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Biopharma
Human Cell-Expressed Proteins
The production of human recombinant proteins from human cells provides
some unique advantages for clinical applications; these are imparted by
human-specific glycosylation patterns and include lower immunogenicity,
greater biological activity and greater stability both in vitro and in vivo
By Denese Marks and Glenn Pilkington at Apollo Life Sciences
Dr Denese Marks received a PhD in Biochemistry from the University of Technology, Sydney (Australia), and has
subsequently carried out postdoctoral research at the University of Vienna (Austria), the University of Melbourne (Australia)
and the Institute of Child Health (London, UK), before joining Apollo Cytokine Research as Head of Cell Biology in 2005.
Glenn Pilkington has spent fourteen years in the biotechnology industry at Diadexus, LLC (South San Francisco, USA),
Intracel Corporation (Frederick, USA), and two years at Apollo Cytokine Research (Sydney, Australia) in the development
of fully human therapeutic proteins. He obtained his MSc from the University of Melbourne (Australia) and is a co-inventor
on 22 patent applications, including 12 relating to Apollo’s hcxTM technology.
The use of human proteins as biopharmaceuticals in
the treatment of disease – including stem cell
transplantation and immunotherapy for cancer – has
increased dramatically over the past three decades.
During the same period, regulatory bodies such as the
FDA and EMEA have indicated a preference for nonanimal derived supplements, recommending that the
industry use well-characterised cell lines for the
production of human protein biopharmaceuticals. The
production of proteins in human cells also provides
some unique advantages for clinical applications,
including greater activity and stability in vitro and
in vivo, and lower immunogenicity imparted by the
human-specific glycosylation. These benefits are often
not available from the non-human expression systems
currently used to produce therapeutic proteins, such as
bacteria, yeast, insect, mouse or hamster cells.
The treatment of several diseases, particularly cancer,
now routinely includes cell-based therapies whereby
human cells are harvested, cultured ex vivo and re-infused
into patients. Ex vivo expansion of human cells
requires chemically defined media containing optimal
combinations of recombinant human cytokines and
growth factors. Most growth factors and cytokines are
highly glycosylated, with glycosylation representing up to
75% of their molecular weight. Glycosylation is a highly
species-specific post-translational modification that
affects the biological properties of proteins. Recombinant
proteins produced from E. coli are not glycosylated, and
growth factors and cytokines produced from other
expression systems – such as insect, yeast or mammalian
cells – exhibit different glycosylation patterns compared
with native human proteins. The importance of humanspecific glycosylation of cytokines and growth factors is
now being recognised, as differences in glycosylation can
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cause the alteration of a protein’s biological properties,
resulting in higher immunogenicity, lower biological
activity and altered pharmacodynamics in vivo.
With the trend to elimination of non-human
components from biopharmaceutical production, human
cell-expressed proteins offer unique advantages. At Apollo
Cytokine Research, we produce human cell-expressed
(hcxTM) proteins with human-specific post-translational
modifications that are as close as possible to the native
protein. Our aim is to identify structural and biological
differences between these proteins and equivalent
proteins produced from non-human expression systems,
and we routinely evaluate differences in cell proliferation,
viability, morphology, cell surface marker expression, and
activation of specific intracellular signalling pathways.
This article provides an overview of how hcxTM proteins
can provide benefits for human cell culture, focusing in
particular on EPO (erythropoietin), G-CSF granulocytecolony stimulating factor, SCF (stem cell factor) and IL-4
(interleukin-4) for culturing haematopoietic stem cells
(HSCs) and dendritic cells (DCs).
CULTURE OF HUMAN
HAEMATOPOIETIC STEM CELLS
All mature blood and immune cells develop from a
population of adult stem cells called haematopoietic stem
cells (HSCs). They are generally characterised by
expression of the CD34 cell surface antigen; these CD34
positive (CD34+) cells have the capacity for self-renewal
and differentiation into a variety of specialised cell types.
The regenerative capacity of these cells is finely controlled
by the cellular micro-environment, and through
molecular signals from cytokines and growth factors.
Ex vivo expanded HSCs are used therapeutically for
stem cell transplantation following myelo-ablative
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chemotherapy or radiotherapy, as well as for the
treatment of blood disorders such as anaemia and
thalassaemia. It is necessary to expand HSCs ex vivo to
generate sufficient cells to be of therapeutic benefit.
Some important benefits of culturing CD34+ HSCs in TM
EPOhcx, G-CSFhcx or SCFhcx are outlined below.
