TNFR1 antagonists and TNFR2 agonists for treating acute pain

Abstract

The present invention relates to modulators of TNF signalling for use in treating and / or preventing acute pain. In particular, a modulator of TNF signalling may be a tumour necrosis factor receptor 2 (TNFR2) agonist comprising (i) a TNFR2 binding domain comprising three TNF homology domains (THD) that specifically bind to TNFR2; and (ii) an Fc domain. The present invention further relates to combinations of a TNFR2 agonist and an inhibitor of tumour necrosis factor receptor 1 (TNFR1) signalling.

Description

[0001] AGONIST POLYPEPTIDE

[0002] FIELD

[0003] The present invention relates to modulators of TNF signalling for use in treating and / or preventing acute pain. In particular, a modulator of TNF signalling may be a tumour necrosis factor receptor 2 (TNFR2) agonist comprising (i) a TNFR2 binding domain comprising three TNF homology domains (THD) that specifically bind to TNFR2; and (ii) an Fc domain. The present invention further relates to combinations of a TNFR2 agonist and an inhibitor of tumour necrosis factor receptor 1 (TNFR1) signalling.

[0004] BACKGROUND

[0005] The Tumor Necrosis Factor (TNF) superfamily is a family of structurally related cytokines with various functions.

[0006] TNF itself is a multifunctional cytokine with pleiotropic functions. It is a master regulator of the immune system and a key player in the initiation and orchestration of inflammation and immunity. TNF, like most ligands of the superfamily, is synthesized as a trimeric type 2 transmembrane protein (tmTNF) that can be proteolytically processed into soluble circulating TNF homotrimers (sTNF). Interestingly, sTNF and tmTNF differ in their capability to activate the two distinct TNF receptors (TNFRs): TNFR1 and TNFR2. Whereas TNFR1 is activated by both sTNF and tmTNF, TNFR2 is dependent on tmTNF to be robustly activated (Muhlenbeck et al, 2000, J. Biol., Chem. 275, 32208-32213; Wajant et al, 2001 , Oncogene 20, 4101-4106). Deregulated TNF expression and signalling can cause chronic inflammation, which may result in the development of autoimmune diseases and tissue damage (Fischer et al., 2015, Antibodies 4, 48-70; Kalliolias & Ivashkiv, 2016, Nat. Rev. Rheumatol. 12, 49-62).

[0007] It has been shown that TNFR1 and TNFR2 induce opposing biologic responses. Whereas TNFR1 signalling promotes inflammation and tissue degeneration, TNFR2 contributes to immune suppression as well as tissue homeostasis and regeneration (Probert et al., 2015, Neuroscience 302, 2-22). Therefore, next-generation therapeutic approaches targeting the TNF system have been developed, including blocking of sTNF-TNFRI interaction or signalling and selective activation of TNFR2 (Shibata et al., 2009, Biomaterials 30, 6638-6647; Steed et al., 2003, Science 301 , 1895-1898; Dong et al., 2016, PNAS 113, 12304-12309). The immunosuppressive activity mediated through TNFR2 is of particular interest for potential therapeutic application in autoimmune diseases. The immunosuppressive properties of TNFR2 have been attributed to its prominent role in expansion and stabilization of Treg cells (Chen et al. , 2007, J. Immunol. 179, 154-161 ; Chen et al., 2013, J. Immunol. 190, 1076-1084), a highly specialized subpopulation of T cells that function to suppress immune responses. According to the prevailing view, Treg cells regulate the self-tolerance of the immune system and help to prevent the development of autoimmune diseases. In addition to CD4+ Treg cells, additional T cell subpopulations with regulatory activity exist (i.e. , CD8+ Treg cells). Similar to CD4+ Treg cells, the most potent CD8+ suppressors are characterized by the expression of TNFR2 (Ablamunits et al., 2010, Eur. J. Immunol. 40, 2891-2901).

[0008] W02020 / 260368 describes TNFR2 agonists with improved stability characteristics. In particular, W02020 / 260368 describes polypeptides comprising three TNF homology domains (THD) of TNF-ligand family members proteins that specifically bind to the extracellular part of TNFR2, wherein C-terminal and N-terminal reference points are defined by consensus sequences. The THDs are linked by short stretches of further C-terminal and / or N-terminal amino acids of the THD or variants thereof as well as by peptide linkers.

[0009] However, there remains a need for improved TNFR2 agonists. In particular, there remains a need for TNFR2 agonists with advantageous properties for use in treating diseases, disorders and conditions associated with TNF signalling. Pharmacological activation of TNFR2 using the TNFR2 agonist EHD2-sc-mTNFR2 in mice was previously shown to promote recovery from chronic neuropathic pain via a Treg-dependent response (Fischer R, et al., Proc Natl Acad Sci U S A. 2019; 116(34): 17045-17050). However, there remains a need for modulators of TNF signalling capable of alleviating other pain types.

[0010] SUMMARY

[0011] The present invention is based, at least in part, on the inventors’ surprising finding that modulators of TNF signalling (such as TNFR2 agonists) are also highly effective in treating acute pain over short time frames, i.e. alleviating pain within hours and enhancing recovery time from acute pain. This was not expected as it was previously thought that the effects of TNF modulators on the pain pathways were mediated through a slow Treg-dependent response. The present invention is based, at least in part, on the inventors’ development of TNFR2 agonists with particularly advantageous and surprising properties for use in treating acute pain. For example, the present inventors have developed TNFR2 agonists with reduced binding to and activation of FcyRlla and beneficial in vivo properties in the context of treating acute pain. Accordingly, the present invention provides a pharmaceutical composition comprising one or more modulators of TNF signalling for use in treating and / or preventing acute pain.

[0012] In some embodiments, the one or more modulators of TNF signalling each independently comprise a polypeptide, an antibody, a nucleic acid, a small molecule, or a combination thereof.

[0013] In some embodiments, the one or more modulators of TNF signalling comprises a tumour necrosis factor receptor 2 (TNFR2) agonist.

[0014] In some embodiments, the TNFR2 agonist is a polypeptide comprising a TNFR2 binding domain comprising three TNF homology domains (THD) that specifically bind to TNFR2.

[0015] In some embodiments, the C-terminus of the first and second THD, respectively, which is in each case defined by the C-terminal consensus sequence:

[0016] V-X1-F-G-X2-X3 (SEQ ID NO: 28); is linked to the N-terminus of the second and third THD, respectively, which is in each case defined by the N-terminal consensus sequence:

[0017] P-X4-A-H-X5 (SEQ ID NO: 29); through a peptide Xa, which is in each case independently selected and has a length of 9 to 12 amino acids, preferably 9 to 11 , more preferably 9 to 10, wherein X1 is F or Y, wherein X2 is A or I, wherein X3 is a non-polar / hydrophobic or polar / neutral amino acid, preferably selected from the group consisting of F and I, wherein X4 is V or A, and wherein X5 is V or L.

[0018] In some embodiments, each THD comprises an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 2

[0019] In some embodiments, one or more THD, preferably each THD, comprises or consists of SEQ ID NO: 3 or an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 3. In some embodiments, one THD comprises or consists of SEQ ID NO: 4 or an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 4.

[0020] In some embodiments, the TNFR2 binding domain comprises or consists of a sequence with at least 80% sequence identity to SEQ ID NO: 5.

[0021] In some embodiments, the polypeptide further comprises a multimerization domain.

[0022] In some embodiments, the multimerization domain is a dimerization domain. In some embodiments, the dimerization domain is an immunoglobulin Fc domain.

[0023] In some embodiments, the polypeptide comprises (i) a TNFR2 binding domain comprising three TNF homology domains (THD) that specifically bind to TNFR2; and (ii) an Fc domain, wherein the Fc domain comprises the following mutations L234A, L235A, A327G, A330S and P331S.

[0024] In some embodiments, the Fc domain does not comprise a proline at position 233.

[0025] In some embodiments, the Fc domain comprises a glycine at position 236.

[0026] In some embodiments, the Fc domain (i) does not comprise a proline at position 233; and (ii) comprises a glycine at position 236.

[0027] In some embodiments, the Fc domain comprises a sequence with at least 85% sequence identity to SEQ ID NO: 1.

[0028] In some embodiments, the Fc domain does not comprise a proline at position 233; and the Fc domain comprises a sequence with at least 85% sequence identity to SEQ ID NO: 1.

[0029] In some embodiments, the Fc domain comprises a glycine at position 236; and the Fc domain comprises a sequence with at least 85% sequence identity to SEQ ID NO: 1.

[0030] In some embodiments, the Fc domain (i) does not comprise a proline at position 233; and (ii) comprises a glycine at position 236; and (iii) comprises a sequence with at least 85% sequence identity to SEQ ID NO: 1. In some embodiments, the TNFR2 agonist is a polypeptide comprising a sequence with at least 80% sequence identity to SEQ ID NO: 7.

[0031] In some embodiments, the TNFR2 agonist is a polypeptide comprising the sequence of SEQ ID NO: 7.

[0032] In some embodiments, the one or more modulators of TNF signalling comprises an inhibitor of tumour necrosis factor receptor 1 (TNFR1) signalling.

[0033] In some embodiments, the inhibitor of TNFR1 signalling comprises a tumour necrosis factor receptor 1 (TNFR1) antagonist or a soluble TNF inhibitor.

[0034] In some embodiments, the TNFR1 antagonist comprises an antibody or fragment thereof capable of specifically binding TNFR1 and inhibiting TNFR1 signalling.

[0035] In some embodiments, the TNFR1 antagonist is selected from the group consisting of Atrosimab, GSK2862277, scRIantTNF, TROS and DS41.

[0036] In some embodiments, the soluble TNF inhibitor comprises an antibody or fragment thereof capable of specifically binding and inhibiting soluble TNF.

[0037] In some embodiments, the soluble TNF inhibitor is selected from the group consisting of XPro1595 and SAR441566.

[0038] In some embodiments, the pharmaceutical composition comprises one or more nucleic acid(s) encoding the one or more modulator(s) of TNF signalling.

[0039] In some embodiments, the acute pain has persisted for less than 6 months, such as for less than 5 months, for less than 4 months, for less than 3 months, for less than 2 months, or for less than 1 month. Preferably, in some embodiments, the acute pain has lasted for less than 2 months. In some embodiments, the acute pain has lasted for less than 1 month.

[0040] In some embodiments, the acute pain has persisted for less than 6 weeks, for less than 5 weeks, for less than 4 weeks, for less than 3 weeks, for less than 2 weeks, or for less than 1 week. In some embodiments, the acute pain has persisted for less than 7 days, for less than 6 days, for less than 5 days, for less than 4 days, for less than 3 days, for less than 2 days, or for less than 1 day.

[0041] In some embodiments, the acute pain arises from injury or trauma.

[0042] In some embodiments, the acute pain arises from surgery and / or health treatment.

[0043] In some embodiments, the acute pain is associated with osteoarthritis.

[0044] In some embodiments, the acute pain is post-operative acute pain.

[0045] In some embodiments, the pharmaceutical composition is administered to a subject systemically.

[0046] In some embodiments, the pharmaceutical composition is administered to a subject subcutaneously.

[0047] In some embodiments, the pharmaceutical composition is administered to a subject intravenously.

[0048] In some embodiments, the pharmaceutical composition is administered to a subject intrathecally.

[0049] In some embodiments, the pharmaceutical composition is administered to a subject intraarticularly, optionally by intra-articular injection.

[0050] The present invention provides a TNFR2 agonist for use in treating and / or preventing acute pain, wherein the TNFR2 agonist is administered in combination with an inhibitor of TNFR1 signalling. In some embodiments, the inhibitor of TNFR1 signalling is a TNFR1 antagonist.

[0051] The present invention provides an inhibitor of TNFR1 signalling for use in treating and / or preventing acute pain, wherein the inhibitor of TNFR1 signalling is administered in combination with a TNFR2 agonist. In some embodiments, the inhibitor of TNFR1 signalling is a TNFR1 antagonist. The present invention provides a TNFR2 agonist and an inhibitor of TNFR1 signalling, for use in treating and / or preventing acute pain. In some embodiments, the inhibitor of TNFR1 signalling is a TNFR1 antagonist.

[0052] In some embodiments, the TNFR2 agonist and the inhibitor of TNFR1 signalling are administered concurrently or sequentially.

[0053] In some embodiments, the TNFR2 agonist is the TNFR2 agonist comprised within the pharmaceutical composition for use according to the invention.

[0054] In some embodiments, the inhibitor of TNFR1 signalling is the inhibitor of TNFR1 signalling comprised within the pharmaceutical composition for use according to the invention.

[0055] The present invention provides a method of treating and / or preventing acute pain, the method comprising administering to a subject a pharmaceutical composition comprising a modulator of TNF signalling.

[0056] In some embodiments, the modulator of TNF signalling is as further defined in the pharmaceutical composition for use according to the invention.

[0057] In some embodiments, the pharmaceutical composition for use according to the invention comprising the TNFR2 agonist is administered in combination with the pharmaceutical composition for use according to the invention comprising the inhibitor of TNFR1 signalling.

[0058] In some embodiments, the pharmaceutical composition for use according to the invention comprising the inhibitor of TNFR1 signalling is administered in combination with the pharmaceutical composition for use according to the invention comprising the TNFR2 agonist.

[0059] BRIEF DESCRIPTION OF THE FIGURES

[0060] Figure 1 - TNFR2 agonism alleviates acute pain after intravenous application:

[0061] Rats underwent paw incision according to the Brennan method. Then, they were treated using the mouse TNFR2 agonist sc-mTNFR2-Fc or the NSAID Naproxen by i.v. injection 80 minutes post-surgery. Pain hypersensitivity was determined using the Von Frey test for mechanical allodynia at the indicated times. (A) Schematic diagram indicating the workflow of the experiment (B) Quantification of the pain hypersensitivity (mechanical allodynia). n=10 animals per group, *p<0.05 vs. PBS.

[0062] Figure 2 - sc-mTNFR2-Fc treatment alleviates pain after subcutaneous application:

[0063] Rats underwent paw incision according to the Brennan method. Then, they were treated using the mouse TNFR2 agonist sc-mTNFR2-Fc or the NSAID Keterolac by s.c. injection 30 minutes post-surgery. Pain hypersensitivity was determined using the Von Frey test for mechanical allodynia at the indicated times. (A) Schematic diagram indicating the workflow of the experiment (B) Quantification of the pain hypersensitivity (mechanical allodynia). n=10 animals per group, *p<0.05, **p<0.01 vs. PBS.

[0064] Figure 3 - Local TNFR2 agonism is superior to system application in alleviating acute pain and functional recovery post surgery:

[0065] (A) Schematic diagram indicating the workflow of the experiment. Mice underwent paw incision according to the Brennan method. They were treated either with (B) systemic i.p. injection (day -1 , day 2 and day 5 after surgery) or (C) the combination of local intrathecal (1 hour before surgery) and systemic i.p. injection (day 2 and day 5 after surgery) of the mouse TNFR2 agonist sc-mTNFR2-Fc. Pain hypersensitivity was determined using the Von Frey test for mechanical allodynia at the indicated times.

