Anti-Human CD137 (4-1BB) (Urelumab) [Clone BMS-66513] — Fc Muted™

Research-grade Urelumab biosimilars are commonly used as calibration standards or reference controls in pharmacokinetic (PK) bridging ELISA assays to enable accurate quantification of drug concentrations in serum samples for both biosimilar and reference products. These biosimilars are selected and validated to ensure they are highly comparable to the reference antibody in terms of binding and performance characteristics.

Key aspects of their use in PK bridging ELISA:

• Single Analytical Standard: The biosimilar (Urelumab biosimilar) is used as the calibrator (standard curve) for quantifying both the test (biosimilar) and reference (originator) drug in serum samples. This practice is common because it minimizes variability and improves comparability when performing bioequivalence studies between biosimilars and reference drugs.

• Assay Validation: Before using a biosimilar as a standard, the assay undergoes rigorous validation to confirm that the biosimilar and reference drug have equivalent analytical response—in other words, the ELISA detects them with similar accuracy and precision across the quantitation range. During validation, both products are tested as quality control (QC) samples and their quantification against the biosimilar standard curve must fall within predefined equivalence criteria.

• Standard Curve Preparation: Serial dilutions of the Urelumab biosimilar are prepared in human serum to generate a standard curve with known concentrations. This standard curve is used to interpolate the concentration of drug (either reference or biosimilar) present in test samples.

• Reference Control Role: In addition to serving as the calibration standard, Urelumab biosimilars can be used as positive controls or reference reagents within the assay workflow, helping confirm consistent assay performance and analytical comparability over time.

• Bridging ELISA Format: The assay is typically a bridging format ELISA, meaning it uses the same or similar capture and detection reagents for both biosimilar and reference drug, allowing direct comparison and minimizing bias.

Essential details:

• The use of a single biosimilar calibrator is supported by regulatory and industry guidance, provided analytical comparability is demonstrated and stringent validation criteria are applied.
• This strategy is applicable regardless of whether the serum samples contain the reference or biosimilar product, so long as the method equivalence is established during assay development and validation.
• Research-grade Urelumab biosimilars (e.g., anti-CD137/4-1BB monoclonal antibodies) are frequently sold for this purpose and their purity and integrity are routinely documented (e.g., >95% by SDS-PAGE, as indicated by some vendors).

In summary, Urelumab biosimilars are integral as standardized references and calibration standards in PK bridging ELISA assays for serum drug quantitation, as long as bioanalytical equivalence between the biosimilar and reference product is established through method validation.

The primary in vivo models used to evaluate research-grade anti-4-1BB (CD137) antibodies for tumor growth inhibition and analysis of tumor-infiltrating lymphocytes (TILs) are syngeneic mouse models and humanized knock-in mouse models. Each has specific applications depending on whether the antibody is mouse-reactive or human-specific.

Key models for anti-4-1BB antibody administration and TIL characterization:

• Syngeneic mouse models (e.g., CT26, MC38, B16):
  These models involve transplanting mouse-origin tumors into immunocompetent mice of the same strain. Mouse-reactive anti-4-1BB antibodies are commonly used. Experimentation in these models allows assessment of antibody-mediated tumor growth inhibition and analysis of TILs, particularly CD8+ T cell and regulatory T cell (Treg) populations, via flow cytometry and immunohistochemistry.

  • Example: In the CT26 and MC38 models, administration of a mouse-reactive 4-1BB agonist or bispecific 4-1BB antibody resulted in significant tumor rejection and enhanced TIL responses (increased CD8+ T cells, reduced Tregs).

• Humanized 4-1BB knock-in mice (e.g., B-h4-1BB mice):
  For research-grade anti-human 4-1BB antibodies (including clones like Urelumab or Utomilumab), humanized knock-in mice engineered to express human CD137 in place of murine CD137 are required. Tumor cells (often MC38) are implanted, and anti-human 4-1BB antibodies are administered. TILs are analyzed at endpoint by flow cytometry and immunohistochemistry to quantify leucocyte populations, total T cells, CD8+ T cells, and Tregs.

  • Example: In B-h4-1BB mice bearing MC38 tumors, anti-human 4-1BB antibody treatment led to increased CD8+ T cell infiltration and reduced Treg cells within the tumor, demonstrating potent immunomodulatory activity in vivo.

Additional context:

• Fully human antibodies like Urelumab and Utomilumab are not mouse-reactive and thus require humanized models for in vivo efficacy and immune profiling.
• These models are routinely used to characterize changes in immune cell subsets (especially CD8+ and regulatory T cells) after antibody treatment, providing mechanistic insight into anti-tumor responses.

Summary Table:

These models represent the gold standard for preclinical testing of anti-4-1BB antibodies, enabling robust analysis of tumor growth inhibition and the modulation of immune cell populations in the tumor microenvironment.

