Note: Descriptions are shown in the official language in which they were submitted.
CA 02901370 2015-08-24
PRODUCTION OF IL-37 IN PLANTS
FIELD
[0001] The present disclosure relates generally to plant molecular
farming and more
specifically to the functional production and purification of human
interleukin-37, and isoforms
thereof, in plants.
BACKGROUND
[0002] Plants can be engineered to produce a variety of types of
commercially
valuable products such as peptides, proteins and secondary metabolites,
(Twyman et al,
2003, Besaran et al., 2008, Xu et al, 2012, Egelkrout et al, 2012). Products
that have been
made include, but are not limited to, enzymes for industrial, (Yang et al,
2005, Zhong et al.,
2006, Grey et al, 2009, Pereira et al., 2013, Kolotilin et al, 2013) or
medical applications,
(Downing et al., 2006, Shaaltiel et al., 2007, Singhabahu et al., 2013, Melnik
et al, 2013),
vaccines, (Tacket, 2010; Rybicki, 2010), antibodies, (Hiatt et al., 1989;
Girtich et al, 2006, De
Muynck et al., 2010, Peters et al., 2011), structural proteins, (Shoseyou et
al, 2013), blood
proteins, (He et al, 2011), and hormones, (Nykiforuk et al, 2006, Gils et al,
2005, Jez et al.,
2013).
[0003] A variety of different plant species have been used for
recombinant protein
production including field crops such as maize, soybean, barley, rice, potato,
and tobacco
(Conley et al, 2011). Additionally, non-field crop plant species have been
adapted for
recombinant protein production such as duckweed, (US 6,815,184), moss, (Decker
et al,
2004) and algae, (Mayfield et al., 2007, Rosales-Mendoza, et al., 2011).
[0004] One factor that influences the effectiveness of plant-based
production systems
is the amount of the recombinant protein that is accumulated. Rates of
accumulation depend
on rates of formation and stability of the recombinant product.
[0005] Cytokines are soluble hormone-like proteins that are primarily
secreted by
white blood cells. More than 100 cytokines have been characterized that
regulate a broad
spectrum of physiological processes, (da Cunha et al, 2014). Cytokines that
have anti-
inflammatory activity are valuable for the treatment of many autoimmune and
inflammatory
diseases (Banchereau et al, 2012). A number of cytokines have been
successfully made in
plants, (reviewed by Sirko et al, 2011, da Cunha et al, 2014), including alpha
interferon, (Zhu
et al., 1994), erythropoietin, (Matsumoto, S., et al, 1993, Conley et al,
2012; Jez et al, 2013),
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IL-2, (Park et al, 2002. Redkiewicz et al, 2012, Zhang et al., 2014), IL-4,
(Magnuson, N. S.,
1988. Ma et al., 2005), IL-10, (Menassa et al., 2001; 2004, 2010; Fujiwara et
al., 2010; Kaldis
et al., 2013) IL-12, (Gutierrez-Ortega, 2004, Elias-Lopes, et al, 2008), IL-
13, (Wang et al,
2008), IL-18, (Zhang et al., 2005), and IL-24, (US 2010/0278775).
[0006] IL-37 is a dual-function cytokine in that in addition to normal
binding to its
cognate cell-surface receptors to exert its anti-inflammatory effect, the
intracellular precursor
forms of IL-37 have the ability to translocate to the nucleus and can
influence subsequent
downstream transcription of inflammatory genes (Bulau et al., 2014). To the
best of the
inventor's knowledge, IL-37 has not previously been produced in plants. It is
therefore
desirable to develop constructs, methods and systems for the production of IL-
37, and
isoforms of IL-37, in plants.
SUMMARY
[0007] It is an object of the present disclosure to obviate or mitigate
at least one
disadvantage of the previous production of IL37.
[0008] In one embodiment there is provided a chimeric nucleic acid
sequence
construct for expression of IL-37 in a plant cell.
[0009] In one embodiment there is provided a chimeric nucleic acid
sequence
construct for expression of IL-37 in a plant cell, comprising: a promoter
nucleic acid
sequence; an enhancer nucleic acid sequence operatively associated with said
promoter (EV
5'UTR); a signal peptide nucleic acid sequence operatively associated with
said 5'UTR
(IL37a; or barley a-amylase); an IL-37 nucleic acid sequence encoding IL-37
operatively
associated with said signal peptide sequence; and a terminator nucleic acid
sequence
operatively associated with said IL-37 nucleic acid sequence.
[0010] In a specific example, said promoter is a constitutive promoter or
an inducible
promoter.
[0011] In a specific example, the promoter is a Cauliflower Mosaic Virus
35S
promoter, a ubiquitin promoter, a tubulin promoter, a Rubisco promoter, a
histone promoter,
an actin promoter, a lipase promoter, a metallothione promoter, an RNA
polymerase
promoter, an elongation factor promoter, an expansin promoter.
[0012] In a specific example, the promoter is an organ specific promoter,
preferably a
seed specific promoter, a tuber specific promoter, a leaf specific promoter, a
fruit specific
promoter, or a root specific promoter.
[0013] In a specific example, said 5'UTR comprises EV 5'UTR.
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[0014] In a specific example, said IL-37 is codon-optimized for
expression in said
plant.
[0015] In a specific example, said IL-37 nucleic acid exhibits from about
70% to about
100% sequence identity to IL-37.
[0016] In a specific example, said IL-37 nucleic acid comprises IL-37a
nucleic acid,
IL-37b nucleic acid; IL-37c nucleic acid; IL-37d nucleic acid; or IL-37e
nucleic acid.
[0017] In another embodiment, said chimeric nucleic acid sequence
construct further
comprises a targeting sequence nucleic acid operative associate with said IL-
37 nucleic acid
sequence
[0018] In a specific example, said targeting sequence comprises a sub-
cellular
targeting sequence, an endoplasmic targeting sequence, a cell cytoplasmic
targeting
sequence, a chloroplast targeting sequence and a cell excretion targeting
sequence.
[0019] In a specific example, said targeting sequence is a KDEL targeting
sequence,
or a barley aleurone DNA signal sequence.
[0020] In another embodiment, the chimeric nucleic acid sequence
construct further
comprises a purification tag nucleic acid sequence construct operatively
associated with said
IL-37 nucleic acid sequence.
[0021] In another embodiment, the chimeric nucleic acid sequence
construct further
comprises a protease cleavage nucleic acid operatively associate with said IL-
37 nucleic acid
or said affinity tag nucleic acid.
[0022] In a specific example, said purification tag encodes Soybean
Agglutinin (SBA),
transferrin, a polyhistidine, a Strepll, glutathione S-transferase.
[0023] In a specific example, said protease cleavage nucleic acid encodes
a tobacco
etch virus (TEV) protease cleavage sequence.
[0024] In another embodiment, the chimeric nucleic acid sequence
construct further
comprises a selectable marker nucleic acid.
[0025] In a specific example, the selectable marker imparts resistance to
kanamycin,
hygromycin, streptomycin, or a herbicide such as phosphinothricin or
glyphosate.
[0026] In a specific example, said IL-37 is human IL-37.
[0027] In a specific example, said IL-37 is optimized for expression in a
plant.
[0028] In another embodiment, said construct is IL-37b-1-a, IL-37-1b, IL-
37a-1, IL-
37d-1, IL-37e-1, IL-37c-1, IL-37b-2, IL-37a-2, IL-37d-2, IL-37e-2, IL-37c-2,
IL-37b-3, IL-37a-
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3, IL-37d-3, IL-37e-3, IL-37c-3, IL-37b-4, IL-37a-4, IL-37d-4, IL-37e-4, IL-
37c-4, IL-37b-5, IL-
37a-5, IL-37d-5, IL-37e-5, or IL-37c-5.
[0029] In another embodiment there is provided a plant, plant cell, or
plant part,
transformed with a chimeric nucleic acid construct as described above, and
herein.
[0030] In a specific example, said plant, plant cell, or plant part, is a
suspension
culture, an embryo, a meristematic region, a callus tissue, a leaf, root,
shoot, gametophyte,
sporophyte, pollen, seed or microspore.
[0031] In a specific example, the plant cell is from a monocot, or a
dicot.
[0032] In a specific example, the monocotyledon is a corn, a cereal, a
grain, a grass,
or a rice.
[0033] In a specific example, the dicot is a tobacco, a tomato, a potato,
a legume,
tarwi or oca.
[0034] In another embodiment, there is provided a method of producing IL-
37 in a
plant comprising the steps of (a) transforming a plant cell with a chimeric
nucleic acid
construct as described herein and above; (b) generating a whole plant
according to the
methods as described above and herein, from said transformed cell under
conditions
resulting in expression of IL-37 in said plant; (c) harvesting at least a
portion of said whole
plant; and (d) purifying said IL-37 from said harvested at least a portion of
said whole plant.
[0035] Other aspects and features of the present disclosure will become
apparent to
those ordinarily skilled in the art upon review of the following description
of specific
embodiments in conjunction with the accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] Embodiments of the present disclosure will now be described, by
way of
example only, with reference to the attached Figures.
[0037] Fig. 1. Illustration of alternative splicing of IL-37 gene
generating 5 different
isoforms;
[0038] Fig. 2. cDNA sequence and encoded amino acids of IL-37b with the
signal
peptide being underlined;
[0039] Fig. 3. cDNA sequence and encoded amino acids of IL-37a with the
signal
peptide being underlined;
[0040] Fig. 4. cDNA sequence and deduced amino acids of IL-37c with
signal
peptide being underlined;
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[0041] Fig 5. cDNA sequence and encoded amino acids of IL-37d with signal
peptide
being underlined;
[0042] Fig 6. cDNA sequence and encoded amino acids of IL-37e with signal
peptide
being underlined;
[0043] Fig 7. Comparison and alignment of the amino acid sequences of
five IL-37
isoforms. Identical amino acids in the five IL-37b isoforms are denoted by
asterisks.
Deletions are shown as dashes within sequences;
[0044] Fig. 8. Schematic illustration of IL-37b plant transformation
plasmid vectors;
[0045] Fig. 9. Schematic illustration of IL-37a plant transformation
plasmid vectors;
[0046] Fig. 10. Schematic illustration of IL-37d plant transformation
plasmid vectors;
[0047] Fig. 11. Schematic illustration of IL-37e plant transformation
plasmid vectors;
[0048] Fig. 12. Schematic illustration of IL-37c plant transformation
plasmid vectors;
[0049] Fig. 13. Plant codon-optimized nucleotide sequence and the deduced
amino
acids of IL-37b with a barley a-amylase peptide underlined;
[0050] Fig. 14. Plant codon-optimized nucleotide sequence and the deduced
amino
acids of IL-37a with a barley a-amylase peptide underlined;
[0051] Fig. 15. Plant codon-optimized nucleotide sequence and the deduced
amino
acids of IL-37d with a barley a-amylase peptide underlined;
[0052] Fig. 16. Plant codon-optimized nucleotide sequence and the deduced
amino
acids of IL-37e with a barley a-amylase peptide underlined;
[0053] Fig. 17. Plant codon-optimized nucleotide sequence and the deduced
amino
acids of IL-37c with a barley a-amylase peptide underlined;
[0054] Fig. 18. Plant codon-optimized nucleotide sequence and the deduced
amino
acids of SBA-IL-37b fusion gene with the TEV cleavage site underlined and the
linker
sequence indicated in italics;
[0055] Fig. 19. Schematic illustration of the procedure used to assemble
SBA and IL-
37a as a fusion gene;
[0056] Fig. 20. Plant codon-optimized nucleotide sequence and the deduced
amino
acids of SBA-IL-37a fusion gene with the TEV cleavage site underlined and the
linker
sequence indicated in italics;
[0057] Fig. 21. Plant codon-optimized nucleotide sequence and the deduced
amino
acids of SBA-IL-37d fusion gene with the TEV cleavage site underlined and the
linker
sequence indicated in italics;
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CA 02901370 2015-08-24
[0058] Fig. 22. Plant codon-optimized nucleotide sequence and the deduced
amino
acids sequence of the SBA-IL-37e fusion gene with the TEV cleavage site
underlined and
the linker sequence indicated in italics;
[0059] Fig. 23. Plant codon-optimized nucleotide sequence and the deduced
amino
acids sequence of the SBA-IL-37c fusion gene with the TEV cleavage site
underlined and the
linker sequence indicated in italics;
[0060] Fig. 24: Western blot detection of IL-37b in leaves of N.
benthamiana
transiently transformed with IL-37b-la. Protein samples (40 pg) were separated
by 10%
SDS-PAGE gel electrophoresis, transferred onto PVDF membranes and probed with
the
anti-hIL-37 antibody. + = commercial recombinant IL-37 protein (30 ng loaded);
dai, days
after infiltration; WT, protein samples from uninfiltrated control N.
benthamiana leaves.
Numbers to the left indicate the sizes of the protein markers in Kilodaltons.
The double-
headed arrow indicates the dimeric form of IL-37b, while the single-headed
arrow indicates
the monomeric form of IL-37b;
[0061] Fig. 25: Western blot detection of IL-37d in leaves of N.
benthamiana
transiently transformed Agrobacterium containing IL-37d. Protein samples (40
pg) were
separated by 10% SDS-PAGE gel electrophoresis, transferred onto PVDF membranes
and
probed with the anti-hIL-37 antibody. WT, protein samples from non-infiltrated
control N.
benthamiana leaves; dai, days after infiltration; Numbers to the left indicate
the sizes of the
protein markers in Kilodaltons. The double-headed arrow indicates the dimeric
form of IL-
37d, while the single-headed arrow indicates the monomeric form of IL-37d;
[0062] Fig. 26. Western blot analysis of IL-37bx6His in stable transgenic
tobacco
plants;Protein samples (40 pg) were separated by 12.5% SDS-PAGE gel
electrophoresis,
transferred onto PVDF membranes and probed with the anti-hIL-37 antibody. 1 to
24,
representatives of the individual transgenic tobacco lines; WT, protein
samples from
untransformed control tobacco; +, commercial recombinant IL-37 protein (30 ng
loaded);
The double-headed arrow indicates the dimeric form of IL-37bx8His, while the
single-headed
arrow indicates the monomeric form of IL-37bx6His;
[0063] Fig.27. Western blot analysis of SBA-IL-37c fusion protein
expression in
stable transgenic tobacco plants. Protein samples (40 pg) were separated by
10% SDS-
PAGE gel electrophoresis, transferred onto PVDF membranes and probed with anti-
hIL-37
antibodies (A). The same Western blot was overexposed to show the formation of
a
multimeric form of E. coli-derived IL-37 standard (B). 1 to 12,
representatives of the individual
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transgenic tobacco lines; WT, protein samples from untransformed control
tobacco; +,
commercial recombinant IL-37 protein (30 ng loaded); The double-headed arrow
indicates
the dimeric form of SBA-IL-37c, whereas the single-headed arrow indicates the
monomeric
form of SBA-IL-37c; The star indicates the degradation products of SBA-IL-37c
caused
during sample preparation, while the triangle indicates the presence of a
multimeric form of
E.coli-derived IL-37 standard;
[0064] Fig. 28. Enzyme-linked immunosorbent assay (ELISA) analysis of IL-
37
protein expression levels in leaf tissue of individual transgenic tobacco
lines carrying IL-37b-
lb. The numbers on the bottom of the figure represent the different tobacco
lines. WT, wild-
type tobacco used as a control. TSP, total soluble protein;
[0065] Fig. 29. Deglycosylation analysis of plant-derived recombinant IL-
37bx6His.
