Note: Descriptions are shown in the official language in which they were submitted.
CA 02427574 2003-05-05
PLAN'C BIOREACTORS
The present invention relates to the use of plants as a bioreactor for the
production of heterologous proteins. More specifically this invention relates
to
the expression of transgenes of interest for oral administration using plants.
BACKGROUND OF THE INVENTION
Expression of mammalian genes in several plants including tobacco and
Arabidopsis has been recognized as efficient, low cost, non-sterile
bioreactors for
the production of proteins valuable to both medicine and industry (Ma and Hein
1995). Recently, evidence was presented that demonstrated that the four chains
of
the secretory immunoglobulin were properly expressed and assembled in plants
and that the antibody was fully functional (Ma et al 1995). Furthermore,
bacterial
(Haq et al 1995), and viral (Mason et al 1996) antigens produced in transgenic
tobacco and potato effectively immunized mice when the transgenic potato was
administered orally. Oral administration of protein antigens can result in a
diminished immune response to subsequent systemic administration (eg., by
injection) of the same antigen, a process known as oral tolerance (Mowat,
1987).
Oral tolerance has recently been explored as a potential antigen-specific
immunotherapeutic strategy for the treatment of organ-specific autoimmune
diseases. In animal models, oral administration of disease inducing
autoantigens
has been shown to inhibit several experimental autoimmune diseases, including
animal NOD diabetes, uveoretinitis, experimental autoimmune encephalomyelitis
(EAE), and rheumatoid arthritis (RA). However, one limitation with clinical
use
of the oral tolerance approach is the cost of protein production, as oral
tolerance
requires the ingestion of relatively large amounts of protein antigens, often
in
excess of quantities that can be provided by conventional protein synthesis or
fermentation systems.
As the immune system is specific in the recognition of protein antigens, it
is preferred that the antigens are mammalian and species specific to elicit
the
desired reduction in antigen directed immune responses. The use of plants as
an
expression system for the production of mammalian antigen proteins offers
unique
CA 02427574 2003-05-05
advantages beyond high production yields at competitive low cost. These
advantages include reduced health risks from possible pathogen contamination
as
would be the case with extraction from mammalian tissues, correct modification
and assembly of protein leading to function of proteins expressed by the plant
when required or desired, optimization of high expression levels, and the
ability to
induce oral immune tolerance without extensive purification of the plant
material.
Using edible plant tissue leads to major cost reductions due to the simplicity
of the
delivery system.
Peripheral immune responses can be downregulated with oral
administration of protein antigens, and this effect is antigen and protein
specific.
Human volunteers fed keyhole limpet hemocyanin (KLH) demonstrated reduced
peripheral immune responses to KLH on recall responses of lymphocytes re-
exposed to that antigen in vitro (Matsui, M. et al., 1996, Ann N Y Acad Sci
778:398-404). Therefore, oral tolerance can be demonstrated in humans. The
magnitude of response may be influenced by several factors, including
expression
levels of the transgenic protein of interest within plants, or the presence of
immune adjuvants or response modifiers present within the composition.
Plants also may represent an ideal expression system for oral immune
tolerance for several additional factors. Firstly, immune tolerance requires
activation events within T lymphocytes, and plants by their composition which
includes lectins may facilitate this. Additionally, the accumulation of
transgenic
proteins within plant cell compartments may protect the antigen from
degradation
in the digestive tract. This could augment oral immune tolerance by delivering
transgenic plant protein to effector sites in the gut distal to the digestive
environment of the stomach and duodenum (e.g. Kong et al, 2001, P.N.A.S.,
98:11539-11544).
The use of plants as an expression system for production of mammalian
antigenic proteins offers several unique advantages, including high production
yields at competitive low cost, reduced health risks from pathogen
contamination,
and correct modification and assembly of foreign proteins. An additional
advantage of a plant production system is that proteins, in the case of oral
immune
tolerance induction, may be used directly without extensive purification
resulting
CA 02427574 2003-05-05
4
in further cost reductions. In this regard it has been noted that proteins
produced in
transgenic tobacco required purification prior to administration. Clearly
steps
involving purification or processing are undesirable if ease of oral
administration
is to be maximized as well as minimizing any associated costs for production.
Numerous plants have proven themselves to be amenable to
transformation with heterologous genes and for some time tobacco has been the
model system for plant transformation. Despite the fact that crop-protection
focused biotechnologies have not found application in non-food crop plant
production, a major role does remain for such plants as bioreactors. An
example
of a non-food crop plant is tobacco, which is capable of producing high levels
of
soluble protein (fraction 1 protein, F1P; Woodleif et al 1981) and pilot
systems
have been developed to purify this fraction for use as a high protein dietary
supplement (Montanari et al 1993).
From both a regulatory and public safety stand point non-food crop plants
are ideal species for the transgenic production of biologically active
proteins.
Non-food crop plants minimize the risk of accidental leakage of transgenic
plant
material expressing genes for biologically active proteins into the human food
chain. Other plant bioreactor systems based on canola (Rooijen et al. 1995),
potato (Manson et al 1996), rice and cassava (Ma and Hein 1995) do not offer
this
advantage. Furthermore, non-food crop plants can be selected so that
production
in areas where there are no naturally occurnng wild species further minimizes
the
risk of gene leakage to the local flora, an example of this would be to grow
tobacco in regions where tobacco does not overwinter, such as Canada. With any
non-food crop plant, transgenic proteins can be produced using any tissue or
organ
of the plant. However if protein production is based on leaves, not seeds or
tubers, and when coupled with the fact that the leaves are harvested before
flowering there is virtually no risk of uncontrolled bioreactor plants
occurnng in
future crop seasons.
However, many non-food crop plants contain high levels of secondary
plant products making plant tissue obtained from these plants unsuitable for
direct
oral administration. Earlier studies have described the administration of
tobacco-
CA 02427574 2003-05-05
derived proteins to mice, however, the proteins were in a partially purified
form.
For example the study by Mason et al ( 1996) involved the direct oral
administration of viral antigens expressed in potato tuber and tobacco. The
potato
tuber samples were directly fed to mice, yet the antigen, when obtained from
5 tobacco, had to be partially purified using sucrose gradients prior to
administration
to mice.
The breeding of low alkaloid-containing tobacco plants has been reported
(Chaplin 19?7), however, use of such a plant as a bioreactor has not been
suggested.
Recently, specific alteration of nicotine levels within tobacco, by either
over
expression (i.e. increasing) or antisense expression (decreasing) of
putrescine N-
methyltransferase, a rate limiting enzyme involved in the nicotine
biosynthetic
pathway, has been suggested (U.S. 5,260,205, issued November 9, 1993 and U.S.
5,369,023, issued November 29, 1994; inventors Nakatani and Malik). However,
these methods are directed to the alteration of nicotine levels so that the
levels of
other alkaloids that affect the flavours and aroma of tobacco are not modified
in
any manner. These plants are not ideal for use as a non-food crop plant as
describe herein, since the levels of only a select group of alkaloids have
been
reduced. Further, no nicotine free plants were actually produced, nor was the
use
of these transgenically modified tobacco plants, as a bioreactor for the
synthesis of
proteins of interest, suggested. However, such methods may be used and
augmented in order to produce non-food crop plants where the total alkaloid
level
is reduced. Such an approach may be useful for the production of low alkaloid
plants as a bioreactor for the synthesis of proteins of interest as
contemplated by
this invention.
US 6,338,850 and EP 720, 484 discloses the expression of glutamic acid
decarboxylase within a plant.
Oral tolerance has been explored as a potential antigen specific
immunotherapeutic strategy for the treatment of organ specific autoimmune
disease and prevention of transplantation and rejection. However, there are
several potential limitations in the success of this approach in human
disease,
CA 02427574 2003-05-05
6
including selection of the appropriate antigen that is specific to disease,
intervention in the disease process prior to onset of marked epitope spreading
in
which the targeting of the initial peptide is extended to associated peptides
within
the triggering protein, intervention in the disease process prior to onset of
advanced clinical symptoms, and the requirement for large amounts of protein
antigen suitable for clinical use. This latter issue leads to large costs of
protein
production from the use of conventional expression systems including
transgenic
expression in tissue culture and fermentation systems, to produce the amounts
of
protein needed. Additionally, expression in bacterial systems incapable of
glycosylation and correct folding of the protein, leads to the formation of
inclusion bodies and marked reduction in levels of recoverable usable protein
antigens.
There is a need in the art for a method of making proteins in large
1 S quantities and at low cost. Further there is a need in the art for making
biologically
active proteins in large quantities and low cost, that do not require
purification or
further processing prior to being administered to a subject. Furthermore,
there is a
need for enhancing the action of an autoantigen, for example by co-
administration
of the autoantigen with a cytokin.
It is an object of the invention to overcome disadvantages of the prior art.
The above object is met by the combinations of features of the main
claims, the sub-claims disclose further advantageous embodiments of the
invention.
CA 02427574 2003-05-05
SUMMARY OF THE INVENTION
The present invention relates to the use of plants as a bioreactor for the
production of heterologous proteins. More specifically this invention relates
to
the expression of transgenes of interest for oral administration using plants.
The present invention provides a nucleotide sequence encoding a cytokine
and an autoantigen, and a plant, or portion thereof, a plant cell, or a seed,
comprising the nucleotide sequence. The cytokine may be selected from the
group consiting of IL-4, IL-10, and TGF-(3, and the autoantigen may be
glutamic
acid decarboxylase (GAD), or a fragment thereof. Furthermore, the nucleotide
sequence encoding the cytokine may, or may not be not genetically linked to
the
nucleotide sequence encoding the autoantigen, or a fragment thereof. Non-
limiting examples of the plant include alfalfa, corn, wheat, soybean, potato,
canola, tobacco, and low-nicotine, low-alkaloid tobacco.
If the plant is a low-nicotine, low alkloid tobacco, then the leaves of the
low nicotine, low alkaloid tobacco plant comprise:
a) a nicotine concentration (wt/wt) which is less than about 10 % the
nicotine concentration (wt/wt) of Delgold tobacco plants grown under the same
conditions, and
b) an alkaloid concentration (wt/wt) which is less than about 10 % the
alkaloid concentration (wt/wt) of Delgold tobacco plants grown under the same
conditions.
The present invention pertains to a vector comprising a nucleotide
sequence, wherein a first nucleotide sequence encodes a cytokine is
operatively
linked to a plant regulatory region and a terminator region, and a second
nucleotide sequence encodes autoantigen, is operatively linked to a plant
regulatory region and a terminator region.
The present invention also provides a method (method A) for the
production of a cytokine and an autoantigen, comprising:
CA 02427574 2003-05-05
i) transforming a plant, or a portion thereof, with a first vector comprising
a nucleotide sequence encoding the cytokine or the autoantigen, the second
nucleotide sequence operatively linked with 5' and 3' regulatory regions;
ii) introducing into the plant, or into a second plant a second vector
comprising a second nucleotide sequence encoding the other of the cytokine or
autoantigen, the second nucleotide sequence operatively linked with 5' and 3'
regulatory regions; and
iii) growing the plant, or the plant and the second plant, and allowing
expression of the cytokine and autoantigen.
Also included in the present invention, is method A as defined above,
wherein after the step of growing (step iii)), there is a step of harvesting
the tissue
from the plant, to produce harvested tissue. Furthermore, after the step of
harvesting, the cytokine and autoantigen, are obtained from the harvested
tissue.
The cytokine may be selected from the group consiting of IL-4, IL-10, and TGF-
[3, and the autoantigen may be glutamic acid decarboxylase (GAD), or a
fragment
thereof.
The present invention also embraces a use of the plant, or the plant and
second plant, produced using the method A, defined above, for the production
of a
medicament for the treatment of a disease state in an animal. The present
invention also provides a use of the harvested material, produced using the
method A as defined above, for the production of a medicament for the
treatment
of a disease state in an animal.
The present invention is directed to a composition comprising a cytokine
and an autoantigen, wherein the cytokine and autoantigen, are produced by the
method A as defined above. The composition as just defined may be used for the
production of a medicament for the treatment of a disease state in an animal.
The
disease state may be selected from the group consisting of an auto-immune
disease, rheumatoid arthritis, diabetes and insulitus. The composition may
also be
used for inducing regulatory T cells. Furthermore, the composition as defined,
CA 02427574 2003-05-05
may be orally administered. The cytokine may be selected from the group
consisting of IL-4, IL-10, and TGF-(3, and the autoantigen may be glutamic
acid
decarboxylase (GAD), or a fragment thereof.
The present invention relates to a method (method B) for the production of
a cytokine and an autoantigen comprising:
i) transforming a plant, or a portion thereof, with a first vector comprising
a nucleotide sequence encoding the cytokine or the autoantigen, first
nucleotide
sequence operatively linked with 5' and 3' regulatory regions, to produce a
first
transformed plant; and
ii) transforming a second plant, or apportion thereof, with a second vector
comprising a second nucleotide sequence encoding the other of the cytokine or
the
autoantigen, the second vector operatively linked with 5' and 3' regulatory
regions,
to produce a second transformed plant; and
iii) crossing the first transformed plant produced in step i) with second
transformed plant produced in step ii), to produce a dual transgenic plant;
iv) growing the dual transgenic plant and allowing expression of the
cytokine and autoantigen, or a fragment thereof;
Also included in the present invention, is method B as defined above,
wherein after the step of growing (step iv)), there is a step of harvesting
the tissue
from the plant, to produce harvested tissue. Furthermore, after the step of
harvesting, the cytokine and autoantigen, or a fragment thereof, may be
obtained
from the harvested tissue. The cytokine may be selected from the group
consiting
of IL-4, IL-10, and TGF-[3, and the autoantigen may be glutamic acid
decarboxylase (GAD), or a fragment thereof.
The present invention also embraces a use of the transformed plant
produced using the method B defined above, for the production of a medicament
for the treatment of a disease state in an animal. The present invention also
provides a use of the harvested material obtained from transformed plant
produced
using the method B as defined above, for the production of a medicament for
the
treatment of a disease state in an animal. The cytokine may be selected from
the
CA 02427574 2003-05-05
group consiting of IL-4, IL-10, and TGF-~3, and the autoantigen may be
glutamic
acid decarboxylase (GAD), or a fragment thereof. Examples of the disease state
include auto-immune disease, rheumatoid arthritis, diabetes and insulitus.
5 The present invention provides a composition comprising a cytokine and
an autoantigen, wherein one, or both of the cytokine and autoantigen, are
plant
expressed. The cytokine may be selected from the group consiting of IL-4, IL-
10,
and TGF-(3, and the autoantigen may be glutamic acid decarboxylase (GAD), or a
fragment thereof. Furthermore, the present invention relates to the use of the
10 composition as just defined, for the preparation of a medicament for the
treatment
of a disease state. The disease state may be selected from the group
consisting of
an auto-immune disease, rheumatoid arthritis, diabetes and insulitus. The
medicament may also be used for inducing regulatory T cells. Furthermore, the
composition as defined, may be orally administered.
The present invention provides for a method (method C) for the production
of a cytokine and an autoantigen, comprising, growing a plant, or a portion
thereof, comprising a first nucleotide sequence encoding the cytokine or the
autoantigen, and a second nucleotide sequence encoding the other of the
cytokine
or autoantigen, or a fragment thereof, and allowing for expression of the
cytokine
and autoantigen. Additionally, tissue may be harvested from the plant, to
produce
harvested tissue, and the cytokine and autoantigen, may be obtained from the
harvested tissue. The cytokine may be selected from the group consiting of IL-
4,
IL-10, and TGF-(3, and the autoantigen may be glutamic acid decarboxylase
(GAD), or a fragment thereof.
The present invention is directed to a composition comprising the cytokine
and the autoantigen, wherein the cytokine and the autoantigen, are produced by
the method C, as defined above, and a use of the composition for the
preparation
of a medicament for the treatment of a disease state.
The present invention relates to a method (method D) for the production of
a cytokine and an autoantigen, comprising, transforming a plant, or a portion
may be orally administered. Th
CA 02427574 2003-05-05
11
thereof, with a vector comprising, a first nucleotide sequence encoding the
cytokine and operatively linked to a plant regulatory region and a terminator
region, and a second nucleotide sequence encoding the autoantigen, operatively
linked to a plant regulatory region and a terminator region, to produce a
transformed plant, growing the transformed plant, and allowing expression of
the
cytokine and the autoantigen. The cytokine may be selected from the group
consiting of IL-4, IL-10, and TGF-(3, and the autoantigen may be glutamic acid
decarboxylase (GAD), or a fragment thereof.
Also included in the present invention is a composition comprising a
cytokine and an autoantigen, wherein the cytokine and autoantigen, are
produced
by the method D, as described above, and a use of the composition for the
preparation of a medicament for the treatment of a disease state.
The present invention embraces a method (method E) for the production of
a cytokine and an autoantigen comprising:
i) transforming a plant, or a portion thereof, with a first vector comprising
a nucleotide sequence encoding the cytokine or autoantigen, or a fragment
thereof,
the first nucleotide sequence operatively linked with 5' and 3' regulatory
regions;
ii) introducing into the plant, or into a second plant, a second vector
comprising a second nucleotide sequence encoding the other of the cytokine or
autoantigen, or a fragment thereof, the second nucleotide sequence operatively
linked with 5' and 3' regulatory regions; and
iii) growing the plant, or the plant and the second plant, and allowing
expression of the cytokine and autoantigen.
Also provided by the present invention as defined above, the IL-4 and
GAD, or a fragment thereof, may comprise sequences from different species, or
the IL-4 and GAD, or a fragment thereof, may comprise sequences from the same
species. The IL-4 and GAD, or a fragment thereof, may be selected from, but
are
not limited to being selected from the group consisting of:
a) marine IL-4 and marine GAD 67;
b) marine IL-4 and marine GAD 65;
CA 02427574 2003-05-05
12
c) marine IL-4 and human GAD 67;
d) marine IL-4 and human GAD 65;
e) human IL-4 and human GAD 67;
f) human IL-4 and human GAD 65;
g) human IL-4 and marine GAD 67;
h) human IL-4 and marine GAD 67;
i) human IL-4, marine IL-4, or a combination thereof and,
j) marine GAD 67, marine GAD 65, human GAD 65, human GAD
67, or a combination thereof:
According to the present invention, there is provided a composition
comprising an plant-expressed cytokine, and an autoantigen. In an embodiment
the cytokine is a contra-inflammatory cytokine and the autoantigen is GAD or a
fragment thereof. The cytokine and autoantigen may be produced in the same
plant or in different plants. For example, but not wishing to be limiting, the
cytokine may be produced in a first plant, and the autoantigen may produced in
a
second plant.
The present invention pertains to a method of treating an auto-immune
disease in a subject comprising, orally administering to the subject a
composition
comprising a plant-expressed contra-inflammatory cytokine, and an autoantigen,
a
contra-inflammatory cytokine, and a plant-expressed autoantigen, or a plant-
expressed contra-inflammatory cytokine, and a plant-expressed autoantigen.
Further, the present invention provides a method of eliciting an immune
response against an antigen in a subject, the method comprising orally
administering a plant or portion thereof comprising an antigen and an cytokine
to
the subject. The antigen may be an autoantigen, for example, but not limited
to
GAD, or a fragment thereof. Further, the cytokine may comprise a contra-
inflammatory cytokine. Preferably the autoantigen and cytokine comprise
sequences naturally present in the subject.
The present invention further contemplates a plant produced according to
the methods, methods A-D as defined above. For example, but not wishing to be
CA 02427574 2003-05-05
13
limiting, the plant may be a potato, tomato, alfalfa, corn, tobacco, or a low-
nicotine, low-alkaloid tobacco plant comprising a transgenic nucleotide
sequence
capable of producing two or more proteins of interest in the plant. In a
embodiment of the present invention the protein of interest may comprise, but
is
not limited a cytokine, for example a contra- inflammatory (anti-inflammatory)
cytokine, IL-4, IL-10, TGF~i, or an autoantigen, for example GAD, or a
combination thereof. In a further aspect of an embodiment, the protein of
interest
comprises IL-4 and GAD. The present invention also contemplates plant cells,
tissues, seeds comprising the protein or proteins of interest.
