Anti-Human CD194 (CCR4) (Mogamulizumab) [Clone KW-0761]

Research-grade Mogamulizumab biosimilars are commonly used as calibration standards or reference controls in pharmacokinetic (PK) bridging ELISAs to quantitatively measure drug concentration in serum samples. Their primary roles are to provide a reliable and representative standard curve and to act as a quality control to ensure assay accuracy and equivalence between the biosimilar and innovator drug.

Key aspects of their use in PK bridging ELISA:

• Calibration Standard:
  Research-grade biosimilars of Mogamulizumab, which use the same protein sequences as the therapeutic antibody, are used to prepare a set of standards at known concentrations. These are serially diluted (often in human serum) to generate a standard curve spanning the relevant range for serum measurements.

• Reference Control:
  The biosimilar is often used both as a reference control and as the analytical standard for the PK assay. This ensures consistency across all measurements and enables direct comparison of concentrations measured in clinical samples from subjects administered the innovator or biosimilar product.

• Assay Validation and Bridging:
  To demonstrate bioanalytical equivalence, a single validated PK ELISA method is developed using the biosimilar as the primary calibrator. This standard is used to quantify concentrations of both the biosimilar and innovator drugs in test samples, allowing comparison of pharmacokinetics between the products. Precision and accuracy of measurement for both the biosimilar and reference products are statistically assessed to confirm suitability as a bridging assay.

• Implementation in ELISA:
  In typical PK ELISA workflows:

  • Plates are coated with anti-idiotypic antibodies specific to Mogamulizumab's idiotype.
  • Serum samples and standards containing known biosimilar concentrations are added.
  • Detection is performed using labeled secondary antibodies.
  • The biosimilar standard generates the calibration curve, and samples are interpolated for quantification.

• Quality Control:
  Quality controls (QCs) prepared using both biosimilar and reference drug help assess inter-assay and intra-assay precision. Standards are often lyophilized for stability and require validation for sensitivity, specificity, recovery, and matrix effects.

• Regulatory Context:
  This approach aligns with industry and regulatory consensus to minimize variability by using one robust method validated for both biosimilar and innovator measurements. It supports bioequivalence studies by establishing analytical comparability in the context of PK bridging.

In summary: Mogamulizumab biosimilars are employed as calibration standards to generate the standard curve in PK ELISA, serve as reference controls to validate assay accuracy, and facilitate the quantitative comparison needed for biosimilar bridging studies. This ensures rigorous and reproducible measurement of drug concentrations in clinical serum samples during biosimilar development and therapeutic drug monitoring.

Primary Models for Anti-CD194 Antibody Studies in Tumor Immunotherapy

Syngeneic Models

Syngeneic mouse tumor models are the most common platforms for preclinical in vivo studies of immunotherapy agents, including research-grade antibodies targeting immune cell markers such as CCR4 (CD194). These models allow for the assessment of tumor growth inhibition and the characterization of tumor-infiltrating lymphocytes (TILs) in the context of a fully intact immune system.

• Diversity of Models: Syngeneic models like MC38, TC-1, CT26, RENCA, and B16F10 are frequently used due to their well-characterized baseline immune profiles, tumor-infiltrating lymphocyte (TIL) populations, and varying levels of immunogenicity. For example, CT26 and RENCA are highly immune-infiltrated, while B16F10 is poorly infiltrated, providing a spectrum for immunotherapy testing.
• Predictive Power: These models are critical for evaluating how immune checkpoint inhibitors (and other immunomodulators) affect TIL dynamics and tumor growth inhibition before clinical translation.
• Mechanistic Insights: Studies using syngeneic models, though not directly citing anti-CD194, demonstrate that manipulation of T cell subsets (e.g., via CSF1R inhibitors or anti-PD-1) leads to measurable changes in CD8⁺ and CD4⁺ T cell infiltration, regulatory T cell (Treg) proportions, and overall TIL composition. This suggests that anti-CD194 (which targets CCR4, a marker enriched on Tregs and Th2 cells) would likely be tested in similar syngeneic settings to assess effects on TILs and tumor control.

