TNF receptor 2-specific agonist fusion proteins comprising TNF homology domains and mutated FC domains

Abstract

The present disclosure relates to a tumour necrosis factor receptor 2 (TNFR2) agonist polypeptide comprising (i) a TNFR2 binding domain comprising three TNF homology domains (THD) that specifically bind to TNFR2; and (ii) an Fc domain. The disclosure further relates to a TNFR2 agonist for use in the treatment and / or prevention of - for example - chronic pain or multiple sclerosis.

Description

[0001] TNF RECEPTOR 2-SPECIFIC AGONIST FUSION PROTEINS COMPRISING TNF HOMOLOGY DOMAINS AND MUTATED FC DOMAINS

[0002] FIELD

[0003] The present disclosure relates to a tumour necrosis factor receptor 2 (TNFR2) agonist polypeptide comprising (i) a TNFR2 binding domain comprising three TNF homology domains (THD) that specifically bind to TNFR2; and (ii) an Fc domain. The disclosure further relates to a TNFR2 agonist for use in the treatment and / or prevention of - for example - chronic pain (e.g., chronic neuropathic pain) or multiple sclerosis.

[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 ak, 2000, J. Biol., Chem. 275, 32208-32213; Wajant et al, 2001, Oncogene 20, 4101-4106).

[0007] There remains a need for improved TNFR2 agonists. In particular, there remains a need for TNFR2 agonists with advantageous properties for in vivo administration and for use as medicaments.

[0008] SUMMARY

[0009] The present disclosure is based, at least in part, on the inventors’ development of TNFR2 agonist polypeptides with particularly advantageous and surprising properties. For example, the present inventors have developed TNFR2 agonist polypeptides with reduced binding to and activation of FcyRlla and beneficial in vivo properties. Further, the present TNFR2 agonist polypeptides have been demonstrated to have notable efficacy on disease-relevant pathways, including direct effects on supraspinal neurons. Without wishing to be bound by theory, the inventors consider that the direct effects on supraspinal neurons - distinct from previously reported effects on Treg cells - may facilitate long-term and preventative effects of the present TNFR2 agonists. Accordingly, the present disclosure provides a tumour necrosis factor receptor 2 (TNFR2) agonist polypeptide comprising (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.

[0010] Suitably, the Fc domain may not comprise a proline at position 233. Suitably, the Fc domain may comprise a glycine at position 236. Suitably, the Fc domain (i) may not comprise a proline at position 233 and (ii) may comprise a glycine at position 236.

[0011] Suitably, the Fc domain may comprise a sequence with at least 85% sequence identity to SEQ ID NO: 1.

[0012] Suitably, each THD may independently 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,

[0013] Q84N / A141 I / E142G / S143D, A141 H / E142S / S143D, A141 H / S143D,

[0014] L25V / A141 D / E142D / S143D, A141 N / E142D / S143D, A141T / E142S / S143D,

[0015] A141Q / E142D / S143D, A141T / E142D / S143D, A141 D / E142G / S143D, A141 D / S143D,

[0016] 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, in the corresponding position of 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.

[0017] Suitably, each THD may comprise an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 2.

[0018] Suitably, the THD according to the disclosure may comprise the mutations D139N and A141 R at the position corresponding to the sequence of SEQ ID NO: 2.

[0019] Suitably, one or more THD may comprise or consist of SEQ ID NO: 3 or an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 3. Suitably, each THD may comprise or consist of SEQ ID NO: 3 or an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 3.

[0020] Suitably, one THD may comprise or consist of SEQ ID NO: 4 or an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 4.

[0021] Suitably, the first and second THD may be directly linked without any intervening peptide linkers. Suitably, the second and third THD may be directly linked without any intervening peptide linkers. Suitably, (i) the first and second THD and (ii) the second and third THD may be directly linked without any intervening peptide linkers.

[0022] Suitably, the TNFR2 binding domain may comprise or consist of a sequence with at least 90% sequence identity to SEQ ID NO: 5.

[0023] Suitably, the TNFR2 binding domain may be joined to the Fc domain by a flexible linker consisting of 3-20 amino acids.

[0024] Suitably, the flexible linker may be a Ser-Gly linker. Suitably, the flexible linker may be a Ser- Gly linker having the sequence: GGSGGGGSGG (SEQ ID NO: 6).

[0025] Suitably, the TNFR2 agonist polypeptide may comprise a sequence with at least 90% sequence identity to SEQ ID NO: 7.

[0026] The present disclosure provides a TNFR2 agonist multimer in the form of a dimer comprising two TNFR2 agonist polypeptides, wherein each TNFR2 agonist polypeptide is independently a TNFR2 agonist polypeptide according to the disclosure.

[0027] Suitably, each TNFR2 agonist polypeptide in the dimer may be identical.

[0028] The present disclosure provides a polynucleotide encoding a TNFR2 agonist polypeptide according to the disclosure.

[0029] The present disclosure provides a vector comprising a polynucleotide according to the disclosure.

[0030] The present disclosure provides a pharmaceutical composition comprising a TNFR2 agonist polypeptide according to the disclosure, TNFR2 agonist multimer according to the disclosure, a polynucleotide according to the disclosure, or a vector according to the disclosure. The present disclosure provides a TNFR2 agonist comprising three TNF homology domains (THD) that specifically bind to TNFR2 and an Fc domain for use in treating and / or preventing chronic pain (e.g., chronic neuropathic pain) or multiple sclerosis.

[0031] The present disclosure provides a method for treating and / or preventing chronic pain (e.g., chronic neuropathic pain) or multiple sclerosis comprising administering a TNFR2 agonist to a subject in need thereof, wherein the TNFR2 agonist comprises three TNF homology domains (THD) that specifically bind to TNFR2 and an Fc domain.

[0032] The present disclosure provides a use of a TNFR2 agonist comprising three TNF homology domains (THD) that specifically bind to TNFR2 and an Fc domain in the manufacture of a medicament for treating and / or preventing chronic pain (e.g., chronic neuropathic pain) or multiple sclerosis.

