WO2012073045A2

WO2012073045A2 - Polypeptide scaffold

Info

Publication number: WO2012073045A2
Application number: PCT/GB2011/052394
Authority: WO
Inventors: Duncan Mcgregor; William Eldridge; Simon Robins; Marie Fernie; Tricia White; Stuart Pritchard; Susan King
Original assignee: Cyclogenix Ltd
Priority date: 2010-12-03
Filing date: 2011-12-02
Publication date: 2012-06-07
Also published as: WO2012073045A3

Abstract

A modified polypeptide is described comprising a whey acidic protein (WAP) domain, suitably a trappin WAP domain. The WAP domain is modified by insertion, deletion or substitution of at least one amino acid residue at a position in the WAP domain selected from the region between the first cysteine residue and the second cysteine residue (loop 1) in the polypeptide sequence and/or the region between the second cysteine residue and the third cysteine residue (loop 2) in the polypeptide sequence. Methods of generating the modified polypeptides and their use in pharmaceutical compositions are also described.

Description

POLYPEPTIDE SCAFFOLD

FIELD

The invention relates to novel polypeptide scaffolds for use in the display of libraries of peptides, particularly peptide aptamers. In addition the invention provides methods for the construction of protein scaffolds and their use in the production of chimaeric polypeptides with therapeutic and commercial utility.

BACKGROUND

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. These 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. Novel methods of purification are readily applied; they are easily conjugated to drugs/toxins, penetrate efficiently into tissues and are readily formatted into mono- or multi-specific binders (Skerra et al 2000, J Mol Recognit. 2000; 13:409-410; Binz and Pluckthun 2005, Nat Biotechnol. 2005; 23(10):1257- 1268).

Desirable physical properties of potential alternative scaffold molecules include high thermal stability and reversibility of thermal folding and unfolding. Several methods have been applied to increase the apparent thermal stability of proteins and enzymes, including rational design based on comparison to highly similar thermostable sequences, design of stabilizing disulfide bridges, mutations to increase [alpha]-helix propensity, engineering of salt bridges, alteration of the surface charge of the protein, directed evolution, and composition of consensus sequences (Lehmann and Wyss, 2001 ,CurrOpinBiotechnol. 12: 371-375).

High thermal stability is a desired property of such scaffolds as it may increase the yield of recombinant protein obtained, and improve solubility of the purified molecule. It is well known that highly structured naturally occurring disulphide-rich microproteins such as knottins or cyclotides, containing 2-3 disulphide bonds possess these properties (Colgrave and Craik, Biochemistry 2004, 43, 5965-5975). Nevertheless, there is a continuing need for new scaffold polypeptides that show particularly high levels of thermal stability and resistance to degradation.

It is an object of the present invention to overcome the deficiencies noted in the prior art and to provide alternative and improved protein scaffolds.

SUMMARY

The present invention is based upon the identification of a novel scaffold platform based on the whey acidic protein domain family (WAP). Scaffolds based upon the WAP domain derived from one or more of this family of proteins have been found by the present inventors to exhibit many of the properties desirable in an alternative scaffold protein. In general terms, the present invention provides a modified, stabilised WAP domain framework or scaffold, which can be used, inter alia, for the selection of de novo binding domains having desired binding characteristics, such as affinity for new target molecules and/or high affinity for known or new ligands.

Accordingly, in a first aspect the invention provides a modified polypeptide comprising a whey acidic protein (WAP) domain, wherein the WAP domain is modified by insertion, deletion or substitution of at least one amino acid residue at a position in the WAP domain selected from the group consisting of: the region between the first cysteine residue and the second cysteine residue (loop 1 ) in the polypeptide sequence; the region between the second cysteine residue and the third cysteine residue (loop 2) in the polypeptide sequence; and the region between the first cysteine residue and the second cysteine residue (loop 1 ) in the polypeptide sequence, and the region between the second cysteine residue and the third cysteine residue (loop 2) in the polypeptide sequence.

A second aspect of the invention provides a modified polypeptide comprising a WAP domain, wherein the WAP domain is modified by insertion, deletion or substitution of at least one amino acid residue at a position in the WAP domain between the first cysteine residue and the second cysteine residue (loop 1 ) in the polypeptide sequence.

A third aspect of the invention provides a modified polypeptide comprising a WAP domain, wherein the WAP domain is modified by insertion, deletion or substitution of at least two amino acid residues at a position in the WAP domain between the second cysteine residue and the third cysteine residue (loop 2) in the polypeptide sequence.

A fourth aspect of the invention provides a protein scaffold comprising a framework substantially based upon the polypeptide sequence of a WAP domain, wherein the region located between the first cysteine residue and the second cysteine residue (loop 1 ) in the polypeptide sequence is replaced with a heterologous sequence of between 4 and 18 amino acid residues, optionally between 6 and 12 amino acid residues. A fifth aspect of the invention provides a modified polypeptide scaffold of general formula I:

A - [X]n - C - [Χ']η' - B I wherein

A is a polypeptide chain comprising Thr-Lys-Pro-Gly-Ser-Cys,

X and X' are oligopeptide sequences of n residues and n' residues in length respectively comprising any amino acid residue and X and X' may be the same or different,

n is at least 1 and at most 18,

n' is at least 1 and at most 18,

C is a Cys residue, and

B is a polypeptide chain comprising the sequence of SEQ ID NO: 8 with the proviso that the modified polypeptide scaffold does not comprise the sequence of wild type elafin or of a modified elafin comprising the wild type sequence and point mutation resulting in a substitution of the wild type Met residue at position 25 to an Iso, Val or Leu residue.

A sixth aspect of the invention provides a modified polypeptide scaffold of general formula II:

A - [X]n - D wherein

A is a polypeptide chain comprising Thr-Lys-Pro-Gly-Ser-Cys, X is an oligopeptide sequence of n residues in length comprising a sequence of any amino acid residue,

n is at least 1 and at most 18, and

D is a polypeptide chain comprising the sequence of SEQ ID NO: 9 with the proviso that the modified polypeptide scaffold does not comprise the sequence of wild type elafin.

A seventh aspect of the invention provides a modified polypeptide scaffold of general formula III:

E - [Χ']η' - B III wherein

E is a polypeptide chain comprising the sequence of SEQ ID NO: 10,

X' is an oligopeptide sequence of n' residues in length comprising a sequence of any amino acid residue,

n' is at least 1 and at most 18, and

B is a polypeptide chain comprising the sequence of SEQ ID NO: 8 with the proviso that the modified polypeptide scaffold does not comprise the sequence of wild type elafin or of a modified elafin comprising the wild type sequence and a point mutation resulting in a substitution of the wild type Met residue at position 25 to an Iso, Val or Leu residue.

A eighth aspect of the invention provides for nucleic acid sequences that encode a modified polypeptide scaffold as defined in any of general formulas I, II or III.