[0066] DETAILED DESCRIPTION

[0067] TUMOR NECROSIS FACTOR (TNF)

[0068] The tumor necrosis factor (TNF) superfamily is a family of structurally related cytokines with various functions. The structural hallmark defining the TNF ligand family is the carboxyterminal TNF homology domain (THD) which is composed of two stacked b-pleated sheets that adopt a conserved jellyroll-like tertiary fold (Bodmer et al, 2000, Trends Biochem. Sci. 27, 19-26; Fesik, 2000, Cell 103, 273-282; Locksley et ak, 2001 , Cell 104, 487-501). This structural composition leads to the self-association of THD monomers into trimers and is necessary for receptor binding. Due to the carboxy-terminal localization of the THD, both the transmembrane form as well as soluble TNF ligands assemble into trimers.

[0069] TNF is synthesized as a trimeric type 2 transmembrane protein (tmTNF) that can be proteolytically processed into soluble circulating TNF homotrimers (sTNF). sTNF and tmTNF differ in their capability to activate the two distinct TNF receptors (TNFRs): TNFR1 and TNFR2. Whereas TNFR1 is activated by both sTNF and tmTNF, TNFR2 is dependent on tmTNF to be robustly activated (Muhlenbeck et ak, 2000, J. Biol., Chem. 275, 32208-32213; Wajant et al, 2001 , Oncogene 20, 4101-4106). TNFR1 is constitutively expressed on most cell types, whereas TNFR2 is restricted primarily to endothelial, epithelial, and subsets of immune cells. The primary role of TNF is in the regulation of immune cells. TNF, as an endogenous pyrogen, is able to induce fever, apoptotic cell death, cachexia, and inflammation, inhibit tumorigenesis and viral replication, and respond to sepsis via IL-1 and IL-6-producing cells. Dysregulation of TNF production has been implicated in a variety of human diseases.

[0070] An illustrative TNF amino acid sequence is the human TNF sequence designated as UniProt P01375.

[0071] An illustrative TNFR1 amino acid sequence is the human TNFR1 sequence designated as UniProt P19438.

[0072] TNFR2 is also known as tumor necrosis factor receptor superfamily member 1 B (TNFRSF1 B) and CD120b. An illustrative TNFR2 amino acid sequence is the human TNFR2 sequence designated as UniProt P20333.

[0073] MODULATORS OF TNF SIGNALLING

[0074] The present invention relates to a pharmaceutical composition comprising one or more modulator(s) of TNF signalling for use in treating and / or preventing acute pain. The present invention also relates to a method of treating and / or preventing acute pain, the method comprising administering to a subject a pharmaceutical composition comprising one or more modulator(s) of TNF signalling.

[0075] The present invention also relates to use of a pharmaceutical composition comprising one or more modulator(s) of TNF signalling in the manufacture of a medicament for treating and / or preventing acute pain.

[0076] It will be understood that a modulator of TNF signalling is an agent that is capable of directly or indirectly activating or inhibiting the TNF signalling pathway. A modulator of TNF signalling may be an agent that directly or indirectly activates the tumour necrosis factor receptor 2 (TNFR2) signalling pathway (i.e. a TNFR2 agonist). A modulator of TNF signalling may be an agent that directly or indirectly inhibits the tumour necrosis factor receptor 1 (TNFR1) signalling pathway (i.e. an inhibitor of TNFR1 signalling).

[0077] In some embodiments, the TNF signalling is TNF alpha and / or TNF beta signalling. In some embodiments, the TNF signalling is TNF alpha signalling.

[0078] In some embodiments, the modulator of TNF signalling is an agent that activates the TNF signalling pathway. In some embodiments, the modulator of TNF signalling is an agent that activates the TNFR2 signalling pathway.

[0079] In some embodiments, the modulator of TNF signalling is an agent that inhibits the TNF signalling pathway. In some embodiments, the modulator of TNF signalling is an agent that inhibits the TNFR1 signalling pathway.

[0080] In some embodiments, the modulator of TNF signalling may be selected from the list consisting of: a TNFR2 agonist, a TNFR1 antagonist, and a combination thereof.

[0081] It will be understood that a modulator of TNF signalling (e.g. a TNFR2 agonist) may increase signalling through TNFR2 by at least 10%, as compared to a control. It will also be understood that a modulator of TNF signalling (e.g. a TNFR2 agonist) may increase signalling through TNFR2 by at least 2-fold, as compared to a control. Such signalling may be assessed using a reporter assay. Indeed, TNFR2 agonists can be identified using a cellular reporter system where an NFkB reporter gene is fused to a luciferase reporter and activation of the NFkB pathway by TNFR2 activation induces luciferase expression in reporter cells. Suitable reporter cell lines for identifying TNFR2 agonists are commercially available, e.g., from InvivoGen (HEK-Blue™ TNF-a cells). Alternatively, a reporter cell line for identifying TNFR2 agonists may be generated, e.g., from the commercially available SBI System Biosciences NF- KB / 293 / GFP-LUC™ Transcriptional Reporter Cell Line (Catalog # TR860A-1), by modifying the cell line to overexpress TNFR2 and to knockout the TNFR1 gene. In such reporter systems, a TNFR2 agonist may increase the luminescence signal by at least 2 fold as compared to a control. Furthermore, assays for demonstrating activation of the NFKB pathway by TNFR2 agonists are also described, e.g., in Fischer R, et al., Cell Signal. 2011 ; 23(1):161-70.

[0082] It will be understood that a modulator of TNF signalling (e.g. an inhibitor of TNFR1 signalling) may decrease (i.e., inhibit) signalling through TNFR1 by at least 10%, as compared to a control. It will also be understood that a modulator of TNF signalling (e.g. an inhibitor of TNFR1 signalling) may decrease (i.e., inhibit) signalling through TNFR1 by at least 2-fold, as compared to a control. Such signalling may be assessed using a reporter assay. Indeed, inhibitors of TNFR1 signalling can be identified using a cellular reporter system where an NFkB reporter gene is fused to a luciferase reporter and activation of the NFkB pathway by TNFR1 activation induces luciferase expression in reporter cells. Suitable reporter cell lines for identifying inhibitors of TNFR1 signalling are commercially available, e.g., from SBI System Biosciences (Catalog # TR860A-1 , NF-KB / 293 / GFP-LUC™ Transcriptional Reporter Cell Line). In such a reporter system, an inhibitor of TNFR1 signalling may decrease the luminescence signal by at least 2 fold as compared to a control.

[0083] In some embodiments, the pharmaceutical composition comprises two or more modulators of TNF signalling.

[0084] In some embodiments, the pharmaceutical composition comprises two modulators of TNF signalling.

[0085] In some embodiments, the pharmaceutical composition comprises three, four, or five modulators of TNF signalling.

[0086] In some embodiments, each modulator of TNF signalling is different.

[0087] In some embodiments, the one or more modulators of TNF signalling each independently comprise a polypeptide, an antibody, a nucleic acid, a small molecule, or a combination thereof. In some embodiments, at least one modulator of TNF signalling is a TNFR2 agonist.

[0088] In some embodiments, at least one modulator of TNF signalling is an inhibitor of TNFR1 signalling.

[0089] In some embodiments, the pharmaceutical composition comprises (i) at least one modulator of TNF signalling that is a tumour necrosis factor receptor 2 (TNFR2) agonist; and (ii) at least one modulator of TNF signalling that is an inhibitor of tumour necrosis factor receptor 1 (TNFR1) signalling.

[0090] TUMOUR NECROSIS FACTOR RECEPTOR 2 (TNFR2) AGONIST

[0091] In some embodiments, the pharmaceutical composition according to the invention comprises one or more modulators of TNF signalling for use in treating and / or preventing acute pain, wherein the one or more modulators of TNF signalling comprises a tumour necrosis factor receptor 2 (TNFR2) agonist.

[0092] It will be understood that the TNFR2 agonist may be capable of inducing signalling via TNFR2.

[0093] In some embodiments, the TNFR2 agonist is a protein or an antibody. In some embodiments, the TNFR2 agonist is a protein. In some embodiments, the TNFR2 agonist is a fusion protein. In some embodiments, the TNFR2 agonist comprises a TNF protein sequence (or fragment or variant thereof) which comprises mutations for selective binding to TNFR2. In some embodiments, the TNFR2 agonist is a fusion protein comprising a TNF protein sequence (or fragment or variant thereof) and a multimerization domain (as further defined herein). In some embodiments, the TNFR2 agonist is an antibody (e.g., a TNFR2 agonist antibody). In some embodiments, the TNFR2 agonist is an antibody that specifically binds TNFR2 and optionally activates TNFR2 signalling. TNFR2 agonist antibodies have been previously described, for example TNFR2-specific antibodies 80M2 (Hycult, Catalog # HM2022) and MR2-1 (Hycult, Catalog # HM2007).

[0094] Suitable assays and techniques for measuring and / or quantifying the activity (e.g. binding capability and / or activity) of the TNFR2 binding domain may include, but are not limited to, ELISA, surface plasmon resonance (SPR), quartz crystal microbalance (QCM), bioluminescence assays and flow cytometry. Functional assays for receptor activation can be performed using reporter cell lines or by measuring downstream signalling molecule (e.g. by Western blot). Systems for quantifying bioactivity of TNFR2 agonists have been demonstrated in Fischer et al, 2017, Scientific Reports, 7(1):6607; and Fischer et al, 2018, Arthritis & Rheumatology, 70(5):722-735. Illustrative methods are provided in the present Examples.

[0095] In some embodiments, the TNFR2 agonist is a polypeptide comprising a TNFR2 binding domain comprising three TNF homology domains (THD) that specifically bind to TNFR2.

[0096] In some embodiments, the TNFR2 agonist may induce TNFR2 signalling in a cell. In some embodiments, the TNFR2 agonist may induce TNFR2 signalling in a nerve cell. In some embodiments, the TNFR2 agonist may induce TNFR2 signalling in neurons. In some embodiments, the TNFR2 agonist may induce TNFR2 signalling in microglia. In some embodiments, the TNFR2 agonist may induce TNFR2 signalling in immune cells.

[0097] TNF homology domain (THD)

[0098] The term THD as used herein refers to a protein domain shared by all tumor necrosis factor (TNF, formerly known as TNFa or TNF alpha) ligand family members. Homology implies evolutionary lineage from a common ancestor. A homology domain is a conserved part of a given protein sequence and (tertiary) structure that can evolve, function, and exist independently of the rest of the protein chain. It is a structural feature shared by all members of a certain protein family. Each domain forms a compact three-dimensional structure and often can be independently stable, folded and critical for biological activity.

[0099] It will be understood that three THDs (i.e. a first, second, and third THD) form the TNFR2 binding domain of the TNFR2 agonist.

[0100] In some embodiments, the C-terminus of the first and second THD, respectively, which is in each case defined by the C-terminal consensus sequence

[0101] V-X1-F-G-X2-X3 (SEQ ID NO: 28), is linked to the N-terminus of the second and third THD, respectively, which is in each case defined by the N-terminal consensus sequence:

[0102] P-X4-A-H-X5 (SEQ ID NO: 29), through a peptide Xa, which is in each case independently selected and optionally has a length of 9 to 12 amino acids, preferably 9 to 11 , more preferably 9 to 10, wherein X1 is F or Y, wherein X2 is A or I, wherein X3 is a non-polar / hydrophobic or polar / neutral amino acid, preferably selected from the group consisting of F and I, wherein X4 is V or A, and wherein X5 is V or L.

[0103] In some embodiments, one or more THD may independently comprise an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (optionally at least 90%, such as at least 95%) sequence identity to SEQ ID NO: 2.

[0104] In some embodiments, one or more THD may independently comprise or consist of the amino acid sequence according to SEQ ID NO: 2.

[0105] In some embodiments, each THD may independently comprise an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (optionally at least 90%, such as at least 95%) sequence identity to SEQ ID NO: 2.

[0106] In some embodiments, each THD may independently comprise or consist of the amino acid sequence according to SEQ ID NO: 2.

[0107] SEQ ID NO: 2 - THD sequence without mutations

[0108] SRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFK GQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLE KGDRLSAEINRPDYLDFAESGQVYFGIIAL

[0109] In some embodiments, the three THDs of the TNFR2 agonist are identical in their amino acid sequence.

[0110] Various mutations have been described to increase the specificity of binding to the extracellular part of TNFR2. Preferably the mutations decrease binding affinity to TNFR1 , while essentially maintaining the affinity for TNFR2, thereby increasing the specificity for TNFR2 (i.e. the Kd for binding to TNFR2 is at least 10-fold, at least 100-fold, at least 1000- fold, preferably at least 5000-fold, higher than the Kd for binding to TNFR1). Exemplary mutations are known in the art and are disclosed in Loetscher et al (JBC, vol 268, no 35, pp. 26350-26357, 1993; see Table 1), Abe et al (Biomaterials 32 (2011) 5498-5504; see Table 1), Ando et al (Biochemistry and Biophysics Reports, 7; 2016; 309-315; see Table 2) and Ban et al (Molecular and Cellular Therapies (2015) 3:7).

[0111] In some embodiments, one or more, or each, THD according to the invention may independently comprise one or more mutations. Such a THD may be considered as a variant. In some embodiments, one or more, or each, THD may independently comprise a contiguous amino acid sequence as outlined in W02020 / 260368 (incorporated herein by reference).

[0112] In some embodiments, the THD may comprise a contiguous amino acid sequence comprising SEQ ID NO: 9, optionally comprising at least one mutation or set of mutations selected from the group consisting of: D143Y, D143F, D143E, D143N, D143T, D143S, E146Q, E146H, E146K, A145R / S147T, Q88N / T89S / A145S / E146A / S147D, Q88N / A145I / E146G / S147D, A145H / E146S / S147D, A145H / S147D, L29V / A145D / E146D / S147D, A145N / E146D / S147D, A145T / E146S / S147D, A145Q / E146D / S147D, A145T / E146D / S147D, A145D / E146G / S147D, A145D / S147D, A145K / E146D / S147T, A145R / E146T / S147D, A145R / S147T, E146D / S147D, E146N / S147, S95C / G148C, K65A, K65W, Q67K, Q67T, Q67Y, L75H, L75W, D143W, D143V, D143V / F144L / A145S, D143N / A145R, D143V / A145S, L29V, L29T, L29S, L29A, L29G, R31 H, R311, R31 L, R32G, R32E, S147L, S147R, S147P S147T, S147A, Q149E, Q149N, E146D, E146N, E146S, E146G, A145R, A145S, A145T, A145H, A145K, A145F, A145D, A145G, A145N, A145P, A145Q, A145Y, A145V and A145W, preferably selected from D143N and A145R, wherein the above position numbering is in respect of the sequence of SEQ ID NO: 9.

