ALMT10 Antibody

What is ADAM10 and why is it an important research target?

ADAM10 is a transmembrane metalloprotease that mediates ectodomain shedding of various transmembrane proteins, including adhesion proteins, growth factor precursors, and cytokines. It plays essential roles in development and tissue homeostasis through its proteolytic activity. ADAM10 cleaves numerous substrates including TNF-alpha, JAM3, heparin-binding epidermal growth-like factor, ephrin-A2, CD44, CDH2, and amyloid precursor protein (APP) . Its involvement in the Notch signaling pathway is particularly significant for developmental processes and cancer progression, making it an important target for antibody-based research and potential therapeutic applications .

What applications are ADAM10 antibodies commonly used for in research?

ADAM10 antibodies are employed in multiple research applications, including:

• Western blotting (WB) for protein expression analysis

• Immunohistochemistry on paraffin-embedded tissues (IHC-P) for localization studies

• Functional studies targeting active vs. inactive ADAM10 conformations

• Cancer research investigating ADAM10's role in tumor development

• Therapeutic development targeting ADAM10 in cancer and other diseases

The selection of a specific ADAM10 antibody depends on the intended application, species reactivity requirements, and whether the research aims to detect total ADAM10 or a specific active conformation .

How do I validate the specificity of an ADAM10 antibody for my research?

Validating ADAM10 antibody specificity is crucial for reliable results. A comprehensive validation approach includes:

• Positive and negative control samples (tissues/cell lines known to express or lack ADAM10)

• Comparison with established reference antibodies

• Knockout/knockdown validation - testing on ADAM10-deficient samples

• Cross-reactivity assessment with related ADAM family proteins

• Evaluation in multiple applications (WB, IHC, etc.) to confirm consistent results

• Analysis of band patterns in Western blots (ADAM10 appears at ~90 kDa mature form and ~60 kDa processed form)

Thorough validation is particularly important when investigating ADAM10 in novel contexts or when developing therapeutic antibodies targeting specific ADAM10 conformations .

How do I optimize Western blot protocols for ADAM10 detection?

For optimal Western blot detection of ADAM10, consider the following methodology:

• Sample preparation: Use non-reducing conditions when epitopes are conformation-dependent; include protease inhibitors during extraction

• Loading concentration: Start with 20-30 μg of total protein, adjusting based on expression level

• Electrophoresis conditions: 8-10% SDS-PAGE gels typically provide good resolution for ADAM10

• Transfer settings: Semi-dry or wet transfer protocols may require optimization (typically 90-100V for 60-90 minutes)

• Blocking: 5% non-fat milk or BSA in TBST (Tris-buffered saline with 0.1% Tween-20)

• Primary antibody dilution: Typically 1:1000 to 1:2000, but optimize based on antibody specifications

• Expected results: Look for bands at approximately 90 kDa (mature form) and 60 kDa (processed form)

Always run appropriate positive controls (e.g., cell lines known to express ADAM10) and consider inclusion of molecular weight markers that span the expected size range .

What are the key considerations for immunohistochemical detection of ADAM10?

For successful immunohistochemical detection of ADAM10 in tissues:

• Fixation: 10% neutral buffered formalin is standard; overfixation may mask epitopes

• Antigen retrieval methods: Heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) should be tested

• Antibody dilution: Begin with manufacturer's recommendation (typically 1:100-1:500) and optimize

• Incubation conditions: Overnight at 4°C or 1-2 hours at room temperature

• Detection system: HRP-polymer or biotin-streptavidin systems are commonly used

• Counterstaining: Hematoxylin provides good nuclear contrast

• Controls: Include positive control tissues (brain, heart, liver express ADAM10), negative controls (primary antibody omission), and isotype controls

When interpreting results, note that ADAM10 typically shows membrane and cytoplasmic staining patterns, with intensity varying based on tissue type and disease state .

How can I differentiate between active and inactive forms of ADAM10 in my experiments?

Differentiating between active and inactive ADAM10 conformations requires specialized approaches:

• Conformation-specific antibodies: Some antibodies (like 8C7) preferentially recognize the active conformation of ADAM10 present on tumor cells by binding to the substrate-binding cysteine-rich domain

• Activity assays: Measure ADAM10 enzymatic activity using fluorogenic peptide substrates

• Substrate cleavage analysis: Monitor cleavage of known ADAM10 substrates like Notch receptors or APP

• Co-immunoprecipitation: Examine interactions with regulatory proteins that affect ADAM10 activity

• Cell surface biotinylation: Assess the proportion of ADAM10 at the cell surface (where it's typically active)

Research shows that the active form of ADAM10 is elevated in tumors in both mice and humans, making this distinction particularly relevant for cancer research applications .

How do ADAM10 antibodies contribute to understanding cancer progression mechanisms?

