immunization regimens (five subcutaneous injections at weekly intervals with one monthly booster or three subcutaneous injections at monthly intervals). T-cell-mediated responses were determined by interferon (IFN)-γ enzyme-linked immunospot (ELISpot) assay and lymphocyte-proliferation assay (LPA). RESULTS Adverse events were reported by 39 of 60 (65%) subjects; all were mild or moderate and none was serious. Adverse events occurred most frequently in the highest dose group, 80YU, and the number of individual events was higher after weekly immunization than monthly. The most common adverse events were injection-site reactions. Most (88%) subjects responded to GS-4774 by at least one of the T-cell assays. Following immunization with GS-4774, IFN-γ-producing T-cells specific for HBV antigens were detectable in 30 (51%) subjects. The ELISpot response was observed at all doses, with the highest frequency of responders occurring at the highest dose (10YU: 45%; 40YU: 35%; 80YU: 74%). Proliferative responses to HBV recombinant antigens were observed in 90% subjects; responses were mainly independent of GS-4774 dose and immunization regimen. CONCLUSIONS GS-4774 was safe and well-tolerated in healthy subjects with injection-site reactions being the most frequently reported adverse events. With both weekly and monthly regimens, GS-4774 provided HBV-specific immune responses at all doses evaluated. Further evaluation of GS-4774 is ongoing in patients with chronic HBV infection. CLINICAL TRIAL REGISTRY Clinicaltrials.gov (NCT01779505).

A Whole Recombinant Yeast-Based Therapeutic Vaccine Elicits HBV X, S and Core Specific T Cells in Mice and Activates Human T Cells Recognizing Epitopes Linked to Viral Clearance

Chronic hepatitis B infection (CHB) is characterized by sub-optimal T cell responses to viral antigens. A therapeutic vaccine capable of restoring these immune responses could potentially improve HBsAg seroconversion rates in the setting of direct acting antiviral therapies. A yeast-based immunotherapy (Tarmogen) platform was used to make a vaccine candidate expressing hepatitis B virus (HBV) X, surface (S), and Core antigens (X-S-Core). Murine and human immunogenicity models were used to evaluate the type and magnitude of HBV-Ag specific T cell responses elicited by the vaccine. C57BL/6J, BALB/c, and HLA-A*0201 transgenic mice immunized with yeast expressing X-S-Core showed T cell responses to X, S and Core when evaluated by lymphocyte proliferation assay, ELISpot, intracellular cytokine staining (ICS), or tumor challenge assays. Both CD4+ and CD8+ T cell responses were observed. Human T cells transduced with HBc18–27 and HBs183–91 specific T cell receptors (TCRs) produced interferon gamma (IFNγ following incubation with X-S-Core-pulsed dendritic cells (DCs). Furthermore, stimulation of peripheral blood mononuclear cells (PBMCs) isolated from CHB patients or from HBV vaccine recipients with autologous DCs pulsed with X-S-Core or a related product (S-Core) resulted in pronounced expansions of HBV Ag-specific T cells possessing a cytolytic phenotype. These data indicate that X-S-Core-expressing yeast elicit functional adaptive immune responses and supports the ongoing evaluation of this therapeutic vaccine in patients with CHB to enhance the induction of HBV-specific T cell responses.

Phase II Study of the GI-4000 KRAS Vaccine After Curative Therapy in Patients With Stage I-III Lung Adenocarcinoma Harboring a KRAS G12C, G12D, or G12V Mutation

