86 A Highly Compact Epitope Based Marker Suicide Gene for Safer and Easier Adoptive T Cell Gene Therapy Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & C[.]
CANCER-TARGETED GENE & CELL THERAPY for tumor-associated antigens (TAAs) preferentially expressed on melanoma cells Recognition of cell-surface TAAs independent of major histocompatibility complex can be achieved by introducing a TAA-specific chimeric antigen receptor (CAR) on T cells using gene therapy This approach is being used in clinical trials to adoptively transfer CD19-specific CAR+ T cells in patients with B-lineage malignancies To generate T-cell therapy for melanoma we targeted a TAA derived from human endogenous retroviruse (HERV), whose genome stably integrated into humans millions of years ago During oncogenesis, biologically active variants of HERV, such as the envelope (env) protein of HERV-K, are expressed on the surface of melanoma, but not normal cells To target HERV-K, T cells were engineered to express a CAR specific for this env protein, by replacing the antigen-binding exodomain of CD19-specific CAR with the scFv sequence of a HERV-K env specific monoclonal antibody This new CAR was cloned as a transposon into our Sleeping Beauty (SB) system which we have adapted for human application DNA plasmids coding for the HERV-K env-specific CAR and SB transposase were electrotransferred into primary human T cells and genetically modified CAR+ T cells were selectively propagated on irradiated artificial antigen presenting cells (aAPC) expressing HERV-K env and desired T-cell co-stimulatory molecules After co-culture on g-irradiated aAPC, 95% of CD3+ T cells expressed the CAR and these CAR+ T cells were able to specifically kill HERV-Kenv+ melanoma targets in vitro in contrast to CARneg T cells Specificity of these CAR+ T cells was proven by expressing the HERV-K env protein in antigen negative EL4 mouse cells which were preferentially killed compared to HERV-Kenvneg EL4 parental cells In aggregate, these data demonstrate for the first time that T cells targeting an active ancient retrovirus can be used as an immunotherapy for melanoma, using an approach that has translational appeal for clinical trials 85 SNS01-T, an eIF5A-Based Gene Therapy Nanoparticle Designed for the Treatment of Multiple Myeloma, Has Anti-Tumoral Activity in Lymphoma Zhongda Liu,1 Terence Tang,1 Catherine A Taylor,1 Qifa Zheng,1 Sarah Francis,1 John E Thompson,1,2 Richard Dondero.2 Dept of Biology, University of Waterloo, ON, Canada; 2Senesco Technologies Inc., Bridgewater, NJ INTRODUCTION: The eukaryotic translation initiation factor 5A (eIF5A) is the only known protein to be regulated by posttranslational addition of a hypusine residue Hypusine-eIF5A has been identified as a marker of neoplastic growth and metastasis, and suppression of hypusinated eIF5A sensitizes multiple myeloma (MM) cells to apoptosis In vitro cell studies and in vivo xenograft Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy studies have demonstrated that simultaneous suppression of eIF5A expression and over-expression of a non-hypusinable pro-apoptotic mutant of eIF5A (eIF5AK50R) potently induces apoptosis in MM cells SNS01-T is a gene therapy nanoparticle targeted to the treatment of MM and is comprised of: a B cell-specific plasmid expressing eIF5AK50R; an siRNA targeting the native eIF5A that promotes growth of cancer cells; and polyethylenimine A multiple ascending dose Phase 1b/2a open-label study has been initiated to evaluate the safety and tolerability of six weeks of twice-weekly administration of SNS01-T by intravenous infusion in patients with relapsed or refractory MM Inasmuch as the B29 promoter driving expression of eIF5AK50R is active in all B cell lineages, the activity of SNS01-T as an anti-cancer agent in non-Hodgkin’s Lymphomas (NHL) of B cell origin was assessed using both mantle cell lymphoma (MCL) and diffuse large B cell lymphoma (DLBCL) xenograft models METHODS: In vitro biological activity of SNS01-T was assessed using RT-qPCR, and uptake of nanoparticles was assessed by FACS analysis of cells treated with fluorescently-labeled SNS01-T Antitumoral activity was evaluated using MCL and DLBCL xenograft models with twice-weekly intravenous injections of SNS01-T at doses ranging from 0.093 