Anti-Human TNF-α Adalimumab [Clone D2E7]

Research-grade Adalimumab biosimilars are used as calibration standards or reference controls in pharmacokinetic (PK) bridging ELISAs by constructing a standard curve against which the unknown drug concentrations in serum samples are measured. This allows the precise quantification of adalimumab (originator or biosimilar) in patient samples and supports cross-comparisons between biosimilar and reference products.

Key aspects:

• Role in Calibration/Standard Curves: Research-grade adalimumab biosimilars are serially diluted in serum or assay buffer to generate a standard curve that spans the expected drug concentration range in clinical samples. This curve forms the basis for quantifying unknowns by interpolating their ELISA signals.

• Reference or Control: The biosimilar can be used as a reference calibrator or quality control sample to ensure assay consistency. PK bridging studies often use both the originator (e.g., Humira®) and biosimilar (e.g., ABP501) as standards to confirm assay equivalence and enable bridging of PK data between the two products.

• Demonstrating Interchangeability and Assay Suitability: Studies validate that both biosimilar and reference standards yield overlapping standard curves and comparable results, ensuring the assay is sensitive and specific for both molecules. For example, the LISA-TRACKER Adalimumab assay demonstrated high correlation (R² ~0.94-0.98) and similar slopes when measuring both ABP501 biosimilar and Humira® in human serum, confirming suitability for quantifying either in clinical samples.

• Assay Harmonization: International standards (e.g., the WHO International Standard for Adalimumab) or well-characterized biosimilar reference preparations are employed to enable assay harmonization across labs. Concordance in assay readouts using different standards (either in-house, commercial kit, or international) demonstrates interchangeability and supports reproducible PK comparisons in bridging studies.

• Serum Matrix Consideration: Calibration standards must be prepared in matrices matched to patient samples—typically pooled human serum spiked with known concentrations of the biosimilar—to account for serum-specific effects on assay signal.

• Assay Validation and Controls: In addition to calibration curves, biosimilars may also serve as positive controls (QCs) in each run. Assay accuracy, precision, specificity, and parallelism between biosimilar and originator standards are key validation criteria.

Summary Table: Use of Adalimumab Biosimilars as Calibration Standards in PK Bridging ELISA

In summary, PK ELISA bridging studies rely on well-characterized biosimilar and/or originator adalimumab standards for accurate, reproducible drug quantification, enabling regulatory and clinical cross-comparison of biosimilar and reference products.

The primary in vivo models where a research-grade anti-TNF-α antibody is administered to study tumor growth inhibition and characterize tumor-infiltrating lymphocytes (TILs) are syngeneic mouse models and, to a lesser extent, humanized mouse models.

Key details:

• Syngeneic models use mouse tumor cell lines (e.g., CT26 colorectal carcinoma) implanted into immunocompetent mice (such as BALB/c or C57BL/6).

  • Anti-TNF-α mAbs are administered to these mice to study effects on tumor growth, immune pathways, stromal response, angiogenesis, and TIL populations such as lymphocytes and macrophages.
  • TILs can be characterized by flow cytometry and immunohistochemistry for specific immune markers after antibody treatment.

• Humanized mouse models involve engrafting immunodeficient mice with human immune cells and sometimes human tumor cells.

  • While less common for anti-TNF-α studies (since most anti-TNF-α mAbs are mouse- or rat-specific in standard preclinical work), these models enable assessment of human TIL responses and cytokine interactions in an in vivo context relevant to human immunotherapies.

• Experimental endpoints commonly include tumor volume/weight, flow cytometry for TIL subsets (e.g., CD8+, CD4+, macrophages), and evaluation of immune gene expression or protein markers.
• These models are considered most relevant for assessing both tumor growth inhibition and TIL phenotype/function in response to anti-TNF-α intervention.

Patient-derived xenograft (PDX) models are rarely used for direct anti-TNF-α mAb studies targeting the immune compartment, as these mice lack fully functional immune systems unless humanized.

Summary:
Syngeneic mouse models are the gold standard for in vivo anti-TNF-α administration to characterize TILs and tumor growth inhibition. Humanized mice are also employed for translational studies involving human immune-tumor interactions, though less frequently for anti-TNF-α mAbs unless human-specific reagents are used.

Current State of Adalimumab Biosimilar Use in Complex Immune-Oncology

Adalimumab is a monoclonal antibody targeting tumor necrosis factor-alpha (TNF-α) and is widely used in autoimmune diseases. Its biosimilars—near-identical copies with similar efficacy and safety profiles—are increasingly available and studied in various contexts. However, there is no published evidence in the provided search results or in the literature that adalimumab or its biosimilars are being combined with checkpoint inhibitors (such as anti-CTLA-4 or anti-LAG-3 antibodies, or their biosimilars) in complex immune-oncology models to study synergistic effects.

