Page 243 - Haematologica Vol. 109 - July 2024

ARTICLE - Application of CAAR T-cell therapy in ITP
sera containing anti-GPlb/lX antibodies revealed that most
autoepitopes are in different sites in GPIbα.25 In addition, anti-GPIbα antibodies can induce profound irreversible thrombocytopenia by an Fc-independent mechanism.26 As the intermembrane distance of the immunologic syn- apse is a critical design parameter of the ligand-binding domain,27 various truncated forms of GPIbα were used as the targeting domain in the study.
In this work, we proposed a new strategy for immunother- apy for ITP in which autoantigen-modified T cells function like a Trojan horse to trap autoreactive B cells and perform specific killing. We constructed the GPIbα fragment into the second-generation CAR structure and verified that GPIbα CAAR T cells function as expected in vitro and in vivo. GPIbα CAAR T is a precision cellular immunotherapy with potential to induce complete and durable remission of refractory and relapsed ITP patients.
Methods
Animal experiments were approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (IACUC no. 3284). Experiments involving humans were conducted per the Declaration of Helsinki and were approved by the Ethics Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (IEC-J (487)).
GPIbα chimeric autoantibody receptor plasmid construction
The native GPIbα ectodomain of various lengths was con- structed into a second-generation CAR structure containing a CD8α hinge/transmembrane region, 4-1BB co-stimulatory domain, and intracellular CD3ζ domain. Lentiviral vectors with or without green fluorescent protein (GFP) expression were used. GFP was linked with the CAAR fragment to fa- cilitate the detection of GPIbα CAAR expression. The GPIbα mutant plasmid was constructed using the QuikChange Site-Directed Mutagenesis Kit.
Modified light transmission analysis
J. Zhou et al.
In vitro cytotoxicity assays
Donor-matched non-transduced T (NTD-T) cells or GPIbα CAAR T cells were co-incubated with control hybridoma (PE) and the target anti-GPIbα hybridoma cells (APC) at an effector to target (E:T) ratio of 5:1, while the ratio of target hybridoma cells to control hybridoma cells was 3:1. Cyto- toxicity was evaluated at 24 hours using flow cytometry based on the changes in the ratio of target hybridoma cells to control hybridoma cells.
Anti-GPIbα hybridoma xenograft models Six-to-eight-week-old NSG mice (NOD-scid IL2Rγnull) were intravenously inoculated with anti-GPIbα B cells (105 cells per mouse), followed 2 days later by injection with LBD-mutg233k T cells or NTD T cells (107 cells per mouse), and monitored for engraftment and therapeutic response by the IVIS® system every few days.
Cytometric bead array
The microbeads carrying different fluorescence (APC) inten- sities were separately coated with monoclonal antibodies: anti-GPIX (clone: sz-1), anti-granule membrane protein (GMP) 140 (clone: sz-51), anti-GPIb (clone: sz-2), anti-GPIIb (clone: sz-22), and anti-GPIIIa (clone: sz-21). Platelets were isolated from ITP patients or healthy controls, then platelet lysate was incubated with the coated microbeads, followed by FITC-conjugated goat-anti-human IgG polyclonal anti- bodies, and finally analyzed with flow cytometry.
GPIbα enzyme-linked immunospot assay
GPIbα CAAR T-cell-specific cytolysis of anti-GPIbα anti- body-producing human B cells was measured using an enzyme-linked immunospot (ELISpot) assay. peripheral blood mononucelar cells isolated from patients and healthy control were stimulated and then incubated with GPIbα CAAR T cells, anti-CD19 CAR T cells, or NTD T cells on the polyvinylidene difluoride-bottomed microplate coated with human GPIbα protein, anti-human IgG antibody, or BSA. Specific antibodies secreted by B cells will form spots on the microplates, which can be counted.
Details on these protocols and additional methods are provided in the Online Supplementary Appendix.
Results
Development of GPIbα chimeric autoantibody receptor plasmids
GPIbα is the largest subunit of the GPIb-IX complex (Figure 1A), and 282 N-terminal membrane-distal residues, which are composed of seven leucine-rich repeats (LRR), make up the ligand-binding domain (LBD), which binds to VWF and other ligands. The VWF binding region is followed by a heavily O-glycosylated macroglycopeptide domain and a mechanosensory domain (MSD).28 Anti-LBD antibodies are
CAAR3 and GPIbα residue 233-mutated CAAR3 (CAAR3-mut- g233k) were expressed on the Jurkat T-cell surface with lenti- virus transduction. Jurkat-CAAR3-T and Jurkat-CAAR3-mut- g233k T cells were resuspended to a concentration of 107 cells/mL, and 300 μL of cell suspension was added to a colorimetric cup. Then, 5 μg/mL human VWF was added in the presence of 0.25 mg/mL ristocetin. Under the shear force and the induction of ristocetin,16 the VWF protein binds to Jurkat T cells expressing extracellular domains of GPIbα (CAAR3) on the surface, eventually triggering an aggregation reaction of the cells, which can be measured as an increase in light transmission collected by a platelet aggregation analyzer.
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