Also provided in the present invention is a method of delivering an
interleukin, an autoantigen, or both an interleukin and an autoantigen to a
subject
in need thereof comprising, orally administering the plant as defined above,
or
portion thereof, to the subject.
The present invention also discloses a method of enhancing the effect of an
immune response against an antigen in a subject, comprising administering a
plant-expressed interleukin and the antigen to the subject.
The present invention also provides a use of plant-expressed IL-4 for the
preparation of a medicament for use as an immunomodulator in oral tolerance.
The present invention also is directed to a use of plant-expressed IL-10 for
the preparation of a medicament for: inducing mast cell proliferation,
inhibiting
the production of a proinflamatory cytokine, treating psorasis, wound healing,
treating liver fibrosis, or treating chronic hepatitis C.
This summary of the invention does not necessarily describe all necessary
features of the invention, but that the invention may also reside in a sub-
combination of the described features.
CA 02427574 2003-05-05
14
BRIEF DESCRIPTION OF THE DRAWINGS
These and other features of the invention will become more apparent from
the following description in which reference is made to the appended drawings
wherein:
FIGURE 1 shows an immunoblot of proteins extracted from leaf tissues of
transgenic tobacco plants transformed with a plant expression vector
comprising human GAD65. Lanes 1-2: protein extracts from individual
transgenic lines (40 ~g total protein was loaded onto the gel); Lane C:
protein extracts from empty vector-transformed tobacco plants;
rmGAD67: recombinant mouse GAD67 purified from E. coli (standard).
FIGURE 2 shows an immunoblot of proteins extracted from leaf tissues of
transgenic tobacco plants transformed with a plant expression vector
comprising mouse IL-4. Lane 1: Standard mouse IL-4 (PharMingen); Lane
2: protein from empty vector-transi:ormed tobacco plants; Lane 3: protein
from IL-4 transgenic tobacco plants (40 ,ug protein was loaded per lane
onto the gel).
FIGURE 3 shows results of an MC/9 cell proliferation assay exhibiting the
biological activity of plant-derived IL-4. The black circle indicates the
background level of 3H incorporation by MC/9 cells. Black triangles
represent plant recombinant IL-4, clear circles represent recombinant IL-4
obtained from Pharmingen, and open triangles represent the international
IL-4 reference obtained from the National Cancer Institute-Frederick
Cancer Research and Development Center
FIGURE 4 shows development of diabetes in NOD-scid mice receiving a single
i.v. injection of 10 X 106 spleen cells from diabetic donors mixed with 10
X 106 spleen cells from nondiabetic NOD mice fed transgenic GAD and
IL-4 plants, in comparison to control mice receiving a single i.v. injection
of 10 X 106 spleen cells from diabetic donors. Splenic mononuclear cells
CA 02427574 2003-05-05
from mice fed GAD plus IL-4 plants were cotransferred with T cells from
diabetic NOD mice to 8-week-old NOD-skid mice. A positive control
group received cells only from diabetic NOD mice.
5 FIGURE 5 graphically depicts the expression of human interleukin-10 (hIL-10)
in plants. Figure SA and Figure SB show the quantity of IL-10 secreted to
the apoplast following transformation of tobacco plants with nuclotide
sequences comprising either a long hIL-10 3'UTR region or a short hIL-10
UTR region respectively. Figure SC shows the quantity of plant
10 recombinant IL-10 expressed in transgenic tobacco plants transformed
with an IL-10 nucleotide sequence which further comprises a KDEL
endoplasmic reticulum retention signal.
FIGURE 6 shows results of the analysis of plant recombinant IL-10 (prIL-10)
15 protein. Non-reducing SDS-PAGE ofprIL-10 and hIL-10. Black arrow
head means dimer; white arrow head means monomer.
FIGURE 7 shows results of reducing SDS-PAGE of prIL-10 and hIL-10, either
digested with thrombin (lanes +thrombin), incubated in thrombin buffer (-
thrombin a) or untreated (-thrombin b). Molecular weight standards kDa.
FIGURE 8 shows results of biological activity determination of prIL-10, hIL-10
and prIL-10 inhibited LPS-induced secretion of IL-6 by
macrophage/monocyte PUS-1.8 cells. 4x105 LPS-stimulated cells/ml were
incubated with increasing concentrations of prIL-10 (plant produced) ,
rIL-10 (insect produced) or sIL-10 (reference standard) for 24 h. Cell
culture supernatants were collected and tested for IL-6 concentrations by
ELISA. Vertical lines indicate standard error bars. Gray triangles indicate
plant recombinant IL-10 (prIL-10), gray squares are control plant extract
(C), open circles are the reference IL-10 standard, black diamonds are
recombinant IL-10 (rIL-10, from insect), the open square represents
unstimulated cells, and the gray circle represents LPS-stimulated cells.
CA 02427574 2003-05-05
16
FIGURE 9 shows results of the co-stimulatory effect of prIL-10 or hIL-10 on
mast cell proliferation. The ability of hIL-10 (2 ng m1'1) or prIL-10 (1.27
ng m11) and rIL-4 (0.08 ng m11) to enhance growth of MC/9 mast cells
was assessed by a [3H] thymidine incorporation assay. mAb: neutralizing
anti-IL-10 monoclonal antibody (l,ug ml''); rIL-4: recombinant murine
IL-4; prIL-10: plant recombinant human IL-10; hIL-10: recombinant
human IL-10 produced in insect.
FIGURE 10 shows the oligonucleotide cassettes for integration of TMV 5'
untranslated region and PR-lb transit peptide sequences into transgene
constructs. Figure 10(A) the TMV leader sequence. Figure 10(B) the PR-
lb signal peptide sequence. Figure 10(C) the amino acid translation of
encoded signal peptide. Identity, sequence, and position of each
oligonucleotide within the cassettes are indicated. TMV sequence is
marked in bold, PR-lb signal peptide sequence in plain letters and
additional sequence in italics. Nucleotides constituting all or part of the
initiation codon are present within boxes. Oligonucleotides #1091 (SEQ
ID NO:1); #1092 (SEQ ID N0:4); and #1309 (SEQ ID N0:2) represent
the TMV oligo set. Oligonucleotides #1091, #1092 (SEQ ID NO:1 and 4),
and #4361 through #4365 (SEQ ID NOs: 3, 5, 6, 8 and 9 respectively)
represent the TMV-PR oligo set.
FIGURE 11 displays the map of the T-DNA region of pCDX-TL-MAFPII.
FIGURE 12 displays the map of the T-DNA region of pMON-TL-MAFPII.
FIGURE 13 is a Western blot showing Type II AFP accumulation in field plants.
Western blot analysis of total soluble protein (2.5 wg) extracts from R1
generation Type II AFP transgenic plants in which the AFP was targeted to
accumulate in the extracellular space. Samples are designated by number
to indicate the plot from which it was collected and by letter to indicate the
transformed parent plant from which the R1 plants were descended.
Records at the Delhi station indicate that A plants were descended from
parent II4c2-#10, B plants from II4cz-#1, C from pII3a-#7, and D plants
CA 02427574 2003-05-05
17
were wild-type controls (4D). Total soluble protein extract from a field-
grown wild type plant was included in the analysis as a negative control
and mixed with mature Type II AFP (25 ng) purified from sea raven sera
to generate the positive control (AFP).
S
FIGURE 14 shows the cDNA sequence of human IL-10. The cDNA sequence is
denoted in small letters, the encoded protein sequence is denoted in capital
letters below the nucleotide sequence with the one letter amino acid code.
The signal peptide sequence is in bold. Oligonucleotides used to amplify
hIL-10 sequences or to fuse other sequences to the hIL-10 gene are shown
in capital letters underlined by an arrow. Restriction sites introduced by
PCR are shaded. Encoded amino acids are shown above the hIL-10-His-
KDEL oligonucleotide sequence.
FIGURE 15 shows a schematic depiction of three IL-10 constructs used in plant
transformation as described herein. Figure 15 (A) shows the full length,
1601 by cDNA of IL-10. Transformed plants comprising this construct are
designated as "F" plants in Figure 16. Figure 15 (B) shows a truncated IL-
10 sequence comprising nucleotides 4-732, with only a portion of the 3'
UTR (168 bp). Transformed plants comprising this construct are
designated as "E" plants in Figure 16. Figure 15 (C) shows an IL-10
construct with the 3' UTR portion removed, and a thrombin recognition
sequence, a His tag and a KDEL sequence added to the 3' end of the IL-10
sequence. The region containing the thrombin-His tag and KDEL sequence
is magnified to show the order of those sequences with respect to the hIL-
10 gene. Transformed plants comprising this construct are designated as
"G" plants in Figure 16. The hIL-10 coding region the signal peptide is
depicted by a dark shaded box, and the cDNA is shown by a straight line.
Oligonucleotides are represented by short arrows.
FIGURE 16 shows the IL-10 protein levels within transgenic plants as
determined by ELISA. The protein concentration was determined and
compared against a hIL-10 standard curve. Individual transformed plants
were analyzed. Plants denoted "E" were transformed with hIL-10 construct
CA 02427574 2003-05-05
18
containing coding sequences and a short 3' UTR; plants denoted "F" were
transformed with hIL-10 construct containing coding sequences and the
complete 3' UTR; plants denoted "G" were transformed with hIL-10
construct containing coding sequences fused to thrombin-His tag-KDEL
S sequence.
FIGURE 17 graphically depicts total pathology scores for the large bowel (1b)
and small bowel (sb) of 5 groups of IL10 null mice fed control chow
(Control), chow plus 10% 81 V9 (81 V9-10), chow plus 20% 81 V9
(81 V9-20), chow plus 10% G7-2-9 (IL10-10), or chow plus 20% G7-2-9
(IL 10-20).
CA 02427574 2003-05-05
19
DESCRIPTION OF PREFERRED EMBODIMENT
The present invention relates to the use of plants as a bioreactor for the
production of heterologous proteins. More specifically this invention relates
to
the expression of transgenes of interest for oral administration using plants.
The following description is of a preferred embodiment by way of
example only and without limitation to the combination of features necessary
for
carrying the invention into effect.
According to an aspect of an embodiment of the present invention, there is
provided a transgenic plant encoding or expressing two or more than two
proteins
of interest. Also provided is a first transgenic plant encoding or expressing
a first
protein of interest, and a second transgenic plant encoding or expressing a
second
protein of interest, so that when these plants are crossed, a transgenic
plant,
encoding or expressing two, or more than two proteins of interest is produced.
The transgenic plant, or portion thereof expressing the two or more proteins
of
interest may be orally administered to a subject with minimal or no prior
processing. In such an example, which is not meant to be limiting in any
manner,
the transgenic plant or portion thereof comprising the two or more proteins of
interest may be used to treat, prevent or both treat and prevent a medical
ailment
in a subj ect.
By "operatively linked" or "operably linked" it is meant that the particular
sequences interact either directly or indirectly to carry out their intended
as
described herein. The interaction of operatively linked sequences may, for
example, be mediated by proteins associating with the sequences. A
transcriptional regulatory region and a sequence of interest (coding region of
interest) are "operatively linked" when the sequences are functionally
connected
so as to permit transcription of the sequence of interest to be mediated or
modulated by the transcriptional regulatory region. Similarly, a terminator
region
and a coding region of interest are operatively linked when a terminator
region
connected to the coding region, results in the termination of a transcript
encoded
by the sequence of interest.
CA 02427574 2003-05-05
By "DNA regulatory region" it is meant a nucleic acid sequence that has
the property of controlling the expression of a DNA sequence that is operably
linked with the regulatory region. Such regulatory regions may include
promoter
S or enhancer regions, and other regulatory elements recognized by one of
skill in
the art. By "promoter" it is meant the nucleotide sequences at the S' end of a
coding region, or fragment thereof that contain all the signals essential for
the
initiation of transcription and for the regulation of the rate of
transcription and
include constitutive promoters, tissue specific promoters or inducible
promoters as
10 would be known to those of skill in the art. :Examples of known
constitutive
regulatory elements include promoters associated with the CaMV 35S transcript.
(Odell et al., 1985, Nature, 313: 810-812), the rice actin 1 (Zhang et al,
1991,
Plant Cell, 3: 1155-1165) and triosephosphate isomerase 1 (Xu et al, 1994,
Plant
Physiol. 106: 459-467) genes, the maize ubiquitin 1 gene (Cornejo et al, 1993,
15 Plant Mol. Biol. 29: 637-646), the Arabidopsis ubiquitin 1 and 6 genes
(Holtorf et
al, 1995, Plant Mol. Biol. 29: 637-646), and the tobacco translational
initiation
factor 4A gene (Mandel et al, 1995 Plant Mol. Biol. 29: 995-1004). The term
"constitutive" as used herein does not necessarily indicate that a gene under
control of the constitutive regulatory element is expressed at the same level
in all
20 cell types, but that the gene is expressed in a wide range of cell types
even though
variation in abundance is often observed. However, it is to be understood that
development, tissue specific, or inducible regulatory elements may also be
used.
If tissue specific expression of the gene is desired, for example seed, or
leaf specific expression, then regulatory regions or promoters specific to
these
tissues may also be employed. Inducible promoters may also be used in order to
regulate the expression of the gene following the induction of expression by
providing the appropriate stimulus for inducing expression. In the absence of
an
inducer the DNA sequences or genes will not be transcribed. Typically the
protein factor, that binds specifically to an inducible promoter to activate
transcription, is present in an inactive form which is then directly or
indirectly
converted to the active form by the inducer. The inducer can be a chemical
agent
such as a protein, metabolite, growth regulator, herbicide or phenolic
compound
or a physiological stress imposed directly by heat, cold, salt, or toxic
elements or
CA 02427574 2003-05-05
21
indirectly through the action of a pathogen or disease agent such as a virus.
A
plant cell containing an inducible promoter may be exposed to an inducer by
externally applying the inducer to the cell or plant such as by spraying,
watering,
heating or similar methods (e.g. Gatz, C. and Lenk, LR.P.,1998, Trends Plant
Sci.
3, 352-358; which is incorporated by reference). Non-limiting examples, of
potential inducible promoters include, but not limited to, teracycline-
inducible
promoter (Gatz, C.,1997, Ann. Rev. Plant Physiol. Plant Mol. Biol. 48, 89-108;
which is incorporated by reference), steroid inducible promoter (Aoyama, T.
and
Chua, N.H.,1997, Plant J. 2, 397-404; which is incorporated by reference) and
ethanol-inducible promoter (Salter, M.G., et al, 1998, Plant Journal 16, 127-
132;
Caddick, M.X., et a1, l 998, Nature Biotech. 16, 177-180, which are
incorporated
by reference) cytokinin inducible IB6 and CKII genes (Brandstatter, I. and
Kieber,
J.J.,1998, Plant Cell 10, 1009-1019; Kakimoto, T., 1996, Science 274, 982-985;
which are incorporated by reference) and the auxin inducible element, DRS
(Ulmasov, T., et al., 1997, Plant Cell 9, 1963-1971; which is incorporated by
reference).
The chimeric constructs of the present invention may further comprise a 3'
untranslated (or terminator) region. A 3' untranslated region refers to that
portion
of a nucleotide sequence comprising a segment that contains a polyadenylation
signal and any other regulatory signals capable of effecting, or terminating
mRNA processing or gene expression. Non-limiting examples of suitable 3'
regions are the 3' transcribed non-translated regions containing a
polyadenylation
signal of Agrobacterium tumour inducing (Ti) plasmid genes, such as the
nopaline
synthase (Nos gene) and plant genes such as the soybean storage protein genes
and the small subunit of the ribulose-1, 5-bisphosphate carboxylase
(ssRUBISCO)
gene.
By "protein of interest" it is meant any protein that is to be expressed in a
transformed plant. Such proteins may include, but are not limited to,
pharmaceutically active proteins, for example one or more than one of IL-1 to
IL-
24, IL-26 and IL-27, cytokines, contra-inflammatory cytokines, Erythropoietin
(EPO), CSF, including G-CSF, GM-CSF, hPG-CSF, M-CSF, TGF~3, Factor VIII,
CA 02427574 2003-05-05
22
Factor IX, tPA, hGH, receptors, receptor agonists, antibodies,
neuropolypeptides,
insulin, vaccines, growth factors for example but not limited to epidermal
growth
factor, keratinocyte growth factor, transformation growth factor, growth
regulators, antigens, autoantigens including, but not limited to GAD, or a
fragment thereof, their derivatives and the like.
The one or more proteins of interest may be expressed within a plant using
an appropriate vector comprising a nucleotide sequence that encodes a protein
of
interest, and that is capable of being expressed within plant tissue. Such a
vector
may also include ubiquitous, development, tissue specific, or inducible
regulatory
regions and other 5' and 3' regulatory elements as would be known to one of
skill
in the art. Other elements that may be included with this vector include
sequences
for targeting the protein of interest to the cytosol or secretory pathway such
as, but
not limited to, the C-terminal KDEL sequence, an endoplasmic reticulum
retention motif (Schouten et al 1966). Other retention signals, as would be
known
to one of skill in the art, may also be used for this purpose. Furthermore,
such a
vector may include marker genes for the detection of expression within the
transgenic plant, or linker sequences, proteolytic cleavage sequences, and
other
sequences that aid in the purification of a protein of interest. An example,
which is
not to be considered limiting in any manner, of a sequence that aids in the
purification of a protein of interest is an affinity tag, for example,
sequences that
encode a HIS tag. However, it is to be understood that other affinity tags, as
are
known within the art, may also be used for the purpose of purification. An
example, of a sequence that aids in proteolytic cleavage may include, but is
not
limited to a thrombin cleavage sequence. Such a sequence may permit a protein
of
interest to be separated from an attached co-translated sequence, such as, but
not
limited to a KDEL or other sequence.
The present invention provides a nucleotide sequence encoding a cytokine
and an autoantigen. Furthermore, there is provided a vector comprising the
nucleotide sequence encoding a cytokine and autoantigen, wherein the
nucleotide
sequence encoding cytokine is operatively linked to a plant regulatory region
and
a terminator region, and the nucleotide sequence encoding the autoantigen, is
operatively linked to a plant regulatory region and a terminator region. The
CA 02427574 2003-05-05
23
present invention is also directed to a plant, or a portion thereof, plant
cell, or
seed, comprising the nucleotide sequence encoding a cytokine and an
autoantigen.
Preferably, the nucleotide sequence is stably integrated within the genome of
the
plant.
By the term "plant matter", it is meant any material derived from a plant.
Plant matter may comprise an entire plant, tissue, cells, or any fraction
thereof.
Further, plant matter may comprise intracellular plant components,
extracellular
plant components, liquid or solid extracts of plants, or a combination
thereof.
Further, plant matter may comprise plants, plant cells, tissue, a liquid
extract, or a
combination thereof, from plant leaves, stems, fruit, roots or a combination
thereof. Plant matter may comprise a plant or portion thereof which has not be
subjected to any processing steps. However, it is also contemplated that the
plant
material, or matter, may be subjected to minimal processing steps as defined
below, or more rigorous processing, including partial or substantial protein
purification using techniques commonly known within the art including, but not
limited to chromatography, electrophoresis and the like. Any plant may be used
to express the protein described herein, for example but not limited to food-
crop
plants for example alfalfa, corn, wheat, soybean, potato, canola, and non food
crop
plants, such as tobacco or a low-nicotine, low alkaloid tobacco.
By the term "minimal processing" it is meant plant matter, for example, a
plant or portion thereof comprising a protein of interest which is partially
purified
to yield a plant extract, homogenate, fraction of plant homogenate or the
like.
Partial purification may comprise, but is not limited to disrupting plant
cellular
structures thereby creating a composition comprising soluble plant components,
and insoluble plant components which may be separated for example, but not
limited to, by centrifugation, filtration or a combination thereof. In this
regard,
proteins secreted within the extracellular space of leaf or other tissues
could be
readily obtained using vacuum or centrifugal extraction, or tissues could be
extracted under pressure by passage through rollers or grinding or the like to
squeeze or liberate the protein free from within the extracellular space.
Minimal
processing could also involve preparation of crude extracts of soluble
proteins,
since these preparations would have negligible contamination from secondary
CA 02427574 2003-05-05
24
plant products. Further, minimal processing may involve methods such as those
employed for the preparation of F 1 P as disclosed in Woodleif et al ( 1981 ).
