Supplementary Figure 5A and 5B from Therapeutic Implications of a Human Neutralizing Antibody to the Macrophage-Stimulating Protein Receptor Tyrosine Kinase (RON), a c-MET Family Member
Supplementary Figure 1 from Therapeutic Implications of a Human Neutralizing Antibody to the Macrophage-Stimulating Protein Receptor Tyrosine Kinase (RON), a c-MET Family Member
Proc Amer Assoc Cancer Res, Volume 46, 2005
6181
The class III receptor tyrosine kinase FLT3 is an attractive therapeutic target as it is overexpressed in blasts of ∼90% of acute myelogenous leukemia (AML) and the majority of B-lymphoid leukemia patients. Internal tandem duplications (ITDs) in the juxtamembrane region and point mutations in the kinase domain of FLT3 are found in ∼37% of AML patients and are associated with a poor prognosis. We have recently developed a fully human, high affinity monoclonal antibody (EB10) which is readily internalized upon binding to FLT3 receptor on human leukemia cells. In the present study, a novel auristatin conjugate of the anti-FLT3 antibody (EB10-MMAF) was prepared using a dipeptide linker that allows for drug release inside the lysosomes of antigen-positive cells. The MMAF conjugates were stable in buffers and plasma. EB10-MMAF (drug/antibody ratio = 8) retained the binding affinity and internalizing capability of EB10. The conjugate was highly potent, and selectively inhibited the growth of FLT3-expressing leukemia cells with an IC50 of 0.19 nM and 0.08 nM for MV4;11 and BaF3-ITD cells (both positive for FLT3-ITD), 1.11 nM, 6.18 nM and 1.82 nM for REH, EOL-1, EM3 cells (all three positive for wild-type FLT3), and 135 nM for JM1 (negative for FLT3). An MMAF conjugate with a control antibody was not active in these cell lines (IC50s > 5.9 uM). Flow cytometric analysis with annexin V indicated that EB10-MMAF treatment induced apoptosis of leukemia cells in vitro . In vivo treatment with EB10-MMAF strongly inhibited leukemia growth and prolonged survival of mice in both EOL-1 (mean survival time ∼81.0 +/− 46.8 days for the 3 mg/kg group compared to 41.5+/−15 days for the untreated group) and BaF3-ITD (mean survival time ∼91.5 +/− 39.5 days for the 5 mg/kg group compared to 32.8 +/− 5.2 days for the untreated group) leukemia models. In summary, immunoconjugates composed of a fully human anti-FLT3 antibody and a potent auristatin drug may provide a valuable therapeutic approach for AML and other FLT3-positive leukemias. In summary, immunoconjugates composed of a fully human anti-FLT3 antibody and a potent auristatin drug may provide a valuable therapeutic approach for AML and other types of FLT3-positive leukemia.
Vascular endothelial growth factor (VEGF) and its receptors (VEGFR) have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. Here we show that certain "liquid" tumors such as acute myeloid leukemia not only produce VEGF but also express functional VEGFR, resulting in an autocrine loop for tumor growth and propagation. In addition, the leukemia-derived VEGF can also stimulate the production of growth factors, including interleukin 6 (IL6) and granulocyte-macrophage colony stimulating factor (GM-CSF), by human endothelial cells, which in turn further promotes the growth of leukemia cells (the paracrine loop). A fully human anti-VEGFR2 (or kinase insert domain-containing receptor, KDR) antibody, IMC-2C6, strongly blocks KDR/VEGF interaction and neutralizes VEGF-stimulated activation of KDR in endothelial cells. In a system where leukemia cells are co-cultured with endothelial cells, IMC-2C6 inhibits both the production of IL6 and GM-CSF by endothelial cells and the growth of leukemia cells. Finally, IMC-2C6 effectively blocks VEGF-induced migration of KDR+ human leukemia cells, and when administered in vivo, significantly prolonged survival of mice inoculated with KDR+ human leukemia cells. Taken together, our data suggest that anti-KDR antibodies may have broad applications in the treatment of both solid tumors and certain types of leukemia.
