CRK6 Antibody

What is CCR6 and why is it significant in immunological research?

CCR6 (C-C chemokine receptor type 6) is a chemokine receptor pivotal for immune cell trafficking during immune responses and host defense. It plays a crucial role in the migration of pathological Th17 cells during the course of certain inflammatory diseases. CCR6 is particularly important in research because compelling evidence suggests that chemokine receptors like CCR6 are critical in the pathogenesis of autoimmune and chronic inflammatory diseases .

The receptor functions as a signaling molecule when bound by its ligand CCL20, facilitating cellular chemotaxis toward inflammatory sites. Understanding CCR6 expression and function allows researchers to develop targeted therapies for inflammatory conditions where CCR6-expressing cells play a pathogenic role.

What cell types express CCR6 and how can I detect them?

CCR6 is predominantly expressed on:

• Th17 cells (CCR6+ CD4+ T cells)

• Some subsets of B cells

• Immature dendritic cells

• Memory T cells

Detection methods include:

• Flow cytometry: The most common method for detecting CCR6-expressing cells in cell suspensions. Cells can be stained with fluorescently-labeled anti-CCR6 antibodies and analyzed .

• Immunohistochemistry/Immunofluorescence: For detecting CCR6 expression in tissue sections.

• RT-qPCR: For quantifying CCR6 mRNA expression levels in cell populations.

How do I validate the specificity of a CCR6 antibody?

Validating CCR6 antibody specificity requires multiple complementary approaches:

• Positive and negative controls: Test antibody binding on cell lines that overexpress CCR6 versus those that do not express CCR6 .

• Blocking experiments: Pre-incubate the cells with unlabeled CCR6 antibody or CCL20 (the natural ligand) before staining with the labeled antibody. Reduction in staining indicates specificity.

• Western blot analysis: Confirm that the antibody recognizes a protein of the expected molecular weight.

• Knockout or knockdown validation: Test antibody on CCR6 knockout cells or cells where CCR6 has been silenced by RNA interference.

• Cross-reactivity testing: Ensure the antibody does not bind to related chemokine receptors by testing on cells expressing other chemokine receptors but not CCR6.

How can I develop and validate an antagonistic monoclonal antibody against CCR6?

Developing antagonistic monoclonal antibodies against CCR6 involves several sophisticated steps:

• Immunization strategy: Whole cell immunization using cells overexpressing human CCR6 receptor has proven effective. This approach overcomes the challenges of purifying stable GPCR proteins while preserving their native conformation .

• Screening approaches: Primary screening by flow cytometry to identify antibodies that bind specifically to CCR6-expressing cells, followed by functional screening to identify those with antagonistic properties.

• Functional validation:

  • β-arrestin recruitment assays (measuring IC50 values)

  • Calcium mobilization assays

  • Chemotaxis inhibition assays using CCL20 as chemoattractant

  • Measurement of downstream signaling effects (e.g., IL-17A expression in Th17 cells)

A successful example from the literature demonstrated that the 1C6 antibody blocked response in β-arrestin recruitment assay with IC50 of 10.23 nM, reduced migration of CCR6-expressing cells toward CCL20, and inhibited IL-17A expression in Th17 cells .

What are the optimal protocols for measuring CCR6 antibody-mediated inhibition of chemotaxis?

The chemotaxis inhibition assay is critical for validating CCR6 antibody functionality:

• Cell preparation:

  • Use cell lines stably transfected with human CCR6 (e.g., L1.2 hCCR6)

  • Maintain cells in appropriate medium (RPMI 1640 with 10% heat-inactivated FCS, 2% L-glutamine and antibiotics)

  • Wash cells in PBS and resuspend at 10^6 cells/ml in assay buffer (RPMI 1640, 1% endotoxin-free BSA, plus antibiotics)

• Antibody preincubation:

  • Preincubate 1×10^5 cells with test antibodies (typically at 1μg/ml) for 30 minutes

• Transwell setup:

  • Place preincubated cells in the upper chamber of a Transwell plate with 5-μm pores

  • Add human CCL20 (typically 100 nM) to the lower chamber

• Incubation and analysis:

  • Incubate at 37°C for 4 hours (5% CO2)

  • Count migrated cells in the lower chamber using flow cytometry

• Data interpretation:

  • Calculate percent inhibition relative to control antibodies

  • Generate dose-response curves if testing multiple antibody concentrations

What are the considerations for designing bispecific antibodies targeting CCR6 and CXCR3?

Designing bispecific antibodies (BsAb) targeting both CCR6 and CXCR3 requires addressing several critical considerations:

• Rationale: Both CXCR3 and CCR6 play crucial roles in the migration of pathological Th1 and Th17 cells during inflammatory diseases. Targeting a single receptor has proven disappointing in clinical trials, suggesting that simultaneous targeting of multiple receptors may be more effective .

