WO2014057284A2 - Translocating peptide - Google Patents

Abstract

A cyclotide sequence defined as anon-naturally occurring linear or cyclic peptide having a cysteine knot backbone moiety and a defined blood-brain barrier translocation moiety, said peptide molecule comprising: i) a peptide having said blood-brain barrier translocation activity, wherein said peptide has an amino acid sequence comprising a plurality of contiguous amino acids, wherein said peptide is about 6to 50 amino acid residues; and ii) a cysteine knot backbone grafted to said peptide of step i).

Description

TRANSLOCATING PEPTIDE

FIELD

The invention relates to novel molecules that have desirable functions, such as the ability to cross membranes or physiological barriers when introduced into an animal. The novel molecules are peptides and may be incorporated into larger polypeptides in order to confer the membrane- translocation activity onto them also. The invention also relates to methods of making and using the peptides and compositions that comprise them as therapeutic or diagnostic agents in humans.

BACKGROUND

The blood-brain barrier (BBB) performs a neuroprotective function by tightly controlling access to the brain; consequently it also impedes access of pharmacological agents to cerebral tissues, necessitating the use of vectors for their transit. BBB permeability is frequently a rate-limiting factor for the penetration of drugs or peptides into the central nervous system (CNS) (see Pardridge, W. M. J. Neurovirol. 5: 556-569 (1999); Bickel, U., Yoshikawa, T. & Pardridge, W. M. Adv. Drug Deliv. Rev. 46: 247-279 (2001 ). The brain is shielded against potentially toxic substances by the BBB, which is formed by brain capillary endothelial cells that are closely sealed by tight junctions. In addition, brain capillaries possess few fenestrae and few endocytic vesicles, compared to the capillaries of other organs (see Pardridge, W. M. J. Neurovirol. 5: 556-569 (1999)). There is little transit across the BBB of large, hydrophilic molecules aside from some specific proteins such as transferrin, lactoferrin and low-density lipoproteins, which are taken up by receptor-mediated endocytosis (see Pardridge, W. M. J. Neurovirol. 5: 556-569 (1999); Tsuji, A. & Tamai, I. Adv. Drug Deliv. Rev. 36: 277-290 (1999); Kusuhara, H. & Sugiyama, Y. Drug Discov. Today 6:150-156 (2001); Dehouck, B. et al. J. Cell. Biol. 138: 877-889 (1997); and Fillebeen, C. et al. J. Biol. Chem. 274: 701 1 -7017 (1999).

A possible solution to this problem would be to use carrier microprotein peptides capable of crossing the BBB, fused to the therapeutic molecule. A proposed example of such a molecule is a member of the naturally occurring family of cysteine-knot microproteins or cyclotides found in various plant species. Cysteine-knot microproteins (cyclotides) are small peptides, typically consisting of about 30- 40 amino acids, which can be found naturally as cyclic or linear forms, where the cyclic form has no free N- or C-terminal amino or carboxyl end. They have a defined structure based on three intramolecular disulfide bonds and a small triple stranded β-sheet (Craik et al., 2001 ; Toxicon 39, 43-60). The cyclic proteins exhibit conserved cysteine residues defining a structure referred to herein as a "cysteine knot". This family includes both naturally occurring cyclic molecules and their linear derivatives as well as linear molecules which have undergone cyclization. These molecules are useful as molecular framework structures having enhanced stability over less structured peptides. (Colgrave and Craik, 2004; Biochemistry 43, 5965-5975). However, these molecules are not themselves capable of crossing the blood-brain barrier to any great extent, and cannot be used as neuro- therapeutic agents themselves or act as carriers of therapeutic peptide or protein agents. The main cyclotide features are a remarkable stability due to the cysteine knot, a small size making them readily accessible to chemical synthesis, and an excellent tolerance to sequence variations. Cyclotides therefore appear as appealing leads or scaffolds for peptide drug design. The cyclotide scaffold is found in almost 30 different protein families among which conotoxins, spider toxins, squash inhibitors, agouti-related proteins and plant cyclotides are the most populated families. Cyclotides from plants in the Rubiaceae and Violaceae families are for the most part found to be head-to-tail cyclic peptides [9-1 1 ]. However, within the squash inhibitor family of cyclotides both cyclic and linear cyclotides have been identified from Momordica cochinchinensis: the cyclic trypsin inhibitors (MCoTI)- I and -II and their linear counterpart MCoTI-lll [13]. It is now clear that both cyclic and linear variants can exist in different cyclotide families, but the impact of the cyclization is poorly understood. Cyclic peptides were expected to display improved stability, better resistance to proteases, and reduced flexibility when compared to their linear counterparts, hopefully resulting in enhanced biological activities. However, linear cyclotides have the advantage of being able to be more easily linked to other peptides or proteins. Also, cyclotides have not proven to readily cross the BBB in their natural forms and it would be desirable to combine the structural stability of the cyclotide scaffold with the ability to cross the blood-brain barrier, and link other biological activities to the cyclotide molecule. Therefore, identification of cyclotides that are amenable to retaining structure and blood-brain transfer capabilities as both linear or cyclic molecules would be desirable. It would be further desirable to be able use such cyclotides to increase the ability to improve the activity and bioavailability of biological therapeutics in the body.

SUMMARY

The inventors have identified a subset of cyclotide molecules that demonstrate particular advantage in crossing the blood-brain barrier. These cyclotides of the invention may be used to deliver therapeutic agents across the BBB for the treatment of neurological conditions.

The invention is directed towards a cyclotide molecular framework comprising a sequence of amino acids or analogues thereof forming a cysteine-knot backbone wherein said cysteine-knot backbone comprises sufficient disulfide bonds or chemical equivalents thereof, to confer a knotted topology on the three-dimensional structure of said cysteine-knot backbone and wherein at least one exposed amino acid residue such as on one or more beta turns and/or within one or more loops, is inserted or substituted relative to the naturally occurring amino acid sequence, such that the cyclotide of the invention has enhanced translocation behaviour compared with the unmodified parental cyclotide. The modified cyclotides of the invention incorporate sufficient modified amino acid sequence information to provide the desired features of high enzymatic stability and good translocation or permeation behaviour, such that novel carrier cyclotides can be identified. A cyclotide sequence of the invention may be defined as a non-naturally occurring linear or cyclic peptide having a cysteine knot backbone moiety and a defined blood-brain barrier translocation moiety, said peptide molecule comprising: i) a peptide having said blood-brain barrier translocation activity, wherein said peptide has an amino acid sequence comprising a plurality of contiguous amino acids, wherein said peptide is about 6 to 50 amino acid residues; and ii) a cysteine knot backbone grafted to said peptide of step i), wherein said cysteine knot backbone comprises the structure (I):

(X^ ... X^d (X, ... Xa)C₂(X'₁... X'_b)C₃(X"i ... Χ"ο)θ4(Χ"Ί ... X'"_d)C₅(X^lv, ... X^IVe)C₆(X^Vi ... X^v _f)

Loop6 Loopl Loop2 Loop3 Loop4 Loop5 Loop6 wherein C.sub.1 to C.sub.6 are cysteine residues; wherein each of C.sub.1 and C.sub.4, C.sub.2 and C.sub.5, and C.sub.3 and C.sub.6 are connected by a disulfide bond to form a cysteine knot; wherein each X represents an amino acid residue in a loop, wherein said amino acid residues may be the same or different; wherein d is about 1 -2; wherein one or more of loops 1 , 2, 3, 5 or 6 have an amino acid sequence comprising the sequence of said peptide of step i), wherein any loop comprising said sequence of said peptide of step i) comprises 2 to about 30 amino acids, and wherein for any of loops 1 , 2, 3, 5, or 6 that do not contain said sequence of said peptide of step i), a, b, c, e, and f, may be the same or different, and may be any number from 1 to 20, from 3 to 20, from 6 to 20, or from 3 to 10. Alternatively, a may be from 3 to 10 and b, c, e and f may be from 1 to 20.

In other embodiments, the peptide having blood-brain barrier translocation activity may comprise from 2 to 50 amino acids, and the one or more loop comprising the peptide may have from 2 to 50 amino acids or from 6 to 50 amino acids; the peptide having blood-brain barrier translocation activity may comprise from 6 to 50 amino acids, and the one or more loop comprising the peptide may have from 6 to 50 amino acids; the peptide having blood-brain barrier translocation activity may comprise from 2 to 30 amino acids, and the one or more loop comprising the peptide may have from 2 to 50 amino acids, or from 6 to 50 amino acids, from 2 to 30 amino acids or from 6 to 30 amino acids; or the peptide having blood-brain barrier translocation activity may comprise from 6 to 30 amino acids, and the one or more loop comprising the peptide may have from 6 to 50 amino acids or from 6 to 30 amino acids residues.

The novel cyclotide peptides may subsequently be used to deliver a biologically active agent, such as a peptide, polypeptide or protein with therapeutic agonist or antagonist activity.

In another embodiment of the present invention the cyclotide comprises a sequence of amino acids or analogues thereof forming a cysteine-knot backbone, wherein said cysteine-knot backbone comprises sufficient disulfide bonds or chemical equivalents thereof, to confer a knotted topology on the three- dimensional structure of said cysteine-knot backbone and wherein at least one exposed amino acid residue such as on one or more beta turns and/or within one or more loops, is inserted or substituted relative to the naturally occurring amino acid sequence, such that the modified cyclotide has the desired properties of high enzymatic stability and translocation, such that oral administration and/or BBB transfer of the cyclotide is feasible. In a further aspect of the invention, the cyclotide sequence may be either linear or cyclic. In another embodiment, a linear or cyclic, cyclotide of the invention is operably linked to the N- or C- terminus of a biologically active agonist or antagonist peptide or protein moiety, where such linkage allows the translocation across the BBB, and entry of the biologically active peptide or protein moiety. In a further aspect of the invention, a BBB transfer linear or cyclic, cyclotide of the invention is operably linked to the N- or C-terminus of a biologically active agonist, or antagonist, peptide or protein moiety, where such linkage allows the translocation across the BBB, and entry of a pharmacologically relevant dose of the biologically active peptide or protein moiety.

In a further embodiment, cyclotides of the invention are derived from linear or cyclic form of cyclotides of the Momordicae, Rubiaceae and Violaceae, plant species. In a preferred aspect, cyclotides of the invention are derived from linear or cyclic form of cyclotides of the Momordicae species including the squash serine protease inhibitor family (Otlewski & Korowarsch Acta Biochim Pol. 1996;43(3):431 - 44), and in a more preferred aspect from Momordica cochinchinensis trypsin inhibitors MCoTI-l [SEQ ID NO: 001 ] and -II [SEQ ID NO: 002] (naturally cyclic) and MCoTI-lll (naturally linear) [SEQ ID NO: 3] below.

