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, Sally J. Denardo

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)359-367
Number of pages9
JournalProtein Engineering, Design and Selection
Volume19
Issue number8
DOIs
StatePublished - Aug 2006

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Cysteine
Tumors
Peptides
Polyethylene glycols
Cells
Epitopes
Imaging techniques
Neoplasms
Molecules
Biopsy
Antigens
Antibodies
Escherichia coli
Amino acids
Immunoglobulin Fragments
Mucins
Sulfhydryl Compounds
Tumor Cell Line
Prostate
Breast

Keywords

  • di-scFv
  • MUC-1
  • PEGylation
  • Pretargeting

ASJC Scopus subject areas

  • Biochemistry
  • Biotechnology

Cite this

Development of tumor targeting anti-MUC-1 multimer : Effects of di-scFv unpaired cysteine location on PEGylation and tumor binding. / Xiong, Cheng Yi; Natarajan, Arutselvan; Shi, Xu Bao; Denardo, Gerald L; Denardo, Sally J.

In: Protein Engineering, Design and Selection, Vol. 19, No. 8, 08.2006, p. 359-367.

Research output: Contribution to journalArticle

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