Progress in transgenic production of human plasma proteins
in mammary bioreactors
W. N. Drohan*l’ and D. B. Clark?
*Plasma Derivatives Department, Jerome H. Holland Laboratory for the Biomedical Sciences, American Red
Cross, Rockville, MD 20855 and ?Plasma Services, American Red Cross, Shawnee, CO 80475
~
ABSTRACT: Safe and effective protein concentrates
proteins, antithrombin I11 and crl-antitrypsin, are currently in clinical trials. However, a number of issues
remain t o be solved, including the low success rate in
producing transgenic animals and differences in posttranslational processing of the expressed proteins compared to their native human counterparts. Overall,
though, the future of transgenic technology for the production of recombinant human plasma proteins appears bright.
produced from transgenic animals should be available
in the future in relatively unlimited quantities and at
reasonable costs. The technology for the production of
recombinant human proteins in the milk of transgenic
animals has advanced markedly in the past few years
to the point where a number of therapeutically useful
proteins have been produced a t levels on the order of
tens of grams of protein per liter of milk. Two such
Key Words: Transgenic, Recombinant, Plasma, Proteins, Bioreactors, Lactation
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02000 American Society of Animal Science. All rights reserved.
Introduction
J. h i m . Sci. 2000. 78(Suppl. 3):l-7
mechanisms for tissue-specific expression of normal
genes (Brinster et al., 1983; Swift et al., 19841, and later
work developed methods for achieving tissue-specific
expression of introduced genes. Tissue-specific expression of transgenes has been achieved in blood cells,
kidneys, salivary glands, and mammary glands, among
many others (Lubon, 1998).
Further development of this technology was made
possible by fundamental discoveries in a number of
fields over several decades. Increased understanding of
the hormonal control of reproduction in mammals; the
refinement of techniques to harvest, manipulate, and
re-implant eggs and early embryos; and the use of recombinant DNA technology to isolate, characterize, and
engineer defined configurations of DNA fragments all
provided a foundation for the development of transgenic
animals. The initial commercial applications envisioned the use of transgenic technology for genetic improvement of livestock. This might be accomplished,
for instance, through manipulation of the endocrine
or immune systems, and modification of biochemical
pathways or body fluid composition. Several technical
problems such as the manipulation of pronuclei for microinjection without affecting subsequent development
and viability of embryos were solved (Hammer et al.,
1985; Wall et al., 1985), and soon transgenic rabbits,
sheep, pigs (Brem et al., 1985; Hammer et al., 1985;
Buhler et al., 19901, goats (Ebert et al., 1991), and cows
(Krimpenfort et al., 1991; Hill et al., 1992) were being developed.
Production of pharmaceutical proteins in transgenic
animals was proposed as early as 1982 by Palmiter et al.
(1982). Their idea was based on a number of significant
advances in molecular biology and animal physiology,
especially the successful transfer of recombinant DNA
into mouse embryos by microinjection (Gordon et al.,
1980). Integration of injected DNA into host chromosomes and germline transmission had already been reported by several groups (e.g., Gordon et al., 1981;
Wagner et al., 1981). The integrated DNA was shown
to be expressed and to have dramatic phenotype effects;
for instance, “giant” mice up to twice as large as their
nontransgenic littermates were created (Palmiter et al.,
1982). The “giant” mice were produced by microinjecting a DNA fragment containing the promoter of the
mouse metallothionein-I gene fused t o the structural
gene of rat growth hormone into the pronuclei of fertilized mouse eggs. Growth hormone levels in some of the
mice were up to 800-fold greater than in normal mice,
resulting in animals up to 1.87 times the weight of their
nontransgenic littermates.
By 1983 appropriate tissue-specific expression of
transgenes in mice had been demonstrated. Early work
focused on using transgenic animals to explore the
‘Correspondence: 15601 Crabbs Branch Way (phone: (301)7380745; fax: (301)738-0708; E-mail: [email protected]).
1
Drohan and Clark
2
Table 1. Protein production in transgenic animalsa
Concentration in
human plasma,
RIL
Protein
Level of
recombinant
protein expression,
g/L
Animal species
Reference
Albumin
35-53
10.0
Mouse
Hunvitz et al.: 1994
cq-Antitrypsin
1.4-3.2
12.5
Mouse
Sheep
Archibald et al., 1990
Wright et al., 1991
Antithrombin 111
.17-.39
Mouse, goat
H. Meade'
Fibrinogen
1.7-4.0
Mouse
Prunkard et al., 1996
60.0
Hemoglobin A
Monoclonal
antibody
LA-tPAb
10.0
7.0
2.0
-
32.0
Pig
Sharma et al., 1994
10.0
5.0
Mouse, goat
H. Meade'
-
3.0
Goat
Ebert et al., 1991
2.6 mglL
Pig
Paleyanda et al., 1997
Mouse
Sheep
Yull et al., 1995
Clark et al., 1989
Mouse
Mouse
Pig
-
H. Meade'
Factor VI11
.1 mg/L.
