Page 244 - Haematologica Vol. 109 - July 2024

ARTICLE - Application of CAAR T-cell therapy in ITP J. Zhou et al. AB
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Figure 1. Construction and validation of the GPIbα chimeric autoantibody receptor structure. (A) The GPIb-IX complex contains GPIbα, GPIbb (green), and GPIX (blue); GPIbα is the largest subunit. GPIbα is composed of seven leucine-rich repeats (LRR), make up the ligand-binding domain (LBD), followed by a heavily O-glycosylated macroglycopeptide domain and a mechanosensory do- main (MSD). (B) Schematic of GPIbα chimeric autoantibody receptor (CAAR) constructs comprising diverse lengths of the native GPIbα ectodomain, followed by the CD8α transmembrane domain and 4-1BB-CD3ζ intracellular co-stimulatory and activation domains, named LBD, CAAR1, CAAR2, CAAR3, and CAAR4. (C) Crystal structures (Protein data bank ID: 1m10) of the NH2-terminal domain of GPIbα (green, residues 1 to 290) and its complex with the von Willebrand Factor (VWF) A1 domain (blue, residues 498 to 705) are shown, and the loss-of-function mutation at residue 233 of GPIbα (red, G233K) is indicated. (D) Ristocetin-induced VWF binding to GPIbα fragments expressed on Jurkat T cells. CAAR3 and residue 233-mutated CAAR3 (CAAR3-mutg233k) were expressed on the surface of Jurkat T cells, and cell aggregation was measured as an increase in light transmission (%) (N=3; *P<0.05). (E) In order to identify the mature conformational epitopes, mutated-GPIbα CAAR plasmids were transfected into HEK293T cells, and then the cells were stained with an anti-human CD42b (GPIbα) antibody (clone: HIP1). (F) At a multiplicity of infection (MOI) of 25, primary human T cells were transduced with LBD-mutg233k-CAAR, CAAR3-mutg233k-CAAR, and CAAR4- mutg233k-CAAR lentivirus, and CAAR expression was determined using an anti-human CD42b antibody (clone: HIP1). The transduction efficiency of GPIbα CAAR+ in T cells from 3 healthy donors was calculated. SSC: side scatter; NTD T: non-trans- duced T cells. ****P<0.0001.
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