Anti-Human CD137 (4-1BB) (Urelumab) [Clone BMS-66513]

Research-grade Urelumab biosimilars are used as calibration standards (analytical standards) or reference controls in pharmacokinetic (PK) bridging ELISAs to measure drug concentrations in serum by providing a consistent, well-characterized antibody to generate the standard curve and as controls in assay validation, ensuring reliable quantitation of both biosimilar and reference versions of the therapeutic.

In a PK bridging ELISA for therapeutic antibodies (such as Urelumab):

• The biosimilar is typically chosen as the assay calibrator (standard): Serial dilutions of the research-grade Urelumab biosimilar are prepared in a matrix that matches the study samples (e.g., human or monkey serum), creating a standard curve for quantification.
• Serum samples from study subjects (preclinical models or patients) are tested in parallel with this standard curve, and the concentration of Urelumab in those samples is interpolated using the biosimilar-derived curve.
• Reference controls (often consisting of known concentrations of the biosimilar and, if available, the originator molecule) are included as quality control (QC) samples to assess precision and accuracy of the assay within each plate and across runs.
• A single PK assay using the biosimilar as calibrator enables quantification of both the biosimilar and reference (originator) product in all samples, directly supporting PK similarity studies and minimizing method-to-method variability.

Key details for best practices:

• Bioanalytical equivalence of the assay for both the biosimilar and the originator must first be demonstrated by testing both molecules across relevant concentrations and showing comparable response, typically within a predefined equivalence range (e.g., 0.8–1.25 for mean measured concentration ratios).
• Once comparability is established, the biosimilar serves as the sole analytical standard for all data generation during method validation and sample analysis.
• Research-grade Urelumab biosimilars are produced to high purity and specificity standards (generally >95% purity, <0.5 EU/mg endotoxin), and are functionally equivalent to the therapeutic antibody for ELISA purposes.

In summary, research-grade Urelumab biosimilars are used:

• As the standard curve calibrator for quantitative PK analysis in bridging ELISAs.
• As reference controls/QC samples during validation to verify assay performance for both biosimilar and originator molecules.
• To ensure methodological consistency and reliable cross-comparison between different product lots and clinical sample types, which is required in regulatory-compliant PK bioanalytical workflows.

The primary in vivo models where research-grade anti-4-1BB (CD137) antibodies are administered to study tumor growth inhibition and analyze tumor-infiltrating lymphocytes (TILs) are syngeneic mouse tumor models. Humanized mouse models are also used to a lesser extent, particularly when evaluating human-specific antibodies or human T cell function.

Key Details and Supporting Evidence:

• Syngeneic mouse tumor models are the standard preclinical platform for testing anti-4-1BB antibodies. In these models, tumor cell lines derived from the same genetic background as the host mouse (most often C57BL/6 or BALB/c) are implanted, preserving a functional mouse immune system. Examples include the CT26 (colon carcinoma), MC38 (colon adenocarcinoma), and B16 (melanoma) models. Administration of mouse-reactive anti-4-1BB antibodies leads to significant tumor growth inhibition, frequently accompanied by increased proliferation and activation of cytotoxic CD8+ T cells and NK cells within the tumor microenvironment, as well as changes in regulatory T cell populations.

• Characterization of TILs in these models is standard. Studies report that anti-4-1BB antibody treatment expands tumor-reactive CD8+ TILs, increases IFN-γ production, and decreases suppressive regulatory T cell infiltration. Some studies use multicolor flow cytometry or single-cell sequencing to detail these immune landscape changes.

• Humanized mouse models—where human immune cells (PBMCs or CD34+ HSCs) are engrafted into immunodeficient mice—are also employed, especially for evaluating human-specific anti-4-1BB antibodies or bispecific antibodies (like anti-4-1BB×PD-L1 BsAbs). Humanized models are particularly beneficial for investigating the effect of these agents on human TILs ex vivo or in vivo, but logistical complexity, cost, and incomplete reconstitution of all immune components often limit their widespread usage compared to syngeneic systems.

Summary of findings:

• Syngeneic mouse models remain the benchmark for in vivo anti-4-1BB research, providing robust settings for immunotherapy evaluation and TIL studies.
• Humanized models are primarily used for testing human antibodies or when human immune function is required, typically in translational studies.
• Both model types permit comprehensive TIL characterization, informing mechanisms of anti-tumor efficacy.

If you need specific tumor models or cell line recommendations, or want details on TIL phenotyping techniques used in these settings, further information can be provided.

Researchers study the synergistic effects of Urelumab biosimilars with other checkpoint inhibitors (such as anti-CTLA-4 or anti-LAG-3 biosimilars) by combining agents with complementary mechanisms in complex immune-oncology models, including both preclinical (e.g., mouse models) and clinical settings.