These
methods include aqueous extraction of soluble protein from green tobacco
leaves
by precipitation with any suitable salt, for example but not limited to KHS04.
Other methods may include large-scale maceration and juice extraction in order
to
permit the direct use of the extract. As an example, which is not meant to be
limiting in any manner, a composition comprising plant matter, IL-4, and GAD
65
proteins of interest may be partially purified to increase the yield of IL-4
and
GAD in relation to the amount of plant matter.
By "oral administration" it is meant the oral delivery of plant matter to a
subject in the form of plant material or tissue. The plant matter may be
administered as part of a dietary supplement, along with other foods, or
encapsulated. The plant matter or tissue may also be concentrated to improve
or
1 S increase palatability, or provided along with other materials,
ingredients, or
pharmaceutical excipients, as required.
By "medical ailment" it is meant a defined medical condition that can be
treated with a specific pharmaceutical suitable for the treatment of the
condition.
Examples of such medical ailments and their corresponding suitable
pharmaceutical include, but are not limited to: diabetes and the
administration of
IL-4 or insulin or a combination thereof; other autoimmune diseases for
example
rheumatoid arthritis, diabetes and the administration of a cytokine, for
example
IL10, IL-4, GAD, or a combination thereof, allergy treatment and
administration
of IL-12, or direct effects, for example, cytokines may also be used for
topical
delivery in the treatment of psoriasis or for wound healing, for the treatment
of
liver fibrosis or chronic hepatitis C and treatment using IL-10, for
conditions
related to blood clotting with the administration of Factor VIII, Factor IX or
tPA
or combinations thereof; medical conditions requiring the stimulation of
progenitor cells to monocytes/macrophages and the administration of G-CSF,
GM-CSF, hPG-CSF, M-CSF or combinations thereof; viral infections and the
administration of interferons e.g. interferon-a, interferon-13, interferon-y
or
combinations thereof.
CA 02427574 2003-05-05
In an aspect of an embodiment of the present invention, the proteins of
interest comprise one or more cytokines, for example contra-inflamatory
cytokines or interleukins, and one or more autoantigens. By the term
5 "autoantigen" it is meant a compound, such as but not limited to, a protein
of
interest that is administered to a subject that otherwise produces this
protein and
this exogenously administered protein is capable of eliciting an immunological
response following oral administration. Preferably, the immunological response
comprises either reducing or eliminating T lymphocytes and other cells capable
of
10 tissue specific injury, or inducing regulatory T lymphocytes or other cells
which
may lead to reduced destruction of tissues expressing the autoantigen either
by
direct effect or by secondary effect on destructive T lymphocytes or other
cells.
As examples, which are not meant to be limiting in any manner, the present
invention provides a transgenic plant expressing, IL-4, IL-10, or IL-4 and
15 glutamic acid decarboxylase (GAD), or a fragment thereof.
Any cytokine, for example a contra-inflammatory cytokine, interleukin, or
biologically active derivative or variant thereof, and any autoantigen, or
biologically active derivative or variant thereof may be produced in the
transgenic
20 plant of the present invention. The cytokine and autoantigen may comprise
an
amino acid sequence as found naturally in the same or different species.
Further,
the cytokine may be produced in the same plant cells, or tissues, as the
autoantigen, or the cytokine and autoantigen may be produced in different
plant
cells or tissues of the same plant. Similarly, the one or proteins of interest
may be
25 expressed under the control of different regulatory regions that are active
at
different stages of the cell cycles, plant or tissue development, or that are
inducible. Without wishing to be considered limiting in any manner the plant
may
express marine IL-4 and marine GAD 67; marine IL-4 and marine GAD 65;
marine IL-4 and human GAD 65; marine IL-4 and human GAD 67; human IL-4
and human GAD 65; and human IL-4 and human GAD 67.
The present invention further contemplates a composition comprising a
plant-expressed cytokine, for example a contra-inflammatory cytokine, an
autoantigen, or a plant expressed cytokine and autoantigen. In an aspect of an
CA 02427574 2003-05-05
26
embodiment of the present invention, which is not to be considered limiting in
any
manner, the composition may comprise plant-expressed IL-10, plant expressed IL-
4, plant expressed GAD, or plant expressed IL-4 and GAD. The IL-10, IL-4, and
GAD may comprise an amino acid sequence which is identical to any IL-10, IL-4,
and GAD amino acid sequence, respectively, as known in the art. Further, the
invention contemplates variations and derivatives of IL-10, IL-4 and GAD that
comprise biological activity. The IL-10, IL-4, and GAD may be produced
together in a single plant or they may be produced in separate plants.
For example the composition of the present invention may comprise a
combination of autoantigen such as GAD from one plant, combined with a plant
expressed adjuvant cytokine such as IL-4, or IL-10 from a second plant.
Production of these proteins in separate plants allows control of the
composition
mix in terms of relative proportions of autoantigen to cytokine. For example,
which is not to be considered limiting, the composition of the present
invention
may comprise a combination of partially purified IL-4 from a first plant and
GAD
from a second plant comprising associated plant matter, or the composition may
comprise IL-4 and GAD obtained from separate plants following purification of
these proteins.
Alternatively, the expression vector used for the transgenic plants may
comprise nucleic acid sequences that express both the autoantigen or
transplantation antigen, and a cytokine or a number of cytokines. Such a
vector
may be used to develop a single plant expressing several proteins of interest.
Similarly, a plant expressing the autoantigen or transplant antigen may be
crossed
using techniques as known in the art, with a plant expressing one or more than
one
cytokines in order to produce a plant expressing both an autoantigen or
transplant
antigen and one or more than one cytokines.
Plants of the present invention may also be useful in oral application to the
gut for modulation of immune responses in the absence of a concurrently
administered autoantigen. An example of such a case, where an autoantigen may
not be required relates to inflammation, for example but not limited to
inflammatory bowel disease (IBD), by which the direct topical application to
the
CA 02427574 2003-05-05
27
gut of anti-inflammatory cytokines mauy be used to prevent and treat disease.
Cytokines such as interleukin-10 and interleukin-4 may be used for this
purpose,
however, other cytokines and growth factors growth factors which have a direct
protective effect on enterocyte epithelial cell survival in any inflammatory
condition as would be determined by one of skill in the art may also be used.
The present invention provides a composition comprising a cytokine or
growth factor, an autoantigen or transplantation antigen, or a combination
therof.
This composition may be used for a variety of purposes, for example but not
limited to, augment the tolerizing capacity of an autoantigen, either to
improve the
tolerance effect of low level expression of autoantigen, or to maximize the
effect
by inducing regulatory cells or deleting harmful cells, or by benefiting oral
tolerance by skewing immune responses at the time of antigen presentation. The
composition may also be used to deliver cytokines, growth factors or other
compounds expressed within the plant delivery system to attenuate or abrogate
inflammatory conditions as would be found in inflammatory bowel disease but
also graft-versus-host disease following transplantation, or as a result of
inflammation secondary to injury caused by infection or physical injury of
epithelial and other parenchymal cells of the gut or skin.
Without wishing to be bound by theory, the use of endogenous proteins
such as cytokines as adjuvants may augment oral immune tolerance by skewing T
helper responses to a less harmful subset. However, these proteins would not
induce neutralizing immune responses since they are endogenous proteins.
Neutralizing immune responses may be observed with the use of foreign
proteins,
for example, mucosal immune adjuvants such as cholera toxin B subunits
(Arakawa T, et al., 1998, Nat Biotechnol. 16:934-8), where with continued
exposure, immune responses and antibodies will be directed against the cholera
toxin B subunits and lead to neutralization of any beneficial effect thus
preventing
long term oral tolerance induction.
The immune response at the time of initial antigen presentation to T
lymphocytes is influenced by the presence of cytokines which act as potent
biological response modifiers at the time of initial activation. Cytokines
such as
CA 02427574 2003-05-05
28
IL-4 and IL-10 may shift T cell responses towards T helper 2 (Th2) subsets,
which
have been shown to be of benefit in autoimmune diseases mediated by
autoaggressive Th-1 helper (Thl) subsets. Alternatively, Th-1 cytokines (eg.
interferon gamma, IL-2, IL-12) might skew the T helper subset to Th-1
responses,
and increase responses which may be useful to augment endogenous protective
responses to pathogens for example, within the gut and skin. Therefore, the
present invention provides plants) expressing both regulatory cytokines and an
autoantigen(s) or transplantation antigens) of interest, as these compositions
would maximize the effect of antigen specific oral immune tolerance.
The use of plants as an expression system for the production of
mammalian antigen proteins offers unique advantages beyond high production
yields at competitive low cost. Immune tolerance requires activation events
within T lymphocytes, and plants that include lectins, in addition to other
compounds, may facilitate such activation events. While the specificity of
oral
immune tolerance depends on antigen selection, the magnitude of response may
be influenced by expression levels of the transgenic protein of interest
within a
plant. It can also be influenced by the presence of immune adjuvants or
response
modifiers present within the composition. Plant lectins for example expressed
within the cell walls of plant cells may generally augment the immune response
by direct activation of T cells. Additionally, the accumulation of transgenic
proteins within plant cell compartments may become protected from degradation
in the digestive tract (Kong et al, 2001, P.N.A.S. 98:11539-11544). This would
augment oral immune tolerance by delivering transgenic plant protein to immune
effector sites more in the gut distal to the digestive environment of the
stomach
and duodenum.
Furthermore, plants may also be useful for direct, oral application of a
cytokine to the gut for modulation of immune responses in the absence of a
concurrently administered autoantigen, for example as in the cases of
inflammation such as in the case of inflammatory bowel disease (IBD). In this
example, the direct topical application to the gut of anti-inflammatory
cytokines
would prevent and treat disease. This approach would include the use of
cytokines such as IL-10 and IL-4 but may also include other cytokines and
growth
CA 02427574 2003-05-05
29
factors, or growth factors that have a direct protective effect on enterocyte
epithelial cell survival in any inflammatory condition.
Inflammatory bowel disease (IBD) represents a spectrum of diseases with
over-lapping clinical presentations, although ulcerative colitis and Crohn's
colitis,
the most widely represented and serious IBD in North America have
characteristic
features that aid diagnosis. Treatments currently used are often inadequate,
poorly
tolerated, associated with serious adverse effects and expensive. It is known
that
the human contra-inflammatory cytokine interleukin-10 (IL10) may have
application in the treatment of inflammatory bowel disease (Leach et al. 1999.
Tox. Path. 27:123-133). IL-10 given systemically has been shown to be of
benefit
in rodent models of inflammatory bowel disease, a similar but less modest
effect
was also observed in human clinical trials (Fedorak et al. 2000. Gastroent.
119:1473-1482; Schreiber et a1. Gastroent. 119:1461-1472). The magnitude of
the
therapeutic benefit in humans was limited by systemic toxicity as patients
treated
with higher amounts of IL-10 demonstrated a reduction in both hemoglobin and
platelet levels. Without wishing to be bound by theory, direct application of
cytokines through oral administration of plant material expressing cytokines
may
avoid this systemic effect in a manner similar to that observed with the
topical
application of drugs.
One new method demonstrated in animal models of Crohn's disease was
intra-gastric inoculation of an animal model of Crohn's disease with
genetically
engineered Lactococci that secrete recombinant IL10 (Steidler et al. Science.
289:1352-1355). The treatment was effective in the test animals, although its
application in humans may be limited because of the containment problems that
would result from the presence of heavy loads of genetically engineered
bacteria
in human faeces. Oral administration of recombinant cytokines has been
suggested and may be a possibility for IL10 (Rollwagen and Baqar 1996.
Immunol. Today 17:548). Although there are concerns about digestive
degradation of oral cytokines, there is evidence that IL10 is biologically
active at
mucosal surfaces, at least when administered with an autoantigen and when
related to oral tolerance (Slavin et al. 2001. Int. Immunol. 13:825-833). The
CA 02427574 2003-05-05
problem that remains is the delivery of a sufficient dose of recombinant IL10
to
lesions in the GI tract so that a direct contra-inflammatory effect is
created.
Crohn's disease is a chronic inflammatory bowel disease with frequent
5 remissions and exacerbations, and several extra-GI manifestations. Lesions
are
typically discontinuous and can occur anywhere within the Gl tract. The most
common locations are ileum and colon, and formation of strictures and fistulas
are
common. In contrast, ulcerative colitis is contiguous and characteristically
affects
the colon. While the etiology of both of these inflammatory bowel diseases
10 remains unknown, there is data demonstrating the participation of immune
cells in
these conditions. Cytokines may direct the intensity, extent, and eventual
outcome
of inflammatory lesions. Interestingly, mice made deficient for IL-10, by
targeted
gene disruption, develop bowel inflammation and lesions resembling
inflammatory bowel disease, which can be improved by systemic administration
15 of IL-10 (Kahn et al. 1993). The balance between proinflammatory cytokines
such
as interleukin-l, interleukin-6, y-interferon and tumor necrosis factor (TNF)
and
anti-inflammatory cytokines such as receptor antagonists, IL-10, IL-4, and
transforming growth factor beta (TGF-Vii) might ultimately determine the
outcome
of inflammatory bowel disease (Fiocchi 1993). By its nature in down regulating
20 pro-inflammatory cytokines, for example TNF-a and up regulation of
interleukin-
1 receptor antagonists, IL-10 may be an effective strategy to treat
inflammatory
bowel disease. The present treatments for inflammatory bowel disease are
empirically designed to reduce inflammation and rely heavily on
corticosteroids
and immuno-solisolates (5-ASA).
Human IL-10 has been cloned, and both mouse and human cDNA encode
functionally and structurally similar proteins (with 178 amino acids)
including a
hydrophobic leader sequence and greater than 70% homology in predicted amino
acid sequences. Interleukin-10 (IL-10) exists in both monomeric and
homodimeric
forms. Biological activity is known to be associated predominantly twith the
homodimeric form of IL-10.
CA 02427574 2003-05-05
31
In addition to production by CD4+ TH-2 clones, IL-10 is produced by B
cells, activated mast cells, macrophages, monocytes and keratinocytes.There
are
multiple roles for IL-10 in the regulation of the immune response, including
inhibition of T cell, monocyte and macrophage function, as well as inhibition
of
cytokines particularly those of the TH-1 T cell subset (interferon gamma) and
inhibition of B cell proliferation. The inhibition of various cytokines such
as
interferon gamma, GM-CSF and TNF following lectin or anti-CD3 activation is
both transcriptional and translational. There is also inhibitory activity on
MHC
class 11 antigen expression, which is a protein central to the recognition of
antigen
presenting cells by T-cell. IL-10 also inhibits the production of hydrogen
peroxide
and nitric oxide following interferon-gamma activation. Therefore there is a
strong basis for the concept that IL-10 naturally occurs to provide negative
regulation to immune responses.
Glutamic acid decarboxylase (GAD), is an enzyme which is found in brain
as well as pancreatic islets. There are several isoforms including GAD65
(molecular weight of 65kDa) and GAD67 (molecular weight of 67kDa ), which
are differentially expressed in various species, within pancreatic islets.
Both
isoforms are implicated in the pathogenesis of IDDM, as immune responses to
GAD appear prior to the onset of clinical disease, and strategies used to re-
establish tolerance to both isoforms of GAD prevent disease in mammals, for
example mice. Fragments of GAD are also known to and assist in oral tolerance
(see for example: US 5,837,812; US 6,211,352; WO 96/26218; US 5,645,998; US
5,762,937; US 6,001,360; US 5,475,086; US 5,674,978; US 5,705,626; US
5,846,740; US 5,998,366; US 6,011,139; all of which are incorporated herein by
reference). Therefore, the present invention contemplates the use of full
length
GAD, fragments thereof, or a combination of full length GAD and fragments
thereof.
The immune response at the time of initial antigen presentation to T
lymphocytes is influenced by the presence of cytokines which act as potent
biological response modifiers at the time of initial activation. Without
wishing to
be bound by theory, contra inflammatory cytokines, for example but not limited
to
IL-4, IL-10, or TGF-(3 may shift T cell responses towards T helper 2 (Th2)
CA 02427574 2003-05-05
32
subsets, which have been shown to be of benefit in autoimmune diseases
mediated
by autoaggressive Th-1 helper (Thl) subsets. Alternatively, Th-1 cytokines
(eg.
interferon gamma, IL-2, interleukin-12) might skew the T helper subset to Th-1
responses, and increase responses which may be useful to augment endogenous
protective responses to pathogens for example, within the gut and skin.
Therefore
plants) expressing both regulatory cytokines and an autoantigen(s) or
transplantation antigens) of interest, would maximize the effect of antigen
specific oral immune tolerance.
IL-4 is a growth and differentiation factor for T-cells. In particular, IL-4
promotes the development of the subset of T helper (Th2) cells from native T
cells
upon antigen stimulation. IL-4 producing Th2 cells generally protect against
the
onset of many organ-specific autoimmune diseases, including type 1 diabetes.
Thus the availability of an abundant source of IL-4 may prove invaluable for
the
immunotherapy of diabetes.
Plant produced IL-4 is biologically active, for example as demonstrated by
the stimulation of mast cell growth in the presence of IL-4 (Figure 3). In
this
example, plant recombinant IL-4 exhibits the same effect as commercially
available IL-4, and IL-4 from other sources. Similarly, plant produced IL-10
is
also biologically active, for example as demonstrated in the inhibition of LPS-
induced secretion of IL-6 by macrophage/monocyte cells (Figure 8). This
example demonstrates that plant recombinant IL-10 exhibits the same biological
activity as recombinant insect IL-10, or IL-10 from other sources.
Therefore, the present invention provides a method of producing a
cytokine and an autoantigen comprising,
i) providing, or transforming, a plant, or portion thereof with a nucleotide
sequence encoding the cytokine and the autoantigen;
ii) expressing the cytokine and the autoantigen in the plant, and if desired;
iii) harvesting the plant or portion thereof.
Furthermore, after the step of harvesting, the cytokine and autoantigen may be
obtained from the harvested tissue.
CA 02427574 2003-05-05
33
The present invention also provides a method of producing a cytokine and
an autoantigen comprising,
i) providing, or transforming, a first plant, or portion thereof with a first
nucleotide sequence encoding the cytokine;
ii) providing, or transforming, a second plant, or portion thereof with a
second nucleotide sequence encoding the autoantigen;
iii) crossing the first and second plant to produce a dual transgenic plant;
iv) expressing the cytokine and autoantigen in the dual transgenic plant;
and if desired
v) harvesting the dual transgenic plant, or a portion thereof.
Furthermore, after the step of harvesting, the cytokine and autoantigen may be
obtained from the harvested tissue.
The present invention also provides a method of producing a cytokine and
an autoantigen comprising,
i) providing, or transforming, a first plant, or portion thereof with a first
nucleotide sequence encoding the cytokine;
ii) providing, or transforming, a second plant, or portion thereof with a
second nucleotide sequence encoding the autoantigen;
iii) expressing the cytokine and autoantigen in the first and second plant;
iv) harvesting the first and second plant, or a portion thereof to obtain
harvested tissue; and
v) combining the harvested tissue to produce a composition comprising a
cytokine and an autoantigen.
Furthermore, after the step of harvesting, the cytokine and autoantigen may be
further processed prior to combining, for example by purifying, or partially
puroifying the cytokine, the autoantigen, or both the cytokine and the
autoantigen.
The steps of transforming and expressing may be performed in any manner
as described within the art and may be readily practised by a person of skill
in the
art. For example, the constructs of the present invention can be introduced
into
plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA
transformation, micro-injection, electroporation, etc. For reviews of such
techniques see for example Weissbach and Weissbach, Methods for Plant
CA 02427574 2003-05-05
34
Molecular Biology, Academy Press, New York VIII, pp. 421-463 (1988);
Geierson and Corey, Plant Molecular Biology, 2d Ed. (1988); and Miki and Iyer,
Fundamentals of Gene Transfer in Plants. In Plant Metabolism, 2d Ed. DT.
