Anti-Human IL-12/23 (Ustekinumab) [Clone CNTO-1275]

Research-grade Ustekinumab biosimilars are commonly used as calibration standards and reference controls in pharmacokinetic (PK) bridging ELISA assays to quantitatively measure drug concentrations in serum samples. Their use is essential to ensure assay accuracy, establish equivalency with the originator drug, and satisfy regulatory criteria for biosimilar assessment.

Key uses in PK bridging ELISA:

• Calibration Standards:
  Biosimilar ustekinumab preparations of known concentration are serially diluted to generate a standard curve. This curve is plotted as absorbance versus concentration and serves as the reference for interpolating the concentration of ustekinumab in unknown serum samples. The biosimilar must be highly similar to the reference product in structure and function for the curve to accurately represent clinical samples.

• Reference Controls:
  Positive controls (reference controls) composed of defined concentrations of research-grade biosimilar ustekinumab are included in each assay run to assess assay validity and precision. Acceptable ranges for these controls are predefined (e.g., 0.8 µg/ml and 7 µg/ml, required to fall within specific limits for the test to be valid). Consistently recovering these controls within range demonstrates reliability and reproducibility of the assay.

• PK Bridging Application:
  Analytical PK bridging studies compare biosimilar drug concentration profiles to those of the reference (originator) product. ELISA kits validated for both biosimilar and originator detection use side-by-side standards curves to confirm assay equivalence. Data from such bridging ELISAs are key for demonstrating that the biosimilar behaves indistinguishably in patient samples, forming part of the regulatory evidence for biosimilarity.

• Technical details:

  • Typical ELISA measurement range covers low ng/mL to several µg/mL concentrations for ustekinumab in serum or plasma.
  • Kits are optimized to minimize cross-reactivity, matrix effects, and ensure high specificity (e.g., through anti-idiotypic antibody use).
  • Both analytical sensitivity (limit of detection) and quantitation range are defined for each kit and must be validated with the chosen biosimilar standard.

Summary Table: Role of Biosimilar Standards in PK ELISA

This use of biosimilar standards ensures robust, reproducible, and regulatory-accepted quantification of ustekinumab in pharmacokinetic studies.

The primary models where a research-grade anti-IL-12/IL-23 p40 antibody is administered in vivo to study tumor growth inhibition and tumor-infiltrating lymphocytes (TILs) are syngeneic mouse models with fully functional immune systems. Use of humanized mouse models for this purpose is rare due to species specificity of the antibody reagents and cytokine biology.

Essential context and details:

• Syngeneic Models: These models involve immunocompetent mice (typically C57BL/6 or BALB/c) bearing mouse-derived tumors such as B16 melanoma, MC38 colon carcinoma, or bladder cancer. The anti-IL-12/IL-23 p40 antibody targets both IL-12 and IL-23 pathways, allowing for assessment of tumor growth and immune cell infiltration changes. Syngeneic models are specifically suited for characterization of murine TILs, as the antibody used is reactive with mouse cytokines, and the mice’s immune response is complete.

• Typical Preclinical Studies: Anti-IL-12/IL-23 p40 antibodies (often from vendors like eBioscience) are administered systemically, and effects on tumor growth, as well as the phenotype and numbers of TILs (CD8+ T cells, NK cells, Tregs, myeloid cells), are quantified via flow cytometry, immunohistology, or gene expression profiling.

• Example: In skin carcinogenesis models and lymphoma induction models, IL-12/23 p40-deficient mice display altered tumor susceptibility and TIL profiles compared to wild-type controls, demonstrating the centrality of this axis in shaping tumor immunity. Additionally, preclinical studies often combine anti-IL-12/IL-23 p40 with other agents (e.g., checkpoint blockade, anti-CD40, or anti-IL-23 alone) and assess synergistic antitumor effects and modification of the tumor immune microenvironment.

• Humanized Models: While some studies use humanized mice to investigate human immune mechanisms, there is minimal published evidence of anti-IL-12/IL-23 p40 antibodies being administered in these systems for tumor inhibition or TIL characterization, primarily due to cross-reactivity issues and the complexity of reconstituting both human cytokines and immune components.

In summary, syngeneic mouse tumor models are the standard for in vivo administration of anti-IL-12/IL-23 p40 antibodies to study tumor growth inhibition and characterize TILs. Studies using humanized models for this specific research question are highly limited.

