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5 Immunogenicity of AAV Gene Therapy Products
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40 Martino, A.T. and Markusic, D.M. (2020). Immune response mechanisms against
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adeno‐associated viral vectors pseudotyped with serotype 2, 5, and 8 capsid
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43 Lai, C.M., Estcourt, M.J., Himbeck, R.P. etal. (2012). Preclinical safety evaluation
of subretinal AAV2.sFlt‐1in non‐human primates. Gene Ther. 19 (10): 999–1009.
44 Amado, D., Mingozzi, F., Hui, D. etal. (2010). Safety and efficacy of subretinal
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Sci. Transl. Med. 2 (21): 21ra16.
45 Calcedo, R., Morizono, H., Wang, L. etal. (2011). Adeno‐associated virus
antibody profiles in newborns, children, and adolescents. Clin. Vaccine Immunol.
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46 Li, C., Narkbunnam, N., Samulski, R.J. etal. (2012). Neutralizing antibodies
against adeno‐associated virus examined prospectively in pediatric patients with
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47 Vandamme, C., Adjali, O., and Mingozzi, F. (2017). Unraveling the complex story
of immune responses to AAV vectors trial after trial. Hum. Gene Ther. 28 (11):
1061–1074.
48 Mingozzi, F., Maus, M.V., Hui, D.J. etal. (2007). CD8(+) T‐cell responses to
adeno‐associated virus capsid in humans. Nat. Med. 13 (4): 419–422.
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50 Mingozzi, F., Meulenberg, J.J., Hui, D.J. etal. (2009). AAV‐1‐mediated gene
transfer to skeletal muscle in humans results in dose‐dependent activation of
capsid‐specific T cells. Blood 114 (10): 2077–2086.
51 Ertl, H.C.J. (2021). T cell‐mediated immune responses to AAV and AAV vectors.
Front. Immunol. 12: 666666.
52 Long, B.R., Veron, P., Kuranda, K. etal. (2021). Early phase clinical
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Section III
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Bioanalysis for Gene Therapy
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6
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Bioanalytical Methods to Detect Preexisting and
Post-administration Humoral Immune Responses
Against AAV Capsid Proteins
Christian Vettermann1 and Boris Gorovits
1
Translational Sciences, Assay Strategy & Development, BioMarin Pharmaceutical, Inc., Novato, CA, USA
2
Gorovits BioSolutions, LLC, Andover, MA , USA
2
6.1 Introduction
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Preexisting and posttreatment humoral anti‐Adeno‐associated virus (AAV)
immunity can significantly impact treatment safety or/and efficacy. The presence
of preexisting antibodies may result in insufficient level of transgene protein
expression[1] while post‐dose immune response is expected and typically is considered as main barrier for repeat administration of AAV‐based therapeutics. AAV
humoral immune responses to AAV capsid can be assessed by methods detecting
either total antibodies (TAb) or neutralizing antibodies (NAb). TAb comprises all
immunoglobulins that bind to a particular AAV serotype, including those that can
neutralize the AAV vector’s ability to transduce cells, i.e. those that are NAb. Due
to the nature of the protocols used to detect the presence of AAV NAb, these methods are often referred to as transduction inhibition (TI) methods, which are
described in more detail later in this chapter. Some level of correlation between
the results from AAV TAb and TI methods can be expected and has been
reported[2, 3]. However, this correlation is complex and not always linear. In
addition to the difference in mechanisms employed by TAb and TI methods, they
can also differ in sensitivity, specificity, and other critical performance parameters.
There is an ongoing debate as to whether preexisting TAb vs. NAb impact
study outcomes, with some evidence for AAV5 suggesting that high‐sensitivity
Drug Development for Gene Therapy: Translational Biomarkers, Bioanalysis, and Companion
Diagnostics, First Edition. Edited by Yanmei Lu and Boris Gorovits.
© 2024 John Wiley & Sons, Inc. Published 2024 by John Wiley & Sons, Inc.
