Receptor agonists

Abstract

The present invention relates to fusion proteins that agonise a somatostatin receptor (SSTR), in particular somatostatin receptor 4 (SSTR4), and methods for producing the same. The fusion proteins of the invention comprise a scaffold and a somatostatin (SST)-derived peptide and, optionally, a partner domain.

Description

[0001] RECEPTOR AGONISTS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to fusion proteins that agonise a somatostatin receptor (SSTR), in particular somatostatin receptor 4 (SSTR4), and methods for producing the same.

[0004] BACKGROUND TO THE INVENTION

[0005] Somatostatin (SST), or somatotropin-release inhibitory factor (SRIF), is a cyclic neuropeptide distributed widely in the human central nervous system and peripheral tissues. SST is produced by multiple cell types including neurons and neuroendocrine, immune and inflammatory cells in response to various factors such as ions, nutrients, neuropeptides, neurotransmitters, hormones, growth factors and cytokines (Patel, 1999, Frontiers in Neuroendocrinology 20(3): 157-198). It is a multifunctional peptide that among many roles is able to regulate exocrine secretion, modulate neurotransmitter release and control cell proliferation. The biological effects of SST are mediated by somatostatin receptors 1 to 5 (SSTR1-5), which are members of the family of G protein-coupled receptors (GPCRs).

[0006] While most SSTRs are involved in homeostatic hormone regulation, SSTR4 has been shown to mediate analgesic, anti-inflammatory, and anti-depressant effects by modulating sensory nerve transmission, without influencing hormone secretion (Sandor et al., 2006, Eur. J. Pharm., 539: 71-75). Recent studies have shown that SSTR4 knockout mice develop acute and chronic inflammation, along with neuropathic hyperalgesia.

[0007] SSTR4 agonists have been proposed as promising drug candidates for non-opioid pain control, especially for chronic neuropathic, inflammatory and mixed pain (Sandor et al., 2006; Kantas et al., 2019, Int. J. Mol. Sci., 2019, 20, 6245). Chronic pain affects millions of people worldwide, with some studies indicating upwards of 30% of all people. It is an important public health concern with its high morbidity, increased mortality, and major health-care costs.

[0008] Eli Lilly & Co. are developing a selective SSTR4 small molecule drug, LY-3556050 (previously referred to as CNTX-0290 and acquired from Centrexion Therapeutics), for oral administration for treating several chronic pain indications, including diabetic peripheral neuropathic pain (DPNP) which is being investigated in phase II clinical trials. WO2023 / 044326 describes such selective SSTR4 agonist small molecule salts.

[0009] However, small molecule drugs have their limitations, including a greater propensity for off-target effects and relatively short half-lives; the latter being a serious hindrance when treating chronic pain.

[0010] Peptide Logic is a Californian-based company developing peptide-antibody conjugates (PACs) as a means of increasing the half-life of peptide therapeutics. They were awarded a $3M grant in October 2019 to develop peripherally restricted SSTR4 agonists for pain. However, antibody conjugates often require complex conjugation chemistry which may be expensive, complicates manufacturing and quality control processes, and may lead to immunogenicity and safety issues.

[0011] Thus, there remains a need for safe and effective pain relief with durable effects. As such, long- acting selective SSTR4 agonists are desirable. SUMMARY OF THE INVENTION

[0012] In a first aspect of the invention, a fusion protein is provided comprising: a scaffold and a somatostatin (SST)-derived peptide; wherein the fusion protein is a somatostatin receptor (SSTR) agonist, in particular a SSTR4 agonist, more particularly a selective SSTR4 agonist.

[0013] In a second aspect of the invention, an isolated nucleic acid comprising a nucleotide sequence encoding a fusion protein according to the first aspect of the invention is provided.

[0014] In a third aspect of the invention, a vector comprising the nucleic acid according to the second aspect of the invention is provided.

[0015] In a fourth aspect of the invention, a host cell comprising the nucleic acid according to the second aspect of the invention or the vector according to the third aspect of the invention is provided.

[0016] In a fifth aspect of the invention, there is provided a method of making a fusion protein comprising culturing the host cell according to the fourth aspect of the invention under suitable conditions.

[0017] In a sixth aspect of the invention, a pharmaceutical composition comprising the fusion protein according to the first aspect of the invention, and at least one pharmaceutically acceptable excipient, vehicle or carrier, is provided.

[0018] In a seventh aspect of the invention, a fusion protein according to the first aspect of the invention, or a pharmaceutical composition according to the sixth aspect of the invention, for use as a medicament, optionally for use in treating pain and / or inflammation, is provided.

[0019] In an eighth aspect of the invention, a method of treating a subject in need thereof is provided, said method comprising administering to the subject a therapeutically effective amount of a pharmaceutically acceptable composition according to the sixth aspect of the invention, or a fusion protein according to the first aspect of the invention, optionally wherein the subject has, or is at risk of developing, pain or inflammation.

[0020] In a ninth aspect of the invention, use of a fusion protein according to the first aspect of the invention in the manufacture of a medicament for the treatment of pain and / or inflammation is provided.

[0021] In a tenth aspect of the invention, there is provided a method of generating a fusion protein comprising a scaffold and a SST-12-derived peptide, wherein the fusion protein is a somatostatin receptor (SSTR) agonist, optionally a SSTR4 agonist, optionally a selective SSTR4 agonist, said method comprising: a) identifying a suitable scaffold comprising a disulphide loop; b) replacing the disulphide loop with said SST-12-derived peptide; and c) determining that the fusion protein is a SSTR agonist, optionally a SSTR4 agonist, optionally a selective SSTR4 agonist.

[0022] BRIEF DESCRIPTION OF THE FIGURES

[0023] FIG. 1 depicts expression vectors used in the Examples. plNT2 plasmids contain digestion sites Ncol and Xhol, CH1-CH2-CH3 Hg1 gene is downstream of an Xhol site for insertion of VH domain encoding inserts. plNT112 plasmids contain digestion sites Nhel and Notl. Two variants of the construct were used that had either CL-lambda or CL-kappa downstream of Notl sequence. pINT 112 with Nhel and BamHI digestions sites was used for cloning of single domain VHH (NANOBODIES). All domains to be inserted are represented by white boxes.

[0024] FIG. 2 is a bar chart showing the results from a cAMP inhibition assay at top concentration for SSTR4 agonist testing of IgG antibodies presenting SST-14 derived peptide ‘pepT (SEQ ID NO: 2) in HCDR3. Horizontal dotted line signifies the upper bounds of the assay (forskolin stimulated cells with buffer only). Samples were run at varying top concentrations: AB1 parent (1800nM), AB1 pep1 (1 OOOnM), AB2 pep1 (2000nM), AB3 parent (3000nM), AB3 pep1 (3000nM), AB4 parent (2700nM), AB4 pep1 (2000nM), AB5 parent (3000nM), and AB5 pep1 (720nM). SST-14 was run at 1000 nM. Error bars - standard deviation, n=2.

[0025] FIG. 3 shows results from a cAMP inhibition assay with concentration-response curves for SSTR4 agonist testing of IgG antibodies presenting SST-14 derived peptide ‘pepT (SEQ ID NO: 2) in HCDR3. Sigmoidal curve fit (three parameter) constrained to 1 and 0 as top and bottom values, respectively. Each concentration was tested in duplicate. (A) - AB1 pep1 , (B) - AB2 pep1 , (C) - AB3 pep1, (D) - AB4 pep1 , (E) - AB5 pep1 , (F) - SST-14.

[0026] FIG. 4 is a structural and primary sequence representation of EETI-II (PDB: 1 MRO) and AGRP (PDB: 2IT7): EETI-II loop 1 and loop 5 highlighted (A); and AGRP knottin with loop 4 highlighted (B). Primary sequences with knottin disulphide bridges (black lines) are shown below.

[0027] FIG. 5 is a bar chart showing results from a cAMP inhibition assay at top concentration for SSTR4 agonist testing of KNOTBODY molecules presenting SST-14 derived peptide ‘pepT (SEQ ID NO: 2) in knottin moieties. Horizontal dotted line signifies the upper bounds of the assay (forskolin stimulated cells with buffer only). Samples were run at varying top concentrations: KB1 (7176 nM), KB2 (1000 nM), KB1 pep1 (2000 nM), KB2 L1 pep1 (5000 nM) and KB2 L5 pep1 (1000 nM). SST-14 was run at 1000 nM. Error bars - standard deviation, n=2.

[0028] FIG. 6 shows results from a cAMP inhibition assay with concentration-response curves for SSTR4 agonist testing of KNOTBODY molecules presenting SST-14 derived peptide ‘pepT (SEQ ID NO: 2) in knottin moieties. Sigmoidal curve fit constrained to 1 and 0 as top and bottom values, respectively. Each concentration tested in duplicate. (A) - KB1 pep1, (B) - KB2 L1 pep1 , (C) - KB2 L5 pep1 , (D) - SST-14.

[0029] FIG. 7 is a bar chart showing results from a cAMP inhibition assay at top concentration for SSTR4 agonist testing of an IgG antibody presenting SST-14 derived peptide ‘pepT (SEQ ID NO: 2) in LCDR2. Horizontal dotted line signifies the upper bounds of the assay (forskolin stimulated cells with buffer only). Samples were run at varying top concentrations: AB6 pep1 (1700 nM), KB1 (7176 nM) and KB2 (1000 nM). SST-14 was run at 1000 nM. Error bars - standard deviation, n=2.

[0030] FIG. 8 is a bar chart showing results from a cAMP inhibition assay at top concentration for SSTR4 agonist testing of single domain VHH (NANOBODIES) presenting SST-14 derived peptide ‘pepT (SEQ ID NO: 2) in CDR3. Horizontal dotted line signifies the upper bounds of the assay (forskolin stimulated cells with buffer only). Samples were run at 4000 nM. SST-14 run at 1000 nM. Error bars - standard deviation, n=2.

[0031] FIG. 9 shows results from a cAMP inhibition assay with concentration-response curves for SSTR4 agonist testing of single domain VHH (NANOBODIES) presenting SST-14 derived peptides in CDR3. Sigmoidal curve fit (three-parameter) constrained to 1 and 0 as top and bottom values, respectively. Each concentration tested in duplicate. (A) - NB1 pep1, (B) - NB2 pepT FIG. 10 is a bar chart showing results from a cAMP inhibition assay at top concentration for SSTR4 agonist testing of an IgG antibody presenting SST-14 derived peptides ‘pep1 ’ (SEQ ID NO: 2), ‘pep2’ (SEQ ID NO: 3), ‘pep3’ (SEQ ID NO: 4), ‘pep4’ (SEQ ID NO: 5), ‘pep5’ (SEQ ID NO: 6) and ‘pep6’ (SEQ ID NO: 7) in HCDR3. Horizontal dotted line signifies the upper bounds of the assay (forskolin stimulated cells with buffer only). Samples were run at varying top concentrations: AB1 parent (1800 nM), AB1 pep1 (1000 nM), AB1 pep2 (900 nM), AB1 pep3 (2700 nM), AB1 pep4 (2000 nM), AB1 pep5 (3000 nM), AB1 pep6 (3000 nM). SST-14 was run at 1000 nM. Error bars - standard deviation, n=2.

[0032] FIG. 11 shows bar chart results from a cAMP inhibition assay at top concentration for SSTR4 / SSTR2 agonism selectivity testing of an IgG antibody presenting SST-14 derived peptides ‘pepT (SEQ ID NO: 2) and ‘pep4’ (SEQ ID NO: 5) in HCDR3. Horizontal dotted line signifies the upper bounds of the assay (forskolin stimulated cells with buffer only). Samples were run at varying top concentrations: AB1 parent (1800 nM), AB1 pep1 (1000 nM) and AB1 pep4 (2000 nM). SST-14 was run at 1000 nM. Error bars - standard deviation, n=2. (A) Data from SSTR2 testing, (B) Data from SSTR4 testing.

[0033] FIG. 12 shows results from a cAMP inhibition assay with concentration-response curves for SSTR4 / SSTR2 agonism selectivity testing of an IgG antibody presenting a SST-14 derived peptide ‘pep4’ (SEQ ID NO: 5) in HCDR3. Construct tested - AB1 pep4. Sigmoidal curve fit (four-parameter) constrained to 1 and 0 as top and bottom values, respectively. Each concentration was tested in duplicate. (A) Data from SSTR2 testing, (B) Data from SSTR4 testing.

[0034] FIG. 13 shows results from a cAMP inhibition assay with concentration-response curves for SSTR4 agonist testing of a panel of IgG antibodies presenting SST-14 derived peptide ‘pepT (SEQ ID NO: 2) across a variety of heavy chain and light chain CDRs. (A) - AB7 pep1 LCDR1 , LCDR2 and LCDR3, (B) - AB10 pep1 HCDR3 and LCDR2, (C) - AB9 pep1 HCDR1 and HCDR2, (D) - AB8 pep1 HCDR1 and HCDR2, and (E) - AB7 pep1 HCDR1 and HCDR2. Sigmoidal curve fit (four-parameter), with mean data points plotted with standard deviation error bars. Each concentration was repeated 3-4 times.

[0035] FIG. 14 shows results from a cAMP inhibition assay with concentration-response curves for SSTR4 agonist testing of two IgG antibodies presenting full-length somatostatin (SST-14 also known as ‘pep7’ (SEQ ID NO: 1 )): AB2 pep7 and AB4 pep7. Sigmoidal curve fit (four-parameter), with mean data points plotted with standard deviation error bars. Each concentration was repeated 3-4 times.

[0036] FIG. 15 is a bar chart showing results from a cAMP inhibition assay for SSTR4 agonist testing of a variety of biologic modalities presenting full-length somatostatin-14 peptide (SST-14 / pep7, SEQ ID NO:1 ) or a somatostatin-14-derived peptide (SST-12 / pep1, SEQ ID NO:2). Mean values are plotted with standard deviation, n=2 repeats per test article.

[0037] DETAILED DESCRIPTION OF THE INVENTION

[0038] DEFINITIONS

[0039] The term ‘antibody’ is used herein in the broadest sense to refer to molecules with an immunoglobulin-like domain (e.g. IgG, IgM, IgA, IgD or IgE) and includes monoclonal, recombinant, polyclonal, chimeric, human, and humanized molecules of this type. The terms ‘full’, ‘whole’ or ‘intact’ antibody, used interchangeably herein, refer to a heterotetrameric glycoprotein with an approximate molecular weight of 150,000 Daltons. An intact antibody is composed of two identical heavy chains (HCs) and two identical light chains (LCs) linked by covalent disulphide bonds. This H2L2 structure folds to form three functional domains comprising two antigen-binding fragments, known as ‘Fab’ fragments, and a ‘Fc’ crystallisable fragment. The Fab fragment is composed of the variable region at the amino-terminus, variable heavy (VH) or variable light (VL), and the constant region at the carboxyl terminus, CH1 (heavy) and CL (light), respectively. The Fc fragment is composed of two domains formed by dimerization of paired CH2 and CH3 regions. The Fc may elicit effector functions by binding to receptors on immune cells or by binding C1q, the first component of the classical complement pathway. The five classes of antibodies IgM, IgA, IgG, IgE and IgD are defined by distinct heavy chain amino acid sequences which are called , a, y, E, and 5, respectively, each heavy chain can pair with either a K or A light chain. The majority of antibodies in the serum belong to the IgG class, there are four isotypes of human IgG, namely lgG1 , lgG2, lgG3 and lgG4, the sequences of which differ mainly in their hinge region.

[0040] The term ‘antibody-derived scaffold’ refers to a scaffold molecule comprising an immunoglobulin- like domain and includes an intact antibody, a single variable domain, a VHH domain antibody, an antigen binding fragment (such as a Fab), a single chain Fv, an Fc domain, a diabody, a minibody, etc. (for a summary of alternative ‘antibody’ formats see Holliger and Hudson, Nature Biotechnology, 2005, Vol 23, No. 9, 1126-1136).

[0041] ‘CDRs’ are defined as the complementarity determining region amino acid sequences of an antibody. These are the hypervariable regions of immunoglobulin heavy and light chains. There are three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) and three light chain CDRs (LCDR1, LCDR2 and LCDR3) in the variable portion of an immunoglobulin. Thus, ‘CDRs’ as used herein refers to all three heavy chain CDRs, all three light chain CDRs, all heavy and light chain CDRs, or at least two CDRs. Throughout this specification, amino acid residues in variable domain sequences are numbered according to the IMGT numbering convention and Fc domain sequences are numbered according to the EU numbering convention. It will be apparent to those skilled in the art that there are alternative numbering conventions for amino acid residues in variable region sequences and full-length antibody sequences. The CDRs referred to herein, in particular in the examples section and assigned SEQ ID NOs, are according to the IMGT numbering convention. There are also alternative numbering conventions for CDR sequences, for example those set out according to Kabat and Chothia et al. (1989) Nature 342: 877-883. The structure and protein folding of the antibody may mean that other residues are considered part of the CDR sequence and would be understood to be so by a skilled person. Other numbering conventions for CDR sequences available to a skilled person include ‘AbM’ (University of Bath) and ‘contact’ (University College London) methods. Table 1 below represents one definition using each numbering convention for each CDR. It should be noted that some of the CDR definitions may vary depending on the individual publication used.

[0042] Table 1 CDR Definitions according to different numbering conventions.

[0043] A ‘cysteine-rich peptide’ as used herein comprises at least two disulphide bonds, preferably three disulphide bonds, as a core structural element and is between 20 and 60 amino acids, preferably 30 and 50 amino acids, in length. A cysteine-rich peptide may comprise a ‘disulphide-directed beta hairpin’ (DDH) structure comprising an anti-parallel beta hairpin stabilised by two disulphide bonds. The DDH core structure is found in the widely studied ‘inhibitory cysteine knot (ICK)’ structure of knottins. Both knottins and related backbone-cyclised peptides known as cyclotides typically comprise three disulphide bonds and are examples of cysteine-rich peptides (Kintzing and Cochran, 2016, Current Opinion in Chemical Biology, 34: 143-150). Cysteine-rich peptides may function as ion-channel inhibitors (i.e. ion channel modulating, ICM, peptides), including those naturally produced by venomous animals and synthetic derivatives thereof (i.e. venom-derived, VD, peptides).

[0044] A ‘domain’ as used herein is a folded protein structure which has tertiary structure independent of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins.

[0045] A ‘fusion protein’ as used herein is a chimeric protein created by joining at least two polypeptide sequences from different sources, e.g. encoded by different genes, together such that they are transcribed and translated as a single unit. As used herein, a fusion protein comprises a scaffold and an SST -derived peptide. The SST-derived peptide may be joined directly (a direct fusion) or via linkers to the N-terminal or C-terminal of the scaffold or inserted at a site within the scaffold with or without linkers.

[0046] A ‘KNOTBODY’ as used herein is an antibody or antigen-binding fragment thereof comprising a knottin inserted into or replacing one or more CDRs. KNOTBODY molecules are described in detail in WO2017 / 118761.

[0047] A ‘knottin’ as used herein is a cysteine-rich peptide that has an interwoven disulfide-bonded framework, triple-stranded p-sheet fold, and one or more solvent exposed loops. Knottins typically comprise at least 3 disulfide bridges and are characterised by a disulphide knot which is achieved when a disulfide bridge between cysteines, e.g. Ill and VI, crosses the macrocycle formed by the two other disulfides (e.g. disulfides l-IV and ll-V) and the interconnecting backbone. These bridges stabilise the common tertiary fold formed of antiparallel p-sheets and in some cases a short 3io helix. The pairs of Roman numerals refer to the order in which each cysteine appears in the sequence and the position of the partner cysteine with which it forms a disulphide bond. Knottins are 20-60 residues long, usually 26- 48 residues, and are found in diverse organisms ranging from arthropods, molluscs, and arachnids to plants. Knottins arising from conus snails (conotoxins) in particular have been widely studied. Many thousands of other knottins have been identified and their sequences and structures are publicly available for example from on-line databases (such as the Knottin on-line database, Centre de Biochimie Structural, CNRS, France).

[0048] A ‘linker’ is used herein to mean one or more amino acid residues that join the scaffold to the SST-derived peptide in the fusion protein.

