Anti-Human CD38 (Daratumumab)

Research-grade Daratumumab biosimilars are commonly used as calibration standards or reference controls in pharmacokinetic (PK) bridging ELISA assays to accurately measure Daratumumab concentrations in serum samples.

• Calibration Standards: Biosimilars with a well-characterized concentration are prepared in serial dilutions and added to the ELISA plate alongside patient serum samples. These standards are critical in generating a standard curve, which correlates ELISA signal (typically absorbance) to known Daratumumab concentrations.
• Reference Controls: Biosimilars can also be used as quality control (QC) samples at defined concentrations to monitor assay accuracy and precision across runs, ensuring reliable sample quantification.

Essential Context and Use in the PK Bridging ELISA

• In a PK bridging ELISA, the assay plate is coated with an anti-Daratumumab capture antibody, and the Daratumumab from standards or serum samples binds during incubation. A detection antibody (often biotinylated or HRP-conjugated) is added, followed by a substrate, producing a colorimetric signal proportional to Daratumumab concentration.
• The standard curve is created by plotting absorbance versus known biosimilar concentrations on the x-axis (often using a four-parameter logistic fit).
• Unknown serum sample concentrations are then interpolated from this curve. The values may need to be multiplied by a dilution factor depending on the sample preparation.

Why Use Biosimilars as Standards/Controls?

• Biosimilar Daratumumab is analytically similar to the originator product and thus behaves identically in the ELISA.
• When biosimilar and reference products demonstrate analytical equivalence in the assay, a single analytical standard (the biosimilar) can be used for both biosimilar and reference product quantification, reducing variability and simplifying the assay setup.
• This approach is industry best practice for PK bridging assays supporting biosimilar development, ensuring data comparability and facilitating regulatory acceptance.

Workflow Overview

1. Prepare Calibration Standards: Serial dilutions of research-grade Daratumumab biosimilar in serum matrix.
2. Prepare Quality Controls: Aliquots at low, medium, and high concentrations using the biosimilar.
3. Add Standards, Controls, and Unknowns: Load into pre-coated ELISA wells.
4. Proceed with Incubation/Washing/Detection: Follow established sandwich ELISA protocol.
5. Plot Standard Curve & Quantify: Use calibration curve to determine unknown sample concentrations.

Supporting Details

• Only use biosimilars or originator/innovator of validated equivalence as calibrators, as cross-reactivity and binding affinity must match the therapy.
• Regulatory and industry guidance recommend this calibration approach to demonstrate PK bioequivalence during biosimilar development.

In summary, research-grade Daratumumab biosimilars are functionally identical analytical standards or reference controls for accurate, reproducible measurement of drug concentration in serum using PK bridging ELISA.

Research on tumor growth inhibition and characterization of tumor-infiltrating lymphocytes (TILs) using a research-grade anti-CD38 antibody is primarily conducted in syngeneic models. These models offer a comprehensive approach to studying tumor immunology by allowing the introduction of specific genetic modifications or treatments in mice with genetically similar tumors. Here's a summary of relevant models and findings:

Syngeneic Models

1. KP and LLC Lung Cancer Models: These models are used to study the effects of CD38 expression on tumor growth and immune cell function. Research has shown that CD38 can suppress CD8+ T cell function, leading to enhanced tumor growth. The administration of anti-CD38 antibodies in these models has been explored to enhance antitumor immunity by inhibiting CD38-mediated immunosuppression.

2. MC38, Hepa1-6, CT-26, and EMT-6 Tumors: While these models are often used in the context of anti-PD-1 therapies, they could also be relevant for studying the effects of CD38 inhibition, given their immunologically heterogeneous nature. However, specific studies involving anti-CD38 antibodies in these models are not detailed in the provided sources.

Humanized Models

Humanized models are less commonly used for studying CD38-specific effects due to the complexity of modeling human immune systems in mice. These models typically involve the introduction of human immune cells or genes into mice to mimic human immune responses. However, they are not specifically mentioned in the context of anti-CD38 antibody research in the provided search results.

Conclusion

The primary models for studying the effects of anti-CD38 antibodies in tumor growth inhibition and TIL characterization are syngeneic models like KP and LLC lung cancer models. These models provide valuable insights into the immunosuppressive role of CD38 and the potential benefits of combining CD38 inhibitors with other immunotherapies. Humanized models are less commonly referenced in this context but could offer additional insights into mechanisms relevant to human tumors.

Researchers studying daratumumab biosimilars in combination with other checkpoint inhibitors such as anti-CTLA-4 or anti-LAG-3 (including biosimilars of these agents) do so to evaluate whether these agents work synergistically to enhance antitumor immune responses in complex immune-oncology models. This research typically involves both preclinical and clinical settings and focuses on leveraging the complementary mechanisms of action of these therapies.

Essential context and details:

• Daratumumab is a monoclonal antibody targeting CD38, primarily used in multiple myeloma, but also under investigation in other hematologic malignancies and preclinical solid tumor models. Its anti-tumor mechanism includes antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and potentially direct induction of apoptosis or modulation of tumor microenvironment.
• Checkpoint inhibitors (e.g., anti-CTLA-4, anti-LAG-3) act by disrupting the "off signals" that tumors exploit to shut down immune responses. For example, CTLA-4 and LAG-3 are immune checkpoint molecules on T cells; blocking them removes inhibitory signals, leading to enhanced T cell activation and antitumor activity.

