CCR4-6 Antibody

What are CCR4 and CCR6, and what cell types predominantly express these chemokine receptors?

CCR4 and CCR6 are chemokine receptors that play crucial roles in immune cell trafficking and function. CCR4 is highly expressed on specific T cell subsets, including T-regulatory cells (Tregs) and cutaneous T-cell lymphoma (CTCL) cells, making it a potential therapeutic target for cancer immunotherapy . This receptor is associated with skin-homing capacity in both normal and malignant T cells . CCR4 is the receptor for chemotactic ligands CCL17 and CCL22 .

CCR6 is another chemokine receptor that, in combination with CCR4 or CXCR3, defines distinct CD4+ T cell subsets with specific lineage commitments. According to comprehensive phenotyping studies, CCR4+CCR6+ T cells express cytokines and transcription factors specific for the Th17 lineage, while CXCR3+CCR6+ cells represent Th1Th17 lineage cells . These different combinations of chemokine receptors serve as useful surface markers to identify functionally distinct T cell populations.

The expression patterns of these receptors have significant implications for understanding disease pathogenesis. For example, in HIV infection, cells expressing CCR6 (both CCR4+CCR6+ and CXCR3+CCR6+ subsets) harbor higher levels of integrated HIV DNA in treatment-naive subjects .

How are anti-CCR4 antibodies developed and humanized for research and therapeutic applications?

Anti-CCR4 antibodies can be developed through several approaches, with phage display technology and hybridoma techniques being predominant methods. One established approach involves immunizing mice with peptides corresponding to human CCR4 epitopes, followed by humanization of the resulting murine antibodies . For example, the therapeutic antibody mogamulizumab (KW-0761) was developed by humanizing murine MAb KM2160, which was generated against a peptide corresponding to N-terminal amino acid residues 2–29 of human CCR4 .

A more direct approach utilizes human non-immune antibody libraries and in vitro display technology. This method involves screening phage-displayed human naïve antibody libraries on intact CCR4+ cells to select CCR4-specific binders . This approach eliminates the need for subsequent humanization steps since the antibodies are derived from human sequences originally.

For humanization of murine antibodies, researchers typically employ complementarity-determining region (CDR) grafting methods. This process involves transferring the antigen-binding regions (CDRs) from a murine antibody onto human antibody framework regions . Following this initial grafting, affinity maturation is performed through techniques such as site-directed mutagenesis to optimize binding properties while maintaining the humanized nature of the antibody .

The development process typically includes validation steps such as binding affinity determination, epitope mapping, and functional assays to ensure that the humanized antibody retains the desired specificity and biological activity of the original murine antibody .

What techniques are most effective for detecting and quantifying CCR4 and CCR6 expression in clinical samples?

Multiple complementary techniques can be employed for detecting and quantifying CCR4 and CCR6 expression in clinical samples:

Flow Cytometry Analysis:
This is the gold standard for cellular phenotyping, allowing researchers to quantify both the percentage of cells expressing these receptors and the expression level (median fluorescence intensity) on individual cells. In clinical studies, flow cytometry has been used to track changes in CCR4+ cell populations before and after therapeutic interventions . For example, in CTCL patients treated with mogamulizumab, flow cytometry revealed reduction in CCR4+ malignant T cells from 83.7% to 25.2% following treatment .

Quantitative Real-Time PCR:
This technique enables measurement of CCR4 and CCR6 mRNA expression levels in peripheral blood mononuclear cells (PBMCs). The methodology typically involves:

• RNA extraction from PBMCs using commercial kits (e.g., RNeasy Mini Kit)

• cDNA synthesis using reverse transcriptase

• Quantitative PCR using preformulated primers and probes

• Data analysis with appropriate housekeeping genes as controls (e.g., GAPDH)

Studies have shown correlation between CCR4 mRNA levels and absolute numbers of circulating CCR4+ T cells in patients, validating this approach .

Immunohistochemical Staining:
For tissue samples, immunohistochemistry provides spatial information about CCR4 and CCR6 expression. The procedure typically employs:

• Formalin-fixed, paraffin-embedded tissue sections

• Antigen retrieval methods

• Primary antibodies specific for CCR4 or CCR6

• Detection systems such as the DAKO EnVision+ System (DAB)

• Counterstaining for cellular context

This technique allows assessment of the percentage of infiltrating lymphocytes expressing these receptors in different tissue compartments.

How do T cell subsets expressing different combinations of CCR4 and CCR6 differ in their functional properties?

T cell subsets defined by different combinations of CCR4 and CCR6 demonstrate distinct functional properties related to cytokine production, susceptibility to viral infection, and tissue-homing capabilities:

Cytokine Production Profiles:

• CCR4+CCR6+ T cells: Express cytokines and transcription factors specific for the Th17 lineage.

• CCR4+CCR6− T cells: Produce cytokines consistent with a Th2 profile.

• CXCR3+CCR6+ T cells: Represent the Th1Th17 lineage and are major producers of TNF-α and CCL20.

• CXCR3+CCR6− T cells: Express a Th1 cytokine profile with a decreased TNF-α/IL-10 ratio compared to other subsets .

