CA2901370A1 - Production of il-37 in plants - Google Patents

Abstract

Described herein is the production, recovery and purification of human interleukin 37 from plants. The invention relates to methods of molecular farming plant, including in tobacco, Tarwi and Oca, wherein both stable and transient expression of the five known variants of human interleukin 37 were made IL-37 variants are additionally produced as fusions with soybean lectin, transferrin and polyhistidine tags to aid in stability and recovery by affinity chromatography.

Description

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), 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 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.

[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-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;

[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;

[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 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-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.

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 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.

[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.

[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

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.

[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.

[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

[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).

[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).

[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.

[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.

[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).

[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 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).

[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 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 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' 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 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 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

[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 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

[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

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

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.

[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 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

[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).

Transformants were selected on MS medium (Murashige and Skoog 1962) containing 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

[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 (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.

[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.

[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 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

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

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

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 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 (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 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:

[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 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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[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.

Claims (27)

CLAIMS:

1. A chimeric nucleic acid sequence construct for expression of IL-37 in a plant cell

2. The chimeric nucleic acid sequence construct of claim 1, comprising:
(i) a promoter nucleic acid sequence;
(ii) an enhancer nucleic acid sequence operatively associated with said promoter (EV 5'UTR);
(iii) a signal peptide nucleic acid sequence operatively associated with said 5'UTR
(IL37a; or barley .alpha.-amylase);
(iv) an IL-37 nucleic acid sequence encoding IL-37 operatively associated with said signal peptide sequence; and (v) a terminator nucleic acid sequence operatively associated with said IL-nucleic acid sequence.

3. The chimeric nucleic acid sequence construct according to claim 2, wherein said promoter is a constitutive promoter or an inducible promoter.

4. The chimeric nucleic acid sequence construct according to claim 2, wherein 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, a RNA polymerase promoter, an elongation factor promoter, an expansin promoter.

5. The chimeric nucleic acid sequence construct according to claim 2, wherein 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 seed specific promoter, a root specific promoter.

6. The chimeric nucleic acid sequence construct according to any one of claims 1-5, wherein said 5'UTR comprises EV 5'UTR.

7. The chimeric nucleic acid sequence construct according to any one of claims 1-6, wherein said IL-37 is codon-optimized for expression in said plant.

8 The chimeric nucleic acid sequence construct according to any one of claims 1-6, wherein is said IL-37 nucleic acid exhibits from about 70% to about 100%
sequence identity to IL-37.

9. The chimeric nucleic acid sequence construct according to any one of claims 1-6, wherein 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

The chimeric nucleic acid sequence construct of claim 2, further comprising.
(vi) a targeting sequence nucleic acid operative associate with said IL-37 nucleic acid sequence

11. The chimeric nucleic acid sequence construct according to claim 10, wherein 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

12. The chimeric nucleic acid sequence construct according to claim 11, wherein said targeting sequence is a KDEL targeting sequence, or a barley aleurone DNA
signal sequence

13 The chimeric nucleic acid sequence construct according to any one of claims 1-12, further comprising:
(vii) a purification tag nucleic acid sequence construct operatively associated with said IL-37 nucleic acid sequence.

14. The chimeric nucleic acid sequence construct of claim 13, further comprising a protease cleavage nucleic acid operatively associate with said IL-37 nucleic acid or said affinity tag nucleic acid.

15. The chimeric nucleic acid sequence construct of claim 13 or 14, wherein said purification tag encodes SBA, transferrin, a polyhistidine, a Strepll, glutathione S-transferase

16. The chimeric nucleic acid sequence construct of claim 14 or 15, wherein said protease cleavage nucleic acid encodes a TEV protease cleavage sequence.

17. The chimeric nucleic acid sequence construct according to any one of claims 1-16, further comprising a selectable marker nucleic acid

18. The chimeric nucleic acid sequence construct according to claim 17, wherein the selectable marker imparts resistance to kanamycin, hygromycin, streptomycin, or a herbicide such as phosphinothricin or glyphosate.

19. The chimeric nucleic acid sequence construct according to any one of claims 1-18, wherein said IL-37 is human IL-37.

20. The chimeric nucleic acid sequence construct according to any one of claims 1-18, wherein said IL-37 is optimize for expression in a plant.

21. A chimeric nucleic acid construct, wherein siad 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-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

22. A plant, plant cell, or plant part, transformed with a chimeric nucleic acid construct according to any one of claims 1-21.

23 The plant, plant cell, or plant part, of claim 22, wherein said plant, plant cell, or plant part, is a suspension culture, an embryo, a merstematic region, a callus tissue, a leaf, root, shoot, gametophyte, sporophyte, pollen, seed or microspore

24. The plant, plant cell, or plant part, of claim 22, wherein the plant cell is from a monocotyledon, or a dicotyledon.

25. The plant, plant cell, or plant part, of claim 24, wherein the monocotyledon is a corn, a cereal, a grain, a grass, or a rice.

26. The plant, plant cell, or plant part, of claim 24 wherein the dicotyledon is a tobacco, a tomato, a potato, a legume, tarwi or oca.

27. A method of producing IL-37 in a plant comprising the steps of (a) transforming a plant cell with a chimeric nucleic acid construct according to any one of claims 11-21 , (b) generating a whole plant according to any one of claims 22-26 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.