Yeast Cytosine Deaminase Research Articles

THER-06. THERAPEUTIC EFFICACY OF RRV-MEDIATED PRODRUG ACTIVATOR GENE THERAPY IN CLINICAL TRIALS OF RECURRENT HIGH-GRADE GLIOMA AND IN MURINE ORTHOTOPIC MODELS OF INTRACEREBRAL GLIOMA AND INTRACEREBELLAR MEDULLOBLASTOMA

Toca 511, a clinical-stage tumor-selective retroviral replicating vector (RRV), encodes optimized yeast cytosine deaminase (CD), which converts the prodrug 5-fluorocytosine (5-FC) to the active drug 5-fluorouracil (5-FU) within infected cancer cells. In preclinical models of intracerebral glioblastoma, 5-FU generated locally by Toca 511 (RRV-CD) prodrug activator gene therapy has also been shown to kill immunosuppressive myeloid cells in the tumor microenvironment, leading to anti-cancer immune activation and long-term survival. Early-phase clinical trials of Toca 511 in recurrent high-grade glioma showed highly promising evidence of therapeutic benefit, leading to a Phase III trial completed in late 2019 (n=400 patients, randomized 1:1 vs. standard chemotherapy), which appeared to show negative results overall. However, additional analysis showed possible efficacy in prespecified subgroups, and further clinical investigation is being pursued. In preclinical studies, we have also evaluated RRV for use in medulloblastoma, the most common malignant tumor of the pediatric nervous system. Both established and primary human medulloblastoma cell lines supported efficient RRV replication in vitro, with spread to >90% of cells by day 10 post-inoculation, and RRV-CD-transduced medulloblastoma cells showed significant dose-dependent reduction of viability upon exposure to 5-FC, compared to controls. In an intracerebellar HDMB03 medulloblastoma model, RRV-CD-treated mice exhibited long-term survival while on sequential cycles of 5-FC prodrug, until prodrug treatment was stopped, after which 25% long-term survival was observed (median survival 110 days) as compared to controls (median survival 28 days, 100% lethality) (p=0.00007). These results support further evaluation of RRV-mediated prodrug activator gene therapy for pediatric brain tumors.

EXTH-44. DEVELOPMENT OF RETROVIRAL REPLICATING VECTORS ENCODING SEQUENCE-OPTIMIZED NITROREDUCTASE FOR PRODRUG ACTIVATOR GENE THERAPY IN HUMAN GLIOMA MODELS

Abstract Early-phase clinical trials of Toca 511, a retroviral replicating vector (RRV) expressing an optimized form of yeast cytosine deaminase for prodrug activator gene therapy, showed highly promising evidence of therapeutic benefit and increased survival in recurrent high-grade glioma, but an international Phase 3 trial failed to meet primary and secondary endpoints. Nonetheless, promising results observed within predetermined subgroups, combined with the highly favorable safety profile of this approach, make it worthwhile to consider alternative prodrug-activating enzymes in order to increase therapeutic efficacy. In this study, we employed RRV to deliver modified versions of another prodrug activator gene, E.coli nitroreductase (NTR), which activates the prodrug CB1954 to generate a potent bifunctional alkylating agent. We constructed RRV encoding two different isoforms of wild-type E.coli NTR genes (NfsA, NfsB) as well as variants of both isoforms extensively modified to optimize human codon usage and vector stability. Further efforts to improve the potency of these prodrug activator enzymes included the construction of additional new vectors incorporating point mutations at the active site which have previously been demonstrated to improve prodrug conversion efficiency. NTR transgene insertion did not affect RRV replication, which resulted in increasing NTR expression over time in human glioma cells in culture. Sequence optimization resulted in enhanced genomic stability of the vectors upon serial passage and also in significantly reduced cell viability in vitro on treatment of fully transduced U87 and U87-EGFRviii glioma cells with CB1954 prodrug. In orthotopic U87-EGFRviii intracerebral tumor models, stereotactic intratumoral injection of sequence-optimized RRV-NTR and repeated cycles of prodrug treatment resulted in rapid and significant reduction of tumor burden as monitored by bioluminescence imaging, leading to prolonged survival benefit. These data indicate that improved therapeutic efficacy can be achieved with optimized NTR prodrug activator gene therapy delivered by RRV in experimental glioma models.