Mcoti-I GGVCPKILQRCRRDSDCPGACICRGNGYCGSGSD [SEQ ID NO: 001]

Mcoti-II GGVCPKILKKCRRDSDCPGACICRGNGYCGSGSD [SEQ ID NO: 002]

Mcoti-III ERACPRILKKCRRDSDCPGACICRGNGYCG [SEQ ID NO: 003]

While such cyclotides retain the structural stability characteristic of the cyclic-cysteine knot, they are not readily amenable to the translocation across mucosal surfaces. Therefore, the inventors have identified cyclotides of structure I, derived from loop replacement libraries based on Mcoti-II (SEQ ID NO: 002) with enhanced blood-brain barrier translocation characteristics which have the amino acid sequences of SEQ ID 004-SEQ ID 026 as set out in Table 1

A further embodiment of the invention provides for a nucleic acid sequence that encodes the amino acid sequence of the second embodiment. Suitably the nucleic acid sequence may comprise the sequence of SEQ ID NO: 002. In particular embodiments of the invention the nucleic acid sequence may be comprised within a vector, suitably an expression vector, optionally a display vector (including phage or cis-display vectors).

A further embodiment of the invention provides a composition comprising the compound of general formula I together with a pharmaceutically acceptable excipient.

Another embodiment of the invention provides a method for identifying a compound of formula I, comprising the steps of: a) constructing a cyclotide sequence display library;

b) expressing the sequence library in order to obtain expressed peptides, or polypeptides that comprise the one or more compounds of formula I;

c) administering the expressed peptides or polypeptides that comprise the compounds of formula I to an animal, optionally via the oral route;

d) recovering any compounds of formula I from the blood, lymph and/or tissues of the animal; e) determining the sequence of the recovered compound of formula I.

DRAWINGS

The invention is illustrated by the following drawings in which, Figure 1 shows the multiple cloning site of pSP1

Figure 2 shows Pe data for selected cyclotide-fusion proteins, demonstrating transfer across an in vitro BBB cell model.

Figure 3 shows a microscope image of a section of rat brain 4 hours after intravenous injection with Flourescein-labelled Cyclotide-Single domain fusion protein C15 (UV microscopy x200 magnification).

Figure 4 shows the 4',6-diamidino-2-phenylindole (DAPI)-stained image of the same section of brain demonstrated in Figure 3 (UV microscopy x200 magnification).

Figure 5 shows a microscope image of a section of rat brain 1 hour after intravenous injection with unlabelled cyclotide-Single domain fusion C15, which has been detected with FITC-labelled anti-V5 antibody (UV microscopy x400 magnification).

Figure 6 shows the DAPI stained image of the same section of brain demonstrated in Figure 5 (UV microscopy x400 magnification).

Figure 7 shows a section of rat brain from a rat injected with Flourescein-labelled TNFR ll-Cyclotide C1 1 fusion at 4 hour post-injection (UV microscopy x200 magnification).

Figure 8 shows rat injected with Flourescein-labelled TNFR II control at 4 hour post-injection (UV microscopy x200 magnification).

Figure 9 shows a section of brain from a rat injected with TNFR ll-Cyclotide C1 1 fusion at 4 hour post- injection (Anti-V5-HRP/DAB detection; normal light microscopy x200 magnification).

Figure 10 shows a section of brain from a rat injected with TNFR II control at 4 hour post-injection (Anti-V5-HRP/DAB detection, normal light microscopy x200 magnification). DETAILED DESCRIPTION

Prior to setting forth the invention, a number of definitions are provided that will assist in the understanding of the invention. All references cited herein are incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "nucleic acid sequence" as used herein, is a single or double stranded covalently-linked sequence of nucleotides in which the 3' and 5' ends on each nucleotide are joined by phosphodiester bonds. The polynucleotide may be made up of deoxyribonucleotide bases or ribonucleotide bases. Nucleic acid sequences may include DNA and RNA, and may be manufactured synthetically in vitro or isolated from natural sources. Sizes of nucleic acid sequences, also referred to herein as "polynucleotides" are typically expressed as the number of base pairs (bp) for double stranded polynucleotides, or in the case of single stranded polynucleotides as the number of nucleotides (nt). One thousand bp or nt equal a kilobase (kb). Polynucleotides of less than around 40 nucleotides in length are typically called "oligonucleotides" and may comprise primers for use in manipulation of DNA such as via polymerase chain reaction (PCR).

The term "vector" is used to denote a DNA molecule that is either linear or circular, into which another nucleic acid (typically DNA) sequence fragment of appropriate size can be integrated. Such DNA fragments) can include additional segments that provide for transcription of a gene encoded by the DNA sequence fragment. The additional segments can include and are not limited to: promoters, transcription terminators, enhancers, internal ribosome entry sites, untranslated regions, polyadenylation signals, selectable markers, origins of replication and such like. A variety of suitable promoters for prokaryotic (e.g. the β-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system, lac, tac, T3, T7 promoters for E. coli) and eukaryotic (e.g. simian virus 40 early or late promoter, Rous sarcoma virus long terminal repeat promoter, cytomegalovirus promoter, adenovirus late promoter, EG-1 a promoter) hosts are available. Expression vectors are often derived from plasmids, cosmids, viral vectors and yeast artificial chromosomes; vectors are often recombinant molecules containing DNA sequences from several sources.

Specific embodiments provide for an expression vector that encodes a modified polypeptide or fragment/domain thereof that comprise a molecule of the invention. Accordingly, the DNA encoding a relevant peptide of the invention can be inserted into a suitable expression vector (e.g. pGEM®, Promega Corp., USA), where it is operably linked to appropriate expression sequences, and transformed into a suitable host cell for protein expression according to conventional techniques (Sambrook J. et a/., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY). Suitable host cells are those that can be grown in culture and are amenable to transformation with exogenous DNA, including bacteria, fungal cells and cells of higher eukaryotic origin, preferably mammalian cells, typically human cells and cell lines. To aid in purifying the peptides of the invention, the polypeptide (and corresponding nucleic acid) of the invention may include a purification sequence, such as a His-tag. In addition, or alternatively, the modified polypeptides may, for example, be grown in fusion with another protein and purified as insoluble inclusion bodies from bacterial cells. This is particularly convenient when the modified polypeptide to be synthesised may be toxic to the host cell in which it is to be expressed. Alternatively, modified polypeptides may be synthesised in vitro using a suitable in vitro (transcription and) translation system (e.g. the E. coli S30 extract system: Promega corp., USA).

The term "operably linked", when applied to DNA sequences, for example in an expression vector or construct indicates that the sequences are arranged so that they function cooperatively in order to achieve their intended purposes, i.e. a promoter sequence allows for initiation of transcription that proceeds through a linked coding sequence as far as the termination sequence.

In one embodiment of the present invention the vector is suitable as a polypeptide library display vector, enabling the polypeptide gene product of the cyclotide encoding gene to remain associated with the vector following transcription. As used herein, the term "polypeptide library display" refers to a system in which a collection of polypeptides or peptides, that may form part or all of a library, are made available for selection based upon a specified characteristic. The specified characteristic may be a physical, chemical or functional characteristic. Suitable display systems utilise a cellular expression system, for instance an expression of a library of nucleic acids in appropriately transformed, infected, transfected or transduced cells and display of the encoded polypeptides on the surface of the cells. Alternative cellular expression systems may include emulsion compartmentalization and display. Optional display systems link the coding function of a nucleic acid and physical, chemical and/or functional characteristics of a polypeptide or peptide encoded by the nucleic acid. When such a display system is employed, polypeptides or peptides that have a desired physical, chemical and/or functional characteristic can be selected and the nucleic acid encoding the selected polypeptide is readily isolated. Several display systems that link the coding functionality of a nucleic acid with the associated polypeptide product are known in the art, for example, bacteriophage display (phage display), ribosome display, emulsion compartmentalization and display, yeast display, puromycin display, bacterial display, display on plasmid, covalent display, CIS display and the like. (See, e.g., EP 0436597 (Dyax), U.S. Pat. No. 6,172,197 (McCafferty et al.), U.S. Pat. No. 6,489,103 (Griffiths et al.).

The term "library" refers to a mixture of heterogeneous polypeptides or nucleic acids. The library is composed of a plurality of members, each of which has a substantially unique polypeptide or nucleic acid sequence. Sequence differences between library members are responsible for the diversity present in the library. In the present invention the library may take the form of a simple mixture of polypeptides or nucleic acids, or may be in the form of organisms or cells, for example bacteria, viruses, animal or plant cells and the like, transformed with a library of nucleic acids. Usually, each individual organism (such as a phage) or cell contains only one or a very limited number of library members. Advantageously, the nucleic acids are incorporated into expression vectors, in order to allow expression of the polypeptides encoded by the nucleic acids. In a preferred aspect, therefore, a library may take the form of a population of host organisms, each organism containing one or more copies of an expression vector containing a single member of the library in nucleic acid form which can be expressed to produce its corresponding polypeptide member - i.e. the polypeptide gene product. Thus, the population of host organisms has the potential to encode a widely diverse number of polypeptides. An embodiment of the present invention provides for a library of polypeptides that are based around modified versions of cyclobody polypeptides, in which the diversity or variance between library members is located in the polypeptide sequences of one or more of the loop or variable regions of one or more functional modules within the protein.

The term "amino acid" in the context of the present invention is used in its broadest sense and is meant to include naturally occurring L a-amino acids or residues. The commonly used one and three letter abbreviations for naturally occurring amino acids are used herein: A=Ala; C=Cys; D=Asp; E=Glu; F=Phe; G=Gly; H=His; l=lle; K=Lys; L=Leu; M=Met; N=Asn; P=Pro; Q=Gln; R=Arg; S=Ser; T=Thr; V=Val; W=Trp; and Y=Tyr (Lehninger, A. L, (1975) Biochemistry, 2d ed., pp. 71 -92, Worth Publishers, New York). The general term "amino acid" further includes D-amino acids, retro-inverso amino acids as well as chemically modified amino acids such as amino acid analogues, naturally occurring amino acids that are not usually incorporated into proteins such as norleucine, and chemically synthesised compounds having properties known in the art to be characteristic of an amino acid, such as β-amino acids. For example, analogues or mimetics of phenylalanine or proline, which allow the same conformational restriction of the peptide compounds as do natural Phe or Pro, are included within the definition of amino acid. Such analogues and mimetics are referred to herein as "functional equivalents" of the respective amino acid. Other examples of amino acids are listed by Roberts and Vellaccio, The Peptides: Analysis, Synthesis, Biology, Gross and Meiehofer, eds., Vol. 5 p. 341 , Academic Press, Inc., N.Y. 1983, which is incorporated herein by reference. Such modifications may be particularly advantageous for increasing the stability of modified knottin domains and/or for improving or modifying solubility, bioavailability and delivery characteristics (e.g. for in vivo applications).

