Activation Of c-Met Pathway Research Articles

An Agonistic Monoclonal Antibody Targeting cMet Attenuates Inflammation and Up-Regulates Collagen Synthesis and Angiogenesis in Type 2 Diabetic Mice Wounds.

Diabetic wounds account for 25 to 50 percent of total diabetic health care costs annually, and present overall healing rates of less than 50 percent. Because delayed diabetic wound healing is associated with impaired fibroblast function, the authors hypothesize that tyrosine kinase Met (cMet) agonistic monoclonal antibody will promote diabetic wound healing by means of stable activation of hepatocyte growth factor/cMet signaling. Two 6-mm dorsal wounds were created in each mouse (6-week-old, male BKS.Cg-Dock7 m +/+Lepr db /J; n = 5). After subcutaneous injections of agonist (20 mg/kg) at 0 and 72 hours, the wound sizes were measured at days 0, 1, 3, 6, and 10. Histologic and immunohistochemical analyses were performed at day 10 (cMet, α-smooth muscle actin, CD68, and transforming growth factor-β). In vitro cytotoxicity and migration tests with diabetic fibroblasts were performed with or without agonist treatment (1 or 10 nM). cMet pathway activation of fibroblasts was confirmed through p-p44/42 mitogen-activated protein kinase, p-mTOR, p-cMet, and ROCK-1 expression. The cMet agonistic monoclonal antibody-treated group showed 1.60-fold lower wound area ( p = 0.027), 1.54-fold higher collagen synthesis ( p = 0.001), and 1.79-fold lower inflammatory cell infiltration ( p = 0.032) than the saline-treated control. The agonist increased cMet (1.86-fold; p = 0.029), α-smooth muscle actin (1.20-fold; p = 0.018), and vascular endothelial growth factor (1.68-fold, p = 0.029) expression but suppressed CD68 (1.25-fold; p = 0.043), transforming growth factor-β (1.25-fold; p = 0.022), and matrix metalloproteinase-2 (2.59-fold; p = 0.029) expression. In vitro agonist treatment (10 nM) of diabetic fibroblasts increased their migration by 8.98-fold ( p = 0.029) and activated the hepatocyte growth factor/cMet pathway. Tyrosine kinase Met agonistic monoclonal antibody treatment improved diabetic wound healing in mice and reduced wound-site inflammatory cell infiltration. These results need to be validated in large animals before piloting human trials. Although further clinical studies are necessary to evaluate its therapeutic efficacy, our study suggested that cMet agonistic monoclonal antibody can be the alternative modality in order to improve wound healing cascade in diabetic foot patients.

Abstract OT-28-04: Neratinib and tepotinib combination in advanced breast cancer and inflammatory breast cancer patients with abnormal HER2 and c-Met pathway activity as measured by the CELsignia signaling pathway activity test

Abstract Background: While HER2 targeted therapeutics have significantly improved outcomes of patients with HER2 overexpressed advanced breast cancer (ABC), c-Met pathway activation serves as a resistance mechanism in about 60% of patients. Neratinib is an irreversible tyrosine kinase inhibitor of the ErbB family of receptors (EGFR, HER2, and HER4) and tepotinib is a tyrosine kinase inhibitor of c-Met. From live extracted cells from patient biopsies, the CELsignia Pathway Activity Test measures in real-time ErbB and c-Met signaling activity and the dynamic response to pathway inhibition by targeted therapy ex vivo. The CELsignia test may identify patients with ABC who would benefit from the combination of neratinib and tepotinib regardless of the molecular breast cancer subtype. Trial Design: We designed a phase Ib/II multi-center, non-randomized, open-label study of combination of neratinib with tepotinib in patients with advanced breast cancer including those with inflammatory breast cancer. The study sites include UT MD Anderson Cancer Center, City of Hope, and The Ohio State University Cancer Center. Four dose levels of neratinib, and two dose levels of tepotinib will be studied in phase Ib to determine the maximum tolerated dose (MTD). Phase II portion of the study will test the early efficacy of neratinib and tepotinib combination in patients with HER2-/HR+ ABC with abnormal HER2 and c-Met signaling activity determined by the CELsignia test. Eligibility: All patients with IBC/ABC will be eligible for the phase Ib. Eligible patients must have measurable metastatic disease per RECIST v1.1 or unresectable disease amenable for biopsy and have had at >1 line of systemic therapy for advanced disease. Patients will have an Eastern Cooperative Oncology Group performance status of 0 to 2. For the phase II component, patients must have hyperactive HER2 and c-Met signaling determined by CELsignia test. Aims: The primary objective of the phase Ib component is to determine the MTD of the combination in previously treated IBC/ABC of any molecular subtype. The primary objective of the phase II component is to determine the overall response rate (ORR) in patients with HER2-/HR+ ABC who have a positive CELsignia test result (described in the Eligibility). Secondary objectives include progression free and overall survival, clinical benefit rate, duration of response, and safety and tolerability. Exploratory objectives will be to assess HER2 and c-Met driven signaling pathways as either hyperactive or abnormally active by CELsignia test. Statistical Methods: The phase I study will be conducted using a Bayesian optimal interval (BOIN) design to establish the MTD and the recommended phase II dose and assess early efficacy of the combination with a maximum of 21 patients. Two dose levels of tepotinib, and four dose levels of neratinib will be studied. The phase II study will assess the ORR using Bayesian optimal phase 2 (BOP2) design to rule out a 10% ORR in favor of a 30% target ORR, with a power of 80% and two-sided 5% significance level. Accrual: Once MTD is determined, up to 29 evaluable CELsignia test positive subjects will be enrolled to the phase II component of the study and an interim analysis will be performed when the total number of enrolled patients reaches 15. We hope to complete the accrual of patients by July 2022 and to schedule the last patient visit by July 2023. Contact information: For more information or to refer a patient, email blim@mdanderson.org, eahobbs@mdanderson.org Citation Format: Evthokia Hobbs, Eric Lindquist, Brian Sullivan, Esther Yoon, Ying Yuan, Angela Marx, Jie Willey, Huiming Sun, Rachel Layman, Daniel Stover, Yuan Yuan, Bora Lim. Neratinib and tepotinib combination in advanced breast cancer and inflammatory breast cancer patients with abnormal HER2 and c-Met pathway activity as measured by the CELsignia signaling pathway activity test [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-28-04.

