Systematic testing and specificity mapping of alloantigen-specific chimeric antigen receptors in regulatory T cells
Nicholas A.J. Dawson, … , Majid Mojibian, Megan K. Levings
Nicholas A.J. Dawson, … , Majid Mojibian, Megan K. Levings
Published February 12, 2019
Citation Information: JCI Insight. 2019;4(6):e123672. https://doi.org/10.1172/jci.insight.123672.
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Research Article Immunology Transplantation

Systematic testing and specificity mapping of alloantigen-specific chimeric antigen receptors in regulatory T cells

Abstract

Chimeric antigen receptor (CAR) technology can be used to engineer the antigen specificity of regulatory T cells (Tregs) and improve their potency as an adoptive cell therapy in multiple disease models. As synthetic receptors, CARs carry the risk of immunogenicity, particularly when derived from nonhuman antibodies. Using an HLA-A*02:01–specific CAR (A2-CAR) encoding a single-chain variable fragment (Fv) derived from a mouse antibody, we developed a panel of 20 humanized A2-CARs (hA2-CARs). Systematic testing demonstrated variations in expression, and ability to bind HLA-A*02:01 and stimulate human Treg suppression in vitro. In addition, we developed a new method to comprehensively map the alloantigen specificity of CARs, revealing that humanization reduced HLA-A cross-reactivity. In vivo bioluminescence imaging showed rapid trafficking and persistence of hA2-CAR Tregs in A2-expressing allografts, with eventual migration to draining lymph nodes. Adoptive transfer of hA2-CAR Tregs suppressed HLA-A2+ cell–mediated xenogeneic graft-versus-host disease and diminished rejection of human HLA-A2+ skin allografts. These data provide a platform for systematic development and specificity testing of humanized alloantigen-specific CARs that can be used to engineer specificity and homing of therapeutic Tregs.

Authors

Nicholas A.J. Dawson, Caroline Lamarche, Romy E. Hoeppli, Peter Bergqvist, Vivian C.W. Fung, Emma McIver, Qing Huang, Jana Gillies, Madeleine Speck, Paul C. Orban, Jonathan W. Bush, Majid Mojibian, Megan K. Levings

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Figure 5

hA2-CAR Tregs are suppressive in vitro and in a model of xenogeneic GvHD in vivo.

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hA2-CAR Tregs are suppressive in vitro and in a model of xenogeneic GvHD...
(A) Cell proliferation dye-e450–labeled (CPD-e450–labeled) HLA-A2neg CD4+ “responder” T cells were stimulated with a 1:1 ratio of mature HLA-A2+ dendritic cells in the absence/presence of varying ratios of the indicated CPD-e660–labeled control or m/hA2-CAR Tregs. After 6 days, the amount of proliferation of the CPD-e450–labeled CD4+ responder T cells was measured by flow cytometry. Top: representative data and gating strategy, with proliferation index (Prolif. index). Bottom: average data for n = 3–7 pooled from at least 3 independent experiments. Statistics were performed using a 2-way ANOVA with Holm-Šídák post hoc test versus a ΔNGFR Treg control. *P < 0.05; mean ± SEM. (B–D) Irradiated NSG mice were injected with PBS (n = 3); HLA-A*02:01pos PBMCs alone (n = 5); HLA-A*02:01pos PBMCs and a 1:2 ratio of H1k2 hA2-CAR Tregs (n = 6); or mA2-CAR Tregs (n = 4). Data were pooled from 2 independent experiments. (B) Survival curve, log-rank (Mantel-Cox) test. (C) Human CD45+ engraftment upon experimental or humane end point (gating strategy in Supplemental Figure 7A). (D) Percent weight change at sacrifice relative to experiment start (day 49 or earlier). Statistical significance determined using a 1-way ANOVA and Holm-Šídák post hoc test; mean ± SEM. *P ≤ 0.05, **P ≤ 0.01.