anti-HGF Antibody Research Articles

In vitro immuno-prevention of nitration/dysfunction of myogenic stem cell activator HGF, towards developing a strategy for age-related muscle atrophy.

In response to peroxynitrite (ONOO-) generation, myogenic stem satellite cell activator HGF (hepatocyte growth factor) undergoes nitration of tyrosine residues (Y198 and Y250) predominantly on fast IIa and IIx myofibers to lose its binding to the signaling receptor c-met, thereby disturbing muscle homeostasis during aging. Here we show that rat anti-HGF monoclonal antibody (mAb) 1H41C10, which was raised in-house against a synthetic peptide FTSNPEVRnitroY198EV, a site well-conserved in mammals, functions to confer resistance to nitration dysfunction on HGF. 1H41C10 was characterized by recognizing both nitrated and non-nitrated HGF with different affinities as revealed by Western blotting, indicating that the paratope of 1H41C10 may bind to the immediate vicinity of Y198. Subsequent experiments showed that 1H41C10-bound HGF resists peroxynitrite-induced nitration of Y198. A companion mAb-1H42F4 presented similar immuno-reactivity, but did not protect Y198 nitration, and thus served as the control. Importantly, 1H41C10-HGF also withstood Y250 nitration to retain c-met binding and satellite cell activation functions in culture. The Fab region of 1H41C10 exerts resistivity to Y250 nitration possibly due to its localization in the immediate vicinity to Y250, as supported by an additional set of experiments showing that the 1H41C10-Fab confers Y250-nitration resistance which the Fc segment does not. Findings highlight the invitro preventive impact of 1H41C10 on HGF nitration-dysfunction that strongly impairs myogenic stem cell dynamics, potentially pioneering cogent strategies for counteracting or treating age-related muscle atrophy with fibrosis (including sarcopenia and frailty) and the therapeutic application of investigational HGF drugs.

Abstract 5848: Unraveling c-MET targeting resistance mechanisms in gastric cancer via single-cell transcriptome analysis; Novel therapeutic target discovery of ERO1A and VEGFA

Abstract Background: Gastric cancer (GC) is the fifth most prevalent malignant tumor worldwide, and the fourth most common cause of mortality. c-MET has been reported to be associated with poor prognosis in GC. Over the decades, treatment approaches targeting HGF/c-MET have been developed including small molecule tyrosine kinase inhibitors, anti-c-MET and anti-HGF monoclonal antibodies. Unfortunately, the results of previous study support the development of acquired resistance to HGF/c-Met inhibitors. They suggest a possible resistance mechanism involving poor MET-status detection, crosstalk between c-MET and compensatory signaling pathways, the occurrence of acquired a new mutation, however, knowledge regarding the mechanism of c-MET inhibitor is limited.2. Purpose: We identify new markers and mechanisms that induce drug resistance when c-MET-targeted inhibitors are treated in c-MET-amplified GC cell lines in 3D culture, and discover new therapeutic targets for GC via single cell-RNA sequencing analysis.3. Material and Methods: Single cell-RNA sequencing is performed on c-MET amplified GC cell line in the 2D and 3D culture methods according to the presence or absence of c-MET inhibitor treatment. Through DEG and clustering analysis, the candidate genes estimated to impart resistance are selected and verified by qPCR. Through over-expression, induction of resistance for c-MET inhibitor treatment with target gene is evaluated in c-MET amplified GC cell line. c-MET inhibitor selected gene is constructed as a xenograft model to reveal the mechanism of drug resistance. Retrospective analysis confirms the relationship between target gene expression level and the resistance for c-MET inhibitor or other TKI treated patients with GC. 4. Results: Treatment of c-MET-amplified GC cell lines with c-MET inhibitors under the conditions of 2D and 3D cultures confirmed that resistance was induced only in the 3D. We performed a high-precision single cell RNA-Seq analysis of 18056 single cells in total from c-MET amplified GC cell line MKN-45 in 2D/3D culture control as well as corresponding samples with volitinib treatment investigate their gene expression signatures. Fifteen selected candidate genes presumed to induce drug resistance were screened and validated by qPCR, with a total of six genes consistent with the sequencing results. After over-expression of six genes to the c-MET amplified gastric cancer cell line, c-MET inhibitors were treated and cell viability was performed to check for resistance to cell viability. Finally, ERO1A and VEGFA were over-expressed to confirm the resistant for the c-MET inhibitor treatment in vitro and in vivo.5. Discussion: We plan to build a xenograft mouse model with candidate genes such as ERO1a and VEGFA to prove whether the candidate gene overcomes drug resistance when they are on or off. Citation Format: Hajung Kim, Jinhyeon Kim, Seung-Jae Myung, Charny Park, Sun young Kim. Unraveling c-MET targeting resistance mechanisms in gastric cancer via single-cell transcriptome analysis; Novel therapeutic target discovery of ERO1A and VEGFA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5848.

Tumor-associated Macrophages Mediate Gefitinib Resistance in Lung Cancer through HGF/c-met Signaling Pathway.

