Abstract
A major goal in antibody design for cancer therapy is to tailor the pharmacokinetic properties of the molecule according to specific treatment requirements. Key parameters determining the pharmacokinetics of therapeutic antibodies are target specificity, affinity, stability, and size. Using the p185HER-2 (HER-2)-specific scFv 4D5 as model system, we analyzed how changes in molecular weight and valency independently affect antigen binding and tumor localization. By employing multimerization and PEGylation, four different antibody formats were generated and compared with the scFv 4D5. First, dimeric and tetrameric miniantibodies were constructed by fusion of self-associating, disulfide-linked peptides to the scFv 4D5. Second, we attached a 20-kDa PEG moiety to the monovalent scFv and to the divalent miniantibody at the respective C terminus. In all formats, serum stability and full binding reactivity of the scFv 4D5 were retained. Functional affinity, however, did change. An avidity increase was achieved by multimerization, whereas PEGylation resulted in a 5-fold decreased affinity. Nevertheless, the PEGylated monomer showed an 8.5-fold, and the PEGylated dimer even a 14.5-fold higher tumor accumulation than the corresponding scFv, 48 h post-injection, because of a significantly longer serum half-life. In comparison, the non-PEGylated bivalent and tetravalent miniantibodies showed only a moderate increase in tumor localization compared with the scFv, which correlated with the degree of multimerization. However, these non-PEGylated formats resulted in higher tumor-to-blood ratios. Both multimerization and PEGylation represent thus useful strategies to tailor the pharmacokinetic properties of therapeutic antibodies and their combined use can additively improve tumor targeting.
Highlights
Antibodies have attracted attention as cancer therapeutics, because of their ability to bind to cell surface antigens selectively expressed on tumor cells [1, 2]
To systematically investigate the correlation of these molecular properties with the in vivo behavior, we constructed and characterized four different antibody formats, all based on the antiHER-2 scFv 4D5 [24, 41, 42], using two strategies
The various miniantibody formats showed no obvious difference in serum stability, and PEGylation and oligomerization did not affect aggregation or degradation. They differed in serum half-life in vivo, suggesting that elimination by metabolism and excretion is a major determinant of their tumor targeting properties
Summary
Antibodies have attracted attention as cancer therapeutics, because of their ability to bind to cell surface antigens selectively expressed on tumor cells [1, 2]. Depending on the desired mode of action, the pharmacokinetic properties of the therapeutic molecule need to meet different requirements. If the mode of action does not include a cytotoxic mechanism, accumulation in normal tissues may be less harmful, and high concentration at the tumor site can be the main criterion. To enhance the efficacy of therapeutic antibodies, understanding how changes of the molecular format may affect the pharmacokinetic behavior is crucial. There seems to be an upper limit in affinity beyond which no further improvement can be achieved [6, 8, 9]: in the “loading phase”, the total dose of antigen-bound antibody molecules accumulating at the tumor is limited by plasma clearance (depletion of the plasma pool) and diffusion, in addition to the total amount injected (bolus size). ScFv PEGylation and Multimerization for Tumor Targeting demonstrated that for a tumor antigen with the turnover rate of HER-2, raising the affinity above 10Ϫ9 M no longer translates into increased tumor accumulation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
