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WO2006056009A1 - Peptides possedant des proprietes adhesives a utiliser dans un dosage de phase solide et des regimes de synthese de polymeres chimiques - Google Patents

Peptides possedant des proprietes adhesives a utiliser dans un dosage de phase solide et des regimes de synthese de polymeres chimiques Download PDF

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Publication number
WO2006056009A1
WO2006056009A1 PCT/AU2005/001775 AU2005001775W WO2006056009A1 WO 2006056009 A1 WO2006056009 A1 WO 2006056009A1 AU 2005001775 W AU2005001775 W AU 2005001775W WO 2006056009 A1 WO2006056009 A1 WO 2006056009A1
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Prior art keywords
amino acid
peptide
adhesive peptide
adhesive
peptides
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PCT/AU2005/001775
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English (en)
Inventor
David Andrew Anderson
Fan Li
Melanie Louise Ladiges
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Hepgenics Pty Ltd
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Priority claimed from AU2004906706A external-priority patent/AU2004906706A0/en
Application filed by Hepgenics Pty Ltd filed Critical Hepgenics Pty Ltd
Publication of WO2006056009A1 publication Critical patent/WO2006056009A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

  • Peptides having adhesive properties for use in solid phase assay and chemical polymer synthesis regimes are provided.
  • the present invention relates generally to the field of solid phase technology such as used in diagnostic assays and solid phase synthesis. More particularly, the present invention provides peptides having adhesive properties. Still more particularly, the peptides of the
  • present invention facilitate the immobilization of molecules or cells or viral particles to solid or semi-solid substrates for use in a wide range of solid phase assays and chemical or polymer synthesis regimes.
  • present peptides further facilitate a wide range of therapeutic and/or diagnostic applications due to their ability to bind to and therefore influence the activities of chemical or biological molecules or cells and tissues in a pre-
  • Solid phase assays are conducted using a variety of formats which vary in the substrate material and form in which it is provided. For example, mictrotitre plates are injection moulded from a variety of plastics to form an array of individual wells. Polystyrene latex microparticles and glass microscope slides are also commonly used. Immunoassay procedures are widely used to detect antigens or host antibodies determined by antigens. Commonly, such assays, for example, enzyme linked immunoabsorbent assays (ELISA), use a solid support, such as a polystyrene microtitre plate coated with a specific antibody or antigen.
  • ELISA enzyme linked immunoabsorbent assays
  • one approach is to covalently couple agents to a solid surface using a bi-functional imidazole coupling agent such as Tl carbonyldiimidazole (CDI-Sigma) as described by Hearn, Methods Enzymology, 735:102-1 17, 1987.
  • a bi-functional imidazole coupling agent such as Tl carbonyldiimidazole (CDI-Sigma) as described by Hearn, Methods Enzymology, 735:102-1 17, 1987.
  • Amino-terminated silanes can be used to coat a solid surface and form a covalent attachment with nucleic acids. This is described, for example, in Rogers K R e/ al, eds,, "Affinity Biosensors: Techniques and Protocols” Humana. Totowa, NJ, 1998.
  • Streptavidin or strepatividin derivative-coated solid surfaces are used in conjunction with biotin modified reagents in order to bind the reagent to a solid surface as described in Reznik G O e/ al, Bioconjug, Chem., 72(6): 1000- 1004, 2001.
  • Another solution is to use compounds such as poly-L-lysine which binds non-covalently to glass or latex and there enhances binding to nucleic acids by electrostatic attraction between the poly-lysine and phosphate groups of the nucleic acid molecule. Aspects of this approach are discussed in Ruiz-Taylor et al., PNAS, 98:852-857, 2001.
  • Heparin coated plates have been described, including heparin directly coupled to activated polystyrene or alternatively coupled to lipids which are passively absorbed to polystyrene plates.
  • reversible non-covalent interactions are employed including antibody-antigen interactions, receptor-ligand, sugar-sugar binding proteins, DNA-DNA interactions and DNA-DNA binding proteins.
  • the physicochemical nature of the solid surface has a profound influence over the methods used to attach molecules of interest.
  • the physicochemical nature of the solid surface also has a profound influence on the behaviour of the molecules once they are attached.
  • the surface characteristics of substrate material influences the outcome of biological assays in unpredictable ways. For example proteins can absorb to latex beads and become denatured. Non-specific protein binding to solid surfaces is also a problem causing reduced assay sensitivity and reproductibility.
  • Phage display peptide libraries are useful for identifying binding partners. See, for example, U.S. Pat. No. 5,223,409 and U.S. Pat. No. 5,498,530.
  • random peptide sequences are displayed by fusion with coat proteins of filamentous phage.
  • the displayed peptides are affinity-eluted against an immobilized potential binding partner.
  • the retained phages may be enriched by successive rounds of affinity purification and repropagation.
  • the best binding peptides may be sequenced to identify key residues within one or more structurally related families of peptides. See, for example, Cwirla et al, Science, 276:1696-1699, 1997, in which two distinct families were identified.
  • the peptide sequences may also suggest which residues may be safely replaced by alanine scanning or by mutagenesis at the DNA level. Mutagenesis libraries may be created and screened to further optimize the sequence of the best binders. Lowman, Ann. Rev. Biophys. Biomol. Struct, 26:401-424, 1997.
  • SEQ ID NOs. Sequence Identity Numbers for the nucleotide and amino acid sequences referred to in the specification are defined after the bibliography. A summary of the SEQ ID NOs is given in Table 1 before the Examples.
  • the present invention provides a family of adhesive peptides in synthetic or recombinant form or homologs, derivatives, mimetics and functional chemical equivalents thereof and methods for their use in a variety of practical applications involving facilitation of attachment of molecules and cells or microbial agents to solid or semi-solid surfaces.
  • the present invention further provides the nucleic acid molecules encoding the subject adhesive sequences in the form of a plasmid, expression vector or other genetic construct or library with optionally sites for incorporation of a nucleic acid molecule encoding a polypeptide of interest.
  • Cells, host cells and viral particles expressing the nucleic acid molecules are also contemplated.
  • the present invention provides an adhesive peptide as herein described bound to or otherwise associated with substrates or molecules of interest such as a polypeptide of interest, a nucleic acid molecule of interest, a chemical or synthetic molecule, cell, microorganism or tissue, device or other solid or semi-solid substrate or support.
  • substrates or molecules of interest such as a polypeptide of interest, a nucleic acid molecule of interest, a chemical or synthetic molecule, cell, microorganism or tissue, device or other solid or semi-solid substrate or support.
  • the present invention provides methods for conducting assays wherein molecules required for the assay are immobilized, attached or otherwise associated to/with a solid or semi-solid substrate via an adhesive peptide of the present invention.
  • immunoassays are contemplated such as enzyme, or other detectable marker-linked, immunosorbent-based assays, including chromatography-based assays.
  • the present invention provides agonists and antagonists of the interaction between the sequence of amino acids constituting the present adhesive peptides and its binding partners, and methods of screening and uses therefor.
  • Figure 1 is a graphical representation showing the results of an ELISA assay using anti- RESA (ring-infected erythrocyte surface antigen of Plasmodium falciparum) monoclonal antibody to detect C-terminally biotinylated RESA peptides bound to streptavidin coated microtitre plates (Chemicon). Examples of recombinant peptides comprising the sequence of amino acids constituting an adhesive peptide and a RESA peptide antigen from P.
  • RESA ring-infected erythrocyte surface antigen of Plasmodium falciparum
  • Figure 2 is a graphical representation showing the results of an ELISA assay using anti- RESA monoclonal antibody to detect C-terminally biotinylated RESA peptides bound to uncoated polystyrene microtitre plates (Nunc MaxiSorp). The peptide concentration is plotted against the OD450/620 indicating the absorbance at 450nm with a reference wavelength of 620nm. Examples of recombinant peptides comprising the sequence of amino acids constituting an adhesive peptides and a RESA peptide antigen from P.
  • Adhesive Peptides 1 and 2 and a control biotinylated peptide of the same length comprising the same RESA amino acid sequence together with a further nine amino acids normally found at this position relative to the RESA epitope in the full length antigen (P3).
  • Peptides were bound at various concentrations to microtitre plates, blocked, and the amount of peptide bound detected using monoclonal antibody (28/2) and HRPO- conjugated anti-mouse IgG.
  • FIG. 3 is a graphical representation showing the results of an ELISA measuring binding of biotinylated peptides (Peptides Pepl (Pl), Pep 2 (P2) and Pep 3 (P3) to polystyrene microtitre plates (Nunc, Maxisorp) and shows enhanced binding by P2 over Pl which shows enhanced binding over control peptide P3. Binding of biotinylated peptides is detected using conjugated streptavidin-HRP.
  • FIG 4 is a table showing the amino acid sequences of adhesive peptides in accordance with the present invention. Different conserved amino acid residues are highlighted. Aromatic amino acids (Tryptophan (Trp), Tyrosine (Tyr) and Phenylalanine (Phe)) are highlighted with a back slash pattern. Histidine (not essential) is highlighted with a trellis pattern. In A, proline is highlighted with a forward slash pattern. Peptides 1 to 14 are a family of related peptides identified in biopanning experiments.
  • Peptides, Pepl and Pe ⁇ 2 correspond to Adhesive Peptides 1 and 2, respectively (supra) and are the same except that aa2 and aa6 of Pepl are aa6 and aa2 of Pep2.
  • Pep3 corresponds to P3 (supra) and demonstrates that histidine alone is not sufficient for binding.
  • Figure 5 is a graphical representation showing binding of Peptide 1, Peptide 2 and Peptide 3 (non-adhesive control peptide) to a range of substrates.
  • Peptides at various concentrations were bound to a variety of multiwell plate surfaces (Greiner Medium, Greiner High, Nunc Maxisorp, Nunc Polysorp, Nunc Medisorp, Polyvinyl chloride) according to the description in the legend to Figure 2.
  • the peptide concentration is plotted against the OD450/620 indicating the absorbance at 450nm with a reference wavelength of 620nm. Bound peptide was detected by ELISA probing with monoclonal antibody 28/2 against the RESA epitope.
