T cell receptor gene therapy targeting WT1 prevents acute myeloid leukemia relapse post-transplant

Abstract

Relapse after allogeneic hematopoietic cell transplantation (HCT) is the leading cause of death in patients with acute myeloid leukemia (AML) entering HCT with poor-risk features^1-3. When HCT does produce prolonged relapse-free survival, it commonly reflects graft-versus-leukemia effects mediated by donor T cells reactive with antigens on leukemic cells⁴. As graft T cells have not been selected for leukemia specificity and frequently recognize proteins expressed by many normal host tissues, graft-versus-leukemia effects are often accompanied by morbidity and mortality from graft-versus-host disease⁵. Thus, AML relapse risk might be more effectively reduced with T cells expressing receptors (TCRs) that target selected AML antigens⁶. We therefore isolated a high-affinity Wilms' Tumor Antigen 1-specific TCR (TCR_C4) from HLA-A2⁺ normal donor repertoires, inserted TCR_C4 into Epstein-Bar virus-specific donor CD8⁺ T cells (T_TCR-C4) to minimize graft-versus-host disease risk and enhance transferred T cell survival^7,8, and infused these cells prophylactically post-HCT into 12 patients ( NCT01640301 ). Relapse-free survival was 100% at a median of 44 months following infusion, while a concurrent comparative group of 88 patients with similar risk AML had 54% relapse-free survival (P = 0.002). T_TCR-C4 maintained TCR_C4 expression, persisted long-term and were polyfunctional. This strategy appears promising for preventing AML recurrence in individuals at increased risk of post-HCT relapse.

Extended Data 1:

Flow diagram of patients enrolled on the clinical study.

Extended Data 2:

Treatment Plan.

Extended Data 3:

Characteristics of treated and comparative group patients.

Extended Data 4:

Normal count recovery after HCT and T_TCR-C4 infusions.

Extended Data 5:

Adverse events.

Extended Data 6:

Timing and grade of GVHD observed after T_TCR-C4 infusion.

Extended Data 7:

In vivo persistence kinetics and clonotype evolution of transferred post-HCT T_TCR-C4.

Extended Data 8:

T_EM Clonotypes composing infused T_TCR-C4 differentiate into T_CM and T_TD in vivo (Figure).

Extended Data 9:

Identification of PBMC sub-populations.

Extended Data 10:

Significantly expressed genes in T_TCR-C4.

Figure 1:. Phenotypic and functional characteristics of EBV-specific donor-derived T_TCR-C4.

(A) Mean effective concentrations of peptide required to achieve 50% lysis (EC₅₀₎ of WT1 peptide-pulsed TAP-deficient HLA-A2⁺ B-cell lymphoblastoid cells (T2 B-LCL) by Jurkat-Nur77 T cells transduced with TCRs isolated from infused clonal cell products (from our previous study) and T_TCR-C4. (B) Lysis of 3 independent HLA-A2⁺ (red shades) primary leukemias versus a HLA-A2⁻ (black) primary leukemia by donor T_TCR-C4 at decreasing effector to target (E:T) ratios. For each independent leukemia, the median (dots) and standard error (bars) of triplicate values (n=3) are shown. (C) Specific downregulation of the endogenous EBV-specific TCR after TCR_C4 transduction during the product generation process shown for a representative patient (Pt 10). These experiments were repeated >3 times with similar results. Binding of TCR_C4-transduced (top panels) and non-transduced EBV-specific cells (lower panels) to WT1 (y axis) and EBV (x axis) tetramers before (left panels) or immediately after day 7 sorting (second to left panels) and after two rounds of expansion with anti-CD3/CD28 stimulus (right-most panels). (D) Percent of WT1-tet⁺ T_TCR-C4 that also bound EBV tetramer, for infused products (n=12). (E) Percent of WT1 tet⁺ T_TCR-C4 within the infusion products (n=12) that produced IFNγ, TNFα or IL-2 in response to WT1_126–134 (red, n=12 individual values) and EBV BMLF1_280–288 (green, n=11 individual values) peptides. (F) Expression of CD27, CD28, CD127, CD62L, CCR7, PD-1 (n=12 individual values), TIM3 (n=11) and LAG3 (n=12) in infused T_TCR-C4 products (n=12), and (G) EBV-specific T cells in donor leukapheresis (n=8). For all figures: box and whisker plots include range (whiskers), interquartile range (box), median (horizontal line) and all individual values.

