Challenges with development of a pharmacokinetics assay to measure a variably glycosylated fusion protein.

Aim: Development of recombinant fusion proteins as drugs poses unique challenges for bioanalysis. This paper describes a case study of a glycosylated fusion protein, where variable glycosylation, matrix interference and high sensitivity needs posed unique challenges. Results: Six different assay configurations, across four different platforms were evaluated for measurement of drug concentrations. Two platforms that achieved the assay requirements were Simoa HD-1 and immune-capture LC-MS/MS-based assay. Conclusion: Both, Simoa HD-1 and the mass spectrometry-based methods were able to detect total drug by providing the adequate matrix tolerance, required sensitivity and detection of all the various glycosylated fusion proteins to support clinical sample analysis. The mass spectrometry-based method was selected due to robustness and ease of method transfer.

Combination immunotherapy of chlorogenic acid liposomes modified with sialic acid and PD-1 blockers effectively enhances the anti-tumor immune response and therapeutic effects.

Melanoma is one of the most common malignant tumors. The anti-PD-1 antibody is used for the treatment of metastatic melanoma. Treatment success is only 35-40% and a range of immune-related adverse reactions can occur. Combination of anti-PD1 antibody therapy with other oncology therapies has been attempted. Herein, we assessed whether chlorogenic acid liposomes modified with sialic acid (CA-SAL) combined with anti-PD1 antibody treatment was efficacious as immunotherapy for melanoma. CA-SAL liposomes were prepared and characterized. In a mouse model of B16F10 tumor, mice were treated with an anti-PD1 antibody, CA-SAL, or combination of CA-SAL + anti-PD1 antibody, and compared with no treatment controls. The tumor inhibition rate, tumor-associated macrophages (TAMs) phenotype, T-cell activity, and safety were investigated. We observed a significant decrease in the proportion of M2-TAMs and CD4+Fop3+ T cells, while there was a significant increase in the proportion of M1-TAMs and CD8+ T cells, and in the activity of T cells, and thus in the tumor inhibition rate. No significant toxicity was observed in major organs. CA-SAL and anti-PD1 Ab combination therapy presented synergistic anti-tumor activity, which enhanced the efficacy of the PD-1 checkpoint blocker in a mouse model of melanoma. In summary, combination immunotherapy of CA-SAL and anti-PD1 Ab has broad prospects in improving the therapeutic effect of melanoma, and may provide a new strategy for clinical treatment.

Exhausting tumor associated macrophages with sialic acid-polyethyleneimine-cholesterol modified liposomal doxorubicin for enhancing sarcoma chemotherapy.

To overstep the dilemma of chemical drug degradation within powerful lysosomes of tumor associated macrophages (TAMs), a sialic acid-polyethylenimine-cholesterol (SA-PEI-CH) modified liposomal doxorubicin (DOX-SPCL) was designed with both TAMs targeting and smart lysosomal trafficking. The modified liposome DOX-SPCL performed particle size as 103.2 ±â€¯3.1 nm and zeta potential as -4.5 ±â€¯0.9 mV with encapsulation efficiency as 95.8 ±â€¯0.5%. In in vitro cell experiments, compared with conventional liposomal doxorubicin (DOX-CL) and PEGylated liposomal doxorubicin (DOX-PL), DOX-SPCL showed a selective binding on TAMs and a mere lysosomal concentration. In pharmacokinetic study, DOX-SPCL effectively impeded/delayed the disposition of mononuclear phagocyte system (MPS) with a value of AUC0-t as 796.03 ±â€¯66.93 mg L-1 h. In S180 sarcomas bearing mice, DOX-SPCL showed the greatest tumor inhibition rate (92.7% ±â€¯3.6%) compared with DOX-CL (46.4% ±â€¯2.0%) or DOX-PL (58.8% ±â€¯7.6%). The <0.5% positive region of TAMs in tumor section indicated a super TAMs exhaustion for DOX-SPCL treatment. Conclusively, DOX-SPCL was supposed as a safe and effective liposomal preparation for clinical sarcoma treatment via TAMs targeting/deletion delivery strategy.

