Gene therapy using genetically modified lymphocytes targeting VEGFR-2 inhibits the growth of vascularized syngenic tumors in mice
Dhanalakshmi Chinnasamy, … , Nicholas P. Restifo, Steven A. Rosenberg
Dhanalakshmi Chinnasamy, … , Nicholas P. Restifo, Steven A. Rosenberg
Published October 11, 2010
Citation Information: J Clin Invest. 2010;120(11):3953-3968. https://doi.org/10.1172/JCI43490.
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Research Article Genetics

Gene therapy using genetically modified lymphocytes targeting VEGFR-2 inhibits the growth of vascularized syngenic tumors in mice

Abstract

Immunotherapies based on adoptive cell transfer are highly effective in the treatment of metastatic melanoma, but the use of this approach in other cancer histologies has been hampered by the identification of appropriate target molecules. Immunologic approaches targeting tumor vasculature provide a means for the therapy of multiple solid tumor types. We developed a method to target tumor vasculature, using genetically redirected syngeneic or autologous T cells. Mouse and human T cells were engineered to express a chimeric antigen receptor (CAR) targeted against VEGFR-2, which is overexpressed in tumor vasculature and is responsible for VEGF-mediated tumor progression and metastasis. Mouse and human T cells expressing the relevant VEGFR-2 CARs mediated specific immune responses against VEGFR-2 protein as well as VEGFR-2–expressing cells in vitro. A single dose of VEGFR-2 CAR-engineered mouse T cells plus exogenous IL-2 significantly inhibited the growth of 5 different types of established, vascularized syngeneic tumors in 2 different strains of mice and prolonged the survival of mice. T cells transduced with VEGFR-2 CAR showed durable and increased tumor infiltration, correlating with their antitumor effect. This approach provides a potential method for the gene therapy of a variety of human cancers.

Authors

Dhanalakshmi Chinnasamy, Zhiya Yu, Marc R. Theoret, Yangbing Zhao, Rajeev K. Shrimali, Richard A. Morgan, Steven A. Feldman, Nicholas P. Restifo, Steven A. Rosenberg

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Figure 1

Construction and characterization of recombinant retroviral vectors expressing CARs targeted against mouse VEGFR-2.

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Construction and characterization of recombinant retroviral vectors expr...
(A) Schematic representation of recombinant retroviral vectors encoding CARs used in this study. In the DC101-CD828BBZ CAR vector, the DC101 ScFv is made up of the VH and VL derived from a rat IgG against mouse VEGFR-2 joined by a 218 linker that is linked to the hinge and transmembrane regions of the mouse CD8α chain and mouse intracellular signaling sequences derived from CD28, 4-1BB, and CD3-z molecules. The DC101-CD828Z vector lacked the 4-1BB signaling domain. The DC101-CD8 construct lacked all the intracellular signaling sequences. The SP6-CD828BBZ construct was derived from SP6, a mouse monoclonal antibody raised against a hapten 2, 4, 6-trinitrophenyl (TNP). (B) Enriched splenic CD3+ T cells were transduced with indicated retroviral vectors shown in A. Four days after transduction, cells were analyzed for transgene expression by flow cytometry. Representative FACS data are presented, showing the percentage of T cells in each quadrant, with the percentage of transgene-positive cells in parentheses. IsoAb, isotype control antibody. GAM, goat anti-mouse antibody. (C and D) Enriched CD3+ mouse T cells were transduced with the indicated retroviral vectors. Four days later, cells were cultured on antigen-coated 96-well microtiter plates for 3 days. (C) The proliferation of T cells was measured as [3H]thymidine incorporation during the last 16 hours. (D) Culture supernatants were assayed for IFN-γ by ELISA. Results are presented as the mean ± SEM of triplicates.