Anti-Human IFNAR-1 (Anifrolumab) [Clone MEDI-546] — Fc Muted™

Research-grade Anifrolumab biosimilars serve as critical calibration standards and reference controls in pharmacokinetic bridging ELISA assays designed to measure drug concentrations in serum and plasma samples. The application of these biosimilars follows established quantitative immunoassay principles with specific adaptations for therapeutic drug monitoring.

ELISA Assay Design and Methodology

The Anifrolumab ELISA employs a quantitative competitive enzyme immunoassay technique where recombinant Human IFNAR1 is pre-coated onto microplates. In this competitive binding format, standards (prepared from research-grade biosimilars) or patient samples are premixed with biotin-labeled antibody before being pipetted into wells. The Anifrolumab in the sample competitively binds to the pre-coated IFNAR1 protein alongside the biotin-labeled Anifrolumab.

The assay utilizes a colorimetric detection method where color development occurs in inverse proportion to the amount of Anifrolumab bound in the initial competitive step. After washing steps and addition of Streptavidin-HRP followed by substrate solution, the color intensity is measured and directly correlates to drug concentration.

Calibration Standards and Quality Control

Standard Curve Generation

Research-grade biosimilars are used to create calibration standards across the validated range of 46.88 - 3,000 ng/mL, with a sensitivity threshold of 27.67 ng/mL. These standards enable accurate quantification of Anifrolumab concentrations in patient serum and plasma samples during pharmacokinetic studies.

Precision Requirements

The assay maintains strict precision criteria with both intra-assay and inter-assay precision requirements of <20%. Reference controls prepared from biosimilars are tested multiple times to validate these precision parameters, with coefficient of variation (CV) values typically ranging from 7.5% to 16.6% for intra-assay precision and 9.5% to 13.6% for inter-assay precision.

Biosimilar Specifications for PK Applications

Research-grade Anifrolumab biosimilars used in these assays are fully human monoclonal antibodies (IgG1, kappa) specifically designed to target IFNAR1. These biosimilars feature engineered modifications including the triple mutation L234F/L235E/P331S, which renders them effector-null while maintaining binding specificity. The molecular weight of 147.2 kDa and purity ≥95% ensure consistent performance as reference standards.

The biosimilars are produced in CHO cells using animal-free production methods and undergo Protein A purification. Each lot is functionally tested and validated to ensure batch-to-batch consistency critical for longitudinal pharmacokinetic studies.

Pharmacokinetic Bridging Applications

In PK bridging studies, these calibrated assays enable researchers to track Anifrolumab concentrations over time following various dosing regimens. The competitive ELISA format is particularly suitable for therapeutic drug monitoring as it can accurately quantify drug levels across the physiologically relevant concentration range encountered in clinical studies, from initial peak concentrations down to trough levels near the sensitivity limit.

The combination of well-characterized biosimilar standards and validated competitive ELISA methodology provides the analytical foundation necessary for robust pharmacokinetic assessments in both preclinical and clinical development programs.

The primary models used for in vivo administration of research-grade anti-IFNAR1 antibody to study tumor growth inhibition and characterize tumor-infiltrating lymphocytes (TILs) are murine syngeneic tumor models. These models allow for intact mouse immune systems and controlled immune-tumor interactions relevant to anti-IFNAR1 therapy.

Key details:

• Syngeneic models involve implantation of mouse-derived tumor cell lines into immunocompetent mice of the same genetic background (e.g., C57BL/6 or BALB/c). Common syngeneic tumor lines include B16F10 (melanoma), CT26 (colon), RENCA (renal), and EMT6 (breast).
• Syngeneic models are essential for studying immune modulation, including effects on TIL composition and activation following immune-targeting therapeutics such as anti-IFNAR1 antibodies.
• These models allow characterization of TILs (e.g., CD8+ T cells, myeloid-derived suppressor cells), changes in interferon signaling in the tumor microenvironment (TME), and direct assessment of tumor growth following antibody administration.
• Studies probe immune changes in vivo under various treatment regimens including anti-IFNAR1 or other immunotherapies, helping define mechanisms of response and resistance.

Humanized mouse models (mice engrafted with human immune components or PBMCs and, sometimes, human tumors) have also been utilized to approximate human immunobiology and are informative for translational research. However, most published in vivo mechanistic characterization, especially regarding anti-IFNAR1, utilizes syngeneic murine models due to:

• Availability of validated mouse-reactive anti-IFNAR1 reagents.
• The need for robust, scalable immune profiling (flow cytometry, TIL isolation, cytokine analysis).
• Immunocompetent host immunity, which is essential for full interpretation of antiviral/antitumor responses induced via type I interferon blockade.

Summary table: (Model features relevant to anti-IFNAR1 studies)

Studies reporting direct use of anti-IFNAR1 for tumor growth inhibition and TIL characterization cite syngeneic models as their primary experimental system. Additional exploration in humanized models may occur for translational validation but is less common for mechanistic immunology work.

If you need specific references or protocol details for anti-IFNAR1 antibody dosing regimens and TIL characterization in these models, please specify the tumor type or therapeutic context.

