MET Antibody

What is the MET receptor and why is it targeted with antibodies?

The MET receptor is a proto-oncoprotein tyrosine kinase that regulates various physiological processes including cell proliferation, scattering, morphogenesis, and survival. It plays a key role in cancer cell growth and invasion by transducing signals from the extracellular matrix into the cytoplasm through binding to hepatocyte growth factor (HGF) . MET is targeted with antibodies because it is frequently dysregulated in multiple tumor types, making it valuable for both diagnostic and therapeutic purposes. Antibodies can be designed to either block HGF/MET interaction, induce MET degradation, or deliver cytotoxic payloads to MET-expressing cells . Understanding the MET signaling pathway is crucial for developing effective antibody-based therapeutics.

What are the different types of MET antibodies available for research purposes?

Researchers have access to several distinct types of MET antibodies, each with specific properties and applications:

• Bivalent antibodies: These contain two binding sites that can cross-link MET receptors, potentially inducing receptor dimerization and activation .

• Monovalent (one-armed) antibodies: These bind to monomeric MET protein without inducing activation, blocking HGF binding without causing receptor dimerization .

• Non-activating antibodies: Antibodies like LY2875358 that can both block HGF interaction and trigger receptor downregulation .

• Biparatopic antibodies: These recognize two distinct epitopes in the MET Sema domain, offering enhanced efficacy over single-epitope antibodies .

• Fluorophore-conjugated antibodies: Including APC-conjugated, Alexa Fluor 488-conjugated, or biotin-conjugated antibodies for visualization of MET localization and tracking .

• Phospho-specific antibodies: These recognize phosphorylated forms of MET, particularly at specific tyrosine residues like Y1234/Y1235, to assess activation status .

• Antibody-drug conjugates: Such as ABBV-400, which combines a MET-targeting antibody with a cytotoxic payload .

The selection of antibody type should be guided by the specific research question, as each type can yield different biological outcomes.

How do MET antibodies differ in their mechanisms of action?

MET antibodies exhibit diverse mechanisms of action that significantly impact their research applications and therapeutic potential:

Researchers should note that the mechanism is highly dependent on antibody structure. Bivalent antibodies can paradoxically activate MET signaling through inducing receptor clustering, as observed with APC-conjugated anti-MET antibodies which cause rapid and transient formation of MET clusters on the plasma membrane . In contrast, monovalent antibodies typically block signaling without activation. Some antibodies like LY2875358 combine HGF blocking with receptor downregulation mechanisms .

How should researchers design experiments to evaluate MET antibody specificity and cross-reactivity?

Evaluating MET antibody specificity requires a multi-faceted approach to eliminate false positives and ensure target engagement:

• Peptide competition assays: Pre-incubate antibodies with phosphopeptides corresponding to MET immunogen, non-phosphopeptides, and generic phosphotyrosine-containing peptides. Only the specific phosphopeptide corresponding to MET should block antibody signal, demonstrating specificity .

• Negative controls: Include isotype-matched control antibodies (e.g., mouse IgG1 conjugated to the same fluorophore) to establish background levels .

• Knockout validation: Compare antibody staining between MET-expressing cells and MET-knockout cells using CRISPR/Cas9 or siRNA knockdown approaches.

• Multiple detection methods: Validate findings using at least two independent techniques, such as immunoblotting, flow cytometry, and immunofluorescence microscopy .

• Cross-species reactivity: Test antibodies against MET from different species if cross-species applications are intended.

For phospho-specific MET antibodies, specificity testing should include treatment with phosphatase to confirm that signal is dependent on phosphorylation status. Additionally, stimulation with HGF can serve as a positive control to increase phosphorylation at specific sites .

What are the best methods for monitoring MET activation using antibodies?

Monitoring MET activation requires detection of specific molecular events that occur during receptor activation:

• Phospho-specific antibodies: Use antibodies targeting phosphorylated tyrosine residues Y1234/Y1235 in the activation loop of MET kinase domain. These phosphorylation events are directly correlated with MET activation . Western blotting analysis and immunofluorescence with these antibodies can assess both the level and localization of activated MET.

