FUSION PROTEINS: APPLICATIONS AND CHALLENGES - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 8

Acrp30 or Fc [57]. Other naturally occurring trimer inducing
molecules are the carboxyl-terminal noncollagenous
domains (NC1 domains) of collagens XV and XVIII coding
for endostatin. This was utilized to generate a multi-
functional anti-angiogenic compound consisting of a scFv
and a NC1 domain. Cleavage of the NC1 domain by tumor-
associated proteases released endostatin from the scFv
trimers, thus multiplying the anti-angiogenic effect [58].
Collagen trimers were also used to increase the binding
strength of scFv 20- to1000-fold though multimerization. It
was shown that this collagen-like scaffold (Gly-Pro-Pro) 10
could either be fused to the N- or C-terminus. Depending on
the addition of cysteins flanking the scaffold even a hex-
americ configuration could be obtained [59]. The trivalent
human plasma protein tetranectin builds the core of a novel
scaffold, the Atrimer TM . So far Atrimers have not been used
as basis for fusion proteins, but their multiple loops have
been engineered for diverse binding applications [60].

aggregates might cross-link B-cell receptors, or increase
protein internalization of antigen-presenting cells (APC),
thus initiating an immune response. However, endocytosis
can also happen if the target of a fusion protein is up taken.
Furthermore, nonhuman modification (e.g., glcosylation)
will also induce an immune reaction. Many of these
reactions can be predicted in silico or analyzed in vitro
with a wide range of assays [65]. Since both B- and T-cell
epitopes contribute to immunogenicity, it is very advisable
to identify and remove these epitopes. Due to the high
polymorphism of the major histocompatibility complex
(MHC) it can be difficult to remove all T-cell epitopes.
B-cell epitopes are not restricted to MHC molecules; there-
fore, it might be easier to eliminate B-cell epitopes [66].
More details on the immunogenicity of fusion proteins can
be seen in Chapter 5 of this topic.

1.4.5 Mutagenesis for Molecule Optimization

A lot of work on the optimization of fusion proteins by side-
directed mutagenesis is focused toward improving parame-
ters beyond immunogenicity. An important factor is the
resistance against proteases, on one hand during manufac-
ture and on the other hand while circulating through the
patient's organism. In both cases, it must be distinguished
between exo- and endopeptidases. For exopeptidases that
can cleave their target from either end, usually a modifica-
tion of the terminal aa abolishes the degradation. For
instance, the extension of a glucagon-like peptide-1
(GLP-1) fusion protein by only a single amino acid at the
N-terminal prevented cleavage by dipeptidyl peptidase-IV
(DPP-IV) [2]. The circulation half-life of an immunotoxin
could be doubled when replacing an endoprotease-sensitive
arginine residue by serine or lysine [67]. However, proteo-
lytic processing during the endosome/lysosome trafficking
is an important step in the mechanism of action of immu-
notoxins. Therefore, engineering of cleavage recognition
sequences must be done very carefully. Recently, it was
demonstrated that a deletion of a protease-sensitive region of
PE38 abolishes lysosomal degradation while maintaining
efficacy and increasing tolerance of high doses [68]. A
combination of increased half-life and improved activity
induced by single amino acid changes could also be
observed with an interleukin-2 (IL-2) immunocytokine.
Here, the linker peptide between the C-terminus of the
antibody heavy chain and the N-terminus of IL-2 was
modified, primarily with the aim to remove protease cleav-
age sites [69]. The nonselective toxicity of immunotoxins
can be avoided by generating in-frame fusions with ubiquitin
that triggers rapid degradation. However, the insertion of a
cancer protease-specific cleavage sequence can stabilize the
immunotoxin by removal of the ubiquitin moiety. For
instance, a saporin-based immunotoxin containing a pros-
tate-specific antigen (PSA) recognition sequence had a

1.4.4 Immunogenicity

Rational design of fusion proteins must also be used in order
to minimize immunogenicity of the new construct. Even if
using only human proteins as starting point for a modular
assembly, still the region between both molecules represent
a novel epitope that could elicit an immune response.
Therefore, it is recommended to use at least in silico analysis
to predict T- and B-cell epitopes. A lot of work on the
removal of T-cell epitopes or de-immunization of fusion
proteins has been conducted on immune toxins. In one
recent example, a truncated version of Pseudomonas exo-
toxin A (PE38) fused to an anti CD22 scFv was scanned for
the presence of B-cell epitopes with a huge set of antibodies.
The identified major epitopes were neutralized by specific
mutations[61].Furthermore, twolysosomalprotease-sensitive
areas were eliminated from PE38. The resulting mutant had a
drastically reduced immunogenicity and an even improved
cytotoxicity [62].
A very special case is the antibody-directed enzyme
prodrug therapy (ADEPT) that utilizes nonhuman enzymes
to metabolize inactive prodrugs into highly potent toxins.
One of the promising enzymes, a beta-lactamase had to be
mutagenized to remove CD4 + T-cell epitopes. Site-directed
mutagenesis replacing individual aa lowered the T-cell
response fivefold [63].
In general, Fc-fusion proteins should also display lower
immunogenicity. This effect can be based on the presence of
inhibitory Fc receptors on B lymphocytes, the Fc g RIIb. This
hypothesis was proven with the injection of DNA coding for
a Exendin-4 Fc-fusion protein that did not result in the
generation of neutralizing antibodies, which was the case
for Exendin-4 alone [64].
A number of factors determine the immunogenicity
potential of proteins in general. For example, protein