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

Blood. 2021 Dec 23;138(25):2655-2669. doi: 10.1182/blood.2020010477.


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).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Bispecific / pharmacology
  • Antibodies, Bispecific / therapeutic use*
  • Antineoplastic Agents, Immunological / pharmacology
  • Antineoplastic Agents, Immunological / therapeutic use*
  • Cell Line, Tumor
  • HLA-A2 Antigen / immunology
  • Humans
  • Leukemia, Myeloid, Acute / drug therapy*
  • Leukemia, Myeloid, Acute / immunology
  • Mice
  • Peptides / pharmacology
  • Peptides / therapeutic use*
  • Receptors, Antigen, T-Cell / immunology
  • T-Lymphocytes, Cytotoxic / drug effects
  • T-Lymphocytes, Cytotoxic / immunology
  • Tumor Cells, Cultured
  • WT1 Proteins / immunology*


  • Antibodies, Bispecific
  • Antineoplastic Agents, Immunological
  • HLA-A*02 antigen
  • HLA-A2 Antigen
  • Peptides
  • Receptors, Antigen, T-Cell
  • WT1 Proteins
  • WT1 protein, human

Associated data