WO2009090268A1 - Peptide mimetics - Google Patents

Abstract

Peptides that bind to Fcγ receptors and may or may not elicit functional activity are provided. Methods of screening, selection, manufacture, and use of such peptides are also provided.

Description

Peptide Mimetics

CROSS-REFERNCE TO RELATED APPLICATIONS This application claims benefit of U.S. provisional application No. 61/021,672, filed January 17, 2008, which is incorporated by reference in its entirety.

The present application relates to peptides that bind to Fcγ receptors and may or may not elicit functional activity, as well as methods for selecting such peptides. It further relates to manufacture and use of these peptides following selection, for example in therapy.

Antibodies of the IgG class are the predominant isotype in serum and interstitial fluids (75%) . The intact format used almost exclusively in therapeutic antibodies is a Y- shaped, multidomain protein with an antigen-binding site located on two Fab tips. Recruitment of effector function is mediated by the stem Fc domain. Effector functions mediated by IgG include antibody-dependent cell-mediated cytotoxicity (ADCC) , phagocytosis, complement-dependent cytotoxicity (CDC) and superoxide generation. ADCC, phagocytosis and superoxide burst are mediated through interaction of complexed IgG with Fcγ receptors (FcγRs) , which are the Fc receptor of IgG, expressed on the surface of leukocytes. CDC is mediated by the interaction of complexed IgG with the complement system. FcγRs are also closely related to other IgG-mediated immunoregulatory functions such as transcription of the genes encoding proinflammatory mediators, internalisation of immune complexes which contributes to antigen presentation on MHC, regulation of immune system cell activation and maturation of dendritic cells (Anderson 1986, Gessner 1998, Amigorena 1999, Machy 2000) .

Three distinct classes of receptor are recognized by the Fc portion of IgG and have been defined according to their immunochemical and physicochemical properties: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16) (Anderson 1986, Unkeless 1988) . Each of them displays distinct IgG isotype binding affinity and specificity. FcγRI interacts with high-affinity to monomeric as well as aggregated human IgGl (hlgGl) or hIgG3 (Ka=IO^"8 to 10^"9 M^"1; K_D = 1 to 1OnM) (Ravetch 1991, Hulett 1994, Takal 2002) . The interaction with polymeric hIgG4 and hIgG2 is much weaker (10 and 100-fold less, respectively) (Burton 1985, Clark 1997) . FcγRI also binds murine IgG2a (mIgG2a) and mIgG3 with an affinity equivalent to that of hIgG3 (Lubeck 1985) . The high-affinity interaction is a distinctive property of FcγRI in comparison with the receptor classes FcγRII and FcγRIII, which have a much lower affinity for monomeric IgG (K_D = 0.1 to 10 μM) . FcγRI is constitutively expressed on mononuclear phagocytes that include monocytes, macrophages, CD34⁺ progenitor cells, and dendritic cells, and can be induced on neutrophils, eosinophils, and mesengial cells (Anderson 1980, Perussia 1983, Ravetch 1991, Uciechowski 1998) . The physiological contributions of FcγRI are unclear. FcγRI is always present in combination with other receptor classes and the functional difference between the distinct types of FcRs expressed on the same cell-type is not yet clear (Ceuppens 1988, van de Winkel 1993) . To deliver a signal to cells, FcγRs need to be aggregated at the cell surface by complexed IgG. Even though the molecular mechanisms that initiate signals remain poorly delineated, it would seem that FcγR aggregation may occur by two mechanisms, which are dependent on receptor affinity (Wolsy 1995, Tamir 1996) .

Since the first construction of a peptide phage library (Scott 1990), over one thousand reports have been published that use this technology to isolate ligands able to bind and/or modulate biological functions of a myriad of targets (Cwirla 1997, Ballinger 1999, Koivunen 1999, Wu 2000, Buhl 2002, Essler 2002, Karasseva 2002, Mori 2004) . Although in theory ligands could bind to a protein anywhere on its solvent- exposed surface, most peptides selected by phage display recognize localised sites that appear to coincide with natural ligand binding sites, and consequently function as agonists or antagonists (Delano 2000).

Understanding of the molecular basis of the interaction between Fc receptors and the Fc portion of an antibody has been the subject of detailed investigations using chimeric IgG isotypes, comparative binding studies from different species and different isotypes, IgG modified by site-directed mutagenesis, binding analysis of fragments or synthetic peptides, and recently the screening of peptide libraries

(Hamburger 1975, Junghans 1990, Goldsmith 1997, Umaήa 1999, Babu 2001, Nakamura 2001, Nakamura 2002, Uray 2004, Lazar 2006) . Earlier studies have attributed the localization of the site for monocyte binding to the CH3 domain of IgG (Yasmeen 1973, Okafor 1974, Ciccimarra 1975) . After successful crystallization of the extracellular portion of three ligand- free receptors: FcγRIIa, lib and FcγRIIIb (Maxwell 1999, Sonderman 1999, Zhang 2000, Sonderman 2001), as well as FcγRIIIb complexed with the Fc fragment of hlgGl (Sondermann 2000, Radaev 2001a) and NMR studies of the interaction of IgG2b with mFcγRII (Kato 2000), three principal regions involved in the binding and activation of the Fc receptors have been proposed. One dominant site comprising residues 234 to 239, located in the N-proximal end of the CH2 domains, was involved in binding to all three FcγRs . The sequence L234LGGPS239 was shared by IgGs binding optimally to the receptors (Ratcliffe 1982, Sondermann 2000, Radeav 2001b, Shields 2001, Sondermann 2001) . The second region involved the sequence Asp265-Pro271 located in the CH2 domains B/C loops (Maxwell 1999, Hulett 1994, Hulett 1995, Tamm 1996, Shields 2001) . The third recognition site, which may be one of the two principal areas of contact, consists of the region Gly316- Ala339, located towards the C-terminal end of the CH2 domains (Sondermann 2000, Wines 2000, Radaev 2001a, Shields 2001, Sondermann 2001) . The existence of additional FcγR-binding sites on the Fc fragment that are more specific to the receptor class and in particular hFcγRII and hFcγRIII, such as Lys274-Arg301, Arg292-Thr299, or Tyr407-Arg416, have also been proposed (Sarmay 1984, Chappel 1991, Haagen 1995, Clark 1997, Sondermann 2000, Shields 2001, Medgyesi 2004, Uray 2004) . However, it is unclear whether these regions contribute directly to the binding site or whether they perturb protein conformation (Shields 2001). Other factors, such as the glycosylation at Asn297, whose composition varies among the IgG subtypes, may also contribute to receptor binding by stabilizing the CH2 domain in conformations conducive to binding (Burton 1985, Tao 1989, Lund 1990, Morrison 1994, Jefferis 1998, Mimura 2001, Krapp 2003) . Studies on the hFcγRI have shown binding by the lower hinge control peptides

G₂₃₃LLGGPYG₂₄₀ (Lund 1991), E₂₃₃LLGGPSVF₂₄₁ (Sheridan 1999), and C₂₃₃LLGGLGC₂₄₀ (Berntzen 2006), which have a linear epitope. Similar studies have been performed for hFcγRII (Uray 2004, Medgyesi 2004) . Despite the extensive research described above to identify peptide binders, there have been no studies using combinatorial peptide libraries that have identified any sequences homologous to the discontinuous functional epitope of the Fc fragment, which as a result could mimic IgG and activate the FcRs (Nakamura 2001, Nakamura 2002, Berntzen 2006, Cendron 2007). In the present application, the identification of such a consensus sequence for a discontinuous epitope, with a structure and/or composition able to mimic partially the conformation of the binding site of the Fc fragment has been achieved. As such, the small structured peptides of the invention can mimic specifically the effect of native Fc on FcγRI and can therefore facilitate the design of specific and potent agonists or antagonists. Due to these properties, peptides of embodiments of the present invention are particularly suitable for use in therapeutic and/or diagnostic treatments of the human or animal body. The peptides may be particularly useful for treating disorders associated with cancer, infectious diseases and autoimmune/inflammatory disorders .

Description of the Figures

Figures Ia and Ib show the effect of peptides on the binding of hlgGl to hFcγRI by competitive ELISA. hFcγRI absorbed onto microwells was incubated with 67nM of hlgGl and serial dilutions of peptides 50μM (light grey shading), 16.6μM (no shading), 5.53μM (dark grey shading) and 1.84μM (shaded black) . Without peptide as inhibitor, the signal binding of hlgGl is normalised to 100%. hlgGl binding was detected by

HRP-conjugated F(ab')2 fragment goat anti-hlgGl. The sequence correspondence of each peptide is indicated in Table 2 and the sequence listing, p Hinge: lower hinge linear peptide (SEQ ID NO:24; PPCPAPELLGGPSVFLFP) corresponds to the fragment 225-244 of hlgGl (EU nomenclature) . cp : soluble cyclic peptide, p: soluble linear peptide. Results shown are the mean of five independent experiments.

Figure 2 shows the effect of Fc mimetic peptide complexed on beads on superoxide production from γ-IFN stimulated U937 cells. 100 μl of 2 x 10⁵ cells were incubated with serial dilutions (10 μl) of either peptide or hlgG complexed on microbeads. After incubation for 5 min at 37°C with lucigenin (10 μl), luminescence emission was measured over a period of 60min. RLU: relative luminescence units, mcp : multimeric soluble cyclic peptide, m: multimeric. Results shown were the mean of five independent experiments.

Figure 3 shows the inhibition by soluble Fc mimetic peptides of superoxide production generated by Fc mimetic peptides complexed on beads from γ-IFN stimulated U937 cells. Cells were activated with complexed peptides mcp 22, 29, 30, 33 or hlgGl. To inhibit superoxide bursts, the corresponding soluble peptide (5OmM) or hlgGl (0.7μM) was added to each complex. Red blood cells (RBCs) (NIP derivatised RBCs sensitized or not with anti-NIP antibody), irrelevant cyclic peptide (SEQ ID NO:26; THFDTCSWMYCWDGWW) , control beads not conjugated with any peptide, mcp: multimeric soluble cyclic peptide, m: multimeric. RLU: relative luminescence units were taken for a 60min period. Results shown were the mean of four independent experiments .

Figure 4 shows inhibition by soluble Fc mimetic peptides or monomeric hlgGl of superoxide bursts mediated by hlgGl complexed on beads from γ-IFN stimulated U937 cells. lOOμl of cells (2 x 10⁵ in lOOμl) were incubated with lOμl of NIP- derivatized RBCs sensitized with anti-NIP hlgGl and a range of concentrations of hlgGl (0.2nM to 105nM) (Fig 4a), or a range of concentration of soluble peptides (3OnM to 25OnM) (Fig 4b) , in order to inhibit superoxide production, (a) hlgGl (D) (b) soluble cyclic peptide 22 (•) , 29 (♦) , 30 (■) , 33 (D) and irrelevant cyclic peptide (σ) (SEQ ID NO:26; THFDTCSWMYCWDGWW) . RLU: Relative luminescence units. Results shown were the mean of four independent experiments.

Figure 5 shows phagocytosis of Fc mimetic peptides complexed on beads by γ-IFN stimulated U937 cells. Cells (2 x 10⁵) were incubated 2h at 37⁰C with lOμl of the FITC-labelled bead- molecule complexes. Cells were then treated with trypan blue for Ih to quench the extracellular fluorescence. The ingested fluorescence was monitored by epifluorescence microscopy and using a FACScalibur flow cytometer. Figure 5 (a) and (b) represent the same cell under fluorescent or non-fluorescent conditions respectively. Internalization of FITC-conjugated bead-peptide complex (peptide 22) in the cell could be seen clearly. Magnification: X40. Figure 5(c) and (d) illustrates flow cytometry profiles showing the fluorescence intensity of the FITC-conjugated beads. Profile A shows beads non-complexed and beads complexed with irrelevant peptide (SEQ ID NO: 26; THFDTCSWMYCWDGWW) . Profile B shows beads complexed with peptide 22. Profile C shows beads complexed with hlgGl . Profile D shows beads non-complexed, beads complexed with irrelevant peptide and beads complexed with peptide 22 in the presence of hlgGl. Profile E shows beads complexed with IgGl in the presence of hlgGl . Results shown were representative of three independent experiments.

Figure 6 shows the specificity of Fc mimetic peptides for hFcγR assessed by Alpha Screen™. Each biotinylated peptide was incubated for 2 hours with 20μg/ml of streptavidin donor beads and the hFcγR pre-incubated with 20μg/ml of nickel chelate acceptor beads. The signal from the peptides at a range of concentrations: 37, 111 and 333 nM, was normalized relative to the signal obtained from the hlgGl binding at a range of concentrations: 5, 15 and 46nM on hFcγRI, hFcγRIIa, hFcγRIIb and 15, 45 and 137nM on hFcγRIIIa, hFcγRIIIb (which represented 100% of binding) for each receptor. Figure 6a shows the binding results to hFcγRI, figure 6b shows the binding results to hFcγRIIa, figure 6c shows the binding results to hFcγRIIb, figure 6d shows the binding results to hFcγRIIIa and figure 6e shows the binding results to hFcγRIIIb. Luminescence was monitored on a Fusion-α analyser. Irrelevant cyclic peptide (SEQ ID NO: 26; THFDTCSWMYCWDGWW) . Results shown were the mean of four independent experiments.

Figure 7 shows the cross-reactivity of Fc mimetic peptides complexed on beads to FcγR bearing cells assessed by rosette formation. Microbeads (6 x lOVml) were sensitised with 10 μM of peptide 22, 29, 30, 33 or 400 nM of hlgGl and incubated in PBS with 2 x 10⁶ cells/ml of primary NK cells, Daudi (Burkitt's B-Lymphoma) , K562 (erythroblastoid) , or U937

(monocyte-like histiocytic lymphoma) at the ratio 100:1 (bead- molecules: leucocyte cells). The peptide or IgG-bead complexes bound to cell surface were examined microscopically through rosette formation. Rosettes were considered as positive if at least 5 beads were clustered around one cell. The rosette percentage indicated the ratio of positive cells to total cells counted, mcp : multimeric soluble cyclic peptide, m: multimeric. Irrelevant cyclic peptide (SEQ ID NO:26; THFDTCSWMYCWDGWW) , control beads were not conjugated with any peptide. The results shown were one of five comparable experiments .

Figure 8 shows binding assay of Fc non-mimetic peptides that are unable to compete with IgG for binding to hFcγRI. Figure 8a: hFcγRI binding assay assessed by Alpha Screen™. Each biotinylated peptide and IgGl was incubated at a range of concentrations for 2 hours with 20μg/ml of streptavidin A donor beads and the hFcγRI pre-incubated with 20μg/ml of nickel chelate acceptor beads. Key: cyclic peptide 6 (σ) , 17

(D), 22 (•), 42 (p) and IgGl (O). Figure 8b: Superoxide competition assay between soluble peptides and peptide 33 complexed on beads. U937 cells were incubated with 20OnM of peptide 33 complexed on beads (lOμl) and with serial dilution of soluble peptides (lOμl) . Key: cyclic peptide 6 (O), 17

(•) , 22 (■) and 42 (p) .

Figure 9 shows the inhibition by soluble Fc non-mimetic peptides of superoxide production generated by peptide 33 complexed on beads from γ-IFN stimulated U937 cells. 100 μl of 2 x 10⁵ cells were pre-incubated with serial dilutions (10 μl) of soluble peptide simultaneously with a suboptimal concentration of peptide 33 complexed on beads. After incubation for 5 min at 37°C with lucigenin (10 μl), luminescence emission was measured over a period of 60min. Key: cyclic peptide 8(D), 21 (p) , 26 (σ) , 27 (■) , 36 (♦) , Hinge-CH₂ (H); linear peptide Hinge-CH₂ (tf), 22 S₄Si₃ (∑) •

Figure 10 shows the activation of superoxide production by dimeric cyclic peptide 33. Key: multimeric cyclic peptide mcp 33 (O) , dimer cyclic peptide (dimer cp 33 (D) linked by a KKKKK linker (SEQ ID NO: 23; VNSCLLLPNLLGCSYEKKKKKESYCGLLNPLLLCSNV) .

