To date, the structure-activity relationship studies of heparin/heparan sulfate with their diverse binding partners such as growth factors, cytokines, chemokines, and extracellular matrix proteins have been limited yet provide early insight that specific sequences contribute to this manifold biological role. This has led to an impetus for the chemical synthesis of oligosaccharide fragments of these complex polysaccharides, which can provide an effective tool for this goal. The synthesis of three heparin mimetic hexasaccharides with distinct structural patterns is described herein, and the influence of the targeted substitution on their bioactivity profiles is studied using in vitro affinity and/or inhibition toward different growth factors and proteins. Additionally, the particularly challenging synthesis of an irregular hexasaccharide is reported, which, interestingly, in spite of being considerably structurally similar with its two counterparts, displayed a unique and remarkably distinct profile in the test assays.
M281 is a fully human, anti-neonatal Fc receptor (FcRn) antibody that inhibits FcRn-mediated immunoglobulin G (IgG) recycling to decrease pathogenic IgG while preserving IgG production. A randomized, double-blind, placebo-controlled, first-in-human study with 50 normal healthy volunteers was designed to probe safety and the physiological maximum for reduction of IgG. Intravenous infusion of single ascending doses up to 60 mg/kg induced dose-dependent serum IgG reductions, which were similar across all IgG subclasses. Multiple weekly doses of 15 or 30 mg/kg achieved mean IgG reductions of ≈85% from baseline and maintained IgG reductions ≥75% from baseline for up to 24 days. M281 was well tolerated, with no serious or severe adverse events (AEs), few moderate AEs, and a low incidence of infection-related AEs similar to placebo treatment. The tolerability and consistency of M281 pharmacokinetics and pharmacodynamics support further evaluation of M281 in diseases mediated by pathogenic IgG.
The importance of IgG glycosylation, Fc-gamma receptor (FcγR) single nucleotide polymorphisms and FcγR copy number variations in fine tuning the immune response has been well established. There is a growing appreciation of the importance of glycosylation of FcγRs in modulating the FcγR-IgG interaction based on the association between the glycosylation of recombinant FcγRs and the kinetics and affinity of the FcγR-IgG interaction. Although glycosylation of recombinant FcγRs has been recently characterized, limited knowledge exists on the glycosylation of endogenous human FcγRs. In order to improve the structural understanding of FcγRs expressed on human cells we characterized the site specific glycosylation of native human FcγRIII from neutrophils of 50 healthy donors and from matched plasma for 43 of these individuals. Through this analysis we have confirmed site specific glycosylation patterns previously reported for soluble FcγRIII from a single donor, identified FcγRIIIb specific Asn45 glycosylation and an allelic effect on glycosylation at Asn162 of FcγRIIIb. Identification of FcγRIIIb specific glycosylation allows for assignment of FcγRIIIb alleles and relative copy number of the two alleles where DNA/RNA is not available. Intriguingly the types of structures found to be elevated at Asn162 in the NA2 allele have been shown to destabilize the Fc:FcγRIII interaction resulting in a faster dissociation rate. These differences in glycosylation may in part explain the differential activity reported for the two alleles which have similar in vitro affinity for IgG. The importance of IgG glycosylation, Fc-gamma receptor (FcγR) single nucleotide polymorphisms and FcγR copy number variations in fine tuning the immune response has been well established. There is a growing appreciation of the importance of glycosylation of FcγRs in modulating the FcγR-IgG interaction based on the association between the glycosylation of recombinant FcγRs and the kinetics and affinity of the FcγR-IgG interaction. Although glycosylation of recombinant FcγRs has been recently characterized, limited knowledge exists on the glycosylation of endogenous human FcγRs. In order to improve the structural understanding of FcγRs expressed on human cells we characterized the site specific glycosylation of native human FcγRIII from neutrophils of 50 healthy donors and from matched plasma for 43 of these individuals. Through this analysis we have confirmed site specific glycosylation patterns previously reported for soluble FcγRIII from a single donor, identified FcγRIIIb specific Asn45 glycosylation and an allelic effect on glycosylation at Asn162 of FcγRIIIb. Identification of FcγRIIIb specific glycosylation allows for assignment of FcγRIIIb alleles and relative copy number of the two alleles where DNA/RNA is not available. Intriguingly the types of structures found to be elevated at Asn162 in the NA2 allele have been shown to destabilize the Fc:FcγRIII interaction resulting in a faster dissociation rate. These differences in glycosylation may in part explain the differential activity reported for the two alleles which have similar in vitro affinity for IgG. Receptors for the Fc region of IgG (FcγRs) 1The abbreviations used are:FcγRreceptor for Fc region of IgGADCPantibody dependent cellular phagocytosisADCCantibody dependent cellular cytotoxicityITAMimmunoreceptor tyrosine-based activation motifGPIglycophosphatidylinositolMLPAmultiplexed ligation-dependent probe amplification. 