Partially purified plant-derived IL-37bx6His was treated with PNGase F. The
treated samples
were separated on 10% SDS¨PAGE gels followed by Western blotting using rabbit
anti-IL-37
antibody followed by addition of goat anti-rabbit secondary antibody
conjugated with
horseradish peroxidase. Lanes a and b, untreated human transferrin standard;
lanes c and d,
human transferrin standard treated with PNGase F; lanes f and g, untreated IL-
37bx6His
samples; lanes h and I, IL-37bx6His samples treated with PNGase F; lane e,
E.coli-derived
commercial IL-37. ¨, untreated samples; +, treated samples;
[0066] Fig. 30. A Coomassie blue stained SDS-PAGE gel used to visualize
purified
SBA-IL-37c samples following one-step affinity chromatography on N-acetyl-D-
galactosamine-linked agarose column. The fusion protein was purified from
total extracts of
Nicotiana benthamiana leaves infiltrated with Agrobacterium containing IL-37c-
5 (Figure 12).
TSP, total soluble protein; FT-H, flow through; W-H, wash through; El to E6,
individual
elution fractions. M, protein molecular weight markers. The arrow indicates
purified SBA-IL-
37c;
[0067] Figure 31. Effect of plant-derived made IL-37 and SBAIL-37 fusion
protein on
the inhibition of TTNFa in LPS-stimulated mouse primary kidney cells. Med,
medium only;
[0068] Figure 32 is a schematic diagram of the construct (pLIL-37d+hTf)
used for
plant transformation to generate plants expressing IL-37d+hTf.
[0069] Figure 33 is a schematic diagram of the construct (p35SIL-37d+hTf)
used for
plant transformation to generate plants expressing I137d+hTf.
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DETAILED DESCRIPTION
[0070] Generally, the present disclosure provides nucleic acids,
constructs, methods
and systems for the production of IL-37, and isoforms of IL-37, in plants.
[0071] IL-37, (formerly IL-1 family member 7) was discovered
independently by
several groups in 2000, (Smith et al, 2000, Busfield et al, 2000, Kumar et al,
2000). The RNA
transcribed from the human IL-37 gene is alternatively spliced resulting in
the expression of
five different isoforms, (Taylor et al., 2002, Boraschi et al., 2011). IL-37b
is the best
characterized of the IL-37 isoforms. It is comprised of 218 amino acids
including a 45 aa
signal sequence, (Pan et al, 2001). IL-37 is synthesized as a precursor and is
processed to
its mature form by caspase-1, (Bulau et at, 2014).
[0072] IL-37 is a fundamental inhibitor of innate immunity, (NoId et al.,
2010) and has
been shown to have powerful anti-inflammatory activities by suppression of pro-
inflammatory
cytokine production, (Teng et al, 2014).
[0073] As shown in Figure 1, the human IL-37 gene undergoes alternative
splicing,
resulting in the expression of five different isoforms (Taylor et al 2002;
Boraschi et al 2011).
[0074] IL-37 isoform b (shown in Figure 2) is the best characterized IL-
37 isoform,
and is the one with the longest sequence (218 amino acids including a 45-amino
acid signal
sequence; MW: 24,126 Dalton). IL-37b is also the most abundant IL-37 isoform
(Pan et al.,
2001). IL-37b shares a similar 13-barrel structure with other members of the
IL-1 family. IL-
37b was reported to form homodimers under experimental conditions.
[0075] The IL-37a isoform (shown in Figure 3) consists of 192 amino acids
including
a 21-amino acid signal peptide sequence (MW: 21,543 Daltons), and has a 13-
barrel structure
like IL-37b or other members of the IL-1 family (Boraschi et al., 2011).
[0076] IL-37c (shown in Figure 4) is identical to IL-37b, except for a
120-base pair in-
frame deletion due to splicing of exon 2 to exon 5. As a result, IL-37c is 40-
amino acids
shorter than IL-37b. It is speculated that the biological activity of IL-37c,
may not be the same
as IL-37b because the protein structure of IL-37c is considered to be
different from that of IL-
37b due to the large deletion of (40) amino acids (Boraschi et al., 2011).
[0077] IL-37d (shown in Figure 5) consists of 197amino acids including a
20-amino
acid signal sequence (MW: 21,950 Daltons) and has a 13-barrel structure like
IL-37b or other
members of the IL-1 family (Boraschi et al., 2011).
[0078] IL-37e (shown in Figure 6) consists of 157 amino acids including a
putative
20-amino acid signal sequence (MW: 17,459 Daltons), and is the shortest form
of IL-37. The
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CA 02901370 2015-08-24
protein structure of IL-37e is considered to be different from that of IL-37b
and as such, it has
been suggested that the biological activity of IL-37e may be different from IL-
37b (Boraschi et
al., 2011).
[0079] Detailed comparison of the amino acid sequences of the five IL-37
isoforms is
shown in Figure 7.
[0080] IL-37 is expressed in a variety of normal human tissues, such as
the lymph
nodes, thymus, and uterus (Pan et al., 2001). Some IL-37 isoforms are
expressed in a
tissue-specific fashion. IL-37c is mainly present in the heart, whereas IL-37d
and IL-37e are
only present in the bone marrow and testes, and IL-37a is the only isoform
expressed in the
brain (Taylor et al., 2002).
[0081] IL-37 is synthesized as a precursor and is processed to its mature
form by the
enzyme called caspase-1 (Boraschi et al., 2011). IL-37 is now recognized as a
dual-function
cytokine in that in addition to normal binding to its cognate cell-surface
receptors to exert its
anti-inflammatory effect, the intracellular precursor forms of IL-37 have the
ability to
translocate to the nucleus and can influence subsequent downstream
transcription of
inflammatory genes (Bulau et al., 2014).
[0082] As mentioned above, in some embodiments, there is described herein
nucleic
acids, constructs, methods and systems for the production of IL-37, and
isoforms of IL-37, in
plants.
[0083] Such production of IL-37, isoforms of IL-37, is also referred to
as molecular
farming.
[0084] In some examples, the IL-37, and isoforms of IL-37, is human IL-
37, and
isoforms of human L-37.
[0085] In other examples, the IL-37, and isoforms of IL-37, is non-human
IL-37 and
isoforms of IL-37.
[0086] It will be appreciated that that degeneracy of the genetic code
provides a large
number of polynucleotide sequences encoding IL-37, or IL-37 isoforms.
Contemplated
herein are those variations of nucleotide sequence that can be made by
selecting
combinations based upon possible codon usage.
[0087] In some examples there may be provided vectors for the expression
of fusion
protein between IL-37 variants and SBA for expression in tobacco, tarwi seed
and oca.
[0088] In some examples, the selection of nucleotide sequence is chosen
to optimize
expression in plants.
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[0089] In some examples, a functional equivalent of IL-37 is provided. As
used
herein a functional equivalent encompasses a protein or nucleic acid molecule
that
possesses functional or structural characteristics that are substantially
similar to IL-37. A
functional equivalent of a protein may contain modification depending upon the
necessity of
such modifications for the performance of a specific function. The term
functional equivalent
is intended to include fragment, mutants, hybrids, variants, analogs, or
chemical derivatives
of a molecule.
[0090] In some example, the functional equivalent may be a fusion
protein.
[0091] Production of proteins in plants
[0092] Molecular farming approaches have been described for the
manufacture of a
variety of individual proteins of industrial and medicinal value, for
examples:, avidin, (US
5,767,379), aprotinin, (US 5,824,870), beta-glucuronidase, (US 5,804,694),
serum albumin,
(US 7,741,536; US 8,158,857), human glucocerebrosidase, (US 6,846,968; US
7,951,557;
US 8,227,230; US 8,449,876), lactoferrin, (US 6,569,831; US 7,138,150; US
7,276,646; US
7,354,902; 8,334,254), alpha galactosidase,(US 6,890,748), laccase, (US
7,071,384), milk
proteins, (US 6,991,824; US 7,417,178; US 7,718,851), blood proteins, (US
6,344,600; US
8,686,225), glutamic acid decarboxylase, (US 6,338,850), somatotropin, (US
6,288,304),
epidermal growth factor, (US 7,091,401), apolipoprotein, (US 7,786,352),
collagen, (US
7,232,886; US 8,455,717), L-iduronidase, (5,929,304), human glycoprotein
hormone, (US
7,655,781), plasminogen, (US 8,017,836), chymosin, (US7,390,936), lysozyme,
(US
6,518,297; US 6,943,244), glucanase, (US 8,558,058), immunogenic proteins, (US
6,761,914), interferon, (US 6,815,184) and insulin, (US 7,547,821).
[0093] Production of cvtokines in plants
[0094] The production of cytokines in plants has been reviewed Sirko et
al, (2011)
and da Cunha et al, (2014). In animals, cytokines are soluble hormone-like
proteins secreted
primarily by white blood cells in very low concentrations, (10-15M to 10-12M).
[0095] Cytokines are important components of the immune system and are
involved
in a wide range of cellular processes.
[0096] Recombinant production of cytokines has been achieved in bacteria
and yeast
however clinical use of cytokines has been limited by the cost of production
in these
systems. Plants offer many advantages for large-scale and cost effective
production of
cytokines.
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[0097] As described in references herein, interleukins IL-2, IL-3, IL-4,
ILI 0, IL-12, IL-
13, IL-18 and IL-24 have been made in plants.
[0098] Generally, the expression levels that were achieved, (Table 1)
were low. A
number of different reasons could account for the low levels of accumulation,
and while not
wishing to be bound by theory, may include innate protein instability and
degradation by
proteinases.
[0099] Table 1. Levels of Interleukins Produced in Plants
IL Plant host Concentration Reference
IL-2 Tob cell 9 x 10-5 g/L Magnuson et al, 1998
culture
IL-2 Potato 115 ng/g FW Park et al, 2002
tuber
IL-2 Tob leaf 0.014 -0.1 % TSP Redkiewicz et al, 2012
virus
IL-2- Tob leaf 0.006 ¨ 0.03% TSP "
CMTI virus
IL-2- Tob leaf 0.078 ¨ 0.03% TSP "
SP12 virus
IL-3 Tob leaf 0.144 mg/g TSP Musiychuk et al. 2013
virus
IL-4 Tob cell 4.5 x 10-4g/L Magnuson et al, 1998
cult
IL-4 Tob 0.1 % TSP Ma et al. 2005
IL-4 Potato 0.08 % TSP Ma et al. 2005
I L-4-ELP Tob 0.1% TSP Patel et al. 2007
IL-4-Hist I Tob
0.001 % TSP
IL-10 Tob 0.27% TSP
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IL-10 Tob 0.43 mg/g
Menassa et al. 2004
--F
IL-10 Tob 0.37 mg/g Bortesi et at. 2009
IL-10 I Rice seed 0.05 mg/g Fujiwara et at. 2010
IL-10- Tob cell 3% TSP Kaldis et al, 2013
ELP cult
IL-12 Tob 4 x 10-4 mg/g Bortesi et al. 2009
IL-12 Tomato 7.3 x 10-3mg/g Gutierrez-Ortega et al. 2005
IL-13 tTob 0.15% TSP Bortesi et at. 2009
IL-18 Tob 5.5 x 10-4mg/g Bortesi et at, 2009
IL-24 Tob cell 0.3% TSP Menassa et at
susp US2010/0278775
[00100] It would be desirable to produce IL-37, and isoforms of IL-37, in
plants.
[00101] In some example, it may be desirable to make high levels of IL-37
in plant to
aid in the purification and recovery of recombinant IL-37 produced in plants.
[00102] Transformation of plants
[00103] The development of plants for use in molecular farming for large
scale
manufacture of recombinant proteins, peptides and metabolites, of commercial
and medical
value and/or interest has evolved progressively from the initial discovery
that plants can be
engineered to express foreign genes by a process known as transformation.
[00104] The initial discovery of transformation of plants was based on the
determination that the pathogenic bacterium Agrobacterium tumefaciens, the
causal agent of
the plant Crown Gall disease, naturally comprises DNA plasmids that are
capable of
transferring specific DNA sequences into plant cells that then become stably
incorporated
within the plant host DNA. (Gelvin, 2003, Barampuram et at, 2011).
[00105] It was subsequently determined that Agrobacterium rhizogenes, the
causal
agent of the plant Hairy Root disease, also transfers plasmid DNA sequences
into plant cells
by an analogous mechanism.
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CA 02901370 2015-08-24
[00106] The transformation of plants for the expression of foreign genes
in plants has
become a well understood technology for a person skilled in the art.
[00107] The possible use of this technology for plant molecular farming
was first
advanced in patents authored by Goodman et al., (US 4,956,282; US 5,550,038;
US
5,629,175, US 6,096,547 and US 6,774,283), however the proposed use of
molecular
farming was narrowly limited to, mouse interferon in transgenic tobacco.
[00108] Many different procedures are well known to workers skilled in the
art that
result in plant transformation and recombinant gene expression in transgenic
plants.
Generally transformation methods can be divided into two basic strategies;
physical methods
to introduce foreign DNA into plant cells and Agrobacterium-mediated
transformation of plant
cells, (Barampuram et al., 2011).
[00109] A common technique for the physical delivery of foreign DNA into
plant cells
has been the "biolistic" acceleration of small dense carrier particles, such
as particles of gold,
that are coated in foreign DNA, by what is known in the art as a "gene gun"
(US 4,945,050;
US 5,036,006; and US 5,371,015). A variety of different "gene guns" for
shooting DNA into
plant cells have been developed (Christou, 1992). Physical carriers such as
tungsten
"whiskers" or silicon carbide crystals have also been used to deliver foreign
DNA by puncture
of the cell wall creating channels for DNA entry (US 5,302,523, US 5,464,765;
US
7,259,016; US 6,350,611).
[00110] Other physical approaches have included the micro-injection of DNA
solutions
directly into cells, (US 4,743,548, US 5,994,624) and production of pores in
cellular
membranes for DNA uptake with electric currents (US 6,022,316). The removal of
the
external cell wall barrier and preparation of protoplasts facilitates the
uptake of DNA directly
from solution but in some instances regeneration of plants from protoplasts is
challenging
(US 4,684,611; and US 5,453,367).
[00111] A widely practiced general method of achieving plant
transformation has been
by the use of Agrobacteria (as reviewed in Gelvin, 2003). Agrobacterium
tumefaciens and
related soil bacteria naturally comprise a DNA plasmid (i.e. a T-DNA plasmid)
that is
physically mobilized into plant cells by bacteria proliferating in a wound
site. The T-DNA
plasmid has left and right border sequences that are required for integration
of DNA into the
plant host genome. Foreign DNA between the border sequences is thus
selectively
introduced into the host genome.