Figure 11 shows the overall molecular modelling of cyclic peptide 33 and FcγRI-peptide complex. Figure 11 (a) Overlay ribbon structure of two peptide models obtained after docking into FcγRI and superimposed to each other. The black backbone represents the A-chain conformer aligned with the Fc-A chain and the dark grey backbone represents the B-chain conformer aligned with the Fc-B chain. LeulO, Leull, Glyl2 and Pro8 from the cyclic peptide are indicated, and the disulphide bridge Cys4:Cysl3 is shown in white, nitrogen atoms are in dark grey and oxygen atoms are in black. Figure 11 (b) Overall view of peptide conformers complexed with FcγRI . The FcγRI model was built and refined on the sequence and the structure of FcγRII I in complex with Fc fragment using the program PRIME. A-chain conformer is shown in black and B-chain conformer in dark grey. FcγRI is shown below the conformers. Figure 11 (c) Overall view of peptide conformers superimposed to the IgG-Fc fragment. A-chain conformer is shown in black and B-chain conformer in dark grey. Figures 11 (d) and (e) show a close-up view of the interaction surfaces of the A-chain conformer (d) and B-chain conformer (e) with FcγRI. The interacting residues from conformers and receptor are marked. The tryptophan- proline sandwich formed by W87 and WHO (d) and the hydrophobic area constituted by Y116, H131 and W132 (e) are maintained in this model. Potential N-glycosylation sites are not shown.

Figure 12 shows the binding of single-chain homodimer peptide 33 to FcγRI by indirect AlphaScreen Assay. Inhibition of biotinylated-IgGl binding to FcγRI was performed in the presence of a concentration range of IgGl (D) or parallel dimeric peptide 33 (♦) . 20 mg/ml of nickel donor beads were pre-incubated with FcγRI (20 nM) for 30 min. The competitors were subsequently added for a further 30 min. Subsequently, 20 mg/ml of streptavidin donor beads treated with a suboptimal concentration of biotinylated-IgGl (10 nM) were added to the preparation for a further 1 h. Luminescence was monitored on a Fusion-α analyzer. RLU: relative luminescence units. Results show representative curves obtained from three independent experiments .

Figure 13 shows the biologic activity of single-chain homodimer peptide 33 by superoxide generation from γ-IFN stimulated U937 cells. Figure 13 (a) 2 x 10⁵ cells were activated with serial dilutions of multimeric cyclic peptide mcp 33 (■) or parallel dimeric peptide 33 (D) . After incubation with 2.5 mM lucigenin, luminescence was measured over a period of 60 min. Results shown are the means of four separate experiments. Figure 13 (b) The dimeric peptide 33 mass was confirmed by SELDI-TOF/MS . Figure 13 (c) U937 cells were incubated using a non-saturating concentration of dimeric peptide 33, and superoxide bursts were inhibited using a concentration range of IgGl (T) or soluble peptide 33 (■) . Incubation and measurements were performed and obtained as described above. Results shown are the means of three separate experiments. Key: parallel dimeric peptide 33(SEQ ID NO: 23; VNSCLLLPNLLGCSYEKKKKKESYCGLLNPLLLCSNV) ; RLU, relative luminescence units.

Summary of the Invention

In the present application phage-display selection has been used to isolate peptides able to selectively bind to the IgG- Fc binding site on FcγRI . One class of peptide (Fc mimetic peptide) mimic IgG-mediated effector properties and therefore trigger effector functions in a complexed form (multimeric) and inhibit them in a soluble form (monomeric) . A second class of peptide (Fc non-mimetic peptide) do not mimic IgG-mediated effector properties and therefore can either trigger or inhibit the effector functions.

In a first embodiment, the present invention provides a biologically active peptide that competes with the Fc fragment of an IgG for binding to Fcγ receptor, wherein on binding the peptide may activate and/or inhibit Fc effector function.

Alternatively, the peptide is an Fc mimetic peptide and in a first, complexed form the peptide activates Fc effector function and in a second, soluble form the peptide inhibits Fc effector function.

Alternatively, the peptide is an Fc non-mimetic peptide and in a first, complexed form does not activate effector function and in a second, soluble form the peptide inhibits Fc effector function .

Alternatively, the peptide is an Fc non-mimetic peptide and in a homodimeric form activates effector function.

The Fcγ receptor may be selected from FcγRI , FcγRII or FcγRIII. Preferably the receptor is a human receptor, more preferably human FcγRI (hFcγRI).

Fc mimetic and non-mimetic peptides bind FcγR in the Fc binding site. Binding of the mimetic peptide to Fcγ receptor in a complexed form may elicit a number of Fc effector functions. For example, once activated, the main roles of the FcγRI lie in facilitating phagocytosis, endocytosis of opsonized particles, antigen presentation, release of inflammatory mediators (e.g. IL-6, TNFα, IL-I), cellular cooperation, superoxide burst and mediating ADCC. In another example, FcγRIII is largely responsible for mediating ADCC.

In contrast, binding of the non-mimetic peptide to Fc receptor in a complexed form may not activate Fcγ receptor and therefore does not elicit effector function. To characterise functionally Fc mimetic and non-mimetic peptides, the peptides may be biotinylated and conjugated to particles such as streptavidin-coated paramagnetic beads therefore forming multimeric complexes. Furthermore, the peptides may be oligomerized either by branching peptides on a polylysine backbone or dimerized by using a linker between two homo or heteropeptides . Such multimeric complexes can mimic immune complex activity. Effector function may be determined using a number of biological assays as set out in the Examples section that measure a superoxide burst or phagocytosis, for example .

By competition elution with hlgGl, a panel of phage-peptides, whose sequences exhibit a hydrophobic core and a highly charged carboxy-terminal end, were identified and characterized. In one example, a peptide according to the present invention competes with IgGl for binding to FcγRI . This binding may be determined using an in vitro assay, such as, for example, an ELISA assay or BIACORE analysis.

In an aspect of the present embodiment, the peptide is constrained by a disulfide bridge and contains a tripeptide motif of LLG within the cyclic peptide sequence delineated by the disulfide bridge. The peptide may also comprise a dipeptide motif ςP within the cyclic peptide sequence, wherein ς represents a hydrophobic residue (and generally a lysine or tryptophan amino acid residue) . In addition, the peptide may also comprise threonine at the N-terminal and/or an acidic region at the C-terminal of the peptide (and mainly a glutamic acid) .

In another embodiment of the present application, the peptide comprises a consensus amino acid sequence of TX₂CXXςPXLLGCφXE, "wherein X is any amino acid residue, ς is a hydrophobic residue and φ is an acidic residue.

The consensus sequence resembles four regions present in the primary sequence of the C_H2 domain of IgGl: sequence 234-236 (with LLG motif), 265-270 (with acidic region), 327-333 (with ςP) and 297-299 (with TXX) . Modelling data indicates that peptides mimic at least two of these epitopes: sequence 234- 236 and 327-333, as numbered by the EU index of Rabat (1987, 1991) .

Peptides of the invention may bind to Fcγ receptors, such as hFcγRI, II or III, in a manner similar to that observed for hlgGl or hIgG3. In an alternative embodiment, peptides of the present invention may be specific for one of the hFcγ receptors. For example, the peptides may be specific for hFcγRI and not exhibit cross-reactivity with low-affinity Fcγ receptors classes, such as hFcγRII or hFcγRIII. In one aspect of the present embodiment, the Fc mimetic and non-mimetic peptides bind specifically to human FcγRI and do not exhibit cross-reactivity with hFcγRII or hFcγRIII, unlike IgG.

In another aspect of the present invention, the Fc mimetic peptide sequences may be selected from one of the following (cp: cyclic peptide) :

In one embodiment, the peptide is selected from SEQ ID No: 1, 5, 13 or 15.

Alternatively, the Fc mimetic peptide may exist as a dimeric or oligomeric peptide. A dimeric peptide may have an amino acid sequence comprising two peptides of the invention linked by a linker. An oligomeric peptides may be three or more peptides of the invention linked by a peptide linker. The linker may comprise an amino acid sequence of less than about 10 amino acid residues, such as about 9, 8, 7, 6, 5, 4 or 3 amino acid residues. Common peptide linkers may comprise the residues glycine (G) and serine (S) in a number of combinations. Alternatively, the amino acid lysine (K) may also be used to form a linker. In one embodiment, a KKKKK linker (SEQ ID NO: 22) may link the dimeric or oligomeric peptides. In another embodiment, the dimeric form may compete with an Fc fragment of an IgG for binding to Fcγ receptors and on binding elicit effector function.

In an alternative embodiment, the dimeric peptide may have the sequence VNSCLLLPNLLGCSYEKKKKKESYCGLLNPLLLCSNV (SEQ ID NO: 23) and may additionally exist as a parallel dimer. Since this dimeric peptide can only activate Fc effector function, it falls within the classification of an Fc-non mimetic peptide although it is composed of Fc mimetic peptides. The unusual ability of this cyclic peptide dimer to trigger effector function even in the absence of formation of multimeric complexes suggests a potential therapeutic application for this peptide as discussed later in the application. In an alternative aspect of the present embodiment, the Fc non-mimetic peptide sequence may be selected from one of the following (cp: cyclic peptide) :

A further embodiment of the present invention relates to a method for identifying a peptide that binds to an FcγR comprising: i) selecting a peptide by IgG competition elution during phage display, for example as determined by ELISA (see Example

6); ii) assessing binding specificity and functionality of the peptide using biotinylated peptide conjugated to particles such as streptavidin-coated paramagnetic beads, for example as determined by Alphascreen^® (see Example 7) .

In one aspect, the functionality of the peptides of the invention is assessed by activation of superoxide burst and phagocytosis in a human leukocyte cell line U937, as described in Examples 8 and 10, although other techniques are known in the art.

Binding specificity of the peptides of the invention may be assessed by determining rosette formation on binding to cells expressing Fcγ receptors, as described in Example 9.

One embodiment of the invention comprises a method of activating FcγRI comprising contacting FcγRI with a composition that comprises an Fc mimetic peptide having an amino acid sequence of TX₂CXXςPXLLGCφXE and wherein, X is any amino acid, ς is a hydrophobic residue and φ is an acidic amino acid. Preferably the receptor is a human Fcγ receptor, more preferably human FcγRI (hFcγRI) . Conjugated to a Fab or scFv or any other scaffolds enclosing at least one antigen binding site, they could stimulate the immune response. In soluble form, they might block FcγR-mediated effector functions .

In an alternative embodiment of the present invention, a peptide that binds FcγR but does not compete with IgG for binding to the receptor is provided. Such a peptide appears to bind the receptor at a site different to that of the mimetic/non-mimetic peptides of the first embodiment since the peptide does not compete with IgG for binding to the FcγR. The peptide does not activate or inhibit Fc effector function on binding and may lack sufficient avidity to trigger the receptor-mediated effector function in complexed form. Such a peptide falls within the class of Fc non-mimetic peptides.

These FcγR peptides may bind to FcγRI, more preferably, human FcγRI .

The sequence of such a peptide, as described above, may be selected from one of the following (cp: cyclic peptide) :

Such peptides may be conjugated to an antibody or antibody fragment such as Fab, Fab', Fab' -SH, scFv, Fv, dAb or Fd. Such antibody or antibody fragment may specifically bind desired target antigens in order to elicit specific biological functions in order to prevent, treat, mitigate or diagnose/screen for diseases such as cancers, infectious diseases or autoimmune/inflammatory disorders as described below.

For small monomeric antigens such as IL-6, administration of antibodies can result in a build-up of antigen-antibody complexes in the blood (Montero-Julian F. A. and others (1995) Blood 85, 917-924), resulting in increased levels of antigen. The rationale is that although effector functions are not triggered because the antibody remains monomeric, immune complexes can have the half-life of free antibody in the serum, mediated via FcRn recycling of IgG. Potential therapeutic applications of the cyclic peptide dimers as described in one embodiment of the present application rely on its unusual ability to trigger effector function even in the absence of formation of multimeric complexes. It is anticipated that a fusion protein of, for example, peptide of SEQ ID NO: 23 with a scFv directed against small monomeric antigens would result in rapid inactivation and clearance of scFv-antigen complexes via FcγRI-bearing macrophages or monocytes, through effector functions including endocytosis or superoxide generation. In one embodiment, the peptides of the invention do not share any homology with the common consensus sequence as described above. Such peptides could be useful as a diagnostic tool and may also have a therapeutic application that is independent of effector functions, such as cell targeting with a conjugated molecule. Additionally the peptides of the invention may be conjugated with a detectable label.

Further aspects and embodiments of the present invention are disclosed herein in and preferred aspects and embodiments are subject to the claims included below.

Detailed Description

A peptide according to an embodiment of the present invention may be used herein to refer to constrained (i.e. having some element allowing cyclisation between two backbone termini, two side chains, or one of the termini and a side chain, as for example, amide or disulfide bonds) or unconstrained (e.g. linear) amino acid sequences of less than about 50 amino acid residues, such as less than about 40, 30, 20 or 10 amino acid residues. This list may also including oligomers, such as 3, 4 or 5 peptides linked together or dimers comprising 2 peptides linked together by means of a peptide linker, for example. Of the peptides of less than about 40 amino acid residues, preferred are the peptides of between about 10 and about 30 amino acid residues and especially the peptides of about 16 to 18 amino acid residues. However, on reading the present disclosure, it will be apparent to the skilled person that it is not the length of a particular peptide but its ability to bind to FcγRI and compete with the binding of IgG described herein that distinguishes the peptide of the invention. For example, amino acid sequences of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 25 amino acid residues are contemplated to be peptide compounds within the context of the present invention. A dimeric peptide may have an amino acid sequence comprising two peptide amino acid sequences linked by a linker. The linker may comprise an amino acid sequence of less than about 10 amino acid residues, such as 9, 8, 7, 6, 5, 4 or 3 amino acid residues. The linker amino acid residues may be of a single amino acid or combinations of different amino acids. For example, combinations of glycine (G) and serine (S) may be used. Alternatively, the linker may comprise residues of glycine, serine or lysine (K) only.

A peptide of the invention that is able to mimic biological activity of an Fc fragment, such as effector function may be termed a ^ΛFc mimetic peptide' . Effector function of an Fc fragment as encompassed by, but not limited by the present invention comprises phagocytosis, CDC and/or ADCC. This biologically active peptide binds to Fcγ receptor. Such a peptide competes with the Fc fragment of IgG for binding to Fcγ receptor and therefore binds to the same or an overlapping binding site on Fcγ receptor as the Fc fragment. Present in a complex form (multimeric) , which may include dimers and oligomers, an Fc mimetic peptide can trigger effector functions. Present in soluble form (monomeric) , an Fc mimetic peptide can inhibit effector functions. These peptides in soluble form may be able to activate effector functions if the IC₅₀ concentration of the soluble form required to inhibit the effector function is lower than 10-20 μM.

