Muscle-specific tyrosine kinase myasthenia gravis (MuSK MG) is an autoimmune neuromuscular syndrome caused by anti-MuSK autoantibodies that interrupt neuromuscular junction signaling and lead to severe muscle weakness. In the muscle, the MuSK transmembrane receptor is localized to the postsynaptic side of the neuromuscular junction and forms a multimolecular complex with lipoprotein receptor-related protein 4 (LPR4) and the neuronal proteoglycan agrin. Anti-MuSK autoantibodies, which are predominantly IgG4, disrupt MuSK-LRP4 signaling, which consequently interferes with vital neuromuscular junction synaptic transmission.

Although no therapies are clinically approved specifically for MuSK MG, current therapeutic approaches to treat MuSK MG are mostly centered on B-cell depletion with rituximab, which has been shown to reduce patients' autoantibodies. However, rituximab induces generalized immunosuppression due to chronic B-cell depletion, which subsequently puts the patient at risk of developing serious infections because of a compromised immune system. Therefore, there is a compelling rationale for the development of precision medicine therapies targeting only the pathogenic anti-MuSK B cells and sparing patients’ healthy B cells to avoid generalized immunosuppression.

In the context of precision medicine, the clinically approved anti-CD19 chimeric antigen receptor (CAR) T-cell therapy has revolutionized the field by genetically engineering patients’ T cells to eliminate previously refractory B-cell cancers. CAR T cell therapy has led to complete and durable remissions of B-cell malignancies and has recently demonstrated early promise that such precision medicine therapies can be extended beyond B-cell cancers to other incurable B-cell-mediated diseases. In the past several years, our laboratory has been re-designing CAR technology for autoimmune disease therapy by utilizing autoantigens as the extracellular domain of a chimeric autoantibody receptor (CAAR). CAARs target T cell cytotoxicity against autoantigen-specific B-cells, which express a surface-bound autoantibody (i.e., B cell receptor) that defines the pathogenic autoimmune cell population.

MuSK is a transmembrane tyrosine kinase whose ectodomain comprises three immunoglobulin-like (Ig1-Ig3) and frizzled-like (Fz) domains. An estimated 100%, 58%, and 23% of MuSK MG patient sera recognize Ig1, Ig2, and Ig3-Fz domains, respectively. Aiming to specifically target anti-MuSK B-cells, we designed a MuSK-CAAR comprising the complete MuSK ectodomain as the extracellular domain of the CAAR, linked to cytoplasmic T cell receptor costimulatory and activation CD137-CD3ζ signaling domains. In our recently published study in Nature Biotechnology,we report the design and functional validation of the MuSK-CAAR expressed in human T cells and its efficacy and safety in preclinical models. 

In this study, we show that MuSK-CAAR T cells demonstrated specific cytotoxicity against anti-MuSK B-cells targeting epitopes physiologically relevant to human MuSK MG. Secretion of the activation cytokine IFN-g was detected only in MuSK-CAART co-cultures with pathogenic B-cells. MuSK autoantibodies demonstrated varying effects on MuSK-CAART activity, but were observed to induce IFN-g  production and MuSK-CAART proliferation in a concentration-related manner. Specific cytotoxicity against monocytes and natural killer cells by MuSK-CAART was not observed with normal human IgG or anti-MuSK monoclonal antibodies. Anti-MuSK BCR-targeted cytolysis demonstrates comparable efficacy as CD19-targeted cytolysis in eliminating anti-MuSK B-cells in vivo. In a syngeneic MuSK experimental autoimmune myasthenia gravis model with rare anti-MuSK target B-cells and circulating anti-MuSK antibodies, MuSK-CAART treatment resulted in antigen-specific IgG depletion without total B-cell depletion and did not require prior lymphodepletion for therapeutic effect. Toxicology screens did not identify specific off-target cytotoxic interactions of MuSK-CAART.

In summary, we have established the preclinical safety and efficacy of MuSK-CAART for MuSK-specific B-cell depletion in various in vitro and in vivo models, which led to the recent FDA clearance of an Investigational New Drug (IND) application of MuSK-CAART for the treatment of MuSK autoantibody-positive MG. The MuSK-CAART phase 1 clinical trial recently opened for recruitment, which shifts focus from preclinical to clinical mechanism of action. We hypothesize that MuSK-CAART will reset immune tolerance in MuSK MG by depleting MuSK-reactive B-cells and normalizing peripheral T-cell subsets, ideally leading to safe and lasting disease remission.