Anti-Human TNF-α Adalimumab [Clone D2E7] — Fc Muted™

Research-grade Adalimumab biosimilars can serve as calibration standards or reference controls in pharmacokinetic (PK) bridging ELISA assays designed to quantify drug concentrations in serum samples, provided their potency, structure, and binding characteristics are comparable to the reference product (e.g., Humira).

Context and Application:

• In a PK bridging ELISA for adalimumab, known concentrations of research-grade adalimumab biosimilars are used to generate a standard curve against which unknown serum sample concentrations are measured.
• The biosimilar or reference standard is diluted serially in matrix-matched conditions (e.g., pooled human serum) to mimic clinical samples, ensuring the calibration curve accurately reflects real assay conditions.
• Calibration standards are usually prepared in the same serum matrix to account for any potential matrix effects that could alter antibody detection or binding. This is crucial for accuracy and reproducibility.
• The WHO International Standard (IS) for adalimumab was validated across multiple platforms and settings, demonstrating that a biosimilar adalimumab IS could harmonize drug quantification regardless of assay or laboratory. Both research-grade biosimilars and the IS were shown to provide comparable standard curves and quantification results, even when different in-house or commercial assay kits were used.
• In such studies, each laboratory receives ampoules of the reference material or biosimilar standard, which they reconstitute and run alongside their own standards and test samples. Dilutions are used to ensure standards cover the expected clinical range of adalimumab concentrations.
• The ELISA itself typically captures adalimumab from the serum using immobilized recombinant human TNF-α (its antigen), then detects bound antibody using a species-specific secondary antibody conjugated to a reporter enzyme. Concentrations are interpolated from the standard curve established by the biosimilar or reference preparation.

Key Points for Use of Biosimilar Standards:

• Equivalence: The biosimilar adalimumab must demonstrate comparable binding and ELISA detection as reference adalimumab.
• Validation: Comparative studies (in-house or multi-center) confirm that using a research-grade biosimilar for calibration yields results indistinguishable from those obtained with commercial standards or the WHO IS.
• Standardization: Adoption of a common biosimilar or international standard across assays promotes result harmonization, allowing for robust inter-laboratory comparison and improved therapeutic monitoring.
• Controls: In addition to calibration standards, research-grade biosimilars may be included as positive controls to monitor assay consistency and performance.

Summary Table: Role of Adalimumab Biosimilars in ELISA PK Assays

Research-grade adalimumab biosimilars are thus integral to ensuring the accuracy, consistency, and comparability of serum drug concentration measurements in PK bridging ELISAs, provided they are appropriately validated against an accepted reference.

The primary models used to study the effects of anti-TNF-α antibodies on tumor growth inhibition and characterize tumor-infiltrating lymphocytes (TILs) include both syngeneic and orthotopic transplant models.

Syngeneic Models

• Definition: Syngeneic models involve using immunocompetent mice with murine cell lines to form tumors. This allows for a better understanding of the interaction between the host immune system and tumors .
• Usefulness: These models are beneficial for studying the role of immune responses in tumor growth and the effects of anti-TNF-α antibodies on these processes. They are particularly useful for solid tumors with immunosuppressive microenvironments.
• Examples: Common syngeneic models include the CT26 (colorectal cancer) and 4T1 (breast cancer) murine cell lines .

Orthotopic Transplant Models

• Definition: Orthotopic transplant models involve transplanting tumor cells into their natural environment within the host. This model is particularly relevant for studying colorectal cancer, where CT26 cells are often used .
• Usefulness: These models are highly relevant for studying tumor growth and the effects of anti-TNF-α antibodies in a more naturalistic setting. They are particularly useful for examining the tumor microenvironment and the immune responses within it.
• Examples: An example is the use of the CT26 model to study the effects of anti-TNF-α monoclonal antibodies on suppressing tumor growth by enhancing apoptosis, inhibiting angiogenesis, and modulating the stromal response .

Humanized Models

• Definition: Humanized models involve using mice with human immune systems to study human tumor cells. While these models are crucial for studying human-specific immune interactions, they are not typically used for anti-TNF-α studies focused on murine systems.
• Usefulness: Humanized models are more relevant for preclinical studies of human-specific treatments and are not primarily used for basic research on anti-TNF-α effects in murine models.

In summary, syngeneic and orthotopic transplant models are the primary approaches for studying the effects of anti-TNF-α antibodies on tumor growth inhibition and characterizing TILs in a murine context. These models provide valuable insights into the immune microenvironment and the mechanisms of tumor growth modulation by anti-TNF-α therapy.

Adalimumab Biosimilars and Checkpoint Inhibitors in Immune-Oncology Research

Adalimumab biosimilars, such as SB5 and CT-P17, are designed to copy the effects of the reference adalimumab, a monoclonal antibody that targets tumor necrosis factor (TNF-α), and are primarily used to treat autoimmune and inflammatory diseases like rheumatoid arthritis, psoriasis, and inflammatory bowel disease. In contrast, immune checkpoint inhibitors (ICIs) such as anti-CTLA-4 (e.g., ipilimumab) and anti-LAG-3 antibodies are primarily used in cancer immunotherapy to enhance anti-tumor immune responses by blocking inhibitory signals on T cells.

