CD146 Monoclonal Antibody

What is CD146 and why is it significant in cancer research?

CD146 (also known as MCAM, MUC18, S-endo 1, or Cell surface glycoprotein P1H12) is an adhesion molecule expressed on multiple tumors including melanoma, kidney, pancreatic, breast, lung, gastric, and hepatic cancers . It is a 646 amino acid single-pass type 1 transmembrane glycoprotein with a calculated molecular mass of approximately 72 kDa, though N-linked glycosylation causes it to migrate at approximately 118 kDa in polyacrylamide gels . As a member of the immunoglobulin superfamily, CD146 contains 2 V-type and 3 C-type Ig-like domains .

CD146 is significant in cancer research because its expression is associated with tumor progression, particularly in melanoma . CD146-positive tumors typically display higher proliferation rates and greater capacity to metastasize, making it both a potential biomarker for poor prognosis and a therapeutic target . In some studies, CD146 has proven to be a better marker of disease progression in melanoma than sentinel lymph node analysis on biopsies .

How does CD146 expression differ between normal and cancer cells?

This differential expression provides the basis for developing tumor-specific antibodies like TsCD146 mAb, which can recognize CD146 expressed on cancer cells but not on vascular cells . Immunofluorescence experiments on human biopsies have demonstrated that such antibodies can bind to tumor cells in melanoma, verrucous skin carcinoma, renal carcinoma, and colonic adenocarcinoma samples without binding to endothelial cells in the same tissues or in normal skin, kidney, and colon samples .

What are the different forms of CD146 and their functional significance?

CD146 exists in multiple forms with distinct biological functions:

• Membrane isoforms:

  • Short CD146: Promotes cellular proliferation, migration, and adhesion in endothelial cells

  • Long CD146: Essential for stabilizing the junctions of neo-vessels necessary for pseudo-capillary formation

• Soluble CD146:

  • Secreted by cancer cells

  • Mediates both autocrine effects on cancer cells and paracrine effects on vascular endothelial cells

  • Increases cancer cell proliferation and production of pro-tumorigenic and angiogenic factors (e.g., VEGF)

  • Promotes an anti-apoptotic phenotype and decreases cellular senescence

  • Activates the c-myc signaling pathway

The complementary roles of short and long CD146 isoforms are particularly important in angiogenesis, as demonstrated in both in vitro and in vivo models . Understanding these distinct forms is crucial for developing targeted therapeutic approaches.

What criteria should be considered when selecting a CD146 monoclonal antibody for cancer research?

When selecting a CD146 monoclonal antibody for cancer research, researchers should consider:

• Target specificity: Determine whether you need an antibody that recognizes:

  • All forms of CD146 (pan-CD146 antibody)

  • Only tumor-specific CD146 (like TsCD146 mAb)

  • Specific isoforms (short, long, or soluble CD146)

• Cross-reactivity profile: Verify whether the antibody shows cross-reactivity with normal tissues expressing CD146, such as endothelial cells .

• Application compatibility: Confirm the antibody's validated applications (Western blot, flow cytometry, immunohistochemistry, PET imaging, etc.) and whether these align with your experimental needs .

• Clone characteristics: Consider the clone's isotype (e.g., IgG1), epitope specificity, and whether it recognizes native or denatured forms of CD146 .

• Functional properties: Assess whether the antibody has neutralizing activity or induces internalization of CD146, which may be relevant for therapeutic applications .

For tumor-specific applications, antibodies like TsCD146 mAb that specifically recognize tumor CD146 without binding to vascular CD146 offer significant advantages for both diagnostic and therapeutic purposes .

How can researchers validate the specificity of CD146 antibodies in their experimental systems?

