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Since development of a serotype‐monospecific TAb assay may not be possible
due to high homology between AAV serotypes, AAV serotype specificity may need
to be further characterized when patients are retreated with a different AAV capsid. To understand whether a particular TAb response in an individual is serotype‐
specific, a confirmatory test can be conducted, whereby excess amount of unlabeled
AAV vector for the serotype of interest is added to determine if the assay signal is
reduced.
6.2.4.3 Precision
Assay precision is a parameter to describe the degree of variability in assay raw
signal, based on results obtained in multiple intra‐ and inter‐run tests[14]. Like
protein biopharmaceutical ADA assays, PC samples prepared at a few concentrations in NC matrix (or surrogate) are tested in replicates (typically two per sample)
and evaluated on several occasions (typically 3) by two or more analysts. As for
regular ADA assays, variability is expressed as coefficient of variation percentage
(CV%) and is expected to be less than 20%.
6.2.4.4 Matrix Interference and Selectivity
Impact from sample matrix is evaluated in interference and selectivity assessments. Several (typically 10) individual matrix samples are analyzed unspiked
and spiked with PC control reagent at the low PC (LPC) and high PC (HPC) concentration. This enables an understanding of whether inter‐individual variations
in matrix components interfere with detection and if the assay can reliably detect
PC at low concentrations across multiple donors. The test needs to be performed
using healthy and disease population samples to understand if any difference in
potential interference can be observed. It is understood that for rare diseases
obtaining indication‐specific matrices may be challenging and alternative solutions, for example, use of sample collected in clinical study, may be required. It is
recommended that at least 80% of unspiked samples should score TAb negative
and the same percentage of LPC/HPC‐spiked samples should score TAb positive.
In addition, the potential impact of known interfering factors, such as lipids,
hemoglobin and bilirubin, and other matrix components that are expected to be
present in the sample (e.g. anti‐coagulation factors) has been discussed in regulatory guidance and needs to be considered[6].
6.2.4.5 Assay Cut-Point
TAb assay cut‐point is arguably one of the most important parameters to be determined during assay development or validation. Assay cut‐point is a threshold
value that is used to determine whether a given sample is positive or negative for
the presence of AAV TAb, an approach that is very similar to the one used for
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protein biotherapeutic ADA assays [14, 29]. Cut‐point values are assay‐ and
laboratory‐specific and are separately defined for screening, confirmatory, and
titration protocols. Frequently, screening and titration protocols utilize the same
cut‐point value. As for the ADA methods, an AAV TAb assay cut‐point is determined based on a statistical analysis of assay signals generated for a collection of
treatment‐naïve individual donor samples that are preferably negative for preexisting AAV immunity. Generally, 50 or more individual samples are tested in 3 or
more runs to obtain a sufficient collection of data. The data are analyzed for statistical and biological outliers that are excluded before determining the 95th or
99th percentile of distribution for screening (as well as titration) and confirmatory
tests, respectively[14, 29].
Due to preexisting AAV antibodies, it may be challenging to obtain TAb negative
treatment‐naïve individual donor samples. It may also be difficult to separate true
TAb positive vs true TAb negative populations. Conducting a confirmatory test
may offer a solution, with an assumption that samples that generate a signal that is
inhibited in the presence of unlabeled AAV material by 50% or more contain specific AAV TAb. Results generated for these reactive samples may be removed from
the data before performing cut‐point data analysis.
6.2.5 TAb Assay Data Interpretation
Information about AAV TAb status of an individual patient may facilitate a decision about AAV treatment eligibility and help understand potential clinical
events of interest, including impact on safety or efficacy of the GTx treatment.
Application of TAb methods for detection of preexisting AAV immunity has
gained popularity since these methods are easy to develop and implement, relatively straightforward for development into a companion diagnostic (CDx) or
similar routine test, and relatively robust in their application. If TAb assay results
are used as treatment eligibility criteria, a threshold TAb titer for enrolment
needs to be pre‐defined. The specific treatment eligibility TAb titer threshold
may be dependent on disease, route of administration, assay sensitivity, nature of
the detected antibodies, and other parameters. It continues to be a subject of
ongoing debate. Examples include using the lowest detectable AAV antibody
titer, 1:1000 titer[30] or no detectable antibody titer[1, 31] as a threshold for
eligibility. For some routes of administration (e.g. intrathecal, intravitreal, and
subretinal), concerns related to the impact of AAV antibodies may be reduced
compared to a systemic, intravenous route of administration. Therefore, the TAb
titer thresholds that would be applied when determining patient eligibility would
also be different.
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6.3 Considerations forCell-based Transduction
Inhibition Assays
6.3.1 Principle and Methodology ofCell-based AAV TI Assays
AAV TI assays are typically performed as cell‐based reporter‐gene assays[3, 18,
32–34]. A permissible cell line, such as HEK293T/17, is seeded in complete media
on white clear‐bottom 96 well plates and grown overnight. On the next day,
plasma or serum test samples are mixed with a viral AAV reporter gene construct
carrying, for example, a CMV promoter‐driven luciferase reporter gene (AAV‐
Luc), in serum‐free media and incubated for 30minutes at room temperature to
allow binding of AAV inhibitors to the vector capsid (Figure6.2).
Following this incubation, samples are added to the cells in replicate wells at a
certain multiplicity of infection (MOI), generally between 5000 and 400,000 vg per
cell[18, 34]. After a 1‐hour incubation at 37
etoposide solution in complete media may be added to stimulate DNA repair
mechanisms that enhance productive transduction. After 2days, the cell culture
medium is removed and a luciferase substrate, such as Steady‐Glo, is added for
10minutes at ambient temperature. Luminescence is measured, for example, by
using a 500‐ms integration time on a VICTOR‐™X microplate reader. The relative
°C, a final concentration of 20 μM
mix sample
w/ AAV-Luc
w/o AAV
inhibitors
Luciferase expression Decreased luciferase
Figure6.2 Principle of cell-based AAV transduction inhibition (TI) assays.
with AAV
inhibitors
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luminescence of a test sample is then normalized to that of a NC sample, which
consists of pooled normal human plasma or serum without any detectable AAV
transduction inhibitors. The results can be further converted to percent transduction values relative to NC. Alternatively, test sample results may be expressed as
percent inhibition values by subtracting the percent transduction value of a test
sample from 100%. Either way, one needs to establish a cut‐point below or above
which a test sample would be considered positive. Positive test samples are usually reported with a semiquantitative TI titer value as a relative measure of their
capacity to inhibit AAV transduction. Titer values are determined by serially diluting a positive test sample to determine the highest sample dilution still detectable
in the TI assay. The reciprocal of the highest detectable sample dilution is equal to
the numerical neutralizing titer value.
6.3.2 AAV TI Assay Development: Designing forClinical Relevance
One of the key parameters to optimize during TI assay development is MOI. MOI
represents the ratio between the quantity of AAV vector and number of plated
cells. Cell lines that require a higher MOI to achieve productive transduction can
be thought of as more difficult to transduce. Difficult to transduce cell lines can
pose challenges for developing sensitive AAV TI assays, since higher test sample
titers will be required to neutralize the higher quantity of AAV vector used in the
assay, resulting in limited detectability of lower titers. Cell lines commonly used
for TI assays are HEK293, HeLa, and HuH7. Depending on AAV serotype, a popular choice is HEK293 and its derivatives, such as HEK293T/17 and 2V6.11[3, 35].
To optimize MOI, AAV vector titrations are performed, similar to how drug‐
response curves are needed to develop cell‐based anti‐protein therapeutic NAb
assays. These titrations are performed to establish the dynamic range, within
which a meaningful decrease in AAV vector concentration generates a distinctly
measurable change in assay signal. The goal is to ensure that– at the selected
MOI– the assay signal, such as measured luminescence, is sufficiently robust yet
not fully saturated. Otherwise, the assay would remain unresponsive to a meaningful decrease in effective AAV vector concentration mediated by neutralizing
factors in a test sample. In conclusion, MOIs should be selected to fall well within
the dynamic range of the assay.
