Anti-Human Nectin-4 (Enfortumab) [Clone AGS-22M6E] — Fc Muted™

Research-grade enfortumab biosimilars serve as critical analytical tools in pharmacokinetic bridging ELISAs, functioning as both calibration standards and reference controls to enable accurate quantification of drug concentrations in serum samples. The use of biosimilars in this context represents a sophisticated bioanalytical approach that addresses the unique challenges of measuring complex biologics like antibody-drug conjugates.

Biosimilar Standards in Single-Method PK Assays

The most optimal approach for PK assays involves developing a single analytical method that uses a single analytical standard for quantitative measurement of both biosimilar and reference products. This strategy offers significant advantages by decreasing inherent variability associated with multiple methods and eliminating the need for crossover analysis during blinded clinical studies. Research-grade enfortumab biosimilars are particularly valuable as they can serve as the primary analytical standard once bioanalytical comparability is established between the biosimilar and reference products.

Calibration Curve Development and Implementation

In PK bridging ELISAs, enfortumab biosimilars are used to create comprehensive calibration curves that span the expected concentration range of clinical samples. For human PK assays, biosimilar standards are typically prepared in human serum at concentrations ranging from 50 to 12,800 ng/mL, with nine independent standard curve points being common practice. Available enfortumab ELISA kits demonstrate sensitivity levels as low as 0.188 ng/mL with assay ranges of 0.313-20 ng/mL for some formats, while others offer 6-8 point calibration curves with sensitivities around 15 ng/mL.

The calibration standards are prepared by serial dilution of the biosimilar reference material in the same matrix as the test samples - typically neat pooled human serum. This matrix matching is crucial for accurate quantification, as it ensures that any matrix effects impact both standards and samples equally. The resulting standard curve establishes the relationship between known concentrations and measurable signals, allowing for interpolation of unknown sample concentrations.

Quality Control and Reference Controls

Enfortumab biosimilars also function as quality control materials prepared at multiple concentration levels within the analytical range. These QC samples, prepared independently from the calibration standards, serve to monitor assay performance and ensure data quality throughout the analysis. Typical QC concentrations include low (150 ng/mL), medium (1,250 ng/mL), and high (9,600-12,800 ng/mL) levels that bracket the expected sample concentrations.

Bioanalytical Comparability Assessment

A critical aspect of using biosimilars as standards involves establishing bioanalytical equivalence between the biosimilar and reference products within the assay method. This process begins with robust method qualification studies that generate precision and accuracy datasets for both products, followed by statistical analysis to determine bioanalytical equivalence. The evaluation typically involves comparing 90% confidence intervals to predefined equivalence criteria (often 0.8-1.25 fold), ensuring that the biosimilar can serve as an appropriate surrogate standard for measuring the reference product.

ELISA Format and Detection Strategy

Most enfortumab PK assays utilize sandwich ELISA formats, where the drug is captured between two antibodies - a capture antibody and a detection antibody. The biosimilar standards are processed identically to test samples, undergoing the same binding, washing, and detection steps. Results are typically collected by measuring absorbance, where higher absorbance values correlate with higher drug concentrations. Background subtraction is performed to obtain corrected absorbance values that are used for quantification.

Validation and Regulatory Compliance

When biosimilars serve as analytical standards, the resulting PK assays must undergo full validation following established bioanalytical guidelines. This includes assessment of accuracy, precision, selectivity, sensitivity, and stability across multiple analysts and days. The validation process specifically evaluates the performance of both biosimilar and reference product samples against the biosimilar-derived calibration curve to ensure equivalent quantification performance.

The use of research-grade enfortumab biosimilars as calibration standards represents a scientifically robust approach that enables reliable pharmacokinetic assessment while minimizing analytical variability and supporting regulatory requirements for biosimilar drug development programs.

The primary in vivo models used to study tumor growth inhibition and characterize tumor-infiltrating lymphocytes (TILs) after administration of a research-grade anti-Nectin-4 antibody are syngeneic mouse models and humanized or xenograft mouse models.

