Ackr3-Venus knock-in mouse lights up brain vasculature.

The atypical chemokine receptor 3, ACKR3, is a G protein-coupled receptor, which does not couple to G proteins but recruits ßarrestins. At present, ACKR3 is considered a target for cancer and cardiovascular disorders, but less is known about the potential of ACKR3 as a target for brain disease. Further, mouse lines have been created to identify cells expressing the receptor, but there is no tool to visualize and study the receptor itself under physiological conditions. Here, we engineered a knock-in (KI) mouse expressing a functional ACKR3-Venus fusion protein to directly detect the receptor, particularly in the adult brain. In HEK-293 cells, native and fused receptors showed similar membrane expression, ligand induced trafficking and signaling profiles, indicating that the Venus fusion does not alter receptor signaling. We also found that ACKR3-Venus enables direct real-time monitoring of receptor trafficking using resonance energy transfer. In ACKR3-Venus knock-in mice, we found normal ACKR3 mRNA levels in the brain, suggesting intact gene transcription. We fully mapped receptor expression across 14 peripheral organs and 112 brain areas and found that ACKR3 is primarily localized to the vasculature in these tissues. In the periphery, receptor distribution aligns with previous reports. In the brain there is notable ACKR3 expression in endothelial vascular cells, hippocampal GABAergic interneurons and neuroblast neighboring cells. In conclusion, we have generated Ackr3-Venus knock-in mice with a traceable ACKR3 receptor, which will be a useful tool to the research community for interrogations about ACKR3 biology and related diseases.

Delivery of membrane impermeable molecules to primary mouse T lymphocytes.

The pore-forming toxin streptolysin-O (SLO) enables intracellular delivery of molecules up to 100 kDa and has been used for short-term delivery of membrane-impermeable substances to assess their effects on cellular activities. A limitation of this technique is the loss of intracellular components and the potential unpredicted alterations of cellular metabolism and signaling. This protocol, optimized for primary mouse T lymphocytes, describes steps for SLO-mediated cell membrane permeabilization and substance supplementation, followed by immunoblotting and immunofluorescent microscopy for assessing cellular effects. For complete details on the use and execution of this protocol, please refer to Xu et al., 2021a, Xu et al., 2021b.

Characterization and Specification of a Trivalent Protein-Based Pneumococcal Vaccine Formulation Using an Adjuvant-Free Nanogel Nasal Delivery System.

We previously developed a safe and effective nasal vaccine delivery system using a self-assembled nanosized hydrogel (nanogel) made from a cationic cholesteryl pullulan. Here, we generated three pneumococcal surface protein A (PspA) fusion antigens as a universal pneumococcal nasal vaccine and then encapsulated each PspA into a nanogel and mixed the three resulting monovalent formulations into a trivalent nanogel-PspA formulation. First, to characterize the nanogel-PspA formulations, we used native polyacrylamide gel electrophoresis (PAGE) to determine the average number of PspA molecules encapsulated per nanogel molecule. Second, we adopted two methods-a densitometric method based on lithium dodecyl sulfate (LDS)-PAGE and a biologic method involving sandwich enzyme-linked immunosorbent assay (ELISA)-to determine the PspA content in the nanogel formulations. Third, treatment of nanogel-PspA formulations by adding methyl-ß-cyclodextrin released each PspA in its native form, as confirmed through circular dichroism (CD) spectroscopy. However, when nanogel-PspA formulations were heat-treated at 80 °C for 16 h, CD spectroscopy showed that each PspA was released in a denatured form. Fourth, we confirmed that the nanogel-PspA formulations were internalized into nasal mucosa effectively and that each PspA was gradually released from the nanogel in epithelial cells in mice. Fifth, LDS-PAGE densitometry and ELISA both indicated that the amount of trivalent PspA was dramatically decreased in the heat-treated nanogel compared with that before heating. When mice were immunized nasally using the heat-treated formulation, the immunologic activity of each PspA was dramatically reduced compared with that of the untreated formulation; in both cases, the immunologic activity correlated well with the content of each PspA as determined by LDS-PAGE densitometry and ELISA. Finally, we confirmed that the trivalent nanogel-PspA formulation induced equivalent titers of PspA-specific serum IgG and mucosal IgA Abs in immunized mice. These results show that the specification methods we developed effectively characterized our nanogel-based trivalent PspA nasal vaccine formulation.

