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-zeta 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.

Anti-HLA-A2-CAR Tregs prolong vascularized mouse heterotopic heart allograft survival

“…The complementarity determining regions of the anti‐HLA‐A2 scFv from a human B‐cell‐derived hybridoma (clone SN607D8) were grafted onto the framework of Herceptin/trastuzumab (clone 4DG) as previously described 14 …”

“…The complementarity determining regions of the anti-HLA-A2 scFv from a human B-cell-derived hybridoma (clone SN607D8) were grafted onto the framework of Herceptin/trastuzumab (clone 4DG) as previously described. 14 To generate an anti-HLA-A2 murine CAR, the grafted scFv was cloned in-frame to the murine CD8 hinge and transmembrane domains followed by CD28 and CD3ζ intracellular domains via isothermal DNA assembly. The A2-CAR was cloned into a murine stem cell virus-based retroviral vector with an N-terminal myc tag and in-frame to a truncated EGFR (EGFRt), separated by a P2A peptide.…”

“…Most published studies evaluated A2‐CAR Treg function in humanized NOD. Cg‐ Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice for protection of HLA‐A2–positive skin grafts or in the context of xenogenic GvHD (graft versus host disease) 9‐14 . However, NSG mice are severely immunodeficient due to the absence of many immune cell types and incompatibilities between ligands and receptors from human T cells and mouse hosts.…”

“…Since donor and recipient major histocompatibility complex (MHC) mismatches are the main trigger of allograft rejection, donor-specific MHCs are attractive targets for CAR Treg therapy. Several groups have developed CARs targeting HLA-A2 (A2-CARs), [9][10][11][12][13][14] because up to 25% of transplantations are characterized by an HLA-A2-positive donor and an HLA-A2-negative recipient. Most published studies evaluated A2-CAR Treg function in humanized NOD.…”

Regulatory T Cells: Liquid and Living Precision Medicine for the Future of VCA

“…Additionally, the authors found that engineered Treg selectively traffic to HLA-A2 expressing transplanted islet cells, thereby underscoring their ability to recognize their cognate antigen in vivo. 80 Their study outlined the need for engineering highly specific Treg without disrupting their functionality by introduction of a single HLA-antigen-specific CAR. Removal of the endogenous TCR and demonstration of stability of Treg function in vivo encourage the potential use of "off-the-shelf" Treg for the treatment of rejection reaction in transplant patients.…”