Phase Ib study of gemcitabine, nab-paclitaxel, and ficlatuzumab in patients with advanced pancreatic cancer.

693 Background: Paired-related homeodomain transcription factor 1 (Prrx1) isoforms are involved in pancreatic development, pancreatitis, and carcinogenesis. Hepatocyte growth factor (HGF) is a transcriptional target of Prrx1b. Ficlatuzumab is a recombinant humanized HGF antibody, that neutralizes HGF/c-Met binding and HGF-induced c-Met phosphorylation. In preclinical pancreatic ductal adenocarcinoma (PDAC) models, inhibition of Prrx1b-HGF signaling using ficlatuzumab and gemcitabine reduced primary tumor volume and eliminated metastatic disease. Methods: Patients (pts) with previously-untreated metastatic PDAC enrolled in a phase Ib dose escalation study with 3+3 design and two dose cohorts of ficlatuzumab (10mg/kg and 20mg/kg) administered intravenously every other week with gemcitabine (G; 1000mg/m2) and nab-paclitaxel (A; 125mg/m2) given 3 weeks on and 1 week off. This was followed by an expansion phase at the maximally tolerated dose (MTD) of the combination. Results: 24 pts (sex, 12M:12F; median age, 69 years [range, 51-82 years]) were enrolled. No dose-limiting toxicities were identified in the phase 1b (N = 6 pts) and ficlatuzumab at 20mg/kg with GA was advanced to the expansion phase (N = 18 pts). By RECISTv1.1 in the full study population, 7 (29%) pts had partial response, 15 (63%) had stable disease, and 2 (8%) could not be evaluated. Median progression-free survival was 8 months (range, 3-16 months), 4 pts are still on study treatment. The primary toxicities attributed to ficlatuzumab included hypoalbuminemia (grade 3, 21%; any grade, 91%) and edema (grade 3, 8%; any grade, 91%). Nine (38%) of the 24 pts discontinued study treatment due to these toxicities prior to disease progression. Conclusions: The combination of ficlatuzumab with gemcitabine and nab-paclitaxel is associated with durable treatment responses but also significant hypoalbuminemia and edema that may impair treatment tolerability. Serial blood samples were collected for circulating HGF measurements, and mandatory pretreatment biopsies were collected for tumor c-MET pathway activation and 3D organoid culture drug sensitivity testing. Clinical trial information: NCT03316599.

Failures in preclinical and clinical trials of c-Met inhibitors: evaluation of pathway activity as a promising selection criterion.

C-Met is a frequently overexpressed or amplified receptor tyrosine kinase involved in metastatic-related functions, including migration, invasion, cell survival, and angiogenesis. Because of its role in cancer progression and metastasis, many inhibitors have been developed to target this pathway. Unfortunately, most c-Met inhibitor clinical trials have failed to show significant improvement in survival of cancer patients. In these trials tumor type, protein overexpression, or gene amplification are the primary selection criteria for patient inclusion. Our data show that none of these criteria are associated with c-Met pathway activation. Hence, it is conceivable that the majority of c-Met inhibitor clinical trial failures are the consequence of a lack of appropriate patient selection. Further complicating matters, c-Met inhibitors are routinely tested in preclinical studies in the presence of high levels of exogenous Hepatocyte Growth Factor (HGF), its activating ligand. In our studies, several tumor cell lines showed sensitivity to a c-Met inhibitor at high HGF concentrations (50 ng/mL). However, when the tumor lines were tested at HGF levels typically detected in human serum (0.4 to 0.8 ng/mL), inhibitor activity was lost. Thus testing c-Met inhibitors at non-physiological concentrations of HGF may lead to incorrect predictions of drug efficacy in vivo.

Generation and Characterization of a Bispecific Antibody Targeting Both PD-1 and c-MET.

Bispecific antibodies, BsAbs, are molecules with the ability to bind to two different epitopes on the same or different antigens. c-MET, cellular-mesenchymal to epithelial transition factor, is deregulated in many types of human malignancies. Abnormal c-MET activation in cancer correlates with poor prognosis. PD-1, programmed death-1, is an additional inhibitory receptor expressed by T cells. Blocking the interactions between PD-1 and PD-L1 has emerged as a promising immunotherapy for treating cancer. The goal of this study was to identify a novel bispecific antibody targeting both c-MET and PD-1 as an anti-cancer therapeutic candidate. The BsAb was produced using 293E expression system and purified by Protein A affinity chromatography. Then the binding specificity and affinity of the BsAb was examined by FACS and biolayer light interferometry. The ability of the BsAb to inhibit the proliferation of tuman cells was measured using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit; the potential signaling pathway involved was identified by Western Blot. Cytokine secreted by PHA-L stimulated PBMC was measured by ELISA. Effects of BsAb on PBMC-mediated lysis of MKN45 cells was measured by LDH cytotoxicity assay. Based on the original sequences of PD-1 and c-MET mAb, a BsAb gene was designed, cloned into pCEP4 vector for expression in 293E cells. The BsAb was obtained after purification of the cell culture supernatant. It can bind to PD-1 and c-MET simultaneously, the calculated affinity was 11.5 nM for PD-1 and 9.09 nM for c-MET. The BsAb enhanced IFN-γ production over control IgG by 2-3 folds. It also inhibit the c-MET pathway activation and the proliferation of tumor cells significantly, comparable to JnJ-38877605. The BsAb showed dose-dependent cytotoxic activity against MKN45 cells. Our results indicated that a novel BsAb recognizing PD-1 and c-MET was successfully generated. It could redirect T cells to kill tumor cells, while retaining its inherent ability to restore T cells and inhibit tumor cells. With this potential, this BsAb could be developed as a therapeutic candidate for the treatment of various solid tumors.