Dennis, DH Turpin, DD Lefebrve, DB Layzell (eds), Addison Wesly, Langmans
Ltd. London, pp. 561-579 (1997). It is preferred that the nucleotide sequences
encoding the proteins of interest, are stably integrated within the genome of
the
plant.
Furthermore, the plant may be any plant known in the art, for example, but
not limited to food-crop plants for example alfalfa, corn, wheat, soybean,
potato,
canola, and non food crop plants, such as tobacco. If the plant is a tobacco
plant,
it is preferred that the tobacco plant is a low-nicotine, low alkaloid tobacco
plant.
Following the step of harvesting, the plant, or portion thereof, may be
orally administered to a subject, or the plant, or portion thereof, may be
partially
purified as described previously prior to being administered to a subject.
The present invention also provides a method of treating, or preventing
diabetes in a subject. The method comprises administering to a subject a plant
or
more than one plant expressing an autoantigen, for example GAD, with a
cytokine, for example, but not limited to IL-4 or IL-10, either singly or in
combination, to augment oral immune tolerance to the autoantigen. Any IL-4, IL-
10, or GAD may be used provided that the protein exhibits IL-4, IL-10 or GAD
activity, respectively. However, it is well known in the art that fragments of
GAD
are biologically active, or that fragments of GAD assist in oral tolerance
(see for
example: US 5,837,812; US 6,211,352; WO 96/26218; US 5,645,998; US
5,762,937; US 6,001,360; US 5,475,086; US 5,674,978; US 5,705,626; US
5,846,740; US 5,998,366; US 6,011,139; all of which are incorporated herein by
reference), therefore the present invention also pertains to a method of
treating, or
preventing diabetes in a subject comprising administering to a subject a
plant, or
more than one plant, expressing an autoantigen, for example GAD or a fragment
thereof, with a cytokine, for example IL-4 or IL-10, either singly or in
combination.
CA 02427574 2003-05-05
Preferably the IL-4, IL-10, GAD, or a fragment of GAD, comprise amino
acid sequences which are substantially identical to the amino acid sequences
of
the proteins in the subject being treated. By substantially identical it is
meant that
the administered amino acids sequences exhibit from about 95% to about 100%
identity with the subject sequence. Preferably, the administered sequence
exhibits
100% identity with the subject sequence. Thus, as will be evident to someone
of
skill in the art, preferably a human IL-4 is employed with human GAD to treat,
prevent or both treat and prevent diabetes in a human subject.
10 Homology determinations may be made as known in the art using
oligonucleotide alignment algorithms for example, but not limited to a BLAST
(GenBank URL: www.ncbi.nlm.nih.gov/cgi-bin/BLAST/, using default
parameters: Program: blastp; Database: nr; Expect 10; filter: default;
Alignment:
pairwise; Query genetic Codes: Standard(1)) or FASTA, again using default
15 parameters.
The present invention provides a vector comprising a nucleotide sequence
encoding an cytokine, a autoantigen or both a cytokine and an autoantigen, and
operativly linked with one, or more than one regulatory regions active in a
plant.
20 An example of the cytokine is an interleukin, for example any one of IL1 to
IL24,
IL-26 or IL-27, or a contra-inflammatory cytokine, for example but not limited
to
IL-4, IL-10 or TGF-(3. Preferably, the interlekin is a member of the IL-10
family
(Fickensher, 2002, Trends in Immunology 23: 89), which includes members that
have conserved helical structures of a homo-dimer, but variable receptor
binding
25 residues which alter their function. Examples of members of the IL-10
family
include IL-10, IL-19, IL-20, IL-22, IL-24 and IL-26. More preferably the
interleukin is IL-4 or IL10. An example of the autoantigen is GAD, or a
fragment
of GAD that is biologically active, or functional in assisting oral tolerance.
The
vector may further comprise a second nucleotide sequence fused to nucleotide
30 sequence, the second nucleotide sequence encoding, an enzyme cleavage site,
an
endoplasmic reticulum retention signal, an affinity tag to aid purification of
said
protein of interest, or a combination thereof. Preferably the endoplasmic
reticulum retention signal comprises the amino acid sequence KDEL. The present
CA 02427574 2003-05-05
36
invention is also directed to a plant comprising the vector, and to plants
expressing
the nucleotide sequence as defined above.
Therefore, the present invention also provides a composition comprising a
plant-expressed cytokine, and an autoantigen. The plant-expressed cytokine can
a
contra-inflammatory cytokine, for example IL-4, IL10, or TGF-Vii. Preferably,
the
contra-inflammatory cytokine, is IL-4. Furthermore, the autoantigen, can be
GAD, or a fragment thereof.
The present invention also provides a method of eliciting an immune
response against an antigen in a subject, the method comprising orally
administering a plant or portion thereof comprising an antigen and an
interleukin
to the subject. The antigen may be an autoantigen, for example, but not
limited to
GAD, or a fragment thereof. However, other autoantigens are also contemplated.
Further, the interleukin may comprise IL-4, but may be one or more other
interleukins. In an embodiment that is not meant to be limiting in any manner,
the
autoantigen is GAD, or a fragment thereof, and the interleukin is IL-4.
Preferably
the autoantigen and interleukin comprise sequences that are naturally present
in
the subject.
The present invention further contemplates a method of treating
inflammatory bowel disease in a subject, the method comprising, administering
a
plant, or portion thereof comprising IL-10 to said subject. The inflammatory
bowel disease may comprise ulcerative colitis or Crohn's colitis, but is not
limited
to these inflammatory bowel diseases.
The present invention also provides a use of a plant, or a first and second
plant comprising a cytokine and an autoantigen for the production of a
medicament for the treatment of a disease state in an animal, for example but
not
limited to a mammal, including a human. If the cytokine and autoantigen are
produced in separate plants, then the plant material comprising the cytokine
and
autoantigen may be combined for use in preparing the medicament. Similarly,
extracts, either crude extracts comprising plant material, partially purified
extracts
CA 02427574 2003-05-05
37
or purified extracts, may be obtained from plants expressing a cytokine, and
an
autoantigen, and these extracts may be combined for preparing the medicament
comprising a cytokine and an autoantigen. The cytokine can be selected from
the
group consisting of IL-4, IL-10, and TGF-(3, and the autoantigen is glutamic
acid
decarboxylase (GAD), or a fragment thereof.
Refernng now to Figure 1, there is shown an immunoblot of Nicotiana
tabacum cv. SR1 tobacco plants transformed with a plant expression vector
comprising the human GAD65 gene. The results show that transgenic plants
producing human GAD65 exhibit a single protein band that is of the correct
size.
Further, the expression level of GAD65, estimated from blot densitometry was
about 0.04% by weight of the total soluble protein. However, the level of
expression may be modulated in plants as would be evident to someone of skill
in
the art, for example, but not limited to, through the use of promoters with
different
activities, or by other accessory nucleotide sequences such as but not limited
to
enhancer, repressor, ER retention, and signal, sequences.
SR1 tobacco is provided as a non-limiting example of a plant that can be
used to express a protein of interest as described herein, for example but not
limited to GAD65. SR1 is a wild type tobacco plant with respect to nicotine
expression, however, GAD65, and other proteins as described herein may be
expressed in low nicotine tobacco as described below. The use of low nicotine
tobacco may be desired if plant matter expressing one or more than one
proteins
described herein is to be orally administered.
Refernng now to Figure 2, there is shown an immunoblot of proteins
extracted from Nicotiaha tabacum cv. SR1) transformed with expression vectors
comprising a nucleotide sequence encoding IL-4. The results indicate that
transgenic IL-4 may be produced in plants. The immunoblot analysis exhibits a
unique protein band recognized by a monoclonal antibody specific for mouse IL-
4
which is comparable in size to standard recombinant mouse IL-4. Further, the
expression level of plant-derived recombinant murine IL-4 (mIL-4), as
determined
by IL-4-specific ELISA, was found to be about 0.02% by weight of the total
leaf
CA 02427574 2003-05-05
38
protein. However, the level of expression may be modulated in plants as would
be
evident to someone of skill in the art, for example, but not limited to,
through the
use of promoters with different activities, or by other accessory nucleotide
sequences such as, but not limited to enhancer, repressor ER retention, and
signal
sequences.
Referring now to Figure 3, there is shown results following stimulation of
a mast cell line with plant recombinant IL-4. As shown in Figure 3,
proliferation
of mast cells occurred with plant recombinant IL-4 demonstrating that plant-
derived recombinant IL-4 is biologically active, and that it may be used as an
immunomodulator in oral tolerance.
Transgenic tobacco leaf tissues comprising GAD and IL-4 were added to
the feed of non-obese diabetic (NOD) mice in an amount of up to about 12%
1 S (w/w) of their diet. The amount of IL-4 was estimated to be about l0,ug to
about
12 ,ug per mouse daily, whereas the amount of human GAD65 was estimated to be
about 8~g to about 10 ~cg per mouse daily. NOD mice receiving a composition of
GAD65 and IL-4 produced within tobacco, exhibited a decrease in the incidence
of developing diabetes compared with mice fed plant-produced GAD65, plant-
produced IL-4, or plant matter comprising an empty vector. These results are
described further in Example 6. The addition of GAD with IL-4 reduced the
incidence of diabetes and as well reduced the severity of insulitus. However,
also
of clinical significance is the prevention of diabetes, as in the case of
inducing
protection with regulatory cells. Insulitus may still be a prominent feature
in
protected mice. Therefore the invention as described does not depend on the
reduction of peri-islet infiltrates, as these infiltrates if present may not
be
destructive, and may represent T cells producing Th2 cytokines themselves.
IL-4 is known to mediate immunoglobulin class switching to IgE
production (Ryan, 1997), and such an event is thought to be associated with
the
induction of an allergic response. As described in Example 6, oral immune
tolerance with Th2 response skewing did not induce IgE response in the mouse
models tested, either with autoantigen GAD or with IL-4.
CA 02427574 2003-05-05
39
To examine whether the protection from diabetes in NOD mice fed both
GAD and IL-4 transgenic plants may be associated with the induction of
GAD-specific regulatory T cells, the ability of T cells from treated mice to
inhibit
S the adoptive transfer of diabetes was tested. As shown in Figure 4, SO% of
the
mice receiving a mixture of splenic mononuclear cells from GAD+IL-4 treated,
diabetic mice developed diabetes (IDDM) at the end of 8 weeks post transfer,
while 100% of the mice receiving only diabetic splenic cells developed IDDM.
These data demonstrate that regulatory cells are only increased in those mice
in
which GAD and IL-4 are used concurrently. These regulatory cells can prevent
the full diabetogenic effect of spleen cells taken from mice with new onset
diabetes. These results also suggest that oral administration of plant
produced
GAD with interleukin-4 or other cytokines could possibly induce regulatory T
cells in those not yet having overt diabetes, and in this pre-diabetic
condition,
1 S regulatory cells may prevent the full onset of disease by blocking an
activated
effector T cell population. The delivery of GAD with IL-4 or other cytokines
may
also lead to protection by deletional mechanisms, with removal of autoreactive
T
cells, along with increased expression of regulatory T cells.
Thus, the present invention contemplates a method for inducing regulatory
T-cells against an autoantigen in a subject, comprising orally administering a
composition comprising IL-4 and an autoantigen. Further, there is provided a
method of inducing regulatory T-cells against GAD in a subject, comprising
orally
administering a plant comprising IL-4 and GAD, or a fragment thereof, or a
composition comprising plant produced IL-4 and GAD, or a fragment thereof. The
subject may be an animal or any mammalian subject but is preferably human. In
the latter embodiment, preferably the IL-4 is human IL-4, and GAD is human
GAD 67, GAD 6S, or a combination thereof, or an active fragment of GAD 67,
GAD 64S or a combination thereof .
The present invention also provides a plant or portion thereof expressing
IL-4, IL-10, GAD, or a combination thereof. The plant may be any plant known
in
the art for example but not limited to, alfalfa, wheat, corn, soybean, canola,
potato
and tobacco. If the plant is a tobacco plant, the tobacco plant may be a low-
CA 02427574 2003-05-05
alkaloid plant, or a low-nicotine tobacco plant. For example, the plant can be
a
low nicotine low alkaloid tobacco plant, such as, but not limited to, 81V-9 or
SR1.
A low nicotine low alkaloid tobacco plant is an example of a non-food crop
plant.
S From both a regulatory and public safety stand point non-food crop plants
are ideal species for the transgenic production of biologically active
proteins since
the risk of leakage of transgenic plant material into the human food chain is
negligible. Even though non-food crop plants are ideal species for use as
bioreactors from both a regulatory and public safety point of view, there are
10 several obstacles that must be overcome prior to their use as bioreactors.
A potential drawback with the use of non-food crop plants, for example
tobacco, as bioreactors is that these plants may contain undesirable secondary
plant products. Secondary plant products are constituents that are generally
toxic
15 or reduce the palatability of the plant tissue or that are addictive in
nature. This is
a significant concern since one of the benefits of preparing proteins of
interest
within a plant is that large quantities of protein may be required for
administration
(Ma and Hein, 1995). Therefore, any toxic, addictive, or otherwise non-
desirable
products should be avoided within the plant tissue. For example, tobacco
plants
20 contain high levels of secondary plant products such as nicotine and
related
alkaloids, making the plant tissue generally unsuitable for the direct oral
administration. Earlier studies have described the administration of tobacco-
derived proteins to mice, however, the proteins were in a partially purified
form.
For example, the study by Mason et al (1996) involved the direct oral
25 administration of viral antigens expressed in potato tuber and tobacco. The
potato
tuber samples were directly fed to mice, yet the antigen, when obtained from
tobacco, had to be partially purified using sucrose gradients prior to
administration
to mice. It is desirable to prepare transgenic proteins within non-food crop
plants
that permit the direct use of plant tissue for oral administration, or
following
30 minimal processing.
By "non-food crop plant" it is meant a plant that has traditionally not been
used, or grown for human food purposes. Examples of non-food crop plants
include, but are not limited to, ornamental plants, tobacco, hemps, weeds,
trees
CA 02427574 2003-05-05
41
and the like. It is contemplated that such non-food crops plants may be used
for
the production of one or more proteins of interest. In order that the non-food
crop
plant be used as a bioreactor for the production of one or more transgenically-
expressed proteins of interest suitable for oral administration with minimal
or no
processing, it is preferably that such a non-food crop plant is characterized
as
being:
a) non-toxic;
b) palatable following minimal or no processing; and
c) characterized as having low levels of non-desirable secondary plant
products such as alkaloids, nicotine, or the like.
However, if it is desired that the protein of interest is not to be orally
administered, then any plant may be used for the production of a protein of
interest, following the methods of the present invention, as described herein.
In
such applications where there is no need for orally administering the protein
of
interest, the protein of interest may be combined with other nucleic acid
sequences
that are useful for the purification of the protein of interest in order that
the protein
be suitable for further use.
Use of the terms "low nicotine" and "low alkaloid" with reference to
plants, such as, but not limited to tobacco, means a plant that contains a
significantly lower concentration of alkaloids when compared with a similar
plant
comprising regular alkaloid content. For example, which is not to be
considered
limiting in any manner, a low alkaloid tobacco plant may be defined as a plant
that
contains less than from about 0.2% to about 70 % of the total alkaloids
present in
plants comprising a regular level of alkaloids. More preferably, a low-
alkaloid
plant is one that contains about 0.2% to about 10% of the total alkaloids
present in
plants comprising a regular level of alkaloids. An example of a low alkaloid
plant, that comprises about 2.8% of the total alkaloid level of a regular
tobacco, is
81 V-9 (see Table 1, Example 4). Similarly, a low nicotine tobacco plant is
one
that contains a significantly lower concentration of nicotine when compared
with
a similar plant comprising regular nicotine content. For example, which is not
to
be considered limiting in any manner, a low nicotine tobacco plant may be
defined
as a plant that contains less than from about 0.2% to about 70 % of the total
CA 02427574 2003-05-05
42
nicotine present in plants comprising a regular level of nicotine. More
preferably
a low-nicotine plant is one that contains from about 0.2% to about 10% of the
total
nicotine present in plants comprising a regular level of nicotine. An example
of a
low nicotine plant, that comprises about 2.6% of the total nicotine level of
regular
S tobacco, is 81 V-9 (Table 1, Example 4). Conversely, an example of a tobacco
plant which may be considered to comprise a regular level of alkaloids and
nicotine is Delgold tobacco plants.
Low alkaloid or low nicotine plants may be obtained through conventional
breeding programs (e.g. Chaplin, 1977), through selective down regulation of
undesired genes (e.g. U.S. 5,260,205, issued November 9, 1993 and U.S.
5,369,023, issued November 29, 1994; inventors Nakatani and Malik), or by any
other means e.g. mutagenesis followed by selection of desired traits. However,
as
is known to one of skill in the art, the ultimate nicotine or alkaloid level
may still
1 S depend upon the environment under which the plant is grown.
Several methods have been proposed for the removal of nicotine from
tobacco, however, these processes typically involve the treatment of post
harvest-
tissue. For example the use of solvents (EP 10,665, published May 14, 1980;
inventors Kurzhalz and Hubert), or potassium metabisulphite, potassium
sulphate
and nitrate (U.5. 4,183,364, issued January 8, 1980; inventor Gumushan) have
been proposed as methods for removing nicotine from tobacco leaves. All of
these treatments are designed to maintain the flavour and aroma of the
tobacco,
and these methods involve extensive post-harvest processing and are therefore
not
suitable for the preparation of products as described in this invention.
Specific alteration of nicotine levels within tobacco, by either over
expression (i.e. increasing) or antisense expression (decreasing) of
putrescine N-
methyltransferase, a rate limiting enzyme involved in the nicotine
biosynthetic
pathway, has been suggested (U.5. 5,260,205, issued November 9, 1993 and U.S.
5,369,023, issued November 29, 1994; inventors Nakatani and Malik). These
methods are directed to the alteration of nicotine levels so that the levels
of other
alkaloids that affect the flavours and aroma of tobacco are not modified in
any
manner. No nicotine free plants were actually produced, nor was the use of
these
CA 02427574 2003-05-05
43
transgenically modified tobacco plants, as a bioreactor for the synthesis of
proteins of interest, suggested. However, such methods, if modified to reduce
total
plant alkaloid levels, may be used in order to produce non-food crop plants
wherein total alkaloid levels are reduced.
In order to establish the efficacy of transgene expression in a low-alkaloid
plant, a low-nicotine plant, or a low-nicotine low-alkaloid plant, such as,
but not
limited to 81 V-9, several proteins of interest were expressed. These proteins
included, but are not limited to IL-4, IL-10, GAD, and a Type II antifreeze
protein
(AFP). However, any protein of interest may be expressed in a low-nicotine,
low-
alkaloid tobacco plant.
Administration of cytokines orally may offer an alternative to systemic
application or topical application and may have local and systemic effects.
Interestingly acid inactivation and protease digestion does not appear to
inactivate
all cytokine effects, in the limited experience to date using an oral
approach.
Bioactivity may be retained even in the presence of trypsin and chymotrypsin,
at
least for IL-6, suggesting heavy glycosylation and other factors confer acids
stability to cytokines (Rollwagen and Baqar 1996). Furthermore, interferons
can
suppress collagen induced arthritis in rats when given in high dose orally.
Oral
cytokines may simplify an approach to delivery and could be useful in models
of
infection, HIV, autoimmunity and transplantation rejection. Transgenic plants
may also protect a transgenic protein of interest that is administered orally,
providing greater resistance to gastrointestinal digestion (e.g. Kong et al.,
2001,
P.N.A.S. 98:11539-11544). This protection includes the "encapsulation" of
transgenic proteins within cytoplasmic micro compartments, as well as the
presence of plant glycosylation and other factors which confers as stability
to
functional proteins such as cytokines and as well the other components of the
plant tissue which may further enable the transgenic proteins of interest to
reach
heights within the GI tract that are responsible for immune modulation, or to
reach
those sites within the GI tract that are undergoing inflammation such as in
inflammatory bowel disease.
