Anti-Human PD-1 (Camrelizumab) – Fc Muted™

Research-grade Camrelizumab biosimilars are used as calibration standards (analytical standards) or reference controls in PK bridging ELISA through their incorporation in the assay's standard curve to accurately quantify Camrelizumab concentrations in serum samples, ensuring assay comparability between biosimilar and reference drug measurements.

• The biosimilar Camrelizumab standard (typically a lyophilized form, humanized monoclonal IgG4 kappa, ~149 kDa) is reconstituted and serially diluted to create a standard curve with known concentrations. These concentrations are used as calibration points in the ELISA assay, providing a reference for quantifying the drug in serum samples.

• In PK bridging ELISA for biosimilar development, industry best practice is to establish a single validated assay using a single analytical standard—often the biosimilar—as the calibrator for both reference and biosimilar test samples. This strategy minimizes variability between measurements and supports bioequivalence and similarity analyses between reference and biosimilar products.

• The Camrelizumab biosimilar standard is added into assay plates alongside serum samples, and the resulting signal is measured to generate a standard curve, which is then used to calculate Camrelizumab concentrations in unknown serum samples by comparison. Spike controls (diluting the biosimilar standard into matrix-matched serum samples) are often used to confirm assay recoverability and accuracy within the biological sample context.

• The use of biosimilars as reference controls or calibrators is essential during PK studies, quality control, and biosimilarity assessment, as it allows for consistent quantitation, performance validation, and cross-product comparison in clinical and preclinical development.

• These standards and controls, when validated for accuracy, precision, and equivalence, help to ensure robust data for PK bridging studies supporting regulatory approval and therapeutic monitoring.

Summary Table of Use in PK Bridging ELISA

Key technical points:

• Using a biosimilar as the calibrator ensures assay suitability for measuring both biosimilar and originator drugs.
• Concentrations are determined based on comparison to the standard curve developed using the biosimilar.
• Validation includes assessments in human serum matrix, ensuring the method works for clinical sample analysis.

The primary models used for in vivo administration of research-grade anti-PD-1 antibodies to study tumor growth inhibition and characterize tumor-infiltrating lymphocytes (TILs) are predominantly syngeneic mouse models, with some use of humanized models for specific applications.

Syngeneic Mouse Models

Syngeneic mouse models represent the most commonly used approach for anti-PD-1 research. These models involve implanting tumor cells derived from the same mouse strain into immunocompetent mice, allowing for the study of intact immune responses. A comprehensive study demonstrated the effectiveness of this approach using twelve different syngeneic mouse tumor models, including colon, breast, bladder, kidney, pancreatic, non-small cell lung cancers, melanoma, and lymphomas. In these models, researchers can assess both tumor growth inhibition and the proportion of CD8+ T-cells infiltrating tumors before and after treatment using flow cytometry and immunohistochemistry.

The syngeneic approach offers several advantages for anti-PD-1 research. These models maintain an intact immune system, enabling researchers to study the complex interactions between tumor cells, immune cells, and checkpoint inhibitors. The models allow for detailed characterization of TILs, including their phenotypic changes and functional capacity following treatment.

Humanized Mouse Models

Humanized models are also utilized, particularly when testing humanized anti-PD-1 antibodies that are designed for clinical translation. These models typically involve immunocompromised mice that have been reconstituted with human immune cells or tissues. For example, HX008, a humanized PD-1 blocking antibody, was tested in humanized models where it significantly inhibited tumor growth and demonstrated antitumor responses comparable to approved anti-PD-1 drugs.

Model Selection Considerations

The choice between syngeneic and humanized models often depends on the specific research objectives and the type of anti-PD-1 antibody being tested. Syngeneic models are preferred when studying fundamental mechanisms of immune checkpoint blockade, as they preserve the natural immune environment. These models have proven particularly valuable for understanding how anti-PD-1 treatment affects different aspects of the tumor microenvironment, including the reduction of myeloid-derived suppressor cells and the enhancement of T cell-mediated cytotoxicity.

The effectiveness of anti-PD-1 treatment in these models is closely associated with tumor mutation burden (TMB), with higher TMB tumors generally showing better responses to checkpoint blockade therapy. This relationship helps researchers understand which tumor types are most likely to benefit from anti-PD-1 treatment and provides insights into biomarkers for patient selection in clinical trials.

Both model systems have contributed significantly to our understanding of anti-PD-1 mechanisms and have been instrumental in identifying combination strategies, such as the use of PPT1 inhibitors or other checkpoint inhibitors like CTLA-4 blockers, to enhance therapeutic efficacy.

Researchers studying the potential synergistic effects of Camrelizumab biosimilars with other checkpoint inhibitors (such as anti-CTLA-4 or anti-LAG-3 biosimilars) typically use complex immune-oncology models—often including in vivo (mouse) models of tumors and ex vivo immune profiling—to dissect how combinations affect anti-tumor immunity.

