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information/roctavian- epar- product-
5
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Immunogenicity of AAV Gene Therapy Products
Vibha Jawa1 and Bonnie Wu
1
Clinical Pharmacology, Pharmacometrics and Bioanalysis (CPPB), Bristol Myers Squibb, Princeton, NJ, USA
2
Biologics Discovery and Development Sciences, Johnson & Johnson Innovative Medicine, Spring
House, PA, USA
Immune response to recombinant AAV (rAAV) based therapies has been well
characterized through preclinical and clinical experience [1]. The immune
response can be mediated by product‐associated risks that include viral capsids,
genome containing the nucleic acid as well as the transgene product. Each of these
structural components can induce either an innate or adaptive phase immune
response. In addition, the clinical risks can be related to the nature of genetic
mutation, disease state, route of administration, and site of injection as well as any
standard of care treatments that can contribute to the risks[1].
2
117
5.1 Innate and Adaptive Immunity Induced by
AAV-Based Gene Therapies
5.1.1 Innate Immune Response
The adeno‐associated virus (AAV) vectors were not considered very strong elicitors of innate or adaptive immune response when compared to Adenovirus
(Adv) [2]. However, preclinical and clinical observations have shown that the
AAV‐derived content (capsids, nucleic acid content, translated gene product, etc.)
may be recognized as foreign. The membrane bound as well as intracellular pattern recognition receptors (PRRs) on innate immune cells like macrophages and
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.
5 Immunogenicity of AAV Gene Therapy Products
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118
dendritic cells can react with AAV and related content if they have structural
motifs/patterns [pathogen associated molecular patterns (PAMPs)] similar to
pathogenic organisms[3].
Multiple mechanisms of action have been identified by which rAAV can activate
innate immune response. This includes (1) AAV capsid inducing Toll‐like receptor
2 (TLR2) present on cell surface or endosomal membrane, (2) AAV packaged
genome is hydrolyzed in lysosome or endosome to release viral nucleic acid content triggering a Toll‐like receptor 9 (TLR9) mediated response, (3) activation of
stimulator of interferon genes (STING)/interferon regulatory factor 3 (IRF3) pathway by mitochondrial DNA that triggers cytosolic sensors leading to upregulation
of type I interferons (IFNs), (4) additional pathways like inflammasome formation
and viral RNA sensors retinoic acid‐inducible gene I (RIG‐1) and anti‐melanoma
differentiation‐associated gene‐5 (MDA‐5) induced IFNs[4, 5]. All pathways lead
to release of proinflammatory chemokines like tumor necrosis factor‐alpha
(TNF‐α) and interleukin (IL‐6) through engagement of myeloid differentiation primary response 88 (MyD88)/interleukin‐1 receptor‐associated kinase (IRAK) pathways while inflammasome maturation leads to IL‐1β and IL‐18 production[6]. An
activation of innate immune response through IF‐stimulated genes (ISGs) can
cause inhibition of viral replication and consequent spreading. In addition, innate
phase cytokines and chemokines can prime a T‐cell‐mediated adaptive immune
response that could also induce a long‐term memory response[7].
An additional pathway that can induce innate response could be complement‐
dependent activation triggered by pre-existing anti‐AAV antibodies (PEA) complexed with AAV capsids[8, 9]. High‐dose AAV complexed with PEA can activate
classical complement cascade leading to cell lysis through membrane attack complex (MAC)[10]. Low‐dose AAV leads to generation of complement factors C3b
and C3d that opsonize AAV and can interact with their respective receptors CR1
and CR3 expressed on macrophages[8]. This leads to an enhanced uptake and
antigen presentation which can further recruit T cells. B cells can also uptake C3d
opsonized viral particles through CR2 and cross‐link with B cells to induce effector and memory B cells (Figure5.1).
