Anti-Human IL-36R (Spesolimab) [Clone BI-655130] — Fc Muted™

Research-grade Spesolimab biosimilars are used as analytical standards to generate calibration curves and as quality control/reference samples in PK bridging ELISA assays for measuring drug concentration in serum samples.

In detail:

• A single PK assay is developed that quantitatively measures both the biosimilar (e.g., research-grade Spesolimab) and its reference product in serum matrices.
• Calibration standards are prepared by spiking known amounts of the biosimilar into human serum at various concentrations (e.g., 50–12,800 ng/mL), which are then used to create the assay’s standard curve. This allows for the quantification of unknown serum samples by interpolating their signal from the calibration curve.
• Reference controls or quality control (QC) samples are also prepared from both the biosimilar and reference product at predetermined concentrations. These samples are assayed in parallel with study samples to ensure assay accuracy, precision, and comparability between the biosimilar and reference.
• With demonstrated bioanalytical equivalence (statistical equivalence between biosimilar and reference in assay performance), the biosimilar (such as research-grade Spesolimab) is often selected as the sole calibrator for all study samples in a PK bridging ELISA.
• This approach minimizes method variability and avoids the need for separate assays for different products. It is considered best practice for biosimilar PK studies, as it enables direct comparison and supports bioequivalence evaluation.

Supporting Points:

• Calibration curves constructed from biosimilar standards allow consistent quantification across both biosimilar and reference study samples, referencing to the same analytical standard.
• Inclusion of QC/reference samples generated from both biosimilar and reference batches ensures assay robustness and supports regulatory requirements for comparability in biosimilar development.
• Method validation includes testing with sets of biosimilar and reference controls over multiple runs, confirming accuracy, precision, and equivalence within defined acceptance criteria (e.g., confidence intervals within [0.8, 1.25]).

This process underpins pharmacokinetic bridging ELISA methodology by using research-grade biosimilars (such as Spesolimab) to ensure accurate, comparable, and regulatory-compliant measurement of drug concentration in serum samples during biosimilar development.

The primary in vivo models where a research-grade anti-IL-36R antibody is administered to study tumor growth inhibition and to characterize tumor-infiltrating lymphocytes (TILs) are mouse syngeneic tumor models such as CT26 (colorectal cancer), RENCA (renal cell carcinoma), and B16F10 (melanoma). These models are most frequently used due to their fully functional murine immune system, which is required for interrogating both tumor growth and immune cell dynamics in the tumor microenvironment following anti-IL-36R treatment.

Key model details:

• Syngeneic Mouse Models:

  • CT26 (colon carcinoma, BALB/c background): Used to assess in vivo effects of modulating IL-36R signaling, including administration of antagonists like IL-36Ra (IL-36 receptor antagonist), which has demonstrated reduction in tumor burden and notable effects on TIL populations.
  • RENCA (renal cell carcinoma, BALB/c): Frequently used for immunotherapy research to evaluate tumor growth and immune infiltration profiles in response to immune modulation.
  • B16F10 (melanoma, C57BL/6): Important for representing less immunogenic ("cold") tumors and assessing changes in TILs and responsiveness to immunotherapy.

• Experimental Manipulation:

  • Administration of anti-IL-36R (or IL-36Ra): Intraperitoneal injection has been used (as in ), directly antagonizing the IL-36R pathway and allowing evaluation of both tumor growth and the characteristics of TILs by flow cytometry and immunophenotyping.
  • Genetic modifications (CRISPR-Cas9 IL-36R knockout lines): Used in parallel with antibody-based approaches to validate target specificity and pathway dependency.

Immunological readouts in these models:

• TIL Profiling: These studies employ flow cytometry and immunohistochemistry to quantify and characterize TIL populations, including CD4+ T cells, CD8+ T cells, NK cells, dendritic cells, and myeloid populations, post anti-IL-36R treatment.
• Tumor Growth Monitoring: Tumor size and burden are tracked to correlate immune changes with antitumor efficacy.

Humanized models: There are currently no widely documented examples of anti-IL-36R being tested in fully humanized tumor models for detailed TIL analysis, likely due to the lack of cross-reactivity of many research-grade antibodies and the technical challenges in establishing robust humanized immune systems in mice for IL-36-related research.

Summary Table: Syngeneic Models for Anti-IL-36R Studies

Syngeneic mouse models remain the gold standard for investigating the effect of anti-IL-36R antibodies on tumor growth and TIL composition in vivo.

Researchers studying synergistic effects in immune-oncology models have explored combining checkpoint inhibitors such as anti-CTLA-4 and anti-LAG-3 antibodies, frequently in combination with PD-1/PD-L1 inhibitors, to potentiate anti-tumor immune responses. However, use of Spesolimab biosimilar in conjunction with checkpoint inhibitors for these synergy studies is not yet well documented in published literature.

