Abstract PD3-07: TEM8 specific CAR T cells serve as a novel targeted therapy for triple negative breast cancer and its supporting endothelium

Abstract

Abstract Background: Triple Negative Breast Cancer (TNBC) refers to an aggressive subtype of breast cancer negative for HER2, estrogen and progesterone receptors. Lacking these receptors, individuals with TNBC do not benefit from many of the targeted therapies for breast cancer. Tumor endothelial marker 8 (TEM8), originally identified as a tumor endothelium associated antigen, has more recently been implicated in TNBC pathogenesis and as a marker of breast cancer stem like cells. Here we report that T cells expressing a TEM8-specific chimeric antigen receptor (CAR) serve as a novel approach to target both TNBC cells and its supporting endothelium. CARs combine the specificity of a monoclonal antibody with the signaling properties of a T cell. Methods: We designed two novel TEM8-specific CAR molecules. A CAR molecule containing an exodomain derived from the anti-TEM8 L2 antibody, followed by CD28 and CD3-zeta signaling domains (second generation CAR) and CD28, 41BB and CD3-zeta signaling domains (third generation CAR), respectively. Retroviral transduction was used to express the TEM8 CAR transgene constructs on HEK 293T cells, then on primary T cells. Results: Immunofluorescence staining revealed that in a panel of primary TNBC breast cancer samples, TEM8 was overexpressed in comparison to normal adjacent breast tissue (6/6). Costaining with the pan-endothelial cell marker CD31 revealed that this overexpression was not confined to the endothelial compartment, but also present on tumor parenchymal cells. The immortalized TNBC cell lines (MDA-MB-231, MDA-MB-436, MDA-MB-468 and Hs578T) expressed endogenous levels of TEM8 protein as revealed by western blot. Greater than 90% transduction of primary human T cells was achieved using both of our CAR constructs, as detected by flow cytometry. TEM8 specific T cells displayed significantly higher killing of TEM8 positive TNBC and tumor endothelial cells (2H11 and bEND.3) in standard four hour chromium release assays when compared to both non-transduced or irrelevant (CD19) CAR T cells and secreted immunomostimulatory cytokines upon encounter of TEM8 positive cells in coculture assays. In a vascularized xenograft model, MDA MB468 cells were injected subcutaneously with matrigel into athymic nude mice and followed via bioluminescence imaging over the course of two months. Established tumors were treated with either, second or third generation TEM8 specific T cells, HER2 specific T cells, non-transduced T cells or left untreated. Relative to non-transduced T cells, TEM8 specific second and third generation CAR T cells significantly delayed tumor growth by 36 days and 50 days, respectively. Conclusion: TEM8 specific CAR T cells could serve as a novel targeted therapy for TNBC and supporting endothelium. Citation Format: Byrd T, Fousek K, Pignata A, Szot C, Bielamowicz K, Wakefield A, Koch J, Landi D, Seaman S, Wels W, Fletcher B, Hegde M, St Croix B, Ahmed N. TEM8 specific CAR T cells serve as a novel targeted therapy for triple negative breast cancer and its supporting endothelium. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD3-07.