Anti-Human IL 12/23 (Briakinumab) [Clone ABT-874]

Use of Research-Grade Briakinumab Biosimilars as Calibration Standards in PK Bridging ELISA

Research-grade briakinumab biosimilars are increasingly used as reference controls or calibration standards in pharmacokinetic (PK) bridging enzyme-linked immunosorbent assays (ELISAs) for measuring drug concentrations in serum samples, particularly when comparing biosimilars to originator biologics. This approach is critical for demonstrating bioequivalence and supporting regulatory approval of biosimilars.

Key Steps in PK Bridging ELISA Using Biosimilar Standards

• Calibration Standards Preparation: In a typical PK bridging ELISA, a set of calibration standards is prepared in human serum matrix, covering a clinically relevant concentration range. These standards can be prepared from the research-grade biosimilar itself, serving as the analytical reference for quantification.
• Method Qualification and Validation: The analytical method is first qualified by demonstrating that both the biosimilar and the reference product (the originator biologic) can be measured with comparable precision and accuracy using the same assay. This involves statistical comparison of precision and accuracy profiles across both products to confirm bioanalytical equivalence.
• Single Assay, Single Standard Strategy: The preferred industry practice is to use a single PK ELISA calibrated with the biosimilar standard to quantify both the biosimilar and the reference product in clinical samples. This minimizes variability and eliminates the need for crossover analysis in bioequivalence studies.
• Assay Validation: The assay is fully validated according to regulatory guidance, including assessment of precision, accuracy, linearity, and robustness. Validation samples prepared from both the biosimilar and the reference product are quantified against the biosimilar standard curve to confirm that the method is suitable for its intended use.
• Analytical Equivalence Assessment: If the 90% confidence intervals for the measured concentrations of both products fall within a predefined equivalence interval (e.g., 0.8–1.25), analytical equivalence is concluded. This approach supports the demonstration of PK similarity required for biosimilar approval.

Rationale for Biosimilar as Reference Control

Using a biosimilar as the reference control or calibration standard in PK bridging ELISAs is scientifically justified as long as bioanalytical equivalence between the biosimilar and the originator is rigorously demonstrated. This ensures that the assay correctly reflects the pharmacokinetic behavior of both products in clinical samples. The reference control’s properties—such as concentration, purity (>95% by SDS-PAGE), and formulation (sterile, preservative-free, BSA and azide free)—must be well-characterized to ensure reliable and reproducible quantification.

Practical Considerations

• Research-Grade vs. Clinical-Grade: Research-grade briakinumab biosimilars, such as those available from specialized vendors, are primarily for assay development, optimization, and validation, not for clinical use.
• Assay Design: Pre-coated ELISA plates with antibodies specific to briakinumab are used to capture the drug from serum samples. Bound drug is then detected using a suitable secondary antibody, with quantification against the biosimilar standard curve.
• Application: These methods are critical for estimating briakinumab concentrations in human serum and plasma, especially during clinical trials or bioequivalence studies for biosimilar development.

Summary Table: Role of Biosimilar Standards in PK Bridging ELISA

Conclusion

Research-grade briakinumab biosimilars are used as calibration standards or reference controls in PK bridging ELISAs to measure drug concentration in serum samples by providing a well-characterized, consistent reference for assay calibration. This approach, when combined with rigorous method validation, ensures that both the biosimilar and originator biologic can be accurately and equivalently quantified, which is essential for demonstrating PK similarity and supporting biosimilar development.

Research-grade anti-IL-12/IL-23 p40 antibodies are primarily studied in syngeneic mouse models for investigating tumor growth inhibition and tumor-infiltrating lymphocyte (TIL) characterization. These models utilize immunocompetent mice with functional immune systems, which is crucial for accurately assessing the immunomodulatory effects of IL-12/IL-23 pathway targeting.

Syngeneic Mouse Models

The most commonly employed syngeneic models use specific antibodies that target the shared p40 subunit of both IL-12 and IL-23. The C17.8 antibody is a well-established research-grade monoclonal antibody that specifically reacts with the mouse p40 subunit (also known as IL-12β), which is a 40 kDa component shared by both IL-12 and IL-23 heterodimeric cytokines. This antibody is widely used in preclinical studies to block both IL-12 and IL-23 signaling pathways simultaneously.