[0049] A ‘loop’ or ‘disulphide loop’ as defined herein with respect to a scaffold that is not derived from an antibody (i.e. not an antibody-derived scaffold) is an amino acid sequence between and including sequential cysteine residues. In the case of knottins, loops are numbered 1-5 based on their natural order. For example, loop 1 of a knottin extends between the first cysteine and the second cysteine, loop 5 extends between the fifth and sixth cysteines, and so forth. A loop may be flexible and connect secondary structure elements without having easily observable regular patterns in structure.

[0050] ‘Percent identity’ or '% identity’ between a query amino acid sequence and a subject amino acid sequence is the ‘Identities’ value, expressed as a percentage, that is calculated using a suitable algorithm or software, such as BLASTP, FAST A, DNASTAR Lasergene, GeneDoc, Bioedit, EMBOSS needle or EMBOSS infoalign, over the entire length of the query sequence after a pair-wise global sequence alignment has been performed using a suitable algorithm / software such as BLASTP, FAST A, ClustalW, MUSCLE, MAFFT, EMBOSS Needle, T-Coffee, and DNASTAR Lasergene. A query sequence may be an amino acid sequence identified herein.

[0051] The term ‘pharmaceutically acceptable’ as used herein pertains to compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc. must also be ‘acceptable’ in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington’s Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990. The precise nature of the carrier or other material will depend on the route of administration and would be readily apparent to a skilled person.

[0052] ‘Scaffold’ or ‘scaffold domain’ as used herein is an independently folding structural domain with a stable tertiary structure which is able to present somatostatin (SST) or a SST-derived peptide and allow the interaction of the SST or SST-derived peptide with a somatostatin receptor (SSTR), such as SSTR4. SST adopts a disulfide-stabilized p hairpin structure with the residues F7-W8-K9-T10 at the tip of the turn. In particular, the scaffold permits presentation of the SST pharmacophore (W8-K9) in the correct orientation for interaction with its binding site on the SSTR. Suitable scaffolds include domains represented within paralogous or orthologous proteins which have been utilized in evolution for driving interactions of said scaffold with other molecules. Scaffolds also include natural domains which have been engineered to display diversity as well as entirely synthetic proteins evolved or designed to form a stable self-folding structure. The scaffold may comprise secondary structural elements which combine to form the stable core structure of the scaffold. For example, the scaffold may comprise multiple contiguous or non-contiguous scaffold residues which form covalent and non-covalent interactions with the side chains or the peptide backbone of other scaffold residues. Interactions may include hydrogen bonds, disulphide bonds, ionic interactions and hydrophobic interactions. The scaffold residues may form secondary structural elements of the domain, such as a helices, strands, p sheets, and other structural motifs. Examples of scaffold domains that may be used according to the invention include immunoglobulin domains, including single domain antibodies and antibody fragments; cysteine-rich peptides, such as knottins and venom toxin peptides; affibodies (engineered Z domain of Protein A domain); monobodies / adnectins (engineered fibronectins); designed ankyrin repeat proteins (DARPins); adhirons; anticalins; thioredoxins; affilins; Kunitz domain-based scaffolds; CTLA-4-based scaffolds; avimers; transferrin-based scaffolds; GroEL; GroES; fynomers; tetranectins; atrimers; and T7 phage gene 2 proteins (Gp2). See, for example, Gebauer and Skerra, 2020, Annual Review of Pharmacology and Toxicology, 60, 391-415.

[0053] The phrase ‘single variable domain’ as used herein refers to an antibody variable domain (for example VH, VHH, VL) that specifically binds an antigen or epitope independently of a different variable region or domain. See, for example, De Meyer et al., 2014, Trends Biotechnol., 32, 263-270 and Shao et al., 2007, Mol. Immunol., 44, 656-665.

[0054] ‘Somatostatin’, ‘SST’, ‘somatostatin-14’ and ‘SST-14’ are used interchangeably herein to mean the naturally occurring human somatostatin peptide A1-G2-C3-K4-N5-F6-F7-W8-K9-T10-F11-T12- S13-C14 (SEQ ID NO:1 ). SST is a cyclic tetra-decapeptide internally stabilised by a disulphide bridge between cysteine residues in positions 3 and 14 (Anoop et al., 2014, J. Biol. Chem. 289(24): 16884-903). SST-14 adopts a disulfide-stabilized p hairpin structure with residues F7-W8-K9-T10 at the tip of the turn. The W8-K9 residue pair at the turn tip of SST mediates its bioactivities, and the tip of the p hairpin engages with a binding pocket in its receptor SSTR4 (Zhao et al., 2022, Cell Research, 32: 761-772). The human somatostatin peptide is encoded by the SST gene (NCBI Gene ID: 6750), located on chromosome

[0055] 3 (3q27.2), which comprises two exons. The amino acid sequence of human preprosomatostatin has the database reference NP_001039.1 and is encoded by the nucleotide sequence of database reference NM_001048.4. Alternate cleavage of the single preproprotein encoded by this gene also results in another active form; an N-terminally extended isoform of 28 amino acids.

[0056] ‘SST-12’ is used herein to mean a truncated form of naturally occurring SST-14 which is missing the first two residues (A1-G2) and, accordingly, has the sequence C3-K4-N5-F6-F7-W8-K9-T10-F11- T12-S13-C14 (SEQ ID NO:2).

[0057] ‘Somatostatin-derived peptide’, ‘SST-derived peptide’, ‘somatostatin-14-derived peptide’ and ‘SST-14-derived peptide’ are used interchangeably herein to mean a peptide sequence that is a variant of naturally occurring somatostatin (SST) comprising the pharmacophore residue pair W8-K9 and two flanking cysteine residues that form a disulphide bridge. An SST-derived peptide is capable of binding to and agonising at least one SST receptor, preferably SSTR4. Typically, an SST-derived peptide comprises F7 / A7-W8-K9-T10 and may further comprise F11 (i.e. F7 / A7-W8-K9-T10-F11). An SST-derived peptide may be the same length or shorter than naturally occurring SST, e.g. 14 amino acids or fewer, for example 13, 12, 11, 10, 9 or 8 amino acids long. An SST-derived peptide may have an amino acid sequence defined by X1-X2-C-X3-X4-F-X5-W-K-T F-Xe-X -C, wherein:Xi is independently absent or any amino acid, optionally A; X2 is independently absent or any amino acid, optionally G; X3 is independently absent or K; X4 is independently absent or N; Xs is A or F; Xe is independently absent or T; and X7 is independently absent or S. An SST-derived peptide includes SST-12 also referred to as pep1 in the Examples section (SEQ ID NO:2), pep4 (SEQ ID NO:5) and pep6 (SEQ ID NO:7). An SST-derived peptide includes an SST-12-derived peptide. In the case of an SST-12-derived peptide, the two cysteine residues are present at the N-terminal and C-terminal of the peptide. An SST-12-derived peptide may be the same length or shorter than SST-12, e.g. 12 amino acids or fewer, for example 11, 10, 9 or 8 amino acids long.

[0058] A ‘somatostatin receptor’ or ‘SSTR’ as used herein is a G-protein-coupled receptor (GPCR) family member that binds to and mediates the biological activity of somatostatin and includes SSTR1 , SSTR2, SSTR3, SSTR4 and SSTR5. In particular, the SSTR is a human SSTR. The human somatostatin receptor 4 (SSTR4) is encoded by SSTR4 (Gene ID: 6754), located on chromosome 20 (20p11.21) and comprises one exon. SSTR4 is highly expressed in the central nervous system and mediates potent analgesic and anti-inflammatory effects (Sandor et al., 2006). SSTR4 controls nociceptive transmission by modulating multiple pathways in dorsal root ganglia neurons (Somvashi and Kumar, 2014, PLoS ONE, 9, e85193). The crystal structure of SSTR4 bound to SST-14 has recently been published in Zhao et al., 2022. SSTR4 has been shown to enhance potassium currents by opening G protein-coupled, inwardly rectifying potassium (GIRK) channels; decrease calcium currents by inhibition of voltage-gated calcium channels; and inhibit transient receptor potential vanilloid-1 (TRPV1) and ankyrin-1 channels (TRPA1).

[0059] A ‘somatostatin receptor (SSTR) agonist’ as used herein is a peptide, in particular a SST-derived peptide as disclosed herein, or protein, in a particular a fusion protein as disclosed herein, that binds to a somatostatin receptor (SSTR) and stimulates its activity. Somatostatin (SST) is a naturally occurring agonist of all SSTRs, whereas a SST-derived peptide or a fusion protein as disclosed herein may be an agonist of one or more or all of SSTRs. Preferably, the SST-derived peptide or fusion protein is a selective SSTR4 agonist. By ‘selective’ is meant that the SST-derived peptide or fusion protein preferentially binds to one or more SSTRs, in this case SSTR4. In particular, where the selective SSTR agonist is a selective SSTR4 agonist it stimulates activity of SSTR4 to a greater degree than any other SSTR, at a given concentration, preferably with negligible or no stimulation of any other SSTR. Assays for determining whether or not a peptide or protein is a SSTR agonist, or a selective SSTR agonist, would be well known to a skilled person and include the cAMP production inhibition assay disclosed in the Examples. Briefly, cells expressing the SSTR in question are stimulated with forskolin (which activates adenylyl cyclase, catalysing the ATP to cAMP reaction, resulting in increased cAMP levels) and then exposed to a SST-derived peptide or fusion protein. If the SST-derived peptide or fusion protein is an agonist, upon binding to the SSTR the receptor undergoes a conformational change that causes the Gi protein alpha subunit to dissociate and inhibit adenylyl cyclase resulting in lowered cAMP levels. Accordingly, the stronger the agonist, the lower the resulting cAMP levels for a given concentration of agonist. For a selective SSTR4 agonist, the IC50 value of the SST-derived peptide or fusion protein in a cAMP production inhibition assay with a SSTR4-expressing cell line is lower than the IC50 value of the SST-derived peptide or fusion protein with respect to any other SSTR (SSTR1, SSTR2, SSTR3 and SSTR5) expressing cell line.

[0060] In reference to a particular condition, ‘treating’, and grammatical variations thereof as used herein, means: (1) to ameliorate the condition or one or more of the biological manifestations of the condition, (2) to interfere with a) one or more points in the biological cascade that leads to or is responsible for the condition or b) one or more of the biological manifestations of the condition, (3) to alleviate one or more of the symptoms, effects or side effects associated with the condition or treatment thereof, (4) to slow the progression of the condition or one or more of the biological manifestations of the condition or (5) to prevent the onset of one or more of the biological manifestations of the condition. Treatment can be therapeutic, prophylactic or preventative. The subject will be one who is in need thereof. Those in need of treatment may include individuals already suffering from a particular medical disease, in addition to those who may develop the disease in the future. Therefore, prophylactic therapy is also contemplated. The skilled artisan will appreciate that ‘prevention’ is not an absolute term. In medicine, ‘prevention’ is understood to refer to the prophylactic administration of a drug to substantially diminish the likelihood or severity of a condition or biological manifestation thereof, or to delay the onset of such condition or biological manifestation thereof.

[0061] A ‘variant’ sequence refers to a sequence in which at least one amino acid residue has been changed with respect to the original sequence, e.g. by way of insertion, deletion or substitution. A substitution is preferably a conservative substitution. As is well recognised in the art, amino acids may be divided into groups based on common side-chain properties and a given amino acid in a sequence may be substituted by another amino acid from the same group without affecting function, i.e. certain amino acid substitutions are regarded as being ‘conservative’. For example, amino acids may be grouped as follows: hydrophobic (Met, Ala, Vai, Leu, lie), neutral hydrophilic (Cys, Ser, Thr), acidic (Asp, Glu), basic (Asn, Gin, His, Lys, Arg), aromatic (Trp, Tyr, Phe), and residues that influence chain orientation (Gly, Pro).

[0062] STATEMENT OF THE INVENTION

[0063] The invention provides a fusion protein comprising: a scaffold, and a somatostatin (SST)-derived peptide, wherein the fusion protein is a somatostatin receptor (SSTR) agonist; as well as methods of making the same.

[0064] It is envisioned that the SST-derived peptide imparts SSTR agonist activity on the fusion protein and is itself an SSTR agonist. In some instances, the scaffold may contribute to SSTR binding and / or agonist activity, for example, if the scaffold is an antibody-derived scaffold, the CDRs may contact the SSTR and contribute to binding and / or agonist activity.

[0065] In an embodiment the SSTR agonist is an agonist of all five SSTRs. In an embodiment, the SSTR agonist is a SSTR4 agonist. In an embodiment, the SSTR agonist is selective for one or more SSTRs. In an embodiment, the SSTR agonist is a SSTR4 agonist and is selective over SSTR2. In other words the SSTR agonist preferentially stimulates activity of SSTR4 and potentially also SSTR1 , SSTR3 and / or SSTR5 to a greater degree than SSTR2, preferably with negligible or no stimulation of SSTR2, even more preferably with negligible or no stimulation of SSTR2, SSTR3 and SSTR5, and most preferably with negligible or no stimulation of SSTR2, SSTR1 , SSTR3 and SSTR5. In an embodiment, the SSTR agonist is selective for a single SSTR. In an embodiment, the SSTR agonist is a selective SSTR4 agonist. In other words, the IC50 value of the SST-derived peptide or fusion protein in a cAMP production inhibition assay with a SSTR4-expressing cell line is lower than the IC50 value of the SST-derived peptide or fusion protein with respect to any other SSTR (SSTR1 , SSTR2, SSTR3 and SSTR5) expressing cell line. In an embodiment, the cAMP production inhibition assay is as described in the Examples.

[0066] In an embodiment, the selective SSTR4 agonist is greater than 5-fold, greater than 6-fold, greater than 7-fold, greater than 8-fold, greater than 9-fold, or greater than 10-fold more selective for SSTR4 than any other SSTR. In an embodiment, the selective SSTR4 agonist is greater than 50-fold, greater than 100-fold, greater than 200-fold, greater than 300-fold, greater than 400-fold, greater than 500-fold, greater than 600-fold, greater than 700-fold, greater than 800-fold, greater than 900-fold, or greater than 1000- fold more selective for SSTR4 than any other SSTR. In an embodiment, the selective SSTR4 agonist is between 5-fold and 1000-fold more selective for SSTR4 than any other SSTR. In an embodiment, the selective SSTR4 agonist is between 100-fold and 1000-fold more selective for SSTR4 than any other SSTR. In an embodiment, the selective SSTR4 agonist is between 250-fold and 1000-fold more selective for SSTR4 than any other SSTR. In an embodiment, the selective SSTR4 agonist is between 500-fold and 1000-fold more selective for SSTR4 than any other SSTR. In an embodiment, the selective SSTR4 agonist is selective over SSTR2. In an embodiment, the selective SSTR4 agonist has minimal to no agonist activity on SSTR2. In an embodiment, the selective SSTR4 agonist has minimal to no agonist activity on SSTR1, SSTR2, SSTR3 and SSTR5.

[0067] The SST-derived peptide may be fused directly to the N-terminal or the C-terminal of the scaffold. Alternatively, the SST-derived peptide may be inserted within the scaffold. The fusion protein may comprise one or more linkers joining the SST-derived peptide to the scaffold. For example, a linker may be used to join the SST-derived peptide to the N-terminal of the scaffold. A linker may be used to join the SST-derived peptide to the C-terminal of the scaffold.

[0068] In the case of the SST-derived peptide being inserted within the scaffold, a linker may be used on one or both sides of the SST-derived peptide. When a linker is used on both sides of the SST-derived peptide it may be the same linker on both sides or a different linker on each side. In an embodiment, the N-terminal of the SST-derived peptide is directly fused to the scaffold and the C-terminal of the SST- derived peptide is joined to the scaffold using a linker. In an embodiment, the C-terminal of the SST- derived peptide is directly fused to the scaffold and the N-terminal of the SST-derived peptide is joined to the scaffold using a linker. In an embodiment, both the N-terminal and the C-terminal of the SST-derived peptide are directly fused to the scaffold. In an embodiment, both the N-terminal of the SST-derived peptide and the C-terminal of the SST-derived peptide are joined to the scaffold using identical linkers. In an embodiment, the N-terminal of the SST-derived peptide and the C-terminal of the SST-derived peptide are joined to the scaffold using different linkers.

[0069] A linker may comprise a short sequence of amino acid residues, for example, 1 to 20 amino acid residues or 1 to 15 amino acid residues. Any suitable linker sequence may be employed. Preferably it is a heterologous sequence and is non-immunogenic. Suitable linker amino acid sequences are well known in the art and may include the amino acid sequences GG, SGG, GGSG, GGGS, AEAAAKEAAAKA, GGS, GGSGGS, GGSGGSGGSGGS or PAPAPAPA or a variant of any of these sequences.

[0070] A relatively short and rigid linker is preferred for terminal fusions of the SST-derived peptide to the scaffold, e.g. an SST-derived peptide fused to the N-terminal or C-terminal of an Fc domain (an ‘Fc fusion’). For example, a linker of between 1 and 5 amino acid residues maybe used, e.g. 2 or 3 or 4 amino acids.

[0071] In the case of integral fusions, where an SST-peptide is inserted within the scaffold sequence, a longer, more flexible linker may be employed. In an embodiment, the linker is 4 to 20 amino acid residues long. In an embodiment, when a linker is used on both sides of the SST-derived peptide to join it to an internal site within the scaffold, each linker is 4 to 11 amino acids long. In an embodiment, each linker is 5 to 10 amino acids long. In an embodiment, each linker is 6 to 9 amino acids long. In an embodiment, each linker is 7 or 8 amino acids long.

[0072] An SST-derived peptide may be the same length or shorter than naturally occurring SST, e.g. 14 amino acids or fewer. In an embodiment, the SST-derived peptide is 6-14 amino acids long. In an embodiment, the SST-derived peptide is 7-14 amino acids long. In an embodiment, the SST-derived peptide is 8-14 amino acids long. In an embodiment, the SST-derived peptide is 6-12 amino acids long. In an embodiment, the SST-derived peptide is 7-12 amino acids long. In an embodiment, the SST-derived peptide is 8-12 amino acids long. In an embodiment, the SST-derived peptide is 13 amino acids long. In an embodiment, the SST-derived peptide is 12 amino acids long. In an embodiment, the SST-derived peptide is 11 amino acids long. In an embodiment, the SST-derived peptide is 10 amino acids long. In an embodiment, the SST-derived peptide is 9 amino acids long. In an embodiment, the SST-derived peptide is 8 amino acids long.

[0073] In an embodiment, the SST-derived peptide of any of the aforementioned lengths comprises F7 / A7-W8-K9-T10. In an embodiment, the SST-derived peptide comprises F7 / A7-W8-K9-T10-F11.

[0074] In an embodiment, the SST-derived peptide has an amino acid sequence defined by X1-X2-C-X3- X4-F-X5-W-K-T F-Xe-XyC, wherein: Xi is independently absent or any amino acid, optionally A; X2 is independently absent or any amino acid, optionally G; X3 is independently absent or K; X4 is independently absent or N; X5 is A or F; Xe is independently absent or T; and X7 is independently absent or S. In an embodiment, Xi and X2 are absent.

[0075] In an embodiment, X1 is A or a conservative substitution thereof. In an embodiment, X2 is G or a conservative substitution thereof. In an embodiment, X3 is K. In an embodiment, X4 is N. In an embodiment, X5 is A or F. In an embodiment, X6 is T. In an embodiment, X7 is S. In an embodiment, X1 and X2 are absent.

[0076] In an embodiment, the SST-derived peptide consists of an amino acid sequence of SEQ ID NO:2. In an embodiment, the SST-derived peptide consists of an amino acid sequence of SEQ ID NO:5 or SEQ ID NO:7 or a selective SSTR4 agonist variant thereof. In an embodiment, the SST-derived peptide consists of an amino acid sequence of SEQ ID NO:5. In an embodiment, the SST-derived peptide consists of an amino acid sequence of SEQ ID 1X10:7.

[0077] In an embodiment, the SST-derived peptide is not an amino acid sequence of SEQ ID NO:2, 3, 4 or 6. In an embodiment, the SST-derived peptide is not an amino acid sequence of SEQ ID NO:2. In an embodiment, the SST-derived peptide is not an amino acid sequence of SEQ ID NO:3. In an embodiment, the SST-derived peptide is not an amino acid sequence of SEQ ID NO:4. In an embodiment, the SST-derived peptide is not an amino acid sequence of SEQ ID NO:6.

[0078] The scaffold may be any suitable scaffold for presenting an SST-derived peptide. The scaffold may be an antibody-derived scaffold. In an embodiment, the antibody-derived scaffold is selected from the group consisting of: an intact antibody, a single variable domain, a VHH domain antibody, an antigen binding fragment (such as a Fab), a single chain Fv, an Fc domain, a diabody, and a minibody.