The biological behavior of cells changes after they develop drug resistance, and the degree of resistance will be affected by the tumor microenvironment. In this study, we aimed to study the effects of M2 macrophages on gefitinib resistance. We polarized THP-1 cells into M0 and M2 macrophages, and conducted various experiments to investigate the effects of M2 macrophages on gefitinib resistance in lung cancer. We found that M2 macrophages promote gefitinib resistance in HCC827 and PC9 cells. In addition, we used ELISA to measure the secretion level of HGF. HGF secretion levels were significantly increased in M2 macrophages. Exogenous HGF remarkably increased the proliferation and invasion in HCC827 and PC9 cells. However, the addition of anti-HGF antibodies abolished the proliferation and invasion of both HCC827 and PC9 cells promoted by M2 macrophages. Furthermore, M2 macrophages or exogenous HGF significantly increased the expression of p-met and p-ERK in HCC827 and PC9 cells, while anti-HGF antibodies diminished the expression of p-met and p-ERK by neutralizing HGF in M2 macrophages. Our results revealed that M2 macrophages promote gefitinib resistance by activating ERK and HGF/c-met signaling pathways in HCC827 and PC9 cells. Our findings provide a new therapeutic strategy for gefitinib resistance in lung cancer.

Telocytes protect against lung tissue fibrosis through hexokinase 2-dependent pathway by secreting hepatocyte growth factor.

Pulmonary fibrosis (PF) is one of the common manifestations of end-stage lung disease. Chronic lung failure after lung transplantation is mainly caused by bronchiolitis obliterans syndrome (BOS) and is mainly characterized by lung tissue fibrosis. Pulmonary epithelial-mesenchymal transformation (EMT) is crucial for pulmonary fibrosis. Telocytes (TCs), a new type of mesenchymal cells, play a protective role in various acute injuries. For exploring the anti-pulmonary fibrosis effect of TCs in the BOS model in vitro and the related mechanism, rat tracheal epithelial (RTE) cells were treated with transforming growth factor-β (TGF-β) to simulate lung tissue fibrosis in vitro. The RTE cells were then co-cultured with TCs primarily extracted from rat lung tissue. Western blot, Seahorse XF Analysers and enzyme-linked immunosorbent assay were used to detect the level of EMT and aerobic respiration of RTE cells. Furthermore, anti-hepatocyte growth factor (anti-HGF) antibody was exogenously added to the cultured cells to explore further mechanisms. Moreover, hexokinase 2 (HK2) in RTE cells was knocked down to assess whether it influences the blocking effect of the anti-HGF antibody. TGF-β could induce lung tissue fibrosis in RTE cells in vitro. Nevertheless, TCs co-culture decreased the level of EMT, glucose metabolic indicators (lactate and ATP) and oxygen levels. Furthermore, TCs released hepatocyte growth factor (HGF). Therefore, the exogenous addition of anti-HGF antibody in the co-culture system blocked the anti-lung tissue fibrosis effect. However, HK2 knockdown attenuated the blocking effect of the anti-HGF antibody. In conclusion, TCs can protect against lung tissue fibrosis by releasing HGF, a process dependent on HK2.

HGF/c-MET: A Potential Target for the Treatment of Various Cancers

Background: Cancer is the abnormal growth of cells in the body due to an imbalance in the normal apoptotic pathways. The abnormality in the cancer cells makes them malignant. Various types of treatment, including chemotherapy, radiation therapy, targeted therapy, and immunotherapy (IMT), are used for cancer. Mesenchymal-epithelial transition factor (c-Met) belongs to the tyrosine kinase receptor family and is overexpressed in various types of cancers. c-Met is a proto-oncogene and facilitates a wide range of biological functions, including cell proliferation, growth, migration, invasion, and angiogenesis, through interaction with its sole ligand hepatocyte growth factor (HGF). Currently, various c-mesenchymal-epithelial transition (c-MET) inhibitors and antibodies are in human trials for their anti-cancer activity. Introduction: The c-MET is a kinase receptor for hepatocyte growth factor (HGF). It is wellrecognized for its tumorigenic potential. HGF binding with c-Met leads to c-Met dimerization and c- Met phosphorylation, which in turn activates many intracellular signalling pathways, including ERK1/2, MAPK, STAT3, Rac1, and PI3K/AKT. These pathways regulate the proliferation, invasion, and migration of cancer cells. Upon binding of HGF to c-MET, a series of phosphorylation reactions get started, which leads to transcription and translation of various proteins, followed by abnormal growth of cancerous tissues due to dysregulation of the cell cycle. The HGF/c-MET signalling pathways have shown their potential in the development of many cancers, including gastric cancer (GC). Several clinical trials have evaluated the therapeutic benefits of MET-targeted therapies involving various agents, such as anti-MET antibodies, anti-HGF antibodies, and tyrosine kinase inhibitors (TKIs). Various c-MET inhibitors are in clinical trials. The current review is focussed on the critical role of the HGF/c-MET pathways in the progression of various cancers, including GC. In addition, this review will also focus on the combination potential of c-MET inhibitors with immuno-oncology drugs, such as programmed cell death protein 1 (PD-1) inhibitors, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitors, tumor necrosis factor (TNF) receptor (OX-40) agonists, etc. Materials and Methods: The method adopted for the study was primarily based on the secondary search through a systematic review of the literature. Results: A study of recent literature and various preclinical and human trials suggests the effectiveness of c-MET inhibitors (alone or in combination) in different cancer types, including GC. In this paper, the results have been elaborated as to how many papers/manuscripts/publications on the topic are present, how many drugs are in Phase I/II/ III/RCT, etc., and that how many papers report on the clinical outcomes of which agent/drug (mentioned in percentage). Conclusion: The use of c-MET inhibitors and antibodies has emerged as a latent therapeutic approach for the treatment of various types of cancer. The c-MET inhibitors can also be used in combination with various immunotherapeutic drugs, like PD-1 inhibitors, OX-40 agonists, etc.

