Fc Fragment Of IgG Research Articles

Adenoviral Delivery of Recombinant Soluble Human Tumor Necrosis Factor Receptor 1 Partially Normalized Mouse Model of Asthma

BackgroundTumor necrosis factor α (TNF-α) is a proinflammatory cytokine that has been implicated in the airway pathology of asthma and result in resistance to hormone therapy. Tumor necrosis factor α...

Post-translational modification of a chimeric EPO-Fc hormone is more important than its molecular size in defining its in vivo hematopoietic activity

BackgroundRecombinant erythropoietin (EPO) has been marketed as biopharmaceutical for anemia and chronic renal failure. Long-acting EPO variants that aimed at achieving less frequent dosing have been generated, either by the addition of glycosylation sites or increasing its molecular weight. MethodsThe hEPO cDNA linked to the human IgG Fc fragment was cloned as a single codifying gene on the pAdtrack-CMV vector, yielding the recombinant adenoviral genome. For in vitro and in vivo expression assays cervical cancer cell line (SiHa) and nulliparous goats were used, respectively. The hematopoietic activity of EPO-Fc, expressed as the differential increment of hematocrit was evaluated in B6D2F1 mice. NP-HPLC of the 2AB-labeled N-glycan was carried out to profile analysis. ResultsThe direct transduction of mammary secretory cells with adenoviral vector is a robust methodology to obtain high levels of EPO of up to 3.5mg/mL in goat's milk. SiHa-derived EPO-Fc showed significant improvement in hematopoietic activity compared to the commercial hEPO counterpart or with the homologous milk-derived EPO-Fc. The role of the molecular weight seemed to be important in enhancing the hematopoietic activity of SiHa-derived EPO-Fc. However, the lack of sialylated multi-antennary glycosylation profile in milk-derived EPO-Fc resulted in lower biological activity. ConclusionsThe low content of tri- or tetra-antennary sialylated N-glycans linked to the chimeric EPO-Fc hormone, expressed in the goat mammary gland epithelial cells, defined its in vivo hematopoietic activity. General significanceThe sialylated N-glycan content plays a more significant role in the in vivo biological activity of hEPO than its increased molecular weight.

Abstract 36: FCGR2A Promoter Methylation and Risks for IVIG Unresponsiveness in Kawasaki Disease

Kawasaki disease (KD) is characterized by pediatric systemic vasculitis of an unknown cause and the Fc Fragment of IgG, Low Affinity IIa, Receptor ( FCGR2A ) gene was reported to involve in increasing susceptibility of KD. Because DNA methylation is one of the epigenetic mechanisms that control gene expression, we hypothesized that methylation status of CpG islands in FCGR2A promoter predisposes an individual to Kawasaki disease. We recruited 36 KD patients and 24 healthy subjects with informed consents. And eleven potential methylation loci within the targeted promoter region (chr1:161474603-161475102) of Fc Fragment of IgG, Low Affinity IIa, Receptor were selected for investigation. Methylation at the CpG sites G, H and J displayed a strongly associations with KD, whereas CpG sites B,C,E,F,H,J and K were found to be correlated with non-responsive to IVIG treatment. In addition, CpG sites G, J and K were predicted as the significant transcription factor binding site for NF-kB, Myc-Max and SP2 respectively. Our study reports a significant association between the promoter methylation of FCGR2A , susceptibility of Kawasaki disease and therapeutic outcomes of IVIG treatment. The methylation levels of CpG sites of FCGR2A gene promoter may be an important marker for optimizing IVIG therapy.

A novel engineered VEGF blocker with an excellent pharmacokinetic profile and robust anti-tumor activity.

