Thomas H. King

Yeasts encoding tumour antigens in cancer immunotherapy

Immunotherapy for cancer represents an attractive therapeutic target because of its specificity and lack of toxicity, but products investigated so far have been limited by neutralisation, complexity of manufacturing and requirement for patient-specific products. Recombinant yeast cells are capable of stimulating the immune system to produce highly specific and potent cellular responses against target protein antigens with little toxicity. Data from animal models suggest that Tarmogens™ (yeast-based immunotherapeutics) can elicit protective immunity against xenografted and chemically induced tumours. This concept is now being tested in a Phase I trial in patients with colorectal, pancreatic and non-small cell lung cancers.

Combination Therapy with a Second-Generation Androgen Receptor Antagonist and a Metastasis Vaccine Improves Survival in a Spontaneous Prostate Cancer Model

Purpose: Enzalutamide, a second-generation androgen antagonist, was approved by the U.S. Food and Drug Administration (FDA) for castration-resistant prostate cancer (CRPC) treatment. Immunotherapy has been shown to be a promising strategy for prostate cancer. This study was performed to provide data to support the combination of enzalutamide and immunotherapy for CRPC treatment. Experimental Design: Male C57BL/6 or TRAMP (transgenic adenocarcinoma of the mouse prostate) prostate cancer model mice were exposed to enzalutamide and/or a therapeutic vaccine targeting Twist, an antigen involved in epithelial-to-mesenchymal transition and metastasis. The physiologic and immunologic effects of enzalutamide were characterized. The generation of Twist-specific immunity by Twist-vaccine was assessed. Finally, the combination of enzalutamide and Twist-vaccine to improve TRAMP mice overall survival was evaluated. Results: Enzalutamide mediated immunogenic modulation in TRAMP-C2 cells. In vivo, enzalutamide mediated reduced genitourinary tissue weight, enlargement of the thymus, and increased levels of T-cell excision circles. Because no changes were seen in T-cell function, as determined by CD4+ T-cell proliferation and regulatory T cell (Treg) functional assays, enzalutamide was determined to be immune inert. Enzalutamide did not diminish the ability of Twist-vaccine to generate Twist-specific immunity. Twist was confirmed as a valid tumor antigen in TRAMP mice by immunohistochemistry. The combination of enzalutamide and Twist-vaccine resulted in significantly increased overall survival of TRAMP mice compared with other treatment groups (27.5 vs. 10.3 weeks). Notably, the effectiveness of the combination therapy increased with disease stage, i.e., the greatest survival benefit was seen in mice with advanced-stage prostate tumors. Conclusions: These data support the combination of enzalutamide and immunotherapy as a promising treatment strategy for CRPC. Clin Cancer Res; 19(22); 6205–18. ©2013 AACR.

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.

Construction and Immunogenicity Testing of Whole Recombinant Yeast-Based T-Cell Vaccines

GlobeImmunes Tarmogen(®) immunotherapy platform utilizes recombinant Saccharomyces cerevisiae yeast as a vaccine vector to deliver heterologous antigens for activation of disease-specific, targeted cellular immunity. The vaccines elicit immune-mediated killing of target cells expressing viral and cancer antigens in vivo via a CD8(+) CTL-mediated mechanism. Tarmogens are not neutralized by host immune responses and can be administered repeatedly to boost antigen-specific immunity. Production of the vaccines yields stable off-the-shelf products that avoid the need for patient-specific manufacturing found with other immunotherapeutic approaches. Tarmogens for the treatment of chronic hepatitis B and C and various cancers were well tolerated and immunogenic in phase 1 and 2 clinical trials encompassing >600 subjects. The platform is being widely utilized in basic vaccine research and the most rapid path to success in these endeavors follows from optimal immunoassay selection and execution. This chapter provides detailed methods for the construction and preclinical immunogenicity testing of yeast-based immunotherapeutic products to support the rapid and efficient use of this versatile technology.

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

GI-19007, a Novel Saccharomyces cerevisiae-Based Therapeutic Vaccine against Tuberculosis

ABSTRACT As yet, very few vaccine candidates with activity in animals against Mycobacterium tuberculosis infection have been tested as therapeutic postexposure vaccines. We recently described two pools of mycobacterial proteins with this activity, and here we describe further studies in which four of these proteins (Rv1738, Rv2032, Rv3130, and Rv3841) were generated as a fusion polypeptide and then delivered in a novel yeast-based platform (Tarmogen) which itself has immunostimulatory properties, including activation of Toll-like receptors. This platform can deliver antigens into both the class I and class II antigen presentation pathways and stimulate strong Th1 and Th17 responses. In mice this fusion vaccine, designated GI-19007, was immunogenic and elicited strong gamma interferon (IFN-γ) and interleukin-17 (IL-17) responses; despite this, they displayed minimal prophylactic activity in mice that were subsequently infected with a virulent clinical strain. In contrast, in a therapeutic model in the guinea pig, GI-19007 significantly reduced the lung bacterial load and reduced lung pathology, particularly in terms of secondary lesion development, while significantly improving survival in one-third of these animals. In further studies in which guinea pigs were vaccinated with BCG before challenge, therapeutic vaccination with GI-19007 initially improved survival versus that of animals given BCG alone, although this protective effect was gradually lost at around 400 days after challenge. Given its apparent ability to substantially limit bacterial dissemination within and from the lungs, GI-19007 potentially can be used to limit lung damage as well as facilitating chemotherapeutic regimens in infected individuals.

