Browse
Submit Data
Databases
API
Help

Dataset Information

27 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

An engineered human IgG1 CH2 domain with decreased aggregation and nonspecific binding.

ABSTRACT: The immunoglobulin (Ig) CH2 domain is a promising scaffold for the development of candidate therapeutics. We have previously shown that the stability of isolated CH2 could be increased by the introduction of an additional disulfide bond and removal of seven N-terminal residues (m01s). However, both isolated CH2 and m01s aggregate, likely due to the existence of aggregation-prone regions (APRs) that we identified by using computational methods. This knowledge was used to generate a phage display library of mutants. The library was incubated at high temperature to remove aggregating CH2 domains, and then panned against a mouse anti-human CH2 monoclonal antibody targeting a conformational epitope to remove misfolded CH2s. After two rounds of panning, one clone, m01s5, with smaller APRs, was identified. After additional mutagenesis one clone, m01s5.4, which aggregated much less than m01s as measured by a turbidity assay and dynamic light scattering, was identified. m01s5.4 also exhibited much lower nonspecific binding than m01s. Engineering of a previously identified m01s-based tumor antigen-specific binder led to a dramatic reduction of its aggregation without affecting its binding. In summary, we describe a new approach for reducing aggregation based on a combination of computational and phage display methodologies, and show that aggregation of CH2-based scaffolds can be significantly reduced by the newly identified mutants, which can improve the developability of potential CH2-based therapeutics.

SUBMITTER: Cao G

PROVIDER: S-EPMC6927756 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

N-terminal truncation of an isolated human IgG1 CH2 domain significantly increases its stability and aggregation resistance.

Project description:Isolated human immunoglobulin G (IgG) CH2 domains are promising scaffolds for novel candidate therapeutics. Unlike other human IgG domains, CH2 is not involved in strong interchain interactions, and isolated CH2 is relatively stable. However, isolated single CH2 is prone to aggregation. In native IgG and Fc molecules, the N-terminal residues of CH2 from the two heavy chains interact with each other and form hinge regions. By contrast, the N-terminal residues are highly disordered in isolated CH2. We have hypothesized that the removal of the CH2 N-terminal residues may not only increase its stability but also its aggregation resistance. To test this hypothesis we constructed a shortened variant of IgG1 CH2 (CH2s) where the first seven residues of the N-terminus were deleted. We found that the thermal stability of CH2s was increased by 5 °C compared to CH2. Importantly, we demonstrated that CH2s is significantly less prone to aggregation than CH2 as measured by Thioflavin T (ThT) fluorescence, turbidity, and light scattering. We also found that the CH2s exhibited pH-dependent binding to a soluble single-chain human neonatal Fc receptor (shFcRn) which was significantly stronger than the very weak binding of CH2 to shFcRn as measured by flow cytometry. Computer modeling suggested a possible mode of CH2 aggregation involving its N-terminal residues. Therefore, deletion of the N-terminal residues could increase drugability of CH2-based therapeutic candidates. This strategy to increase stability and aggregation resistance could also be applicable to other Ig-related proteins.

| S-EPMC3795862 | biostudies-literature

Engineered antibody CH2 domains binding to nucleolin: Isolation, characterization and improvement of aggregation.

Project description:Smaller recombinant antibody fragments are now emerging as alternatives of conventional antibodies. Especially, immunoglobulin (Ig) constant CH2 domain and engineered CH2 with improved stability are promising as scaffolds for selection of specific binders to various antigens. We constructed a yeast display library based on an engineered human IgG1 CH2 scaffold with diversified loop regions. A group of CH2 binders were isolated from this yeast display library by panning against nucleolin, which is a tumor-associated antigen involved in cell proliferation, tumor cell growth and angiogenesis. Out of 20 mutants, we selected 3 clones exhibiting relatively high affinities to nucleolin on yeasts. However, recombinant CH2 mutants aggregated when they were expressed. To find the mechanism of the aggregation, we employed computational prediction approaches through structural homology models of CH2 binders. The analysis of potential aggregation prone regions (APRs) and solvent accessible surface areas (ASAs) indicated two hydrophobic residues, Val264 and Leu309, in the β-sheet, in which replacement of both charged residues led to significant decrease of the protein aggregation. The newly identified CH2 binders could be improved to use as candidate therapeutics or research reagents in the future.

| S-EPMC6957259 | biostudies-literature

Engineered Soluble Monomeric IgG1 Fc with Significantly Decreased Non-Specific Binding.

