CLN - Ask The Expert
Recombinant adeno-associated virus (AAV) vectors are most commonly used as a delivery system for in vivo gene therapies. Of all the AAV subtypes, AAV9 is one of the most studied and widely used in clinical trials.
One limitation of these treatments is that pre-existing antibodies against AAVs can bind to the capsid of an AAV-based gene therapy, potentially reducing its benefits or even causing adverse events. Consequently, AAV9 antibody tests are used to prescreen gene therapy candidates to identify those who might not be suitable for AAV9-based therapies. These tests are also used to monitor ongoing treatment, to guide dosage adjustments, and to personalize treatment plans.
Multiplex assays simultaneously detect antibodies against multiple AAV serotypes, including AAV9, saving time and providing a comprehensive immunological profile. This is useful for screening and understanding cross-reactivity among different AAV vectors. Neutralizing antibody tests measure the ability of antibodies to inhibit the AAV9 vector, offering insights into the potential efficacy of gene therapy. In contrast, ELISA-based total AAV9 antibody tests provide a comprehensive view of all AAV9 antibodies, including both neutralizing and non-neutralizing types, aiding in the evaluation of a patient's overall response and gene therapy candidate prescreening.
First and foremost, labs must determine which method is most appropriate based on their technical capabilities and requirements. Titer cutoff is another criterion to consider. AAV9 antibody tests semi-quantify antibody titers by sequentially diluting the serum or plasma until an endpoint titer is reached. The greater the dilution, the higher the antibody levels. For example, a dilution or endpoint titer of 1:400 indicates a higher antibody concentration than an endpoint titer of 1:200. The chosen titer cutoff can differ between assays. Variations in titer cutoff may also arise within the same assay depending on how the test is optimized. It is therefore crucial to standardize assay conditions when choosing an AAV9 antibody test. Additionally, sensitivity and specificity are crucial factors in selecting an AAV9 antibody test. In particular, the test should be sensitive enough to detect low levels of antibodies, especially for the purpose of prescreening patients who may have minimal pre-existing immunity.
Variations in assay conditions, reagents, and equipment can impact the comparability of data across different studies and clinical trials. Addressing these challenges requires rigorous assay development, thorough validation, and interdisciplinary collaboration.
Labs should develop comprehensive protocols detailing AAV9 antibody testing procedures, including sample preparation, assay methodologies, and data analysis. These protocols should be standardized to ensure consistent execution across different operators and test batches. Assay parameters such as antibody dilution and incubation time should be optimized to enhance sensitivity and specificity. Additionally, labs should evaluate the analytic performance characteristics of a test to ensure that they adhere to predefined quality criteria.
ELISA assays in particular come with additional considerations. These tests detect antibodies in serum or plasma samples using the AAV capsid protein as an antigen. The concentration of this capsid protein directly influences these assays’ sensitivity and specificity, making it crucial that this parameter be standardized across labs. Using an appropriate capsid concentration through accurate capsid titration ensures that there’s a sufficient amount of antigen for antibody binding, thereby maximizing the signal-to-noise ratio and enabling reliable detection even at low antibody levels.
Jingcai Wang, MD, PhD, CC(NRCC), MLS(ASCP) SH, is assistant director of clinical chemistry and immunology at Nationwide Children’s Hospital and an assistant professor (clinical) of pathology at The Ohio State University in Columbus, Ohio. +Email: [email protected]