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PEDS Advance Access originally published online on June 7, 2006
Protein Engineering Design and Selection 2006 19(8):359-367; doi:10.1093/protein/gzl020
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Development of tumor targeting anti-MUC-1 multimer: effects of di-scFv unpaired cysteine location on PEGylation and tumor binding

Cheng-Yi Xiong, Arutselvan Natarajan, Xu-Bao Shi, Gerald L. Denardo and Sally J. Denardo1

Department of Internal Medicine, Division of Hematology/Oncology, Section of Radiodiagnosis and Therapy, University of California Davis Cancer Center 1508 Alhambra Boulevard, Sacramento, CA 95816, USA

1To whom correspondence should be addressed. Radiodiagnosis and Therapy, Molecular Cancer Institute, University of California Davis Medical Center, 1508 Alhambra Boulevard, Room 3100, Sacramento, CA 95816, USA E-mail: sjdenardo{at}ucdavis.edu

MUC1 mucin expressed in epithelial cancer, such as prostate and breast, is aberrantly glycosylated providing unique targets for imaging and therapy. In order to create a broadly applicable construct to target these unique epitopes on metastatic cancer, we selected an antibody fragment (scFv) that binds both synthetic MUC1 core peptide and epithelial cancer cell-expressed MUC1, and developed a recombinant bivalent molecule (di-scFv). Genetically engineered modifications of the di-scFv were constructed to create five molecular versions, each having a free cysteine (di-scFv-c) at different locations for site-specific conjugation. The effects of the engineered cysteine in the varied sites were studied relative to tumor binding and polyethylene glycol-maleimide (PEG-Mal) conjugation (PEGylation). Escherichia coli production as well as binding to MUC1 core peptide, human tumor cell lines and human tumor biopsies, were comparable. However, the location of the engineered cysteine in these di-scFv-c did influence PEGylation efficiency of this free thiol; higher PEGylation efficiency occurred with this cysteine in the inter-scFv linkage. Di-scFv-c PEG, with the cysteine engineered after the fifth amino acid in the linker, was used as an example to demonstrate comparable antigen-binding to non-PEGylated di-scFv-c. In summary, novel anti-MUC1 di-scFv-c molecules can be efficiently produced, purified and conjugated by site-specific PEGylation without loss of immunoreactivity, thus providing flexible multidentate constructs for cancer-targeted imaging and therapy.

Keywords: di-scFv/MUC-1/PEGylation/pretargeting

Received October 31, 2005; revised March 21, 2006; accepted March 27, 2006.


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