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CA 02901370 2015-08-24
[00112] Naturally occurring plasmids have been modified, "disarmed" by
removal of
genes that cause tumor formation and support bacterial growth. The Ti plasmid
was also
modified to remove so-called virulence factors needed for DNA transfer. These
factors were
placed on a separate plasmid so that only selective recombinant DNA is added
to the host
plant cells and not the Vir genes. The technique of removal of the virulence
factor DNA to a
separate plasmid is known as "disarming" and resulted in the development of
the preferred,
so-called, binary transformation method (US. 4,940,838).
[00113] Generally, methods for plant transformation are well known in the
art and
detailed procedures have been published and described in patents and patent
applications,
for transformation of well-known plant species such as tobacco, (Conley et al.
2011), canola,
(US 5,188,958), Arabidopsis, (US 6,353,155), corn, (US 5,981,840), cotton, (US
5,998,207),
oil palm, (US 8,017,837), rice, (US 7,939, 328), soybean, (US 5,024,944) and
wheat, (US
7,026,528).
[00114] The transformed plants used herein may be monocotyledonous or
dicotyledonous plant or plant cell.
[00115] The monocotyledonous plants include, but are not limited to, corn,
cereals,
grains, grasses, and rice.
[00116] The dicotyledonous plants may include, but are not limited to,
tobacco,
tomatoes, potatoes, and legumes including soybean, lupines and alfalfa.
[00117] The expression in plants also encompasses plant cells.
[00118] Plant cells, includes suspension cultures, embryos, meristematic
regions,
callus tissue, leaves, roots, shoots, gametophytes, sporophytes, seeds, pollen
and
microspores. Plants may be at various stages of maturity and may be grown in
liquid or on
solid culture, or in soil or suitable medium in pots, greenhouses or fields.
Expression in plants
may be transient or permanent. Plants also refers to any clone of such a
plant, seed, selfed
or hybrid progeny, propagule whether generated sexually or asexually, and
descendants of
any of these, such as cuttings or seed.
[00119] In specific examples, there is provided methods and compositions
for
recombinant IL-37, and IL-37 isoforms, in transgenic tobacco, oca tubers,
(Oxalis tuberose),
and tarwi seed, (Lupinus mutabilis).
[00120] Vectors for expression and production of IL-37, and isoforms of IL-
37, in
plant cells
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CA 02901370 2015-08-24
[00121] Vectors used for transformation of plants may also include one or
more
elements for the control of gene expression, such as promoters, enhancers,
terminators,
selectable markers, targeting sequences and intervening sequences.
[00122] promoters
[00123] Promoters are elements that regulate gene expression, that are
located
upstream from the coding gene sequences. Promoters are typically located
directly
upstream of the coding gene sequence.
[00124] Promoters that are suitable for use in in molecular farming
activities may be
constitutive, cell specific, tissue specific or organ specific, inducible or
developmentally
regulated, promoters.
[00125] There are a variety of examples of constitutive promoters which
many be used
for gene expression in plants, plant tissues, and plant organs. For example,
the constitutive
Cauliflower Mosaic Virus 35S Promoter (Odell et al., 1985; Ow et al., 1987, US
5,164,316;
US 5,352,605), has been used to provide high rates of foreign gene expression
in many plant
systems. In another example, the CaMV 19S promoter is used. In another
example, the
figwort mosaic virus (FMV) 34S (US 6,051753) promoter is used.
[00126] Other highly expressed constitutive promoters have similar
functions, (US
6,987,179, Christensen et al., 1996; Ishige et al., 1999). Constitutive
promoters have been
developed from viral, (US 5,378,619; US 5,466,788; US 5,824,857; US
5,850,019), and
bacterial plasmid, (US 4,771,002; US 5,102,796; US 5,106,739; US 5,182,200; US
5,366,887; US 5,428,147) genes. For some applications plant gene derived
constitutive
promoters may be preferred and have been cloned, for example, from ubiquitin,
(US
5,510,474; US 6,638,766), tubulin, (US 5,635,618; US 6,903,247), Rubisco, (US
4,962,028),
histone, (US 5,491,288), actin, (US 7,838,652), lipase, (US 7,964,393),
metallothione, (US
8,026,412), RNA polymerase, (US 5,891,681), elongation factor, (US 5,117,011)
and
expansin, (US 6,566,586) genes.
[00127] Organ specific promoters, for example, those that direct
expression to tubers
and seeds are useful for molecular farming applications as recombinant
proteins are housed
in organs naturally evolved for protein storage and stability. Organs such as
seeds, tubers
and roots may be stored until recovery of the recombinant protein is required.
In addition to
the ocatin tuber storage gene promoter, (Doshi et al., US201030347144) tuber
specific
promoter elements have been described from potato, (US 5,436,393; US
5,723,757; US
6,184,443) and sweet potato, (US 7,273,967; US 7,411,115).
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CA 02901370 2015-08-24
[00128] In some examples, promoters have been described that are expressed
in
different organs such as leaves, roots, fruit and flowers and in seeds at
various stages of
development, (i.e. US 5,420,034; US 5,530,194;; US 5,824,865; US 5,955,649; US
6,541,222). In particular, promoters that are active in legume seed are
relevant to the
methods of molecular farming in tarwi described herein, (US 5,504,200; Lycett
et al., 1984,
1985; Jaiswal et al., 2007).
[00129] Thus, depending upon the desired tissue, expression may be
targeted to the
endosperm, aleurone layer, embryo (or its parts such as scutellum and
cotyledons), pericarp,
stem, leaves, tuber, roots, etc.
[00130] In other examples, promoters that can be induced by physiological
manipulations such as heat shock, light or harvest injury, (Saidi et al, 2005,
Shimizu-Sato et
al., 2002, US 5,670,349; US 5,689,056; US 7,329,789; US 7,388,091) or by
hormone, growth
regulator or chemical induction (Martinez et al., 1999, Zou et al., 2000,
Roslan et al.,
2001,Tang et al, 2004, US 5,139,954; US 5,364,780; US 5,589.614; US 5,614,395;
US
5,965,387; US 6,504.032; US 6,617,498; US 6,784,340; US 6,958,236) can be
used.
Inducible systems can also be developed whereby a gene is activated by the
induced
removal of a blocking DNA sequence by a site specific recombinase (Hoff et
al., 2001).
[00131] In some specific examples, the promoter is a Cauliflower Mosaic
Virus 35S
promoter, an ubiquitin promoter, a tubulin promoter, a Rubisco promoter, a
histone promoter,
an actin promoter, a lipase promoter, a metallothione promoter, a RNA
polymerase promoter,
an elongation factor promoter, an expansin promoter.
[00132] In some specific examples, the promoter is an organ specific
promoter,
preferably a seed specific promoter, a tuber specific promoter, a leaf
specific promoter, a fruit
specific promoter, a root specific promoter.
[00133] enhancers
[00134] It is known in the art that gene activity can be increased by a
variety of means,
including the use of enhancer sequences. Enhancer sequences are short DNA
regions (50-
100 bp) of DNA that can bind proteins that activate transcription of a gene or
genes. Non
limiting examples of enhancers include sequences related to physiological
induction such as
heat, pathogen attack, (Mitsuhara et a)., 1996), and viral promoter sequences
such as double
35S, (Kay et al., 1987), and AMV RNA4, (Datla et a)., 1993). A database of cis-
acting
regulatory elements has been created, (Lescot et al., 2002). Gene expression
can also be
increased by intron sequences, (Parra et al., 2011).
- 16-
CA 02901370 2015-08-24
[00135] In one example, the enhancer is a tobacco etch virus 5'
untranslated leader
sequence (TEV 5'-UTR).
[00136] selectable markers
[00137] Transgenic plants may be selectively recovered from transformed
cells using
selectable markers. Transformation vectors may comprise a gene that encodes
such a
selectable marker, for example a product that allows the identification and/or
selection of
transformed plants.
[00138] Examples of gene products that can be used as visual reporter
genes to
recognize transformants include GUS, (Beta-glucuronidase) that imparts a blue
pigment, or
GFP, the green fluorescent protein that can be detected via fluorescence.
[00139] It will be appreciated that gene products that impart a negative
or positive
selection can also be used effectively to select transformed cell (Miki et
al., 2004). Examples
of selectable markers that have been used extensively include those that
impart antibiotic
resistance such as to kanamycin, hygromycin or streptomycin, or provide
tolerance to
herbicides such as phosphinothricin or glyphosate.
[00140] A variety of strategies have also been used to remove selectable
marker
genes because of concerns that such genes may have a negative environmental
impact
(Gleave et al., 1999).
[00141] Additionally, methods have been developed to limit gene flow in
the
environment (Hills et al., 2007) or equip transgenic plants with conditionally
lethal genes such
that transgenic plants can be selectively eliminated from any natural site
(Schernthaner et al,
2003, US 8,124,843).
[00142] terminator
[00143] Transcription termination sequences may be positioned downstream
of the
transcription initiation region, and may be derived from the same gene as the
transcription
initiation region, or from a different gene. The termination sequence may be
chosen for
stability of the mRNA to enhance expression.
[00144] In some examples, the terminator sequence is the NOS terminator
from
Agrobacterium Ti plasmid or the rice alpha-amylase terminator.
[00145] targeting sequence
[00146] In some examples, a targeting sequence is used in the expression
of IL-37, or
the isoforms of IL-37.
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CA 02901370 2015-08-24
[00147] Such targeting sequences (also referred to as signal sequences)
permit the
processing and translocation of the protein as appropriate. Targeting
sequences may be
derived from mammals, or plants. The targeting sequences will direct the IL-37
to a sub-
cellular location, such as the cytosol, endoplasmic reticulum (ER), plastid,
or chloroplast.
[00148] In specific examples, the targeting sequence is a subcellular
targeting
sequence, an endoplasmic targeting sequence, a cell cytoplasmic targeting
sequence, a
chloroplast targeting sequence, or a cell excretion targeting sequence.
[00149] In a specific example, the targeting sequence is a KDEL targeting
sequence.
[00150] In a specific example, the targeting sequence is a barley aleurone
DNA signal
sequence.
[00151] In some example, a protease site or self-processing site may be
included to
facilitate the release of the targeting sequence from the recombinant IL-37,
or isoform of IL-
37.
[00152] Studies of transgene expression in plants have demonstrated a
positive role
for introns in gene expression. lntrons that contain enhancement sequences
such as CGATT
are found prominently in highly expressed genes and intron splicing may be
needed for
efficient nuclear export and stability of transcripts, (Parra et al., 2011)
[00153] Production in plants
[00154] It is known to the skilled worker that most plants may be
regenerated from
cultured cells or tissues including protoplasts derived from plant cells.
[00155] Methods for plant regeneration vary with species, but generally a
cell capable
of being cultured alone or part of a tissue and containing copies of the
vectors described
herein is provided. Callus tissue may be formed and shoots may be induced from
callus and
subsequently rooted, or shoots may be induced directly from a cell within a
meristem.
Alternatively, embryo formation can be induced from the cell suspension. These
embryos
may be germinated to form plants.
[00156] Following cultivation, the transgenic plant is harvested to
recover the IL-37.
Harvesting may include harvesting the entire plant, or only the leaves, or
roots of the plant.
Harvesting may not kill the plant is if only a portion of the transgenic plant
is harvested.
[00157] The transgenic plants described herein may also be used to develop
hybrids
or novel varieties.
[00158] The mature transgenic plants are selfed and non-segregating.
Alternatively,
an outcross may be performed to move the gene into another plant.
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CA 02901370 2015-08-24
[00159] Recovery and purification of proteins produced in plants.
[00160] Different strategies have been described to enhance the recovery
of
transgenic products from the transformed host cells, (Besaran et al., 2008;
Davies, 2010;
Huang et al., 2012; Xu et al, 2012, Melnik et al., 2013, Sabalza et al, 2013).
[00161] Broadly, any process which separates the recombinant IL-37 from
other
elements or compounds, for example, on the basis of charge, molecular size,
and/or binding
affinity, and/or the like, may be used.
[00162] Specialized protein production strategies have been tried, such as
attachment
of recombinant peptides of interest via oleosin fusions to oil-bodies and
separation of the
target protein by floatation, (US 6,924,363), or formation of dense virus like
particles,
(US20130344100, D'Aoust et al, 2010) or protein bodies, (US 5,990,384; US
7,575,898; US
7,732,569, US 7,794,979; US 7,795,382;) that may be separated by
sedimentation.
Additional methods include use of cross-linked solubility tags, (US 8,617,843)
and acid-
cleavable linkers, (US 8,609,621).
[00163] Procedures for the recovery of recombinant peptides and proteins
from plants
also involve purification from crude biological extracts of cells and tissues.
The need to
remove large amounts of impurities and contaminants results in multiple
operations. Each
step in the recovery process affects the overall efficiency of the process,
increasing costs
and processing time.
[00164] One method that to increase the efficiency of recovery of a
recombinant
protein is to fuse the recombinant protein of interest to an affinity
purification tag that has
specific binding properties to a ligand which can be used to capture the
recombinant protein
of interest. Affinity purification methods have been developed for many
different applications,
and are known to the skilled worker.
[00165] Affinity-tagged fusion proteins are separated from the bulk of
other cellular
components and proteins by selective adhesion to chromatographic materials.
Typically, a
crude mixture is passed through a column where the fusion protein containing
the affinity
purification tag selectively bind to the column matrix, and are thus captured
by the column
and separated from the bulk of other materials that pass through the column
without binding.
Affinity tag-based chromatography methods allow fast, simple purification of
recombinant
proteins and thus remain an efficient purification approach regardless of the
level of
expression in the host plant, (Terpe, 2003).
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CA 02901370 2015-08-24
[00166] Immobilized Metal Ion Affinity Chromatography (IMAC) is a well-
known
approach for affinity tagged purification of recombinant proteins. IMAC for
fractionating
proteins was first disclosed by Porath, J. et al., (1975). It was discovered
that proteins could
be immobilized in a column which contained chelated metal ions in an IDA
support resin. The
proteins bind to the metal ion(s) through amino acid residues capable of
donating electrons.
Amino acids with potential electron donor groups are cysteine, histidine, and
tryptophan.
Proteins interact with metal ions through one or more of these amino acids
with electron
donating side chains. A number of factors play a role in binding; for example,
conformation of
the particular protein, number of available coordination sites on the
immobilized metal ion,
accessibility of protein side chains to the metal ion, and number of available
amino acids for
coordination with the immobilized metal ion. It is difficult to predict which
proteins will bind
and with what affinity.
[00167] A widely used improvement of this concept is the poly-histidine
affinity tag,
(His-tag), system wherein the recombinant protein comprises an extension of
six, (or more)
histidine amino acids. The histidine tag has a strong affinity to bind to
chelated nickel ions as
the affinity ligand. The column matrix is comprised of nickel-nitrilotriacetic
acid chelate [Ni-
NTA] which specifically binds the imidazole side chain of histidine. The His-
tag purification
system has achieved high levels of purification of bacterially produced
recombinant proteins
but only lower levels of purity have been achieved in plants (Sharma et al.,
2009).
