Selection of monoclonal antibodies which induce internalization and phosphorylation of p185HER2 and growth inhibition of cells with HER2/NEU gene amplification.
In order to obtain further information on the biological role of the HER2/neu oncoprotein monoclonal antibodies (MAbs) were produced against the p185 extracellular domain. To immunize the mice and screen the hybridoma supernatants we selected a lung adenocarcinoma cell line (Calu-3), which demonstrated an over-expression of p185HER2 measured as the reactivity with polyclonal rabbit serum to the 14-amino-acid carboxy-terminal-HER2/neu. Two MAbs, designated MGR2 (IgG1) and MGR3 (IgG2), selected for reactivity on Calu-3 and negativity on A43I live cells, the reference target cell for EGF receptor expression, were found to immunoprecipitate a 185-kDa molecule. Immunodepletion experiments with the polyclonal antiserum and cross-competition experiments indicated that the 2 reagents recognized 2 different epitopes located on the p185HER2 molecule. One of the 2 MAbs, MGR3, was found to internalize, induce p185HER2 phosphorylation and inhibit tumor cell growth in vitro. These results indicate that MGR3 is directed against a determinant located in the p185HER2 ligand binding site and may compete with the p185HER2 ligand, but is incapable of inducing a complete mitotic signal.
Tagliabue
E
E
Division of Experimental Oncology E, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy.
Centis
F
F
Campiglio
M
M
Mastroianni
A
A
Martignone
S
S
Pellegrini
R
R
Casalini
P
P
Lanzi
C
C
Ménard
S
S
Colnaghi
M I
MI
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification / Q000494:pharmacology*; D001692:Biological Transport; D002455:Cell Division; D002460:Cell Line; D005784:Gene Amplification; D006801:Humans; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D009369:Neoplasms; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D011519:Proto-Oncogenes; D018719:Erb-b2 Receptor Tyrosine Kinases
D000911:Antibodies, Monoclonal; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases