CCR4-4 Antibody

What is CCR4 and why is it an important research target?

CCR4 (CD194) is a C-C Chemokine Receptor 4 belonging to the G protein-coupled receptor family, characterized by seven transmembrane alpha helical domains. It serves as a receptor for the chemokines CCL17 (TARC) and CCL22 (MDC), which direct the migration of target cells . CCR4 has emerged as a significant research and therapeutic target due to its expression pattern on specific immune cell subsets and certain cancer types. It plays crucial roles in T cell migration, particularly to the skin, and has been implicated in various pathological conditions including T-cell lymphomas . The receptor's selective expression on regulatory T cells (Tregs) and tumor-infiltrating immune cells, along with its involvement in metastasis, makes it a compelling target for cancer immunotherapy approaches .

Which cell types express CCR4 and what methodologies can detect this expression?

CCR4 exhibits a specific cellular distribution pattern that makes it particularly relevant for immunological research. Although initially thought to be restricted to Th2 cells, CCR4 expression has since been identified in several T cell subsets including certain Th17, Treg, and Th1 populations . Importantly, naive T cells do not express CCR4, indicating its role in memory or effector functions . Beyond T cells, CCR4 expression has been documented on activated natural killer (NK) cells, platelets, monocytes, and basophils .

For detection methods, flow cytometric analysis remains the gold standard when using anti-CCR4 antibodies. Pre-titrated antibodies such as the D8SEE clone can be used at approximately 5 μL (0.125 μg) per test in a final volume of 100 μL, with optimal cell numbers ranging from 10^5 to 10^8 cells per test . For immunohistochemistry applications, polyclonal antibodies (such as HPA031613 from Sigma-Aldrich) can be utilized on paraformaldehyde-fixed and paraffin-embedded samples . Additionally, fluorophore-conjugated antibodies like mogamulizumab labeled with Alexa Fluor 680 can provide sensitive detection in fluorescence-based assays .

How should researchers optimize flow cytometry protocols when using anti-CCR4 antibodies?

When designing flow cytometry experiments with anti-CCR4 antibodies, several methodological considerations are essential for robust results:

• Antibody selection and titration: Pre-titrated antibodies such as the D8SEE clone conjugated to APC have been validated for flow cytometric analysis. These can typically be used at 5 μL (0.125 μg) per test in a final volume of 100 μL .

• Spectral parameters: For APC-conjugated anti-CCR4 antibodies, use appropriate excitation (633-647 nm) and emission (660 nm) filters, typically with a red laser .

• Sample preparation: When working with peripheral blood, use viability dyes such as LIVE-DEAD violet to exclude dead cells. For optimal detection of CCR4-expressing subsets, include appropriate markers for T cell subpopulations .

• Controls: Include proper isotype controls and FMO (fluorescence minus one) controls to accurately determine CCR4-positive populations, especially when analyzing heterogeneous samples.

• Analysis strategies: For detecting chimeric antigen receptor (CAR) expression in anti-CCR4 CAR T cells, biotinylated anti-human Fab antibodies can be employed, while control CD19-CAR detection can utilize anti-mouse Fab antibodies .

Data should be collected using appropriate analyzers (e.g., LSRFortessa) and analyzed with software such as FlowJo version 10 .

What are the key considerations for developing novel anti-CCR4 antibodies?

The development of novel anti-CCR4 antibodies requires careful consideration of several critical factors:

• Target epitope selection: Given CCR4's seven transmembrane structure, selecting accessible epitopes on extracellular domains is essential for antibody binding .

• Generation strategies: Successful approaches include phage display with human antibody libraries and affinity maturation techniques. Cell-based antibody selection strategies have proven effective for generating antibodies against G-protein coupled receptors like CCR4 .

• Functional validation: Novel antibodies should be evaluated for:

  • Competitive binding with natural ligands (CCL17/CCL22)

  • Blockade of ligand-induced signaling

  • Inhibition of chemotaxis

  • Effector functions such as ADCC or phagocytosis when applicable

• Specificity assessments: Cross-reactivity testing against related chemokine receptors is essential to ensure specificity for CCR4.

• Format considerations: Developing various formats (whole IgG, Fab, scFv) may be necessary depending on the intended application, particularly for CAR T cell development .