Targeting HGF/c-Met Axis Decreases Circulating Regulatory T Cells Accumulation in Gastric Cancer Patients.

Simple SummaryRestoring an effective immune response is the key goal of immunotherapy. One of the major mechanisms of tumor-induced immunosuppression is regulatory T cells (Treg) accumulation. In this study, using in vitro and in vivo analysis, we assessed the impact of the HGF/c-Met pathway, involved notably in tumor angiogenesis, on Treg accumulation in patients with gastric cancer. First, we reported that c-Met is expressed on circulating monocytes of gastric cancer patients and this expression seems to be associated with the worst outcome. Secondly, during in vitro cultures, c-Met+ monocytes differentiate into dendritic cells with tolerogenic properties able to induce the proliferation of Treg. Finally, rilotumumab, an anti-HGF antibody, decreases the percentage of circulating Treg in gastric cancer patients. Using HGF/c-Met inhibitors to partially reverse immunosuppression could lead to the development of new treatment associations, for example with immune checkpoint blockers.Elucidating mechanisms involved in tumor-induced immunosuppression is of great interest since it could help to improve cancer immunotherapy efficacy. Here we show that Hepatocyte Growth Factor (HGF), a pro-tumoral and proangiogenic factor, and its receptor c-Met are involved in regulatory T cells (Treg) accumulation in the peripheral blood of gastric cancer (GC) patients. We observed that c-Met is expressed on circulating monocytes from GC patients. The elevated expression on monocytes is associated with clinical parameters linked to an aggressive disease phenotype and correlates with a worse prognosis. Monocyte-derived dendritic cells from GC patients differentiated in the presence of HGF adopt a regulatory phenotype with a lower expression of co-stimulatory molecules, impaired maturation capacities, and an increased ability to produce interleukin-10 and to induce Treg differentiation in vitro. In the MEGA-ACCORD20-PRODIGE17 trial, GC patients received an anti-HGF antibody treatment (rilotumumab), which had been described to have an anti-angiogenic activity by decreasing proliferation of endothelial cells and tube formation. Rilotumumab decreased circulating Treg in GC patients. Thus, we identified that HGF indirectly triggers Treg accumulation via c-Met-expressing monocytes in the peripheral blood of GC patients. Our study provides arguments for potential alternative use of HGF/c-Met targeted therapies based on their immunomodulatory properties which could lead to the development of new therapeutic associations in cancer patients, for example with immune checkpoint inhibitors.

Local administration of anti-hepatocyte growth factor-neutralizing antibody reverts naturally occurring periodontitis

ObjectivesPeriodontitis is a chronic inflammatory process associated with the loss of tooth-supporting tissue. The imbalance of epithelial-mesenchymal signaling is considered to drive disease progression, and hepatocyte growth factor (HGF) is one of the main mediators of this interaction. The aim of this study was to validate the role of HGF in the pathogenesis of periodontitis and to evaluate the effects of anti-HGF neutralizing antibodies. MethodsGingival tissues from cynomolgus monkeys, which naturally develop severe periodontitis, were isolated to establish an in vitro periodontitis model. Periodontitis-affected monkeys were treated by gingival injection of anti-HGF neutralizing antibodies. The therapeutic effects were documented by clinical examination (probing depth and bleeding on probing), histological examination of tissue, and reevaluation of gingival fibroblasts in the in vitro model. ResultsPeriodontitis-affected monkeys contain periodontitis-associated fibroblasts (PAFs) with a pro-inflammatory phenotype that induced pronounced collagen degradation in vitro. This degradation was effectively inhibited by anti-HGF-neutralizing antibodies. Locally administered anti-HGF antibody to monkey gingiva clinically improved the severity of periodontitis. This was also reflected in the tissue histology with lower inflammatory cell infiltrates in treated gingiva than in non-treated gingiva. Moreover, fibroblasts isolated from anti-HGF-treated gingiva demonstrated reduced collagen degradation capacity. ConclusionsOur study confirmed the central role of HGF in the pathogenesis of severe periodontitis in relevant in vitro and in vivo models. The positive effect of anti-HGF treatment provides a strong rationale for the use of anti-HGF-neutralizing antibodies for the treatment of human periodontitis.

