Elucidation of the antagonistic mechanisms of various anti-FLT3 antibodies via binding epitope dissection

AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA 4110 FMS-like tyrosine kinase 3 (FLT3), a receptor consisting of 5 immunoglobulin (Ig)-like extracellular domains, a trans-membrane domain and an intracellular kinase domain, is highly expressed in an array of hematological malignancies including ~90% of acute myelogenous leukemia. FLT3 ligand (FL) stimulation of the receptor promotes the survival and proliferation of leukemia cells. We previously identified several antibodies to FLT3, including EB10, NC7 and D4-3, and demonstrated that these antibodies could effectively block FLT3/FL interaction and inhibit the growth of leukemia cells, irrespective of whether the cells express the ligand-dependent wild-type receptor or the ligand-independent receptor mutants. Here we perform studies in an attempt to elucidate the mechanisms of the inhibitory activity of these antibodies, via dissecting the binding epitope(s) of the antibodies on the receptor. While EB10 and D4-3 only bind to recombinant FLT3 protein that was indirectly coated on a plastic surface via a capturing anti-tag antibody, NC7 binds to the receptor protein that was either indirectly coated or directly immobilized on the plastic surface. Both EB10 and D4-3 monovalent Fab fragments, but not NC7 Fab, block FLT3 from binding to its ligand. However, all three block FLT3/ligand interaction when in the form of bivalent IgG. Epitope-mapping studies using a series of domain-deletion mutants revealed that extracellular Ig domain 1 to 4 of FLT3 is required for FL binding. The binding epitope(s) for both EB10 and D4-3 was mapped to Ig domain 4, and that for NC7 was located in Ig domain 5. These results indicate that EB10 and D4-3 inhibit FLT3 receptor activation through direct competition with ligand and/or blocking of receptor dimerization whereas NC7 IgG antagonistic activity is by steric hindrance. Taken together, our study not only yields significant insight on the mechanism(s) of action of the three anti-FLT3 antibodies, but may also provide strong guidance in the search for more potent and specific FLT3 antagonists.