Human CD83-targeted chimeric antigen receptor T cells prevent and treat graft-versus-host disease

Abstract

Graft-versus-host disease (GVHD) remains an important cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (allo-HCT). For decades, GVHD prophylaxis has included calcineurin inhibitors, despite their incomplete efficacy and impairment of graft-versus-leukemia (GVL). Distinct from pharmacologic immune suppression, we have developed what we believe is a novel, human CD83-targeted chimeric antigen receptor (CAR) T cell for GVHD prevention. CD83 is expressed on allo-activated conventional CD4+ T cells (Tconvs) and proinflammatory dendritic cells (DCs), which are both implicated in GVHD pathogenesis. Human CD83 CAR T cells eradicate pathogenic CD83+ target cells, substantially increase the ratio of regulatory T cells (Tregs) to allo-activated Tconvs, and provide durable prevention of xenogeneic GVHD. CD83 CAR T cells are also capable of treating xenogeneic GVHD. We show that human acute myeloid leukemia (AML) expresses CD83 and that myeloid leukemia cell lines are readily killed by CD83 CAR T cells. Human CD83 CAR T cells are a promising cell-based approach to preventing 2 critical complications of allo-HCT - GVHD and relapse. Thus, the use of human CD83 CAR T cells for GVHD prevention and treatment, as well as for targeting CD83+ AML, warrants clinical investigation.

Keywords: Cancer immunotherapy; Oncology; Stem cell transplantation; T cells; Transplantation.

Figure 1. Human CD83-targeted CAR T construct and functional characteristics.

(A) An anti-CD83 single-chain variable fragment is followed by a CD8 hinge and transmembrane domain, as well as a 41BB costimulatory domain and CD3ζ activation domain. The CAR is tagged with a fluorescence reporter at the 3′ end. The CAR reporter gene is cloned into an SFG retroviral vector. (B) Graph shows CAR gene transfer among T cells (mean ± SEM) by expression of the intracellular EGFP reporter whereas mock-transduced cells are EGFP negative and CD83 CAR T cells are EGFP positive. (C) Graph demonstrates the relative amount of CD4⁺ or CD8⁺ subsets among the mock-transduced or CD83 CAR T cells at day +7 after production (n = 2–3 independent donor experiments). (D and E) The amount of IFN-γ and IL-2 released by mock-transduced or CD83 CAR T cells after stimulation with CD83⁺ DCs. (F) CD83 CAR T cells or mock-transduced T cells were cocultured with CD83⁺ DCs and cytotoxicity was measured on a real-time cell analysis system. The data are presented (mean ± SEM) as the average normalized cell index over time for duplicate wells. Normalized cell index is calculated as cell index at a given time point divided by cell index at the normalized time point, which is day 1 after addition of T cells. One representative experiment of 2 is shown. (G) CD83 CAR T cells or mock-transduced T cells were stimulated by CD83⁺ DCs and the absolute number of T cells (mean ± SEM) was calculated weekly over a 14-day period. One representative experiment of 2 shown. ANOVA (D–G). ***P = 0.0001–0.001; ****P < 0.0001.

Figure 2. CD83 is differentially expressed on human activated conventional CD4⁺ T cells compared with regulatory T cells.

Human T cells were stimulated by allogeneic moDCs (DC/T cell ratio, 1:30) or CD3/CD28 beads (bead/T cell ratio, 1:30). CD83 expression on activated Tconvs (CD4⁺, CD127⁺, CD25⁺) or Tregs (CD4⁺, CD127^–, CD25⁺, Foxp3⁺) was measured at baseline, 4 hours, 8 hours, 24 hours, and 48 hours after stimulation. Graphs show the amount of CD83⁺ Tconvs or Tregs (mean ± SEM) after (A) allogeneic DC or (B) CD3/CD28 bead stimulation (n = 5 independent experiments). (C) Graph shows the frequency (mean ± SEM) of CD83⁺ Th1 (CD4⁺, T-bet⁺), Th2 (CD4⁺, GATA3⁺), or Th17 (CD4⁺, RORγt⁺) cells after 8 hours of DC-allostimulation (n = 8 independent experiments). Human CD83 CAR or mock T cells were cultured with DC-allostimulated PBMCs at a ratio of 1:10 over 48 hours. (D) Graph shows the frequency of CD83⁺, CD3⁺, and CD3^– target cells when cultured with CD83 CAR or mock-transduced T cells (n = 7 independent experiments). (E) Contour plots show the expression of CD83 among EGFP⁺ CAR T cells over time. One representative experiment of 2 is shown. (F) Human CD4⁺ or CD8⁺ T cells were activated with CD3/CD28 beads for 8 hours, then removed from the beads and cocultured with autologous CD83 CAR T cells or mock-transduced T cells for 24 hours (CAR T–to–T cell ratio, 5:1). Graph shows the triplicate mean ± SEM of live CD4⁺ or CD8⁺ T cells at the end of culture. One representative experiment of 2 is shown. ANOVA (A–D and F). *P < 0.05, **P = 0.001–0.01; ***P = 0.0001–0.001; ****P < 0.0001.

Figure 3. Human CD83 CAR T cells reduce alloreactivity.

Human T cells were cultured with allogeneic cytokine-matured moDCs at a DC/T cell ratio of 1:30 (i.e., 100,000 T cells and 3333 moDCs). CD83 CAR T cells were added at specific ratios to the moDCs (3:1 to 1:10, where the lowest amount of CAR T cells added was 333 cells). T cell proliferation was measured by Ki-67 expression at day +5. CAR T cells were gated out by their expression of GFP. Controls included T cells alone (i.e., no proliferation), mock-transduced T cells, and CD19 CAR T cells. These mock-transduced T cells did not express a CAR but were treated in an identical fashion as the transduced CD83 CAR T cells. The CD19 CAR T cells used an identical 41BB costimulation domain as the CD83 CAR T cells, but targeted an irrelevant antigen. One of 2 representative experiments is shown.