The generation of anti-CCR4 antibodies using human non-immune libraries and phage display on GPCR-expressing cells has been successfully demonstrated, offering a template for future development efforts .

How do anti-CCR4 antibodies function in cancer immunotherapy applications?

Anti-CCR4 antibodies demonstrate multiple mechanisms of action in cancer immunotherapy settings:

• Direct inhibition of CCR4 signaling: These antibodies can compete with CCR4's natural ligands (CCL17/TARC and CCL22/MDC), effectively blocking ligand-induced signaling pathways and subsequent cell migration . This mechanism is particularly relevant for tumors that utilize CCR4-mediated pathways for growth or metastasis.

• Antibody-dependent cellular cytotoxicity (ADCC): Humanized anti-CCR4 antibodies like mogamulizumab can recruit effector cells (primarily NK cells) to eliminate CCR4-expressing tumor cells. This ADCC activity has been demonstrated against CCR4-positive cell lines and in xenograft models of EBV-positive NK-cell lymphomas .

• Phagocytosis induction: Some anti-CCR4 antibodies can stimulate phagocytosis of tumor cells by macrophages, providing an additional mechanism for tumor cell clearance .

• Modulation of the tumor microenvironment: By targeting CCR4-positive regulatory T cells (Tregs) within the tumor microenvironment, these antibodies may reduce immunosuppression and potentially enhance other immunotherapeutic approaches .

In vivo studies have demonstrated significant survival benefits in mouse models of human T-cell lymphoma treated with anti-CCR4 antibodies, supporting their therapeutic potential .

What are the specific considerations for treating EBV-associated lymphoproliferative diseases with anti-CCR4 antibodies?

The treatment of Epstein-Barr virus (EBV)-associated lymphoproliferative diseases (LPDs) with anti-CCR4 antibodies requires specific considerations based on current research:

• Expression profiling: CCR4 expression has been confirmed in most EBV-positive T and NK cell lines, with 8 of 17 patients with EBV-associated T/NK-LPDs showing CCR4 expression on EBV-infected cells . Notably, all five patients with hydroa vacciniforme (a photodermatosis caused by clonal expansion of EBV-infected γδT cells) demonstrated CCR4 positivity .

• Therapeutic mechanism: In these diseases, anti-CCR4 antibodies like mogamulizumab primarily function through ADCC. Ex vivo studies have demonstrated that CCR4-positive EBV-infected γδT cells from hydroa vacciniforme patients can be effectively killed by mogamulizumab through this mechanism .

• Patient selection: Given the heterogeneous expression of CCR4 across patients (8/17 in one study), immunophenotyping to confirm CCR4 expression on the malignant cells is critical before initiating therapy .

• Therapeutic resistance considerations: EBV-infected cells may exhibit alterations in signaling pathways or immune evasion mechanisms that could potentially impact responses to anti-CCR4 therapy, necessitating careful monitoring during treatment.

These findings suggest that anti-CCR4 antibody therapy may be particularly beneficial for specific subsets of EBV-associated T/NK-LPDs, especially those with confirmed CCR4 expression.

How can anti-CCR4 CAR T cells be designed to overcome fratricide effects in T-cell lymphoma treatment?

• scFv optimization: Testing multiple antibody configurations (8 different anti-CCR4 CARs with varying spatial combinations of heavy and light chain variable regions) is crucial for identifying constructs with optimal activity. These designs incorporate second-generation backbone CAR elements containing 4-1BB and CD3ζ domains .

• Exploiting selective fratricide: Rather than viewing fratricide as entirely detrimental, research has shown that anti-CCR4 CAR T cells specifically deplete Th2 and Tregs while sparing CD8+ and Th1 T cells. This selective depletion actually increases the percentage of CAR+ T cells in the final product and may enhance anti-tumor efficacy .

• Transduction and expansion protocols: Optimal protocols include:

  • T cell stimulation with anti-CD3/CD28 beads

  • Lentiviral transduction at MOI of 4 one day after stimulation

  • Bead removal 5 days post-stimulation

  • Regular cell counting and media replenishment

  • Harvesting when T-cell size decreases to approximately 350 μm³

• Functional validation: Assessment of CAR T cell functionality through:

  • Degranulation assays

  • Intracellular cytokine production

  • Cytotoxicity against CCR4+ tumor targets

This approach has demonstrated superior antitumor efficacy and long-term remission in murine models of human T-cell lymphoma, particularly with mogamulizumab-based CCR4-CAR T cells .