Hypoxia theranostics of a human prostate cancer xenograft and the resulting effects on the tumor microenvironment

Hypoxia is frequently observed in human prostate cancer, and is associated with chemoresistance, radioresistance, metastasis, and castrate-resistance. Our purpose in these studies was to perform hypoxia theranostics by combining in vivo hypoxia imaging and hypoxic cancer cell targeting in a human prostate cancer xenograft. This was achieved by engineering PC3 human prostate cancer cells to express luciferase as well as a prodrug enzyme, yeast cytosine deaminase, under control of hypoxic response elements (HREs). Cancer cells display an adaptive response to hypoxia through the activation of several genes mediated by the binding of hypoxia inducible factors (HIFs) to HRE in the promoter region of target gene that results in their increased transcription. HIFs promote key steps in tumorigenesis, including angiogenesis, metabolism, proliferation, metastasis, and differentiation. HRE-driven luciferase expression allowed us to detect hypoxia in vivo to time the administration of the nontoxic prodrug 5-fluorocytosine that was converted by yeast cytosine deaminase, expressed under HRE regulation, to the chemotherapy agent 5-fluorouracil to target hypoxic cells. Conversion of 5-fluorocytosine to 5-fluorouracil was detected in vivo by 19F magnetic resonance spectroscopy. Morphological and immunohistochemical staining and molecular analyses were performed to characterize tumor microenvironment changes in cancer-associated fibroblasts, cell viability, collagen 1 fiber patterns, and HIF-1α. These studies expand our understanding of the effects of eliminating hypoxic cancer cells on the tumor microenvironment and in reducing stromal cell populations such as cancer-associated fibroblasts.

Successful gene therapy requires targeting the vast majority of cancer cells

ABSTRACT Suicide gene therapy using gene-directed enzyme prodrug therapy (GDEPT) is based on delivering a gene-encoded enzyme to cells that converts a nontoxic prodrug into its toxic metabolite. The bystander effect is thought to compensate for inefficiencies in delivery and expression because the produced toxic metabolite can spread to adjacent non-expressing cells. The purpose of this study was to assess the significance of bystander effect in GDEPT over the long term in vivo. We performed experiments using mixtures of yeast cytosine deaminase (yCD) expressing and empty vector (EV) containing cells. First, the bystander effect was assessed in various ratios of colon cancer cell lines RKO with yCD/EV in 2D and 3D culture. Next, tumors raised from RKO with yCD/EV in mice were treated with the prodrug 5-fluorocytosine (5-FC) for 42 days to assess bystander effect in vivo. Cell types constituting relapsed tumors were determined by 5-FC treatment and PCR. We were able to demonstrate bystander effect in both 2D and 3D. In mice, tumors initially regressed, but they all eventually recurred including those produced from 80% yCD expressing cells. Cells explanted from the recurrent tumors demonstrated that suicide gene expressing cells had been selected against during in vivo treatment with 5-FC. We conclude that gene therapy of malignant tumors in patients using the yCD/5-FC system will require targeting well over 80% of the malignant cells, and therefore will likely require improved bystander effect or repeated treatment.

A facile and scalable in production non-viral gene engineered mesenchymal stem cells for effective suppression of temozolomide-resistant (TMZR) glioblastoma growth

BackgroundMesenchymal stem cells (MSCs) serve as an attractive vehicle for cell-directed enzyme prodrug therapy (CDEPT) due to their unique tumour-nesting ability. Such approach holds high therapeutic potential for treating solid tumours including glioblastoma multiforme (GBM), a devastating disease with limited effective treatment options. Currently, it is a common practice in research and clinical manufacturing to use viruses to deliver therapeutic genes into MSCs. However, this is limited by the inherent issues of safety, high cost and demanding manufacturing processes. The aim of this study is to identify a facile, scalable in production and highly efficient non-viral method to transiently engineer MSCs for prolonged and exceptionally high expression of a fused transgene: yeast cytosine deaminase::uracil phosphoribosyl-transferase::green fluorescent protein (CD::UPRT::GFP).MethodsMSCs were transfected with linear polyethylenimine using a cpg-free plasmid encoding the transgene in the presence of a combination of fusogenic lipids and β tubulin deacetylase inhibitor (Enhancer). Process scalability was evaluated in various planar vessels and microcarrier-based bioreactor. The transfection efficiency was determined with flow cytometry, and the therapeutic efficacy of CD::UPRT::GFP expressing MSCs was evaluated in cocultures with temozolomide (TMZ)-sensitive or TMZ-resistant human glioblastoma cell lines. In the presence of 5-fluorocytosine (5FC), the 5-fluorouracil-mediated cytotoxicity was determined by performing colometric MTS assay. In vivo antitumor effects were examined by local injection into subcutaneous TMZ-resistant tumors implanted in the athymic nude mice.ResultsAt > 90% transfection efficiency, the phenotype, differentiation potential and tumour tropism of MSCs were unaltered. High reproducibility was observed in all scales of transfection. The therapeutically modified MSCs displayed strong cytotoxicity towards both TMZ-sensitive and TMZ-resistant U251-MG and U87-MG cell lines only in the presence of 5FC. The effectiveness of this approach was further validated with other well-characterized and clinically annotated patient-derived GBM cells. Additionally, a long-term suppression (> 30 days) of the growth of a subcutaneous TMZ-resistant U-251MG tumour was demonstrated.ConclusionsCollectively, this highly efficient non-viral workflow could potentially enable the scalable translation of therapeutically engineered MSC for the treatment of TMZ-resistant GBM and other applications beyond the scope of this study.