Liposome-aided metabolic engineering of tumor surface immunogenicity.

Approaches to increase tumor immunogenicity are of therapeutic potentials. We herein reported the use of liposomes for covalent incorporation of neoantigen on tumor surfaces with DNP-conjugated sialic acid (DNPSia). Relative to free DNPSia, sugar-encapsulated biotinylated liposomes (DNPSia@LP@biotin) enables effective cell surface expression of DNPSia on biotin receptor (BR)-expressing cells over BR-free cells in vitro, and on tumor cell surfaces with high tumor-to-normal tissue contrast in a mice model. These findings suggest the potentials of targetable liposomes for modulating tumor surface immunity via metabolic oligosaccharide engineering.

Evaluation of Sialic Acid in Infant Feeding: Contents and Bioavailability.

Sialic acid (Sia) contents and bioaccessibility (BA) in human milk (HM) and infant formulas (IFs) were determined, and Sia intakes by infants between 0 and 6 months of age were evaluated. Total Sia contents in HM decreased during lactation from 136.14 to 24.47 mg/100 mL. The total Sia contents in IFs (13.15-25.78 mg/100 mL) were lower than in HM and were not related to the addition of ingredients acting as sources of Sia in their formulation. The Sia intakes derived from IF consumption were lower than in HM, and only one IF reached the intakes provided by HM from the age of 2 months. Despite the lower total Sia content in IFs, the BA of Sia in IFs (88.08-92.96%) was significantly greater than in mature HM (72.51%) and similar to that found in colostrum (96.43%). However, the Sia contents in the available soluble fraction of IFs did not reach those provided by HM.

Exploring the Potential of Norbornene-Modified Mannosamine Derivatives for Metabolic Glycoengineering.

Metabolic glycoengineering (MGE) allows the introduction of unnaturally modified carbohydrates into cellular glycans and their visualization through bioorthogonal ligation. Alkenes, for example, have been used as reporters that can react through inverse-electron-demand Diels-Alder cycloaddition with tetrazines. Earlier, norbornenes were shown to be suitable dienophiles; however, they had not previously been applied for MGE. We synthesized two norbornene-modified mannosamine derivatives that differ in the stereochemistry at the norbornene (exo/endo linkage). Kinetic investigations revealed that the exo derivative reacts more than twice as rapidly as the endo derivative. Through derivatization with 1,2-diamino-4,5-methylenedioxybenzene (DMB) we confirmed that both derivatives are accepted by cells and incorporated after conversion to a sialic acid. In further MGE experiments the incorporated sugars were ligated to a fluorophore and visualized through confocal fluorescence microscopy and flow cytometry.

Preferential Accumulation of 14C-N-Glycolylneuraminic Acid over 14C-N-Acetylneuraminic Acid in the Rat Brain after Tail Vein Injection.

The two main molecular species of sialic acid existing in nature are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Neu5Ac is abundant in mammalian brains and plays crucial roles in many neural functions. In contrast, Neu5Gc is present only at a trace level in vertebrate brains. The brain-specific suppression of Neu5Gc synthesis, which is a common feature in mammals, suggests that Neu5Gc has toxicity against brain functions. However, in vivo kinetics of Neu5Gc in the whole body, especially in the brain, has not been studied in sufficient detail. To determine the in vivo kinetics of Neu5Gc, 14C-Neu5Gc was enzymatically synthesized and injected into rat tail veins. Although most of 14C-Neu5Gc was excreted in urine, a small amount of 14C-Neu5Gc was detected in the brain. Brain autoradiography indicated that 14C-Neu5Gc was accumulated predominantly in the hippocampus. 14C-Neu5Gc transferred into the brain was incorporated into gangliosides including GM1, GD1a, GD1b, GT1b and GQ1b. Reduction of 14C-Neu5Gc after intracerebroventricular infusion was slower than that of 14C-Neu5Ac in the brain and hippocampus. The results suggest that Neu5Gc is transferred from blood into the brain across the blood brain barrier and accumulates in the brain more preferentially than does Neu5Ac.

