Biomedical Engineering Reference
In-Depth Information
proteins [19], the implications of extrinsic factors that are
particular to the therapeutic protein or its administration
should also be considered in this context. These include
protein modifications, aggregation, and innate immune sys-
tem activation. Modifications in recombinant proteins that
occur in the in vitro expression and purification processes,
including post-translational modifications and denaturation,
could render them sufficiently different from their natural
“self” analogs to be immunogenic. Aggregates of a thera-
peutic protein can be recognized by the immune system in a
number of ways. One way that aggregates may trigger
immune responses is through the recurrence of B-cell epit-
opes on the surfaces of the aggregate that could simulate the
presence of highly repetitive arrays, such as those seen on
the surfaces of bacteria and viruses, facilitating cross-linking
of the B-cell receptor to activate proliferation. Alternatively,
aggregates are, similar to the virus-like particles (VLPs) that
so effectively induce immune responses in the vaccine
context, more likely to be taken up by APCs, where degra-
dation and presentation of T-cell epitopes are then able to
drive a T
d
response [20,21]. Finally, engagement of Toll-like
receptor (TLR) family members by products of microbial
degradation has been shown to stimulate B-cell responses
and antibody production in the absence of T-cell help [22].
Thus, even low-level contaminants or impurities such as
endotoxins, microbial agents, or host cell proteins present
along with the therapeutic proteins could contribute to their
immunogenicity.
from the native protein components, resulting in greater
immunogenicity. De-immunization of the new immuno-
genic epitopes generated by fusion at the junctional region
can be used to reduce their interaction with a T-cell receptor
(TCR) [26]. Owing to these potential concerns of immuno-
genicity due to the fusion of two or more protein compo-
nents, a robust immunogenicity evaluation strategy is often
required as a part of risk assessment to support the drug
development at preclinical and clinical stages [27]. Swanson
et al. have reviewed the various assay platforms and their
suitability for immunogenicity assessment [28]. From this
comparative analysis, surface plasmon resonance-based
Biacore assays emerge as ideal for testing the immunoge-
nicity of fusion proteins. With the Biacore approach, each
component of the fusion protein, such as the peptide portion,
Fc portion, or any linker regions, can be independently
assessed for antibody reactivity simultaneously on parallel
immobilized surfaces [28].
5.3 TOOLS FOR IMMUNOGENICITY SCREENING
5.3.1
In Silico
Analysis Tools
As discussed earlier, immunogenicity of a therapeutic pro-
tein depends largely on its ability to trigger either a cellular
or humoral immune response. In the case of cellular
responses, T cells recognize small linear peptides derived
from protein antigens upon their uptake and proteolytic
processing by an APC. These peptides are then presented
to T cells as a complex of epitope in the binding groove of
the MHC on the APC surface. One of the critical determi-
nants of T-cell epitope immunogenicity is the affinity of that
epitope for the groove of the MHC; the higher the affinity the
greater the likelihood that an epitope will be recognized by a
T cell [29]. In contrast to T-cell epitope recognition, which is
restricted by presentation on MHC molecules, B cells and
the antibodies that they produce generally recognize con-
formational epitopes directly on the surface of native pro-
teins. Compared to MHC-T-cell epitope interactions, those
between the B-cell receptor and B-cell epitope are much
more difficult to describe mathematically for use in predic-
tion algorithms [30,31]. Thus, the groundbreaking work
toward establishing reliable methods for immunogenicity
prediction appears somewhat biased toward the definition of
T-cell epitopes; accessible, high throughput, and accurate
prediction of B-cell epitopes may be beyond the reach of
current computational algorithms.
There are a number of factors inherent in the binding
between a T-cell epitope and an MHC-binding groove that
make this relationship amenable to computer-based predic-
tion algorithms. Because several common HLA-DR types
share largely overlapping peptide-binding repertoires, anal-
ysis focused on as few as eight MHC molecules can “cover”
5.2.1 Relevance to Fusion Proteins
Full-length Fc-fusion proteins are normally generated by
fusing the N-terminus of the protein of interest containing
the biologically active site to murine or human Fc (hinge-
CH2-CH3). The fusion of proteins with Fc allows for large-
scale manufacturing (milligram to gram quantities), easy
purification, and an increased in vivo half-life, which proves
useful in therapeutic applications [23,24]. Taking what we
know about general protein immunogenicity, we can envi-
sion a number of Fc-fusion protein attributes that should be
given special consideration in preclinical development.
For example, the joining of two nonimmunogenic or self-
proteins together can form junctions never before “seen”
by the immune system that could comprise new
T- and B-cell epitopes. Likewise, Fc-fusion proteins may
be taken up and processed differently than either of their
native counterparts, contributing to higher immunogenicity
than predicted for either component. Immunogenicity is
enhanced by binding to Fc receptors on the surface of
APCs thereby targeting the antigen to these cells [25]. If
this new combination increases uptake or differentially
stimulates APCs, then the host immune system may respond
differently. Also, these novel protein sequences could be
processed and presented in a way that is sufficiently distinct
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