[0113] It will be understood that a “set of mutations” as used above refers to a combination of mutations indicated by the use of 7”, where all of the mutations in the combination are selected, e.g. if “Q88N / T89S / A145S / E146A / S147D” as used above is selected, all of the indicated mutations will be present in the amino acid sequence.

[0114] It will be understood that SEQ ID NO. 5 of WQ2020 / 260368 corresponds to SEQ ID NO: 8 of the present disclosure. Thus, the amino acids at positions 77 to 233 of SEQ ID NO: 5 of WQ2020 / 260368 are the same as the amino acids at positions 77 to 233 of present SEQ ID NO: 8. Suitably, the amino acid positions and mutations listed above for SEQ ID NO. 9 equally apply to present SEQ ID NO: 8 (or SEQ ID NO: 5 of WQ2020 / 260368), where it will be understood that the amino acid at position 77 of both present SEQ ID NO: 8 and SEQ ID NO: 5 of WQ2020 / 260368 equates to position 1 in the above position numbering.

[0115] SEQ ID NO. 5 of WQ2020 / 260368 is presented below as SEQ ID NO: 8. SEQ ID NO: 8 - SEQ ID NO. 5 of WQ2020 / 260368 (positions 77 to 233 are underlined) MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEF PRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQ LVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAE AKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL

[0116] The amino acids at position 77 to 233 of SEQ ID NO: 8 (i.e. which is the same as SEQ ID NO. 5 of W02020 / 260368) are presented below as SEQ ID NO: 9, where position 77 of SEQ ID NO: 8 equates to position 1 of SEQ ID NO: 9.

[0117] SEQ ID NO: 9 - sequence of amino acids from position 77 to 233 of SEQ ID NO. 5 of WQ2020 / 260368 (which is SEQ ID NO: 8 of the present disclosure)

[0118] VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYS QVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGG VFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL

[0119] In some embodiments, the THD may comprise a contiguous amino acid sequence consisting of the amino acids sequence according to SEQ ID NO: 9, optionally comprising at least one mutation or set of mutations selected from the group consisting of: D143Y, D143F, D143E, D143N, D143T, D143S, E146Q, E146H, E146K, A145R / S147T,

[0120] Q88N / T89S / A145S / E146A / S147D, Q88N / A145I / E146G / S147D, A145H / E146S / S147D,

[0121] A145H / S147D, L29V / A145D / E146D / S147D, A145N / E146D / S147D, A145T / E146S / S147D, A145Q / E146D / S147D, A145T / E146D / S147D, A145D / E146G / S147D, A145D / S147D,

[0122] A145K / E146D / S147T, A145R / E146T / S147D, A145R / S147T, E146D / S147D, E146N / S147, S95C / G148C, K65A, K65W, Q67K, Q67T, Q67Y, L75H, L75W, D143W, D143V, D143V / F144L / A145S, D143N / A145R, D143V / A145S, L29V, L29T, L29S, L29A, L29G, R31 H, R31 I, R31 L, R32G, R32E, S147L, S147R, S147P S147T, S147A, Q149E, Q149N, E146D, E146N, E146S, E146G, A145R, A145S, A145T, A145H, A145K, A145F, A145D, A145G, A145N, A145P, A145Q, A145Y, A145V and A145W, preferably selected from D143N and A145R. It will be understood that a “set of mutations” as used above refers to a combination of mutations indicated by the use of 7”, where all of the mutations in the combination are selected, e.g. if “Q88N / T89S / A145S / E146A / S147D” as used above is selected, all of the indicated mutations will be present in the amino acid sequence.

[0123] In the context of the present invention, the mutations may be described by reference to SEQ ID NO: 2. In some embodiments, the THD may comprise at least one mutation or set of mutations selected from the group consisting of: D139Y, D139F, D139E, D139N, D139T, D139S, E142Q, E142H, E142K, A141 R / S143T, Q84N / T89S / A141S / E142A / S143D,

[0124] Q84N / A141 I / E142G / S143D, A141 H / E142S / S143D, A141 H / S143D,

[0125] L25V / A141 D / E142D / S143D, A141 N / E142D / S143D, A141T / E142S / S143D,

[0126] A141Q / E142D / S143D, A141T / E142D / S143D, A141 D / E142G / S143D, A141 D / S143D,

[0127] A141 K / E142D / S143T, A141 R / E142T / S143D, A141 R / S143T, E142D / S143D, E142N / S143, S91C / G144C, K61A, K61W, Q63K, Q63T, Q63Y, L71 H, L71W, D139W, D139V, D139V / F140L / A141S, D139N / A141 R, D139V / A141S, L25V, L25T, L25S, L25A, L25G, R27H, R27I, R27L, R28G, R28E, S143L, S143R, S143P S143T, S143A, Q145E, Q145N, E142D, E142N, E142S, E142G, A141 R, A141S, A141T, A141 H, A141 K, A141 F, A141 D, A141G, A141 N, A141 P, A141Q, A141Y, A141V and A141W, at the position corresponding to the sequence of SEQ ID NO: 2, preferably selected from D139N and A141 R at the positions corresponding to the sequence of SEQ ID NO: 2. It will be understood that a “set of mutations” as used above refers to a combination of mutations indicated by the use of 7”, where all of the mutations in the combination are selected, e.g. if “Q84N / T89S / A141S / E142A / S143D” as used above is selected, all of the indicated mutations will be present in the amino acid sequence. In some embodiments, one or more THD according to the invention may independently comprise an amino acid sequence comprising the mutations D139N and A141 R at the positions corresponding to the sequence of SEQ ID NO: 2.

[0128] In some embodiments, each THD according to the invention may comprise an amino acid sequence comprising the mutations D139N and A141 R at the positions corresponding to the sequence of SEQ ID NO: 2.

[0129] It will be understood that the sequence of SEQ ID NO: 2 with the mutations D139N and A141 R is represented by the amino acid sequence of SEQ ID NO: 3.

[0130] In some embodiments, one or more THD according to the invention may independently comprise the amino acid sequence according to SEQ ID NO: 3 or a variant thereof with at least 80% sequence identity to SEQ ID NO: 3. In some embodiments, one or more THD according to the invention may independently comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (optionally at least 90%, such as at least 95%) sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, one or more THD according to the invention may independently consist of the amino acid sequence according to SEQ ID NO: 3. It will be understood that in these embodiments, regardless of the % sequence identity to SEQ ID NO: 3, the amino acid sequence of the one or more THD will include the D139N and A141 R mutations.

[0131] In some embodiments, each THD according to the invention may independently comprise the amino acid sequence according to SEQ ID NO: 3 or a variant thereof with at least 80% sequence identity to SEQ ID NO: 3. In some embodiments, each THD according to the invention may independently comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (optionally at least 90%, such as at least 95%) sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, each THD according to the invention may consist of the amino acid sequence according to SEQ ID NO: 3. It will be understood that in these embodiments, regardless of the % sequence identity to SEQ ID NO: 3, the amino acid sequence of each THD will include the D139N and A141 R mutations.

[0132] SEQ ID NO: 3 - THD sequence with D139N and A141 R

[0133] SRTPSDKPVAHWANPQAEGQLQWLNRRANALLANGVELRDNQLWPSEGLYLIYSQVLFK GQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLE KGDRLSAEINRPDYLNFRESGQVYFGIIAL

[0134] Without wishing to be bound by theory, the D139N and A141 R mutations may confer TNFR2 selectivity.

[0135] In some embodiments, the three THDs of the TNFR2 binding domain are identical in their amino acid sequence. In some embodiments, each THD may independently comprise or consist of the amino acid sequence according to SEQ ID NO: 3 or a variant thereof.

[0136] In some embodiments, at least one of the THDs may comprise an additional “S” at the N terminus. In some embodiments, at least one of the THDs may comprise or consist of the amino acid sequence according to SEQ ID NO: 3 or a variant thereof, and may further comprise an additional “S” at the N terminus.

[0137] In some embodiments, only one of the THD (e.g. the first THD) may comprise an additional “S” at the N terminus.

[0138] In some embodiments, the TNFR2 binding domain may comprise an additional “S” at the N terminus. In other words, the first THD of the TNFR2 binding domain may comprise an additional “S” at the N terminus. In some embodiments, each THD of the TNFR2 binding domain according to the invention may independently comprise or consist of the amino acid sequence according to SEQ ID NO: 3 or a variant thereof, but the first THD of the TNFR2 binding domain may further comprise an additional “S” at the N terminus.

[0139] SEQ ID NO: 4 is an amino acid sequence comprising the amino acid sequence according to SEQ ID NO: 3 with an additional “S” at the N terminus.

[0140] In some embodiments, a THD according to the invention may comprise the amino acid sequence according to SEQ ID NO: 4 or a variant thereof with at least 80% sequence identity to SEQ ID NO: 4. In some embodiments, a THD according to the invention may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (optionally at least 90%, such as at least 95%) sequence identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, a THD according to the invention may consist of the amino acid sequence according to SEQ ID NO: 4. It will be understood that in these embodiments, regardless of the % sequence identity to SEQ ID NO: 4, the amino acid sequence of the THD will include the equivalent D139N and A141 R mutations.

[0141] SEQ ID NO: 4 - THD sequence with equivalent D139N and A141 R and additional S

[0142] SSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLF KGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQL EKGDRLSAEINRPDYLNFRESGQVYFGIIAL

[0143] In some embodiments, the first THD of the TNFR2 binding domain according to the invention may comprise or consist of the amino acid sequence according to SEQ ID NO: 4 or a variant thereof, and the second THD and third THD of the TNFR2 binding domain according to the invention may each comprise or consist of the amino acid sequence according to SEQ ID NO: 3 or a variant thereof.

[0144] In some embodiments, each THD according to the invention may independently comprise the amino acid sequence according to SEQ ID NO: 30 or a variant thereof with at least 80% sequence identity to SEQ ID NO: 30. In some embodiments, each THD according to the invention may independently comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (optionally at least 90%, such as at least 95%) sequence identity to the amino acid sequence of SEQ ID NO: 30. In some embodiments, each THD according to the invention may independently comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 30. In some embodiments, each THD according to the invention may consist of the amino acid sequence according to SEQ ID NO: 30. It will be understood that in these embodiments, regardless of the % sequence identity to SEQ ID NO: 30, the amino acid sequence of each THD will include the D139N and A141 R mutations.

[0145] SEQ ID NO: 41- THD sequence with D132N and A134R

[0146] PVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPS THVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSA EINRPDYLNFRESGQVYFGIIAL

[0147] In some embodiments, the first and second THD may be directly linked without any intervening peptide linkers. In some embodiments, the second and third THD may be directly linked without any intervening peptide linkers. In some embodiments, (i) the first and second THD and (ii) the second and third THD may be directly linked without any intervening peptide linkers.

[0148] In other embodiments, the first and second THD and / or second and third THD may be linked by intervening peptide linkers. In some embodiments, the intervening peptide linkers may be from 1 to 50, optionally from 1 to 20 amino acids in length. In some embodiments, the intervening peptide linkers may be from 8 to 12, optionally from 9 to 10 amino acids in length. The intervening peptide linkers may comprise any sequence, for example a flexible GS-rich sequence. In some embodiments, the intervening peptide linkers may be independently selected from a peptide which consists of XC-XL-XN; wherein:

[0149] Xc is selected from the group consisting of A, A-L, L, preferably A-L;

[0150] XL is absent or is an amino acid linker consisting of 1-11 amino acids,

[0151] XN is absent or selected from the group consisting of K, D-K, S-D-K, P-S-D-K (SEQ ID NO: 31), T-P-S-D-K (SEQ ID NO: 32), R-T-P-S-D-K (SEQ ID NO: 33), S-R-T-P-S-D-K (SEQ ID NO: 34), S-S-R-T-P-S-D-K (SEQ ID NO: 35), T-K, S-T-K, H-S-T-K (SEQ ID NO: 36), A-H-S-T-K (SEQ ID NO: 37), L-A-H-S-T-K (SEQ ID NO: 38), H-L-A-H-S-T-K (SEQ ID NO: 39), L-H-L-A-H-S-T-K (SEQ ID NO: 40). In some embodiments, each intervening peptide linker may be A-L.

[0152] XL when present may comprise any amino acid sequence. XL when present may comprise a G / S linker sequence (i.e., 1-11 glycine and / or serine amino acids). SEQ ID NO: 5 is an amino acid sequence comprising (in sequential order) the amino acid sequence according to SEQ ID NO: 4 and two amino acid sequences according to SEQ ID NO: 3.

[0153] In some embodiments, the TNFR2 binding domain may comprise the amino acid sequence according to SEQ ID NO: 5 or a variant with at least 80% sequence identity to SEQ ID NO: 5. In some embodiments, the TNFR2 binding domain according to the invention may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (optionally at least 90%, such as at least 95%) sequence identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the TNFR2 binding domain according to the invention may consist of the amino acid sequence according to SEQ ID NO: 5. It will be understood that in these embodiments, regardless of the % sequence identity to SEQ ID NO: 5, the amino acid sequence of the TNFR2 binding domain will include the equivalent D139N and A141 R mutations for each THD.

[0154] SEQ ID NO: 5 - illustrative full trimeric THD TNFR2 binding domain

[0155] SSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLF KGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQL EKGDRLSAEINRPDYLNFRESGQVYFGIIALSRTPSDKPVAHVVANPQAEGQLQWLNRRAN ALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIK SPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLNFRESGQVYFGIIALSRT PSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQ GCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKG DRLSAEINRPDYLNFRESGQVYFGIIAL

[0156] Fc domain

[0157] In some embodiments, the TNFR2 agonist further comprises a multimerization domain. The multimerization domain may be selected from the group consisting of: an antibody, an antibody heavy chain, an immunoglobulin Fc domain, a heavy chain domain 2 (CH2) of IgM (MHD2), a heavy chain domain 2 (CH2) of IgE (EHD2), or the tetramerization domain of p53.

[0158] In some embodiments, the multimerization domain is a dimerization domain.

[0159] In some embodiments, the dimerization domain is an immunoglobulin Fc domain. The immunoglobulin Fc domain may be an lgG1 , lgG2 or lgG4 immunoglobulin Fc domain. The immunoglobulin Fc domain may be an lgG1 immunoglobulin Fc domain comprising an N297A mutation. In some embodiments, the dimerization domain is an immunoglobulin Fc domain mutant without FcR and / or C1q binding, optionally wherein the Fc domain comprises one or more of the following mutations: FcAab, LALA, LALA-GP, lgG2, lgG2o, aglycosylated lgG1 , lgG1 (L234F / L235E / P331S), lgG2m4, lgG4 ProAlaAla.

[0160] A fragment crystallizable (Fc) domain is the “tail” region of an antibody that typically interacts with Fc receptors expressed on the surface of cells. Fc domains also interact with other proteins, such as proteins of the complement system.

