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Induction of Three-Dimensional Growth of Human Liver Cells in Simulated Microgravity
We previously reported that a NASA-developed bioreactor that simulates microgravity environment and creates the unique environment of low shear force and high-mass transfer is conducive for maintaining long term 3-D cell cultures of functional hepatocytes (60 days). However, significant further expansion of liver mass, or the remodeling of liver in vitro was jeopardized by the appearance of apoptotic zones in the center of large cell aggregates. To optimize oxygenation and nutritional uptake within growing cellular aggregates we cultured primary human liver cells (HLC) in a bioreactor in the presence or absence of microcarriers and biodegradable scaffolds. Also, to promote angiogenesis, HLC were cultured with or without microvascular endothelial cells. HLC were harvested from human livers by collagenase perfusion. While microcarriers did not affect cell growth, HLC cultured with biodegradable scaffolds made from polyglycolic acid (PGA) formed aggregates up to 3 cm in length. Culturing cells with PGA scaffolds increased the efficiency of cell self-assembly and the formation of larger cell aggregates. Based on histological evaluation it appears that the degree of apoptotic cells was diminished as compared to cultures without scaffolds. Histology of HLC with PGA-scaffolds revealed cell distribution between the fibers of the scaffolds, and cell-cell and cell-fiber interactions. Analyses of HLC spheroids revealed tissue-like structures comprised of hepatocytes, biliary epithelial cells and/or progenitor liver cells that were arranged as bile duct-like structures along nascent vascular sprouts. Electron microscopy revealed groups of cohesive hepatocytes and bile canaliculi with multiple microvilli and tight cellular junctions. Hepatocytes were further organized into tight clusters surrounded by complex stromal structures and reticulin fibers. Also, we observed higher levels of albumin mRNA expression when hepatocytes were co-cultured with endothelial cells. To evaluate viability and microsomal deethylation activity of hepatocytes we assessed the metabolism of midazolam by gas chromatography. Samples that were collected from HLC cultured in the bioreactor contained higher levels of midazolam metabolites (1-OH-midazolam and 4-OH-midazolam) than samples collected from conventional cultures. In summary, we have shown that co-culture of HLC with endothelial cells and/or culturing in the presence of PGA scaffolds provides additional advantages in maintaining functional activity of 3-D hepatocyte cultures that resemble liver tissue. This cell culture system may facilitate studies of liver regeneration and cell-to-cell interactions that occur in vivo and suggest the feasibility of using this approach for pre-clinical metabolic screening of novel drug candidates.
Second-Phase Hepatitis C Plasma Viral Kinetics Directly Reflects Reduced Intrahepatic Burden of Hepatitis C Virus
Mathematical models explain how antivirals control viral infections. Hepatitis C virus (HCV) treatment results in at least 2 phases of decline in viremia. Here, the first phase reflects clearance of rapidly produced virions. The second phase is hypothesized to derive from loss of infected cells but has been challenging to prove. Using single-cell methods, we quantified the number of hepatitis C virus (HCV)-infected hepatocytes in liver biopsies taken before and within 7 days of initiating direct-acting antivirals (DAAs) in a double-blinded randomized controlled trial testing 2 (sofosbuvir-velpatasvir) versus 3 (sofosbuvir-velpatasvir-voxilaprevir) DAAs. We employed thousands of intrahepatic measurements in 10 persons with chronic genotype 1a HCV infection: median proportion of infected hepatocytes declined from 11.3% (range, 1.3%–59%) to 0.6% (range, <0.3%–5.8%), a loss of 75%–95% infected hepatocytes. Plasma viremia correlated with numbers of HCV-infected hepatocytes (r = 0.77; P < .0001). Second-phase plasma dynamics and changes in infected hepatocytes were indistinct (P = .16), demonstrating that second-phase viral dynamics derive from loss of infected cells. DAAs led to a decline in intracellular HCV RNA and interferon-stimulated gene expression (P < .05 for both). We proved that second-phase viral dynamics reflect decay of intrahepatic burden of HCV, partly due to clearance of HCV RNA from hepatocytes.