• Receptor clustering visualization: Apply fluorophore-conjugated anti-MET antibodies (e.g., APC-conjugated or biotin-conjugated followed by fluorescent streptavidin) to live serum-starved cells to monitor the rapid formation of MET clusters on the plasma membrane, which occurs within 2-5 minutes of activation .

• Multi-color immunofluorescence: Combine multiple detection channels to simultaneously monitor:

  • Total MET distribution (using anti-MET antibody conjugated to one fluorophore)

  • Activated MET (using phospho-specific antibody with a different fluorophore)

  • Antibody-bound MET (using a third fluorescent channel)

• Downstream signaling analysis: Monitor phosphorylation of key downstream effectors including AKT, ERK1/2, and GAB1 by western blotting to confirm functional activation of the pathway .

• Temporal analysis: Track the kinetics of MET activation by sampling at multiple timepoints (e.g., 2, 5, 7, 10, 15, and 30 minutes) after antibody or HGF addition to capture the typically transient nature of receptor activation .

For accurate results, researchers should perform these assays in serum-starved cells to reduce background activation from serum components.

What are the critical controls when using fluorescently labeled MET antibodies for imaging studies?

When designing imaging experiments with fluorescently labeled MET antibodies, the following controls are essential:

• Isotype controls: Include fluorophore-conjugated control IgG of the same isotype to establish background fluorescence levels. For example, when using APC-tagged mouse monoclonal anti-MET antibody, include APC-tagged mouse IgG controls .

• Blocking controls: Pre-incubate cells with unlabeled antibodies targeting the same epitope to confirm signal specificity through competitive binding.

• Fixation controls: Compare live-cell staining (which detects only cell surface MET) with fixed and permeabilized cell staining (which detects total MET) to distinguish surface from intracellular pools .

• Cross-channel bleed-through controls: When using multiple fluorophores, include single-label controls to assess spectral overlap, particularly important when simultaneously detecting total MET, phosphorylated MET, and antibody localization .

• Temporal controls: Image at multiple timepoints to track the dynamic processes of receptor clustering, internalization, and degradation, as MET antibodies can induce rapid changes in receptor distribution .

• Cell type controls: Include both MET-expressing and low/non-expressing cell lines to validate antibody specificity and sensitivity across different expression levels .

• HGF stimulation control: Compare antibody-induced MET clustering patterns with those induced by the natural ligand HGF to distinguish physiological from artificial effects .

These controls enable researchers to differentiate between specific antibody binding to MET and non-specific fluorescence, ensuring the reliability of imaging data for quantitative analyses of receptor dynamics.

How do bivalent versus monovalent MET antibodies differ in their effects on receptor trafficking and degradation?

The valency of MET antibodies dramatically influences receptor trafficking and degradation through distinct mechanisms:

Bivalent Antibodies:

• Induce rapid receptor clustering on the plasma membrane within 2-5 minutes of exposure

• Create highly polarized MET clusters that become progressively more concentrated into a single bright spot or patched area by 7-10 minutes

• Trigger internalization of antibody-MET complexes within 15-30 minutes, resulting in disappearance from cell surface and strong intracellular staining

• Promote rapid degradation of the receptor through altered intracellular processing

• Render cells refractory to further HGF stimulation due to antibody-mediated MET depletion

Monovalent (One-Armed) Antibodies:

• Bind to monomeric MET without inducing receptor clustering or dimerization

• Block HGF binding without activating downstream signaling

• Allow MET to remain on the cell surface for longer periods without significant internalization

• Preserve MET availability for potential physiological functions while blocking pathological activation

• Effectively inhibit HCC cell proliferation and migration without triggering MET activation

This fundamental difference explains why monovalent antibodies like the one-armed 5D5 derivative have been pursued for therapeutic development, as they avoid the potentially counterproductive receptor activation and subsequent signaling that can occur with bivalent antibodies . The biparatopic approach offers another mechanism, where binding to two distinct epitopes inhibits MET recycling, thereby promoting lysosomal trafficking and degradation more effectively than either parental antibody alone .

