Short constrained peptides derived from phage display libraries as epitope models: the case of mAb 2F5

In this manuscript, we developed a Two-fold Symmetric Linchpin (TSL) that converts readily available phage display peptides libraries made of 20 common amino acids to genetically-encoded libraries of bicyclic peptides displayed on phage. TSL combines an aldehyde-reactive group and two thiol-reactive groups; it bridges two side chains of cysteine [C] with an N-terminal aldehyde group derived from the N-terminal serine [S], yielding a novel bicyclic topology that lacks a free N-terminus. Phage display libraries of SX1CX2X3X4X5X6X7C sequences, where Xi is any amino acids but Cys, were converted to a library of bicyclic TSL-[S]X1[C]X2X3X4X5X6X7[C] peptides in 45 ± 15% yield. Using this library and protein morphogen NODAL as a target, we discovered bicyclic macrocycles that specifically antagonize NODAL-induced signaling in cancer cells. At a 10 µM concentration, two discovered bicyclic peptides completely suppressed NODAL-induced phosphorylation of SMAD2 in P19 embryonic carcinoma. The TSL-[S]Y[C]KRAHKN[C] bicycle inhibited NODAL-induced proliferation of NODAL-Tky-nu ovarian carcinoma cells with apparent IC50 1 µM. The same bicycle at 10 µM concentration did not affect the growth of the control Tky-nu cells. TSL-bicycles remained stable over the course of the 72 hour-long assays in a serum-rich cell-culture medium. We further observed general stability in mouse serum and in a mixture of proteases (PronaseTM) for 33 diverse bicyclic macrocycles of different ring sizes, amino acid sequences, and cross-linker geometries. TSL-constrained peptides expand the previously reported repertoire of phage display bicyclic architectures formed by cross-linking Cys side chains. We anticipate that it will aid the discovery of proteolytically stable bicyclic inhibitors for a variety of protein targets.

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Genetically-Encoded Discovery of Proteolytically Stable Bicyclic Inhibitors of Morphogen NODAL

10.26434/chemrxiv.13870592.v1 ◽

2021 ◽

Author(s):

Jeffrey Wong ◽

Raja Mukherjee ◽

Olena Bilyk ◽

Jiayuan Miao ◽

Vivian Triana Guzman ◽

...

Keyword(s):

Amino Acids ◽

Phage Display ◽

Aldehyde Group ◽

Amino Acid Sequences ◽

Mouse Serum ◽

Side Chains ◽

Protein Targets ◽

Phage Display Libraries ◽

Reactive Groups ◽

Constrained Peptides

In this manuscript, we developed a Two-fold Symmetric Linchpin (TSL) that converts readily available phage display peptides libraries made of 20 common amino acids to genetically-encoded libraries of bicyclic peptides displayed on phage. TSL combines an aldehyde-reactive group and two thiol-reactive groups; it bridges two side chains of cysteine [C] with an N-terminal aldehyde group derived from the N-terminal serine [S], yielding a novel bicyclic topology that lacks a free N-terminus. Phage display libraries of SX1CX2X3X4X5X6X7C sequences, where Xi is any amino acids but Cys, were converted to a library of bicyclic TSL-[S]X1[C]X2X3X4X5X6X7[C] peptides in 45 ± 15% yield. Using this library and protein morphogen NODAL as a target, we discovered bicyclic macrocycles that specifically antagonize NODAL-induced signaling in cancer cells. At a 10 µM concentration, two discovered bicyclic peptides completely suppressed NODAL-induced phosphorylation of SMAD2 in P19 embryonic carcinoma. The TSL-[S]Y[C]KRAHKN[C] bicycle inhibited NODAL-induced proliferation of NODAL-Tky-nu ovarian carcinoma cells with apparent IC50 1 µM. The same bicycle at 10 µM concentration did not affect the growth of the control Tky-nu cells. TSL-bicycles remained stable over the course of the 72 hour-long assays in a serum-rich cell-culture medium. We further observed general stability in mouse serum and in a mixture of proteases (PronaseTM) for 33 diverse bicyclic macrocycles of different ring sizes, amino acid sequences, and cross-linker geometries. TSL-constrained peptides expand the previously reported repertoire of phage display bicyclic architectures formed by cross-linking Cys side chains. We anticipate that it will aid the discovery of proteolytically stable bicyclic inhibitors for a variety of protein targets.

