Engineering Aggregation Resistance in IgG by Two Independent Mechanisms: Lessons from Comparison of Pichia pastoris and Mammalian Cell Expression

Schaefer, Jonas V.; Plückthun, Andreas

doi:10.1016/j.jmb.2012.01.027

Cited by 47 publications

(32 citation statements)

References 87 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Schaefer and Pluckthun outline a strategy of fusing a negatively charged peptide to the N terminus of human antibodies to increase aggregation resistance (37). While their approach is clearly compatible with antigen binding, the observed effect is not positional and can be attributed to increased net charge of the molecule (37). It may, however, provide synergies with the strategy outlined here.…”

Section: Discussionmentioning

confidence: 96%

“…The authors do not provide evidence on antigen binding, repertoire diversity, or V H -V L pairing, making their findings difficult to assess. Schaefer and Pluckthun outline a strategy of fusing a negatively charged peptide to the N terminus of human antibodies to increase aggregation resistance (37). While their approach is clearly compatible with antigen binding, the observed effect is not positional and can be attributed to increased net charge of the molecule (37).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

General strategy for the generation of human antibody variable domains with increased aggregation resistance

Dudgeon

Rouet

Kokmeijer

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

176

164

View full text Add to dashboard Cite

The availability of stable human antibody reagents would be of considerable advantage for research, diagnostic, and therapeutic applications. Unfortunately, antibody variable heavy and light domains (V H and V L ) that mediate the interaction with antigen have the propensity to aggregate. Increasing their aggregation resistance in a general manner has proven to be a difficult and persistent problem, due to the high level of sequence diversity observed in human variable domains and the requirement to maintain antigen binding. Here we outline such an approach. By using phage display we identified specific positions that clustered in the antigen binding site (28, 30-33, 35 in V H and 24, 49-53, 56 in V L ). Introduction of aspartate or glutamate at these positions endowed superior biophysical properties (non-aggregating, well-expressed, and heat-refoldable) onto domains derived from common human germline families (V H 3 and V κ 1). The effects of the mutations were highly positional and independent of sequence diversity at other positions. Moreover, crystal structures of mutant V H and V L domains revealed a surprising degree of structural conservation, indicating compatibility with V H ∕V L pairing and antigen binding. This allowed the retrofitting of existing binders, as highlighted by the development of robust high affinity antibody fragments derived from the breast cancer therapeutic Herceptin. Our results provide a general strategy for the generation of human antibody variable domains with increased aggregation resistance.biotechnology | monoclonal antibodies | protein aggregation | protein engineering | antibody therapeutics P rotein aggregation represents a key bottleneck in the generation of antibody-based reagents and hinders the development and production of human therapeutics (1). It is generally believed that the aggregation propensity of larger antibody reagents (such as immunoglobulin G and Fab) is mostly determined by their variable domain components (V H and V L ), although there is currently little understanding of the mechanisms involved (1, 2). Indeed, significant differences of aggregation rates have been reported for antibodies that differ exclusively in their variable domains (1, 2). Aggregation propensity is even more pronounced for smaller antibody reagents, which lack the interdomain stabilization of their larger counterparts (3, 4). This is a major problem in biotechnology due to an increasing trend toward smaller antibody formats for imaging and tumor targeting applications (4). Common formats include human single chain fragments (scFv) and human single domain antibodies, both of which frequently display poor biophysical properties (1, 3).The aggregation propensity of human variable domains is in marked contrast to the variable heavy domains of camels and llamas, which are generally nonaggregating and soluble ("V HH domains") (5-7). More favorable properties have also been described for "camelized" and other engineered human V H model domains (8-12) but not for human V L domains. Their p...

show abstract

Section: Discussionmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

General strategy for the generation of human antibody variable domains with increased aggregation resistance

Dudgeon

Rouet

Kokmeijer

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

176

164

View full text Add to dashboard Cite

show abstract

“…8,21 Moreover, a general relationship has been observed in which antibodies with enhanced thermal stability are less susceptible to aggregation. 22 Studies have attempted to improve antibody aggregation and solubility while minimizing the effect on antigen binding using a variety of engineering approaches, including the addition of negative and positive charges within the V region, 15,23 the addition of charged residues at antibody termini, 24,25 and the introduction of hydrophilic residues that improve VH/VL pairing and reduce exposure of the hydrophobic interface. 26 Our study sought to demonstrate the general applicability of a stable IgG framework for the grafting of diverse antibody CDRs and specificities, simultaneously providing improvement in thermal stability and retention of high-affinity antigen binding affinity.…”

Section: Discussionmentioning

confidence: 99%

A general approach to antibody thermostabilization

McConnell

Zhang

Macomber

et al. 2014

mAbs

View full text Add to dashboard Cite

“…These regions typically contain hydrophobic patches (e.g., Fc receptor-binding sites) and oppositely charged domains that can interact with themselves or with the CDRs, leading to poor solubility. Glycans also significantly impact solubility, typically in a positive manner (24, 63, 64). …”

Section: Antibody Colloidal Stability (Solubility)mentioning

confidence: 99%

Advances in Antibody Design

Tiller

Tessier

2015

Annu. Rev. Biomed. Eng.

206

152

View full text Add to dashboard Cite

The use of monoclonal antibodies as therapeutics requires optimizing several of their key attributes. These include binding affinity and specificity, folding stability, solubility, pharmacokinetics, effector functions, and compatibility with the attachment of additional antibody domains (bispecific antibodies) and cytotoxic drugs (antibody–drug conjugates). Addressing these and other challenges requires the use of systematic design methods that complement powerful immunization and in vitro screening methods. We review advances in designing the binding loops, scaffolds, domain interfaces, constant regions, post-translational and chemical modifications, and bispecific architectures of antibodies and fragments thereof to improve their bioactivity. We also highlight unmet challenges in antibody design that must be overcome to generate potent antibody therapeutics.

show abstract

Engineering Aggregation Resistance in IgG by Two Independent Mechanisms: Lessons from Comparison of Pichia pastoris and Mammalian Cell Expression

Cited by 47 publications

References 87 publications

General strategy for the generation of human antibody variable domains with increased aggregation resistance

General strategy for the generation of human antibody variable domains with increased aggregation resistance

A general approach to antibody thermostabilization

Advances in Antibody Design

Contact Info

Product

Resources

About