Recombinant Human Nectin-2 (NECTIN2), partial (Active)

What is Recombinant Human Nectin-2 and what is its primary biological function?

Recombinant Human Nectin-2 (NECTIN2), partial (Active) is a laboratory-produced version of the native Nectin-2 protein, which functions as a Ca²⁺-independent cell-cell adhesion molecule. Nectin-2 is one of the critical plasma membrane components of adherens junctions that mediates cellular adhesion . The protein plays essential roles in intercellular junction formation, immune synapse organization, and viral receptor activity.

To study this protein effectively, researchers typically use recombinant forms expressed in mammalian expression systems such as CHO cells. Methodologically, the recombinant protein can be produced by cloning Nectin-2 cDNA into mammalian expression vectors (such as pcDNA3.1, pEE12.4, or pEF1/myc-HisA), followed by transfection into appropriate cell lines and subsequent protein purification .

How can Nectin-2 expression be accurately detected and quantified in experimental samples?

Several complementary methodologies can be employed to detect and quantify Nectin-2 expression:

• Gene Expression Profile Analysis: RT-PCR and microarray techniques can detect Nectin-2 mRNA levels in tissue samples and cell lines.

• Immunohistochemistry (IHC): For tissue sections, IHC using specific anti-Nectin-2 antibodies allows visualization of protein expression patterns and localization within the tissue architecture.

• Flow Cytometry Analysis: For cell suspensions and cultured cell lines, flow cytometry using fluorescently-labeled anti-Nectin-2 antibodies provides quantitative data on cell surface expression levels .

• Cell ELISA: This method can detect Nectin-2 expression in adherent cells and is particularly useful for screening hybridomas producing anti-Nectin-2 antibodies .

For reliable quantification, researchers should incorporate appropriate positive and negative controls, including Nectin-2-overexpressing cell lines (like Nectin-2/CHO) and knockdown/knockout samples.

What cell lines are suitable for studying Nectin-2 function and developing research models?

Based on research findings, several cell lines are appropriate for Nectin-2 studies:

For establishing research models, stable Nectin-2-expressing cell lines can be generated by transfecting expression vectors (like pEF1/myc-HisA-Nectin-2) into cells using electroporation methods, followed by selection in media containing appropriate antibiotics such as Geneticin . Expression levels should be verified by flow cytometry before experimental use.

What is the relationship between Nectin-2 overexpression and cancer pathogenesis?

Nectin-2 demonstrates significant overexpression in various cancer types, particularly breast and ovarian cancers, as confirmed through gene expression profile analysis and immunohistochemistry studies . This overexpression pattern suggests a potential role in cancer pathogenesis through several possible mechanisms:

• Altered Cell Adhesion Properties: Disruption of normal adherens junction function may contribute to increased cancer cell invasion and metastasis.

• Cell Proliferation Signaling: Research indicates that antibodies blocking Nectin-2 can partially inhibit cancer cell proliferation, suggesting its involvement in growth signaling pathways .

• Immune Evasion: Nectin-2 interactions with immune receptors may modulate anti-tumor immune responses.

The functional significance of Nectin-2 overexpression has been demonstrated experimentally, where polyclonal antibodies specific to Nectin-2 suppressed in vitro proliferation of OV-90 ovarian cancer cells by 10-15% . Additionally, the consistent overexpression across multiple cancer types (including neuroblastoma, myeloid and lymphoblastic leukemias, gastric cancer, and colon cancer) suggests a broader oncogenic role beyond just breast and ovarian cancers .

For researchers investigating this relationship, comparing Nectin-2 expression levels with clinical outcomes and tumor characteristics provides valuable insights into its role in cancer progression.

How should epitope binning be conducted for anti-Nectin-2 monoclonal antibodies?

Epitope binning is a critical process for classifying antibodies based on their binding sites on the target protein. For anti-Nectin-2 antibodies, a systematic approach involves:

• Generation of Diverse Antibody Panel: Develop a broad panel of antibodies using recombinant Nectin-2 protein and/or Nectin-2-overexpressing cells as immunogens .

• Biotinylation of Representative Antibodies: Chemically label selected antibodies using a biotin labeling kit (e.g., Biotin Labeling Kit-NH2) .

