Biomedical Engineering Reference
In-Depth Information
for pharmaceutical purpose have been expressed as ELP
fusions in plants. First attempts were done with human and
murine interleukins. An increase in the accumulation of hIL-
10 and mIL-4 was reported but biological activity of the
plant-derived cytokines was not determined [104]. The first
ELPylated, biologically active recombinant protein from
transgenic plants was a fragment of naturally occurring
soluble glycoprotein 130 (sgp130), which is the natural
inhibitor of IL-6 trans-signaling [109,110] and has been
shown to be a potent candidate for therapy of rheumatoid
arthritis and colon cancer [111,112]. The mini-sgp130-ELP
was expressed in transgenic tobacco plants and purified via
ITC followed by size-exclusion chromatography. This leads
to a yield of 141
m
g purified protein per gram fresh leaf
material. The plant-derived recombinant mini-sgp130-ELP
inhibited IL-6 trans-signaling but not classical IL-6 signal-
ing [113].
Two antibodies, mAb 2F5 and mAb 2G12, with neutral-
izing capacity of the human immunodeficiency virus 1 (HIV-
1) were expressed in tobacco leaves and seeds as ELP-fusion
proteins with ELP on either the light or heavy chains, on
both or on neither. Floss and co-workers confirmed the
increased accumulation of independent antibody chains
due to C-terminal fusion with 100 repeats of the penta-
peptide Val-Pro-Gly-Xaa-Gly for both 2F5 and 2G12 immu-
noglobulin chains. In addition, they verify a higher
accumulation of the full-length IgGs with ELP either on
the light or heavy chains or on both [114,115]. Detailed
characterization of the purified antibodies revealed that ELP
fusion to the light chain, heavy chain or both chains had no
adverse effects on protein quality, and ELPylation did not
interfere with folding, assembly, trafficking in the secretory
pathway or post-translational modification, but enhance
stability while at the same time simplifying recovery
[114]. In seeds, however, N-glycans on the naked antibody
were extensively trimmed compared to those on the ELP-
fusion formats, and were localized to a different subcellular
compartment [115]. Surface plasmon resonance (SPR) spec-
troscopy showed that the kinetic binding parameters of the
antibody formats were identical to the immunoglobulins
lacking ELP produced in Chinese hamster ovary (CHO)
cells. Purification of antibody ELP fusions by ITC was
demonstrated and importantly, protein purification from
seeds by ITC did not affect the binding kinetics
[114,115]. The in vitro HIV-neutralization properties of
the tobacco leaf-derived 2G12 and ELP fusions were equiv-
alent to or better than those of the CHO counterpart [115].
Recently, the ELP-fusion technology has been used for
the ELPylation of an anti-human TNF Camelidae heavy
chain monoclonal antibody (anti-TNF-V
H
H) [116]. This
single-domain antibody (anti-TNF-V
H
H
ELP
) was produced
in transgenic tobacco plants (Figure. 14.4A) [117]. A cell-
based cytotoxicity assay revealed that the inhibitory
properties of the plant-derived anti-TNF-V
H
H
ELP
were
indistinguishable from the E. coli produced anti-TNF-
V
H
H without ELP. This demonstrates that neither the in
planta production, purification via ITC nor the fusion of 100
repeats of Val-Pro-Gly-Xaa-Gly (Xaa: Ala, Val or Gly) to the
antibody format compromised the biological activity. In
contrast, the serum half-life of the plant-produced V
H
H
was dramatically increased compared to the traditional E.
coli produced anti-TNF-V
H
H [117]. Furthermore, the
authors could show for the first time that an antibody
ELP-fusion protein was biologically active in vivo as dem-
onstrated in the LPS/D-gal septic shock model applied to
humanized TNF mice (Figure 14.4B).
The production of recombinant human erythropoietin
(rhEPO) for therapeutically applications is currently done
in mammalian cell culture, which is technically complex and
expensive. The fusion of ELP (28xVal-Pro-Gly-Val-Gly) to
EPO had a negligible effect on many of the reported
expression constructs, although the presence of an ELP-
fusion partner significantly increased the accumulation of
specific EPO constructs. The authors argued that his limited
effect of ELP fusion on EPO accumulation may be due to the
potential toxicity of EPO-ELP to the plant [118]. However,
the presence of an ELP-fusion partner appears to have no
effect on the receptor binding of prEPO, demonstrating that
ELP has no effect on the biological activity of recombinant
proteins [118].
Fusion of (Val-Pro-Gly-Xaa-Gly)
100
to a mycobacterial
antigen and the expression of the ELPylated protein in
J
FIGURE 14.4 (Continued)
human TNF. Mice were intravenously injected with 100
m
g plant
derived
Nt
TNF-V
H
H
ELP
or with 100
m
g
Ec
TNF-V
H
H from E. coli as control. Serum samples were
prepared and levels of the respective nanobodies were quantified by ELISA. The in vivo persistence of
the ELPylated nanobody from tobacco plants was
24-fold longer than that of non-ELPylated TNF-
V
H
H produced in bacteria. (C) The therapeutic application of the tobacco produced and ITC purified
TNF-V
H
H-ELP-fusion protein was tested in humanized TNF mice using a LPS/D-Gal sepsis model.
Mice treated with LPS/D-Gal died during the first 24 h (PBS), but mice deficient for human TNF
(TNF KO) survived. All mice treated with either bacterial produced TNF-V
H
H(
Ec
TNF-V
H
H) or
plant-expressed TNF-V
H
H
ELP
(
Nt
TNF-V
H
H
ELP
) survived, demonstrating the in vivo ability of the
ELPylated nanobody from transgenic plants to block the activity of human TNF. Source: (A), (B)
and (C) are reprinted from Reference [117]. Copyright (2011), with permission from Wiley.
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