Dynamic ranges can be broad, and this is where strategic optimization may be
employed to maximize clinical relevance of a cell‐based TI assay. The lower the MOI
for a given number of plated cells, the more sensitive the assay will be, since a lower
quantity of AAV vector will need to be neutralized by a particular volume of test
sample. The question is how sensitive does the assay need to be? To answer this with
clinical efficacy in mind, it can be helpful to determine the volume of test sample
(plasma or serum) that is incubated together with a particular quantity of AAV
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vector in the assay, based on the selected MOI and number of plated cells. The AAV
vector‐to‐sample ratio can then be compared to the maximal AAV vector plasma
concentration anticipated in study participants, based on intended clinical dose
level. For example, an adult clinical trial participant who is administered an AAV
vector dose level of 6 E12 vector genome per kilogram (vg/kg) will receive a total
vector dose of 4.2 E14
a theoretical maximal plasma concentration of 1.4 E11
total plasma volume of 3000
may be incubated with 42microliters of undiluted plasma test sample, resulting in
a vector‐to‐sample ratio of 3.36 E09 vg/0.042 mL=8 E10 vg/mL, which is about two
times less than theoretical maximal plasma concentration of 1.4 E11
To be relevant for clinical efficacy evaluations, the vector‐to‐sample ratio in a TI
assay should be equal to or lower than the maximal plasma concentration of the
AAV GTx theoretically achieved in study participants, as illustrated in the example above. Otherwise, the neutralizing potential associated with a particular volume of plasma or serum could completely neutralize clinically administered GTx
quantities, while barely inhibiting transduction of the cell line. Therefore, cell
lines requiring exceedingly high MOIs may not be suitable for use in TI assays. In
conclusion, to develop a TI assay with clinically relevant sensitivity, it is useful to
concurrently optimize cell density per well, MOI, and test sample volume, while
comparing vector‐to‐sample ratio invitro to anticipated GTx plasma levels invivo.
Similar considerations have been successfully applied to interpolate clinical relevance of conventional anti‐biologics NAb assays[36].
vg, assuming average body weight of 70 kg. This would lead to
vg/mL, assuming average
mL. In a cell‐based TI assay, 3.36 E09 vg AAV vector
vg/mL.
6.3.3 Key Assay Validation Parameters
This section has been written in consideration of available regulatory guidance for
immunogenicity assays[6, 37] and industry white papers[38, 39].
6.3.3.1 Screening and Titer Cut-Points
Test samples that contain AAV inhibitors are expected to decrease transduction in
a cell‐based TI assay compared to NC. To adjudicate a sample as positive or negative for AAV inhibitors, a cut‐point is required to define a meaningful decrease in
transduction. This analytical cut‐point is established in reference to cell‐based
assay procedures; it does not directly translate to the anticipated impact on clinical efficacy. This will be discussed further below, refer to: Data Interpretation:
Determining a Meaningful Titer Threshold for Clinical Efficacy.
Two main strategies exist to establish TI assay cut‐points: (1) using a threshold of
50% transduction at a predetermined sample dilution compared to a NC sample[35];
(2) using a statistical cut‐point derived from treatment‐naïve, AAV inhibitor‐negative
individual donor samples [3, 40], as recommended by regulatory guidance [6].
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A 50% transduction cut‐point is commonly used in antiviral and vaccine research
to report neutralizing titers against target viruses, and thus this strategy is oftentimes applied for measuring AAV inhibitors. However, this approach could miss
less potent but potentially impactful inhibitors that only partially decrease transduction to 60–70%.