Abstract 4149: Increased migration ability of osimertinib-resistant EGFR-T790M mutant non-small-cell lung cancer cells

Abstract Background: A third generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) osimertinib is effective against gefitinib/erlotinib-resistant non-small-cell lung cancer (NSCLC) harboring EGFR T790M mutation. Although the majority of these patients initially responds to osimertinib, they eventually develop resistance. Acquired mutations such as EGFR C797S mutation have been reported to confer resistance to osimertinib, but mechanisms of resistance to osimertinib or biological behaviors of osimertinib-resistant NSCLC have not been fully elucidated. Materials and methods: Osimertinib-resistant PC-9/ZD/OR1 and PC-9/ZD/OR3 cells were generated after continuous exposure of gefitinib-resistant PC-9/ZD cells harboring both EGFR exon 19 deletion and T790M mutations to increasing concentrations of osimertinib. Growth-inhibitory effect of inhibitors gefitinib and osimertinib was evaluated by MTT assay. Common EGFR mutational status was examined by RainDrop Digital PCR System (RainDance Technologies). Immunoblot analysis was performed to investigate the modulation of molecules relevant to EGFR signaling pathways. Cell-adhesion assay, wound closure assay and transwell assay were performed to evaluate the ability of cell adhesion and migration. Results: Both PC-9/ZD/OR1 and PC-9/ZD/OR3 cells showed 50- to 120-fold more resistant to osimertinib than parental PC-9/ZD cells, and maintained resistance to gefitinib as well. PC-9/ZD/OR cells maintain EGFR exon 19 deletion and T790M mutations and C797S mutation was not detected. Although phosphorylation of EGFR and ERK1/2 were inhibited by osimertinib treatment in PC-9/ZD/OR cells, phosphorylation of Akt was not inhibited. PC-9/ZD/OR cells express the elevated level of c-MET and its phosphorylated form, suggesting that bypass signaling is activated to evade EGFR pathway blockade. PC-9/ZD/OR cells also express marked level of E-cadherin which parental PC-9/ZD cells do not express. PC-9/ZD/OR cells exhibited enhanced adherent activity, cell motility and migration ability compared to PC-9/ZD cells. Notably, osimertinib treatment significantly reduced the number of migrated cells not only in PC-9/ZD cells but also in PC-9/ZD/OR cells. These results suggest that osimertinib-resistant PC-9/ZD/OR cells acquired enhanced migration ability through c-MET pathway activation and/or induction of E-cadherin. Osimertinib treatment may be still effective to inhibit cell migration, suggesting that cell migration and cell growth are driven by different signaling pathways in PC-9/ZD/OR cells. Conclusion: c-MET overexpression was suggested to confer resistance to osimertinib and the resistant cells exhibited enhanced cell migration. Continuative osimertinib treatment may be beneficial in preventing metastasis even after the failure and involvement of c-MET and E-cadherin in osimertinib resistance should be further investigated. Citation Format: Satoshi Kambayashi, Yasuhiro Koh, Maiha Ohyama, Ayaka Tanaka, Hiroaki Akamatsu, Nahomi Tokudome, Hiroki Ueda, Nobuyuki Yamamoto. Increased migration ability of osimertinib-resistant EGFR-T790M mutant non-small-cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4149. doi:10.1158/1538-7445.AM2017-4149

A viral microRNA downregulates metastasis suppressor CD82 and induces cell invasion and angiogenesis by activating the c-Met signaling.

Kaposi’s sarcoma (KS) is the most common AIDS-associated malignancy etiologically caused by Kaposi’s sarcoma-associated herpesvirus (KSHV). KS is a highly disseminated and vascularized tumor comprised of poorly differentiated spindle-shaped endothelial cells. KSHV encodes 12 pre-microRNAs (pre-miRNAs) that yield 25 mature miRNAs, but their roles in KSHV-induced tumor dissemination and angiogenesis remain largely unknown. KSHV-encoded miR-K12-6 (miR-K6) can produce two mature miRNAs, miR-K6-3p and miR-K6-5p. Recently, we have shown that miR-K6-3p promoted cell migration and angiogenesis by directly targeting SH3 domain binding glutamate-rich protein (SH3BGR) (PLoS Pathog. 2016;12(4):e1005605). Here, by using mass spectrometry, bioinformatics analysis and luciferase reporter assay, we showed that miR-K6-5p directly targeted the coding sequence (CDS) of CD82 molecule (CD82), a metastasis suppressor. Ectopic expression of miR-K6-5p specifically inhibited the expression of endogenous CD82 and strongly promoted endothelial cells invasion in vitro and angiogenesis in vivo. Overexpression of CD82 significantly inhibited cell invasion and angiogenesis induced by miR-K6-5p. Mechanistically, CD82 directly interacted with c-Met to inhibit its activation. MiR-K6-5p directly repressed CD82, relieving its inhibition on c-Met activation and inducing cell invasion and angiogenesis. Deletion of miR-K6 from KSHV genome abrogated KSHV suppression of CD82 resulting in compromised KSHV activation of c-Met pathway, and KSHV-induced invasion and angiogenesis. In conclusion, these results show that by inhibiting CD82, KSHV miR-K6-5p promotes cell invasion and angiogenesis by activating the c-Met pathway. Our findings illustrate that KSHV miRNAs may play an essential role in the dissemination and angiogenesis of KSHV-induced malignancies.