  • Peptide 1 and Peptide 2 were found to have greatly enhanced binding relative to Peptide 3 on all plate surfaces, although the absolute level of binding varied between plates.
  • Figure 6 is a graphical representation showing binding of Peptide 1, Peptide 2 and Peptide
  • Figure 7 is a graphical representation showing binding of Peptide 1, Peptide 2 and Peptide 3 (non-adhesive control peptide) to a range of substrates.
  • Peptides were bound at various dilutions to a variety of membrane surfaces (PVDF, Nitrocellulose, Nylon) using a Bio DotBlot apparatus (BioRad) according to the description in the legend to Figure 3, with bound peptide detected by probing with Alexa 680-Streptavidin. Results were quantitated by exposure to the Odyssey infrared scanner (LiCor Inc.). The peptide concentration is plotted against relative fluorescence on a scale of 0 or negative to hundreds. Peptide 1 and Peptide 2 were found to have greatly enhanced binding relative to Peptide 3 on all surfaces, however binding to Nylon was very low.
  • FIG 8 is a photographic representation showing binding of Peptide 1, Peptide 2 and Peptide 3 (non-adhesive control peptide) to Porex membrane substrate.
  • Peptides were bound at various dilutions to PorexTM polyethylene membrane (T3 hydrophilic membrane) using a Bio DotBlot apparatus (BioRad) according to the description in the legend to Figure 3, with bound peptide detected by probing with Alexa 680-Streptavidin. Results were scanned by exposure to the Odyssey infrared scanner (LiCor Inc.).
  • Peptide 1 and Peptide 2 were found to have greatly enhanced binding relative to Peptide 3 with Peptide 2 being approximately 25-fold higher binding than Peptide 1 on this membrane, with Peptide 3 being undetectable.
  • Figure 9 is a graphical representation showing adhesive properties of peptides with Trytophan mutations.
  • Peptides were prepared based on the first 9 amino acids of Peptide 2 plus the RESA epitope and a C-terminal Biotin residue. Individual peptides were prepared with substitution of 0, 1, 2, or 3 of the 4 Tryptophan residues (W) from the C-terminus of the adhesive peptide sequence, to yield P2 (WTWQWHPWS) SEQ ID NO: 22, P4 (WTWQWHPLS) SEQ ID NO: 23, P5 (WTWQLHPLS) SEQ ID NO: 24, and P6 (WTTQLHPLS) SEQ ID NO: 25, P3 is equivalent to Peptide 3 specification (ENVPEHVQH) SEQ ID NO: 26.
  • Peptides were bound to Nunc Maxisorp plates according to the description in the legend to Figure 3, with bound peptide detected by probing with Streptavidin-peroxidase.
  • the peptide concentration is plotted against the OD450/620 indicating the absorbance at 450nm with a reference wavelength of 620nm.
  • the results show that the progressive replacement of Tryptophan residues leads to a corresponding, progressive decrease in the binding of peptides to the plate, demonstrating that all of the tryptophan residues contribute to the total level of binding.
  • the peptide shows greatly enhanced binding compared to control (P3).
  • Figure 10 is a graphical representation showing relative binding of adhesive peptides.
  • Peptides were prepared with addition of 1 or 2 glycine residues to the N-terminus, or progressive truncations from the C-terminus or the N-terminus of the 8 amino acid sequence of (Peptide 2 (WTWQWHPW)). All peptides have an additional sequence (GGKPLAQGSG-biotin) at their C-terminus.
  • Peptides were bound to Nunc Maxisorp plates according to the description in the legend to Figure 3, with bound peptide detected by probing with Streptavidin-peroxidase. The peptide concentration is plotted against the OD450/620 indicating the absorbance at 450nm with a reference wavelength of 620nm.
  • Figure 10 (C) the equivalent peptides prepared with the modification of the N-terminus by biotinylation. AU N-terminally biotinylated peptides showed greatly reduced levels of binding compared to the non-acetylated peptides shown in Figure 10 (A). The binding of the adhesive peptide is reduced by some modifications of the N-terminus (such as acetylation or biotinylation), but is not reduced by some other modifications (such as addition of one or two glycine residues).
  • Figure 11 is a graphical representation of data showing the utility of the adhesive peptides in enhancing the binding of macromolecules to solid substrates.
  • Peptide 1 and Peptide 2 (each of which contains a C-terminal biotin) were used to bind Streptavidin to microtitre plates, allowing the subsequent binding of other biotinylated macromolecules via the remaining (approximately 3) biotin-binding sites on the tetrameric Streptavidin protein.
  • Peptide 1, Peptide 2 and Peptide 3 non-adhesive control peptide were bound to Nunc Maxisorp plates at a concentration of 0.1 ⁇ g/ml.
  • Figure 12 is a photographic representation of data showing the utility of the adhesive peptides in enhancing the binding of macromolecules to solid substrates.
  • Peptide 1 and Peptide 2 (each of which contains a C-terminal biotin) were used to bind Streptavidin to Porex membrane (B).
  • Peptide 1, Peptide 2 and Peptide 3 (non-adhesive control peptide) were bound to Porex T3 membrane at a concentration of 0.1 ⁇ g/ml using a Bio DotBlot apparatus. After washing, serial dilutions of Streptavidin were added to peptide-coated wells for 1 hour at room temperature, or to uncoated wells overnight at 4°C as a control for passive binding of Streptavidin.
  • Figure 13 is a photographic representation of data showing the utility of the adhesive peptides in enhancing the binding of macromolecules to solid substrates.
  • the adhesive peptide WGWQWGPW was prepared as an N-Hydroxysuccinimide (NHS) active ester, and conjugated to monoclonal antibody (MAb) K2-4F2 (antibody to hepatitis A virus) or MAb PH-315 (antibody to human IgM) by mixing at 4°C for two days.
  • Serial dilutions of the adhesive peptide-conjugated MAbs were prepared in PBS, pH 7.4, and serial dilutions of the equivalent unconjugated MAbs were prepared in 0.1 M sodium bicarbonate buffer, pH 9.6.
  • Figure 14 is a diagrammatic representation showing functional analogs of aromatic amino acids, tryptophan, tyrosine and phenylalanine; 60 (L-1-beta-Naphthylalanine), 103 (Beta- (3-Pyridyl)-L-alanine), 161 (Nz-Dabcyl-L-lysine), 264 (beta-(2-quinolyl)-alanine) and 389 (Phenyl-phenylalanine; Biphenylalanine).
  • the present invention is predicated in part by the discovery via phage display experiments of a family of related peptides which exhibit binding to solid surfaces. Peptide mutagenesis studies have been used to identify amino acid sequences which are important for binding activity. Various adhesive peptides (polypeptides) based on these sequences have been tested for their ability to adhere to a variety of surfaces and were found to bind to a plurality of polymeric surfaces and to enhance the binding of molecules of interest to a plurality of polymeric surfaces.
  • the present invention contemplates a variety of practical uses for the adhesive peptides of the present invention, in synthetic or recombinant form.
  • the adhesive peptides enable immobilization of molecules or cells or microorganisms to coat or modify solid or semi-solid substrates for use in a wide range of solid or semi-solid phase assays.
  • the present peptides will facilitate a wide range of therapeutic and/or diagnostic applications due to their ability to bind to and therefore influence the activities of chemical or biological molecules or cells and tissues in a pre-determined manner.
  • solid supports for use in assays including immunoassays, mass spectrometry and surface plasmon resonance can be made comprising the adhesive peptides of the present invention as a coating or component of a coating for any solid or semi-solid substrate.
  • the composition of solid supports may be modified to enhance binding by the subject adhesive peptides.
  • one member of a binding pair such as streptavidin is immobilized to a polymeric substrate (surface) via an adhesive peptide comprising a C-terminal biotin.
  • Any target molecule of interest having a complementary binding site for streptavidin, such as a biotin can then be immobilized on the surface of a substrate via the adhesive peptide.
  • Analyte binding molecules typically antigens, antibodies or small epitope antibodies in immunoassays, can be made to bind to immobilized streptavidin through the remaining biotin-binding sites on the tetrameric streptavidin (see, for example, Example 8 and Figure 11). It will be appreciated that this concept and variations thereof will be readily applied to other applications and target molecules and polymeric substances which it is desirable to coat or modify.
  • binding partner or "binding pair” is a reference to complementary molecules which bind or interact with each other via a reversible non-covalent or covalent attachment determined by their structure.
  • exemplary proteinaceous binding partners include antibody- antigen, enzyme-substrate, biotin-streptavidin, biotin-antibiotin antibodies, digoxigenin - anti-digoxigenin antibodies mannose/maltose/amylose-mannose/maltose/amylose-binding protein and cytokine or ligand receptor interactions.
  • Other binding relationships are known to those skilled in the art, such as for example those employing glutathione, nickel- chelators and leucine zipper binding pairs (c-Jun and vFos) and any such binding relationship is included herein.
  • adhesive peptides may be conjugated to a molecule to be immobilized such as a proteinaceous or non-proteinaceous molecule and used to immobilize these, via the adhesive peptide, to a polymeric surface. See, for example, Example 9 and Figure 13.
  • the target molecule to be applied to the surface is expressed recombinantly along with the adhesive peptide sequence as a fusion or chimeric polypeptide.
  • the present invention provides a method of coating or modifying a polymeric surface with a molecule of interest comprising (a) directly or indirectly attaching the molecule of interest to an adhesive peptide as described herein and (b) contacting the polymeric surface with sufficient adhesive peptide and under conditions suitable to coat the surface with the molecule of interest.
  • the method further comprises (c) washing the polymeric surface to remove unbound adhesive peptide.
  • the adhesive peptide is detectably modified and the method further optionally comprises (d) detecting the detectable modification to assess or quantify the binding of adhesive peptide or the molecule of interest to the polymeric surface.
  • step (a) comprises modifying the adhesive peptide to introduce an attachable modification where by the molecule of interest is attached to the adhesive peptide.
  • the attachable modification is attached to the C-terminal end of the adhesive peptide.
  • the attachable modification may be made by any suitable technique routinely used by those of skill in the art.
