Anti-Human IL-4Rα (CD124) (Dupilumab) [Clone REGN668]

Research-grade Dupilumab biosimilars are used as calibration standards or reference controls in pharmacokinetic (PK) bridging ELISA assays by serving as the quantitative benchmark to generate the standard curve against which the concentration of Dupilumab in test serum samples is measured.

Context and Supporting Details:

• Single analytical standard: In PK bridging ELISA for biosimilars, the current industry best practice is to develop a single PK assay that uses one analytical standard (often the research-grade biosimilar itself) for quantifying both the biosimilar and the reference product in serum samples. This reduces variability and avoids the need for separate assays for each product.

• Preparation of calibration curve: Serial dilutions of the research-grade Dupilumab biosimilar are spiked into the same matrix as test samples (usually blank human serum) to generate a calibration (standard) curve. These known concentrations are run in parallel with study samples in every assay.

• Measurement process:

  • The ELISA plate typically contains a capture reagent (often an anti-Dupilumab antibody or anti-IgG4).
  • The serum samples and standard curve samples containing known Dupilumab concentrations are introduced.
  • After incubation and washing steps, a detection reagent (secondary antibody) is applied, and a substrate is added for color development.
  • The optical density (OD) at a specific wavelength (e.g., 450 nm) correlates with Dupilumab concentration.
  • Unknown concentrations in test samples are interpolated from the standard curve generated with the biosimilar standard.

• Establishing comparability: Before using a biosimilar as the standard, the assay method is qualified and validated for precision, accuracy, and equivalence between the biosimilar and the reference product. Side-by-side analysis ensures that both are measured comparably in the assay.

• Reference controls: Quality control (QC) samples are also prepared using either the biosimilar or reference product at multiple concentrations to ensure ongoing assay performance.

Summary Table: Use of Biosimilars as PK Bridging ELISA Standards

Key Points:

• Using a single, well-characterized biosimilar as the standard is recommended to support PK bioequivalence assessments.
• Method validation is required to demonstrate that the biosimilar and the originator produce equivalent signals across concentrations in the context of the assay.
• This approach is aligned with regulatory expectations for minimizing assay variability in biosimilar development.

This procedure ensures accuracy, robustness, and regulatory compliance in measuring Dupilumab concentrations in clinical and preclinical PK studies.

The primary models where a research-grade anti-IL-4Rα antibody is administered in vivo to study tumor growth inhibition and characterize tumor-infiltrating lymphocytes (TILs) are syngeneic mouse models, particularly with murine tumor cell lines, and occasionally genetically engineered mouse models employing allografts.

Essential context:

• Syngeneic mouse models involve implanting mouse tumor cells into immunocompetent mice of the same genetic background, allowing for full immune system analysis, including TIL profiling. Commonly used lines include B16F10 (melanoma), RENCA (renal carcinoma), CT26 (colon carcinoma), MC38 (colon adenocarcinoma), Hepa1-6 (liver carcinoma), and EMT-6 (breast carcinoma).
• In published studies, anti-IL-4Rα antibodies such as RegN1103 (the murine homolog of the human therapeutic dupilumab) have been tested in genetically engineered or allograft models, such as the alveolar rhabdomyosarcoma (aRMS) orthotopic allograft in SHO mice and models derived from breast cancer cells.

Supporting details:

• In the referenced study, murine SHO mice (deficient in B and T cells for allograft acceptance) were orthotopically implanted with murine aRMS tumor cells and treated with a research-grade anti-IL-4Rα antibody (RegN1103). Tumor growth and metastasis were assessed; TIL composition can be characterized as described in related syngeneic model studies.
• Tumor-immune profiling of syngeneic models (e.g., B16F10, RENCA, CT26) allows immune cell composition—including TILs like CD8+ T cells, CD4+ T cells, NK cells, and myeloid-derived suppressor cells—to be studied in response to immunotherapies, which may include cytokine pathway inhibitors like anti-IL-4Rα.
• Genetically engineered or allograft mouse models may be needed for humanized antibody testing, but true “humanized” models (with partially human immune systems, e.g., NSG mice reconstituted with human PBMCs or HSCs) are rarely used for mouse-specific IL-4Rα antibodies unless the antibody cross-reacts with mouse and human IL-4Rα.

Additional relevant information:

• There is currently limited evidence of anti-IL-4Rα use specifically in humanized mouse models for TIL characterization due to receptor species specificity. Most published in vivo work employs syngeneic mouse models with anti-mouse IL-4Rα antibodies.
• Key experimental endpoints commonly include tumor growth inhibition, survival, lung and lymph node metastasis rates, and immunophenotyping of TILs via flow cytometry and histology.

Summary Table: Common Model Types Used

In summary:
Most in vivo studies administering research-grade anti-IL-4Rα antibodies to study tumor growth and TILs use syngeneic mouse models with murine tumor cell lines. Humanized models are seldom used unless the antibody is cross-reactive. TILs—including CD8+ T-cells, CD4+ T-cells, NK cells, and myeloid-derived suppressor cells—are routinely analyzed in these studies.