[0161] In some embodiments, the TNFR2 agonist comprises an Fc domain, wherein the Fc domain comprises the following mutations L234A, L235A, A327G, A330S and P331S.

[0162] In some embodiments, the Fc domain does not comprise a proline at position 233.

[0163] In some embodiments, the Fc domain comprises a glycine at position 236.

[0164] In some embodiments, the Fc domain (i) does not comprise a proline at position 233 and (ii) comprises a glycine at position 236.

[0165] It will be understood that the amino acid numbering and residues for L234A, L235A, A327G, A330S, P331S, proline at position 233 and glycine at position 236, may be relative to the lgG1 positions and residues described in Armour et al., (Eur. J. Immunol. 1999. 29: 2613-2624; incorporated herein by reference), in particular in Table 1 therein.

[0166] It will also be understood that the amino acid numbering and residues for L234A, L235A, A327G, A330S, P331S, proline at position 233 and glycine at position 236, may be relative to the IgG constant region residues and positions according to the Ell numbering system (found in Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S. and Foeller, C., Sequences of proteins of immunological interest. US Department of Health and Human services, NIH, Bethesda 1991).

[0167] It will also be understood that the positions of 233, 234, 235, 236, 327, 330, and 331 described above for the Fc domain may correspond to the corresponding positions in SEQ ID NO: 1 as outlined in Table 1 below. Table 1 :

[0168] In some embodiments, the Fc domain of the TNFR2 agonist comprises the following mutations: L14A, L15A, A107G, A110S and P111S, wherein the amino acid numbering is relative to the sequence of SEQ ID NO: 1. It will be understood that SEQ ID NO: 1 already comprises “A” at position 14, “A” at position 15, “G” at position 107, “S” at position 110 and “S” at position 111.

[0169] It thus will be understood that L14A, L15A, A107G, A110S and P111S, wherein the amino acid numbering is relative to the sequence of SEQ ID NO: 1 , correspond to the L234A, L235A, A327G, A330S and P331S mutations and positions relative to the lgG1 positions and residues in the Ell numbering system / as described in Armour et al.

[0170] It will be understood that the Fc domain of the TNFR2 agonist, comprising the L234A, L235A, A327G, A330S and P331S mutations, may be defined as a “Fc[LALA-Aa]”.

[0171] It will also be understood that the Fc domain of the TNFR2 agonist, comprising L14A, L15A, A107G, A110S and P111S relative to amino acid positions of SEQ ID NO: 1 , may be defined as a “Fc[LALA-Aa]”.

[0172] In some embodiments, the Fc domain according to the invention may comprise the amino acid sequence according to SEQ ID NO: 1 or a variant thereof with at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, the Fc domain according to the invention may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (optionally at least 90%, such as at least 95%) sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the Fc domain according to the invention may consist of the amino acid sequence according to SEQ ID NO: 1. It will be understood that in these embodiments, regardless of the % sequence identity to SEQ ID NO: 1 , the amino acid sequence of the Fc domain will include the L14A, L15A, A107G, A110S and P111S mutations relative to the sequence of SEQ ID NO: 1 , or the equivalent mutations at corresponding positions (e.g. L234A, L235A, A327G, A330S and P331S).

[0173] SEQ ID NO: 1

[0174] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0175] (L14A, L15A, A107G, A110S and P111S are shown in bolded and underlined in the above sequence)

[0176] As defined above, an Fc domain comprising the sequence of SEQ ID NO: 1 (with the L14A, L15A, A107G, A110S and P111S mutations) may be defined as a “Fc[LALA-Aa]”.

[0177] Without wishing to be bound by theory, it is considered that the Fc domain comprising the amino acid sequence of SEQ ID NO: 1 may have reduced, or essentially no, binding to Fc gamma receptors or C1q. In particular, the Fc domain (i.e. “Fc[LALA-Aa]”) of the TNFR2 agonist of the invention (e.g. an Fc domain comprising the sequence of SEQ ID NO: 1) may have reduced binding to FcyRlla. The ‘reduced’ binding may be in comparison to a corresponding Fc domain which does not comprise the L14A, L15A, A107G, A110S and P111S mutations. In some embodiments, the reduced binding to FcyRlla may be in comparison to the Fc[Aab] construct described by Armour et al., (Eur. J. Immunol. 1999. 29: 2613-2624).

[0178] Without wishing to be bound by theory, it is also considered that the Fc domain comprising the amino acid sequence of SEQ ID NO: 1 may have a longer or extended half-life due to FcRn-mediated recycling of the Fc domain.

[0179] Linker

[0180] In some embodiments, the TNFR2 binding domain is joined to the Fc domain by a linker. Without wishing to be bound by theory, the linker may spatially separate the TNFR2 binding domain and the Fc domain.

[0181] In some embodiments, the linker may be a peptide linker. In some embodiments, the linker may be a flexible linker. Without wishing to be bound by theory, a flexible linker allows the TNFR2 binding domain and the Fc domain to orient in different directions to enable binding to their respective ligands / binding partners.

[0182] In some embodiments, the TNFR2 binding domain is joined to the Fc domain by a flexible linker consisting of 3-20 amino acids, such as 8-15 amino acids.

[0183] In some embodiments, the linker may be 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in length. In some embodiments, the linker may be more than 20 amino acids in length, such as 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 or more than 30 amino acids in length.

[0184] Suitable linkers will be known in the art and may be used in the context of the present invention.

[0185] In some embodiments, the flexible linker may be a Ser-Gly linker. In some embodiments, the linker may comprise a plurality of glycine and serine residues. The glycine (denoted as Gly or G) and serine (denoted as Ser or S) residues may be present in any combination. For example, the linker may comprise the format (Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 41))n or (Gly-Ser)n, where n indicates the number of repeats. Suitable Ser-Gly linkers will be known in the art and may be used in the context of the present invention. In some embodiments, the flexible linker may be a Ser-Gly linker of 8-15 amino acids in length.

[0186] In some embodiments, the flexible linker may comprise or consist of the amino acid sequence: GGSGGGGSGG (SEQ ID NO: 6).

[0187] Full length TNFR2 agonist

[0188] In some embodiments, the TNFR2 agonist comprises a TNFR2 binding domain comprising three TNF homology domains (THD) that specifically bind to TNFR2.

[0189] In some embodiments, the TNFR2 agonist comprises (i) a TNFR2 binding domain comprising three TNF homology domains (THD) that specifically bind to TNFR2; and (ii) an Fc domain.

[0190] In some embodiments, the TNFR2 agonist comprises (i) a TNFR2 binding domain comprising three TNF homology domains (THD) that specifically bind to TNFR2; and (ii) an Fc domain, wherein the Fc domain comprises the following mutations L234A, L235A, A327G, A330S and P331S. It will be understood that the TNFR2 agonist may be considered as a fusion protein.

[0191] In some embodiments, the TNFR2 agonist has the following structure in a N-terminus to C- terminus orientation:

[0192] TNFR2 binding domain - linker - Fc domain.

[0193] In some embodiments, the TNFR2 agonist comprises the following amino acid sequences in a N-terminus to C-terminus orientation:

[0194] SEQ ID NO: 5 - SEQ ID NO: 6 - SEQ ID NO: 1

[0195] In some embodiments, variation within SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 1 may independently be selected from any of the variation permitted above.

[0196] It will be understood that the combined amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 1 in a N-terminus to C-terminus orientation is shown as the amino acid sequence according to SEQ ID NO: 7.

[0197] In some embodiments, the TNFR2 agonist may comprise the amino acid sequence according to SEQ ID NO: 7 or a variant with at least 80% sequence identity to SEQ ID NO: 7. In some embodiments, the TNFR2 agonist according to the invention may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the TNFR2 agonist according to the invention may comprise an amino acid sequence having at least 90% (such as at least 95%) sequence identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the TNFR2 agonist according to the invention may comprise an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the TNFR2 agonist according to the invention may comprise the amino acid sequence according to SEQ ID NO: 7. In some embodiments, the TNFR2 agonist according to the invention may consist of the amino acid sequence according to SEQ ID NO: 7. It will be understood that in these embodiments, regardless of the % sequence identity to SEQ ID NO: 7, the amino acid sequence of each of the THDs will include the equivalent D139N and A141 R mutations, and the amino acid sequence of the Fc domain will include the equivalent L234A, L235A, A327G, A330S and P331S mutations. SEQ ID NO: 7 (full length molecule)

[0198] SSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLF KGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQL EKGDRLSAEINRPDYLNFRESGQVYFGIIALSRTPSDKPVAHWANPQAEGQLQWLNRRAN ALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIK

[0199] SPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLNFRESGQVYFGIIALSRT PSDKPVAHWANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQ GCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKG DRLSAEINRPDYLNFRESGQVYFGIIALGGSGGGGSGGDKTHTCPPCPAPEAAGGPSVFLF

[0200] PPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRW SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPG

[0201] TNFR2 agonist multimer

[0202] The TNFR2 agonist according to the invention may combine with one or more other TNFR2 agonist(s) according to the invention to form a multimeric molecule.

[0203] It will be understood that the multimeric molecule may be referred to as a TNFR2 agonist multimer.

[0204] It will be understood that each individual TNFR2 agonist according to the invention can be considered as a TNFR2 agonist monomer.

[0205] It will also be understood that multiple TNFR2 agonist monomers can bind together by proteinprotein interactions which allow multimerisation of the TNFR2 agonist monomers, such as by non-covalent bonds or covalent bonds.

[0206] In some embodiments, the TNFR2 agonist multimer may be in the form of a dimer.

[0207] In some embodiments, the TNFR2 agonist multimer may comprise six or more, optionally six, TNF homology domains (THD) that specifically bind to TNFR2.

[0208] In some embodiments, each TNFR2 agonist monomer may bind together through the interactions of each of the Fc domains to one another. For example, in a dimer, the Fc domain of one TNFR2 agonist may bind to the Fc domain of the other TNFR2 agonist. It will be understood that the Fc domain of each TNFR2 agonist monomer may interact with the Fc domain(s) of one or more other TNFR2 agonist monomer(s) through any interaction that is suitable to allow the Fc domains to bind together. As such, the binding of the Fc domains of the TNFR2 agonist monomers to each other is not limited to any one particular type of interaction.

[0209] Accordingly, in some embodiments, the TNFR2 agonist multimer is in the form of a dimer comprising two TNFR2 agonists, wherein each TNFR2 agonist is independently a TNFR2 agonist according to the invention.

[0210] In some embodiments, each TNFR2 agonist in the dimer may be identical (e.g. by having the same amino acid sequence).

[0211] Without wishing to be bound by theory, it is also considered that the advantageous effects of the TNFR2 agonist of the invention are at least in part due to the presence of an Fc domain described herein, which allows the formation of a dimer of the TNFR2 agonist of the invention. In particular, the dimer of the TNFR2 agonist of the invention is considered to unexpectedly provide improved efficacy in vivo, compared to known TNFR2 agonist molecules of the prior art comprising higher orders of THD domains, such as tetrameric TNFR2 agonist molecules comprising p53.

[0212] INHIBITOR OF TUMOUR NECROSIS FACTOR RECEPTOR 1 (TNFR1) SIGNALLING

[0213] In some embodiments, the pharmaceutical composition according to the invention comprises one or more modulators of TNF signalling for use in treating and / or preventing acute pain, wherein the one or more modulators of TNF signalling comprises an inhibitor of tumour necrosis factor receptor 1 (TNFR1) signalling.

[0214] It will be understood that the inhibitor of TNFR1 signalling may be capable of blocking signalling via TNFR1.

[0215] It will also be understood that an inhibitor of TNFR1 signalling may function by binding to TNFR1 and preventing TNFR1 agonists from binding to and / or activating TNFR1.

[0216] It will also be understood that an inhibitor of TNFR1 signalling may function by binding to TNFR1 ligands and preventing the TNFR1 ligands from binding to and / or activating TNFR1.

[0217] Suitable assays and techniques for measuring and / or quantifying the activity (e.g. binding capability and / or activity) of the inhibitor of TNFR1 signalling may include, but are not limited to, ELISA, surface plasmon resonance (SPR), quartz crystal microbalance (QCM), bioluminescence assays and flow cytometry. Functional assays for receptor activation can be performed using reporter cell lines or by measuring downstream signalling molecule (e.g. by Western blot).

[0218] In some embodiments, the inhibitor of TNFR1 signalling comprises a TNFR1 antagonist or a soluble TNF inhibitor.

[0219] In some embodiments, the inhibitor of TNFR1 signalling comprises a TNFR1 antagonist.

[0220] In some embodiments, the TNFR1 antagonist comprises an anti-TNFR1 antagonistic antibody or fragment thereof. In some embodiments, the TNFR1 antagonist comprises an antibody or fragment thereof capable of specifically binding TNFR1 and inhibiting TNFR1 signalling.

[0221] In some embodiments, the TNFR1 antagonist is selected from the group consisting of Atrosimab, Atrosab, GSK2862277, scRIantTNF, TROS and DS41.

[0222] In some embodiments, the TNFR1 antagonist is Atrosimab.

[0223] In some embodiments, the inhibitor of TNFR1 signalling comprises a soluble TNF inhibitor.

[0224] In some embodiments, the soluble TNF inhibitor comprises an antibody or fragment thereof capable of specifically binding and inhibiting soluble TNF.

[0225] Without wishing to be bound by theory, it will be understood that soluble TNF is capable of binding to TNFR1.

[0226] Accordingly, it will be understood that an inhibitor of soluble TNF is an inhibitor of TNFR1 signalling.

[0227] In some embodiments, the soluble TNF inhibitor is selected from the group consisting of XPro1595 and SAR441566.

[0228] Illustrative inhibitors of TNFR1 signalling include Atrosab (as described, for example, in Zettlitz, K. A., et al., MAbs, 2010, 2(6):639-47) and Atrosimab (as described, for example, in Richter, F., et al., Front. Immunol., 2021 , 12:705485); for example. In some embodiments, the inhibitor of TNFR1 signalling is Atrosab. The complementaritydetermining regions (CDRs) CDRH1 , CDRH2, CDRH3, CDRL1 , CDRL2, and CDRL3 sequences of Atrosab are presented below as SEQ ID NOs: 10-15 respectively. The heavy chain variable (VH) and light chain variable (VL) sequences of Atrosab are presented below as SEQ ID NO: 16 and SEQ ID NO: 17 respectively.

