Page 254 - Haematologica Vol. 109 - July 2024

ARTICLE - Application of CAAR T-cell therapy in ITP J. Zhou et al.
globulin (Ig)G antibodies (APC). (B) A cytometric bead array was applied to detect platelet-specific antibodies in ITP patient 3. Cutoff values were determined with reference to negative controls (mean + 3 standard deviations). Autoantibodies targeting human platelet GPIX/GPIb/GPIIb/GPIIIa/GMP140 were detected. GMP140, granule membrane protein 140. (C) Peripheral blood mononucelar cells (PBMC) from ITP patient 3 or healthy controls were stimulated with interleukin (IL)-2 and R848 for 2 days and then co-cultured with GPIbα CAAR T, anti-CD19CAR T, or non-transduced T (NTD T) cells for 1 day on plates coated with GPIbα protein and anti-human IgG antibody. Bovine serum albumin (BSA) served as a negative control antigen. One spot represents IgG antibodies secreted by a single B cell. The number of spots detected in each well is shown in the top right corner. (D) The con- ceptual graph of GPIbα CAAR T therapy in ITP. GPIbα CAAR T cells act like a “Trojan horse”, trapping autoreactive B cells and performing specific killing. PLT: platelet; BCR: B-cell receptor.
macroglycopeptide sequences could form large steric hindrances, interfering with antibodies’ binding to CAAR structures (CAAR3-mutg233k-CAAR and CAAR4-mutg233k- CAAR). Furthermore, the structure of MSD is unstable,28 and we can’t tell whether the MSD expressed in the CAAR structure is folded or expanded, which may also interfere with the binding of CAAR3-mutg233k-CAAR and autoan- tibodies. The changes that occurred in the O-glycosylated macroglycopeptide domain and MSD in the CAAR expressed in the cells are worth exploring. An exciting and gratifying result was that GPIbα CAAR T cells successfully eliminated anti-GPIbα B cells from a patient with refractory ITP, and normal B cells were not destroyed compared to anti-CD19 CAR T cells, supporting further clinical application.
As anti-GPIIb/IIIa(αIIb/b3)5,47 is the most commonly detected autoantibody in ITP patients, we are also trying to construct GPIIb/IIIa-CAAR T cells. However, platelet-associated an- ti-GPIIb/IIIa autoantibodies from chronic ITP patients mainly depend on conformationally intact GPIIb/IIIa and divalent cations for maximal binding.5,48 The N-terminal globular head of GPIIb-IIIa seems to play an essential role as a hot spot for autoantigenic epitopes in chronic ITP.49 Integrating both αIIb and b3 into the CAAR structure is difficult, and it is chal- lenging to ensure that the conformational epitopes created by the two subunits are consistent with the native epitopes. Though difficult, related work is still being done by our team. In summary, a novel GPIbα CAAR was constructed, and we demonstrated GPIbα CAAR T-cell’s efficacy and safety in vitro and in vivo models. GPIbα CAAR T-cell therapy is a
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Disclosures
No conflicts of interest to disclose.
Contributions
HM, JL, JZ and YX designed the experiments. HM, JL and YH analyzed the data. ZJ and HM wrote the paper. JZ and YX performed the experiments. JS, HJ, LH and MX helped with the experiments.
Acknowledgments
The authors thank Jun Peng and Ming Hou from Qilu Hos- pital, Shandong University, for providing specimens and for their helpful comments and suggestions.
Funding
This work was supported by the National Natural Science Foundation of China (grant 82070124 to HM, and grant 82330005 to HM), the Technology Innovation Plan key re- search and development projects of Hubei Province (grant 2023BCB019 to HM), and the Basic Research Support Pro- grams Foundation of Huazhong University of Science and Technology (grant 2023BR033 to HM).
Data-sharing statement
Data that support the findings of this study are available from the corresponding author upon reasonable request.
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