6 Bioanalytical Methods to Detect Preexisting and Post-administration
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TI protocols may not only be reporting NAb but also the presence of non‐
immunoglobulin‐based inhibitors without considerable biological significance
[2, 4]. Nonetheless, TI methods enjoy wide popularity, sometimes in combination
with TAb methods, for pretreatment evaluation of patient eligibility[1]. Contrary
to the practice used for protein‐based biotherapeutics, a tier‐based approach for
humoral immunogenicity assessment (first‐tier TAb, second‐tier Nab only if TAb
positive) is not commonly followed when determining eligibility of patients for
gene therapy (GTx)[5]. Instead, sponsors select either one or both AAV prescreening methods for pretreatment evaluation.
In this chapter, we present information related to the principle of TAb and TI
methods, critical details of the protocols, key performance parameters, and
aspects of data interpretation. Finally, value and challenges associated with standardization of TAb and TI methods are briefly discussed.
6.2 Considerations forAAV Total Antibody Assays
6.2.1 Nature ofAAV TAb Assay Analyte
A diverse antibody response against capsid proteins occurs after natural exposure
to AAV or after therapeutic administration of AAV‐based GTx vectors. Such
responses vary by epitope specificity, immunoglobulin isotype, and functional
characteristics of the antibodies. Because AAV‐neutralizing antibodies are a subset of total antibodies, the correlation between TAb and NAb levels is complex,
with the general expectation of a higher prevalence of TAb[2, 3]. The TAb to NAb
response comparison is further complicated by different assay formats used to
detect the two analytes and an oftentimes higher sensitivity of TAb assays as compared to common NAb protocols. Even in the absence of a measurable NAb,
detection of TAb can indicate prior exposure to AAV through natural infections.
Based on the definition of TAb responses, all AAV antibody isotypes are expected
to be detected in the TAb analytical protocol, with most attention generally paid to
the detection of serum IgG and IgM classes. Detection of serum IgE typically falls
into a unique category and is only requested based on specific clinical observations, as defined in regulatory guidance [6]. Understanding whether the TAb
assay detects both or only one of the IgM and IgG classes of immunoglobulins is
viewed as critical and is based on the assay format and reagents used in the
method. It is commonly expected that low affinity IgM responses which develop
early after exposure will then mature into IgG‐based responses. Information about
detailed isotype composition of an immune response at a given timepoint could
be informative and may facilitate the interpretation of immune‐associated toxicities or hypersensitivity reactions[7]. Because a preexisting TAb response is likely
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Labeled AAV capsid
(a) (b)
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to be mature, it is expected to be primarily composed of IgG isotypes, with a
smaller fraction of samples containing IgM class antibodies[8, 9].
6.2.2 Primary Analytical Methodologies applied forAAV
TAb Detection
Based on the definition of the AAV TAb analyte, assay formats, and analytical
platforms used are similar to those applied when detecting anti‐drug antibody
(ADA) responses to protein therapeutics. Two of the most common protocols are
antigen‐capture (aka sandwich) and bridging assay formats (Figure6.1). In the
antigen‐capture format, the capture reagent, most commonly the AAV vector,
defines the specificity of the detected TAb analyte. Antibodies in the sample will
bind to the capture reagent on the plate and are detected using, for example, a
labeled species‐specific anti‐immunoglobulin antibody. Isotype specificity of the
detector reagent defines whether the method can detect one or more AAV antibody classes and should be carefully evaluated [10]. Alternatively, broadly
immunoglobulin‐binding proteins A, G, or L can serve as detector reagents, which
is typically done in combination to account for differential binding to various antibody isotypes[4]. The detector reagents are labeled with either an enzyme (for
enzyme‐linked immunosorbent assays, ELISA) or ruthenium (for electro‐
chemiluminescent assays, ECL). Final assay signal generation is triggered by the
addition of enzyme‐substrate (ELISA) or read buffer (ECL).