[0033] Suitably, the TNFR2 agonist may be a TNFR2 agonist polypeptide according to the disclosure, TNFR2 agonist multimer according to the disclosure, a polynucleotide according to the disclosure, a vector according to the disclosure, or provided as a pharmaceutical composition according to the disclosure.

[0034] Accordingly, the present disclosure provides a TNFR2 agonist polypeptide according to the disclosure, TNFR2 agonist multimer according to the disclosure, a polynucleotide according to the disclosure, a vector according to the disclosure, or a pharmaceutical composition according to the disclosure, for use in treating and / or preventing chronic pain (e.g., chronic neuropathic pain) or multiple sclerosis.

[0035] Accordingly, the present disclosure provides a method for treating and / or preventing chronic pain (e.g., chronic neuropathic pain) or multiple sclerosis comprising administering a TNFR2 agonist polypeptide according to the disclosure, TNFR2 agonist multimer according to the disclosure, a polynucleotide according to the disclosure, a vector according to the disclosure, or a pharmaceutical composition according to the disclosure, to a subject in need thereof.

[0036] Accordingly, the present disclosure provides a use of a TNFR2 agonist polypeptide according to the disclosure, TNFR2 agonist multimer according to the disclosure, a polynucleotide according to the disclosure, a vector according to the disclosure, or a pharmaceutical composition according to the disclosure, in the manufacture of a medicament for treating and / or preventing chronic pain (e.g., chronic neuropathic pain) or multiple sclerosis.

[0037] In some embodiments, the chronic pain is associated with osteoarthritis. Accordingly, the present disclosure provides a TNFR2 agonist polypeptide according to the disclosure, TNFR2 agonist multimer according to the disclosure, a polynucleotide according to the disclosure, a vector according to the disclosure, or a pharmaceutical composition according to the disclosure, for use in treating and / or preventing chronic pain associated with osteoarthritis.

[0038] Accordingly, the present disclosure provides a method for treating and / or preventing chronic pain associated with osteoarthritis, comprising administering a TNFR2 agonist polypeptide according to the disclosure, TNFR2 agonist multimer according to the disclosure, a polynucleotide according to the disclosure, a vector according to the disclosure, or a pharmaceutical composition according to the disclosure, to a subject in need thereof.

[0039] In some embodiments, the TNFR2 agonist, TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition according to the disclosure is administered to a subject systemically.

[0040] In some embodiments, the TNFR2 agonist, TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition according to the disclosure is administered to a subject subcutaneously.

[0041] In some embodiments, the TNFR2 agonist, TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition according to the disclosure is administered to a subject intravenously.

[0042] In some embodiments, the TNFR2, TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition according to the disclosure is administered to a subject intrathecally.

[0043] In some embodiments, the TNFR2 agonist, TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition according to the disclosure is administered to a subject intraarticularly.

[0044] In some embodiments, the intraarticular administration is by intraarticular injection.

[0045] Suitably, the TNFR2 agonist induces TNFR2 signalling in neurons.

[0046] Suitably, the neurons are supraspinal neurons, preferably Nex+ supraspinal neurons. Suitably, the TNFR2 agonist is administered to the subject systemically. Suitably, the TNFR2 agonist is administered to the subject systemically and induces TNFR2 signalling in neurons, such as supraspinal neurons, optionally Nex+ supraspinal neurons.

[0047] BRIEF DESCRIPTION OF THE FIGURES

[0048] Figure 1 : Biochemical and functional characterization (A) schematic showing the architecture of scTNFR2-Fc[LALA-Aa]. (B) scTNFR2-Fc[LALA-Aa] was purified by protein A affinity chromatography followed by preparative size exclusion chromatography (SEC). 5 pg SCTNFR2- Fc[LALA-Aa] and a protein standard were analyzed by analytical SEC. Dotted lines indicate the molecular weight of standard proteins in kD. (C) 2 pg scTNFR2-Fc[LALA-Aa] and a protein standard were dissolved by a 4-12% Criterion TGX Stain-Free gel under non-reducing and reducing conditions followed by protein visualization using UV light activation. (D) Binding of scTNFR2-Fc[LALA-Aa] and recombinant human TNF (rhTNF) to immobilized TNFR2 was analyzed by enzyme linked immunosorbent assay (ELISA). Data are shown as % binding relative to the highest concentration of scTNFR2-Fc[LALA-Aa] n=3, mean±SD.

[0049] Figure 2: Binding of TNFR2 agonists to FcyRlla - Binding of (A) FcyRlla (H 131 ) to immobilized SCTNFR2-FC fusion proteins or (B) SCTNFR2-FC fusion proteins to immobilized FcyRlla (R131) was analyzed by ELISA. Data are shown as % binding relative to the highest concentration of SCTNFR2-FC[WT], n=1 , mean±range of technical duplicates.

[0050] Figure 3: Affinity of TNFR2 agonists to FcyRlla - (A) Recombinant FcyRlla (H 131 ) receptor was captured on a C1 sensor chip. Affinity of rituximab and scTNFR2-Fc[Aab] to FcyRlla (H131) was quantified using SPR analysis. Data were processed applying a Langmuir 1 :1 binding model including bulk refractive index (Rl) contribution using the Biacore T200 Evaluation Software version 3.2. (B) Recombinant rituximab or scTNFR2-Fc[LALA-Aa] was captured on a C1 sensor chip. Affinity of FcyRlla (H 131 ) to ligands was quantified using SPR analysis. Data were processed applying a Langmuir 1 :1 binding model including bulk refractive index (Rl) contribution using the Biacore T200 Evaluation Software version 3.2.

[0051] Figure 4: Activation of FcyRlla by TNFR2 agonists - Activation of FcyRlla was quantified using transgenic BW5147 reporter cell lines expressing chimeric FcyR2a haplotypes (A) H131 and (B) R131 using ELISA technology; mean ± SD n=2. (C) Activation of FcyRlla using the FcyRlla-H (H131) expressing reporter cells (Promega Cat.# G9901) was measured by luminescence; mean ± range of two technical replicates n=1. Figure 5: sc-mTNFR2-Fc induced alleviation of motor disease in EAE mice - (A) Female and male EAE mice were treated using the TNFR2 agonist prototype p53-scTNFR2 by i.p. injection at days 10, 13 and 16 post immunization. Motor behaviour was scored daily. B) Female and male EAE mice were treated using sc-mTNFR2-Fc by i.p. injection at days 10, 13 and 16 post immunization. Motor behaviour was scored daily.