An ninth aspect of the invention provides a nucleic acid vector comprising a nucleic acid sequence that encodes a modified polypeptide scaffold as defined in any of general formulas I, II or III in operative combination with a promoter sequence. A tenth aspect of the invention provides for a method of constructing a genetic library that comprises a multiplicity of chimaeric modified WAP domain-containing polypeptides, said method comprising: a) obtaining a template nucleic acid sequence that encodes a WAP domain polypeptide when expressed; b) identifying one or more loop regions within the WAP domain and generating a plurality of sequences that correspond to the one or more loop regions and that are capable of replacing the one or more identified loop regions with a randomized nucleic acid sequence;

c) replacing the one or more loop regions within the WAP domain template nucleic acid with the plurality of sequences of (b) so as to generate a plurality of recombinant chimaeric WAP domain nucleic acid sequences;

d) isolating the plurality of recombinant chimaeric WAP domain nucleic acid sequences; e) inserting the recombinant sequences of (d) into a plurality of nucleic acid expression vectors;

f) transforming host cells with the vectors of step (e); and

g) culturing the host cells of step (f) under conditions suitable for expression of said chimaeric proteins.

Optionally, the expression vector may be a display system vector (such as those described above), in which case step (g) would result in the construction of a peptide display library.

An eleventh aspect of the invention provides for a method for identifying a polypeptide, wherein said polypeptide comprises a modified WAP scaffold polypeptide, the method comprising

(i) constructing a genetic library according to the methods described herein; and

(ii) screening the library to identify the polypeptide.

The method may be used to screen a library of the invention in order to identify polypeptides that exhibit at least one property - optionally more than one - selected from the group consisting of: antibody binding capacity; antigen binding capacity; therapeutic activity; enzymic catalytic activity; enzymic inhibitory activity; receptor binding capacity; substrate binding capacity; and membrane translocation activity.

A twelfth aspect of the invention provides a method for preparing a pharmaceutical composition comprising identifying a polypeptide as described by the methods described herein, and combining the polypeptide so identified with a suitable pharmaceutical excipient.

DRAWINGS

The invention is further illustrated in the accompanying drawings in which:

Figure 1 shows a schematic of the elafin WAP domain sequence loop structure, showing Loop 1 and 2, with the elastase inhibitory P1 position in Loop 2 indicated. Figure 2 shows the parental elafin scaffold sequence synthesized by GeneArt. The elafin DNA sequence is underlined and italicized.

Figure 3 shows a pSPI phagemid vector multiple cloning site. DNA can be cloned as Ncol-Notl fragments, in-frame with full-length pill, separated by a short linker and a supE TAG codon. The pelB leader and beginning of the pill gene are indicated.

Figure 4 shows photographs of blood-brain transfer of Elafin#91 or GLP-1 (7-36). Four hour tissue sections of brain cortex showing the presence of (a) fluorescein labelled GLP-1 (7-36) peptide; and (b) fluorescein labelled Elafin#91.

Figure 5 shows photographs of blood-brain transfer of orally administered Elafin#91 or GLP-1 (7-36). Four hour tissue sections of brain cortex showing (a) the absence of any fluorescein labelled GLP-1 (7-36) peptide; and (b) the presence of fluorescein labelled Elafin#91.

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 "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 WAP domain polypeptides, in which the diversity or variance between library members is located in the polypeptide sequences of one or more of the loop regions of the WAP domain 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 macromolecule comprising one or more polypeptide chains.

A "domain" as referred to herein, is a tertiary polypeptide structure independent of the rest of a protein to which the domain may be linked, either covalently or otherwise. Domains can be responsible for specific functional properties of proteins or protein complexes, and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein or of the domain itself.

Whey acidic protein domain (WAP) was first characterised in relation to the whey acidic protein, which is the most abundant protein in rodent milk. The WAP domain contains a distinct four- disulphide core structure and is found in a wide variety of proteins that include whey proteins, proteinase inhibitors, neurophysins, plant agglutinin, adhesion molecules, scorpion toxins, bactericidal peptides and pollen proteins. Human members of the WAP domain family include whey acidic protein, elafin (elastase-specific inhibitor), caltrin-like protein (a calcium transport inhibitor), eppin, and other extracellular proteinase inhibitors. The disulphide-bonding pattern has been resolved via X-ray crystallography in relation to members of the trappins (Grutter et al. EMBO J. (1988) 7: 345-351 ; Tsunemi et al. J. Mol. Biol. (1993) 232: 310-11 ; Tsunemi et al. Biochemistry (1996) 35: 11570-6). Particular WAP domain containing proteins that are suited to modification according to the present invention include the Trappin family of proteins. Trappin (TRansglutaminase substrate and wAP domain containing Protein) proteins contain the characteristic WAP domain including a four-disulfide bond core peptide in the C-terminus of the protein (Schalkwijk et al. Biochem J. (1999) 340: 569-77). Trappins are protease inhibitors and it is the WAP domain which occupies the C-terminus that is thought to contain the protein's anti- protease active site.

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 fragment(s) 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 [beta]-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- 1a promoter) hosts are available. Expression vectors are often derived from plasm ids, cosmids, viral vectors and yeast artificial chromosomes; vectors are often recombinant molecules containing DNA sequences from several sources. Specific embodiments of the present invention provide for an expression vector that encodes a modified WAP domain protein, typically a WAP domain protein modified by the insertion of a randomised sequence in one of the loop regions. In one embodiment of the present invention the vector is suitable as a polypeptide library display vector, enabling the polypeptide gene product of the modified WAP domain gene to remain associated with the vector following transcription.

The term "operably linked", when applied to DNA sequences, for example in an nucleic acid vector, 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 an associated coding sequence as far as a termination sequence.

As described above the vectors of the invention may be linked to a polypeptide display system. As used herein, the term "display system" 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.).

In a specific embodiment of the invention the WAP domain DNA library is a phage display library. Phage display is based on DNA libraries fused to the N-terminal end of filamentous bacteriophage coat proteins and their expression in a bacterial host resulting in the display of foreign peptides on the surface of the phage particle with the DNA encoding the fusion protein packaged in the phage particle (Smith G. P., 1985, Science 228: 315-1317). Libraries of fusion proteins incorporated into phage, can then be selected for binding members against targets of interest (ligands). Bound phage can then be allowed to re-infect Escherichia coli (E. coli) bacteria and then amplified and the selection repeated, resulting in the enrichment of binding members (Parmley, S. F., & Smith, G. P. 1988., Gene 73: 305-318; Barrett R. W. et al., 1992, Analytical Biochemistry 204: 357-364 Williamson et al., Proc. Natl. Acad. Sci. USA, 90: 4141- 4145; Marks et al., 1991 , J. Mol. Biol. 222: 581 -597).

In another embodiment, the WAP domain library is a Lacl fusion library. Lacl fusion plasmid display is based on the DNA binding ability of the lac repressor. Libraries of random peptides are fused to the C-terminal end of the lacl repressor protein. Linkage of the Lacl-peptide fusion to its encoding DNA occurs via the lacO sequences on the plasmid, forming a stable peptide- Lacl-peptide complex. These complexes are released from their host bacteria by cell lysis, and peptides of interest isolated by affinity purification on an immobilised receptor target. The plasmids thus isolated can then be reintroduced into E. coli by electroporation to amplify the selected population for additional rounds of screening (Cull, M. G. et al. 1992. Proc. Natl. Acad. Sci. U.S.A. 89:1865-1869).

