Structure-based Stability Engineering of the Mouse IgG1 Fab Fragment by Modifying Constant Domains

Teerinen, Tuija; Valjakka, Jarkko; Rouvinen, Juha; Takkinen, Kristiina

doi:10.1016/j.jmb.2006.06.073

Cited by 24 publications

(23 citation statements)

References 38 publications

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“…4). Peak 1 was redox-enriched without using GuHCl, and peak 3 was enriched using ϳ1 M GuHCl, a concentration well below that known to affect overall secondary or tertiary structure of the antibody (11,22). The major RP-HPLC peaks in the redox-treated materials eluted at approximately the same retention times as peaks in the untreated (no redox) IgG2 control material.…”

Section: Structural Heterogeneity Of Human Igg2 and Homogeneity Of Igmentioning

confidence: 94%

Structural and Functional Characterization of Disulfide Isoforms of the Human IgG2 Subclass

Dillon

Ricci

Vezina

et al. 2008

Journal of Biological Chemistry

258

327

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we have identified that the human IgG2 subclass exists as an ensemble of distinct isoforms, designated IgG2-A, -B, and -A/B, which differ by the disulfide connectivity at the hinge region. In this report, we studied the structural and functional properties of the IgG2 disulfide isoforms and compared them to IgG1. Human monoclonal IgG1 and IgG2 antibodies were designed with identical antigen binding regions, specific to interleukin-1 cell surface receptor type 1. In vitro biological activity measurements showed an increased activity of the IgG1 relative to the IgG2 in blocking interleukin-1␤ ligand from binding to the receptor, suggesting that some of the IgG2 isoforms had lower activity. Under reduction-oxidation conditions, the IgG2 disulfide isoforms converted to IgG2-A when 1 M guanidine was used, whereas IgG2-B was enriched in the absence of guanidine. The relative potency of the antibodies in cell-based assays was: IgG1 > IgG2-A > IgG2 Ͼ Ͼ IgG2-B. This difference correlated with an increased hydrodynamic radius of IgG2-A relative to IgG2-B, as shown by biophysical characterization. The enrichment of disulfide isoforms and activity studies were extended to additional IgG2 monoclonal antibodies with various antigen targets. All IgG2 antibodies displayed the same disulfide conversion, but only a subset showed activity differences between IgG2-A and IgG2-B. Additionally, the distribution of isoforms was influenced by the light chain type, with IgG2 composed mostly of IgG2-A. Based on crystal structure analysis, we propose that IgG2 disulfide exchange is caused by the close proximity of several cysteine residues at the hinge and the reactivity of tandem cysteines within the hinge. Furthermore, the IgG2 isoforms were shown to interconvert in whole blood or a "bloodlike" environment, thereby suggesting that the in vivo activity of human IgG2 may be dependent on the distribution of isoforms.

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Section: Structural Heterogeneity Of Human Igg2 and Homogeneity Of Igmentioning

confidence: 94%

Structural and Functional Characterization of Disulfide Isoforms of the Human IgG2 Subclass

Dillon

Ricci

Vezina

et al. 2008

Journal of Biological Chemistry

258

327

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“…This conforms to an earlier finding that cavities are crucial for the conformational flexibility of photosynthetic proteins (9). Knowing the position of cavities allows to predict the effects of mutations (10), and thus to engineer proteins with increased stability (11). In neurotransmitter receptors, interior cavities are believed to form the site of action for some anesthetics (12).…”

Section: Introductionmentioning

confidence: 99%

Voronoia: analyzing packing in protein structures

Rother

Hildebrand

Goede

et al. 2009

Nucleic Acids Research

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The packing of protein atoms is an indicator for their stability and functionality, and applied in determining thermostability, in protein design, ligand binding and to identify flexible regions in proteins. Here, we present Voronoia, a database of atomic-scale packing data for protein 3D structures. It is based on an improved Voronoi Cell algorithm using hyperboloid interfaces to construct atomic volumes, and to resolve solvent-accessible and -inaccessible regions of atoms. The database contains atomic volumes, local packing densities and interior cavities calculated for 61 318 biological units from the PDB. A report for each structure summarizes the packing by residue and atom types, and lists the environment of interior cavities. The packing data are compared to a nonredundant set of structures from SCOP superfamilies. Both packing densities and cavities can be visualized in the 3D structures by the Jmol plugin. Additionally, PDB files can be submitted to the Voronoia server for calculation. This service performs calculations for most full-atomic protein structures within a few minutes. For batch jobs, a standalone version of the program with an optional PyMOL plugin is available for download. The database can be freely accessed at: http://bioinformatics.charite.de/voronoia.

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“…Previous studies in which hydrophobic cores were altered also did not obtain enhanced variants (20–22), and introducing mutations at locations other than hydrophobic cores may be more effective. For example, there has been one report in which mouse IgG1 Fab fragments with increased stability were obtained when a hydrophilic residue in the cavity of the constant region was replaced with a hydrophobic residue (9). A random mutagenesis strategy throughout the whole constant region would be compatible with in vitro selection of stable variants.…”

Section: Resultsmentioning

confidence: 99%

“…Compared to the commonly used scFv, Fab fragments have no artificial sequence and contain the constant region, being more antibody-like. Also, because the constant region stabilizes the whole Fab fragment (9,10), the Fab fragment is more stable than the scFv in most cases (10,11). Therefore, a novel display method for totally in vitro selection of Fab fragments would be very useful.…”

Section: Introductionmentioning

confidence: 99%

Bicistronic DNA display for in vitro selection of Fab fragments

Sumida

Doi

Yanagawa

2009

Nucleic Acids Research

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In vitro display methods are superior tools for obtaining monoclonal antibodies. Although totally in vitro display methods, such as ribosome display and mRNA display, have the advantages of larger library sizes and quicker selection procedures compared with phage display, their applications have been limited to single-chain Fvs due to the requirement for linking of the mRNA and the nascent protein on the ribosome. Here we describe a different type of totally in vitro method, DNA display, that is applicable to heterodimeric Fab fragments: in vitro compartmentalization in water-in-oil emulsions allows the linking of an oligomeric protein and its encoding DNA with multiple ORFs. Since previously used emulsions impaired the synthesis of functional Fab fragments, we modified conditions for preparing emulsions, and identified conditions under which it was possible to enrich Fab fragments 106-fold per three rounds of affinity selection. Furthermore, we confirmed that genes encoding stable Fab fragments could be selected from a Fab fragment library with a randomized hydrophobic core in the constant region by applying heat treatment as a selection pressure. Since this method has all advantages of both phage display and totally in vitro display, it represents a new option for many applications using display methods.

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Structure-based Stability Engineering of the Mouse IgG1 Fab Fragment by Modifying Constant Domains

Cited by 24 publications

References 38 publications

Structural and Functional Characterization of Disulfide Isoforms of the Human IgG2 Subclass

Structural and Functional Characterization of Disulfide Isoforms of the Human IgG2 Subclass

Voronoia: analyzing packing in protein structures

Bicistronic DNA display for in vitro selection of Fab fragments

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