Background: Chimeric antigen receptor T cells (CARTs) hold promising therapeutic potential for refractory tumors. GD2 is a tumor antigen expressed on neuroblastoma and osteosarcoma. In initial studies, T cells expressing 1st generation GD2-CARTs were shown to be safe and mediated modest antitumor activity in some patients with refractory neuroblastoma.

Methods: We developed a 3rd generation GD2-CART (GD2-CAR.OX40.28.z.ICD9) and conducted a phase I trial (NCT02107963) to determine the feasibility of producing and safety of administering escalating doses of GD2-CARTs in children and young adults with GD2+ solid tumors, including neuroblastoma and osteosarcoma, following cyclophosphamide-based lymphodepletion. Patient samples were evaluated for cytokine profile kinetics, immune phenotype analysis with mass cytometry (CyTOF), transcriptomic evaluation with RNA-sequencing (RNA-seq), epigenetic determination with Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), and functional studies with flow cytometry.

Results: 15 patients aged 8-28 years were enrolled on four dose levels, of which 13 patients were infused. No dose-limiting toxicities were observed and administration of up to 1x107 GD2-CART/kg was feasible and safe for children and young adults with neuroblastoma and osteosarcoma. 15.4% (2/13) of patients experienced grade-1 cytokine release syndrome (CRS) and no neurological toxicity was observed. We measured the expansion and persistence of adoptively transferred GD2-CARTs in the peripheral blood. GD2-CARTs expanded in all patients receiving treatment, half of whom had expansion similar to that seen in clinically active CD19 and CD22 CARTs, but the GD2-CARTs had limited persistence. At Day 28 following GD2-CART infusion, 23.1% (3/13) of evaluable patients had progressive disease and 76.9% (10/13) had stable disease (SD). 3/10 SD patients remained stable at 60 days post-GD2-CART, but all patients eventually progressed. Since a major barrier to CART efficacy is inadequate CART expansion, we comprehensively evaluated for phenotypic, transcriptomic, and epigenetic immune profiles of patient apheresis, CART product, and post-treatment peripheral blood samples to identify determinants of CART expansion. GD2-CART expansion is significantly correlated with several T cell markers, and a larger baseline naïve and central memory T cell pool. Unique myeloid populations are associated with CART expansion. ATACseq identifies epigenetic differences in pre-treatment apheresis that may predict good CAR expansion in patients.

Conclusions: GD2-CART therapy following cyclophosphamide conditioning was well tolerated at all four dose levels in pediatric and young adult patients with neuroblastoma and osteosarcoma. Subsequent multi-dimensional analyses suggest key mechanisms underlying CART biology and function and highlight the potential of defining and applying molecular signatures in apheresis and CART product as biomarkers and prognostic indicators of CART expansion, with promise for advancing immunotherapies for solid tumor patients in the future.

Citation Format: Sneha Ramakrishna, Sabina Kaczanowska, Tara Murty, Cristina F. Contreras, Melinda Merchant, John Glod, Norma Gutierrez, Ahmad Alimadadi, David Stroncek, Steven Highfill, Caroline Duault, Priyanka B. Subrahmanyam, Tyson Holmes, Warren Reynolds, Reema Baskar, Antoine Barge, Hayley Lyon, Radim Moravec, Srinika Ranasinghe, Joyce Yu, Roshni Biswas, Samuel Pollack, Stephen Van Nostrand, James Lindsay, Mina Pichavant, Bita Sahaf, Sean C. Bendall, Andrew J. Gentles, Holden Maecker, Catherine C. Hedrick, Crystal Mackall, Rosandra Kaplan. GD2.Ox40.CD28.z CAR T cell trial in neuroblastoma and osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT142.