title: Cloning and expression of human V-genes derived from phage display libraries as fully assembled human anti-TNF alpha monoclonal antibodies.
abstract: BACKGROUND
  With the advent of phage antibody libraries, access to completely human antibody fragments is feasible, either by direct selection from human antibody libraries, or by guided selection. After selection, Fabs and scFvs may need to be expressed as complete antibodies in mammalian cells for further characterisation, or if effector functions are required.
  
  
  OBJECTIVE
  To rebuild and express the human anti-TNF alpha antibody Fab-P3A2 (isolated as a Fab fragment from phage display libraries by guided selection) as a fully assembled, functional human antibody (gamma-1, lambda) in Sp2/0 myeloma cells, and to perform preliminary characterisation studies of the secreted IgG1 molecule. A further objective was to investigate the kinetics of human antibody production and the stability of antibody secretion in transfectomas cultured in various media formulations.
  
  
  METHODS
  A tripartite strategy was employed for cloning heavy chain gene (VH)-P3 and light chain gene V lambda-A2-C lambda into mammalian cell expression vectors p alpha Lys-30 and p alpha Lys-17 respectively. The cell line P3A2.B5 was isolated after co-transfection of Sp2/0 mouse myelomas with the constructs, expanded and weaned into a protein free medium. Fully assembled Ig-P3A2 antibody was purified by Protein A affinity chromatography and characterised with respect to size of antibody chains, and affinity for human TNF alpha. Stability of secretion was investigated by extended serial sub-culture and analysis of P3A2.B5 sub-clones. Strategies of media enrichment were tested for any effect on antibody productivity by selected P3A2.B5 sub-clones.
  
  
  RESULTS
  The cell line P3A2.B5 secreted an assembled, human antibody Ig-P3A2, with heavy and light chains of molecular weight 55 and 28 KD respectively. Equilibrium capture studies showed Ig-P3A2 to have a dissociation constant of approximately 1.5 x 10(-8) M. The mean specific productivity of the cell line increased from 1.2 pg/cell/day to 7.8 pg/cell/day by a combination of medium enrichment and serum reduction. Prolonged serial sub-culture of P3A2.B5 showed the cell line to be unstable with respect to antibody secretion.
  
  
  CONCLUSIONS
  We have outlined a method for expression of human V genes as assembled antibodies in Sp2/0 myeloma cells. A cloning strategy for the stable expression of scFv or Fab genes isolated from phage display libraries as assembled human antibodies of the IgGl subclass in Sp2/0 myeloma cells has been described. For maximising specific productivity of antibody-producing cell lines, supplementation of culture media with glucose, glutamine and amino acids increases antibody yield significantly compared to that in conventional media, indicating the latter is stoichiometrically limiting for production purposes.
authors.affiliation: Department of Biotechnology, University of New South Wales, Kensington, Sydney, Australia. s.mahler@unsw.edu.au
authors.forename: S M
authors.initials: SM
authors.lastname: Mahler
authors.forename: C P
authors.initials: CP
authors.lastname: Marquis
authors.forename: G
authors.initials: G
authors.lastname: Brown
authors.forename: A
authors.initials: A
authors.lastname: Roberts
authors.forename: H R
authors.initials: HR
authors.lastname: Hoogenboom
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D002455:Cell Division; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D019151:Peptide Library; D015183:Restriction Mapping; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D013205:Staphylococcal Protein A; D014409:Tumor Necrosis Factor-alpha