Researchers studying synergistic effects of Anifrolumab biosimilars with other checkpoint inhibitors (such as anti-CTLA-4 or anti-LAG-3 biosimilars) in complex immune-oncology models typically combine these agents to target multiple immune pathways and overcome resistance mechanisms in tumor immunity.

Combining different checkpoint inhibitors allows exploitation of their distinct mechanisms:

• Anifrolumab biosimilar blocks type I interferon (IFN) receptor signaling by binding to IFNAR1, thereby inhibiting downstream immune activation driven by type I IFNs (e.g., IFN-α, IFN-β), which are involved in immune cell maturation and cytokine production.
• Anti-CTLA-4 biosimilar inhibits CTLA-4-mediated negative regulation, enhancing T cell activation in lymphoid organs and facilitating priming and expansion of effector T cells.
• Anti-LAG-3 biosimilar targets LAG-3, which is often co-expressed with PD-1 on exhausted T cells; blocking LAG-3 can further restore T cell function and potentiate anti-tumor immunity when used with other checkpoint inhibitors.

In preclinical and translational research, these biosimilars are used together to:

• Simulate complex tumor microenvironments where multiple immune suppressive pathways operate.
• Examine additive or synergistic effects on T cell activation, proliferation, and infiltration into tumors.
• Assess the impact on cytokine profiles, immune cell phenotypes, and gene expression signatures (such as interferon signatures modulated by Anifrolumab).
• Model resistance mechanisms by combining agents that relieve different checkpoints, often leading to improved anti-tumor responses compared to monotherapy, as seen in preclinical models and early-phase studies.

Experimental Protocols Typically Involve:

• Using mouse models engrafted with human tumors and immune cells (humanized mice).
• Administering biosimilar antibodies alone and in combination, monitoring tumor growth, T cell function, and adverse events.
• Analyzing immune cell populations (flow cytometry), cytokine assays, and transcriptional profiling to determine synergistic action.

Key Insights:

• Combination approaches can overcome limits of single-agent therapies by addressing redundant or compensatory immune regulatory pathways.
• Synergy depends on the model and agents; combinations may increase efficacy, but also risk additive toxicity and immune-related adverse effects.

While the literature mainly discusses branded compounds, biosimilars are used in preclinical studies for mechanistic and comparative purposes—to ensure safety and efficacy profiles match originators before clinical evaluation.

In summary, researchers use Anifrolumab biosimilars with checkpoint inhibitors like anti-CTLA-4 or anti-LAG-3 in immune-oncology models to investigate potential synergy by co-targeting the interferon pathway and additional immune checkpoints, leading to enhanced anti-tumor immune responses in mechanistically robust experimental settings.

An Anifrolumab biosimilar can be used as a capture or detection reagent in a bridging ADA ELISA to detect anti-drug antibodies (ADAs) that a patient may develop in response to the therapeutic drug Anifrolumab. In this assay, the biosimilar, which matches the drug's antigen-binding regions, serves as a surrogate for the actual therapeutic, allowing the detection of immune responses without using clinical-grade material.

Context and Supporting Details:

• Bridging ADA ELISA design:
  This assay exploits the bivalent nature of ADAs produced in patient serum. Typically, one molecule of the biosimilar Anifrolumab (e.g., biotinylated) is used to coat a streptavidin plate or serve as a capture reagent, while a differently labeled molecule of the same biosimilar (e.g., HRP-conjugated or fluorophore-labeled) serves as the detection reagent. If a patient sample contains anti-Anifrolumab antibodies (ADAs), those ADAs "bridge" between the capture and detection biosimilar molecules, forming a complex that generates a measurable signal.

• Workflow example:

  • The patient sample is incubated in an ELISA plate where biotinylated Anifrolumab biosimilar is captured on a streptavidin-coated well.
  • Serum ADAs, if present, bind to this biosimilar.
  • HRP-labeled (or otherwise detectable) Anifrolumab biosimilar is added, binding the other arm of the ADA, completing the "bridge."
  • After washing, signal substrate is added, and the amount of reaction product indicates ADA presence and level.

• Why use a biosimilar as reagent:
  Using an Anifrolumab biosimilar ensures that the detection reagents have the same antigenic specificity as the clinical product. This is important for reliable detection of antibodies elicited against any region of the therapeutic molecule (e.g., the variable region). Biosimilars, being research-grade and engineered for high purity and validated activity, provide a practical alternative to using clinical-grade drug, which may be limited or regulated for research use only.

• Assay considerations:

  • The assay’s sensitivity depends on the quality and labeling of biosimilar reagents, as well as how well they replicate the original drug's structure.
  • Specificity controls are needed, as human sera can contain interfering substances or pre-existing antibodies that might bind non-specifically.

• Clinical utility:
  Detecting ADAs is crucial because they can impact the pharmacokinetics (how the drug is processed by the body), safety, and efficacy of Anifrolumab treatment. Anifrolumab generally shows low immunogenicity, but regular ADA testing enables clinicians to monitor for rare immune responses which could affect therapy.

Summary Table: Anifrolumab Biosimilar in ADA Bridging ELISA

By using an Anifrolumab biosimilar as both capture and detection reagents in a bridging ELISA, laboratories can effectively monitor patient immune response to the therapeutic without depleting valuable clinical drug supplies.