Anti-Human IL-36R (Spesolimab) [Clone BI-655130]

Research-grade Spesolimab biosimilars are commonly used as calibration standards or reference controls in pharmacokinetic (PK) bridging ELISAs to ensure accurate and comparable measurement of drug concentration in serum samples from both biosimilar and reference products.

Key roles and processes:

• In PK bridging ELISAs for biosimilars, a single analytical standard (often the biosimilar itself) is typically used to generate the calibration curve for quantification of both the biosimilar and the reference (originator) antibody product.
• The selection of either the biosimilar or the reference as the calibrator is based on demonstration of bioanalytical equivalency, meaning that both the biosimilar and reference produce comparable assay responses (parallelism and similar recovery) within the method.
• Calibration standards are prepared by spiking known concentrations of the biosimilar into blank serum to generate a standard curve, which is then used to quantify drug concentrations in study samples.
• Reference controls, which can include both the biosimilar and reference Spesolimab, are also spiked across the standard curve range as Quality Control (QC) samples to confirm the comparability of assay performance for both products.

Benefits and essential practices:

• Using a single analytical standard in the ELISA reduces assay variability and avoids the need for complex cross-comparison or bridging between separate standard curves for each product.
• Regulatory and bioanalytical best practice is to establish analytical equivalence by comparing the results for both products against the biosimilar-based standard curve and statistical testing (e.g., ensuring the 90% confidence interval of relative accuracy falls within a predefined range such as [0.8, 1.25]).
• Only after this equivalency is established is the biosimilar accepted as the sole assay calibrator for routine PK analysis, with continued inclusion of drug-product and reference QC samples in every run to monitor ongoing comparability.
• This approach is specifically described as optimal for bridging PK assays in biosimilar development and has been demonstrated to provide data suitable for pharmacokinetic bioequivalence assessments, as outlined in regulatory and industry guidance.

Regulatory context:

• Authorities such as EMA and FDA require demonstration that the assay can robustly and accurately measure both biosimilar and reference drugs, justifying the use of a single assay and standard for both, contingent on demonstrated equivalence.

Example specific to Spesolimab:

• Studies measuring Spesolimab PK in serum have described similar practices, using validated ligand-binding assays (such as electrochemiluminescence or ELISA), with attention to sensitivity and specificity, though exact details of the standard used are not always disclosed.

Summary Table: How Research-Grade Biosimilar is Used in PK ELISA

This approach ensures robust, accurate, and comparable measurement of Spesolimab (or its biosimilar) in clinical serum samples, meeting regulatory and scientific demands for pharmacokinetic bridging studies.

The primary in vivo models where a research-grade anti-IL-36R antibody is used to study tumor growth inhibition and characterize tumor-infiltrating lymphocytes (TILs) are murine syngeneic tumor models, with the most notable examples being CT26 (colon carcinoma), RENCA (renal carcinoma), B16F10 (melanoma), and MC38 (colon adenocarcinoma).

• CT26 (colon carcinoma, BALB/c mice): The use of IL-36R antagonists (including IL-36Ra, which mimics an antibody-mediated blockade of IL-36R) in this model has shown a reduction in tumor burden and cell proliferation, demonstrating that blocking IL-36R signaling can inhibit tumor growth.

• RENCA (renal cell carcinoma, BALB/c mice): RENCA tumors are highly infiltrated by immune cells and frequently used for immunotherapy studies, providing a rich context for analyzing changes to the TIL compartment following IL-36R pathway manipulation.

• B16F10 (melanoma, C57BL/6 mice): Though this model is poorly infiltrated ("immune cold"), it is nonetheless used to probe responses to immune modulators like anti-IL-36R due to its relevance for studying immune resistance mechanisms.

• MC38 (colon adenocarcinoma, C57BL/6 mice): Also used for modulating IL-36 signaling in vivo, known for its robust immune responses and suitability for immunotherapy mechanistic studies.

Experimental usage details:

• In these models, syngeneic implantation of tumor cells in immunocompetent mice allows assessment of both tumor growth and the immune infiltrate (including TILs) after administration of anti-IL-36R antagonists or antibodies.
• These studies often include comprehensive immunophenotyping of TILs, documenting changes in CD8+ T cells, CD4+ T cells, myeloid-derived suppressor cells, neutrophils, and other immune cell populations in the tumor microenvironment following IL-36R blockade.
• For IL-36R pathway blockade, both genetic ablation (CRISPR) and pharmacological antagonism (such as IL-36Ra as a surrogate for antibody blockade) have been used in mouse models. Direct administration of a research-grade anti-IL-36R antibody would generally follow similar protocols, with analysis of TILs by flow cytometry and functional assays.

Humanized Models:

• Humanized mouse models (where components of the human immune system are engrafted into immunodeficient mice) have not been specifically described in the cited search results for anti-IL-36R tumor studies; published literature and standard practice indicate that such studies are less common, primarily due to the species specificity of IL-36R and available blocking antibodies. Most in vivo characterization so far has been conducted in mouse syngeneic models.

In summary, the syngeneic mouse tumor models CT26, RENCA, B16F10, and MC38 are the primary platforms for evaluating anti-IL-36R antibodies in vivo for tumor growth inhibition and TIL characterization, with CT26 being particularly well-documented for the use of IL-36R antagonism. Humanized models have not yet been prominently used in this context based on available data.