Context and Supporting Details:

• Syngeneic Models:
  These involve implanting mouse tumor cells expressing human Nectin-4 into immunocompetent mice (such as C57BL/6 or BALB/c). This setup permits detailed study of immune interactions and TIL profiles because the mouse’s immune system is intact. For example, in one study, C57BL/6 mice were inoculated with engineered hNectin4-Luc MC38 tumor cells and treated with Nectin-4-targeted CAR-T cells to assess both tumor growth inhibition and changes in TIL populations, especially when combined with chemotherapy-induced lymphodepletion. Commercial platforms like TD2 also highlight their experience with syngeneic models in immunotherapy research, including the characterization of TIL baseline populations and gene signatures. These models are preferred for mechanistic immune studies.

• Humanized/Xenograft Models:
  Human tumor cells expressing Nectin-4 (e.g., bladder, breast, pancreatic, or lung cancers) are implanted into immunodeficient mice (e.g., NSG, nude mice) or mice reconstituted with a human immune system. In these models, anti-Nectin-4 antibodies (such as those used in antibody-drug conjugates like enfortumab vedotin) demonstrated significant tumor growth inhibition and even regression, while also allowing some assessment of human-derived TILs if using humanized mice. The NSG mouse model was used to demonstrate tumor eradication and prolonged survival after administration of Nectin-4-targeted CAR-T cells in lung metastasis settings.

Model Comparison and Application:

Additional Information:

• Syngeneic models, while immunologically complete, require genetic manipulation for human antigen expression and may not fully recapitulate human immune context.
• Humanized models better approximate human TIL responses but are limited by incomplete immune reconstitution and higher cost.

In summary, for in vivo studies focusing on tumor growth inhibition and TIL characterization with anti-Nectin-4 antibodies, syngeneic tumor models are fundamental for immune profiling, while humanized/xenograft models are crucial for translational relevance and efficacy evaluation.

Researchers are exploring the combination of Enfortumab biosimilar with various checkpoint inhibitors to study synergistic effects in immune-oncology models, though the specific combinations with anti-CTLA-4 or anti-LAG-3 biosimilars require understanding the broader framework of combination immunotherapy research.

Mechanism of Action and Research Applications

Enfortumab biosimilars are designed to mirror the therapeutic benefits of Enfortumab vedotin, providing researchers with a cost-effective alternative for studying biologic therapies in preclinical and clinical research settings. These biosimilars enable studies without the high costs associated with the reference product while maintaining similar efficacy in research applications.

The antibody-drug conjugate (ADC) mechanism of Enfortumab involves targeting Nectin-4 expressing cancer cells through a monoclonal antibody connected to a cytotoxic agent via a stable linker. Once inside the cancer cell, the linker is cleaved, releasing the chemotherapy agent that disrupts microtubules, leading to cell cycle arrest and apoptosis. This targeted approach minimizes damage to healthy cells compared to traditional chemotherapy treatments.

Combination Research Strategies

Multiple Checkpoint Inhibitor Combinations

Research on combining multiple checkpoint inhibitors is based on the principle that targeting multiple pathways can increase the activity of each therapy while overcoming individual monotherapy limitations. The logic behind these combinations stems from their different mechanisms of action - for example, anti-CTLA-4 agents primarily act in the lymph node compartment to restore induction and proliferation of activated T cells, while anti-PD-1 agents mainly function at the tumor periphery to prevent neutralization of cytotoxic T cells.

Current Clinical Applications

Enfortumab vedotin is already being investigated in combination with immune checkpoint inhibitors like pembrolizumab (anti-PD-1). This combination therapy aims to enhance the immune system's ability to recognize and destroy cancer cells, potentially improving survival rates and reducing tumor progression in patients with advanced or metastatic urothelial carcinoma. Clinical trials examining this combination have shown promising early results.

Research Methodology and Considerations

Preclinical Model Studies

In preclinical immune-oncology models, researchers can use Enfortumab biosimilars alongside other checkpoint inhibitor biosimilars to:

• Study mechanism interactions: Investigating how the direct cytotoxic effects of the ADC enhance the immune activation provided by checkpoint inhibition
• Evaluate timing and sequencing: Determining optimal dosing schedules and treatment sequences for maximum synergistic benefit
• Assess immune microenvironment changes: Monitoring how the combination affects tumor-infiltrating lymphocytes, regulatory T cells, and myeloid-derived suppressor cells

Research-Only Applications

It's important to note that Enfortumab biosimilars are intended strictly for research purposes and not for direct patient administration. This allows researchers to conduct extensive preclinical studies to understand combination effects before advancing to clinical trials.