Inhibition of MET Signaling with Ficlatuzumab in Combination with Chemotherapy in Refractory AML: Clinical Outcomes and High-Dimensional Analysis.

Acute myeloid leukemia patients refractory to induction therapy or relapsed within one year have poor outcomes. Autocrine production of hepatocyte growth factor by myeloid blasts drives leukemogenesis in pre-clinical models. A phase Ib trial evaluated ficlatuzumab, a first-in-class anti-HGF antibody, in combination with cytarabine in this high-risk population. Dose-limiting toxicities were not observed, and 20 mg/kg was established as the recommended phase II dose. The most frequent treatment-related adverse event was febrile neutropenia. Among 17 evaluable patients, the overall response rate was 53%, all complete remissions. Phospho-proteomic mass cytometry showed potent on-target suppression of p-MET after ficlatuzumab treatment and that attenuation of p-S6 was associated with clinical response. Multiplexed single cell RNA sequencing using prospectively acquired patient specimens identified interferon response genes as adverse predictive factors. The ficlatuzumab and cytarabine combination is well-tolerated with favorable efficacy. High-dimensional analyses at single-cell resolution represent promising approaches for identifying biomarkers of response and mechanisms of resistance in prospective clinical studies.

YYB-101, a Humanized Antihepatocyte Growth Factor Monoclonal Antibody, Inhibits Ovarian Cancer Cell Motility and Proliferation.

Hepatocyte growth factor (HGF) acts as a key regulator in promoting ovarian cancer metastasis. Previously, we observed that YYB-101, a humanized anti-HGF antibody, effectively inhibits ovarian cancer cell migration, invasion, and progression. Here, we evaluated the signaling mechanisms affected by YYB-101 that are important in ovarian cancer cell progression. Using cell migration, invasion and proliferation assays, we evaluated the effects of YYB-101 on A2780/luc and SKOV3 cells. The effects of YYB-101 on signaling molecules were determined by immunocytochemistry and immunoblot analysis. YYB-101 inhibited HGF-induced ovarian cancer cell motility by down-regulating paxillin phosphorylation and actin-cytoskeleton rearrangement. Also, YYB-101 inhibited ovarian cancer cell proliferation by reducing c-MET phosphorylation and activating apoptosis in vitro and in vivo. These effects were significantly enhanced by combining YYB-101 treatment with paclitaxel, a standard chemotherapy drug. YYB-101 can be examined as a new therapeutic agent for the treatment of patients with ovarian cancer.

Inhibition of hepatocyte growth factor/c-Met signalling abrogates joint destruction by suppressing monocyte migration in rheumatoid arthritis

To determine the expression of hepatocyte growth factor (HGF) in RA biological fluids, the role of HGF in monocyte migration and the therapeutic effect of the c-Met inhibitor savolitinib in an arthritis model mice. HGF/c-Met expression in serum, SF and synovial tissues (STs) obtained from RA patients and controls, as well as RA fibroblast-like synoviocytes (FLSs), was evaluated by ELISA and immunostaining. To determine the function of HGF in RA SF, we preincubated RA SF with a neutralizing anti-HGF antibody and measured the chemotactic ability of a human acute monocytic leukaemia cell line (THP-1). Additionally, examinations were conducted of SKG mice treated with savolitinib for 4 weeks. HGF levels in serum from RA patients were significantly higher than those in the controls and were decreased by drug treatment for 24 weeks. Additionally, the HGF level in SF from RA patients was higher than that in SF from OA patients. HGF and c-Met expression was also noted in RA STs. Stimulation of RA FLSs with TNF-α increased HGF/c-Met expression in a concentration-dependent manner, and c-Met signal inhibition suppressed production of fractalkine/CX3CL1 and macrophage inflammatory protein-1α/CCL3. When HGF was removed by immunoprecipitation, migration of THP-1 in RA SF was suppressed. In SKG mice, savolitinib significantly suppressed ankle bone destruction on µCT, with an associated reduction in the number of tartrate-resistant acid phosphatase-positive osteoclasts. HGF produced by inflammation in synovium of RA patients activates monocyte migration to synovium and promotes bone destruction via a chemotactic effect and enhanced chemokine production.

