OSMR Antibody

What is OSMR and what signaling pathways does it activate in cancer?

OSMR (oncostatin M receptor) is a single-pass membrane protein belonging to the type I cytokine receptor family. It plays a critical role in cancer biology, particularly in ovarian cancer where it promotes cell proliferation and metastasis through STAT3 signaling activation . OSMR functions by forming a heterodimeric complex with IL6ST (gp130) upon binding of its ligand oncostatin M (OSM), which is predominantly expressed by tumor-associated macrophages in the cancer microenvironment . This receptor complex formation triggers downstream signaling cascades that result in prolonged STAT3 activation, leading to enhanced cancer cell proliferation and migration .

Additionally, OSMR can associate with IL31RA to form the heterodimeric IL31 receptor, demonstrating its versatility in different signaling pathways . OSMR mRNA is expressed at relatively high levels in various cell types including neural cells, fibroblasts, epithelial cells, and multiple tumor cell lines, making it a potentially valuable therapeutic target .

How are OSMR antibodies characterized for research applications?

OSMR antibodies are characterized through multiple validation techniques to ensure specificity and functionality before application in research settings. Standard characterization methods include:

• Western blot analysis: Performed across multiple human tissues and cell lines to evaluate antibody specificity. For OSMR antibodies like 10982-1-AP, positive signals have been detected in HeLa, A375, HEK-293T, Raji, and HepG2 cells at the expected molecular weight of 110 kDa .

• Immunohistochemistry validation: Comparing antibody staining patterns with known expression profiles in tissues.

• Enhanced validation techniques:

  • Genetic validation using siRNA knockdown to demonstrate specificity

  • Recombinant expression validation

  • Independent antibody validation using multiple antibodies targeting different epitopes

  • Orthogonal validation comparing protein and RNA expression data

The characterization process ensures that OSMR antibodies used in research provide reliable and reproducible results across various experimental applications .

What are the key applications of OSMR antibodies in cancer research?

OSMR antibodies serve multiple critical functions in cancer research, particularly in studying tumor biology and developing potential therapeutic approaches:

• Diagnostic applications: OSMR antibodies are used in immunohistochemistry (IHC) and immunofluorescence (IF) to detect OSMR expression levels in tumor tissues, which may correlate with disease progression or prognosis .

• Mechanistic studies: Western blot (WB) applications using OSMR antibodies help elucidate the molecular pathways involved in OSMR signaling by detecting protein expression, phosphorylation, and interaction with other signaling molecules .

• Therapeutic development: Human monoclonal antibodies targeting OSMR, such as clones B14 and B21, have demonstrated preclinical efficacy in suppressing cancer cell growth by disrupting OSM-induced OSMR-IL6ST heterodimerization and blocking oncogenic signaling .

• Functional assays: OSMR antibodies can be used to study the biological consequences of OSMR blockade on cancer cell proliferation, migration, and invasion in vitro .

• In vivo models: Anti-OSMR antibodies have been employed in mouse models to evaluate their efficacy in suppressing tumor growth, providing crucial preclinical data for potential translation to human therapies .

Research has shown that OSMR targeting antibodies can effectively inhibit ovarian cancer growth both in vitro and in vivo, highlighting their potential as immunotherapeutic agents .

How are human monoclonal antibodies against OSMR developed and screened?

The development of human monoclonal antibodies against OSMR involves sophisticated molecular biology techniques and screening protocols. Based on the search results, the process typically follows these methodological steps:

• Library construction and phage display: Large human scFv phage display antibody libraries are constructed from cDNA extracted from PBMCs and tonsils of multiple donors. This diversity ensures broad coverage of potential binding epitopes .

