Toward aggregation-resistant antibodies by design

Lee, Christine C.; Perchiacca, Joseph M.; Tessier, Peter M.

doi:10.1016/j.tibtech.2013.07.002

Cited by 83 publications

(69 citation statements)

References 85 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…From these data (Supplementary Information S3), an optimal working concentration of 50 mg/mL was determined for the experimental aggregation assays. In our experimental conditions, a 50 mg/mL bevacizumab solution led to »25% and 32% aggregation after incubation for 24 h and 48 h, respectively, at 52 C. These findings are in relative agreement with previous results; we found »2.8-fold more aggregation than when commercial Avastin was incubated at 50 C for 24 h (8.8% aggregation observed). 22 Given that we are working at twice the formulation concentration and at 52 C (vs. 50 C), this result was expected (a 3.2-fold difference in the amount of aggregates was observed between commercial Avastin and bevacizumab incubated at 40 C).…”

Section: Mutation Of High Sap Residues Increases Bevacizumab Stabilitysupporting

confidence: 92%

“…32,39,50,51 Negatively charged amino acids are suggested to locally modify the charge of the protein, enhancing protein-protein repulsion. The addition of aspartic residues in the bevacizumab sequence slightly decreased the protein charge at pH 6.0 from 14 to 12 units (as calculated with PROPKA 3.0) 52 present on the mAb Fab domain surface, whereas the introduction of lysine increased the charge by 2 units.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Rational design of therapeutic mAbs against aggregation through protein engineering and incorporation of glycosylation motifs applied to bevacizumab

Courtois

Agrawal

Lauer³

et al. 2015

mAbs

View full text Add to dashboard Cite

The aggregation of biotherapeutics is a major hindrance to the development of successful drug candidates; however, the propensity to aggregate is often identified too late in the development phase to permit modification to the protein's sequence. Incorporating rational design for the stability of proteins in early discovery has numerous benefits. We engineered out aggregation-prone regions on the Fab domain of a therapeutic monoclonal antibody, bevacizumab, to rationally design a biobetter drug candidate. With the purpose of stabilizing bevacizumab with respect to aggregation, 2 strategies were undertaken: single point mutations of aggregation-prone residues and engineering a glycosylation site near aggregation-prone residues to mask these residues with a carbohydrate moiety. Both of these approaches lead to comparable decreases in aggregation, with an up to 4-fold reduction in monomer loss. These single mutations and the new glycosylation pattern of the Fab domain do not modify binding to the target. Biobetters with increased stability against aggregation can therefore be generated in a rational manner, by either removing or masking the aggregation-prone region or crowding out protein-protein interactions.

show abstract

Section: Mutation Of High Sap Residues Increases Bevacizumab Stabilitysupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Rational design of therapeutic mAbs against aggregation through protein engineering and incorporation of glycosylation motifs applied to bevacizumab

Courtois

Agrawal

Lauer³

et al. 2015

mAbs

View full text Add to dashboard Cite

show abstract

“…The utility of antibodies can be limited by solution properties such as solubility, aggregation and thermal stability, which are often related to each other. 18 Poor thermal stability may affect both antibody solubility and aggregation, although antibody aggregation may also be independent of thermal stability. Rational engineering and selection-based methods have demonstrated that solubility and aggregation can be improved independently of thermal stability.…”

Section: Discussionmentioning

confidence: 99%

A general approach to antibody thermostabilization

McConnell

Zhang

Macomber

et al. 2014

mAbs

View full text Add to dashboard Cite

“…21,22,31 Previous studies have found that inclusion of a number of design modifications, including variations in CH3-scFv linker length and type, and also scFv stability engineering via addition of disulfide bonds, improved construct stability. [70][71][72][73][74][75] A concern with the Fc-linked dimeric design was whether the presence of 2 anti-LPS scFvs would result in EDV TM nanocell aggregation via binding of multiple nanocells rather than high avidity binding of a single nanocell. An alternative Fc-linked BsAb was engineered consisting of 2 EGFR targeting scFvs at the N-terminus (high avidity) and a single anti-LPS scFv at the C-terminus of the final product.…”

Section: Wwwtandfonlinecom 59 Mabsmentioning

confidence: 99%

Nanocell targeting using engineered bispecific antibodies

Taylor

Howard

Jones

et al. 2015

mAbs

View full text Add to dashboard Cite

There are many design formats for bispecific antibodies (BsAbs), and the best design choice is highly dependent on the final application. Our aim was to engineer BsAbs to target a novel nanocell (EnGeneIC Delivery Vehicle or EDV TM nanocell) to the epidermal growth factor receptor (EGFR). EDV TM nanocells are coated with lipopolysaccharide (LPS), and BsAb designs incorporated single chain Fv (scFv) fragments derived from an anti-LPS antibody (1H10) and an anti-EGFR antibody, ABX-EGF. We engineered various BsAb formats with monovalent or bivalent binding arms and linked scFv fragments via either glycine-serine (G4S) or Fc-linkers. Binding analyses utilizing ELISA, surface plasmon resonance, bio-layer interferometry, flow cytometry and fluorescence microscopy showed that binding to LPS and to either soluble recombinant EGFR or MDA-MB-468 cells expressing EGFR, was conserved for all construct designs. However, the Fc-linked BsAbs led to nanocell clumping upon binding to EDV TM nanocells. Clumping was eliminated when additional disulfide bonds were incorporated into the scFv components of the BsAbs, but this resulted in lower BsAb expression. The G4S-linked tandem scFv BsAb format was the optimal design with respect to EDV binding and expression yield. Doxorubicin-loaded EDV TM nanocells actively targeted with tandem scFv BsAb in vivo to MDA-MB-468-derived tumors in mouse xenograft models enhanced tumor regression by 40% compared to passively targeted EDV TM nanocells. BsAbs therefore provide a functional means to deliver EDV TM nanocells to target cells.

show abstract

Toward aggregation-resistant antibodies by design

Cited by 83 publications

References 85 publications

Rational design of therapeutic mAbs against aggregation through protein engineering and incorporation of glycosylation motifs applied to bevacizumab

Rational design of therapeutic mAbs against aggregation through protein engineering and incorporation of glycosylation motifs applied to bevacizumab

A general approach to antibody thermostabilization

Nanocell targeting using engineered bispecific antibodies

Contact Info

Product

Resources

About