Abstract 914: Preinfusion polyfunctional profiles and cytokine secretion activity of transgenic TCR-T cells, and serum cytokine profiles, are associated with clinical outcomes in sarcoma and melanoma
Authors: Peters Cole W. , Quiros Crystal , Han Edward X. , Kawakami Moe , Kidd Conner , Klomhaus Alexandra , Ribas Antoni , Nowicki Theodore S. 
Published: April 2023

Abstract
Background: Transgenic TCR-T cells targeting MART-1 and NY-ESO-1 for the treatment of melanoma and sarcoma, respectively, are associated with robust initial clinical responses. However, these responses are often not durable, and a significant proportion of patients do not respond to therapy at all. Therefore, there is a significant need to understand the factors driving success or failure of these therapeutics.
Methods: We analyzed the functional single-cell and bulk cytokinetic secretome profiles of TCR-T products infused into 26 patients (MART-1 n = 13, NY-ESO-1 n = 13) under associated TCR-T cell clinical trials at UCLA (NCT00910650, NCT02070406, NCT01697527, NCT03240861) using the IsoPlexis proteomic barcode chip platform. TCR-T cells were separated into CD4 and CD8 compartments and stimulated with their cognate antigen presented by transgenic HLA-A:02:01 K562 cells in vitro at an effector:target ratio of 1:1. Stimulated cells were loaded into IsoCode chips for single-cell polyfunctionality analysis while culture media supernatants from the antigen-specific stimulation were loaded into CodePlex chips for bulk analysis. All chips were automatically processed using an IsoSpark machine and analyzed using IsoSpeak software. Patients’ baseline preinfusion serum cytokine levels were analyzed via Luminex Immunoassay.
Results: Single-cell secretome analysis showed that polyfunctional CD4 TCR-T cells producing TNF-a and IL-17A were significantly associated with improved progression-free survival and overall survival, respectively (p = 0.036; p = 0.014). Polyfunctional CD8 TCR-T cells producing TNF-a and MIP-1a were significantly lower in patients with progressive disease at end-of-study compared to patients without progressive disease (p = 0.0498; p = 0.0468). Bulk secretome analysis demonstrated that aggregate CD4 production levels of IL-4 and IL-5 were both significantly lower in clinical responders compared to non-responders (286.9 vs 1088.5 pg/mL, p = 0.0073; 811.8 vs 1957.9 pg/mL, p = 0.034). Preinfusion patient serum IL-15 levels examined by Luminex were significantly greater in clinical responders compared to non-responders (19.65 vs 4.74 pg/mL p = 0.0122).
Conclusions: In clinical samples of preinfusion TCR-T cells targeting MART-1 and NY-ESO-1, polyfunctional CD4 TCR-T cells producing TNF-a and IL-17A, CD8 TCR-T cells producing TNF-a and MIP-1a, as well as preinfusion serum IL-15 levels, are significantly correlated with improved clinical outcomes in TCR-T cell therapy targeting MART-1 and NY-ESO-1. Furthermore, aggregate TCR-T cell production of the Th2-associated cytokines IL-4 and IL-5 were significantly associated with non-response to therapy. These results have important implications for the design of future generations of transgenic cellular therapies.
Citation Format: Cole W. Peters, Crystal Quiros, Edward X. Han, Moe Kawakami, Conner Kidd, Alexandra Klomhaus, Antoni Ribas, Theodore S. Nowicki. Preinfusion polyfunctional profiles and cytokine secretion activity of transgenic TCR-T cells, and serum cytokine profiles, are associated with clinical outcomes in sarcoma and melanoma [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 914.