LCR8 Antibody

What is CCR8 and why is it a significant target for antibody development?

CCR8 is a seven-transmembrane chemokine receptor that plays a crucial role in immune regulation. Its significance as a therapeutic target stems from its selective expression on tumor-infiltrating regulatory T cells (Tregs) . While Tregs are essential for maintaining immune homeostasis in normal tissues, tumor-infiltrating Tregs promote tumor growth by suppressing antitumor immunity .

CCR8 has been identified as a molecule selectively expressed in tumor-infiltrating Tregs, making it suitable for targeted cancer immunotherapy . Research indicates that selective depletion of CCR8+ Tregs can potentially restore antitumor immunity without affecting systemic Tregs, thereby reducing the risk of autoimmune side effects .

For researchers evaluating CCR8 as a therapeutic target, methodological approaches should include:

• Assessment of CCR8 expression profiles across different immune cell populations

• Correlation of CCR8 expression with tumor progression and patient outcomes

• Evaluation of effects following CCR8 blockade or depletion in preclinical models

How does CCR8 expression correlate with cancer prognosis?

High CCR8 expression on tumor-infiltrating Tregs has been reported to correlate with poor prognosis in several types of cancers . This correlation suggests that CCR8+ Tregs contribute significantly to tumor immune evasion and disease progression.

To methodically investigate this correlation, researchers should:

• Employ flow cytometry and immunohistochemistry to quantify CCR8 expression in tumor samples

• Conduct survival analyses correlating expression levels with clinical outcomes

• Perform multivariate analyses to account for confounding factors

• Compare expression patterns across different cancer types and stages

What is known about CCR8 ligands and their signaling mechanisms?

CCL1 is one of the primary ligands of CCR8 and plays a major role in potentiating Treg-suppressive activity . The functional involvement of CCR8 signaling in tumor-infiltrating Tregs is still being elucidated, with some research indicating that CCR8 signaling may not be necessary for suppressing tumor immunity .

Researchers investigating CCR8-ligand interactions should consider:

• Calcium mobilization assays to assess receptor activation following ligand binding

• Structural studies to visualize receptor-ligand complexes

• Signaling pathway analyses to determine downstream effects of CCR8 activation

• Competitive binding assays to characterize binding kinetics and affinities

How can researchers verify CCR8 expression in experimental samples?

Verification of CCR8 expression is critical for both basic research and therapeutic development. Based on established methodologies, researchers should:

• Use fluorescently-labeled anti-CCR8 antibodies for flow cytometric analysis

• Employ rat anti-human CCR8 antibody (clone: 3-3F) followed by secondary detection systems

• Utilize Transcription Factor Buffer Set for intracellular staining when required

• Include appropriate positive controls (CCR8-expressing cells) and negative controls (cells expressing other chemokine receptors)

It's essential to verify that antibodies recognize the native conformation of CCR8, as demonstrated with mAb1, which binds specifically to Treg cells in human peripheral blood mononuclear cells (PBMCs) and dissociated tumor cells (DTCs) .

What is the structural basis of antibody-mediated CCR8 inhibition?

The structural basis of antibody-mediated CCR8 inhibition has been elucidated through studies examining antibody-CCR8 complexes. Current data reveals that:

Anti-CCR8 antibodies like mAb1 bind to the extracellular region of human CCR8 through multiple interface interactions . The binding interface involves:

• Engagement with the receptor's extracellular loops (ECLs)

• Interaction between CDRH3, CDRL1, CDRL3 and the ECL2b region of CCR8

• Additional stabilization through CDRH1 and CDRH2 interactions with CCR8 ECL1

This binding mode differs from structures of other class A GPCRs in complex with antibodies engaging receptor extracellular loops , suggesting unique structural properties that can be exploited for selective targeting.

How does the CCL1-CCR8 interaction mechanism differ from other chemokine receptor-ligand pairs?

The CCL1-CCR8 interaction exhibits distinctive characteristics compared to other chemokine receptor-ligand pairs:

• Structural studies reveal interaction modes that are unique to this particular receptor-ligand combination

• The binding process follows a specific two-step, two-site sequence that differs from other chemokine receptor interactions

• These unique interaction patterns contribute to the specific signaling properties and potential targeting strategies for CCR8

Understanding these differences provides critical insights for developing highly selective antagonists that can disrupt CCR8 signaling without affecting other chemokine receptors.