In further embodiments the WAP domain is expressed in in vitro display systems such as ribosome display. An entirely in vitro ribosome system has been described based on the linkage of peptides to the NA encoding them through the ribosome. Ribosome display has also been used for the selection of single-chain Fv antibody fragments (scFv) (Matheakis, L. C. et al., 1994 Proc. Natl. Acad. Sci. USA, 91 : 9022-9026; Hanes, J. &Pluckthun, A. 1997 Proc. Natl. Acad. Sci. USA, 94: 4937-4942).

Once selected and isolated from a display library of modified WAP domain polypeptides, any given modified WAP domain polypeptide of the present invention can be produced in recombinant host cells according to conventional techniques (Sambrook J. et al, Molecular Cloning: a Laboratory Manual, (2001 ) 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. A specified modified WAP domain polypeptide of the present invention shows particular utility in a variety of fields including novel biological therapeutics The polypeptides of the present invention can be used to identify other proteins and polypeptides that interact with the modified WAP domain protein in the cellular environment. Conventional techniques for determining protein-protein interactions, such as the yeast two- hybrid screen, can be used to identify potential binding partners. Alternative protein-protein interactions or protein-small molecule interactions can be investigated using technologies such as a BIAcore^® which detects molecular interactions using surface plasmon resonance (BIAcore, Inc., Piscataway, NJ; see also www.biacore.com).

In a specific embodiment of the invention, the WAP domain polypeptide that has been modified according to the methods of the present invention is human elafin (see SEQ ID NO: 16). Elafin (trappin-2, SKALP, ESI) is a serine protease inhibitor and a member of the trappin family of WAP domain-containing proteins. This protein was isolated initially in a variety of settings around the same time, hence, the diverse nomenclature. It is has subsequently been defined as a member of the Trappin family of genes for which there is only one form in humans, trappin-2. In addition, the N-terminus of Trappin-2 contains a transglutaminase substrate domain (residues 23-60) composed of a repeating consensus sequence (Gly-Gln-Asp-Pro-Val-Lys) GQDPVK. This domain is thought to confer upon Trappin-2 an ability to form polymers and mediate interactions with the extracellular matrix. Trappin-2 is a secreted protein found primarily at mucosal surfaces and is thought to be a potent tissue-bound inhibitor of inflammation, responsible for maintaining the epithelial integrity. The protein is 117 amino acids in length, which includes a 22 amino acid hydrophobic signal peptide. In its full-length form, the protein is 12.3 kDa in size. Cleavage of the signal peptide yields a 9.9 kDa mature form of the protein. A further cleaved product is the 6 kDa form which includes the WAP domain. The elafin WAP domain has been defined as the C-terminal 57 amino acids of full-length elafin residing between Ala 1 and Gin 57, with protease inhibition occuring by insertion of the elafin WAP domain inhibitory loop (CAMLNPPNRC) into the active site pocket and interference with the catalytic residues of the protease (Tsunemi et al. Biochemistry 1996, 35, 11570-11576; Schalwijketal. Biochem.J. 1999, 340: 569-577; Kato et al. BMC Evolutionary Biology 2010, 10: 1471-2148).

Wild type Trappin-2, although not normally expressed in the epidermis of skin, is expressed in inflammatory conditions such as psoriasis (Wiedow et al. J. Biol. Chem (1990) 265 (25): 14791- 95). Other sites of expression include the oesophagus, pharynx, vagina, and oral epithelium. Specifically, production is thought to occur in stratified epithelial tissues, although some evidence exists for production by macrophages as well (in lung tissue). It has also been found in sputum and broncho-alveolar lavage fluid. Trappin-2 has been found to be a potent and specific inhibitor of a restricted set of proteases, specifically leukocyte elastase and leukocyte proteinase-3, both derived from neutrophils. In addition, it is a substrate for transglutaminases which mediate the covalent binding to extracellular matrix proteins. The gene for Trappin-2 is approximately 2.3 kb long and is composed of three exons and two introns. Due to its robust nature (small size, resistance to extreme pH, heat and oxidation), it has been tested as an anti- inflammatory agent for a number of conditions including lung emphysema, cystic fibrosis, reperfusion injury from myocardial infarction and resistance and susceptibility to HIV infection.

The scaffolds of the invention 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 other non-trappin members of the WAP domain family.

The WAP domain polypeptides of the invention are modified typically by insertion, deletion or substitution of a region within the polypeptide sequence that does not contribute to structural integrity of the domain. By "structural integrity" it is meant that the characteristic tertiary structure of the WAP domain, including the distinct four-disulphide core, is not disrupted by virtue of the modifications made to the polypeptide. Hence, DNA encoding a particular WAP domain can be used as a template to build a library that inserts, replaces or randomises one or more WAP domain loop while maintaining the cysteine residues that define the WAP domain. In accordance with embodiments of the present invention, modifications may be made within one or both of the two loop regions present in WAP domain. The loop regions are surface exposed and have been found by the present inventors to be particularly suited to mutation or complete replacement with sequences, allowing display of a plurality of potential peptide sequences. The first loop sequence is characterised as the region of the polypeptide sequence that is located between the first cysteine residue and the second cysteine residue. In the elafin polypeptide sequence this is between Cys₁₆ and Cys₂3. The second loop sequence is characterised as the region of the polypeptide sequence that is located between the second cysteine residue and the third cysteine residue. In the elafin polypeptide sequence this is between Cys₂3 and Cys₃₂. The amino acid residues at each of the mutated positions may be non-selectively randomized, i.e. by replacing each of the specified amino acids with one of the other 19 naturally occurring amino acids; or may be selectively randomized, i.e. by replacing each of the specified amino acids with one from a defined sub-group of the remaining 19 naturally occurring amino acids. It will be appreciated that one convenient way of creating a library of mutant peptides with randomized amino acids at each selected location, is to randomise the nucleic acid codon of the corresponding nucleic acid sequence that encodes the selected amino acid. In this case, in any individual peptide expressed from the library, any of the 20 naturally occurring amino acids may be incorporated at the randomised position. Therefore, in some instances (e.g. approximately 5%), the wild-type amino acid residue may be 'randomly' incorporated by chance.

Typically the amino acid residues present in the loop regions of the WAP domain polypeptide will be subject to mutation by way of insertion, deletion or substitution with a sequence typically between 1 and 18 amino acid residues in length. It is optional that the replacement sequence is longer than 18 amino acid residues in length. In specific embodiments of the in invention the sequence is between 1 and 16 amino residues in length, more suitably between 2 and 14 amino acid residues in length, or even around 6, 8, 10 or 12 amino acid residues in length.

