Anti-Human CD3 x DLL3 (Tarlatamab) [Clone AMG 757]

Research-grade Tarlatamab biosimilars are typically used as calibration standards or reference controls in PK bridging ELISA by serving as the analytical standard against which both biosimilar and reference product drug concentrations in serum samples are measured.

In PK bridging ELISA assays for biosimilars:

• A single analytical standard is selected—usually the biosimilar product with established analytical comparability to the reference drug, here Tarlatamab.
• The method is robustly qualified to confirm that the biosimilar and reference product behave similarly in the assay (demonstrating bioanalytical equivalence within predefined parameters, such as precision, accuracy, and equivalence interval).
• Quality Control (QC) samples are prepared with both the biosimilar and the reference drug, and their performance within the assay is assessed relative to the standard curve generated from the biosimilar calibration standards.
• The standard curve is constructed by spiking increasing concentrations of the biosimilar Tarlatamab into human serum to cover the assay’s intended dynamic range (nominal concentrations may range from low ng/mL to tens of μg/mL, e.g., 50–12800 ng/mL).

Key steps:

• Confirm that the biosimilar and reference Tarlatamab are analytically equivalent in the ELISA, minimizing confounding variability by relying on a single standard.
• Use the biosimilar standard to calibrate the assay, and then quantify serum sample concentrations based on their signal compared to this standard.
• This approach enables direct comparison of drug concentrations for both reference and biosimilar products in clinical PK studies, supporting bioequivalence and regulatory requirements.

Essential context:

• The use of a single biosimilar standard avoids variability inherent in multiple calibration curves and enables blinded PK studies, as recommended by regulatory and industry best practices.
• Analytical method validation involves extensive robustness testing, often over multiple days, with independent analyst sets to ensure reliable performance across different lots and runs.
• This paradigm ensures scientifically rigorous, unbiased determination of pharmacokinetic profiles for biosimilar and reference Tarlatamab in study serum samples.

The primary in vivo models used to evaluate research-grade anti-CD3 x DLL3 bispecific antibodies for tumor growth inhibition and analysis of tumor-infiltrating lymphocytes (TILs) are humanized mouse models—specifically, mice implanted with DLL3-expressing human tumor cell lines and reconstituted with human immune cells.

Key models include:

• Subcutaneous xenograft models in CD3+ T-cell humanized mice:

  • SHP-77 (small cell lung cancer, SCLC) human tumor cells are implanted subcutaneously in immune-deficient mice, typically NSG strains. These mice are "humanized" by engraftment of human PBMCs (peripheral blood mononuclear cells) or T cells.
  • The DLL3/CD3 bispecific antibody is administered intravenously. Tumor growth is monitored, and tumor regression is evaluated; analysis of TILs is performed post-treatment. The bispecific induces tumor infiltration by human T cells and converts "cold" (noninflamed) tumors into "hot" (inflamed) states, facilitating analysis of TILs and cytokine responses.

• PBMC-reconstituted B-NDG mice implanted with human tumor cell lines:

  • Solid tumor models, such as SHP-77, are used; B-NDG mice are engrafted with human PBMCs to allow human T-cell mediated responses. Administration of an anti-CD3 x DLL3 bispecific antibody or analog results in robust tumor growth inhibition and allows characterization of the immune infiltrate within the tumor.

Other relevant approaches:

• Orthotopic and metastatic xenograft models: Some studies use orthotopic delivery (directly into the lung) or systemic (tail vein) injection of DLL3-positive tumor cells (e.g., SCLC cell line H82) into NSG or NSG-SGM3 mice reconstituted with human T cells, followed by treatment with DLL3-targeting bispecific antibodies or CAR T cells. These models support the analysis of tumor growth inhibition and immune cell dynamics in appropriate tissue contexts.

Traditional syngeneic (fully immunocompetent murine tumor in mouse) models are generally not employed for anti-CD3 x DLL3 studies. This is because bispecific antibodies that engage human CD3 require a humanized immune system for activity; murine T cells do not respond to human CD3-targeting antibodies.

Summary Table: Models for anti-CD3 x DLL3 Bispecific Antibody Studies

Note: TIL analysis feasibility depends on the study design and tissue harvest timing.

No evidence supports the routine use of fully syngeneic mouse models (with mouse DLL3 and native mouse CD3) for anti-CD3 x DLL3 bispecifics, since these would not recapitulate human antibody/antigen/CD3 biology.

For all of these models, tumor growth inhibition and detailed characterization of TILs—including T-cell infiltration, activation, cytokine production, and exhaustion—can be systematically evaluated.

Researchers use the Tarlatamab biosimilar, a bispecific T-cell engager targeting DLL3 (on tumor cells) and CD3 (on T cells), to directly redirect T-cell cytotoxicity toward DLL3-expressing cancer cells—commonly in small cell lung cancer—within preclinical immune-oncology models. To study synergistic effects with other checkpoint inhibitors such as anti-CTLA-4 or anti-LAG-3 biosimilars, they often combine Tarlatamab with these agents in vitro (cell cultures) and in vivo (mouse or humanized mouse) models to assess additive or synergistic immune responses.