[0079] In an embodiment, the scaffold is a single variable domain. In an embodiment the single variable domain is a VH domain. In an embodiment, the single variable domain scaffold is any one of the VH domains as set out in SEQ ID NOs:8, 10, 12, 14, and 16. In an embodiment, the single variable domain scaffold is the VH domain as set out in SEQ ID NO:8. In an embodiment, the single variable domain scaffold is the VH domain as set out in SEQ ID NO: 14.

[0080] In an embodiment, the single variable domain is a VL domain. In an embodiment, the single variable domain scaffold is any one of the VL domains as set out in SEQ ID NOs:9, 11 , 13, 15, and 17. In an embodiment, the single variable domain scaffold is the VL domain as set out in SEQ ID NO:9. In an embodiment, the single variable domain scaffold is the VL domain as set out in SEQ ID NO: 15.

[0081] In an embodiment, the single variable domain is a VHH domain.

[0082] In an embodiment, the scaffold is an Fc domain. In an embodiment, the Fc domain comprises or consists of the CH2-CH3 sequence in SEQ ID NO:18 or a variant thereof. In an embodiment, the SST- derived peptide is directly fused to the N-terminal of an Fc domain. In an embodiment, the SST-derived peptide is directly fused to the C-terminal of an Fc domain. In an embodiment a linker is used to join the SST-derived peptide to the N-terminal of an Fc domain. In an embodiment a linker is used to join the SST-derived peptide to the C-terminal of an Fc domain.

[0083] In an embodiment, the scaffold is a monoclonal antibody. In an embodiment, the antibody is an IgG antibody. In an embodiment, the IgG antibody is an lgG1 antibody. In an embodiment, the IgG antibody is an lgG4 antibody.

[0084] In the case of an antibody-derived scaffold, the SST-derived peptide may be inserted into or replace a CDR. In an embodiment, the CDR is a heavy chain CDR. In an embodiment, the CDR is HCDR3. In an embodiment, the CDR is a light chain CDR. In an embodiment, the CDR is LCDR2. In an embodiment, the CDR is LCDR3. In an embodiment, the scaffold is VHH and the CDR is CDR3.

[0085] In an embodiment, the CDR in which the SST-derived peptide is inserted or replaces is up to 35 amino acids long, up to 34 amino acids long, up to 33 amino acids long, up to 32 amino acids long, up to 31 amino acids long or up to 30 amino acids long.

[0086] In an embodiment, the SST-derived peptide replaces the amino acid sequence between and including two non-canonical cysteine residues in a CDR. In an embodiment, the two non-canonical cysteine residues are within HCDR3. In such an embodiment, the SST-derived peptide is preferably a SST-12-derived peptide. In an embodiment, the SST-12-derived peptide is pep4 (SEQ ID NO:5) or pep6 (SEQ ID NO:7) or a selective SSTR4 agonist variant thereof.

[0087] In some embodiments, a fusion protein as described herein comprises a SST-derived peptide fused to a VH domain, preferably in or replacing a CDR, that is associated with a VL domain (i.e. a partner domain) through a covalent or non-covalent bond. In some embodiments, a fusion protein as described herein comprises a SST-derived peptide fused to a VL domain, preferably in or replacing a CDR, that is associated with a VH domain (i.e. a partner domain) through a covalent or non-covalent bond. For example, a fusion protein as described herein may be in the form of a whole antibody, such as an IgG, or an antibody-derived molecule such as a Fab, a Fab', a F(ab')2, or a scFv. In an embodiment, the IgG antibody is an IgG 1 or an lgG4 antibody.

[0088] In an embodiment, the fusion protein comprises or consists of a i) a VH domain comprising the SST-derived peptide of SEQ ID NO:5, or a selective SSTR4 agonist variant thereof, in a HCDR or replacing a HCDR, and ii) a partner VL domain. In an embodiment, the fusion protein comprises or consists of a i) a VH domain comprising the SST-derived peptide of SEQ ID NO:5, or a selective SSTR4 agonist variant thereof, in or replacing a HCDR3, and ii) a partner VL domain.

[0089] In an embodiment, the fusion protein comprises or consists of a i) a VL domain comprising the SST-derived peptide of SEQ ID NO:5, or a selective SSTR4 agonist variant thereof, in a LCDR or replacing a LCDR, and ii) a partner VH domain. In an embodiment, the fusion protein comprises or consists of a i) a VL domain comprising the SST-derived peptide of SEQ ID NO:5, or a selective SSTR4 agonist variant thereof, in or replacing a LCDR3, and ii) a partner VH domain. In an embodiment, the fusion protein comprises or consists of a i) a VL domain comprising the SST-derived peptide of SEQ ID NO:5, or a selective SSTR4 agonist variant thereof, in or replacing a LCDR2, and ii) a partner VH domain.

[0090] In an embodiment, the fusion protein comprises or consists of the VH domain as set out in SEQ

[0091] ID NO:36 and the VL domain as set out in SEQ ID NO:15. In an embodiment, the fusion protein comprises or consists of the VH domain as set out in SEQ ID NO:44, or a selective SSTR4 agonist variant thereof, and the VL domain as set out in SEQ ID NO:9.

[0092] In an embodiment, the fusion protein is an intact antibody comprising the VH domain as set out in SEQ ID NO:44, or a selective SSTR4 agonist variant thereof, and the VL domain as set out in SEQ ID NO:9.

[0093] In other embodiments, a fusion protein as described herein that comprises a VH domain or a VL domain is a nanobody, a single domain antibody or a VHH that is not associated with a partner domain. In an embodiment, the fusion protein comprises or consists of any one of the amino acid sequences set out in SEQ ID NOs:33-37 or 41-44. In an embodiment, the fusion protein comprises or consists of the amino acid sequence set out in SEQ ID NO:36. In an embodiment, the fusion protein comprises or consists of the amino acid sequence set out in SEQ ID NO:44.

[0094] The CDRs that do not comprise an SST-derived peptide may have no or minimal interaction or binding to the SSTR. Alternatively, the CDRs may contact the SSTR. In an embodiment, the CDRs contact the SSTR. In an embodiment, the CDRs contribute to SSTR agonist activity. In an embodiment, the CDRs contribute to selective SSTR agonist activity. In an embodiment, the CDRs contribute to selective SSTR4 agonist activity.

[0095] In another embodiment, the fusion protein is an Fc-fusion protein. In an embodiment, the Fc- fusion protein comprises as SST-derived protein as disclosed herein, in particular SST-12-derived peptide pep4 (SEQ ID NO:5) or pep6 (SEQ ID NO:7), or a selective SSTR4 agonist variant thereof.

[0096] In an embodiment when the fusion protein comprises an Fc domain (e.g. an antibody, a KNOTBODY, an Fc fusion, an Fcab, etc.), the Fc domain is “effector null”, “attenuated”, “disabled” or “silenced” (these terms being used interchangeably herein), i.e. the Fc does not mediate or has significantly reduced effector functions compared to a wild-type Fc. Suitable constant regions may be selected accordingly, optionally including mutations that alter (e.g., reduce or eliminate) one or more Fc effector functions and these are well known in the art. Thus, for example, a fusion protein may comprise a human lgG1 , lgG2, lgG3, lgG4 Fc region or an Fc disabled variant thereof. Suitable Fc disabling mutations include LALA (L234A and L235A), LALA-PG (L234A, L235A and P329G), LALA-KA (L234A, L235A and K322A), GSR (L234G, L235S and G236R), STR (L234S, L235T and G236R); SVR (L234S, L235V and G236R); TQR (L234T, L235Q and G236R); and TTR (L234T, L235T and G236R). Where a fusion protein comprises an lgG4 Fc region, it may also comprise hinge stabilising mutations S228P and L235E.

[0097] The long half-life of IgG antibodies is reported to be dependent on their binding to FcRn. Therefore, substitutions that increase the binding affinity of IgG to FcRn at pH 6.0 while maintaining the pH dependence of the interaction with target, by engineering the constant region, have been extensively studied (Ghetie et al, 1997, Nature Biotech, 15: 637-640; Hinton et al, 2004, J Biol Chem, 279: 6213- 6216; Dall'Acqua et al, 2002, J Immunol 169(9):5171-5180). The in-vivo half-life of fusion proteins of the present invention comprising an Fc domain may be altered by modification of a heavy chain constant domain or an FcRn binding domain therein.

[0098] In adult mammals, FcRn, also known as the neonatal Fc receptor, plays a key role in maintaining serum antibody levels by acting as a protective receptor that binds and salvages antibodies of the IgG isotype from degradation. IgG molecules are endocytosed by endothelial cells and, if they bind to FcRn, are recycled out of the cells back into circulation. In contrast, IgG molecules that enter the cells and do not bind to FcRn are targeted to the lysosomal pathway where they are degraded.

[0099] FcRn is believed to be involved in both antibody clearance and the transcytosis across tissues (see Junghans, 1997, Immunol Res, 16:29-57 and Ghetie and Ward, 2000, Annu Rev Immunol 18:739- 766). Human lgG1 residues that interact directly with human FcRn include Ile253, Ser254, Lys288, Thr307, Gln311 , Asn434 and His435. Mutations at any of these positions may enable increased serum half-life and / or altered effector properties of fusion proteins of the invention.

[0100] Fusion proteins of the present invention may have amino acid modifications that increase the affinity of the constant domain or fragment thereof for FcRn. Increasing the half-life (i.e. , serum half-life) of therapeutic and diagnostic IgG antibodies and other bioactive molecules has many benefits including reducing the amount and / or frequency of dosing of these molecules. In one embodiment, the fusion protein of the invention comprises all or a portion (an FcRn binding portion) of an IgG constant domain having one or more half-life extending amino acid modifications. Such mutations include, with reference to lgG1 , M252Y / S254T / T256E (commonly referred to as YTE mutations), M428L / N434S (commonly referred to as LS mutations), H433K and N434F (commonly referred to as HN or NHance mutations), and L309D / Q311H / N434S (commonly referred to as DHS).

[0101] The scaffold may be a scaffold that is not antibody-derived. In an embodiment, the scaffold is selected from the group consisting of: a cysteine-rich peptide, a CTLA-4-based binder, lipocalin, an anticalin, SpA, an avimer, an affibody, GroEL, GroES, transferrin, fibronectin, an adnectin, an fynomer, a monobody, tetranectin, an atrimer, a Kunitz domain-based binder, a designed ankyrin repeat protein (DARPin), an adhiron, an affilin, a thioredoxin, and a T7 phage gene 2 protein (Gp2).

[0102] In an embodiment, the cysteine-rich peptide is a knottin. In an embodiment the knottin is selected from the group consisting of EETI-II (SEQ ID NO:21 ), AGRP (SEQ ID NO:23), Huwentoxin-IV, ProTx-ll, Ssm6a, Kaliotoxin, mokatoxin-1 , Conotoxin-co, MCoTI-ll, Shk, PcTX1, mambalgin, and a variant of any of the foregoing that retains the correct fold structure. Suitable sequences also include variants of these reference sequences. In an embodiment the knottin is EETI-II (SEQ ID NO:21). In an embodiment, the knottin is AGRP (SEQ ID NO:23). In an embodiment, the knottin is not ProTx-ll.

[0103] In an embodiment, the cysteine-rich peptide is an ion channel-modulating peptide. In an embodiment, the cysteine-rich peptide is a venom toxin.

[0104] Ion channel-modulating peptides (both agonistic and antagonistic) comprising multiple disulphide bonds are well-known in the art. Examples include venom toxin peptides from venomous species, such as spiders, snakes, scorpions and venomous snails. The structural conformations and disulphide linkage patterns of venom toxin peptides are also well known in the art. For example, an analysis of venoms of spiders and other animals reveals a multitude of conformations and patterns of disulphide linkage. For example, spider toxin huwentoxin-ll has a disulphide linkage pattern of l-lll, ll-V, IV-VI and ‘Janus-faced atracotoxins’ (J-ACTXs) has a disulphide linkage pattern of l-IV, ll-VII, lll-IV and V-VIII (including an unusual ‘vicinal’ disulphide bond between 2 neighbouring cysteines), where the pairs of Roman numerals refer to the order where each cysteine appears in the sequence and the position of the partner cysteine with which it forms a disulphide bond. A disulphide loop of a cysteine-rich peptide scaffold may be replaced with a SST-derived peptide as disclosed herein, in particular a SST-12-derived peptide as disclosed herein. The cysteine-rich peptide may itself be located in a CDR of an antibody-derived scaffold.

[0105] When the scaffold is a knottin, the SST-derived peptide may be inserted into or replace a knottin loop, e.g. loop 1 , loop 2, loop 3, loop 4 or loop 5. In an embodiment, the SST-derived peptide is inserted into or replaces knottin loop 1. In an embodiment, the SST-derived peptide is inserted into or replaces knottin loop 2. In an embodiment, the SST-derived peptide is inserted into or replaces knottin loop 3. In an embodiment, the SST-derived peptide is inserted into or replaces knottin loop 4. In an embodiment, the SST-derived peptide is inserted into or replaces knottin loop 5.

[0106] In an embodiment, the knottin is inserted into or replaces a CDR of an antibody-derived scaffold. In an embodiment, the knottin is inserted into or replaces CDR1, CDR2, or CDR3 in a single variable domain. In an embodiment, the knottin is inserted into or replaces HCDR1 , HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 in a monoclonal antibody or derivative thereof. Accordingly, the insertion of an SST- derived peptide into or replacing a knottin loop in a KNOTBODY is contemplated, such that a fusion protein of the invention may comprise an SST-derived peptide inserted into a KNOTBODY scaffold. In an embodiment, the knottin displaying the SST-derived peptide is fused to the N-terminal or C-terminal of an Fc domain.

[0107] In an embodiment, the KNOTBODY scaffold has an amino acid sequence as set forth in SEQ ID NO:22 or 24. In an embodiment, the KNOTBODY comprises any one of the amino acids as set forth in SEQ ID NOs:38-39, or a SSTR agonist variant sequence thereof. In an embodiment, the KNOTBODY comprises any one of the amino acids as set forth in SEQ ID NOs:38-39, or a SSTR agonist variant sequence thereof; and a partner domain. In an embodiment, the fusion protein comprises any one of the amino acids as set forth in SEQ ID NOs:38-39, or a SSTR agonist variant sequence thereof; and a partner domain of SEQ ID NO:28.

[0108] A SST-derived peptide according to the invention may be a variant sequence of a SST-derived peptide sequence disclosed herein. A SST-derived peptide according to the invention may differ from a reference sequence shown herein by addition, substitution, or deletion of 1 amino acid residue, 2, 3, 4, 5 or 6 amino acid residues. For example, variants may differ from a reference SST-derived peptide sequence disclosed herein by the substitution of 1 , 2, 3, 4, 5 or 6 amino acids. In an embodiment, these substitutions are conservative substitutions.

[0109] In an embodiment, the SST-derived peptide does not comprise any D-amino acids. In a particular embodiment, the SST-derived peptide does not comprise D-Tryptophan. In an embodiment, the SST- derived peptide comprises only L-amino acids and, optionally glycine which is achiral. In an embodiment, the SST-derived peptide does not comprise hydroxy proline.

[0110] In an embodiment, the fusion protein does not comprise any D-amino acids. In a particular embodiment, the fusion protein does not comprise D-Tryptophan. In an embodiment, the fusion protein comprises only L-amino acids and, optionally glycine which is achiral. In an embodiment, the fusion protein does not comprise hydroxyproline.

[0111] A fusion protein or scaffold according to the invention may be a variant sequence of a fusion protein sequence or scaffold sequence disclosed herein. A variant sequence may include post- translational modifications. In an embodiment, the variant amino acid sequence may have at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the reference sequence.

[0112] In an embodiment, the scaffold comprises or consists of an amino acid sequence at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set out in any one of SEQ ID NOs:8-24 or 28-30.

[0113] In an embodiment, the fusion protein comprises or consists of an amino acid sequence at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set out in any one of SEQ ID NOs:33-44.

[0114] A fusion protein or scaffold sequence that is a variant of a reference sequence may have 1 or more amino acid residues altered relative to the reference sequence. For example, 50 or fewer amino acid residues may be altered relative to the reference sequence. In an embodiment, 45 or fewer, 40 or fewer, 35 or fewer, 30 or fewer, 25 or fewer, 20 or fewer, 15 or fewer, 10 or fewer, 5 or fewer, 3 or fewer, or 2 or 1 amino acid residues are altered relative to the reference sequence.

[0115] In an embodiment, the scaffold comprises or consists of an amino acid sequence having 50 or fewer, 45 or fewer, 40 or fewer, 35 or fewer, 30 or fewer, 25 or fewer, 20 or fewer, 15 or fewer, 10 or fewer, 5 or fewer, 3 or fewer, or 2 or 1 amino acid residues altered relative to an amino acid sequence set out in any one of SEQ ID NOs:8-24 or 28-30.

[0116] In an embodiment, the fusion protein comprises or consists of an amino acid sequence having 50 or fewer, 45 or fewer, 40 or fewer, 35 or fewer, 30 or fewer, 25 or fewer, 20 or fewer, 15 or fewer, 10 or fewer, 5 or fewer, 3 or fewer, or 2 or 1 amino acid residues altered relative to an amino acid sequence set out in any one of SEQ ID NOs:33-44.

[0117] The binding affinity (KD) of the fusion protein-SSTR interaction may be, for example, from about 100 picomolar (pM) to about 100 micromolar (pM). In an embodiment, the binding affinity of the fusion protein-SSTR interaction is from about 1 nM to about 1 micromolar (pM). In an embodiment, the binding affinity of the fusion protein-SSTR interaction is from about 100 pM to about 1 nM). In some embodiments, the fusion protein can bind to an SSTR with a KD less than or equal to 1 micromolar. In some embodiments, the fusion protein can bind to an SSTR with a KD less than or equal to 1 nanomolar.

[0118] The binding affinity of the fusion protein is determined by the association constant (Ka) and the dissociation constant (Kd) (KD = Kd / Ka). The binding affinity may be measured by BIACORE (surface plasmon resonance). Alternatively, the binding affinity can be measured by FORTEBIO.

[0119] A fusion protein described herein may further comprise one or more heterologous amino acid sequences in addition to the scaffold, SST-derived peptide and optional linker. For example, the fusion protein may further comprise one or more additional domains which improve the stability, pharmacokinetic, targeting, affinity, purification and production properties of the fusion protein described herein.

[0120] The invention also provides a method of generating a fusion protein, comprising a scaffold and a SST-12-derived peptide, wherein the fusion protein is a somatostatin receptor (SSTR) agonist, said method comprising: a) identifying a suitable scaffold comprising a disulphide loop; b) replacing the disulphide loop in the scaffold with said SST-12-derived peptide; and c) determining that the fusion protein is a SSTR agonist. Step c) may comprise or consist of step c-i) of determining if the fusion protein is a SSTR agonist and step c-ii) of selecting a SSTR agonist.

[0121] In an embodiment, the SSTR agonist is a SSTR4 agonist. In an embodiment, the SSTR4 agonist is a selective SSTR4 agonist.

[0122] Any suitable scaffold as disclosed herein may be used. In an embodiment, the scaffold is an antibody-derived scaffold as disclosed herein. In an embodiment, the antibody derived scaffold is a single variable domain. In an embodiment, the single variable domain is a VH domain. In an embodiment, the single variable domain is a VHH domain. In an embodiment, the single variable domain is a VL domain. In an embodiment, the scaffold is a monoclonal antibody. In an embodiment, the scaffold is a human antibody. In an embodiment, the scaffold is a humanised antibody.

[0123] In an embodiment, the disulphide loop in the scaffold, including the cysteine residues, is 10 to 20 amino acids long. In an embodiment, the disulphide loop in the scaffold, including the cysteine residues, is 12 to 20 amino acids long. In an embodiment, the disulphide loop in the scaffold, including the cysteine residues, is 12 to 19 amino acids long. In an embodiment, the disulphide loop in the scaffold, including the cysteine residues, is 12 to 17 amino acids long. In an embodiment, the disulphide loop in the scaffold, including the cysteine residues, is about 12 amino acids long.