A Randomized Phase I Study to Evaluate the Safety, Tolerability, and Pharmacokinetics of Recombinant Erwinia Asparaginase (JZP-458) in Healthy Adult Volunteers.

L-asparaginase has been an important component of acute lymphoblastic leukemia (ALL) therapy for over 40 years, and is standard therapy during ALL induction and consolidation treatment. L-asparaginases are immunogenic and can induce hypersensitivity reactions; inability to receive asparaginase has been associated with poor patient outcomes. There are L-asparaginases of varied bacterial origins, with the most commonly used being Escherichia coli (E. coli); therefore, to ensure that patients who develop hypersensitivity to E. coli-derived asparaginases receive an adequate therapeutic course, alternative preparations are warranted. JZP-458 is a recombinant Erwinia asparaginase produced using a novel Pseudomonas fluorescens expression platform that yields an enzyme with no immunologic cross-reactivity to E. coli-derived asparaginases. To evaluate the safety, tolerability, and pharmacokinetics (PK) of a single dose of JZP-458, a randomized, single-center, open-label, phase I study was conducted with JZP-458 given via i.m. injection or i.v. infusion to healthy adult volunteers. At the highest doses tested for each route of administration (i.e., 25 mg/m2 i.m. and 37.5 mg/m2 i.v.), JZP-458 achieved serum asparaginase activity (SAA) levels ≥ 0.1 IU/mL at 72 hours postdose for 100% of volunteers. Bioavailability for i.m. JZP-458 was estimated at 36.8% based on SAA data. All dose levels were well-tolerated, with no unanticipated adverse events (AEs), no serious AEs, and no grade 3 or higher AEs. Based on PK and safety data, the recommended JZP-458 starting dose for the pivotal phase II/III study in adult and pediatric patients is 25 mg/m2 i.m. and 37.5 mg/m2 i.v. on a Monday/Wednesday/Friday dosing schedule.

Fusion of Lysostaphin to an Albumin Binding Domain Prolongs Its Half-Life and Bactericidal Activity in the Systemic Circulation.

Antibacterial lysins are promising proteins that are active against both antibiotic-susceptible and antibiotic-resistant bacterial strains. However, a major limitation of antibacterial lysins is their fast elimination from systemic circulation. PEGylation increases the plasma half-life of lysins but renders them inactive. Here we report the construction of a fusion protein of lysostaphin, a potent anti-staphylococcal lysin, and an albumin-binding domain from streptococcal protein G. The resulting fusion protein was less active than the parent enzyme lysostaphin, but it still retained significant antibacterial activity even when bound to serum albumin. The terminal half-life of the fusion protein in rats was five-fold greater than that of lysostaphin (7.4 vs. 1.5 h), and the area under the curve increased more than 115 times. Most importantly, this increase in systemic circulation time compensated for the decrease in activity. The plasma from rats that received an injection of the fusion protein retained bactericidal activity for up to 7 h, while plasma from rats that received plain lysostaphin lacked any detectable activity after 4 h. To the best of our knowledge, this is the first report of an antibacterial lysin with both improved pharmacokinetic parameters and prolonged bactericidal activity in the systemic circulation.

Quantitative determination and pharmacokinetic study of fusaricidin A in mice plasma and tissues using ultra-high performance liquid chromatography-tandem mass spectrometry.

Fusaricidins are a family of cyclic lipodepsipeptides that convey antifungal and antibacterial activity. Fusaricidin A (FA) is one of the Fusaricidins major compounds and it is showing promising activity against fungi and bacteria. In the present study, a fast and sensitive ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-MS/MS) method was developed for the analysis of FA in mice plasma, liver, kidney and brain tissues. The instrument was operated in positive electrospray ionization mode. Multiple reaction monitoring (MRM) mode was performed with ion pairs of m/z: 883.5→256.3, 883.5→197.2 and 883.5→72.1 for FA. The method was validated for linearity, repeatability, accuracy, stability, limits of detection (LOD) and limits of quantification (LOQ). The LOD and LOQ were 0.01 and 0.05 ng/mL for plasma and tissues, respectively. The calibration curve (10-200 ng/mL) was linear ( r2 = 0.99). Precision and accuracy values were found to be < 10% (within acceptable limit). The pharmacokinetic and tissue distribution characteristics of FA were determined in plasma, liver, kidney and brain of CD1 mice after I.V. administration of a single dose of 15 mg/kg body weight. Highest plasma concentration (Cmax) was calculated to be 4169.97 ± 50 ng/mL with a tmax of 0.08 h. The plasma clearance rate of FA was 397.6 ± 203 mL/h with a t1/2 of 2.2 ± 0.5 h and apparent volume of distribution during the terminal phase (Vz) of 979.2 ± 318 mL. The highest tissue concentration (Cmax) was found in the liver (219 ± 14 ng/mg) at a tmax of 0.08 h followed by the kidneys (38.6 ± 16 ng/mg) at tmax of 0.2 h. FA was poorly distributed to the brain with a Cmax of 0.45 ± 0.2 ng/mg and a tmax of 0.08 h. The method for quantitative analysis and pharmacokinetic data provided will support the development of various formulation approaches and therapeutic application for future clinical studies.

Sortagged anti-EGFR immunoliposomes exhibit increased cytotoxicity on target cells.

PURPOSE: Conventional chemotherapy is associated with therapy-limiting side effects, which might be alleviated by targeted chemotherapeutics such as immunoliposomes. The targeting ligands of immunoliposomes are commonly attached by unspecific chemical conjugation, bearing risk of structural heterogeneity and therewith related biological consequences. Chemoenzymatic methods may mitigate such risks through site-specific conjugation. METHODS: The formulation parameters for pentaglycine-modified, doxorubicin-loaded liposomes and the reaction conditions for a site-specific, Sortase-A mediated conjugation with monoclonal antibodies were thoroughly evaluated. The cytotoxicity of such sortagged, epidermal growth factor receptor (EGFR)-specific immunoliposomes was tested on human breast cancer cells. RESULTS: Sortaggable liposomes with a defined size (140 nm, PDI < 0.25) and high encapsulation efficiency (>90%) were obtained after manufacturing optimization. A ratio of 1.0-2.5 µM mAb/100 µM pentaglycine yielded stable dispersions and circumvented carrier precipitation during ligand grafting. The cytotoxicity on EGFR+ MDA-MB-468 was up to threefold higher for EGFR-specific immunoliposomes than for the nontargeted controls. CONCLUSIONS: Sortase-A is suitable to generate immunoliposomes with a site-specific ligand-carrier linkage and hence improves chemical homogeneity of targeted therapeutics. However, the sweet spot for manufacturability utilizing mAbs with two Sortase-A recognition sites is narrow, making mono-reactive binders such as scFvs or Fab's preferable for a further development. Despite this, the immunoliposomes demonstrated a targeted delivery of doxorubicin, indicating the potential to increase the therapeutic window during the treatment of EGFR+ tumors.

Comparative evaluation of dimeric and monomeric forms of ADAPT scaffold protein for targeting of HER2-expressing tumours.

