cART Restores Transient Responsiveness to IFN Type 1 in HIV-Infected Humanized Mice.

The lack of a human immunodeficiency virus (HIV) cure has heightened interest in immunotherapy. As such, type I interferons (IFNs), in particular, IFN alpha (IFN-α), have gained renewed attention. However, HIV pathogenesis is driven by sustained IFN-mediated immune activation, and the use of IFNs is rather controversial. The following questions therein remain: (i) which IFN-α subtype to use, (ii) at which regimen, and (iii) at what time point in HIV infection it might be beneficial. Here, we used IFN-α14 modified by PASylation for its long half-life in vivo to eventually treat HIV infection. We defined the IFN dosing regimen based on the maximum increase in interferon-stimulated gene (ISG) expression 6 h after its administration and a return to baseline of ubiquitin-specific protease 18 (USP18) prior to the next dose. Notably, USP18 is the major negative regulator of type I IFN signaling. HIV infection resulted in increased ISG expression levels in humanized mice. Intriguingly, high baseline ISG levels correlated with lower HIV load. No effect was observed on HIV replication when PASylated IFN-α14 was administered in the chronic phase. However, combined antiretroviral therapy (cART) restored responsiveness to IFN, and PASylated IFN-α14 administered during analytical cART interruption resulted in a transiently lower HIV burden than in the mock-treated mice. In conclusion, cART-mediated HIV suppression restored transient IFN responsiveness and provided a potential window for immunoenhancing therapies in the context of analytical cART interruption. IMPORTANCE cART is highly efficient in suppressing HIV replication in HIV-infected patients and has resulted in a dramatic reduction in morbidity and mortality in HIV-infected people, yet it does not cure HIV infection. In addition, cART has several disadvantages. Thus, the HIV research community is exploring novel ways to control HIV infection for longer periods without cART. Here, we explored novel, long-acting IFN-α14 for its efficacy to control HIV replication in HIV-infected humanized mice. We found that IFN-α14 had no effect on chronic HIV infection. However, when mice were treated first with cART, we observed a transiently restored responsiveness to INF and a transiently lower HIV burden after stopping cART. These data emphasize (i) the value of cART-mediated HIV suppression and immune reconstitution in creating a window of opportunity for exploring novel immunotherapies, (ii) the potential of IFNs for constraining HIV, and (iii) the value of humanized mice for exploring novel immunotherapies.

PASylation improves pharmacokinetic of liposomes and attenuates anti-PEG IgM production: An alternative to PEGylation.

PASylation, which was recently reported as the conjugation of pharmacologically active compounds with polypeptide sequences mainly made of proline, alanine and serine, has been proposed as an alternative to PEGylation. In this study, we designed PAS-modified liposomes (PASylated liposomes) and studied the effect of the incorporation of PAS-lipid on the stability and pharmacokinetic properties of liposomes, and compared them both in vitro and in vivo to PEGylated liposomes. Results showed that PASylated liposomes modified with single-chained PAS-lipid C16-(PA3)7 (SC-PAS-Lip) showed comparable storage and serum stability to PEGylated liposomes (PEG-Lip), and a significantly decreased macrophage uptake compared with unmodified liposomes. SC-PAS-Lip displayed long circulating pharmacokinetic profile which was not impacted by the repeated administration of liposomes, and they were less likely to induce the production of anti-PEG IgM compared with PEGylated liposomes, presenting PASylation as an alternative liposome modification strategy to PEGylation.

Efficient secretory production of proline/alanine/serine (PAS) biopolymers in Corynebacterium glutamicum yielding a monodisperse biological alternative to polyethylene glycol (PEG).

