Binding Moieties Based On Shark IgNAR Domains

Abstract

The present invention relates to immunoglobulin new antigen receptors (IgNARs) from fish and uses thereof. In particular, the present invention relates to modified IgNAR variable domains and to domains from members of the immunoglobulin superfamily that have been modified to include structural features derived from IgNAR variable domains.

1 . A method of altering a property of an IgNAR variable domain comprising eight β-strand regions, designated A, A′, B, C, D, E, F and G according to  FIG. 2 , and nine loop regions, designated 1 to 9 according to  FIG. 2 , said method comprising modifying the IgNAR variable domain within at least one of the β-strand regions or loop regions. 
     
     
         2 . The method according to  claim 1 , wherein the modification alters the binding characteristics of the IgNAR variable domain. 
     
     
         3 . The method according to  claim 1 , wherein the modification improves the solubility of the IgNAR variable domain. 
     
     
         4 . The method according to  claim 1 , wherein the modification improves the stability of the IgNAR variable domain. 
     
     
         5 . The method according to  claim 1 , wherein the modification increases or decreases the propensity for the IgNAR variable domain to form homodimers. 
     
     
         6 . The method according to any preceding claim, in which the unmodified β-strand regions and loop regions have the amino acid residue numbering according to Table 3. 
     
     
         7 . The method according to any preceding claim, in which the unmodified loop region 5 comprises two β-strand regions designated C′ and D′ according to  FIG. 3  and three loop regions designated 5a, 5b and 5c according to  FIG. 3 . 
     
     
         8 . The method according to  claim 7 , in which the unmodified β-strand regions C, C′ and D′ have the amino acid residue numbering according to Table 3A. 
     
     
         9 . The method according to  claim 7  or  claim 8 , in which the C α  trace of loop region 5b in the unmodified IgNAR variable domain is no more than 5 Å above the plane formed by the C α  trace of residues 22, 83 and 36 as defined in Table 1. 
     
     
         10 . The method according to any preceding claim, in which the amino acid sequence of the unmodified β-strand regions A, A′, B, C, D, E, F and G and loop regions 1, 2, 3, 6, 7 and 9 comprises an amino acid sequence according to  FIG. 1  and/or Table 1. 
     
     
         11 . The method according to any preceding claim, in which the unmodified IgNAR is a Type 2 or Type 3 IgNAR. 
     
     
         12 . The method according to any preceding claim, in which the unmodified IgNAR is derived from a shark. 
     
     
         13 . The method according to any one of  claims 1  to  10 , in which the amino acid sequence of the unmodified IgNAR variable domain is a sequence shown in  FIG. 1 . 
     
     
         14 . The method according to any one of  claims 1  to  10 , in which the unmodified IgNAR variable domain is 12Y-1, 12Y-2, 12A-9 or 1A-7. 
     
     
         15 . The method according to any preceding claim, in which the modification is a substitution, insertion, deletion or combination thereof. 
     
     
         16 . The method according to any preceding claim, in which the modification comprises the substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids. 
     
     
         17 . The method according to any preceding claim, in which the modification comprises deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids. 
     
     
         18 . The method according to any preceding claim, in which the modification comprises insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids. 
     
     
         19 . The method according to any preceding claim, in which one or more loop regions of the IgNAR are modified. 
     
     
         20 . The method according to any preceding claim, in which loop region 4, or part thereof, and/or loop region 8, or part thereof, are modified. 
     
     
         21 . The method according to  claim 20 , in which loop region 8 is modified by point mutations. 
     
     
         22 . The method according to  claim 20 , in which loop region 8 is modified by randomisation. 
     
     
         23 . The method according to  claim 20 , in which loop region 8 is modified so as to facilitate the adoption of β-strand configurations at the C- and/or N-terminal ends. 
     
     
         24 . The method according to  claim 23 , in which loop region 8 is modified by addition or substitution of one or more amino acid residues at the C- and/or N-terminal ends of the loop region 
     
     
         25 . The method according to  claim 24 , in which from 2 to 10 amino acid residues are added or substituted at the C- and/or N-terminal ends of the loop region. 
     
     
         26 . The method according to any one of  claims 1  to  20  and  23  to  25 , in which the IgNAR variable domain is modified by grafting a CDR loop or portion thereof from a V-set or an I-set domain into a loop region of the IgNAR variable domain. 
     