SO023HEPATOCYTE GROWTH FACTOR (HGF) PRECLUDE HIGH-FAT DIET-INDUCED OBESITY AND IMPROVED INSULIN RESISTANCE IN MICE

Abstract Background and Aims Obesity and its associated chronic inflammation in adipose tissue initiate insulin resistance, which is related to several pathologies including hypertension and atherosclerosis. Previous reports demonstrated that circulating hepatocyte growth factor (HGF) level was associated with obesity and type 2 diabetes. However, its precise role in obesity and related-pathology is unclear. Method In this experiment, cardiac-specific over-expression of human HGF in mice (HGF-Tg mice) which showed 4-5 times higher serum HGF levels than wild-type mice were used. We chose cardiac specific HGF overexpression, as other strain of HGF transgenic mice such as liver and kidney specific HGF overexpression mice develop cancer and cystic diseases, which are rare in the heart. In the present study, using HGF-Tg mice and anti-HGF neutralizing antibody (HGF-Ab), we explored the role of HGF in obese and insulin resistance induced by high fat diet (HFD) for 14 weeks (200 or 400ug/week). Results With normal chow diet (ND), there were no significant changes in body weight between WT and HGF-Tg mice. While body weight in wild-type mice fed with HFD for 14 weeks was significantly increased accompanied with insulin resistance, HGF-Tg mice prevented body weight gain and insulin resistance. Insulin resistance in obesity arises from the combination of altered functions of insulin target cells (e.g., liver, skeletal muscle, and adipose tissue) and the accumulation of macrophages that secrete pro-inflammatory mediators in adipose tissue. The accumulation of macrophages and elevated levels of inflammatory mediators in adipose tissue were significantly inhibited in HGF-Tg mice as compared to wild-type mice. In the gWAT, the mRNA levels of the mature macrophage marker F4/80, the chemoattractants, MCP-1 and CXCL2, and the inflammatory cytokines, such as TNF-α and iNOS, were significantly increased in WT mice fed with HFD. However, these levels were markedly reduced in HGF-Tg mice fed with HFD. Additionally, activation of Akt by insulin administration was significantly reduced in the gWAT SM, and liver by HFD; however, this activation was restored in HGF-Tg mice. Moreover, insulin-induced Akt signaling was decreased in HGF-Ab groups as compared to saline group under HFD condition. Importantly, HFD significantly increased the level of HGF mRNA by approximately 2 fold in gWAT, SM, and liver without changing cMet expression. All together, these data indicate that the HGF as one of the systemic gWAT, SM, and liver-derived growth factor plays a role in compensatory mechanism against insulin-resistance through the at least anti-inflammatory effect in adipose tissue. The HFD-induced obesity in wild-type mice treated with HGF-neutralizing antibody showed an exacerbated response to the glucose tolerance test. Conclusion HGF suppresses inflammation in adipose tissue induced by a high-fat diet, and as a result improves systemic insulin resistance. These gain-of-function and loss-of-function studies demonstrated that the elevated HGF level induced by HFD have protective role against obesity and insulin resistance.

Mesenchymal stem cells regulate the Th17/Treg cell balance partly through hepatocyte growth factor in vitro

IntroductionMesenchymal stem cells (MSCs) exert immunomodulatory functions by inducing the development and differentiation of naive T cells into T cells with an anti-inflammatory regulatory T cell (Treg) phenotype. Our previous study showed that hepatocyte growth factor (HGF) secreted by MSCs had immunomodulatory effects in the context of lipopolysaccharide (LPS) stimulation. We hypothesized that HGF is a key factor in the MSC-mediated regulation of the T helper 17 (Th17) cell/regulatory T (Treg) cell balance.MethodsWe investigated the effects of MSCs on the differentiation of CD4+ T cells and the functions of Th17/Treg cells in response to LPS stimulation by performing in vitro coculture experiments. MSCs were added to the upper chambers of cell culture inserts, and CD4+ T cells were plated in the lower chambers, followed by treatment with LPS or an anti-HGF antibody. Th17 (CD4+CD3+RORrt+) and Treg (CD4+CD25+Foxp3+) cell frequencies were analysed by flow cytometry, and the expression of Th17 cell- and Treg cell-related cytokines in the CD4+ T cells or culture medium was measured by quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Neutrophil functions were determined by flow cytometry after a coculture with Th17/Treg cells.ResultsThe percentage of CD4+CD25+Foxp3+ cells was significantly increased in the CD4+ T cell population, while the percentage of CD4+CD3+RORrt+ cells was significantly decreased after MSC coculture. However, the MSC-induced effect was significantly inhibited by the anti-HGF antibody (p < 0.05). Furthermore, MSCs significantly inhibited the CD4+ T cell expression of IL-17 and IL-6 but increased the expression of IL-10 (p < 0.05 or p < 0.01); these effects were inhibited by the anti-HGF antibody (p < 0.05). In addition, CD4+ T cells cocultured with MSCs significantly inhibited neutrophil phagocytic and oxidative burst activities (p < 0.05 or p < 0.01); however, these MSC-induced effects were inhibited by the anti-HGF antibody (p < 0.05).ConclusionThese data suggested that MSCs induced the conversion of fully differentiated Th17 cells into functional Treg cells and thereby modulated the Th17/Treg cell balance in the CD4+ T cell population, which was partly attributed to HGF secreted by the MSCs.

Humanized Anti-hepatocyte Growth Factor Monoclonal Antibody (YYB-101) Inhibits Ovarian Cancer Progression.

