CCR6 Recombinant Monoclonal Antibody

What is CCR6 and why is it a significant target for monoclonal antibody development?

CCR6 is a G-protein-coupled receptor highly expressed in B lymphocytes, effector and memory T cells, regulatory T cells, and immature dendritic cells. It has gained significant attention as a therapeutic target because:

• CCR6 and its ligand CCL20 are upregulated in tissues of patients with chronic inflammatory conditions

• It serves as the main chemokine receptor for Th17 cells, which are implicated in many chronic inflammatory conditions

• The CCL20/CCR6 axis plays critical roles in cancer, intestinal bowel disease, psoriasis, and autoimmune diseases

• Inhibition of CCR6 in preclinical models has shown promising results in reducing disease activity in models of skin inflammation, arthritis, and colitis

How do researchers validate the specificity of anti-CCR6 recombinant monoclonal antibodies?

Validation of anti-CCR6 antibody specificity involves multiple complementary approaches:

• Transfected cell line testing: Testing antibody binding to CCR6-transfected cells (e.g., L1.2, CHO-K1) versus non-transfected parental cell lines

• Cross-reactivity assessment: Evaluating binding to CCR6 from different species (human, cynomolgus monkey, mouse) to determine species specificity

• Flow cytometry with known CCR6-expressing cells: Testing reactivity with cell populations known to express CCR6, such as B cells and specific T cell subsets

• Comparison with isotype controls: Using appropriate isotype control antibodies to confirm specific staining

• Functional assays: Demonstrating inhibition of CCL20-mediated effects, such as chemotaxis or calcium flux

For example, one validated antibody (C6Mab-19) demonstrated specific binding to hCCR6-overexpressed CHO-K1 cells with an extremely high affinity (KD of 3.0 × 10^-10 M) .

What are the common applications for CCR6 recombinant monoclonal antibodies in research?

CCR6 recombinant monoclonal antibodies have diverse research applications:

How do different anti-CCR6 recombinant monoclonal antibodies compare in their ability to block CCL20-mediated signaling?

Anti-CCR6 antibodies vary significantly in their ability to block CCL20-mediated signaling. Key factors affecting blocking efficacy include:

• Binding epitope: Antibodies targeting the CCL20 binding region generally show superior blocking activity

• Binding affinity: Higher affinity antibodies typically demonstrate more effective blocking

• Antibody format: Different isotypes and modifications can affect functional activity

Experimental data demonstrates these differences:

• The humanized 6H12 (h6H12) antibody showed high-affinity binding to human CCR6 and effectively blocked CCL20-mediated chemotaxis

• MSM-R605, a fully human antibody, demonstrated dose-dependent inhibition of CCL20-induced calcium flux in Chem-1 cells expressing human CCR6, providing quantitative measurement of functional blocking

• GSK3050002, a humanized IgG1κ antibody with 48 pM binding affinity to human CCL20, showed dose-dependent decreases in CCR6+ cell recruitment to skin blisters with maximal effects at doses of 5 mg/kg and higher

When evaluating blocking antibodies, researchers should conduct dose-response studies and use appropriate functional readouts relevant to their specific research questions.

What methodological approaches allow researchers to overcome species cross-reactivity limitations when developing anti-CCR6 therapeutic antibodies?

Species cross-reactivity represents a significant challenge in CCR6-targeted therapeutic development. Several methodological approaches can address this limitation:

• Transgenic animal models: Development of humanized CCR6 transgenic mice (hCCR6-Tg/mCCR6-/-) where mouse CCR6 is replaced with human CCR6, allowing testing of human-specific antibodies in mouse models

• Multi-species binding antibodies: Identification and optimization of antibodies that recognize conserved epitopes across species, though this is challenging due to sequence variability

• Surrogate antibodies: Development of paired antibodies - one targeting human CCR6 for clinical development and a surrogate antibody targeting the corresponding species CCR6 for preclinical studies

• Epitope grafting: Engineering target animals to express the human epitope recognized by the therapeutic antibody

For example, researchers developed hCCR6-Tg/mCCR6-/- mice to test an anti-human CCR6 antibody (h6H12) that did not cross-react with mouse CCR6. This model demonstrated that "following immunization with recombinant mouse myelin oligodendrocyte glycoprotein, a significant increase in hCCR6 expression was observed on CD4+ and CD8+ T cells isolated from lymph nodes and spleen," enabling preclinical testing of the human-specific antibody .

How can researchers optimize experimental design when using anti-CCR6 antibodies for therapeutic development in inflammatory disease models?

