WO2007099346A1

WO2007099346A1 - Binding members for ghrelin

Info

Publication number: WO2007099346A1
Application number: PCT/GB2007/000741
Authority: WO
Inventors: Bryan Michael Edwards; Fraser Ewing Welsh; Melanie Boyle; Steven Godfrey Lane; Philip Antony Bland-Ward; Matthew Alexander Sleeman
Original assignee: Cambridge Antibody Technology Ltd
Current assignee: MedImmune Ltd
Priority date: 2006-03-03
Filing date: 2007-03-05
Publication date: 2007-09-07
Anticipated expiration: 2008-09-03

Abstract

This invention relates to binding members for ghrelin, in particular anti-acyl ghrelin antibody molecules, especially human antibody molecules comprising the sequences set out herein, and especially those that neutralise acyl-ghrelin activity. Anti-ghrelin antibody molecules of the invention may be used in the diagnosis or treatment of ghrelin-related disorders, including obesity.

Description

BINDING MEMBERS FOR GHRELIN

FIELD OF INVENTION

The present invention relates to binding members for ghrelin, in particular anti-acyl ghrelin antibody molecules, especially human antibody molecules, and especially those that neutralise acyl-ghrelin activity. It further relates to methods for using anti-ghrelin antibody molecules in diagnosis or treatment of ghrelin-related disorders, including obesity.

The present invention provides antibody molecules of particular value in binding and neutralising ghrelin, and thus of use in any of a variety of therapeutic treatments, as indicated by the experimentation contained herein and further by the supporting technical literature.

BACKGROUND

Obesity is becoming increasingly prevalent in modern society and there is an urgent need for novel treatments that induce weight loss. The cost of treating obesity-related disease such as diabetes, cardiovascular disease and osteoarthritis will continue to rise until safe and effective methods of achieving significant and sustained weight loss become available. Current drug treatments offer only modest efficacy in terms of weight loss and duration of effect, and are associated with undesirable side effects. Significant long term weight loss is achievable with surgical treatment but such methods carry significant risk of mortality and are reserved for the morbidly obese.

Hormones and neurotransmitter substances that physiologically modulate feeding behaviour and energy homeostasis are key targets for the development of novel anti-obesity therapies. Therefore, pharmacological agents that disrupt the action of the hormone ghrelin could provide an effective method of controlling body weight. Ghrelin is a peptide hormone produced principally by the endocrine cells of the stomach and lower gastrointestinal tract. The predominant active form of ghrelin, a 28 amino acid peptide, is cleaved from a 117 amino acid precursor protein. A shorter 27 amino acid ghrelin isoform has also been described (Hosoda, Kojima et al, 2000) . This 27 amino acid splice variant lacks residue Glutamine 14 and is also known as Des-Gln Ghrelin. Functional activity at the ghrelin receptor is conferred by post-translational modification at the serine residue in position 3 (Ser3) of the ghrelin molecule. This modification is also seen in the splice variant Des-Gln Ghrelin. Thus, acyl-ghrelin (octanoyl-ghrelin) is the primary active form of modified endogenous ghrelin in humans and represents approx 10% of total circulating ghrelin in humans, with the majority present in unmodified (desacylated) form. However, decanoylated and decenoylated ghrelins have also been described but these represent only small fraction of modified ghrelin (Hosoda, Kojima et al, 2003) . Structure-activity studies have shown that unmodified ghrelin has negligible affinity for the ghrelin receptor, whilst modification at Ser3 with bulky aliphatic additions is optimal for agonistic activity (Bednarek et al., 2000). The endogenous enzyme (s) responsible for conversion of des-acyl ghrelin to acyl ghrelin is currently unknown. Ghrelin is produced mainly in the GI tract where the concentration of ghrelin is greatest in the stomach fundus and decreases progressively toward the colon. Ghrelin immunoreactive cells have also been identified in the arcuate nucleus, an area of the hypothalamus known to regulate appetite.

The ghrelin receptor is the growth hormone secretagogue receptor type Ia (GHSRIa) . GHSRla is a 7TM protein localised in the hypothalamus, pituitary and hippocampus (Guan, Yu et al. 1997) . There is lower expression in the pancreas, thyroid, spleen, myocardium and adrenal gland. GHSRla is also abundantly expressed on afferent vagal neurons (Date, Murakami et al. 2002) . It is likely that the orexigenic effects of ghrelin are mediated by activation of GHSRla on hypothalamic arcuate neurones expressing NPY and AgRP, with additional effects mediated by activation of vagal afferents. In rodent studies antagonists of NPY and AgRP inhibit the orexigenic action of ghrelin

(Nakazato, Murakami et al . 2001; Asakawa, Inui et al. 2001; Shintani, Ogawa et al. 2001), and vagotomy abrogates the stimulatory effects of ghrelin on feeding behaviour (Date, Murakami et al. 2002).

The physiological effects of ghrelin include stimulation of appetite, and increased gastric motility and gastric acid secretion (Kojima and Kangawa, 2001) . In humans, serum levels of ghrelin increase before meals and fall rapidly after food ingestion (Cummings, Purnell et al. 2001). In rats, ghrelin suppresses fat utilisation and increases glycolysis (Tschop, Smiley et al. 2000). Administration of exogenous ghrelin results in weight gain in rodents (Tschop, Smiley et al. 2000) and stimulates food intake in healthy humans whilst reducing postprandial energy expenditure (Wren, Seal et al. 2001). Certain clinical observations also support an association between body weight and the level of circulating ghrelin. Ghrelin levels are lowered in morbidly obese patients who have undergone gastric by-pass surgery (Cummings, Weigle et al. 2002). Individuals with Prader-Willi Syndrome, a condition characterised by hyperphagia and obesity, display levels of serum ghrelin considerably higher than those of appropriately matched control subjects Cummings, Clement et al., 2002) .

Detailed study of ghrelin null mice has shown that ghrelin deletion has no impact on appetite, size, growth rate, food intake, body composition or gross behaviour when compared to wild-type controls (Sun et al. 2003) . However, mice lacking the ghrelin receptor do not develop diet-induced obesity and, when fed a high fat diet, store less energy and preferentially burn fat as an energy substrate (Zigman et al, 2005) . GHSR antagonists reduce food intake in lean and obese mice (Asakawa et al., 2003) and polyclonal anti-ghrelin IgG have been shown to suppress feeding in rodents (Nakazato, Murakami et al. 2001).

WO 2005/016951A2 describes the use of murine, chimeric, humanised and deimmunised monoclonal antibodies. US 6,291,653 Bl describes generally antibodies to ghrelin with affinities of InM or greater and their uses in a range of therapeutic indications including obesity. Purified rabbit anti-ghrelin sera have been used to suppress feeding in rats (Nakazato, Murakami et al . 2001 Nature. 2001 Jan 11; 409(6817): 194- 8) .

SUMMARY OF INVENTION

The present invention provides binding members for ghrelin, in particular acyl-ghrelin i.e. ghrelin which is octanoylated at serine 3. Preferably, the binding members bind human ghrelin. Thus, a binding member of the invention may bind human ghrelin or non-human ghrelin (e.g. non-human primate ghrelin and/or rat ghrelin and/or mouse ghrelin) .

Binding members of the invention may be antibodies to human acyl- ghrelin, especially human antibodies, which may be cross-reactive with non-human acyl-ghrelin, including non-human primate acyl-ghrelin and/or mouse acyl-ghrelin and/or rat acyl-ghrelin.

A binding member in accordance with the present invention preferably neutralises ghrelin. Neutralisation means reduction or inhibition of biological activity of ghrelin, e.g. reduction or inhibition of ghrelin binding to one or more of its receptors (preferably, growth hormone secretagogue receptor Ia (GHSRIa) ) . The reduction in biological activity may be partial or total. The degree to which an antibody neutralises ghrelin is referred to as its neutralising potency. Potency may be determined or measured using one or more assays known to the skilled person and/or as described or referred to herein. For example, a suitable assay may comprise determining the ability of a binding member to modulate, for example promote or inhibit, Ca²⁺ signalling evoked by acyl ghrelin in cells expressing GHSRl. Other suitable assays may comprise determining the ability of a binding member to modulate the acyl ghrelin mediated differentiation of adipocytes, proliferation of hypothalamic neurons, inpsitol phosphate accumulation or beta lactamase activity.

Assays and potencies are described in more detail elsewhere herein.

Binding members of the present invention may be optimised for neutralising potency. Generally, potency optimisation involves mutating the sequence of a selected binding member (normally the variable domain sequence of an antibody) to generate a library of s binding members, which are then assayed for potency and the more potent binding members are selected. Thus selected "potency- optimised" binding members tend to have a higher potency than the binding member from which the library was generated. Nevertheless, high potency binding members may also be obtained without optimisation, for example a high potency binding member may be obtained directly from an initial screen e.g. a biochemical neutralisation assay. The present invention provides both potency- optimised and non-optimised binding members, as well as methods for potency optimisation from a selected binding member. The present invention thus allows the skilled person to generate binding members having high potency.

A binding member may inhibit the ability of acyl-ghrelin to induce Ca signaling and/or inhibit the binding of acyl-ghrelin to GHSRIa.

A binding member in accordance with the present invention preferably has one or more of the following biological activities in a mammal: appetite suppression, gastric motility reduction, gastric acid secretion reduction, fat utilization increase and glycolysis reduction.

In some embodiments, a binding member of the invention comprises an antibody molecule. In other embodiments, a binding member of the invention comprises an antigen-binding site within a non-antibody molecule, e.g. a set of CDRs in a non-antibody protein scaffold, as discussed further below.

In various aspects and embodiments of the invention there is provided the subject-matter of the claims included below.

Preferred embodiments within the present invention are antibody molecules, whether whole antibody (e.g. IgG, such as IgG4) or antibody fragments (e.g. scFv, Fab, dAb) . Preferably, an antibody molecule of the invention is a human antibody molecule. Antibody molecules comprising antibody antigen-binding sites are provided, as are antibody VH and VL domains . Within VH and VL domains are provided complementarity determining regions, ("CDRs") , and framework regions, ("FRs") , to form VH or VL domains as the case may be. An antibody antigen-binding site may consist of an antibody VH domain and/or a VL domain. All VH and VL sequences, CDR sequences, sets of CDRs and sets of HCDRs and sets of LCDRs disclosed herein represent aspects and embodiments of the invention. A "set of CDRs" comprises CDRl, CDR2 and CDR3. Thus, a set of HCDRs means HCDRl, HCDR2 and HCDR3, and a set of LCDRs means LCDRl, LCDR2 and LCDR3. Unless otherwise stated, a "set of CDRs" includes HCDRs and LCDRs.

Examples of antibody VH and VL domains and CDRs according to the present invention are as listed in the appended sequence listing. A number of antibody lineages are disclosed herein, defined with reference to sequences, e.g. a set of CDR sequences, optionally with one or more, e.g. one or two substitutions. In some embodiments, the preferred parent lineage is the CPlAl lineage. The present inventors have identified the CPlAl lineage as providing human antibody antigen- binding sites against acyl-ghrelin that are of particular value.

The CPlAl lineage is defined with reference to a set of six CDR sequences of CPlAl as follows: HCDRl SEQ ID NO: 3, HCDR2 SEQ ID NO: 4, HCDR3 SEQ ID NO: 5, LCDRl SEQ ID NO: 8, LCDR2 SEQ ID NO: 9, and LCDR3 SEQ ID NO: 10. The set of CDRs wherein the HCDRl has the amino acid sequence of SEQ ID NO: 3, the HCDR2 has the amino acid sequence of SEQ ID NO: 4, the HCDR3 has the amino acid sequence of SEQ ID NO: 5, the LCDRl has the amino acid sequence of SEQ ID NO: 8, the LCDR2 has the amino acid sequence of SEQ ID NO: 9, and the LCDR3 has the amino acid sequence of SEQ ID NO: 10, are herein referred to as the "CPlAl set of CDRs". The HCDRl, HCDR2 and HCDR3 within the CPlAl set of CDRs are referred to as the "CPlAl set of HCDRs" and the LCDRl, LCDR2 and LCDR3 within the CPlAl set of CDRs are referred to as the "CPlAl set of LCDRs". A set of CDRs with the CPlAl set of CDRs, CPlAl set of HCDRs or CPlAl LCDRs, or one or two substitutions therein, is said to be of the CPlAl lineage.

Other lineages and sets of CDRs are defined with reference to the analogous CDRs set out herein, where HCDRl is SEQ ID NO: 10N+3, HCDR2 is SEQ ID NO: 10N+4, HCDR3 is SEQ ID NO: 10N+5, LCDRl is SEQ ID NO: 10N+8, LCDR2 is SEQ ID NO: 10N+9, and LCDR3 is SEQ ID NO: 10N+ 10, and N is any interger from 0 to 11.