ADAM10 antibodies have revealed critical insights into cancer progression mechanisms:

• Notch signaling modulation: ADAM10 antibodies have helped establish that ADAM10-mediated Notch cleavage maintains cancer stem cells, which contribute to metastasis and chemoresistance

• Identifying overexpression patterns: Research using ADAM10 antibodies has demonstrated ADAM10 overexpression correlates with aggressive metastatic phenotypes in multiple cancers including colon, gastric, prostate, breast, ovarian, uterine, and leukemia

• Signaling network analysis: Antibody-based studies have revealed aberrant signaling from Notch, erbBs, and other receptors associated with ADAM10 overactivity

• Tumor microenvironment studies: ADAM10 antibodies help track how ADAM10 activity influences interactions between tumor cells and surrounding stroma

These insights support ADAM10 inhibition as an important approach to deter the progression of advanced cancers, particularly colorectal cancer .

What methodological approaches can be used to target active ADAM10 in cancer research?

Several methodological approaches can specifically target active ADAM10 in cancer research:

• Conformation-specific antibodies: Antibodies like 1H5 and 8C7 that bind to activated ADAM10 conformations allow selective targeting of tumor cells

• Substrate-binding region targeting: Focus on the substrate-binding cysteine-rich domain rather than the catalytic domain avoids off-target effects

• Antibody-drug conjugates: Cytotoxic drugs conjugated to ADAM10-specific antibodies enable tumor-selective targeting

• Combination therapies: Test ADAM10 antibodies in combination with established chemotherapeutics (e.g., Irinotecan)

• Xenograft models: Evaluate the efficacy of ADAM10-targeting approaches in animal models

Studies show that antibodies targeting the ADAM10 substrate-binding region can inhibit Notch cleavage and proliferation of cancer cell lines both in vitro and in mouse models, with promising results for tumor growth inhibition .

How does the 1H5 human anti-ADAM10 monoclonal antibody work, and what are its research applications?

The 1H5 human anti-ADAM10 monoclonal antibody represents an innovative approach for ADAM10-targeted cancer therapy research:

Mechanism of Action:

• Binding target: Specifically binds to the substrate-binding cysteine-rich domain of ADAM10

• Conformational specificity: Recognizes an activated ADAM10 conformation present on tumor cells

• Functional effects: Inhibits Notch cleavage while augmenting catalytic activity towards small peptide substrates

• Therapeutic potential: When combined with Irinotecan, causes effective tumor growth inhibition without discernible toxicity

Research Applications:

• Colon cancer research: Validated in COLO205 and other colon cancer cell lines

• Notch signaling studies: Tool for investigating ADAM10-mediated Notch processing

• Combination therapy research: Model for studying synergistic effects with chemotherapeutic agents

• Cancer stem cell investigations: Means to target cancer stem cell populations maintained by Notch signaling

This approach of targeting the substrate-binding region overcomes limitations of previous small molecule inhibitors that exhibited musculoskeletal toxicity by targeting the active site .

How do TspanC8 tetraspanins regulate ADAM10 function, and how can this be studied?

The regulation of ADAM10 by TspanC8 tetraspanins represents a complex research area:

Regulatory Mechanisms:
TspanC8 tetraspanins (six members of the tetraspanin superfamily) regulate ADAM10 through multiple mechanisms:

• Endoplasmic reticulum exit control: TspanC8s facilitate ADAM10 trafficking from the ER

• Substrate selectivity modulation: Different TspanC8s direct ADAM10 toward specific substrates

• Compartmentalization: TspanC8s influence ADAM10 localization within membrane microdomains

• Conformational changes: May affect ADAM10 active/inactive conformational states

Experimental Approaches:

• Co-immunoprecipitation assays to detect ADAM10-TspanC8 interactions

• Fluorescence microscopy for co-localization studies

• TspanC8 knockdown/overexpression combined with ADAM10 activity assays

• Analysis of substrate cleavage patterns with different TspanC8 expressions

• Proximity ligation assays to confirm direct protein interactions

Understanding these interactions provides insights into tissue-specific ADAM10 functions and potential therapeutic targeting strategies .

What factors should be considered when developing antibody-drug conjugates targeting ADAM10?

Developing effective antibody-drug conjugates (ADCs) targeting ADAM10 requires consideration of multiple technical factors:

• Epitope selection: Target epitopes specific to active ADAM10 conformations found on tumor cells

• Antibody characteristics:

  • Human or humanized antibodies preferred to minimize immunogenicity

  • Binding affinity optimization (Kd typically in nanomolar range)

  • Internalization efficiency assessment

• Linker chemistry:

  • Cleavable vs. non-cleavable linkers based on internalization mechanism

  • Stability in circulation to minimize off-target effects

• Cytotoxic payload selection:

  • Match potency to expected target expression levels

  • Consider bystander effect requirements

• Drug-to-antibody ratio (DAR) optimization:

  • Typically 2-4 drug molecules per antibody

  • Higher DARs may affect pharmacokinetics

• Validation approaches:

  • In vitro cytotoxicity against ADAM10-expressing cell lines

  • Selectivity assessment using cell lines with varying ADAM10 expression

  • In vivo xenograft models for efficacy and toxicity evaluation

Research with ADCs targeting active ADAM10 has demonstrated preferential killing of cells displaying the target epitope and tumor growth inhibition in mouse models .

How can potential off-target effects of ADAM10 antibodies be identified and minimized?

