A combinatorial caar-T cell strategy to eliminate factor VIII inhibitors in preclinical models of Hemophilia A

Abstract Background Chimeric Autoantibody Receptor T (CAAR-T) cell therapy, an extension of chimeric antigen receptor T cell (CAR-T) technology, presents a promising therapeutic strategy for autoimmune diseases. It achieves this by selectively eliminating autoreactive immune cells responsible for producing pathogenic antibodies or mediating cytotoxicity. This approach has achieved encouraging outcomes, as evidenced by the successful application of CD19-directed CAR-T therapy in systemic lupus erythematosus. In hemophilia A, a major clinical challenge is the formation of anti-factor VIII (FVIII) neutralizing antibodies, termed inhibitors, following recombinant FVIII therapy, which renders hemorrhage control ineffective and necessitates more intensive or alternative treatment strategies. Methods The FVIII CAAR construct was designed by linking FVIII C1 or A2 domain, which were the dominant fragments targeted by the inhibitors, with CD28 and CD3zeta intracellular domains via the CD28 transmembrane domain, and then T cells were transduced with a lentiviral system to enable the CAAR-T cells to recognize FVIII inhibitor-producing B cells for their selective elimination. In vitro cytotoxicity assays were conducted to evaluate the killing efficacy of CAAR-T cells. To mimic inhibitor development in hemophilia A and assess in vivo efficacy, the FVIII knockout (F8KO) mice were immunized with FVIII protein to induce inhibitor production, followed by the CAAR-T treatment and efficacy assessment. Additionally, a humanized mouse model was developed to simulate the clinical context and evaluate the therapeutic potential of CAAR-T cells within a human immune system environment. Results In vitro cytotoxicity assays demonstrated that C1 domain-containing CAAR-T cells exhibited comparable killing efficiency to A2 domain-containing CAAR-T cells against their respective target cells. In the F8KO hemophilia A inhibitor mouse model, both C1 and A2 CAAR-T cells significantly reduced circulating FVIII inhibitor levels, and the combination of C1 and A2 CAAR-T cells showed superior efficacy in suppressing inhibitor re-emergence upon FVIII rechallenge with good safety. In the humanized NCG mouse model, the combinatorial CAAR-T therapy also potently eliminated inhibitor-generating cells, effectively inhibiting the in vivo expansion of target cells. Conclusion The study demonstrates that CAAR-T cell therapy shows significant potential for treating hemophilia A by effectively reducing FVIII inhibitors. In vitro and in vivo experiments demonstrated that both C1 and A2 CAAR-T cells robustly eliminate FVIII inhibitor-producing B cells, with the combined therapy exhibiting superior efficacy in preventing inhibitor re-emergence and inhibiting target cell expansion. This novel approach offers a promising therapeutic strategy for addressing the clinical challenges associated with hemophilia A, particularly the formation of FVIII inhibitors following recombinant FVIII therapy.