Phase 2 clinical trial of rapamycin-resistant donor CD4+ Th2/Th1 (T-Rapa) cells after low-intensity allogeneic hematopoietic cell transplantation

Abstract

In experimental models, ex vivo induced T-cell rapamycin resistance occurred independent of T helper 1 (Th1)/T helper 2 (Th2) differentiation and yielded allogeneic CD4(+) T cells of increased in vivo efficacy that facilitated engraftment and permitted graft-versus-tumor effects while minimizing graft-versus-host disease (GVHD). To translate these findings, we performed a phase 2 multicenter clinical trial of rapamycin-resistant donor CD4(+) Th2/Th1 (T-Rapa) cells after allogeneic-matched sibling donor hematopoietic cell transplantation (HCT) for therapy of refractory hematologic malignancy. T-Rapa cell products, which expressed a balanced Th2/Th1 phenotype, were administered as a preemptive donor lymphocyte infusion at day 14 post-HCT. After T-Rapa cell infusion, mixed donor/host chimerism rapidly converted, and there was preferential immune reconstitution with donor CD4(+) Th2 and Th1 cells relative to regulatory T cells and CD8(+) T cells. The cumulative incidence probability of acute GVHD was 20% and 40% at days 100 and 180 post-HCT, respectively. There was no transplant-related mortality. Eighteen of 40 patients (45%) remain in sustained complete remission (range of follow-up: 42-84 months). These results demonstrate the safety of this low-intensity transplant approach and the feasibility of subsequent randomized studies to compare T-Rapa cell-based therapy with standard transplantation regimens.

Trial registration: ClinicalTrials.gov NCT00077480.

Figures

Figure 1

Phase 2 clinical trial design of ex vivo and in vivo Sirolimus for low-intensity allogeneic HCT. Donors underwent steady-state apheresis #1, whereby CD4+ T cells were purified by positive selection; costimulated with anti-CD3 and anti-CD28; and exposed to IL-4, IL-2, and rapamycin in culture for 12 days. The resultant “T-Rapa” cell product was cryopreserved and administered as a preemptive DLI at day 14 post-HCT. Donors underwent apheresis #2 after granulocyte–colony-stimulating factor (G-CSF) mobilization; the peripheral blood stem cell (PBSC) product was unmanipulated (T-cell replete). The recipient was treated for 4 consecutive days (days −6 through −3) with concomitant fludarabine (Flu, 30 mg/m2 per day) and cyclophosphamide (Cy, 300 mg/m2 per day). GVHD prophylaxis consisted of short-course Sirolimus (from day −2 until day +14 post-HCT) and cyclosporine A (from day −1 until day +100 post-HCT).

Figure 2

T-Rapa cell products have relatively equal numbers of genes that are up- or downregulated in expression relative to culture input CD4+ T cells. T-Rapa cell products were manufactured ex vivo (n = 6) and compared with the purified CD4+ T cells used to initiate the cultures (n = 6). RNA was purified from each paired sample, with further analysis by gene expression microarray. The heat map illustrates that genes were consistently differentially expressed, with 18.1% of genes (6147 of 34 051) being differentially expressed in T-Rapa products relative to input CD4 cells (P < .001); the number of upregulated genes in T-Rapa cells was relatively equal to the number of downregulated genes.

Figure 3

T-Rapa cells express a balanced Th2/Th1 cytokine phenotype. (A) T-Rapa cell clinical products were analyzed by intracellular flow cytometry for expression of GATA-3 (Th2 cell transcription factor), T-bet (Th1 cell transcription factor), and FoxP3 (TREG cell transcription factor) (left). The intraproduct ratio of GATA-3:T-bet in T-Rapa cells was >1:1 (*P < .05). (B) T-Rapa cell clinical products were evaluated at the time of DLI (day 12 of culture) and after an additional culture interval intended to evaluate effector function and differentiation plasticity (day 18 of culture). At each time point, the T-Rapa cells were costimulated, and the resultant supernatants were tested for cytokine content by Luminex (mean ± standard error of the mean; n = 40; *, increase from day 12 to day 18, P < .05). Values are expressed as pg/mL (1 × 106 cells per mL per 24 hours).

Figure 4

T-Rapa cell infusion results in predominate donor CD4+ T-cell reconstitution. (A) Percent donor T lymphoid chimerism for each patient at days 14, 28, and 100 after allogeneic hematopoietic stem cell transplant (SCT) (top; ***, day 28 > day 14 and day 100 > day 28; P < .0001). Post-SCT numbers of donor vs host CD4+ or CD8+ T cells were estimated by multiplying CD4 and CD8 cell absolute numbers by percent CD3 chimerism values. The figure shows median estimated values for absolute numbers of donor and host CD4+ T cells (left) and CD8+ T cells (right) at days 14, 28, and 100 post-SCT (comparisons are day 28 vs day 14 and day 100 vs day 28; ***P < .001; **P < .01; *P < .05; between n = 23 and n = 33 evaluated for each paired analysis). (B) Percent donor myeloid chimerism for each patient at days 14, 28, and 100 post-SCT (***, day 28 > day 14 and day 100 > day 28; P < .0001). NS, not significant.

Figure 5

T-Rapa cell recipients have immune reconstitution of a mixed Th2 and Th1 cytokine phenotype. (A) Percentage of CD4+ T cells expressing GATA-3, T-bet, and FoxP3 as measured by intracellular flow cytometry at day +14 posttransplant (just before T-Rapa cell DLI) and at 1 month and 3 months post-HCT (comparisons are 1 month vs day 14 and 3 months vs day 14; **P < .001; *P < .05). Intrapatient ratio of GATA-3 to T-bet expressing CD4+ T cells is shown for each time point. (B) At 1, 2, 4, and 8 weeks post-HCT, peripheral blood mononuclear cells were costimulated, and the 24-hour supernatant was tested for cytokine content by Luminex (mean ± standard error of the mean; between n = 32 and n = 34 evaluated for each paired analysis; comparisons are week 4 vs week 2 and week 8 vs week 2; **P < .001; *P < .01).