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 6

Expression of m/hA2 CARs endows Tregs with rapid and persistent homing to HLA-A2:01+ skin allografts.

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Expression of m/hA2 CARs endows Tregs with rapid and persistent homing t...
Tregs were cotransduced with lentivirus encoding luciferase and either a control HER2-CAR, mA2-CAR, or hA2-CAR (H1k2) and expanded for 7 days. Dual transduced cells were FACS sorted, expanded for 5 more days, then injected into NSG mice that had previously been transplanted with juxtaposed skin transplants from both NSG and NSG-HLA-A*02:01 transgenic mice. (A) Schematic representation of the experimental setup. (B) Representative luciferase imaging of skin grafts from 2 different experiments (A and B) at the indicated time points after Treg injection. Red solid circle denotes location of NSG skin graft; black dotted circle denotes location of A2-positive NSG skin graft; green square denotes location of draining lymph node with a visually detectable luciferase signal. Amount of luciferase radiance was quantified using the average amount of photons/s/cm2/steradian and plotted as a ratio between the HLA-A*02:01-NSG and NSG skin grafts (C) 72 hours after Treg injection or (D) over time. n = 6–7 per group pooled from 3 independents experiments; mean ± SEM. Repeated-measures ANOVA with Bonferroni’s correction. *P < 0.05, **P < 0.01.