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 Cloughesy4

1Henry Ford Hospital; 2University of California, Irvine; 3Cleveland Clinic Foundation; 4University of California, Los Angeles; 5Ontario Brain Institute; 6Tocagen Inc.

© 2017 Tocagen Inc.

Introduction and BackgroundToca 511 (Vocimagene amiretrorepvec) is an investigational retroviral replicating vector (RRV) thatencodes the transgene cytosine deaminase (CD) not present in human cells1. Toca 511 can be deliveredby multiple routes and selectively infects and spreads in tumor cells. Subsequent oral administrationof investigational extended-release 5-FC (Toca FC) results in formation of 5-FU within infected tumorcells expressing CD2. 5-FU kills cancer cells and Myeloid Derived Suppressor Cells (MDSCs) leading toimmune activation against the tumor via a combination of mechanisms. This sequence of events isamplified with multiple cycles of Toca FC. Treatment with Toca 511 and Toca FC selectively destroyscancer 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-FUSTEP 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. MolTher, 2012. 20:1689-16982. Ostertag, D., et al. Brain tumor eradication and prolonged survival from intratumoral conversion of 5-fluorocytosine to 5-fluorouracil using a nonlyticretroviral replicating vector. Neuro Oncol, 2012. 14(2): p. 145-593. 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 108 to 4.8 x 1010

• 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

SURGERY

IntracranialToca 511

Cyclic Toca FC

IntravenousToca 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

DAPICytosine

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 50

2

4

61 01 21 41 6

n u m b e r o f IV a d m in is tra tio n s

CD

pro

tein

ex

pre

ss

ion

% a

rea

sta

ine

d p

os

itiv

e

T Cell Infiltration in Tumor Does Not Limit Cytosine Deaminase Expression After IV Delivery of Toca 511

X axis log transformed T cell (CD3)

Cyto

sine

Dea

min

ase

R2=0.20Data 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 511CD3 – 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-FUAnticancer

Drug

5-FC (Toca FC)Antifungal

Prodrug

Structural RRV genesRegulatory 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

Cyto

sine

Dea

min

ase

Increased Regulatory T Cell Infiltration is Not Associated With Increased Cytosine Deaminase Expression

Treg (CD3+Foxp3+)

R2=0.11

Y axis log transformed

Cyto

sine

Dea

min

ase

R2=0.11Data 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 511Foxp3 – 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

Perc

enta

ge o

f Sur

vivi

ng P

atie

nts

Time to Death (months)Number of Patients at Risk17 17 13 11 9 6 5 2 11 11 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 3rd 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 1st 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

7/11/2014Preop

5/15/2014Baseline

6/16/2014 2/11/2015 7/20/2015 1/4/2016 11/28/2016

Lesio

n 1

Lesio

n 2

04-302

Treatment-Related Adverse Events

*No treatment-related deaths.

MTD not defined• DLT at 1.5 x 1010 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)