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284
Number of IFN-gamma spots in response to
600
500
400
300
200
100
0
PBMC separated fresh, tested frozen PBMC separated >24 h, tested
the CMV peptide pool
frozen
donor 14
donor 1
donor 16
donor 17
donor 8
donor 19
donor 20
donor 22
donor 24
donor 7
donor 27
donor 11
donor 30
Figure11.4 Fresh and cryopreserved PBMC perform equally well in recall antigen-
specific ELISPOT assay (CMV peptide pool).
Of note, the TB test is an invitro diagnostic ELISPOT test for the detection of
effector T cells that respond to stimulation by Mycobacterium tuberculosis. This
diagnostic ELISPOT kit that is approved by the FDA (T‐SPOT TB test) allows shipment of whole blood samples for up to 54 hours post venipuncture before PBMC
isolation and testing[66].
For the standardization of separation and cryopreservation of PBMCs from
whole blood for use in recall, antigen‐specific assays such as ELISPOT and serum‐
free solutions are recommended for washing, cryopreservation, thawing, and
testing. Even if serum‐containing solutions have been screened and qualified for
cell culture use, an approximately 30% effect on the human sample population can
still be expected due to toxicity or mitogenic effects of serum on the PBMC samples
used in the recall antigen‐specific ELISPOT assay, which can affect the results. It
has been established that, while reproducible results among laboratories can be
obtained using serum‐free solutions, different qualified sera used in the different
laboratories can introduce variability in the ELISPOT assay results[44].
Some laboratories prefer to let the thawed cryopreserved PBMCs rest, which
+
was established for CD8
T cells that do not require APCs, using the CEF peptide
pool (derived from CMV, Epstein–Barr virus, and flu virus epitopes), presented by
11 Class 1 HLA‐A and HLA‐B alleles[67–70].
However, it has been shown that resting of thawed PBMCs is not generally beneficial in comparison to direct exvivo ELISPOT assays performed[71]. It doubles
the required cell numbers for the assay, increases complexity of work, and with it
the cost. It might lead to loss of effector cells, as these are sticky and have thin
285
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membranes. The activation of the cells increases the cell size and thins the membrane, making them more vulnerable when handled. Accordingly, effector
T cells or plasma B cells might be selectively lost over memory T and B cells,
which have a thicker membrane and are smaller, and thus more resistant to
handling. In addition, if antigen processing by APCs is required for more complex
recall antigens (proteins, viruses, virus particles, etc.) then the results of the
ELISPOT testing might be more impacted by resting. The scientific community is
split on this direct exvivo and resting topic.
Investigators should consider all these factors and test if resting would be beneficial or not for their given study and the given recall antigens to be used in the
recall antigen‐specific ELISPOT assay.
11.3.2.2 Antigen Concentration and Number ofReplicates
Besides the standardization of the sample material, that is, the PBMCs, the antigen
intended to be used needs to be optimized to qualify and validate the recall
antigen‐specific ELISPOT assay. The optimal antigen concentration will need to
be determined, as the concentration of the antigen will influence the obtained
results[57]. The antigen concentration will influence the number of spots recalled
from the PBMCs (i.e. whether all cells that are present or only a fraction of the
cells respond), as well as the quality of the spots measured, leading to distinct
spots with reduced signal‐to‐noise ratio.
The number of replicates required for the ELISPOT assay depends on the assay
system, the antigen, the affinity with which the T cells are assumed to respond to
the antigen, and the frequency of the specific T‐cell population within the PBMC
sample[54].
The FDA‐approved T‐SPOT kit uses single wells for their recall antigen in the
IFN‐gamma ELISPOT assay[66]. The usage of triplicates is more common in the
scientific community, and sometimes even six‐well replicates have been used if
the frequency of the recall antigen‐specific T cells was very low. The decision on
the required number of replicates for testing of clinical samples should be made
during the optimization phase of the recall antigen‐specific ELISPOT assay and
tested with the decision made after Phase 1 of the validation/qualification have
been completed and acceptance criteria have been generated (see Section3.1)
since this provides the precision data.
In summary, recall antigen‐specific ELISPOT assays are very robust and can be
qualified and validated to support clinical trials in all phases of the drug lifecycle.
The most crucial component for the ELISPOT assay is the quality of the PBMCs
provided, which should be carefully considered.
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286
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12
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Detection ofHumoral Response toTransgene
Protein and Gene Editing Reagents
George Buchlis1 and Boris Gorovits
1
Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA
2
Gorovits BioSolutions, LLC, Cambridge, MA, USA
2
12.1 Pre- and Post-dose Humoral Immunity
toTransgene-expressed Proteins
Much emphasis on the development of gene therapies has been placed on
measuring, avoiding, and/or limiting immune responses to the viral capsid. While
anti‐capsid immune responses can indeed render the therapeutic ineffective,
anti‐transgene immune responses can prevent patients from benefiting from
either gene therapies or existing protein therapeutics. Humoral responses to
secreted transgene proteins can neutralize an enzyme’s activity, block its uptake,
and reduce its availability. In addition, antibody responses to non‐secreted
proteins can potentially lead to the destruction of transduced cells through
complement‐dependent or antibody‐dependent cellular cytotoxicity (ADCC).
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12.1.1 Risk-based Analysis ofResponse Probability and Impact
12.1.1.1 Route ofAdministration
One of the greatest determinants of humoral responses to transgene proteins is the
route of administration of the gene therapy. The most well‐documented route of
administration resulting in anti‐transgene antibodies is intramuscular injection.
