Intravenous Delivery of Toca 511 in Patients With High Grade Glioma Results in Quantifiable Expression of Cytosine Deaminase in Tumor Tissue Tobias Walbert, 1 Daniela Bota, 2 Michael A Vogelbaum, 3 Steven N Kalkanis, 1 Linda M Liau, Tom Mikkelsen, 1,5 Leah A Mitchell, 6 Maria E Rodriguez-Aguiree, 6 Derek Ostertag, 6 Douglas J Jolly, 6 Harry E Gruber, 6 Jolene S Shorr, 6 Timothy F Cloughesy 4 1 Henry Ford Hospital; 2 University of California, Irvine; 3 Cleveland Clinic Foundation; 4 University of California, Los Angeles; 5 Ontario Brain Institute; 6 Tocagen Inc. © 2017 Tocagen Inc. Introduction and Background Toca 511 (Vocimagene amiretrorepvec) is an investigational retroviral replicating vector (RRV) that encodes the transgene cytosine deaminase (CD) not present in human cells 1 . Toca 511 can be delivered by multiple routes and selectively infects and spreads in tumor cells. Subsequent oral administration of investigational extended-release 5-FC (Toca FC) results in formation of 5-FU within infected tumor cells expressing CD 2 . 5-FU kills cancer cells and Myeloid Derived Suppressor Cells (MDSCs) leading to immune activation against the tumor via a combination of mechanisms. This sequence of events is amplified with multiple cycles of Toca FC. Treatment with Toca 511 and Toca FC selectively destroys cancer cells within the body, while leaving healthy cells unharmed. Toca 511- optimized RRV expressing CD, a prodrug activator gene Conclusion SCDT-06 Research supported by: Tumor STEP 1 - Toca 511 & CD Brain and tumor samples from Tocagen clinical trial patients selectivity of cancer cells 5-FU CD 5-FU CD 5-FU STEP 2 - Toca FC CD Proposed MOA: Tumor killing and anti-cancer immune activation immune activation Toca FC- investigational extended-release oral formulation of 5-FC • 5-FC crosses blood-brain barrier and is approved for fungal infections of the brain • CD converts 5-FC to 5-FU within infected cells • GBM cell lines and MDSCs are sensitive to 5-FU • 5-FU inhibits thymidylate synthase, perturbs RNA synthesis, and affect glycosylation of proteins and lipids • 5-FU mediated killing triggers anticancer immunity from within tumor with systemic benefit • 5-FU has a very short half-life so systemic toxicity is not observed REFERENCES: 1. Perez, O. D. et al. Design and selection of Toca 511 for clinical use: modified retroviral replicating vector with improved stability and gene expression. Mol Ther, 2012. 20:1689-1698 2. Ostertag, D., et al. Brain tumor eradication and prolonged survival from intratumoral conversion of 5-fluorocytosine to 5-fluorouracil using a nonlytic retroviral replicating vector. Neuro Oncol, 2012. 14(2): p. 145-59 3. Mitchell LA et al. Toca 511 gene transfer and treatment with the prodrug, 5-fluorocytosine, promotes durable antitumor immunity in a mouse glioma model. Neuro Oncol, 2017. 19(7): p. 930-939 4. Cloughesy TF. et. al. Phase 1 trial of vocimagene amiretrorepvec and 5-fluorocytosine for recurrent high-grade glioma. Sci Transl Med. 2016 Jun 1;8(341) Many thanks to all of the patients, their families and caregivers, and to individuals and groups providing financial support Toca 511 & Toca FC – Clinical Development in Primary and Metastatic Brain Tumors Indication Preclinical Phase 1 Phase 2/3 Recurrent high grade glioma (rHGG) Advanced solid tumors (including brain metastases) Newly diagnosed high grade glioma Clinical Results Phase 1 Ascending Dose Trials in rHGG Explored Different Delivery Techniques for Toca 511 Injection into cavity wall after removal of tumor Resection NCT01470794 Direct injection into tumor NCT01156584 Intratumoral Intravenous NCT01985256 Injection IV prior to resection and into cavity wall at resection • Open label, ascending dose • Toca 511 – 4.8 x 10 8 to 4.8 x 10 10 • Toca FC – 135 to 300 mg/kg/day x 7 days, q 4-6 weeks Design Objectives • Vector deposition in tumor • MTD; safety and tolerability • Overall survival, objective response Modified from T.F. Cloughesy, MD, SNO, November 20, 2015 Study Population (n = 17) • Recurrent HGG • Planned resection ≥ 80% • 18-80 years old • KPS ≥ 70 S U R G E R Y Intracranial Toca 511 Cyclic Toca FC Intravenous Toca 511 (1, 3, or 5 days) Analysis of cytosine deaminase expression in tumor Study Overview for the Intravenous Trial Characteristic n = 17 Age, median (range) 52.0 years (28-77) Male, n (%) 8 (47.1) White, n (%) 17 (100) KPS, n (%) 70-80 3 (17.6) 90 11 (64.7) 100 3 (17.6) Demographic & Baseline Characteristics Neuro-Oncology History Tissue Processing for Analysis of Toca 511 IHC analysis performed on resected tumor tissue after IV delivery of Toca 511 to determine if T cell infiltrates altered viral entry and/or spread • Portions of resected