• Antibody format selection:

  • IgG-like formats preserve effector functions (ADCC)

  • (scFv)₄-IgG formats may face expression challenges

  • Morrison format (parent IgG with scFv fused to C-terminal region) shows promise

• Stability optimization:

  • Introduction of disulfide bonds between VH and VL domains of scFv portions

  • Optimization of linker length to reduce aggregation

  • SEC profiling to monitor aggregation products

• Functional validation requirements:

  • Simultaneous binding to both receptors (demonstrated by flow cytometry and SPR)

  • Inhibition of chemotaxis mediated by both receptors

  • Maintenance of effector functions like ADCC

• Humanization considerations:

  • Grafting murine CDRs onto human germline framework sequences

  • Back-mutation of framework residues in the "Vernier zone"

  • Structural modeling to confirm proper folding

How do I evaluate CCR6 antibody-mediated effects on IL-17 production in Th17 cells?

Evaluating the effects of CCR6 antibodies on IL-17 production in Th17 cells involves:

• Th17 cell generation:

  • Isolate CD4+ T cells from peripheral blood

  • Culture with appropriate cytokine cocktail (IL-6, TGF-β, IL-23, IL-1β) and anti-CD3/CD28 stimulation

  • Verify Th17 polarization by flow cytometry (CCR6+, CD161+)

• Antibody treatment:

  • Add CCR6 antibody at varying concentrations to Th17 cultures

  • Include isotype control antibodies as negative controls

• Evaluation methods:

  • RT-qPCR for IL-17A mRNA quantification

  • ELISA for IL-17A protein in culture supernatants

  • Intracellular cytokine staining and flow cytometry for single-cell IL-17A detection

• Analysis considerations:

  • Normalize expression levels to reference genes

  • Calculate percent inhibition compared to untreated controls

  • Determine dose-response relationship and IC50 values

What assays should I use to comprehensively evaluate CCR6 antibody signaling pathway inhibition?

A comprehensive evaluation of CCR6 antibody signaling pathway inhibition requires multiple complementary assays:

• β-arrestin recruitment assay:

  • Utilize cells co-expressing CCR6 and β-arrestin-reporter construct

  • Treat with antibody (various concentrations) before CCL20 stimulation

  • Measure luminescence and calculate normalized percent inhibition

  • Generate IC50 values using dose-response curves

• Calcium mobilization assay:

  • Plate cells (e.g., RBL-hCCR6) at appropriate density

  • Add antibody dilutions followed by CCL20 stimulation

  • Include calcium-sensitive fluorescent dye or substrate

  • Measure fluorescence/luminescence using plate reader

• ERK phosphorylation:

  • Western blot or flow cytometry-based detection of phospho-ERK

  • Pretreat cells with antibody before CCL20 stimulation

• Chemotaxis assay:

  • Transwell migration of CCR6+ cells toward CCL20 gradient

  • Quantify cell migration by flow cytometry

• Downstream gene expression:

  • RT-qPCR analysis of CCR6-regulated genes (e.g., IL-17)

  • RNA-seq for comprehensive pathway analysis

What are the optimal methods for characterizing antibody-dependent cell-mediated cytotoxicity (ADCC) of CCR6 antibodies?

ADCC is an important effector function for therapeutic antibodies targeting CCR6+ pathogenic cells:

• Target cell preparation:

  • Label CCR6-expressing target cells with membrane dye (e.g., PKH26)

  • Wash cells thoroughly and resuspend at 1×10^6 cells/ml

  • Dispense in round-bottom 96-well plates (1×10^5 cells/well)

  • Preincubate with antibody at various concentrations (0.1-5 μg/ml)

• Effector cell preparation:

  • Isolate PBMCs from healthy donor blood using Ficoll gradient centrifugation

  • Further isolate NK cells using CD56 microbeads and magnetic separation

  • Alternatively, use whole PBMCs as effector cells

• ADCC assay procedure:

  • Add NK cells to antibody-treated target cells at appropriate E:T ratio (e.g., 4:1)

  • Incubate at 37°C for 3-4 hours

  • Add cell death indicator (e.g., TO-PRO 3 iodide)

  • Include counting beads for absolute quantification

• Analysis by flow cytometry:

  • Gate on target cells (PKH26+)

  • Determine percent cell death (TO-PRO 3 iodide+)

  • Calculate specific lysis by comparing to controls

How do I resolve discrepancies between different CCR6 signaling assays?

When different assays yield conflicting results about CCR6 antibody functionality, consider:

• Biased signaling phenomena: CCR6, like other GPCRs, can exhibit biased signaling where certain pathways are selectively modulated. For example, the 1C6 antibody blocked β-arrestin recruitment but did not inhibit calcium mobilization . This is not necessarily a discrepancy but may reflect the biological complexity of GPCR signaling.