     
         27 . The method according to  claim 26 , in which the CDR loop or portion thereof is grafted into loop region 4 and/or 8 of the IgNAR variable domain. 
     
     
         28 . The method according to  claim 27 , in which amino acids from loop region 8 are replaced by a CDR3 loop or portion thereof from a V-set or an I-set domain. 
     
     
         29 . The method according to any one of  claim 28 , in which amino acids 86 to 103 as defined in Table 1 or a portion thereof are replaced. 
     
     
         30 . The method according to  claim 27 , in which amino acids from loop region 4 are replaced by a CDR1 loop or portion thereof from a V-set or an I-set domain. 
     
     
         31 . The method according to  claim 30 , in which amino acids 28 to 33 as defined in Table 1 or a portion thereof are replaced. 
     
     
         32 . The method according to any preceding claim, in which one or more amino acid residues within the patch defined by residues 33, 37, 46, 48, 50, 51, 59, 61, 86, 94, 95, 96, 98, 99 and 101 as shown in Table 1 are modified. 
     
     
         33 . The method according to any preceding claim, in which at least one of the β-strand regions or loop regions 1-3, 5-7 or 9 is modified when loop region 4 and/or loop region 8 of the IgNAR is modified. 
     
     
         34 . The method according to  claim 33 , in which at least one of β-strand regions C, D, E or F or loop regions 5, 6 or 7 is modified. 
     
     
         35 . The method according to  claim 34 , in which at least one of β-strand regions C or D or loop region 5 is modified. 
     
     
         36 . The method according to  claim 35 , in which loop region 5 is modified. 
     
     
         37 . The method according to any preceding claim, in which the IgNAR is a Type 2 or Type 3. 
     
     
         38 . The method or binding moiety according to any preceding claim, in which the IgNAR is derived from a shark. 
     
     
         39 . A binding moiety comprising a modified IgNAR variable domain produced by a method according to any one of  claims 1  to  38 . 
     
     
         40 . A binding moiety comprising an IgNAR variable domain comprising eight β-strand regions, designated A, A′, B, C, D, E, F and G according to  FIG. 2 , and nine loop regions, designated 1 to 9 according to  FIG. 2 , wherein the IgNAR variable domain has been modified within at least one of the β-strand regions or loop regions. 
     
     
         41 . A method of modifying an I- or V-set domain, said method comprising inserting and/or substituting one or more structural features from an IgNAR variable domain into the I- or V-set domain. 
     
     
         42 . The method according to  claim 41 , in which the structural feature is a loop region from an IgNAR variable domain according to  FIG. 2 . 
     
     
         43 . The method according to  claim 42 , in which the loop region is loop region 4 or 8 from an IgNAR variable domain. 
     
     
         44 . The method according to  claim 43 , in which loop region 8 and/or loop region 4 are grafted onto the I-set or V-set domain. 
     
     
         45 . The method according to  claim 44 , in which suitably predetermined amino acids of the I- or V-set domain are replaced with an insert comprising amino acids 86 to 103 from the IgNAR variable domain as defined by Table 1. 
     
     
         46 . The method according to any one of  claims 41  to  45 , in which all or a portion of the CDR2 loop of the I-set or V-set domain is removed. 
     
     
         47 . The method according to  claim 41 , in which the structural feature is the solvent exposed face at the C-terminus of the loop region 4 and in the C and D β-strands from an IgNAR variable domain according to  FIG. 2 . 
     
     
         48 . The method according to any one of  claims 41  to  47 , in which amino acids of the I- or V-set domain equivalent to amino acids 32, 33, 34, 35, 55, 57 and 58 as defined in Table 1 or a portion thereof are modified. 
     
     
         49 . The method according to  claim 48 , in which the modification introduces charged or polar amino acids at these positions. 
     
     
         50 . The method according to any one of  claims 41  to  49 , in which the modification improves the solubility of the I- or V-set domain. 
     
     
         51 . The method of modifying an I-set domain according to any one of  claims 41  to  50 . 
     
     
         52 . The method according  claim 51 , in which the I-set domain is selected from NCAM, VCAM, ICAM, Telokin, MADCAM-1, Twitchin and Titin. 
     
     
         53 . The method of modifying a V-set domain according to any one of  claims 41  to  50 . 
     
     
         54 . The method according  claim 53 , in which the V-set domain is selected from antibodies, T cell receptors (TCRs), CTLA-4, CD28, ICOS, CD2, CD4, Cd7, CD22, CD33, CD80, CD86, CD48 and CD58. 
     