Whether reduced detectability of less potent AAV inhibitors poses a significant
hurdle for clinical data interpretation will depend on the assay setup, for example,
on how much lower the vector‐to‐sample ratio is compared to the theoretical maximal vector plasma concentration in patients: If the vector‐to‐sample ratio is 10
times lower than the anticipated vector plasma concentration, then using an analytical cut‐point of 50% transduction could be justified, simply because the quantity of AAV vector neutralized invitro would translate to as little as 5% of the
maximal clinical vector concentration anticipated in the tested sample volume. It
will also be critical when implementing a 50% transduction cut‐point to account
for assay variability: Variability needs to be empirically evaluated and may be
caused by different reagent lots, composition of individual sample matrices, passage number of cells, and operators.
Statistical cut‐points have been discussed elsewhere[6, 14, 39] and are commonly used for detection of anti‐drug antibodies to therapeutic proteins. This bioanalytical strategy has also been successfully implemented for clinical AAV TI
assays[3, 40]. Statistical cut‐points are generally considered to be more objective,
even though they can be difficult to establish due to a varying degree of preexisting AAV antibodies in healthy humans and patient populations alike [3, 41].
Ahigh number of antibody‐positive samples may confound the statistical analysis
of data generated during cut‐point establishment. To mitigate cut‐point inflation
resulting from using samples with preexisting AAV antibodies, samples may be
prescreened prior to the cut‐point experiments to exclude those associated with
less than 50% transduction, indicative of preexisting AAV immunity [3]. To be
more rigorous, prescreening could include a confirmatory assay (described below)
and any sample with more twofold increase in transduction following immunoglobulin depletion would be considered AAV NAb‐positive and excluded from
cut‐point experiments. These approaches may require an initial analysis of a
larger number of commercial samples to find 50individual samples without preexisting AAV immunity. Alternative methods to deal with antibody‐positive samples and analyze data generated during cut‐point establishment are discussed in
the literature[29, 42–45].
6.3.3.2 Limit ofDetection
To characterize the analytical sensitivity of a cell‐based AAV TI assay, a limit of
detection (LOD) can be determined using a surrogate positive‐control AAV antibody. Both polyclonal and monoclonal antibodies may serve as PC, with polyclonal
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antibodies better reflecting the nature of humoral immune responses in future
study samples. The LOD is defined as the concentration of a positive‐control antibody in neat (undiluted) sample matrix that is detectable at the cut‐point of the TI
assay (Figure6.3). Serial 1:2 or 1:3 dilutions of a positive‐control antibody are
typically performed and tested. Dilutions expected to fall above and below cut‐
point should be included to accurately interpolate the LOD. The LOD may be interpolated using either a fitted curve, as shown in Figure6.3, or a linear curve interval
between the two antibody concentrations whose transduction values flank the
cut‐point.
Regulatory agencies recommend a detection limit of 100
ng/mL or less for the
positive‐control antibody in conventional anti‐protein therapeutic TAb assays and
acknowledge that cell‐based NAb assays may not always meet this mark [6].
Meeting this specification for AAV TI assay will depend on the type and affinity of
the positive‐control antibody and hence does not necessarily reflect the level of
AAV inhibitors in study samples. Furthermore, it is presently unknown what level
of AAV inhibitors in study participants will have a considerable impact on GTx
efficacy or safety. This level may also depend on GTx vector‐specific factors, such
as AAV serotype and route of administration. In conclusion, a clinically relevant
LOD target value cannot be specified for AAV TI assays at this time.
Therefore, suitable assay sensitivity may need to be ascertained through alternative avenues, such as (1) consideration of the MOI and vector‐to‐sample ratio, as
discussed above; (2) adequate resolution of AAV inhibitor titers in a large number
of individual subjects with pre‐existing immunity, which should range from low to
high titers; (3) empirical verification of predicted dose‐neutralization curves, as
described in the last paragraph; (4) correlation with efficacy and/or safety signals
in nonclinical studies.