• Assay sensitivity differences:

  • β-arrestin assays often have higher sensitivity than calcium flux assays

  • Functional assays like chemotaxis may require higher antibody concentrations

• Receptor expression levels:

  • Verify consistent CCR6 expression across different cell systems

  • Quantify receptor numbers by saturation binding or flow cytometry

• Resolution strategies:

  • Perform full dose-response curves for all assays

  • Include positive control inhibitors (e.g., small molecule CCR6 antagonists)

  • Validate findings in primary cells in addition to cell lines

  • Test multiple antibody clones or formats

• Interpretation framework:

  • Consider which readout is most relevant to the biological context of interest

  • Determine if differences reflect biased antagonism that might be therapeutically advantageous

What are the critical quality control parameters for humanized CCR6 antibodies?

Quality control for humanized CCR6 antibodies requires assessment of:

• Binding specificity and affinity:

  • Flow cytometry on CCR6+ and CCR6- cells

  • Surface Plasmon Resonance (SPR) for binding kinetics

  • Competition binding with natural ligand CCL20

• Structural integrity:

  • Size-exclusion chromatography (SEC) to assess aggregation

  • SDS-PAGE under reducing and non-reducing conditions

  • Mass spectrometry for identity confirmation

• Stability assessments:

  • Thermal stability (DSC, nanoDSF)

  • Freeze-thaw stability

  • Long-term storage stability testing

• Functional tests:

  • Maintenance of antagonistic function after humanization

  • Comparison with original murine antibody

  • Functional assays (β-arrestin, chemotaxis, ADCC)

• Immunogenicity risk assessment:

  • In silico T-cell epitope analysis

  • Evaluation of framework "back mutations"

  • Removal of sequence liabilities (deamidation, oxidation sites)

How can I distinguish between cytotoxic versus signaling blockade effects of CCR6 antibodies in experimental systems?

Distinguishing between direct cytotoxic effects and signaling blockade requires careful experimental design:

• Temporal separation experiments:

  • Short-term assays (minutes to hours) typically reflect signaling blockade

  • Longer assays (>24 hours) may include cytotoxic components

  • Compare kinetics of response for different endpoints

• Selective inhibition controls:

  • Include F(ab')2 fragments (lacking Fc region) to eliminate ADCC

  • Use Fc-mutated antibodies with reduced effector functions

  • Compare with small molecule CCR6 antagonists (signaling-only effects)

• Direct measurements:

  • Apoptosis/viability assays (Annexin V/PI staining)

  • ADCC assays with varying E:T ratios

  • Real-time monitoring of cell health during signaling assays

• Genetic approaches:

  • Compare effects in NK cell-depleted systems

  • Use effector cells from donors with specific FcγR polymorphisms

  • CRISPR/Cas9 disruption of downstream signaling components

• Combined readout systems:

  • Simultaneous measurement of signaling and cell death

  • Single-cell approaches to correlate receptor occupancy with cell fate

What are emerging strategies for enhancing CCR6 antibody therapeutic efficacy?

Cutting-edge approaches to enhance CCR6 antibody therapeutics include:

• Bispecific and multispecific formats:

  • Co-targeting CXCR3 and CCR6 to simultaneously block Th1 and Th17 migration

  • Trispecific antibodies incorporating additional inflammatory targets

  • Development of stabilized formats to prevent aggregation and improve pharmacokinetics

• Engineering enhanced effector functions:

  • Fc engineering for increased or selective ADCC

  • Complement-dependent cytotoxicity (CDC) enhancement

  • pH-dependent binding for improved tissue penetration

• Novel delivery strategies:

  • Tissue-targeted delivery to inflammatory sites

  • Controlled-release formulations

  • Cell-penetrating antibody formats

• Combination approaches:

  • CCR6 antibodies with immune checkpoint inhibitors

  • Combination with small molecule chemokine receptor antagonists

  • Integration with emerging cytokine-targeting approaches

• Therapeutic applications beyond autoimmunity:

  • Oncology applications (targeting CCR6+ lymphoma cells)

  • Transplantation (preventing CCR6-mediated graft rejection)

  • Metabolic disease (targeting CCR6+ cells in adipose inflammation)

How can single-cell analysis technologies enhance CCR6 antibody research?

Single-cell technologies offer powerful new approaches for CCR6 antibody research:

• Single-cell RNA sequencing applications:

  • Identifying heterogeneity within CCR6+ cell populations

  • Discovering novel CCR6+ pathogenic cell subsets

  • Elucidating complete transcriptional changes after antibody treatment

• CyTOF/mass cytometry:

  • High-dimensional phenotyping of CCR6+ cells

  • Simultaneous assessment of multiple signaling pathways

  • In vivo tracking of CCR6+ cells after antibody treatment

• Spatial transcriptomics:

  • Mapping CCR6+ cells within tissue microenvironments

  • Understanding niche-specific effects of CCR6 blockade

  • Correlating CCR6 expression with disease pathology

• Single-cell CRISPR screens:

  • Identifying genes that modify CCR6 antibody sensitivity

  • Discovering synthetic lethal interactions with CCR6 blockade

  • Mapping CCR6 signaling networks at single-cell resolution

• Multiomics integration:

  • Correlating CCR6 protein levels with transcriptome and epigenome

  • Predicting therapeutic response based on multi-parameter analysis

  • Developing personalized approaches to CCR6-targeted therapy