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Table 3b.
SakSTAR epitopes (alanine-modified sequence)
Alanine Substituted version
DRB1*0701 score: 82
nd
percentile
73
AAEFAVVAL
81
DRB1*0301 score: 88
th
percentile
76
FAVVALAPS
84
DRB1*0301 score: 90
th
percentile
79
VALAPSAKI
87
then be targeted for modification. Modifications of these regions may potentially
reduce the immunogenicity of therapeutic proteins, as described for the humanized
monoclonal antibody and the staphylokinase protein examples. The next section
describes the process of deimmunization by epitope mapping and modification of
protein sequences.
6.4 A Step-by-Step Approach to Deimmunization
The approach to deimmunization of functional therapeutics described in the next
paragraphs is a multistep process involving (1) analysis of the therapeutic protein
for the presence of MHC binding motifs; (2) synthesis and testing of the target
peptides for MHC Class II binding and immunogenicity
in vitro;
(3) development
of “de-immunized” versions of the regions where the MHC binding motifs have
been modified; (4) synthesis and testing of the deimmunized counterparts
in vitro
;
and (5) testing of the recombinant, deimmunized protein
in vivo
for immunogenicity.
Evaluation of the effect of protein modification on protein function is characterized
by structural modeling (
in silico)
, at step (3) following the resynthesis of the protein.
6.4.1 Initial Screen for Class II Epitopes and Epitope Clusters
The first step in the deimmunization analysis involves screening the target protein
for Class II epitopes. We have used EpiMatrix matrices corresponding to eight com-
mon HLA alleles (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701,
DRB1*0801, DRB1*1101, DRB1*1301, DRB1*1501), which are well represented
in most human populations. EpiMatrix identifies clusters of putative epitopes re-
stricted by at least one of these eight common alleles in any given protein. For ex-
ample, EpiMatrix (Class II, eight alleles) epitope mapping of human beta-interferon
demonstrates seven potential epitope cluster (Fig. 4). Collectively these clusters
contain almost all of the predicted epitopes. This type of clustering of MHC binding
motifs is typical of promiscuous epitopes (Meister, Roberts, Berzofsky, and
De Groot 1995).