mg/kg to 0.75 mg/kg RESULTS: SNS01-T transfected MM, MCL, and DLBCL cell lines and resulted in suppression of endogenous eIF5A mRNA and accumulation of the eIF5AK50R transgene Twice-weekly dosing of SNS01-T at doses > 0.18 mg/kg resulted in statistically significant inhibition of tumor growth in both MCL and DLBCL xenograft models In the MCL xenograft model, tumor growth inhibition of 52.7 %, 79.1 %, and 89.0 % was observed for SNS01-T doses of 0.18 mg/kg, 0.375 mg/ kg, and 0.75 mg/kg, respectively Similar results were obtained in the DLBCL model Median survival in the MCL xenograft model was 21 days for control mice and up to 42 days (p = 0.002; logrank test) for mice treated with SNS01-T (0.75 mg/kg) SNS01-T also increased survival in the DLBCL model from 20 days for control mice to 45 days (p = 0.002; logrank test) for mice treated with 0.375 mg/kg SNS01-T CONCLUSION: SNS01-T is a gene therapy nanoparticle that modulates expression of eIF5A to kill cancers of B cell origin and is being investigated in the clinic in patients with multiple myeloma SNS01-T is biologically active in other B cell cancers, including MCL and DLBCL MCL and DLBCL xenograft tumor growth was significantly inhibited following intravenous administration of SNS01-T, and treatment provided a significant survival advantage The results support the clinical investigation of SNS01-T in NHL patients 86 A Highly Compact Epitope-Based Marker Suicide Gene for Safer and Easier Adoptive T-Cell Gene Therapy Brian Philip,1 Barry Flutter,1 Ronjon Chakraverty,1 Amit Jathoul,1 Martin Pule.1 Haematology, UCL Cancer Institute, London, United Kingdom Introduction: A compact marker-suicide gene reliant on off-theshelf reagents would offer considerable utility for adoptive T-cell therapy Marker genes enable measurement of transduction efficiency and allow sorting of transduced cells while suicide genes allow deletion of administered T-cells in case of toxicity Previous marker genes include the Neomycin resistance gene, truncated nerve growth factor receptor and CD34; however all of these possess limitation to application including immunogenicity, unexpected biological activity and long coding regions respectively Similarly, several suicide genes have been described with Herpes Simplex Virus Thymidine Kinase and inducible Caspase in clinical use Here limitations include either immunogenicity or limited availability of the inducing drug Aim: We sought to generate a compact marker-suicide gene that enables both clinical grade sorting and effective deletion using CD34 cliniMACS and Rituximab respectively By using minimal epitopes required for S35 CANCER-TARGETED GENE & CELL THERAPY binding, we hoped to greatly reduce the size of the construct as well as rendering it biologically inert Results: We first sought to locate the epitope from CD34 which binds QBEnd10, the monoclonal antibody used in Miltenyi cliniMACS CD34 selection system By epitope mapping, we determined that this antibody binds a linear fragment of CD34 16 amino acids long which demonstrates equal binding of QBEnd10 as the native CD34 antigen Next we sought to incorporate Rituximab binding epitopes to engender Rituximab triggered cellular killing After much optimisation we determined that a construct designated (RQR8), composed of two Rituximab binding epitopes flanking a single QBEnd10 epitope on the CD8 stalk demonstrates effective sorting using cliniMACS CD34 system and efficient deletion by both CDC and ADCC with Rituximab We have demonstrated functional co-expression of RQR8 with both chimeric antigen receptors and transgenic T-cell receptors Finally, we tested RQR8 in vivo using a C57BL/6 to Balb/c murine model of GvHD We demonstrated almost complete in vivo depletion of RQR8 expressing T-cells within days of antibody administration and subsequent resolution of GvHD 87 Preclinical and Clinical Evaluation of Oncolytic Immunotherapy with Ad5/3-E2F1Δ24-GMCSF (CGTG-602), a GM-CSF Producing Adenovirus Targeted to Tumors on Four Levels Tuuli Ranki,1 Lotta Kangasniemi,1 Petra Ahokas,1 Minna Oksanen,2 Kaarina Partanen,3 Kalevi Kairemo,3 Leena Laasonen,3 Anna Kanerva,2 Vincenzo Cerullo,2 Akseli Hemminki.2 R&D, Oncos Therapeutics, Helsinki, Finland; 2Cancer Gene Therapy Group, University of Helsinki, Finland; 3International