A semi-synthetic repertoire of intrinsically stable antibody fragments derived from a single-framework scaffold 1 1Edited by J. Kahn

Desiderio, Angiola; Franconi, Rosella; López, Marián; Villani, Maria Elena; Viti, Francesca; Chiaraluce, Roberta; Consalvi, Valerio; Neri, Dario; Benvenuto, Eugenio

doi:10.1006/jmbi.2001.4756

Cited by 72 publications

(40 citation statements)

References 53 publications

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“…Numerous studies on the use of mutagenesis to stabilize antibodies have appeared. Using an aggregation propensity mapping algorithm, a number of more stable mutants of full-length antibodies were designed and prepared (533)(534)(535)(536).…”

Section: Site-directed Mutagenesismentioning

confidence: 99%

Stability of Protein Pharmaceuticals: An Update

Manning¹,

Chou²,

Murphy³

et al. 2010

Pharm Res

989

782

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In 1989, Manning, Patel, and Borchardt wrote a review of protein stability (Manning et al., Pharm. Res. 6:903-918, 1989), which has been widely referenced ever since. At the time, recombinant protein therapy was still in its infancy. This review summarizes the advances that have been made since then regarding protein stabilization and formulation. In addition to a discussion of the current understanding of chemical and physical instability, sections are included on stabilization in aqueous solution and the dried state, the use of chemical modification and mutagenesis to improve stability, and the interrelationship between chemical and physical instability.

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Section: Site-directed Mutagenesismentioning

confidence: 99%

Stability of Protein Pharmaceuticals: An Update

Manning¹,

Chou²,

Murphy³

et al. 2010

Pharm Res

989

782

View full text Add to dashboard Cite

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“…Moreover, by means of an evolutionary approach, we obtained a synthetic repertoire ('F8 library'), built upon intracellular antibody consensus frameworks, combinatorially mutating four amino acid residues in the complementarity-determining region 3 (CDR3) of both V H and V L chains. From this repertoire, we isolated intrinsically stable antibodies with new specificities and expressed as soluble molecules in the bacterial cytoplasm (Desiderio et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

Immunomodulation of cucumber mosaic virus infection by intrabodies selected in vitro from a stable single-framework phage display library

et al. 2005

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Immunomodulation by the ectopic expression of intracellular antibodies ('intrabodies') has a great potential for interfering with physiological or pathological functions in vivo in a highly specific manner. One of the major obstacles of this technology is the inability of most antibodies to properly fold and function in the reducing environment of the cytoplasm, which prevents the formation of essential disulfide bonds. We wished to assess the intracellular performance of antibodies derived from a semi-synthetic single-chain variable fragment (scFv) phage display library ('F8 library') built on a thermodynamically stable single-framework scaffold. To this purpose, we chose to modulate the infection of a pandemic plant pathogen, the cucumber mosaic virus (CMV). After in vitro 'biopanning' on immobilized virions, two scFvs were biochemically characterized, showing high affinity toward the antigen. They were transiently expressed at high yields as soluble molecules in the cytoplasm of Nicotiana benthamiana plants. Subsequently, they were expressed in the cytoplasm of transgenic tomato plants. Challenge with high viral dose showed that both scFvs were able to elicit a phenotypic effect and led to the identification of a transgenic line fully resistant to infection. In these plants, the scFv binds the virus in the inoculated leaves preventing viral long distance movement. This work represents the first demonstration that the 'F8 library' can be directly screened in vitro to rapidly isolate antigen-specific scFvs that act as effective intrabodies in vivo. These antibodies represent powerful tools to interfere with several intracellular targets, modulating pathogen infectivity and/or cellular metabolism.

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“…The successful expression of a trxscfv fusion is also notable since single-chain antibodies are notoriously difficult to express in the cytoplasm and generally reserved to special cases with select framework sequences in phage display vectors (Desiderio et al, 2001) or that have been selected from randomized libraries (Martineau et al, 1998). Successful expression of intrabodies (cytoplasmic antibodies) has also been demonstrated, for example, by fusion with maltose-binding protein at the car- Figure 2.…”

Section: Resultsmentioning

confidence: 99%

Expression and characterization of a humanized cocaine‐binding antibody

Redwan

Larsen

Zhou

et al. 2003

Biotech & Bioengineering

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The murine immunoglobulin G (IgG) cocaine-binding monoclonal antibody (mAb), GNC92H2, is notable for its exquisite specificity for cocaine, as opposed to chemically-related cocaine metabolites, and for its moderately high affinity (K(d) approximately 200 nM) for cocaine. Recently, we described the crystal structure of a mouse/human chimeric Fab construct at 2.3 A resolution. Herein, we report the successful framework humanization of a single-chain Fv (scFv) GNC92H2 construct without loss of affinity for cocaine. In brief, we compared the mAb GNC92H2 sequence to human antibody sequences, and used structure-based design to incorporate mutations (total = 49) that would humanize the framework region without affecting the overall shape of the binding pocket or the key cocaine-contact residues. The codons of the rationally designed sequence were optimized for E. coli expression, and the gene was synthesized by a de novo PCR reaction using 14 overlapping primers. Expression of the scFv construct was significantly improved in E. coli by fusion to thioredoxin. Intriguingly, this construct apparently refolds to form soluble active antibody in the reducing environment of the cytoplasm. Competitive ELISA and equilibrium dialysis demonstrated comparable binding activity between the humanized scFv and the whole IgG. The successful humanization of mAb GNC92H2 should enhance its potential therapeutic value by reducing its overall. immunogenicity.

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A semi-synthetic repertoire of intrinsically stable antibody fragments derived from a single-framework scaffold 1 1Edited by J. Kahn

Cited by 72 publications

References 53 publications

Stability of Protein Pharmaceuticals: An Update

Stability of Protein Pharmaceuticals: An Update

Immunomodulation of cucumber mosaic virus infection by intrabodies selected in vitro from a stable single-framework phage display library

Expression and characterization of a humanized cocaine‐binding antibody

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