A highly efficient non-viral process for programming mesenchymal stem cells for gene directed enzyme prodrug cancer therapy

Mesenchymal stem cells (MSCs) driven gene-directed enzyme prodrug therapy has emerged as a potential strategy for cancer treatment. The tumour-nesting properties of MSCs enable these vehicles to target tumours and metastases with effective therapies. A crucial step in engineering MSCs is the delivery of genetic material with low toxicity and high efficiency. Due to the low efficiency of current transfection methods, viral vectors are used widely to modify MSCs in preclinical and clinical studies. We show, for the first time, the high transfection efficiency (> 80%) of human adipose tissue derived-MSCs (AT-MSCs) using a cost-effective and off-the-shelf Polyethylenimine, in the presence of histone deacetylase 6 inhibitor and fusogenic lipids. Notably, the phenotypes of MSCs remained unchanged post-modification. AT-MSCs engineered with a fused transgene, yeast cytosine deaminase::uracil phosphoribosyltransferase (CDy::UPRT) displayed potent cytotoxic effects against breast, glioma, gastric cancer cells in vitro. The efficiency of eliminating gastric cell lines were effective even when using 7-day post-transfected AT-MSCs, indicative of the sustained expression and function of the therapeutic gene. In addition, significant inhibition of temozolomide resistant glioma tumour growth in vivo was observed with a single dose of therapeutic MSC. This study demonstrated an efficient non-viral modification process for MSC-based prodrug therapy.

Intracellular prodrug gene therapy for cancer mediated by tumor cell suicide gene exosomes.

Recently, we reported about exosomes possessing messenger RNA (mRNA) of suicide gene secreted from mesenchymal stem/stromal cells (MSCs) engineered to express the suicide gene-fused yeast cytosine deaminase::uracil phosphoribosyltransferase (yCD::UPRT). The yCD::UPRT-MSC exosomes are internalized by tumor cells and intracellularly convert prodrug 5-fluorocytosine (5-FC) to cytotoxic drug 5-fluorouracil (5-FU). Human tumor cells with the potential to metastasize release exosomes involved in the creation of a premetastatic niche at the predicted organs. We found that cancer cells stably transduced with yCD::UPRT gene by retrovirus infection released exosomes acting similarly like yCD::UPRT-MSC exosomes. Different types of tumor cells were transduced with the yCD::UPRT gene. The homogenous cell population of yCD::UPRT-transduced tumor cells expressed the yCD::UPRT suicide gene and secreted continuously exosomes with suicide gene mRNA in their cargo. All tumor cell suicide gene exosomes upon internalization into the recipient tumor cells induced the cell death by intracellular conversion of 5-FC to 5-FU and to 5-FUMP in a dose-dependent manner. Most of tumor cell-derived suicide gene exosomes were tumor tropic, in 5-FC presence they killed tumor cells but did not inhibit the growth of human skin fibroblast as well as DP-MSCs. Tumor cell-derived suicide gene exosomes home to their cells of origin and hold an exciting potential to become innovative specific therapy for tumors and potentially for metastases.

Antitumor efficacy of cytosine deaminase-armed vaccinia virus plus 5-fluorocytosine in colorectal cancers

BackgroundVaccinia viruses have emerged as attractive therapeutic candidates for cancer treatment due to their inherent ability of tumor tropism and oncolytic property. Cytosine deaminase (CD), which is derived from bacteria or yeast, can convert a relatively nontoxic prodrug 5-fluorocytosine (5-FC) into the active anticancer drug 5-Fluorouracil (5-FU). Vaccinia virus armed with the prodrug-activator CD gene would result in augmented antitumor effects that combined the effect of vaccinia virus and 5-FU together, and particularly limited the anticancer drug to tumor regions.MethodsThe attenuated vaccinia Tian Tan strain Guang 9 (VG9), with active yeast CD expression and thymidine kinase (TK) deficiency, was successfully constructed. Then, in vitro and in vivo antitumor efficacy of vaccinia VG9-CD plus 5-FC administration was evaluated in colorectal cancer cells.ResultsVaccinia viruses displayed different oncolytic potency in vitro cells, no relationship with whether they were cancer cells or normal cells. In colorectal tumor models, mice treated with vaccinia VG9-TK− showed better tumor remission ability and prolonged survival. Moreover, vaccinia VG9-CD in combination with gavage administration of 5-FC displayed the best antitumor efficacy, especially for the prolongation of survival.ConclusionsVaccinia VG9-CD in combination with 5-FC plays combined effect of vaccinia virus and chemotherapy, and becomes a promising virotherapy for cancer.

