WO2010133893A1 - Procédé - Google Patents

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Publication number
WO2010133893A1
WO2010133893A1 PCT/GB2010/050838 GB2010050838W WO2010133893A1 WO 2010133893 A1 WO2010133893 A1 WO 2010133893A1 GB 2010050838 W GB2010050838 W GB 2010050838W WO 2010133893 A1 WO2010133893 A1 WO 2010133893A1
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Prior art keywords
protein
artibody
binding molecule
binding
exposed portion
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PCT/GB2010/050838
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English (en)
Inventor
Mikhail Soloviev
Bazbek Davletov
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Royal Holloway And Bedford New College
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Publication of WO2010133893A1 publication Critical patent/WO2010133893A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/75—Fibrinogen
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins

Definitions

  • the present invention relates to a method of producing a binding molecule and binding molecules produced by the method.
  • affinity reagents such as antibodies, antibody mimics, their fragments, aptamers and all other types of affinity reagents
  • an affinity reagent must be complementary to the surface of the antigen molecule at the interface of the affinity reagent and antigen.
  • affinity reagents are generally sought and developed against folded proteins, folded fragments, folded protein products of post-translational modification, or any other complete structure of the protein or protein fragment, including peptides or even haptens. In this way, proteins or protein fragments are generally considered to be "complete" unchangeable units against which affinity reagents are developed.
  • the aim is to achieve a high degree of complementarity between the surfaces of the two molecules at their interface.
  • Such an interface is stabilised through a combination of hydrogen bonds (with or without additional water molecules), ionic and van der Waals interactions, and through spatial complementarity between the two molecules. Devising such an interface is the main challenge in the field of antibody research and development.
  • the general direction in antibody research and development is to ensure that the antigenic protein (or protein fragment) structure remains intact through the antibody development process. This is to develop antibodies, etc. against the native conformation of a protein.
  • affinity reagents when binding to a particular part of a protein, may compete against the individual structural fragments of the protein itself. For example, where a native protein has two regions of secondary structure which bind together, an affinity reagent could compete with the first region of secondary structure to bind to the second region of secondary structure. This takes advantage of the fact that protein folding is not an irreversible process and that the elements of the protein secondary or supersecondary structure, or protein motifs and small domains already possess the appropriate structure to enable a very good fit with the remaining part of the protein structure.
  • Protein structure is not permanently fixed but is adaptable and individual elements of the protein's own primary, secondary, tertiary, or quaternary structures are labile, i.e. they can move apart from other parts of the protein and are not fixed in the final native completely folded state (unless covalently cross-linked, for example, with disulphide bonds). Therefore, even after a protein is completely folded into its final native state, portions of the protein may unfold and re-fold to a certain extent. For example, the so called "assisted protein folding" in nature relies on a similar phenomenon to unfold and re-fold imperfectly folded proteins. Protein re-folding is also being widely used in research, e.g.
  • fragments of a protein's own sequence and structure if presented to the otherwise complete and folded protein, will compete with the corresponding fragments of that protein molecule and will bind to that protein.
  • the inventors have termed this use of fragments of the protein of interest as binding molecules as the artificial antibody approach or "artibody" approach.
  • the artibody approach is principally different from any existing approaches in that the artibody itself is a fragment of the protein which is used for recognising the complementary elements of that same protein.
  • the artibody approach allows an affinity reagent or binding molecule to be designed based solely on knowledge of the target antigen structure/sequence. This approach is fundamentally different to the affinity reagent generation approaches used previously and has never before been considered as a suitable method for the rational design of new affinity reagents or binding molecules.
  • J. G. Hall & C. Frieden. P.N.A.S. USA, Vol. 86, pp. 3060-3064 (1989) also relates to inhibitors of protein folding.
  • the inhibitors disclosed in this document bind to internal regions of the protein, not near the surface of the target protein in place of a part of the target protein. The inhibitors are not used on the completely folded protein.
  • beta breaker peptide simply binds to the surface of the target protein. It does not bind in place of a portion of the target protein.
  • WO 96/39834 relates to the use of peptides to bind to proteins to stop amyloid formation such as in Alzheimer's disease. These peptides bind to the surface of the target protein rather than binding in place of a portion of the target protein near the surface of the target protein. Dwyer, Life Sciences, Vol. 45, pp. 421-429 (1989) discloses two proteins binding to each other at the surface of the protein. This document does not disclose a peptide binding to the target protein in place of a surface exposed portion of the target protein.
  • the present invention provides a method of producing a binding molecule, the method comprising the steps of: selecting a surface exposed portion of a fully folded target protein; and producing a binding molecule to the target protein, wherein the binding molecule comprises a peptide having a sequence which has substantial identity with at least a substantial part of the sequence of the surface exposed portion of the fully folded target protein thereby allowing the binding molecule to bind to the target protein in place of the surface exposed portion of the target protein.
  • the sequence of the peptide binding molecule is based on the sequence of the portion of the target protein which is displaced when the binding molecule binds to the remaining portion of the target protein. This means that the conformation of the binding molecule in the binding region will be similar or identical to the conformation of the binding region of the surface exposed portion.
  • the binding molecule competes with and displaces the fragment of the target protein to which it corresponds. It displaces the solvent exposed portion of the target protein upon which the peptide of the binding molecule is based.
  • the binding molecule should be close to the target protein and should be able to mimic the structure of the target protein.
  • the peptide of the binding molecule In order for the peptide of the binding molecule to interact with the target protein by displacing the original fragment (i.e. the solvent exposed portion), the peptide of the binding molecule should assume the spatial position and orientation of the displaced fragment of the target protein.
  • binding region when used with respect to the binding molecule means the region defined by the amino acids which are exposed on the surface of the binding molecule and which interact with the target protein in order to cause the binding molecule to bind to the target protein. This is the region of the binding molecule which interacts with the target protein in place of the surface exposed portion.
  • binding region when used with respect to the surface exposed portion of the target protein means the region defined by the amino acids which interact with the target protein in order to cause the surface exposed portion to bind to the target protein. This is the region of the surface exposed portion which forms interactions with the target protein and is normally buried when the surface exposed portion is bound to the target protein in its native state.
  • the target protein that is selected in the method can be any protein to which it is desired to produce a binding molecule.
  • the target protein must have a surface exposed portion upon which the binding molecule can be based.
  • the target protein may be a protein which is formed from a number of subunits, i.e. it is a multimer. In such an embodiment, the protein as a whole is considered to be the target protein. Therefore, the solvent exposed portion should be at the surface of the protein when in its multimeric configuration. An internal portion of one of the subunits, which is on the outside surface of one of the subunits but which is on the inside of the protein in its multimeric state, is not a solvent exposed portion.
  • the target protein is a protein which is a single peptide or monomer. For example, this may be a single polypeptide chain or multiple polypeptide chains chemically linked together so that a single protein unit is formed.
  • the target protein does not have any quaternary structure.
  • the term "surface exposed portion” means a portion of the sequence of the target protein which is at the surface of the target protein so that at least one side of the surface exposed portion is on the outside of the protein.
  • the surface exposed portion is at the surface of the target protein when the target protein is in a fully folded state. This may be the native, fully folded conformation of the target protein.
  • Some proteins form dimers where the two proteins bind together at their surfaces. In such a dimer, the areas where the two monomers bind cannot be considered to be a surface exposed portion because, although the area is at the surface of one of the monomers, it is not surface exposed in the native dimer protein. Therefore, the surface exposed portion should be at the surface of the fully folded target protein when it is in its native state.
  • the binding molecule will not be able to bind to the target protein if its binding site is sterically hindered by the presence of, for example, another protein monomer.
  • the surface exposed portion is generally partially embedded in the surface of the protein but must be able to be displaced from the rest of the target protein.
  • the surface exposed portion may form a defined structure at the surface of the target protein.
  • the surface exposed portion may form a stable structure.
  • the surface exposed portion is a discreet structural element such as a portion of secondary structure.
  • the sequence at the C-terminal end of the protein may form a helix which lies on the surface of the protein in its native state. Such a helix is one possible surface exposed portion of the protein.
  • This helix may be partially embedded in that it lies in a groove formed by the rest of the target protein. However, at least one side of the helix will be at the surface of the protein and the helix can be displaced from the rest of the target protein.
  • a protein may have a ⁇ -strand which lies at the surface of the protein in its native state and is partially embedded in the protein.
  • Such a ⁇ -strand is another possible surface exposed portion of the protein provided that it can be displaced from the rest of the protein to allow a binding molecule, which is based on the ⁇ -strand, to bind to the protein.
  • the surface exposed portion contains at least one polar amino acid. More preferably, the surface exposed portion contains a mixture of polar and non-polar amino acids.
  • the surface exposed portion of the target protein may be any suitable structure.
  • the surface exposed portion may be a portion of primary structure, secondary structure, supersecondary structure, motif, domain or tertiary structure element.