ADAPTs are small engineered non-immunoglobulin scaffold proteins, which have demonstrated very promising features as vectors for radionuclide tumour targeting. Radionuclide imaging of human epidermal growth factor 2 (HER2) expression in vivo might be used for stratification of patients for HER2-targeting therapies. ADAPT6, which specifically binds to HER2, has earlier been shown to have very promising features for in vivo targeting of HER2 expressing tumours. In this study we tested the hypothesis that dimerization of ADAPT6 would increase the apparent affinity to HER2 and accordingly improve tumour targeting. To find an optimal molecular design of dimers, a series of ADAPT dimers with different linkers, -SSSG- (DiADAPT6L1), -(SSSG)2- (DiADAPT6L2), and -(SSSG)3- (DiADAPT6L3) was evaluated. Dimers in combination with optimal linker lengths demonstrated increased apparent affinity to HER2. The best variants, DiADAPT6L2 and DiADAPT6L3 were site-specifically labelled with 111In and 125I, and compared with a monomeric ADAPT6 in mice bearing HER2-expressing tumours. Despite higher affinity, both dimers had lower tumour uptake and lower tumour-to-organ ratios compared to the monomer. We conclude that improved affinity of a dimeric form of ADAPT does not compensate the disadvantage of increased size. Therefore, increase of affinity should be obtained by affinity maturation and not by dimerization.

Half-life extension of porcine interferon-α by fusion to the IgG-binding domain of streptococcal G protein.

Recombinant interferon-α (rIFN-α) has been widely used for treating viral infections. However, the clinical efficacy of unmodified rIFN-α is limited due to small molecular size and rapid clearance from circulation. In this study we developed a novel strategy for half-life extension of porcine IFN-α (PoIFN-α) by fusion to the immunoglobulin (Ig)-binding C2 domain of streptococcal protein G (SPG). The coding sequences for PoIFN-α6 and SPG C2 domain, with a tobacco etch virus (TEV) protease recognition sequence introduced at the 5-end, were cloned into an elastin-like polypeptide (ELP) fusion expression vector and expressed as an ELP-PoIFNα-C2 fusion protein. After optimization of the conditions for soluble protein expression and purification, the fusion protein was purified to more than 90% purity by two rounds of inverse transition cycling (ITC) in the presence of 0.5% Triton X-100. After cleavage with self-aggregating peptide ELK-16-tagged tobacco etch virus protease, the protease was removed by quick centrifugation and PoIFNα-C2 protein was recovered by an additional round of ITC with 98% purity. Western blotting analysis showed that PoIFNα-C2 protein had the specific affinity for pig IgG binding. The antiviral assay showed that PoIFNα-C2 protein had potent antiviral activities against vesicular stomatitis virus and porcine pseudorabies virus. After single intravenous or subcutaneous injection into rats, PoIFNα-C2 protein showed 16- or 4-fold increase in serum half-life with significantly improved bioavailability.

Influence of the Membrane Dye R18 and of DMSO on Cell Penetration of Guanidinium-Rich Peptides.

A quantitative analysis by confocal fluorescence microscopy of the entry into HEK293 and MCF-7 cells by fluorescein-labeled octaarginine (1) and by three octa-Adp derivatives (2 - 4, octamers of the ß-Asp-Arg-dipeptide, derived from the biopolymer cyanophycin) is described, including the effects of the membrane dye R18 and of DMSO on cell penetration.

Crystal structures and kinetic analyses of N-acetylmannosamine-6-phosphate 2-epimerases from Fusobacterium nucleatum and Vibrio cholerae.

Sialic acids are nine-carbon sugars that are found abundantly on the cell surfaces of mammals as glycoprotein or glycolipid complexes. Several Gram-negative and Gram-positive bacteria have the ability to scavenge and catabolize sialic acids to use as a carbon source. This gives them an advantage in colonizing sialic acid-rich environments. The genes of the sialic acid catabolic pathway are generally present as the operon nanAKE. The third gene in the operon encodes the enzyme N-acetylmannosamine-6-phosphate 2-epimerase (NanE), which catalyzes the conversion of N-acetylmannosamine 6-phosphate to N-acetylglucosamine 6-phosphate, thus committing it to enter glycolysis. The NanE enzyme belongs to the isomerase class of enzymes possessing the triose phosphate isomerase (TIM) barrel fold. Here, comparative structural and functional characterizations of the NanE epimerases from two pathogenic Gram-negative bacteria, Fusobacterium nucleatum (Fn) and Vibrio cholerae (Vc), have been carried out. Structures of NanE from Vc (VcNanE) with and without ligand bound have been determined to 1.7 and 2.7 Šresolution, respectively. The structure of NanE from Fn (FnNanE) has been determined to 2.2 Šresolution. The enzymes show kinetic parameters that are consistent with those of Clostridium perfringens NanE. These studies allowed an evaluation of whether NanE may be a good drug target against these pathogenic bacteria.

