AU2010200432A1

AU2010200432A1 - V-like domain binding molecules

Info

Publication number: AU2010200432A1
Application number: AU2010200432A
Authority: AU
Inventors: Gregory Coia; Maria Galanis; Peter John Hudson; Robert Alexander Irving; Stewart Douglas Nuttall
Original assignee: Diatech Pty Ltd
Current assignee: Diatech Pty Ltd
Priority date: 1998-03-06
Filing date: 2010-02-08
Publication date: 2010-02-25
Also published as: AU2006252050A1

Description

Feb-2010 16:25 WATERMARK (F) 61398196010 36/147 POO Second 29 AUSTRLIA Rgulation32(2) AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Application Number: Lodged: Invention Title: V-like domain binding molecules The following statement is a full description of this invention, Including the best method of performing it known to us: P111AiAUh127 COMS ID No: ARCS-265561 Received by IP Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:25 VATERMARK (F) 61398196010 37/147 1A V-like Domain Binding Molecules Field of the Invention The present invention relates to V-like Domain binding molecules with 5 affinities for target molecules. The present invention also relates to compositions comprising these V-like domain binding molecules and to methods of diagnosis or treatment which involve the use of these molecules. The present invention also relates to a method for selecting V-like Domain binding molecules with novel binding affinities and/or specificities. 10 Background of the Invention Immunoglobulin Superfamily - Antibody Variable (V Domains Antibodies are the paradigm of specific high-affinity binding reagents and provide an antigen binding site by interaction of variable heavy (Vs)! and 15 variable light (V) immunoglobulin domains. The binding interface is formed by six surface polypeptide loops, termed complementarity determining regions (CDRs), three from each variable domain, which are highly variable and combined provide a sufficiently large surface area for interaction with antigen. Specific binding reagents can be formed by association of only the 20 V and VL domains into an Fv module. Bacterial expression is enhanced by joining the V-domains with a linker polypeptide into a single-chain scFv molecule. "Humanisation" of recombinant antibodies by grafting murine CDR loop structures onto a human Fv framework is disclosed by Winter et al EP-239400. 25 Methods to improve the expression and folding characteristics of single-chain Fv molecules were described by Nieba et al (1997). The properties of single V-domains, derived from natural mammalian antibodies, have been described by Gussow et at in WO/90/05144 and EP 0368684B1 and by Davis et al in WO/91/08482. Single camelid V-domains have been 30 described by Hamers et al in WO/96/34103 and in WO/94/25591. A method for reducing the hydrophobioity of the surface of a human Va domain by replacing human amino acid sequences with camelid amino acid sequences was described by Davies and Riechmann (1994). Methods to exchange other regions of human V, sequences with camel sequences to further enhance 35 protein stability, including the insertion of cysteine residues in CDR loops, were described by Davies and Riechmann (1996). COMS ID No: ARCS-265561 Received by IP Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:26 WATERMARK (F) 61398196010 38/147 2 Several attempts to engineer high-affinity single domain binding reagents using either the Va or VL domains alone have been unsuccessful, due to lack of binding specificity and the inherent insolubility of single domains in the absence of the hydrophobic face where the VII and VL 5 domains interact (Kortt et at, 1995). T-cell Receptor Variable MV Domains The T-oell receptor has two V-domains that combine into a structure similar to the Nv module of an antibody that results from combination of the VII and VL domains. k~ovotny et al (1991) described how the two V-domains 10 of the T-cell receptor (termed alpha and beta] can be fused and expressed as a single chain polyjeptide end, further, how to alter surface residues to reduce the hydrophobicity directly analogous to an antibody scFv. Other publications describe the expression characteristics of single-chain T-cel receptors comprising two V-alpha and V-beta domains (Wulfing and 15 Pluckthun,1994; Ward, 1991). Non-antibody ligands - CTLA-4 and CD)28 V-like Domains There are a class of non-antibody ligands which bind to specific binding partners which also comprise V-like domains. These V-like domains are distinguished from those of antibodies or T-cell receptors because tWay 20 have no propensity to join together into Fv-type molecules. These non antibody ligands provide an alternative framework for the development of novel binding moieties with high affinities for target molecules. Single whmih VBe binding molecules derived from these non-antibody ligands wihare soluble are- therefore desirable. Examples of suitable non-antibody 25 ligands are CTLA-4, CD28 and 1005 (Hutloff et al, 1999). Cytotoxic T-lymphocyte associated antigen 4 (GT IA-4) and the homologous cell-surface proteins CD28 and 1005, are involved in 7-cell regulation during the imune response. CTLA-4 is a 4 kfla homnodimer expressed primarily and transiently on the surface of activated T-cells, where 30 it interacts with CD8o and CD86 surface antigens on antigen presenting cells to effect regulation of the immune response '(Waterhouse et al. 1990, van der Merwe at al. 1997): GD28 is a 44kDa homodimer expressed predominantly on T-cells and, like CTLA-4, interacts with 01D80 and CD86 surface antigens. on antigen presenting cells to effect regulation of the immune response 35 (Linsley et aLi199o). Current theory suggests that competition between OTLA 4 and 01D28 for available ligan:ds controls the level of immune response, for COMS ID No: ARcS-265561 Received by IP Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-OS Feb-2010 16:26 WATERMARK (F) 61398196010 39/147 3 example, gene deletion of CTLA,-4 in knock-out mice results in a massive aver-proliferation of activated T-cells (Waterhouse et al. 1995), Each CTLA-4 monomeric subunit consists of an N-terminal extracelniar domain, transmembrane region and C-terminal intracellular 5 domain. The extracellular domain comprises an N-terminal V-ike domain (W±D; of approximately 14 kfln predicted molecular weight by homology to the inununoglobulin superfamily) and a stalk of about *10 residues connecting the VLD to the transmeffbrane region. The VLD comprises surface loops corresponding to GDR-1, CDjR-2 and CDR-3 of an antibody V-domain (Matzler 10 1997). Recent structural and mutational studies on GTLA-4 suggest that binding to CD)80 and CD8B occurs via thq VLD surface formed from A'GFCC' V-like beta-strands and also from the highly consented MYPPPY sequence in the CDR3-like surface loop (Peach et al, 1994; Morton at a!. 1906; Metzler et a!. 1997). Dimerisation between CTLA-4 monomers occurs through a 15 disulphide bond between cystine residues (Cys'") in the two stalks, which results in tethering of the two extracellular domains,, but without any apparent direct association between V-like domains (Metzler et al. 1997). Dimerisation appears to contribute exclusively to increased avidity for the ligands. 20 In vitro Expression of Solubl 'a Forms of CTLA-4. Neither the extracellullar domains nor V-like domains (VLDs] of human GTLA-4 molecule have been successfully expressed as soluble monomers in bacterial cells, presumably d~ue to aggregation of tihe expressed proteins (Linsley at al, 1995). Expression of the extracellular N-terminal domain (Mae 25 to Aspu 4 , comprising Gys tm ) in E.coli results in production of a dimeric 28 kDa MIX protein, in which two CTLA-4 V-like domains are joined by a disulphide linkage at Cys 1 ". Truncation at Va 114 removes these cysteines and was intended to enable expression of a 14 lcDa VLD in soluble, monomeric form. However, the product aggregated and it was concluded that 30 hydrophobic sites, which were normally masked by glycosylation. were now exposed and caused aggregation (Linsley et al, 1995). There have been some reports of successful expression of monomeric, glycosylated CTLA-4 extracellular domains in eukaryotic expression systems (ie CHO cells and the yeast Pichia pastoris; Linsley et a!. 19959; Metzler et al. 35 1997; Gerstmayer et al. 19M7. Glycosylation in these eukaryotic expression

