The c-MET receptor tyrosine kinase contributes to neutrophil-driven pathology in cutaneous leishmaniasis.

Neutrophils are the first line of defence against invading pathogens. Although neutrophils are well-known professional killers, some pathogens including Leishmania (L.) parasites survive in neutrophils, using these cells to establish infection. Manipulation of neutrophil recruitment to the infection site is therefore of interest in this cutaneous disease. The c-MET tyrosine kinase receptor was shown to promote neutrophil migration to inflamed sites. Here, we investigated the importance of c-MET expression on neutrophils in their recruitment to the infection site and the role of c-Met expression in the pathology of leishmaniasis. Following infection with L. mexicana, mice with conditional deletion of c-MET in neutrophils controlled significantly better their lesion development and parasite burden compared to similarly infected wild type mice. Our data reveal a specific role for c-MET activation in Leishmania-induced neutrophil infiltration, a process correlating with their negative role in the pathology of the diseases. We further show that c-MET phosphorylation is observed in established cutaneous lesions. Exposure to L. mexicana upregulated c-Met expression predominantly in infected neutrophils and c-Met expression influenced ROS release by neutrophils. In addition, pharmacological inhibition of c-MET, administrated once the lesion is established, induced a significant decrease in lesion size associated with diminished infiltration of neutrophils. Both genetic ablation of c-MET in neutrophils and systemic inhibition of c-MET locally resulted in higher levels of CD4+T cells producing IFNγ, suggesting a crosstalk between neutrophils and these cells. Collectively, our data show that c-MET activation in neutrophils contributes to their recruitment following infection, and that L. mexicana induction of c-MET on neutrophils impacts the local pathology associated with this disease. Our results suggest a potential use for this inhibitor in the control of the cutaneous lesion during this parasitic infection.

MET overexpression contributes to STAT4-PD-L1 signaling activation associated with tumor-associated, macrophages-mediated immunosuppression in primary glioblastomas.

BACKGROUND: Dysregulated receptor tyrosine kinases, such as the mesenchymal-epidermal transition factor (MET), have pivotal role in gliomas. MET and its interaction with the tumor microenvironment have been previously implicated in secondary gliomas. However, the contribution of MET gene to tumor cells' ability to escape immunosurveillance checkpoints in primary gliomas, especially in glioblastoma (GBM), which is a WHO grade 4 glioma with the worst overall survival, is still poorly understood. METHODS: We investigated the relationship between MET expression and glioma microenvironment by using multiomics data and aimed to understand the potential implications of MET in clinical practice through survival analysis. RNA expression data from a total of 1243 primary glioma samples (WHO grades 2-4) were assembled, incorporating The Cancer Genome Atlas, Chinese Glioma Genome Atlas, and GSE16011 data sets. RESULTS: Pearson's correlation test from the three data sets indicated that MET showed a robust correlation with programmed death-ligand 1 (PD-L1) and STAT pathways. Western blot analysis revealed that in GBM cell lines (N33 and LN229), PD-L1 and phosphorylated STAT4 were upregulated by MET activation treatment with hepatocyte growth factor and were downregulated on MET suppression by PLB-1001. Tumor tissue microarray analysis indicated a positive correlation between MET and PD-L1 and macrophage-associated markers. Chromatin immunoprecipitation-PCR assay showed enrichment of STAT4 in the PD-L1 DNA. Transwell co-culture and chemotaxis assays revealed that knockdown of MET in GBM cells inhibited macrophage chemotaxis. Moreover, we performed CIBERSORTx and single-cell RNA sequencing data analysis which revealed an elevated number of macrophages in glioma samples with MET overexpression. Kaplan-Meier survival analysis indicated that activation of the MET/STAT4/PD-L1 pathway and upregulation of macrophages were associated with shorter survival time in patients with primary GBM. CONCLUSIONS: These data indicated that the MET-STAT4-PD-L1 axis and tumor-associated macrophages might enforce glioma immune evasion and were associated with poor prognosis in GBM samples, suggesting potential clinical strategies for targeted therapy combined with immunotherapy in patients with primary GBM.

Discovery of amivantamab (JNJ-61186372), a bispecific antibody targeting EGFR and MET.