Phase II study of the c-MET inhibitor tivantinib (ARQ 197) in patients with relapsed or relapsed/refractory multiple myeloma

The hepatocyte growth factor/c-MET pathway has been implicated in the pathobiology of multiple myeloma, and c-MET inhibitors induce myeloma cell apoptosis, suggesting that they could be useful clinically. We conducted a phase II study with the c-MET inhibitor tivantinib in patients with relapsed, or relapsed and refractory myeloma whose disease had progressed after one to four prior therapies. Tivantinib, 360 mg orally per dose, was administered twice daily continuously over a 4-week treatment cycle without a cap on the number of allowed cycles, barring undue toxicities or disease progression. Primary objectives were to determine the overall response rate and the toxicities of tivantinib in this patient population. Sixteen patients were enrolled in a two-stage design. Notable grade 3 and 4 hematological adverse events were limited to neutropenia in five and four patients, respectively. Nonhematological adverse events of grade 3 or higher included hypertension (in four patients); syncope, infection, and pain (two each); and fatigue, cough, and pulmonary embolism (one each). Four of 11 evaluable patients (36%) had stable disease as their best response, while the remainder showed disease progression. Overall, tivantinib as a single agent did not show promise for unselected relapsed/refractory myeloma patients. However, the ability to achieve stable disease does suggest that combination regimens incorporating targeted inhibitors in patients with c-MET pathway activation could be of interest.

Abstract 5027: Detection of soluble protein biomarkers in NSCLC serum samples by meso scale discovery electrochemiluminescence platform

Abstract Identification of soluble biomarkers has become a critical non-invasive approach for disease diagnosis and monitoring in the treatment of solid tumors. Non-Small Cell Lung Cancer (NSCLC) is the most common form of lung cancer, accounting for approximately 85% of all lung cancers worldwide. The EGFR family of genes is known to be highly expressed in NSCLC tumors and play a critical role in the progression of the disease. Although patients initially respond well to EGFR TKI therapy, acquired resistance occurs through several mechanisms, including compensatory cMET pathway activation (Robinson et al. 2013). Previous reports have demonstrated that soluble forms of EGFR, Her2 and cMET are generated through either alternate splicing of mRNA or proteolytic cleavage of the full length receptors (Wilken et al. 2013). Serum concentrations of these receptors can be correlated with prognosis as well as treatment response (Gregorc, 2004, Kasahara, 2010 Heinmoller, 2003). Soluble receptor ligands, including Hepatocyte growth factor (HGF), have also been evaluated in NSCLC and a strong correlation has also been observed between the levels of serum HGF and disease outcome in patients treated with EGFR-TKIs (Kasahara et al. 2010). In this study, we evaluated concentrations of sEGFR, sMET, sHER2,, and HGF in NSCLC versus healthy normal serum samples via meso scale discovery (MSD) platform. Inventoried and custom MSD assays were optimized to measure these proteins in a training set consisting of 19 NSCLC and 16 normal healthy serum samples. Significantly lower concentrations of sEGFR and sHER2 were observed in the NSCLC serum samples compared to normal (p value = 0.0092 for both receptors), whereas, in contrast, the levels of HGF were significantly higher in the NSCLC patient samples (p value <0.0001). No significant difference in sMET concentration was observed between NSCLC and normal serum. Based on this data, it was determined that a ratio of protein concentration of each soluble receptor (EGFR, Her2 and cMET) to HGF yielded a greater differentiation between NSCLC and normal samples (p value <0.0001, = 0.0041,and 0.0327 respectively). Cut-off values to define NSCLC versus normal patients were established for each ratio and validated in a blinded independent cohort, consisting of 50 NSCLC and 12 healthy normal serum samples. PPV and NPV were then calculated to determine sensitivity and specificity for each protein ratio. Results from the validation study show an almost complete separation between healthy and NSCLC patient samples (p value <0.0001, AUC = 1)for all three ratios tested (sEGFR/HGF, sHER2/HGF and sMET/HGF), with a PPV = 100%, 98%, 100% and NPV = 100%, 100%, 85.7%, respectively. Overall, these findings indicate potential clinical applications for these protein pairs as non-invasive biomarkers of NSCLC diagnosis, prognosis and treatment monitoring to support emerging TKI therapies Citation Format: Dana S. Gaffney, Katherine Bell, Gabriela Martinez, Jayaprakash Karkera, Suso Platero. Detection of soluble protein biomarkers in NSCLC serum samples by meso scale discovery electrochemiluminescence platform. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5027.