What methodologies are recommended for investigating MET antibody-induced receptor downregulation?

Investigating MET receptor downregulation by antibodies requires methodologies that track receptor levels, localization, and degradation pathways:

• Quantitative time-course studies: Monitor surface MET levels at multiple timepoints (0, 15, 30, 60, 120, 240 minutes) after antibody treatment using:

  • Flow cytometry with non-competing fluorescently labeled MET antibodies

  • Cell surface biotinylation followed by precipitation and immunoblotting

  • Immunofluorescence microscopy with membrane markers

• Total vs. surface MET discrimination: Compare total MET levels (by western blot of whole cell lysates) with surface MET levels to determine if receptors are being internalized or degraded .

• Lysosomal tracking: Co-localize MET with lysosomal markers (LAMP1/2) using dual-color immunofluorescence to confirm lysosomal trafficking, and employ lysosomal inhibitors (bafilomycin A1, chloroquine) to determine if degradation is lysosome-dependent .

• Proteasomal involvement: Test proteasome inhibitors (MG132, bortezomib) to assess potential proteasomal degradation pathways.

• Recycling assays: Use antibody feeding and acid wash techniques to distinguish between receptor degradation and recycling back to the plasma membrane .

• Degradation kinetics: Perform cycloheximide chase experiments to measure MET half-life with and without antibody treatment, revealing differences in degradation rates.

• Ubiquitination analysis: Immunoprecipitate MET and probe for ubiquitin to determine if antibody binding promotes receptor ubiquitination prior to degradation.

The biparatopic antibody approach illustrates how these methodologies can reveal mechanism distinctions, as researchers demonstrated that biparatopic antibodies inhibit MET recycling more effectively than parental antibodies, promoting lysosomal trafficking and degradation . These techniques can identify whether an antibody induces CBL-dependent or CBL-independent degradation pathways, which is critical for predicting efficacy.

How can researchers differentiate between agonistic and antagonistic effects of MET antibodies?

Distinguishing between agonistic and antagonistic effects of MET antibodies requires assessment of both immediate signaling events and downstream biological consequences:

• Phosphorylation kinetics analysis: Measure the phosphorylation of Y1234/Y1235 in the MET activation loop at multiple early timepoints (2-30 minutes). Agonistic antibodies typically induce rapid but transient phosphorylation, while antagonistic antibodies show minimal to no increase in phosphorylation .

• Downstream signaling cascade evaluation: Assess phosphorylation of key downstream effectors:

  • AKT (survival pathway)

  • ERK1/2 (proliferation pathway)

  • GAB1 (scaffolding protein)

  True antagonists will prevent HGF-induced phosphorylation of these proteins while not inducing phosphorylation themselves .

• Functional biological assays:

  • Cell scattering assays: Measure dispersal of epithelial colonies (agonists induce scattering)

  • Cell migration assays: Quantify cell movement in transwell or wound healing assays

  • Proliferation assays: Assess BrdU incorporation or cell counting over time

  • Angiogenesis assays: Evaluate tube formation using HUVECs

• Receptor clustering visualization: Observe MET distribution using fluorescent antibodies. Agonistic antibodies induce visible receptor clustering on the membrane, while antagonistic antibodies typically do not alter receptor distribution patterns .

• Competition with HGF: Determine if the antibody competes with HGF for binding to MET using ELISA-based competition assays. Antagonistic antibodies typically compete with HGF, while some agonistic antibodies may bind to different epitopes .

• Sequential stimulation test: Pretreat cells with the test antibody, then challenge with HGF. If cells remain responsive to HGF (showing additional phosphorylation of MET and downstream targets), the antibody likely has minimal agonistic activity. If cells become refractory to HGF stimulation due to receptor downregulation, this suggests agonistic effects followed by desensitization .

