Immunogenicity testing is essential in the bioanalysis of cell and gene therapies, vaccines, and biologics. It is conducted during preclinical studies to ensure that efficacy data is not biased by immune-mediated degradation of the drug product. In clinical studies, immunogenicity testing is used as an inclusion/exclusion criterion, and post-administration immunogenicity testing is used to assess safety, efficacy, and persistence.
Consequences of immunogenicity
Pre-existing antibodies against adenoviruses, vaccines, and biologics can impact therapeutic efficacy, degrade the drug product, and, in the case of AAV therapeutics, hinder efficient cell transduction and lead to the elimination of transduced cells. The immunogenicity must be considered in all bioanalyses, including pharmacokinetics (PK), biodistribution, and shedding studies. If pre-existing antibodies are present, they can affect transgene protein expression, potentially compromising the reliability of PK, biodistribution, and shedding data.
For gene therapies, understanding the immune status of each treated individual is crucial for determining efficacy, safety, and dosing strategies. In clinical trials, pre-existing antibodies should be monitored to predict therapeutic response and post-dose immune responses should be assessed to evaluate safety, efficacy, and persistence.
What is immunogenicity?
The immune system and its response to foreign substances are highly complex. If an individual has been previously exposed to an agent, they may have acquired adaptive immunity, which includes a humoral B-cell-dependent antibody response and a T-cell-mediated cellular response. Upon encountering new foreign agents, the immune system first initiates a reaction through the innate immune system, often involving cytokine cascades to recruit and activate immune cells. Over time, this leads to the development of adaptive immunity.
A significant portion of the population has been infected with non-pathogenic adenoviruses and has acquired adaptive immunity. In AAV therapeutics, AAVs are generally not considered potent elicitors of innate or adaptive immune responses compared to natural adenoviruses. This is due to strategies such as codon optimization, CpG reduction, incorporation of regulatory elements, lower dosing, and immunomodulation to evade the immune response. However, the immune response to AAV therapeutics remains complex due to their multi-component nature, including the capsid, nucleic acid content, and translated gene product, which may be recognized as foreign by the immune system.
Immunogenicity testing with TCR/BCR sequencing
We perform next-generation sequencing (NGS) to analyze T-cell receptors (TCRs) and B-cell receptors (BCRs) in the adaptive immune system, providing insights into the response to therapeutic agents such as gene therapies, vaccines, or immunotherapies. TCR/BCR sequencing is performed on RNA to track active expression and on DNA to detect all T-cell and B-cell clones present in the sample, regardless of transcriptional activity. DNA-based sequencing also allows for the identification of rearranged V(D)J regions.
Affinity-based proteomics for immunogenicity testing
Cytokine profiling and immune protein quantification are efficiently measured using Olink panels, which can simultaneously analyze 48, 96, or 384 proteins using only microliters of plasma or blood. The workflow is highly automated and optimized for high throughput.
The smaller panels, such as Olink Cytokine 48, measure cytokine levels both relatively and absolutely. These panels are available for both mouse preclinical applications and human clinical applications. Olink also offers various panels focused on immune response, immuno-oncology, and inflammation.
As a service provider for Olink for over a decade, we perform these assays to support drug development. Protocols are also available for non-human primates (NHPs), other biofluids, and tissues. Reach out to us, and we’ll be happy to assist.
Immunogenicity testing with qPCR/dPCR assays
qPCR and dPCR are highly sensitive techniques with short turnaround times, high throughput capacity, scalability, and a high degree of automation, making them excellent tools for analyzing large sample numbers and conducting large cohort studies. These assays can be designed to detect single (singleplex) or multiple (multiplex) immunogenicity biomarkers, such as the expression of cytokines like IL-6, TNF-α, and IFN-γ, which serve as indicators of immune activation.