Efficient Prodrug Activator Gene Therapy by Retroviral Replicating Vectors Prolongs Survival in an Immune-Competent Intracerebral Glioma Model.

Prodrug activator gene therapy mediated by murine leukemia virus (MLV)-based retroviral replicating vectors (RRV) was previously shown to be highly effective in killing glioma cells both in culture and in vivo. To avoid receptor interference and enable dual vector co-infection with MLV-RRV, we have developed another RRV based on gibbon ape leukemia virus (GALV) that also shows robust replicative spread in a wide variety of tumor cells. We evaluated the potential of GALV-based RRV as a cancer therapeutic agent by incorporating yeast cytosine deaminase (CD) and E. coli nitroreductase (NTR) prodrug activator genes into the vector. The expression of CD and NTR genes from GALV-RRV achieved highly efficient delivery of these prodrug activator genes to RG-2 glioma cells, resulting in enhanced cytotoxicity after administering their respective prodrugs 5-fluorocytosine and CB1954 in vitro. In an immune-competent intracerebral RG-2 glioma model, GALV-mediated CD and NTR gene therapy both significantly suppressed tumor growth with CB1954 administration after a single injection of vector supernatant. However, NTR showed greater potency than CD, with control animals receiving GALV-NTR vector alone (i.e., without CB1954 prodrug) showing extensive tumor growth with a median survival time of 17.5 days, while animals receiving GALV-NTR and CB1954 showed significantly prolonged survival with a median survival time of 30 days. In conclusion, GALV-RRV enabled high-efficiency gene transfer and persistent expression of NTR, resulting in efficient cell killing, suppression of tumor growth, and prolonged survival upon CB1954 administration. This validates the use of therapeutic strategies employing this prodrug activator gene to arm GALV-RRV, and opens the door to the possibility of future combination gene therapy with CD-armed MLV-RRV, as the latter vector is currently being evaluated in clinical trials.

Immune modulation after Toca 511 and Toca FC treatment of colorectal cancer patients.

186 Background: Toca 511 (vocimagene amiretrorepvec) is a cancer-selective, retroviral replicating vector encoding yeast cytosine deaminase that converts 5-fluorocytosine (5-FC) into 5-fluorouracil in the tumor microenvironment (TME). In animal models, Toca 511 and 5-FC kill dividing cancer and nearby immunosuppressive cells, leading to antitumor immune activity. A Phase 1 study of Toca 511 & Toca FC (extended-release 5-FC) in patients with recurrent high grade glioma revealed results consistent with this proposed mechanism. A Phase 3 trial is ongoing. Methods: Toca 6 (NCT02576665) is a Phase 1b, single-arm, multicenter study designed to investigate immunological changes after Toca 511 & Toca FC treatment in patients with advanced solid tumors, including colorectal cancer (CRC). Patients received intravenous (IV) Toca 511 for 3 days, and underwent biopsy of metastatic tumor before and ~ 4 weeks after starting oral Toca FC. Toca FC was repeated every 4-6 weeks. Peripheral blood mononuclear cells and tumor biopsies were evaluated for treatment related immune responses. Results: 17 CRC patients with a median 5 lines of prior chemotherapy were enrolled. At last data cut-off, 9 patients were alive and the median overall survival was 9.4 months. A patient receiving concomitant panitumumab had a partial response. IV Toca 511 led to viral expression in tumor, which decreased post-Toca FC while maintaining a reservoir of virus in the remaining tumor. T cells shifted from naïve to effector phenotypes, CD4 memory T cells expanded, and/or B cells increased after Toca FC treatment in 36% of patients. Marked changes in tumor infiltrating cells (CD11b myeloid cells, Tregs, exhausted T cells and CD8 T cells) occurred after Toca FC treatment. Treatment has been generally well tolerated. We also plan to report insights gained from RNA analysis of TME and update on clinical finding. Conclusions: Clinical data suggest a signal of activity in these heavily pretreated CRC patients warranting further exploration. IV Toca 511 administration showed viral infection of CRC metastatic tumor. Toca 511 & Toca FC may be associated with T cell mediated immune activity in peripheral blood and metastatic tumor, consistent with pre-clinical data in multiple tumor types. Clinical trial information: NCT02576665.