[00168] Another frequently used purification scheme is based on
streptavidin (StepII)
which comprises a tag of eight amino acids (WSHPQFEK) that bind specifically
to the
vitamin, biotin, (US 5,506,121; US 6,841,359; US 7,138,253; US 7,981,632).
Columns where
biotin is fixed are used for affinity chromatography. The Strepll tag has been
used in plants
to isolate recombinant membrane-anchored protein kinase, NtCDPK2, from leaf
extracts of
N. benthamiana, (Witte et al. 2004) and recombinant Arabidopsis glutathione S-
transferase
expressed in N. benthamiana, (Dixon et al, 2009).
[00169] As affinity chromatography approaches are based on selective
binding of a
protein tag and a ligand affixed to a column or bead the use of antigenic tags
and related
antibodies for trapping fusion proteins has been used to purify certain
recombinant proteins.
Application of antibody binding to trap a protein of interest is limited
however by the very tight
association between the antigen and antibody that makes recovery of the target
protein from
the column problematic as conditions required to dissociate the bound
molecules may be
damaging to the protein of interest. The antibody approach was used to a
limited degree for
- 20 -
CA 02901370 2015-08-24
FLAG peptide, (Hopp et al, 1988), the KT3 epitope peptide, (Martin et al.,
1992) and the
alpha tubulin epitope protein, (Skinner et al., 1991).
[00170] Additional protein based affinity tags include phosphoprotein
affinity resins,
(US 7,294,614), carbonic anhydrase tags, (US 5,595,887), a zein-based
polypeptide tag, (US
7,732,569) and elastin, (US 7,928,191).
[00171] Other affinity tags that have been used to recover recombinant
proteins from
plants include the ability of peptide tags to bind to various types of
carbohydrates. Examples
include binding to starch, (US 7,135,619; US 7,662,918), maltose, (US
5,643,758; US
7,883,867), cellulose, (US 5,137,819) and chitin, (US 6,897,285; US
7,732,565). Affinity
fusion-based chromatography methods allow fast and one-step purification.
[00172] Once a fusion protein has been separated from a complex mixture
and
purified by any affinity chromatography method it may be necessary to remove
the affinity
purification tag to achieve desired function. It may be possible to achieve
this chemically by
use of cyanogen bromide that cleaves at a methionine residue, (US 4,366,246),
but this
approach has limited utility.
[00173] In some examples, cleavage is accomplished via the action of a
protease. The
protease may be an exopeptidase, or an amino peptidase that trims unwanted
amino acids
from the end of peptide chains. However of greater utility are endopeptidases
that cleave a
specific peptide sequence placed between the protein of interest and the
affinity tag. A wide
variety of different proteases could be considered for this function,
preference being those
that are highly specific for the target cleavage sequence.
[00174] Various proteases that have been used to cleave affinity tags or
fusion
proteins include Factor Xa, (that cleaves the tetrapeptide: ile, glu, gly,
arg), collagenase,
(that cleaves pro-xyz-gly ! pro) viral protease, V8 peptidase, ubiquitin
hydrolase, IgA
protease, potyvirus protease, Ulp protease, cornanvirus 30-like protease,
picornavirus
protease, subtilisin, trypsin, rennin and tobacco etch virus protease.
[00175] Methods for the generation of proteolytic enzymes that are
specific against
selected peptide sequences have been described in US 5,602,021, US 6,383,775,
US
5,780,279.
[00176] In planta, chymosin and pepsin have been used to cleave
recombinant
proteins that have been fused to oil-body oleosins, (US 5,650,554; US
7,501,265; US
7,531,315).
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CA 02901370 2015-08-24
[00177] In a preferred embodiment the protein fusion tag for affinity
chromatographic
recovery of a fusion protein is soybean agglutinin, (SBA), (Tremblay et al,
2010; 2011).
Soybean agglutinin is a tetrameric lectin glycoprotein from soybean seed. SBA
binds to N-
acetyl-D-galactosamine and is able to agglutinate cells with this glycan on
their surface. SBA
represents approximately 2% of the soluble protein in soybean seed and can be
isolated
from soybean flour following purification using N-acetyl-D-galactosamine
beads, (Percin et
al., 2009).
[00178] Soybean agglutinin (SBA) is a specific N-acetylgalactosamine-
binding plant
lectin that is a novel and a superior affinity tag for high-quality
purification of tagged proteins
expressed in planta. The preferred cleavage sequence for the removal of the
SBA affinity
chromatography tag from fusion proteins is the TEV protease sequence that has
been shown
to provide a highly specific and dependable cleavage reaction, (Carrington et
al, 1988;
Kapust et al, 2002). The TEV protease can be conveniently produced on a large
scale,
(Blommel et al., 2007).
[00179] The highly specific interaction of the SBA tag with a
galactosamine affinity
column represents a preferred mechanism of achieving efficient and
commercially scalable
transgenic protein recovery and the soybean agglutinin affinity
chromatographic separation
of fusion proteins is superior to other affinity separation systems.
[00180] It will be appreciate that it is not always necessary to remove
the affinity
purification tag. In some examples, the recombinant protein containing the
affinity
purification tag retains (or sufficiently retains) the function of the
recombinant protein as
compared to a recombinant protein which does not include the affinity
purification tag.
[00181]
[00182] In one example herein, IL-37 is made as a fusion with soybean
lectin. The
lectin fusion proved to be stable, effective as an affinity tag and with equal
to or greater
activity than un-fused interleukins.
[00183] Uses
[00184] Plant production systems have been described for the production of
therapeutic antibodies (Hiatt et al.1989, US 5,202,422; US 5,639,947; US
5,959,177; US
5,639,947; US 6,852,319; US 6,995,014; US 7,037,732). Production by Hiatt et
al was
undertaken in transgenic tobacco. The further refinement of antibody
manufacture included
secretion from whole plants such as duckweed, (US 7,632,983) and tobacco cell
suspension
cultures, (US 6,080,560; US 6,140,075). Antibody production technologies were
further
- 22 -
CA 02901370 2015-08-24
refined by association with seed oil-bodies, (US 7,098,383) and modification
of plant
glycosylation reactions to produce antibodies with a glycosylation pattern
more similar to
mammalian systems, (US 7,632,983).
[00185] Edible plant products
[00186] Another application of plant molecular farming that has been
described is the
development of plant made vaccines, including those that can be delivered by
oral
consumption of the transgenic plant materials, (reviewed by Rybicki, 2010),
and US
5,654,184; US 5,679,880; US 5,679,679; and US 5,686,079. They describe tobacco
derived
antigens that are incorporated into foods to combat bacterial disease agents
such as
Streptococcus mutans, (dental caries), E. coli and Salmonella (food
poisoning). The concept
that the antigenic substance can be incorporated in an edible food such as a
banana or
tomato was developed by Arntzen et al. (US 5,484,719; US 5,612,487; US
5,914,123; US
6,034,298; US 6,136,320; US 7,504,560,). A wide variety of different disease
applications
have been considered including virus-mediated disease, (US 7,985,891; US
8,158,855; US
7,527,810; US 8,513,397, US 8,535,930, Joensuu, et alõ 2009), bacteria-
mediated disease,
(US 6,261,561; US 6,406,889; US 6,881,411; US 7,354,760, Kolotilin et al,
2012) and
medical conditions such as senile dementia, (US 7,700,837).
[00187] Methods of the invention are conveniently practiced by providing
the
compounds and/or compositions used in such method in the form of a kit. Such a
kit
preferably contains the composition. Such a kit preferably contains
instructions for the use
thereof.
[00188] Methods of the invention are conveniently practiced by providing
the
compounds and/or compositions used in such method in the form of a commercial
packaging. Such a commercial packaging preferably contains the composition.
Such a
commercial packaging preferably contains instructions for the use thereof.
[00189] The invention can be further illustrated by the following
examples, although it
will be understood that these examples are included merely for the purposes of
illustration
and are not intended to limit the scope of the invention unless otherwise
specifically
indicated.
[00190] EXAMPLES
[00191] Construction of IL-37 expression vectors
[00192] Human IL-37 has 5 isoforms (IL-37a to e) with IL-37b being the
longest, (218
amino acids). IL-37a, IL-37c, IL-37d and IL-37e are deletion variants of IL-
37b that result
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CA 02901370 2015-08-24
from alternative pre-mRNA splicing (Figure 1). The native nucleotide sequence
and the
deduced amino acid sequences of IL-37 isoforms a to e are shown in Figures 2
to 6,
respectively. Detailed comparison of the amino acid sequences of five IL-37
isoforms is
shown in Figure 7. A cDNA clone comprising the human IL-37b DNA sequence was
purchased from OriGene Technologies, (Rockville, Maryland, USA). This cDNA
clone was
used as a common template in polymerase chain reactions (PCR) to obtain the
coding
sequence of IL-37b as well as to obtain the coding sequences for IL-37a, IL-
37d and IL-37e.
[00193] To optimize the expression of IL-37 in plant cells, synthetic
genes encoding
each of the five isoforms of IL-37, with codon usage optimized for expression
in plant cells,
were created based on the relative codon usage frequencies of abundant
proteins in tobacco
(Sardana et al., 1996).
[00194] Fusion genes comprising IL-37 coding sequence fused in-frame to
the
soybean agglutinin (SBA) coding sequence were also produced. Constructed IL-37
expression vectors were transferred into Agrobacterium for Agrobacterium-
mediated plant
transformation.
[00195] IL-37b-la
[00196] IL-37b-la is a vector designed to express the native form of human
IL-37b in a
constitutive manner (see Fig. 8). The IL-37b-1a vector consists of the
unmodified native DNA
of IL-37b (Figure 3) flanked by a cauliflower mosaic virus (CaMV) 35S
promoter, that drives
constitutive expression, followed by a tobacco etch virus 5' untranslated
leader sequence
(TEV 5'-UTR) that enhances protein expression levels (Carrington and Freed
1990) and a
nopaline synthase (nos) terminator sequence from Agrobacterium tumefaciens to
ensure
proper transcription termination. To construct this vector, the coding region
of IL-37b was
amplified from the IL-37 cDNA clone (clone No.SC122809-0R, OriGene
Technologies) by
PCR using designed forward (5'-ATATACATGTCAGGCTGTGATAGGAGGG-3'; SEQ ID NO:
1) and reverse (5'-ATATTCTAGATTACT AATCGCTGACCTCACTGGGGCTC-3'; SEQ ID
NO: 2) primers. The forward primer contains a Pci1 restriction endonuclease
site (underlined)
as part of the translation start region, whereas the reverse primer contains
an Xba1
restriction endonuclease site (underlined) immediately after the stop codon.
After
amplification, the PCR product was digested with Pci1 and Xba1 endonucleases,
and ligated
into the Nco1 and Xba1 digested sites of the intermediate plasmid pTRL2-GUS
replacing the
GUS reporter gene. Plasmid pRTL-GUS contains an enhanced double CaMV 35S
promoter
(35S), that drives constitutive expression of a GUS reporter gene, a tobacco
etch virus 5'
- 24 -
CA 02901370 2015-08-24
untranslated leader sequence (TEV 5'-UTR) and a nos terminator sequence
(Carrington and
Freed, 1990). The 1L-37b-la expression cassette comprising the 35S promoter,
the TEV 5'-
UTR, the native coding sequence of IL-37b and the nos terminator was then
released as a
single Hindi!l fragment from pRTL-IL-37b and ligated into the same site of the
binary plant
transformation vector pB1101.
[00197] IL-37b-lb
[00198] The IL-37b-lb vector, as shown in Figure 8, is identical to 1L-37b-
la vector
except for a poly-histidine (6xHis) tag placed at the C-terminus for the
convenience of
downstream processing. To construct this vector, the coding region of IL-37b
was amplified
from the IL-37 cDNA clone (OriGene Technologies) by PCR using the reverse
primer (5'-
ATATTCTAGATTAGTGATGATGATGATGATGATCGCTGACCTCACTGGGGCTC-3'; SEQ
ID NO: 3) that contains extra nucleotides encoding the 6xHis tag. The PCR
product was
digested with Pci1 and Xba1endonucleases, ligated into pTRL2-GUS and then into
pB1101
using the procedures described above for the construction of IL-37b-la.
[00199] IL-37a-1
[00200] IL-37a-1 is a vector designed to express the native form of human
IL-37a in a
constitutive manner as shown in Figure 9. The IL-37a-1 clone consists of
unmodified native
nucleotide sequence of IL-37a (Figure 4) flanked by a 35S promoter followed by
a TEV 5'-
UTR and a nos terminator sequence. To construct this clone, the coding
sequence of IL-37a
was amplified from the OriGene Technologies clone (clone No.SC122809-0R) by
PCR using
designed forward (5'-ATACATGTCAGGCTGTGATAGGAGGGAAACAGAAACCAAAGGAAA
GAACAGCTTTAAGAAGCGCTTAAGAGGTCCAAAGGTGAAGAACTTAAACCCGAAG-3';
SEQ ID NO 4) and reverse (5'-ATATTCTAGATTACTAATCGCTGACCTCACTGGGGCTC-3';
SEQ ID NO 5) primers. The forward primer contains a Pcil site (underlined) as
part of the
translational start site, whereas the reverse primer contains an Xba1 site
(underlined)
immediately after the stop codon. After amplification, the PCR product was
digested with
Pci1 and Xba1 endonucleases, and ligated into pTRL2-GUS digested with Nco1 and
Xba1.
The IL-37a.1 expression cassette was then released as a single HindIllfragment
and ligated
into the binary vector pB1101.
[00201] IL-37d-1
[00202] IL-37d.1 is a vector designed to express the native form of human
IL-37d in a
constitutive manner as shown in Figure 10. To construct this clone, the native
coding
sequence of IL-37d was amplified from the IL-37a-1 vector constructed above by
PCR using
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CA 02901370 2015-08-24
forward (5'-TTACATGTCCTTTGTGGGGGAGAACTCAGGAGTGAAAATGGGC
TCTGAGGACTGGGAAAAAGATGAACCCCAGTGCTGCTTAGAAGGTCCAAAGGTG
AAGAACTTAAACCCG-3'; SEQ ID NO: 6) and reverse (5'-
ATATTCTAGATTACTAATCGCTGACCTC ACTGGGGCTC-3'; SEQ ID NO: 7) primers. The
forward primer contains a Pci1 site (underlined) as part of the translational
start site, while
the reverse primer contains an Xba1 site (underlined) immediately after the
stop codon. After
amplification, the PCR product was digested with Pci1 and Xba1 endonucleases,
and ligated
into pTRL2-GUS, and then into binary plasmid pB1101 as described above.