Context and Mechanism

• Spesolimab is an IL-36 receptor antagonist whose primary clinical application is in treating generalized pustular psoriasis (GPP) by reducing pro-inflammatory IL-36 signaling. Its mechanism is distinct from classical immune checkpoint inhibitors, which target T-cell exhaustion and immune suppression in the tumor microenvironment.
• Checkpoint inhibitors (e.g., anti-CTLA-4, anti-LAG-3) act by blocking inhibitory signals on T cells, thereby enhancing anti-tumor immunity. Dual blockade (CTLA-4 and LAG-3) has shown synergistic inhibition of T-cell proliferation and suppression of pathological immune responses in transplant and cancer models.

Combination Strategies and Synergy Study Techniques

• Although studies have combined checkpoint inhibitors (anti-CTLA-4, anti-LAG-3, and PD-1/PD-L1) to examine synergy—such as the ongoing phase II trial using ipilimumab (CTLA-4), nivolumab (PD-1), and relatlimab (LAG-3)—there is no documented evidence of Spesolimab biosimilars being incorporated in these triple or dual immune checkpoint inhibitor combinations in oncology models within existing literature.
• For synergy evaluation in immune-oncology, combinations typically use:
  • Syngeneic mouse tumor models, genetically engineered models, or humanized immune models.
  • Monitoring tumor growth, immune cell infiltration, cytokine profiles, and survival rates.
  • Molecular analysis of signaling pathways impacted by each agent.

Potential Rationale for Future Combinations

• Spesolimab’s ability to suppress inflammation via IL-36 pathway inhibition suggests a possible utility in modulating the tumor microenvironment if future preclinical evidence demonstrates IL-36’s role in cancer progression or immune evasion.
• Synergistic studies with checkpoint inhibitors depend on robust immune activation and reversal of immune suppression; thus, adding Spesolimab may be hypothesized to alter myeloid or stromal compartments, but this is speculative and not yet supported by published models.

Summary Table: Comparison of Approaches

Key Insights

• Checkpoint inhibitor combinations are standard for synergy studies in immune-oncology.
• Spesolimab biosimilars are not yet documented in such combinations for immune-oncology models, and their inclusion remains theoretical absent further mechanistic evidence.
• The technical setup for such studies involves well-characterized animal models, immune marker profiling, and detailed molecular analysis.

Further research is needed to clarify whether IL-36 pathway modulation using Spesolimab could yield synergy with checkpoint inhibitors in cancer immunity contexts, as this concept is not supported by current published data.

A Spesolimab biosimilar can be effectively used as either the capture or detection reagent in a bridging anti-drug antibody (ADA) ELISA to monitor immunogenicity in patients treated with Spesolimab or its biosimilars.

In a bridging ADA ELISA, the core principle leverages the bivalent nature of ADAs:

• The ELISA plate is first coated with the Spesolimab biosimilar, which acts as the capture reagent.
• Patient serum (which may contain ADAs if the patient’s immune system has responded to the therapeutic Spesolimab) is incubated on the plate, allowing any ADAs in the sample to bind to the immobilized biosimilar.
• After washing, a detection reagent—typically the same Spesolimab biosimilar labeled with a detection tag such as biotin, HRP, or another suitable label—is added. This binds to the other “arm” of the ADA, resulting in the formation of a “drug-ADA-drug” bridge.
• After additional washing, a substrate is added, and the resulting signal (e.g., colorimetric or chemiluminescent) is proportional to the amount of ADA present in the sample.

Key steps in application:

• Both capture and detection reagents are the Spesolimab biosimilar; one is immobilized, the other is labeled.
• The bridging format allows for detection of antibodies regardless of isotype, as long as they can bind two Spesolimab molecules simultaneously.
• Use of a biosimilar, rather than the reference product, ensures safety (for the laboratory operator and supply chain), can be convenient for assay standardization, and may improve availability. Biosimilars are typically structurally and antigenically very close to the original molecule, making them suitable for this application as long as their epitope structure mirrors that of the original therapeutic.

Context on Spesolimab immunogenicity detection:

• Clinical trials monitoring Spesolimab immunogenicity have used validated bridging ELISA or bridging electrochemiluminescence immunoassays, following these general principles.
• Bridging formats are the industry standard for initial ADA screening due to sensitivity and their ability to detect antibodies of multiple isotypes.

Limitations:

• The bridging ELISA detects only bivalent antibodies and might miss monovalent ADA.
• High circulating drug levels can interfere with the assay by occupying ADA binding sites, reducing detectability (“drug tolerance”).
• Use of highly pure biosimilar is important to avoid false positives due to impurities or differences in the presentation of antigenic sites.

In summary, using a Spesolimab biosimilar as both capture and detection reagent in a bridging ADA ELISA enables sensitive detection of patient immune responses against the therapeutic in clinical monitoring and immunogenicity assessment.