Biomedical Engineering Reference
In-Depth Information
develop specific and sensitive in vitro methods to analyze
the potential immunogenicity of new therapeutic proteins.
The challenge in developing these types of assays is to
provide an appropriate milieu in culture that accurately
represents in vivo conditions of immune stimulation
[52-54]. In this respect, bulk cultures of peripheral blood
lymphocytes, either with or without co-stimulatory signals
(anti-CD28 antibody, IL-2, IL-7, etc.), have been utilized to
assess immunogenicity of therapeutic proteins. A number of
biological outcomes for T-cell activation can be measured in
these in vitro assays, including cytokine secretion (IFN-
g
,
IL-2, IL-4, etc.), regulation of cell surface markers of
activation, signal transduction events, and proliferation. If
blood from individuals participating in clinical trials is
available, peptides predicted as T-cell epitopes and validated
in MHC-binding assays can be tested for their reactivity with
T cells in vitro. A positive immune response, measured by
peptide-specific cytokine production, proliferation, and so
on, is a strong indicator that the parent protein is immuno-
genic. Supported by this type of evidence, these peptides can
be considered validated T-cell epitopes with the potential to
contribute to an antidrug Ab response.
Enzyme-linked immunosorbent assay (ELISA) and ELI-
Spot are two related methods, both of which measure
cytokines secreted by T cells (i.e., IFN-
g
, IL-2, and IL-
4). ELISAs are a means to measure cytokine levels in culture
supernatants generated under conditions of T-cell stimula-
tion. Cytokine levels measured in an ELISA can provide
information about the magnitude of the response (how much
cytokine is secreted into the supernatant) as well as the
quality of the response (which cytokines are or are not
secreted into the supernatant). ELISpot assays provide
information regarding the numbers of cytokine-producing
cells within a cell population stimulated ex vivo. ELISpots
are sensitive because they are able to identify cells that
produce relatively low quantities of the cytokine of interest.
The proliferation of T cells in response to stimulation by a
peptide-MHC complex can bemeasured by (1) the dilution of
a fluorescent dye, CFSE, that will decrease in fluorescence
intensity by half with each round of cell division, a property
measured by flow cytometry and (2) the incorporation of a
radioactive nucleotide, tritiated thymidine, into the DNA of
dividing but not resting cells. Some of the most precise
methodologies available for measuring and defining T-cell
responses are measured by flow cytometry. For example, T
cells bearing TCR specific for a particular epitope can be
directly enumerated using fluorescent-labeled tetrameric
complexes of MHC class II molecules loaded with the peptide
of interest. Antigen-specific T cells detected in this manner
can be further examined for phenotypic and activation mark-
ers as well as for intracellular cytokine expression.
HLA-binding assays can be used to measure the affinity
of peptides derived from immunoinformatics analyses for
binding to soluble MHC class II molecules in vitro.
Evaluation of MHC binding can be performed as a com-
petition assay, where a “test” peptide added over a range of
concentrations is measured for its ability to inhibit the
binding of a labeled “reference” peptide with a well-
characterized affinity for a particular MHC allele [55].
This standardized method is cell-free and can be adapted
for high throughput [56]. The advantage in using in vitro
HLA binding assays is that detailed information about a
peptide's HLA affinity may be obtained prior to perform-
ing experiments on live cells. Data acquired from these
assays may be indicative of immune responses in later
experiments, such as ELISpots or HLA transgenic mouse
studies, providing a reliable framework to interpret results.
It is important to note, however, that the conditions under
which candidate epitopes are tested in vitro are different
from the cellular environment in ways that make a 1:1
comparison difficult. Artificially synthesized peptides may
contain impurities or sequence errors. Solvents used in the
experiment may interact negatively with certain amino
acids, causing oxidation or forming unwanted disulfide
bonds. Quality-controlled peptide manufacturing and stor-
age along with proper reagent selection can minimize the
impact of these problems.
Because competition-binding assays yield indirect mea-
sures of affinity on the basis of how effectively a peptide
inhibits the binding of a labeled reference peptide, only
relative measurements proportionate to specific assay
parameters can be made. A set of peptides can be “ranked”
for binding to a given HLA allele provided they have been
tested under the same conditions, but comparisons across
alleles are not as straightforward. By fine-tuning the protocol
for each allele through repeated experiments, a consensus
among data points can be approached.
T-cell restimulation assays can be utilized to identify and
measure a recall or memory response in peripheral blood
mononuclear cells (PBMCs) derived from subjects who have
been exposed to a given biological product within the
context of a clinical trial. As the precursor frequency of
the antigen-specific cells in a recall response is relatively
high, whole PBMC assays are generally both rapid and
sensitive with no pretreatment or enrichment required. Epit-
ope mapping of the recall response can be performed using
specific peptides from the whole protein at the time of
challenge. T-cell responses after restimulation ex vivo can
include ELISA, ELISpot, proliferation, and flow cytometric
analysis of activation markers of intracellular cytokine
expression.
Na
ıve PBMC assays have been used to predict immu-
nogenicity of therapeutic proteins [54,57]. Compared to a
recall response, the precursor frequency of antigen-specific
cells in a na
ıve population is quite low. Thus, multiple
rounds of antigen stimulation, sometimes over several
weeks, are required to expand sufficient numbers of
antigen-specific T cells for reliable measurement. By
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