The innate immune response mediated by complement activation (complement
proteins C3 and its cleaved products C3b, C3bi, C3d) can be activated by direct
interaction with AAV capsid or through pre-existing anti‐capsid antibodies complexed with low doses of AAV capsid. The complement cascade can eventually
lead to formation of the MAC that can lyse the cell. The anaphylactic fragments
C3a and C5a generated during cleavage can induce inflammatory responses.
Other innate pathways include engagement and activation of the toll‐like receptors TLR 3,7,8 TLR 9 by viral capsid and genome respectively that further prime
the adaptive phase immune response.
Innate immune system
Complexed capsid/
pre-existing anticapsid antibodies
C3b
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High AAV
C3a
5.2 Preclinical Immunogenicity Risk Assessment 119
C6-9/MAC
C5b
C5b
C5a
dose
TLR 9
TLR 2, 3 7 8
Proinflammatory cytokines stimulate adaptive immune system
C3
C3
Opsonization
C3b
C3b
Factor I/Factor H
C3a
Low AAV dose
C3bi
Adaptive immune system
Target cell
MHC I
TCR
MHC II
APC
CR1
CR2
CR3
C3d
TCR
CD8 T cell
CD4 T cell
Granzymes and perforins
MHC I
Anti-capsid Abs
TCR
CD8 T cell
Target cell
Cellular immune response to target tissue
Anti-transgene antibodies
Humoral immune response to transgene and capsid
Figure5.1 AAV-mediated activation of innate and adaptive phase immune responses.
APC, antigen-presenting cell (Macrophage/Dendritic cell/B-cell); CR1, complement
receptor recognizing complement fragment C3b on macrophages; CR2, complement
receptor recognizing complement fragment C3d on B-cells and dendritic cells; CR3,
complement receptor recognizing the inactivated C3b (C3bi) on macrophages; and
dendritic cells; TCR: T-cell receptor; MHC: major histocompatibility complex;
MAC: membrane attack complex.
5.1.2 Adaptive Immune Response
Both humoral and cell‐mediated immune responses can occur following the administration of AAV‐based gene therapies. The humoral response includes pre‐existing
antibodies (PEAs) to AAV capsids that can limit the viral transduction as well as
treatment emergent antibody response to capsid‐specific epitopes[11]. The transgene
expression in the targeted cells or tissues can also be associated with an antibody
response. Both capsid proteins and transgene product can also elicit MHC Class I
and Class II‐driven cellular response. MHC Class 1 activates a cytotoxic CD8+ driven
T‐cell response while MHC Class 2leads to a CD4+ driven Teffector response.
5.2 Preclinical Immunogenicity Risk Assessment
The immune response to AAV gene therapies (GTs) can occur at any stage of
development. Hence, a comprehensive risk assessment is needed that will identify
the relevant risk factors. The risk factors can be product, process, and patient
derived as summarized in sections below.
5 Immunogenicity of AAV Gene Therapy Products
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120
5.2.1 Product-related Risk Factors
Product‐related risk factors can arise from the vector genome, viral capsid serotype,
and transgene product. The capsid‐associated risk factors include post‐
modified capsid‐derived peptides and aggregation[12–14]. Similarly, vector genome‐
related risk factors include unmethylated CpG motifs, self‐complementary DNA, as
vector DNA and viral dsDNA that can engage PRRs like TLR9[15–17]. The serotype
of capsids and their related transduction efficiencies in different tissues can also
contribute to the risk[18–20]. The choice of serotypes can impact clinical outcomes
such as safety, vector clearance, treatment eligibility, occurrence of transaminase
elevations, activation of capsid‐directed cytotoxic T‐cell responses, and clinical efficacy. There is a similarity in several structural features responsible for tissue specificity and transduction across serotypes. However, there are distinct features that
can also impact gene transfer and immunogenicity. Among the 13 AAV serotypes
that have been identified, AAV5 is the most distinct and has the least homology to
more common serotypes like AAV2, AAV8, and AAV10. Based on these homologies,
the risk of cross‐reactive sero‐reactivity also varies [21] and selection of capsids
where the least pre-existing immunogenicity risk is anticipated can help mitigate
any potential immune‐mediated vector clearance.