Nanoscale bubble delivered YCD-TK/Cx26 gene therapeutic system suppresses tumor growth by inducing necrosis of tumor tissues in mouse Xenograft bladder cancer models.

Bladder cancer is considered as the fifth most common cancer in the whole world. This study aimed to investigate the anti-tumor effects of Nanoscale bubbles delivered yeast cytosine deaminase thymidine kinase/connexin 26 (YCD-TK/Cx26) on tumor cell proliferation and tumor growth. Nanoscale bubble was prepared using thin-film hydration-sonication method. Nanoscale bubble-LV5-YCD-TK+PCD-Cx26 was generated and transfected into BIU-87 cells. MTT assay was employed to detect cell viability. Apoptosis was determined using a flow cytometry assay. YCD-TK and Cx26 expressions were detected using Western blot and Real Time-PCR (RT-PCR). BIU-87 cells were transplanted into mice to establish Xenograft models. The tumor volume was recorded. HE staining was used to examine necrosis areas in tumor tissues. Nanoscale bubble (Nanoscale bubble-LV5-YCD-TK+PCD-Cx26) successfully mediated YCD-TK and Cx26 gene expression in BIU-87 cells. Nanoscale bubble delivered YCD-TK/Cx26 expression significantly inhibited cell viability and induced apoptosis compared to Nanoscale bubble-LV5-YCD-TK and Nanoscale bubble group (p<0.05). Nanoscale bubble delivered YCD-TK/Cx26 expression triggered significantly higher levels of bystander effect compared to single YCD-TK or single Cx26 gene (p<0.05). Nanoscale bubble delivered YCD-TK/Cx26 expression significantly reduced tumor volume in mouse Xenograft bladder cancer model compared to LV5-YCD-TK and 5-FC+GCV group (p<0.05). Nanoscale bubble delivered YCD-TK/Cx26 expression significantly reduced the necrosis of tumor tissues in mouse Xenograft bladder cancer model compared to LV5-YCD-TK group and 5-FC+GCV group (p<0.05). Nanoscale bubble delivered YCD-TK/Cx26 gene therapeutic system efficiently reduced BIU-87 cell proliferation in vitro, and suppressed tumor growth by inducing necrosis of tumor tissues in mouse Xenograft bladder cancer models.

Novel Semi-Replicative Retroviral Vector Mediated Double Suicide Gene Transfer Enhances Antitumor Effects in Patient-Derived Glioblastoma Models.

As glioblastomas are mostly localized infiltrative lesions, gene therapy based on the retroviral replicating vector (RRV) system is considered an attractive strategy. Combinations of multiple suicide genes can circumvent the limitations associated with each gene, achieving direct and synergistic cytotoxic effects, along with bystander cell killing. In this study, we constructed a semi-and pseudotyped-RRV (sp-RRV) system harboring two suicide genes—herpes simplex virus type 1 thymidine kinase (TK) and yeast cytosine deaminase (CD)—to verify the dissemination and antitumor efficacy of our sp-RRV system (spRRVe-sEF1α-TK/sRRVgp-sEF1α-CD) in seven patient-derived glioblastoma stem-like cells (GSCs). Flow cytometry and high-content analysis revealed a wide range of transduction efficiency and good correlation between the delivery of therapeutic genes and susceptibility to the prodrugs ganciclovir and 5-fluorocytosine in patient-derived GSCs in vitro. Intra-tumoral delivery of spRRVe-sEF1α-TK/sRRVgp-sEF1α-CD, combined with prodrug treatment, synergistically inhibited cell proliferation and angiogenesis while increasing apoptosis and the depletion of tumor-associated macrophages in orthotopic glioblastoma xenografts. Genomic profiling of patient-derived GSCs revealed that the key genes preventing sp-RRV infection and transmission were associated with cell adhesion, migration, development, differentiation, and proliferation. This is the first report demonstrating that a novel sp-RRV-mediated TK/CD double suicide gene transfer system has high oncolytic power against extremely heterogeneous and treatment-refractory glioblastomas.