Factor IX
.005
.06
Factor X
Protein C
.01
,004
25 MVL
.7
1.6
2.0
Drews et al., 1995
Van Cott et al., 1997
"Only the highest levels of protein detected are cited. Also, protein levels may not correlate with the level
of active forms.
bLonger-acting tissue plasminogen activator.
CPersonalcommunication.
Extension of this technology to production ofheterologous proteins came from the demonstration that known
regulatory elements of several milk protein genes such
as the mouse whey acidic protein (Campbell et al., 1984)
and sheep B-lactoglobulin (Ali and Clark, 19881, could
direct the tissue-specific expression of transgenes (Andres et al., 1987; Simons et al., 1987). Pharmaceutical
proteins were soon shown to be secreted in the milk of
transgenic animals, the first being tissue plasminogen
activator in mice (Gordon et al., 1981; Pittius et al.,
1988) followed by coagulation factor IX in sheep (Clark
et al., 1989). Isolation and characterization of human
globulin gene regulatory sequences led to the evaluation of hemoglobin production in animal erythrocytes
(Behringer et al., 1989; Hanscombe et al., 1989).
Current Issues
After more than 15 yr of development, transgenic
animals producing foreign proteins in their milk are
not uncommon, as shown in Table 1;however, a number
of issues remain t o be solved. The creation of transgenic
livestock is still extremely inefficient, current technology does not provide control over transgene integration
sites, and regulatory control of transgene expression
is still not fully understood. Only a low percentage of
modified embryos lead to transgenic animals, and some
of these have mosaic integration of the transgene that
may preclude transmission to future generations. However, when transmission from founder animals does
occur, stable inheritance of the transgene is observed
over several generations in sheep (Carver et al., 19931,
pigs (Purse1 et al., 1989; Van Cott et al., 1997) and
goats (Denman et al., 1991).
Expression of the transgene can be affected by the
integration site, and insertional mutagenesis of endogenous genes can occur. Transgenes are apparently inserted into the genome in a random fashion, and other
sequences around the integration site are often seen to
have an effect on expression (Palmiter and Brinster,
1986). A large number of insertional mutations have
been identified in transgenic mice, many of which affect
development (Mark et al., 1992). It has been estimated
that approximately 10% of mouse transgenic cell lines
carry recessive insertional mutations that produce obvious phenotypic changes (Palmiter and Brinster, 1986;
Jaenisch, 1988).
Therefore, an excess of founder animals must be generated and culled t o select those with appropriate expression characteristics. The regulatory sequences that
control expression levels, or that direct precise tissuespecific and developmentally regulated expression, are
still not fully understood, nor are possible interactions
between regulatory and coding or iatrogenic sequences.
Ectopic expression due to interaction of regulatory and
coding sequences and aberrant splicing of transgenes
have been observed. For example, Shani et al. (1992)
showed that the introns of the human serum albumin
gene markedly affect the level of expression of the gene
in transgenic mice. Barash et al. (1994) found that although the sheep B-lactoglobulin gene was expressed
only in mammary tissue in transgenic mice, a human
serum albumin gene fused with the sheep Blactoglobulin promoter was expressed ectopically in muscle, kid-
Transgenic production of plasma proteins
ney, brain, spleen, salivary, and skin tissue, suggesting
a n interaction between the promoter and the human
serum albumin gene. Yull et al. (1995) were able to
markedly increase the expression of human factor M
in the milk of transgenic mice and sheep by removing
a cryptic 37 splice site in the factor IX gene. The previously low levels of expression of the factor IX gene
had been caused by aberrant splicing of the transgene
RNA at the cryptic splice site. Because of these uncertainties, new gene constructs are routinely tested in
mice before moving to larger animals.
