Anti-Human Mesothelin (Amatuximab) – Fc Muted™

Research-grade Amatuximab biosimilars are used as calibration standards or reference controls in pharmacokinetic (PK) bridging ELISAs by serving as the known reference material against which unknown serum concentrations of Amatuximab are quantified. The most accepted practice is to use a single, well-characterized biosimilar standard to generate the standard curve, enabling accurate quantitation of both biosimilar and reference (originator) drug levels in test samples.

Supporting Context and Details:

• Use in Standard Curve Generation: A series of known concentrations of the Amatuximab biosimilar are prepared—typically through serial dilution—to create a standard (calibration) curve. This curve is used to interpolate the concentration of Amatuximab in unknown serum samples by comparing their ELISA signals (e.g., absorbance values) to the standards.

• Bridging ELISA Principle: PK bridging ELISAs are designed to capture both the reference and biosimilar drug by bridging between drug and anti-drug reagent pairs (e.g., using anti-idiotype or anti-Fc antibodies), ensuring that both products are measured equivalently. The assay is first qualified/validated to confirm that the biosimilar and the reference behave in a bioanalytically equivalent manner (e.g., similar affinity, recovery, and curve response in various matrix conditions).

• Selection of Calibration Standard: If biosimilar and reference show equivalent bioanalytical performance, the biosimilar is commonly selected as the single calibrator (analytical standard) for the assay. This simplifies quantification, reduces variability, and removes the need for parallel standard curves.

• Reference Controls: Quality control (QC) samples prepared from the biosimilar may also be included at low, medium, and high concentrations to monitor assay performance, typically generated from separate batches than the calibration standards.

• Analytical Validation: The assay must be validated according to regulatory guidelines (e.g., FDA, EMA) for parameters including specificity, accuracy, precision, range, recovery, and stability—demonstrating robust measurement irrespective of source (originator or biosimilar) within the validated range.

• Research-use Limitation: Most Amatuximab biosimilar reagents and ELISA kits are intended for research use only—not for diagnostic or therapeutic applications.

Summary Table: Amatuximab Biosimilar Usage in PK Bridging ELISA

When implemented properly, the result is a single, robust PK assay capable of accurate Amatuximab concentration measurement in serum, regardless of whether the drug in patient samples is reference or biosimilar product.

The primary in vivo models used for administering research-grade anti-Mesothelin antibodies to study tumor growth inhibition and to characterize tumor-infiltrating lymphocytes (TILs) are:

• Humanized mouse models expressing human mesothelin in an immune-competent background.
• Syngeneic models employing murine tumor cell lines engineered to express human mesothelin.
• Human tumor xenograft models (often in immune-deficient mice), primarily for assessing direct anti-tumor activity and, in some studies, TIL characterization.

Model Details and Rationale:

• Humanized Mesothelin Transgenic Mouse Models:
  These mice express human mesothelin in a physiological pattern, overcoming the issue that antibodies targeting human mesothelin do not cross-react with the murine protein. Researchers implant syngeneic murine tumor cell lines engineered to express human mesothelin, enabling the study of antibody therapies in a fully immune-competent host, including analysis of TILs and immune-mediated tumor rejection.

  • Example: Models where human mesothelin is expressed either systemically (serosal membranes) or tissue-specifically (thyroid).
  • Utility: Allows evaluation of tumor response and immune cell infiltration relevant to human antigen targeting.

• Syngeneic Models (Mouse Tumors Expressing Human Mesothelin):
  Immunocompetent mice are injected with murine cell lines (e.g., pancreatic, ovarian, or mesothelioma lineages) that express human mesothelin, creating a system compatible with human-specific antibody therapies.

  • This setup enables both anti-tumor efficacy assessment and immune profiling, including TIL analysis.

• Human Tumor Xenograft Models (NSG/Nude Mice):
  While these mice lack a functional immune system and therefore do not allow full TIL characterization, they are widely used to test direct tumor growth inhibition effects of anti-mesothelin antibodies on human tumor cells.

  • Example: Administration of anti-mesothelin antibodies or CAR-T cells targeting mesothelin in NSG mice bearing human ovarian, pancreatic, or mesothelioma xenografts.
  • TIL analysis is limited due to immune compromise, but tumor-infiltrating CAR T cells and changes to the grafted tumor microenvironment can be assessed.

Supporting Context:

• Mouse syngeneic models are broadly used to evaluate immunotherapeutic activity, immune infiltration, and profiling of TILs in response to antibody treatment.
• Humanized mesothelin models offer the advantage of recapitulating human tumor antigen presentation in an immune-competent setting, necessary to fully assess TILs and immune modulation after antibody administration.
• Standard xenograft models enable robust quantification of tumor inhibition but do not permit full immune analysis except for engineered effectors such as CAR-T cells.

Summary Table:

In summary, humanized mesothelin transgenic models and syngeneic tumor models with engineered human mesothelin expression are the primary models for both tumor growth inhibition and TIL characterization, while human xenograft models primarily serve tumor growth studies with limited capacity for TIL analysis.

