Contribution of Variable Domains to the Stability of Humanized IgG1 Monoclonal Antibodies

Ionescu, Roxana; Vlasak, Josef; Price, Colleen; Kirchmeier, Marc

doi:10.1002/jps.21104

Cited by 287 publications

(310 citation statements)

References 36 publications

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“…The melting temperature (T m ) of the wild type human CH2 and CH3 domain, as measured by DSF, has been reported to be approximately 70°C and 81.5°C, respectively. 37 Our DSF experiments yielded comparable results with a CH2 and CH3 T m of 68.8°C and 80.8°C, respectively. In our experiments, the T m of the CH2 domain of the previously described YTE mutant Fc domain was 7.2°C lower than that of wild type, in line with previous reports.…”

Section: Resultssupporting

confidence: 74%

Extending human IgG half-life using structure-guided design

Booth

Ramakrishnan

Narayan

et al. 2018

mAbs

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Engineering of antibodies for improved pharmacokinetics through enhanced binding to the neonatal Fc receptor (FcRn) has been demonstrated in transgenic mice, non-human primates and humans. Traditionally, such approaches have largely relied on random mutagenesis and display formats, which fail to address related critical attributes of the antibody, such as effector functions or biophysical stability. We have developed a structure- and network-based framework to interrogate the engagement of IgG with multiple Fc receptors (FcRn, C1q, TRIM21, FcγRI, FcγRIIa/b, FcγRIIIa) simultaneously. Using this framework, we identified features that govern Fc-FcRn interactions and identified multiple distinct pathways for enhancing FcRn binding in a pH-specific manner. Network analysis provided a novel lens to study the allosteric impact of half-life-enhancing Fc mutations on FcγR engagement, which occurs distal to the FcRn binding site. Applying these principles, we engineered a panel of unique Fc variants that enhance FcRn binding while maintaining robust biophysical properties and wild type-like binding to activating receptors. An antibody harboring representative Fc designs demonstrates a half-life improvement of > 9 fold in transgenic mice and > 3.5 fold in cynomolgus monkeys, and maintains robust effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity.

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Section: Resultssupporting

confidence: 74%

Extending human IgG half-life using structure-guided design

Booth

Ramakrishnan

Narayan

et al. 2018

mAbs

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“…The major endothermic transition is observed at 83.5 C. As previously described, the DSC peak with the largest peak area can be assigned to the unfolding of the Fab part of antibodies. 32,33 Therefore, we concluded from the data shown in Figure 3A that the T M for the antibody Fab unfolding increases about 16 K after removal of predicted liabilities in HC plus LC (from 68 C for WT to 83.5 C for mAb1_Hv2_Lv3).…”

Section: Biophysical Characterizationmentioning

confidence: 87%

Boosting antibody developability through rational sequence optimization

Seeliger

Schulz

Litzenburger

et al. 2015

mAbs

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(2015) Boosting antibody developability through rational sequence optimization , mAbs, 7:3, 505-515, DOI: 10.1080DOI: 10. /19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 The application of monoclonal antibodies as commercial therapeutics poses substantial demands on stability and properties of an antibody. Therapeutic molecules that exhibit favorable properties increase the success rate in development. However, it is not yet fully understood how the protein sequences of an antibody translates into favorable in vitro molecule properties. In this work, computational design strategies based on heuristic sequence analysis were used to systematically modify an antibody that exhibited a tendency to precipitation in vitro. The resulting series of closely related antibodies showed improved stability as assessed by biophysical methods and longterm stability experiments. As a notable observation, expression levels also improved in comparison with the wild-type candidate. The methods employed to optimize the protein sequences, as well as the biophysical data used to determine the effect on stability under conditions commonly used in the formulation of therapeutic proteins, are described. Together, the experimental and computational data led to consistent conclusions regarding the effect of the introduced mutations. Our approach exemplifies how computational methods can be used to guide antibody optimization for increased stability.

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“…[29][30][31] CH2 and CH3 comprise the constant fragment (Fc) of the antibody. The denaturation of Rituximab was always irreversible, and the T m values were also scan rate dependent (results not shown), which precluded full thermodynamic analysis.…”

Section: Thermal Denaturationmentioning

confidence: 99%

“…32 This is a common property of large, complex proteins such as mAbs and has been described earlier. 30,33 However, the use of the denaturation enthalpy is still permissible according to the first law of thermodynamics, even if the process is irreversible. 34 In addition, no distortion, which is sometimes observed when the samples aggregate irreversibly, was seen on the shape of the heat capacity profiles for the Rituximab samples.…”

Section: Thermal Denaturationmentioning

confidence: 99%

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Aggregation of a multidomain protein: A coagulation mechanism governs aggregation of a model IgG1 antibody under weak thermal stress

et al. 2010

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Using an IgG1 antibody as a model system, we have studied the mechanisms by which multidomain proteins aggregate at physiological pH when incubated at temperatures just below their lowest thermal transition. In this temperature interval, only minor changes to the protein conformation are observed. Light scattering consistently showed two coupled phases: an initial fast phase followed by several hours of exponential growth of the scattered intensity. This is the exact opposite of the lagtime behavior typically observed in protein fibrillation. Dynamic light scattering showed the rapid formation of an aggregate species with a hydrodynamic radius of about 25 nm, which then increased in size throughout the experiment. Theoretical analysis of our light scattering data showed that the aggregate number density goes through a maximum in time providing compelling evidence for a coagulation mechanism in which aggregates fuse together. Both the analysis as well as size-exclusion chromatography of incubated samples showed the actual increase in aggregate mass to be linear and reach saturation long before all molecules had been converted to aggregates. The CH2 domain is the only domain partly unfolded in the temperature interval studied, suggesting a pivotal role of this least stable domain in the aggregation process. Our results show that for multidomain proteins at temperatures below their thermal denaturation, transient unfolding of a single domain can prime the molecule for aggregation, and that the formation of large aggregates is driven by coagulation.

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Contribution of Variable Domains to the Stability of Humanized IgG1 Monoclonal Antibodies

Cited by 287 publications

References 36 publications

Extending human IgG half-life using structure-guided design

Extending human IgG half-life using structure-guided design

Boosting antibody developability through rational sequence optimization

Aggregation of a multidomain protein: A coagulation mechanism governs aggregation of a model IgG1 antibody under weak thermal stress

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