• Panning process:

  • Recombinant OSMR protein (typically 50μg) is coated on MaxiSorp immune tubes and blocked with 8% milk

  • Pre-blocked phages are incubated with the antigen-coated tubes

  • After washing with PBST and PBS, bound phages are eluted using triethylamine (TEA)

  • Eluted phages are tittered and used to infect host bacteria for amplification

• Primary screening by Phage ELISA:

  • Individual colonies (e.g., 1504 as described in the study) are picked to make phage for ELISA binding with OSMR

  • ELISA plates are coated with OSMR antigen (1μg/mL in PBS) overnight at 4°C

  • After blocking and incubation with phages, HRP-conjugated Mouse-anti-M13 secondary antibody is used for detection

  • Positive clones are identified by colorimetric development using TMB substrate

• Affinity measurement using BLI (Bio-Layer Interferometry):

  • Antibody (20 μg/mL) is loaded onto protein G biosensors

  • Loaded biosensors are exposed to a series of recombinant OSMR concentrations (typically 0.41–900 nM)

  • Association and dissociation rates are measured to calculate binding affinity (Kd)

This systematic approach has successfully yielded therapeutic candidates like B14 and B21 antibody clones, which demonstrated high efficacy in disrupting OSM-induced OSMR-IL6ST dimerization and oncogenic signaling .

What methodologies are used to evaluate OSMR antibody efficacy in preclinical models?

Evaluating OSMR antibody efficacy in preclinical models involves a comprehensive array of in vitro and in vivo methodologies to assess their therapeutic potential. The following approaches are typically employed:

• In vitro functional assays:

  • Cell proliferation and viability assays to measure antibody effects on cancer cell growth

  • Migration and invasion assays to assess metastatic potential

  • Signaling pathway analysis using phospho-protein detection to confirm STAT3 inhibition

  • Receptor dimerization studies to evaluate disruption of OSMR-IL6ST complex formation

• In vivo tumor models:

  • Athymic female nude mice (Nu/Nu) bearing human ovarian cancer xenografts (e.g., Heya8-Luc+ cells)

  • Syngeneic models using immunocompetent FVB/NJ-Homozygous mice with murine cancer cells

  • Treatment protocols typically involve antibody administration (e.g., 10 mg/kg body weight) twice weekly for 5-6 weeks

  • Tumor burden is monitored weekly using bioluminescence imaging with Xenogen IVIS100 imaging system

• Sample analysis methodologies:

  • Collection of ascites from tumor-bearing mice and filtration through 40μm filters

  • Peritoneal wash with sterile PBS for non-tumor bearing control mice

  • Centrifugation of filtrate containing cells at 1500 rpm and washing with PBS

  • Downstream analysis of collected cells for various biological parameters

• Comparative studies:

  • Parallel evaluation of different antibody clones (e.g., B14 and B21)

  • Combination with OSM treatment (e.g., 250ng/kg body weight) to assess inhibition of ligand-induced effects

  • Comparison with isotype control antibodies to confirm specificity

These methodological approaches collectively provide robust evidence for the efficacy of anti-OSMR antibodies in preclinical settings, demonstrating their potential as immunotherapeutic agents for ovarian cancer treatment .

How can OSMR antibodies be optimized for improved specificity and reduced off-target effects?

Optimizing OSMR antibodies for improved specificity and reduced off-target effects requires a multifaceted approach combining rational design, advanced screening methods, and rigorous validation:

• Epitope-focused design strategies:

  • Target regions with the lowest possible sequence identity to other human proteins

  • Select epitopes with maximum percent sequence identity of less than 60% to minimize cross-reactivity

  • Utilize sliding window approaches (10 aa or 50 aa residues) to identify unique regions

  • Avoid membrane regions and signal peptides predicted through MDM and MDSEC majority decision methods

• Antigenicity optimization:

  • Select regions with high predicted antigenicity (tendency to generate immune response)

  • Utilize proprietary antigenicity prediction tools based on sliding window approaches

  • Focus on peak regions in antigenicity curves that are also unique to OSMR

• Validation approaches to confirm specificity:

  • Perform cross-reactivity testing against a panel of 384 different antigens

  • Conduct genetic validation through siRNA knockdown experiments

  • Evaluate consistency between immunohistochemistry data and consensus RNA levels

  • Compare staining patterns of multiple independent antibodies targeting different epitopes

• Structural modifications for improved performance:

  • Engineer antibody framework regions to improve stability

  • Modify complementarity-determining regions (CDRs) to enhance binding affinity

  • Consider humanization for antibodies intended for therapeutic applications

  • Evaluate various antibody formats (full IgG, Fab, scFv) for specific research applications

By implementing these optimization strategies, researchers can develop OSMR antibodies with enhanced specificity, higher affinity, and reduced off-target effects, making them more valuable for both research and potential therapeutic applications.