[0052] Figure 6: sc-mTNFR2-Fc treatment impacts development of motor disease in EAE mice - Female and male EAE mice were treated using (A,B) the TNFR2 agonist prototype p53- SCTNFR2 or (C,D) sc-mTNFR2-Fc by i.p. injection at days 10, 13 and 16 post immunization. Motor behaviour was scored daily. Shown is the incidence of motor disease development and the day of motor disease onset.

[0053] Figure 7: sc-mTNFR2-Fc agonist treatment alleviates pain in EAE mice - Female and male EAE mice were treated using (A) the TNFR2 agonist prototype p53-scTNFR2 or (B) sc- mTNFR2-Fc by i.p. injection at days 10, 13 and 16 post immunization. Pain hypersensitivity was determined using the Von Frey test for mechanical allodynia.

[0054] Figure 8: sc-mTNFR2-Fc treatment alleviates pain after SCI - Female mice were undergoing moderate spinal cord contusion injury (SCI). Treatment with (A) the TNFR2 agonist prototype EHD2-SCTNFR2 or (B) sc-mTNFR2-Fc was started 1 week post-injury on days 7, 10 and 13. Pain hypersensitivity was determined using the Von Frey test for mechanical allodynia.

[0055] Figure 9: sc-mTNFR2-Fc treatment alleviates pain after SCI - Female mice were undergoing moderate spinal cord contusion injury (SCI). Treatment with (A) the TNFR2 agonist prototype EHD2-sc-mTNFR2 or (B) sc-mTNFR2-Fc was started 1-week post-injury on days 7, 10 and 13. Pain behaviour was determined using the mechanical conflict avoidance test.

[0056] Figure 10: sc-mTNFR2-Fc treatment promotes pain recovery after peripheral nerve injury via microglial-TNFR2 - Tamoxifen was administered to animals to induce recombination resulting in deletion of TNFR2 in CX3CR1-expressing cells. TNFR2fl / flmice were used as controls. Mice were undergoing chronic constriction injury (CCI) at week 0. Treatment with sc-mTNFR2-Fc was started 1-week post-injury on days 7, 10 and 13. Pain hypersensitivity was determined using the Von Frey test for mechanical allodynia.

[0057] Figure 11 : sc-mTNFR2-Fc agonist treatment promotes pain recovery after peripheral nerve injury via neuronal TNFR2 - Tamoxifen was administered to animals to induce recombination resulting in deletion of TNFR2 in Nex-expressing cells. TNFR2fl / flmice were used as controls. Mice were undergoing chronic constriction injury (CCI) at week 0. Treatment with different doses of sc-mTNFR2-Fc was started 1-week post-injury on days 7, 10 and 13. Pain hypersensitivity was determined using the Von Frey test for mechanical allodynia.

[0058] Figure 12: Activation of FcyRllla by TNFR2 agonists. (A) Hela cells were stably transfected with TNFR2 expression constructs. Surface expression of TNFR2 was quantified by flow cytometry. (B) Activation of FcyRllla was quantified using transgenic U937-HiBiT reporter cells and TNFR2-overexpressing Hela-TNFR2 cells; mean ± SD n=3.

[0059] Figure 13: sc-mTNFR2-Fc treatment alleviates pain in a chemically induced OA model. The experimental outline is shown in (A). Rats were locally injected with monoiodoacetate (MIA) to induce chemical destruction of joint tissue. Systemic s.c. application of the TNFR2 agonist sc-mTNFR2-Fc or the control Pregabalin was started 7 days post-MIA injection and repeated twice weekly until end of observation period. (B) Pain hypersensitivity was determined using the static weight bearing test (n=10 animals / group + / - SEM).

[0060] Figure 14: sc-mTNFR2-Fc treatment alleviates pain in post-traumatic OA model after local application. The experimental outline is shown in (A). Mice underwent DMM surgery of the right knee leading to development of OA pain. Local i.a. application of the TNFR2 agonist sc- mTNFR2-Fc was started 4 weeks post-MIA injection and repeated once weekly until end of observation period. (B) Pain hypersensitivity was determined using the Von Frey test for mechanical allodynia (n=4 animals / group + / - SEM).

[0061] Figure 15: sc-mTNFR2-Fc treatment alleviates pain in post-traumatic OA model after systemic application. The experimental outline is shown in (A). Mice underwent DMM surgery of the right knee leading to development of OA pain. Systemic i.p. application of the TNFR2 agonist sc-mTNFR2-Fc was started 4 weeks post-MIA injection and repeated once weekly until end of observation period. (B) Pain hypersensitivity was determined using the Von Frey test for mechanical allodynia (n=14-16 animals / group + / - SEM).

[0062] Figure 16: scTNFR2-Fc treatment alleviates motor disease and pain in EAE model of multiple sclerosis. The experimental outline is shown in (A). EAE was induced in huTNFR2-k / i mice, where the endogenous mouse TNFR2 is exchanged with a chimeric TNFR2 consisting of the extracellular human TNFR2 domain fused to the transmembrane and intracellular domain of mouse TNFR2. Mice were treated using the TNFR2 agonist SCTNFR2-FC by s.c. injection starting day 10, 13 and 16 post immunization. Motor behaviour was scored daily. (B) Shown is the incidence of motor disease development and the day of motor disease onset. (C) Pain hypersensitivity was determined using the Von Frey test for mechanical allodynia (n=11-12 animals / group + / - SEM).

[0063] DETAILED DESCRIPTION

[0064] TNF / TNFR2

[0065] 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 ak, 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.

[0066] TNF is synthesized as a trimeric type 2 transmembrane protein (tmTNF) that can be proteolytical ly 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.