  • it may be a helix, a ⁇ -strand, a ⁇ -turn, a random coil, or a motif such as a helix-turn- helix, ⁇ -hairpin motif, or another motif comprising one or more helices and/or ⁇ - strands.
  • the surface exposed portion may simply be a portion of secondary structure.
  • the surface exposed portion may be a helix, ⁇ -strand or ⁇ -hairpin motif.
  • the sequence of the surface exposed portion can be located anywhere in the sequence of the target protein as long as it forms a structure which is exposed on the surface of the target protein.
  • the surface exposed portion may be located at or near the C- terminal or N-terminal of the target protein. Alternatively, it may be located within the sequence of the target protein, i.e. not at or near the C-terminal or N-terminal of the target protein. Preferably, the surface exposed portion is not located at the N- terminal of the target protein.
  • the surface exposed portion may be located at or near the C-terminal of the target protein.
  • about 90% of proteins have a surface exposed C-terminal domain.
  • C-terminal and N-terminal mean that the surface exposed portion is located at the C-terminal end or at the N-terminal end of the target protein.
  • the surface exposed portion of the target protein may be any suitable structure. This may be formed from a continuous stretch of amino acids in the sequence of the target protein, for example, a helix, a ⁇ -strand or a ⁇ -hairpin motif. Alternatively, it may be formed from distinct stretches of amino acids in the sequence of the target protein which are adjacent in the native conformation; for example, two ⁇ -strands which are at a distance from each other in the sequence of the protein but are adjacent in the native protein to form a ⁇ -sheet.
  • the surface exposed portion is formed by a continuous stretch of amino acids in the sequence of the target protein. This allows the easy design of the binding molecule as it is based on the sequence of the surface exposed portion.
  • the surface exposed portion has at least one flexible end capable of forming a hinge. This allows the surface exposed portion to be displaced from its native position in the target protein relatively easily so that the surface exposed portion allows the binding molecule to bind to the target protein. Having a flexible hinge allows the surface exposed portion to be displaced or to move relatively easily so that the binding molecule can bind in its place.
  • the surface exposed portion has two flexible ends.
  • An end of a surface exposed portion may be flexible as a result of being connected to an amino acid or amino acids which allow a large degree of movement, for example, glycine, serine or alanine, and/or as a result of being connected to another flexible amino acid or acids, for example, lysine, arginine, asparagine, glutamic acid, aspartic acid, glutamine or threonine. It will be readily apparent to a person skilled in the art whether or not the surface exposed portion has a flexible end. For example, the flexibility of amino acid chains is discussed in Karplus and Schulz, Naturwissenschaften, 72, 212 (1985); Huang et al.
  • an end of a surface exposed portion may be flexible as a result of being close to an end of the protein (such as the N-terminus or C-terminus) so that the end is not constrained by interactions with other parts of the protein.
  • An end of the surface exposed portion can be considered to be flexible if its movement is not constrained.
  • the movement of an end of the surface exposed portion might be considered to be constrained if it is prevented from leaving its natively folded position. This may be because of strong non-covalent interactions with surrounding amino acids. Alternatively, it may be because the end is physically restricted from moving, e.g. by being blocked by other elements of the protein structure, because of covalent interactions with other amino acids (for example, through disulphide bonds) or by having insufficient size, such as length, to allow any movement.
  • the surface exposed portion preferably has a sequence containing between about 3 and about 100 amino acids. More preferably, the surface exposed portion has a sequence which contains between about 5 and about 50 amino acids, more preferably still, between about 8 and about 40 amino acids and, even more preferably, the surface exposed portion has a sequence which contains between about 10 and about 30 amino acids. As will be appreciated by one skilled in the art, the number of amino acids contained in the sequence of the surface exposed portion will depend on the nature of the surface exposed portion itself. For example, if the surface exposed portion is a helix, the sequence may contain between about 8 and about 40 amino acids whereas if the surface exposed portion is an intramolecular loop, the sequence may contain between about 3 and about 15 amino acids.
  • the target protein is a protein having a known structure.
  • regions potentially suitable for the generation of binding molecules can be predicted using a variety of approaches which are well known to those skilled in the art. Many of these can be found at http://www.expasy.org/tools/. For example, some suitable approaches are as follows:
  • SWISS-MODEL Workspace A web-based environment for protein structure homology modelling. Bioinformatics, 22,195-201; Kopp J. and Schwede T. (2004) The SWISS-MODEL Repository of annotated three-dimensional protein structure homology models Nucleic Acids Research 32, D230-D234; Schwede T, Kopp J, Guex N, and Peitsch MC (2003) SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Research 31 : 3381-3385; Guex, N. and Peitsch, M. C.
  • SWISS-MODEL and the Swiss-PdbViewer An environment for comparative protein modelling. Electrophoresis 18: 2714-2723; Peitsch, M. C. (1995) Protein modeling by E-mail Bio/Technology 13: 658-660; Bates, P.A., Kelley, L.A., MacCallum, R.M. and Sternberg, M.J.E. (2001) Enhancement of Protein Modelling by Human Intervention in Applying the Automatic Programs 3D-JIGSAW and 3D-PSSM. Proteins: Structure, Function and Genetics, Suppl 5:39-46; Bates, P.A. and Sternberg, M.J.E. (1999) Model Building by Comparison at CASP3: Using Expert Knowledge and Computer Automation.
  • Geno3D Automatic comparative molecular modelling of protein. Bioinformatics, 2002, 18, 213-214; and Geourjon C, Combet C, Blanchet C, Deleage G. Identification of related proteins with weak sequence identity using secondary structure information. Protein Sci 2001 Apr;10(4):788-97.
  • Such methods include: SOPM (Geourjon and Deleage, 1994); SOPMA (Geourjon and Deleage, 1995); HNN (Guermeur, 1997); MLRC (Guermeur et al, 1999); DPM (Deleage and Roux, 1987); DSC (King and Sternberg, 1996); GOR I (Gamier et al, 1978); GOR nUGibrat ef a/.. 1987); GOR IV (Gamier et al.. 1996); PHD (Rost and Sander, 1993); PREDATOR (Frishman and Argos. 1996); SIMPA96 (Levin, 1997).
  • the need to determine or to predict protein structure may be obviated by making of a set of partially overlapping peptides covering the whole or part of the target protein sequence and experimentally testing each peptide for its binding propensity. Even without the knowledge of the protein structure, such an approach will provide a significantly faster and cheaper way to identify a binding molecule compared to traditional immunisations or in vitro screening techniques.
  • the sequence of the binding molecule is based on at least a substantial part of the sequence of the surface exposed portion of the target protein.
  • the binding region of the binding molecule possesses the same or a similar structure as the surface exposed portion or adopts this structure on binding to the target protein by displacing the surface exposed portion. This allows the binding molecule to bind to the rest of the target protein in place of the surface exposed portion with relatively high selectivity and specificity since the binding molecule has the same or a similar sequence compared to the surface exposed portion of the target protein.
  • the sequence of the binding molecule does not have to be identical to the sequence of the surface exposed portion of the target protein or a part of the surface exposed portion. Not all of the amino acids in the surface exposed portion of the target protein will be involved in interacting or fitting with the rest of the target protein in order to allow the surface exposed portion to bind to the rest of the target protein. Since the surface exposed portion is located at the surface of the target protein, some of the amino acids in the sequence of the surface exposed portion will be on the surface of the protein and, therefore, they will not interact with the rest of the target protein. Therefore, these amino acids do not have to be identical in the binding molecule in order for the binding molecule to have an identical conformation to the surface exposed portion in the binding region.
  • the surface exposed portion of the target protein is a helix which lies in a groove in the rest of the target protein
  • only approximately 50% of the amino acids in the helix may interact with and bind to the rest of the protein.
  • These will be the amino acids down one side of the helix which fit into the groove in the rest of the target protein.
  • the amino acids on the other side of the helix may not interact and bind with the groove in the rest of the protein. Therefore, when producing a binding molecule, it is not always necessary for these amino acids in the binding molecule to be identical to the ones in the surface exposed portion because they do not interact with the groove and so may not play a role in binding.
  • amino acids of the binding molecule which are identical to the amino acids of the surface exposed portion are in the binding region of the binding molecule.
  • Amino acids in other areas of the binding molecule can also be identical and this may also be beneficial, for example, it may help to ensure the structure of the binding molecule remains similar to that of the surface exposed portion.
  • the conformation of the binding molecule in the binding region should be substantially the same as the conformation of the displaced surface exposed portion in the binding region so that the binding molecule can bind to the target protein in place of the surface exposed portion.
  • the conformation of the binding molecule and the surface exposed portion do not have to be identical in their binding regions for the binding molecule to be able to bind to the target protein.
  • the conformation of the binding molecule in the binding region can differ slightly and the binding molecule will still be able to bind to the target protein.