Synergy in Combination:

• Rationale for combination: Daratumumab's mechanism, which depletes CD38-positive cells (including some immunosuppressive cells), may enhance the tumor-targeting capacity of T cells. Checkpoint blockade (by anti-CTLA-4 or anti-LAG-3) further unmasks T cell activity. Combining the two could potentially yield additive or synergistic effects by:
  • Increasing cytotoxic immune cell infiltration and activity at the tumor site.
  • Modulating the suppressive tumor microenvironment.
  • Overcoming resistance mechanisms observed with single-agent therapies.

How combinations are studied:

• Preclinical models (e.g., xenograft mouse models):
  • Daratumumab biosimilars are administered with or without additional checkpoint inhibitor biosimilars to tumor-bearing mice.
  • Immunological and tumor response endpoints are measured, such as tumor growth inhibition, immune cell composition analysis (flow cytometry for activated CD8+ T cells, NK cells, regulatory T cells), cytokine profiles, and biomarker studies.
• These models allow researchers to measure:
  • Changes in immune cell populations (e.g., reduction in suppressive NK or regulatory T cells by daratumumab; increased T cell activation by checkpoint blockade).
  • Functional synergy, such as increased cytolytic activity, improved tumor clearance, and changes in biomarkers of immune activation.
• Clinical translation—the preclinical data guide early-phase clinical trials combining anti-CD38 antibodies with checkpoint inhibitors, observing safety, efficacy, immune-related adverse events, and early signs of immune synergy.

Examples from checkpoint inhibitor combinations:

• Combination regimens (e.g., PD-1 plus CTLA-4, or PD-1 plus LAG-3) have already shown improved results over monotherapies in models and clinical trials.
• In the absence of direct preclinical publications pairing daratumumab specifically with anti-CTLA-4 or anti-LAG-3, the combinatorial rationale is borrowed from robust preclinical evidence with other checkpoint inhibitor pairs and the distinct but complementary targets each antibody addresses.

Additional notes:

• Effects on immune cell subsets: Daratumumab reduces peripheral NK cell counts (as NKs also express CD38), but remaining NK and T cells retain cytotoxic functionality, so combining with checkpoint blockade is still rational.
• Biomarkers and stratification: Recent research identifies immune signatures (presence of LAG-3, CTLA-4 expression) that can help predict which patients may benefit most from such combinations.

In summary, researchers combine daratumumab biosimilars with checkpoint inhibitor biosimilars in models to probe potentially synergistic antitumor immunity, using a mix of tumor growth, immunophenotyping, and translational biomarker studies to understand the mechanisms and optimize future therapy regimens.

Role of Daratumumab Biosimilar in Bridging ADA ELISA

Bridging ELISA (Enzyme-Linked Immunosorbent Assay) is a standard method for detecting anti-drug antibodies (ADAs), which are produced by a patient’s immune system in response to a therapeutic drug such as daratumumab or its biosimilar. In the context of immunogenicity testing for a daratumumab biosimilar, the biosimilar itself can serve as both the capture and detection reagent in a bridging ADA ELISA, enabling specific identification of ADAs directed against the therapeutic agent.

Technical Approach

• Capture Step: The biosimilar daratumumab (usually biotinylated for immobilization) is coated onto a streptavidin-coated ELISA plate. This setup captures any ADAs in the patient serum that recognize the biosimilar.
• Detection Step: After washing away unbound components, a second version of the biosimilar (often labeled with horseradish peroxidase, HRP, or another detectable moiety) is added. If ADAs are present, they will ‘bridge’ the captured and labeled biosimilar, resulting in a signal that can be quantified.
• Signal Generation: Subsequent addition of a substrate produces a measurable signal proportional to the amount of ADA present, allowing for high-throughput, sensitive detection of immunogenicity.

Analytical Considerations

• Specificity and Sensitivity: The use of the biosimilar as both capture and detection reagent enhances assay specificity for antibodies against the biosimilar structure, but may miss non-overlapping epitopes or antibodies unique to the reference product. This specificity is particularly important in biosimilar development, where the goal is to demonstrate that the immune response elicited by the biosimilar is comparable to that of the reference product.
• Comparative Immunogenicity: Regulatory agencies recommend head-to-head comparative immunogenicity testing between biosimilars and their reference products, often using a single validated assay (the “one-assay” approach), where the biosimilar reagent detects ADAs for both the biosimilar and the reference product. This minimizes bias and facilitates direct comparison of immunogenicity profiles.
• Assay Limitations: Matrix effects (e.g., serum components), soluble targets, or residual drug can interfere with ADA detection, necessitating careful optimization, use of high-quality reagents, and possibly sample pre-treatment (e.g., acid dissociation) to improve sensitivity.

Practical Implications

• Clinical Monitoring: The bridging ELISA using the biosimilar as both capture and detection reagent provides a practical tool for monitoring patient immune responses during treatment, helping to identify individuals at risk for reduced efficacy or adverse events due to ADA development.
• Biosimilar Development: For regulatory approval, demonstrating that the biosimilar elicits an equivalent immune response to the reference product is critical. The bridging ADA assay format, with the biosimilar as reagent, is central to this comparative immunogenicity assessment.

Summary Table

Conclusion

In immunogenicity testing for a daratumumab biosimilar, the biosimilar itself is employed as both the capture and detection reagent in a bridging ADA ELISA. This setup allows sensitive, specific, and comparative monitoring of patient immune responses, which is essential for both clinical safety assessment and regulatory approval of biosimilars. The assay format must be carefully optimized to minimize interference and ensure reliable detection of clinically relevant ADAs.