BackgroundRelatively poor penetration and retention in tumor tissue has been documented for large molecule drugs including therapeutic antibodies and recombinant immunoglobulin constant region (Fc)-fusion proteins due to their large size, positive charge, and strong target binding affinity. Therefore, when designing a large molecular drug candidate, smaller size, neutral charge, and optimal affinity should be considered.MethodsWe engineered a recombinant protein by molecular engineering the second domain of VEGFR1 and a few flanking residues fused with the Fc fragment of human IgG1, which we named HB-002.1. This recombinant protein was extensively characterized both in vitro and in vivo for its target-binding and target-blocking activities, pharmacokinetic profile, angiogenesis inhibition activity, and anti-tumor therapeutic efficacy.ResultsHB-002.1 has a molecular weight of ~80 kDa, isoelectric point of ~6.7, and an optimal target binding affinity of <1 nM. The pharmacokinetic profile was excellent with a half-life of 5 days, maximal concentration of 20.27 μg/ml, and area under the curve of 81.46 μg · days/ml. When tested in a transgenic zebrafish embryonic angiogenesis model, dramatic inhibition in angiogenesis was exhibited by a markedly reduced number of subintestinal vessels. When tested for anti-tumor efficacy, HB-002.1 was confirmed in two xenograft tumor models (A549 and Colo-205) to have a robust tumor killing activity, showing a percentage of inhibition over 90% at the dose of 20 mg/kg. Most promisingly, HB-002.1 showed a superior therapeutic efficacy compared to bevacizumab in the A549 xenograft model (tumor inhibition: 84.7% for HB-002.1 versus 67.6% for bevacizumab, P < 0.0001).ConclusionsHB-002.1 is a strong angiogenesis inhibitor that has the potential to be a novel promising drug for angiogenesis-related diseases such as tumor neoplasms and age-related macular degeneration.

IGF-IR determines the fates of BCR/ABL leukemia.

BackgroundThe tyrosine kinase receptor insulin-like growth factor 1 receptor (IGF-IR) contributes to the initiation and progression of many types of malignancies. We previously showed that IGF-2, which binds IGF-IR, is an extrinsic factor that supports the ex vivo expansion of hematopoietic stem cells (HSCs). We also demonstrated that IGF-IR is not required for HSC activity in vivo.Methods and resultsHere we investigated the role of IGF-IR in chronic myeloid leukemia (CML) using the retroviral BCR/ABL transplantation mouse model. Existing antibodies against IGF-IR are not suitable for flow cytometry; therefore, we generated a fusion of the human IgG Fc fragment with mutant IGF-2 that can bind to IGF-IR. We used this fusion protein to evaluate mouse primary hematopoietic populations. Through transplantation assays with IGF-IR+ and IGF-IR− cells, we demonstrated that IGF-IR is expressed on all mouse HSCs. The expression of IGF-IR is much higher on CML cells than on acute lymphoblastic leukemia (ALL) cells. The depletion of IGF-IR expression in BCR/ABL+ cells led to the development of ALL (mostly T cell ALL) but not CML. Lack of IGF-IR resulted in decreased self-renewal of the BCR/ABL+ CML cells in the serial replating assay.ConclusionIGF-IR regulates the cell fate determination of BCR/ABL+ leukemia cells and supports the self-renewal of CML cells.

Insulin receptor binding motif tagged with IgG4 Fc (Yiminsu) works as an insulin sensitizer to activate Akt signaling in hepatocytes.

Insulin resistance is a key feature of obesity and type 2 diabetes mellitus (T2DM). Interaction of insulin with the insulin receptor (IR) leads to both its auto-phosphorylation and phosphorylation of tyrosine residues on the IR substrate (IRS) proteins, initiating the activation of intracellular signaling cascades. The metabolic effects of IRS are known to be mediated through pathways involving phosphatidyl-inositol 3-kinase (PI-3K), which result in the activation of Akt signaling. The C-terminal region of the IR ectodomain is required to facilitate the conformational changes that are required for high-affinity binding to insulin. Furthermore, the CH2 and CH3 domains in the Fc fragments of immunoglobulins are responsible for their binding to the Fc receptor, which triggers transcytosis. In this study, we created a fusion peptide of the C-terminal end of the human IR ectodomain with the IgG4 Fc fragment, including an intervening polyG fragment to ensure enough space for insulin binding. We named this new peptide "Yiminsu", meaning an insulin sensitizer. The results of our analyses show that Yiminsu significantly facilitates insulin signaling via the activation of Akt in hepatocytes in a dose- and time-dependent manner. Further studies are required to determine whether Yiminsu can act as an insulin sensitizer.