A bispecific antibody (BsAb) targeting the epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition factor (MET) pathways represents a novel approach to overcome resistance to targeted therapies in patients with non-small cell lung cancer. In this study, we sequentially screened a panel of BsAbs in a combinatorial approach to select the optimal bispecific molecule. The BsAbs were derived from different EGFR and MET parental monoclonal antibodies. Initially, molecules were screened for EGFR and MET binding on tumor cell lines and lack of agonistic activity toward MET. Hits were identified and further screened based on their potential to induce untoward cell proliferation and cross-phosphorylation of EGFR by MET via receptor colocalization in the absence of ligand. After the final step, we selected the EGFR and MET arms for the lead BsAb and added low fucose Fc engineering to generate amivantamab (JNJ-61186372). The crystal structure of the anti-MET Fab of amivantamab bound to MET was solved, and the interaction between the two molecules in atomic details was elucidated. Amivantamab antagonized the hepatocyte growth factor (HGF)-induced signaling by binding to MET Sema domain and thereby blocking HGF ß-chain-Sema engagement. The amivantamab EGFR epitope was mapped to EGFR domain III and residues K443, K465, I467, and S468. Furthermore, amivantamab showed superior antitumor activity over small molecule EGFR and MET inhibitors in the HCC827-HGF in vivo model. Based on its unique mode of action, amivantamab may provide benefit to patients with malignancies associated with aberrant EGFR and MET signaling.

Development of Antigen-specific Chimeric Antigen Receptor KHYG-1 Cells for Glioblastoma.

BACKGROUND/AIM: Glioblastoma is the most common cancer among primary brain tumors, however, its prognosis and treatment advances are very poor. Here, we investigated whether c-Met, FOLR1, and AXL proteins are promising targets for chimeric antigen receptor (CAR) T-cell therapy, for they are known to be over-expressed in a variety of solid tumors. MATERIALS AND METHODS: CAR constructs were prepared and CAR KHYG-1 cells targeting c-Met, FOLR1, or AXL were made by lentiviral transduction. The activity of CAR KHYG-1 cells against cancer cells was measured by cytokine secretion and cell lysis assays. RESULTS: c-Met and AXL were over-expressed in most glioblastoma cell lines (11/13), but not in neuroblastoma cell lines (0/8). FOLR1 was over-expressed only in one among 16 glioblastoma cell lines. Our antigen-specific CAR KHYG-1 cells eradicated target positive glioblastoma cells selectively. CONCLUSION: Anti-c-Met and anti-AXL CAR NK or T cells could be effective in glioblastoma cells.

Anti-tumor efficacy of human anti-c-met CAR-T cells against papillary renal cell carcinoma in an orthotopic model.

Chimeric antigen receptor (CAR)-T cell therapy has shown salient efficacy in cancer immunotherapy, particularly in the treatment of B cell malignancies. However, the efficacy of CAR-T for solid tumors remains inadequate. In this study, we displayed that c-met is an appropriate therapeutic target for papillary renal cell carcinoma (PRCC) using clinical samples, developed an anti-human c-met CAR-T cells, and investigated the anti-tumor efficacy of the CAR-T cells using an orthotopic mouse model as pre-clinical research. Administration of the anti-c-met CAR-T cells induced marked infiltration of the CAR-T cells into the tumor tissue and unambiguous suppression of tumor growth. Furthermore, in combination with axitinib, the anti-tumor efficacy of the CAR-T cells was synergistically augmented. Taken together, our current study demonstrated the potential for clinical application of anti-c-met CAR-T cells in the treatment of patients with PRCC.

Down-regulation of the tumor suppressor miR-34a contributes to head and neck cancer by up-regulating the MET oncogene and modulating tumor immune evasion.

