Novel Biomarkers of Immune Toxicity from CAR-T Cell Therapy Using Ultrasensitive NULIS Proteome Technology

Translational relevanceUsing serial samples from patients undergoing anti-CD19 CAR T-cell therapy and NULISATM, an assay with attomolar sensitivity, we identify novel plasma proteins associated with severe treatment-associated toxicities. This study not only reveals the evolution of proteomic biomarkers before and during therapy and their association with toxicities, it distinguishes between the induction and recovery phases, and provides insights into factors that may mediate chronic sequelae of CAR T-cell therapy. As the use of CAR T-cell therapy is limited by toxicities, especially in elderly patients or those with pre-existing comorbidities, being able to identify the patients who are at the highest risk for severe toxicities will allow for stratification strategies that would ultimately enable more widespread application of this cutting-edge therapy. Finally, this investigation reveals several potential therapeutic candidates to enable better management and pre-emptive mitigation of acute toxicities. PurposeThe 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 DesignPlasma 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. ResultsOur 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. ConclusionsThis 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.