Current chemotherapy regimens have certain limitations in improving the survival rates of patients with advanced ovarian cancer. Hepatocyte growth factor (HGF) is important in ovarian cancer cell migration and invasion. This study assessed the effects of YYB-101, a humanized monoclonal anti-HGF antibody, on the growth and metastasis of ovarian cancer cells. YYB-101 suppressed the phosphorylation of the HGF receptor c-MET and inhibited the migration and invasion of SKOV3 and A2780 ovarian cancer cells. Moreover, the combination of YYB-101 and paclitaxel synergistically inhibited tumor growth in an in vivo ovarian cancer mouse xenograft model and significantly increased the overall survival (OS) rate compared with either paclitaxel or YYB-101 alone. Taken together, these findings suggest that YYB-101 has therapeutic potential in ovarian cancer when combined with conventional chemotherapy agents.

Abstract CT078: Cyfi: Results from a Phase Ib expansion cohort of anti-hepatocyte growth factor and cytarabine in relapsed and refractory AML

Abstract Objective: Patients with AML who are refractory to induction therapy or relapse within 1 year have poor outcomes. Elevated serum hepatocyte growth factor (HGF) level is an adverse prognostic factor. Pre-clinical models have shown that myeloid blasts produce HGF in an autocrine fashion and pharmacologic blockade of the HGF/c-Met axis sensitizes blasts to cell death. We initiated a Phase Ib study with dose expansion cohort study to assess the safety and tolerability of the anti-HGF antibody ficlatuzumab with cytarabine in AML patients who are refractory to 7+3 or have relapsed within 1 year of induction. Methodology: The 3 x 3 design was used for the Phase I with an expansion cohort of an additional 13 patients accrued and treated at the MTD. Ficlatuzumab was administered in escalated dosing of 10, 15, or 20 mg/Kg for 4 doses every 2 weeks starting on day 1. 20 mg/Kg dose was used for the expansion cohort. Cytarabine was administered at a fIxed dose of 2 g/m2 on days 2-7. PBMCs were collected at defined time points. Differential expression from multiplexed single cell RNA sequencing (scRNAseq) was used to assess biomarkers predictive of response. Results: Of the total 18 patients accrued thus far, 16 are evaluable. 5 had progressive disease, and 11 responded, all complete responses. Most frequent grade 3/4 TEAEs were febrile neutropenia, LFT abnormalities, and electrolyte disturbance. There was 1 death from sepsis and multi-organ failure on day 23, following ANC recovery, from the disease, and 1 patient who withdrew from the study due to grade 4 gastrointestinal bleed, likely ficlatuzumab related, both prior to response assessment. scRNA sequencing identified a TNF alpha and IFN gamma inflammatory signature that correlates with response to ficlatuzumab at count recovery. Conclusion: Cytarabine and ficlatuzumab is a safe combination with promising efficacy in high risk relapsed and refractory AML. This combination is warranted in further Phase II studies. scRNAseq may be used to identify biomarkers of response. Citation Format: Victoria E. Wang, Charalambos Andreadis. Cyfi: Results from a Phase Ib expansion cohort of anti-hepatocyte growth factor and cytarabine in relapsed and refractory AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT078.

First-in-human phase I trial of anti-hepatocyte growth factor (HGF) antibody (YYB101) in refractory solid tumor patients: Integrative pathologic-genomic analysis and the final results.

3104 Background: It has been demonstrated in vivo that HGF-MET signaling axis is a key molecular determinant in tumor invasion and there is a significant association in HGF expression and mesenchymal phenotype in addition to immune cell recruitment. We have developed a HGF neutralizing humanized monoclonal antibody antibody, YYB-101. The aim of this study was to determine the maximum tolerate dose (MTD), safety, pharmacokinetics (PK), and pharmacodynamics (PD) of YYB101, in patients with refractory solid tumors. Methods: YYB101 was administered intravenously at once every 2 weeks doses of 0.3, 1, 3, 5, 10, 20, 30 mg/kg, according to a 3+3 dose escalation design. Enrolled patients were planned to receive YYB101 until disease progression or intolerable toxicity. The escalation and expansion cohorts (20mg/kg) were completed. Pre-planned biomarker analysis was performed in parallel. Results: 39 heavily pre-treated refractory cancer patients were enrolled and received YYB101. No DLT was observed. YYB101 demonstrated dose proportional PK up to the dose of 30 mg/kg. No patients discontinued treatment because of adverse events. Based on PK analysis and toxicity data, the recommended dose was determined as 20 mg/kg. Of 39 evaluation patients, there was 1 confirmed partial response for &gt; +14months (2.5%, N = 1; 1 (of 2) sebaceous carcinoma) and 17 stable disease as best response (43.5%, N = 17; 7 (of 13) CRC, 3 (of 4) melanoma, 1 (of 2) sebaceous carcinoma, 1 (of 3) gastric, 1 (of 1) basal cell carcinoma, 2 (of 10) ovarian cancer, 1 (of 1) HCC, 1 (of 1) lung cancer). Of note, 1 sebaceous carcinoma patient who have failed to ≥2+ lines of chemotherapy, have been responding to YYB for 14 months. The MET and HGF expressions by immunohistochemistry (IHC) were evaluated in 19 and 17 tumor specimens, respectively. Neither protein expressions were significant predictors for treatment response to anti-HGF antibody. However, we have observed significant reduction in HGF in responders to YYB. Two long-term responders had mesenchymal signature in RNA sequencing. Conclusions: YYB101 has a favorable safety profile in patients with refractory solid tumors and a dose-proportional PK. Efficacy data are encouraging and phase II combination therapy with YYB101 is planned to be open in metastatic CRC patients as salvage treatment. The predictive power of mesenchymal signature in YYB responders will be defined prospectively. Clinical trial information: 02499224.