100
90
80
70
60
50
40
30
% Transduction
20
10
0
050 100 150 200 250 300 350400
Figure6.3 LOD interpolation at the assay cutpoint set for 50% transduction.
Antibody concentration (ng/mL)
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6.3.3.3 Precision
Precision of cell‐based TI assays is generally lower than that of ligand‐binding
assays, resulting in higher coefficients of variance (CV). Intra‐replicate, intra‐
assay, and inter‐assay precision of TI assays can be assessed using low (LPC) and
high (HPC) PC AAV antibody‐spiked samples. CVs can be evaluated for raw luminescence signals or normalized transduction values and should generally remain
below 30%. Inter‐assay CV is also expected to remain below 30%, even though this
limit could be elevated to 50%, if such higher variance does not affect the reproducibility of qualitative results (negative/positive) for low‐positive samples.
Titer precision is also important to consider, which can be assessed as the range
of titer values measured repeatedly for the same sample. Titer precision is primarily dependent on the dilution scheme applied to the test samples and will typically
fall within ±1 of the mean log
(titer) derived from repeated measurements if a
2
1:2 dilution scheme is used. This corresponds to the last detectable sample dilution shifting one step upwards or downwards.
6.3.3.4 Specificity
Specificity for AAV TI assay addresses: (1) detection of neutralizing AAV antibodies versus non‐antibody‐based neutralizing factors (antibody specificity); (2) no
detection of AAV inhibitors that exclusively react with other AAV serotypes (serotype specificity); (3) detection of AAV inhibitors to both reporter gene construct
and GTx (GTx specificity). For (1), refer to section: Confirmatory Steps to Ensure
Specific Detection of Neutralizing AAV Antibodies. Serotype specificity (2) can be
assessed by testing samples spiked with monoclonal antibodies that exhibit exclusive reactivity against related AAV serotypes, which should remain undetectable.
This assessment is, however, of limited value and merely confirms that the
reporter gene construct is not contaminated with another AAV serotype. Clinical
test samples typically contain polyclonal mixtures of AAV antibodies that broadly
cross‐react with multiple AAV serotypes[41].
The underlying question of (3) is whether the reporter gene construct adequately represents the GTx. Even though reporter gene construct and GTx have
the same AAV serotype, they may have been produced by different manufacturing
processes and differ in the size of packaged DNA, both of which could impact
capsid antigenicity. One way to confirm GTx specificity in TI assays is to spike
excess GTx into a sample containing AAV inhibitors. The GTx will bind to AAV
inhibitors, preventing them from neutralizing the reporter gene construct, thus
leading to increased transduction.
6.3.3.5 Confirmatory Steps toEnsure Specific Detection ofNeutralizing
AAV Antibodies
While TI assays detect AAV inhibitors, these assays do not routinely distinguish
between neutralizing antibodies (NAbs) and non‐antibody‐based neutralizing
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factors[3]. This could obfuscate GTx patient eligibility when investigating preexisting AAV immunity, since some non‐antibody‐based neutralizing factors
decrease transduction invitro but are not impactful invivo, as demonstrated for
an AAV5 GTx in nonhuman primates[4].
The nature of non‐antibody‐based neutralizing factors in plasma is heterogeneous and poorly understood[46–49]. To ensure specific detection of NAbs in AAV
TI assays, a confirmatory procedure may be implemented that depletes antibodies
using resins coupled with immunoglobulin‐binding proteins, such as protein A,
G, and L[33, 40, 50]. Samples that screened positive for AAV inhibitors are retested
following immunoglobulin depletion, which restores transduction if and only if
NAbs were present. A mock depletion of the same sample is performed in parallel
to account for non‐specific depletion by unconjugated resin. Transduction following protein AGL depletion can be normalized to that observed following mock
depletion, establishing an AGL/mock ratio[40]. For regulated studies, a statistical
confirmatory cutpoint can be derived for the AGL/mock ratio; sensitivity, precision, and selectivity of the confirmatory assay could also be validated.
