Anti-Human TNF-α (Infliximab) [Clone ABP-710]

Research-grade Infliximab biosimilars are used as calibration standards or reference controls in pharmacokinetic (PK) bridging ELISA assays to enable accurate and interchangeable measurement of infliximab concentrations in serum, regardless of the specific product (originator or biosimilar) administered.

In PK bridging ELISA for infliximab, the main steps involving biosimilars as standards are:

• Calibration Curve Preparation:
  The biosimilar is serially diluted in a matrix (often serum or buffer that mimics matrix effects) to generate calibration standards at known concentrations. These standards establish the calibration curve against which unknown sample concentrations are assessed.

• Reference Standard Interchangeability:
  Studies report that both originator infliximab (Remicade) and its biosimilars (e.g., Remsima) can be used interchangeably for assay calibration in ELISA and LC-MS/MS-based bioanalytical methods. Cross-verification shows nearly identical quantitation across products, supporting their use as reference controls in clinical PK assays. This interchangeability is essential for therapeutic drug monitoring in patients treated with either the originator or biosimilar.

• Assay Design:
  In the bridging ELISA, the solid phase is typically coated with TNFα, which specifically binds infliximab (originator or biosimilar) from patient serum. Detection is achieved using anti-human IgG or other suitable antibody conjugates. The measured signal (e.g., absorbance) from patient samples is then compared to the calibration curve generated from the biosimilar-reference standards to quantify drug levels.

• Control and Quality Verification:
  Biosimilars are used as positive controls to validate assay performance in each run. Their concentration and recovery help ensure robustness and reproducibility of results.

Key Supporting Insights

• Robust Quantitation:
  Biosimilars show linear, accurate concentration-response in calibration curves (e.g., 0.293 to 300 µg/ml), with high precision in quantitation matching the originator performance.
• Assay Harmonization:
  The use of biosimilar standards facilitates harmonization and mutual comparability between different assay platforms and labs, addressing differences in TNFα coating, immune complex formation, and potential for interference from anti-drug antibodies.
• Clinical Relevance:
  Dose adjustment and therapy decisions typically rely on trough infliximab levels (often <3–5 µg/ml), which can be accurately determined using biosimilar calibration curves.

In summary, research-grade infliximab biosimilars are pivotal for preparing calibration standards and reference controls in PK bridging ELISA assays, ensuring reliable quantitation of drug concentration in serum samples and facilitating consistent clinical monitoring whether patients receive originator or biosimilar therapy.

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

Key context and details:

• Syngeneic Mouse Models:
  These models use mouse tumor cell lines grafted into immunocompetent mice of matching strains, such as CT26 colon carcinoma in BALB/c mice. Anti-TNF-α antibodies are administered to evaluate tumor growth inhibition and the immune microenvironment, allowing analysis of TILs using immunohistochemistry and flow cytometry.

  • For example, anti-TNF-α mAb was administered in a CT26 orthotopic colorectal cancer model (BALB/c mice), with characterization of immune infiltrates, apoptosis, and stromal responses.
  • Other frequent tumor cell lines used include B16F10 (melanoma), RENCA (renal carcinoma), and 4T1 (breast cancer).
  • These models support detailed TIL characterization, benefiting from fully functioning mouse immune systems.

• Humanized Mouse Models (less common specifically for anti-TNF-α studies):
  Humanized mice are immunodeficient strains engrafted with human immune cells and/or human tumors. While most anti-TNF-α studies use syngeneic models, humanized models are sometimes used to study the effects of anti-TNF-α antibody in a setting that more closely mimics the human immune-tumor interaction, including assessments of TILs.

• Experimental Workflow in Syngeneic Models:

  1. Tumor cells are implanted (subcutaneous or orthotopic) into inbred mice.
  2. Anti-TNF-α antibody is administered systemically.
  3. Tumor growth rates are measured, and tumors are harvested for immunohistochemical and flow cytometric analysis of TILs, such as CD8+ and CD4+ T cells, myeloid populations, and NK cells.
  4. The effect on apoptosis, stromal response, and angiogenesis is often assessed in parallel.

• Humanized Models:
  These models can allow the evaluation of human-specific antibodies and the study of human TILs but are less commonly used for anti-TNF-α preclinical research compared to syngeneic models. They are particularly relevant when the antibody does not cross-react with mouse TNF-α.

Summary Table:

Most studies, including those exploring anti-TNF-α effects on tumor immunity and TILs, rely on syngeneic mouse models such as CT26 in BALB/c or B16F10 in C57BL/6.

If you require protocols or detailed experimental setups, please specify the tumor type or antibody species (murine, human, chimeric).

Researchers use infliximab biosimilars in combination with checkpoint inhibitors (such as anti-CTLA-4 or anti-LAG-3 biosimilars) to investigate their potential synergistic effects in complex immune-oncology models, particularly by examining how TNF-α blockade can modulate both therapeutic efficacy and immune-mediated toxicity (irAEs).