A further advantage of an elafin WAP domain as a scaffold is that it has been subject to accelerated evolution and that the most likely selective forces for the accelerated evolution are extrinsic proteinases produced by invasive microorganisms (Tamechika et al. 1996, J. Biol Chem, 271 : 7012-7018). This resistance to proteolytic attack makes the elafin scaffold useful for designing or selecting novel microproteins capable of (a) surviving in the intestinal tract of a human, and (b) crossing mucosal surfaces through interaction with specific epithelial receptors. Thus the elafin WAP domain scaffold library is likely to be an excellent source for deriving orally available microprotein sequences. Elafin's ability to block enzyme activity and its lack of toxicity in humans makes it an even more promising candidate as a scaffold for building new biological activities by replacing the native elastase inhibition within loop 2 (Quinn et al. 2010. Respiratory Medicine Journal, 4, 20-31 ). The modified WAP domain polypeptides of the invention are characterised as chimaeric proteins that comprise the underling tertiary structure characteristic of a WAP domain protein but with novel sequences comprised within the loop regions. In this way, the underling tertiary structure is defined as a "scaffold" suitable for the display of peptide libraries within the loop regions. The peptides displayed in the loop regions may have value in diverse areas including therapeutics, diagnostics, and industrial biochemistry. By way of example, the modified WAP domain polypeptides of the invention may be modified within one or both of the loop regions to include a peptide sequence that is an antigen. By "antigen" we denote a molecule that triggers an immune response. An antigen may be in the form of an oligopeptide sequence that bears specific epitopes that allow antibodies raised against such fragments to also bind to the full- length wild-type polypeptide. Substructures of antigens are generally referred to as "epitopes" (e.g. B-cell epitopes, T-cell epitopes), as long as they are immunologically relevant, i.e. are also recognisable by natural or monoclonal antibodies. Hence, the described oligopeptide sequences may be comprised within a library of sequences inserted into the scaffold WAP protein of the invention. In this way the modified scaffold protein of the invention may function as a vaccine component. The structure of the modified WAP domain proteins of the invention is particularly stable under extremes of temperature and pH, making them highly suitable for use in vaccine compositions.

In a further embodiment of the invention, the modified WAP domain polypeptide scaffold functions as a delivery vector or structural support for a therapeutic peptide. Peptides with particular therapeutic properties, such as receptor antagonist activity, may be inserted into the loop regions of a modified WAP domain polypeptide in order to form a chimaeric protein. Alternatively, a therapeutic agent my be attached or conjugated via another linkage point (with or without an intervening spacer group) on the scaffold, including at the N or C terminus. These modified therapeutic polypeptides are resistant to degradation in vivo and can be further targeted to particular sites within the body by optional addition of further targeting or membrane translocation sequences to free N or C termini of the modified WAP domain polypeptide. A modified polypeptide of the invention may be conjugated 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.

In a further embodiment of the invention, the modified WAP domain polypeptide provides an alternative scaffold framework for an engineered non-antibody protein. In this way the first and/or second loops of the WAP domain can be altered to define antigen-binding regions akin to those of the F_ab domain of an antibody. The antigen refers to a molecular structure which may completely make up a specific binding partner or be part of a specific binding partner to a binding-region of the modified WAP domain of the present invention. In a specific embodiment of the invention, a display system can be used to generate a library of modified WAP domain proteins in which either or both of the loop regions are randomized and optionally either increased in size or reduced in size (i.e. by varying the number of amino-acid residues in the respective loops) without substantially altering the core tertiary structure of the protein. The display library can be screened with labelled antigens, fragments or epitopes in order to identify and isolate individual members of the library that exhibit the desired antigen-binding characteristics. The isolated clones from the display library can be further screened to optimise the antigen-binding characteristics. As mentioned previously, the improved stability of modified WAP domains both in vitro and in vivo means that half-life and potential for high biological tolerance (low toxicity) of the antigen-binding modified WAP domain polypeptides is an attractive feature of this technology. Suitable antigens (including epitopes) may include cell surface antigens, including receptors, markers of solid tumours or haematologic cancer cells (e.g. lymphoma or leukaemia), viral antigens, bacterial antigens, protazoal antigens, allergens, allergy related molecules, albumin (e.g. human, rodent, or bovine), fluorescent molecules (including fluorescein), blood group antigens, small molecules, drugs, enzymes, catalytic sites of enzymes or enzyme substrates, and transition state analogues of a enzyme substrates.

Further embodiments of the invention provide that the WAP domain polypeptide is suitably modified by insertion or substitution of a peptide sequence that encodes a number of functional polypeptides including, but not limited to: a therapeutic peptide; a receptor binding sequence; a proteolytic cleavage site; a catalytic active site; a glycosylation site; a phosphorylation site; a ubiquitylation or sumoylation site; a methylation or acetylation site; an antigenic sequence; and a nucleic acid binding sequence.

An embodiment of the invention provides a method of constructing a genetic library that comprises a multiplicity of chimaeric modified WAP domain-containing polypeptides, said method comprising: a) obtaining a template nucleic acid sequence that encodes a WAP domain polypeptide when expressed;

b) identifying one or more loop regions within the WAP domain and generating a plurality of sequences that correspond to the one or more loop regions and that are capable of replacing the one or more identified loop regions with a randomized nucleic acid sequence;

c) replacing the one or more loop regions within the WAP domain template nucleic acid with the plurality of sequences of (b) so as to generate a plurality of recombinant chimaeric WAP domain nucleic acid sequences; d) isolating the plurality of recombinant chimaeric WAP domain nucleic acid sequences; e) inserting the recombinant sequences of (d) into a plurality of nucleic acid expression vectors;

f) transforming host cells with the vectors of step (e); and

Optionally, the expression vector may be a display system vector (such as those described above), in which case step (g) would result in the construction of a peptide display library. In an example of the invention in use, described in more detail below, a phage display library is produced. The peptide display library may be screened to select only those peptide display packages that display a target peptide portion having the characteristics required.

In a specific embodiment of the invention the library of modified WAP domain proteins is screened to identify individual members of the library that exhibit membrane translocation activity, and in particular oral availability when delivered to an animal. By "oral availability" it is meant that the polypeptide is delivered to an animal orally (such as via feeding) and is capable of passing into the bloodstream of the animal by transiting the intestinal wall. A method for screening for oral availability, according to one embodiment, includes the steps of: a) constructing a modified WAP domain polypeptide sequence display library;

b) expressing the modified WAP domain sequence library in order to obtain expressed modified WAP domain polypeptides;

c) administering the expressed modified WAP domain polypeptides to an animal via the oral proute;

d) recovering any modified WAP domain polypeptide from the blood, lymph and/or tissues of the mammal;

e) determining the sequence of the modified WAP domain polypeptide. 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 modified WAP domain polypeptides may be suitably administered by oral gavage. Recovery of the modified WAP domain polypeptides from the body of the animal may be via biopsy, sample of the blood 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 modified WAP domain polypeptides that exhibit tissue specificity and/or the availability to cross various additional barriers within the body of the animal. By way of example, modified WAP domain polypeptides that are orally available and that are subsequently found in a brain biopsy of a screened non-human animal would be considered as demonstrating the additional ability of being able to cross the blood-brain barrier (BBB).

Modified WAP domain polypeptides, including modified Trappin polypeptides, of the invention that demonstrate the potential to act as stable scaffolds with oral availability (and/or tissue specificity) may be comprised within pharmaceutical compositions or utilised as targeting moieties for other pharmaceutical agents.

