Production and binding analyses of a humanised scFv against a cryptic epitope on tumour-associated fibronectin

Nor Adzimah Johdi, Ruth Harman, Irene Sanjuan, Christina Kousparou, Nigel Courtenay-Luck, Mahendra P. Deonarain

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Tumour-associated splice variants of fibronectin are a major source of tumour-matrix associated targets and are proving very successful in the development of clinical agents to treat cancer. One of the first monoclonal antibodies to be produced to this target, murine BC-1, recognises a cryptic epitope in domain 7 of the B-form splice variant (EDB-FN). Antibody fragments based on this immunoglobulin (IgG) were unstable, but BC-1 humanisation provided an opportunity to produce a more stable single-chain Fv (scFv). The variable domains of the humanized BC-1 IgG were sub-cloned and constructed into a scFv (HuBC-1 scFv) which was successfully expressed in Escherichia coli. The scFv retained its conformationally-sensitive epitope recognition and demonstrated a good affinity to the target of around 50 nM as measured by ELISA, Surface Plasmon Resonance and Flow Cytometry. Furthermore, the scFv was thermostable and stable in serum allowing substantial localisation to human tumours grown in mouse xenograft models. This scFv could form the basis of future tumour-specific biopharmaceuticals.

Original languageEnglish
Pages (from-to)157-163
Number of pages7
JournalProtein Expression and Purification
Volume88
Issue number1
DOIs
Publication statusPublished - 2013

Fingerprint

Single-Chain Antibodies
Fibronectins
Epitopes
Neoplasms
Immunoglobulin G
Immunoglobulin Fragments
Surface Plasmon Resonance
Heterografts
Immunoglobulins
Flow Cytometry
Enzyme-Linked Immunosorbent Assay
Monoclonal Antibodies
Escherichia coli
Serum

Keywords

  • Binding
  • EDB-fibronectin
  • Flow cytometry
  • Humanized
  • Single-chain Fv
  • Surface plasmon resonance

ASJC Scopus subject areas

  • Biotechnology

Cite this

Production and binding analyses of a humanised scFv against a cryptic epitope on tumour-associated fibronectin. / Johdi, Nor Adzimah; Harman, Ruth; Sanjuan, Irene; Kousparou, Christina; Courtenay-Luck, Nigel; Deonarain, Mahendra P.

In: Protein Expression and Purification, Vol. 88, No. 1, 2013, p. 157-163.

Research output: Contribution to journalArticle

Johdi, Nor Adzimah ; Harman, Ruth ; Sanjuan, Irene ; Kousparou, Christina ; Courtenay-Luck, Nigel ; Deonarain, Mahendra P. / Production and binding analyses of a humanised scFv against a cryptic epitope on tumour-associated fibronectin. In: Protein Expression and Purification. 2013 ; Vol. 88, No. 1. pp. 157-163.
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