Page 250 - Haematologica Vol. 109 - July 2024

ARTICLE - Application of CAAR T-cell therapy in ITP J. Zhou et al.
Figure 4. Evaluation of soluble anti-GPIbα antibody effects on GPIbα chimeric autoantibody receptor T-cell cytotoxicity. (A) Non-transduced T cells (NTD T) or LBD-mut223k T cells were co-incubated with a mixture of the 4 anti-GPIbα target cells at the indicated effector to target (E:T) ratios in the presence or absence of soluble anti-GPIbα immunoglobulin (Ig)G polyclonal antibody (a mixture of Gvb1, Gvb2, Gvb3, and Gvb4, and the concentration of each antibody is 5 μg/mL). Cytotoxicity was evaluated at 16 hours by lactate dehydrogenase (LDH) release assay. Meanwhile, human interferon (IFN)-γ (B) in cell culture supernatants was quantitated by enzyme-linked immunosorbant assay (ELISA) after 16 hours. (C-D) NTD T or LBD-mutg233k T cells were incubat- ed with each anti-GPIbα IgG monoclonal antibody (0, 10, 20, or 40 μg/mL) at an E:T ratio of 5. Cytotoxicity was evaluated at 16 hours by LDH release assay, and human IFN-γ in cell culture supernatants was quantitated by ELISA after 16 hours. NS: not sig- nificant P>0.05; *P<0.05; **P<0.01; ***P<0.005; ****P<0.0001.
sponse. Mice treated with NTD T cells were used to monitor the allogenic effects. Bioluminescence imaging indicated that GPIbα CAAR T cells significantly reduced anti-GPIbα hybridoma outgrowth compared to NTD T cells (Figure 5A). From the 14th day to the 21st day, the autoreactive B-cell burden (mean ROI) of the LBD-mutg233k T-treated mice 3 and 4 (T3 and T4) decreased from 4,096.406 to 1,028.801 and 28,029.28 to 2,671.87 (photons/s/cm2/sr), respectively (Figure 5B). Total human T cells and CAAR T cells in periph- eral blood (PB) were monitored every few days (Figure 5C). PB T cells of the CAAR T-treated mice proliferated rapidly from day 18 to day 24, far exceeding the proliferation of the NTD T group, which encounters with target cells can explain. Control and LBD-mutg233k T-cell-treated mice were euthanized 21-28 days after hybridoma cell/T-cell injection, and T-cell persistence and penetration in bone marrow, spleen, and blood samples were assessed by flow cytometry. Consistent with the PB results, T cells of the CAAR T-treated mice isolated from the spleen and bone marrow exhibited a more potent ability to persist and proliferate in vivo than T cells in the control group, as shown in Figure 5D, E. Immunofluorescence imaging (Online Supplementary Figure S4A) also indicated lympho- cyte infiltration and persistence in the liver and spleen. The plasma anti-GPIbα antibody titer increased in mice treated with NTD T cells from day 7 to 21. In contrast, the titers in GPIbα CAAR T-cell-treated mice were significantly reduced compared to those in NTD T-cell-treated mice by day 21 after T-cell injection (Figure 5F). The reduced serum anti-GPIbα ELISA results also reflected hybridoma control in LBD-mutg233k T-treated mice. Off-target cytotoxic ef- fects of GPIbα-CAAR T cells on mice were not observed, as hematoxylin and eosin (H&E) staining revealed that the morphology of tissues and organs in mice did not change significantly, and serum biochemical levels were normal (Online Supplementary Figure S4B, C). In conclusion, GPIbα CAAR T cells demonstrated in vivo persistence and specific cytolytic capacity with no apparent organ toxicity.
GPIbα chimeric autoantibody receptor T cells showed potential in eradicating autoreactive B cells of immune thrombocytopenia patients
In order to test the ability of GPIbα CAAR T cells to inter- act with native GPIbα autoantibodies from ITP patients, a plasma antibody binding assay (Figure 6A; Online Supple- mentary Figure S5A) was first applied. Patients diagnosed
with primary ITP were included and tested for specific platelet autoantibodies using a cytometric bead array (Figure 6B; Online Supplementary Figure S5B). Sera from three ITP patients with anti-GPIb antibodies and healthy controls were diluted at 1:10 and 1:20 and then incubated with HEK293T cells expressing the LBD-mutg233k-CAAR structure tagged with GFP. After incubation, the cells were stained with APC-anti-human immunoglobulin (Ig)G anti- bodies. The flow cytometry results (Figure 6A) showed that the serum anti-GPIbα antibodies of patient 3 could react with LBD-mutg233k-CAAR, but no binding of patient 1 and patient 2 plasma antibodies to LBD-mutg233k-CAAR was detected. A possible explanation for this negative result is that platelet lysates were used to screen patients with anti-GPIb antibodies, and the level of platelet antibodies in the lysates is much higher than the platelet antibodies in the plasma. At the same time, we examined the binding reaction of plasma from patient 3 to CAAR3-mutg233k- CAAR and CAAR4-mutg233k-CAAR. Interestingly, although CAAR3-mutg233k-CAAR and CAAR4-mutg233k-CAAR con- tained fragments of LBD-mutg233k-CAAR, they did not react with serum antibodies from patient 3, and the results are presented in Online Supplementary Figure S5A. The ELISpot assay was employed to verify GPIbα CAAR T-cell potential in eradicating autoreactive B cells in ITP patients. ELISpot analysis (Figure 6C) showed that anti-GPIbα IgG B cells, but not total IgG B cells from ITP patients, were depleted by GPIbα CAAR T cells. Meanwhile, anti-CD19 CAR T cells eliminated all IgG B cells from ITP patients and healthy con- trols. In summary, we confirmed the viability of the “Trojan hypothesis” for the treatment of ITP patients. GPIbα CAAR T cells function like a “Trojan horse”, trapping autoreactive B cells and performing specific killing. (Figure 6D)
Discussion
Our study presents a novel concept for the treatment of refractory and relapsed ITP patients with autoantigen-mod- ified T cells based on CAR T cells. Since patients with anti-GPIbα antibodies have a poor response to standard immunosuppressive therapy, GPIbα was constructed into the ligand-binding domain of the CAAR structure in this study. Anti-GPIbα antibodies can cause platelet desialyla- tion, mediate Fc-independent platelet eradication,34,35 affect thrombopoietin production in the liver, and account for
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