Precision Engineering of an Anti-HLA-A2 Chimeric Antigen Receptor in Regulatory T Cells for Transplant Immune Tolerance

Abstract

Infusion of regulatory T cells (Tregs) engineered with a chimeric antigen receptor (CAR) targeting donor-derived human leukocyte antigen (HLA) is a promising strategy to promote transplant tolerance. Here, we describe an anti-HLA-A2 CAR (A2-CAR) generated by grafting the complementarity-determining regions (CDRs) of a human monoclonal anti-HLA-A2 antibody into the framework regions of the Herceptin 4D5 single-chain variable fragment and fusing it with a CD28-ζ signaling domain. The CDR-grafted A2-CAR maintained the specificity of the original antibody. We then generated HLA-A2 mono-specific human CAR Tregs either by deleting the endogenous T-cell receptor (TCR) via CRISPR/Cas9 and introducing the A2-CAR using lentiviral transduction or by directly integrating the CAR construct into the TCR alpha constant locus using homology-directed repair. These A2-CAR+TCRdeficient human Tregs maintained both Treg phenotype and function in vitro. Moreover, they selectively accumulated in HLA-A2-expressing islets transplanted from either HLA-A2 transgenic mice or deceased human donors. A2-CAR+TCRdeficient Tregs did not impair the function of these HLA-A2+ islets, whereas similarly engineered A2-CAR+TCRdeficientCD4+ conventional T cells rejected the islets in less than 2 weeks. A2-CAR+TCRdeficient Tregs delayed graft-versus-host disease only in the presence of HLA-A2, expressed either by co-transferred peripheral blood mononuclear cells or by the recipient mice. Altogether, we demonstrate that genome-engineered mono-antigen-specific A2-CAR Tregs localize to HLA-A2-expressing grafts and exhibit antigen-dependent in vivo suppression, independent of TCR expression. These approaches may be applied towards developing precision Treg cell therapies for transplant tolerance.

Chimeric Antigen Receptor (CAR) Regulatory T-Cells in Solid Organ Transplantation

“…Bioluminescence imaging demonstrated preferential migration of HLA-A2 CAR Tregs towards the HLA-A2+ skin allograft (64), indicating that HLA-specific CAR Tregs can migrate towards the location where regulation is required. Muller et al demonstrated similar effects where HLA-A2-CAR Tregs migrate to the HLA-A2+ transgenic islets in a model of induced diabetes in immunodeficient mice (68). In addition, Noyan et al studied a different HLA-A2 CAR, in a model where immunodeficient NOD rag gamma (NRG) mice were transplanted with human HLA-A2+ skin grafts (26).…”

“…MacDonald et al demonstrated a very low proportion of induced cell death in HLA-A2-positive cells when co-cultured with high ratios of CAR Tregs, but similar experiments with the presence of HLA-A2-positive PBMCs show that cytolytic activity was negligible (62). Another study in the setting of murine allogeneic islet transplantation indicated that despite the accumulation of CAR Tregs in allografts, no islet destruction could be observed, which suggested that CAR Tregs are not cytotoxic to allogeneic islets (68). Since these studies have described Treg stability directly after generation and infusion, long-term stability still remains to be investigated in order to establish safety.…”

“…HLA-A2 specificity of the scFv was confirmed by cytokine production and cytotoxicity of CAR Teff cells after stimulation with HLA-A2 positive and HLA-A2 negative cells (27). In addition, Muller et al used the variable domain sequences of a well-characterized human B-cell derived hybridoma (clone SN607D8) (67) to produce an HLA-A2 specific scFv (68). Importantly, they found that the original anti-HLA-A2 sequences of the SN607D8 hybridoma could not successfully be expressed in the scFv.…”

“…Additionally, CAR Treg specificity can be assessed with a panel of APCs expressing different single HLA alleles by measuring activation marker expression (64). Furthermore, tetramer staining (26,62,63), proliferation or cytokine secretion upon coculture with antigen-positive cells (26,27,63,68) can be used. To move from the experimental setting to the clinical reality, the possibility to target the most prevalent HLA antigens should be explored, in order to serve the vast majority of transplant recipients.…”

“…To ensure safety, Treg phenotype should not be affected by the transduction or any alterations performed on these cells. Several studies demonstrated that expression levels of key regulatory T-cell markers and/or homing receptors, for example, FoxP3, CTLA-A4, CD39, CD45RA, and CCR7 remain unchanged in CAR Tregs (26,27,62,68). Other studies have established that Tregs preserved their in vitro capacity to suppress via the endogenous TCR (62) or CAR (63).…”