Optimization of colorectal cancer vaccine candidate protein GA733‐Fc expression in a baculovirus–insect cell system

AbstractThe baculovirus–insect cell expression system has been used to produce functional recombinant proteins. The antigen GA733 is a cell‐surface glycoprotein highly expressed on most human colorectal carcinoma cells. Conditions for the expression of GA733 fused to the human immunoglobulin IgG Fc fragment (GA733‐Fc) were optimized in the baculovirus expression system. Several variable factors were adjusted to optimize expression, including the cell line (Sf9 and High Five), multiplicity of infection (MOI) value (0.05, 0.1, 0.5, 1 and 3), post‐infection time (48, 72 and 96 h) and harvested sample (cell culture media (CM) or cell lysate (CL)). In addition, two pFastBac Dual vectors carrying the GA733‐Fc gene were constructed to express GA733‐Fc with or without an endoplasmic reticulum (ER) retention sequence KDEL and used to generate recombinant baculoviruses. Western blot showed that expression depended on the conditions used to express the recombinant proteins. The protein production level and secretion capability differed in each cell line. In Sf9 cells, the highest expression in the CM and CL was obtained with GA733‐Fc at 96 h post‐infection at 0.1 MOI and with GA733‐FcK at 96 h post‐infection at 3 MOI, respectively. In High Five cells, the highest expression in the CM and CL was obtained with GA733‐Fc at 48 h post‐infection at 1 MOI and with GA733‐FcK at 48 h post‐infection at 3 MOI, respectively. These results suggest that the MOI value, post‐infection time and subcellular localization affect expression, and that these conditions can be modified to optimize protein expression in the baculovirus–insect cell system.

Assessment of the Influence of Inflammation and FCGR3A Genotype on Infliximab Pharmacokinetics and Time to Relapse in Patients with Crohn's Disease.

Infliximab is a monoclonal anti-tumor necrosis factor-α (anti-TNFα) antibody that profoundly modified the treatment of Crohn's disease (CD). The polymorphism of Fc fragment of IgG, low affinity IIIa, receptor (CD16a) [FCGR3A] influences the biological response to infliximab in patients with CD. Our aim was to study its influence on infliximab pharmacokinetics and risk of relapse after infliximab discontinuation. In 111 CD patients in remission, infliximab was discontinued and its concentrations were measured for 30months or until relapse. Infliximab pharmacokinetics were described using monocompartmental population modeling. The elimination rate of infliximab increased with C-reactive protein (CRP) [p=0.00018] and was 16% higher in FCGR3A-158V/V patients than in F carriers (p=0.0028). Risk of relapse was higher in patients with baseline CRP≥5mg/L than in those with a lower value (p=0.0000029). In addition, there was a first-order interaction between CRP and the FCGR3A genotype; in patients with high CRP, risk of relapse was higher for V/V patients than for F carriers (hazard ratio 4.80 and 2.84 for V/V and F carriers, respectively; p=0.013). Both increased inflammation and FCGR3A-158V/V genotype are associated with increased infliximab elimination and risk of relapse after infliximab discontinuation in patients with CD.

