US20150004137A1

US20150004137A1 - Novel forms of plant defensins

Info

Publication number: US20150004137A1
Application number: US14/352,380
Authority: US
Inventors: Mark Darren Hulett; Ivan Ka Ho Poon
Original assignee: BALMORAL AUSTRALIA Pty Ltd
Current assignee: BALMORAL AUSTRALIA Pty Ltd
Priority date: 2011-10-19
Filing date: 2012-10-19
Publication date: 2015-01-01
Also published as: US9540423B2; CA2851144A1; NZ622284A; EP2768849B1; AU2012325676A1; AU2012325675A1; EP2768849A4; EP2768849A1; AU2012325676B2; BR112014009277A2; WO2013056308A1; ZA201401800B; US20150158918A1; WO2013056309A1

Abstract

The present invention relates to heterogeneous or artificially created forms of plant defensins, and to uses of such heterogenous or artificially created defensins including methods for preventing or treating proliferative diseases. Compositions for use as animal and human medicaments are also provided. The present invention also relates to associated methods, uses, systems and kits.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 61/548,825 filed on 19 Oct. 2011, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to heterogeneous forms of defensins, and to uses of such heterogeneous defensins including methods for preventing or treating proliferative diseases. Animal and human medicaments are also provided. The present invention also relates to associated methods, uses, systems and kits.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

Not applicable.

BACKGROUND TO THE INVENTION

Plants are known to produce a variety of chemical compounds, either constitutively or inducibly, to protect themselves against environmental stresses, wounding, or microbial invasion.
Of the plant antimicrobial proteins that have been characterized to date, a large proportion share common characteristics. They are generally small (<10 kDa), highly basic proteins and often contain an even number of cysteine residues (typically 4, 6 or 8). These cysteines all participate in intramolecular disulfide bonds and provide the protein with structural and thermodynamic stability (Broekaert et al. (1997)). Based on amino acid sequence identities, primarily with reference to the number and spacing of the cysteine residues, a number of distinct families have been defined. They include the plant defensins (Broekaert et al., 1995, 1997; Lay et al., 2003a), thionins (Bohlmann, 1994), lipid transfer proteins (Kader, 1996, 1997), hevein (Broekaert at al., 1992) and knottin-type proteins (Cammue et al., 1992), as well as antimicrobial proteins from Macadamia integrifolia (Marcus et al., 1997; McManus at al., 1999) and Impatiens balsamina (Tailor et al., 1997; Patel at al., 1998) (Table 1). All these antimicrobial proteins appear to exert their activities at the level of the plasma membrane of the target microorganisms, although it is likely that the different protein families act via different mechanisms (Broekaert et al., 1997). The cyclotides are a new family of small, cysteine-rich plant peptides that are common in members of the Rubiaceae and Violaceae families (reviewed in Craik et al., 1999, 2004; Craik, 2001). These unusual cyclic peptides (Table 1) have been ascribed various biological activities including antibacterial (Tam, et al., 1999), anti-HIV (Gustafson et al., 1994) and insecticidal (Jennings et al., 2001) properties.

TABLE 1

Small, cysteine-rich antimicrobial proteins in plants.

	Repre-	No. of
Peptide	sentative	amino
family	member	acids	Consensus sequence

Plant defensins	Rs-AFP2	51

α/β-Thionin (8-Cys type)	α- Purothionin	45

Lipid transfer protein	Ace-AMPI	93

Hevein- type	Ac-AMP2	30

Knottin- type	Mj-AMP1	36

Macadamia	MiAMP1	76

Impatiens	Ib-AMP1	20

Cyclotide	Kalata B1	29

The size of the mature protein and spacing of cysteine residues for representative members of plant antimicrobial proteins is shown in Table 1. The numbers in the consensus sequence represent the number of amino acids between the highly conserved cysteine residues in the representative member, but other members of the family may vary slightly in the inter-cysteine lengths. The disulfide connectivities are given by connecting lines. The cyclic backbone of the cyclotides is depicted by the broken line (from Lay and Anderson, 2005).

Defensins

The term “defensin” has previously been used in the art to describe a diverse family of molecules that are produced by many different species and which function in innate defense, against pathogens including bacteria, fungi, yeast and viruses.

Plant Defensins

Plant defensins (also termed γ-thionins) are small (˜5 kDa, 45 to 54 amino acids), basic proteins with eight invariant cysteine residues that form four strictly conserved disulfide bonds with a Cys_I-Cys_VIII, Cys_II-Cys_IV, Cys_III-Cys_VIand Cys_V-Cys_VIIconfiguration. As well as these four strictly conserved disulfide bonds, some plant defensins have an additional disulfide bond (Lay et al., 2003a, 2003b; Janssen et al., 2003).
The name “plant defensin” was coined in 1995 by Terras and colleagues who isolated two antifungal proteins from radish seeds (R5-AFP1 and R5-AFP2) and noted that at a primary and three-dimensional structural level these proteins were distinct from the plant α-/β-thionins but shared some structural similarities to insect and mammalian defensins (Terras et al., 1995; Broekaert et al., 1995).
Plant defensins exhibit clear, although relatively limited, sequence conservation. Strictly conserved are the eight invariant cysteine residues and a glycine at position 32 (numbering relative to the continuous NaD1 sequence; see for example SEQ ID NO: 22 and the sequence alignments shown in any of FIGS. 11 to 15) or position 34 (numbering relative to R5-AFP2). With reference to the numbering of amino acids residues in R5-AFP2, in most of the sequences a serine at position 8 (position 7 in NaD1), an aromatic residue at position 11 (position 10 in NaD1), a glycine at position 13 (position 12 in NaD1) and a glutamic acid at position 29 (position 27 in NaD1) are also conserved (Lay et al., 2003a; Lay and Anderson, 2005).
The three-dimensional solution structures of the first plant defensins were elucidated in 1993 by Bruix and colleagues for γ1-P and γ1-H (also referred to herein as “g1-H”). Since that time, the structures of other seed-derived and two flower-derived (NaD1 and PhD1) defensins have been determined (Lay et al., 2003b; Janssen et al., 2003). All these defensins elaborate a motif known as the cysteine-stabilized αβ (CSαβ) fold and share highly superimposable three-dimensional structures that comprise a well-defined α-helix and a triple-stranded antiparallel β-sheet. These elements are organized in a βαββ arrangement and are reinforced by four disulfide bridges.
The CSαβ motif is also displayed by insect defensins and scorpion toxins. In comparing the amino acid sequences of the structurally characterized plant defensins, insect defensins and scorpion toxins, it is apparent that the CSαβ scaffold is highly permissive to size and compositional differences.
The plant defensin/γ-thionin structure contrasts to that which is adopted by the α- and β-thionins. The α- and β-thionins form compact, amphipathic, L-shaped molecules where the long vertical arm of the L is composed of two α-helices, and the short arm is formed by two antiparallel β-strands and the last (˜10) C-terminal residues. These proteins are also stabilized by three or four disulfide bonds (Bohlmann and Apel, 1991).
Plant defensins have a widespread distribution throughout the plant kingdom and are likely to be present in most, if not all, plants. Most plant defensins have been isolated from seeds where they are abundant and have been characterized at the molecular, biochemical and structural levels (Broekaert et al., 1995; Thomma et al., 2003; Lay and Anderson, 2005). Defensins have also been identified in other tissues including leaves, pods, tubers, fruit, roots, bark and floral tissues (Lay and Anderson, 2005).
An amino acid sequence alignment of several defensins that have been identified, either as purified protein or deduced from cDNAs, has been published by Lay and Anderson (2005). Other plant defensins have been disclosed in U.S. Pat. No. 6,911,577, International Patent Publication No. WO 00/11196 and International Patent Publication No. WO 00/68405, the entire contents of which are incorporated herein by reference.

Two Classes of Plant Defensins

In the first and largest class, Class I, the precursor protein is composed of an endoplasmic reticulum (ER) signal sequence and a mature defensin domain. These proteins enter the secretory pathway and have no obvious signals for post-translational modification or subcellular targeting (FIG. 10A).
The second class of defensins are produced as larger precursors with C-terminal prodomains or propeptides (CTPPs) of about 33 amino acids (FIG. 10B). Class II defensins have been identified in solanaceous species where they are expressed constitutively in floral tissues (Lay et al., 2003a; Gu et al., 1992; Milligan et al., 1995; Brandstadter et al., 1996) and fruit (Aluru et al., 1999) and in salt stressed leaves (Komori et al., 1997; Yamada et al., 1997). The CTPP of the solanaceous defensins from Nicotiana alata (NaD1) and Petunia hybrida (PhD1 and PhD2) is removed proteolytically during maturation (Lay et al., 2003a).

Biological Activity of Plant Defensins

The best characterized activity of some but not all plant defensins is their ability to inhibit, with varying potencies, a number of fungal species (for examples, see Broekaert et al., 1997; Lay et al., 2003a; Osborn et al., 1995). Growth inhibitory effects on Gram-positive and Gram-negative bacteria have also been described (Segura et al., 1998; Moreno et al., 1994; Zhang and Lewis, 1997).
The inventors have previously disclosed in International Patent Publication No. WO 02/063011 certain defensins and their use in inducing resistance in plants or parts of plants to pathogen infestation. The entire content of WO 02/063011 is incorporated herein by reference.
Plant Defensins with Antiproliferative Activity in Cancer Cells
The inventors have previously disclosed that Solanaceous Class II plant defensins have activity in preventing or treating proliferative diseases in mammals. Methods of treating or preventing proliferative disease by administering plant defensins, together with associated uses, kits and pharmaceutical compositions, are disclosed in international patent application no. PCT/AU2011/000760 filed on 23 Jun. 2011. Such methods, uses, kits and pharmaceutical compositions are also disclosed in U.S. patent application Ser. No. 13/166,960, also filed on 23 Jun. 2011. The entire content of both PCT/AU2011/000760 and U.S. Ser. No. 13/166,960 is incorporated herein by reference.
Previous attempts to modify the defensins to improve and broaden their activity have hitherto been largely unsuccessful. Thus, despite the utility of the inventions disclosed in WO 02/063011, PCT/AU2011/000760 and U.S. Ser. No. 13/166,960 there remains a great need in the relevant art for plant defensins that display enhanced anti-proliferative activity and/or broader spectrum of activity.
Further studies by the inventors into the mechanism of action of plant defensins by the inventors have surprisingly revealed that Class II Solanaceous plant defensins, such as NaD1 may target cells through the recognition of a ‘phospholipid pattern’ by a specific region of the defensin sequence.
This information provides basis for developing new defensins with advantageous qualities and functions, including but not limited to functionalisation of defensins that are inactive or have low activity in relation to treatment of proliferative diseases, variation of, or addition of new, plant defensin effector functions, and development of defensins with enhanced cytotoxic potency. These new plant defensins therefore also provide for new methods of treatment, uses and kits suitable for the prevention or treatment of diseases, disorders and ailments such as proliferative diseases.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, there is provided a heterogeneous plant defensin, wherein the plant defensin comprises a first polypeptide sequence and a second polypeptide sequence, wherein the second polypeptide sequence is derived from a plant defensin other than the plant defensin from which the first polypeptide sequence is derived.
In some embodiments, the heterogeneous plant defensin comprises a third polypeptide sequence.
In some embodiments, the heterogeneous plant defensin contains eight invariant cysteine amino acids.
In some embodiments, the heterogeneous plant defensin contains at least five loop regions.
In some embodiments, the heterogeneous plant defensin comprises the following amino acid sequence:
(SEQ ID NO: 1)

X_A-C1-X_B-C2-X_C-C3-X_D-C4-X_E-C5-X_F-C6-X_G-C7-X_H-C8-

X_I;

wherein C1, C2, C3, C4, C5, C6, C7 and C8 is cysteine, said cysteine being an invariant cysteine, X_A, X_B, X_C, X_D, X_E, X_F, X_G, X_H, X_Iis any naturally or non-naturally occurring amino acid and;
X_Ais 1 to 7 amino acids in length;
X_Bis 9, 10 or 11 amino acids in length;
X_Cis 3 to 8 amino acids in length;
X_Dis 3 amino acids in length;
X_Eis 9 to 13 amino acids in length;
X_Fis 4, 5, 6, 7 or 8 amino acids in length;
X_Gis 1 amino acid in length;
X_His 1 to 4-amino acids in length; and
X_Iis 0 or 1 amino acid in length.
In some embodiments, the heterogeneous plant defensin comprises the following amino acid sequence:
(X)₁₋₇-C-(X)₃₋₄-S-(X)₂-o-(X)₁-g-(X)₁-C-(X)₃₋₈-C-

(X)₃-C-(X)₂₋₄-e-(X)₄₋₆-g-(X)₁-C-(X)₄₋₈-C-(X)₁-C-

(X)₁₋₄-C-(X)₀₋₁

wherein C is cysteine, said cysteine being an invariant cysteine; S is serine, o represents an aromatic amino acid (phenylalanine, tryosine, tryptophan or histidine), e represents glutamate, g represents glycine and (X)_nrepresents any naturally occurring or non-naturally occurring amino acid and the integer represents the number of amino acids in that may comprise the specific region of the amino acid sequence.
In some embodiments, the second polypeptide sequence is positioned between the fourth and eighth invariant cysteine residues.
In some embodiments, the second polypeptide is positioned between the fifth and sixth invariant cysteine residues.
In preferred embodiments the first polypeptide sequence is derived from a Class I plant defensin.
In preferred embodiments the second polypeptide sequence is derived from a Class II Solanaceous plant defensin.
In certain embodiments, the first polypeptide sequence is derived from a Class I plant defensin and the second polypeptide sequence is derived from a Class II Solanaceous plant defensin.
In some embodiments the third polypeptide sequence is derived from a Class I plant defensin.
In some embodiments, the second polypeptide sequence comprises a loop 5 region derived from a Class II Solanaceous plant defensin, or a fragment or variant thereof.
In some embodiments, the loop 5 region comprises the entire loop 5 region of a Class II Solanaceous plant defensin, said loop 5 defined as by being flanked by the fifth and sixth invariant cysteine (Cys) amino acid residues of a Class II Solanaceous plant defensin amino acid sequence.
In certain embodiments, the loop 5 region of the heterogeneous plant defensin may begin with a serine amino acid positioned adjacent to the fifth invariant cysteine amino acid residue, wherein the serine amino acid is positioned to the C-terminal side of the fifth invariant cysteine residue.
In certain embodiments, loop 5 region comprises an amino acid sequence beginning at the end of the second β-strand and ending at the N-terminal side of the sixth invariant cysteine amino acid residue of a Class II Solanaceous plant defensin amino acid sequence.
In some embodiments, the second polypeptide sequence corresponds to an amino acid sequence comprising X₁-X₂-X₃-X₄-X₅-X₆, wherein X₁is serine or arginine, X₂is lysine, arginine or histidine, X₃and X₄are each a hydrophobic amino acid, X₅is arginine, lysine or histidine and X₆is arginine, lysine, histidine or asparagine.
In some embodiments, the second polypeptide sequence corresponds to an amino acid sequence comprising X₁-X₂-X₃-X₄-X₅-X₆, wherein X₁is serine or arginine, X₂is lysine, X₃is isoleucine, leucine or valine, X₄is leucine or glutamine, X₅is arginine and X₆is arginine, lysine or asparagine.
In some embodiments, the second polypeptide sequence corresponds to an amino acid sequence comprising X₂-X₃-X₄-X₅-X₆, wherein X₂is lysine, arginine or histidine, X₃and X₄are each a hydrophobic amino acid, X₅is arginine, lysine or histidine and X₆is arginine, lysine, histidine or asparagine.
In some embodiments, the second polypeptide sequence corresponds to an amino acid sequence comprising X₂-X₃-X₄-X₅-X₆, wherein X₂is lysine, X₃is isoleucine, leucine or valine, X₄is leucine or glutamine, X₅is arginine and X₆is arginine, lysine or asparagine.
In some embodiments, the second polypeptide sequence corresponds to an amino acid sequence selected from the group comprising (i) SKILRR (SEQ ID NO: 2), (ii) SKLLRR (SEQ ID NO: 4), (iii) SKILRK (SEQ ID NO: 6), (iv) SKVLRR (SEQ ID NO: 8), (v) SKVLRK (SEQ ID NO: 10), (vi) SKLQRK (SEQ ID NO: 12), (vii) SKLLRN (SEQ ID NO: 14), (viii) SKLLRK (SEQ ID NO: 16), (ix) SKIQRN (SEQ ID NO: 18), (x) RKLQRK (SEQ ID NO: 20) or (xi) KILRR (SEQ ID NO: 89), (xii) KLLRR (SEQ ID NO: 91), (xiii) KILRK (SEQ ID NO: 93), (xiv) KVLRR (SEQ ID NO: 95), (xv) KVLRK (SEQ ID NO: 97), (xvi) KLQRK (SEQ ID NO: 99), (xvii) KLLRN (SEQ ID NO: 101), (xviii) KLLRK (SEQ ID NO: 103), (xix) KIQRN (SEQ ID NO: 105) or (xx) KLQRK (SEQ ID NO: 107).
In some embodiments, the second polypeptide sequence corresponds to an amino acid sequence comprising S-K-I-L-R-R (SEQ ID NO:2).
In some embodiments, the second polypeptide sequence corresponds to an amino acid sequence comprising K-I-L-R-R (SEQ ID NO:89).
In some embodiments, the first and the third polypeptide sequences are both derived from the same plant defensin.
In some embodiments, the first polypeptide sequence and optionally the third polypeptide sequence contain one or more amino acid substitutions, deletions or modifications, wherein the substitution, deletion or modification amino acid does not naturally occur at the substituted, deleted or modified position in the plant defensin from which the first polypeptide sequence and optionally the third polypeptide sequence is derived.
In particular embodiments, there is provided a heterogeneous plant defensin, wherein said defensin comprises a first polypeptide sequence and a second polypeptide sequence and at least eight invariant cysteine amino acid residues, wherein the first polypeptide sequence is derived from a Class I plant defensin and the second polypeptide is derived from a Class II Solanaceous plant defensin, and wherein the second polypeptide sequence comprises a loop 5 region, said loop 5 region being positioned between the fifth and sixth invariant cysteine residues of the heterogeneous plant defensin amino acid sequence, and wherein the second polypeptide sequence corresponds to an amino acid sequence comprising X₁-X₂-X₃-X₄-X₅-X₆, wherein X₁is serine or arginine, X₂is lysine, arginine or histidine, X₃and X₄are each a hydrophobic amino acid, X₅is arginine, lysine or histidine and X₆is arginine, lysine, histidine or asparagine.
In particular embodiments, there is provided a heterogeneous plant defensin, wherein said defensin comprises a first polypeptide sequence and a second polypeptide sequence and at least eight invariant cysteine amino acid residues, wherein the first polypeptide sequence is derived from a Class I plant defensin and the second polypeptide is derived from a Class II Solanaceous plant defensin, and wherein the second polypeptide sequence comprises a loop 5 region, said loop 5 region being positioned between the fifth and sixth invariant cysteine residues of the heterogeneous plant defensin amino acid sequence, and wherein the second polypeptide sequence corresponds to an amino acid sequence comprising X₁-X₂-X₃-X₄-X₅-X₆, wherein X₁is serine or arginine, X₂is lysine, X₃is isoleucine, leucine or valine, X₄is leucine or glutamine, X₅is arginine or lysine and X₆is arginine or lysine.
In particular embodiments, there is provided a heterogeneous plant defensin, wherein said defensin comprises a first polypeptide sequence and a second polypeptide sequence and at least invariant eight cysteine amino acid residues, wherein the first polypeptide sequence is derived from a Class I plant defensin and the second polypeptide is derived from a Class II Solanaceous plant defensin, and wherein the second polypeptide sequence comprises a loop 5 region of the said loop 5 region being positioned between the fifth and sixth invariant cysteine amino acid residues of the heterogeneous plant defensin amino acid sequence, and wherein the second polypeptide sequence corresponds to an amino acid sequence comprising X₁-X₂-X₃-X₁-X₅-X₆, wherein X₁is serine or arginine, X₂is lysine, X₃is isoleucine, X₁is leucine, X₅is arginine and X₆is arginine.
In particular embodiments, there is provided a heterogeneous plant defensin, wherein said defensin comprises a first polypeptide sequence and a second polypeptide sequence and at least eight invariant cysteine amino acid residues, wherein the first polypeptide sequence is derived from a Class I plant defensin and the second polypeptide is derived from a Class II Solanaceous plant defensin, and wherein the second polypeptide sequence comprises a loop 5 region positioned between the fifth and sixth invariant cysteine residues, and wherein the second polypeptide sequence corresponds to an amino acid sequence comprising X₂-X₃-X₄-X₅-X₆, wherein X₂is lysine, arginine or histidine, X₃and X₄are each a hydrophobic amino acid, X₅is arginine, lysine or histidine and X₆is arginine, lysine, histidine or asparagine.
In particular embodiments, there is provided a heterogeneous plant defensin, wherein said defensin comprises a first polypeptide sequence and a second polypeptide sequence and at least eight invariant cysteine amino acid residues, wherein the first polypeptide sequence is derived from a Class I plant defensin and the second polypeptide is derived from a Class II Solanaceous plant defensin, and wherein the second polypeptide sequence comprises a loop 5 region positioned between the fifth and sixth invariant cysteine residues, and wherein the second polypeptide sequence corresponds to an amino acid sequence comprising X₂-X₃-X₄-X₅-X₆, wherein X₂is lysine, X₃is isoleucine, leucine or valine, X₄is leucine or glutamine, X₅is arginine or lysine and X₆is arginine or lysine.
In particular embodiments, there is provided a heterogeneous plant defensin, wherein said defensin comprises a first polypeptide sequence and a second polypeptide sequence and at least eight invariant cysteine amino acid residues, wherein the first polypeptide sequence is derived from a Class I plant defensin and the second polypeptide is derived from a Class II Solanaceous plant defensin, and wherein the second polypeptide sequence comprises a loop 5 region positioned between the fifth and sixth invariant cysteine residues, and wherein the second polypeptide sequence corresponds to an amino acid sequence comprising X₂-X₃-X₄-X₅-X₆, wherein X₂is lysine, X₃is isoleucine, X₄is leucine, X₅is arginine and X₆is arginine.
In particular embodiments, the heterogeneous plant defensin is selected from SEQ ID NO:29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO:69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO:30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58 SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86 or SEQ ID NO: 88
In certain embodiments, the heterogeneous plant defensin has enhanced and/or broader spectrum anti-proliferative disease activity, or enhanced and/or broader spectrum cytotoxic activity relative to the defensin from which the first polypeptide sequence is derived.
In particular embodiments, there is provided a heterogeneous plant defensin comprising an amino acid backbone derived from or corresponding to a Class I defensin having at least eight invariant cysteine amino acid residues and comprising a loop region located between the fifth and sixth invariant cysteine residues, the loop region on the backbone being subjected to one or more of a substitution, addition and/or deletion and/or replacement by a loop region, or modified form thereof, from a Class II Solanaceous plant defensin, said Class II Solanaceous plant defensin having at least eight invariant cysteine amino acid residues and wherein the substitute loop from the Class II Solanaceous plant defensin is derived from the region between the fifth and sixth invariant cysteine residues of the Class II Solanaceous plant defensin, wherein the heterogeneous defensin exhibits enhanced anti-proliferative activity and/or broader spectrum of activity compared to the Class I defensin used as the backbone or the Class II Solanaceous plant defensin from which the substitute loop is derived.
In some embodiments the heterogeneous defensin binds to a phospholipid, preferably PIP2 (phosphatidylinositol 4,5-bisphosphate or Ptdlns(4,5)P2).
In certain embodiments the second polypeptide sequence, or part thereof, binds to PIP2.
In some embodiments the PIP2 is located in a cell membrane, where said cell membrane is a tumour or cancer cell membrane.
In particular embodiments the heterogeneous defensin has cell membrane permeabilisation activity.
In a second aspect of the present invention, there is provided a nucleic acid encoding the plant defensin of the first aspect.
In a third aspect of the present invention, there is provided a vector comprising the nucleic acid of the second aspect.
In a fourth aspect of the present invention, there is provided a host cell comprising the vector of the third aspect.
In a fifth aspect of the present invention, there is provided a plant defensin produced by the host cell of the fourth aspect.
In a sixth aspect of the present invention, there is provided a pharmaceutical composition for use in preventing or treating a proliferative disease, wherein the pharmaceutical composition comprises the plant defensin of the first aspect, the nucleic acid of the second aspect, the vector of the third aspect, the host cell of the fourth aspect or the expression product of the fifth aspect, together with a pharmaceutically acceptable carrier, diluent or excipient.
In a seventh aspect of the present invention, there is provided a method for preventing or treating a proliferative disease, wherein the method comprises administering to a subject a therapeutically effective amount of the plant defensin of the first aspect, the nucleic acid of the second aspect, the vector of the third aspect, the host cell of the fourth aspect, the expression product of the fifth aspect or the pharmaceutical composition of the sixth aspect, thereby preventing or treating the proliferative disease.
In an eighth aspect of the present invention, there is provided use of the plant defensin of the first aspect, the nucleic acid of the second aspect, the vector of the third aspect, the host cell of the fourth aspect, the expression product of the fifth aspect or the pharmaceutical composition of the sixth aspect in the preparation of a medicament for preventing or treating a proliferative disease.
In a ninth aspect of the present invention, there is provided a kit for preventing or treating a proliferative disease, wherein the kit comprises a therapeutically effective amount of the plant defensin of the first aspect, the nucleic acid of the second aspect, the vector of the third aspect, the host cell of the fourth aspect, the expression product of the fifth aspect or the pharmaceutical composition of the sixth aspect.
In a tenth aspect of the present invention, there is provided use of the kit of the ninth aspect for preventing or treating a proliferative disease, wherein the therapeutically effective amount of the plant defensin of the first aspect, the nucleic acid of the second aspect, the vector of the third aspect, the host cell of the fourth aspect, the expression product of the fifth aspect or the pharmaceutical composition of the sixth aspect is administered to a subject, thereby preventing or treating the proliferative disease.