Abstract 752: The effect of food on the pharmacokinetics of tivozanib

Background: Tivozanib, a potent, selective, long-half-life tyrosine kinase inhibitor of vascular endothelial growth factor receptors 1, 2, and 3, has demonstrated antitumor activity in a Phase II study in patients with renal cell carcinoma (RCC), and is currently being studied in clinical trials in patients with RCC and other solid tumors. The goal of this study was to investigate the effect of food on the pharmacokinetics (PK) of a single 1.5 mg dose of tivozanib in healthy subjects. Methods: This was a single-center, open-label, randomized, two-period, crossover Phase I trial. Subjects were admitted to the clinical research unit (CRU) 1 day before dosing, fasted ∼10 hours, and were randomized to fed (standard high-fat breakfast)/fasted or fasted/fed sequence. In each phase of the sequence, subjects received a single oral dose of tivozanib 1.5 mg with a 6-week washout period between doses. Subjects remained at the CRU for at least 48 hours post dose for blood sample collection and safety monitoring, and were assessed on an outpatient basis for up to 504 hours post dose. PK data were analyzed by non-compartmental methods. The effect of food on PK was assessed using standard criteria for bioequivalence based on the exposure parameters AUC 0-α and C max . If the 90% confidence intervals for the fed/fasted AUC 0-α and C max fell within the range of 80% to 125%, it was concluded that food had no effect on exposure. Results: Thirty healthy volunteers were enrolled (19M/11F; mean age 39 years [range 22-53 years]). There was no significant difference in AUC 0-α of serum tivozanib between the fed and fasted states (107.4%; Table 1). Food caused a significant decrease in serum tivozanib levels vs fasted state (C max : 77.5%). Conclusions: These results indicate that dosing tivozanib with food decreases maximal concentrations by ∼ 23%, but does not affect overall exposure. As tivozanib is dosed chronically in oncology patients and accumulates ∼6 to 7 times single-dose levels when at steady-state, these results are not likely to affect dosing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 752. doi:1538-7445.AM2012-752

Abstract 2561: Yeast vector-encoding multiple MUC1 agonist epitopes (yeast-MUC1) can induce MUC1-specific T-cell immune responses

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The tumor-associated antigen MUC1 is overexpressed on various human hematological and epithelial malignancies. The MUC1 molecule, which has an N-terminal (MUC1-N) and a C-terminal (MUC1-C, which has been shown to act as an oncogene), is an attractive target for cancer immunotherapy. We have identified and reported 10 agonist epitopes (7 in the C-terminus, 2 in the N-terminus VNTR region, and 1 in the non-VNTR region) that enhance production of CD8 cytotoxic T lymphocytes. These epitopes span HLA-A2, HLA-A3, and HLA-A24 MHC class I alleles, thus encompassing the majority of the North American population. Compared to native epitopes, these agonist epitopes more efficiently generate MUC1-specific T cells, induce production of IFN-gamma by MUC1-specific T cells, and lyse human tumor cells expressing MUC1 native epitopes in an MHC-restricted manner. We have previously shown that heat-killed recombinant Saccharomyces cerevisiae yeast genetically modified to express carcinoembryonic antigen can efficiently activate human dendritic cells (DCs) and stimulate CEA-specific CD8+ T cells. Here, we investigated the ability of a Saccharomyces cerevisiae vector containing the MUC1 transgene (yeast-MUC1) and encoding MUC1 with 8 agonist epitopes to activate human DCs, stimulate MUC1-N- and MUC1-C-specific T cells, and generate MUC1-specific T cells. We show here for the first time that human DCs treated with yeast-MUC1 vectors can activate MUC1-N and MUC1-C agonist-specific T-cell lines and can act as antigen-presenting cells to generate MUC1-N- and MUC1-C-specific T cells specific to each agonist epitopes, and that these T cells are capable of lysing MUC1-expressing tumor cells. Together, these findings provide a rationale for further clinical evaluation of yeast-MUC1 constructs encoding MUC1 agonist epitopes in cancer vaccine immunotherapy. Citation Format: Kwong Y. Tsang, Benjamin Boyerinas, Caroline Jochems, Tim Rodell, Thomas King, Jeffey Schlom. Yeast vector-encoding multiple MUC1 agonist epitopes (yeast-MUC1) can induce MUC1-specific T-cell immune responses. [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 2561. doi:10.1158/1538-7445.AM2014-2561