[0229] In some embodiments, the inhibitor of TNFR1 signalling is an antibody comprising (a) a heavy chain variable (VH) domain that comprises the CDR sequences CDRH1 , CDRH2, and CDRH3, and (b) a light chain variable (VL) domain that comprises the CDR sequences CDRL1 , CDRL2, and CDRL3; wherein: a) the CDRH1 sequence is GYTFTDFYIN (SEQ ID NO: 10); b) the CDRH2 sequence is WIGEIYPYSGHAYYNEKFKA (SEQ ID NO: 11); c) the CDRH3 sequence is WDFLDY (SEQ ID NO: 12); d) the CDRL1 sequence is RSSQSLLHSNGNTYLHWY (SEQ ID NO: 13); e) the CDRL2 sequence is LLIYTVSNRFS (SEQ ID NO: 14); and f) the CDRL3 sequence is SQSTHVPYT (SEQ ID NO: 15).

[0230] In some embodiments, the inhibitor of TNFR1 signalling is an antibody comprising (a) a heavy chain variable (VH) domain that comprises the CDR sequences CDRH1 , CDRH2, and CDRH3, and (b) a light chain variable (VL) domain that comprises the CDR sequences CDRL1 , CDRL2, and CDRL3; wherein: the VH comprises or consists of the sequence:

[0231] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDFYINWVRQAPGQGLEWIGEIYPYSGHAYY NEKFKARVTITADKSTSTAYMELSSLRSEDTAVYYCARWDFLDYWGQGTTVTVSS (SEQ ID NO: 16); and the VL comprises or consists of the sequence:

[0232] DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGNTYLHWYLQKPGQSPQLLIYTVSNRFSG VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGGGTKVEIKR (SEQ ID NO: 17).

[0233] In some embodiments, the inhibitor of TNFR1 signalling is Atrosimab. The CDRH1 , CDRH2, CDRH3, CDRL1 , CDRL2, and CDRL3 sequences of Atrosimab are presented below as SEQ ID NOs: 18-23 respectively. The VH and VL sequences of Atrosimab are presented below as SEQ ID NO: 24 and SEQ ID NO: 25 respectively. The Atrosimab full light and heavy chain sequences are presented below as SEQ ID NO: 26 and SEQ ID NO: 27 respectively In some embodiments, the inhibitor of TNFR1 signalling is an antibody comprising (a) a heavy chain variable (VH) domain that comprises the CDR sequences CDRH1 , CDRH2, and CDRH3, and (b) a light chain variable (VL) domain that comprises the CDR sequences CDRL1 , CDRL2, and CDRL3; wherein: a) the CDRH1 sequence is DFYIN (SEQ ID NO: 18); b) the CDRH2 sequence is EIVPSQGEAKYNDKFK (SEQ ID NO: 19); c) the CDRH3 sequence is WDFLDY (SEQ ID NO: 20); d) the CDRL1 sequence is RSSQSLLHSNGNTYLH (SEQ ID NO: 21); e) the CDRL2 sequence is TVSNRFS (SEQ ID NO: 22); and f) the CDRL3 sequence is SQSTHVPYT (SEQ ID NO: 23).

[0234] In some embodiments, the inhibitor of TNFR1 signalling is an antibody comprising (a) a heavy chain variable (VH) domain that comprises the CDR sequences CDRH1 , CDRH2, and CDRH3, and (b) a light chain variable (VL) domain that comprises the CDR sequences CDRL1 , CDRL2, and CDRL3; wherein: the VH comprises or consists of the sequence:

[0235] HVQLVQSGAEVKKPGSSVKVSCKASGYTFTDFYINWVRQAPGQGLEWIGEIVPSQGEAKY NDKFKARVTITADKSTSTAYMELSSLRSEDTAVYYCARWDFLDYWGQGTTVTVSS (SEQ ID NO: 24); and the VL comprises or consists of the sequence:

[0236] DVQMTQSPSSLSASVGDRVTITCRSSQSLLHSNGNTYLHWYQQKPGKAPKLLIYTVSNRFS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCSQSTHVPYTFGGGTKVEIK (SEQ ID NO: 25).

[0237] In some embodiments, the inhibitor of TNFR1 signalling is an antibody comprising or consisting of:

[0238] (i) a light chain of the sequence:

[0239] DVQMTQSPSSLSASVGDRVTITCRSSQSLLHSNGNTYLHWYQQKPGKAPKLLIYTVSNRFS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCSQSTHVPYTFGGGTKVEIKGTGGGSGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPSVFPLAPSSKSTSGGT AALGCLVKDYFPSDIAVEWESGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYSC

[0240] SVMHEALHNHYTQKSVEPKSC (SEQ ID NO: 26), and

[0241] (ii) a heavy chain of the sequence:

[0242] HVQLVQSGAEVKKPGSSVKVSCKASGYTFTDFYINWVRQAPGQGLEWIGEIVPSQGEAKY NDKFKARVTITADKSTSTAYMELSSLRSEDTAVYYCARWDFLDYWGQGTTVTVSSGTGGG SGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPSVFIFPPSDEQ LKSGTASVVCLVNNFYPRDIAVEWEVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYSCSVMHEALHNHYTQKSFNRGEC (SEQ ID NO: 27).

[0243] In some embodiments, the inhibitor of TNFR1 signalling described herein comprises a variant of the amino acid sequence according to any one of SEQ ID NOs: 10-27 with at least 80% sequence identity thereto.

[0244] In some embodiments, the inhibitor of TNFR1 signalling described herein comprises a variant of the amino acid sequence according to any one of SEQ ID NOs: 10-27 with at least 85% sequence identity thereto.

[0245] In some embodiments, the inhibitor of TNFR1 signalling described herein comprises a variant of the amino acid sequence according to any one of SEQ ID NOs: 10-27 with at least 90% sequence identity thereto.

[0246] In some embodiments, the inhibitor of TNFR1 signalling described herein comprises a variant of the amino acid sequence according to any one of SEQ ID NOs: 10-27 with at least 95% sequence identity thereto.

[0247] In some embodiments, the inhibitor of TNFR1 signalling described herein comprises a variant of the amino acid sequence according to any one of SEQ ID NOs: 10-27 with at least 96% sequence identity thereto.

[0248] In some embodiments, the inhibitor of TNFR1 signalling described herein comprises a variant of the amino acid sequence according to any one of SEQ ID NOs: 10-27 with at least 97% sequence identity thereto.

[0249] In some embodiments, the inhibitor of TNFR1 signalling described herein comprises a variant of the amino acid sequence according to any one of SEQ ID NOs: 10-27 with at least 98% sequence identity thereto.

[0250] In some embodiments, the inhibitor of TNFR1 signalling described herein comprises a variant of the amino acid sequence according to any one of SEQ ID NOs: 10-27 with at least 99% sequence identity thereto. POLYNUCLEOTIDE

[0251] In some embodiments, the pharmaceutical composition according to the invention comprises one or more polynucleotide(s) each comprising a nucleic acid sequence encoding the one or more modulator(s) of TNF signalling according to the invention.

[0252] In some embodiments, the one or more modulator(s) of TNF signalling comprise(s) a nucleic acid encoding the TNFR2 agonist according to the invention or the inhibitor of TNFR1 signalling according to the invention.

[0253] In some embodiments, the pharmaceutical composition according to the invention comprises one or more polynucleotide(s) each comprising a nucleic acid sequence encoding the TNFR2 agonist according to the invention.

[0254] In some embodiments, the one or more polynucleotides encode two or more TNFR2 agonist molecules that form a TNFR2 agonist multimer as described herein when expressed in a cell, wherein each TNFR2 agonist molecule is independently a TNFR2 agonist according to the invention.

[0255] In some embodiments, the pharmaceutical composition according to the invention comprises one or more polynucleotide(s) each comprising a nucleic acid sequence encoding the inhibitor of TNFR1 signalling according to the invention.

[0256] As used herein, the terms “polynucleotide”, “nucleotide”, and “nucleic acid” are intended to be synonymous with each other.

[0257] Due to the redundancy of the genetic code, variations in nucleic acid sequences are possible that encode for the same polypeptide. These sequences are encompassed by the present invention. Therefore multiple polynucleotides are envisaged, each with a different nucleic acid sequence but which encodes a polypeptide according to the invention or a further polypeptide as described herein. It is possible to design and produce such nucleic acid sequences without difficulty.

[0258] The nucleic acid sequence may be an RNA or DNA sequence or a variant thereof. The term "polynucleotide" includes an RNA or DNA sequence. It may be single or double stranded. It may, for example, be genomic, recombinant, mRNA or cDNA. The polynucleotide or nucleic acid sequence may comprise synthetic nucleotides and / or modified nucleotides. These synthetic nucleotides and / or modified nucleotides may enhance the in vivo activity and / or stability of the polynucleotide.

[0259] The polynucleotide may be codon optimised for production in the host cell of choice.

[0260] In some embodiments, the polynucleotide encoding the one or more modulator(s) of TNF signalling of the present invention is a DNA polynucleotide. In some embodiments, the polynucleotide encoding the one or more modulator(s) of TNF signalling of the present invention is an RNA polynucleotide. In some embodiments, the polynucleotide encoding the one or more modulator(s) of TNF signalling of the present invention is an mRNA polynucleotide. In some embodiments, the polynucleotide encoding the one or more modulator(s) of TNF signalling of the present invention is a cDNA polynucleotide.

[0261] In some embodiments, the polynucleotide encoding the TNFR2 agonist of the present invention is a DNA polynucleotide. In some embodiments, the polynucleotide encoding the TNFR2 agonist of the present invention is an RNA polynucleotide. In some embodiments, the polynucleotide encoding the TNFR2 agonist of the present invention is an mRNA polynucleotide. In some embodiments, the polynucleotide encoding the TNFR2 agonist of the present invention is a cDNA polynucleotide.

[0262] In some embodiments, the polynucleotide encoding the inhibitor of TNFR1 signalling of the present invention is a DNA polynucleotide. In some embodiments, the polynucleotide encoding the inhibitor of TNFR1 signalling of the present invention is an RNA polynucleotide. In some embodiments, the polynucleotide encoding the inhibitor of TNFR1 signalling of the present invention is an mRNA polynucleotide. In some embodiments, the polynucleotide encoding the inhibitor of TNFR1 signalling of the present invention is a cDNA polynucleotide.

[0263] As used herein, “variant” is synonymous with “mutant” and refers to a polynucleotide or amino acid sequence which differs in comparison to the corresponding wild-type sequence. The term “wild-type” is used to mean a gene or protein having a polynucleotide or amino acid sequence respectively, which is identical with the native gene or protein respectively.

[0264] In some embodiments, the nucleic acid sequence may be operably linked to a heterologous sequence, such as a promoter or regulatory sequence, forming an expression cassette.

[0265] The expression cassette may comprise one or more control sequences. Control sequences are sequences that control and regulate transcription and, where appropriate, translation, and include promoter sequences, transcriptional regulators encoding sequences, ribosome binding sequences (RBS) and / or transcription terminating sequences. The expression cassette may additionally include an enhancer, which may be adjacent to or distant from the promoter sequence and can function to increase transcription from the same. The expression control sequence may be functional in prokaryotic cells or in eukaryotic cells and organisms, such as mammalian cells. The expression cassette may comprise a promoter. Any promoter may be used in this methodology. In general, it is advantageous to employ a strong promoter functional in eukaryotic cells. The strong promoter may be, but not limited to, the immediate early cytomegalovirus promoter (CMV-IE) of human or murine origin, or optionally having another origin such as the rat or guinea pig.

[0266] In more general terms, the promoter has either a viral, or a cellular origin. A strong viral promoter other than CMV-IE that may be usefully employed in the practice of the invention is the early / late promoter of the SV40 virus or the LTR promoter of the Rous sarcoma virus. A strong cellular promoter that may be usefully employed in the practice of the invention is the promoter of a gene of the cytoskeleton, such as e.g. the desmin promoter (Kwissa et al., 2000), or the actin promoter (Miyazaki et al., 1989).

[0267] The promoter may be a constitutive promoter. The promoter may be a tissue specific promoter.

[0268] VECTOR

[0269] In some embodiments, the one or more polynucleotide(s) may be comprised within one or more vector(s).

[0270] Such a vector may be used to introduce the nucleic acid sequence into a cell so that the cell expresses and / or produces the one or more modulator(s) of TNF signalling according to the invention, such as the TNFR2 agonist according to the invention and / or the inhibitor of TNFR1 signalling according to the invention.

[0271] As used herein, the term “vector”, which may also be termed an “expression vector” or and “expression construct”, is usually a plasmid or virus designed for protein expression in cells. The vector is used to introduce a specific gene into a target cell and can use the cell's mechanism for protein synthesis to produce the protein encoded by the gene. The vector may be engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector. The goal of a well-designed vector is the production of significant amount of stable messenger RNA, and therefore proteins. Examples of suitable vectors include but are not limited to plasmids, cosmids, phages, viruses or artificial chromosomes.

[0272] The vector according to the invention may be any agent capable of delivering a polynucleotide to a cell and / or maintaining a polynucleotide in a cell. In some embodiments, the vector may be selected from the list consisting of: viral vectors, plasmids, naked nucleic acids, transposonbased vectors, nucleic acids complexed with polypeptide or other molecules, and nucleic acids immobilised onto solid phase particles.

[0273] In some embodiments, the vector may be a plasmid or a viral vector. In some embodiments, the vector may be a retroviral vector or a lentiviral vector.

[0274] The vector may be capable of transfecting or transducing a cell.

[0275] In some embodiments, a cell may comprise a polynucleotide according to the invention or a vector according to the invention. The cell may be capable of producing and / or expressing the one or more modulator(s) of TNF signalling according to the invention, such as the TNFR2 agonist according to the invention and / or the inhibitor of TNFR1 signalling according to the invention. The cell may be a bacterial, fungal, yeast, plant or animal cell. The cell may be a mammalian cell or an insect cell. The cell may be a human cell.

[0276] In some embodiments, the polynucleotide according to the invention or vector according to the invention may, for example, be introduced into a cell by transduction or transfection in vitro or ex vivo.

[0277] The cell is then capable of expressing and / or producing the one or more modulator(s) of TNF signalling according to the invention, such as the TNFR2 agonist according to the invention and / or the inhibitor of TNFR1 signalling according to the invention, when the cell is cultured under suitable conditions. The one or more modulator(s) of TNF signalling according to the invention, such as the TNFR2 agonist according to the invention and / or the inhibitor of TNFR1 signalling according to the invention can be harvested from the cell or supernatant of the cell.

[0278] Accordingly, described herein is a method of making the one or more modulator(s) of TNF signalling according to the invention, such as the TNFR2 agonist according to the invention and / or the inhibitor of TNFR1 signalling according to the invention, which comprises the steps of: (i) introducing a polynucleotide according to the invention or a vector according to the invention into a cell as described above; (ii) culturing the cell under suitable conditions to express and / or produce the one or more modulator(s) of TNF signalling according to the invention, such as the TNFR2 agonist according to the invention and / or the inhibitor of TNFR1 signalling according to the invention; and (iii) harvesting the one or more modulator(s) of TNF signalling according to the invention, such as the TNFR2 agonist according to the invention and / or the inhibitor of TNFR1 signalling according to the invention.

