Prolonged Circulating Lives of Single-Chain Fv Proteins Conjugated with Polyethylene Glycol:  A Comparison of Conjugation Chemistries and Compounds

Abstract

The utility of single-chain Fv proteins as therapeutic agents would be substantially broadened if the circulating lives of these minimal antigen-binding polypeptides were both prolonged and adjustable. Poly(ethylene glycol) (PEG) bioconjugate derivatives of the model single-chain Fv, CC49/218 sFv, were constructed using six different linker chemistries that selectively conjugate either primary amines or carboxylic acid groups. Activated PEG polymers with molecular weights of 2000, 5000, 10 000, 12 000, and 20 000 were included in the sFv bioconjugate evaluation. Additionally, the influence of PEG conjugate geometry in branched PEG strands (U-PEG) and the effect of multimeric PEG-sFv bioconjugates on circulating life and affinity were examined. Although random and extensive PEG polymer conjugations have been achievable in highly active derivatives of the prototypical PEG-enzymes, PEGylation of CC49/218 sFv required stringent adjustment of reaction conditions in order to preserve antigen-binding affinity as measured in either mucin-specific or whole cell immunoassays. Purified bioconjugates with PEG:sFv ratios of 1:1 through 2:1 were identified as promising candidates which exhibit sFv affinity (K(d)) values within 2-fold of the unmodified sFv protein. Interestingly, PEG conjugation to carboxylic acid moieties, using a PEG-hydrazide chemistry, achieved significant activity retention in bioconjugates at a higher PEG:sFv ratio (5:1) than with any of the amine-reactive activated PEG 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 total 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 increased serum half-life, respectively, relative to the unmodified sFv. The demonstrated suitability of established random conjugation chemistries for PEGylation of sFv proteins, in conjunction with innovative site-specific conjugation methods, indicates that production of a panoply of sFv proteins with both engineered affinity and tailored circulating life may now be achievable.

Protein PEGylation Decreases Observed Target Association Rates via a Dual Blocking Mechanism

“…Nevertheless, we found a decrease in apparent affinity when comparing the binding properties of the PEGylated constructs with those of their unPEGylated equivalents. This is in contradiction to some earlier reports (Chapman et al, 1999;Lee et al, 1999;Chapman, 2002) but consistent with a more recent study (Yang et al, 2003). Earlier reports had suggested that PEGylated antibody fragments, which carry the PEG tail at a site where direct interference with the binding region is unlikely, retain full binding activity.…”

“…This nonimmunogenic polymer (Caliceti and Veronese, 2003) can increase the hydrodynamic radius of the conjugated protein to a huge extent, leading to significantly decreased renal clearance (Chapman et al, 1999;Lee et al, 1999;Batra et al, 2002;Chapman, 2002). Furthermore, it can act to shield protein sites from recognition by the immune system or serum proteases (Cunningham-Rundles et al, 1992;Tsutsumi et al, 2000).…”

“…Successful applications of this technique have been reported (Chapman et al, 1999;Lee et al, 1999;Weir et al, 2002), showing that PEG tails from 2 kDa up to 40 kDa can be coupled to antibody fragments (scFv or Fab). However, conflicting conclusions about changes in affinity have been reached (see below).…”

PEGylation and Multimerization of the Anti-p185HER-2 Single Chain Fv Fragment 4D5

“…The 5-fold reduction in apparent affinity upon PEGylation of the antibody fragments was observed in the kinetic BIAcore measurements (Table 2) as well as in the equilibrium binding assays on SK-OV-3 tumor cells (Table 1). In earlier reports (69,71,72) it was stated that antibody conjugates with a PEG polymer attached at a single engineered cysteine distal to the antigen binding site retain full binding activity. In a more recent study (50), however, similar findings of reduced affinity as we have demonstrated here were also reported for other scFv fragments that were site-specifically conjugated with PEG polymers of different sizes.…”

Modulating the pharmacokinetics of therapeutic antibodies

“…Amine-directed chemical polysialylation (see below) was successfully used to modify an anti-tumour Fab (Constantinou et al 2008), but the same process damaged an anti-tumour scFv which was resolved by site-specific modification (Constantinou et al 2009). Similarly, conjugation of larger polymer chains using amine-reactive conjugation of an anti-TAG-72 scFv was also found to be more detrimental on antibody activity than conjugation to carboxylic acid moieties using PEG-hydrazine chemistry (Lee et al 1999). Unfortunately, in the absence of structural information it is difficult to predict which residues may be important for bioactivity.…”