An antibody lineage of the invention may, for example, comprise a set of CDRs wherein the HCDRl has the amino acid sequence of any one of SEQ ID NOS: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103 and 113, the HCDR2 has the amino acid sequence of any one of SEQ ID NOS: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104 and 114, the HCDR3 has the amino acid sequence any one of SEQ ID NOS: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, and 115, the LCDRl has the amino acid sequence of any one of SEQ ID NOS: 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, and 118, the LCDR2 has the amino acid sequence of any one of SEQ ID NOS: 9, 19, 29, 39, 49, 59, 69, 79, 89, 99, 109, and 119, and the LCDR3 has the amino acid sequence of any one of SEQ ID NOS: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, and 120; or may comprise a set of CDRs which contains one or more amino acid substitutions, deletions or insertions, for example one or two amino acid substitutions, compared with such a set of CDRs.

Other lineages may for example include the DP47G7, CPlCl, CPlBl, BMV1D8, BMV2D6, BMV2E4, G008H05, G001C06, G041C10, GOOlElI and GOOlCIl lineages.

The DP47G7 lineage is defined with reference to a set of six CDR sequences of DP47G7 as follows: HCDRl SEQ ID NO: 13, HCDR2 SEQ ID NO: 14, HCDR3 SEQ ID NO: 15, LCDRl SEQ ID NO: 18, LCDR2 SEQ ID NO: 19 and LCDR3 SEQ ID NO: 20.

The CPlCl lineage is defined with reference to a set of six CDR sequences of CPlCl as follows: HCDRl SEQ ID NO: 23, HCDR2 SEQ ID NO: 24, HCDR3 SEQ ID NO: 25, LCDRl SEQ ID NO: 28, LCDR2 SEQ ID NO: 29, and LCDR3 SEQ ID NO: 30.

The CPlBl lineage is defined with reference to a set of six CDR sequences of CPlBl as follows: HCDRl SEQ ID NO: 33, HCDR2 SEQ ID NO: 34, HCDR3 SEQ ID NO: 35, LCDRl SEQ ID NO: 38, LCDR2 SEQ ID NO: 39 and LCDR3 SEQ ID NO: 40.

The BMV1D8 lineage is defined with reference to a set of six CDR sequences of BMV1D8 as follows: HCDRl SEQ ID NO: 43, HCDR2 SEQ ID NO: 44, HCDR3 SEQ ID NO: 45, LCDRl SEQ ID NO: 48, LCDR2 SEQ ID NO: 49 and LCDR3 SEQ ID NO: 50.

The BMV2D6 lineage is defined with reference to a set of six CDR sequences of BMV2D6 as follows: HCDRl SEQ ID NO: 53, HCDR2 SEQ ID NO: 54, HCDR3 SEQ ID NO: 55, LCDRl SEQ ID NO: 58, LCDR2 SEQ ID NO: 59, and LCDR3 SEQ ID NO: 60.

The BMV2E4 lineage is defined with reference to a set of six CDR sequences of BMV2E4 as follows: HCDRl SEQ ID NO: 63, HCDR2 SEQ ID NO: 64, HCDR3 SEQ ID NO: 65, LCDRl SEQ ID NO: 68, LCDR2 SEQ ID NO: 69, and LCDR3 SEQ ID NO: 70.

The G008H05 lineage is defined with reference to a set of six CDR sequences of G008H05 as follows: HCDRl SEQ ID NO: 73, HCDR2 SEQ ID NO: 74, HCDR3 SEQ ID NO: 75, LCDRl SEQ ID NO: 78, LCDR2 SEQ ID NO: 79, and LCDR3 SEQ ID NO: 80.

The G001C06 lineage is defined with reference to a set of six CDR sequences of G001C06 as follows: HCDRl SEQ ID NO: 83, HCDR2 SEQ ID NO:

84, HCDR3 SEQ ID NO: 85, LCDRl SEQ ID NO: 88, LCDR2 SEQ ID NO: 89, and LCDR3 SEQ ID NO: 90.

The G041C10 lineage is defined with reference to a set of six CDR sequences of G041C10 as follows: HCDRl SEQ ID NO: 93, HCDR2 SEQ ID NO: 94, HCDR3 SEQ ID NO: 95, LCDRl SEQ ID NO: 98, LCDR2 SEQ ID NO: 99, and LCDR3 SEQ ID NO: 100.

The GOOlElI lineage is defined with reference to a set of six CDR sequences of GOOlElI as follows: HCDRl SEQ ID NO: 103, HCDR2 SEQ ID NO: 104, HCDR3 SEQ ID NO: 105, LCDRl SEQ ID NO: 108, LCDR2 SEQ ID NO: 109 and LCDR3 SEQ ID NO: 110. The GOOlClI lineage is defined with reference to a set of six CDR sequences of GOOlCIl as follows: HCDRl SEQ ID NO: 113, HCDR2 SEQ ID NO: 114, HCDR3 SEQ ID NO: 115, LCDRl SEQ ID NO: 118, LCDR2 SEQ ID NO: 119 and LCDR3 SEQ ID NO: 120.

Sets of CDRs of the above lineages are provided, as indicated, as are sets of CDRs with the disclosed sequences containing one or two amino acid substitutions.

Antibody lineages which are specific for acyl ghrelin (i.e. they bind to acyl ghrelin but not des acyl ghrelin) include, for example CPlAl, CPlBl, CPlCl, DP47G07, BMV1D8, BMV2D6, BMV2E4, G008H05 and G041C10 lineage.

Antibody lineages which show binding to des acyl ghrelin as well as acyl ghrelin include, for example, G001C06, GOOlEIl and GOOlCIl.

The present invention also provides binding members and antibody molecules comprising the defined sets of CDRs, set of HCDRs or set of LCDRs, as disclosed herein, and sets of CDRs of with one or two substitutions within the disclosed set of CDRs. The relevant set of CDRs is provided within an antibody framework or other protein scaffold, e.g. fibronectin or cytochrome B (Koide et al., 1998; Nygren et al., 1997), as discussed below. Preferably, antibody framework regions are employed. For example, one or more CDRs or a set of CDRs of an antibody may be grafted into a framework (e.g. human framework) to provide an antibody molecule or different antibody molecules . For example, an antibody molecule may comprise CDRs of an antibody of the CPlAl lineage and framework regions of human germline gene segment sequences. An antibody of a lineage may be provided with a set of CDRs within a framework which may be subject to "germlining", where one or more residues within the framework are changed to match the residues at the equivalent position in the most similar human germline framework (for example a framework from the VHl family, such as DPlO, or a framework from the λl family, such as DPL5) . Thus, antibody framework regions are preferably germline and/or human.

The invention provides an isolated human antibody specific for acyl- ghrelin (i.e. they bind to acyl ghrelin but not des acyl ghrelin) , having a VH domain comprising a set of HCDRs in a human germline framework, for example a VHl framework such as DPlO. Normally, the binding member also has a VL domain comprising a set of LCDRs, preferably in a human germline framework comprising a Vλl, e.g. DPL5. Preferably, the CDRs are a set of CDRs disclosed herein.

In one aspect, the present invention provides a binding member for acyl-ghrelin, comprising an antibody antigen-binding site which is composed of a human antibody VH domain and a human antibody VL domain and which comprises a set of CDRs, wherein the VH domain comprises HCDRl, HCDR2 and HCDR3 and the VL domain comprises LCDRl, LCDR2 and LCDR3, wherein the HCDRl has the amino acid sequence of SEQ ID NO: 3, the HCDR2 has the amino acid sequence of SEQ ID NO: 4, the HCDR3 has the amino acid sequence of SEQ ID NO: 5, the LCDRl has the amino acid sequence of SEQ ID NO: 8, the LCDR2 has the amino acid sequence of SEQ ID NO: 9, and the LCDR3 has the amino acid sequence of SEQ ID NO: 10; or wherein the set of CDRs contains one or more amino acid substitutions, deletions or insertions, for example, one or two amino acid substitutions, compared with this set of CDRs.

Thus, the invention provides a binding member for acyl-ghrelin, comprising an antibody antigen-binding site which is composed of a human antibody VH domain and a human antibody VL domain and which comprises a set of CDRs, wherein the set of CDRs is the CPlAl set of CDRs or other set of CDRs disclosed herein, or a set of CDRs containing one or more amino acid substitutions, deletions or insertions, for example, one or two amino acid substitutions, compared with the CPlAl set of CDRs or other set of CDRs disclosed herein.

Preferred embodiments have the CPlAl or DP47G7, CPlCl, CPlBl, BMV1D8, BMV2D6, BMV2E4, G008H05, G001C06, G041C10, GOOlEIl or GOOlCIl set of CDRs.

Any set of HCDRs of the lineages disclosed herein can be provided in a VH domain that is used as a binding member alone or in combination with a VL domain. A VH domain may be provided with a set of HCDRs of a CPlAl, DP47G7 or other lineage antibody, and if such a VH domain is paired with a VL domain, then the VL domain may be provided with a set of LCDRs of a CPlAl, DP47G7 or other lineage antibody.

The VH and VL domain frameworks comprise framework regions, one or more of which may be a germlined framework region, normally human germline . The VH domain framework is preferably human heavy chain germ-line framework and the VL domain framework is preferably human light chain germ-line framework. Framework regions of the heavy chain domain may, for example, be selected from the VH-I family, including for example VHl DP-5 and VHl DP-8 frameworks, and the VH3 family, including, for example, VH3 DP49, VH3 DP47 and VH3 Dp31 frameworks. Framework regions of the light chain may, for example, be selected from the λl family, including, for example, VLl DPL2, DPL3, DPL5 or DPL8 frameworks.

One or more CDRs may be taken from the CPlAl VH or VL domain and incorporated into a suitable framework. This is discussed further herein. CPlAl HCDRs 1, 2 and 3 are shown in SEQ ID NO: 3, 4, and 5 respectively. CPlAl LCDRs 1, 2 and 3 are shown in SEQ ID NO: 8, 9, 10, respectively. All this applies the same for other CDRs and sets of CDRs as disclosed herein, especially for DP47G7, CPlCl, CPlBl, BMV1D8, BMV2D6, BMV2E4, G008H05, G001C06, G041C10, GOOlEIl and GOOlCIl. HCDRs and LCDRs for these lineages are described in more detail above.

Preferred embodiments of the present invention employ the antibody VH and/or VL domain of an antibody molecule of the CPlAl antibody molecule. A binding member comprising an antibody antigen-binding site comprising such a VH and/or VL domain is also provided by the present invention.

Other embodiments are as follows:

A VH domain, VL domain, set of HCDRs, set of LCDRs, or set of CDRs of: DP47G7 (VH SEQ ID NO: 12; VL SEQ ID NO: 17), CPlCl (VH SEQ ID NO: 22; VL SEQ ID NO: 27), CPlBl (VH SEQ ID NO: 32; VL SEQ ID NO: 37), BMV1D8 (VH SEQ ID NO: 42; VL SEQ ID NO: 47), BMV2D6 (VH SEQ ID NO: 52; VL SEQ ID NO: 57), BMV2E4 (VH SEQ ID NO: 62; VL SEQ ID NO: 67), G008H05 (VH SEQ ID NO: 72; VL SEQ ID NO: 77), G001C06 (VH SEQ ID NO: 82; VL SEQ ID NO: 87), G041C10 (VH SEQ ID NO: 92; VL SEQ ID NO: 97), GOOlEIl (VH SEQ ID NO: 102; VL SEQ ID NO: 107) and GOOlCIl (VH SEQ ID NO: 112; VL SEQ ID NO: 117) .

In a highly preferred embodiment, a VH domain is provided with the amino acid sequence of SEQ ID NO: 2, this being termed "CPlAl VH domain". In a further highly preferred embodiment, a VL domain is provided with the amino acid sequence of SEQ ID NO: 7, this being termed "CPlAl VL domain". A highly preferred antibody antigen-binding site provided in accordance with the present invention is composed of the CPlAl VH domain, SEQ ID NO: 2, and the CPlAl VL domain, SEQ ID NO: 7. This antibody antigen-binding site may be provided within any desired antibody molecule format, e.g. scFv, Fab, IgG, IgG4 etc., as is discussed further elsewhere herein. In a further highly preferred embodiment, the present invention provides an IgG4 antibody molecule comprising the CPlAl VH domain, SEQ ID NO: 2, and the CPlAl VL domain, SEQ ID NO: 7. This is termed herein "CPlAl IgG4".

Other IgG or other antibody molecules comprising the CPlAl VH domain, SEQ ID NO: 2, and/or the CPlAl VL domain, SEQ ID NO: 1, are provided by the present invention, as are other antibody molecules comprising the CPlAl set of HCDRs (SEQ ID NOS: 3, 4 and 5) within an antibody VH domain, and/or the CPlAl set of LCDRs (SEQ ID NOS: 8, 9 and 10) within an antibody VL domain.

As noted, the present invention provides a binding member which binds acyl-ghrelin, preferably human acyl-ghrelin, and which comprises the CPlAl VH domain (SEQ ID NO: 2) and/or the CPlAl VL domain (SEQ ID NO: 7) . Properties of such a binding member are disclosed herein.

Generally, a VH domain is paired with a VL domain to provide an antibody antigen-binding site, although as discussed further below a VH domain alone may be used to bind antigen. In one preferred embodiment, the CPlAl VH domain (SEQ ID NO: 2) is paired with the CPlAl VL domain (SEQ ID NO: 7), so that an antibody antigen-binding site is formed comprising both the CPlAl VH and VL domains. Analogous embodiments are provided for the other VH and VL domains disclosed herein. In other embodiments, the CPlAl VH is paired with a VL domain other than the CPlAl VL. Light-chain promiscuity is well established in the art. Again, analogous embodiments are provided by the invention for the other VH and VL domains disclosed herein.