[0067] 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. 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).

[0068] 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).

[0069] 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.

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

[0071] 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.

[0072] TUMOUR NECROSIS FACTOR RECEPTOR 2 (TNFR2) AGONIST

[0073] The present disclosure provides a TNFR2 agonist polypeptide comprising (i) a TNFR2 binding domain comprising three TNF homology domains (THDs) that specifically bind to TNFR2; and (ii) an Fc domain, wherein the Fc domain comprises the following mutations L234A, L235A, A327G, A330S and P331S.

[0074] The present TNFR2 agonist polypeptide may be capable of inducing signalling via TNFR2.

[0075] 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.

[0076] TNF homology domain (THD)

[0077] The present TNFR2 agonist polypeptide comprises a TNFR2 binding domain comprising three TNF homology domains (THDs) that specifically bind to TNFR2. In other words, it will be understood that three THDs (e.g. a first, second, and third THD) form the TNFR2 binding domain of the TNFR2 agonist polypeptide.

[0078] The term THD as used in the present specification 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.

[0079] 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.

[0080] Suitably, one or more THD may independently comprise or consist of the amino acid sequence according to SEQ ID NO: 2.

[0081] 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.

[0082] Suitably, each THD may independently comprise or consist of the amino acid sequence according to SEQ ID NO: 2.

[0083] SEQ ID NO: 2 - THD sequence without mutations

[0084] SRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPST HVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPD YLDFAESGQVYFGI IAL

[0085] In some embodiments of the present disclosure, the three THDs of the TNFR2 agonist polypeptide of the present disclosure are identical in their amino acid sequence. 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).

[0086] Suitably, one or more, or each, THD according to the disclosure may independently comprise one or more mutations. Such a THD may be considered as a variant.

[0087] Suitably, one or more, or each, THD may independently comprise a contiguous amino acid sequence as outlined in W02020 / 260368 (incorporated herein by reference).

[0088] Suitably, 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,

[0089] 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.

[0090] It will be understood that a “set of mutations” as used above refers to a combination of mutations indicated by the use of 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.

[0091] 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 W02020 / 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.

[0092] SEQ ID NO. 5 of WQ2020 / 260368 is presented below as SEQ ID NO: 8.

[0093] SEQ ID NO: 8 - SEQ ID NO. 5 of WQ2020 / 260368 (positions 77 to 233 are underlined)

[0094] MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEFPRDLS L I SPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYS QVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEK GDRLSAEINRPDYLDFAESGQVYFGIIAL

[0095] 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 WQ2020 / 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.

[0096] SEQ ID NO: 9 - sequence of amino acids from position 77 to 233 of SEQ ID NO. 5 of WQ2020 / 260368 (SEQ ID NO: 8 of the present disclosure)

[0097] VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQG CPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEI NRPDYLDFAESGQVYFGIIAL

[0098] Suitably, 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,

[0099] D143S, E146Q, E146H, E146K, A145R / S147T, Q88N / T89S / A145S / E146A / S147D,

[0100] Q88N / A145I / E146G / S147D, A145H / E146S / S147D, A145H / S147D,

[0101] L29V / A145D / E146D / S147D, A145N / E146D / S147D, A145T / E146S / S147D,

[0102] A145Q / E146D / S147D, A145T / E146D / S147D, A145D / E146G / S147D,

[0103] 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. 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.

[0104] In the context of the present disclosure, the mutations may be described by reference to SEQ ID NO: 2.

[0105] Suitably, 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, Q84N / A141 I / E142G / S143D,

[0106] A141 H / E142S / S143D, A141 H / S143D, L25V / A141 D / E142D / S143D, A141 N / E142D / S143D, A141T / E142S / S143D, A141Q / E142D / S143D, A141T / E142D / S143D, A141 D / E142G / S143D, A141 D / S143D, 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.

[0107] Suitably, one or more THD according to the disclosure 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.

[0108] Suitably, each THD according to the disclosure 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.

[0109] 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.

[0110] In some embodiments, one or more THD according to the disclosure 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. Suitably, one or more THD according to the disclosure 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. Suitably, one or more THD according to the disclosure may independently consist of the amino acid sequence according to SEQ ID NO: 3. It will be understood that regardless of the % sequence identity, the amino acid sequence of the one or more THD will always include the D139N and A141 R mutations.

[0111] In some embodiments, each THD according to the disclosure 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. Suitably, each THD according to the disclosure 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. Suitably, each THD according to the disclosure may consist of the amino acid sequence according to SEQ ID NO: 3. It will be understood that regardless of the % sequence identity, the amino acid sequence of each THD will always include the D139N and A141 R mutations.

[0112] SEQ ID NO: 3 - THD sequence with D139N and A141 R

[0113] SRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPST HVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPD YLNFRESGQVYFGI IAL

[0114] Without wishing to be bound by theory, the D139N and A141 R mutations may confer TNFR2 selectivity.

[0115] In some embodiments, the three THDs of the TNFR2 binding domain of the present disclosure 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.

[0116] In some embodiments, at least one of the THDs may comprise an additional “S” at the N terminus. Suitably, 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.

[0117] In some embodiments, only one of the THD (e.g. the first THD) may comprise an additional “S” at the N terminus.

[0118] Suitably, 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. Suitably, each THD of the TNFR2 binding domain according to the disclosure 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.

[0119] 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.

[0120] In some embodiments, a THD according to the disclosure 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. Suitably, a THD according to the disclosure 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. Suitably, a THD according to the disclosure may consist of the amino acid sequence according to SEQ ID NO: 4. It will be understood that regardless of the % sequence identity, the amino acid sequence of the THD will always include the D139N and A141 R mutations.

[0121] SEQ ID NO: 4 - THD sequence with D139N and A141 R and additional S

[0122] SSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPS THVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRP DYLNFRESGQVYFGIIAL

[0123] Suitably, the first THD of the TNFR2 binding domain according to the disclosure 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 disclosure may each comprise or consist of the amino acid sequence according to SEQ ID NO: 3 or a variant thereof.

[0124] 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.