BACKGROUND: MicroRNAs (miRs) have been shown to play an important role in tumorigenesis, including in head and neck squamous cell carcinoma (HNSCC). The miR-34 family is thought to play a role in tumor suppression, but the exact mechanism of their action in HNSCC is not well understood. Moreover, the impact of chromosomal changes and mutation status on miR-34a expression remains unknown. METHODS: Differential expression of miR-34a, MET, and genomic alterations were assessed in the Cancer Genome Atlas (TCGA) datasets as well as in primary HNSCC and adjacent normal tissue. The biological functions of miR-34a in HNSCC were investigated in samples derived from primary human tumors and HNSCC cell lines. The expression of MET was evaluated using immunohistochemistry, and the molecular interaction of miR-34a and MET were demonstrated by RNA pulldown, RNA immunoprecipitation, luciferase reporter assay, and rescue experiments. Lastly, locked nucleic acid (LNA) miRs in mouse xenograft models were used to evaluate the clinical relevance of miR-34a in HNSCC tumor growth and modulation of the tumor microenvironment in vivo. RESULTS: Chromosome arm 1p loss and P53 mutations are both associated with lower levels of miR-34a. In HNSCC, miR-34a acts as a tumor suppressor and physically interacts with and functionally targets the proto-oncogene MET. Our studies found that miR-34a suppresses HNSCC carcinogenesis, at least in part, by downregulating MET, consequently inhibiting HNSCC proliferation. Consistent with these findings, administration of LNA-miR-34a in an in vivo model of HNSCC leads to diminished HNSCC cell proliferation and tumor burden in vitro and in vivo, represses expression of genes involved in epithelial-mesenchymal transition, and negates the oncogenic effect of MET in mouse tumors. Consistently, LNA-miR-34a induced a decreased number of immunosuppressive PDL1-expressing tumor-associated macrophages in the tumor microenvironment. In HNSCC patient samples, higher levels of miR-34a are significantly associated with a higher frequency of Th1 cells and CD8 naïve T cells. CONCLUSIONS: Our results demonstrate that miR-34a directly targets MET and maintains anti-tumor immune activity. We propose miR-34a as a potential new therapeutic approach for HNSCC.

A MET Targeting Antibody-Drug Conjugate Overcomes Gemcitabine Resistance in Pancreatic Cancer.

PURPOSE: Pancreatic cancer is an aggressive disease associated with a poor 5-year overall survival. Most patients are ineligible for surgery due to late diagnosis and are treated primarily with chemotherapy with very limited success. Pancreatic cancer is relatively insensitive to chemotherapy due to multiple factors, including reduced bioavailability of drugs to tumor cells. One strategy to improve drug efficacy with reduced toxicity is the development of antibody-drug conjugates (ADC), which have now been used successfully to treat both solid and liquid tumors. Here, we evaluate the efficacy of TR1801-ADC, a newly developed ADC composed of a MET antibody conjugated to the highly potent pyrrolobenzodiazepine toxin-linker, tesirine. EXPERIMENTAL DESIGN: We first evaluated MET expression and subcellular localization in pancreatic cancer cell lines, human tumors, and patient-derived xenografts (PDX). We then tested TR1801-ADC efficacy in vitro in pancreatic cancer cell lines. Preclinical evaluation of TR1801-ADC efficacy was conducted on PDXs selected on the basis of their MET expression level. RESULTS: We show that MET is highly expressed and located at the plasma membrane of pancreatic cancer cells. We found that TR1801-ADC induces a specific cytotoxicity in pancreatic cancer cell lines and a profound tumor growth inhibition, even in a gemcitabine-resistant tumor. We also noted synergism between TR1801-ADC and gemcitabine in vitro and an improved response to the combination in vivo. CONCLUSIONS: Together, these results suggest the promise of agents such as TR1801-ADC as a novel approach to the treatment of pancreatic cancer.

A receptor-antibody hybrid hampering MET-driven metastatic spread.

BACKGROUND: The receptor encoded by the MET oncogene and its ligand Hepatocyte Growth Factor (HGF) are at the core of the invasive-metastatic behavior. In a number of instances genetic alterations result in ligand-independent onset of malignancy (MET addiction). More frequently, ligand stimulation of wild-type MET contributes to progression toward metastasis (MET expedience). Thus, while MET inhibitors alone are effective in the first case, combination therapy with ligand inhibitors is required in the second condition. METHODS: In this paper, we generated hybrid molecules gathering HGF and MET inhibitory properties. This has been achieved by 'head-to-tail' or 'tail-to-head' fusion of a single chain Fab derived from the DN30 MET antibody with a recombinant 'ad-hoc' engineered MET extracellular domain (decoyMET), encompassing the HGF binding site but lacking the DN30 epitope. RESULTS: The hybrid molecules correctly bind MET and HGF, inhibit HGF-induced MET downstream signaling, and quench HGF-driven biological responses, such as growth, motility and invasion, in cancer cells of different origin. Two metastatic models were generated in mice knocked-in by the human HGF gene: (i) orthotopic transplantation of pancreatic cancer cells; (ii) subcutaneous injection of primary cells derived from a cancer of unknown primary. Treatment with hybrid molecules strongly affects time of onset, number, and size of metastatic lesions. CONCLUSION: These results provide a strategy to treat metastatic dissemination driven by the HGF/MET axis.

