An official website of the United States government
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.
more »
« less
Structure of an anti-PEG antibody reveals an open ring that captures highly flexible PEG polymers
Abstract Polyethylene glycol (PEG) is a polymer routinely used to modify biologics and nanoparticles to prolong blood circulation and reduce immunogenicity of the underlying therapeutic. However, several PEGylated therapeutics induce the development of anti-PEG antibodies (APA), leading to reduced efficacy and increased adverse events. Given the highly flexible structure of PEG, how APA specifically bind PEG remains poorly understood. Here, we report a crystal structure illustrating the structural properties and conformation of the APA 6-3 Fab bound to the backbone of PEG. The structure reveals an open ring-like sub-structure in the Fab paratope, whereby PEG backbone is captured and then stabilized via Van der Waals interactions along the interior and exterior of the ring paratope surface. Our finding illustrates a strategy by which antibodies can bind highly flexible repeated structures that lack fixed conformations, such as polymers. This also substantially advances our understanding of the humoral immune response generated against PEG.
more »
« less
- Award ID(s):
- 1810168
- PAR ID:
- 10191282
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Communications Chemistry
- Volume:
- 3
- Issue:
- 1
- ISSN:
- 2399-3669
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Nonhormonal products for on-demand contraception are a global health technology gap; this unmet need motivated us to pursue the use of sperm-binding monoclonal antibodies to enable effective on-demand contraception. Here, using the cGMP-compliant Nicotiana -expression system, we produced an ultrapotent sperm-binding IgG antibody possessing 6 Fab arms per molecule that bind a well-established contraceptive antigen target, CD52g. We term this hexavalent antibody “Fab-IgG-Fab” (FIF). The Nicotiana -produced FIF had at least 10-fold greater sperm-agglutination potency and kinetics than the parent IgG, while preserving Fc-mediated trapping of individual spermatozoa in mucus. We formulated the Nicotiana -produced FIF into a polyvinyl alcohol–based water-soluble contraceptive film and evaluated its potency in reducing progressively motile sperm in the sheep vagina. Two minutes after vaginal instillation of human semen, no progressively motile sperm were recovered from the vaginas of sheep receiving FIF Film. Our work supports the potential of multivalent contraceptive antibodies to provide safe, effective, on-demand nonhormonal contraception.more » « less
-
Abstract As upstream product titers increase, the downstream chromatographic capture step has become a significant “downstream bottleneck.” Precipitation becomes more attractive under these conditions as the supersaturation driving force increases with the ever‐increasing titer. In this study, two precipitating reagents with orthogonal mechanisms, polyethylene glycol (PEG) as a volume excluder and zinc chloride (ZnCl2) as a cross linker, were examined as precipitants for two monoclonal antibodies (mAbs), one stable and the other aggregation‐prone, in purified drug substance and harvested cell culture fluid forms. Manual batch solubility and redissolution experiments were performed as scouting experiments. A high throughput (HTP) liquid handling system was used to investigate the design space as fully as possible while reducing time, labor, and material requirements. Precipitation and redissolution were studied by systematically varying the concentrations of PEG and ZnCl2to identify combinations that resulted in high yield and good quality for the stable mAb; PEG concentrations in the range 7–7.5 wt/vol% together with 10 mM ZnCl2gave a yield of 97% and monomer contents of about 93%. While yield for the unstable mAb was high, quality was not acceptable. Performance at selected conditions was further corroborated for the stable mAb using a continuous tubular precipitation reactor at the laboratory scale. The HTP automation system was a powerful tool for locating desired (customized) conditions for antibodies of different physicochemical properties.more » « less
-
null (Ed.)As upstream product titers increase, the downstream chromatographic capture step has become a significant “downstream bottleneck.” Precipitation becomes more attractive under these conditions as the supersaturation driving force increases with the ever-increasing titer. In this study, two precipitating reagents with orthogonal mechanisms, polyethylene glycol (PEG) as a volume excluder and zinc chloride (ZnCl2) as a cross linker, were examined as precipitants for two monoclonal antibodies (mAbs), one stable and the other aggregation-prone, in purified drug substance and harvested cell culture fluid forms. Manual batch solubility and redissolution experiments were performed as scouting experiments. A high throughput (HTP) liquid handling system was used to investigate the design space as fully as possible while reducing time, labor, and material requirements. Precipitation and redissolution were studied by systematically varying the concentrations of PEG and ZnCl2 to identify combinations that resulted in high yield and good quality for the stable mAb; PEG concentrations in the range 7–7.5 wt/vol% together with 10 mM ZnCl2 gave a yield of 97% and monomer contents of about 93%. While yield for the unstable mAb was high, quality was not acceptable. Performance at selected conditions was further corroborated for the stable mAb using a continuous tubular precipitation reactor at the laboratory scale. The HTP automation system was a powerful tool for locating desired (customized) conditions for antibodies of different physicochemical properties.more » « less
-
Abstract Hydrogels are often synthesized through photoinitiated step‐, chain‐, and mixed‐mode polymerizations, generating diverse network topologies and resultant material properties that depend on the underlying network connectivity. While many photocrosslinking reactions are available, few afford controllable connectivity of the hydrogel network. Herein, a versatile photochemical strategy is introduced for tuning the structure of poly(ethylene glycol) (PEG) hydrogels using macromolecular monomers functionalized with maleimide and styrene moieties. Hydrogels are prepared along a gradient of topologies by varying the ratio of step‐growth (maleimide dimerization) to chain‐growth (maleimide‐styrene alternating copolymerization) network‐forming reactions. The initial PEG content and final network physical properties (e.g., modulus, swelling, diffusivity) are tailored in an independent manner, highlighting configurable gel mechanics and reactivity. These photochemical reactions allow high‐fidelity photopatterning and 3D printing and are compatible with 2D and 3D cell culture. Ultimately, this photopolymer chemistry allows facile control over network connectivity to achieve adjustable material properties for broad applications.more » « less