• Competitive Binding Assay Setup:

  • Pre-incubate Nectin-2-expressing cells (e.g., 3 × 10³ Nectin-2/CHO cells) with unlabeled anti-Nectin-2 mAb (5 μg/mL) and Streptavidin-Alexa Fluor 647 (330 ng/mL)

  • Add biotinylated anti-Nectin-2 mAb (100 ng/mL)

  • Measure fluorescence intensity using an appropriate detection system

• Binding Inhibition Calculation: Calculate the percentage of binding inhibition using the formula:
  Binding inhibition (%) = [1 - (A/B)] × 100
  Where A represents fluorescence with unlabeled antibody and B represents fluorescence without unlabeled antibody .

• Hierarchical Clustering Analysis: Employ Ward's hierarchical clustering method using appropriate software (e.g., SpotFire DecisionSite) to classify antibodies into distinct epitope bins .

This approach has successfully classified anti-Nectin-2 mAbs into 7 distinct epitope bins, with different bins demonstrating unique functional properties .

What are the mechanisms behind antibody-dependent cellular cytotoxicity (ADCC) in anti-Nectin-2 therapeutic approaches?

ADCC represents a primary mechanism of action for anti-Nectin-2 antibodies in cancer therapy. The process involves:

• Recognition and Binding: Anti-Nectin-2 antibodies specifically bind to Nectin-2 overexpressed on cancer cell surfaces.

• Immune Cell Recruitment: The Fc region of bound antibodies engages Fc receptors on effector immune cells (primarily NK cells).

• Cytolytic Activity: Activated immune cells release perforin and granzymes that induce target cell apoptosis.

Research has demonstrated that anti-Nectin-2 mAbs exhibit varying ADCC activities depending on their epitope bin classification, with those in bin VII (like Y-443) demonstrating the strongest ADCC activity . This epitope-dependent ADCC efficacy suggests that the specific binding orientation or affinity influences Fc region accessibility for immune cell interaction.

In vivo studies with Y-443 showed significant anti-tumor effects against OV-90 ovarian cancer cells and MDA-MB-231 breast cancer cells in mouse therapeutic models, with ADCC being identified as the principal mechanism of action . These findings highlight the potential of targeting Nectin-2 with antibodies optimized for ADCC activity as a promising therapeutic strategy.

How do different anti-Nectin-2 monoclonal antibodies compare in their functional properties and therapeutic potential?

Anti-Nectin-2 monoclonal antibodies demonstrate significant functional diversity based on their epitope specificity and structural characteristics:

The functional diversity is epitope-dependent, with different antibodies exhibiting various capabilities:

• Cell Proliferation Inhibition: Antibodies in bin VI showed direct inhibitory effects on OV-90 cell proliferation, albeit with modest efficacy (similar CDR sequences suggest a specific mechanism) .

• Nectin-2 Interaction Inhibition: Some antibodies can block Nectin-2–Nectin-2 or Nectin-2–Nectin-3 interactions, potentially disrupting cellular adhesion and signaling functions.

• ADCC Potency: Y-443 from epitope bin VII demonstrated the strongest ADCC activity and most promising in vivo anti-tumor effects, suggesting this epitope region as optimal for therapeutic development .

These findings indicate that epitope selection is critical when developing anti-Nectin-2 therapeutic antibodies, with bin VII epitopes offering the strongest ADCC-mediated anti-tumor effects.

What experimental approaches are most effective for evaluating anti-Nectin-2 antibody efficacy in preclinical models?

A comprehensive evaluation of anti-Nectin-2 antibody efficacy requires multiple complementary approaches:

• In Vitro Functional Assays:

  • Cell Proliferation Assays: Measure direct growth inhibition of Nectin-2-expressing cancer cell lines (e.g., OV-90)

  • ADCC Assays: Quantify antibody-mediated cytotoxicity using appropriate effector cells

  • Interaction Inhibition Assays: Assess disruption of Nectin-2–Nectin-2 or Nectin-2–Nectin-3 binding

• In Vivo Tumor Models:

  • Xenograft Studies: Evaluate tumor growth inhibition in immunodeficient mice bearing human cancer cell lines (e.g., OV-90 ovarian or MDA-MB-231 breast cancer cells)

  • Dosing Regimens: Test various dosing schedules (e.g., weekly administration) to determine optimal therapeutic protocols

  • Combination Studies: Assess synergy with standard-of-care treatments

• Mechanism of Action Investigations:

  • ADCC-Deficient Antibody Variants: Compare wild-type antibodies with Fc-mutated versions to confirm ADCC contribution

  • Immune Cell Depletion: Selectively deplete NK cells or other effector populations to validate their role in therapeutic efficacy

Research has demonstrated that Y-443 (administered at 10 mg/kg weekly) significantly inhibited tumor growth in both OV-90 and MDA-MB-231 models, with ADCC identified as the primary mechanism through mechanistic studies .