In combination strategies:

• Urelumab is an agonist antibody targeting CD137 (4-1BB), providing a potent costimulatory signal that boosts T-cell activation, cytokine production, survival, and proliferation, and enhances NK cell-mediated cytotoxicity.
• When combined with checkpoint inhibitors such as anti-PD-1 (e.g., nivolumab), Urelumab increases the expression of immune and inflammatory genes in tumors, especially upregulating interferon-γ and key cytotoxic T cell (CD8) and NK cell signatures, which reflect a more inflamed and immunoactive tumor microenvironment.
• Checkpoint inhibitors like anti-CTLA-4 and anti-LAG-3 block inhibitory signals on T cells, but with different immunological foci:
  • Anti-CTLA-4 mainly acts in lymphoid tissues, enhancing the priming and proliferation of activated T cells.
  • Anti-LAG-3 and anti-PD-1/PD-L1 act more within the tumor, releasing exhaustion "brakes" on effector T cells.

Methodology in synergy studies:

• In vitro assays: Use co-culture systems with tumor cells and primary immune cells to assess T-cell activation, cytokine secretion, and tumor killing.
• In vivo mouse models: Transplant tumors into immunocompetent mice and treat with combinations (e.g., Urelumab + anti-CTLA-4). Measure tumor regression, T-cell infiltration, cytokine profiles, and survival.
  • For example, models have shown that anti-PD-1/LAG-3 combinations depend on the presence of CD4 helper T cells, whereas anti-PD-1/CTLA-4 drives more direct CD8 cytotoxic activity.
• Clinical and translational studies: Analyze tumor biopsies and blood before and after therapy in patients treated with combination regimens to profile gene expression changes, immune cell subsets, and cytokine/chemokine levels.
  • Increase in interferon-responsive genes, CD8 T cell markers (e.g., CD8, GZMB), and inflammatory cytokines after Urelumab+Nivolumab or Urelumab+Cetuximab therapy indicates immune system activation.

Rationale for synergy:

• These combinations are hypothesized to have additive or synergistic antitumor effects by simultaneously:
  • Augmenting T-cell activation (via CD137 agonism with Urelumab).
  • Releasing inhibitory signals (via CTLA-4, PD-1, or LAG-3 blockade).
• Targeting distinct checkpoints can overcome the limitations of monotherapies, increase tumor immunogenicity, and improve clinical outcomes.

Safety considerations:

• Dose selection and toxicity, particularly risk of liver toxicity (transaminitis), remain important for combination regimens; Urelumab exhibits dose-dependent adverse events and is studied at doses balancing efficacy and tolerability.

In summary, Urelumab biosimilar combinations with other checkpoint inhibitors are tested in experimental and clinical models to harness complementary immune-modulating pathways, enhance T-cell and NK cell responses, and improve antitumor activity beyond what single agents achieve.

A Urelumab biosimilar can be used as a capture or detection reagent in a bridging anti-drug antibody (ADA) ELISA to monitor a patient’s immune response by leveraging its structural similarity to the therapeutic drug, Urelumab. The bridging ELISA format is widely used for this purpose because it is highly sensitive for the detection of antibodies against biologic drugs.

Essential context and mechanism:

• In a bridging ADA ELISA, the principle relies on the ability of bivalent (two-armed) anti-drug antibodies (ADAs) in patient serum to simultaneously bind two identical drug molecules: one immobilized on the plate (capture) and another labeled for detection.
• A biosimilar of Urelumab, which has the same antigen-binding region as the therapeutic antibody but is made specifically for research use and not therapeutic purposes, is ideal because it mimics the therapeutic target and interacts identically with ADAs generated in response to Urelumab treatment.

Typical steps using a Urelumab biosimilar in a bridging ELISA:

• The Urelumab biosimilar is immobilized on the ELISA plate as the capture reagent.
• Patient serum is added. If anti-Urelumab antibodies (ADAs) are present, they will bind to the immobilized Urelumab biosimilar.
• After washing, biotinylated or HRP-labeled Urelumab biosimilar is added as the detection reagent. This will bind to another site on the captured ADA, forming a “bridge” between the capture and detection reagents (hence the name “bridging ELISA”).
• The presence of these complexes is revealed by adding a substrate for HRP (if HRP-labeled detection is used), generating a measurable signal proportional to the amount of ADA in the sample.

Advantages:

• The use of a biosimilar ensures that any antibody raised against the original drug will also recognize the capture/detection molecule, ensuring high assay sensitivity and cross-reactivity—crucial for reliable monitoring in patient samples.
• Since the biosimilar is engineered for research, it is not typically subject to the regulatory restrictions of the original therapeutic but functions equivalently for assay purposes.

Summary Table: Use of Urelumab Biosimilar in Bridging ADA ELISA

Using a Urelumab biosimilar in this way ensures that the assay specifically detects anti-drug antibodies targeting the therapeutic and allows researchers or clinicians to monitor the patient's immune response to Urelumab therapy.