Abstract 4771: The addition of toca 511 and 5-FC to temozolomide improves response in a temozolomide-resistant murine glioblastoma model and correlates with toca 511 dose

Abstract Background: Toca 511 (vocimagene amiretrorepvec), an amphotropic retroviral replicating vector (RRV), can successfully and safely deliver a functional, optimized yeast cytosine deaminase (CD) gene to tumors. Within infected cells, CD converts 5-fluorocytosine (5-FC) to the anti-cancer drug 5-fluorouracil (5-FU). The combination of Toca 511 with oral extended release 5-FC (Toca FC), is currently being evaluated in a randomized phase III clinical trial for recurrent high grade glioma (glioblastoma (GBM) and anaplastic astrocytoma) (NCT02414165, Toca 5). Temozolomide (TMZ), in combination with radiation therapy, is the standard of care used for first-line chemotherapy treatment of patients with GBM, the most common and aggressive form of primary brain cancer. Previously, we have shown that: (1) Toca 511/5-FC treatment provides durable response in a syngeneic murine glioma model and supports anti-tumor immune memory; (2) The combination of TMZ and Toca 511/5-FC had synergistic efficacy in a TMZ-sensitive human glioma nude mouse model; (3) TMZ did not inhibit the efficacy of Toca 511/5-FC in a TMZ resistant murine glioma syngeneic model; (4) Toca 511/5-FC caused significant radiosensitization in a radioresistant murine glioma model. Results: To assess the interaction of TMZ with escalating doses of Toca 511 (as defined by percent of tumor transduction by RRV), an orthotopic TMZ-resistant murine glioma model, Tu-2449, was utilized. These results show that moderate levels of tumor transduction of Toca 511 (30% - 50%) with 5-FC treatment prolongs survival in the presence of TMZ compared with lower transduction rates (10%). Additionally, mice treated with Toca 511/5-FC and TMZ are being tested for the establishment of anti-tumor immune memory. Conclusion: These results demonstrate that (1) survival correlates with the transduction levels of Toca 511 when combined with temozolomide in the Tu-2449 orthotopic glioma model and (2) that this combination may support anti-tumor immune memory. These studies along with prior work support evaluation of the combination of Toca 511/5-FC with temozolomide in patients with newly diagnosed GBM (NRG-BN006). Citation Format: Maria E. Rodriguez-Aguirre, Sophie Viaud, Daniel Mendoza, Tiffany Montellano, Derek G. Ostertag, Harry Gruber, Douglas Jolly, Cornelia Bentley. The addition of toca 511 and 5-FC to temozolomide improves response in a temozolomide-resistant murine glioblastoma model and correlates with toca 511 dose [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4771.

Comprehensive mutagenesis on yeast cytosine deaminase yields improvements in 5‐fluorocytosine toxicity in HT1080 cells

AbstractImproved prodrug‐activating enzymes have the potential to increase the therapeutic efficacy of gene‐directed enzyme prodrug therapy (GDEPT). Yeast cytosine deaminase (yCD) is commonly used to convert the prodrug 5‐fluorocytosine (5‐FC) to the chemotherapeutic 5‐fluorouracil for GDEPT. Mutagenesis studies on yCD aimed at improving its application in GDEPT have been limited to subsets of residues or have sought to improve a single property of the enzyme. We performed comprehensive site‐saturation mutagenesis (CSM) on yCD designed to create all 2,983 possible unique protein mutants with a single amino acid substitution. We identified active variants through Escherichia coli genetic complementation and screened these mutants, and combinations thereof, for increased ability to sensitize E. coli and HT1080 fibrosarcoma cells to 5‐FC. Several mutants identified in this study showed increased sensitization ability for both E. coli and HT1080 cells indicating that CSM is an effective directed evolution tool for identifying unexpectedly beneficial mutations.

A highly efficient non-viral process for engineering theranostic mesenchymal stem cells for gene directed enzyme prodrug cancer therapy

Background & Aim Mesenchymal stem cells (MSC) are emerging as promising vehicles for Gene-directed enzyme prodrug therapy (GDEPT). The inherent tumor-trophic migratory properties of MSC enable these vehicles to deliver effective, targeted therapy to tumors and metastatic diseases. A critical step in modifying MSC is the delivery of genes with high efficiency and low cytotoxicity. Due to the poor efficiency of transfection approaches, viral methods are used extensively to transduce MSC in preclinical and clinical studies. This study aims to develop a scalable non viral gene delivery method to efficiently modify MSC. Methods, Results & Conclusion Methods TrafEn (Trafficking Enhancer), to enhance transfection using off-the-shelf, cost-effective polymers. TrafEn was rationally developed to facilitate efficient trafficking of the genetic material inside cells. Polyplex transfection was enhanced by including a simultaneous treatment with DOPE/CHEMS lipid suspension and a microtubule stabilizer, a histone deacetylase-6 inhibitor. Result We demonstrate, for the first time, the efficient transfection (>90%) of human adipose tissue derived MSCs (AT-MSCs) using an off-the shelf and cost-effective cationic polymer, polyethylenimine, in the presence of fusogenic lipids and histone deacetylase 6 inhibitor (HDAC6i) (Figure 1). Importantly, the cell phenotypes of MSCs remained unchanged after modification, a key requisite for clinical application of MSCs. AT-MSCs modified with a fused transgene, yeast cytosine deaminase::uracil phosphoribosyltransferase (CDy::UPRT), exhibited strong cytotoxic effects towards glioma, breast and gastric cancer cells in vitro. The efficiency of killing of gastric MKN1 and MKN28 cell lines were greater than 80% even with 7 days post-transfected AT-MSCs, indicative that the expression period of the therapeutic gene was sustainable. Conclusion This study describes a useful tool for polymer based ex vivo MSC modification which is highly scalable and cost-effective. The proposed workflow bypasses the restriction in viral vector supply and expedites MSC modification, without compromising the quality and anti-cancer efficacy of the theranostic MSCs.