Comprehensive Cancer Center Docrates, Helsinki, Finland Oncolytic adenoviruses that express granulocyte-macrophage colony-stimulating factor (GM-CSF) induce anti-cancer immunity while acting directly on cancer cells by oncolysis Viral oncolysis releases tumor epitopes for antigen presenting cells (APC), GMCSF stimulates APC proliferation, maturation and migration APCs induce tumor-specific cytotoxic T-cells and, thus, an oncolytic virus coding for GM-CSF can induce potent antitumor immunity Systemically elevated cytokine levels can result to toxic side effects, however, and therefore GM-CSF expression has to be effectively restricted to tumor tissue To this end, we engineered a serotype 5-based GMCSF producing oncolytic adenovirus, CGTG-602 (Ad5/3-E2F1-Δ24GMCSF), with E2F-1 promoter controlled expression of constant region mutant E1A, resulting in replication dependent production of GMCSF from E3 CGTG-602 has dual mechanisms for p16-Rb pathway selectivity and features a chimeric 5/3 fiber that allows CAR-independent entry to tumor cells CGTG-602 was found to be a specific and potent anti-cancer agent when tested preclinically in vitro and in an immunocompetent Syrian hamster model 13 patients with solid tumors refractory to standard treatments were treated with CGTG-602 Treatments were well tolerated and tumor-specific and adenovirus-specific T-cell immunity was seen Frequent changes in serum antibody levels against tumor associated antigens were seen after treatment with CGTG-602 and these changes seemed to correlate with other signs of efficacy Overall, with regard to tumor marker or radiological responses, signs of antitumor efficacy were seen in 9/12 evaluable patients (75%) Median survival was 135 days which is unusually high for a chemotherapy refractory population CGTG-602 is an attractive candidate for further clinical development 88 A Phase II Clinical Trial of Recombinant Human Endostatin Adenovirus (E10A) in Combination with Paclitaxel and Cisplatin in Patients with Head and Neck Carcinomas Wen Ye, Ranyi Liu, Jiangxue Wu, Lixia Li, Zhongzhen Guan, Wenlin Huang State Key Laboratory of Oncology, Cancer Center, Sun Yat-Sen University, Guangzhou, Guangdong, China Conclusions: RQR8 is a 136 amino acid combined marker-suicide gene This enables clinical grade selection and cellular deletion with off-the shelf GMP reagents and pharmaceutical respectively Its minimal size allows co-expression of multiple additional genes We hope RQR8 will make therapy with engineered T-cells simpler, cheaper and safer S36 Background Recombinant adenovirus encoding human Endostatin gene (E10A) has been proved to be an efficient and low-toxic drug candidate against solid tumors by inhibiting VEGF-induced tumor angiogenesis in preclinical and phase I clinical trials Here we investigated the efficacy and safety of E10A in combination with paclitaxel and cisplatin for the treatment of patients with advanced head and neck squamous cell carcinoma (HNSCC) or nasopharyngeal carcinoma (NPC) in an open-label, phase II, multicenter clinical trial with a randomize manner Methods We randomly assigned 70 of 140 eligible patients with recurrent or metastatic HNSCC or NPC into test group to receive E10A (at a dose of E10A of 1.0×1012 VP (virus particles) intratumoral injection on day1 and 8) plus cisplatin (at a dose of 25 mg/m2 per day on day to day 5) and paclitaxel (at a dose of 160 mg/m2 on day 3) every weeks for a maximum of cycles, while other 70 eligible patients in the control group to receive chemotherapy (cisplatin and paclitaxel) only Tumors were evaluated every cycles during treatment and every months in the follow-up Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy ... anti-cancer agent when tested preclinically in vitro and in an immunocompetent Syrian hamster model 13 patients with solid tumors refractory to standard treatments were treated with CGTG-602 Treatments... with paclitaxel and cisplatin for the treatment of patients with advanced head and neck squamous cell carcinoma (HNSCC) or nasopharyngeal carcinoma (NPC) in an open-label, phase II, multicenter... evaluable patients (75%) Median survival was 135 days which is unusually high for a chemotherapy refractory population CGTG-602 is an attractive candidate for further clinical development 88 A