[0279] PHARMACEUTICAL COMPOSITION

[0280] In some embodiments, the pharmaceutical composition according to the invention may further comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant, salt, and optionally one or more further pharmaceutically active polypeptides and / or compounds.

[0281] In some embodiments, the pharmaceutical composition according to the invention may comprise one or more additional compounds, components and / or active agents.

[0282] In some embodiments, the pharmaceutical composition may comprise one or more TNFR2 agonist(s) according to the present invention in combination with one or more inhibitor(s) of TNFR1 signalling according to the invention.

[0283] In some embodiments, the pharmaceutical composition may comprise one or more inhibitor(s) of TNFR1 signalling according to the invention in combination with one or more TNFR2 agonist(s) according to the present invention.

[0284] In some embodiments, a first pharmaceutical composition comprising one or more TNFR2 agonist(s) according to the present invention may be administered in combination with a second pharmaceutical composition comprising one or more inhibitor(s) of TNFR1 signalling according to the invention.

[0285] In some embodiments, a first pharmaceutical composition comprising one or more inhibitor(s) of TNFR1 signalling according to the present invention may be administered in combination with a second pharmaceutical composition comprising one or more TNFR2 agonist(s) according to the invention.

[0286] Pharmaceutical compositions typically should be sterile and stable under the conditions of manufacture and storage. The pharmaceutical composition according to the invention may be produced using current good manufacturing practices (CGMP). The term “pharmaceutical composition” as used in the present specification refers to a substance and / or a combination of substances being used for the identification, prevention or treatment of a tissue status or disease. The pharmaceutical composition is formulated to be suitable for administration to a patient in order to prevent and / or treat disease. Further a pharmaceutical composition refers to the combination of an active agent with a carrier, inert or active, making the composition suitable for therapeutic use. Pharmaceutical compositions can be formulated for oral, parenteral, topical, inhalative, rectal, sublingual, transdermal, subcutaneous or vaginal application routes according to their chemical and physical properties.

[0287] Pharmaceutical compositions comprise solid, semisolid, liquid, transdermal therapeutic systems (TTS). Solid compositions are selected from the group consisting of tablets, coated tablets, powder, granulate, pellets, capsules, effervescent tablets or transdermal therapeutic systems. Also comprised are liquid compositions, selected from the group consisting of solutions, syrups, infusions, extracts, solutions for intravenous application, solutions for infusion or solutions of the carrier systems of the present invention. Semisolid compositions that can be used in the context of the invention comprise emulsion, suspension, creams, lotions, gels, globules, buccal tablets and suppositories.

[0288] The term “carrier”, as used herein, refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as saline solutions in water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.

[0289] A sterile saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously.

[0290] Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0291] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. The compounds of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[0292] In some embodiments, the salt may comprise a metal cation, such as a sodium salt or a potassium salt.

[0293] In some embodiments, the pharmaceutical composition may comprise an aqueous diluent or solvent. In some embodiments, the aqueous diluent or solvent may be a phosphate buffered saline solution, such as a sterile phosphate buffered saline solution.

[0294] In some embodiments, the pharmaceutical composition may be, for example, in a form suitable for oral, parenteral administration, intravenous (such as intravenous infusion), intramuscular, subcutaneous, topical, inhalative, rectal, sublingual, transdermal, or vaginal administration.

[0295] In some embodiments, the pharmaceutical composition according to the invention may be in a form suitable for intravenous infusion.

[0296] In some embodiments, the pharmaceutical composition according to the invention may be in a form suitable for systemic administration.

[0297] In some embodiments, the pharmaceutical composition according to the invention may be in a form suitable for subcutaneous administration.

[0298] In some embodiments, the pharmaceutical composition according to the invention may be in a form suitable for intravenous administration.

[0299] In some embodiments, the pharmaceutical composition according to the invention may be in a form suitable for intrathecal administration. In some embodiments, the pharmaceutical composition according to the invention may be in a form suitable for intra-articular administration.

[0300] ACUTE PAIN

[0301] The present invention provides a pharmaceutical composition comprising one or more modulator(s) of TNF signalling for use in treating and / or preventing acute pain.

[0302] In some embodiments, the acute pain may be associated with any disease, disorder, or condition associated with and / or involving TNF, such as aberrant TNF signalling.

[0303] Acute pain is typically understood to be distinct from chronic pain, in that it is usually sudden in onset and usually caused by something specific (e.g., an injury, surgery or particular diseases). Acute pain is typically sharp or intense in quality, at least at the onset. Acute pain typically lasts for a short period of time (e.g., from a few minutes to less than six months) and may disappear when the underlying cause is treated / healed. Acute and chronic pain are different clinical entities and typically require different approaches to treatment. In some instances, acute pain may be understood to be pain that is not chronic pain. Both chronic and acute pain are well-defined in the art and have been acknowledged as distinct conditions, see e.g., Bonezzi C, et al., Pain Ther. 2020; 9(Suppl 1): 1 -15; Grichnik KP, Ferrante FM. Mt Sinai J Med. 1991 ; 58(3):217-20; International Association for the Study of Pain; Acute Pain; 2023 (https: / / www.iasp-pain.org / resources / topics / acute-pain / ).

[0304] In some embodiments, the acute pain may arise from injury or trauma.

[0305] In some embodiments, the acute pain may arise from surgery and / or health treatments. In some embodiments, the acute pain may be post-operative pain.

[0306] In some embodiments, the acute pain may arise from osteoarthritis and related diseases.

[0307] In some embodiments, the acute pain is associated with osteoarthritis.

[0308] In some embodiments, the acute pain is post-operative acute pain.

[0309] Acute pain includes, but is not limited to, acute herpes zoster pain, acute inflammatory pain, acute intermittent pain, acute musculoskeletal pain, acute obstetric pain, acute tendonitis pain, acute visceral pain, post-traumatic pain, burns, myocardial infarction, acute pancreatitis, cancer-associated acute pain, acute pain associated with tumor-related pain such as bone pain, headache and facial pain, visceral pain, or cancer treatment, such as post-chemotherapy symptoms, acute pain that may be attributed to a herniated or ruptured intervertabral disc.

[0310] In some embodiments, acute pain may be defined as pain that persists for about 6 months. In some embodiments, acute pain may be defined as pain that persists for less than 6 months.

[0311] In some embodiments, acute pain may be defined as pain that persists for a period of time selected from the list consisting of: about 5 months, about 4 months, about 3 months, about 2 months, or about 1 month. Preferably, acute pain may be defined as pain that persists for about 2 months or about 1 month.

[0312] In some embodiments, acute pain may be defined as pain that persists for a period of time selected from the list consisting of: less than 5 months, less than 4 months, less than 3 months, less than 2 months, or less than 1 month. Preferably, acute pain may be defined as pain that persists for a period of time of less than 2 months or less than 1 month.

[0313] In some embodiments, acute pain may be defined as pain that persists for about 6 weeks. In some embodiments, acute pain may be defined as pain that persists for less than 6 weeks.

[0314] In some embodiments, acute pain may be defined as pain that persists for a period of time selected from the list consisting of: about 5 weeks, about 4 weeks, about 3 weeks, about 2 weeks, or about 1 week.

[0315] In some embodiments, acute pain may be defined as pain that persists for a period of time selected from the list consisting of: less than 5 weeks, less than 4 weeks, less than 3 weeks, less than 2 weeks, or less than 1 week.

[0316] In some embodiments, acute pain may be defined as pain that persists for about 7 days. In some embodiments, acute pain may be defined as pain that persists for less than 7 days.

[0317] In some embodiments, acute pain may be defined as pain that persists for a period of time selected from the list consisting of: about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day. In some embodiments, acute pain may be defined as pain that persists for a period of time selected from the list consisting of: less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days, or less than 1 day.

[0318] In some embodiments, acute pain may be defined as pain that persists for about 24 hours. In some embodiments, acute pain may be defined as pain that persists for less than 24 hours.

[0319] In some embodiments, acute pain may be defined as pain that persists for a period of time selected from the list consisting of: about 23 hours, about 22 hours, about 21 hours, about 20 hours, about 19 hours, about 18 hours, about 17 hours, about 16 hours, about 15 hours, about 14 hours, about 13 hours, about 12 hours, about 11 hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, or about 1 hour.

[0320] In some embodiments, acute pain may be defined as pain that persists for a period of time selected from the list consisting of: less than 23 hours, less than 22 hours, less than 21 hours, less than 20 hours, less than 19 hours, less than 18 hours, less than 17 hours, less than 16 hours, less than 15 hours, less than 14 hours, less than 13 hours, less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, or less than 1 hour.

[0321] In some embodiments, acute pain may be defined as pain that persists for up to 12 weeks.

[0322] In some embodiments, acute pain may be defined as pain that persists for (i.e., lasts for) less than 3 months. In some embodiments, the pharmaceutical compositions described herein may be useful in the treatment of acute pain that has persisted (or is expected to persist, e.g., where surgery is planned) for more than an hour but less than 3 months. In some embodiments, the pharmaceutical compositions described herein may be useful in the treatment of acute pain that has persisted (or is expected to persist) for more than 24 hours but less than 3 months. In some embodiments, the pharmaceutical compositions described herein may be useful in the treatment of acute pain that has persisted (or is expected to persist) for more than 1 week but less than 3 months.

[0323] In some embodiments, acute pain may be defined as pain that persists for about 6 weeks to about 3 months. In some embodiments, acute pain may be defined as pain that persists for about 6 weeks to about 2 months.

[0324] The term "disease" and "disorder" are used interchangeably herein, referring to an abnormal condition, especially an abnormal medical condition such as an illness or injury, wherein a cell, a tissue, an organ, or an individual is not able to efficiently fulfil its function anymore. Typically, but not necessarily, a disease is associated with specific symptoms or signs indicating the presence of such disease. The presence of such symptoms or signs may thus, be indicative for a cell, a tissue, an organ, or an individual suffering from a disease. An alteration of these symptoms or signs may be indicative for the progression of such a disease. A progression of a disease is typically characterised by an increase or decrease of such symptoms or signs which may indicate a "worsening" or "bettering" of the disease. The "worsening" of a disease is characterised by a decreasing ability of a cell, tissue, organ or individual / patient to fulfil its function efficiently, whereas the "bettering" of a disease is typically characterised by an increase in the ability of a cell, tissue, an organ or an individual / patient to fulfil its function efficiently.

[0325] The terms “treat”, “treatment” and “treating” refers to lessening, reducing or improving at least one symptom associated with an existing disease or condition and / or to slow down, reduce or block the progression of the disease or condition and / or to delay or prevent the onset of symptoms (such as further symptoms) of the disease or condition.

[0326] The terms “prevent”, “prevention” and “preventing” refers to preventing the onset of symptoms of a disease or condition, and as such encompasses prophylactic treatment.

[0327] In some embodiments, the one or more modulator(s) of TNF signalling may result in a lowered incidence of disease or symptoms, delayed onset of disease or symptoms, and / or reduced severity of disease or symptoms, compared to other therapies that are known in the art.

[0328] It will be understood that the one or more modulator(s) of TNF signalling according to the invention may alleviate acute pain faster than known therapies. It will also be understood that the one or more modulator(s) of TNF signalling according to the invention may promote improved recovery after surgery, compared to known therapies.

[0329] In particular, without wishing to be bound by theory, the TNFR2 agonist according to the invention may have the advantageous effect of alleviating acute pain and improving recovery following surgery compared to known therapies. The TNFR2 agonist according to the invention may also reduce or obviate reliance on the use of opiates to treat acute pain.

[0330] In some embodiments, the one or more modulator(s) of TNF signalling may alleviate acute pain faster, compared to other therapies that are known in the art. In some embodiments, the TNFR2 agonist may alleviate acute pain faster, compared to other therapies that are known in the art.

[0331] In some embodiments, the one or more modulator(s) of TNF signalling may reduce or obviate the need to treat acute pain by administering opiates. In some embodiments, the TNFR2 agonist may reduce or obviate the need to treat acute pain by administering opiates.

[0332] In some embodiments, the one or more modulator(s) of TNF signalling may reduce the dosage of opiates needed to treat acute pain. In some embodiments, the TNFR2 agonist may reduce the dosage of opiates needed to treat acute pain.

[0333] COMBINATION THERAPY

[0334] The medical uses and methods of treatment described herein may be used in combination with additional treatments and / or medicaments. For example, the medical uses and methods of treatment described herein may be combined with known treatments for acute pain, known chemotherapeutic treatments, known immunomodulatory treatments and / or known radiotherapy treatments in the form of a combination therapy or treatment.

[0335] In some embodiments, treatments for acute pain may include the administration of nonsteroidal anti-inflammatory drugs (NSAIDs) and / or benzodiazepines.

[0336] The present invention provides a TNFR2 agonist for use in treating and / or preventing acute pain, wherein the TNFR2 agonist is administered in combination with an inhibitor of TNFR1 signalling. In some embodiments, the inhibitor of TNFR1 signalling is a TNFRI antagonist.

[0337] The present invention provides an inhibitor of TNFR1 signalling for use in treating and / or preventing acute pain, wherein the inhibitor of TNFR1 signalling is administered in combination with a TNFR2 agonist. In some embodiments, the inhibitor of TNFR1 signalling is a TNFR1 antagonist. The present invention provides a TNFR2 agonist and an inhibitor of TNFR1 signalling, for use in treating and / or preventing acute pain. In some embodiments, the inhibitor of TNFR1 signalling is a TNFR1 antagonist.

[0338] In some embodiments, the TNFR2 agonist and the inhibitor of TNFR1 signalling are administered concurrently.

[0339] In some embodiments, the TNFR2 agonist and the inhibitor of TNFR1 signalling are administered sequentially.

[0340] In some embodiments, the TNFR2 agonist is the TNFR2 agonist as described herein.

[0341] In other words, in some embodiments the TNFR2 agonist is comprised within the pharmaceutical composition as described herein.

[0342] In some embodiments, the inhibitor of TNFR1 signalling is the inhibitor of TNFR1 signalling as described herein.

[0343] In other words, in some embodiments the inhibitor of TNFR1 signalling is comprised within the pharmaceutical composition as described herein.

[0344] In some embodiments, a first pharmaceutical composition comprising one or more TNFR2 agonist(s) according to the present invention may be administered in combination with a second pharmaceutical composition comprising one or more inhibitor(s) of TNFR1 signalling according to the invention.

[0345] In some embodiments, a first pharmaceutical composition comprising one or more inhibitor(s) of TNFR1 signalling according to the present invention may be administered in combination with a second pharmaceutical composition comprising one or more TNFR2 agonist(s) according to the invention.

[0346] It will be understood that the combination of one or more TNFR2 agonist(s) according to the invention and one or more inhibitor(s) of TNFR1 signalling according to the present invention may alleviate acute pain faster than known therapies and / or may promote improved recovery after surgery, compared to known therapies. It will be understood that the combination of one or more TNFR2 agonist(s) according to the invention and one or more inhibitor(s) of TNFR1 signalling according to the present invention may also reduce or obviate reliance on the use of opiates to treat acute pain.

