Anti-Human Nectin-4 (Enfortumab) [Clone AGS-22M6E]

Research-grade Enfortumab biosimilars are commonly used as calibration standards or reference controls in pharmacokinetic (PK) bridging ELISA assays to quantitatively measure Enfortumab concentrations in serum samples during biosimilar development and PK studies.

In this context, the key approach involves:

• Single Analytical Standard Approach: Industry best practice for PK bridging ELISAs is to develop a single assay using either the biosimilar or the reference product as the analytical (calibration) standard. The standard is prepared at known concentrations to generate a calibration curve.

• Quantification of Both Products: The assay is validated to ensure it quantifies both the biosimilar and the reference product equivalently in serum, establishing bioanalytical comparability according to regulatory guidance. This involves method qualification studies where both the biosimilar and reference are tested in parallel to confirm similar recovery, linearity, precision, and accuracy.

• Preparation and Use in ELISA:

  • Standards (calibrators) and quality control samples are prepared from the research-grade biosimilar (or reference product) at a range of concentrations typically spanning the expected drug levels in serum (see typical range details in commercial assay kits).
  • These standards are run alongside patient or subject serum samples in the ELISA, providing a standard curve for quantifying unknown Enfortumab concentration by interpolation.
  • In a competitive ELISA, the Enfortumab in sample competes with labeled Enfortumab for binding to pre-coated NECTIN4 (the drug target); signal intensity is inversely proportional to drug concentration.

• Reference Controls: Separately, both the biosimilar and the reference drug can be used as quality control reagents or bridging controls to verify assay consistency and comparability.

The rationale is to reduce assay variability and avoid the confounding effects of using multiple calibration curves. Regulatory expectations require demonstration that the ELISA is equally sensitive and accurate for both the biosimilar and the originator drug.

Summary of workflow:

• Biosimilar Enfortumab is used to prepare:
  • Calibration standards: establish the standard curve for drug quantification.
  • Quality controls/reference standards: serve as consistent positive controls to monitor assay performance.
• The assay is validated to ensure equivalence in measuring both biosimilar and reference product, supporting PK bridging for biosimilar approval.

This approach aligns with current bioanalytical and regulatory practice for biosimilar pharmacokinetic evaluation.

Based on the research literature, several primary animal models have been used to study anti-Nectin-4 antibody therapy in vivo, focusing on tumor growth inhibition and immune cell characterization.

Syngeneic Models

C57BL/6 Mouse Model with MC38 Colorectal Cancer

The most well-characterized syngeneic model for anti-Nectin-4 therapy utilizes C57BL/6 mice implanted with human Nectin-4-expressing MC38 colorectal cancer cells (hNectin4-Luc.MC38). In this fully immune-competent system, researchers administered murine Nectin-4-targeted CAR-T cells (Nectin4 mCAR-T) intravenously at varying doses. The model demonstrated dose-dependent anti-tumor effects, with high-dose treatment achieving complete remission in some mice. Importantly, when combined with lymphodepletion using cyclophosphamide (CPA), the treatment resulted in dramatic tumor burden reduction and complete remission lasting more than 60 days without recurrence.

This model is particularly valuable because it maintains a fully functional immune system, allowing researchers to study the complex interactions between anti-Nectin-4 therapy and tumor-infiltrating lymphocytes in an immune-competent environment. The use of luciferase-tagged tumor cells also enables real-time monitoring of tumor progression and response to therapy.

Humanized and Immunodeficient Models

NSG Mouse Model for Lung Metastases

NSG (NOD-SCID-gamma) mice have been employed to study lung metastatic disease using fourth-generation Nectin4-7.19 CAR-T cells that secrete IL-7 and CCL19. In this model, the engineered CAR-T cells demonstrated the ability to eradicate metastatic tumors and significantly prolong survival. The model becomes particularly powerful when Nectin4-7.19 CAR-T cells are combined with FAP-12 CAR-T cells targeting cancer-associated fibroblasts, showing synergistic anti-tumor effects.

Xenograft Models for Multiple Cancer Types

Extensive xenograft studies have been conducted using mouse models implanted with human cancer cell lines from various tumor types. These models included:

• Breast cancer xenografts - showed significant growth inhibition and tumor regression with enfortumab vedotin treatment
• Bladder cancer xenografts - demonstrated tumor regression following anti-Nectin-4 ADC therapy
• Pancreatic cancer xenografts - exhibited significant growth inhibition
• Lung cancer xenografts - showed marked anti-tumor activity

The enfortumab vedotin antibody-drug conjugate (comprising anti-Nectin-4 antibody conjugated to MMAE) was administered in these models, resulting in dose-dependent cell death and significant tumor growth inhibition across all four cancer types.

Model Characterization and TIL Analysis

The research emphasizes the importance of comprehensive tumor microenvironment characterization in these models. Syngeneic models are particularly valuable because they encompass a range of immunogenicities, from highly immune-infiltrated tumors to poorly infiltrated ones. The tumor-immune profiling reveals that each model possesses unique baseline populations of tumor-infiltrating lymphocytes, which can be systematically characterized for:

• CD8+ T cell populations and their activation status
• Myeloid-derived suppressor cell enrichment
• Overall immune infiltration patterns
• Response to checkpoint inhibitors and other immunotherapies

These well-characterized baseline immune profiles are essential for interpreting how anti-Nectin-4 therapy modulates the tumor microenvironment and affects TIL composition and function.

