Pharmacokinetics and biodistribution of engineered single-chain antibody constructs of MAb CC49 in colon carcinoma xenografts

Monoclonal antibodies (MAbs) have been proven useful in clinical studies fo r both diagnostic and therapeutic applications. The single-chain Fv (scFv) construct made from MAbs has potential applications for improved cancer dia gnosis and therapy. A new CC49 scFv construct recognizing a tumor-associate d mucin, TAG-72, was engineered and evaluated by immunological, pharmacokin etic and biodistribution analysis. Methods: The CC49 scFv construct was gen erated in which the V-L and V-H variable region genes were joined together with a 25-amino acid helical linker (205C). The new CC49 scFv(205C) was exp ressed as a monomer as well as a stable noncovalent dimer ([scFv](2)). The pharmacokinetic, biodistribution and tumor targeting characteristics of rad iolabeled CC49 scFv were compared with CC49 IgG and enzymatically derived f ragments F(ab')(2) and Fab', using the athymic mice bearing human colon can cer xenografts. Results: The association constant (KA) for the intact CC49, dimeric scFv (scFv), and monomeric scFv were 1.7 x 10(9), 1.99 x 10(9) and 0.52 x 10(9) M-1 by Scatchard analysis and 1.14 x 10(8), 4.46 x 10(7) and 1.5 x 10(7) M-1, respectively, by BIAcore analysis. Pharmacokinetic studies showed that more than 50% of monomeric scFv (similar to 27 kDa) was cleare d from the blood in less than 10 min. The CC49 Fab' generated enzymatically from the parent murine Mab' (50 kDa) had a blood clearance that was faster than that of the (scFv), (60 kDa) with half of the activity cleared from t he serum within 30 and 50 min, respectively. The CC49 dimeric scFv(205C) sh owed a two-fold higher tumor uptake (than scFv or Fab') reaching 10 %ID/g a t 60 min after injection. The scFv dimer also showed an excellent stability and increased avidity in vivo compared with the monomer, as demonstrated b y the longer retention in tumor with 3 %ID/g remaining at 48 h. Conclusion: The rapid clearance from the blood, higher tumor uptake and longer retenti on of the stable dimer of CC49 scFv make it an important agent for potentia l imaging and therapeutic applications.