title: In vitro and in vivo characterisation of a recombinant carboxypeptidase G2::anti-CEA scFv fusion protein.
abstract: BACKGROUND
  There is considerable interest in the specific targeting of therapeutic agents to cancer cells. Of particular promise is a technique known as Antibody-Directed Enzyme Prodrug Therapy (ADEPT). In this approach an enzyme is targeted to the tumour by its conjugation to a tumour specific-antibody tumour. After allowing sufficient time for the conjugate to localise at the tumour and clear from the circulatory system, a relatively non-toxic prodrug is administered. This prodrug is converted to a highly cytotoxic drug by the action of the targeted enzyme localised at the tumour site.
  
  
  OBJECTIVE
  To construct gene fusions between the pseudomonad carboxypeptidase G2 (CPG2) gene and DNA encoding MFE-23 (an anti-carcinoembryonic antigen (CEA) single-chain Fv (scFv) molecule), derived from a phage display library. To overexpress the resultant gene fusions in Escherichia coli, and assess the in vitro and in vivo properties of the purified fusion proteins.
  
  
  METHODS
  To introduce unique cloning restriction sites into the 5'-end of the CPG2 gene by site-directed mutagenesis to facilitate fusion to the 3'-end of the gene encoding MFE-23 (constructs with or without a flexible (Gly4Ser)3 linker-encoding sequence were designed). To overexpress the resultant gene fusions under transcriptional control of the lac promoter and to direct the fusion proteins produced to the periplasmic space of E. coli through translational coupling to the pelB signal peptide.
  
  
  RESULTS
  Biologically active recombinant CPG2::MFE-23 scFv fusion proteins were produced in E. coli and shown to possess enzyme and anti-CEA activity. Affinity chromatography followed by size exclusion gel filtration yielded approximately 0.7-1.4 mg/l from shake flask culture. The fusion protein in which the enzyme and antibody moieties were joined by a linker peptide was shown to be effectively localised in nude mice bearing human colon tumour xenografts, giving favourable tumour to blood ratios.
  
  
  CONCLUSIONS
  MFE-23 scFv serves as an ideal candidate for the antibody arm of a bacterially expressed fusion protein with CPG2. The biological properties of this recombinant protein suggest that it may be employed for tumour specific prodrug activation. However, further assessment of its stability and pharmokinetics is required if genetic fusion is to be considered as an alternative to chemical conjugation.
authors.affiliation: Department of Molecular Microbiology, Centre for Applied Microbiology and Research, Wiltshire, UK.
authors.forename: N P
authors.initials: NP
authors.lastname: Michael
authors.forename: K A
authors.initials: KA
authors.lastname: Chester
authors.forename: R G
authors.initials: RG
authors.lastname: Melton
authors.forename: L
authors.initials: L
authors.lastname: Robson
authors.forename: W
authors.initials: W
authors.lastname: Nicholas
authors.forename: J A
authors.initials: JA
authors.lastname: Boden
authors.forename: R B
authors.initials: RB
authors.lastname: Pedley
authors.forename: R H
authors.initials: RH
authors.lastname: Begent
authors.forename: R F
authors.initials: RF
authors.lastname: Sherwood
authors.forename: N P
authors.initials: NP
authors.lastname: Minton
mesh_terms: D000818:Animals; D019677:Artificial Gene Fusion; D015153:Blotting, Western; D002272:Carcinoembryonic Antigen / Q000235:genetics* / Q000276:immunology; D002846:Chromatography, Affinity; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003090:Coliphages / Q000235:genetics; D015179:Colorectal Neoplasms / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015723:Gene Library; D005998:Glycine / Q000235:genetics; D006801:Humans; D007763:Lac Operon; D051379:Mice; D008819:Mice, Nude; D016297:Mutagenesis, Site-Directed; D009368:Neoplasm Transplantation; D011133:Polysaccharide-Lyases / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D012694:Serine / Q000235:genetics; D014183:Transplantation, Heterologous; D011623:gamma-Glutamyl Hydrolase / Q000235:genetics* / Q000378:metabolism
chemical_list: D002272:Carcinoembryonic Antigen; D011993:Recombinant Fusion Proteins; D012694:Serine; D011623:gamma-Glutamyl Hydrolase; D011133:Polysaccharide-Lyases; C074124:pectin lyase B; D005998:Glycine