Transferred WT1-reactive CD8+ T cells can mediate antileukemic activity and persist in post-transplant patients

Abstract

Relapse remains a leading cause of death after allogeneic hematopoietic cell transplantation (HCT) for patients with high-risk leukemias. The potentially beneficial donor T cell-mediated graft-versus-leukemia (GVL) effect is often mitigated by concurrent graft-versus-host disease (GVHD). Providing T cells that can selectively target Wilms tumor antigen 1 (WT1), a transcription factor overexpressed in leukemias that contributes to the malignant phenotype, represents an opportunity to promote antileukemic activity without inducing GVHD. HLA-A*0201-restricted WT1-specific donor-derived CD8 cytotoxic T cell (CTL) clones were administered after HCT to 11 relapsed or high-risk leukemia patients without evidence of on-target toxicity. The last four treated patients received CTL clones generated with exposure to interleukin-21 (IL-21) to prolong in vivo CTL survival, because IL-21 can limit terminal differentiation of antigen-specific T cells generated in vitro. Transferred cells exhibited direct evidence of antileukemic activity in two patients: a transient response in one patient with advanced progressive disease and the induction of a prolonged remission in a patient with minimal residual disease (MRD). Additionally, three treated patients at high risk for relapse after HCT survive without leukemia relapse, GVHD, or additional antileukemic treatment. CTLs generated in the presence of IL-21, which were transferred in these latter three patients and the patient with MRD, all remained detectable long-term and maintained or acquired in vivo phenotypic and functional characteristics associated with long-lived memory CD8 T cells. This study supports expanding efforts to immunologically target WT1 and provides insights into the requirements necessary to establish potent persistent T cell responses.

Figure 1. Phenotypic and functional characteristics of WT1-specific CD8⁺T-cell clones isolated and expanded for infusions

(A and B) From the left: Lysis by WT1-specific CD8⁺T-cell clones of TAP-deficient HLA-A*0201⁺ B-LCL (T2 B-LCL) pulsed with decreasing concentrations of WT1 peptide in a ⁵¹Cr-release assay, and expression of CD27, CD28 and CD127 by WT1-specific CD8⁺T-cell clones (bold line) compared to isotype control (grey area) for clones generated without IL-21 (A) and with exposure to IL-21 (B). Inset values represent percentages of CD27⁺, CD28⁺ and CD127⁺CD8⁺T-cells respectively. Median fluorescent intensity (MFI) of staining for CD27 (C), CD28 (D), and CD127 (E). Mean effective concentrations of peptide required to achieve 50% lysis (EC₅₀₎ (F), and percent maximal lysis at an effector to target ratio (E:T) of 10:1 (G) of clones generated without (left) or with IL-21 (right). An unpaired two-tailed equal variance test was used for statistical analysis.

Figure 2. Kinetics of in vivo persistence of WT1-specific CTL clones and leukemia disease burden

Percent multimer⁺CD8⁺ T-cells (left y-axis) in PBMCs (solid circles) and BM (open circles), and percent leukemic blasts (right y-axis) in PBMCs (solid red diamonds) and BM (open red diamonds) collected 7 days (+/−2 days) before and at defined timepoints after infusions are shown. (A) The 5 Pts who received clones generated without IL-21 with detectable leukemia at the time of treatment, (B) The 2 Pts who received clones generated without IL-21 without detectable leukemia at the time of treatment, and (C) the 4 patients who received clones generated in the presence of IL-21, including 1 with MRD (Pt 27) and 3 without detectable leukemia at the time of treatment. Infusion schedule indicated by downward arrows: (a) 3.3 × 10⁸ WT1-specific CTL/m²; (b) 1 × 10⁹ CTL/m²; (c) 3.3 × 10⁹ CTL/m²; (d) 3.3 × 10⁹ CTL/m² followed by low dose s.c IL-2 × 14 days; (e) 1 × 10¹⁰ CTL/m²; (f) 1 × 10¹⁰ CTL/m² followed by low dose s.c IL-2 × 14 days.

Figure 3. Localization of adoptively transferred WT1-specific CTL to the BM

(A) Percent multimer⁺ CD8⁺ cells in blood (left) and BM (right) at all time points from the 11 patients in whom blood and BM were analyzed at the same time. (B) Same analysis as above performed at all time points on the 4 patients who received CTL clones that were generated with exposure to IL-21 and persisted long-term in vivo. Only samples in which at least one site showed detectable transferred cells are shown. Horizontal bars indicate medians. A two-tailed paired signed-rank test was used for statistical analysis.

Figure 4. Phenotypic characteristics of transferred WT1-specific CD8⁺ T-cells persisting in vivo

(A) Expression of CD27 (y-axis) and CD45RO (x-axis) (upper plots), CD28 (y-axis) and CD62L (x-axis) (middle plots), and CD127 (y-axis) and CCR7 (x-axis) (lower plots) on gated multimer⁺ cells for CD8⁺ CTL clones generated without IL-21 for Pt 15 (representative) immediately before infusion and after 6 days in vivo; and (B) CTL clones generated with exposure to IL-21 for Pts 21, 24, 27 and 28 immediately before infusions, and 280, 250, 160 and 84 days respectively in vivo after the first infusion.

Figure 5. Functional characteristics of persisting transferred WT1-specific CTL

(A) left-most plot: Percent cells producing IFNγ by the CTL clone generated without IL-21 for Pt 20 (representative) in response to WT1-pulsed T2 B-LCL at an E:T ratio of 10:1; next plot to the right: TNFα (y axis) and IL-2 (x-axis) production of IFNγ⁺ cells. Right-most plot: the same analysis performed on PBMC 1 day after transfer in vivo. (B) Plots to the left: Percent IFNγ production for CTL clones generated with exposure to IL-21 (Pts 21, 24, 27 and 28), and the respective TNFα and IL-2 production. Plots to the right: The same analysis performed on PBMC obtained after 160, 250, 160 and 84 days respectively in vivo. (C) Intranuclear Ki-67 expression on pre-infusion CTL clones harvested on day 14 of the ex vivo expansion cycle generated without (striped/grey column) or in the presence of IL-21 (solid column). Intranuclear Ki-67 expression on post-infusion CD8⁺multimer⁺ cells averaged from PBMCs of patients who received CTL clones generated without (open circles, dashed lines) or in the presence of IL-21 (solid circles, solid lines). Open squares and grey dotted lines represent average Ki-67 expression on patient endogenous CD8⁺multimer^- cells for all patients combined.

Figure 6. Evidence of anti-leukemic activity

(A) Percent WT1-specific multimer⁺ cells detected in PBMC (solid black circles, black line) of Pt 15 after infusion of 3.3 × 10⁹ cells/m² (left y-axis), concurrent percent leukemic blasts in the blood (red area) (inner right y-axis). Total white blood cells (solid grey line) and lymphocytes (dashed grey line) (outer right y-axis) in samples collected before and after the infusion (x-axis). (B) Percent multimer⁺CD8⁺ T-cells (y-axis) among PBMCs (solid circles) and in BM (open circles) collected before and after infusions for Pt 27. Arrows indicate time of infusions. Below the graph are characteristics of the patient’s primary B-ALL at diagnosis (left, below B-ALL), and at timepoints at which clonal B cells or the abnormal karyotype were analyzed and detected (+) in BM. (C) Mean effective concentrations of peptide required to achieve 50% lysis (EC₅₀₎ of pulsed T2 BLCL at an effector to target ratio (E:T) of 10:1 by the CTL clones infused for each patient.