Human single-domain neutralizing intrabodies directed against Etk kinase: a novel approach to impair cellular transformation.
Etk, the 70-kDa member of the Tec family of nonreceptor protein tyrosine kinases, is expressed in a variety of hematopoietic, epithelial, and endothelial cells and was shown to be involved in several cellular processes, including proliferation, differentiation, and motility. In this study, we describe a novel approach using a human single-domain antibody phage display library for the generation of intrabodies directed against Etk. These single-domain antibodies bind specifically to recombinant Etk and efficiently block its kinase activity. When expressed in transformed cells, these antibodies associated tightly with Etk, leading to significant blockade of Etk enzymatic activity and inhibition of clonogenic cell growth in soft agar. Our results indicate that Etk may play a role in Src-induced cellular transformation and thus may represent a good target for cancer intervention. Furthermore, our single-domain antibody-based intrabody system proves to be an excellent tool for future intracellular targeting of other signaling molecules.
Paz
Keren
K
Department of Antibody Technology and Protein Sciences, ImClone Systems, 180 Varick Street, New York, New York 10014, USA. keren.paz@imclone.com
Brennan
Laura A
LA
Iacolina
Michelle
M
Doody
Jacqueline
J
Hadari
Yaron R
YR
Zhu
Zhenping
Z
D000362:Agar / Q000737:chemistry; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D015153:Blotting, Western; D002454:Cell Differentiation; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic / Q000378:metabolism*; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D041681:NIH 3T3 Cells; D019151:Peptide Library; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000502:physiology*; D034622:RNA Interference; D011994:Recombinant Proteins / Q000737:chemistry; D015398:Signal Transduction; D014162:Transfection
D000970:Antineoplastic Agents; D019151:Peptide Library; D011994:Recombinant Proteins; D000362:Agar; D004247:DNA; D005982:Glutathione Transferase; C492367:BMX protein, human; D011505:Protein-Tyrosine Kinases