Page 247 - Haematologica Vol. 109 - July 2024

ARTICLE - Application of CAAR T-cell therapy in ITP J. Zhou et al.
Gvb4 to mutated GPIbα chimeric autoantibody receptor (CAAR) structures. GPIbα CAAR were expressed on the surface of HEK293T cells, which were incubated with each hybridoma antibody (APC-conjugated). Green fluorescent protein (GFP) was linked with the CAAR fragment to facilitate the detection of GPIbα CAAR expression. The binding force between each hybridoma antibody and the CAAR was calculated as the ratio of cells bound to hybridoma antibodies in total GPIbα CAAR-GFP-expressing cells (Q2/(Q2+Q3)). NS: not significant P>0.05; *P<0.05; **P<0.01; ***P<0.005; ****P< 0.0001.
ciencies to these CAAR; this suggested that the different CAAR structures had different spatial conformations that may affect the binding force of the hybridoma antibody. In summary, we obtained four hybridomas with different binding sites that could simulate B cells from ITP patients with different anti-GPIbα epitopes for further CAAR T-cell cytolytic assays. As Gvb4 could bind to all the CAAR (Figure 2F), the transduction efficiencies of GPIbα CAAR T cells were determined using the APC-conjugated hybridoma antibody Gvb4, as shown in Online Supplementary Figure S2C.
In vitro, GPIbα chimeric autoantibody receptor T cells exhibited specific cytolysis of autoreactive B cells
In order to verify the specific lysis of autoreactive B cells by GPIbα CAAR T cells, NTD T or GPIbα CAAR T cells were incubated with each anti-GPIbα hybridoma various at ra- tios ranging from 1:1 to 10:1. At 16 hours postincubation, the medium supernatants were collected and measured immediately for LDH activity. GPIbα CAAR T cells demon- strated specific lysis of autoreactive B cells (Gvb1/Gvb2/ Gvb3/Gvb4) targeting different GPIbα domains in a man- ner dependent on the E:T ratio and NTD T cells showed no cytotoxicity (Figure 3A). Notably, the pernicious effects of GPIbα CAAR T cells varied due to variations in binding epitopes of the target cell to CAAR, and LBD-mutg233k T cells exhibited the most robust cytolysis function against the four hybridomas. We also performed cytotoxicity assays with flow cytometry to further prove the specific cytolytic function of the CAAR T cells. NTD T or LBD-mutg233k T cells were coincubated with hybridoma control and the target anti-GPIbα hybridoma cells. The original ratio of target hybridoma cells to control hybridoma cells was 3:1. As shown in Figure 3B-E, due to the specific lysis of target hybridoma cells by LBD-mutg233k T cells, the ratio of target cells to control cells in the co-culture system decreased significantly compared with that in the NTD T group. Similar results for CAAR3-mutg233k T, CAAR4-mut- g233k T, CAAR1 T, and CAAR2 T cells were obtained and are presented in Online Supplementary Figure S3. High levels of the pro-inflammatory cytokines (Figure 3F) IL-2 and in- terferon (IFN)-γ were secreted by T cells after stimulation with each target hybridoma and varied among different GPIbα CAAR T cells and tested extremely low in the NTD T groups. Consistent with the binding assay results (Figure 2C-F), even though the binding sites of GPIbα CAAR and each hybridoma were similar, the difference brought about by the spatial conformation of the CAAR eventually resulted in differences in the binding forces and cytolytic functions of the GPIbα CAAR T cells. LBD-mutg233k T cells showed
a stable spatial conformation and robust cytolytic ability. CAAR4-mutg233k T cells, comprising the LBD and a heavily O-glycosylated macroglycopeptide domain, exhibited the most potent binding with hybridoma antibodies, and the cytolytic efficiency was inferior to that of LBD-mutg233k T cells. CAAR3-mutg233k-CAAR has an extra MSD structure and a poorer killing effect than CAAR4-mutg233k-CAAR. The MSD structure’s instability28 may influence the binding of CAAR3-mutg233k T cells and anti-GPIbα hybridomas, possibly accounting for the lower killing efficiency.
Soluble anti-GPIbα antibodies slightly affect GPIbα chimeric autoantibody receptor T-cell cytotoxicity Soluble autoantibodies can either prevent CAAR contact with anti-CAAR BCR or enhance cytotoxicity by activat- ing CAAR T cells; therefore, their impact on CAAR T-cell cytotoxicity is erratic.14,15 In order to determine soluble anti-GPIbα antibody effects on GPIbα CAAR T-cell activi- ty, we performed cytotoxicity assays in the presence of a wide range of concentrations (0-40 μg/mL) of monoclonal antibodies (mAb) or mixed mAb, as no quantitative data on plasma anti-GPIbα IgG concentration in ITP patients were currently available. GPIbα CAAR T-cell cytotoxicity against hybrid target cells (Gvb1/Gvb2/Gvb3/Gvb4) was mildly increased with higher effector-to-target ratios in the presence of soluble anti-GPIbα polyclonal IgG (a mixture of Gvb1, Gvb2, Gvb3, and Gvb4, and the concentration of each antibody is 5 μg/mL) (Figure 4A), as were IFN-γ lev- els (Figure 4B). Then, NTD T or LBD-mutg233k T cells and mixed target cells were incubated with each anti-GPIbα mAb at various concentrations (0, 10, 20, or 40 μg/mL). Cytotoxicity (Figure 4C) and IFN-γ levels (Figure 4D) were slightly affected in a concentration-related manner and varied among the anti-GPIbα mAb. The cytolytic efficien- cy of CAAR T cells and the IFN-γ levels were marginally enhanced or somewhat reduced with increasing antibody concentration in different groups. Therefore, we conclude that the impact of soluble anti-GPIbα mAb on CAAR T-cell function generally varies and is minimal.
GPIbα chimeric autoantibody receptor T cells demonstrated in vivo persistence and specific cytolytic activity with no apparent organ toxicity
A xenograft model was developed to assess the cytolytic efficiency and safety of GPIbα CAAR T cells in vivo. Luciferase (Luc)/GFP-expressing anti-GPIbα hybridoma cells (105 cells per mouse) and LBD-mutg233k T cells (107 cells per mouse) were intravenously injected into 6–8-week-old NSG mice, which were examined for engraftment and therapeutic re-
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