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2l Over-polifration56 ofectived tci: ell (~ 11 aterhouse et al.15).0 Feb-2010 16:27 WATERMARK (F) 61398196010 40/147 4 systems is presumed to occur at the two N-linked glycosylation sites in the VID (Asn76 and AsnIO8 ). However, high yields have only been described for expression of a gene encoding a CTLA-4 VLD fused to Ig-CH2/CH3 domains which produces a dimeric recombinant protein with 2 CTLA-4 VLDs 5 attached to an Fc subunit (WO 95/01994 and AU 16458/95). AU 60590/96 describes mutated forms of CTLA-4 VLDs with single amino acid replacements of the first tyrosine (Y) in the MYPPFY surface loop which retain and modifies the affinity for the natural CD80 and CD86 ligands. AU 60590/96 describes the preferred soluble form of CTLA-4 VLDs as a 10 recombinant CTIA-4/Ig fusion protein expressed in eukaryotic cells and does not solve the aggregation problem in prokaryote expression systems. EP 0757099A2 describes the use of CTLA-4 mutant molecules, for example the effect of changes on ligand binding of mutations in the CDRS-like loop. 15 Summary of the Invention The present inventors have now developed novel binding molecules derived from the V-like domains (VLDs) of non-antibody ligands such as CTLA-4, CD28 and ICOS. Replacement of CDR loop structures within the VLDs results unexpectedly in the production of monomeric, correctly folded 20 molecules with altered binding specificities and improved solubility. Accordingly, in a first aspect the present invention provides a binding moiety comprising at least one monomeric V-like domain (VLD) derived from a non-antibody ligand, the atileast one monomeric V-like domain being characterised in that at least one CDR loop structure or part thereof is 25 modified or replaced such that the solubility of the modified VLD is improved when compared with the unmodified VLD. Within the context of the present invention, the modification or replacement may involve any change to one or more physical characteristics (such as size, shape, charge, hydrophobicity etc) of the at least one CDR loop 30 structure. The modification or replacement may result in a reduction in the size of the at least one CDR loop structure. In a preferred embodiment however, at least one CDR loop structure or part thereof is modified or replaced such that (1) the size of the CDR loop structure is increased when compared 35 with corresponding CDR loop structure in the unmodified VLD; and/or COMS ID No: ARCS-265561 Received by IP Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:28 WATERMARK (F) 61398196010 41/147 5 (iH) the modification ior replacement results in the formation of a disulphide bond within or between one or more of the CDR loop structures. Inna second aspect, the present invention provides a binding moiety comprising at least one monomeric V-like domain [VLD) derived from a non 5 antibody ligand, the at least one monomeric V-like domain being. characterised in that at leastione CDR loop structure or part thereof is modified or replaced such tlat (1) the size of the CUjR loop structure is altered when compared with corresponding CDR loop structure in the unmodified VLED; and/or 10 (ii) the modification or replacement results in the formation of a disulphide band within or between one or more of the CDR loop structures. In a preferred embodiment of the second aspect the size of the CDR loop structure is increased by at least two, more preferably at least three, more preferably at least six and more preferably at least nine amino acid 15 residues. In a further preferred embodimentr the modified binding moiety a the first or second aspect of the present invention also exhibits an altered binding affinity or specificity when compared with the unmodified binding moiety. Preferably, the effect of replacing or modifying the CDR loop 20 structure is to reduce or abolish the affinity of the VI] to one or more natural ligands of the unmodified VLD. Preferably, the effect of replacing or modifying the CsR loop structure is also to change the binding specificity of the VLD . Thus it is preferred that the modified VLD binds to a specific binding partner which is dierent to thatof the unmodified VL D. 25 The phrase V-le domain or "VL " is intended torefer to a domain which has similar structural features to the variable heavy () or variable light [Vn antibody. These similar structural features include CUR loop structures. By "CDR loop structures" we mean surface polypeptide loop structures or regions like the complementarity determining regions in SO antibody V-domains. The phrase "non-antibody ligand is intended to refer to any ligand which binds to a specific binding partner and which is not an antibody or a T-cel receptor. Examples of suitable non-antibody ligands are r-cell surface proteins such as CTLA-4, CD 8 and ICOS. It will be appreciated by those 35 skilled in the art that other non-antibody ligands which may provide V-like domains suitable for the invention are other T-cell surface proteins such as COMSIDNo ARCS-265561 Received by IF Australia: Time (H:m) 1711 Date (Y-M-d) 2010-02-a Feb-2010 16:29 WATERMARK (F) 61398196010 42/147 6 CD2, CD4, CD7 and CD16; B cell surface proteins such as CD19, CD79a, CD22, CD33, CD80 and CD86; adhesion molecules such as CD48, CD541CAM and CD58. These molecules, which are listed in Table 1, provide a non exhaustive list of structures which may form the basis for the single domain 5 binding molecules of the present invention. The phrase "V-like domain derived from a non-antibody ligand" is intended to encompass chimeric V-like domains which comprise at least part of a V-like domain derived from a non-antibody ligand. 10 TABLE 1: NoN-ANTBoDy LIGANDS Molecule Size Structure Tbell Surface Proteins GD2 45-58kDa VCI domains CD4 55kDa V2C2 CD7 4okDa V domain CD16 5D-65kDa 2x C domains B cell Surface Proteins CD19 95kna 2x C domains CD79a 33kDa CD22 130-14ekDa lxV oxC domains CDSS 67kDa VC domain CD80 6OkDa VCdomain CD86 60kDa VC domain Adhesion molecules CD48 45kDa VC domain CD541CAM 85-110kDa CD58 55-7OkDa VC domain 1 V = variable Ig domain, C w constant domain 15 These molecules are discussed in (1) The Leucocyte Antigen Facts Book, 1993, Eds Barclay et al., Academic Press, London; and (2) CD Antigens 1996 (1997) Immunology Today 18, 100-101, the entire contents of which are Incorporated herein by reference. COMS ID No: ARCS-265561 Received by IP Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:29 WATERMARK (F) 61398196010 43/147 7 The "solubility" of modified binding moieties of the present invention correlates with the production of correctly folded, monomeric domains. The solubility of the modified VLDs may therefore be assesed by RPLC. For example, soluble (monomeric) VLDs will give rise to a single peak on the 5 HPLC chromatograph, whereas insoluble (eg. multimeric and aggregated) VLDs will give rise to a plurality of peaks. A person skilled in the art will therefore be able to detect an increase in solubility of modified VLDs using routine HPLC techniques. It will be appreciated that the binding moieties of the present 10 invention may be coupled together, either chemically or genetically, to form multivalent or multifunctional reagents. For example, the addition of C terminal tails, such as in the native CTLA-4 with Cyst will result in a dimer. The binding moieties of the present invention may also be coupled to 15 other molecules for various diagnostic formulations. For example, the VILDs may comprise a C-terminal polypeptide tail or may be coupled to streptaividin or biotin for multi-site in vitro assays. The VLDs may also be coupled to radioisotopes,. dye markers or other imaging reagents for in vivo detection and/or localisation of cancers, blood clots, etc. The VLDs may also be 20 immobilised by coupling onto insoluble devices and -platforms for diagnostic and biosensor applications. In a most preferred embodiment of the first aspect of the present invention, the V-like domain is derived from the extracellular domain of the CTLA-4 molecule or the CD28 molecule. In a further preferred embodiment 25 one or more surface loops of the CTLA-4 V-like domain and preferably the CDR-1, CDR-2 or CDR-3 loop structures are replaced with a polypeptide which has a binding affinity for a target molecule of interest. Target molecules of interest comprise, but are not limited to, drugs, steroids, pesticides, antigens, growth factors, tumour markers, cell surface proteins or 30 viral coat proteins. It will be appreciated that these VLDs may be polyspecific, having affinities directed by both their natural surfaces and modified polypeptide loops. In a further preferred embodiment the effect of replacing or modifying the CTLA-4, CD28 and ICOS V-like domain surface loops is to abolish the 35 natural affinity to CD80 and CD86. COMS ID No ARCS-265561 Received by IP Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:30 WATERMARK (F) 61398196010 44/147 8 In one preferred embodiment, one or more of the CDR loop structures of the VLD are replaced with one or more CDR loop structures derived from an antibody. The antibody may be derived from any species. In a preferred embodiment, the antibody Is derived from a human, rat, mouse, camel, llama 5 or shark, The antibody or antibodies maybe selected from the camel antibody cAB-Lys3 and the human anti-melanoma antibody V86. In a further preferred embodiment, one or more of the CDR loop structures are replaced with a binding determinant derived from a non antibody polypeptide. For example, one or more of the CDR loop structures 10 may be replaced with a polypeptide hormone, such as somatostatin which is a 14 residue intra-disulphide bonded polypeptide important in cancer cell recognition, or with a viral protein such as the human influenza virus haemagglutinin protein. In a further preferred embodiment the V-lil domain of the binding 15 moiety comprises CDR loop structures homologous in character to CDR loop structures found in camelid or llama antibodies. For example, the CDR loop structures may contain one or more non-conventional substitutions (eg. hydrophobic to polar in nature). In another preferred embodiment the CUR 1 and CDR-3 loop structures may adopt non-canonical conformations which 20 are extremely heterologous in length. The V-like domain may also possess a disulphide linkage interconnecting the CDR-1 and CDR-3 loop structures (as found in some camel VIH antibodies) or the CDR-2 and CDR-3 loop structures (as found in some llama VEH antibodies). In a third aspect the present invention provides a polynucleotide 25 encoding a binding moiety of the first or second aspect of the present invention. The polynucleotide may be incorporated Into a plasmid or expression vector. In a fourth aspect