Variants of the VH and VL domains and CDRs of the present invention, including those for which amino acid sequences are set out herein, and which can be employed in binding members for. ghrelin can be obtained by means of methods of sequence alteration or mutation and screening. Such methods are also provided by the present invention.

In accordance with further aspects of the present invention there is provided a binding member which competes for binding to antigen with any binding member which both binds the antigen and comprises a binding member, VH and/or VL domain disclosed herein, or HCDR3 disclosed herein, or variant of any of these. Competition between binding members may be assayed easily in vitro, for example using ELISA and/or by tagging a specific reporter molecule to one binding member which can be detected in the presence of one or more other untagged binding members, to enable identification of binding members which bind the same epitope or an overlapping epitope.

Binding members preferably bind an epitope comprising serine 3 of acyl-ghrelin. A suitable epitope may, for example, be located within residues 1 to 5, residues 1 to 8, or residues 1 to 14 of acyl-ghrelin.

Thus, a further aspect of the present invention provides a binding member comprising a human antibody antigen-binding site that competes with an antibody molecule, for example especially CPlAl or other scFv and/or IgG4 described herein for binding to acyl-ghrelin. In further aspects, the present invention provides a binding member comprising a human antibody antigen-binding site which competes with an antibody antigen-binding site for binding to acyl-ghrelin, wherein the antibody antigen-binding site is composed of a VH domain and a VL domain, and wherein the VH and VL domains comprise a set of CDRs of the CPlAl or other lineage, disclosed herein.

Various methods are available in the art for obtaining antibodies against acyl-ghrelin and which may compete with a CPlAl or other antibody molecule described herein, an antibody molecule with a CPlAl or other set of CDRs described herein, for binding to acyl-ghrelin. In a further aspect, the present invention provides a method of obtaining one or more binding members able to bind the antigen, the method including bringing into contact a library of binding members according to the invention and said antigen, and selecting one or more binding members of the library able to bind said antigen.

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, or covalent, ribosomal or other in vitro display systems, each particle or molecular complex containing nucleic acid encoding the antibody VH variable domain displayed on it, and optionally also a displayed VL domain if present.

Following selection of binding members able to bind the antigen 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 binding member. Such nucleic acid may be used in subsequent production of a binding member or an antibody VH or VL variable domain by expression from nucleic acid with the sequence of nucleic acid taken from a bacteriophage or other particle or molecular complex displaying a said selected binding member.

An antibody VH variable domain with the amino acid sequence of an antibody VH variable domain of a said selected binding member may be provided in isolated form, as may a binding member comprising such a VH domain.

Ability to bind acyl-ghrelin may be further tested, also ability to compete with e.g. CPlAl (e.g. in scFv format and/or IgG format, e.g. IgG4) for binding to ghrelin. Ability to neutralise ghrelin may be tested, as discussed further below. A binding member according to the present invention may bind acyl- ghrelin with the affinity of a CPlAl or other antibody molecule described herein, which may for example be an scFv or IgG4, or with an affinity that is better.

A binding member according to the present invention may neutralise ghrelin with the potency of a CPlAl or other antibody molecule described herein, which may, for example, be an scFv or IgG4, or with a potency that is better.

Binding affinity and neutralisation potency of different binding members can be compared under appropriate conditions.

The antibodies of the present invention have a number of advantages over existing commercially available anti-ghrelin antibodies. For example, the present invention provides human antibodies, which are expected to display a lower degree of immunogenicity when chronically or repeatedly administered to humans for therapeutic or diagnostic use. Further, the present invention provides antibodies that have greater affinity and are more potent neutralisers of ghrelin, and therefore a desired therapeutic or diagnostic effect may be achieved using less antibody material. The antibodies also have an in vivo half life in mice (e.g. 6-7 days) that is consistent with previous observations for human antibodies in rodents. This supports the view that the antibodies of the present invention are typical for their class and are therefore suitable for therapeutic applications in man.

The invention also provides heterogeneous preparations comprising anti-acyl-ghrelin antibody molecules. For example, such preparations may be mixtures of antibodies with full-length heavy chains and heavy chains lacking the C-terminal lysine, with various degrees of glycosylation and/or with derivatized amino acids, such as cyclization of an N-terminal glutamic acid to form a pyroglutamic acid residue.

In further aspects, the invention provides an isolated nucleic acid which comprises a sequence encoding a binding member, VH domain and/or VL domains according to the present invention, and methods of preparing a binding member, a VH domain and/or a VL domain of the invention, which comprise expressing said nucleic acid under conditions to bring about production of said binding member, VH domain and/or VL domain, and recovering it.

A further aspect of the present invention provides nucleic acid, generally isolated, encoding an antibody VH variable domain and/or VL variable domain disclosed herein.

Another aspect of the present invention provides nucleic acid, generally isolated, encoding a VH CDR or VL CDR sequence disclosed herein, especially a VH CDR selected from: CPlAl (VH CDRl SEQ ID NO: 3, VH CDR2 SEQ ID NO: 4, and VH CDR3 SEQ ID NO: 5), or a VL CDR selected from: CPlAl (VL CDRl SEQ ID NO: 8, VL CDR2 SEQ ID NO: 9, and VL CDR3 SEQ ID NO: 10) . Nucleic acid encoding the CPlAl set of CDRs, nucleic acid encoding the CPlAl set of HCDRs and nucleic acid encoding the CPlAl set of LCDRs are also provided by the present invention, as are nucleic acids encoding individual CDRs, HCDRs, LCDRs and sets of CDRs, HCDRs, LCDRs of the CPlAl lineage and other lineages described herein.

A further aspect provides a host cell transformed with nucleic acid of the invention.

A yet further aspect provides a method of production of an antibody VH variable domain, the method including causing expression from encoding nucleic acid. Such a method may comprise culturing host cells under conditions for production of said antibody VH variable domain.

Analogous methods for production of VL variable domains and binding members comprising a VH and/or VL domain are provided as further aspects of the present invention.

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.

Further aspects of the present invention provide for compositions containing binding members of the invention, and their use in methods of inhibiting or neutralising ghrelin, including methods of treatment of the human or animal body by therapy.

Binding members according to the invention may be used in a method of treatment or diagnosis of the human or animal body, such as a method of treatment (which may include prophylactic treatment) of a disease or disorder in a human patient which comprises administering to said patient an effective amount of a binding member of the invention. Conditions treatable in accordance with the present invention include any in which ghrelin plays a role, especially obesity and obesity- related conditions, including for example, Type II non-insulin dependent diabetes mellitus (NIDDM) , Prader-Willi syndrome, hyperphagia, impaired satiety, hypertension, coronary heart disease, osteoarthritis, metabolic syndrome, cancer, in particular breast, endometrial, prostate and bowel cancer, and gastric motility disorders such as irritable bowel syndrome and functional dyspepsia.

These and other aspects of the invention are described in further detail below. BRIEF DESCRIPION OF FIGURES

Figure 1 shows the inhibition of binding of human 32pM ¹²⁵I acyl- ghrelin to cell membranes prepared from HEK293 cells transfected with GHSRIa by CPlAl (IgG₄) .

Figure 2 shows Ca²⁺ signalling in HEK293 cells expressing GHSRIa as evoked by human or rat acyl ghrelin. In this experiment the EC₅₀ values were 2.7and 4.6nM for human and rat ghrelin respectively.

Figure 3 shows the inhibition of Ca²⁺ signalling evoked by 5nM human acyl ghrelin in HEK293 cells expressing GHSRIa by CPlAl (IgG₄) . In this experiment the CPlAl IC₅₀ value was 1.3nM and the Dlys3 GHRP-6 IC₅₀ value was 3.3nM

Figure 4 shows the inhibition of Ca²⁺ signalling evoked by 5nM rat acyl ghrelin in HEK293 cells expressing GHSRla by CPlAl (IgG₄) . In this experiment the CPlAl IC₅₀ value was 0.7nM and the Dlys3 GHRP-6 IC₅₀ value was 1.7nM

Figure 5 shows the inhibition of binding of CPlAl (IgG) to acyl ghrelin by peptide fragments of des-ghrelin and Neurotensin.

Figure 6 shows the inhibition of binding of CPlAl (IgG) to acyl ghrelin by peptide fragments of acyl ghrelin, Neurotensin and Motilin.

Figure 7 shows the binding of human ¹²⁵I acyl ghrelin to cell membranes prepared from HEK293 cells transfected with GHSRla In this experiment the K_D was calculated to be 32pM Figure 8 shows the inhibition of binding of human ¹²⁵I acyl ghrelin to cell membranes prepared from HEK293 cells transfected with GHSRIa by CPlAl (scFv)

5 Figure 9 shows the inhibition of human ghrelin induced Ca²⁺ signalling in HEK293 cells expressing GHSRla by CPlAl (scFv)

Figure 10 shows the inhibition of rat ghrelin -induced Ca²⁺ signalling in HEK293 cells expressing GHSRla by CPlAl (scFv) 10

Figure 11 shows a pharmacokinetic study of CPlAl in male C57BL6 mice.

TERMINOLOGY

15 It is convenient to point out here that "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

20

Ghrelin

Ghrelin is a peptide hormone which binds GHSRla. Ghrelin exists in two forms: a 28 aa peptide and a 27-aa splice variant (termed ^Λdes-Gln Ghrelin' ) . Both forms may be octanoylated at amino acid Serine 3

25 (Hosoda, Kojima et al. 2000) . Both forms of ghrelin also exist in unoctanoylated form, which is termed ^λdes-acyl' or ^Λdes-octanoylated' ghrelin.

The term ^Λacyl-ghrelin' as used herein refers to both the octanoylated 30 28aa peptide an^ the 27aa splice variant. The term ^Λghrelin' as used herein also refers to the octanoylated forms, unless context dictates otherwise In the context of the present invention, acyl-ghrelin is normally human acyl-ghrelin, although it may be non-human acyl-ghrelin (e.g. non-human primate acyl-ghrelin and/or rat acyl-ghrelin and/or mouse acyl-ghrelin) . Acyl-ghrelin is also referred to in places as "the antigen" .

Obesity

Obesity is a condition of severe overweight in which excess fat has accumulated in an individual.

The most common measurement of obesity is the Body Mass Index (BMI) scale. The BMI is calculated as the ratio of weight over height squared (kg/m²) . A BMI of 20 to 25 is considered to be within ideal body weight range. Most medical sources define a BMI of 27 or higher to be "obese." Morbid obesity is an extreme obesity phenotype and is defined as a BMI of 40 or more.

Obesity is associated with a range of conditions including Type II non-insulin dependent diabetes mellitus (NIDDM) , Prader-Willi syndrome, hyperphagia, impaired satiety, hypertension, coronary heart disease, osteoarthritis, metabolic syndrome, cancer, in particular breast, endometrial, prostate and bowel cancer, and gastric motility disorders such as irritable bowel syndrome and functional dyspepsia.

Binding member

This describes a member of a pair of molecules that have binding specificity for one another. The members of a binding pair may be naturally derived or wholly or partially synthetically produced. One member of the pair of molecules has an area on its surface, or a cavity, which specifically binds to and is therefore complementary to a particular spatial and polar organisation of the other member of the pair of molecules. Thus, the members of the pair have the property of binding specifically to each other. In some contexts, a binding member may be referred to as a specific binding member. Examples of types of binding pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. The present invention is concerned with antigen-antibody type reactions.

A binding member normally comprises a molecule having an antigen- binding site. For example, a binding member may be an antibody molecule or a non-antibody protein that comprises an antigen-binding site. An antigen binding site may be provided by means of arrangement of CDRs on non-antibody protein scaffolds such as fibronectin or cytochrome B etc. (Haan & Maggos, 2004; Koide et al., 1998; Nygren et al., 1997), or by randomising or mutating amino acid residues of a loop within a protein scaffold to confer binding specificity for a desired target. Scaffolds for engineering novel binding sites in proteins have been reviewed in detail by Nygren et al. (1997).

Protein scaffolds for antibody mimics are disclosed in WO/0034784 in which the inventors describe proteins (antibody mimics) that include a fibronectin type III domain having at least one randomised loop. A suitable scaffold into which to graft one or more CDRs, e.g. a set of HCDRs, may be provided by any domain member of the immunoglobulin gene superfamily. The scaffold may be a human or non-human protein.

An advantage of a non-antibody protein scaffold is that it may provide an antigen-binding site in a scaffold molecule that is smaller and/or easier to manufacture than at least some antibody molecules. Small size of a binding member may confer useful physiological properties such as an ability to enter cells, penetrate deep into tissues or reach targets within other structures, or to bind within protein cavities of the target antigen.