What are the critical quality attributes for recombinant Nectin-2 protein production for research applications?

Producing high-quality recombinant Nectin-2 protein requires attention to several critical parameters:

• Expression System Selection: Mammalian expression systems (particularly CHO cells) are preferred over bacterial systems to ensure proper folding and post-translational modifications. Research has successfully used CHOK1SV cells maintained in CD-CHO medium with L-glutamine supplementation .

• Construct Design Considerations:

  • Include the complete extracellular domain (a.a. 1-361) for full functionality

  • Consider fusion tags (Fc, FLAG, His) based on experimental requirements

  • Optimize signal sequences for efficient secretion

• Purification Strategy:

  • Multi-step chromatography approach (Protein A for Fc-fusion proteins)

  • Size exclusion chromatography to isolate monomeric fraction

  • Endotoxin removal using specialized columns (e.g., ActiClean Etox)

• Quality Control Metrics:

  • Purity assessment by SDS-PAGE and size exclusion chromatography

  • Functional validation through binding assays with known interaction partners

  • Endotoxin level determination (<1 EU/mg for cell-based assays)

For specialized applications like antibody generation, maintaining native conformation of the recombinant protein is essential, which may require additional validation steps such as circular dichroism analysis or epitope accessibility testing.

What approaches are most effective for developing Nectin-2 knockout or knockdown models?

Creating reliable Nectin-2 loss-of-function models requires careful consideration of experimental design:

• CRISPR/Cas9 Knockout Approach:

  • Design guide RNAs targeting early exons of the Nectin-2 gene

  • Screen for complete protein loss using Western blot and flow cytometry

  • Validate clones by sequencing to confirm frameshift mutations

• RNA Interference Strategies:

  • Design multiple siRNA/shRNA sequences targeting different regions of Nectin-2 mRNA

  • Optimize transfection/transduction protocols for each cell line

  • Establish stable knockdown lines using lentiviral vectors for long-term studies

• Validation Requirements:

  • Quantify knockdown/knockout efficiency at both mRNA and protein levels

  • Assess phenotypic changes in cell adhesion, morphology, and proliferation

  • Include rescued expression controls to confirm specificity of observed effects

• Considerations for Cancer Cell Lines:

  • Some cancer cell lines may have altered dependency on Nectin-2 for survival

  • Compensatory mechanisms may emerge in stable knockout lines

  • Inducible systems may be preferable for studying essential functions

These approaches provide complementary information about Nectin-2 function, with transient knockdown revealing immediate consequences of Nectin-2 loss and stable knockout models demonstrating long-term adaptation and functional redundancy in adhesion pathways.

How might Nectin-2-targeted therapies be optimally combined with other cancer treatment modalities?

The development of effective combination strategies requires understanding potential synergistic interactions:

• Combination with Immune Checkpoint Inhibitors:

  • Anti-Nectin-2 antibodies inducing ADCC could enhance immune infiltration in tumors

  • Sequential administration (anti-Nectin-2 followed by checkpoint inhibitors) might maximize efficacy

  • Monitoring changes in immune cell populations before and after treatment is essential

• Integration with Conventional Chemotherapy:

  • Chemotherapy can increase antigen presentation and enhance ADCC

  • Nectin-2-targeted ADCs may synergize with DNA-damaging agents

  • Staggered dosing schedules may reduce overlapping toxicities

• Radiation Therapy Combinations:

  • Radiation can upregulate cell adhesion molecules including Nectin family proteins

  • Local radiation may enhance efficacy of systemically administered anti-Nectin-2 antibodies

  • Fractionated radiation schedules may optimize combinatorial effects

• Experimental Design Considerations:

  • Establish clear mechanisms of action for each treatment modality

  • Determine optimal sequencing through systematic in vitro and in vivo studies

  • Monitor resistance mechanisms unique to combination approaches

Preliminary research suggests that antibody-dependent cellular cytotoxicity as the main mechanism of anti-Nectin-2 mAbs makes them potentially complementary to treatments that enhance immune cell recruitment or activation in the tumor microenvironment .