The benefit of applying the antigen‐capture assay format is the ability to distinguish between IgG and IgM class responses. Understanding isotype composition
of antibody responses may be insightful, since it can inform about the status of
immune response maturity. Early immune responses are primarily IgM‐based
with a gradual transition to IgG. Human immunoglobulin isotypes have various
ability to engage and activate the complement pathway or trigger various types of
Labeled anti-species
immunoglobulin antibody
Anti-AAV capsid antibody
AAV capsid
Figure6.1 Principles of antigen capture (a) and bridging (b) assay formats designed to
detect presence of anti-AAV antibodies.
Anti-AAV capsid
antibody
AAV capsid
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hypersensitivity reactions[7, 11]. The broadly discussed possibility of re‐dosing
patients previously treated with AAV GTx may greatly depend on the nature and
isotype composition of the AAV antibody response.
The potential challenges of the antigen‐capture assay format include selecting a
detector reagent capable of specific identification of isotypes of interest and the ability to apply a single detector reagent that can detect both species‐specific (e.g.
human) AAV antibody responses as well as the positive control (PC) used in the
assay. A non‐human PC antibody reagent is frequently chosen based on the ease of
generation and lack of supply concerns. The assay detector reagent therefore should
either be cross‐reactive to both human antibody and non‐human PC reagent or two
separate detector reagents need to be applied[12]. This approach may be less favorable as it does not provide information on the integrity of the antihuman detector
used for detection of actual sample responses. To monitor performance of the
human immunoglobulin detector reagent, additional controls may thus be considered and applied during routine sample testing. For example, a previously tested,
confirmed‐positive incurred study sample may be used as a surrogate PC.
The TAb bridging assay format offers a robust alternative and has been broadly
applied to detect ADA responses against protein‐based biotherapeutics[13, 14]. In
this format, the multi‐valent nature of a typical immunoglobulin is utilized to create a bridge between AAV capture and labeled AAV detector reagents [3]. The
bridging assay format is therefore isotype‐agnostic and expected to determine the
combined presence of IgG, IgM, and other isotypes without distinguishing
between them. A subset of monovalent IgG4 antibodies that underwent Fab arm
exchange may remain undetectable[15]. Like for the antigen‐capture assay format, ELISA and ECL analytical platforms can be applied.
Detection of ADA responses against protein‐based biotherapeutics commonly
includes assessment of signal specificity, which is conducted in competition‐
based confirmatory tests [6]. In this assessment, samples are spiked with unlabeled biotherapeutic at a concentration that is sufficient to block antibody binding
to the capture reagent used in the assay. A significant reduction in assay signal
indicates the presence of biotherapeutic‐specific antibodies. While this approach
has been implemented for many protein biotherapeutics, it has not found broad
application in the AAV GTx field, though exceptions exist [1, 3]. High material
cost and the multivalent nature of the capture and detector reagents (AAV capsids), which results in potentially high amounts of unlabeled AAV reagent for
successful competition of antibody binding, are two likely reasons.
6.2.3 Tab Assay Critical Reagent Considerations
6.2.3.1 Positive and Negative Control Selection
Like ADA assay protocols, a positive‐control AAV antibody reagent is used to
determine the quality of TAb assay runs, i.e. it serves as the assay suitability
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control material. Several commercial AAV antibody reagents are available with
various AAV serotype specificities. These monoclonal and polyclonal antibodies
can be successfully applied in a proprietary AAV TAb method after it is demonstrated that the commercial antibody reagent sufficiently binds to the AAV serotype of interest. Alternatively, an AAV serotype‐specific PC reagent may be
developed[16]. The need for serotype‐specific AAV PC reagent is debatable since
the degree of homology between various AAV serotypes is high, leading to a significant degree of antibody cross‐reactivity between serotypes[1]. Hence, even if
specificity to a particular AAV serotype was demonstrated for a PC reagent during
method validation, it does not rule out the assay’s ability to detect antibodies that
are cross‐reactive to other AAV serotypes in clinical test samples.
Nonetheless, a serotype‐specific PC might bind with higher affinity to a particular AAV capsid and thus offer an advantage when characterizing the sensitivity of
the method for detecting antibodies to the serotype of interest. While PC reagent
is used in characterizing assay sensitivity, precision, selectivity, and robustness,
other critical parameters, such as matrix interference and cut‐point, are defined
based on the analysis of negative control matrix and treatment‐naïve individual
donor samples.
