CCR4 Antibody

What is CCR4 and why is it considered a valuable target for antibody development in cancer research?

CCR4 (C-C chemokine receptor type 4, also known as CD194) is a G-protein coupled chemokine receptor expressed on various cell types, most notably on T-regulatory cells (Tregs) and certain cancer cells. It represents a compelling target for cancer immunotherapy for several reasons. First, CCR4 is highly expressed on cutaneous T-cell lymphoma (CTCL) cells, where it contributes to their skin-homing capacity . Second, CCR4 is prominently expressed on Tregs that migrate to tumors expressing CCL17 and CCL22 (CCR4 ligands), facilitating tumor evasion from immune surveillance . This dual expression pattern on both malignant cells and immunosuppressive Tregs makes CCR4 an ideal target for antibody-based cancer therapeutics that can simultaneously target the tumor and modulate the tumor microenvironment .

Research approaches typically involve immunophenotyping of patient samples to establish CCR4 expression levels, correlation with clinical outcomes, and in vitro studies demonstrating antibody effects on both malignant cells and Tregs.

How are anti-CCR4 antibodies utilized in flow cytometry for research applications?

Flow cytometry represents a critical application for anti-CCR4 antibodies in research settings. When designing flow cytometry experiments with anti-CCR4 antibodies, researchers should employ the following methodological approach:

• Cell preparation: Isolate target cells (e.g., peripheral blood mononuclear cells) using density gradient separation.

• Antibody staining: Use a multi-color panel including anti-CCR4 antibodies alongside other relevant markers (e.g., CD4, CD25 for Treg identification).

• Control selection: Include appropriate isotype controls to establish specific binding, as demonstrated with Mouse IgG2B isotype control in the scientific data for MAB1567 .

• Secondary detection: If using unconjugated primary antibodies, apply appropriate fluorophore-conjugated secondary antibodies like PE-conjugated anti-mouse IgG .

• Analysis: Gate on relevant populations first (e.g., CD4+ T cells) before analyzing CCR4 expression.

For example, scientific data shows that human PBMCs can be stained with Mouse Anti-Human CD4 APC-conjugated Monoclonal Antibody and Mouse Anti-Human CCR4 Monoclonal Antibody (MAB1567), followed by Phycoerythrin-conjugated Anti-Mouse IgG Secondary Antibody for effective detection . This approach allows for precise identification and characterization of CCR4-expressing cell populations within complex samples.

What mechanisms of action do anti-CCR4 antibodies employ against CCR4-positive tumor cells?

Anti-CCR4 antibodies demonstrate multiple mechanisms of action against CCR4-positive tumor cells, making them versatile therapeutic agents. Based on published research, these mechanisms include:

• Complement-dependent cytotoxicity (CDC): Anti-CCR4 antibodies can activate the complement cascade, leading to the formation of membrane attack complexes and subsequent lysis of target cells. In experimental protocols, this is typically measured using LDH release assays after incubating target cells with antibodies in the presence of rabbit or mouse serum as a complement source .

• Antibody-dependent cellular cytotoxicity (ADCC): These antibodies engage Fc receptors on effector cells such as NK cells and neutrophils, facilitating targeted killing of CCR4-expressing tumor cells. The efficacy of ADCC can be quantified using cytotoxicity assays with isolated effector cells and CCR4+ target cells in the presence of the antibody .

• Inhibition of chemokine-induced signaling: Anti-CCR4 antibodies can block the interaction between CCR4 and its ligands (CCL17/CCL22), thereby inhibiting downstream signaling pathways critical for tumor cell survival and migration .

• Phagocytosis: Some anti-CCR4 antibodies can promote phagocytosis of antibody-coated tumor cells by macrophages and other phagocytic cells .

Research has shown that humanized versions of anti-CCR4 antibodies, such as the affinity-optimized variant mAb2-3 derived from mAb1567, demonstrate enhanced CDC and ADCC activities against CCR4+ tumor cells compared to their murine counterparts .