the present invention provides a prokaryotic or eukaryotic host cell transformed with a polyhucleotide according to the third 30 aspect of the present invention. In a fifth aspect the present invention provides a method of producing a binding moiety which comprises culturing a host cell according to the fourth aspect of the present invention under conditions enabling expression of the binding moiety and optionally recovering the binding moiety. COMS ID No: ARcS-265561 Received by IP Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-0s Feb-2010 16:30 WATERMARK (F) 61398196010 45/147 9 In a preferred embodiment of the present invention the binding moiety is produced by expression in a bacterial host Preferably, the binding moiety is unglycosylated. -In a sixth aspect the present invention provides a pharmaceutical 5 composition comprising a binding moiety of the first or second aspect of the present invention and a pharmaceutically acceptable carrier or diluent In a seventh aspect tha present invention provides a method of treating a pathological condition in a, subject, which method comprises administering to the subject a binding moiety according to the first or second aspect of the 10 present invention. For in vivo applications it is preferable that VIDs are homologous to the subject of treatment or diagnosis and that any possible xenoantigens are removed. Accordingly it is preferred that Vt]) molecules for use in clinical applications are substantially homologous to naturally occurring human 15 immunoglobuhn supeifaiily members. In an eighth aspect the present invention provides a method of selecting a binding moiety with an affinity for a target molecule which comprises screening a library of polynucleotides for expression of a binding moiety with an affinity for the target molecule, the polyaucleotides encoding 20 VLDs derived from one or more non-antibody ligands, wherein the polynucleotides have been subjected to inutagenesis which results in a modification or replacement in at least one CDR loop structure in at least one VLD and wherein the solubility of the isolated modified VLD is improved when compared with the isolated unmodified VLD. 25 It will be appreciated by those skilled in the art that within the context of the eighth aspect of the present invention, any method of random or targeted mutagenesis may bpr used to introduce modifications into the V-like domains. In a preferred embodiment the mutagenesis is targetted mutagenesis. Preferably, the targetted mutagenesis involves replacement of 30 at least one sequence within at least one CDR ioop structure using splice overlap FOR technology. It will also be appreciated by those slled in the art that the polynucleotide library may contain sequences which encode VLDs comprising CDR loop structures which are substantially identical to CDR 35 loop structures found in natully occurring immunoglobulins as well as COMS ID No: ARpS-26551 Received by I Australia: Time (H:m) 17a11 Date (Y-M-d) 201-02-c8 Feb-2010 16:31 WATERMARK (F) 61398196010 46/147 10 sequences which encode VLDs comprising non-naturally occurring CDR loop structures. In a preferred embodiment of the eighth aspect of the present invention, the screening process involves displaying the modified V-like 5 domains as gene III protein fusions on the surface of bacteriophage particles. The library may comprise bacteriophage vectors such as pHFA, fd-tet-dog or pFAB.5c containing the polytiucleotides encoding the V-like domains. In a further preferred embodiment of the eighth aspect, the screening process involves displaying the modified V-like domains in a ribosomal 10 display selection system. Throughout this specification, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, 15 integers or steps. Brief Description of the Drawings Figure 1: CTLA-4 VLD-Specific Oligonucletides. 20 Figure 2: Polynucleotide sequence of complete cDNA encoding human CTLA-4 and polypeptide sequence of the VLD of human CTLA-4. Figure 3& Display of CTLA-4 VLD STMs as gene 3 fusions on the surface of 25 phage or phagemid. CTLA-4 VLD STMs are depicted as black spheroids; gene 3 protein is depicted as white spheroids; FLAG polypeptide is depicted in grey; genes are marked in a similar colour code and are depicted in an oval phage(mid) vector. 30 Figure 4: Schematic representation of the somatostatin polypeptide. Somatostatin (somatotropin telease-inhibiting factor SRIF) is a cyclic 14 amino acid polypeptide. Thd cyclic nature is provided by a disulphide linkage between the cysteine residues at positions 3 and 14. The four residues which constitute the tip of the loop (Phe-Trp-Lys-Thr) are 35 implicated in binding to menribers of the somatostatin receptor family. COMS ID No: ARCS-265561 Received by IP Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:32 WATERMARK (F) 61398196010 47/147 11 Figure 5: Size exclusion HPLC profiles of affinity purified CTLA-4 VLD and CTLA-4-Som3 STM. Recombinant human CTLA-4 proteins were expressed in E. coli host TG1 from vector pGC, purified from periplasmic extracts by anti-FLAG affinity chromatography and subjected to size exclusion 5 chromatography on a calibrated Superose 12 HR column. The elation profiles of purified CTLA-4 VLD and CTLA-4-Som3 STM are overlayed in this graph. CTLA-4 VLD comprises tetramer (21.86 min), dimer (26.83) and monomer (29.35 min). CTLA-4-Som3 STM comprises dimer (26.34) and monomer (29.28). Traces represent absorbance at 214 nm and are given in 10 arbitrary units. Figure 6: Schematic diagram of CTLA-4 VLD loop replacements. Construct A (CTLA-4 VLD: S2) represents the wild-type CTLA-4 extracellular V domain, spanning residues 1-115. Constructs B (CTLA-Soml; PP2) and C 15 (CTLA-4-Som1-Cys20; PP5) both contain the 14 residue somatostain polypeptide in CDRI. PP5 also carries a C-terminal extension containing Cys120. Construct D (CTLA-4-Som3; PPS) contains the 14 residue somatostatin polypeptide in place of CDR3. In construct E (CTLA-4-HA2: XX4), CDR2 has been replaced with a haemagglutinin tag. In construct F 20 (CTLA-4-SomI-Som3: VV3), both CDRl and CDR3 have been replaced with the somatostain polypeptide. In construct G (CTLA-4-Som-HA2-Som3: ZZ3) CDR1 and CDR3 are replaced with the somatostatin polypeptide whilst CDR2 is replaced with a haemagglutinin tag. In construct H (CTLA-4-anti-lys:V8), all three CDR loop structures have been replaced with the CDR loops from a 25 camel anti-lysozyme VaH molecule. Construct I (CTLA-4-anti-mel: 3E4) represents CTLA-4 VLD in which all three CDRs have been replaced by the VH CDR loops from anti-melanoma antibody V86 (Cai And Garen, 1997). PeiB, cleavable pectate lyase secretion sequence (22 aa); flag, dual flag tag (AAADYKDDDDKAADYKDDDDK). 30 Figure 7: HPLC profiles of purified recombinant human CTLA-4 STMs. Recombinant CTLA-4 VLDs were expressed in E. coli host TG1 from vector pGC, purified from periplasric extracts by anti-FLAG affinity chromatography and subjected to size exclusion chromatography on a 35 calibrated Suparose 12 HR column. The elution profiles of the purified proteins are shown. Panel A, CTLA-4 DIMER (PP5); Panel B, CTLA-4R (S2); COMS ID No: ARCS-265561 Received by IP Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:32 WATERMARK (F) 61398196010 48/147 12 Panel C, CTLA-4-HA2 (XX4); Panel D, CTLA-4-Som3 (PPB); Panel E, CTLA 4-Somi (PP2); Panel F, CTLA-4-Soml-Som3 (VV3); Panel G, CTLA-4-Som HA2-Som3 (ZZ3); Panel H, CTLA-4-anti-lys (2V); Panel , CTLA-4-anti-mel (3E4). ). Traces represent absorbance at 214 nm and are given in arbitrary 5 units. Figure 8: Comparison by size exclusion FPLC analysis of affinity purified CTLA-4 constructs synthesised using bacterial expression vector pGC or pPOW. Recombinant human CTLA-4R or its loop variants were expressed in 10 E, coli host TOP10F, purified from periplasmic extracts by anti-FLAG affinity chromatography and.subjected to size exclusion chromatography on a calibrated Superose 12 HR column. The elution profiles of proteins expressed from vector pGC are shown on the left, whilst proteins expressed from vector pPOW are shown on the right. Panel A, wild-type CTLA-4 VILD 15 (S2); B, CTLA-4-Som1(PP2); C, CTLA-4-Som3(PP8); D, CTLA-4-Anti lys(2V8); E, CTLA-4-Som1-HA2-Som3(ZZ3), Figure 9: Protein stability analysis. Stability of monomer preparations of CTLA-4 VID and loop variant constructs was analysed by size exclusion fplc 20 chromatography on a precalibrated superose 12 hr (Pharmacia) column following several cycles of freeze/thawing. Aliquots of each protein were tested immediately after peak purification and following two cycles of freeze/thawing. A, CTLA-4 VLD (S2); B, CTLA-4-Soml (PP2); C, CTLA-4 Som3 (PP8); D, CTLA-4-anti-lys (2V); E, CTLA-4-Som-HA2-Som3 (ZZ3). 25 Figure 10: Lysozyme binding characteristics of CTLA-4-anti-lys construct 2V8. Figure 11: Screening of CTLA-4 VLD phagemid library on immobilised Sh 30 bleomycin. Figure 12: Screening of CTLA-4 VLD libraries in solution Detailed Description of the Inivention 35 The present invention relates to the design of novel soluble VLD binding molecules derived from the V-like domain of immunoglobulin COMS ID No: ARCS-265561 Received by IP Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:33 WATERMARK (F) 61398196010 49/147 13 superfamily members, such as the human CTLA-4 molecule. The preferred binding molecules of the present invention provide the following advantages (i) use of a native human protein obviates the need for subsequent humanisation of the recombinant molecule, a step often required to protect 5 against immune system response if used in human treatment; (ii) the domain is naturally monomeric as described above (incorporation of residue Cys120 in a C-terminal tall results in production of a dimeric molecule); and (iii) structural modifications have resulted in improved E.coli expression levels. Prior to publication of -the first CTLA-4 structure determination, 10 available sequence data and mutational analyses of both this molecule and CD28 were analysed. This allowed modelling and prediction of the regions corresponding to antibody CDRI, 2 and 3 regions. It was hypothesised that such areas would be susceptible to mutation or substitution without substantial effect upon the molecular framework and hence would allow 15 expression of a correctly folded molecule. The subsequently published structure (Metzler et al. 1997) showed these predictions to be accurate, despite the unexpected separation of CDRl from the ligand-binding site, and the extensive bending of CDR3 to form a planar surface contiguous with the ligand binding face. 20 In an initial set of experiments the V-like domain of the human CTLA 4 molecule was modified by replacement of CDR loop structures with either of two defined polypeptides. The two