[0124] In an embodiment, the disulphide loop is in a CDR. In an embodiment, the CDR is up to 35 amino acids long, up to 34 amino acids long, up to 33 amino acids long, up to 32 amino acids long, up to 31 amino acids long or up to 30 amino acids long. In an embodiment, the CDR is up to 25 amino acids long. In an embodiment, the CDR is up to 22 amino acids long. In an embodiment, the CDR is 8 to 22 amino acids long. In an embodiment, the CDR is 14 to 16 amino acids long.

[0125] In an embodiment, the CDR is a heavy chain CDR. In an embodiment, the CDR is HCDR3. In an embodiment, the CDR is a light chain CDR. In an embodiment, the CDR is LCDR2. In an embodiment, the CDR is LCDR3. In an embodiment, the scaffold is VHH and the CDR is CDR3.

[0126] In an embodiment, the disulphide loop is formed by two non-canonical cysteine residues in a CDR. In an embodiment, the two non-canonical cysteines are in HCDR3. In an alternative embodiment, the disulphide loop has been engineered into a CDR. In an embodiment, the engineered disulphide loop is in a light chain CDR. In an embodiment, the engineered disulphide loop is in LCDR1. In an embodiment, the engineered disulphide loop is in LCDR2. In an embodiment, the engineered disulphide loop is in LCDR3. In an embodiment, the engineered disulphide loop is in a heavy chain CDR. In an embodiment, the engineered disulphide loop is in HCDR1. In an embodiment, the engineered disulphide loop is in HCDR2. In an embodiment, the engineered disulphide loop is in HCDR3.

[0127] In an embodiment, the scaffold is not an antibody-derived scaffold, as described herein. In an embodiment, the scaffold is a cysteine-rich peptide. In an embodiment, the scaffold is a knottin and the disulphide loop is a knottin loop. In an embodiment, the knottin loop is loop 1. In an embodiment, the knottin loop is loop 2. In an embodiment, the knottin loop is loop 3. In an embodiment, the knottin loop is loop 4. In an embodiment, the knottin loop is loop 5.

[0128] In an embodiment, the cysteine-rich peptide is inserted into a CDR of an antibody or antibody- derived molecule.

[0129] In an embodiment, the fusion protein is a KNOTBODY. In an embodiment, the SST-12 derived peptide has an amino acid sequence defined by C-X3-X4- F-Xs-W-K-T F-X6-X7-C, wherein:

[0130] X3 is independently absent or K;

[0131] X4 is independently absent or N;

[0132] Xs is A or F;

[0133] Xe is independently absent or T; and

[0134] X7 is independently absent or S.

[0135] In an embodiment, the SST-12-derived peptide is pep4 (SEQ ID NO:5) or pep6 (SEQ ID NO:7) or a selective SSTR4 agonist variant thereof. In an embodiment, the SST-12-derived peptide is pep4 (SEQ ID NO:5) or a selective SSTR4 agonist variant thereof. In an embodiment, the SST-12-derived peptide is pep4 (SEQ ID NO:5).

[0136] In an embodiment, step c) of the method involves determining that the SSTR agonist is a SSTR4 agonist. In an embodiment, step c) of the method involves determining that the SSTR agonist is a selective SSTR4 agonist.

[0137] In vitro assays and / or in vivo animal models may be used in step c) of the method.

[0138] In an embodiment, determining that the fusion protein is a SSTR agonist is achieved using a cAMP production inhibition assay, e.g. as disclosed in the Examples and commercially available. Such assays utilise cells overexpressing a given SSTR receptor and involve stimulating them via the addition of forskolin, which in turn stimulates adenylyl cyclase enzyme to catalyse the ATP to cAMP reaction, thus overproducing cAMP. Upon agonist binding to the SSTR, which is usually coupled to Gi protein alpha subunit, the SSTR undergoes a conformational change that causes Gi protein alpha subunit to dissociate and inhibit the function of adenylyl cyclase resulting in lowered cAMP levels. Accordingly, the lower the cAMP levels for a given concentration of agonistic fusion protein, the stronger the agonist for the given SSTR. If a fusion protein is not an agonist of the SSTR, cAMP levels will remain high and comparable to control levels, i.e. in the absence of fusion protein. Alternatively, rather than measuring the level of cAMP directly a fluorescence resonance energy transfer (FRET) based assay tracking the dissociation of G proteins upon activation of a given SSTR may be used.

[0139] In an alternative embodiment, determining that the fusion protein is a SSTR4 agonist is achieved using dorsal root ganglia (DRG)-based assays which are well known in the art. SSTR4 activation results in a dissociation of the coupled G beta-gamma complex, which in turn activates G protein-coupled inwardly rectifying K+channels (GIRK). The activation of GIRK results in hyperpolarisation of DRG cells. Rat and human SSTR4 are highly homologous. Since, rat SSTR4 is expressed in 45 % of DRG neurons, these can be used for agonist screening by the whole-cell voltage clamp technique (Gorham et al, European Journal of Pharmacology, 2014, 736:101-6). Good agonist molecules will induce more cell hyperpolarisation via activation of GIRK.

[0140] The in vivo activity of the fusion protein may be determined by animal models known in the art. For example, the ability of the fusion protein to reduce, inhibit or reverse mechanical hyperalgesia or allodynia in the murine Chronic Constriction Injury (CCI) model may be determined. The CCI model is a model for neuropathic pain. Alternatively, the Complete Freund's adjuvant (CFA) mouse model, which results in the development of injury-induced, persistent inflammatory pain, may be used to determine the effect of the fusion protein on nociceptive pain (Gould et al, Pain. 2000, 85(1-2): 301-3). Fusion proteins as described herein may be produced using synthetic or recombinant techniques which are standard in the art.

[0141] In some embodiments, the fusion protein described herein may be produced with an affinity tag, which may, for example, be useful for purification. An affinity tag is a heterologous peptide sequence which forms one member of a specific binding pair. Polypeptides containing the tag may be purified by the binding of the other member of the specific binding pair to the polypeptide, for example in an affinity column. For example, the tag sequence may form an epitope which is bound by an antibody molecule.

[0142] Suitable affinity tags include for example, glutathione-S-transferase, (GST), maltose binding domain (MBD), MRGS(H)e, DYKDDDD’K (FLAG), T7-, S- (KETAAAKFERQHMDS), poly-Arg (Rs-e), poly- His (H2-10), poly-Cys (C4) poly-Phe(Fn) poly-AspfDs-ie), SUMO tag (Invitrogen Champion pET SUMO expression system), Strept-tag II (WSHPQFEK), c-myc (EQKLISEEDL), Influenza-HA tag (Murray, P. J. et al (1995) Anal Biochem 229, 170-9), Glu-Glu-Phe tag (Stammers, D. K. et al (1991 ) FEBS Lett 283, 298-302), Tag.100 (Qiagen; 12 aa tag derived from mammalian MAP kinase 2), Cruz tag 09™ (MKAEFRRQESDR, Santa Cruz Biotechnology Inc.) and Cruz tag 2 (MRDALDRLDRLA, Santa Cruz Biotechnology Inc.). Known tag sequences are reviewed in Terpe (2003) Appl. Microbiol. Biotechnol. 60 523-533. In preferred embodiments, a poly-His tag such as (H)e, His-SUMO tag (Invitrogen Champion pET SUMO expression system), or MRGS(H)e may be used. The affinity tag sequence may be separated from the fusion protein described herein after purification, for example, using site-specific proteases.

[0143] In some embodiments, the fusion protein described herein may be coupled to a leader peptide to direct secretion of the fusion protein from cell into the culture medium as a precursor fusion protein. A range of suitable leader peptides are known in the art. For example, an a-factor secretion signal or BiP leader sequence may be employed. The leader peptide is located at the N terminus of the precursor fusion protein. The leader peptide is then removed by post-translational processing after expression of the precursor to generate the fusion protein.

[0144] Fusion proteins as described herein may be isolated, in the sense of being free from contaminants, such as other polypeptides and / or cellular components.

[0145] The invention provides in another aspect a nucleic acid encoding a fusion protein disclosed herein and a vector comprising such a nucleic acid.

[0146] Nucleic acid 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.

[0147] Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Preferably, the vector contains appropriate regulatory sequences to drive the expression of the nucleic acid in mammalian cells. Suitable regulatory sequences to drive the expression of heterologous nucleic acid coding sequences in expression systems are well- known in the art and include constitutive promoters, for example viral promoters such as CMV or SV40, and inducible promoters, such as Tet-on controlled promoters. A vector may also comprise sequences, such as origins of replication, promoter regions and selectable markers, which allow for its selection, expression and replication in bacterial hosts such as E. coli. Vectors may be plasmids, viral e.g. phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 3rd edition, Russell et al., 2001, Cold Spring Harbor Laboratory Press. 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, are described in detail in Current Protocols in Molecular Biology, Ausubel et al. eds. John Wiley & Sons, 1992.

[0148] In the case of fusion proteins comprising antibody-derived scaffolds including a heavy chainsequence and a light-chain sequence, these sequences may be on the same or different vectors. In an embodiment, one vector comprises a heavy chain sequence and a second vector comprises a light chain sequence.

[0149] A nucleic acid or vector as described herein may be introduced into a host cell. Another aspect of the invention provides a recombinant cell comprising a nucleic acid or vector that expresses a fusion protein as described above. In the case of fusion proteins comprising antibody-derived scaffolds, when the heavy chain sequence and the light chain sequence are in separate vectors, both vectors are introduced into the same host cell.

[0150] A range of host cells suitable for the production of recombinant fusion proteins are known in the art. Suitable host cells may include prokaryotic cells, in particular bacteria such as Escherichia coli and Lactococcus lactis and eukaryotic cells, including mammalian cells such as Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NS0 mouse melanoma cells, YB2 / 0 rat myeloma cells, human embryonic kidney cells (e.g. HEK293 cells), human embryonic retina cells (e.g. PerC6 cells), amphibian cells such as Xenopus oocytes, insect cells such as Trichoplusia ni, Sf9 and Sf21 and yeast cells, such as Pichia pastoris.

[0151] Techniques for the introduction of nucleic acid into cells are well established in the art and any suitable technique may be employed, in accordance with the particular circumstances. 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. adenovirus, AAV, lentivirus or vaccinia. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage.

[0152] Marker genes such as antibiotic resistance or sensitivity genes may be used in identifying clones containing nucleic acid of interest, as is well-known in the art.

[0153] The introduced nucleic acid may be on an extra-chromosomal vector within the cell, or the nucleic acid may be integrated into the genome of the host cell. Integration may be promoted by inclusion of sequences within the nucleic acid or vector which promote recombination with the genome, in accordance with standard techniques.

[0154] The introduction may be followed by expression of the nucleic acid to produce the encoded fusion protein described herein. In some embodiments, host cells (which may include cells actually transformed although more likely the cells will be descendants of the transformed cells) may be cultured in vitro under conditions for expression of the nucleic acid, so that the encoded polypeptide is produced. When an inducible promoter is used, expression may require the activation of the inducible promoter.

[0155] The expressed polypeptide comprising or consisting of the fusion protein may be isolated and / or purified, after production. This may be achieved using any convenient method known in the art. Techniques for the purification of recombinant polypeptides are well known in the art and include, for example HPLC, FPLC and affinity chromatography. In some embodiments, purification may be performed using an affinity tag on the polypeptide as described above.

[0156] After production, the fusion protein may be investigated further, for example the pharmacological properties and / or activity may be determined. Methods and means of protein analysis are well-known in the art.

[0157] Post-translational modifications may take place during the manufacturing processes for the fusion proteins of the invention. Some post-translational modifications result in a chemical change which does not change the sequence (e.g. Met and oxidized Met; or Asp and isomerised / iso-Asp; or aggregation) while others result in a sequence change such as the conversion of one amino acid residue into another (e.g. Asn conversion to Asp via deamidation; or lysine deletion). Further post-translational modification variants are described below. A fusion protein or scaffold which comprises a sequence change may be the result of a designed sequence change or a post-translational modification.

[0158] Post-translational modifications are chemical changes to the fusion protein or scaffold that may be the result from production of the antibody in a host cell, upstream and / or downstream manufacturing processes, and / or length of storage and storage conditions (e.g. effect of exposure to light, temperature, pH, water, or by reaction with an excipient and / or the immediate container closure system). Therefore, the composition of the invention may be formed from the manufacture or storage of fusion proteins of the invention. Exemplary post-translational modifications comprise antibody-related sequence changes, cleavage of certain leader sequences, the addition of various sugar moieties in various glycosylation patterns including non-enzymatic glycosylation or glycation; deamidation; oxidation; disulfide bond scrambling and other cysteine variants, such as free sulfhydryls, racemized disulfides, thioethers and trisulfide bonds; isomerization; C-terminal lysine cleavage or clipping; and / or N-terminal glutamine cyclisation.

[0159] A fusion protein as described herein may be useful in therapy.

[0160] Whilst the fusion protein may be administered alone, it will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the fusion protein. Thus, pharmaceutical compositions may comprise, in addition to the fusion protein itself, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art.

[0161] In some embodiments, the fusion protein may be provided in a lyophilised form for reconstitution prior to administration. For example, a lyophilised fusion protein may be re-constituted in sterile water and mixed with saline prior to administration to an individual.

[0162] In some embodiments, the pharmaceutical composition comprises a nucleic acid encoding a fusion protein of the invention. In an embodiment, the nucleic acid is a mRNA therapy.

[0163] The fusion proteins and pharmaceutical compositions of the invention may be administered by any appropriate route. For parenteral, for example sub-cutaneous or intra-venous administration, e.g. by injection, the pharmaceutical composition comprising the fusion protein described herein, nucleic acid or cell may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles, such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be employed as required including buffers such as phosphate, citrate and other organic acids; antioxidants, such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3’- pentanol; and m-cresol); low molecular weight polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions, such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants, such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington’s Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990.

[0164] In some embodiments, the fusion protein or pharmaceutical composition is administered intravenously (e.g. by intravenous (IV) infusion).

[0165] In some embodiments, the fusion protein or pharmaceutical composition is administered by injection, for example subcutaneous (SC) injection. Therefore, in one aspect there is provided an injection device comprising the fusion protein or pharmaceutical composition of the invention. The injection device may comprise a pen injector device or an autoinjector device. In one embodiment, the fusion protein or pharmaceutical composition is contained in a prefilled syringe.

[0166] The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.

[0167] Pharmaceutical compositions and formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the fusion protein described herein with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary, shaping the product.

[0168] A pharmaceutical composition comprising a fusion protein described herein may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

[0169] A fusion protein or pharmaceutical composition described herein may be used in a method of treatment of the human or animal body. A method of treatment may comprise administering a fusion protein or pharmaceutical composition described herein to an individual in need thereof. In an embodiment the subject has, or is at risk of developing, pain or inflammation. In an embodiment, the pain is chronic pain. In an embodiment, the pain is acute pain. In an embodiment the subject has an inflammatory disease. In an embodiment the subject has, or is at risk of developing, a disorder of the central nervous system (CNS). In an embodiment the subject has, or is at risk of developing, a depressive disorder. In an embodiment the subject has, or is at risk of developing, epilepsy. In an embodiment the subject has, or is at risk of developing, Alzheimer’s disease. In an embodiment the subject has, or is at risk of developing, cancer. In an embodiment the subject has, or is at risk of developing, a lung disorder, such as asthma or chronic obstructive pulmonary disease (COPD).

[0170] Related aspects of the invention provide a fusion protein or pharmaceutical composition as disclosed herein for use in the treatment of a disease or disorder as disclosed herein, and the use of a fusion protein or pharmaceutical composition described herein in the manufacture of a medicament for the treatment of a disorder or disease as disclosed herein.

[0171] Pain can be split into both nociceptive pain, induced by activation of nociceptors by specific stimuli, and neuropathic pain, induced by damage to the somatosensory system. Nociceptive pain is associated with inflammatory diseases such as rheumatoid arthritis and osteoarthritis, whereas neuropathic pain is associated with nerve damage as well as metabolic diseases including diabetes (Schmader, 2002, Clin. J. Pain., 18(6): 350-354). Neuropathic pain may be peripherally or centrally mediated neuropathic pain.

[0172] In an embodiment, the pain is peripherally mediated neuropathic pain, centrally mediated neuropathic pain or nociceptive pain. In an embodiment, the pain is peripherally mediated neuropathic pain.

[0173] Inflammatory diseases include diseases characterised by increased, excessive or aberrant levels of inflammatory response relative to healthy non-pathological controls, such as chronic inflammation, gout, lupus, endometriosis, type 1 diabetes, fatty liver disease, autoimmune diseases, such as rheumatoid arthritis, cardiovascular disorders, gastrointestinal disorders, such as inflammatory bowel disease, such as Crohn’s disease and ulcerative colitis, and lung diseases, such as asthma and COPD.

[0174] In an embodiment, the pain is associated with an inflammatory disease. In an embodiment, the inflammatory disease is rheumatoid arthritis (RA), osteoarthritis (OA), or an inflammatory bowel disease (IBD), e.g. Crohn’s disease or ulcerative colitis. In an embodiment, the inflammatory disease is osteoarthritis (OA).

[0175] In an embodiment, the pain is back pain. In an embodiment, the pain is lower back pain.

[0176] Pain may, for example be cancer-, surgery-, visceral damage-, headache- or trauma-associated pain.

[0177] In an embodiment, the pain is acute pain. In an embodiment, the pain is post-operative pain.

[0178] In some embodiments, pain that may be treated as described herein may include the symptoms of allodynia; hyperalgesia; spontaneous pain; or expansion of the receptive field. Causes of these pain- related symptoms may include diabetic peripheral neuropathy, arthritic pain, post-herpetic neuralgia, trigeminal neuralgia, post-stroke pain, multiple sclerosis-associated pain, neuropathy-associated pain such as in idiopathic or post-traumatic neuropathy and mononeuritis, HIV-associated neuropathic pain, cancer-associated neuropathic pain, carpal tunnel-associated neuropathic pain, hyperalgesia, for example opioid-induced hyperalgesia, sciatica, spinal cord injury-associated pain, complex regional pain syndrome, fibromyalgia-associated neuropathic pain, lumbar and cervical pain, reflex sympathetic dystrophy, phantom pain, phantom limb syndrome, peripheral nerve or spinal cord trauma, entrapment neuropathy, nerve transection including surgery, Lissauer tract section, limb amputation, stump pain, neuroma / tumour compression, arteriovenous malformation, Vitamin B12 deficiency, alcoholic neuropathy, pain caused by the side effects of anti-cancer and anti-AIDS therapies, pain associated with inflammation or infection of a tooth (toothache), visceral pain, pain caused by chemical burns, pain caused by local or systemic infection, or pain caused by connective tissue disease, such as rheumatoid arthritis, Wallenberg's syndrome, systemic lupus erythematosus, multiple sclerosis, and polyarteritis nodosa.

[0179] An individual suitable for treatment as described above may be a mammal, such as a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), canine (e.g. a dog), feline (e.g. a cat), equine (e.g. a horse), a primate, simian (e.g. a monkey or ape), a monkey (e.g. marmoset, baboon), an ape (e.g. gorilla, chimpanzee, orang-utan, gibbon), or a human.

[0180] In a preferred embodiment, the individual is a human. In other preferred embodiments, nonhuman mammals, especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g. murine, primate, porcine, canine, or rabbit animals) may be employed.

[0181] Administration is normally in a ‘therapeutically effective amount’ or ‘prophylactically effective amount’, this being sufficient to show benefit to a patient. Such benefit may be at least amelioration of pain in the patient. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the method of administration, the scheduling of administration and other factors known to medical practitioners.

[0182] In some embodiments, the fusion protein may fail to cross the BBB or penetrate the CNS and so may fail to activate SSTR, in particular SSTR4, in the CNS. This is particularly true for larger fusion proteins of the invention, for example antibody or KNOTBODY therapeutics. The fusion protein may therefore be a peripheral SSTR agonist, in particular a SSTR4 agonist, i.e. it activates SSTR, in particular SSTR4, in peripheral neurons, but does not activate or results in substantially no activation of SSTR, in particular SSTR4, in CNS neurons.

[0183] In some embodiments, where treatment of a CNS-related disorder is required, for example when the subject has, or is at risk of developing, a depressive disorder, the fusion protein penetrates the BBB and activates SSTR in the CNS. Such BBB-penetrating molecules may be multi-specific molecules, e.g. bispecific molecules, that target in addition to targeting SSTR4 also target the BBB, for example by binding to a BBB receptor (e.g. transferrin receptor or CD98hc), which enables receptor-mediated transcytosis into the CNS. Alternatively, or in addition, such molecules may be formulated in such a way to allow passage through the BBB or they may be administered directly into the CNS (e.g. via intrathecal injection).