Use of antigen binding sites in non-antibody protein scaffolds is reviewed in Wess, 2004. Typical are proteins having a stable backbone and one or more variable loops, in which the amino acid sequence of the loop or loops is specifically or randomly mutated to create an antigen-binding site having specificity for binding the target antigen. Such proteins include the IgG-binding domains of protein A from S. aureus,, transferrin, tetranectin, fibronectin (e.g. 10th fibronectin type III domain) and lipocalins . Other approaches include synthetic "Microbodies" (Selecore GmbH) , which are based on cyclotides - small proteins having intra-molecular disulphide bonds.

In addition to antibody sequences and/or an antigen-binding site, a binding member according to the present invention may comprise other amino acids, e.g. forming a peptide or polypeptide, such as a folded domain, or to impart to the molecule another functional characteristic in addition to ability to bind antigen. Binding members of the invention may carry a detectable label, or may be conjugated to a toxin or a targeting moiety or enzyme (e.g. via a peptidyl bond or linker) . For example, a binding member may comprise a catalytic site (e.g. in an enzyme domain) as well as an antigen binding site, wherein the antigen binding site binds to the antigen and thus targets the catalytic site to the antigen. The catalytic site may inhibit biological function of the antigen, e.g. by cleavage.

Although, as noted, CDRs can be carried by scaffolds such as fibronectin or cytochrome B (Haan & Maggos, 2004; Koide et al., 1998; Nygren et al . , 1997), the structure for carrying a CDR or a set of CDRs of the invention will generally be of an antibody heavy or light chain sequence or substantial portion thereof in which the CDR or set of CDRs is located at a location corresponding to the CDR or set of CDRs of naturally occurring VH and VL antibody variable domains encoded by rearranged immunoglobulin genes . The structures and locations of immunoglobulin variable domains may be determined by reference to Kabat, et al . , 1987, and updates thereof. A number of academic and commercial on-line resources are available to query this database. For example, see Martin, A. C. R. Accessing the Kabat Antibody Sequence Database by Computer PROTEINS: Structure, Function and Genetics, 25 (1996) , 130-133 and the associated on-line resource, currently at the web address of http: //www.bioinf . org.uk/abs/simkab.html.

Antibody molecule

This describes an immunoglobulin whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein comprising an antibody antigen-binding site. Antibody fragments that comprise an antibody antigen-binding site are molecules such as Fab, scFv, Fv, dAb, Fd, and diabodies.

It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the CDRs, of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. See, for instance, EP-A-184187, GB 2188638A or EP-A-239400, and a large body of subsequent literature. A hybridoma or other cell producing an antibody may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.

As antibodies can be modified in a number of ways, the term "antibody molecule" should be construed as covering any binding member or substance having an antibody antigen-binding site with the required specificity. Thus, this term covers antibody fragments and derivatives, including any polypeptide comprising an antibody antigen- binding site, whether natural or wholly or partially synthetic. Chimeric molecules comprising an antibody antigen-binding site, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP-A- 0120694 and EP-A-0125023, and a large body of subsequent literature.

Further techniques available in the art of antibody engineering have made it possible to isolate human and humanised antibodies. For example, human hybridomas can be made as described by Kontermann & Dubel (2001) . Phage display, another established technique for generating binding members has been described in detail in many publications such as Kontermann & Dubel (2001) and WO92/01047 (discussed further below) . Transgenic mice in which the mouse antibody genes are inactivated and functionally replaced with human antibody genes while leaving intact other components of the mouse immune system, can be used for isolating human antibodies (Mendez et al., 1997) .

Synthetic antibody molecules may be created by expression from genes generated by means of oligonucleotides synthesized and assembled within suitable expression vectors, for example as described by Knappik et al. (2000) or Krebs et al. (2001).

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, 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; Holliger et al., 1993) . Fv, scFv or diabody molecules may be stabilised 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) .

A dAb (domain antibody) is a small monomeric antigen-binding fragment of an antibody, namely the variable region of an antibody heavy or light chain (Holt et al. 2003). VH dAbs occur naturally in camelids (e.g. camel, llama) and may be produced by immunising a camelid with a target antigen, isolating antigen-specific B cells and directly cloning dAb genes from individual B cells. dAbs are also producible in cell culture. Their small size, good solubility and temperature stability makes them particularly physiologically useful and suitable for selection and affinity maturation. A binding member of the present invention may be a dAb comprising a VH or VL domain substantially as set out herein, or a VH or VL domain comprising a set of CDRs substantially as set out herein.

Where bispecific antibodies are to be used, these may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Holliger & Winter, 1993), e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above. Examples of bispecific antibodies include those of the BiTE™ technology in which the binding domains of two antibodies with different specificity can be used and directly linked via short flexible peptides. This combines two antibodies on a short single polypeptide chain. Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction.

Bispecific diabodies, as opposed to bispecific whole antibodies, may also be particularly useful because they can be readily constructed and expressed in E.colϊ. Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94/13804) from libraries. if one arm of the diabody is to be kept constant, for instance, with a specificity directed against acyl-ghrelin, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected. Bispecific whole antibodies may be made by knobs-into-holes engineering (Ridgeway et al., 1996).

Antigen-binding site

This describes the part of a molecule that binds to and is complementary to all or part of the target antigen. In an antibody molecule it is referred to as the antibody antigen-binding site, and comprises the part of the antibody that specifically binds to and is complementary to all or part of the target antigen. Where an antigen is large, an antibody may only bind to a particular part of the antigen, which part is termed an epitope. An antibody antigen-binding site may be provided by one or more antibody variable domains. Preferably, an antibody antigen-binding site comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH) .

Specific

This may be used to refer to the situation in which one member of a binding pair will not show any significant binding to molecules other than its specific binding partner (s). The term is also applicable where, e.g. an antigen-binding site, is specific for a particular epitope that is carried by a number of antigens, in which case the binding member carrying the antigen-binding site will be able to bind to the various antigens carrying the epitope.

For example, an antibody antigen-binding domain specific for acyl- ghrelin may bind to acyl-ghrelin (i.e. 28aa or 27aa ghrelin octanoylated at serine 3) and show no binding or substantially no binding to des-acyl-ghrelin (i.e. 28aa or 27aa ghrelin which is not octanoylated at serine 3) .

Isolated

This refers to the state in which binding members of the invention, or nucleic acid encoding such binding members, will generally be in accordance with the present invention. Isolated members and isolated nucleic acid will be free or substantially free of material with which they are naturally associated such as other polypeptides or nucleic acids with which they are found in their natural environment, or the environment in which they are prepared (e.g. cell culture) when such preparation is by recombinant DNA technology practised in vitro or in vivo. Members and nucleic acid may be formulated with diluents or adjuvants and still for practical purposes be isolated - for example, the members will normally be mixed with gelatin or other carriers if used to coat microtitre plates for use in immunoassays, or will be mixed with pharmaceutically acceptable carriers or diluents when used in diagnosis or therapy. Binding members may be glycosylated, either naturally or by systems of heterologous eukaryotic cells (e.g. CHO or NSO (ECACC 85110503) cells, or they may be (for example if produced by expression in a prokaryotic cell) unglycosylated.

DETAILED DESCRIPTION

As noted above, a binding member in accordance with the present invention preferably neutralises ghrelin. The degree to which an antibody neutralises ghrelin is referred to as its neutralising potency.

Potency is normally expressed as an IC50 value, in nM unless otherwise stated. IC50 is the median inhibitory concentration of an antibody molecule. In functional assays, IC50 is the concentration that reduces a biological response by 50 % of its maximum. In ligand- binding studies, IC50 is the concentration that reduces receptor binding by 50 % of maximal specific binding level.

IC50 may be calculated by plotting % biological response (represented e.g. by Fluorometric Imaging Plate Reader or FLIPR) or % specific receptor binding as a function of the log of the binding member concentration, and using a software program such as Prism (GraphPad) to fit a sigmoidal function to the data to generate IC50 values, for example as described in the Examples herein.

A binding member in accordance with the present invention preferably inhibits human ghrelin-evoked calcium signaling in cells expressing GHSRIa receptor, e.g. cells recombinantly transfected with a GHSRla gene, for instance HEK cells.

In a "FLIPR" calcium signaling assay as described in Example 2 herein, a binding member according to the invention preferably has a potency (IC50) for neutralising human ghrelin of or less than 1000, 600, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 nM. Normally, a binding member of the invention has a potency of 5 nM or less, preferably 2.5 nM or less, more preferably 1 nM or less. In particularly preferred embodiments, the potency is 0.5 nM or less, e.g. 0.4 nM or less; 0.3 nM or less; 0.2 nM or less; or 0.15 nM or less. -In some embodiments, the potency may be about 0.1 nM.

Potency may be between 0.1-100 nM, 0.1-50 nM, 0.1-10 nM, or 0.1-1.0 nM. For example, potency may be 0.1-5.0 nM, 0.2-5.0 nM, 0.3-5.0 nM, or 0.3-4.0 nM.

In some embodiments of the invention, the neutralising potency of a non-potency-optimised binding member in a HEK cell assay as described herein is about 1.4 nM for human acyl ghrelin. However, this is only an example and higher potencies may be achieved. Although potency optimisation may be used to generate higher potency binding members from a given binding member, it is also noted that high potency binding members may be obtained even without potency optimisation.

Preferably, a binding member according to the invention binds human acyl ghrelin and/or rat acyl-ghrelin with an affinity of or less than 1, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3 or 0.2 nM. For example, a binding member may bind human acyl ghrelin and/or rat acyl-ghrelin with an affinity of about 708pM.

As noted above, variants of antibody molecules disclosed herein may be produced and used in the present invention. Following the lead of computational chemistry in applying multivariate data analysis techniques to the structure/property-activity relationships (Wold, et al. 1984), quantitative activity-property relationships of antibodies can be derived using well-known mathematical techniques such as statistical regression, pattern recognition and classification (Norman et al. 1998; Kandel & Backer, 1995; Krzanowski, 2000; Witten & Frank, 1999; Denison (Ed), 2002; Ghose & Viswanadhan) . The properties of antibodies can be derived from empirical and theoretical models (for example, analysis of likely contact residues or calculated physicochemical property) of antibody sequence, functional and three- dimensional structures and these properties can be considered singly'i and in combination.

An antibody antigen-binding site composed of a VH domain and a VL domain is formed by six loops of polypeptide: three from the light chain variable domain (VL) and three from the heavy chain variable domain (VH) . Analysis of antibodies of known atomic structure has elucidated relationships between the sequence and three-dimensional structure of antibody combining sites (Chothia et al. 1992; Al- Lazikani, et al. 1997). These relationships imply that, except for the third region (loop) in VH domains, binding site loops have one of a small number of main-chain conformations: canonical structures. The canonical structure formed in a particular loop is determined by its size and the presence of certain residues at key sites in both the loop and in framework regions (Chothia et al. and Al-Lazikani et al., supra) .

This study of sequence-structure relationship can be used for prediction of those residues in an antibody of known sequence, but of an unknown three-dimensional structure, which are important in maintaining the three-dimensional structure of its CDR loops and hence maintain binding specificity. These predictions can be backed up by comparison of the predictions to the output from lead optimization experiments. In a structural approach, a model can be created of the antibody molecule (Chothia, et al. 1986) using any freely available or commercial package such as WAM (Whitelegg & Rees, 2000). A protein visualisation and analysis software package such as Insight II (Accelerys, Inc.) or Deep View (Guex & Peitsch, 1997) may then be used to evaluate possible substitutions at each position in the CDR. This information may then be used to make substitutions likely to have a minimal or beneficial effect on activity.

The techniques required to make substitutions within amino acid sequences of CDRs, antibody VH or VL domains and binding members are generally 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 activity, and tested for ability to bind and/or neutralise ghrelin and/or for any other desired property.

Variable domain amino acid sequence variants of any of the VH and VL domains whose sequences are specifically disclosed herein may be employed in accordance with the present invention, as discussed. Particular variants may include one or more amino acid sequence alterations (addition, deletion, substitution and/or insertion of an amino acid residue) , may be less than about 20 alterations, less than about 15 alterations, less than about 10 alterations or less than about 5 alterations, maybe 5, 4, 3, 2 or 1. Alterations may be made in one or more framework regions and/or one or more CDRs.

Preferably alterations do not result in loss of function, so a binding member comprising a thus-altered amino acid sequence preferably retains an ability to bind and/or neutralise acyl-ghrelin. More preferably, it retains the same quantitative binding and/or neutralising ability as a binding member in which the alteration is not made, e.g. as measured in an assay described herein. Most preferably, the binding member comprising a thus-altered amino acid sequence has an improved ability to bind or neutralise acyl-ghrelin.

Alteration may comprise replacing one or more amino acid residue 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. Preferred 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, 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 in some embodiments 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 binding members of the invention may comprise non-natural or non-standard amino acids described above. In some embodiments non-standard amino acids (e.g. D-amino acids) may be incorporated into an amino acid sequence during synthesis, while in other embodiments the non-standard amino acids may be introduced 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 ghrelin binding and neutralising properties in a binding member 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.

