Attachment of poly(ethylene glycol) (PEG) to proteins can greatly alter their pharmacological properties, including extending the plasma half-life and reducing immunogenicity, both of which are potentially beneficial to tumour targeting. IgG, F(ab')2 and Fab' fragments of the anti-CEA antibody A5B7 were chemically modified with PEG (M(r) 5,000), labelled with 125I and their pharmacokinetics compared with the unmodified forms in the LS174T colonic xenograft in nude mice. PEG modification of the intact antibody had little effect on biodistribution, although tumour localisation was slightly reduced. In contrast, similar modification of F(ab')2 and Fab'A5B7 significantly prolonged plasma half-life and increased radioantibody accumulation in the tumour and to a lesser extent in normal tissues, but reduced tissue to blood ratios. Prior to modification, Fab' A5B7 (M(r) 50,000) cleared more rapidly from the circulation than F(ab')2 (M(r) 100,000), but after PEG attachment their biodistributions converged, while the tumour to blood ratios were reduced and resembled that of the intact antibody. The enhanced tumour accumulation, reduced normal tissue to blood ratios and potentially reduced immunogenicity of fragments after PEG attachment may therefore prove superior to either unmodified fragments or intact antibody for antibody-targeted therapy, although the increased plasma half-life may necessitate the use of a clearance mechanism.
Summary Polyethylene glycol (PEG) modification of a chimeric Fab' fragment (F9) of A5B7 (a-CEA), using an improved coupling method, increases its specificity for subcutaneous LS174T tumours. PEGylation increased the area under the concentration-time curve (AUCo-144) in all tissues but there were significant differences (variance ratio test, F=27.95, P<0.001) between the proportional increases in AUCo144, 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 AUCbl..d 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 tissuespecific fashion.
Single-chain antibodies (scFvs) can be derived from a monoclonal antibody (MAb) or produced directly using filamentous phage technology, where antibodies with desired binding and purification characteristics can be readily selected from libraries. To test the hypothesis that the latter approach is more useful, we compared 2 anti-carcinoembryonic antigen (CEA) scFvs produced by these 2 different approaches. Our study showed that, both in the purification process and in the biodistribution pattern, MFE-23, produced by filamentous phage technology, gave favourable results compared to A5-SC, which is derived from the A5B7 MAb. This indicates the value of the filamentous phage approach for obtaining tumour-targeting scFvs.
The enzyme carboxypeptidase G2 (CPG2) can be targeted to tumors by antibodies and used to activate prodrugs in a treatment called antibody-directed enzyme prodrug therapy (ADEPT). Different doses of CPG2 conjugated to the anti-CEA antibody A5B7 were administered i.v. to nude mice bearing the LS174T human colon adenocarcinoma xenograft, and the biodistribution of conjugate activity 48 and 72 h later was determined using a novel high-performance liquid chromatography (HPLC) method. Conjugate doses of 2,500 and 625 U/kg gave tumor enzyme levels of 0.5-0.6 U/g. Lower doses of 300 and 150 U/kg gave tumor enzyme levels of 0.1-0.3 U/g. Intriguingly, the best tumor:blood ratio of conjugate activity at both 48 and 72 h was achieved after administration of the 625-U/kg dose, not the 2,500-U/kg dose. After 48 h this ratio was 3.8, whereas after 72 h the value was 5.5. This conjugate dose also gave the greatest tumor:tissue ratios in all other tissues examined. After 72 h the tumor:colon ratio was 105, whereas the tumor:kidney ratio was 36. In ADEPT, to obtain maximal tumor damage to LS174T xenografts in nude mice with minimal systemic toxicity using the A5B7-CPG2 conjugate, prodrug should therefore be administered at least 72 h after a conjugate dose of 625 U/kg.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.