CCR4-5 Antibody

What is CCR4 and why is it a target for cancer immunotherapy?

CCR4 is a chemokine receptor that plays a significant role in immune cell trafficking and tumor progression. It becomes a compelling target for cancer immunotherapy for several reasons. First, elevated expression of CCR4 in tumors correlates with poor prognosis in several cancer types. Second, CCR4 is highly expressed on certain malignant cells, particularly in cutaneous T-cell lymphoma (CTCL) and renal cell carcinoma (RCC). Third, CCR4 is prominently expressed on regulatory T cells (Tregs) that can migrate to tumors expressing the CCR4 ligands CCL17 and CCL22, facilitating tumor evasion from immune surveillance . These characteristics make CCR4 a valuable therapeutic target not only for malignancies where tumor cells directly express CCR4 but also for modulating the tumor microenvironment in other cancer types.

What are the main anti-CCR4 antibodies currently used in research?

Research on anti-CCR4 antibodies has focused on several key candidates. Mogamulizumab (KW-0761) is an anti-CCR4 antibody with enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity that has been approved in Japan for treating relapsed adult T cell leukemia (ATL) and has been tested in patients with peripheral and cutaneous T cell lymphomas . The mouse anti-CCR4 monoclonal antibody mAb1567 recognizes both the N-terminal and extracellular domains of CCR4 and has been humanized for potential therapeutic development . Affi-5 is a fully human antagonistic anti-CCR4 antibody that has shown antitumor activity in preclinical renal cancer models . Each antibody has distinct binding properties and mechanisms of action that make them suitable for different research and therapeutic approaches.

How does the CCR4-5 (Affi-5) antibody differ from other anti-CCR4 antibodies?

Affi-5 is distinguished from other anti-CCR4 antibodies by several key characteristics. Unlike mogamulizumab, which primarily works through ADCC, Affi-5 functions as an antagonistic antibody that directly blocks CCR4 signaling by inhibiting the binding of its ligands CCL17 and CCL22 . This antagonistic activity has been demonstrated to inhibit the migration of RENCA cells (a renal cell carcinoma line) to murine CCL17 and CCL22. Furthermore, Affi-5 has cross-reactivity between human and murine CCR4, making it valuable for preclinical studies in mouse models . Rather than primarily targeting the malignant cells directly, Affi-5 appears to exert its antitumor effects by modulating the tumor microenvironment, particularly altering the phenotype of tumor-associated macrophages and increasing NK cell infiltration in a T cell-dependent manner .

What are the primary mechanisms of action for anti-CCR4 antibodies?

Anti-CCR4 antibodies operate through multiple mechanisms depending on their specific properties. The predominant mechanisms include:

• Antagonistic activity: Antibodies like Affi-5 block the interaction between CCR4 and its ligands (CCL17 and CCL22), inhibiting chemotaxis of CCR4-positive cells .

• Antibody-dependent cellular cytotoxicity (ADCC): Some anti-CCR4 antibodies, particularly mogamulizumab, are engineered to enhance ADCC activity, triggering immune cell-mediated killing of CCR4-positive cells through interaction with Fc receptors on natural killer cells and neutrophils .

• Complement-dependent cytotoxicity (CDC): Certain anti-CCR4 antibodies can activate the complement system, leading to the formation of membrane attack complexes and cell lysis .

• Immunomodulation: Anti-CCR4 antibodies can alter the composition and phenotype of immune cells in the tumor microenvironment, shifting the balance toward anti-tumor immunity .

These mechanisms may operate simultaneously or to varying degrees depending on the specific antibody, target cell type, and tumor microenvironment.

How does Affi-5 affect the tumor microenvironment in preclinical models?

What is the role of T cells in mediating the effects of anti-CCR4 antibodies?

T cells, particularly CD4+ T cells, play a crucial role in mediating the antitumor effects of anti-CCR4 antibodies. In studies with Affi-5, the antibody's antitumor activity was completely abolished in T cell-deficient nude mice, indicating that the adaptive immune system is essential for its efficacy . Moreover, the antibody's effects on macrophage phenotype and NK cell infiltration were also absent in these mice, suggesting that T cells are upstream mediators of these changes in the innate immune response .

When combined with a neutralizing antibody to MHCII, which disrupts CD4+ T cell function, the antitumor actions of Affi-5 were completely abrogated, further emphasizing the critical role of CD4+ T cells . Interestingly, Affi-5 treatment increased the number of CD4+ effector cells positive for IFN-γ expression in treated tumors, indicating enhanced Th1 responses that could drive the observed changes in the tumor microenvironment . These findings highlight the complex interplay between the adaptive and innate immune systems in mediating the therapeutic effects of anti-CCR4 antibodies.