Project description:Due to the long serum half-life provided by the neonatal Fc receptor (FcRn) recycling, the IgG1 Fc has been pursued as the fusion partner to develop therapeutic Fc-fusion proteins, or as the antibody-derived scaffold that could be engineered with antigen-binding capabilities. In previous studies, we engineered the monomeric Fc by mutating critical residues located on the IgG1 Fc dimerization interface. Comparing with the wild-type dimeric Fc, monomeric Fc might possess substantial advantages conferred by its smaller size, but also suffers the disadvantage of non-specific binding to some unrelated antigens, raising considerable concerns over its potential clinical development. Here, we describe a phage display-based strategy to examine the effects of multiple mutations of IgG1 monomeric Fc and, simultaneously, to identify new Fc monomers with desired properties. Consequently, we identified a novel monomeric Fc that displayed significantly decreased non-specificity. In addition, it exhibited higher thermal stability and comparable pH-dependent FcRn binding to the previous reported monomeric Fc. These results provide baseline to understand the mechanism underlying the generation of soluble IgG1 Fc monomers and warrant the further clinical development of monomeric Fc-based fusion proteins as well as antigen binders.

| S-EPMC5693891 | biostudies-literature

Shortened engineered human antibody CH2 domains: increased stability and binding to the human neonatal Fc receptor.

Project description:The immunoglobulin (Ig) constant CH2 domain is critical for antibody effector functions. Isolated CH2 domains are promising scaffolds for construction of libraries containing diverse binders that could also confer some effector functions. We have shown previously that an isolated human CH2 domain is relatively unstable to thermally induced unfolding, but its stability can be improved by engineering an additional disulfide bond (Gong, R., Vu, B. K., Feng, Y., Prieto, D. A., Dyba, M. A., Walsh, J. D., Prabakaran, P., Veenstra, T. D., Tarasov, S. G., Ishima, R., and Dimitrov, D. S. (2009) J. Biol. Chem. 284, 14203-14210). We have hypothesized that the stability of this engineered antibody domain could be further increased by removing unstructured residues. To test our hypothesis, we removed the seven N-terminal residues that are in a random coil as suggested by our analysis of the isolated CH2 crystal structure and NMR data. The resulting shortened engineered CH2 (m01s) was highly soluble, monomeric, and remarkably stable, with a melting temperature (T(m)) of 82.6 °C, which is about 10 and 30 °C higher than those of the original stabilized CH2 (m01) and CH2, respectively. m01s and m01 were more resistant to protease digestion than CH2. A newly identified anti-CH2 antibody that recognizes a conformational epitope bound to m01s significantly better (>10-fold higher affinity) than to CH2 and slightly better than to m01. m01s bound to a recombinant soluble human neonatal Fc receptor at pH 6.0 more strongly than CH2. These data suggest that shortening the m01 N terminus significantly increases stability without disrupting its conformation and that our approach for increasing stability and decreasing size by removing unstructured regions may also apply to other proteins.

| S-EPMC3149322 | biostudies-literature

Engineered CH2 domains (nanoantibodies).

