Biomedical Engineering Reference
In-Depth Information
Over two decades ago, B cells with immunoglobulin (Ig)
fusion proteins was used as tolerogens. Immunoglobulins,
especially IgG subclasses, had long been known to be
excellent tolerogenic carriers by virtue of long half-life,
favorable pharmacodymamics, and their ability to cross-link
inhibitory Fc receptors [66]. The fusion protein per se was
shown to be tolerogenic [67], a point that we shall discuss
further. Animal studies demonstrated that retroviral expres-
sion of this Ig fusion in stem cells and B cells induced
tolerance [68] and that T
reg
cells were induced by tolero-
genic B-cell presentation of epitopes, an effect that was
enhanced by the presence of the IgG heavy chain scaffold
[69]. Thus, Fc-fusion proteins are one option to consider for
accessing tolerogenic mechanisms and for potentially
decreasing the immunogenicity of a biologic drug.
5.5 CASE STUDY AND CLINICAL EXPERIENCE
5.5.1 Observed Versus Predicted Immunogenicity
of 21 Antibodies in Clinical Use
The accuracy of in silico methods for immunogenicity
prediction has been tested on more than 20 antibodies in
clinical use, in vitro or in a retrospective study. Protein
sequence information for the variable regions of heavy and
light chains was obtained from GenBank and the United
States Patent and Trademark Office. All sequences were
analyzed and scored for immunogenicity according to an
immunogenicity scale. Observed immunogenicity from
multiple studies was obtained, averaged, and regressed
against the predicted overall immunogenicity score of the
combined heavy and light chains (Figure 5.2). The results of
this regression analysis showed that the immunogenicity
score was a statistically significant predictor of observed
immunogenicity (correlation coefficient
5.4.4 Regulatory T-Cell Epitopes in IgG Framework
Regions: Tregitopes
0.67). Notable in
this analysis is the relatively small data set, limited by the
numbers of therapeutic antibodies in clinical trials, with only
20-30 participants on average per study. With recent evi-
dence suggesting that observed immunogenicity for some
therapeutics has been under-reported due to inadequate
assays [86], this retrospective study supports the use of in
silico immunogenicity assessment as part of a comprehen-
sive preclinical development strategy [9].
¼
“Tregitopes” are one potential explanation for the toleroge-
nicity of Fc-fusion proteins as described in 2008 [70]. Tre-
gitopes are highly conserved, tolerogenic peptides discovered
during epitope mapping and de-immunization programs with
human protein (especially IgG) therapeutics. It was observed
that all IgG subclasses contained epitopes in constant regions
that were predicted to have strong binding to MHC class II
and that were promiscuous and conserved in multiple mam-
malian species. It was predicted that these epitopes may be
recognized by T
reg
[70]. Further experimental data provided
evidence that Tregitopes not only activate FoxP3-expressing
T cells, they also suppress effector T-cell reactions in vitro
and in vivo [70]. In their natural state, Tregitopes are inte-
grated into the arms and stem of human antibodies and may
serve to balance the inflammatory triggers that are present in
the rearranged segments (variable loops) of the antibody
arms. Tregitopes are therefore present in intravenous immu-
noglobulins (IVIGs), a blood-derived product that is used
clinically to control autoimmune conditions [71]. Further
studies may provide some insight into the mechanisms by
which IVIG acts as an immunosuppressive agent in the
clinical setting [71-74]. Immunoglobulin therapies have
previously been shown to induce expansion of T
reg
in vitro
and in vivo [72,74]. Consistent with this observation, co-
incubation of donor PBMCwith Tregitopes derived from IgG
has been shown to lead to suppression of immune response to
bystander antigens. The corresponding murine epitopes sup-
press in vivo immune response in HLA DR4 transgenic mice
[70-72]. De Groot and collaborators have now shown that the
Tregitopes are immunosuppressive, dampen inflammation,
and induce tolerance in animal models of T1D, gene therapy,
and organ transplants [75-81]. These observations have been
corroborated by other researchers who have identified similar
peptides in IgG but have not recognized them as being T
reg
epitopes [82-85].
5.5.2 Correlation of Clinical Immunogenicity of Fusion
Proteins with
In Silico
Risk Estimates
In a prospective study, the sequences from four different
peptibody-based fusion proteins were assessed in silico for
binding to HLA-DR alleles using the EpiMatrix algorithm,
which assigns a Z-score to overlapping 9-mer sequences
based on their predicted affinity for eight class II MHC alleles
[52,54]. All four molecules were also administered in clinical
trials, where an independent immunogenicity assessment was
conducted. Table 5.2 summarizes the EpiMatrix-generated
scores associated with each protein and their respective rates
of antibody incidence (binding and neutralizing). An assess-
ment of Tregitope content in each molecule was also per-
formed and the Z-scores adjusted accordingly. FPX1, which
TABLE 5.2 Correlation of Clinical Immunogenicity with
In Silico
Risk Estimates
Protein Therapeutics
FPX 1
FPX 2
FPX 3
FPX 4
EpiMatrix score
21.97
1.76
0.76
1.63
Tregitope-adjusted
EpiMatrix score
21.97
1.62
1.76
111.25
Binding antibodies
(%)
37
7.8
5.6
4.5
Neutralizing
antibodies (%)
40
0.5
n.a.
0
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