Bacterial expression and characterization of recombinant biologically active anti-tyrosine kinase receptor antibody forms.
Homomeric and heteromeric interactions among cell-surface tyrosine kinase receptors belonging to the ErbB family lead to intracellular signaling cascades which are involved in cell activation, cytoskeletal interactions, and cellular transformation leading to neoplasia. Monoclonal antibodies which specifically bind to p185neu or epidermal growth factor receptor (EGFR), such as 7.16.4 and 225, respectively, can elicit tumor growth-inhibitory effects on transformed cells which overexpress either or both of these receptors. In order to better understand these receptor-receptor and receptor-antibody interactions and to gain insights that may be useful in the production and design of an antibody-based anticancer therapeutic, novel small recombinant 7.16.4 and 225 single-chain Fv fragments (scFv) were constructed, expressed, and characterized. We showed that these recombinant antibody fragments, which retain binding affinity, can be produced and purified from bacterial cell lysates. Our analyses further demonstrate that fusion of a 61 amino-acid dimerization domain with 7.16.4 and 225 scFv (7.16.4hth and 225hth) is sufficient to restore biological activity to these recombinant proteins.
Peterson
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NC
Department of Pathology & Laboratory Medicine, University of Pennsylvania, Philadelphia 19104, USA.
Greene
M I
MI
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D001419:Bacteria / Q000235:genetics; D002460:Cell Line; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D015536:Down-Regulation; D066246:ErbB Receptors / Q000276:immunology*; D051379:Mice; D011956:Receptors, Cell Surface / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D066246:ErbB Receptors