Protein-Programmed Accumulation of Yeast Cytosine Deaminase in Cancer Cells in Response to Mock-Hypoxia.

One limitation of gene-directed enzyme prodrug therapy (GDEPT) is the difficulty in selectively and efficiently transducing cancer cells with the gene encoding a prodrug-converting enzyme. To circumvent this issue, we sought to move the selectivity from the gene delivery level to the protein level. We developed fusion proteins of the prodrug-activating enzyme yeast cytosine deaminase (yCD) and the oxygen-dependent degradation domain (ODDD) of HIF-1α, a domain that regulates the accumulation of HIF-1α in an oxygen-dependent manner. We called these HOPE fusions for HIF1-α Oxygen-dependent degradation domain/Prodrug-converting Enzyme. The HOPE fusions were designed to selectively accumulate in cells experiencing hypoxia and thus selectively cause conversion of the prodrug 5-fluorocytosine (5-FC) to the chemotherapeutic 5-fluorouracil (5-FU) where oxygen levels are low (e.g., at the center of a tumor). Consistent with our hypothesis, HT1080 fibrosarcoma cells transduced with HOPE fusion genes exhibited increased fusion protein accumulation and increased sensitization to 5-FC in mock-hypoxia.

Construction of replication-competent oncolytic retroviral vectors expressing R peptide-truncated 10A1 envelope glycoprotein

Replication-deficient retroviral (RDR) vectors have been generally used for gene therapy, but clinically beneficial transduction efficiency is difficult to achieve with these vectors. In recent times, attention has been focused on the use of murine leukemia virus (MLV)-based replication-competent retroviral (RCR) vectors. RCR vectors have been shown to achieve efficient tumor reduction in a wide variety of cancer models. Most RCR vectors have been developed from amphotropic 4070 A MLV env, which is broadly applied in basic research. In this study, we generated RCR vectors based on Moloney MLV by replacing the native env gene in a full-length viral genome with the 10A1 env gene. 10A1 MLV can infect a wide variety of cells. Unlike amphotropic MLV, the 10A1 MLV can use amphotropic MLV receptor Pit2 or gibbon ape leukemia virus (GaLV) receptor Pit1. The resulting construct MoMLV-10A1-EGFP was able to replicate in 293 T, NIH3T3, and Mus dunni cells. To evaluate the potential of MoMLV-10A1 vector as a therapeutic agent, we incorporated the yeast cytosine deaminase (CD) suicide gene into vectors. The resulting vector MoMLV-10A1-CD could inhibit the growth of human 293T cells upon 5-fluorocytosine (5-FC) administration. In addition, to lyse tumor cells by syncytium, MoMLV-10A1-R(-)-EGFP was generated by replacing wild-type 10A1 env with the 16-amino acid R peptide-truncated 10A1 env gene. Syncytium formation was observed in the TE671 human tumor cells, 293 T and PG13 cells upon transfection of the MoMLV-10A1-R(-)-EGFP vector. This result suggests that replication of this vector could be oncolytic in itself. We also found that syncytium could contribute to enhance cell-to-cell transmission of the retroviral vectors. Our results thus show that the MoMLV-10A1 vectors can be potentially useful for cancer gene therapy.

Abstract A018: Effects of Toca 511 and Toca FC on tumor microenvironment and peripheral blood populations in patients with advanced malignancies

Abstract Toca 511 (vocimagene amiretrorepvec) is an investigational, conditionally lytic, retroviral replicating vector that selectively infects cancer cells due to cell division requirements for virus integration into the genome, and defects in innate and adaptive immune responses found in malignant tissues that support virus replication. Toca 511 spreads through cancer cells and stably delivers optimized yeast cytosine deaminase (CD) that, upon administration of the prodrug, Toca FC (an investigational, extended-release version of 5-fluorocytosine [5-FC]), converts 5-FC into 5-fluorouracil (5-FU). 5-FU kills infected dividing cancer cells and diffuses to and kills surrounding cells in the tumor microenvironment, including immunosuppressive myeloid cells. In animal models, this depletion of immunosuppressive myeloid cells leads to therapeutically active immunity against tumors. A similarly derived antitumor response may occur in cancer patients, as local injection of Toca 511 into the tumor bed after resection of recurrent high-grade glioma followed by treatment with Toca FC has been associated with prolonged survival and durable complete responses (median duration of follow-up: 37.4+ months); responses were delayed in onset, consistent with time to response for immuno-oncology agents. The current phase 1b, multicenter, open-label study (Toca 6; NCT02576665) is designed to investigate changes in immune activity after treatment with Toca 511 and Toca FC in patients with advanced malignancies. Toca 511 is administered intravenously (IV) daily for 3 days and then as a single injection into metastatic or recurrent tumor. Oral Toca FC is started ~4 weeks later and repeated every 4-6 weeks. Biopsies are obtained prior to and following exposure to Toca 511 and Toca FC treatment to evaluate changes in immune activity, and peripheral blood is obtained contemporaneously for evaluation. The study has enrolled 19 patients to date (colorectal cancer: 15; sarcoma: 2; non-small cell lung and pancreas cancer: 1 each). Treatment has been well tolerated. Viral RNA, DNA, and CD protein expression are observed in tumor after IV delivery of Toca 511. We plan to report on tumor microenvironment remodeling that follows treatment with Toca 511 and Toca FC. Infiltrating T-cell subpopulations, B cells, and monocytes quantified by immunofluorescence from stained formalin-fixed, paraffin-embedded samples will be presented. Additionally, changes in peripheral blood, including T-cell effector, helper/memory, and regulatory populations, and myeloid lineage cells following exposure to Toca 511 alone and following subsequent exposure to Toca FC will be reported. Data from this study will inform future development of Toca 511 and Toca FC alone or in combination with other therapies in patients with solid tumors. Citation Format: Jaime Merchan, Jordi Rodon, Derek Ostertag, Shree Venkat, Arthur Donahue, Peder Horner, Dalissa Tijera, Thian Kheoh, Douglas J. Jolly, Harry E. Gruber, Jolene S. Shorr, Gerald S. Falchook. Effects of Toca 511 and Toca FC on tumor microenvironment and peripheral blood populations in patients with advanced malignancies [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A018.

Controlled Epidermal Growth Factor Receptor Ligand Display on Cancer Suicide Enzymes via Unnatural Amino Acid Engineering for Enhanced Intracellular Delivery in Breast Cancer Cells.

Proteins are ideal candidates for disease treatment because of their high specificity and potency. Despite this potential, delivery of proteins remains a significant challenge due to the intrinsic size, charge, and stability of proteins. Attempts to overcome these challenges have most commonly relied on direct conjugation of polymers and peptides to proteins via reactive groups on naturally occurring residues. While such approaches have shown some success, they allow limited control of the spacing and number of moieties coupled to proteins, which can hinder bioactivity and delivery capabilities of the therapeutic. Here, we describe a strategy to site-specifically conjugate delivery moieties to therapeutic proteins through unnatural amino acid (UAA) incorporation, in order to explore the effect of epidermal growth factor receptor (EGFR)-targeted ligand valency and spacing on internalization of proteins in EGFR-overexpressing inflammatory breast cancer (IBC) cells. Our results demonstrate the ability to enhance targeted protein delivery by tuning a small number of EGFR ligands per protein and clustering these ligands to promote multivalent ligand-receptor interactions. Furthermore, the tailorability of this simple approach was demonstrated through IBC-targeted cell death via the delivery of yeast cytosine deaminase (yCD), a prodrug converting enzyme.

Mesenchymal Stem Cell Exosome-Mediated Prodrug Gene Therapy for Cancer.

Exosomes derived from human mesenchymal stem cells (MSCs) engineered to express the suicide gene yeast cytosine deaminase::uracil phosphoribosyl transferase (yCD::UPRT) represent a new therapeutic approach for tumor-targeted innovative therapy. The yCD::UPRT-MSC-exosomes carry mRNA of the suicide gene in their cargo. Upon internalization by tumor cells, the exosomes inhibit the growth of broad types of cancer cells in vitro, in the presence of a prodrug. Here we describe the method leading to the production and testing of these therapeutic exosomes. The described steps include the preparation of replication-deficient retrovirus possessing the yCD::UPRT suicide gene, and the preparation and selection of MSCs transduced with yCD::UPRT suicide gene. We present procedures to obtain exosomes possessing the ability to induce the death of tumor cells. In addition, we highlight methods for the evaluation of the suicide gene activity of yCD::UPRT-MSC-exosomes.