Identifying and minimizing off-target effects of ADAM10 antibodies requires systematic approaches:

Identification Methods:

• Cross-reactivity screening:

  • Test against related ADAM family proteins

  • Screen against tissue panels from relevant species

• Cellular phenotype analysis:

  • Compare effects in ADAM10 knockout cells vs. wild-type

  • Rescue experiments with ADAM10 re-expression

• Proteomic profiling:

  • Analysis of cleavage patterns of known substrates

  • Unbiased proteomic approaches to identify unexpected changes

• Transcriptomic analysis:

  • RNA-seq to identify unexpected pathway alterations

  • Compare with known ADAM10 inhibition signatures

Minimization Strategies:

• Epitope refinement:

  • Target unique ADAM10 regions with minimal homology to other proteins

  • Consider conformation-specific antibodies that recognize active ADAM10 only

• Domain-specific targeting:

  • Target the substrate-binding region rather than the catalytic domain

  • Focus on regulatory domains specific to ADAM10

• Dosing optimization:

  • Establish dose-response relationships to determine minimal effective dose

  • Consider combination therapy to allow lower antibody concentrations

Research indicates that targeting the substrate-binding region of ADAM10 rather than the catalytic domain may reduce off-target effects compared to small molecule inhibitors that target the active site .

How are ADAM10 antibodies being used to investigate neurodegenerative diseases?

ADAM10 antibodies are increasingly employed in neurodegenerative disease research, particularly for Alzheimer's disease investigations:

• Amyloid precursor protein (APP) processing studies:

  • ADAM10 functions as an α-secretase in APP processing

  • ADAM10 antibodies help track how alterations in ADAM10 expression/activity affect amyloidogenic vs. non-amyloidogenic APP processing

  • Quantitative analysis of α-secretase vs. β-secretase cleavage products

• Notch signaling in neuronal development and maintenance:

  • ADAM10 antibodies reveal how Notch processing influences neuronal health

  • Analysis of lateral inhibition during neurogenesis

  • Investigation of adult neuroplasticity mechanisms

• Prion protein cleavage:

  • ADAM10 contributes to normal cellular prion protein cleavage

  • Antibodies help track how disrupted ADAM10 activity may contribute to prion pathogenesis

• Therapeutic development approaches:

  • ADAM10 activation strategies to promote non-amyloidogenic processing

  • Selective ADAM10 modulation to avoid disruption of essential physiological functions

ADAM10 antibodies with high specificity are essential for distinguishing between ADAM10 and related metalloproteases (ADAM17) that may have overlapping functions in the central nervous system .

What are the latest approaches for developing function-blocking ADAM10 antibodies?

Recent advancements in developing function-blocking ADAM10 antibodies include:

• Structure-guided design strategies:

  • Targeting specific functional domains based on crystal structure data

  • Rational selection of epitopes that interfere with substrate binding

  • Computer-aided design to optimize binding and inhibitory properties

• Allosteric modulation approaches:

  • Antibodies that bind outside the active site but induce conformational changes

  • Stabilization of inactive conformations of ADAM10

  • Interference with TspanC8 tetraspanin interactions that regulate ADAM10

• Substrate-selective inhibition:

  • Antibodies designed to block specific substrate interactions while preserving others

  • Targeting substrate binding pockets with varying geometries/properties

  • Domain-specific antibodies that affect only subsets of ADAM10 functions

• Combination strategies:

  • Bispecific antibodies targeting ADAM10 and key substrates simultaneously

  • Cocktails of antibodies targeting different ADAM10 domains for comprehensive inhibition

  • Antibody combinations with small molecule inhibitors for synergistic effects

The 1H5 antibody exemplifies a successful approach by targeting the substrate-binding cysteine-rich domain rather than the catalytic domain, demonstrating effective inhibition of Notch cleavage and tumor growth in colon cancer models when combined with chemotherapy .

How can ADAM10 antibodies contribute to personalized cancer therapy research?

ADAM10 antibodies offer several approaches for advancing personalized cancer therapy research:

• Patient stratification methodologies:

  • Immunohistochemical analysis of tumor biopsies for ADAM10 expression levels

  • Assessment of ADAM10 activation state using conformation-specific antibodies

  • Correlation of ADAM10 patterns with treatment response and survival outcomes

• Biomarker development:

  • ADAM10 substrate shedding measurement in patient samples

  • Quantification of active vs. total ADAM10 ratios in tumors

  • Analysis of downstream signaling pathway activation states

• Therapeutic targeting strategies:

  • Patient-specific antibody selection based on ADAM10 expression/activity profiles

  • Combination therapy design incorporating ADAM10 inhibition with conventional treatments

  • Monitoring of therapy response through serial ADAM10 activity assessment

• Resistance mechanism investigation:

  • Tracking changes in ADAM10 expression/activity during treatment

  • Analysis of compensatory proteolytic pathways that emerge during ADAM10 inhibition

  • Evaluation of tumor evolution in response to ADAM10-targeted therapies

Research demonstrates that antibodies like 1H5 that recognize activated ADAM10 conformations present on tumor cells can effectively inhibit tumor growth in combination with chemotherapeutic agents, suggesting potential for personalized treatment approaches based on tumor-specific ADAM10 activation patterns .