polypeptides were human somatostatin (Som) and a portion of the human influenza virus haemagglutinin protein (HA-tag). Somatostatin (SRIF: somatotropin release 25 inhibiting factor) is a 14 residue polypeptide comprising a disulphide bond that forces the central 10 residues into a loop. Human somatostatin is biologically widespread within the body and mediates a number of diverse physiological functions such as regulation of growth hormone secretion etc (Reisne, 1995). Human somdtostatin binds a number of specific receptors so (there are at least five subtyples) which have differing tissue specificities and affinities (Schonbrunn et al. 995). These aspects of binding and activation are as yet poorly understood,ibut one salient feature is the high density of somatostatin receptors present on a number of cancerous cell lines, for example cancers of the neuro-endocrine system and small lung cancers 35 (Reubi 1997). Artificial analogues of somatostatin have been produced for COMS ID No: ARcS-265561 Received by 1P Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:34 WATERMARK (F) 61398196010 50/147 14 imaging of such tumours which are resistant to degradation compared with the highly labile somatostatin polypeptide. The haemagglutinin epitope sequence consists of the 9 residues YPYDVPDYA. A commercially produced antibody is available which 5 specifically recognises this sequence. The epitope tag can be detected when randomly or directionally incorporated within the structure of proteins (Canfield et al. 1996). Replacement of one or more CDR loop structures in the CTLA-4 VIike domain with somatostatin or the HA-tag resulted in the production of 10 soluble, monomeric, unglycosylated binding molecules using different bacterial expression systems. This surprising finding shows that V-like domains provide a basic framework for constructing soluble, single domain molecules, where the binding specificity of the molecule may be engineered by modification of the CDR loop structures. 15 The basic framework residues of the V-like domain may be modified in accordance with structural features present in camelid antibodies. The camel heavy chain immunoglobulins differ from "conventional" antibody structures by consisting of only a single VH domain (Hamers-Casterman et al. 1993). Several unique features allow these antibodies to overcome the dual 20 problems of solubility and inability to present a sufficiently large antigen binding surface. First, several non-conventional substitutions (predominantly hydrophobic to polar in nature) at exposed framework residues reduce the hydrophobic surface, while maintaining the internal beta-sheet framework 25 structure (Desmyter et al. 1996). Further, within the three CDR-loops several structural features compensate for the loss of antigen binding-surface usually provided by the VL domain. While the CDR2 loop does not differ extensively from other VH domains, the CDR-1 and -3 loops adopt non-canonical conformations which are extremely heterologous in length. For example, the 30 HI loop may contain anywhere between 2-8 residues compared to the usual five in Ig molecules. However, it is the CDR3 loop which exhibits greatest variation: in 17 camel antibody sequences reported, the length of this region varies between 7 and 21 residues (Muyldermans et al. 1994). Thirdly, many camelid VH domains possess a disulphide linkage interconnecting CDRs -1 35 and -3 in the case of camels and interconnecting CDRs -1 and -2 in the case of llamas (Vu et al. 1997). The function of this structural feature appears to COMS ID No: ARCS-265561 Received by IP Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:35 WATERMARK (F) 61398196010 51/147 15 be maintenance of loop stability and providing a more contoured, as distinct from planar, loop conformation which both allows binding to pockets within the antigen and gives an increased surface area. However, not all camelid antibodies possess this disulphide bond suggesting that it is not an absolIte 5 structural requirement These foregoing features have enabled camelid V-domains to present as soluble molecules in vivo and with sufficiently high affinity to form ah effective immune response against a wide variety of target antigens. .In contrast, cell surface receptors of the Ig superfamily (such as CD4 and CP2) 10 comprise V-like binding domains and appear to bind cognate receptors with surface features other than the CDR loops. These V-like domains bind to cognate receptors with very low affinity. Physiological signalling between two cells are mediated by the avidity of multi-point binding, when two cell surfaces connect and each contains multiple receptors. CD2 is a well 15 characterised example: binding to CD58 is mediated by a highly constrained set of minor electrostatic interactions generated by charged and polar residues located in the GFCCG'C" face (not the antibody type CDR-1, CDR2 or CDR-3 loops). This results in a low affinity but highly specific interaction (Bodian et al 1994). 20 The present invention also relates to a method for generating and selecting single VLD molecules with novel binding affinities for target molecules. This method involves the application of well known molecular evolution techniques to V-like domains derived from members of the immunoglobulin superfamily. The method may involve the production of 25 phage or ribosomal display libraries for screening large numbers of mutated V-like domains. Filamentous fd-bacteriophage genomes are engineered such that the phage display, on their surface, proteins such as the Ig-like proteins (scFv, Fabs) which are encoded by the DNA that is contained within the phage 30 (Smith, 1985; Huse et al., 1989; McCafferty et al., 1990; Hoogenboom et al. 1991). Protein molecules can be displayed on the surface of Fd bacteriophage, covalently coupled to phage coat proteins encoded by gene III, or less commonly gene VII. Insertion of antibody genes into the gene III coat protein gives expression of 3-5 recombinant protein molecules per phage, 35 situated at the ends. In contrast, insertion of antibody genes into gene VIII has the potential to display about 2000 copies of the recombinant protein per COMS ID No: ARCS-265561 Received by IP Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:35 WATERMARK (F) 61398196010 52/147 16 phage particle, however this is a multivalent system which could mask the affinity of a single displayed protein. Fd phagamid vectors are also used; since they can be easily switched from the display of functional Ig-lilce fragments on the surface of Fd-bacteriophage to secreting soluble Ig-like 5 fragments in E. coli. Phage-diisplayed recombinant protein fusions with the N-terminus of the gene In coat protein are made possible by an amber codon strategically positioned between the two protein genes. In amber suppressor strains of E. coli, the resulting Ig domain-gene III fusions become anchored. in the phage coat. 10 A selection process based on protein affinity can be applied to any high-affinity binding reagents such as antibodies, antigens, receptors and ligands (see, for example, Winter and Milstein, 1991, the entire contents iof which are incorporated herein by reference). Thus the selection of the highest affinity binding protein displayed on bacteriophage is coupled to the 15 recovery of the gene encoding that protein. Ig-displaying phage can be affinity selected by binding to cognate binding partners covalently coupled to beads or adsorbed to plastic suraces in a manner similar to ELISA or solid phase radloinununoassays. While almost any plastic surface will adsorb protein antigens, some commercial products are especially formulated for 20 this purpose, such as Nunc Imniunotubes. Ribosomal display libraries involve polypeptides synthesised de novo in cell-free translation systems and displayed on the-surface of ribosomes for selection purposes ([Hanes and Pluckthun, 11997; He and Taussig, 1997). The "cell-free translation system"l comprises ribosomes, soluble eniyxnes required 25 for protein synthesis (usually from the same cell as the ribosomes), transfer RNAs, adenosine triphosphate, guanosine triphosphate, a ribonucleoside triphosphate regenerating system (such as phosphoenol pyruvate and pyruvate kinaseJ, and the salts and buffer required to synthesize a protein encoded by an exogenous rnIRNA. The translation of polypeptides can be s0 made to occur under conditions which maintain intact polysomes, i.e. where ribosomes, mRNTA molecule and translated polypeptides are associated in a single complex. This effectively leads to "ribosome display" of the translated polypeptide. For selection, the translated polypeptides, in association with the 35 corresponding ribosome complex: are mixed with a target molecule which is bound to a matrix (e.g. Dynabeads). The target molecule may be any COMS ID No: ARr-S-265561 Received by IP Austreaa Time (HNm) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:36 WATERMARK (F) 61398196010 53/147 17 compound of interest (or a portion thereof) such as a DNA molecule, a protein, a receptor, a cell surface molecule, a metabolite, an antibody, a hormone or a virus. The ribosomes displaying the translated polypeptides will bind the target molecule and these complexes can be selected and the 5 mRNA re-amplified using RT-PCR, Although there are several alternative approaches to modify binding molecules the general approach for all displayed proteins conforms to a pattern in which individual binding reagents are selected from display libraries by affinity to their cognate receptor. The genes encoding these 10 reagents are modified by any one or combination of a number of in vivo and in vitro mutation strategies and constructed as a new gene pool for display and selection of the highest affinity binding molecules. In order that the nature of the present invention may be more clearly understood preferred forms thereof will now be described with reference to 15 the following examples. Example 1 Gene Construction and Cloning CTLA-4 STM (STM: solluble truncated mutants of CTLA-4, used herein 20 to describe CTLA-4 chimaeric V-like domain proteins) gene construction and cloning was by standard and well-described techniques (Polymerase chain reaction with specifically designed oligonucleotide primers, splice overlap extension, restriction enzyme digests etc). A list of oligonucleotide printers used is given in Figure 1. 25 The wild-type STM construct was amplified from cloned human CTLA-4 DNA (Figure 2) (and could be similarly amplified from reverse transcribed human cDNA by a competent worker in the field) using the oligonucleotide primers #3553 and #4316, which incorporated Sfi and Not! restriction sites at the 5' and 3' ends respectively. These terminal primers 30 were used in all further constructions except: (i) where #4851 or #5443 was used to incorporate an ApaL1 site at the 5' end; (ii) where #4486 was used to add a C-terminal tail Including residue Cys120; (iii) where #5467 was used to incorporate an EcoRi site at the 5' end; and (iv) where the specific set of extension primers were used for ribosomal display. 