Role of sialylation in the anti-inflammatory activity of intravenous immunoglobulin - F(ab′)2versus Fc sialylation

Immunoglobulin (Ig)G antibodies play an important role in the defence against pathogenic microorganisms, but are also responsible for tissue destruction and inflammation during autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Paradoxically, pooled IgG preparations from thousands of donors [intravenous immunoglobulin (IVIg)] are also an efficient treatment to suppress several autoimmune diseases and chronic inflammation. Research has highlighted the role of the sugar domain attached to the IgG Fc-fragment as a molecular switch, which can enhance or block the pro- and anti-inflammatory effector functions of the antibody molecule. Indeed, deglycosylation of serum IgG in mice with active autoimmune diseases results in an amelioration of autoantibody-dependent inflammation 1. Of note, altered IgG glycosylation patterns, containing low levels of terminal galactose and sialic acid residues, and single nucleotide polymorphisms in glycosyltransferase genes have been associated with active autoimmune disease in RA, SLE and Crohn's disease, for example 2–5. Furthermore, IVIg infusion was shown to induce sialylated IgG glycovariants in patients with Kawasaki disease and correlated with a prolonged treatment response 6. Together with the results of several studies showing that sialylated IgG glycovariants are critical for the anti-inflammatory activity of IVIg in several in vivo model systems, this has led to proposing a model in which one possible mechanism of IVIg therapy may be to replenish these active anti-inflammatory and immunomodulatory IgG glycovariants, thereby re-establishing immune homeostasis. However, sialic acid-containing sugar moieties can attach to the IgG Fc- or F(ab′)2-fragment, and evidence showing the relevance of both these sialylated sugar domains for IVIg activity has been reported recently.

Abstract LB-238: Antitumor activity of mini-IGFBP-3, a protease-resistant derivative of human IGF binding protein-3

Abstract Insulin-like growth factors (IGF) are polypeptide hormones with potent anabolic and mitogenic properties that regulate cell growth and differentiation. Dysregulation of the IGF axis has been well documented in the development and progression of multiple types of cancer. Consequently, targeting the IGF axis has become an area of intense preclinical and clinical research through the development of 3 major classes of therapeutic compounds: antibodies that target IGF-IR, dual IGF-IR and IR tyrosine kinase inhibitors (TKI), and IGF ligands (IGF-I and IGF-II) neutralizing antibodies. Due to their high affinities for IGF-I and IGF-II, six IGFBPs regulate IGF bioavailability and IGFBP-3 is the main carrier of circulating IGFs in postnatal serum. In order to efficiently neutralize the excess of free IGF ligand maintaining tumor progression, we have produced, using the baculovirus insect cell system, a protease-resistant mutein of human IGFBP-3 with conserved affinities towards both IGF-I and IGF-II that was fused to the human IgG1 Fc fragment. In vitro, the resulting immunoadhesin (mIGFBP-3-Fc), evaluated in both regular and 3D culture, was found to block proliferation, arrest cell cycle and induce apoptosis in various types of cancer cell lines (Colon Adenocarcinoma, Ewing's Sarcoma, Adrenocortical Carcinoma and Multiple Myeloma (MM). mIGFBP-3-Fc antitumor activity was evaluated in vivo using RPMI-8226 luciferase-expressing MM cells xenografts. When MM cells were grafted subcutaneouly, daily mIGFBP-3-Fc injections drastically reduced tumor growth and induced tumor cell apoptosis. Similarly, after iv tumor cell injection, bone marrow localization of MM cells was substantially reduced in the mIGFBP-3-Fc-treated group. Furthermore, when used in association with the proteasome inhibitor Bortezomib, mIGFBP-3-Fc was shown to enhance the antitumor effect of Bortezomib used as single agent. Altogether, these data support the concept that mIGFBP-3-Fc could improve MM treatment either as a single agent or in association with proteasome inhibitors. Citation Format: François Godeau, Patricia Leneuve, Laetitia Da Costa, Yves Le Bouc, Jack-Michel Renoir. Antitumor activity of mini-IGFBP-3, a protease-resistant derivative of human IGF binding protein-3. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-238. doi:10.1158/1538-7445.AM2014-LB-238