Optimizing experimental design for anti-CCR6 therapeutic studies requires careful consideration of several factors:

• Target validation: Confirm CCR6 expression and relevance in the disease model

  • Analyze CCR6 expression patterns in relevant tissues using flow cytometry and IHC

  • Compare expression between healthy and disease states

• Dosing strategy optimization:

  • Establish clear dose-response relationships as demonstrated with GSK3050002, where "doses of 5 mg/kg and higher" showed maximal effects

  • Determine optimal dosing frequency based on antibody half-life (e.g., approximately 2 weeks for GSK3050002)

• Pharmacokinetic/Pharmacodynamic correlation:

  • Use suction skin blister or similar models to assess target engagement in tissues

  • Correlate serum and tissue levels with biological effects

  • Monitor GSK3050002/CCL20 complex levels as a biomarker of target engagement

• Appropriate controls:

  • Include isotype control antibodies

  • Consider Fc-modified variants to distinguish Fc-mediated from target-blocking effects

• Multiple readouts:

  • Clinical scores (e.g., EAE scoring)

  • Cellular analysis (flow cytometry of infiltrating cells)

  • Histopathological assessment

  • Molecular markers of inflammation

A comprehensive example from GSK3050002 clinical studies demonstrates this approach: researchers established a relationship between pharmacokinetics, target engagement, and pharmacodynamics by measuring both serum and blister fluid levels of GSK3050002/CCL20 complex alongside functional effects on CCR6+ cell recruitment .

What are the key considerations for developing flow cytometry panels to accurately identify and quantify CCR6-expressing cell populations?

Developing optimized flow cytometry panels for CCR6+ cell identification requires addressing several technical challenges:

• Antibody clone selection:

  • Choose validated anti-CCR6 clones with demonstrated specificity

  • For human samples, clones like 53103R have been validated for detecting CCR6 in CD19+ human PBMCs

  • For mouse samples, clones like 140706 have shown good specificity

• Panel design considerations:

  • Include markers to identify relevant cell populations (e.g., CD3, CD4, CD19, B220)

  • For Th17 cell identification, consider the marker combination: CD3+CD4+CXCR3-CCR6+

  • For Th1/17 cells: CD3+CD4+CXCR3+CCR6+

• Technical optimization:

  • Titrate antibodies to determine optimal concentration

  • Consider fluorochrome brightness when assigning to markers of different expression levels

  • Include appropriate FMO (fluorescence minus one) controls

• Sample preparation considerations:

  • Optimize fixation conditions that preserve CCR6 epitope

  • Minimize time between sample collection and staining as surface receptors may internalize

  • Consider the impact of enzymatic dissociation on CCR6 epitope integrity

For example, a successful panel used in one study included: "T-cells (CD3+), Th1 (CD3+CD4+CXCR3+CCR6−), Th2 (CD3+CD4+CXCR3−CCR6−), Th17 (CD3+CD4+CXCR3−CCR6+) and Th1/17 (CD3+CD4+CXCR3+CCR6+) T-cells, while monocytes and granulocytes were gated based on forward and side scatter" .

How can researchers address epitope masking or modulation when detecting CCR6 in different experimental contexts?

Epitope masking or modulation presents challenges when detecting CCR6 across different experimental contexts. Researchers can implement these methodological solutions:

• For flow cytometry applications:

  • Use multiple anti-CCR6 antibody clones targeting different epitopes

  • Optimize staining buffer composition (calcium concentration can affect chemokine receptor conformation)

  • Evaluate the impact of cell activation state on CCR6 detection

  • Consider using indirect staining approaches for amplification when epitope accessibility is limited

• For immunohistochemistry applications:

  • Optimize antigen retrieval methods (as demonstrated for anti-CCR6 antibody EPR22259, which used "heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 mins")

  • Test multiple fixation protocols to determine optimal epitope preservation

  • Consider the impact of tissue processing on epitope accessibility

• For Western blotting applications:

  • Compare reducing vs. non-reducing conditions

  • Optimize detergent selection for membrane protein extraction

  • Consider native vs. denatured protein detection approaches

• Ligand-induced receptor modulation:

  • Be aware that CCL20 binding may induce receptor internalization or conformational changes

  • Design experiments to account for potential ligand-induced epitope masking

  • Consider time-course experiments to track receptor dynamics after ligand exposure

• Validation across methods:

  • Confirm CCR6 detection using complementary techniques (e.g., flow cytometry, immunohistochemistry, and Western blotting)

  • Use genetic approaches (knockout/knockdown) to validate antibody specificity

Some antibodies have demonstrated robust performance across multiple applications. For example, anti-CCR6 antibody EPR22259 has been validated for immunoprecipitation, Western blotting, and immunohistochemistry, suggesting reliable epitope recognition across different experimental conditions .

