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 1

Expression of a panel of hA2-CARs on human Tregs.

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Expression of a panel of hA2-CARs on human Tregs. (A) Schematic represen...
(A) Schematic representation of CAR humanization. CDRs from the BB7.2-derived scFv were determined using Kabat or Chothia definitions for each heavy and light chain and grafted onto suitable human framework sequences. mm, Mus musculus; hs, Homo sapiens. (B) Human Tregs were transduced with lentivirus encoding the indicated constructs. After 7 days of expansion, the ability of ΔNGFR+ cells to bind to HLA-A*02:01 tetramers was measured by flow cytometry. Left: Representative flow cytometry plots. Right: Summarized data of percent or MFI of A*02:01-tetramer binding. Data represent n = 2–4 for each construct pooled from at least 2 independent experiments. Mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.