1The abbreviations used are:FcγRreceptor for Fc region of IgGADCPantibody dependent cellular phagocytosisADCCantibody dependent cellular cytotoxicityITAMimmunoreceptor tyrosine-based activation motifGPIglycophosphatidylinositolMLPAmultiplexed ligation-dependent probe amplification. are critical in modulating the adaptive immune response. Interaction between the receptors and IgG in immune complexes or on opsonized cells promotes downstream effector function such as antibody dependent cellular phagocytosis (ADCP) and antibody dependent cellular cytotoxicity (ADCC). In humans, there are five activating FcγRs specifically FcγRI (CD64), FcγRIIa (CD32A), FcγRIIc (CD32c), FcγRIIIa (CD16A), and FcγRIIIb (CD16B) as well as the inhibitory FcγRIIb (CD32B) (1Dearon M. Fc receptor biology.Ann. Rev. Immunol. 1997; 15: 203-234Crossref PubMed Scopus (1038) Google Scholar). FcγRIIIa and FcγRIIIb are two closely related proteins with at least 95% homology in the amino acid sequence of the extracellular domains which are nearly indistinguishable when considering the common variants (Fig. 1). receptor for Fc region of IgG antibody dependent cellular phagocytosis antibody dependent cellular cytotoxicity immunoreceptor tyrosine-based activation motif glycophosphatidylinositol multiplexed ligation-dependent probe amplification. receptor for Fc region of IgG antibody dependent cellular phagocytosis antibody dependent cellular cytotoxicity immunoreceptor tyrosine-based activation motif glycophosphatidylinositol multiplexed ligation-dependent probe amplification. FcγRIIIa is expressed on NK cells, and subsets of monocytes, macrophages and dendritic cells. The cytoplasmic domain of FcγRIIIa associates with the immunoreceptor tyrosine-based activation motif (ITAM) containing common FcRγ chain which drives intracellular signaling events (2Ravetch J.V. Perussia B. Alternative membrane forms of Fc gamma RIII (CD16) on human natural killer cells and neutrophils. Cell type-specific expression of two genes that differ in single nucleotide substitutions.J. Exp. Med. 1989; 170: 481-497Crossref PubMed Scopus (493) Google Scholar). The V158F polymorphism which is found in the extracellular domain of FcγRIIIa results in increased affinity between the V158 variant and all IgG subclasses (3Bruhns P. Iannascoli B. England P. Mancardi D.A. Fernandez N. Jorieux S. Daëron M. Specificity and affinity of human Fcγ receptors and their polymorphic variants for human IgG subclasses.Blood. 2009; 113: 3716-3725Crossref PubMed Scopus (981) Google Scholar). Functionally, NK cells bearing FcγRIIIa with the V158 variant exhibit enhanced response to immune complex stimulation (4Wu J. Edberg J.C. Redecha P.B Bansal V Guyre P.M Coleman K Salmon J.E Kimberly R.P. A novel polymorphism of FcgammaRIIIa (CD16) alters receptor function and predisposes to autoimmune disease.J. Clin. Invest. 1997; 100: 1059-1070Crossref PubMed Scopus (595) Google Scholar). FcγRIIIb is a glycophosphatidylinositol (GPI) linked protein expressed primarily on neutrophils and basophils (2Ravetch J.V. Perussia B. Alternative membrane forms of Fc gamma RIII (CD16) on human natural killer cells and neutrophils. Cell type-specific expression of two genes that differ in single nucleotide substitutions.J. Exp. Med. 1989; 170: 481-497Crossref PubMed Scopus (493) Google Scholar). FcγRIIIb is highly polymorphic with three common alleles differing at 5 sites in the protein (Fig. 1). Alleles named NA1, NA2 and SH or alternately HNA-1a, HNA-1b and HNA-1c have been described (2Ravetch J.V. Perussia B. Alternative membrane forms of Fc gamma RIII (CD16) on human natural killer cells and neutrophils. Cell type-specific expression of two genes that differ in single nucleotide substitutions.J. Exp. Med. 1989; 170: 481-497Crossref PubMed Scopus (493) Google Scholar), (5Bux J. Stein E.L. Bierling P. Fromont P. Clay M. Stroncek D. Santoso S. Characterization of a new alloantigen (SH) on the human neutrophil Fc gamma receptor IIIb.Blood. 1997; 89: 1027-1034Crossref PubMed Google Scholar) and additional variants have been detected (6Matsuo K. Procter J.L. Stroncek D. Variations in genes encoding neutrophil antigens NA1 and NA2.Transfusion. 