Targeting intracellular WT1 in AML with a novel RMF-peptide-MHC-specific T-cell bispecific antibody

Abstract

Antibody-based immunotherapy is a promising strategy for targeting chemoresistant leukemic cells. However, classical antibody-based approaches are restricted to targeting lineage-specific cell surface antigens. By targeting intracellular antigens, a large number of other leukemia-associated targets would become accessible. In this study, we evaluated a novel T-cell bispecific (TCB) antibody, generated by using CrossMAb and knob-into-holes technology, containing a bivalent T-cell receptor-like binding domain that recognizes the RMFPNAPYL peptide derived from the intracellular tumor antigen Wilms tumor protein (WT1) in the context of HLA-A*02. Binding to CD3ε recruits T cells irrespective of their T-cell receptor specificity. WT1-TCB elicited antibody-mediated T-cell cytotoxicity against AML cell lines in a WT1- and HLA-restricted manner. Specific lysis of primary acute myeloid leukemia (AML) cells was mediated in ex vivo long-term cocultures by using allogeneic (mean ± standard error of the mean [SEM] specific lysis, 67 ± 6% after 13-14 days; n = 18) or autologous, patient-derived T cells (mean ± SEM specific lysis, 54 ± 12% after 11-14 days; n = 8). WT1-TCB-treated T cells exhibited higher cytotoxicity against primary AML cells than an HLA-A*02 RMF-specific T-cell clone. Combining WT1-TCB with the immunomodulatory drug lenalidomide further enhanced antibody-mediated T-cell cytotoxicity against primary AML cells (mean ± SEM specific lysis on days 3-4, 45.4 ± 9.0% vs 70.8 ± 8.3%; P = .015; n = 9-10). In vivo, WT1-TCB-treated humanized mice bearing SKM-1 tumors exhibited a significant and dose-dependent reduction in tumor growth. In summary, we show that WT1-TCB facilitates potent in vitro, ex vivo, and in vivo killing of AML cell lines and primary AML cells; these results led to the initiation of a phase 1 trial in patients with relapsed/refractory AML (#NCT04580121).

© 2021 by The American Society of Hematology.

Figures

Graphical abstract

Figure 1.

Analysis of WT1 expression in primary AML cells and tumor cell lines by real-time qPCR. (A) WT1 expression in primary AML cells at different time points during the course of the disease (primary diagnosis, n = 65; posttherapy, n = 26; relapsed/refractory, n = 21). (B) WT1 expression in tumor cell lines (n = 37). (C) Correlation of blast count and WT1 expression in bone marrow (BM) samples of patients with AML at primary diagnosis (n = 38) and posttherapy (n = 24). (D) Correlation of WT1 expression and NPM1 (wild-type [wt], n = 37; mutant [mut], n = 25), FLT3-ITD (wt, n = 45; mut, n = 17), and CEBPA (wt, n = 34; mut, n = 6) mutational status. Bars represent mean ± SEM. Statistical analysis: Mann-Whitney U test. **P < .01, ****P < .0001.

Figure 2.

Structure of WT1-TCB, mode of action, binding motif, and crystallography. (A) Scheme of WT1-TCB–mediated T-cell engagement. (B) Binding motif as determined by alanine scanning and pMHC array. Anchor residues Met2 and Leu9 (marked in blue) can be replaced by few amino acids, whereas Arg1, Phe3, Asn5, and Ala6 (red) are important for recognition of the RMF–pMHC complex by 11D06. Residues Pro4, Pro7, and Tyr8 (black) are less critical for binding. (C) Single-substitution plot for RMF peptide in the context of HLA-A*02–β2m heterodimer bound by 11D06 antibody. Each bar corresponds to substitution with 1 of 20 amino acids, and a deletion is shown by the last bar. The height of each bar indicates the signal intensity. Bars are ordered as follows: the 8 nonpolar residues (AFILMVWP; orange); the 7 polar/neutral residues (GSYCQTN; green); the 3 positively charged residues (RKH; blue); and the 2 negatively charged residues (DE; pink). (D) Crystal structures of the 11D06–RMF–pMHC (pdb entry 7BBG) and ESK1–RMF-pMHC (pdb entry 4WUU) complexes. (E) View of the peptide-binding site of pMHC in the Fab 11D06–HLA-A*02 RMF–pMHC complex. Heavy and light chains of Fab 11D06 are colored in blue and cyan, respectively, and the RMF peptide is colored in magenta. The HLA-A*02 pMHCI is shown as a transparent surface in white, with selected side chains highlighted in green. (F) Quantification of endogenous RMF peptide purified from a primary AML patient sample. Mass spectrometry chromatogram overlay of dioxidized endogenous RMF peptide precursor ion [m/z = 570.7790 (2+); red] and dioxidized internal standard 13C6, 15N4-labeled peptide precursor ion [m/z = 575.7831 (2+); blue]. Endogenous RMF peptide was immunoprecipitated by using WT1-TCB. Figure 2A created with BioRender.com.

Figure 3.

Dose–response relationship after treatment with WT1-TCB in cocultures of tumor cell lines with HD T cells. (A) Transporter associated with antigen processing–deficient HLA-A*02+ T2 cells, unpulsed (black) vs RMF peptide pulsed (red) after treatment with Ctrl-TCB (left) or WT1-TCB* (second from left). Second from right: control with HLA-A02− HL-60 cells and Ctrl-TCB (black) or WT1-TCB* (red). Right: T2 cells pulsed with increasing doses of RMF peptide and treatment with 10−8 M Ctrl-TCB (black) or WT1-TCB* (red). (B) Killing assay performed on AML cell lines (OCI-AML3 and SKM-1) and treated with varying concentrations of WT1-TCB (red) or Ctrl-TCB (black). Tumor cell killing was evaluated after 44 hours (SKM-1) or 68 hours (OCI-AML3); n = 10. CD25 expression (C) and proliferation of CD4+ and CD8+ T cells (D) after 3 days in killing assays using AML cell lines (OCI-AML3 and SKM-1) at varying concentrations of WT1-TCB (red) or Ctrl-TCB (black); n = 6. gMFI, geometric mean fluorescence intensity.