Synthesis of a fluorescently tagged sialic acid analogue useful for live-cell imaging.

A cytidine 5'-monophosphate (CMP)-sialic acid analogue carrying a fluorescent reporter group, an inhibitor of sialyltransferase, was synthesised in order to investigate glycan synthesis events in cells. The compound was found to be a substrate of a CMP-sialic acid transporter, and specific Golgi vesicles were visualised in the cells.

From a library of MAG antagonists to nanomolar CD22 ligands.

Siglec-2, also known as CD22, is involved in the regulation and survival of B-cells and has been successfully targeted in cell depletion therapies with antibody-based approaches. Sialic acid derivatives, already known to bind with high affinity to myelin-associated glycoprotein (MAG, Siglec-4), were screened for their binding affinity for CD22 by surface plasmon resonance. The best compound identified was further modified with various hydrophobic substituents at the 2-, 5-, and 9-positions of the sialic acid scaffold, leading to nanomolar derivatives, of which ligand 17 b shows the most promising pharmacodynamic and pharmacokinetic profiles. Isothermal titration calorimetry measurements demonstrate that the binding is enthalpy driven. Interestingly, the thermodynamic fingerprints reveal an excellent correlation between gains in enthalpy and compensation by increased entropy costs. Moreover, 17 b exhibits a residence time in the range of a few seconds, clearly prolonged relative to residence times typically observed for carbohydrate-lectin interactions. Finally, initial tests regarding drug-like properties of 17 b demonstrate the required high plasma protein binding yet a lack of oral availability, although its distribution coefficient (log D) is in the required range.

Effect of simulated gastrointestinal digestion on sialic acid and gangliosides present in human milk and infant formulas.

The effects of simulated gastrointestinal digestion upon sialic acid and gangliosides in infant and follow-on formulas and human milk, as well as their bioaccessibility, have been evaluated. The gastric stage is the step that causes a greater decrease in sialic acid and ganglioside contents. The intestinal stage only decreases the total and individual contents of gangliosides. After gastrointestinal digestion, neither sialic acid nor gangliosides were found in the nonbioaccessible fraction. The highest bioaccessibility (100 × content in soluble fraction after gastrointestinal digestion/total content) of sialic acid is found in human milk (87%), followed by infant formula (77%) and follow-on formula (16%). In the case of gangliosides, the highest bioaccessibility is present in the follow-on formula (51%), followed by human milk (29%) and infant formula (5%).

Glycosylation-modified erythropoietin with improved half-life and biological activity.

Erythropoietin (EPO) controls the production of red blood cells, so it is important to maintain high levels of EPO activity and half-life. Here, we modified glycosylation sites in human erythropoietin (HuEPO) gene, resulting in proteins with addition of 1-4 glycosylation sites. The modified gene was introduced into CHO cells. The expressed EPO analogs were analyzed by SDS-PAGE. Half-life of the analogs was determined by sialic acid content test. In vivo potencies of analogs were evaluated by reticulocyte count and haematocrit level. The metabolic clearance of recombinant human erythropoietin (rHuEPO) and its analogs were determined by EPO immunoradiometrics assay. We have shown that the carbohydrate content in modified EPO molecules is increased. The modified EPO, [Val(3)Asn(4)Thr(6)Asn(30)Thr(32)Val(87)Asn(88)Thr(90)]EPO, increases 3.3 times in elimination half-life, 2.1 times in activity and prolongs 2 days functional time in vivo in comparison to rHuEPO. These findings suggest that the addition of glycosylation sites in EPO enhances half-life and biological activity of EPO, duration of action of EPO anlogues positively correlated with the number of glycosylated sites, while addition of 4 glycosylation sites does not further enhance the erythropoietic potency.

Low molecular weight antagonists of the myelin-associated glycoprotein: synthesis, docking, and biological evaluation.