Cytosolic Delivery of Multidomain Cargos by the N Terminus of Pasteurella multocida Toxin.

The zoonotic pathogen Pasteurella multocida produces a 146-kDa modular toxin (PMT) that enters host cells and manipulates intracellular signaling through action on its Gα protein targets. The N terminus of PMT (PMT-N) mediates cellular uptake through receptor-mediated endocytosis, followed by the delivery of the C-terminal catalytic domain from acidic endosomes into the cytosol. The putative native cargo of PMT consists of a 710-residue polypeptide with three distinct modular subdomains (C1-C2-C3), where C1 contains a membrane localization domain (MLD), C2 has an as-yet-undefined function, and C3 catalyzes the deamidation of a specific active-site glutamine residue in Gα protein targets. However, whether the three cargo subdomains are delivered intact or undergo further proteolytic processing during or after translocation from the late endosome is unclear. Here, we demonstrate that PMT-N mediates the delivery of its native C-terminal cargo as a single polypeptide, corresponding to C1-C2-C3, including the MLD, with no evidence of cleavage between subdomains. We show that PMT-N also delivers nonnative green fluorescent protein (GFP) cargo into the cytosol, further supporting that the receptor-binding and translocation functions reside within PMT-N. Our findings further show that PMT-N can deliver C1-C2 alone but that the presence of C1-C2 is important for the cytosolic delivery of the catalytic C3 subdomain by PMT-N. In addition, we further refine the minimum C3 domain required for intracellular activity as comprising residues 1105 to 1278. These findings reinforce that PMT-N serves as the cytosolic delivery vehicle for C-terminal cargo and demonstrate that its native cargo is delivered intact as C1-C2-C3.

NADPH oxidase activation in neutrophils: Role of the phosphorylation of its subunits.

Neutrophils are key cells of innate immunity and during inflammation. Upon activation, they produce large amounts of superoxide anion (O2 -. ) and ensuing reactive oxygen species (ROS) to kill phagocytized microbes. The enzyme responsible for O2 -. production is called the phagocyte NADPH oxidase. This is a multicomponent enzyme system that becomes active after assembly of four cytosolic proteins (p47phox , p67phox , p40phox and Rac2) with the transmembrane proteins (p22phox and gp91phox , which form the cytochrome b558 ). gp91phox represents the catalytic subunit of the NADPH oxidase and is also called NOX2. NADPH oxidase-derived ROS are essential for microbial killing and innate immunity; however, excessive ROS production induces tissue injury and prolonged inflammatory reactions that contribute to inflammatory diseases. Thus, NADPH oxidase activation must be tightly regulated in time and space to limit ROS production. NADPH oxidase activation is regulated by several processes such as phosphorylation of its components, exchange of GDP/GTP on Rac2 and binding of p47phox and p40phox to phospholipids. This review aims to provide new insights into the role of the phosphorylation of the NADPH oxidase components, that is gp91phox , p22phox , p47phox , p67phox and p40phox , in the activation of this enzyme.

Safety, immunogenicity, pharmacokinetics, and efficacy of degradation of anti-HLA antibodies by IdeS (imlifidase) in chronic kidney disease patients.