[0184] In an embodiment, the CNS disorder is a depressive disorder. In an embodiment, the CNS disorder is Alzheimer’s disease or another form of dementia (i.e., major or mild neurocognitive disorders) associated with one or more medical conditions, including frontotemporal lobar degeneration, Lewy body disease, vascular disease, traumatic brain injury, substance or medication use, HIV infection, prion disease, Parkinson’s disease, and Huntington’s disease. In an embodiment, the CNS disorder is selected from the group consisting of Alzheimer’s disease, depression, anxiety, schizophrenia, bipolar disorder, autism, epilepsy, and hyperactivity disorder. In an embodiment, the CNS disorder is Alzheimer’s disease. In an embodiment, the CNS disorder is epilepsy. In an embodiment, the CNS disorder is depression. A composition may be administered alone or in combination with other treatments, for example treatment with other analgesics, such as paracetamol, non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen, opioids such as tramadol or dihydrocodeine, anti-epileptics such as gabapentin, pregabalin or carbamazepine or anti-depressants such as amitriptyline or duloxetine, either simultaneously or sequentially dependent upon the circumstances of the individual to be treated. In certain embodiments, a composition of the invention can be co-administered to a subject with one or more additional therapeutic agents.

[0185] In an embodiment, a fusion protein or composition of the invention is administered in combination with a non-opioid pain medication.

[0186] In an embodiment, a fusion protein or composition of the invention is administered in combination with an NSAID. In an embodiment, the NSAID is ibuprofen, naproxen, diclofenac or aspirin. In an embodiment, a fusion protein or composition of the invention is administered in combination with ibuprofen and / or paracetamol.

[0187] In an embodiment, a fusion protein or composition of the invention is administered in combination with gabapentin or pregabalin.

[0188] In an embodiment, a fusion protein or composition of the invention is administered in combination with a Nav1.8 inhibitor. In an embodiment, a fusion protein or composition of the invention is administered in combination with suzetrigine (VX-548).

[0189] In an embodiment, the fusion protein or composition of the invention is administered in combination with a beta-secretase inhibitor, a gamma-secretase inhibitor, a HMG-CoA reductase inhibitor, a nonsteroidal anti-inflammatory drug, vitamin E, an anti-amyloid antibody, an antidepressant, an antipsychotic, an anxiolytic, or an anticonvulsant.

[0190] Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners, hospital consultants, and other medical doctors and may depend on the severity of the symptoms and / or progression of a disease being treated. Appropriate doses of therapeutic polypeptides are well known in the art (Ledermann J. A. et al. (1991 ) Int. J. Cancer 47: 659-664; Bagshawe K.D. et al. (1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922). Specific dosages may be indicated herein or in the Physician's Desk Reference (2003) as appropriate for the type of medicament being administered. A therapeutically effective amount or suitable dose of a fusion protein described herein may 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 the disease to be treated, the size and location of the area to be treated, the precise nature of the fusion protein described herein, and the nature of any detectable label or other molecule attached to the fusion protein described herein.

[0191] An initial higher loading dose, followed by one or more lower doses, may be administered. This is a dose for a single treatment of an adult patient, which may be proportionally adjusted for children and infants. Treatments may be repeated at daily, twice-weekly, weekly or monthly or longer intervals, at the discretion of the physician. The treatment schedule for an individual may be dependent on the pharmacokinetic (PK) and pharmacodynamic (PD) properties of the fusion protein described herein, the composition, the route of administration and the nature of the condition being treated. Treatment may be periodic, and the period between administrations may be about 12 hours or more, 24 hours or more, 36 hours or more, 48 hours or more, 96 hours or more, one week or more, or one month or more. Fusion proteins described herein are shown to exert long lasting effects in vivo. In some preferred embodiments, the period between administrations may be one week or more, two weeks or more, or one month or more, for example, administration may be weekly, biweekly or monthly.

[0192] Suitable formulations and routes of administration are described above.

[0193] A kit-of-parts comprising the pharmaceutical composition as disclosed herein together with instructions for use may be provided.

[0194] The invention includes the following numbered embodiments.

[0195] 1. A fusion protein comprising: a scaffold; and a somatostatin (SST)-derived peptide; wherein the fusion protein is a somatostatin receptor (SSTR) agonist, optionally a SSTR4 agonist, optionally a selective SSTR4 agonist.

[0196] 2. The fusion protein according to embodiment 1 , wherein the SST-derived peptide is a SSTR agonist peptide, optionally a SSTR4 agonist peptide, optionally a selective SSTR4 agonist peptide.

[0197] 3. The fusion protein according to embodiment 1 or embodiment 2, further comprising one or more linkers joining the SST-derived peptide to the scaffold.

[0198] 4. The fusion protein according to any one of embodiments 1 to 3, wherein the SST-derived peptide is fused to the N-terminal or the C-terminal of the scaffold.

[0199] 5. The fusion protein according to any one of embodiments 1 to 3, wherein the SST-derived peptide is inserted within the scaffold.

[0200] 6. The fusion protein according to any one of embodiments 1 to 5, wherein the SST-derived peptide has an amino acid sequence defined by X1-X2-C-X3-X4-F-X5-W-K-T F-Xe-X -C, wherein:

[0201] Xi is independently absent or any amino acid, optionally A;

[0202] X2 is independently absent or any amino acid, optionally G;

[0203] X3 is independently absent or K;

[0204] X4 is independently absent or N;

[0205] Xs is A or F;

[0206] Xe is independently absent or T; and X7 is independently absent or S.

[0207] 7. The fusion protein according to embodiment 6, wherein Xi and X2 are absent. 8. The fusion protein according to any one of the preceding embodiments, wherein the SST-derived peptide consists of an amino acid sequence of SEQ ID NO:5 or SEQ ID NO:7 or a selective SSTR4 agonist variant thereof.

[0208] 9. The fusion protein according to any one of the preceding embodiments wherein the scaffold is an antibody-derived scaffold.

[0209] 10. The fusion protein according to any one of the preceding embodiments, wherein the scaffold is a single variable domain, a scFv, a Fab domain, an Fc domain, or a monoclonal antibody.

[0210] 11. The fusion protein according to embodiment 9 or embodiment 10, wherein the SST-derived peptide is inserted into or replaces a CDR.

[0211] 12. The fusion protein according to embodiment 11, wherein the CDR is up to 35 amino acids long, optionally up to 30 amino acids long.

[0212] 13. The fusion protein according to embodiment 11 or embodiment 12, wherein the CDR is a heavy chain CDR, optionally HCDR3.

[0213] 14. The fusion protein according to embodiment 11 or embodiment 12, wherein the CDR is a light chain CDR, optionally LCDR2.

[0214] 15. The fusion protein according to any one of embodiments 11 to 14, wherein the SST-derived peptide replaces the amino acid sequence between and including two non-canonical cysteine residues in a CDR, optionally HCDR3.

[0215] 16. The fusion protein according to any one of embodiments 1 to 8, wherein the scaffold is not an antibody-derived scaffold.

[0216] 17. The fusion protein according to embodiment 16, wherein the scaffold is selected from the group consisting of: a cysteine-rich peptide, a CTLA-4-based binder, lipocalin, an anticalin, SpA, an avimer, an affibody, GroEL, GroES, transferrin, fibronectin, an adnectin, a fynomer, a monobody, tetranectin, an atrimer, a Kunitz domain-based binder, a designed ankyrin repeat protein (DARPin), an adhiron, an affilin, a thioredoxin, and a T7 phage gene 2 protein (Gp2).

[0217] 18. The fusion protein according to embodiment 17, wherein the cysteine-rich peptide is selected from the group consisting of EETI-II, AGRP, Huwentoxin-IV, ProTx-l I, Ssm6a, Kaliotoxin, mokatoxin-1 , Conotoxin-co, MCoTI-ll, Shk, PcTX1 , mambalgin, and a variant of any of the foregoing that retains the correct fold structure. 19. The fusion protein according to embodiment 17 or embodiment 18, wherein the cysteine-rich peptide is a knottin and the SST peptide is inserted into or replaces a knottin loop.

[0218] 20. The fusion protein according to embodiment 19, wherein the knottin is inserted into or replaces a CDR in a single variable domain of an antibody-derived scaffold or wherein the knottin is fused to the N-terminal or C-terminal of an Fc domain.

[0219] 21. An isolated nucleic acid comprising a nucleotide sequence encoding a fusion protein according to any one of the preceding embodiments.

[0220] 22. A vector comprising the nucleic acid according to embodiment 21.

[0221] 23. A host cell comprising the nucleic acid according to embodiment 21 or the vector according to 22.

[0222] 24. A method of making a fusion protein comprising culturing the host cell according to embodiment 23 under suitable conditions.

[0223] 25. A pharmaceutical composition comprising the fusion protein according to any one of embodiments 1 to 20, and at least one pharmaceutically acceptable excipient, vehicle, or carrier.

[0224] 26. A fusion protein according to any one of embodiments 1 to 20, or a pharmaceutical composition according to embodiment 25, for use as a medicament, optionally for use in treating pain, optionally chronic pain, and / or inflammation.

[0225] 27. A method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutically acceptable composition according to embodiment 25, or a fusion protein according to any one of embodiments 1 to 20, optionally wherein the subject has, or is at risk of developing, pain, optionally chronic pain, or inflammation.

[0226] 28. Use of a fusion protein according to any one of embodiments 1 to 20, in the manufacture of a medicament for the treatment of pain, optionally chronic pain, and / or inflammation.

[0227] 29. A method of generating a fusion protein comprising a scaffold and a SST-12-derived peptide, wherein the fusion protein is a somatostatin receptor (SSTR) agonist, optionally a SSTR4 agonist, optionally a selective SSTR4 agonist, said method comprising: a) identifying a suitable scaffold comprising a disulphide loop; b) replacing the disulphide loop with said SST-12-derived peptide; c) determining that the fusion protein is a SSTR agonist, optionally a SSTR4 agonist, optionally a selective SSTR4 agonist. 30. The method according to embodiment 29, wherein the scaffold is an antibody-derived scaffold and the disulphide loop is formed by two non-canonical cysteine residues in a CDR, optionally HCDR3.

[0228] 31. The method according to embodiment 29, wherein the scaffold is an antibody-derived scaffold and the disulphide loop has been engineered into a CDR, optionally wherein the CDR is LCDR2.

[0229] 32. The method according to embodiment 30 or embodiment 31 , wherein the CDR is up to 22 amino acids long, optionally 8 to 22 amino acids long, optionally 14 to 16 amino acids long.

[0230] 33. The method according to embodiment 29, wherein the scaffold is a cysteine-rich peptide, optionally wherein the cysteine rich-peptide is a knottin and the disulphide loop is a knottin loop.

[0231] 34. The method according to any one of embodiments 29 to 33, wherein the SST-12-derived peptide has an amino acid sequence defined by C-X3-X4-F-X5-W-K-T-F-X6-X7-C, wherein:

[0232] X3 is independently absent or K;

[0233] X4 is independently absent or N;

[0234] Xs is A or F;

[0235] Xe is independently absent or T; and

[0236] X7 is independently absent or S.

[0237] 35. The method according to any one of embodiments 29 to 34, wherein the SST-12-derived peptide consists of an amino acid sequence of SEQ ID NO:5 or SEQ ID NO:7 or a selective SSTR4 agonist variant thereof.

[0238] 36. The method according to any one of embodiments 29 to 35, wherein determining that the fusion protein is a SSTR agonist is achieved using a cAMP production inhibition assay.

[0239] 37. The method according to embodiment 36, wherein the IC50 value of the fusion protein in a cAMP production inhibition assay with a SSTR4-expressing cell line is lower than the IC50 value of the fusion protein with respect to a SSTR2-expressing cell line.

[0240] 38. The method according to embodiment 36, wherein the IC50 value of the fusion protein in a cAMP production inhibition assay with a SSTR4-expressing cell line is lower than the IC50 value of the fusion protein with respect to any other SSTR (SSTR1, SSTR2, SSTR3 and SSTR5) expressing cell line.

[0241] 39. The method according to any one of embodiments 29 to 35, wherein determining that the fusion protein is a SSTR agonist is achieved using a dorsal root ganglion (DRG)-based assay. 40. The method according to any one of embodiments 29 to 39, wherein the SST-12-derived peptide is 8 to 12 amino acids long.

[0242] 41. The method according to any one of embodiments 29 to 40, wherein the SST-12-derived peptide is 12 amino acids long.

[0243] 42. The method according to any one of embodiments 29 to 41 , wherein the disulphide loop is 8 to 20 amino acids long, optionally 10 to 20 amino acids long, optionally 12 to 19 amino acids long, optionally 12 to 17 amino acids long.

[0244] 43. The method according to any one of embodiments 29 to 42, wherein the disulphide loop is about 12 amino acids long.

[0245] 44. The method according to any one of embodiments 29 to 43, wherein step c) comprises or consists of step c-i) of determining if the fusion protein is a SSTR agonist and step c-ii) of selecting a SSTR agonist.

[0246] 45. The fusion protein according to any one of embodiments 1 to 7, wherein the SST-derived peptide is 12 amino acids long.

[0247] 46. The fusion protein according to embodiment 20, wherein the fusion protein is a KNOTBODY.

[0248] Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term ‘comprising’ replaced by the term ‘consisting of’ and the aspects and embodiments described above with the term ‘comprising’ replaced by the term ‘consisting essentially of’.

[0249] It is to be understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and / or optional features either singly or together with any of the other aspects, unless the context demands otherwise.

[0250] Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure and, as such, these are within the scope of the present invention.

[0251] All documents and sequence database entries mentioned in this specification are incorporated herein by reference in their entirety for all purposes.

[0252] ‘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 (Hi) A and B, just as if each is set out individually herein.

[0253] EXAMPLES

[0254] Summary

[0255] The following experiments demonstrate the successful insertion of somatostatin 14 (Example 7) and somatostatin 14-derived peptides (Examples 1-6 and 8) into a variety of scaffolds; including the HCDR3 of antibody VH scaffolds (Example 1 ); the knottin domain of KNOTBODY VL scaffolds (Example 2); the LCDR2 of an antibody VL scaffold (Example 3); the CDR3 of a VHH / NANOBODY scaffold (Example 4), as well as additional modalities (Example 8) to produce biologically functioning fusion proteins. The insertion of a somatostatin 14-derived peptide into each of these scaffolds resulted in fusion proteins with strong SSTR4 agonistic function. A series of novel somatostatin 14-derived peptides were inserted into HCDR3 of an antibody VH scaffold resulting in an SSTR4 subtype specific agonist antibody (Example 5). Further, Example 6 demonstrates that successful IgG SSTR4 agonist generation is not dependent on which CDR the SST-14 derived peptide is inserted into.

[0256] Abbreviations

[0257] 95% Cl 95 per cent confidence interval

[0258] °C degrees Celsius

[0259] AGRP Agouti-related protein

[0260] ATP adenosine tri-phosphate bp base pair cAMP cyclic adenosine mono-phosphate

[0261] CDR complementarity determining region

[0262] CHO-K1 Chinese hamster ovary K1 cell line

[0263] DNA deoxyribonucleic acid

[0264] E. coli Escherichia coli

[0265] EETI-II Ecballium elaterium trypsin inhibitor II

[0266] HCDR1 complementarity determining region one of the heavy chain

[0267] HCDR2 complementarity determining region two of the heavy chain

[0268] HCDR3 complementarity determining region three of the heavy chain

[0269] HEK293 Human Embryonic Kidney (HEK) 293 cell line HPLC-SEC size exclusion high-performance liquid chromatography

[0270] IC50 half-maximal inhibitory concentration

[0271] IgG Immunoglobulin G

[0272] LCDR1 complementarity determining region one of the light chain

[0273] LCDR2 complementarity determining region two of the light chain

[0274] LCDR3 complementarity determining region three of the light chain min minute ml millilitre mM millimolar mm millimetre pg microgram pl microlitre pM micromolar

[0275] MWCO molecular weight cut off ng nanogram nM nanomolar

[0276] PCR polymerase chain reaction

[0277] SST-12 somatostatin-12

[0278] SST-14 somatostatin-14

[0279] SSTR2 somatostatin receptor 2

[0280] SSTR4 somatostatin receptor 4

[0281] VH antibody heavy chain variable heavy domain

[0282] VL antibody light chain variable domain

[0283] Example 1 - Design, production and SSTR4 agonist function testing of IgG antibodies presenting a somatostatin-14 derived peptide (SST-12 / pep1) in HCDR3

[0284] Methods

[0285] A number of previously published VH sequences (with accompanying VL sequences) containing a pair of non-canonical cysteine residues present within the HCDR3 region were identified from anti-SARS-CoV- 21-3antibody and anti-cobrotoxin4antibody sources. The resulting IgG molecules together with the naming convention used in this work are summarised in Table 2, but briefly the different antibody scaffolds were labelled as ‘AB1 parent’, ‘AB2 parent’, ‘AB3 parent’, ‘AB4 parent’ and ‘AB5 parent’.

[0286] Table 2. Sourcing of IgG VH / VL sequences

[0287] The VH domains of the IgG molecules described above were used as a presentation scaffold for a human somatostatin-14 derived peptide. The native human somatostatin-14 (SST-14) peptide sequence is ‘AGCKNFFWKTFTSC’5(SEQ ID NO:1 ). The N-terminal amino acid residues ‘AG’ are not expected to be involved in agonist function6and were thus removed resulting in a somatostatin-12 (SST- 12) peptide sequence ‘CKNFFWKTFTSC’ (SEQ ID NO:2), also referred to herein as ‘pepT.

[0288] The non-canonical cysteine regions (including the cysteine resides) in VH constructs described in Table 2 were replaced with the whole sequence of ‘pepT. The modifications are summarised in Table 3, the scaffolds with a peptide insertion were labelled as ‘AB1 pep1’ (SEQ ID NO:33), ‘AB2 pep1 ’ (SEQ ID NO:34), ‘AB3 pepT (SEQ ID NO:35), ‘AB4 pepT (SEQ ID NO:36) and ‘AB5 pepT (SEQ ID NO:37).

[0289] Table 3. Summary of modifications to IgG VH domains (only H0DR3 regions are shown)

[0290] Methods for expressing antibody heavy chains and light chains are well established in the art. For example, EP3137602B1 describes the pINT series of mammalian expression plasmids including plNT2 for heavy chain expression in HEK293 cells (see FIG. 1 ). VH genes encoding fused SST-14 derived peptides, i.e.SST-12 peptides, in HCDR3 were cloned into plNT2 using flanking Ncol and Xhol sites.

[0291] Mammalian expression vector plNT2 containing the Hg1 gene for CH1-CH2-CH3 domains (SEQ ID NO: 18) was linearised using restriction enzymes Ncol and Xhol (NEB, standard reaction conditions) and purified using a standard gel extraction protocol (MinElute PCR Purification Kit, Qiagen: 28004). The synthetic genes encompassing VH sequences with desired modifications were designed to contain >21 bp complementary DNA regions upstream and downstream of the gene of interest, respectively, for assembly with linearised plNT2 expression vector using NEBuilder HiFi DNA Assembly method, and were ordered from Integrated DNA Technologies (IDT, eBlock Gene Fragments). DNA assembly was carried out using manufacturer’s instructions, but briefly, 6 ng of synthetic gene DNA was mixed with 20 ng of purified linearised vector, the mixture was then mixed in a 1:1 ratio with NEBuilder HiFi DNA Assembly master mix (NEB #E2621 ) and incubated for 60 minutes at 50 °C. The assembled DNA was then used to transform NEB 10-beta Competent E. coli (NEB # C3019H). Successful sequence confirmed transformants were isolated and plasmid DNA was prepared using QIAGEN Plasmid Plus Midi Kit (Qiagen, Cat. No 12945, standard manufacturer’s instructions). Correct sequences were confirmed using standard Sanger sequencing procedures (Eurofins and Genewiz) using primers 5’(CAACGGGACTTTCCAAAATGTCGTAAC)3’ (SEQ ID NO:45) and 5’(CAGTAGCAGGCTTGAGGTC TG)3’ (SEQ ID NO:46).