Abstract 1907: Wnt pathway antagonist OMP-54F28 (FZD8-Fc) inhibits tumor growth and reduces tumor-initiating cell frequency in patient-derived hepatocellular carcinoma and ovarian cancer xenograft models

Abstract The Wnt/beta-catenin pathway, which signals through the Frizzled (FZD) receptor family and several co-receptors, has long been implicated in cancer. We have developed OMP-54F28, a recombinant fusion protein consisting of the ligand-binding domain of FZD8 and a human IgG1 Fc fragment. OMP-54F28 acts as a decoy receptor in sequestering Wnts and preventing them from binding to FZD receptors and thereby inhibiting Wnt signaling. The Wnt pathway is important for stem cell self renewal, differentiation, tumorigenicity, and epithelial-mesenchymal transition (EMT). Using minimally passaged human patient-derived xenograft tumors, we demonstrate that OMP-54F28 is efficacious as a single agent and in combination with standard of care in four hepatocellular carcinoma (HCC) and two ovarian cancer models. In the HCC models, OMP-54F28 shows tumor growth inhibition (TGI) as a single agent (average of 46%, p&amp;lt;0.05 vs. control) and displays additive TGI in combination with Sorafenib (average of 78%, p&amp;lt;0.05; 48% Sorafenib alone). Also, in the ovarian cancer models, treatment with OMP-54F28 results in TGI as a single agent (average of 32% vs. control) and shows additive TGI in combination with Paclitaxel (average of 78%, p&amp;lt;0.05; 48% with Paclitaxel alone). We also performed in vivo serial transplantation assays and found that OMP-54F28 as a single agent and in combination with standard of care reduces tumor-initiating cell frequency in both HCC and ovarian cancer xenografts. The anti-tumor effect was associated with a decrease in cell proliferation, induction of cell differentiation, and modulation of target Wnt pathway genes. Our data demonstrate the potential therapeutic benefit of targeting Wnt signaling in HCC and ovarian cancer. OMP-54F28 is being tested in Phase 1b clinical studies in these indications. Citation Format: Pete Yeung, Lucia Beviglia, Belinda Cancilla, Cristina Dee-Hoskins, James W. Evans, Marcus M. Fischer, Wan-Ching Yen, Austin Gurney, John Lewicki, Timothy Hoey, Ann M. Kapoun. Wnt pathway antagonist OMP-54F28 (FZD8-Fc) inhibits tumor growth and reduces tumor-initiating cell frequency in patient-derived hepatocellular carcinoma and ovarian cancer xenograft models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1907. doi:10.1158/1538-7445.AM2014-1907

Screening genes crucial for pediatric pilocytic astrocytoma using weighted gene coexpression network analysis combined with methylation data analysis.

To identify novel genes associated with pediatric pilocytic astrocytoma (PA) for better understanding the molecular mechanism underlying the pediatric PA pathogenesis. Gene expression profile data of GSE50161 and GSE44971 and the methylation data of GSE44684 were downloaded from Gene Expression Omnibus. The differentially expressed genes (DEGs) between PA and normal control samples were screened using the limma package in R, and then used to construct weighted gene coexpression network (WGCN) using the WGCN analysis (WGCNA) package in R. Significant modules of DEGs were selected using the clustering analysis. Function enrichment analysis of the DEGs in significant modules were performed using the WGCNA package and clusterprofiler package in R. Correlation between methylation sites of DEGs and PA was analyzed using the CpGassoc package in R. Totally, 3479 DEGs were screened in PA samples. Thereinto, 3424 DEGs were used to construct the WGCN. Several significant modules of DEGs were selected based on the WGCN, in which the turquoise module was positively related to PA, whereas blue module was negatively related to PA. DEGs (for example, DOCK2 (dedicator of cytokinesis 2), DOCK8 and FCGR2A (Fc fragment of IgG, low affinity IIa)) in blue module were mainly involved in Fc gamma R-mediated phagocytosis pathway and natural killer cell-mediated cytotoxicity pathway. Methylations of 14 DEGs among the top 30 genes in blue module were related to PA. Our data suggest that DOCK2, DOCK8 and FCGR2A may represent potential therapeutic targets in PA that merits further investigation.

