PD1 Expression in EGFRvIII-Directed CAR T Cell Infusion Product for Glioblastoma Is Associated with Clinical Response

Abstract

The epidermal growth factor receptor variant III (EGFRvIII) has been investigated as a therapeutic target for chimeric antigen receptor (CAR) T cell therapy in glioblastoma. Earlier research demonstrated that phenotypic and genotypic characteristics in T cells and CAR T product predicted therapeutic success in hematologic malignancies, to date no determinants for clinical response in solid tumors have been identified. We analyzed apheresis and infusion products from the first-in-human trial of EGFRvIII-directed CAR T for recurrent glioblastoma (NCT02209376) by flow cytometry. Clinical response was quantified via engraftment in peripheral circulation and progression-free survival (PFS), as determined by the time from CAR T infusion to first radiographic evidence of progression. The CD4⁺CAR T cell population in patient infusion products demonstrated PD1 expression which positively correlated with AUC engraftment and PFS. On immune checkpoint inhibitor analysis, CTLA-4, TIM3, and LAG3 did not exhibit significant associations with engraftment or PFS. The frequencies of PD1⁺GZMB⁺ and PD1⁺HLA-DR⁺ CAR T cells in the CD4⁺ infusion products were directly proportional to AUC and PFS. No significant associations were observed within the apheresis products. In summary, PD1 in CAR T infusion products predicted peripheral engraftment and PFS in recurrent glioblastoma.

Keywords: CAR T cells; GBM; PD-1; glioblastoma; immunotherapy.

Figure 1

Summary of Clinical Outcomes for EGFRvIII-Directed CAR T Clinical Trial and Flow Cytometric Analysis of Patient Plasma Products. Summary of clinical outcomes for clinical trial on EGFRvIII-directed CAR T for recurrent glioblastoma (NCT02209376). Patient 209 was ultimately not included for analysis due to having an outlier PFS value. (A) Plot of days following CAR T infusion and engraftment in peripheral blood. Following earlier studies, peripheral engraftment was quantified as log₁₀copies/μg of genomic DNA. (B) PFS plotted as Kaplan-Meier estimator for all subjects. (C) Summary values of patient characteristics, performance of resection, total AUC, 30-day AUC, and peak engraftment for all subjects. ^* Patient 209 was excluded due to having an outlier progression-free survival (p<0.001 on Grubbs’ test). (D) Comparison of PD1 expression for CD3⁺CD4⁺CAR⁺ cells for transfusion products from all nine recurrent GBM patients receiving EGFRvIII-directed CAR T. Mean fluorescence intensity (MFI) is quantified on the table to the right. (E) Correlation of PD1 MFI and peak CAR T engraftment levels.

Figure 2

PD1 Correlations for CD4⁺CAR⁺ Cells in Patient Transduction Products. Correlation of PD1 expression with clinical outcomes for CD4⁺CAR⁺ cells in patient transduction products. (A) Association between PD1 expression and total AUC. (B) Association between PD1 expression and PFS. Error bars shown are 95% confidence intervals.

Figure 3

ICI Correlations for CD4⁺CAR⁺ Cells in Patient Transduction Products. Correlation of ICI expression (CTLA4, TIM3, LAG3) with clinical outcomes for CD4⁺CAR⁺ cells in patient transduction products. (A) Association between ICI expression and total AUC. (B) Association between ICI expression and PFS. (C) Correlation of PD1-ICI co-expression (PD1⁺CTLA4⁺, PD1⁺TIM3⁺, and PD1⁺LAG3⁺) and AUC. (D) Correlation of PD1-ICI co-expression and PFS. Error bars shown are 95% confidence intervals.

Figure 4

PD1 Correlations for CD4⁺CAR^- Cells in Patient Transduction Products. Correlation of PD1 expression with clinical outcomes for CD4⁺CAR^- cells in patient transduction products. (A) Association between PD1 expression and total AUC. (B) Association between PD1 expression and PFS. Error bars shown are 95% confidence intervals.

Figure 5

Activation Marker Correlations for CD4⁺CAR⁺ Cells in Patient Transduction Products. Correlation of activation marker expression (GRZB, HLA-DR) with clinical outcomes for CD4⁺CAR⁺ cells in patient transduction products. (A) Association between activation marker expression and total AUC. (B) Association between activation marker expression and PFS. (C) Correlation of PD1-activation marker co-expression (PD1⁺GRZB⁺ and PD1⁺HLA-DR⁺) and AUC. (D) Correlation of PD1-activation marker co-expression and PFS. Error bars shown are 95% confidence intervals.