[0292] Plasmids for expression of the VL genes for each IgG were cloned in the same manner with the following exceptions: mammalian expression vector plNT112 containing either CL-kappa (SEQ ID NO:19) or CL-lambda kappa (SEQ ID NO:20) was linearised using restriction enzymes Nhel and Notl (FIG. 1). The synthetic genes encompassing VL genes were designed to contain >21 bp complementary DNA regions 5’(TGTTTGCTGTATATTTTCAGCTAGC)3’ (SEQ ID NO:47) and 5’(GCCGCCCCTTCCGTGTTCATC)3’ (SEQ ID NO:48) upstream and downstream of the gene of interest, respectively, for assembly with linearised plNT112 expression vector. Correct sequences were confirmed using standard Sanger sequencing procedures (Eurofins and Genewiz) using a primer 5’(TTCATGTCTAGCAAGATTAAAGGTG)3’ (SEQ ID NO:49).

[0293] The constructs described in this and subsequent examples were produced using HEK293F protein expression system. For each construct, transfection quality DNA was prepared using Plasmid Plus Kit (Qiagen, Cat. No 12945). 33 pg of plNT112 plasmid DNA (Light Chain) was mixed with 16.5 pg of plNT2 plasmid DNA (Heavy Chain) and incubated with ExpiFectamine 293 Reagent (2.7 pl per ml of cell culture, Thermo Fisher Scientific, #A14524) in Opti-MEM Medium (50 pl per ml of cell culture, Thermo Fisher Scientific, #A1435101) for 20 minutes. After the incubation, the mixture was added to 50 ml of HEK-293F cells (Thermo Fisher Scientific, Cat. No. R790-07) seeded at a density of 2.5 x 106cells / ml in 125 ml tissue culture flasks. Culture flasks were incubated at 37 °C for 5 days (with 5 % CO2, 70 % humidity and shaking at 125 rpm, 25 mm throw). All transfections successfully expressed the designed IgG molecules. Expressed proteins were purified from the cell culture supernatants using Protein A Beads (Generon, Cat. No. PC-A100) according to manufacturer's instructions. Purified proteins were buffer exchanged into modified EC000 buffer (145 mM NaCI, 4 mM KCI, 2 mM CaCh, 1 mM MgCL, 10 mM HEPES; the pH was adjusted to 7.4 with NaOH) using Zeba Spin Desalting Columns, 7K MWCO, 5 mL (ThermoFisher, #89883). Where possible, proteins were further purified by performing preparative SEC using the AKTA Express (Cytiva). S200 10 / 300 column was equilibrated in EC000 buffer, samples were injected using a 2 ml loop, and fractionation was carried out at a flow rate of 0.25 ml / min. Each protein was analysed via HPLC-SEC (Agilent 1260) in 1x and 2x PBS with 0.24 mL / min flow rate on a Superdex 200 5 / 150 column (Cytiva) to confirm successful production of the antibody molecules.

[0294] Agonist function testing of the constructs generated was performed using a commercially available cyclic adenosine monophosphate (cAMP) production inhibition assay (cAMP Hunter express SSTR4 CHO-K1 GPCR Assay, Eurofins, # 95-0059E2CP2L). The assay kit provides a stable CHO-K1 cell line expressing the human SSTR4 receptor. The agonist testing is performed by first stimulating the cells to overproduce cAMP via addition of forskolin that stimulates adenylyl cyclase enzyme that catalyses the ATP to cAMP reaction. SSTR4 receptor is coupled to Gi protein alpha subunit, upon binding to an agonist, SSTR4 undergoes a conformational change that causes Gi protein alpha subunit to dissociate. The dissociated Gi protein alpha subunit then inhibits the function of adenylyl cyclase resulting in lowered cAMP levels. Following this, the assay window can be defined by comparing cAMP levels of forskolin stimulated and un-stimulated cells.

[0295] Here, the assay was carried out using manufacturer’s instructions. Briefly, SSTR4 CHO-K1 cells were plated on clear bottom 96 well plates at approx. 20,000 cells per well. Cells were allowed to recover and adhere for 24 hours at 37 °C (at 5% CO2 atmosphere). Growth medium was then removed and cells were incubated with 1 :3 serially diluted IgG samples premixed with 20 M of forskolin. Each protein concentration was applied to at least two cell plated wells (n=2). Wells with forskolin only and no forskolin were also prepared to define the assay window. All above incubations were in EC000 buffer at 37 °C (at 5% C02atmosphere) for 30 minutes. After the incubation, 15 L of cAMP Antibody Reagent was added to all wells followed by addition of 60 L of working Solution. This was then incubated for 1 hour at room temperature (approx. 23 °C) in the dark followed by addition of 60 pL of cAMP Solution D (cAMP Antibody Reagent, Working Solution and cAMP Solution Dare proprietary components of the cAMP Hunter express SSTR4 CHO-K1 GPCR Assay, Eurofins, # 95-0059E2CP2L assay). A final incubation of 3 hours at room temperature in the dark was carried out before reading the luminescence signal for 1 second per well using ClarioStar (BMG) or ID5 (Molecular Devices) plate readers. Readouts were normalized to average top value of buffer only and average bottom value of no forskolin wells. The duplicate values were averaged, and top concentration readouts were plotted. To obtain concentration-response curves for select constructs, the cAMP assay was run at varying test compound concentrations. The data was fit using sigmoidal three-parameter inhibition model on Prism 10 (GraphPad).

[0296] Results

[0297] The constructs described above were successfully purified. The cAMP assay to test SSTR4 agonism function revealed that all the antibodies containing SST-12 / pep1 (SEQ ID NO:2) insertions tested at top concentration showed agonist activity as signified by lowered cAMP levels (FIG. 2). Four out of five antibodies tested (AB1 pep1 , AB2 pep1, AB4 pep1 and AB5 pep1 ) managed to inhibit cAMP production at 100 % signifying full SSTR4 agonism. None of the tested parental versions of the antibody scaffolds showed any cAMP inhibition, meaning that only antibodies with SST-12 / pep1 (SEQ ID NO:2) insertions induced SSTR4 agonism. Surprisingly, four out of five tested antibodies showed SSTR4 agonism comparable to SST-14 control. The cAMP based SSTR4 agonism assay was also employed to test each of the agonist antibodies at varying concentrations (FIG.3). The cAMP levels recorded revealed that SSTR4 agonism was achieved in a concentration-dependent fashion. The data was fit using a sigmoidal model and IC50 values were determined (Table 4). This data revealed a broad range of agonist potency achieved. Four out of five tested antibodies showed agonism below IC50 values of 350 nM with one of the antibodies (AB4 pep1 ) showing IC50 as low as 43 nM, which is less than 20-fold higher than the SST- 14 small cyclical peptide control.

[0298] Table 4. SSTR4 agonist antibody testing summary. IC50 values obtained from cAMP inhibition concentration-response curves shown in FIG 3. 95 % confidence intervals are also listed. Conclusions

[0299] Taken together, the data in this example demonstrates that potent SSTR4 agonists can be generated through insertion of a SST-14-derived peptide, in particular a SST-12 peptide, into an antibody-derived scaffold, in particular into the HCDR3 loop.

[0300] Example 2 - Design, production and SSTR4 agonist function testing of KNOTBODY molecules with a somatostatin-14 derived peptide (SST-12 / pep1) in Knottin domains

[0301] This example describes the construction, expression and functional validation of KNOTBODY-based SSTR4 agonist molecules. The KNOTBODY-based molecules were imparted with SSTR4 agonist function through insertion of a novel SST-14-derived peptide into the knottin domain of the KNOTBODY molecules.

[0302] Methods

[0303] In previous work described in WO2017 / 118761 knottins were presented on the surface loops (complementarity determining regions, CDRs) of antibodies. These insertions exemplified by insertion in light chain CDRs 1, 2 or 3 (LCDR1 , LCDR2, or LCDR3) or heavy chain CDRs 1 , 2 or 3 (HCDR1, HCDR2, HCDR3) are referred to as KNOTBODY molecules and this KNOTBODY format was used to exemplify insertion of a SST-14-derived peptide (‘pepT, also referred to as SST-12, SEQ ID NO:12) directly into knottin scaffolds.

[0304] As a starting point, a previously described KNOTBODY termed KBA12 EETI-II7was used which contains the EETI-II8(SEQ ID NO:21 ) knottin inserted into VL LCDR2 (SEQ ID NO:17). This example also describes the design of a novel KNOTBODY molecule based on the human Agouti-Related Protein (AGRP)9and subsequent engineering of the novel KNOTBODY molecule to present a SST-14 derived peptide for SSTR4 agonist generation.

[0305] The amino acid sequence for human AGRP was sourced from literature10(SEQ ID NO:23), this sequence covers the amino acid region 87-120 residues of the full-length AGRP and contains a cysteine to alanine mutation at position 105 (relative to full-length sequence). The three-dimensional structures for EETI-II and AGRP knottins together with primary sequences annotated with di-sulphide bridges are presented in FIG. 4A and 4B, respectively. A novel AGRP-based KNOTBODY construct was designed to replace the EETI-II peptide (SEQ ID NO:21) with the AGRP peptide (SEQ ID NO:23) in VL domain of KBA12 EETI-II (SEQ ID NO:22). The final construct of KBA12 AGRP (VL SEQ ID NO:24) inserts the AGRP amino acid sequence (SEQ ID NO:23) between framework linker sequences ‘AGR’ (at N-terminus relative to the insertions) and ‘ANS’ (at C-terminus relative to the insertions).

[0306] In the case of KBA12 AGRP the SST-14-derived peptide (SEQ ID NO:2) replaced ‘loop 4’ of AGRP (SEQ ID NO:25), this KNOTBODY construct was termed ‘KB1 pepT (VL SEQ ID NO:38). In the case of KBA12 EETI-II the SST-14 derived peptide (SEQ ID NO:2) was designed to replace ‘loop T or ‘loop 5’ of EETI-II (SEQ ID NO:26 and SEQ ID NO:27, respectively), the final KNOTBODY constructs corresponding to the two different loop insertions were labelled ‘KB2 L1 pepT (SEQ ID NO:39) and ‘KB2 L5 pepT (SEQ ID NO:40). The parental KNOTBODY constructs were termed ‘KBT and ‘KB2’ for AGRP and EETI-II based KNOTBODY molecules, respectively. The cloning was carried out as described for antibody light chains in Example 1 , with the VL domains cloned into target plNT112 vectors containing CL-lambda domains (SEQ ID NO:20). Each KNOTBODY was co-expressed with a previously described heavy chain, termed D1A12 (SEQ ID NO:28). The preparation of plasmids for expression of D1A12 is described in WO 2019 / 012015. The D1A12 heavy chain was cloned with Hg1 constant domains (SEQ ID NO:18).

[0307] KNOTBODY purification and functional testing was carried out in the same manner as described in Example 1.

[0308] Results

[0309] The KNOTBODY constructs described above were successfully purified. The cAMP assay to test SSTR4 agonism function revealed that all the KNOTBODY molecules containing pep1 (SEQ ID NO:2) insertions tested at top concentration showed agonist activity as signified by lowered cAMP levels (FIG. 5). Neither parental KNOTBODY (KB1 , KB2) showed SSTR4 agonist activity as shown by cAMP levels remaining high. The AGRP-based construct KB1 pep1 showed the strongest agonism of the three SST-14-derived peptide displaying KNOTBODY molecules tested, this was also confirmed by the cAMP based SSTR4 agonism assay at varying KNOTBODY concentrations (FIG. 6), where a concentration-dependent SSTR4 agonist activity was shown for all three KNOTBODY molecules containing pep1 (SEQ ID NO:2). The data was fit using a sigmoidal model and IC50 values were determined (Table 5). KB1 pep1 was revealed to have an IC50 value of 1309 nM. Both KNOTBODY molecules containing the EETI-II knottin (KB2 L1 and KB2 L5) showed SSTR4 agonist function regardless of which loop pep1 (SEQ ID NO:2) was inserted to.

[0310] Table 5. SSTR4 agonist KNOTBODY testing summary. IC50 values obtained from cAMP inhibition concentration-response curves shown in Figure 3. 95 % confidence intervals are also listed.

[0311] Conclusions

[0312] The data in this example demonstrates that SSTR4 agonists can be generated through insertion of a SST-14-derived peptide into various knottins. Furthermore, the demonstrated agonism through SST-14- derived peptide insertion was shown to be knottin loop independent.

[0313] Example 3 - Design, production and SSTR4 agonist function testing of IgG antibody presenting somatostatin-14 derived peptide (SST-12 / pep1) in LCDR2

[0314] Methods

[0315] SST-14-derived peptide (SEQ ID NO:2) replaced the knottin region of a KNOTBODY light chain termed KBA12 EETI-II (SEQ ID NO:22) described in Example 2. The original knottin sequence motif (SEQ ID NO:26) of the VL gene was replaced with a SST-14-derived peptide (SEQ ID NO:2) in a way that replaced the whole original sequence (except for the final ‘G’ residue EETI-II of the knottin which was inadvertently kept) with the whole SST-14-derived peptide sequence including cysteine residues. The resulting construct was termed ‘AB6 pepT (SEQ ID NO:41).

[0316] The cloning, antibody production and SSTR4 agonist testing was carried out as described in Examples 1 and 2, with the VL domain cloned into a plNT112 vector containing CL-lambda (SEQ ID NO:20). The cloned light chain construct was co-expressed with the D1A12 (SEQ ID NO:28) heavy chain, the cloning of which is described in Example 2.

[0317] Results

[0318] AB6 pep1 was successfully produced and tested for SSTR4 agonism using the cAMP assay. The data for a single concentration point test is presented in FIG. 7. Here, the SSTR4 agonism of AB6 pep1 was compared to two KNOTBODY constructs KB1 and KB2 (negative controls) and free cyclical peptide SST- 14 (positive control). KB1 and KB2 were not expected to induce SSTR4 agonism as they did not possess an agonist peptide insertion. AB6 pep1 showed near complete depletion of cAMP levels at the concentration of 1700 nM suggesting strong SSTR4 agonist function, whereas the control antibodies showed no reduction of cAMP levels.

[0319] Conclusions

[0320] The results of this example show that a potent SSTR4 agonist antibody molecule can be produced through insertion of a SST-14 derived peptide (SEQ ID NO: 2) into LCDR2. This exemplifies that the SSTR4 agonist generation methods described in this patent are not CDR dependant, in other words, any of the six antibody CDR loops are expected to be amenable to such insertion of a SST-14 derived peptide.

[0321] Example 4 - Design, production and SSTR4 agonist function testing of VHH (NANOBODY) with a somatostatin-14 derived peptide (SST-12 / pep1) in CDR3

[0322] Methods

[0323] VHH sequences containing a CDR3 with two non-canonical cysteine residues were derived from ‘CoV- AbDab: the Coronavirus Antibody Database’3; specifically, VHH antibodies aRBD_42 (SEQ ID NO:29) and Nb63 (as described in CN112094342A, SEQ ID NQ:30 herein) were selected. In each case, parental CDR3 motifs ‘CSGLGC’ (SEQ ID NO:31 ) and ‘CSGSGC’ (SEQ ID NO:32) of aRBD_42 and Nb63, respectively, were replaced with SST-14 derivative peptide - ‘pepT (SEQ ID NO:2). The parental constructs were termed ‘NB1 parent’ and ‘NB2 parent’, whereas pep1 (SEQ ID NO:2) containing constructs were termed ‘NB1 pepT (SEQ ID NO:42) and ‘NB2 pepT (SEQ ID NO:43), respectively. All VHH constructs were modified with a C-terminal tri-alanine linker, TriFlag peptide tag and six histidine tag for detection and purification purposes.

[0324] The cloning of VHH described above was performed as described in Example 1 with the following exceptions: mammalian expression vector plNT112 (FIG. 1C) was linearised using restriction enzymes Nhel and BamHI (NEB, standard reaction conditions), and the synthetic VHH genes were designed to contain >21 bp complementary DNA regions 5’(TGCCTTTCTCTCCACAGGCGCCATGGCC)3’ (SEQ ID NO:50) and 5’(TAATAATAATAAGGATCCTTTAAGCTTACGACGTGATCA)3’ (SEQ ID NO:51 ) upstream and downstream of the VHH gene of interest, respectively.

[0325] For VHH expression in HEK293 cells, the transfection protocol was identical to that described in Example 1. Following successful transfections, the obtained cell culture supernatants for the four constructs were then harvested, and dialysed (Thermo Scientific SnakeSkin Dialysis Tubing, #68035) three times in 1 x PBS pH 7.4 at 4 °C (>4 hours each). The expressed proteins were purified from the dialysed cell culture supernatants using Ni-NTA Agarose (Qiagen Cat #30210) according to manufacturer's instructions. Briefly, 50 ml of each supernatant was incubated overnight at 4 °C with 300 pl Ni-NTA resin, the supernatant was removed, and resin was washed two times with 1 x PBS + 20 mM Imidazole pH 8.0. The VHH nanobodies were then eluted in 1 x PBS + 300 mM Imidazole pH 8.0. Purified proteins were buffer exchanged into modified EC000 buffer (same recipe as Example 1) using Zeba Spin Desalting Columns, 7K MWCO, 5 ml_ (ThermoFisher, #89883). Proteins were further purified by performing preparative size exclusion chromatography as described in Example 1. Each protein was analysed via HPLC-SEC (Agilent 1260) in 1x and 2x PBS with 0.24 mL / min flow rate on a Superdex 200 5 / 150 column (Cytiva). The prepared nanobodies showed chromatographic profiles corresponding to intact VHH molecules.

[0326] Results

[0327] Both parental and agonist peptide inserted VHH constructs were successfully produced and tested for SSTR4 agonism using the cAMP assay as described before. When tested at top concentration (1.7 M) both NB1 pep1 and NB2 pep1 show strong SSTR4 agonism (FIG. 8). NB2 pep1 shows complete dampening of cAMP levels, even potentially exceeding the somatostatin-14 control levels (tested at 1 M). None of the parental versions of these constructs showed SSTR4 agonism as signified by the cAMP remaining high. The SSTR4 agonism assay at varying agonist VHH concentrations was also carried out (FIG. 9). Same as for molecules described in Examples 1 and 2, a concentration-dependent SSTR4 agonism was confirmed. The data was fit using a sigmoidal three-parameter inhibition model and IC50 values of 3245 and 256 nM were determined for NB1 pep1 and NB2 pep1, respectively.

[0328] Conclusions

[0329] The results of this example, show that the approach taken in Examples 1 , 2, 3 and 4, with respect to antibodies and KNOTBODY molecules, is equally applicable for single variable domains, with potent VHH-derived SSTR4 agonist molecules being generated. What is more, at least one of the agonist VHH constructs shows potency comparable to agonist IgG constructs described in Example 1.

[0330] Example 5 - Design, production and SSTR4 subtype specific agonist function testing of IgG antibodies presenting various SST-14-derived peptides (SST-12 / pep2, pep3, pep4, pep5 and pep6) in HCDR3

[0331] Example 1 showed generation of antibody agonist molecules based on SST-14-derived peptide (SEQ ID NO:2) which targeted SSTR4, but were also expected to agonise other SSTR family subtypes (SSTR1, SSTR2, SSTR3, SSTR5). Here, a series of SST-14-derived peptides were inserted into one of the IgG scaffolds described in Example 1 to generate novel SSTR4 subfamily specific agonists. Methods

[0332] Literature was consulted11to obtain two human SST-14 derived peptides with SSTR4 agonist selectivity: pep2: ‘CFAWKTFC’ (SEQ ID NO:3) pep3: ‘CFAWKTAC’ (SEQ ID NO:4).

[0333] These peptides were previously shown to induce agonism in SSTR4 and not any other SSTR family subtype when tested as cyclical peptides without scaffolding. Additional SST-14-derived novel peptides were also designed: pep4: ‘CKNFAWKTFTSC’ (SEQ ID NO:5) pep5: ‘CGGFAWKTFGGC’ (SEQ ID NO:6) pep6: ‘CFFWKTFC’ (SEQ ID NO:7)

[0334] Peptide ‘pep4’ (SEQ ID NO:5) was predicted to allow for maintained agonist function against SSTR4, however the change of the pharmacophore ‘FWKT’ to ‘AWKT’ was predicted to prevent agonist function against other SSTR family members, since phenylalanine in position 7 (relative to SST-14) is considered less important for SSTR4 agonism than other SSTR family members11. Peptide ‘pep5’ (SEQ ID NO:6) was included as a variant of ‘pep4’ with flexible residues ‘GG’ replacing regions proximal to the outer cysteine residues to test the importance of non-pharmacophore residues for SSTR4 agonism. Lastly, peptide ‘pep6’ (SEQ ID NO:7) was included to test the importance of length for SSTR4 agonism, this peptide was predicted not to be selective for SSTR4 over other SSTR family members due to maintaining the ‘FWKT’ motif in sequence.