The role and mechanism of mesenchymal stem cell in modulating human pulmonary microvascule endothelial cell permeability via paracrine hepatocyte growth factor

Objective: To explore the role and mechanism of mesenchymal stem cell (MSC) in modulating human pulmonary microvascular endothelial cell (HPMECs) permeability via hepatocyte growth factor (HGF). Methods: The study introduced a co-cultured model between HPMECs and human mesenchymal stem cell conditioned media (MSC-CM) collected from MSCs after 24 h hypoxia culture, and meanwhile HGF was neutralized in MSC-CM by anti-HGF antibody respectively, followed by lipopolysaccharide (LPS) stimulation. Finally, the following measurements were performed: the permeability of HPMECs, the protein expression of vascular endothelial cadherin (VE-cadherin), Occludin in HPMECs by Western blot, HPMECs apoptosis by Annexin V-FITC/PI and HPMECs proliferation by 3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di- phenytetrazoliumromide(MTT). Results: Compared to LPS group (4.15±0.88), MSC-CM reduced endothelial paracellular permeability injured by LPS(1.56±0.36, P<0.01), however, the MSC-CM effect was significantly blocked by anti-HGF antibody(3.11±0.74, P<0.05). Furthermore MSC-CM significantly increased the expression of VE-cadherin(0.71±0.05 vs. 0.38±0.19, P<0.05)and Occludin protein(0.96±0.05 vs. 0.51±0.02, P<0.05) in HPMECs, which was significantly blocked by anti-HGF antibody (P<0.05). MSC-CM significantly reduced the number of early apoptotic cells (6.82±1.80 vs. 17.09±1.89, P<0.05). However, the effect of MSC-CM was significantly blocked by neutralizing HGF (12.07±0.98, P<0.01). The cell viability results by MTT assay confirmed that MSC-CM(6.82±1.80, P<0.05)restored cell viability to a greater extent than LPS stimulation only(0.47±0.09), and meanwhile the MSC-CM effect was significantly inhibited by neutralizing HGF from MSC-CM with anti-HGF antibody (0.69±0.29, P<0.05). Conclusion: HGF secreted by MSCs reduces endothelial cell paracelluar permeability induced by LPS, and the possible mechanisms include remodelling of endothelial intercellular adherence junction, promoting endothelial cell proliferation and restraining endothelial cell apoptosis.

MET Tyrosine Kinase Inhibition Enhances the Antitumor Efficacy of an HGF Antibody.

Receptor tyrosine kinase therapies have proven to be efficacious in specific cancer patient populations; however, a significant limitation of tyrosine kinase inhibitor (TKI) treatment is the emergence of resistance mechanisms leading to a transient, partial, or complete lack of response. Combination therapies using agents with synergistic activity have potential to improve response and reduce acquired resistance. Chemoreagent or TKI treatment can lead to increased expression of hepatocyte growth factor (HGF) and/or MET, and this effect correlates with increased metastasis and poor prognosis. Despite MET's role in resistance and cancer biology, MET TKI monotherapy has yielded disappointing clinical responses. In this study, we describe the biological activity of a selective, oral MET TKI with slow off-rate and its synergistic antitumor effects when combined with an anti-HGF antibody. We evaluated the combined action of simultaneously neutralizing HGF ligand and inhibiting MET kinase activity in two cancer xenograft models that exhibit autocrine HGF/MET activation. The combination therapy results in additive antitumor activity in KP4 pancreatic tumors and synergistic activity in U-87MG glioblastoma tumors. Pharmacodynamic characterization of biomarkers that correlate with combination synergy reveal that monotherapies induce an increase in the total MET protein, whereas combination therapy significantly reduces total MET protein levels and phosphorylation of 4E-BP1. These results hold promise that dual targeting of HGF and MET by combining extracellular ligand inhibitors with intracellular MET TKIs could be an effective intervention strategy for cancer patients who have acquired resistance that is dependent on total MET protein. Mol Cancer Ther; 16(7); 1269-78. ©2017 AACR.