  • the adhesive peptide is biotinylated in order to coat the polymeric surface with streptavidin.
  • the adhesive peptide is synthesised with one of a leucine zipper binding pair and applied to a polymeric surface in order to facilitate binding of the second leucine zipper binding peptide which is itself attached to a molecule of interest.
  • the method further comprises (d) contacting the polymeric surface with the second member of the binding pair attached to the molecule of interest; and optionally (e) a further washing step to remove unbound reagents.
  • the present invention provides an adhesive peptide comprising the amino acid sequence:
  • Ai to A 4 is independently selected from a hydrophobic amino acid selected from; Ala, GIy, He, Phe, Pro, Met, Trp, Tyr, VaI; D or L isomers thereof; and a functional analog thereof as substituted with a non-polar substituent such as, for example, an alkyl, alkenyl, alkynyl, aryl or heterocyclyl substituent;
  • amino acid sequence generally means a sequence of two or more amino acid residues. As illustrated herein in Example 9, an amino acid sequence comprising nine amino acids with four interspersed tryptophans showed the greatest amount of binding to particular polymeric substrates. Accordingly, where small adhesive peptides are required less than but about 9 amino acids provides a useful starting point. However, peptides of longer length may be preferred for some applications and the adhesive peptide amino acid sequence may be of any length such as from about 10 to about 50 amino acids or from about 10 to about 1000 amino acids or more. This nine amino acid sequence or a part of this sequence may also be repeated in order to confer enhanced adhesiveness.
  • amino acid sequence of the adhesive peptide portion of a peptide or polypeptide is generally about 6 to 20 amino acids and the remainder of the molecule may include a proteinaceous or non proteinaceous molecule of any convenient size.
  • the non-adhesive peptide portion of the molecule may be attached by covalent or non-covalent linkage.
  • the term "about” refers to a quantity, level, value, dimension, size, or amount that varies by as much as 30%, 20%, or 10% to a reference quantity, level, value, dimension, size, or amount.
  • alkyl used either alone or in compound words, denotes saturated straight chain, branched or cyclic hydrocarbon groups, preferably C 1-2O alkyl, eg Ci -10 or C]. 6 .
  • straight chain and branched alkyl include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, n-pentyl and branched isomers thereof, n-hexyl and branched isomers thereof, n-heptyl and branched isomers thereof, n-octyl and branched isomers thereof, n-nonyl and branched isomers thereof, and n-decyl and branched isomers thereof.
  • cyclic alkyl examples include mono- or polycyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl and the like.
  • alkynyl denotes groups formed from straight chain, branched or cyclic hydrocarbon residues containing at least one carbon-carbon triple bond including ethynically mono-, di- or poly- unsaturated alkyl or cycloalkyl groups as previously defined.
  • the term preferably refers to C 2-20 alkynyl. Examples include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, and butynyl isomers, and pentynyl isomers.
  • aryl denotes a C 6 -Ci 4 aromatic hydrocarbon group.
  • Suitable aryl groups include • phenyl, biphenyl, naphthyl, tetrahydronaphthyl, anthracenyl, dihydroanthracenyl and phenanthrenyl.
  • Preferred aryl groups include phenyl, biphenyl and naphthyl.
  • heterocyclyl denotes monocyclic, polycyclic or fused, saturated, unsaturated or aromatic hydrocarbon residues, wherein one or more carbon atoms (and where appropriate, hydrogen atoms attached thereto) are replaced by a heteroatom.
  • Suitable heteroatoms include, O, N, S, and Se. Where two or more carbon .atoms are replaced, this may be by two or more of the same heteroatom or by different heteroatoms.
  • heterocyclic groups may include pyrrolidinyl, pyrrolinyl, piperidyl, piperazinyl, morpholino, indolinyl, imidazolidinyl, pyrazolidinyl, thiomorpholino, dioxanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyrrolyl, pyridyl, thienyl, furyl, pyrrolyl, indolyl, pyridazinyl, pyrazolyl, pyrazinyl, thiazolyl, pyrimidinyl, quinolinyl, isoquinolinyl, benzofuranyl, benzothienyl, purinyl, quinazolinyl, phenazinyl, acridinyl, benzoxazolyl, benzothiazolyl and the like.
  • Preferred heterocyclyl groups ' include but are not limited
  • Ai to A 4 is independently selected from one of Trp, Tyr, Phe, Ala, DY, DW, DF, 60 (L-1-beta-Naphthylalanine), 103 (Beta-(3-Pyridyl)-L-alanine), 161 (Nz-Dabcyl-L-lysine), 264 (beta-(2-quinolyl)-alanine) and 389 (Phenyl -phenylalanine; Biphenylalanine).
  • X 2 is His
  • n is a sequence of n amino acids wherein nl + n2 is from 0 to 50 amino acids and wherein the sequence X j may comprise the same or different amino acids selected from any naturally or non-naturally occurring amino acid residue;
  • sequence X j comprises at least one proline.
  • nl + n2 is from 2 to 50, or any number from 0 to 50 i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50.
  • the present invention provides an adhesive peptide comprising the amino acid sequence:
  • Xi is Phe, Tyr or Trp; X 2 is His;
  • X 3 is Phe, Tyr or Trp
  • n is a sequence of n amino acids wherein nl + n2 is from 0 to 50 amino acids and wherein the sequence X j may comprise the same or different amino acids selected from any naturally or non-naturally occurring amino acid residue; or a functional derivative thereof comprising at least about 20% amino acid sequence similarity thereto or a homolog, mimetic or analog thereof.
  • the present invention provides an adhesive peptide comprising an amino acid sequence:
  • Xi is Phe, Tyr or Trp; X 2 is His;
  • X 3 is Phe, Tyr or Trp
  • X 4 is any naturally or non-naturally occurring amino acid
  • X 5 is Phe, Tyr or Trp
  • n is a sequence of n amino acids wherein nl + n2 is from 0 to 50 amino acids and wherein the sequence X j may comprise the same or different amino acids selected from any naturally or non-naturally occurring amino acid;
  • the adhesive peptide comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1 to 19 and SEQ ID NOs: 21 to 58. or a functional derivative thereof having at least 20% similarity to any one of SEQ ID NOS: 1 to 19 and SEQ ID Nos: 21 to 58.
  • the adhesive peptide is selected from the group comprising SEQ ID NOs: 27, 28, 29, 30, 35, 39, 43, 44, 47 and 49 or a part thereof or a functional form thereof having at least about 40% sequence identity over the full length of the adhesive peptide. In other embodiments, the adhesive peptide is selected from the group consisting SEQ ID NOs: 27, 29, 44, 45, 47, 49, 50, 54, 55 and 57 or a part thereof or a functional form thereof having at least about 40% sequence identity over the full length of the adhesive peptide.
  • a non-polar substituent such as, for example, an alkyl, alkenyl, alkynyl, aryl or heterocyclyl substituent.
  • the substituted hydrophobic amino acid is alanine.
  • the D isomer is D-tryptophan or D-tyrosine.
  • the adhesive peptides are further modified in accordance with the present invention in order to serve a wide range of functions. For example, for different chemical or physical properties (solubility, strength, length weight etc) or in order to immobilize attached molecules or to serve to identify the adhesive peptides or the attached molecule or even the attached molecule bound or otherwise attached to a further one ore more molecules.
  • the adhesive peptide comprises one or more amino acids which are detectably modified to allow detection of the adhesive peptide.
  • the adhesive peptide comprises an N-terminal modification and/or a C-terminal modification.
  • the N- terminal modification is typically one or two glycine residues or an equivalent addition.
  • the C-terminal modification comprises a genetic or a proteinaceous or non-proteinaceous molecule, a detectable and/or an attachable modification.
  • Non- proteinaceous molecules include large or small chemical compounds.
  • a “detectable modification” refers broadly to any modification to the peptide that facilitates its detection, either directly or indirectly.
  • the detectable modification is conveniently selected from: a fluorescence molecule, a chromogen, a catalyst, an enzyme, a dye such as an infrared dye, a flurochrome, a chemiluminescent, bioluminescent or phosphorescent moiety, a lanthanide ion, a radioisotope or a visual label such as gold or silver nanoparticles.
  • Fluorescent molecules are particularly well established however, this is a rapidly moving field and the present invention is in no way limited to the use of any particular detectable modification.
  • distinguishable compounds such as fluorophores, dyes or particles are used to facilitate combinatorial analyses.
  • a direct visual label use may be made of a colloidal metallic or non-metallic particle, a dye particle, bioluminescent enzymes, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like.
  • Especially preferred labels of this type include large colloids, for example, metal colloids such as those from gold, selenium, silver, tin and titanium oxide.
  • an enzyme is used as a direct visual label, biotinylated residues are incorporated.
  • Suitable fluorochromes include, but are not limited to, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), R-Phycoerythrin (RPE), and Texas Red.
  • FITC fluorescein isothiocyanate
  • TRITC tetramethylrhodamine isothiocyanate
  • RPE R-Phycoerythrin
  • Texas Red Texas Red
  • Other exemplary fluorochromes include those discussed by Dower et al. (International Publication WO 93/06121). Reference also may be made to the fluorochromes described in U.S. Patents 5,573,909 (Singer et al), 5,326,692 (Brinkley et al). Alternatively, reference may be made to the fluorochromes described in U.S. Patent Nos.
  • fluorescent labels include, for example, fluorescein phosphoramidites such as Fluoreprime (Pharmacia), Fluoredite (Millipore) and FAM (Applied Biosystems International), Texas Red, NBD, coumarin, dansyl chloride and rhodamine.
  • Radioactive reporter molecules include, for example, 32 P, which can be detected by an X-ray or phosphoimager techniques.
  • an "attachable modification” is a broad reference to a modification made to the peptide in order to facilitate, directly or indirectly, its attachment to, or detachment from, another molecule.
  • the peptide can be biotin labelled to facilitate attachment to streptavidin (a molecule of interest).
  • the peptide may be designed to comprise a cleavage site to facilitate detachment from another molecule.
  • the present invention furthermore provides recombinant or synthetic fusion peptides or polypeptides comprising a first amino acid sequence having adhesive properties and a second sequence constituting a heterologous polypeptide.