tissue representing various regions of tumor were formalin fixed and paraffin embedded (FFPE) • Adjacent portions of tumor were frozen for PCR analysis of cytosine deaminase expression PCR IHC IV Administration of Toca 511 Results in Expression of Cytosine Deaminase Protein in Tumor DAPI Cytosine Deaminase gag Merge gag protein localizes with cytosine deaminase expression after IV delivery of Toca 511 Nuclear stain Target protein encoded by Toca 511 1 of 3 major proteins encoded by the retroviral genome IV Injection of Toca 511 on 3 Consecutive Days Trends With Higher Expression of Cytosine Deaminase Protein in Tumors 1 3 5 0 2 4 6 10 12 14 16 number of IV administrations CD protein expression % area stained positive T Cell Infiltration in Tumor Does Not Limit Cytosine Deaminase Expression After IV Delivery of Toca 511 X axis log transformed T cell (CD3) Cytosine Deaminase R 2 =0.20 Data points of a given color represent samples from the same patient – 1 “cytosine deaminase high” and 1 “cytosine deaminase low” sample analyzed/patient Cytosine deaminase – target protein encoded by Toca 511 CD3 – pan T cell marker Data suggest immunologically “hot” tumors with high T cell infiltrate will not limit Toca 511 entry and spread Tumor selectivity and replication in cancers cells is driven by: • Defects in the innate immune system of cancer cells • Virus enters some normal cells, but is rapidly eliminated by innate and acquired immunity • Virus spreads through tumor without triggering immune system • Virus only infects dividing cells Optimized CD (cytosine deaminase) 5-FU Anticancer Drug 5-FC (Toca FC) Antifungal Prodrug Structural RRV genes Regulatory genes CD gene Regulatory genes 5-FU has a very short half-life with direct cell killing localized to cancer microenvironment RRV = Retroviral replicating vector Increased Regulatory T Cell Infiltration is Not Associated With Increased Cytosine Deaminase Expression Treg (CD3+Foxp3+) R 2 =0.11 Y axis log transformed Cytosine Deaminase R 2 =0.11 Data points of a given color represent samples from the same patient – 1 “cytosine deaminase high” and 1 “cytosine deaminase low” sample analyzed/patient Cytosine deaminase – target protein encoded by Toca 511 Foxp3 – transcription factor identifying regulatory T cells Data suggest Toca 511 does not rely on Treg-mediated immune suppressive microenvironment for entry and spread Median Overall Survival Percentage of Surviving Patients Time to Death (months) Number of Patients at Risk 17 17 13 11 9 6 5 2 1 1 1 1 0 Censored observation Patients censored at last date of contact. Median OS = 13.6 months (95% CI 5.8, 19.7) Data cut-off 17 April 2017. Best Overall Response – Independent Radiologic Review • Best response (Macdonald criteria) – stable disease in 3 of 17 patients (17.6%) • 2 of 17 patients achieved radiologic responses with delayed onset following clinical progression – Anaplastic astrocytoma (IDH1 wt) at 3 rd recurrence • Completed 5 cycles of Toca FC; no subsequent anticancer therapy • PR noted on routine follow-up MRI, 9 months after discontinuing Toca FC • Patient died (PD) > 2.5 years after initiation of treatment – GBM (IDH1 mt) at 1 st recurrence • Completed 12 cycles of Toca FC at time of response • CR onset 13 months after initiation of Toca FC • Remains in response as of last assessment – duration 16+ months • Toca FC ongoing; no other anticancer therapy Data cut-off 17 April 2017. Durable Complete Radiologic Response in GBM (IDH1 mt) Patient Treated With Toca 511 & Toca FC 7/11/2014 Preop 5/15/2014 Baseline 6/16/2014 2/11/2015 7/20/2015 1/4/2016 11/28/2016 Lesion 1 Lesion 2 04-302 Treatment-Related Adverse Events *No treatment-related deaths. MTD not defined • DLT at 1.5 x 10 10 TU Toca 511 – Vasogenic cerebral edema (Grade 3) • No additional DLTs observed Data cut-off 17 April 2017. • Following IV delivery of Toca 511, cytosine deaminase protein is detectable and quantifiable in resected HGG tumor tissue • Toca 511 can infect and spread in “hot” and “cold” tumors, with activity independent of high levels of immunosuppressive T cells in the tumor microenvironment • mOS of 13.6 months is consistent with other Toca 511 & Toca FC ascending dose studies – 14.4 months for resection followed by Toca 511 injection – 13.8 months for delivery of Toca 511 via stereotactic biopsy needle • Late onset (> 12 months) radiologic responses in 2 patients (1 GBM, IDH1 mt; 1 anaplastic astrocytoma, IDH1 wt), consistent with immunologic response • Toca 511 & Toca FC appeared to be well tolerated – few Grade ≥ 3 treatment-related adverse events • Results support evaluation of Toca 511 & Toca FC in other solid tumors, including metastatic brain tumors (Toca 6)