Prediction of adeno-associated virus neutralizing antibody activity for clinical application

Abstract

Patients with neutralizing antibodies (Nab) against adeno-associated virus (AAV) are usually excluded from treatment with AAV vectors. To develop a standard assay for detecting Nab inhibition activity, we systematically studied current AAV Nab assays in vitro and in vivo. Several factors were found that influence the Nab titers based on the in vitro assay, including: sera volume, AAV dose/cell, cell number and choice of transgenes. When the Nab titer assay was performed in vivo via intramuscular (IM) or systemic administration, a 4-fold increase in sensitivity for measurement of Nab titers was observed compared to an identical in vitro test. To better mimic the clinical setting, after passively transferring human Nabs into mice, blood was collected before systemic injection of AAV vector and used for Nab titer analysis in vitro or via IM injection. The results showed that AAV delivered via IM injection had a similar inhibition pattern to systemic administration. These studies indicate critical parameters necessary for optimizing Nab sensitivity and that an in vivo Nab assay is more sensitive than an in vitro assay for inclusion/exclusion criteria. The variables identified by this study may explain some of the compounding clinical data seen to date with respect to efficiency of AAV transduction in various Phase I clinical trials.

Structure-guided evolution of antigenically distinct adeno-associated virus variants for immune evasion

“…Here 25 μL of antibody or antiserum (as specified for individual experiments) was mixed with an equal volume containing recombinant AAV vectors (1,000-10,000 vg/cell) in tissue culture-treated, black, glass-bottom 96-well plates (Corning) and then incubated at room temperature for 30 min. Assay parameters were carefully controlled as outlined previously (30). A total of 5 × 10 4 HEK293 cells in 50 μL of medium was then added to each well, and the plates were incubated in 5% CO 2 at 37°C for 48 h. Cells were then lysed with 25 μL of 1× passive lysis buffer (Promega) for 30 min at room temperature.…”

Effective Depletion of Pre-existing Anti-AAV Antibodies Requires Broad Immune Targeting

“…Various clinically relevant models have been established to characterize pre-existing anti-AAV humoral immunity and cross-reactivity 11, 12, 13, 14, 15, 16. Efforts have been made to develop strategies to overcome pre-existing anti-AAV Abs, including AAV capsid modification and decoys,17, 18, 19, 20 transient pharmacological immunomodulation,21, 22, 23, 24, 25 and plasmapheresis,24, 26 demonstrating potential for improving rAAV gene delivery by circumventing or reducing the pre-existing Abs in animal models and in humans.…”

Oracle or false prophet? Can we predict AAV efficacy based on preexisting antibody titers?

“…15 Others have suggested that in vivo neutralization assays, in which Nabs are passively transferred to mice following human serum injection to the animals, are more sensitive than those neutralization assays performed in vitro and thus better suited for inclusion/exclusion criteria. 16 However, neutralizing assays (both in vivo and in vitro) rely on the ability of a reporter vector to transduce the target cells and mediate quantifiable expression levels that decrease proportionally to the amount of circulating transduction inhibitors. This poses a number of significant limitations to their standardization, as transduction efficiency is highly serotypedependent and, in general, the sensitivity of the assay decreases as the AAV dose increases, compromising the comparison of NAb titers between serotypes with distinct transduction efficiencies.…”

“…This notion, perhaps prudent in principle, has been recently challenged by Mingozzi and colleagues on the grounds that binding antibodies may in fact increase capsid internalization and transgene expression and thus NAb assays are better predictors of the outcome of gene transfer . Others have suggested that in vivo neutralization assays, in which Nabs are passively transferred to mice following human serum injection to the animals, are more sensitive than those neutralization assays performed in vitro and thus better suited for inclusion/exclusion criteria . However, neutralizing assays (both in vivo and in vitro) rely on the ability of a reporter vector to transduce the target cells and mediate quantifiable expression levels that decrease proportionally to the amount of circulating transduction inhibitors.…”

“…As an example, the assay used by the authors to measure anti‐AAV5 NAbs requires an MOI of 25 000, supplemented with etoposide, an agent that promotes transduction, whereas the anti‐AAV8 NAb assay uses an MOI of 200 with no requirement for agents like etoposide . Other characteristics that impact NAb titers when evaluated using in vitro assays include the amount of serum used, the cell number on the plate and the reporter transgene . In this regard, use of assays that do not rely on transduction performance, such as total antibody assays or the assay recently developed by Guo and colleagues, which relies on quantification of AAV binding to the target cells in vitro using a qPCR assay …”