Abstract 1790: Exploring the efficacy of allogeneic gamma delta T cell adoptive cell therapy against the pediatric brain tumor medulloblastoma
Authors: Lowry Benjamin S. , Jonus Hunter C. , Lee Jasmine , Spencer Trent H. , Kenney Anna M. , Goldsmith Kelly 
Published: April 2023

Antigenic heterogeneity and limited immune cell infiltration has historically hindered immunotherapy approaches to treat the pediatric brain tumor medulloblastoma (MB). Therefore, modern MB treatment modalities rely on broadly cytotoxic chemotherapy and/or radiation, leaving survivors with side effects that diminish quality of life, stressing an urgent need for novel approaches. Gamma delta (γδ) T cells represent an emerging class of cellular immunotherapy with preclinical potency against the CNS tumor glioblastoma and pediatric solid tumors. The ability of γδ T cells to recognize tumor stress antigens, alongside their MHC independence, supports γδ T cell usage as an off-the-shelf allogeneic cellular immunotherapy for MB and other immunologically cold cancers. Our team recently identified protein tyrosine kinase 7 (PTK7) as a novel immunotherapy target highly expressed in neuroblastoma, with low pediatric healthy tissue expression. Alpha beta T cells engineered with CARs targeting PTK7 demonstrated impressive antitumor efficacy against PTK7+ neuroblastoma xenografts. In that study, we also noted MB and other pediatric solid tumors express elevated levels of PTK7 mRNA. This work therefore serves to test the susceptibility of MB to γδ T cell immunotherapy and validate if targeting PTK7 can further enhance γδ T cell potency.

Six MB cell lines representing three out of the four MB molecular subgroups were chosen: DAOY, ONS-76, UW228, D341, D425 and D283. Western blot analysis showed five of six MB cell lines express PTK7 protein, and flow cytometry confirmed PTK7 membrane localization. PTK7high (DAOY) and PTK7low (ONS-76) cells were chosen to test for the antigen-specific killing potential of PTK7-targeted CAR γδ T cells against MB. To generate PTK7-targeted γδ T cells, previously cryopreserved γδ T cells were electroporated with mRNA encoding a PTK7-targeted CAR construct containing the CD28 co-stimulatory domain. After a four hour co-culture, both ONS-76 and DAOY showed moderate rates of apoptosis from exposure to mock electroporated or naïve γδ T cells at 1:1, 2:1 and 5:1 effector:target (E:T) ratios, with approximately 30-40% cell death at 5:1. There was no change in susceptibility of ONS-76 cells to PTK7-CAR γδ T cells when compared to mock electroporated or naïve γδ T cell controls. However, co-culture of DAOY cells with PTK7-CAR γδ T cells markedly increased tumor cell death by as much as 40% compared to the mock controls at E:T ratios as low as 1:1.

In summary, naïve expanded γδ T cells induce pronounced MB tumor cell death. Cytotoxicity is further increased against PTK7high MB cells following the introduction of an anti-PTK7 CAR. These results provide a strong rationale for additional preclinical studies regarding the feasibility of γδ T cell-based immunotherapy against MB.

Citation Format: Benjamin S. Lowry, Hunter C. Jonus, Jasmine Lee, Trent H. Spencer, Anna M. Kenney, Kelly Goldsmith. Exploring the efficacy of allogeneic gamma delta T cell adoptive cell therapy against the pediatric brain tumor medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1790.