The injured adult mammalian central nervous system is an inhibitory environment for axon regeneration due to specific inhibitors, among them the myelin-associated glycoprotein (MAG), a member of the Siglec family (sialic-acid binding immunoglobulin-like lectin). In earlier studies, we identified the lead structure 5, which shows a 250-fold improved in vitro affinity for MAG compared to the tetrasaccharide binding epitope of GQ1balpha (1), the best physiological MAG ligand described so far. By modifying the 2- and 5-position, the affinity of 5 could be further improved to the nanomolar range (-->19a). Docking studies to a homology model of MAG allowed the rationalization of the experimental binding properties. Finally, pharmacokinetic parameters (stability in the cerebrospinal fluid, logD and permeation through the BBB) indicate the drug-like properties of the high-affinity antagonist 19a.

Metabolic fate of intravenously administered N-acetylneuraminic acid-6-14C in newborn piglets.

BACKGROUND: Sialic acid (N-acetylneuraminic acid), a component of gangliosides and sialylglycoproteins, may be a conditional nutrient in early life because endogenous synthesis is limited. The aim of this study was to investigate the metabolic fate of intravenously administrated N-acetylneuraminic acid-6-14C (sialic acid) in piglets. METHOD: Three-day-old male domestic piglets (Sus scrofa) were injected via the jugular vein with 5 microCi (11-12 x 10(6) cpm) of N-acetylneuraminic acid-6-14C (specific activity of 55 mCi/mmol). Blood samples were collected at regular intervals over the next 120 min. The organs were then removed and the urine collected for determination of residual radioactivity. RESULTS: Within 2 min of injection, 80% of the activity was removed from the blood and by 120 min the remaining activity approached 8%. At 120 min, the brain contained significantly more radioactivity (cpm/g tissue) than the liver, pancreas, heart and spleen, but less than the kidneys. Within the brain, the percentage of total injected activity was highest in the cerebrum (0.175 +/-0.008) followed by the cerebellum (0.0295 +/-0.006, p=0.00006) and the thalamus (0.029 +/- 0.006, p =0.00003). CONCLUSIONS: An exogenous source of sialic acid is capable of crossing the blood-brain barrier and being taken up into various tissues. The findings suggest that dietary sources of sialic acid may contribute to early brain development in newborn mammals.

Molecular characterization of pig ST8Sia IV--a critical gene for the formation of neural cell adhesion molecule and its response to sialic acid supplement in piglets.

ST8Sia IV (polysialyltransferase IV gene) encodes a key enzyme that is required for polysialic acid synthesis. Polysialic acid is a component of the neural cell adhesion molecule and is necessary for synaptic plasticity of neural cells. We characterized 5.3 kb of pig ST8Sia IV cDNA and determined its expression profile in different organs. In hippocampus, ST8Sia IV mRNA levels were increased approximately 4.5-fold in piglets with sialic acid as a milk supplement, which suggested that exogenous sialic acid is a conditionally essential nutrient for early brain development. Extensive analyses were also performed among its orthologs from human, mouse, rat, chicken, frog and zebrafish. Our results supported that the piglet is a better animal model than other nonprimate species in the studies of ST8Sia IV related metabolism and nutrition in human infants. This pig cDNA provides a basis for uncovering the roles of ST8Sia IV during piglet development and maturation.

The asialoglycoprotein receptor clears glycoconjugates terminating with sialic acid alpha 2,6GalNAc.

Endogenous ligands have not, to date, been identified for the asialoglycoprotein receptor (ASGP-R), which is abundantly expressed by parenchymal cells in the liver of mammals. On the basis of the rapid clearance of BSA bearing multiple chemically coupled sialic acid (Sia)alpha2,6GalNAcbeta1,4GlcNAcbeta1,2Man tetrasaccharides (SiaGGnM-BSA) from the circulation, and the ability of the ASGP-R hepatic lectin-1 subunit to bind SiaGGnM-BSA, we previously proposed that glycoproteins modified with structures terminating with Siaalpha2,6GalNAc may represent previously unrecognized examples of endogenous ligands for this receptor. Here, we have taken a genetic approach using wild-type and ASGP-R-deficient mice to determine that the ASGP-R in vivo does indeed account for the rapid clearance of glycoconjugates terminating with Siaalpha2,6GalNAc. We have also determined that the ASGP-R is able to bind core-substituted oligosaccharides with the terminal sequence Siaalpha2,6Galbeta1,4GlcNAc but not those with the terminal Siaalpha2,3Galbeta1,4GlcNAc. We propose that glycoproteins bearing terminals Siaalpha2,6GalNAc and Siaalpha2,6Gal are endogenous ligands for the ASGP-R, and that the ASGP-R helps to regulate the relative concentration of serum glycoproteins bearing alpha2,6-linked Sia.

