The in vitro resistance of IgG2 to proteolytic attack concurs with a comparative paucity of autoantibodies against peptide analogs of the IgG2 hinge

Brezski, Randall J.; Oberholtzer, Allison; Strake, Brandy; Jordan, Robert E.

doi:10.4161/mabs.3.6.18119

Cited by 36 publications

(36 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To assess protease susceptibility of the newly generated variants, mAbs were incubated with proteases capable of cleaving human IgG1 (29). Similar to previous studies, IgG1 was cleaved by MMP-3, MMP-12, GluV8, and IdeS to varying degrees after a 24-h incubation (Fig.…”

Section: Detection Of Cleaved Iggs In Human Squamous Cell Carcinoma-mentioning

confidence: 98%

“…Kinetic digests were performed with a 10% molar ratio for MMP-3 and 0.1% (w/w) IdeS and were quenched at the indicated time points using a final concentration of 10 mM EDTA for the MMP-3 digest and 10 mM iodoacetamide for the IdeS digest. The percentage of intact IgG remaining was calculated as done previously (29).…”

Section: Methodsmentioning

confidence: 99%

“…This is typically accomplished by co-incubating macrophages with mAb-opsonized tumor cells and assessing internalization of the tumor cells within macrophages over a time frame typically no longer than 4 h (40, 46 -48). Our group previously developed a flow cytometry and microscopy-based ADCP assay where the target tumor cells (MDA-MB-231) expressed GFP to obviate the need to label the tumor cells with a dye (29,46). Macrophage internalization of MDA-MB-231 cells opsonized with a tumor targeting anti-CD142 mAb was assessed by either flow cytometry or fluorescence microscopy (29,40).…”

Section: Antibody-dependent Macrophage Killing Of Human Tumor Cell LImentioning

confidence: 99%

“…Sequence alignments comparing the lower hinge of IgG1 and IgG2 suggested that the resistance to cleavage may be due to amino acid differences between IgG1 and IgG2 (29). However, the IgG2 subclass has very weak binding to Fc␥RIIIa (30) and thus low to undetectable ADCC capacity.…”

mentioning

confidence: 99%

“…Previously, we have shown that the human IgG2 subclass is resistant to cleavage by a number of physiologically relevant proteases, including MMP-3, MMP-7, MMP-12, and MMP-13 as well as GluV8 (29). Sequence alignments comparing the lower hinge of IgG1 and IgG2 suggested that the resistance to cleavage may be due to amino acid differences between IgG1 and IgG2 (29).…”

mentioning

confidence: 99%

See 4 more Smart Citations

Engineered Protease-resistant Antibodies with Selectable Cell-killing Functions

Kinder

Greenplate

Grugan

et al. 2013

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Background: Proteases can cleave human IgG1 antibodies, resulting in loss of cell-killing functions. Results: Mutation of the lower hinge of IgG1 confers protease resistance but disrupts Fc effector functions. Conclusion: Compensating mutations in the C H 2 domain can selectively restore Fc effector functions on a protease-resistant backbone. Significance: Protease-resistant antibodies may be desirable for microenvironments with high protease content and/or when selected cell-killing functions are needed.

show abstract

Section: Detection Of Cleaved Iggs In Human Squamous Cell Carcinoma-mentioning

confidence: 98%

Section: Methodsmentioning

confidence: 99%

Section: Antibody-dependent Macrophage Killing Of Human Tumor Cell LImentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Engineered Protease-resistant Antibodies with Selectable Cell-killing Functions

Kinder

Greenplate

Grugan

et al. 2013

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

Antibodies to IgG4 Hinge Can Be Found in Rheumatoid Arthritis Patients During All Stages of Disease and May Exacerbate Chronic Antibody‐Mediated Inflammation

Stadt

Vrieze

Derksen

et al. 2014

Arthritis & Rheumatology

View full text Add to dashboard Cite

Objective. In rheumatoid arthritis (RA), autoantibodies such as anti-citrullinated protein antibodies (ACPAs) develop in response to neoepitopes that are formed under conditions of chronic inflammation. These autoantibodies may subsequently be fragmented by inflammation-associated proteases, leading to the formation of F(ab) 2 fragments. The hinge of F(ab) 2 fragments can serve as a neoepitope, and so-called antihinge antibodies (AHAs) can be found in RA patients, which might modulate the function of (fragmented) autoantibodies. We undertook this study to investigate the presence and specificities of AHAs in different stages of RA and to study their function.Methods. The presence of AHAs was assessed by radioimmunoassay in healthy controls, blood donors who later developed RA, patients with arthralgia, patients with early RA, and patients with established RA. Specificity of the AHAs was analyzed with inhibition assays, and complement-activating ability was studied with a C4b deposition assay.Results. Antibodies to IgG1 hinge, IgG2 hinge, and IgG4 hinge were detected in patients with established RA, with anti-IgG4 hinge antibodies being most specific (appearing in 1% of healthy controls, 3.8% of blood donors who later developed RA, 13% of arthralgia patients, 19% of early RA patients, and 16% of established RA patients). Anti-IgG4 hinge antibodies were subclass specific and were able to restore C4b deposition by IgG4 F(ab) 2 fragments. In patients with arthralgia and patients with early RA, anti-IgG4 hinge antibodies were associated with rheumatoid factors and ACPAs.

show abstract

Engineering a human IgG2 antibody stable at low pH

et al. 2020

View full text Add to dashboard Cite

IgG2 subclass antibodies have unique properties that include low effector function and a rigid hinge region. Although some IgG2 subclasses have been clinically tested and approved for therapeutic use, they have a higher propensity than IgG1 for aggregation, which can curtail or abolish their biological activity and enhance their immunogenicity. In this regard, acid-induced aggregation of monoclonal antibodies during purification and virus inactivation must be prevented. In the present study, we replaced the constant domain of IgG2 with that of IgG1, using anti-2,4-dinitrophenol (DNP) IgG2 as a model antibody, and investigated whether that would confer greater stability. While the anti-DNP IgG2 antibody showed significant aggregation at low pH, this was reduced for the IgG2 antibody containing the IgG1 CH2 domain. Substituting three amino acids within the CH2 domain-namely, F300Y, V309L, and T339A (IgG2_YLA)-reduced aggregation at low pH and increased CH2 transition temperature, as determined by differential scanning calorimetric analysis. IgG2_YLA exhibited similar antigen-binding capacity to IgG2, low affinity for FcγRIIIa, and low binding ability to C1q. The same YLA substitution also reduced the aggregation of panitumumab, another IgG2 antibody, at low pH. Our engineered human IgG2 antibody showed reduced aggregation during bioprocessing and provides a basis for designing improved IgG2 antibodies for therapeutic applications. K E Y W O R D Sacid-induced aggregate, ADCC, aggregation, antibody, CDC, IgG2 subclass, subclass change

show abstract

The in vitro resistance of IgG2 to proteolytic attack concurs with a comparative paucity of autoantibodies against peptide analogs of the IgG2 hinge

Cited by 36 publications

References 38 publications

Engineered Protease-resistant Antibodies with Selectable Cell-killing Functions

Engineered Protease-resistant Antibodies with Selectable Cell-killing Functions

Antibodies to IgG4 Hinge Can Be Found in Rheumatoid Arthritis Patients During All Stages of Disease and May Exacerbate Chronic Antibody‐Mediated Inflammation

Engineering a human IgG2 antibody stable at low pH

Contact Info

Product

Resources

About