BACKGROUND: PAS biopolymers are recombinant polypeptides comprising the small uncharged L-amino acids Pro, Ala and/or Ser which resemble the widely used poly-ethylene glycol (PEG) in terms of pronounced hydrophilicity. Likewise, their random chain behaviour in physiological solution results in a strongly expanded hydrodynamic volume. Thus, apart from their use as fusion partner for biopharmaceuticals to achieve prolonged half-life in vivo, PAS biopolymers appear attractive as substitute for PEG-or other poorly degradable chemical polymers-in many areas. As a prerequisite for the wide application of PAS biopolymers at affordable cost, we have established their highly efficient biotechnological production in Corynebacterium glutamicum serving as a well characterized bacterial host organism. RESULTS: Using the CspA signal sequence, we have secreted two representative PAS biopolymers as polypeptides with ~ 600 and ~ 1200 amino acid residues, respectively. Both PAS biopolymers were purified from the culture supernatant by means of a simple downstream process in a truly monodisperse state as evidenced by ESI-MS. Yields after purification were up to ≥ 4 g per liter culture, with potential for further increase by strain optimization as well as fermentation and bioprocess development. Beyond direct application as hydrocolloids or to exploit their rheological properties, such PAS biopolymers are suitable for site-specific chemical conjugation with pharmacologically active molecules via their unique terminal amino or carboxyl groups. To enable the specific activation of the carboxylate, without interference by the free amino group, we generated a blocked N-terminus for the PAS(1200) polypeptide simply by introducing an N-terminal Gln residue which, after processing of the signal peptide, was cyclised to a chemically inert pyroglutamyl group upon acid treatment. The fact that PAS biopolymers are genetically encoded offers further conjugation strategies via incorporation of amino acids with reactive side chains (e.g., Cys, Lys, Glu/Asp) at defined positions. CONCLUSIONS: Our new PAS expression platform using Corynex® technology opens the way to applications of PASylation® technology in multiple areas such as the pharmaceutical industry, cosmetics and food technology.

Post-VX exposure treatment of rats with engineered phosphotriesterases.

The biologically stable and highly toxic organophosphorus nerve agent (OP) VX poses a major health threat. Standard medical therapy, consisting of reactivators and competitive muscarinic receptor antagonists, is insufficient. Recently, two engineered mutants of the Brevundimonas diminuta phosphotriesterase (PTE) with enhanced catalytic efficiency (kcat/KM = 21 to 38 × 106 M-1 min-1) towards VX and a preferential hydrolysis of the more toxic P(-) enantiomer were described: PTE-C23(R152E)-PAS(100)-10-2-C3(I106A/C59V/C227V/E71K)-PAS(200) (PTE-2), a single-chain bispecific enzyme with a PAS linker and tag having enlarged substrate spectrum, and 10-2-C3(C59V/C227V)-PAS(200) (PTE-3), a stabilized homodimeric enzyme with a double PASylation tag (PAS-tag) to reduce plasma clearance. To assess in vivo efficacy, these engineered enzymes were tested in an anesthetized rat model post-VX exposure (~ 2LD50) in comparison with the recombinant wild-type PTE (PTE-1), dosed at 1.0 mg kg-1 i.v.: PTE-2 dosed at 1.3 mg kg-1 i.v. (PTE-2.1) and 2.6 mg kg-1 i.v. (PTE-2.2) and PTE-3 at 1.4 mg kg-1 i.v. Injection of the mutants PTE-2.2 and PTE-3, 5 min after s.c. VX exposure, ensured survival and prevented severe signs of a cholinergic crisis. Inhibition of erythrocyte acetylcholinesterase (AChE) could not be prevented. However, medulla oblongata and diaphragm AChE activity was partially preserved. All animals treated with the wild-type enzyme, PTE-1, showed severe cholinergic signs and died during the observation period of 180 min. PTE-2.1 resulted in the survival of all animals, yet accompanied by severe signs of OP poisoning. This study demonstrates for the first time efficient detoxification in vivo achieved with low doses of heterodimeric PTE-2 as well as PTE-3 and indicates the suitability of these engineered enzymes for the development of highly effective catalytic scavengers directed against VX.

Direct Affinity Purification of Long-Acting PASylated Proteins with Therapeutic Potential Using L-Prolinamide for Mild Elution.

Both insufficient plasma half-life (circulation for only few hours or less) and laborious downstream purification can be bottleneck for biological drug development. We report a novel strategy for the efficient and gentle affinity purification of pharmacologically relevant proteins modified by PASylation for prolonged action in vivo. We previously described antibodies specific for Pro/Ala-rich sequences (PAS) covering a range of binding characteristics. Our present approach relies on a chromatography matrix functionalized with a low-affinity PAS-specific antibody Fab fragment for specific adsorption of the PASylated protein from a macromolecular mixture. With the complete absence of hydrophobic/aromatic or ionic groups in the PAS sequence epitope, binding is mediated by Van der Waals contacts and distinct hydrogen bonds only. Surprisingly, selective competitive elution is achieved by application of the highly soluble and biologically inactive imino acid derivative L-prolinamide. Based on the specific but strongly dynamic biomolecular interaction, our procedure allows the direct one-step purification of PASylated proteins from a cell extract or culture supernatant while avoiding harsh elution conditions as they are often needed for conventional affinity chromatography.