The combination of these models provides researchers with a comprehensive platform to evaluate anti-Nectin-4 antibody efficacy across different immunological contexts, from fully immune-competent syngeneic systems to humanized models that allow for human-specific antibody testing.

Researchers utilize Enfortumab biosimilars in combination with checkpoint inhibitor biosimilars to explore synergistic mechanisms that could enhance therapeutic efficacy in complex immune-oncology models. This approach leverages the complementary mechanisms of action between antibody-drug conjugates and immune checkpoint inhibitors to potentially overcome the limitations of monotherapy approaches.

Mechanistic Rationale for Combination Studies

The combination of Enfortumab biosimilars with checkpoint inhibitor biosimilars is based on the principle that targeting multiple pathways can increase the activity of each agent while overcoming individual limitations. Enfortumab vedotin operates through a targeted delivery mechanism, where the monoclonal antibody component binds specifically to Nectin-4 expressing cancer cells, delivering cytotoxic agents directly to tumor sites while minimizing damage to healthy tissue. When the linker is cleaved inside the cancer cell, it releases potent chemotherapy agents that disrupt microtubules, leading to cell cycle arrest and apoptosis.

Checkpoint inhibitors work through different mechanisms, with anti-CTLA-4 agents primarily acting in the lymph node compartment to restore induction and proliferation of activated T cells, while anti-PD-1/PD-L1 agents mainly function at the tumor periphery, preventing neutralization of cytotoxic T cells by PD-L1 expressing tumor cells and plasmacytoid dendritic cells in the tumor microenvironment.

Research Applications and Study Design

Enfortumab biosimilars provide researchers with cost-effective alternatives for studying biologic therapies in preclinical settings, enabling expanded availability of treatments in clinical trials without the high costs associated with reference products. These research-use-only biosimilars are designed to mirror the action and therapeutic benefits of Enfortumab vedotin while maintaining similar efficacy in preclinical and clinical research settings.

Researchers are investigating how Enfortumab vedotin's ability to stimulate immune responses against tumors could be enhanced when combined with checkpoint inhibitors. The combination aims to leverage the direct cytotoxic effects of the antibody-drug conjugate while simultaneously removing immunosuppressive brakes that prevent effective anti-tumor immune responses.

Clinical Evidence and Promising Results

Clinical trials have demonstrated the potential of such combination approaches. The combination of enfortumab vedotin with pembrolizumab (an anti-PD-1 checkpoint inhibitor) has shown remarkable results, with median survival time extended from 16 to 31.5 months in patients with metastatic cancer - effectively doubling patient survival. This represents an unprecedented improvement, as cancer drug trials typically show only modest increases in survival measured in weeks or months.

Research is also exploring combinations with other checkpoint inhibitors beyond PD-1/PD-L1 blockade. Studies focusing on multiple checkpoint inhibitor combinations suggest that targeting different immune regulatory pathways simultaneously can overcome resistance mechanisms and enhance overall therapeutic efficacy.

Advantages in Complex Immune-Oncology Models

The use of biosimilars in combination studies offers several research advantages. They enable comprehensive investigation of synergistic mechanisms without prohibitive costs, allowing researchers to explore various combination ratios, dosing schedules, and treatment sequences. The highly selective mechanism of Enfortumab vedotin, which minimizes collateral damage to healthy cells, makes it an appealing partner for combination studies with checkpoint inhibitors that can sometimes cause significant immune-related adverse events.

These combination approaches are particularly valuable for studying advanced or treatment-resistant cancers, where single-agent therapies have shown limited efficacy. The potential for Enfortumab biosimilars to enhance immune recognition of cancer cells, combined with checkpoint inhibitors that unleash existing immune responses, represents a promising strategy for overcoming tumor immune evasion mechanisms.

A biosimilar form of Enfortumab can be utilized as either the capture or detection reagent in a bridging anti-drug antibody (ADA) ELISA to monitor a patient’s immune response by specifically detecting antibodies generated against Enfortumab.

In a bridging ELISA for ADA detection:

• The biosimilar Enfortumab is used in two distinct forms:
  • One form is biotinylated and immobilized onto streptavidin-coated plates to act as the capture reagent.
  • The other is labeled (for example, with HRP or a fluorescent dye) to serve as the detection reagent.
• When a patient’s serum is added, any anti-Enfortumab antibodies (ADAs) present will simultaneously bind to both the immobilized and labeled Enfortumab. This “bridges” the capture and detection reagents, allowing for specific identification and quantification of ADAs.
• This approach leverages the bivalent binding capability of immunoglobulins, improving specificity and sensitivity in ADA detection.

When using a biosimilar as the reagent:

• A validated one-assay approach—using the biosimilar Enfortumab for both capture and detection—is generally favored for head-to-head immunogenicity assessment in comparative studies against the originator (reference) product.
• It ensures consistent detection of antibodies against both biosimilar and reference Enfortumab, facilitating regulatory comparisons and enabling monitoring of immune responses in patients treated with either version.
• The choice of the biosimilar reagent is important for high assay specificity and to account for any subtle sequence or structural differences that could affect immunogenicity profiles.

Key considerations:

• High-quality reagent preparation (biotinylation, labeling, purity) is critical due to potential interference from serum matrix components.
• Assay design should mitigate cross-reactivity and non-specific binding to optimize sensitivity and reliability.

In summary, a biosimilar Enfortumab is employed in the bridging ADA ELISA as both capture and detection agent to monitor anti-drug antibodies, providing an accurate measure of a patient’s immune response to the drug in clinical immunogenicity assessments.