MON-LB033 Unleashing the Anti-Inflammatory Potential of Treg Cells Against Type I Diabetes Using Advanced Chimeric Antigen Receptor Technology

Abstract

Regulatory T (Treg) cells play a critical role in maintenance of tolerance and are ideal for development of therapies designed to suppress inflammation and autoimmunity. Emerging data indicate that, compared to polyclonal Tregs, adoptive transfer of disease-relevant antigen-specific Tregs is advantageous in terms of reduced risk of nonspecific immune suppression and need for fewer cells. The successful use of chimeric antigen receptor (CARs) technology for the generation of antigen-specific effector T cells suggests that a similar approach can be used to generate antigen-specific Tregs. Here, we aimed at developing pancreatic Beta cell-specific CAR Tregs and explore their therapeutic application against Type 1 diabetes (T1D). Methods: Two GAD65 B cell epitopes known to interact with two immunodominant regions in the N-terminal (CAR-N), and Middle (CAR-M) regions were selected for assembly onto T cell receptors (1). These GAD65 CAR Tregs were used to prevent/treat diabetes in our humanized mouse model that closely resembles human T1D (2). Autologous, amplified Tregs were transduced with CAR-M and CAR-N Treg constructs. Treg expression of CAR-M/N was confirmed by PCR and western blot. First, a group of humanized mice was infused with 5 million GAD65 specific CAR-M/N Tregs co-expressing GFP, 24 hr before sacrifice. CAR Treg cells were then processed into single cell suspensions and tracked in peripheral blood, pancreas (PN), peri-pancreatic lymph nodes (PLN), and spleen using flow cytometry. Also, 3D iDISCO image was collected from the same sites for visualization of CAR Treg cells 24 post-infusion. Second, three groups of humanized mice were treated with 5 million either GAD65 specific CAR-M/N Tregs, or untreated (normal control) Tregs or EPCAM (CAR Treg control). These mice were euthanized 30 days after treatment. Again, Tregs were tracked in PN, PLN, and spleens using flow cytometry. Also, glucose tolerance tests (GTT) were carried out before cell infusion and at 30 days. Results: GAD65 specific CAR-M/N Tregs homed to pancreatic islets 24 hours after infusion. The Treg population was also significantly increased in PN and spleen of CAR-M/N Treg treated groups as compared to untreated Treg and EPCAM treated groups at 30 days. GTT showed that humanized mice had significantly lower blood glucose at 60 min after being treated with CAR-M Tregs as compared to controls. CAR-N Treg treated mice had lower blood glucose at 90 and 120 min time points as compared to controls. Untreated Tregs group showed no significant differences as compared to EPCAM. Conclusion: The results demonstrated the potential use of CAR technology to generate potent, functional, and stable Beta cell-specific CAR Tregs as therapeutic against T1D in humans. This project sets a stage for making antigen-specific CAR Treg to treat other autoimmune diseases. 1) U.S. Provisional Patent #62/757,313 2) U.S. Provisional Patent #62/713,827 Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.