A Modular Method for the High-Yield Synthesis of Site-Specific Protein-Polymer Therapeutics.

A versatile method is described to engineer precisely defined protein/peptide-polymer therapeutics by a modular approach that consists of three steps: 1) fusion of a protein/peptide of interest with an elastin-like polypeptide that enables facile purification and high yields; 2) installation of a clickable group at the C terminus of the recombinant protein/peptide with almost complete conversion by enzyme-mediated ligation; and 3) attachment of a polymer by a click reaction with near-quantitative conversion. We demonstrate that this modular approach is applicable to various protein/peptide drugs and used it to conjugate them to structurally diverse water-soluble polymers that prolong the plasma circulation duration of these proteins. The protein/peptide-polymer conjugates exhibited significantly improved pharmacokinetics and therapeutic effects over the native protein/peptide upon administration to mice. The studies reported here provide a facile method for the synthesis of protein/peptide-polymer conjugates for therapeutic use and other applications.

Site-Specific Zwitterionic Polymer Conjugates of a Protein Have Long Plasma Circulation.

Many proteins suffer from suboptimal pharmacokinetics (PK) that limit their utility as drugs. The efficient synthesis of polymer conjugates of protein drugs with tunable PK to optimize their in vivo efficacy is hence critical. We report here the first study of the in vivo behavior of a site-specific conjugate of a zwitterionic polymer and a protein. To synthesize the conjugate, we first installed an initiator for atom-transfer radical polymerization (ATRP) at the N terminus of myoglobin (Mb-N-Br). Subsequently, in situ ATRP was carried out in aqueous buffer to grow an amine-functionalized polymer from Mb-N-Br. The cationic polymer was further derivatized to two zwitterionic polymers by treating the amine groups of the cationic polymer with iodoacetic acid to obtain poly(carboxybetaine methacrylate) with a one-carbon spacer (PCBMA; C1 ), and sequentially with 3-iodopropionic acid and iodoacetic acid to obtain PCBMA(mix) with a mixture of C1 and C2 spacers. The Mb-N-PCBMA polymer conjugates had a longer in vivo plasma half-life than a PEG-like comb polymer conjugate of similar molecular weights (MW). The structure of the zwitterion plays a role in controlling the in vivo behavior of the conjugate, as the PCBMA conjugate with a C1 spacer had significantly longer plasma circulation than the conjugate with a mixture of C1 and C2 spacers.

Ring-opening polymerization of prodrugs: a versatile approach to prepare well-defined drug-loaded nanoparticles.

The synthesis of polymer-drug conjugates from prodrug monomers consisting of a cyclic polymerizable group that is appended to a drug through a cleavable linker is achieved by organocatalyzed ring-opening polymerization. The monomers polymerize into well-defined polymer prodrugs that are designed to self-assemble into nanoparticles and release the drug in response to a physiologically relevant stimulus. This method is compatible with structurally diverse drugs and allows different drugs to be copolymerized with quantitative conversion of the monomers. The drug loading can be controlled by adjusting the monomer(s)/initiator feed ratio and drug release can be encoded into the polymer by the choice of linker. Initiating these monomers from a poly(ethylene glycol) macroinitiator results in amphiphilic diblock copolymers that spontaneously self-assemble into micelles with a long plasma circulation, which is useful for systemic therapy.

From hit to vial: Precision discovery and development of an imidazopyrimidine TLR7/8 agonist adjuvant formulation.

Vaccination can help prevent infection and can also be used to treat cancer, allergy, and potentially even drug overdose. Adjuvants enhance vaccine responses, but currently, the path to their advancement and development is incremental. We used a phenotypic small-molecule screen using THP-1 cells to identify nuclear factor-κB (NF-κB)-activating molecules followed by counterscreening lead target libraries with a quantitative tumor necrosis factor immunoassay using primary human peripheral blood mononuclear cells. Screening on primary cells identified an imidazopyrimidine, dubbed PVP-037. Moreover, while PVP-037 did not overtly activate THP-1 cells, it demonstrated broad innate immune activation, including NF-κB and cytokine induction from primary human leukocytes in vitro as well as enhancement of influenza and SARS-CoV-2 antigen-specific humoral responses in mice. Several de novo synthesis structural enhancements iteratively improved PVP-037's in vitro efficacy, potency, species-specific activity, and in vivo adjuvanticity. Overall, we identified imidazopyrimidine Toll-like receptor-7/8 adjuvants that act in synergy with oil-in-water emulsion to enhance immune responses.

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.

Semi-synthetic terpenoids with differential adjuvant properties as sustainable replacements for shark squalene in vaccine emulsions.

Synthetic biology has allowed for the industrial production of supply-limited sesquiterpenoids such as the antimalarial drug artemisinin and ß-farnesene. One of the only unmodified animal products used in medicine is squalene, a triterpenoid derived from shark liver oil, which when formulated into an emulsion is used as a vaccine adjuvant to enhance immune responses in licensed vaccines. However, overfishing is depleting deep-sea shark populations, leading to potential supply problems for squalene. We chemically generated over 20 squalene analogues from fermentation-derived ß-farnesene and evaluated adjuvant activity of the emulsified compounds compared to shark squalene emulsion. By employing a desirability function approach that incorporated multiple immune readouts, we identified analogues with enhanced, equivalent, or decreased adjuvant activity compared to shark squalene emulsion. Availability of a library of structurally related analogues allowed elucidation of structure-function relationships. Thus, combining industrial synthetic biology with chemistry and immunology enabled generation of sustainable terpenoid-based vaccine adjuvants comparable to current shark squalene-based adjuvants while illuminating structural properties important for adjuvant activity.