What strategies can researchers employ to develop CCR6-expressing cell lines for antibody screening and characterization?

Creating reliable CCR6-expressing cell lines requires careful consideration of expression systems and validation approaches:

• Selection of appropriate host cell lines:

  • CHO-K1 cells: Successfully used for stable expression of human CCR6

  • L1.2 murine pre-B cell lymphoma: Demonstrated high-level expression of transfected human CCR6

  • HEK293: Commonly used for transient expression of GPCRs

• Expression vector design considerations:

  • Codon optimization for host cell line

  • Selection of appropriate promoters (e.g., CMV for high expression)

  • Inclusion of selection markers (e.g., antibiotic resistance)

  • Addition of epitope tags if needed for detection/purification

• Validation of CCR6 functionality:

  • Calcium mobilization assays in response to CCL20

  • Migration/chemotaxis assays

  • Receptor internalization studies

  • Binding assays with labeled CCL20

• Quantification of expression levels:

  • Flow cytometry with anti-CCR6 antibodies

  • Quantitative Western blotting

  • Radioligand binding assays for precise quantification

For example, Chem-1 cells expressing human CCR6 were successfully used to demonstrate the inhibitory activity of MSM-R605 on CCL20-induced calcium mobilization, providing a functional readout system for antibody characterization .

How do CCR6 knockout and transgenic mouse models contribute to understanding the therapeutic potential of anti-CCR6 antibodies?

CCR6 knockout and transgenic mouse models have provided critical insights into CCR6 biology and therapeutic targeting:

• CCR6 knockout mice observations:

  • Show greater resistance to autoimmune and inflammatory diseases including psoriasis and arthritis

  • Demonstrate reduced disease severity in experimental autoimmune encephalomyelitis (EAE)

  • Provide validation of CCR6 as a therapeutic target

• Human CCR6 transgenic models (hCCR6-Tg/mCCR6-/- mice):

  • Enable testing of human-specific anti-CCR6 antibodies in mouse disease models

  • Allow evaluation of human CCR6 biology in vivo

  • Provide translational insights for clinical development

• Applications in therapeutic development:

  • Anti-CCR6 polyclonal antibody treatment prevented progression of EAE in mouse models

  • Humanized anti-CCR6 antibody (h6H12) showed similar binding and neutralizing behavior to the parental mouse antibody

  • These models allow assessment of both efficacy and potential adverse effects of CCR6 targeting

• Expression pattern insights:

  • Interestingly, CCR6 expression patterns differ between humans and mice: "all human B cells expressed CCR6, compared with approximately 55% of B cells in mice. A larger proportion of human CD3+ cells expressed the receptor (~14%) compared with mouse CD3+ cells"

  • These differences highlight the importance of humanized models for translational research

These models have established that "CCR6 plays a critical role in Th17 type inflammatory reactions, and CCR6 inhibition may offer an alternative approach for the treatment of these lesions" .

What methodological approaches can researchers use to evaluate the therapeutic efficacy of anti-CCR6 antibodies in inflammatory disease models?

Evaluating therapeutic efficacy of anti-CCR6 antibodies requires comprehensive assessment strategies:

• Disease model selection:

  • Experimental autoimmune encephalomyelitis (EAE): Well-established model where "hCCR6-Tg/mCCR6–/– mice develop EAE in a manner similar to wild-type C57BL/6 mice"

  • Skin inflammation models: Relevant for psoriasis research

  • Colitis models: Important for inflammatory bowel disease studies

  • Arthritis models: For rheumatoid arthritis applications

• Treatment timing strategies:

  • Preventive protocols: Administering antibody before disease induction

  • Therapeutic protocols: Treating established disease

  • Both approaches should be tested as demonstrated with anti-CCR6 antibodies in EAE where "anti-CCR6 treatment prevents and inhibits clinical progression in established EAE"

• Comprehensive assessment metrics:

  • Clinical scoring systems specific to each model

  • Histopathological analysis of tissue inflammation and damage

  • Flow cytometric analysis of infiltrating immune cells

  • Cytokine/chemokine profiling in serum and affected tissues

  • Functional assessments relevant to the disease model

• Mechanistic investigations:

  • CCR6+ cell trafficking analysis

  • Examination of lymphoid organ architecture and germinal center formation

  • Assessment of antigen-specific T and B cell responses

  • Evaluation of CCL20/CCR6 axis inhibition in target tissues

• Translational biomarkers:

  • Identification of biomarkers that correlate with clinical efficacy

  • Development of assays to monitor target engagement (e.g., measuring GSK3050002/CCL20 complex)

  • Correlation of pharmacokinetics with pharmacodynamic endpoints

In clinical studies, the experimental skin suction blister model has proven valuable for assessing pharmacokinetics, target engagement, and the ability of anti-CCL20 antibody (GSK3050002) to inhibit recruitment of inflammatory CCR6-expressing cells .

