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

Purpose: The selection and characterization of antiMUC-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 antiMUC-1 phage display library from hyperimmunized mice, with 5 10 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 (Ka) range between 8 10 7 and 10 M . 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. Introduction Although radioimmunotherapy using intact MAbs has been studied in clinical trials for the treatment of breast cancer and other solid tumors, therapeutic success has been limited by the large size of the MAb (150 kDa) molecules which retards blood clearance and decreases the accumulation of the radiopharmaceutical in the tumor (1). Recombinant antibodies and their fragments in combination with new therapy approaches such as pretargeting have become a promising resource for the design of high-affinity specific targeting drugs (2, 3). Recent construction of engineered targeting molecules has demonstrated the potential for multivalent high affinity reagents built from small binding fragments (25 kDa), which can potentially target tumor more efficiently than large MAbs (4). Antibody fragment units have also been engineered for gene therapy, imaging, immunotherapy, radioimmunotherapy, chemotherapy, and prodrug therapy (5–13). ScFvs corresponding to the VH and VL immunoglobulin domains connected by a biologically inert flexible linker create the smallest antibody fragments that usually retain specific binding characteristics. ScFv molecules can be produced from existing MAb hybridoma clones, however, phage display libraries can provide a multitude of scFvs from a more diverse antibody gene pool and thus allow for the selection of scFvs to various epitopes and with a range of binding characteristics (14–19). One of the epithelial mucin family of molecules, MUC-1, has received considerable interest as a cancer antigen target. It is abundantly expressed on a number of epithelial cancers, where its aberrant distribution and glycosylation make it structurally and antigenically distinct from MUC-1 expressed by nonmalignant cells (20–23). The dominant form of MUC-1 is a high molecular weight molecule, comprised of an extracellular mucin domain with tandem repeats of 20 amino acids, a transmembrane region, and a cytoplasmic tail. Within the extracellular domain, novel epitopes of shortened carbohydrate chains and portions of the 20 amino acid tandem repeats are exposed in malignant cells and hence provide specific targets for antibodies (24–26). In normal epithelial tissue, MUC-1 is localized to the 1 Presented at the “Ninth Conference on Cancer Therapy with Antibodies and Immunoconjugates,” October 24–26, 2002, Princeton, NJ. This work was supported by U.S. Department of Energy Grant DE-FG0100NE22944, California Breast Cancer Research Program Postdoctoral Fellowship Grant [M. D. W. ], a Society of Nuclear Medicine Education and Research Foundation Fellowship [M. D. W. ], and National Cancer Institute Grant CA-47829. 2 To whom requests for reprints should be addressed, at Radiodiagnosis and Therapy, Molecular Cancer Institute, University of California, Davis Medical Center, 1508 Alhambra Boulevard, Room 3100, Sacramento, CA 95816. Phone: 916-734-3787; Fax: 916-451-2857; E-mail: sjdenardo@ucdavis.edu. 3 The abbreviations used are: MAb, monoclonal antibody; Ka, binding affinity; PDB, Protein Data Bank; scFv, single-chain antibody fragment; VH, variable heavy chain; VL, variable light chain; ITPG, isopropyl-D-thiogalactopyranoside; TES, 0.2 M Tris-HCl (pH 8.0), 0.5 mM EDTA, 0.5 M sucrose. 3845s Vol. 9, 3845s–3853s, September 1, 2003 (Suppl.) Clinical Cancer Research