A "polypeptide" is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or in vitro by synthetic means. Polypeptides of less than around 12 amino acid residues in length are typically referred to as "peptides" and those between about 12 and about 30 amino acid residues in length may be referred to as "oligopeptides". The term "polypeptide" as used herein denotes the product of a naturally occurring polypeptide, precursor form or proprotein. Polypeptides can also undergo maturation or post-translational modification processes that may include, but are not limited to: glycosylation, proteolytic cleavage, lipidization, signal peptide cleavage, propeptide cleavage, phosphorylation, and such like. The term "protein" is used herein to refer to a large polypeptide molecule as well as a macromolecule comprising one or more polypeptide chains.

The term "peptide" as used herein (e.g. in the context of a therapeutic peptide or framework) refers to a plurality of amino acids joined together in a linear or circular chain. The term oligopeptide is typically used to describe peptides having between 2 and about 50 or more amino acids. Peptides larger than about 50 are often referred to as polypeptides or proteins. For purposes of the present invention, the term "peptide" is not limited to any particular number of joined amino acids, and the term "peptide" is thus used interchangeably with the terms "oligopeptide", "polypeptide" and "protein".

By "derived from" it is meant that the peptide concerned includes one or more mutations in comparison to the primary amino acid sequence of the peptide on which it is based. Thus, a derivative of a modified polypeptide of the invention may have one or more (e.g. 1 , 2, 3, 4, 5 or more) chemically modified amino acid side chains compared to the modified polypeptide from which it is derived. Suitable modifications may include pegylation, sialylation and glycosylation. In addition or alternatively, a derivative of a modified polypeptide may contain one or more (e.g. 1 , 2, 3, 4, 5 or more) amino acid mutations, substitutions or deletions to the primary sequence of a selected modified polypeptide. Accordingly, the invention encompasses the results of maturation experiments conducted on a modified polypeptide to improve or alter one or more characteristics of the initially identified molecule. By way of example, one or more amino acid residues of a selected modified polypeptide sequence may be randomly or specifically mutated (or substituted) using procedures known in the art (e.g. by modifying the encoding DNA or RNA sequence). The resultant library or population of derivatised polypeptides may be selected - by any known method in the art - according to predetermined requirements: such as improved specificity against particular target ligands; or improved drug properties (e.g. solubility, bioavailability, immunogenicity etc.).

The molecules of the present invention, suitably referred to as "cyclotides", are typically peptides or peptide derivatives that possess a specific biological activity in vivo. The term "cyclotides" as used herein, comprises a linear or cyclic amino acid sequence containing a structure referred to herein as a "cysteine knot". A cysteine knot occurs when a disulfide bond passes through a closed cyclic loop formed by two other disulfide bonds and the amino acids in the backbone. However, reference herein to a "cysteine knot" includes reference to structural equivalents thereof which provide similar constraints to the three-dimensional structure of the cyclic backbone. For example, appropriate turns and loops in the "cysteine-knot" backbone may also be achieved by engineering suitable covalent bonds or other forms of molecular associations. All such modifications to the cyclic backbone which result in retention of the three-dimensional knotted topology conferred by the cysteine knot are encompassed by the present invention. Furthermore, although a cysteine knot is characterized by a knot formed by three disulfide bonds, the present invention extends to molecules comprising only two disulfide bonds. In such a case, the molecular framework may need to be further stabilized using other means or the molecular framework may retain suitable activity despite a change in three- dimensional structure caused by the absence of a third disulfide bond. In yet a further modification, the cysteine knot backbone may comprise more than three disulfide bonds such as occurring in a double or multiple cysteine knot arrangement or in a single cysteine knot arrangement supplement by one or two additional disulfide bonds.

The cyclotides may be conjugated to another agent or polypeptide, or in certain embodiments one or more cyclotides are incorporated into another polypeptide to form a synthetic chimaeric (or fusion) polypeptide/protein. Suitably, the cyclotides may be comprised within a loop region of a scaffold polypeptide. The resultant modified scaffold can then itself be conjugated or comprised within a larger molecule. Suitably cyclotides of the invention may be enriched for cysteine residues. A specific embodiment of the present invention described in more detail below provides for cyclotides that exhibit the ability to traverse the blood-brain barrier, suitably rendering the cyclotide or any molecule comprising or conjugated thereto available to the brain.

In some cases it may be desirable to conjugate a modified or derivatised cyclotide of the invention to one or more additional modified polypeptides or fragments thereof in order to create a modified polypeptides. The term "conjugate" is used in its broadest sense to encompass all methods of attachment or joining that are known in the art. For example, the cyclotide moiety can be an amino acid extension of the C- or N-terminus of the modified polypeptide. In addition, a short amino acid linker sequence may lie between the modified polypeptide and the cyclotide moiety. Suitable linker groups may comprise an amide, an ester, a disulfide, a sulfide, a ketal, a succinate, an oxime, a carbamate, a carbonate, a sialyl ether, or a triazole. The invention further provides for molecules where the modified polypeptide is linked, e.g. by chemical conjugation to the cyclotide moiety optionally via a linker sequence. Typically, the modified polypeptide will be linked to the other moiety via sites that do not interfere with the activity of either moiety. The term "conjugated" is used interchangeably with terms such as "linked", "bound", "associated", "fused" or "attached". A wide range of covalent and non-covalent forms of conjugation are known to the person of skill in the art, and fall within the scope of the invention. For example, disulphide bonds, chemical linkages and peptide chains are all forms of covalent linkages. Where a non-covalent means of conjugation is preferred, the means of attachment may be, for example, a biotin-(strept)avidin link or the like. Antibody (or antibody fragment)-antigen interactions may also be suitably employed to conjugate a cyclotide of the invention to another moiety, such as a non-polypeptide moiety, small molecule, or biological drug.

A "non-polypeptide moiety" as used herein, refers to an entity that does not contain an polypeptide sequence or three-dimensional fold. The person of skill in the art understands and can determine whether a polypeptide molecule is an polyprotein or peptide sequence, for example, by way of sequence homology or structure prediction or determination. Such non-polypeptide moieties include nucleic acids and other polymers, peptides, proteins, peptide nucleic acids (PNAs), antibodies, antibody fragments, and small molecules, amongst others. Suitably, a non-polypeptide moiety is a biological molecule (e.g. comprising a polynucleotide or peptide), and advantageously is a therapeutic or targeting molecule.

A cyclotide of the invention may also be conjugated or otherwise linked to a therapeutic selected from the group consisting of: a small molecule; an antibody or antibody fragment; a cytokine; a nucleic acid; a bioactive peptide; a glycosylated peptide; an imaging agent; and a radioactive compound.

The cyclotide molecules of the invention may comprise a specific functionality which typically includes at least some type of membrane translocation activity. In a specific embodiment of the invention, the membrane translocation activity comprises an activity selected from one or more of: an ability to translocate across the gut wall (e.g. intestinal mucosa); an ability to translocate across the blood brain barrier; an ability to translocate across a cell membrane; an ability to translocate into a sub cellular compartment; an ability to translocate into the nucleus of a cell; and an ability to translocate into an organelle, including a mitochondrion. The membrane translocation activity may be suitably comprised within a peptide or polypeptide that resides within the functional module a larger protein. Hence, in an embodiment of the invention the cyclotide may be comprised within a synthetic polypeptide scaffold. Desirable physical properties of potential scaffold molecules include high thermal stability and reversibility of thermal folding and unfolding.

The technique of using a protein "scaffold" and the engineering of loops or regions within the scaffold to alter activity is most notable with regard to the field of antibodies and antibody fragments, which have a natural repertoire of variable regions or loops. The variable loops of antibodies have been extensively engineered to produce peptides having improved binding (e.g. affinity and/or specificity) to known ligands, and also to expand the binding substrates for particular antibody frameworks (see for example, Knappik et al., (2000) J. Mol. Biol., 296, 57-86; and EP 1025218). The engineering of non- antibody frameworks has been reviewed, for example, by Hosse et al., (2006), Protein Sci., 15, 14-27. Such non-antibody or alternative scaffold proteins have considerable advantages over traditional antibodies due to their small size, high stability, and ability to be expressed in prokaryotic hosts. Suitable scaffolds may include modified whey acidic protein (WAP) domain containing polypeptides, such as those described in the inventors' co-pending International patent application published as WO-A-2012073045. It will be appreciated by the skilled person that suitable scaffolds are not limited solely to the WAP domain of human elafin (trappin-2) but may include other human trappins (e.g. SLPI) or non-human trappins (e.g. from porcine, bovine or simian sources). In addition, the invention extends to cyclotides comprised within other non-trappin members of the WAP domain family.

In a specific embodiment of the invention a library of cyclotides is screened to identify individual members of the library that exhibit a desired biological activity such as membrane translocation activity, and in particular transfer across the BBB when delivered to an animal. By "transfer across the blood-brain barrier" it is meant that the polypeptide is delivered to an animal and is capable of passing into the brain of the animal by traversing blood vessel walls in the brain. A method for screening for BBB transfer, according to one embodiment, includes the steps of: a) constructing a cyclotide sequence display library;

b) expressing the sequence library in order to obtain expressed cyclotides, or polypeptides that comprise the cyclotides;

c) administering the expressed cyclotides or polypeptides that comprise the cyclotides to an animal;

d) recovering any cyclotide from the brain of the mammal;

e) determining the sequence of the recovered cyclotide.

The animal used in such a screen is typically a bird or mammal, and may be selected from humans, primates, cattle, sheep, rodents, cats, dogs, and rabbits. In the case of non-human animals the library of cyclotides may be suitably administered by oral gavage or inclusion within normal animal feed. Recovery of the cyclotides from the body of the animal may be via biopsy, or in the case of non- human animals via sacrifice of the animal and histological and pathological analysis of the tissues in the body. In this way it is also possible to identify members of the library of cyclotides that exhibit tissue specificity and/or the availability to cross various additional barriers within the body of the animal. By way of example, modified cyclotides that are found in a brain biopsy of a screened non- human animal would be considered as demonstrating the ability of being able to cross the BBB. In a specific embodiment of the invention the cyclotides of the invention are comprised within a phage display library and the determination of BBB transfer is made by analysing which cyclotides are capable of facilitating transport of an associated phage particle as a whole into the brain of an animal which has been fed or intravenously injected with at least a part of the phage display library.

In other embodiments of the invention cyclotides are conjugated to or comprised within desirable biological therapeutic agents which are then administered to a test animal in order to determine the BBB transfer of the modified biological therapeutic agent.