The modified WAP domain polypeptides of the present invention may be comprised within pharmaceutical compositions in certain embodiments. Typically, a specified WAP-domain polypeptide will be isolated from a library and characterised for its desired therapeutic potential. Suitably the isolated modified WAP domain polypeptide will be utilised in purified form together with one or more pharmacologically approved carriers. Typically, these carriers will include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, any including saline and/or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's. Suitable physiologically-acceptable adjuvants, if necessary to keep a polypeptide complex in suspension, may be chosen from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates. Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, may also be present. A variety of suitable formulations can be used, including extended release formulations where there is particular need for such a mode of administration. In specific embodiments of the present invention, the modified WAP domain polypeptides 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 modified WAP domain polypeptides of the present invention, or even combinations of different modified WAP domain polypeptides according to the present invention having different specificities and which may or may not be pooled prior to administration. The modified WAP domain polypeptides of the present invention may be also combined for administration (or at least co-administered) with other biological therapeutics including Adalimumab, Infliximab, Bevacizumab, Cetuximab and Trastuzumab, by way of non-limiting example.

The route of administration of pharmaceutical compositions according to the invention may be any of those commonly known to those of ordinary skill in the art. For therapy, including without limitation immunotherapy, the selected ligands thereof of the invention can be administered to any patient in accordance with standard techniques. The administration can be by any appropriate mode, including parenterally, intravenously, intramuscularly, intraperitoneally, transdermal^, via the pulmonary route, or also, appropriately, by direct infusion with a catheter. The dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counterindications and other parameters to be taken into account by the clinician. Administration can be local (e.g., local delivery to the lung by pulmonary administration, e.g., intranasal administration) or systemic as indicated. The modified WAP domain polypeptides of the invention will be suitably preserved in order to be in a form appropriate for administration to human or animal patients. Preservation may also involve chemical or other modification so as to stabilise the polypeptides for in-vivo use. Stabilisation may include PEGylation or other appropriate chemical processing. In addition, the modified WAP domain polypeptides can be lyophilised for storage and reconstituted in a suitable carrier prior to use.

Pharmaceutical compositions containing the present modified polypeptides or a combination thereof with other drugs or biologicals can be administered for prophylactic and/or therapeutic treatments. In certain therapeutic applications, an adequate amount to accomplish at least partial inhibition, suppression, modulation, killing, or some other measurable parameter, of a population of selected cells is defined as a therapeutically effective dose.

The peptide display libraries generated according to the methods of the invention may be screened by a variety of different methods in order to identify desired clones containing insertions, substitutions or deletions that confer particular properties.

In one embodiment of the invention, substrate binding activity can be determined by methods such as surface plasmon resonance (SPR), in which a test substrate compound is immobilized on a surface and the library is screened to identify those clones with highest binding activity to the immobilized compound (i.e. above a threshold K_D). In a specific embodiment of the invention, the immobilized compound may comprise a cell surface receptor, or at least the ligand binding regions of a cell surface receptor. By way of example, a cell surface receptor comprising a C-terminal C9 tag (a nine-amino acid sequence of the rhodopsin carboxyl tail) may be utilised. This tag is recognised by the monoclonal antibody 1 D4, which in turn is immobilized on a CM4 Biacore^® (SPR) sensor chip using standard amine-coupling chemistry. Solubilised tagged receptor proteins can be captured by the 1 D4 antibody and immobilized on surfaces within the CM4 chip. The interaction of orthosteric and allosteric binding interactions with the solubilized and immobilized receptor can then be observed. Other binding interactions may also be monitored using this approach, including antibody-antigen type binding interactions. In a further embodiment of the invention the library may be screened for enzyme inhibitory activity by assessing if one or more clones from the library are capable of binding to a target enzyme and inhibiting its ability to convert an substrate into a reaction product, typically a detectable reaction product. Many enzymic reactions are coupled to consumption/conversion of one or more cofactors including ATP, s-adenosyl methionine, NADH, FADH or a coenzyme. Consumption can be monitored either by incorporation or release of a radiolabel comprised within the cofactor, such as tritium, P³² or C¹⁴. Alternatively, conversion of the cofactor or the substrate may result in release of a detectable molecule. Various assays suitable for identifying interactions between enzymes and potential modulators, including inhibtors, are known in the art, see for example Handbook of assay development in drug discovery, Ed. Lisa K. Minor (2006) CRC Press, USA.

The invention is further exemplified in the following non-limiting examples. EXAMPLES

DNA encoding a particular WAP domain can be used as a template to build a library that inserts, replaces or randomises one or more WAP domain loop while maintaining the cysteine residues that define the WAP domain. A preferred WAP domain library is derived from the Trappin sub-family of WAP domains, and in even more preferred embodiment the WAP domain library is derived from human elafin. Isolating an unknown gene which encodes a desired peptide from a recombinant DNA library can be a difficult task. The use of hybridisation probes may facilitate the process, but their use is generally dependent on knowing at least a portion of the sequence of the gene which encodes the protein. When the sequence is not known, DNA libraries can be expressed in an expression vector, and antibodies have been used to screen plaques or colonies for the desired protein antigen. This procedure has been useful in screening small libraries, but rarely occurring sequences which are represented in less than about 1 in 10⁵ clones, as is the case with rarely occurring cDNA molecules or synthetic peptides, can be easily missed, making screening libraries larger than 10⁶ clones at best laborious and difficult. Screening larger libraries has required the development of methods designed to address the isolation of rarely occurring sequences, which are based on the co-selection of molecules, along with the nucleic acids that encode them. In a more preferred embodiment, libraries are built using methods that linked the expressed WAP domain scaffold polypeptide to the nucleic acid encoding that polypeptide.

Example 1

Construction of elafin WAP domain scaffold peptide display library. Two initial libraries were prepared, replacing either L00P1 or LOOP2 (see Figure 1 ) in the elafin sequence. The elafin WAP domain library template sequence was synthesized and supplied in a vector by GeneArt AG, Regensburg, Germany (Figure 2). Both randomized loops were PCR amplified and cloned as Ncol-Notl digested fragments into similarly digested pSP1 phagemidplll fusion vector derived from the pHEN1 pill vector (Hoogenboom et al, Nucleic Acids Research, 19: 4133-4137 (1991 )). (The pSP1 multiple cloning site is shown Figure 3). Detailed library build was carried out according to the following procedure:

(a) Primary PCRs.

For the primary PCR amplifications 10x 50μΙ amplifications were set up for each of ELAFBLOOP1 FOR (SEQ ID 001 ) or ELAFLOOP2FOR (SEQID 002) oligonucleotide primer and ELAFREV primer (SEQ ID 003). Each 50μΙ reaction mixture contained 10ng elafinGeneArt DNA vector, 25pmol of the appropriate FOR and REV primers, 0.1 mM dNTPs, 2.5 units Taq DNA polymerase, and 1x NEB PCR reaction buffer (20 mMTris-HCI pH 8.8, 10 mM (NH₄)₂S0₄, 10 mM KCI, 2 mM MgS0₄, 0.1 % Triton X-100) (NEB Ltd, Cambridge, U.K.). Reactions were performed for 30 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 50μΙ water per column.

(b) Pull-through re-amplification.