Production and Characterization of Monoclonal Antibodies against Horse Immunoglobulins Useful for the Diagnosis of Equine Diseases

Monoclonal antibodies (MAbs) against horse IgG were produced by immunizing Balb/c mice with purified horse IgG and were characterized in indirect ELISA versus purified immunoglobulins from donkey, cow, buffalo, sheep, pig, and chicken. Three MAbs (1B10B6C9, 1B10B6C10, 1B10B6E9) reacted only with horse and donkey IgG and IgM and, in western blotting, were specific for the Fc fragment of equine IgG. MAb 1B10B6E9 was used in chemiluminescent immunoblotting assay for the diagnosis of dourine and in indirect immunofluorescence assay (IFA) for the diagnosis of African horse sickness and dourine.

P.1.g.031 The neuroprotective and anti-amnestic effects of carbamylated erythropoietin derivates in ischemic brain injury

Purpose: To compare the neuroprotection of carbamylated erythropoietin (CEPO) and EPO fusion protein containing TR domain from glycoprotein MUC1 (CEPO-TR), and EPO fusion protein with modified Fc fragments of IgG1 (CEPO-Fc) against ischemic brain injury, including behavioral disturbances in a bilateral focal ischemic infarction of the rat prefrontal cortex. Methods: The carbamylated erythropoietin and erythropoietin derivates were produced by treatment of purified proteins with potassium cyanate in borate buffer. The resulting carbamylated erythropoietin and derivates exhibit no erythropoietic activity in UT-7/EPOR cell viability assay. Bilateral focal ischemic infarction of the prefrontal cortex (areas Frl and Fr2) was induced by the method of photochemical thrombosis in rats. After passive avoidance training and testing rats were treated, respectively, with following regimens: saline, 50mg/kg CEPO, 50mg/kg CEPO-TR, 50mg/kg CEPO-Fc. The substance was injected intraperitoneally in 1 h after operation. Neurological deficit scores and infarct volume were assessed at 4 and 7 days after operation. Functional state of CNS was determined by conditioned passive avoidance response (PA), i.e. by the latency of transition from light compartment to dark compartment (in sec). Morphometric measurements of the areas and volumes of the ischemic focus on serial slices were carried out on animal brain fixed by plunging into formalinethanol-acetic acid mixture (2:7:1). The data were statistically processed using Statistica 6.0 software. Summary of results: Additional oligosaccharides, linked to the erythropoietin, prolong its half-life and increase bioactivity in vivo. For this purpose we, for the first time, used TR domain from glycoprotein MUC1, bearing 5 additional sites for O-glycosylation. Our result indicated that ligation of TR-domain to the coding sequence of EPO did not affect secretion of the chimeric protein into the medium, receptor binding affinity in vitro bioactivity, compared with EPO wild type. However, both the in vitro potency and half-life in circulation of EPO bearing TR or Fc fragments were significantly enhanced. After photothrombosis and injection of saline, the latency of entry into the dark compartment decreased from 300 s to 76 s. Treatment of rats with CEPO, CEPOTR, and CEPO-Fc after photothrombosis restored latency of PA to 243, 258, 227 s, respectively, on day 4. Only injection of CEPOTR retains passive avoidance latency 207 s on the day 7. Ischemic damage volume in animals treated with CEPO-TR on 7th day after operation was 15.8±5.4 against 24.22±4.7 with control injection of saline (P< 0.05). This phenomenon on 7th day was observed only with CEPO-TR. Protection efficiency coefficient, calculated from these experiments represented 34%. Another erythropoietin derivate did not give statistically significant results. Conclusion: Treatment of rats with CEPO and CEPO fusion proteins after photothrombosis of cortex resulted in restoration of passive avoidance response and diminishing of volume of ischemic damage. Those, our study indicates that CEPO-Fc and CEPO-TR display neuroprotectionand anti-amnestic activity, while CEPO-TR demonstrates prolongation ability.