EGFR-mediated intracellular delivery of Pc 4 nanoformulation for targeted photodynamic therapy of cancer: in vitro studies.

In photodynamic therapy (PDT), the light activation of a photosensitizer leads to the generation of reactive oxygen species that can trigger various mechanisms of cell death. Harnessing this process within cancer cells enables minimally invasive yet targeted cancer treatment. With this rationale, here we demonstrate tumor-targeted delivery of a highly hydrophobic photosensitizer Pc 4 loaded within biocompatible poly(ethylene glycol)-poly(ɛ-caprolactone) block co-polymer micelles. The micelles were surface-modified with epidermal growth factor receptor (EGFR)-targeting GE11 peptides for active targeting of EGFR-overexpressing cancer cells, in vitro. Pc 4-loaded EGFR-targeted micelles were incubated with EGFR-overexpressing A431 epidermoid carcinoma cells for various time periods, to determine Pc 4 uptake by epifluorescence microscopy. The cells were subsequently photoirradiated, and PDT-induced cell death for various incubation periods was determined by MTT assay and fluorescence Live/Dead assay. Our results indicate that active EGFR targeting of the Pc 4-loaded micelles accelerates intracellular uptake of the drug. Consequently, this enhances the PDT-induced cytotoxicity within shorter time periods. FROM THE CLINICAL EDITOR: Photodynamic cancer therapy using Pc 4, a light activated and highly hydrophobic photosensitizer is demonstrated in this paper in vitro. Pc 4 was delivered in block-copolymer micelles surface-modified with GE11 peptides targeting EGFR-overexpressing cancer cells.

A Two-Step Orthogonal Chromatographic Process for Purifying the Molecular Adjuvant QS-21 with High Purity and Yield.

QS-21 is a triterpene glycoside saponin found in the bark of the Chilean soap bark tree Quillaja saponaria. It is a highly potent vaccine adjuvant that is included in two approved vaccines and has shown promise in numerous other vaccine candidates in the research and clinical pipelines. One major hurdle to the widespread use of this adjuvant is the difficulty of obtaining it in high yield and purity. Previously reported purification approaches either showed suboptimal purity and/or yield, lacked efficiency, or had strict requirement on the composition of the starting material. Here, we report the development of a new two-step orthogonal chromatographic process, consisting of a polar reversed-phase (RP) chromatography step followed by a hydrophilic interaction chromatography (HILIC) step, for purifying QS-21 from a commercially available Quillaja saponaria bark extract with high yield and > 97% purity. This process makes available a simple and efficient method for obtaining highly pure QS-21 from saponin-enriched bark extract.

Development of thermostable vaccine adjuvants.

INTRODUCTION: The importance of vaccine thermostability has been discussed in the literature. Nevertheless, the challenge of developing thermostable vaccine adjuvants has sometimes not received appropriate emphasis. Adjuvants comprise an expansive range of particulate and molecular compositions, requiring innovative thermostable formulation and process development approaches. AREAS COVERED: Reports on efforts to develop thermostable adjuvant-containing vaccines have increased in recent years, and substantial progress has been made in enhancing the stability of the major classes of adjuvants. This narrative review summarizes the current status of thermostable vaccine adjuvant development and looks forward to the next potential developments in the field. EXPERT OPINION: As adjuvant-containing vaccines become more widely used, the unique challenges associated with developing thermostable adjuvant formulations merit increased attention. In particular, more focused efforts are needed to translate promising proof-of-concept technologies and formulations into clinical products.

A brush-polymer conjugate of exendin-4 reduces blood glucose for up to five days and eliminates poly(ethylene glycol) antigenicity.

The delivery of therapeutic peptides and proteins is often challenged by a short half-life, and thus the need for frequent injections that limit efficacy, reduce patient compliance and increase treatment cost. Here, we demonstrate that a single subcutaneous injection of site-specific (C-terminal) conjugates of exendin-4 (exendin) - a therapeutic peptide that is clinically used to treat type 2 diabetes - and poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA) with precisely controlled molecular weights lowered blood glucose for up to 120 h in fed mice. Most notably, we show that an exendin-C-POEGMA conjugate with an average of 9 side-chain ethylene glycol (EG) repeats exhibits significantly lower reactivity towards patient-derived anti-poly(ethylene glycol) (PEG) antibodies than two FDA-approved PEGylated drugs, and that reducing the side-chain length to 3 EG repeats completely eliminates PEG antigenicity without compromising in vivo efficacy. Our findings establish the site-specific conjugation of POEGMA as a next-generation PEGylation technology for improving the pharmacological performance of traditional PEGylated drugs, whose safety and efficacy are hindered by pre-existing anti-PEG antibodies in patients.

Protein-polymer conjugation-moving beyond PEGylation.

In this review, we summarize-from a materials science perspective-the current state of the field of polymer conjugates of peptide and protein drugs, with a focus on polymers that have been developed as alternatives to the current gold standard, poly(ethylene glycol) (PEG). PEGylation, or the covalent conjugation of PEG to biological therapeutics to improve their therapeutic efficacy by increasing their circulation half-lives and stability, has been the gold standard in the pharmaceutical industry for several decades. After years of research and development, the limitations of PEG, specifically its non-degradability and immunogenicity have become increasingly apparent. While PEG is still currently the best polymer available with the longest clinical track record, extensive research is underway to develop alternative materials in an effort to address these limitations of PEG. Many of these alternative materials have shown promise, though most of them are still in an early stage of development and their in vivo distribution, mechanism of degradation, route of elimination and immunogenicity have not been investigated to a similar extent as for PEG. Thus, further in-depth in vivo testing is essential to validate whether any of the alternative materials discussed in this review qualify as a replacement for PEG.