[0347] In some embodiments, the combination of one or more TNFR2 agonist(s) according to the invention and one or more inhibitor(s) of TNFR1 signalling according to the present invention may alleviate acute pain faster, compared to other therapies that are known in the art.

[0348] In some embodiments, the combination of one or more TNFR2 agonist(s) according to the invention and one or more inhibitor(s) of TNFR1 signalling according to the present invention may reduce or obviate the need to treat acute pain by administering opiates.

[0349] In some embodiments, the combination of one or more TNFR2 agonist(s) according to the invention and one or more inhibitor(s) of TNFR1 signalling according to the present invention may reduce the dosage of opiates needed to treat acute pain.

[0350] SUBJECT

[0351] As used herein, the terms “patient” and “subject” may be used interchangeably.

[0352] In some embodiments, the subject of the medical uses and methods of treatment according to the present invention may be a mammal.

[0353] In some embodiments, the subject is human. In some embodiments, the subject may alternatively be a non-human mammal, including for example, a dog, a cat, a horse, a cow, a sheep or a pig.

[0354] In some embodiments, the subject is male. In some embodiments, the subject is female.

[0355] In some embodiments, the subject is a human male. In some embodiments, the subject is a human female.

[0356] ADMINISTRATION

[0357] In some embodiments, the pharmaceutical composition is administered orally, parenterally, intravenously (such as intravenous infusion), locally, intrathecally, intra-articularly, intramuscularly, subcutaneously, topically, by inhalation, rectally, sublingually, by a transdermal route, or vaginally. In some embodiments, the pharmaceutical composition is administered to a subject systemically.

[0358] In some embodiments, the pharmaceutical composition is administered to a subject subcutaneously.

[0359] In some embodiments, the pharmaceutical composition is administered to a subject intravenously.

[0360] In some embodiments, the pharmaceutical composition is administered to a subject intrathecally.

[0361] In some embodiments, the pharmaceutical composition is administered to a subject intraarticularly. In some embodiments, the pharmaceutical composition is administered to a subject by intra-articular injection.

[0362] In some embodiments, the pharmaceutical composition may be administered one or more (e.g., multiple) times to a subject. In some embodiments, the pharmaceutical composition may be administered locally and systemically to a subject. In some embodiments, the pharmaceutical composition may be administered intrathecally, and intravenously or subcutaneously, to a subject. In some embodiments, the pharmaceutical composition may be administered intrathecally to a subject prior to or during surgery. In some embodiments, the pharmaceutical composition may be administered intrathecally prior to or during surgery, and intravenously or subcutaneously after surgery, to a subject. In some embodiments, the pharmaceutical composition may be administered intrathecally or systemically prior to or during surgery, and systemically (e.g., intravenously, subcutaneously or intramuscularly) after surgery, to a subject.

[0363] In some embodiments, the pharmaceutical composition is injected into a subject.

[0364] In some embodiments, the pharmaceutical composition is administered to a subject once.

[0365] In some embodiments, the pharmaceutical composition is administered to a subject over a period of hours, days, weeks, months or years. In some embodiments, the pharmaceutical composition treats and / or prevents acute pain within 2 hours after the pharmaceutical composition is administered to a subject (e.g., where the pharmaceutical composition is administered intravenously).

[0366] In some embodiments, the pharmaceutical composition treats and / or prevents acute pain within 3 hours after the pharmaceutical composition is administered to a subject intravenously.

[0367] In some embodiments, the pharmaceutical composition treats and / or prevents acute pain within 6 hours after the pharmaceutical composition is administered to a subject subcutaneously.

[0368] NUMBERED PARAGRAPHS

[0369] The present invention may be described by way of the following numbered paragraphs:

[0370] 1 . A pharmaceutical composition comprising one or more modulators of TNF signalling for use in treating and / or preventing acute pain.

[0371] 2. The pharmaceutical composition for use according to paragraph 1 , wherein the one or more modulators of TNF signalling each independently comprise a polypeptide, an antibody, a nucleic acid, a small molecule, or a combination thereof.

[0372] 3. The pharmaceutical composition for use according to paragraph 1 or paragraph 2, wherein the one or more modulators of TNF signalling comprises a tumour necrosis factor receptor 2 (TNFR2) agonist.

[0373] 4. The pharmaceutical composition for use according to paragraph 3, wherein the TNFR2 agonist is (i) a polypeptide comprising a TNFR2 binding domain comprising three TNF homology domains (THD) that specifically bind to TNFR2; or (ii) a TNFR2 agonist antibody.

[0374] 5. The pharmaceutical composition for use according to paragraph 4, wherein the C- terminus of the first and second THD, respectively, which is in each case defined by the C- terminal consensus sequence:

[0375] V-X1-F-G-X2-X3 (SEQ ID NO: 28); is linked to the N-terminus of the second and third THD, respectively, which is in each case defined by the N-terminal consensus sequence:

[0376] P-X4-A-H-X5 (SEQ ID NO: 29); through a peptide Xa, which is in each case independently selected and has a length of 9 to 12 amino acids, preferably 9 to 11 , more preferably 9 to 10, wherein X1 is F or Y, wherein X2 is A or I, wherein X3 is a non-polar / hydrophobic or polar / neutral amino acid, preferably selected from the group consisting of F and I, wherein X4 is V or A, and wherein X5 is V or L.

[0377] 6. The pharmaceutical composition according to paragraph 4 or paragraph 5, wherein each THD comprises an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 2

[0378] 7. The pharmaceutical composition according to any of paragraphs 4 to 6, wherein one or more THD, preferably each THD, comprises or consists of SEQ ID NO: 3 or an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 3.

[0379] 8. The pharmaceutical composition according to any of paragraphs 4 to 7, wherein one THD comprises or consists of SEQ ID NO: 4 or an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 4.

[0380] 9. The pharmaceutical composition according to any of paragraphs 4 to 8, wherein the TNFR2 binding domain comprises or consists of a sequence with at least 80% sequence identity to SEQ ID NO: 5.

[0381] 10. The pharmaceutical composition for use according to any of paragraphs 4 to 9, wherein the polypeptide further comprises a multimerization domain.

[0382] 11. The pharmaceutical composition for use according to paragraph 10, wherein the multimerization domain is a dimerization domain, optionally wherein the dimerization domain is an immunoglobulin Fc domain. 12. The pharmaceutical composition for use according to any one of paragraphs 4 to 11, wherein the polypeptide comprises (i) a TNFR2 binding domain comprising three TNF homology domains (THD) that specifically bind to TNFR2; and (ii) an Fc domain, wherein the Fc domain comprises the following mutations L234A, L235A, A327G, A330S and P331S.

[0383] 13. The pharmaceutical composition for use according to paragraph 11 or paragraph 12, wherein:

[0384] (a) the Fc domain:

[0385] (i) does not comprise a proline at position 233 and / or

[0386] (ii) comprises a glycine at position 236; and / or

[0387] (b) the Fc domain comprises a sequence with at least 85% sequence identity to SEQ ID NO: 1.

[0388] 14. The pharmaceutical composition for use according to any one of paragraphs 3 to 13, wherein the TNFR2 agonist is a polypeptide comprising a sequence with at least 80% sequence identity to SEQ ID NO: 7.

[0389] 15. The pharmaceutical composition for use according to paragraphs 3 to 14, wherein the TNFR2 agonist is a polypeptide comprising the sequence of SEQ ID NO: 7.

[0390] 16. The pharmaceutical composition for use according to any one of paragraphs 1 to 15, wherein the one or more modulators of TNF signalling comprises an inhibitor of tumour necrosis factor receptor 1 (TNFR1) signalling.

[0391] 17. The pharmaceutical composition for use according to paragraph 16, wherein the inhibitor of TNFR1 signalling comprises a tumour necrosis factor receptor 1 (TNFR1) antagonist or a soluble TNF inhibitor.

[0392] 18. The pharmaceutical composition for use according to paragraph 17, wherein the TNFR1 antagonist comprises an antibody or fragment thereof capable of specifically binding TNFR1 and inhibiting TNFR1 signalling.

[0393] 19. The pharmaceutical composition for use according to paragraph 17 or 18, wherein the TNFR1 antagonist is selected from the group consisting of Atrosimab, GSK2862277, scRIantTNF, TROS and DS41. 20. The pharmaceutical composition for use according to paragraph 17, wherein the soluble TNF inhibitor comprises an antibody or fragment thereof capable of specifically binding and inhibiting soluble TNF.

[0394] 21. The pharmaceutical composition for use according to paragraph 17 or 20, wherein the soluble TNF inhibitor is selected from the group consisting of XPro1595 and SAR441566.

[0395] 22. The pharmaceutical composition for use according to any preceding paragraph, wherein the pharmaceutical composition comprises one or more nucleic acid(s) encoding the one or more modulator(s) of TNF signalling.

[0396] 23. The pharmaceutical composition for use according to any preceding paragraph, wherein the acute pain has persisted for less than 2 months, or for less than 1 month.

[0397] 24. The pharmaceutical composition for use according to any preceding paragraph, wherein the acute pain has persisted, for less than 6 weeks, for less than 5 weeks, for less than 4 weeks, for less than 3 weeks, for less than 2 weeks, or for less than 1 week.

[0398] 25. The pharmaceutical composition for use according to any preceding paragraph, wherein the acute pain has persisted, for less than 7 days, for less than 6 days, for less than 5 days, for less than 4 days, for less than 3 days, for less than 2 days, or for less than 1 day.

[0399] 26. The pharmaceutical composition for use according to any one of paragraphs 1-25, wherein the acute pain arises from injury or trauma.

[0400] 27. The pharmaceutical composition for use according to any one of paragraphs 1-25, wherein the acute pain arises from surgery and / or health treatment.

[0401] 28. The pharmaceutical composition for use according to any one of paragraphs 1-25, wherein the acute pain is associated with osteoarthritis.

[0402] 29. The pharmaceutical composition for use according to any one of paragraphs 1-25, wherein the acute pain is post-operative acute pain.

[0403] 30. The pharmaceutical composition for use according to any one of paragraphs 1-29, wherein the pharmaceutical composition is administered to a subject systemically. 31. The pharmaceutical composition for use according to any one of paragraphs 1-29, wherein the pharmaceutical composition is administered to a subject subcutaneously.

[0404] 32. The pharmaceutical composition for use according to any one of paragraphs 1-29, wherein the pharmaceutical composition is administered to a subject intravenously.

[0405] 33. The pharmaceutical composition for use according to any one of paragraphs 1-29, wherein the pharmaceutical composition is administered to a subject intrathecally.

[0406] 34. The pharmaceutical composition for use according to any one of paragraphs 1-29, wherein the pharmaceutical composition is administered to a subject intra-articularly, optionally by intra-articular injection.

[0407] 35. A TNFR2 agonist for use in treating and / or preventing acute pain, wherein the TNFR2 agonist is administered in combination with an inhibitor of TNFR1 signalling.

[0408] 36. An inhibitor of TNFR1 signalling for use in treating and / or preventing acute pain, wherein the inhibitor of TNFR1 signalling is administered in combination with a TNFR2 agonist.

[0409] 37. A TNFR2 agonist and an inhibitor of TNFR1 signalling, for use in treating and / or preventing acute pain.

[0410] 38. The TNFR2 agonist for use and / or the inhibitor of TNFR1 signalling for use according to any one of paragraphs 35-37, wherein the inhibitor of TNFR1 signalling is a TNFR1 antagonist.

[0411] 39. The TNFR2 agonist for use and / or inhibitor of TNFR1 signalling for use of any one of paragraphs 35 to 38, wherein the TNFR2 agonist and the inhibitor of TNFR1 signalling are administered concurrently or sequentially.

[0412] 40. The TNFR2 agonist for use and / or the inhibitor of TNFR1 signalling for use according to any one of paragraphs 35-39, wherein the TNFR2 agonist is the TNFR2 agonist comprised within the pharmaceutical composition for use according to any of paragraphs 3- 15 or 22-34. 41 . The TNFR2 agonist for use and / or the inhibitor of TNFR1 signalling for use according to any one of paragraphs 35-39 wherein the inhibitor of TNFR1 signalling is the inhibitor of TNFR1 signalling comprised within the pharmaceutical composition for use according to any of paragraphs 16-34.

[0413] 42. A method of treating and / or preventing acute pain, the method comprising administering to a subject a pharmaceutical composition comprising a modulator of TNF signalling.

[0414] 43. The method of paragraph 42, wherein the modulator of TNF signalling is as further defined in the pharmaceutical composition for use according to any one of paragraphs 2-34.

[0415] 44. The pharmaceutical composition for use according to any of paragraphs 3-15 or 22-34 comprising the TNFR2 agonist, wherein the pharmaceutical composition is administered in combination with the pharmaceutical composition for use according to any of paragraphs 16- 34 comprising the inhibitor of TNFR1 signalling.

[0416] 45. The pharmaceutical composition for use according to any of paragraphs 16-34 comprising the inhibitor of TNFR1 signalling, wherein the pharmaceutical composition is administered in combination with the pharmaceutical composition for use according to any of paragraphs 3-15 or 22-34 comprising the TNFR2 agonist.

[0417] GENERAL TERMS AND DEFINITIONS

[0418] The term “polypeptide” is used in the conventional sense to mean a series of amino acids, typically L-amino acids, connected one to the other, typically by peptide bonds between the a- amino and carboxyl groups of adjacent amino acids. The term “polypeptide” is used interchangeably with the terms “amino acid sequence”, “peptide” and / or “protein”. The term “residues” is used to refer to amino acids in an amino acid sequence.

[0419] The term "variant" refers to a polypeptide that has an equivalent function to the amino acid sequences described herein, but which includes one or more amino acid substitutions, insertions or deletions.

[0420] As used herein, “variant” is synonymous with “mutant” and refers to a polynucleotide or amino acid sequence which differs in comparison to the corresponding wild-type sequence. The term “wild-type” is used to mean a gene or protein having a polynucleotide or amino acid sequence respectively, which is identical with the native gene or protein respectively. The nucleic acid sequence may be an RNA or DNA sequence or a variant thereof. The term "polynucleotide" includes an RNA or DNA sequence. It may be single or double stranded. It may, for example, be genomic, recombinant, mRNA or cDNA.

[0421] This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0422] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

[0423] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0424] The terms "comprising", "comprises" and "comprised of' as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms "comprising", "comprises" and "comprised of' also include the term "consisting of.

[0425] The terms “identity” and “% sequence identity” as used herein, may refer to the proportion of nucleotides or amino acids (expressed in percent) of a contiguous nucleotide sequence or contiguous amino acid sequence respectively which across the sequence, are identical to a reference sequence. The identity is calculated by counting the number of aligned nucleobases or amino acids that are identical (a Match) between the sequence of interest and a reference sequence, and dividing that number by the total number of nucleotides amino acids respectively and multiplying by 100.