CA 02427574 2003-05-05
44
Gene constructs were transformed into N. tabacum cv. Xanthi and 81 V9-4
tobacco strains (available from Agricuture and Agri-Food Canada, Pest
Management Research Center, Delhi Farm, Genetics Section) . 81 V9-4 is a flue-
cured tobacco line containing only trace amounts of alkaloids and is an ideal
component for tobacco-based molecular farming applications. Gene constructs
were built for production of pro and mature forms of a Type II antifreeze
protein
(AFP), IL-4, IL-10 or GAD to be accumulated in the cell cytosol and
extracelluar
space. Several different constructs comprising IL-10 were constructed in order
to
optimize expression of the gene within a non-food crop plant.
Using the methods described in Example 3, low-nicotine, low-alkaloid
tobacco plants expressing plant recombinant human interleukin-10 (prIL-10)
comprising a (thrombin)-(His tag)-(KDEL) sequence were created. Refernng now
to Figure 5, there is shown results of the expression of human interleukin-10
(hIL-
10) in plants. Figure 5A and Figure 5B show the quantity of IL-10 secreted to
the
apoplast following transformation of tobacco plants with nucleotide sequences
comprising either a long hIL-10 3'UTR region or a short hIL-10 UTR region
respectively. Figure SC shows the quantity of plant recombinant IL-10
expressed
in transgenic tobacco plants transformed with an IL-10 nucleotide sequence
which
further comprises a KDEL endoplasmic reticulum retention signal. The results
indicate that human IL-10 may be produced in plants, such as, but not limited
to
low-nicotine, low-alkaloid tobacco plants.
Referring now to Figure 6, there is shown an immunoblot following non
denaturing SDS-PAGE of recombinant human IL-10 (hIL-10) produced in insect
cells and plant recombinant IL-10 (prIL-10). The results indicate that plant
recombinant IL-10 is correctly assembled into dimers. The difference between
the
migration of the prIL-10 andhIl-10 is due to the presence of the (thrombin)-
(His
tag)-(KDEL) sequence in the prIL-10 protein.
Refernng now to Figure 7, there is shown results of digesting plant
recombinant IL-10 that comprises the (thrombin)-(His tag)-(KDEL) with
thrombin. As shown in Figure 7, thrombin digestion of the protein indicated
that
the signal peptide was properly processed in plants.
CA 02427574 2003-05-05
One important biological role of IL-10 is in sepsis, which is a serious and
often fatal consequence of exposure to endotoxins or LPS (Howard et al.,
(1993)
J.Exp. Med. 4, 1205-1208 (Appendix B)). IL-10 inhibits the production of
5 proinflammatory cytokines such as TNF-a and IL-6 by LPS stimulated cells.
Complete inhibition requires the intact IL-10 protein, as small synthesized
peptides taken from the C and N terminus of the IL-10 protein are ineffective
(Gesser, 1997 PNAS 94, 14620-14625). Biological activity was tested by
determining its ability to inhibit the LPS induced secretion of IL-6 by the
marine
10 monocyte cell line PU51.8. As shown by the results depicted in Figure 8,
plant
recombinant IL-10 inhibits LPS induced secretion of IL-6 in a dose dependent
manner similar to insect derived human IL-10 (hIL-10). For both proteins, a
linear
inhibition of IL-6 secretion occurred at concentrations of between about 0.039
and
about 2.5 ng per mL. Control protein from an untransformed plant has no
effect.
15 These results suggest that plant recombinant IL-10 produced in plants such
as
low-nicotine, low-alkaloid tobacco plants is biologically active.
Human IL-10 is known to induce proliferation of MC/9 marine mast cells.
Further, proliferation can be blocked by a monoclonal antibody (mAB) specific
20 for the IL-10 soluble receptor. Refernng now to Figure 9, there is shown
results
demonstrating that plant recombinant IL-10 induced proliferation of mast
cells.
Further, the proliferation was blocked by the addition of neutralizing anti-IL-
10
mAb, confirming that the proliferation was due to the plant recombinant IL-10
and not other factors present in the purified plant fraction. These results
25 demonstrate that plants are able to express IL-10 , and can process and
assemble
the protein into biologically active dimeric form.
IgG expression in plants lends support to the idea that passage through the
endoplasmic reticulum may enhance protein accumulation. In plants bred to
30 simultaneously express mouse IgG light and heavy chain proteins targeted to
the
extracelluar space, the amount of each protein increased up to 60-fold over
that
seen when expressed individually into the same compartment (Hiatt et al 1989).
Expression of both protein components into the cytosol, however, did not
affect
CA 02427574 2003-05-05
46
their level of accumulation. IgG processing and assembly in lymphocytes occurs
through the action of heavy-chain binding proteins present in the endoplasmic
reticulum (ER). The observed increase in yield when both proteins were
targeted
to the extracelluar space has been attributed to an enhanced stability
contingent on
IgG assembly. Consistent with this hypothesis, active antibody complexes were
observed when the proteins were targeted to the extracelluar space but not
when
targeted to the cytosol.
To further maximize expression levels and transgene protein production
the gene encoding the protein of interest, the codon usage within the non-crop
plant of interest should be determined. Also, it has been shown that
endoplasmic
reticulum retention signals can dramatically increase transgene protein
levels, for
example the KDEL motif (Schouten et al 1996). Replacing any secretory signal
sequence with a plant secretory signal will also ensure targeting to the
endoplasmic reticulum (Denecke et al 199U).
Thus, according to an aspect of an embodiment of the present invention,
there is provided a method for the production of a protein of interest, the
method
comprising,
i) selecting a low-nicotine, low alkaloid tobacco plant;
ii) transforming the plant with a vector containing a gene encoding the
protein of interest and operatively linked with 5' and 3' regulatory regions
allowing expression of the protein of interest, thereby producing a
transformed
plant;
iii) growing and harvesting the transformed plant containing the protein of
interest, and;
iv) obtaining plant tissues from the transformed plant that comprises the
protein of interest.
The, present invention also pertain to a method for the production of a
protein of interest, the method comprising,
i) providing a low-nicotine, low alkaloid tobacco plant containing a gene
encoding the protein of interest that is operatively linked with 5' and 3'
regulatory
regions and allowing expression of the protein of interest;
CA 02427574 2003-05-05
47
ii) growing and harvesting the plant containing the protein of interest, and;
iii) obtaining plant tissues from the plant.
Preferably, the low-nicotine, low-alkaloid tobacco plant exhibits an
alkaloid concentration and a nicotine concentration which is less than about
10%
the alkaloid concentration and less than about 10% the nicotine concentration
of
Delgold tobacco plants grown under the same conditions. More preferably, the
low-nicotine, low-alkaloid tobacco plant exhibits an alkaloid concentration
and a
nicotine concentration which is less than about S% the alkaloid concentration
and
less than about S% the nicotine concentration of Delgold tobacco plants grown
under the same conditions. Still more preferably, the low-nicotine, low-
alkaloid
tobacco plant exhibits an alkaloid concentration and a nicotine concentration
which is less than about 0.28% the alkaloid concentration and less than about
2.6% the nicotine concentration of Delgold tobacco plants grown under the same
conditions.
The protein of interest may be any protein. In an aspect of an embodiment,
which is not meant to be limiting in any manner, the protein may be an
interleukin, for example, but not limited to IL-4, IL-10, or both. The
interleukin
may comprise a human interleukin, but is not limited to being human. Further,
the
interleukin may be genetically modified for example by site-directed
mutagenesis
or other methods which are known to those skilled in the art.
In another aspect of the present invention which is not meant to be limiting
in any manner, the plant may comprise GAD, for example, but not limited to
GAD67, GAD65, or a combination thereof. As described previously for plant
produced interleukins, the GAD may comprise a human GAD, but is not limited
to being human. Further, GAD may be genetically modified for example by site-
directed mutagenesis or other methods which are known to those skilled in the
art.
The protein of interest may also comprise a protease cleavage sequence,
for example but not limited to a thrombin cleavage sequence, a localization
signal,
for example but not limited to an endoplasmic reticulum retention sequence,
and
an affinity tag sequence to aid in the purification of the protein of
interest, for
CA 02427574 2003-05-05
48
example a HIS tag. Methods of modifying proteins to comprising such sequences
are known in the art and within the capability of a person of skill in the
art.
The above description is not intended to limit the claimed invention in any
manner, furthermore, the discussed combination of features might not be
absolutely necessary for the inventive solution.
The present invention will be further illustrated in the following examples.
However it is to be understood that these examples are for illustrative
purposes
only, and should not be used to limit the scope of the present invention in
any
manner.
Examples
1 S Example 1: AFP Gene Construction and Expression
To maximize potential AFP production at the translational level, the native
5'-untranslated region of the fish cDNA was replaced with the 5'-untranslated
leader region of the tobacco mosaic virus (TMV) (Richards et al 1977 & 1978,
Sleat et al 1988). The fish signal peptide sequence was also replaced with
that of
the tobacco pathogenesis-related protein 1b (PR-lb) (Cornelissen et al 1986,
Sijmons et al 1990, Denecke et al 1990). Oligonucleotide cassettes were
designed: three oligonucleotides, #1091, (SEQ ID NO:l); #1092 (SEQ ID N0:4);
and #1309 (SEQ ID N0:2), which would hybridize to generate the complete TMV
leader sequence (TMV Cassette: Figure 10A); and five oligonucleotides #4361-
#4365 (SEQ ID NOs: 3, 5, 6, 8 and 9, respetively) which in concert with
oligonucleotides #1091 and #1092 (SEQ ID NO:1 and 4) would hybridize to
generate a linked TMV leader and PR-lb signal peptide DNA sequence (TMV-PR
Cassette: Figure 1 OB). The 5' end of these cassettes corresponded to a BamH I
sticky end restriction site. The 3' end of the TMV cassette was blunt and
incorporated an additional adenine nucleotide intended to form the first
nucleotide
of the start codon while the 3' end of the TMV-PR cassette was compatible with
Nde I-cut DNA but would not be able to re-generate the restriction site
following
ligation.
CA 02427574 2003-05-05
49
To facilitate addition of the TMV and TMV-PR cassettes, sea raven Type
II AFP cDNA was site-specifically mutated according to the method described by
Kunkel (1985) to incorporate an Nde I restriction site at either by 157 or
207.
These mutations also introduced appropriate in-frame methionine start codons
at
the junctions between sequences encoding pre and pro, or pro and mature
portions
of the AFP. To build the gene construct for accumulation of AFP into the
cytosol,
plasmids containing the mutated Type II AFP cDNAs were initially cut with NdeI
and made blunt-ended with mung bean nuclease. The cDNA fragments were
released by cleavage with Sal I, were isolated and directionally ligated into
a
BamH IlSal I-cut pTZl8 vector together with annealed TMV oligonucleotides
#1091, #1092 and #1309. The TMV-proAFP and TMV-mAFP constructs were
then cut with BamH I and Hinc II, and the isolated AFP gene fragments ligated
separately into pMON 893. For gene constructs targeting AFP to the
extracelluar
space, the mutated Type II AFP cDNAs were initially subcloned from their pTZ
19 vectors into Hind IIIlSaI I-cut pBluescript vectors to position a BamH I
restriction site S' to the AFP coding sequence. The pBluescript-AFP clones
were
cut with BamH I and Nde I restriction enzymes to remove the sea raven signal
peptide sequence or signal peptide and pro-region DNA. The plasmid portions of
these digests were isolated and ligated with annealed oligonucleotides #1091,
#1092, and #4361 through #4365. The gene constructs were then cut with Xba I
and Kpn I, and separately cloned into pCDX-1.
Cloning of the Type II AFP gene constructs into pMON 893 or pCDX-1
oriented the gene cassette into the vector between the double CaMV 35S
promoter
(Kay et al 1987) and the NOS polyadenylation sequence. AFP encoded by the
transgene constructs was identical to the pro and mature AFP forms present in
fish
with the exception of one additional methionine at the N-terminal end of the
proteins. The T-DNA portion of the final constructs are depicted in Figures 11
and 12. Cleavage of the signal peptide components from the expressed AFPs was
predicted (von Heijne 1986) to occur immediately prior to the added methionine
in both the pro and mature AFP gene constructs so the AFP accumulating in the
cytosol and extracelluar space should be identical.
CA 02427574 2003-05-05
Gene constructs were transformed into N. tabacum cv. Xanthi and 81V9-4
tobacco strains according to the method described by Horsch et al (1988).
Attempts to generate plants carrying transgenes for cytosolic accumulation of
the
5 AFP were made intermittently over a period of several years. Discs infected
with
A. tumefaciens carrying these gene constructs showed little tendency to form
callus
and seemed more subject to bacterial infection than comparably treated discs
transformed with other transgene constructs. Eventually six transgenic plants
were
regenerated: one proAFP transgenic in each tobacco strain, three mature AFP
10 transgenics in Xanthi and one mature AFP transgenic in 81V9-4. By contrast,
within six months, eight transgenic plants were regenerated carrying gene
constructs for AFP accumulation into the extracellular space: two proAFP
transgenics in each strain and four mature AFP transgenics in 81 V-9.
Transformed and regenerated plants were initially selected for kanamycin
15 resistance. Transgenic status was subsequently determined by direct PCR
screening for the AFP transgene using primers #3339:
5'-TATTTTTTACAACAATTACCAACAAC-3' (SEQ ID NO:10)
and #4708:
5'-CAGCAGTCATCTGCATACAGCAC-3' (SEQ ID NO:11 )
20 which hybridize to the TMV leader and at the 3' end of the sea raven AFP
coding
sequence, respectively. Type II AFP gene constructs were amplified in 30
cycles
of denaturation for 1 min at 95 °C, annealing for 1 min at 55
°C, and elongation for
2 min at 72°C. This generated amplification products of 440 and 388 by
from the
gene constructs for cytosolic accumulation of the pro and mature AFPs, and 532
25 and 480 by fragments from gene constructs designed for accumulation of the
same
AFPs into the extracellular space.
Analysis of Trans en~pression
30 Plants were removed from the growth chamber and maintained at room
temperature under a grow light for a minimum 48 h prior to RNA or protein
extraction. RNA was prepared by selective precipitation according to the
method
of Palmiter (1974). Total RNA (40 fig) was analysed by Northern blots
according
CA 02427574 2003-05-05
51
to the method of Lehrach et al (1977) and probed with [oc 32P]-labelled sea
raven
AFP cDNA sequence.
Total soluble protein extracts were prepared from three to four leaves
taken from the upper half of a healthy plant according to the method described
by
Gengenheimer ( 1990). Extracts were dialysed at 4 ° C against 0.1 %,
0.01 % and
0.001 % ascorbic acid in SpectraPor~ #3 dialysis tubing (MWCO 3.5 kDa) and
centrifuged for 1 S min at 12,OOOxg to remove precipitate formed during
dialysis
prior to lyophilization. Samples were resuspended in Millipore~-filtered water
and
their protein concentrations determined by Bradford assay (16) relative to a
BSA
standard.
To extract protein present in the apoplast by vacuum infiltration (Sijmones
et al 1990), leaves were cut longitudinally into 4 cm x 0.8 cm strips, and
rolled
1 S lengthwise in 5 cm x 1.8 cm strips of Parafilm~' so that the bottom of the
leaf was
exposed to view. The leaf strip was exposed to vacuum derived from a water
aspirator twice for 2 min or until the exposed leaf surface was dark green.
Each
treated leaf strip yielded 10 ~ 2.5 u1 of extract. Larger volume samples were
too
dilute for use and were discarded. Samples which contained green pellets were
also discarded to reduce the possibility of cytosolic protein contamination.
All
remaining extracts from a given plant were pooled and the amount of protein
present determined by Bradford (1976) protein assay relative to a BSA
standard.
Extract concentrations were generally between 0.1-0.2 pg/pl.
Western Analysis
Lyophilized protein was resuspended directly in loading buffer (60 mM
Tris-HC1, pH 6.8/10% glycerol/5% ~3-mercaptoethanol/2% (w/v) SDS/ 1.3 x 10-
3% (w/v) bromophenol blue), and were electrophoresed through a 17%
polyacrylamide/0.1 M sodium phosphate, pH 6.8/4 M Urea/0.1% SDS gel using
0.1 M sodium phosphate, pH 6.8/0.1% SDS running buffer. Electrophoresis was
conducted at 50 V for 3-~ h or until the 6 kDa pre-stained marker was at the
bottom of the gel. Western blots were prepared according to the method
described
CA 02427574 2003-05-05
52
by Burnette et al (1981) and probed with polyclonal antibody to the mature sea
raven AFP. A chemiluminescence detection kit (Amersham) for probing of
Western blots was used to detect bound first antibody. All reactions were
earned
out according to the manufacturer's directions.
AFP Gene Expression and Protein Accumulation
Western blot analysis of total soluble protein extracted from plants
carrying genes for cytosolic AFP expression did not fmd evidence of either pro
or
mature AFP accumulation even when up to 80 pg of protein extract were
analysed. In contrast, a protein which co-migrated with mature AFP from sea
raven and cross-reacted with antibody to Type II AFP was detected in as little
as
2.5 pg total soluble protein extract of plants in which the AFP was targeted
to the
extracellular space (Figure 13). This protein was unique to the transgenic
plants
1 S and was absent in extracts of a wild-type plant and a plant, pII3a-#9,
which had
been transformed and regenerated but determined to be non-transgenic by PCR.
Insufficient resolution was obtained with these extracts to differentiate size
differences between the AFPs produced by the plants carrying transgenes for
production of pro or mature AFPs. This was probably due to the viscosity of
the
total soluble protein extracts which often caused distortion of samples during
the
running of gels. For this reason, positive control Type II AFP was mixed with
total soluble protein extract from a lab-grown wild-type plant to generate a
suitable size standard. Wild-type protein extract was not mixed with the
molecular weight markers, so the size of plant-produced AFP could not
accurately
estimated from these extracts. AFP accumulation varied between plants and
appeared slightly higher in plants II4cz-#1 and 10 which earned genes for
mature
AFP production. Based on Western blot comparison with a known amount of
Type II AFP, the amount of AFP produced by the plants has been estimated to be
approximately 0.5-1% of the total soluble protein.
mRNA Transcription from Transgenes for Cytosolic AFP Accumulation
RNA extracts of plants transgenic with gene constructs for cytosolic AFP
accumulation were analysed by Northern blot to determine if the AFP transgene
CA 02427574 2003-05-05
53
was transcribed. Two plants, Xanthi TmSR #1 and 81V9-4 TmSR #1, carrying
transgenes for AFP accumulation into the cell cytosol were found to produce a
0.96 kb RNA transcript which hybridized with the AFP cDNA used as a probe: on
longer exposure of the blots, the same RNA transcript was also seen in extract
from Xanthi TmSR #2 and slightly larger one of approximately 1.02 kb was
detected in extract from 81 V9-4 TpSR #3. Transcripts from pro and mature AFP
transgene constructs were expected to differ by 51 nucleotides. Both plant-
produced AFP gene transcripts were both considerably larger than a PCR-
generated control sample estimated to be 0.36 kb and representing the size of
the
mature AFP transgene between the TMV leader and the 3' end of the coding
sequence. This was consistent with the size difference expected based on the
additional coding sequence and 3'-untranslated region (155 nt total) present
in the
transgene construct, and with the use of the NOS polyadenylation sequence to
obtain polyadenylated transcripts. Both transcripts were smaller than an
equivalent gene transcript of 1.06 kb produced in a plant carrying the
transgene
for targeted mature AFP accumulation into the extracellular space. This size
difference was consistent with the addition oi~the 90 nt signal peptide coding
sequence to the transgene construct for secretion of the AFP. Based on EtBr
staining, more RNA was loaded onto the gel from the plant producing AFP for
accumulation into the extracellular space compared to the amount of RNA from
the plants targeting the AFP to the cytosol. No comparison could be made
regarding the relative amounts of AFP mRNA transcribed from these constructs.
However, similar loadings were achieved between the plants targeting the AFP
to
the cytosol. The difference in amount of transcript observed, therefore,
suggests
that this transgene construct was being expressed at variable levels in the
different
plants.
AFP Present in the Apo~last
Vacuum infiltration extracts were prepared from AFP-producing plants to
determine if the expressed AFP was present in the extracellular space. These
extracts were not subject to the viscosity and sample distortion associated
with
total soluble protein extracts, so were also considered more suitable to
estimate
CA 02427574 2003-05-05
54
the sizes of the plant AFPs. Western blot analysis confirmed the presence of
AFP.