[00203] IL-37e-1
[00204] IL-37e.1 is a vector designed to express the native form of human
IL-37e in a
constitutive manner as shown in Figure 11. To construct this vector, the
coding sequence of
IL-37e was amplified from the vector IL-37a-1 by PCR using forward (5'-
TTACATGTCCTTTGTGGGGGAGAACTCAGGAGTGAAAATGGGCTCTGAGGACTGGGAA
AAAGATGAACCCCAGTGCTGCTTAGAAGAGATCTTCTTTGCATTAGCCTCATCC -3'; SEQ
ID NO: 8) and reverse (5'-ATATTCTAGATTACTAATCGCTGACCTCACTGGGGCTC-3';
SEQ ID NO: 9) primers. The forward primer contains a Pci1 site (underlined),
while the
reverse primer contains an Xba1 site (underlined) immediately after the stop
codon. The
PCR product was digested with Pci1 and Xba1endonucleases, and ligated into
plasmid
pTRL2-GUS, and then into binary plasmid pB1101 as described above.
[00205] IL-37c-1
[00206] IL-37c-1 is a vector designed to express the native form of human
IL-37c in a
constitutive manner as shown in Fig. 12. To construct this vector, a DNA
fragment based on
the native nucleotide sequence of IL-37c was synthesized using a commercial
service (Bio
Basic Canada Inc., Markham, Ontario, Canada), with a poll endonuclease site
being
introduced at the translation start and an Xba1 nuclease site introduced
immediately after the
stop codon. The synthesized IL-37c DNA was digested with pci1 and Xba1, and
cloned into
plasmid pTRL2-GUS and then into plant binary vector pB1101 as described above.
[00207] IL-37b-2
[00208] IL-37b-2 is a vector designed to increase the expression of a
plant, (tobacco)
codon-optimized human IL-37b gene in plant cells. The IL-37b-2 vector (shown
in Figure 8)
consists of 35S promoter including TEV 5'-UTR, plant codon-optimized human IL-
37b gene
with the native signal peptide replaced by a barley a-amylase signal peptide
(Figure 13), and
nos terminator. To construct this vector, a tobacco codon-optimized nucleotide
sequence
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CA 02901370 2015-08-24
encoding IL-37b with a barley a-amylase signal peptide with the codon usage
frequencies of
abundant proteins of tobacco plant was designed and subsequently generated
using a
commercial service (Bio Basic Canada Inc., Markham, Ontario, Canada). To
facilitate sub-
cloning, an Nco1 endonuclease site was introduced at the translation start and
an Xba1
endonuclease site introduced immediately after the stop codon. The plant codon-
optimized
IL-37b gene was digested with Nco1 and Xba1 endonucleases, and cloned into
pTRL2-GUS
replacing the GUS reporter. The resulting IL-37b.1.1 expression cassette was
then released
as a HindlIl fragment and cloned into plant transformation vector pB1101.
[00209] IL-37a-2
[00210] IL-37a-2 is a vector designed to increase the expression of a
plant codon-
optimized human IL-37a gene in plant cells. The IL-37a-2 vector (shown in
Figure 9) consists
of 35S promoter including TEV 5'-UTR, plant (tobacco) codon-optimized human IL-
37a gene
containing the signal peptide coding sequence of barley a-amylase, and nos
terminator (Fig.
14). To construct this vector, a tobacco codon-optimized synthetic IL-37a gene
containing a
signal peptide coding sequence of barley a-amylase was designed and
subsequently created
using a commercial service (Bio Basic Canada Inc., Markham, Ontario, Canada).
To facilitate
sub-cloning, an Nco1 endonuclease site was introduced at the translation
start, while an
Xba1 endonuclease site was added at the C-terminal immediately after the stop
codon. The
plant-optimized synthetic IL-37b gene was digested with Nco1 and Xba1
endonucleases, and
cloned into pTRL2-GUS replacing the GUS reporter gene. The resulting IL-37a-2
expression
cassette was then released and cloned into plant transformation vector pBI101.
[00211] IL-37d-2
[00212] IL-37d-2 is a vector designed to increase the expression of a
plant codon-
optimized IL-37d gene in plant cells. The IL-37d-2 vector, (shown in Figure
10), consists of
35S promoter including TEV 5'-UTR, plant (tobacco) codon-optimized human IL-
37d gene
containing the signal peptide coding sequence of barley a-amylase, and nos
terminator. To
construct this vector, a tobacco codon-optimized synthetic IL-37d gene
containing a signal
peptide coding sequence of barley a-amylase (shown in Figure 15) was designed
and
subsequently created using a commercial service (Bio Basic Canada Inc.,
Markham, Ontario,
Canada). Procedures for the sub-cloning of plant-optimized synthetic IL-37d
gene into
pTRL2-GUS and then into pB1101 were equivalent to those used in the
construction of IL-
37b-2.
[00213] IL-37e-2
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CA 02901370 2015-08-24
[00214] IL-37e-2 is a vector designed to increase the expression of a
plant codon-
optimized IL-37e gene in plant cells. The IL-37e-2 vector, (shown in Figure
11) consists of
35S promoter including TEV 5'-UTR, plant (tobacco) codon-optimized human IL-
37e gene
containing the signal peptide coding sequence of barley a-amylase, and nos
terminator. To
construct this vector, a tobacco codon-optimized synthetic IL-37e gene
containing the signal
peptide coding sequence of barley a-amylase (shown in Fig. 16) was designed
and
subsequently created using a commercial service (Bio Basic Canada Inc.,
Markham, Ontario,
Canada). Procedures for the sub-cloning of the plant-optimized synthetic IL-
37e gene into
pTRL2-GUS and then into pB1101 were equivalent to those used in the
construction of IL-
37b-2.
[00215] IL-37c-2
[00216] IL-37c-2 is a vector designed to increase the expression of a
plant codon-
optimized IL-37c gene in plant cells. The IL-37e-2 vector, (shown in Figure
12), consists of
35S promoter including TEV 5'-UTR, plant (tobacco) codon-optimized human IL-
37e gene
containing the signal peptide coding sequence of barley a-amylase, and nos
terminator. To
construct this vector, a tobacco codon-optimized synthetic IL-37e gene
containing the signal
peptide coding sequence of barley a-amylase (shown in Figure 17) was designed
and
subsequently created using a commercial service (Bio Basic Canada Inc.,
Markham, Ontario,
Canada). Procedures for the sub-cloning of the plant-optimized synthetic 1L-
37c gene into
pTRL2-GUS and then into pB1101 were equivalent to those used in the
construction of IL-
37b-2.
[00217] IL-37b-3
[00218] IL-37b-3 is a vector designed to target the expression of plant-
optimized
human IL-37b to the endoplasmic reticulum (ER) compartment in plant cells. As
shown in
Figure 8, vector IL-37b-3 is identical to IL-37b-2, except for the addition of
a 6xHis tag and an
ER-targeting KDEL motif at the C-terminus. To construct this vector, IL-37b-2
was subjected
to PCR modification using forward (5'- ATATCCATGGGAAAGAACGGTTCACTATGCT G -3';
SEQ ID NO: 10) and reverse (5'-
ATATCTAGATTATAACTCATCTTTGTGATGATGATGATGGT
GGTCTGACACCTCTGACGGAGACATTTCG -3'; SEQ ID NO: 11) primers. The forward
primer contains a Nco1 endonuclease site (underlined) as part of the
translational start site,
whereas the reverse primer contains the sequence encoding the 6xHis tag and an
ER
retention KDEL motif before the stop codon and a Xba1 endonuclease site
(underlined). The
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CA 02901370 2015-08-24
PCR product was digested with Nco1 and Xba1, sub-cloned into pTRL2-GUS,
replacing the
GUS gene and then into the pB1101 plant transformation vector using the
procedures
equivalent to those used for the construction of IL-37b-2.
[00219] IL-37a-3
[00220] IL-37a-3 is a vector designed to target the expression of plant-
optimized
human IL-37a to the plant cell ER compartment. As shown in Figure 9, the IL-
37a-3 vector is
identical to IL-37a-2, except for a C-terminal 6xHis tag and ER-targeting KDEL
signal motif.
To construct this vector, IL-37a-2 was subjected to PCR modification using
forward (5'-
ATATCCATGGGAAAGAACGGITCACTATGCTG -3'; SEQ ID NO: 12) and reverse (5'-
ATATCTAGATTATAACTCATCTTTGTGATGATGATGATGGTGGTCTGACACCTCTGACGG
AGACA TTTCG -3'; SEQ ID NO: 13) primers. The forward primer contains a Nco1
endonuclease site (underlined) as part of the translational start site,
whereas the reverse
primer contains the sequence encoding a 6xHis tag, and an ER targeting KDEL
signal motif
and a Xba1 endonuclease site (underlined). The PCR product was digested with
Nco1 and
Xba1, sub-cloned into pTRL2-GU5, replacing the GUS gene and then into the
pB1101plant
transformation vector using the procedures equivalent to those used for the
construction of
IL-37a-2.
[00221] IL-37d-3
[00222] IL-37d-3 is a vector designed to target the expression of plant-
optimized
human IL-37d to the plant cell ER compartment. As shown in Figure 10, the IL-
37d-3 vector
is identical to IL-37d-2, except for a C-terminal 6xHis tag and ER-targeting
KDEL signal motif
To construct this vector, IL-37d-2 was subjected to PCR modification using
forward (5'-
ATATCCATGGGTAAAAACGGATCTCTATGC-3'; SEQ ID NO: 14) and reverse (5'-
ATATCTAGATTACAACTCATCTTTGTGATGATGATGATGGTGGTCAGACACCTCAGAGGG
GGACATC-3'; SEQ ID NO: 15) primers. The forward primer contains a Nco1
endonuclease
site (underlined) as part of the translational start site, whereas the reverse
primer contains
the sequence encoding a 6xHis tag, and an ER targeting KDEL signal motif and a
Xba1
endonuclease site (underlined). The PCR product was digested with Nco1 and
Xba1, sub-
cloned into pTRL2-GUS, replacing the GUS gene and then into the pB1101 plant
transformation vector using the procedures equivalent to those used for the
construction of
IL-37d-2.
[00223] IL-37e-3
- 29 -
CA 02901370 2015-08-24
[00224] IL-37e-3 is a vector designed to target the expression of plant-
optimized
human IL-37d to the plant cell ER compartment. As shown in Figure 11, the IL-
37e-3 vector
is identical to IL-37e-2, except for a C-terminal 6xHis tag and ER-targeting
KDEL signal
motif. To construct this vector, IL-37e-2 was subjected to PCR modification
sing forward (5'-
ATATCCATGGGTAAAAACGGAAGCCTTTGTTG-3'; SEQ ID NO: 16) and reverse (5'-
ATATCTAGATTAAAGTTCATCTTTGTGATGGTGATGGTGATGATCTGAAACCTCACTTGG
ACTCATCTC -3'; SEQ ID NO: 17) primers. The forward primer contains a Nco1
endonuclease site (underlined) as part of the translational start site,
whereas the reverse
primer contains the sequence encoding a 6xHis tag, and an ER targeting KDEL
signal motif
and an Xba1 endonuclease site (underlined). The PCR product was digested with
Nco1 and
Xba1, sub-cloned into pTRL2-GUS, replacing the GUS gene and then into the
pB1101 plant
transformation vector using the procedures equivalent to those used for the
construction of
IL-37d-2.
[00225] IL-37c-3
[00226] 1L-37c-3 is a vector designed to target the expression of plant-
optimized
human IL-37c to the plant cell ER compartment. As shown in Figure12, the IL-
37c-3 clone is
identical to 1L-37c-2, except for a C-terminal 6xHis tag and ER-targeting KDEL
signal motif.
To construct this clone, IL-37c-2 was subjected to PCR modification using
forward (5'-
ATATCCATGGGTAAAAACGGAAGCCTTTGTTG -3'; SEQ ID No: 18) and reverse (5'-
ATATCTAGATTAAAGTTCATCTTTGTGATGGTGATGGTGATGATCTGACACTTCAGACGG
TGAC-3'; SEQ ID NO: 19) primers. The forward primer contains a Nco1
endonuclease site
(underlined) as part of the translational start site, whereas the reverse
primer contains the
sequence encoding a 6xHis tag, and an ER targeting KDEL signal motif and a
Xba1
endonuclease site (underlined). The PCR product was digested with Nco1 and
Xba1, sub-
cloned into pTRL2-GUS, replacing the GUS gene and then into the pB1101 plant
transformation vector using the procedures equivalent to those used for the
construction of
IL-37c-2.
[00227] IL-37b-4
[00228] IL-37b-4 is a vector designed to express constitutively a plant
codon
,(tobacco) optimized human IL-37b gene as a fusion with soybean agglutinin
(SBA), as
shown in Figure 8. To construct this vector, (see Figure 8) a tobacco codon-
optimized DNA
encoding SBA, a linker region of 15 amino acids (SGGGGSGGGGSGGGG; SEQ ID NO:
20), a Cla1 recognition site (ATCGAT), a TEV protease cleavage site (ENLYFQG;
SEQ ID
- 30 -
CA 02901370 2015-08-24
NO: 21) and the mature form of IL-37b was synthesized (Figure 18) by a
commercial DNA
sequence service provider (Bio Basic Canada Inc., Markham, Ontario, Canada).
To facilitate
sub-cloning, an Nco1 endonuclease site was introduced at the translation start
and an Xba1
endonuclease site introduced immediately after the stop codon. The SBA-IL-37b
synthetic
fusion gene was isolated from pUC-SBA-1L-37b following digestion with Nco1 and
Xba1
endonucleases, and sub-cloned into the same sites of pTRL2-GUS replacing the
GUS
reporter gene. The resulting SBA-1L-37b expression cassette was then cloned
into the plant
transformation vector pB1101 as a single HindlIl fragment.
[00229] IL-37a-4
[00230] IL-37a-4 is a vector designed to express plant codon, (tobacco)
optimized IL-
37a gene as a fusion with soybean agglutinin (SBA) as shown in Figure 9. To
construct this
vector, a DNA sequence encoding the mature form of IL-37a was amplified by PCR
using
clone IL-37a-2 as a template in the presence of forward (5'-
ATATCGATGAAAATTTGTATTTTCAAGGCAGAGGTCCTAAGGTAAAGAATCTTAACC -3';
SEQ ID NO: 22) and reverse (5'-
TAATCTAGATTAGTCGGAAACTTCAGACGGGGACATTTCG -3'; SEQ ID NO: 23) primers.
The forward primer contains a Cla1 site (underlined) for in-frame fusion with
the fusion
partner SBA and also contains a sequence encoding the TEV protease cleavage
site
ENLYFQG. The reverse primer contains an Xba1 endonuclease cleavage site
(underlined)
immediately after the stop codon. As illustrated in Figure 19, the PCR product
was digested
with Cla1 and Xba1, and ligated into pUC-SBA-1L-37b replacing the IL-37b
portion. The
resulting SBA-1L-37a fusion gene (Figure 20) was then released as an Nco1 and
Xba1
fragment and ligated into pTRL2-GUS replacing GUS reporter. The SBA-1L-37a
expression
cassette was isolated as a single Hindi!l fragment and ligated into plant
transformation vector
pB1101.