Antiretroviral therapy for HIV and intrahepatic hepatitis C virus replication
HIV alters host responses to hepatitis C virus (HCV). However, the impact of antiretroviral therapy (ART) on HCV is rarely understood in relevant tissues and never before within individual hepatocytes. HIV and HCV kinetics were studied before and after ART initiation among 19 HIV/HCV co-infected persons. From five persons with the largest decline in plasma HCV RNA, liver tissues collected before and during ART, when plasma HIV RNA was undetectable, were studied. Here we used single-cell laser capture microdissection and quantitative PCR to assess intrahepatic HCV. Immunohistochemistry was performed to characterize intrahepatic immune cell populations. Plasma HCV RNA declined by 0.81 (0.52–1.60) log 10 IU/ml from a median (range) 7.26 (6.05–7.29) log 10 IU/ml and correlated with proportions of HCV-infected hepatocytes (r = 0.89, P = 2 × 10 -5 ), which declined from median (range) of 37% (6–49%) to 23% (0.5–52%) after plasma HIV clearance. Median (range) HCV RNA abundance within cells was unchanged in four of five participants. Liver T-cell abundance unexpectedly decreased, whereas natural killer (NK) and NK T-cell infiltration increased, correlating with changes in proportions of HCV-infected hepatocytes (r = -0.82 and r = -0.73, respectively). Hepatocyte expression of HLA-E, an NK cell restriction marker, correlated with proportions of HCV-infected hepatocytes (r = 0.79). These are the first data to show that ART control of HIV reduces the intrahepatic burden of HCV. Furthermore, our data suggest that HIV affects the pathogenesis of HCV infection by an NK/NK T-cell-mediated mechanism that may involve HLA-E and can be rescued, at least in part, by ART.
Induction of three-dimensional assembly of human liver cells by simulated microgravity
The establishment of long-term cultures of functional primary human liver cells (PHLC) is formidable. Developed at NASA, the Rotary Cell Culture System (RCCS) allows the creation of the unique microgravity environment of low shear force, high-mass transfer, and 3-dimensional cell culture of dissimilar cell types. The aim of our study was to establish long-term hepatocyte cultures in simulated microgravity. PHLC were harvested from human livers by collagenase perfusion and were cultured in RCCS. PHLC aggregates were readily formed and increased up to 1 cm long. The expansion of PHLC in bioreactors was further evaluated with microcarriers and biodegradable scaffolds. While microcarriers were not conducive to formation of spheroids, PHLC cultured with biodegradable scaffolds formed aggregates up to 3 cm long. Analyses of PHLC spheroids revealed tissue-like structures composed of hepatocytes, biliary epithelial cells, and/or progenitor liver cells that were arranged as bile duct-like structures along nascent vascular sprouts. Electron microscopy revealed groups of cohesive hepatocytes surrounded by complex stromal structures and reticulin fibers, bile canaliculi with multiple microvilli, and tight cellular junctions. Albumin mRNA was expressed throughout the 60-d culture. A simulated microgravity environment is conducive to maintaining long-term cultures of functional hepatocytes. This model system will assist in developing improved protocols for autologous hepatocyte transplantation, gene therapy, and liver assist devices, and facilitate studies of liver regeneration and cell-to-cell interactions that occur in vivo.
A Long Non-coding RNA, LOC157273, Is an Effector Transcript at the Chromosome 8p23.1-PPP1R3B Metabolic Traits and Type 2 Diabetes Risk Locus
Causal transcripts at genomic loci associated with type 2 diabetes (T2D) are mostly unknown. The chr8p23.1 variant rs4841132, associated with an insulin-resistant diabetes risk phenotype, lies in the second exon of a long non-coding RNA (lncRNA) gene, LOC157273, located 175 kilobases from PPP1R3B, which encodes a key protein regulating insulin-mediated hepatic glycogen storage in humans. We hypothesized that LOC157273 regulates expression of PPP1R3B in human hepatocytes. We tested our hypothesis using Stellaris fluorescent in situ hybridization to assess subcellular localization of LOC157273; small interfering RNA (siRNA) knockdown of LOC157273, followed by RT-PCR to quantify LOC157273 and PPP1R3B expression; RNA-seq to quantify the whole-transcriptome gene expression response to LOC157273 knockdown; and an insulin-stimulated assay to measure hepatocyte glycogen deposition before and after knockdown. We found that siRNA knockdown decreased LOC157273 transcript levels by approximately 80%, increased PPP1R3B mRNA levels by 1.7-fold, and increased glycogen deposition by >50% in primary human hepatocytes. An A/G heterozygous carrier (vs. three G/G carriers) had reduced LOC157273 abundance due to reduced transcription of the A allele and increased PPP1R3B expression and glycogen deposition. We show that the lncRNA LOC157273 is a negative regulator of PPP1R3B expression and glycogen deposition in human hepatocytes and a causal transcript at an insulin-resistant T2D risk locus.