The efficacy and safety of onartuzumab in patients with solid cancers: A meta-analysis of randomized trials.

BACKGROUND: Onartuzumab, a humanized monovalent monoclonal antibody to the MET protein, has been tested in various cancers. We conducted a meta-analysis of randomized phase II and III clinical trials to investigate the efficacy and safety of onartuzumab in solid cancers. METHODS: We searched PubMed, PMC, EMBASE, and the Cochrane library databases. We included randomized phase II or III trials that evaluated the additional benefits of onartuzumab in comparison with the standard treatments. Data on progression-free survival (PFS), overall survival (OS), and adverse events (AEs) were pooled and analyzed. RESULTS: From nine studies, a total of 2,138 patients were included in the meta-analysis. The addition of onartuzumab to the standard treatment resulted in no improvement of PFS (hazard ratio (HR) = 1.00 [95% confidence interval (CI), 0.90-1.11], P = 0.93) and OS (HR = 1.08 [95% CI, 0.94-1.23], P = 0.29). In the subgroup analysis with patients with non-small-cell lung cancer (NSCLC), onartuzumab was not associated with a significant improvement of OS (HR = 1.12 [95% CI, 0.93-1.34], P = 0.23) and PFS (HR = 1.05 [95% CI, 0.91-1.21], P = 0.52). With respect to AEs, onartuzumab increased the incidence of hypoalbuminemia (odds ratio (OR) = 14.8 [95% CI, 3.49-62.71], P < 0.001), peripheral edema (OR = 6.52 [95% CI, 3.60-11.81], P < 0.001), neutropenia (OR = 1.36 [95% CI, 1.03-1.79], P = 0.03), thrombocytopenia (OR = 1.98 [95% CI, 1.03-3.81], P = 0.04), and venous thrombotic events (OR = 3.05 [95% CI, 1.39-6.71], P = 0.006). CONCLUSION: This meta-analysis indicates that the addition of onartuzumab to the standard treatments had no definite survival benefit with increased severe toxicities in patients with solid cancer.

Pharmaceutical strategies in the emerging era of antibody-based biotherapeutics for the treatment of cancers overexpressing MET receptor tyrosine kinase.

Pharmaceutical innovation in the development of novel antibody-based biotherapeutics with increased therapeutic indexes makes MET-targeted cancer therapy a clinical reality.

The AKT-independent MET-V-ATPase-MTOR axis suppresses liver cancer vaccination.

Despite recent progress in hepatitis treatment, there have been no significant advances in the development of liver cancer vaccines in recent years. In this study, we investigated the regulatory effect and potential mechanism of hepatocyte growth factor receptor (MET, also known as HGFR) on tumor vaccinations for liver cancer in mice. Herein, we demonstrate that MET expression is significantly associated with the immunogenicity of liver cancer in mice and humans, and that MET depletion dramatically enhances the protective efficacy of chemotherapy-based anti-liver cancer vaccination. Mechanistically, MET repressed liver cancer immunogenicity independent of the traditional PI3K-AKT cascade, and MET interacted with vacuolar ATP synthase (V-ATPase) and mediated the activation of mammalian target of rapamycin (MTOR), thus suppressing liver cancer immunogenicity. The efficacy of chemotherapy-based liver cancer vaccination was markedly enhanced by targeting the MET-V-ATPase-MTOR axis, highlighting a translational strategy for identifying MET-associated drug candidates for cancer prevention.

Phase I Dose-Escalation and -Expansion Study of Telisotuzumab (ABT-700), an Anti-c-Met Antibody, in Patients with Advanced Solid Tumors.