[00231] IL-37d-4
[00232] IL-37d-4 is a vector designed to express plant codon optimized IL-
37d as a
fusion with SBA as shown in Fig.10. To construct this vector, the same
procedure described
above for the construction of SBA-1L-37a was used. The DNA sequence encoding a
mature
form of IL-37d was obtained by PCR amplification of clone IL-37d-2 using the
forward (5'-
ATATCGATGAAAATTTGTATTTTCAAGGGAACCTCAATGTTGTTTAGA AGG-3'; SEQ ID
NO: 24) and reverse (5'-TAATCTAGATTAGTCAGACACCTCAGAGGGGGAC -3'; SEQ ID
- 31 -
CA 02901370 2015-08-24
NO 25). Nucleotide sequence and the deduced amino acids of SBA-IL-37d fusion
gene were
shown in Figure 21.
[00233] IL-37e-4
[00234] IL-37e-4 is a vector designed to express plant, (tobacco) codon
optimized IL-
37d as a fusion with SBA as shown in Figure11. To construct this vector, the
same
procedure as described above for the construction of SBA-IL-37a was used. The
DNA
sequence encoding a mature form of IL-37e was obtained by PCR amplification of
clone IL-
37e-2 using the forward (5'- ATATCGATGAAAATTTGTATTTTCAAGGCGAGCCACAG
TGTTGCTTGGAAGAG -3'; SEQ ID NO: 26) and reverse (5'-
TAATCTAGATTAATCTGAAACCTCAC TTGGACTC -3'; SEQ ID NO: 27) primers. Nucleotide
sequence and the deduced amino acid sequence of the SBA-IL-37e fusion gene are
shown
in Figure 22.
[00235] IL-37c-4
[00236] IL-37c-4 is a vector designed to express plant, (tobacco) codon
optimized IL-
37c as a fusion with soybean agglutinin (SBA) as shown in Fig.12. To construct
this clone,
the same procedure as described above for the construction of SBA-IL-37a was
used. The
DNA sequence encoding a mature form of IL-37c was obtained by PCR
amplification of
clone IL-37c-2 using the forward (5'-ATATCGATGAAAATTTGTATTTTCAAGGCGTA
CATACTATATTCTTCGCTCTAG -3'; SEQ ID NO: 28) and reverse (5'-
TAATCTAGATTAATCTGA CACTTCAGACGGTGAC -3'; SEQ ID NO: 29) primers. The
nucleotide sequence and the deduced amino acids of the SBA-IL-37e fusion gene
are shown
in Figure 23.
[00237] IL-37b-5
[00238] IL-37b-5 is a vector designed for seed-specific expression of a
plant-preferred
codon optimized SBA-IL-37b fusion protein as shown in Figure 8. IL-37b-5 is
identical to IL-
37b-4, except that the 35S promoter in the IL-37b-4 construct was removed and
replaced
with a seed-specific LegA2 promoter from pea (P/sum sativum L.) legumin
protein A
encoding the legA gene. The 857-bp LegA2 promoter was amplified by PCR from
plasmid
pLPhA (Lycett, et al., 1985) using forward (5'-TATAATAAGCTTGTCGACAATT
CCTTCTTAATGGTAGTCTAGTTTAC-3'; SEQ ID NO: 30) and reverse (5'-
TATATTAAGCTTGAGCTCG
GATCCATGGCTCGAGTGGTTGGATAGAATATATGTTTGTGACGCG-3'; SEQ ID NO: 31)
primers.
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CA 02901370 2015-08-24
[00239] IL-37a-5
[00240] IL-37a-5 is a vector designed for seed-specific expression of a
plant-preferred
codon optimized SBA-IL-37a fusion protein as shown in Figure 9. IL-37a-5 is
identical to IL-
37a-4, except that the 35S promoter in IL-37a-4 construct was replaced with
seed-specific
LegA2 promoter as described above.
[00241] IL-37d-5
[00242] IL-37d-5 is a vector designed for seed-specific expression of a
plant-preferred
codon optimized SBA-IL-37d fusion protein as shown in Figure 10. IL-37d-5 is
identical to IL-
37d-4, except that the 35S promoter in IL-37d-4 construct was replaced with
seed-specific
LegA2 promoter as described above.
[00243] IL-37e-5
[00244] IL-37ed-5 is a vector designed for seed-specific expression of a
plant-
preferred codon optimized SBA-IL-37e fusion protein as shown in Figure 11. IL-
37e-5 is
identical to IL-37e-4, except that the 35S promoter in IL-37e-4 construct was
replaced with
seed-specific LegA2 promoter as described above.
[00245] IL-37c-5
[00246] IL-37c-5 is a vector designed for seed-specific expression of a
plant-preferred
codon optimized SBA-IL-37c fusion protein as shown in Figure 12. IL-37c-5 is
identical to IL-
37c-4, except that the 35S promoter in IL-37c-4 construct was replaced with
seed-specific
LegA2 promoter as described above.
[00247] Example 2
[00248] Transient Transformation of Nicotiana benthamiana with
Agrobacterium
Containing IL-37 Gene Constructs
[00249] The IL-37 gene constructs described above were transferred into
Agrobacterium tumefaciens by tri-parental mating (Ma et al., 2005). The
Agrobacterium strain
used was LBA4404 carrying the disarmed Ti plasmid pAL 4404. Transient
expression of the
IL-37 constructs was achieved by infection of 6-8-week-old leaves of Nicotiana
benthamiana
by the method of Sparkes et al. (2006) with minor modifications. Overnight
cultures of
Agrobacterium containing an individual IL-37 gene construct were grown until a
density of an
A600 0.5 to 1 was reached, at which time the cells were centrifuged at 800g
for 10 min, rinsed
four times with infiltration medium (0.5% D-glucose, 50 mM MES, 2 mM Na3PO4,
0.0001 M
acetosyringone), and then re-suspended at a cell density of A600=0.5. A second
Agrobacterium strain containing the gene for p19, a viral protein that
inhibits RNA silencing
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CA 02901370 2015-08-24
and is known to boost the yield of transiently expressed proteins (Lakatos et
al., 2004;
Tremblay et al., 2011) was also similarly prepared. The two Agrobacterial
cultures were
mixed at equal concentration and the mixture was then infiltrated into the
abaxial side of the
leaves using a 1-mL needle-free syringe and infected tissue was harvested at
day 2 through
day 5 post-infection.
[00250] Example 3
[00251] Analysis of IL-37 Expression in Transiently Transformed Nicotiana
benthamiana
[00252] Total Leaf Protein Preparation
[00253] Total soluble leaf protein was extracted using an extraction
buffer consisting
of: 200 mM Tris pH 8.0, 100 mM NaCI, 400 mM Sucrose, 1 mM phenylmethylsulfonyl
fluoride, 10 mM EDTA, 14 mM b-mercaptoethanol, 0.05% Tween-20, 2 pg/mL
leupeptin, 2
pg/mL aprotinin). Agrobacterium infiltrated leaf tissue was ground with a
pestle in liquid
nitrogen and successively homogenized in cold extraction buffer at a leaf
tissue: buffer ratio
of 1:3. The mixture was incubated on ice for 30 min and centrifuged for 20 min
at 20,000 x g
at 4 C. The supernatant was transferred to 1.5-mL tubes and re-centrifuged for
an additional
min at 40C to remove remaining leaf debris. The supernatant was then decanted
to new
1.5-ml tubes and stored at 4 degree C. on ice.
[00254] The expression of IL-37 in total extract of Agrobacterium-
infiltrated Nicotiana
benthamiana leaves was determined by Western blot. Protein samples were boiled
for 10
min prior to loading on a 10% SDS¨PAGE gel and resolved. Gels were then
transferred to
polyvinylidene difluoride (PVDF) membranes as described previously (Tremblay
et al. 2011).
The blot was then blocked for overnight at 4 C degree with 5% w/v milk in TBS-
T and
washed 3 times, each time for 5 min, with TBS-T. The blots were incubated
overnight at 4 C
in 1:3000 rabbit anti-IL-37 (Abcam #ab116282) in 1/3 blocking buffer 2/3 wash
buffer. The
blots were washed 3 times, each time for 10 min, in wash buffer and incubated
in 1:5,000
goat anti-rabbit- IgG (H+L) (KPL #074-1506) for 1 h at room temperature. The
blots were
washed 3 times, each time for 10 min, in wash buffer and then incubated with
Bio-Rad Clarity
western ECL substrate (#170-5060). Blots were exposed and then developed using
a film
processor.
[00255] Detection of recombinant native IL-37b protein in Nicotiana
benthamiana
Transiently Transformed with IL-37b-1a
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CA 02901370 2015-08-24
[00256] As seen in Example 1, IL-37b-la expresses a native IL-37b with its
native
signal peptide from the unaltered native DNA sequence (Figure 8). A 35S
promoter and Nos
terminator are used for constitutive expression of the construct. Leaf samples
were collected
from three independent plants on different days post-infiltration. The results
of Western blot
analysis showed the expression of recombinant native IL-37 in leaf samples
collected from
day 2 to day 5 (Figure 24). There are two bands of approximately 25 and 50 kDa
that
showed strong reactivity with the IL-37 antibody. The band of 25 kDa
corresponds to the size
of the monomeric form of IL-37 (indicated by a single-headed arrow), whereas
the band of 50
kDa corresponds to the size of the dimeric form of IL-37 (indicated by a
double-headed
arrow). A third fainter band of about 19 kDa was also seen in Western blotting
and this band
may be a degradation product of plant-made IL-37 probably caused during sample
preparation. As expected, no protein bands of similar sizes were detected from
total leaf
extract prepared from non-infiltrated leaves of N. benthamiana plants under
identical
conditions.
[00257] Detection of recombinant native IL-37d protein in Nicotiana
benthamiana
Transiently Transformed with IL-37d-1
[00258] As seen in Example 1, IL-37d-1 expresses a native IL-37d with its
native
signal peptide from the unaltered native DNA sequence (Figure 10). A 35S
promoter and
Nos terminator are used for constitutive expression of the construct. Leaf
samples were
collected from three independent plants on different days post-infiltration.
The results of
Western blot analysis showed the expression of recombinant native IL-37d in
leaf samples
collected from day 2 to day 5 (Figure 25). The anti-IL-37 antibody recognized
a dominant
band of approximately 44 to 45 kDa (indicated by a double-headed arrow),
corresponding to
the size of the dimeric form of IL-37d. A second band specifically recognized
by the same
anti-IL-37 antibody had a molecular weight of approximately 21 kDa,
corresponding to the
size of the monomeric form of IL-37 (indicated by a single-headed arrow). As
expected, no
protein bands of similar sizes were detected from total leaf extract prepared
from non-
infiltrated leaves of N. benthamiana plants under identical conditions.
[00259] Example 4
[00260] Stable transformation of low-nicotine and low-alkaloid Nicotiana
tabacum
Plants with Agrobacterium- Containing IL-37 Gene Constructs
[00261] Stable Agrobacterium-mediated transformation of low-nicotine, low-
alkaloid
tobacco cv. 81V9-4 was performed using a standard protocol (Horsch et al.,
1985).
- 35 -
CA 02901370 2015-08-24
Transformants were selected on MS medium (Murashige and Skoog 1962) containing
100
mg/L kanamycin, 500 mg/L carbenicillin, 1 mg/L BAP and 0.1 mg/L NAA. Shoots
that
developed were transferred to a phytohormone-free MS medium containing 100
mg/L
kanamycin, and 250 mg/L carbenicillin for root formation. Regenerated plants
were
transferred from Magenta TM boxes to pots, and grown further under greenhouse
conditions.
Over 25 transgenic plants were regenerated for each of IL-37 construct
transformed into
plant cells. Regenerated plants were analyzed for expression of the IL-37
protein by Western
blot and ELISA.
[00262] Example 5
[00263] Analysis of IL-37 Expression in stable transgenic Nicotiana
tabacum tobacco
plants
[00264] Total Leaf Protein Preparation
[00265] Total leaf soluble protein from stable transgenic tobacco plants
was extracted
using the same procedures as used for total protein extraction from
Agrobacterium-infiltrated
Nicotiana benthamiana leaf samples described above.
[00266] The expression of IL-37 in total extract of transgenic tobacco
leaves was
determined by Western blot as described above.
[00267] Detection of IL-37bx6His protein in stable transgenic tobacco
plants
harbouring IL-37b-1b
[00268] IL-37b-lb expresses His-tagged IL-37b with a native signal peptide
(see
Example 1 and Figure 8). A 35S promoter and Nos terminator are used for
constitutive
expression of the construct. Over 25 individual transgenic tobacco lines were
generated
following leaf disc transformation with Agrobacterium containing IL-37b-lb.
The expression
of IL-37bx6His protein in transgenic tobacco plants was analysed analyzed by
Western blot
using anti-IL-37 antibodies. As shown in Figure 26, the anti-IL-37 antibody
detected two
major bands. The smaller band with a molecular weight of approximately 24 kDa
corresponds to the size of the monomeric form of IL-37bx6His (indicated by a
single-headed
arrow). The larger band of approximately 50 kDa corresponds to the size of the
dimeric form
of IL-37bx6His (indicated by a double-headed arrow). As expected, no protein
bands of
similar sizes were detected in extracts from wild-type plants.
[00269] Detection of SBA-IL-37c fusion protein in stable transgenic
tobacco plants
harbouring IL-37c-4
- 36 -
CA 02901370 2015-08-24
[00270] IL-37c-4 expresses plant codon optimized IL-37c as a fusion with
SBA (see
Example 1 and Figure 12).
[00271] A 35S promoter and Nos terminator were used for constitutive
expression of
the construct. Over 25 individual transgenic tobacco lines were generated
following leaf disc
transformation with Agrobacterium containing IL-37c-4. The expression of SBA-
IL-37c fusion
protein in transgenic tobacco plants was analysed analyzed by Western blot
using an anti-IL-
37 antibody. As shown in Figure 27, the anti-IL-37 antibody detected several
specific bands.
The protein band with a molecular weight of approximately 50 kDa corresponds
to the size of
the monomeric form of the fusion protein (indicated by a single-headed arrow).
Those protein
bands having molecular weights above 50 kDa likely represent various
multimeric forms of
the fusion protein (indicated by a double-headed arrow), given that SBA is a
tetrameric
protein. As expected, no protein bands of similar sizes were detected in
extracts from wild-
type tobacco plants. Two additional protein bands with molecular weights of
smaller than 50
kDa that were also recognized by anti-IL-37 antibody (indicated by a star) are
likely the result
of degradation of the SBA-IL-37c fusion protein during sample preparation.
[00272] Example 6
[00273] Quantification of IL-37 expression in tobacco plants
[00274] Total soluble protein (TSP) from tobacco leaves was extracted
using the
procedures described above. The levels of IL-37 expression in total extract
were quantified
by enzyme linked immunosorbent assay (ELISA). E. coli-derived IL-37 commercial
IL-37
(R&D Systems) and triplicate plant IL-37 samples were bound to 96 well plates
by incubation
in phosphate buffer overnight at 4 C. The plates were then washed with PBS-T
and blocked
with 3% BSA in PBS-T for 1 h at room temperature. The plates were washed 3
times and
then incubated overnight at 4oC in 1:2,000 rabbit anti-IL-37 antibody (Abcam
Cat. NO.
