Abstract
BackgroundOverexpression of the RON receptor tyrosine kinase contributes to epithelial cell transformation, malignant progression, and acquired drug resistance. RON also has been considered as a potential target for therapeutic intervention. This study determines biochemical features and inhibitory activity of a mouse monoclonal antibody (mAb) Zt/f2 in experimental cancer therapy.ResultsZt/f2 is a mouse IgG2a mAb that is highly specific and sensitive to human RON and its oncogenic variants such as RON160 (ED50 = 2.3 nmol/L). Receptor binding studies revealed that Zt/f2 interacts with an epitope(s) located in a 49 amino acid sequence coded by exon 11 in the RON β-chain extracellular sequences. This sequence is critical in regulating RON maturation and phosphorylation. Zt/f2 did not compete with ligand macrophage-stimulating protein for binding to RON; however, its engagement effectively induced RON internalization, which diminishes RON expression and impairs downstream signaling activation. These biochemical features provide the cellular basis for the use of Zt/f2 to inhibit tumor growth in animal model. Repeated administration of Zt/f2 as a single agent into Balb/c mice results in partial inhibition of tumor growth caused by transformed NIH-3T3 cells expressing oncogenic RON160. Colon cancer HT-29 cell-mediated tumor growth in athymic nude mice also was attenuated following Zt/f2 treatment. In both cases, ~50% inhibition of tumor growth as measured by tumor volume was achieved. Moreover, Zt/f2 in combination with 5-fluorouracil showed an enhanced inhibition effect of ~80% on HT-29 cell-mediated tumor growth in vivo.ConclusionsZt/f2 is a potential therapeutic mAb capable of inhibiting RON-mediated oncogenesis by colon cancer cells in animal models. The inhibitory effect of Zt/f2 in vivo in combination with chemoagent 5-fluorouracil could represent a novel strategy for future colon cancer therapy.
Highlights
Overexpression of the RON receptor tyrosine kinase contributes to epithelial cell transformation, malignant progression, and acquired drug resistance
Immunofluorescent analysis indicated that Zt/f2 binds to an epitope(s) on RON extracellular sequences (Figure 1A) and displays similar binding affinity as Zt/g4 at a defined concentration
It binds to an epitope(s) on the RON extracellular sequences, which differs from macrophage-stimulating protein (MSP) and Zt/g4
Summary
Overexpression of the RON receptor tyrosine kinase contributes to epithelial cell transformation, malignant progression, and acquired drug resistance. Fortynine amino acids (from Tyr884 to Gln930) in this sequence are coded by exon 11, which often is deleted through the splicing process [23,24] This deletion results in formation of a single-chain precursor RON165, which is retained in cytoplasm [23,24]. Considering the importance of extracellular domains in ligand binding, receptor maturation, and activation, it is believed that biological or chemical agents that interact with these domains should regulate RON activation and control its downstream signaling events. Such studies should provide a basis for the development of potential therapeutics designed to inhibit RONmediated tumorigenesis
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