Anti-Human IL-1RAP (IL-1R3) (Nadunolimab) – Fc Muted™

Research-grade Nadunolimab biosimilars are used as analytical standards (calibrators) or reference controls in ELISA-based PK bridging assays by serving as the known concentration reference against which drug levels in serum samples are quantified.

In a pharmacokinetic (PK) bridging ELISA, the process typically involves the following steps:

• Preparation of Calibration Standards: Serial dilutions of the biosimilar Nadunolimab are made in human (or relevant species) serum to cover the expected range of concentrations encountered in the PK study, such as 50, 100, 200, 400, 800, 1600, 3200, 6400, and 12800 ng/mL.

• Assignment of Analytical Standard: After performing method qualification to demonstrate bioanalytical equivalence between biosimilar and reference products, one standard (usually the biosimilar itself) is selected as the single analytical calibrator for the assay. This ensures consistent quantification and minimizes variability that could arise from using different standards.

• Measurement Protocol: The ELISA plate is coated with anti-Nadunolimab capture reagent; serum samples, standards (diluted biosimilar), positive controls, and negative controls are added to the wells. Detection reagents (such as a labeled anti-Nadunolimab antibody) are then used to create a measurable signal proportional to the drug concentration.

• Standard Curve Construction: The biosimilar calibration standards generate a standard curve on each plate, which is used to interpolate the concentration of Nadunolimab in unknown samples. Internal controls (positive and negative) are also included to adjust for run-to-run variability and ensure assay robustness and reproducibility.

• PK Quantification and Bioanalytical Comparability: The same standard (biosimilar Nadunolimab) is used to quantify both biosimilar and reference product concentrations in blinded serum samples—this is critical for regulatory PK bridging studies. The method is validated to ensure that it measures both products equivalently, supporting PK equivalence and bioanalytical comparability.

Key Points:

• Biosimilar Nadunolimab serves as the calibrator/reference standard to make the drug concentration measurements traceable and comparable in the ELISA.
• Method validation with appropriate controls (positive/negative, QC samples) ensures assay accuracy, precision, and reproducibility for regulatory submission.
• A single analytical standard (biosimilar) eliminates variation that could result from using more than one calibrator, providing consistent PK data for dose-response and bioequivalence assessment.

This approach meets regulatory and industry best practices by ensuring measurable equivalence of biosimilar and originator products, enabling the reliable use of research-grade Nadunolimab biosimilars for calibration and control in PK ELISA assays intended for serum sample analysis.

The primary in vivo models used to study research-grade anti-IL-1RAP (Nadunolimab) antibody for tumor growth inhibition and characterization of tumor-infiltrating lymphocytes (TILs) are mainly human tumor xenograft models (including patient-derived xenografts, PDX) in immunodeficient mice, as well as humanized mouse models. Syngeneic mouse models are generally not used because Nadunolimab is specific for human IL-1RAP, which is not present in mouse syngeneic tumor lines.

Key models:

• Human tumor xenograft models (including PDX):

  • Immunodeficient mice (such as NSG or NOD/SCID mice) are implanted with human tumor cells or tumor fragments.
  • Nadunolimab efficacy has been studied for tumor growth inhibition in these models, since only human IL-1RAP is targeted and human tumor cells express this antigen.
  • For example, strong anti-leukemic effects were shown in AML xenograft models using anti-IL-1RAP monoclonal antibodies.
  • PDX models are especially relevant for evaluating anti-tumor effects alongside standard therapies such as chemotherapy.

• Humanized immune system mouse models:

  • For meaningful characterization of TILs, models must allow for interactions between human immune effectors and tumor cells.
  • Humanized mice (mice engrafted with human hematopoietic stem cells to develop a human immune system) are sometimes used to allow for evaluation of antibody-dependent cellular cytotoxicity (ADCC) and profiling of lymphocytic and myeloid populations within tumors after Nadunolimab treatment.
  • In these models, effects on NK cells, CD8+ T cells, and myeloid cells can be analyzed in the tumor microenvironment, consistent with the reported mechanisms of Nadunolimab.

Syngeneic models:

• Murine syngeneic tumor models are not typically suitable because mouse IL-1RAP is not recognized by the humanized Nadunolimab.
• Studies often focus on human tumor/immune cell combinations for functional readouts relevant to the antibody’s specificity.

Additional details:

• Tumor-infiltrating lymphocyte characterization in these models demonstrates enhanced NK cell and T cell infiltration and activation, which is central to Nadunolimab’s mechanism via ADCC and possible synergy with checkpoint inhibitors.
• Clinical samples from treated patients are also analyzed for TILs and immune modulation, complementing preclinical in vivo results.

In summary, research-grade anti-IL-1RAP (Nadunolimab) is most commonly evaluated in immunodeficient mouse xenograft models (PDX or cell line-derived) and, less frequently, humanized mouse models, not standard murine syngeneic models, due to antibody specificity for the human target. These models are used to assess both tumor growth inhibition and TIL composition after in vivo administration.

Researchers use nadunolimab biosimilar, an anti-IL1RAP antibody, in combination studies with other checkpoint inhibitors—such as anti-CTLA-4 or anti-LAG-3 biosimilars—to investigate whether simultaneous blockade of multiple immunosuppressive pathways yields enhanced or synergistic antitumor effects in complex immune-oncology models. While direct results on the combined use of nadunolimab with these specific checkpoint inhibitors are limited, the central approach draws from the mechanistic rationale underlying multi-checkpoint immunotherapy:

• Rationale for Combination:
  Nadunolimab blocks IL1RAP, impeding IL-1–driven immunosuppressive signaling and activating NK cell-mediated tumor destruction via Antibody-Dependent Cellular Cytotoxicity (ADCC). Other checkpoint inhibitors (e.g., anti-CTLA-4 or anti-LAG-3) target distinct pathways that restrain T cell activation or function, broadening immune response profiles in tumor microenvironments.

• Experimental Design in Models:
  Researchers typically employ:

  • In vitro co-culture systems with tumor cells, immune effector cells, and combinations of antibodies to measure cytokine release, immune cell proliferation, or cytotoxic activity.
  • In vivo mouse tumor models evaluating tumor growth, immune cell infiltration, and survival outcomes following administration of nadunolimab and other checkpoint blockers.
  • Multiplex biomarker profiling (e.g., cytokines such as IL-8, immune cell markers, exhaustion markers like PD-1/LAG-3/TIM3) to dissect synergistic immune modulation and correlate with therapeutic efficacy.

• Synergistic Effects and Key Readouts:
  Studies of multi-checkpoint inhibitor combinations show that blockade of CTLA-4 and PD-1, or PD-1 and LAG-3, can enhance T cell activation and overcome tumor-mediated immunosuppression, leading to improved antitumor responses. Nadunolimab adds a complementary mechanism by targeting stromal-driven IL-1 signaling and enhancing innate immunity, making it a candidate for synergistic combinations:

  • Enhanced tumor cell killing
  • Reduction of immunosuppressive cell infiltration
  • Prolonged progression-free and overall survival in preclinical/clinical models
  • Correlation of efficacy with immune marker changes (e.g., serum IL-8 reduction, increased T cell/NK cell activation)

• Translational and Clinical Implications:
  Early-phase clinical data (e.g., nadunolimab plus pembrolizumab, an anti-PD-1), and mechanistic preclinical work, shows manageable safety and promising efficacy in disease control, supporting further exploration with other checkpoint inhibitors.

In summary, nadunolimab biosimilar is used in preclinical and emerging clinical models as part of combinatorial immunotherapy strategies, with checkpoint inhibitors such as anti-CTLA-4 or anti-LAG-3, to test for additive or synergistic immune activation and antitumor effects across complex, heterogeneous tumor microenvironments.

A Nadunolimab biosimilar can serve as either the capture or detection reagent in a bridging ADA ELISA to monitor a patient’s immune response (i.e., anti-drug antibodies, ADA) against Nadunolimab by exploiting the ability of ADAs to bind two molecules of the drug (or biosimilar) simultaneously.

Bridging ADA ELISA Basic Principle:

• In a bridging ELISA, both the capture and detection reagents are forms of the therapeutic antibody (here, Nadunolimab or its biosimilar), each tagged differently.
• Patient serum potentially containing ADA is incubated with a biotinylated Nadunolimab biosimilar (capture) and an HRP-labeled or dye-labeled Nadunolimab biosimilar (detection).
• If ADA is present, it will form a "bridge" between the capture and detection Nadunolimab, enabling quantification.

How a Nadunolimab biosimilar is used:

• Capture Reagent: The biosimilar is biotinylated and coated onto a streptavidin plate or directly adhered, to "capture" any ADA from the patient sample that recognizes Nadunolimab.
• Detection Reagent: The same or another batch of the biosimilar, labeled (e.g., with HRP), detects the ADA that was bound by the capture reagent.

Why use the biosimilar?

• Biosimilars are equivalent in epitope recognition to the reference therapeutic and are validated to perform identically in immunogenicity assessments.
• Using a biosimilar ensures consistent, reproducible reagent supply and can address cases where the original reference drug is limited or unavailable.

Assay Format Example (for Nadunolimab):

1. Plate preparation: Streptavidin-coated plates bind the biotinylated Nadunolimab biosimilar (capture).
2. Sample incubation: Patient serum is added; ADA, if present, binds the capture Nadunolimab.
3. Detection: HRP- or dye-labeled Nadunolimab biosimilar is added; if ADA bridges the two, a complex forms.
4. Signal development: Substrate is added for colorimetric or chemiluminescent detection.

Key Points:

• This format is highly sensitive for ADA with bivalent or multivalent binding capacity, which is typical.
• The use of a biosimilar must be validated to ensure comparable assay performance versus the originator.
• Specificity controls are essential to account for potential assay interference from serum components.

Summary Table: Components in Bridging ADA ELISA with Nadunolimab biosimilar

This approach directly supports immunogenicity monitoring as recommended in biosimilar development and regulatory guidelines.