Platelet-derived growth factor receptor beta (PDGFRbeta) is upregulated in most of solid tumors. It is expressed by pericytes/smooth muscle cells, fibroblast, macrophage, and certain tumor cells. Several PDGF receptor-related antagonists are being developed as potential antitumor agents and have demonstrated promising antitumor activity in both preclinical and clinical settings. Here, we produced a fully human neutralizing antibody, IMC-2C5, directed against PDGFRbeta from an antibody phage display library. IMC-2C5 binds to both human and mouse PDGFRbeta and blocks PDGF-B from binding to the receptor. IMC-2C5 also blocks ligand-stimulated activation of PDGFRbeta and downstream signaling molecules in tumor cells. In animal studies, IMC-2C5 significantly delayed the growth of OVCAR-8 and NCI-H460 human tumor xenografts in nude mice but failed to show antitumor activities in OVCAR-5 and Caki-1 xenografts. Our results indicate that the antitumor efficacy of IMC-2C5 is primarily due to its effects on tumor stroma, rather than on tumor cells directly. Combination of IMC-2C5 and DC101, an anti-mouse vascular endothelial growth factor receptor 2 antibody, resulted in significantly enhanced antitumor activity in BxPC-3, NCI-H460, and HCT-116 xenografts, compared with DC101 alone, and the trend of additive effects to DC101 treatment in several other tumor models. ELISA analysis of NCI-H460 tumor homogenates showed that IMC-2C5 attenuated protein level of vascular endothelial growth factor and basic fibroblast growth factor elevated by DC101 treatment. Finally, IMC-2C5 showed a trend of additive effects when combined with DC101/chemotherapy in MIA-PaCa-2 and NCI-H460 models. Taken together, these results lend great support to the use of PDGFRbeta antagonists in combination with other antiangiogenic agents in the treatment of a broad range of human cancers.
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.
Abstract First- , second- , and third-generation EGFR tyrosine kinase inhibitors (TKIs) demonstrate profound tumor responses in EGFR mutation-positive NSCLC. Nevertheless, most patients invariably develop acquired resistance. The objective of the present study was to evaluate the effects of dual EGFR blockade using a ligand-blocking antibody (necitumumab) and second- or third-generation TKIs (afatinib or osimertinib) in preclinical NSCLC models with EGFR mutations. Afatinib and osimertinib (AZD9291), second- and third-generation EGFR TKIs, respectively, are irreversible covalent inhibitors of the EGFR tyrosine kinase, with the latter specifically targeting a mutant form of the receptor (EGFR-T790M). We sought to investigate whether combination of necitumumab (LY3012211) with afatinib and osimertinib would provide any benefit over the monotherapies in preclinical mouse models of lung cancer. Using PC-9 and NCI-H1975 xenograft models of NSCLC that harbor EGFR mutations, we observed that dual EGFR blockade of ligand binding and receptor tyrosine kinase activity improved the antitumor efficacy and, more importantly, resulted in durable tumor remissions compared to either single-agent therapy. Additionally, we explored two dosing schedules of necitumumab/osimertinib combination treatment (concurrent versus phased). Concurrent administration of both agents for four weeks was significantly more efficacious compared to the phased schedule (e.g., administration of osimertinib for one week followed by necitumumab and osimertinib combination for additional three weeks). To provide mechanistic insights into the combinatorial activity observed, we employed high-content gene expression analysis using nCounter Pan-Cancer Pathways assay. Pathway analysis of the combination and single-agent treatment groups identified multiple shared and treatment-specific pathway alterations. Genes implicated in FGFR, PI3K pathways and extracellular remodeling appeared to be upregulated by combination and monotherapies and suggested potential common mechanisms of acquired resistance to EGFR inhibitors, whereas compromised DNA repair mechanisms were identified in PC-9 and NCI-H1975 tumors upon treatment with both combinations. In conclusion, these data demonstrate that combination of necitumumab with second- or third-generation EGFR TKIs results in an improved antitumor efficacy compared to the respective monotherapies; a more profound inhibition of EGFR pathway and compromised DNA repair mechanisms may underlie this effect. A phase I clinical trial of necitumumab and osimertinib in EGFR mutation-positive stage IV or recurrent NSCLC who have progressed on a previous EGFR TKI is currently ongoing (NCT02496663). Citation Format: Amelie Forest, Erik R. Rasmussen, Thompson N. Doman, Michael Amatulli, Rajiv Bassi, Gerald E. Hall, Jason R. Manro, Manisha Brahmachary, David Surguladze, Yung-mae M. Yao, Michael D. Kalos, Ruslan D. Novosiadly. Combination of necitumumab with second- or third-generation EGFR tyrosine kinase inhibitors downregulates DNA repair mechanisms and induces durable tumor remissions in xenograft NSCLC models with EGFR mutations [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A160.
Supplementary Figure 1 from Therapeutic Implications of a Human Neutralizing Antibody to the Macrophage-Stimulating Protein Receptor Tyrosine Kinase (RON), a c-MET Family Member
<div>Abstract<p>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 <i>in vivo</i> 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 (ED<sub>50</sub> = 0.15 nmol/L) to RON and effectively blocks interaction with its ligand, macrophage-stimulating protein (MSP; IC<sub>50</sub> = 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 ∼100 cancer cell lines and ∼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. (Cancer Res 2006; 66(18): 9162-70)</p></div>