[0125] In other embodiments, the first and second THD and / or second and third THD may be linked by intervening peptide linkers. Suitably, the intervening peptide linkers may be independently selected from a peptide which consists of XC-XL-XN; wherein:

[0126] Xcis selected from the group consisting of A, A-L, L, preferably A-L;

[0127] XL is absent or is an amino acid linker consisting of 1-11 amino acids, XNis absent or selected from the group consisting of K, D-K, S-D-K, P-S-D-K, T-P-S- D-K, R-T-P-S-D-K, S-R-T-P-S-D-K, S-S-R-T-P-S-D-K, T-K, S-T-K, H-S-T-K, A-H-S-T- K, L-A-H-S-T-K, H-L-A-H-S-T-K, L-H-L-A-H-S-T-K. Suitably, each intervening peptide linker may be A-L.

[0128] 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.

[0129] 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. Suitably, the TNFR2 binding domain according to the disclosure 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. Suitably, the TNFR2 binding domain according to the disclosure may consist of the amino acid sequence according to SEQ ID NO: 5. It will be understood that regardless of the % sequence identity, the amino acid sequence of the TNFR2 binding domain will always include the D139N and A141 R mutations.

[0130] SEQ ID NO: 5 - illustrative full trimeric THD TNFR2 binding domain

[0131] SSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPS THVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRP DYLNFRESGQVYFGIIALSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEG LYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGG VFQLEKGDRLSAEINRPDYLNFRESGQVYFGI IALSRTPSDKPVAHVVANPQAEGQLQWLNRRANALL ANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRET PEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLNFRESGQVYFGI IAL

[0132] Fc domain

[0133] 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.

[0134] The present TNFR2 agonist polypeptide comprises an Fc domain, wherein the Fc domain comprises the following mutations L234A, L235A, A327G, A330S and P331S.

[0135] In some embodiments, the Fc domain may not comprise a proline at position 233. In some embodiments, the Fc domain may comprise a glycine at position 236. In some embodiments, the Fc domain (i) may not comprise a proline at position 233 and (ii) may comprise a glycine at position 236.

[0136] Suitably, 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 1 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.

[0137] Suitably, 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).

[0138] Suitably, 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.

[0139] Table 1 :

[0140] Suitably, the Fc domain of the TNFR2 agonist polypeptide may comprise 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 has “A” at position 14, “A” at position 15, “G” at position 107, “S” at position 110 and “S” at position 111.

[0141] 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 IgG 1 positions and residues in the Ell numbering system / as described in Armour et al.

[0142] It will be understood that the Fc domain of the TNFR2 agonist polypeptide, comprising the L234A, L235A, A327G, A330S and P331S mutations, may be defined as a “Fc[LALA-Aa]”, as used in the appended Examples.

[0143] It will also be understood that the Fc domain of the TNFR2 agonist polypeptide, 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]”, as used in the appended Examples.

[0144] In some embodiments, the Fc domain according to the disclosure 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. Suitably, the Fc domain according to the disclosure 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. Suitably, the Fc domain according to the disclosure may consist of the amino acid sequence according to SEQ ID NO: 1. It will be understood that regardless of the % sequence identity, the amino acid sequence of the Fc domain will always include the L14A, L15A, A107G, A110S and P111S mutations relative to the sequence of SEQ ID NO: 1 , or the mutations at corresponding positions (e.g. L234A, L235A, A327G, A330S and P331S).

[0145] SEQ ID NO: 1

[0146] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPG

[0147] (L14A, L15A, A107G, A110S and P111S are shown in bolded and underlined in the above sequence)

[0148] 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]”, as used in the appended Examples.

[0149] 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, as demonstrated in the appended Examples, the Fc domain (i.e. “Fc[LALA-Aa]”) of the TNFR2 agonist polypeptide of the disclosure (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 P111 S mutations. Suitably, 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).

[0150] 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.

[0151] Linker

[0152] 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.

[0153] Suitably, the linker may be a peptide linker. Suitable, 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.

[0154] 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.

[0155] Suitably, 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. Suitably, 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.

[0156] Suitable linkers will be known in the art and may be used in the context of the present disclosure.

[0157] In some embodiments, the flexible linker may be a Ser-Gly linker. Suitably, 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)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 disclosure. In some embodiments, the flexible linker may be a Ser-Gly linker of 8-15 amino acids in length. In some embodiments, the flexible linker may comprise or consist of the amino acid sequence: GGSGGGGSGG (SEQ ID NO: 6).

[0158] TNFR2 agonist polypeptide

[0159] The present TNFR2 agonist 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.

[0160] Suitably, the TNFR2 agonist polypeptide according to the disclosure may be considered as a fusion protein.

[0161] In some embodiments, the TNFR2 agonist polypeptide has the following structure in a N- terminus to C-terminus orientation:

[0162] TNFR2 binding domain - linker - Fc domain.

[0163] In some embodiments, the TNFR2 agonist polypeptide comprises the following amino acid sequences in a N-terminus to C-terminus orientation:

[0164] SEQ ID NO: 5 - SEQ ID NO: 6 - SEQ ID NO: 1

[0165] 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.

[0166] In some embodiments, the TNFR2 agonist polypeptide 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. Suitably, the TNFR2 agonist polypeptide according to the disclosure 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. For example, the TNFR2 agonist polypeptide according to the disclosure 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. The TNFR2 agonist polypeptide according to the disclosure may comprise an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 7. Suitably, the TNFR2 agonist polypeptide according to the disclosure may comprise the amino acid sequence according to SEQ ID NO: 7. Suitably, the TNFR2 agonist polypeptide according to the disclosure may consist of the amino acid sequence according to SEQ ID NO: 7. Suitably, each of the three THDs of the TNFR2 agonist polypeptide may comprise the D139N and A141 R mutations. Suitably, regardless of the % sequence identity, the amino acid sequence of each of the THDs may include the D139N and A141 R mutations, and the amino acid sequence of the Fc domain will include the L234A, L235A, A327G, A330S and P331S mutations.