[0335] IgG scaffold AB1 was chosen for testing the SST-14-derived peptides described above. Each peptide was inserted into HCDR3 as described in Example 1 , where non-canonical cysteine regions of parental molecules (including the cysteine resides) in resulting VH constructs were replaced with the whole sequence of each peptide. All the cloning, protein production and SSTR4 agonist testing was carried out as described in Example 1. A commercial SSTR2 agonist assay - cAMP Hunter express SSTR2 CHO-K1 GPCR Assay (Eurofins, 95-0095E2) was used for SSTR subtype specificity testing. The assay followed the same principle and procedure as the SSTR4 assay described in Example 1. SSTR4 and SSTR2 comparison experiments were carried out in parallel with identical sample preparations split between assay plates coated with either SSTR4 or SSTR2 cells. Four parameter concentration-response inhibition model was used to fit the data.

[0336] Results

[0337] The series of putative agonist peptides in the AB1 IgG scaffold - AB1 pep1, AB1 pep2, AB1 pep3, AB1 pep4, AB1 pep5 and AB1 pep6 were successfully produced.

[0338] First, an SSTR4 agonist experiment was carried out (FIG. 10). Again, AB1 pep1 showed good agonism (as shown in Example 1). Surprisingly, the two constructs with literature derived SSTR4 selective agonist peptides pep2 and pep3 did not show SSTR4 agonism as cAMP levels remained high (these two peptides were also tested in other antibody and KNOTBODY scaffolds, with none of the constructs showing any agonism - data not shown).

[0339] Of the three constructs containing novel peptides (pep4, pep5, pep6), AB1 pep4 (SEQ ID NO:44) showed the highest degree of SSTR4 agonism (showing cAMP levels decreasing to 15 % relative to complete inhibition). AB1 pep6 showed partial SSTR4 agonism (50 % cAMP inhibition), while AB1 pep5 showed no SSTR4 agonism (no cAMP inhibition).

[0340] Due to AB1 pep4 having the highest potency for SSTR4, this construct was used in the SSTR4 to SSTR2 comparison experiment shown in FIG. 11. As expected, AB1 pep1 showed good agonism of SSTR2 (FIG. 11 A) and SSTR4 (FIG. 11 B) as is signified by decreased cAMP levels. AB1 pep4, however, showed good agonism for SSTR4 (FIG. 11B), but none for SSTR2. This shows that AB1 pep4 is selective for SSTR4 over SSTR2 (and by extrapolation - other SSTR family subtypes). The difference in agonist potency was quantified by carrying out a concentration titrated agonism experiment on SSTR2 and SSTR4 (FIG. 12). AB1 pep4 showed very weak (if any) concentration dependent agonism of SSTR2 (FIG. 12A), through extrapolation of the fit curve an IC50 value of 6500 nM could be determined. In clear contrast, AB1 pep4 showed a clear concentration dependent agonism of SSTR4 (FIG. 12B), with IC50 value determined to be 660 nM. Comparing the two IC50 values obtained >10-fold selectivity for SSTR4 over SSTR2 was calculated.

[0341] Conclusions

[0342] Example 5 showed the possibility to build on the learnings from previous examples to engineer agonist molecules with additional functionality, namely, molecules with selectivity for specific receptor subtypes in closely related receptor families (e.g. AB1 pep4).

[0343] It was surprising that there was a lack of SSTR4 agonism with insertions of SST-14 derived peptides pep2 and pep3 containing the ‘AWKT’ pharmacophore, which, according to literature11, should be sufficient for specific SSTR4 agonism. Given these peptides are less than two-thirds the length of native SST-14, each comprising 8 amino acids, and were specifically designed to be active in free cyclic form, it is possible that directly engineering them into HCDR3 of an antibody scaffold results in limited flexibility of the peptide and / or incorrect folding resulting in the pharmacophore being unable to access the receptor binding site sufficiently. However, the successful detection of SSTR4 agonism (albeit partial) with AB1 pep6 (pep6 is also 8 amino acids long), suggests that shorter peptide insertions can also enable SSTR4 agonism, but these shorter peptides are likely to exhibit lower potency in the context of a scaffold.

[0344] Pep5 is the same length as pep4 (both 12 amino acids) and both contain the ‘AWKT’ pharmacophore. Yet AB1 pep5 showed no SSTR4 agonism, whereas AB1 pep4 did. Amino acid sequence outside of the pharmacophore is clearly also important.

[0345] We have identified molecules that show increased selectivity for SSTR4 over SSTR1 / 2 / 3 / 5 (data not shown) as well as molecules that show increased selectivity for SSTR2 over SSTR1 / 3 / 4 / 5 (data not shown) via sequence modifications.

[0346] Example 6 - Design, production and SSTR4 agonist function testing of a panel of IgG antibodies presenting a somatostatin-14 derived peptide (SST-12 / pep1) across each heavy chain and light chain CDR in turn

[0347] In the above examples, successful insertion of SST-14 derived peptide into HCDR3 or LCDR2 to produce functional SSTR4 agonists has been shown. Here we show that insertion of SST-14 derived peptide (SEQ IS NO:2) into any of the CDRs of an IgG can produce functional SSTR4 agonists. Methods

[0348] The pairing of the IgG heavy and light chains of the IgG constructs tested, together with the naming convention used in this example is summarised in Table 6, but briefly the different antibody scaffolds were labelled as ‘AB7 parent’, ‘AB8 parent’, ‘AB9 parent’ and ‘AB10 parent’.

[0349] Table 6. IgG heavy chain and light chain sequences

[0350] The SST-14-derived peptide (SST-12 / pep1, SEQ ID NO:2, bold and underlined in Table 7) was either 1 ) inserted into one of the heavy chain CDRs or ones of the light chain CDRs of the above- described scaffolds, or 2) replaced one of the LCDRs or one of the HCDRs of the above-described scaffolds, with the respective CDR being fully removed and a number of linker amino acids on the N- and C- termina of SST-12 added (linker amino acids are only underlined in Table 7). This latter format is herein termed “replacement with linker”.

[0351] The sequences of the original CDRs, together with the sequences of the ‘new’ CDRs following SST-14-derived peptide insertion / replacement into the various scaffolds is summarised in Table 7.

[0352] Table 7. Summary of modifications to IgG CDRs

[0353] The cloning, antibody production and SSTR4 agonist testing of all of the molecules was carried out as described in Examples 1 and 2, in two sets of experiments (experiment 1 or 2 as shown in Table 7). All transfections were carried out in a 2:1 heavy chain: light chain ratio.

[0354] Results

[0355] The IgG constructs described above were in the most part successfully purified, apart from a few “replacement with linker” constructs which failed to express. The failure in expression could be attributed to a shorter CDR length which may be problematic for protein folding and hence a lack of detectable expression, or that the specific linkers used are not amenable to the particular scaffold.

[0356] The cAMP assay to test for SSTR4 agonism function revealed that all expressed IgG constructs containing SST-12 showed agonist activity ranging from <1 nM to 104 nM (Table 8). A number of parental antibody molecules (not including SST-12 / 14) have previously shown no SSTR4 agonism effect (data not shown), AB10 parent is shown to have no agonist activity against SSTR4.

[0357] Table 8. SSTR4 agonist antibody testing summary. IC50 values obtained from cAMP inhibition concentration-response curves shown in FIG 13. 95 % confidence intervals are also listed

[0358] Conclusions

[0359] Successful IgG SSTR4 agonist generation is not dependent on which CDR the SST-14-derived peptide is inserted into.

[0360] Example 7 - Design, production and SSTR4 agonist function testing of IgG antibodies presenting full-length somatostatin-14 peptide (SST-14 / pep7) in HCDR3

[0361] The aim of this example is to confirm that insertion of SST-14 (SEQ ID NO:1 ), also referred to herein as ‘pep7’, into an IgG CDR results in a functional SSTR4 agonist. As postulated in example 1, whether or not the N-terminal somatostatin amino acid residues ‘AG’ are present is not critical for agonist function.

[0362] Methods

[0363] IgG antibodies comprising full-length human somatostatin peptide, SST-14 / pep7 (SEQ ID NO:1 ), in HCDR3 were generated. SST-14 replaced the whole sequence of the naturally occurring cysteine constrained loop present in HCDR3 of two lgG1 scaffolds: AB2 and AB4 (see Example 1 ), i.e. the resultant constructs included the N-terminal “AG” outside of the cysteine loop. The constructs containing the ‘new’ HCDR3 were termed ‘AB2 pep7’ and ‘AB4 pep7’, respectively, and the sequences of the original HCDR3s (cysteine constrained loop shown in bold underlined) and resultant HCDR3s (SST-14 shown in bold underlined) are shown in Table 9.

[0364] Table 9. Summary of modifications to HCDR3 The cloning of expression vectors for AB2 Pep7 and AB4 Pep7 was carried out via site directed mutagenesis (SDM) of AB2 Pep1 and AB4 Pep1 expression vectors, respectively. Sequences of the primers used are shown in Table 10 [SDM primers].

[0365] Table 10. SDM primer sequences

[0366] Thermococcus kodakaraenis (KOD) Hot Start Master Mix (71842, Sigma-Aldrich) was used to initiate 50 pl mutagenesis reactions with 10 ng of original expression vectors using the mutation carrying primers. The thermal cycling conditions were set as follows: 95 °C for 2 minutes followed by thirty cycles of 95 °C for 20 seconds, 70 °C for 2 minutes 30 seconds, followed by 70 °C for 5 minutes. The reactions were then digested with Dpnl enzyme (R0176S, NEB) to remove methylated template DNA for 1 hour at 37 °C with 20-minute deactivation at 80 °C. The rest of the cloning procedure was carried out the same way as described in Example 1 .

[0367] Antibody production and SSTR4 agonist testing was carried out as described in Example 1 and Example 2.

[0368] Results

[0369] The cAMP assay to test for SSTR4 agonism function revealed that both IgG constructs containing SST- 14 / pep7 showed agonist activity (Table 11 ).

[0370] Table 11. SSTR4 agonist antibody testing summary. IC50 values obtained from cAMP inhibition concentration-response curves shown in FIG 14. 95 % confidence intervals are also listed

[0371] Conclusions

[0372] The data in this example demonstrates that SSTR4 agonists can be generated through insertion of the full naturally occurring somatostatin sequence, SST-14 / pep7 (SEQ ID NO:1 ) into HCDR3 of IgG. Example 8 - Design, production and SSTR4 agonist function testing of a variety of biologic modalities presenting full-length somatostatin-14 peptide (SST-14 / pep7) or a somatostatin-14- derived peptide (SST-12 / pep1)

[0373] Previous examples have shown the successful generation of fusion molecules with SSTR4 agonist activity, including selective SSTR4 activity in a number of formats: IgG antibodies (Examples 1 , 3 and 5- 7), KNOTBODY molecules (Example 2) and NANOBODIES (Example 4). The aim of this example is to assess the feasibility of fusing full-length somatostatin (SST-14 / pep7, SEQ ID NO:1)) or a SST-14- derived peptide (SST-12 / pep1, SEQ ID NO:2) into a greater variety of different biologic modalities to produce functioning SSTR4 agonist molecules.

[0374] Methods

[0375] The following different types of constructs were made as summarised in Table 12.

[0376] MODI was based on fibronectin (Fn3) as disclosed by Chandler and Buckle13with SST-12 replacing the FG loop therein (equivalent to L3 loop in our nomenclature).

[0377] MOD2 was based on ADHIRON 4N6T described by Tiede et al14with the site 1 and site 2 nomenclature used therein being referred to as L1 and L2 herein. In MOD2 L1 PEP1 , SST-12 replaced site 1 (L1 ) and in MOD2 L2 PEP1 , SST-12 replaced site 2 (L2).

[0378] Design of MOD3 involved fusing full-length somatostatin to the N-terminus of an Fc domain of lgG1 near the “hinge” region defined by sequence “DKTHTCPPCPAP” (SEQ ID NO:108) via a flexible linker “GGGGSGGGGSGGGGS” (SEQ ID NO:109), whereas MOD4 fused full-length somatostatin to the C-terminus of an Fc domain of IgG 1 via an identical linker sequence.

[0379] In MOD5, SST-12 has been fused to the N-terminal of a trastuzumab light chain using a flexible linker “GGGGSGGGGSGGGGS” (SEQ ID NO: 109), which was paired with a trastuzumab heavy chain comprising the Fc LAGA mutations and a C-terminal lysine deletion.

[0380] In MOD6, SST-12 has been fused to the N-terminal of a trastuzumab heavy chain using a flexible linker “GGGGSGGGGSGGGGS” (SEQ ID NQ:109), which was paired with a trastuzumab light chain.

[0381] Table 12. Modalities tested

[0382] The cloning of constructs M0D6 PEP1 and M0D7 PEP1 was performed as described in Example 1. The cloning of constructs M0D1 PEP1 , M0D2 L1 PEP1, M0D2 L2 PEP1 , M0D3 PEP7, M0D4 PEP7, M0D6 PEP1 and M0D7 PEP1 was performed as described in Example 4. The protein production of M0D1 PEP1 , M0D2 L1 PEP1 and M0D2 L2 PEP1 was performed as described in Example 4. The protein production of M0D5 PEP1, M0D6 PEP1 , M0D3 PEP7 and M0D4 PEP7 was carried out as described in Examples 1 and 2.

[0383] All agonist testing was performed as described in Example 1. Results

[0384] Full-length somatostatin (SST-14 / pep7, SEQ ID NO:1 ) or SST-14-derived peptide (SST-12 / pep1, SEQ NO:2) were inserted into a variety of modalities. Constructs were expressed successfully and functional testing using SSTR4 over-expressing cell lines and a cAMP readout showed functional agonism for all modalities. A limited amount of inhibition was also detected with parental modalities without SST-14- derived peptide inserted, however this could possibly be due to high concentrations of sample in the assay interfering with cAMP detection. However, entities inserted with SST-14-derived peptide were superior causing 93 to 120% inhibition of cAMP. We note that over 100% inhibition may be due to a lowering of endogenous cAMP.

[0385] Table 13. SSTR4 agonist modality testing summary. IC50 values obtained from cAMP inhibition concentration-response curves shown in FIG. 15.

[0386] Conclusions

[0387] This example demonstrates that SSTR4 agonists can be generated through insertion or fusion of SST-14 (SEQ ID NO:1) or SST-14-derived peptides (SEQ ID NO:2) into a wide variety of biologic entities, such as MONOBODIES, ADHIRONS, and N-terminal IgG fusions.

[0388] References

[0389] (1 ) Oho, A.; Muecksch, F.; Schaefer-Babajew, D.; Wang, Z.; Finkin, S.; Gaebler, C.; Ramos, V.; Cipolla, M.; Mendoza, P.; Agudelo, M.; Bednarski, E.; DaSilva, J.; Shimeliovich, I.; Dizon, J.; Daga, M.; Millard, K. G.; Turroja, M.; Schmidt, F.; Zhang, F.; Tanfous, T. B.; Jankovic, M.; Oliveria, T. Y.; Gazumyan, A.; Caskey, M.; Bieniasz, P. D.; Hatziioannou, T.; Nussenzweig, M. C. Anti-SARS-CoV-2 Receptor-Binding Domain Antibody Evolution after mRNA Vaccination. Nature 2021 , 600 (7889), 517-522..

[0390] (2) Karatt A. K.; Bullen G.; Moreels L; Villar E. P.; Mccafferty J. Sars-Cov2 Antibodies. WO 2022 / 058618 A2, September 21 , 2021.

[0391] (3) Raybould, M. I. J.; Kovaltsuk, A.; Marks, C.; Deane, C. M. CoV-AbDab: The Coronavirus Antibody Database. Bioinformatics 2021 , 37 (5), 734-735.

[0392] (4) Laustsen, A. H.; Jensen, Line L; McCafferty, J.; Vellatt, A. K.; Lomonte, B.; Gutierrez, J. M. Neutralisation Of A-Neurotoxins Using Human Recombinant Igg Antibodies. WO 2023 / 209212 A1, April 28, 2023.

[0393] (5) Patel, Y. C.; Greenwood, M. T.; Panetta, R.; Demchyshyn, L; Niznik, H.; Srikant, C. B. The Somatostatin Receptor Family. Life Sci. 1995, 57 (13), 1249-1265. (6) Zhao, W.; Han, S.; Qiu, N.; Feng, W.; Lu, M.; Zhang, W.; Wang, M.; Zhou, Q.; Chen, S.; Xu, W.; Du, J.; Chu, X.; Yi, C.; Dai, A.; Hu, L; Shen, M. Y.; Sun, Y.; Zhang, Q.; Ma, Y.; Zhong, W.; Yang, D.; Wang, M.-W.; Wu, B.; Zhao, Q. Structural Insights into Ligand Recognition and Selectivity of Somatostatin Receptors. Cell Res. 2022, 32 (8), 761-772.

[0394] (7) VELLATT, A. K.; McCafferty, J.; SURADE, S. B.; LUETKENS, T.; MASTERS, E. W.; Dyson, M. R.; BELL, D. C. Potassium Channel Inhibitors. W02019012015A1, January 17, 2019.

[0395] (8) Heitz, A.; Avrutina, O.; Le-Nguyen, D.; Diederichsen, LI.; Hernandez, J.-F.; Gracy, J.; Kolmar, H.; Chiche, L. Knottin Cyclization: Impact on Structure and Dynamics. BMC Struct. Biol. 2008, 8, 54.

[0396] (9) Patel, M. P.; Fabersunne, C. S. C.; Yang, Y.; Kaelin, C. B.; Barsh, G. S.; Millhauser, G. L. Loop Swapped Chimeras of the Agouti-Related Protein (AgRP) and the Agouti Signaling Protein (ASIP) Identify Contacts Required for Melanocortin 1 Receptor (MC1 R) Selectivity and Antagonism. J. Mol. Biol. 2010, 404 (1 ), 45-55.

[0397] (10) Silverman, A. P.; Levin, A. M.; Lahti, J. L; Cochran, J. R. Engineered Cystine-Knot Peptides That Bind Avp3 Integrin with Antibody-Like Affinities. J. Mol. Biol. 2009, 385 (4), 1064-1075.

[0398] (11) Grace, C. R. R.; Koerber, S. C.; Erchegyi, J.; Reubi, J. C.; Rivier, J.; Riek, R. Novel Sst 4 - Selective Somatostatin (SRIF) Agonists. 4. Three-Dimensional Consensus Structure by NMR. J. Med. Chem. 2003, 46 (26), 5606-5618.

[0399] (12) Jackson, P. J.; McNulty, J. C.; Yang, Y.-K.; Thompson, D. A.; Chai, B.; Gantz, I.; Barsh, G. S.; Millhauser, G. L. Design, Pharmacology, and NMR Structure of a Minimized Cystine Knot with Agouti- Related Protein Activity. Biochemistry 2002, 41 (24), 7565-7572.

[0400] (13) Chandler, P. G. and Buckle, A. M. Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain. Cells, 2020, 9(3), PMID: 32143310.

[0401] (14) Tiede, C.; Tang, A. A. S.; Deacon, S. E.; Mandal, LI.; Nettleship, J. E.; Owen. R. L; George, S. E.; Harrison, D. J.; Owens, R. J.; Tomlinson, D. C.; McPherson, M. J. Adhiron: a stable and versatile peptide display scaffold for molecular recognition applications. Protein Eng. Des. Sei., 2014, 27(5): 145- 55, PMID: 24668773.

