Fig. 1: CAR construct design and production of CAR-iMAC cells.
From: A second-generation M1-polarized CAR macrophage with antitumor efficacy
a, Constructs of EGFRvIII/GPC3-targeting CARs. The CARs mainly comprise an extracellular signal peptide, a scFv recognizing EGFRvIII/GPC3, a transmembrane (TM) domain and a hinge region from CD8α, and either without an intracellular domain (truncated CAR), or with an intracellular CD3ζ signaling domain (CD3ζ-CAR), or cytoplasmic TIR domain from TLR4 (TIR-CAR), or both CD3ζ and TIR domains in tandem (CD3ζ-TIR-CAR). The hinge was added between scFv and CD8α TM domains to endow the CARs with the flexibility to target antigens. All the CAR transgenes were linked to the EGFP-expressing sequence via a T2A element. b, Overview of the differentiation process of EGFRvIII-targeting CAR-iMACs from the CAR-expressing iPSCs. EGFP-labeled CAR-iPSCs were differentiated into CAR-iMACs by mimicking the in vivo process of mesoderm induction, hematopoietic stem cell specification and myeloid cell production. Confocal imaging was performed to observe the morphology of CAR-iMACs by detecting the EGFP protein. The experiment was performed more than five times. c, Flow cytometry analysis of EGFP-positive populations of EGFRvIII-CAR-iMACs. d, Flow cytometry analysis was performed to determine the transduction efficiency of the four types of EGFRvIII-CARs in iMACs by detecting the expression of CARs via an allophycocyanin (APC)-conjugated rabbit antibody specific to F(ab′)2 of human IgG. Results in c and d are representative plots from three biological replicates. The results were processed using FlowJo.
