Anti-Human EGFR x MET (Amivantamab)

Research-grade Amivantamab biosimilars are commonly used as calibration standards (analytical standards) or reference controls in pharmacokinetic (PK) bridging ELISA assays to quantitatively measure Amivantamab concentrations in serum samples, ensuring comparability between test and reference (originator) products in biosimilar development.

Essential context and key methodological details:

• Single PK Assay Approach:
  Best practice in biosimilar PK assay development is to employ a single ligand-binding (ELISA) assay using a single analytical standard (usually the biosimilar or reference product), ensuring the assay can accurately measure both the biosimilar and the reference Amivantamab in the same manner. This minimizes assay variability and avoids the need for multiple crossover calibrations.

• Calibration Standards:
  The standard curve in the ELISA is generated by spiking known concentrations of the research-grade Amivantamab biosimilar into blank human serum. These standards are then used to create the calibration curve against which unknown sample concentrations are quantified.

• Reference Controls and Quality Controls (QCs):
  QC samples can be prepared using the biosimilar, the reference (originator) product, or both, at several concentration levels to test the assay's performance and confirm bioanalytical equivalence. During assay validation, both the biosimilar and reference product are typically measured to ensure the method gives comparable and reliable results for both.

• Assay Validation and Equivalence Testing:
  The assay must be qualified to confirm it has sufficient precision, accuracy, and robustness for both products. Analytical equivalence is statistically demonstrated (for example, assessing whether the ratio of measured concentrations between products falls within a predefined equivalence window, e.g., 0.8–1.25). If equivalence is established, a single analytical standard (commonly the biosimilar) is selected for all subsequent quantitation.

• Operational Details from Commercial Kits:
  ELISA kits like the KRIBIOLISATM Amivantamab ELISA specify that standards (prepared from research-grade Amivantamab) and test samples should be assayed in duplicate, and samples at high concentrations may need to be diluted to avoid non-linear effects (hook effect).

Summary Table: Use of Biosimilar Amivantamab in PK ELISA

In summary:
Research-grade Amivantamab biosimilars serve as the benchmark standard curve calibrator and/or reference control in PK ELISA bridging assays, providing quantitative comparability for bioanalytical PK studies crucial to biosimilar and reference product equivalence assessment.

The primary in vivo models used to administer research-grade anti-EGFR x MET bispecific antibodies for studying tumor growth inhibition and characterization of tumor-infiltrating lymphocytes (TILs) are syngeneic mouse models and humanized mouse xenograft models. Each model type serves distinct research goals related to antibody efficacy and immune response characterization.

Model Types and Their Use:

• Syngeneic Mouse Models:
  These models involve implanting mouse-derived tumor cell lines into immunocompetent mice of the same genetic background. They provide a fully functional immune system, enabling accurate assessment of TIL responses and immune-mediated mechanisms of tumor growth inhibition. Syngeneic models are a standard for immunotherapy studies, permitting detailed profiling of immune cell infiltration and phenotypic changes following antibody treatment. Common syngeneic models (e.g., RENCA, CT26, EMT6, B16F10) are widely used to compare differences in immunogenicity and immune cell infiltration, as well as to test mechanistic hypothesis regarding drug-induced immune changes.

• Humanized Mouse Xenograft Models:
  In these models, human tumor cells are implanted into immunodeficient mice that have been reconstituted with components of a human immune system, allowing assessment of anti-EGFR x MET antibody efficacy in a human cellular context. Humanized models better mimic human TIL phenotypes and dynamics and are necessary when studying bispecific antibodies that target human EGFR and MET, since mouse HGF does not interact strongly with human MET. For example, studies employing a bispecific EGFR/MET antibody (MetHer1) tested its efficacy using a non-small cell lung cancer xenograft in mice bearing an autocrine HGF loop, requiring human HGF expression for relevant pathway engagement.

Experimental Context:

• Syngeneic models are ideal for dissecting immunotherapy mechanisms and immune cell composition after treatment, especially for immune checkpoint inhibitors or bispecific antibodies when the goals include comprehensive TIL characterization using flow cytometry and gene expression.
• Humanized models are used when the antibody specifically requires human EGFR and MET targets, or when the immune response needs to be studied in a human (rather than purely murine) context. These experiments may combine antibody administration with profiling of TIL populations in tumor tissue derived from human patients or cell lines.

Key Considerations:

• If the primary aim is TIL characterization and immune system interactions, then syngeneic mouse models are preferred due to the fully functional and genetically matched murine immune system.
• For direct testing of human-specific antibody constructs (e.g., anti-EGFR x MET bispecifics targeting human epitopes), humanized xenograft models are necessary, but these have limitations in immune system completeness and may require further engineering for robust lymphocyte reconstitution and HGF signaling.