What are the emerging strategies for improving anti-Nectin-2 antibody efficacy beyond conventional formats?

Several innovative approaches are being explored to enhance anti-Nectin-2 therapeutic efficacy:

• Antibody-Drug Conjugates (ADCs):

  • Conjugation of cytotoxic payloads to anti-Nectin-2 antibodies

  • Selection of linkers optimized for cancer cell-specific release

  • Recent research indicates promising results with novel ADCs targeting Nectin-2

• Bispecific Antibody Formats:

  • Dual targeting of Nectin-2 and immune cell activating receptors (CD3, CD16)

  • Simultaneous binding to Nectin-2 and complementary tumor antigens

  • Enhanced tumor selectivity through avidity effects

• Antibody Engineering Approaches:

  • Fc engineering to enhance ADCC (afucosylation, specific amino acid modifications)

  • Optimization of binding kinetics for improved tumor penetration

  • Development of antibody fragments with enhanced tissue distribution

• Immune Cell Engagers:

  • Nectin-2-targeted CAR-T cell approaches

  • NK cell engagers leveraging the strong ADCC mechanism observed with Y-443

  • Combinations with cytokine therapy to enhance immune cell function

These approaches build upon the demonstrated efficacy of conventional anti-Nectin-2 antibodies while addressing limitations such as tumor penetration, immune cell availability, and potential resistance mechanisms.

What factors should be considered when inconsistent results are observed in Nectin-2 detection assays?

Inconsistent results in Nectin-2 detection may stem from several technical factors:

• Antibody Selection Issues:

  • Different epitope specificities may detect distinct Nectin-2 conformations or isoforms

  • Solution: Use multiple antibodies targeting different epitope bins

  • Validate antibody specificity using Nectin-2 knockout controls

• Expression Heterogeneity Challenges:

  • Nectin-2 expression varies between cell populations and with cell density

  • Solution: Standardize cell culture conditions and harvest protocols

  • Consider single-cell analysis methods for heterogeneous samples

• Technical Variables in Flow Cytometry:

  • Buffer composition affects epitope accessibility

  • Solution: Optimize fixation protocols and include appropriate blocking steps

  • Use a consistent gating strategy across experiments

• Tissue Processing Effects in IHC:

  • Antigen retrieval efficiency impacts detection sensitivity

  • Solution: Develop optimized protocols for each tissue type

  • Include positive control tissues with known expression patterns

When troubleshooting, systematic evaluation of each variable using appropriate controls (including Nectin-2-overexpressing cell lines like Nectin-2/CHO) helps isolate the source of inconsistency .

How can researchers effectively address the challenge of developing antibodies against conformational epitopes of Nectin-2?

Generating antibodies against conformational epitopes requires specialized approaches:

• Immunization Strategy Optimization:

  • Use a combination of recombinant protein and cell-based immunization

  • Alternate between soluble protein and Nectin-2-expressing cells to maintain conformational epitopes

  • Consider DNA immunization to express native protein in vivo

• Screening Methodology Refinement:

  • Implement cell-based ELISA rather than plate-bound protein screening

  • Develop competition assays with known ligands to identify functionally relevant epitopes

  • Include conformational disruption controls (reducing agents, denaturation) to identify conformation-sensitive antibodies

• Production and Purification Considerations:

  • Optimize recombinant protein production to maintain native conformation

  • Include stabilizing agents during purification to preserve structural integrity

  • Consider membrane preparations or detergent-solubilized protein formats

• Validation Approaches:

  • Confirm epitope conformational dependence through mutagenesis studies

  • Evaluate binding under different conditions that affect protein structure

  • Assess functional blockade of Nectin-2 interactions with natural binding partners

These strategies have proven successful in generating diverse antibody panels targeting conformational epitopes of Nectin-2, as demonstrated by the development of antibodies capable of blocking specific Nectin-2 interactions .