These combined approaches provide a comprehensive assessment of whether a MET antibody acts as an agonist, antagonist, or has mixed effects depending on context and concentration.

What criteria should be used to select MET antibodies for targeted cancer therapy development?

Selection of MET antibodies for cancer therapy development should be based on the following evidence-based criteria:

• Binding specificity and affinity: Select antibodies with high affinity (sub-nanomolar Kd) and specificity for MET, confirmed through comprehensive cross-reactivity testing against related receptor tyrosine kinases .

• Mechanism of action relevance: Consider whether the therapeutic goal is:

  • Blocking ligand-receptor interaction (antagonistic antibodies)

  • Inducing receptor degradation (downregulating antibodies)

  • Delivering cytotoxic payloads (antibody-drug conjugates)

• Structural considerations: Monovalent antibodies generally avoid the paradoxical agonism seen with bivalent antibodies, making one-armed constructs like emibetuzumab potentially safer .

• Epitope selection: Antibodies targeting the:

  • Sema domain can block HGF binding (e.g., 107_A07)

  • Ig domains can affect receptor conformation (compact vs. open)

  • Biparatopic designs targeting multiple epitopes show enhanced efficacy

• Effect on tumor models: Prioritize antibodies demonstrating:

  • Inhibition of MET-driven xenograft growth

  • Prevention of metastasis in relevant models

  • Activity in models resistant to other therapies

• Patient selection biomarkers: Develop companion diagnostics to identify patients likely to respond:

  • MET amplification (≥5 copies preferred)

  • MET exon 14 skipping mutations

  • High MET protein expression (IHC 2+ or 3+ in ≥50% of tumor cells)

• Pharmacological properties: Evaluate:

  • Half-life suitable for practical dosing schedules

  • Acceptable toxicity profile

  • Tissue penetration capabilities

The clinical disappointments of some MET antibodies highlight the importance of proper patient selection. For instance, using the MET/CEN7 ratio or next-generation sequencing to detect true MET amplification has proven more effective than protein expression alone for identifying patients likely to respond .

How can researchers address the challenge of acquired resistance to MET antibody therapies?

Addressing acquired resistance to MET antibody therapies requires systematic investigation of resistance mechanisms and development of innovative strategies:

• Resistance mechanism identification:

  • Perform genomic and proteomic profiling of resistant versus sensitive models

  • Develop resistant cell lines through chronic exposure to MET antibodies

  • Analyze patient samples pre-treatment versus at progression

  • Investigate bypass pathway activation (EGFR, HER2, AXL, FGFR)

• Combination therapy approaches:

  • Simultaneously target MET and known resistance pathways (e.g., EGFR)

  • Combine MET antibodies with MET tyrosine kinase inhibitors to prevent both ligand-dependent and ligand-independent activation

  • Use multi-targeting bispecific antibodies that engage MET and a resistance-mediating receptor

• Novel antibody designs:

  • Develop biparatopic antibodies that bind two distinct epitopes on MET, showing greater efficacy and potentially overcoming resistance mechanisms

  • Create antibody-drug conjugates like ABBV-400 that deliver cytotoxic payloads, adding a direct cell-killing mechanism independent of signaling blockade

• Intermittent dosing strategies:

  • Investigate drug holiday approaches to prevent or delay resistance development

  • Implement adaptive dosing based on pharmacodynamic biomarkers

• Combination with immune checkpoint inhibitors:

  • Explore potential synergies between MET inhibition and immunotherapy

  • Investigate whether MET inhibition alters the tumor immune microenvironment

Research suggests that resistance to MET-targeted therapies often involves activation of alternative signaling pathways. For example, MET amplification has been found to confer resistance to EGFR-targeting therapies in NSCLC and colorectal cancer, suggesting bidirectional resistance mechanisms between these pathways . Understanding such crosstalk is essential for designing more effective combination strategies.