Antibodies to transgene expressed in muscle fibers have been observed in multiple preclinical animal models, including mice [1–7], rabbit [8], and non‐human
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.
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292
primates (NHP) [9, 10]. However, there are ways to mitigate transgene‐elicited
antibody responses to muscle gene transfer. One approach is to administer the vector intravenously but in an isolated region of the anatomy. In a method termed
locoregional intravenous administration or regional administration, transvenular
extravasation of the vector is achieved under high pressure in a cannulated limb
vein isolated from the systemic circulation via tourniquet[9, 11, 12]. Similarly, in a
technique termed afferent transvenular retrograde extravasation (ATVRX), a similar isolated vector delivery achieves substantial muscle transduction without
accompanying transgene antibody responses[13, 14].
One method to address the antigenicity of muscle‐directed gene transfer is the
use of immunomodulation or immunosuppression. These approaches in preclinical studies have involved the use of cyclophosphamide[15], anti‐CD4 antibody
with cyclosporin[16], cyclosporin with mycophenolate mofetil (MMF) and anti‐
thymocyte globulin (ATG)[17], cyclosporin with rituximab[18], MMF with sirolimus [19], and rapamycin with ibrutinib [20]. Another interesting approach is
downregulating expression in antigen‐presenting cells via miRNA inclusion in
the transgene cassette[21]. Restricting expression to myofibers by employing a
muscle‐specific promoter has shown efficacy in human gene therapy trials of
limb‐girdle muscular dystrophy type 2D[22, 23].
Intravenous administration of a gene therapy vector is commonly employed for
transduction of well‐vascularized organ systems or to access broad areas of the
anatomy. For many transgene proteins that are expressed in the liver, this route of
gene transfer does not provoke deleterious antibody responses. This phenomenon
has been observed in mice[24–36], dogs[37, 38], and humans[39, 40]. Of course,
there are instances where antibodies do form against the therapeutic protein in
non‐disease models of IV gene therapy. This can often be due to the immunogenic
nature of the transgene protein [41–44], or the propensity of NHP to mount
humoral immune responses to foreign transgene products[41, 45].
Monogenic diseases of the nervous system are a frequent target for gene therapy
research approaches. However, anti‐transgene antibody responses have been
observed in preclinical animal models against nonself proteins when the gene
therapy vector is infused into brain parenchyma[45, 46]. This nonself antibody
response can be reduced with administration of rapamycin[47].
Intraocular gene transfer of a subretinal‐administered Adeno‐associated virus
(AAV) expressing RPE65 became the first FDA‐approved gene therapy in the
United States. In this limited setting, anti‐transgene immune responses are rarely
observed, even while detectable yet non‐consequential anti‐capsid humoral
responses are measured in the circulation. This has been observed in small animals[48], large animals [49, 50], and human patients[51, 52]. In fact, ocular
readministration of the gene therapy has proven efficacious in human
subjects[53].
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12.1.1.2 Biodistribution ofVector, Vector Serotype, Dose, and
Expression Level
While the administration route often dictates the location (biodistribution) of tissue transduction, intrinsic properties of the gene therapy vector itself, serotype,
dose, promoter specificity, and promoter strength can have a strong impact on the
formation of antibodies against the transgene product.
Importantly, the transgene genome itself can play a role in enhanced antibody
production. Unmethylated CpG sequences in the transgene cassette can stimulate
TLR9 and MyD88‐dependent type I interferon production shown to be essential
in anti‐transgene responses[5, 7]. Along with the sequence of the gene therapy
transgene, the structure of the packaged DNA can also impact responses to the
expressed protein. Self‐complementary AAV vectors were developed to package
the genome as a double‐stranded structure, overcoming the rate‐limiting step of
second‐strand synthesis thought to result in less efficient protein expression from
the traditionally single‐stranded AAV genome [54, 55]. However, this has been
shown to elicit stronger immune responses against the transgene product[56, 57].
The promoter that drives gene expression of the transgene also factors into
humoral responses against the translated protein. Studies that have restricted
expression to the liver via a hepatocyte‐specific promoter have generally shown a
lack of anti‐transgene antibody responses[25, 26, 39, 40, 58].
The AAV capsid serotype can also influence whether responses are mounted against
the transgene as well[2]. This will vary in different species based on tissue tropism
variation. While serotype, biodistribution, and route of administration are all intimately related and responsible for anti‐transgene responses, the dose of vector influences humoral immunity to the protein. In general terms, the higher the dose, the
higher the expression, and greater the potential for eliciting anti‐transgene antibody
responses[59]. This was shown in mice to be correlated to the amount of expression
on a per‐cell basis and not simply overall expression in the target organ[60].
12.1.1.3 Patient Immune Status: Age, Prior Exposure, No Endogenous
Production, Immunosuppression, and Autoimmunity
After understanding how the gene therapy vector can influence transgene antibody formation, it’s important to consider the immune status and antigenic exposure of the recipient. An interesting phenomenon occurs when administering
gene therapy very early in life: in utero, perinatally, or neonatally. This early therapeutic administration of a vector leads to immunological tolerance to the transgene
product. This effect has been observed in mice[61–65], dogs[66], sheep[67], and
nonhuman primates[66, 68].
Another factor influencing antibody responses to the transgene is the immune
state of the patient (i.e. immunosuppressed, autoimmune condition). Mice strains
prone to autoimmunity developed anti‐transgene antibodies after gene transfer[69].
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