1. ¹Department of Antibody Technology, ImClone Systems Incorporated, New York, NY, USA
2. ²Department of Experimental Therapeutics, ImClone Systems Incorporated, New York, NY, USA
3. ³Department of Molecular Biology, ImClone Systems Incorporated, New York, NY, USA
4. ⁴Department of Protein Science, ImClone Systems Incorporated, New York, NY, USA

Correspondence: Dr Z Zhu, Department of Antibody Technology, ImClone Systems Incorporated, 180 Varick Street, New York, NY 10014, USA. Fax: +1 212 645 2054; E-mail: Zhenping@imclone.com

Received 18 October 2004; Accepted 2 May 2005; Published online 2 June 2005.

Abstract

FMS-like tyrosine kinase 3 (FLT3) receptor is highly expressed in an array of hematological malignancies including 90% of acute myelogenous leukemia (AML). Ligand stimulation of the receptor promotes the survival and proliferation of leukemia cells. Strategies targeting FLT3 using monoclonal antibodies may therefore constitute an effective therapeutic approach 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 receptor internalization upon cell surface binding. Phage antibodies were screened first for their ability to bind to cell surface receptor and induce receptor 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, which bound specifically to both soluble and cell surface-expressed FLT3. All three antibodies were shown to be internalized upon binding to cell surface-expressed receptor in a time-dependent fashion. EB10 and D4-3 blocked ligand binding to the receptor with IC₅₀s 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, these results suggest that both EB10 and D4-3 may represent excellent therapeutic candidates for the treatment of FLT3-expressing human leukemia, both as unmodified antibodies and as conjugates of cytotoxic agents.