Ensuring drug safety is a central requirement in innovative drug development, and immunotoxicity has become an increasingly important focus for both regulators and developers. Immunotoxicity refers to unintended drug effects on the immune system, which may lead to immunosuppression, abnormal immune activation, hypersensitivity, cytokine release, or developmental immune effects. Nonclinical immunotoxicity assessment should follow a risk-based, case-by-case strategy using a Weight of Evidence (WoE) approach. This means integrating drug-related properties, mechanism of action, target expression in immune tissues, existing data from similar products, in vitro or ex vivo findings, routine toxicology results, ADME and exposure information, clinical context, patient population, dosing regimen, and any early clinical immune-related signals. Pivotal safety studies intended to support regulatory submissions should be conducted under GLP conditions to ensure data reliability and traceability.
A key area of immunotoxicity assessment is immunosuppression, which occurs when a drug reduces immune function or interferes with immune surveillance. This may result from direct effects on immune cells, inhibition of critical signaling pathways such as TCR, BCR, or cytokine signaling, or indirect effects on immunoregulatory mechanisms. Routine toxicology studies can provide early warning signals, including changes in thymus, spleen, lymph nodes, bone marrow, immune organ histopathology, lymphocyte counts, leukocyte differentials, and serum globulin levels. When these findings suggest potential immunosuppression, additional functional assays may be needed, such as T-cell-dependent antibody response (TDAR), natural killer cell activity assays, macrophage phagocytosis assays, and lymphocyte subset analysis by flow cytometry. Severe or broad immunosuppression may also impair tumor immune surveillance, so carcinogenicity risk should be evaluated using a WoE-based approach.
Another major concern is immunoenhancement or immune activation, in which a drug abnormally increases host immune responses. This may occur through direct immune pathway stimulation, inhibition of negative immune regulation, or mechanisms that mimic endogenous immunostimulatory signals. Excessive immune activation can disrupt immune tolerance and potentially trigger or worsen autoimmune disease. For biologics, cell therapies, and other products expected to activate immune responses, cytokine release syndrome is a critical risk. In vitro cytokine release assays using human peripheral blood mononuclear cells or whole blood are often used to evaluate cytokines such as IL-6, TNF-α, and IFN-γ across relevant drug concentrations. For high-risk products, first-in-human starting dose selection may rely on MABEL or pharmacologically active dose approaches, supported by robust pharmacology data such as EC50, ECmax, and receptor occupancy. Hypersensitivity and pseudo-allergic reactions should also be considered based on drug structure, mechanism of action, and animal observations.
Developmental immunotoxicity should be evaluated when a drug has potential immunotoxic effects and may be used in pregnant or lactating women, children, or situations where offspring exposure is expected. These assessments may be incorporated into enhanced pre- and postnatal development studies or conducted as dedicated developmental immunotoxicity studies. Key endpoints can include immune organ development, immune cell populations, and immune responses to standard antigens in offspring. More broadly, the selection of immunotoxicity methods and animal models should be driven by WoE analysis, drug characteristics, and the specific immune questions being addressed. Standard toxicology studies provide the foundation, while supplemental assays such as TDAR, NK cell activity, flow cytometry, and cytokine release testing allow deeper evaluation of specific immune risks.
Animal model selection is especially important for immunotoxicity assessment. The selected species should have relevant target expression, pharmacological response, and metabolic characteristics comparable to humans. Rodents are commonly used for standard toxicology studies and assays such as TDAR, but they may not always be suitable for complex biologics, antibody drugs, fusion proteins, gene therapies, or cell therapies due to species-specific target biology and immune response differences. In these cases, non-human primates or other large animal models may provide more predictive information, particularly for complex immunomodulatory effects, cytokine release risk, and immunogenicity. Overall, nonclinical immunotoxicity assessment should be conducted in phases, from early in vitro screening and preliminary toxicology during discovery, to routine and supplemental studies before IND submission, and to more extensive evaluations such as developmental immunotoxicity or carcinogenicity studies during later development when justified by emerging risks.