Integrative biomarker analyses indicate etiological variations in hepatocellular carcinoma

The purpose of this study was to determine whether biomarkers from baseline plasma and archival tissue specimens collected from patients enrolled in the EVOLVE-1 trial - a randomized phase 3 study of everolimus in hepatocellular carcinoma (HCC) - were associated with prognosis, etiology or ethnicity. Circulating plasma levels of bFGF, PLGF, VEGF, VEGF-D, c-Kit, collagen IV, sVEGFR1 and VEGFR2 were measured by ELISA (N=503). Protein levels of IGF-1R, c-Met, mTOR, Tsc2 were assayed by immunohistochemistry (N=125). Genomic DNA sequencing was conducted on a panel of 287 cancer-related genes (N=69). Patients with baseline plasma concentrations of VEGF or sVEGFR1 above the cohort median had significantly shorter overall survival. These plasma biomarkers retained prognostic significance in a multivariate Cox regression model with geographic region, macroscopic vascular invasion and alpha fetoprotein AFP levels. Membranous c-Met protein levels were significantly lower for Asian patients, as well as for hepatitis B viral etiology. The prevalence of genetic changes were similar to previous reports, along with a trend towards higher PTEN and TSC2 mutations among Asians. The angiogenesis biomarkers VEGF and sVEGFR1 were independent prognostic predictors of survival in patients with advanced HCC. Potential differences in c-Met and mTOR pathway activation between Asian and non-Asian patients should be considered in future clinical trials. Our study demonstrates that circulating angiogenesis biomarkers can predict the survival outcome in patients with advanced hepatocellular carcinoma independent of the clinical variables. There is etiology and ethnicity variation in molecular pathway activation in hepatocellular carcinoma, which should be considered for future clinical trial design of targeted therapy. NCT01035229.

Abstract A11: Activity of a bispecific antibody targeting EGFR and cMet with enhanced Fc effector function in EGFR mutant setting with cMet pathway activation

Abstract Non-small cell lung cancers (NSCLCs) with activating mutations in the Epidermal Growth Factor Receptor (EGFR) gene are associated with high response rates (70-80%) to EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib and gefitinib, but most acquire resistance over time through numerous mechanisms. In these patients, the cMet pathway is often activated to compensate and provide resistance to the EGFR targeted monotherapy; this activation can occur by MET gene amplification, overexpression of cMet protein, or an increase in the ligand HGF. We have designed a bispecific EGFR-cMet antibody (JNJ-61186372) with multiple mechanisms of action resulting in anti-tumor activity in the EGFR mutant setting, with or without cMet pathway activation. Controlled Fab-arm exchange was used to produce JNJ-61186372, a technique that allows for efficient large-scale preparation of bispecific antibodies with a regular IgG1 structure. JNJ-61186372 was shown to bind EGFR and cMet and efficiently inhibited ligand-induced phosphorylation of both receptors. In addition to this important mechanism of action, we have engineered the antibody to contain lower than normal fucose levels to increase Fc-dependent effector mechanisms. JNJ-61186372 exhibited antibody dependent cellular cytotoxicity (ADCC) activity in vitro in a range of NSCLC cell lines with EGFR mutations, KRas mutation, and/or amplified MET gene. Furthermore, the low fucose form of JNJ-61186372 demonstrated more effective ADCC activity compared to its normal fucose counterpart. The bispecific JNJ-61186372 antibody showed increased potency (2-3 fold) compared to the combination of monovalent EGFR and monovalent cMet antibodies, demonstrating the beneficial effects of dual targeting in a single molecule. Antibody dependent cell-mediated phagocytosis (ADCP) activity of JNJ-61186372 was also confirmed in vitro. We have also demonstrated that Fc-dependent effector functions contributed to in vivo anti-tumor growth activity of JNJ-61186372, in a xenograft model with EGFR mutations and cMet activation. Our data demonstrate that the bispecific antibody JNJ-61186372, generated using controlled Fab-arm exchange, has in vitro ADCC and ADCP activity in EGFR mutant settings, either with or without cMet pathway activation, and with KRas mutation. In addition, the Fc-dependent effector mechanisms contributed to in vivo anti-tumor efficacy. The dual signaling inhibition of EGFR and cMet pathways by JNJ-61186372, combined with enhanced Fc effector function, may provide multiple mechanisms to combat resistance in EGFR mutant NSCLC patients. Citation Format: Keri L. Soring, Katharine D. Grugan, Randall J. Breszki, Jose Pardinas, Leopoldo Luistro, Barbara Bushey, Joost Neijssen, Paul Parren, Janine Schuurman, Mark Anderson, Ricardo Attar, Matthew V. Lorenzi, Mark Chiu, Sheri Moores. Activity of a bispecific antibody targeting EGFR and cMet with enhanced Fc effector function in EGFR mutant setting with cMet pathway activation. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A11.

Abstract 794: Prognostic value of high c-Met expression in patients with poor prognosis pancreatic adenocarcinoma following surgical resection: comparison of three c-Met scoring methods and exploration of underlying mechanisms of c-Met overexpression

