Anti-Human PD-L1 (CD274) (Avelumab) [Clone MSB0010718C]

Research-grade Avelumab biosimilars are commonly used as calibration standards (reference standards) and quality controls in pharmacokinetic (PK) bridging ELISAs, enabling the accurate quantification of Avelumab drug concentrations in serum samples in biosimilar development studies.

Essential context and details:

• Purpose in PK Bridging ELISA:

  • In biosimilar development, PK bridging ELISAs must reliably measure both the reference product and the biosimilar in patient serum, to demonstrate pharmacokinetic similarity between products.
  • Using a single research-grade biosimilar standard as the calibrant (analytical standard) reduces analytical variability compared to using multiple product-specific calibrators and allows direct analytical comparability.

• Assay Setup:

  • The ELISA plate is coated with reactant (commonly an anti-Avelumab antibody or the target antigen such as PD-L1).
  • Serial dilutions of the biosimilar standard are prepared in serum or plasma matrix to generate a standard curve, spanning the quantitation range (e.g., 10–12,800 ng/mL).
  • Unknown serum samples are incubated in parallel wells.
  • After binding and washing steps, a detection reagent—often HRP-conjugated—binds any captured Avelumab.

• Interpretation and Assessment:

  • The colorimetric signal is directly proportional to the Avelumab concentration.
  • Sample concentrations are interpolated from the standard curve generated using the research-grade biosimilar calibrator.
  • Multiple sets of standards and controls using both the biosimilar and reference product may be included during method validation to demonstrate that the assay quantifies both products equivalently.

• Regulatory and Best Practice Notes:

  • Regulatory guidance and scientific consensus recommend validating that the biosimilar standard and the reference product are bioanalytically equivalent in the assay; that is, the measured concentrations for both products at equivalent nominal concentrations should fall within predefined acceptance criteria (e.g., 80–125% relative recovery).
  • If equivalence is established, the biosimilar is used as the sole calibrator in subsequent PK bridging assays.

• Research-Grade Biosimilar for Controls:

  • In routine analysis, the same biosimilar is used to make quality control samples (QCs) at low, medium, and high concentrations to monitor assay precision and accuracy.

Summary Table: Usage of Avelumab Biosimilars in PK ELISA

In summary, research-grade Avelumab biosimilars serve a critical role as analytical standards and controls for sensitive, comparative PK measurements in ELISAs, ensuring analytical rigor and regulatory compliance in biosimilar assessments.

Research-grade anti-PD-L1 antibodies are primarily tested in syngeneic mouse models for studying tumor growth inhibition and characterizing tumor-infiltrating lymphocytes (TILs), with humanized models serving as complementary systems for translational validation.

Syngeneic Mouse Models

Primary Tumor Models

The most commonly utilized syngeneic models include MC38, Hepa1-6, CT-26, and EMT-6 tumors, which represent diverse cancer types with varying intrinsic tumor immunity profiles. These models are particularly valuable because they maintain intact immune systems that allow for comprehensive TIL characterization. Additional syngeneic models frequently employed include NS-1 multiple myeloma cells in BALB/c mice, where anti-PD-L1 treatment achieved 60% tumor-free rates at 24 days post-challenge.

Experimental Approaches

Syngeneic models demonstrate remarkable consistency in anti-PD-L1 responses regardless of initial tumor size. Studies show that tumor size increments of approximately 2 mm occur at the end of the first treatment cycle, with this effect being independent of initial tumor burden (small, medium, or large). The therapeutic response typically requires at least three doses to observe significant antitumor effects, with responders achieving either delayed tumor growth or complete regression.

TIL Characterization Capabilities

These models excel at enabling detailed TIL analysis through systematic depletion studies. Researchers can perform targeted depletion of CD8+ or CD4+ T cell populations to dissect their individual contributions to anti-PD-L1 efficacy. Flow cytometry and immunohistochemistry are routinely used to assess CD8+ T cell infiltration before and after treatment, providing mechanistic insights into treatment responses.

Humanized Mouse Models

Clinical Translation Models

Humanized models, particularly non-obese diabetic scid gamma (NSG) mice engrafted with human immune cells, serve as critical translational platforms. These models utilize human tumor cell lines such as PC-3 and HCT-116 following engraftment with allogeneic human T cells and monocyte-derived dendritic cells. Single-dose anti-PD-L1 treatment in these models significantly inhibits tumor growth when administered either at inoculation or after tumor establishment.

Advantages and Applications

Humanized models offer unique advantages for testing humanized antibodies and assessing human-specific immune responses. The development of humanized target knock-in mouse tumor models has proven instrumental for evaluating immunotherapy bioactivity in more clinically relevant contexts.

Comparative Effectiveness

Sensitivity Profiles

Among the various models tested, small molecule PD-1/PD-L1 inhibitors demonstrate broader effectiveness compared to antibodies alone. In comprehensive screenings across 12 syngeneic tumor types including colon, breast, bladder, kidney, pancreatic, and non-small cell lung cancers, melanoma, and lymphomas, small molecule inhibitors showed sensitivity in 11 out of 12 tumor types, while anti-PD-1 antibodies were effective in only 8 out of 12 models.

Mechanistic Insights

The effectiveness of anti-PD-L1 treatment correlates strongly with tumor mutation burden (TMB), particularly in syngeneic models where this relationship is more pronounced than with traditional antibody approaches. This correlation provides valuable predictive biomarkers for treatment response and helps guide model selection for specific research objectives.

Both syngeneic and humanized models remain essential for anti-PD-L1 research, with syngeneic models providing robust mechanistic insights and humanized models offering critical translational validation for clinical development.

Researchers use Avelumab biosimilars in combination with other checkpoint inhibitors—such as anti-CTLA-4 or anti-LAG-3 agents—to explore synergistic anti-tumor effects, particularly in preclinical and translational immune-oncology models. These studies aim to evaluate how blocking multiple non-redundant immune regulation pathways can enhance immune system-mediated tumor clearance and overcome resistance to single-agent therapies.

Key approaches and experimental models:

• In Vitro Combinatorial Studies: Researchers often use co-culture systems containing tumor cells, peripheral blood mononuclear cells (PBMCs), or natural killer (NK) cells to investigate how dual or triple blockade (e.g., Avelumab + anti-CTLA-4 or anti-LAG-3) modulates cytotoxicity, cytokine production, and immune cell activation. These studies can delineate direct anti-tumor effects and immune effector functions such as antibody-dependent cellular cytotoxicity (ADCC) specific to Avelumab biosimilars.

• Synergy in Blocking Parallel Pathways: Avelumab blocks the PD-L1/PD-1 interaction—thereby reinvigorating T cell activity—while CTLA-4 and LAG-3 inhibitors release other brakes on T cells at different points in their activation and proliferation cycles. Using biosimilars of these antibodies in combination allows researchers to test if dual checkpoint blockade produces additive or synergistic immune responses compared to monotherapy, especially where tumors adapt by upregulating alternative checkpoints.

• Animal Model Combinations: In syngeneic or humanized mouse models, Avelumab biosimilars (when engineered to be cross-reactive) are combined with anti-CTLA-4 or anti-LAG-3 agents to monitor tumor growth, immune infiltrate dynamics, and overall survival. While Avelumab’s unique ADCC capacity can’t be fully evaluated in regular murine models due to species-specific Fc interactions, models with human immune cells (e.g., PBMC-engrafted mice) enable partial recapitulation of combined therapeutic effects.

• Immunophenotyping and Biomarker Analysis: Combinatorial treatments are used to assess changes in tumor-infiltrating lymphocytes, expression of exhaustion markers, and cytokine profiles. This helps identify mechanisms underlying synergy and potential biomarkers predictive of response to multi-checkpoint inhibition.

• Rational Design for Overcoming Resistance: By combining Avelumab biosimilars with checkpoint inhibitors targeting CTLA-4 or LAG-3, researchers aim to prevent immune evasion—where tumors upregulate alternative checkpoints under selective pressure from PD-L1 blockade. This rational, multi-target blockade addresses tumor heterogeneity and plasticity observed with monotherapies.

Relevance of Biosimilars: Biosimilars are crucial in preclinical exploratory settings due to their availability, reduced cost relative to branded originators, and use in model optimization before advancing to clinical trials.

Summary Table:
| Agent | Checkpoint Targeted | Unique Properties | Role in Combination Studies ||----------------|---------------------|-------------------------------------|-------------------------------------------------|| Avelumab biosimilar | PD-L1 | Induces ADCC; blocks PD-L1/PD-1 | Core backbone in multi-checkpoint blockade || Anti-CTLA-4 biosimilar | CTLA-4 | Blocks early T cell inhibition | Enhances priming, prevents tolerance || Anti-LAG-3 biosimilar | LAG-3 | Relieves T cell exhaustion | Boosts rescue of exhausted T cells |

In summary, Avelumab biosimilars are combined with other checkpoint inhibitors to study whether simultaneous pathway blockade results in increased tumor immunogenicity, more robust T cell activation, and resistance mitigation, using in vitro assays, humanized models, and in-depth immune profiling.

In the context of immunogenicity testing, a biosimilar of Avelumab (or any monoclonal antibody) can be used as a capture or detection reagent in a bridging ADA ELISA to monitor a patient's immune response against the therapeutic drug. While specific details on Avelumab biosimilars are not provided in the search results, the following general approach applies:

Bridging ADA ELISA Protocol

1. Capture Reagent Preparation: The Avelumab biosimilar is biotinylated to serve as the capture reagent. This step involves modifying the drug with biotin, which allows it to bind to streptavidin-coated plates.

2. Plate Coating: Streptavidin-coated plates are used to capture the biotinylated Avelumab biosimilar. The biotinylated drug is added to the plates and allowed to bind via the streptavidin-biotin interaction.

3. Sample Addition: Patient serum samples are added to the wells containing the captured biotinylated Avelumab. Any anti-drug antibodies (ADAs) present in the serum will bind to the immobilized drug.

4. Detection Reagent: A second, differently labeled version of the Avelumab biosimilar is used as the detection reagent. This could be labeled with horseradish peroxidase (HRP) or a fluorescent dye, allowing for the detection of bound ADAs.

5. Detection: The detection reagent is added to the wells, and the ADAs are detected based on the label used. For HRP, a chromogenic substrate like TMB is used to provide a colorimetric signal proportional to the amount of ADAs present.

Role of Biosimilars in ADA Detection

• High Sensitivity: Biosimilars can be used to enhance the sensitivity of ADA detection by ensuring that the capture and detection reagents closely mimic the original drug, reducing potential differences in antigenicity.
• Comparability: The use of biosimilars as reagents can help ensure that the assay is compatible with both the originator drug and its biosimilars, making it useful for comparing immunogenicity across different formulations.

However, specific applications or research directly involving Avelumab biosimilars in ADA ELISA tests are not detailed in the provided search results. The general approach outlined above can be adapted based on the specific characteristics of Avelumab and its biosimilars.