Rigorous validation of CD146 antibody specificity should include:

• Cell panel testing: Test the antibody against:

  • Multiple CD146-positive cancer cell lines (e.g., melanoma, pancreatic cancer)

  • CD146-negative cell lines as negative controls (e.g., Lovo colorectal cancer cells)

  • Normal CD146-expressing cells (e.g., HUVEC, HMEC-1, HUA-SMC)

• Expression correlation: Verify CD146 expression in test cells at both:

  • mRNA level using RT-PCR with primers directed against the extracellular portion of CD146

  • Protein level using ELISA on cell lysates

• Multi-technique confirmation: Confirm antibody specificity using complementary techniques:

  • Flow cytometry to assess cell surface binding

  • Immunofluorescence to evaluate tissue section staining patterns

  • Western blot to confirm recognition of the appropriate molecular weight band (~118-140 kDa)

• Co-staining experiments: For tissue sections, perform co-staining with established markers (e.g., CD31 for endothelial cells) to confirm cell-type specificity .

• Competitive binding: Perform blocking experiments with recombinant CD146 to confirm binding specificity .

For research focused on tumor-specific CD146, validation should specifically demonstrate differential binding between tumor and normal tissues expressing CD146 .

How can CD146 monoclonal antibodies be optimized for immunohistochemical detection of tumors?

Optimizing CD146 monoclonal antibodies for immunohistochemical detection of tumors requires:

• Tissue preparation considerations:

  • Fixation method: Formalin-fixed, paraffin-embedded (FFPE) tissues may require antigen retrieval to expose CD146 epitopes

  • Fresh frozen sections may better preserve native CD146 conformation for certain antibody clones

• Antibody selection:

  • For general CD146 detection, standard CD146 antibodies like clone OJ79c can be used

  • For tumor-specific detection, specialized antibodies like TsCD146 mAb are recommended to avoid cross-reactivity with vascular CD146

• Protocol optimization:

  • Titrate antibody concentration to determine optimal signal-to-noise ratio

  • Include positive controls (known CD146-positive tumors) and negative controls (CD146-negative tissues)

  • Use double immunofluorescence with endothelial markers (e.g., CD31) to differentiate tumor CD146 from vascular CD146

• Detection system selection:

  • For brightfield microscopy: HRP-conjugated secondary antibodies with DAB substrate

  • For fluorescence: Fluorophore-conjugated secondary antibodies suitable for the specific tumor type being studied

• Counterstaining:

  • Nuclear counterstains help visualize tissue architecture

  • For multi-color immunofluorescence, include DAPI for nuclear visualization

Research has validated that TsCD146 mAb can successfully detect CD146-positive tumor cells in human biopsies of melanoma, verrucous skin carcinoma, renal carcinoma, and colonic adenocarcinoma without binding to endothelial cells or normal tissue counterparts .

What are the critical parameters for successful detection of CD146 by Western blot?

For optimal Western blot detection of CD146, researchers should consider these critical parameters:

• Sample preparation:

  • Use appropriate lysis buffers containing protease inhibitors to prevent degradation

  • For membrane proteins like CD146, include detergents suitable for membrane solubilization

• Electrophoresis conditions:

  • Use reducing conditions for most CD146 antibodies

  • Account for the apparent molecular weight discrepancy (calculated ~72 kDa vs. observed ~118-140 kDa) due to glycosylation

• Transfer considerations:

  • PVDF membranes are effective for CD146 detection

  • For high molecular weight glycoproteins like CD146, longer transfer times or specialized transfer methods may be required

• Blocking and antibody incubation:

  • Use appropriate blocking buffers (e.g., Immunoblot Buffer Group 1 has been validated)

  • Optimal primary antibody concentration (e.g., 1 μg/mL for MAB932)

  • HRP-conjugated secondary antibodies specific to the primary antibody isotype

• Detection methods:

  • Enhanced chemiluminescence (ECL) provides sensitive detection

  • For quantitative analysis, consider fluorescent secondary antibodies

• Controls:

  • Positive control: HeLa cell lysates have been validated for CD146 detection

  • Loading control: Use housekeeping proteins to normalize expression levels

Research has demonstrated successful detection of CD146 as a specific band at approximately 140 kDa in HeLa human cervical epithelial carcinoma cell lysates using appropriately optimized Western blot protocols .