One of two general paths is followed in targeting the
production site for a transgenic protein. One approach
is to express the protein in tissues homologous to the
tissue of origin of the native protein. A good example
is expression of human hemoglobin in animal erythrocytes (Swanson et al., 1992; Sharma et al., 1994). This
approach allows the use of isologous promoter sequences and generally gives correct posttranslational
modification (Rao et al., 1994). It also offers the advantages of developmental control of transgene expression
and correct cotranslational processing of globin chains
(Rao et al., 1994). Sequestration of hemoglobin in erythrocytes also increases the ease and efficiency of recovery
and purification. This system has evolved into the synthesis of fusion proteins consisting of human ru-endorphin or magainin peptides as C-terminal extensions of
human a-globin, at levels of more than 25% of the total
hemoglobin in transgenic erythrocytes (Sharma et
al., 1994).
The other approach is to produce proteins in heterologous secretory organs such as the mammary glands,
salivary glands, or urinary tract. A number of examples
are shown in Table 1. In these cases promoters and
regulatory sequences have been identified that produce
greater than normal physiological levels of expression
(Lubon, 1998). In addition, the products are easily collected in the excreted fluids. Many proteins, such as
those normally found in plasma, are difficult to recover
and purify from their native milieu, and changing their
site of production can be advantageous for downstream
purification. Disadvantages of this approach lie in the
unpredictable effects of combining heterologous promoters and genes as described above in the work of
Barash et al. (1994) and Shani et al. (1992) and in
the possible physiological effects of foreign proteins on
specific organs. Such effects could include disrupted
lactation and leakage of proteins into body fluids. Because of this, it may be necessary to produce proteins
that have potent biological effects as inactive fusion
proteins with subsequent activation in vitro. Finally,
posttranslational modification of the human protein
may be altered in heterologous cells.
Despite these issues, major progress has been made
in the production of recombinant proteins in the milk of
transgenic animals, as shown in Table 1.For instance,
striking results have been obtained with human 01antitrypsin. With a n optimized hybrid gene consisting
of the sheep /?-lactoglobulin promoter and the human
3
cul-antitrypsin gene, protein levels up to 60 g/L were
secreted into the milk of sheep (Wright et al., 1991).
This recombinant protein has been characterized by
isoelectric focusing, peptide mapping, amino acid sequencing, mass spectrometry, and glycan analysis and
found to be biologically active and very similar to the
human plasma-derived protein (Colman and Garner,
1995). Transgenic 01-antitrypsin is currently being
evaluated clinically for the treatment of al-antitrypsin deficiency.
Another example of a human protein being produced
in a transgenic animal is human antithrombin 111. The
caprine [j-casein promoter and the human antithrombin
111 gene were used to obtain more than 7 g/L in the
milk of transgenic goats (H. Meade, personal communication). Again, the protein obtained was biologically
active. Recombinant antithrombin I11 has the distinction of being the first transgenically produced protein
to enter clinical trials. After successful Phase I (acute
safety) trials in 20 volunteers, the protein is now in
Phase I1 (safety and dose-setting) trials in patients undergoing coronary artery bypass grafting.
Other examples include human fibrinogen produced
in milk at levels at least two orders of magnitude
greater than those observed in other recombinant production systems (Prunkard et al., 19961, and longeracting tissue plasminogen activator, a modified version
of the native human protein, successfully produced in
goat milk at levels of 3 g/L. However, a seemingly obvious candidate for transgenic production, human serum
albumin, is still problematic. Even after enormous efforts to increase the level of expression in the mammary
gland, 10 g/L, the maximum amount obtained in mouse
milk is still lower than that present in human plasma
(Shani et al., 1992; Hurwitz et al., 1994).Although this
seems comparable to the production levels of the other
proteins described, therapeutic doses of albumin are on
the order of tens of grams, whereas doses of the other
proteins are generally in the milligram range.
Posttranslational Modifications
The above-described proteins require few posttranslational modifications, mainly removal of the signal
peptide and N-linked glycosylation. However, the composition and structure of the carbohydrate side chains
attached to these transgenic proteins may differ from
those of the same recombinant protein produced in the
other systems, and from those of their native human
counterparts. When produced in goat milk, longer-acting tissue plasminogen activator contained a population of small, very heterogeneous oligosaccharides that
had significantly less sialic acid, N-acetylglucosamine,
and galactose than the same recombinant protein when
it is produced in C127 cells (Denman et al., 1991). It
also contained N-acetylgalactosamine, which is absent
in the C127 cell-derived protein. Likewise, Protein C
from pig milk also contained N-acetylgalactosamine, a
sugar not observed in human Protein C (W. Velander,
4
Drohan and Clark
personal communication). The more basic PI values of
recombinant al-antitrypsin polypeptides from sheep
milk may also reflect differences in sialylation (Carver
et al., 1992).
