Information Technology Reference
In-Depth Information
primarily responsible for the peptide's binding affinity with respect to multiple
alleles. Substitutions at these key amino acids may then be considered and re-
evaluated in a reiterative manner.
When modifying a protein sequence, it is important to consider the effect of the
substitutions on the function of the protein. This process may involve the analysis of
multiple substitutions and comparative
in silico
and
in vitro
analyses of the substitu-
tions' effects on protein function (see Structural Modeling, above).
6.5.3 Reducing Immunogenicity Following Clinical Trials
The final scenario involves the evaluation and deimmunization of protein therapeutics
that have already been tested in human subjects. Blood samples from exposed subjects
are obtained, restimulation is performed in order to activate and expand the relevant T
memory cells, and T-cell response in ELISpot assays is measured. T-cell response to
the native protein and to immunogenic peptides can then be contrasted with response to
peptides that have been modified to reduce immunogenicity. The protein can then be
modified to reduce immunogenicity while carefully preserving function.
6.6 Conclusions
The approach to deimmunization of functional therapeutics described in this article is
a multistep process involving (1) analysis of the therapeutic protein for presence of
MHC binding motifs; (2) synthesis and testing of the target peptides in the MHC
Class II binding and immunogenicity
in vitro
; (3) development of “deimmunized”
versions of these regions for which 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 and function
following natural translation.
In summary, epitope-mapping tools can be used to predict Class II restricted
T-cell epitopes contained in therapeutic protein sequences. These tools can also be
used to accurately discriminate between immunogenic and nonimmunogenic pep-
tides. The demand for preclinical methods for evaluating the immunogenic potential
of therapeutic proteins is expected to increase as the number of therapeutic proteins
and monoclonal antibodies entering the preclinical pipeline increases. While more
extensive validation is needed, this chapter provides a road map for deimmunization
that may be worth pursuing as it may accelerate the development of improved thera-
peutic proteins.
References
Braun, A., Kwee, L., Labow, M.A., and Alsenz, (1997) Protein aggregates seem to play a key
role among the parameters influencing the antigenicity of interferon alpha (IFN-alpha) in
normal and transgenic mice. J. Pharm. Res. 14:1472-1478.
Carroll, M.C. (2004) The complement system in B cell regulation. Mol. Immunol. 41:141-146.