[0167] SEQ ID NO: 7 (full length molecule)

[0168] SSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPS THVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRP DYLNFRESGQVYFGIIALSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEG LYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGG VFQLEKGDRLSAEINRPDYLNFRESGQVYFGI IALSRTPSDKPVAHVVANPQAEGQLQWLNRRANALL ANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRET PEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLNFRESGQVYFGI IALGGSGGGGSGGDKTHTC PPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQ

[0169] VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG

[0170] TNFR2 AGONIST MULTIMER

[0171] The TNFR2 agonist polypeptide according to the disclosure may combine with one or more other TNFR2 agonist polypeptide(s) according to the disclosure to form a multimeric molecule.

[0172] Suitably, the multimeric molecule may be referred to as a TNFR2 agonist multimer.

[0173] It will be understood that each TNFR2 agonist polypeptide according to the disclosure can be considered as a TNFR2 agonist monomer.

[0174] 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.

[0175] Suitably, the TNFR2 agonist multimer may be in the form of a dimer.

[0176] Suitably, 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 polypeptide may bind to the Fc domain of the other TNFR2 agonist polypeptide. 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.

[0177] Accordingly, the present disclosure provides a TNFR2 agonist multimer in the form of a dimer comprising two TNFR2 agonist polypeptides, wherein each TNFR2 agonist polypeptide is independently a TNFR2 agonist polypeptide according to the disclosure.

[0178] Suitably, each TNFR2 agonist polypeptide in the dimer may be identical (e.g. by having the same amino acid sequence).

[0179] Without wishing to be bound by theory, it is also considered that the advantageous effects of the TNFR2 agonist polypeptide of the disclosure 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 polypeptide of the disclosure. In particular, the dimer of the TNFR2 agonist polypeptide of the disclosure is considered to unexpectedly provide improved efficacy in vivo, as demonstrated in the appended Examples, compared to known TNFR2 agonist molecules of the prior art comprising higher orders of THD domains, such as tetrameric TNFR2 agonist molecules comprising p53.

[0180] POLYNUCLEOTIDE

[0181] The present disclosure provides a polynucleotide comprising a nucleic acid sequence which encodes the TNFR2 agonist polypeptide of the present disclosure. The present disclosure also provides one or more polynucleotides encoding a TNFR2 agonist multimer in the form of a dimer comprising two TNFR2 agonist polypeptides, wherein each TNFR2 agonist polypeptide is independently a TNFR2 agonist polypeptide according to the disclosure.

[0182] As used herein, the terms “polynucleotide”, “nucleotide”, and “nucleic acid” are intended to be synonymous with each other.

[0183] 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 disclosure. Therefore multiple polynucleotides are envisaged, each with a different nucleic acid sequence but which encodes a polypeptide according to the disclosure or a further polypeptide as described herein. It is possible to design and produce such nucleic acid sequences without difficulty. 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.

[0184] The polynucleotide may be codon optimised for production in the host cell of choice.

[0185] In some embodiments, the polynucleotide encoding the TNFR2 agonist polypeptide of the present disclosure is a DNA polynucleotide.

[0186] In some embodiments, the polynucleotide encoding the TNFR2 agonist polypeptide of the present disclosure is a RNA polynucleotide.

[0187] In some embodiments, the polynucleotide encoding the TNFR2 agonist polypeptide of the present disclosure is a mRNA polynucleotide.

[0188] In some embodiments, the polynucleotide encoding the TNFR2 agonist polypeptide of the present disclosure is a cDNA polynucleotide.

[0189] 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.

[0190] Suitably, the nucleic acid sequence may be operably linked to a heterologous sequence, such as a promoter or regulatory sequence, forming an expression cassette.

[0191] The expression cassette may comprise one or more control sequences. Control sequences are sequences that control and regulate transcription and, where appropriate, the translation of the TNFR2 agonist polypeptide of the present disclosure, 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.

[0192] 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 disclosure 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 disclosure 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).

[0193] The promoter may be a constitutive promoter. The promoter may be a tissue specific promoter.

[0194] VECTOR

[0195] The present disclosure provides a vector which comprises the polynucleotide according to the present disclosure.

[0196] Accordingly, the vector may comprise a polynucleotide comprising a nucleic acid sequence which encodes the TNFR2 agonist polypeptide of the present disclosure.

[0197] Such a vector may be used to introduce the nucleic acid sequence into a cell so that the cell expresses and / or produces the TNFR2 agonist polypeptide of the present disclosure and / or the TNFR2 agonist multimer of the present disclosure.

[0198] 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.

[0199] The vector according to the disclosure may be any agent capable of delivering a polynucleotide to a cell and / or maintaining a polynucleotide in a cell. Suitably, the vector may be selected from the list consisting of: viral vectors, plasmids, naked nucleic acids, transposon- based vectors, nucleic acids complexed with polypeptide or other molecules, and nucleic acids immobilised onto solid phase particles.

[0200] Suitably, the vector may be a plasmid or a viral vector. Suitably, the vector may be a retroviral vector or a lentiviral vector.

[0201] The vector may be capable of transfecting or transducing a cell.

[0202] Suitably, a cell may comprise a polynucleotide according to the disclosure or a vector according to the disclosure. The cell may be capable of producing and / or expressing the TNFR2 agonist polypeptide of the present disclosure and / or the TNFR2 agonist multimer of the present disclosure. 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.

[0203] Suitably, the polynucleotide according to the disclosure or vector according to the disclosure may, for example, be introduced into a cell by transduction or transfection in vitro or ex vivo.

[0204] The cell is then capable of expressing and / or producing the TNFR2 agonist polypeptide of the present disclosure and / or the TNFR2 agonist multimer of the present disclosure, when the cell is cultured under suitable conditions. The TNFR2 agonist polypeptide of the present disclosure and / or the TNFR2 agonist multimer of the present disclosure can be harvested from the cell or supernatant of the cell.

