282. A scFv-Based CAR To Redirect T Cells To IL13Ra2-Positive Glioma

Cancer – Targeted Gene and Cell Therapy II 282. A scFv-Based CAR To Redirect T Cells To IL13Ra2-Positive Glioma

Giedre Krenciute,1 Simone Krebs,1 David Torres,1 Maciej Lesniak,2 Irina Balyasnikova,2 Stephen Gottschalk.1 1 Department of Pediatrics, Hematology and Oncology Section, Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX; 2Department of Surgery, The Brain Tumor Center, University of Chicago, Chicago, IL. BACKGROUND: Glioblastoma (GBM) is the most aggressive primary brain tumor in humans, and is virtually incurable with conventional therapies. Immunotherapy with T cells expressing chimeric antigen receptors (CARs) is an attractive approach to improve outcomes. IL13Ra2 is aberrantly expressed in GBM therefore it is a promising target for CAR T-cell immunotherapy. The antigen recognition domain of CARs normally consists of a single chain variable fragment (scFv), however current IL13Ra2-specific CARs use IL13 muteins as an antigen recognition domain. We have previously shown that IL13 mutein-based CARs also recognize IL13Ra1, raising significant safety concerns. To overcome this obstacle we have recently generated a high affinity IL13Ra2-specific scFv. The goal of this project is now to develop a scFv-based IL13Ra2specific CAR (IL13Ra2-CAR) and evaluate the effector function of IL13Ra2-CAR T cells. METHODS: We constructed a panel of IL13Ra2-CARs that contain our new IL13Ra2-specific scFv (m47) as an ectodomain, a short hinge (SH) or long hinge (LH), a CD28 transmembrane domain, and endodomains that contain signaling domains derived from CD3ζ and co-stimulatory molecules (CD28.ζ, CD137.ζ, CD28.CD137.ζ, CD28.CD134.ζ). IL13Ra2-CAR T cells were generated by retroviral transduction, and we determined their effector function in vitro, in co-culture and cytotoxicity assays, and in vivo, in the U373 brain xenograft model. RESULTS: Expression of all CARs in T cells was similar as judged by Western blot analysis. However, cell surface expression varied depending on used hinge and endodomain. In cytotoxicity assays, T cells expressing the different IL13Ra2-CARs only killed target cells expressing IL13Ra2, but not IL13Ra1 thus confirming the specificity of receptor-CAR interaction. While all IL13Ra2-CAR T cells secreted significant levels of IFNγ in co-culture assays with the IL13Ra2+ glioma cell line U373, only short hinge CAR T cells secreted significant amounts of IL2. T cells expressing IL13Ra2-CARs with a deleted endodomain (IL13Ra2.∆-CAR) secreted no cytokines confirming that cytokine production depends on the presence of a functional IL13Ra2-CAR. In vivo, injection of IL13Ra2.SH.CD28.ζCAR T cells into U373-bearing mice resulted in regression of gliomas as judged by bioluminescence imaging. IL13Ra2.LH.CD28.ζ- or IL13Ra2.∆-CAR T cells had no antitumor effects. CONCLUSION: For the first time we have generated a CAR that only recognizes IL13Ra2 and not IL13Ra1. Comparison of several IL13Ra2-CARs revealed so far that a CAR with a SH and a CD28.ζ endodomain results in optimal T-cell activation as judged by IL2 production and in vivo anti-glioma activity. Our results warrant further active exploration of IL13Ra2-specific scFv-based CAR T cells for the adoptive immunotherapy of high grade glioma.

Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy

283. Targeted Delivery of C/EBPα-Small Activation RNA (saRNA) by RNA Aptamer Inhibits the Growth of Pancreatic Ductal Adenocarcinoma (PDAC) In Vivo

Sorah Yoon,1 Kai-Wen Huang,2 Paul Mintz,3 Nagy Habib,3 John J. Rossi.1,4 1 Molecular and Cellular Biology, Beckman Research Institute of City of Hope, Duarte, CA; 2Surgery and Hepatitis Research Center, National Taiwan University College of Medicine, Taipei, Taiwan; 3 Surgery and Cancer, Imperial College London, London, United Kingdom; 4Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, Duarte. Transcriptional factor C/EBPα (CCAAT/enhancer-binding protein-a) is downregulated and silenced in pancreatic ductal adenocarcinoma (PDAC). In this study, we tested the feasibility of targeting promoter regions of transcriptional factor C/EBPα to induce transcriptional activation by small activation RNA (saRNA) for cancer therapeutics in PDAC. C/EBPα is known to up-regulate p21, a strong inhibitor of cell proliferation. C/EBPα-saRNA inhibited cell proliferation in PDAC. For the targeted delivery into cells, small structured single-stranded RNAs known as aptamers, recognizing targets specifically, were employed for the effective delivering vehicles. 2’-fluropyrimidine RNA aptamers (2’F-RNA) were selected to acquire nuclease resistance based on the whole cell SELEX. The cell internalization was evaluated through flow cytometry and confocal microscopy. The selected 2’F-RNA aptamers got internalized into a variety of pancreatic cancer cell lines. Human pancreatic cancer specimen histopathology and cell binding assays indicated that they recognized cancer cells specifically, not to normal cells. The conjugates, aptamer with C/EBPα-saRNA, were constructed for the targeted delivery. The conjugates escaped endosome and localized in nuclear. The conjugates of aptamer with C/EBPα-saRNA inhibited cell proliferation and upregulated gene expression. Xenograft mice model was used to test tumor cell growth inhibition in vivo throughout the transcriptional activation of C/EBPα. The conjugates of aptamer with C/EBPα-saRNA reduced tumor burden significantly in vivo, showing its anti-tumor effects. The aptamer target expressed on cell membrane was identified mitochondrial Hsp70. For the first time, results from these studies provide a new therapeutics strategy through transcriptional activation of C/EBPα by aptamer mediation induce anti-tumor effects in pancreatic cancer.

284. High-Efficiency Targeted Introduction of an Anti-CD19 CAR Into the CCR5 Locus in Primary Human T Cells

Malika Hale,1 Guillermo S. Romano Ibarra,1 Iram F. Khan,1 Jaya Sahni,1 Andrew M. Scharenberg,1,2 David J. Rawlings.1,2 1 Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children’s Research Institute, Seattle, WA; 2Departments of Pediatrics and Immunology, University of Washington School of Medicine, Seattle, WA.

The safety and efficacy of anti-CD19 chimeric antigen receptor (CD19-CAR) therapeutic cell transfer for treatment of CD19+ B cell malignancies is currently being evaluated in multiple clinical trials. Current protocols employ randomly integrating lentiviral or gammaretroviral vectors to introduce the CAR into patient- or donor-derived T cells. This strategy carries with it an inherent risk of insertional mutagenesis as well as variable CAR expression. Here we have developed a method of targeting a CD19-CAR construct directly into the CCR5 locus by homology directed recombination (HDR). Using a CCR5 megaTAL nuclease (an engineered homing endonuclease and TALEN chimera) and an AAV donor template (comprised of an MND promoter-CD19CAR-T2A-BFP expression cassette between CCR5 S113