What optimization strategies should researchers employ when using anti-CCR6 antibodies for immunohistochemistry of formalin-fixed paraffin-embedded tissues?

Optimizing immunohistochemistry protocols for CCR6 detection in FFPE tissues requires systematic approach:

• Antigen retrieval optimization:

  • Test multiple antigen retrieval solutions: For anti-CCR6 antibody EPR22259, "heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins" was effective

  • Optimize retrieval duration and temperature

• Antibody selection and validation:

  • Select antibodies specifically validated for IHC-P applications

  • C6Mab-19 successfully stained "formalin-fixed paraffin-embedded lymph node tissues from a patient with non-Hodgkin lymphoma"

  • Clone 53103R effectively detected CCR6 in "immersion fixed paraffin-embedded sections of human spleen"

• Signal amplification and detection system selection:

  • For EPR22259, researchers used "Anti-Mouse IgG VisUCyte™ HRP Polymer Antibody" with "DAB (brown) and counterstained with hematoxylin (blue)"

  • For MAB195R, researchers used "Anti-Mouse IgG VisUCyte™ HRP Polymer Antibody" with similar detection methodology

• Protocol optimization:

  • Titrate primary antibody concentration

  • Optimize incubation time and temperature (e.g., "5 µg/mL for 1 hour at room temperature" for MAB195R or "for 30 mins at 37°C" for EPR22259 )

  • Include appropriate blocking steps to reduce background

• Controls and validation:

  • Include positive control tissues with known CCR6 expression

  • Incorporate negative controls (isotype control or secondary antibody only)

  • Consider dual staining with lineage markers to confirm cell type-specific expression

For troubleshooting purposes, researchers should note that "specific staining was localized to cytoplasm in splenocytes" when using MAB195R in human spleen sections , providing a reference for expected staining patterns.

How can researchers address potential artifacts and ensure specificity when using anti-CCR6 antibodies in flow cytometry?

Ensuring specific CCR6 detection in flow cytometry requires addressing several technical challenges:

• Antibody validation strategies:

  • Test on CCR6-transfected vs. non-transfected cell lines

  • Include biological positive controls (e.g., B cells) and negative controls

  • Compare staining patterns with multiple anti-CCR6 clones

• Control implementation:

  • Include appropriate isotype controls matched for species, isotype, and fluorochrome

  • For mouse anti-human CCR6 clone 53103R, use "Mouse IgG2B Isotype Control"

  • Perform fluorescence-minus-one (FMO) controls to set accurate gates

• Addressing non-specific binding:

  • Optimize blocking protocols using serum or FcR blocking reagents

  • Titrate antibody to minimize background while maintaining specific signal

  • Consider the impact of dead cell binding (include viability dye)

• Sample preparation considerations:

  • Minimize time between sample collection and staining

  • Optimize fixation protocols that preserve CCR6 epitope

  • Evaluate the impact of different buffer compositions on staining quality

• Functional validation:

  • Confirm that CCR6+ sorted cells respond to CCL20 in migration assays

  • Demonstrate that antibody pretreatment can block CCL20-induced responses

For example, when detecting "CCR6 in CD19+ Human PBMCs by Flow Cytometry," researchers successfully used "Mouse Anti-Human CD19 PE-conjugated Monoclonal Antibody" alongside "Mouse Anti-Human CCR6 Monoclonal Antibody" and compared to isotype control, followed by "Goat anti-Mouse IgG APC-conjugated secondary antibody" .

What methodological approaches can researchers use to evaluate anti-CCR6 antibody-mediated inhibition of CCL20-induced functional responses?