  • the heterologous polypeptide of interest is an antigen, epitope or mimetope.
  • the present invention provides methods of immobilizing a substance of interest including a chemical or biological molecule, cell or viral particle to a solid or semi-solid support, the method comprising attaching, binding or otherwise associating the substance to an adhesive peptide and contacting the associated adhesive peptide with the solid or semi-solid support for a time and under conditions to permit immobilization of the substance, wherein the adhesive peptide comprises the amino acid sequence:
  • Ai to A 4 is independently selected from a hydrophobic amino acid selected from; Ala, GIy, He, Phe, Pro, Met, Trp, Tyr, VaI; D or L isomers thereof; and a functional analog thereof as substituted with a non-polar substituent such as, for example, an alkyl, alkenyl, alkynyl, aryl or heterocyclyl substituent;
  • the heterocyclyl group is selected from indolinyl, pyridyl, indollyl, quinolinyl, isoquinolinyl and quinazolinyl.
  • Ai to A 4 is independently selected from one of Trp, Tyr, Phe, Ala, DY, DW, DF, 60 (L-l-beta- Naphthylalanine), 103 (Beta-(3-Pyridyl)-L-alanine), 161 (Nz-Dabcyl-L-lysine), 264 (beta- (2-quinolyl)-alanine) and 389 (Phenyl-phenylalanine; Biphenylalanine).
  • the adhesive peptide comprises a sequence selected from the group comprising any one of SEQ ID NOs: 1 to 19 and SEQ ID NOs: 21 to 58.
  • the adhesive peptide is preferably SEQ ID NOs: 27, 28, 29, 30, 35, 39, 43, 44, 47 and 49 or a part thereof or a functional form thereof having at least about 40% sequence identity over the full length of the adhesive peptide.
  • the adhesive peptide is preferably selected from SEQ ID NOs: 27, 29, 44, 45, 47, 49, 50, 54, 55 and 57 or a part thereof or a functional form thereof having at least about 40% sequence identity over the full length of the adhesive peptide.
  • the present invention provides a method " for optimising coatings for a selected polymeric substance using adhesive peptides.
  • the method comprises (a) contacting a selected polymeric surface with a plurality (separately or together) of amino acid sequences of SEQ ID NO: 27 or a part thereof and derivatives of SEQ ID NO: 27 in which X aa is any amino acid and wherein Ai to A 4 is independently selected from a hydrophobic amino acid selected from: Ala, GIy, He, Phe, Pro, Met, Trp, Tyr, VaI; L and D isomers of any of these; and any of these hydrophobic amino acids as substituted with a non-polar substituent such as, for example, an alkyl, alkenyl, alkynyl, aryl or heterocyclyl substituent and (b) identifying which of the peptides demonstrates the greatest level of binding to the polymeric surface and (c) coating the polymeric surface using the adhesive peptide or a proteinaceous or non-
  • the adhesive peptide is detectably or attachably modified.
  • the same procedure would also be used to optimise the sequence of amino acids in an adhesive peptide for use with one or more selected solid or semi solid supports.
  • the method would comprise steps (a) and (b).
  • the present invention provides a method of optimising coatings for a polymeric substance using an adhesive peptide, the method comprising: (a) contacting a selected polymeric surface with a plurality (separately or together) of peptides comprising the amino acid sequence:
  • X aa any amino acid
  • Ai to A 4 is independently selected from a hydrophobic amino acid selected from; Ala, GIy, lie, Phe, Pro, Met, Tip, Tyr, VaI; D or L isomers thereof; and a functional analog thereof as substituted with a non-polar substituent such as, for example, an alkyl, alkenyl, alkynyl, aryl or heterocyclyl substituent; and (b) identifying which of the amino acid sequences demonstrates the greatest level of binding to the polymeric surface; and (c)coating the polymeric surface using the adhesive peptide or a proteinaceous or non-proteinaceous molecule comprising the adhesive peptide identified in step (b).
  • the present invention provides a method for optimising coatings for a selected polymeric substance using adhesive peptides.
  • the method comprises (a) contacting a selected polymeric surface with a plurality (separately or together) of amino acid sequences of SEQ ID NO: 27 or a part thereof and derivatives of SEQ ID NO: 27 in which Xaa is any amino acid and wherein Ai to A 4 is independently selected from a hydrophobic amino acid selected from: Ala, GIy 3 He, Phe, Pro, Met, Trp, Tyr, VaI; L and D isomers of any of these; and any of these hydrophobic amino acids as substituted with a non-polar substituent such as, for example, an alkyl, alkenyl, alkynyl, aryl or heterocyclyl substituent and (b) identifying which of the peptides demonstrates the greatest level of binding to the polymeric surface and (c) coating the polymeric surface using the adhesive peptide or a proteinaceous or non-proteinace
  • the same procedure would also be used to optimise the sequence of amino acids in an adhesive peptide for use with one or more selected solid or semi solid supports.
  • the method would comprise steps (a) and (b).
  • the present invention provides methods of immobilizing a substance of interest including a chemical or biological molecule, cell or viral particle to a solid or semi-solid support, the method comprising attaching, binding or otherwise associating the substance to an adhesive peptide and contacting the associated adhesive peptide with the solid or semi-solid support for a time and under conditions to permit immobilization of the substance, wherein the adhesive peptide comprises the amino acid sequence:
  • X aa any amino acid
  • Aj to A 4 is independently selected from a hydrophobic amino acid selected from; Ala, GIy, He, Phe, Pro, Met, Trp, Tyr, VaI; D or L isomers thereof; and a functional analog thereof as substituted with a non-polar substituent such as, for example, an alkyl, alkenyl, alkynyl, aryl or heterocyclyl substituent; or a functional derivative thereof comprising at least about 20% amino acid sequence similarity thereto.
  • a non-polar substituent such as, for example, an alkyl, alkenyl, alkynyl, aryl or heterocyclyl substituent; or a functional derivative thereof comprising at least about 20% amino acid sequence similarity thereto.
  • the method uses adhesive peptides wherein Ai to A 4 is independently selected from one of Tip, Tyr, Phe, Ala, DY, DW, DF, 60 (L-l-beta- Naphthylalanine), 103 (Beta-(3-Pyridyl)-L-alanine), 161 (Nz-Dabcyl-L-lysine), 264 (beta- (2-quinolyl)-alanine) and 389 (Phenyl-phenylalanine; Biphenylalanine).
  • Ai to A 4 is independently selected from one of Tip, Tyr, Phe, Ala, DY, DW, DF, 60 (L-l-beta- Naphthylalanine), 103 (Beta-(3-Pyridyl)-L-alanine), 161 (Nz-Dabcyl-L-lysine), 264 (beta- (2-quinolyl)-alanine)
  • the adhesive peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 19 and SEQ ID NOs: 21 to 58 or a part thereof or a functional derivative thereof comprising at least about 40% sequence identity over the full length of the adhesive peptide.
  • the present invention provides methods of immobilizing a substance of interest including a chemical or biological molecule, cell or viral particle to a solid or semi-solid support, the method comprising attaching, binding or otherwise associating the substance to an adhesive peptide and contacting the associated adhesive peptide with the solid or semi-solid support for a time and under conditions to permit immobilization of the substance, wherein the adhesive peptide comprises the amino acid sequence:
  • Xi is Phe, Tyr or Trp; X 2 is His;
  • X 3 is Phe, Tyr or Trp
  • n is a sequence of n amino acids wherein nl + n2 is from 0 to 50 amino acids and wherein the sequence X j may comprise the same or different amino acids selected from any naturally or non-naturally occurring amino acid residue;
  • the adhesive peptide is incorporated into and/or on to the solid or semi-solid support during or subsequent to manufacture of the support and the substance of interest is attached, bound or otherwise associated with the solid or semi-solid support thereafter.
  • the substance of interest is a polypeptide or a nucleic acid molecule.
  • the substance or molecule of interest is a polypeptide or peptide of interest and adhesive peptides are produced recombinantly as fusion polypeptides or peptides.
  • the polypeptide or peptide of interest is an immunological binding partner such as an antigen or an antibody.
  • the polypeptide of interest is a blocking agent such as BSA or a milk protein.
  • the substance or molecule of interest is a nucleic acid molecule, lipid or carbohydrate.
  • the resulting molecule is a hybrid (for example a peptide :nucleic acid, peptide:lipid, peptide carbohydrate molecule as well as a peptide ipeptide/polypeptide) molecule.
  • the molecule of interest may also be one member of a binding pair such as biotin or streptavidin or as described further herein.
  • polypeptide or molecule or substance of interest includes a single polypeptide or molecule or substance of interest, as well as two or more polypeptides, molecules or substances of interest; reference to a “sample” includes two or multiple samples; and so forth.
  • solid or semi-solid substrate, support or surface includes without limitation any particular support or carrier having suitable surface or sub-surface chemistries, shape or size for its use in assays, for analysis, treatment or diagnosis or other applications.
  • Exemplary substrates or surfaces include plates, particles, membranes, filters, chips, pins, needles, hollow fibres, capillaries, pellets, disks, medical and other devices or semi-solid surfaces such as gels, certain membranes, micelles, liposomes and matrices.
  • Particular substrates (surfaces or supports) may comprise plastics (latex), glass, metal, or other colloidal particle, teflon or other components. In this regard, reference may be made to International Publication Nos.
  • polymeric supports may be formed from polystyrene cross-linked with 1-5% divinylbenzene or from polyamides such as nylon.
  • Polymeric supports may also be formed from hexamethylenediamine-polyacryl resins and related polymers, poly[N- ⁇ 2-(4-hydroxylphenyl)ethyl ⁇ ] acrylamide (i.e.