PASylation of IL-1 receptor antagonist (IL-1Ra) retains IL-1 blockade and extends its duration in mouse urate crystal-induced peritonitis.

Interleukin-1 (IL-1) is a key mediator of inflammation and immunity. Naturally-occurring IL-1 receptor antagonist (IL-1Ra) binds and blocks the IL-1 receptor-1 (IL-1R1), preventing signaling. Anakinra, a recombinant form of IL-1Ra, is used to treat a spectrum of inflammatory diseases. However, anakinra is rapidly cleared from the body and requires daily administration. To create a longer-lasting alternative, PASylated IL-1Ra (PAS-IL-1Ra) has been generated by in-frame fusion of a long, defined-length, N-terminal Pro/Ala/Ser (PAS) random-coil polypeptide with IL-1Ra. Here, we compared the efficacy of two PAS-IL-1Ra molecules, PAS600-IL-1Ra and PAS800-IL-1Ra (carrying 600 and 800 PAS residues, respectively), with that of anakinra in mice. PAS600-IL-1Ra displayed markedly extended blood plasma levels 3 days post-administration, whereas anakinra was undetectable after 24 h. We also studied PAS600-IL-1Ra and PAS800-IL-1Ra for efficacy in monosodium urate (MSU) crystal-induced peritonitis. 5 days post-administration, PAS800-IL-1Ra significantly reduced leukocyte influx and inflammatory markers in MSU-induced peritonitis, whereas equimolar anakinra administered 24 h before MSU challenge was ineffective. The 6-h pretreatment with equimolar anakinra or PAS800-IL-1Ra before MSU challenge similarly reduced inflammatory markers. In cultured A549 lung carcinoma cells, anakinra, PAS600-IL-1Ra, and PAS800-IL-Ra reduced IL-1α-induced IL-6 and IL-8 levels with comparable potency. In human peripheral blood mononuclear cells, these molecules suppressed Candida albicans-induced production of the cancer-promoting cytokine IL-22. Surface plasmon resonance analyses revealed significant binding between PAS-IL-1Ra and IL-1R1, although with a slightly lower affinity than anakinra. These results validate PAS-IL-1Ra as an active IL-1 antagonist with marked in vivo potency and a significantly extended half-life compared with anakinra.

Catalytic activity and stereoselectivity of engineered phosphotriesterases towards structurally different nerve agents in vitro.

Highly toxic organophosphorus nerve agents, especially the extremely stable and persistent V-type agents such as VX, still pose a threat to the human population and require effective medical countermeasures. Engineered mutants of the Brevundimonas diminuta phosphotriesterase (BdPTE) exhibit enhanced catalytic activities and have demonstrated detoxification in animal models, however, substrate specificity and fast plasma clearance limit their medical applicability. To allow better assessment of their substrate profiles, we have thoroughly investigated the catalytic efficacies of five BdPTE mutants with 17 different nerve agents using an AChE inhibition assay. In addition, we studied one BdPTE version that was fused with structurally disordered PAS polypeptides to enable delayed plasma clearance and one bispecific BdPTE with broadened substrate spectrum composed of two functionally distinct subunits connected by a PAS linker. Measured kcat/KM values were as high as 6.5 and 1.5 × 108 M-1 min-1 with G- and V-agents, respectively. Furthermore, the stereoselective degradation of VX enantiomers by the PASylated BdPTE-4 and the bispecific BdPTE-7 were investigated by chiral LC-MS/MS, resulting in a several fold faster hydrolysis of the more toxic P(-) VX stereoisomer compared to P(+) VX. In conclusion, the newly developed enzymes BdPTE-4 and BdPTE-7 have shown high catalytic efficacy towards structurally different nerve agents and stereoselectivity towards the toxic P(-) VX enantiomer in vitro and offer promise for use as bioscavengers in vivo.

Challenges and advancements in the pharmacokinetic enhancement of therapeutic proteins.