A peptide that is unable to mimic the effector functions of an Fc fragment of an IgG to an Fcγ receptor, namely to trigger effector functions in multimeric form and inhibit effector functions in soluble form may be referred to as an ^ΛFc non- mimetic peptide' . Using this terminology, four groups of peptides may be defined: i) The first group comprises a peptide that competes with the Fc fragment for binding to the Fcγ receptor and therefore binds to the same binding site on the Fcγ receptor as the Fc fragment. However such peptides cannot activate effector functions in a complexed form but can activate effector functions when in soluble form. These peptides may have lost their ability to trigger effector functions if the IC₅₀ concentration of the soluble form required to inhibit the effector function is higher than 10-20 μM and hence the avidity of the complexed peptide is sufficiently low. ii) The second group comprises a peptide that competes with the Fc fragment of an IgG for binding to the Fcγ receptor and therefore binds to the same binding site on the Fcγ receptor as the Fc fragment. This peptide can activate effector functions when in a dimeric as well as when in a complexed form i.e. as a soluble cyclic peptide dimer. Such peptides may also be known as committed agonists. iii) The third group comprises a peptide that does not compete with the Fc fragment of an IgG for binding to the Fcγ receptor. These peptides however can activate the Fcγ receptor when in complexed form (Berntzen et al, 2006) . iv) The fourth group comprises a peptide that does not compete with the Fc fragment of an IgG for binding to the Fcγ receptor but is able to bind to the FcγR at a site distinct from the Fc binding site. These peptides cannot activate or inhibit effector functions in complexed or soluble form. These peptides may have an affinity and avidity that is too high to activate effector function.

Specific peptides within the context of the present invention may comprise both naturally and non-naturally occurring amino acid sequences. By non-naturally occurring is meant that the amino acid sequence is not found in nature. Example non- naturally occurring amino acid sequences have between about 10 and 30 amino acid residues, alternatively about 20 amino acid residues. These include peptides, peptide analogs, peptoid and peptidomimetics containing naturally as well as non-naturally occurring amino acids. In a specific aspect, the peptides of the invention comprise amino acid residues consisting of only naturally occurring amino acids.

A C-terminal region of an immunoglobulin heavy chain that also comprises the hinge region between the two constant domains CHl and CH2 may be referred to as a ^ΛFc fragment' This fragment of the C-terminal region may a native sequence Fc fragment or a variant Fc fragment. Although the boundaries of the Fc fragment of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc fragment is usually defined to stretch from an amino acid residue at position 231 to the carboxyl-terminus thereof. With the upper and core hinge, the ^ΛFc fragment' starts from position 216 (EU nomenclature according to Rabat (1987, 1991)). The Fc fragment of an immunoglobulin generally comprises two constant domains, CH2 and CH3. The CH2 domain of a human IgG Fc fragment usually extends from about amino acid 231 to about amino acid 340. The CH3 domain of a human IgG Fc fragment usually extends from about amino acid 341 to about amino acid residue 447 of a human IgG (i.e. comprises the residues C-terminal to a CH2 domain) . In embodiments of the present invention, the variant IgG Fc fragment may be selected from IgGl, IgG2, IgG3 or IgG4, preferably the IgG Fc fragment of IgGl . IgGl Fc may be written in the alternative as Fcγl . A ^Λhinge fragment' is generally defined as stretching from GIu 216 to Pro 230 of human IgGl, or the equivalent positions in IgG2, IgG3 or IgG4 (Burton,

1985). A functional Fc fragment possesses an effector function of a native sequence Fc fragment for example: CIq binding, CDC, Fc receptor binding, phagocytosis, endocytosis of opsonized particles, antigen presentation, release of inflammatory mediators (e.g. IL-6, TNFα, IL-I), cellular cooperation, superoxide burst, ADCC, down regulation of cell surface receptors (e.g. B cell receptor), etc. Effector function of an Fc fragment as encompassed by, but not limited by the present invention comprises phagocytosis, CDC and/or ADCC.

As is generally known in the art, an Fcγ receptor (FcγR) is a receptor that binds an IgG antibody and includes receptors of the FcγRI , FcγRII and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIa (an activating receptor) and FcγRIIb (an inhibiting receptor), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Fc receptors are reviewed in Ravetch and Kinet (1991, Annu. Rev. Immunol 9: 457-92); Capel et al . ,

(1994, Immunomethods 4: 25-34); and de Haas et al . , (1995, J. Lab. Clin. Med. 126: 330-41) .

A peptide according to an embodiment of the present invention may be obtained from a library of peptides that are able to bind to an Fcγ receptor such as FcγRI . The library may be displayed on particles or molecular complexes, e.g. replicable genetic packages, such as yeast, bacterial or bacteriophage (e.g. T7) particles, viruses, cells or covalent, ribosomal, microbead or other in vitro display systems, each particle or molecular complex containing nucleic acid encoding the peptide. Phage display is described in WO92/01047 and e.g. US patents US5969108, US5565332, US5733743, US5858657, US5871907, US5872215, US5885793, US5962255, US6140471, US6172197, US6225447, US6291650, US6492160 and US6521404, each of which is herein incorporated by reference in their entirety.

Following selection of peptides of the invention able to bind an FcγRI receptor and displayed on bacteriophage or other library particles or molecular complexes, nucleic acid may be taken from a bacteriophage or other particle or molecular complex displaying a said selected peptide. Such nucleic acid may be used in subsequent production of a peptide by expression from nucleic acid with the sequence of nucleic acid taken from a bacteriophage or other particle or molecular complex displaying a said peptide.

A peptide of the invention in soluble form can bind to the FcγRI without eliciting an effector response. In order to activate the receptor it is necessary for receptor aggregation to occur.

Ability to bind FcγRI may be further tested, also ability to compete with e.g. an Fc fragment of an IgG for binding to

FcγRI . A peptide according to the present invention may bind

FcγRI with the affinity of a functional Fc fragment or with an affinity that is better or lower, as measured by, for example,

BIACORE.

Binding affinity of different peptides can be compared under appropriate conditions.

The techniques required to make substitutions within amino acid sequences of peptides of the invention are available in the art. Variant sequences may be made, with substitutions that may or may not be predicted to have a minimal or beneficial effect on effector function, and tested for ability to bind Fc receptors and/or for any other desired property.

A further aspect of the invention is a polypeptide comprising a sequence that has at least 60, 70, 80, 85, 90, 95, 98 or 99% amino acid sequence identity with a sequence of any of the peptides shown in the appended sequence listing. The alignment tools ALIGN (Myers and Miller, CABIOS 4:11-17, 1989) or LFASTA (Pearson and Lipman, 1988) may be used to perform sequence comparisons (Internet Program © 1996, W. R. Pearson and the University of Virginia, fasta20u63 version 2.0u63, release date December 1996) . ALIGN compares entire sequences against one another, while LFASTA compares regions of local similarity. These alignment tools and their respective tutorials are available on the Internet at the NCSA Website. Alternatively, for comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function can be employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1) . When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties) . The BLAST sequence comparison system is available, for instance, from the NCBI web site; see also Altschul et al . , J. MoI. Biol. 215:403-410, 1990; Gish. & States, Nature Genet. 3:266-272, 1993; Madden et al . Meth. Enzymol. 266:131-141, 1996; Altschul et al . , Nucleic Acids Res. 25:3389-3402, 1997; and Zhang & Madden, Genome Res. 7:649-656, 1997.

Orthologs of proteins are typically characterized by possession of greater than 75% sequence identity counted over the full-length alignment with the amino acid sequence of specific protein using ALIGN set to default parameters. Proteins with even greater similarity to a reference sequence will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, or at least 98% sequence identity. In addition, sequence identity can be compared over the full length of specific domain (s) of the disclosed polypeptides. When significantly less than the entire sequence is being compared for sequence identity, homologous sequences will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85%, at least 90%, at least 95%, or at least 99% depending on their similarity to the reference sequence. Sequence identity over such short windows can be determined using LFASTA; methods are described at the NCSA Website .

Particular variants may include one or more amino acid sequence alterations (addition, deletion, substitution and/or insertion of an amino acid residue) .

Alteration may comprise replacing one or more amino acid residues with a non-naturally occurring or non-standard amino acid, modifying one or more amino acid residue into a non- naturally occurring or non-standard form, or inserting one or more non-naturally occurring or non-standard amino acid into the sequence. Examples of numbers and locations of alterations in sequences of the invention are described elsewhere herein. Naturally occurring amino acids include the 20 "standard" L-amino acids identified as G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, C, K, R, H, D, E by their standard single-letter codes. Non-standard amino acids include any other residue that may be incorporated into a polypeptide backbone or result from modification of an existing amino acid residue. Non-standard amino acids may be naturally occurring or non-naturally occurring. Several naturally occurring non- standard amino acids are known in the art, such as 4- hydroxyproline, 5-hydroxylysine, 3-methylhistidine, N- acetylserine, etc. (Voet & Voet, Biochemistry, 2nd Edition, (Wiley) 1995). Those amino acid residues that are derivatised at their N-alpha position will only be located at the N- terminus of an amino-acid sequence. Normally in the present invention an amino acid is an L-amino acid, but it may be a D- amino acid. Alteration may therefore comprise modifying an L- amino acid into, or replacing it with, a D-amino acid. Methylated, acetylated and/or phosphorylated forms of amino acids are also known, and amino acids in the present invention may be subject to such modification.

Amino acid sequences in antibody domains and peptides of the invention may comprise non-natural or non-standard amino acids described above. Non-standard amino acids (e.g. D-amino acids) may be incorporated into an amino acid sequence during synthesis, or by modification or replacement of the "original" standard amino acids after synthesis of the amino acid sequence .

Use of non-standard and/or non-naturally occurring amino acids increases structural and functional diversity, and can thus increase the potential for achieving desired properties in a peptide of the invention. Additionally, D-amino acids and analogues have been shown to have better pharmacokinetic profiles compared with standard L-amino acids, owing to in vivo degradation of polypeptides having L-amino acids after administration to an animal e.g. a human.

Peptides of the invention can be further modified or derivatized to contain additional nonproteinaceous moieties that are known in the art and readily available. Such derivatives may improve the solubility, absorption and/or biological half-life of the compounds. The moieties may alternatively eliminate or attenuate any undesirable side- effect of the compounds.

Exemplary derivatives include compounds in which: The compound is cross-linked or is rendered capable of cross- linking between molecules. For example, the peptide portion may be modified to contain one Cys residue and thereby be able to form an intermolecular disulfide bond with a like molecule. The compound may also be cross-linked through its C-terminus .

The N-terminus may be acylated or modified to a substituted amine. Exemplary N-terminal derivative groups include -NRRl (other than -NH2 ) , -NRC(O)Rl, -NRC(O)ORl, -NRS(O) 2Rl, -NHC (O)NHRl, succinimide, or benzyloxycarbonyl-NH- (CBZ-NH-) , wherein R and Rl are each independently hydrogen or lower alkyl and wherein the phenyl ring may be substituted with 1 to 3 substituents selected from the group consisting of C1-C4 alkyl, C1-C4 alkoxy, chloro, and bromo .

The C-terminus may be esterified or amidated. For example, methods described in the art may be used to add (NH-CH2-CH2- NH2)2 to peptides of this invention. Likewise, methods described in the art may be used to add-NH2 to peptides of this invention. Exemplary C-terminal derivative groups include, for example, -C(O)R2 wherein R2 is lower alkoxy or- NR3R4 wherein R3 and R4 are independently hydrogen or C1-C8 alkyl (preferably C1-C4 alkyl) .

A disulfide bond may be replaced with another, preferably more stable, cross-linking moiety (e.g., an alkylene) . See, for example: Bhatnagar et al . (1996), J. Med. Chem. 39: 3814 9; Alberts et al . (1993) Thirteenth Am. Pep. Symp., 357-9.

One or more individual amino acid residues may be modified. Various derivatizing agents are known to react specifically with selected side chains or terminal residues, as described in detail below:

Lysinyl residues and amino terminal residues may be reacted with succinic or other carboxylic acid anhydrides, which reverse the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.

Arginyl residues may be modified by reaction with any one or combination of several conventional reagents, including phenylglyoxal, 2 , 3-butanedione, 1, 2-cyclohexanedione, and ninhydrin. Derivatization of arginyl residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.

Specific modification of tyrosyl residues has been studied extensively, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane . Most commonly, N- acetylimidizole; and tetranitromethane are used to form 0- acetyl tyrosyl species and 3-nitro derivatives, respectively.

Carboxyl sidechain groups (aspartyl or glutamyl) may be selectively modified by reaction with carbodiimides (R' -N=C=N- R') such as l-cyclohexyl-3- (2-morpholinyl- (4-ethyl) carbodiimide or l-ethyl-3- (4-azonia-4, 4-dimethylpentyl) carbodiimide . Furthermore, aspartyl and glutamyl residues may be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues may be deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention. Cysteinyl residues can be replaced by amino acid residues or other moieties either to eliminate disulfide bonding or, conversely, to stabilize cross-linking. See, e.g., Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9.

Derivatization with bifunctional agents is useful for cross- linking the peptides of the invention or their functional derivatives to a water-insoluble support matrix or to other macromolecular vehicles. Commonly used cross-linking agents include, e.g., 1 , 1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'- dithiobis (succinimidylpropionate) , and bifunctional maleimides such as bis-N-maleimido-1 , 8-octane . Derivatizing agents such as methyl-3- [ (p-azidophenyl) dithio] propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide- activated carbohydrates and the reactive substrates described in U.S. Patents: US3, 969, 287 ; US3, 691, 016; US4 , 195, 128 ; US4,247, 642; US4,229,537; and US4,330,440 are employed for protein immobilization.

Carbohydrate (oligosaccharide) groups may conveniently be attached to sites that are known to be glycosylation sites in proteins. Generally, O-linked oligosaccharides are attached to serine (Ser) or threonine (Thr) residues while N-linked oligosaccharides are attached to asparagine (Asn) residues when they are part of the sequence Asn-X-Ser/Thr, where X can be any amino acid except proline. X is preferably one of the 19 naturally occurring amino acids other than proline. The structures of N-linked and O-linked oligosaccharides and the sugar residues found in each type are different. One type of sugar that is commonly found on both is N-acetylneuraminic acid (referred to as sialic acid) . Sialic acid is usually the terminal residue of both N-linked and 0-linked oligosaccharides and, by virtue of its negative charge, may confer acidic properties to the glycosylated compound.

Such site(s) may be incorporated in the linkers contemplated for the peptides of the invention and are preferably glycosylated by a cell during recombinant production of the polypeptide compounds (e.g., in mammalian cells such as CHO, BHK, COS) . However, such sites may further be glycosylated by synthetic or semi-synthetic procedures known in the art.

Other possible modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, oxidation of the sulphur atom in Cys, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains. Creighton, Proteins: Structure and Molecule Properties (W. H. Freeman & Co., San I Francisco), pp. 79-86 (1983) .

Preferably, the moieties suitable for derivatization of the peptides of the invention are water soluble polymers. Non- limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG) , copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3- dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The polymer may be linked to the peptide in the manner set forth in US patents: US4640835, US4496689, US4301144, US4670417, US4791192 or US4179337. WO 93/00109 also describes methods of linking amino acid residues in polypeptides to PEG molecules. The number of polymers attached to the peptide may vary, and if more than one polymers are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the peptide to be improved or whether it will be used in a therapy under defined conditions, for example.

In an alternative embodiment, peptides of the invention can be further modified to contain a serum carrier protein in order to extend the half life in vivo. The serum carrier protein may be a naturally occurring serum carrier protein or a fragment thereof. Particular examples include thyroxine-binding protein, transthyretin, αl-acid glycoprotein, transferrin, fibrinogen and especially, albumin, together with fragments thereof. Preferably the carrier proteins are of human origin. Where desired each may have one or more additional or different amino acids to the naturally occurring sequence providing that the resulting sequence is functionally equivalent with respect to half-life. Fragments include any smaller part of the parent protein that retains the carrier function of the mature sequence. The peptide and carrier protein components may be directly or indirectly covalently linked. Indirect covalent linkage is intended to mean that an amino acid in a peptide is attached to an amino acid in a carrier protein through an intervening chemical sequence, for example a bridging group. Particular bridging groups include for example aliphatic, including peptide. Direct covalent linkage is intended to mean that an amino acid in a peptide is immediately attached to an amino acid in a carrier protein without an intervening bridging group. Particular examples include disulphide (--S--S--] and amide [--CONH--] linkages, for example when a cysteine residue in one component is linked to a cysteine residue in another through the thiol group in each, and when the C-terminal acid function of one component is linked to the N-terminal amine of the other.

