Subject(s)
CD4 Antigens/history , CD8 Antigens/history , Intracellular Membranes/enzymology , Intracellular Membranes/immunology , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/history , Animals , CD4 Antigens/chemistry , CD4 Antigens/physiology , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/enzymology , CD4-Positive T-Lymphocytes/immunology , CD8 Antigens/chemistry , CD8 Antigens/physiology , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/enzymology , CD8-Positive T-Lymphocytes/immunology , Cell Communication/immunology , History, 20th Century , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/chemistry , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/physiology , Major Histocompatibility Complex/immunology , Mice , Mice, Inbred C57BL , Signal Transduction/immunologyABSTRACT
The members of the epidermal growth factor receptor (EGFR) family are over expressed in a variety of malignancies and are frequently linked to aggressive disease and a poor prognosis. Although clinically effective monoclonal antibodies (MAbs) have been developed to target HER2 and EGFR, the remaining two family members, HER3 and HER4, have not been the subject of significant efforts. In this paper, we have taken the initial steps required to generate antibodies with potential clinically utility that target the members of the EGFR family. The genes for the extracellular domains (ECDs) of all four members of the EGFR family were cloned and used to stably transfect 293 (HEK) cells. Milligram quantities of each ECD were produced and characterized. The HER3, HER4, and EGFR ECDs were then employed as targets for the selection of antibodies from naïve human scFv (single-chain Fv) phage display libraries. Six unique scFv clones were isolated that bound specifically to HER3, 13 unique clones were isolated with specificity for HER4 and 52 unique anti-EGFR clones were isolated. These scFvs provide a valuable and potentially clinically relevant panel of agents to target the members of the EGFR family.
Subject(s)
ErbB Receptors/genetics , Immunoglobulin Variable Region/isolation & purification , Base Sequence , Cell Line , Cell Line, Tumor , Cloning, Molecular , DNA Primers , Female , Humans , Kidney , Molecular Sequence Data , Ovarian Neoplasms , Receptor, ErbB-4 , TransfectionABSTRACT
Antitumor diabody molecules are noncovalent single-chain Fv dimers that recapitulate the divalent binding properties of native IgG antibodies. Diabodies are capable of substantial accumulation in tumor xenografts expressing relevant antigens in immunodeficient mouse models. With a Mr of approximately 55,000, diabodies are rapidly cleared from the circulation, resulting in tumor-to-blood ratios that significantly exceed those achieved early after the administration of monoclonal antibodies. We have evaluated the therapeutic potential of the beta-emitting isotope yttrium-90 (t1/2, 64 hours) conjugated to the C6.5K-A diabody that specifically targets the HER2/neu human tumor-associated antigen. We have found that a single intravenous dose of 150 microCi (200 microg) 90Y-CHX-A"-C6.5K-A diabody substantially inhibits the growth rates of established MDA-361/DYT2 human breast tumor xenografts in athymic nude mice. In contrast, 300 microCi (300 microg) 90Y-CHX-A"-C6.5K-A diabody resulted in only a minor delay in the growth of SK-OV-3 human ovarian cancer xenografts. The maximum tolerated dose was also dependent on the tumor xenograft model used. These studies indicate that genetically engineered antitumor diabody molecules can be used as effective vehicles for radioimmunotherapy.