     
         55 . The method according  claim 53  or  claim 54 , in which the V-set domain is a TCRVα or Vβ domain and the equivalent amino acids to the solvent exposed surface of an IgNAR variable domain are Gly30, Ser31, Phe32, Phe33, Phe62, Thr63, Ala64 and Gln65. 
     
     
         56 . The method according  claim 53  or  claim 54 , in which the V-set domain is a TCRVα or Vβ domain and the one or more residues located at the interface between the Vα and Vβ domains are modified. 
     
     
         57 . The method according  claim 56 , in which the one or more amino acid residues are selected from the group consisting of Ser31, Pro43, Leu89 and Phe106 and combinations thereof. 
     
     
         58 . A binding domain comprising a modified I- or V-set domain produced according to a method of any one of  claims 41  to  57 . 
     
     
         59 . A binding moiety comprising an I- or V-set domain, wherein the I- or V-set domain has been modified by substitution or insertion of one or more structural features from an IgNAR variable domain into the I- or V-set domain. 
     
     
         60 . A binding moiety comprising an I- or V-set domain, wherein the I- or V-set domain has been modified by introducing a modification into a region equivalent to loop region 4 or loop region 8 of an IgNAR variable domain. 
     
     
         61 . A binding moiety comprising a multimer comprising:
 (i) at least two IgNAR domains, which may be the same or different, and at least one of which is a IgNAR variable domain;   (ii) at least two I-set domains, which may be the same or different, and at least one of which is a I-set domain according to the present invention; or   (iii) at least two V-set domains, which may be the same or different, and at least one of which is a V-set domain according to the present invention.   
     
     
         62 . A binding moiety according to the invention linked to a diagnostic reagent. 
     
     
         63 . A binding moiety according to the invention immobilised on a solid support or coupled to a biosensor surface. 
     
     
         64 . A polynucleotide encoding a binding moiety according to the invention. 
     
     
         65 . A vector comprising a polynucleotide according to the present invention. 
     
     
         66 . A host cell comprising a vector according to the invention. 
     
     
         67 . A method of producing a binding moiety according to the invention which comprises culturing a host cell of the present invention under conditions enabling expression of the binding moiety according to the invention and optionally recovering the a binding moiety. 
     
     
         68 . A pharmaceutical composition comprising a binding moiety according to the invention and a pharmaceutically acceptable carrier or diluent. 
     
     
         69 . A method of treating a pathological condition in a subject, which method comprises administering to the subject a binding moiety according to the invention. 
     
     
         70 . A method of selecting a binding moiety according to the invention with an affinity for a target molecule which comprises screening a library of polynucleotides of the present invention for expression of a binding moiety according to the invention with an affinity for the target molecule. 
     
     
         71 . A polynucleotide library comprising a plurality of polynucleotides encoding binding moieties according to the invention, which polynucleotides comprise one or more modifications in the IgNAR variable domain, I set domain or V-set domain. 
     
     
         72 . A crystal of a variable domain of a Type 2 IgNAR that effectively diffracts X-rays for the determination of the atomic coordinates of the variable domain of the IgNAR to a resolution of better than 4.0 Å, wherein the variable domain of the Type 2 IgNAR consists of 105 to 125 amino acid residues and comprises an amino acid sequence according to Table 1 and/or  FIG. 1 . 
     
     
         73 . A crystal of a variable domain of a Type 2 IgNAR comprising a structure defined by all or a portion of the coordinates of Appendix I(a), (b), (c) or (d)±a root mean square deviation from the Cα atoms of less than 0.5 Å. 
     
     
         74 . A method of homology modelling comprising the steps of:
 (a) aligning a representation of an amino acid sequence of an IgSF domain with the amino acid sequence of 12Y-1, 12Y-2, 12A-9 or 1A-7 as shown in  FIG. 1  to match homologous regions of the amino acid sequences;   (b) modelling the structure of the matched homologous regions of said IgSF domain on the corresponding regions of the 12Y-1, 12Y-2, 12A-9 or 1A-7 structure as defined by Appendix I(a), (b), (c) or (d); and   (c) determining a conformation (e.g. so that favourable-interactions are formed within the IgSF domain and/or so that a low energy conformation is formed) for said IgSF domain which substantially preserves the structure of said matched homologous regions.   
     
