Page 252 - Haematologica Vol. 109 - July 2024

ARTICLE - Application of CAAR T-cell therapy in ITP J. Zhou et al.
Figure 5. GPIbα chimeric autoantibody receptor T cells mediated autoreactive B-cell depletion in vivo. (A) Anti-GPIbα B cells and ligand-binding domain LBD-mutg233k T cells were intravenously inoculated into 7-week-old female NSG mice. Autoreactive B-cell burden was monitored via bioluminescence imaging every few days. (B) Quantification of the autoreactive B-cell burden indicat- ed by the radiance detected in the region of interest with group values or individual values. T1, 2, 3, 4 mean mice 1, 2, 3, 4. (C) Flow cytometry analysis of total human T cells in the peripheral blood (PB) of non-transduced T cell (NTD T)-treated mice (N=4) and LBD-mutg233k T-treated mice (N=4) monitored every few days. T cells were stained with anti-human CD45 antibody (APC) and anti-human CD3 antibody (BV421). Chimeric autoantibody receptor (CAAR) expression in T cells was detected with an an- ti-human CD42b antibody (FITC). T-cell penetration and persistence in the spleen (D) and bone marrow (BM) (E) of the mice were analyzed with flow cytometry. T cells were stained with anti-human CD45 antibody (APC) and anti-human CD3 antibody (BV421). (F) The serum anti-GPIbα antibody titer of the mice on day 7 and day 21 was detected by enzyme-linked immunosorbant assay at a dilution ratio of 20. NS: not significant P>0.05; *P<0.05; **P<0.01; ***P<0.005. ROI: region of interest; SSC: side scatter.
the inactivated negative feedback mechanism of mega- karyocyte-generated platelets that leads to peripheral platelet destruction.30,36 In this work, site-mutated GPIbα ectodomain of varying lengths were incorporated into second-generation CAR structures to direct specific T-cell cytolysis against autoreactive B cells.
In ITP, no cell immunotherapy targeting B cells is cur- rently used. Compared to CAR T-cell therapy, CAAR T-cell therapy ultimately and precisely removes autoreactive B cells and has fewer side effects due to a lower “tumor” burden.14 This work demonstrated the in vitro cytolytic capacity and persistence of GPIbα CAAR T cells and their ability to eliminate GPIbα-specific B cells in vivo precisely. B-cell depletion therapy not only reduces autoantibody production but also reduces splenic CD8+ T-cell prolif- eration in vitro, as well as the ability of CD8+ T cells to activate and mediate ITP 37 and T-follicular helper cells in both the spleen and the blood.38 It is anticipated that B-cell clearance mediated by CAAR T-cell therapy may have an additional impact on moderating immunological dysregulation in ITP patients.
The N-terminus of GPIbα contains binding sites for VWF, and anti-GPIbα antibodies can interfere with normal plate- let function by inhibiting GPIbα-VWF-mediated platelet aggregation, which may increase the severity of the pa- tient’s hemorrhage at the same time.39 In order to prevent the off-target effect caused by the competitive binding of GPIbα CAAR T cells with VWF, we generated mutations (Figure 1B; Online Supplementary Figure S6) at residues 231, 232, and 233 of GPIbα (K231V/Q232V/G233k/G233D). The results demonstrated that GPIbα mutations strongly inhibited the binding of GPIbα to VWF, which could allevi- ate possible off-target effects after CAAR T-cell infusion in vivo.
The binding force between CAR T cells and cancer cells and the size of the CAR antigen are related to CAR T-cell efficacy, which may explain the varied cytolytic capacity of the CAAR,40,41 as shown by the results of the hybridoma antibody binding assay (Figure 2) and in vitro cytotoxic assay (Figure 3) in our study. GPIbα CAAR T cells with various truncated forms of GPIbα all exhibited cytotoxicity against anti-GPIbα hybridomas. Meanwhile, the epitopes of autoantibodies are also diverse in ITP patients, so it is feasible to choose the most suitable GPIbα CAAR T-cell
therapy for different patients. The MSD of GPIbα is critical in sensing shear stress and converts this mechanical in- formation into a protein-mediated signal in platelets,28 the role and structural changes of which were also evaluated in this work. CAAR4-mutg233k-CAAR did not contain the MSD; CAAR4-mutg233k T cells showed a better response to anti-GPIbα autoantibodies and exhibited better killing efficiency than CAAR3-mutg233k T cells, which contain MSD. MSD does affect the spatial conformation of the CAAR and negatively affects subsequent binding and cyto- lytic functions, which did not seem to be necessary in the CAAR constructs. The same results were also confirmed in cytolytic assays of CAAR1 and CAAR2 (Figure 3F; Online Supplementary Figure S3).
In the in vivo study, a hybridoma xenograft model was used, referring to existing research.14 The four hybridomas were obtained by immunizing BALB/C mice with human platelets. The hybridoma antibodies could only target human GPIbα and could not bind to mouse platelets to mediate platelet destruction in mice; thus, we could not assess whether platelets increase after infusion of CAAR T cells. The humanized mouse model was considered, but previous research42,43 has shown that human platelets are low in the PB of humanized mouse models, limiting their applicability. Although we were unable to monitor the therapeutic effect of the CAAR T cells, we did show that the CAAR T cells could selectively lyse target cells, reduce autoantibody titers, and result in a lower human platelet clearance rate (Online Supplementary Figure S4D) in an in vivo xenograft mouse model. In addition, control of the anti-GPIbα hybridoma burden in LBD-mutg233k T-treated mice validated CAAR T-cell persistence in vivo. In order to verify the potential of GPIbα CAAR T cells in clinical applications, we first demonstrated their reactivity with sera from ITP patients with anti-GPIb antibodies. We found that anti-GPIb antibodies from patient 3 could bind to the LBD of GPIbα (LBD-mutg233k-CAAR) but did not react with CAAR3-mutg233k-CAAR or CAAR4-mutg233k- CAAR, which also contained the LBD. We hypothesized that the expression of the LBD is more stable than that of other extracellular domains of GPIbα, as it can medi- ate rapid downstream action.29 In addition, studies have shown that the macroglycopeptide domain can affect the binding of LBD to ligands.44-46 We anticipated that
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