Peptides of the invention may be labelled with a detectable or functional label. Thus, a peptide can be present in the form of a peptide conjugate so as to obtain a detectable and/or quantifiable signal. A peptide conjugate may comprise a peptide of the invention conjugated with a detectable or functional label. A label can be any molecule that produces or can be induced to produce a signal, including but not limited to fluorescers, radiolabels, enzymes, chemiluminescers or photosensitizers . Thus, binding may be detected and/or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or light absorbance.

Suitable labels include, by way of illustration and not limitation,

- enzymes, such as alkaline phosphatase, glucose-6-phosphate dehydrogenase ("G6PDH"), alpha-D-galactosidase, glucose oxydase, glucose amylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase and peroxidase e.g. horseradish peroxidase;

- dyes;

- fluorescent labels or fluorescers, such as fluorescein and its derivatives, fluorochrome, rhodamine compounds and derivatives, GFP (GFP for "Green Fluorescent Protein") , dansyl, umbelliferone, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine; fluorophores such as lanthanide cryptates and chelates e.g. Europium etc (Perkin Elmer and Cis Biointernational) , - chemoluminescent labels or chemiluminescers, such as isoluminol, luminol and the dioxetanes;

- bio-luminescent labels, such as luciferase and luciferin;

- sensitizers; - coenzymes;

- enzyme substrates;

- radiolabels including but not limited to bromineW, carbonl4, cobalt57, fluorineδ, gallium67, gallium 68, hydrogen3 (tritium), indiumlll, indium 113m, iodinel23m, iodinel25, iodinel26, iodinel31, iodinel33, mercurylO7, mercury203, phosphorous32, rhenium99m, rheniumlOl, rheniumlO5, ruthenium95, ruthenium97, rutheniumlO3 , rutheniumlO5, scandium47, selenium75, sulphur35, technetium99, technetium99m, telluriuml21m, telluriuml22m, telluriuml25m, thuliuml65, thuliuml67, thuliuml68, yttriuml99 and other radiolabels mentioned herein;

- particles, such as latex or carbon particles; metal sol; crystallite; liposomes; cells, etc., which may be further labelled with a dye, catalyst or other detectable group; - molecules such as biotin, digoxygenin or 5- bromodeoxyuridine;

- toxin moieties, such as for example a toxin moiety selected from a group of Pseudomonas exotoxin (PE or a cytotoxic fragment or mutant thereof) , Diptheria toxin or a cytotoxic fragment or mutant thereof, a botulinum toxin A, B, C, D, E or F, ricin or a cytotoxic fragment thereof e.g. ricin A, abrin or a cytotoxic fragment thereof, saporin or a cytotoxic fragment thereof, pokeweed antiviral toxin or a cytotoxic fragment thereof and bryodin 1 or a cytotoxic fragment thereof.

Suitable enzymes and coenzymes are disclosed in Litman, et al., US4275149, and Boguslaski, et al . , US4318980, each of which are herein incorporated by reference in their entireties. Suitable fluorescers and chemiluminescers are disclosed in Litman, et al . , US4,275,149, which is incorporated herein by reference in its entirety. Labels further include chemical moieties, such as biotin that may be detected via binding to a specific cognate detectable moiety, e.g. labelled avidin or streptavidin . Detectable labels may be attached to peptides of the invention using conventional chemistry known in the art, or by gene fusion.

Peptide conjugates or their functional fragments can be prepared by methods known to the person skilled in the art. Peptides can be coupled to enzymes or to fluorescent labels directly or by the intermediary of a spacer group or of a linking group, such as a polyaldehyde, like glutaraldehyde, ethylenediaminetetraacetic acid (EDTA) , diethylene- triaminepentaacetic acid (DPTA) , or in the presence of coupling agents, such as those mentioned above for the therapeutic conjugates. Conjugates containing labels of fluorescein type can be prepared by reaction with an isothiocyanate .

The methods known to the person skilled in the art existing for coupling the therapeutic radioisotopes to the peptides either directly or via a chelating agent, such as EDTA, DTPA mentioned above can be used for the radioelements which can be used in diagnosis. It is likewise possible to perform labelling with sodiuml25 by the chloramine T method [i] or else with technetium99m by the technique of Crockford et al . , (US4424200, herein incorporated by reference in its entirety) or attached via DTPA as described by Hnatowich (US4479930, herein incorporated by reference in its entirety) .

There are numerous methods by which the label can produce a signal detectable by external means, for example, by visual examination, electromagnetic radiation, heat, and chemical reagents. The label can also be bound to another antibody that binds the peptide of the invention, or to a support.

The label can directly produce a signal, and therefore, additional components are not required to produce a signal.

Numerous organic molecules, for example fluorescers, are able to absorb ultraviolet and visible light, where the light absorption transfers energy to these molecules and elevates them to an excited energy state. This absorbed energy is then dissipated by emission of light at a second wavelength. This second wavelength emission may also transfer energy to a labelled acceptor molecule, and the resultant energy dissipated from the acceptor molecule by emission of light for example fluorescence resonance energy transfer (FRET) . Other labels that directly produce a signal include radioactive isotopes and dyes.

Alternately, the label may need other components to produce a signal, and the signal producing system would then include all the components required to produce a measurable signal, which may include substrates, coenzymes, enhancers, additional enzymes, substances that react with enzymic products, catalysts, activators, cofactors, inhibitors, scavengers, metal ions, and a specific binding substance required for binding of signal generating substances. A detailed discussion of suitable signal producing systems can be found in Ullman, et al . US5185243, which is herein incorporated herein by reference in its entirety.

In an alternative embodiment, the peptide of the present invention may be modified to form a chimeric molecule comprising the peptide fused or linked to another heterologous polypeptide or amino acid sequence. Such fusions may be recombinantly produced by methods well known in the art. Alternatively, such fusion may be produced by chemical conjugations (including both covalent and non-covalent conjugations) .

In another embodiment, the peptide may be fused or linked with an immunoglobulin or a particular region of an immunoglobulin (an immunoadhesion) . Such a fusion could be to the binding domain of an antibody or antibody fragment, such that the Fc mimetic peptide comprises an antibody antigen-binding site and is therefore directed to a particular antigen. Antibody fragments that comprise an antibody antigen-binding site include, but are not limited to, molecules such as Fab, Fab' , Fab' -SH, scFv, Fv, dAb and Fd. Various other antibody molecules including one or more antibody antigen-binding sites have been engineered, including for example Fab2 , Fab3, diabodies, triabodies, tetrabodies and minibodies. Antibody molecules and methods for their construction and use are described in Hollinger & Hudson (2005) .

It has been shown that fragments of a whole antibody can perform the function of binding antigens. Examples of binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CHl domains; (ii) the Fd fragment consisting of the VH and CHl domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward et al 1989, McCafferty et al 1990, Holt et al 2003), which consists of a VH or a VL domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al 1988, Huston et al 1988); (viii) bispecific single chain Fv dimers (PCT/US92/09965) and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO94/13804; Hollinger et al 1993) . Fv, scFv or diabody molecules may be stabilized by the incorporation of disulphide bridges linking the VH and VL domains (Reiter et al 1996) . Minibodies comprising a scFv joined to a CH3 domain may also be made (Hu et al 1996) . Other examples of binding fragments are Fab' , which differs from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CHl domain, including one or more cysteines from the antibody hinge region, and Fab' -SH, which is a Fab' fragment in which the cysteine residue (s) of the constant domains bear a free thiol group.

The present invention provides a method comprising causing or allowing binding of a peptide as provided herein to, for example, FcγRI . As noted, such binding may take place in vivo, e.g. following administration of a peptide, or nucleic acid encoding a peptide, or it may take place in vitro, for example in ELISA, Western blotting, immunocytochemistry, immunoprecipitation, affinity chromatography, and biochemical or cell-based assays.

Those skilled in the art are able to choose a suitable mode of determining binding of the peptide to FcγRI according to their preference and general knowledge, in light of the methods disclosed herein. Such methods may include inter alia competitive ELISA and alpha screen.

A kit comprising a peptide according to any aspect or embodiment of the present invention is also provided as an aspect of the present invention. In the kit, the peptide may be labelled to allow its reactivity in a sample to be determined, e.g. as described further below. Further the peptide may or may not be attached to a solid support. Components of a kit are generally sterile and in sealed vials or other containers. Kits may be employed in diagnostic analysis or other methods for which peptides are useful. A kit may contain instructions for use of the components in a method, e.g. a method in accordance with the present invention. Ancillary materials to assist in or to enable performing such a method may be included within a kit of the invention. The ancillary materials include a second, different peptide or an antibody which binds to the first peptide and is conjugated to a detectable label (e.g., a fluorescent label, radioactive isotope or enzyme) . Each component of the kits is generally in its own suitable container. Thus, these kits generally comprise distinct containers suitable for each peptide. Further, the kits may comprise instructions for performing the assay and methods for interpreting and analyzing the data resulting from the performance of the assay.

In various aspects and embodiments, the present invention extends to a peptide that competes for binding to, for example, hFcγRI with hlgGl. Competition between peptides may be assayed in vitro, for example by tagging a specific reporter molecule to hlgGl which can be detected in the presence of other untagged peptide (s), to enable identification of peptides which bind hFcγRI. Competition may be determined for example using ELISA in which hFcγRI is immobilized to a plate and a first tagged or labelled hlgGl along with one or more other untagged or unlabelled peptides is added to the plate. Presence of an untagged peptide that competes with the tagged hlgGl is observed by a decrease in the signal emitted by the tagged hlgGl .

For example, the present invention includes a method of identifying a peptide, comprising (i) immobilizing hFcγRI to a support, (ii) contacting said immobilized hFcγRI simultaneously or in a step-wise manner with at least one tagged or labelled hlgGl according to the invention and one or more untagged or unlabelled test peptides, and (iii) identifying a functional peptide by observing a decrease in the amount of bound tag from the tagged hlgGl. Such methods can be performed in a high-throughput manner using a multiwell or array format. Such assays may also be performed in solution. See, for instance, US 5,814,468, which is herein incorporated by reference in its entirety. As described above, detection of binding may be interpreted directly by the person performing the method, for instance, by visually observing a detectable label, or a decrease in the presence thereof. Alternatively, the binding methods of the invention may produce a report in the form of an autoradiograph, a photograph, a computer printout, a flow cytometry report, a graph, a chart, a test tube or container or well containing the result, or any other visual or physical representation of a result of the method.

A peptide having been identified may be made in transformed host cells using recombinant DNA techniques. If the vehicle component is a polypeptide, the peptide-vehicle fusion product may be expressed as one. To do so, a recombinant DNA molecule encoding the peptide is first prepared using methods well known in the art. For instance, sequences coding for the peptides could be excised from DNA using suitable restriction enzymes. Alternatively, the DNA molecule could be synthesized using chemical synthesis techniques, such as the phosphoramidate method. Also, a combination of these techniques could be used.

Therefore, the present invention further provides an isolated nucleic acid encoding a peptide of an embodiment of the present invention. Nucleic acid may include DNA and/or RNA.

The present invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise at least one polynucleotide as above. The present invention also provides a recombinant host cell that comprises one or more constructs as above. A nucleic acid encoding any peptide as provided, itself forms an aspect of the present invention, as does a method of production of the encoded product, which method comprises expression from encoding nucleic acid therefor. Expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. Following production by expression a peptide may be isolated and/or purified using any suitable technique, then used as appropriate .

Nucleic acid according to the present invention may comprise DNA or RNA and may be wholly or partially synthetic. Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA molecule with the specified sequence in which U is substituted for T, unless context requires otherwise.

A method of production may comprise a step of isolation and/or purification of the product. A method of production may comprise formulating the product into a composition including at least one additional component, such as a pharmaceutically acceptable excipient.

Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids e.g. phagemid, or viral e.g. 'phage', as appropriate. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Ausubel et al (1999) (Short Protocols in Molecular Biology, John Wiley & Sons, 4^th edition) .

A further aspect of the present invention provides a host cell containing nucleic acid as disclosed herein. Such a host cell may be in vitro and may be in culture. Such a host cell may be in vivo. In vivo presence of the host cell may allow intra-cellular expression of the peptides of the present invention as intra-cellular peptides which may be used for gene therapy.

Systems for cloning and expression of a peptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, plant cells, filamentous fungi, yeast and baculovirus systems and transgenic plants and animals. A common bacterial host is E. coli.

Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for production of a polypeptide. Mammalian cell lines available in the art for expression of a heterologous polypeptide include, for example, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, YB2/0 rat myeloma cells, human embryonic kidney cells, human embryonic retina cells and many others known in the art.

A still further aspect provides a method comprising introducing nucleic acid of the invention into a host cell. The introduction may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus. Introducing nucleic acid in the host cell, in particular a eukaryotic cell may use a viral or a plasmid based system. The plasmid system may be maintained episomally or may be incorporated into the host cell or into an artificial chromosome. Incorporation may be either by random or targeted integration of one or more copies at single or multiple loci. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage.

The introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene. The purification of the expressed product may be achieved by methods known to one of skill in the art.

Nucleic acid of the invention may be integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences that promote recombination with the genome, in accordance with standard techniques.

The present invention also provides a method that comprises using a construct as stated above in an expression system in order to express a peptide as above. Alternatively, the peptides of the present invention may be made by synthetic methods, such as solid phase synthesis.

Peptides of an embodiment of the present invention may be used in methods of diagnosis, prevention, treatment or mitigation of disease in human or animal subjects, e.g. human.

Peptides used in a method of diagnosis may be conjugated to a detectable label so that binding to Fcγ receptor can be detected. Suitable detectable labels are discussed above. Peptides may be used to diagnose or treat, prevent or mitigate disorders associated with cancer, infection or autoimmune/inflammatory disorders. The peptides of the invention are particularly suited for use in the treatment of these conditions since the small structured peptides can mimic specifically the effect of native Fc on FcγRI and can therefore facilitate the design of specific and potent agonists or antagonists. Tumour cells expressing Fcγ receptor, such as non-hematopoietic tumour cells, may be particularly susceptible to treatment by peptides of the present invention.

Regarding disorders associated with infection, bacteria or parasite cells infected by a pathogen may express Fcγ receptor. Such expression could short-circuit the normal immune response by trapping IgG and consequently the immune response against the invader may be weakened. For example, in instances where the pathogen is a viral pathogen, the virus- infected cells may express Fcγ receptor and this could reduce the exposure of the virus to immune cells. Binding of a peptide of the present invention to these expressed Fcγ receptors could therefore inhibit this non-specific binding. Such down regulation of effector function may occur in conditions such as herpes simplex virus, ebola virus or human cytomegalovirus .

Alternatively, viral IgG that binds Fcγ receptor may enhance viral infectivity and in some cases the replication of virus into monocytes/macrophages and granulocytic cells. This phenomenon is known as antibody dependent enhancement (ADE; Cancel Tirado & Yoon, 2003) and has been reported in vitro and in vivo for numerous viruses of importance to public health and veterinary medicine. For example, such enhancement of effector function may occur in conditions such as dengue fever or HIV. Inflammatory disorders may include allergic diseases such as asthma and autoimmune diseases in which autoantibodies are produced during the differentiation of B cells into plasma cells. Examples of B cell disorders include autoimmune thyroid disease, including Graves' disease and Hashimoto's thyroiditis, rheumatoid arthritis, systemic lupus erythematosus (SLE), Sjogrens syndrome, immune thrombocytopenic purpura (ITP), multiple sclerosis (MS), myasthenia gravis (MG), psoriasis, scleroderma, insulin- dependent diabetes mellitus, and inflammatory bowel disease, including Crohn's disease and ulcerative colitis.

