201 A New Epithelial Junction Opener for Cancer Therapy Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy S78 CANCER IMMUNOTHERAPY I 199 Phase[.]
CANCER - IMMUNOTHERAPY I 199 Phase II Study of FANG™ (Autologous Tumor Cell – GMCSF/pbi-shRNA™ Furin DNA Plasmid) Vaccine in Advanced Melanoma John Nemunaitis,1,2,3,4 Neil Senzer,1,4 Minal Barve,3 Padmasini Kumar,4 Donald D Rao,4 Gladice Wallraven,4 Beena O Pappen,4 Joseph Kuhn,5 Peter Beitsch,2 Robert Steckler,2 Brian M Gogel,2 Walton Taylor,2 Phillip B Maples.4 Mary Crowley Cancer Research Centers, Dallas; 2Medical City HCA, Dallas; 3Texas Oncology, P.A., Dallas; 4Gradalis, Inc., Dallas; 5WLS Surgical Associates, P.A., Dallas The Phase I trial of FANG™ vaccine (Senzer et al Mol Ther 2012; 20:679-686) provided sufficient safety data to proceed to Phase II efficacy studies This decision was bolstered by the correlation of duration of survival with induced immune response at months (in patients with serial analysis) Specifically, metastatic melanoma patients in the Phase I trial, who having failed standard therapy had an expected survival prognosis of 6 months, received a combined total of 25 vaccinations (i.e patients at dose level x 107 and patient at 2.5 x 107 cells / injection (database review revealed no evidence of dose related response)) These patients have thus far experienced a median survival of 698 days since starting treatment (967, 835, 560, and 490 days, each) Two of these patients demonstrated induced T cell activation via IFN gamma ELISPOT assessment by Month post treatment start One patient did not show positive T cell activation at the Month ELISPOT assessment but did so by Month One of the patients who, coming off treatment early and not having a Month assessment, was ELISPOT positive at Month 16 These patients also showed knockdown of the endogenous immunosuppressive proteins TGF1 (mean 99% knockdown) and TGF2 (mean 86% knockdown) mediated by targeted pbi-shRNA™ furin transfected vaccine product Based on these results, the Phase II trial (CL-PTL 114, BB-IND 14205) was activated following FDA, RAC, IBC and IRB acceptance Design of the ongoing Phase II study involves an intradermal injection of the FANG™ vaccine (1.0 x 107 cells/injection/ month; maximum of 12 vaccinations) Four patients with Stage IV melanoma with biopsy accessible lesions have been treated Neither hematologic function, liver enzyme, renal function, or electrolyte assays nor protocol specified physical examinations demonstrated any toxic effects Immune function analyses, including intratumoral immune phenotype and ELISPOT analysis of cytotoxic T cell function to autologous tumor antigens, are ongoing and will be reported Stable disease has been observed in of treated patients In conclusion, results support continuation of accrual 200 Renal Cell Tumor-Mediated Reprogramming of Natural Killer Cells by Soluble Immune Modulators Yue Guan,1 Purba Singh,1 Trenae Mann,1 Christopher B Chambers,1 Donald S Torry,1 Andrew Wilber.1 Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, IL Natural killer (NK) cells classically are associated with immune surveillance and destruction of tumor cells via cytotoxicity Recent studies indicate that NK cells with non-classical CD phenotypes (CD56brightCD16dim-negative) lose their cytotoxic capacity, release pro-angiogenic factors, and facilitate tumor growth Mechanisms driving these changes are not established but soluble factors are thought to direct the unique phenotypic and functional differentiation of these CD56brightCD16- NK cells We used a combination of clinical specimens, primary cell culture, and a novel animal model to evaluate the effects of transforming growth factor beta (TGF) and prostaglandin E2 (PGE2) on NK cell phenotype and function in renal cell carcinoma (RCC) Plasma levels of PGE2 for RCC patients (N=7) were 92 + 39 pg/mL before surgery and 45 + 11 pg/ S78 mL after nephrectomy (P=0.02, T-test) Post-operative levels of PGE2 were approaching levels observed for healthy volunteers (27 + 11 pg/mL), suggesting that PGE2 is a RCC