AFP was detected in plants pII3a-#7 and II4c2-#1 which earned mature and
proAFP transgenes respectively, as a diffuse band of approximately 14.7 kDa
which migrated at a similar rate to mature AFP isolated from fish sera. A
small
mobility difference was observed between the AFPs produced by the two plants,
however, proAFP isolated from fish sera was distinctly larger than either
plant-
produced AFP.
Having established the presence of AFP in vacuum infiltration extracts of
those plants carrying transgenes for extracellular AFP accumulation, the
extracts
were concentrated ten-fold and tested for thermal hysteresis activity and
effects on
ice crystal morphology using the nanolitre osmometer. Extracts from plants
expressing gene constructs for either the pro or mature AFP both showed
evidence
of AFP activity. Ice crystals grown in these plant extracts were seen as the
characteristic eye-shaped bipyramids produced in the presence of native Type
II
AFP. Thermal hysteresis activity was measured and based on comparison with a
standard activity curve the amount of active AFP present in the extracellular
space
was estimated as 2% of the total protein present in the compartment.
RNA analysis of the transgenic plants has shown that they are able to
transcribe the integrated transgene constructs, and that the size of the plant
AFP
mRNA from both construct types is consistent with that expected for a full-
length,
polyadenylated transcript. mRNA produced from the transgene for AFP
accumulation into the extracellular space was obviously translatable.
The presence of plant-produced AFPs in vacuum infiltration extracts
indicates that the plant recognized the PR-lb signal peptide component and
correctly targeted the attached AFPs to the extracellular space. The predicted
size
of the AFPs for export to the extracellular space is 18.7 or 17 kDa with the
signal
peptide component attached and 15.8 or 14.1 kDa without the signal peptide for
the pro and mature AFPs, respectively. Western blot analysis of the plant
vacuum
infiltration extracts showed that the AFPs produced from the proAFP and mature
AFP transgene constructs and present in the extracellular space both co-
migrated
with mature Type II AFP and were approximately 14.7 kDa. This suggests that
CA 02427574 2003-05-05
not only is the plant capable of cleaving the signal peptide from the
expressed
AFP but that it can remove most or all of the pro-region of the AFP.
Example 2: IL-4 expression and determination of biological activity.
5
Mouse IL-4 is a glycoprotein of 140 amino acid residues including a
putative 20-amino acid signal peptide, which has a molecular weight of 19 to
21
kDa, depending on the degree of glycosylation (Proc. Natl. Acad. Sci. USA
83:2061, 86). Mouse IL-4 cDNA was first amplified with PCR using the primers:
10 5'- AAT CTCGAGCATATC CAC GGATGC,'GAC-3' (SEQ ID NO: 13) and
S'-ATAGGTACCGTAATCCATTTGC ATGATGC-3' (SEQ ID N0:14),
and the PCR product encoding the full protein was isolated and ligated to a
signal
peptide encoding sequence from peanut peroxidase and the KDEL sequence (SEQ
ID NO: 22). To facilitate subsequent purification of the recombinant product
15 from plant cells, a DNA sequence encoding 6 consecutive histidines was also
included in the reconstituted IL-4 gene (SEQ ID N0:21). The reconstituted IL-4
cDNA was then used to replace the b-glucuronidase (GUS) gene in plasmid
pTRL2-GUS composed of a CaMV35S promoter with a double enhancer
sequence (Ehn-35S) linked to a 5' untranslated tobacco etch virus leader
sequence,
20 GUS and a nonpaline synthase (NOS) terminator. The resulting IL-4
expression
cassette was inserted into the binary plant expression vector pBinl9, and then
transferred into the Agrobacterium tumefaciens strain LBA4404 (pAL4404) for
plant transformation.
25 Transformation of Nicotiana tabacum cv. SR1 was by leaf disc
cocultivation with A. tumefaciens LBA4404 harbouring IL-4 using procedures
described previously (Ma et al, 1997). Mature plants were generated from
regenerated shoots under antibiotic selection in hormone-free rooting medium,
and then subjected to screening for recombinant protein expression. Transgenic
30 plants expressing the highest levels of transgene proteins were selected
for
long-term maintenance.
Western blot analysis and chemiluminescent ELISA.
CA 02427574 2003-05-05
56
Tobacco plants (Nicotiana tabacum cv. SR1) were transformed with the
plant expression vectors comprising a mouse IL-4 gene by A.
tumefaciens-mediated leaf explant transformation method. The regenerated
tobacco plants were then analyzed for the presence of mIL-4 DNA, its specific
mRNA transcripts and protein products. The leaf tissue from IL-4 transgenic
tobacco was homogenized on ice in extraction-buffer (25 mM Tris-HCI, pH 8.0,
50 mM NaCI, 0.1% SDS, 2 mM 2-mercaptoethanol, 1 mM PMSF, 2 mg/ml
Leupeptin, 2 mg/ml Aprotitin, and 2 mg/ml Pepstatin A). The tissue homogenates
were centrifuged at 4 C for 15 min at 14,000 g and the concentration of
proteins in
the supernatants was determined by protein assay (Bio-Rad, Canada). Samples of
proteins were separated by SDS polyacrylamide gel electrophoresis, proteins
were
transferred onto PDF membrane by electroblotting and the recombinant protein
was detected by incubation with anti-mouse IL-4 antibody (PharMingen) and a
horseradish peroxidase-conjugated secondary antibody and ECL detection
reagents (Amersham). Immunoblot analysis of protein homogenates from IL-4
transformed tobacco leaf tissues showed a unique protein band recognized by
monoclonal antibody specific for mouse IL-4 (Figure 2, lane 3), which is
comparable in length to standard recombinant mouse IL-4 (Figure 2, lane 1 ).
The level of IL-4 production in trangenic tobacco leaf tissues was
determined by chemiluminescent ELISA. Briefly, for quantification of plant-
derived recombinant mouse IL-4 (prIL-4), the 96-well microtiter plates (Nunc,
Roskilde) were coated overnight at 4°C with 2 mg/ml anti-IL-4
capture mAb
(clone 11131 l; PharMingen) in 100 ml binding buffer (0.1 M Na2HP04, pH 9.0).
The plates were then washed three times with PBS containing 0.5% Tween 20,
blocked with 1% BSA in PBS, washed and incubated with leaf extracts and mouse
rIL-4 standard (Pharmingen), overnight at 4 ° C. The plates were washed
again and
incubated with biotinylated mouse anti-IL-4 detection mAb (clone BVD6-2462,
PharMingen; 1 mg/ml) for 1 h at room temperature, followed by avidin-
horseradish peroxidase (HRP) and ABTS substrate (both from Sigma Chemical
Co., Oakville, Ontario) for 30 min at room temperature for colour development.
ODs were read by a Microplate reader (ML3000, Dynatech Laboratories) at 405
nm to determine the relative amounts of recombinant IL-4 in the extracts. The
CA 02427574 2003-05-05
57
expression level of plant-derived recombinant mIL-4, as determined by IL-4-
specific ELISA, was found to be approximately 0.02% of total leaf proteins.
Determination of biological activity of plant-derived recombinant IL-4.
Although recombinant IL-4 could be detected by both western blot and
ELISA, it was important to demonstrate its biological activity. IL-4 was first
described as a T-cell-derived B-cell growth factor (Howard et aL, 1982), and
has
now been shown to mediate a wide array of biological effects on the immune
system including growth stimulation for B cells, T cells and mast cells (Hu-Li
et
al., 1987; Lee et al., 1986). Measurement of IL-4 dependent cell proliferation
is
considered to be a sensitive bioassay for IL-4 activity. The in vitro T-cell
proliferation and cytokine induction were determined as previously described.
Briefly, a spleen cell suspension was prepared from individual mice in ice-
cold
1 S PBS. The spleen cell suspension was cultured in 96-well flat-bottomed
plates at 2
X lOS cells/well in RPMI (BioWhittaker, Walkersville, MD) supplemented with
10% FCS (Life Technologies, Grand Island, NY).
Cultures were stimulated with purified hGAD (10-20 mg/ml) or OVA (10
mg/ml, Sigma) as a negative control. Cells were harvested after 72 h, and were
then assayed for the incorporation of [3H]thymidine (1 mCi/well; Amersham,
Oakville, Ontario, Canada) added during the last 18 h of culture. Accordingly,
plant-derived recombinant IL-4 was tested with the MC/9 murine mast cell line
(obtained from the American Type Culture Collection, Rockville, MD) which
requires IL-4 for proliferation. As shown in Figure 3, proliferation of mast
cells
occurred with both plant-derived recombinant IL-4 (prIL-4) and control
standard
rIL-4 (Pharmingen), suggesting that plant-derived recombinant IL-4 is
biologically active, a function that is desirable for use as an
immunomodulator in
oral tolerance.
Oral administration of low-dose IL-4 does not significantly alter serum IL-4
levels.
The in-vivo activity of plant produced IL-4 is determined in NOD mice.
NOD and control strain (insulitis- and diabetes-free) female mice (20
mice/group)
CA 02427574 2003-05-05
58
are fed either transgenic low nicotine tobacco leaves expressing recombinantly
produced IL-4. At various times during the 4 week treatment, circulating serum
levels of plant recombinant IL-4-fed and un-fed NOD and control mice are
analyzed by ELISA as described above. Both NOD and control mice are
monitored for any potential toxic effects arising from the plant recombinant
IL-4
feeding, and their serum concentrations of IgE and IgG quantified by ELISA.
Briefly, serum samples were added at appropriate dilutions to microtiter
plates
coated with 2 mg/ml of purified anti-mouse IgE capature mAb (clone R35-72;
PharMingen). Biotinylated anti-mouse IgE mAb (2 mg/ml, clone R35-118;
PharMingen) together with avidin-peroxidase and ABTS substrate were used for
detection. No serum IgE concentrations were found to be significantly elevated
in IL-4 treated mice compared to that in control-treated mice under the
conditions
tested. Thus, administration of low dose IL-4 does not induce an appreciable
increase in serum IgE concentration.
Feeding of plant recombinant IL-4 to protect against the onset of insulitis
and/or Type 1 diabetes in NOD mice is also determined. Pre-diabetic female
NOD mice (4 weeks of age) fed or not fed with plant recombinant IL-4 are
maintained in a specific pathogen free animal facility. Mice are monitored
weekly
for their blood glucose levels (BGL), and mice that are hyperglycemic (i.e.
BGL >
11.1 mmol/L) for two consecutive weeks are diagnosed as Type 1 diabetic. The
onset of insulitis is monitored by immunohistochemical analysis of pancreatic
tissue by sacrificing mice at various times (2 weeks to 2 months) after plant
recombinant IL-4-feeding is initiated.
Example 3: Interleukin-10 (IL-10) expression and determination of biological
activity
Full length mRNA encoding human IL-10 (hIL-10) is 1601 nucleotides
long, with a short 5' UTR of 30 nucleotides (nt) and a long 3' UTR of 1037 nt.
The
hIL-10 coding sequence is 534 base pairs long and codes for a 178 amino acid
preprotein with a 18 amino acid secretory signal (Vieira et al., 1991). The
full
length human IL10 cDNA is isolated from polyA RNA by RT-PCR and fully
sequenced to ensure fidelity. In order to maximize expression levels and
transgene
CA 02427574 2003-05-05
59
protein production, the IL10 cDNA is examined for codon usage patterns and
optimized as necessary (Perlak et al. 1991 ).
Since ER retention signals can increase the concentration of disulfide
bonded transgene proteins, therefore the KDEL ER retention motif may be added
to the IL-10 cDNA using site directed mutagenesis (see results in Figure 5
(C)).
For comparative purposes an identical synthetic gene, without the KDEL signal,
is
prepared and used in the transformation studies (SEQ ID N0:12).
The expression system adopts a duplicated 35S enhancer-promoter plus
AMV leader sequence (Kay et al. 1987; Jobling and Gehrke 1987). However
other constitutive promoters may also be employed, for example, T1276 a
constitutive promoter from tobacco (see co-pending application US application
serial No.08/593,121). The IL-10 gene is cloned into a T-DNA vector between
the
T-DNA border sequences using the Binl9 derived plant expression vector
pCamTerX. The synthetic IL-10 gene is transformed to tobacco using
Agrobacterium mediated transformation (Horsch et al. 1989). The Agrobacterium
strain used is EHA101 carrying the disarmed Ti plasmid pEHA104. The plant
selectable marker is neomycin phosphotransferase.
To maximize transgene expression and ensure stable field performance a
large number of primary transformants need to be generated, selected for
expression levels, screened for copy number, and single-copy high-expressing
lines evaluated for field performance. Standard methods (e.g. Northern and
Western blotting) are used to evaluate expression levels (Sambrook et al.
1989).
ELISA is used to quantify transgene protein yields.
A sequence containing the hIL-10 open reading frame and a truncated 3'
UTR (nt 4-732) is amplified by PCR using oligonuleotides hIL-10-5'-BamHI and
hIL-10-3'-EcoRI (Figure 15 (B); transformed plants comprising this contruct
are
denoted "E" in Figure 16, and see Figure S(A). The 5' oligonucleotide is
designed
with a BamHI site at the 5' end, and the 3' oligo is designed with an EcoRI
site at
the 5' end. These restriction sites enable the easy directional cloning of the
construct in the phagemid pBluescript (Stratagene) and in the Bin 19-derived
CA 02427574 2003-05-05
binary vector pCaMter X (Frisch et al., 1995). To determine the effect of the
3'
UTR on expression of hIL-10 in plants, the complete cDNA (4-1601; Figure 15
(A); transformed plants comprising this contruct are denoted "F" in Figure 16,
and
Figure S(B) is also cloned into pBluescript and pCaMter X.
To maximize protein accumulation in the plant cell, the endoplasmic
reticulum (ER) retention signal, KDEL (Schauten et al., 1996), is added to the
3'
end of hIL-10 coding sequence (Figure 14); transformed plants comprising this
contruct are denoted "G" in Figure 16, and Figure 5 (C). A His tag is also
added
10 before the KDEL signal to facilitate purification of the transgenic protein
(see
Figure 15 (C), and SEQ ID N0:12, nucleotides 544-630). A thrombin recognition
sequence is inserted immediately preceding the His tag that allows the
cleavage of
the His tag and the KDEL peptides. This third construct encodes a preprotein
of
197 amino acids. A single oligonucleotide is used to fuse these sequences to
the
15 end of hIL-10 reading frame (see Figures 14 and 15). This oligonucleotide
is used
in conjunction with oligonucleotide hIL-10-S'-BamHI in a PCR to generate the
fragment which was then cloned both in pBluescript and in pCaMter X.
Cloning of the IL-10 gene constructs into pCaMter X orientes the gene
20 between the double CaMV double 35S promoter (Kay et al., 1987) and the NOS
(Nopaline synthase) polyadenylation sequence (Frisch et al., 1995). The plant
recombinant IL-10 (prIL-10) encoded by the native transgene constructs was
identical to the native human IL-10. After purification and thrombin cleavage,
the
prIL-10 encoded by the tagged construct differs in only its two terminal amino
25 acids from the native hIL-10. .
Gene constructs are transformed into N. tabacum cv. 81 V9-4 low alkaloid
tobacco strains according to the method described by Miki et al. (1998).
Transformed and regenerated plants are initially selected for kanamycin
30 resistance. Regenerated plants are analyzed for expression of the IL10
protein by
solid phase sandwich Enzyme-Linked ImmunoSorbent Assay (ELISA). Briefly,
the JES3-9D7 anti-IL-10 mAB (PharMingen) is used as a capture antibody and is
paired with the biotinylated JES3-1268 anti-IL-10 mAB (PharMingen) as the
detecting antibody. Recombinant human IL-10 (PharMingen) is used as standard.
CA 02427574 2003-05-05
61
Leaf tissue ( 1 g of fresh weight) was ground in 2 ml of extraction buffer
(phosphate buffered saline (PBS) pH 7.4, 0.05% (v/v) Tween 20, 2% (w/v)
polyvinylpolypyrrolidone (PVPP), 1 mM phenylmethylsulfonylfluoride (PMSF),
S 1 ug/ml Leupeptin) and clarified by centrifugation at 13,OOOg for 15 min at
4*C.
The supernatant containing total soluble proteins was analyzed directly in a
hIL-10 ELISA using standard protein and antibodies as per the supplier's
protocol
(Pharmingen). The results are presented in Figure 16.
Leaf tissue from transgenic plants containing HIS-tagged phIL-l OC (plant
human IL-l OC) was ground ( 1 g fresh weight in 3.5 ml buffer) in 20mM Tris pH
8,
100 mM NaCI, 0.1% (v/v) Tween 20. The extract was clarified by centrifugation,
and the supernatant was filtered through a 0.22 ~m membrane. prIL-lOC was
purified by IMAC on a chelating Hi-Trap column (Amersham Pharmacia). The
presence of phIL-l OC in the eluted fractions was assessed by immunoblotting
using biotinylated polyclonal anti-IL-10 antibodies (1:1000 R&D systems),
Streptavidin-HRP (1:3000 Genzyme) and EC".L detection reagents (Amersham
Pharmacia). Protein was quantitated using the Bradford method. Thrombin (1
unit, Novagen) was used to digest 1 ng of insect recombinant IL-10 or phIL-l
OC
in a 20 p1 reaction in 20 mM Tris-HCl pH 8.4, 0.15 M NaCI, 2.5 mM CaCl2 for 16
hours at 23°C.
Eighteen transgenic plants are regenerated from the native construct
containing only a short stretch of 3' UTR (called E6 and E15 in Figure 16),
and
nineteen plants are regenerated from the native construct containing the
entire 3'
UTR (called F10 in Figure 16). All of those and the first two plants
regenerated
from the construct containing the His tag and KDEL ER retention signal (called
G7 in Figure 16) are tested for presence of IL-10 protein by ELISA.
A variable level of plant recombinant IL-10 (prIL-10) is observed in the
regenerated plants. There were no marked differences between plants
transformed
with the full-length hIL-10 cDNA and those transformed with 3' truncated cDNA.
CA 02427574 2003-05-05
62
However, the highest level of prIL-10 is obtained in one of the two plants
transformed with the construct containing the ER retention signal, G7-2.
The results in Figure 5(A) and Figure 5(B) show the quantity of IL-10
secreted to the apoplast following transformation of tobacco plants with
nuclotide
sequences comprising either a long hIL-10 3'UTR region or a short hIL-10 UTR
region respectively. Figure 5(C;) shows the quantity of plant recombinant IL-
10
expressed in transgenic tobacco plants transformed with an IL-10 nucleotide
sequence which further comprises a KDEL endoplasmic reticulum retention
signal. The results demonstrate that human IL-10 may be produced in plants.
Western analysis of extracts of recombinant human IL-10 (hIL-10)
produced in insect cells and plant recombinant IL-10 (prIL-10) are shown in
Figure 6. The results indicate that plant recombinant IL-10 is correctly
assembled
into dimers. The difference between the migration of the prIL-10 and hIl-10 is
due
to the presence of the (thrombin)-(His tag)-(KDEL) sequence in the prIL-10
protein.
Western analysis of digested plant recombinant IL-10 that comprises
thrombin cleavage site-His tag-KDEL (hIL-10) with thrombin is shown in Figure
7 and indicates that the signal peptide was properly processed in plants. No
digestion was observed in the insect produced IL-10 lacking the thrombin
cleavage site.
Functional activity of prIL-10 was assessed in two independent assays.
