A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties

Tiller, Thomas; Schuster, Ingrid; Deppe, Dorothée; Siegers, Katja; Strohner, Ralf; Herrmann, Tanja; Berenguer, Marie; Poujol, Dominique; Stehle, Jennifer; Stark, Yvonne; Heßling, Martin; Daubert, Daniela; Felderer, Karin; Kaden, Stefan A.; Kölln, Johanna; Enzelberger, Markus; Urlinger, Stefanie

doi:10.4161/mabs.24218

Cited by 181 publications

(155 citation statements)

References 76 publications

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“…Domains from the V H 1 and V 2 families have been associated with stable scFv v-domain pairing (31,32), and both V H 1-69 and V 2-14 are highly represented in the human antibody repertoire (33,34). Indeed, an extensive study of V H -V L pairing found V 2-14 to be the most highly represented V germline in the repertoire, where it is often paired with V H 1-69 (34). Despite this, 3B4 was found to suffer from very low stability and solubility in an scFvbased bispecific format (13).…”

Section: Discussionmentioning

confidence: 99%

A Combination of Structural and Empirical Analyses Delineates the Key Contacts Mediating Stability and Affinity Increases in an Optimized Biotherapeutic Single-chain Fv (scFv)

Terraube

Tam

et al. 2016

Journal of Biological Chemistry

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Background: Antibody v-domains in scFv format often suffer from aggregation and stability issues that restrict formulation. Results: Structural and empirical analyses of an optimized scFv revealed that three V L -CDR3 mutations were sufficient to mediate significant stability and affinity improvements. Conclusion: scFv issues were resolved via removal of side-chain clashes at the V L /V H interface. Significance: CDR-restricted mutagenesis delivers stability-optimized molecules for high concentration dosing.

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Section: Discussionmentioning

confidence: 99%

A Combination of Structural and Empirical Analyses Delineates the Key Contacts Mediating Stability and Affinity Increases in an Optimized Biotherapeutic Single-chain Fv (scFv)

Terraube

Tam

et al. 2016

Journal of Biological Chemistry

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“…[2][3][4] To avoid such setbacks, risk mitigation strategies increasingly center around "Quality-by-Design" (QbD) approaches, which consider developability-related issues early in the drug discovery phase and involve rationally engineering antibodies toward favorable Chemistry, Manufacturing and Control (CMC) and pharmacokinetics (PK) properties. [5][6][7] Choosing molecules that exhibit low intrinsic immunogenicity and that are robust against the formation of immunogenic degradation products can also reduce safety risks. However, the large number of different antibodies typically considered in early projects, and the small amount of each antibody that is typically available, precludes extensive experimental characterization and determination of CMC properties such as yield, chemical and physical stability.…”

Section: Introductionmentioning

confidence: 99%

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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“…With Fab libraries, the heavy and light chains are expressed as separate gene products from the same vector with constant heavy 1 and constant light domains, respectively (5). The establishment of modern, optimized, human antibody phage display libraries supports the contemporary role of this combinatorial technology in ongoing antibody discovery (19)(20)(21)(22).…”

Section: Antibody Phage Display Formatsmentioning

confidence: 97%

“…For this semi-synthetic antibody library, donorsourced fragments comprise the V H CDR3 and complete V L , while in vitro synthesis of V segment DNA introduces designed diversity in V H CDR1 and CDR2 (19). Another group developed synthetic phage display libraries by modifying the sequence and length of all six CDRs based upon their context in natural human antibody gene families (20)(21)(22).…”

Section: Synthetic Phage Antibody Librariesmentioning

confidence: 99%

“…Modern phage libraries displaying Fab fragments are well established and engineered for improved biochemical and expression properties for therapeutic discovery (19)(20)(21)(22). For displaying Fabs on the phage surface, either the heavy or light chain constant domain-preferably the constant heavy 1 to avoid anchored V L dimers-is C-terminally fused to pIII.…”

Section: Fab-piii Displaymentioning

confidence: 99%

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Phage and Yeast Display

Sheehan

Marasco

2015

Microbiol Spectr

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Despite the availability of antimicrobial drugs, the continued development of microbial resistance-established through escape mutations and the emergence of resistant strains-limits their clinical utility. The discovery of novel, therapeutic, monoclonal antibodies (mAbs) offers viable clinical alternatives in the treatment and prophylaxis of infectious diseases. Human mAb-based therapies are typically nontoxic in patients and demonstrate high specificity for the intended microbial target. This specificity prevents negative impacts on the patient microbiome and avoids driving the resistance of nontarget species. The in vitro selection of human antibody fragment libraries displayed on phage or yeast surfaces represents a group of well-established technologies capable of generating human mAbs. The advantage of these forms of microbial display is the large repertoire of human antibody fragments present during a single selection campaign. Furthermore, the in vitro selection environments of microbial surface display allow for the rapid isolation of antibodies-and their encoding genes-against infectious pathogens and their toxins that are impractical within in vivo systems, such as murine hybridomas. This article focuses on the technologies of phage display and yeast display, as these strategies relate to the discovery of human mAbs for the treatment and vaccine development of infectious diseases.

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A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties

Cited by 181 publications

References 76 publications

A Combination of Structural and Empirical Analyses Delineates the Key Contacts Mediating Stability and Affinity Increases in an Optimized Biotherapeutic Single-chain Fv (scFv)

A Combination of Structural and Empirical Analyses Delineates the Key Contacts Mediating Stability and Affinity Increases in an Optimized Biotherapeutic Single-chain Fv (scFv)

Boosting antibody developability through rational sequence optimization

Phage and Yeast Display

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