2000; 40: 645-653Crossref PubMed Scopus (30) Google Scholar). These variants do not influence the affinity of the FcγR-IgG interaction (3Bruhns P. Iannascoli B. England P. Mancardi D.A. Fernandez N. Jorieux S. Daëron M. Specificity and affinity of human Fcγ receptors and their polymorphic variants for human IgG subclasses.Blood. 2009; 113: 3716-3725Crossref PubMed Scopus (981) Google Scholar) but have been reported to influence neutrophil activity (7Van der Heijden J. Zhao X. Geissler J. Rispens T. Van den Berg T.K. Kuijpers T.W. Haplotypes of FcγRIla and FcγRIIIb Polymorphic Variants Influence IgG-Mediated Responses in Neutrophils.J. Immunol. 2014; 192: 2715-2721Crossref PubMed Scopus (29) Google Scholar), (8Salmon J.E. Edberg J.C. Kimberly R.P. Fcy receptor III on human neutrophils. Allelic variants have functionally distinct capacities.J. Clin. Invest. 1990; 85: 1287-1295Crossref PubMed Scopus (203) Google Scholar), and (9Salmon J.E. Edberg J.C. Brogle N.L. Kimberly R.P. Allelic polymorphisms of human Fcy receptor IIA and Fcy receptor IIIB. Independent mechanisms for differences in human phagocyte function.J. Clin. Invest. 1992; 89: 1274-1281Crossref PubMed Scopus (250) Google Scholar). Both FcγRIIIa and FcγRIIIb are heavily glycosylated. FcγRIIIa contains five potential sites of glycosylation at N38, N45, N74, N162 and N169. The NA1 and NA2/SH alleles are distinguished by amino acid differences at four sites specifically R19S, N47S, D65N and V89I for the NA1 and NA2 alleles respectively. The SH allele is distinguished from the NA2 alleles by A61D substitution (5Bux J. Stein E.L. Bierling P. Fromont P. Clay M. Stroncek D. Santoso S. Characterization of a new alloantigen (SH) on the human neutrophil Fc gamma receptor IIIb.Blood. 1997; 89: 1027-1034Crossref PubMed Google Scholar). The NA2 allele of FcγRIIIb is potentially glycosylated at the five sites found in FcγRIIIa and has an additional consensus site at N65. The NA1 allele on the other hand has only four potential sites of glycosylation because of allelic variation. The N45 and N65 sites are not glycosylated in the NA1 allele because of the presence of N47 and D65 respectively. Glycosylation has long been established as a critical parameter influencing the FcγR-IgG interaction with core fucosylation (10Umaña P. Jean-Mairet J. Moudry R. Amstutz H. Bailey J.E. Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity.Nat. Biotechnol. 1999; 17: 176-180Crossref PubMed Scopus (647) Google Scholar) and sialylation (11Kaneko Y. Nimmerjahn F. Ravetch J.V. Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation.Science. 2006; 313: 670-673Crossref PubMed Scopus (1379) Google Scholar) of the Fc domain of IgG being the best studied. FcγRIII glycosylation has additionally been reported to play a role in the Fc:FcγRIII interaction. Point mutations targeting each of the N-linked glycans from FcγRIII demonstrated that the glycans at N45 and N162 play a role in the formation of the Fc:FcγRIII complex. Removal of the N45 glycosylation site increased the affinity of the interaction between the Fc bearing nonfucosylated glycans and FcγRIIIa (12Shibata-Koyama M. Iida S. Okazaki A. Mori K. Kitajima-Miyama K. Saitou S. Kakita S. Kanda Y. Shitara K. Kato K. Satoh M. The N-linked oligosaccharide at FcγRIIIa Asn-45: an inhibitory element for high FcγRIIIa binding affinity to IgG glycoforms lacking core fucosylation.Glycobiology. 2009; 19: 126-134Crossref PubMed Scopus (92) Google Scholar). A separate study using a similar approach demonstrated that the N-glycans at N162 were required for the higher affinity interaction seen for nonfucosylated glycans on the Fc (13Ferrara C. Stuart F. Sondermann P. Brünker P. Umaña P. The carbohydrate at FcγRIIIa Asn-162. An element required for high affinity binding to non-fucosylated IgG glycoforms.J. Biol. Chem. 2006; 281: 5032-5036Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). The crystal structure of the complex between FcγRIIIa bearing high mannose type glycans (14Ferrara C. Grau S. Jäger C. Sondermann P. Brçnker P. Waldhauer I. Hennig M. Ruf A. Rufer A.C. Stihle M. Umaña P. Benz J. Unique carbohydrate-carbohydrate interactions are required for high affinity binding between FcgammaRIII and antibodies lacking core fucose.Proc. Natl. Acad. Sci. USA. 2011; 108: 12669-12674Crossref PubMed Scopus (542) Google Scholar) or neutral complex glycans (15Mizushima T. Yagi H. Takemoto E. Shibata-Koyama M. Isoda Y. Iida S. Masuda K. Satoh M. Kato K. Structural basis for improved efficacy of therapeutic antibodies on defucosylation of their Fc glycans.Genes Cells. 