Daniel S. Pereira

Therapeutic implications of a human neutralizing antibody to the macrophage-stimulating protein receptor tyrosine kinase (RON), a c-MET family member.

RON is a member of the c-MET receptor tyrosine kinase family. Like c-MET, RON is expressed by a variety of epithelial-derived tumors and cancer cell lines and it is thought to play a functional role in tumorigenesis. To date, antagonists of RON activity have not been tested in vivo to validate RON as a potential cancer target. In this report, we used an antibody phage display library to generate IMC-41A10, a human immunoglobulin G1 (IgG1) antibody that binds with high affinity (ED50 = 0.15 nmol/L) to RON and effectively blocks interaction with its ligand, macrophage-stimulating protein (MSP; IC50 = 2 nmol/L). We found IMC-41A10 to be a potent inhibitor of receptor and downstream signaling, cell migration, and tumorigenesis. It antagonized MSP-induced phosphorylation of RON, mitogen-activated protein kinase (MAPK), and AKT in several cancer cell lines. In HT-29 colon, NCI-H292 lung, and BXPC-3 pancreatic cancer xenograft tumor models, IMC-41A10 inhibited tumor growth by 50% to 60% as a single agent, and in BXPC-3 xenografts, it led to tumor regressions when combined with Erbitux. Western blot analyses of HT-29 and NCI-H292 xenograft tumors treated with IMC-41A10 revealed a decrease in MAPK phosphorylation compared with control IgG-treated tumors, suggesting that inhibition of MAPK activity may be required for the antitumor activity of IMC-41A10. To our knowledge, this is the first demonstration that a RON antagonist and specifically an inhibitory antibody of RON negatively affects tumorigenesis. Another major contribution of this report is an extensive analysis of RON expression in approximately 100 cancer cell lines and approximately 300 patient tumor samples representing 10 major cancer types. Taken together, our results highlight the potential therapeutic usefulness of RON activity inhibition in human cancers.

Involvement of the VEGF receptor 3 in tubular morphogenesis demonstrated with a human anti-human VEGFR-3 monoclonal antibody that antagonizes receptor activation by VEGF-C

In this report we utilize a novel antagonist antibody to the human VEGFR-3 to elucidate the role of this receptor in in vitro tubular morphogenesis of bovine and human endothelial cells (EC cells) induced by VEGF-C. The antibody hF4-3C5 was obtained by panning a human phage display library on soluble human VEGFR-3. The binding affinity constant of hF4-3C5 significantly exceeds that of the interaction of VEGFR-3 with VEGF-C. hF4-3C5 strongly inhibits the binding of soluble VEGFR-3 to immobilized VEGF-C and abolishes the VEGF-C-mediated mitogenic response of cells that expresses a chimeric human VEGFR-3-cFMS receptor. In fluorescence experiments, hF4-3C5 reactivity is observed with human lymphatic endothelial cells (LECs) and human umbilical vein endothelial cells (HUVECs). Binding of hF4-3C5 shows that about half of bovine aortic endothelial (BAE) cells express VEGFR-3 and cells in this subpopulation are primarily responsible for the chemotactic response to the mature form of VEGF-C (VEGF-CΔNΔC). This response was strongly inhibited by the addition of hF4-3C5. In vitro tube formation by BAE cells induced by VEGF-CΔNΔC was reduced by greater than 60% by hF4-3C5 whereas the response to VEGF165 was unaffected. Addition of hF4-3C5 together with an antagonist antibody to VEGFR-2 completely abolished the response to VEGF-CΔNΔC. Similar results were obtained with HUVECs. Together, these findings point to a role for VEGFR-3 in vascular tubular morphogenesis and highlight the utility of hF4-3C5 as a tool for the investigation of the biology of VEGFR-3.

Overexpression of the h-ras oncogene in primary primitive human hematopoietic cells alters proliferation and differentiation

Abstract The Ras pathway plays a central role in the control of cellular proliferation and differentiation. In order to examine the role of Ras signaling in hematopoietic regulation, we transduced CD34 + -enriched human cord blood cells with an MSCV-based retroviral vector encoding R12 activated H-Ras. The effects upon proliferation and differentiation were then examined in vitro. Compared to cells transduced with a control vector, H-Ras expressing cells had an elevated frequency of CFU-M (76 ± 16% vs. 22 ± 13%) and increased monocyte/macrophage cell frequency in long-term suspension cultures (69 ± 10% CD14 + vs. 28 ± 8% CD14 + ). H-Ras expression also impaired CFC survival/expansion in suspension culture relative to controls. To determine the effects of decreasing the levels of H-Ras signaling, the farnesyltransferase inhibitor 66177 (Schering-Plough) was used. At low doses, the inhibitor did not significantly reduce the frequency of monocyte/macrophage lineage cells, but led to the appearance of primitive/blastic monocytic colonies and cells with extended proliferative and self-renewal capacities. Upon removal of the inhibitor, these cells rapidly differentiated into large, adherent macrophages. These results suggest that the level of Ras pathway signaling is an important determinant of myeloid cell fate, and may illustrate a manner in which Ras activation in primitive hematopoietic cells contributes to leukemogenesis.