A humanized single-chain variable fragment antibody against beta3 integrin in Escherichia coli. Academic Article uri icon

Overview

MeSH

  • Animals
  • Blood Platelets
  • Cloning, Molecular
  • Codon, Terminator
  • Electrophoresis, Polyacrylamide Gel
  • Flow Cytometry
  • Humans
  • Integrin beta3
  • Mice
  • Mice, Inbred C57BL
  • Mutagenesis, Site-Directed
  • Onium Compounds
  • Oxidation-Reduction
  • Peptide Library
  • Plasmids
  • Platelet Activation
  • Recombinant Proteins
  • Solubility

MeSH Major

  • Antibodies, Monoclonal, Humanized
  • Antibody Specificity
  • Escherichia coli
  • Peptide Fragments
  • Platelet Glycoprotein GPIIb-IIIa Complex
  • Single-Chain Antibodies

abstract

  • Patients with HIV-1 immune-related thrombocytopenia (HIV-1-ITP) have a unique antibody (Ab) against platelet GPIIIa49-66, which is capable of inducing oxidative platelet fragmentation in the absence of complement activation. By screening a human phage antibody library with the GPIIIa49-66 peptide as bait, we have developed several humanized phage Abs, which act similarly to the parental Ab. However, the presence of a stop codon in the heavy chain of the obtained phage clones limits their expression in soluble recombinant form. To circumvent this problem, we mutated the stop codon inside clone 11 that exhibits the highest binding activity to platelet GPIIIa49-66, resulting in a soluble scFv format (named A11) in Escherichia coli Rosseta. In in vitro binding assay, A11 exhibited similar binding specificity to parental Ab at various concentrations. Moreover, A11 is able to induce oxidative platelet fragmentation by preferentially binding to activated versus resting platelets. These findings provide a proof-of-principle for the development of a novel approach to inhibit arterial thrombosis by generating a selective scFv for the lysis of platelet-rich thrombi.

publication date

  • December 2011

has subject area

  • Animals
  • Antibodies, Monoclonal, Humanized
  • Antibody Specificity
  • Blood Platelets
  • Cloning, Molecular
  • Codon, Terminator
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli
  • Flow Cytometry
  • Humans
  • Integrin beta3
  • Mice
  • Mice, Inbred C57BL
  • Mutagenesis, Site-Directed
  • Onium Compounds
  • Oxidation-Reduction
  • Peptide Fragments
  • Peptide Library
  • Plasmids
  • Platelet Activation
  • Platelet Glycoprotein GPIIb-IIIa Complex
  • Recombinant Proteins
  • Single-Chain Antibodies
  • Solubility

Research

keywords

  • Journal Article

Identity

Language

  • eng

PubMed Central ID

  • PMC3278807

Digital Object Identifier (DOI)

  • 10.1089/hyb.2011.0056

PubMed ID

  • 22149280

Additional Document Info

start page

  • 543

end page

  • 548

volume

  • 30

number

  • 6