Integrated Transcriptomic and Proteomic Analysis Identifies Plasma Biomarkers of Hepatocellular Failure in Alcohol-Associated Hepatitis
Alcohol-associated hepatitis (AH) is a form of liver failure with high short-term mortality. Recent results have shown that HNF4a defective function and systemic inflammation are major disease drivers of AH. Plasma biomarkers of hepatocyte function could be useful for diagnostic and prognostic purposes. Herein an integrative analysis of hepatic RNAseq and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to identify plasma protein signatures for mild and severe AH patients. Alcohol-related liver disease cirrhosis (ALD)(AC), non-alcoholic fatty liver disease (NALFD), and healthy subjects (HC) were used as comparator groups. Identified proteins primarily involved in hepatocellular function were decreased in AH patients which included hepatokines, clotting factors, complement cascade components, and hepatocyte growth activators. A protein signature of AH disease severity was identified including thrombin (THRB), hepatocyte growth factor alpha (HGFA), clusterin (CLUS), human serum factor H-related protein (FHR1) and kallistatin (KAIN), which exhibited large abundance shifts between severe and non-severe AH. The combination of THRB and HGFA discriminated between severe and non-severe AH with high sensitivity and specificity. These findings were correlated with the liver expression of genes encoding secreted proteins in a similar cohort, finding a highly consistent plasma protein signature reflecting HNF4A and HNF1A functions. This unbiased proteomic-transcriptome analysis identified plasma protein signatures and pathways associated with disease severity, reflecting HNF4A/1A activity useful for diagnostic assessment in AH.
Ligand dependent interaction between PC-TP and PPARδ mitigates diet-induced hepatic steatosis in male mice
Phosphatidylcholine transfer protein (PC-TP; synonym StarD2) is a soluble lipid-binding protein that transports phosphatidylcholine (PC) between cellular membranes. To better understand the protective metabolic effects associated with hepatic PC-TP, we generated a hepatocyte-specific PC-TP knockdown (L-Pctp -/- ) in male mice, which gains less weight and accumulates less liver fat compared to wild-type mice when challenged with a high-fat diet. Hepatic deletion of PC-TP also reduced adipose tissue mass and decreases levels of triglycerides and phospholipids in skeletal muscle, liver and plasma. Gene expression analysis suggest that the observed metabolic changes are related to transcriptional activity of peroxisome proliferative activating receptor (PPAR) family members. An in-cell protein complementation screen between lipid transfer proteins and PPARs uncovered a direct interaction between PC-TP and PPARδ that was not observed for other PPARs. We confirmed the PC-TP– PPARδ interaction in Huh7 hepatocytes, where it was found to repress PPARδ-mediated transactivation. Mutations of PC-TP residues implicated in PC binding and transfer reduce the PC-TP-PPARδ interaction and relieve PC-TP-mediated PPARδ repression. Reduction of exogenously supplied methionine and choline reduces the interaction while serum starvation enhances the interaction in cultured hepatocytes. Together our data points to a ligand sensitive PC-TP– PPARδ interaction that suppresses PPAR activity.
