The Wilms' tumor oncogene protein (WT1) is an attractive target for immunotherapy for a wide range of leukemias and cancers. WT1 is an intracellular transcription factor that is widely expressed in acute and chronic leukemias, but not appreciably in normal adult tissues. WT1 has been implicated in oncogenesis and appears to be expressed in the CML stem cell. Thus far, the intracellular location of WT1 has limited its use as a cancer target to T cell-based vaccine therapies. Several such vaccines directed to WT1 peptides are in clinical trials worldwide. However, such approaches is likely to be weaker and slower acting than typical antibody therapies that act directly to kill cells, especially for a leukemia that is not in remission. The WT1 peptide (RMFPNAPYL) is presented by HLA-A0201 and induces cytotoxic CD8 T cells capable of killing WT1+ leukemia cells. Therefore, we hypothesized that a monoclonal antibody (mAb) specific for WT1 peptide/HLA-A2 complexes on the cell surface (a “TCR-like antibody”) could be an effective direct therapeutic agent alone, or armed with potent anti-cancer agents. In addition, such an antibody would provide a potential tool for identifying appropriate patients for clinical trials of both WT1 vaccine and WT1 mAb therapies, or as a prognostic tool. Using phage-display technology, we selected a number of human scFv phage mAbs specific for WT1 peptide RMFPNAPYL, when bound in the context of HLA-A0201 molecules. Bivalent, full-length human IgG1 mAbs were next constructed and tested for biochemical and immunological functions. The scFv phage mAbs bound specifically to T2 cells (TAP deficient) pulsed with WT1 peptide RMFPNAPYL, but not other HLA-A0201-binding peptides, demonstrating specific recognition of the WT1peptide/HLA-A0201 complex. Flow cytometry and radio-immunoassay was used to quantify sites, determine avidity, and show specificity. The IgG1 mAb showed high binding affinity (Kd= 0.2 nM) for the WT1 peptide/A0201 complex on live cells. The IgG1 mAb also recognized leukemia and tumor cell lines (n= 8 of 12) that expressed WT1, in an HLA-A0201, WT1 peptide-restricted manner, but not WT1-negative or HLA-A02-negative cells (n=13). The mAb was capable of killing WT1 peptide-pulsed T2 cells, as well as un-pulsed WT1+, A0201 + cancer cell lines, demonstrating presentation of the epitope in sufficient numbers for therapeutic attack. Complement mediated killing of cells was not seen. Alanine scanning of the epitope pointed to peptide position #1 as critical. In conclusion, cytotoxic human monoclonal antibodies targeting WT1 peptide/HLA complexes represent a novel therapeutic approach to target leukemias and solid tumors that over-express the intracellular WT1 oncoprotein.
Yan:eureka therapeutics: Employment. Zhou:eureka therapeutics: Employment. Liu:eureka therapeutics: Employment.
Asterisk with author names denotes non-ASH members.