Therapeutic uses and applications for the cyclotides of the invention therefore include any disease or condition that requires repetitive treatment regimes or the frequent administration of a biological therapeutic agent. This includes, therapeutic applications that may benefit from conjugation of a cyclotide to the N- or C-terminus of the therapeutic agent that so renders the therapeutic agent more readily available to the brain or cerebrospinal fluid of the recipient. Diseases that are suitable for treatment include but are not limited to: the treatment of various neoplastic and non-neoplastic diseases and disorders (e.g. cancers / neoplastic diseases and related conditions); chronic degenerative and neurodegenerative diseases or disorders (e.g. multiple sclerosis, Parkinson's disease and Alzheimer's disease), strokes or other brain damaging conditions. In a further embodiment, cyclotides of the invention are fused to a therapeutic agent and used as an intravenously administered therapeutic treatment of Alzheimer's disease or intracranial neoplasms. In a further embodiment, a cyclotide of the invention is conjugated to a therapeutic agent capable of enhancing cognitive ability such as memory.

In a further aspect, the present invention relates to a conjugate which may comprise a carrier selected from the group consisting of any one of the peptides of the present invention, and a bioactive or therapeutic agent selected from the group consisting, for example, of a drug (e.g., a small molecule drug, e.g., an antibiotic), a medicine, a detectable label, a protein (e.g., an enzyme), protein-based compound (e.g., a protein complex comprising one or polypeptide chain) and a polypeptide (peptide). The agent may be more particularly, a molecule which is active at the level of the central nervous system. The agent may be any agent for treating or detecting a neurological disease.

In accordance with the present invention, the detectable label may be a radio imaging agent. Example of a label which may be conjugated with the carrier of the present invention and which is encompassed herein includes, for example and without limitation, an isotope, a fluorescent label (e.g., rhodamine), a reporter molecule (e.g., biotin), etc. Other examples of detectable labels include, for example, a green fluorescent protein, biotin, a his tag protein and .beta.-galactosidase.

Example of a protein or protein-based compound which may be conjugated with the carrier of the present invention and which is encompassed herein includes, without limitation, an antibody, an antibody fragment (e.g., an antibody binding fragment such as a Fv fragment, F(ab)2, F(ab)2' and Fab and the like), a single domain antibody (e.g. such as a camelid VHH domain, a shark novel antigen receptor (NAR), or a human VH or VL domain), a peptidic- or protein-based drug (e.g., a positive pharmacological modulator (agonist) or an pharmacological inhibitor (antagonist)) etc. Other examples of agent which are encompassed herein include growth factors (e.g. Fibroblast Growth Factor and related proteins, Nerve Growth Factor, Glial Cell-derived Neurotrophic Factor, Brain- Derived Neurotrophic Factor, Neurotrophin-3 and Neurotrophin-4), cellular toxins (e.g., monomethyl auristatin E (MMAE), toxins from bacteria endotoxins and exotoxins; diphtheria toxins, botunilum toxins, tetanus toxins, pertussis toxins, staphylococcus enterotoxins, toxic shock syndrome toxin TSST-1 , adenylate cyclase toxin, Shiga toxin, cholera enterotoxin, and others), soluble receptors (such as TNF receptor 1 or 2) and anti-angiogenic compounds (endostatin, catechins, nutriceuticals, chemokine IP-10, inhibitors of matrix metalloproteinase (MMPIs), anastellin, vironectin, antithrombin, tyrosine kinase inhibitors, VEGF inhibitors, antibodies against receptor, herceptin, avastin and panitumumab and others).

In specific embodiments of the present invention, the cyclotide-conjugated therapeutic molecules of the present invention are utilised as separately administered compositions or in conjunction with other therapeutic agents. These additional agents can include various immunotherapeutic drugs, such as cyclosporine, methotrexate, adriamycin or cisplatinum, and immunotoxins, or chemotherapeutic drugs such as tamoxifen, paclitaxel, oxaliplatin, vincristine and fluorouracil. Pharmaceutical compositions can include combinations of various cytotoxic or other agents in conjunction with the polypeptides of the present invention. One or more additional pharmaceutically acceptable carrier (such as diluents, adjuvants, excipients or vehicles) may be combined with therapeutic molecules comprising cyclotides of the invention in a pharmaceutical composition. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Pharmaceutical formulations and compositions of the invention are formulated to conform to regulatory standards and can be administered orally, intravenously, topically, or via other standard routes. Administration can be systemic or local.

In a further aspect the present invention provides a biologically active polypeptide which may be able to cross (i.e., crossing) a cell layer mimicking (which mimics) a mammalian blood brain barrier in an in vitro assay, the polypeptide may be selected, for example, from the group of; a cyclotide of from 4 to 25 (e.g., 10-50) amino acid long, which may comprise SEQ ID NO. 004, an cyclotide consisting of SEQ ID NO. 004, a biologically active analogue of SEQ ID NO. 004 of from about 10 to 50 amino acids long, and; a biologically active fragment of SEQ ID NO. 004 (of from 10 to 40 amino acids) or biologically active fragment of a SEQ ID NO. 004 analogue (of from about 10 to 40 amino acids). In accordance with the present invention there is provided a biologically active analogue of SEQ ID NO. 004 which may be selected, for example, from the group consisting of a SEQ ID NO. 004 analogue which may comprise at least 35% identity with the amino acid sequence of SEQ ID NO. 004, a SEQ ID NO. 004 analogue which may comprise at least 40% identity with the amino acid sequence of SEQ ID NO. 004, a SEQ ID NO. 004 analogue which may comprise at least 50% identity with the amino acid sequence of SEQ ID NO. 004, a SEQ ID NO. 004 analogue which may comprise at least 60% identity with the amino acid sequence of SEQ ID NO. 004, a SEQ ID NO. 004 analogue which may comprise at least 70% identity with the amino acid sequence of SEQ ID NO. 004, a SEQ ID NO. 004 analogue which may comprise at least 80% identity with the amino acid sequence of SEQ ID NO. 004, a SEQ ID NO. 004 analogue which may comprise at least 90% identity with the amino acid sequence of SEQ ID NO. 004 and; a SEQ ID NO. 004 analogue which may comprise at least 95% (i.e., 96%, 97%, 98%, 99% and 100%) identity with the amino acid sequence of SEQ ID NO. 004. For example, the biologically active analogue of SEQ ID NO. 004 may comprise an amino acid sequence selected from the group consisting of an amino acid sequence defined in any one of SEQ ID NO. 004 to SEQ ID NO.: 026.

The medicaments and pharmaceutical compositions of the invention can take the form of liquids, solutions, suspensions, lotions, gels, tablets, pills, pellets, powders, modified-release formulations (such as slow or sustained-release), suppositories, emulsions, aerosols, sprays, capsules (for example, capsules containing liquids or powders), liposomes, microparticles or any other suitable formulations known in the art. Other examples of suitable pharmaceutical vehicles are described in Remington's Pharmaceutical Sciences, Alfonso R. Gennaro ed., Mack Publishing Co. Easton, Pa., 19th ed., 1995, see for example pages 1447-1676.

To aid dissolution of the therapeutic agent (comprising the cyclotide) into the aqueous environment a surfactant might be added as a wetting agent. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents might be used and could include benzalkonium chloride or benzethomium chloride. Potential nonionic detergents that could be included in the formulation as surfactants include: lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 20, 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants, when used, could be present in the formulation of the peptide or nucleic acid or derivative either alone or as a mixture in different ratios.

Additives may be included to further enhance cellular uptake of the cyclotide of the invention, such as the fatty acids oleic acid, linoleic acid and linolenic acid.

An "analogue" is to be understood herein as a polypeptide originating from an original sequence or from a portion of an original sequence and which may comprise one or more modification; for example, one or more modification in the amino acid sequence (e.g., an amino acid addition, deletion, insertion, substitution etc.), one or more modification in the backbone or side-chain of one or more amino acid, or an addition of a group or another molecule to one or more amino acids (side-chains or backbone). An "analogue" is therefore understood herein as a molecule having a biological activity and chemical structure (or a portion of its structure) similar to that of a polypeptide described herein. An analogue comprises a polypeptide which may have, for example, one or more amino acid insertion, either at one or both of the ends of the polypeptide and/or inside the amino acid sequence of the polypeptide. An "analogue" may have sequence similarity and/or sequence identity with that of an original sequence or a portion of an original sequence and may also have a modification of its structure as discussed herein. The degree of similarity between two sequences is based upon the percentage of identities (identical amino acids) and of conservative substitution.

Similarity or identity may be compared, for example, over a region of 2, 3, 4, 5, 10, 19, 20 amino acids or more (and any number there between). Identity may include herein, amino acids which are identical to the original peptide and which may occupy the same or similar position when compared to the original polypeptide. An analogue which have, for example, 50% identity with an original polypeptide may include for example, an analogue comprising 50% of the amino acid sequence of the original polypeptide and similarly with the other percentages. It is to be understood herein that gaps may be found between the amino acids of an analogues which are identical or similar to amino acids of the original peptide. The gaps may include no amino acids, one or more amino acids which are not identical or similar to the original peptide. Biologically active analogues of the carriers (polypeptides) of the present invention are encompassed herewith.

Per cent identity may be determined, for example, with an algorithm GAP, BESTFIT, or FASTA in the Wisconsin Genetics Software Package Release 7.0, using default gap weights.

For example an analogue may comprise or have 50% identity with an original amino acid sequence and a portion of the remaining amino acid which occupies a similar position may be for example a non-conservative or conservative amino acid substitution.

Therefore, analogues of the present invention comprises those which may have at least 90% sequence similarity with an original sequence or a portion of an original sequence. An "analogue" may have, for example at least 35%, 50%, 60%, 70%, 80%, 90% or 95% (96%, 97%, 98%, 99% and 100%) sequence similarity with an original sequence or a portion of an original sequence. Also, an "analogue" may also have, for example, at least 35%, 50%, 60%, 70%, 80%, 90% or 95% (96%, 97%, 98%, 99% and 100%) sequence similarity to an original sequence with a combination of one or more modification in a backbone or side-chain of an amino acid, or an addition of a group or another molecule, etc. Exemplary amino acids which are intended to be similar (a conservative amino acid) to others are known in the art and includes, for example, those listed in Table 1 .

Analogues of the present invention also comprises those which may have at least 35%, 50%, 60%, 70%, 80%, 90% or 95% (96%, 97%, 98%, 99% and 100%) sequence identity with an original sequence or a portion of an original sequence. Also, an "analogue" may have, for example, 35%, 50%, 60%, 70%, 80%, 90% or 95% (sequence) identity to an original sequence (i.e., an analogue that is at least 35%, 50%, 60%, 70%, 80%, 90% or 95% identical to an original peptide) with a combination of one or more modification in a backbone or side-chain of an amino acid, or an addition of a group or another molecule, etc.