  • one Q) 3 silica cellulose, polystyrene, poly(halomethylstyrene), poly(halostyrene), poly(acetoxystyrene), latex, grafted copolymers such as polyethylene glycol/polystyrene, porous silicates, for example, controlled pore-glass supports, polyacrylamide, for example, poly(acryloylsarcosine methyl ester), dimethylacrylamide optionally cross-linked with N,N'-bis-acrylolyl ethylene diamine, glass particles coated with a hydrophobic polymer inclusive of cross-linked polystyrene or a fluorinated ethylene polymer which provides a material having a rigid or semi-rigid surface, poly(N-acryloylpyrrolidine) resins, Wang (trade mark) resins, Pam resins, Merrifield (trade mark) resins, PAP and SPARE polyamide resins, polyethylene functionalized with acrylic acid, kieselguhr/polyamide (Pepsy
  • Preferred polymeric supports are plastics, polypropylene, polystyrene, polyethylene, nitrocellulose, nylon, polyvinyl chloride and functional equivalents or derivatives thereof.
  • Naturally occurring polymeric surfaces include matrix proteins such as those comprising fibrinogen, fibronectin, fibrin, collagen.
  • the solid or semi-solid support may be synthesized or generated or modified using processes which incorporate the adhesive peptide in the support or coating therefore.
  • a coating includes discrete areas of support coverage.
  • Oligonucleotide or nucleic acid array are generally provided on, and/or in, a solid substrate where oligonucleotides or nucleic acids with different known sequences are deposited at discrete known locations associated with its surface.
  • the substrate can be in the form of a two dimensional substrate as described in U.S. Patent No. 5,424,186. Such substrate may be used to synthesise two-dimensional spatially addressed oligonucleotide
  • the substrate may be characterized in that it forms a tubular array in which a two dimensional planar sheet is rolled into a three-dimensional tubular configuration.
  • the substrate may also be in the form of a microsphere or bead connected to the surface of an optic fibre as, for example, disclosed by Chee et al. in WO 00/39587.
  • Oligonucleotide arrays have at least two different features and a density of at least 400 features per cm 2 .
  • the arrays can have a density of about 500, at least one thousand, at least 10 thousand, at least 100 thousand, at least one million or at least 10 million features per cm 2 .
  • the substrate may be silicon or glass and can have the thickness of a glass microscope slide or a glass cover slip, or may be composed of other synthetic polymers. Substrates that are transparent to light are useful when the method of performing an assay on the substrate involves optical detection.
  • supports for arrays are generated which have adhesive peptide binding activity. These supports are used to generate arrays of nucleic acid molecules attached at discrete locations associated with the surface via adhesive peptides.
  • the adhesive peptide is modified at its C-terminal end with nucleic acid binding molecules or an attachable modification suitable for attachment to an oligonucleotide.
  • an analyte of interest is detected by interaction with an antibody to the analyte which antibody is also linked to a detection marker.
  • Analogous enzyme-based assays use an enzyme reaction in place of an antigen-antibody interaction.
  • Various modifications of immunochromatographic methods are described in Published US Patent Application Nos. 20010006821, 20040087036 and 20040214347 which are incorporated herein in their entirety. Immunogold filtration methods for analysis of multiple analytes are described in Published US Patent Application No. 20030165970 incorporated herein.
  • a "molecule” refers to any chemical, synthetic and/or biological molecule including a nucleic acid molecule, polypeptide, carbohydrate, lipid and includes complex mixtures of these, naturally occurring forms or derivatized forms thereof.
  • peptide and polypeptide are used herein to refer to a polymer of amino acids of and its equivalent connected by peptide bonds and do not refer to a specific length. Thus peptides, oligopeptides and proteins are included within the definition.
  • Amino acids comprise an amino group, a carboxyl group, a hydrogen atom and a distinctive R group bonded to a carbon atom which is called the ⁇ -carbon.
  • the R group is referred to as a side chain.
  • glycine which has a side chain consisting of hydrogen.
  • Alanine (Ala), valine (VaI), leucine (Leu) and isoleucine (He) have a hydrocarbon group side chain.
  • Proline (Pro) differs from other common amino acids by having a secondary amino group. Two amino acids, serine and threonine contain aliphatic hydroxyl groups. There are three common amino acids having an aromatic side chain, namely phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp). In contrast to the amino acids referred to previously, which are uncharged at physiological pH, lysine and arginine are positively charged under neutral conditions.
  • Negatively charged amino acids include glutamate (GIu) (glutamine (GIn) when uncharged) and aspartate (Asp) (asparagine (Asn) when uncharged).
  • Amino acids having a side chain comprising a sulphur atom include methionine (Met) and cysteine (Cys). Peptide bonds are formed between the ⁇ -carboxyl group of one amino acid with the ⁇ -amino group of another by hydrolysis.
  • “Derivatives" of the recited amino acid sequences are also contemplated. These molecules are designed to retain the functional activity (binding) of the reference peptide or to exhibit enhanced activity.
  • Parts include fragments comprising from about 20% i.e., 2 residues of a 10 amino acid sequence to 30% or more of the sequence i.e., 40%, 50%, 60%, 70%, 80%, 85%, 90% and 95% of the reference sequence.
  • Polypeptide derivatives according to the invention can be identified either rationally, or via established methods of mutagenesis (see, for example, Watson, J. D. et al, "Molecular Biology of the Gene", Fourth Edition, Benjarnin/Cummings, Menlo Park, Calif., 1987). Random mutagenesis approaches require no a priori information about the sequence that is to be mutated.
  • Substantial changes in function are generally made by selecting substitutions that are less conservative than those shown in Table A.
  • the substitutions which are likely to produce the greatest changes in the properties of a peptide are those in which (a) a hydrophilic residue (eg, Ser or Thr) is substituted for, or by, a hydrophobic residue (eg, Ala, Leu, He, Phe or VaI); (b) a cysteine or proline is substituted for, or by, any other residue; (c) a residue having an electropositive side chain (eg, Arg, His or Lys) is substituted for, or by, an electronegative residue (eg, GIu or Asp) or (d) a residue having a bulky side chain (eg, Phe or Trp) is substituted for, or by, one having a smaller side chain (eg, Ala, Ser) or Glycine having a hydrogen atom only as a side "chain”.
  • a hydrophilic residue eg, Ser or Thr
  • Tryptophan, Tyrosine and Alanine appear in different peptides in the same relative positions and maintain the functional characteristics of enhanced binding to these surfaces.
  • Derivatives include adhesive peptides that comprise amino acids which are analogs of naturally occurring amino acids. Such analogs are typically substituted with non-polar substituents. Those skilled in the art can routinely generate sets of derivatives comprising different amino acids, altered amino acids or synthetic analogs of amino acids, which are tested for their ability to bind to polymeric substrates of interest. For the avoidance of doubt, the term derivatives is in no way limited to molecules which are directly derived from a reference peptide and include peptides generated independently but with reference to information derived from a reference peptide.
  • Peptide analogues or derivatives may contain conservative amino acid substitutions at various locations along their sequence, as compared to the reference amino acid sequence.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub-classified as follows:
  • Acidic The residue has a negative charge due to loss of H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH.
  • Amino acids having an acidic side chain include glutamic acid and aspartic acid.
  • the residue has a positive charge due to association with H ion at physiological pH or within one or two pH units thereof (e.g., histidine) and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH.
  • Amino acids having a basic side chain include arginine, lysine and histidine.
  • the residues are charged at physiological pH and, therefore, include amino acids having acidic or basic side chains (i.e., glutamic acid, aspartic acid, arginine, lysine and histidine).
  • amino acids having acidic or basic side chains i.e., glutamic acid, aspartic acid, arginine, lysine and histidine.
  • Hydrophobic The residues are not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium.
  • Amino acids having a hydrophobic side chain include tyrosine, valine, isoleucine, leucine, methionine, phenylalanine and tryptophan.
  • Neutral/polar The residues are not charged at physiological pH, but the residue is not sufficiently repelled by aqueous solutions so that it would seek inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium.
  • Amino acids having a neutral/polar side chain include asparagine, glutamine, cysteine, histidine, serine and threonine.
  • amino acids having a small side chain include glycine, serine, alanine and threonine.
  • amino acid residues may fall in two or more classes.
  • sub-classification according to this scheme is presented in the Table B. TABLE B
  • Conservative amino acid substitution also includes groupings based on side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine.
  • amino acid substitutions falling within the scope of the invention are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. After the substitutions are introduced, the variants are screened for biological activity.
  • similar amino acids for making conservative substitutions can be grouped into three categories based on the identity of the side chains.
  • the first group includes glutamic acid, aspartic acid, arginine, lysine, histidine, which all have charged side chains;
  • the second group includes glycine, serine, threonine, cysteine, tyrosine, glutamine, asparagine;
  • the third group includes leucine, isoleucine, valine, alanine, proline, phenylalanine, tryptophan, methionine, as described in Zubay, G., Biochemistry, third edition, Wm.C. Brown Publishers (1993).
  • a predicted non-essential amino acid residue in a peptide is typically replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of a polynucleotide coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for an activity of the parent polypeptide to identify mutants which retain that activity.
  • the encoded peptide can be expressed recombinantly and the activity of the peptide can be determined.
  • derivatives will display at least about 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 % similarity to a functional reference sequence as, for example, set forth in any one of SEQ ID NO: 1 and 19 and 21 to 58.
  • variants will have at least 30, 40, 50, 55, 60, 65, 70, 75, 80 s 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity to a functional reference sequence as, for example, set forth in any one of SEQ ⁇ D NOs: 1 to 19 and 21 to 58.
  • Suitable peptides also include peptides that are encoded by polynucleotides that hybridise under stringency conditions as defined herein, especially high stringency conditions, to adhesive peptide encoding sequences, or the non- coding strand thereof.
  • derivative encompasses modifications of the peptide or polypeptide, for example, glycosylation, aceylation, phosphorylation, biotinylation and the like. Common modifications are hydroxylated proline, hydroxyproline; ⁇ -carboxylutamate and O- phosphoserine. Included in the definition therefore are, for example, polypeptides or peptides containing one or more analogs of an amino acid including for example, unnatural amino acids such as those given in Table 3 before the Examples, or peptides with substituted linkages. Generally in relation to adhesive peptides, modifications are to directly or indirectly enhance the adhesive qualities or modify the binding capacity or capabilities of the peptide.
  • One particular modification contemplated herein is to express or synthesise peptides or polypeptides as a polymer comprising one or several or multiple repeats of the herein-recited amino acid sequences or parts thereof.