Cytoskeletal filament assembly and the control of cell spreading and function by extracellular matrix
This study was undertaken to analyze how cell binding to extracellular matrix produces changes in cell shape. We focused on the initial process of cell spreading that follows cell attachment to matrix and, thus, cell 'shape' changes are defined here in terms of alterations in projected cell areas, as determined by computerized image analysis. Cell spreading kinetics and changes in microtubule and actin microfilament mass were simultaneously quantitated in hepatocytes plated on different extracellular matrix substrata. The initial rate of cell spreading was highly dependent on the matrix coating density and decreased from 740 microns 2/h to 50 microns 2/h as the coating density was lowered from 1000 to 1 ng/cm2. At approximately 4 to 6 hours after plating, this initial rapid spreading rate slowed and became independent of the matrix density regardless of whether laminin, fibronectin, type I collagen or type IV collagen was used for cell attachment. Analysis of F-actin mass revealed that cell adhesion to extracellular matrix resulted in a 20-fold increase in polymerized actin within 30 minutes after plating, before any significant change in cell shape was observed. This was followed by a phase of actin microfilament disassembly which correlated with the most rapid phase of cell extension and ended at about 6 hours; F-actin mass remained relatively constant during the slow matrix-independent spreading phase. Microtubule mass increased more slowly in spreading cells, peaking at 4 hours, the time at which the transition between rapid and slow spreading rates was observed. However, inhibition of this early rise in microtubule mass using either nocodazole or cycloheximide did not prevent this transition. Use of cytochalasin D revealed that microfilament integrity was absolutely required for hepatocyte spreading whereas interference with microtubule assembly (using nocodazole or taxol) or protein synthesis (using cycloheximide) only partially suppressed cell extension. In contrast, cell spreading could be completely inhibited by combining suboptimal doses of cytochalasin D and nocodazole, suggesting that intact microtubules can stabilize cell form when the microfilament lattice is partially compromised. The physiological relevance of the cytoskeleton and cell shape in hepatocyte physiology was highlighted by the finding that a short exposure (6 hour) of cells to nocodazole resulted in production of smaller cells 42 hours later that exhibited enhanced production of a liver-specific product (albumin). These data demonstrate that spreading and flattening of the entire cell body is not driven directly by net polymerization of either microfilaments or microtubules.(ABSTRACT TRUNCATED AT 400 WORDS).
Apolipoprotein A5 reduces clearance of VLDL by altering apolipoprotein E content
Apolipoprotein A-V (APOA5) is a critical regulator of circulating triglyceride (TG) levels. Its deletion leads to elevated plasma TG concentrations by altering the metabolism of very low-density lipoprotein (VLDL) particles in vivo. One way APOA5 exerts its effects is through modulation of lipoprotein lipase (LPL) activity, specifically by disrupting inhibitory interactions between LPL and angiopoietin-like proteins (ANGPTLs). However, the impact of APOA5 on VLDL composition and its potential to alter VLDL metabolism in other ways remains poorly understood. To address this, we investigated the influence of APOA5 on the VLDL proteome, LPL activation, and hepatic remnant uptake. Using VLDL from Apoa5 knockout (KO) and wild-type (WT) mice, we found no evidence that APOA5 directly enhances LPL activity in purified or plasma systems. However, VLDL from Apoa5 K mice was cleared significantly more slowly by cultured hepatocytes. Proteomics experiments from two independent laboratories identified consistent depletion of 17 proteins involved in lipoprotein metabolism, inflammation, and immune response in Apoa5 KO VLDL, including APOE and serum amyloid A1 (SAA1). Remarkably, reintroduction of recombinant mouse APOA5 to the KO plasma partially restored the WT VLDL proteome, including APOE, and normalized VLDL uptake by hepatocytes without altering LPL lipolysis. These findings reveal that APOA5 influences hepatic clearance of VLDL remnants by modulating particle composition, particularly APOE content. This study expands the functional scope of APOA5 in TG metabolism and underscores its role in VLDL remodeling and remnant clearance, offering new insights with implications for understanding hypertriglyceridemia and its roles in inflammation and immune response.