A "fragment" is to be understood herein as a polypeptide originating from a portion of an original or parent sequence or from an analogue of said parent sequence. Fragments encompass polypeptides having truncations of one or more amino acids, wherein the truncation may originate from the amino terminus (N-terminus), carboxy terminus (C-terminus), or from the interior of the protein. A fragment may comprise the same sequence as the corresponding portion of the original sequence. Biologically active fragments of the carrier (polypeptide) described herein are encompassed by the present invention.

Thus, biologically active polypeptides in the form of the original polypeptides, fragments (modified or not), analogues (modified or not), derivatives (modified or not), homologues, (modified or not) of the carrier described herein are encompassed by the present invention.

Therefore, any polypeptide having a modification compared to an original polypeptide which does not destroy significantly a desired biological activity is encompassed herein. It is well known in the art, that a number of modifications may be made to the polypeptides of the present invention without deleteriously affecting their biological activity. These modifications may, on the other hand, keep or increase the biological activity of the original polypeptide or may optimize one or more of the particularity (e.g. stability, bioavailability, etc.) of the polypeptides of the present invention which, in some instance might be needed or desirable. Polypeptides of the present invention comprises for example, those containing amino acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are known in the art. Modifications may occur anywhere in a polypeptide including the polypeptide backbone, the amino acid side-chains and the amino- or carboxy-terminus. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from posttranslational natural processes or may be made by synthetic methods. Modifications comprise for example, without limitation, pegylation, acetylation, acylation, addition of acetomidomethyl (Acm) group, ADP-ribosylation, alkylation, amidation, biotinylation, carbamoylation, carboxyethylation, esterification, covalent attachment to fiavin, covalent attachment to a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of drug, covalent attachment of a marker (e.g., fluorescent, radioactive, etc.), covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation and ubiquitination, etc. It is to be understood herein that more than one modification to the polypeptides described herein are encompassed by the present invention to the extent that the biological activity is similar to the original (parent) polypeptide.

As discussed above, polypeptide modification may comprise, for example, amino acid insertion (i.e., addition), deletion and substitution (i.e., replacement), either conservative or non-conservative (e.g., D-amino acids, desamino acids) in the polypeptide sequence where such changes do not substantially alter the overall biological activity of the polypeptide.

Example of substitutions may be those, which are conservative (i.e., wherein a residue is replaced by another of the same general type or group) or when wanted, non-conservative (i.e., wherein a residue is replaced by an amino acid of another type). In addition, a non-naturally occurring amino acid may substitute for a naturally occurring amino acid (i.e., non-naturally occurring conservative amino acid substitution or a non-naturally occurring non-conservative amino acid substitution).

EXAMPLES

The following non-limiting examples are provided to further illustrate the present invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the invention, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

A. Library Construction

Two primary libraries based on the MCOTI II native sequence (SEQ ID NO. 027) cyclotide scaffolds were built containing mutant sequences between cysteines 1 and 2, (termed "Loopl ").

To create the libraries, oligonucleotides were designed to insert the mutant sequences into Loop 1 ("Loopl for" or "Loopl fori 2" SEQ ID NOs 028 or 0). All randomised amino acid positions were encoded in the library by a "Trinucleotide" nucleic acid sequence, termed "TRN", where TRN represents an equal mixture of codons for 19 of the naturally occurring amino acids excluding cysteine.

PCR products were cloned as Nco\-Not\ digested fragments into similarly digested pSP1 phagemid pill fusion vector derived from the pHEN1 pill vector (Hoogenboom et a/. , 1991 , Nucleic Acids Res., 19: 4133-4137). The pSP1 multiple cloning site is shown in Figure 1 .

(i) PCR amplification of Cyclotide loop libraries

For the primary PCR amplifications 10x 50 μΙ amplifications were set up for the Loop libraries using the appropriate oligonucleotide primers (("Loopl for" or "Loop1 for12" SEQ ID NOs: 028 or 029 and Plllseqrev SEQ ID NO 030). Each 50 μΙ reaction mixture contained 10 ng pCyclol , 25 pmol of the appropriate forward and reverse primers, 0.1 mM dNTPs, 2.5 units Taq DNA polymerase, and 1x NEB PCR reaction buffer (20 mM Tris-HCI pH 8.8, 10 mM (NH₄)₂SO₄,10 mM KCI, 2 mM MgS0₄, 0.1 % Triton X-100; NEB Ltd, Cambridge, UK). Reactions were performed for 30 PCR cycles of 94°C, 20s; 60°C, 40s; 72°C, 30s, followed by 5 minutes at 72°C. Reaction products were purified using two Wizard PCR clean-up columns per repertoire (Promega Ltd, Southampton, UK), and eluted into 50 μΙ water per column.

(ii) Pull-through re-amplification of selected DNA

To prepare the final Cyclotide library DNA products, 40x 50 μΙ amplifications were set up for each Cyclotide loop library, using oligonucleotide primers "Loopl PTrev" and "Loop5PTrev" (SEQ ID NOs: 031 and 032). Each 50 μΙ reaction mixture contained approximately 25 ng primary Cyclotide Loop library, 25 pmol of the appropriate forward and reverse primers, 0.1 mM dNTPs, 2.5 units Taq DNA polymerase, and 1x NEB PCR reaction buffer (20 mM Tris-HCI pH 8.8, 10 mM (NH₄)₂SO₄,10 mM KCI, 2 mM MgS0₄, 0.1 % Triton X-100; NEB Ltd, Cambridge, UK). Reactions were performed for 25 cycles of 94°C, 20s; 60°C, 40s; 72°C, 30s, followed by 5 minutes at 72°C. Reaction products were purified using four Wizard PCR clean-up columns per library (Promega Ltd, Southampton, UK), and eluted into 100 μΙ water per column.

(Hi) Cloning into vector pSP1

Each of the libraries, and 250 μg pSP1 vector DNA were digested with enzymes Nco\ and Not\ (100 units each enzyme) for 5 hours at 37°C (NEB, Cambridge, UK), and purified using one Wizard PCR clean-up column per library, and four Wizard PCR clean-up columns for the digested vector DNA (Promega Ltd, Southampton, UK). Each DNA sample was then eluted into 100 μΙ water. Half of each digested library DNA was ligated overnight at 16°C in 400 μΙ with 50 μg of Nco\-Not\ cut pSP1 vector and 4000U of T4 DNA ligase (NEB Ltd, Southampton, UK). After incubation the ligations were adjusted to 200 μΙ with nuclease free water, and DNA precipitated with 1 μΙ 20 mg/ml glycogen, 100 μΙ 7.5M ammonium acetate and 900 μΙ ice-cold (-20°C) absolute ethanol, vortex mixed and spun at 13,000 rpm for 20 minutes in a microfuge to pellet DNA. The pellets were washed with 500 μΙ ice-cold 70% ethanol by centrifugation at 13,000 rpm for 2 minutes, then vacuum dried and re-suspended in 100 μΙ DEPC-treated water. 1 μΙ aliquots of each library were electroporated into 80 μΙ E. coli (TG1). Bacterial cells were grown in 1 ml SOC medium per cuvette for 1 hour at 37°C, and plated onto 2x TY agar plates supplemented with 2% glucose and 100 μg/ml ampicillin. 10^~4, 10^~5 and 10^~6 dilutions of the electroporated bacteria were also plated to assess library size. Colonies were allowed to grow overnight at 30°C. Combined library size was approximately 2x 10¹⁰ clones with >95% with in-frame inserts.

(iv) Phage amplification

Separate phage stocks were prepared for each cyclotide library. The bacteria were then scraped off the plates into 50 ml 2x TY broth supplemented with 20% glycerol, 2% glucose and 100 μg/ml ampicillin. 1 ml of bacterial medium was added to a 50 ml 2x TY culture broth supplemented with 1 % glucose and 100 μg/ml ampicillin and infected with 10¹¹ kanamycin resistance units (km) M13K07 helper phage at 37°C for 30 minutes without shaking, then for 30 minutes with shaking at 200 rpm. Infected bacteria were transferred to 200 ml 2x TY broth supplemented with 25 μg/ml kanamycin, 100 μg/ml ampicillin, and 20 μΜ IPTG, then incubated overnight at 30°C, shaking at 200 rpm. Bacteria were pelleted at 4000 rpm for 20 minutes in 50 ml Falcon tubes, and 40 ml 2.5M NaCI / 20% PEG 6000 was added to 400 ml of particle supernatant, mixed vigorously and incubated on ice for 1 hour to precipitate phage particles. Particles were pelleted at 1 1000 rpm for 30 minutes in 250 ml Oakridge tubes at 4°C in a Sorvall RC5B centrifuge, then resuspended in 40 ml water and 8 ml 2.5M NaCI / 20% PEG 6000 added to reprecipitate particles, then incubated on ice for 20 minutes. Particles were again pelleted at 1 1000 rpm for 30 minutes in 50 ml Oakridge tubes at 4°C in a Sorvall RC5B centrifuge, then resuspended in 5 ml PBS buffer, after removing all traces of PEG / NaCI with a pipette. Bacterial debris was removed via centrifugation for 5 minutes at 13500 rpm in a microcentrifuge. The supernatant was filtered through a 0.45 μηι polysulfone syringe filter, adjusted to 20% glycerol and stored at -70°C.

B. Selection for Delivery into Brain Parenchyma

In order to select members of the cyclotide libraries that are capable not only of crossing the blood- brain barrier, but also of carrying a biological cargo with them, cyclotide phage libraries were administered to the leg vein of Hooded Lister rats. After four hours, Rats were euthanased, brains were perfused to remove any blood-associated phage and infective phage were recovered via infection of TG1 E.coli.

(i) Library selections

Before the experiment, a baseline blood sample was taken from the tip of the study rat. All blood samples were taken up in a final concentration of 10mM Heparin sulphate. For each library, an aliquot of approximately 10¹³ a.r.u. pooled Cyclotide library phage stock was taken up in 100μΙ PBS (pH 7.4). A final volume of 0.8ml was administered to each rat via intravenous injection into the leg vein. A 200μΙ blood sample was taken at 30 minutes, 60 minutes, 240 minutes and. A final volume of approximately 200μΙ of blood was taken at 4 hours after administration.

In order to determine titres of phage in the bloodstream of the rat, 50μΙ heparinised blood was added to 10ml log phase TG1 E. coli bacteria and incubated at 37°C without shaking for 30 minutes, then with shaking at 200 rpm for 30 minutes. 10 ³, 10^~4 and 10^~5 dilutions of the infected culture were prepared to estimate the number of particles recovered; the remainder was then centrifuged at 4000 rpm for 10 minutes, supernatant was removed and the pellet resuspended in 300 μΙ 2x TY medium by vortex mixing. Bacteria were plated onto 2x TY agar plates supplemented with 2% glucose and 100 μg/ml ampicillin, and colonies allowed to grow overnight at 30°C. The remaining volume of blood was divided into 500ul aliquots and added to individual 10ml aliquots of log phase TG1 E. coli bacteria and the infection and recovery process repeated.

