title: Synthesis and secretion of a functional antibody in a vaccinia virus expression system.
abstract: A humanized rat monoclonal antibody (Campath 1H) has been expressed in HeLa cells using recombinant vaccinia viruses. Heavy and light chain recombinant viruses were constructed separately and when grown independently produced proteins of the expected molecular weights. Expressed heavy chain was entirely intracellular but light chain was mainly excreted and processed. When cells were infected at high multiplicity with both heavy and light chain recombinants a proportion of the heavy chain was then found in the extracellular medium. This secreted heavy chain was shown to be associated with light chain as judged by co-electrophoresis in non-reducing SDS polyacrylamide gels and by co-purification on protein-A sepharose. The secreted heavy and light chain complexes were functionally active as an antibody, with activity comparable to authentic Campath 1H antibody as assessed by ELISA, T-cell binding and antigen binding assays. Production of antibody in this system was achieved in the absence of serum, which is an important consideration in the production of monoclonal antibodies (MAbs). The amount of antibody produced was 0.2-0.4 micrograms/10(6) cells without optimization of expression levels. The wide host cell range of vaccinia virus together with the recently developed methods for increasing expression levels make this an attractive candidate as a flexible general vehicle for producing MAbs.
authors.affiliation: Department of Molecular Sciences, Wellcome Research Laboratories, Beckenham, Kent, U.K.
authors.forename: A R
authors.initials: AR
authors.lastname: Carroll
authors.forename: D J
authors.initials: DJ
authors.lastname: Rowlands
authors.forename: B E
authors.initials: BE
authors.lastname: Clarke
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics; D001483:Base Sequence; D001666:Binding Sites, Antibody; D003001:Cloning, Molecular; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007147:Immunoglobulin Light Chains / Q000235:genetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D051381:Rats; D011993:Recombinant Fusion Proteins; D013601:T-Lymphocytes / Q000378:metabolism; D014616:Vaccinia virus / Q000235:genetics
chemical_list: D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins