CCR4-3 Antibody

What is CCR4 and why is it a significant target for antibody development?

CCR4 is a chemokine receptor that binds ligands CCL17 (TARC) and CCL22 (MDC). It plays a crucial role in the migration of T cells, particularly to the skin. CCR4 is highly expressed on cutaneous T-cell lymphoma (CTCL) cells and on regulatory T cells (Tregs). Its expression profile makes it an attractive target for both research and therapeutic applications. The significance of CCR4 stems from its involvement in T-cell trafficking to tissues, regulation of immune responses, and its overexpression in certain malignancies .

How does the specificity of CCR4-3 antibodies compare to other anti-CCR4 antibodies?

Anti-CCR4 antibodies vary in their binding epitopes and characteristics. Some antibodies like mAb1567 recognize both the N-terminal and extracellular domains of CCR4, while others may target specific regions. When selecting an antibody, researchers should consider that epitope specificity affects the antibody's ability to inhibit ligand binding, trigger receptor internalization, or mediate effector functions. For instance, mAb1567 binds to both N-terminal and extracellular loop regions, contributing to its high potency in inhibiting chemotaxis . Specificity testing using chimeric receptors (like CCR4/CCR8 chimeras) can help determine the exact binding epitopes and potential cross-reactivity with related chemokine receptors .

What are the recommended storage conditions for maintaining CCR4-3 antibody activity?

CCR4-3 antibodies should typically be stored at -20°C or -80°C for long-term storage . For working solutions, storage between 2°C and 8°C is recommended, with protection from prolonged exposure to light, particularly for fluorophore-conjugated antibodies . It's crucial to avoid repeated freeze-thaw cycles as mentioned in preparation guidelines . For reconstituted antibodies, they can generally be stored at 2-8°C for approximately 1 month or at -20 to -70°C for up to 6 months under sterile conditions .

What are the optimal protocols for using CCR4-3 antibodies in flow cytometry experiments?

For flow cytometry applications, CCR4 antibodies should be titrated to determine optimal concentrations, typically in the range of 0.2-1 μg per test. A recommended protocol includes:

• Prepare single-cell suspensions from your sample (e.g., PBMCs, tissue-derived cells)

• Block Fc receptors with appropriate blocking reagent (5-10 minutes)

• Surface staining: Incubate cells with fluorophore-conjugated anti-CCR4 antibody at 4°C for 30 minutes in the dark

• For multi-color panels, include appropriate markers to identify cell populations of interest (e.g., CD3, CD4, CD25 for Tregs)

• Wash twice with flow cytometry buffer

• Analyze on a flow cytometer with appropriate compensation controls

For intracellular staining, following surface staining, fix cells with 2% paraformaldehyde for 10 minutes, then permeabilize with 0.5% saponin before intracellular antibody staining . The antibody has been validated for detection of CCR4 in human PBMCs, showing specific staining of CCR4-expressing T cell subsets .

How can I validate the specificity of CCR4-3 antibody in my experimental system?

To validate antibody specificity, employ multiple approaches:

• Positive and negative controls: Test the antibody on cell lines known to express CCR4 (e.g., CTCL cell lines like Mac-1) and those that don't

• Blocking experiments: Pre-incubate the antibody with recombinant CCR4 protein before staining to confirm specific binding is inhibited

• siRNA knockdown: Reduce CCR4 expression in a positive cell line and demonstrate reduced antibody binding

• Western blot analysis: Confirm detection of a band at the expected molecular weight (~42 kDa for CCR4)

• Chimeric receptor experiments: Use CCR4/CCR8 chimeras to map epitope specificity

• Cross-species reactivity testing: If working with mouse models, confirm whether your anti-human CCR4 antibody cross-reacts with mouse CCR4 (only ~85% sequence identity)

What methods are most effective for studying CCR4-dependent chemotaxis in vitro?

For studying CCR4-dependent chemotaxis, the Transwell migration assay is most commonly used:

• Place CCR4-expressing cells (1×10^6/well) in the upper chamber of Transwell plates (typically 5-8 μm pore size)

• Add CCR4 ligands (CCL17 or CCL22, 50-100 ng/ml) to the lower chamber

• For inhibition studies, pre-incubate cells with anti-CCR4 antibody (varied concentrations)

• Incubate for 3-4 hours at 37°C

• Collect cells from the bottom chamber and enumerate by flow cytometry or cell counting

• Calculate migration index: (number of migrated cells with chemokine)/(number of migrated cells without chemokine)

This method has been effectively used to demonstrate how anti-CCR4 antibodies like mAb1567 inhibit chemotaxis of both CCR4+ tumor cells and CD4+CD25high Tregs toward CCL17 and CCL22 .

How is CCR4 expression distributed across different T cell subsets, and what techniques best characterize this distribution?

CCR4 shows differential expression across T cell subsets:

For characterization, a multiparameter flow cytometry approach is most effective:

• Use antibody combinations targeting lineage markers (CD3, CD4)

• Include subset markers (CD25, CD127 for Tregs; CCR7, CD45RA for memory/naïve)

• Quantify absolute receptor numbers using QuantiBRITE PE beads and PE-labeled anti-CCR4 antibody

• Correlate with functional studies (migration, cytokine production)

This approach has revealed that Tregs express approximately 2.5-fold higher surface density of CCR4 compared to effector T cells .

What is the functional significance of CCR4 expression on regulatory T cells?

CCR4 expression on Tregs has several important functional implications:

• Tissue trafficking: CCR4 enables Tregs to migrate to tissues expressing CCL17/CCL22, particularly skin and sites of inflammation

• Tumor infiltration: Tumors and tumor-associated macrophages secrete CCL22, recruiting CCR4+ Tregs to establish an immunosuppressive microenvironment

• Immune regulation: CCR4+ Tregs maintain peripheral tolerance and are present in tissues under normal conditions

• Transplantation: CCR4+ Tregs are involved in allograft tolerance, with recruitment dependent on CCL22

• Therapeutic target: Anti-CCR4 antibodies can block Treg migration and abrogate their suppressive function, potentially enhancing anti-tumor immunity

Experimentally, it has been shown that CCR4+ Tregs strongly suppress effector T cell proliferation, and that anti-CCR4 antibodies can inhibit both Treg migration toward CCL22 and their suppressive activity in T cell proliferation assays .

How does CCR4 expression change during T cell activation and differentiation?

During T cell activation and differentiation, CCR4 expression undergoes dynamic regulation:

• Naïve T cells: Generally low CCR4 expression

• Activation: Upregulation of CCR4 upon TCR stimulation, particularly in cells primed in skin-draining lymph nodes

• Differentiation: Preferential expression on Th2 and Th17 cells, with variable expression on Th1 cells

• Memory formation: Maintained on certain memory T cell subsets, especially skin-homing memory cells

• Disease states: Further upregulation during inflammation or in malignant transformation

To study these changes, researchers should use time-course experiments with flow cytometry analysis following T cell activation with anti-CD3/CD28 or antigen-specific stimulation. This should be combined with analysis of other homing receptors (like CLA for skin-homing) to understand the coordinated regulation of migration potential .

What role does CCR4 play in experimental autoimmune encephalomyelitis (EAE), and how can anti-CCR4 antibodies be used to study this disease model?

CCR4 contributes significantly to EAE pathogenesis, as demonstrated in CCR4−/− mice which show:

• Delayed disease onset: CCR4−/− mice exhibit significantly delayed EAE development

• Reduced disease incidence and severity: Lower mean cumulative scores and fewer inflammatory lesions

• Decreased CNS infiltrate: Reduced numbers of infiltrating lymphocytes and monocytes

• Normal peripheral T cell responses: No alterations in Th1 or Th17 responses to myelin antigen

Interestingly, the mechanism appears to involve CD11b+Ly6Chi inflammatory macrophages (iMϕ) rather than direct effects on T cells. To study CCR4's role using antibodies:

• Use flow cytometry to monitor CCR4 expression on different immune cell populations during disease progression

• Administer anti-CCR4 antibodies prophylactically or therapeutically in the EAE model

• Assess clinical scores, CNS infiltration, and composition of infiltrating cells

• Analyze peripheral immune responses to distinguish between effects on priming versus trafficking

This approach can help determine whether CCR4 primarily regulates iMϕ tissue accumulation or inflammatory function in EAE .

What are the mechanisms of action for therapeutic anti-CCR4 antibodies in cutaneous T-cell lymphoma (CTCL)?

Therapeutic anti-CCR4 antibodies employ multiple mechanisms to target CTCL cells:

• Complement-dependent cytotoxicity (CDC): Antibodies like mAb1567 activate the complement system to form membrane attack complexes on CCR4+ tumor cells

• Antibody-dependent cellular cytotoxicity (ADCC):

  • NK cell-mediated ADCC: Natural killer cells recognize antibody-coated tumor cells and induce apoptosis

  • Neutrophil-mediated ADCC: Neutrophils can also contribute to tumor cell killing

• Inhibition of chemotaxis: Blocking CCR4 prevents tumor cell migration toward CCL17/CCL22, potentially limiting disease spread

• Depletion of suppressive Tregs: Anti-CCR4 antibodies deplete CCR4+ Tregs, enhancing anti-tumor immune responses

• Disruption of tumor microenvironment: Reduced recruitment of CCR4+ cells can alter the supportive microenvironment

These mechanisms have been demonstrated through in vitro assays including CDC (with rabbit or mouse complement), ADCC (using PBMCs, isolated NK cells, or neutrophils as effectors), and chemotaxis inhibition assays. Humanized antibodies like mAb2-3 show enhanced ADCC and CDC activities compared to their murine counterparts .

How can I design in vivo experiments to evaluate anti-CCR4 antibody efficacy in tumor models?

For evaluating anti-CCR4 antibody efficacy in tumor models:

• Model selection:

  • Xenograft models using CCR4+ human tumor cell lines (e.g., luciferase-expressing Mac-1 CTCL cells) in immunodeficient mice

  • Syngeneic models with mouse CCR4+ tumor cells in immunocompetent mice to assess impact on immune microenvironment

• Experimental design:

  • Group size: Typically 8-10 mice per group for statistical power

  • Treatment regimen: Begin when tumors are established (50-100 mm³)

  • Dosing: 3-5 mg/kg antibody administered intraperitoneally 2-3 times per week

  • Controls: Isotype control antibodies at equivalent doses

• Monitoring parameters:

  • Tumor volume measurements using calipers (calculate as length × width² × 0.52)

  • Bioluminescence imaging for luciferase-expressing tumors

  • Survival analysis

  • Body weight and adverse events monitoring

• Mechanistic analyses:

  • Flow cytometry of tumor infiltrating lymphocytes

  • Immunohistochemistry for complement deposition

  • Analysis of Treg numbers and function

  • Cytokine profiling in tumor microenvironment

This approach has been successfully used to demonstrate the anti-tumor efficacy of anti-CCR4 antibodies like mAb1567 in CTCL models .

How can single-cell technologies be integrated with anti-CCR4 antibodies to better understand heterogeneity in CCR4-expressing immune populations?