To recover infective phage particles from brain parenchyma, rats were euthanased at 4 hours after administration and brains were perfused with DMEM at 37°C via injection into the carotid artery (10ml per rat). Brains were then excised from rats and homogenised (using Bio Masher homogenizer, Omni International, 1000 Williams Drive, Suite 1024, Marietta, GA, USA) in 800μΙ sterile PBS. Homogenised material was centrifuged at 14,000g in a microfuge. Supernatant was then added to 10ml culture of mid log phase TG1 E.coli. The infection and recovery process described above was repeated. Individual colonies were picked into 1 ml 2x TY medium supplemented with 2% glucose and 100 μg/ml ampicillin and grown overnight at 37°C. Plasmid DNA was then prepared and sequences of individual cyclotide clones was determined via sequencing (examples of DNA sequences are shown in Table 1 , SEQ IDs: 037 to 059).

C. Blood-Brain Barrier (BBB) analysis of cyclotide fusions

(i) Expression of CYCLOTIDE-Single domain fusions

Cyclotide DNA sequences were cloned into a modified version of pSecTag2 (Invitrogen), containing the sequence for a β-galactosidase-binding single domain antibody with a C-terminal 6xHis and V5 tag (SEQ ID NO. 033) which was cloned into the Not1 and Xho1 sites in the vector. Cyclotide sequences were PCR amplified from pSP1 vector using oligonucleotide primers "Loop5PTrev" and "Cyc Bam for" (SEQ ID NOs 032 and 035). For each clone, a 50 μΙ reaction mixture was set up containing approximately 25 ng Cyclotide clone in pSP1 , 25 pmol of the appropriate forward and reverse primers, 0.1 mM dNTPs, 2.5 units Taq DNA polymerase, and 1x NEB PCR reaction buffer (20 mM Tris-HCI pH 8.8, 10 mM (NH₄)₂SO₄,10 mM KCI, 2 mM MgS0₄, 0.1 % Triton X-100; NEB Ltd, Cambridge, UK). Reactions were performed for 25 cycles of 94°°C, 20s; 58°C, 30s; 72°C, 30s, followed by 5 minutes at 72°C. Reaction products were purified using four Wizard PCR clean-up columns per library (Promega Ltd, Southampton, UK), and eluted into 100 μΙ water per column. Both pSecTag2 vector and purified products were then digested with enzymes BamH1 and Not\ (10 units each enzyme) for 90 minutes at 37°C (NEB, Cambridge, UK), and purified using one Wizard PCR clean-up column per clone or vector DNA (Promega Ltd, Southampton, UK). Each DNA sample was then eluted into 50 μΙ water. 9 μΙ of each digested Cyclotide clone DNA was ligated with 1 μΙ of BamH1-Not\ cut pSecTag2 vector and 40U of Quick Ligase (NEB Ltd, Southampton, UK) for 5 minutes at room temperature. After incubation the ligations were adjusted to 200 μΙ with nuclease free water, and DNA precipitated with 1 μΙ 20 mg/ml glycogen, 100 μΙ 7.5M ammonium acetate and 900 μΙ ice-cold (-20°C) absolute ethanol, vortex mixed and spun at 13,000 rpm for 20 minutes in a microfuge to pellet DNA. The pellets were washed with 500 μΙ ice-cold 70% ethanol by centrifugation at 13,000 rpm for 2 minutes, then vacuum dried and re-suspended in 100 μΙ DEPC-treated water. 1 μΙ aliquots of each library were electroporated into 80 μΙ E. coli (NEB5). Bacterial cells were grown in 1 ml SOC medium per cuvette for 1 hour at 37°C, and plated onto 2x TY agar plates supplemented with 100 μg/ml ampicillin. Plates were incubated over night at 37°C. Individual colonies were picked into 1 ml 2x TY medium supplemented with 100 μg/ml ampicillin and grown overnight at 37°C. Plasmid DNA was then prepared and sequences of individual cyclotide fusion clones were determined via sequencing.

(ii) Transient transfection of fusion DNA into HEK 293 EBNA cells

T75 flasks were seeded with15ml of HEK 293 EBNA cells at 8 x 10⁵ cells/ml in Optimem medium (Invitrogen) and incubated over night at 37°C in a humidified incubator supplemented with 5% C0₂. 180 μΙ of purified plasmid DNA at 1 .0mg/ml in sterile water was added to 600 μΙ of Optimem. 66 μΙ Lipofectamine 2000 (Invitrogen) was added to 660 μΙ Optimem, mixed briefly and incubated at room temperature for 5 minutes. The two solutions were then combined and incubated for a further 20 minutes at room temperature. Medium from T75 flasks was removed carefully and replaced with 15ml fresh Optimem. DNA/Lipofactamine/Optimem solution was added to the flasks, which were then placed in at 37°C in a humidified incubator supplemented with 5% C0₂. Medium was harvested after 7 days and His-tagged proteins were purified via immobilised Metal Anion Chromatography (IMAC).

(Hi) IMAC purification of cyclotide-Single domain fusions

Harvested medium was centrifuged at 4000 g to remove particulate matter. Imidazole was added to the supernatant to a final concentration of 10mM. 300μΙ of His-Select Nickel Affinity gel beads (Sigma) were added to approximately 30ml of medium and incubated overnight on a blood mixer. Beads were centrifuged briefly and supernatant was removed. Beads were washed with 1 ml of PBS supplemented with 10mM imidazole. Beads were centrifuged briefly and supernatant was removed. Beads were washed twice more. Bound proteins were then eluted via the addition of 0.5ml PBS supplemented with 250mM imidazole. Imidazole was removed via dialysis in 2L PBS (pH 7.4) overnight at room temperature, using Slidealyzer dialysis cassettes (10,000 mw cut-off) (Thermo Fisher Scientific Ltd, Unit 9 Atley Way, North Nelson Industrial Estate Cramlington, Northumberland, UK). (iv) In vitro BBB model cell assay

The in vitro model of BBB was established by using a coculture of BBCECs obtained from Cellial Technologies SA (Faculte Jean Perrin, Rue Jean Souvraz - BP133, 62303 Lens Cedex France) and new-born rat astrocytes as described previously (Culot M et al Toxicol In Vitro. Apr;22(3):799-81 1 , 2008). Ready to use co-culture plates were prepared according to the manufacturer's instructions: briefly, frozen plates were thawed at 37°C. BBB complete culture medium was then added to the lower compartment. Cells were resuspended in each well via pipetting. Cells were allowed to adhere to the membrane for 4 hours at 37°C, after which, cells were washed once with complete culture medium. Cells were then incubated in complete culture medium for 72 hours at 37°C. Complete culture medium was replaced with BBB inducing medium and cells were incubated for a further 72 hours at 37°C. Medium was removed from the upper chamber and replaced with 400 μΙ Ringer- Hepes buffer (RH buffer: NaCI, 150 mM ; KCI, 5.2mM ; CaCI2, 2.2 mM ; MgCI2, 0.2 mM ; NaHC03, 6 mM ; HEPES, 5 mM ; Glucose 2.8 mM. pH 7.2-7.4) containing Lucifer Yellow (Sigma) at 20μΜ and approximately 100ng of cyclotide-Single domain fusion. Upper chamber plate was then placed in a "receiving plate" containing 800 μΙ RH buffer per well. After 1 hour at 37°C, Upper chambers and receiving wells were separated for analysis. Lucifer Yellow concentrations were determined via fluorescence analysis (Exc 425nm - Em 538 nm) using a Spectra MAX Gemini XS fluorescent plate reader (Molecular Devices, Sunnyvale, California, USA). cyclotide-Single domain fusion fusion concentrations were determined via β-galactosidase binding ELISA. Briefly, β-galactosidase (SIGMA) was adsorbed to 96 well ELISA plates (Maxisorb, Nunc, Kamstrupvej 90, Postbox 280, DK-4000 Roskilde, Denmark) at 5μg/ml in PBS over night at 4°C. Plates were blocked for 1 hour 4% milk protein in PBS at room temperature and washed once with PBS. Supernatants from upper and lower chambers were added and incubated for 1 hour at room temperature. Plates were washed twice with PBS/0.1 % tween 20 (SIGMA) and twice with PBS. Horse radish peroxidase conjugated anti-6 x His tag (Ab1 187, Abeam, 330 Cambridge Science Park, Cambridge, CB4 0FL, UK), diluted to 1 :5000 in 2% milk protein: PBS/0.1 % tween 20 was added and incubated for 1 hour at room temperature. Plates were washed three times with PBS/0.1 % tween 20 and twice with PBS. Plates were developed for 5 minutes at room temperature with freshly prepared TMB (3, 3', 5,5'- Tetramethylbenzidine) substrate buffer (0.005% H₂0₂, 0.1 mg/ml TMB in 24 mM citric acid / 52 mM sodium phosphate buffer pH 5.2). The reaction was stopped with 12.5% H₂S0₄ and read at 450 nm.

(v) In vivo BBB analysis

To determine if cyclotide-Single domain fusion fusions were capable of traversing the BBB, selected cyclotide-Single domain fusion fusions chosen from the panel assayed in the in vitro cell assay were either Flourescein-labelled or unlabelled and injected into the tail vein of Hooded Lister rats. Brains from the sample rats were excised and sectioned for analysis under UV microscopy. Labelled material was observed directly, whereas unlabelled material was detected via Flourescein-labelled anti-V5 antibody. To Flourescein-label cyclotide-Single domain fusion fusions, NHS-Flourescein (Thermo Scientific, 3747 N. Meridian Rd, Rockford, IL, USA) was used according to the manufacturer's instructions. Briefly, approximately 1 mg of NHS-Flourescein was reconstituted in 10ΟμΙ of DMSO. 7μΙ of this solution was added to 1 ml of cyclotide-Single domain fusion fusion at 1 mg/ml. This solution was incubated at room temperature for 2 hours. Excess NHS-Flourescein was removed by applying a Zeba Spin desalting column (10k mwco, Thermo Scientific).