  • the identity must be sufficient so that the binding region of the binding molecule can bind to the target protein.
  • at least about 80% of the amino acids in the binding region of the binding molecule are identical to those in the surface exposed portion, more preferably, at least about 85%, more preferably still, at least about 90%, even more preferably, at least about 95% and, most preferably, 100%.
  • one or more amino acids may be removed from the sequence of the surface exposed portion.
  • amino acids in the binding region of the binding molecule which should remain unchanged compared to the surface exposed portion of the target protein are those amino acids which are responsible for the interactions and which provide the complementarity of the shape and surface of the interacting groups, hence causing the surface exposed portion to bind to the rest of the target protein.
  • amino acids are on the whole the ones with buried side chains and are likely to be, for example, large aliphatic, aromatic or sulphur containing amino acids. They are likely to be mostly hydrophobic amino acids.
  • the binding molecule has at least about 50% identity with the surface exposed portion or a substantial part thereof. More preferably, the binding molecule has at least about 60% identity with the surface exposed portion or a substantial part thereof, even more preferably, at least about 70% identity, more preferably still, at least about 80% identity, even more preferably, at least about 85% identity, more preferably still, at least about 90% identity, even more preferably, at least about 95% identity, and most preferably, 100% identity. These percentages refer to the identity between the surface exposed portion and the binding molecule based on the sequence of the surface exposed portion.
  • the binding molecule is complexed or conjugated to another moiety, for example, another protein
  • the conjugated or complexed moiety is not taken into consideration when assessing the overall identity between the binding molecule and the surface exposed portion of the target protein.
  • this additional amino acid or acids are added at the end of the binding region of the binding molecule.
  • the additional amino acid or acids may be added in internal regions of the binding region of the binding molecule.
  • the additional amino acid or acids provide strong interactions. Suitable amino acids are aliphatic, aromatic, sulphur containing or polar amino acids. This helps to increase the number of interactions which take place between the binding molecule and the target protein thereby helping to improve the binding affinity of the binding molecule.
  • the binding affinity of the binding molecule may also be improved by increasing the rigidity of the binding molecule. This may be done by introducing cysteine residues in appropriate positions in the sequence to form disulphide bridges which help to stabilise the structure of the binding molecule.
  • the binding molecule can be incorporated into a suitable protein scaffold to help increase the rigidity of the binding molecule.
  • suitable protein scaffolds can be engineered from any protein having stable tertiary structure and a large number of examples have been reported and are well known to those skilled in the art. For example, suitable protein scaffolds are discussed in Hosse et al. A new generation of protein display scaffolds for molecular recognition. Protein Science (2006), 15:14-27 and Binz et al. Engineering novel binding proteins from nonimmunoglobulin domains. Nature Biotechnology, v.23 n.lO (2005) pp 1257-1268.
  • the amino acids of the binding molecule may be modified to allow them to interact with the displaced surface exposed portion.
  • the amino acids that can be modified are those which are exposed on the surface of the binding molecule once the binding molecule has bound to the target protein. These amino acids will not be in the binding region of the binding molecule.
  • the displaced portion of the target protein (the surface exposed portion), which was partially buried, will become exposed upon binding of the binding molecule.
  • This displaced surface exposed portion is likely to be in close proximity to the binding molecule. Therefore, the binding of the binding molecule can be further stabilised if the formally buried amino acids of the surface exposed portion can be made to interact with the 'unused' amino acids on the surface of the binding molecule.
  • This modification process can be done using rational design or affinity selection and saturation. This helps to increase the affinity of the binding molecule for the target protein as the binding molecule binds to the target protein as well as interacting with the displaced surface exposed portion.
  • the binding molecule has a sequence which is substantially identical with at least a substantial part of the sequence of the surface exposed portion of the target protein.
  • the expression "a substantial part of the sequence of the surface exposed portion” means that the binding molecule may be based on a substantial part of the surface exposed portion. For example, if a helix is selected as the surface exposed portion on which the binding molecule is based, the sequence of the binding molecule does not have to be based on the whole sequence of the helix.
  • the binding molecule may be based on the sequence of the first half of the helix. In this way, the sequence of the binding molecule has identity with a substantial part (i.e. the first half) of the sequence of the surface exposed portion.
  • the binding molecule must have a sequence which is substantially identical to a large enough portion of the surface exposed portion so that it can displace at least some of the surface exposed portion and bind to the target protein in place of the surface exposed portion.
  • the part of the sequence of the surface exposed portion on which the binding molecule is based must be large enough so that the binding molecule produced is able to bind to the target protein and displace the surface exposed portion.
  • the binding molecule has identity with at least 50%, more preferably at least 70%, even more preferably at least 90%, even more preferably at least 95%, and most preferably 100% of the surface exposed portion of the target protein. It is most preferred that the binding molecule is based on the whole sequence of the surface exposed portion.
  • the sequence of the binding molecule which allows it to bind to the target protein is preferably between about 3 and about 100 amino acids. More preferably, the sequence of the binding molecule is between about 3 and about 50 amino acids, more preferably still, the sequence of the binding molecule is between about 5 and about 40 amino acids and, even more preferably, between about 10 and about 30 amino acids.
  • the number of amino acids contained in, for example, the elements of secondary or supersecondary structure, or short motifs will vary between different proteins, domains, motifs and secondary structure elements. Therefore, the sequence and the number of amino acids in the sequence of the binding molecule will partly depend on the nature and size of the surface exposed portion that has been selected.
  • the sequence of the binding molecule may contain anywhere between about 3 and about 50 amino acids.
  • the sequence of the binding molecule may contain anywhere between about 3 and about 100 amino acids.
  • the sequence of the binding molecule may be between about 3 and about 10 amino acids.
  • the binding molecule is preferably specific for the protein target.
  • the binding molecule is a relatively short polypeptide, it can be produced relatively easily using chemical synthesis. Suitable methods are well known to those skilled in the art.
  • the binding molecule of the invention discussed above may optionally be conjugated to or complexed with an additional moiety.
  • the moiety may be any suitable moiety and the identity of the moiety will depend on the intended function of the conjugate or complex.
  • the binding molecule may be conjugated to or complexed with a molecule, such as a polypeptide, which causes homo- or hetero-oligomerisation.
  • the molecule may cause dimerisation, trimerisation, tetramerisation, pentamerisation or polymerisation.
  • the binding molecule may be conjugated to or complexed with a molecule which allows attachment to other molecules or surfaces such as biotin which allows attachment to avidin coated surfaces.
  • the binding molecule may be conjugated to or complexed with functional molecules such as inhibitors, enzymes, toxins, and regulators. Further, the binding molecule may be conjugated to or complexed with peptides, other binding molecules (with the same or different specificity), antibodies, antibody mimics, antibody fragments or antibody mimic fragments which retain their binding functionality, aptamers, functional proteins, structural proteins, nucleic acids, lipids, carbohydrates, natural cell metabolites, chemically synthesised unnatural moieties, aromatic compounds, organic compounds, inorganic compounds, pharmaceutically active ingredients, chemical groups, metals, non-metals, particles, gels, reporter molecules or groups, or labels such as fluorescent, luminescent, quenchers, radioactive, magnetic, non-magnetic, isotopic or spin labels (as in EPR or ESR).
  • functional molecules such as inhibitors, enzymes, toxins, and regulators.
  • the binding molecule may be conjugated to or complexed with peptides, other binding molecules (with the same or different specificity),
  • the additional moiety may optionally be conjugated via a linker.
  • Suitable linkers are well known to those skilled in the art.
  • the binding molecule may comprise other modifications which can be used to improve binding or physical properties of the binding molecule. These include but are not limited to: acylation, acetylation, deacetylation, alkylation, amidation, biotinylation, carboxylation, glutamylation, glycosylation, glycation, glycylation, hydroxylation, iodination, isoprenylation, lipoylation (e.g.
  • prenylation myristoylation, farnesylation, geranylgeranylation
  • oxidation palmitoylation, pegylation, phosphopantetheinylation, phosphorylation, polysialylation, pyroglutamate formation, ubiquitination, citrullination, deamination, deamidation, eliminylation, dehydration, and decarboxylation.
  • binding molecules produced by the invention have a wide range of uses and display a number of advantages over affinity reagents described in the prior art.
  • the binding molecules of the invention do not rely on any pre-defined protein scaffold, for example, as in the case of antibodies which have a very particular immunoglobulin protein scaffold which holds the complementarity determining regions (CDRs) in place, or in the case of numerous known antibody mimics, for example, as discussed in Hosse et al. A new generation of protein display scaffolds for molecular recognition. Protein Science (2006), 15:14-27 and Binz et al. Engineering novel binding proteins from non-immunoglobulin domains. Nature Biotechnology, v.23 n.lO (2005) pp 1257-1268.
  • CDRs complementarity determining regions
  • the structure of the binding molecule is much simpler which means that the binding molecule can be produced much more easily.