What are the key technical considerations when using OSMR antibodies for detecting heterodimerization with IL6ST?

Detecting OSMR-IL6ST heterodimerization using antibodies presents several technical challenges requiring specific methodological approaches:

• Antibody selection considerations:

  • Choose antibodies that recognize epitopes outside the dimerization interface to avoid interference with the biological interaction

  • For therapeutic applications, select antibodies that specifically disrupt the heterodimerization, such as clones B14 and B21 which were shown to abrogate OSM-induced OSMR-IL6ST heterodimerization

  • Consider using pairs of antibodies targeting OSMR and IL6ST simultaneously for co-detection studies

• Recommended detection methods:

  • Proximity ligation assays (PLA): Allows visualization of protein interactions within 40nm distance

  • Co-immunoprecipitation: Using anti-OSMR antibodies to pull down the complex followed by IL6ST detection

  • FRET/BRET assays: For real-time monitoring of receptor interactions in living cells

  • Immunofluorescence co-localization: Using differently labeled antibodies against OSMR and IL6ST

• Experimental design factors:

  • Timing: OSM stimulation typically induces rapid receptor dimerization, requiring precise timing for detection

  • Fixation methods: Use gentle fixation protocols to preserve protein-protein interactions

  • Positive controls: Include OSM treatment (known to induce heterodimerization)

  • Negative controls: Include antibodies known to block the interaction or use cells with OSMR/IL6ST knockdown

• Troubleshooting strategies:

  • If no interaction is detected, verify individual receptor expression levels

  • Ensure antibody compatibility with chosen detection method

  • Consider membrane permeabilization protocols that preserve receptor complex integrity

  • Optimize OSM concentration and treatment duration for maximum complex formation

Researchers studying OSMR-IL6ST heterodimerization should consider using antibodies at the recommended dilutions (e.g., 1:1000-1:4000 for Western blot applications for some commercial antibodies) and may need to titrate the antibodies in each testing system to obtain optimal results .

How can researchers quantitatively assess OSMR internalization and degradation induced by therapeutic antibodies?

Quantitative assessment of OSMR internalization and degradation induced by therapeutic antibodies requires sophisticated methodological approaches to track receptor dynamics. Based on the available research, the following techniques provide robust quantitative data:

• Flow cytometry-based approaches:

  • Surface receptor labeling before and after antibody treatment

  • Time-course analysis to determine internalization kinetics

  • Dual-color flow cytometry using fluorescently labeled anti-OSMR antibodies that don't compete with therapeutic antibodies

  • Quantification by calculating the percentage decrease in mean fluorescence intensity

• Confocal microscopy techniques:

  • Live-cell imaging using fluorescently labeled OSMR antibodies

  • Co-localization studies with endosomal/lysosomal markers (EEA1, LAMP1)

  • Z-stack acquisition to track receptor movement from membrane to intracellular compartments

  • Quantitative image analysis measuring membrane vs. cytoplasmic signal ratio over time

• Biochemical assessment methods:

  • Surface biotinylation assays to specifically track cell surface proteins

  • Western blot analysis of total OSMR levels at various time points after antibody treatment

  • Cycloheximide chase experiments to distinguish between degradation and reduced synthesis

  • Proteasome and lysosome inhibitor studies to determine degradation pathways

• Advanced molecular techniques:

  • OSMR tagging with pH-sensitive fluorescent proteins to track endosomal trafficking

  • CRISPR-Cas9 editing of OSMR to introduce trackable tags without disrupting function

  • Receptor ubiquitination analysis to correlate with degradation kinetics

  • Pulse-chase experiments using metabolic labeling to track receptor half-life

How do researchers determine optimal antibody concentrations for in vivo OSMR targeting studies?