Nowadays, proteins are frequently administered as therapeutic agents in human diseases. However, the main challenge regarding the clinical application of therapeutic proteins is short circulating plasma half-life that leads to more frequent injections for maintaining therapeutic plasma levels, increased therapy costs, immunogenic reactions, and low patient compliance. So, the development of novel strategies to enhance the pharmacokinetic profile of therapeutic proteins has attracted great attention in pharmaceuticals. So far, several techniques, each with their pros and cons, have been developed including chemical bonding to polymers, hyper glycosylation, Fc fusion, human serum albumin fusion, and recombinant PEG mimetics. These techniques mainly classify into three strategies; (i) the endosomal recycling of neonatal Fc receptor which is observed for immunoglobulins and albumin, (ii) decrease in receptor-mediated clearance, and (iii) increase in hydrodynamic radius through chemical and genetic modifications. Recently, novel PEG mimetic peptides like proline/alanine/serine repeat sequences are designed to overcome pitfalls associated with the previous technologies. Biodegradability, lack of or low immunogenicity, product homogeneity, and a simple production process, currently make these polypeptides as the preferred technology for plasma half-life extension of therapeutic proteins. In this review, challenges and pitfalls in the pharmacokinetic enhancement of therapeutic proteins using PEG-mimetic peptides will be discussed in detail.

PASylated Thymosin α1: A Long-Acting Immunostimulatory Peptide for Applications in Oncology and Virology.

Thymosin α1 (Tα1) is an immunostimulatory peptide for the treatment of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections and used as an immune enhancer, which also offers prospects in the context of COVID-19 infections and cancer. Manufacturing of this N-terminally acetylated 28-residue peptide is demanding, and its short plasma half-life limits in vivo efficacy and requires frequent dosing. Here, we combined the PASylation technology with enzymatic in situ N-acetylation by RimJ to produce a long-acting version of Tα1 in Escherichia coli at high yield. ESI-MS analysis of the purified fusion protein indicated the expected composition without any signs of proteolysis. SEC analysis revealed a 10-fold expanded hydrodynamic volume resulting from the fusion with a conformationally disordered Pro/Ala/Ser (PAS) polypeptide of 600 residues. This size effect led to a plasma half-life in rats extended by more than a factor 8 compared to the original synthetic peptide due to retarded kidney filtration. Our study provides the basis for therapeutic development of a next generation thymosin α1 with prolonged circulation. Generally, the strategy of producing an N-terminally protected PASylated peptide solves three major problems of peptide drugs: (i) instability in the expression host, (ii) rapid degradation by serum exopeptidases, and (iii) low bioactivity because of fast renal clearance.

Surface protein engineering increases the circulation time of a cell membrane-based nanotherapeutic.

Mammalian cell membranes are often incompatible with chemical modifications typically used to increase circulation half-life. Using cellular nanoghosts as a model, we show that proline-alanine-serine (PAS) peptide sequences expressed on the membrane surface can extend the circulation time of a cell membrane derived nanotherapeutic. Membrane expression of a PAS 40 repeat sequence decreased protein binding and resulted in a 90% decrease in macrophage uptake when compared with non-PASylated controls (P ≤ 0.05). PASylation also extended circulation half-life (t1/2 = 37 h) compared with non-PASylated controls (t1/2 = 10.5 h) (P ≤ 0.005), resulting in ~7-fold higher in vivo serum concentrations at 24 h and 48 h (P ≤ 0.005). Genetically engineered membrane expression of PAS repeats may offer an alternative to PEGylation and provide extended circulation times for cellular membrane-derived nanotherapeutics.

Selection of an Anticalin® against the membrane form of Hsp70 via bacterial surface display and its theranostic application in tumour models.

We describe the selection of Anticalins against a common tumour surface antigen, human Hsp70, using functional display on live Escherichia coli cells as fusion with a truncated EspP autotransporter. While found intracellularly in normal cells, Hsp70 is frequently exposed in a membrane-bound state on the surface of tumour cells and, even more pronounced, in metastases or after radiochemotherapy. Employing a recombinant Hsp70 fragment comprising residues 383-548 as the target, Anticalins were selected from a naïve bacterial library. The Anticalin with the highest affinity (KD=13 nm), as determined towards recombinant full-length Hsp70 by real-time surface plasmon resonance analysis, was improved to KD=510 pm by doped random mutagenesis and another cycle of E. coli surface display, followed by rational combination of mutations. This Anticalin, which recognises a linear peptide epitope located in the interdomain linker of Hsp70, was demonstrated to specifically bind Hsp70 in its membrane-associated form in immunofluorescence microscopy and via flow cytometry using the FaDu cell line, which is positive for surface Hsp70. The radiolabelled and PASylated Anticalin revealed specific tumour accumulation in xenograft mice using positron emission tomography (PET) imaging. Furthermore, after enzymatic coupling to the protein toxin gelonin, the Anticalin showed potent cytotoxicity on FaDu cells in vitro.