What methodologies best assess the neutralizing activity of anti-CCR8 antibodies?

Rigorous assessment of neutralizing activity is essential for characterizing anti-CCR8 antibodies. Based on established protocols:

• FLIPR calcium mobilization assays with CCR8-expressing cells provide quantitative measurements of inhibition efficacy

• Experimental design should include:

  • Human CCR8-expressing HEK293T cells loaded with calcium indicators

  • Addition of test antibodies at various concentrations

  • Stimulation with CCL1 (typically 200 nmol/L)

  • Measurement of calcium influx using fluorometric plate readers

Dose-response curves should be generated to determine IC50 values, and comparison with isotype control antibodies is essential to confirm specificity of the neutralizing effect.

How can researchers measure and optimize ADCC activity of anti-CCR8 antibodies?

Antibody-dependent cellular cytotoxicity (ADCC) is a crucial mechanism for anti-CCR8 antibodies like S-531011. To measure and optimize ADCC:

• Use CCR8-expressing target cells and appropriate effector cells (NK cells)

• Quantify cell lysis using appropriate readouts (fluorescence, luminescence)

• Calculate EC50 values from dose-response curves

• Compare activity against different cell types expressing varying levels of CCR8

For optimization, researchers should consider:

• Antibody engineering to enhance Fc receptor binding

• Testing different antibody isotypes and subclasses

• Assessing the impact of glycosylation patterns on ADCC potency

• Evaluating ADCC in the presence of the tumor microenvironment components

What in vivo models are most appropriate for testing anti-CCR8 antibody efficacy?

Selection of appropriate in vivo models is critical for translational research on anti-CCR8 antibodies:

• Human CCR8 knock-in (hCCR8 KI) mice represent the gold standard model, as they express human CCR8 instead of mouse CCR8

• Tumor models commonly used with hCCR8 KI mice include:

  • CT26.WT cells (3-3.5 × 10⁵ cells subcutaneously implanted)

  • EMT6 cells (3 × 10⁵ cells subcutaneously implanted)

Treatment protocols typically involve:

• Intravenous administration via tail vein

• Multiple dosing (e.g., days 4 and 11 post-implantation)

• Inclusion of appropriate controls (isotype antibodies, buffer)

• Measurement of tumor volume over time

For combination therapy studies, researchers should consider co-administration with checkpoint inhibitors such as anti-PD-1 antibodies, which has shown enhanced efficacy in preclinical models .

What approaches can identify the binding epitopes of anti-CCR8 antibodies?

Identification of binding epitopes provides crucial insights for antibody development and optimization:

• X-ray crystallography or cryo-electron microscopy of antibody-CCR8 complexes

• Mutagenesis studies targeting specific residues in CCR8 extracellular domains

• Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

• Competition binding studies with known ligands or antibodies

Structural studies have revealed that anti-CCR8 antibodies like mAb1 interact with CCR8 through:

• Multiple interfaces involving complementarity-determining regions (CDRs)

• Specific interactions with extracellular loops (ECLs)

• Both polar and hydrophobic contacts with receptor domains

How can researchers differentiate between antagonistic and neutralizing effects of anti-CCR8 antibodies?

Distinguishing between antagonistic and neutralizing effects requires careful experimental design:

• Antagonism refers to direct blocking of ligand binding without necessarily affecting receptor levels

• Neutralization encompasses broader inhibitory effects, including receptor internalization or signaling disruption

Methodological approaches should include:

• Comparison of antibody effects on ligand binding versus receptor expression

• Assessment of receptor internalization following antibody binding

• Evaluation of downstream signaling pathways with and without ligand stimulation

• Analysis of receptor conformation changes using biophysical techniques

Understanding these distinctions is crucial for developing antibodies with desired mechanistic properties.

What methods best evaluate the selectivity of CCR8 targeting in tumor microenvironments?

Evaluating the selectivity of CCR8 targeting in tumor microenvironments requires sophisticated approaches:

S-531011 demonstrated selective depletion of tumor-infiltrating CCR8+ Tregs without affecting Tregs derived from human peripheral blood mononuclear cells , highlighting the importance of comprehensive selectivity assessment.

