title: IgM secretory tailpiece drives multimerisation of bivalent scFv fragments in eukaryotic cells.
abstract: BACKGROUND
  The monoclonal antibody (mAb) TP-3 binds selectively to human and canine osteosarcoma (OS) cells and is therefore a potential candidate for use as a targeting agent in radioimmunoimaging and therapy of OS metastases. However, intact murine mAbs have several drawbacks such as large size, delayed blood clearance and high immunogenicity, all of which can be overcome by genetic engineering.
  
  
  OBJECTIVE
  To construct and express bivalent and multivalent TP-3 scFv fragments from the mammalian expression vector, pLNO. This vector has unique restriction sites for simple cassette cloning of any individual variable (V) and constant (C) genes and has previously been used for expression of intact chimeric TP-3 mAbs and Fab fragments. Furthermore, it is also suitable for expression of any modified V region, such as a scFv fragment, fused to any modified C region or to non-immunoglobulin protein sequences.
  
  
  METHODS
  Six different constructs were made; three scFv-CH3 fragments that differed in the design of linker between the scFv fragment and the IgG CH3 domain. These constructs were also made with the IgM secretory tailpiece (microtp) attached to the C terminus.
  
  
  RESULTS
  All constructs were secreted as bivalent antibody fragments with a molecular weight of about 100 kDa. A band corresponding to a dimer appeared in all the supernatants from TP-3 scFv-CH3 producing cells, whether microtp was present or not, whereas higher orders of multimers were not seen. However, pulse chase analyses of the cells revealed that a small fraction of higher order polymers was formed from genes including the fragment encoding microtp and that microtp conferred retention both to monomers and intermediate polymers. The recombinant TP-3 antibody fragments were shown to bind human OS cells.
  
  
  CONCLUSIONS
  Recombinant mAb fragments can be designed and cloned into the mammalian expression vector, pLNO. This vector is flexible in the sense that the genes encoding such fragments can be expressed from either cDNA or from genomic DNA. A microtp attached to the CH3 domain in these fragments was sufficient to drive polymerization, however inefficiently and intracellular retention of both monomers and intermediate polymers was observed.
authors.affiliation: Department of Biology, University of Oslo, Norway.
authors.forename: T
authors.initials: T
authors.lastname: Olafsen
authors.forename: I B
authors.initials: IB
authors.lastname: Rasmussen
authors.forename: L
authors.initials: L
authors.lastname: Norderhaug
authors.forename: O S
authors.initials: OS
authors.lastname: Bruland
authors.forename: I
authors.initials: I
authors.lastname: Sandlie
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018033:Antibodies, Bispecific / Q000276:immunology / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D001859:Bone Neoplasms / Q000276:immunology; D019281:Dimerization; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005803:Genes, Immunoglobulin; D005818:Genetic Engineering; D005822:Genetic Vectors; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D007075:Immunoglobulin M / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D012516:Osteosarcoma / Q000276:immunology; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism; D012635:Secretory Component / Q000378:metabolism*; D014162:Transfection
chemical_list: D018033:Antibodies, Bispecific; D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007075:Immunoglobulin M; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D012635:Secretory Component