Integrating single-cell technologies with anti-CCR4 antibodies can provide deeper insights into the heterogeneity of CCR4-expressing cells:

• Single-cell RNA-seq with protein detection:

  • Use CITE-seq or REAP-seq methods, which combine anti-CCR4 antibodies conjugated to oligonucleotide tags with single-cell transcriptomics

  • This allows correlation of CCR4 protein levels with global gene expression profiles

  • Can identify novel CCR4+ subpopulations with distinct transcriptional programs

• Mass cytometry (CyTOF):

  • Label anti-CCR4 antibodies with rare earth metals

  • Combine with 30+ other markers to deeply phenotype CCR4+ cells

  • Enables high-dimensional analysis of CCR4 co-expression with other chemokine receptors and functional markers

• Spatial transcriptomics with immunofluorescence:

  • Use fluorescently-labeled anti-CCR4 antibodies alongside spatial transcriptomics methods

  • Map CCR4+ cell locations in tissues relative to other cell types and ligand-producing cells

  • Understand tissue microniches where CCR4-dependent interactions occur

• Live cell imaging with labeled antibodies:

  • Use non-blocking fluorescent anti-CCR4 Fab fragments

  • Track CCR4+ cell migration and interactions in real-time

  • Quantify receptor dynamics, internalization, and signaling

These approaches can reveal functionally distinct CCR4+ cell subsets that may respond differently to therapeutic targeting .

What strategies can address the challenge of antibody penetration into solid tumors when targeting CCR4+ cells in the tumor microenvironment?

Addressing antibody penetration into solid tumors requires multifaceted approaches:

• Antibody engineering strategies:

  • Use smaller formats: scFv, Fab, or nanobody derivatives of anti-CCR4 antibodies

  • Engineer antibodies with optimized isoelectric points and reduced hydrophobicity

  • Develop bispecific antibodies targeting both CCR4 and tumor-specific antigens

• Combination approaches:

  • Pair anti-CCR4 antibodies with agents that normalize tumor vasculature (e.g., anti-VEGF therapy)

  • Combine with ECM-modifying enzymes (hyaluronidase, collagenase) to reduce physical barriers

  • Use ultrasound or radiation to temporarily enhance vascular permeability

• Alternative delivery methods:

  • Intratumoral injection of anti-CCR4 antibodies when accessible

  • Nanoparticle-conjugated antibodies for enhanced EPR effect

  • Antibody-drug conjugates to increase potency against the cells that are reached

• Monitoring strategies:

  • Use radioisotope-labeled antibodies with PET imaging to quantify tumor penetration

  • Multiplex immunohistochemistry on serial biopsies to assess penetration depth

  • Implement mathematical modeling to predict optimal dosing for maximal tumor penetration

These strategies could enhance the effectiveness of anti-CCR4 antibodies against solid tumors with CCR4+ infiltrating cells .

How can we address potential resistance mechanisms to anti-CCR4 antibody therapy in clinical applications?

Addressing resistance to anti-CCR4 antibody therapy requires understanding and targeting multiple mechanisms:

• Receptor downregulation or mutation:

  • Monitor CCR4 expression levels before and during treatment

  • Sequence CCR4 to detect emerging mutations in the antibody binding epitope

  • Develop antibodies targeting different epitopes for sequential or combination therapy

• Alternative chemokine receptor usage:

  • Profile expression of other chemokine receptors (CCR8, CCR10) that might compensate for CCR4 blockade

  • Consider dual receptor targeting strategies

  • Use chemokine receptor antagonists in combination with anti-CCR4 antibodies

• Fc receptor polymorphisms affecting ADCC:

  • Genotype FcγR polymorphisms to predict ADCC efficacy

  • Engineer Fc regions for enhanced binding to activating FcγRs regardless of polymorphisms

  • Use defucosylated antibodies (like mogamulizumab) to enhance ADCC potency

• Complement inhibition:

  • Assess expression of membrane complement inhibitors (CD55, CD59)

  • Combine with inhibitors of complement regulatory proteins

  • Engineer antibodies with enhanced C1q binding

• Immunosuppressive microenvironment adaptations:

  • Monitor changes in cytokine profiles during treatment

  • Analyze emergence of alternative immunosuppressive cell populations

  • Combine with checkpoint inhibitors to address multiple immune evasion mechanisms

Implementation of regular molecular monitoring and adaptive combination strategies can help mitigate resistance development .