DEFINITIONS

The term “derivable” includes, and may be used interchangeably with, the terms “obtainable” and “isolatable”. Compositions or other matter of the present invention that is “derivable”, “obtainable” or “isolatable” from a particular source or process include not only compositions or other matter derived, obtained or isolated from that source or process, but also the same compositions or matter however sourced or produced.
The term “derived” includes, and may be used interchangeably with, the terms “obtained” and “isolated”. Compositions or other matter of the present invention that is “derived” from a particular source or process include not only compositions or other matter derived directly from that source or process, but also the same compositions or matter derived indirectly, for example, by way of recombinant DNA technology. In the case of heterogeneous plant defensins comprising two or more polypeptides, each polypeptide may be correctly described as “derived” from a particular plant defensin if that polypeptide has been expressed by an expression vector or cassette, such expression vector or cassette having had inserted into it a cloned version of the polypeptide. Accordingly, a polypeptide that is “derived” from a plant defensin need not be the actual polypeptide sourced directly from that plant defensin, but may be an expressed clone of a polypeptide sourced from a plant defensin.
The terms “heterogeneous”, “heterogeneous plant defensin”, “heterogeneous defensin” “modified plant defensin” or “modified defensin” and related terms may be used interchangeably and as used herein refer to a defensin amino acid or polypeptide sequence that has been modified by the introduction, addition, deletion or substitution of, for example, one or more naturally or non-naturally occurring amino acids, polynucleotides or polypeptides. It is to be understood that the amino acids, polynucleotides or polypeptides that may be introduced, added or substituted to produce the heterogeneous defensin, can be derived or obtained or are obtainable from, for example, a defensin such as a Class II Solanaceous plant defensin. For example, a Class I plant defensin may be used as a backbone wherein the loop region between fifth and sixth invariant cysteines of the backbone is modified by an amino acid substitution, addition and/or deletion or substitution or addition of a loop region that is derived from the region between the fifth and sixth invariant cysteines of a Class II Solanaceous defensin, or a modified form thereof, to replace all or part of this loop region in the Class I defensin sequence. The backbone Class I defensin may also optionally comprise additional mutations outside this loop region. When present, from 1 to about 50 additional mutations in the form of an amino acid substitution, addition and/or deletion may be present in the backbone Class I defensin.
The terms “enhanced” and “improved” and related terms in the context of the invention mean a quantitative or qualitative change, improvement, modulation, increase or modification, in one or more of anti-proliferative disease activity, cytotoxic activity, spectrum of activity, stability and/or membrane permeabilization capacity as relative a naturally occurring plant defensin or the plant defensin which is the subject of modification or relative to the one or more defensins from which the sequences for the hetereogenous defensin are derived. “Enhancing” and similar terms are intended to encompass any increase, change, modification or modulation of anti-proliferative disease activity, cytotoxic activity or spectrum of activity, stability, solubility and/or membrane permeabilization capacity, whether brought about by increase in the activity of the defensin itself, or by increase in the amount of defensin present, or both.
The term “wild-type” as used herein in relation to defensins includes polypeptides in their native form.
As used herein the term “polypeptide” means a polymer made up of amino acids linked together by peptide bonds, and includes fragments or analogues thereof. The terms “polypeptide”, “protein” and “amino acid” are used interchangeably herein, although for the purposes of the present invention a “polypeptide” may constitute a portion of a full length protein.
The term “nucleic acid” as used herein refers to a single- or double-stranded polymer of deoxyribonucleotide, ribonucleotide bases or known analogues of natural nucleotides, or mixtures thereof. The term includes reference to the specified sequence as well as to the sequence complementary thereto, unless otherwise indicated. The terms “nucleic acid”, “polynucleotide” and “nucleotide sequence” are used herein interchangeably. It will be understood that “5′ end” as used herein in relation to a nucleic acid corresponds to the N-terminus of the encoded polypeptide and “3′ end” corresponds to the C-terminus of the encoded polypeptide.
The term “purified” means that the material in question has been removed from its natural environment or host, and associated impurities reduced or eliminated such that the molecule in question is the predominant species present. The term “purified” therefore means that an object species is the predominant species present (ie., on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 30 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 to 90 percent of all macromolecular species present in the composition. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. The terms “purified” and “isolated” may be used interchangeably. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein or nucleic acid that is the predominant species present in a preparation is substantially purified. The term “purified” in some embodiments denotes that a protein or nucleic acid gives rise to essentially one band in an electrophoretic gel.
The term “fragment” refers to a polypeptide or nucleic acid that encodes a constituent or is a constituent of a polypeptide or nucleic acid of the invention thereof. Typically the fragment possesses qualitative biological activity in common with the polypeptide or nucleic acid of which it is a constituent. A peptide fragment may be between about 5 to about 150 amino acids in length, between about 5 to about 100 amino acids in length, between about 5 to about 50 amino acids in length, or between about 5 to about 25 amino acids in length. Alternatively, the peptide fragment may be between about 5 to about 15 amino acids in length. The term “fragment” therefore includes a polypeptide that is a constituent of a full-length plant defensin polypeptide and possesses qualitative biological activity in common with a full-length plant defensin polypeptide. A fragment may be derived from a full-length plant defensin polypeptide or alternatively may be synthesised by some other means, for example chemical synthesis.
The term “fragment” may also refer to a nucleic acid that encodes a constituent or is a constituent of a polynucleotide of the invention. Fragments of a nucleic acid do not necessarily need to encode polypeptides which retain biological activity. Rather the fragment may, for example, be useful as a hybridization probe or PCR primer. The fragment may be derived from a polynucleotide of the invention or alternatively may be synthesized by some other means, for example chemical synthesis. Nucleic acids of the present invention and fragments thereof may also be used in the production of antisense molecules using techniques known to those skilled in the art.
The term “recombinant” when used with reference, for example, to a cell, nucleic acid, protein or vector, indicates that the cell, nucleic acid, protein or vector has been modified by the introduction of a heterologous nucleic acid or protein or by the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Accordingly, “recombinant” cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. By the term “recombinant nucleic acid” is meant a nucleic acid, originally formed in vitro, in general, by the manipulation of a nucleic acid, for example, using polymerases and endonucleases, in a form not normally found in nature. In this manner, operable linkage of different sequences is achieved. Thus an isolated nucleic acid, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined, are both considered “recombinant” for the purposes of this invention. It is understood that once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e., using the in vivo cellular machinery of the host cell rather than in vitro manipulations. However, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes of the invention. Similarly, a “recombinant protein” is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid as depicted above.
The term “variant” as used herein refers to substantially similar sequences. Generally, polypeptide sequence variants possess qualitative biological activity in common. Further, these polypeptide sequence variants may share at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity. Also included within the meaning of the term “variant” are homologues of polypeptides of the invention. A homologue is typically a polypeptide from a different species but sharing substantially the same biological function or activity as the corresponding polypeptide disclosed herein.
Further, the term “variant” also includes analogues of the polypeptides of the invention, wherein the term “analogue” means a polypeptide which is a derivative of a polypeptide of the invention, which derivative comprises addition, deletion, substitution of one or more amino acids, such that the polypeptide retains substantially the same function. The term “conservative amino acid substitution” refers to a substitution or replacement of one amino acid for another amino acid with similar properties within a polypeptide chain (primary sequence of a protein).
As for polypeptides discussed above, the term “variant” as used herein refers to substantially similar sequences. Generally, polynucleotide sequence variants encode polypeptides which possess qualitative biological activity in common. Further, these polynucleotide sequence variants may share at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity. Also included within the meaning of the term “variant” are homologues of polynucleotides of the invention. A homologue is typically a polynucleotide from a different species but sharing substantially the same activity.
The term “invariant” refers to one or more amino acids or polynucleotides that do not substantially vary, for example, in relative position or charge, in, for example, a class, sub-class, group or family of amino acid, polypeptide or polynucleotide sequences.
The terms “identical” or percent “identity” in the context of two or more polypeptide (or nucleic acid) sequences, refer to two or more sequences or sub-sequences that are the same or have a specified percentage of amino acid residues (or nucleotides) that are the same over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region, as measured using sequence comparison algorithms, or by manual alignment and visual inspection, such techniques being well known to the person skilled in the art.
Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence”, “comparison window”, “similarity”, “sequence similarity”, “sequence identity”, “percentage of sequence similarity”, “percentage of sequence identity”, “substantially similar” and “substantial identity”. A “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e. only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al. (Nucl. Acids, Res. 25: 3389, 1997). A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. (In: Current Protocols in Molecular Biology, John Wiley & Sons Inc. 1994-1998).
The terms “sequence similarity” and “sequence identity” as used herein refers to the extent that sequences are identical or functionally or structurally similar on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity”, for example, is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, I) or the identical amino acid residue (e.g. Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present disclosure, “sequence identity” will be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, Calif., USA) using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity.
The nucleic acid molecules taught herein are also capable of hybridizing to other genetic molecules including the nucleic acid molecule encoding the heterogeneous defensin.
Stringency conditions can be defined by, for example, the concentrations of salt or formamide in the pre-hybridization and hybridization solutions, or by the hybridization temperature, and are well known in the art. For example, stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature, altering the time of hybridization, as described in detail, below. In alternative aspects, nucleic acids of the present disclosure are defined by their ability to hybridize under various stringency conditions (e.g., high, medium, and low).
Reference herein to a “low stringency” includes and encompasses from at least about 0 to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions. Generally, low stringency is at from about 25-30° C. to about 42° C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions. Alternative stringency conditions may be applied where necessary, such as “medium stringency”, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions, or “high stringency”, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization, and at least about 0.01 M to at least about 0.15 M salt for washing conditions. In general, washing is carried out T_m=69.3+0.41 (G+C) % (Marmur and Doty, J Mol Biol 5:109-118, 1962). However, the Tm of a duplex nucleic acid molecule decreases by 1° C. with every increase of 1% in the number of mismatch base pairs (Bonner and Laskey, Eur J Biochem 46:83-88, 1974). Formamide is optional in these hybridization conditions. Accordingly, particularly preferred levels of stringency are defined as follows: low stringency is 6×SSC buffer, 0.1% w/v SDS at 25-42° C.; a moderate stringency is 2×SSC buffer, 0.1% w/v SDS at a temperature in the range 20° C. to 65° C.; high stringency is 0.1×SSC buffer, 0.1% w/v SDS at a temperature of at least 65° C.
As used herein the term “treatment”, refers to any and all uses which remedy a disease state or symptoms, prevent the establishment of disease, or otherwise prevent, hinder, retard, ameliorate or reverse the progression of disease or other undesirable symptoms in any way whatsoever.
The terms “Class I defensin” includes reference to defensins from various organisms such as mammals, plants and insects. Class I plant defensins sequences may be identified by the presence of an endoplasmic reticulum (ER) signal sequence followed by a mature defensin domain as illustrated in FIG. 10. Examples of Class I defensins as used herein include, but are not limited to; NaD2 (Acc No. AF509566); g1-H (Acc No. P20230); Psd1 (Acc No. P81929); Ms-Def1 (Acc No. AAV85437); Dm-AMP1 (Acc No. AAB34972); R5-AFP2 (Acc No. AAA69540) or g-zeathionin 2 (Acc No. ABG78829) see FIG. 11 and FIGS. 13 to 15 for exemplary Class I defensin sequences, origins and Accession numbers.
The terms “Class II defensins” “Class II plant defensins” and “Class II Solanaceous plant defensins” as used herein refer to defensins that are produced as larger precursors with C-terminal pro-domains or pro-peptides (CTPPs) of about 33 amino acids. Most of the Class II defensins identified to date have been found in Solanaceous plant species such as Nicotiana spp., Petunia spp., Capsicum spp. Sequence alignments of exemplary Class II Solanaceous plant defensins of the invention are provided in FIGS. 11 to 15. A Class II Solanaceous defensin can be generally distinguished from other defensins by a relatively conserved C-terminal domain as shown in FIG. 10. Examples of Class II Solanaceous defensins as used herein include, but are not limited to; NaD1 (SEQ ID NO: 22; NCBI database accession no. AF509566), NsD1 (SEQ ID NO 109 and 110), NsD2 (SEQ ID NO: 111 and 112), NoD173 (SEQ ID NO: 113 and 114), PhD1A (Sol Genomics Network database accession no. SGN-U207537), TPP3 (NCBI database accession no. SLU20591), FST (NCBI database accession no. Z11748), NatD1 (NCBI database accession no. AY456268), NeThio1 (NCBI database accession no. AB005265), NeThio2 (NCBI database accession no. AB005266), NpThio1 (NCBI database accession no. AB005250), CcD1 (NCBI database accession no. AF128239), PhD1 (NCBI database accession no. AF507975), PhD2 (NCBI database accession no. AF507976), any defensin with an amino acid or nucleic acid sequence corresponding to any of the sequences set forth under NCBI database accession numbers EU367112, EU560901, AF112869 or AF112443, or any defensin with an amino acid or nucleic acid sequence corresponding to any of the sequences set forth under Sol Genomics Network database accession numbers SGN-U448338, SGN-U449253, SGN-U448480, SGN-U447308, SGN-U578020, SGN-U577258, SGN-U286650, SGN-U268098, SGN-U268098, SGN-U198967, SGN-U196048, SGN-U198968 or SGN-U198966.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g. in cell biology, chemistry, molecular biology and cell culture). Standard techniques used for molecular and biochemical methods can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed. (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons, Inc.—and the full version entitled Current Protocols in Molecular Biology).
Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
Throughout this specification, reference to numerical values, unless stated otherwise, is to be taken as meaning “about” that numerical value. The term “about” is used to indicate that a value includes the inherent variation of error for the device and the method being employed to determine the value, or the variation that exists among the study subjects.
The reference to any prior art in this specification is not, and should not be taken as an acknowledgement or any form of suggestion that prior art forms part of the common general knowledge of the person skilled in the art.
The entire content of all publications, patents, patent applications and other material recited in this specification is incorporated herein by reference.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 is an exemplary amino acid sequence of a heterogeneous defensin.
SEQ ID NOS: 2-21 and 89-108 are exemplary nucleotide sequences and corresponding amino acid sequences for the second polynucleotide sequence of the heterogeneous defensin.
SEQ ID NO: 22 is an exemplary full length amino acid sequence for the plant defensin NaD1.
SEQ ID NO: 23 is an exemplary full length amino acid sequence for the plant defensin Dm-AMP 1 and SEQ ID NO:24 is the corresponding nucleotide sequence.
SEQ ID NO: 25 is an exemplary full length amino acid sequence for the plant defensin g1-H and SEQ ID NO:26 is the corresponding nucleotide sequence.
SEQ ID NO: 27 is an exemplary full length amino acid sequence for the plant defensin NaD2 and SEQ ID NO:28 is the corresponding nucleotide sequence.
SEQ ID NO:29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63: SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO:69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87 are exemplary full length amino acid sequences of heterogeneous defensin.
SEQ ID NO:30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58 SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID 76, SEQ ID NO: 78, SEQ ID 80, SEQ ID NO: 82, SEQ ID 84, SEQ ID NO: 86 and SEQ ID 88 are the corresponding nucleic acid sequences of the exemplary full length amino acid sequences of heterogeneous defensin.
SEQ ID NO: 109 is an exemplary full length amino acid sequence for the plant defensin NsD1, with SEQ ID NO: 110 being the corresponding nucleic acid sequence.
SEQ ID NO: 111 is an exemplary full length amino acid sequence for the plant defensin NsD2, with SEQ ID NO: 112 being the corresponding nucleic acid sequence.
SEQ ID NO:113 is an exemplary full length amino acid sequence for the plant defensin NoD173 with SEQ ID NO: 114 being the corresponding nucleic acid sequence.

