Biomedical Engineering Reference
In-Depth Information
conditioning na ıve blood samples ex vivo through
prolonged and/or repeated exposure to experimental
antigen, immune responses can be accurately character-
ized. A kinetic analysis of responses following stimulation
with the protein antigens of interest can be useful for
detecting both low and high frequency antigen-specific
effector cells.
Both whole proteins and peptides can be used to measure
T-cell responses in vitro; peptides can be of variable length
(9-25 residues). Peptides presented in the context of class I
MHC are generally limited to 9 or 10 amino acids in length,
although some processing is believed to occur during the
T-cell assay and so 15-mers are also used for class I assays.
In contrast with class I epitopes, which are short and fit
tightly in the MHC molecule to which they bind, class II
(T helper) epitopes are displayed on an open-ended groove
in the MHC II. As such, longer peptides of 20 or more amino
acids in length can shift within this groove, thereby increas-
ing the number of discreet 9-mer epitopes able to interact
with the MHC molecule. The only limit on the size of the
peptide is its ability to remain in a linear conformation in the
groove. Similar to peptides, whole antigens too can be used to
measure T-cell responses in vitro. The recognition of these
antigens requires the presence of an APC that is capable of
processing and presenting peptides derived from the antigen.
Advantages of whole PBMC assays include the ability to set
up several assays and/or assay conditions with a limited
blood sample volume and the ease of assay performance,
features which lend themselves to high-throughput assay
development. However, since PBMC contain several cell
types capable of secreting IFN- g (NK cells, NKT cells,
CD4, or CD8 T cells), assays employing PBMC to measure
this cytokine may encounter obstacles. As not all IFN-
g -producing cells utilize the HLA class II-TCR activation
pathway, the variety of cell types can contribute to a
nonspecific signal. This bystander effect can be minimized
by use of CD8
3. select the optimal set of markers for the identification
of activated T cells and distinguishing between T reg
and T eff (T-effector cells) subsets;
4. identify parameters that define memory versus na
ıve
T-cell populations; and
5. standardize methods for cell harvest and preparation
from whole blood.
ıve and presensitized blood can differ in the content
of antigen-specific T eff and T reg . Hence, immediate han-
dling of whole blood with a stringent freezing process is
essential to ensure viability and functionality. On the basis
of the nature of response (primed vs stimulated and
recalled), stimulation methods and the amount of antigen
required for challenge can also differ. The na
Na
ıve cells will
require multiple challenges while antigen-specific recall
responses can be elicited even with a single challenge.
Similarly, stimulation can be performed with peptides or
whole proteins alone, peptides in context of tetramers or
APCpulsedwithwholeproteins.
5.4 APPROACHES FOR RISK ASSESSMENT
AND MINIMIZATION
5.4.1 Importance to Product Development
The extent to which one characterizes immunogenicity of a
new protein therapeutic is determined in part by the potential
risk of immune-mediated sequelae in the human population
targeted for its use. Factors to consider include, but are not
limited to, inherent potential immunogenicity of the protein,
its biological role, indication of use, route of administration,
duration of treatment, and health status of the patient (e.g.,
immune competence).
The immunogenicity of therapeutic protein products is
an important issue in product development as it can pose a
serious safety risk to the health of patients if not thor-
oughly addressed during the drug development process.
Indeed, serious and unexpected immune responses
observed in clinical trial patients can derail a promising
therapeutic otherwise on the path to successful licensing
and marketing. To preclude these events, strategies for
assessing immunological risk of a potential protein thera-
peutic should be incorporated into the earliest stages of the
drug development process. Even before the clinical trial
stage of development, immunogenicity should be consid-
ered for its impact on the ability to accurately measure
pharmacokinetics, pharmacodynamics, bioavailability, and
efficacy during preclinical testing. Implementation strate-
gies might include the use of current methods for immu-
nogenicity prediction, the development of in vitro
techniques to test the immunogenicity of predicted epito-
pes as well as validated and sensitive in vivo assays for
T-cell-depleted PBMCs. The optimized in
vitro concentration of protein required for challenge may
be nonphysiologic, potentially because of a limited antigen
presenting population and/or co-stimulation. Moreover,
there is a limitation in the number of APCs when either
whole PBMCs or enriched PBMCs are used in a long-term
assay. The response rate of T cells could be further
improvedbycocultureofoptimalAPC:T-cellratios.
While the practice of inducing T-cell responses in vitro
through MHC-bound peptides on APCs can generate valu-
able information, the complete nature of the immune
response cannot be fully replicated in these experiments.
Future considerations for developing effective in vitro
assays include the ability to
þ
1. characterize responders by setting statistically derived
criteria such as fold increase or stimulation index;
2. distinguish responders from nonresponders;
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