Immunoglobulin G binding protein (IGBP) from Rhipicephalus haemaphysaloides: identification, expression, and binding specificity.

As the second most important human ectoparasite, ranked only after mosquitoes, the tick threatens the development of husbandry and even the health of humans worldwide. Immunoglobulin G binding proteins (IGBPs) are considered to be the major factors used by ticks to evade the host immune system and the damage caused by host antibodies. In this study, an IGBP-MB homologue was identified in the tick Rhipicephalus haemaphysaloides, which was predominantly detected in the salivary glands and hemolymph of male ticks. Recombinant IGBP (rIGBP/His) displayed significant binding activity to IgGs from rabbits and pigs, and bound to the F(ab)'2 but not the Fc fragment of rabbit IgG. Although the silencing of IGBP expression in ticks had no obvious effect on their blood-feeding and subsequent oviposition, antibodies raised to rIGBP/GST reduced the replete body weight (218.9 ± 20mg in the control group vs. 142.5 ± 43.3mg in the test group, P < 0.05 by Student's t test) and increased the mortality of the ticks. This study extends our understanding of the immunoevasive function of IGBPs and is a step towards the development of a vaccine against ticks.

CAPACITY OF GROUP A (TYPE M12) STREPTOCOCCI TO BIND IMMUNE COMPLEXES AND THEIR ROLE IN PATHOGENESIS OF POST-STREPTOCOCCAL GLOMERULONEPHRITIS

Abstract. In former studies, using an experimental rabbit model of acute post-streptococcal glomerulonephritis (GAS), we have proven a role of M-like Fc-binding streptococcal proteins (IgG FcBPs) for initiating destructive/degenerative lesions of renal glomeruli that are characteristic to membranous/proliferative and destructive glomerulonephritis. This activity was shown for the strains of types 1, 22 and 49. Their clinical isolates are able to bind Fc fragment of human and rabbit IgG, and are considered as an etiological agent of glomerulonephritis. It is well known that GAS strains of M12 serotype (commonly nephritogenic) are not able to interact with IgG monomers, and usually bind aggregated IgG, in spite of participation of IgG FcBPs in the both events. In present study, the GAS reference strain M12(1800) and twenty-one clinical strains of M12 type isolated from the patients with acute poststreptococcal glomerulonephritis (APSGN) were tested for binding with two artificial immune complexes (ICs): (i) peroxidase – antiperoxidase rabbit IgG (PAP) and (ii) tetanus toxoid – anti-tetanus human IgG (TAT). Streptococcal strain M12 (1800), as well as the majority of clinical isolates (19 strains) were strongly positive for the binding of both ICs tested. Using previously described model of experimental streptococcal glomerulonephritis rabbits were injected with heat-killed M12(1800) and each of two clinical isolates M12(257) and M12(305), positive and negative for the binding of ICs, respectively. Renal tissue material of rabbits injected with M12(1800) and M12(257), but not M12(305), showed strong inflammatory and degenerative changes compatible with pattern observed in APSGN. Streptococcal strains M12(1800) and M12(257), in contrast to strain M12(305), induced circulating anti-IgG, tissue deposition of IgG and C3 as well as secretion of IL-1β, Il-6 and TNF-α by the glomerular mesangial and endothelial cells. Our experimental data suggest that the IC-binding ability of type M12 streptococci should be of importance for the nephritogenic potential of these GAS serotype strains.

Characterization of asparagine 330 deamidation in an Fc-fragment of IgG1 using cation exchange chromatography and peptide mapping.

