Directed Evolution of an Anti-prion Protein scFv Fragment to an Affinity of 1 pM and its Structural Interpretation

Luginbühl, Béatrice; Kanyo, Z.F.; Jones, R. Mark; Fletterick, Robert J.; Prusiner, Stanley B.; Cohen, Fred E.; Williamson, R. Anthony; Burton, Dennis R.; Plückthun, Andreas

doi:10.1016/j.jmb.2006.07.027

Cited by 93 publications

(51 citation statements)

References 60 publications

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“…High affinity and selectivity are critical issues for antibody therapeutic capacity. Many different approaches have been reported for improving the affinity of scFvs derived from phage antibody libraries, including error-prone PCR (27), CDR walking (28), hot-spot mutagenesis (29), parsimonious mutagenesis (30), etc. None of them is based on a rational approach to accelerate and direct the maturation process.…”

Section: Discussionmentioning

confidence: 99%

Affinity maturation of antibodies assisted by in silico modeling

Barderas

Desmet²,

Timmerman³

et al. 2008

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

126

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Rational engineering methods can be applied with reasonable success to optimize physicochemical characteristics of proteins, in particular, antibodies. Here, we describe a combined CDR3 walking randomization and rational design-based approach to enhance the affinity of the human anti-gastrin TA4 scFv. The application of this methodology to TA4 scFv, displaying only a weak overall affinity for gastrin17 (K D ‫؍‬ 6 M), resulted in a set of nine affinity-matured scFv variants with near-nanomolar affinity (KD ‫؍‬ 13.2 nM for scFv TA4.112). First, CDR-H3 and CDR-L3 randomization resulted in three scFvs with an overall affinity improvement of 15-to 35-fold over the parental. Then, the modeling of two scFv constructs selected from the previous step (TA4.11 and TA4.13) was followed by a combination of manual and molecular dynamics-based docking of gastrin17 into the respective binding sites, analysis of apparent packing defects, and selection of residues for mutagenesis through phage display. Nine scFv mutants were obtained from the second maturation step. A final 454-fold improvement in affinity compared with TA4 was obtained. These scFvs showed an enhanced potency to inhibit gastrin-induced proliferation in Colo 320 WT and BxPc3 tumoral cells. In conclusion, we propose a structure-based rational method to accelerate the development of affinity-matured antibody constructs with enhanced potential for therapeutic use.antibody engineering ͉ gastrin ͉ in vitro affinity maturation ͉ pancreatic cancer

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Section: Discussionmentioning

confidence: 99%

Affinity maturation of antibodies assisted by in silico modeling

Barderas

Desmet²,

Timmerman³

et al. 2008

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

126

View full text Add to dashboard Cite

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“…Along with combinatorial library technology, a variety of screening systems, such as phage display, ribosome display, etc., have been constructed. [1][2][3][4][5] Although a large library around 10 12 can be prepared using phage and mRNA display systems, the number of all sequences of proteins using 20 amino acids at each position is too large to be covered. For example, the number of possible protein sequences resulting from 100 amino acid residues is 10 130 .…”

Section: Introductionmentioning

confidence: 99%

Selection and structural analysis of de novo proteins from an α3β3 genetic library

et al. 2009

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The construction of novel functional proteins has been a key area of protein engineering. However, there are few reports of functional proteins constructed from artificial scaffolds. Here, we have constructed a genetic library encoding a3b3 de novo proteins to generate novel scaffolds in smaller size using a binary combination of simplified hydrophobic and hydrophilic amino acid sets. To screen for folded de novo proteins, we used a GFP-based screening system and successfully obtained the proteins from the colonies emitting the very bright fluorescence as a similar intensity of GFP. Proteins isolated from the very bright colonies (vTAJ) and bright colonies (wTAJ) were analyzed by circular dichroism (CD), 8-anilino-1-naphthalenesulfonate (ANS) binding assay, and analytical size-exclusion chromatography (SEC). CD studies revealed that vTAJ and wTAJ proteins had both a-helix and b-sheet structures with thermal stabilities. Moreover, the selected proteins demonstrated a variety of association states existing as monomer, dimer, and oligomer formation. The SEC and ANS binding assays revealed that vTAJ proteins tend to be a characteristic of the folded protein, but not in a molten-globule state. A vTAJ protein, vTAJ13, which has a packed globular structure and exists as a monomer, was further analyzed by nuclear magnetic resonance. NOE connectivities between backbone signals of vTAJ13 suggested that the protein contains three a-helices and three b-strands as intended by its design. Thus, it would appear that artificially generated a3b3 de novo proteins isolated from very bright colonies using the GFP fusion system exhibit excellent properties similar to folded proteins and would be available as artificial scaffolds to generate functional proteins with catalytic and ligand binding properties.

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“…To increase the affinities of the library members, it is best to select for those having the lowest dissociation rate constant (off-rate) from the target (7,13,15,28,29). This off-rate selection can be applied for the improvement of known binders (after mutagenizing the gene for defined binders and thus creating a new library), but also during the initial selection from the original library.…”

Section: Selection In Platesmentioning

confidence: 99%

“…By using very stringent selections for affinity, antibody scFv fragments have been evolved to affinities as high as 5 pM or even 1 pM (12,13). By choosing an appropriate selection pressure, properties other than affinity could be optimized, such as, for example, stability (7).…”

mentioning

confidence: 99%

Ribosome Display: A Technology for Selecting and Evolving Proteins from Large Libraries

Dreier

Plückthun

2010

Methods in Molecular Biology

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The selection and concomitant affinity maturation of proteins to bind to user-defined target molecules have become a key technology in biochemical research, diagnostics, and therapy. One of the most potent selection technologies for such applications is ribosome display. It works entirely in vitro, and this has two important consequences. First, since no transformation of any cells is required, libraries with much greater diversity can be handled than with most other techniques. Second, since a library does not have to be cloned and transformed, it is very convenient to introduce random errors in the library by PCR-based methods and select improved binders. Thus, a true directed evolution, an iteration between randomization and selection over several generations, can be conveniently carried out, e.g., for affinity maturation. Ribosome display has been used successfully for the selection of antibody fragments and other binding proteins, such as Designed Ankyrin Repeat Proteins (DARPins). Birgit Dreier and Andreas Plückthun AbstractThe selection and concomitant affinity maturation of proteins to bind to user-defined target molecules have become a key technology in biochemical research, diagnostics, and therapy. One of the most potent selection technologies for such applications is ribosome display. It works entirely in vitro, and this has two important consequences. First, since no transformation of any cells is required, libraries with much greater diversity can be handled than with most other techniques. Second, since a library does not have to be cloned and transformed, it is very convenient to introduce random errors in the library by PCRbased methods and select improved binders. Thus, a true directed evolution, an iteration between randomization and selection over several generations, can be conveniently carried out, e.g., for affinity maturation. Ribosome display has been used successfully for the selection of antibody fragments and other binding proteins, such as Designed Ankyrin Repeat Proteins (DARPins).

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Directed Evolution of an Anti-prion Protein scFv Fragment to an Affinity of 1 pM and its Structural Interpretation

Cited by 93 publications

References 60 publications

Affinity maturation of antibodies assisted by in silico modeling

Affinity maturation of antibodies assisted by in silico modeling

Selection and structural analysis of de novo proteins from an α3β3 genetic library

Ribosome Display: A Technology for Selecting and Evolving Proteins from Large Libraries

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