What are the most sensitive and specific methods for detecting MET expression to guide antibody therapy selection?

Optimal patient selection for MET antibody therapies requires sensitive and specific detection methods:

For clinical application, a tiered testing approach is recommended:

• Start with IHC screening for MET overexpression

• Follow positive cases with FISH or NGS to confirm true MET amplification

• Include testing for MET exon 14 skipping mutations in relevant cancer types (e.g., NSCLC)

Studies have demonstrated that patient selection based on true MET amplification (≥5 copies) rather than protein overexpression alone correlates better with response to MET-targeted therapies . The combined use of multiple detection methods provides the most comprehensive assessment to guide therapy selection.

How might novel antibody engineering approaches enhance the efficacy of anti-MET therapeutics?

Advanced antibody engineering offers promising avenues to overcome current limitations of MET-targeted therapies:

• Biparatopic antibodies: Antibodies that simultaneously bind two distinct epitopes on MET have demonstrated significantly enhanced efficacy. Research has shown that biparatopic MET×MET antibodies outperform either parental antibody alone by inhibiting MET recycling and promoting lysosomal degradation . This approach addresses the challenge of transient effects seen with conventional antibodies.

• Antibody-drug conjugates (ADCs): The ABBV-400 conjugate combines the c-Met-targeting antibody telisotuzumab with a novel topoisomerase 1 inhibitor payload, showing promising efficacy in colorectal cancer patients with c-Met overexpression. In patients with higher c-Met expression, objective response rates exceeding 30% were observed at doses ≥2.4 mg/kg .

• pH-dependent binding antibodies: Engineering antibodies that bind MET strongly at neutral pH (cell surface) but release at acidic pH (endosomes) could prevent recycling while avoiding lysosomal degradation of the antibody itself, improving pharmacokinetics.

• Bispecific antibodies: Creating antibodies that simultaneously target MET and complementary pathways (EGFR, HER3, etc.) could prevent bypass resistance and enhance efficacy through simultaneous pathway inhibition.

• Intracellular antibody delivery: Developing methods to deliver antibodies intracellularly could target MET signaling complexes within the cytoplasm, potentially disrupting signaling nodes inaccessible to conventional antibodies.

• Immune-engaging antibodies: Bispecific T-cell engagers (BiTEs) or Fc-engineered antibodies that enhance antibody-dependent cellular cytotoxicity (ADCC) against MET-expressing tumors could add immune-mediated killing mechanisms to direct signaling inhibition.

• Antibody fragments and alternative scaffolds: Smaller binding molecules may offer improved tumor penetration, particularly in solid tumors where conventional antibodies have limited distribution.

These approaches represent significant advancements beyond traditional antagonistic antibodies, potentially addressing the limitations that have hindered clinical success of earlier generation MET antibodies.

What are the most promising methodologies for investigating antibody-induced conformational changes in the MET receptor?

Investigating antibody-induced conformational changes in MET requires sophisticated biophysical and structural approaches:

• X-ray crystallography: This method has successfully revealed the binding mode of antibody Fab fragments to MET receptor fragments. The crystal structure of the 107_A07 Fab in complex with MET (519-740) elucidated binding to the Ig1 domain and provided insights into how antibody binding affects the "compact" versus "open" conformations of MET .

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This technique can map conformational changes and dynamics across the entire MET receptor upon antibody binding, identifying regions that become more protected or exposed, suggesting conformational shifts.

• Single-molecule Förster resonance energy transfer (smFRET): By labeling different domains of MET with donor and acceptor fluorophores, researchers can directly observe conformational changes in real-time upon antibody binding.

• Cryo-electron microscopy (Cryo-EM): This emerging approach can visualize full-length MET in complex with antibodies at near-atomic resolution, particularly valuable for understanding large conformational changes that may be difficult to crystallize.

• Molecular dynamics simulations: Computational approaches can model the dynamic interactions between antibodies and MET, predicting conformational changes and providing hypotheses for experimental validation.

