Abstract 3867: Identification of novel biomarkers of immune toxicity from CAR T-cell therapy using ultrasensitive NULISA™ proteome technology
Authors: Kirkpatrick Riley , Beer Joanne , Patel Manishkumar S. , Jalota Akansha , Mian Agrima , Ariyapala Ishara S. , Hao Qinyu , Feng Wei , Ma Xiao-Jun , Luo Yuling , Hill Brian T. , Gupta Neetu 
Published: March 2024

Abstract
Introduction: The efficacy of anti-CD19 chimeric antigen receptor (CAR) T-cell therapy in patients with relapsed/refractory large B-cell lymphoma (LBCL) is limited by acute toxic events, most notably cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Identification of biomarkers of toxicities can allow for selection of high-risk patients and elucidate targetable mechanisms for mitigation. Previous biomarker studies have been constrained by relatively narrow panels of potential mediators and limited time points. Here, we employed NULISA, a novel ultrasensitive assay capable of simultaneously quantifying 204 inflammatory proteins from a single sample, to identify temporal proteome associations with acute toxicities in anti-CD19 CAR T-cell treated patients.
Methods: Baseline and post-treatment peripheral blood samples from 80 patients with LBCL who underwent anti-CD19 CAR-T cell therapy were collected within specific time intervals. Plasma samples (n=480) were analyzed with NULISA. CRS and ICANS were graded according to ASTCT consensus criteria. Patients were grouped according to their maximum CRS and ICANS scores. Differential protein abundance across the toxicity groups was assessed using linear mixed effects models fit for each protein, including a time by toxicity group interaction. Proteins showing significant associations were subjected to pathway enrichment and network analysis.
Results: Severe CRS and ICANS patient groups showed proteome upregulation beginning on day 1-2 and peaking at day 6-9, followed by extensive downregulation on day 11-16. The strongest upregulated pathway associations of severe CRS and ICANS were inflammatory response, IL-17 signaling, non-genomic action of vitamin D3, regulation of leukocyte proliferation, and cellular extravasation. Downregulated pathways included anti-microbial humoral response and TNFs binding to receptors. In addition to the previously identified inflammatory proteins IL-6, IFNγ, sIL-2Rα, CXCL8, CCL2, our analysis revealed significant association of several novel immune regulators and mediators. These included upregulated Th2 cytokines, IL-17A, IL-22, GZMB, CTLA4, IFNA1;IFNA13, and downregulated S100A12, IL-12B, CCL22, BDNF and TNFSFs, revealing exquisite temporal orchestration of lymphoid and myeloid activities during CRS and ICANS development.
Conclusions: This study represents the most comprehensive characterization of immune response to CAR T-cell therapy to date. Using the novel NULISA technology, we identified new proteins and functional pathways associated with CAR T-cell-induced toxicity, implicating them as potential biomarkers. These previously unidentified factors also provide a platform to further investigate the causative immune mechanisms of acute toxicities in CAR-T cell therapy.
Citation Format: Riley Kirkpatrick, Joanne Beer, Manishkumar S. Patel, Akansha Jalota, Agrima Mian, Ishara S. Ariyapala, Qinyu Hao, Wei Feng, Xiao-Jun Ma, Yuling Luo, Brian T. Hill, Neetu Gupta. Identification of novel biomarkers of immune toxicity from CAR T-cell therapy using ultrasensitive NULISA™ proteome technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3867.