eChalk Talk: Bioanalytical Strategy for Immunogenicity Assessments of ATMPs

Advanced therapy medicinal products (ATMPs), comprising cell therapies, gene therapies, and tissue-engineered products, are innovative biotherapeutics that offer groundbreaking opportunities to address unmet clinical needs. Due to their complexity, ATMPs pose an enhanced risk of inducing patient immune responses. Consequently, assessment and monitoring of immunogenicity are crucial for ensuring the safety and efficacy of these products. 

In this webinar, we explore common immunogenicity assessments for ATMPs, detailing their applications, methodologies, and potential opportunities and challenges. 

Watch the full webinar now or continue reading:

Immunogenicity of ATMPs 

Immune activation in response to treatment with ATMPs can occur via two primary pathways: 

1. Humoral immunogenicity involves the generation of anti-drug antibodies (ADAs) that can bind to therapeutic proteins or peptide fragments and block the mechanism of action of the ATMP. For example, in chimeric antigen receptor (CAR)-T cell therapy, CARs can be shed and then taken up by antigen presenting cells (APCs), which process that CAR into peptide fragments that are presented on the surface of MHC Class II. CD4+ T cells with a T-cell receptor that recognizes that peptide-MHC Class II complex as foreign will activate and induce B-cell activation, resulting in ADA production. Binding of the ADA to the CAR-T cell may interfere with its ability to engage with tumor cells, decreasing therapeutic efficacy. Moreover, substantial amounts of circulating ADA can potentially lead to rare but serious adverse events such as systemic anaphylaxis. 
2. Cellular immunogenicity arises when there is a T cell response against the therapeutic agent. For instance, in CAR-T cell therapy, the CAR T cell expresses peptides from the CAR on the surface of MHC Class I. CD8+ T-cells that recognize that peptide-MHC Class I complex can become activated by the complex and release inflammatory cytokines that not only recruit other immune cell mediators, but also release lytic granules that cause CAR-T cell lysis. Thus, the cellular immune response can directly impact ATMP efficacy by destroying the cells that express the functional gene product.  

Assessing Humoral Immunogenicity 

There are two main approaches to humoral immunogenicity assessments—ligand binding assays (LBAs) and cell-based assays. 

Ligand-binding assays 

LBAs are commonly used to detect ADAs in patient serum samples when the recombinant protein product is available. These assays can be performed in different formats: 

• Bridging assay. In this format, the therapeutic protein product is biotinylated and ruthenylated labelled and then exposed to the patient serum sample. If ADAs are present, they will bridge the labelled drug product, and that bridged complex can be captured onto an immunoassay plate.
• Stepwise assay. In cases where the nature of the therapeutic protein product precludes the use of a bridging assay, a stepwise assay offers an alternative approach to humoral immunogenicity assessment. In this format, the drug product is immobilized on the surface of a plate and patient serum sample is added. Bound ADAs are detected with a species-specific detection antibody. Sensitivity can be increased by adding a secondary detection antibody to heighten the signal-to-noise ratio.  

For in vivo gene therapies, humoral immune responses must be monitored for both the protein product and the vector used to deliver that product to target cells.  

Cell-based assays 

When the therapeutic protein product is not available as a recombinant protein, a cell-based assay can be used to assess humoral immunogenicity. In this format, a cell line expressing the functional protein product is exposed to a patient serum sample, which may contain both antibodies directed against that protein product and background antibodies. Bound ADAs are detected with a secondary fluorescent detection protein such as Protein A/G using flow cytometry. 

Choosing the right assay format 

Both LBAs and cell-based assays have their respective advantages and drawbacks for detecting ADAs. LBAs utilize standard bridging assay format and validation parameters and often provide a more sensitive and robust high-throughput assay. However, LBAs rely on the availability of recombinant protein product and use of that recombinant version of the protein compromises on properly folded protein. Cell-based assays allow for the detection of ADAs against possible nonsequential conformational epitopes. However, the use of cells increases the potential for background matrix effects. Cell-based assays may also be challenging for autologous cell therapies where each individual CAR-T cell is different. 

The bottom line is there is no right or wrong answer. Instead, assay selection may be driven by which critical reagents are available.  

Neutralizing antibody assays 

Neutralizing antibodies (NAbs) are a subset of ADAs that block the mechanism of action of a drug. For adeno-associated virus (AAV) vectors, NAbs inhibit the ability of the AAV to infect its target cell and deliver its genetic payload. A common format for measuring NAbs against AAVs is a cell-based luciferase assay. In this assay, the AAV has a luciferase reporter and, in the absence of NAbs, the AAV will bind to its target receptor on the cell surface, incorporate into the cell, and express luciferase. In the presence of NAbs, AAV will be blocked from uptake, resulting in decreased luciferase detection.  

This cell-based luciferase is commonly called a transduction inhibition assay since it detects not only NAb activity, but also the activity of any other non-antibody factor that blocks the ability of the AAV to enter the target cell.  

Assessing Cellular Immunogenicity 

Cellular immunogenicity can be measured by evaluating either T-cell activation or cytotoxicity. While humoral immunogenicity assessments utilize patient serum, cellular immunogenicity assessments use peripheral blood mononuclear cells (PBMCs) containing potentially reactive T cells. Typically, assays are performed on cryopreserved PBMCs collected at baseline and at different times following ATMP administration.  

Evaluation T-cell activation 

T-cell activation can be assessed via: 

• Evaluating cytokine release from activated T cells using an ELISpot assay
• Measuring intracellular cytokine production and directly phenotyping T cell subsets using flow cytometry 

For ELISpot assays, PBMCs are stimulated with overlapping peptide pools comprised of the sequence of the viral vector used to deliver the functional gene product or the expressed functional protein itself. A negative control is needed for establishing baseline stimulation of the PBMCs, and a positive control is necessary for determining the viability and reactivity of that PBMC preparation. The PBMCs are captured onto a 96-well plate that is pre-coated with an anti-cytokine antibody that will detect cytokines released by activated T cells. Interferon gamma (IFN-γ) is a common cytokine used as a surrogate readout of CD8 T cell activation and cellular immunogenicity. 

To measure intracellular cytokine production, PBMCs are stimulated with overlapping peptide pools and then multi-parameter flow cytometry is used to gate specific cell subsets and evaluate the cytokine those cells are producing. The multiplexing capability of flow cytometry allows for simultaneous evaluation of multiple cytokines.  

Evaluating cytotoxicity 

Target cell lysis assays assess the direct killing of target cells expressing the functional gene product by CD8 T cells in a PBMC preparation.  

Technical challenges 

Challenges associated with the development and validation of cellular immunogenicity assessments include: 

• Assay background 
• Sample quality, handling, and volume requirements 
• Reagent availability and selection, including positive samples for qualification and validation; design, synthesis, and pooling strategy of peptides; and generation of target cell lines 
• Issues related to multiplexing 

Key Takeaway 

ATMPs are complex molecules associated with a heightened risk of triggering immune responses that can impact both efficacy and safety. Designing and implementing a robust bioanalytical strategy that incorporates multiple assay formats for investigating both humoral and cellular immune responses is essential for optimizing therapeutic efficacy and minimizing adverse events.  

To learn more about how BioAgilytix can help with design, development, and validation of ATMP immunogenicity assays, speak to one of our scientists.