35 A splice overlap PCR strategy using combinations of the oligonucleotides primers listed in Figure 1 was used to produce variations of COMS ID No: ARCS-265561 Received by IP Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:37 WATERMARK (F) 61398196010 54/147 18 CDR-1, CDR-2 and/or GDR-3 loop structure replacements. The variations, which are described in greater detail in the following examples are listed in Table 2. 5 TABLE 2 CDR-i combination CUDR-1 CTLA-4 VLD S" 9 FVCEYA.SPGKATE............ VRV... 10 Anti-lysozyme .S'FVCEYA.SGYTIGPYCMG.......... VRV... Somatostatln-14 S"FVCSYA.AGCKNFWKTFTSCATE. VRV... Anti-melanoma S'FVCYASGFTSSYAMS.......... VRV... 15 Randomisation I S 19 FVCBYA.XXXXXXXG ............. VRV... Randomsation 2 SmFVCEYA.XXXXX X G .......... VRV... Randomisation 3 S"VCEYA.XXarXarXXarCXG....... VRV,.. Randomisation 4 S'aFVCEYA.SPGXXXX.............. VRV... Randomisation 5 S"FVCEYA.SPGXCXX ............ VRV. 20 Randomisation 6 S' 9 FVCEYA.XXXXX XTE........... VRV... Randomisation 7 SlFVCEYA.fXXXXCXATE......... V... Randomisation 8 S"FVCBYA.AGCKNXXXOXTSC&TR. VRV.. 25 CDR-2 combinations CDR-Z CTLA4 VLD Q"VTEVCAA.TYMMGNELTF.LDDSICT... 30 Anti-lysozyme Q 4 VTEVCAA.AINMGGGITF.LDDSICT.., Haemaggutinin tag Q"VTEVCAA.TYPYDVPDYA.LDDSICr.. Anti-melanoma Q'VTEVCAA.AISGSGGSTY.LDDSICT... Randomisation 1 Q"VTEVCAA.TYXXGXELTF.LDDSICr... 35 Randomisation 2 Q"VTEVCAA.CYXXGXELTF.LDDSICr... COMS ID No: ARCS-265561 Received by IP Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:37 WATERMARK (F) 61398196010 55/147 19 CDR-3 combiations CDR-3 CTLA-4 VLD C"KV.ELMYPPPYYL ............. GIG... 5 Anti-lysozyme C"KV.DSTIYASYYECGHGLSTGGYGYDS. GIG... Somatostatin-14 C"KV.EACKNFFWKTFTSQ......... GIG... Anti-melanoma C"KV.GWtLRGEEGDYYMDV.......... GIG... Randomisation 1 C"KV.XXXXX XX.............. GIG.. 10 Randomisation 2 C V. XXX ....... GfG.. Randomisation 3 C"KV.XXXXXC.XXXX ............ GIG... Randomisation 4 C"KV.XXXXXXX.C.XXX.......... GIG... Randomisation 5 C"KV.XXXXXXXX.C.XXXX......... GIG.. Randomisation 6 C"IKV.XXXXXXXXX.C.XXXXXXX..... GIG... 15 Randomisation 7 C"KV.EXXXXXXXXX............... GIG... Randomisation 8 C"KV.EXXXXXX.CXXXXXX....... GIG... Randomisation 9 C"KV.EAGCKNXXXXXXTS.......... GIG... 20 For generation of randomised sections of the CDR loop structures, similar splice-overlap techniques were used with oligonucleotides where a given triplet(s) were encoded by the sequence NNgfI where N represents any of the four possible nucleotide bases. This combination covers all possible amino acid residues. Alternatively, randomisation was biased towards 25 certain subsets of amino acids (for example aromatic residues, Figure 1, #5452). In some instances, a variant technique was used for STM gene construction, where randondsed oligonucleotide primers were designed which incorporated restriction sites for direct cloning into the similarly 30 modified (with complementary restriction sites) CTLA-4 VLD framework (for example Figure 1, #4254). Completed constructs were cut with approrlate combinations of restriction enzymes (for example Sfil,Notl, ApaLl, EcoRI) and cloned into like sites in appropriate expression vectors. These vectors comprise: (i) for as production of soluble protein expression vectors pGC (Coia et al, 1996) and pPOW (Power et al, 1992; Kortt et al. 1995) (ii) for bacteriophage and COMS ID No: ARCS-265561 Received by IP Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:38 WATERMARK (F) 61398196010 56/147 20 phagemid display, completed STM constructs were cut with the restriction enzymes Sfil and Nod or ApaLI and Not and cloned into the vectors pHiFA, and pFAB.5c (phagemid) or pfd-Tet-DOG (phage). These vectors allow display of the STMs as gene3 protein fusions on the surface of bacteriophage 5 in 1-2 (phagemid) or 3-5 (phage) copies per bacteriophage particle (Figure 3). All DNA constructs were verified by restriction analysis and DNA sequencing and tested for expression of recombinant protein by standard and well-understood techniques (Polyacrylamide gel electrophoresis, Western blot etc). Example 2 Production and Isolation of Recombinant STM Proteins Recombinant proteins were produced using vectors which represent different protocols for periplasmic expression systems. These vectors were 15 (i) pGC: this vector allows high level expression of heterologous proteins by chemical (IPTG) induction, which are targeted to the periplasmic space by means of a leader sequence. The leader sequence is subsequently cleaved to produce the mature protein. In addition, this vector contains two in-frame 8 residue tag sequences (FLAG tags) which allow affinity purification of the 20 recombinant protein. (ii) pPOW, which, like pGC, allows high level heat inducible expression of proteins targeted to the periplasmic space by means of a cleavable leader sequence and two in-frame 8 residue tag sequences (FLAG tags). Recombinant proteins were purified by the following methods, which 25 are but two variations of well established techniques. (i) Bacterial clones in vector pGC were grown overnight in 2YT medium/37"C /200 rpm/100mg/ml ampicillin, 196 glucose (final). Bacteria were diluted 1/100 into either 0.5 ot 21 of 2YT medium supplemeited with 100mg/ml ampicillin, 0.196 glucose (final), and grown at 28*C/ 200 rpm. These cultures were grown to an optical 30 density of between 0.2-0.4, at which stage they were induced with 1mM IPTG (final). Cultures were allowed to grow for 16 hours (overnight) before harvesting. Bacteria were collected by centrifugation (Beckman JA-14 rotor or equivalent/6K/10min/*C) and the periplasmic fraction collected by standard techniques. Briefly, this involved resuspension of bacterial pellets 35 in a 1/25th volume of spheroplast forming buffer consisting of 100mM Tris HC1/0.5M sucrose/0.5 mM EDTA (pH8.0), followed by addition of 1/500&t COMS ID No: ARCS-265561 Received by P Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:39 WATERMARK (F) 61398196010 57/147 21 volume of hen egg lysozyme (2mgtmliIn water) and incubation for 10min. A 0.5x solution of the above spheroplasting buffer was then added to a final volume of 115th of the original culture, and the incubation continued fat a further 20min. The cell debris was then pelleted by centrifugation (Beckman 5 JA-14 rotor or equivalent/K/5minJ400) and the supernatant containing the periplasmic fraction collected. All of the above procedures were performed at 4VC. Samples were processed immediately by sonication, filtration through a 0.45J±t nitrocellulose membrane and processed immediately or stored at 4 0 C in the presence of sodium azide (0.05%]. If freezing was 10 required, no more then one freeze-thaw cycle was allowed. (ii) Bacterial clones in plOW were grown overnight at 30 0 C in 100 ml ZxYT broth containing 100 4mgml (w/v) ainpicillin. On the following day cultures were used to inoculate 900 ml fresh 2xYT broth containing 100 pig/mi (wlv) ampicillin, to ODoflaum = 0.2-0.5, and grown at 30CC with shaking (150-200 15 rpm) until OD600nra = 4 i.e. late log phase. To induce recombinant protein expression, the temperature was raised to 42 0 C for 1 hour and then dropped to 2006 for a further hour. Cells were harvested by centrifuigation (Beckinan JA-14 /6K rpm/5 rnin/4"C), the cell pellet resuspended in 100 ml extraction buffer (20mM hris pH 8.0/ 0.2mg/nil (w/v) lysozyme/0.i% (v/v) Tween-20] 20 and incubated at V~C overnight. Samples ware sonicated for 30 seconds and cellular debris collected by centrifugation (Beckman JA-14 /14K rpm/iD0 znin/4 0 0). The aqueous phase, containing the "lysozyme wash, was retained. Cell pellets were then washed twice with ice-cold water and this "osmotic, shock' wash was retained. Each wash consisted of resuspending the pellet in 25 100 ml ice-cold water followed by incubation on ice for 10 minutes in the first instance followed by 1 hour in the second instance. Following centrifugation (Beckmaan. JA-14 /14K rpm/tO min/4 0 C), the pH of the aqueous phase was adjusted by addition of 10 ml 1OxTBS, pH 8. The "lysozyme" and "osmotic shock!'washes were pooled and constitute the soluble or 30 "periplasinic" protein fraction. Periplasmic fractions were sonicated, filtered through a 0.45gp nitrocellulose membrane and processed immediately or stored at 4*C in the presence of sodium aside (0.05%), PMSF (23 pg/nil) and EDTrA (50 mNvf. Recombinant proteins were purified by affinity chromatography 35 through a divinyl sulphone activated agarose (Mil-Leak)-linked anti-FLAG antibody column. Periplasnmic extracts were directly loaded onto a 10 ml COMS ID No: ARCS-265561 Received by IP Australia: lime (H:m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:39 WATERMARK (F) 61398196010 58/147 22 anti-FLAG column which had been pre-equilibrated in TBS (pH 8) containing 0.0596 (w/v) sodium azide, Bound proteins were eluted with Immunopure Gentle Ag/Ab Elution Buffer (Pierce). Samples were then dialysed against TBS/0.05% (wlv) azide (pH 8), concentrated by ultrafiltration over a 3 kDa 5 cut-off membrane (YM3, Diaflo), and analysed by HPLC on a pre-calibrated Superose 12 HR or Superdex 200 HR column (Pharmacia Biotech), at a flow rate of 0.5 ml/min. Fractions corresponding to monomeric, dimeric and tetrameric species were collected, concentrated as above, and stored at 4*C prior to analysis. Protein concentration was determined 10 spectrophotometrically using an extinction coefficient at A280 of 1.27 for the CTLA-4R extracellular domain, 0.92 for CTLA-4-Somi, 1.13 for CTLA-4 Som3, 1.05 for CTLA-4-Anti-Lys. All of the above protein chemistry methods are standard techniques within the field. Purified proteins were analysed by standard techniques for example polyacrylamide gel electrophoresis, western 15 blot, dot blot etc. Cloning and expression in the bacteriophage expression vectors pHFA, pFAB.5c and fd-tat dog, and subsequent production of recombinant bacteriophage, were by standard and well-established techniques. Screening of libraries of randomised CTLA-4 STMs was by standard and well 20 established techniques (Galanis et al 1997). Example 3 CTLA-4 STMs incorporating Somatostatin and Haemagglutinin Peptides. Initially the-CDRl or the CDR3 loop structures of the CTLA-4 STM 25 were replaced with the somatostatin polypeptide, This 14 residue polypeptide is conformationally constrained by an intra-disulphide linkage between Cys3 and Cys14 (Figure 4). This was reasoned to form a discrete protein