DMG-01. Protein changes in CSF as biomarkers of CAR activity and disease response in patients with DIPG treated on the BrainChild03 trial

Objective To identify biomarkers of response/toxicity in cerebrospinal fluid (CSF) to B7H3-targeting CAR-T infusions in patients with diffuse intrinsic pontine glioma (DIPG), and to quantify response to repeat locoregional CAR-T infusions. Methods CSF samples were collected pre- and post- locoregional infusion of CAR-T cells from patients with DIPG enrolled on the BrainChild-03 trial. Standard workflow was followed for LC-mass spectrometry (MS)/MS profiling of TMT-labeled CSF biospecimens. Linear mixed effects models were run to identify the proteins associated with pre-/post-treatment status. Pathway enrichment analyses were conducted to identify pathways significantly (adjusted p-values< 0.05) associated with treatment. A time trend analysis was run using the linear mixed effects model, coding time points as ordinal entities. Results One-hundred-forty-one CSF samples were collected from 15 pediatric patients enrolled in the BrainChild-03 trial. In total, 3774 proteins were detected and quantified. Within the first two treatment courses (C1W1, C1W3, C2W1 and C2W3, W is week), 171 proteins showed significant association with pre-/post- treatment, of which 106 proteins exhibited higher abundance post-treatment compared to pre. Pathway enrichment analysis further revealed >600 pathways associated with pre-/post- treatment status. If directly comparing C1W1-pre to C2W3-post, 625 proteins differed, of which 355 boosted proteins by treatment were enriched for immune, cell cycle and translation pathways; and 270 proteins were enriched for synapse, neuron and development pathways. Conclusions This exploratory work demonstrates changes in protein enrichment in CSF across multiple timepoints during locoregional CAR T therapy. These data demonstrate that proteins related to immune, cell cycle and translation pathways may be boosted post treatment, while that of the neuronal system, metabolic process may be suppressed post treatment. These changes in CSF will be further studied as they correlate to disease response and toxicity and results should be considered in the development of future immunotherapy trials.