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Screening of potent neutralizing antibodies against SARS-CoV-2 using convalescent patients-derived phage-display libraries

Cell Discovery ◽

10.1038/s41421-021-00295-w ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Yongbing Pan ◽

Jianhui Du ◽

Jia Liu ◽

Hai Wu ◽

Fang Gui ◽

...

Keyword(s):

Phage Display ◽

Neutralizing Antibodies ◽

Variable Region ◽

Neutralizing Antibody ◽

Chain Gene ◽

Prophylactic Efficacy ◽

Heavy Chains ◽

Effective Interventions ◽

Phage Display Libraries

AbstractAs the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to threaten public health worldwide, the development of effective interventions is urgently needed. Neutralizing antibodies (nAbs) have great potential for the prevention and treatment of SARS-CoV-2 infection. In this study, ten nAbs were isolated from two phage-display immune libraries constructed from the pooled PBMCs of eight COVID-19 convalescent patients. Eight of them, consisting of heavy chains encoded by the immunoglobulin heavy-chain gene-variable region (IGHV)3-66 or IGHV3-53 genes, recognized the same epitope on the receptor-binding domain (RBD), while the remaining two bound to different epitopes. Among the ten antibodies, 2B11 exhibited the highest affinity and neutralization potency against the original wild-type (WT) SARS-CoV-2 virus (KD = 4.76 nM for the S1 protein, IC50 = 6 ng/mL for pseudoviruses, and IC50 = 1 ng/mL for authentic viruses), and potent neutralizing ability against B.1.1.7 pseudoviruses. Furthermore, 1E10, targeting a distinct epitope on RBD, exhibited different neutralization efficiency against WT SARS-CoV-2 and its variants B.1.1.7, B.1.351, and P.1. The crystal structure of the 2B11–RBD complexes revealed that the epitope of 2B11 highly overlaps with the ACE2-binding site. The in vivo experiment of 2B11 using AdV5-hACE2-transduced mice showed encouraging therapeutic and prophylactic efficacy against SARS-CoV-2. Taken together, our results suggest that the highly potent SARS-CoV-2-neutralizing antibody, 2B11, could be used against the WT SARS-CoV-2 and B.1.1.7 variant, or in combination with a different epitope-targeted neutralizing antibody, such as 1E10, against SARS-CoV-2 variants.

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Selection of Peptides Targeting Helix 31 of Bacterial 16S Ribosomal RNA by Screening M13 Phage-Display Libraries

Molecules ◽

10.3390/molecules16021211 ◽

2011 ◽

Vol 16 (2) ◽

pp. 1211-1239 ◽

Cited By ~ 18

Author(s):

Tek N. Lamichhane ◽

N. Dinuka Abeydeera ◽

Anne-Cécile E. Duc ◽

Philip R. Cunningham ◽

Christine S. Chow

Keyword(s):

Phage Display ◽

Ribosomal Rna ◽

16S Ribosomal Rna ◽

Phage Display Libraries ◽

M13 Phage ◽

Selection Of

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Peptides derived from phage display libraries as potential neutralizers of Shiga toxin-induced cytotoxicity in vitro and in vivo

Journal of Applied Microbiology ◽

10.1111/jam.12451 ◽

2014 ◽

Vol 116 (5) ◽

pp. 1322-1333 ◽

Cited By ~ 5

Author(s):

R.A. Bernedo-Navarro ◽

M.M. Miyachiro ◽

M.J. da Silva ◽

C.F. Reis ◽

R.A. Conceição ◽

...

