Introduction: The development of inhibitory antibodies (inhibitors) against coagulation factor VIII (FVIII) is a critical complication in the treatment of hemophilia A, as hemostasis can no longer be re-established by FVIII replacement therapy. Cellular Treg therapy is an attractive candidate for targeted treatment of B and T cells involved in inhibitor formation, as it can suppress both cell types through direct and indirect mechanisms. Here, we generated antigen specific Tregs by engineering and retrovirally transducing a chimeric antigen receptor (CAR) molecule with specificity to human FVIII. Antigen recognition via the single chain antibody variable regions triggers signaling via the CD28 and CD3z co-stimulatory domains, which induces CAR-Treg activation and proliferation independent of MHC recognition. We show that human FVIII specific CAR-Tregs with second generation mouse signaling domains are activated and proliferate in response to antigen recognition, which can cause contact dependent or cytokine dependent suppression of inhibitor producing cells. Methods:A single chain variable fragment specific for human FVIII was cloned (Creative Biolabs, NY) out of an EBV transformed B cell from a hemophilia A patient (BO2C11), which produces IgG4 directed against residues 2125-2332 of huFVIII, corresponding to the carboxy-terminal end of C1 and the complete highly immunogenic C2 domain. This was fused to a 2nd generation CAR construct expressing CD28 and CD3ζ signaling molecules and packaged in a retroviral system (pMys-IRES-eGFP). CD3/CD28 activated polyclonal Tregs were retrovirally transduced to generate FVIII CAR-Tregs. Binding to Fc-FVIII was tested using an A647 conjugated Fc antibody. Upregulation of activation and exhaustion markers, specifically CD69, CD44, LAP, GARP and PD1 was tested 24 h after stimulation with either Fc-FVIII or B domain deleted (BDD) FVIII. Proliferation of CellTrace Violet labeled and FVIII stimulated CAR-Tregs was tested both in vitro and in vivo upon stimulation with BDD FVIII. 1x106 GFP+ FVIII CAR-Tregsorted cells were adoptively transferred into F8 e16−/− hemophilia A mice, and recipients were challenged, starting 1 day later, with weekly IV injections of 1 IU BDD-FVIII for 4 weeks. Plasma was tested for inhibitor formation at 4 and 8 weeks using the Bethesda assay and IgG1 ELISA. Results:FVIII CAR-Tregs bound Fc-FVIII (Eloctate) in a specific manner, with no binding observed to control Fc-FIX (Alprolix). Stimulation for 24 hours with either Fc-FVIII or BDD-FVIII resulted in upregulation of activation and exhaustion markers such as CD69, CD44, GARP and PD-1. No non-specific activation with the unrelated Fc-FIX antigen was observed. CellTrace Violet labeled CAR-Treg and CAR transduced conventional T cells underwent antigen-specific proliferation upon 72 hours of in vitro stimulation with FVIII. In vivo, persistence of adoptively transferred CAR-Tregs was observed only when antigen was concurrently administered. We are currently testing for the suppression of inhibitor development in BALB/c F8 e16−/− hemophilia A mice following adoptive immunotherapy with FVIII CAR-Tregs and continued weekly administrations of BDD-FVIII. Conclusions: Here, we demonstrate that CAR-Tregs directed to human FVIII show antigen-specific binding, activation and proliferation. We found persistence of adoptively transferred FVIII CAR-Tregs in response to antigen stimulation in mice. The suppressive activity of FVIII CAR-Treg on inhibitory antibody formation is now being evaluated in hemophilia A mice that are given factor replacement therapy with FVIII. CAR-Tregs represent an effective way to generate a large pool of antigen-specific cells, with no requirement for MHC restriction.


Biswas: Bayer Hemophilia Awards Program: Research Funding. Herzog: Novo Nordisk: Research Funding; Applied Genetic Technologies Corporation: Consultancy; Spark Therapeutics: Patents & Royalties.

Author notes


Asterisk with author names denotes non-ASH members.