Abstract Context: The HGF/c-Met pathway is an hypoxia-inducible pathway involved in tumor-stroma interactions and invasion in pancreatic ductal adenocarcinoma (PDAC). Assessment of c-Met expression is a critical issue as c-Met inhibitors are under clinical development and are suggested to display antitumor activity only in high c-Met tumors. We aimed to assess the prognostic value of c-Met overexpression in PDAC and explore underlying mechanisms (hypoxia, gene amplification, post transcriptional deregulation). Patients and methods: Patients (Pts) with resected PDAC who had received no perioperative chemo/radiotherapy were retrospectively selected. c-Met immunostaining was graded using a simplified score (high c-Met: ≥20% of cancer cells with 3+ staining) and compared to a standard scale combining staining surface and intensity (SI score) and to the MetMab score. Concordance between entire section and tissue microarray (TMA) was assessed and a computer-assisted quantification algorithm was developed (Aperio® software). Hypoxia was assessed by visual grading of HIF-1α/CA9 immunostaining, necrosis, and automated microvascular density (Aperio®). c-Met gene copy number was assessed by fluorescent in situ hybridization (FISH) and Taqman® based copy number variation (CNV) assay. c-Met mRNA levels were quantified using reverse transcription PCR (RT-PCR). c-Met pathway activation was assessed by immunostaining of phospho-c-Met, phospho-GAB1 and downstream signaling (pAKT, pERK, and pSTAT3) and graded visually. Clinical, pathological, and molecular biomarkers were correlated with disease-free (DFS) and overall (OS) survivals. Results: Thirty-seven Pts were analyzed. The simplified c-Met score displayed the best prognostic value and reproducibility vs both the SI score and the MetMab score; using this score: (a) high c-Met (7/37) was associated with shorter DFS (6.3 vs 33.0 months, HR: 3.456, p = 0.0035) and OS (10.8 vs 39.0 months, HR: 4.257, p = 0.0006); and (b) the kappa index was 0.773±0.122. In multivariate analysis, high c-Met was independently associated with both DFS and OS. c-Met expression was concordant on entire section and TMA in 87.9% of cases, and quantifiable using a specific computer-assisted algorithm. There was no correlation between hypoxia-related markers and c-Met expression. FISH and CNV analysis did not show c-Met gene amplification. mRNA quantification and downstream pathway activation data will be presented. Conclusion: c-Met is an independent prognostic marker in resected PDAC that may help identifying Pts at high risk of recurrence and poor survival. High c-Met expression was not associated with hypoxia or gene amplification in this study. The simplified c-Met scale is a robust and reproducible scoring method that could be used for ongoing larger studies on TMA. Citation Format: Cindy Neuzillet, Annemilaï Tijeras-Raballand, Jérôme Raffenne, Armand de Gramont, Pierre Bedossa, Valérie Paradis, Alain Sauvanet, Jean-Baptiste Bachet, Eric Raymond, Pascal Hammel, Anne Couvelard, Jérôme Cros. Prognostic value of high c-Met expression in patients with poor prognosis pancreatic adenocarcinoma following surgical resection: comparison of three c-Met scoring methods and exploration of underlying mechanisms of c-Met overexpression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 794. doi:10.1158/1538-7445.AM2015-794

Abstract 1690: Mechanistic characterization of MM-131, a bispecific antibody that blocks c-Met signaling through concurrent targeting of EpCAM

Abstract Purpose: MM-131 is a purely antagonistic, bispecific antibody that potently inhibits HGF/c-Met signaling by co-targeting the widely expressed tumor antigen EpCAM. The purpose of these studies is to uncover the mechanism by which MM-131 exhibits potent inhibition of both HGF-dependent and HGF-independent c-Met signaling in EpCAM positive tumor cells. Methods: To assess the role of EpCAM in mediating avid binding of MM-131 to c-Met, we quantified the cell surface levels of c-Met and EpCAM in a panel of cancer cell lines using flow cytometry and determined how potently MM-131 inhibits HGF-dependent c-Met signaling in each cell line. Using these data, we built a computational model to explain and quantify the effect of EpCAM targeting. We then tested this model by (1) predicting the activity of MM-131 in other cell lines, based on their EpCAM:c-Met ratios; (2) knocking down EpCAM in cell lines by RNA interference; and (3) comparing MM-131 to a variant of MM-131 in which its EpCAM-targeting arm was mutated to impair binding. To uncover the mechanism by which MM-131 inhibits HGF-independent c-Met signaling, we monitored the effect of MM-131 on c-Met levels, using quantitative imaging and immunoblotting. Results: Consistent with its design, we found that MM-131 is more potent at inhibiting HGF-dependent c-Met signaling, cell viability, and cell migration in EpCAM-high cells than in EpCAM-low cells. In addition, MM-131 potency is reduced when EpCAM levels are knocked down by RNA interference. By mutating the EpCAM-targeting arm of MM-131, the observed potency in EpCAM-high cells is noticeably reduced. Further cell biological characterization of MM-131 revealed two distinct mechanisms of action: (1) MM-131 blocks ligand binding to c-Met; and (2) MM-131 induces downregulation of c-Met. In side-by-side comparison studies, MM-131 was found to be more potent at inhibiting HGF-dependent signaling, cell migration, and cell viability than one-armed-5D5 (MetMab), and uniquely effective at inhibiting HGF-independent signaling through downregulation of c-Met. Consistent with the design criteria, MM-131 did not exhibit any discernable agonistic activity characteristic of bivalent c-Met antibodies such as LY2875358. The molecular effects of MM-131 observed in vitro were also observed in vivo: MM-131 inhibited tumor growth in models of ligand-dependent and ligand-independent c-Met signaling and induced downregulation of c-Met. Conclusions: MM-131 is a bispecific antibody designed to overcome HGF-dependent and HGF-independent c-Met pathway activation by concurrently targeting EpCAM. MM-131 shows no agonistic activity in preclinical models and exhibits two principal mechanisms of action: it antagonizes HGF binding and it induces downregulation of c-Met. These findings support the clinical development of MM-131 in c-Met-driven epithelial tumors that also express EpCAM. Citation Format: Adnan O. Abu-Yousif, Jessica B. Casaletto, Kristina Masson, Aaron Fulgham, Melissa Geddie, Birgit Schoeberl, Ulrik Nielsen, Gavin MacBeath. Mechanistic characterization of MM-131, a bispecific antibody that blocks c-Met signaling through concurrent targeting of EpCAM. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1690. doi:10.1158/1538-7445.AM2015-1690

Involvement of activation of C-met signaling pathway in CD151-induced HUVECs angiogenesis.