tumor-derived factor Phenotype analysis revealed that peripheral blood NK (pNK) cells from healthy volunteers were characteristically CD56dimCD16bright (95%); however, RCC tumor-derived NK cells exhibited the CD56brightCD16- phenotype The tumor-derived NK cells had elevated vascular endothelial growth factor (VEGF) transcripts (3fold) compared to freshly isolated pNK cells Trans-differentiation of CD56dimCD16bright pNK cells to the CD56brightCD16dim phenotype was achieved after culture with TGF; this conversion was coincident with impaired NK cytotoxicity (>50% reduction) and a modest augmentation of VEGF mRNA upon crosslinking of the activating receptor NKp46 A Balb/c mouse renal adenocarcinoma cell line (Renca) expressing moderate levels of TGF (300-350 pg/mL) was engineered for PGE2 expression with murine Cox2 in antisense (Cox2-) or sense (Cox2+) orientations ELISA demonstrated that Cox2- cells produced background levels of PGE2 (35 pg/mL) while Cox2+ cells produced >5,000 pg/mL PGE2 Exposure of freshly isolated human pNK cells to conditioned supernatants from Cox2(TGF only) or Cox2+ (TGF and PGE2) cells reduced cytotoxicity by 20% and 30%, respectively In vivo studies demonstrated rapid tumor growth and metastasis over three weeks following kidney capsule injection in Balb/c mice Lymphocytes isolated from these RCC-like tumors were infiltrated with NK-cells (CD3-p46+), nearly 60% of which demonstrated diminution or absence of CD16 Collectively, these studies support a role for TGF and PGE2 in trans-differentiation of CD56dimCD16bright pNK cells to CD56brightCD16dim-negative cells which have lost their normal destructive capacities and, instead, support tumor growth and metastasis Studies designed to inhibit the immune suppressive functions of these molecules are planned and may provide new therapeutic avenues for future cancer treatments 201 A New Epithelial Junction Opener for Cancer Therapy Ines Beyer,1 Hua Cao,1 Jonas Persson,1 Roma Yumul,1 Andre Lieber.1 Medicine, University of Washington, Seattle Most solid tumors are of epithelial origin Epithelial cancers maintain intercellular junctions that link cancer cells together and prevent the penetration of therapeutics deep into the cancer Several studies demonstrated that the upregulation of epithelial junction proteins correlated with increased resistance to therapy, including therapy with monoclonal antibodies and chemotherapeutics One of the epithelial junction proteins is desmoglein (DSG2) Recently, we demonstrated that a group of human adenoviruses (Ad) (Ad serotype 3, 7, 11, and 14) use DSG2 as a primary attachment receptor for the infection of cells DSG2 is overexpressed in epithelial malignancies We have created a small recombinant protein derived from Ad serotype This protein binds to DSG2 and triggers transient opening of epithelial junctions We therefore named the protein “JO-1” (“junction opener -1”) JO-1 is produced in E.coli and can be easily purified In a number of xenograft tumor models, we have shown that intravenous injection of JO-1 increased the efficacy of monoclonal antibody (e.g trastuzumab/Herceptin and cetuximab/ Erbitux) therapy Many chemotherapy drugs are larger than 500 Da, the upper limit of molecules that are able to pass through tight junctions We have demonstrated that the therapeutic effects of several of these drugs, including paclitaxel, nab-paclitaxel, docetaxel, irinotecan, and liposomal doxorubicin are increased by co-therapy with JO-1 Furthermore, we have shown that JO-1 leads to massive accumulation of chemotherapy drugs in the tumor, decreasing the drug concentrations in the rest of the body This resulted in the elimination of chemotherapy-related toxicities to bone marrow, liver, and intestine Toxicology studies carried out in mice and monkeys have Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy CANCER - IMMUNOTHERAPY I shown that intravenous injection of JO-1 is safe and well-tolerated Additional in vivo studies have demonstrated that JO-1 remains active in the presence of anti-JO-1 antibodies In immunocompetent