Construction and optimization of a CC49-based scFv-beta-lactamase fusion protein for ADEPT.
CC49 is a clinically validated antibody with specificity for TAG-72, a carbohydrate epitope that is over-expressed and exposed on a large fraction of solid malignancies. We constructed a single chain fragment (scFv) based on CC49 and fused it to beta-lactamase. The first generation fusion protein, TAB2.4, was expressed at low levels in Escherichia coli and significant degradation was observed during production. We optimized the scFv domain of TAB2.4 by Combinatorial Consensus Mutagenesis (CCM). An improved variant TAB2.5 was identified that resulted in an almost 4-fold improved expression and 2.5 degrees higher thermostability relative to its parent molecule. Soluble TAB2.5 can be manufactured in low-density E.coli cultures at 120 mg/l. Our studies suggest that CCM is a rapid and efficient method to generate antibody fragments with improved stability and expression. The fusion protein TAB2.5 can be used for antibody directed enzyme prodrug therapy (ADEPT).
Roberge
Martin
M
Genencor International, a Danisco company, 925 Page Mill Road, Palo Alto, CA 94304, USA.
Estabrook
Melodie
M
Basler
Joshua
J
Chin
Regina
R
Gualfetti
Pete
P
Liu
Amy
A
Wong
Stephanie B
SB
Rashid
M Harunur
MH
Graycar
Tom
T
Babé
Lilia
L
Schellenberger
Volker
V
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000627:therapeutic use; D000912:Antibodies, Neoplasm / Q000235:genetics* / Q000627:therapeutic use; D000951:Antigens, Neoplasm / Q000276:immunology; D020650:Combinatorial Chemistry Techniques / Q000379:methods; D016384:Consensus Sequence / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D006023:Glycoproteins / Q000276:immunology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019151:Peptide Library; D011355:Prodrugs / Q000627:therapeutic use*; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis* / Q000627:therapeutic use; D001618:beta-Lactamases / Q000235:genetics* / Q000627:therapeutic use
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; C091202:B72.3 antibody; D006023:Glycoproteins; D019151:Peptide Library; D011355:Prodrugs; D011993:Recombinant Fusion Proteins; C047052:tumor-associated antigen 72; D001618:beta-Lactamases