| The Wilms' tumor 1 (WT1) oncoprotein is an intracellular, oncogenic transcription factor that is over-expressed in a wide range of leukemias and solid cancers. RMFPNAPYL (RMF), a WT1-derived CD8+ T cell HLA-A*02:01 epitope, is a validated target for T cell--based immunotherapy. Using phage display technology, we discovered a fully human "T cell receptor (TCR) mimic" (TCRm) monoclonal antibody (mAb), ESK1, specific for the WT1 RMF peptide/HLA-A* 02:01 (RMF/A2) complex. ESKI bound to several leukemia and solid tumor cell lines, and primary leukemia cells, in a WT1- and HLA-A* 02:01--restricted manner, with high avidity (Kd = 0.1 nM). Low doses of naked ESK1 antibody cleared established, disseminated, human acute lymphocytic leukemia and Ph+ leukemia in NOD/SCID Fcgammac-/- (NSG) mouse models. At therapeutic doses, no toxicity was seen in HLA-A*02:01 transgenic mice. ESK1 mediated antibody-dependent human effector cell cytotoxicity (ADCC) in vitro, and no other mechanisms were observed in vitro or with isolated murine effectors ex vivo.;The RMF/A2 epitope levels are far lower than other therapeutic mAb targets, so enhanced ADCC activity could be more valuable clinically. ESKM, a construct with altered Fc glycosylation, demonstrated increased ADCC activity in vitro and greater potency and efficacy in mice. ESKM demonstrated similar pharmacokinetic properties and biodistribution pattern to ESK1, supporting its clinical utility. Despite increased potency against WT1+ HLA-A* 02:01+ cancer targets, ESKM was not toxic to human HLA-A* 02:0.1 transgenic mice.;Finally, we identified surface HLA-A2 level, intracellular WT1, and immunoproteasome activation as major processes driving presentation of the RMF peptide in the context of HLA-A*02:01. IFNgamma treatment, which increases total HLA-A2 expression and induces immunoproteasome subunits, increased ESK mAb binding to a panel of cancer cells lines. Despite doubling ESK binding, we could not demonstrate an improvement in ADCC activity in vitro after IFNgamma treatment. Conversely, hsp90 inhibition proportionally decreased surface HLA-A2 and ESK binding, and diminished ADCC activity in vitro. This is a proof of concept that binding of a TCRm mAb can be modulated, in some cases affecting mAb activity, and warrants further investigation into clinically relevant drugs that modulate peptide/HLA epitope level. |