Summary Table

Syngeneic models provide a rich environment for TIL profiling and response prediction to immunotherapeutics, while humanized xenografts are often deployed when the antibody requires human target engagement and the immune context must approximate that of human patients.

Researchers use the Amivantamab biosimilar in combination with other checkpoint inhibitors, such as anti–PD-1 (e.g., pembrolizumab), to investigate potential synergistic anti-tumor effects in complex immune-oncology models, focusing on mechanisms like immune cell activation, tumor microenvironment remodeling, and overcoming acquired resistance.

Amivantamab is a fully human IgG1 bispecific antibody targeting EGFR and MET, and is available as a biosimilar for research use to study receptor blockade, downmodulation, and immune effector activation in EGFR- and MET-driven tumor models. In preclinical studies, combining Amivantamab biosimilar with PD-1 blockade (pembrolizumab) in humanized patient-derived xenograft (PDX) models of squamous cell carcinomas resulted in significant tumor growth reduction compared to monotherapies.

Key mechanistic insights from combination studies include:

• Enhanced infiltration of CD8+ T cells: Combination therapy led to increased numbers of granzyme B–producing CD8+ cytotoxic T cells within the tumor, associated with more effective tumor cell killing.

• Remodeling the tumor microenvironment (TME): Amivantamab reduced glycolytic markers (SLC16A3, LDHA) in EGFR/MET-high tumor cell subpopulations, mitigating the immunosuppressive effects of lactate accumulation and facilitating better immune cell infiltration.

• Stimulation of central memory T cells: Combination approaches promoted the presence of central memory CD8+ T cells, which mediate stronger and longer-lasting anti-tumor immunity.

While published research specifically discusses synergistic effects with PD-1/PD-L1 inhibitors (e.g., pembrolizumab), the basic methodology—using amivantamab biosimilar in conjunction with checkpoint inhibitors—can in principle be extended to other classes such as anti–CTLA-4 or anti–LAG-3 biosimilars, to delineate their impact on T cell subsets, immune cell activation, and TME modulation. Experimental designs typically involve:

• Treating tumor-bearing humanized mouse models or in vitro tumor co-cultures with amivantamab biosimilar alone, checkpoint inhibitor alone, and combinations.
• Measuring tumor growth, quantifying immune cell infiltration and activation (e.g., flow cytometry for CD8/GZMB+ T cells).
• Profiling transcriptomic or metabolic changes in the TME to reveal mechanistic underpinnings of any observed synergy.

Clinical studies are ongoing to further explore combinations of Amivantamab with different immune checkpoint inhibitors (e.g., cetrelimab in NSCLC), emphasizing the translational potential of these synergistic strategies.

To date, published results provide strong preclinical evidence for synergy between amivantamab biosimilar and anti–PD-1 blockade, particularly in models showing resistance to single-agent immunotherapy, with enhanced immune infiltration and tumor cell killing as central findings. Equivalent studies with anti–CTLA-4 or anti–LAG-3 biosimilars are rational next steps following this methodology.

An Amivantamab biosimilar can be used as both the capture and detection reagent in a bridging anti-drug antibody (ADA) ELISA to detect patient antibodies developed against Amivantamab during immunogenicity testing.

How it works in a bridging ADA ELISA:

• Assay Principle: In a bridging ELISA, the ADA (antibody) in patient serum acts as a bridge between an immobilized (captured) version of the therapeutic (here, the Amivantamab biosimilar) and a labeled version of the same drug for detection.
• Use as Capture Reagent: The plate is coated with the Amivantamab biosimilar (or a biotinylated version is captured on streptavidin-coated plates).
• Sample Incubation: Patient serum containing potential ADA is added. If ADA is present, it binds to the immobilized Amivantamab.
• Detection Reagent: A second, labeled preparation of the Amivantamab biosimilar (e.g., HRP- or dye-labeled) is then added. This labeled biosimilar will bind to another epitope on the ADA, forming a "bridge" between the capture and detection reagents.
• Signal Generation: Substrate is added to produce a measurable signal, which indicates the presence of ADA in the patient sample.

Why use a biosimilar as reagent?

• The biosimilar is employed because it is structurally equivalent to the reference product, binds the same ADAs, and is suitable for detecting patient antibodies against the therapeutic drug.
• Using both the capture and detection reagents from the same biosimilar ensures high specificity and sensitivity for antibodies that recognize drug epitopes, regardless of minor differences between biosimilar and original drug.

Key points for ADA bridging ELISA using an Amivantamab biosimilar:

• Detection is based on the ability of patient ADA to simultaneously bind two molecules of the biosimilar: one immobilized (capture) and one labeled (detection).
• Assay sensitivity and specificity depend on the quality and epitope coverage of the biosimilar reagent, as well as optimization for matrix effects and drug interference.

This strategy is standard for immunogenicity testing against monoclonal antibody therapeutics and their biosimilars, including bispecific agents like Amivantamab.