Context and Supporting Details:

• Experimental Design:

  • Tarlatamab biosimilar is available for both in vitro and in vivo research use, specifically for modeling immune-mediated tumor killing.
  • In combination studies, researchers co-administer Tarlatamab biosimilar with checkpoint inhibitors (e.g., anti-CTLA-4 or anti-LAG-3 biosimilars) and evaluate endpoints such as T-cell activation, cytokine production, tumor lysis, and overall survival in animal models.

• Immunological Mechanisms:

  • Tarlatamab acts by bridging T cells and DLL3-positive tumor cells, triggering targeted cytotoxic T-cell activation and tumor cell apoptosis, even in immunosuppressive tumor environments where traditional checkpoint blockade is less effective.
  • Checkpoint inhibitors, like anti-CTLA-4 and anti-LAG-3, enhance antitumor immunity by removing inhibitory signals from T cells (releasing immune "brakes").
    • For example, anti-CTLA-4 promotes direct activation of cytotoxic CD8+ T cells.
    • Anti-LAG-3 therapy increases CD4+ helper T-cell activity while reducing regulatory T-cell suppression, indirectly boosting CD8+ cytotoxic responses.

• Rationale for Combining Modalities:

  • DLL3-targeting BiTEs like Tarlatamab force immune engagement with cancer cells, but efficacy may be hampered by exhaustion or other immune suppression within the tumor microenvironment.
  • Checkpoint inhibitors relieve distinct immunosuppressive pathways, potentially amplifying Tarlatamab-induced T-cell cytotoxicity.
  • By combining these, researchers can determine if checkpoint blockade potentiates the BiTE-induced tumor killing and identify which immune cell subsets are essential for synergy.

• Assessment of Synergy:

  • Commonly measured outcomes include enhanced T-cell activation (CD4+ and CD8+), greater tumor cell killing, delayed tumor growth, and increased survival compared to single-agent therapy.
  • Use of biosimilars allows for preclinical combination studies without the regulatory or access limitations associated with clinical-grade drugs.

Summary Table: Research Roles of Tarlatamab and Checkpoint Inhibitor Biosimilars

No direct preclinical study of "Tarlatamab plus anti-CTLA-4 or anti-LAG-3 biosimilar" is cited in these sources, but the underlying methodology is established by combining BiTEs with checkpoint inhibitors in complex immune-oncology models to measure potentially synergistic immune responses and antitumor outcomes. This approach enables translation to future clinical trial designs if synergy is established preclinically.

A Tarlatamab biosimilar is used as a capture or detection reagent in a bridging anti-drug antibody (ADA) ELISA by leveraging its ability to bind to anti-Tarlatamab antibodies formed in patient serum, thus monitoring the patient’s immune response against the therapeutic drug.

Essential assay mechanism:

• In a bridging ADA ELISA, the therapeutic biosimilar (Tarlatamab) is chemically labeled in two formats—commonly biotinylated for capture and enzyme/dye-labeled (e.g., HRP) for detection.
• Capture reagent:
  • Biotinylated Tarlatamab biosimilar is immobilized on a streptavidin-coated plate.
  • Patient serum (potentially containing anti-Tarlatamab antibodies) is added; any ADA present forms a “bridge” by binding to both the immobilized (capture) and labeled (detection) Tarlatamab reagent, since the ADA recognizes two epitopes on the drug—typical of immunoglobulins.
• Detection reagent:
  • The second (detection) Tarlatamab biosimilar molecule is conjugated to HRP or another reporter. It binds to the opposite paratope of the ADA, completing the bridge.
  • After incubation and washing, the presence of ADA is revealed by substrate conversion (e.g., TMB for peroxidase), measured as optical density.

Key details for immunogenicity monitoring:

• Biosimilar Tarlatamab reagents ensure the assay measures immune response to the actual therapeutic (including relevant epitopes).
• The ELISA is sensitive and allows screening of large sample volumes for immune responses against the drug itself.
• The use of biosimilar reagents is crucial in clinical research where the originator drug may be limited or for manufacturing robust reagent supply. Both capture and detection reagents must retain native epitope conformation for ADA recognition.

Assay workflow summary:

• Biotinylated Tarlatamab biosimilar coats the plate (capture reagent).
• Patient serum is added; ADAs, if present, bind this coated drug.
• HRP- or dye-labeled Tarlatamab biosimilar (detection reagent) is added, completing a bridge between two drug molecules via the ADA.
• Wash steps remove unbound components; color development measures ADA binding quantitatively.

Additional notes:

• Assay customization: Labeling protocols and blocking solutions are optimized to minimize matrix interference and maximize specificity in clinical samples.
• A validated 3-tiered approach is often used (screening, confirmation, titer), with competitive inhibition by excess drug to confirm ADA specificity against Tarlatamab.

Summary Table: Bridging ADA ELISA using Tarlatamab biosimilar

This approach enables sensitive and specific monitoring of immunogenicity to Tarlatamab by directly detecting ADA responses against the therapeutic drug in patient samples.