How can flow cytometry protocols be optimized for CD146 detection in heterogeneous samples?

Optimizing flow cytometry for CD146 detection in heterogeneous samples requires:

• Sample preparation considerations:

  • Single-cell suspensions: For solid tumors, optimize tissue dissociation to maintain CD146 epitope integrity

  • Viability staining: Include viability dyes to exclude dead cells that may cause non-specific binding

  • Fc receptor blocking: Reduce non-specific binding, especially in immune cell-containing samples

• Antibody panel design:

  • Include cell-type specific markers alongside CD146 (e.g., CD31 for endothelial cells, tumor-specific markers for cancer cells)

  • Select appropriate fluorophores based on instrument configuration and expected expression levels

  • If detecting tumor-specific CD146, include TsCD146 mAb to differentiate from vascular CD146

• Staining protocol optimization:

  • Titrate antibody concentration to determine optimal signal separation

  • Include FMO (fluorescence minus one) controls for accurate gating

  • For intracellular CD146, use appropriate permeabilization reagents

• Analytical considerations:

  • Implement hierarchical gating strategies to identify specific cell populations

  • Consider CD146 expression as both percentage positive and mean fluorescence intensity

  • For rare cell populations, collect sufficient events for statistical significance

• Validation:

  • Compare staining patterns with isotype control antibodies

  • Confirm specificity using CD146-positive (e.g., HeLa) and CD146-negative cell lines

Research has demonstrated successful flow cytometry detection of CD146 in various cell types, including HeLa cells, using anti-CD146 monoclonal antibodies followed by fluorophore-conjugated secondary antibodies .

How can CD146 monoclonal antibodies be used for in vivo tumor detection and imaging?

CD146 monoclonal antibodies offer promising applications for in vivo tumor detection and imaging:

• PET imaging applications:

  • Radiolabeled CD146 antibody fragments (Fab'2) can be used for positron emission tomography (PET)

  • TsCD146 mAb has been successfully radiolabeled and used to detect human melanoma cells via PET imaging in murine xenograft models

  • This approach provides non-invasive detection of CD146-positive tumors with high specificity

• Antibody modification considerations:

  • Using antibody fragments (Fab, Fab'2) rather than whole antibodies improves tumor penetration and reduces background

  • Selection of radiolabels depends on the desired imaging modality (PET vs. SPECT)

  • For tumor-specific imaging, antibodies like TsCD146 mAb that don't bind to vascular CD146 provide higher specificity

• Study design parameters:

  • Timing between injection and imaging should be optimized based on antibody pharmacokinetics

  • Control studies should include radiolabeled isotype-matched antibodies

  • Biodistribution studies complement imaging to quantify tumor-to-background ratios

• Validation approaches:

  • Correlate imaging results with ex vivo analysis of tumor tissue

  • Confirm specificity by comparing CD146-positive and CD146-negative tumors

• Clinical translation considerations:

  • Humanized or human antibodies reduce immunogenicity concerns

  • Safety profiles must be established through appropriate preclinical studies

The specific binding properties of TsCD146 mAb to tumor CD146 but not vascular CD146 make it particularly valuable for targeted imaging approaches, potentially enabling more accurate tumor detection while avoiding effects on normal vasculature .

What mechanisms explain the anti-proliferative effects of CD146 antibodies on cancer cells?

The anti-proliferative effects of CD146 antibodies on cancer cells involve several mechanisms:

• CD146 internalization:

  • TsCD146 mAb has been shown to induce internalization of cell surface CD146 in cancer cells

  • This internalization leads to decreased membrane expression of CD146 (approximately 20-25% reduction) after 72 hours of treatment

  • Importantly, this effect is specific to cancer cells and does not occur in endothelial cells

• Reduced total CD146 protein:

  • Treatment with TsCD146 mAb reduces total CD146 protein levels in cancer cells (by approximately 50% in UACC-1273 and 25% in Panc-1 cells)

  • This reduction was not observed in endothelial cells, further demonstrating tumor specificity