Researchers investigating synergistic immune-oncology effects use ustekinumab biosimilars, which target the interleukin (IL)-12/23 pathway, in combination with other checkpoint inhibitors like anti-CTLA-4 or anti-LAG-3 agents primarily through preclinical models and early-phase clinical studies. These studies aim to leverage the distinct, non-overlapping mechanisms of each agent to boost immune responses against tumors.

Context and Methodology:

• Combination Rationale: Ustekinumab biosimilars inhibit IL-12/23, modifying the tumor microenvironment and immune cell activity. Checkpoint inhibitors like anti-CTLA-4, anti-PD-1, or anti-LAG-3 unblock T cell activation or reinvigorate exhausted T cells by targeting inhibitory pathways. Using both simultaneously can address multiple mechanisms that tumors exploit to evade the immune system.
• Model Systems: Researchers employ:
  • Preclinical murine tumor models engineered to express relevant immune and cytokine profiles.
  • Patient-derived xenografts or ex vivo human immune cell/tumor co-cultures to mimic complex human immune-tumor interactions.
• Study Designs:
  • Experimental groups receive either monotherapy (each agent alone) or combinations of ustekinumab biosimilar and checkpoint inhibitors.
  • Key endpoints include tumor regression, immune infiltration, cytokine profiles, and toxicity.
  • Combinations can reveal synergistic, additive, or antagonistic effects on tumor control and immune response.
  • Mechanistic endpoints often include T cell phenotyping, cytokine analysis, and biomarkers of immune activation or suppression.
• Clinical Translation: Promising combinations from preclinical studies progress to early-phase clinical trials, where similar synergistic principles are tested in humans.
  • For example, the combination of CTLA-4 and PD-1 blockade has improved durable response rates in some cancers but also increased toxicity compared to monotherapy; similar principles are applied to LAG-3 and other checkpoints.
• Current Status: While most clinical and real-world use focus is on anti-tumor combinations involving PD-1/PD-L1 or CTLA-4 with other checkpoint inhibitors, research into combining cytokine pathway inhibition (ustekinumab or its biosimilars) with checkpoints like LAG-3 is ongoing, seeking to broaden the subset of responsive patients.
• Biosimilar Use: The role of ustekinumab biosimilars in such research follows their demonstration of equivalent pharmacokinetics and safety compared to the reference product. Cost-effectiveness and increased accessibility of biosimilars facilitate larger, more inclusive studies and may accelerate the exploration of novel combinations.

In summary, researchers use ustekinumab biosimilars in synergy studies by evaluating mechanistic and therapeutic interactions with other checkpoint inhibitors in controlled experimental systems, aiming ultimately to design more effective and widely accessible cancer immunotherapies.

A Ustekinumab biosimilar is used in a bridging anti-drug antibody (ADA) ELISA as both the capture and detection reagent to monitor the immune response by detecting ADAs in patient samples directed against Ustekinumab.

In the standard bridging ADA ELISA format for Ustekinumab (or its biosimilar):

• Microplate wells are coated with the Ustekinumab biosimilar, serving as the capture reagent.
• A serum sample from a patient (potentially containing ADAs) is added; if ADAs are present, they will bind to the immobilized biosimilar on the plate.
• After washing, the same biosimilar molecule—labeled with an enzyme (e.g., HRP) or biotin—is added as the detection reagent. If ADAs are present in the sample, they will have “bridged” between the capture and detection biosimilar molecules; otherwise, there will be no signal.
• After further washes, a substrate is added, and the signal (commonly colorimetric) is proportional to the amount of ADA present in the patient’s serum.

Key points specific to Ustekinumab biosimilars as reagents:

• The biosimilar used as the reagent must be as similar as possible to the therapeutic (reference) drug, as epitope differences could affect ADA detection.
• This assay detects antibodies regardless of isotype and is sensitive to both free ADAs and, depending on protocol (e.g., acid dissociation), drug-bound ADAs.
• Using a biosimilar as both capture and detector is valid as long as it mimics the reference drug’s epitopes with high fidelity—a regulatory expectation when evaluating biosimilarity and comparative immunogenicity.

Summary Table: Role of Ustekinumab Biosimilar in Bridging ADA ELISA

This assay allows monitoring of patient immunogenicity by quantifying the ADA response to the therapeutic drug.

For clinical and regulatory comparability studies, employing the biosimilar in this fashion is essential to evaluate its relative immunogenicity against the reference product.