G-CSF and SCF
Granulocyte colony stimulating factor (G-CSF)
stimulates differentiation and maturation of
granulocytic cells, and activates neutrophils in the
circulation. G-CSF has one O-linked glycosylation site,
which is known to increase the stability of G-CSF (1),
resulting in higher biological activity and greater ability
to stimulate CD34+ stem cell proliferation (2). G-CSF is
used in combination with other cytokines and growth
factors for ex vivo expansion of CD34+ stem cells to treat
post-chemotherapy neutropenia in cancer patients (3,4).
G-CSF is also used to mobilise stem cells from cancer
patients or normal donors for stem cell transplantation
and reconstitution (5). Mobilisation with glycosylated
G-CSF results in neutrophils with normal morphology
and function, whereas mobilisation with nonglycosylated G-CSF has resulted in neutrophils with
defective chemotaxis and increased actin polymerisation
(6,7), emphasising the importance of glycosylation on
therapeutic proteins.
Stem cell factor (SCF) is another glycoprotein that
augments proliferation of erythroid, myeloid and
lymphoid haematopoietic progenitors ex vivo. SCF acts
synergistically with other cytokines and is known to
protect progenitor cells from apoptosis (8) and to prime
stem cells for differentiation. SCF is heavily glycosylated,
with N- and O-linked glycans on multiple residues. SCF
is routinely used clinically in combination with other
cytokines to expand stem cells ex vivo, and in
combination with G-CSF to mobilise CD34+ cells into
the circulation of cancer patients (4).
To examine the benefits of glycosylation, the ability of
glycosylated G-CSFhcx was tested in combination with
SCFhcx for expansion of CD34+ human HSCs in vitro and
compared with commercially available non-glycosylated
recombinant growth factors produced in E. coli. The
glycosylated G-CSFhcx and SCFhcx induced enhanced
proliferation, producing more and larger colonies in
colony-forming assays, compared with G-CSF and SCF
from E. coli (see Figure 1). When used in combination in
liquid culture, the glycosylated G-CSFhcx and SCFhcx
promoted significantly more cellular proliferation than
E. coli-expressed G-CSF and SCF. Preliminary evidence
suggests that G-CSFhcx was a more potent activator of
STAT3, which may be the underlying cell signalling
mechanism responsible for the increased cell
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Number of colonies
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40
30
20
10
0
E coli
Small
hcx
Large
Total
Colony size
proliferation. G-CSFhcx and SCFhcx, when used together,
form a more potent cocktail for HSC expansion in vitro.
The increased biological activity of these growth
factors could translate into more effective clinical
therapies. For example, the enhanced ex vivo
amplification of CD34+ cells using hcxTM growth factors
could reduce the period of neutropenia following stem
cell transplantation, subsequently reducing the incidence
of infection and increasing the survival rates of patients.
Erythropoietin
Erythropoietin (EPO) is one of the most studied
molecules in terms of the requirement for glycosylation
for correct protein function. It is used predominantly to
treat anaemia resulting from kidney disease or
chemotherapy/radiotherapy as it induces erythroid
differentiation of CD34+ cells, leading to the generation
of mature red blood cells in vivo. EPO is heavily
glycosylated with one O-linked and 3 N-linked
glycosylation sites, and an average oligosaccharide content
of 40% by weight. Glycosylation of EPO is important for
cellular secretion, and protein conformation, solubility,
stability and biological activity (9). In particular, terminal
sialylation on the N-glycans of EPO is necessary for
increased circulatory half-life, as asialo-erythropoietin is
rapidly cleared from the plasma (10). Until recently, most
commercially available recombinant human EPO was
purified from Chinese Hamster Ovary (CHO) or Baby
Hamster Kidney (BHK) cells. However, some humanspecific oligosaccharide structures are not synthesised by
CHO or BHK cells, as they lack several glycosyl
transferases including sialyl-α 2-6 transferase and α 1-3/4
fucosyl transferase (11). The absence of these enzymes in
non-human cell lines may be detrimental to the activity
of EPO. These potential differences thus make structural
analysis of glycans and their subsequent correlation with
biological activity of great importance.
Our research indicated that EPOhcx, in combination
with stem cell factor (SCFhcx), induced over two-fold more
erythroid differentiation of CD34+ HSCs, compared with
Figure 1: Clonogenic
activity of CD34+ expanded
cells by G-CSF and SCF.