2011; 16: 1071-1080Crossref PubMed Scopus (187) Google Scholar) and the Fc domain for human IgG1 showed the interaction was stabilized by carbohydrate-protein and carbohydrate-carbohydrate interactions primarily involving FcγRIII glycans at N162. A recent study utilizing NMR to characterize the solution phase dynamics of glycoengineered FcγRIIIa expressed in HEK cells identified unexpected contacts between the glycans at N45 and the polypeptide backbone (16Subedi G.P. Falconer D.J. Barb A.W. Carbohydrate–Polypeptide Contacts in the Antibody Receptor CD16A Identified through Solution NMR Spectroscopy.Biochemistry. 2017; 56: 3174-3177Crossref PubMed Scopus (31) Google Scholar). Initial studies examining influence of FcγR glycosylation on the FcγRIII:Fc interaction relied on mutagenesis to selectively remove entire glycan chains. Subsequent studies utilized recombinant proteins produced in different cell types to examine the influence of the nature of the glycans present on the receptor on Fc:FcγRIII binding. Several groups have published data on recombinant FcγR glycosylation from BHK (17Takahashi N. Cohen-Solal J. Galinha A. Fridman W.H. Sautès-Fridman C. Kato K. N-glycosylation profile of recombinant human soluble Fcgamma receptor III.Glycobiology. 2002; 12: 507-515Crossref PubMed Scopus (19) Google Scholar), NS0 (18Cosgrave E.F. Struwe W.B. Hayes J.M. Harvey D.J. Wormald M.R. Rudd P.M. N-Linked glycan structures of the human Fcgamma receptors produced in NS0 cells.J. Proteome Res. 2013; 12: 3721-3737Crossref PubMed Scopus (24) Google Scholar), CHO and HEK (19Zeck A. Pohlentz G. Schlothauer T. Peter-Kataliniæ J. Regula J.T. Cell type-specific and site directed N-glycosylation pattern of FcγRIIIa.J. Proteome Res. 2011; 10: 3031J-3039JCrossref PubMed Scopus (64) Google Scholar), (20Hayes J.M. Frostell A. Karlsson R. Mçller S. Martín S.M. Pauers M. Reuss F. Cosgrave E.F. Anneren C. Davey G.P. Rudd P.M. Identification of Fc gamma receptor glycoforms that produce differential binding kinetics for rituximab.Mol. Cell. Proteomics. 2017; 16: 1770-1788Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The FcγRIII glycosylation pattern varied significantly among expression systems and was demonstrated to influence the kinetics but not the affinity of the interaction when comparing CHO and HEK expressed protein (19Zeck A. Pohlentz G. Schlothauer T. Peter-Kataliniæ J. Regula J.T. Cell type-specific and site directed N-glycosylation pattern of FcγRIIIa.J. Proteome Res. 2011; 10: 3031J-3039JCrossref PubMed Scopus (64) Google Scholar). A recent publication compared the effect of expression system on glycosylation pattern of recombinant FcγRI and FcγRIIIa and reproduced the differential kinetics reported by Zeck et al. (20Hayes J.M. Frostell A. Karlsson R. Mçller S. Martín S.M. Pauers M. Reuss F. Cosgrave E.F. Anneren C. Davey G.P. Rudd P.M. Identification of Fc gamma receptor glycoforms that produce differential binding kinetics for rituximab.Mol. Cell. Proteomics. 2017; 16: 1770-1788Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The authors proposed that glycosylation differences, principally branching and sialylation, destabilized the interaction resulting in more rapid dissociation of the complex. These studies complement those utilizing point mutations and provide detail on the influence of FcγR glycan structure on the Fc-FcγR interaction. The advances in LC-MS based characterization of glycopeptides in the past decade (21Wuhrer M. Catalina M.I. Deelder A.M. Hokke C.H. Glycoproteomics based on tandem mass spectrometry of glycopeptides.J. Chromatography B. 2007; 849: 115-128Crossref PubMed Scopus (355) Google Scholar), provide a means for monitoring site specific glycosylation changes (22Segu Z.M. Mechref Y. Characterizing protein glycosylation sites through higher-energy C-trap dissociation.Rapid Commun. Mass Spectrometry. 2010; 24: 1217-1225Crossref PubMed Scopus (107) Google Scholar) of proteins from complex biological systems (23Thaysen-Andersen M. Packer N.H. Advances in LC–MS/MS-based glycoproteomics: Getting closer to system-wide site-specific mapping of the N- and O-glycoproteome.Biochim. Biophys. Acta. 2014; 1844: 1437-1452Crossref PubMed Scopus (170) Google Scholar). Characterization of site-specific glycosylation patterns of endogenous human FcγRs can help to advance an understanding of the impact of FcγR glycosylation on immune cell activation. Here we present the characterization of native FcγRIIIb glycosylation from isolated human neutrophils as well as soluble FcγRIII, which is a mixture of FcγRIIIa and FcγRIIIb, isolated from matched plasma. Through this analysis we identified FcγRIIIb specific glycosylation at N45 and an allelic influence on glycosylation of N162, which are consistent with and expand upon recent reports. Matched plasma and neutrophils were obtained from healthy donors after informed consent through a combination of an internal blood donor program as well as a commercial source (Sanguine Bio; Sherman Oaks CA). The collection, handling and biomolecular analysis of healthy human neutrophils per experimental protocol 102013–001 was approved by the Western Institutional