loop, analogous to the CDR loops found in antibodies, and particularly analogous to the long CDRs found in camelid antibodies which 30 are also stabilised by a disulphide linkage. The effect of substituting CDR1 in the presence or absence of Cys120 ie. whether a dimer could be produced, was also tested. These experiments produced an unexpected and surprising result Substitution of either CDR-i or -3 with somatostatin significantly enhanced the production of monomeric protein. This is illustrated in Figure 35 5 where replacement of the CDR-3 loop structure with somatostatin COMS ID No: ARCS-265561 Received by IP Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:40 WATERMARK (F) 61398196010 59/147 23 significantly increased the ratio of monomeric to dimeric/tetrameric protein species. In further experiments, simultaneous replacement of both CDR-1 and 3 loop structures by somatostatin resulted in production of high-levels of 5 monomeric protein. This shows that extensive loop structure replacements can be accommodated by the CTLA-4 scaffolding. Structurally, one of the somatostatin loops may lie flat against the face of the molecule in a manner analogous to that of the CDR-3 loop structure of CTLA4 VID. In a further extension 6f the CDR loop structure-replacement strategy, 10 a region corresponding to CDR-2 was replaced with the 8-residue haemagglutinin (HA) tag sequence. Use of the conformationally constrained somatostatin loop in this position was considered unsuitable as this region partially encompasses the C" beta strand running the length of the molecule. The HA tag could be detected upon this CTLA-4 STM by use of an anti-HA 15 antibody. Gel filtration experiments showed the presence of a range of protein species, from monomeric through to aggregated species suggesting that CDR-2-only substitutions were not stable (Figures 6,7). Final proof of principle that the CTLA-4 CDR loop structures could be replaced with other polypeptides to produce monomeric, soluble, STMs was 20 by simultaneous replacement of all three CDR loop structures with two somatostatin and one HA epitope respectively. This STM produced a correctly folded and monomeric protein upon gel filtration chromatography (Figures 6,7). The positions of CDR loop structure substitutions within the CTLA-4 25 VLD for the various STMs are shown in figure 6. HPLC profiles of affinity purified STM proteins are shown in figure 7. Identical results were obtained for proteins produced in two different protein expression systems: pGC where protein expression is chemically induced, and pPOW where protein expression is temperature induced (see Example 2)(Figure 8). 30 Polyacrylamide gel electropbloresis followed by western blot analysis indicated that the CTLA-4 STMs could be reduced and ran at the expected molecular weights and absent of glycosylation. Testing of isolated monomeric STM proteins showed that they remained monomeric after zero, one, or two freeze-thaw cycles (figure 9). 35 CTLA-4 STMs retained the correct conformation since a conformationally-specific anti-CTLA-4 antibody recognised STMs with both COMS ID No: ARCS-265561 Received by IP Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:41 WATERMARK (F) 61398196010 60/147 24 CDRI and -3 loop structure replacements. Interestingly, this antibody recognised the wild type monomer and the dimer (CDRl replaced) poorly, contrasting with the strong reaction observed for the modified protein species. This suggests that in the wild type STM some form of local 5 interaction is occurring that occludes the antibody binding site, and that this interaction is similar to the result when two CTLA-4 molecules are tethered together (presumably blocking access to the antibody). Example 4 10 CTLA-4 STMs Based Upon a Camel anti-Lysozyme Antibody. The camel. YH antibody cAb-Lys3 isolated from immunised camels specifically binds within the active site cleft of hen egg lysozyme (Desmyter et al. 1996). To illustrate the ability of CTLA-4 STMs to function in a similar fashion, the three CDR loop structures of CTLA-4 VLD STM were replaced 15 with the three CDR loop regions from cAb-Lys3. Positions and sequence of the substitutions are shown in figure 6. Expression of this STM (2V8) in either pGC or pPOW based expression systems resulted in production of predominantly monomeric soluble protein (Figures 7, 8). Protein solubility of this CTLA-4 STM was superior to native CTLA-4 VLD. ELISA analysis 20 showed that (pGC produced) purified monomeric protein specifically bound hen egg lysozyme compared to non-specific antigens and compared to the CTLA-4 STM with somatostalin substituted within the CDRI loop structure (PP2) (Figure 10A). Real-time binding analysis by BIAcore showed that the lysozyme specifically bound to immobilised anti-lysozyme STM (Figure 10B). 25 The CTLA-4 STM framework is thus folding correctly and presenting the CDR loop structures in a manner in which they can bind lysozyme antigen. To further enhance expression of the CTLA-4 VLD anti-lysozyme, the coding sequence was adjusted by splice overlap PCR to comprise codons preferential for E. col expression. 30 Example 5 CTLA-4 STMs Based Upon aiHuman anti-Melanoma Antibody. The human-derived aniti-melanoma antibody V86 specifically binds human melanoma cells. This antibody is unusual in that binding affinity 35 resides entirely within the VH region, addition of a cognate V. decreases binding efficiency, and that the V 5 domain expressed with a small segment of COMS ID No: ARCS-265561 Received by IP Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:42 VATERMARK (F) 61398196010 61/147 25 the VL domain has a high degree of solubility (Cai and Garen, 1997). To further illustrate that replacement of CTLA-4 VID CDR loop structures enhances solubility and that the resultant STMs can be produced in bacterial expression systems, the three CDR loop structures of CTLA-4 were replaced 5 with the three CDR loop regions from V86. Positions and sequence of this substitutions are shown in Figure 6. Expression of this STM (3E4) in pGC again resulted in production of predominantly monomeric soluble protein (Figure 7) with enhanced solubility compared to the CTLA-4 VLD. 10 Example 6 Construction of CTLAA STMs as Libraries of Bindig Molecules To select CTLA-4 STMs with novel binding specificities, VLD libraries were produced containing randomised CDR1 and CDR3 loop structures. Oligonucleotide primers used for library construction are listed in figure 1. 15 Combinations of oligonucleotide primers used for library construction are shown in Table 3. Table 3. CTLA-4 STM Library Combinations 20 CDRI CDR3 14483* 4254 5449 5451 5452 5450 5446 4835 "852 +1 +1 ifihli1111 1111 /11111 lII/l /1111 1111/ 5470 1111/111111+2 +2 +2 + 2 +2 /1111 5474 1/1////// //1/ +2 +2 +2 +2 +2 / 5471 1111/1 f1111+2 +2 +2 +2 +2 11111 5472 1/11111/// 11//////11 +2 +2 +2 +2 +2 / 5475 1111111111 1111111111 +2 +2 +2 +2 +2 S/1/// 85473 1111_ 1/111_+2 +2 +2 +2 +2 11|11/ 4836 ////// ///////////1//////////////// /////+3 *; oligonucleotide number. +: library combination. 1,2,3: describes library number. 25 COMS ID No: ARCS-265561 Received by IP Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:42 WATERMARK (F) 61398196010 62/147 26 DNA constructs encoding the resultant libraries were cloned into vectors pHFA or pFAB.5c for production of fd-phagemid based libraries and into pfd-Tet-Dog for production of fd-phage based libraries (see examples 1 and 2). Library I was cloned into vector pHFA and consisted of 2.1 x 10' 5 independent clones. Library 3 was cloned into vectors pHFA (5.7 x 10 independent clones) and pfd-Tet-Dog (2.2 x 10' independent clones). Library 2 was cloned into pFAB.5c (1.7 x 10' independent clones) and into pfd-Tet-Dog (1.8 x 105 independent clones). Numbers of independent clones were determined by counting gross numbers of transformed colonies 10 constituting the library, followed by assaying for the presence and proportion of CTLA-4 STM-specific DNA. For library 2, the variability of the full library was tested by sequencing of representative clones. These results are presented in Table 4. The expected heterogeneity of insert size and sequence was observed. A high 15 proportion of UAG termination codons were observed, consistent with the oligonucleotide randomisation strategy. To prevent these codons causing premature termination of the CTLA-4 STM gene protein fusions, libraries were transferred into the R.coli strains Tg-1 and JM109, which suppress this termination codon by insertion of a glutamic acid residue. Cysteine residues 20 were present in the high numbers expected from the desgn of the oligonucleotides,and were in positions capable of forming Intra- and inter CDR loop structure disulphide bonds. COMS ID No: ARCS-265561 Received by IP Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:43 WATERMARK (F) 61398196010 63/147 27 Table 4 CDRI and CDR3 Inserts from a Representative Series of Library 2 Clones CLONE CDR1 CDR3 3M-2 ND1 LPSSDTRAYS 3M-3 QESGGRPG LPRGPPLLSL 3M-5 SPGRCLN ND 3M-6 EWKR*HGG LCPGACGCVY 3M-7 NSG*NEGG ND 3M-11 DKPVTKSG ND M3-17 SPGACP* ND 3M-18 SPGKCDQ ND 3M-19 SPGMCAR LMYPPPYYL 3M-20 ND PFLFLPC*FFF 3N-1 WTLGHHKLCEG LFTCLLALCS 3N-2 SPGECYG SWLSTTXCLSSCS 3N-3 SPG*CQD LLGSLLSCFASLS 3N-4 SPG*CQD SPGSLLSCFASXS 3N-5 SPGRCTD VICHSSVCLSD/EVC 3N-6 ND DLPSYLACSI 3N-7 SPGRCDA ALCWDVFYCSFPSY 3N-8 ELFGHARYCKG VSITSP*LCPVKVFD 3N-9 SPGKV*N LFVPFVSP 3N-12 SPGDLWV ESGLSPVSPCSLYSL 3N-13 TSANGPYG PWAYRFLAVL 3N-14 RKTREKYG ELMYPPPYYLGI 3N-15 SPGQELT ELFFLLYAPCYLFQR 5 ND: Not Done *: UAG termination codon COMS ID No: ARCS-265561 Received by P Australia: Time (Hm) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:43 WATERMARK (F) 61398196010 64/147 28 Bacteriophage particles displaying CTLA-4 STMs as gene 3 protein fusions were rescued from E.coli cells by standard protocols and panned against antigens presented in a number of contexts as described in the 5 following examples. Example 7 CTLA-4 STM Libraries: Selection against Antigens on Solid Supports Four different antigens falling into a class of proteins with clefts or 10 crevices within their structures were selected for screening. It was anticipated that the CTLA-4 VLD STMs, being of smaller size than antibodies, and possessing elongated CDR loop structures (especially CPR-3) would be able to access these cleft regions. The antigens selected were:-(i) hen egg lysozyme (EC 3.2.1.17); (ii) bovine carbonic anhydrase (EC 4.2.1.1); 15 (iii) fungal a-amylase (EC 3.2.1.1); and (iv) Streptoalloteichus hindustanis resistance protein ShBle (Gatignol et al. 1988). For binding to plates, antigens in coating buffer (1mg/ml in 0.1M NaHCO3 pHB.5) were boundto Costar ELISA plates by standard procedures. Rescued phage and phagemid derived libraries were panned by standard and well-understood procedures 20 except that lower than standard number of washes were employed to allow low affinity binding phage to be selected. Figure 11 shows titres of libraries selected against ShBle. After round 4, recovered bacteriophage titres were higher than controls. To those skilled in the art, this represents selection of specific binding moieties, and it is then a routine process to produce those 25 selected CTLA-4 VLD STMs using expression vectors such as pGC or pPOW (as described in example 2). Example 8 CTLA-4 STM Libraries: Selection against Antigens in Solution. 