As noted above, a CDR amino acid sequence substantially as set out herein is preferably carried as a CDR in a human antibody variable domain or a substantial portion thereof. The HCDR3 sequences substantially as set out herein represent preferred embodiments of the present invention and it is preferred that each of these is carried as a HCDR3 in a human heavy chain variable domain or a substantial portion thereof. Variable domains employed in the invention may be obtained or derived from any germ-line or rearranged human variable domain, or may be a synthetic variable domain based on consensus or actual sequences of known human variable domains. A CDR sequence of the invention (e.g. CDR3) may be introduced into a repertoire of variable domains lacking a CDR (e.g. CDR3) , using recombinant DNA technology.

For example, Marks et al. (1992) describe methods of producing repertoires of antibody variable domains in which consensus primers directed at or adjacent to the 5' end of the variable domain area are used in conjunction with consensus primers to the third framework region of human VH genes to provide a repertoire of VH variable domains lacking a CDR3. Marks et al. further describe how this repertoire may be combined with a CDR3 of a particular antibody. Using analogous techniques, the CDR3-derived sequences of the present invention may be shuffled with repertoires of VH or VL domains lacking a CDR3, and the shuffled complete VH or VL domains combined with a cognate VL or VH domain to provide binding members of the invention. The repertoire may then be displayed in a suitable host system such as the phage display system of WO92/01047 or any of a subsequent large body of literature, including Kay, Winter & McCafferty (1996) , so that suitable binding members may be selected. A repertoire may consist of from anything from 10⁴ individual members upwards, for example from 10⁶ to 10^s or 10¹⁰ members. Other suitable host systems include yeast display, bacterial display, T7 display, ribosome display and covalent display.

Analogous shuffling or combinatorial techniques are also disclosed by Stemmer (1994) , who describes the technique in relation to a β- lactamase gene but observes that the approach may be used for the generation of antibodies. A further alternative is to generate novel VH or VL regions carrying CDR-derived sequences of the invention using random mutagenesis of one or more selected VH and/or VL genes to generate mutations within the entire variable domain. Such a technique is described by Gram et al. (1992) , who used error-prone PCR. In preferred embodiments one or two amino acid substitutions are made within a set of HCDRs and/or LCDRs.

Another method that may be used is to direct mutagenesis to CDR regions of VH or VL genes. Such techniques are disclosed by Barbas et al. (1994) and Schier et al. (1996).

All the above-described techniques are known as such in the art and the skilled person will be able to use such techniques to provide binding members of the invention using routine methodology in the art.

A further aspect of the invention provides a method for obtaining an antibody antigen-binding site specific for ghrelin antigen, the method comprising providing by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a VH domain set out herein, a VH domain which is an amino acid sequence variant of the VH domain, optionally combining the VH domain thus provided with one or more VL domains, and testing the VH domain or VH/VL combination or combinations to identify a binding member or an antibody antigen-binding site specific for ghrelin antigen and optionally with one or more preferred properties, preferably ability to neutralise ghrelin activity. Said VL domain may have an amino acid sequence which is substantially as set out herein.

An analogous method may be employed in which one or more sequence variants of a VL domain disclosed herein are combined with one or more VH domains . In a preferred embodiment, CPlAl VH domain (SEQ ID NO: 2) may be subject to mutation to provide one or more VH domain amino acid sequence variants, optionally combined with CPlAl VL (SEQ ID NO: 7) .

A further aspect of the invention provides a method of preparing a binding member specific for acyl-ghrelin, which method comprises:

(a) providing a starting repertoire of nucleic acids encoding a VH domain which either include a CDR3 to be replaced or lack a CDR3 encoding region; (b) combining said repertoire with a donor nucleic acid encoding an amino acid sequence substantially as set out herein for a VH CDR3 such that said donor nucleic acid is inserted into the CDR3 region in the repertoire, so as to provide a product repertoire of nucleic acids encoding a VH domain;

(c) expressing the nucleic acids of said product repertoire;

(d) selecting a binding member specific for acyl-ghrelin; and

(e) recovering said binding member or nucleic acid encoding it.

A preferred donor VH CDR3 is the CPlAl HCDR3 (SEQ ID NO: 5) .

Again, an analogous method may be employed in which a VL CDR3 of the invention is combined with a repertoire of nucleic acids encoding a VL domain that either include a CDR3 to be replaced or lack a CDR3 encoding region. A preferred donor VH CDR3 is the CPlAl LCDR3 (SEQ ID NO: 10) .

Similarly, one or more, or all three CDRs may be grafted into a repertoire of VH or VL domains that are then screened for a binding member or binding members specific for acyl-ghrelin.

In some preferred embodiments, one or more of CPlAl HCDRl (SEQ ID NO: 3), HCDR2 (SEQ ID NO: 4) and HCDR3 (SEQ ID NO: 5) and/or one or more of CPlAl LCDRl (SEQ ID NO: 8), LCDR2 (SEQ ID NO: 9) and LCDR3 (SEQ ID NO: 10) or the CPlAl set of LCDRs may be employed.

Similarly, other VH and VL domains, sets of CDRs, sets of HCDRs and/or sets of LCDRs, and HCDR3 and LCDR3 sequences disclosed herein may be employed.

A substantial portion of an immunoglobulin variable domain will comprise at least the three CDR regions, together with their intervening framework regions. Preferably, the portion will also include at least about 50% of either or both of the first and fourth framework regions, the 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. Additional residues at the N-terminal or C-terminal end of the substantial part of the variable domain may be those not normally associated with naturally occurring variable domain regions. For example, construction of binding members of the present invention made by recombinant DNA techniques may result in the introduction of N- or C-terminal residues encoded by linkers introduced to facilitate cloning or other manipulation steps. Other manipulation steps include the introduction of linkers to join variable domains of the invention to further protein sequences including antibody constant regions, other variable domains (for example, in the production of diabodies) or detectable/functional labels as discussed in more detail elsewhere herein.

Although in some aspects of the invention, binding members comprising a pair of VH and VL domains are preferred, single binding domains based on either VH or VL domain sequences form further aspects of the invention. It is known that single immunoglobulin domains, especially VH domains, are capable of binding target antigens in a specific manner. For example, see the discussion of dAbs above. In the case of either of the single binding domains, these domains may be used to screen for complementary domains capable of forming a two- domain binding member able to bind acyl-ghrelin.

This may be achieved by phage display screening methods using the so- called hierarchical dual combinatorial approach as disclosed in WO92/01047, in which an individual colony containing either an H or L chain clone is used to infect a complete library of clones encoding the other chain (L or H) and the resulting two-chain binding member is selected in accordance with phage display techniques such as those described in that reference. This technique is also disclosed in Marks et al, ibid.

In some embodiments, phage display screening methods may be used for the initial rounds of screening (e.g. the first and/or second rounds) and ribosome display methods may be used for subsequent rounds of screening.

Binding members of the present invention may further comprise antibody constant regions or parts thereof, preferably human antibody constant regions or parts thereof. For example, a VL domain may be attached at its C-terminal end to antibody light chain constant domains including human CK or Cλ chains, preferably Cλ chains. Similarly, a binding member based on a VH domain may be attached at its C-terminal end to all or part (e.g. a CHl domain) of an immunoglobulin heavy chain derived from any antibody isotype, e.g. IgG, IgA, IgE and IgM and any of the isotype sub-classes, particularly IgGl and IgG4. IgG4 is preferred. IgG4 is preferred because it does not bind complement and does not create effector functions. Any synthetic or other constant region variant that has these properties and stabilizes variable regions is also preferred for use in embodiments of the present invention. Binding members of the invention may be labelled with a detectable or functional label. Detectable labels include radiolabels such as ¹³¹I or ⁹⁹Tc, which may be attached to antibodies of the invention using conventional chemistry known in the art of antibody imaging. Labels also include enzyme labels such as horseradish peroxidase. Labels further include chemical moieties such as biotin that may be detected via binding to a specific cognate detectable moiety, e.g. labelled avidin.

Binding members of the present invention are designed to be used in methods of diagnosis or treatment in human or animal subjects, preferably human.

Accordingly, further aspects of the invention provide methods of treatment comprising administration of a binding member as provided, pharmaceutical compositions comprising such a binding member, and use of such a binding member in the manufacture of a medicament for administration, for example in a method of making a medicament or pharmaceutical composition comprising formulating the binding member with a pharmaceutically acceptable excipient.

Clinical indications in which an anti-ghrelin antibody may be used to provide therapeutic benefit include obesity and obesity-related conditions, including for example, Type II non-insulin dependent diabetes mellitus (NIDDM) , Prader-Willi syndrome, hyperphagia, impaired satiety, anxiety, hypertension, coronary heart disease, osteoarthritis, metabolic syndrome, cancer, in particular breast, endometrial, prostate and bowel cancer, and gastric motility disorders such as irritable bowel syndrome and functional dyspepsia.

Anti-ghrelin treatment may be given orally, by injection (for example, subcutaneously, intravenously, intraperitoneal or intramuscularly) , by inhalation, or topically (for example intraocular, intranasal, rectal, on skin) . The route of administration can be determined by the physicocheitiical characteristics of the treatment, by special considerations for the disease or by the requirement to optimise efficacy or to minimise side effects.

It is envisaged that anti-ghrelin treatment will not be restricted to use in the clinic. Therefore, subcutaneous injection using a needle free device is also preferred.

Combination treatments may be used to provide significant synergistic effects, particularly the combination of an anti-acyl-ghrelin binding member with one or more other drugs . A binding member according to the present invention may be provided in combination or addition to other weight control agents, such as sibutramine (Meridia; Abbott) or orlistat (Xenical; Roche) , for the treatment of obesity and associated disorders .

In accordance with the present invention, compositions provided may be administered to individuals. Administration is preferably 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. 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. Appropriate doses of antibody are well-known in the art; see Ledermann et al. (1991) and Bagshawe (1991) . Specific dosages indicated herein, or in the Physician's Desk Reference (2003) as appropriate for the type of medicament being administered, may be used. A therapeutically effective amount or suitable dose of a binding member 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 antibody is for diagnosis or for treatment, the size and location of the area to be treated, the precise nature of the antibody (e.g. whole antibody, fragment or diabody) , and the nature of any detectable label or other molecule attached to the antibody. A typical antibody dose will be in the range lOOμg to Ig for systemic applications, and lμg to lmg for topical applications. Typically, the antibody will be a whole antibody, preferably the IgG4 isotype. This is a dose for a single treatment of an adult patient, which may be proportionally adjusted for children and infants, and also adjusted for other antibody formats in proportion to molecular weight.

Treatments may be repeated at daily, twice-weekly, weekly or monthly intervals, at the discretion of the physician. In preferred embodiments of the present invention, treatment is periodic, and the period between administrations is about two weeks or more, preferably about three weeks or more, more preferably about four weeks or more, or about once a month.

Binding members 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 binding member.

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, stabiliser 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, or by injection, e.g. intravenous .

Pharmaceutical compositions for oral administration 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 intravenous injection, 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, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

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

Binding members 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 antibody concentrations and pH. Solid formulations may be produced by lyophilisation, spray drying, or drying by supercritical fluid technology, for example. Formulations of anti-ghrelin 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 may include viscosity modifying agents, which prolong the time that the drug is resident at the site of action. In certain embodiments, the binding member may be prepared with a carrier that will protect the binding member 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. See, e.g. Robinson, 1978.

The present invention provides a method comprising causing or allowing binding of a binding member as provided herein to acyl-ghrelin. As noted, such binding may take place in vivo, e.g. following administration of a binding member, or nucleic acid encoding a binding member, or it may take place in vitro, for example in ELISA, Western blotting, immunocytochemistry, immuno-precipitation, affinity chromatography, or cell based assays such as a GHSRIa binding assay.

The amount of binding of binding member to ghrelin may be determined. Quantitation may be related to the amount of the antigen in a test sample, which may be of diagnostic interest.

A kit comprising a binding member or antibody molecule according to any aspect or embodiment of the present invention is also provided as an aspect of the present invention. In a kit of the invention, the binding member or antibody molecule may be labelled to allow its reactivity in a sample to be determined, e.g. as described further below. 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 antibody molecules 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 reactivities of antibodies in a sample may be determined by any appropriate means. Radioimmunoassay (RIA) is one possibility.

Radioactive labelled antigen is mixed with unlabelled antigen (the test sample) and allowed to bind to the antibody. Bound antigen is physically separated from unbound antigen and the amount of radioactive antigen bound to the antibody determined. The more antigen there is in the test sample the less radioactive antigen will bind to the antibody. A competitive binding assay may also be used with non-radioactive antigen, using antigen or an analogue linked to a reporter molecule. The reporter molecule may be a fluorochrome, phosphor or laser dye with spectrally isolated absorption or emission characteristics. Suitable fluorochromes include fluorescein, rhodamine, phycoerythrin and Texas Red. Suitable chromogenic dyes include diaminobenzidine .

Other reporters include macromolecular colloidal particles or particulate material such as latex beads that are coloured, magnetic or paramagnetic, and biologically or chemically active agents that can directly or indirectly cause detectable signals to be visually observed, electronically detected or otherwise recorded. These molecules may be enzymes, which catalyse reactions that develop, or change colours or cause changes in electrical properties, for example. They may be molecularly excitable, such that electronic transitions between energy states result in characteristic spectral absorptions or emissions. They may include chemical entities used in conjunction with biosensors. Biotin/avidin or biotin/streptavidin and alkaline phosphatase detection systems may be employed.