Review Board. Plasma was collected in EDTA tubes. Neutrophils were isolated from lysed whole blood by negative selection using the Neutrophil Enrichment Kit (StemCell Technologies, Vancouver, BC, Canada; Catalogue #19257) following manufacturer's instructions. Freshly isolated neutrophils were pelleted and frozen at −80 °C until they were used for analysis. Paired neutrophils and plasma from a patient were from the same draw. For 33 of these donors FcγR polymorphisms and copy number were determined using Multiplexed Ligation-dependent Probe Amplification (MLPA). MLPA assays were performed using commercially available kits from MRC-Holland (P110–100R and P111–100R) using methods similar to those previously described (24Breunis W.B. van Mirre E. Geissler J. Laddach N. Wolbink G. van der Schoot E. de Haas M. de Boer M. Roos D. Kuijpers T.W. Copy number variation at the FCGR locus includes FCGR3A, FCGR2C and FCGR3B but not FCGR2A and FCGR2B.Human Mutation. 2009; 30: E640-E650Crossref PubMed Scopus (118) Google Scholar), (25Nagelkerke S. Tacke C.E. Breunis W.B. Geissler J. Sins J.W. Appelhof B. van den Berg T.K. de Boer M Kuijpers T.W. Nonallelic homologous recombination of the FCGR2/3 locus results in copy number variation and novel chimeric FCGR2 genes with aberrant functional expression.Genes Immunity. 2015; 16: 422-429Crossref PubMed Scopus (23) Google Scholar). This kit contains probes for determining copy number variation (CNV) and single nucleotide polymorphisms (SNPs) in the FCGR2/C locus. Genomic DNA was extracted from neutrophils from healthy donors using the QIAamp DNA Mini Blood Kit (QIAgen, Hilden, Germany). DNA was denatured at 95 °C and incubated with MLPA probes at 60 °C for a minimum of 18 h. Ligation and PCR amplification were performed according to MLPA manufacturer instructions. Fragment analysis was performed on an ABI-3730XL capillary electrophoresis instrument using POP7 polymer. Data were analyzed using the Coffalyser software (MRC-Holland) according to software manufacturer instructions. FCGR3B allele types and copy number were determined using probes 06639-L06203, 03616-L02983, and 03616-L02990; copy number determination was confirmed by FCGR3B allele-independent probes 03618-L02985 and 03615-L12809. Neutrophil FcγRIIIb was isolated from ∼5 million neutrophils. Plasma FcγRIII was isolated from 50 μl of plasma. Proteins were immunoprecipitated using biotinylated goat polyclonal antibodies against human FcγRIII (R&D Systems, Minneapolis, MN; BAF1597). The proteins were isolated from neutrophils by first spinning down cells at 300 × g for 2 min and then washing 3 × 500 μl of ice cold PBS. Then 75 μl of IP Lysis Buffer (ThermoFisher Scientific, Waltham, MA; 87787) was added to each sample and cells were lysed by sonication and cell debris spun out at 10,000 × g for 5 min. PBS was added to the supernatant to bring the volume to 500 μl then the biotinylated antibody was added and allowed to incubate for 18 h at 4 °C. The antibody-FcγR complex was isolated using streptavidin magnetic beads (ThermoFisher Scientific 88816). The beads were washed two times 500 μl of IP Lysis buffer and two times with 500 μl of ice-cold PBS. The bound protein was eluted by incubating the beads in 50 μl of 6 m guanidine HCl. The eluted protein was reduced for 30 min at 65 °C with DTT at a concentration of 25 mm. Free cysteine residues were alkylated with iodoacetamide at a concentration of 75 mm. The isolated proteins were dialyzed across a 10kDa membrane against 4L of 25 mm ammonium bicarbonate for 18 h at 4 °C before proteolysis. Soluble FcγRs were isolated from 50 μl of plasma as described above omitting the cell lysis and centrifugation step. The glycosylation pattern of FcγRIII N45 was characterized using a chymotrypsin (Sequencing Grade Promega, Madison, WI; V1061) which cleaves C-terminal to hydrophobic residues such as tyrosine, tryptophan and phenylalanine to generate the glycopeptides shown in Table I. A sequential digestion with endoproteinase GluC (Sequencing Grade Promega V1651) which cleaves C-terminal to glutamic acid followed by chymotrypsin was used to characterize the N162 glycopeptides. We have focused on site specific glycosylation at N45 and N162 because removal of these two sites of glycosylation was demonstrated to influence the interaction between FcγRIII and IgG. The glycopeptide at N45 is common to FcγRIIIa and the NA2 and SH alleles of FcγRIIIb whereas the NA1 allele is nonglycosylated at this site. The glycopeptide at N162 is common to FcγRIIIa and all alleles of FcγRIIIb (Table I).Table ISpecies-specific peptide sequences generated from proteolysis of FcγRIIISequenceSpeciesVariantFIDA(61)ATVD(65)DSGEYFcγRIIIb NA1/FcγRIIIaFcγRIII A61/D65FIDA(61)ATVN(65)DSGEYFcγRIIIb NA2FcγRIII A61/N65FIDD(61)ATVN(65)DSGEYFcγRIIIb SHFcγRIII D61/N65FHN(45)ES(47)LISSQASSYFcγRIIIb NA2/SH/FcγRIIIaFcγRIII