Safety, immunogenicity, pharmacokinetics, and efficacy of the IgG-degrading enzyme of Streptococcus pyogenes (IdeS [imlifidase]) were assessed in a single-center, open-label ascending-dose study in highly sensitized patients with chronic kidney disease. Eight patients with cytotoxic PRAs (median cytotoxic PRAs of 64%) at enrollment received 1 or 2 intravenous infusions of IdeS on consecutive days (0.12 mg/kg body weight ×2 [n = 3]; 0.25 mg/kg ×1 [n = 3], or 0.25 mg/kg ×2 [n = 2]). IgG degradation was observed in all subjects after IdeS treatment, with <1% plasma IgG remaining within 48 hours and remaining low up to 7 days. Mean fluorescence intensity values of HLA class I and II reactivity were substantially reduced in all patients, and C1q binding to anti-HLA was abolished. IdeS also cleaved the IgG-type B cell receptor on CD19+ memory B cells. Anti-IdeS antibodies developed 1 week after treatment, peaking at 2 weeks. A few hours after the second IdeS infusion, 1 patient received a deceased donor kidney offer. At enrollment, the patient had a positive serum crossmatch (HLA-B7), detected by complement-dependent cytotoxicity, flow cytometry, and multiplex bead assays. After IdeS infusion (0.12 mg/kg ×2) and when the HLA-incompatible donor (HLA-B7+ ) kidney was offered, the HLA antibody profile was negative. The kidney was transplanted successfully.

Cell-penetrating peptides derived from Clostridium difficile TcdB2 and a related large clostridial toxin.

Clostridium difficile TcdB (2366 amino acid residues) is an intracellular bacterial toxin that binds to cells and enters the cytosol where it glucosylates small GTPases. In the current study, we examined a putative cell entry region of TcdB (amino acid residues 1753-1851) for short sequences that function as cell-penetrating peptides (CPPs). To screen for TcdB-derived CPPs, a panel of synthetic peptides was tested for the ability to enhance transferrin (Tf) association with cells. Four candidate CPPs were discovered, and further study on one peptide (PepB2) pinpointed an asparagine residue necessary for CPP activity. PepB2 mediated the cell entry of a wide variety of molecules including dextran, streptavidin, microspheres, and lentivirus particles. Of note, this uptake was dramatically reduced in the presence of the Na+/H+ exchange blocker and micropinocytosis inhibitor amiloride, suggesting that PepB2 invokes macropinocytosis. Moreover, we found that PepB2 had more efficient cell-penetrating activity than several other well-known CPPs (TAT, penetratin, Pep-1, and TP10). Finally, Tf assay-based screening of peptides derived from two other large clostridial toxins, TcdA and TcsL, uncovered two new TcdA-derived CPPs. In conclusion, we have identified six CPPs from large clostridial toxins and have demonstrated the ability of PepB2 to promote cell association and entry of several molecules through a putative fluid-phase macropinocytotic mechanism.

Radionuclide Tumor Targeting Using ADAPT Scaffold Proteins: Aspects of Label Positioning and Residualizing Properties of the Label.

