Retroviral Replicating Vectors Deliver Cytosine Deaminase Leading to Targeted 5-Fluorouracil-Mediated Cytotoxicity in Multiple Human Cancer Types

Abstract

Toca 511 is a modified retroviral replicating vector based on Moloney c-retrovirus with an amphotropic envelope. As an investigational cancer treatment, Toca 511 preferentially infects cancer cells without direct cell lysis and encodes an enhanced yeast cytosine deaminase that converts the antifungal drug 5-fluorocytosine to the anticancer drug, 5-fluorouracil. A panel of established human cancer cell lines, derived from glioblastoma, colon, and breast cancer tissue, was used to evaluate parameters critical for effective anticancer activity. Gene transfer, cytosine deaminase production, conversion of 5-fluorocytosine to 5-fluorouracil, and subsequent cell killing occurred in all lines tested. We observed >50% infection within 25 days in all lines and 5-fluorocytosine LD 50 values between 0.02 and 6 lg/ml. Although we did not identify a small number of key criteria, these studies do provide a straightforward approach to rapidly gauge the probability of a Toca 511 and 5-fluorocytosine treatment effect in various cancer indications: a single MTS assay of maximally infected cancer cell lines to determine 5-fluorocytosine LD 50 . The data suggest that, although there can be variation in susceptibility to Toca 511 and 5-fluorocytosine because of multiple mechanistic factors, this therapy may be applicable to a broad range of cancer types and individuals.INTRODUCTION THE IDEA OF USING replication-competent viruses as a direct therapy for the treatment of cancer emerged more than a century ago.1 The c-retrovirus murine leukemia virus (MLV) is an attractive tool for tumor gene therapy, as its nonlytic life cycle and requirement for host cell division allow for tumor-selective enhanced gene transfer 2 and spread throughout the tumor. This MLV-based approach to gene therapy has been used to deliver and express the cytosine deaminase (CD) gene into tumor cells, providing a specific conversion of the well-tolerated antifungal prodrug, 5-fluorocytosine (5-FC), into the potent chemotherapeutic 5-fluorouracil (5-FU), which has been well established in preclinical studies. [2][3][4][5][6] Building on this concept, Tocagen's retroviral replicating vector (RRV) named Toca 511 7 (vocimagene amiretrorepvec) preferentially infects tumors without immediate cell killing and encodes an optimized yeast cytosine deaminase (yCD) that converts 5-FC into 5-FU within infected tumors. 3,4,7,8 We are currently investigating the clinical utility, in recurrent high-grade glioma, of Toca 511 in combination with Toca FC, an investigational orally administered extended-release formulation of 5-FC (NCT01470794, NCT01156584, NCT01985256, and NCT02414165).CD is lacking or poorly expressed in most human cells but is often expressed in yeast and bacteria as part of the pyrimidine salvage pathway and is responsible for converting cytosine to uracil and ammonia. CD also catalyzes, by means of a deamination step, the conversion of the prodrug 5-FC to the chemotherapeutic drug 5-FU 9,10 within cancer cells expressing gene therapy-delivered CD. CDbased prodru...

Therapeutic activity of retroviral replicating vector-mediated prodrug activator gene therapy for pancreatic cancer

“…As shown previously in a variety of other malignancies [27,28,36], here we show that RRV also shows efficient and selective infection of all human and murine PDAC cells tested in vitro. After inoculation of RRV-GFP, the percentage of GFP positive cells increased logarithmically and remained persistently high, at levels of 90% or more, through serial passages.…”

Toca 511 gene transfer and treatment with the prodrug, 5-fluorocytosine, promotes durable antitumor immunity in a mouse glioma model

“… 2–4 We have shown that this treatment modality has broad applicability in multiple tumor types. 5 , 6 Additionally, we and our colleagues (Hiraoka et al, accompanying article) show that this therapeutic approach also exerts an extended immunotherapeutic effect. These data are further supported by an abundance of evidence that cytotoxic anticancer drugs, including 5-FU, can promote antitumor immune responses.…”

Delayed Progression of Lung Metastases Following Delivery of a Prodrug-activating Enzyme

“…Breton and colleagues performed a study using a modified Ankara virus to express yeast CD in situ, reducing tumor growth in a glioblastoma mouse model (10). After introducing a viral vector for in vitro transformation of several cancer cell lines (12,31) and evaluation in preclinical models (32) not detected significant immunogenicity of bCD in animals (33,34). We do not anticipate that bCD 1525 would have immunogenic properties different from those of the regular protein, but this should be evaluated before translation to human use.…”

“…To minimize sideeffects of 5-FU, targeted delivery of bCD to tumor cells coupled with systemic administration of 5-FC is also being considered. Monoclonal antibodies conjugated to the enzyme, as well as viral vectors and non-viral vectors, have been used to achieve this goal (6)(7)(8)(9)(10)(11)(12)(13). Recent studies have demonstrated prostate cancer cell-specific nanoparticle delivery of bCD to tumor xenografts by specific targeting of prostate-specific membrane antigen that resulted in improved tumor control (13,14) following localized conversion of 5-FC to 5-FU.…”