To prepare the final elafin Loopl or Loop2 DNA products, 40x 50μΙ amplifications were set up for each repertoire, using either ELAFBPTFOR (SEQ ID 004) for LOOP1 , or ELAFBFOR (SEQ ID 005) for LOOP2, and ELAFBREV primer (SEQ ID 006). Each 50μΙ reaction mixture contained approximately 25ng primary LOOP1 or 2 DNA product, 25pmol of the appropriate FOR and REV primers, 0.1 mM dNTPs, 2.5 units Taq DNA polymerase, and 1x NEB PCR reaction buffer (20 mMTris-HCI pH 8.8, 10 mM (NH₄)₂S0₄, 10 mM KCI, 2 mM MgS0₄, 0.1 % Triton X-100) (NEB Ltd, Cambridge, U.K.). 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 repertoire (Promega Ltd, Southampton, UK), and eluted into 100μΙ water per column.

(c) Cloning into pSP1.

Each of the two repertoires, and 250pg pSP1 vector DNA were Ncol-Notl digested with 100 units of each enzyme for 5 hours at 37°C (NEB, Cambridge, UK), and purified using one Wizard PCR clean-up columns per repertoire, and four for the digested vector DNA (Promega Ltd, Southampton, UK), and eluted into 100μΙ water. Half of each repertoire was ligated overnight at 16°C in 400μΙ with 50μg of Ncol-Notl cut pSP1 and 4000u of T4 DNA ligase (NEB Ltd, Southampton, UK). After incubation the ligations were adjusted to 200μΙ with nuclease free water, and DNA precipitated with 1 Ι 20mg/ml glycogen, 10ΟμΙ 7.5M ammonium acetate and 900μΙ ice-cold (-20°C) absolute ethanol, vortex mixed and spun at 13,000rpm for 20 minutes in a microfuge to pellet DNA. The pellets were washed with 500μΙ ice-cold 70% ethanol by centrifugation at 13,000rpm for 2 minutes, then vacuum dried and re-suspended in 100μΙ DEPC-treated water. 1 μΙ aliquots of each repertoire was electroporated into 80μΙ E. coli (TG1 ). Cells were grown in 1 ml SOC medium per cuvette used for 1 hour at 37°C, and plated onto 2xTY agar plates supplemented with 2% glucose and 100μg/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 of the order of 4x10⁹ clones with >95% with in-frame inserts.

(d) Phage amplification. Separate phage stocks were prepared for each repertoire library. The bacteria were then scraped off the plates into 50ml 2xTY broth supplemented with 20% glycerol, 2% glucose and 100μg/ml ampicillin. 1 ml was added to a 50ml 2xTY culture broth supplemented with 1 % glucose and 100μg/ml ampicillin and infected with 10¹¹ kanamycin resistance units (kru) M13K07 helper phage at 37°C for 30 minutes without shaking, then for 30 minutes with shaking at 200rpm. Infected bacteria were transferred to 200ml 2xTY broth supplemented with 25μg/ml kanamycin, 100μg/ml ampicillin, and 20μΜ IPTG, then incubated overnight at 30°C, shaking at 200rpm. Bacteria were pelleted at 4000rpm for 20 minutes in 50ml Falcon tubes, and 40ml 2.5M NaCI/20% PEG 6000 was added to 400ml of particle supernatant, mixed vigorously and incubated on ice for 1 hour to precipitate phage particles. Particles were pelleted at 11000rpm for 30 minutes in 250ml Oakridge tubes at 4°C in a Sorvall RC5B centrifuge, then resuspended in 40ml water and 8ml 2.5M NaCI/20% PEG 6000 added to reprecipitate particles, then incubated on ice for 20 minutes. Particles were again pelleted at 11000rpm for 30 minutes in 50ml Oakridge tubes at 4°C in a Sorvall RC5B centrifuge, then resuspended in 5ml PBS buffer, after removing all traces of PEG/NaCI with a pipette. Bacterial debris was removed by a 5 minute 13500rpm spin in a microcentrifuge. The supernatant was filtered through a 0.45μιη polysulfone syringe filter, adjusted to 20% glycerol and stored at -70°C.

Table 1

Nucleic acid sequences

SEQ ID Sequence

001 ACTAAGCCTGGCTCCTGCCCCNNKNNKNNKNNKNNKNNKNNKNNKTGCGCCAT

GTTGAATCCCCCTAACC

002 CCCCATTATCTTGATCCGGTGCNNKNNKNNKNNKNNKNNKNNKNNKNNKTGCTT

GAAAGATACTGAC 003 CTG GGG AAC GAA ACA GGC C

004 GCC CAG CCG GCC ATG GCC ACT AAG CCT GGC TCC TGC CCC

005 GCC CAG CCG GCC ATG GCC ACT AAG CCT GGC TCC TGC CCC ATT ATC

TTG ATC CGG TGC

006 TTT TTT TGC GGC CGC CTG GGG AAC GAA ACA GGC CAT CCC G

Table 2

Polypeptide sequences

SEQ ID Sequence

007 TKPGSC

008 CLKDTDCPGIKKCCEGSCGMACFVPQ

009 CAMLNPPNRCLKDTDCPGIKKCCEGSCGMACFVPQ

010 TKPGSCPIILIRC

Example 2 Selection of anti-HA antibody specific elafin WAP domain scaffold

In this example a phage display library of clones generated as per Example 1 was screened with an antibody that binds specifically with human haemagglutinin (HA) antigen. Hence, clones that bind specifically with anti-HA antibody were identified.

Selection.

5pg biotinylated anti-HA antibody was diluted to in 500μΙ PBS. 50 μΙ M280 streptavidin-coated paramagnetic beads were added to the solution and mixed for 15 minutes. Beads were then washed twice with PBS. Beads were then blocked for 1 hour with 4% skimmed milk powder in PBS. 100μΙ of each of the PEG/NaCI precipitated phage solutions were added to 800μΙ of 4% skimmed milk powder in PBS. To remove non anti-HA-specific library members, 50μΙ uncoated M280 beads were added and mixed for 30 minutes before being discarded. This process was repeated two more times. Blocked, coated beads were then added to the solution and mixed for 1 hour.Beads were then washed six times with PBS/tween 20 and twice with PBS. Bound phage were eluted by addition of 0.75ml of 0.1 M triethylamine, incubated for 10 minutes followed by addition of 0.25ml of 0.5M TRIS-HCI (pH 8.0). Supernatant was then added to 10ml TG1 E.coli in 2xTY culture broth at O.D. 600nm = 0.6 and mixed. Culture was incubated at 37°C without shaking for 30 minutes and for a further 30 minutes at 37°C with shaking (200rpm orbital shaking incubator). 100μΙ culture was taken to generate a dilution series to determine cfu titre, the remaining culture was centrifuged at 1 ,500xg for 5 minutes. Supernatant was discarded and pelleted cells were resuspended in 300μΙ of 2xTY culture broth and then coated on 2xTY agar plates supplemented with 2% glucose and 10Q^g/ml ampiciHin. Colonies were allowed to grow overnight at 30°C. cfu titres were determined and phage amplification was carried out as outlined in the previous example. The selection process was repeated once.

ELISA screen of selection output phage clones.