[0426] Therefore, Percentage of Identity = (Matches x 100) / Length of aligned region. Insertions and deletions are not allowed in the calculation the percentage of identity. Chemical modifications of nucleotides may be disregarded provided that the functional capacity to form Watson Crick base pairing is retained.

[0427] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0428] This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0429] The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

[0430] EXAMPLES

[0431] Example 1 - TNFR2 aqonism alleviates acute pain in the Brennan paw incision model

[0432] To evaluate the therapeutic potential of TNFR2 agonism, the impact of a mouse TNFR2- specific sc-mTNFR2-Fc surrogate molecule on acute post-operative pain was evaluated using the Brennan paw incision rodent model.

[0433] To this end, rats were anesthetized using isoflurane. Under anaesthesia, a 1-cm longitudinal incision was made through the skin and fascia. The plantaris muscle was elevated and incised longitudinally using hemostasis. Then, the wound was apposed with two sutures and the rats were allowed to recover from general anaesthesia. To assess the impact of TNFR2 agonism, rats were administered 80 minutes post incision using intravenous route with sc-mTNFR2-Fc mutein (1 mg / kg) or PBS or using oral route with the NSAID naproxen (30 mg / kg). Pain response was evaluated using the von Frey methodology. The von Frey test is based on applying short pulses of pressure that are not painful to a naive animal. To this end, the rat was placed in an enclosure and positioned on a metal mesh surface but allowed to move freely. The test began after the cessation of exploratory behaviour. The set of Von Frey monofilaments provides an approximate logarithmic scale of actual force and a linear scale of perceived intensity. When the tip of a fiber of a given length and diameter was pressed against the skin at right angles, the force of application increased as long as the researcher continues to advance the probe until the fiber bends. After the fiber bends, the probe continues to advance, causing the fiber to bend more, but without additional force being applied to the paw.

[0434] Therefore, fiber length and dimeter are directly correlated with the force applied to the hind paw. Rodents exhibit a paw withdrawal reflex when the paw is unexpectedly touched. The Touch Test Sensory Evaluator was used on the plantar surfaces of the rat’s foot. The animal indicated sensation by pulling back its paw. The minimal force needed to elevate the withdrawal reflex is considered as the value of reference. A decrease in force needed to induce withdrawal was indicative of allodynia. The von Frey test was performed on the right hind paw, on study day -2 (baseline), then on study days 0 (3 and 6 hours post treatment) and day 1 post incision.

[0435] As expected, the NSAID naproxen significantly alleviated pain 3 hours post treatment. Surprisingly, sc-mTNFR2-Fc alleviated mechanical allodynia 3 hours post administration (Fig. 1 B). In contrast to the naproxen-treated mice, which exhibited significantly increased acute pain after 24 hours, sc-mTNFR2-Fc intravenous administration non-significantly alleviated pain hypersensitivity after 24 hours. This indicates a direct analgesic effect of TNFR2 agonism on surgery induced acute pain.

[0436] Next, the therapeutic effect of subcutaneous administration of sc-mTNFR2-Fc on acute pain was investigated. Sprague-Dawley rats were therefore treated after induction of post-surgery pain using the Brennan paw incision model in an independent follow-up study.

[0437] To this end, under anaesthesia with isoflurane, the planter aspect of the left hind paw was first cleansed with 4% chlorhexidine solution. A 1 cm longitudinal incision was made 0.5 cm from the edge of the heel. The underlying plantaris muscle was elevated and incised longitudinally. Following haemostasis, the skin is apposed with two single sutures of 5-0 vicryl. The wound site was then cleansed with ethanol solution and sterile saline. Animals were returned to their home cages containing soft white post-surgical bedding. 30 minutes post-surgery animals were administered with sc-mTNFR2-Fc (5 mg / kg) or the NSAID Keterolac (10 mg / kg) via subcutaneous administration. The injection was repeated on day 1 (24 hours post-surgery).

[0438] Measurement of the paw withdrawal threshold was achieved using calibrated (force; g) von Frey monofilaments (Touch-Test Sensory Evaluator; Scientific Marketing Associates) applied to the plantar surface of the hind paw. Withdrawal threshold was determined by increasing and decreasing stimulus intensity and estimated using the Dixon’s up-down method (Dixon, 1980; Chaplan et al., 1994). Therefore, the animals were placed on an elevated mesh bottom platform with a 0.5 cm2grid to provide access to the ventral side of the hind paws. An inverted plexiglass container was placed on top of each rat and testing was performed after an initial 15-20 minute acclimatisation / habituation period. The von Frey filaments were placed perpendicular to the plantar surface of the ipsilateral hind paw, from below the mesh floor. The monofilaments were held at the position for approximately 8 seconds with enough force to cause a slight bend of the filament. Only immediate sharp withdrawal responses from the stimulus (or flinching) were considered to represent a positive response. An initial filament (4 g) was applied to the animal's paw or other target area response observed. A positive response (e.g., withdraws its paw), triggered a decrease the stimulus intensity (use of weaker filament). A negative (or lack of response), triggered an increase in the stimulus intensity (use of stronger filament). Filaments of varying intensities were applied, adjusting the intensity based on the animal's response to the previous stimulus. Each response directed the selection of the next stimulus intensity. This process continued until there was a change in response (i.e. if animal had a positive response to initial, 4 g, filament, this is a change a lack of response from weaker filaments). After this change, four more assessments occurred to conclude the measurement. The pattern of the last five responses was used to identify predetermined adjustment factors which inform the calculation of the appropriate threshold.

[0439] Baseline thresholds: Three baseline BL measurements were taken prior to treatment (on day -1 , -2 and -3). The average of the last two (day -2 and -1) readings was considered the baseline. Animals were then ranked and randomised (based on a Latin square design) to treatment groups according to PWT baseline readings. Following compound dosing, animals had von Frey measurements taken on day 0 (1 , 3 and 6 hours after dosing) and day 1 animals were injected again with Keterolac, or PBS (sc-mTNFR2-Fc group) (1 hour after dosing).

[0440] As expected, the NSAID Ketorolac significantly alleviated pain after 6 and 24 hours post treatment. Surprisingly, sc-mTNFR2-Fc alleviated mechanical allodynia also 6 and 24 hours post administration (Fig. 2B). Importantly, in contrast to Keterolac, sc-mTNFR2-Fc was not reapplied on day 1 after surgery. The delayed therapeutic effect exhibited after subcutaneous administration, as compared to intravenous administration, indicated that bioavailability of the TNFR2 agonist impacts its therapeutic effect on acute pain.

[0441] Example 2 - Route of administration impacts the therapeutic efficacy of TNFR2 agonists

[0442] To evaluate the impact of the route of administration on the therapeutic effect of TNFR2 agonism on acute pain, local intrathecal to systemic intraperitoneal (i.p.) administration was compared.

[0443] A Brennan paw incision was performed on 8-week-old male and female C57BL / 6 mice. To induce anesthesia, 4-5% isoflurane was administered, and maintained with 1.5-2% isoflurane during the procedure. Prior to the procedure, ophthalmic ointment was applied to the mice's eyes and the plantar skin was swabbed with a chlorhexidine solution. A midline longitudinal incision was made through the skin and fascia of the proximal edge of the plantar aspect of the left hind paw. The plantaris muscle was gently separated from the underlying tissue, and a 4-mm longitudinal incision was made through the midline of the muscle using a no. 11 scalpel blade without damaging the origin or insertion end of the muscle. The skin was then closed with two 6-0 PDSII sutures (Ethicon).

[0444] Mice were administered either systemic injections (10 mg / kg, i.p) or a combination of intrathecal injections (10 pg) and systemic injections (10 mg / kg, i.p) of the mouse TNFR2 agonist sc-mTNFR2-Fc. Time points for systemic administration at day -1 , day 2 and 5 after surgery were selected. Similarly, the combination of local intrathecal, administered 1 hour prior to surgery and systemic i.p. injection at day 2 and day 5 after surgery were selected. For intrathecal injections, a small patch of fur (20 x 20 mm) was shaved over the lumbar spine. The mice were acclimated to manual restraint at the pelvic girdle within a towel to minimize stress. A 30-gauge needle (attached to a 25 mL Hamilton syringe) was inserted between the L5 and L6 vertebrae, and successful entry was indicated by a tail flick. The drug or its vehicle (5 mL) was slowly injected over a period of 20 seconds into the intrathecal space, and the needle was held in place for an additional 10 seconds to prevent backflow.

[0445] Von Frey assessments were performed to evaluate mechanical allodynia at multiple time points, starting 2 hours after surgery and continuing until Day 14 post-operatively. Using a predetermined set of eight von Frey (vF) monofilaments (0.008-6 g, Stoelting) with the up- down method, the hind paw 50% mechanical withdrawal thresholds were measured (Chaplan et al., 1994). The mice were acclimated for at least 30 minutes in an opaque acrylic box positioned atop an elevated wire mesh platform. The vF hair was applied perpendicularly to the proximal region of the glabrous skin on the plantar surface of the hindpaw, adjacent to the incision site. Each trial commenced with the application of an intermediate filament (0.16 g) for 3 seconds, causing a slight bending of the filament. In the event of a positive response (i.e. , rapid withdrawal or licking of the paw within 3 seconds of removing the filament), the next smallest filament was tested. If there was a negative response, the next larger filament was tested. The trial was continued until four measurements beyond the initial change in response (i.e., no response followed by a response, or vice versa) were recorded. The 50% mechanical withdrawal threshold was calculated using the statistical method described by Dixon (1965).

[0446] As shown previously, systemic administration of the TNFR2 agonist alleviated pain within the first 24 hours after administration. Furthermore, the group treated with sc-mTNFR2-Fc continuously showed reduced pain levels throughout the observation time of 14 days postsurgery, indicating an increased TNFR2-mediated pain recovery (Fig. 3B). Interestingly, local intrathecal administration 1 hour prior surgery, followed by systemic intraperitoneal administration on days 2 and 5 post surgery was highly superior in alleviating pain compared to continuous systemic administration (Fig. 3C). The initial local administration dramatically improved the analgesic effect directly after surgery within 2 hours post-surgery. Furthermore, animals in the TNFR2 agonist treatment group recovered quicker than animals in the control treatment group. This indicated that TNFR2 agonist treatment directly promotes analgesic effects and accelerated recovery from acute pain.

[0447] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Claims

CLAIMS1 . A pharmaceutical composition comprising one or more modulators of TNF signalling for use in treating and / or preventing acute pain.

2. The pharmaceutical composition for use according to claim 1 , wherein the one or more modulators of TNF signalling each independently comprise a polypeptide, an antibody, a nucleic acid, a small molecule, or a combination thereof.

3. The pharmaceutical composition for use according to claim 1 or claim 2, wherein the one or more modulators of TNF signalling comprises a tumour necrosis factor receptor 2 (TNFR2) agonist; optionally wherein the TNFR2 agonist is a polypeptide comprising a TNFR2 binding domain comprising three TNF homology domains (THD) that specifically bind to TNFR2.

4. The pharmaceutical composition for use according to claim 3, wherein the polypeptide further comprises a multimerization domain; optionally wherein the multimerization domain is a dimerization domain, further optionally wherein the dimerization domain is an immunoglobulin Fc domain.

5. The pharmaceutical composition for use according to claim 3 or claim 4, wherein the polypeptide comprises (i) a TNFR2 binding domain comprising three TNF homology domains (THD) that specifically bind to TNFR2; and (ii) an Fc domain, wherein the Fc domain comprises the following mutations L234A, L235A, A327G, A330S and P331S.

6. The pharmaceutical composition for use according to any one of claims 3 to 5, wherein the TNFR2 agonist is a polypeptide comprising a sequence with at least 80% sequence identity to SEQ ID NO: 7.

7. The pharmaceutical composition for use according to any one of claims 1 to 6, wherein the one or more modulators of TNF signalling comprises an inhibitor of tumour necrosis factor receptor 1 (TNFR1) signalling; optionally wherein the inhibitor of TNFR1 signalling comprises a tumour necrosis factor receptor 1 (TNFR1) antagonist or a soluble TNF inhibitor.

8. The pharmaceutical composition for use according to claim 7, wherein(i) the TNFR1 antagonist comprises an antibody or fragment thereof capable of specifically binding TNFR1 and inhibiting TNFR1 signalling; optionally wherein the TNFR1 antagonist is selected from the group consisting of Atrosimab, GSK2862277, scRIantTNF, TROS and DS41; or(ii) the soluble TNF inhibitor comprises an antibody or fragment thereof capable of specifically binding and inhibiting soluble TNF; optionally wherein the soluble TNF inhibitor is selected from the group consisting of XPro1595 and SAR441566.

9. The pharmaceutical composition for use according to any preceding claim, wherein the acute pain has persisted for less than 2 months, or for less than 1 month; optionally wherein the acute pain has persisted, for less than 6 weeks, for less than 5 weeks, for less than 4 weeks, for less than 3 weeks, for less than 2 weeks, or for less than 1 week; further optionally wherein the acute pain has persisted, for less than 7 days, for less than 6 days, for less than 5 days, for less than 4 days, for less than 3 days, for less than 2 days, or for less than 1 day.

10. The pharmaceutical composition for use according to any one of claims 1 to 9, wherein:(i) the acute pain arises from injury or trauma;(ii) the acute pain arises from surgery and / or health treatment;(iii) the acute pain is associated with osteoarthritis; or(iv) the acute pain is post-operative acute pain.

11. The pharmaceutical composition for use according to any one of claims 1 to 10, wherein the pharmaceutical composition is administered to a subject:(i) systemically;(ii) subcutaneously;(iii) intravenously;(iv) intrathecally; or(v) intra-articularly; optionally by intra-articular injection.

12. A TNFR2 agonist for use in treating and / or preventing acute pain, wherein the TNFR2 agonist is administered in combination with an inhibitor of TNFR1 signalling.

13. An inhibitor of TNFR1 signalling for use in treating and / or preventing acute pain, wherein the inhibitor of TNFR1 signalling is administered in combination with a TNFR2 agonist.

14. A TNFR2 agonist and an inhibitor of TNFR1 signalling, for use in treating and / or preventing acute pain.

15. The TNFR2 agonist for use and / or the inhibitor of TNFR1 signalling for use according to any one of claims 12 to 14, wherein: (i) the inhibitor of TNFR1 signalling is a TNFRI antagonist;(ii) the TNFR2 agonist and the inhibitor of TNFR1 signalling are administered concurrently or sequentially;(iii) the TNFR2 agonist is the TNFR2 agonist comprised within the pharmaceutical composition for use according to any of claims 3-6 or 9-11 ; and / or (iv) the inhibitor of TNFR1 signalling is the inhibitor of TNFR1 signalling comprised within the pharmaceutical composition for use according to any of claims 7-11.

Images