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

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 p185 HER-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.

N-Glycosylation as Novel Strategy to Improve Pharmacokinetic Properties of Bispecific Single-chain Diabodies

“…We found that conjugation of a 40-kDa branched mPEG chain strongly increased the hydrodynamic radius of the protein from 2.7 to 7.9 nm as determined by SEC. Similar observations were made for other PEGylated recombinant antibodies (42). Thus, the increased circulation time is likely caused by a reduced renal clearance.…”

Efficient Tumor Targeting with High-Affinity Designed Ankyrin Repeat Proteins: Effects of Affinity and Molecular Size

“…S3-S4). The PEG20-containing proteins eluted at an apparent molecular weight of >300 kDa, consistent with the well-known effect of PEG20 of greatly increasing the hydrodynamic radius (26,28). PEGylation had previously been shown to have no effect on the off-rate and only a small effect on the on-rate (28).…”

“…For PEGylation of the DARPins, a cysteine was introduced at the penultimate position and the protein was allowed to react with maleimide-PEG of 20, 40, or 60 kDa. The scFv fragment 4D5 was expressed and purified in E. coli SB536 as previously described (26). HER2 (first 631 amino acids of the mature protein) was kindly provided by Dr. Tim Adams and coworkers (CSIRO, Melbourne, Australia).…”

Fragments d’anticorps à domaine unique

“…Cependant, les nanobodies étant plus petits que la limite de filtration des glomérules rénaux, ils sont rapidement éliminés dans les urines. Pour éviter ce problème, une première approche consiste à coupler chimiquement une molécule de PEG (polyéthylène glycol) au dAb (PEGylation) [16]. Une solution bien plus élégante consiste à fusionner un dAb d'intérêt à un autre dAb affin pour l'albumine, protéine très abondante dans le sérum, conférant ainsi à la protéine de fusion un temps de demi-vie bien plus long, dépendant de l'affinité du dAb anti-albumine [17].…”