ELP-FUSION TECHNOLOGY FOR BIOPHARMACEUTICALS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 214

transgenic tobacco plants was initiated to obtain an efficient
vaccine against Mycobacterium tuberculosis, which is the
main tuberculosis (TB) causing microorganism in humans.
Two mycobacterial antigens, Ag85B and ESAT-6, which are
common in M. tuberculosis and M. bovis and might there-
fore be effective for both human and bovine TB, have been
connected to the fusion antigen TBAg [119]. Effective
vaccination of mice and guinea pigs was achieved with
this fusion protein [120-122]. ELPylated TBAg was pro-
duced in transgenic tobacco plants and immunogenicity of
the recombinant protein was assessed in mice, as a reference
laboratory model, and piglets, as a preclinical model of
human vaccination based on similarities between swine and
human immune responses [19]. Both mice and piglets,
injected with the ITC-purified ELPylated TBAg or crude
leaf extract, produced antibodies against the plant-derived
antigen and showed no side effects. Furthermore, a T-cell
immune response able to recognize the native antigens,
Ag85B and ESAT-6 contained in CFP (culture filtrate pro-
teins) and PPD (purified protein derivatives) was detected in
mice [19].
Avian flu hemagglutinin H5 (HA1) and neuraminidase
(NA) N1 fused to (Val-Pro-Gly-Xaa-Gly) 100 have been
produced in the ER of transgenic tobacco plants and purified
using the salt- and temperature-dependent phase transition
of ELP. Here, an effective membrane-based ITC method
based on Ge et al. [32] was developed to overcome
the proteolytic cleavage and/or partial denaturation of
the ELPylated protein. This procedure resulted in a
large improvement of the protein purity of recombinant
ELPylated NA. Enzymatic activity of plant-derived
NA-ELP, assessed on the basis of its ability to cleave
2 0 -(4-methylumbelliferyl)- a - D -N-acetylneuraminic acid,
was enhanced compared to the classical centrifugation-
based ITC purification method. In addition, the efficiency
of the purification was significantly improved as shown for
hemagglutinin, neuraminidase, and anti-TNF-V H H ELP [33].
Recently, a detailed study of the ELP-fusion technology
demonstrated the effect of ELP size, orientation, and amino
acid sequence composition on the accumulation of different
recombinant proteins (GFP, IL-10, EPO, scFv) as well as the
effects of ELP size on the efficiency of ITC purification from
plant extracts and the biological activity of EPO were inves-
tigated [123]. The C-terminal ELP 28 (28xVal-Pro-Gly-Val-
Gly) significantly increased the concentration of IL-10 and
EPO in the transient system. A random rearrangement of ELP
amino acids (rELP tag) showed a significant reduction of
protein yield suggesting that the proper protein sequence and
structure of ELP is important for its beneficial effects on the
accumulation of recombinant proteins [123]. The concentra-
tion of all four recombinant proteins tested aswell as nonfused
ELP was reduced when utilizing an ELP tag comprising 80
repeats of Val-Pro-Gly-Val-Gly. In contrast, a 4.25-fold
enhancement of the recombinant proteins in leaves was

demonstrated when comparing the ELP 80 to the ELP 20 fusion
protein levels. Furthermore, the size of ELP has a significant
impact on the purity of the target proteins. The highest
concentration of purified recombinant proteins was obtained
when using ELP 20 ,ELP 30 , or ELP 40 as fusion partner but
larger ELP tags (ELP 80 ,ELP 120 , ELP 240 ) resulted in the
highest recovery rates indicating that ELP size is more
important than ELP concentration for effective ITC purifica-
tion [123]. For the investigation of effects of ELP tags on the
biological activity of recombinant proteins different EPO
expression constructs were evaluated. The resulting recom-
binant EPO-ELP variants were tested for their ability to bind
the human EPO receptor (EPOR). The presence of ELP tags
appeared to have no adverse effect on biological activity of
recombinant proteins as demonstrated by the receptor binding
capacity of recombinant EPO derived from plants [123].

14.5 CHALLENGES AND FUTURE
PERSPECTIVES

The development of ELPylation in the 90s of the last century
has opened a novel way of laboratory-scale and presumably
industrial-scale protein expression and purification. The
attractiveness of ELPylation is based on the thermally stimu-
lated inverse phase transition, which can be used for non-
chromatographic purification of recombinant proteins.
However, the combination of ELPylation and intein-technol-
ogy and the ELP-mediated affinity capture (EMAC) allows
the production of ELP-free target proteins. The good bio-
compatibility of ELPs and their controllable aggregation in
vitro and in vivo will enable novel medical applications, such
as hyperthermia-induced locally extravascular accumulation,
ELP-based drug depot formation or materials for tissue repair
or engineering. In the near future, we will see systematic
studies of relations between ELP size and steric influences on
antigenicity (vaccine), functionality (recognition by antibod-
ies), and enzymatic activity. Moreover, more sophisticated
mouse studies with ELPylated proteins are needed to evaluate
the potential of ELPylation for therapeutic application.
Plant-made industrial and pharmaceutical proteins are still
in their early days, with no protein products being in the clinic.
However, current clinical studies are on the way to demon-
strate the general feasibility of application of plant made
pharmaceuticals with current state-of-the-art production sys-
tems. The adaptation of ELPylation to plant biotechnology
has revealed a general increase in accumulation rate, with
good ITC-based recovery and high bioactivity of the final
target protein. Future work is needed to fully explore the
potential of the ELPylation technology on plant bio-
technology for the in planta production of industrial and
pharmaceutical proteins. Scale-up experiments for purifica-
tion and processing have to be performed andmass production
systems have to be developed.