S47/N45 glycopeptidesFHN(45)EN(47)LISSQASSYFcγRIIIb NA1FcγRIII N47SDPVQLEVHI(89)GWFcγRIIIb NA2/SH/FcγRIIIaFcγRIII I89SDPVQLEVHV(89)GWFcγRIIIb NA1FcγRIII V89VGSKN(162)VSSEFcγRIII N162FcγRIII N162 glycopeptides Open table in a new tab The peptides and glycopeptides were analyzed by nLC-MS/MS on a Dionex Ultimate 3000 nano RSLC (ThermoFisher Scientific) coupled to a QExactive mass spectrometer (ThermoFisher Scientific) equipped with and EasySpray nano-LC source (ThermoFisher Scientific). Peptides were separated on an EasySpray C18 column (0.75 × 250 mm 2 μm particle size ThermoFisher Scientific ES802). A data dependent acquisition was run initially to identify glycopeptides from each site. Glycopeptides were identified by searching the high-resolution accurate mass MS/MS spectra for Y1 ions which tend to form readily under HCD fragmentation of N-glycopeptides. Nonreducing end oxonium ions diagnostic for a variety of structural features including N-acetyllactosamine extensions and antennary fucose including Lewis and sialyl-Lewis structures were used to differentiate isomeric species. The structures of selected isomeric glycans were confirmed using low energy CID fragmentation on an Orbitrap Velos (ThermoFisher Scientific). Glycopeptide fragmentation for structural characterization was visualized using GlycoWorkbench (26Ceroni A. Maass K. Geyer H. Geyer R. Dell A. Haslam S.M. GlycoWorkbench: a tool for the computer-assisted annotation of mass spectra of glycans.J. Proteome Res. 2008; 7: 1650-1659Crossref PubMed Scopus (753) Google Scholar). Isomers arising from differences in sialic acid linkages were characterized using sialidase S according to manufacturer's instruction. This enzyme preferentially cleaves α2–3 linked sialic acid under the conditions used. Multiple chromatographic peaks were identified for each of the sialylated species across all sites. After initial identification a targeted Tier 3 nLC-MS/MS method was applied for the quantitation of site specific glycosylation as well as assignment of allelic variants based on peptide sequence information. The quadrupole isolation width was set to ±1.5 Da for the isolation of the parent ion of each of the species for the chymotryptic digest (Table II) and the chymotryptic+GluC digest (Table III). The AGC was on the QExactive was set to 1,000,0000 ions and a normalized collision energy of 27 was used for glycopeptide fragmentation. Targeted species were confirmed based on the full MS/MS and quantified based on the extracted ion abundance for the most abundant fragment. Glycopeptides fragmented by HCD give rise to a characteristic Y1 ion which when detected with HR/AM provides a marker fragment unique to each glycopeptide backbone. Identification of the glycopeptides was dependent upon the presence of the Y1 ion in the MS/MS spectrum as well as the presence of at least two specific peptide fragments and the characteristic oxonium ions (supplemental Fig. S2, supplemental Fig. S3). The relative abundance of each glycopeptide was determined using the extracted ion abundance of the Y1 ion relative to the summed extracted ion abundance of this fragment from all glycopeptides at each site of glycosylation. The specific precursor and fragment ions monitored for each species are shown in supplemental information (supplemental Fig. S2).Table IICharacterization of alleleic variants and N45 glycosylation. Species monitored for targeted MS/MS analysis of FcγRIII chymotryptic digest. Isolation width was set at 3 Da. The species monitored are described in the comments column. The species with glycan compositions represent glycopeptide while those with the FcγRIIIb alleles are peptide specific markers for those allelesMass [m/z]Start [min]End [min](N)CESpecies701.3425025FcγRIII N65D NA1/NA2798.9354125FcγRIII N47 NA1723.8306025FcγRIII D61 SH683.3475325FcγRIII V89 NA1690.5505625FcγRIII I89 NA21394.1323825FcγRIIIb S47 N45 M51475.1323825FcγRIIIb S47 N45 M61556.1313725FcγRIIIb S47 N45 M71094.8364225FcγRIII S47 N45 M4A1G1S11143.5364225FcγRIII S47 N45 FM4A1G1S11637.1313725FcγRIIIb S47 N45 M81718.2303725FcγRIIIb S47 N45 M91148.8364225FcγRIII S47 N45 M5A1G1S11040.0364225FcγRIII S47 N45 A1G1S11088.8364225FcγRIII S47 N45 FA1G1S1 Open table in a new tab Table IIICharacterization of N162 glycosylation. Species monitored for targeted MS/MS analysis of FcγRIII GluC+chymotrypsin digest. Isolation width was set at 3 DaMass [m/z]Composition1038.8N162 FA2G2S1F11111.4N162 FA3G3S11160.1N162 FA3G3S1F11209.2N162 FA3G3S2868.0N162 FA1G1S1989.7N162 FA2G2S1892.7N162 FA2G21086.7N162 FA2G2S21135.4N162 FA2G2S2F11257.2N162 FA3G3S2F1941.5N162 FA2G2F11306.2N162 FA3G3S3453.7N162 Agycosyl Open table in a new tab Data analysis was performed using Xcalibur software Qual browser. Each glycopeptide species was quantified based on the MS/MS extracted ion current (XIC) for the ion corresponding