How do anti-CCR4 antibodies affect T-regulatory cell function in cancer models?

Anti-CCR4 antibodies impact T-regulatory (Treg) cell function through multiple mechanisms that collectively reduce immunosuppression in the tumor microenvironment:

• Inhibition of Treg migration: Anti-CCR4 antibodies effectively block the chemotaxis of CD4+CD25high Tregs toward CCL22, a CCR4 ligand often produced by tumors . In experimental settings, this can be assessed using transwell migration assays measuring the movement of isolated Tregs in response to CCL22 gradients in the presence or absence of anti-CCR4 antibodies.

• Abrogation of suppressive activity: Research demonstrates that anti-CCR4 antibodies can directly interfere with the suppressive function of Tregs in T-cell proliferation assays . This is typically evaluated by co-culturing CD4+CD25high Tregs with CD4+CD25- effector T cells in the presence of stimulatory antibodies (anti-CD3/anti-CD28) and measuring proliferation using 3H-thymidine incorporation .

• Selective depletion of Tregs: Due to the high expression of CCR4 on Tregs, anti-CCR4 antibodies with enhanced ADCC activity can selectively deplete these cells from the tumor microenvironment, shifting the balance toward effector T-cell responses.

The functional consequence of these effects is the restoration of anti-tumor immunity through reduction of Treg-mediated immunosuppression. For researchers investigating these mechanisms, it is essential to establish appropriate in vitro suppression assays and in vivo models that can accurately measure changes in Treg function and their impact on anti-tumor responses.

What strategies are employed to generate and optimize anti-CCR4 antibodies with enhanced therapeutic potential?

Several sophisticated strategies have been developed to generate and optimize anti-CCR4 antibodies with enhanced therapeutic potential:

• Humanization of murine antibodies: Researchers have successfully humanized mouse anti-CCR4 antibodies like mAb1567 by grafting murine complementarity-determining regions (CDRs) onto human antibody frameworks . This process preserves the binding specificity while reducing immunogenicity for clinical applications.

• Affinity maturation: After initial humanization, antibodies undergo affinity optimization through techniques such as:

  • CDR mutagenesis

  • Phage display selection with stringent washing conditions

  • Competitive elution strategies

  This approach has yielded variants like mAb2-3 with improved binding characteristics and enhanced effector functions .

• Cell-based antibody selection strategies: Fully human antibodies against CCR4 have been generated using phage display on GPCR-expressing cells, allowing selection of antibodies that recognize native conformations of the receptor . This methodology is particularly valuable for membrane proteins like CCR4 where proper folding is critical for epitope recognition.

• Fc engineering: Modification of the Fc region of anti-CCR4 antibodies can enhance effector functions like ADCC and CDC, improving their therapeutic efficacy .

• Bispecific antibody development: Some research approaches involve generating bispecific antibodies that simultaneously target CCR4 and another relevant antigen to enhance therapeutic efficacy.

These optimization strategies have collectively led to the development of anti-CCR4 antibodies with dual mechanisms of action: blocking receptor signaling and mediating antibody-directed tumor cell killing through Fc-dependent effector mechanisms .

How can researchers effectively determine the epitope specificity of anti-CCR4 antibodies?

Determining epitope specificity is crucial for understanding antibody function and optimizing therapeutic applications. For anti-CCR4 antibodies, researchers can employ these methodological approaches:

• Domain swapping and chimeric receptor construction: Generating chimeric receptors by swapping domains between CCR4 and related chemokine receptors (e.g., CCR8) helps identify binding regions. For example, studies with mAb1567 used CCR4/CCR8 chimeras (Chi#1 and Chi#2) to demonstrate that this antibody recognizes epitopes involving both the N-terminal domain and extracellular loops of CCR4 .

• Alanine scanning mutagenesis: Systematically replacing amino acids in potential epitope regions with alanine can identify critical residues required for antibody binding.

• Peptide mapping: Synthesizing overlapping peptides spanning the extracellular domains of CCR4 and testing them for antibody binding in ELISA or similar assays.