Visualization of cancer-associated alterations of molecular phenotype using radionuclide imaging is a noninvasive approach to stratifying patients for targeted therapies. The engineered albumin-binding domain-derived affinity protein (ADAPT) is a promising tracer for radionuclide molecular imaging because of its small size (6.5 kDa), which satisfies the precondition for efficient tumor penetration and rapid clearance. Previous studies demonstrated that the human epidermal growth factor receptor type 2 (HER2)-targeting ADAPT6 labeled with radiometals at the N terminus is able to image HER2 expression in xenografts a few hours after injection. The aim of this study was to evaluate whether the use of a nonresidualizing label or placement of the labels at the C terminus would further improve the targeting properties of ADAPT6. Methods: Two constructs, Cys2-ADAPT6 and Cys59-ADAPT6, having the (HE)3DANS sequence at the N terminus were produced and site-specifically labeled using 111In-DOTA or 125I-iodo-((4-hydroxyphenyl)ethyl) maleimide (HPEM). The conjugates were compared in vitro and in vivo. HER2-targeting properties and biodistribution were evaluated in BALB/C nu/nu mice bearing ovarian carcinoma cell (SKOV-3) xenografts. Results: Specific HER2 binding and high affinity were preserved after labeling. Both Cys2-ADAPT6 and Cys59-ADAPT6 were internalized slowly by HER2-expressing cancer cells. Depending on the label position, uptake at 4 h after injection varied from 10% to 22% of the injected dose per gram of tumor tissue. Regardless of terminus position, the 125I-HPEM label provided more than 140-fold lower renal uptake than the 111In-DOTA label at 4 after injection. The tumor-to-organ ratios were, in contrast, higher for both of the 111In-DOTA-labeled ADAPT variants in other organs. Tumor-to-blood ratios for 111In-labeled Cys2-ADAPT6 and Cys59-ADAPT6 did not differ significantly (250-280), but 111In-DOTA-Cys59-ADAPT6 provided significantly higher tumor-to-lung, tumor-to-liver, tumor-to-spleen, and tumor-to-muscle ratios. Radioiodinated variants had similar tumor-to-organ ratios, but 125I-HPEM-Cys59-ADAPT6 had significantly higher tumor uptake and a higher tumor-to-kidney ratio. Conclusion: Residualizing properties of the label strongly influence the targeting properties of ADAPT6. The position of the radiolabel influences targeting as well, although to a lesser extent. Placement of a label at the C terminus yields the best biodistribution features for both radiometal and radiohalogen labels. Low renal retention of the radioiodine label creates a precondition for radionuclide therapy using 131I-labeled HPEM-Cys59-ADAPT6.

Outcome of pediatric patients with acute lymphoblastic leukemia/lymphoblastic lymphoma with hypersensitivity to pegaspargase treated with PEGylated Erwinia asparaginase, pegcrisantaspase: A report from the Children's Oncology Group.

BACKGROUND: Erwinia asparaginase is a Food and Drug Administration approved agent for the treatment of acute lymphoblastic leukemia (ALL) for patients who develop hypersensitivity to Escherichia coli derived asparaginases. Erwinia asparaginase is efficacious, but has a short half-life, requiring six doses to replace one dose of the most commonly used first-line asparaginase, pegaspargase, a polyethylene glycol (PEG) conjugated E. coli asparaginase. Pegcristantaspase, a recombinant PEGylated Erwinia asparaginase with improved pharmacokinetics, was developed for patients with hypersensitivity to pegaspargase. Here, we report a series of patients treated on a pediatric phase 2 trial of pegcrisantaspase. PROCEDURE: Pediatric patients with ALL or lymphoblastic lymphoma and hypersensitivity to pegaspargase enrolled on Children's Oncology Group trial AALL1421 (Jazz 13-011) and received intravenous pegcrisantaspase. Serum asparaginase activity (SAA) was monitored before and after dosing; immunogenicity assays were performed for antiasparaginase and anti-PEG antibodies and complement activation was evaluated. RESULTS: Three of the four treated patients experienced hypersensitivity to pegcrisantaspase manifested as clinical hypersensitivity reactions or rapid clearance of SAA. Immunogenicity assays demonstrated the presence of anti-PEG immunoglobulin G antibodies in all three hypersensitive patients, indicating a PEG-mediated immune response. CONCLUSIONS: This small series of patients, nonetheless, provides data, suggesting preexisting immunogenicity against the PEG moiety of pegaspargase and poses the question as to whether PEGylation may be an effective strategy to optimize Erwinia asparaginase administration. Further study of larger cohorts is needed to determine the incidence of preexisting antibodies against PEG-mediated hypersensitivity to pegaspargase.

CagL from Helicobacter pylori has ADP-ribosylation activity and exerts partial protective efficacy in mice.