First, the ability of prIL-10 to inhibit the secretion of IL-6 by
lipopolysaccharide
(LPS) -stimulated (20 ,ug ml-~) macrophages was evaluated. ELISA was used to
measure IL-6 (Pharmingen) levels in the culture supernatent of the marine
monocyte /macrophage cell line PUS-1.8 (ATCC) stimulated by varying doses of
rIL-10 (Pharmingen), prIL-10 purified by IMAC, or control plant proteins
(Fiorentino et al. 1991. J. Immunol. 147:3815-3822 1991). Second, the ability
of
IL-10 to enhance the IL-4 dependent proliferation of the mast cell line MC/9
(ATCC) was measured. MC/9 cells were grown for 2 days in varying doses of
rIL-10 and phIL-l OC in the presence or absence of rIL-4 and in the presence
or
61
Leaf tissue ( 1 g of fr
CA 02427574 2003-05-05
63
absence of 1 pg of neutralizing anti-human IL-10 mAB (Pharmingen) (Zheng et
al. 1995. J. Immunol. 154:5590-5600). Cells were pulsed for 16 h with 1 ~Ci
well~l [3H] thymidine and cultures were harvested in glass fiber filters and
counted
in a liquid scintillation counter. Proliferation was assessed by measuring the
incorporation of [3H] thymidine. In both bioassays, the commercial rIL-10 was
calibrated to an IL-10 reference reagent (5000 reference units (RU) per
microgram) obtained from the National Cancer Institute.
IL-10 inhibits the production of proinflammatory cytokines such as TNF-a
and IL-6 by LPS stimulated cells, however, complete inhibition requires the
intact
IL-10 protein. Biological activity of recombinant IL-10 was tested by
determining
its ability to inhibit the LPS induced secretion of IL-6 by the murine
monocyte
cell line PU51.8. Plant recombinant IL-10 (prIL-10) inhibits LPS induced
secretion of IL-6 in a dose dependent manner similar to insect derived human
IL-
10 (hIL-10) as shown in Figure 8. For both proteins (insect and plant produced
IL-10), a linear inhibition of IL-6 secretion occurred at concentrations of
between
about 0.039 and about 2.5 ng per mL. Control protein from an untransformed
plant has no effect. These results demonstrate that prIL-10 produced in plants
is
biologically active.
Human IL-10 is known to induce proliferation of MC/9 murine mast cells.
As shown in Figure 9, addition of plant recombinant IL-10 (hIL-10), or rIL-4
and
hIL-10 induced proliferation of mast cells.
Cell proliferation can be blocked by a monoclonal antibody (mAb) specific
for the IL-10 soluble receptor. As shown in Figure 9, cell proliferation was
blocked by the addition of neutralizing anti-IL-10 mAb (Pharmingen), in the
presence or absence of rIL-4, (see Example 2) confirming that the increase in
observed cell proliferation was due to the plant recombinant IL-10 and not
other
factors present in the purified plant fraction. Similar results were observed
for
insect-produced IL-10 (hIL-10).
CA 02427574 2003-05-05
64
Collectively, these results demonstrate that plant-produced IL-10 is
processed and assemble into a biologically active dimeric form of IL-10.
Following the detection of IL-10 transcripts and protein in the transgenic
plants crude protein extracts containing prlL-10 the demonstration of
bioactivity
of human IL-10 is determined in vitro by its effect on T cell proliferation in
response to lectins (PHA) as well as in standard mixed lymphocyte responses
(MLR). Plant extract or purified IL-10 from plant extract is added to media
containing human lymphocytes, at a concentration ranging from 1-100 ngm/ml.
Proliferation is determined in PHA cultures and mixed lymphocyte reaction
cultures at days 3-6 by standard 3H- thymidine incorporation proliferation
assays.
Surface expression of MHC class II proteins is determined on human
lymphocytes activated by either PHA or ConA in the presence of increasing
concentrations of IL-10. And compared to control plant extracts.
Peripheral blood Iyrnphocytes are stimulated with LPS (1-10 p/ml) for 24-
48 hours, and levels of cytokines determined by ELISA or bioassay. Inhibition
is
determined as well, at the RNA level, by Northern blot analysis.
IL-10 is beneficial in prevention of lethal endotoxemia. Following
appropriate approval the effect of IL-10 purified from plant extract on
endotoxic
injury in BALB/c mice is tested. E.coli is administered interperitoneally or
intravenously in the presence of various amounts of recombinant munne IL Z0.
Plant extract is given to mice orally in addition to purified IL-10 from
plants given
intravenously. Outcomes are determined by survival of mice, which is
essentially
zero percent in untreated mice. Appropriate controls include PBS treated mice
as
well as mice given standard animal chow.
IL-10 deficient mice develop bowel inflammation and lesions resembling
inflammatory bowel disease, which is improved by systemic IL-10. To test
whether oral plant IL-10 can prevent bowel disease in an IL-10 deficient mouse
model of IBD, transgenic plant material is administered to IL-10 null mice
daily
CA 02427574 2003-05-05
from age 4 weeks and mice are assessed for the presence of bowel lesions from
age 8 to 30 weeks by weight gain and histology of tissue.
Creation of leaf material for inclusion in mouse chow
5 Seed from single high expressing homozygous line, G7-9-2 (low nicotine
tobacco) created using standard techniques (Mild et al. 1999. Biotechnol Agric
For 45:336-354) as described earlier, was germinated and seedlings transferred
to
pots filled with soil. The seedlings were grown in controlled environment
chambers (Conviron) set at 21 EC with 16h day-length until approximately 10
10 weeks old. Leaves were removed from plants, quick frozen in liquid N2, and
lyophilized.
Concentration of IL10 in tissue (~~/,g dry wei t)
The concentration of recombinant IL10 in the leaf tissue was determined
1 S as follows: lyophillized leaf tissue (1 g of dry weight) was ground in 2
ml of
extraction buffer (phosphate buffered saline (PBS) pH 7.4, 0.05% (v/v) Tween
20,
2% (w/v) polyvinylpolypyrrolidone (PVPP), 1 mM phenylmethylsulfonylfluoride
(PMSF), 1 ug/ml Leupeptin) and clarified by centrifugation at 13,OOOg for 15
min
at 4°C. The supernatant containing total soluble proteins was analyzed
directly in
20 a commercial human IL-10 ELISA using standard protein and antibodies as per
the supplier's protocol (Pharmingen). Protein was quantitated using the
Bradford
method (Bradford. 1976. Anal. Biochem. 72:248-254). The lyophilized tissue
was found to contain 2.234 mg plant recombinant IL10 per gram of dry tissue.
25 Ingredients of diet
Mouse chow with either 10 % (w/w) or 20 % (w/w) of lyophilized plant
tissue (G7-9-2) with a concentration of 2.234 mg /g dry weight of plant
recombinant IL10 was prepared. In addition similar chow was prepared with
either 10 % (w/w) or 20 % (w/w) of untransformed (81 V9) lyophilized tobacco
30 tissue. Control chow with no plant tissue was also prepared. One hundred
grams
of 10 % plant tissue chow contained 20 g lyophilized tobacco tissue , 44 g of
dextrose, 12 g of corn starch, 17 g of casein, S g cellulose, 4 g minerals, 2
g salt
supplement, 1 g of vitamins, 4.5 g of fat and and 0.5 g of corn oil. One
hundred
CA 02427574 2003-05-05
66
grams of 20 % plant tissue chow contained 20 g lyophilized tobacco tissue , 40
g
of dextrose, 11 g of corn starch, 17 g of casein, 4 g minerals, 2 g salt
supplement,
1 g of vitamins, 4.5 g of fat and 0.5 g of corn oil. One hundred grams of
control
chow contained 50 g of dextrose, 15 g of corn starch, 18 g of casein, 5 g
cellulose,
4 g minerals, 2 g salt supplement, 1 g of vitamins, 4.5 g of fat and 0.5 g of
corn
oil. The amount of chow eaten by each group and the amount of water consumed
was recorded.
Mouse feeding trials
An experiment was conducted for 30 days with 5 groups of 6 week old
IL10 null mice (Kuhn et al. 1993. Cell 75:263-270), with four males and four
females in each group. Body weight of the mice ranged between 17 and 21
grams. All mice were fed 5 g of chow per day and were housed in micro-isolator
cages. The first group of mice received control chow, the second group chow
with
1 S 10 % 81 V9, the third group chow with 20 % 81 V 9, the fourth group chow
with 10
G7-9-2, the fifth chow with 20 % G7-9-2. The drinking water of all mice
contained 1.16 m1/1 of an H2 antihistamine (ranitidine) to reduce gastric
acidity
and limit acid induced loss of biological activity of the IL10 in the plant
tissue
(Grimley et al. 1997. Aliment Pharmacol. Ther. 11:875-879; Syto et al. 1998.
Biochem. 37:16943-16951). All mice were sacrificed at the end of 30 days.
Pathology
Colon sections were taken and sections prepared. Colon sections were
randomized and interpreted blindly. Grading system for mice given IL-10 or IL-
4
includes the presence of ulceration, epithelial injury, infiltration and the
presence
of lymphoid follicles. Each variable was scored from 0 (which is normal
pathology without infiltrate or injury), to 4 (which represents severe
ulceration,
extensive epithelial injury with loss of crypts, infiltration of the lamina
sub-mucosa as well as the lamina muscularis and >3 lymphoid follicles). Scores
from individual mice were decoded and analysed using analysis of variance. The
results are presented in Figure 17. The results suggest that mice fed G7-9-2
tobacco (comprising IL-10) at a concentration of 20 % (w/w) had significantly
lower histological scores than mice fed 81V9 tobacco (P=0.003) or control chow
(P<0.001 ).
CA 02427574 2003-05-05
67
The predominant pathology in IL-10 deficient mice appears to be within
the small bowel, with large bowel involvement increasing in mice housed in
S facilities in which there is a higher rate of pathogen infections within
various
colonies. Consistent with this, given the clean facilities that the mice were
treated
in, the predominant pathology was found in the small bowels of mice, and
greatest
in those mice given control chow or vector control chow. The greatest benefit
was
found in the small bowel of mice given 20% of their total daily protein in the
form
of interleukin-10 expressing low nicotine tobacco plant material. In these
treated
mice, the level of pathology was essentially equivalent to normal mice without
inflammatory bowel disease.
Example 4: Alkaloid levels in leaf tissue of tow-nicotine, low-alkaloid
tobacco
1 S plants.
In order to establish concentrations of tobacco alkaloids in normal and
low-nicotine, low-alkaloid tobacco plants a field trial was conducted during
the
summer of 1997. Transplants of Delgold, a tobacco cultivar with normal
alkaloid
concentrations and 81 V-9 the non-food bioreactor genotype used for this
present
invention were started in mid April in a cold frame greenhouse located at the
Delhi Research Station in Ontario Canada. The two cultivars were transplanted
into a 1.2 x 2 m plots at a density of approximately 133,000 plants per
hectare.
The trial employed a randomized complete design with four replications and was
fertilized with 60 kg nitrogen per hectare. The trial was harvested 40 days
after
planting, the tissue was roughly chopped, quick frozen in liquid nitrogen and
held
at -70 °C until ready for analysis. The tissue was then lyophillized
and tobacco
alkaloid concentrations in the tissue determined by gas chromatography. For
this
analysis approximately 1 gram of lyophilized tobacco tissue was ground to pass
a
20 mesh screen and was then extracted with dichloromethane and aqueous sodium
hydroxide by shaking for 10 min on a wrist action shaker. The dichloromethane
layer was filtered and collected for alkaloid analysis. The internal standard
method was used with anethole being that standard. All gas chromatographic
analyses were performed on an Hewlett Packard 5890 series II using a DBS 60m
CA 02427574 2003-05-05
68
x.25mm i.d. column with splitless injection and a 2 p1 sample volume. Total
tobacco alkaloid concentrations were found to be 35 fold lower in the low-
nicotine, low-alkaloid tobacco plants compared tothe normal tobacco cultivar.
A
similar pattern is found for the level of nicotine (see Table 1).
Table 1.
Concentration (mg/g) of the various tobacco alkaloids found in the leaves of
normal tobacco and in the non-food bioreactor.
CultivarNicotineMyosmine AnabasineAnatabineTotal % of % of
total total
nicotinealkaloid
____________________________________
mg~g
-____________________________
Delgold 100 100
7.892a*
O.OOOa
0.013a
O.OOOa
7.923a
81 V-9 2.6 0.28
0.209b
O.OOOa
0.018a
0.020b
0.227b
*means followed by a different letter are significantly from different from
each
other at P=0.05
Example 5: Glutamic acid decarboxylase (GAD) expression
Cloning of Genes Encoding Human Glutamic Acid Decarboxylase,
GAD65 and GAD67 from Human Brain Tissue was performed as follows:
Preparation of RNA
Tissue of frozen human brain sufficent to produce cDNA using PCR, was
obtained from autopsy material. Total RNA was prepared from these samples
using RNA TRIZOL kit (Life Technologies, Inc.) according to the manufacturer's
instructions. Poly(A)+ mRNA was selected by oligo(dT)-cellulose
CA 02427574 2003-05-05
69
chromatography (Pharmacia). The concentration and purity of isolated Poly(A)+
mRNA were determined at A2~o and AZgo.
RT-PCR of RNA.
Human mRNA (250 ng used as template) for GAD65 was reverse-
transcribed using a random primer (oligo (dT),Z_1g) and Superscript II reverse
transcriptase (Life Technologies, Inc.) according to the manufacturer's
instructions in a total reaction volume of 20 ,u1. For isolation of human
GAD65,
single-stranded cDNA (1 ~l) was amplified with GAD65 gene-specific
oligonucleotide primers;
Forward primer:
5'- AATTCCATGGCATCTCCGGGCTCT GGC-3' (SEQ ID NO:15) and
NcoI
Reverse Primer:
5'- ATAATCTAGATTATAAATCTTGTCCAAGGCGTTC -3'(SEQ ID
N0:16).
XbaI
(NcoI and XbaI were the two new restriction enzyme cleavage sites created to
facilitate subsequent cloning)
in a total reaction volume of 100 ,u1 containing NH4 buffer, 1.5 mM MgCl2,
200,uM of dNTP, 0.2 ~M of each primer and 2.5 units of high fidelity DNA
polymerase Pwo (Boehringer Mannheim). The PCR reaction was performed as
follows: 3 min at 94°C, followed by 30 cycles at 94°C for 1 min,
55°C for 1 min
and 72°C for 2 min, followed by a final extension at 72°C for 10
min. The PCR
products were purified by agarose-gel electrophoresis and extracted using a
gel
extraction kit (QIAGEN Inc.) The purified cDNA was digested with both NcoI
and XbaI, and ligated to modified plasmid vector pBluescript at the same
sites.
For the isolation of human GAD67, single-stranded cDNA (1 ~cl) was
amplified with GAD67 gene-specific oligonucleotide primers:
Forward primer:
CA 02427574 2003-05-05
7~
5'- TTAATCTAGA CCATGGCGTCTTCGACCCCATCTTCG-3' (SEQ ID
N0:17)
XbaI NcoI
Reverse primer:
S 5'- TTAA TCTAGATTACAGATCCTGGCCCAGTCTTTC-3'(SEQ ID N0:18)
XbaI
(XbaI and NcoI were the new restriction enzyme cleavage sites created to
facilitate subsequent cloning).
In a total reaction volume of 100 ,u1 containing NHa buffer, 1.5 mM MgCl2,
200,uM of dNTP, 0.2 ,uM of each primer and 2.5 units of high fidelity DNA
polymerase Pwo (Boehringer Mannheim). The PCR reaction was as follows: 3
min at 94°C, followed by 30 cycles at 94°C for 1 min,
55°C for 1 min and 72°C for
2 min, followed by a final extension at 72°C for 10 min. The PCR
products were
1 S purified by agarose-gel electrophoresis and extracted by using gel
extraction kit
(QIAGEN Inc.) The purified cDNA was digested with XbaI, and Iigated to
modified plasmid vector PBluescript at the same site.
DNA Seauencin~
The sequence of both human GAD65 and human GAD67 cDNA was
determined in both directions by the dideoxynucleotide chain-termination
method
using DNA oligonucleotide primers complementary to different sequences of the
coding region. The sequence was compared with published sequence of human
brain and islet GAD65 and GAD67. A comparison of the sequence of our isolated
human brain GAD65 and GAD67 gene with published sequence of human brain
GAD65 and GAD67 demonstrated that they are identical.
Construction of,~lant e~ression vectors for human GAD65.
To construct a plant expression vector for human GAD65, human GAD65
cDNA, was amplified with PCR using the primers
5'-AATCCATGGCATCTCCGGGCTCTG-3' (SEQ ID N0:19) and
CA 02427574 2003-05-05
71
5'-ATA TCTAGATAAATCTTGTCCAAGGCG-3', (SEQ ID N0:20)
and the purified PCR product was then ligated to plasmid pTRL-GUS in
replacement of GUS gene. The resulting GAD65 expression cassette was then
cloned into vector pBinl9 and then transferred into A. tumefaciens LBA4404.
Transformation of Nicotiana tabacum cv. SR1 was by leaf disc cocultivation
with
A. tumefaciens LBA4404 harbouring hGAD65 using the procedures described
previously (Ma et al, 1997). Mature plants were generated from regenerated
shoots under antibiotic selection in hormone-free rooting medium, and then
subjected to screening for recombinant protein expression. Transgenic plants
expressing the highest levels of transgene proteins were selected for long-
term
maintenance.
Western blot analysis and chemiluminescent ELISA.
Tobacco plants (Nicotiana tabacum cv. SRl) were transformed with the
plant expression vectors comprising a human GAD65 gene by A.
tumefaciens-mediated leaf explant transformation method. The regenerated
tobacco plants were then analyzed for the presence of hGAD65 DNA, its specific
mRNA transcripts and protein products. Integration of human GAD65 cDNA into
the plant genome was confirmed by polymerase chain reaction (PCR) using
GAD65 specific primers of genomic DNA, and mRNA transcripts were
demonstrated by northern blot analysis using P32-labeled DNA probes
representing the structural gene of hGAD65. Immunoblot analysis of plant
recombinant GAD65 was performed according to the following procedure. The
leaf tissue from GAD65 transgenic tobacco was homogenized on ice in extraction-
buffer (25 mM Tris-HCI, pH 8.0, 50 mM NaCI, 0.1 % SDS, 2 mM 2-
mercaptoethanol, 1 mM PMSF, 2 mg/ml Leupeptin, 2 mg/ml Aprotitin, and 2
mg/ml Pepstatin A). The tissue homogenates were centrifuged at 4°C for
15 min
at 14,000 g and the concentration of proteins in the supernatants was
determined
by protein assay (Bio-Rad, Canada). Samples of proteins were separated by SDS
polyacrylamide gel electrophoresis, proteins were transferred onto PDF
membrane
by electroblotting and the recombinant protein was detected by incubation with
anti-human GAD65 antibody (Cedarlane, Hornsby) and a horseradish peroxidase-
conjugated secondary antibody and ECL detection reagents (Amersham).
CA 02427574 2003-05-05
72
Immunoblot analysis of protein homogenates from transgenic tobacco leaf
tissues
showed a single protein band of the correct size (kDa 65, Figure 1, lanes l
and 2),
while no protein band was recognized with the same GAD antibody in extracts
from control tobacco plants transformed with empty vector (lane C) (Fig.l). An
E.
coli-derived recombinant GAD67 standard was included for the purpose of
densitometry comparison. The level of plant-derived recombinant GAD65
expression was determined by scanning the signal strength of the applied GAD
leaf extracts and purified E. coli-derived recombinant GAD67 of known
concentration on the obtained blots using a pdi white light scanner (Raytest).
The
expression level of GAD65, estimated from blot densitometry, was found to be
approximately 0.04% of total soluble proteins.
Example 6: Transgenic plant feeding trials
NOD mice were housed according to guidelines of the Canadian Council
on Animal Care and were screened for bacterial and viral pathogens. Parental
colony incidence of diabetes in female mice is 70-80% by 30 weeks. Female pre-
diabetic NOD mice were started at 4 weeks of age. Fresh mIL-4 and hGAD65
transgenic tobacco leaf tissues were homogenized in liquid nitrogen and the
tissue
homogenates were added to feed up to 12% (w/w) of diet. The amount of mouse
IL-4 was calculated to deliver approximately 10-12 ~cg per mouse daily if
completely digested, whereas the amount of human GAD65 fed per mouse daily
was approximately 8-10 ,ug. Mice were caged as a group of 4. Control mice
received an equivalent amount of corresponding leaf tissue from vector-
transformed tobacco. Mice were monitored for glucose levels in urine weekly by
using TES-TAPE Lilly, Indianapolis, 1N), with serum testing to confirm
diabetes
(> 16.7 mmol/1 on two consecutive days (Glucoscan 2000 Strips, Lifescan,
Milpitas, CA).