DSG2 transgenic mice with syngeneic tumors, JO-1 significantly enhanced PEGylated liposomal doxorubicin (Doxil) therapy when given in multiple cycles with an interval of weeks More recently, we have used immunocompetent animal models to demonstrate that JO-1 treatment also enhances the intratumoral penetration and efficacy of anti-tumor T-cells We are now focused on the clinical translation of JO-1 in combination with Doxil for the treatment of ovarian cancer 202 A Chemotherapy-Selective Method for Expansion of Transgenic T Cells David Rushworth,1 Laurence Cooper.1 Pediatrics, MD Anderson Cancer Center, Houston, TX Adoptive transfer of T cells as a therapy for cancer is developing rapidly, and the use of transgenic T cells has shown promise in early clinical trials for multiple purposes related to cancer therapy However, these therapies have severe side effects and high costs related to conditioning the patient and infusing enough T cells into the patient to stably engraft cancer-specific T cells We have developed a novel method to selectively grow T cells in the presence of cytotoxic chemotherapeutics These chemotherapy resistant T cells provide an opportunity for novel T cell expansion and infusion regimens that could treat cancer with chemotherapeutic agents as well as chemotherapy resistant T cells potentially decreasing the cost and time required to effectively treat patients with T cells Here we describe the unique properties of transgenically modified methotrexate and 5-fluorouracil resistant T cells that have also been redirected to target B cell leukemia antigen CD19 using a chimeric antigen receptor 203 Phase I Study of the “Triad” Autologous (FANG™) Vaccine, Incorporating Bifunctional shRNAfurin and GMCSF Transgene Expression, in Advanced Ewing’s Sarcoma: Preliminary Data in Pediatrics Patients Maurizio Ghisoli,1 Carl Lenarsky,1 Gladice Wallraven,2 Padmasini Kumar,2 Phillip B Maples,2 John Nemunaitis.1,2,3,4 Texas Oncology, P.A, Dallas; 2Gradalis, Inc., Dallas; 3Mary Crowley Cancer Research Centers, Dallas; 4Medical City HCA, Dallas The FANG™ vaccine contains a plasmid encoding both GMCSF and an RNA interference (RNAi) moiety, bifunctional shRNAfurin (pbi-shRNA™ furin), that targets and down regulates the proprotein convertase Furin resulting in knockdown of both TGFb1 and b2 The pbi-shRNA™ technology is designed to maximize target inhibition via translational repression through a process involving simultaneous mRNA cleavage and p-body sequestration GMCSF further activates and expands the cytotoxic T-cell population in the immune afferent arm This “triad” concept promotes immune detolerization, enhances antigen presentation and antigen processing Based on initial encouraging results in adult cancer patients at Mary Crowley Cancer Research Center with mechanistic demonstration of the technology (mean Furin knockdown was 85.09% and mean TGFb1 and -b2 knockdown were 93.5% and 92.5%, respectively), survival advantage (median survival was 554 days, FANG™ versus 255 days, non FANG™, (p=.006)) and with the lack of serious adverse events, a Phase I study was amended to include pediatric patients ( 12 y.o.) with advanced/relapsed Ewing’s sarcoma who had failed standard treatment options Briefly, the vaccine is cGMP manufactured at Gradalis, Inc following autologous tumor tissue harvest Vaccine is produced at a dose of x cells/injection The vaccine is administered intradermally once a month (minimum of to a maximum of 12 doses) ELISPOT analysis of cytotoxic T cell function to autologous Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy tumor antigens are monitored at baseline, Months 2, 4, and EOT To date, pediatric patients entered the screening process for entry into trial, with relapsed Ewing’s Sarcoma and with progressive disease Vaccine manufacturing was successful in of the harvested patients, patient had insufficient viable tumor tissue Two patients were registered into the protocol, one died from progressive disease (received one FANG™ vaccine injection) One patient who developed pulmonary