Modulating immune checkpoint pathways with agonist or antagonistic therapeutic antibodies has been a recent major breakthrough for cancer therapy. Targeting cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1) or its ligand (PD-L1) have demonstrated striking clinical responses with durable disease control resulting in improved survival in some patients across a broad spectrum of cancer types. Agonist antibodies targeting T cell activation pathways such as OX40, CD40, or CD27 are also in active clinical development. Given the power of these new therapies, it is important to evaluate the complex and dynamic immune responses using appropriate in-vitro functional assays as well as understanding the particular nuances associated with target of interest.
Recent assessment reports from the Food and Drug Administration (FDA) or European Medicines Agency (EMA) of approved checkpoint therapies to PD-1 have demonstrated that non-antigen specific functional assays, including Staphylococcal enterotoxin B (SEB) or Phytohaemagglutinin (PHA) stimulation, have an integral part in non-clinical characterization of these immune checkpoint therapies. These assays were used primarily for in-vitro pharmacodynamic assessment of: 1) potentiation of T cell responses in the presence of the biologic, and 2) PK/PD relationship between ex-vivo stimulated patient samples with treatment and correlating receptor occupancy levels on T cells.
With drug development companies understanding the critical role of these non-antigen specific functional assays, many are starting to use them earlier in the drug discovery process by employing them as a primary screening approach with their candidate therapeutics. Other trends include using these assays for translational research where patient’s samples from active clinical trials are stimulated ex-vivo in the presence of other checkpoint inhibitors to explore potential additive responses. Data derived from these clinical research efforts can provide a powerful assessment real-time or post-hoc when evaluating non-responding or responding patients by combining the data with expression data derived from cell profiling panels, such as nanoString Technologies, or sequencing data of T-cell and B-cell receptors from Adaptive Biotechnologies.
How Non-Antigen Specific Functional Assays are performed at iQ
iQ applies our experience and knowledge performing these assays to work closely with your team to provide a roadmap for evaluating your test articles using our non-antigen-specific functional assays. Our immunologists carefully consider the biological nature of your target of interest, such as target expression kinetics and cell subset expression profile, and then triage the appropriate functional readouts, which could include flow cytometric measurement of the target of interest on cell subsets, proliferation, or detection of cytokine responses. iQ also incorporates the big picture perspective when executing these studies as different research groups have different end project goals ranging from practical assessment of a few candidate molecules to high-throughput screening of libraries of candidates.
iQ fully leverages our collective knowledge and experience in performing these assays (PHA, SEB, or CD3/CD28) to provide robust and reproducible results. We have identified and optimized critical factors that are crucial to properly execute non-antigen specific functional assays. This includes the use of human samples that have undergone rigorous characterization and are used across a variety of our projects. This accumulation of experience working with known samples allow us to apply this knowledge to your assay. We also use premium grade stimulants procured from reputable manufacturers who operate under cGMP (21 CFR 211, Q7A) conditions or provide these materials to the global diagnostics industry.
Example Non-Antigen Specific Functional Assay Results
Figure 1. Example: Kinetic Assessment of Checkpoint Target on CD8+ T cells stimulated with PHA. PBMCs from a normal healthy donor were stimulated in-vitro with PHA. A kinetic time course was performed to monitor the expression of a novel checkpoint target on CD8+ T cells by flow cytometry. Day 5 data is shown. Flow Dot Plot.
Figure 2. Example: Enhanced Proliferation in Response CD3 Stimulation in the presence of checkpoint inhibitor. CFSE
The iQ Experience
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