[0205] Accordingly, described herein is a method of making the TNFR2 agonist polypeptide of the present disclosure and / or the TNFR2 agonist multimer of the present disclosure, which comprises the steps of (i) introducing a polynucleotide according to the disclosure or a vector according to the disclosure into a cell as described above; (ii) culturing the cell under suitable conditions to express and / or produce the TNFR2 agonist polypeptide of the present disclosure and / or the TNFR2 agonist multimer of the present disclosure; and (iii) harvesting the TNFR2 agonist polypeptide and / or the TNFR2 agonist multimer.

[0206] PHARMACEUTICAL COMPOSITION

[0207] The present disclosure provides a pharmaceutical composition comprising the TNFR2 agonist polypeptide of the present disclosure. Suitably, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant, salt, and optionally one or more further pharmaceutically active polypeptides and / or compounds. Suitably, the pharmaceutical composition according to the disclosure may comprise one or more compounds, components and / or active agents, in addition to the TNFR2 agonist polypeptide according to the present disclosure. Suitably, the pharmaceutical composition may comprise the TNFR2 agonist polypeptide according to the present disclosure in combination with one or more TNFR1 antagonist(s).

[0208] The present disclosure provides a pharmaceutical composition comprising the TNFR2 agonist multimer of the present disclosure. Suitably, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant, salt, and optionally one or more further pharmaceutically active polypeptides and / or compounds.

[0209] Suitably, the pharmaceutical composition according to the disclosure may comprise one or more compounds, components and / or active agents, in addition to the TNFR2 agonist multimer according to the present disclosure. Suitably, the pharmaceutical composition may comprise the TNFR2 agonist multimer according to the present disclosure in combination with one or more TNFR1 antagonist(s).

[0210] The present disclosure provides a pharmaceutical composition comprising the polynucleotide according to the present disclosure. Suitably, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant, salt, and optionally one or more further pharmaceutically active polypeptides and / or compounds.

[0211] Suitably, the pharmaceutical composition according to the disclosure may comprise one or more compounds, components and / or active agents, in addition to the polynucleotide according to the present disclosure. Suitably, the pharmaceutical composition may comprise the polynucleotide according to the present disclosure in combination with one or more TNFR1 antagonist(s).

[0212] The present disclosure provides a pharmaceutical composition comprising the vector according to the present disclosure. Suitably, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant, salt, and optionally one or more further pharmaceutically active polypeptides and / or compounds.

[0213] Suitably, the pharmaceutical composition according to the disclosure may comprise one or more compounds, components and / or active agents, in addition to the vector according to the present disclosure. Suitably, the pharmaceutical composition may comprise the vector according to the present disclosure in combination with one or more TNFR1 antagonist(s). Pharmaceutical compositions typically should be sterile and stable under the conditions of manufacture and storage. The pharmaceutical composition according to the disclosure may be produced using current good manufacturing practices (CGMP).

[0214] 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.

[0215] 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 disclosure. Semisolid compositions that can be used in the context of the disclosure comprise emulsion, suspension, creams, lotions, gels, globules, buccal tablets and suppositories.

[0216] 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.

[0217] A sterile saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously.

[0218] 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. 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 disclosure 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.

[0219] Suitably, the salt may comprise a metal cation, such as a sodium salt or a potassium salt.

[0220] Suitably, the pharmaceutical composition may comprise an aqueous diluent or solvent. Suitably, the aqueous diluent or solvent may be a phosphate buffered saline solution, such as a sterile phosphate buffered saline solution.

[0221] In some embodiments, the pharmaceutical composition according to the disclosure 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.

[0222] Suitably, the pharmaceutical composition according to the disclosure may be in a form suitable for intravenous infusion. In some embodiments, the pharmaceutical composition according to the disclosure may be in a form suitable for intravenous administration. Suitably, the pharmaceutical composition according to the disclosure may be administered intravenously.

[0223] In some embodiments, the pharmaceutical composition according to the disclosure may be in a form suitable for intraparenteral administration. In some embodiments, the pharmaceutical composition according to the disclosure may be administered intraparenterally. In some embodiments, the pharmaceutical composition according to the disclosure may be in a form suitable for systemic administration. In some embodiments, the pharmaceutical composition according to the disclosure may be administered systemically.

[0224] In some embodiments, the pharmaceutical composition according to the disclosure may be in a form suitable for subcutaneous administration. In some embodiments, the pharmaceutical composition according to the disclosure may be administered subcutaneously.

[0225] In some embodiments, the pharmaceutical composition according to the disclosure may be in a form suitable for local administration. In some embodiments, the pharmaceutical composition according to the disclosure may be administered locally. For example, the pharmaceutical composition may be administered locally (e.g., directly) at or adjacent to the site of chronic pain (e.g., chronic pain associated with osteoarthritis), for example to a particular joint or muscle.

[0226] In some embodiments, the pharmaceutical composition according to the disclosure may be in a form suitable for intrathecal administration. In some embodiments, the pharmaceutical composition according to the disclosure may be administered intrathecally.

[0227] In some embodiments, the pharmaceutical composition according to the disclosure may be in a form suitable for intraarticular administration. In some embodiments, the pharmaceutical composition according to the disclosure may be administered intraarticularly.

[0228] In some embodiments, the pharmaceutical composition according to the disclosure may be in a form suitable for intraarticular administration to a knee. In some embodiments, the pharmaceutical composition according to the disclosure may be administered to a knee intraarticularly.

[0229] In some embodiments, the intraarticular administration is by intraarticular injection.

[0230] MEDICAL USE

[0231] The present disclosure provides a TNFR2 agonist comprising three TNF homology domains (THD) that specifically bind to TNFR2 and an Fc domain for use in treating and / or preventing chronic pain (e.g., chronic neuropathic pain) or multiple sclerosis. The present disclosure provides a TNFR2 agonist comprising three TNF homology domains (THD) that specifically bind to TNFR2 and an Fc domain for use in treating and / or preventing chronic pain (e.g., chronic neuropathic pain).

[0232] The present disclosure provides a TNFR2 agonist comprising three TNF homology domains (THD) that specifically bind to TNFR2 and an Fc domain for use in treating and / or preventing chronic pain associated with osteoarthritis.