BRIEF DESCRIPTION OF THE FIGURES

The present invention will now be described, by way of example only, with reference to the following figures.

FIG. 1A is a series of micrographs showing the effect of NaD1 on human HeLa and U937 cells. HeLa cells cultured as adherent monolayers on coverslips or U937 cells immobilized onto 10% poly-L-lysine-coated coverslips, were stained with the membrane dye PKH67 (20 μM) and treated with 1001 NaD1 in the presence of 1 μg/ml propidium iodide (PI) for 25 minutes. Cells were then imaged by normal light microscopy or confocal laser scanning microscopy (CLSM). Scale bars represent 10 μm.

FIG. 1B is a series of micrographs comparing the kinetics of bleb formation and permeabilisation in U937 cells mediated by NaD1. U937 cells immobilised onto 10% poly-L-lysine coverslips were treated with 200 NaD1 in the presence of both 100 μg/ml 4 kDa FITC-dextran and 1 μg/ml PI and cells imaged over a period of 6 minutes. Cells were then imaged by light microscopy and confocal laser scanning microscopy (CLSM). The arrow indicates the first site of PI entry. Scale bars represent 10 μm.

FIG. 2A is a graphical representation of a flow cytometry dot plot showing BODIPY-NaD1 binding to viable and NaD1-permeabilised U937 cells. U937 cells at 10⁶ml⁻¹in RPMI medium containing 0.1% BSA were treated with 10 μM NaD1 or BODIPY-NaD1 at 37° C. for 30 min prior to addition of 7AAD (1 μg ml⁻¹) and flow cytometry analysis. The percentage of cells in each population (boxed) is indicated.

FIG. 2B is a series of micrographs examining the subcellular localisation of BODIPY-NaD1 in U937 and PC3 cells. U937 cells immobilised onto 10% poly-L-lysine coverslips or PC3 cells cultured as adherent monolayers on coverslips, were treated with 1001 BODIPY-NaD1 in the presence of 1 μg/ml PI for 12 minutes and cells imaged by CLSM. Scale bars represent 10 μm.

FIG. 2C is a series of micrographs showing the effect of NaD1 on GFP-only transfected Hela cells. HeLa cells were cultured as adherent monolayers on coverslips and transfected with pEGFP-N1. Cells were then treated with 10 μM NaD1 in the presence of 1 μg/ml PI and imaged by normal light microscopy and CSLM over a period of 6 minutes. The arrow indicates the first site of PI entry. Scale bars represent 10 μm.

FIG. 2D is a series of micrographs showing the effect of NaD1 on GFP-PH(PLCδ) transfected Hela cells. HeLa cells were cultured as adherent monolayers on coverslips and transfected with pEGFP-GFP-PH(PLCδ). Cells were then treated with 10 μM NaD1 in the presence of 1 μg/ml PI and imaged by normal light microscopy and CSLM over a period of 6 minutes. The arrow indicates the first site of PI entry on a GFP-PH(PLCδ) expressing cell. Scale bars represent 10 μm.

FIG. 3A is a diagrammatic representation showing the superimposition of the NaD1 NMR structure (PDB: 1MR4) with the NaD1 monomer crystal structure.

FIG. 3B is a series of diagrammatic representations of the NaD1:PIP2 (phosphatidylinositol 4,5-bisphosphate or Ptdlns(4,5)P2) oligomer. The top two panels show two orthogonal views of the NaD1:PIP2 oligomer comprising 14 NaD1 monomers (shown as ribbons) and 14 PIP2 molecules (shown as sticks), with the surface of the complex shown in translucent form. The bottom panel shows a surface representation of the NaD1 14-mer, displaying the extended binding groove on the inside of the arch. For clarity the 14 bound PIP2 molecules were omitted.

FIG. 3C is a series of diagrammatic representations showing the molecular basis of the monomer-monomer and dimer-dimer interactions. In the top left panel, the interface of two NaD1 monomers revealing the hydrogen bonding pattern is shown. Key residues involved in interactions in the monomer-monomer interface are labelled. For clarity bound PIP2 molecules are omitted. Main chain-only hydrogen bonds are formed between K4 residues from each monomer, main chain:side chain hydrogen bonds are found between the backbone oxygen of E6 and R1, and the backbone oxygen of C47 and K45 respectively. Salt bridges are found between R1 and E27 from each monomer. In the top right panel, four molecules of NaD1 forming a dimer of dimers is shown. Monomer I N8 forms a hydrogen bond with K17 from monomer III. Monomer II E2 and D31 form hydrogen bonds with the backbone nitrogen of monomer III R1, with the nitrogen of monomer II R1 forming a hydrogen bond with monomer III D31. A monomer II:monomer IV hydrogen bond is found between K17 and N8. In the bottom left panel the PIP2 binding site on monomer I is shown. PIP2 interactions with monomer I are mediated by K4, H33, K36, I37, L38 and R40 with all three phosphate groups of PIP2. Additional hydrogen bonds are contributed by monomer II R40 and monomer IV′ K36. The bottom right panel shows the PIP2 binding site on monomer II. PIP2 interactions with monomer II are mediated by K4, H33, K36, I37, L38 and R40 with all three phosphate groups of PIP2. Additional hydrogen bonds are contributed by monomer I R40 and monomer III K36.

FIG. 3D is a schematic diagram illustrating the molecular interactions of the NaD1:PIP2 complex. The amino acid residues from neighbouring NaD1 monomers involved in binding two PIP2 molecules are illustrated as boxes and their interactions with PIP2 indicated with lines.

FIG. 4A is a series of transmission electron microscopy (TEM) images of the NaD1-PIP2 oligomer. Shown are images of NaD1 alone (left panel), PIP2 alone (middle panel) and NaD1-PIP2 (right panel). Scale bars represent 100 nm.

FIG. 4B is an immunoblot depicting the lipid-mediated oligomerisation of the class II defensins NaD1 and TPP3, and the class I defensin NaD2. Each of the defensins at 250 ng/ml were incubated with 0.5 mM PIP2 or PA at room temperature for 30 minutes and samples then treated with the cross-linker bis[sulfosuccinimidyl] suberate (BS³) at 12.5 mM for a further 30 minutes, Samples were reduced and denatured, separated by SDS-PAGE, and examined by immunoblotting with an anti-NaD1 (NaD1 and TPP3) or anti-NaD2 (NaD2) antibody. Molecular weight markers are indicated.

FIG. 5A is a diagram depicting the amino acid sequence alignment of recombinant NaD1 (rNaD1) and the two NaD1 loop swap forms D2L4A and D2L5. D2L4A and D2L5 differ from rNaD1 in that their loop 4 and loop 5 regions have been replaced with the equivalent regions of the class I defensins NaD2, respectively.

FIG. 5B is a graphical representation showing the effect of rNaD1, rNaD1(D2L4A), or rNaD1(D2L5) on the permeabilisation of U937 cells. Cells were incubated with 10 μM of each defensin for 30 minutes at 37° C. upon which 1 μg/ml PI propidium iodide (PI) was added. The number of cells that stained positively for PI was determined by flow cytometry.

FIG. 5C is an immunoblot depicting the lipid binding profile of rNaD1, rNaD1(D2L4A), or rNaD1(D2L5). Echelon™ membrane lipid strips were probed with the defensins and binding detected with a rabbit anti-NaD1 antibody followed by a horseradish peroxidase (HRP) conjugated donkey anti-rabbit IgG antibody.

FIG. 5D is a graphical representation of the densitometric quantification of the lipid binding profile of rNaD1 or rNaD1(D2L4A) or NaD1(D2L5) to Echelon™ membrane lipid strips shown in FIG. 5C. Data is the mean of three replicate experiments±SEM.

FIG. 6A is a diagram depicting the amino acid sequence of NaD1 and the NaD1 with a loop swap DmAMP1L5, γ1-hordoL5, RsAFP2L5 and VrD1L5. The four loop swap NaD1s differ from NaD1 in that their loop 5 regions have been replaced with the equivalent regions of the class I defensins DmAMP1, γ1-hordothionin, RsAFP2 and VrD1.

FIG. 68 is a graphical representation showing the effect of NaD1, DmAMP1L5, γ1-hordoL5, RsAFP2L5 and VrD1L5 on the permeabilisation of U937 cells. Cells were incubated with 10 μM of each defensin for 30 minutes at 37° C. upon which 1 μg/ml PI propidium iodide (PI) was added. The number of cells that stained positively for PI was determined by flow cytometry.

FIG. 6C is an immunoblot depicting the lipid binding profile of NaD1, DmAMP1L5, γ1-hordoL5, RsAFP2L5 and VrD1L5. Echelon™ membrane lipid strips were probed with the defensins and binding was detected with a rabbit anti-NaD1 antibody followed by a horseradish peroxidase (HRP) conjugated donkey anti-rabbit IgG antibody.

FIG. 6D is a graphical representation of the densitometic quantification of the lipid binding profile of NaD1, NaD1(DmAMP1L5), NaD1(γ1-hordoL5), NaD1(RsAFP2L5) and NaD1(VrD1L5) to Echelon™ membrane lipid strips shown in FIG. 6C. Data is the mean of three replicate experiments±SEM.

FIG. 7 is a diagrammatic representation of the proposed molecular mechanism by which NaD1 induces membrane blebbing/permeabilisation and oligomerisation with PIP2. The proposed four main steps are shown as (i) entry, (ii) dimerisation and competition, (iii) oligomerisation and bleb, and (iv) permeabilisation. The proposed order of assembly of NaD1:PIP2 oligomer is also shown and can potentially be formed by the sequential recruitment of a NaD1 monomer followed by a PIP2 molecule or the dimerisation of two single NaD1:PIP2 complex followed by the recruitment of NaD1:PIP2 dimers.

FIG. 8 is a graphical representation showing the effect of rNaD1, K36E and R40E NaD1 mutants on the permeabilisation of U937 cells. Cells were incubated with 5 μM of each defensin for 30 minutes at 37° C. upon which 1 mg/ml PI propidium iodide (PI) was added. The number of cells that stained positively for PI was determined by flow cytometry.

FIG. 9 Part A is a ribbon representation of solution structure of NaD1 (PDB code 1MR4). The N- and C-termini, all secondary structure elements and the side-chains of the four disulfide bonds are shown. The Loop 5 region is indicated. FIG. 9 Part B is a diagrammatic representation of the amino acid sequence of NaD1. The secondary structure elements and the disulfide bonds (dashed lines) are given above the sequence. Loops (L1-L7), as defined by the amino acids between two neighbouring invariant cysteine residues, are given below the sequence.

FIG. 10 diagrammatically illustrates the two classes of plant defensins. Part A: Class I: All plant defensins are produced with an ER signal sequence in addition to the mature defensin domain. Part B: Class II: In some plants, particularly those from the Solanaceae, cDNA clones have been isolated that encode plant defensins with an additional C-terminal prodomain. The four strongly conserved disulfide bonds in the defensin domain are illustrated by connecting lines.

FIG. 11 is a representation of exemplary amino acid sequence alignments of Class II Solanaceous plant defensins and Class I plant defensins, showing the eight conserved or invariant cysteine residues and other highly conserved amino acid residues as shaded regions.

FIG. 12A is a representation of an alignment of exemplary amino acid sequences of Class II Solanaceous plant defensins demonstrating the extremely high level of conservation in the amino acid sequence located between the fifth and sixth invariant Cysteine residues corresponding to the loop 5 region (note: CcD1 from Capsicum chinense is identified as Cc-gth in this figure).

FIG. 12B is the same representation as shown in FIG. 12A without shading.

FIG. 13 is a representation of an alignment of exemplary amino acid sequences of Class I defensins aligned against the Class II Solanaceous defensin NaD1. It demonstrates the variability amongst Class I defensins of the amino acid sequence located between the fifth and sixth invariant cysteine residues corresponding to the loop 5 region.

FIG. 14A is a representation of an alignment of exemplary amino acids of Class II Solanaceous defensins and Class I defensins based on an alignment of the corresponding positions of the eight invariant Cysteine (Cys) residues demonstrating that certain amino acid residues outside of the loop 5 region are also conserved. The eight conserved or invariant cysteine residues and other conserved amino acid residues are shown as shaded regions. For the sake of comparison, the numbering of all the amino acid sequences in the alignment is based on the NaD1 amino acid sequence whereby the first amino acid residue of NaD1 is designated as residue 1.

FIG. 14B is the same representation as shown in FIG. 14A without shading.

FIG. 15A is a representation of an alignment of exemplary amino acids of Class II Solanaceous defensins and Class I defensins based on an alignment of the eight invariant Cysteine residues again demonstrating that certain amino acid residues outside of the loop 5 region are also conserved. For the sake of comparison, the numbering of all the amino acid sequences in the alignment is based on the NaD1 amino acid sequence whereby the first amino acid residue of NaD1 is designated as residue 1.

FIG. 15B is the same representation as shown in FIG. 14A without shading.

DETAILED DESCRIPTION OF THE INVENTION

As a result of the findings of the inventors as described herein, heterogeneous forms of plant defensins with advantageous qualities and functions, including but not limited to functionalisation of defensins that were previously inactive or had low activity in relation to treatment of proliferative disease, new or varied plant defensin functions, defensins with improved or enhanced cytotoxic potency, membrane permeabilisation activity and/or broader spectrum of activity can be produced.
The heterogeneous defensins as described herein are proposed to be useful in the manufacture of animal and human medicaments as well as associated uses, systems and kits. These findings also provide for methods for the prevention or treatment of proliferative diseases such as cancer, as well as associated uses, systems, kits.
Through investigations into the mechanism of action of the defensins, the present inventors have surprisingly found that the Class II Solanaceous plant defensin loop 5 region, as exemplified by NaD1, is involved in the anti-proliferative activity of Class II Solanaceous defensins. Without being bound to any mechanism or theory, the inventors have identified that specific amino acid residues located in the loop 5 region of Class II Solanaceous plant defensins interact with cellular lipids, particularly PIP2 (phosphatidylinositol 4,5-bisphosphate or Ptdlns(4,5)P2) in cell membranes.
For example, the inventors observed that addition of 10 μM NaD1 to the human tumour cell lines HeLa and PC3 resulted in profound plasma membrane blebbing and cell permeabilsation (FIG. 1A). Significantly, the site of cell permeabilisation corresponds with the site of plasma membrane blebbing, demonstrating NaD1 causes membrane blebbing that results in weakening of the plasma membrane and cell permeabilisation (FIG. 1B). NaD1 was found to accumulate on the surface of U937 cells prior to membrane permeabilisation (FIG. 2A) and inside permeabilised cells at the membrane bleb(s), cytoplasm, nucleolus and possibly at specific cytoplasmic organelles (FIG. 2B). Without wishing to be bound by a specific mechanism or theory, the inventors observed that the binding of NaD1 to the phosphoinositide PIP2 is involved in cell permeabilisation and tumour cell killing. Further, HeLa cells overexpressing the PIP2 binding protein PH(PLCδ) tagged with GFP were protected from permeabilisation by NaD1, whereas cells overexpressing GFP alone were sensitive to NaD1 (FIGS. 2C and 2D). These data demonstrate that the binding of PIP2 by NaD1 initiates membrane blebbing and cell permeabilisation and represents a novel mechanism by which tumour cells can be killed. The understanding of the mechanism of action of Class II Solanaceous plant defensins, such as NaD1, provides important information to assist in the design of novel defensins with enhanced or transformed functions for therapeutic use in the prevention or treatment of proliferative diseases.
The structural determination of the NaD1-PIP2 complex by crystallization and X-ray diffraction was used to define the precise molecular basis of how NaD1 interacts with PIP2 (FIG. 3A-3C). The region of NaD1 that is involved for the binding of PIP2 was identified as loop 5, amino acids Ser³⁵-Arg⁴⁰(FIG. 9). The binding of PIP2 by NaD1 also mediates the formation of an oligomeric structure (FIGS. 4A and 4B). Oligomer formation by NaD1 is lipid-dependent, with the binding of only PIP2 but not PA mediating the efficient formation of oligomers. The oligomerisation by PIP2 was also conserved in another class II defensin, TPP3. In contrast, the Class I defensin NaD2 forms oligomers with PA but not PIP2 (FIG. 4B). Without wishing to be bound by theory, as Class II Solanaceous plant defensins but not Class I defensins are able to kill tumour cells, these data suggest that the specificity of lipid-mediated oligomerisation of defensins is involved in tumour cell killing (FIG. 7).
The inventors further observed that the amino acid sequences of a range of Class II Solanaceous plant defensins, as shown in FIGS. 11 and 12A and 12B, are very highly conserved in the loop 5 region flanked by the fifth and sixth invariant cysteines. However, in contrast, there is very little consensus across the Class I defensin group as a whole in the loop 5 region. There is also very little, to no consensus between the loop 5 region sequence in the Class II Solanaceous defensins and the loop 5 sequence region in the Class I defensins (see FIGS. 11 to 15).
Without being bound to any specific mechanism or pathway, the inventors have found that the Class II Solanaceous plant defensin loop 5 region, as defined by being flanked by the fifth and sixth (invariant) cysteine (Cys) amino acid residues of a Class II Solanaceous plant defensin amino acid sequence, is involved in cytoxicity by replacing of Ser³⁵-Arg⁴⁰of NaD1 with the equivalent region of the class I defensins NaD2, DmAMP1, γ1-hordothionin, RsAFP2 or VrD1, and observing a loss in cytotoxic activity (FIGS. 5A-C and 6A-C). In addition, the replacement of two different residues in the loop 5 region lysine-(K)36 and arginine-(R)40 with glutamic acid (E) also resulted greatly reduced cell permeabilisation (FIG. 8), further demonstrating the involvement of the loop 5 region of Class II plant defensins in tumour cell killing and suggesting that a Class I defensin could be transformed into a defensin with cytotoxic activity by replacing its loop 5 region with a loop 5 region (for example Ser³⁵-Arg⁴⁰of NaD1) from a Class II Solanaceous defensin.
In further studies, the present inventors have identified additional amino acid residues outside of the Class II Solanaceous defensin loop 5 region that are important for the cytoxic activity of the defensin and which are involved in, for example any one or more of the formation of H-bonds and/or salt bridges, protein-PIP2 interactions and/or PI uptake, plasma membrane blebbing and/or cell permeabilisation. Specifically, based on numbering relative to the continuous NaD1 amino acid sequence (SEQ ID NO. 22), the inventors have observed that, in addition to the loop 5 region, any one, or more of the following additional amino acid residues outside of the Class II Solanaceous defensin loop 5 are important for cytoxic activity of Class II Solanaceous defensins: Lysine-(K)4, Lysine-(K)28, Aspartic Acid (D)31, Histidine-(H)33 and Lysine (K)45. FIGS. 14 and 15 illustrate the position of these additional amino acids in NaD1 relative to other exemplary Class II and Class I plant defensins. Without wishing to be bound by theory, based on this observation, the inventors have found that, in addition to the loop 5 region as mentioned above, the presence of one or more of the following amino acids, at the positions noted, is important for cytotoxic activity:
a K (Lysine) at or around +1 amino acid residue relative to the first invariant Cysteine;
a K (Lysine) at or around +5 amino acid residues relative to the fourth invariant Cysteine;
a D (Aspartic Acid) at or around −3 amino acids residues relative to the fifth invariant Cysteine;
a H (Histidine) at or around −1 amino acid residue relative to the fifth invariant Cysteine; and/or
a K (Lysine) at or around +2 amino acid residues relative to the seventh invariant Cystine.
As would be clear to the person skilled in the art, the nomenclature used herein to describe the invariant Cysteines as, for example, “the first invariant Cysteine”, “the second invariant Cysteine”, “the fourth invariant Cysteine”, etc, refers to the first, second or fourth occurrence, respectively, of a Cysteine in the relevant sequence when viewed from left to right as presented herein. The person skilled in the art would therefore understand that reference to a K (Lysine) at or around +1 amino acid residue relative to the first invariant Cysteine refers to a K occurring at 1 position to the right of the first invariant Cysteine, as presented herein, and similarly, reference to a D (Aspartic Acid) at or around −3 amino acids residues relative to the fifth invariant Cysteine refers to a D occurring at 3 positions to the left of the fifth invariant Cysteine, as presented herein.
As a result of these studies into plant defensins, a new family of mutant forms of plant defensins may be produced with advantageous qualities and functions, including but not limited to functionalisation of defensins that were previously inactive or had low activity in relation to treatment of proliferative diseases, variation of, or addition of new, plant defensin effector functions, and development of defensins with higher cytotoxic potency, by, for example, alteration, substitution or modification of amino acids inside and, optionally; outside of the loop 5 region of a Class I defensin so as to produce a novel heterogeneous defensin, with, for example, the added ability to interact with phospholipids such as PIP2.

