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Selection and Characterization of Anti-MUC-1 scFvs Intended for Targeted Therapy¹

Michelle D. Winthrop, Sally J. DeNardo², Huguette Albrecht, Gary R. Mirick, Linda A. Kroger, Kathleen R. Lamborn, eslovas Venclovas, Michael E. Colvin, Patricia A. Burke and Gerald L. DeNardo

University of California Davis Medical Center, Sacramento, California 95816 [M. D. R., S. J. D., H. A., G. R. M., L. A. K., P. A. B., G. L. D.]; University of California San Francisco, San Francisco, California 94143 [K. R. L.]; Lawrence Livermore National Laboratory, Livermore, California 94550 [. V., M. E. C.]; and Institute of Biotechnology, 2028 Vilnius, Lithuania [. V.]

Purpose: The selection and characterization of anti-MUC-1 single-chain antibody fragments (scFv) is a first step toward the construction of new anticancer molecules designed for optimal blood clearance and tumor penetration. The mucin MUC-1 was chosen as an antigen because it is abundantly expressed on epithelial cancers in an aberrantly glycosylated form, making it structurally and antigenically distinct from MUC-1 expressed on normal cells.

Experimental Design: A previously constructed anti-MUC-1 phage display library from hyperimmunized mice, with 5 x 10⁵ calculated variants, was screened for the selection of anti-MUC-1 scFvs. Selection criteria were high binding to a MUC-1 peptide containing 4 tandem repeats of 20 amino acids and to MUC-1-positive MCF-7 (human breast cancer) cell lysates in ELISA.

Results: Six anti-MUC-1 scFv clones were selected and characterized. Nucleotide sequencing showed that four of them were full length scFv genes (variable heavy chain + variable light chain), whereas the remaining two contained either a variable heavy chain or a variable light chain alone. Their binding affinities (K_a) range between 8 x 10⁷ and 10⁹ M^-1. Immunohistopathology demonstrated reactivity with breast cancer cells (MCF-7 and BT20) and human breast biopsy tissue. Molecular modeling revealed high structural similarity of the anti-MUC-1 scFvs with the X-ray-determined structure of the anti-CEA scFv (MFE-23).

Conclusions: In vitro antigen binding was demonstrated for the selected anti-MUC-1 scFvs. The binding affinities of these scFvs are in a promising range for efficient in vivo antigen binding. These anti-MUC-1 scFvs will be evaluated as antigen-binding modules in new multifunctional agents for the detection and therapy of cancer.

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