Modulation of antibody pharmacokinetics by chemical polysialylation.

Chemical coupling of a variety of polymers to therapeutic proteins has been studied as a way of improving their pharmacokinetics and pharmacodynamics in vivo. Conjugates have been shown to possess greater stability, lower immunogenicity, and a longer blood circulation time due to the chemicophysical properties of these hydrophilic long chain molecules. Naturally occurring colominic acid (polysialic acid, PSA) has been investigated as an alternative to synthetic polymers such as poly(ethylene glycol) (PEG) due to its lower toxicity and natural metabolism. Antibodies and their fragments are a good example of the types of proteins which benefit from pharmacokinetic engineering. Here, we chemically attached differing amounts and differing lengths of short (11 kDa) and longer (22 kDa) chain colominic acid molecules to the antitumor monoclonal antibody H17E2 Fab fragment. Different coupling ratios and lengths were seen to alter the electrophoretic mobility of the Fab fragment but have a minor effect on the antibody immunoreactivity toward the placental alkaline phosphatase (PLAP) antigen. Polysialylation generally increased Fab fragment blood half-life resulting in higher tumor uptake in a KB human tumor xenograft mouse model. One H17E2 Fab-PSA conjugate had over a 5-fold increase in blood exposure and over a 3-fold higher tumor uptake with only a marginal decrease in tumor/blood selectivity ratio compared to the unconjugated Fab. This conjugate also had a blood bioavailability approaching that of a whole immunoglobulin.

Polysialylated insulin: synthesis, characterization and biological activity in vivo.

Polysialic acids (PSA) (colominic acid; CA) of 22 and 39 kDa average molecular weight were oxidized with sodium periodate at carbon 7 of the nonreducing end to form an aldehyde group. The oxidized CAs (96-99% oxidation) were then reacted with the amino groups of recombinant human insulin at various CA/insulin molar ratios (25:1 to 150:1 range) for up to 48 h in the presence of sodium cyanoborohydride (reductive amination). Polysialylated insulin conjugates were precipitated (together with intact nonreacted insulin, if any) at time intervals from the reaction mixtures with ammonium sulfate, further purified by size exclusion chromatography and/or ion exchange chromatography (IEC), and the final conjugates assayed for PSA and protein. Results showed an initial rapid conjugation rate peaking at about 12 h, to form a plateau over a period of 12-48 h. Moreover, the extent of polysialylation (CA/insulin molar ratios in the conjugate) was dependent on the PSA used, the initial CA/insulin molar ratios in the reaction mixture and the time of the coupling reaction. Thus at 48 h of incubation, CA/insulin molar ratios in the conjugates were 1.60-1.74 for the 22-kDa CA and 2.37-2.45 for the 39-kDa CA. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of intact insulin and insulin reacted with non-oxidized CA for 48 h revealed well-resolved single bands which migrated similar distances in the gel. On the other hand, polysialylated (22-kDa CA) insulin yielded multiple diffused bands suggesting heterogenicity as a result of differential polysialylation. The pharmacological activity of polysialylated insulin was compared with that of intact insulin in normal female outbred T/O mice. After subcutaneous injection of intact insulin (0.3 units per mouse), blood glucose levels were reduced to nadir values at 1 h to return to normal at 3 h. In contrast, blood glucose levels in animals injected with polysialylated insulin (0.3 units or protein equivalence for polysialylated insulin), having attained nadir values also at 1 h, returned to normal levels after 6 h (39 kDa) and 9 h (22 kDa CA-insulin). It is concluded that polysialylation offers a promising strategy for the enhancement of the therapeutic value of insulin and other pharmacologically active peptides.