Weiping Gao

In situ growth of a stoichiometric PEG-like conjugate at a protein's N-terminus with significantly improved pharmacokinetics

The challenge in the synthesis of protein-polymer conjugates for biological applications is to synthesize a stoichiometric (typically 1:1) conjugate of the protein with a monodisperse polymer, with good retention of protein activity, significantly improved pharmacokinetics and increased bioavailability, and hence improved in vivo efficacy. Here we demonstrate, using myoglobin as an example, a general route to grow a PEG-like polymer, poly(oligo(ethylene glycol) methyl ether methacrylate) [poly(OEGMA)], with low polydispersity and high yield, solely from the N-terminus of the protein by in situ atom transfer radical polymerization (ATRP) under aqueous conditions, to yield a site-specific (N-terminal) and stoichiometric conjugate (1:1). Notably, the myoglobin-poly(OEGMA) conjugate [hydrodynamic radius (Rh): 13 nm] showed a 41-fold increase in its blood exposure compared to the protein (Rh: 1.7 nm) after IV administration to mice, thereby demonstrating that comb polymers that present short oligo(ethylene glycol) side chains are a class of PEG-like polymers that can significantly improve the pharmacological properties of proteins. We believe that this approach to the synthesis of N-terminal protein conjugates of poly(OEGMA) may be applicable to a large subset of protein and peptide drugs, and thereby provide a general methodology for improvement of their pharmacological profiles.

In situ growth of a PEG-like polymer from the C terminus of an intein fusion protein improves pharmacokinetics and tumor accumulation

This paper reports a general in situ method to grow a polymer conjugate solely from the C terminus of a recombinant protein. GFP was fused at its C terminus with an intein; cleavage of the intein provided a unique thioester moiety at the C terminus of GFP that was used to install an atom transfer radical polymerization (ATRP) initiator. Subsequent in situ ATRP of oligo(ethylene glycol) methyl ether methacrylate (OEGMA) yielded a site-specific (C-terminal) and stoichiometric conjugate with high yield and good retention of protein activity. A GFP-C-poly(OEGMA) conjugate (hydrodynamic radius (Rh): 21 nm) showed a 15-fold increase in its blood exposure compared to the protein (Rh: 3.0 nm) after intravenous administration to mice. This conjugate also showed a 50-fold increase in tumor accumulation, 24 h after intravenous administration to tumor-bearing mice, compared to the unmodified protein. This approach for in situ C-terminal polymer modification of a recombinant protein is applicable to a large subset of recombinant protein and peptide drugs and provides a general methodology for improvement of their pharmacological profiles.

Sortase‐Catalyzed Initiator Attachment Enables High Yield Growth of a Stealth Polymer from the C Terminus of a Protein

Conventional methods for synthesizing protein/peptide-polymer conjugates, as a means to improve the pharmacological properties of therapeutic biomolecules, typically have drawbacks including low yield, non-trivial separation of conjugates from reactants, and lack of site- specificity, which results in heterogeneous products with significantly compromised bioactivity. To address these limitations, the use of sortase A from Staphylococcus aureus is demonstrated to site-specifically attach an initiator solely at the C-terminus of green fluorescent protein (GFP), followed by in situ growth of a stealth polymer, poly(oligo(ethylene glycol) methyl ether methacrylate) by atom transfer radical polymerization (ATRP). Sortase-catalyzed initiator attachment proceeds with high specificity and near-complete (≈95%) product conversion. Subsequent in situ ATRP in aqueous buffer produces 1:1 stoichiometric conjugates with >90% yield, low dispersity, and no denaturation of the protein. This approach introduces a simple and useful method for high yield synthesis of protein/peptide-polymer conjugates.

