Isolation of scFv antibody fragments against HER2 and CEA tumor antigens from combinatorial antibody libraries derived from cancer patients.

Tumor cells expressing HER-2/neu and CEA antigens are potentially ideal targets for antibody-targeted therapy. In this study, two large human combinatorial libraries have been generated from the lymph nodes of breast cancer patients that express HER2 and CEA antigens in their tumors. These 'immune' libraries have been constructed in two different formats of scFv, differing in the length of the peptide linker connecting the two variable VH and VL domains. Libraries derived from these patients may contain a larger pool of anti-tumor antigen antibodies and are useful repertoire for isolating scFvs against any tumor markers. The results of this study showed that we were successful in obtaining human scFvs against HER-2/neu and CEA. For HER-2, cell-panning strategy was performed and resulted in two scFv binders that detected the complete HER-2 receptor on the cell membrane and internalized to the cells. Also, preliminary ELISA data showed that several anti-CEA scFv binders were isolated by panning.

Green fluorescent-conjugated anti-CEA single chain antibody for the detection of CEA-positive cancer cells.

According to World Health Organization (WHO), cancer is a leading cause of death worldwide, accounting for 7.4 million deaths (around 13% of all deaths) in 2004. Monoclonal/recombinant antibodies, which specifically target clinical biomarkers of disease, have increasingly been applied as powerful tools in cancer imaging and therapy, a fact that is highlighted by some nine FDA-approved monoclonal antibodies (MAbs) or their immunoconjugates (as of December 2008) for use in cancer treatment. In this study, five monoclonal antibodies (MAbs) were generated and characterized against carcinoembryonic antigen (CEA), which is widely used clinically as both a blood and tissue tumor marker of epithelial malignancy. Variable domains (VH and VL) of one the stable MAbs with highest affinity were PCR-amplified and assembled as single-chain antibody fragment (scFv). Following the cloning and expression of scFv antibody fragments in Escherichia coli, the functional binding and specificity of the recombinant antibody were confirmed by ELISA. To develop a direct in vitro detection of CEA-positive cancer cells, scFv DNA was genetically fused to enhanced green fluorescent protein (EGFP) gene and expressed in bacteria. The chimeric fluorescent protein is able to specifically detect CEA-positive cell lines; no cross-reactivity was observed with a negative control cell line. This strategy will likely allow the establishment of a rapid, single-step detection assay of CEA, which is considered to be one of the best predictors of malignancy among all other tumor markers.

Isolation of BNYVV coat protein-specific single chain Fv from a mouse phage library antibody.

Beet necrotic yellow vein virus (BNYVV) infects sugar beet plants worldwide and is responsible for the rhizomania disease and severe economic losses. Disease severity and lack of naturally occurring resistant plants make it very difficult to control the virus, both from epidemiological and economic standpoints. Therefore, early detection is vital to impose hygiene restrictions and prevent further spread of the virus in the field. Immunoassays are one of the most popular methodologies for the primary identification of plant pathogens including BNYVV since they are robust, sensitive, fast, and inexpensive. In this study, the major coat protein (CP21) of BNYVV was cloned and expressed in Escherichia coli. Thereafter, mice were immunized with purified CP21 and a phage antibody library was constructed from their PCR-amplified immunoglobulin repertoire. Following filamentous phage rescue of the library and four rounds of panning against recombinant CP21 antigen, several specific single chain Fv fragments were isolated and characterized. This approach may pave the way to develop novel immunoassays for a rapid detection of viral infection. Moreover, it will likely provide essential tools to establish antibody-mediated resistant transgenic technology in sugar beet plants.

Development of a bacterial surface display of hexahistidine peptide using CS3 pili for bioaccumulation of heavy metals.

A novel cell surface display system for metal uptake was developed using CS3 pili of enterotoxigenic Escherichia coli, which is a suitable system for display of heterologous peptides. The recombinant bacteria producing the hybrid pili containing the hexahistidine peptide accumulated high concentrations of Cd(2+) and Ni(2+) at 656.2 and 276.5 nmol per mg dry weight of bacterial cell, respectively. The recombinant bacteria may be useful in water and waste water treatment.