HIV Permissiveness:
Different CCR4/CCR6 subsets show varied permissiveness to HIV infection, correlating with their expression of HIV co-receptors:

• CCR4+CCR6+ and CXCR3+CCR6+ T cells express both CCR5 and CXCR4 HIV co-receptors and are highly permissive to both R5 and X4 HIV replication.

• CCR4+CCR6− T cells express CXCR4 but not CCR5 and are permissive to X4 HIV only.

• CXCR3+CCR6− T cells express both co-receptors but demonstrate relative resistance to both R5 and X4 HIV in vitro .

Tissue-Homing Potential:

• CCR4+CCR6+ and CXCR3+CCR6+ T cells exhibit gut- and lymph node-homing potential .

• CCR4 expression is particularly associated with skin-homing capacity, which is relevant in conditions like cutaneous T-cell lymphoma .

Role in Viral Persistence:
CCR6+ T cells (including both CCR4+CCR6+ and CXCR3+CCR6+ subsets) harbor higher levels of integrated HIV DNA in treatment-naive HIV-infected subjects compared to CCR6- T cells, suggesting their role as viral reservoirs .

These functional differences make these T cell subsets important targets for therapeutic interventions in various disease contexts, from HIV infection to T cell malignancies.

What experimental approaches can be used to evaluate antibody-dependent cellular cytotoxicity (ADCC) of anti-CCR4 antibodies?

Evaluating ADCC of anti-CCR4 antibodies requires well-designed experimental systems that assess the ability of these antibodies to engage immune effector cells for targeted killing of CCR4-expressing cells. The following approaches are methodologically robust:

Standard ADCC Assay with Primary NK Cells:

• Target cell preparation: CCR4+ tumor cell lines (e.g., Mac-1 cells) are labeled with calcein-AM or alternative viability indicators.

• Effector cell isolation: Primary NK cells are isolated from peripheral blood of healthy donors using negative selection techniques.

• Co-culture setup: Target and effector cells are co-cultured at various effector-to-target (E:T) ratios (typically ranging from 5:1 to 50:1) in the presence of different concentrations of anti-CCR4 antibodies.

• Cytotoxicity quantification: After 4-hour incubation, cell lysis is measured using either:

  • Release of intracellular labels (e.g., calcein-AM, 51Cr)

  • Lactate dehydrogenase (LDH) release assay

• Calculation: Specific lysis percentage is calculated using the formula:
  % specific lysis = [(E - SE - S)/(TM - S)] × 100
  Where E = released LDH from E/T culture with antibody; SE = spontaneous released LDH from effectors; S = spontaneous released LDH from targets; TM = maximum released LDH from lysed targets .

Neutrophil-Mediated ADCC:
Similar protocol to NK cell ADCC, but using isolated neutrophils as effector cells, which provides insights into an alternative cellular mechanism of ADCC .

Antibody Engineering Considerations:
The Fc region of anti-CCR4 antibodies can be modified to enhance ADCC activity:

• Production of defucosylated IgG1 (as in the case of mogamulizumab/KW-0761)

• Expression in mammalian cells with kifunensine, which inhibits glycosylation processing .

In Vivo Models:

• Human tumor xenograft models in immunodeficient mice reconstituted with human NK cells

• Assessment of tumor growth inhibition and survival benefit

• Correlation with in vitro ADCC potency .

These approaches provide comprehensive evaluation of the ADCC potential of anti-CCR4 antibodies, which is crucial for their development as cancer therapeutics.

How can researchers optimize affinity maturation for anti-CCR4 antibodies?

Affinity maturation for anti-CCR4 antibodies involves systematic optimization strategies to enhance binding properties while maintaining specificity and functional activity. Based on successful approaches in the field, the following methodological framework can be employed:

Phage Display-Based Affinity Maturation:

• Creation of focused mutant libraries:

  • Target complementarity-determining regions (CDRs) for mutagenesis, particularly CDR3 regions which often contribute most significantly to binding

  • Use site-directed mutagenesis with degenerate primers or error-prone PCR with controlled mutation rates

  • Create libraries with diversity focused on key binding residues identified through structural or computational analysis

• Stringent selection strategies:

  • Implement competitive elution using increasing concentrations of unlabeled CCR4 ligands (CCL17, CCL22)

  • Employ decreasing concentrations of target antigen across selection rounds

  • Use cell-based selection on intact CCR4+ cells rather than isolated protein to maintain native conformation of the receptor

• High-throughput screening:

  • Develop rapid screening assays using flow cytometry to evaluate binding to CCR4+ cell lines

  • Compare median fluorescence intensity values across antibody variants

  • Analyze dose-response curves using appropriate binding equations (e.g., "one site - total binding")

Functional Validation of Improved Variants:
Each affinity-matured variant should be evaluated for:

• Competitive inhibition of natural ligand binding (CCL17 and CCL22)

• Inhibition of ligand-induced signaling and chemotaxis

• Maintenance or enhancement of effector functions (ADCC, CDC)

• Cross-reactivity with non-human primate and mouse CCR4 (if relevant for preclinical studies)

Structural Considerations:

• Maintain the structural integrity of the antibody

• Avoid introducing destabilizing mutations

• Consider the impact of mutations on manufacturability parameters like expression level and stability

This systematic approach to affinity maturation was successfully demonstrated in the development of anti-CCR4 antibodies like mAb2-3, which showed improved affinity and stronger CDC and ADCC activities against CCR4+ tumor cells following affinity maturation of humanized mAb1567 .

