Site-Specific Polysialylation of an Antitumor Single-Chain Fv Fragment

Abstract

Protein pharmacokinetic modulation is becoming an important tool in the development of biotherapeutics. Proteins can be chemically or recombinantly modified to alter their half-lives and bioavailability to suit particular applications as well as improve side effect profiles. The most successful and clinically used approach to date is chemical conjugation with poly(ethylene glycol) polymers (PEGylation). Here, therapeutic protein half-life can be increased significantly while retaining biological function, reducing immunogenicity and cross-reaction. Naturally occurring alternatives to such synthetic polymers could have major advantages such as lower side effects due to biodegradability and metabolism. Polysialic acid (PSA) has been investigated as a pharmacokinetic modulatory biopolymer with many successful examples in preclinical and clinical development. Single-chain Fvs (scFvs) are a choice antibody format for human therapeutic antibody discovery. Because of their small size, they are rapidly eliminated from the circulation and often are rebuilt into larger proteins for drug development and a longer half-life. Here we show that chemical polysialylation can increase the half-life of an antiplacental alkaline (PLAP) and anticarcinoembryonic antigen (CEA) scFv (F1 and MFE-23, respectively) 3.4-4.9-fold, resulting in a 10.6-15.2-fold increase in blood exposure. Amine-directed coupling of the MFE-23 scFv reduced its immunoreactivity 20-fold which was resolved by site-specific polysialylation through an engineered C-terminal thiol residue. The site-specifically polysialylated MFE-23 scFv demonstrated up to 30-fold improved tumor uptake while displaying favorable tumor:normal tissue specificity. This suggests that engineering antibody fragments for site-specific polysialylation could be a useful approach to increase the half-life for a variety of therapeutic applications.

Biodistribution of a Bispecific Single-chain Diabody and Its Half-life Extended Derivatives

“…Thus, C-terminal conjugation of the scFv 4D5 with a 20-kDa PEG resulted in a 5-fold loss of affinity (14), while, for example, a PEGylated tandem scFv directed against MUC-1 showed a similar binding to MUC-1 as the wild-type tandem scFv (15). A recent study of polysialylated anti-CEA scFv MFE-23 molecules revealed that the conjugation chemistry has a strong effect on immunoreactivity (16). Random polysialylation of in average 1.4 11-kDa PSA chains per scFv resulted as compared with the unmodified scFv in a 20-fold reduction of binding in ELISA.…”

Glycoengineering of plants yields glycoproteins with polysialylation and other defined N-glycoforms

“…Clinical studies with polysialylated insulin and erythropoietin have shown that polySia is a superb alternative to polyethyleneglycol (PEG) in endowing protein drugs with longer circulatory half-lives and reduced immunogenicity (11). Furthermore, chemically polysialylated antitumor mAb fragments (scFvs) have shown significant increase in bioavailability and tumor uptake, suggesting scFvs with polySia are a viable alternative to whole IgGs (12). The highly hydrophilic nature of polySia renders a hydration pattern similar to PEG, thus creating a protective microenvironment to increase the active life of biologics and prevent them from being recognized by the immune system.…”

Site-specific enzymatic polysialylation of therapeutic proteins using bacterial enzymes

“…PSA has been chemically conjugated to a few clinically relevant therapeutic proteins and was shown to improve their circulating half-life without adversely affecting their function (10,11). Recently, site-specific chemical coupling of PSA was demonstrated on an antitumor single-chain variable region fragment (12), but this requires engineering a terminal Cys, which is not always an option for other therapeutic proteins. To our knowledge, site-specific enzymatic addition of PSA has not yet been reported.…”