Tumor infiltrating lymphocytes expanded from pediatric neuroblastoma display heterogeneity of phenotype and function.

Adoptive transfer of ex vivo expanded tumor infiltrating lymphocytes (TILs) has led to clinical benefit in some patients with melanoma but has not demonstrated convincing efficacy in other solid cancers. Whilst the presence of TILs in many types of cancer is often associated with better clinical prognosis, their function has not been systematically evaluated across cancer types. Responses to immunological checkpoint inhibitors in a wide range of cancers, including those for which adoptive transfer of expanded TILs has not shown clinical benefit, has clearly delineated a number of tumor type associated with tumor-reactive lymphocytes capable of effecting tumor remissions. Neuroblastoma is an aggressive childhood solid cancer in which immunotherapy with GD2-directed antibodies confers a proven survival advantage through incompletely understood mechanisms. We therefore evaluated the feasibility of ex vivo expansion of TILs from freshly resected neuroblastoma tumors and the potential therapeutic utility of TIL expansions. TILs were successfully expanded from both tumor biopsies or resections. Significant numbers of NKT and γδT cells were identified alongside the mixed population of cytotoxic (CD8+) and helper (CD4+) T cells of both effector and central memory phenotypes. Isolated TILs were broadly non-reactive against autologous tumor and neuroblastoma cell lines, so enhancement of neuroblastoma killing was attained by transducing TILs with a second-generation chimeric antigen receptor (CAR) targeting GD2. CAR-TILs demonstrated antigen-specific cytotoxicity against tumor cell lines. This study is the first to show reproducible expansion of TILs from pediatric neuroblastoma, the high proportion of innate-like lymphocytes, and the feasibility to use CAR-TILs therapeutically.

Chimeric Antigen Receptor-Engineered Human Gamma Delta T Cells: Enhanced Cytotoxicity with Retention of Cross Presentation.

Gamma delta T (γδT) lymphocytes are primed for rapid function, including cytotoxicity toward cancer cells, and are a component of the immediate stress response. Following activation, they can function as professional antigen-presenting cells. Chimeric antigen receptors (CARs) work by focusing T cell function on defined cell surface tumor antigens and provide essential costimulation for robust activation. Given the natural tropism of γδT cells for the tumor microenvironment, we hypothesized that their transduction with CARs might enhance cytotoxicity while retaining their ability to migrate to tumor and act as antigen-presenting cells to prolong the intratumoral immune response. Using a GD2-targeting CAR as a model system, we showed that γδT cells of both Vδ1 and Vδ2 subsets could be expanded and transduced to sufficient numbers for clinical studies. The CAR added to the cells' innate cytotoxicity by enhancing GD2-specific killing of GD2-expressing cancer cell lines. Migration toward tumor cells in vitro was not impaired by the presence of the CAR. Expanded CAR-transduced Vδ2 cells retained the ability to take up tumor antigens and cross presented the processed peptide to responder alpha beta T (αßT) lymphocytes. γδ CAR-T cell products show promise for evaluation in clinical studies of solid tumors.