Figure 4.

WT1-TCB–mediated T-cell cytotoxicity and T-cell activation. (A) Induction of downstream TCR signaling in NFAT Jurkat Reporter cells upon recognition of RMF peptide–MHC complexes on primary AML cells. Statistical analysis, Mann-Whitney U test (n = 10). (B) Dose-dependent specific lysis of primary HLA-A*02+ AML cells by allogeneic (black solid line; n = 9) or autologous (black dashed line; n = 3) PBMCs after 48 hours in short-term cytotoxicity assays with WT1-TCB. Controls: HLA-A*02+ WT1– CD33+ HD cells (gray solid line; n = 6) and primary HLA-A*02− AML cells (gray dashed line; n = 10). CD25 (C) and CD69 (D) expression on CD3+ T cells after 48 hours in killing assays with allogeneic PBMCs and primary HLA-A*02+ AML cells (black solid line; n = 9), primary HLA-A*02− AML cells (gray dashed line; n = 10), or HLA-A*02+ WT1− CD33+ HD cells (gray solid line; n = 6). (E) T-cell proliferation after 96 hours in killing assays with allogeneic PBMCs and primary HLA-A*02+ AML cells (black solid line; n = 8), primary HLA-A*02− AML cells (gray dashed line; n = 7), or HLA-A*02+ WT1− CD33+ HD cells (gray solid line; n = 3). (F) Secretion of interferon-γ (IFN-γ) and granzyme B in allogeneic and autologous settings after 72 hours in a short-term cytotoxicity assay with primary HLA-A*02 AML cells. (G) Specific lysis of primary AML cells by allogeneic T cells (left: HLA-A*02− AML, n = 11; center: HLA-A*02+ AML, n = 19) or autologous T cells (right: HLA-A*02+ AML, n = 8) after 10 to 13 days in long-term cocultures and treatment with WT1-TCB*. (H) Representative dot plots from flow cytometry analysis showing CD2 and CD33 expression in long-term coculture samples. (I) Upregulation of activation and surrogate exhaustion markers on CD3+ T cells on days 3 to 4 in long-term cocultures with primary HLA-A*02+ and HLA-A*02− AML cells (n = 14-22). Bars represent the mean ± SEM. Statistical analysis: Kruskal-Wallis test and subsequent Dunn’s test. **P < .01, ****P < .0001. MFI, mean fluorescence intensity; n.s., not significant; RLU, relative luminescence units.

Figure 5.

WT1-TCB* vs WT1-specific T cells. (A) Specific lysis of unpulsed and RMF peptide–pulsed primary AML cells from HLA-A*02− and HLA-A*02+ patients by WT1RMF-specific T-cell clone after 3 days in feeder layer–based long-term cocultures. Specific lysis was calculated by subtraction of unspecific lysis by HD T cells under the same conditions (n = 6-9). (B) Specific lysis of primary AML cells after 3 days in feeder layer–based long-term cocultures with WT1RMF-specific T-cell clone and treatment with WT1-TCB* compared with HD T cells (n = 9). (C) Specific lysis of primary AML cells (all NPM1 wild-type) after 3 days in feeder layer–based long-term cocultures with WT1RMF-specific T-cell clone and treatment with WT1-TCB*. Specific lysis was calculated by subtraction of unspecific lysis by a T-cell clone targeting an unrelated epitope of mutated NPM1 (n = 6 ). (D) Representative dot plots from flow cytometry analysis showing CD2 and CD33 expression in lysis experiments. Bars represent mean ± SEM. Statistical analysis: Mann-Whitney U test. *P < .05, **P < .01. n.s, not significant.

Figure 6.

WT1-TCB*–mediated cytotoxicity in combination with lenalidomide (Lena). (A) WT1-TCB–mediated specific lysis of primary AML cells significantly increased in combination with Lena after 4 days of coculture with HD T cells (n = 9-10). Lena was added at a concentration of 10 μM together with 10 nM WT1-TCB* in ex vivo cytotoxicity assays with primary AML cells and HD T cells at an E:T ratio of 1:2 for 4 days. (B) Levels of proinflammatory cytokines increase after combination of WT1-TCB* with Lena and levels of the anti-inflammatory interleukin 10 (IL-10) decrease (n = 9). (C) Representative example of CD45RA and CCR7 expression analysis. (D) Percentages of central memory T cells after 7 to 10 days of treatment (n = 8). Bars represent the mean ± SEM. Statistical analysis: Wilcoxon matched-pairs signed-rank test. *P < .05, **P < .01. n.s., not significant; TNF-α, tumor necrosis factor α.

Figure 7.

Antitumor efficacy of WT1-TCB in humanized mouse models. (A-B) Tumor growth curves of the SKM-1 model in hSC-NSG mice treated once weekly with vehicle control or 0.05, 0.1, or 1 mg/kg of WT1-TCB. (C) Cytometric analysis of the CD34+CD133+ stem cell population in the bone marrow of SKM-1 tumor-bearing hSC-NSG mice engrafted with cord blood from HLA-A*02+ donors after 54 days of treatment with WT1-TCB*. (D-E) Tumor growth in AML PDX mice (AML573) treated once weekly with 3 mg/kg WT1-TCB or Ctrl-TCB beginning on day 30, assessed by in vivo imaging. One WT1-TCB–treated mouse died during imaging on day 32.