Influence of N-glycosylation on effector functions and thermal stability of glycoengineered IgG1 monoclonal antibody with homogeneous glycoforms

Glycosylation of the conserved asparagine residue in each heavy chain of IgG in the CH2 domain is known as N-glycosylation. It is one of the most common post-translational modifications and important critical quality attributes of monoclonal antibody (mAb) therapeutics. Various studies have demonstrated the effects of the Fc N-glycosylation on safety, Fc effector functions, and pharmacokinetics, both dependent and independent of neonatal Fc receptor (FcRn) pathway. However, separation of various glycoforms to investigate the biological and functional relevance of glycosylation is a major challenge, and existing studies often discuss the overall impact of N-glycans, without considering the individual contributions of each glycoform when evaluating mAbs with highly heterogeneous distributions. In this study, chemoenzymatic glycoengineering incorporating an endo-β-N-acetylglucosaminidase (ENGase) EndoS2 and its mutant with transglycosylation activity was used to generate mAb glycoforms with highly homogeneous and well-defined N-glycans to better understand and precisely evaluate the effect of each N-glycan structure on Fc effector functions and protein stability. We demonstrated that the core fucosylation, non-reducing terminal galactosylation, sialylation, and mannosylation of IgG1 mAb N-glycans impact not only on FcγRIIIa binding, antibody-dependent cell-mediated cytotoxicity, and C1q binding, but also FcRn binding, thermal stability and propensity for protein aggregation.     

Leishmania infantum: mixed T-helper-1/T-helper-2 immune response in experimentally infected BALB/c mice

The main goal of the present study was to characterise the course of infection and immunological responses developed by Leishmania infantum infected BALB/c mice. Parasite load was determined by Real-time TaqMan PCR while cytokine and Immunoglobulin G (IgG) production were assessed by ELISA. Leishmania DNA was detected in spleen and liver as soon as day 1 post-inoculation (pi) and the parasitism was sustained until the end of the experiment. The cytokine kinetics in spleen and liver was generally associated with the oscillations of parasite load. Overall, it was not observed a distinct Th1 or Th2 pattern of cytokine production during the time of experiment. The infected mice developed a mixed immune response, with concomitant production of IFN-gamma, IL-4 and IL-10, both in spleen and liver, and both IgG isotypes. However, our results suggest that, compared to liver, the spleen is more susceptible to L. infantum infection.     

Plasmodium yoelii: the effect of second blood meal and anti-sporozoite antibodies on development and gene expression in the mosquito vector, Anopheles stephensi

The sporogonic development of the malaria parasite takes place in the mosquito and a wide range of factors modulates it. Among those, the contents of the blood meal can influence the parasite development directly or indirectly through the mosquito response to the infection. We have studied the effect of a second blood meal in previously infected mosquitoes and the effect of anti-sporozoite immune serum on parasite development and mosquito response to the infection. The prevalence and intensity of infection and gene expression of both Plasmodium yoelii and Anopheles stephensi was analyzed. We verified that a second blood meal and its immune status interfere with parasite development and with Plasmodium and mosquito gene expression.     

Floating culture promotes the maintenance of hematopoietic stem cells

In this study we examined the effect of the specific gravity of culture medium on the frequency of hematopoietic stem cell (HSC) maintenance. We used a newly developed high-specific-gravity media. Bone marrow cells were isolated and cultured, and HSC activity was evaluated. The number of hematopoietic progenitor/stem cells was markedly higher in the medium with high specific gravity. In high-specific-gravity media, cells did not precipitate, maintenance of HSCs was increased, and there was a concomitant accumulation of beta-catenin. This novel technique for maintaining HSC populations provides an important new tool for studies in regenerative medicine.     

Assignment of 1H, 13C and 15N resonances of the reduced human IgG1 CH3 domain

Here we report the NMR resonance assignments for the reduced form of human IgG1 C_H3 domain, a 26 kDa dimer in solution (residues 341–447). The assignments have been deposited in the BioMagResBank with a BMRB accession number of 15204.     