Deamidation is one of the most common degradation pathways for proteins and frequently occurs at "hot spots" with Asn-Gly, Asn-Ser or Asn-Thr sequences. Occasionally, deamidation may occur at other motifs if the local protein structure can participate or assist in the formation of the succinimide intermediate. Here we report the use of a chymotryptic peptide mapping method to identify and characterize a deamidated form of an IgG1 which was observed as an acidic peak in the cation exchange chromatography (CEX). The antibody was formulated in sodium acetate buffer, pH 5.3 and this deamidated form was observed mainly under thermal stress conditions. It was found that the IgG1 molecule with deamidation in the Fc region at asparagine residue 330 (in a Val-Ser-Asn-Lys motif) is the predominant form in this CEX peak, and was missed by tryptic mapping because the peptides are hydrophilic and elute near the void volume. In addition, a domain-based CEX method using papain digestion was developed to monitor the Asn 330 deamidation. These methods revealed that the Fc deamidation occurs mainly at Asn 330 in the VSNK motif at pH 5.3, whereas at pH 7.5, deamidation occurs predominantly at Asn 389 and Asn 394 in the NGQPENNYK motif.

FRI0345 Autoantibodies in Rheumatoid Arthritis Specifically Recognize Igg Heavy Chain Complexed with Hla-Dr, Which is Strongly Associated with Rheumatoid Arthritis Susceptibility

BackgroundParticular HLA class II alleles are strongly associated with susceptibility to rheumatoid arthritis; however, how HLA class II molecules regulate susceptibility to autoimmune diseases has remained unclear. Rheumatoid factor (RF)...

Expression of Aβ-Fc Fusion Protein in Transgenic Potato

Transgenic potato was generated to express recombinant 5 repeated β-amyloid (Aβ) peptides, potential antigens to be applied as a preventive accine for Alzheimer’s disease using Agrobacterium mediated transformation. The Aβ peptides were fused to the human IgG Fc fragment enhancing protein and KDEL, which is the endoplasmic reticulum (ER) retention signal (5Aβ-FcK). The 5Aβ-FcK, was expressed under the control of the duplicated 35S promoter. PCR analysis confirmed the presence of the transgene in several transgenic potato lines. Southern blot analysis showed only a single gene copy number in transgenic line 22, whereas multiple gene copy numbers were shown for transgenic lines 31 and 44. Northern blot analysis showed that line 22 had stronger mRNA levels when compared to lines 31 and 44. Immunoblot analysis confirmed that the 5Aβ-FcK protein was expressed in the transgenic potato plant. These results indicate that 5Aβ fused to Fc can be expressed in potato plants.

A first-in-human phase 1 study of anticancer stem cell agent OMP-54F28 (FZD8-Fc), decoy receptor for WNT ligands, in patients with advanced solid tumors.

2505 Background: The WNT/FZD signaling pathway is implicated in tumor cell de-differentiation and cancer stem cell (CSC) function in numerous cancer types. As a first-in-class recombinant fusion protein, OMP-54F28 binds WNT ligands and blocks WNT signaling through its domain of an extracellular part of human Frizzled 8 receptor (fused to a human IgG1 Fc fragment). In patient-derived xenograft models, OMP-54F28 inhibits growth and CSC frequency, promotes differentiation of tumor cells, and synergizes with chemotherapy in a broad spectrum of malignancies. Methods: A 3+3 design was used; OMP-54F28 was given intravenously every 3 weeks. Objectives were determination of maximum tolerated dose (MTD), safety, pharmacokinetics (PK), pharmacodynamics (PD), and efficacy. Results: 25 patients were treated in 7 dose-escalation cohorts (0.5, 1, 2.5, 5, 10, 15 and 20 mg/kg). No further dose escalation was pursued as animal data and PK modeling indicated 10 mg/kg as the target efficacious dose. Most common related Grade...