• Surface plasmon resonance (SPR) with conformational-specific probes: Using secondary antibodies that recognize specific MET conformations after primary antibody binding can reveal induced conformational changes.

• Limited proteolysis coupled with mass spectrometry: Changes in protease accessibility upon antibody binding can map conformational alterations in the receptor structure.

Evidence suggests that MET exists in multiple conformations, including "compact" and "open" states. Overlay of the Fab-MET crystal structure with the InternalinB-MET crystal structure showed that the 107_A07 Fab comes into close proximity with the HGF/SF-binding SEMA domain only when MET is in the "compact" conformation . This supports the hypothesis that antibody binding can stabilize specific receptor conformations, influencing ligand binding and downstream signaling.

How can spatial biology techniques enhance our understanding of MET antibody mechanisms in the tumor microenvironment?

Emerging spatial biology techniques offer unprecedented insights into MET antibody mechanisms within the complex tumor microenvironment:

• Multiplexed immunofluorescence (mIF): This technique allows simultaneous detection of multiple proteins, enabling visualization of:

  • MET expression and activation state

  • Antibody penetration and binding in different tumor regions

  • Co-localization with other signaling molecules

  • Spatial relationships with immune cells and stromal components

• Imaging mass cytometry (IMC) and Multiplex ion beam imaging (MIBI): These technologies can detect 40+ protein markers simultaneously on a single tissue section, revealing how MET antibody treatment affects various cell populations and signaling pathways throughout heterogeneous tumors.

• Spatial transcriptomics: Combining in situ hybridization with next-generation sequencing enables mapping of gene expression patterns across tissue sections, revealing how MET antibody treatment affects transcriptional programs in tumor cells and the surrounding microenvironment.

• In vivo imaging using fluorescently labeled antibodies: Real-time tracking of antibody distribution, penetration, and retention can be achieved using techniques like:

  • Intravital microscopy in window chamber models

  • Near-infrared fluorescence imaging

  • Positron emission tomography (PET) with radiolabeled antibodies

• Digital spatial profiling (DSP): This allows quantitative analysis of protein and RNA expression with spatial resolution, enabling precise measurement of MET pathway components and their modulation by antibody treatment across different tumor regions.

• Ex vivo tumor slice culture with spatial readouts: This approach maintains the native tissue architecture while allowing controlled antibody treatment and high-resolution imaging to track MET dynamics in near-physiological conditions.

These technologies can address critical questions about how MET antibodies function in the complex tumor setting, including:

• Whether antibody penetration is sufficient throughout the tumor

• How heterogeneous MET expression affects response

• Whether antibody-induced MET signaling changes differ between tumor centers and invasive margins

• How MET modulation affects interactions with immune cells and stromal components

This spatial context is particularly important given MET's role in invasion and metastasis, processes highly dependent on interactions with the surrounding microenvironment.

What are the most significant unresolved questions in MET antibody research?

Despite significant advances, several critical questions remain unanswered in MET antibody research:

• Predictive biomarkers: While MET amplification, mutations, and protein overexpression are associated with response to MET-targeted therapies, the precise thresholds and combinations of biomarkers that predict antibody efficacy remain unclear. Research is needed to develop more precise patient selection strategies that go beyond simple expression levels .

• Resistance mechanisms: The molecular basis of primary and acquired resistance to MET antibodies is incompletely understood. Systematic studies of resistant models and patient samples are needed to identify bypass pathways and develop rational combination strategies .

• Cellular trafficking determinants: What molecular factors determine whether MET receptors are recycled or degraded following antibody binding, and how can antibodies be engineered to consistently promote degradation over recycling ?

• Conformation-specific targeting: How can antibodies be designed to selectively recognize and stabilize inactive MET conformations while avoiding active conformations that might trigger paradoxical signaling ?

• Tumor microenvironment effects: How do MET antibodies affect the broader tumor ecosystem, including cancer-associated fibroblasts, immune cells, and vasculature, and how do these effects contribute to therapeutic efficacy or resistance?