  • Analogs contemplated herein include but are not limited to modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogs.
  • the N-terminus of the adhesive peptides is unaceylated and unbiotinylated. As demonstrated herein, the presence of Glycine and/or Proline is acceptable.
  • side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH 4 .
  • the guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
  • the carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide.
  • Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4- chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2- chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
  • Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.
  • Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
  • Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethy lpyrocarbonate .
  • Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine,- norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine, listed all numbers 60 (L-1-beta-Naphthylalanine), 103 (Beta-(3-Pyridyl)-L- alanine), 161 (Nz-Dabcyl-L-lysine), 264 (beta-(2-quinolyl)-alanine) or 389 (Phenyl- phenylalanine; Biphenylalanine), D-isomers of amino acids and specifically DW and
  • peptides can be conformationally constrained by, for example, incorporation of C ⁇ and N ⁇ -methylamino acids, introduction of double bonds between C ⁇ and Cp atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
  • Recombinant polypeptides are generally generated with a more limited range of modifications however there have been significant advances in protein formulation and chemical modification in the area of recombinant therapeutic or diagnostic polypeptides.
  • Modifications include those which protect therapeutic proteins, primarily by blocking their exposure to proteolytic enzymes. Further modifications may also increase the polypeptide stability, circulation time, and biological activity.
  • a review article describing protein modification and fusion proteins is Francis, Focus on Growth Factors, 5:4-10 Mediscript,
  • polypeptides of interest include, without limitation antigens, epitope and mimetopes.
  • the antigen may comprise epitope regions from two or more polypeptides from different organisms, species or subspecies or their derivatives.
  • the subject invention provides a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding an adhesive peptide or a functional derivative or a nucleotide sequence which hybridises thereto under low stringency conditions wherein the peptide comprises an amino acid sequence: Xa a i Aj X aa2 A 2 X aa3 A 3 X aa4 X aa5 A 4 , or a part thereof
  • Ai to A 4 is independently selected from a hydrophobic amino acid selected from; Ala, GIy, He, Phe, Pro, Met, Trp, Tyr, or a functional derivative thereof comprising at least about 20% amino acid sequence similarity thereto.
  • the nucleic acid sequence encodes a sequence selected from the group comprising any one of SEQ ID NOs: 1 to 19 and SEQ ID NOs: 21 to 58 or a part thereof or a functional form thereof having at least about 40% sequence identity over the full length of the adhesive peptide.
  • the preferred sequences to encode are SEQ ID NOs: 27, 28, 29, 30, 35, 39, 43, 44, 47 and 49 or a part thereof or a functional form thereof having at least about 40% sequence identity over the full length of the adhesive peptide.
  • sequences to encode are selected from the group comprising SEQ ID NOs: 27, 29, 44, 45, 47, 49, 50, 54, 55 and 57 or a part thereof or a functional form thereof having at least about 40% sequence identity over the full length of the adhesive peptide.
  • the subject invention provides a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding an adhesive peptide or a functional derivative or a nucleotide sequence which hybridises thereto under low stringency conditions wherein the peptide comprises an amino acid sequence:
  • X] is Phe, Tyr or Trp
  • X 2 is His; [X j ] n is a sequence of n amino acids wherein nl + n2 is from about 2 to about 50 amino acids and wherein the sequence X j may comprise the same or different amino acids selected from any amino acid residue.
  • nl + n2 is from 2 to 50, or any number from 0 to 50 i.e., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50.
  • amino acid sequence comprises:
  • Xj is Phe, Tyr or Trp
  • X 2 is His
  • X 3 is Phe, Tyr or Trp
  • n is a sequence of n amino acids wherein nl + n2 is from 0 to 50 amino acids and wherein the sequence X j may comprise the same or different amino acids selected from any naturally or non-naturally occurring amino acid residue;
  • amino acid sequence comprises:
  • Xj is Phe, Tyr or Trp; X 2 is His; X 4 is any amino acid; X 5 is Phe, Tyr or Trp; [X j ] n is a sequence of n amino acids wherein nl + n2 is from 0 to 50 amino acids and wherein the sequence X j may comprise the same or different amino acids selected from any amino acid.
  • the nucleic acid is in the form of a plasmid, phage display or other genetic construct or library and further comprises one or more further nucleic acid sequences of interest.
  • sequence similarity and “sequence identity” as used herein refer to the extent that sequences are identical or functionally or structurally similar on a nucleotide-by- nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
  • a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g.
  • A, T, C, G, I) or the identical amino acid residue e.g, Ala, Pro, Ser, Thr, GIy, VaI, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, GIu, Asn, GIn, Cys and Met
  • amino acid residue e.g, Ala, Pro, Ser, Thr, GIy, VaI, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, GIu, Asn, GIn, Cys and Met
  • sequence identity will be understood to mean the "match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity which counts as identical, substitutions involving conservative substitutions.
  • the percentage similarity between a particular sequence and a reference sequence is at least about 20% to 40% or at least about 30% to 60% or at least about 70% or at least about 80% or at least about 90% or at least about 95% or above such as at least about 96%, 97%, 98%, 99% or greater.
  • Percentage similarities or identities between 60% and 100% are also contemplated such as 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.
  • kits may be in the form of vectors, libraries, cells etc.
  • low stringency includes and encompasses from at least about 0 to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions.
  • low stringency is at from about 25-30°C to about 42 0 C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions.
  • Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0,5 M to at least about 0.9 M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization, and at least about 0.01 M to at least about 0.15 M salt for washing conditions.
  • medium stringency which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0,5 M to at least about 0.9 M salt for washing conditions
  • high stringency which includes and encompasses from at least about 31% v/v to at least about 50% v/v form
  • T m of a duplex DNA decreases by TC with every increase of 1% in the- number of mismatch base pairs (Bonner et al, Eur. J. Biochem., 46:83, 1974).
  • Formamide is optional in these hybridization conditions. Accordingly, particularly preferred levels of stringency are defined as follows: low stringency is 6 x SSC buffer, 0.1% w/v SDS at 25-42°C; a moderate stringency is 2 x SSC buffer, 0.1% w/v SDS at a temperature in the range 20°C to 65°C; high stringency is 0.1 x SSC buffer, 0.1 % w/v SDS at a temperature of at least 65 0 C.
  • similarity includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non- identity at the nucleotide level, “similarity” includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level, “similarity” includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels (exemplary conservative substitutions are listed in Table A). In a particularly preferred embodiment, nucleotide and amino acid sequence comparisons are made at the level of identity rather than similarity.
  • references to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence”, “comparison window”, “sequence similarity”, “sequence identity”, “percentage of sequence similarity”, “percentage of sequence identity”, “substantially similar” and “substantial identity”.
  • a “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e.
  • sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window” refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence.
  • the comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
  • GAP Garnier et al.
  • FASTA Altschul et al.
  • TFASTA Pearson-Prot al.
  • flow cytometry is particularly convenient in high throughput systems.
  • flow cytometry is a high throughput technique which involves rapidly analyzing the physical and chemical characteristics of particles as they pass through the path of one or more laser beams while suspended in a fluid stream. As each cell or particle intercepts the laser beam, the scattered light and fluorescent light emitted by each cell or particle is detected and recorded using any suitable tracking algorithm.
  • fluorophores is particularly useful
  • Other detectable markers for use in this format include luminescence and phosphorescence as well as infrared dyes as mentioned above.
  • an “analyte” includes any molecule of biological interest and includes without limitation: cytokines, hormones, antigens (including one or more epitopes or mimetopes), forensic samples, antibodies, haptens, enzymes, natural products, components of chemical libraries, drugs including those of veterinary or pharmaceutical interest, environmental constituents and the like.
  • fusion polypeptide or “chimeric polypeptide” or “hybrid polypeptide” are interchangeably used to mean a polypeptide comprising two or more associated polypeptides which are expressed as part of the same expression product, or which are generated by synthetic means. Fusion polypeptides may comprise two or more polypeptides and intervening regions such as, for example, linker or spacer regions. In particular, regions which permit or directly or indirectly facilitate a particular surface topology may be selected. Polypeptide topology in a viral particle may be assessed for example by protease protection assay or by determining interactivity with antibodies.
  • Subject refers to an animal, preferably a mammal and more preferably human.
  • a patient regardless of whether a human or non-human animal may be referred to as an individual, subject, animal, host or recipient.
  • the molecules and methods of the present invention have applications in human medicine, veterinary medicine as well as in general, domestic or wild animal husbandry.
  • an "animal” includes an avian species such as a poultry bird, an aviary bird or game bird.
  • the preferred animals are humans or other primates, livestock animals, laboratory test animals, companion animals or captive wild animals.
  • laboratory test animals include ducks, snow geese, mice, rats, rabbits, guinea pigs and hamsters.
  • Rabbits and rodent animals, such as rats and mice provide a convenient test system or animal model.
  • Livestock animals include sheep, cows, pigs, goats, horses and donkeys.
  • Non-mammalian animals such as avian species, zebrafish and amphibians are also contemplated.
  • sample is used in its broadest context to include purified or unpurified compositions from a subject, laboratory or environment.
  • the sample is a biological sample collected from an antibody containing fluid from a subject and may include without limitation tissue or cells from any tissue such as blood, plasma, lymph, saliva or other mucous secretions, tears, spinal fluid and so forth. It should be understood that reference to a sample includes samples which have undergone some form of processing as well as samples taken directly from a subject, environment or laboratory.
  • Processing may include such steps as dilution, filtration or other separation techniques or maceration.
  • Fusion proteins may be produced using well known techniques by those of skill in the art, such as those summarised in molecular biology laboratory manuals for example, Sambrook and Russell "Molecular Cloning - A Laboratory Manual” Cold spring Harbour Press, 2001 (incorporated herein by reference). Expression systems and vectors are also described in Sambrook and Russell (supra) together with purification and re-folding protocols. Specifically, expression systems may use for example, bacterial, mammalian, yeast or insect host cells depending on the size and nature of the molecule to be expressed. A wide range of plasmids are commercially available for the expression of fusion proteins. Chimeric proteins and multimeric molecules are generated using equivalent procedures.