metastasis one year post completion of treatment has stable disease month after treatment initiation and showed activation of the ELISPOT assay There have been no treatment-related serious adverse effects; one patient had a grade treatment-related AEs limited to the skin erythema at the injection site This limited initial data shows the safety and feasibility of this study Updated results will be presented 204 Dissecting the Human T Cell Receptor Repertoire in Different Immunological Conditions by Deep Sequencing Eliana Ruggiero,1 Jan P Nicolay,2,3 Anne Arens,1 Raffaele Fronza,1 Anna Paruzynski,1 Ali Nowrouzi,1 Gửkỗe ĩrenden,1 Sergij Goerdt,3 Hanno Glimm,1 Peter H Krammer,2 Manfred Schmidt,1 Christof von Kalle.1 Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center, Heidelberg, Germany; 2Division of Immunogenetics, German Cancer Research Center, Heidelberg, Germany; 3Department of Dermatology, Venereology and Allergology, University Medical Center, Mannheim, Germany T cell ability to recognize foreign antigens relies on the high variety of T cell receptors (TCR), heterodimeric or glycoproteins Each chain is originated from the somatic rearrangement of non contiguous genes belonging to different families (V, (D, only for and chains) and J) Addition and deletion of non templated nucleotides at the rearranging loci further increase the diversity of the TCR molecule By deep sequencing of the CDR3 region, the hypervariable portion of the TCR responsible for antigen recognition, it is possible to identify the clonality and the functional status of a T cell population We have established TCR-LAM direct sequencing, a new unbiased, highly specific and sensitive RNA-based assay for the characterization of the TCR repertoire diversity in healthy and in diseased conditions Analysis of the TCR repertoire in healthy HLAunrelated individuals led to the dissection of the events generating the TCR molecules A not uniform V and J gene usage and the occurrence of preferred VJ pairings were observed A deep insight in the CDR3 sequence down to the single nucleotide level, displayed a high degree of variability in the center of the sequence with a preferential usage of guanines These results hold true for both TCR chains and for every donor Convergent recombination, the generation of identical TCR amino acid (aa) specificities through different VJ pairings and/ or different nucleotide sequences, was observed in 11% and 7% chain CDR3 aa sequences A sharing of 2.4% and 1% TCR aa sequences between any donors (public clones) was identified in our dataset Sequencing results from a blinded analysis of the TCR repertoire in 10 patients with severe and mild form of cutaneous T cell lymphoma (Sézary disease) showed the ability of TCR-LAM to dissect different stages of the disease We observed a distorted oligoclonal repertoire with a dominating TCR clonotype in the severe disease form and a less restricted oligoclonal repertoire in the mild disease form Moreover, imunomonitoring of the TCR diversity was performed on peripheral blood mononuclear cells (PBMC) in CMV seropositive donors after restimulation of the CMV-specific T cell response with a viral peptide pool Overtime (day 0, day and day 15 after restimulation) analysis showed the emergence of specific TCR clonotypes already at day CMV-specific sequences were detected at day even with a very low frequency (0.01%) A high degree of convergent recombination and the presence of TCR motifs, showed S79 ... Oncology, P .A, Dallas; 2Gradalis, Inc., Dallas; 3Mary Crowley Cancer Research Centers, Dallas; 4Medical City HCA, Dallas The FANG™ vaccine contains a plasmid encoding both GMCSF and an RNA interference... in Advanced Ewing’s Sarcoma: Preliminary Data in Pediatrics Patients Maurizio Ghisoli,1 Carl Lenarsky,1 Gladice Wallraven,2 Padmasini Kumar,2 Phillip B Maples,2 John Nemunaitis.1,2,3,4 Texas... survival advantage (median survival was 554 days, FANG™ versus 255 days, non FANG™, (p=.006)) and with the lack of serious adverse events, a Phase I study was amended to include pediatric patients