     
         75 . A method for determining the structure of a protein, which method comprises providing the co-ordinates of Appendix I(a), (b), (c) or (d), and either
 (a) positioning the co-ordinates in the crystal unit cell of said protein so as to provide a structure for said protein; or   (b) assigning NMR spectra Peaks of said protein by manipulating the coordinates of Appendix I(a), (b), (c) or (d).   
     
     
         76 . A system containing at least one of the following:
 (a) atomic coordinate data according to Appendix I, said data defining the three-dimensional structure of 12Y-1, 12Y-2, 12A-9 or 1A-7 or at least selected coordinates thereof;   (b) structure factor data (where a structure factor comprises the amplitude and phase of the diffracted wave) for 12Y-1, 12Y-2, 12A-9 or 1A-7, said structure factor data being derivable from the atomic coordinate data of Appendix I;   (c) atomic coordinate data of an IgSF domain generated by homology modelling of the IgSF domain based on the data of Appendix I;   (d) atomic coordinate data of the IgSF domain generated by interpreting X-ray crystallographic data or NMR data by reference to the data of Appendix I; and   (e) structure factor data derivable from the atomic coordinate data of (c) or (d).   
     
     
         77 . A computer-readable storage medium, comprising a data storage material encoded with computer readable data, wherein the data are defined by all or a portion of the structure coordinates of 12Y-1, 12Y-2, 12A-9 or 1A-7, or a variant of 12Y-1, 12Y-2, 12A-9 or 1A-7, wherein said variant comprises backbone atoms that have a root mean square deviation from the Cα or backbone atoms (nitrogen-carbonα-carbon) of Appendix I of less than 2 Å 
     
     
         78 . A computer-readable storage medium according to  claim 77 , in which the root mean square deviation from the Cα or backbone atoms of Appendix I is not more than 1.5 Å, preferably less than 1.5 Å, more preferably less than 1.0 Å, even more preferably less than 0.74 Å, even more preferably less than 0.72 Å and most preferably less than 0.5 Å. 
     
     
         79 . A computer-readable data storage medium comprising a data storage material encoded with a first set of computer-readable data comprising a Fourier transform of at least a portion (e.g. selected coordinates as defined herein) of the structural coordinates for 12Y-1, 12Y-2, 12A-9 or 1A-7 according to Appendix I; which, when combined with a second set of machine readable data comprising an X-ray diffraction pattern of a molecule or molecular complex of unknown structure, using a machine programmed with the instructions for using said first set of data and said second set of data, can determine at least a portion of the structure coordinates corresponding to the second set of machine readable data. 
     
     
         80 . A computer readable media with at least one of:
 (a) atomic coordinate data according to Appendix I recorded thereon, said data defining the three-dimensional structure of 12Y-1, 12Y-2, 12A-9 or 1A-7, or at least selected coordinates thereof;   (b) structure factor data for 12Y-1, 12Y-2, 12A-9 or 1A-7 recorded thereon, the structure factor data being derivable from the atomic coordinate data of Appendix I;   (c) atomic coordinate data of a target IgSF domain generated by homology modelling of the IgSF domain based on the data of Appendix 1;   (d) atomic coordinate data of a modified IgSF domain generated by interpreting X-ray crystallographic data or NMR data by reference to the data of Appendix I; and   (e) structure factor data derivable from the atomic coordinate data of (c) or (d).   
     
     
         81 . A method of providing data for generating structures and/or performing rational drug design for IgSF domains, the method comprising:
 (i) establishing communication with a remote device containing computer-readable data comprising at least one of
 (a) atomic coordinate data according to Appendix I, said data defining the three-dimensional structure of 12Y-1, 12Y-2, 12A-9 or 1A-7, at least one sub-domain of the three-dimensional structure of 12Y-1, 12Y-2, 12A-9 or 1A-7, or the coordinates of a plurality of atoms of 12Y-1, 12Y-2, 12A-9 or 1A-7; 
 (b) structure factor data for 12Y-1, 12Y-2, 12A-9 or 1A-7, said structure factor data being derivable from the atomic coordinate data of Appendix I; 
 (c) atomic coordinate data of a modified IgSF domain generated by homology modelling of the domain based on the data of Appendix I; 
 (d) atomic coordinate data of a protein generated by interpreting X-ray crystallographic data or NMR data by reference to the data of Appendix I; and 
 (e) structure factor data derivable from the atomic coordinate data of (c) or (d); and 
   (ii) receiving said computer-readable data from said remote device.