Figure 2:. Prophylactic effect, kinetics of in vivo persistence and clonotype evolution of T_TCR-C4 in vivo.

(A) Swimmer plot of the 12 patients who received T_TCR-C4 prophylactically after HCT (grey horizontal bars) - timing of marrow assessments (red lines) prior to and after T_TCR-C4-infusions, the first T cell infusion (black lines) and continuing follow-up in CR (black arrows) are shown. (B) Overall survival, relapse-free survival and (C) relapse probability for treated patients (n=12), from time of T_TCR-C4 infusion. (D) Overall survival (black line), relapse-free survival (grey line) and (E) relapse probability for comparative-group patients (n=88), from time of documented NED. For accuracy, RFS/OS and relapse probabilities are shown in years from T cell infusion for treated patients and years from NED for T_TCR-C4-treated and comparative-group patients respectively. (F) Percent tet⁺CD8⁺ T cells (left y-axis) in PBMCs collected after the first infusion for all patients (n=12), and (G) for selected patients (n=6) who received a second infusion within 365 days of their first. Pt 11’s second infusion is not depicted here. Black asterisks indicate T_TCR-C4 infusion and blue asterisks indicate the infusion was followed by low-dose s.c. IL-2 (250,000 IU/m² twice daily for 14 days). (H) Frequencies of individual clonotypes (y-axis) in infused cells (left-most bar-graph) and their frequencies in blood after infusions (bar-graphs to the right) for 8 select patients (Patients 10, 11, 12, 18, 20, 21, 24 and 25) whose tet⁺ frequency was detectable for >84 days. Dotted line indicates 20%.

Figure 3.. Phenotypic, functional characteristics and transcriptome of T_TCR-C4 in vivo.

(A) Expression of CD27, CD28, CD127, CD62L, CCR7 (top row) and PD1, TIM3 and LAG3 (bottom row) (y-axis) on gated tet⁺ cells from T_TCR-C4 products (n=12), immediately before infusion (n=12) and after 7 (n=12), ~30 (n=12), ~100 (n=10) and 200–300 (n=7) days in vivo. (B) Percent of WT1 tet⁺ T_TCR-C4 within the infusion products (n=12), and after ~30 (n=9) and ~100 (n=8) days in vivo in patients who had >1% detectable T_TCR-C4 tet⁺ cells, which produced IFNγ (top graph), TNFα (middle graph) and/or IL-2 (bottom graph) in response to 1μM WT1 peptide. Box and whisker plots include range (whiskers), interquartile range (box), median (horizontal line) and all individual values. Two-sided paired t-tests were used for statistical analysis. n.s.: not significant. (C) EBV (top graph) and CMV (bottom graph) viremia (IU/ml, y-axis) measured after each infusion for all patients (EBV) and in the subset in whom the patient and/or donor were CMV⁺ respectively (Supplementary Table 2). The number of values (n) obtained after infusions is indicated above the timepoint. Median and interquartile range are shown. Two-sided paired t-tests were used for statistical analysis. n.s.: not significant. (D) tSNE visualization of clustering of PBMC for selected Patient 10 (top) and Patient 18 (bottom) after 320 and 253 days in vivo, respectively. Cells clustered into populations, as indicated. Representative marker genes shown in Extended Data 9. (E) Location in clusters of CD8⁺ lymphocytes carrying the TCR_C4 transgene (blue) for each respective patient. Consistent with flow cytometry data (Figure 3A), most T_TCR-C4 clustered with endogenous CCR7^– populations and a small subpopulation clustered with CCR7⁺ populations (for patients 10 and 18, respectively, 1.44% and 2.8% in the scRNAseq CCR7⁺ cluster, compared to 2.86% and 3.2% by flow cytometry). Note: T cells and monocytes sometimes form doublets attached to the same gel-bead, which can result in some T_TCR-C4 clustering with monocytes.