Individual colonies from selection output plates were picked into 10ΟμΙ 2xTY supplemented with 2% glucose and 100pg/ml ampiciHin and allowed to reach stationary phase of growth via incubation overnight at 37 °C with shaking (200rpm orbital shaking incubator). 10μΙ culture was added to 1 ml 2xTY supplemented with 2% glucose and lOO g/ml ampiciHin. Culture was allowed to reach O.D. 600nm = 0.6 at 37 °C with shaking. Cultures were then innoculated with M13 K07 helper phage at M.O.I of 10. Cultures were incubated at37°C without shaking for 30 minutes and for a further 30 minutes at 37°C with shaking. Cultures were then centrifuged at 2,500xg for 10 minutes. Supernatant was replaced with an equal volume of 2xTY broth supplemented with 25Mg/ml kanamycin, 100 g/ml ampiciHin, and 20μΜ IPTG, then incubated overnight at 30°C, shaking at 200rpm. Cultures were centrifuged at 2,500xg for 10 minutes. Phage-containing supernatant was then used for phage ELISA. Immobilon 2 96-well ELISA plates were coated with anti-HA antibody at ^g/ml in PBS (ΙΟΟμΙ/well) via incubation overnight at 4°C. Plates were blocked with 4% skimmed milk powder in PBS for 1 hour and then washed once with PBS. Phage supernatant was diluted 1 :1 with 4% skimmed milk powder in PBS and added to blocked plates (100pl/well) and incubated for 1 hour. Plates were washed twice with PBS/tween20 and twice with PBS. Anti-M13-HRP conjugated antibody, diluted at 1 :5000 in 2% skimmed milk powder in PBS/tween20 was added to plates (100pl/well). Plates were incubated for 1 hour. Plates were washed three times with PBS/tween20 and twice with PBS. Signals were detected with TMB. Clones generating signal:background of greater than 5:1 were selected for sequencing to determine binding motif for anti-HA antibody.

The selected clones carried the sequences set out in Table 3. Table 3

SEQ ID Sequence

011 MATKPSCPWDYPYDQPCAMLNPPNRCLKDTDCPGIKKCCEGSCGMACFVPQAAA

012 MATKPSCWGPFDVPDYCAMLNPPNRCLKDTDCPGIKKCCEGSCGMACFVPQAAA

013 MATKPSCAWPFDVPDSCAMLNPPNRCLKDTDCPGIKKCCEGSCGMACFVPQAAA

014 MATKPSCPIILIRCWPFDVPDSCLKDTDCPGIKKCCEGSCGMACFVPQAAA The underlined regions of the sequences indicate the variation from the wild type sequence. It can be seen that for the polypeptides shown in SEQ ID NOS: 011-013 the variation occurs by way of a mutation in loop 1 of the scaffold, whereas for SEQ ID NO: 014 the mutation is in loop 2 of the scaffold.

This Example demonstrates how the peptide display library can be screened to identify unique novel polypeptides with specific properties.

Example 3

The libraries generated in Example 1 can also be screened to identify clones that demonstrate the ability to translocate across membranes. In so doing specific clones encode peptides that can demonstrate the ability to cross the gut lining and enter the bloodstream when fed to an animal or patient, hence classifying such peptides as Orally available'. Certain of these clones are also able to cross the blood brain barrier (BBB) rendering them particularly suitable as vectors for therapeutic agents.

Selection of orally available modified Elafin clones Approximately 10¹³ members of each pill phage loop library (1 & 2, Example 1 above) were administered to Hooded Lister rats via oral gavage in 500μΙ PBS. To identify orally available phage clones blood samples were taken at 0, 30, 60 and 120 min. To identify orally available elafin library phage clones, also capable of also crossing the blood-brain barrier, rats were sacrificed after two hours, and homogenised brain tissue was examined for the presence of infectious phage. Blood or brain samples were added to TG1 E.coli at mid-exponential growth and infection was allowed to proceed. Cells were then plated out onto 2xTY/ampicillin/glucose agar plates and incubated over night at 32°C. Titres of output phage were calculated. Individual colonies from output plates were then grown up and sequenced. Peptide Synthesis Elafin clone #91 was identified as having the following sequence:

TKPGSCPIILIRCSFRSWWFLCLKDTDCPGIKKCCEGSCGMACFVPQ [SEQ ID NO: 021] Clone #91 is modified in the loop 2 region (see underlined portion above). This library sequence was chosen as a representative output library member and synthesized as a N-terminally fluorescein-labelled peptide by Alta Bioscience (Birmingham, UK). GLP-1 (7-36) (SEQ ID 022: HAEGTFTSDVSSYLEGQAAKEFIAWLVK) was chosen as a control peptide, known to cross the blood-brain barrier, and similarly synthesized as a N-terminally fluorescein-labelled peptide. Peptides were oxidised as follows: To stirred peptide solution in water, 0.4 M NH₄HC0₃ added dropwise over 20 min to give final peptide concentration of 10 mg/ml. Peptides were stirred at room temperature for a further 24 h and freeze dried.

Blood-brain barrier transfer

200pg of either Elafin #91 (SEQ ID NO: 021 ) or GLP-1 (7-36; SEQ ID NO: 022) in water, were administered to Lister hooded rats (~200g) via i.v. injection. After 4 hours, rats were sacrificed and tissues excised and snap-frozen on dry ice. 10 μιη sections of frozen tissues were then examined under a microscope using UV illumination. Fluorescence was observed in brain sections for both Elafin #91 and GLP-1 (7-36), and is approximately 5-1 Ox more intense for Elafin #91 (see Figure 4).

Oral availability & blood-brain barrier transfer 2mg of Elafin #91 or GLP-1 (7-36) in water was administered to Lister hooded rats (~200g) via oral gavage. After 4 hours, rats were sacrificed and tissues excised and snap-frozen on dry ice. 10 pm sections of frozen tissues were then examined under a microscope using UV illumination. Fluorescence is observed in brain sections for Elafin #91 but not GLP-1 (7-36) (see Figure 5), indicating that this peptide of the invention can be administered orally and enter the brain. In addition, the experiment shows that the properties of the peptide are not perturbed by N-terminal attachment to the fluorescein molecule. Hence, the peptide can function in vivo as a orally available tissue targeting vector for other conjugated factors including therapeutic agents.

Although particular embodiments of the invention have been disclosed herein in detail, this has been done by way of example and for the purposes of illustration only. The aforementioned embodiments are not intended to be limiting with respect to the scope of the appended claims, which follow. The choice of nucleic acid starting material, the clone of interest, or type of library used is believed to be a routine matter for the person of skill in the art with knowledge of the presently described embodiments. It is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims.

Claims

WE CLAIM:

1. A modified polypeptide comprising a whey acidic protein (WAP) domain, wherein the WAP domain is modified by insertion, deletion or substitution of at least one amino acid residue at a position in the WAP domain selected from the group consisting of: the region between the first cysteine residue and the second cysteine residue (loop 1 ) in the polypeptide sequence; the region between the second cysteine residue and the third cysteine residue (loop 2) in the polypeptide sequence; and the region between the first cysteine residue and the second cysteine residue (loop 1 ) in the polypeptide sequence, and the region between the second cysteine residue and the third cysteine residue (loop 2) in the polypeptide sequence.