  • the binding molecule may be produced by chemical synthesis.
  • the binding molecule is capable of folding correctly without any help or scaffold since the protein structure on which the binding molecule is based can fold correctly to give a functional native protein.
  • binding molecules are based on the sequence of known proteins, it is not necessary to use complicated techniques to produce them such as animal immunisation or in vitro affinity selection using large libraries and a "display system" (e.g. cell display, phage display, ribosome display, mRNA display, ARM, yeast-two- hybrid or similar systems). Further, affinity maturation can be avoided to develop the binding molecules. If affinity maturation is used, it is greatly simplified using the binding molecules of the invention. Further, the binding molecules do not require library screening for the design of the affinity reagent against a known protein target.
  • display system e.g. cell display, phage display, ribosome display, mRNA display, ARM, yeast-two- hybrid or similar systems.
  • the method allows a "rational" approach to complete de novo design of binding molecules based on the target protein structure or even the sequence alone, and does not rely on any other proteins, protein scaffolds or any other scaffolds and does not require the knowledge of other protein sequences or structures.
  • TPR tetratricopeptide
  • PBP E.coli periplasmic binding protein
  • binding molecules are based on the sequence of a known protein and therefore adopt a similar or identical conformation to that known protein, they are naturally relatively highly selective for their target protein because the interactions between the binding molecule and the target protein are the same or similar as those found in the native protein. Therefore, this produces a relatively stable structure. Further, the binding molecules can be denatured without the loss of their binding ability and will subsequently refold to their correct functional state. This makes them very robust so that they are likely to have a relatively long storage life and can survive being subjected to relatively harsh conditions. This means that binding molecules can be regenerated and reused and are therefore advantageous over traditional antibodies in affinity-based assays and especially in such applications where multiple use of the affinity reagent may be required.
  • Such examples include but are not limited to blots, arrays, affinity chromatography, chromatography resins, other immunosorbent substrates including ELISA, beads, particles microparticles, nanoparticles, microfluidic and chip-based applications, Lab-on-a-Chip, Biacore, other SPR based assays, QCM based assays, optical and spectrometric fluorescence and mass spectrometry detection, scanning microscopy and other instrumental measurements.
  • the robust character of the binding molecules makes them especially suitable for therapeutics applications because they are more stable against extreme pH conditions, denaturing media and can be easily made protease-resistant.
  • a further advantage of the binding molecules of the invention is that the binding molecule may affect the function of the target protein as it may cause its conformation to change to a non-native state. This is as a result of the surface exposed portion of the target protein being displaced from its original position and the associated changes in the target protein conformation. This allows the binding molecules to be used to affect protein function. This can be used to down regulate protein function or disrupt protein-protein interaction networks (especially useful in cancer therapies), thus providing definitive advantage over traditional approaches, e.g. therapeutic antibodies.
  • the ability to affect the function of the target protein is the intrinsic ability of the binding molecule, due to the mechanism of interaction with the target protein. This mechanism is principally different form protein-protein interaction involved in traditional antibody-antigen or antibody mimics-antigen interaction. Since the binding molecules resemble fragments of native proteins which originate from the protein surface (e.g. normally exposed to the immune system), there is a reduced risk of developing an immune response against such a fragment as these portions of protein are likely to have been exposed to the immune system previously.
  • binding molecules are relatively small so higher immobilisation densities can be achieved (e.g. on arrays) and they show increased permeability which is advantageous for use in imaging, cytochemistry, histochemistry, and therapeutic applications.
  • the binding molecules have a defined position within the target protein, hence multiple interaction sites and multiple binding molecules are possible, so improved affinity/specificities are achievable (this is not always possible with large IgG antibodies which, because of their size, will not always be able to bind to more than one binding site on their antigen simultaneously, especially on smaller antigens).
  • the present invention provides a method of producing a binding molecule, the method comprising: producing a binding molecule to a fully folded target protein, wherein the binding molecule comprises a peptide having a sequence which has substantial identity with at least a substantial part of the sequence of a surface exposed portion of the fully folded target protein thereby allowing the binding molecule to bind to the target protein in place of the surface exposed portion of the target protein.
  • the present invention also provides a binding molecule obtained by the method of the first or second aspects of the invention.
  • the present invention provides a binding molecule comprising one of the sequences selected from SEQ ID NOS: 1-39, 41-53 and 55-86, wherein the binding molecule can bind to a target protein displacing a surface exposed portion of the target protein.
  • the binding molecule according to the third aspect of the present invention has a maximum length of 100 amino acids, more preferably less than 50 amino acids, more preferably still, less than 30 amino acids and most preferably less than 22 amino acids.
  • binding molecule according to the third aspect of the present invention consists of one of the sequences selected from SEQ ID NOS: 1-39, 41-53 and 55-86.
  • binding molecules can be used in a number of different ways.
  • the binding molecules may be used in pharmaceutical, medical, veterinary, biological and biotechnology research and development applications.
  • the binding molecules can also be used in research, medical diagnostics, therapy and biotechnology.
  • the invention provides the binding molecule described above for use in therapy or diagnostics.
  • the present invention also provides the use of the binding molecule described above.
  • the present invention provides a pharmaceutical composition comprising the binding molecule described above in combination with a pharmaceutically acceptable excipient.
  • binding molecule is extensively used. The skilled person will appreciate that the features of the binding molecule are equally applicable to the peptide which forms the binding molecule. In the majority of cases the terms "binding molecule” and "peptide” are exchangeable.
  • the binding molecule is a peptide which has a sequence that has substantial identity with at least a substantial part of the sequence of the surface exposed portion of the fully folded target protein thereby allowing the binding molecule to bind to the target protein in place of the surface exposed portion of the target protein.
  • Figure 1 shows the selected target protein structure - a fragment of human Fibrinogen sequence shown in the form of a double-D dimer from human fibrin (PDB ID: IFZE, panel A) and the devised artibody (named "FBB204", panel B).
  • the dashed line indicates the position of the selected artibody within the target protein structure.
  • Panel C shows the alignment of the artibody sequence with that of the target protein (Fibrinogen).
  • Panel D shows the specific binding of FBB204 artibody as measured using ELISA assay and Peroxidase conjugated FBB204 (2 measurements). Open bars show the binding of FBB204 artibody to Fibrinogen, filled bars show displacement of that binding by the excess of unlabelled FBB204 (filled bars) and therefore prove the specificity of the interaction.
  • Figure 2 shows the selected target protein structure - a single subunit of human C- reactive protein (PDB ID: 1LJ7, panel A) and the devised artibody (named "CRP206", panel B).
  • the dashed line indicates the position of the selected artibody within the target protein structure.
  • Panel C shows the alignment of the artibody sequence with that of the target protein (CRP).
  • Panel D shows the specific binding of CRP206 artibody as measured using ELISA assay and Peroxidase conjugated CRP206 (2 experiments). Open bars show the binding of CRP206 artibody to CRP, filled bars show displacement of that binding by the excess of unlabelled CRP206 (filled bars) and dashed bars show the binding of CRP206 artibody to an irrelevant target protein (BSA). Both of the negative controls show nearly identical values and either can be used in the calculation of the specific artibody binding.
  • FIG. 3 shows human C-reactive protein (PDB ID: 1LJ7, panel A) and the devised artibodies (named “CRP206” and “CRP205", panel B).
  • the dashed lines indicate the position of the selected artibodies within the target protein structure.
  • Panel C shows the alignment of the CRP206 artibody sequence with that of the target protein (CRP).
  • Panel D shows the alignment of the CRP205 artibody sequence with that of the target protein (CRP).
  • Panel E shows the structure of the face-to-face decamer multisubunit CRP. The position of the artibodies are indicated with arrows.
  • CRP205 is prevented from binding to the multimerised CRP, whilst CRP206 binds (means of two measurements, panel F).
  • FIG 4 shows human myoglobin (PDB ID: 2MM 1) and the devised binding molecules (artibodies named "MG202", “MG201”, “MG301”, “NlOl” and “MGlOl", panel A).
  • the dashed lines indicate the position of the selected artibodies within the target protein structure.
  • Panels B,C,D and E show the alignment of the "MG202", “MG201”, “MG301” and “NlOl” artibody sequences (respectively) with that of the target protein (myoglobin).
  • Artibody "MGlOl” is constructed from two identical "NlOl” fragments.
  • Panel F specific binding of MGlOl, MG201, MG202 and MG301 artibodies to human myoglobin (all artibodies were conjugated to peroxidase and were used in a standard ELISA type assay). Open bars show normalised values for total binding. Nonspecific binding was determined using the excess of unconjugated artibodies (filled bars) or by measuring binding to an irrelevant protein BSA (dashed bars).
  • Figure 5 shows that binding affinity of artibodies is comparable with that of monoclonal antibodies.
