Advances in protein therapy are hindered by the poor stability, inadequate pharmacokinetic (PK) profiles, and immunogenicity of many therapeutic proteins. Polyethylene glycol conjugation (PEGylation) ...is the most successful strategy to date to overcome these shortcomings, and more than 10 PEGylated proteins have been brought to market. However, anti-PEG antibodies induced by treatment raise serious concerns about the future of PEGylated therapeutics. Here, we demonstrate a zwitterionic polymer network encapsulation technology that effectively enhances protein stability and PK while mitigating the immune response. Uricase modified with a comprehensive zwitterionic polycarboxybetaine (PCB) network exhibited exceptional stability and a greatly prolonged circulation half-life. More importantly, the PK behavior was unchanged, and neither anti-uricase nor anti-PCB antibodies were detected after three weekly injections in a rat model. This technology is applicable to a variety of proteins and unlocks the possibility of adopting highly immunogenic proteins for therapeutic or protective applications.
Revealing the Immunogenic Risk of Polymers Li, Bowen; Yuan, Zhefan; Hung, Hsiang‐Chien ...
Angewandte Chemie International Edition,
October 15, 2018, Letnik:
57, Številka:
42
Journal Article
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Poly(ethylene glycol) (PEG) conjugation has been the gold standard to ameliorate the pharmacokinetic (PK) and immunological profiles of proteins. PEG polymer does become immunogenic once attached to ...proteins, evoking PEG‐specific antibody (Ab) responses. The anti‐PEG Abs could cause PEGylated biologic treatments to fail and even result in lethal adverse reactions. Thus the zwitterionic poly(carboxybetaine) (PCB) has been introduced as a PEG substitute for protein modification. Addressed herein is anti‐polymer Ab induction by conjugating PEG and PCB polymers to a series of carrier proteins with escalating immunogenicity. Results indicate that titers of PEG‐specific Abs were quantitatively correlated to the immunogenicity of carrier proteins, whereas the generation of PCB‐specific Abs was minimal and insensitive to increased protein immunogenicity. This work provides insight into the immunological properties of PEG and PCB and has far‐reaching implications for the development of polymer‐protein conjugates.
Risk management: Poly(ethylene glycol) (PEG) and zwitterionic poly(carboxybetaine) (PCB) as a promising PEG substitute were explored for their immunogenic risks. A strong quantitative correlation between the level of PEG‐specific antibodies (Abs) and the immunogenicity of proteins carrying PEG was observed, revealing the propensity of PEG to become immunogenic. In contrast, PCB manifested low immunogenic risk, as PCB‐specific Abs were negligible.
Medical devices face nonspecific biofouling from proteins, cells, and microorganisms, which significantly contributes to complications and device failure. Imparting these devices with nonfouling ...capabilities remains a major challenge, particularly for those made from elastomeric polymers. Current strategies, including surface coating and copolymerization/physical blending, necessitate compromise among nonfouling properties, durability, and mechanical strength. Here, a new strategy is reported to achieve both high bulk mechanical strength and excellent surface nonfouling properties, which are typically contradictory, in one material. This is realized through a nonfouling polymeric elastomer based on zwitterionic polycarboxybetaine derivatives. By hiding both charged moieties of the zwitterionic compounds with hydrocarbon ester and tertiary amine groups, the bulk polymer itself is elastomeric and hydrophobic while its superhydrophilic surface properties are restored upon hydrolysis. This coating‐free nonfouling elastomer is a highly promising biomaterial for biomedical and engineering applications.
A stable nonfouling polymeric elastomer is demonstrated. This elastomer, based on zwitterionic polycarboxybetaine derivatives, requires no hydrophilic surface coating to achieve excellent nonfouling surface properties. By hiding both charged moieties of the zwitterionic compounds, a hydrophobic elastomer is fabricated; hydrolysis of its outermost surface then restores superhydrophilic and nonfouling surface properties, while maintaining excellent bulk mechanical characteristics.