How are anti-CCR4 antibodies being used in renal cell carcinoma research?

In renal cell carcinoma (RCC) research, anti-CCR4 antibodies like Affi-5 have demonstrated significant potential through several key experimental applications:

• Target validation: Studies have confirmed elevated expression of CCR4 in human RCC biopsies and abnormal levels of CCR4 ligands in RCC patient plasma, establishing CCR4 as a relevant therapeutic target .

• Preclinical efficacy testing: Using the RENCA orthotopic mouse model, researchers have demonstrated that anti-CCR4 antibody treatment significantly inhibits tumor burden as measured by tumor weight and bioluminescence compared to isotype controls . This efficacy has been documented at both 10 mg/kg and 20 mg/kg doses of Affi-5.

• Mechanism exploration: Anti-CCR4 antibodies have been used to investigate how modulating the CCR4 pathway affects the tumor microenvironment, revealing significant alterations in immune cell phenotypes and cytokine profiles .

• Combination therapy assessment: Researchers are exploring how anti-CCR4 antibodies might work in combination with other immune modulators in RCC models, based on their ability to alter the tumor microenvironment .

These applications provide valuable insights into both the therapeutic potential of anti-CCR4 antibodies in RCC and the fundamental immunological mechanisms operating in the tumor microenvironment.

What experimental approaches are used to study anti-CCR4 antibodies in T-cell lymphomas?

The study of anti-CCR4 antibodies in T-cell lymphomas employs several sophisticated experimental approaches:

• Flow cytometry analysis: Researchers use multi-parameter flow cytometry to detect CCR4 expression on malignant T-cells and to evaluate the effects of anti-CCR4 antibodies on different T-cell subpopulations .

• Cytotoxicity assays: Complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) assays are employed to assess the cell-killing potential of anti-CCR4 antibodies against lymphoma cells. These typically involve measuring the release of lactate dehydrogenase (LDH) or other markers of cell death .

• Humanization and affinity optimization: For antibodies like mAb1567, researchers perform humanization and affinity optimization to enhance therapeutic potential while reducing immunogenicity, using techniques such as CDR grafting and phage display .

• CAR T-cell development: More recently, the anti-CCR4 single-chain variable fragments (scFvs) derived from anti-CCR4 monoclonal antibodies are being used to construct chimeric antigen receptor (CAR) T cells for targeting T-cell lymphomas .

• Immunohistochemistry: This technique is used to assess CCR4 expression in patient samples and experimental models, providing insight into the distribution and level of the target .

These approaches collectively provide a comprehensive understanding of the therapeutic potential and mechanisms of anti-CCR4 antibodies in T-cell lymphomas.

How can researchers assess the potential fratricide in CCR4-targeted therapies?

Assessing potential fratricide (self-killing of therapeutic T cells) in CCR4-targeted therapies is crucial, especially when developing treatments like CAR T cells that target antigens potentially expressed on the therapeutic cells themselves. Researchers employ several methodologies:

• Subset analysis of T cells: Flow cytometry analysis to characterize which T-cell subsets express CCR4 and might be susceptible to fratricide. This includes identifying Th2, Th17, and Treg cells that typically express higher levels of CCR4 .

• Monitoring T-cell expansion: Tracking the expansion kinetics of transduced T cells (e.g., with CCR4-CAR) compared to control constructs to detect potential self-limitation of growth due to fratricide .

• Phenotypic analysis of expanding populations: Characterizing the phenotype of successfully expanding T cells to determine if certain subsets are selectively depleted during the manufacturing process .

• Functional assays post-expansion: Testing the functional capacity of the expanded T-cell products to ensure that despite potential fratricide, the remaining cells maintain their therapeutic potential .

• In vivo models: Using animal models to assess whether the fratricide observed in vitro translates to limited persistence or efficacy in vivo .

What are the key methodologies for humanizing and optimizing anti-CCR4 antibodies?

The humanization and optimization of anti-CCR4 antibodies involve several sophisticated techniques to enhance therapeutic potential while minimizing immunogenicity:

• ScFv construction: Researchers generate single-chain variable fragments (scFvs) by fusing the heavy chain variable region (VH) and light chain variable region (VL) in different orientations (H-to-L or L-to-H) connected by standard peptide linkers. This approach allows for testing multiple configurations to identify optimal binding .

