Stability engineering of antibody single-chain Fv fragments

Wörn, Arne; Plückthun, Andreas

doi:10.1006/jmbi.2000.4265

Cited by 534 publications

(387 citation statements)

References 130 publications

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“…2,8 Introduction of disulfide bridges or improved interface interactions stabilize Fv dimerization in specific cases; however, such strategies remain associated with loss of antigen affinity or increased aggregation propensity. [9][10][11][12] Asymmetric bispecific antibodies including Fc domains give rise to improperly paired side-products even when optimized. 2 Thus, they generally require further engineering, extensive purification, or special production systems imposing specific limitations.…”

Section: Introductionmentioning

confidence: 99%

CODV-Ig, a universal bispecific tetravalent and multifunctional immunoglobulin format for medical applications

Steinmetz

Vallée

Beil

et al. 2016

mAbs

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Bispecific immunoglobulins (Igs) typically contain at least two distinct variable domains (Fv) that bind to two different target proteins. They are conceived to facilitate clinical development of biotherapeutic agents for diseases where improved clinical outcome is obtained or expected by combination therapy compared to treatment by single agents. Almost all existing formats are linear in their concept and differ widely in drug-like and manufacture-related properties. To overcome their major limitations, we designed cross-over dual variable Ig-like proteins (CODV-Ig). Their design is akin to the design of circularly closed repeat architectures. Indeed, initial results showed that the traditional approach of utilizing (G4S) x linkers for biotherapeutics design does not identify functional CODV-Igs. Therefore, we applied an unprecedented molecular modeling strategy for linker design that consistently results in CODV-Igs with excellent biochemical and biophysical properties. CODV architecture results in a circular self-contained structure functioning as a self-supporting truss that maintains the parental antibody affinities for both antigens without positional effects. The format is universally suitable for therapeutic applications targeting both circulating and membrane-localized proteins. Due to the full functionality of the Fc domains, serum half-life extension as well as antibody-or complement-dependent cytotoxicity may support biological efficiency of CODV-Igs. We show that judicious choice in combination of epitopes and paratope orientations of bispecific biotherapeutics is anticipated to be critical for clinical outcome. Uniting the major advantages of alternative bispecific biotherapeutics, CODV-Igs are applicable in a wide range of disease areas for fast-track multi-parametric drug optimization.

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Section: Introductionmentioning

confidence: 99%

CODV-Ig, a universal bispecific tetravalent and multifunctional immunoglobulin format for medical applications

Steinmetz

Vallée

Beil

et al. 2016

mAbs

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“…However, they also have the disadvantages of high cost, varying stability, and challenging production and purification (Worn and Pluckthun 2001, Quintero-Hernandez et al 2007, Malpiedi et al 2013. Antibody fragments obtained by chemical reduction are an  2-MEA -2-mercaptoethylamine EDTA -ethylenediaminetetraacetic acid disodium dihydrate TCEP -(2-carboxyethyl)phosphine attractive alternative as their production is inexpensive and uncomplicated.…”

Section: Introductionmentioning

confidence: 99%

Considerations in producing preferentially reduced half-antibody fragments

Makaraviciute

Jackson

Millner

et al. 2016

Journal of Immunological Methods

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Half-antibody fragments are a promising reagent for biosensing, drug-delivery and labeling applications, since exposure of the free thiol group in the Fc hinge region allows oriented Preferential reduction of rabbit anti-myoglobin IgG antibodies was optimized and the highest half-antibody yield was obtained with 35 mM TCEP. Finally, it has been demonstrated that produced anti-myoglobin half-IgG fragments retained their binding activity.

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“…8 Understanding the intramolecular interactions that govern the formation of these intermediates would represent an important advance towards solving the protein-folding problem. [9][10][11][12] Reductive unfolding, the converse of oxidative folding, is the process by which a disulfidebond-containing protein loses its native structure (unfolds) through the (successive) reduction of its disulfide bonds. [13][14][15][16][17][18][19][20][21] In reductive unfolding, protein disulfide bonds scattered throughout the macromolecular architecture can serve as reporter groups for probing local fluctuations within the folded polypeptide; these fluctuations eventually lead to the unfolding of the whole molecule.…”

Section: Introductionmentioning

confidence: 99%

A Localized Specific Interaction Alters the Unfolding Pathways of Structural Homologues

Narayan

Kurinov

et al. 2006

J. Am. Chem. Soc.

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Reductive unfolding studies of proteins are designed to provide information about intramolecular interactions that govern the formation (and stabilization) of the native state and about folding/ unfolding pathways. By mutating Tyr92 to G, A, or L in the model protein, bovine pancreatic ribonuclease A, and through analysis of temperature factors and molecular dynamics simulations of the crystal structures of these mutants, it is demonstrated that the markedly different reductive unfolding rates and pathways of ribonuclease A and its structural homologue onconase can be attributed to a single, localized, ring-stacking interaction between Tyr92 and Pro93 in the bovine variant. The fortuitous location of this specific stabilizing interaction in a disulfide-bond-containing loop region of ribonuclease A results in the localized modulation of protein dynamics which, in turn, enhances the susceptibility of the disulfide bond to reduction leading to an alteration in the reductive unfolding behavior of the homologues. These results have important implications for folding studies involving topological determinants to obtain folding/unfolding rates and pathways, for protein structure-function prediction through fold recognition, and for predicting proteolytic cleavage sites.

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Stability engineering of antibody single-chain Fv fragments

Cited by 534 publications

References 130 publications

CODV-Ig, a universal bispecific tetravalent and multifunctional immunoglobulin format for medical applications

CODV-Ig, a universal bispecific tetravalent and multifunctional immunoglobulin format for medical applications

Considerations in producing preferentially reduced half-antibody fragments

A Localized Specific Interaction Alters the Unfolding Pathways of Structural Homologues

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