How should researchers interpret changes in Treg populations following anti-CCR8 antibody treatment?

Interpreting changes in Treg populations requires rigorous analytical approaches:

• Quantitative assessment should include:

  • Absolute numbers and percentages of Tregs within CD4+ T cells

  • Foxp3 expression levels as a marker of Treg identity

  • Functional suppression assays to assess remaining Treg activity

  • Analysis of Treg subsets (CCR8+ vs. CCR8-)

• Spatial distribution analysis should evaluate:

  • Central tumor versus invasive margin distribution

  • Proximity to effector T cells and other immune populations

  • Changes in clustering patterns before and after treatment

Distinguishing between depletion effects and functional modulation is critical for understanding the mechanism of action of anti-CCR8 antibodies.

What biological and technical factors affect reproducibility in CCR8 antibody research?

Multiple factors can impact reproducibility in CCR8 antibody research:

Researchers should implement:

• Detailed reporting of all experimental conditions

• Validation across multiple experimental systems

• Appropriate statistical analyses accounting for biological variability

• Independent replication studies

How can researchers distinguish on-target from off-target effects of anti-CCR8 antibodies?

Distinguishing on-target from off-target effects requires comprehensive experimental approaches:

• Control experiments should include:

  • Isotype-matched control antibodies

  • CCR8-knockout or knockdown systems

  • Competitive binding with known CCR8 ligands

  • Testing in CCR8-negative cell populations

• Mechanistic studies should evaluate:

  • Dose-dependency of observed effects

  • Correlation between CCR8 expression levels and antibody effects

  • Comparison of multiple antibodies targeting different CCR8 epitopes

  • Effects in the presence of specific pathway inhibitors

Thorough characterization of antibody specificity, as demonstrated for mAb1 which binds selectively to CCR8 but not to other chemokine receptors , is essential for accurate interpretation of experimental results.

What combinatorial approaches with anti-CCR8 antibodies show the most promise?

Combination therapy represents a promising direction for enhancing anti-CCR8 antibody efficacy:

• Anti-CCR8 antibodies combined with immune checkpoint inhibitors (anti-PD-1) have demonstrated synergistic antitumor effects in preclinical models

• Potential rational combinations include:

  • Other Treg-targeting approaches (CTLA-4 inhibitors)

  • Innate immune stimulators (TLR agonists, STING agonists)

  • Conventional therapies (chemotherapy, radiation)

  • Cancer vaccines

Research priorities should include:

• Mechanistic studies to understand synergistic interactions

• Optimization of dosing and scheduling

• Identification of predictive biomarkers for combination response

• Evaluation of combination-specific toxicity profiles

What are the challenges in translating CCR8 antibody research to clinical applications?

Translation of CCR8 antibody research faces several challenges:

• Biological challenges include:

  • Potential differences in CCR8 biology between humans and preclinical models

  • Heterogeneity of CCR8 expression across different tumor types and patients

  • Redundancy in immunosuppressive mechanisms within tumors

  • Development of resistance mechanisms

• Technical and development challenges include:

  • Manufacturing antibodies with consistent ADCC activity

  • Developing predictive biomarkers for patient selection

  • Designing appropriate clinical trial endpoints

  • Monitoring on-target, off-tumor effects

Addressing these challenges requires:

• Thorough preclinical validation in humanized models

• Development of companion diagnostics for CCR8 expression

• Adaptive clinical trial designs

• Comprehensive immune monitoring during clinical studies

How might next-generation anti-CCR8 antibodies be engineered for enhanced efficacy?

Engineering next-generation anti-CCR8 antibodies offers opportunities for enhanced efficacy:

• Structural modifications may include:

  • Fc engineering to optimize ADCC activity

  • Bispecific formats targeting CCR8 and another relevant target

  • Antibody-drug conjugates for enhanced Treg depletion

  • pH-dependent binding to improve tumor selectivity

• Novel formats to consider:

  • Smaller antibody fragments with improved tumor penetration

  • Extended half-life variants for reduced dosing frequency

  • Conditionally active antibodies responsive to the tumor microenvironment

Guided by structural insights from antibody-CCR8 complexes , rational design approaches can enhance binding affinity, specificity, and functional properties of next-generation anti-CCR8 therapeutics.