  • Synthetic fusions are also routinely generated by those of skill in the art using conventional liquid or solid phase chemistries. Reference may be made in this regard to synthesis as described in "Synthetic Vaccines” edited by Nicholson, Blackwell, Scientific Publications and particularly Chapter 9 entitled “Peptide Synthesis”. Synthetic peptide combinatorial libraries are also produced, for example, as described by Houghten et ⁇ l, Nature, 354:84- 86, 1991. E-Boc or f-moc chemistries are described in Stewart and Young, “Solid Phase Peptides Synthesis", 2nd ed., Pierce Chemical Co., Rockford, 111., 1984.
  • peptide libraries are synthesized with adhesive peptide sequences to enhance subsequent immobilization strategies.
  • Antigens may generally be identified using well known techniques, such as those summarized in Paul, “Fundamental Immunology", 3rd ed., 243-247, Raven Press, 1993 and references cited therein. Such techniques include screening polypeptides and overlapping fragments for the ability to react with antigen-specific antibodies, antisera and/or T-cell lines or clones. As used herein, antisera and antibodies are "antigen-specific” if they specifically bind to an antigen (i.e., they react with the protein in an ELISA or other immunoassay, and do not react detectably with unrelated proteins). Antigen fragments may react at a level that is similar to or greater than the reactivity of the full length polypeptide.
  • a polypeptide may be immobilized via adhesive peptides on a solid support and contacted with patient sera to allow binding of antibodies within the sera to the immobilized polypeptide. Unbound sera may then be removed and bound antibodies detected, for example using a labeled Protein A.
  • Monoclonal antibodies are conveniently prepared in pure form and in large quantities.
  • the preparation of hybridoma cell lines for monoclonal antibody production derived by fusing sensitized lymphocytes with an immortal cell line and selecting specific antibody producers is well known in the art by now standard procedures such as those described in Harlow and Lane (supra); and Kohler and Milstein, European Journal of Immunology, 6:511-519, 1976.
  • the present invention also provides methods of screening for agonists or antagonists of the interaction between the amino acid sequences of the subject invention and binding partners comprising contacting the peptide sequence with a compound and assaying for (i) the presence of a complex between the peptide sequence and a compound or (ii) for the presence of complex between the peptide sequence and a ligand, by methods well known in the art. In such competitive binding assays the peptide sequence or the ligand is labelled in order to assess the activity of the compound.
  • the present invention also provides a method for screening for agonists or antagonists of the activity of peptides having adhesive properties identified herein comprising exposing the peptide sequences to a compound and assaying for:-
  • a change in the level of an indicator of the activity of the adhesive peptide sequence may be tested in in vitro assays.
  • Target molecule may be expressed recombinantly or occur naturally or be upregulated in cells or cell lines which are useful in in vitro screens for agonists or antagonists.
  • Natural products include those from coral, soil, plant or the ocean or antarctic environments.
  • Two-hybrid screening is another useful method for identifying binding partners.
  • the present invention provides a method for the treatment or prophylaxis of a disease or condition comprising administering a therapeutic amount of a compound which modulates the activity of a herein described adhesive peptide sequences in genetic or proteinaceous form.
  • the disease is caused by a pathogenic mycobacterium such as M. tuberculosis.
  • the present invention is directed to the treatment of a disease or condition in any animal of commercial or humanitarian interest including plants, primates, livestock animals including fish and birds, laboratory test animals, companion animals, or captive wild animals.
  • compositions which are prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18 th Ed., Mack Publishing, Company, Easton, PA, U.S.A., 1990.
  • the composition may contain the active agent or pharmaceutically acceptable salts of the active agent.
  • These compositions may comprise, in addition to one of the active substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g. intravenous, oral, intrathecal, epineural or parenteral.
  • the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, powders, suspensions or emulsions.
  • any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets).
  • tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques.
  • the active agent can be encapsulated to make it stable to passage through the gastrointestinal tract while at the same time allowing for passage across the blood brain barrier. See for example, International Patent Publication No. WO 96/11698.
  • the compound may be dissolved in a pharmaceutical carrier and administered as either a solution or a suspension.
  • suitable carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin.
  • the carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like.
  • the compounds When the compounds are being administered intrathecally, they may also be dissolved in cerebrospinal fluid.
  • the active agent is preferably administered in a therapeutically effective amount.
  • the actual amount administered and the rate and time-course of administration will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage, timing, etc. is within the responsibility of general practitioners or specialists and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington's Pharmaceutical Sciences, supra.
  • targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibodies or cell specific ligands. Targeting may be desirable for a variety of reasons, e.g. if the agent is unacceptably toxic or if it would otherwise require too high a dosage or if it would not otherwise be able to enter the desired cells.
  • the vector could be targeted to the desired cells or expression of expression products could be limited to specific cells, stages of development or cell cycle stages.
  • the cell based delivery system is designed to be implanted in a patient's body at the desired site and contains a coding sequence for the target agent.
  • the agent could be administered in a precursor form for conversion to the active form by an activating agent produced in, or targeted to, the cells to be treated. See, for example, European Patent Application No. 0 425 73 IA and International Patent Publication No. WO 90/07936.
  • Non-conventional Code Non-conventional Code amino acid amino acid
  • D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm
  • D-N-methylcysteine Dnmcys N-(3 ,3 -diphenylpropyl)glycine Nbhe
  • D-N-methyllysine Dnmlys N-methyl- ⁇ -aminobutyrate Nmgab ⁇ N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet
  • Phage display was used to identify peptide with adhesive properties using technology known to those skilled in the art. A number of 12-mers were identified independently by this process as set out below.
  • Trp His Trp Ser Trp lie GIn Asn Ala Ala Pro Asn (1)
  • Exemplary peptides No. 1 and No. 3-8 ⁇ supra were from one bio-panning, exemplary peptides No. 2 and No. 9-12 were from a second bio-panning, exemplary peptides No. 13 and 14, plus one isolate of exemplary peptide No. 1, were from a third bio-panning.
  • Adhesive Peptide 1 is the "normal" motif (equivalent to exemplary peptide 1 ⁇ supra in Example 1) while Adhesive Peptide 2 has a histidine transposition.
  • the underlined sequence is the RESA (ring-infected erythrocyte surface antigen) epitope from Plasmodium falciparum.
  • Control peptide (P3) has the amino acid sequence that is normally found in this position adjacent to the RESA epitope in the full-length antigen.
  • each of these peptides were bound at various concentrations to streptavidin- coated microtitre plates, plates were then blocked and the amount of peptide bound was detected using monoclonal antibody 28/2 and HRPO-conjugated anti-mouse IgG. All peptides had similar antigenic reactivity when bound to plates via biotin, giving maximal absorbances of around 0.5 ( Figure 1).
  • these adhesive peptides demonstrated enhanced binding even to polyvinylchloride. This suggests that the adhesive properties of the peptides are not limited to any one surface, and may be useful for many other surfaces including (but not limited to) glass, plastics, teflon, metals such as gold and the like.
  • the sequence is from Protein bpoB of Mycobacterium tuberculosis. There was no equivalent sequence in M. bovis or M. leprae indicating that the sequence is adhesive for pathogenesis.
  • Peptides 1 to 14 correspond to the family of related peptides identified in biopanning experiments described in Example 1 (SEQ ID NO: 1 to 14).
  • Peptides, Pepl and Pep2 correspond to Adhesive Peptide 1 (SEQ ID NO: 18) and Adhesive Peptide 2 (SEQ ID NO: 19), respectively (supra) and are the same except that aa2 and aa6 of Pepl are aa6 and aa2 of Pep2.
  • Pep3 corresponds to P3 (supra) (SEQ ID NO: 20) and demonstrates that histidine alone is not sufficient for binding.
  • Adhesive peptides to bind to a range of different substrates and surfaces
  • the adhesive peptides were shown to bind strongly to a wide variety of plastic and membrane surfaces, demonstrating their utility for the great majority of diagnostic or analytical applications.
  • Peptide 1, Peptide 2 and Peptide 3 (non-adhesive control peptide) according to Example 2 were prepared as dilutions and bound to a variety of multiwell plate surfaces (Greiner Medium, Greiner High, Nunc Maxisorp, Nunc Polysorp, Nunc Medisorp, Polyvinyl chloride) according to the description in Example 2, with bound peptide detected by ELISA probing with monoclonal antibody 28/2 against the RESA epitope.
  • Peptide 1 and Peptide 2 were found to have greatly enhanced binding relative to Peptide 3 on all plate surfaces, although the absolute level of binding varied between plates with Polysorp having very low binding. The results are shown in Figure 5.
  • Peptide 1, Peptide 2 and Peptide 3 (non-adhesive control peptide) according to Example 2 were prepared as dilutions and bound to a variety of multiwell plate surfaces (Greiner Medium, Greiner High, Nunc Maxisorp, Polyvinyl chloride) according to the description in the legend to Figure 3, with bound peptide detected by probing with Streptavidin- peroxidase.
  • Peptide 1 and Peptide 2 were found to have greatly enhanced binding relative to Peptide 3 on all plate surfaces, although the absolute level of binding varied between plates with Greiner medium having lower binding. The results are shown in Figure 6.
  • Peptide 1, Peptide 2 and Peptide 3 (non-adhesive control peptide) according to Example 2 were prepared as dilutions and bound to a variety of membrane surfaces (PVDF,
  • Peptide 1 , Peptide 2 and Peptide 3 (non-adhesive control peptide) according to Example 2 were prepared as dilutions and bound to PorexTM polyethylene membrane (T3 hydrophilic membrane) using a Bio DotBlot apparatus (BioRad) according to the description in the legend of Figure 3, with bound peptide detected by probing with Alexa 680-Streptavidin. Results were scanned by exposure to the Odyssey infrared scanner (LiCor Inc.). Peptide 1 and Peptide 2 were found to have greatly enhanced binding relative to Peptide 3 with Peptide 2 being approximately 25-fold higher binding than Peptide 1 on this membrane. The results are shown in Figure 8.