  • Panel A binding of a monoclonal antibody clone [40Fl 1] (from Abeam) to human fibrinogen using ELISA assay.
  • Panel B binding of FBB204 artibody to human fibrinogen using ELISA assay. Both plots show Ln(binding), arbitrary units (the vertical axes) vs. time in seconds (horizontal axes). The slope of the fitted linear curve shows the dissociation rate kd for each of the affinity reagents tested.
  • Figure 6 shows that binding of an artibody results in structural changes in the target protein, as indicated by the disappearance of binding of a monoclonal antibody [40Fl 1] to the same protein (in the presence of an excess of an artibody FBB204). Both values for measured using identically loaded, assayed and washed wells.
  • FIG. 7 shows green fluorescent protein GFP (PDB ID: IEMA, Panel A) and the devised artibody (named "GFP307", panel B).
  • the dashed line indicate the position of the selected artibody within the target protein structure.
  • Panel C shows the alignment of the "GFP307” artibody sequence with that of the target protein (GFP).
  • Panel D shows fluorescence spectra of the GFP incubated with and without GFP307 artibody. The excitation was at 387 nm in both cases.
  • Thinner dotted line shown the fluorescence emission spectrum of GFP incubated without artibody, and the thicker dotted line shows the emission spectrum of GFP incubated with GFP307 artibody.
  • Figure 8 shows the selected target protein structure - a single subunit of human C- reactive protein (PDB ID: 1LJ7, panel A) and the devised artibody (named "CRP206", panel B).
  • the dashed line indicates the position of the selected artibody within the target protein structure.
  • Panel C shows design of the CRP306 artibody, which consists of a 16 amino acid long binding region (consisting of two beta strands and a beta turn), followed by an 8 amino acid long linker, followed by 55 amino acid long fragment of a homo-oligomeric pentamerising peptide from Phe-14 protein (which forms a helix, shown as a cylinder on panel C), followed by a three amino acid long C-terminal fragment to allow labelling, cross-linking or immobilisation of the artibody on surfaces.
  • Panel D shows homo-oligopentamerisation mechanism of the CRP306 artibody.
  • Figure 9 shows the selected target protein structure - a human leptin (PDB ID: 1AX8, panel A) and the devised artibody (named "LP302", panel B).
  • the position of the disulphide bond is indicated on Panel A.
  • the dashed line indicates the position of the selected artibody within the target protein structure.
  • Panel C shows the alignment of the LP302 artibody sequence with that of the target protein (Leptin).
  • Figure 10 shows the selected target protein structure - a fragment of human Fibrinogen sequence shown in the form of a double-D dimer from human fibrin (PDB ID: IFZE, panel A), the devised binding region (Panel B, this is similar to the FBB204 artibody, as in Figure 1).
  • Panel C shows the artibody "BIA311", which consists of a binding region (underlined) a linker region and a C-terminal region containing four lysine residues (doubly underlined). The dashed line indicates the position of the selected artibody within the target protein structure.
  • Figure 11 is a surface plasmon resonance curve showing the binding kinetics of Artibody BIA311, immobilised on BIAcore chip CM5, with Fibrinogen (the target antigen). Horizontal axis - time in seconds, vertical axis - specific binding (RU, arbitrary units).
  • Figure 12A is a surface plasmon resonance curve showing the binding kinetics of Artibody BIA311, immobilised on BIAcore chip CM5. The Artibody is contacted with an irrelevant target (4180 - 4480 seconds), washed with Gly/NaCl solution (4623-4923 seconds), and then contacted with Fibrinogen (5100-5400) with 0.1% n- octyl- ⁇ -D-glucoside added. Horizontal axis - time in seconds, vertical axis - specific binding (RU, arbitrary units).
  • Figure 12B shows an enlarged portion of the graph of Figure 12 A.
  • Figure 13A is a surface plasmon resonance curve showing the binding kinetics of Artibody BIA311, immobilised on BIAcore chip CM5, and contacted with progressively higher concentrations of Fibrinogen. A wash cycle is carried out between each Fibrinogen sample.
  • Figure 13B shows association and dissociation curves for each sample of Fibrinogen (as shown in Figure 13A). The curves are overlaid onto each other; washing/regeneration curves are not shown.
  • Figure 13C shows fitting of the dissociation curves using commercially available software (provided with the Biacore2000 instrument).
  • Figure 13D shows fitting of the dissociation curves using commercially available software (provided with the Biacore2000 instrument). Horizontal axis in all figures - time in seconds, vertical axis - specific binding (RU, arbitrary units).
  • Figure 14A is a surface plasmon resonance curve showing the binding kinetics of Artibody FBB204, immobilised on BIAcore chip CM5 through the SH- group of an internal cysteine using EDC/NHS and PDEA/Na-borate chemistry, when contacted with 0.016% n-octyl- ⁇ -D-glucoside (1760 - 1940 seconds), washed with 1% SDS/0.1M NaOH solution (2060 - 2180 seconds), then contacted with 1 mg/ml Fibrinogen in the presence of 0.016% n-octyl- ⁇ -D-glucoside (2300 - 2480 seconds), and followed by the next wash/regeneration cycle (2600 - 2720 seconds).
  • FIG. 14B shows binding of Fibrinogen to Artibody FB204 depending on the n-octyl- ⁇ -D- glucoside concentration. Horizontal axis - concentration of n-octyl- ⁇ -D-glucoside in %, vertical axis - specific binding (RU, arbitrary units).
  • Figure 15 is a surface plasmon resonance curve showing the immobilisation of TBA Artibodies on a BIAcore chip using EDC/NHS and PDEA/Na-borate chemistry, followed by 3 x 1 min washes with 1%SDS/1M NaOH. First channel was loaded with TBA405, second - with TBA406, third - TBA 407, fourth - TBA 408. Horizontal axis - time in seconds, vertical axis - specific binding (RU, arbitrary units).
  • Figures 16 A, 16B, 16C and 16D are surface plasmon resonance curves showing real time binding of the recombinant Iy expressed Botulinum toxin A protein to four different TBA Artibodies immobilise in a BIacore chip.
  • time 5920 seconds corresponds to the end of the chip regeneration/wash cycle (SDS/NaOH wash);
  • 6040 seconds is the start of application and binding of the Botulinum toxin to each of the TBA Artibodies;
  • 6160 seconds correspond to the end of association, beginning of the dissociation stage; 6160-8060 seconds is the dissociation stage.
  • Timepoint 8060 seconds is the start of the next regeneration stage (SDS/NaOH wash).
  • Artibody TBA405 is shown in Figure 16A.
  • Artibody TBA406 is shown in Figure 16B.
  • Artibody TBA407 is shown in Figure 16C.
  • Artibody TBA408 is shown in Figure 16D.
  • the term “artibody” is used which is comparable to the term “binding molecule” used above. However, the meaning of these words should not be considered to be identical. Further, the terms “artibody forming region” and “artibody forming sequence” are used which are comparable to the term “surface exposed portion” used above.
  • an artibody molecule can be designed against a blood protein
  • an artibody molecule can be designed against a glycoprotein
  • an artibody molecule can be designed against a cardiovascular disease marker; - a single secondary structure element can be used to make a binding artibody molecule;
  • beta strand structure can be used to make a binding artibody molecule
  • a C-terminal fragment of a target protein can be used to make a binding artibody molecule
  • a non-identical variant of the target protein can be used to design an artibody against the target protein if required.
  • the structure used to design the FBB204 artibody was of a fibrin dimer - a shorter processed and dimerised form of fibrinogen - but the artibody can bind unprocessed fibrinogen;
  • - artibody can be covalently attached to another protein (e.g. Peroxidase) through one of the artibody ends;
  • another protein e.g. Peroxidase
  • - artibody can be used instead of traditional antibodies or antibody mimics to bind selected protein targets in an affinity assay
  • - that artibody can be used to bind selected protein targets in an enzyme linked affinity assays, such as ELISA;
  • artibody can be designed in a way that quaternary structure of the target protein will not affect artibody binding, (i.e. D-dimer vs. fibriogen)
  • Example 1 Detailed description of Example 1 :
  • Selected protein target Fibrinogen
  • MSMKIRPFFPQQ (SEQ ID NO: 1);
  • MSMKIRPFFPQQGSC SEQ ID NO: 2;
  • MSMKIRPFFPOOGSC SEQ ID NO: 2 to introduce SH- group for labelling.
  • Peroxidase was attached covalently to the FBB204 through C-terminal Cysteine
  • FBB204 artibody binds Fibrinogen, the binding is specifically displaced by the excess of unlabelled FBB204, thus proving the specificity of the interaction.