• CDR grafting: The complementarity-determining regions (CDRs) from a murine antibody are transferred onto a human antibody framework, preserving the binding specificity while reducing immunogenicity .

• Framework modification: After CDR grafting, researchers may need to make strategic modifications to the human framework regions to restore the proper antibody conformation and binding affinity .

• Affinity maturation: Techniques like phage display may be employed to generate antibody variants with improved binding characteristics through directed evolution approaches .

• Format optimization: Testing different antibody formats, including full IgG constructs and scFv-Fc fusions, to identify formats with optimal binding and effector functions .

• Functional screening: Assessing humanized antibody variants for retention of binding specificity and functional properties such as antagonistic activity, CDC, and ADCC potential .

These methodologies were successfully applied to antibodies like mAb1567, resulting in humanized variants that maintained high affinity for CCR4 while exhibiting reduced immunogenicity .

How can researchers effectively evaluate the antagonistic activity of anti-CCR4 antibodies?

Evaluating the antagonistic activity of anti-CCR4 antibodies requires specific assays that assess the antibody's ability to block CCR4-ligand interactions and downstream functional consequences:

• Migration inhibition assays: Researchers can assess the antibody's ability to inhibit the migration of CCR4-positive cells (like RENCA cells) toward CCR4 ligands (CCL17 and CCL22) using transwell migration chambers. The degree of migration inhibition directly correlates with the antagonistic potency of the antibody .

• Ligand binding competition assays: Flow cytometry-based assays where fluorescently labeled CCL17 or CCL22 binding to CCR4-positive cells is measured in the presence or absence of increasing concentrations of the anti-CCR4 antibody .

• Calcium flux inhibition: Since chemokine receptor activation typically triggers calcium signaling, researchers can measure the antibody's ability to inhibit CCL17/CCL22-induced calcium flux in CCR4-expressing cells .

• Signaling pathway inhibition: Western blotting or phospho-flow cytometry to detect inhibition of downstream signaling molecules activated by CCR4, such as MAPK, Akt, or STAT proteins .

• Receptor internalization assays: Evaluating whether the antibody affects CCR4 internalization, which can be another mechanism of functional antagonism .

For Affi-5 specifically, migration inhibition assays demonstrated its ability to inhibit the movement of RENCA cells toward both murine CCL17 and CCL22, confirming its cross-species antagonistic activity .

What are the considerations for developing CCR4-targeted CAR T-cell therapies?

Developing CCR4-targeted chimeric antigen receptor (CAR) T-cell therapies involves several unique considerations due to CCR4's expression pattern and role in T-cell biology:

• ScFv selection and orientation: Researchers must test multiple anti-CCR4 scFvs in different orientations (H-to-L vs. L-to-H) to identify constructs with optimal binding and functional properties. This is critical as subtle differences in CAR design can significantly impact efficacy .

• Fratricide management: Since CCR4 is expressed on certain T-cell subsets (Th2, Th17, and Tregs), researchers must address potential fratricide during CAR T-cell manufacturing. This may involve strategies to transiently suppress CCR4 expression or selective expansion protocols .

• Costimulatory domain selection: The choice between CD28, 4-1BB, or other costimulatory domains can affect CAR T-cell persistence, cytokine release, and efficacy against CCR4+ malignancies .

• Target cell heterogeneity: Researchers must consider the heterogeneous expression of CCR4 across different T-cell malignancies and patient samples, which may necessitate combination approaches or patient selection strategies .

• On-target/off-tumor effects: Given CCR4's expression on normal T-cell subsets, researchers must carefully assess the potential for on-target/off-tumor toxicity and develop strategies to mitigate these effects .

• Functional assessment: Beyond cytotoxicity, comprehensive functional assessment of CCR4-CAR T cells should include persistence, cytokine production, exhaustion markers, and in vivo efficacy in relevant preclinical models .

These considerations highlight the complexity of developing CCR4-targeted CAR T-cell therapies and the need for careful optimization at multiple levels.

How do different anti-CCR4 antibodies compare in their mechanisms and efficacy profiles?

Different anti-CCR4 antibodies exhibit distinct mechanisms and efficacy profiles that reflect their structural and functional properties:

• Mogamulizumab (KW-0761): This defucosylated humanized antibody was designed to enhance ADCC activity and has demonstrated clinical efficacy in adult T-cell leukemia and other CCR4-positive T-cell malignancies. Its primary mechanism involves direct killing of CCR4-positive malignant cells through enhanced NK cell-mediated ADCC, with potential secondary effects on regulatory T cells .

