Systematic testing and specificity mapping of alloantigen-specific chimeric antigen receptors in regulatory T cells
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
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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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 3

Cross-reactivity of humanized anti–HLA-A2 CARs with common HLA-A allelic variants.

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Cross-reactivity of humanized anti–HLA-A2 CARs with common HLA-A allelic...
(A) Schematic diagram of experimental setup and gating strategy for the FlowPRT cell assay. ΔNGFR or CAR Tregs were incubated with a cocktail of single HLA FlowPRA beads for 30 minutes, and bead-CAR Treg interactions were quantified as the loss of beads in a bead singlet gate based on FSC/SSC profile. (B) Binding to HLA-A*02:01–coated beads for each m/hA2-CAR Treg relative to binding of a ΔNGFR Treg control. Statistical significance determined by 1-way ANOVA and Holm-Šídák post hoc test comparing with mA2-CAR; mean ± SEM; **P < 0.01. (C and D) Correlation between the mean of HLA-A*02:01 binding measured by the FlowPRT cell assay and either (C) HLA-A*02:01 tetramer MFI evaluated by flow cytometry or (D) increase in the proportion of CD69+ cells 16 hours after coculture with HLA-A*02:01 versus negative control HLA-A*24:01 K562 cells. (E) Percent binding of each m/hA2-CAR Treg to the indicated HLA-A alleles after normalization to an ΔNGFR Treg control from the same donor. Dotted line represents 2 SDs from the mean of the bead-only control. For a summary of statistical results in E, see Supplemental Table 1. n = 3–6 from at least 3 independent experiments.