Journal: Nature Communications

Article Title: Enhancing anti-EGFRvIII CAR T cell therapy against glioblastoma with a paracrine SIRPγ-derived CD47 blocker

doi: 10.1038/s41467-024-54129-w

Figure Lengend Snippet: a Mechanism of action of conventional aEGFRvIII CAR T cell monotherapy in GBM. b Proposed aEGFRvIII-SGRP CAR T cell combination therapy whereby SGRP-mediated CD47 blockade induces phagocytic modulation of GAMs in the context of EGFRvIII-heterogenous GBM and its immunosuppressive iTME. c Outline of the SGRP engineering strategy, including specific AA substitutions to the endogenous human SIRPγ-V1 sequence and addition of an N-terminal IL-2 signal sequence (IL2sig) leading to constitutive SGRP secretion. d Polycistronic lentiviral constructs encoding mCherry (mC)-labeled aCD19 CAR or aEGFRvIII CAR under the control of EF1A promoter ± SGRP secretion. e Workflow of CAR T cell production applied throughout the study. a – e Created in BioRender. Hutter, G. (2022) BioRender.com/u48r093. Representative plots of CAR:target protein binding by aCD19 CAR T cells ( f ) or aEGFRvIII CAR T cells ( g ) to CAR-bound biotinylated (bt)-CD19 (top plots) or bt-EGFRvIII (bottom plots); n = 2 healthy donors (HDs) assessed per CAR. h TATA-box binding protein (TBP)-normalized expression of mCherry and SGRP detected by real-time quantitative PCR (RT-qPCR) in aEGFRvIII CAR or aEGFRvIII-SGRP CAR T cells, showing mCherry expression in CARs transduced with either construct and SGRP expression specifically in aEGFRvIII-SGRP CARs; n = 4 HDs. Data are presented as scatter plots with mean values ± SD. Statistical differences were assessed by two-sided unpaired t tests with Welch’s correction. i Differentially secreted proteins in aEGFRvIII-SGRP CAR- vs aEGFRvIII CAR-conditioned media, highlighting the presence of SGRP exclusively in aEGFRvIII-SGRP CAR; n = 2 HDs. Mean SGRP expression: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$-\log 10{{{\rm{qValue}}}}/\log 2{{{\rm{foldchange}}}}=8.60/6.65$$\end{document} − log 10 qValue / log 2 foldchange = 8.60 / 6.65 . Source data are provided as a Source Data file. Source data for ( i ) are provided as Supplementary Data .

Article Snippet: Experiments involving ELISA assays were performed using a CD47:SIRP alpha Biotinylated Inhibitor Screening ELISA Assay Pair (#EP-102, ACROBiosystems, USA) or Human SIRP alpha DuoSet ELISA (#DY4546-05, R&D Systems, USA).

Techniques: Sequencing, Construct, Labeling, Control, Protein Binding, Binding Assay, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Transduction