  • Example 2 Artibodv design against human C-reactive protein ( Figure 2) Example 2 further shows that:
  • an artibody molecule can be designed against an acute-phase protein
  • an artibody molecule can be designed against an inflammatory marker protein
  • an artibody molecule can be designed against a protein linked to diabetes, hypertension, myocardial and cerebral infarcts;
  • an artibody can be designed against a protein which is differentially expressed in some forms of cancer, for example colon cancer or in response to the diet, smoking, physical exercise, and behavioural traits;
  • an artibody molecule can be designed against a protein which has more than one subunit
  • - more than one secondary structure element can be used to make a binding artibody molecule
  • an artibody molecule may contain a beta turn
  • - artibody forming region does not have to be at the C-terminus and can be in the middle of the target protein sequence
  • - binding part of the artibody sequence may differ slightly from the target protein sequence and does not have to match the target protein sequence 100%;
  • - artibody can be attached to another protein (e.g. Peroxidase) through one of the internal (not terminal) amino acids;
  • another protein e.g. Peroxidase
  • the value of the specific artibody binding can be obtained either by comparing total binding and the binding to the same target but measured in the presence of an excess of unlabelled artibody, or alternatively, by comparing the total binding and the binding to an irrelevant protein target.
  • CRP C-reactive protein
  • KDIGYSFTVGGSEILFE SEQ ID NO: 3
  • KDIGYSFTVGGCEILFE SEQ ID NO: 4;
  • CRP206 artibody binds CRP, the binding is specifically displaced by the excess of unlabelled CRP206.
  • artibody binding to BSA protein can be used as the negative control. The above two negative controls prove the specificity of the interaction.
  • Example 3 further shows that:
  • - more than one artibody can be designed against a selected protein target
  • - that artibody binding can be used to discriminate between different folding states of the target protein, or ligand binding by the target protein, such as for example the structural changes (face-to-face decamer multisubunit formation by CRP from two independent pentamers) associated with Ca 2+ binding by CRP;
  • artibody ELISA assays are not limited of a single wavelength and can be read at different wavelength, for example 660 nm;
  • CRP C-reactive protein
  • KDIGYSFTVGGSEILFE SEQ ID NO: 3
  • CRP206 artibody binds decamer multisubunit CRP, whilst CRP205 artibody is prevented from binding due to the shielding of the binding site by the complex quaternary structure of the decamer multisubunit CRP, showing that artibodies can be used to study structural traits of target proteins.
  • Example 4 Artibodies against human myoglobin ( Figure 4)
  • Example 4 further shows that:
  • an artibody molecule can be designed against a protein which is not normally present in the bloodstream;
  • an artibody molecule can be designed against a protein which is normally expressed and present in solid tissues;
  • an artibody molecule can be designed against a tissue marker
  • an artibody molecule can be designed against a protein which normally has only one subunit
  • an alpha helix structure can be used to make a binding artibody molecule
  • - N-terminal fragment of a target protein can be used to make an artibody molecule
  • - artibodies can be designed against discontinuous epitopes ;
  • - artibody may contain more than one copy of the binding fragment in one artibody molecule
  • a multimeric artibody can be designed by joining multiple artibodies in a single molecule, i.e. covalently.
  • Selected protein target Myglobin
  • GLSDGEWQL VLNVW (SEQ ID NO: 7) (N- terminal artibody forming fragment) and KDMASNYKELGFQG (SEQ ID NO: 9) (C- terminal artibody forming fragment);
  • KDMASNYKELGFQGC (SEQ ID NO: 10) (MG201), ASNYKELGFQGSSSC (SEQ ID NO: 10) (MG201), ASNYKELGFQGSSSC (SEQ ID NO: 10) (MG201), ASNYKELGFQGSSSC (SEQ ID NO: 10) (MG201), ASNYKELGFQGSSSC (SEQ ID NO: 10) (MG201), ASNYKELGFQGSSSC (SEQ ID NO: 10) (MG201), ASNYKELGFQGSSSC (SEQ ID NO: 10) (MG201), ASNYKELGFQGSSSC (SEQ ID NO: 10) (MG201), ASNYKELGFQGSSSC (SEQ ID NO: 10) (MG201), ASNYKELGFQGSSSC (SEQ ID NO: 10) (MG201), ASNYKELGFQGSSSC (SEQ ID NO: 10) (MG201), ASNYKELGFQGSSSC (SEQ ID NO: 10) (MG201), ASNY
  • MG202 C-terminal Cysteine added to introduce SH- group for labelling
  • MG301 three amino acid-long linker and one Cysteine added to introduce SH- group for labelling
  • NlOl two and five amino acid-long fragments added (N-terminal and C- terminal to the artibody binding site, one amino acid removed from the middle of the artibody forming region and one Cysteine added to introduce SH- group for labelling
  • NlOl a dimer of two NlOl artibodies, to which two and five amino acid-long fragments added (N-terminal and C-terminal to the first binding region, one amino acid removed from the middle of each of the two binding regions, a Cysteine is used to introduce SH- group for labelling (MGlOl).
  • NlOl and MGlOl are based on discontinuous sequence epitopes of the target protein. Peroxidase was attached covalently to the cysteines in all the artibodies.
  • Assay direct binding ELISA assay, read at 450 nm;
  • Result shown on Figure 4 include the MG301 and MGlOl binding to myoglobin.
  • MGlOl artibody which have only 7 amino acid long binding region(s) and -13% sequence difference with the target protein artibody forming region (one amino acid omitted form the 8 amino acid-long fragment) still bind myoglobin protein.
  • an artibody molecule possesses binding affinity comparable to that of a monoclonal antibody
  • Selected protein target Fibrinogen
  • MSMKIRPFFPQQ (SEQ ID NO: 14);
  • MSMKIRPFFPQQGSC SEQ ID NO: 15;
  • MSMKIRPFFPOOGSC SEQ ID NO: 15 to introduce SH- group for labelling.
  • Peroxidase was attached covalently to the FBB204 through C-terminal Cysteine
  • Assay direct binding ELISA assay, various incubation times, read at 450 nm; Monoclonal antibody used for comparison - clone [40Fl 1] (from Abeam), peroxidase conjugated antibody;
  • an artibody molecule can be designed to disrupt or change the structure of the target protein.
  • Selected protein target Fibrinogen
  • MSMKIRPFFPQQ (SEQ ID NO: 14);
  • MSMKIRPFFPQQGSC SEQ ID NO: 15;
  • MSMKIRPFFPOOGSC SEQ ID NO: 15 to introduce SH- group for labelling.
  • Peroxidase was attached covalently to the FBB204 through C-terminal Cysteine
  • Monoclonal antibody used - clone [40Fl 1] (from Abeam), peroxidase conjugated antibody.
  • FBB204 artibody abolishes binding of the monoclonal antibody [40Fl 1] to human fibrinogen.
  • FBB204 is designed to displace the corresponding artibody-forming region form the target protein. The only shielding effect therefore can come from the displaced fragment of the native fibrinogen (which proves the change on the target protein structure). Another alternative could be the disappearance of the epitope due to changes in the epitope structure. This would also directly prove the change in the target protein structure in response to the binding of FBB204 artibody.
  • - N-terminal fragment of a target protein can be used to make an artibody molecule
  • - Artibody can be designed against a protein of animal origin
  • - Artibody can be designed against a jellyfish protein
  • - Artibody can be designed against a protein which is naturally fluorescent
  • - Artibody can be designed against a protein which acquires ability to fluoresce following its recombinant production
  • - Artibody can be used to affect function of recombinantly produced protein
  • an artibody molecule can be designed to disrupt or change the function of the target protein, such as for example, but not limited to R&D and therapeutic applications.
  • GFP Green fluorescent protein
  • GFP fluorescent GFP protein was produced using in vitro transcription and translation system RTS 100 E. coli HY Kit from Roche. Assay: GFP was incubated with or without GFP307 artibody and with an irrelevant artibody; following an incubation fluorescent spectra were taken.
  • GFP307 artibody causes changes in the fluorescent properties of GFP, this confirms that artibodies can be used to modulate functional properties of the target proteins.
  • Example 8 further shows that:
  • a multimeric artibody can be designed by joining multiple artibodies non-covalently, for example by self-assembly or polymerisation; this can be achieved using molecules, such as but not limited to the elements of protein tertiary and quaternary structure, self-assembling proteins or other molecules capable of self-assembly such as nucleic acids, small molecules, and their combinations;
  • - protein helixes can be used to produce oligomeric artibodies.
  • CRP C-reactive protein
  • KDIGYSFTVGGSEILFE SEQ ID NO: 3
  • Example 9 further shows that:
  • an artibody molecule can be designed against a hormone; - an artibody molecule can be designed against a protein which is processed and has the signal peptide removed by signal peptidases;
  • an artibody molecule can be designed against a protein involved in metabolism
  • an artibody molecule can be designed against a protein involved in modulating immune response, angiogenesis, and reproduction;
  • an artibody molecule can be designed against a protein produced by adipose tissue, placenta ovaries, skeletal muscle, stomach, mammary epithelial cells, bone marrow, pituitary and liver;
  • an artibody molecule can be designed against a protein structure which contains intramolecular disulphide bonds.