CD151 is a member of the tetraspanin family that is implicated as a promoter of pathological or physiological angiogenesis. C-Met is expressed on a variety of cells including vascular endothelial cells (VECs) and up-regulated during angiogenesis. In this study, we investigated whether CD151 regulated migration, proliferation, tube formation and angiogenesis of human umbilical VECs (HUVECs) with activation of C-Met. Moreover, we studied whether CD151 could affect the angiogenic molecules such as nitric oxide (NO), vascular cell adhesion molecule-1 (VCAM-1) and vascular endothelial growth factor (VEGF). The expression of CD151 was determined by Western blotting. The cell proliferation assay was performed using the cell counting kit-8 (CCK-8) method and cell migration was assessed in microchemotaxis chambers by using fetal bovine serum (FBS) as the chemotactic stimulus. The angiogenic molecules were evaluated using ELISA. The NO level was detected using NO detection kit. The potential involvement of various signaling pathways was explored using relevant antibodies. We found that proliferation, migration and tube formation of HUVECs were promoted by CD151 with activation of C-Met, FAK and CDC42, while they were suppressed with CD151 knockdown by RNAi. Similarly, the levels of NO, VCAM-1 and VEGF in HUVECs were increased by CD151, but they were inhibited with CD151 knockdown by RNAi. These data suggested that CD151 could promote migration, proliferation, tube formation and angiogenesis of HUVECs, which was possibly related to the C-Met signaling pathways.

Abstract DDT01-03: Discovery and preclinical pharmacology of JNJ-61186372: A novel bispecific antibody targeting EGFR and cMET

Abstract NSCLC with activating mutations in the EGFR gene are associated with high response rates to EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib and gefitinib, but invariably acquired resistance emerges over time. A primary cause of resistance is the emergence of secondary mutations in EGFR which neutralize the effectiveness of TKIs. In addition, the cMet pathway is often activated, either through MET gene amplification, overexpression of cMet protein, or an increase in the ligand HGF, to provide a compensatory survival pathway conferring resistance to EGFR TKIs. We have designed a bispecific EGFR-cMet antibody (JNJ-61186372) with a unique set of mechanisms of action resulting in anti-tumor activity in the EGFR mutant setting, with or without cMet pathway activation. We have demonstrated three mechanisms of action that contribute to the activity of JNJ-61186372: 1) inhibition of ligand-induced phosphorylation of both EGFR and cMet, 2) receptor degradation in vivo, and 3) enhanced ADCC activity. JNJ-61186372 inhibited EGF-induced phosphorylation of EGFR in cell lines with either wild-type (WT) EGFR or activating mutations in EGFR. In the same cell lines, JNJ-61186372 inhibited HGF-induced phosphorylation of cMet. JNJ-61186372 also blocked pERK and pAkt with similar IC50 values in EGFR-WT and EGFR mutant cell lines, indicating that downstream signaling pathways were inhibited. Total protein levels of both EGFR and cMet were decreased in xenograft tumor models following treatment with JNJ-61186372 compared to tumors from mice treated with PBS control suggesting that one mechanism by which JNJ-61186372 suppresses EGFR and cMet activity in vivo is through degradation of both receptors. The third mechanism of action is directing immune cells to kill tumor cells. JNJ-61186372 is produced with low levels of fucosylation, which translates to an enhanced antibody-dependent cellular cytotoxicity (ADCC). These three mechanisms of action of JNJ-61186372 provide a distinct preclinical profile for targeting both EGFR and cMET in a single bispecific antibody. JNJ-61186372 demonstrated efficacy in multiple in vivo tumor models with EGFR mutations, including both cell line and patient-derived xenografts. Importantly, JNJ-61186372 effectively inhibited tumor growth in models with mutant EGFR and cMet activation, whereas single agent EGFR inhibitors were less effective. The preclinical data support the clinical development of JNJ-61186372 in patients with lung cancer and other malignancies associated with aberrant EGFR and cMET signaling. Citation Format: Sheri L. Moores, Mark Chiu, Barbara Bushey, Kristen Chevalier, Peter Haytko, Joost Neijssen, Paul Parren, Janine Schuurman, Mark Anderson, Ricardo Attar, Robert Kramer, Matthew V. Lorenzi. Discovery and preclinical pharmacology of JNJ-61186372: A novel bispecific antibody targeting EGFR and cMET. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr DDT01-03. doi:10.1158/1538-7445.AM2014-DDT01-03

MiR-210 over-expression enhances mesenchymal stem cell survival in an oxidative stress environment through antioxidation and c-Met pathway activation.

microRNA-210 (miR-210) has generally been reported to be associated with cell survival under hypoxia. However, there are few data regarding the role of miR-210 in the survival of mesenchymal stem cells (MSCs) under oxidative stress conditions. Thus, we sought to investigate whether miR-210 over-expression could protect MSCs against oxidative stress injury and what the primary mechanisms involved are. The results showed that over-expression of miR-210 significantly reduced the apoptosis of MSCs under oxidative stress, accompanied by obvious increases in cell viability and superoxide dismutase activity and remarkable decreases in malonaldehyde content and reactive oxygen species production, resulting in a noticeable reduction of apoptotic indices when compared with the control. Moreover, the above beneficial effects of miR-210 could be significantly reduced by c-Met pathway repression. Collectively, these results showed that miR-210 over-expression improved MSC survival under oxidative stress through antioxidation and c-Met pathway activation, indicating the potential development of a novel approach to enhance the efficacy of MSC-based therapy for injured myocardium.

GW24-e3739 MiR-210 Over-expression Enhances Mesenchymal Stromal Cells Survival in an Oxidative Stress Circumstance though Anti-oxidation and c-Met Pathways Activation

ObjectivesWe aimed to investigate whether over-expression of miR-210, an oxide-related miR, could protect mesenchymal stromal cells (MSCs) against apoptosis induced by oxidative stress, and if so, what the potential mechanisms...