Expanded CD34+ HSCs
were inoculated into
semisolid cultures of
methylcellulose containing
G-CSFhcx and SCFhcx or
E. coli-expressed G-CSF
and SCF. Cultures were
incubated at 37ºC and
the number and sizes of
colonies scored 14 days
later using an inverted
microscope
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90
a) Glycophorin A expression
250
EPOhcx
CHO EPO
200
Events
Mean fluorescence units
300
150
100
50
0
0
100
101
102
103
104
Fluorescence intensity
1,800
EPOhcx
1,600
CHO EPO
1,400
1,200
1,000
800
Events
Mean fluorescence units
2,000
90
b) CD71 expression
600
400
for therapeutic applications. Hence DCs must be
expanded ex vivo from precursor cells, such as peripheral
blood monocytes, in order to generate vaccines.
Production of ‘clinical grade’ DCs for large-scale cancer
vaccine development follows current good manufacturing
practice (cGMP) guidelines. These protocols use serumfree medium to eliminate non-human proteins, but
continue to use cytokines and growth factors such as
GM-CSF, IL-4 and TNF-α produced from non-human
cells. The use of hcxTM proteins in these processes would
eliminate all animal-derived products and provide other
benefits, such as greater stability in cell culture.
Glycosylated IL-4hcx is more stable in cell culture medium
than the non-glycosylated IL-4 currently used. Increased
stability in culture has the advantage of reducing cost and
the number of medium changes, thereby reducing the risk
of contamination.
0
0
200
100
101
102
103
104
Fluorescence intensity
Figure 2: EPO-induced
erythroid differentiation.
CD34+ HSCs were
cultured with EPOhcx
and SCFhcx or CHO cellexpressed EPO and SCF
for 7 days, and the
expression of erythroid
markers (a) glycophorin
A and (b) CD71 was
determined
CHO-expressed EPO. This was demonstrated by higher
expression of both glycophorin A (see Figure 2a) and
CD71 (see Figure 2b), which are markers of more mature
erythroid differentiation. We also observed that CD34+
HSCs cultured in EPOhcx produced morphologically
more differentiated cells than CD34+ HSCs grown in
CHO-expressed EPO, which remained blast-like.
Similarly, haemoglobin expression was higher in HSCs
grown in EPOhcx, compared with CHO-expressed EPO.
Cellular proliferation and viability was, however,
equivalent with both sources of EPO – indicating that the
increased cellular differentiation using EPOhcx did not
compromise cell number or viability in these cultures.
Thus, our in vitro results using EPOhcx demonstrated
more rapid induction of erythroid differentiation of
HSCs compared with non-human cell-expressed EPO,
again suggesting potential clinical benefits such as the
production of more functionally mature erythroid cells
in EPOhcx-treated patients.
DENDRITIC CELL CULTURE
Dendritic cells (DCs) are potent antigen-presenting cells
of the immune system and are actively involved in
immune responses, including graft rejection and T-cell
dependent responses, as well as in autoimmune diseases
and the pathogenesis of HIV infection (12). DCs are used
clinically to generate vaccines against several cancers,
whereby autologous DCs are generated ex vivo and then
pulsed with tumour-related peptides, proteins or tumour
cell lysates to boost antigen-specific T-cell mediated
tumour immunity. DCs represent a small percentage of
leukocytes and cannot be isolated in sufficient numbers
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CONCLUSION
Human cell-expressed proteins offer the benefits of fully
human proteins with human-specific post-translational
modifications, reducing the potential for immunogenicity.
Other benefits include elimination of the risk of
transmissible spongiform encephalopathies (TSE) and
other infectious diseases, a longer half-life in vitro and in
vivo, and often greater biological activity. The growing
requirement for chemically defined, animal protein-free
media in the production of biopharmaceuticals suggests
that human cell-expressed proteins will provide the next
generation of growth factors and cytokines for ex vivo and
in vivo therapeutic applications.
The authors can be contacted at
[email protected]
References
1.
Carter et al, Biologicals. 32, pp37-47, 2004
2.
Qerol et al, Hematologica. 84, pp493-498, 1999
3.
Reiffers et al, Lancet. 354, pp1,092-1,093, 1999
4.
McNeice et al, Blood. 96, pp3,001-3,007, 2000
5.
Anderlini and Champlin, Drugs. 62 Suppl. 1,
pp79-88, 2002
6.
Azzara et al, Am. J. Hematol. 66, pp306-307, 2001
7.
Carulli et al, Am. J. Hematol. 81, pp318-323, 2006
8.
Zeuner et al, Blood. 102, pp87-93, 2003
9.
Egrie and Brown, 2001
10. Fukuda et al, Blood. 73, pp84-89, 1989
11. Skibeli et al, Blood. 98, pp3,626-3,634, 2001
12. Steinman, Annu. Rev. Immunology. 9,
pp274-296, 1991
13. Casadevall, Nephrol. Dial. Transplant. 16 (suppl. 3),
pp3-13, 2005
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