30 For selection in solution, the antigens bovine carbonic anhydrase and fungal a-amylase were biotinylated and selections performed in solution using capture by streptavidin coated magnetic beads. Throughout these experiments washes were kept constant at either 2 or 5 washes per selection round. Titres of recovered bacteriophage post-elution are shown In Figure 35 12. After round 4, recovered bacteriophage titres were higher than controls. To those skilled in the art, this represents selection of specific binding COMS ID No: ARCS-265561 Received by IP Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:44 WATERMARK (F) 61398196010 65/147 29 moieties, and it is then a routine process to produce these selected CTLA-4 VLD STMs using expression vectors such as pGC or pPOW (as described in example 2). 5 Example 9 CTLA-4 STM Libraries: Selection in an Alternative Display and Selection System. To allow further maturation and selection of antigen binding STMs, the CTLA-4 STM library was ligated into a plasmid to add a downstream C 10 terminal spacer polypeptide (heavy constant domain). Upstream transcriptional and translational initiation sequences were added by PCR amplification using specific oligonucleotides (Figure 1). This PCR DNA was used as a template for production of RNA followed by translation and display of the library on ribosomes in a coupled cell free translation system as 15 described by He and Taussig (1997). To demonstrate binding, CTLA-4 STM ribosome complexes were panned on hepatitis B surface antigen (hbsa), glycophorin (glyA) and bovine serum albumin (BSA) coated dynabeads. RNA from ribosome complexes bound to hbsa, glyA and BSA was recovered by RT-PCR. It is then a routine process to clone these RT-PCR products into an 20 expression vector such as pGC or pPOW (as described in example 2) allowing production of CTLA-4 STMs. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention allowing display of libraries of CTLA-4 STMs as ribosome complexes (as in this example) as well as display on the surface of live cells 25 (which may be derived from a eukaryotic or prokaryotic background) and may include bacteria, yeast, mammalian or insect cells. Example 10 CTLA4 STMs: AffinLty Maturation and CDR2 Mutation. 30 To allow further maturation and selection of antigen-binding STMs, and the construction of randomised CDR-1, -2 and -3 libraries, CDR-2 randomised oligonucleotide primers were produced (Figure 1). A variation of these primers contained conserved cysteine residues to allow construction of STMs with CDR2-CDR3 disuiphide linkages mimicing those found in. llama 35 single domain antibodies. Splice overlap PCR allowed creation of libraries containing all three CDR loop structures randomised. COMS ID No: ARCS-265561 Received by IP Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:45 WATERMARK (F) 61398196010 66/147 30 It will be appreciated >y persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefo , to 5 be considered in all respects as illustrative and not restrictive. COMS ID No: ARCS-265561 Received by P Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:45 WATERMARK (F) 61398196010 67/147 31 REFERENCES Bodian DL, Jones EY, Harlos K, Stuart DI, Davis SJ (1994) Crystal structure of the extracellular region of the human cell adhesion molecule CD2 at 2.5 A 5 resolution. Structure 2: 755-766 Cai X and Garen A (1997) Comparison of fusion phage libraries displayinIg VHI or single-chain Fv antibody fragments derived from the antibody repertoire of a vaccinated melanoma patient as a source of melanoma-specific 10 targeting molecules Immunil 94: 9261-9266 Canfield VA, Norbeck L, Levenson R (1996) Localization of cytoplasmic land extracellular domains of Na,K-ATPase by epitope tag insertion Biochem 85: 14165-14172 15 Coia, G., Hudson, P.J., Lilley, G.G. (1996) Construction of recombinant extended single-chain antibody peptide conjugates for use in the diagnosis of HIV-1 and HIV-2. J. Immunol. 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Gatignol A, Durand H, and Tiraby G (1988) Bleomycin resistance conferred 35 by a drug-binding protein FEBS Lett 230: 171-175 COMS ID No: ARCS-265561 Received by P Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:45 VATERMARK (F) 61398196010 68/147 32 Gerstmayer B, Pessara U, Wels W (1997) Construction and expression in the yeast Pichia pastoris of functionally active soluble forms of the human costimulatory molecules B7-4 and B7-2 and the B7 counter-receptor CThA-4. FEBS Left 407: 63-68 5 Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R (1993) Naturally occurring antibodies devoid of light chains. Nature 363: 446-448 10 Hanes J and Pluckthun A (1997) In vitro selection and evolution of functional proteins by using ribosome display Proc.Natl.Acad. Sci. USA. 94: 4937-4942 He lf and Taussig MJ (1997) Antibody-ribosome-mRNA (ARM) complexes as efficient selection particles for in vitro display and evolution of antibody 15 combining sites. Nucl. Acids Res. 25: 5132-5134 Hoogenboom, KR., Griffiths, AD., Johnson, KS., Chiswell, D.J., Hudson, P., and Winter, G. 1991. Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. 20 Nucleic Acids Res. 19:4133-4137. House, W. D.,Sastry, L.,Iverson, S. A.Kang, A. S.,Alting, M. M.,Burton, D. R.,Benkovic, S. J., & Lerner, R. A. (1989). Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. Science, 246: 25 1275-81. Hutloff, A., Dittrich, A.M., Beier, K.C., ElijaschewitSch, B., Kraft, R., Anagnostopoulos, L and Kroczek, R.A. (1999). ICOS is an inducible T-cell co-stimulator structurally and functionally related t. CD28. Nature 397: 263 30 266. Kortt, A.A., Guthrie, RE., Hinds, M.G., Power, B.E., Ivancic, N., Caldwell, J.B,, Gruen, L.C., Norton, R.S. and Hudson, P.J. (1995) Solution properties of E. coli expressed VH domain of anti-neuraminidase antibody NC41. J. 35 Protein Chemistry. 14, 167-178. COS..N:ACS25.. Rciedb.P.utaia.iie(.).71.at.YM-)21...0...-.......02 COMS 10No:ARC-S-265561 Received by IP Australia: Time (N~m) 17:11 Date (Y-M-d) 2010-02-as Feb-2010 16:46 WATERMARK (F) 61398196010 69/147 33 Linsley, PS, Clark EA, and Ledbetter JA (1990) The T cell antigen CD28 mediates adhesion with B cells by interacting with the activation antigen, B7/BB1 Proc. Natl. Acad. Sci USA 87: 5031, 5 Linsley PS, Nadler SG, Bajorath J, Peach R, Leung HT, Rogers J, Bradshaw J, Stebbins M, Leytze G, Brady W, et al (1995) Binding stoichiometry of the cytotoxic T lymphocyte-associated molecule-4 (CTLA-4). A disulfide-linked homodimer binds two CD88.molecules. J-Biol Chem 270: 15417-24 10 McCafferty, J.,Griffiths, A. D,,Winter, G., & Chiswell, D. J. (1990). Phage antibodies: filamentous phage displaying antibody variable domains. Nature, 348:552-4. Metzler WJ, Bajorath J, Fenderson W, Shaw SY, Constantine KL, Naemura J, 15 Leytze G, Peach RJ, Lavoie TB, Mueller L, Linsley PS (1997) Solution structure of human CTLA-4 and delineation of a CD80/CD86 binding site conserved in CD28 Nat Struct Biol 4:527-531 Morton PA, Fu XT, Stewart JA, Giacoletto KS, White SL, Leysath CE, Evans 20 RJ, Shieh JJ, Karr RW (1996) Differential effects of CTLA-4 substitutions on the binding of human CD80 (B7-1) and CD86 (B7-2) J Immunol 156: 1047 1054 Muyldermans S, Atarhouch T, Saldanha J, Barbosa JA, Hamers R (1994) 25 Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains. Protein Eng 7: 1129-1135. Nieba L, Honegger A, Krebber C, Pluckthun A (1997) Disrupting the hydrophobic patches at the antibody variable/constant domain interface: 30 improved in vivo folding and physical characterization of an engineered scFv fragment Protein Engineering (4):435-44 Novotny J, Ganju RK, Smiley ST, Hussey RE, Luther MA, Recny MA, Siliciano RF, Reinherz EL (1991) A soluble, single-chain T-cell receptor 35 fragment endowed with antigen-combining properties Proc Natl Acad Sci U S A 88(19):8646-8650. COMS ID No: ARCS-265561 Received by IP Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:46 WATERMARK (F) 61398196010 70/147 34 Peach RJ, Bajorath J, Brady W, Leytze G, Greene J, Naemura J, Linsley PS (1994) Complementarity determining region 1 (CDRi)- and CDR3-analogous regions in CTLA-4 and CD28 determine the binding to B7-1 J Exp Med 180: 5 2049-2058 Power, B.E., Ivancic, N., Harley, V.R., Webster, R.G., Kortt, A.A, Irving, R.A. and Hudson, P.J. (1992) High-level temperature-induced synthesis of an antibody VH-domain in Escherichia coli using the PeiB secretion signal gene 10 113:95-99. Reisine, T. (1995) Somatostatin. Cell Molec. Neurobiol. 15: 597-614 Reubi, J. C. (1997) Regulatory peptice receptors as molecular targets for 15 cancer diagnosis and therapy. Q. J. Nucl. Med. 41: 63-70 Schonbrunn A, Gu YZ, Brown PJ, Loose-Mitchell D (1995) Function and regulation of somatostatin receptor subtypes. Ciba Found. Symp. 190: 204 217 20 Smith, G. P. (1985). Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science, 228, 1315-1317 van der Merwe PA, Bodian DL, Daenke S, Linsley P, Davis SJ (1997) CD80 25 (B7-1) binds both CD28 and CTLA-4 with a low affinity and very fast kinetics. J Exp Med 185: 393-403 Vu KB, Ghahroudi MA, WynsL, Muyldermans S (1997) Comparison of llama VH sequences from conventional and Heavy Chain Antibodies. Molec 30 Immunol 34: 1121-1131 Ward ES (1991) Expression and secretion of T-cell receptor V alpha and V beta domains using Escherichia col as a host Scand J Immunol 34(2):215-220. 35 Waterhouse P, Marengere LE, Mittricker HW, Mak TW (1996) CTLA-4, a negative regulator of T-lymphocyte activation. Immunol Rev 153: 183-207 COMS ID No: ARCS-265561 Received by P Australia: Time (H m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:47 WATERMARK (F) 61398196010 71/147 35 Waterhouse P, Penninger JM, Timms E, Wakeham A, Shahinian A. Lee KP, Thompson CB, Griesser H, Mak TW (1995) Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4 Science 270: 985-988 5 Winter G, Milstein C. (1991). Man-made antibodies. Nature. 349: 293-299. Wulfing C, Pluckthun A and in (1994) Correctly folded T-cell receptor fragments in the periplasm of Escherichia coBl -Influence of folding catalysts 10 J Mol Biol 242(5):655-69. COMS ID No: ARCS-265561 Received by P Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08