Heterogeneous Plant Defensins

The present invention provides a heterogeneous plant defensin, wherein the plant defensin comprises a first polypeptide sequence and a second polypeptide sequence, wherein the second polypeptide sequence is derived from a plant defensin other than the plant defensin from which the first polypeptide sequence is derived.
In some embodiments, the heterogeneous plant defensin comprises a third polypeptide sequence.
In some embodiments, the heterogeneous plant defensin comprises the sequence:
(SEQ ID NO: 1)

X_A-C1-X_B-C2-X_C-C3-X_D-C4-X_E-C5-X_F-C6-X_G-C7-X_H-C8-X_I;

wherein C1, C2, C3, C4, C5, C6, C7 and C8 is cysteine, said cysteine being an invariant cysteine,
X_A, X_B, X_C, X_D, X_E, X_F, X_G, X_H, X_Iis any naturally occurring amino acid and;
X_Ais 1 to 7 amino acids in length;
X_Bis 9, 10 or 11 amino acids in length;
X_Cis 3 to 8 amino acids in length;
X_Dis 3 amino acids in length;
X_Eis 9 to 13 amino acids in length;
X_Fis 4, 5, 6, 7 or 8 amino acids in length;
X_Gis 1 amino acid in length;
X_His 1 to 4 amino acids in length; and
X_Iis 0 or 1 amino acid in length.
In some embodiments, the second polypeptide sequence is positioned between the fourth and eighth invariant cysteine amino acid residues corresponding to the sequence as set forth in SEQ ID NO:1
In some embodiments, the second polypeptide is positioned between the fifth and sixth invariant cysteine residues corresponding to the sequence as set forth in SEQ ID NO:1.
In preferred embodiments the first polypeptide sequence is derived from a Class I plant defensin.
In preferred embodiments the second polypeptide sequence is derived from a Class II Solanaceous plant defensin.
In certain embodiments, the first polypeptide sequence is derived from a Class I plant defensin and the second polypeptide sequence is derived from a Class II Solanaceous plant defensin.
In some embodiments the third polypeptide sequence is derived from a Class I plant defensin.
In some embodiments, the second polypeptide sequence comprises a loop 5 region derived from a Class II plant defensin, or a fragment or variant thereof. The loop 5 region may begin with a serine.
In some embodiments, the loop 5 region derived from the Class II Solanaceous plant defensin comprises the entire loop 5 region of a Class II Solanaceous plant defensin, said loop 5 defined as by being flanked by the fifth and sixth invariant cysteine (Cys) amino acid residues of a Class II Solanaceous plant defensin amino acid sequence.
In some embodiments, the second polypeptide sequence comprises a fragment or modified form of the loop 5 region of a Class II Solanaceous plant defensin.
In certain embodiments, loop 5 region derived from the Class II Solanaceous plant defensin comprises an amino acid sequence beginning at the fifth invariant cysteine amino acid residue, or end of the second β-strand, and ending at the N-terminal side of the sixth invariant cysteine amino acid residue of a Class II Solanaceous plant defensin amino acid sequence. The loop 5 region is referred to as “L5” in FIG. 9 Part B. Exemplary Class II Solanaceous plant defensin loop 5 regions and amino acid sequences are shown in FIGS. 11, 12A and 12B.
In some embodiments, the second polypeptide sequence corresponds to an amino acid sequence comprising X₁-X₂-X₃-X₄-X₅-X₆, wherein X₁is serine or arginine, X₂is lysine, arginine or histidine, X₃and X₄are each a hydrophobic amino acid, X₅is arginine, lysine or histidine and X₆is arginine, lysine, histidine, asparagine.
In some embodiments, the second polypeptide sequence corresponds to an amino acid sequence comprising X₁-X₂-X₃-X₄-X₅-X₆, wherein X₁is serine or arginine, X₂is lysine, X₃is isoleucine, leucine or valine, X₄is leucine or glutamine, X₅is arginine and X₆is arginine, lysine or asparagine.
In some embodiments, the second polypeptide sequence corresponds to an amino acid sequence comprising X₂-X₃-X₄-X₅-X₆, wherein X₂is lysine, arginine or histidine, X₃and X₄are each a hydrophobic amino acid, X₅is arginine, lysine or histidine and X₆is arginine, lysine, histidine or asparagine.
In some embodiments, the second polypeptide sequence corresponds to an amino acid sequence comprising X₂-X₃-X₄-X₅-X₆, wherein X₂is lysine, X₃is isoleucine, leucine or valine, X₄is leucine or glutamine, X₅is arginine and X₆is arginine, lysine or asparagine.
In some embodiments, the second polypeptide corresponds to an amino acid sequence comprising (i) SKILRR (SEQ ID NO: 2), (ii) SKLLRR (SEQ ID NO: 4), (iii) SKILRK (SEQ ID NO: 6), (iv) SKVLRR (SEQ ID NO: 8), (v) SKVLRK (SEQ ID NO: 10), (vi) SKLQRK (SEQ ID NO: 12), (vii) SKLLRN (SEQ ID NO: 14), (viii) SKLLRK (SEQ ID NO: 16), (ix) SKIQRN (SEQ ID NO: 18), (x) RKLQRK (SEQ ID NO: 20) or (xi) KILRR (SEQ ID NO: 89), (xii) KLLRR (SEQ ID NO: 91), (xiii) KILRK (SEQ ID NO: 93), (xiv) KVLRR (SEQ ID NO: 95), (xv) KVLRK (SEQ ID NO: 97), (xvi) KLQRK (SEQ ID NO: 99), (xvii) KLLRN (SEQ ID NO: 101), (xviii) KLLRK (SEQ ID NO: 103), (xix) KIQRN (SEQ ID NO: 105) or (xx) KLQRK (SEQ ID NO: 107).
In some embodiments, the second polypeptide sequence corresponds to an amino acid sequence comprising S-K-I-L-R-R (SEQ ID NO: 2).
In some embodiments, the second polypeptide sequence corresponds to an amino acid sequence comprising K-I-L-R-R (SEQ ID NO: 89).
In some embodiments, the first and the third polypeptide sequences are both derived from the same plant defensin.
In some embodiments the heterogeneous defensin may display enhanced and/or broader spectrum anti-proliferative disease activity, and/or cytotoxic activity, and/or cell membrane permeabilisation, and/or enhanced PIP2 binding activity relative to a defensin prior to modification or relative to the defensin from which the first polypeptide is derived.
In certain embodiments the heterogeneous defensin binds to a phospholipid, preferably PIP2.
In some embodiments the second polypeptide sequence, or part thereof, binds to PIP2 in a cell membrane.
In preferred embodiments, the heterogeneous plant defensin binds PIP2 in a cell membrane.
In certain embodiments, the heterogeneous plant defensin has cell membrane permeablisation activity.
In specific embodiments, the cell membrane is a tumour cell membrane.
In some embodiments, first polypeptide sequence and optionally the third polypeptide sequence contain one or more amino acid or nucleotide substitutions, deletions or modifications, wherein the substitution, deletion or modification does not naturally occur at the substituted, deleted or modified position in the plant defensin from which the first polypeptide sequence and optionally the third polypeptide sequence is derived.
In particular embodiments, there is provided a heterogeneous plant defensin, wherein said defensin comprises a first polypeptide sequence and a second polypeptide sequence and at least eight invariant cysteine amino acid residues, wherein the first polypeptide sequence is derived from a Class I plant defensin and the second polypeptide is derived from a Class II Solanaceous plant defensin, and wherein the second polypeptide sequence comprises a loop 5 region, said loop 5 region being positioned between the fifth and sixth invariant cysteine residues of the heterogeneous plant defensin amino acid sequence, and wherein the second polypeptide sequence corresponds to an amino acid sequence comprising X₁-X₂-X₃-X₄-X₅-X₆, wherein X₁is serine or arginine, X₂is lysine, arginine or histidine, X₃and X₄are each a hydrophobic amino acid, X₅is arginine, lysine or histidine and X₆is arginine, lysine, histidine or asparagine.
In particular embodiments, there is provided a heterogeneous plant defensin, wherein said defensin comprises a first polypeptide sequence and a second polypeptide sequence and at least eight invariant cysteine amino acid residues, wherein the first polypeptide sequence is derived from a Class I plant defensin and the second polypeptide is derived from a Class II Solanaceous plant defensin, and wherein the second polypeptide sequence comprises a loop 5 region, said loop 5 region being positioned between the fifth and sixth invariant cysteine residues of the heterogeneous plant defensin amino acid sequence, and wherein the second polypeptide sequence corresponds to an amino acid sequence comprising X₁-X₂-X₃-X₄-X₅-X₆, wherein X₁is serine or arginine, X₂is lysine, X₃is isoleucine, leucine or valine, X₄is leucine or glutamine, X₅is arginine or lysine and X₆is arginine or lysine.
In particular embodiments, there is provided a heterogeneous plant defensin, wherein said defensin comprises a first polypeptide sequence and a second polypeptide sequence and at least invariant eight cysteine amino acid residues, wherein the first polypeptide sequence is derived from a Class I plant defensin and the second polypeptide is derived from a Class II Solanaceous plant defensin, and wherein the second polypeptide sequence comprises a loop 5 region of the said loop 5 region being positioned between the fifth and sixth cysteine residues of the heterogeneous plant defensin amino acid sequence, and wherein the second polypeptide sequence corresponds to an amino acid sequence comprising X₁-X₂-X₃-X₄-X₅-X₆, wherein X₁is serine or arginine, X₂is lysine, X₃is isoleucine, X₄is leucine, X₅is arginine and X₆is arginine.
In particular embodiments, there is provided a heterogeneous plant defensin, wherein said defensin comprises a first polypeptide sequence and a second polypeptide sequence and at least eight invariant cysteine amino acid residues, wherein the first polypeptide sequence is derived from a Class I plant defensin and the second polypeptide is derived from a Class II Solanaceous plant defensin, and wherein the second polypeptide sequence comprises a loop 5 region positioned between the fifth and sixth invariant cysteine residues, and wherein the second polypeptide sequence corresponds to an amino acid sequence comprising X₂-X₃-X₄-X₅-X₆, wherein X₂is lysine, arginine or histidine, X₃and X₄are each a hydrophobic amino acid, X₅is arginine, lysine or histidine and X₆is arginine, lysine, histidine or asparagine.
In particular embodiments, there is provided a heterogeneous, plant defensin, wherein said defensin comprises a first polypeptide sequence and a second polypeptide sequence and at least eight invariant cysteine amino acid residues, wherein the first polypeptide sequence is derived from a Class I plant defensin and the second polypeptide is derived from a Class II Solanaceous plant defensin, and wherein the second polypeptide sequence comprises a loop 5 region positioned between the fifth and sixth invariant cysteine residues, and wherein the second polypeptide sequence corresponds to an amino acid sequence comprising X₂-X₃-X₄-X₅-X₆, wherein X₂is lysine, X₃is isoleucine, leucine or valine, X₄is leucine or glutamine, X₅is arginine or lysine and X₆is arginine or lysine.
In particular embodiments, there is provided a heterogeneous plant defensin, wherein said defensin comprises a first polypeptide sequence and a second polypeptide sequence and at least eight invariant cysteine amino acid residues, wherein the first polypeptide sequence is derived from a Class I plant defensin and the second polypeptide is derived from a Class II Solanaceous plant defensin, and wherein the second polypeptide sequence comprises a loop 5 region positioned between the fifth and sixth invariant cysteine residues, and wherein the second polypeptide sequence corresponds to an amino acid sequence comprising X₂-X₃-X₄-X₅-X₆, wherein X₂is lysine, X₃is isoleucine, X₄is leucine, X₅is arginine and X₆is arginine.
In some embodiments, the heterogeneous plant defensin is produced by replacing the amino acids substantially comprising a loop 5 region positioned between the fifth and sixth invariant cysteine residues of a Class I defensin with an amino acid sequence substantially comprising a loop 5 region positioned between the fifth and sixth invariant cysteine residues of a Class II Solanaceous plant defensin.
In particular embodiments, the heterogeneous plant defensin is selected from the following SEQ ID NO:29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO:69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87 SEQ ID NO:30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58 SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86 or SEQ ID NO: 88.
In certain embodiments, the heterogeneous plant defensin has enhanced and/or broader spectrum anti-proliferative disease, and/or enhanced and/or broader spectrum cytotoxic activity relative to the defensin from which the first polypeptide is derived,
In some embodiments, the heterogeneous plant defensin further comprises one or more additional amino acid substitutions, deletions or additions,
In some embodiments, the heterogeneous plant defensin further comprises one or more amino acid substitutions, deletions or additions in the first polypeptide.
In particular embodiments, the heterogeneous plant defensin further comprises one or more amino acids selected from the group consisting of: a K (Lysine) at or around +1 amino acid residue relative to the first invariant Cysteine; a K (Lysine) at or around +5 amino acid residues relative to the fourth invariant Cysteine; a D (Aspartic Acid) at or around −3 amino acids residues relative to the fifth invariant Cysteine; a H (Histidine) at or around −1 amino acid residue relative to the fifth invariant Cysteine; and/or a K (Lysine) at or around +2 amino acid residues relative to the seventh invariant Cystine; and/or conservative substitutions or functional and/or structural equivalents thereof.
In yet further embodiments, the heterogeneous plant defensin comprises a first polypeptide derived from a Class I defensin from Aesculus hippocastanum, Arabidopsis halleri, Arachis diogoi, Brassica campestris, Brassica juncea, Brassica napus, Brassica oleracea, Brassica rapa subsp., Beta vulgaris, Bupleurum kaoi, Cajanus cajan, Capsicum annuum, Capsicum chinense, Cassia fistula, Cicer arietinum, Clitoria ternatea, Dahlia merckii, Echinochloa crus-galli, Elaeis guineensis, Ginkgo biloba, Glycine max, Hardenbergia violacea, Helianthus annuus, Heuchera sanguinea, Hordeum vulgare, Jatropha curcas, Lepidium meyenii, Medicago sativa, Medicago truncatula, Nicotiana alata, Nicotiana tabacum, Oryza sativa Japonica, Pachyrhizus erosus, Petunia integrifolia, Petunia hybrida, Picea abies, Pinus sylvestris, Pisum sativum, Plantago major, Prunus persica, Pyrus pyrifolia, Raphanus sativus, Saccharum officinarum, Sinapis alba, Solanum lycopersicum, Solanum tuberosum, Sorghum bicolor, Spinacia oleracea, Tephrosia villosa, Trigonella foenum-gr, Triticum aestivum, Triticum kiharae, Triticum monococcum, Triticum turgidum, Vicia faba, Vigna radiate, Vigna unguiculata, Wasabi japonica, Zea mays, Zea mays subsp. Mays.
In yet further embodiments, the heterogeneous plant defensin comprises a first polypeptide derived from a Class I defensin from Nicotiana alata, Nicotiana suaveolens, Hordeum vulgare, Pisum sativum, Medicago saliva, Dahlia merckii, Raphanus sativus, Zea mays.
In particular embodiments, the first polypeptide is derived from a Class I defensin defensin from the group comprising; NaD2 (SEQ ID NO: 27), NsD3, TGAS118 (Acc AJ133601), P322 (Acc ACJ26760), PPT (Acc AAA64740), SE60 (Acc Q0752), 10 kDa (Acc P18646), Cp-thionin, (Acc P83399), VrD1 (vrCRP) (Acc AAR08912), Psd1 (P81929), MsDef1 (alfAFP)(Acc AF319468), R5-AFP2 (AccP30230), Ah-AMP1 (Acc AAB34970), Hs-AFP1 (Acc POC8Y5), Dm-AMP1 (Acc AAB34972), Ct-AMP1 (AAB34971), SPI1 (Acc CAA62761), M2A (Acc P30232), SD2 (AAF72042, γ1-H (g1-H) (Acc P20230), γ1-P (Acc P20158), Tad1 (Acc BAC10287), Slα2 (Acc P21923), Slα3 (Acc P2192), γ1-Z (Acc P81008), γ2-Z (Acc P81009), Fabatin-1 (Acc P81456), So-D2 (Acc P81571) or WT1 (Acc AB012871) (See FIG. 11).
In particular embodiments, the first polypeptide is derived from a Class I defensin defensin from the group comprising; NaD2, Dm-AMP1, g1-H, Psd1, Ms-Deft, R5-AFP2 or g-zeathionin 2 (γ2-Z).
In particular embodiments, the second polypeptide is derived from a Class II Solanaceous defensin from Nicotiana spp., Solanum spp., Petunia spp., or Capsicum spp.
In particular embodiments, the second polypeptide is derived from a Class II Solanaceous defensin from Nicotiana alata, Nicotiana suaveolens, Nicotiana occidentalis, Petunia hybrida, Solanum lycopersicum or Capsicum chinense.
In preferred embodiments, the second polypeptide is derived from NaD1, NsD1, NsD2, NoD173, TPP3, PhD1, PhD1A, PhD2, TPP3, FST, NeThio1, NeThio2, NpThio1, Na-gth or CcD1.

Polynucleotides

In embodiments where the compositions of the present invention comprise polypeptides, the present invention also provides nucleic acids encoding such polypeptides, or fragments or complements thereof. Such nucleic acids may be naturally occurring or may be synthetic or recombinant.
In some embodiments, the nucleic acids may be operably linked to one or more promoters.
In particular embodiments the nucleic acids encode a heterogeneous plant defensin or a functional fragment thereof.
In more particular embodiments, the heterogeneous plant defensin comprises the amino acid sequence set forth as any one of SEQ ID NO:29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO:69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87.
In particular embodiments, the heterogeneous plant defensin is encoded by the nucleic acid sequence set forth as any one of SEQ ID NO:30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58 SEQ, ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86 or SEQ ID NO: 88
In some embodiments, the heterogeneous plant defensin comprises a serine amino acid positioned adjacent to the fifth invariant cysteine amino acid corresponding to the sequence set forth as SEQ ID NO:1, wherein the serine amino acid is positioned to the C-terminal side of the fifth invariant cysteine.
In still other embodiments, the heterogeneous plant defensin comprises an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%_;90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% identical to the amino acid sequence set forth as SEQ ID NOs: SEQ ID NO:29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO:69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87 or a fragment thereof.