Current Research Context

Adalimumab biosimilars are not typically used in combination with ICIs like anti-CTLA-4 or anti-LAG-3 in complex immune-oncology (IO) models. The primary focus in IO research is on combining different classes of checkpoint inhibitors (e.g., anti-CTLA-4 plus anti-PD-1/PD-L1) to leverage their distinct mechanisms of action and potentially achieve synergistic anti-tumor effects. These combinations are extensively studied in both preclinical models and clinical trials, with evidence showing improved outcomes in some cancers, though often at the cost of increased toxicity.

TNF-α inhibitors such as adalimumab and its biosimilars, however, have a different mechanism: they suppress inflammation by neutralizing TNF-α, a cytokine implicated in autoimmune and inflammatory pathways. There is little evidence in the current literature that researchers are combining TNF-α inhibitors (or their biosimilars) with checkpoint inhibitors to study synergistic effects in IO models. The rationale for such combinations is not well established, as TNF-α blockade might theoretically counteract the pro-inflammatory, immune-activating effects desired from checkpoint inhibition.

Potential Research Directions

If researchers were to explore the combination of adalimumab biosimilars with checkpoint inhibitors in IO models, they would likely do so to address specific hypotheses, such as:

• Modulating the tumor microenvironment (TME): TNF-α can influence the TME, potentially affecting immune cell infiltration and function. Combining TNF-α inhibition with checkpoint blockade could be tested to see if it alters the immune contexture of tumors in a way that enhances or dampens anti-tumor immunity.
• Managing immune-related adverse events (irAEs): Checkpoint inhibitors can cause severe inflammatory side effects. TNF-α inhibitors are sometimes used to treat these irAEs, so their biosimilars could be studied for preventing or mitigating toxicity while maintaining anti-tumor efficacy.
• Exploring paradoxical effects: There is some preclinical evidence that TNF-α can have both pro- and anti-tumor effects depending on context. A research question could be whether TNF-α blockade, via adalimumab biosimilars, unexpectedly synergizes with checkpoint inhibition in certain settings.

Current Evidence Gaps

No published studies or reviews in the provided literature describe the use of adalimumab biosimilars together with checkpoint inhibitors in IO models. The main focus of biosimilar development has been on demonstrating equivalence to the reference product in their primary indications (autoimmune/inflammatory diseases), not on novel combinations in cancer immunotherapy.

Combination strategies in IO are instead focused on other drug classes—such as chemotherapy, targeted therapies, epigenetic drugs, and various small molecule inhibitors—alongside ICIs. The rationale for these combinations is based on mechanistic synergies that enhance T cell activation, overcome resistance, or modulate the TME in favor of anti-tumor immunity.

Summary Table: Adalimumab Biosimilars vs. Checkpoint Inhibitors in Research

Conclusion

There is no current evidence that researchers are using adalimumab biosimilars in conjunction with checkpoint inhibitors (e.g., anti-CTLA-4 or anti-LAG-3) to study synergistic effects in complex immune-oncology models. The primary research focus remains on combining different classes of ICIs or ICIs with other immunomodulatory agents to enhance anti-tumor immunity. Any exploration of adalimumab biosimilars in this context would be highly speculative and would require strong preclinical rationale, given the divergent mechanisms and clinical indications of these agents.

Using Adalimumab Biosimilar in Bridging ADA ELISA

In the context of immunogenicity testing, adalimumab biosimilars can be used as capture or detection reagents in bridging ADA (Anti-Drug Antibody) ELISA assays to monitor a patient's immune response against the therapeutic drug. This approach is crucial for detecting antibodies that may be generated against adalimumab, affecting its efficacy and safety.

Principle of Bridging ADA ELISA

1. Preparation of Plates: The ELISA plate is coated with adalimumab biosimilar, which serves as the capture reagent. This coating allows the adalimumab or its biosimilar to capture ADA present in patient samples.

2. Sample Incubation: Patient serum or plasma samples are added to the wells. Any ADA present in these samples will bind to the adalimumab biosimilar coated on the plate.

3. Detection Reagent: A labeled version of the adalimumab biosimilar (e.g., HRP-conjugated) is then added as the detection reagent. This labeled adalimumab will bind to the ADA that has been captured by the adalimumab biosimilar on the plate, forming a "bridge" between the two adalimumab molecules.

4. Detection: After washing to remove unbound components, a chromogenic substrate is added. The enzymatic activity of the HRP conjugate converts the substrate into a colored product, which is proportional to the amount of ADA present in the sample.

Why Use Adalimumab Biosimilar?

• Consistency and Similarity: Adalimumab biosimilars are highly similar to the reference product in terms of structure and function, ensuring consistent results across different batches and assays.
• Cost-Effectiveness: Biosimilars may be more cost-effective than using the original adalimumab, which can reduce the overall cost of the assay.
• Versatility: Biosimilars can be used in both capture and detection roles, providing flexibility in assay design.

However, ensuring the biosimilar is of high quality and truly similar to the original adalimumab is crucial for accurate ADA detection.

Examples and Applications

• Detection of ADA: Bridging ELISA using adalimumab biosimilars can detect ADA in patient samples treated with adalimumab or its biosimilars, helping in assessing immune responses and making informed treatment decisions.

• Clinical Utility: The presence of ADA can impact the efficacy of adalimumab, and monitoring these antibodies can guide treatment adjustments or the selection of alternative therapies.