The polypeptide biophysics of proline/alanine-rich sequences (PAS): Recombinant biopolymers with PEG-like properties.

PAS polypeptides comprise long repetitive sequences of the small L-amino acids proline, alanine and/or serine that were developed to expand the hydrodynamic volume of conjugated pharmaceuticals and prolong their plasma half-life by retarding kidney filtration. Here, we have characterized the polymer properties both of the free polypeptides and in fusion with the biopharmaceutical IL-1Ra. Data from size exclusion chromatography, dynamic light scattering, circular dichroism spectroscopy and quantification of hydrodynamic and polar properties demonstrate that the biosynthetic PAS polypeptides exhibit random coil behavior in aqueous solution astonishingly similar to the chemical polymer poly-ethylene glycol (PEG). The solvent-exposed PAS peptide groups, in the absence of secondary structure, account for strong hydrophilicity, with negligible contribution by the Ser side chains. Notably, PAS polypeptides exceed PEG of comparable molecular mass in hydrophilicity and hydrodynamic volume while exhibiting lower viscosity. Their uniform monodisperse composition as genetically encoded polymers and their biological nature, offering biodegradability, render PAS polypeptides a promising PEG mimetic for biopharmaceutical applications.

PASylation technology improves recombinant interferon-ß1b solubility, stability, and biological activity.

Recombinant interferon-ß1b (IFN-ß1b) is an effective remedy against multiple sclerosis and other diseases. However, use of small polypeptide (molecular weight is around 18.5 kDa) is limited due to poor solubility, stability, and short half-life in systemic circulation. To solve this problem, we constructed two variants of PASylated IFN-ß1b, with PAS sequence at C- or N-terminus of IFN-ß1b. The PAS-modified proteins demonstrated 4-fold increase in hydrodynamic volume of the molecule combined with 2-fold increase of in vitro biological activity, as well as advanced stability and solubility of the protein in solution as opposed to unmodified IFN-ß1b. Our results demonstrate that PASylation has a positive impact on stability, solubility, and functional activity of IFN-ß1b and potentially might improve pharmacokinetic properties of the molecule as a therapeutic agent.

Treatment of diet-induced lipodystrophic C57BL/6J mice with long-acting PASylated leptin normalises insulin sensitivity and hepatic steatosis by promoting lipid utilisation.

AIMS/HYPOTHESIS: Recombinant leptin offers a viable treatment for lipodystrophy (LD) syndromes. However, due to its short plasma half-life, leptin replacement therapy requires at least daily subcutaneous (s.c.) injections. Here, we optimised this treatment strategy in LD mice by using a novel leptin version with extended plasma half-life using PASylation technology. METHODS: A long-acting leptin version was prepared by genetic fusion with a 600 residue polypeptide made of Pro, Ala and Ser (PASylation), which enlarges the hydrodynamic volume and, thus, retards renal filtration, allowing less frequent injection. LD was induced in C57BL/6J mice by feeding a diet supplemented with conjugated linoleic acid (CLA). Chronic and acute effects of leptin treatment were assessed by evaluating plasma insulin levels, insulin tolerance, histological liver sections, energy expenditure, energy intake and body composition. RESULTS: In a cohort of female mice, 4 nmol PAS-leptin (applied via four s.c. injections every 3 days) successfully alleviated the CLA-induced LD phenotype, which was characterised by hyperinsulinaemia, insulin intolerance and hepatosteatosis. The same injection regimen had no measurable effect when unmodified recombinant leptin was administered at an equivalent dose. In a cohort of LD males, a single s.c. injection of PAS-leptin did not affect energy expenditure but inhibited food intake and promoted a shift in fuel selection towards preferential fat oxidation, which mechanistically substantiates the metabolic improvements. CONCLUSIONS/INTERPRETATION: The excellent pharmacological properties render PASylated leptin an agent of choice for refining both animal studies and therapeutic strategies in the context of LD syndromes and beyond.

Development of a Clonal and High-Yield Mammalian Cell Line for the Manufacturing of a Hyperactive Human DNase I with Extended Plasma Half-Life Using PASylation® Technology.