Accordingly, further aspects of the invention provide methods of treatment comprising administration of a peptide as provided, pharmaceutical compositions comprising such a peptide, and use of such a peptide in the manufacture of a medicament for administration, for example in a method of making a medicament or pharmaceutical composition comprising formulating the peptide with a pharmaceutically acceptable excipient. A pharmaceutically acceptable excipient may be a compound or a combination of compounds entering into a pharmaceutical composition not provoking secondary reactions and which allows, for example, facilitation of the administration of the active compound(s), an increase in its lifespan and/or in its efficacy in the body, an increase in its solubility in solution or else an improvement in its conservation. These pharmaceutically acceptable vehicles are well known and will be adapted by the person skilled in the art as a function of the nature and of the mode of administration of the active compound(s) chosen.

Peptides of the present invention will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the peptide. Thus pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, inhaled, intra-tracheal, topical, intra-vesicular or by injection, as discussed below.

Pharmaceutical compositions for oral administration are also envisaged in the present invention. Such oral formulations may be in tablet, capsule, powder, liquid or semi-solid form. A tablet may comprise a solid carrier, such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier, such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols, such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

For intra-venous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles, such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be employed as required including buffers such as phosphate, citrate and other organic acids; antioxidants, such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3' -pentanol; and tricresol); low molecular weight polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagines, histidine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions, such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants, such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG) .

Peptides of the present invention may be formulated in liquid, semi-solid or solid forms depending on the physicochemical properties of the molecule and the route of delivery. Formulations may include excipients, or combinations of excipients, for example: sugars, amino acids and surfactants. Liquid formulations may include a wide range of concentrations and pH . Solid formulations may be produced by lyophilisation, spray drying, or drying by supercritical fluid technology, for example. Formulations of peptides will depend upon the intended route of delivery: for example, formulations for pulmonary delivery may consist of particles with physical properties that ensure penetration into the deep lung upon inhalation; topical formulations (e.g. for treatment of scarring, e.g. dermal scarring) may include viscosity modifying agents, which prolong the time that the drug is resident at the site of action. A peptide may be prepared with a carrier that will protect the peptide against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are known to those skilled in the art (Robinson, J. R. ed., Sustained and Controlled Release Drug Delivery Systems, Marcel Dekker, Inc., New York, 1978) .

Treatment may be given orally, by injection (for example, subcutaneously, intra-articular, intra-venously, intra- peritoneal, intra-arterial or intra-muscularly) , by inhalation, intra-tracheal, by the intra-vesicular route (instillation into the urinary bladder) , or topically (for example intra-ocular, intra-nasal, rectal, into wounds, on skin) . The treatment may be administered by pulse infusion, particularly with declining doses of the peptide. The route of administration can be determined by the physicochemical characteristics of the treatment, by special considerations for the disease or by the requirement to optimize efficacy or to minimize side-effects. One particular route of administration is intra-venous . Another route of administering pharmaceutical compositions of the present invention is subcutaneously. It is envisaged that treatment will not be restricted to use in the clinic. Therefore, subcutaneous injection using a needle-free device is also advantageous.

A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

A peptide of the invention may be used as part of a combination therapy in conjunction with an additional medicinal component. Combination treatments may be used to provide significant synergistic effects, particularly the combination of a peptide of the invention with one or more other drugs, for example an anti-cancer agent. A peptide of the invention may be administered concurrently or sequentially or as a combined preparation with another therapeutic agent or agents, for the treatment of one or more of the conditions listed herein.

A peptide according to the present invention may be provided in combination or addition with one or more of the following agents: chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal agent, and/or cardioprotectant . Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

Other therapeutic regimens may be combined with the administration of an anticancer agent, anti-bacterial, antiviral or anti-inflammatory agent. The combination therapy may be administered as a simultaneous or sequential regimen. When administered sequentially, the combination may be administered in two or more administrations. The combined administration includes co-administration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities.

A peptide of the invention and one or more of the above additional medicinal components may be used in the manufacture of a medicament. The medicament may be for separate or combined administration to an individual, and accordingly may comprise the peptide and the additional component as a combined preparation or as separate preparations. Separate preparations may be used to facilitate separate and sequential or simultaneous administration, and allow administration of the components by different routes e.g. oral and parenteral administration. In accordance with the present invention, compositions provided may be administered to mammals. Administration is normally in a "therapeutically effective amount", this being sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the type of peptide, the method of administration, the scheduling of administration and other factors known to medical practitioners. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors and may depend on the severity of the symptoms and/or progression of a disease being treated. A therapeutically effective amount or suitable dose of a peptide of the invention can be determined by comparing its in vitro activity and in vivo activity in an animal model. Methods for extrapolation of effective dosages in mice and other test animals to humans are known. The precise dose will depend upon a number of factors, including whether the peptide is for diagnosis, prevention or for treatment, the size and location of the area to be treated and the nature of any detectable label or other molecule attached to the peptide. An initial higher loading dose, followed by one or more lower doses, may be administered. The dose for a single treatment of an adult patient may be proportionally adjusted for children and infants. Treatments may be repeated at daily, twice- weekly, weekly or monthly intervals, at the discretion of the physician. Treatments may, for example, be every two to four weeks for subcutaneous administration and every four to eight weeks for intra-venous administration. Treatment may be periodic, and the period between administrations is about two weeks or more, e.g. about three weeks or more, about four weeks or more, or about once a month. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. Treatment may be given before, and/or after surgery, and/or may be administered or applied directly at the anatomical site of surgical treatment.

Further aspects and embodiments of the present invention will be apparent to those skilled in the art in the light of the present disclosure, including the following experimental exemplification .

All documents mentioned anywhere in this specification are incorporated by reference. In addition, this application claims benefit of U.S. provisional application No. 61/021,672, filed January 17, 2008, which is incorporated by reference in its entirety.

Experimental Examples

Example 1

Cloning procedures, construction libraries and Phage libraries synthesis

Three cyclic phage-peptide libraries X₂CXi₀CX₂, X₃CX₈CX₃ and

X₅CX₄X₅ were constructed by site-directed mutagenesis according to the method of Kunkel from three oligonucleotides 5'- GCTAAACAACTTTCAACAGTTTCTGCGGCCGC (SNN) ₂ACA ( SNN) _I0ACA ( SNN) ₂CTGTGC ACTGTGAGAATAGAAGG-3' , 5' GCTAAACAACTTTCAACAGTTTCTGCGGCCGC (SNN) ₃ ACA(SNN) ₈ACA (SNN) ₃CTGTGCACTGTGAGAATAGAAGG-3' , 5 ' GCTAAACAACTTT

CAACAGTTTCTGCGGCCGC(SNN) ₅ACA (SNN) ₄ACA (SNN) sCTGTGCACTGTGAGAATAGA AGG-3', where N = T, C, G or A, and S = G or C (Kunkel 1987) . Briefly, Fd-tet-Dogl phage vector was transformed into CJ236 E.coli cells (New England BioLabs, MA, USA), which was grown on 2 x TY medium containing 12.5 μg tetracycline/ml plate for 16 h at 37°C. One single colony was grown in 30 ml of 2 x TY medium containing 12.5 μg tetracycline/ml plate for 16h at 37°C and subsequently the supernatant with phage production was precipitated with 0.15 vol. of 20% (w/v) PEG-8000, 2.5 M NaCl for 10 min at 24°C. The final pellet was resuspended in

0.5 ml of PBS. dU-ssDNA template was extracted using a Qiaprep Spin M13 kit (Qiagen, Hilden, Germany) . 0.7 μg of oligonucleotide was phosphorylated for 1 h at 37°C, using T4 polynucleotide kinase (20U) (New England BioLabs, MA, USA) . Denaturation and annealing steps were carried out from 0.7 μg of phosphorylated oligonucleotide and 20 μg of dU-ssDNA template (90⁰C for 2 min, 50⁰C for 3 min, 20⁰C for 5 min) . The elongation step was performed with T4 DNA ligase (30 U) and T7 DNA polymerase (30 U) after 20⁰C for 3 h. Covalent closed circular DNA (ccc-DNA) was purified using a Roche DNA purification kit (Roche Diagnostics GmbH, Mannheim, Germany) in a final volume of 35 μl. 4 x 4.5 μl of ccc-DNA was electroporated in 4 x 400 μl of fresh competent E.coli TGl cells with a micropulser (Bio-Rad, Ca, USA) at 2.5 kV, 200 Ω, 25 μF in 0.1 cm cuvette. Electroporated TGl cells were then incubated at 37°C for 1 h in 1 ml of 2 x TY medium containing 12.5 μg tetracycline/ml . A dilution series of each library was then plated to determine the number of transformants . The remaining cells were plated on large 2 x TY plates containing 12.5 μg tetracycline/ml plate and incubated for 16 h at 31°C. The diversity of clones was determined by sequencing the random region of one hundred bacterial clones with the primer FdTetSeq (5'-GTCGTCTTTCCAGACGTTAGT-S') in an ABI Prism analyser (Applied Biosystems, CA, USA) . Phage libraries were then prepared by PEG precipitation (0.3 vol. of 20% (w/v) PEG- 8000, 2.5 M NaCl) for 1 h at 4°C and purified by CsCl gradient ultra-centrifigation for 24 h. After extraction of the band containing phages, the remaining CsCl in the sample was removed twice by dialysis against TE buffer. Finally, to validate the functionality of libraries, three rounds of selection were performed on human serum albumin (HSA) .

Example 2

Phage-peptide panning selection

An aliquot from each production was subjected to three rounds of selection using three different conditions (cond.l, cond.2 and cond.3) . In each round, binding selections were performed on the extracellular domain of hFcγRIa/CD64 (R&D Systems Inc, Minneapolis, USA) at 10 μg/ml in 0.1 M NaHCO₃, pH 8.6 at 4°C for 16 h for the first round, and at 10 μg/ml for cond.l and 5 μg/ml for cond.2 and cond.3 conditions for the further rounds, immobilized on Maxisorp microplates (Nunc A/S, Roskilde, Denmark) . The coated plates were then washed once with PBS (0.14 M NaCl, 0.01 M Phosphate Buffer pH 7.4) and alternatively incubated at each selection round either with 300 μl of blocking solution containing 4% (w/v) powdered milk/0.1 M NaHCO3, 0.1 μg/ml FcγRI or with 0.5% BSA (w/v) /0.1 M NaHCO3, 0.7 nM hFcγRI for 2 h at 4°C with gentle shaking. Phage-peptides from each library (3.0 x 10¹¹ transducing units (TUs) ) were first equilibrated in 100 μl of PBS/4% powdered milk or 0.5% BSA for 1 h at 20⁰C with gentle rotation. Then, coated plates were washed once with PBS and each phage sample was added to microwells for 2 h with gentle shaking following of 1 h without shaking at 24°C. After washing ten times with PBS/0.1 % Tween-20 and ten times with PBS in cond.l, twenty times with PBS/0.1 % Tween-20 and twenty times with PBS in cond.2 and five times with PBS/0.1% Tween-20 and five times with PBS in cond.3, bound phage-peptides were eluted by hlgGl competition (2 μM in PBS/4% powdered milk) for 1 h with gentle shaking. Specific phage-peptide elutions were then amplified by infecting a logarithmic phase culture of the E. coli TGl strain (Δ(lac-pro), supE, thi, hsdD5/F' , traD36, proAB, lacl^q, lacZΔM15) . Infected cells were grown in 20 ml of 2 x TY medium containing 12.5 μg tetracycline/ml for 16 h at 31°C, while a small aliquot was titred. The culture was then centrifuged twice at 6000 x g and 8000 x g for 10 min at 4°C. The supernatant containing phage particles was precipitated twice with 0.15 vol. of 20% (w/v) PEG-8000, 2.5 M NaCl at 4°C, with slight agitation for 16 h and 1 h respectively. After each precipitation the solution was centrifuged twice at 12,000 x g for 20 min and 10 min at 4°C respectively. Eventually, the final pellet was resuspended in 1 ml PBS buffer. All phage samples were filtered through a 0.45 μm filter and stored at 4°C. The titre of each production was estimated after counting between 1.0 x 10¹² and 1 x 10¹³ TUs per ml. The second round of selection was performed as previously, but with 5.0 x 10¹⁰ TUs in cond.l, and 2.5 x 10¹⁰ TUs in cond.2 and cond.3 of amplified phages from the first selection. The third and forth final round were performed with 2.0 x 10¹⁰ TUs in cond.l, 5.0 x 10⁹ TUs in cond.2, and 1.0 x 10¹⁰ TUs in cond.3. One hundred clones of each production were sequenced after two and three rounds of biopanning.

After four rounds of biopanning and in the three experimental conditions, a significant increase in the number of eluted phages occurred only with the CPEP-8 library. The greatest amplification (~2000-fold) was obtained after round 2 and for cond.l, where the pressure of selection was weakest (Table

1.) . It is known that for selections from cyclic libraries it is desirable to employ peptides of varying shapes, since binders can be preferentially isolated from specific cycle sizes in a given library and not from other cyclic libraries (Koivunen 1995, Adey 1996, Nakamura 2001, Nakamura 2002) .

Table 1. Enrichment of phage-peptides after four rounds of biopanning

Round of selection n=4 cond . 1 cond . 2 cond . 3

CPEP- 4 X.₅OX.₄OX.₅ ND ND ND

Phage peptide libraries were selected using three separate conditions (cond. 1, cond. 2 and cond. 3) . The values obtained correspond to the ratio of phage enrichment [(Input n/Output n) / (Input n-1/Output n-1)] and were determined by counting the number of TUs on agar plates after infection with E.coli bacteria. ND: not determined

The amino acid sequences of phage-peptides selected from the

CPEP-8 library revealed seventeen unique sequences that shared a strong homology and corresponded to the general consensus sequence TX₂CXXςPXLLGCΦXE (using the amino acid single letter code, ς represents a hydrophobic residue often of type L or W, and Φ is usually an acid amino acid) (Table 2.) . According to the conditions of selection, between 70% and close to 100% of phage-peptides selected exhibited the XXςPXLLG motif. Close to 60% of amino acids enclosed between both cysteine residues were of hydrophobic nature. Some of them exhibited a hydrophobic core sequence rich in leucine residues (up to five residues in the peptides 22 and 33) . Interestingly, 90% of phage-peptides displayed in the hydrophobic core a constant proline residue. The composition of the tripeptide sequence flanking the cyclic ring in the carboxy-terminal end, comprised more than 30% of negatively charged side chains. These acid residues could occupy one or more of three positions. The sequencing of many of the clones selected from the CPEP-4 and CPEP-10 libraries did not show a similarity with the consensus sequence. The greatest clone diversity (11 in all) was obtained with cond.l, where the stringency conditions were weaker. In contrast, in cond.2 and 3, only six different clones were isolated, which represented around 97% and 74% respectively of the total diversity. Interestingly, in each condition of selection, the sequences identified were all different .

To identify a homology with the primary sequence of the IgG Fc fragment, the consensus sequence was aligned with hlgGl (EU numbering) . This comparison revealed a strong similarity with the fragment 225-240 present in the lower hinge sequence, which joins the Cγl and Cγ2 domains. Twelve peptides exhibited the Pro-Xxx-Leu-Leu-Gly motif, present in the Fc fragment sequence of hlgGl. One peptide (peptide 26) displayed the full heptapeptide Pro-Glu-Leu-Leu-Gly of the hlgGl lower hinge sequence. Some other homologies can also be inferred from the consensus within the sequence 265-270 (with acidic region) , 327-333 (with ςP) and 297-299 (with TXX).