Researchers have not yet reported studies using Spesolimab biosimilars in combination with checkpoint inhibitors (such as anti-CTLA-4 or anti-LAG-3 biosimilars) in immune-oncology models as of the most recent literature. However, based on established scientific principles and analogous research, the potential approach for studying such synergistic effects can be outlined.

Key context and supporting details:

• Spesolimab is a monoclonal antibody targeting the IL-36 receptor, primarily developed and approved for severe inflammatory diseases, particularly generalized pustular psoriasis, by blocking IL-36-mediated proinflammatory pathway signaling. It is not itself a conventional checkpoint inhibitor like anti-PD-1, anti-CTLA-4, or anti-LAG-3; rather, it modulates innate and adaptive immune responses through cytokine pathway inhibition.

• Checkpoint inhibitors (e.g., anti-CTLA-4, anti-LAG-3, anti-PD-1) have demonstrated synergistic efficacy in oncology models by targeting different immune regulatory pathways. For example, combining anti-CTLA-4 (enhances T cell priming in lymph nodes) and anti-PD-1 (restores T cell cytotoxicity at tumor sites) can overcome resistance and amplify antitumor immunity, albeit with increased toxicity. Anti-LAG-3, in particular, targets T cell exhaustion and can further rejuvenate antitumor responses, especially in combination regimes.

• Combination rationale in immune-oncology: Synergistic studies often use mouse tumor models or humanized immune systems, administering antibodies that target different axes, such as checkpoint pathways and inflammatory cytokines, to model potential cooperative or additive effects on T cell activation, tumor rejection, and immune cell infiltration. The culmination of these combinatorial strategies is typically evaluated through tumor growth inhibition, flow cytometry of immune populations, gene expression profiling, and survival analyses.

Potential experimental approach (based on methodology from analogous studies):

• Researchers would administer a Spesolimab biosimilar (IL-36R antagonist) with one or more checkpoint inhibitor biosimilars (e.g., anti-CTLA-4, anti-LAG-3) to tumor-bearing animals or complex 3D in vitro models.
• The study would monitor immune cell activation status, cytokine release, T cell exhaustion markers, tumor growth curves, and overall survival.
• The hypothesis would be that IL-36R blockade might attenuate immunosuppressive or pro-tumor inflammation, potentially augmenting checkpoint inhibitor efficacy by reprogramming the tumor microenvironment or reducing inhibitory feedback loops, thereby enabling more effective T cell–mediated tumor eradication.
• Comprehensive immune profiling post-treatment would reveal mechanistic insights, such as changes in regulatory T cell populations, interferon-gamma production, and disruption of exhaustion markers, as has been seen when combining anti-LAG-3 with anti-PD-1 or anti-CTLA-4, albeit not specifically with Spesolimab.

Current limitations:

• There is no direct published evidence or model system for combinations explicitly using Spesolimab with checkpoint inhibitors in oncology as of the latest research.

• Most combination checkpoint inhibitor research emphasizes anti-PD-1, anti-CTLA-4, and anti-LAG-3 in cancer models, not anti-IL-36R agents such as Spesolimab, which are still focused on inflammatory and dermatological indications.

In summary, while checkpoint inhibitor combinations (anti-CTLA-4, anti-LAG-3, etc.) are well established in preclinical and clinical immuno-oncology, the combination with Spesolimab biosimilars is still theoretical and not yet reported in peer-reviewed oncology studies. Researchers would likely employ established immune-oncology model systems, leveraging immune cell and tumor microenvironment endpoints to detect synergy if such studies were undertaken in the future.

A Spesolimab biosimilar can be used as either the capture or detection reagent in a bridging ADA ELISA to monitor a patient’s immune response (development of anti-drug antibodies, ADAs) against spesolimab by exploiting the ability of ADAs to bridge between two molecules of the drug (or its biosimilar) via their antigen-binding sites.

In a typical bridging ELISA for ADA detection:

• The ELISA plate is coated with spesolimab biosimilar (capture reagent).
• Patient serum is added; any ADA present will bind to the immobilized spesolimab biosimilar.
• After washing, a labeled spesolimab biosimilar (detection reagent), often conjugated with an enzyme such as horseradish peroxidase (HRP) or biotin, is added. This binds to the other antigen-binding site of the ADA, forming a "bridge" between the immobilized and labeled drug molecules.
• Signal generation (via enzyme substrate reaction) signifies ADA presence.

Purpose of using a biosimilar:

• Biosimilars are structurally and functionally very similar to the original therapeutic drug (spesolimab), allowing recognition by ADAs generated in response to the originator drug.
• Using a biosimilar avoids interference from the therapeutic drug present in the patient sample, improves specificity (if the biosimilar has subtle sequence or tag differences), and may be more accessible or economical for assay production.

Key Considerations:

• The specific ELISA described in related literature uses the therapeutic drug (or its analogs/biosimilars) for both capture and detection, which is the hallmark of a bridging ADA assay for monitoring immune response to biologics like spesolimab.
• Assay sensitivity and drug tolerance (ability to detect ADA in the presence of circulating drug) are important and should be validated in the context of clinical sample immunogenicity testing.

Clinical Application:

• This format allows detection of binding ADAs in patients treated with spesolimab during clinical trials, and the assay performance (sensitivity, drug tolerance) must be verified and justified for regulatory submission and monitoring.

In summary, a spesolimab biosimilar used as both capture and detection reagent in a bridging ELISA enables sensitive monitoring of anti-spesolimab immune responses, crucial for evaluating drug immunogenicity in clinical settings.