Comprehensive Serum Proteome Profiling of Cytokine Release Syndrome and Immune Effector Cell-Associated Neurotoxicity Syndrome Patients with B-Cell ALL Receiving CAR T19

Abstract

Purpose: To study the biology and identify markers of severe cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) in children after chimeric antigen receptor T-cell (CAR T) treatment.

Experimental design: We used comprehensive proteomic profiling to measure over 1,400 serum proteins at multiple serial timepoints in a cohort of patients with B-cell acute lymphoblastic leukemia treated with the CD19-targeted CAR T CTL019 on two clinical trials.

Results: We identified fms-like tyrosine kinase 3 (FLT3) and mast cell immunoglobulin-like receptor 1 (MILR1) as preinfusion predictive biomarkers of severe CRS. We demonstrated that CRS is an IFNγ-driven process with a protein signature overlapping with hemophagocytic lymphohistiocytosis (HLH). We identified IL18 as a potentially targetable cytokine associated with the development of ICANS.

Conclusions: We identified preinfusion biomarkers that can be used to predict severe CRS with a sensitivity, specificity, and accuracy superior to the current gold standard of disease burden. We demonstrated the fundamental role of the IFNγ pathway in driving CRS, suggesting CRS and carHLH are overlapping rather than distinct phenomena, an observation with important treatment implications. We identified IL18 as a possible targetable cytokine in ICANS, providing rationale for IL18 blocking therapies to be translated into clinical trials in ICANS.

Figure 1.. Identification and validation of pre-infusion biomarkers for cytokine release syndrome.

(A) Differential expression analysis of proteins at the pre-infusion timepoint (N=22) comparing patients with severe and minimal cytokine release syndrome (CRS) identified a single significant marker, Mast Cell Immunoglobulin-like Receptor 1 (MILR1). An absolute fold change threshold of 2 and a false discovery rate (FDR) threshold of 0.05 were used. (B) MILR1 levels in patients with severe (N=13) and minimal CRS (N=13) over time. Patients with severe CRS had high levels of MILR1 at pre-infusion that decreased over time, where those with minimal CRS had consistent levels over time. (C) Receiver operative characteristic (ROC) curve of MILR1 as a binary predictor of severe CRS (N=22). (D) FLT3 levels and MILR1 levels at the pre-infusion timepoint are highly correlated (R=0.86, p=1.3x10⁶, N=22). Dots are colored by CRS category with higher levels of both biomarkers in patients with severe CRS compared to those with minimal CRS. (E) Mean levels of FLT3 and FLT3-ligand (FLT3LG) in patients with severe (green lines) and minimal (purple lines) CRS. Patients with severe CRS have high levels of FLT3 at pre-infusion that decline over time in an inverse manner to levels of FLT3LG. (F) ROC curve of FLT3 and MILR1 in an expanded validation cohort (N=39) predicted the development of severe CRS with a sensitivity of 0.78 and a specificity of 0.71 (AUC=0.78). Cytosig outputs of proteins known to be associated with MILR1 (G) and FLT3 (H) from previously reported RNA sequencing data sets. Y-axis represents mean log fold change of significantly different cytokines (p ≤0.05) listed on the X-axis.

Figure 2.. Insights into the pathophysiology of cytokine release syndrome at the peak timepoint.

(A) Differential expression analysis at the peak timepoint for patients with severe (N=13) compared to minimal (N=13) CRS. Colored dots meet the threshold of significance. Orange dots are dots known to be associated with IFNγ signaling. Pink dots are not known to be associated with IFNγ signaling. A fold change threshold of 2 and a false discovery rate (FDR) threshold of 0.05 were used to define significance. (B) Pathway analysis of significantly differentially expressed proteins is presented. Notably, the TNF signaling pathway was significantly perturbed. (C) Protein-protein clustering at pre-infusion, prior to peak, peak and last day timepoints in severe patients in whom all 4 timepoints were available (N=8). The cluster with the highest degree of correlation between the proteins and the cluster centroid (0.96) is presented. Notably IFNγ, and its canonical responders CXCL9 and CXCL10 were highly clustered together, as was the T-cell exhaustion marker CD274 (PDL1). (D) Heatmap of 29-protein signature associated with HLH clusters patients in to severe vs. minimal CRS when unsupervised clustering is applied at the peak timepoint (N=26). Data for the heatmap was mean-centered. Dot plot demonstrating per patient levels cytokines previously shown to be elevated (E) or decreased (F) in the setting of HLH. Patients with severe CRS (N=13) demonstrate a pattern very similar to that seen in patients with HLH. Y axis represents NPX log2 scale, *represents significantly different proteins with p-value ≤0.05 calculated using a Wilcoxon test. Green dots indicate patients with minimal CRS and blue dots represent patients with severe CRS.

Figure 3.. Immune effector cell associated neurotoxicity syndrome (ICANS) associated cytokines.

Longitudinal levels for patients who did (left; N=14) and did not (right; N=12) develop ICANS are demonstrated. Yellow coloration represents the onset of ICANS symptoms for each patient. The cytokines IL-1A (A) and IL-1B (B) were not associated with the development of ICANS symptoms when comparing the area under the curve (AUC) (p=0.38 and p=0.54, respectively) or peak values during ICANs (p=0.55 and p=0.8) in those who did (left) vs did not (right) develop ICANS by Wilcoxon. IL-18 levels (C) were associated with the onset of ICANS symptoms in most patients (AUC: p<0.01; peak values during ICANS: p<0.01). X-axis demonstrates day, Y-axis represents NPX for each protein. Expression of proteins associated with both CRS and ICANS (Y-axis) or with ICANS alone (X-axis) is presented in (D). Purple dots are statistically significant proteins with a nominal p-value ≤ 0.05 computed with a t-test and have an absolute fold change ≥ 1.5. IL-18 is colored in pink. (E) Longitudinal levels of CELA3A, the highest expressed protein from (D) is shown associated with onset of ICANS symptoms (AUC: p<0.01; peak values during ICANS: p<0.01). X-axis demonstrates day, Y-axis represents NPX for each protein.

Figure 4.. Evidence of complement dysregulation related to the development of CRS and ICANS.

Severe cytokine release syndrome (CRS) is associated with renal dysfunction as demonstrated by increases in the renal dysfunction marker Cystatin C (A), particularly at the peak timepoint (p < 0.001). (B) Top twenty linear correlates of Cystatin C at the peak timepoint. Proteins related to complement are denoted with purple rectangles. (C) A heatmap of all complement related proteins measured by Olink with unsupervised hierarchical clustering applied to all patients at the peak CRS timepoint (N=26). Patients with minimal CRS are colored in green and patients with severe CRS are colored in blue. Color bar represents variable range of NPX value. (D) Soluble c5b9 (sc5b9) levels (ng/mL) in patients with (N=14) and without (N=12) ICANS symptoms measured over time. Yellow lines represent occurrence of ICANS symptoms with grey lines denoting absence of ICANS symptoms. Dashed horizontal line represents upper limit of normal (ULN) on sC5b9 assay (247 ng/mL). Area under the curve (AUC) comparison of those who did vs did not develop ICANS showed a trend toward a significant difference (p=0.07 by Wilcoxon), but there were outliers (E) Comparing the highest sC5b9 level in patients who did (N=14) and did not (N=12) develop ICANS compared with t-test, p=0.046 did demonstrate a difference between groups.