HIV influences clustering and intracellular replication of hepatitis C virus
We report HCV and HIV coinfection is common and HIV leads to increased HCV viraemia and accelerated disease progression. However, the biological basis of this interaction remains poorly understood and little is known about the impact of HIV on HCV replication at the cellular level. We analysed HCV RNA, based on single-cell laser-capture microdissection, in liver biopsies from monoinfected (n = 4) and HCV/HIV-coinfected (n = 5) participants. HCV RNA was assayed in 3200 hepatocytes with information of spatial position. We compared HCV RNA levels and clustering properties of infection between mono- and coinfected participants, and developed a mathematical model of infection. Although the median plasma HCV RNA level and the fraction of infected cells were comparable in monoinfected (7.0 log 10 IU/mL and ~ 30%) and coinfected (7.3 log 10 IU/mL and ~ 40%) participants, the median HCV RNA per infected hepatocyte in monoinfected (2.8IU) was significantly lower than in coinfected (8.2IU) participants (p = .03). Clustering of infected cells was more prominent in monoinfected participants (91% of samples) than in coinfected participants (~48%), p = .0045, suggesting that spatial spread may be influenced by HIV coinfection. Interestingly, when clustering does occur, the size of clusters is similar in both types of infection. A mathematical model of infection suggested that HIV allows higher intracellular accumulation of HCV RNA by impeding the export of HCV RNA. Our observations show that HIV coinfection impacts intracellular accumulation of HCV RNA and the clustering of HCV-infected cells, but to a less extent the fraction of HCV-infected cells.
Modeling hepatitis C virus kinetics during liver transplantation highlights the role of the liver in virus clearance
While the liver, specifically hepatocytes, are widely accepted as the main source of hepatitis C virus (HCV) production, the role of the liver/hepatocytes in clearance of circulating HCV remains unknown. Frequent HCV kinetic data were recorded and mathematically modeled from five liver transplant patients throughout the anhepatic (absence of liver) phase and for 4 hr post-reperfusion. During the anhepatic phase, HCV remained at pre-anhepatic levels (n = 3) or declined (n = 2) with t 1/2 ~1 hr. Immediately post-reperfusion, virus declined in a biphasic manner in four patients consisting of a rapid decline (t 1/2 = 5 min) followed by a slower decline (t 1/2 = 67 min). Consistent with the majority of patients in the anhepatic phase, when we monitored HCV clearance at 37°C from culture medium in the absence/presence of chronically infected hepatoma cells that were inhibited from secreting HCV, the HCV t 1/2 in cell culture was longer in the absence of chronically HCV-infected cells. The results suggest that the liver plays a major role in the clearance of circulating HCV and that hepatocytes may be involved.
Targeted protein degradation systems to enhance Wnt signaling
Molecules that facilitate targeted protein degradation (TPD) offer great promise as novel therapeutics. The human hepatic lectin asialoglycoprotein receptor (ASGR) is selectively expressed on hepatocytes. We have previously engineered an anti-ASGR1 antibody-mutant RSPO2 (RSPO2RA) fusion protein (called SWEETS) to drive tissue-specific degradation of ZNRF3/RNF43 E3 ubiquitin ligases, which achieved hepatocyte-specific enhanced Wnt signaling, proliferation, and restored liver function in mouse models, and an antibody–RSPO2RA fusion molecule is currently in human clinical trials. In the current study, we identified two new ASGR1- and ASGR1/2-specific antibodies, 8M24 and 8G8. High-resolution crystal structures of ASGR1:8M24 and ASGR2:8G8 complexes revealed that these antibodies bind to distinct epitopes on opposing sides of ASGR, away from the substrate-binding site. Both antibodies enhanced Wnt activity when assembled as SWEETS molecules with RSPO2RA through specific effects sequestering E3 ligases. In addition, 8M24-RSPO2RA and 8G8-RSPO2RA efficiently downregulate ASGR1 through TPD mechanisms. These results demonstrate the possibility of combining different therapeutic effects and degradation mechanisms in a single molecule.
Hepatic pathology in mice after continuous inhalation exposure to 1, 1, 1-trichloroethane
Mice exposed to either 250ppm or 1,000ppm 1,1,1-trichloroethane in air continuously for 14 weeks demonstrated significant changes in the centrilobular hepatocytes for the 1,000ppm group. Moderate liver triglyceride accumulation was evident in the 1,000ppm group and peaked at 40mg/gm of tissue after 7 weeks of exposure. Focal hepatocyte necrosis occurred in 40% of the mice exposed to 1,000ppm for 12 weeks. This necrosis was associated with an acute inflammatory infiltrate and hypertrophy of Kupffer cells. These findings indicate that the pathological alternations observed with 1,1,1-trichloroethane are similar to those observed with dichloromethane except for different time courses of the effects and different degrees of recovery. The toxic effects of 1,1,1-trichloroethane are of a similar type to those produced by carbon tetrachloride but appear much less severe.