Mono ADP-ribosyltransferases are a class of functionally conserved enzymes present in prokaryotic and eukaryotic organisms. In prokaryotes, mono ADP-ribose transfer enzymes often represent a family of exotoxins that display activity in a variety of bacteria responsible for causing disease in plants and animals. A bioinformatic approach has allowed us to identify that CagL gene from some Helicobacter pylori strains shares a sequence pattern with ADP-ribosylating toxins of the CT-group. In this manuscript we show that recombinant CagL from Shi470 is catalytically active showing ADP-ribosyltransferase, NAD-glycohydrolase, and auto-ADP-ribosylation activities. This is the first time that a catalytically active member of the ADP-ribosyltransferase family is identified in Helicobacter pylori. This observation may lead to the discovery of novel functions exerted by CagL in the pathogenesis of Helicobacter pylori. Indeed, we have shown that vaccination with CagL has protective efficacy in mice indicating that CagL may be considered as potential component of a Helicobacter pylori vaccine.

Arginase purified from endophytic Pseudomonas aeruginosa IH2: Induce apoptosis through both cell cycle arrest and MMP loss in human leukemic HL-60 cells.

Arginase is a therapeutic enzyme for arginine-auxotrophic cancers but their low anticancer activity, less proteolytic tolerance and shorter serum half-life are the major shortcomings. In this study, arginase from Pseudomonas aeruginosa IH2 was purified to homogeneity and estimated as 75 kDa on native-PAGE and 37 kDa on SDS-PAGE. Arginase showed optimum activity at pH 8 and temperature 35 °C. Mn2+ and Mg2+ ions enhanced arginase activity while, Li+, Cu2+, and Al3+ ions reduced arginase activity. In-vitro serum half-life of arginase was 36 h and proteolytic half-life against trypsin and proteinase-K was 25 and 29 min, respectively. Anticancer activity of arginase was evaluated against colon, breast, leukemia, and prostate cancer cell lines and lowest IC50 (0.8 IU ml-1) was found against leukemia cell line HL-60. Microscopic studies and flow cytometric analysis of Annexin V/PI staining of HL-60 cells revealed that arginase induced apoptosis in dose-dependent manner. Cell cycle analysis suggested that arginase induced cell cycle arrest in G0/G1 phase. The increasing level of MMP loss, ROS generation and decreasing level of SOD, CAT, GPx and GSH suggested that arginase treatment triggered dysfunctioning of mitochondria. The cleavage of caspase-3, PARP-1, activations of caspase-8, 9 and high expression of proapoptotic protein Bax, low expression of anti-apoptotic protein Bcl-2 indicated that arginase treatment activates mitochondrial pathway of apoptosis. Purified arginase did not exert cytotoxic effects on human noncancer cells. Our study strongly supports that arginase could be used as potent anticancer agent but further studies are required which are underway in our lab.

Albumin-binding domain from Streptococcus zooepidemicus protein Zag as a novel strategy to improve the half-life of therapeutic proteins.

Recombinant antibody fragments belong to the promising class of biopharmaceuticals with high potential for future therapeutic applications. However, due to their small size they are rapidly cleared from circulation. Binding to serum proteins can be an effective approach to improve pharmacokinetic properties of short half-life molecules. Herein, we have investigated the Zag albumin-binding domain (ABD) derived from Streptococcus zooepidemicus as a novel strategy to improve the pharmacokinetic properties of therapeutic molecules. To validate our approach, the Zag ABD was fused with an anti-TNFα single-domain antibody (sdAb). Our results demonstrated that the sdAb-Zag fusion protein was highly expressed and specifically recognizes human, rat and mouse serum albumins with affinities in the nanomolar range. Moreover, data also demonstrated that the sdAb activity against the therapeutic target (TNFα) was not affected when fused with Zag ABD. Importantly, the Zag ABD increased the sdAb half-life ∼39-fold (47min for sdAb versus 31h for sdAb-Zag). These findings demonstrate that the Zag ABD fusion is a promising approach to increase the half-life of small recombinant antibodies molecules without affecting their therapeutic efficacy. Moreover, the present study strongly suggests that the Zag ABD fusion strategy can be potentially used as a universal method to improve the pharmokinetics properties of many others therapeutics proteins and peptides in order to improve their dosing schedule and clinical effects.