Ab153889) in 1/2 blocking:1/2 wash buffer. The plates were washed 3 times and
incubated for
1 h at room temperature with 1:5,000 goat anti-rabbit-HRP in 1/2 blocking:1/2
wash buffer
and then rinsed 3 times. The plates were incubated with Substrate Reagent Pack
(DY999,
R&D Systems) according to the manufacturer's instructions. The color was
developed for 20
min and then stopped by addition of an equal volume of 2 M H2SO4. The plate
was then
read using a Thermomax microplate reader (Molecular Devices, USA). Standard
curves were
calculated and used to determine protein concentrations of the individual
samples. The
negative control was protein extract prepared from wild-type plant tissue.
- 37 -
CA 02901370 2015-08-24
[00275] The results of ELISA for quantification of levels of IL-37bx6His
protein
expression in individual transgenic plant lines transformed with IL-37b-1b
were are shown in
Figure 28. As can be seen, expression levels of IL-37bx6His were variable
among individual
transgenic tobacco lines. Transgenic line T4 expresses the highest level of IL-
37bx6His,
accounting for approximately 0.47% TSP.
[00276] Example 7
[00277] Glycosylation analysis of plant-derived IL-37
[00278] Glycosylation analysis was executed on the partially purified His-
tagged IL-
37b (IL-37bx6His) samples obtained from transgenic plants
[00279] His-tagged IL-37b (IL-37bx6His) was partially purified from total
soluble leaf
extracts of stable transgenic tobacco plants carrying plasmid IL-37-1 b using
HiTrapTM
Chelating HP Columns (GE Healthcare Life Sciences, Baie d'Urfe, Quebec,
Canada)
according to the manufacturer's instructions. Eluted IL-37bx6His fractions
were dialysed
dialyzed extensively against phosphate-buffered saline (PBS) and concentrated
using a
speed vacuum at 4 C.
[00280] The glycosylation status of plant-derived IL-37b was evaluated by
enzymatic
deglycosylation using peptide-N-glycosidase F (PNGase F; Sigma-Aldrich cat.
No. P7367).
PNGase F is capable of removing both high-mannose and complex type N-glycans
except
those containing a-(1,3)-linked core fucose. Five or ten micrograms of
partially purified plant-
derived IL-37bx6His were added to 45 pl of 50 mM sodium phosphate buffer (pH
7.5), and
the protein was then denatured by adding 5 pl of denaturation solution (0.2%
SDS with 100
mM 2-mercaptoethanol) followed by heating the protein solution at 100 C for
10 minutes.
Five pl of PNGase F enzyme (500 units/ml) was added and the reaction mixture
was
incubated overnight at 37 C. Deglycosylation was assessed by SDS-PAGE followed
by
Western blotting using anti-IL-37 antibody. Digestion of glycosylated human
transferrin
standard (Sigma-Aldrich) with PNGase F was used as a positive control.
[00281] As shown in Figure 29, there were no detectable band shifts seen
in partially
purified plant IL-37bx6His samples following treatment with PNGase F when
compared to
untreated samples, suggesting that plant-derived recombinant IL-37b is not
glycosylated. In
contrast, digestion of glycosylated human transferrin control with PNGase F
under identical
conditions reduced its size from original molecular weight of 78 kDa to a
smaller molecular
weight of 73 kDa, the expected size for the nonglycosylated human transferrin.
The
experiments were repeated.
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CA 02901370 2015-08-24
[00282] Example 8
[00283] One-step purification of plant-derived SBA-IL-37 fusion protein
using acetyl-D-
galactosamine-linked agarose affinity chromatography
[00284] Total soluble proteins from Agrobacterium-infiltrated leaves of
Nicotiana
benthamiana or from leaves of transgenic Nicotiana tabacum tobacco plants were
extracted
as described above. The extract was subjected to (NH4)2SO4 cut (50%), and
centrifuged at
10,000 rpm for 30 min at 4 C, and the protein pellet was re-suspended in PBS
and dialyzed
extensively against PBS. The crude protein was loaded on an N-acetyl-D-
galactosamine-
linked agarose (Sigma-Aldrich, Product NO. A2278-5ML) packed column that had
been
equilibrated with 0.1 M NaCI. The column was then washed extensively with 0.1
M NaCI until
the absorbance at 280 nm of the effluent had fallen below 0.01. The affinity-
adsorbed SBA-
IL-37 fusion protein was eluted with 0.5 M galactose in 0.1 M NaCI and
collected in a series
of fractions. The collected fractions were analysed by SDS-PAGE and visualized
by
Coomassie blue staining. SBA-IL-37 positive fractions were pooled.
[00285] Figure 30 shows a Coomassie blue stained SDS-PAGE gel to visualize
purified SBA-IL-37c samples following one-step affinity chromatography on N-
acetyl-D-
galactosamine-linked agarose column. The fusion protein was purified from
total extracts of
Nicotiana benthamiana leaves infiltrated with Agrobacterium containing IL-37c-
5 (Figure 12).
[00286] Example 9
[00287] Demonstration of biological activity of plant-derived IL-37 using
mouse kidney
primary cells
[00288] Both plant-derived IL-37bx6His and SBA-IL-37c fusion protein were
tested for
their biological activity using mouse kidney primary cells.
[00289] IL-37bx6His protein was partially purified from total soluble leaf
extracts of
stable transgenic tobacco plants transformed with IL-37-1b by using HiTrapTM
Chelating HP
Columns as described above.
[00290] SBA-IL-37 fusion protein was purified from total extracts of
Nicotiana
benthamiana leaves infiltrated with Agrobacterium containing IL-37c-5
following one-step
affinity chromatography on N-acetyl-D-galactosamine-linked agarose column as
described
above.
[00291] Mouse primary kidney cells used to test biological activity of
plant-made
human IL-37 were isolated from B6 mice (Jackson Laboratory, Maine, USA). Mice
were killed
by asphyxiation using carbon dioxide and kidneys were harvested. Primary cells
are isolated
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CA 02901370 2015-08-24
from harvested kidneys according to the protocols described by Du et al
(2007), and cultured
and maintained in k1 medium [(50:50 DMEM and Ham's F12; Invitrogen)
supplemented with
10% (v/v) FBS, hormone mix (5 Ig/mL insulin, 34 pg/mL triiodothyronine, 5
Ig/mL transferrin,
1.73 ng/mL sodium selenite, 8 ng hydrocortisone and 25 ng/mL epidermal growth
factor),
100 U/mL penicillin and 0.1mg/mL streptomycin). To conduct the assay, cells
were seeded in
triplicate in 96-well tissue culture plates in complete K1 medium at a density
of 1-5x104 cells
per well and grown overnight at 37 C to allow cell attachment to the plate.
Cells were then
treated with IL-37b-6xHis, SBA-IL-37e or commercial IL-37 at various
concentrations (0, 500,
400 or 200 ng/ml) for 24 hours. After incubation, culture media were removed
and cells were
treated with 1pg/m1 lipopolysaccharide (LPS) for 24 hours. Cells treated with
LPS alone were
used as controls. Culture media was collected and analyzed via ELISA kit for
the content of
tumor necrosis factor alpha (TNFa).
[00292] The biological activity of plant-derived IL-37bx6His and SBA-IL-
37c fusion
protein was assessed by their ability to inhibit the production of
inflammatory cytokines such
as tumor necrosis factor (TNF) in LPS-stimulated mouse primary kidney cells.
As shown in
Figure 31, LPS-stimulated mouse primary kidney cells produced high levels of
TNFa.
Addition of plant-made IL-37bx6His, SBA-IL-37c or commercial IL-37
significantly and dose
dependently reduced the production of TNFa. These results show that plant-
derived IL-
37bx6His and SBA-IL-37c fusion protein are biologically active. SBA-IL-37c
fusion protein
appeared to be much more effective than an equal concentration of commercial
IL-37 at
inhibiting LPS-induced TNFa production, suggesting improved function was
related to
increased stability of IL-37 when fused to SBA. Partially purified IL-36b-
6xHis showed some
toxicity in mouse primary kidney cells due to contamination with plant-
endogenous proteins.
[00293] EXAMPLE 10
[00294] Construction of IL-37d+hTf expression vector for Tarwi
[00295] The pLIL-37d+hTf expression vector was constructed to express the
fusion
gene (human IL-37d+human Transferrin) in tarwi seed. This vector contains the
LegA2 seed
specific promoter from pea (Rerie et al. 1991) and a CaMV 35S terminator. The
amino acid
sequences of a human IL-37d (NCB! Reference Sequence: NP_775294.1) and human
Transferrin (GenBank: ABI97197.1) were back-translated using the codon
preference of
legumes, and the resulting artificial ORF was synthesized (GenScript USA
Inc.,NJ, USA).
Two DNA fragments (attB4-LegA2-attB1 and attB1-IL37d+hTf-attB3) were amplified
by
polymerase chain reaction (PCR) using specific pairs of primers which carries
specific attB
- 40 -
CA 02901370 2015-08-24
recombinase site according to the protocol of the MultiSite Gateway Three
Fragment Vector
Construction Kit (Invitrogen; Catalogue # 12537-023). The vector pER598 was
derived from
pKm43GW (Karimi et al. 2005) used as a destination clone. A binary vector pIL-
37d-hTf was
generated by inserting a single gene cassette into pER598 using LR clonase
reactions to
transfer the gene cassette from the entry clone to the destination vector were
performed
according to the protocol of the MultiSite Gateway Three Fragment Vector
Construction Kit
(Invitrogen). The vector was electroporated into disarmed Agrobacterium
tumefaciens strain
GV3101-pMP90 (Koncz and Schell, 1986) prior to plant transformation
experiments. Binary
vector; Nucleotide sequence and the deduced amino acids of IL-37d+hTf fusion
gene are
shown below:
[00296] Figure 32 shows a schematic diagram of the construct (pLIL-
37d+hTf) used
for plant transformation to generate plants expressing IL-37d+hTf. The
construct contains a
fusion gene encoding IL-37d+hTf under the control of Leg A2 seed specific
promoter and
terminator (t35S). The marker gene neomycin phosphotransferase II (nptI1),
regulated by
Nopaline synthase promoter (p-nos) and terminator (t-nos), was integrated into
the construct
(RB, right border of theT-DNA; LB, left border of the T-DNA)
[00297] SEQ ID 32: Nucleotide sequence of IL37+hTf (2634 bp)
[00298] ATGAGTTTTGTTGGAGAGAATAGTGGAGTGAAGATGGGTTCCGAAGATT
GGGAGAAAGATGAGCCACAGTGTTGCCTTGAGGGTCCTAAAGTTAAGAATCTTAACCCA
AAGAAATTTTCTATCCATGATCAAGATCACAAGGTTTTAGTGCTCGATTCAGGAAATCTTA
TTGCTGTGCCAGATAAGAACTACATCAGACCTGAAATATTTTTCGCTCTTGCATCTTCATT
GAGTTCCGCTTCTGCAGAAAAGGGATCACCTATACTTTTGGGAGTTTCAAAAGGAGAGT
TCTGTCTTTACTGCGATAAGGATAAAGGTCAAAGTCATCCATCCCTTCAGTTGAAGAAAG
AAAAACTTATGAAACTTGCTGCACAAAAAGAGTCTGCTAGAAGGCCTTTTATATTCTATA
GAGCACAGGTTGGAAGTTGGAATATGTTGGAATCCGCTGCACATCCAGGTTGGTTTATC
TGTACTAGTTGTAATTGCAACGAGCCTGTTGGTGTGACTGATAAGTTCGAAAACAGGAA
ACACATTGAGTTTTCTTTCCAGCCAGTGTGCAAGGCAGAGATGTCTCCTTCAGAGGTGT
CCGATGTGCCAGATAAGACAGTTAGATGGTGCGCAGTTTCCGAACACGAAGCAACAAAA
TGCCAGAGTTTCAGGGATCACATGAAGTCCGTTATTCCAAGTGATGGTCCTTCCGTTGC
TTGTGTGAAGAAAGCATCTTATCTTGATTGCATCAGAGCTATTGCTGCAAATGAGGCTGA
TGCAGTTACTTTAGATGCAGGACTTGTTTATGATGCTTACTTAGCACCAAATAACCTCAA
ACCTGTTGTGGCTGAGTTTTACGGAAGTAAAGAAGATCCACAGACTTTCTATTACGCTGT
TGCAGTTGTGAAGAAAGATTCCGGTTTTCAGATGAATCAACTTAGAGGAAAGAAAAGTTG
- 41 -
CA 02901370 2015-08-24
TCATACAGGATTGGGTAGATCCGCTGGTTGGAACATTCCAATAGGACTTTTGTATTGCG
ATCTTCCAGAGCCTAGGAAACCTCTCGAAAAGGCTGTTGCAAATTTCTTTTCTGGTTCAT
GTGCTCCTTGCGCAGATGGAACTGATTTTCCACAGCTTTGTCAATTGTGCCCTGGATGT
GGTTGCTCTACACTTAACCAATATTTTGGATACTCAGGTGCTTTCAAATGTTTGAAGGAT
GGAGCTGGAGATGTTGCATTCGTGAAACATTCTACTATCTTCGAGAATCTTGCTAACAAG
GCAGATAGAGATCAGTACGAACTCCTCTGTCTTGATAACACTAGGAAACCAGTTGATGA
GTACAAGGATTGCCATTTGGCTCAAGTGCCTAGTCACACAGTTGTGGCAAGATCTATGG
GAGGTAAAGAAGATCTTATTTGGGAGCTTTTGAACCAGGCTCAAGAGCACTTCGGAAAG
GATAAGTCTAAGGAATTCCAACTTTTCTCTTCACCACATGGTAAAGATCTTCTCTTTAAGG
ATTCAGCTCACGGATTCTTGAAAGTTCCACCTAGGATGGATGCTAAGATGTATTTGGGTT
ATGAATACGTGACAGCAATTAGAAATCTTAGGGAAGGAACTTGTCCAGAGGCTCCTACA
GATGAATGTAAACCTGTTAAGTGGTGCGCATTATCACATCACGAGAGGCTCAAATGCGA
TGAATGGTCTGTTAATTCAGTGGGAAAGATTGAGTGTGTTAGTGCTGAAACTACAGAGG
ATTGCATAGCAAAGATCATGAACGGAGAAGCTGATGCAATGTCCTTGGATGGAGGTTTT
GTTTACATTGCTGGAAAATGTGGTCTTGTTCCAGTGTTGGCAGAAAACTACAACAAGTCA
GATAACTGCGAAGATACTCCTGAGGCTGGATACTTTGCAGTGGCTGTTGTGAAGAAAAG
TGCATCCGATCTTACATGGGATAATTTGAAGGGAAAGAAGTCTTGTCATACTGCTGTTGG
TAGAACAGCAGGATGGAACATACCAATGGGACTTTTGTACAATAAGATAAACCACTGTA
GGTTCGATGAATTTTTCTCTGAGGGTTGCGCTCCAGGATCAAAGAAAGATAGTTCCTTAT
GTAAACTCTG CATGGGATCTGGTTTAAATCTCTGTGAG CCTAATAACAAGGAAGGATATT
ACGGTTACACTGGAGCTTTTAGATGCTTGGTTGAGAAAGGAGATGTGGCTTTCGTGAAG
CATCAGACTGTGCCTCAAAATACAGGAGGAAAGAATCCAGATCCTTGGGCTAAGAATCT
TAACGAAAAGGATTACGAGTTACTCTGTOTTGATGGAACTAGAAAGCCAGTTGAAGAGT
ACGCTAATTGCCATCTTGCTAGAGCACCTAACCACGCTGTTGTGACAAGGAAGGATAAG
GAAGCATGTGTTCATAAGATCCTTAGGCAACAGCAACACTTGTTTGGTTCTAATGTGACT
GATTGTTCAGGAAACTTTTGCCTTTTCAGATCAGAGACTAAAGATCTTTTGTTCAGGGAT
GATACAGTTTGTCTTGCTAAGTTGCATGATAGGAACACTTATGAAAAGTACCTTGGAGAA
GAGTATGTTAAGGCAGTGGGAAACTTGAGGAAATGCTCCACATCATCACTTTTAGAGGC
TTGCACTTTTAGAAGACCATGATAG
[00299] SEQ ID 33: Amino acid sequence of IL37+hTf (876 amino acids)
[00300] MSFVGENSGVKMGSEDWEKDEPQCCLEGPKVKNLNPKKFSIHDQDHKVLV
LDSGNLIAVPDKNYIRPEIFFALASSLSSASAEKGSPILLGVSKGEFCLYCDKDKGQSHPSLQ
LKKEKLMKLAAQ KESARRPF I FYRAQVGSWNMLESAAH PGWF I CTSC NC NEPVGVTDKFEN
- 42 -
CA 02901370 2015-08-24
RKHIEFSFQPVCKAEMSPSEVSDVPDKTVRWCAVSEHEATKCQSFRDHMKSVIPSDGPSV
ACVKKASYLDCIRAIAANEADAVTLDAGLVYDAYLAPNNLKPWAEFYGSKEDPQTFYYAVA
VVKKDSGFQMNQLRGKKSCHTGLGRSAGWNIPIGLLYCDLPEPRKPLEKAVANFFSGSCAP
CADGTDFPQLCQLCPGCGCSTLNQYFGYSGAFKCLKDGAGDVAFVKHSTIFENLANKADR
DQYELLCLDNTRKPVDEYKDCHLAQVPSHTVVARSMGGKEDLIWELLNQAQEHFGKDKSK
EFQLFSSPHGKDLLFKDSAHGFLKVPPRMDAKMYLGYEYVTAIRNLREGTCPEAPTDECKP
VKWCALSHHERLKCDEWSVNSVGKIECVSAETTEDCIAKIMNGEADAMSLDGGFVYIAGKC
GLVPVLAENYNKSDNCEDTPEAGYFAVAVVKKSASDLTVVDNLKGKKSCHTAVGRTAGWNI
PMGLLYNKINHCRFDEFFSEGCAPGSKKDSSLCKLCMGSGLNLCEPNNKEGYYGYTGAFR
CLVEKGDVAFVKHQTVPQNTGGKNPDPWAKNLNEKDYELLCLDGTRKPVEEYANCHLARA
PNHAVVTRKDKEACVHKILRQQQHLFGSNVTDCSGNFCLFRSETKDLLFRDDTVCLAKLHD
RNTYEKYLGEEYVKAVGNLRKCSTSSLLEACTFRRP
[00301] Example 11
[00302] Stable Transformation of Tarwi (Lupinus mutabilis) with IL-37
isoforms
[00303] Two binary vectors IL-37b-5 (as described in earlier example of
tobacco) and
pLIL-37d+hTf were transformed into tarwi via Agrobacterium mediated
transformation.