200μg labelled or unlabelled material was injected into Hooded Lister rats via the tail vein. After 4 hours, rats were euthanased and brains excised. Brains were frozen on dry ice. Ι Ομηι sections of frozen tissues were then placed onto microscope slides and allowed to air-dry for 30 minutes at room temperature. Slides were placed in methanol at -20°C for 10 minutes. Slides were air-dried for 1 hour at room temperature and washed twice with PBS. Tissues were blocked with 10% Foetal Bovine Serum (FBS) for 30 minutes at room temperature. If detection of unlabelled material was required, anti-V5-FITC (Invitrogen) was added at 1 :500 dilution in 10% FBS and incubated at room temperature for 1 hour. Slides were then washed five times with PBS. Vectashield mounting medium with DAPI (Vector Laboratories, 3 Accent Park, Bakewell Road, Peterborough, UK) was applied and sections were covered with a coverslip. Sections were then analysed using UV microscopy.

D. Blood-Brain Barrier (BBB) analysis of cyclotide fusions

(i) Expression of TNFR ll-Cyclotide fusions

Cyclotide C1 1 and C13 DNA sequences (SEQ ID No. 042 and 044) were cloned into a modified version of pSecTag2 (Invitrogen, 3 Fountain Drive, Inchinnan Business Park, Paisley PA4 9RF, UK), containing the sequence for human Tumour Necrosis Factor Receptor II extracellular domain (TNFR II), a V5 tag and a HIS tag (SEQ ID NO. 034) which was cloned into the BamH1 and Xho1 sites in the vector. Cyclotide C1 1 and C13 sequences were PCR amplified from pSP1 vector using oligonucleotide primers "Loop5PTrev" and "Cyc Eco For" (SEQ ID NOs 032 and 036). A 50 μΙ reaction mixture was set up containing approximately 25 ng Cyclotide C1 1 or C13 in pSP1 , 25 pmol of the appropriate forward and reverse primers, 0.1 mM dNTPs, 2.5 units Taq DNA polymerase, and 1x NEB PCR reaction buffer (20 mM Tris-HCI pH 8.8, 10 mM (NH₄)₂SO₄,10 mM KCI, 2 mM MgS0₄, 0.1 % Triton X-100; NEB Ltd, Cambridge, UK). Reactions were performed for 25 cycles of 94°°C, 20s; 58°C, 30s; 72°C, 30s, followed by 5 minutes at 72°C. Reaction products were purified using four Wizard PCR clean-up columns per library (Promega Ltd, Southampton, UK), and eluted into 100 μΙ water per column. Both pSecTag2 vector and purified products were then digested with enzymes EcoR1 and Not\ (10 units each enzyme) for 90 minutes at 37°C (NEB, Cambridge, UK), and purified using one Wizard PCR clean-up column per clone or vector DNA (Promega Ltd, Southampton, UK). Each DNA sample was then eluted into 50 μΙ water. 9 μΙ of each digested Cyclotide clone DNA was ligated with 1 μΙ of EcoR1-Not\ cut pSecTag2 vector and 40U of Quick Ligase (NEB Ltd, Southampton, UK) for 5 minutes at room temperature. After incubation the ligations were adjusted to 200 μΙ with nuclease free water, and DNA precipitated with 1 μΙ 20 mg/ml glycogen, 100 μΙ 7.5M ammonium acetate and 900 μΙ ice-cold (-20°C) absolute ethanol, vortex mixed and spun at 13,000 rpm for 20 minutes in a microfuge to pellet DNA. The pellets were washed with 500 μΙ ice-cold 70% ethanol by centrifugation at 13,000 rpm for 2 minutes, then vacuum dried and re-suspended in 100 μΙ DEPC-treated water. 1 μΙ aliquots of each library were electroporated into 80 μΙ E. coli (NEB5). Bacterial cells were grown in 1 ml SOC medium per cuvette for 1 hour at 37°C, and plated onto 2x TY agar plates supplemented with 100 μg/ml ampicillin. Plates were incubated over night at 37°C. Individual colonies were picked into 1 ml 2x TY medium supplemented with 100 μg/ml ampicillin and grown overnight at 37°C. Plasmid DNA was then prepared and sequences of individual cyclotide fusion clones were determined via sequencing.

(ii) Transient transfection of TNFR II control and TNFR ll-Cyclotide fusion DNA into HEK 293 EBNA cells

TNFR ll-Cyclotide fusion and TNFR II "No Cyclotide" control protein were expressed in HEK 293 EBNA cells as follows: T75 flasks were seeded withl 5ml of HEK 293 EBNA cells at 8 x 10⁵ cells/ml in Optimem medium (Invitrogen, 3 Fountain Drive, Inchinnan Business Park, Paisley PA4 9RF, UK) and incubated over night at 37°C in a humidified incubator supplemented with 5% C0₂. 180 μΙ of purified plasmid DNA at 1 .Omg/ml in sterile water was added to 600 μΙ of Optimem. 66 μΙ Lipofectamine 2000 (Invitrogen) was added to 660 μΙ Optimem, mixed briefly and incubated at room temperature for 5 minutes. The two solutions were then combined and incubated for a further 20 minutes at room temperature. Medium from T75 flasks was removed carefully and replaced with 15ml fresh Optimem. DNA/Lipofactamine/Optimem solution was added to the flasks, which were then placed in at 37°C in a humidified incubator supplemented with 5% C0₂. Medium was harvested after 7 days and His-tagged proteins were purified via immobilised Metal Anion Chromatography (IMAC).

(Hi) IMAC purification of TNFR II control and TNFR ll-Cyclotide fusions

Harvested medium was centrifuged at 4000 g to remove particulate matter. Imidazole was added to the supernatant to a final concentration of 10mM. 300μΙ of His-Select Nickel Affinity gel beads (Sigma) were added to approximately 30ml of medium and incubated overnight on a blood mixer. Beads were centrifuged briefly and supernatant was removed. Beads were washed with 1 ml of PBS supplemented with 10mM imidazole. Beads were centrifuged briefly and supernatant was removed. Beads were washed twice more. Bound proteins were then eluted via the addition of 0.5ml PBS supplemented with 250mM imidazole. Imidazole was removed via dialysis in 2L PBS (pH 7.4) overnight at room temperature, using Slidealyzer dialysis cassettes (10,000 mw cut-off) (Thermo Fisher Scientific Ltd, Unit 9 Atley Way, North Nelson Industrial Estate Cramlington, Northumberland, UK). (iv) In vivo BBB analysis

To determine if TNFR ll-Cyclotide fusions were capable of traversing the BBB, TNFR ll-Cyclotide fusions and TNFR II control were either Flourescein-labelled or unlabelled and injected into the tail vein of Hooded Lister rats. Brains from the sample rats were excised and sectioned for analysis. Flourescein-labelled material was observed directly via UV microscopy, whereas unlabelled material was detected via anti-V5-HRP conjugated antibody, using 3,3' Diaminobenzidine (DAB) as a substrate and hematoxylin (both Vector Laboratories, 3 Accent Park, Bakewell Road, Peterborough, UK) as a counter stain.

To flourescein-label sample proteins, NHS-Flourescein (Thermo Fisher Scientific Ltd, Unit 9 Atley Way, North Nelson Industrial Estate Cramlington, Northumberland, UK) was used according to the manufacturer's instructions. Briefly, approximately 1 mg of NHS-Flourescein was reconstituted in 10ΟμΙ of DMSO. 7μΙ of this solution was added to 1 ml of sample protein at 1 mg/ml. This solution was incubated at room temperature for 2 hours. Excess NHS-Flourescein was removed by applying a Zeba Spin desalting column (10k mwco, Thermo Fisher Scientific Ltd, Unit 9 Atley Way, North Nelson Industrial Estate Cramlington, Northumberland, UK).

200μg labelled or unlabelled material was injected into Hooded Lister rats via the tail vein. After 4 hours, rats were euthanased and brains excised. Brains were frozen on dry ice. Ι Ομηι sections of frozen tissues were then placed onto microscope slides and allowed to air-dry for 30 minutes at room temperature. Slides were placed in methanol at -20°C for 10 minutes. Slides were air-dried for 1 hour at room temperature and washed twice with PBS. Vectashield mounting medium with DAPI (Vector Laboratories, 3 Accent Park, Bakewell Road, Peterborough, UK) was applied and sections were covered with a coverslip. Sections were then analysed using UV microscopy.

If anti-V5 detection of unlabelled material was required, tissues were blocked with 10% Foetal Bovine Serum (FBS) for 30 minutes at room temperature. Anti-V5-HRP (Invitrogen, 3 Fountain Drive, Inchinnan Business Park, Paisley PA4 9RF, UK) was added at 1 :500 dilution in 10% FBS and incubated at room temperature for 1 hour. Slides were then washed three times with PBS/0.1 % tween and two times with PBS. Bound anti-V5-HRP was detected via incubation with DAB substrate. Precipitation was stopped by washing twice with PBS. Counterstaining was carried out by incubating for 40 seconds in hematoxylin. Slides were washed with water and then incubated in 75% Ethanol for 2 minutes. Slides were incubated for a further two minutes in 90% Ethanol and finally two minutes in 95% Ethanol. Slides were allowed to air dry for 1 hour at room temperature. Slides were then incubated in Xylene for 10 minutes at room temperature and allowed to air dry. DPX mounting medium (Sigma-Aldrich Company Ltd. The Old Brickyard, New Road, Gillingham, Dorset, SP8 4XT) was added to each slide before adding a coverslip and allowing to set over night. Sections were then observed via light microscopy. SEQ ID Name Sequence