2. The modified polypeptide of claim 1 , wherein the WAP domain is obtained from a proteinase inhibitor protein.

3. The modified polypeptide of claims 1 or 2, wherein the WAP domain is obtained from a trappin protein.

4. The modified polypeptide of claims 1 to 3, wherein the WAP domain comprises the WAP domain of elafin (trappin-2, SKALP, ESI).

5. The modified polypeptide of any previous claim, wherein the WAP domain is modified by substitution of one or more amino acid residues.

6. The modified polypeptide of claim 5, wherein the WAP domain is modified by substitution of at least two amino acid residues.

7. The modified polypeptide of any previous claim, wherein the WAP domain is modified by insertion or substitution of a peptide sequence that encodes an antigen-binding site.

8. The modified polypeptide of any of claims 1 to 6, wherein the WAP domain is modified by insertion or substitution of a peptide sequence selected from one or more of the group consisting of: a therapeutic peptide; a receptor binding sequence; a proteolytic cleavage site; a catalytic active site; a glycosylation site; a phosphorylation site; a methylation site; an acetylation site; a ubiquitylation or sumoylation site; an antigenic sequence; and a nucleic acid binding sequence.

9. The modified polypeptide of any previous claim with the proviso that the modified polypeptide scaffold does not comprise the sequence of wild type elafin or of a modified elafin comprising the wild type sequence and point mutation resulting in a substitution of the wild type Met residue at position 25 to an Iso, Val or Leu residue.

10. The modified peptide of any of claims 1 to 8, wherein the peptide comprises the sequence of SEQ ID NO: 021.

11. A modified polypeptide of any of claims 1 to 10 for use in medicine.

12. A polypeptide comprising a WAP domain, wherein the WAP domain is modified by insertion, deletion or substitution of at least one amino acid residue at a position in the WAP domain between the first cysteine residue and the second cysteine residue (loop 1 ) in the polypeptide sequence.

13. A polypeptide comprising a WAP domain, wherein the WAP domain is modified by insertion, deletion or substitution of at least two amino acid residues at a position in the WAP domain between the second cysteine residue and the third cysteine residue (loop 2) in the polypeptide sequence.

14. The polypeptide of claims 12 or 13, wherein prior to modification the polypeptide comprises the sequence of SEQ ID NO: 6.

15. A polypeptide scaffold comprising a framework substantially based upon the polypeptide sequence of a WAP domain, characterized in that:

(a) the region located between the first cysteine residue and the second cysteine residue (loop 1 ) in the polypeptide sequence is replaced with a heterologous sequence of between 4 and 18 amino acid residues; and/or

(b) the region located between the second cysteine residue and the third cysteine residue (loop 2) in the polypeptide sequence is replaced with a heterologous sequence of between 4 and 18 amino acid residues.

16. The polypeptide of claim 14, wherein WAP polypeptide upon which the scaffold is based comprises the sequence of SEQ ID NO: 16.

17. A modified polypeptide scaffold of general formula I: - [X]n - C - [Χ']η' wherein

A is a polypeptide chain comprising Thr-Lys-Pro-Gly-Ser-Cys,

n is at least 1 and at most 18,

n' is at least 1 and at most 18_:

C is a Cys residue, and

18. The modified peptide of claim 16, wherein the peptide comprises the sequence of SEQ ID NO: 021.

19. A nucleic acid encoding a modified polypeptide scaffold of claim 17.

20. A nucleic acid vector comprising the nucleic acid of claim 18 in operative combination with a promoter sequence, and optionally one or more additional sequences selected from the group consisting of: a selection marker; an origin of replication; and a reporter gene.

21. The nucleic acid vector of claim 20, wherein the vector is selected from the group consisting of: a phage display (phagemid) vector; a Lacl fusion plasmid vector; a ribosome display vector; and a CIS display vector.

22. A modified polypeptide scaffold of general formula II:

A - [X]n - D II wherein

n is at least 1 and at most 18, and

23. A nucleic acid encoding a modified polypeptide scaffold of claim 22.

24. A nucleic acid vector comprising the nucleic acid of claim 23 in operative combination with a promoter sequence, and optionally one or more additional sequences selected from the group consisting of: a selection marker; an origin of replication; and a reporter gene.

25. The nucleic acid vector of claim 24, wherein the vector is selected from the group consisting of: a phage display (phagemid) vector; a Lacl fusion plasmid vector; a ribosome display vector; and a CIS display vector.

26. A modified polypeptide scaffold of general formula III:

E - [Χ']η' - B III wherein

E is a polypeptide chain comprising the sequence of SEQ ID NO: 10,

X' is an oligopeptide sequences of n' residues in length comprising a sequence of any amino acid residue,

n' is at least 1 and at most 18, and

27. A nucleic acid encoding a modified polypeptide scaffold of claim 26.

28. A nucleic acid vector comprising the nucleic acid of 27 in operative combination with a promoter sequence and optionally one or more additional sequences selected from the group consisting of: a selection marker; an origin of replication; and a reporter gene.

29. The nucleic acid vector of claim 28, wherein the vector is selected from the group consisting of: a phage display (phagemid) vector; a Lacl fusion plasmid vector; a ribosome display vector; and a CIS display vector.

30. A modified polypeptide of any of claims 17, 22 or 26 for use in medicine.

31. A modified polypeptide of any of claims 17, 22 or 26 for use as a transfer vector when conjugated to a therapeutic agent.

32. The modified polypeptide of claim 31 , wherein the therapeutic agent is 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.

33. A method of constructing a genetic library that comprises a multiplicity of chimaeric modified WAP domain-containing polypeptides, said method comprising: a) obtaining a template nucleic acid sequence that encodes a WAP domain polypeptide when expressed;

b) identifying one or more loop regions within the WAP domain and generating a plurality of sequences that correspond to the one or more loop regions and that are capable of replacing the one or more identified loop regions with a randomized nucleic acid sequence; c) replacing the one or more loop regions within the WAP domain template nucleic acid with the plurality of sequences of (b) so as to generate a plurality of recombinant chimaeric WAP domain nucleic acid sequences;

f) transforming host cells with the vectors of step (e); and

34. The method of claim 33, wherein the expression vectors are peptide display system vectors.

The method of claim 34, wherein the expression vectors are phage display vectors.

36. A method for identifying a polypeptide, wherein said polypeptide comprises a modified WAP scaffold polypeptide, the method comprising: (i) constructing a genetic library according to the method of any of claims 33 to 35; and

(ii) screening the library to identify the polypeptide.

37. The method of claim 36, wherein the method comprises screening the library in order to identify polypeptides that exhibit at least one property selected from the group consisting of: antibody binding capacity; antigen binding capacity; therapeutic activity; enzymic catalytic activity; enzymic inhibitory activity; receptor binding capacity; substrate binding capacity; and membrane translocation activity including oral availability .

38. A method for preparing a pharmaceutical composition comprising identifying polypeptide as described in the method of claims 36 or 37, and combining the polypeptide identified with a suitable pharmaceutical excipient.