Abstract B74: Co-targeting the tumor and its associated vasculature in glioblastoma
Authors: Byrd Tiara T. , Fousek Kristen , Grada Zakaria , Aviles-Padilla Kevin , Bielamowicz Kevin , Gottschalk Stephen , Croix Bradley St , Fletcher Bradley , Hegde Meenakshi , Ahmed Nabil 
Published: May 2015

Abstract
Background: Glioblastoma (GBM) is the most common and most aggressive primary brain tumor in adults. With the current standard treatment, survival is poor with substantial treatment related toxicities underscoring the need for new targeted therapies. Preclinical and early clinical data demonstrate the efficacy of chimeric antigen receptor (CAR)-grafted T-cells as a novel approach against cancer. Previous work from our lab has demonstrated that HER2-specific T cells can induce tumor regression in an orthotopic xenogeneic model. However, tumors recurred in a subset of mice treated with HER2-specific T cells. It is now evident that tumors are heterogeneous cellular complexes whose growth is dependent upon reciprocal interactions between the tumor and its heterogeneous microenvironment. In particular, glioblastomas are highly vascularized tumors, known for their invasive phenotype. Tumor Endothelium Marker 8 (TEM8) is upregulated in tumor angiogenesis and conserved in mice, thus making it a target which can be studied pre-clinically for both safety and efficacy.
Objective: The purpose of this study is to determine whether co-targeting the tumor antigen, HER2 and Tumor Endothelium Marker 8 (TEM8) using bispecific CAR T cells will result in improved tumor control in GBM.
Methods: In order to generate TEM8 and HER2 bispecific CAR T cells we employed two methods. For the first method we designed a TEM8 CAR, to be coexpressed along with a separate HER2 CAR on the same T cells (TEM8-HER2 BiCAR T cells). For the second method we designed a bispecific tandem CAR (TEM8-HER2 TanCAR) in which TEM8 and HER2 specific single chain variable fragments have been placed in-tandem and are connected by a glycine-serine linker to make a single CAR to be expressed on T cells. CAR constructs were assembled on clone manager, synthesized, then sequence verified and force expressed on T cells using a retroviral system. CAR T cells were expanded in IL-2 in parallel with the control T cells from the same donor. Flow cytometry was then used to confirm CAR expression on T cells and binding specificity. ELISA and flow-based cytotoxicity assays were used to assess selectivity and killing potential of TEM8 and HER2 bispecific T cells.
Results: We have successfully cloned in the TEM8 and TEM8-HER2 TanCAR constructs and generated TEM8-HER2 BiCAR T cells TEM8-HER2 specific TanCAR T cells, respectively. Up to 70% of T cells expressed the specific CAR on the cell surface. TEM8 and HER2 specific CAR T cells selectively recognize and kill TEM8 and HER2 positive targets and release the immuno-stimulatory cytokines interferon-gamma and interleukin-2 in vitro. Further, cytokine release was augmented when bispecific T cells were simultaneously exposed to both target targets.
Conclusion: Heterogeneous nature of GBM favors co-targeting the tumor and its microenvironment. We have successfully generated the first TEM8 specific CAR T cell product to specifically target the tumor associated vasculature. Co-targeting tumor and its endothelium using bispecific CAR T cells could enhance T cell activation and can potentially be used to improve tumor control and have therapeutic application in GBM patients.
Citation Format: Tiara T. Byrd, Kristen Fousek, Zakaria Grada, Kevin Aviles-Padilla, Kevin Bielamowicz, Stephen Gottschalk, Bradley St Croix, Bradley Fletcher, Meenakshi Hegde, Nabil Ahmed. Co-targeting the tumor and its associated vasculature in glioblastoma. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B74. doi:10.1158/1538-7445.CHTME14-B74