Increased expression of the selectin ligand sialyl-Lewis(x) by biochemical engineering of sialic acids.

Sialylation of glycoproteins and glycolipids plays an important role during development, regeneration and pathogenesis of several diseases. The precursor of all physiological sialic acids is N-acetyl-d-mannosamine. Using N-propanoyl mannosamine, a novel precursor of sialic acid, we showed earlier that sialic acids with a prolonged N-acyl side chain (e.g., N-propanoyl neuraminic acid) are incorporated into cell surface glycoconjugates. In this study, we report the structural and functional consequences of the incorporation of the nonphysiological sialic acid, N-propanoyl neuraminic acid, into glycoconjugates of HL60-I cells. These cells do not express UDP-GlcAc-2-epimerase, the key enzyme of the biosynthesis of N-acetyl-d-mannosamine. Therefore, they do not express sialyl-Lewis(x) structures and consequently do not bind to selectins. Application of N-acetyl-d-mannosamine leads to the expression of sialyl-Lewis(x) structures and to binding to selectins. Surprisingly, incorporation of N-propanoyl neuraminic acid into glycoconjugates of these cells leads to a dramatic increase of sialyl-Lewis(x) structures and to increased adhesion to selectins.

N-Acetylneuraminic acid uptake in Pasteurella (Mannheimia) haemolytica A2 occurs by an inducible and specific transport system.

The N-acetylneuraminic acid (NeuAc) transport system of Pasteurella (Mannheimia) haemolytica A2 was studied when this bacterium was grown in both complex and chemically defined media. Kinetic measurements were carried out at 37 degrees C in 50 mM Tris-HCl buffer, pH 8.0, containing 50 microg/ml bovine serum albumin. Under these conditions, the uptake rate was linear for at least 3 min and the calculated K(m) for NeuAc was 0.1 microM. The transport rate was increased by the addition of several cations and was inhibited by sodium arsenite (95%), N,N'-dicyclohexyl-carbodiimide (50%), and 2,4-dinitrophenol (40%) at final concentration of 1 mM (each). These results support the notion that NeuAc uptake is an active sugar cation symporter. Study of specificities showed that glucosamine, mannose and mannosamine inhibited the transport of NeuAc in this bacterium. Analysis of expression revealed that the NeuAc transport system was induced by NeuAc and by the simultaneous presence of glucose and galactose in the growth medium.

Role of sialylation in determining the pharmacokinetics of neutrophil inhibitory factor (NIF) in the Fischer 344 rat.

1. Recombinant neutrophil inhibitory factor (NIF) is a glycoprotein. Its amino acid sequence remains constant and has a molecular weight of 28.9 kD. However, approximately 40% of the total molecular weight consists of glycans with variable structure. 2. The pharmacokinetics of 11 different NIF batches with varying extents and patterns of sialylation have been investigated in the Fischer 344 rat following intravenous administration. These data indicate that reducing the extent of NIF sialylation reduces the half-life of the molecule due to an increase in the systemic clearance. Also, an increase in the number of unsialylated or neutral glycans may increase the volume of distribution of NIF, although this effect is marginal. 3. Isolated perfused rat liver (IPRL) investigations have shown that sialylated NIF has a low hepatic extraction (< 1%), while asialo NIF has an extraction that is > 20-fold higher. Co-administration of asialo NIF with asialo fetuin (a protein cleared by hepatic asialoglycoprotein receptor (possibly galactose)-mediated uptake reduced the hepatic extraction of asialo NIF. 4. These data suggest that NIF molecules that have free sugar moieties (possibly galactose) interact with an asialoglycoprotein receptor (possibly galactose-mediated) in the liver (parenchymal cells/hepatocytes). Interaction with this receptor leads to cellular internalization and degradation.