• Competition binding assays: Determining whether the antibody competes with natural ligands (CCL17/CCL22) or other antibodies with known binding sites can provide insight into epitope location.

• X-ray crystallography or cryo-EM: For definitive epitope mapping, structural analysis of antibody-CCR4 complexes can reveal precise binding interfaces, though this approach is technically challenging for membrane proteins like CCR4.

Research has shown that some antibodies, such as mAb1567, recognize conformational epitopes involving multiple domains of CCR4, rather than linear epitopes confined to a single region . This complex epitope recognition may contribute to the functional properties of these antibodies, including their ability to block ligand binding and receptor signaling.

What are the optimal assays for evaluating the functional effects of anti-CCR4 antibodies on chemotaxis and cell signaling?

When evaluating the functional effects of anti-CCR4 antibodies, researchers should employ comprehensive assay panels that assess both chemotaxis inhibition and disruption of signaling pathways:

• Chemotaxis Assays:

  • Transwell migration: Place CCR4+ cells (tumor cells or Tregs) in the upper chamber of a transwell system with CCL17/CCL22 in the lower chamber. Pretreatment with anti-CCR4 antibodies should inhibit migration toward the chemokine gradient .

  • Real-time cell migration: More sophisticated systems allow real-time quantification of migration using impedance-based or video microscopy approaches.

  • 3D migration in collagen matrices: This better simulates in vivo environments compared to 2D transwell systems.

• Signaling Assays:

  • Calcium flux: Measure intracellular Ca2+ mobilization following CCL17/CCL22 stimulation using fluorescent indicators like Fluo-4 AM. Effective anti-CCR4 antibodies should block this response .

  • Phosphorylation of downstream mediators: Assess activation of signaling molecules like ERK1/2, Akt, or PKC using phospho-specific antibodies in western blots or phospho-flow cytometry.

  • G-protein activation: Measure GTPγS binding or cAMP modulation in response to CCL17/CCL22 with and without antibody pretreatment.

  • β-arrestin recruitment: Using BRET or FRET-based assays to measure receptor desensitization and internalization pathways.

• Receptor Internalization Assays:

  • Flow cytometry-based measurement of surface CCR4 downregulation following ligand exposure, with or without antibody pretreatment.

Research has shown that fully human antagonistic antibodies against CCR4 can effectively compete with ligand binding, block ligand-induced signaling pathways, and inhibit chemotaxis of CCR4+ cells . When designing these assays, it's critical to include appropriate positive controls (known CCR4 antagonists) and negative controls (isotype-matched antibodies) to validate specificity.

How should researchers design in vivo studies to evaluate the efficacy of anti-CCR4 antibodies in cancer models?

Designing rigorous in vivo studies for anti-CCR4 antibodies requires careful consideration of model selection, treatment regimens, and multifaceted endpoints:

Research has demonstrated that anti-CCR4 antibodies can provide significant survival benefits in mouse models of human T-cell lymphoma, with mechanisms involving both direct tumor killing and modulation of the immune microenvironment . The experimental design should incorporate appropriate controls, including isotype-matched antibodies, to distinguish specific from non-specific effects.

How can researchers enhance the complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) of anti-CCR4 antibodies?

Enhancing the effector functions of anti-CCR4 antibodies requires strategic modifications at multiple levels:

• Fc Engineering Approaches:

  • Glycoengineering: Modifying the glycosylation pattern of the Fc region, particularly by reducing core fucosylation, significantly enhances ADCC activity by increasing binding affinity to FcγRIIIa on NK cells.

  • Amino acid substitutions: Specific mutations in the Fc region (e.g., S239D/I332E or G236A/S239D/I332E) can dramatically increase binding to activating Fc receptors while decreasing binding to inhibitory receptors.

  • Isotype selection: Human IgG1 isotype generally demonstrates stronger ADCC and CDC activities compared to other isotypes, making it the preferred backbone for therapeutic antibodies requiring these functions .