• Disruption of proliferation pathways:

  • CD146 is involved in cellular proliferation pathways

  • Antibody-mediated reduction in CD146 expression likely disrupts these signaling cascades

  • In UACC-1273, C81-61, and Panc-1 cells, a 20-25% decrease in proliferation was observed after 72 hours of treatment with TsCD146 mAb

• Impact on downstream signaling:

  • CD146 affects epithelial-mesenchymal transition

  • CD146 signaling can influence c-myc pathway activation

  • Disruption of these pathways by antibody binding contributes to anti-proliferative effects

• Increased apoptosis:

  • TsCD146 mAb treatment increases apoptosis in CD146-positive cancer cells

  • This occurs after TsCD146-mediated internalization of cell surface CD146

These mechanisms collectively explain how CD146-targeting antibodies like TsCD146 mAb can reduce tumor growth in preclinical models while sparing normal vascular tissues that also express CD146 .

How can researchers distinguish between different CD146 isoforms in their experimental systems?

Distinguishing between different CD146 isoforms requires specific methodological approaches:

• PCR-based differentiation:

  • Design primers specific to unique regions of short and long CD146 isoforms

  • Use RT-PCR or qPCR with isoform-specific primers to quantify relative expression levels

  • For soluble CD146, identify unique sequences or junctions not present in membrane-bound forms

• Protein-level detection methods:

  • Western blot: Membrane and soluble forms may show slight molecular weight differences

  • Develop antibodies against isoform-specific epitopes

  • For soluble CD146, analyze culture supernatants or biological fluids like plasma using ELISA

• Functional assays to distinguish isoforms:

  • Short CD146: Assess proliferation, migration, and adhesion functions

  • Long CD146: Evaluate junction stabilization and vessel formation capacities

  • Soluble CD146: Measure paracrine effects on endothelial cells or autocrine effects on cancer cells

• Genetic manipulation approaches:

  • Use siRNA specifically targeting individual isoforms

  • Overexpress specific isoforms and observe functional consequences

  • Researchers have demonstrated that siRNA against short CD146 decreases cellular proliferation, migration, and adhesion, while siRNA against long CD146 destabilizes neo-vessel junctions

• Subcellular localization analysis:

  • Perform fractionation studies to separate membrane, cytoplasmic, and nuclear components

  • Use confocal microscopy with isoform-specific antibodies to visualize distinct cellular distributions

Research has demonstrated that these different isoforms have complementary effects in processes like angiogenesis, making their distinction crucial for understanding CD146 biology in both normal and disease contexts .

How should researchers interpret apparent discrepancies in CD146 molecular weight across different experimental systems?

When encountering CD146 molecular weight discrepancies, researchers should consider these factors:

Understanding these factors helps researchers correctly interpret CD146 molecular weight variations and avoid misattributing technical artifacts to biological differences.

What are the common challenges in detecting circulating CD146-positive cancer microparticles, and how can they be overcome?

Detecting circulating CD146-positive cancer microparticles presents several challenges that can be addressed through specific methodological approaches:

• Sensitivity limitations:

  • Challenge: Low abundance of cancer microparticles in circulation

  • Solution: Implement pre-enrichment steps such as ultracentrifugation or size-exclusion chromatography

  • Utilize high-sensitivity detection methods like TsCD146 mAb, which has been validated for detecting CD146-positive cancer microparticles in patient plasma

• Specificity concerns:

  • Challenge: Distinguishing cancer-derived from endothelial-derived CD146-positive microparticles

  • Solution: Use tumor-specific CD146 antibodies like TsCD146 mAb that do not bind to vascular CD146

  • Implement multi-marker approaches combining CD146 with other tumor markers

• Sample handling issues:

  • Challenge: Microparticle integrity can be compromised during collection and processing

  • Solution: Standardize collection protocols (anticoagulant type, processing time, temperature)

  • Minimize freeze-thaw cycles and validate microparticle stability in storage conditions

• Quantification challenges:

  • Challenge: Accurate enumeration of microparticles

  • Solution: Use calibrated flow cytometry with size reference beads

  • Implement digital PCR for nucleic acid quantification from microparticles

• Analytical variability:

  • Challenge: Inter-laboratory variation in detection methods

  • Solution: Establish standardized protocols and participate in proficiency testing

  • Include appropriate controls (spiked samples, reference materials)

Research has demonstrated that TsCD146 mAb can specifically detect CD146-positive cancer microparticles in the plasma of patients, providing a potential liquid biopsy approach for CD146-positive tumors .