Humanized Models

Humanized mouse models (e.g., NSG mice engrafted with human immune cells and tumors) represent another approach, particularly for testing human-specific antibodies. However, there is no direct evidence in the provided literature that research-grade anti-human CD194 antibodies have been administered to humanized mice for tumor studies. Humanized models are more technically challenging and expensive, and their use is typically reserved for antibodies or therapies with strict human specificity.

Rationale for Model Selection

• Syngeneic models are preferred for initial in vivo mechanistic studies of anti-CD194 (CCR4) antibodies because they permit the interrogation of tumor-immune interactions in a genetically matched, immunocompetent host.
• TIL characterization—including changes in CD8⁺, CD4⁺, and Treg populations—is routinely performed in these models using flow cytometry, immunohistochemistry, and transcriptional profiling.
• Perturbation of CCR4⁺ cells (e.g., depletion or modulation) is hypothesized to alter the balance of TIL subsets, potentially enhancing antitumor immunity by reducing Treg-mediated suppression or shifting T helper polarization.

Summary Table

Conclusion

Syngeneic mouse tumor models (e.g., MC38, CT26, RENCA) are the primary in vivo systems where research-grade antibodies (such as anti-CD194) would be administered to study tumor growth inhibition and TIL dynamics, given their established use in immunotherapy research and robust capacity for immune profiling. Humanized models are theoretically possible but are not documented in the current literature for this specific application. Syngeneic models allow for detailed, reproducible analysis of how targeted immune modulation (e.g., anti-CD194) reshapes the tumor immune microenvironment and influences therapeutic outcomes.

Mogamulizumab in Combination with Checkpoint Inhibitors: Research Strategy and Findings

Mechanistic Rationale

Mogamulizumab is a monoclonal antibody targeting CCR4, a receptor expressed on regulatory T-cells (Tregs) and certain malignant T-cells. By binding CCR4, Mogamulizumab not only blocks T-cell migration but also depletes Tregs via antibody-dependent cellular cytotoxicity (ADCC), potentially reducing immune suppression in the tumor microenvironment and enhancing antitumor immunity. Checkpoint inhibitors, such as anti-CTLA-4 or anti-LAG-3 antibodies, act by releasing brakes on cytotoxic T-cells, further amplifying immune responses against tumors.

The hypothesis behind combining Mogamulizumab with checkpoint inhibitors is that simultaneous depletion of immunosuppressive Tregs (via Mogamulizumab) and reinvigoration of cytotoxic T-cells (via checkpoint blockade) could lead to synergistic antitumor effects—enhancing response rates and overcoming resistance seen with monotherapies.

Experimental Approaches

Preclinical and Clinical Models

• In vitro and in vivo studies: Researchers use murine or humanized mouse models of solid tumors or hematologic malignancies to test the combination. Tumor growth, immune cell infiltration (e.g., CD8+ T-cells, Tregs), and cytokine profiles are analyzed to assess synergy.
• Clinical trials: Phase I/II trials in humans, often in neoadjuvant or advanced settings, evaluate safety, pharmacodynamics (e.g., Treg depletion, CD8+ T-cell influx), and preliminary efficacy (response rates, progression-free survival).
• Biomarker-driven studies: Flow cytometry, immunohistochemistry, and gene expression profiling are employed to correlate immune modulation with clinical outcomes.

Example from Recent Research

A phase I trial combined Mogamulizumab with checkpoint inhibitors in the neoadjuvant setting. While Treg depletion was consistently observed in blood and tumor tissue, the clinical benefit was modest, with partial responses in a minority of patients and stable disease in others. Interestingly, increases in tumor-infiltrating lymphocytes correlated with better pathological response, suggesting that immune contexture matters. However, synergistic enhancement of therapeutic efficacy was not clearly demonstrated, possibly due to off-target depletion of CCR4-expressing antitumor effector cells or induction of other immunosuppressive populations.

Observed Effects and Limitations

Key Findings

• Treg depletion: Mogamulizumab effectively reduces Tregs in peripheral blood and tumors, which is a prerequisite for relieving immune suppression.
• Increased CD8+ T-cell infiltration: In some models, combining Mogamulizumab with checkpoint inhibitors leads to higher CD8+ T-cell presence in tumors, associated with improved outcomes.
• Modest clinical synergy: Despite strong immunological rationale, clear synergistic clinical efficacy (e.g., dramatically improved response rates) has not been consistently observed. This may be due to complex feedback mechanisms, such as depletion of CCR4-positive antitumor T-cells or compensatory induction of alternative immunosuppressive cells.