Chemically-Induced Cancer Models represent a major category of syngeneic systems where anti-p40 antibodies have demonstrated significant effects. In DMBA-initiated and TPA-promoted two-stage skin carcinogenesis models, mice with genetic deficiencies in IL-12/23p40 showed significantly decreased numbers of carcinogen-induced papillomas compared to wild-type mice. Similarly, in N-methyl-N-nitrosourea (MNU)-induced lymphoma models, the IL-12/23 axis plays a critical role in tumor susceptibility.

Orthotopic Brain Tumor Models have shown particular promise for studying localized anti-IL-12/IL-23 therapy. Preclinical studies using intratumoral IL-12 application combined with systemic checkpoint blockade resulted in complete eradication of orthotopic gliomas in mice. These models are especially valuable for characterizing TIL responses because they allow for detailed flow cytometric analysis of immune cell infiltration and activation states.

TIL Characterization Outcomes

The administration of anti-IL-12/IL-23 p40 antibodies in these syngeneic models produces distinct patterns of TIL modulation. Studies have demonstrated increased infiltration of CD4+ and CD8+ T cells, with enhanced effector functions as evidenced by higher frequencies of IFNγ and TNFα expression. Importantly, regulatory T cells (Tregs) also show increased cytokine expression, suggesting heightened IFNγ-induced Treg fragility, which can contribute to enhanced anti-tumor immunity.

Adoptive Cell Therapy Models represent another important application where syngeneic systems are used to study IL-12/IL-23 modulation effects on TILs. In these models, tumor-specific CD8+ T cells are expanded ex vivo in the presence of IL-12, then adoptively transferred back into syngeneic hosts. These IL-12-conditioned T cells demonstrate enhanced antitumor responses, increased persistence, and higher expression of activation markers including CD25, ICOS, OX40, granzyme B, and IFNγ. Crucially, these cells express lower levels of PD-1 and show decreased susceptibility to IFNγ-induced apoptosis.

Combination Therapy Models

Syngeneic models have been particularly valuable for testing combination approaches that simultaneously target the IL-12/IL-23 axis. For example, studies combining agonistic anti-CD40 monoclonal antibodies to drive IL-12 production with anti-IL-23 antibodies to counter tumor-promoting effects have shown greater antitumor activity than either agent alone. These combination studies are especially relevant for cancers with rich myeloid infiltrates and upregulated IL-23, such as sarcomas.

The preference for syngeneic over humanized models in this research area stems from the need to study the complex interplay between different immune cell populations in an intact immune system. The IL-12/IL-23 pathway involves multiple immune cell types including T cells, natural killer cells, dendritic cells, and myeloid-derived suppressor cells, making syngeneic models the optimal choice for comprehensive TIL characterization and tumor growth inhibition studies.

Researchers use the Briakinumab biosimilar—an antibody targeting IL-12 and IL-23—in immune-oncology models to investigate how modulating inflammatory cytokines might synergize with other checkpoint inhibitors such as anti-CTLA-4 or anti-LAG-3 biosimilars. These combination studies are designed to dissect the interplay between cytokine signaling and T cell checkpoint modulation on tumor immunity and therapeutic efficacy.

Key methodologies and rationale:

• Combination Rationale: The logic of combining multiple immune modulators is based on the fact that checkpoint inhibitors (e.g., targeting CTLA-4, LAG-3, or PD-1) act primarily by modulating T cell activation or exhaustion, whereas Briakinumab affects pathways (IL-12/IL-23) that are central to both T cell differentiation (Th1/Th17) and the chronic inflammatory milieu in the tumor microenvironment.

• Synergy Hypothesis: Suppressing IL-12/IL-23 with Briakinumab can potentially:

  • Reduce pro-tumorigenic chronic inflammation (mediated by Th17/IL-23 axis).
  • Shift immune cell populations towards phenotypes more responsive to checkpoint blockade (e.g., increased CD8+ T cell cytotoxicity or reduced regulatory T cell (Treg) immunosuppression).
  • Augment the impact of checkpoint inhibitors by lessening the recruitment and survival of immunosuppressive or exhausted immune cells in tumors.