PLA2R-IgG间接荧光定量免疫层析分析的构建与应用

目的 血清抗磷脂酶A2受体抗体IgG（PLA2R-IgG）水平是诊断和治疗特发性膜性肾病（IMN）的重要依据,而目前国内外常规检测手段主要是酶免法。为提升检测的便捷性,同时满足灵敏、宽量程分析需求,本研究构建了一种新的PLA2R-IgG检测技术。方法 采用包裹铕元素的微球示踪,对反应步骤进行选择,对微球制备液的pH、微球-抗体反应比例和反应时间优化,本文基于间接法构建了PLA2R-IgG的荧光定量免疫层析检测方法,并进行了初步临床评价。结果 本方法的灵敏度达0.7 RU/ml,标准曲线方程为y=0.771x-1.437,相关系数0.995,线性测量范围为0.7~1 500 RU/ml,回收率为86.27%~98.98%,平均批内变异系数为8.13%,交叉反应率均小于0.1%,试剂37℃储存10 d稳定。本方法与市售酶免试剂盒相关性为0.953,阴阳性判断一致,对IMN的检出率为76.9%。结论 采用两步法反应的PLA2R-IgG间接荧光定量免疫层析分析,快速、灵敏、准确,具有临床实用性。     

Usefulness of IgG4 subclass antibodies for diagnosis of human clonorchiasis

The present study analyzed serum IgG subclass antibody reaction to major antigenic bands of Clonorchis sinensis to investigate improvement of its serodiagnosis. Of the four subclass antibodies, IgG1 and IgG2 antibodies were produced but not specific, IgG3 antibody was least produced, and IgG4 antibody was prominent and specific. The serum IgG antibody reaction to any of 43-50, 34-37, 26-28, and 8 kDa bands was found in 65.5% of 168 egg positive cases while IgG4 antibody reaction was found in 22.0% of them. The positive rates of IgG and IgG4 antibodies were directly correlated with the intensity of infection. All of the sera from heavily infected cases over EPG 5,000 showed positive reaction for specific IgG and IgG4 antibodies. The specific serum IgG4 antibody disappeared within 6 months after treatment. The bands of 35 kDa and 67 kDa cross-reacted with IgG antibodies but not with IgG4 antibodies in sera of other trematode infections. The present findings suggest that serum IgG4 antibody reaction to 8 kDa band is specific but not sensitive. Any method to increase its sensitivity is required for improved serodiagnosis.     

Calculation of NMR relaxation, covolume, and scattering-related properties of bead models using the SOLPRO computer program

The hydrodynamic properties of macromolecules and bioparticles, represented by bead models, can be calculated using methods implemented in the computer routine HYDRO. Recently, a new computer routine, SOLPRO, has been presented for the calculation of various SOLution PROperties. These include (1) time-dependent electro-optic and spectroscopic properties related to rotational diffusion, (2) non-dynamic properties like scattering curves, and (3) dimensionless quantities that combine two or more solution properties in a form which depends on the shape of the macromolecule but not on its size. In the present work we describe the inclusion of more of those types of properties in a new version of SOLPRO. Particularly, we describe the calculation of relaxation rates in nuclear magnetic resonance (NMR). For dipolar coupling, given the direction of the dipole the program calculates values of the spectral density, from which the NMR relaxation times can be obtained. We also consider scattering-related properties, namely the distribution of distances for the bead model, which is directly related to the angular dependence of scattered intensity, and the particle's longest distance. We have devised and programmed a procedure to calculate the covolume of the bead model, related to the second virial coefficient and, in general, to the concentration dependence of solution properties. Various shape-dependent dimensionless quantities involving the covolume are calculated. In this paper we also discuss some aspects, namely bead overlapping and hydration, that are not explicitely included in SOLPRO, but should be considered by the user. Received: 25 May 1998 / Revised version: 30 July 1998 / Accepted: 30 July 1998     

Screening Neutral and Acidic IgG N-Glycans by High Density Electrophoresis

IgG carries bi-antennary N-linked glycans which differ in degrees of galactosylation, core fucosylation and bisecting N-acetyl glucosamine. The majority of these are non-sialyated closely related neutral structures which can be resolved by HPLC analysis, but which are difficult to separate in techniques such as fluorophore-coupled carbohydrate electrophoresis. Derivatisation with the singly charged fluorophore, 2-amino benzoic acid and separation in gels with a 30% monomer content in tris/glycine buffer enabled separation of neutral glycans. In particular, agalactosyl glycans with either a core fucose substitution or bisecting N-acetyl galactosamine could be resolved. Good separation of mono- and di-galactosylated glycans was also achieved with this system. It was shown that IgG can be separated from serum by size-exclusion and anion exchange chromatography with minimal contamination, with complete glycan release accomplished by the enzyme peptide-N-glycosidase F (F. meningosepticum). This method of resolving IgG glycans could be used to monitor patients in which glycosylation changes may have a diagnostic value, as in rheumatoid arthritis. It could also be used to monitor recombinant IgG glycosylation where routine screening is required in the biotechnology industry.