Figure 4. Human CD83 CAR T cells prevent xenogeneic GVHD.

(A) NSG mice received 25 × 10⁶ human PBMCs and were inoculated with low (1 × 10⁶) or high dose (10 × 10⁶) CD83 CAR or (1 × 10⁶ to 10 × 10⁶) mock-transduced T cells. The CARs were autologous to the PBMC donor. An additional control group of mice received PBMCs alone. (A) Survival and (B) GVHD clinical scores are shown. Clinical scores incorporate an aggregate assessment of activity, fur and skin condition, weight loss, and posture. Pooled data from 3 independent experiments, up to 9 mice per experimental arm for survival, with a representative experiment depicting long-term GVHD clinical scores. In separate experiments, recipient mice were humanely euthanized at day +21 and tissue GVHD severity was evaluated by an expert blinded pathologist. Xenogeneic GVHD path scores, representative H&E images, amount of Ki-67⁺ CD3⁺ T cells/HPF, and representative IHC images (CD3, red; Ki-67, brown) are shown for recipient lung (C–F) and liver (G–J). Original magnification, ×100. Pooled data from 2 independent experiments, up to 6 mice per experimental arm. Log-rank test (A), ANOVA (C and G), Mann-Whitney (E and I). **P = 0.001–0.01 and ***P = 0.0001–0.001.

Figure 5. Human CD83-targeted CAR T cells significantly reduce CD83⁺ DCs.

NSG mice received 25 × 10⁶ human PBMCs plus 1 × 10⁶ CD83 CAR or mock-transduced T cells as described. Mice were humanely euthanized on day +21 and the spleens were harvested. (A) Representative contour plots show the frequency of human CD1c⁺ CD14^– DCs; CD1c⁺ CD83⁺ DCs; and CD1c⁺ MHC class II⁺ DCs in the mouse spleens at day +21. Graph shows the absolute number (mean ± SEM) of human (B) CD1c⁺ CD83⁺ DCs and (C) CD1c⁺ MHC class II⁺ DCs in the mouse spleens at day +21. Pooled data from 2 independent experiments, up to 6 mice per experimental arm. ANOVA (B and C). *P < 0.05; **P = 0.001–0.01.

Figure 6. Human CD83-targeted CAR T cells significantly reduce CD4⁺ and CD83⁺ T cells while increasing the Treg/activated Tconv ratio in vivo.

NSG mice received 25 × 10⁶ human PBMCs plus 1 × 10⁶ CD83 CAR or mock-transduced T cells as described. Mice were humanely euthanized on day +21 and the spleens were harvested. (A) Representative contour plots show the amount of EGFP⁺ CD83 CAR T cells in the inoculated mice at day +21, compared with mice that received mock-transduced T cells. (B) Representative contour plots show the frequency of human CD4⁺ T cells in the recipient spleens. Graphs show the absolute numbers (mean ± SEM) of (C) CD4⁺ and (D) CD4⁺ CD83⁺ T cells in the mouse spleens at day +21. (E) Contour plots depict the percentage of CD4⁺, CD127^–, CD25⁺, Foxp3⁺ Tregs in the mouse spleens at day +21. Graphs show the amount (mean ± SEM) of (F) Tregs and the (G) Treg/activated Tconv ratio at day +21 in the recipient mice. (H) Contour plots depict the frequency of CD4⁺ IFN-γ⁺ Th1 cells and CD4⁺ IL-4⁺ Th2 cells in the mouse spleens at day +21. Graphs demonstrate the absolute numbers (mean ± SEM) of (I) Th1 and (J) Th2 cells in the recipient spleens. Pooled data from 2 independent experiments, up to 6 mice per experimental arm. ANOVA (C, D, F, G, I, and J). *P < 0.05, **P = 0.001–0.01.

Figure 7. CD83 is a cellular target for human AML.

Histograms show CD83 expression among proliferating (A) K562 and (B) Thp-1 cells with MFI noted in the lower right-hand corner (FMO, unfilled). Human CD83 CAR or mock-transduced T cells were cocultured with fresh K562 or Thp-1 cells at an E/T ratio of 10:1. (C and D) Target cell killing was monitored using the xCELLigence RTCA system. A representative experiment for each is shown (triplicate mean ± SEM). NSG-SGM3 mice were injected with MOLM-13 EGFP/luciferase⁺ 1 × 10⁶ cells. Bioluminescence (BLI) was performed (IVIS Systems) on day +9 and then the mice were treated with 2.5 × 10⁶ CD83 CAR T or mock-transduced T cells. Mice were then imaged weekly. (E) Graph shows fold change (mean ± SEM) for average radiance (p/sec/cm²/Sr) at 1 week after injection of CD83 CAR or mock-transduced T cells. (F) Representative image shows weekly BLI intensities among each mouse per group over 4 weeks (n = 2 independent experiments, with 7–8 mice per experimental arm). (G) Graph shows the expression (mean ± SEM) of CD83 compared with CD33 or CD123 among freshly acquired human CD34⁺ AML blasts. (H) Representative histogram shows CD83 expression among human CD34⁺ AML blasts (FMO, unfilled) (n = 15 patient samples). ANOVA (C, D, E, and G). *P < 0.05, **P = 0.001–0.01, ***P = 0.0001–0.001, and ****P < 0.0001.