Thursday, November 21, 2024
11/21/2024
Project Summary The objective of this proposal is to develop a T cell-based immunotherapy for the treatment of high-risk neuroblastoma (NB), for which outcomes remain poor. The adoptive transfer of chimeric antigen receptor (CAR) T cells specific for CD19 has been highly effective for CD19+ hematological malignancies. However, the efficacy of CAR T cells for the treatment of solid tumors, including NB, has been limited. Glypican 2 (GPC2) is a promising CAR target for NB since it is closely linked to its malignant phenotype. I have successfully generated a GPC2- CAR with a 41BBζ signaling domain and have shown in preliminary studies that T cells expressing this CAR (GPC2.41BBζ-CAR T cells) recognize and kill GPC2+ NB cells. In this project I now propose to optimize the effector function of GPC2.41BBζ-CAR T cells. Optimal CAR T cell activation and expansion requires a distinct set of signals, which consist of antigen-specific CD3ζ activation (signal 1) and costimulation (signal 2). Upon proper costimulation, T cells produce cytokines (signal 3), which is critical for their expansion. We and other investigators have shown that CAR T cells have a limited ability to sequentially eradicate tumor cells. In this application I therefore propose to investigate if provision of signal 3 or improving signal 1 enhances the antitumor activity of GPC2.41BBζ-CAR T cells. Specifically, I hypothesize that provision of signal 3 via transgenic expression of IL-15 or improving signal 1 by knocking out the negative regulator Src homology domain 2-containing phosphatase-1 (SHP-1) will bolster the antitumor activity of GPC2-CAR T cells for the treatment of NB. This hypothesis will be tested in two interrelated research aims. Aim 1 compares the effector function of GPC2.41BBζ-CAR and IL-15 secreting GPC2.41BBζ-CAR T cells, and Aim 2 will determine whether CRISPR/Cas9-mediated knockout of SHP-1 improves the effector function of GPC2.41BBζ-CAR T cells. State- of-the-art immunological assays will be utilized in both aims including phenotypic analysis, and their ability to kill tumor cells, expand, and produce cytokines after repeated exposure to tumor cells. Additionally, these CAR T cells will be compared in vivo to assess their ability to expand, persist, and eliminate established tumors. This project was carefully designed to provide me with a training opportunity to familiarize myself with the rapidly evolving field of cellular immunotherapy for cancer and state of the art techniques to characterize tumor-specific T cells. At the conclusion of the grant I expect to have optimized GPC2-CAR T cells for future clinical testing in patients with recurrent/refractory NB.
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