Putative transformed and regenerated plants were initially selected for
kanamycin resistance.
Over twenty transgenic plants were regenerated for each of constructs IL-37b-5
(LegA2
prom::SBA::linker:TEV cleavage site::137b::nos terminator) and pIL-37d-hTf
(LegA2
prom::IL-37d::human Transferrin::nos terminator). Regenerated plants were
analyzed for
expression of the IL-37b and IL-37d protein by solid phase sandwich Enzyme-
Linked
ImmunoSorbent Assay (ELISA).
[00304] Example 12
[00305] Total tarwi seed extract preparation
[00306] Five mature T1 seeds from three tarwi transgenic lines transformed
with IL-
37b-5 and four transgenic tarwi lines transformed with pLIL-37d+hTf were
randomly collected
and about 25 mg of seed cotyledon tissues were chipped from each seed. Seed
tissues were
immediately snap-frozen upon harvesting, homogenized in liquid nitrogen.
Homogenized
plant material was ground in ice-cold extraction buffer (50 mM Tris-buffered
saline pH = 7.4,
100 mM NaCI, and insoluble polyvinylpolypyrrolidone (PVPP) was used (0.1g/g
fresh
weight). Crude extract was clarified by centrifugation at 13000rpm for 10 min
at 4C . The
total protein concentration in each sample was determined by the Bradford
Assay method
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CA 02901370 2015-08-24
using bovine serum albumin (BSA) as a standard. Protein supernatant was
directly used in
an ELISA assay.
[00307] Quantitative ELISA for detection of IL-37 expression in transgenic
tarwi seeds
[00308] IL-37 protein concentration in crude extract was determined by
comparison
with an IL-37 standard curve in an IL-37 ELISA kit (Catalog # MBS705712,
MyBioSource-
Canada), according to the manufacturers' instruction using the Victor3V plate
reader
(PerkinElmer) to measure the OD at 450 nm with correction filter of 690 nm. A
4-parameter
logistic fit curve was obtained by plotting the absorbance versus the
corresponding
concentration of the standards. Values are expressed in pg/ml. Quantitative
data are
presented as average of duplicate for samples and standards. All statistical
analyses were
performed by use of (MyAssays) software.
[00309] Table: IL37 expression in transgenic tarwi seed by ELISA
No. Transgenic line Transformation Protein content IL37
expression by
binary vector (mg/g) ELISA(ng/g)
1 NL43-A IL-37b-5 79.2 8.8
2 NL43-B 82.3 4.9
3 NL43-C 81.4 3.2
4 NL42-A pLIL-37d+hTf 83.2 12.8
NL42-B 77.3 13.9
6 NL42-C 81.4 10.2
7 NL42-D 79.5 9.1
4 Wild type tarwi seed Non 80.2 0.0
(negative control) transformed
[00310]
[00311] Example 13
[00312] Construction of IL-37d+hTf expression vector for Oca
[00313] The p35SIL-37d+hTf expression vector was constructed to express
the fusion
gene (human IL-37d+ human transferrin) in oca (Oxalis tuberosa). This vector
contains the
CaMV35S constitutive promoter and terminator too. The amino acid sequences of
a human
IL-37d (NCBI Reference Sequence: NP_775294.1) and human transferrin (GenBank:
ABI97197.1) were back-translated using the plant codon preference, and the
resulting
artificial ORF was synthesized (GenScript USA Inc.,NJ, USA). Two DNA fragments
(attB4-
CaMV35S-attB1 and attB1-IL37d+hTf-attB3) were amplified by polymerase chain
reaction
- 44 -
CA 02901370 2015-08-24
(PCR) using specific pairs of primers which carries specific attB recombinase
site according
to the protocol of the MultiSite Gateway Three Fragment Vector Construction
Kit (Invitrogen;
Catalogue # 12537-023). The vector pER598 was derived from pKm43GW (Karimi et
al.
2005) used as a destination clone. A binary vector pIL-37d-hTf was generated
by inserting a
single gene cassette into pER598 using LR clonase reactions to transfer the
gene cassette
from the entry clone into the destination vector were performed according to
the protocol of
the MultiSite Gateway Three Fragment Vector Construction Kit (Invitrogen;
Catalogue #
12537-023). The vector was electroporated into disarmed Agrobacterium
tumefaciens strain
GV3101-pMP90 (Koncz and Schell, 1986) prior to plant transformation
experiments.
[00314] Figure 33: Schematic diagram of the construct (p35SIL-37d+hTf)
used for
plant transformation to generate plants expressing I137d+hTf. The construct
contain a fusion
gene encoding IL-37d+hTf under the control of CaMV35S constitutive promoter
and
terminator (t35S). The selectable gene neomycin phosphotransferase II (nptI1),
regulated by
Nopaline synthase promoter (p-nos) and terminator (t-nos), was integrated into
the construct
(RB, right border of the T-DNA; LB, left border of the T-DNA)
[00315] Example 14
[00316] Stable transformation of oca (Oxalis tuberosa) Plants with IL-37
isoforms
[00317] Three binary vectors IL-37b-lb and IL-37b-3 (as described in
earlier example
of tobacco) as well as p35S-1-37d+hTf were transformed into oca via
Agrobacterium
mediated transformation (our filed patent # CA2808845 Al and US20130347144).
Putative
transformed and regenerated plants were initially selected for kanamycin
resistance. Single
transgenic events were regenerated for each of the constructs. Regenerated
plants were
analyzed by regular PCR to check for the presence of the transgene and for
expression of
the IL-37b protein, a solid phase sandwich Enzyme-Linked Immuno Sorbent Assay
(ELISA)
was performed.
[00318] Example 15
[00319] Total oca leaf extract preparation
[00320] Leaves and stem were randomly sampled from oca plants that were
over 15
cm in height. Six leaves were sampled, with one leaf disc (1.5 cm in diameter)
taken from
each leaf. The six leaf discs and 3 pieces of stem (1.5 cm in size) were added
to 2 ml
microfuge tubes containing 3 ceramic disposable beads (BioSpec Products, Inc.
Cat. No.
11079125z), flash frozen in liquid nitrogen and stored at -80 degree C. The 2
ml tubes were
loaded onto a prechilled freezer blocks for the automatic tissue grinder
(Retsch, Inc.). The
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CA 02901370 2015-08-24
blocks were shaken at a frequency of 30 times/sec for 2 min. The blocks were
then turned
inside out, and the grinding was repeated a second time. Blocks were then
removed and
centrifuged at 3700 rpm for 1 min to remove tissue powder from lids of the
tubes. Frozen
powdered tissue (approx. 60 mg of fresh weight) was vortexed for 5 sec in 400
ul of
extraction buffer (phosphate buffered saline (PBS) pH 7.4, 0.05% (v/v) Tween
20, and 2%
(w/v) polyvinylpolypyrrolidone (PVPP). The supernatant was clarified by
centrifugation at
13,000 g for 10 min at 4 degree C. The supernatant was removed to 1.5 ml tubes
and re-
centrifuged for an additional 10 min at 4 degree C. to remove remaining leaf
and stem debris.
The supernatant was then removed to new 1.5 ml tubes and stored at 4 degree C.
on ice and
used in an ELISA assay.
[00321] Quantitative ELISA for detection of IL-37 expression in transgenic
oca leaves
[00322] IL-37 protein concentration in crude extract was determined by
comparison
with an IL-37 standard curve in an IL-37 ELISA kit (Catalog # MBS705712,
MyBioSource-
Canada), according to the manufacturers' instruction using the Victor3V plate
reader
(PerkinElmer) to measure the OD at 450 nm with correction filter of 690 nm. A
4-parameter
logistic fit curve was obtained by plotting the absorbance versus the
corresponding
concentration of the standards. Values are expressed in pg/ml. Quantitative
data are
presented as average of duplicate for samples and standards. All statistical
analyses were
performed by use of (MyAssays) software.
[00323] Table: IL37 expression in transgenic oca through ELISA transformed
with
vector IL-37b-1b
No. Transgenic line Tissue Protein content IL37 expression
by
(mg/g fresh weight)
ELISA(ng/g fresh weight)
1 CM-0-8-D Leaf 1.01 40.5
2 Stem 0.73 65.6
3 Wild type Oca (negative Leaf 1.05 0.0
4 control) Stem 0.81 0.0
[00324] Example 16
[00325] Construction of INPACT-IL-37b expression vector and generation of
stable
transgenic tobacco plants:
- 46 -
CA 02901370 2015-08-24
[00326] A set of two In-Plant Activation (IN PACT) expression vectors
consisting of a
uniquely designed split-gene cassette (human IL-37b gene) incorporating the
cis replication
elements of Tobacco yellow dwarf geminivirus (TYDV) and an ethanol-inducible
activation
cassette encoding the TYDV Rep and RepA proteins (Dugdale et al., 2014). These
vectors
were constructed under contract at Queensland University of Technology,
Brisbane,
Australia as per patent # AU2001243939. Stable Agrobacterium-mediated
transformation of
tobacco (Nicotiana benthamiana) was performed using a standard protocol
(Horsch et al.,
1985). Transformants were selected on MS medium (Murashige and Skoog 1962)
containing
25 mg/L kanamycin, 25 mg/L hygromycin, 1 mg/L BAP and 0.1 mg/L NAA. Shoots
that
developed were transferred to a phytohormone-free MS medium containing 25 mg/L
kanamycin, and 25 mg/L hygromycin for root formation. Regenerated plants were
transferred
from Magenta TM boxes to pots, and grown further under greenhouse conditions.
Over 25
transgenic plants were regenerated for the INPACT-IL37b construct transformed
into plant
cells. Regenerated plants were analyzed for expression of the IL-37b protein
by ELISA.
[00327] Example 17
[00328] Analysis of IL-37b Expression in stable transgenic Nicotiana
benthamiana
tobacco plants
[00329] Activation of INPACT-IL-37b construct; total Leaf Protein
Preparation and
quantification of IL-37b by ELISA:
[00330] Transgenic plants were sprayed and soil drenched with 5% ethanol
to activate
the INPACT-IL-37b construct and leaf material was collected after 5 dpei (days
post ethanol
induction). Total soluble leaf protein was extracted using an extraction
buffer and procedure
described in example 3 of oca and the same procedure was followed for
quantification of IL-
37b by ELISA.
[00331] Table: IL-37b expression in selected transgenic tobacco lines by
ELISA
No. Transgenic line Protein content IL37 expression by ELISA
(mg/g fresh weight) (ng/g fresh weight)
1 CM-T-2-1 1.43 83.4
2 CM-T-2-3 1.51 88.9
3 CM-T-2-4 2.12 80.5
4 CM-T-2-5 2.20 135.7
CM-T-2-7 1.45 81.5
- 47 -
CA 02901370 2015-08-24
6 CM-T-2-9 1.62 65.4
7 CM-T-2-11 1.78 78.5
8 CM-T-2-13 1.67 83.0
9 CM-T-2-21 1.97 90.5
CM-T-2-22 1.89 76.6
11 Wild type tobacco 1.76 0.0
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CA 02901370 2015-08-24
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[00650] The above-described embodiments are intended to be examples only.
Alterations, modifications and variations can be effected to the particular
embodiments by
those of skill in the art. The scope of the claims should not be limited by
the particular
embodiments set forth herein, but should be construed in a manner consistent
with the
specification as a whole.
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