Claims

1. A binding moiety comprising at least one monomeric V-like domain (VLD) derived from a non-antibody ligand, the at least one monomeric V-like 5 domain being characterized in that at least one CDR loop structure or part thereof is modified or replaced such that the solubility of the modified VLD is improved when compared with the unmodified VLD.

2. A binding moiety according to claim 1 in which at least one CDR loop 10 structure or part thereof is modified or replaced such that (i) the size of the CDR loop structure is increased when compared with the corresponding CDR loop structure in the unmodified VID; and/or (ii) the modification or replacement results in the formation of a disulphide bond within or between one or more of the CDR loop structures. 15

3. A binding moiety comprising at least one monomeric V-like domain (VL D) derived from a non-antibody ligand, the at least one monomeric Valike domain being characterised in that at least one CDR loop structure or part thereof is modified or replaced such that 20 (i) the size of the CDR loop structure is altered when compared with the corresponding CDR loop structure in the unmodified VLD; and/or (ii) the modification or replacement results in the formation of a disulphide bond within or between one or more of the CDR loop structures. 25

4. A binding moiety according to claim 3 in which the size of the CDR loop structure is increased by at least two amino acid residues.

5. A binding moiety according to claim 3 in which the size of the CDR loop structure is increased by at least six amino acid residues. 30

6. A binding moiety according to claim 3 in which the size of the CDR loop structure is increased by at least nine amino acid residues.

7. A binding moiety according to any one of claims 1 to 6 in which the 35 binding affinity of the modified VID is altered when compared with the unmodified VLD. COMS ID No: ARCS-265561 Received by IP Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:48 VATERMARK (F) 61398196010 73/147 37

8. A binding moiety according to claim 7 in which the affinity of the modified VLD to at least one natural ligand of the unmodified VLD is reduced. 5

9. A binding moiety according to any one of claims 1 to 8 in which the binding specificity of the modified VLD is different to that of the unmodified VLD.

10 10. A binding moiety according to any one of claims 1 to 9 in which the non-antibody ligand is a T-cell surface protein.

11. A binding moiety according to claim 10 in which the non-antibody ligand is CTLA-4, CD28 or ICOS. 15

12. A binding moiety according to claim 11 in which the non-antibody ligand is CTLA-4.

13. A binding moiety according to any one of claims 1 to 12 in which one 20 or more of the CDR loop structures is replaced with a binding determinant derived from a non-antibody polypeptide.

14. A binding moiety according to claim 13 in which the binding determinant is derived from somatostatin or haemagglutinin. 25

15. A binding moiety according to any one of claims 1 to 12 in which one or more of the CDR loop structures is replaced with one or more CDR loop structures derived from an antibody or antibodies, 30

16. A binding moiety according to claim 15 in which the antibody or antibodies are derived from a rat, mouse, human, camel, llama or shark

17. A binding moiety according to claim 15 or claim 16 in which the antibody or antibodies are selected from the camel antibody cAB-Lys3 and 35 the human anti-melanoma antibody V86. COMS ID No: ARCS-265561 Received by IP Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:48 WATERMARK (F) 61398196010 74/147 38

18. A binding moiety according to any one of claims 1 to 17 linked to a diagnostic reagent.

19. A binding moiety according to claim 18 in which the diagnostic 5 reagent is selected from the group consisting of streptavidin, biotin, a radioisotope, dye marker or other imaging reagent

20. A multivalent reagent comprising two or more binding moieties as claimed in any one of claims 1 to 19. 10

21. A binding moiety or multivalent reagent according to any one of claims 1 to 20 immobilised on a solid support or coupled to a biosensor surface;

22. A polynucleotide encoding a binding moiety or multivalent reagent as 15 claimed in any one of claims 1 to 20.

23. A vector comprising a polynucleotide according to claim 22.

24. A host cell transformed with a vector as claimed in claim 23. 20

25. A host cell according to claim 24 in which the cell Is a bacterial cell.

26. A method of producing a binding moiety which comprises culturing a host cell as claimed in claim 24 or claim 25 under conditions enabling 25 expression of the binding moiety and optionally recovering the binding moiety.

27. A method according to claim 26 in which the binding moiety is unglycosylated. 30

28. A pharmaceutical composition comprising a binding moiety as claimed in any one of claims 1 to 20 and a pharmaceutically acceptable carrier or diluent COMS ID No: ARCS-265561 Received by IP Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08 Feb-2010 16:49 WATERMARK (F) 61398196010 75/147 39

29. A method of treating a pathological condition in a subject which method comprises administering to the subject a binding moiety as claimed in any one of claims 1 to 20. 5

30. A method of selecting a binding moiety with an affinity for a target molecule which comprises screening a library of polynucleotides for expression of a binding moiety with an affinity for the target molecule, the polynucleotides encoding VLDs derived from one or more non-antibody ligands, wherein the polynucleotides have been subjected to mutagenesis 10 which results In a-modification or replacement in at least one CDR loop structure in at least one VID and wherein the solubility of the isolated modified VLD is improved when compared with the isolated unmodified VLD. 15

31. A method according to claim 30 in which the screening process involves displaying the modified V-like domains as gene IIl protein fusions on the surface of bacteriophage particles.

32. A method according to claim 30 in which the screening process 20 involves displaying the modified V-like domains in a ribosomal display selection system.

33. A binding moiety according to any one of claims 1 to 20 produced by a method according to any one of claims 30 to 32. COMS ID No: ARCS-265561 Received by P Australia: Time (H:m) 17:11 Date (Y-M-d) 2010-02-08