Keyword(s):

Phage Display ◽

Shiga Toxin ◽

Phage Display Libraries

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Development of Anti-Infectives Using Phage Display: Biological Agents against Bacteria, Viruses, and Parasites

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00567-12 ◽

2012 ◽

Vol 56 (9) ◽

pp. 4569-4582 ◽

Cited By ~ 50

Author(s):

Johnny X. Huang ◽

Sharon L. Bishop-Hurley ◽

Matthew A. Cooper

Keyword(s):

Phage Display ◽

Significant Contribution ◽

Biological Agents ◽

Preclinical Development ◽

Phage Display Technology ◽

Whole Cells ◽

Drug Candidates ◽

Phage Display Libraries ◽

Wide Range ◽

Display Technology

ABSTRACTThe vast majority of anti-infective therapeutics on the market or in development are small molecules; however, there is now a nascent pipeline of biological agents in development. Until recently, phage display technologies were used mainly to produce monoclonal antibodies (MAbs) targeted against cancer or inflammatory disease targets. Patent disputes impeded broad use of these methods and contributed to the dearth of candidates in the clinic during the 1990s. Today, however, phage display is recognized as a powerful tool for selecting novel peptides and antibodies that can bind to a wide range of antigens, ranging from whole cells to proteins and lipid targets. In this review, we highlight research that exploits phage display technology as a means of discovering novel therapeutics against infectious diseases, with a focus on antimicrobial peptides and antibodies in clinical or preclinical development. We discuss the different strategies and methods used to derive, select, and develop anti-infectives from phage display libraries and then highlight case studies of drug candidates in the process of development and commercialization. Advances in screening, manufacturing, and humanization technologies now mean that phage display can make a significant contribution in the fight against clinically important pathogens.

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Oxford nanopore sequencing enables rapid discovery of single-domain antibodies from phage display libraries

BioTechniques ◽

10.2144/btn-2018-0123 ◽

2018 ◽

Vol 65 (6) ◽

pp. 351-356 ◽

Cited By ~ 1

Author(s):

Michael J Lowden ◽

Kevin A Henry

Keyword(s):

Phage Display ◽

Single Domain ◽

Nanopore Sequencing ◽

Phage Display Libraries ◽

Oxford Nanopore ◽

Single Domain Antibodies

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Generation of Lamprey Monoclonal Antibodies (Lampribodies) Using the Phage Display System

Biomolecules ◽

10.3390/biom9120868 ◽

2019 ◽

Vol 9 (12) ◽

pp. 868 ◽

Cited By ~ 1

Author(s):

Khan M. A. Hassan ◽

John D. Hansen ◽

Brantley R. Herrin ◽

Chris T. Amemiya

Keyword(s):

Monoclonal Antibodies ◽

Phage Display ◽

Genomic Rearrangements ◽

Filamentous Phage ◽

Human Cell Lines ◽

Display System ◽

Phage Display Libraries ◽

Humoral Antibodies ◽

Leucine Rich Repeats ◽

Lymphocyte Receptors

The variable lymphocyte receptors (VLRs) consist of leucine rich repeats (LRRs) and comprise the humoral antibodies produced by lampreys and hagfishes. The diversity of the molecules is generated by stepwise genomic rearrangements of LRR cassettes dispersed throughout the VLRB locus. Previously, target-specific monovalent VLRB antibodies were isolated from sea lamprey larvae after immunization with model antigens. Further, the cloned VLR cDNAs from activated lamprey leukocytes were transfected into human cell lines or yeast to select best binders. Here, we expand on the overall utility of the VLRB technology by introducing it into a filamentous phage display system. We first tested the efficacy of isolating phage into which known VLRB molecules were cloned after a series of dilutions. These experiments showed that targeted VLRB clones could easily be recovered even after extensive dilutions (1 to 109). We further utilized the system to isolate target-specific “lampribodies” from phage display libraries from immunized animals and observed an amplification of binders with relative high affinities by competitive binding. The lampribodies can be individually purified and ostensibly utilized for applications for which conventional monoclonal antibodies are employed.

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