The TAb assay negative control (NC) is used to calculate plate‐specific assay
cut‐points and further serves as diluent for PC reagent and individual test samples
if titered, as well as a general assay suitability control. The NC reagent is commonly generated by combining several individual samples that had shown no or
limited AAV antibody reactivity. Selection of individual matrix samples without
any AAV reactivity is complicated by high AAV antibody prevalence in treatment‐
naïve human populations [9, 17, 18]. Screening and confirmatory tests may be
required to determine which of the samples can be pooled to generate NC reagent.
Alternative solutions include depletion of AAV antibodies from matrix by applying nonspecific (e.g. Protein G‐based[19]) or specific (using AAV capsid of interest [20]) affinity pull‐down. However, these immune‐depletion protocols may
potentially produce matrix that is no longer representative of study samples,
resulting in a high potential for over‐reporting AAV TAb positive responses.
Finally, NC matrix needs to match that of the study samples tested for the presence of AAV TAb. The use of surrogate matrix may need to be evaluated in cases
where access to natural negative matrix is limited (e.g. aqueous humor)[21].
6.2.3.2 Capture and Detection Reagents
Like in protein biotherapeutic ADA methods, AAV capsid‐derived capture reagent is used for TAb assays in both antigen‐capture and bridging formats. AAV
capsid is also needed as a detector reagent in the bridging format (Figure 6.1).
AAV capsid‐derived material can be the drug substance or product of interest (i.e.
unaltered AAV vector), or surrogate AAV vector encoding another transgene, or a
capsid protein preparation representative of investigated AAV serotype. Although
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using a capsid protein preparation may seem attractive, as it avoids working with
transduction‐competent AAV vectors, epitopes found on capsid proteins may not
be representative of antigenic epitopes on intact AAV capsids. Therefore, it is generally recommended to use intact AAV capsids of the same serotype as the
investigated GTx vector. If the AAV vector is identical to a naturally occurring
serotype, commercially available AAV preparations may be applied. Similarly,
commercially sourced AAV preparations may be used in a confirmatory step, if
applied to demonstrate specificity of AAV antibody responses.
6.2.3.3 Sample Testing Strategy
A typical approach to ADA sample analysis may be applied when detecting an
AAV TAb. This tier‐based approach includes initial screening, confirmatory, and
titration testing[14]. For a typical protein biotherapeutic ADA response, the reason to apply a tier‐based approach is to reduce the number of samples that are
evaluated in the final titration test, since only a fraction of samples is expected to
score positive in both screening and confirmatory assays. In contrast, a robust
AAV response with high incidence rate, in particular following therapeutic GTx
vector administration, is expected. This could support a proposal to directly apply
titration analysis and avoid delays related to initial testing of samples in screening
and/or confirmatory steps.
6.2.4 Key Assay Qualification/Validation Parameters
6.2.4.1 Assay Sensitivity
Considerations for assay sensitivity that align with expectations for anti‐protein
biotherapeutic ADA protocols based on current FDA guidance[6] are relevant to
AAV TAb assays. Regulatory guidance suggests that at least 100 ng/mL sensitivity
based on a suitable PC reagent is expected, and it has been typically relatively easy
to achieve this goal[22]. The sensitivity parameter is assessed based on performance of the assay PC that may need to be reconsidered, together with assay conditions, if the desired sensitivity is not reached.
6.2.4.2 Serotype Specificity
Based on high homology between AAV serotypes, it can be expected and has been
demonstrated that AAV antibodies detected in GTx treatment‐naïve populations
are highly cross‐reactive[17, 23]. The level of homology between serotypes may
determine the degree of cross‐reactivity observed[24]. Importantly, a lower degree
of cross‐reactivity was reported for TI methods, which is likely due to the complexity of the viral transduction process and requirement for high specificity and
affinity of immunoglobulins with neutralizing activity[25–28].
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