• Enhancing CDC Activity:

  • Hexamer formation promotion: Mutations that enhance Fc-Fc interactions promote hexamer formation upon antigen binding, significantly improving C1q recruitment and CDC activity.

  • C1q binding optimization: Specific mutations in the C1q binding site can enhance complement activation.

  • Membrane proximity optimization: Selecting epitopes that position the Fc region closer to the cell membrane can improve complement activation.

• Optimizing ADCC Enhancement:

  • NK cell engagement: Bispecific formats incorporating anti-CD16 binding domains can enhance NK cell recruitment and activation.

  • Effector cell activation: Combining anti-CCR4 treatment with cytokines that activate NK cells (IL-15, IL-2) can potentiate ADCC in vivo.

• Experimental Evaluation Methods:

  • CDC assays: Use LDH release assays with rabbit or mouse serum as complement sources to quantify cell lysis .

  • ADCC assays: Employ primary NK cells or neutrophils as effector cells and CCR4+ target cells at various effector:target ratios to measure antibody-mediated killing .

Research has demonstrated that affinity-optimized variants of humanized anti-CCR4 antibodies, such as mAb2-3 derived from mAb1567, show enhanced CDC and ADCC activities against CCR4+ tumor cells compared to their parent antibodies .

What approaches can be used to overcome potential resistance mechanisms to anti-CCR4 antibody therapy?

Addressing resistance to anti-CCR4 antibody therapy requires multifaceted strategies targeting various escape mechanisms:

• Receptor Downregulation or Mutation:

  • Bispecific antibodies: Develop antibodies targeting multiple epitopes on CCR4 or targeting CCR4 plus another relevant tumor marker.

  • Alternative chemokine receptor targeting: Combine anti-CCR4 with antibodies against other chemokine receptors expressed on target cells to prevent alternative migration pathways.

  • Epitope mapping and selection: Choose antibodies targeting conserved, functionally critical epitopes less likely to tolerate mutations.

• Immunosuppressive Microenvironment:

  • Checkpoint inhibitor combination: Pair anti-CCR4 therapy with PD-1/PD-L1 or CTLA-4 inhibitors to overcome multiple immunosuppressive mechanisms simultaneously.

  • Targeting additional immunosuppressive cells: Combine with therapies targeting other immunosuppressive populations (MDSCs, M2 macrophages).

  • Cytokine modulation: Add cytokines that promote effector T-cell function (IL-2, IL-15) or block immunosuppressive cytokines (TGF-β).

• Effector Cell Dysfunction:

  • NK cell activating strategies: Combine with IL-15 superagonists or anti-KIR antibodies to enhance NK cell responsiveness.

  • Fc optimization: Use antibody variants with enhanced Fc receptor binding as described in question 5.1.

  • Macrophage polarization: Add agents that promote M1 polarization to enhance phagocytosis of antibody-coated tumor cells.

• Monitoring Resistance Development:

  • Serial liquid biopsies: Monitor CCR4 expression and potential mutations in circulating tumor DNA.

  • Immune monitoring: Track changes in immune cell populations, particularly Tregs and effector T cells, during treatment.

  • Functional assays: Periodically assess ADCC activity with patient serum and autologous effector cells against autologous or surrogate target cells.

Research has shown that anti-CCR4 antibodies with dual mechanisms of action (signaling inhibition and immune effector engagement) may be less susceptible to resistance development compared to single-mechanism agents . Additionally, the targeting of both tumor cells and Tregs by anti-CCR4 antibodies provides multiple pathways to therapeutic efficacy, potentially reducing the impact of any single resistance mechanism.

How should researchers correlate CCR4 expression levels with anti-CCR4 antibody efficacy in preclinical models?

Establishing robust correlations between CCR4 expression and antibody efficacy requires comprehensive analytical approaches:

• Quantitative Assessment of CCR4 Expression:

  • Flow cytometry: Determine both the percentage of CCR4+ cells and the mean fluorescence intensity (MFI) to quantify receptor density . This should be performed on both tumor cells and relevant immune populations (Tregs).

