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Volume 2 Supplement 3

Abstracts of the 29th Annual Scientific Meeting of the Society for Immunotherapy of Cancer (SITC)

Dual targeting of the tumor and its associated vasculature using a single bispecific chimeric antigen receptor molecule


We have previously demonstrated the efficacy of HER2 specific chimeric antigen receptor (CAR) T cells in animal models of human cancer. While HER2 CAR T cells induced tumor regression, tumors recurred in a subset of animals. Lytic co-targeting of the tumor endothelium could represent a strategy that enhances tumor control. Tumor endothelium marker 8 (TEM8) is a recently described tumor endothelium-restricted antigen conserved in both mice and humans that represents an attractive antigen for vascular targeting [1].


To test the advantage of co-targeting TEM8 in conjunction with HER2 using a T cell product expressing a novel TEM8/HER2 bispecific CAR molecule.


We designed, in silico, a single CAR molecule with both TEM8 and HER2-specific exodomains joined together in tandem (thus termed TanCAR). A retroviral construct encoding a TEM8 specific single chain variable fragment (scFv) from the mAb SB5, a Glycine/Serine linker and a HER2 specific scFv (mAb FRP5) exodomain, followed by a CD28 transmembrane domain, and a CD28.CD3-zeta signaling endodomain was transduced CD3/CD28-activated T cells with RD114-pseudotyped retroviral particles to generate TEM8/HER2 bispecific TanCAR T cells. CAR expression was confirmed by flow cytometry. Standard immunoassays were used to test the CAR T cell functionality.


TEM8/HER2 TanCAR molecules were expressed on the surface of up to 90% of primary T cells. Staining specific for FRP5 and SB5 ensured the expression of the TanCAR molecule in its entirety. TanCAR T cells selectively recognized and killed TEM8 and HER2 positive targets distinctly, as evidenced by the release of the immuno-stimulatory cytokines interferon-gamma and interleukin-2 in vitro and standard 4 hour 51Cr release cytotoxicity assays. Cytokine release and killing were significantly enhanced when TanCAR T cells encountered both target antigens simultaneously. The kinetics of T cell activation followed a second order kinetic equation denoting a superadditive or synergistic effect upon recognition of a second antigen. Minimal activation or cytolytic activity occurred with target negative controls or with CAR null T cells.


Co-targeting the tumor and its vasculature using bispecific TanCAR T cells could enhance activation of these cells and potentially be used to improve tumor control with therapeutic application in cancer patients.


  1. Chaudhary A, Hilton MB, Seaman S, Haines DC, Stevenson S, Lemotte PK, Tschantz WR, Zhang XM, Saha S, Fleming T, St Croix B: TEM8/ANTXR1 blockade inhibits pathological angiogenesis and potentiates tumoricidal responses against multiple cancer types. Cancer Cell. 2012, 21 (2): 212-26. 10.1016/j.ccr.2012.01.004.

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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.

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Byrd, T., Fousek, K., Pignata, A. et al. Dual targeting of the tumor and its associated vasculature using a single bispecific chimeric antigen receptor molecule. j. immunotherapy cancer 2 (Suppl 3), P6 (2014).

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