PMID: 9194170Apr 1, 1997Paper

Two amino acid mutations in an anti-human CD3 single chain Fv antibody fragment that affect the yield on bacterial secretion but not the affinity

Protein Engineering
S M KipriyanovM Little

Abstract

Recombinant antibody fragments directed against cell surface antigens have facilitated the development of novel therapeutic agents. As a first step in the creation of cytotoxic immunoconjugates, we constructed a single-chain Fv fragment derived from the murine hybridoma OKT3, that recognizes an epitope on the epsilon-subunit of the human CD3 complex. Two amino acid residues were identified that are critical for the high level production of this scFv in Escherichia coli. First, the substitution of glutamic acid encoded by a PCR primer at position 6 of VH framework 1 by glutamine led to a more than a 30-fold increase in the production of soluble scFv. Second, the substitution of cysteine by a serine in the middle of CDR-H3 additionally doubled the yield of soluble antibody fragment without any adverse effect on its affinity for the CD3 antigen. The double mutant scFv (Q,S) proved to be very stable in vitro: no loss of activity was observed after storage for 1 month at 4 degrees C, while the activity of scFv containing a cysteine residue in CDR-H3 decreased by more than half. The results of production yield, affinity, stability measurements and analysis of three-dimensional models of the structure suggest that the sixth amino acid...Continue Reading

Citations

Apr 16, 2002·Journal of Molecular Recognition : JMR·Marianne WeidenhauptThierry Vernet
Nov 26, 2002·Journal of Molecular Recognition : JMR·Parisa AsvadiRobert L Raison
May 30, 2008·European Journal of Nuclear Medicine and Molecular Imaging·Matthias EderMichael Eisenhut
Nov 23, 2006·Biotechnology Letters·Brigitte Gasser, Diethard Mattanovich
Jan 13, 2006·Cancer Metastasis Reviews·Mehdi Arbabi-GhahroudiRoger MacKenzie
Feb 12, 2004·Journal of Immunological Methods·Fabrice Le GallSergey M Kipriyanov
May 16, 2002·Journal of Immunological Methods·Jamshid TanhaC Roger MacKenzie
Oct 16, 2002·Journal of Immunological Methods·Corinna D Tuckey, Christopher J Noren
Mar 4, 2000·Journal of Immunological Methods·Y LiG C Whitelam
Dec 25, 2002·Journal of Molecular Biology·Stefan EwertAndreas Plückthun
Jun 24, 2003·Journal of Molecular Biology·Sergey M KipriyanovMelvyn Little
Jun 6, 2003·Trends in Biotechnology·Andrew BradburyPeter Pavlik
Apr 6, 1999·Trends in Biotechnology·A Cattaneo, S Biocca
Aug 28, 1998·Current Opinion in Biotechnology·P J Hudson
Jan 13, 2009·Protein Engineering, Design & Selection : PEDS·Annemarie HoneggerAndreas Plückthun
Jan 11, 2011·Protein Engineering, Design & Selection : PEDS·Jennifer C PaiJennifer A Maynard
Oct 17, 2002·Protein Science : a Publication of the Protein Society·Nicolas HugoDanièle Altschuh
Feb 24, 2001·Protein Science : a Publication of the Protein Society·P Saviranta PJauriaT Lövgren
Nov 10, 2001·Annual Review of Biomedical Engineering·J Maynard, G Georgiou
Jan 20, 1999·Proceedings of the National Academy of Sciences of the United States of America·A A McCormickR Levy
Sep 19, 2012·Journal of Molecular Biology·Fabrice Le GallSergej M Kiprijanov
Oct 21, 2003·International Journal of Cancer. Journal International Du Cancer·Michaela A E ArndtSusanna M Rybak
Dec 2, 2006·Journal of Immunological Methods·Daniel X JohanssonMats A A Persson
Jul 11, 2006·Advanced Drug Delivery Reviews·Kim L Wark, Peter J Hudson

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