Anti-Human IL-12/23 (Ustekinumab) [Clone CNTO-1275] — Fc Muted™

Research-grade Ustekinumab biosimilars are used as calibration standards or reference controls in pharmacokinetic (PK) bridging ELISA by serving as the known reference material to generate the standard curve, enabling accurate quantification of Ustekinumab concentrations in serum samples.

Essential context and supporting details:

• Calibration standards in a PK ELISA are solutions of known concentration, typically prepared by serially diluting a reference-grade Ustekinumab biosimilar (or the original reference product) in a biological matrix similar to study samples (like pooled human serum).

• These standards are run on each ELISA plate to generate a standard curve (OD versus concentration), against which unknown serum sample concentrations are interpolated.

• According to the KRIBIOLISA™ Ustekinumab ELISA kit, the calibrators/standards used for calibration are typically validated and calibrated against a commercially sourced reference Ustekinumab product (such as STELARA™), ensuring equivalence between the measured biosimilar and the reference during quantification.

• Biosimilar-grade material must have demonstrated high similarity in terms of binding and immunoreactivity to the reference product to be suitable as a standard or reference control.

• In a PK bridging ELISA assay, serum samples are processed alongside the standards. The sandwich ELISA format uses a capture antibody specific to Ustekinumab (including biosimilars) and a detection antibody targeting the Fc region, ensuring that the standard curve derived from the biosimilar calibrator accurately reflects the characteristics of the Ustekinumab in patient samples.

• Results calculation: The unknown sample concentrations are interpolated from the standard curve, which has been generated using the biosimilar-based calibration standards.

• Controls: Reference controls may include samples with low, medium, and high concentrations of the biosimilar to monitor accuracy and precision across the working range.

Key points for use in PK bridging ELISA:

• Use of a research-grade Ustekinumab biosimilar as a calibration standard ensures consistency, traceability, and comparability of results across studies and laboratories.
• Kits and protocols often specify that calibration standards should match or be cross-validated against the reference product to confirm assay validity, especially given regulatory requirements for biosimilar comparability studies.
• PK similarity studies may use both biosimilar and reference Ustekinumab (EU or US-sourced) as standards/controls to ensure assay detects no meaningful differences, further supporting biosimilarity.

In summary, biosimilar Ustekinumab is calibrated and validated against the reference product and used to generate standard curves and QC controls in PK ELISA, facilitating accurate drug quantitation in bridging and comparability studies.

The primary in vivo models where a research-grade anti-IL-12/IL-23 p40 antibody is administered to investigate tumor growth inhibition and tumor-infiltrating lymphocytes (TILs) are syngeneic mouse tumor models. Humanized models are less common for this specific application due to the species specificity of the p40 subunit recognized by such antibodies.

Key context and experimental details:

• Syngeneic mouse models use immunocompetent mice implanted with mouse-derived tumors (e.g., B16 melanoma, MC38 colon carcinoma, MB49 bladder carcinoma), allowing for full immune interactions including TIL responses.
• Anti-IL-12/IL-23 p40 antibodies such as clone C17.8 (or commercial equivalents from eBioscience, Bio X Cell, etc.) specifically block endogenous mouse IL-12 and IL-23, affecting both Th1 and Th17/Tc17 responses in the tumor microenvironment.
• Studies using these models typically administer the anti-p40 antibody systemically (often IP or IV) during tumor establishment or growth, monitoring effects on tumor size and characterizing TIL populations (CD8+, CD4+, Tregs, myeloid cells) by flow cytometry at endpoint.

Supporting examples:

• Application of anti-IL-12/IL-23 p40 neutralizing antibody in syngeneic mouse models, such as DMBA/TPA-induced skin tumors or orthotopic gliomas, has helped dissect the role of these cytokines in tumor immunity, modulating both tumor progression and TIL composition.
• Syngeneic bladder and sarcoma mouse models have been used to show that blocking IL-23 (using p40 antibodies) enhances the antitumor efficacy of therapies that boost IL-12 or T cell responses.

About humanized models:

• Use of anti-IL-12/IL-23 p40 antibodies in humanized mouse models (mice engrafted with human immune cells and human tumors) is rare for studies targeting endogenous cytokine signaling, because standard research antibodies for mouse p40 do not cross-react with human p40, and few studies have monitored both tumor control and TILs in this context.
• Humanized models would require a human-specific anti-IL-12/IL-23 p40 antibody and appropriate humanized tumor/immune cell combinations, which is more typical in translational exploratory research or drug development and less in the standard academic setting.

In summary:

• Syngeneic mouse tumor models (using murine anti-p40 antibodies) are the canonical approach for these studies.
• These models facilitate full immune analyses, including effects on tumor-infiltrating lymphocyte populations under IL-12/23 blockade.
• Humanized models have theoretical utility but far fewer empirical studies using anti-IL-12/IL-23 p40 blockade to dissect TIL phenotypes in vivo.