The signals generated by individual antibody-reporter conjugates may be used to derive quantifiable absolute or relative data of the relevant antibody binding in samples (normal and test) .

The present invention also provides the use of a binding member as above for measuring antigen levels in a competition assay, that is to say a method of measuring the level of antigen in a sample by employing a binding member as provided by the present invention in a competition assay. This may be where the physical separation of bound from unbound antigen is not required. Linking a reporter molecule to the binding member so that a physical or optical change occurs on binding is one possibility. The reporter molecule may directly or indirectly generate detectable, and preferably measurable, signals. The linkage of reporter molecules may be directly or indirectly, covalently, e.g. via a peptide bond or non-covalently . Linkage via a peptide bond may be as a result of recombinant expression of a gene fusion encoding antibody and reporter molecule.

The present invention also provides for measuring levels of antigen directly, by employing a binding member according to the invention for example in a biosensor system.

The mode of determining binding is not a feature of the present invention and those skilled in the art are able to choose a suitable mode according to their preference and general knowledge.

As noted, in various aspects and embodiments, the present invention extends to a binding member that competes for binding to ghrelin with any binding member defined herein, e.g. CPlAl IgG4. Competition between binding members may be assayed easily in vitro, for example by tagging a specific reporter molecule to one binding member which can be detected in the presence of other untagged binding member (s), to enable identification of binding members which bind the same epitope or an overlapping epitope.

Competition may be determined for example using ELISA in which ghrelin is immobilised to a plate and a first tagged binding member along with one or more other untagged binding members is added to the plate. Presence of an untagged binding member that competes with the tagged binding member is observed by a decrease in the signal emitted by the tagged binding member.

In testing for competition a peptide fragment of the antigen may be employed, especially a peptide including or consisting essentially of an epitope of interest. A peptide having the epitope sequence plus one or more amino acids at either end may be used. Binding members according to the present invention may be such that their binding for antigen is inhibited by a peptide with or including the sequence given. In testing for this, a peptide with either sequence plus one or more amino acids may be used.

Binding members that bind a specific peptide may be isolated for example from a phage display library by panning with the peptide (s) .

The present invention further provides an isolated nucleic acid encoding a binding member of the present invention. Nucleic acid may include DNA and/or RNA. In a preferred aspect, the present invention provides a nucleic acid that codes for a CDR or set of CDRs or VH domain or VL domain or antibody antigen-binding site or antibody molecule, e.g. scFv or IgG4, of the invention as defined above. 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 CDR or set of CDRs or VH domain or VL domain or antibody antigen- binding site or antibody molecule, e.g. scFv or IgGA 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 VH or VL domain, or binding member may be isolated and/or purified using any suitable technique, then used as appropriate.

Binding members, VH and/or VL domains, and encoding nucleic acid molecules and vectors according to the present invention may be provided isolated and/or purified, e.g. from their natural environment, in substantially pure or homogeneous form, or, in the case of nucleic acid, free or substantially free of nucleic acid or genes of origin other than the sequence encoding a polypeptide with the required function. 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.

Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, plant cells, yeast and baculovirus systems and transgenic plants and animals. The expression of antibodies and antibody fragments in prokaryotic cells is well established in the art. For a review, see for example Plϋckthun (1991) . A common, preferred 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 binding member for example Chadd & Chamow (2001) , Andersen & Krummen (2002) , Larrick & Thomas (2001) . Mammalian cell lines available in the art for expression of a heterologous polypeptide include 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.

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. For further details see, for example, Sambrook & Russell (2001). 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., 1988 and Ausubel et al., 1999.

Thus, 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 intracellular expression of the binding members of the present invention as "intrabodies" or intracellular antibodies. Intrabodies may be used for gene therapy. A still further aspect provides a method comprising introducing such nucleic acid 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 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.

In one embodiment, the nucleic acid of the invention is 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 binding member or polypeptide as above.

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. All documents mentioned in this specification are incorporated herein by reference in their entirety. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

Certain aspects and embodiments of the invention will now be illustrated by way of example, with reference to the following experimentation and the accompanying drawings .

EXAMPLE 1

Isolation of anti-GHRELIN scFv

ScFv antibody repertoire

Large single chain Fv (scFv) human antibody libraries derived from spleen lymphocytes from 20 donors and peripheral blood lymphocytes tonsil B cells and bone marrow B cells from 43 donors (Vaughan et al

1996) and cloned into a phagemid vector were used for selections.

Phage display selections

ScFv which recognise ghrelin were isolated from large phage display libraries in a series of repeated selection cycles on acyl (Ser3) ghrelin (Phoenix Pharma Inc) essentially as described in Vaughan et al 1996. In brief, antigen was modified by biotinylation (Lys24) . The phage libraries were incubated with the biotinylated antigen prior to capture with streptavidin-coated beads (Dynabeads M-280) according to manufacturer's protocols (Dynal) . Bound phage were recovered by magnetic separation whilst unbound phage were washed away. Bound phage were then rescued as described in Vaughan et al 1996 and the selection process repeated. A representative proportion of clones from the outputs of selection rounds were subjected to DNA sequencing as described in Vaughan et al 1996 and Osbourn et al 1996. Unique scFv were assessed for their ability to neutralise acyl ghrelin as purified scFv preparations in acyl ghrelin dependent biochemical and cell based assays. To promote isolation of acyl ghrelin specific scFv, the libraries were depleted of des ghrelin binders prior to selection by incubation with solid phase des ghrelin.

Ribosome display selections Ribosome display libraries were created and screened for scFv that specifically recognise acyl (ser3) ghrelin (Phoenix Pharma Inc) essentially as described in Hanes et al 2000. Phage display outputs (see above) were converted to ribosome display format. On the DNA level, a T7 promoter was added at the 5' -end for efficient transcription to mRNA. On the mRNA level, the construct contained a prokaryotic ribosome-binding site (Shine-Dalgarno sequence) . At the 3' end of the single chain, the stop codon was removed and a portion of gill (gene III) was added to act as a spacer (Hanes et al 2000) .

Affinity-based selections were performed whereby, following incubation with the library, biotinylated acyl ghrelin was captured by streptavidin-coated paramagnetic beads (Dynal M280) . Bound tertiary complexes (mRNA-ribosome-scFv-ghrelin) were recovered by magnetic separation whilst unbound complexes were washed away. The mRNA encoding the bound scFv were then recovered by RT-PCR as described in Hanes et al and the selection process repeated with decreasing concentrations (25OnM - 1OnM over 3 rounds) of biotinylated acyl ghrelin present during the selection. Unique scFv were assessed for their ability to neutralise acyl ghrelin as purified scFv preparations in acyl ghrelin dependent biochemical and cell based assays

EXAMPLE 2

Biochemical assay characterisation

The neutralisation potency of purified scFv preparations was assessed in a biochemical assay which monitored the interaction of acyl ghrelin with cell membranes expressing GHSRIa, as presented on SPA beads. HEK293 cells expressing GHSRIa were produced in house. WGA PVT SPA beads were obtained from Amersham Biosciences. Assay buffer comprised 25mM Hepes pH7.4, 5mM MgCl₂, ImM CaCl₂ and 0.4% BSA. In brief, SPA beads were coated with, cell membranes prepared from HEK293 cells expressing GHSRIa. Beads at a concentration of βmg per ml were mixed with cell membrane (25μg membrane per mg of beads) . These were mixed well then incubated on ice (10 minutes) and at 4°C (one hour) . The coated beads were resuspended in assay buffer. Three-fold dilution series of purified scFv samples in assay buffer were added to the wells of a 96 well Optiplate at 25μl per well. Coated beads were added to each well at 50μl per well. To all wells 25μl of ¹²⁵I acyl ghrelin (Amersham Biosciences) was added at a final concentration of 32pM in assay buffer. Plates were incubated for one hour or more at room temperature before reading on a Topcount plate reader. IC₅₀ values were determined by curve fitting data to a four parameter logistic equation using PRISM software.

The results are shown in Figures 1, 7 and 8. The IC₅₀ value for the inhibition of binding of human 32pM ¹²⁵I acyl- ghrelin to cell membranes prepared from HEK293 cells transfected with GHSRIa by CPlAl (IgG₄) was found to be 0.2nM (figure 1).

The K_D for binding of human ¹²⁵I acyl ghrelin to cell membranes prepared from HEK293 cells transfected with GHSRla was calculated to be 32pM (figure 7) .

CPlAl (scFv) was also shown to inhibit binding of human ¹²⁵I acyl ghrelin to cell membranes prepared from HEK293 cells transfected with GHSRla (figure 8) . EXAMPLE 3 Conversion to IgG

Clones were converted from scFv to IgG format by sub-cloning the V_H and V_L domains into vectors expressing whole antibody heavy and light chains respectively. The V_H domain is cloned into a vector (pEU8.2) containing the human heavy chain constant domains and regulatory elements to express whole IgG heavy chain in mammalian cells. Similarly, the V_L domain is cloned into a vector (pEU4.2) for the expression of the human light chain (lambda) constant domains and regulatory elements to express whole IgG light chain in mammalian cells. Vectors for the expression of heavy chains and light chains were originally described in Persic et al 1997 The CAT vectors have been engineered simply by introducing an OriP element. To obtain IgGs, the heavy and light chain IgG expressing vectors are transfected into EBNA-HEK293 mammalian cells. IgGs are expressed and secreted into the medium. Harvests were pooled and filtered prior to purification. The IgG was purified using a HiTrap Protein A HP column (Amersham, 17- 0402-01) following the manufacturers recommendations. IgG was eluted from the column using 0.1 M Citrate (pH 3.0) and neutralised by the addition of Tris-HCl (pH 9.0). Fractions containing IgG were buffer exchanged into PBS using NaplO columns (Amersham, #17-0854-02) and the concentration of IgG was determined spectrophotometrically using an extinction coefficient based on the amino acid sequence of the IgG Mach et al 1992. The purified IgG were analysed for aggregation or degradation using SEC-HPLC.

EXAMPLE 4

Cell based assay characterisation

The ability of antibodies to neutralise the interaction of acyl ghrelin and GHSRla was measured in a cellular assay system where the intracellular Ca²⁺ release resulting from the receptor ligand interaction is visualised by means of a fluorescent calcium indicator Briefly, HEK293 cells transfected with GHSRla were produced in house. Assay buffer comprised 125mM NaCl, 5mM KCl, ImM MgCl₂, 1.5mM CaCl₂, 3OmM Hepes, 2.5mM Probenicid, 5mM Glucose, 1% FBS, pH7.4, Dye loading solution comprised 10ml DMEM + 0.1% FBS, 2OmM Hepes, 2.5mM Probenecid, 0.03% pluronic acid and 20μM Dye FLUO-4 AM (TEF labs). HEK 293 cells transfected with GHSRla were seeded at 1 x 10⁵ per well in lOOμl on black-walled Poly D-Lysine treated plates (Costar) and incubated overnight at 37°C and 5% CO2 in DMEM. Media was aspirated and lOOμl per well of dye loading solution added. The cells were incubated for 60 minutes at 37°C in 5% CO₂. The cells were then washed twice in lOOμl PBS. To each well 70μl of FLIPR buffer was added. The cells were left to rest for 10 mins at 37°C in 5% CO₂. Inhibitor titrations (1:3) were prepared in a V bottom plate (Greiner) in assay buffer in a volume of 20μl. Acyl ghrelin (20μl of 33nM stock) was added to each well of the inhibitor plate. Six wells with ghrelin only (no inhibitors) were included to determine the maximum response. Positive control wells for the dye loading process were prepared using Ionomycin (Calbiochem) diluted 2.4μl per lOOOμl in FLIPR buffer. Negative control wells were prepared without cells and with cells but no dye. The inhibitor plate was incubated for 30mins before addition of 30μl to each well of the cell plate (final assay concentration of 5nM acyl ghrelin) . The maximum ghrelin response was generally between 10,000 and 16,000 counts. Data were normalised to the maximum ghrelin response and expressed as a percentage of total response. Analysis was by non-linear regression (with constraints) in Prism to calculate IC₅₀ values.

The EC₅₀ values of 2.7 and 4.6nM for human and rat ghrelin respectively, were determined for Ca²⁺ signalling in HEK293 cells expressing GHSRla (figure 2) . IC₅₀ values of 1.3nM for CPlAl (IgG₄) and 3.3nM for Dlys3 GHRP-6 were determined for the inhibition of Ca²⁺ signalling evoked by 5nM human acyl ghrelin in HEK293 cells expressing GHSRIa (figure 3) . IC₅₀ values of 0.7nM for CPlAl (IgG₄) and 1.7nM for Dlys3 GHRP-6 were determined for the inhibition of Ca²⁺ signalling evoked by 5nM rat acyl ghrelin in these cells (figure 4) .

CPlAl (scFv) was also shown to inhibit Ca²⁺ signalling in HEK293 cells expressing GHSRIa induced by human (figure 9) and rat (figure 10) ghrelin.