Project description:Currently, almost all FDA approved therapeutic antibodies (except ReoPro, Lucentis and Cimzia which are Fabs), and the vast majority of those in clinical trials are full-size antibodies mostly in IgG1 format of about 150 kDa size. A fundamental problem for such large molecules is their poor penetration into tissues (e.g., solid tumors) and poor or absent binding to regions on the surface of some molecules (e.g., on the HIV envelope glycoprotein) which are fully accessible only by molecules of smaller size. Therefore, much work especially during the last decade has been aimed at developing novel scaffolds of much smaller size and high stability. Here I briefly describe a proposition to use the immunoglobulin (Ig) constant CH2 domain (CH3 for IgE and IgM) as a scaffold. CH2 is critical for the Ig effector functions. Isolated CH2 is stable monomer in contrast to all other constant domains and most of the variable domains. CH2 and engineered CH2 domains with improved stability can be used as scaffolds for construction of libraries containing diverse binders to various antigens. Such binders based on a CH2 scaffold could also confer some effector functions. Because the CH2 domains are the smallest independently folded antibody domains that can be engineered to contain simultaneously antigen-binding sites and binding sites mediating effector and stability functions, and to distinguish them from domain antibodies which are used to denote engineered VH or VL domains or nanobodies which are used to denote camelid VHH, I termed them nanoantibodies (nAbs).

| S-EPMC2715188 | biostudies-literature

Pharmacokinetics of engineered human monomeric and dimeric CH2 domains.

Project description:Therapeutic monoclonal antibodies have several advantages over small molecule drugs and small proteins and peptides, including a long serum half-life. The long serum half-life of IgG is due, in part, to its molecular weight (150kDa) and its ability to bind FcRn. Both the CH2 and CH3 domains of Fc are involved in FcRn binding. Antibody fragments and antibody-like scaffolds have improved penetration into tissues due to their small size, yet suffer from a short serum half-life of less than one hour. The human CH2 domain (CH2D) of IgG1 retains a portion of the FcRn binding site, is amenable to modification for target binding, and may represent the smallest antibody-like scaffold retaining a relatively long serum half-life. Here we describe the generation of a dimeric CH2D (dCH2D) and determination of its pharmacokinetics (PK), as well as the PK of wild-type monomeric CH2D (mCH2D) and a short stabilized CH2D variant (ssCH2D) in normal B6 mice, human FcRn transgenic mice and cynomolgus macaques. The elimination half-life of dCH2D was 9.9, 10.4 and 11.2 hours, and that of ssCH2D was 13.1, 9.9 and 11.4 hours, in B6 mice, hFcRn mice and cynomolgus macaques, respectively. These half-lives were slightly longer than that of mCH2D (6.9 and 8.8 hours) in B6 and hFcRn mice, respectively. These data demonstrate that engineered CH2D-based variants have relatively long serum half-lives, making them a unique scaffold suitable for development of targeted therapeutics.

| S-EPMC3499341 | biostudies-literature

Interactions of IgG1 CH2 and CH3 Domains with FcRn.

Project description:Antibody fragments are emerging as promising biopharmaceuticals because of their relatively small-size and other unique properties. However, when compared to full-size antibodies, most of the current antibody fragments of VH or VL display greatly reduced half-lives. A promising approach to overcome this problem is through the development of novel antibody fragments based on IgG Fc region, which contributes to the long half-life of IgG through its unique pH-dependent association with the neonatal Fc receptor (FcRn). The IgG Fc region comprises two CH2 and two CH3 domains. In this report, we present a comparative study of the FcRn binding capability of the CH2 and CH3 domains. The stability and aggregation resistance of these domains were also investigated and compared. We found that monomeric CH2 and CH3 domains exhibited the pH-dependent FcRn binding while the dimeric forms of CH2 and CH3 domains did not. Although all of these domains had high serum stability, they had aggregation tendencies as measured by dynamic light scattering. By providing a better understanding of the structure-activity relationship of the Fc fragment, these results guide further approaches to generate novel Fc-based small-size antibody fragments that possess pH-dependent FcRn binding capability, desired in vivo half-lives, and other favorable biophysical properties for their druggability.

| S-EPMC3980117 | biostudies-literature

A stable engineered human IgG3 antibody with decreased aggregation during antibody expression and low pH stress.