Challenges and Considerations

• Sample size and follow-up: Many studies have small cohorts and short follow-up, limiting statistical power and ability to detect rare synergistic effects.
• Tumor heterogeneity: Response may depend on the specific immune context of the tumor, highlighting the need for biomarker stratification.
• Dosing and sequencing: Optimal timing and dosing of combination therapies remain to be defined to maximize benefit and minimize toxicity.

Future Directions

Researchers continue to explore Mogamulizumab-based combinations in more complex immune-oncology models, including:

• Multi-omics approaches to identify predictive biomarkers of synergy.
• Novel checkpoint targets (beyond PD-1/PD-L1/CTLA-4) to test in combination.
• Mechanistic studies to disentangle the effects of Treg depletion versus off-target immune modulation.

Conclusion

Researchers use Mogamulizumab biosimilars with checkpoint inhibitors (e.g., anti-CTLA-4, anti-LAG-3) to test whether simultaneous Treg depletion and T-cell activation can yield synergistic antitumor activity. While preclinical and early clinical data show immune modulation (Treg depletion, CD8+ T-cell influx), clear clinical synergy remains elusive, underscoring the complexity of immune-oncology interactions and the need for further mechanistic and biomarker-driven studies.

In the context of immunogenicity testing, using a Mogamulizumab biosimilar as a capture or detection reagent in a bridging ADA ELISA involves several steps to monitor a patient's immune response against the therapeutic drug. However, the specific use of Mogamulizumab in this context is not detailed in the provided search results. Instead, I will outline a general process for using a biosimilar monoclonal antibody in a bridging ADA ELISA, which can be adapted for Mogamulizumab:

General Steps for Using a Biosimilar in a Bridging ADA ELISA

1. Preparation of the Biosimilar Reagent: The Mogamulizumab biosimilar would need to be prepared for use as either a capture or detection reagent. This involves conjugating the biosimilar to a suitable label, such as biotin or enzyme (e.g., horseradish peroxidase), depending on the desired detection method.

2. ELISA Setup:

   • Capture Reagent: If the biosimilar is used as a capture reagent, it would be immobilized onto an ELISA plate. This step allows the ELISA to capture antibodies or immune complexes that bind to the biosimilar.
   • Detection Reagent: If the biosimilar is used as a detection reagent, it would be added after the capture step. The detection reagent, labeled with a marker like HRP, would bind to the captured antibodies or immune complexes, allowing for detection.

3. Assay Procedure:

   • Sample Preparation: Patient serum or plasma samples would be prepared and added to the ELISA plate to allow anti-drug antibodies (ADAs) to bind to the immobilized capture reagent.
   • Washing and Blocking: Unbound materials are washed away, and any non-specific binding sites are blocked to reduce background noise.
   • Detection: The labeled detection reagent (if using the biosimilar for detection) is added, and after incubation, the ELISA substrate (e.g., TMB) is added to produce a colorimetric signal that indicates the presence and concentration of ADAs.

4. Interpretation: The signal intensity corresponds to the amount of ADAs present in the patient's samples. This information is crucial for assessing the patient's immune response to the therapeutic drug.

For specific details on using Mogamulizumab or its biosimilar in such an assay, additional research or specific protocols would be necessary, as the search results do not provide direct information on this application.

Considerations for Mogamulizumab Biosimilar

Mogamulizumab is a humanized monoclonal antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) activity. If used in an ADA ELISA, considerations should include:

• Specificity and Sensitivity: The assay should be optimized to detect specific ADAs against Mogamulizumab while minimizing cross-reactivity with other antibodies.
• Expression System: Differences in the expression system used for the biosimilar compared to the originator could affect immunogenicity and need to be carefully considered.
• Post-translational Modifications: Variations in glycosylation or other post-translational modifications could influence both the drug's activity and its recognition by the immune system.