EXAMPLE 5

Antibody specificity analysis

Specificity analysis was carried out using a soluble inhibition ELISA. Briefly, Nunc plates were coated with 50μl Neutravidin per well, diluted 1:1000 in PBS and incubated overnight at 4°C. Biotinylated acyl ghrelin at Iμgml^"1 in PBS was added to each well and incubated for 30 minutes at room temperature. The wells were rinsed with PBS and blocked with 3% Marvel PBS for 30 minutes at room temperature.

IgG samples were prepared by diluting in 3% Marvel PBS to 20μgml^~1. Soluble antigens were prepared at double the required final concentration. IgG and antigens were mixed and incubated for 1 hour at room temperature before transfer to the acyl ghrelin plate. Plates were washed with PBS-tween and PBS. IgG binding was detected by HRP conjugated anti Lambda or Kappa IgG' s. Plates were incubated for 1 hour then washed with PBS-tween and PBS. TMB substrate (50μl) was added to each well. The colorimetric reaction was quenched by the addition of 25μl sulphuric acid. Plates were read at 45OnM. Data was analysed as the percentage of ELISA signal compared with that of a no soluble antigen control. The ability of peptide fragments of des-ghrelin and Neurotensin to inhibit binding of CPlAl (IgG) to acyl ghrelin was determined. No inhibition of binding of CPlAl to acyl ghrelin by des ghrelin peptides was observed (figure 5) . No binding of CAT-OOl to acyl ghrelin was observed.

The ability of peptide fragments of acyl ghrelin, Neurotensin and Motilin to inhibit binding of CPlAl (IgG) to acyl ghrelin was determined. Acyl ghrelin and acyl ghrelin fragments 1-5 and 1-14 were observed to inhibit binding of CPlAl to acyl ghrelin. No inhibition of binding was observed by acyl (ser 18) ghrelin, Motilin or neurotensin. No binding of CAT-001 to acyl ghrelin was observed (figure 6) .

EXAMPLE 6 Affinity of GHRELIN binding antibodies .

The BIAcore 2000 System (Pharmacia Biosensor) was used to assess the kinetic parameters of the interaction of IgGs with acyl Ghrelin. The Biosensor uses the optical effects of surface plasmon resonance to study changes in surface concentration resulting from the interaction of an analyte molecule with a ligand molecule that is linked to a chip surface. Typically the analyte species in free solution is passed over the coupled ligand and any binding is detected as an increase in local SPR signal. This is followed by a period of washing, during which dissociation of the analyte species is seen as a decrease in SPR signal. After which any remaining analyte is stripped from the ligand and the procedure repeated at several different analyte concentrations. A series of controls are usually employed during an experiment to ensure that neither the absolute binding capacity or kinetic profile of the coupled ligand change significantly. A proprietary hepes buffer saline (HBS-EP) is typically used as the main diluent of analyte samples and dissociation phase solvent. The experimental data is recorded in resonance units (directly corresponding to the SPR signal) with respect to time. The resonance units are directly proportional to the size and quantity of analyte bound. The BIAevaluation software package can then be used to fit to the data according to pre-determined models in order to assign a rate constant to the dissociation phase (dissociation rate units s^"1) and association phase (association rate units M^"1 s^"1) . These figures then allow calculation of the Association and Dissociation Affinity Constants .

The affinity of the IgG clone, CPlAl was estimated using a single experiment in which biotinylated acyl Ghrelin was non-covalently captured by streptavidin surface. A series of IgG dilutions, from 100 to 0.78nM were then sequentially passed over the acyl Ghrelin ligand. The molarity of IgG was calculated using the concentration (assessed by measurement of absorbance at 280nm) and the estimated non post- translationally modified mature polypeptide mass (150 kDa) . A flow rate of 30 μl inin^{^1} was used and the CPlAl analyte injected into the flow cell for 60 s and the changes in resonance i.e. dissociation from the chip, measured for 1200 s. The chip surface was regenerated fully by a 30 s injection of 10 mM Glycine pH 1.5 followed by a 60 s pause before injection of the proceeding sample.

Reference cell corrected data was subject to fitting using the standard BIAcore bivalent analyte model for simultaneous global calculation of the association and dissociation rates, with the Rmax value set to local. The level of acyl ghrelin captured during each cycle was assessed to ensure that the quantity captured remained stable during the entire experiment. Additionally, the dissociation rate of acyl ghrelin was assessed to determine if a correction for baseline drift was required. However, these interactions proved to be sufficiently reproducible and stable. The validity of the data was constrained by the calculated chi² and T value (parameter value/offset), which were <2 and >50 respectively. The calculated K_D value of CPlAl is approximately 708pM constituting a ka value of 4.73XlO⁵M^-1S^"1 and a kd value of 3.35xlO^"4 s^"1. The chi² value for the fit was 0.0656 with T values of 79.2 (ka) and 144 (kd) .

EXAMPLE 7

Pharmacokinetic analysis of GHRELIN binding antibodies in mice. Exposure to CPlAl following a single I. P. dose was determined in C57/BL6 mice. Forty male mice (25g) received CPlAl (10mg/kg i.p.) and were killed in batches of five animals for plasma sampling at the following time points: 2, 6, 24, 48, 96, 240, 460 and 504hr. Individual plasma samples were analysed by ELISA for total CPlAl IgG using CPlAl for the standard curve. Human antibody was detected in mouse plasma using an anti-human Fc capture antibody and an anti-human Lambda light chain detection antibody. Data was analysed using Excel to determine the pharmacokinetic (PK) profile of the CPlAl antibody.

The Antibody absorbtion phase was between 0 & 6 hours with redistribution phase being estimated between 6-96 hours (figure 11) . The elimination phase of CPlAl was calculated from time points post 240 hours (CPlAl T 1/2 app. 6 days) .

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Claims

CLAIMS :

1. An isolated binding member for acyl-ghrelin, comprising an antibody antigen-binding site which is composed of a human antibody VH domain and a human antibody VL domain and which comprises a set of CDRs HCDRl, HCDR2, HCDR3, LCDRl, LCDR2 and LCDR3, wherein the VH domain comprises HCDR 1, HCDR2, HCDR3 and a framework and the VL domain comprises LCDRl, LCDR2, LCDR3 and a framework, wherein the set of CDRs is selected from the group consisting of: i) a set of CDRs defined wherein the HCDRl has the amino acid sequence of any one of SEQ ID NOS: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, and 113, the HCDR2 has the amino acid sequence of any one of SEQ ID NOS: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, and 114, the HCDR3 has the amino acid sequence any one of SEQ ID NOS: 5, 15, 25,

35, 45, 55, 65, 75, 85, 95, 105, and 115, the LCDRl has the amino acid sequence of any one of SEQ ID NOS: 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, and 118, the LCDR2 has the amino acid sequence of any one of SEQ ID NOS: 9, 19, 29, 39, 49, 59, 69, 79, 89, 99, 109, and 119, and the LCDR3 has the amino acid sequence of any one of SEQ ID NOS: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, and 120; or,

(ii) a set of CDRs which contains one or more amino acid substitutions, deletions or insertions compared with a set of CDRs of (i) •

2. A binding member according to claim 1 wherein the set of VH CDRs is defined wherein;

HCDRl, HCDR2 and HCDR3 have the sequence of SEQ ID NOS: 3 to 5 respectively, HCDRl, HCDR2 and HCDR3 have the sequence of SEQ ID NOS: 13 to 15 respectively,

HCDRl, HCDR2 and HCDR3 have the sequence of SEQ ID NOS: 23 to 25 respectively, HCDRl, HCDR2 and HCDR3 have the sequence of SEQ ID NOS: 33 to 35 respectively,

HCDRl, HCDR2 and HCDR3 have the sequence of SEQ ID NOS: 43 to 45 respectively, HCDRl, HCDR2 and HCDR3 have the sequence of SEQ ID NOS: 53 to 55 respectively,

HCDRl, HCDR2 and HCDR3 have the sequence of SEQ ID NOS: 63 to 65 respectively,

HCDRl, HCDR2 and HCDR3 have the sequence of SEQ ID NOS: 73 to 75 respectively,

HCDRl, HCDR2 and HCDR3 have the sequence of SEQ ID NOS: 83 to 85 respectively,

HCDRl, HCDR2 and HCDR3 have the sequence of SEQ ID NOS: 93 to 95 respectively, HCDRl, HCDR2 and HCDR3 have the sequence of SEQ ID NOS: 103 to 105 respectively, or;

HCDRl, HCDR2 and HCDR3 have the sequence of SEQ ID NOS: 113 to 115 respectively.

3. A binding member according to claim 1 or claim 2 wherein the set of VL CDRs is defined wherein;

LCDRl, LCDR2 and LCDR3 have the sequence of SEQ ID NOS: 8 to 10 respectively,

LCDRl, LCDR2 and LCDR3 have the sequence of SEQ ID NOS: 18 to 20 respectively,

LCDRl, LCDR2 and LCDR3 have the sequence of SEQ ID NOS: 28 to 30 respectively,

LCDRl, LCDR2 and LCDR3 have the sequence of SEQ ID NOS: 38 to 40 respectively, LCDRl, LCDR2 and LCDR3 have the sequence of SEQ ID NOS: 48 to 50 respectively,

LCDRl, LCDR2 and LCDR3 have the sequence of SEQ ID NOS: 58 to 60 respectively, LCDRl, LCDR2 and LCDR3 have the sequence of SEQ ID NOS: 68 to 70 respectively,

LCDRl, LCDR2 and LCDR3 have the sequence of SEQ ID NOS: 78 to 80 respectively, LCDRl, LCDR2 and LCDR3 have the sequence of SEQ ID NOS: 88 to 90 respectively,

LCDRl, LCDR2 and LCDR3 have the sequence of SEQ ID NOS: 98 to 100 respectively,

LCDRl, LCDR2 and LCDR3 have the sequence of SEQ ID NOS: 108 to 110 respectively, or

LCDRl, LCDR2 and LCDR3 have the sequence of SEQ ID NOS: 118 to 120 respectively.

4. A binding member according to claim 1 or claim 2 wherein the set of CDRs is defined wherein;

HCDRl, HCDR2 and HCDR3 have the sequence of SEQ ID NOS: 3 to 5 respectively and LCDRl, LCDR2 and LCDR3 have the sequence of SEQ ID

NOS: 8 to 10 respectively,

HCDRl, HCDR2 and HCDR3 have the sequence of SEQ ID NOS: 13 to 15 respectively and LCDRl, LCDR2 and LCDR3 have the sequence of SEQ ID

NOS: 18 to 20 respectively,

HCDRl, HCDR2 and HCDR3 have the sequence of SEQ ID NOS: 23 to 25 respectively and LCDRl, LCDR2 and LCDR3 have the sequence of SEQ ID

NOS: 28 to 30 respectively, HCDRl, HCDR2 and HCDR3 have the sequence of SEQ ID NOS: 33 to 35 respectively and LCDRl, LCDR2 and LCDR3 have the sequence of SEQ ID

NOS: 38 to 40 respectively,

HCDRl, HCDR2 and HCDR3 have the sequence of SEQ ID NOS: 43 to 45 respectively and LCDRl, LCDR2 and LCDR3 have the sequence of SEQ ID NOS: 48 to 50 respectively,

HCDRl, HCDR2 and HCDR3 have the sequence of SEQ ID NOS: 53 to 55 respectively and LCDRl, LCDR2 and LCDR3 have the sequence of SEQ ID

NOS: 58 to 60 respectively, HCDRl, HCDR2 and HCDR3 have the sequence of SEQ ID NOS: 63 to 65 respectively and LCDRl, LCDR2 and LCDR3 have the sequence of SEQ ID

NOS: 68 to 70 respectively,

HCDRl, HCDR2 and HCDR3 have the sequence of SEQ ID NOS: 73 to 75 respectively and LCDRl, LCDR2 and LCDR3 have the sequence of SEQ ID

NOS: 78 to 80 respectively,

HCDRl, HCDR2 and HCDR3 have the sequence of SEQ ID NOS: 83 to 85 respectively and LCDRl, LCDR2 and LCDR3 have the sequence of SEQ ID

NOS: 88 to 90 respectively, HCDRl, HCDR2 and HCDR3 have the sequence of SEQ ID NOS: 93 to 95 respectively and LCDRl, LCDR2 and LCDR3 have the sequence of SEQ ID

NOS: 98 to 100 respectively,

HCDRl, HCDR2 and HCDR3 have the sequence of SEQ ID NOS: 103 to 105 respectively and LCDRl, LCDR2 and LCDR3 have the sequence of SEQ ID NOS: 108 to 110 respectively, or

HCDRl, HCDR2 and HCDR3 have the sequence of SEQ ID NOS: 113 to 115 respectively and LCDRl, LCDR2 and LCDR3 have the sequence of SEQ ID

NOS: 118 to 120 respectively.

5. A binding member according to any one of claims 1 to 4 wherein the set of CDRs consists of a set of CDRs selected from the group consisting of: the CPlAl set of CDRs, defined wherein the HCDRl has the amino acid sequence of SEQ ID NO: 3, the HCDR2 has the amino acid sequence of SEQ ID NO: 4, the HCDR3 has the amino acid sequence of SEQ ID NO:

5, the LCDRl has the amino acid sequence of SEQ ID NO: 8, the LCDR2 has the amino acid sequence of SEQ ID NO: 9, and the LCDR3 has the amino acid sequence of SEQ ID NO: 10; and, a set of CDRs which contains one or more amino acid substitutions, deletions or insertions compared with the CPlAl set of

CDRs.

