Anti-Human CD3 x CD19 (Blinatumomab) [Clone AMG103] — Fc Muted™

Research-grade Blinatumomab biosimilars serve as calibration standards or reference controls in pharmacokinetic (PK) bridging ELISA assays by providing a defined, quantifiable amount of drug that is serially diluted to generate a standard curve against which unknown serum concentrations of Blinatumomab are measured.

In the PK ELISA:

• Blinatumomab biosimilar (research grade) is used to prepare a series of known concentrations (the calibration standards) by dilution in a protein buffer intended to mimic the serum matrix.
• These standards, along with study samples, are assayed together on plates pre-coated with a capture reagent specific for Blinatumomab (e.g., Human CD19/Leu-12 in the KRIBIOLISA ELISA).
• Typically, detection is based on a competitive assay format: both the unlabeled standard/sample and a constant amount of labeled Blinatumomab (e.g., Blinatumomab:HRP conjugate) compete for binding to the capture antigen on the plate.
• After washing, the amount of labeled Blinatumomab bound is inversely proportional to the amount present in the standards/samples. Detection substrate is added, producing a quantifiable signal (e.g., colorimetric), which is measured, usually at 450 nm.
• Reference controls (high, medium, and low concentrations) are included in each run to monitor assay performance, ensure accuracy, and verify consistency between assays and across plates.

Calibration standards are essential for:

• Establishing the standard curve relating signal (e.g., absorbance) to Blinatumomab concentration.
• Ensuring quantitative results for unknown samples by interpolation.
• Confirming assay specificity and sensitivity for the drug, not for closely related proteins or endogenous immunoglobulins.

Matrix matching: For pharmacokinetic applications, the calibrators (Blinatumomab biosimilar standards) are often prepared in blank (drug-free) human serum to closely match the biological matrix of test samples, thus accounting for matrix effects on assay performance.

In summary:

• Research-grade Blinatumomab biosimilars are critical for generating the standard curves and quality controls required for accurate, reproducible quantification of drug levels in serum by bridging ELISA in PK studies.
• Consistency and calibration against these reference materials enable direct inter-study and inter-lab comparability.

Key ELISA components and roles:

• Calibration standard: Research-grade Blinatumomab biosimilar (known quantity).
• Reference/control: Aliquots at defined concentrations (e.g., low, high) for QC.
• Sample: Patient serum for PK assessment.
• Detection system: Competitive binding, colorimetric or ECL readout.

This design ensures that pharmacokinetic data derived from serum samples accurately reflect in vivo drug concentrations, enabling robust PK analysis.

The primary in vivo models for administering anti-CD3 x CD19 antibodies to study tumor growth inhibition and tumor-infiltrating lymphocytes (TILs) characterization are humanized xenograft models using immunodeficient mice engrafted with human immune cells and tumor cells, and syngeneic murine models when using murine-targeted bispecific constructs.

Key model types:

• Humanized Xenograft Models

  • Description: Immunodeficient mice (e.g., NOG, SCID) are engrafted with human tumor cells (such as SU-DHL-10 [DLBCL] or Nalm-6 [ALL]) and human PBMCs (peripheral blood mononuclear cells).
  • Usage: Research-grade anti-CD3 x CD19 bispecific antibodies (e.g., TNB-486, blinatumomab analogs) are administered to evaluate tumor growth inhibition (TGI) and TIL profiles in a system containing human tumor antigens and human immune cells.
  • Advantages: Allows direct testing of human-specific reagents and assessment of human TIL population dynamics, including CD4+, CD8+ T cells and suppressor/regulatory subsets.
  • Limitations: Host murine microenvironment may limit full human immune interactions beyond the engrafted PBMCs.

• Syngeneic Murine Models (for mouse-specific bispecific antibodies)

  • Description: Murine tumor cell lines expressing a human target (e.g., B16F10 cells engineered to express human antigens like EpCAM) are implanted into immunocompetent mice, which are then treated with a murine-specific anti-CD3 x target bispecific antibody.
  • Usage: These models enable study of TILs within a fully functional mouse immune system and permit immune intervention mechanism analysis.
  • Advantages: Intact murine immunity (including Tregs and myeloid-derived suppressor cells), useful for mechanistic investigation and biomarker discovery.
  • Limitations: Cannot be used with human-specific anti-CD3 x CD19 antibodies unless targets are engineered or fully murinized reagents are available.

Supporting details:

• Studies using humanized xenograft models have demonstrated dose-dependent inhibition of tumor growth and allowed analysis of TIL dynamics, including depletion of CD19+ cells and cytokine profiles following anti-CD3 x CD19 administration.
• Syngeneic models are particularly valuable for investigating immunomodulatory mechanisms, assessing efficacy across immune cell subsets, and identifying biomarkers correlated with therapeutic response or resistance.

Summary Table:

In summary, anti-CD3 x CD19 antibodies are primarily tested in humanized xenograft models for human tumor growth inhibition and TIL characterization, while syngeneic models are used for mechanistic and biomarker studies with murine-targeted constructs.

Researchers studying synergistic effects between Blinatumomab biosimilars and other checkpoint inhibitors (such as anti-CTLA-4 or anti-LAG-3 biosimilars) use complex immune-oncology models—primarily in preclinical (such as mouse) models and, less commonly, in clinical studies—designed to assess how these combinations modulate the immune response against tumors.