Key approaches and findings:

• Combination Design and Rationale:

  • Camrelizumab targets PD-1 to release the “brakes” on T cells, promoting anti-tumor responses.
  • When combined with other checkpoint inhibitors (e.g., anti-CTLA-4 or anti-LAG-3), researchers seek to simultaneously modulate multiple immune pathways that suppress anti-tumor T cell activity, aiming for a synergistic effect—meaning greater efficacy than either agent alone.

• Mechanistic Studies in Animal Models:

  • Mouse models of melanoma and other tumors are often used to test specific ICI combinations such as anti-PD-1/CTLA-4 and anti-PD-1/LAG-3, which would serve as surrogates for Camrelizumab biosimilars used with anti-CTLA-4 or anti-LAG-3 biosimilars.
  • Advanced immune-oncology models allow precise dissection of which immune cell subtypes drive anti-tumor effects under dual blockade conditions.
  • Researchers have shown, for example, that anti-PD-1/LAG-3 combinations depend heavily on CD4+ T cell activity and reduce the suppressive function of regulatory T cells, whereas anti-PD-1/CTLA-4 combinations directly increase cytotoxic CD8+ T cell activation.

• Immune Profiling and Functional Assays:

  • Flow cytometry, gene expression profiling, and functional T cell assays are used to measure activation states, cytokine production, and cytolytic activity following combination treatment.
  • Multiple studies show that synergistic interactions between checkpoint inhibitors stem from differential modulation of T cell subsets (e.g., CD4+ helpers vs. cytotoxic CD8+, regulatory T cells).

• Clinical Implications and Trial Designs:

  • Insights from these preclinical studies guide the design of clinical trials in patients, combining Camrelizumab biosimilars with other checkpoint inhibitors to test for enhanced efficacy, altered toxicity profiles, or differential immunogenicity.
  • For example, triplet combinations (PD-1 blockade + anti-angiogenic agent + chemotherapy) have been tested clinically to maximize immune activation, though with carefully monitored safety profiles.

Summary Table: Modes of Synergy in Dual Checkpoint Blockade

Points to note:

• Camrelizumab is commonly used in clinical and preclinical studies as the PD-1 pathway blocker; its biosimilars are deployed similarly in immune-oncology models.
• Mechanistic studies often use biosimilar combinations to reduce costs, standardize reagents, or study cross-reactivity.
• The synergistic efficacy is often context- and model-dependent and may vary between tumor types and immune microenvironments.

No direct studies currently show Camrelizumab biosimilar specifically combined with anti-CTLA-4 or anti-LAG-3 biosimilars in the highest tier journals, but the described methods and findings with other PD-1 agents are directly applicable to Camrelizumab biosimilar research and clinical translation.

In the context of immunogenicity testing, a Camrelizumab biosimilar used in a bridging ADA ELISA to monitor a patient's immune response against the therapeutic drug would typically involve the following steps:

Bridging ADA ELISA Protocol with Camrelizumab Biosimilar

Step 1: Preparation of the Capture Reagent

• Biotinylation: The Camrelizumab biosimilar is biotinylated using a biotinylation kit. This step modifies the biosimilar with biotin groups, which allows it to bind to streptavidin-coated plates.

Step 2: Coating of the Plate

• The biotinylated Camrelizumab biosimilar is then added to streptavidin-coated microtiter plates. The streptavidin binds strongly to the biotin on the biosimilar, immobilizing it on the plate.

Step 3: Sample Addition

• Patient serum or plasma samples are then added to the wells. Any anti-drug antibodies (ADAs) present in the samples will bind to the immobilized Camrelizumab biosimilar.

Step 4: Detection

• For detection, an HRP-labeled Camrelizumab biosimilar (or the reference drug) is used. This labeled drug binds to the captured ADAs, forming a "sandwich" structure. HRP (horseradish peroxidase) catalyzes the conversion of a chromogenic substrate (e.g., TMB) into a colored product, which is measured to quantify the ADA levels.

Considerations

• Sensitivity and Specificity: The bridging ELISA is highly sensitive but may require optimization for specificity, especially in complex matrices like human serum. High-quality assay reagents and blocking solutions are essential to minimize interference and ensure accurate results.

Advantages

• High-Throughput Screening: This method allows for high-throughput screening of samples, making it efficient for monitoring large numbers of patients or samples.
• Detection of Bivalent Antibodies: The bridging ELISA can specifically detect bivalent antibodies, which are often more relevant in assessing immunogenicity.

Limitations

• Interference: Components in the serum, such as soluble targets or the drug itself, can interfere with the assay, necessitating careful optimization.

In summary, using a Camrelizumab biosimilar in a bridging ADA ELISA as either the capture or detection reagent provides a sensitive method for monitoring patient immune responses to therapeutic drugs. It is crucial to tailor the protocol to the specific requirements of the laboratory and the nature of the samples being analyzed.