Express Mutagenesis

Regulatory T cells (Tregs) are a subpopulation of T cells capable of suppressing immune responses and thereby can be harnessed for tolerance induction in replacement therapy, a conventional therapy for deficiency diseases. Rana et al. have engineered two types of antigen-specific Tregs: chimeric antigen receptor (CAR)-Treg and T cell receptor (TCR) fusion construct (TRuC)-Treg. In mouse models of hemophilia A, they observed that CAR-Treg engagement could not suppress inhibitory antibody responses to replacement factor VIII (FVIII) therapy. In contrast, TRuC-Tregs induced functional suppression on the development of adaptive immune responses to FVIII. These findings indicate that TRuC can be a desired platform to improve the suppressive activity of Tregs against inhibitory antibodies in replacement therapy.

Background

Hemophilia A is a genetic disorder caused by a deficiency of blood clotting factor VIII (FVIII). Replacement of FVIII has been used for decades as an efficacious treatment to prevent bleeding and support normal coagulation in individuals with hemophilia A. Nevertheless, alloantibodies develop and neutralize exogenously infused FVIII, affecting more than one-third of patients with severe hemophilia A. Therefore, Rana et al. sought to harness engineered Tregs redirected by CAR (CAR-Treg) or TRuC (TRuC-Treg) to induce specific tolerance to extraneous therapeutic proteins in replacement FVIII therapy.

Experiment

To generate a FVIII-specific CAR, a single chain variable fragment (scFv), specific against human FVIII, was complexed to second-generation murine 28z CAR signaling sequences. A Myc tag was cloned into the construct using recombinant DNA technology. Through site directed mutagenesis, single amino acid changes were introduced in the immune receptor tyrosine-based activation motifs and signaling domains of CD 3 and CD28, respectively. The FVIII scFv sequence was fused to the N terminus of murine CD3ε by GenScript in order to develop FVIII-directed TRuC Tregs. Transfer plasmids were generated through inserting FVIII CAR or TRuC sequences into the pMYs-IRES-mScarlet retroviral backbone and transfected into the cell line with PlatE ecotropic retroviral packaging.

To explore whether CAR- and TRuC-redirected Tregs were able to suppress inhibitor formation, naive recipient mice were infused with 5 × 105-sorted FVIII CAR Tregs or TRuC Tregs (5 × 105 or 1 × 106) followed by 4 weekly i.v. injections of 1.5 international units B domain-deleted-FVIII. Plasma from mice was analyzed for inhibitor formation by the Bethesda assay and anti-FVIII IgG1 ELISA.

Results

Infusion of FVIII CAR Tregs increased the formation of inhibitors in recipient animals; in fact, mice that received CAR Treg developed high-titer inhibitors compared to those in the control group that only received FVIII injections. Targeted mutations in the CD3z or CD28 signaling motifs or interleukin-10 overexpression were not sufficient to restore CAR Treg function.

On the other hand, TRuC-Tregs exhibited controlled antigen-specific signaling via engagement of the entire TCR complex, therefore inducing functional suppression of the FVIII-specific antibody response. Most recipient animals in the TRuC Treg group did not develop detectable inhibitors at 4 weeks, whereas high titer inhibitors developed in all control mice.

Reference

Rana, Jyoti, et al. "CAR and TRuC redirected regulatory T cells differ in capacity to control adaptive immunity to FVIII." Molecular Therapy (2021).

Powell, Jerry S. "Recombinant factor VIII in the management of hemophilia A: current use and future promise." Therapeutics and clinical risk management 5 (2009): 391.

Wight, J., and S. Paisley. "The epidemiology of inhibitors in haemophilia A: a systematic review." Haemophilia 9.4 (2003): 418-435.

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