Evaluating functional inhibition by anti-CCR6 antibodies requires robust assay systems:

• Calcium mobilization assays:

  • Use cells expressing human CCR6 (e.g., Chem-1/hCCR6 cells)

  • Load cells with calcium-sensitive dyes

  • Measure fluorescence changes after CCL20 stimulation

  • As demonstrated with MSM-R605, measure "inhibition of the increase in the intracellular Ca concentration in response to addition of human CCL20 to Chem-1 cells expressing human CCR6"

  • Generate dose-response curves for the antibody

• Chemotaxis/migration assays:

  • Use transwell systems with CCL20 as chemoattractant

  • Pretreat cells with varying concentrations of anti-CCR6 antibody

  • Quantify migration inhibition

  • The humanized 6H12 antibody demonstrated ability to "block CCL20-mediated chemotaxis"

• Receptor internalization assays:

  • Assess CCR6 surface expression after CCL20 stimulation

  • Determine if antibody prevents receptor internalization

  • Use flow cytometry or imaging approaches for quantification

• Signaling pathway analysis:

  • Examine downstream pathways (e.g., MAPK, Akt activation)

  • Use phospho-specific antibodies in Western blot or flow cytometry

  • Determine IC50 values for inhibition of specific signaling events

• Ex vivo cell recruitment models:

  • Skin suction blister model: Assess recruitment of CCR6+ cells

  • GSK3050002 demonstrated "dose-dependent decreases in CCR6+ cell recruitment to skin blisters with maximal effects at doses of 5 mg/kg and higher"

These functional assays should include appropriate controls and dose-response analyses to fully characterize the inhibitory potential of anti-CCR6 antibodies.

How can researchers quantitatively assess CCR6 receptor occupancy and target engagement by therapeutic antibodies?

Quantitative assessment of receptor occupancy and target engagement is critical for therapeutic antibody development:

• Direct binding measurement approaches:

  • Flow cytometry-based methods:

    • Compare binding of a labeled detection antibody before and after treatment

    • Use competing antibodies targeting non-overlapping epitopes

  • Radiolabeled ligand binding studies:

    • Measure displacement of radiolabeled CCL20

• Complex formation quantification:

  • GSK3050002/CCL20 complex measurement:

    • "Total CCL20 was captured on streptavidin MSD plates using biotinylated goat anti-CCL20 polyclonal antibody and GSK3050002/CCL20 detected using ruthenylated mouse anti-idiotype antibody"

    • Results showed "complex of GSK3050002/CCL20 increased in serum and blister fluid with increasing doses"

• Pharmacokinetic/Pharmacodynamic correlation:

  • Relate antibody levels in serum and tissues to functional effects

  • For GSK3050002, "dose-dependent decreases in CCR6+ cell recruitment" correlated with complex levels in serum and blister fluid

• Ex vivo functional assays:

  • Collect patient samples after antibody administration

  • Test responsiveness to CCL20 stimulation

  • Correlate functional inhibition with antibody exposure

• Biomarker development:

  • Identification of downstream markers that reflect target engagement

  • Monitoring changes in CCR6+ cell trafficking patterns

  • Evaluation of inflammatory markers in affected tissues

For translational development, the GSK3050002 study demonstrated that "levels of GSK3050002/CCL20 complex appeared to increase in a dose-dependent manner and reach maximum levels at doses of 5 mg/kg and higher both in serum and interstitial fluid, suggesting that a saturable target engagement has been achieved at the high doses" .

What approaches can researchers use to develop bispecific antibodies targeting CCR6 and complementary inflammatory pathways?

Development of bispecific antibodies targeting CCR6 and other inflammatory pathways represents an advanced therapeutic strategy:

• Target selection considerations:

  • Complementary inflammatory pathways to CCR6/CCL20:

    • Th17-related cytokines (IL-17, IL-23)

    • Other chemokine receptors on CCR6+ cells (e.g., CXCR3 for Th1/17 cells)

    • Co-stimulatory molecules involved in T cell activation

  • Consider pathways with demonstrated synergy in disease models

• Bispecific antibody format selection:

  • Evaluate tandem scFv formats (e.g., BiTE)

  • Consider IgG-based bispecifics with different valencies

  • Assess asymmetric bispecific designs

  • For CCR6 targeting, maintaining high-affinity binding is critical as demonstrated with multiple antibodies showing pM range affinities

• Functional characterization approaches:

  • Compare monospecific vs. bispecific effects in:

    • Calcium flux assays

    • Chemotaxis inhibition

    • Cell activation/cytokine production

  • Test for synergistic vs. additive effects

  • Evaluate potential antagonism between targeting arms

• In vivo testing strategies:

  • Use disease models where both pathways contribute

  • For CCR6 assessment, consider EAE models where "anti-CCR6 treatment prevents and inhibits clinical progression"

  • Compare bispecific to combination of monospecific antibodies

  • Evaluate safety and potential unexpected effects

• Manufacturing considerations:

  • Assess expression levels and stability

  • Implement appropriate quality control testing

  • Consider strategies to minimize heterogeneity

While no specific examples of CCR6-targeting bispecific antibodies were described in the provided search results, the approach builds on established expertise in CCR6 targeting through monospecific antibodies with demonstrated efficacy in inflammatory disease models.