Page 245 - Haematologica Vol. 109 - July 2024

ARTICLE - Application of CAAR T-cell therapy in ITP
J. Zhou et al.
associated with refractoriness to IVIg or steroids in ITP pa- tients,23,26,29 and the MSD juxtamembrane portion of GPIbα is related to ligand binding and signal transduction.30 Native GPIbα ectodomains of various lengths (LBD/CAAR1/CAAR2/ CAAR3/CAAR4) were constructed into a second-generation CAR structure containing a CD8α hinge/transmembrane region, 4-1BB co-stimulatory domain, and intracellular CD3ζ domain, as shown in Figure 1B and Online Supplementary Figure S1. LBD CAAR contains the LRR domain; CAAR1 con- tains the macroglycopeptide domain; CAAR2 contains the macroglycopeptide domain plus MSD; CAAR3 comprises the LRR domain, the macroglycopeptide domain, and MSD; and CAAR4 consists of the LRR domain and the macro- glycopeptide domain. The five CAAR structures were first expressed on the surface of HEK293T cells, and the proper conformation of the CAAR structures was verified by the anti-human platelet GPIbα antibody (clone: HIP1). As shown in Online Supplementary Figure S2A, CAAR expression was successfully detected in LBD CAAR, CAAR3, and CAAR4 but not in CAAR structures that did not contain LBD (CAAR1, CAAR2), probably because this antibody recognizes a site in the LBD. The above results preliminarily proved that the conformational epitope of GPIbα CAARs is consistent with that of native human platelet GPIbα. CAAR expression in primary human T cells was also detected. T cells were transduced with GPIbα-CAAR lentivirus (multiplicity of infection 25 to 100), and the transduction efficiencies of GPIbα CAAR T cells were determined with an anti-human platelet GPIbα antibody (clone: HIP1) by flow cytometry (Online Supplementary Figure S2B).
GPIbα G233K mutation inhibited von Willebrand factor binding to GPIbα chimeric autoantibody receptor T cells The platelet surface membrane protein GPIbα participates in platelet plug formation by binding to the A1 domain of VWF, which is already attached to the subendothelium.16 The b-switch region (AAs 227-241) of GPIbα plays an essential role in forming the GPIbα-VWF complex, and residue 233 plays a critical role in regulating VWF binding.31-33 The crystal structures of the NH2-terminal domain of GPIbα (residues 1 to 290) and its complex with the VWF-A1 domain (resi- dues 498 to 705) are shown in Figure 1C. In order to avoid affecting the regular role of the GPIbα-VWF complex in the process of hemostasis after CAAR T-cell infusion, we chose residue 233 (G233K) of GPIbα for mutation in the CAAR structure. CAAR3 and GPIbα residue 233-mutated CAAR3 (CAAR3-mutg233k) were expressed on the Jurkat T-cell surface with lentivirus transduction. As shown in the aggregometry measurements in Figure 1D, under shear force and induction by ristocetin, the VWF protein bound to Jurkat-CAAR3 T cells, triggering a cell aggregation reac- tion, and an increase in light transmission was observed, which was not detected in the Jurkat-CAAR3-mutg233k T-cell group. The results demonstrated that the G233K mutation of GPIbα expressed on the cell surface strongly
inhibits its binding to VWF, which could alleviate possible off-target effects after CAAR T-cell infusion in vivo. The mutated GPIbα CAAR structures were named LBD-mut- g233k, CAAR3-mutg233k, and CAAR4-mutg233k, and the integrity of the CAAR epitopes was confirmed with HIP1 (Figure 1E). The transduction efficiencies of the mutated GPIbα CAAR T cells are presented in Figure 1F. At a mul- tiplicity of infection (MOI) of 25, LBD-mutg233k T cells exhibited the highest transduction efficiency, followed by CAAR4-mutg233k T and CAAR3-mutg233k T cells. The size of the CAAR fragment was an essential factor affecting the transfection efficiency as indicated, and the results from three healthy donor T cells are shown (Figure 1F).
Generation of four anti-GPIbα hybridomas with different chimeric autoantibody receptor binding sites
In order to generate hybridomas and antibodies that bind to human platelet GPIbα, washed human platelet lysate was used as the antigen for mouse immunization, and mouse serum was screened by GPIbα enzyme-linked immunsorbant assay (ELISA). The four selected anti-GPIbα hybridomas were named Gvb1, Gvb2, Gvb3, and Gvb4. Human platelets were incubated with each APC-conjugated hybridoma antibody (Gvb1/Gvb2/Gvb3/Gvb4) and FITC-anti-human CD42b anti- body simultaneously to analyze the ability of the acquired antibodies to bind to human platelets. The flow cytom- etry results (Figure 2A) showed that the four anti-GPIbα antibodies could bind to human platelets, demonstrating their ability to bind with the native conformation epitope of GPIbα. As target cell surface antigen density might af- fect CAR T-cell cytolytic efficiency,27 the density of BCR on the surface of each hybridoma was then measured with an anti-mouse IgG antibody (APC-conjugated). The BCR mean fluorescence intensity (MFI) of the four hybridomas is presented in Figure 2B, varying among the anti-GPIbα hybridomas, and the Gvb3 hybridoma showed the highest surface BCR density, followed by the Gvb1, Gvb2, and Gvb4 hybridomas.
In order to verify the binding ability of the hybridoma an- tibodies to mutated GPIbα CAAR and identify the binding sites between them, we further expressed mutated GPIbα CAAR in HEK293T cells. The proportion of cells that bound to the hybridoma antibodies in total GPIbα CAAR-GFP-ex- pressing cells (Q2/(Q2+Q3)) was determined and represented the binding force between the hybridoma antibodies and CAAR. As shown in Figure 2C-F, Gvb1, Gvb2, and Gvb3 could bind to LBD-mutg233k-CAAR, CAAR3-mutg233k-CAAR, and CAAR4-mutg233k-CAAR, while Gvb4 could bind to all GPIbα CAAR. The locus recognized by Gvb4 is thus in the overlapping part of LBD-mutg233k-CAAR and CAAR1/CAAR2. The findings revealed that though the binding epitopes of each hybridoma antibody to the CAAR should be similar, the binding efficiencies varied. Gvb4 could bind to all GPIbα CAAR, proving that its binding site is the same site shared by the above CAAR but exhibited different binding effi-
Haematologica | 109 July 2024
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