Mining a human transcriptome database for chemical modulators of NRF2
Nuclear factor erythroid-2 related factor 2 (NRF2) encoded by the NFE2L2 gene is a transcription factor critical for protecting cells from chemically-induced oxidative stress. We developed computational procedures to identify chemical modulators of NRF2 in a large database of human microarray data. A gene expression biomarker was built from statistically-filtered gene lists derived from microarray experiments in primary human hepatocytes and cancer cell lines exposed to NRF2-activating chemicals (oltipraz, sulforaphane, CDDOIm) or in which the NRF2 suppressor Keap1 was knocked down by siRNA. Directionally consistent biomarker genes were further filtered for those dependent on NRF2 using a microarray dataset from cells after NFE2L2 siRNA knockdown. The resulting 143-gene biomarker was evaluated as a predictive tool using the correlation-based Running Fisher algorithm. Using 59 gene expression comparisons from chemically-treated cells with known NRF2 activating potential, the biomarker gave a balanced accuracy of 93%. The biomarker was comprised of many well-known NRF2 target genes (AKR1B10, AKR1C1, NQO1, TXNRD1, SRXN1, GCLC, GCLM), 69% of which were found to be bound directly by NRF2 using ChIP-Seq. NRF2 activity was assessed across ~9840 microarray comparisons from ~1460 studies examining the effects of ~2260 chemicals in human cell lines. A total of 260 and 43 chemicals were found to activate or suppress NRF2, respectively, most of which have not been previously reported to modulate NRF2 activity. Using a NRF2-responsive reporter gene in HepG2 cells, we confirmed the activity of a set of chemicals predicted using the biomarker. The biomarker will be useful for future gene expression screening studies of environmentally-relevant chemicals.
Wogonoside attenuates liver fibrosis by triggering hepatic stellate cell ferroptosis through SOCS1 / P53 / SLC7A11 pathway
Abstract Wogonoside (WG) is a flavonoid chemical component extracted from Scutellaria baicalensis, which exerts therapeutic effects on liver diseases. Ferroptosis, a novel form of programmed cell death, regulates diverse physiological/pathological processes. In this study, we attempted to investigate a novel mechanism by which WG mitigates liver fibrosis by inducing ferroptosis in hepatic stellate cells (HSCs). A CCl 4 ‐induced mouse liver fibrosis model and a rat HSC line were employed for in vivo and in vitro experiments, both treated with WG. Firstly, the levels of the fibrotic markers α‐smooth muscle actin (α‐SMA) and α1(I)collagen (COL1α1) were effectively decreased by WG in CCl 4 ‐induced mice and HSC‐T6 cells. Additionally, mitochondrial condensation and mitochondrial ridge breakage were observed in WG‐treated HSC‐T6 cells. Furthermore, ferroptotic events including depletion of SLC7A11, GPX4 and GSH, and accumulation of iron, ROS and MDA were discovered in WG‐treated HSC‐T6 cells. Intriguingly, these ferroptotic events did not appear in hepatocytes or macrophages. WG‐elicited HSC ferroptosis and ECM reduction were dramatically abrogated by ferrostatin‐1 (Fer‐1), a ferroptosis inhibitor. Importantly, our results confirm that SOCS1/P53/SLC7A11 is a signaling pathway which promotes WG attenuation of liver fibrosis. On the contrary, WG mitigated liver fibrosis and inducted HSC‐T6 cell ferroptosis were hindered by SOCS1 siRNA and pifithrin‐α (PFT‐α). These findings demonstrate that SOCS1/P53/SLC7A11‐mediated HSC ferroptosis is associated with WG alleviating liver fibrosis, which provides a new clue for the treatment of liver fibrosis.
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 β-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.