• Dosing and scheduling optimization: What are the optimal dosing strategies for different MET antibody classes to maximize efficacy while minimizing adaptive resistance?

• Combination strategy prioritization: With numerous potential combination partners, which combinations should be prioritized for clinical development based on mechanistic rationale and preliminary evidence?

Addressing these questions will require interdisciplinary approaches combining structural biology, cell signaling analysis, advanced imaging, and comprehensive clinical correlative studies.

What methodological recommendations can improve the reproducibility of MET antibody research?

To enhance reproducibility in MET antibody research, investigators should adopt these methodological practices:

• Standardized antibody characterization:

  • Report complete information about antibodies including clone, source, lot number, and validation method

  • Perform and document specificity testing using multiple approaches (peptide competition, knockout controls)

  • Validate phospho-specific antibodies with phosphatase treatment controls

• Consistent cell line models:

  • Use well-characterized cell lines with documented MET expression levels

  • Report passage number and growth conditions

  • Regularly authenticate cell lines to prevent misidentification

  • Consider testing multiple cell lines to account for genetic background effects

• Standardized experimental conditions:

  • For signaling studies, precisely document serum starvation protocols

  • Report antibody concentrations in molar terms rather than μg/ml

  • Include positive controls (HGF stimulation) and negative controls (isotype antibodies)

• Comprehensive time-course analysis:

  • Avoid single time-point measurements that may miss transient effects

  • Include early (2-30 minutes) and late (hours-days) time points to capture both immediate signaling and adaptive responses

• Complementary methodological approaches:

  • Combine biochemical (western blot), cellular (immunofluorescence), and functional (migration, proliferation) assays

  • Validate key findings using orthogonal techniques

  • Include in vivo validation where feasible

• Transparent reporting of experimental details:

  • Document antibody purification methods

  • Report buffer compositions and storage conditions

  • Disclose image acquisition parameters and processing steps

• Quantitative analysis and statistics:

  • Report sample sizes and replicate structure

  • Use appropriate statistical tests and report exact p-values

  • Quantify western blot results with normalization to loading controls

Adherence to these recommendations will facilitate comparison across studies and accelerate progress in developing effective MET antibody therapeutics.

What emerging research directions have the greatest potential to advance MET antibody applications?

Several innovative research directions show exceptional promise for advancing MET antibody applications:

• Multi-specific antibody engineering: Beyond biparatopic constructs, developing antibodies that simultaneously target MET and complementary targets (HER family receptors, AXL, RON) could overcome resistance mechanisms and enhance efficacy. Trispecific formats that additionally engage immune effectors represent a particularly promising approach .

• In vivo antibody evolution: Using directed evolution techniques to optimize antibody properties directly in tumor models could yield antibodies with superior tumor penetration, stability, and efficacy compared to conventional in vitro selection methods.

• Intracellular antibody delivery systems: Developing technologies to deliver antibodies to the cytoplasm could enable targeting of intracellular MET signaling complexes, potentially disrupting scaffolding functions that are inaccessible to conventional antibodies.

• Antibody combinations targeting distinct epitopes: Rather than designing single biparatopic antibodies, exploring optimized combinations of multiple monospecific antibodies may enable more flexible and personalized therapy approaches.

• Reversible masking technologies: Engineering antibodies with conditional binding domains that become activated only in the tumor microenvironment could enhance tumor-specific activity while reducing on-target, off-tumor effects.

• MET conformation-selective antibodies: Developing antibodies that specifically recognize and stabilize inactive MET conformations could prevent paradoxical activation while blocking pathological signaling .

• Computational antibody design: Using artificial intelligence and machine learning to design antibodies with optimized binding properties, tissue penetration, and pharmacokinetics could accelerate development of next-generation therapeutics.

• Single-cell response analysis: Mapping the heterogeneity of responses to MET antibodies at the single-cell level within tumors could identify resistance-associated cell states and inform more effective therapeutic strategies.