• Affi-5: This fully human antagonistic antibody functions primarily by blocking CCR4 signaling and modulating the tumor microenvironment. Unlike mogamulizumab, Affi-5's antitumor activity in the RENCA model depends on CD4+ T cells and leads to phenotypic changes in tumor-associated macrophages and increased NK cell infiltration .

• mAb1567 and its humanized variants: This antibody recognizes both the N-terminal and extracellular domains of CCR4 and exhibits multiple mechanisms including direct cytotoxicity, CDC, and neutrophil- and NK-mediated ADCC. It has shown efficacy in CTCL models through these combined mechanisms .

The efficacy of these antibodies varies across different cancer types. Mogamulizumab has proven efficacy in hematological malignancies where malignant cells express CCR4. Affi-5 shows promise in solid tumors like RCC where modulating the immune microenvironment is crucial. The humanized variants of mAb1567 might offer advantages in settings where multiple killing mechanisms are beneficial .

What is the scientific rationale for combining anti-CCR4 antibodies with other immunotherapies?

The scientific rationale for combining anti-CCR4 antibodies with other immunotherapies is founded on their complementary mechanisms of action:

• Enhanced T-cell functionality: Anti-CCR4 antibodies can alter the balance of T-cell subsets in the tumor microenvironment, potentially enhancing the efficacy of checkpoint inhibitors like anti-PD-1/PD-L1 or anti-CTLA-4 antibodies by creating a more favorable environment for T-cell activation .

• Treg depletion: CCR4 is highly expressed on regulatory T cells (Tregs), and anti-CCR4 antibodies can potentially deplete or functionally inhibit these immunosuppressive cells. This could synergize with other immunotherapies by removing a major source of immunosuppression in the tumor microenvironment .

• Macrophage repolarization: As demonstrated with Affi-5, anti-CCR4 antibodies can shift tumor-associated macrophages from an immunosuppressive M2 phenotype toward a pro-inflammatory M1 phenotype. This could potentiate the efficacy of other immunotherapies that depend on innate immune activation .

• Cytokine profile modulation: Anti-CCR4 treatment increases the ratio of Th1 to Th2 cytokines in the tumor microenvironment, creating conditions more favorable for antitumor immunity that could enhance other immunotherapy approaches .

• Increased NK cell activity: The observed increase in NK cell infiltration following anti-CCR4 antibody treatment suggests potential synergy with therapies that enhance NK cell function, such as IL-15 agonists or antibodies targeting inhibitory NK cell receptors .

Research has suggested that combining anti-CCR4 antibodies with other immune modulators may be a valuable therapeutic approach, particularly in cancers with high CCR4 expression in the tumor microenvironment and abnormal plasma CCR4 ligand levels .

What are the emerging strategies for enhancing the specificity and safety of CCR4-targeted therapies?

Several innovative strategies are being developed to enhance the specificity and safety of CCR4-targeted therapies:

• Bispecific antibody approaches: Designing antibodies that simultaneously target CCR4 and another tumor-associated antigen could increase specificity for malignant cells while sparing normal CCR4+ cells .

• Conditional activation strategies: Developing antibody-drug conjugates or engineered T cells that require dual antigen recognition or tumor microenvironment-specific signals (such as hypoxia or acidic pH) to become fully active .

• Affinity tuning: Adjusting the binding affinity of anti-CCR4 antibodies or CAR constructs to preferentially target cells with high CCR4 expression (malignant cells) while sparing cells with lower expression (normal T cells) .

• Transient expression systems: For CCR4-CAR T-cell approaches, using RNA electroporation or other transient expression methods to limit the duration of CAR expression and associated off-tumor toxicity .

• Epitope selection: Targeting specific epitopes of CCR4 that might be differentially accessible or modified in malignant versus normal cells .

• Combination with inhibitory receptors: Incorporating inhibitory modules that can be triggered by molecules expressed on normal cells but not tumor cells, creating a safety switch to prevent damage to healthy tissues .

• Selective subset depletion: Taking advantage of the differential expression of CCR4 across T-cell subsets to selectively deplete certain populations (like Tregs) while preserving others (like cytotoxic T cells) .

These emerging strategies reflect a sophisticated understanding of CCR4 biology and immunotherapy mechanisms, aiming to maximize therapeutic benefit while minimizing unwanted toxicities.