Blinatumomab is a bispecific T-cell engager that links CD3 on T cells with CD19 on B cells, resulting in potent T cell–mediated cytotoxicity of B-cell malignancies by forming a cytolytic immune synapse between T cells and target cells. While its main clinical application is in B-cell acute lymphoblastic leukemia (B-ALL), researchers extend its investigation to combinatory approaches due to the growing interest in synergizing T-cell engagers with checkpoint inhibition to overcome immune evasion and boost anti-tumor activity.

Study Design and Rationale:

• Combining Mechanisms: Researchers combine Blinatumomab biosimilars (which directly stimulate T cell killing of CD19+ cells) with checkpoint inhibitors (e.g., anti-CTLA-4 or anti-LAG-3), which relieve inhibitory signals on T cells, to determine if dual pathway activation leads to enhanced tumor eradication versus monotherapy.
• Immune Cell Analysis: Studies often dissect the effects on different T cell subsets (CD8+, CD4+ helper, and regulatory T cells [Tregs]), since checkpoint inhibitors can alter the activity, abundance, or function of these populations. Research with anti-PD-1/CTLA-4 and anti-PD-1/LAG-3 combinations in melanoma has revealed distinct reliance on specific T cell populations: Anti-LAG-3/PD-1 synergy required CD4+ cells, while anti-CTLA-4/PD-1 acted more directly on CD8+ cytotoxic T cells.

Experimental Approaches:

• In Vitro Models: Tumor cell and immune cell co-culture assays are used to assess T cell activation, cytokine release, and tumor cell lysis when both agents are present.
• In Vivo Models: Humanized mouse models or syngeneic mouse tumor models allow investigation of tumor control, survival outcomes, and immune infiltration after administration of the drug combinations. These studies can be further dissected with immune profiling (flow cytometry, single-cell RNA sequencing) to pinpoint changes in T cell activation states and the emergence/resistance of tumor subclones.
• Mechanistic Studies: Researchers measure
  • Cytokine profiles,
  • Upregulation of activation or exhaustion markers on T cells,
  • Tumor cell death in the presence of both agents, and
  • Reprogramming of the tumor microenvironment.

Clinical Translation:

• While most published results are preclinical, the rationale involves leveraging Blinatumomab's ability to redirect T cells toward leukemic blasts and the checkpoint inhibitors’ capacity to prevent or reverse T cell exhaustion and increase persistence, ultimately aiming for more durable remissions or responses in otherwise refractory cancers.

Summary Table: Mechanisms and Outcomes in Combination

In summary, researchers use these combinations to dissect and exploit non-redundant immune pathways, seeking to maximize anti-tumor efficacy by simultaneously increasing direct tumor cytotoxicity and relieving T cell inhibition. These efforts inform translational strategies for relapsed/refractory B-cell malignancies and potentially solid tumors, though published clinical data for these specific biosimilar combinations remain limited.

A Blinatumomab biosimilar is typically used in a bridging anti-drug antibody (ADA) ELISA as either the capture or detection reagent to monitor the patient’s immune response, specifically the formation of anti-blinatumomab antibodies in patient serum.

Bridging ADA ELISA Overview:

• In the bridging ELISA format, the therapeutic drug or its biosimilar is used on both sides of the "bridge"—one as a capture reagent immobilized on the plate and the other as a detection reagent, often labeled (e.g., with HRP or biotin).
• This design exploits the bivalency of ADAs: ADA molecules can simultaneously bind two identical drug molecules, one attached to the plate, and another labeled for detection.

Practical Use of a Blinatumomab Biosimilar in ADA ELISA:

• Plate Coating (Capture): The biosimilar blinatumomab is immobilized on the ELISA plate.
• Sample Addition: Patient serum, possibly containing anti-blinatumomab antibodies (i.e., ADA), is added. If present, the ADAs bind to the plate-bound blinatumomab.
• Detection: A labeled (e.g., HRP-conjugated) blinatumomab biosimilar is then added. If an ADA is present, it bridges between the capture (plate-bound) and detection (labeled) blinatumomab molecules.
• Signal Development: The detection reagent's label yields a measurable signal (e.g., color change), indicating the presence and, often, quantity of ADA.

Why Use a Biosimilar?

• Biosimilars are structurally and functionally comparable to the original therapeutic and reliably present the same antigenic epitopes recognized by ADA.
• This allows for robust, reproducible ADA detection, even if the branded drug is unavailable for research use or prohibitively expensive.

Key Considerations:

• The assay's specificity hinges on the biosimilar’s ability to faithfully mimic the original drug’s antigenic sites, ensuring accurate ADA detection.
• Labeling (e.g., with HRP or biotin) must not compromise the biosimilar’s ability to interact with patient ADAs.

Examples from Literature:

• Similar strategies are widely employed for other monoclonal antibody drugs and biotherapeutics: the drug (or biosimilar) is used on both sides of the ELISA to detect patient-generated ADAs through the bridging format.
• Such assays have a history of reliable use in immunogenicity testing for biotherapeutic monitoring and regulatory compliance.

In summary, a Blinatumomab biosimilar is used as both capture and detection reagent in a bridging ADA ELISA to sensitively and specifically monitor ADA formation in patients treated with blinatumomab. This aids clinicians and researchers in evaluating immunogenicity, which can impact drug efficacy and safety.