Targeting antigen-specific B cells in immune mediated thrombotic thrombocytopenic purpura using modular CAR T cells

Introduction: Treatment of refractory immune-mediated thrombotic thrombocytopenic purpura (iTTP) patients remains an unmet clinical need. Chimeric Antigen Receptor T (CAR T) cell therapy, targeting CD19 expressing B cells, has been successfully used in autoimmune diseases such as refractory systemic lupus erythematosus. However, this approach targets both the pathogenic and non-pathogenic B cells. Additionally, conventional CAR T cell therapy, lacking regulatory mechanisms, is associated with severe and live-threatening adverse events. A safer approach could involve a modular variant of CAR, like the Universal CAR (UniCAR), which allows better control. UniCAR T cells express a single-chain Fv (scFv) on their surface that binds to a target module (TM). The TM consists of a human La-derived peptide, linked to a protein binding the target cell. TM infusion into the patient allows precise control of UniCAR T cell activity. Moreover, if the TM contains the autoantigen (ADAMTS13 in iTTP), only pathogenic B cells are targeted, sparing the rest of the immune cells.The project aims to test the efficacy of the UniCAR T system in killing target cells expressing an anti-ADAMTS13 antibody or scFv.

Method: Human T cells were transduced with lentiviral vectors expressing the UniCAR construct. Lentiviral vectors encoding MDTCS (N-terminal domains of ADAMTS13) linked to the human La-derived peptide were used to transduce fibroblast 3T3 cells. The TM was purified using Ni²⁺-NTA affinity chromatography. UniCAR T cytotoxicity was assessed via a flow cytometry viability assay. Target cells were the anti-spacer 15D1 hybridoma cells and engineered Nalm-6 cells expressing patient-derived anti-ADAMTS13 scFv on their surface. UniCAR T cells (effector cells) were incubated at different effector-to-target (E:T) ratios and TM concentrations. As a negative control (unspecific killing), UniCAR T cells were incubated with the target cells without TM. Cytotoxicity was evaluated after 24h.

Results: UniCAR T cells were co-cultured with the 15D1 hybridoma cells using four TM concentrations: 0.5, 2.5, 5 and 25 nM. For an E:T ratio of 1:1, a maximum killing of 25.7% was reached at the highest TM concentration with 4.0% of unspecific killing (n=3). At an E:T ratio of 5:1, a maximum killing of 48.0% was reached at the three highest TM concentrations, with unspecific killing of 14.0% (n=3). Next, cytotoxicity was assessed in Nalm-6 cells expressing patient-derived anti-ADAMTS13 scFv at E:T ratio 1:1. Maximum killing of 91.5% was reached at all tested TM concentrations, with 23.8% in the absence of the TM. In all cases, cytotoxicity was significantly higher than unspecific killing.

Conclusion: The UniCAR T cell strategy can be used to specifically kill hybridoma cells expressing 15D1 anti-ADAMTS13 antibody and also engineered Nalm-6 cells expressing patient-derived anti-ADAMTS13 scFv on their surface. Finally, the efficacy of this therapy will also be studied in a mouse model of iTTP.

Publikationsverlauf

Artikel online veröffentlicht:
13. Februar 2025