What methodologies are most effective for assessing the impact of anti-CCR4 antibodies on chemokine-induced cell migration?

Assessing the impact of anti-CCR4 antibodies on chemokine-induced cell migration requires robust methodologies that can quantify both the inhibitory potency of the antibodies and their mechanism of action. The following approaches represent effective research strategies:

Transwell Migration Assays:
This is the gold standard for quantifying chemotaxis inhibition:

• Setup: Use transwell chambers with appropriate pore size (typically 5-8 μm for T cells)

• Cell preparation: Label CCR4+ cells with fluorescent dyes or use cells expressing fluorescent proteins

• Antibody pre-treatment: Incubate cells with various concentrations of anti-CCR4 antibodies

• Chemokine gradient: Add CCR4 ligands (CCL17 or CCL22) to the lower chamber

• Quantification: After 2-4 hours, count migrated cells using flow cytometry or fluorescence plate readers

• Analysis: Calculate percent inhibition compared to control conditions and determine IC50 values

Real-time Cell Migration Monitoring:
More advanced approaches include:

• Time-lapse microscopy to track individual cell movements in response to chemokine gradients

• Automated image analysis to quantify migration parameters including:

  • Velocity

  • Directionality

  • Persistence of movement

  • Morphological changes during migration

Signaling Inhibition Assays:
To understand the mechanism of migration inhibition:

• Calcium flux assays: Measure rapid intracellular calcium mobilization triggered by CCL17/CCL22 binding to CCR4

• Phosphorylation analysis: Western blotting or phospho-flow cytometry to assess inhibition of downstream signaling molecules (e.g., Akt, ERK1/2)

• Receptor internalization: Flow cytometry-based assessment of CCR4 surface expression following ligand exposure

In Vivo Migration Models:
For more physiologically relevant assessment:

• Adoptive transfer of labeled CCR4+ cells in mouse models

• Treatment with anti-CCR4 antibodies that cross-react with mouse CCR4

• Analysis of tissue distribution, particularly to sites with high expression of CCL17/CCL22

These methods collectively provide comprehensive evaluation of how anti-CCR4 antibodies affect cellular migration, which is particularly relevant for therapeutic applications targeting T regulatory cells or malignant T cells that rely on CCR4-mediated homing .

How do anti-CCR4 antibodies affect T regulatory cells in different disease contexts?

Anti-CCR4 antibodies have significant and context-dependent effects on T regulatory cells (Tregs) across various disease settings. Understanding these effects is crucial for therapeutic applications:

Effects in Cutaneous T-Cell Lymphoma (CTCL):
In CTCL patients treated with the anti-CCR4 antibody mogamulizumab:

• Significant reduction in circulating Tregs is observed regardless of clinical response

• The percentage of Tregs decreases from 3.4% to 0.7% of CD4+ T cells (p < 0.01)

• Absolute Treg counts decrease from 68.3/μL to 6.9/μL (p < 0.01)

• CCR4 expression on remaining Tregs is markedly reduced, with CCR4+ Tregs decreasing from 87.9% to 31.7% (p < 0.01)

Mechanism of Treg Depletion:
Anti-CCR4 antibodies deplete Tregs through multiple mechanisms:

• Antibody-dependent cellular cytotoxicity (ADCC) mediated by NK cells against CCR4+ Tregs

• Complement-dependent cytotoxicity (CDC)

• Inhibition of CCR4-mediated trafficking, preventing Tregs from reaching tumor sites

Functional Impact on Immune Responses:
Depletion of CCR4+ Tregs by anti-CCR4 antibodies results in:

• Reduced immunosuppressive activity in the tumor microenvironment

• Enhanced anti-tumor immune responses

• Abrogation of Treg-mediated suppression in T-cell proliferation assays

Disease-Specific Considerations:

• In CTCL: Dual benefit from direct targeting of malignant CCR4+ T cells and depletion of immunosuppressive Tregs

• In solid tumors: Primarily beneficial through reduction of tumor-infiltrating Tregs, which may facilitate tumor cell evasion from immune surveillance

• In inflammatory conditions: Potential risk of exacerbating inflammation through Treg depletion

Monitoring Treg Effects:
Comprehensive assessment includes:

• Flow cytometric quantification of CD4+CD25+Foxp3+ Tregs

• Measurement of Foxp3 mRNA expression in peripheral blood

• Immunohistochemical assessment of Foxp3+ cells in tissue specimens

• Functional suppression assays to assess residual Treg activity

This multi-faceted effect on Tregs represents an important mechanism by which anti-CCR4 antibodies may exert therapeutic effects beyond direct targeting of CCR4+ malignant cells, particularly in the context of enhancing anti-tumor immunity.