  • Quantitative immunohistochemistry: Use digital pathology approaches to quantify CCR4 expression in tissue sections with spatial context.

  • Transcript analysis: Employ qRT-PCR or RNA-seq to quantify CCR4 mRNA levels, though protein expression correlation should be verified.

• Systematic Efficacy Evaluation:

  • Cell line panels: Test anti-CCR4 antibody effects across cell lines with variable CCR4 expression levels to establish threshold levels for response.

  • Patient-derived xenografts (PDXs): Evaluate efficacy across PDX models with varying CCR4 expression to capture tumor heterogeneity.

  • Dose-response relationships: For each model, determine EC50 values for functional endpoints (growth inhibition, ADCC, CDC) and correlate with CCR4 expression levels.

• Multivariate Analysis:

  • Integration of multiple factors: Analyze how CCR4 expression interacts with other variables (e.g., immune infiltration, ligand levels) to predict response.

  • Machine learning approaches: Apply computational methods to identify patterns and biomarker signatures that predict antibody efficacy beyond simple CCR4 expression.

• Dynamic Assessment:

  • Longitudinal sampling: Monitor changes in CCR4 expression over time during treatment to identify adaptive responses.

  • Functional receptor status: Assess not only expression but signaling competence of CCR4 through calcium flux or phosphorylation assays.

Research with anti-CCR4 antibodies has shown that efficacy correlates not only with CCR4 expression levels but also with functional characteristics of the antibody, such as its ability to induce CDC and ADCC . Studies have demonstrated that optimized antibodies like mAb2-3 show enhanced activity against CCR4+ cells compared to parent antibodies, highlighting the importance of both target expression and antibody engineering in determining efficacy .

What biomarkers should be evaluated to monitor anti-CCR4 antibody activity in research models?

Comprehensive biomarker assessment is essential for monitoring anti-CCR4 antibody activity across multiple dimensions:

• Target Engagement Biomarkers:

  • Receptor occupancy: Flow cytometry-based methods using competing antibodies or labeled ligands to determine the percentage of CCR4 receptors bound by therapeutic antibody.

  • Receptor downregulation: Quantify changes in surface CCR4 expression following antibody treatment.

  • Ligand displacement: Measure the antibody's ability to prevent CCL17/CCL22 binding to CCR4+ cells in ex vivo samples.

• Pharmacodynamic Biomarkers:

  • Signaling inhibition: Assess phosphorylation status of downstream signaling molecules (ERK, Akt) in response to CCL17/CCL22 stimulation of samples from treated subjects.

  • Chemotaxis inhibition: Ex vivo migration assays using cells from treated subjects to determine functional blockade of CCR4-mediated migration.

  • Complement activation markers: Measure C3a, C5a, or membrane attack complex components in serum or on CCR4+ cells to assess CDC activity.

• Immune Response Biomarkers:

  • Treg enumeration and function: Quantify changes in Treg frequency, phenotype, and suppressive function in peripheral blood and tumor tissue .

  • Effector T cell activation: Measure proliferation, cytokine production, and activation markers on effector T cells.

  • NK cell activity: Assess NK cell activation status and ex vivo ADCC activity against CCR4+ targets .

  • Cytokine/chemokine profiling: Multiplex analysis of serum cytokines and chemokines to detect shifts in immune response patterns.

• Tumor Response Biomarkers:

  • Apoptosis markers: Quantify cleaved caspase-3, PARP, or Annexin V staining in tumor samples.

  • Proliferation indices: Measure Ki-67 or BrdU incorporation to assess changes in tumor cell proliferation.

  • Immune infiltration: Characterize changes in tumor-infiltrating lymphocytes using multiplexed immunohistochemistry or flow cytometry.

Research has demonstrated that anti-CCR4 antibodies can simultaneously affect multiple biological processes, including direct tumor cell killing via CDC/ADCC and modulation of Treg function . Therefore, monitoring biomarkers across these different domains provides a more complete picture of antibody activity than focusing on any single mechanism.