Vectors, Host Cells and Expression Products

The present invention also provides vectors comprising the nucleic acids as set forth herein. The vector may be a plasmid vector, a viral vector, or any other suitable vehicle adapted for the insertion of foreign sequences, its introduction into cells and the expression of the introduced sequences. The vector may be a eukaryotic expression vector and may include expression control and processing sequences such as a promoter, an enhancer, ribosome binding sites, polyadenylation signals and transcription termination sequences. In preferred embodiments, the vector comprises one or more nucleic acids operably encoding any one or more of the plant defensins set forth herein.
The present invention further provides host cells comprising the vectors as set forth herein. Typically, a host cell is transformed, transfected or transduced with a vector, for example, by using electroporation followed by subsequent selection of transformed, transfected or transduced cells on selective media. The resulting heterologous nucleic acid sequences in the form of vectors and nucleic acids inserted therein may be maintained extrachromosomally or may be introduced into the host cell genome by homologous recombination. Methods for such cellular transformation, transfection or transduction are well known to those of skill in the art. Guidance may be obtained, for example, from standard texts such as Sambrook of al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989 and Ausubel of al., Current Protocols in Molecular Biology, Greene Publ. Assoc. and Wiley-Intersciences, 1992.
The present invention moreover provides expression products of the host cells as set forth herein.
The present invention also provides a vector comprising a nucleic acid encoding the heterogeneous plant defensin or a functional fragment thereof and a host cell comprising such a vector. In further aspects of the invention there is provided a heterogeneous plant defensin produced by the host cell
In some embodiments, the expression product may be polypeptides that prevent or treat proliferative diseases. In preferred embodiments, the expression product is any one or more of the plant defensins disclosed herein.
In other embodiments, the isolated nucleic acid molecule may also be in a vector including an expression or transfer vector suitable for use in microbial cells and non-human animal cells.
In some embodiments, the polynucleotides as described herein may be inserted within a coding region expressing another protein to form a heterogeneous defensin fusion protein or may be used to replace a domain of a protein to give that protein, anti-proliferative disease activity or cytotoxicity. The nucleic acid sequence may be placed under the control of a homologous or heterologous promoter which may be a constitutive or an inducible promoter (stimulated by, for example, presence of a chemical). The transit peptide may be homologous or heterologous to the modified defensin and is chosen to ensure secretion to the desired organelle or to the extracellular space. The transit peptide may be naturally associated with a particular defensin. Such a DNA construct may be cloned or transformed into a biological system which allows expression of the encoded heterogeneous defensin, an active part of the defensin or fragment thereof, Suitable biological systems include microorganisms (for example, the Pichia pastoris expression system, Escherichia coli, Pseudomonas, yeast; viruses; bacteriophages; etc) and cultured cells (such as insect cells, mammalian cells). In some cases, the expressed defensin is subsequently extracted and isolated for use.

Compositions

The present invention also provides pharmaceutical compositions, wherein the pharmaceutical compositions comprise the heterogeneous plant defensin, a nucleic acid, a vector, a host cell or an expression product as disclosed herein, together with a pharmaceutically acceptable carrier, diluent or excipient.
Compositions of the present invention may therefore be administered therapeutically. In such applications, compositions may be administered to a subject already suffering from a condition, in an amount sufficient to cure or at least partially arrest the condition and any complications. The quantity of the composition should be sufficient to effectively treat the patient. Compositions may be prepared according to methods which are known to those of ordinary skill in the art and accordingly may include a cosmetically or pharmaceutically acceptable carrier, excipient or diluent. Methods for preparing administrable compositions are apparent to those skilled in the art, and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa., incorporated by reference herein.
The composition may incorporate any suitable surfactant such as an anionic, cationic or non-ionic surfactant such as sorbitan esters or polyoxyethylene derivatives thereof. Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.
The compositions may also be administered in the form of liposomes. Liposomes may be derived from phospholipids or other lipid substances, and may be formed by mono- or multi-lamellar hydrated liquid crystals dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolisable lipid capable of forming liposomes may be used. The compositions in liposome form may contain stabilisers, preservatives and excipients. Preferred lipids include phospholipids and phosphatidyl cholines (lecithins), both natural and synthetic. Methods for producing liposomes are known in the art, and in this regard specific reference is made to: Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq., the contents of which are incorporated herein by reference.
In some embodiments, the composition may be in the form of a tablet, liquid, lotion, cream, gel, paste or emulsion.
In some embodiments, the composition may further comprise pharmaceutically or veterinarily acceptable carriers, diluents or excipients.
In certain embodiments the composition is in the form of a spray, mist, micro- or nano-particles, aqueous solution, powder, cream, ointment, gel, impregnated bandage, liquid, formulation, paint or other suitable distribution medium including oral forms of the composition.
In particular embodiments the composition further comprises one or more additional anti-proliferative disease or cytotoxic agents.
In certain embodiments, the heterogeneous defensin may have been chemically synthesized or extracted from microorganisms or plants genetically modified to express the heterogeneous defensin.

Dosages

The “therapeutically effective” dose level for any particular patient will depend upon a variety of factors including the condition being treated and the severity of the condition, the activity of the compound or agent employed, the composition employed, the age, body weight, general health, sex and diet of the patient, age, size, growth phase, species of plant, the time of administration, the route of administration, the rate of sequestration of the heterogeneous plant defensin or composition, the duration of the treatment, and any drugs or agents used in combination or coincidental with the treatment, together with other related factors well known in the art. A physician, clinician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the defensin required to prevent or treat applicable conditions.
In certain embodiments, the treatment would be for the duration of the disease state. Slow release formulations are also contemplated herein.
Further, it will be apparent to one of ordinary skill in the art that the optimal quantity and spacing of individual dosages of the composition will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the nature of the particular individual being treated. Also, such optimum conditions can be determined by conventional techniques.
It will also be apparent to one of ordinary skill in the art that the optimal course of treatment, such as the number of doses of the composition given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.
In terms of weight, a therapeutically effective dosage of a composition for administration to a patient is expected to be in the range of about 0.01 mg to about 150 mg per kg body weight per 24 hours; typically, about 0.1 mg to about 150 mg per kg body weight per 24 hours; about 0.1 mg to about 100 mg per kg body weight per 24 hours; about 0.5 mg to about 100 mg per kg body weight per 24 hours; or about 1.0 mg to about 100 mg per kg body weight per 24 hours. More typically, an effective dose range is expected to be in the range of about 5 mg to about 50 mg per kg body weight per 24 hours.
Alternatively, an effective dosage may be up to about 5000 mg/m². Generally, an effective dosage is expected to be in the range of about 10 to about 5000 mg/m², typically about 10 to about 2500 mg/m², about 25 to about 2000 mg/m², about 50 to about 1500 mg/m², about 50 to about 1000 mg/m², or about 75 to about 600 mg/m².
In particular embodiments, the composition as described herein can be used as part of a soil or growth medium preparation program.
In some embodiments, the composition is formulated as dragee cores with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
In particular embodiments, the composition is formulated as push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.

Routes of Administration

The compositions of the present invention can be administered by standard routes. In general, the compositions may be administered by the parenteral (e.g., intravenous, intraspinal, subcutaneous or intramuscular), oral or topical route.
In other embodiments, the compositions may be administered by other enteral/enteric routes, such as rectal, sublingual or sublabial, or via the central nervous system, such as through epidural, intracerebral or intracerebroventricular routes. Other locations for administration may include via epicutaneous, transdermal, intradermal, nasal, intraarterial, intracardiac, intraosseus, intrathecal, intraperitoneal, intravesical, intravitreal, intracavernous, intravaginal or intrauterine routes.
In preferred embodiments, topical compositions can be used. In preparing the compositions, usual media may be employed such as, for example, water, glycols, oils, alcohols, preservatives and/or coloring agents.
In other embodiments, the modified defensins herein may be administered directly to the skin, hair or fur of an animal including a mammal such as a human.
When administered by aerosol or spray, the compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons and/or other solubilizing or dispersing agents known in the art.

Carriers, Excipients and Diluents

Carriers, excipients and diluents must be “acceptable” in terms of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof. Such carriers, excipients and diluents may be used for enhancing the integrity and half-life of the compositions of the present invention. These may also be used to enhance or protect the biological activities of the compositions of the present invention.
Examples of pharmaceutically acceptable carriers or diluents are demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oils, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrolidone; agar; gum tragacanth or gum acacia, and petroleum jelly. Typically, the carrier or carriers will form from 10% to 99.9% by weight of the compositions.
The compositions of the invention may be in a form suitable for administration by injection, in the form of a formulation suitable for oral ingestion (such as capsules, tablets, caplets, elixirs, for example), in the form of an ointment, cream or lotion suitable for topical administration, in an aerosol form suitable for administration by inhalation, such as by intranasal inhalation or oral inhalation, in a form suitable for parenteral administration, that is, subcutaneous, intramuscular or intravenous injection.
For administration as an injectable solution or suspension, non-toxic acceptable diluents or carriers can include Ringer's solution, isotonic saline, phosphate buffered saline, ethanol and 1,2 propylene glycol.

Methods for Preventing or Treating Disease

The present invention provides methods for preventing or treating a proliferative disease, wherein the method comprises administering to a subject a therapeutically effective amount of the heterogeneous plant defensin a nucleic acid, a vector, a host cell, an expression product, or a pharmaceutical composition as disclosed herein, thereby preventing or treating the proliferative disease.
The present invention also provides use of plant defensins, nucleic acids, vectors, host cells and expression products as herein disclosed in the preparation of medicaments for preventing or treating a proliferative disease.
In some embodiments, the proliferative disease may be a cell proliferative disease selected from the group comprising an angiogenic disease, a metastatic disease, a tumourigenic disease, a neoplastic disease and cancer.
In some embodiments, the proliferative disease may be cancer.
In other embodiments, the cancer may be selected from the group comprising acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, anal cancer, appendix cancer, astrocytoma, B-cell lymphoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, bowel cancer, brainstem glioma, brain tumour, breast cancer, bronchial adenomas/carcinoids, Burkitt's lymphoma, carcinoid tumour, cerebral astrocytoma/malignant glioma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumour, endometrial cancer, ependymoma, esophageal cancer, extracranial germ cell tumour, extragonadal germ cell tumour, extrahepatic bile duct cancer, eye cancer, intraocular melanoma/retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumour, gastrointestinal stromal tumour (GIST), germ cell tumour, gestational trophoblastic tumour, glioma, gastric carcinoid, head and/or neck cancer, heart cancer, hepatocellular (liver) cancer, hypopharyngeal cancer, hypothalamic and visual pathway glioma, Kaposi sarcoma, kidney cancer, laryngeal cancer, leukemia (acute lymphoblastic/acute myeloid/chronic lymphocytic/chronic myelogenous/hairy cell), lip and/or oral cavity cancer, liver cancer, non-small cell lung cancer, small cell lung cancer, lymphoma (AIDS-related/Burkitt/cutaneous T-Cell/Hodgkin/non-Hodgkin/primary central nervous system), macroglobulinemia, malignant fibrous histiocytoma of bone/osteosarcoma, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases, myelogenous leukemia, myeloid leukemia, myeloproliferative disorders, nasal cavity and/or paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma/malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumour, pancreatic cancer, islet cell cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma and/or supratentorial primitive neuroectodermal tumours, pituitary adenoma, plasma cell neoplasia/multiple myeloma, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Ewing sarcoma, Kaposi sarcoma, soft tissue sarcoma, uterine sarcoma, Sezary syndrome, skin cancer (non-melanoma), skin cancer (melanoma), skin carcinoma (Merkel cell), small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer with metastatic occult primary, stomach cancer, supratentorial primitive neuroectodermal tumour, T-cell lymphoma, testicular cancer, throat cancer, thymoma and/or thymic carcinoma, thyroid cancer, transitional cancer, trophoblastic tumour, ureter and/or renal pelvis cancer, urethral cancer, uterine endometrial cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulva cancer, Waldenstrom macroglobulinemia or Wilms tumour.
In particular embodiments the cancer is selected from the group comprising basal cell carcinoma, bone cancer, bowel cancer, brain cancer, breast cancer, cervical cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer or thyroid cancer.
In preferred embodiments, a heterogeneous plant defensin, a nucleic acid, a vector, a host cell, an expression product, or a pharmaceutical composition as disclosed herein, is used in the preparation of a medicament for preventing or treating a proliferative disease,
In preferred embodiments the heterogeneous plant defensin as hereindescribed is used for preventing or treating a proliferative disease.

Kits

The present invention provides kits for preventing or treating a proliferative disease, wherein the kits comprise a therapeutically effective amount of a heterologous plant defensin, a nucleic acid, a vector, a host cell, an expression product or a pharmaceutical composition as herein disclosed.
The present invention also provides use of the kits disclosed herein for preventing or treating a proliferative disease, wherein the therapeutically effective amount of a plant defensin, a nucleic acid, a vector, a host cell, an expression product or a pharmaceutical composition as herein disclosed is administered to a subject, thereby preventing or treating the proliferative disease,
Kits of the present invention facilitate the employment of the methods of the present invention. Typically, kits for carrying out a method of the invention contain all the necessary reagents to carry out the method. For example, in one embodiment, the kit may comprise a plant defensin, a polypeptide, a polynucleotide, a vector, a host cell, an expression product or a pharmaceutical composition as herein disclosed.
Typically, the kits described herein will also comprise one or more containers. In the context of the present invention, a compartmentalised kit includes any kit in which compounds or compositions are contained in separate containers, and may include small glass containers, plastic containers or strips of plastic or paper. Such containers may allow the efficient transfer of compounds or compositions from one compartment to another compartment whilst avoiding cross-contamination of samples, and the addition of agents or solutions of each container from one compartment to another in a quantitative fashion.
Typically, a kit of the present invention will also include instructions for using the kit components to conduct the appropriate methods.
Methods and kits of the present invention are equally applicable to any animal, including humans and other animals, for example including non-human primate, equine, bovine, ovine, caprine, leporine, avian, feline and canine species. Accordingly, for application to different species, a single kit of the invention may be applicable, or alternatively different kits, for example comprising compounds or compositions specific for each individual species, may be required.
Methods and kits of the present invention find application in any circumstance in which it is desirable to prevent or treat a proliferative disease.
In preferred embodiments, the proliferative disease is cancer.
In particular embodiments, the cancer is selected from the group comprising basal cell carcinoma, bone cancer, bowel cancer, brain cancer, breast cancer, cervical cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer or thyroid cancer.

Combination Therapies

Those skilled in the art will appreciate that the polypeptides, nucleic acids, vectors, host cells, expression products and compositions disclosed herein may be administered as part of a combination therapy approach, employing one or more of the polypeptides, nucleic acids, vectors, host cells, expression products and compositions disclosed herein in conjunction with other therapeutic approaches to the methods disclosed herein. For such combination therapies, each component of the combination may be administered at the same time, or sequentially in any order, or at different times, so as to provide the desired therapeutic effect. When administered separately, it may be preferred for the components to be administered by the same route of administration, although it is not necessary for this to be so. Alternatively, the components may be formulated together in a single dosage unit as a combination product. Suitable agents which may be used in combination with the compositions of the present invention will be known to those of ordinary skill in the art, and may include, for example, chemotherapeutic agents, radioisotopes and targeted therapies such as antibodies.
Chemotherapeutic agents to be used in combination with the polypeptides, nucleic acids, vectors, host cells, expression products and compositions disclosed herein may include alkylating agents such as cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil and ifosfamide, anti-metabolites such as purine or pyramidine, plant alkaloids and terpenoids such as vinca alkaloids (including vincristine, vinblastine, vinorelbine and vindesine), and taxanes (including paclitaxel and docetaxel), podophyllotoxin, topoisomerase inhibitors such as irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate and teniposide, anti-neoplastics such as doxorubicin, epirubicin and bleomycin, and tyrosine kinase inhibitors.
Targeted therapies to be used in combination with the polypeptides, nucleic acids, vectors, host cells, expression products and compositions disclosed herein may include, for example, imatinib mesylate, dasatinib, nilotinib, trastuzumab, lapatinib, gefitinib, erlotinib, cetuximab, panitumumab, temsirolimus, everolimus, vorinostat, romidepsin, bexarotene, alitretinoin, tretinoin, bortezomib, pralatrexate, bevacizumab, sorafenib, sunitinib, pazopanib, rituximab, alemtuzumab, ofatumuab, tositumomab, 131I-tositumomab, ibritumomab tiuxetan, denileukin diftitox, tamoxifen, toremifene, fulvestrant, anastrozole, exemestane and letrozole.
Other therapies may also be used in combination with the polypeptides, nucleic acids, vectors, host cells, expression products and compositions disclosed herein, including, for example, surgical intervention, dietary regimes and supplements, hypnotherapy, alternative medicines and physical therapy.

Timing of Therapies

Those skilled in the art will appreciate that the polypeptides, polynucleotides, vectors, host cells, expression products and compositions disclosed herein may be administered as a single agent or as part of a combination therapy approach to the methods disclosed herein, either at diagnosis or subsequently thereafter, for example, as follow-up treatment or consolidation therapy as a compliment to currently available therapies for such treatments. The polypeptides, polynucleotides, vectors, host cells, expression products and compositions disclosed herein may also be used as preventative therapies for subjects who are genetically or environmentally predisposed to developing such diseases.
The person skilled in the art will understand and appreciate that different features disclosed herein may be combined to form combinations of features that are within the scope of the present invention.
The present invention will now be further described with reference to the following examples, which are illustrative only and non-limiting.

Examples

Materials and Methods

Purification of Defensins from Solanaceous Flowers
To isolate Class II Solanaceous defensins from their natural source, whole flowers up to the petal coloration stage of flower development were ground to a fine powder and extracted in dilute sulfuric acid as previously described previously (Lay et al, 2003a supra). Briefly, flowers (760 g wet weight) were frozen in liquid nitrogen, ground to a fine powder in a mortar and pestle, and homogenized in 50 mM sulfuric acid (3 mL per g fresh weight) for 5 min using an Ultra-Turrax homogenizer. The homogenate was transferred to a beaker and stirred for 1 h at 4° C. Cellular debris was removed by filtration through Miracloth (Calbiochem, San Diego, Calif.) and centrifugation (25,000×g, 15 min, 4° C.). The pH was then adjusted to 7.0 by addition of 10 M NaOH and the extract was stirred for 1 h at 4° C. before centrifugation (25,000×g, 15 min, 4° C.) to remove precipitated proteins. The supernatant was applied to an SP-Sepharose™ Fast Flow (GE Healthcare Bio-Sciences) column (2.5×2.5 cm) pre-equilibrated with 10 mM sodium phosphate buffer. Unbound proteins were removed by washing with 20 column volumes of 10 mM sodium phosphate buffer (pH 6.0) and bound proteins were eluted in 3×10 mL fractions with 10 mM sodium phosphate buffer (pH 6.0) containing 500 mM NaCl.
Fractions from the SP Sepharose column were subjected to reverse-phase high performance liquid chromatography (RP-HPLC) using either an analytical Zorbax 300SB-C8 RP-HPLC column and an Agilent Technologies 1200 series system or a preparative Vydac C8 RP-HPLC column on a Beckman Coulter System Gold HPLC. Protein samples were loaded in buffer A (0.1% (v/v) trifluoroacetic acid) and eluted with a linear gradient of 0-100% (v/v) buffer B (60% (v/v) acetonitrile in 0.089% (v/v) trifluoroacetic acid. Eluted proteins were detected by monitoring absorbance at 215 nm. Protein peaks were collected and defensins were identified using SDS-PAGE, immunoblotting and mass spectrometry.
Purification of Plant Defensins from Pichia pastoris
The Pichia pastoris expression system is well-known and commercially available from Invitrogen (Carlsbad, Calif.; see the supplier's Pichia Expression Manual disclosing the sequence of the pPIC9 expression vector).
A single pPIC9-NaD1 P. pastoris GS115 colony was used to inoculate 10 mL of BMG medium (described in the Invitrogen Pichia Expression Manual) in a 100 mL flask and was incubated overnight in a 30° C. shaking incubator (140 rpm). The culture was used to inoculate 500 mL of BMG in a 2 L baffled flask which was placed in a 30° C. shaking incubator (140 rpm). Once the OD600 reached 2.0 (˜18 h), cells were harvested by centrifugation (2,500×g, 10 min) and resuspended into 1 L of BMM medium (OD600 =1.0) in a 5 L baffled flask and incubated in a 28° C. shaking incubator for 3 days. The expression medium was separated from cells by centrifugation (4750 rpm, 20 min) and diluted with an equal volume of 20 mM potassium phosphate buffer (pH 6.0). The medium was adjusted to pH 6.0 with NaOH before it was applied to an SP Sepharose column (1 cm×1 cm, Amersham Biosciences) pre-equilibrated with 10 mM potassium phosphate buffer, pH 6.0. The column was then washed with 100 mL of 10 mM potassium phosphate buffer, pH 6.0 and bound protein was eluted in 10 mL of 10 mM potassium phosphate buffer containing 500 mM NaCl. Eluted proteins were subjected to RP-HPLC using a 40 minute linear gradient as described herein below. Protein peaks were collected and analyzed by SDS-PAGE and immunoblotting with the appropriate anti-defensin antibodies. Fractions containing defensin were lyophilized and resuspended in sterile milli Q ultrapure water. The protein concentration of Pichia-expressed defensin was determined using the bicinchoninic acid (BCA) protein assay (Pierce Chemical Co.) with bovine serum albumin (BSA) as the protein standard.

