Noel Smith, head of immunology, Lonza Pharma & Biotech

Presented by: Noel Smith, head of immunology, Lonza Pharma & Biotech

Biopharmaceutical developers are keenly aware of high attrition during preclinical drug development stages. Immunogenicity and immunotoxicity concerns account for many program failures. Thus, developers strive to identify safety concerns as early as possible. By detecting problems early, companies can reengineer their molecules, investigate process-related solutions, or select alternative lead candidates. Smith reviewed methods that Lonza has developed to enhance early immunogenicity and immunotoxicity studies.

Smith noted that several factors drive immunogenicity risks, including factors that cannot be determined until clinical evaluation. Thus, preclinical immunogenicity studies evaluate a protein’s epitope structure and posttranslational modifications to explore its propensities for target-mediated and off-target binding. Drug developers also need to consider host-cell proteins and aggregates that could result from the cell lines they intend to use to express a product.

Lonza applies assays based on human peripheral blood mononuclear cells (PBMCs) and primary human cells to conduct immunogenicity and immunotoxicity studies, respectively. The cells are isolated carefully from whole-blood gathered from hundreds of highly characterized donors, then washed, counted, vialed, and frozen at controlled rates for onsite storage. All PBMC preparations undergo high-resolution human leukocyte antigen (HLA) karyotyping to ensure robust recovery, viability, and naïve/memory functionality. Although such assays are not required for investigational new drug (IND) filing, a growing body of literature suggests that human-cell systems can provide better indications of clinical-response risks than preclinical animal studies can. Thus, drug sponsors are finding that human systems simultaneously bolster their risk-management activities and demonstrate due diligence to regulatory agencies.

Lonza’s screening begins with PBMC-activation assays that gauge innate immune system responses. Frozen PBMC samples are thawed, washed, and seeded onto 96-well plates, then treated with a product for 24–48 hours. Supernatant is harvested for testing in 12- or 48-plex Luminex panels that are customized for a product’s mechanism of action (MoA) and potential process-related impurities. If a product activates the seeded PBMCs, then cytokines and chemokines are released, indicating an immunogenicity concern. Smith noted that these assays enable broad assessment of product- and process-related risks. He added that the assays are especially useful for generic peptide and biosimilar antibody products, which can generate slightly different impurity and immunogenicity profiles than those produced by reference-listed drugs.

Assays for adaptive immune responses are more complex than those that assess innate immunity because they must identify activation of T cells within PBMCs. First, CD14-expressing dendritic cells are isolated from PBMCs, differentiated, then treated with a product protein or peptide and induced to present HLA-bound peptides from the product. Those cells are cocultured with CD4-presenting T cells, which will activate and expand upon recognizing any of the HLA–peptide complexes. Resulting immune responses are augmented, then quantified. Such screening enables complete analysis of T-cell epitope content during lead-selection stages of drug discovery.

Smith continued that in vitro immunotoxicity testing has increased in complexity as the number of therapies based on T cells has proliferated. Thus, Lonza has developed assays that identify risks of target-mediated and off-target binding for cancer therapies. One method determines whether a product’s MoA stimulates cytokine release. A second, cancer-specific method involves coculture of PBMCs with primary cells isolated from heart, lung, liver, kidney, brain, intestinal, vascular, skin, or bone marrow tissue. By determining cell viability in the resulting culture, analysts can quantify MoA-related destruction of healthy tissue. The assay also can identify T-cell activation and proliferation within the resulting PBMC population.

Lonza has validated all those assays clinically using marketed biologics with well-characterized safety profiles, including products that have demonstrated risks for antidrug antibodies and cytotoxic events. Smith noted that the assays can be customized depending on a program’s development stage. But performing rigorous immunogenicity and immunotoxicity assessment early helps to derisk subsequent process development and support IND applications.