This first-in-human phase I study evaluated the pharmacokinetics, safety, and preliminary efficacy of telisotuzumab, formerly called ABT-700, an antagonistic antibody directed against c-Met. For dose escalation (3+3 design), 3 to 6 patients with advanced solid tumors were enrolled into four dose cohorts (5-25 mg/kg). In the dose-expansion phase, a subset of patients was prospectively selected for MET amplification (FISH screening). Patients received telisotuzumab intravenously on day 1 every 21 days. For dose expansion, 15 mg/kg was chosen as the dose on the basis of safety, pharmacokinetics, and other data from the escalation cohorts. Forty-five patients were enrolled and received at least one dose of telisotuzumab (dose escalation, n = 15; dose expansion, n = 30). Telisotuzumab showed a linear pharmacokinetics profile; peak plasma concentration was proportional to dose level. There were no acute infusion reactions and no dose-limiting toxicities were observed. The most common treatment-related adverse events included hypoalbuminemia (n = 9, 20.0%) and fatigue (n = 5, 11.1%). By Response Evaluation Criteria In Solid Tumors (RECIST), 4 of 10 (40.0%) patients with MET-amplified tumors had confirmed partial response in target lesions (one ovarian, two gastric, and one esophageal), two (20.0%) had stable disease, three (30.0%) had progressive disease; one patient was unable to be evaluated. Among patients with nonamplified tumors (n = 35), no objective responses were observed; however, 11 patients had stable disease per RECIST criteria. In conclusion, telisotuzumab has an acceptable safety profile with clinical activity observed in patients with MET-amplified advanced solid tumors.

Targeting c-Met on gastric cancer cells through a fully human fab antibody isolated from a large naive phage antibody library.

PURPOSE: The aberrant Hepatocyte growth factor (HGF)/ mesenchymal-epithelial transition factor (c-Met) signaling pathway in various malignancies and its correlation with tumor invasion and poor prognosis has validated c-Met as a compelling therapeutic target. Up to now, several monoclonal antibodies and small molecule inhibitors targeting c-Met have been introduced with different outcomes, none are yet clinically approved. Toward the generation of novel fully human anti-c-Met molecules, we generated a large naïve Fab antibody library using phage display technology, which subsequently screened for novel Fabs against c-Met. METHODS: A phage library, with a functional size of 5.5 × 1010 individual antibody clones, was prepared using standard protocols and screened for c-Met-specific Fabs by successive rounds of panning. A panel of Fabs targeting c-Met were isolated, from which four clones were selected and further characterized by DNA sequencing. The c-Met binding ability of our selected Fabs was evaluated by c-Met ELISA assay and flow cytometry techniques. RESULTS: Among the confirmed anti-c-Met Fabs, clone C16, showed the highest affinity (Kaff: 0.3 × 109 M-1), and 63% binding to MKN45 cells (a human gastric adenocarcinoma cell-line) as compared to c-Met negative T47D cell-line (9.03%). CONCLUSION: Together, our study presents a single-pot antibody library, as a valuable source for finding a range of antigen-specific Fab antibodies, and also, a fully human, high affinity and specific anti c-Met Fab antibody, C16, which has the potential of developing as a therapeutic or chemotherapeutic delivery agent for killing c-Met-positive tumor cells.

A novel non-agonist c-Met antibody drug conjugate with superior potency over a c-Met tyrosine kinase inhibitor in c-Met amplified and non-amplified cancers.

c-Met is a well-characterized oncogene that is associated with poor prognosis in many solid tumor types. While responses to c-Met inhibitors have been observed in clinical trials, activity appears to be limited to those with MET gene amplifications or mutations. We developed a c-Met targeted antibody-drug conjugate (ADC) with preclinical activity in the absence of MET gene amplification or mutation, and activity even in the context of moderate protein expression. The ADC utilized a high-affinity c-Met antibody (P3D12), that induced c-Met degradation with minimal activation of c-Met signaling, or mitogenic effect. P3D12 was conjugated to the tubulin inhibitor toxin MMAF via a cleavable linker (vc-MMAF). P3D12-vc-MMAF demonstrated potent in vitro activity in c-Met protein-expressing cell lines regardless of MET gene amplification or mutation status, and retained activity in cell lines with medium-low c-Met protein expression. In contrast, the c-Met tyrosine kinase inhibitor (TKI) PHA-665752 slowed tumor cell growth in vitro only in the context of MET gene amplification or very high protein expression. This differential activity was even more marked in vivo. P3D12-vc-MMAF demonstrated robust inhibition of tumor growth in the MET gene amplified MKN-45 xenograft model, and similar results in H1975, which expresses moderate levels of wild type c-Met without genomic amplification. By comparison, the c-Met TKI, PHA-665752, demonstrated modest tumor growth inhibition in MKN-45, and no inhibition at all in H1975. Taken together, these data suggest that P3D12-vc-MMAF may have a superior clinical profile in treating c-Met positive malignancies in contrast to c-Met pathway inhibitors.

GD2-directed CAR-T cells in combination with HGF-targeted neutralizing antibody (AMG102) prevent primary tumor growth and metastasis in Ewing sarcoma.