Wheatgerm agglutinin-mediated toxicity in pancreatic cancer cells.

Lectin binding specificities for carbohydrate allow phenotypic and functional characterization of membrane-associated glycoproteins expressed on cancer cells. This analysis examined wheatgerm agglutinin binding to pancreatic cancer cells in vitro and the resulting toxicity. Membrane preparations of nine human pancreatic carcinoma cell lines were studied for lectin binding using wheatgerm agglutinin (WGA), concanavalin A (ConA) and phytohaemagglutinin-L (PHA-L) in a lectin blot analysis. Cell proliferation in vitro was measured by thymidine incorporation in the absence or presence of lectins at various concentrations. Sialic acid binding lectins or succinyl-WGA (succWGA) served as controls. WGA toxicity was tested after swainsonine or neuraminidase pretreatment. Binding and uptake of fluorescein-labelled lectins was studied under fluorescence microscopy. All pancreatic cell lines displayed high WGA membrane binding, primarily to sialic acid residues. Other lectins were bound with weak to moderate intensity only. Lectin toxicity corresponded to membrane binding intensity, and was profound in case of WGA (ID50 at 2.5-5 microg ml(-1)). WGA exposure induced chromatin condensation, nuclear fragmentation and DNA release consistent with apoptosis. Important steps for WGA toxicity included binding to sialic acid on swainsonine-sensitive carbohydrate and lectin internalization. There was rapid cellular uptake and subsequent nuclear relocalization of WGA. In contradistinction to the other lectins studied, WGA proved highly toxic to human pancreatic carcinoma cells in vitro. WGA binding to sialic acid residues of N-linked carbohydrate, cellular uptake and subsequent affinity to N-acetyl glucosamine appear to be necessary steps. Further analysis of this mechanism of profound toxicity may provide insight relevant to the treatment of pancreatic cancer.

Differential clearance of glycoforms of IgG in normal and autoimmune-prone mice.

In order to understand better the origins of the elevated levels of the glycoform of IgG that lacks galactose on both arms of the oligosaccharide chain (G0%) located in the Fc, which occurs in man and mouse with age, and in particular in autoimmune disease, we investigated the clearance of two glycosylated forms of IgG2a and IgG1 in normal (BALB/c) and autoimmune-prone (MRL/1pr, MRL/+, and non-obese diabetic (NOD)) mice. To investigate the possibility of different rates of catabolism, enzymatically generated glycoforms of monomeric IgG1 and IgG2a (fully glycosylated or G0%), were iodinated and injected into the tail vein of the mice. We found that the G0% IgG2a remained in circulation significantly longer than the fully glycosylated variants, in all of the mouse strains tested. In contrast, the two forms of IgG1 had similar kinetics in all the autoimmune-prone mice, whereas in BALB/c, there was a longer half-life (t1/2) for G0% IgG1. These data suggest that there may be differences in the ability of the IgG glycoforms to bind to the Fc gamma receptors, in particular Fc gamma RI. The clearance rates were found to vary among the strains studied, with MRL/1pr having the fastest catabolic rates for all glycoforms and IgG subclasses tested. This appeared to be due to the presence of circulating IgG and IgM rheumatoid factors (RF). There were significantly increased frequencies and titres for both IgM and IgG RF in MRL/1pr mice compared with the other strains. In contrast, interferon-gamma, known to induce the Fc gamma RI, was found to be similar in the sera, in all of the strains of mice examined. These results suggest that RF probably play an important biological function in the MRL/1pr mice and aid in the clearance of circulating IgG. Our study shows that the state of glycosylation of IgG affects the t1/2 in vivo, and that by removing the terminal sugars (sialic acid and galactose), the antibody (IgG2a) will remain in circulation significantly longer. These observations may thus provide a partial explanation for the increase in relative percentage of this glycoform that occurs with age.