Synergistic Studies in Immune-Oncology: Focus on Checkpoint Inhibitor Combinations

Current research on immune-oncology combination therapies primarily explores combinations of immune checkpoint inhibitors (ICIs) themselves (e.g., anti-CTLA-4 plus anti-PD-1/PD-L1), or ICIs with chemotherapy, targeted therapies, radiotherapy, and other agents that modulate the tumor microenvironment. These combinations aim to overcome resistance, enhance T-cell infiltration, and improve clinical outcomes in various cancers.

• Double checkpoint blockade (e.g., ipilimumab + nivolumab) is well-studied, demonstrating synergy in some cancers, but with increased toxicity.
• Novel checkpoint targets like LAG-3, TIGIT, and others are under investigation, often in combination with PD-1/PD-L1 or CTLA-4 inhibitors.
• Combining ICIs with other pathways—such as PI3K/AKT/mTOR inhibitors, BRAF/MEK inhibitors, or drugs targeting myeloid-derived suppressor cells—is a growing field, with preclinical and early-phase clinical data suggesting potential benefits.

Scientific Rationale for TNF-α Inhibition in Immuno-Oncology

While TNF-α inhibitors like adalimumab are not standard in cancer immunotherapy, there is theoretical interest in their potential to modulate the tumor microenvironment and inflammation. However, clinical and preclinical evidence supporting the combination of TNF-α inhibitors with checkpoint inhibitors is lacking. In fact, TNF-α inhibitors are primarily used to manage immune-related adverse events (irAEs) from checkpoint inhibitors rather than as direct anti-cancer agents.

Challenges and Considerations

• Lack of Precedent: No published studies describe the combination of adalimumab (or its biosimilars) with checkpoint inhibitors (or their biosimilars) for synergistic anti-tumor effects.
• Mechanistic Concerns: TNF-α blockade may suppress certain inflammatory processes necessary for effective anti-tumor immunity.
• Safety: Combining biologic agents increases the risk of immune suppression, infections, and other adverse events.
• Regulatory and Clinical Hurdles: Given the absence of preclinical rationale and safety data, such combinations are not being actively pursued in clinical trials.

Conclusion

Current research in immune-oncology focuses on synergistic combinations of checkpoint inhibitors with each other or with other classes of anti-cancer agents, not with TNF-α inhibitors like adalimumab. There is no evidence from the literature that researchers are using adalimumab biosimilars in conjunction with checkpoint inhibitor biosimilars to study synergistic effects in complex immune-oncology models. Any future exploration of such combinations would require strong preclinical rationale, careful safety assessment, and rigorous clinical evaluation.

In a bridging ADA ELISA for immunogenicity testing, a biosimilar Adalimumab can serve as both the capture and detection reagent to specifically measure anti-adalimumab antibodies (ADAs) generated in patients undergoing therapy with Adalimumab or its biosimilars.

Key methodology details:

• Capture reagent: The biosimilar Adalimumab is immobilized on the microtiter plate. When patient serum is added, any ADA present will bind to this immobilized biosimilar Adalimumab.
• Detection reagent: After washing away unbound substances, a second molecule of Adalimumab (labeled, e.g., with HRP or biotin) is added as the detection reagent. Because ADAs are typically bivalent, they can simultaneously bind both the immobilized and the labeled Adalimumab, forming a “bridge”.
• Result interpretation: The formation of this bridged complex allows for enzyme-mediated colorimetric detection, where the signal intensity is proportional to the amount of ADA present in the sample.

Why use a biosimilar as both capture and detection reagent?

• Biosimilar Adalimumab is structurally and functionally nearly identical to the originator, making it appropriate for ADA assays against both forms.
• It ensures reagent availability and cost-effectiveness, particularly for commercial ADA test kits.
• The use of biosimilar mitigates issues of supply restrictions or high cost associated with the originator biologic, while maintaining assay specificity.

Bridging ELISA advantages:

• It reliably detects bivalent ADAs, the most immunogenic and clinically-relevant species.
• Assay specificity comes from the use of authentic drug (here, a biosimilar) for both capture and detection, reducing cross-reactivity.

Context of patient monitoring:

• By quantitatively measuring ADA levels, clinicians can evaluate the immunogenicity of Adalimumab therapy.
• The technique allows monitoring for loss of drug efficacy, hypersensitivity reactions, and other immune-mediated adverse effects.

Summary table: Biosimilar Adalimumab in bridging ADA ELISA

This approach represents a standardized and sensitive method for monitoring a patient’s immune response against therapeutic Adalimumab or its biosimilars during treatment.