For biotherapeutics that require multiple administrations to fully cure diseases, the induction of undesirable immune response is one common cause for the failure of their treatment. Covalent binding ...of hydrophilic polymers to proteins is commonly employed to mitigate potential immune responses. However, while this technique is proved to partially reduce the antibodies (Abs) reactive to proteins, it may induce Abs toward their associated polymers and thus result in the loss of efficacy. Zwitterionic poly(carboxybetaine) (PCB) is recently shown to improve the immunologic properties of proteins without inducing any antipolymer Abs against itself. However, it is unclear if the improved immunologic profiles can translate to better clinical outcomes since improved immunogenicity cannot directly reflect amelioration in efficacy. Here, a PCB nanocage (PCB NC) is developed, which can physically encase proteins while keeping their structure intact. PCB NC encapsulation of uricase, a highly immunogenic enzyme, is demonstrated to eradicate all the immune responses. To bridge the gap between immunogenicity and efficacy studies, the therapeutic performance of PCB NC uricase is evaluated and compared with its PEGylated counterpart in a clinical‐mimicking gouty rat model to determine any loss of efficacy evoked after five administrations.
A zwitterionic poly(carboxybetaine) (PCB) nanocage (PCB NC) is developed to physically encase proteins while keeping their structure intact. PCB NC encapsulation of uricase is manifested to eliminate all the possible immune responses. A high therapeutic performance of PCB‐NC‐shrouded uricase is demonstrated in a gouty rat model without evoking efficacy loss even after five repetitive administrations, greatly outperforming the industry‐standard PEGylated counterpart.
Although PEGylation reduces the immunogenicity of protein drugs to some extent, its limitations for highly immunogenic biotherapeutics have been demonstrated. Herein, a proactive strategy to ...alleviate the development of anti‐drug antibodies (ADAs) against protein drugs by immunomodulatory bioconjugation is reported. Rapamycin was conjugated to a PEGylated protein therapeutic via a cleavable disulfide linker. The conjugated rapamycin can be released from the bioconjugate and prevent immune responses once the bioconjugate is uptaken by antigen‐presenting cells. The immunomodulatory bioconjugate significantly reduced the titers of ADAs compared with a PEGylated protein. The inhibition of immune responses was specific to the conjugated antigen, avoiding systemic immune suppression and the risk of increased susceptibility to infections. The reported approach breaks the limitations of PEGylation by the proactive prevention of ADAs.
Immunomodulatory bioconjugation: An immunomodulator rapamycin was conjugated to a PEGylated protein therapeutic via a cleavable disulfide linker. This immunomodulatory bioconjugation effectively reduces anti‐drug antibody (ADA) generation by inducing antigen‐specific immune tolerance. The reported approach breaks the limitations of PEGylation by the proactive prevention of the generation of ADAs.
Pulmonary delivery of protein drugs into the systemic circulation is highly desirable as the lung provides a large absorption surface area and a more favorable environment for biologics compared to ...other delivery routes. However, pulmonary systemic delivery of proteins presents several challenges such as poor protein stability and limited bioavailability, especially for large proteins (molecular weight > 50 kDa), which exhibit an average bioavailability of 1% to 5% when delivered via the pulmonary route. Here, we demonstrated that with the conjugation of zwitterionic poly(carboxybetaine) (pCB) polymer, the bioavailability of organophosphate hydrolase (OPH) was significantly increased from 5% to 53%. OPH conjugated with pCB delivered through intubation-assisted intratracheal instillation (IAIS) into the lung exhibited improved pharmacokinetic properties and prophylactic efficacy against organophosphate poisoning, showing its application potential. Zwitterionic polymer conjugation provides the possibility for a favorable, non-invasive delivery of biological therapeutics into the systemic circulation.
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•Delivery of large proteins via inhalation has been limited due to their low bioavailability through the pulmonary route.•Conjugation of poly(carboxybetaine) (pCB) polymers to organophosphate hydrolase (OPH) improves its pharmacokinetics.•Pulmonary-delivered pCB-conjugated OPH protects animals from organophosphate poisoning for up to 24h
Protein, nucleic acid, and small-molecule drugs frequently require chemical modification with polymers such as polyethylene glycol (PEG), to increase their in vivo circulation time and reduce their ...immunogenicity. Previously, we developed low-immunogenic zwitterionic polycarboxybetaine (PCB) to replace PEGylation. Herein, we delineate the design principles of zwitterionic polypeptide to mimic PCB. These studies establish an EKP polypeptide composed of glutamic acid (E), lysine (K), and proline (P) as a low-immunogenic, unstructured, zwitterionic peptide. Then, we demonstrate EKP peptide as a genetically encodable fusion protein platform. The unique zwitterionic design ensures high hydration, thereby increasing hydrodynamic size and improving stability. EKP fusion proteins achieve prolonged circulation, low immunogenicity, and maintained circulation time over multiple injections, thus avoiding accelerating blood clearance (ABC). Its performance substantiates EKP as a valuable platform technology for drugs and biologics.
