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EXAMPLE OF ABSTRACT FORMATTING
Construction and expression of anti-Tn-antigen-specific single chain antibody (scFv) genes from
hybridoma producing MLS128 monoclonal antibody.
Yoko Fujita-Yamaguchi1, Tomoyuki Koizumi1, Kazuhiro Tukamoto1, Haruhiko Ogawa, Ayano Matsumoto-Takasaki1,
Hideki Asanuma1, Hiroshi Nakada2, and Noriaki Yuasa1
1Department of Applied Biochemistry, Tokai University School of Engineering, Kanagawa, Japan; 2Department of
Molecular Bioscience, Faculty of Life Sciences, Kyoto Sangyo University, Kyoto, Japan.
Oncogenic transformation is associated with dysregulation of glycosylation processes that then leads to altered
patterns of carbohydrate functionalization on the surface of cancer cells. These tumor-associated carbohydrate
antigens are often involved in metastatic processes and are associated with a poor prognosis, thus representing
excellent targets for cancer intervention. MLS128 is a monoclonal antibody which recongnizes an epitope
consisting of three or two consecutive Tn-antigens (GalNAcα-Ser/Thr): Tn3 or Tn2. We previously published that
MLS128 treatment inhibited colon and breast cancer cell growth, and that MLS128-inhibited cancer cell growth is in
part mediated by down-regulation of growth factor receptors. This provided the first insights into the potential use of
this particular type of anti-Tn antigen antibodies as a cancer therapeutic (Morita et al. Biosci. Trends 3:32-37, 2009). In this
study, cDNA encoding VH or VL constructed from hybridoma were assembled into VL-Linker-VH, and subcloned
into pCANTAB5E. Phages produced were subjected to panning and screening using Tn3-peptide. DNA sequences
revealed 6 independent clones which showed similar binding activities to Tn3-peptide. One MLS128 scFv gene
(F11) was subcloned into pET22b to generate a pET/MLS128-F11scFv expression vector. E. coli BL21 transformed
with pET/MLS128-F11scFv was induced by IPTG to overexpress MLS128 scFv proteins. Inclusion bodies prepared
from bacterial cells were solubilized in a buffer containing 3.5M Gdn-HCl. From the supernatant, denatured
MLS128 scFv proteins were purified by Ni2+-Sepharose chromatography, and then subjected to refolding by
stepwise dialyses. Nearly 100% of MLS128 scFv proteins were recovered as soluble proteins as judged by SDSPAGE and protein assays. The purified MLS128 scFv protein exhibited Tn3- and Tn-2 binding activities.
Furthermore, to establish stable expression of scFv-Fc proteins in CHO-DHFR cells, the MLS128 scFv gene was
inserted into a template plasmid signal-human Fc-pCI-neo (pCI-Fc), as described previously (Zhang et al.
Biochemistry 46: 263-270, 2007). The MLS128 scFv-Fc protein purified by protein A-Sepharose chromatography
exhibited Tn-antigen binding activity. These results confirmed that the cloned MLS128 variable domain genes can
be used for developing cancer diagnostics and therapeutics.