Researchers currently use Dupilumab biosimilars primarily to manage cutaneous immune-related adverse events (irAEs) such as eczema and bullous pemphigoid induced by checkpoint inhibitor therapies in cancer patients, rather than as direct agents for studying synergy in immune-oncology. There is limited evidence in the published literature regarding the use of Dupilumab biosimilars in combination with checkpoint inhibitors such as anti-CTLA-4 or anti-LAG-3 biosimilars to study synergistic antitumor effects in preclinical or clinical oncological models.

Context and Supporting Details:

• Primary role of Dupilumab: Dupilumab, an antibody targeting the IL-4/IL-13 pathways, is mainly used to treat atopic dermatitis and pruritus, including in cancer survivors who develop these conditions as side effects of immune checkpoint inhibitor treatment. Its non-immunosuppressive profile makes it preferable to corticosteroids, as steroids could potentially interfere with the efficacy of checkpoint inhibitors.
• Management of irAEs: In case series and reviews, Dupilumab has shown efficacy in treating immune-related cutaneous events triggered by therapies such as anti-CTLA-4, anti-PD-1, and anti-PD-L1. This has led to its consideration as a safer alternative to systemic steroids which could blunt antitumor immunity.
• Checkpoint inhibitor combinations: Synergistic antitumor effects are more rigorously studied with combinations of checkpoint inhibitors themselves (e.g., anti-PD-1/CTLA-4, anti-PD-1/LAG-3), with research focusing on their different mechanisms of immune activation and effects on populations such as CD4 and CD8 T cells. These studies often use animal models to characterize how these combinations modulate specific immune cell subsets and resistance mechanisms.

Limitations and Inference:

• There is no reported use, from available sources, of Dupilumab biosimilars combined directly with anti-CTLA-4 or anti-LAG-3 biosimilars in immune-oncology models to study synergistic efficacy against tumors. Instead, Dupilumab is used to control adverse cutaneous side effects arising from checkpoint inhibitor treatment, enabling patients to continue immunotherapy with reduced toxicity.
• The absence of data about Dupilumab's synergy with checkpoint inhibitors in tumor models likely reflects its role in affecting Th2-driven pathways, rather than direct modulation of antitumor immunity (which is driven by Th1 and cytotoxic responses targeted by checkpoint inhibitors).

Summary Table: Use of Dupilumab vs. Checkpoint Inhibitor Combinations in Immuno-Oncology Research

In conclusion, Dupilumab biosimilars are valuable for managing immunotherapy-induced skin side effects in oncology patients, supporting continued checkpoint inhibitor therapy, but are not currently used experimentally in combination with CTLA-4 or LAG-3 biosimilars to investigate antitumor synergy in immune-oncology models.

A Dupilumab biosimilar can be used as the capture or detection reagent in a bridging anti-drug antibody (ADA) ELISA to monitor a patient's immune response by taking advantage of the bivalent nature of ADAs generated against the therapeutic drug.

Key steps in a bridging ADA ELISA:

• Coating or labeling: The Dupilumab biosimilar is either immobilized on the ELISA plate (capture) or conjugated with a detection label (such as biotin or HRP).
• Sample incubation: Patient serum samples, which may contain anti-Dupilumab ADAs, are incubated so that the ADAs can bind to the immobilized Dupilumab biosimilar.
• Detection: A labeled Dupilumab biosimilar is added as the detection reagent, which binds to the remaining free antigen-binding sites of the patient's ADA, forming a "bridge" between capture and detection reagents via the ADA in the sample.
• Signal generation: The signal from the detection reagent is proportional to the amount of ADA present in the patient sample.

This assay design is termed "bridging" because the bivalent ADAs simultaneously bind to both the capture and detection molecules, producing a measurable signal only if patient anti-drug antibodies are present. Biosimilars are appropriate for this use because they possess the same or highly similar antigenic epitopes as the reference Dupilumab, ensuring ADAs generated against the drug bind equivalently to the biosimilar forms used in the assay.

Typical workflow example:

• Biotinylated Dupilumab biosimilar is captured via streptavidin on the plate.
• Patient serum (potentially containing ADAs) is added.
• HRP-labeled Dupilumab biosimilar is applied; if ADAs are present, a sandwich forms between the immobilized and HRP-conjugated biosimilars via the patient's bivalent ADA.
• Addition of chromogenic substrate leads to a color change proportional to ADA concentration.

Assay advantages:

• High specificity for patient-generated ADAs.
• Sensitivity suitable for both IgM and IgG responses.
• Relevance: The biosimilar, being highly similar to the originator product, is ideal for capturing a clinically meaningful immune response.

Take-home point: Dupilumab biosimilars provide a reliable and equivalent antigenic source compared to the reference drug, allowing them to serve as either the capture or detection agent in a bridging ELISA, ensuring robust and specific detection of patient anti-Dupilumab antibodies in immunogenicity monitoring.