Cumulative evidence from several pre-clinical studies suggests that restoration of plasma DNase activity in a thrombo-inflammatory state may improve clinical outcomes. Following injury, hyperactivated immune cells release large amounts of granular proteins together with DNA, which often accumulate in the surrounding environment in so-called neutrophil extracellular traps (NETs). Degradation of excess NETs by systemic DNase administration offers a promising therapeutic approach to ameliorate inflammation and dissolve intravascular clots. In order to expand the therapeutic utility of human DNase I, a variant of the enzyme was developed that has both a prolonged systemic half-life and a higher catalytic activity compared to Dornase alfa (Pulmozyme®), the recombinant form of DNase I approved for inhaled therapy of cystic fibrosis. The hyperactive enzyme was "PASylated" by genetic fusion with a strongly hydrophilic and biodegradable PAS-polypeptide to increase its hydrodynamic volume and retard kidney filtration. A stable TurboCell™ CHO-K1-based cell line was generated which is suitable for the future production of PASylated DNase I according to good manufacturing practice (GMP). Furthermore, a robust bioprocess strategy was devised and an effective downstream process was developed. The final protein product is characterized by excellent purity, favorable physicochemical properties, a 14-fold higher DNA-degrading activity than Dornase alfa and a sustained pharmacokinetic profile, with a 22-fold slower clearance in rats.

Half-life extension via ABD-fusion leads to higher tumor uptake of an affibody-drug conjugate compared to PAS- and XTENylation.

A critical parameter during the development of protein therapeutics is to endow them with suitable pharmacokinetic and pharmacodynamic properties. Small protein drugs are quickly eliminated by kidney filtration, and in vivo half-life extension is therefore often desired. Here, different half-life extension technologies were studied where PAS polypeptides (PAS300, PAS600), XTEN polypeptides (XTEN288, XTEN576), and an albumin binding domain (ABD) were compared for half-life extension of an anti-human epidermal growth factor receptor 2 (HER2) affibody-drug conjugate. The results showed that extension with the PAS or XTEN polypeptides or the addition of the ABD lowered the affinity for HER2 to some extent but did not negatively affect the cytotoxic potential. The half-lives in mice ranged from 7.3 h for the construct including PAS300 to 11.6 h for the construct including PAS600. The highest absolute tumor uptake was found for the construct including the ABD, which was 60 to 160% higher than the PASylated or XTENylated constructs, even though it did not have the longest half-life (9.0 h). A comparison of the tumor-to-normal-organ ratios showed the best overall performance of the ABD-fused construct. In conclusion, PASylation, XTENylation, and the addition of an ABD are viable strategies for half-life extension of affibody-drug conjugates, with the best performance observed for the construct including the ABD.

In vivo neutralization of colchicine toxicity by a PASylated anticalin in a rat model.

We have investigated the pharmacokinetics (PK) and in vivo activity of an Anticalin exhibiting picomolar affinity towards colchicine, a plant toxin with low tolerable dose in humans. PK analysis of the 20-kDa "Colchicalin" protein in male Sprague Dawley rats (n = 3) revealed a very short plasma half-life (3.5 min), which was prolonged 21-fold via genetic fusion with a 200-residue Pro/Ala sequence (PASylation). The scavenging activity of the PASylated Colchicalin was investigated over 3.5 h via stoichiometric application following a sub-toxic i.v. dose of colchicine on anesthetized rats (n = 2) leading to a rapid rise in total plasma colchicine concentration. We then established a 14-day intoxication model in rats (n = 3) at a 30 mg/kg p.o. colchicine dose which was characterized by severe weight loss, elevated neutrophil-to-lymphocyte ratio and shortened survival. PASylated Colchicalin administration at 4.2% of the neutralizing dose (125 mg/kg/day daily for 12 consecutive days) resulted in faster relief of the symptoms in 2/3 of animals (n = 6) compared to the control group without Colchicalin treatment (n = 5). Nevertheless, 1/3 of the rats died suddenly after the first Colchicalin injection, probably due to a steep rise in the total colchicine plasma concentration, which suggests further improvement of the dosing scheme prior to potential application in acute human colchicine poisoning.

The anticancer effect of PASylated calreticulin-targeting L-ASNase in solid tumor bearing mice with immunogenic cell death-inducing chemotherapy.

