Abstract
Polyethylene glycol (PEG) modification of a chimeric Fab' fragment (F9) of A5B7 (alpha-CEA), using an improved coupling method, increases its specificity for subcutaneous LS174T tumours. PEGylation increased the area under the concentration-time curve (AUC0-144) in all tissues but there were significant differences (variance ratio test, F = 27.95, P < 0.001) between the proportional increases in AUC0-144, with the tumour showing the greatest increase. The increase in AUCtumour from F9 to PEG-F9 was similar to the reported increase from Fab' to F(ab')2 while the increase in AUCblood by PEGylation of F9 was only 21% of the reported increase from Fab' to whole IgG. A two sample t-test showed no significant differences between maximal tumour/tissue ratios for PEG-F9 and F9 while the tumour/tissue ratios for PEG-F9 remained high over a longer period, with tumour levels at least double those for F9. PEG-F9 emerges as a new generation antibody with potential advantages for both radioimmunotherapy and tumour imaging. Since there was a reduction in antigen binding, optimisation of PEGylation might further improve tumour specificity. The latter resulted from complex effects on both the entry into and exit rates from tumour and normal tissues in a tissue-specific fashion.
Highlights
Covalent modification of proteins with poly(ethylene glycol)(PEG) has emerged as the method of choice to overcome the major problems associated with protein therapeutics (Francis et al, 1991; Delgado et al, 1992a)
Incubation of recombinant chimeric F(ab') fragment (F9) with tresylated monomethoxypoly(ethylene glycol) (TMPEG) resulted in the formation of species with larger molecular sizes as shown by the appearance of faster eluting peaks in gel permeation chromatography (Figure 1)
The proportion of larger molecular size species increased with the concentration of TMPEG in the reaction mixture, while the proportion of material eluting at the location of the unmodified F9 decreased (Figure 1)
Summary
Covalent modification of proteins with poly(ethylene glycol)(PEG) has emerged as the method of choice to overcome the major problems associated with protein therapeutics (Francis et al, 1991; Delgado et al, 1992a). Increased plasma half-life, increased resistance to proteolysis and substantial reduction in antigenicity/immunogenicity have been found in almost all recombinant and native proteins after PEGylation. The success of this technology is indicated by the number of PEG proteins already in clinical trials (Francis et al, 1991)
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