Design and Construction of Synthetic Phage-Displayed Fab Libraries

Boström, Jenny; Fuh, Germaine

doi:10.1007/978-1-60327-302-2_2

Cited by 9 publications

(10 citation statements)

References 25 publications

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“…Phage library cloning and production were performed as has been described . E. coli CJ236 (NEB, Ipswich, MA) were transformed with template phagemid derived from the phage‐display vector pAS38 encoding Fab 11E fused to gene III of M13 phage.…”

Section: Methodsmentioning

confidence: 99%

A polar ring endows improved specificity to an antibody fragment

2016

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Engineering monovalent Fab fragments into bivalent formats like IgGs or F(ab') 2 can lead to aggregation presumably because of nonspecific off-target interactions that induce aggregation. In an effort to further understand the molecular determinants of nonspecific interactions for engineered antibodies and natively folded proteins in general, we focused on a synthetic Fab with low nanomolar affinity to histone chaperone Anti-silencing factor 1 (Asf1) that demonstrates off-target binding through low solubility (~5 mg/mL) in the multivalent F(ab') 2 state. Here, we generated phage display-based shotgun scanning libraries to introduce aspartate as a negative design element into the antibody paratope. The antibody-combining site was amenable to aspartate substitution at numerous positions within the antigen binding loops and one variant, Tyr Asp, possessed high solubility (>100 mg/ml). Furthermore, the mutations decreased nonspecific interactions measured by column interaction chromatography and ELISA in the multivalent antibody format while maintaining high affinity to the antigen. Structural determination of the antibody-antigen complex revealed that the aspartate-permissive residues formed a polar ring around the structural and functional paratope, recapitulating the canonical feature of naturally occurring protein-protein interactions. This observation may inform future strategies for the design and engineering of molecular recognition.

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Section: Methodsmentioning

confidence: 99%

A polar ring endows improved specificity to an antibody fragment

2016

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“…Antibodies are typically generated against a target antigen of interest using one of several well-established techniques [22,23]. In some instances however, an antibody with ideal characteristics for pre-clinical or clinical consideration may be present only at the protein level and cannot be fabricated by means other than amino acid level sequencing and reverse engineering.…”

Section: 0 Discussionmentioning

confidence: 99%

Resurrection of a clinical antibody: Template proteogenomic de novo proteomic sequencing and reverse engineering of an anti‐lymphotoxin‐α antibody

Castellana

McCutcheon²,

Pham³

et al. 2011

Proteomics

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A mouse hybridoma antibody directed against a member of the TNF-superfamily, lymphotoxin alpha (LT-α), was isolated from stored mouse ascites and purified to homogeneity. After more than a decade of storage the genetic material was not available for cloning, however biochemical assays with the ascites showed this antibody against LT-α (LT-3F12) to be a pre-clinical candidate for the treatment of several inflammatory pathologies. We have successfully rescued the LT-3F12 antibody by performing mass spectrometric analysis, primary amino acid sequence determination by template proteogenomics, and synthesis of the corresponding recombinant DNA by reverse engineering. The resurrected antibody was expressed, purified and shown to demonstrate the desired specificity and binding properties in a panel of immuno-biochemical tests. The work described herein demonstrates the powerful combination of high throughput informatic proteomic de novo sequencing with reverse engineering to re-establish monoclonal antibody expressing cells from archived protein sample, exemplifying the development of novel therapeutics from cryptic protein sources.

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“…The framework of the library was based on the single-chain variable fragment (scFv) sequence of the humanized 4D5 antibody (trastuzumab), generated against the protein encoded by ERBB2 (23). This framework was chosen by virtue of its stability on phage and its ease of conversion to a soluble scFv, fragment antigen-binding (Fab), or antibody (22,24). High-resolution crystal structures of the humanized 4D5 have identified the residues within the highly variable complementarity-determining regions (CDRs) that play the most significant role in antigen binding (25).…”

Section: Significancementioning

confidence: 99%

Generation of MANAbodies specific to HLA-restricted epitopes encoded by somatically mutated genes

Skora

Douglass

Hwang

et al. 2015

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

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Mutant epitopes encoded by cancer genes are virtually always located in the interior of cells, making them invisible to conventional antibodies. We here describe an approach to identify single-chain variable fragments (scFvs) specific for mutant peptides presented on the cell surface by HLA molecules. We demonstrate that these scFvs can be successfully converted to full-length antibodies, termed MANAbodies, targeting "Mutation-Associated Neo-Antigens" bound to HLA. A phage display library representing a highly diverse array of single-chain variable fragment sequences was first designed and constructed. A competitive selection protocol was then used to identify clones specific for mutant peptides bound to predefined HLA types. In this way, we obtained two scFvs, one specific for a peptide encoded by a common KRAS mutant and the other by a common epidermal growth factor receptor (EGFR) mutant. The scFvs bound to these peptides only when the peptides were complexed with HLA-A2 (KRAS peptide) or HLA-A3 (EGFR peptide). We converted one scFv to a fulllength antibody (MANAbody) and demonstrate that the MANAbody specifically reacts with mutant peptide-HLA complex even when the peptide differs by only one amino acid from the normal, WT form.antibody engineering | personalized | oncogene | immunotherapy | therapy

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Design and Construction of Synthetic Phage-Displayed Fab Libraries

Cited by 9 publications

References 25 publications

A polar ring endows improved specificity to an antibody fragment

A polar ring endows improved specificity to an antibody fragment

Resurrection of a clinical antibody: Template proteogenomic de novo proteomic sequencing and reverse engineering of an anti‐lymphotoxin‐α antibody

Generation of MANAbodies specific to HLA-restricted epitopes encoded by somatically mutated genes

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