Prolonged circulating lives of single-chain Fv proteins conjugated with polyethylene glycol: A comparison of conjugation chemistries and compounds

The utility of single-chain Fv proteins as therapeutic agents would be subs tantially broadened if the circulating lives of these minimal antigen-bindi ng polypeptides were both prolonged and adjustable. Poly(ethylene glycol) ( PEG) bioconjugate derivatives of the model single-chain Fv, CC49/218 sFv, w ere constructed using six different linker chemistries that selectively con jugate either primary amines or carboxylic acid groups. Activated PEG polym ers with molecular weights of 2000, 5000, 10 000, 12 000, and 20 000 were i ncluded in the sFv bioconjugate evaluation. Additionally, the influence of PEG conjugate geometry in branched PEG strands (U-PEG) and the effect of mu ltimeric PEG-sFv bioconjugates on circulating life and affinity were examin ed. Although random and extensive PEG polymer conjugations have been achiev able in highly active derivatives of the prototypical PEG-enzymes, PEGylati on of CC49/218 sFv required stringent adjustment of reaction conditions in order to preserve antigen-binding affinity as measured in either mucin-spec ific or whole cell immunoassays. Purified bioconjugates with PEG:sFv ratios of 1:1 through 2:1 were identified as promising candidates which exhibit s Fv affinity (K-d) values within 2-fold of the unmodified sFv protein. Inter estingly, PEG conjugation to carboxylic acid moieties, using a PEG-hydrazid e chemistry, achieved significant activity retention in bioconjugates at a higher PEG:sFv ratio (5:1) than with any of the amine-reactive activated PE G polymers. Prolonged circulating life in mice was demonstrated for each of the PEG conjugates. An increase in PEG polymer length was found to be more effective for serum half-life extension than a corresponding increase in t otal PEG mass. For example, CC49/218 sFv conjugated to either one strand of PEG-20000, or four strands of PEG-5000, displayed about 20- or 14-fold inc reased serum half-life, respectively, relative to the unmodified sFv. The d emonstrated suitability of established random conjugation chemistries for P EGylation of sFv proteins, in conjunction with innovative site-specific con jugation methods, indicates that production of a panoply of sFv proteins wi th both engineered affinity and tailored circulating life may now be achiev able.