High-throughput Generation of Synthetic Antibodies from Highly Functional Minimalist Phage-displayed Libraries

Fellouse, Frédéric A.; Esaki, Kaori; Birtalan, Sara; Raptis, Demetrios A.; Cancasci, Vincenzo J.; Koide, Akiko; Jhurani, Parkash; Vasser, Mark; Wiesmann, Christian; Kossiakoff, Anthony A.; Koide, Shohei; Sidhu, Sachdev S.

doi:10.1016/j.jmb.2007.08.005

Cited by 327 publications

(389 citation statements)

References 47 publications

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“…The phage library was constructed and screened for scFvs specific for mutant KRAS and EGFR peptides bound to HLA molecules using a series of sequential positive and negative selections based on ref. 26 with additional steps of competitive binding to enhance specificity. Free scFvs and a full-length antibody were then generated based on sequences from the candidate phage-displayed scFvs (23).…”

Section: Methodsmentioning

confidence: 99%

“…Free scFvs and a full-length antibody were then generated based on sequences from the candidate phage-displayed scFvs (23). Candidate phage, free scFvs, and the full-length antibody were analyzed by ELISA, flow cytometry after cell staining, and complement-dependent cytotoxicity (26,44).…”

Section: Methodsmentioning

confidence: 99%

“…This allowed us to focus variability on the most important residues for antigen binding rather than backbone residues. In our library, amino acid substitutions were limited to defined paratope residues in four CDRs: CDR-L3, CDR-H1, CDR-H2, and CDR-H3 (26). We skewed the library to include specific amino acids within the CDRs that either were previously demonstrated to play a significant role in antigen binding or were enriched in naturally occurring antibodies (SI Appendix, Table S1).…”

Section: Significancementioning

confidence: 99%

“…We skewed the library to include specific amino acids within the CDRs that either were previously demonstrated to play a significant role in antigen binding or were enriched in naturally occurring antibodies (SI Appendix, Table S1). One important randomization was at CDR-L3, which contained a mixture of serines and tyrosines, two amino acids previously shown to facilitate a minimalist approach to library design (26,27). CDRs in the heavy chain have been shown to play a more significant role in antigen-binding diversity (28)(29)(30), and we therefore introduced more degeneracy in the heavy-chain CDRs than in the light-chain CDRs (SI Appendix, Table S1).…”

Section: Significancementioning

confidence: 99%

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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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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Section: Significancementioning

confidence: 99%

See 2 more Smart Citations

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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“…2 Numerous antibody fragment libraries for phage display have been created, and the creation of new libraries continues as knowledge and technology expands the possibilities. [3][4][5][6][7][8][9][10] Improvements concern stability and functionality of the various antibody fragments themselves, regulation of the expression, the fusion partner utilized in the display system and not least the peptide tag sequences applied (Fig. 1).…”

Section: Introductionmentioning

confidence: 99%

Degradation of C-terminal tag sequences on domain antibodies purified fromE. colisupernatant

Lykkemark

Mandrup

Friis

et al. 2014

mAbs

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The Use of Phage Display in Neurobiology

Bradbury

2010

CP Neuroscience

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Phage display has been extensively used to study protein-protein interactions, receptor- and antibody-binding sites, and immune responses, to modify protein properties, and to select antibodies against a wide range of different antigens. In the format most often used, a polypeptide is displayed on the surface of a filamentous phage by genetic fusion to one of the coat proteins, creating a chimeric coat protein, and coupling phenotype (the protein) to genotype (the gene within). As the gene encoding the chimeric coat protein is packaged within the phage, selection of the phage on the basis of the binding properties of the polypeptide displayed on the surface simultaneously results in the isolation of the gene encoding the polypeptide. This unit describes the background to the technique, and illustrates how it has been applied to a number of different problems, each of which has its neurobiological counterparts. Although this overview concentrates on the use of filamentous phage, which is the most popular platform, other systems are also described.

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High-throughput Generation of Synthetic Antibodies from Highly Functional Minimalist Phage-displayed Libraries

Cited by 327 publications

References 47 publications

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

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

Degradation of C-terminal tag sequences on domain antibodies purified fromE. colisupernatant

The Use of Phage Display in Neurobiology

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