INTRODUCTION Patients with early-stage lung cancer have a high risk of recurrence despite multimodality therapy. KRAS-mutant lung adenocarcinomas are the largest genetically defined subgroup, representing 25% of patients. GI-4000 is a heat-killed recombinant Saccharomyces cerevisiae yeast-derived vaccine expressing mutant KRAS proteins. The present phase II study assessed the feasibility, immunogenicity, and safety of the GI-4000 vaccine in patients with early-stage, KRAS-mutant lung cancer. MATERIALS AND METHODS Patients with stage I-III KRAS-mutant lung cancer who completed curative therapy were enrolled. The patients received the genotype matched GI-4000 vaccine for ≤ 3 years or until intolerance, disease recurrence, or death. The KRAS antigen T-cell response was assessed using the interferon-gamma enzyme-linked immunospot assay in peripheral blood mononuclear cells. The study was powered to detect an immune response in ≥ 25% of patients. RESULTS A total of 24 patients were enrolled over 28 months. No vaccine-related serious adverse events occurred. One patient withdrew consent because of pain at the injection site. The study met its primary endpoint, with 50% of patients developing an immune response to mutant KRAS. The median number of vaccinations received was 15 (range, 1-19). Ten patients experienced disease recurrence, and 6 died. Compared with the genotypically matched historical controls, the recurrence rates were equivalent but overall survival showed a favorable trend. CONCLUSION GI-4000 was well tolerated and immunogenic when used as consolidation therapy in patients with stage I-III KRAS-mutant lung cancer. The patterns of recurrence and death observed in the present study can be used to design a randomized study of GI-4000 with overall survival as the primary endpoint.

Abstract A28: Immune responses to mutated Ras - development of a yeast-based immunotherapeutic

Background: While mutations in the ras gene and corresponding product are known to be etiologic in a number of human cancers, efforts to block the mutated protein have been largely unsuccessful leading to its characterization as “undruggable.” We have taken an alternative approach involving generation of T cell responses to the mutated protein. GI-4000 is a series of proprietary immunotherapeutics designed to target cells with activating ras mutations using heat-killed Saccharomyces cerevisiae yeast (named Tarmogens: Targeted Molecular Immunogens) genetically engineered to express Ras G12 and Q61 mutations. Tarmogens activate antigen-specific T cell-mediated immune responses that kill target cells expressing a number of cancer antigens including mutated Ras. Activating mutations in ras occur in > 90% of pancreas cancer cases and >20% cases of NSCLC. GI-4000 has been evaluated in phase 2 trials in both these indications. GI-4000 has demonstrated: i) protection in a murine model of lung cancer (1), ii) in the pancreas trial, improvements in median RFS and OS vs placebo in subjects with a favorable proteomic signature (2), iii) also in the pancreas trial, 3 month improvement in median OS (p=NS) in R1 subjects, with 5 month improvement for immune responders (2), iv) improved OS in subjects with NSCLC treated with GI-4000 compared to case matched controls: HR=0.577, p=NS. In addition, Tarmogens specific for other oncologic targets decreased human regulatory T cells (Tregs) in vitro with a reciprocal increase in effector T cells (3). Here we discuss immunologic outcomes in R0 subjects enrolled in the pancreas cancer trial. Methods: In the pancreas cancer study 176 subjects with Ras mutant + adenocarcinoma of the pancreas post resection were randomized 1:1 to GI-4000/gemcitabine or placebo/gemcitabine (stratified by resection status: R0/R1). Three weekly injections of GI-4000 or placebo were followed by 6 cycles of gemcitabine 1000 mg/m 2 iv (day 1, 8, 15, then every 28 days). Monthly GI-4000 or placebo were administered on gemcitabine off-weeks and continued monthly until disease recurrence, intolerable toxicity or death. R0 subjects (n=102) with adequate blood samples available from timepoints throughout the study were assayed for immune response by interferon-γ (IFNγ) ELISpot assay using Ras peptide pools containing the G12 mutation present in the subject9s tumor and a mismatched peptide set identical to the mutation-specific set except at G12. Frequencies of Tregs at baseline and pre-gemcitabine were measured by flow cytometry (n=76). Results: In contrast to the R1 group, there was no increase in Ras mutation-specific IFNγ responses in the R0 GI-4000 group compared to placebo: 16/52 (30.8%) vs 22/50 (44.0%) based on pre-specified criteria. However, naive Tregs (CD4 + /CD45RA + /Foxp3 low ) in the GI-4000 treated group were significantly decreased compared to placebo: 11/42 (26.2%) vs 3/34 (8.8%) subjects had a >2-fold decrease in this fraction (p=0.048 Fisher9s exact test). IFNγ responses and OS were strikingly influenced by G12 mutation and associations of specific Ras mutations with outcome will be discussed. Conclusions: GI-4000 decreases frequencies of Tregs, which could be an important attribute for an immunotherapeutic in the treatment of cancer. GI-4000 also generates mutation-specific immune responses and appears to have clinical activity in pancreas cancer and NSCLC. Immune targeting of the activating mutation may be a promising approach to Ras mutated cancers. References: 1. Lu Y., et al. Cancer Research 64: 5084-88, 2004. 2. Richards D.A., et al. ESMO, Vienna, Austria, Sept 2012. 3. Cereda V., et al. Vaccine 29: 4992-99, 2011. Citation Format: Claire Coeshott, Tom Holmes, Alicia Mattson, Tom King, Zhimin Guo, Allen Cohn, Timothy C. Rodell. Immune responses to mutated Ras - development of a yeast-based immunotherapeutic. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A28. doi: 10.1158/1557-3125.RASONC14-A28

