Thursday, April 3, 2025
4/3/2025
Antigen-Specific Immunosuppression of Myasthenia Gravis by CAR-Engineered Tregs - Project summary/abstract Myasthenia gravis (MG) is caused by an antibody-mediated autoimmune response to muscle acetylcholine receptors (AChRs) that impairs neuromuscular transmission. There is no cure for MG or therapy specifically suppressing only the pathological autoimmune response to AChRs. We wish to develop an antigen-specific cell therapy for MG by engineering regulatory T cells (Tregs) with AChR-specific chimeric antigen receptors (CARs) to specifically suppress autoreactive T/B cells. Pathological mechanisms impairing neuromuscular transmission in MG and chronic experimental autoimmune MG (EAMG) are similar. The dysfunction of Tregs to suppress T effector cells and the decrease of Treg population are found in both untreated MG patients and EAMG rats. These suggest that rodent EAMG is a suitable model for evaluating the effectiveness of cell therapies. Adoptive transfer of polyclonal expanded non-specific Tregs reduces EAMG in rats. We hypothesize that AChR-specific CAR-engineered Tregs have greater efficacy and specificity than non-specific Tregs in suppressing the pathological autoimmune response to AChRs in EAMG. To address this hypothesis, we will optimize the transduction efficiency in rat T cells and characterize cellular phenotypes, cytokine profiles, and suppressive function of CAR Tregs in vitro (Aim 1). We have constructed AChR-specific CARs from single chain variable fragments derived from the parental anti-AChR rat hybridoma cell lines. Rat Tregs transduced with an AChR- specific CAR were activated and expanded in vitro in response to antigen-specific stimulation and suppressed T cell proliferation in response to polyclonal stimulation in a dose-dependent manner. We will enhance CAR transduction in rat T cells by producing high-titer retroviral particles using packaging cell lines. Alternatively, we will purify CAR-positive Tregs using the QBend-10 epitope incorporated into the CARs within the CD8 hinge. We will try to increase the yield of CAR-Treg by converting rat conventional T cells into Tregs by enforcing the expression of FoxP3. We will investigate transduced cells for cellular phenotypes, cytokine profiles, and in vitro suppressive function in an antigen-specific manner. Aim 2 is to investigate the safety and efficacy of AChR- specific CAR-transduced Tregs as a potential treatment for MG in EAMG rats. We have developed a novel EAMG model in which the autoimmune response is driven by a feed-forward cycle of autoimmune stimulation to endogenous AChRs released from damaged muscle in the autoimmune assault, allowing to investigate the impact of our CAR-Treg therapy on the self-sustaining autoimmune response to AChRs. Using this novel model, we will investigate prevention of EAMG by administering CAR-transduced Tregs immediately after the induction of EAMG and treatment of ongoing EAMG by administering CAR-transduced Tregs after the onset of chronic EAMG. The latter is the clinically relevant model for treating MG. We will investigate whether and how the epitope specificities of the CARs influence the efficacy and safety profiles of the CAR-Tregs. This work will provide the basis and rationale to investigate the mechanisms by which the CAR Tregs act and create CARs for human use.
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