  • Selected protein target Leptin
  • KVQDDTKTLIKTIVTRIN SEQ ID NO: 19
  • Example 10 further shows that:
  • an artibody molecule can be devised and modified to change the distribution of polar residues along the artibody molecule;
  • an artibody molecule can be devised and modified to change the distribution of charged residues along the artibody molecule;
  • an artibody molecule can be devised and modified to change the distribution of charge along the artibody molecule;
  • an artibody molecule can be devised and modified to achieve directional immobilisation on surfaces
  • an artibody molecule can be immobilised covalently on a solid surface
  • an artibody molecule can be immobilised on a Biacore chip surface; - an artibody molecule can be immobilised using NHS / EDC chemistry;
  • an artibody molecule can be covalently linked to carboxylic group
  • an artibody molecule can be used in real time affinity assays
  • an artibody molecule can be used in SPR based affinity assays.
  • Selected protein target Fibrinogen
  • MSMKIRPFFPQQ (SEQ ID NO: 1);
  • Step l
  • C-terminal (or N-terminal or intra-molecular) peptide preferably corresponding to a discrete element of existing structure, flanked by flexible hinges (e.g. GIy,) or small amino acids (Ala, Ser) - these will be the likeliest places where the native structure will "let go” and bend away.
  • GIy flexible hinges
  • small amino acids Al, Ser
  • Devise linkers, adapters, labels, other proteins or biologically relevant molecules are defined by:
  • Artibody BIA311 was immobilised on BIAcore chip CM5 using NHS/EDC coupling chemistry. This was contacted with Fibrinogen (the target antigen).
  • Fibrinogen the target antigen.
  • the polypeptide sequence of the BIA311 Artibody is MSMKIRPFFPQQGSGSKKKK (SEQ ID NO: 21) (the same as that of Example 10).
  • Figure 11 shows that fibrinogen binds to the BIA311 Artibody with fast kinetics.
  • - Artibody molecules can be used instead of traditional polyclonal or monoclonal antibodies, or other affinity reagents;
  • - Artibody molecules can be immobilised on planar surfaces without the loss of binding activity
  • - Artibody molecules can be immobilised by covalent binding
  • - Artibody molecules can be immobilised by covalent binding through reactive groups such as amino acid side chains;
  • Artibody molecules can be immobilised by covalent binding through an amino acid or amino acids located at or close to the termini of Artibody sequence (not situated at the position away from the termini of the Artibody molecule);
  • - Artibody molecules can be immobilised by covalent binding through lysine amino acid side chains
  • - Artibody molecules can be immobilised by covalent binding through lysine amino acids added to the main sequence of the Artibody polypeptide with the aim of covalent cross-linking;
  • - Artibody molecules can be directed towards the chip surface through the engineering of the desired physico-chemical properties, such as but not limited to their pi;
  • SPR surface plasmon resonance
  • Artibody BIA311 was immobilised on BIAcore chip CM5 and contacted with an irrelevant target (4180 - 4480 seconds), washed with Gly/NaCl solution (4623-4923 seconds) and then with Fibrinogen (5100-5400) to which 0.1% n-octyl- ⁇ -D-glucoside was added.
  • the polypeptide sequence of the BIA311 Artibody is given in Example 10.
  • Figure 12A shows no specific binding to the irrelevant target (FBB204), that the chip withstands a wash cycle, and that the Artibody binds Fibrinogen.
  • the same sensor surface was used, with the same number of immobilised BIA311 molecules, as in Example 12.
  • FBB204 is an Artibody devised against Fibrinogen and both BIA311 and FBB204 have the same active sequence based on the target Fibrinogen protein. FBB204 does not bind to the sensor surface, indicating that any interaction seen with Fibrinogen is not because the protein possesses the same sequence fragment, but because the Artibody binds to the complimentary protein structure, i.e. the rest of the Fibrinogen, by displacing the targeted region.
  • Figure 12B shows a zoomed in section of the binding graph (Figure 12A). This figure shows the increased binding of Fibrinogen to the BIA311 Artibody in the presence of n-octyl- ⁇ -D-glucoside (compared to binding without the additive as shown in Figure 11).
  • - Artibody molecules could be regenerated by standard washing buffers suitable for washing antigens bound to standard antibodies, such buffers as acidic glycine and/or high salt;
  • - Artibody molecules can be used to bind their targets in the presence of binding modulators
  • - Artibody molecules can be used to bind their targets in the presence of detergents
  • Artibody BIA311 was immobilised on BIAcore chip CM5 and contacted with a number of Fibrinogen samples (the target antigen) having different concentrations.
  • Figure 13 A the large peaks just below 21,500 correspond to wash cycles (Gly/NaCl solution).
  • Figure 13A shows that BIA311 is regenerated following each binding cycle. Progressively higher concentrations of Fibrinogen result in stronger binding.
  • Figure 13B shows association and dissociation curves overlaid onto each other; washing/regeneration curves are not shown.
  • Figure 13C shows fitting of the dissociation curves using commercially available software (provided with the Biacore2000 instrument).
  • Figure 13D shows fitting of the dissociation curves using commercially available software (provided with the Biacore2000 instrument).
  • Fitting of the binding curves results in the following binding rates for the BI A311 Artibody: kd ⁇ 7 x 10 ⁇ 3 ; ka ⁇ 2 x 10 3 ; KD ⁇ 3.5 x 10 "6 .
  • - Artibody molecules can bind their protein target and be regenerated/washed in a repetitive manner
  • Artibody FBB204 was immobilised on BIAcore chip CM5 through the SH- group of an internal cysteine using EDC/NHS and PDEA/Na-borate chemistry and contacted with 0.016% n-octyl- ⁇ -D-glucoside (1760 - 1940 seconds), washed with 1% SDS/0.1M NaOH solution (2060 - 2180 seconds), then contacted with 1 mg/ml Fibrinogen in the presence of 0.016% n-octyl- ⁇ -D-glucoside (2300 - 2480 seconds), and followed by the next wash/regeneration cycle (2600 - 2720 seconds).
  • the polypeptide sequence of the FBB204 Artibody is given in Example 1. The binding kinetics are shown in Figure 14A.
  • Figure 14B shows binding of Fibrinogen to Artibody FB204 depending on the n-octyl- ⁇ -D-glucoside concentration.
  • - Artibody molecules can be immobilised by covalent binding through Cysteine amino acid side chains
  • - Artibody molecules can be immobilised by covalent binding through an amino acid internal to the Artibody sequence (not situated at the position close to the C- or N- termini of the Artibody molecule);
  • - Artibody molecules can be stable and do not loose their binding ability after treatment with very harsh regeneration/washing buffers, such as SDS and NaOH;
  • Binding modulators can be used to exert a different effect on Artibody binding depending on their concentration
  • Table 1 below shows the fragments of the Botulinum toxin A protein which were used to design Artibodies against Botulinum toxin A (TBA405-408). Numbers in the left hand column refer to the amino acid position in the sequence of the toxin and the original Botulinum sequence (database Ace. No. A5HZZ9 or P 10845) is shown in parenthesis. Artibody sequences are shown on the right. These were synthesised chemically and used for binding Botulinum toxin A protein in ELISA and on BIAcore CN5 chip.
  • TBA Artibodies were immobilised on a BIAcore chip using EDC/NHS and PDEA/Na-borate chemistry, followed by 3 x 1 min washes with 1%SDS/1M NaOH. This is shown in Figure 15. (First channel was loaded with TBA405, second - with TBA406, third - TBA 407, fourth - TBA 408).
  • an Artibody molecule can be designed against a bacterial protein
  • an Artibody molecule can be designed against a protein target which is produced by expression in standard expression systems, such as a bacterial expression system;
  • an Artibody molecule can be designed against a toxin
  • - different Artibody molecules can be treated in sequential manner, for example, being loaded one by one; - different Artibody molecules can be treated in parallel, for example, being washed in a single washing step, i.e. in parallel;
  • a designed Artibody molecule can differ in its sequence from the respective sequence of the target region of the target protein
  • - Artibodies can be washed with stringent washing buffers such as 1%SDS 0.1M NaOH.
  • TBA Artibodies were immobilise in the same BIacore chip (as described in Example 16) and contacted with the recombinantly expressed Botulinum toxin A protein.
  • Figure 16 shows real time binding of the recombinantly expressed Botulinum toxin A protein to the four different TBA Artibodies immobilise in the BIacore chip.
  • time 5920 seconds corresponds to the end of the chip regeneration/wash cycle (SDS/NaOH wash);
  • 6040 seconds is the start of application and binding of the Botulinum toxin to each of the TBA Artibodies;
  • 6160 seconds correspond to the end of association, beginning of the dissociation stage; 6160-8060 seconds is the dissociation stage. No significant dissociation is detectable during that time indicating high affinity of interaction.
