P06.03.A Combination of EGFRvIII CAR T cell therapy and paracrine GAM modulation for the treatment of GBM

Abstract

Abstract Background The glioblastoma (GBM) immune microenvironment mainly consists of protumoral glioma-associated microglia and macrophages (GAMs). Previously, we showed that blockade of the don't-eat-me signal CD47, overexpressed by GBM cells, rescued GAMs' phagocytic function in mice. However, CD47 blockade monotherapy has been ineffective in treating human solid tumors to date. Thus, we propose a combinatorial approach of local chimeric antigen receptor (CAR) T cell therapy with paracrine GAM modulation for a synergistic elimination of GBM. Material and Methods We lentivirally transduced healthy donor human T cells to generate humanized EGFRvIII-directed CAR T that constitutively secrete a SIRPγ-related protein (SGRP) with high affinity to CD47. Killing assays were performed with endogenous EGFRvIII-expressing BS153 or EGFRvIII-overexpressed U251 GBM cells and assessed by Incucyte time-lapse imaging analysis. CAR T cell activation was confirmed by flow cytometry (FC) and IFNγ was detected from co-culture supernatants or mouse plasma by ELISA. The CAR T cell secretome was analyzed by liquid chromatography-mass spectrometry (LC-MS) to confirm the secretion of SGRP. NSG mice were orthotopically implanted with either EGFRvIII+ BS153 or U251 cells and treated intratumorally with one or two CAR T cell infusions. Results EGFRvIII and EGFRvIII-SGRP CAR T proliferated and killed tumor cells in vitro in a dose-dependent manner within 72h with complete cytotoxicity at E:T 1:1 compared to CD19 CAR T. CAR T cells specifically co-expressed CD25 and CD107a and secreted IFNγ in the presence of tumor antigen (24h CD25/CD107a co-expression: EGFRvIII=59.3±3.00%, EGFRvIII-SGRP=52.6±1.42%, CD19=0.1±0.07%; 24h IFNγ secretion: EGFRvIII=173.6±2.50%, EGFRvIII-SGRP=113.8±6.42%, CD19=4.5±1.49%). Differential expression analysis of the CAR T cell secretome identified SGRP in EGFRvIII-SGRP CAR T supernatants (-Log10qValue/Log2fold-change=13.72/6.62). Consistent with studies of systemic EGFRvIII CAR T cell therapy, our data suggest that intratumoral EGFRvIII CAR T were insufficient to eliminate BS153 tumors with endogenous EGFRvIII (Overall survival; EGFRvIII: 20%, CD19: 0%, n=5/group). Conclusion Here, we show that EGFRvIII CAR T specifically targeted and killed EGFRvIII+ GBM cells in vitro, but failed to control tumor growth in vivo without GAM modulation. EGFRvIII-SGRP CAR T secretome analysis identified SGRP from the supernatants of unstimulated monocultures. SGRP impaired the binding of SIRPα-Fc to CD47-upregulated GBM cells in vitro, but did not elicit macrophage-mediated phagocytosis of GBM cells in our current in vitro experimental setup. Future work will focus on the functional characterization of SGRP and on further investigating the additive effect of CAR T cell therapy and GAM modulation using translational in vivo and ex vivo models.