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the assays cannot predict which peptides are actually expressed on the surface
following natural processing of a protein antigen
in vivo
.
6.4.4 Confirming the Potential of Epitope Clusters Using T Cells
from Donors
The only way to accurately evaluate whether a peptide epitope is processed and
presented, or not (following modification of key amino acid residues) is to measure
T-cell responses to the peptides, using peripheral blood T cells from exposed sub-
jects or splenocytes from exposed mice (see next section). Alternatively, blood from
unexposed donors can be primed
in vitro
. T-cell lines can be developed by incubat-
ing blood samples drawn from unexposed volunteers with the candidate protein and
selected stimulatory molecules such as GMCSF, CD80, or CD86.
While there are many assays for evaluating T-cell response, one of the most accu-
rate means of measuring the response of the individual T-cell is to perform an ELIS-
pot assay. In most cases, the number of T cells responding to a given protein is ex-
tremely few; therefore, an
in vitro
T-cell restimulation assay is used to increase the
assay's sensitivity by expanding the number of T cells present that will respond to a
given peptide. Restimulation assays are usually performed using blood from exposed
subjects (i.e., persons who have been treated with the therapeutic protein and/or may
have participated in clinical trials.)
No matter which cells are used, the first task is to establish an overall level of
response by performing an ELISpot T-cell assay using the candidate protein as
the antigen. Peptides representing the predicted epitopes can also be assessed at
this time. Peptide epitopes that are observed to be associated with a strong T-cell
response can then be modified at critical amino acid residues. Next, in an itera-
tive approach, these modified peptides can be reevaluated for both binding and
immunogenicity.
The goal of these
in vitro
experiments is to demonstrate that the target protein
and peptides representing the clustered epitope regions do engender immune re-
sponses from exposed donors i
n vitro
; that the derivative clustered epitope region
peptides account for the majority of the immunogenicity engendered by the target
protein
in vitro
; and that the deimmunized proteins do not engender immune re-
sponses
in vitro
and
in vivo
.
6.4.5 Confirming Epitope Clusters
in Vivo
Another means of evaluating the impact of epitope modifications on
de novo
T-cell
response is to measure the immunogenicity of the original epitopes and the modified
epitopes in HLA-transgenic mice, either in the context of the whole protein (the
modifications are engineered in) or as peptides.
A number of transgenic mouse strains that express the most common HLA A,
HLA B, and HLA DR molecules have been developed. These mice process and
present epitopes in the context of human HLA. In order to compare the immuno-
genicity of wild-type and modified epitopes, immunizations are carried out with
preparations of the peptide epitopes in adjuvant. The standard regimen is to immunize