Potential Synergistic Mechanisms

The combination of Enfortumab biosimilar with checkpoint inhibitors may create synergistic effects through several mechanisms:

• Enhanced antigen presentation: Cell death induced by the ADC can release tumor antigens, potentially improving immune recognition when combined with checkpoint blockade
• Immune priming: The targeted cell killing may create an inflammatory environment that enhances the effectiveness of immune checkpoint inhibition
• Overcoming resistance: Dual targeting may help overcome resistance mechanisms that limit the effectiveness of single-agent therapies

While specific research on Enfortumab biosimilar combinations with anti-CTLA-4 or anti-LAG-3 biosimilars isn't detailed in current literature, the established framework for combination immunotherapy research provides the foundation for such investigations. Researchers can apply similar methodologies used in successful combinations like nivolumab plus ipilimumab to explore novel ADC-checkpoint inhibitor combinations in complex immune-oncology models.

An Enfortumab biosimilar can be used as either the capture or detection reagent in a bridging anti-drug antibody (ADA) ELISA to monitor a patient’s immune response to Enfortumab by exploiting the antibody’s ability to bind ADAs formed against the therapeutic drug.

How a Bridging ADA ELISA Works with Enfortumab Biosimilar:

• In a typical bridging ELISA for immunogenicity:
  • The same drug (or its biosimilar) is used in two forms: one (often biotinylated) to capture ADAs from patient serum on the plate, and another (often conjugated to a detection label like HRP or a fluorophore) to detect the captured ADA, thereby “bridging” the complex via the bivalent nature of the ADA.
  • If the patient has developed ADAs against Enfortumab, those antibodies will bind to both the capture and detection forms of Enfortumab (biosimilar), yielding a measurable signal.

Specifically using a biosimilar as reagent:

• Enfortumab biosimilar can substitute for the reference (originator) Enfortumab in the ELISA if it is structurally and chemically similar enough to provide equivalent binding to the target ADAs. This is often done in biosimilar clinical development to assess whether immunogenicity (ADA response) is comparable between the biosimilar and reference drug.
• There are two approaches for biosimilar ADA assay design:
  • One-assay approach: The same biosimilar is used as both capture and detection reagent for all samples (patients treated with biosimilar or reference drug), maximizing comparability and assay sensitivity for the biosimilar ADAs.
  • Two-assay approach: Separate assays are conducted, one with the reference product, one with the biosimilar as reagents, allowing side-by-side immunogenicity profiling at the cost of assay complexity.

Typical steps in the protocol (summarized):

1. Coat microplate wells with Enfortumab biosimilar (e.g., biotinylated version) for ADA capture.
2. Incubate with patient serum; if ADAs are present, they will bind to the coated biosimilar.
3. Add enzyme- or fluorophore-labeled Enfortumab biosimilar for detection. If ADA is present, it bridges both capture and detection antibodies.
4. Develop signal: Substrate is added and read; signal intensity correlates to the amount of ADA in the patient serum.

Key considerations:

• The choice of using a biosimilar (vs the reference drug) as reagent depends on assay validation and regulatory guidance, especially since the regulatory goal is to demonstrate no clinically meaningful differences in immunogenicity profile.
• Comparative immunogenicity using biosimilar reagents frequently uses a one-assay bridging ELISA, as this approach is generally recommended to minimize bias and facilitate sensitive, direct comparison of ADA responses.

Additional context:

• Bridging ELISAs are highly sensitive and specifically suited for detecting bivalent ADAs against monoclonal antibody drugs such as Enfortumab, but optimization is needed to address specificity and matrix interferences, especially in serum-based assays.
• Resulting data help assess if the biosimilar and reference Enfortumab elicit similar immune responses, which is a regulatory requirement for biosimilar approval.

In summary, using Enfortumab biosimilar as the capture or detection reagent in a bridging ADA ELISA is an established practice to sensitively monitor and compare immunogenicity by detecting ADAs against the therapeutic drug in patient samples.