Researchers use Urelumab biosimilars—which target and activate CD137 (4-1BB), a potent T-cell costimulatory receptor—in combination with other checkpoint inhibitors such as anti-CTLA-4 or anti-LAG-3 biosimilars to investigate synergistic antitumor immune responses in complex immune-oncology models. These studies are typically performed in both preclinical (animal, cell-based) and clinical (patient-based) settings.

Key approaches and mechanistic insights include:

• Rationale for Combination: CD137 activation by Urelumab enhances T cell responses (increasing cytokine production, proliferation, and survival) and boosts NK cell-mediated ADCC, while checkpoint inhibitors like anti-CTLA-4 or anti-LAG-3 relieve immune suppression by blocking inhibitory pathways. The combinatorial logic is that CD137 agonism amplifies effector cell function, whereas checkpoint blockade prevents T cell exhaustion or restores T cell activation—potentially yielding stronger, more durable antitumor responses than either agent alone.

• Mechanistic Study Designs:

  • In preclinical models, researchers often use murine tumor systems genetically engineered to mimic human immune checkpoint pathways. These allow for detailed measurement of immune cell activation, gene expression changes, cytokine release, and tumor regression in response to various dual- or triple-combination regimens.
  • For example, research on anti-PD-1/CTLA-4 and anti-PD-1/LAG-3 combinations in mice demonstrates differing immune cell dependencies: anti-PD-1/LAG-3 regimens require CD4+ T-cells for antitumor effects, reduce regulatory T cell (Treg) activity, and enhance CD8+ cytotoxic T cell function via increased CD4 helper activity; anti-PD-1/CTLA-4 primarily increases the direct activation and proliferation of cytotoxic CD8+ T cells.

• Measured Outcomes:

  • Molecular and cellular endpoints include upregulation of IFN-γ-induced genes, markers of T-cell and NK-cell activation (e.g., CD3, CD8, CXCL9, GZMB), infiltration of immune effector cells into tumors, and decreased gene signatures associated with immunosuppression.
  • Synergy is evaluated by comparison to monotherapies, seeking additive or multiplicative increases in tumor regression or immune activation (e.g., increased cytokine release, CD8+ T cell cytotoxicity).

• Clinical Translation and Limitations:

  • Early-phase clinical trials of Urelumab in combination with PD-1 inhibitors (e.g., nivolumab) or other checkpoint inhibitors often show increased expression of immune activation genes and cytokines. However, despite robust mechanistic immunologic activity, clinical antitumor responses may be modest and are sometimes limited by toxicity, especially hepatotoxicity at higher doses of Urelumab.
  • Optimal dose selection and the identification of patient subgroups most likely to benefit are ongoing challenges.

Summary Table: Mechanistic Features in Combination Regimens

Preclinical and early clinical studies thus use these combinations to dissect synergistic immune mechanisms, aiming to overcome resistance and improve therapy outcomes through rational combination strategies, though successful translation to strong clinical efficacy remains a key focus.

A Urelumab biosimilar is used in a bridging anti-drug antibody (ADA) ELISA as either the capture or detection reagent to monitor a patient's immune response against the therapeutic anti-4-1BB antibody Urelumab. This is possible because the biosimilar contains the same antigen-binding regions as the therapeutic drug, allowing it to specifically bind to any antibodies the patient has developed against Urelumab.

How it works in a bridging ADA ELISA:

• The bridging ELISA format is designed to detect ADAs by using the therapeutic antibody (here, the Urelumab biosimilar) in two roles:

  • As the capture reagent, the biosimilar is coated on the plate and will bind patient-derived ADAs if present.
  • As the detection reagent, the biosimilar is labeled (typically with an enzyme like HRP or with biotin for later detection) and will bind to another epitope on the ADA, forming a "bridge."

• When a serum sample from a treated patient (potentially containing ADAs against Urelumab) is added, any ADAs present will bridge between the immobilized biosimilar and the labeled biosimilar, allowing detection.

Key steps:

• Plate coating: The biosimilar is immobilized on wells as the capture antibody.
• Sample addition: Patient serum is applied; any ADA present will bind the biosimilar.
• Detection: A labeled version of the biosimilar is added, which binds to another ADA epitope, forming a sandwich or bridge.
• Signal development: Enzyme or dye on the labeled biosimilar generates a measurable signal, proportional to ADA quantity.

Rationale for using a Urelumab biosimilar:

• The biosimilar has identical variable regions as therapeutic Urelumab, ensuring specific detection of anti-Urelumab antibodies.
• The biosimilar is formulated for research (not therapeutic) use, ensuring no patient risk and consistent supply for immunogenicity monitoring.

Application example:

• The process is identical to bridging ELISA workflows described generally for other therapeutic antibodies—where the drug (or biosimilar) serves both as capture and detection reagents (sometimes labeled differently for each role).

In summary, by using a Urelumab biosimilar as both a capture and detection reagent in a bridging ADA ELISA, laboratories can sensitively and specifically detect anti-Urelumab immune responses in patient samples, which is critical for immunogenicity monitoring during therapy.