Additionally, some peptides that did not share any homology with the common consensus sequence were also been identified. These sequences showed a stronger frequency than the background. Notable peptides amongst them are the cyclic peptides 6 (SEQ ID NO: 18; FKALLCKSQLCSRYLK) , 8 (SEQ ID NO: 21; ELLSYCWDQWCWWQDG) , 17 (SEQ ID NO : 19 ;

GHCHFPPERQRYTCLQ) and 42 (SEQ ID NO : 20 ; IPLCVLLPSFTKCRAR) , which were selected from three different libraries. With reference to Table 2 below, the frequency (%) of phage- peptide sharing homology was determined after sequencing of 100 clones from round 2 and 3. The total frequency of phage- peptides identified in each condition is also indicated. Homologous amino acids in each position and those that contribute to generate the consensus sequence are shown in bold. The consensus was aligned with the sequence 225-240 of hlgGl (EU residue number), ς represents a hydrophobic residue, Φ represents an acidic residue, cp: cyclic peptide, p: linear peptide . Table 2: Amino acid sequence and frequency of phage-peptides obtained after selection and amplification with library CPEP-

Clone number Peptide sequence n=3 (%) n=2 (%) cond.l cp 22 TDT C LMLPLLLG C DEE 31.16 0.00 cp 21 DPI C WYFPRLLG C TTL 11.48 0.00 cp 23 WYP C YIYPRLLG C DGD 18.01 0.00 cp 24 GNI C MLIPGLLG C SYE 6.56 0.00 cp 33 VNS C LLLPNLLG C GDD 4.92 0.00 cp 25 TPV C ILLPSLLG C DTQ 13.12 0.00 cp 26 TVL C SLWPELLG C PPE 1.64 0.00 cp 27 TFS C LMWPWLLG C ESL 8.20 1.33 cp 32 FGT C YTWPWLLG C EGF 1.64 0.00 cp 34 SLF C RLLLTPVG C VSQ 0.00 2.66

P 35 HLL V LPRGLLG C TTLA 1.64 0.00

98.37 3.99 cond.2 cp 28 TSL C SMFPDLLG C FNL 83 .72 5 .00 cp 29 SHP C GRLPMLLG C AES 9 .66 5 .00 p 37 TST C SMVPGPLGAV STW 3 .22 0 .00

96.60 10.00 cond.3 cp 30 KDP C TRWAMLLG C DGE 32 .34 0 .00 cp 31 IMT C SVYPFLLG C VDK 38 .22 0 .00 cp 36 IHS C AHVMRLLG C WSR 2 .94 3 .13

73.50 3.13

Consensus TXX C XXCPxLLG C ΦXE

IgGl 225-236 TCP P CPAPELLG

Example 3

Biotinylation of IgGl antibody

Biotinylation was conducted in 200 μl of 100 mM NaHCO₃ (pH 8.2) with 60 μM of hlgGl and EZ-link NHS-LC-Biotin (Perbio/Pierce, Rockford, IL) at a protein: biotin molar ratio of 1:4 for 15 min at 24°C. Biotinylation efficiency was ascertained by MALDI-TOF-MS analysis (Ciphergen Biosystems Ltd, Guilford, UK) . The reaction was stopped by removal of excess biotin reagent via gel filtration over a PD-IO column (Amersham Biosciences, Uppsala, Sweden) and the biotinylated protein concentration was determined by spectrophotometry at

.

Example 4

Synthesis of soluble peptides

Synthetic 18-mer peptides were produced in 1-3 mg amounts and characterized by analytical HPLC and LC/MS (Pepscan Systems, Lelytad, The Netherlands). Peptides were synthesized with a free N-terminus and a biotinylated C-terminus in the general form NH₂-AQX₃CX₈CX₃K-biot in an oxidised state. Cyclic hinge-

CH2 peptide (SEQ ID NO: 25; TAPCAPAPELLGCPSV) corresponding to the cyclic hinge sequence of native IgGl constructed according to the format for cyclic octa-peptides and peptide 22 (peptide 22 S₄Si₃) with Ser substitutions replacing both Cys in peptide 22 were also synthesized as described previously. For the assays, peptides were solubilized in either PBS or in 2.5-50 % CH₃CN or DMF to a final concentration of 5 mM. 10 mg of synthetic dimer cyclic peptide 33 (sequence VNSCLLLPNLLGCSYEKKKKKEYSCGLLNPLLLCNV; SEQ ID NO: 23) (Cysβ- Cysl5, Cys27-Cys36) was purified by HPLC and the quality assured and checked by HPLC and MS (AMS Biotechnology, Oxon, England) .

Example 5

Production of soluble extracellular domains of hFcγRIIa, hFcγRIIb and hFcγRIIIa Human FcγRIIa and FcγRIIIa extracellular domains were cloned in fusion with a Flag-His-tag sequence in pDEST12.2 (Invitrogen, Paisley, UK) and expressed in soluble form in HEK-293 EBNA cells. 6 x 10⁶ cells were transfected with 60 μg DNA complexed with 100 μg PEI in DMEM containing 2% FSB, 100 U/ml penicillin, and 100 μg/ml streptomycin. After 24 h, the media was changed to serum-free CD-CHO. The supernatant was harvested and pooled after 72, 120 and 192 h. The secreted proteins were then purified from cell supernatants by loading onto a 5 ml HisTrap HP column (Amersham Biosciences, Uppsala, Sweden) and implemented on an AKTA Explorer (Amersham Pharmacia Biotech, Uppsala, Sweden) . The column was washed initially with 300 mM PBS, 300 mM NaCl (pH 7.2) until the A₂₈o_nm reading returned to the baseline. The column was washed with 10 column volumes of both 16 mM and 40 mM imidazole. The proteins were eluted with 20 column volumes of a linear gradient of imidazole (40-400 mM) . Fractions (1 ml) eluted between 250 mM and 400 mM imidazole were collected and analysed by SDS-PAGE on NuPAGE Novex 4-12% Bis-Tris gels (Invitrogen, Paisley, UK). The fractions containing the proteins were pooled (20 ml) and samples were concentrated using an Amicon Ultra-15 30 kDa filter (Amicon, Millipore, Watford, UK) . The protein samples were then loaded onto a Superdex S75 HR 10/30 gel Filtration Column (Amersham Pharmacia Biotech) and implemented on an AKTA Explorer. The fractions (1 ml) were analysed using NuPAGE Novex 4-12% Bis- Tris gels. The final pure fractions were pooled, concentrated and the protein concentration was estimated by spectrophotometry at A₂₈o_nm- The purity was analysed by SDS-PAGE and the protein mass was confirmed by MALDI-TOF-MS.

Example 6

Competitive ELISA to hFcγRI 100 μl of hFcγRI (10 μg/ml) in 0.1 M NaHCO₃ (pH 8.6) were immobilised on Microtitre plates (Maxisorp, Nunc) by incubation at 4°C for 16 h. Coated plates were washed once with PBS and then incubated with 300 μl of blocking solution containing 4% (w/v) powdered milk/0.1 M NaHCO₃, 0.7 nM hFcγRI for 2 h at 4°C with gentle shaking. Coated plates were then washed once with PBS. 1.3 μM of hlgGl was incubated with four different concentrations of peptides at 50, 16.6, 5.53 and 1.84 μM (100 μl/well), equilibrated with PBS/4% powdered milk and added to the microwells for 2 h at 24°C with gentle shaking. After washing three times with PBS/0.1 % Tween-20 and once with PBS, bound hlgGl were detected by incubation with 100 μl of horseradish peroxidase-conjugated F(ab')₂ fragment goat anti-hlgG antibody (Jackson ImmunoResearch, Baltimore Pike, USA) diluted 1:5000 in PBS/4% powdered milk, for 1 h at 24°C with gentle shaking. After washing three times with PBS/0.1 % Tween-20 and once with PBS, bound antibodies were revealed with 50 μl of 3, 3', 5, 5' tetramethylbenzidine (Sigma, Missouri, USA) used as substrate. The reaction was stopped by addition of 50 μl of 0.5M sulphuric acid and the absorbance read at A₄₅onm- The absorbance measured in the absence of peptide competitor was taken as 100%, and the absorbance measured in the presence of each peptide was used to calculate the percentage of inhibition.

Among the seventeen peptides studied, thirteen significantly displaced the binding of hlgGl to hFcγRI in a dose-dependent manner (Fig. 1.) . Inactive peptides were either uncyclised (peptides 35 and 37) or did not contain the dipeptide sequence Leu-Leu in the motif core (peptide 34) . It is likely that the absence of the proline residue in the core and/or charged C- terminal end in these peptides contributed to their inactivity as well. The peptide with the corresponding linear hinge sequence to hlgGl (SEQ ID NO:24; PPCPAPELLGGPSVFLFP) did not exhibit detectable binding in the concentration range tested. The same observations have already been reported from studies on the hFcγRI using the lower hinge control peptides G233LLGGPYG240 (Lund 1991), E₂₃₃LLGGPSVF₂₄I (Sheridan 1999), and C₂₃₃LLGGLGC₂₄₀ (Berntzen 2006), and also on the hFcγRII (Uray 2004, Medgyesi 2004). For FcγRIII, plgGl (CPAPELLGGPSV) , pIgG2 (PPVAGPSV) and pIgG4 (PEFLGGPSV) peptides bound the receptor with a Kd in the range between 350 and 500 μM (Radaev 2001b) . In our assay, at the highest concentrations used (50 μM) , peptides 30 and 33 inhibited hlgGl binding by 54% and 64% respectively. Consequently, the apparent IC₅₀ values of peptides 30 and 33 were estimated at around 45 μM and 20 μM respectively.

However, in the same assay the apparent Kd of hlgGl was of the order of 200 nM, a value roughly one hundred fold higher than that reported in the literature (Burton 1985, Lund 1991) . Consequently, the IC₅₀ values may not represent the real affinity of the peptides to the receptor. However, these data do indicate that the peptides identified in the present invention were able to inhibit the binding of hlgGl to hFcγRI.

Example 7

Multimeric binding assays to hFcγRI, hFcγRIIa, hFcγRIIb, hFcγRIIIa and hFcγRIIIb

The AlphaScreen™ technology ("Amplified Luminescent Proximity Homogeneous Assay", Perkin-Elmer BioSignal, Montreal, Canada) was used to measure a direct interaction between biotinylated peptides 22, 29, 30 and 33 (concentration range between 0.4 nM and 1 μM) or biotinylated hlgGl (concentration range between 0.19 nM and 137 nM) bound to streptavidin donor beads (20 μg/ml) , and Fcγ receptors (20 nM) (hFcγRI, hFcγRIIIb, R&D Systems Inc, Minneapolis, USA, hFcγRIIa, hFcγRIIb, hFcγRIIIa see above) bound to nickel chelate acceptor beads (20 μg/ml) . The reaction mixtures were incubated for 2 h in the dark and the subsequent luminescence was monitored on a Fusion-α microplate analyzer with excitation at 680 nm and emission at 600 nm (Perkin-Elmer BioSignal, Montreal, Canada) . The three stronger signals due to peptide binding were normalized relative to the three stronger signals due to hlgGl binding to the corresponding FcγR.

In this assay a direct interaction between the peptides or hlgGl bound to streptavidin-conjugated donor-bead and hFcγRs conjugated to nickel-chelate acceptor-bead was measured. The apparent EC₅₀ values for hlgGl were estimated at 2 nM, 2 nM,

1OnM, 8 nM and 20 nM for hFcγRI, FcγRIIa, FcγRIIb, FcγRIIIa and FcγRIIIb respectively (data not shown) . Because the AlphaScreen™ assay is based on avidity interactions, these values represent relative affinities. Nevertheless, these results were consistent with the binding avidity values of hFcγRs for aggregated hlgGl, required to activate the low- affinity receptors (Gessner 1998). To determine the level of cross-reactivity of peptides to the different hFcγR isoforms, the three highest binding values of hlgGl on each receptor (which were similar in magnitude for each receptor) served to normalise the three strongest binding values of the peptides (signal peptide/signal hlgGl xlOO) (Fig. 6) . The binding signals of peptides 22, 30 and 33 to hFcγRI were stronger than those of peptide 29 to hFcγRI, which correlated with the ranking order found with the superoxide inhibition assay (Fig. 4) . For the other receptors, the four peptides were unable to recognize these different isoforms. These data indicated that in contrast to hlgGl, the four peptides were specific for hFcγRI . Furthermore, neither linear Hinge peptide nor cyclic Hinge peptide were able to bind any receptors at 10 μM (data not shown) . These observations are in accordance with the functional assay results of Example 8, described below.

In a further experiment, peptides 6, 8, 17 and 42, were shown to bind hFcγRI using the AlphaScreen™ assay. These peptides recognised hFcγRI with a lower avidity than peptides described above (Fig. 8a) .

A direct binding assay between biotinylated peptides 6, 8, 17 and 42 (concentration range between 0.5 nM and 300 μM) and FcγRI was also performed as previously described. To inhibit the interaction between biotinylated IgGl and FcγRI the dimeric peptide 33 competitor were used in a range of concentrations from 200 nM to 45 μM. Monomeric IgGl competitor was used in a range from 2 nM to 1.5 μM. As suggested in the manufacter's instructions, initially acceptor beads (20 mg/ml) and FcγRI (20 nM) were pre-incubated with each other for 30 minutes. Then, peptide or IgGl were added to this preparation for a further 30 minutes. Subsequently, biotinylated IgG (10 nM) and donor beads (20 mg/ml) were added to the reaction mixture for a final incubation of 1 hour. All the manipulations and the measurement were done as previously described.

Example 8

Superoxide burst activation and inhibition assay

To generate multimeric complexes, an excess of 5 nmoles of peptide or 0.5 nmoles of biotinylated-hlgGl were incubated with 1 mg of streptavidin-coated paramagnetic beads (Dynabeads M-280 Streptavidin, Invitrogen, Paisley, UK) in PBS for 30 min at 24°C with gentle shaking. After three washes with PBS, the beads were resuspended in 100 μl of PBS. The final concentration was estimated from the theoretical binding capacity of beads and was approximately 10 μM for the peptides and 400 nM for IgGl. Additionally, one sample containing beads alone was prepared as a control. Superoxide bursts were measured as lucigenin-enhanced chemiluminescence (Pound 1993) . U937 cells were preincubated with γ-IFN (1000 UmI^"1) in RPMI medium supplemented with 10% low IgG FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin for 48 h, to induce differentiation and the capacity to generate superoxide. The cells were washed three times in HBSS (pH 7.4) containing 20 mM HEPES, 0.15 mM BSA and then resuspended at 2 x 10⁶ cells/ml in HBSS/BSA. For the superoxide burst activation assay, 100 μl of U937 cells (2 x 10⁵ cells), 10 μl of lucigenin (2.5 mM) and 10 μl of peptide-bead complexes (1.5 nM to 3.0 μM) or hlgGl-bead complexes (61 pM to 133 nM) were incubated for 5 min at 37°C on order to initiate the assay. Chemiluminescence was monitored in 96-well plate format over a 60 min period using a Berthold LB940 Luminometer. In parallel, 10 μl of soluble peptides (50 μM) or IgGl (0.7 μM) were also added to the previous mixtures.

For the inhibition assay, either 10 μl of SRBC (10⁹ cell/ml) derivatized using NIP-caproate-o-succinimide (0.23 mM)

(Pierce, Rockford, USA) in BBS (100 mM boric acid, 25 mM sodium tetraborate, 75 mM NaCl, pH 8.1) for 1 h at 24°C and sensitised (1 x 10⁸ NIP-RBCs) with 450 nM α-NIP IgGl for 1 h at 37°C or 10 μl of a suboptimal concentration of peptide 33 complexed on beads were incubated with 100 μl of cells and 10 μl of serially diluted soluble peptides (3OnM to 300μM) or IgGl (0.2 nM to 100 nM) . The luminescence was measured as previously. From the seventeen peptide-bead complexes tested, some of them have the ability to activate effector function, and some of them cannot activate (Fig. 2) . Peptides 22, 30 and 33 were the most active. Peptides 24, 29, 31 and peptides 23, 25 and 32 exhibited a weaker activity. All the other peptides tested, as well as linear Hinge peptide did not show any detectable biological activities over the range of concentrations used (1.52 nM to 3.33 μM) . Similar observations were reported for the effect of multimeric control peptide P₂₃oAP ELLGGPSV₂₄O on production of TNF and IL-6 (Medgyesi 2004) . Interestingly, the cyclic Hinge peptide (SEQ ID NO: 25; TAPCAPAPELLGCPSV) corresponding to the cyclic hinge sequence of hlgGl constructed according to the CPEP-8 model, failed to induce superoxide release. In addition, peptide 26, which showed the greatest homology with the lower hinge sequence (the only peptide to harbour the full motif PELLG), was also inactive. These data show that the amino acid composition from the original hinge sequence in a structured scaffold was not sufficient to bind and induce hFcγRI mediated functions.