Alternative strategies to confirm the specificity of AAV TI assays include depletion of test samples using resins conjugated with the AAV vector of interest
[20,51], or the addition of empty or generic AAV vectors of the same serotype to
absorb and functionally deplete AAV inhibitors in test samples[52]. Even though
these strategies confirm the interaction of AAV inhibitors with a particular AAV
serotype of interest, they do not immediately provide for a molecular classification of the involved neutralizing factors as antibodies, since any AAV inhibitor
would be physically or functionally depleted.
6.3.3.6 Selectivity/Matrix Interference
Selectivity assessments for AAV TI assays are performed as described for TAb
assays. At least 80% of spiked donor samples should test positive, while at least
80% of unspiked donor samples should test negative. A related assessment is
interference from individual matrix components. For AAV TI assays, it is not
advisable to use endogenously lipemic, hemolytic, and icteric test samples for
interference studies, since results may be confounded by preexisting AAV inhibitors, falsely indicating additional interference. It may be more conclusive to add
interfering factors under investigation (lipids, hemoglobin, or bilirubin) exogenously to individual or pooled samples previously confirmed to be negative for
AAV inhibitors.
6.3.3.7 Stability
Stability studies can be performed by spiking low and high concentrations of positive‐
control AAV antibodies into negative test samples. Stability of non‐antibody‐based
neutralizing factors could alternatively be assessed by retesting a positive donor
sample over time. Neutralizing AAV antibodies are the most relevant AAV
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inhibitors, and antibodies are generally stable if stored at −70 °C or colder[53, 54].
Thus, there is limited value in performing long‐term stability studies at low temperatures. Short‐term and freeze/thaw stability assessments may be more applicable; for example, samples may be stored at higher temperatures, such as −20
°C, or ambient temperature, given logistical constraints, or be subject to repeated
4
freeze–thaws to allow for retesting.
6.3.4 Sample Testing Strategy and Monitoring Assay Performance
Samples from GTx treatment‐naïve subjects can be positive for AAV inhibitors but
seroprevalence typically does not reach 100%[3, 41]. This could justify tiered testing for pre‐dose samples: initial screening at minimally required dilution (MRD),
followed by titering of screen‐positive samples only. Though for rare diseases,
where sample batches are small, screening and titer assays are typically performed
simultaneously to enable timely eligibility decisions. Screen‐positive samples may
also be tested in a confirmatory assay, if deemed suitable for the serotype of
interest.
In contrast, samples collected following systemic GTx administration have high
AAV TI titers due to the formation of neutralizing AAV antibodies, usually with
an incidence rate of 100% and persisting for at least 15 years[55–58]. Therefore, in
most clinical scenarios, post‐dose samples could be titered directly. While post‐
dose samples might also contain non‐antibody‐based neutralizing factors, their
titers will be negligible compared to those from treatment‐emergent neutralizing
AAV antibodies. Hence, confirmatory assays for post‐dose samples may not be
necessary or be restricted to a subset of samples to demonstrate that neutralizing
AAV antibodies have indeed formed as expected.
TI assay performance can be monitored long‐term by trending luminescence
signals and/or transduction/inhibition values for LPC and HPC, as well as titer
values for HPC. Appropriate trending ranges may be established based on results
from at least 10 assay runs.
°C,
6.3.5 Data Interpretation: Preexisting TI Titer and Clinical Efficacy
Preexisting AAV immunity detected in TI assays does not always correlate with
decreased GTx efficacy. Lack of biological impact has been linked to low TI
titers[2, 59] as well as non‐antibody‐based neutralizing factors, which are typically present at low TI titers[3, 4]. This raises the question of whether there is a
clinically meaningful correlation between preexisting TI titers and the likelihood
of clinical success, which could inform GTx patient eligibility.
For TI data interpretation, it can be useful to determine the theoretical neutralizing capacity of plasma/serum in relation to a particular TI titer, based on assay
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