PASylation: a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins

A major limitation of biopharmaceutical proteins is their fast clearance from circulation via kidney filtration, which strongly hampers efficacy both in animal studies and in human therapy. We have developed conformationally disordered polypeptide chains with expanded hydrodynamic volume comprising the small residues Pro, Ala and Ser (PAS). PAS sequences are hydrophilic, uncharged biological polymers with biophysical properties very similar to poly-ethylene glycol (PEG), whose chemical conjugation to drugs is an established method for plasma half-life extension. In contrast, PAS polypeptides offer fusion to a therapeutic protein on the genetic level, permitting Escherichia coli production of fully active proteins and obviating in vitro coupling or modification steps. Furthermore, they are biodegradable, thus avoiding organ accumulation, while showing stability in serum and lacking toxicity or immunogenicity in mice. We demonstrate that PASylation bestows typical biologics, such as interferon, growth hormone or Fab fragments, with considerably prolonged circulation and boosts bioactivity in vivo.

Histological analysis of mouse organs after treatment with PAS#1(600)-hGH After completion of the mouse pharmacodynamic study, two mice from each group treated daily either with the unfused hGH or PAS#1(600)-hGH were subjected to histological analysis of kidney, liver, and spleen (mfd Diagnostics, Wendelsheim, Germany). Animals were sacrificed and the corresponding organs were isolated and stored in 10 % formalin at 4 °C. Paraffin-fixed tissue sections were prepared and stained with haematoxylin/eosin solution. Microscopic analysis was performed using an AXIO Imager A1/M1 (Carl Zeiss, Jena, Germany).
Analysis of mouse sera after PAS#1(600)-hGH treatment for immune reactivity 0.33 µg of different recombinant test proteins, all produced in E. coli and purified as described, were applied to lanes of the same 12 % SDS gel (reduced) and blotted on a nitrocellulose membrane. This Western blot was incubated with a 1:2500 dilution of sera from individual mice of the mouse pharmacodynamic study treated daily either with the unfused hGH or PAS#1(600)-hGH. Bound murine antibodies were detected with a 1:1000 dilution of goat anti-mouse polyvalent Ig/AP conjugate (A0162; Sigma/Aldrich).
On day 21 the immune response was boosted (this time using incomplete Freund's adjuvans in the second immunization experiment with PAS#1(200)-IFN) and additional plasma samples were taken 2 and 7 days thereafter. These samples were analysed by ELISA for the presence of antibodies against the respective immunogen. Briefly, a Maxisorb microtitre plate (NUNC, Roskilde, Denmark) was coated overnight with 50 µl of the corresponding immunogen at a concentration of 25 µg/ml in PBS. After washing, a dilution series of each serum sample was applied and bound murine antibodies were detected with an anti-mouse polyvalent Ig/AP conjugate (A0162; Sigma-Aldrich).

PASylation p. 3
For further analysis, mouse sera were tested for the presence of anti-PAS antibodies by Western blotting. To this end, a set of different test proteins (PAS#1(200)-IFN; PAS#1(200)-IL-1ra; PAS#5(192)-IFN; PAS#5(192)-IL-1ra; IFN; each 0.6 µg; not reduced) was subjected to SDS-PAGE and Western blotting, followed by incubation with appropriately diluted sera collected on day 28. Bound murine antibodies were detected with a anti-mouse polyvalent     After completion of the in vivo PD study, involving administration of PASylated hGH over 10 days (cf. Fig. 5C), mice were subjected to histochemical analysis of kidney, liver, and spleen.
As an example, typical tissue sections of one mouse are shown. There was no abnormality detected, neither in the mice treated with PAS#1(600)-hGH nor in those receiving the unfused recombinant hGH.

Kidney Liver Spleen
PASylation p. 14 In both cases, the strongest signals (see arrows) were observed for the unfused IFN and its PASylated versions (lanes 1, 3, and 5). In contrast, much weaker signals appeared for PAS#1 or PAS#5 if fused to the unrelated protein IL-1ra (lanes 2 and 4), indicating low crossreactivity with either PAS sequence alone. In fact, these residual signals can be explained by antibodies directed against the Strep-tag II, which were specifically detected in Fig. S13 only probed with the anti-mouse Ig/AP-conjugate, served as a negative control. Taken together with the finding from Fig. S12, no immune response was directed against epitopes within the (permuted) PAS#1 and PAS#5 polypeptides, even though the immunized mice had developed antibodies against the human IFN (and also the Strep-tag II) -as expected.