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•A low-immunogenic zwitterionic EKP peptide is identified and confirmed in vivo.•Therapeutic proteins fused with genetically encoded EKP improve their biophysical properties and in vivo performance.•EKP is established as a platform technology for drugs and biologics.
Although PEGylation reduces the immunogenicity of protein drugs to some extent, its limitations for highly immunogenic biotherapeutics have been demonstrated. Herein, a proactive strategy to ...alleviate the development of anti‐drug antibodies (ADAs) against protein drugs by immunomodulatory bioconjugation is reported. Rapamycin was conjugated to a PEGylated protein therapeutic via a cleavable disulfide linker. The conjugated rapamycin can be released from the bioconjugate and prevent immune responses once the bioconjugate is uptaken by antigen‐presenting cells. The immunomodulatory bioconjugate significantly reduced the titers of ADAs compared with a PEGylated protein. The inhibition of immune responses was specific to the conjugated antigen, avoiding systemic immune suppression and the risk of increased susceptibility to infections. The reported approach breaks the limitations of PEGylation by the proactive prevention of ADAs.
Immunmodulatorische Biokonjugation: Der Immunmodulator Rapamycin wurde über einen spaltbaren Disulfid‐Linker mit einem PEGylierten Proteintherapeutikum konjugiert. Diese immunmodulatorische Biokonjugation reduziert effektiv die Anti‐Wirkstoff‐Antikörper(ADA)‐Bildung durch Induktion einer Antigen‐spezifischen Immuntoleranz. Der Ansatz überwindet die Einschränkungen der PEGylierung durch proaktive Verhinderung der Bildung von ADAs.
The commonly used “stealth material” poly(ethylene glycol) (PEG) effectively promotes the pharmacokinetics of therapeutic cargos while reducing their immune response. However, recent studies have ...suggested that PEG could induce adverse reactions, including the emergence of anti‐PEG antibodies and tissue histologic changes. An alternative stealth material with no or less immunogenicity and organ toxicity is thus urgently needed. We designed a polypeptide with high zwitterion density (PepCB) as a stealth material for therapeutics. Neither tissue histological changes in liver, kidney, or spleen, nor abnormal behavior, sickness or death was induced by the synthesized polymer after high‐dosage administration for three months in rats. When conjugated to a therapeutic protein uricase, the uricase–PepCB bioconjugate showed significantly improved pharmacokinetics and immunological properties compared with uricase–PEG conjugates.
Stealth material: A zwitterionic polypeptide (PepCB) was synthesized and conjugated to therapeutics as a stealth material. PepCB exhibited superior biocompatibility and non‐immunogenicity in animal experiments compared to poly(ethylene glycol) (PEG). The new material holds great potential as a PEG alternative for formulating safer and more effective therapeutics.
Butyrylcholinesterase (BChE) is the most promising bioscavenger candidate to treat or prevent organophosphate (OP) poisoning. However, the clinical application of BChE is limited by two obstacles: an ...inadequate circulation half-life and limited sources for production. Although several modification technologies including glycosylation and PEGylation have been developed to improve its pharmacokinetics, none of them have been able to outperform blood-derived native BChE. In this work, we designed a long-circulating bioscavenger nanogel by coating equine serum-derived BChE with a zwitterionic polymer gel layer. This zwitterionic gel coating protected BChE from denaturation and degradation under harsh conditions. Notably, the nanocapsule exhibited a long circulation half-life of ~45h, a three-fold increase from the unmodified native version, enabling both therapeutic and prophylactic applications. In addition, the gel coating reduced the immunogenicity of equine BChE, unlocking the possibility to use non-human derived BChE as an OP bioscavenger in humans.
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