Purpose: The efficacy of anti-CD19 chimeric antigen receptor (CAR) T-cell therapy in large B-cell lymphoma (LBCL) patients is limited by acute toxicities, most notably cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Previous biomarker studies have been constrained by narrow protein panels and limited time points. We employed NULISATM, a novel ultrasensitive assay capable of simultaneously quantifying 204 proteins, to identify temporal proteome associations with acute toxicities in LBCL patients treated with anti-CD19 CAR T-cell therapy. Experimental Design: Plasma samples from 80 LBCL patients who underwent anti-CD19 CAR T-cell therapy, collected before and after cell infusion, were analyzed with NULISATM. Baseline demographics and treatment toxicities, including CRS and ICANS, were graded according to ASTCT consensus criteria. Differential protein abundance, pathway enrichment, and network analysis were performed. Results: Our analysis revealed higher levels of the chemokines CXCL1, CX3CL1, and CCL8 associated with severe toxicity within the first two days of treatment. Thereafter, severe toxicity was marked by more abundant Th2 cell effector cytokines, IL4, IL5 and IL-13, markers of exhaustion, and TNF receptor superfamily proteins CTLA4, PDCD1, CD274, LAG3, TNFRSF1A, TNFRSF1B, and CD40. Finally, patients with severe toxicities showed lower levels of cell growth factors PDGFA and EGF, and the neuronal repair protein BDNF at the resolution stage. Conclusions: This study represents the most comprehensive characterization of proteomic immune response to CAR T-cell therapy to date and identifies novel proteins, pathways, and networks associated with acute toxicity in the initiation and resolution phases, implicating them as potential biomarkers.

B.T. H. has received research funding from Takeda; consultancy, honoraria, research funding from Genentech, Karyopharm, Celgene, Abbvie, Pharmacyclics, Beigene, AstraZenica, Kite, a Gilead Company, BMS; consultancy, honoraria from Novartis., though not used for this study. J.C.B., I.S.A., Q.H., W.F., X.-J.M, and Y.L. are employees and stockholders of Alamar Biosciences, Inc. Y.L. and W.F. were named inventors in a patent application for the NULISATM technology filed by Alamar Biosciences, Inc.

Funding for this study was provided by VeloSano Bike to Cure, Cleveland Clinic Center of Excellence in Lymphoid Malignancies Research and Taussig Cancer Institute.

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For original data, please contact the corresponding author (guptan@ccf.org)