Session 7 : Molecular biology ( III )

Single-chain variable fragments (scFvs) are small tumor-recognition units that hold enormous potential in antibody-based therapeutics. Advantages of scfv over whole antibodies are now explored for radioimmunodetection and for insitu radiotherapy of cancer due to potentially better tumor penetration and blood clearance and reduced immunogenicity. Their clinical applications, however, require the large scale production and purification of biologically active recombinant scFvs. RM2 IgG1.k is a human monoclonal antibody recognizing melanoma, pancreatic and nonsmall cell lung carcinoma. The variable light (VL) and variable heavy (VH) chain domains which form the antigen binding site for the RM2 Mab were rescued using RT-PCR and expressed as a monomeric scfv fragment in which the VL and the VH domains are connected by a (Gly4Ser)3 linker. scfv derived were constructed in both orientations, i.e., Vh-linker-Vl and Vllinker-Vh, but only the latter form could be expressed and secreted in the recombinant Pichia pastoris system. The vectors were derived from plasmid pPIC9 (Invitrogen) where the expression cassette is under the control of the strong AOX I promoter and downstream of the alpha-mating signal sequence. For proper processing and expression of the RM2 scfv gene the vector was digested with XhoI/SnaB1 and KEX2 site recreated by the addition of glu-lys-arg at the 3’ end of VL using oligonucleotide GTCTCGAGAAAAGATCCTATGAGC. The RM2 scfv was also cloned and expressed as his-tag fusion protein in E.coli expression vector Pet24a. For the removal of extra gene sequences the vector was digested with NdeI/SalI and directional cloning of the gene was done at HindIII/XhoI site. Both the Pichia expressed scfv and E.coli expressed scfvhistag was analyzed for purity and immunoreactivity by western blotting, FACS, and competitive ELISA. Western blotting and FACS was done using a mouse anti-linker Mab which clearly showed that as the parent antibody, the RM2scfv and RM2scfv-histag recognized a 52kD ImSF antigen expressed on the surface of live melanoma, pancreatic and nonsmall cell lung carcinoma cells. The expression of scFvs in P. pastoris was 30 to 40-fold higher than in Escherichia coli. Protein expression, culture conditions, and purification were optimized in 1-L cultures. The large-scale production of RM2 scfv in a 10 L fermenter gave rough yields of about 1g/L. The scFv antibody was purified using a hydrophobic column which gave good yields and purity. Biodistribution studies in athymic mice bearing human carcinoma xenografts showed tumortargeting of RM2 scfv. In conclusion,improved yields of monovalent scFvs were achieved using the P. pastoris expression/secretion system. The in vitro and in vivo properties of these scFvs suggest specific tumor targeting activity thereby indicating possible therapeutic applications.