IMPE Abstracts

Objectives/Background: Type 1 Diabetes (T1D) manifests as a loss of insulin secretion due to autoimmune destruction of β-cells by self-reactive T effector lymphocytes. Suppression of autoreactive T effectors by regulatory T cells (Tregs) is a key mechanism of peripheral self-tolerance. T1D subjects have documented numerical and functional Tregs impairment. Recent early-stage clinical trials with adoptively transferred ex vivo-expanded autologous Tregs showed promise, transiently stabilizing C-peptide production. However, success of Tregs-based T1D therapies is limited by the rare frequency of endogenous β-cell-specific Tregs, which need home to the islets and deliver local suppression. We equipped natural Tregs with the engineered β-cell-specific chimeric antigen receptor (CAR) aiming to expedite their islet trafficking and enhance intra-islet antigen-specific immunosuppression.

Methods/Results: Binding of engineered CARs is driven by a single chain variable fragment (scFv-B3s) from the mAb highly species-specific for human β-cell surface marker, NTPDase3. Sequences of VH and VL regions of α-NTPDase3 mAb were used to assemble scFv-B3s. Expressed in E. coli scFv-B3s, tested for the functional NTPDase3 inhibition, showed Ki 1.26-1.52 nM proving its explicit NTPDase3 targeting. CAR constructs contained scFv-B3s cDNA built into conventional second-generation CAR backbone with IRES-ZsGreen assembly, and were generated with and without P2A self-cleavage site-containing cassette driving constitutive expression of FoxP3, the master-regulator of Tregs differentiation and function. Transduction of primary human CD4+ cells with CAR-containing retroviruses produced ~55% ZsGreen+ cells confirmed by Western Blotting and FACS to express CARs. CAR-transduced Tregs expanded for 4wks and tested in co-cultures with HEK-293 cells expressing either mouse or human NTPDase3 displayed antigen-specific proliferation, preserved Treg phenotype (Foxp3+, Eos+, Helios+, GARP+, GITR+), secretion of regulatory cytokines (IL-10, TGFβ), and showed no exhaustion signs. In suppression assays, CAR-Tregs halted both, proliferation and cytotoxicity of human diabetogenic clonal CD8+ effectors. Finally, live human pancreatic slices cultures were used to assess CAR-Tregs migration towards β-cells. 16hrs after CAR-Tregs addition to cell cloning rings on top of slices, CAR-Tregs accumulated in the islets to significantly greater extent than GFP-transduced Tregs or primary CD4+ cells.

Conclusions: We engineered CARs stringent to hNTPDase3, unique live human β-cells marker. In vitro, CAR-Tregs displayed survivability, prolonged maintenance of suppressive phenotype, functional antigen recognition without exhaustion, and subduction of diabetogenic human β-cell-specific T effectors. In situ, on live pancreatic slices, CAR-Tregs efficiently trafficked, and formed stable clusters within the islets. These results warrant further pre-clinical B3s-CAR-Tregs testing, interrogating their protective potency in vivo, in human islet transplants models.