Cell Lines and Culture

Mammalian cell lines used in this study are as follows: human leukemia monocyte lymphoma U937 cells and human transformed cervical cancer HeLa cells. The cells are grown in tissue culture flasks at 37° C. under a humidified atmosphere of 5% CO₂/95% air, and sub-cultured routinely two to three times a week according to the rate of proliferation. All mammalian cells are cultured in RPMI-1640 medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Invitrogen), 100 U/mL penicillin (Invitrogen) and 100 μg/mL streptomycin (Invitrogen). Adherent cell lines are detached from the flask by adding 3-5 mL of a mixture containing 0.25% trypsin and 0.5 μM EDTA (Invitrogen).

Cell Viability and Membrane Permeability Assays

Unless otherwise stated, cells are re-suspended at a cell concentration of 4×10⁵cells/mL in complete culture RPMI-1640 medium supplemented with 10% FBS, 100 μ/mL penicillin and 100 μg/mL streptomycin, and are added to either a V-bottom 96-well plate or microfuge tubes. Cells are kept at 37° C., unless otherwise stated, during protein addition (5 μL) at various concentrations or the set concentration of 10 μM. Typically, cells are mixed with the heterogeneous defensin of interest and are incubated at 37° C. for 30 min. In certain experiments, cells are also incubated at either 4° C. or 37° C. for 2-60 min prior to flow cytometry analysis. Cells are added to an equal volume of complete culture medium containing 2 μg/mL propidium iodide (PI, Annexin V-FITC Apoptosis Detection Kit, Invitrogen) and are analysed immediately by flow cytometry using a FACSCanto cell sorter (Becton Dickson, Fanklin Lakes, N.J.) and Cell Quest Pro Software (Becton Dickson). Typically, 5000-10000 events per sample are collected and the resultant data is analysed using FlowJo software (Tree Star, Ashland, Oreg.). Cells are gated appropriately based on forward scatter (FSC) and side scatter (SSC), with the viable cells is determined by their ability to exclude PI. For analysis purposes, all data is standardised relative to control (normal cell % ranged from approx. 0-7%).
FITC-dextran binding assays are performed as per the PI uptake assay, with the exception that 100 μg ml⁻¹of FITC-dextran (Sigma-Aldrich) is present during the assay and samples are washed twice with PBS containing 0.1% BSA prior to addition of 7AAD (1 μg ml⁻¹) and flow cytometry analysis.

MIT Cell Viability Assays

Tumour cells are seeded in quadruplicate into wells of a flat-bottomed 96-well microtitre plate (50 μL) at various densities starting at 2×10⁶cells/mL. Four wells containing complete culture medium alone are included in each assay as a background control. The microtitre plate are incubated overnight at 37° C. under a humidified atmosphere containing 5% CO₂/95% air, prior to the addition of complete culture medium (100 μL) to each well and further incubated at 37° C. for 48 h. Optimum cell densities (30-50% confluency) for cell viability assays are determined for each cell line by light microscopy.
Tumour cells are seeded in a 96-well microtitre plate (50 μL/well) at an optimum density determined in the cell optimisation assay as above. Background control wells (n=8) containing the same volume of complete culture medium were included in the assay. The microtitre plate are incubated overnight at 37° C., prior to the addition of proteins at various concentrations and the plate is incubated for a further 48 h. The cell viability 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma-Aldrich) assay is as follows: the MTT solution (1 mg/mL) is added to each well (100 μL) and the plate is incubated for 2-3 h at 37° C. under a humidified atmosphere containing 5% CO₂/95% air. Subsequently, for adherent cell lines, the media is removed and is replaced with dimethyl sulfoxide (100 μL, DMSO, Sigma-Aldrich), and placed on a shaker for 5 min to dissolve the tetrazolium salts. In the case of suspension cells, prior to the addition of DMSO the cells are spun at 1500 rpm for 5 min. Absorbance of each well is measured at 570 nm and the IC₅₀values (the protein concentration to inhibit 50% of cell growth) is determined using the Origin Software Program.

Confocal Laser Scanning and Transmission Electron Microscopy

Live imaging was performed on a Zeiss LSM-510 confocal microscope using a 40× oil immersion objective in a 37° C./5% CO₂atmosphere. Adherent cells were cultured on coverslips prior to imaging while non-adherent cells were immobilized onto 10% poly-L-lysine-coated coverslips. All cell types were prepared for imaging in RPMI medium containing 0.1% BSA and 1-2 mg ml⁻¹PI. NaD1, BODIPY-NaD1, FITC-Dextran (100 μg ml⁻¹) was added directly to imaging chamber via capillary tube. In certain experiments, cells were either, stained with PKH67 (Sigma-Aldrich) or transfected with plasmid construct for free GFP or GFP-PH(PLCδ) using Lipofectamine™ 2000 Reagent (Invitrogen) as per manufacturer's instructions prior to imaging. Transmission electron microscopy (TEM) imaging was performed according to a previously described procedure (Adda et al. 2009).

Lipid Binding Assays

Membrane Lipid Strips™, PIP Strips™ and Sphingo Strips™ (Echelon Biosciences, Salt Lake City, Utah) were incubated with PBS/3% BSA for 1-2 h at RT to block non-specific binding. The membrane strips were then incubated with NaD1 (0.12 μM) diluted in PBS/1% BSA overnight at 4° C., prior to thorough washing for 60 min at RT with PBS 10.1% Tween-20. Membrane-bound protein was detected by probing the membrane strips with a rabbit anti-NaD1 polyclonal antibody (diluted 1:2000 with PBS/1% BSA) for 1 h at 4° C., followed by a HRP-conjugated donkey anti-rabbit IgG antibody (diluted 1:2000 with PBS/1% BSA) for 1 h at 4° C. After each antibody incubation, the membrane strips were washed extensively for 60 min at RT with PBS/0.1% Tween-20. Chemiluminescence was detected using the enhanced chemiluminescence (ECL) western blotting reagent (GE Healthcare BioSciences, NSW Australia) and exposed to Hyperfilm (GE Healthcare BioSciences, NSW, Australia) and developed using an Xomat (All-Pro-Imaging).
Densitometry analysis was performed on images obtained from lipid strips using ImageJ (National Institute of Health, Bethesda, Md.). Briefly, circles of equivalent size were traced around areas of interest. A background circle of equal size was also placed in the area on the membrane where there is no lipid and set as the background. The areas of interest were quantified as the average pixel intensity subtracted from the background. Generation of liposomes and liposome pull-down experiments were conducted using a method as described previously (Zhang et al. 2001; Patki et al. 1997).

Cross-Linking Studies

Protein samples at 1 mg ml⁻¹(2.5 μl) were incubated with 0.5 mM PIP2 or PA (2.5 μl) at room temperature for 30 min. Protein complexes were cross-linked through primary amino groups by the addition of 12.5 mM bis[sulfosuccinimidyl] suberate (BS³; 5 μl) at room temperature for 30 min. Samples were reduced and denatured, and subjected to SDS-PAGE.

Crystallographic Methods

NaD1, purified by RP-HPLC, was lyophilised and resuspended in sterile water to a final concentration of 15 mg ml⁻¹. Crystals were grown in sitting drops at 20° C. in 20% PEG 1500 and 10% succinate-phosphate-glycine buffer pH 9 (Newman et al. 2004). Two crystal forms were obtained, the first (form A) belonging to space group P2₁with a=32.697 Å, b=32.685 Å, c=41.977 Å, α=90°, β=100.828°, γ=90° and the second (form B) belonging to P3 ₂21 with a=b=33.091 Å, c=128.77 Å, α=β=90°, γ=120°. The asymmetric units in both forms contain two NaD1 molecules. Diffraction data were collected from crystals flash frozen in mother liquor supplemented with 10% ethylene glycol at 100 K at the Australian Synchrotron (beamline 3ID1) and processed with XDS (Kabsch 2010). For NaD1 crystal form A a heavy atom derivative was obtained by soaking crystals in mother liquor supplemented with 0.2 M 5-amino-2,4,6-triiodoisophthalic acid (I3C) (Beck et al. 2008) for 5 min. I3C sites were found with SheIX and refined using Phenix (Adams et al. 2010). Clear and continuous electron density was obtained for residues the entire molecule. NaD1 crystal form B was solved by molecular replacement using PHASER (Storoni et al. 2004). The final model for both crystal forms was built with Coot (Emsley and Cowtan 2004), refined with Phenix to a resolution of 1.4 Å (form A) and 1.6 (form B).
The NaD1:PIP2 complexes were generated by mixing NaD1 at 10 mg ml⁻¹and PIP2 at a molar ration of 1:1.2. Crystals were grown in sitting drops at 20° C. in 0.2 M ammonium sulfate, 7% PEG 3350, 32% MPD and 0.1 M imidazole pH 7. The structure was solved by molecular replacement with PHASER using the NaD1 structure as a search model. The final model was built with Coot (Emsley and Cowtan 2004) and refined with Phenix to a resolution of 1.6 Å. Figures were prepared using PyMol (Kvansakul et al. 2010)

Transmission Electron Microscopy (TEM)

Samples (10 μl) were applied to 400 mesh copper grids coated with a thin layer of carbon for 2 min. Excess material was removed by blotting and samples were negatively stained twice with 10 μl of a 2% uranyl acetate solution (w/v; Electron Microscopy Services). The grids were air-dried and viewed using a transmission electron microscope, either a JEOL 2000FX operated at 120 kV or a JEOL JEM-2010 operated at 80 kV.
Circular Dichroism Spectrum of rNaD1
To examine whether NaD1 purified from P. pastoris (rNaD1) was correctly folded, its far UV circular dichroism (CD) spectrum was recorded and compared with that of native NaD1. The similarity of the two spectra indicates the structure of rNaD1 was not significantly altered compared to native NaD1.

PCR Mutagenesis of NaD1

Site directed mutagenesis of NaD1 was carried out using the Phusion (Registered Trademark) site-directed mutagenesis kit (Finnzymes). Oligonucleotide primers phosphorylated at the 5′ end were designed to incorporate the desired mutation. The entire template plasmid (pPIC9-NaD1) was amplified in a PCR reaction of 30 cycles with the following temperature profile; 98° C., 30 s; 55° C., 20 s; 72° C., 4 min with a final extension cycle of 72° C. for 10 min. The linear PCR product was then circularized using T4 DNA Quick Ligase for 5 min at RT and transformed into chemically competent TOP10 cells according to the manufacturer's instructions. Constructs were sequenced using the AOX3′ primer to ensure the mutation had been correctly incorporated.
Preparation of Electrocompetent P. pastoris
Electrocompetent P. pastoris GS115 cells (Invitrogen) were prepared as described by Chang et al., Mol Biol Cell 16(10):4941-4953, 2005. Briefly, cells grown overnight in YPD (1% w/v Bacto yeast extract, 2% w/v Bacto peptone extract, and 2% w/v dextrose) were harvested and treated with YPD containing 10 mM DTT, 25 mM HEPES, pH 8, for 15 min at 30° C. with shaking. Cells were washed twice in water and once in ice-cold 1 M sorbitol, before they were resuspended in 1 M sorbitol and divided into 80 μL aliquots for storage at −80° C.
Transformation of P. pastoris GS115 with pPIC9 Constructs
Single E. coli TOP10 colonies transformed with each pPIC9 construct are used to inoculate 10 mL of LB containing 100 μg/mL ampicillin and incubated overnight at 37° C. in a shaking incubator. Plasmid DNA is isolated using the Qiaprep (Registered Trademark) miniprep kit (Qiagen) and linearized overnight using the restriction enzyme SalI. Competent P. pastoris GS115 cells (80 μL) are thawed on ice and 1 μg of linearized DNA is added in an ice-cold Gene Pulser (Registered Trademark) electroporation cuvette with a 0.2 cm gap. DNA is introduced by electroporation at 1.5 kV, 25 μF, 400Ω (Gene Pulser, Bio-Rad Laboratories). Ice-cold 1 M sorbitol (1 mL) is added to the cells before they are plated onto MD plates (1.34% w/v yeast nitrogen base, without amino acids and with ammonium sulfate [US Biological, YNB], 4×10⁻⁵% w/v biotin, 2% w/v dextrose) and incubated at 30° C. for 5 days. Positive colonies are then selected and re-plated onto fresh MD plates.

Generation of a Heterogeneous Plant Defensin

DNA encoding the mature protein of a Class I defensin, for example, NaD2 (SEQ ID NO: 27), DmAMP1 (SEQ ID NO: 23) or γ1-H (SEQ ID NO: 25), as backbone are modified to incorporate a region comprising a loop 5 regions (for example NaD1 equivalent residues 34-40) derived from a Class II Solanaceous plant defensin. Exemplary loop 5 regions that are incorporated into the backbone sequence include the following sequences (i) SKILRR, (SEQ ID NO: 2) (ii) SKLLRR, (SEQ ID NO: 4) (iii) SKILRK (SEQ ID NO: 6), (iv) SKVLRR (SEQ ID NO: 8), (v) SKVLRK(SEQ ID NO: 10), (vi) SKLQRK(SEQ ID NO: 12), (vii) SKLLRN(SEQ ID NO: 14), (viii) SKLLRK(SEQ ID NO: 16), (ix) SKIQRN(SEQ ID NO: 18), (x) RKLQRK (SEQ ID NO: 20) or (xi) KILRR(SEQ ID NO: 89), (xii) KLLRR(SEQ ID NO: 91), (xiii) KILRK(SEQ ID NO: 93), (xiv) KVLRR(SEQ ID NO: 95), (xv) KVLRK(SEQ ID NO: 97), (xvi) KLQRK(SEQ ID NO: 99), (xvii) KLLRN(SEQ ID NO: 101), (xviii) KLLRK(SEQ ID NO: 103), (xix) KIQRN (SEQ ID NO: 105) or (xx) KLQRK(SEQ ID NO: 107). Heterogenous defensin sequences with loop 5 regions as hereindescribed are generated by GenScript USA Inc (Piscataway, N.J.).
The heterogeneous defensin DNA sequences incorporate additional DNA sequences at the 5′ end encoding the amino acids Leu-Glu-Lys-Arg-Ala and including a XhoI restriction endonuclease site (CTCGAG) and the KEX2 cleavage site at the start of the protein. At the 3′ end, DNA is included encoding a stop codon and a NotI restriction endonuclease site. The restriction sites are added so that the genes are subclonable into the equivalent sites in the pPIC9 expression vector (Invitrogen).
The cloned DNA sequence in the pPIC9 vector, plasmid DNA is isolated using a miniprep kit (Invitrogen). The DNA is linearized overnight using the restriction enzyme SalI. Electrocompetent P. pastoris GS115 cells (Invitrogen) are prepared as described by Chang et al. (2005). Briefly, cells grow overnight in YPD (1% Bacto yeast extract, 2% Bacto peptone extract, and 2% dextrose) cells are harvested and are treated with YPD containing 10 mM DTT, 25 mM HEPES, pH 8, for 15 min at 30° C. with shaking. Cells are washed twice in water and once in ice-cold 1 M sorbitol, and are resuspended in 1 M sorbitol and are divided into 80 μL aliquots for storage at −80° C. For transformation of the gene constructs, the electrocompetent P. pastoris GS115 cells (80 μL) are thawed on ice and 1 μg of linearized DNA is added in an ice-cold Gene Pulser® electroporation cuvette (Bio-Rad Laboratories) with a 0.2 cm gap. DNA is introduced by electroporation at 1.5 kV, 25 μF, 400Ω (Gene Pulser, Bio-Rad Laboratories). Ice-cold 1 M sorbitol (1 mL) is added to the cells and then the cells are plated onto MD plates (1.34% yeast nitrogen base, without amino acids and with ammonium sulfate [US Biological, YNB], 4×10-5% biotin, 2% dextrose) and are incubated at 30° C. for 3 days. Positive colonies are selected and are replated onto fresh MD plates. Plasmids are isolated from colonies using a miniprep kit (Invitrogen). Heterogeneous defensins are expressed using a Pichia pastoris expression system and isolated as described above.

Example 1

Class II Solanaceous Plant Defensin-Induced Permeabilisation of Tumour Cells Involves Membrane Blebbing

Example 1

Introduction

It has been shown previously in both PCT/AU2011/000760 and U.S. Ser. No. 13/166,960, incorporated herein by reference, that Class II defensins from solanaceous plants selectively kill mammalian tumour cells by inducing membrane permeabilisation. To investigate the mechanism of action the inventors examined the change in cell morphology when tumour cells are treated with Class II Solanaceous plant defensin NaD1.

Example 1

Results

Live confocal laser scanning microscopy (CLSM) revealed rapid changes on the cell surface of NaD1 permeabilised cells and showed the formation of large bleb-like structures, with (i) an adherent cancer cell line (HeLa—immortal cells derived from human cervical cancer) forming multiple blebs of different sizes and (ii) a non-adherent cancer cell line (U937—human leukaemia) forming typically 1 to 2 large blebs (FIG. 1A). Moreover, bleb size was frequently larger than the actual cell (diameter>20 μm) and did not retract over a period of 20 minutes. To determine whether membrane blebbing occurs prior to, during or following membrane permeabilisation, we treated U937 cells with NaD1 in the presence of propidium iodide (PI) and 4 kDa FITC-dextran to monitor the entry of these molecules into NaD1-sensitive cells (FIG. 1B). FITC-dextran and NaD1 were added at 00:35 min, with FITC-dextran being excluded from cells with intact membrane. Bleb formation was first observed for the cell located at the centre of the panel at 03:25 min, with PI staining appearing at a specific point at the edge of the bleb. From 03:25 to 04:15 min, PI staining was observed in the bleb and the cytoplasm (possibly staining RNA), with FITC-dextran also entering the cell from the bleb site. At 04:20 min, PI-stained molecules were ‘expelled’ out of the cell, possibly at the same region that PI first entered the bleb (FIG. 1B). These data suggest that (i) small molecules such as PI can enter the cell initially at a ‘weakened’ point at the membrane bleb, (ii) the bleb continues to enlarge while PI and 4 kDa FITC-dextran enters, and (iii) intracellular contents are released at the bleb site, representing cytolysis.

Example 2

NaD1 Induces Membrane Blebbing Through Interacting with PIP2

Example 2

Introduction

To further investigate the mechanism of NaD1 action, the binding of BODIPY-labelled NaD1 to tumour cells was tested.