A better understanding of posttranslational glycosylation was provided by a study of recombinant y-interferon produced in mouse milk (James et al., 1995,1996).
Considerable site-specific variation was observed with
complex sialylated and core-fucosylated glycans at one
N-linked site and primarily oligomannose a t the second
site. GalNAc, NeuGc, and Galn 1,3Gal-,Jll,4GlcNAc residues were not detected, in contrast to the same proteins
produced in mouse cell lines grown in culture. The sitespecific addition of oligomannose t o certain asparagine
residues of recombinant antithrombin I11 was also observed in the goat mammary gland. Differences in species- and protein-specific glycosylation patterns may
affect therapeutic efficacy, binding to cellular receptors,
and clearance of the recombinant proteins in patients.
The therapeutic relevance of such effects should become
apparent as these proteins enter clinical trials.
The ability of transgenic cells to perform various posttranslational modifications may also affect the maximum possible rate of production of fully functional
protein. Proteins with fewer posttranslational modifications such as antithrombin 111, tissue plasminogen activator, and ril-antitrypsin are produced in active form
at greater concentrations with greater ease than are
more complex proteins, such as protein C, factor IX,
and factor X, as can be seen in Table 1. Secretion of
protein C and factor IX in active form at .2 t o .4g/L in
milk shows that transgenic production of such proteins
is feasible (Lubon and Paleyanda, 1997). However, as
the number of proteins tested for transgenic production
increases, it has become clear that the mammary gland
epithelium cannot perform all protein modifications efficiently. For example, the hydroxylation of human collagen I (D. Toman, personal communication), the 0glycosylation of bile salt-stimulated lipase (Stromqvist
et al., 1996),and the proteolytic processing of surfactant
protein C (Wei et al., 1995) were essentially nonexistent
in recent studies. In the case of anticoagulant protein
C, at a level of expression of .4 mg/mL, 30 to 40’2%of
the protein C was not cleaved to the two-chain form of
the native protein (Lubon and Paleyanda, 1997).Apparently the mammary gland’s ability to perform this
cleavage could not keep up with the production rate.
This was successfully solved by co-expressing the enzyme furin with protein C to improve the mammary
glands capacity to make this modification (Drews et
al., 1995).Similarly, the expression of human nl-2 fucosyltransferase enzyme altered the oligosaccharide composition of milk and led to the modification of some
glycoproteins to contain the H-Antigen (Prieto et al.,
1995).In pigs, the cell surface Galal-3,Gal-Residue epitope was partially converted to the human-like H-Antigen by the expression of the same enzyme (Koike et al.,
1996). As our understanding of the posttranslational
machinery of mammary epithelial cells grows, our abil-
ity to provide the necessary posttranslational modifications should continue to increase.
Economic Considerations
It is likely that proteins with more extensive posttranslational modifications, as described above, are the
future of the transgenic bioreactor field. As indicated
in Table 1, proteins that are present only in trace
amounts in human plasma can be produced in
transgenics at 100- to 500-fold greater levels. Even proteins as difficult to produce as human Factor VI11 have
been successfully synthesized in the mammary gland
(Paleyanda et al., 1997). Thus, today, we know that we
can produce large volumes of recombinant proteins, for
example, metric tons of nl-antitrypsin, by this technology. This opens the door to an unlimited supply of proteins that were previously produced from limited
human materials such as plasma.
The common expectation is that recombinant proteins will be less expensive to produce in transgenic
animals due to the low capital investment required for
raw material production. This holds true for most protein biologicals when compared to existing yeast, fungi,
and mammalian cell culture processes. For certain
other proteins, however, purification from plasma will
likely remain more economical. Human albumin, which
is present in high concentration in plasma, costs less
than $1.00 per gram to produce, and in spite of all the
technical advances it will be difficult to produce it as
cost-effectively from recombinant systems. Also, many
plasma-derived biologicals need not be purified to the
same extent as transgenic proteins, which may offset
some or all of the cost advantage of transgenically derived materials. For therapeutic safety, the purity of
recombinant proteins from animal fluids must be much
greater than that of proteins from human plasma. Although this will tend to increase the cost and decrease
the recovery of the protein, the greater purity may have
clinical benefit in some cases. However, if fully active
factor VI11 (Paleyanda et al., 1997), factor IX, or erythropoietin can be produced at gramsniter levels, the reduction in the cost of those products could be dramatic.
