Session 7: Molecular biology (III)

2002 Human Antibodies  
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
more » ... n 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 pPIC-9 (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 GTCTCGAGAAAAGATC-CTATGAGC. 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 scfv-
doi:10.3233/hab-2002-111-207 fatcat:qn6vl2ovx5cx3nkdqjxuhtjcyq