to the Peptide+N-acetylglucosamine fragment. The relative abundance was calculated by dividing the XIC area of each species by the summed XIC area for each site. FcγRIIIb alleles were identified based on the presence of marker peptides and specific glycosylation at N45; the specific markers used at each site for the assignment the FcγRIIIb alleles are shown below (Table IV). For assignment of alleles and comparison to copy numbers the abundance of N45 high mannose type glycopeptides was calculated relative to the sum of all high mannose glycopeptides plus the NA1 specific aglycosyl peptide. For presentation of N-glycopeptide distribution the aglycosyl NA1 peptide was not included in the calculation. In order to estimate site occupancy at N65 from the NA2 allele the area of the nonglycosylated peptide was normalized to the area of the D65 peptide from the NA1 allele for heterozygotes. Similarly, the area of the NA2 specific I89 peptide was normalized to the NA1 specific V89 peptide. The ratio of these normalized areas was calculated for donors having both alleles. Because the normalized area N65 to D65 but not the normalized area of I89 to V90 is affected by the presence of glycosylation at N65 a ratio close to 1 is indicative of low site occupancy and ratios ≪1 indicative of relatively high site occupancy.Table IVMarker peptides and glycopeptide sequences for assignment of FcγRIII allelesFcγRIIIb NA1FcγRIIIb NA2FcγRIIIb SHFcγRIIIaFHN(45)EN(47)LISSQASSY AglycosylatedFHN(45)ES(47)LISSQASSY GlycosylatedFHN(45)ES(47)LISSQASSY GlycosylatedFHN(45)ES(47)LISSQASSY GlycosylatedFIDAA(61)TVD(65)DSGEYFIDA(61)ATVN(65)DSGEYFIDD(61)ATVN(65)DSGEYFIDAA(61)TVD(65)DSGEYSDPVQLEVHV(89)GWSDPVQLEVHI(89)GWSDPVQLEVHI(89)GWSDPVQLEVHI(89)GW Open table in a new tab The genotypes of the subjects included in this study were 29% (n = 14) NA1/NA1, 35% (n = 17) NA1/NA2 and 35% (n = 17) NA2/NA2 and 4% (n = 2) NA1/SH genotype. FcyR glycosylation from neutrophils and plasma was analyzed one time for each donor. Statistical comparisons between the alleles were performed using unpaired two-sided t test. Associations between glycoforms and copy number variants were assessed using one-way ordinary ANOVA. p < 0.05 was considered significant. The FcγRIIIb alleles for 50 donors were determined from FcγRIIIb protein isolated from neutrophils via LC-MS/MS. The alleles were ass
Abstract Introduction: Heparan sulphate proteoglycans (HSPGs) play a central role in tumor progression and metastasis by presenting and modulating growth factors, cytokines, and other soluble factors. A novel heparin sulphate mimetic (M402), engineered from heparin to have low anti-coagulant activity, has shown promising anti-tumor efficacy in several pre-clinical tumor models. This study was designed to probe the efficacy and mechanism of action of M402 in a genetically engineered mouse (GEM) model for pancreatic cancer. Methods: Mice that spontaneously develop pancreatic cancer (LSL-KRASG12D/+; Trp53 LSL-R172H/flox; pdx-CRE) were treated with twice weekly i.p. doses of saline or gemcitabine (50 mg/kg) starting at Day 30, or with saline or M402 (40 mg/kg/day) administered by a subcutaneous osmotic minipump from Day 30-90, or with a combination of gemcitabine plus M402. Results: Treatment with M402 alone did not prolong survival and gemcitabine alone showed only a modest improvement in survival; however the combination of M402 and gemcitabine significantly improved survival. Moreover, mice treated with the combination of M402 and gemcitabine showed a substantially lower incidence of metastasis. RT-qPCR analysis revealed that M402 treated mice had significantly lower levels of TGF-alpha mRNA than the saline control group, which is corroborated by a corresponding decrease in tumor cell proliferation. Immunohistochemical analysis revealed that M402 treated mice developed reduced areas of epithelial-to-mesenchymal transition (EMT), as defined by negative staining for E-Cadherin, strongly positive staining for vimentin and positive nuclear staining for SNAIL. As there is a direct link between EGFR activation and the nuclear localisation of SNAIL we propose that M402 affects gemcitabine sensitivity and metastasis formation by reducing the expression of TGF-alpha. Discussion: M402 increased the anti-tumor efficacy of gemcitabine in a GEM model for pancreatic cancer resulting in increased survival and, interestingly, decreased incidence of metastasis. One potential mechanism is that the observed reduction in EMT may be due to the reduced expression of TGF-alpha, a cognate ligand for EGFR. These data suggest that the EGFR pathway is active in KRAS mutant tumors. Overall, these results provide a rationale for investigating the clinical use of M402 in combination with gemcitabine in the treatment of human pancreatic cancer. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-43.
