Targeting the intracellular WT1 oncogene product with a therapeutic human antibody

Abstract

The Wilms tumor 1 (WT1) oncoprotein is an intracellular, oncogenic transcription factor that is overexpressed in a wide range of leukemias and solid cancers. RMFPNAPYL (RMF), a WT1-derived CD8+ T cell human leukocyte antigen (HLA)-A0201 epitope, is a validated target for T cell-based immunotherapy. Using phage display technology, we discovered a fully human "T cell receptor-like" monoclonal antibody (mAb), ESK1, specific for the WT1 RMF peptide/HLA-A0201 complex. ESK1 bound to several leukemia and solid tumor cell lines and primary leukemia cells, in a WT1- and HLA-A0201-restricted manner, with high avidity [dissociation constant (Kd)=0.1 nM]. ESK1 mediated antibody-dependent human effector cell cytotoxicity in vitro. Low doses of naked ESK1 antibody cleared established, disseminated, human acute lymphocytic leukemia and Philadelphia chromosome-positive leukemia in nonobese diabetic/severe combined immunodeficient γc-/- (NSG) mouse models. At therapeutic doses, no toxicity was seen in HLA-A0201 transgenic mice. ESK1 is a potential therapeutic agent for a wide range of cancers overexpressing the WT1 oncoprotein. This finding also provides preclinical validation for the strategy of developing therapeutic mAbs targeting intracellular oncogenic proteins.

Fig. 1

Binding of the phage clones and mAb to WT1 RMFp/HLA-A0201 complexes on live cells measured by flow cytometry. (a) Phage clone of ESK1 binds to T2 cells pulsed with RMF peptide (green), but not to T2 cells alone (pink), T2 cells pulsed with control EW peptide (light blue), or mutant heteroclitic peptide WT1-A1 YMFPNAPLY (orange), compared to secondary goat anti-human-FITC only (dark blue). (b–d) Binding of: isotype control human IgG1 (brown), full-length IgG1 mAbs ESK1 (orange), ESK3 (dark blue), ESK5 (green), ESK15 (light blue) and ESK23 (pink) at 1 ug/ml, followed by secondary PE-goat anti-hum an, on T2 cells pulsed with 50ug/ml RMF peptide (b), without peptide (c), or 50ug/ml irrelevant RHAMM-R3 peptide (d). (e–f) T2 cells pulsed with 50 ug/ml RMF peptide were stained with full-length ESK1 (e) or isotype control human IgG1 (d) at: 0.01 ug/ml (pink), 0.1 ug/ml (light blue), 1ug/ml (orange) or 10ug/ml (green), or secondary alone (red). (g–h) Binding of ESK1, ESK3 and ESK5 at 1ug/mL on T2 cells pulsed with 1.6–50 ug/mL RMF peptide (g), or irrelevant RHAMM-R3 peptide (h).

Fig. 2

Recognition of the naturally presented RMF/A2 complex on the cell surface by ESK1 in a HLA-A0201 and WT1 restricted manner. Binding of ESK1 to human mesothelioma cell lines JMN (a) and MSTO (b), to human leukemia cell lines BV173 (c). K562 (d), BA25 (e), and SET2 (f) with 10 ug/ml ESK1. Binding of ESK1 to AML blasts was measured on gated CD34+/CD33+ cells from HLA-A2 positive (g) or HLA-A2 negative (h) patients. Isotype human IgG1 was used as negative control. HLA and WT1 phenotype is shown for each cell type. Indirect flow cytometry was used in a–e and direct flow cytometry in f–h.

Fig. 3

Radioimmunoassay was used to measure number of antibody bound per cell. (a) ESK1 was labeled with I-125 using chloramine-T to a specific activity of 4.3 mCi per mg and used for Scatchard analysis. (b) ESK1 binding to mesothelioma and leukemia cell lines, with HLA-A2 and WT1 phenotype indicated. Because we cannot determine whether the bivalent mAb is binding to 1 or 2 epitopes on the surface, total epitopes per cell could be as high as twice the number of mAb binding sites.

Fig. 4

ESK1 mediates ADCC with human PBMC effectors at indicated concentrations. (a) T2 cells alone, or pulsed with RMF or irrelevant RHAMM-R3 peptides at 50 ug/ml, incubated with human PBMC effectors at E:T of 35:1 with cytotoxicity measured by LDH assay. (b) Similarly, JMN and MSTO cells at E:T of 30:1. (c) ADCC against BV173 was measured by 4hr-⁵¹Cr-release assay at E:T of 50:1, and HL-60 cells by LDH assay at E:T of 12.5:1. (d) ADCC against SKOV and CC228 cells was measured by 4hr-⁵¹Cr-release assay at E:T of 60:1. (e) ADCC against primary AML blasts from an HLA-A2 positive patient and an HLA-A2 negative patient, measured by LDH assay at E:T of 15:1. The data are representative of 1–3 experiments each. Killing was consistently observed at 1ug/ml ESK1 with multiple donors. Each data point was the average of triplicates.

Fig 5

ESK1 effectively treats human BV173 leukemia in NSG mice. Tumor burden was calculated by summing the luminescent signal of each mouse in four positions, and average signal for each group (n=5) is plotted. Where noted, human effectors were given intravenously on day 6, and antibody (ESK1 or human IgG1 isotype control) was administered intravenously on days 6 and 10. (a). 100μg ESK1 alone or in combination with human effectors significantly reduces tumor burden at early times. (b). After one month, mice treated with ESK1 alone begin to relapse, while ESK1 with human effectors had prolonged durability. 1 of 5 mice was alive without leukemia signal at 70 days. (c) ESK1 significantly reduced tumor burden in a dose-dependent manner. One mouse died of an anesthesia accident in the 100ug group. (d) Survival of mice with disseminated BV173 leukemia treated with three different doses (25, 50, or 100 ug twice) of ESK1 alone on days 6 and 10 after leukemia engrafting. All treatment showed significantly prolonged survival as compared to untreated control animals or animals treated with isotype control hIgG (p< 0.01 by Log rank Mantel Cox test).

Fig. 6. ESK1 specifically treats a second A2+ leukemia, but not A2− or WT1-tumors

(a–b) Three million BA25 human acute lymphocytic leukemia cells (luciferase positive) were injected IV into NSG mice. On day 3, tumor engraftment was confirmed by firefly luciferase imaging in all mice that were to be treated; mice were then randomly divided into different treatment groups. On day 4 and day 8, 100ug mAb ESK1 or the isotype control mAb were injected IV. Tumor growth was assessed by luminescence imaging once to twice a week, and clinical measurements were assessed daily. Significant reductions in the growth of the ALL cells by the ESK1 mAb was seen relative to animals given the isotype control (a) by quantitation of luminescence from the supine animals (p < 0.04 and p < 0.02 on days 13 and 17, respectively.) These data are represented in a day 17 image (b). Treatment with 100ug mAb on days 6 and 10 after injection of 5 million Daudi ALL3. Leukemia cells (c) or 2 million T2 leukemia cells was followed and quantitated by bioluminescent imaging.