Ewing sarcoma (EWS) is the second most common and aggressive type of metastatic bone tumor in adolescents and young adults. There is unmet medical need to develop and test novel pharmacological targets and novel therapies to treat EWS. Here, we found that EWS expresses high levels of a p53 isoform, delta133p53. We further determined that aberrant expression of delta133p53 induced HGF secretion resulting in tumor growth and metastasis. Thereafter, we evaluated targeting EWS tumors with HGF receptor neutralizing antibody (AMG102) in preclinical studies. Surprisingly, we found that targeting EWS tumors with HGF receptor neutralizing antibody (AMG102) in combination with GD2-specific, CAR-reengineered T-cell therapy synergistically inhibited primary tumor growth and establishment of metastatic disease in preclinical models. Furthermore, our data suggested that AMG102 treatment alone might increase leukocyte infiltration including efficient CAR-T access into tumor mass and thereby improves its antitumor activity. Together, our findings warrant the development of novel CAR-T-cell therapies that incorporate HGF receptor neutralizing antibody to improve therapeutic potency, not only in EWS but also in tumors with aberrant activation of the HGF/c-MET pathway.

TR1801-ADC: a highly potent cMet antibody-drug conjugate with high activity in patient-derived xenograft models of solid tumors.

cMet is a well-characterized oncogene that is the target of many drugs including small molecule and biologic pathway inhibitors, and, more recently, antibody-drug conjugates (ADCs). However, the clinical benefit from cMet-targeted therapy has been limited. We developed a novel cMet-targeted 'third-generation' ADC, TR1801-ADC, that was optimized at different levels including specificity, stability, toxin-linker, conjugation site, and in vivo efficacy. Our nonagonistic cMet antibody was site-specifically conjugated to the pyrrolobenzodiazepine (PBD) toxin-linker tesirine and has picomolar activity in cancer cell lines derived from different solid tumors including lung, colorectal, and gastric cancers. The potency of our cMet ADC is independent of MET gene copy number, and its antitumor activity was high not only in high cMet-expressing cell lines but also in medium-to-low cMet cell lines (40 000-90 000 cMet/cell) in which a cMet ADC with tubulin inhibitor payload was considerably less potent. In vivo xenografts with low-medium cMet expression were also very responsive to TR1801-ADC at a single dose, while a cMet ADC using a tubulin inhibitor showed a substantially reduced efficacy. Furthermore, TR1801-ADC had excellent efficacy with significant antitumor activity in 90% of tested patient-derived xenograft models of gastric, colorectal, and head and neck cancers: 7 of 10 gastric models, 4 of 10 colorectal cancer models, and 3 of 10 head and neck cancer models showed complete tumor regression after a single-dose administration. Altogether, TR1801-ADC is a new generation cMet ADC with best-in-class preclinical efficacy and good tolerability in rats.

Molecular Epitope Determination of Aptamer Complexes of the Multidomain Protein C-Met by Proteolytic Affinity-Mass Spectrometry.

C-Met protein is a glycosylated receptor tyrosine kinase of the hepatocyte growth factor (HGF), composed of an α and a ß chain. Upon ligand binding, C-Met transmits intracellular signals by a unique multi-substrate docking site. C-Met can be aberrantly activated leading to tumorigenesis and other diseases, and has been recognized as a biomarker in cancer diagnosis. C-Met aptamers have been recently considered a useful tool for detection of cancer biomarkers. Herein we report a molecular interaction study of human C-Met expressed in kidney cells with two DNA aptamers of 60 and 64 bases (CLN0003 and CLN0004), obtained using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure. Epitope peptides of aptamer-C-Met complexes were identified by proteolytic affinity-mass spectrometry in combination with SPR biosensor analysis (PROTEX-SPR-MS), using high-pressure proteolysis for efficient digestion. High affinities (KD , 80-510 nM) were determined for aptamer-C-Met complexes, with two-step binding suggested by kinetic analysis. A linear epitope, C-Met (381-393) was identified for CLN0004, while the CLN0003 aptamer revealed an assembled epitope comprised of two peptide sequences, C-Met (524-543) and C-Met (557-568). Structure modeling of C-Met-aptamers were consistent with the identified epitopes. Specificities and affinities were ascertained by SPR analysis of the synthetic epitope peptides. The high affinities of aptamers to C-Met, and the specific epitopes revealed render them of high interest for cellular diagnostic studies.