  • Timepoint 8060 seconds is the start of the next regeneration stage (SDS/NaOH wash).
  • Artibody TBA405 is shown in Figure 16 A.
  • Artibody TBA406 is shown in Figure 16B.
  • Artibody TBA407 is shown in Figure 16C.
  • Artibody TBA408 is shown in Figure 16D.
  • - multiple Artibody molecules can be washed and regenerated in a single washing step, where the same regeneration or washing buffer is passed over each of the different sensor surfaces; - Artibodies can bind their target and do not wash away easily, indicating good binding affinity.
  • Table 2 shows the designed Artibodies against the ionotropic Glutamate [NMDA] receptor protein, subunits NRl and NR2.
  • Receptor sequences used to design the Artibodies are shown on the left. The location of the target regions is near the agonist and antagonist or co-agonist binding sites of this channel- forming receptor.
  • PDB database entries "2A5T” and "3BTA” were used to model the Artibodies. Numbers refer to the amino acid position in the sequence of the receptor and the receptor protein sequence (database numbers are shown on the right of the species names).
  • Artibody names and the sequences of the chemically synthesised Artibodies are shown on the right. Underlined amino acids indicate spacers, linkers and amino acids with chemically reactive groups for subsequent chemical modifications. Control sequences (negative controls for relevant Artibodies also shown).
  • - Artibodies can be designed using existing protein structure, for example, available from protein structure databases;
  • - Artibodies can be designed against human proteins
  • - Artibodies can be designed against animal proteins
  • - Artibodies can be designed against animal proteins but can be used on human proteins;
  • - Artibodies can be designed against human proteins but can be used on animal proteins;
  • - Artibodies can be designed against one species and can be used for a different species
  • - Multiple Artibodies can be designed against matching human and non-human proteins or against matching proteins from different species; - The same Artibody can be designed to target multiple species within the same Genus;
  • Artibody can be designed to have different spacers, linkers and groups suitable for downstream uses, for example, chemical covalent labelling through an - SH group of an introduced cysteine or amino groups of introduced Lysines;
  • An Artibody may have one or more chemically reactive site introduced specifically for labelling or cross-linking and immobilisation (e.g. R1N401, R1N403);
  • An Artibody can be designed based on the regions of high sequence similarity between different proteins
  • a negative "control" Artibody can be designed based on the same target sequence
  • a negative "control" Artibody can be designed to have an amino acid sequence differing from that of the specific Artibody;
  • a negative "control" Artibody can be designed to have the same amino acid composition as that of the specific Artibody;
  • a negative "control" Artibody can be designed by reversing the amino acid sequence of the specific artibody
  • An Artibody can be designed to target a membrane protein
  • An Artibody can be designed to target a receptor
  • - Artibodies can be designed to target multiple subunits of the same protein target
  • An Artibody can be designed to target an agonist binding site of a receptor
  • An Artibody can be designed to target a co-agonist binding site of a receptor
  • An Artibody can be designed to have one or multiple sites suitable for subsequent covalent labelling of a molecular load with the aim of targeting or visualising the protein; these could be for example a fluorescent or radioactive label or a drug;
  • Glutamate [NMDA] receptor subunit zeta-1 (NR1 or R1N401 QSSVDIYFRRQVELSGC
  • NMDA-R1 (SEQ ID NO: 30)
  • TropC3 or TRP-3 Design of Artibodies against Transient receptor potential channel 3 (TrpC3 or TRP-3), for example, see Becker EB, Oliver PL, Glitsch MD, Banks GT, Achilli F, Hardy A,
  • Artibodies were designed against a protein which at the moment of Artibody design did not have any quaternary structure (3D structure) available. The exact transmembrane topology of the protein was also not known.
  • the Artibodies were designed after the structure of this protein was modelled using bioinformatics tools available on-line. At various stages of the Artibody design, the following tools were used. These are listed as examples and the Artibody design is not limited to the use of listed tools. Those with ordinary skill in the art will understand that to practice this invention alternative tools of protein structure analysis and modelling tools could be used. These tool were:
  • Artibodies TR3-701 to 706 are designed to bind to the region containing the third Ankyrin repeat and the labile fragment of TRCP3 structure between Ank3 and Ank4 domains. These Artibodies (701, 702, 703, 704, 705, 796, 707) are designed to disrupt the interaction of TRPC3 with its binding partners and affect protein Io causation/targeting .
  • Artibody TR3-707 is designed to target the region situated after the fourth Ank repeat which is predicted to match TRP 2 superfamily domain, which partially overlaps with the N-term of the predicted coiled-coil domain.
  • This Artibody (TR3-707) is designed to bind to TRPC3 as well as disrupt its interactions with binding partners and affect protein localisation/targeting.
  • Artibody TR3-708 is designed to target the region situated between two of the eight predicted transmembrane or membrane-associated domains. At the time of TR3-708 design no accurate knowledge of TRPC protein topology existed. This artibody (TR3- 708) is also designed to help to reveal protein topology and affect TRCP channel properties.
  • Artibodies are designed specifically to target the region shown as situated between the assumed transmembrane domains "3" and "4" shown on Figure 3 from Becker EB, Oliver PL, Glitsch MD, Banks GT, Achilli F, Hardy A, Nolan PM, Fisher EM, Davies KE. Proc Natl Acad Sci U S A. 2009 106(16):6706-l 1.
  • Artibody TR3-709 is designed to target the region situated immediately after the proline-rich region and extending nearly to the predicted coiled-coil / IP3R3 binding region.
  • Artibody PR-709 is designed to affect binding of interacting proteins in this region and to affect function of either of the proteins.
  • All Artibodies have been made synthetically, all Artibodies were synthesised to contain one fluorescent group (TAMRA) and have protected C-termini. All Artibodies have d-Serine at their C-termini and all but one artibody are amidated at their C- temini. All Artibodies should therefore be suitable for both in vitro and in vivo applications. Most of the Artibodies are strongly acidic, TR3-704 is mildly acidic, TR3-706/707 are basic. TR3-709 is prone to oxidation.
  • NHPGFAASKRLTLSPCEQE Sbjct 123 NHPGFAASKRLTLSPCEQE 141 (SEQ ID NO: 64)
  • this Artibody contains fluorescent label (TAMRA) attached to the side chain of the only “Lys” in the middle of the peptide (underlined).
  • TAMRA fluorescent label
  • DSFSHSRSRINAYK Sbjct 206 DSFSHSRSRINAYK 219 (SEQ ID NO: 70)
  • TAMRA_GGCRRRRLQKDIEMGMGNSK (SEQ ID NO: 73)
  • Example 19 further shows that Artibodies can be designed using existing on-line tools and databases, JavaScript programs and published scientific papers.
  • PSA Prostate-specific antigen
  • An Artibody can be designed to target PSA
  • An Artibody can be designed to target a human protein which is a forensic marker
  • An Artibody can be designed to target a human protein which is a disease marker
  • An Artibody can be designed to target a human protein which is a marker of cancer
  • An Artibody can be designed to target a human protein which is a known marker of prostate cancer
  • An Artibody can be designed to target a protein produced by the cells in a gland
  • An Artibody can be designed to target a protein produced by the cells in an exocrine gland such as the prostate gland;
  • An Artibody can be designed to target a protein produced by the cells in female breast;
  • An Artibody can be designed to target a protein which is present in small quantities in the serum under normal conditions;
  • An Artibody can be designed to target a protein which is present in small quantities in the urine under normal conditions;
  • An Artibody can be designed to target a protein which is in larger quantities in semen;
  • An Artibody can be designed to target a protein which is elevated in response to healthy physiological activity, such as sexual intercourse, followed by ejaculation, which usually accompanies male orgasm.
  • An Artibody can be used in the existing tests available for the early detection of prostate cancer
  • An Artibody can be designed to target a glycoprotein
  • An Artibody can be designed to target an enzyme
  • An Artibody can be designed to target a protease
  • An Artibody can be designed to target a serine protease
  • GEVVHYRKWIKDTICANP SEQ ID NO: 75
  • Acet-S VILLGRHSLCHPEDTGQ VFQG- Amid (SEQ ID NO: 78) note, Acetylated N-term; Amidated C-term

Abstract

La présente invention concerne un procédé de production d'une molécule de liaison qui se lie à une protéine d'intérêt cible. Le procédé comprend la production d'une molécule de liaison se liant à une protéine cible totalement repliée, la molécule de liaison comprenant un peptide ayant une séquence qui a une identité substantielle avec au moins une partie substantielle de la séquence d'une partie exposée en surface de la protéine cible totalement repliée de manière à permettre à la molécule de liaison de se lier à la protéine cible à la place de la partie exposée en surface de la protéine cible. Le procédé peut comprendre en outre l'étape de sélection d'une partie exposée en surface d'une protéine cible totalement repliée.
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