Similarly, serotype tropism can limit the non‐specific uptake of the vector and
ensures the targeting and concentration in the relevant tissue. While primary
receptors influence binding, the secondary receptors support internalization and
can influence tropism across the viral vector serotypes[22]. One way to ensure the
targeted delivery of the vector is through use of tissue specific promoters as
observed in some recent clinical trials where liver‐specific promoters were used to
treat FVIII and FIX gene defects.
Such strategies can reduce the overall risk of pre-existing antibody cross‐
reactivity that can lead to elimination of transduced vector and loss of efficacy.
Additionally, limited and targeted high level of transgene expression using the
tissue‐specific serotype can ensure less toxicity due to complement activation and
cytotoxic T‐cell mediated elimination of off‐target cells. There may be differences
in capsid‐specific T‐cell responses based on serotypes. While AAV2, 8, and 10were
associated with elevated liver transaminases, increased capsid‐specific cytotoxic
T‐lymphocyte (CTL) responses, and elimination of transduced gene product in
liver cells, AAV5 did not elicit any CTL responses even with or without increase of
liver ALT (amino alanine transferase) and AST (aspartate aminotransferase) levels.
translationally
5.2.2 Process and Manufacturing-Related Risk Factors
The empty capsid content in the final packaged AAV‐GT product can change from
lot to lot as part of purification process[23–26]. Additional impurities include the
biosynthetic intermediates and incorrectly composed viral particles, AAV
5.2 Preclinical Immunogenicity Risk Assessment 121
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encapsidated host DNA fragment from different species like insect cell DNA,
nuclease‐
and plasmid DNA, etc.[27, 28]. New analytical characterization approaches have
pointed to presence of partially packaged viral genomes even in fully packaged
capsids.
sensitive nucleic acids, helper components, viruses relevant to cell lines
5.2.3 Patient-Related Risk Factors
The capsid‐derived peptides are presented in the context of Class I and Class II
human leukocyte antigen (HLA)/major histocompatibility complex (MHC) that
are used interchangeably. The risk of such peptides driving a CTL (CD8+) or
Teffector (CD4+) response depends on the HLA alleles of the patients and their
affinity to these peptides[29]. The genetic associations with an inflammatory
diseased state can also enhance the risk[30].
The transgene‐driven protein expression and the consequent immune response
could differ based on the nature of gene defects (null vs. point mutation vs.
spliced). The engineered protein expressed by transgene has a much higher risk
compared to the endogenous protein that is close to self [31]. Based on regional
ex
posure and patient demographics, the pre-existing capsid‐specific sero‐ reactivity
can be different. The disease severity and the immune robustness of the patient
can also change the risk due to underlying disease that can exacerbate the immune
response to the AAV‐GT vectors. The highly vascularized nature of site where the
GT vector is delivered vs. immune suppressed/immune deficient site can change
the risk of immunogenicity. Based on the age of patient (pediatric vs. adult vs.
seniors), seroprevalence to the capsid serotypes may differ and can change the
risk for reduction of transgene expression due to complement fixing anti‐capsid
immune complexes. The high dose of AAV genomes has also been associated with
adverse events, especially in patients with a severe disease that cannot tolerate the
high viral burden[32]. Lastly, the administration through an IV route compared
to a local delivery into the subretinal space or liver or muscle may change the risk
and is dependent on the immune‐privileged state of the site and presence of
tissue‐associated professional antigen‐presenting cells [33]. The administration
route will be further discussed in clinical section below.
5.2.4 Nonclinical Assessment ofImmunogenicity
Nonclinical studies to assess immune response to rAAV‐based GTs can provide an
understanding of immune‐mediated adverse events and pharmacology as well as
biodistribution. These observations also provide a better understanding of the
adverse events due to different routes of administration (intra‐thecal, subcutaneous, etc.), dose‐dependent immune‐ and geno‐toxicities due to viral vector capsids
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