Development of c­MET­specific chimeric antigen receptor­engineered natural killer cells with cytotoxic effects on human liver cancer HepG2 cells.

In recent years, cellular immunotherapy has served an important role in the combined treatment of hepatocellular carcinoma. The possibility of specific cell therapies for the treatment of solid tumours has been further explored following the success of chimeric antigen receptor (CAR)­T cell therapy in the treatment of haematological tumours. The present study aimed to evaluate the specificity and efficiency of c­MET­targeted CAR­NK cell immunotherapy on human liver cancer in vitro. A CAR structure that targeted and recognised a c­MET antigen was constructed. c­MET­CAR was transferred into primary NK cells using lentiviral infection. c­MET­positive HepG2 cells were used as an in vitro study model. The cytotoxicity assay results revealed that c­MET­CAR­NK cells exhibited more specific cytotoxicity for HepG2 cells with high c­MET expression compared with the lung cancer cell line H1299, which has low levels of c­MET expression. The results of the present study demonstrated that c­MET may be a specific and effective target for human liver cancer cell CAR­NK immunotherapy. Based on these results, CAR­NK cell­based immunotherapy may provide a potential biotherapeutic approach for liver cancer treatment in the future.

Signals of the Neuropilin-1-MET Axis and Cues of Mechanical Force Exertion Converge to Elicit Inflammatory Activation in Coherent Endothelial Cells.

The neuropilin-1 (NRP1)-MET signaling axis regulates the motility of individual endothelial cells (ECs). It is unknown how this signaling pathway affects the endothelial barrier in coherent ECs forming a tight monolayer. We hypothesized that it is involved both in modulation of the endothelial barrier and in EC activation. To investigate the role of NRP1-MET signaling in inflammatory processes (e.g., systemic inflammatory response syndrome [SIRS] or snakebite-induced SIRS-like conditions), we employed the C-type lectin-related protein rhodocetin-αß (RCαß) as a specific trigger of this signal axis in ECs in vitro. In coherent HUVECs, RCαß reinforced the actin cytoskeleton and increased cell stiffness, thus favoring vascular endothelial cadherin-mediated transmission of intercellular forces. Increased cell stiffness was associated with enhanced activation of RhoA and nuclear translocation of NF-κB. Simultaneously, RCαß-triggered signaling via the NRP1-MET axis increased EC monolayer permeability, induced transcription of proinflammatory genes such as ICAM-1 and, consequently, leukocyte tethering. The RCαß-induced transcriptome differed from that induced by hepatocyte growth factor, although in both cases the same tyrosine kinase, MET, was involved. This was due to RCαß-mediated recruitment of the MET coreceptor NRP1 and additional Rho-mediated activation of the actomyosin system. RCαß induced similar transcriptional and cellular changes if external shear forces were applied. These data highlight the modulatory role of NRP1 as MET coreceptor, and they explain how some snake venoms induce SIRS-like conditions. Additionally, this study demonstrates that inflammatory activation of coherent ECs is triggered by converging signals that are induced by NRP1-MET signaling and influenced by intercellular forces.

SHR-A1403, a novel c-Met antibody-drug conjugate, exerts encouraging anti-tumor activity in c-Met-overexpressing models.

Emerging evidence demonstrates that a c-Met antibody-drug conjugate (ADC) has superior efficacy and safety profiles compared with those of currently available small molecules or antibody inhibitors for the treatment of c-Met-overexpressing cancers. Here we described both the in vitro and in vivo efficacies of SHR-A1403, a novel c-Met ADC composed of a humanized IgG2 monoclonal antibody against c-Met conjugated to a novel cytotoxic microtubule inhibitor. SHR-A1403 showed high affinity to c-Met proteins derived from human or monkey and potent inhibitory effects in cancer cell lines with high c-Met protein expression. In mice bearing tumors derived from cancer cell lines or patient HCC tissues with confirmed c-Met overexpression, SHR-A1403 showed excellent anti-tumor efficacy. Antibody binding with c-Met contributed to SHR-A1403 endocytosis; the subsequent translocation to lysosomes and cytotoxicity of the released toxin are speculated to be predominant mechanisms underlying the anti-tumor activity of SHR-A1403. In conclusion, SHR-A1403 showed significant anti-tumor activity in cancer cell lines, xenograft mouse models and an HCC PDX model, which all have high c-Met levels. These data provide references for SHR-A1403 as a potential therapy for the treatment of cancers with c-Met overexpression.