L-Asparaginase (L-ASNase), a bacterial enzyme that degrades asparagine, has been commonly used in combination with several chemical drugs to treat malignant hematopoietic cancers such as acute lymphoblastic leukemia (ALL). In contrast, the enzyme was known to inhibit the growth of solid tumor cells in vitro, but not to be effective in vivo. We previously reported that two novel monobodies (CRT3 and CRT4) bound specifically with calreticulin (CRT) exposed on tumor cells and tissues during immunogenic cell death (ICD). Here, we engineered L-ASNases conjugated with monobodies at the N-termini and PAS200 tags at the C-termini (CRT3LP and CRT4LP). These proteins were expected to possess four monobody and PAS200 tag moieties, which did not disrupt the L-ASNase conformation. These proteins were expressed 3.8-fold more highly in E. coli than those without PASylation. The purified proteins were highly soluble, with much greater apparent molecular weights than expected ones. Their affinity (Kd) against CRT was about 2 nM, 4-fold higher than that of monobodies. Their enzyme activity (∼6.5 IU/nmol) was similar to that of L-ASNase (∼7.2 IU/nmol), and their thermal stability was significantly increased at 55 °C. Their half-life times were > 9 h in mouse sera, about 5-fold longer than that of L-ASNase (∼1.8 h). Moreover, CRT3LP and CRT4LP bound specifically with CRT exposed on tumor cells in vitro, and additively suppressed the tumor growth in CT-26 and MC-38 tumor-bearing mice treated with ICD-inducing drugs (doxorubicin and mitoxantrone) but not with a non-ICD-inducing drug (gemcitabine). All data indicated that PASylated CRT-targeted L-ASNases enhanced the anticancer efficacy of ICD-inducing chemotherapy. Taken together, L-ASNase would be a potential anticancer drug for treating solid tumors.

Improved stability and pharmacokinetics of wogonin through loading into PASylated ferritin.

Wogonin (Wog) plays an important role in human diseases, especially cancer and inflammatory diseases, but its poor solubility, unstable metabolism and low bioavailability greatly limit its application in biomedical fields. Therefore, we developed a temperature-dependent method to encapsulate wogonin into a novel ferritin-based nanocarrier. To improve the loading capacity and stability, the human H chain ferritin (HFtn) was functionalized with a repetitive polypeptide sequence composed of proline (Pro), alanine (Ala), and serine (Ser) in different residues lengths (PAS10 and PAS30). Wogonin loading and release studies demonstrated that the encapsulation efficiency and stability of the PASylated nanocarriers were significantly higher than those of the wild type. PAS-HFtn-Wog exhibited enhanced cytotoxicity to MCF-7 breast cancer cells and HepG2 liver cancer cells. Notably, the PASylated HFtn, especially PAS30-HFtn greatly prolonged the pharmacokinetics of wogonin in the mice bloodstream. Therefore, wogonin-loaded PAS-HFtn may be a promising drug candidate for cancer therapy.

The Effective Control of Hyperuricemia in Cancer Patients: A New Recombinant Conjugated Variant of Urate Oxidase.

OBJECTIVE: Management of hyperuricemia is crucial to controlling tumor lysis syndrome (TLS) during cancer therapy. Urate oxidase (UOX) that catalyzes the enzymatic oxidation of uric acid into allantoin, is effective in lowering plasma uric acid levels and controlling hyperuricemia. Recently, we developed a new recombinant conjugate variant of UOX therapeutic drug using PASylation technology. This study was designed to evaluate the stability, plasma half-life and immunogencity of PASylated UOX. METHODS: A recombinant variant of PASylated UOX from the Aspergillus flavus was manufactured using bioinformatics and experimental techniques. Ex vivo evaluation of stability of PASylated UOX was done in 50% human serum. For half-life test, recombinant PASylated UOX and rasburicase were administered at 1.5 mg/kg to 10 rats in two different groups and samples were collected after injection Production of antibodies against PASylated drug was also assayed. RESULTS: Residual activity of PASylated UOX in 50% human serum was higher than rasburicase and native UOX. Stability of PASylated UOX at 25°C and 37°C was also higher than rasburicase and native UOX. The PASylated half-life was ~32.1 hours, whereas half-life for rasburicase and native UOX was ~25.1 and ~22.8 hours, respectively. In immunogenicity examination, there is 33% and 36% decrease in the absorbance of native UOX and rasburicase, respectively when compared with that of PASylated UOX. CONCLUSION: Our data confirmed the efficacy and stability of PASylated UOX in comparison to the rasburicase. In summary, the results indicated that PASylated UOX drug is effective at lowering plasma uric acid levels with prolonged plasma half-life and decreased cost.
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