The onset of insulitis is monitored by immunohistochemical analysis of
pancreatic tissue. The pancreata of NOD mice were harvested at 10 weeks of
age.
Pancreatic tissue was removed, fixed with 10% buffered formalin, embedded in
paraffin and sectioned at S ,um intervals. The incidence and severity of
insulitis
CA 02427574 2003-05-05
73
was examined by hematoxylin and eosin staining. A minimum of 15 islets from
each mouse was examined, and the degree insulitis was evaluated based on the
following semi-quantitative rating system: score 0, normal (no mononuclear
cells
infiltrating islets); score 1, peri-insulitis only (mononuclear cells
surrounding
islets, but no infiltration of the islet); score 2, moderate insulitis
(mononuclear
cells infiltrating <50% of the islet); and score 3, severe insulitis (>50% of
the islet
tissue infiltrated by lymphocytes). The results indicate that there was
reduction,
but not absence of insulitis in treated mice.
Suppression of diabetes
To determine whether combined administration of GAD and IL-4 plant
prevented diabetes, mice were supplemented with hGAD65 plus mIL-4 plant leaf
tissues in their diet from 5 weeks to 30 weeks of age. The amount of hGAD65
delivered daily per mouse was estimated to be approximately 8 to 10 ,ug,
whereas
the amount of IL-4 delivered daily per mouse was approximately 10 to 12 ,ug.
Control groups of mice were fed tobacco leaves transformed with empty vector
or
hGAD65 or mIL-4 transgenic leaf tissues alone. The mice were followed for the
development of hyperglycemia. NOD mice receiving GAD65 plus IL-4 transgenic
plant tissues showed a significant decrease in the incidence of developing
diabetes
(<30%, 2 out of 7 NOD mice at 30 weeks of age), compared with the GAD65
plant alone fed mice (80%, 8 out of 10 NOD developed diabetes) or IL-4 plant
alone fed mice ( 100%, 10 out of 10 NOD mice developed diabetes) or empty
vector transgenic plants fed mice (>60%, S out of 8 developed diabetes).
Serum IgE Ab quantification.
IL-4 mediates immunoglobulin class switch to IgE production (Ryan,
1997), which is associated with the induction of an allergic response. To
determine whether oral administration of IL-4 plants elevates the level of IgE
Ab
production in NOD mice treated with both GAD and IL-4 plants or IL-4 plants
alone, GAD plus IL-4 treated, IL-4 treated and control plant-treated mice were
monitored for their relative serum IgE concentrations. The levels of IgE Abs
in
serum samples collected from female NOD mice were determined by ELISA.
CA 02427574 2003-05-05
74
Briefly, serum samples were added at appropriate dilutions to microtiter
plates
coated with 2 ~,g/ml of purified anti-mouse IgE capture mAb (clone R35-72;
PharMingen). Biotinylated anti-mouse IgE mAb (2 p,g/ml, clone R35-118;
PharMingen) together with avidin-peroxidase and ABTS substrate were used for
detection. No serum IgE concentrations were found to be significantly elevated
in
GAD plus IL-4 treated or IL-4 treated mice compared to that in control-treated
mice. Thus, administration of low dose IL-4 does not induce an appreciable
increase in serum IgE concentration.
Treatment with GAD and IL-4 transgenic plants prevents adoptive transfer of
IDDM.
NOD-scid mice 8-10 weeks of age received a single i.p. injection of 10 X
106 spleen cells from diabetic donors mixed with 10 X 106 spleen cells from
nondiabetic NOD mice fed transgenic GAD and IL-4 plants. Control mice
received a single i.p. injection of 10 X 106 spleen cells from diabetic
donors.
Recipient mice were monitored for diabetes up to 8 weeks. Results showed that
50% of the mice receiving a mixture of splenic mononuclear cells from
GAD+IL-4 treated and diabetic mice developed diabetes at the end of 8 weeks
post transfer, while 100% of the mice receiving only diabetic splenic cells
developed IDDM (Figure 4). This suggests that treatment with both GAD and
IL-4 plants induces regulatory T cells in prediabetic NOD mice that are
capable of
blocking tissue destruction by a diverse, activated effector T-cell
population.
All scientific publications and patent documents are incorporated herein by
reference.
The present invention has been described with regard to preferred
embodiments. However, it will be obvious to persons skilled in the art that a
number of variations and modifications can be made without departing from the
scope of the invention as described herein. In the specification the word
"comprising" is used as an open-ended term, substantially equivalent to the
phrase
"including but not limited to", and the word "comprises" has a corresponding
CA 02427574 2003-05-05
meaning. Citation of references is not an admission that such references are
prior
art to the present invention.
CA 02427574 2003-05-05
76
References
Abrams, J.S., M.G. Roncarolo, H. Yasel, U. Anderson, G. Gleich, J. Silver
(1992)
Strategies of anti-cytokine monoclonal antibody development. Immunol.
Rev. 127, S-24.
Bradford M.M. (1976) A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein dye
binding. Anal. Biochem. 72, 248-254.
Burnette W.H. (1981) Western blotting: electrophoretic transfer of proteins
from
SDS-polyacrylamide gels to unmodified nitrocellulose and radio-graphic
detections with antibody and radioiodinated protein A. Anal. Biochem.
112, 195-203.
Chaplin J.F. (1977) Breeding for varying levels of nicotine in tobacco. In:
Proc
Amer Chem Soc, 173rd meeting, New Orleans.
Cornelissen B.J.C, R.A.M Hooft van Huijsduijnen, L.D.Van Loon & J.F.BoI
( 1986) Molecular characterization of messenger mRNAs for
"pathogenesis-related" proteins 1 a, 1 b and 1 c, induced by TMV infection
of tobacco. EMBO J. 5, 37-40.
Denecke J., J.Botterman & R.Deblaere (1990) Protein secretion in plant cells
can
occur via a default pathway. Plant Cell 2, 51-59.
Fourney R.M., J.Miyakoshi, R.S.Day III & M.C.Paterson (1988) Northern
blotting: efficient RNA staining and transfer. Focus 10:1, 5-7.
Fiocchi C. (1993) Cytokines and animal models: a combined path to inflammatory
bowel disease pathogenesis. Gastroenterology 104:1202-1219.
CA 02427574 2003-05-05
77
Frisch, D. A., Harris-Hailer, L.W., Yokubaitis, N.T., Thomas, T.L., Hardin,
S.H.,
and Hall, T.C. (1995). Complete sequence of the binary vector Bin 19.
Plant Mol. Biol. 27, 405-409.
Gengenheimer P (1990) Preparation of Extracts from Plants. Methods of
Enzymology 182, 184-185.
Haq T.A., H.S. Mason, J.D. Clements, C.J. Artzen (1995) Oral immunization with
a recombinant bacterial antigen produced in plants. Science 268, 714-716.
Hiatt A., R.Cafferkey & K.Bowdish (1989) Production of antibodies in
transgenic
plants. Nature 342, 76-78.
Horsch R.B., J.Fry, N.Hofmann, J.Neidermeyer, S.G.Rogers & R.T.Fraley. (1988)
Leaf disc transformation. Plant Molecular Biology Manual A5, 1-9. Ed.
S.B.Gelvin, R.A.Schilperoort, D.P.S.Verma. Kluwer Academic Publishers.
DordrechtBoston/London.
Kay R., A.Chan, M.Daly & J.McPherson (1987) Duplication of CaMV 355
promoter sequences creates a strong enhancer for plant genes. Science
236, 1299-1302.
Kuhn R. LohlerJ, Rennick D, Rajewsky K, Muller W (1993) Interleukin-10
deficient mice develop chronic enterocolitis. Cell 75:263-274.
Kunkel T.A. (1985) Rapid and efficient site-specific mutagenesis without
phenotypic selection. Proc. Natl. Acad. Sci. USA 82, 488-492.
Lehrach H., D.Diamond, J.M.Wozney & H. Boedtker. (1977) RNA molecular
weight determinations by gel electrophoresis underdenaturing conditions;
a critical reexamination. Biochemistry 16, 4743.
CA 02427574 2003-05-05
78
Ma J.K.C., M. Hein (1995) Immunotherapeutic potential of antibodies produced
in plants. TibTech 13, 522-527.
Mason H.S., J.M. Ball, J-J. Shi, X. Jiang, M.K. Estes, C.J. Arntzen (1996)
Expression of Norwalk virus capsid protein in transgenic tobacco and
potato and its oral immunogenicity in mice. Proc. Nat. Acad. Sci. 93,
5335-5340.
Miki, B.L.A., McHugh, S.G., Labbe, H., Ouellet, T., Tolman, J.H., and Brandle,
J.E. (1998) Transgenic tobacco: gene expression and applications.
Biotechnology in Agriculture and Forestry (in press)
Montanari L., P. Fantozzi, S. Pedone (1993) Tobacco fraction 1 (F1P
utilization of
oral feeding and enteral feeding of patients. 1 Heavy Metal evaluation.
Food SAci. Tech. 26, 259-263.
Otsuka T., D. Vallaret, T. Yokota, Y. Takebe, F. Lee, N. Arai, K. Arai (1987)
Structural analysis of the mouse chromosomal gene encoding interleukin-4
which expresses B cell, T cell and mast cell stimulating activities. Nucl.
Acid Res. 15, 333-334
Palmiter R.D. (1974) Magnesium precipitation of ribonucleoprotein complexes:
Expedient techniques for the isolation of undegraded polysomes and
messenger ribonucleic acid. Biochemistry 13, 3606-3615.
Perlak FJ, Fuchs RL, Dean DA, McPherson SL, Fischoff DA (1991) Modification
of the coding sequence enhances plant expression of insect control protein
genes. Proc Natl Acad Sci 88:3324-3328.
Rapoport M.J., D. Zipris, A.H. Lazarus, A. Jaramillo, D.V. Serreze, E.H.
Leiter,
P. Cyopick, J.S. Danska, T.L. Delovitch (1993) IL-4 reverses thymic T cell
proliferative unresponsiveness and prevents diabetes in NOD mice. J. Exp.
Med. 178, 87-99.
CA 02427574 2003-05-05
79
Richards K., H.Guilley, G.Jonard & G.Keith (1977) Leader sequence of 71
nucleotides devoid of G in tobacco mosaic virus RNA. Nature 267, 548-
550.
Richards K., H.Guilley, G.Jonard & L.Hirth (1978) Nucleotide sequence at the
5'
extremity of tobacco mosaic virus RNA. Eur. J. Biochem. 84, 513-519.
Schouten A et al. (1966) The C-terminal KDEL sequence increases the expression
level of a single-chain antibody designed to be targeted to both the cytosol
and the secretory pathway in transgenic tobacco. Plant Molec. Biol. 30,
781-793.
Sleat D.E., R.Jull, P.C.Turner & T.M.A.Wilson (1988) On the mechanism of
translational enhancement by the S'-leader sequence of tobacco mosaic
virus RNA. Eur. J. Biochem. 175, 75-86.
Sijmons P.C., B.M.M.Dekker, B.Schrammeijer, T.C.Verwoerd, P.J.M.van den
Elzen & A.Hoekema (1990) Production of correctly processed human
serum albumin in transgenic plants. Bio/Technology 8, 217-221.
Vieira, P., de Waal-Maleyfyt, R., Dang, M.N., Johnson, K.E., Kastelein, R.,
Fiorentino, D.F., deVries, J.E., Roncarolo, M.G., Mosmann, T.R., and
Moore, K.W. (1991) Isolation and expression of human cytokine synthesis
inhibitory factor cDNA clones: homology to Epstein-Barr virus open
reading frame BCRF1. Proc. Natl. Acad. Sci. U.S.A. 88, 1172-1176.
von Heijne G. (1986) A new method for predicting signal cleavage sites. Nucl.
Acids Res. 14, 4683-4690.
Woodleif W.G., J.F. Chaplin, C.R. Campbell, D.W. DeJong (1981) Effect of
variety and harvest treatments on protein yield of close grown tobacco
Tobacco Sci. 25, 83-86.
CA 02427574 2003-05-05
SEQUENCE LISTING
<110> Her Majesty the Queen in Right of Canada as represented by the
Minister of Agriculture and Agri Food Canada and London Health Sciences
Centre Research Inc.
<120> Plant Bioreactors
<130> 08-873996CA1
<140> not available
<141> not available
<150> 02257531.0
<151> 2002-10-30
<150> 10/137,647
<151> 2002-05-03
<160> 22
<170> PatentIn version 3.0
<210> 1
<211> 20
<212> DNA
<213> Primer
<400> 1
gatcctcgag tatttttaca 20
<210> 2
<211> 58
1
CA 02427574 2003-05-05
<212> DNA
<213> Primer
<400> 2
acaattacca acaacaacaa acaacaaaca acattacaat tactatttac aattacaa 58
<210> 3
<211> 49
<212> DNA
<213> Primer
<400> 3
acaattacca acaacaacaa acaacaaaca acattacaat tactattta 49
<210>4
<211>74
<212>DNA
<213>Primer
<400> 4
ttgtaattgt aaatagtaat tgtaatgttg tttgttgttt gttgttgttg gtaattgttg 60
taaaaatact cgag 74
<210> 5
<211> 51
<212> DNA
<213> Primer
<400> 5
caattacaat gggatttttt ctcttttcac aaatgccaag cttttttctt g 51
<210> 6
2
CA 02427574 2003-05-05
<211>46
<212>DNA
<213>Primer
<400> 6
tcagtactct tctcttattc ctgatcatat ctcactcttc gcatgc 46
<210> 7
<211> 30
<212> PRT
<213> Primer
<400> 7
Met Gly Phe Phe Leu Phe Ser Gln Met Pro Ser Phe Phe Leu Val Ser
1 5 10 15
Thr Leu Leu Leu Phe Leu Ile Ile Ser His Ser Ser His Ala
20 25 30
<210>8
<211>56
<212>DNA
<213>Primer
<400> 8
gagaagagta ctgacaagaa aaaagcttgg catttgtgaa aagagaaaaa atccca 56
<210>9
<211>34
<212>DNA
<213>Primer
<400> 9
3
CA 02427574 2003-05-05
tagcatgcga agagtgagat atgatcagga ataa 34
<210>10
<211>26
<212>DNA
<213>Primer
<400> 10
tattttttac aacaattacc aacaac 26
<210> 11
<211> 23
<212> DNA
<213> Primer
<400> 11
cagcagtcat ctgcatacag cac 23
<210> 12
<211> 1645
<212> DNA
<213> Primer
<400>
12
aaaccacaagacagacttgcaaaagaaggcatgcacagctcagcactgctctgttgcctg 60
gtcctcctgactggggtgagggccagcccaggccagggcacccagtctgagaacagctgc 120
acccacttcccaggcaacctgcctaacatgcttcgagatctccgagatgccttcagcaga 180
gtgaagactttctttcaaatgaaggatcagctggacaacttgttgttaaaggagtccttg 240
ctggaggactttaagggttacctgggttgccaagccttgtctgagatgatccagttttac 300
ctggaggaggtgatgccccaagctgagaaccaagacccagacatcaaggcgcatgtgaac 360
tccctgggggagaacctgaagaccctcaggctgaggctacggcgctgtcatcgatttctt 420
4
CA 02427574 2003-05-05
ccctgtgaaaacaagagcaaggccgtggagcaggtgaagaatgcctttaataagctccaa480
gagaaaggcatctacaaagccatgagtgagtttgacatcttcatcaactacatagaagcc540
tacatgacaatgaagatacgaaactgagacatcagggtggcgactctatagactctagga600
cataaattagaggtctccaaaatcggatctggggctctgggatagctgacccagcccctt660
gagaaaccttattgtacctctcttatagaatatttattacctctgatacctcaaccccca720
tttctatttatttactgagcttctctgtgaacgatttagaaagaagcccaatattataat780
ttttttcaatatttattattttcacctgtttttaagctgtttccatagggtgacacacta840
tggtatttgagtgttttaagataaattataagttacataagggaggaaaaaaaatgttct 900
ttggggagccaacagaagcttccattccaagcctgaccacgctttctagctgttgagctg 960
ttttccctgacctccctctaatttatcttgtctctgggcttggggcttcctaactgctac 1020
aaatactcttaggaagagaaaccagggagcccctttgatgattaattcaccttccagtgt 1080
ctcggagggattcccctaacctcattccccaaccacttcattcttgaaagctgtggccag 1140
cttgttatttataacaacctaaatttggttctaggccgggcgcggtggctcacgcctgta 1200
atcccagcactttgggaggctgaggcgggtggatcacttgaggtcaggagttcctaacca 1260
gcctggtcaacatggtgaaaccccgtctctactaaaaatacaaaaattagccgggcatgg 1320
tggcgcgcacctgtaatcccagctacttgggaggctgaggcaagagaattgcttgaaccc 1380
aggagatggaagttgcagtgagctgatatcatgcccctgtactccagcctgggtgacaga 1440
gcaagactctgtctcaaaaaaataaaaataaaaataaatttggttctaatagaactcagt 1500
tttaactagaatttattcaattcctctgggaatgttacattgtttgtctgtcttcatagc 1560
agattttaattttgaataaataaatgtatcttattcacatcaaaaaaaaaaaaaaaaaaa 1620
ctcgagggggggccggtacccaatg 1645
<210> 13
<211> 27
<212> DNA
<213> Primer
<400> 13
aatctcgagc atatccacgg atgcgac 27
CA 02427574 2003-05-05
<210> 14
<211> 29
<212> DNA
<213> Primer
<400> 14
ataggtaccg taatccattt gcatgatgc 29
<210> 15
<211> 27
<212> DNA
<213> Primer
<400> I5
aattccatgg catctccggg ctctggc 27
<210> 16
<211> 34
<212> DNA
<213> Primer
<400> 16
ataatctaga ttataaatct tgtccaaggc gttc 34
<210> 17
<211> 36
<212> DNA
<213> Primer
<400> 17
ttaatctaga ccatggcgtc ttcgacccca tcttcg 36
fi
CA 02427574 2003-05-05
<210> 18
<211> 34
<212> DNA
<213> Primer
<400> 18
ttaatctaga ttacagatcc tggcccagtc tttc 34
<210> 19
<211> 24
<212> DNA
<213> Primer
<400> 19
aatccatggc atctccgggc tctg 24
<210> 20
<211> 27
<212> DNA
<213> Primer
<400> 20
atatctagat aaatcttgtc caaggcg 27
<210>21
<211>477
<212>DNA
<213>Primer
<400> 21
atggcacttc caattagcaa agttgatttc ttaatattta tgtgtcttat aggattaggg 60
7
CA 02427574 2003-05-05
tcagctctcgagctaatccacggatgcgacaaaaatcacttgagagagatcatcggcatt120
ttgaacgaggtcacaggagaagggacgccatgcacggagatggatgtgccaaacgtcctc180
acagcaacgaagaacaccacagagagtgagctcgtctgtagggcttccaaggtgcttcgt240
atattttatttaaaacatgggaaaactccatgcttgaagaagaactctagtgttctcatg300
gagctgcagagactctttcgggcttttcgatgcctggattcatcgataagctgcaccatg360
aatgagtccaagtccacatcactgaaagacttcctggaaagcctaaagagcatcatgcaa420
atggattactcgggtacccatcatcatcatcatcattctgagaaagatgagctatga 477
<210> 22
<211> 459
<212> DNA
<213> Primer
<400> 22
atggcacttc caattagcaa agttgatttc ttaatattta tgtgtcttat aggattaggg 60
tcagctctcg agctaatcca cggatgcgac aaaaatcact tgagagagat catcggcatt 120
ttgaacgaggtcacaggagaagggacgccatgcacggagatggatgtgccaaacgtcctc180
acagcaacgaagaacaccacagagagtgagctcgtctgtagggcttccaaggtgcttcgt240
atattttatttaaaacatgggaaaactccatgcttgaagaagaactctagtgttctcatg300
gagctgcagagactctttcgggcttttcgatgcctggattcatcgataagctgcaccatg360
aatgagtccaagtccacatcactgaaagacttcctggaaagcctaaagagcatcatgcaa420
atggattactcgggtacctctgagaaagatgagctatga 459
g