References for models and methodology:

Researchers investigating the synergistic effects of ustekinumab biosimilars in combination with other checkpoint inhibitors (such as anti-CTLA-4 or anti-LAG-3 biosimilars) in immune-oncology models typically use preclinical studies and early-phase clinical trials to evaluate potential interactions and therapeutic benefits.

Context and Methodology:

• Checkpoint Inhibitors and Mechanistic Rationale: The foundation of such combination studies lies in the distinct mechanisms of various checkpoint inhibitors. For example, anti-CTLA-4 agents mainly activate T cells in lymph nodes, while anti-PD-1/PD-L1 agents work at the tumor site to overcome local immunosuppression.
• Ustekinumab’s Role: Ustekinumab (and its biosimilars) targets the IL-12 and IL-23 cytokine pathways, modulating the immune response in ways that complement checkpoint blockade mechanisms. By altering the tumor microenvironment and reducing immunosuppressive signals, ustekinumab may potentiate T-cell-mediated anti-tumor effects when used with checkpoint inhibitors.

Practical Study Designs:

• Preclinical Models: Researchers use murine models bearing syngeneic or patient-derived tumors. They administer biosimilar ustekinumab with checkpoint inhibitors (anti-CTLA-4, anti-LAG-3, or anti-PD-1/PD-L1 biosimilars) in various combinations and dosing regimens to map out synergistic effects.
• Outcome Measures: They monitor tumor growth, survival, changes in tumor-infiltrating lymphocytes (especially CD8+ T cells and regulatory T cells), cytokine profiles, and markers of immune activation or exhaustion.
• Mechanism Elucidation: These studies often employ flow cytometry, RNA-sequencing of tumor tissue, immunohistochemistry, and cytokine assays to detail the molecular and cellular mechanisms underlying any observed synergy.

Current Knowledge and Limitations:

• There is extensive support for combining checkpoint inhibitors (e.g., anti-CTLA-4 and anti-PD-1) in both preclinical and clinical settings, where such combinations have increased efficacy but also higher toxicity.
• For ustekinumab biosimilars specifically, most available research focuses on biosimilar equivalency, pharmacokinetics, and approvals for autoimmune diseases rather than direct immune-oncology combinations. Translational and preclinical oncology studies with ustekinumab (originator or biosimilar) in conjunction with checkpoint inhibitors are an active area of research, but large-scale, published results on these specific combinations are currently limited.

Summary Table: Mechanistic Rationale for Combination

Conclusion:
Researchers use ustekinumab biosimilars in combination with checkpoint inhibitors, exploiting their complementary mechanisms to enhance anti-tumor immunity in complex preclinical models, often using murine systems, deep immunophenotyping, and functional assays to establish synergy and inform future clinical trials. Direct clinical or real-world data with ustekinumab biosimilars in these specific combinations for oncology are still emerging, and most evidence is derived from analogous combinations or mechanistic studies.

In a bridging ADA ELISA to monitor a patient’s immune response against ustekinumab, a biosimilar version of ustekinumab can be used as either the capture or detection reagent due to its structural similarity to the reference drug. The typical process is as follows:

• Capture phase: Microtiter plate wells are first coated with the ustekinumab biosimilar, which acts as the capture reagent to bind any anti-drug antibodies (ADAs) present in the patient's serum that recognize ustekinumab.

• Detection phase: After incubation and washing, a labeled form of ustekinumab (for example, horseradish peroxidase [HRP]-conjugated or biotinylated biosimilar ustekinumab) is added to the wells. This labeled drug acts as the detection reagent and will also bind to the captured patient ADAs due to its identical epitope presentation, thus forming a "bridge" via bivalent binding (one arm of the ADA binds to plate-coated biosimilar, and the other arm to the labeled detection biosimilar).

This sandwich or "bridging" format is particularly suitable for the detection of bivalent antibodies – especially IgG-class ADAs – which must simultaneously bind to two identical antigen molecules (here, the biosimilar ustekinumab).

Key details for this format:

• The biosimilar ustekinumab is functionally interchangeable with the reference product in the assay, provided it is shown to be analytically highly similar, especially in relevant epitopes recognized by ADAs.
• Using the biosimilar as both the capture and detection reagent is acceptable so long as there is no clinically meaningful difference in immunogenic epitopes between biosimilar and reference.
• The assay can detect the presence and sometimes the specificity (e.g., domain mapping) of ADAs produced in response to therapy, which may impact drug efficacy and safety.

Summary Table: Use of Ustekinumab Biosimilar in Bridging ADA ELISA

In this context, the biosimilar's use is justified by stringent analytical and functional comparability requirements, ensuring results reflect the true immune response to the therapeutic drug rather than assay artifacts. This approach provides sensitive and specific detection of patient anti-drug antibodies in pharmacovigilance and clinical monitoring settings.