To show the role of the disulfide bridge in the maintenance of a functional structure, peptides 22 was tested in the absence of this constraint by substitution of Cys residues by Ser residues. In this condition the activity of peptides 22 S₄Si₃ was fully abolished. Based on these results, the strongest peptides 22, 29, 30 and 33 were selected for further studies.

In addition to the peptides described above, peptides 6, 8, 17, 42 complexed on beads were also tested for their ability to trigger superoxide burst. However, in the range of concentrations tested (10 μM was the higher estimated concentration) , no stimulation could have been detected (data not shown) . To show that the activities measured were closely linked to the hFcγR-dependent pathways, we investigated whether superoxide production triggered by multimeric complexes could be inhibited specifically with an excess of the corresponding soluble peptides or hlgGl (Fig. 3) .

The activation by hlgGl-bead complexes could be completely inhibited in the presence of 0.7 μM of soluble hlgGl . The same inhibition profile was obtained with suboptimal activity concentration of peptides 22, 29, 30 and 33 complexed on beads in the presence of 50 mM of soluble peptides 22, 29, 30 and 33 respectively or in presence of hlgGl. Taken together, these results showed that the peptide-bead complexes had the capacity to cross-link hFcγRI at the monocyte cell surface and activate hFcγRI-dependent superoxide production. Hence, when used in complexed form the peptides acted as functional agonists able to activate the hFcγRI; when used in soluble form the peptides could compete efficiently with IgGl on the hFcγRI and inhibit effector function.

As expected, immune complex mediated superoxide production could be inhibited in a dose-dependent manner by addition of soluble hlgGl (IC₅₀ ~5 nM) (Fig. 4a) . The competition inhibition experiments carried out with the four soluble Fc mimetic peptides revealed that at 250 μM, they were all able to fully inhibit superoxide production (Fig. 4b) . In contrast, at the same concentration no inhibition was observed with the irrelevant cyclic peptide. The sigmoid competition curves revealed two sets of peptides: peptide 29 displayed an IC₅₀ value of 20 μM and peptides 22, 30 and 33 showed stronger inhibition with IC₅₀ values included between 1 and 2 μM. These data promoted the hypothesis that the four Fc mimetic peptides bind an identical epitope or overlap the Fc binding site on hFcγRI. These peptides were defined as Fc mimetic peptides. Peptides 6, 17 and 42 were unable to inhibit the superoxide production generated by suboptimal concentration of peptide 33 complexed on beads (Fig. 8b) . This is in contrast to the Fc mimetic soluble peptides that could inhibit superoxide production triggered by multimeric peptide 33. These data suggest that these three peptides are not able to recognise hFcγRI at the same binding site as IgG and Fc mimetic peptides. They correspond to one group of Fc non-mimetic peptides.

The peptides 8, 21, 26, 27, 35, 36 and 37 were unable to trigger effector functions (see above) . They were also tested in their soluble form for their ability to inhibit superoxide, production triggered by a suboptimal concentration of peptide 33 complexed on beads (Fig. 9) . These peptides could inhibit the superoxide production in the range of concentration tested. The IC₅₀ value for peptide 27 was 10 μM, namely in the same order of activity as the active peptide. These peptides correspond to another group of Fc non-mimetic peptides.

In order to assess the minimum structural unit able to trigger effector function, peptide 33 was generated in parallel dimer form comprised of two identical cyclic peptides linked to each other by a short (5-mer) lysine spacer (KKKKK; SEQ ID NO: 22) (sequence AQVNSCLLLPNLLGCSYEKKKKKEYSCGLLNPLLLCNVQA; SEQ ID NO: 23) . We showed that this peptide was able to activate a superoxide burst with an EC₅₀ value « 10 μM (Fig. 10) . SDS- PAGE analysis showed the predominant band at the anticipated molecular weight (4,500), and a minor band (9,000) revealed the presence of a cross-linked species (data not shown) . These data suggested that small multimeric cyclic peptide molecules can cross-link receptors on the monocyte cell surface and consequently trigger effector function. Additionally, to investigate whether the dimer construct retained its ability to recognize FcγRI , an indirect AlphaScreen assay was used to assess the ability of dimer to inhibit IgGl binding to FcγRI. Like IgGl, the dimeric construct retained the ability to abrogate the binding between biotinylated-IgG and FcγRI in a dose-dependent manner (Figure 12) . Then, we showed that the dimeric peptide was efficiently able to induce a superoxide burst with an apparent EC₅₀ value around 2 μM (Figure 13a) . Mass spectrometric analysis confirmed the presence of dimeric peptide 33 at the anticipated molecular mass (4494 Da) (Figure 13b). Furthermore, as expected, IgGl and soluble peptide 33 were both able to inhibit effector functions mediated by dimeric peptide 33 (Figure 13c) . These data suggested that small dimeric cyclic peptide molecules can cross-link receptors on the monocyte cell surface and consequently trigger effector function. Unlike IgGl where the stoichiometry with FcγRI is 1:1, it is plausible that the dimeric peptide binds FcγRI with a ratio 1:2 and so can elicit functional cross-linking of two FcγRI monomers .

It appears that the activity against superoxide production depends closely on the hydrophobic nature of residues present in the core and also on the presence of the proline residue. The net negative charge of the tripeptide sequence flanking the disulfide ring at the carboxy-terminal end seems to play an important function as well. Among the seventeen peptides isolated, ten exhibited at least one acid residue in the carboxy-terminal region, and nine of them were able to trigger the superoxide production. A specific site mutagenesis analysis (Ala residue scanning for example) could be used to determine the functional contribution of each residue at each position (Shields 2001) . Table 3: Summary of superoxide burst activity for Fc mimetic and non-mimetic peptides

Example 9

Rosetting assay

Peptide-bead complexes were used to mimic the immune complexes. Binding of immune complexes on hFcγRs expressed on three human leukocyte cell lines, U937 (previously incubated with γ-IFN at 1000 U ml^"1), K562, Daudi, and primary NK cells was determined by the formation of rosettes. All human leukocyte cell lines were obtained from ATCC (Rockville, USA) . The cell lines were incubated in RPMI-1640 medium with GlutaMAX 1 (supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin) at 37°C under a humidified atmosphere of 5% CO₂/air. Peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coats by centrifugation on a Ficoll gradient (Histopaque-1077; Sigma-Aldrich Company Ltd., Dorset, UK) . Mononuclear cells were removed and gently resuspended in 40 ml of PBS containing 2 mM EDTA. After two successive centrifugations in PBS/EDTA, the residual red blood cells were lysed by resuspending the pellet in ice cold 34 mM NaCl and leaving for 30 sec on ice. After adding an equal volume of ice cold 270 mM NaCl to restore osmolarity and centrifugation, PBMCs were pelleted by centrifugation and resuspended in PBS containing 2mM EDTA and 0.5% BSA (w/v) . NK cells were purified from PBMCs by negative selection using a magnetic bead isolation kit (human NK isolation kit II; Miltenyi Biotech Ltd, Surrey, UK) according to the recommendations of the manufacturer. After elution on an LS column (Miltenyi Biotech), the NK cells were pelleted by centrifugation, counted and re-suspended at 2 x 10⁶ cells/ml in RPMI containing 10% low IgG FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. The cell lines were washed three times in HBSS (pH 7.4) containing 20 mM Hepes and 1% BSA (w/v) and re-suspended at 2 x 10⁶ cells/ml in HBSS/BSA. Molecule-bead complexes (6 x 10⁸ bead/ml) described in the previous section were incubated with each cell type (2 x 10⁶ cells/ml) at a ratio of 100:1 of beads to cells in a final volume of 100 μl of PBS. After 20 min at 24°C, 10 μl of acridine orange (0.002%, w/v) were added and the suspension was gently transferred on a hematocytometer . Rosette formation was assessed under UV/visible illumination (Olympus BX61, Olympus, Tokyo, Japan) and the ability of the cells to bind beads was expressed as the percentage of cells that each bound five or more beads. The assay was measured by counting 200 cells in three replicate analyses.

In this assay, beads formed clusters around cells that could easily be detected with an optical microscope. Since hlgGl- bead complexes can recognize each receptor isoform, they were used as positive controls for each cell interaction. The rosetting profiles with hlgGl-bead complexes correlated well with the results reported for antibody-sensitized RBC (Lund 1991) . Hence, the number of rosettes formed with hlgGl-bead complexes varied between 60% and 100% according to the cell lines used. In this assay, we found that the four peptide-bead complexes were potent and specific effectors of rosette formation mediated via hFcγRI binding (Fig. 6) . Binding for U937 approached 100% of rosettes formed and was as effective as that obtained with IgGl. Taken together, these data showed that the four peptides tested were highly specific for hFcγRI among the different Fcγ receptor classes. Such specific binding may be advantageous to prevent any unwanted effects that could occur as a result of non-specific binding to other FcγRs .

Example 10

Phagocytosis assay and Flow cytometry analysis

U937 cells were preincubated with γ-IFN (1000 unit/ml) in

RPMI-1640 medium with GlutaMAX 1 (supplemented with 10% low IgG FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin) for 48 h. Then, the cells were washed three times in HBSS (pH 7.4) containing 20 mM HEPES, 0.15 mM BSA and resuspended at 2 x 10⁶ cells/ml in HBSS/BSA. 5 nmoles of peptides or 500 nmoles of biotinylated-hlgGl were incubated with 1 mg of streptavidin- coated paramagnetic beads and 0.5 nmoles of Fluorescein-Biotin or 50 nmoles of Fluorescein-Biotin respectively (Invitrogen, Paisley, UK) in PBS for 30 min at 24°C with gentle shaking. After three washes with PBS, the beads were resuspended in 100 μl of PBS. 100 μl of U937 cells (2 x 10⁵ cells), 10 μl of fluorescent molecule-bead complexes, were incubated for 2 h at 37°C in PBS with gentle resuspension every 30 min. In parallel the same samples were also incubated under the same conditions in presence of 10 μl of hlgGl (67 μM) . After vigorous homogenisation, trypan blue (0.4%) was added to the tubes for 1 h at 4°C in order to quench the extracellular fluorescence without affecting that due to ingested complexes. The proportion of cells loaded with fluorescent molecule-bead complexes was determined by using a FACSCalibur flow cytometer (BD Biosciences, Oxford, UK) and the data processed by CellQuest Pro Software. The relative fluorescence intensity of 10,000 cells was measured for each sample. Additionally, internalisation of peptide-bead complexes was visualized using an epifluorescence microscope.

The internalized complexes could easily be visualized under fluorescent and non-fluorescent conditions (Fig. 5A and 5B) . The unquenched fluorescence was monitored by flow cytometry analysis. The strong difference in fluorescence intensity measured between the cells treated with bead FITC-conjugated complexed with IgGl and those treated with bead FITC- conjugated uncomplexed or complexed with irrelevant peptide attested the specificity of phagocytic function. The fluorescence intensity measured for the cells treated with beads complexed with peptide 22 exhibited a signal markedly stronger than that obtained with the irrelevant peptide (SEQ

ID NO:26; THFDTCSWMYCWDGWW) . This specific signal was close to the value obtained with hlgGl-bead complexes. Consequently, these data indicated that the complexes formed with peptide 22 could significantly stimulate the phagocytosis of monocytes. The beads treated with peptides 29, 30, and 33 exhibited exactly the same intensity profiles as that obtained with peptide 22 (data not shown) . In addition, in the presence of 0.7 μM hlgGl, the signal intensity obtained with beads complexed with peptide 22 could be abolished and reduced to that of the negative control. This same inhibition profile was seen with the other peptide-bead complexes (data not shown) . The signal generated from hlgGl-bead complexes was also significantly diminished in the presence of soluble hlgGl. Thereby, these data indicated that the peptide-bead complexes could specifically bind FcγRI and activate FcγRI-mediated phagocytosis in the U937 monocyte cell line.

Example 11

Molecular Modelling of an Fc mimetic peptide

A sequence similarity search was carried out for peptide 33 in the PDB database using the algorithm BLAST2 (Ryckaert 1977) . The most similar cyclic peptide (code lsβw) corresponded to the synthetic hepcidin peptide from Hybrid White Striped Bass (Lauth 2005) . The NMR structure of this peptide was used for building a homology model using the program PRIME

(Schrδdinger, LLC: Portland, OR) . After the initial model was built the program suite AMBER8 (Case 2004) was used to perform a short molecular dynamics simulation (MD) in explicit water. The system was solvated by adding a cubic box of pre- equilibrated TIP3P water, and then minimized (conjugate gradient) and equilibrated using the Langevin temperature equilibration scheme and SHAKE (Ryckaert 1977) constraints on hydrogen atoms. During the simulation temperature and pressure were maintained constant at 300 K and 1 atm. The simulation was run for a total of 2ns, and the structure reached stability after 1.3 ns . The average structure over the final 700ps was calculated and minimized. Residues LeulO to Glyl2 of the peptide were superimposed onto the motif Leu234-Gly237 of the B-chain of the Fc fragment, while Leull and Pro8 were superimposed onto Leu235 and Pro239 of the A-chain of the Fc fragment. In both cases we used the crystal structure of Fc in complex with FcγRIII (Sondermann 2000) . A homology model of FcγRI was built and refined using the program PRIME based on the sequence and structure of hFcγRIII-Fc complex. Receptor- peptide complexes were then generated and their free energy of binding was minimized using Macromodel .

The FcγR family shares a high degree of homology in the sequence and the structure of its ectodomains. Based on the published crystal structure for the FcγRIII-Fc complex (Sondermann 2000) a model for the FcγRI-peptide complex has been generated. Peptide 33 was modelled based on the structure of a homologous cysteine-containing peptide and subsequently minimised for free energy following superposition onto the predominant recognition motif Leu234-Gly237 from Fc-A and Leu235 and Pro329 from Fc-B chains. The energy minimisation of the peptide complexed within receptors generated two slightly different conformers (Fig. 11a and lib) .

Pro8 residue of the A-chain conformer makes multiple hydrophobic contacts with residues Trp87 and TrpllO from FcγRI, resembling the interaction between Pro329 of Fc with these two tryptophan residues in both the FcγRIII/Fc-A complex and in the FcγRI/Fc-A model of Sonderman (2001) (Fig. lid) . Additionally, the interaction of Leull with Leull4 resembles the interaction reported between Leu235 of the lower hinge and Leull4 in the FcγRI/Fc-A model and Leu235 and Alall4 for the FcγRIII/Fc-A complex (Sonderman 2001) . In the case of the B- chain conformer, residue Leull makes hydrophobic interactions with Tyrllβ, Hisl31 and Trpl32 of FcγRI (Fig. lie) . A hydrophobic region of FcγRIII defined by histidine residues 116, 131 and 132 that contacts Leu235 of Fc-B has been reported previously (Sonderman, 2001). Additionally there are some potential hydrogen bonds for Pro8 and LeulO of the peptide with Hisl58 and Asnll7 of FcγRI respectively. The lack of favourable interactions that Leull from both conformers makes with FcγRI II may contribute to the absence of binding of peptide 33 with FcγRII I (data not shown) .

The models give insight into the specificity observed for preferential binding of the cyclic peptides to FcγRI rather than to FcγRIII. By comparison with FcγRI, the docking of each cyclic peptide conformer with FcγRIII did not reveal such energetically favourable interactions.

