ORIGINAL ARTICLES   

Minerva Biotecnologica 2008 September;20(3):103-15

Copyright © 2008 EDIZIONI MINERVA MEDICA

lingua: Inglese

Biochemical characterization and experimental tumor targeting properties of an anti-CEA antibody fragment produced in E. coli

Pérez L. ¹, Pimentel G. ², Ravelo R. ², Ayala M. ¹, Bell H. ¹, Miranda M. ², Martínez Y. R. ¹, Sánchez I. ², Musacchio A. ³, Morera V. ³, Canaán L. ³, Oliva J. P. ², Gavilondo J. V. ¹

¹ Recombinant Antibody Laboratory Cancer Research Department Center for Genetic Engineering and Biotechnology Havana, Cuba 2 Nuclear Medicine Department National Institute of Oncology and Radiobiology Havana, Cuba 3 Chemistry and Physics Division Center for Genetic Engineering and Biotechnology Havana, Cuba

Aim. The authors study biochemical and biological properties of bacterially produced scFv antibody fragments constructed with 5- or 15-aa linkers, that couple the VH and VL domains of a mouse monoclonal antibody specific for carcinoembryonic antigen (CEA).
Methods. The polymerase chain reaction (PCR) constructed antibody fragments were cloned into an Escherichia coli (E. coli) periplasm expression vector. Antibody fragments were purified using immobilized metal ion affinity chromatography (IMAC), and size exclusion gel chromatography (SEGC). Recognition of CEA+ LS 174T human tumor cells was assessed by florescence-activated cell sorting (FACS). Specific absorptivity coefficients and light scattering properties were also determined. Nude mice bearing human LS 174T tumors were injected with a 131I radiolabelled scFv.
Results. The expressed scFvs were purified by IMAC and studied by SEGC. The 5-aa scFv preparation was formed by two main peaks (M3-1, and M3-2), with different retention times (equivalent to ca. 66 kDa and 30 kDa, respectively). The 15-aa scFv (L-15) displays a slightly lower molecular weight (ca 25 Kda) in SEGC. All three scFv recognize human tumor LS 174T cells by FACS. Based on this, and after specific absorptivity coefficient and light scattering determinations, we suggest that M3-1 is a divalent antibody fragment (diabody), while M3-2 and L-15 display properties more suggestive of a monomeric scFv. M3-1 was labelled with 131I, injected iv into nude mice bearing human LS 174T tumors. Preferential tumor radioactivity uptake was seen during the 72 hours of the experiment.
Conclusion. The results are encouraging for the further development of the divalent M3-1 antibody fragment as a human tumor targeting vehicle.