Determining optimal antibody concentrations for in vivo OSMR targeting studies involves a systematic approach that balances efficacy with safety considerations:

• Dose-finding strategies:

  • Pilot dose-escalation studies: Begin with a range of doses (e.g., 1, 5, 10, 20 mg/kg) to establish dose-response relationships

  • Pharmacokinetic (PK) analysis: Measure antibody clearance rates and half-life in the target organism to determine dosing frequency

  • Biodistribution studies: Use labeled antibodies to track tissue penetration and tumor accumulation

  • Target occupancy assessment: Evaluate receptor saturation at different dose levels

• Evidence-based dosing protocols:

  • Research has demonstrated efficacy of anti-OSMR antibodies (B14 and B21 mAbs) at 10 mg/kg body weight administered twice weekly for five weeks in athymic female nude mice bearing Heya8-Luc+ ovarian cancer cells

  • This established protocol resulted in significant inhibition of tumor growth as monitored by bioluminescence imaging

  • Comparative studies with varying doses can help identify the minimum effective dose

• Monitoring parameters for dose optimization:

  • Tumor growth inhibition efficacy

  • Body weight changes and general health indicators

  • Biomarker analysis (e.g., STAT3 phosphorylation in tumor samples)

  • Adverse events documentation

  • Serum antibody levels to confirm exposure

• Translational considerations:

  • Allometric scaling for cross-species translation

  • Species differences in OSMR biology and antibody interactions

  • Pharmacodynamic biomarker selection for clinical translation

  • Integration of mechanism-based pharmacokinetic-pharmacodynamic (PK-PD) modeling

The established protocol of 10 mg/kg body weight twice weekly has shown promising results in preclinical models, providing a benchmark starting point for researchers designing new in vivo studies targeting OSMR in cancer models .

What are the current challenges and future directions in developing OSMR antibodies as cancer therapeutics?

The development of OSMR antibodies as cancer therapeutics shows promise but faces several challenges that define future research directions:

• Current technical challenges:

  • Specificity optimization: While current antibodies show good specificity, further engineering may reduce potential off-target effects in diverse tissue types

  • Heterogeneous expression: OSMR expression varies across tumor types and even within the same tumor, requiring strategies to address heterogeneity

  • Resistance mechanisms: Understanding potential compensatory signaling pathways that may emerge following OSMR blockade

  • Combination therapy design: Determining optimal partners for OSMR antibodies to prevent resistance development

• Translational challenges:

  • Species differences: Human OSMR antibodies may not cross-react with murine OSMR, complicating preclinical testing

  • Biomarker development: Identifying patients most likely to benefit from OSMR-targeted therapy

  • Delivery optimization: Ensuring sufficient antibody penetration into solid tumors

  • Safety profile characterization: OSMR has physiological roles that must be considered for long-term inhibition

• Promising future directions:

  • Antibody-drug conjugates (ADCs): Leveraging OSMR internalization to deliver cytotoxic payloads specifically to cancer cells

  • Bispecific antibodies: Developing antibodies targeting both OSMR and IL6ST simultaneously or OSMR and immune effector cells

  • Patient stratification approaches: Developing companion diagnostics to identify high OSMR expressors

  • Combination with immunotherapy: Exploring synergies between OSMR blockade and immune checkpoint inhibitors

• Emerging research areas:

  • Investigating OSMR's role in cancer stem cell maintenance

  • Understanding OSMR contribution to the tumor microenvironment

  • Exploring OSMR in cancers beyond ovarian cancer

  • Developing novel formats of OSMR-targeting molecules beyond conventional antibodies

The preclinical success of human monoclonal antibody clones B14 and B21 in inhibiting ovarian cancer growth provides strong proof-of-principle for OSMR antibody therapeutics, but addressing these challenges will be critical for successful clinical translation .