Abstract 1512: Sunitinib-induced metastasis is tumor dependent and is mediated by c-Met/HGF pathway

Abstract Recent studies in several tumor models indicated that treatment with angiogenic inhibitors may trigger induction of metastasis to other organs. Here we investigated modes of resistance and invasion in several tumors including 4T1 (breast), H460 (lung) and Colo205 (colorectal) using sunitinib at doses comparable to clinically utilized regimen. In comparison with vehicle-treated tumors, sunitinib increased metastasis to lung in 4T1 tumors and to peritoneal lymph node in Colo205 tumors. However, the same treatment did not induce invasiveness in H460 tumors, further suggesting that accelerating metastasis during treatment with angiogenic inhibitors is tumor dependent. Interestingly, Crizotinib (a dual inhibitor of c-Met and ALK pathways) as single agent or in combination with sunitinib reduced metastasis in all models tested suggesting a role for c-Met/HGF pathway in intrinsic- or sunitinib-induced -metastasis. Moreover, ELISA data showed that while c-Met is highly enriched in tumor cells, HGF is secreted mainly by the stroma (mouse HGF) suggesting a paracrine fashion for c-Met pathway activation in the tumors. In conclusion, our findings indicate that sunitinib-induced metastasis is tumor dependent and further supports a rationale for combination of anti-angiogenesis and c-Met inhibition in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1512. doi:1538-7445.AM2012-1512

Abstract 1787: The c-Met inhibitors EMD 1214063 and EMD 1204831 are effective in combination with EGFR and VEGF inhibitors in NSCLC models

Abstract c-Met is a receptor tyrosine kinase that has hepatocyte growth factor as its ligand. Evidence from biochemical and human genetic studies indicate that c-Met is one of the most frequently activated tyrosine kinases in human cancer. Dysregulated c-Met signaling can result in the development of tumors highly dependent on c-Met. Furthermore, enhanced c-Met signaling is likely involved in conferring resistance to epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) inhibitors. The orally available EMD 1214063 and EMD 1204831 inhibit c-Met activity in a potent and highly selective fashion, and are currently being evaluated in cancer patients. Here we report the activity of these compounds in combination with inhibitors of EGFR and VEGF in various tumor xenograft models. EMD 1214063 was tested alone and in combination with EGFR inhibitors (erlotinib and cetuximab) in xenografts of erlotinib-resistant tumor cells, namely the lung cancer H1975 and the NCI-H441 non-small-cell lung cancer (NSCLC) cell lines. The H1975 cell line expresses moderate amounts of c-Met, and EMD 1214063 was inactive as a single agent (treatment group/control group [T/C] 78%; tumor growth delay [TGD] 2 days). Cetuximab was active with a T/C of 13% inducing a TGD of 21 days. The combination of EMD 1214063 and cetuximab was active with a T/C of α1% and a TGD of 42 days. The NCI-H441 cell line is characterized by constitutive c-Met overexpression and is sensitive to c-Met inhibitors in vivo. EMD 1214063 (100 mg/kg) was active inducing a T/C of 20%, while erlotinib alone (30 mg/kg) was not efficacious. However, the combination of EMD 1214063 with erlotinib enhanced tumor growth inhibition, induced partial tumor regression in 2/9 mice, and delayed tumor re-growth. These data are compatible with the hypothesis that cMet pathway activation confers resistance to EGFR inhibitors. We additionally investigated the combination of either EMD 1214063 or EMD 1204831 with VEGF inhibitors in NSCLC models. In NCI-H441 xenografts, EMD 1204831 produced a modest T/C (66%) at the suboptimal dose of 25 mg/kg/bid, similarly to aflibercept at 40 mg/kg (T/C 55%). In contrast, the combination of both compounds yielded a T/C of 1% with partial responses in 2/10 mice. In the EBC-1 model, treatment with EMD 1214063 (10 mg/kg) or with the antiVEGF antibody B20-4.1 (20 mg/kg) as single agents resulted in a T/C of β21% (with partial regression in 1/10 mice) and 19%, respectively. In contrast, the combination of EMD 1214063 and B20-4.1 exhibited enhanced anti-tumor activity with a T/C of −72% and partial regressions in 10/10 mice. These data support the hypothesis that EMD 1214063 or EMD 1204831 may be effective in NSCLC patients in combination with VEGF-targeting agents. In conclusion, our findings provide a rationale to evaluate the efficacy of the combination of EMD 1214063 or EMD 1204831 with EGFR and VEGF inhibitors in NSCLC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1787. doi:1538-7445.AM2012-1787

HGF/c-Met pathway is one of the mediators of sunitinib-induced tumor cell type-dependent metastasis

Recent studies in several tumor models indicated that treatment with angiogenic inhibitors may trigger induction of metastasis to other organs. Here we investigated modes of resistance and invasion in several tumor cell lines including 4T1 (breast), H460 (lung) and Colo205 (colorectal) using sunitinib at doses comparable to clinically utilized regimen. In comparison with vehicle-treated tumors, sunitinib increased metastasis to lung in 4T1 tumors and to peritoneal lymph node in Colo205 tumors. However, the same treatment did not induce invasiveness in H460 tumors, further suggesting that accelerating metastasis during treatment with angiogenic inhibitors is tumor cell-type dependent. Interestingly, Crizotinib (a dual inhibitor of c-Met and ALK pathways) as single agent or in combination with sunitinib reduced metastasis in all models tested suggesting a role for c-Met/HGF pathway in intrinsic- or sunitinib-induced-metastasis. Moreover, ELISA data showed that while c-Met is highly enriched in tumor cells, HGF is secreted mainly by the stroma (mouse HGF) suggesting a paracrine fashion for c-Met pathway activation in the tumors. In conclusion, our findings indicate that sunitinib-induced metastasis is tumor cell-type dependent and further supports a rationale for combination of anti-angiogenics and c-Met inhibition in the clinic.