What methodological approaches can assess the potential off-target effects of anti-CCR4 therapies?

Thorough assessment of potential off-target effects is critical for anti-CCR4 therapies due to CCR4 expression in various normal tissues. Recommended methodological approaches include:

• Comprehensive expression profiling:

  • Analyze CCR4 RNA expression across normal human tissues using public databases like the Human Protein Atlas

  • Conduct immunohistochemical analysis comparing CCR4 expression in target pathologies (e.g., CTCL skin lesions) with normal tissues using tissue microarrays

  • Employ flow cytometry to quantify CCR4 expression levels across immune cell subsets

• In vitro cross-reactivity testing:

  • Assess antibody binding to CCR4-negative cell lines

  • Evaluate potential binding to related chemokine receptors

  • Test reactivity against primary normal cells from various tissues

• Ex vivo toxicity assessments:

  • Perform cytotoxicity assays against normal CCR4-expressing cells (specific T cell subsets)

  • Measure cytokine release in mixed cell populations

• Animal model validation:

  • Monitor for tissue-specific toxicities in relevant animal models

  • Assess hematological parameters, including specific T cell subsets

  • Evaluate for potential immunological adverse events

Fortunately, clinical studies with mogamulizumab have reported manageable safety profiles with primarily hematological adverse effects such as thrombocytopenia, suggesting that despite low-level CCR4 expression in some normal tissues, severe organ-specific adverse effects are uncommon .

What are the optimal storage and handling conditions for maintaining anti-CCR4 antibody functionality?

Maintaining optimal functionality of anti-CCR4 antibodies requires adherence to specific storage and handling protocols:

• Storage temperature: Store fluorophore-conjugated antibodies (e.g., APC-conjugated) at 2-8°C and protected from light to prevent photobleaching. Avoid repeated freeze-thaw cycles which can compromise antibody structure and function .

• Dilution considerations: When preparing working solutions, use appropriate buffers (typically PBS with 0.5-1% BSA) to maintain antibody stability. For flow cytometry applications, final concentrations of approximately 0.125 μg per test in 100 μL have been validated .

• Filtration requirements: Some protocols recommend 0.2 μm post-manufacturing filtration to remove potential aggregates that could interfere with experimental results .

• Transport conditions: During transport between laboratories or facilities, maintain the cold chain and protect from light exposure to preserve antibody functionality.

• Quality control: Periodically verify antibody binding and specificity using positive control samples (e.g., CCR4-expressing cell lines) to ensure continued functionality throughout the antibody's shelf life.

Following these handling practices will help ensure consistent experimental results and maximize the utility of anti-CCR4 antibodies in research applications.

How can researchers troubleshoot inconsistent results in anti-CCR4 antibody experiments?

When facing inconsistent results in experiments utilizing anti-CCR4 antibodies, researchers should systematically address potential sources of variability:

• Antibody quality assessment:

  • Verify antibody lot consistency and stability

  • Confirm proper storage conditions were maintained

  • Consider re-titration of antibody to validate optimal concentrations

• Target expression variables:

  • CCR4 expression can vary with cellular activation status; standardize cell stimulation protocols

  • Account for heterogeneous expression across T cell subsets

  • Consider cytokine or activation-induced changes in receptor expression and internalization

• Methodological considerations:

  • For flow cytometry: Standardize sample preparation, fixation protocols, and instrument settings

  • For functional assays: Ensure consistent effector:target ratios and incubation conditions

  • For imaging applications: Maintain consistent fixation, permeabilization, and staining protocols

• Cell source variability:

  • When using primary human cells, account for donor variability

  • Standardize isolation procedures to minimize activation-induced changes in CCR4 expression

  • Consider using established cell lines as controls alongside primary samples

• Validation strategies:

  • Use alternative detection antibodies targeting different CCR4 epitopes

  • Employ genetic approaches (e.g., qPCR) to correlate protein expression with transcript levels

  • Include positive and negative control samples in each experimental run

Addressing these factors systematically will help identify sources of variability and improve experimental reproducibility when working with anti-CCR4 antibodies.