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A Genetically Engineered Anti-CD45 Single-Chain Antibody-Streptavidin Fusion Protein for Pretargeted Radioimmunotherapy of Hematologic Malignancies

¹ The Fred Hutchinson Cancer Research Center; ² Division of Oncology, University of Washington School of Medicine; and ³ Aletheon Pharmaceuticals, Inc., Seattle, Washington

Requests for reprints: Oliver W. Press, 1100 Fairview Avenue North, Mailstop D3-190, Seattle, WA 98109. Phone: 206-667-1872; Fax: 206-667-1874; E-mail: press{at}u.washington.edu.

Acute myelogenous leukemia (AML) currently kills the majority of afflicted patients despite combination chemotherapy and hematopoietic cell transplantation (HCT). Our group has documented the promise of radiolabeled anti-CD45 monoclonal antibodies (Ab) administered in the setting of allogeneic HCT for AML, but toxicity remains high, and cure rates are only 25% to 30% for relapsed AML. We now show the superiority of pretargeted radioimmunotherapy (PRIT) compared with conventional radioimmunotherapy using a recombinant tetravalent single-chain Ab-streptavidin (SA) fusion protein (scFv₄SA) directed against human CD45, administered sequentially with a dendrimeric N-acetylgalactosamine–containing clearing agent and radiolabeled 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic (DOTA)-biotin. The scFv₄SA construct was genetically engineered by fusing Fv fragments of the human CD45-specific BC8 Ab to a full-length genomic SA gene and was expressed as a soluble tetramer in the periplasmic space of Escherichia coli. The fusion protein was purified to >95% homogeneity at an overall yield of 50% using iminobiotin affinity chromatography. The immunoreactivity and avidity of the fusion protein were comparable with those of the intact BC8 Ab, and the scFv₄SA construct bound an average of 3.9 biotin molecules out of four theoretically possible. Mouse lymphoma xenograft experiments showed minimal toxicity, excellent tumor-specific targeting of the fusion protein and radiolabeled DOTA-biotin in vivo, marked inhibition of tumor growth, and cured 100% of mice bearing CD45-expressing tumors. These promising results have prompted large-scale cGMP production of the BC8 fusion protein for clinical trials to be conducted in patients with hematologic malignancies. (Cancer Res 2006; 66(7): 3884-92)

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