[0402] SEQUENCES

[0403] SEQ ID N0:1

[0404] AGCKNFFWKTFTSC SEQ ID N0:2 CKNFFWKTFTSC SEQ ID NO:3 CFAWKTFC

[0405] SEQ ID NO:4

[0406] CFAWKTAC

[0407] SEQ ID NO:5

[0408] CKNFAWKTFTSC

[0409] SEQ ID NO:6

[0410] CGGFAWKTFGGC

[0411] SEQ ID NO:7

[0412] CFFWKTFC

[0413] SEQ ID NO:8

[0414] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVT

[0415] ITADESTSTAYMELRSLRSDDTAVYYCARDNLGYCSGGSCYSDYYYYYMDVWGQGTLVTVSS

[0416] SEQ ID NO:9

[0417] SYELTQPPSVSVAPGRTATITCEGDNIGQQIVHWYQQKPGQAPVAVISSDSDRPSGIPERFSGSNSGNTA

[0418] TLTISRVEAGDEADYYCQVWDSGSDHVVFGGGTKVTVLGQPKAAPSVTLF

[0419] SEQ ID NQ:10

[0420] EVQLLESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGR

[0421] FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQIAPYCSGGSCYSYFDYWGQGTLVTVSS

[0422] SEQ ID NO:11

[0423] AIQMTQSPSSLSASVGDRVTITCQASQSISNYLNWYQQKPGKAPKLLIYGASNLETGVPSRFSGSGSGTD

[0424] FTFTISSLQPEDIATYYCQQYNNLPLTFGGGTKVEIKRTVAAPSVFI

[0425] SEQ ID NO:12

[0426] EVQLVQSGAEVKKPGASVKVSCKASGFTFTSSAVQWVRQARGQRLEWIGWIWGSGNTNYAQKFQER

[0427] VTITRDMSTSTAYMELSSLRSEDTAVYYCAAPSCSSTSCYDAFDIWGQGTTVTVSS

[0428] SEQ ID NO:13

[0429] DIVMTQSPGTLSLSPGERATLSCGASQSISSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGT

[0430] DFALTISSLEPEDFAVYYCQQYGNSPWSFGQGTKVEIKRTVAAPSVFI

[0431] SEQ ID NO:14

[0432] QVQLVQSGGGWQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGR

[0433] FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKMGGGPYCSGGSCYSNYFDYWGQGTLVTVSS

[0434] SEQ ID NO:15

[0435] DIQMTQSPSSLSASVGDTVTITCRASQSISTYLNWYQQKPGKAPKLLIYGASNLETGVPSRFSGSGSGTD

[0436] FTLTISSLQPEDFATYYCQQSYSTPQTFGQGTKVDIKRTVAAPSVFI

[0437] SEQ ID NO:16

[0438] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAKTWVRQAPGKGLEWVSGISGSGGSTYYADSVKGRF

[0439] TISSDNSKNTLYLQMNSLRAEDTAVYYCAGRYCSSNTYSGCYYYGMDVWGQGTTVTVSS

[0440] SEQ ID NO:17

[0441] DIQMTQSPSSLSASVGDRVTITCRASQSISSFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTD

[0442] FTLTISSLQPEDFATYFCQQSYITPQTFGQGTKVEIKRTVAAPSVFI

[0443] SEQ ID NO:18

[0444] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT

[0445] VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP

[0446] EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN

[0447] KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP

[0448] PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:19

[0449] RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS

[0450] STLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGEC

[0451] SEQ ID NQ:20

[0452] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASS

[0453] YLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0454] SEQ ID NO:21

[0455] CPRILMRCKQDSDCLAGCVCGPNGFCG

[0456] SEQ ID NO:22

[0457] QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAGRCPRILMRCKQDSDCLA

[0458] GCVCGPNGFCGANSGVSDRFSAAKSGTSASLAINGLRSEDEADYYCAAWDDSLNGYVFGTGTKLTVL

[0459] SEQ ID NO:23

[0460] CVRLHESCLGQQVPCCDPAATCYCRFFNAFCYCR

[0461] SEQ ID NO:24

[0462] QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAGRCVRLHESCLGQQVPC

[0463] CDPAATCYCRFFNAFCYCRANSGVSDRFSAAKSGTSASLAINGLRSEDEADYYCAAWDDSLNGYVFGT

[0464] GTKLTVL

[0465] SEQ ID NO:25

[0466] RFFNAF

[0467] SEQ ID NO:26

[0468] PRILMR

[0469] SEQ ID NO:27

[0470] GPNGF

[0471] SEQ ID NO:28

[0472] EVQLVESGGGLVRPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRF

[0473] TISRDNTKNSLYLQMTSLRADDTAFYYCVKDFGPGYGTGWFDYWGPGTLVTVSS

[0474] SEQ ID NO:29

[0475] QLQLVESGGGLVQAGGSLRLSCAASGRTFSSATMGWFRQAPGKEREFVAAISWSGLSRYYADSVKGRF

[0476] TISRDNAENTVYLQMNSLKTEDTAVYYCAADSWGCSGLGCYDARQYDVWGQGTQVTVSS

[0477] SEQ ID NQ:30

[0478] QVQLVESGGGLVQAGGSLRLSCVASGSGFEPNAMGWYRQAPGKQRELVAGITSGGNANYADSVKGRF

[0479] TISRDNVKNTVYLQMNSLKPEDTAVYYCQGPACSGSGCRNYWGQGTQVTVSS

[0480] SEQ ID NO:31

[0481] CSGLGC

[0482] SEQ ID NO:32

[0483] CSGSGC

[0484] SEQ ID NO:33

[0485] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVT

[0486] ITADESTSTAYMELRSLRSDDTAVYYCARDNLGYCKNFFWKTFTSCYSDYYYYYMDVWGQGTLVTVSS

[0487] SEQ ID NO:34

[0488] EVQLLESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGR

[0489] FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQIAPYCKNFFWKTFTSCYSYFDYWGQGTLVTVSS

[0490] SEQ ID NO:35 EVQLVQSGAEVKKPGASVKVSCKASGFTFTSSAVQWVRQARGQRLEWIGWIVVGSGNTNYAQKFQER

[0491] VTITRDMSTSTAYMELSSLRSEDTAVYYCAAPSCKNFFWKTFTSCYDAFDIWGQGTTVTVSS

[0492] SEQ ID NO:36

[0493] QVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKMGGGPYCKNFFWKTFTSCYSNYFDYWGQGTLVTVSS

[0494] SEQ ID NO:37

[0495] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAKTWVRQAPGKGLEWVSGISGSGGSTYYADSVKGRF

[0496] TISSDNSKNTLYLQMNSLRAEDTAVYYCAGRYCKNFFWKTFTSCYYYGMDVWGQGTTVTVSS

[0497] SEQ ID NO:38

[0498] QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAGRGCVRLHESCLGQQVP CCDPAATCYCKNFFWKTFTSCYCRANSGVSDRFSAAKSGTSASLAINGLRSEDEADYYCAAWDDSLNG YVFGTGTKLTV

[0499] SEQ ID NO:39

[0500] QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAGRCKNFFWKTFTSCKQD SDCLAGCVCGPNGFCGANSGVSDRFSAAKSGTSASLAINGLRSEDEADYYCAAWDDSLNGYVFGTGTK LTV

[0501] SEQ ID NQ:40

[0502] QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAGRCPRILMRCKQDSDCLA GCVCKNFFWKTFTSCGANSGVSDRFSAAKSGTSASLAINGLRSEDEADYYCAAWDDSLNGYVFGTGTK LTV

[0503] SEQ ID NO:41

[0504] QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAGRCKNFFWKTFTSCGAN SGVSDRFSAAKSGTSASLAINGLRSEDEADYYCAAWDDSLNGYVFGTGTKLTVL

[0505] SEQ ID NO:42

[0506] QLQLVESGGGLVQAGGSLRLSCAASGRTFSSATMGWFRQAPGKEREFVAAISWSGLSRYYADSVKGRF

[0507] TISRDNAENTVYLQMNSLKTEDTAVYYCAADSWGCKNFFWKTFTSCDARQYDVWGQGTQVTVSS

[0508] SEQ ID NO:43

[0509] QVQLVESGGGLVQAGGSLRLSCVASGSGFEPNAMGWYRQAPGKQRELVAGITSGGNANYADSVKGRF

[0510] TISRDNVKNTVYLQMNSLKPEDTAVYYCQGPACKNFFWKTFTSCRNYWGQGTQVTVSS

[0511] SEQ ID NO:44

[0512] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVT ITADESTSTAYMELRSLRSDDTAVYYCARDNLGYCKNFAWKTFTSCYSDYYYYYMDVWGQGTLVTVSS

[0513] SEQ ID NO:45

[0514] CAACGGGACTTTCCAAAATGTCGTAAC

[0515] SEQ ID NO:46

[0516] CAGTAGCAGGCTTGAGGTC

[0517] SEQ ID NO:47

[0518] TGTTTGCTGTATATTTTCAGCTAGC

[0519] SEQ ID NO:48

[0520] GCCGCCCCTTCCGTGTTCATC

[0521] SEQ ID NO:49

[0522] TTCATGTCTAGCAAGATTAAAGGTG

[0523] SEQ ID NQ:50

[0524] TGCCTTTCTCTCCACAGGCGCCATGGCC

[0525] SEQ ID NO:51 TAATAATAATAAGGATCCTTTAAGCTTACGACGTGATCA

[0526] SEQ ID NO:52

[0527] ARDNLGYCSGGSCYSDYYYYYMDV

[0528] SEQ ID NO:53

[0529] ARDNLGYCKNFFWKTFTSCYSDYYYYYMDV

[0530] SEQ ID NO:54

[0531] AKQIAPYCSGGSCYSYFDY

[0532] SEQ ID NO:55

[0533] AKQIAPYCKNFFWKTFTSCYSYFDY

[0534] SEQ ID NO:56

[0535] AAPSCSSTSCYDAFDI

[0536] SEQ ID NO:57

[0537] AAPSCKNFFWKTFTSCYDAFDI

[0538] SEQ ID NO:58

[0539] AKMGGGPYCSGGSCYSNYFDY

[0540] SEQ ID NO:59

[0541] AKMGGGPYCKNFFWKTFTSCYSNYFDY

[0542] SEQ ID NQ:60

[0543] AGRYCSSNTYSGCYYYG M D V

[0544] SEQ ID N0:61

[0545] AGRYCKNFFWKTFTSCYYYG M DV

[0546] SEQ ID NO:62

[0547] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFT

[0548] ISADTSKNTAYLQMNSLRAEDTAVYYCAKQGGPYCSGGNCYSGYFDYYWGQGTLVTVSSASTKGPSVF

[0549] PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT

[0550] QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD

[0551] VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK

[0552] TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF

[0553] FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0554] SEQ ID NO:63

[0555] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGT

[0556] DFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFY

[0557] PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC

[0558] SEQ ID NO:64

[0559] QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLADIWWDDKKDYNPSLKSRL

[0560] TISKDTSKNQVVLKVTNMDPADTATYYCARSMITNWYFDVWGAGTTVTVSSASTKGPSVFPLAPSSKST

[0561] SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK

[0562] PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF

[0563] NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR

[0564] EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0565] SEQ ID NO:65

[0566] DIQMTQSPSTLSASVGDRVTITCKCQLSVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTE

[0567] FTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYP

[0568] REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC SEQ ID NO:66

[0569] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIHDASSLQSGVPSRFSGSGSGTD

[0570] FTLTISSLQPEDFATYYCQQSFSIPLTFGGGTKMDIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPR

[0571] EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR

[0572] GEO

[0573] SEQ ID NO:67

[0574] QSVSSSY

[0575] SEQ ID NO:68

[0576] QSVCKNFFWKTFTSCSSSY

[0577] SEQ ID NO:69

[0578] GASCKNFFWKTFTSC

[0579] SEQ ID NQ:70

[0580] QQYGSSPLT

[0581] SEQ ID NO:71

[0582] QQYGCKNFFWKTFTSCSSPLT

[0583] SEQ ID NO:72

[0584] QSISSY

[0585] SEQ ID NO:73

[0586] ISCKNFFWKTFTSCHM

[0587] SEQ ID NO:74

[0588] DRVCKNFFWKTFTSCDD

[0589] SEQ ID NO:75

[0590] QQSFSIPLT

[0591] SEQ ID NO:76

[0592] EKCKNFFWKTFTSCSN

[0593] SEQ ID NO:77

[0594] SYAMS

[0595] SEQ ID NO:78

[0596] AVCKNFFWKTFTSCTK

[0597] SEQ ID NO:79

[0598] AISGSGGSTYYADSVKG

[0599] SEQ ID NQ:80

[0600] GTCKNFFWKTFTSCSD

[0601] SEQ ID NO:81 FGPGYGTGWFDY

[0602] SEQ ID NO:82

[0603] KGCKNFFWKTFTSCQG

[0604] SEQ ID NO:83

[0605] GFSLSTSGMS

[0606] SEQ ID NO:84

[0607] GFSLCKNFFWKTFTSCSTSGMS

[0608] SEQ ID NO:85 IWWDDKK SEQ ID NO:86

[0609] IWWCKNFFWKTFTSCDDKK

[0610] SEQ ID NO:87

[0611] GFNIKDTY

[0612] SEQ ID NO:88

[0613] GFNCKNFFWKTFTSCI KDTY

[0614] SEQ ID NO:89

[0615] IYPTNGYT

[0616] SEQ ID NQ:90

[0617] IYPTCKNFFWKTFTSCNGYT

[0618] SEQ ID N0:91

[0619] AKQIAPYAGCKNFFWKTFTSCYSYFDY

[0620] SEQ ID NO:92

[0621] AKMGGGPYAGCKNFAWKTFTSCYSNYFDY

[0622] SEQ ID NO:93

[0623] GCCAAGCAGATTGCGCCTTACGCTGGCTGCAAAAACTTCTTCTGGAAAACCTTTACC

[0624] SEQ ID NO:94

[0625] GGTAAAGGTTTTCCAGAAGAAGTTTTTGCAGCCAGCGTAAGGCGCAATCTGCTTGGC

[0626] SEQ ID NO:95

[0627] GCTAAGATGGGCGGAGGACCGTATGCTGGCTGCAAAAACTTCTTCTGGAAAACCTTTACC

[0628] SEQ ID NO:96

[0629] GGTAAAGGTTTTCCAGAAGAAGTTTTTGCAGCCAGCATACGGTCCTCCGCCCATCTTAGC

[0630] SEQ ID NO:97

[0631] MVSDVPRDLEWAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTI

[0632] TVYAVTGRGDSPASSKPISINYRT

[0633] SEQ ID NO:98

[0634] MVSDVPRDLEWAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTI

[0635] TVYAVCKNFFWKTFTSCSKPISINYRT

[0636] SEQ ID NO:99

[0637] ENSLEIEELARFAVDEHNKKENALLEFVRVVKAKEQVVAGTMYYLTLEAKDGGKKKLYEAKVWVKPWEN FKELQEFKPVGD

[0638] SEQ ID NO:1 QO

[0639] ENSLEIEELARFAVDEHNKKENALLEFVRVVKAKEQCKNFFWKTFTSCTMYYLTLEAKDGGKKKLYEAKV

[0640] WVKPWENFKELQEFKPVGD

[0641] SEQ ID N0:1 Q1

[0642] ENSLEIEELARFAVDEHNKKENALLEFVRWKAKEQWAGTMYYLTLEAKDGGKKKLYEAKVWVKCKNFF

[0643] WKTFTSCNFKELQEFKPVGD

[0644] SEQ ID NQ:102

[0645] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKT

[0646] KPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT

[0647] KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV

[0648] MHEALHNHYTQKSLSLSPGK

[0649] SEQ ID NQ:103 AGCKNFFWKTFTSCGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV

[0650] TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0651] SEQ ID NQ:104

[0652] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV

[0653] MHEALHNHYTQKSLS LSPGKGGGGSGGGGSGGGGSAGCKNFFWKTFTSC

[0654] SEQ ID NQ:105

[0655] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGT

[0656] DFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFY

[0657] PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC

[0658] SEQ ID NQ:106

[0659] CKNFFWKTFTSCGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKP

[0660] GKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVA

[0661] APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL

[0662] SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0663] SEQ ID NQ:107

[0664] CKNFFWKTFTSCGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQA

[0665] PGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMD

[0666] YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL

[0667] QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

[0668] PKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQD

[0669] WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE

[0670] SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0671] SEQ ID NQ:108

[0672] DKTHTCPPCPAP

[0673] SEQ ID NQ:109

[0674] GGGGSGGGGSGGGGS

Claims

1. CLAIMS1. A fusion protein comprising: a scaffold; and a somatostatin (SST)-derived peptide; wherein the fusion protein is a somatostatin receptor 4 (SSTR4) agonist, optionally a selective SSTR4 agonist.

2. The fusion protein according to claim 1 , further comprising one or more linkers joining the SST- derived peptide to the scaffold.

3. The fusion protein according to claim 1 or claim 2, wherein the SST-derived peptide is fused to the N-terminal or the C-terminal of the scaffold or is inserted within the scaffold.

4. The fusion protein according to any one of claims 1 to 3, wherein the SST-derived peptide has an amino acid sequence defined by X1-X2-C-X3-X4-F-X5-W-K-T-F-X6-X7-C, wherein:Xi is independently absent or any amino acid, optionally A;X2 is independently absent or any amino acid, optionally G;X3 is independently absent or K;X4 is independently absent or N;Xs is A or F;Xe is independently absent or T; andX7 is independently absent or S, optionally wherein: a. Xi and X2 are absent; or b. the SST-derived peptide consists of an amino acid sequence of SEQ ID NO:5 or SEQ ID NO:7 or a selective SSTR4 agonist variant thereof.

5. The fusion protein according to any one of the preceding claims wherein the scaffold is an antibody-derived scaffold, optionally a single variable domain, a scFv, a Fab domain, an Fc domain, or a monoclonal antibody.

6. The fusion protein according to claim 5, wherein the SST-derived peptide is inserted into or replaces a CDR, optionally wherein the CDR is: a. up to 35 amino acids long, optionally up to 30 amino acids long; and / or b. a heavy chain CDR, optionally HCDR3; or c. a light chain CDR, optionally LCDR2.

7. The fusion protein according to any one of claims 1 to 4, wherein the scaffold is not an antibody- derived scaffold, optionally wherein the scaffold is selected from the group consisting of: a cysteine-rich peptide, a CTLA-4-based binder, lipocalin, an anticalin, SpA, an avimer, an affibody,GroEL, GroES, transferrin, fibronectin, an adnectin, a fynomer, a monobody, tetranectin, an atrimer, a Kunitz domain-based binder, a designed ankyrin repeat protein (DARPin), an adhiron, an affilin, a thioredoxin, and a T7 phage gene 2 protein (Gp2).

8. The fusion protein according to claim 7, wherein the cysteine-rich peptide is a knottin and the SST-derived peptide is inserted into or replaces a knottin loop, optionally wherein the knottin is inserted into or replaces a CDR in a single variable domain of an antibody-derived scaffold or wherein the knottin is fused to the N-terminal or C-terminal of an Fc domain.

9. An isolated nucleic acid comprising a nucleotide sequence encoding a fusion protein according to any one of the preceding claims.

10. A vector comprising the nucleic acid according to claim 9.

11. A host cell comprising the nucleic acid according to claim 9 or the vector according to claim 10.

12. A method of making a fusion protein comprising culturing the host cell according to claim 11 under suitable conditions.

13. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 8, and at least one pharmaceutically acceptable excipient, vehicle, or carrier.

14. A fusion protein according to any one of claims 1 to 8, or a pharmaceutical composition according to claim 13, for use as a medicament, optionally for use in treating pain, optionally chronic pain, and / or inflammation.

15. A method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutically acceptable composition according to claim 13, or a fusion protein according to any one of claims 1 to 8, optionally wherein the subject has, or is at risk of developing, pain, optionally chronic pain, or inflammation.

16. A method of generating a fusion protein comprising a scaffold and a SST-12-derived peptide, wherein the fusion protein is a somatostatin receptor (SSTR) agonist, optionally a SSTR4 agonist, optionally a selective SSTR4 agonist, said method comprising: a) identifying a suitable scaffold comprising a disulphide loop; b) replacing the disulphide loop with said SST-12-derived peptide; c) determining that the fusion protein is a SSTR agonist, optionally a SSTR4 agonist, optionally a selective SSTR4 agonist.

17. The method according to claim 16, wherein the scaffold is an antibody-derived scaffold and the disulphide loop:a. is formed by two non-canonical cysteine residues in a CDR, optionally HCDR3; or b. has been engineered into a CDR, optionally wherein the CDR is LCDR2.

18. The method according to claim 16, wherein the scaffold is a cysteine-rich peptide, optionally wherein the cysteine rich-peptide is a knottin and the disulphide loop is a knottin loop.

19. The method according to any one of claims 16 to 18, wherein determining that the fusion protein is a SSTR agonist is achieved using a cAMP production inhibition assay.

Images