How might CD146 monoclonal antibodies be incorporated into personalized cancer treatment approaches?

CD146 monoclonal antibodies offer several promising avenues for incorporation into personalized cancer treatment strategies:

• Patient stratification based on CD146 expression:

  • Screen tumors for CD146 expression to identify patients likely to benefit from CD146-targeted therapies

  • Quantify CD146 levels to potentially tailor antibody dosing

  • CD146 has been shown to be a marker of poor prognosis in melanoma and other cancers, allowing risk stratification

• Therapeutic antibody applications:

  • Direct administration of CD146-targeting antibodies like TsCD146 mAb, which has demonstrated ability to reduce xenograft tumor growth

  • Mechanism involves decreased proliferation and increased apoptosis after antibody-mediated CD146 internalization

  • Tumor-specific antibodies like TsCD146 mAb offer targeted effects without impacting vascular CD146, potentially reducing side effects

• Diagnostic companion applications:

  • Use radiolabeled CD146 antibodies for PET imaging to monitor treatment response

  • Detect circulating tumor microparticles using TsCD146 mAb for liquid biopsy approaches

  • Serial monitoring of CD146 expression could guide treatment decisions

• Combination therapy strategies:

  • Pair CD146 antibodies with conventional therapies based on individual tumor characteristics

  • Target both CD146 and related pathways (e.g., angiogenesis via VEGF) based on molecular profiling

  • Sequence therapies based on dynamic changes in CD146 expression

• Emerging approaches:

  • Develop antibody-drug conjugates targeting CD146 for tumor-specific drug delivery

  • Engineer chimeric antigen receptor (CAR) T cells targeting tumor CD146

  • Create bispecific antibodies targeting CD146 and other tumor antigens

TsCD146 mAb represents a promising tool for personalized medicine approaches against CD146-positive tumors, as it specifically targets tumor CD146 without affecting vascular CD146 .

What are the key considerations for developing therapeutic CD146 antibodies with optimal tumor specificity and minimal off-target effects?

Developing therapeutic CD146 antibodies with optimal tumor specificity requires careful consideration of several factors:

• Epitope selection and antibody engineering:

  • Target epitopes specific to tumor CD146 that are absent or inaccessible in normal cellular CD146

  • The TsCD146 mAb approach demonstrates the feasibility of generating antibodies that recognize structural features unique to tumor CD146

  • Consider antibody format (whole IgG, Fab, scFv) based on desired tissue penetration and pharmacokinetics

• Comprehensive cross-reactivity profiling:

  • Test against multiple normal tissues expressing CD146 (endothelial cells, smooth muscle cells, T cells)

  • Evaluate binding to multiple cancer types expressing CD146

  • Include negative controls (CD146-negative cell lines)

  • Perform tissue cross-reactivity studies across major organs

• Functional characterization:

  • Assess antibody internalization properties, as this affects therapeutic mechanisms

  • Evaluate direct effects on cellular proliferation and apoptosis

  • TsCD146 mAb demonstrates 20-25% reduction in cancer cell proliferation while sparing endothelial cells

• Mechanism of action studies:

  • Understand whether efficacy depends on Fc-mediated functions or direct signaling effects

  • Characterize changes in downstream signaling pathways

  • Determine if effects require CD146 internalization, as seen with TsCD146 mAb

• In vivo validation approaches:

  • Test in models with both tumor and normal vascular CD146 expression

  • Evaluate biodistribution to confirm tumor-specific targeting

  • Monitor potential toxicity to vasculature or other CD146-expressing tissues

The development of TsCD146 mAb demonstrates that it is possible to generate antibodies specifically targeting tumor CD146 without affecting normal vascular CD146, providing a model for developing therapeutic antibodies with high tumor specificity and minimal off-target effects .