Example 2

Results

Initially, BODIPY-NaD1 was found to mediate membrane permeabilisation of U937 of a comparable level to unlabelled NaD1 and bound to both viable (7AAD negative) and permeabilised (7AAD positive) U937 cells, with more BODIPY-NaD1 bound to membrane-damaged cells (FIG. 2A). These data suggest that NaD1 can interact with tumor cells prior to membrane permeabilisation and accumulate on NaD1-sensitive cells. We next determined the subcellular localisation of BODIPY-NaD1 on permeabilised tumor cells and showed accumulation of BODIPY-NaD1 at the membrane bleb(s), cytoplasm, nucleolus and possibly at certain cytoplasmic organelles (FIG. 2B). As we have previously shown that the ligands for NaD1 and other class II defensins from solanaceous plants are phosphoinositides, particularly Ptdlns(4,5)P2 (designated PIP2 as disclosed PCT/AU2011/000760 and U.S. Ser. No. 13/166,960 incorporated herein by reference) in together with the observation that PIP2 is a key mediator of cytoskeleton-membrane interactions and its sequestration or enzymatic modification can cause blebbing (Sheetz et al., 2001; Raucher et al., 2000), we then asked whether the binding of NaD1 to PIP2 at the inner leaflet of the plasma membrane could lead to the formation of large blebs. HeLa cells overexpressing GFP-PH(PLCδ), which binds specifically to PIP2, were treated with NaD1 and showed a marked delay from the initiation of blebbing (rapid small membrane blebbing) to membrane permeabilisation when compared to cells expressing free GFP (FIGS. 2C and 2D). These results suggest that the expression of GFP-PH(PLCδ) competes with NaD1 for PIP2 binding at the inner leaflet of the plasma membrane and interferes with NaD1-induced blebbing.

Example 3

Structure of the NaD1:PIP2 Complex

Example 3

Introduction

To gain insight into the NaD1:PIP2 interaction at the atomic level, crystal structures of NaD1 on its own as well as in complex with PIP2 were determined. The structure of NaD1 was solved using single isomorphous replacement with anomalous scattering using crystals soaked with I3C and refined to a resolution of 1.4 Å with a value of R_work/R_freeof 0.19/0.137.

Example 3

Results

The structure of NaD1 in isolation is identical to our previously solved NMR structure (FIG. 3A) (Lay et al. 2003b). The monomeric NaD1 model was then used to solve the structure of a NaD1:PIP2 complex by molecular replacement, and we build a model containing 14 molecules of NaD1 and 14 PIP2 molecules in the asymmetric unit. The final NaD1:PIP2 complex was refined to a resolution of 1.6 Å with a value of R_work/R_freeof 0.155/0.184.
Upon PIP2 binding NaD1 forms an arch composed of 14 NaD1 molecules, with a final arch diameter of 90 Å and a width of 35 Å. 14 PIP2 molecules are bound in an extended binding groove on the inside of the arch (FIG. 3B). The entire oligomeric complex is held together by a complex network of interactions, which include numerous NaD1:NaD1 as well as NaD1:PIP2 interactions (FIG. 3C). The assembly of the oligomer indicates two distinct interfaces. The first interface is formed by an anti-parallel alignment of beta-strand 1 from two NaD1 molecules (monomers I and II) and exhibits two-fold symmetry between the associated monomers (FIG. 3C). It comprises an average buried surface area of 430 Å²and is formed by a network of six hydrogen bonds involving R1, K4, E6, E27, K45 and C47. A second interface is formed by the dimeric NaD1 (comprising monomers I and II) and adjacent NaD1 monomers III and IV (FIG. 3C). This interface is formed by hydrogen bonds involving N8 of monomer I, R1, E2, K17 and D31 of monomer II, R1, K17 and D31 of monomer III and N8 of monomer IV, effectively forming a dimer of dimers. The full 14-mer is thus constructed using two different interfaces. In addition to NaD1:NaD1 interactions, oligomer formation requires the presence of PIP2. NaD1 binds PIP2 in a distinct binding site formed by K4 together with residues 33-40, which comprise a characteristic KILRR motif (FIG. 3C). PIP2 forms a dense network of hydrogen bonds involving K4, H33, K36, I37, L38 and R40 of a single NaD1 monomer. In oligomeric NaD1:PIP2, a single PIP2 binding site also contains interactions with neighbouring NaD1 monomers (FIG. 3C). Bound PIP2 forms additional hydrogen bonds with R40 from monomer II and K36 from monomer IV′, with the full PIP2 binding site in the oligomer comprising contributions from three different NaD1 molecules (FIG. 3C). Consequently oligomer formation appears to be highly cooperative, with multiple interactions between adjacent NaD1 and PIP2 molecules required to form the observed 14-mer (FIG. 3D).

Example 4

NaD1:PIP2 can Form Large Oligomeric Complexes

Example 4

Introduction

To confirm that oligomer formation of NaD1:PIP2 is not a crystallisation artefact, NaD1:PIP2 complexes formed in solution by TEM were examined.

Example 4

Results

It was observed the formation of long string-like structures only when both NaD1 and PIP2 are present (FIG. 4A). Furthermore, using a cross-linking approach of NaD1 with PIP2 in solution, we have also confirmed the formation of NaD1-PIP2 multimers (FIG. 4B). The formation of NaD1 multimers was lipid specific, with PIP2 (binds strongly to NaD1) mediating efficient multimer formation, as apposed to phosphatidic acid (PA) (binds weakly to NaD1) that mediated very low levels of multimerisation (FIG. 4B). As described for NaD1, the class II defensin TPP3 preferentially binds PIP2 over PA (described in PCT/AU2011/000760 and U.S. Ser. No. 13/166,960 incorporated herein by reference) was also efficiently multimerised by PIP2 and not PA. In contrast, the class I defensin NaD2, which preferentially binds PA over PIP2-see PCT/AU2011/000760 and U.S. Ser. No. 13/166,960 specifically incorporated herein by reference, was efficiently multimerised by PA and not PIP2. These data demonstrate the specificity of lipid-mediated multimerisation of defensins. It should also be noted the crosslinking studies indicate that in the absence of lipid, a proportion of NaD1, NaD2 and TPP3, all appear to exist in dimeric as well as monomeric forms (FIG. 4B).

Example 5

Replacement of NaD1 Loop 5 with that of NaD2 Changes Lipid-Binding Specificity and Abolishes Tumour Cell Killing

Example 5

Introduction

The NaD1:PIP2 structure suggests that the loop 5 region (residues 35-40) of NaD1 is involved in the binding of PIP2, as this region is involved in multiple contacts with PIP2 (FIGS. 3C and 3D). As the binding of PIP2 by NaD1 is involved in the permeabilisation of tumour cells, the inventors propose that loop 5 of Class II Solanaceous plant defensins are involved in defensin activity related to the killing of tumour cells. To investigate the involvement of loop 5 of Class II Solanaceous plant defensins in lipid binding and tumour cell killing, the inventors replaced selected loops of NaD1 with the equivalent regions of NaD2, a class I defensin that does not bind PIP2 and does not kill tumour cells as previously disclosed in PCT/AU2011/000760 and U.S. Ser. No. 13/166,960 specifically incorporated herein by reference. Two recombinant NaD1 defensins were generated where NaD2 residues 25-29 (loop 4A, a region of NaD1 not involved in lipid binding) and NaD2 residues 35-40 (loop 5, a region of NaD1 that forms the extended lipid binding groove) were inserted into the corresponding loops of NaD1 (termed rNaD1(D2L4A) and rNaD1(D2L5) (FIG. 5A).

Example 5

Results

Strikingly, rNaD1 and rNaD1(D2L4A) but not rNaD1(D2L5) permeabilised U937 cells (FIG. 5B). In terms of lipid binding, similar to native NaD1, rNaD1 and rNaD1(D2L4A) bound most strongly to PIP2, whereas the specificity of rNaD1(D2L5) was altered in that it bound most strongly to PA (FIGS. 5C and 5D). These results are consistent with our mechanistic model and structure data that preferential binding to PIP2 is key to mediate tumor cell lysis and the specificity of lipid recognition appears to be largely determined by residues located in loop 5 of NaD1. As the residues that define the loop 5 region of Class II Solanaceous defensins are highly conserved, it is proposed that the loop 5 region as defined by being flanked by the fifth and sixth (invariant) cysteine (Cys) amino acid residues of a Class II Solanaceous plant defensin amino acid sequence may be involved in the binding of phospholipids, including PIP2, and therefore proliferative disease cytotoxicity, for example, through cell membrane permeabilisation activity.

Example 6

NaD1 Loop 5 is Involved in the Interaction with Lipid

Example 6

Introduction

To further investigate the involvement of loop 5 of Class II Solanaceous defensins in lipid binding and tumour cell killing/cell permeabilisation the inventors replaced residues 35-40 of NaD1 with the equivalent regions of other class I defensins, DmAMP1 (Dahlia merckii), γ1-hordothionin (Hordeum vulgare), RsAFP2 (Raphanus sativus) and VrD1 (Vigna radiata), to generate rNaD1-DmAMP1L5, rNaD1-ghordoL5, rNaD1-RsAFP2L5, rNaD1-VrD1L5, respectively (FIG. 6A).

Example 6

Results

In contrast to rNaD1, each of the recombinant NaD1 molecules had greatly reduced or no ability to permeabilise U937 cells (FIG. 6B) and showed a preference for binding to PA as opposed to PIP2 (FIGS. 6C and 6D). These results further support the proposal that preferential binding to PIP2 is involved in tumour cell lysis and the specificity of lipid recognition may be largely determined by residues located in the Class II Solanaceous loop 5 region as defined by being flanked by the fifth and sixth invariant cysteine amino acid residues of a Class II Solanaceous plant defensin amino acid sequence.

Example 7

Generating Heterogeneous Plant Defensins

Example 7

Introduction

Exemplary heterogeneous defensins according to the invention are produced by introducing a modified loop 5 region, or variant or fragment thereof, into a backbone amino acid sequence of a defensin, for example a Class I defensin. The modified loop 5 region may be synthesised according to the following structure or sequence: X₁-X₂-X₃-X₄-X₅-X₆, wherein X₁is serine or arginine, X₂is lysine, arginine or histidine, X₃and X₄are each a hydrophobic amino acid, X₅is arginine, lysine or histidine and X₆is arginine, lysine, histidine, asparagine.
Exemplary amino acid sequences that may be suitable for introduction into the backbone sequence to generate a heterogeneous defensin as described herein includes the following range of amino acid sequences with corresponding polynucleotide sequences indicated in brackets;

	(i)
	(SEQ ID NO: 2)
	SKILRR

	(SEQ ID NO: 3)
	(tcc aag att ttg aga aga);

	(ii)
	(SEQ ID NO: 4)
	SKLLRR

	(SEQ ID NO: 5)
	(tcc aag att ttg aga aga);

	(iii)
	(SEQ ID NO: 6)
	SKILRK

	(SEQ ID NO: 7)
	(tcc aag att ttg aga aag);

	(iv)
	(SEQ ID NO: 8)
	SKVLRR

	(SEQ ID NO: 9)
	(tcc aag gtc ttg aga aga);

	(v)
	(SEQ ID NO: 10)
	SKVLRK

	(SEQ ID NO: 11)
	(tcc aag gtc ttg aga aag);

	(vi)
	(SEQ ID NO: 12)
	SKLQRK

	(SEQ ID NO: 13)
	(tcc aag ttg caa aga aag);

	(vii)
	(SEQ ID NO: 14)
	SKLLRN

	(SEQ ID NO: 15)
	(tcc aag ttg ttg aga aat);

	(viii)
	(SEQ ID NO: 16)
	SKLLRK

	(SEQ ID NO: 17)
	(tcc aag ttg ttg aga aag);

	(ix)
	(SEQ ID NO: 18)
	SKIQRN

	(SEQ ID NO: 19)
	(tcc aag att caa aga aat);

	(x)
	(SEQ ID NO: 20)
	RKLQRK

	(SEQ ID NO: 22)
	(aga aag ttg caa aga aag);

	(xi)
	(SEQ ID NO: 89)
	KILRR

	(SEQ ID NO: 90)
	(aag att ttg aga aga);

	(xii)
	(SEQ ID NO: 91)
	KLLRR

	(SEQ ID NO: 92)
	(aag att ttg aga aga);

	(xiii)
	(SEQ ID NO: 93)
	KILRK

	(SEQ ID NO: 94)
	(aag att ttg aga aag);

	(xiv)
	(SEQ ID NO: 95)
	KVLRR

	(SEQ ID NO: 96)
	(aag gtc ttg aga aga);

	(xv)
	(SEQ ID NO: 97
	KVLRK

	(SEQ ID NO: 98)
	(aag gtc ttg aga aag);

	(xvi)
	(SEQ ID NO: 99)
	KLQRK

	(SEQ ID NO: 100)
	(aag ttg caa aga aag);

	(xvii)
	(SEQ ID NO: 101)
	KLLRN

	(SEQ ID NO: 102)
	(aag ttg ttg aga aat);

	(xviii)
	(SEQ ID NO: 103)
	KLLRK

	(SEQ ID NO: 104)
	(aag ttg ttg aga aag);

	(xix)
	(SEQ ID NO: 105)
	KIQRN

	(SEQ ID NO: 106)
	(aag att caa aga aat );
	or

	(xx)
	(SEQ ID NO: 107)
	KLQRK

	(SEQ ID NO: 108)
	(aag ttg caa aga aag).

For example, to produce a heterogeneous defensin, according to the invention, one of the exemplary loop 5 region sequences (i) to (xx) is introduced as a substitute loop 5 region into, for example a Class I defensin amino acid sequence. Exemplary Class I defensin sequences that are modified to produce a heterogeneous defensin include NaD2 ((SEQ ID NO: 27 and 28), Dm-AMP1 (SEQ ID NO: 23 and 24) and γ1-H (SEQ ID NO: 25 and 26). The heterogeneous defensins are produced using standard molecular biology techniques as described above.

For example, Class I defensin Dm-AMP1:
(SEQ ID No: 23)

ELCEKASKTWSGNCGNTGHCDNQCKSWEGAAHGACHVRNGKHMCFC

YFNC

is modified as described herein to produce a heterogeneous defensin according to the invention with the following amino acid sequences (corresponding nucleotide sequences in brackets):

(SEQ ID NO: 29)

ELCEKASKTWSGNCGNTGHCDNQCKSWEGAAHGACSKILRRCFCYFNC

(SEQ ID NO: 30)

(gaattgtgtgaaaaagcttctaaaacttggtctggtaattgtggtaa

tactggtcattgtgataatcaatgtaaatcttgggaaggtgctgctca

tggtgcttgttccaagattttgagaagatgtttctgttatttcaattg

ttaa)

(SEQ ID NO: 31)

ELCEKASKTWSGNCGNTGHCDNQCKSWEGAAHGACSKLLRRCFCYFNC

(SEQ ID NO: 32)

(gaattgtgtgaaaaagcttctaaaacttggtctggtaattgtggtaa

tactggtcattgtgataatcaatgtaaatcttgggaaggtgctgctca

tggtgcttgttccaagattttgagaagatgtttctgttatttcaattg

ttaa)

(SEQ ID NO: 33)

ELCEKASKTWSGNCGNTGHCDNQCKSWEGAAHGACSKILRKCFCYFNC

(SEQ ID NO: 34)

(gaattgtgtgaaaaagcttctaaaacttggtctggtaattgtggtaat

actggtcattgtgataatcaatgtaaatcttgggaaggtgctgctcatg

gtgcttgttccaagattttgagaaagtgtttctgttatttcaattgtta

a)

(SEQ ID NO: 35)

ELCEKASKTWSGNCGNTGHCDNQCKSWEGAAHGACSKVLRRCFCYFNC

(SEQ ID NO: 36)

(gaattgtgtgaaaaagcttctaaaacttggtctggtaattgtggtaata

ctggtcattgtgataatcaatgtaaatcttgggaaggtgctgctcatggt

gcttgttccaaggtcttgagaagatgtttctgttatttcaattgttaa)

(SEQ ID NO: 37)

ELCEKASKTWSGNCGNTGHCDNQCKSWEGAAHGACSKVLRKCFCYFNC

(SEQ ID NO: 38)

(gaattgtgtgaaaaagcttctaaaacttggtctggtaattgtggtaata

ctggtcattgtgataatcaatgtaaatcttgggaaggtgctgctcatggt

gcttgttccaaggtcttgagaaagtgtttctgttatttcaattgttaa)

(SEQ ID NO: 39)

ELCEKASKTWSGNCGNTGHCDNQCKSWEGAAHGACSKLQRKCFCYFNC

(SEQ ID NO: 40)

(gaattgtgtgaaaaagcttctaaaacttggtctggtaattgtggtaata

ctggtcattgtgataatcaatgtaaatcttgggaaggtgctgctcatggt

gcttgttccaagttgcaaagaaagtgtttctgttatttcaattgttaa)

(SEQ ID NO: 41)

ELCEKASKTWSGNCGNTGHCDNQCKSWEGAAHGACSKLLRNCFCYFNC

(SEQ ID NO: 42)

(gaattgtgtgaaaaagcttctaaaacttggtctggtaattgtggtaata

ctggtcattgtgataatcaatgtaaatcttgggaaggtgctgctcatggt

gcttgttccaagttgttgagaaattgtttctgttatttcaattgttaa)

(SEQ ID NO: 43)

ELCEKASKTWSGNCGNTGHCDNQCKSWEGAAHGACSKLLRKCFCYFNC

(SEQ ID NO: 44)

(gaattgtgtgaaaaagcttctaaaacttggtctggtaattgtggtaata

ctggtcattgtgataatcaatgtaaatcttgggaaggtgctgctcatggt

gcttgttccaagttgttgagaaagtgtttctgttatttcaattgttaa)

(SEQ ID NO: 45)

ELCEKASKTWSGNCGNTGHCDNQCKSWEGAAHGACSKIQRNCFCYFNC

(SEQ ID NO: 46)

(gaattgtgtgaaaaagcttctaaaacttggtctggtaattgtggtaata

ctggtcattgtgataatcaatgtaaatcttgggaaggtgctgctcatggt

gcttgttccaagattcaaagaaattgtttctgttatttcaattgttaa)

(SEQ ID NO: 47)

ELCEKASKTWSGNCGNTGHCDNQCKSWEGAAHGACRKLQRKCFCYFNC

(SEQ ID NO: 48)

(gaattgtgtgaaaaagcttctaaaacttggtctggtaattgtggtaata

ctggtcattgtgataatcaatgtaaatcttgggaaggtgctgctcatggt

gcttgtagaaagttgcaaagaaagtgtttctgttatttcaattgttaa)

In another example, Class I defensin g-1H:
(SEQ ID NO: 25)

RICRRRSAGFKGPCVSNKNCAQVCMQEGWGGGNCDGPLRRCK

CMRRC

is modified as described herein to produce a heterogeneous defensin according to the invention with the following amino acid sequences (corresponding nucleotide sequences in brackets):

(SEQ ID NO: 49)

RICRRRSAGFKGPCVSNKNCAQVCMQEGWGGGNCSKILRRCKCMRRC

(SEQ ID NO: 50)

(agaatttgtagaagaagatctgctggtttcaaaggtccatgtgttt

ctaataaaaattgtgctcaagtttgtatgcaagaaggttggggtggt

ggtaattgttccaagattttgagaagatgtaaatgtatgagaagatg

ttaa)

(SEQ ID NO: 51)

RICRRRSAGFKGPCVSNKNCAQVCMQEGWGGGNCSKLLRRCKCMRRC

(SEQ ID NO: 52)

(agaatttgtagaagaagatctgctggtttcaaaggtccatgtgttt

ctaataaaaattgtgctcaagtttgtatgcaagaaggttggggtggt

ggtaattgttccaagattttgagaagatgtaaatgtatgagaagatg

ttaa)

(SEQ ID NO: 53)

RICRRRSAGFKGPCVSNKNCAQVCMQEGWGGGNCSKILRKCKCMRRC

(SEQ ID NO: 54)

(agaatttgtagaagaagatctgctggtttcaaaggtccatgtgtttc

taataaaaattgtgctcaagtttgtatgcaagaaggttggggtggtgg

taattgttccaagattttgagaaagtgtaaatgtatgagaagatgtta

a)

(SEQ ID NO: 55)

RICRRRSAGFKGPCVSNKNCAQVCMQEGWGGGNCSKVLRRCKCMRRC

(SEQ ID NO: 56)

(agaatttgtagaagaagatctgctggtttcaaaggtccatgtgtttc

taataaaaattgtgctcaagtttgtatgcaagaaggttggggtggtgg

taattgttccaaggtcttgagaagatgtaaatgtatgagaagatgtta

a)

(SEQ ID NO: 57)

RICRRRSAGFKGPCVSNKNCAQVCMQEGWGGGNCSKVLRKCKCMRRC

(SEQ ID NO: 58)

(agaatttgtagaagaagatctgctggtttcaaaggtccatgtgtttc

taataaaaattgtgctcaagtttgtatgcaagaaggttggggtggtgg

taattgttccaaggtcttgagaaagtgtaaatgtatgagaagatgtta

a)