6. A binding member according to claim 5 comprising the CPlAl set of CDRs.

7. A binding member according to any one of claims 1 to 6 wherein the VH domain framework is human heavy chain germline framework and/or the VL domain framework is human light chain germline framework.

8. A binding member according to any one of claims 1 to 7 which binds human, rat and/or mouse acyl-ghrelin.

9 A binding member according to any one of claims 1 to 8 comprising the CPlAl VH domain (SEQ ID NO: 2) .

10. A binding member according to any one of claims 1 to 9 comprising the CPlAl VL domain (SEQ ID NO: 7) .

11. A binding member according to any one of claims 1 to 10 that binds human acyl-ghrelin with affinity equal to or better than the affinity of an antigen-binding site for human acyl-ghrelin formed by the CPlAl VH domain (SEQ ID NO: 2) and the CPlAl VL domain (SEQ ID NO: 7), the affinity of the binding member and the affinity of the antigen-binding site being as determined under the same conditions.

12. An isolated binding member for acyl-ghrelin, comprising an antibody antigen-binding site which is composed of a human antibody VH domain and a human antibody VL domain and which comprises a set of CDRs HCDRl, HCDR2, HCDR3, LCDRl, LCDR2 and LCDR3, wherein the HCDRl has the amino acid sequence of SEQ ID NO: 3, the HCDR2 has the amino acid sequence of SEQ ID NO: 4, the HCDR3 has the amino acid sequence of SEQ ID NO: 5, the LCDRl has the amino acid sequence of SEQ ID NO: 8, the LCDR2 has the amino acid sequence of SEQ ID NO: 9, and the LCDR3 has the amino acid sequence of SEQ ID NO: 10.

13. An isolated binding member for acyl-ghrelin, comprising an antibody antigen-binding site which competes for binding to acyl- ghrelin with an antibody antigen-binding site which is composed of the CPlAl VH domain (SEQ ID NO: 2) and the CPlAl VL domain (SEQ ID NO: 7) .

5

14. A binding member according to any one of claims 1 to 13 that binds to and/or neutralises human acyl-ghrelin.

15. A binding member according to claim 14 that neutralizes human 10 acyl-ghrelin, with a potency equal to or better than the potency of a acyl-ghrelin antigen-binding site formed by the CPlAl VH domain (SEQ ID NO: 2) and the CPlAl VL domain (SEQ ID NO: 7), the potency of the binding member and the potency of the antigen-binding site being as determined under the same conditions . 15

16. A binding member according to any one of claims 1 to 15, which binds to human acyl-ghrelin with an affinity (KD) of 708 pM or less in a surface plasmon resonance assay.

20 17. A binding member according to any one of claims 1 to 16, wherein the binding member has an IC50 of not more than 2 nM in an "FLIPR" calcium signaling assay for neutralization of binding of acyl ghrelin to GSHRIa, with a final concentration of 5nM acyl ghrelin.

25 18. A binding member according to claim 17, wherein the IC50 is not more than 1.5nM.

19. An isolated antibody for acyl-ghrelin comprising an antibody antigen-binding site which is composed of the CPlAl VH domain (SEQ ID

30 NO: 2) and the CPlAl VL domain (SEQ ID NO: 7) .

20. A binding member according to any one of claims 1 to 18 that comprises an scFv antibody molecule.

21. A binding member according to any one of claims 1 to 18 that comprises an antibody constant region.

22. A binding member according to claim 21 that comprises a whole antibody.

23. A binding member according to claim 22 wherein the whole antibody is IgG4.

24. An isolated VH domain of an antibody molecule according to any one of claims 1 to 23.

25. An isolated VL domain of an antibody molecule according to any one of claims 1 to 23.

26. A composition comprising an isolated binding member, an antibody, an antibody VH domain or an antibody VL according to any one of claims 1 to 25 and at least one additional component.

27. A composition according to claim 26 comprising a pharmaceutically acceptable excipient, vehicle or carrier.

28. An isolated nucleic acid which comprises a nucleotide sequence encoding a binding member, antibody, or antibody VH or VL domain of a binding member according to any one of claims 1 to 25.

29. A host cell in vitro transformed with nucleic acid according to claim 28.

30. A method of producing a binding member or an antibody VH or VL domain, the method comprising culturing host cells according to claim 29 under conditions for production of said binding member or antibody VH or VL domain.

5

31. A method according to claim 30 further comprising isolating and/or purifying the binding member or antibody VH or VL variable domain.

10 32. A method according to claim 30 or claim 31 further comprising formulating the binding member or antibody VH or VL variable domain into a composition including at least one additional component.

33. A method for producing an antibody antigen-binding domain for

15 acyl-ghrelin, the method comprising; providing, by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a parent VH domain comprising HCDR 1, HCDR2 and HCDR3, wherein the parent VH domain HCDRl, HCDR2 and HCDR3 are the CPlAl set of HCDRs,

20 defined wherein the HCDRl has the amino acid sequence of SEQ ID NO: 3, the HCDR2 has the amino acid sequence of SEQ ID NO: 4 and the HCDR3 has the amino acid sequence of SEQ ID NO: 5, a VH domain which is an amino acid sequence variant of the parent VH domain, and optionally combining the VH domain thus provided with one or more VL domains to

25 provide one or more VH/VL combinations; and testing said VH domain which is an amino acid sequence variant of the parent VH domain or the VH/VL combination or combinations to identify an antibody antigen binding domain specific for acyl-ghrelin.

30 34. A method according to claim 33 wherein the parent VH domain amino acid sequence is SEQ ID NO: 2.

35. A method according to claim 33 or claim 34 wherein said one or more VL domains is provided by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a parent VL domain comprising LCDR 1, LCDR2 and LCDR3, wherein the

5 parent VL domain LCDRl, LCDR2 and LCDR3 are the CPlAl set of LCDRs, defined wherein the LCDRl has the amino acid sequence of SEQ ID NO: 8, the LCDR2 has the amino acid sequence of SEQ ID NO: 9, and the LCDR3 has the amino acid sequence of SEQ ID NO: 10, producing one or more VL domains each of which is an amino acid sequence variant of the 10 parent VL domain.

36. A method according to claim 35 wherein the parent VL domain amino acid sequence is SEQ ID NO: 7.

15 37. A method according to any of claims 33 to 36, wherein the acyl- ghrelin is human acyl-ghrelin.

38. A method of obtaining a binding member that binds acyl-ghrelin, the method comprising

20 providing by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of the CPlAl VH domain (SEQ ID NO. 2) one or more VH domains each of which is an amino acid sequence variant of the CPlAlVH domain, optionally combining one or more VH domain amino acid sequence variants thus provided with one

25 or more VL domains to provide one or more VH/VL combinations; and/or providing by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of the CPlAl VL domain (SEQ ID NO. 7) a VL domain which is an amino acid sequence variant of the CPlAl VL domain, and

30 combining one or more VL domain amino acid sequence variants thus provided with one or more VH domains to provide one or more VH/VL domain combinations; and testing the VH domain amino acid sequence variants or VH/VL combination or combinations for to identify a binding member that binds acyl-ghrelin.

39. A method according to any one of claims 33 to 38 wherein said VH domain which is an amino acid sequence variant of the parent VH domain is provided by CDR mutagenesis.

40. A method according to any one of claims 33 to 39 further comprising producing the antibody antigen-binding site as a component of an IgG, scFv or Fab antibody molecule.

41. A method for producing a binding member that binds acyl-ghrelin, which method comprises: providing starting nucleic acid encoding a VH domain or a starting repertoire of nucleic acids each encoding a VH domain, wherein the VH domain or VH domains either comprise a HCDRl, HCDR2 and/or HCDR3 to be replaced or lack a HCDRl, HCDR2 and/or HCDR3 encoding region; combining said starting nucleic acid or starting repertoire with donor nucleic acid or donor nucleic acids encoding or produced by mutation of the amino acid sequence of HCDRl SEQ ID NO: 3, HCDR2 SEQ ID NO: 4, and/or HCDR3 SEQ ID NO: 5, such that said donor nucleic acid is or donor nucleic acids are inserted into the CDRl, CDR2 and/or CDR3 region in the starting nucleic acid or starting repertoire, so as to provide a product repertoire of nucleic acids encoding VH domains; expressing the nucleic acids of said product repertoire to produce product VH domains; optionally combining said product VH domains with one or more VL domains; selecting a binding member for acyl-ghrelin, which binding member comprises a product VH domain and optionally a VL domain; and recovering said binding member or nucleic acid encoding it.

42. A method according to claim 41 wherein the donor nucleic acids are produced by mutation of said HCDRl and/or HCDR2.

5 43. A method according to claim 41 wherein the donor nucleic acid is produced by mutation of HCDR3.

44. A method according to claim 43 comprising providing the donor nucleic acid by mutation of nucleic acid encoding the amino

10 acid sequence of CPlAl HCDR3 (SEQ ID NO: 5) .

45. A method according to claim 41 comprising providing the donor nucleic acid by random mutation of nucleic acid.

15 46. A method of obtaining a binding member that binds acyl-ghrelin, which method comprises: providing starting nucleic acids encoding one or more VH domains which either comprise a CDR3 to be replaced or lack a CDR3 encoding region, and combining said starting nucleic acid with a donor nucleic

20 acid encoding the VH CDR3 amino acid sequence of SEQ ID NO. 5 such that said donor nucleic acid is inserted into the CDR3 region in the starting nucleic acid, so as to provide product nucleic acids encoding VH domains; or providing starting nucleic acids encoding one or more VL domains

25 which either comprise a CDR3 to be replaced or lack a CDR3 encoding region, and combining said starting nucleic acid with a donor nucleic acid encoding the VL CDR3 amino acid sequence of SEQ ID NO. 10 such that said donor nucleic acid is inserted into the CDR3 region in the starting nucleic acid, so as to provide a product nucleic acids

30 encoding VL domains; expressing the nucleic acids of said product nucleic acids encoding VH domains and optionally combining the VH domains thus produced with one or more VL domains to provide VH/VL combinations, and/or expressing the nucleic acids of said product nucleic acids encoding VL domains and combining the VL domains thus produced with one or more VH domains to provide VH/VL combinations; 5 selecting a binding member comprising a VH domain or a VH/VL combination that binds acyl-ghrelin; and recovering said binding member that binds acyl-ghrelin and/or nucleic acid encoding the binding member that binds acyl-ghrelin.

10 47. A method according to any one of claims 33 to 46 further comprising attaching a product VH domain that is comprised within the recovered binding member to an antibody constant region.

48. A method according to any one of claims 33 to 46 comprising

15 providing an IgG, scFv or Fab antibody molecule comprising the product VH domain and a VL domain.

49. A method according to any of claims 33 to 48, wherein the acyl- ghrelin is human acyl-ghrelin.

20

50. A method according to any one of claims 33 to 49, further comprising testing the antibody antigen-binding domain or binding member that binds acyl-ghrelin for ability to neutralize acyl-ghrelin.

25 51. A method according to claim 50 wherein a binding member comprising an antibody fragment that binds and neutralizes acyl- ghrelin is obtained.

52. A method according to claim 51 wherein the antibody fragment is 30 an scFv antibody molecule.

53. A method according to claim 51 wherein the antibody fragment is an Fab antibody molecule.

54. A method according to claim 52 or claim 53 further comprising providing the VH domain and/or the VL domain of the antibody fragment in a whole antibody.

55. A method according to any one of claims 33 to 54 further comprising formulating the binding member that binds acyl-ghrelin, antibody antigen-binding site or an antibody VH or VL variable domain of the binding member or antibody antigen-binding site that binds acyl-ghrelin, into a composition including at least one additional component .

56. A method according to any one of claims 33 to 55 further comprising binding a binding member that binds acyl-ghrelin to acyl- ghrelin or a fragment of acyl-ghrelin.

57. A method comprising binding a binding member or antibody that binds acyl-ghrelin according to any one of claims 1 to 23 to human acyl-ghrelin or a fragment of human acyl-ghrelin.

58. A method according to claim 56 or claim 57 wherein said binding takes place in vitro.

59. A method according to any of claims 56 to 58, comprising binding the binding member or antibody to human acyl-ghrelin or a fragment of human acyl-ghrelin.

60. A method according to any one of claims 56 to 59 comprising determining the amount of binding of binding member or antibody to acyl-ghrelin or a fragment of acyl-ghrelin.

61. A method according to any one of claims 33 to 60 further comprising use of the binding member or antibody in the manufacture of a medicament for treatment of an acyl-ghrelin associated disease or disorder.

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62. Use of a binding member or antibody according to any one of claims 1 to 23 in the manufacture of a medicament for treatment of an acyl-ghrelin associated disease or disorder.

10 63. A method of treatment of an acyl-ghrelin associated disease or disorder, the method comprising administering a binding member or antibody according to any one of claims 1 to 23 to a patient with the disease or disorder or at risk of developing the disease or disorder.

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