• Typical Experimental Models:

  • Syngeneic or humanized mouse tumor models: Briakinumab biosimilar is administered with checkpoint inhibitors to evaluate changes in tumor growth, immune cell infiltration, and cytokine profiles.
  • Immune cell profiling: Flow cytometry or single-cell sequencing is often used to monitor the activation status of CD4+ and CD8+ T cells, Tregs, and other myeloid populations before and after combination therapy.
  • Mechanistic studies: Researchers use knockout or depletion models (e.g., lacking CD4 or CD8 T cells) to determine which immune subsets are responsible for observed synergistic effects.
  • Readouts: These can include tumor regression, survival benefit, changes in T cell exhaustion markers (LAG-3, CTLA-4), and inflammatory cytokine levels.

• Supporting Evidence:

  • Studies of checkpoint inhibitor combinations (anti-CTLA-4 with anti-PD-1 or anti-LAG-3) have shown that each mechanism recruits different immune cell subsets and acts at distinct stages of T cell activation or tumor infiltration.
  • Even though direct published reports on Briakinumab biosimilar combined with anti-CTLA-4 or anti-LAG-3 are sparse, analogous strategies using cytokine modulators with checkpoint inhibitors have demonstrated additive or synergistic antitumor effects and are justified by mechanistic insights.

Limitations and Considerations:

• Most evidence derives from preclinical models; clinical translation requires careful toxicity profiling as combination immunotherapies can significantly increase immune-related adverse events.
• Precise results may vary depending on tumor type, immune contexture, and the particular checkpoint/cytokine targets being co-inhibited.

In summary, the use of Briakinumab biosimilar alongside checkpoint inhibitors in complex immune-oncology models allows researchers to probe synergistic interactions between cytokine signaling and T cell checkpoint regulation, aiming to identify more effective and durable combination immunotherapy regimens.

A Briakinumab biosimilar can be used as both the capture and detection reagent in a bridging ADA (anti-drug antibody) ELISA to monitor patient immunogenicity by exploiting the bivalent binding capability of ADAs: patient sera containing ADAs will cross-link Briakinumab molecules labeled differently for capture and detection, generating a signal proportional to ADA concentration.

Essential context and methodology:

• In a bridging ELISA for ADA detection, patient serum is incubated with two forms of the therapeutic drug (in this case, a Briakinumab biosimilar): one is biotinylated for capture and immobilized on a streptavidin-coated plate, the other is conjugated to a detection molecule (commonly HRP).
• If anti-Briakinumab ADAs are present in the patient’s serum, they act as a molecular bridge, simultaneously binding to both the capture (biotinylated) and detection (HRP-conjugated) Briakinumab reagents.

Stepwise mechanism:

• The biotinylated Briakinumab biosimilar is immobilized on the plate.
• Patient serum is added: if ADAs are present, their two binding sites (Fab arms) will attach to the immobilized Briakinumab and to the labeled detection Briakinumab, forming a sandwich/bridge.
• The plate is washed, then detection reagent is added—HRP- or dye-conjugated Briakinumab biosimilar—that binds with the ADA if present.
• After a final wash, substrate is added, and a colorimetric or chemiluminescent signal develops, quantifiable by plate reader, and proportional to ADA levels in the sample.

Why a biosimilar is used:

• Biosimilars (such as a Briakinumab biosimilar) are typically highly similar in structure and immunogenic epitopes to the originator drug, allowing them to replace the originator in assay formats to detect class-specific ADAs.
• Using a biosimilar as the assay reagent can reduce cost, ensure consistency of supply, and demonstrate immunogenicity equivalence in regulatory settings.
• Key to the assay’s validity is the biosimilar’s structural similarity, ensuring all ADAs that would bind the clinical product also bind the biosimilar.

Considerations:

• The sensitivity and specificity of bridging ELISA depend on reagent quality, dissociation of drug-ADA complexes in the sample, and potential interference from endogenous serum components.
• The structure and bivalency of both the ADA and the biosimilar are crucial for this assay type, distinguishing it from non-bridging or direct ELISA formats.

In summary, a Briakinumab biosimilar is labeled and used to capture and detect anti-Briakinumab antibodies in a patient’s sample via their ability to bridge two Briakinumab molecules, allowing monitoring of the patient’s immune response to therapy through a high-throughput, sensitive bridging ELISA.