In situ growth of a thermoresponsive polymer from a genetically engineered elastin-like polypeptide

We report the in situgrowth of a thermoresponsive dumbbell-like polymer conjugate of a genetically engineered triblock elastin-like polypeptide (tELP). Atom transfer radical polymerization (ATRP) was used to directly grow a PEG-like polymer selectively from the first and third blocks of the tELP to form a poly(oligo(ethylene glycol) methyl ether methacrylate) (poly(OEGMA)) brush conjugate with quantitative yield. We found that in situgrowth of poly(OEGMA) from tELP significantly changed the inverse phase transition and rheological behaviors of tELP. Dynamic light scattering (DLS) and turbidity measurements as a function of temperature showed that the inverse phase transition behavior of the conjugate was not determined by the tELP but by poly(OEGMA). Oscillatory rheological measurements indicated that the conjugate started to form a physical hydrogel at a temperature of 55 °C. In vitro enzymatic degradation studies showed that the conjugate could be degraded by collagenase. These results suggest that this class of conjugates may be potentially useful as an injectable, thermoresponsive drug carrier for local drug delivery and as a scaffold for tissue engineering.

Tumor-homing, pH- and ultrasound-responsive polypeptide-doxorubicin nanoconjugates overcome doxorubicin resistance in cancer therapy

&NA; Nanomedicines hold promise in overcoming drug resistance in cancer therapy, but the in vivo therapeutic efficacy is limited by their inefficient tumor targeting, poor tumor penetration, low cellular uptake and insufficient drug release. Here we report tumor‐homing, pH‐ and ultrasound‐responsive polypeptide‐doxorubicin nanoconjugates for overcoming doxorubicin resistance. These nanoconjugates show accelerated cellular uptake and doxorubicin release and thus enhanced cytotoxicity to doxorubicin‐resistant cancer cells when exposed to ultrasound. In a doxorubicin‐resistant breast cancer mouse model, they exhibited improved tumor accumulation and penetration following exposure to ultrasound. More importantly, they displayed significantly improved in vivo anticancer efficacy without appreciable side effects post ultrasound irradiation. These findings suggest that these nanoconjugates are promising as a new class of intelligent nanomedicines for overcoming drug resistance in cancer therapy. Graphical abstract Ultrasound accelerates hydrolysis of the pH‐responsive hydrazone bond, promotes drug release and enhances tumor penetration of tumor‐homing, pH and ultrasound‐responsive polypeptide‐doxorubicin nanoconjugates for overcoming doxorubicin resistance. Figure. No caption available.

In situ growth of a C-terminal interferon-alpha conjugate of a phospholipid polymer that outperforms PEGASYS in cancer therapy.

Conjugating therapeutic proteins and peptides to poly(ethylene glycol) (PEG) can improve their pharmacokinetics and therapeutic potential. However, PEGylation suffers from non-specific conjugation, low yield and immunogenicity. Herein we report a new and general methodology to synthesize a protein-polymer conjugate with site-specificity, high yield and activity, long circulation half-life and excellent therapeutic efficacy. A phospholipid polymer, poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), was grown solely from the C-terminus of interferon-alpha to form a site-specific (C-terminal) and stoichiometric (1:1) PMPC conjugate of interferon-alpha in high yield. Notably, the PMPC conjugate showed 194- and 158-fold increases in systemic exposure and tumor uptake as compared with interferon-alpha, respectively. The in vitro antiproliferative bioactivity of the PMPC conjugate was 8.7-fold higher than that of PEGylated interferon-alpha (PEGASYS). In a murine cancer model, the PMPC conjugate completely inhibited tumor growth and cured 75% mice, whereas at the same dose, no mice treated with interferon-alpha or PEGASYS survived. We believe that this new approach to synthesize C-terminal protein conjugates of PMPC may be applicable to a large subset of protein and peptide drugs, thereby providing a general platform for the development of next-generation protein therapeutics.

Site-selective protein modification with polymers for advanced biomedical applications

Protein modification with polymers has led to intriguing and new types of bioconjugates. They combine the tunable physicochemical properties of the polymers with the specific biological activity of the proteins. These unique attributes of protein-polymer conjugates render them interesting and useful in biomedicine. However, the application potential of protein-polymer conjugates is limited by the mostly non-selective protein modification with polymers due to the lack of site-selective protein modification technology. Recent advances in site-selective protein modification and controlled polymerization have made it possible to modify proteins with polymers in a site-selective and controlled manner. In this review, recent advances in site-selective protein modification with polymers are depicted in five parts as follows: site-selective protein modification; site-selective polymer modification; site-selective in situ growth of polymers from proteins; biosafety of polymers; and biomedical applications. Site-selective protein-polymer conjugates are superior to non-selective ones in precise control of structures and functions, which makes them more interesting for advanced biomedical applications ranging from protein delivery to diagnostics. Particularly, important examples in this regard are highlighted in this review. Additionally, major challenges and future directions in this emerging research field are also discussed in the perspective section of this review.

