Inducible caspase-9 suicide gene controls adverse effects from alloreplete T cells after haploidentical stem cell transplantation

Abstract

To test the feasibility of a single T-cell manipulation to eliminate alloreactivity while sparing antiviral and antitumor T cells, we infused 12 haploidentical hematopoietic stem cell transplant patients with increasing numbers of alloreplete haploidentical T cells expressing the inducible caspase 9 suicide gene (iC9-T cells). We determined whether the iC9-T cells produced immune reconstitution and if any resultant graft-versus-host disease (GVHD) could be controlled by administration of a chemical inducer of dimerization (CID; AP1903/Rimiducid). All patients receiving >10(4) alloreplete iC9-T lymphocytes per kilogram achieved rapid reconstitution of immune responses toward 5 major pathogenic viruses and concomitant control of active infections. Four patients received a single AP1903 dose. CID infusion eliminated 85% to 95% of circulating CD3(+)CD19(+) T cells within 30 minutes, with no recurrence of GVHD within 90 days. In one patient, symptoms and signs of GVHD-associated cytokine release syndrome (CRS-hyperpyrexia, high levels of proinflammatory cytokines, and rash) resolved within 2 hours of AP1903 infusion. One patient with varicella zoster virus meningitis and acute GVHD had iC9-T cells present in the cerebrospinal fluid, which were reduced by >90% after CID. Notably, virus-specific T cells recovered even after AP1903 administration and continued to protect against infection. Hence, alloreplete iC9-T cells can reconstitute immunity posttransplant and administration of CID can eliminate them from both peripheral blood and the central nervous system (CNS), leading to rapid resolution of GVHD and CRS. The approach may therefore be useful for the rapid and effective treatment of toxicities associated with infusion of engineered T lymphocytes. This trial was registered at www.clinicaltrials.gov as #NCT01494103.

Figure 1

The kinetics of T-cell subsets after iC9-T-cell infusion in patients not treated with AP1903. Counts of circulating CD3⁺ (A), CD4⁺ T cells (B) and CD8⁺ T cells (C) in 8 patients who did not receive AP1903. Black line with filled circle represents CD19⁺ T cells, and gray dashed line with diamond represents CD19⁻ T cells. The number of evaluable patients at each point is 8 (from 0 to month 1), 6 (months 2-4), 4 (months 5 and 6), 2 (month 7), and 1 (month 8, 9, and 12). Data show means ± standard error of mean of patients infused with iC9-T cells.

Figure 2

iC9-T-cell engraftment and in vivo allodepletion by dimerizer drug. Counts of T-cell subsets in 4 patients who received AP1903. CD3⁺CD19⁺ T cells (A, Pt. 6; B, Pt. 8; C, Pt. 9; D, Pt. 12) and CD3⁺CD19⁻ T cells (E, Pt. 6; F, Pt. 8; G, Pt. 9; H, Pt. 12). ●, □, and ▲ represent the CD3⁺, CD4⁺, and CD8⁺ subtypes, respectively. (I) The copy number of the iC9 transgene per microgram of DNA extracted from PBMC, evaluated by Q-PCR. Arrow indicates the time at which the patient was treated with AP1903. Pt., patient.

Figure 3

Antiviral immune reconstitution after iC9–T-cell infusion. Quantification of pathogen-specific T cells detected by IFN-γ ELISPOT at multiple time points for each patient who did not receive AP1903. Pts. 5 (A) and 7 (B) had EBV reactivations, Pt. 12 had CMV reactivation (C), and Pt. 11 had HHV6 reactivation (D) before iC9–T-cell infusion. Pt. 7 had HHV6 infection (E), and Pt. 11 had VZV infection (F) after iC9–T-cell infusion. Black histograms represent response from total T cells and striped histograms represent response from endogenous T cells. The value of their difference represents the response from infused iC9-T cells. The gray dashed line indicates the viral load at multiple time points.

Figure 4

Virus-specific T cells are retained and remain functional after administration of AP1903. Quantification of pathogen-specific T cells detected by IFN-γ ELISPOT in each patient who received AP1903 to control acute GVHD. Patients had viral infection and/or reactivation for: VZV (A, Pt. 6), EBV (B, Pt. 8 and C, Pt. 9), CMV (D, Pt. 8 and E, Pt. 9), BKV (F, Pt. 9). Black histograms represent response from total T cells and striped histograms represent response from endogenous T cells. The value of their difference represents the response from infused iC9-T cells. The gray dashed line indicates the viral load at multiple time points.

Figure 5

Administration of AP1903 rapidly resolves GVHD symptoms and reduces cytokine release. In Pt. 8, (A) the highest body temperature to the time of AP1903 infusion. (B) Pictures of the skin rash were taken prior to and 25 minutes after beginning the AP1903 infusion. (C) Cytokine production in plasma measured from samples collected 4 hours prior to, 2.5 hours after beginning, and 48 hours after the infusion of AP1903.

Figure 6

Administration of AP1903 affects iC9-T cells in CSF. Detection of iC9-T cells in CSF and PB by flow cytometry before and after administration of AP1903 (Pt. 6). Nine days before CID treatment in CSF (A) and in PB (B), and 14 days after treatment in CSF (C) and in PB (D). The percentage of CD3⁺CD19⁺ T cells and CD3⁺CD19⁻ T cells was calculated under the gate of CD3⁺ T cells.