  • the phage display process used to discover the original peptides yields peptide inserts of 12 amino acids. However, it is apparent from Figure 4 that the first 5 to 8 amino acids show the highest level of conservation between the individual phage displayed peptides.
  • a series of synthetic peptides were prepared based on the first 8 or 9 amino acids of Peptide 2 (WTWQWHPW or WTWQWHPWS, respectively).
  • Peptides were prepared based on the first 9 amino acids of Peptide 2 plus the RESA epitope and a C-terminal Biotin residue. Individual peptides were prepared with substitution of 0, 1, 2, or 3 of the 4 Tryptophan residues (W) from the C-terminus of the adhesive peptide sequence, to yield P2 (WTWQWHPWS) SEQ ID NO: 22, P4 (WTWQWHPLS) SEQ ID NO: 23, P5 (WTWQLHPLS) SEQ ID NO: 24, and P6 (WTTQLHPLS) SEQ ID NO: 25.
  • P3 is equivalent to Peptide 3 of Example 2 (ENVPEHVQH) SEQ ID NO: 26.
  • Peptides were prepared with addition of 1 or 2 glycine residues to the N-terminus, or progressive truncations from the C-terminus or the N-terminus of this 8 amino acid sequence (Peptide 2 (WTWQWHPW)), as shown in the legend to Figure 10. All peptides had an additional sequence (GGKPLAQGSG-biotin) at their C-terminus. Peptides were bound to Nunc Maxisorp plates according to the description in Figure 3 of the original specification, with bound peptide detected by probing with Streptavidin-peroxidase (Figure 10).
  • the adhesive peptide sequence is more effective when placed at the N-terminus of the protein/peptide, much less effective when placed at the C-terminus of the peptide. We presume that it will also be ineffective when placed internally, since this is not adjacent to the N-terminus. Note that the 2 glycine addition at N-terminus is acceptable.
  • Individual peptides were prepared by substitution of the first and/or second W residues in this sequence (Wi and W 2 respectively), using the alternative naturally-occurring amino acids Y, F, or A, or the synthetic (non-conventional) amino acids DW, DY and DF (D- amino acids of W, Y and F respectively) or synthetic amino acids denoted as 60 (L-l-beta- Naphthylalanine), 103 (Beta-(3-Pyridyl)-L-alanine), 161 (Nz-Dabcyl-L-lysine), 264 (beta- (2-quinolyl)-alanine) or 389 (Phenyl-phenylalanine; Biphenylalanine).
  • Peptides were bound to Nunc Maxisorp plates or Titertek polystyrene plates according to the description in the legend to Figure 3, with bound peptide detected by probing with Streptavidin-peroxidase. Results are expressed as optical density of the reaction (mean of 3 determinations). Peptides were ranked by descending order of reactivity for each plate. See Table 4 and 5.
  • 264 appear suitable on Titertek plates, with Peptide 31 (A 1 A 2 ) and Peptide 29 (264[264 2 ) being ranked 1 and 8 respectively on these plates, however they are much less suitable on
  • Maxisorp being ranked 12 and 28 respectively.
  • this method of substitution can be used with any chosen plate or membrane surface to identify the optimal adhesive peptide sequence by experimental testing of each substitution for the chosen plate surface.
  • Comparison of the ranked reactivity of peptides within each plate (Maxisorp or Titertek) demonstrates that some amino acids are equivalent to, or better than W in the first (Wi) and/or second (W 2 ) positions.
  • Peptide 18 having W in the first position and 103 in the second position (Wi IOS 2 ) demonstrates the second-ranked binding to both Maxisorp and Titertek plates, better than the W]W 2 peptide (Peptide 1) which is ranked 4 on Maxisorp, and 9 on Titertek.
  • Tyrosine is also suitable, with YiY 2 (Peptide 23) being ranked 5 on Maxisorp (compared to 4 for W]W 2 ), and 3 on Titertek (compared to 9 for W]W 2 ).
  • Phenylalanine (F) is generally less suitable than Y or W, with Fi or F 2 -containing peptides being ranked no higher than 11 on Maxisorp, and 14 on Titertek.
  • each of the D-amino acids shows reduced binding compared to the corresponding L-amino acids.
  • Peptide 27 (60i60 2 ) and Peptide 30 (389]389 2 ) demonstrate the two lowest levels of binding on both plate surfaces, and peptides with 389 in either position (Peptides 10, 20 and 30) are ranked no higher than 27 on either plate. This result demonstrates that the presence of aromatic or hydrophobic amino acid side-chains is not sufficient for the adhesive peptide properties.
  • Peptide 1 and Peptide 2 were used to bind Streptavidin to microtitre plates (A) or Porex membrane (B), allowing the subsequent binding of other biotinylated macromolecules via the remaining (approximately 3) biotin-binding sites on the tetrameric Streptavidin protein.
  • Peptide 1, Peptide 2 and Peptide 3 (non-adhesive control peptide) were bound to Nunc Maxisorp plates at a concentration of 0.1 ⁇ g/ml. After washing, serial dilutions of Streptavidin were added to peptide-coated wells for 1 hour at room temperature, or to uncoated wells overnight at 4°C as a control for passive binding of Streptavidin. Wells were then washed, blocked, and probed with biotinylated monoclonal antibody (MAb) K3- 4C8 (0.1 ⁇ g/ml).
  • MAb biotinylated monoclonal antibody
  • Peptide 1, Peptide 2 and Peptide 3 (non-adhesive control peptide) were bound to Porex T3 membrane at a concentration of 0.1 ⁇ g/ml using a Bio DotBlot apparatus. After washing, serial dilutions of Streptavidin were added to peptide-coated wells for 1 hour at room temperature, or to uncoated wells overnight at 4°C as a control for passive binding of Streptavidin. Wells were then washed, blocked, and probed with biotinylated monoclonal antibody (MAb) K3-4C8 (0.1 ⁇ g/ml).
  • MAb biotinylated monoclonal antibody
  • the amount of MAb bound (reflecting the binding capacity of Streptavidin bound directly or via peptides to the plate) was quantitated by probing with Alexa 680-labelled anti-mouse IgG and scanning using the Odyssey infrared imager.
  • Peptide 1 and Peptide 2 greatly enhanced the binding capacity for biotinylated macromolecules compared to Peptide 3 and to passive binding of Streptavidin. The results are shown in Figure 12.
  • the adhesive peptide WGWQWGPW (SEQ ID NO: 58) was prepared as an iV-Hydroxysuccinimide (NHS) active ester, and conjugated to monoclonal antibody (MAb) K2-4F2 (antibody to hepatitis A virus) or MAb PH-315 (antibody to human IgM) by mixing at 4°C for two days.
  • MAb monoclonal antibody
  • MAb PH-315 antibody to human IgM
  • Binding of the unconjugated MAbs to Porex membrane was inefficient, requiring 20 ⁇ g/ml of each MAb to yield a strong fluorescent signal.
  • Conjugation of the MAbs to adhesive peptide resulted in an approximate 5-fold increase in the total amount of binding, requiring only 4 ⁇ g/ml of each MAb to yield a strong fluorescent signal, equivalent to that seen with 20 ⁇ g/ml of unconjugated MAb.

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Abstract

L'invention concerne un peptide adhésif renfermant la séquence d'acides aminés Xaa1 A1 Xaa2 A2 Xaa3 A3 Xaa4 Xaa5 A4, ou une partie correspondante dont Xaa représente un quelconque acide aminé, A1 à A4 sont sélectionnés indépendamment parmi un acide aminé hydrophobe choisi parmi Ala, Gly, Ile, Phe, Pro, Met, Trp, Tyr, Val, des isomères D ou L associés et un analogue fonctionnel substitué par un substituant non polaire tel que, par exemple, un alkyle, un alcényle, un alkynyle, un aryle ou un substituant hétérocyclique ou un dérivé fonctionnel associé contenant au moins environ 20 % d'une similitude de séquences d'acides aminés.
PCT/AU2005/001775 2004-11-23 2005-11-23 Peptides possedant des proprietes adhesives a utiliser dans un dosage de phase solide et des regimes de synthese de polymeres chimiques WO2006056009A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102713629A (zh) * 2009-11-20 2012-10-03 俄勒冈健康科学大学 用于检测结核分枝杆菌感染的方法
DE102012110664A1 (de) * 2012-11-07 2014-05-08 Henkel Ag & Co. Kgaa In Beschichtungsmitteln, Haftvermittlern oder Klebstoffen für oxidische Oberflächen einsetzbare Peptide
WO2017034211A1 (fr) * 2015-08-26 2017-03-02 윤원준 Adhésif protéique pour adhérence ou revêtement de surface inorganique
WO2018048290A1 (fr) * 2016-09-12 2018-03-15 윤원준 Composition et procédé de culture permettant de cultiver des cellules souches sans cellules nourricières

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5223409A (en) * 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
US5498530A (en) * 1991-10-16 1996-03-12 Affymax Technologies, N.V. Peptide library and screening method
WO2005049852A2 (fr) * 2003-11-17 2005-06-02 University Of Florida Procedes et compositions destines a induire l'apoptose

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5223409A (en) * 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
US5498530A (en) * 1991-10-16 1996-03-12 Affymax Technologies, N.V. Peptide library and screening method
WO2005049852A2 (fr) * 2003-11-17 2005-06-02 University Of Florida Procedes et compositions destines a induire l'apoptose

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102713629A (zh) * 2009-11-20 2012-10-03 俄勒冈健康科学大学 用于检测结核分枝杆菌感染的方法
DE102012110664A1 (de) * 2012-11-07 2014-05-08 Henkel Ag & Co. Kgaa In Beschichtungsmitteln, Haftvermittlern oder Klebstoffen für oxidische Oberflächen einsetzbare Peptide
WO2017034211A1 (fr) * 2015-08-26 2017-03-02 윤원준 Adhésif protéique pour adhérence ou revêtement de surface inorganique
WO2018048290A1 (fr) * 2016-09-12 2018-03-15 윤원준 Composition et procédé de culture permettant de cultiver des cellules souches sans cellules nourricières

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