Abstract 5314: A proteomic signature predicts response to a therapeutic vaccine in pancreas cancer; analysis from the GI-4000-02 trial

Background: We have previously reported that adjuvant treatment with a therapeutic vaccine targeting the mutated Ras oncogene product generated mutation-specific T cell responses associated with a trend toward improved survival in patients with post-operative residual disease (R1 resections) but no improvement in the overall population 1 . Initial analysis of 90 pretreatment plasma samples using matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) showed the potential to predict improved RFS and OS for treatment with GI-4000/gemcitabine, but not placebo/gemcitabine. Methods: We have developed a novel technique, combining methods used in recent advances in learning theory (‘deep learning’) with newly-refined MS techniques that allow exploration deeper into the proteome to create diagnostic tests. Using 500,000 laser shot Deep MALDI spectra 2 more than 700 mass spectral features were identified. A subset of these was used to create many multivariate classifiers that were filtered for performance and combined using dropout regularization. This method allows the use of smaller training sets and so left a test set with which performance of the signature could be independently assessed. This new methodology was used to create a test (BDX-001) to identify patients likely to benefit from the addition of GI-4000 to gemcitabine. Results: Using BDX-001 for stratification, subjects who are BDX-001(+) demonstrated a 499 day advantage in median OS when treated with GI-4000/gemcitabine vs. placebo/gemcitabine. Additionally, these subjects demonstrated a 351 day improvement in median RFS. BDX-001 did not predict response for placebo/gemcitabine treated subjects. These results were obtained using only test set data, and although the small sample size prohibited statistical significance, it should give an unbiased test performance estimate to be validated independently. Conclusions: BDX-001 is a test developed using novel proteomic and learning theory methods that appears to predict treatment response to GI-4000 in resected pancreas cancer patients, potentially identifying patients with improved RFS and OS in the GI-4000/gemcitabine arm. We plan to prospectively validate BDX-001 as a companion diagnostic in a future study of GI-4000 in pancreas cancer. References 1. Richards et al, ESMO GI. Annals of Oncology, June 2012 23 (suppl 4) 2. Duncan et al, ASMS 2013, http://asms.inmerge.com/Proceedings/2013Proceedings.aspx. Citation Format: Donald A. Richards, Peter Muscarella, Tanios Bekaii-Saab, Lalan S. Wilfong, Vic Velanovich, Julian Raynov, Patrick J. Flynn, William E. Fisher, Samuel H. Whiting, Constana Timcheva, Tom Holmes, Claire Coeshott, Alicia Mattson, Heinrich Roder, Joanna Roder, Allen Cohn, Timothy C. Rodell. A proteomic signature predicts response to a therapeutic vaccine in pancreas cancer; analysis from the GI-4000-02 trial. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5314. doi:10.1158/1538-7445.AM2014-5314