Sequences

Sequences of peptides are shown in the appended sequence listing, in which SEQ ID NOs correspond as follows:

PRT = amino acid sequence

1 cp22 amino acid

2 cp21 amino acid

3 cp23 amino acid

4 cp24 amino acid

5 cp33 amino acid

6 cp25 amino acid

7 cp26 amino acid

8 cp27 amino acid

9 cp32 amino acid

10 cp34 amino acid

11 p35 amino acid

12 cp28 amino acid

13 cp29 amino acid

14 p37 amino acid

15 cp30 amino acid

16 cp31 amino acid

17 cp36 amino acid

18 cp6 amino acid

19 cpl7 amino acid

20 cp42 amino acid

21 cp8 amino acid

22 lysine linker

23 cp33 dimer amino acid

24 p hinge amino acid

25 cyclic hinge CH2 peptide amino acid

26 irrelevant peptide amino acid

27 primer 1

28 primer 2

29 primer 3 30 primer FdTetSeq

31 hlgGl residues 225-240

32 lower hinge control peptide (Lund 1991)

33 lower hinge control peptide (Sheridan 1999) 34 lower hinge control peptide (Berntzen 2006)

35 consensus peptide sequence

36 peptide motif

37 peptide motif

38 peptide motif 39 peptide plgGl (Radaev 2001b)

40 peptide pIgG2 (Radaev 2001b)

41 peptide p!gG4 (Radaev 2001b)

Specific embodiments:

1. A peptide that competes with an Fc fragment of an IgG for binding to Fcγ receptor, wherein the peptide may activate or inhibit Fc effector function.

2. The peptide according to embodiment 1, wherein the peptide is an Fc mimetic peptide and wherein in a first, complexed form the peptide activates Fc effector function and in a second, soluble form the peptide inhibits Fc effector function.

3. The peptide according to embodiment 1, wherein the peptide is an Fc non-mimetic peptide and wherein in a first, complexed form does not activate Fc effector function and in a second, soluble form the peptide inhibits Fc effector function.

4. The peptide according to any one of the preceding embodiments, wherein the Fcγ receptor is selected from FcγRI , FcγRI I or FcγRIII.

5. The peptide according to any one of the preceding embodiments, wherein the Fcγ receptor is a human receptor.

6. The peptide according to any one of the preceding embodiments, wherein the Fcγ receptor is human FcγRI (hFcγRI) .

7. The peptide according to any one of the preceding embodiments, wherein the Fc effector function is phagocytosis.

8. The peptide according to any one of the preceding embodiments, wherein the peptide competes with the Fc portion of an IgG3 or IgGl. 9. The peptide according to any one of the preceding embodiments, wherein the peptide competes with the Fc portion of IgGl.

10. The peptide according to any one of the preceding embodiments, wherein the peptide is conjugated to a particle.

11. The peptide according to any one of the preceding embodiments, wherein the peptide comprises a disulfide bridge for generation of a cyclic peptide ring.

12. The peptide according to any one of the preceding embodiments, wherein the peptide comprises a tripeptide motif of LLG.

13. The peptide according to any one of the preceding embodiments, wherein the peptide comprises a dimer of motif ςP and wherein ς represents a hydrophobic residue.

14. The peptide according to any one of the preceding embodiments, wherein the peptide comprises threonine at an N- terminus of the peptide.

15. The peptide according to any one of the preceding embodiments, wherein the peptide comprises glutamic acid at a C-terminus of the peptide.

16. The peptide according to any one of the preceding embodiments wherein the peptide comprises an amino acid sequence of TX₂CXXςPXLLGCφXE and wherein, X is any amino acid, ς is a hydrophobic residue and φ is an acidic amino acid.

17. The peptide according to any one of the preceding embodiments, wherein the peptide binds specifically to hFcγRI and does not exhibit cross-reactivity with hFcγRII or hFcγRIII.

18. The Fc mimetic peptide according to any one of embodiments 1, 2, 4-17, selected from one of the following:

SEQ ID No Clone ID Sequence

1 cp 22 TDT C LMLPLLLG C DEE

3 cp 23 WYP C YIYPRLLG C DGD

4 cp 24 GNI C MLIPGLLG C SYE

5 cp 33 VNS C LLLPNLLG C GDD

6 cp 25 TPV C ILLPSLLG C DTQ

9 cp 32 FGT C YTWPWLLG C EGF

13 cp 29 SHP C GRLPMLLG C AES

15 cp 30 KDP C TRWAMLLG C DGE

16 cp 31 IMT C SVYPFLLG C VDK

19. The peptide according to embodiment 18, having an amino acid sequence selected from SEQ ID NOs: 1, 5, 13 and 15.

20. A peptide comprising two or more peptides according to embodiments 1, 4-9, 11-13, 16 and 17, linked to each other to generate a dimeric or oligomeric peptide.

21. The dimeric peptide according to embodiment 20, wherein the peptide binds to two hFcγRIs and activates Fc effector function.

22. The dimeric peptide according to embodiment 20 or 21, comprising two Fc mimetic peptides linked by a KKKKK linker (SEQ ID NO: 22) . 23. The dimeric peptide according to any one of embodiments 20 to 22 having amino acid sequence AQVNSCLLLPNLLGCSYEKKKKKESYCGLLNPLLLCSNVQA (SEQ ID NO: 23) .

24. The Fc non-mimetic peptide according to any one of embodiments 1, 3-17, selected from one of the following:

25. A method for identifying a peptide that binds to an FcγR comprising: i) selecting a peptide by IgG competition elution during phage display; ii) assessing binding specificity and functionality of the peptide using biotinylated peptide conjugated to streptavidin- coated paramagnetic beads.

26. The method according to embodiment 25, wherein the functionality of the peptide is assessed by activation of a superoxide burst or phagocytosis in a human leukocyte cell line .

27. The method according to embodiment 25 or embodiment 26, wherein binding specificity of the peptide is assessed by determining rosette formation on binding to cells displaying Fcγ receptors. 28. A nucleic acid encoding the Fc mimetic or non-mimetic peptide according to any one of embodiments 1 to 24.

29. A vector comprising the nucleic acid according to embodiment 28.

30. A host cell comprising the vector according to embodiment 29.

31. A method of activating FcγRI comprising contacting FcγRI with a composition that comprises an Fc mimetic peptide having an amino acid sequence of TX₂CXXςPXLLGCφXE and wherein, X is any amino acid, ς is a hydrophobic residue and φ is an acidic amino acid.

32. A peptide conjugate comprising a peptide according to any one of embodiments 1 to 24 conjugated to an antibody or antibody fragment.

33. A peptide conjugate according to embodiment 32, wherein the antibody fragment comprises a Fab, Fab', Fab' -SH, scFv, Fv, dAb or Fd.

34. A composition comprising a peptide according to any one of embodiments 1 to 24 or a peptide conjugate according to embodiments 32 or 33 and a pharmaceutically acceptable excipient .

35. A composition comprising a peptide according to any one of embodiments 1 to 24 or a peptide conjugate according to embodiments 32 or 33 for use in a method of treatment of the human or animal body by surgery or therapy. 36. The composition according to embodiment 35 for use in treating a disorder associated with Fc effector function.

37. Use of a peptide according to any one of embodiments 1 to 24 or a peptide conjugate according to embodiments 32 or

33, for the manufacture of a medicament for treating a disorder associated with Fc effector function.

38. The composition according to embodiment 36 or use according to embodiment 37, wherein the disorder is a tumour or cancer.

39. The composition according to embodiment 36 or use according to embodiment 37, wherein the disorder is infection.

40. The composition or use according to embodiment 39, wherein the infection is a viral infection responsible for antibody dependent enhancement or causes pathogen expression of Fc receptor in an infected cell.

41. The composition according to embodiment 36 or use according to embodiment 37, wherein the disorder is an inflammatory, allergic or autoimmune disorder.

42. The composition according to embodiment 36 or use according to embodiment 37, for modulation of the balance between activation and inhibition of effector function.

43. A method of treating a disorder associated with Fc effector function, comprising administering a peptide according to any one of embodiments 1 to 24 or a peptide conjugate according to embodiments 32 or 33, to an individual.

44. The method according to embodiment 43, wherein the disorder is a tumour or cancer. 45. The method according to embodiment 43, wherein the disorder is infection.

46. The method according to embodiment 45, wherein the infection is a viral infection responsible for antibody dependent enhancement or causes pathogen expression of Fc receptor in an infected cell.

47. The method according to embodiment 43, wherein the disorder is an inflammatory, allergic or autoimmune disorder.

48. The method according to embodiment 43, for modulation of the balance between activation and inhibition of effector function.

49. The peptide according to any one of embodiments 1 to 24 or the peptide conjugate of embodiments 32 or 33 for use in a method of diagnosis of a disorder of the human or animal body.

50. The peptide or peptide conjugate according to embodiment 49, wherein the disorder is a tumour or cancer.

51. The peptide or peptide conjugate according to embodiment 49, wherein the disorder is an infection.

52. The peptide or peptide conjugate according to embodiment 49, wherein the disorder is an inflammatory, allergic or autoimmune disorder. References

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Claims

Claims

1. A peptide that competes with an Fc fragment of an IgG for binding to Fcγ receptor, wherein the peptide may activate or inhibit Fc effector function.

2. The peptide according to claim 1, wherein the peptide is an Fc mimetic peptide and wherein in a first, complexed form the peptide activates Fc effector function and in a second, soluble form the peptide inhibits Fc effector function.

3. The peptide according to claim 1, wherein the peptide is an Fc non-mimetic peptide and wherein in a first, complexed form does not activate Fc effector function and in a second, soluble form the peptide inhibits Fc effector function.

4. The peptide according to any one of the preceding claims, wherein the Fcγ receptor is selected from FcγRI, FcγRII or FcγRII I.

5. The peptide according to any one of the preceding claims, wherein the Fcγ receptor is a human receptor.

6. The peptide according to any one of the preceding claims, wherein the Fcγ receptor is human FcγRI (hFcγRI) .

7. The peptide according to any one of the preceding claims, wherein the Fc effector function is phagocytosis.

8. The peptide according to any one of the preceding claims, wherein the peptide competes with the Fc portion of an IgG3 or IgGl.

9. The peptide according to any one of the preceding claims, wherein the peptide competes with the Fc portion of IgGl.

10. The peptide according to any one of the preceding claims, wherein the peptide is conjugated to a particle.

11. The peptide according to any one of the preceding claims, wherein the peptide comprises a disulfide bridge for generation of a cyclic peptide ring.

12. The peptide according to any one of the preceding claims, wherein the peptide comprises a tripeptide motif of LLG.

13. The peptide according to any one of the preceding claims, wherein the peptide comprises a dimer of motif ςP and wherein ς represents a hydrophobic residue.

14. The peptide according to any one of the preceding claims, wherein the peptide comprises threonine at an N- terminus of the peptide.

15. The peptide according to any one of the preceding claims, wherein the peptide comprises glutamic acid at a C- terminus of the peptide.

16. The peptide according to any one of the preceding claims wherein the peptide comprises an amino acid sequence of TX₂CXXςPXLLGCφXE and wherein, X is any amino acid, ς is a hydrophobic residue and φ is an acidic amino acid.

17. The peptide according to any one of the preceding claims, wherein the peptide binds specifically to hFcγRI and does not exhibit cross-reactivity with hFcγRII or hFcγRI II.

18. The Fc mimetic peptide according to any one of claims 1, 2, 4-17, selected from one of the following:

19. The peptide according to claim 18, having an amino acid sequence selected from SEQ ID NOs: 1, 5, 13 and 15.

20. A peptide comprising two or more peptides according to claims 1, 4-9, 11-13, 16 and 17, linked to each other to generate a dimeric or oligomeric peptide.

21. The dimeric peptide according to claim 20, wherein the peptide binds to two hFcγRIs and activates Fc effector function.

22. The dimeric peptide according to claim 20 or 21, comprising two Fc mimetic peptides linked by a KKKKK linker (SEQ ID NO: 22) .

23. The dimeric peptide according to any one of claims 20 to 22 having amino acid sequence AQVNSCLLLPNLLGCSYEKKKKKESYCGLLNPLLLCSNVQA (SEQ ID NO: 23) .

24. The Fc non-mimetic peptide according to any one of claims 1, 3-17, selected from one of the following:

25. A method for identifying a peptide that binds to an FcγR comprising: i) selecting a peptide by IgG competition elution during phage display; ii) assessing binding specificity and functionality of the peptide using biotinylated peptide conjugated to streptavidin- coated paramagnetic beads.

26. The method according to claim 25, wherein the functionality of the peptide is assessed by activation of a superoxide burst or phagocytosis in a human leukocyte cell line .

27. The method according to claim 25 or claim 26, wherein binding specificity of the peptide is assessed by determining rosette formation on binding to cells displaying Fcγ receptors .

28. A nucleic acid encoding the Fc mimetic or non-mimetic peptide according to any one of claims 1 to 24.

29. A vector comprising the nucleic acid according to claim 28.

30. A host cell comprising the vector according to claim 29.

31. A method of activating FcγRI comprising contacting FcγRI with a composition that comprises an Fc mimetic peptide having an amino acid sequence of TX₂CXXςPXLLGCφXE and wherein, X is any amino acid, ς is a hydrophobic residue and φ is an acidic amino acid.

32. A peptide conjugate comprising a peptide according to any one of claims 1 to 24 conjugated to an antibody or antibody fragment.

33. A peptide conjugate according to claim 32, wherein the antibody fragment comprises a Fab, Fab', Fab' -SH, scFv, Fv, dAb or Fd.

34. A composition comprising a peptide according to any one of claims 1 to 24 or a peptide conjugate according to claims

32 or 33 and a pharmaceutically acceptable excipient.

35. A composition comprising a peptide according to any one of claims 1 to 24 or a peptide conjugate according to claims 32 or 33 for use in a method of treatment of the human or animal body by surgery or therapy.

36. The composition according to claim 35 for use in treating a disorder associated with Fc effector function.

37. Use of a peptide according to any one of claims 1 to 24 or a peptide conjugate according to claims 32 or 33, for the manufacture of a medicament for treating a disorder associated with Fc effector function.

38. The composition according to claim 36 or use according to claim 37, wherein the disorder is a tumour or cancer.

39. The composition according to claim 36 or use according to claim 37, wherein the disorder is infection.

40. The composition or use according to claim 39, wherein the infection is a viral infection responsible for antibody dependent enhancement or causes pathogen expression of Fc receptor in an infected cell.

41. The composition according to claim 36 or use according to claim 37, wherein the disorder is an inflammatory, allergic or autoimmune disorder.

42. The composition according to claim 36 or use according to claim 37, for modulation of the balance between activation and inhibition of effector function.

43. A method of treating a disorder associated with Fc effector function, comprising administering a peptide according to any one of claims 1 to 24 or a peptide conjugate according to claims 32 or 33, to an individual.

44. The method according to claim 43, wherein the disorder is a tumour or cancer.

45. The method according to claim 43, wherein the disorder is infection.

46. The method according to claim 45, wherein the infection is a viral infection responsible for antibody dependent enhancement or causes pathogen expression of Fc receptor in an infected cell.

47. The method according to claim 43, wherein the disorder is an inflammatory, allergic or autoimmune disorder.

48. The method according to claim 43, for modulation of the balance between activation and inhibition of effector function .

49. The peptide according to any one of claims 1 to 24 or the peptide conjugate of claims 32 or 33 for use in a method of diagnosis of a disorder of the human or animal body.

50. The peptide or peptide conjugate according to claim 49, wherein the disorder is a tumour or cancer.

51. The peptide or peptide conjugate according to claim 49, wherein the disorder is an infection.

52. The peptide or peptide conjugate according to claim 49, wherein the disorder is an inflammatory, allergic or autoimmune disorder.