FMS-like tyrosine kinase 3 (FLT3) receptor, a member of the class III receptor tyrosine kinase family, is highly expressed in an array of hematological malignancies including ∼90% of acute myelogenous leukemia. Ligand stimulation of the receptor promotes the survival and proliferation of leukemia cells. Strategies targeting FLT3 may therefore constitute effective therapeutic approaches for these leukemia. Towards this, we selected a naïve antibody phage display library on both recombinant FLT3 receptor protein and FLT3-expressing leukemia cells using a tailored selection scheme that was designed to isolate antagonistic phage antibodies that not only interfere with receptor/ligand binding but also trigger internalization upon cell surface binding. Phage antibodies were screened first for their ability to bind to cell surface receptor and induce internalization, followed by their activity in blocking ligand-receptor interaction and neutralizing ligand-stimulated receptor activation and cell proliferation. We identified three fully human antibodies, EB10, A2IN, and D4-3, that bound with high affinity (2.7 to 7.3 x 10-10 M) to both soluble and cell surface-expressed FLT3. All three antibodies were shown to be internalized in a time dependent fashion, with a maximum amount of internalization, between ∼45 to 70%, reached at about 1 to 4 h of incubation at 37°C. EB10 and D4-3 blocked ligand binding to the receptor with IC50’s of 14 and 7 nM, respectively. Further, EB10 and D4-3 inhibited FLT3 ligand-induced receptor phosphorylation and cell proliferation in EOL-1 leukemia cells. Taken together, our results suggest that EB10 and D4-3 may represent excellent therapeutic candidates for the treatment of FLT3-expressing human leukemia, either as unmodified, naked antibody or as a conjugate for intracellular delivery of a toxic payload.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]