Researchers use Amatuximab biosimilars in combination with other checkpoint inhibitors such as anti-CTLA-4 or anti-LAG-3 biosimilars to investigate synergistic anti-tumor effects in complex immune-oncology models, leveraging their distinct mechanisms to enhance immune-mediated tumor clearance.

In experimental models, the Amatuximab biosimilar targets mesothelin, a protein highly overexpressed in several cancers such as mesothelioma, ovarian, pancreatic, and lung cancers. By binding mesothelin, Amatuximab can disrupt tumor cell adhesion and enhance immune recognition. This monoclonal antibody is especially useful in research settings due to its high specificity, sensitivity, and accessibility for studying mesothelin expression and function.

Checkpoint inhibitors such as anti-CTLA-4 and anti-LAG-3 biosimilars work by blocking inhibitory pathways that regulate T cell activation:

• CTLA-4 inhibitors block the interaction between CTLA-4 and its ligands, preventing downregulation of T cell activity, thereby enhancing immune responses against tumors.
• LAG-3 inhibitors prevent LAG-3 from binding to MHC class II molecules, revitalizing exhausted T cells and contributing to antitumor responses, particularly when combined with other immune checkpoint blockers.

Synergistic studies typically involve:

• Creating in vitro co-culture systems of human immune cells and tumor cells or utilizing animal tumor models that express mesothelin.
• Treating these models with Amatuximab biosimilar alone, with checkpoint inhibitors alone, and in combination.
• Assessing outcomes such as tumor cell lysis, T cell activation, immune infiltration, and changes in cytokine profiles.

When used together, Amatuximab biosimilars and checkpoint inhibitors can potentiate the anti-tumor effects by:

• Increasing antigen-specific T cell responses through mesothelin targeting.
• Disrupting tumor cell adhesion and immune evasion mechanisms.
• Removing inhibitory signals via checkpoint blockade (CTLA-4/LAG-3), allowing stronger T cell-mediated tumor killing.

Researchers report these combinations are especially valuable for:

• Elucidating the molecular basis of immune synergy in cancer therapy.
• Screening drug candidates and understanding resistance mechanisms in preclinical models.
• Optimizing future immunotherapy strategies, potentially improving outcomes for patients with mesothelin-expressing malignancies.

In summary, Amatuximab biosimilars serve as precise tools for mesothelin-targeted cancer research, and their combination with checkpoint inhibitors in immune-oncology models enables detailed mechanistic and efficacy studies of synergistic antitumor immune responses.

In immunogenicity testing, an Amatuximab biosimilar can be used as both the capture and detection reagent in a bridging ADA ELISA to monitor a patient’s immune response to the therapeutic drug by detecting anti-Amatuximab antibodies (ADAs) in patient samples.

Context and methodology:

• Bridging ADA ELISA principle: This assay exploits the bivalency of ADAs: one antibody arm binds the capture reagent (e.g., immobilized Amatuximab biosimilar on the plate), while the other arm binds the detection reagent (e.g., labeled Amatuximab biosimilar in solution).
• Reagent forms: The Amatuximab biosimilar can be immobilized directly or conjugated to biotin (for streptavidin-coated plates) as the capture reagent and labeled (biotinylated, HRP, etc.) for detection.
• Assay workflow:
  • The plate is coated with Amatuximab biosimilar (capture).
  • Patient serum containing potential ADAs is added, allowing ADAs to bridge between two Amatuximab molecules.
  • After washing, a labeled Amatuximab biosimilar (detection) is added, binding to the other ADA paratope.
  • Signal development (e.g., via HRP/TMB substrate) enables quantification of ADA levels.

Why use a biosimilar in this setup?

• Biosimilars are highly similar in structural, functional, and immunogenic features to the reference product, ensuring ADAs against the reference will also be detected by the biosimilar-based assay.
• Using a biosimilar facilitates comparative immunogenicity studies, ensuring clinical relevance and regulatory compliance.

Detection targets:

• The assay detects anti-Amatuximab antibodies (which may be generated in response to Amatuximab therapy), indicating a patient’s immunogenic response to the drug.
• Results enable monitoring for immune-mediated adverse effects and guiding therapeutic decisions.

Additional relevant points:

• The approach is similar to bridging ELISAs developed for other mAb therapies (e.g., adalimumab, infliximab, metuzumab), capable of measuring both free ADAs and, with adapted protocols, drug-ADA immune complexes.
• The sensitivity and specificity of this method depend on the drug’s quality and the precise nature of assay optimization.

Summary of key process steps:

• Immobilize Amatuximab biosimilar on ELISA plate (capture).
• Add serum sample to allow ADA binding.
• Add labeled Amatuximab biosimilar (detection).
• Detect and quantify ADAs indicating immunogenic response.

This method ensures reliable detection of patient-derived anti-Amatuximab antibodies, vital for tracking immunogenicity and therapeutic safety.