Hepatocyte growth factor produced in lung fibroblasts enhances non-small cell lung cancer cell survival and tumor progression

BackgroundThe influence of lung fibroblasts on lung cancer progression is not fully understood.MethodsLung fibroblasts (HFL1, MRC5, and IMR90 cells) and non-small cell lung cancer (NSCLC)-derived cell lines (A549, EBC1, and HI1017) were cultured under serum-free conditions, and the resulting culture media were designated “cell-conditioned media”. Cell survival (viability) was assessed by WST-1 assay. Concentrations of hepatocyte growth factor (HGF) were measured by ELISA. The BALB/c-nu mouse strain was used for the xenograft model.ResultsLung fibroblast-conditioned media enhanced the survival of the three NSCLC cell lines tested. HGF was produced to a greater extent by lung fibroblasts than NSCLC cells. Exogenous HGF enhanced the survival of NSCLC cells. Either an anti-HGF neutralizing antibody or the Met inhibitor PHA-665752 inhibited the fibroblast-conditioned media-enhanced survival of NSCLC cells. The co-inoculation of mice with NSCLC cells and fibroblasts enhanced tumorigenicity and tumor progression in a mouse xenograft model. PHA-665752 significantly inhibited tumor progression that occurred after the co-inoculation of NSCLC cells and fibroblasts. In addition, HGF production by fibroblasts was stimulated by NSCLC cells.ConclusionsThe current study provides evidence for an interaction between fibroblasts and NSCLC cells via the HGF/Met signaling pathway, which affects NSCLC cell survival and tumor progression. These findings may contribute to the development of anti-cancer-associated fibroblast therapeutic strategies.Trial registrationNo trial registration is required because this study is not a clinical trial. This study does not include any participants or patients.

Phase I study of the anti-HGF monoclonal antibody (mAb), ficlatuzumab, and cetuximab in cetuximab-resistant, recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC).

6038 Background: Cetuximab, an anti-EGFR mAb, is approved for R/M HNSCC but only a minority benefits. Activation of cMet, the receptor for hepatocyte growth factor (HGF), overcomes EGFR inhibition in preclinical models and high serum HGF is associated with resistance in patients (pts). We conducted a phase I trial evaluating the combination of cetuximab and ficlatuzumab, an IgG1 anti-HGF mAb, in pts with cetuximab-resistant, R/M HNSCC. Methods: In this Narayana k-in-a-row phase I design, cetuximab 500 mg/m2 was administered every 2 weeks. Ficlatuzumab dose tiers were 10 mg/kg (tier 1) or 20 mg/kg IV every 2 weeks (tier 2), with inter-patient escalation or de-escalation based on cumulative dose-limiting toxicities (DLT). The recommended phase II dose (RP2D) was set at tier 2 if no DLTs were observed after 8 enrolled pts; expansion continued to n = 12. Key eligibility criteria: R/M HNSCC; recurrence within 6 months of cetuximab-radiation or progression during/within 6 months of palliative cetuximab; ECOG PS 0-1. Candidate biomarkers included serum Veristrat, a proteomic classifier in lung cancer where “good” predicts benefit from anti-EGFR therapy and “poor” indicates resistance and poor prognosis. Results: From Sept 2015–June 2016, 12 pts were enrolled and treated. Primary site: 1 oral cavity; 3 oropharynx (1 p16+); 2 hypopharynx; 5 larynx; 1 external auditory canal. Platinum-refractory: 11/12. Veristrat: 8 poor; 4 good. Three pts were treated at tier 1 and 9 at tier 2. No DLTs were observed. Grade 3 adverse events included: edema (1), hypoalbuminemia (1), infection (2), thromboembolic event (2). Median PFS and OS at RP2D were 6.0 mos (90% CI = 2.0 mos–not reached) and 8.2 mos (90% CI = 2.7 mos–not reached), respectively. Response rate was 17% (90% CI = 0–28%): 2/12 partial response (PR); 1/3 at 10 mg/kg; 1/9 at 20mg/kg. Clinical benefit rate (PR + stable disease) was 67%. Veristrat was not associated with PFS. Conclusions: The RP2D is cetuximab 500 mg/m2 and ficlatuzumab 20 mg/kg every 2 weeks. This well-tolerated combination demonstrated promising activity in pts with poor prognosis, cetuximab-resistant R/M HNSCC. Phase II testing is justified. Clinical trial information: NCT02277197.

Fibronectin and Hepatocyte Growth Factor Produced by Lung Fibroblasts Augment Migration and Invasion of Malignant Pleural Mesothelioma Cells.

The interaction between fibroblasts and malignant pleural mesothelioma (MPM) cells is not well understood. Lung fibroblasts (HFL1, MRC5 and IMR90) and MPM cells (H28, H226 and H2052) were cultured under serum-free conditions and the resulting culture media were collected. Migration and invasion of MPM cells were assessed by chemotaxis and Matrigel assays, respectively. Lung fibroblast-derived media enhanced the migration and invasion of the three tested MPM cell lines. Fibronectin and hepatocyte growth factor (HGF) were produced by lung fibroblasts. Exogenous fibronectin and HGF also enhanced the migration and invasion of the three MPM cells, respectively. Neutralizing anti-HGF antibody inhibited the invasion of H28 cells enhanced by fibroblast-derived media. In addition, the production of fibronectin and HGF was stimulated by MPM cell-derived media. The current study provides additional evidence that might contribute to the development of antitumor-associated fibroblast therapeutic strategies.