(SEQ ID NO: 59)

RICRRRSAGFKGPCVSNKNCAQVCMQEGWGGGNCSKLQRKCKCMRRC

(SEQ ID NO: 60)

agaatttgtagaagaagatctgctggtttcaaaggtccatgtgtttc

taataaaaattgtgctcaagtttgtatgcaagaaggttggggtggtg

gtaattgttccaagttgcaaagaaagtgtaaatgtatgagaagatgt

taa

(SEQ ID NO: 61)

RICRRRSAGFKGPCVSNKNCAQVCMQEGWGGGNCSKLLRNCKCMRRC

(SEQ ID NO: 62)

(agaatttgtagaagaagatctgctggtttcaaaggtccatgtgtttc

taataaaaattgtgctcaagtttgtatgcaagaaggttggggtggtgg

taattgttccaagttgttgagaaattgtaaatgtatgagaagatgtta

a)

(SEQ ID NO: 63)

RICRRRSAGFKGPCVSNKNCAQVCMQEGWGGGNCSKLLRKCKCMRRC

(SEQ ID NO: 64)

(agaatttgtagaagaagatctgctggtttcaaaggtccatgtgtttc

taataaaaattgtgctcaagtttgtatgcaagaaggttggggtggtgg

taattgttccaagttgttgagaaagtgtaaatgtatgagaagatgtta

a)

(SEQ ID NO: 65)

RICRRRSAGFKGPCVSNKNCAQVCMQEGWGGGNCSKIQRNCKCMRRC

(SEQ ID NO: 66)

(agaatttgtagaagaagatctgctggtttcaaaggtccatgtgtttct

aataaaaattgtgctcaagtttgtatgcaagaaggttggggtggtggta

attgttccaagattcaaagaaattgtaaatgtatgagaagatgttaa)

(SEQ ID NO: 67)

RICRRRSAGFKGPCVSNKNCAQVCMQEGWGGGNCRKLQRKCKCMRRC

(SEQ ID NO: 68)

(agaatttgtagaagaagatctgctggtttcaaaggtccatgtgtttct

aataaaaattgtgctcaagtttgtatgcaagaaggttggggtggtggta

attgtagaaagttgcaaagaaagtgtaaatgtatgagaagatgttaa)

In yet another example, Class I defensin NaD2:
(SEQ ID NO: 27)

RTCESQSHRFKGPCARDSNCATVCLTEGFSGGDCRGFRRRCF

CTRPC

is modified as described herein to produce a heterogeneous defensin according to the invention with the following amino acid sequences (corresponding nucleotide sequences in brackets):

(SEQ ID NO: 69)

RTCESQSHRFKGPCARDSNCATVCLTEGFSGGDCSKILRRCFCTRPC

(SEQ ID NO: 70)

(agaacttgcgagtctcagagccaccgtttcaagggaccatgcgcaa

gagatagcaactgtgccaccgtctgtttgacagaagggtggcgactg

ctccaagattttgagaagatgttctgtaccaggccttgctaa)

(SEQ ID NO: 71)

RTCESQSHRFKGPCARDSNCATVCLTEGFSGGDCSKLLRRCFCTRPC

(SEQ ID NO: 72)

(agaacttgcgagtctcagagccaccgtttcaagggaccatgcgcaa

gagatagcaactgtgccaccgtctgtttgacagaaggattttccggt

ggcgactgctccaagattttgagaagatgtttctgtaccaggccttg

ctaa)

(SEQ ID NO: 73)

RTCESQSHRFKGPCARDSNCATVCLTEGFSGGDCSKILRKCFCTRPC

(SEQ ID NO: 74)

(agaacttgcgagtctcagagccaccgtttcaagggaccatgcgcaa

gagatagcaactgtgccaccgtctgtttgacagaaggattttccggt

ggcgactgctccaagatttgagaaagtgtttctgtaccaggccttgc

taa)

(SEQ ID NO: 75)

RTCESQSHRFKGPCARDSNCATVCLTEGFSGGDCSKVLRRCFCTRPG

(SEQ ID NO: 76)

(agaacttgcgagtctcagagccaccgtttcaagggaccatgcgcaa

gagatagcaactgtgccaccgtctgtttgacagaaggattttccggt

ggcgactgctccaaggtcttgagaagatgtttctgtaccaggccttg

ctaa)

(SEQ ID NO: 77)

RTCESQSHRFKGPCARDSNCATVCLTEGFSGGDCSKVLRKCFCTRPC

(SEQ ID NO: 78)

(agaacttgcgagtctcagagccaccgtttcaagggaccatgcgcaa

gagatagcaactgtgccaccgtctgtttgacagaaggattttccggt

ggcgactgctccaaggtcttgagaaagtgtttctgtaccaggccttg

ctaa)

(SEQ ID NO: 79)

RTCESQSHRFKGPCARDSNCATVCLTEGFSGGDCSKLQRKCFCTRPC

(SEQ ID NO: 80)

(agaacttgcgagtctcagagccaccgtttcaagggaccatgcgcaa

gagatagcaactgtgccaccgtctgtttgacagaaggattttccggt

ggcgactgctccaagttgcaaagaaagtgtttctgtaccaggccttg

ctaa)

(SEQ ID NO: 81)

RTCESQSHRFKGPCARDSNCATVCLTEGFSGGDCSKLLRNCFCTRPC

(SEQ ID NO: 82)

(agaacttgcgagtctcagagccaccgtttcaagggaccatgcgcaa

gagatagcaactgtgccaccgtctgtttgacagaaggattttccggt

ggcgactgctccaagttgttgagaaattgtttctgtaccaggccttg

ctaa)

(SEQ ID NO: 83)

RTCESQSHRFKGPCARDSNCATVCLTEGFSGGDCSKLLRKCFCTRPC

(SEQ ID NO: 84)

(agaacttgcgagtctcagagccaccgtttcaagggaccatgcgcaa

gagatagcaactgtgccaccgtctgtttgacagaaggattttccggt

ggcgactgctccaagttgttgagaaagtgtttctgtaccaggccttg

ctaa)

(SEQ ID NO: 85)

RTCESGSHRFKGPCARDSNCATVCLTEGFSGGDCSKIQRNCFCTRPC

(SEQ ID NO: 86)

(agaacttgcgagtctcagagccaccgtttcaagggaccatgcgcaa

gagatagcaactgtgccaccgtctgtttgacagaaggattttccggt

ggcgactgctccaagattcaaagaaattgtttctgtaccaggccttg

ctaa)

(SEQ ID NO: 87)

RTCESQSHRFKGPCARDSNCATVCLTEGFSGGDCRKLQRKCFCTRPC

(SEQ ID NO: 88)

(agaacttgcgagtctcagagccaccgtttcaagggaccatgcgcaa

gagatagcaactgtgccaccgtctgtttgacagaaggattttccggt

ggcgactgcagaaagttgcaaagaaagtgtttctgtaccaggccttg

ctaa)

Example 7

Results

Using the methods described above, the resulting heterogeneous defensins are tested for lipid binding specificity and the ability to kill tumour cells by permeabilisation. It is expected that the heterogeneous defensins display the ability of any one or more of inter alia, binding of PIP2, ability to permeabilise tumour cells.

Example 8

Single Amino Acid Changes in Class II Solanaceous Defensin NaD1 Loop 5 Abolish Killing of Tumour Cells

Example 8

Introduction

To investigate the role of amino acids K36 and R40 in loop 5 of the Class II Solanaceous defensin NaD1 for tumour cell killing, each of these residues were replaced with an amino acid of the opposite charge, glutamic acid (E), to generate K36E and R40E mutants.

Example 8

Results

In contrast to rNaD1, the K36E and R40E mutant NaD1 molecules had greatly reduced ability to permeabilise U937 cells (FIG. 8). These results further support the involvement of loop 5 of NaD1 by identifying two residues in this region that appear to be required for tumour cell killing.

Example 9

In Vitro Anti-Tumour Activity of Heterogeneous Defensin

The effect of the heterogeneous defensin on the viability of tumour cell lines and primary human cell isolates is determined using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) in vitro cell culture viability assay. The tumour cell lines tested are HCT116 (human colon cancer), MCF-7 (human breast cancer), MM170 (human melanoma), PC3 (human prostate cancer), B16-F1 (mouse melanoma), CASMC (human coronary artery smooth muscle cells) and HUVEC (human umbilical vein endothelial cells). Cells are seeded into 96-well flat-bottomed microtitre plates at the following cell numbers: MM170 (2×10⁴/well), MCF-7 (2×10⁴/well), HCT-116 (5×10³/well), PC3 (5×10³/well), B16-F1 (2×10³/well), HUVEC (3×10³/well), CASMC (5×10³/well) and are cultured overnight. The heterogeneous defensin is added to cells to final concentrations ranging from 1 to 100 μM and is incubated for 48 h, upon which MTT assays are carried out as described in the Materials and Methods.

Example 10

Effect of the Heterogeneous Defensin on the Permeabilisation of Human Cell In Vitro

The ability of the heterogeneous defensin to permeabilise tumour cells is assessed using flow cytometry to determine the uptake of the fluorescent dye propidium iodide (PI) (2 mg/mL) by U937 and MM170 cells (4×10⁵/mL) following the treatment of cells with increasing concentrations of the heterogeneous defensin (0 to 100 μM) for 30 min.
Those skilled in the art will appreciate that the disclosure described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

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Claims

1. A heterogeneous plant defensin comprising a first polypeptide sequence and a second polypeptide sequence, wherein the second polypeptide sequence is derived from a plant defensin other than the plant defensin from which the first polypeptide sequence is derived.

2. The heterogeneous plant defensin of claim 1, further comprising a third polypeptide sequence, wherein the third polypeptide sequence is derived from the same plant defensin as the first polypeptide sequence.

3. The heterogeneous plant defensin of claim 1 or 2, comprising the sequence:

(SEQ ID NO: 1) X_A-C1-X_B-C2-X_C-C3-X_D-C4-X_E-C5-X_F-C6-X_G-C7-X_H-C8-X_I,;

wherein C1, C2, C3, C4, C5, C6, C7 and C8 is cysteine, said cysteine being an invariant cysteine,

X_A, X_B, X_C, X_D, X_E, X_F, X_G, X_H, X_Iis any naturally occurring amino acid and;

X_Ais 1 to 7 amino acids in length;

X_Bis 9, 10 or 11 amino acids in length;

X_Cis 3 to 8 amino acids in length;

X_Dis 3 amino acids in length;

X_Eis 9 to 13 amino acids in length;

X_Fis 4, 5, 6, 7 or 8 amino acids in length;

X_Gis 1 amino acid in length;

X_His 1 to 4 amino acids in length; and

X_Iis 0 or 1 amino acid in length.

4. The heterogeneous plant defensin of claim 3, wherein the second polypeptide sequence is positioned between the fourth and eighth invariant cysteine amino acids corresponding to the sequence set forth as SEQ ID NO:1.

5. The heterogeneous plant defensin of claim 3, wherein the second polypeptide sequence is positioned between the fifth and sixth invariant cysteine amino acids corresponding to the sequence set forth as SEQ ID NO:1.

6. The heterogeneous plant defensin of any one of claims 1 to 5, wherein the second polypeptide sequence corresponds to an amino acid sequence comprising X₁-X₂-X₃-X₄-X₅-X₆, wherein X₁is serine or arginine, X₂is lysine, arginine or histidine, X₃and X₄are each a hydrophobic amino acid, X₅is arginine, lysine or histidine and X₆is arginine, lysine, histidine or asparagine

7. The heterogeneous plant defensin of any one of claims 1 to 5, wherein the second polypeptide sequence corresponds to an amino acid sequence comprising X₁-X₂-X₃-X₄-X₅-X₆, wherein X₁is serine or arginine, X₂is lysine, X₃is isoleucine, leucine or valine, X₄is leucine or glutamine, X₅is arginine and X₆is arginine, lysine or asparagine.

8. The heterogeneous plant defensin of any one of claims 1 to 5, wherein the second polypeptide sequence corresponds to an amino acid sequence comprising X₂-X₃-X₄-X₅-X₆, wherein X₂is lysine, arginine or histidine, X₃and X₄are each a hydrophobic amino acid, X₅is arginine, lysine or histidine and X₆is arginine, lysine, histidine or asparagine.

9. The heterogeneous plant defensin of any one of claims 1 to 5, wherein the second polypeptide sequence corresponds to an amino acid sequence comprising X₂-X₃-X₄-X₅-X₆, wherein X₂is lysine, X₃is isoleucine, leucine or valine, X₄is leucine or glutamine, X₅is arginine and X₆is arginine, lysine or asparagine.

10. The heterogeneous plant defensin of any one of claims 1 to 9, wherein the second polypeptide sequence corresponds to an amino acid sequence selected from the group comprising SEQ ID NO:29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO:69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87.

11. The heterogeneous plant defensin of claim 10, wherein the second polypeptide sequence corresponds to an amino acid sequence comprising S-K-I-L-R-R (SEQ ID NO: 2).

12. The heterogeneous plant defensin of claim 10, wherein the second polypeptide sequence corresponds to an amino acid sequence comprising K-I-L-R-R (SEQ ID NO: 89).

13. The heterogeneous plant defensin of any one of claims 1 to 12, wherein the first polypeptide sequence is derived from a Class I defensin.

14. The heterogeneous plant defensin of any one of claims 1 to 13, wherein the second polypeptide sequence is derived from a Class II Solanaceous plant defensin.

15. The heterogeneous plant defensin of any one of claims 1 to 14, further comprising one or more amino acid substitutions, deletions or additions.

16. The heterogeneous plant defensin of any one of claims 1 to 15, further comprising one or more amino acids selected from the group consisting of:

K (Lysine) at or around +1 amino acid residue relative to the first invariant Cysteine;

K (Lysine) at or around +5 amino acid residues relative to the fourth invariant Cysteine;

D (Aspartic Acid) at or around −3 amino acids residues relative to the fifth invariant Cysteine;

H (Histidine) at or around −1 amino acid residue relative to the fifth invariant Cysteine; and/or

K (Lysine) at or around +2 amino acid residues relative to the seventh invariant Cystine, and/or conservative substitutions, functional and/or structural equivalents thereof.

17. The heterogeneous plant defensin of any one of claims 1 to 16, wherein the second polypeptide is derived from a Class II Solanaceous plant defensin from Nicotiana spp., Petunia spp., Solanum spp., or Capsicum spp.

18. The heterogeneous plant defensin of any one of claims 1 to 17, wherein the second polypeptide is derived from NaD1, NsD1, NsD2, NoD173, TPP3, PhD1, PhD1A, PhD2, TPP3, FST, NeThio1, NeThio2, NpThio1, Na-gth or CcD1.

19. The heterogeneous plant defensin of any one of claims 1 to 18, wherein the first polypeptide is derived from a Class I plant defensin from Nicotiana spp., Hordeum spp., Pisum spp., Medicago, Dahlia spp., Raphanus spp or Zea spp.

20. The heterogeneous plant defensin of any one of claims 1 to 19, wherein the first polypeptide is derived from the Class I defensin NaD2, g1-H, Psd1, Ms-Def1, Dm-AMP1, R5-AFP2 or g-zeathionin2.

21. The heterogeneous plant defensin of any one of claims 1 to 20, comprising the amino acid sequence set forth as any one of SEQ ID NO:29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO:69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87.

22. The heterogeneous plant defensin of any one of claims 1 to 20, encoded by the nucleic acid sequence set forth as any one of SEQ ID NO:30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58 SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86 or SEQ ID NO: 88.

23. The heterogeneous plant defensin of any one of claims 3 to 20, comprising a serine amino acid positioned adjacent to the fifth invariant cysteine amino acid corresponding to the sequence set forth as SEQ ID NO:1, wherein the serine amino acid is positioned to the C-terminal side of the fifth invariant cysteine amino acid residue.

24. The heterogeneous plant defensin of any one of claims 1 to 23, wherein the heterogeneous plant defensin displays enhanced anti-proliferative disease and/or cytotoxic activity relative to the defensin from which the first polypeptide sequence is derived.

25. The heterogeneous plant defensin of any one of claims 1 to 24, wherein the heterogeneous defensin binds to a phospholipid.

26. The heterogeneous plant defensin of any one of claims 1 to 24, wherein the second polypeptide sequence, or part thereof, binds to a phospholipid.

27. The heterogeneous plant defensin of any one of claims 1 to 24, wherein the heterogeneous defensin binds to phosphatidylinositol 4,5-bisphosphate or Ptdlns(4,5)P2 (PIP2).

28. The heterogeneous plant defensin of any one of claims 1 to 24, wherein the second polypeptide sequence, or part thereof, binds to phosphatidylinositol 4,5-bisphosphate or Ptdlns(4,5)P2 (PIP2).

29. The heterogeneous plant defensin of claim 25 or 26, wherein the phospholipid is located in a cell membrane.

30. The heterogeneous plant defensin of claim 27 or 28, wherein phosphatidylinositol 4,5-bisphosphate or Ptdlns(4,5)P2(PIP2) is located in a cell membrane.

31. The heterogeneous plant defensin of any one of claims 1 to 30, wherein the heterogeneous plant defensin has cell membrane permeabilisation activity.

32. The heterogeneous plant defensin of any one of claims 29 to 31, wherein the cell membrane is a tumour or cancer cell membrane.

33. A nucleic acid encoding the heterogeneous plant defensin of any one of claims 1 to 32, or a functional fragment thereof.

34. A heterogeneous plant defensin encoded by the nucleic acid of claim 33.

35. A vector comprising the nucleic acid of claim 34.

36. A host cell comprising the vector of claim 35.

37. A heterogeneous plant defensin produced by the host cell of claim 36.

38. A pharmaceutical composition comprising the heterogeneous plant defensin of any one of claim 1 to 32, 34 or 37, the nucleic acid of claim 33, the vector of claim 35, or the host cell of claim 36, together with a pharmaceutically acceptable carrier, diluent or excipient.

39. A method for preventing or treating a proliferative disease, wherein the method comprises administering to a subject a therapeutically effective amount of the heterogeneous plant defensin of any one of claim 1 to 32, 34 or 37, the nucleic acid of claim 33, the vector of claim 35, or the host cell of claim 36 or the pharmaceutical composition of claim 38, thereby preventing or treating the proliferative disease.

40. The method according to claim 39, wherein the proliferative disease is cancer.

41. The method according to claim 40 wherein the cancer is selected from the group comprising basal cell carcinoma, bone cancer, bowel cancer, brain cancer, breast cancer, cervical cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer or thyroid cancer.

42. Use of the heterogeneous plant defensin of any one of claim 1 to 32, 34 or 37, the nucleic acid of claim 33, the vector of claim 35, or the host cell of claim 36 or the pharmaceutical composition of claim 38 in the preparation of a medicament for preventing or treating a proliferative disease.

43. A kit for preventing or treating a proliferative disease, wherein the kit comprises a therapeutically effective amount of the heterogeneous plant defensin of any one of 1 to 32, 34 or 37, the nucleic acid of claim 33, the vector of claim 35, or the host cell of claim 36 or the pharmaceutical composition of claim 38.

44. Use of the kit of claim 43 for preventing or treating a proliferative disease, wherein the therapeutically effective amount of the heterogeneous plant defensin of any one of 1 to 32, 34 or 37, the nucleic acid of claim 33, the vector of claim 35, or the host cell of claim 36 or the pharmaceutical composition of claim 38 is administered to a subject, thereby preventing or treating the proliferative disease.

45. The use of claim 42 or 44, wherein the proliferative disease is cancer.

46. The use of claim 45 wherein the cancer is selected from the group comprising basal cell carcinoma, bone cancer, bowel cancer, brain cancer, breast cancer, cervical cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer or thyroid cancer.

47. A heterogeneous plant defensin of any one of claims of any one of claim 1 to 32, 34 or 37 when used for preventing or treating a proliferative disease.

48. The heterogeneous plant defensin of claim 47, wherein the proliferative disease is cancer.

49. The heterogeneous plant defensin of claim 48 wherein the cancer is selected from the group comprising basal cell carcinoma, bone cancer, bowel cancer, brain cancer, breast cancer, cervical cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer or thyroid cancer.