Poly(Oxanorbornene)‐Protein Conjugates Prepared by Grafting‐to ROMP as Alternatives for PEG

Abstract

PEGylation is the gold standard in protein‐polymer conjugation, improving circulation half‐life of biologics while mitigating the immune response to a foreign substance. However, preexisting anti‐PEG antibodies in healthy humans are becoming increasingly prevalent and elicitation of anti‐PEG antibodies when patients are administered with PEGylated therapeutics challenges their safety profile. In the current study, two distinct amine‐reactive poly(oxanorbornene) (PONB) imide‐based water‐soluble block co‐polymers are synthesized using ring‐opening metathesis polymerization (ROMP). The synthesized block‐copolymers include PEG‐based PONB‐PEG and sulfobetaine‐based PONB‐Zwit. The polymers are then covalently conjugated to amine residues of lysozyme (Lyz) and urate oxidase (UO) using a grafting‐to bioconjugation technique. Both Lyz‐PONB and UO‐PONB conjugates retained significant bioactivities after bioconjugation. Immune recognition studies of UO‐PONB conjugates indicated a comparable lowering of protein immunogenicity when compared to PEGylated UO. PEG‐specific immune recognition is negligible for UO‐PONB‐Zwit conjugates, as expected. These polymers provide a new alternative for PEG‐based systems that retain high levels of activity for the biologic while showing improved immune recognition profiles.

Study of uricase-polynorbornene conjugates derived from grafting-from ring-opening metathesis polymerization

“…It is worth highlighting that the optimized UO modification and all the grafting-from polymerizations that followed were carried out in the presence of an excess of UA, serving as a “site-protecting agent” (Scheme 1). 69–71 The presence of UA molecules temporarily occupying the active sites of UO effectively reduces the likelihood of modification of lysine residues within the active sites.…”

“…ELISAs were designed as previously described. 71 The antibody lot information is provided: For uricase detection, goat-derived anti-uricase antibody from Rockland (200-101-0925, Lot 37209) was used as a primary antibody and donkey anti-goat IgG (H + L) horseradish peroxidase (HRP) conjugates from Invitrogen, (A15999, Lot 967125) was used as a secondary antibody. For PEG detection, rat-derived anti-PEG antibody (MABS1962, clone rAGP6, Lot 3724609 from EMD Millipore Sigma) was used as a primary antibody and goat anti-rat IgM HRP from Invitrogen (31476, Lot XF3603641) was used as a secondary antibody.…”

Grafting-from Synthesis of Plant–Polynorbornene Biohybrid Materials

“…Biohybrid systems that combine synthetic macromolecules with biological systems, such as proteins or cells, have helped to overcome limitations, mainly in the biomedical field. − Engineered for the realm of cell-based therapies, cell–polymer biohybrids have been designed to enhance cellular protection and imbue new functionalities to cells and tissues. , Furthermore, biohybrids of proteins and polymers have demonstrated increased protein stability and a lowered potential for protein immunogenicity, making them advantageous for the development of protein therapeutics. , …”

“…We have recently pioneered the biocompatible synthesis of polynorbornene-derived polymers through ring-opening metathesis polymerization (ROMP) under physiological conditions . This approach has facilitated the rapid integration of polymers with controlled molecular weight distribution and well-defined functional block copolymers at biological interfaces. ,,,− We envisioned that employing this strategy would enable us to introduce block copolymers onto the surface of leaves of living plants without compromising their survival and preserving their versatility for future modifications. Thus, in the future, we could envision complex polymers which could serve as antimicrobial or antifungal agents, superhydrophilic polymers for moisture retention, superhydrophobic polymers for excess water protection or polymeric layers which actively respond to external factors serving as biosensors.…”