Product Safety
One of the major advantages of recombinantly produced proteins is their apparent freedom from contamination with endogenous human viruses. Transgenic
products are likewise apparently free from human infectious agents. The long-standing experience with
therapeutics, such as vaccines produced in animals,
suggests that transmission of infectious agents from
animals should not be a problem. General product
safety is also enhanced by good husbandry of production
herds under specific pathogen-free conditions, and by
compliance with FDA Good Manufacturing Practices
(U.S. Government, 1998) and other FDA regulatory
5
Transgenic production of plasma proteins
guidance specifically focusing on therapeutic protein
production in transgenic animals (FDA, 1995).
Viral safety can be further ensured by viral inactivation steps in the purification processes for these proteins. In addition to methods such as pasteurization
and solvent/detergent treatment, which are routinely
used for plasma-derived products, two new viral inactivation processes particularly useful for the treatment
of transgenically produced proteins have recently been
described: chromatography through an iodine-Sephadex column (S. Miekka, personal communication) and
treatment with gamma irradiation at very low rates
(W. Drohan, IBC Viral Clearance Conf., June 1998,
Philadelphia, PA). Both of these procedures are effective in inactivating all classes of viruses (lipid and nonlipid enveloped) and have minimal or no effect on protein activity.
Future Directions
Dynamic progress in the understanding of gene regulation and recombinant DNA technology will soon allow
us t o design hybrid genes that can be very precisely
regulated. Yeast artificial chromosome technology has
recently been used to produce pigs transgenic for human membrane co-factor gene, overcoming limitations
on transgene size and site of integration (Langford et
al., 1996).Also, new methods of detecting embryos with
successfully incorporated transgenes before transfer to
the foster mothers will make the production of
transgenic animals more efficient (Seo et al., 1997;
Menck et al., 1998). The use of embryonic stem cells
offers another solution for targeted recombination of
transgenes (Piedrahita et al., 1997; Pirity et al., 1998).
Transgenic technology has a number of advantages
for production of modified human proteins, such as
longer-acting tissue plasminogen activator, that have
improved biologic properties compared to their wildtype counterparts. Many of these proteins undergo complex posttranslational modification requiring their production in animal cells. With the development of
techniques for engineering a cell’s posttranslational
processing capabilities, the ability to modify a protein’s
structure is further enhanced. Further, protein structure/function studies continue to elucidate the domains
of protein molecules responsible for various biological
activities. Chimeric molecules comprising multiple domains of the same molecule, or mixing functional domains of several molecules, can be produced that will
potentially have biological properties superior to the
native proteins. For instance, domains of human factor
VI11 that contain the epitopes targeted by most inhibitory antibodies may be able to be replaced by porcine
factor VI11 domains that are less immunogenic (Lubin
et al., 1994).By definition such molecules will have to be
produced by recombinant technology and will be ideal
candidates for transgenic production.
Transgenic animals should not be considered only for
production of recombinant proteins. They can also be
used as donors of cells and organs for xenotransplantation. Several groups are attempting to generate pigs
transgenic for a human complement-inhibitor or decayaccelerating factor (Fodor et al., 1994; Cozzi et al.,
19961, membrane cofactor protein (Langford et al.,
19961, or CD59 protein (Byrne et al., 1996; Kroshus et
al., 1996) as solutions to the worldwide shortage of organs for human transplantation. Success in engineering organs may open up the possibility of
implanting nonautologous cells into human hosts, as
another approach to somatic gene therapy.
The use of transgenic animals to produce recombinant proteins does raise ethical concerns in regard to
the health and welfare of the transgenic animals
(Thompson, 1993; Masood, 1997). However, the commercialization of this technology has been associated
with every effort to develop healthy founder animals.
In fact, given the potential commercial value of a
transgenic herd, these animals receive extremely good
care (Dixon, 1995). ol-Antitrypsin-producing sheep,
antithrombin 111-producing goats, and protein C-producing pigs appear as healthy as normal livestock (W.
Velander, personal communication). Methods of generating transgenic animals will improve, avoiding random transgene integration, unpredictable expression,
mosaicism, and variable germline transmission, thus
drastically reducing the number of animals used. Soon
transgenic techniques will not be more aggressive or
invasive than those used in animal husbandry today.
Transgenic cows are already being generated from embryos collected from slaughterhouses, and the microinjected embryos are introduced into foster mothers
without surgical procedures. Healthy livestock and the
increased benefit to human patients will determine the
ultimate acceptance of this technology.
The potential safety, relatively unlimited supply, and
economical cost of production are major factors fueling
the development of transgenic therapeutics. New indications and treatment methodologies, such as prophylaxis, topical application, novel delivery techniques,
and oral induced-tolerance regimens will provide additional impetus for this growth and development.
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