Project description:Human IgG comprises four subclasses with different biological functions. The IgG3 subclass has a unique character, exhibiting high effector function and Fab arm flexibility. However, it is not used as a therapeutic drug owing to an enhanced susceptibility to proteolysis. Antibody aggregation control is also important for therapeutic antibody development. To date, there have been few reports of IgG3 aggregation during protein expression and the low pH conditions needed for purification and virus inactivation. This study explored the potential of IgG3 antibody for therapeutics using anti-CD20 IgG3 as a model to investigate aggregate formation. Initially, anti-CD20 IgG3 antibody showed substantial aggregate formation during expression and low pH treatment. To circumvent this phenomenon, we systematically exchanged IgG3 constant domains with those of IgG1, a stable IgG. IgG3 antibody with the IgG1 CH3 domain exhibited reduced aggregate formation during expression. Differential scanning calorimetric analysis of individual amino acid substitutions revealed that two amino acid mutations in the CH3 domain, N392K and M397V, reduced aggregation and increased CH3 transition temperature. The engineered human IgG3 antibody was further improved by additional mutations of R435H to obtain IgG3KVH to achieve protein A binding and showed similar antigen binding as wild-type IgG3. IgG3KVH also exhibited high binding activity for FcγRIIIa and C1q. In summary, we have successfully established an engineered human IgG3 antibody with reduced aggregation during bioprocessing, which will contribute to the better design of therapeutic antibodies with high effector function and Fab arm flexibility.

| S-EPMC6459999 | biostudies-literature

Engineered Human Monoclonal scFv to Receptor Binding Domain of Ebolavirus.

Project description:(1) Background: Ebolavirus (EBOV) poses as a significant threat for human health by frequently causing epidemics of the highly contagious Ebola virus disease (EVD). EBOV glycoprotein (GP), as a sole surface glycoprotein, needs to be cleaved in endosomes to fully expose a receptor-binding domain (RBD) containing a receptor-binding site (RBS) for receptor binding and genome entry into cytoplasm for replication. RBDs are highly conserved among EBOV species, so they are an attractive target for broadly effective anti-EBOV drug development. (2) Methods: Phage display technology was used as a tool to isolate human single-chain antibodies (HuscFv) that bind to recombinant RBDs from a human scFv (HuscFv) phage display library. The RBD-bound HuscFvs were fused with cell-penetrating peptide (CPP), and cell-penetrating antibodies (transbodies) were made, produced from the phage-infected E. coli clones and characterized. (3) Results: Among the HuscFvs obtained from phage-infected E. coli clones, HuscFvs of three clones, HuscFv4, HuscFv11, and HuscFv14, the non-cell-penetrable or cell-penetrable HuscFv4 effectively neutralized cellular entry of EBOV-like particles (VLPs). While all HuscFvs were found to bind cleaved GP (GPcl), their presumptive binding sites were markedly different, as determined by molecular docking. (4) Conclusions: The HuscFv4 could be a promising therapeutic agent against EBOV infection.

| S-EPMC8147973 | biostudies-literature

Expression, glycoform characterization, and antibody-binding of HIV-1 V3 glycopeptide domain fused with human IgG1-Fc.

Project description:The third variable (V3) domain of HIV-1 gp120 envelope glycoprotein is critical for HIV-1 entry and represents an attractive target for vaccine design. There are three conserved N-glycans within or around the V3 loop. The N295 and N332 glycans at the base of V3 are usually characterized as high-mannose type in gp120, and the N301 glycan is a complex type. We report in this paper the expression and characterization of glycosylated, full-size V3 domain derived from HIV-1(Bal) strain as an IgG1-Fc fusion protein, including its binding to two broadly HIV-neutralizing antibodies 2G12 and 447-52D. It was found that expressing the V3-Fc fusion protein in the HEK293T cells resulted in the production of a glycoform in which all the N-glycans were complex type, in contrast to the glycosylation pattern of V3 in the context of gp120, where the N295 and N332 glycans are high-mannose type. Controlling the glycosylation to restore an epitope of antibody 2G12 was achieved by using an inhibitor of glycan processing enzymes. Mutational studies indicate that the glycan at N301 slightly decreases the binding of V3-Fc to antibody 447-52D, but it can significantly enhance the binding of the V3-Fc to antibody 2G12 when it is changed to a high-mannose type N-glycan. The high-mannose type V3-Fc fusion protein that includes both the 2G12 and 447-52D epitopes represents an interesting immunogen that may be able to raise anti-HIV neutralizing antibodies.

| S-EPMC2901909 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data