Polymerization Induced Self-Assembly of a Site-Specific Interferon α-Block Copolymer Conjugate into Micelles with Remarkably Enhanced Pharmacology

Conjugating a hydrophilic and protein-resistant polymer to a protein is a widely used strategy to extend the in vivo half-life of the protein; however, the benefit of the half-life extension is usually limited by the bioactivity decrease. Herein we report a supramolecular self-assembly strategy of site-specific in situ polymerization induced self-assembly (SI-PISA) to address the dilemma. An amphiphilic block copolymer (POEGMA-PHPMA) was directly grown from the C-terminus of an important therapeutic protein interferon-α (IFN) to in situ form IFN-POEGMA-PHPMA conjugate micelles. Notably, the in vitro bioactivity of the micelles was 21.5-fold higher than that of the FDA-approved PEGylated interferon-α PEGASYS. Particularly, the in vivo half-life of the micelles (83.8 h) was 1.7- and 100-fold longer than those of PEGASYS (49.5 h) and IFN (0.8 h), respectively. In a tumor-bearing mouse model, the micelles completely suppressed tumor growth with 100% animal survival, whereas at the same dose, PEGASYS and IFN were much less effective. These findings suggest that SI-PISA is promising as a next-generation technology to remarkably enhance the pharmacological performance of therapeutic proteins with short circulation half-lives.

Tuning the molecular size of site-specific interferon-polymer conjugate for optimized antitumor efficacy

The covalent attachment of protein-resistant polymers to therapeutic proteins is a widely used method for extending their in vivo half-lives; however, the effect of molecular weight of polymer on the in vitro and in vivo functions of protein-polymer conjugates has not been well elucidated. Herein we report the effect of molecular weight of poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) on the in vitro and in vivo properties of C-terminal interferon-alpha (IFN)-POEGMA conjugates. Increasing the molecular weight of POEGMA decreased the in vitro activity of IFN-α but increased its thermal stability and in vivo pharmacokinetics. Intriguingly, the in vivo antitumor efficacy of IFN-α was increased by increasing the POEGMA molecular weight from ca. 20 to 60 kDa, but was not further increased by increasing the molecular weight of POEGMA from ca. 60 to 100 kDa due to the neutralization of the improved pharmacokinetics and the reduced in vitro activity. This finding offers a new viewpoint on the molecular size rationale for designing next-generation protein-polymer conjugates, which may benefit patients by reducing administration frequency and adverse reactions, and improving therapeutic efficacy.摘要治疗性蛋白质共价结合蛋白质抗性聚合物是被广泛使用的延长其体内半衰期的方法. 然而, 聚合物的分子量对蛋白质- 聚合物偶 联物体外和体内性能的影响尚未得到很好的阐明. 本文报道了聚(寡聚乙二醇甲基丙烯酸酯) (POEGMA)的分子量对C末端修饰干扰素-α (IFN)-POEGMA偶联物的体外和体内性能的影响. 增加POEGMA的分子量会降低IFN-α的体外活性, 但改善了其热稳定性和体内药代动力 学. IFN-α的体内抗肿瘤功效随着POEGMA分子量从20增加至60 kDa而增强, 但进一步提高POEGMA的分子量到100 kDa, 由于其生物活性 的降低中和了药代动力学的改善作用, 并不能进一步增强其体内抗肿瘤功效. 该发现为蛋白质- 聚合物偶联物分子尺寸效应提供了新的佐 证, 可为设计下一代蛋白质-聚合物偶联物提供参考, 通过减少给药频率和不良反应并改善治疗效果而使患者受益.