Key points and supporting details:

• Model Rationale: Infliximab (and its biosimilars) targets TNF-α, a central cytokine in inflammation that is also involved in immune therapy-associated adverse events (irAEs) and tumor biology. Preclinical and translational studies have shown that TNF-α blockade can improve the safety profile of immune checkpoint inhibitor (ICI) therapies (e.g., anti-CTLA-4, anti-PD-1/PD-L1), resolving irAEs without significantly compromising the antitumor immune response.

• Combination and Mechanisms Studied: Researchers combine infliximab biosimilars with checkpoint inhibitors in animal models (like mouse melanoma or colon cancer models) to examine:

  • Whether TNF-α inhibition can reduce ICI-induced adverse effects (notably colitis and other autoimmune toxicity).
  • Whether TNF-α blockade permits or potentiates stronger antitumor T cell infiltration and improved tumor regression when combined with ICIs, enhancing overall efficacy.
  • The molecular and cellular mechanisms underlying any observed synergy or antagonism, such as changes in T cell activation, apoptosis, cytokine profiles, and tumor immune microenvironment composition.

• Role of Biosimilars: Using biosimilars (like ABP 710) provides functionally equivalent substitutes for the originator infliximab, meeting rigorous comparability standards—making them suitable for both preclinical synergy studies and for expanding availability in translational research or clinical trials.

• Checkpoint Inhibitor Combinations: Synergistic effects are of particular interest with anti-CTLA-4 or anti-LAG-3 biosimilars, as these enhance T cell responses but may also cause off-target inflammation. The combined administration helps distinguish mechanisms of tumor immunity from those of autoimmunity, allowing researchers to optimize dosing or sequencing to balance efficacy and safety.

• Observed Outcomes: In mouse studies, the combination of TNF-α blockade (via infliximab or biosimilar) with anti-CTLA-4/anti-PD-1 therapy led to better tumor control and lower rates of severe immune adverse events. These results have prompted retrospective and prospective studies in humans to further delineate safety and potential augmentation of antitumor effects.

• Immunogenicity Considerations: Combinatorial approaches may also affect the immunogenicity (anti-drug antibody formation) of protein therapeutics, an important parameter that is actively monitored in these studies.

In summary, infliximab biosimilars are combined with checkpoint inhibitors in immune-oncology models to study how TNF-α blockade modulates both therapeutic efficacy and toxicity, providing critical insights into the mechanisms of synergy, with significant translational potential for optimizing cancer immunotherapy regimens.

In a bridging anti-drug antibody (ADA) ELISA for monitoring a patient's immune response to infliximab, an infliximab biosimilar can serve as both the capture and detection reagent to identify anti-infliximab antibodies in patient samples.

How Infliximab Biosimilar Is Used:

• Capture Reagent: The ELISA plate is coated with the infliximab biosimilar, which acts as the antigen to capture any anti-infliximab antibodies (ADA) present in the patient's serum or plasma.
• Detection Reagent: After washing, a detection reagent—typically the same or a labeled form of infliximab biosimilar (e.g., biotinylated, enzyme-, or ruthenium-labeled)—is added. This tagged infliximab binds to another epitope on the ADA, forming a "bridge" between the plate-bound and the detection molecule, because each ADA is bivalent and can bind two infliximab molecules.

Assay Principle (Bridging ELISA Format):

1. Coating: Wells are coated with the infliximab biosimilar.
2. Sample Addition: Patient samples are added. If anti-infliximab antibodies are present, they will bind to the immobilized infliximab.
3. Detection: Labeled (e.g., biotinylated or enzyme-conjugated) infliximab biosimilar is added. This binds to the other arm of the ADA, creating a "bridge" structure.
4. Signal Generation: A secondary reagent (e.g., streptavidin-HRP if the detection antibody is biotinylated, or detection of enzyme activity) is added to generate a measurable signal proportional to the amount of ADA present.

Why Use a Biosimilar?

• Biosimilars are structurally and functionally highly similar to the originator infliximab and have the same epitopes recognized by ADA, making them suitable for use as capture and detection reagents in the assay.

Application:

• This method specifically detects antibodies that bind infliximab (including both originator and biosimilar versions) and is widely used to monitor immunogenicity, which can affect drug efficacy and safety in patients receiving infliximab therapy.

Important Considerations:

• Bridging ELISAs require bivalent antibodies, so they may not detect monovalent anti-drug antibodies.
• Free (circulating) infliximab in patient serum can interfere with ADA detection, so samples may need to be taken at trough drug levels or use drug-tolerant ADA assays.

Summary:
An infliximab biosimilar, properly labeled, is used both to coat assay plates (capture) and as a detection reagent in a bridging ADA ELISA, enabling sensitive monitoring of anti-infliximab immune responses in patients.