Abstract Background: Pancreatic ductal adenocarcinoma (PDA) is an almost uniformly lethal disease. The current treatment for resectable disease consists of surgery and adjuvant gemcitabine therapy. However, for more than 80 percent of the patients with PDA surgery is not an option. For these patients palliative chemotherapy using gemcitabine, or most recently FOLFIRINOX, is considered standard of care. Even with palliative chemotherapy the median overall survival for patients with metastatic PDA is dismal and improved therapeutic strategies are a clear unmet medical need. Heparins may be of interest for the treatment of PDA. They play an important role in tumor progression and metastasis by binding at heparin binding domains, thereby preventing growth factor gradients created along proteoglycan heparan sulfates and thus disrupting signaling. We used a novel heparan sulfate mimetic, M402, which binds to multiple growth factors, adhesion molecules, and chemokines. We previously showed that M402 decreases both epithelial-to-mesynchymal transition and chemotherapy resistance in PDA. Here we further evaluated the underlying mechanisms of M402 in EMT. Results: We studied the effects of M402 on PDA using a genetically engineered mouse model (GEMM) for PDA (LSL-KRASG12D/+; Trp53 LSL-R172H/flox; pdx-CRE), which recapitulates human PDA. Combination therapy of M402 with gemcitabine significantly prolonged the average survival of the mice when compared to mice treated with gemcitabine monotherapy (87 days versus 78 days) and significantly reduced metastasis and local invasion into the small intestine. These data suggest an effect of M402 on invasiveness and EMT. Gemcitabine treatment increased EMT as determined by staining for E-cadherin and Fsp1 double positive cells. In contrast, M402 treatment resulted in a decrease in E-cadherin and Fsp1 double positive cells in pancreatic tumors. We next sought to determine whether M402 affects cancer cells or stromal cells. In vitro experiments show that cell lines derived from our model respond to M402 by decreasing their invasive behavior in 3D culture and scratch assays. Moreover, we found that M402 augments the gemcitabine effect in vitro. These data suggest that M402 affects both EMT and gemcitabine response. Further identification by whole transcriptome microarray analysis of treated tumors hints towards a role of M402 in affecting multiple signaling pathways involved in the regulation of EMT. These data will undoubtedly lead to better insight into the mechanism of action of M402 and will increase our understanding of the pathways PDA cells use to evade the effects of chemotherapy. M402 is currently being investigated in a phase 1/2 M402 gemcitabine combination study to assess if these findings can be translated into a clinical benefit in pancreatic cancer patients. Conclusion: These data suggest that M402 reduces acquired chemotherapy resistance in a GEMM for PDA by decreasing EMT. Citation Format: Ilse Oosterom, Birgit C. Schultes, Chia Lin Chu, Zoya Galcheva-Gargova, Elma Kurtagic, He Zhou, Jay Duffner, Emile E. Voest, David A. Tuveson, Martijn Lolkema. Characterization of effects of M402 on EMT in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 302. doi:10.1158/1538-7445.AM2013-302
To characterize the effect of nipocalimab, a fully human, effectorless IgG1 anti-neonatal Fc receptor (FcRn) monoclonal antibody on immune function.
Background
Nipocalimab binds to FcRn with high affinity which prevents IgG recycling, leading to reduced serum levels of total IgGs, including pathogenic IgG autoantibodies. Rapid, sustained lowering of IgG was observed in the phase 2 VIVACITY study in generalized myasthenia gravis (gMG) and in phase 1 healthy volunteers. In gMG patients, nipocalimab induced rapid and sustained lowering of anti-AChR autoantibodies and MG-ADL scores, but no serious adverse events including clinically significant infections.
Design/Methods
Nipocalimab was evaluated extensively in vitro and in nonhuman primate-based chronic toxicology studies to evaluate selectivity, tolerability, safety and immunopharmacology. Safety, tolerability and immune-focused assessments in clinical phase 1 and Phase 2 MG studies were also completed (NCT02828046, NCT03772587).
Results
Nipocalimab binds specifically in vitro to FcRn without activation of effector function or inhibition of antigen presentation. In nonhuman primates administered up to 300 mg/kg nipocalimab QW for up to 6 months, sustained lowering of IgG was observed without adverse effects. Immunotoxicology identified no effect on immune cell phenotypes; CD8 T cell, NK or innate cell functions; T-dependent neoantigen IgM responses. Neoantigen IgG production was observed, but with lowered peak IgG titers consistent with the anticipated increase in IgG clearance with nipocalimab. In clinical studies, nipocalimab demonstrated a reproducible selective decrease in total serum IgG, including all subclasses of IgG, with no effect on IgM, IgA, IgE, CH50, C3, C4, inflammatory cytokines or acute phase proteins including, C-reactive protein (CRP).
Conclusions
These data suggest that nipocalimab can selectively lower IgG and IgG autoantibodies while preserving cellular immunity, complete IgM response and IgG production after neoantigen challenge. Overall, nipocalimab9s selective effect on IgG recycling provides a mechanistic rationale for potentially decreased infection risk despite substantial IgG lowering.
A series of size-defined low-molecular-weight heparins were generated by regioselective chemical modifications and profiled for their in vitro and in vivo activities. The compounds displayed reduced anti-coagulant activity, demonstrated varying affinities toward angiogenic growth factors (fibroblast growth factor-2, vascular endothelial growth factor and stromal cell-derived factor-1α), inhibited the P-selectin/P-selectin glycoprotein ligand-1 interaction and, notably, exhibited anti-tumor efficacy in a murine melanoma experimental metastasis model. Our results demonstrate that modulating specific sequences, especially the N-domains (-NS or -NH(2) or -NHCOCH(3)) in these polysaccharide sequences, has a major impact on the participation in a diverse range of biological activities. These results also suggest that the 6-O-sulfates, but not the 2-O-sulfates, critically affect the binding of a desulfated derivative to certain angiogenic proteins as well as its ability to inhibit P-selectin-mediated B16F10 melanoma metastases. Furthermore, N-desulfation followed by N-acetylation regenerates the affinity/inhibition properties to different extents in all the compounds tested in the in vitro assays. This systematic study lays a conceptual foundation for detailed structure function elucidation and will facilitate the rational design of targeted heparan sulfate proteoglycan-based anti-metastatic therapeutic candidates.
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