001 MCOTI I

GGVCPKILQRCRRDSDCPGACICRGNGYCGSGSD

002 MCOTI II

GGVCPKILKKCRRDSDCPGACICRGNGYCGSGSD

003 Mcoti-III

ERACPRILKKCRRDSDCPGACICRGNGYCG

004 C15

GGVCNNNATPSLKVCRRDSDCPGACICRGNGYCGSGSD

005 C7

GGVCGMDLFEESPYCRRDSDCPGACICRGNGYCGSGSD

006 C8

GGVCWLRDEHPFKNCRRDSDCPGACICRSNGYCGSGSD

007 C9

GGVCTYWYLYHTKGCRRDSDCPGACICRGNGYCGSGSD

008 CIO

GGVCNLDVANWTVWCRRDSDCPGACICRGNGYCGSGSD

009 Cll

GGVCRHSYSQIPLWCRRDSDCPGACICRGNGYCGSGSD

010 C12

GGVCLELAKAYFQMCRRDSDCPGACICRGNGYCGSGSD

Oil C13

GGVCQQMHFRVMVHCRRDSDCPGACICRGNGYCGSGSD

012 C14

GGVCTHWRWRSTIWCRRDSDCPGACICRGNGYCGSGSD

013 C16

GGVCFVTDHWEHAPCRRDSDCPGACICRGNGYCGSGSD

014 C17

GGVCFDHHSHYIRRCRRDSDCPGACICRGNGYCGSGSD

015 C18

GGVCQWWLHMINAVCRRDSDCPGACICRGNGYCGSGSD

016 C19

GGVCPFLPTEWWNSCRRDSDCPGACICRGNGYCGSGSD

017 C20

GGVCVRKWWYTESICRRDSDCPGACICRGNGYCGSGSD

018 C21

GGVCYDDETPPHETQHCRRDSDCPGACICRGNGYCGSGSD

019 C22

GGVCQRRKWYWKESIQCRRDSDCPGACICRGNGYCGSGSD

020 C23

GGVCQYTKPFVKGPHHCRRDSDCPGACICRGNGYCGSGSD

021 C24

GGVCSKKRKMSSWHPCRRDSDCPGACICRGNGYCGSGSD

022 C25

GGVCEVYVWNGELKAWCRRDSDCPGACICRGNGYCGSGSD

023 C26

GGVCRFQQGKWWEPHQCRRDSDCPGACICRGNGYCGSGSD

024 C27

GGVCHMQHPWSAFAWYCHRDSDCPGACICRGNGYCGSGSD

025 C28

GGVCESDPFTEEFMHHCRRDSDCPGACICRGNGYCGSGSD

026 C29

GGVCHKHGYDPVYVWSCRRDSDCPGACICRGNGYYGSGSD

027 MCOTI II native sequence

ATGGGTGGTGTTTGTCCTAAAATTCTCAAAAAATGTCGTCGTGATTCTGATTGTCCTGGAGCGTG TATTTGTCGTGGTAATGGTTATTGCGGTTCTGGTTCTGATGCGGCCGCCGAG

028 LOOPIFOR

ATG GCC GGT GGT GTT TGT (TRN) 10 TGT CGT CGT GAT TCT GAT TG

045 CI4 DNA Sequence

GGTGGTGTTTGTACTCATTGGCGTTGGCGTTCTACTATCTGGTGTCGTCGTGATTCTGATTGTCC TGGAGCGTGTATTTGTCGTGGTAATGGTTATTGCGGTTCTGGTTCTGAT

046 CI6 DNA Sequence

GGTGGTGTTTGTTTCGTTACTGACCATTGGGAACATGCTCCGTGTCGTCGTGATTCTGATTGTCC TGGAGCGTGTATTTGTCGTGTAAATGGTTATTGCGGTTCTGGTTCTGAT

047 C17 DNA Sequence

GGTGGTGTTTGTTTCGACCATCATTCTCATTACATCCGTCGTTGTCGTCGTGATTCTGATTGTCC TGGAGCGTGTATTTGTCGTGGTAATGGTTATTGCGGTTCTGGTTCTGAT

048 C18 DNA Sequence

GGTGGTGTTTGTCAGTGGTGGCTGCATATGATCAACGCTGTTTGTCGTCCTGATTCTGATTGTCC TGGAGCGTGTATTTGTCCTGGTAATGGTTATTGCGGTTCTGGTTCTGAT

049 CI9 DNA Sequence

GGTGGTGTTTGTCCGTTCCTGCCGACTGAATGGTGGAACTCTTGTCGTCGTGATTCTGATTGTCC TGGAGCGTGTATTTGTCGTGGTAATGGTTATTGCGGTTCTGGTTCTGAT

050 C20 DNA Sequence

GGTGGTGTTTGTGTTCGTAAATGGTGGTACACTGAATCTATCTGTCGTCGTGATTCTGATTGTCC TGGAGCGTGTATTTGTCGTGGTAATGGTTATTGCGGTTCTGGTTCTGAT

051 C21 DNA Sequence

GGTGGTGTTTGTTACGACGACGAAACTCCGCCGCATGAAACTCAGCATTGTCGTCGTGATTCTGA TTGTCCTGGAGCGTGTATTTGTCGTGGTAATGGTTATTGCGGTTCTGGTTCTGAT

052 C22 DNA Sequence

GGTGGTGTTTGTCAGCGTCGTAAATGGTACTGGAAAGAATCTATCCAGTGTCGTCGTGATTCTGA TTGTCCTGGAGCGTGTATTTGTCGTGGTAATGGTTATTGCGGTTCTGGTTCTGAT

053 C23 DNA Sequence

GGTGGTGTTTGTCAGTACACTAAACCGTTCGTTAAAGGTCCGCATCATTGTCGTCGTGATTCTGA TTGTCCTGGAGCGTGTATTTGTCGTGGTAATGGTTATTGCGGTTCTGGTTCTGAT

054 C24 DNA Sequence

GGTGGTGTTTGTTCTAAAAAACGTAAAATGTCTTCTGTTGTTCATCCGTGTCGTCGTGATTCTGA TTGTCCTGGAGCGTGTATTTGTCGTGGTAATGGTTATTGCGGTTCTGGTTCTGAT

055 C25 DNA Sequence

GGTGGTGTTTGTGAAGTTTACGTTTGGAACGGTGAACTGAAAGCTTGGTGTCGTCGTGATTCTGA TTGTCCTGGAGCGTGTATTTGTCGTGGTAATGGTTATTGCGGTTCTGGTTCTGAT

056 C26 DNA Sequence

GGTGGTGTTTGTCGTTTCCAGCAGGGTAAATGGTGGGAACCGCATCAGTGTCGTCGTGATTCTGA TTGTCCTGGAGCGTGTATTTGTCGTGGTAATGGTTATTGCGGTTCTGGTTCTGAT

057 C27 DNA Sequence

GGTGGTGTTTGTCATATGCAGCATCCGTGGTCTGCTTTCGCTTGGTACTGTCATCGTGATTCTGA TTGTCCTGGAGCGTGTATTTGTCGTGGTAATGGTTATTGCGGTTCTGGTTCTGAT

058 C28 DNA Sequence

GGTGGTGTTTGTGAATCTGACCCGTTCACTGAAGAATTCATGCATCATTGTCGTCGTGATTCTGA TTGTCCTGGAGCGTGTATTTGTCGTGGTAATGGTTATTGCGGTTCTGGTTCTGAT

059 C29 DNA Sequence

GGTGGTGTTTGTCATAAACATGGTTACGACCCGGTTTACGTTTGGTCTTGTCGTCGTGATTCTGA TTGTCCTGGAGCGTGTATTTGTCGTGGTAATGGTTATTACGGTTCTGGTTCTGAT

Table 1 Amino acid and nucleic acid sequences of selected Cyclotides

Claims

CLAIMS:

1 . A non-naturally occurring linear or cyclic peptide having a cysteine knot backbone moiety and a defined blood-brain barrier translocation moiety, said peptide molecule comprising: i) a peptide having said blood-brain barrier translocation activity, wherein said peptide has an amino acid sequence comprising a plurality of contiguous amino acids, wherein said peptide is about 6 to 50 amino acid residues; and ii) a cysteine knot backbone grafted to said peptide of step i), wherein said cysteine knot backbone comprises the structure (I):

(X^ ... X^d (X, ... Xa)C₂(X'₁... X'_b)C₃(X"i ... Χ"ο)θ4(Χ"Ί ... X'"_d)C₅(X^lv, ... X^IVe)C₆(X^Vi ... X^v _f)

Loop6 Loopl Loop2 Loop3 Loop4 Loop5 Loop6 wherein C.sub.1 to C.sub.6 are cysteine residues; wherein each of C.sub.1 and C.sub.4, C.sub.2 and C.sub.5, and C.sub.3 and C.sub.6 are connected by a disulfide bond to form a cysteine knot; wherein each X represents an amino acid residue in a loop, wherein said amino acid residues may be the same or different; wherein d is about 1 -2; wherein one or more of loops 1 , 2, 3, 5 or 6 have an amino acid sequence comprising the sequence of said peptide of step i), wherein any loop comprising said sequence of said peptide of step i) comprises 2 to about 30 amino acids, and wherein for any of loops 1 , 2, 3, 5, or 6 that do not contain said sequence of said peptide of step i), a, b, c, e, and f, may be the same or different, and may be any number from 1 to 20.

2. A peptide of claim 1 , wherein for any of loops 1 , 2, 3, 5, or 6 that do not contain said sequence of said peptide of step i), a, b, c, e, and f, may be the same or different, and may be any number from 3 to 10.

3. A peptide of claim 1 which is derived from a cyclic cysteine knot backbone selected from SEQ ID 001 , SEQ ID 002 or SEQ ID 003.

4. A peptide of claim 1 selected from any one of SEQ ID 004, SEQ ID 005, SEQ ID 006, SEQ ID 007, SEQ ID 008, SEQ ID 009, SEQ ID 010, SEQ ID 01 1 , SEQ ID 012, SEQ ID 013, SEQ ID 014, SEQ ID 015, SEQ ID 016, SEQ ID 017, SEQ ID 018, SEQ ID 019, SEQ ID 020, SEQ ID 021 , SEQ ID 022, SEQ ID 023, SEQ ID 024, SEQ ID 025, SEQ ID 026.

5. A conjugate comprising; a) a carrier selected from the group consisting of the non-naturally occurring linear or cyclic peptide of claim 1 , and; b) a therapeutically active agent.

6. A conjugate of claim 5 where the therapeutically active agent is selected from the group consisting of an antibody or an antibody fragment thereof.

7. A conjugate of claim 5 where the therapeutically active agent is selected from the group consisting of a growth factor or hormone.

8. A method for transporting a therapeutically active agent across a blood brain barrier of an individual, the method comprising administering the conjugate of claim 5 in a mammal in need thereof.

9. The method of claim 8, wherein the mammal has a neurological disease.

10. The method as defined in claim 9, wherein said neurological disease is selected from the group consisting of Alzheimer's, Parkinson's disease, a brain tumor and a brain metastasis.

1 1 . A method for treating a patient having a neurological disease comprising administering the conjugate of claim 5 to said patient.

12. A method for diagnosing a neurological disease in a patient in need thereof comprising administering the conjugate of claim 5 to said patient and wherein said conjugate comprises a radiolabel.

13. A pharmaceutical composition comprising a) the conjugate of claim 5 and; b) a pharmaceutically acceptable carrier.

14. The pharmaceutical composition of claim 13, wherein said pharmaceutical composition is used for the treatment of a neurological disease.

15. The pharmaceutical composition of claim 13, wherein said pharmaceutical composition is used for the diagnosis of a neurological disease.

16. A nucleotide sequence encoding the any one of the peptides of claim 4.

17. A method for identifying a compound of Claim 1 comprising the steps of:

a) constructing a variegated cyclotide sequence display library;

b) expressing the sequence library in order to obtain expressed peptides, or polypeptides that comprise the one or more compounds of structure (I);

c) administering the expressed peptides or polypeptides that comprise the compounds of structure (I) to an animal, optionally via the oral route;

d) recovering any compounds of structure (I) from the blood, lymph and/or tissues of the animal;

e) determining the sequence of the recovered compound of structure (I).