How might advances in glycobiology inform the development of next-generation CD146 monoclonal antibodies?

Advances in glycobiology could significantly impact next-generation CD146 monoclonal antibody development:

• Targeting cancer-specific glycoforms:

  • CD146 is heavily glycosylated, migrating at ~118-140 kDa despite a calculated protein mass of ~72 kDa

  • Cancer-specific alterations in glycosylation patterns may contribute to the ability of antibodies like TsCD146 mAb to distinguish tumor CD146 from normal CD146

  • Characterizing these differential glycosylation patterns could enable development of antibodies specifically targeting cancer-associated glycoforms

• Glycoengineering approaches:

  • Modify antibody glycosylation to enhance effector functions (ADCC, CDC) for improved therapeutic efficacy

  • Optimize antibody glycosylation for extended half-life or improved tissue penetration

  • Engineer antibodies specifically recognizing aberrantly glycosylated regions of tumor CD146

• Analytical advances:

  • Implement advanced mass spectrometry and glycomics approaches to characterize site-specific CD146 glycosylation changes in cancer

  • Develop glycopeptide-specific antibodies targeting unique tumor-associated CD146 glycopeptides

  • Create glycoproteomic maps of CD146 across different cancer types and stages

• Functional studies:

  • Investigate how glycosylation affects CD146's role in cell adhesion, migration, and signaling

  • Determine if glycosylation changes contribute to CD146's pro-tumorigenic effects

  • Explore how glycan alterations affect CD146 interactions with binding partners

• Translational applications:

  • Develop diagnostic tools detecting cancer-specific CD146 glycoforms in liquid biopsies

  • Create therapeutic antibodies specifically targeting aberrantly glycosylated CD146

  • Implement companion diagnostics identifying patients with specific CD146 glycoforms

Understanding the extensive N-linked glycosylation of CD146 and how it differs between normal and cancer contexts may reveal new epitopes for more specific therapeutic targeting.

What emerging technologies might enhance the detection and characterization of CD146 in clinical and research settings?

Several emerging technologies show promise for advancing CD146 detection and characterization:

• Single-cell analysis approaches:

  • Single-cell RNA sequencing to map CD146 isoform expression at cellular resolution

  • Mass cytometry (CyTOF) for high-dimensional analysis of CD146 alongside dozens of other markers

  • Imaging mass cytometry to visualize CD146 distribution in spatial context within heterogeneous tissues

• Advanced imaging technologies:

  • Super-resolution microscopy to visualize CD146 distribution and co-localization at nanoscale resolution

  • Multiplexed ion beam imaging (MIBI) for simultaneous detection of CD146 and dozens of other proteins in tissue sections

  • Intravital microscopy to monitor CD146-expressing cells in vivo in real-time

• Liquid biopsy enhancements:

  • Microfluidic platforms for isolation and characterization of CD146-positive circulating tumor cells

  • Digital PCR and NGS-based approaches for ultra-sensitive detection of CD146 in circulation

  • Development of sensitive assays for soluble CD146 isoforms with clinical relevance

• Molecular imaging advances:

  • PET tracers with improved pharmacokinetics based on engineered CD146 antibody fragments

  • Multimodal imaging approaches combining PET with MRI or optical imaging

  • TsCD146 mAb has already demonstrated potential for PET imaging of CD146-positive tumors

• Artificial intelligence applications:

  • Machine learning algorithms for automated quantification of CD146 in imaging studies

  • Predictive models integrating CD146 expression with other biomarkers

  • Computer-aided diagnosis systems incorporating CD146 detection

These technologies could enhance both the sensitivity and specificity of CD146 detection, enabling earlier diagnosis, more precise monitoring, and better-informed therapeutic decision-making for CD146-positive cancers.