[0233] The present disclosure provides a TNFR2 agonist comprising three TNF homology domains (THD) that specifically bind to TNFR2 and an Fc domain for use in treating and / or preventing multiple sclerosis.

[0234] The present disclosure provides a method for treating and / or preventing chronic pain (e.g., chronic neuropathic pain) or multiple sclerosis comprising administering a TNFR2 agonist to a subject in need thereof, wherein the TNFR2 agonist comprises three TNF homology domains (THD) that specifically bind to TNFR2 and an Fc domain.

[0235] The present disclosure provides a method for treating and / or preventing chronic pain (e.g., chronic neuropathic pain) comprising administering a TNFR2 agonist to a subject in need thereof, wherein the TNFR2 agonist comprises three TNF homology domains (THD) that specifically bind to TNFR2 and an Fc domain.

[0236] The present disclosure provides a method for treating and / or preventing chronic pain associated with osteoarthritis comprising administering a TNFR2 agonist to a subject in need thereof, wherein the TNFR2 agonist comprises three TNF homology domains (THD) that specifically bind to TNFR2 and an Fc domain.

[0237] The present disclosure provides a method for treating and / or preventing multiple sclerosis comprising administering a TNFR2 agonist to a subject in need thereof, wherein the TNFR2 agonist comprises three TNF homology domains (THD) that specifically bind to TNFR2 and an Fc domain.

[0238] The present disclosure provides a use of a TNFR2 agonist comprising three TNF homology domains (THD) that specifically bind to TNFR2 and an Fc domain in the manufacture of a medicament for treating and / or preventing chronic pain (e.g., chronic neuropathic pain) or multiple sclerosis. The present disclosure provides a use of a TNFR2 agonist comprising three TNF homology domains (THD) that specifically bind to TNFR2 and an Fc domain in the manufacture of a medicament for treating and / or preventing chronic pain (e.g., chronic neuropathic pain).

[0239] The present disclosure provides a use of a TNFR2 agonist comprising three TNF homology domains (THD) that specifically bind to TNFR2 and an Fc domain in the manufacture of a medicament for treating and / or preventing chronic pain associated with osteoarthritis.

[0240] The present disclosure provides a use of a TNFR2 agonist comprising three TNF homology domains (THD) that specifically bind to TNFR2 and an Fc domain in the manufacture of a medicament for treating and / or preventing multiple sclerosis.

[0241] In some embodiments, the chronic pain may arise from osteoarthritis. In some embodiments, the chronic pain may arise from rheumatoid arthritis. In some embodiments, the chronic pain may arise from arthritis.

[0242] In some embodiments, the chronic pain is associated with osteoarthritis. In some embodiments, the chronic pain is associated with rheumatoid arthritis. In some embodiments, the chronic pain is associated with arthritis.

[0243] In some embodiments, the TNFR2 agonist may be a TNFR2 agonist polypeptide according to the disclosure.

[0244] In some embodiments, the TNFR2 agonist may be a TNFR2 agonist multimer according to the disclosure.

[0245] In some embodiments, the TNFR2 agonist may be a polynucleotide according to the disclosure.

[0246] In some embodiments, the TNFR2 agonist may be a vector according to the disclosure.

[0247] In some embodiments, the TNFR2 agonist may be a pharmaceutical composition according to the disclosure.

[0248] The present disclosure also provides a TNFR2 agonist polypeptide according to the disclosure, TNFR2 agonist multimer according to the disclosure, a polynucleotide according to the disclosure, a vector according to the disclosure, or a pharmaceutical composition according to the disclosure, for use as a medicament in the treatment and / or prevention of a disease in a subject.

[0249] The present disclosure also provides a method for treating and / or preventing a disease comprising administering a TNFR2 agonist polypeptide according to the disclosure, TNFR2 agonist multimer according to the disclosure, a polynucleotide according to the disclosure, a vector according to the disclosure, or a pharmaceutical composition according to the disclosure, to a subject in need thereof.

[0250] The present disclosure also provides a use of a TNFR2 agonist polypeptide according to the disclosure, TNFR2 agonist multimer according to the disclosure, a polynucleotide according to the disclosure, a vector according to the disclosure, or a pharmaceutical composition according to the disclosure, in the manufacture of a medicament for the treatment and / or prevention of a disease in a subject.

[0251] Suitably, the disease may be a disease or condition associated with and / or involving TNF, such as aberrant TNF signalling.

[0252] Suitably, the disease may be selected from the list consisting of: chronic pain, chronic neuropathic pain, and multiple sclerosis.

[0253] Suitably, the disease may be chronic pain (e.g., chronic neuropathic pain) or multiple sclerosis.

[0254] Suitably, the disease may be chronic pain (e.g., chronic neuropathic pain). Suitably, the disease may be multiple sclerosis.

[0255] In some embodiments, the chronic pain may arise from osteoarthritis.

[0256] In some embodiments, the chronic pain may arise from rheumatoid arthritis.

[0257] In some embodiments, the chronic pain may arise from arthritis.

[0258] In some embodiments, the chronic pain is associated with osteoarthritis.

[0259] In some embodiments, the chronic pain is associated with rheumatoid arthritis.

[0260] In some embodiments, the chronic pain is associated with arthritis. Suitably, chronic pain may be described as pain that typically persists for longer than would be normally expected despite the use of medication or treatment. Suitably, chronic pain may be defined as pain that persists for more than 3 months, such as for more than 4 months, for more than 5 months, or for more than 6 months. Chronic pain may be continuous (i.e. it may be always present) or may be transient (i.e. it may be felt “on” and “off” over a period of time as defined above (e.g. more than 3 months).

[0261] In some embodiments, a patient to be treated with the TNFR2 agonist, TNFR2 agonist polypeptide, TNFR2 agonist multimer, a polynucleotide, vector, or pharmaceutical composition of the present disclosure may have been suffering from or experiencing pain for at least 3 months (preferably at least 6 months), thereby making it chronic pain. In some embodiments, a patient to be treated with the TNFR2 agonist, TNFR2 agonist polypeptide, TNFR2 agonist multimer, a polynucleotide, vector, or pharmaceutical composition of the present disclosure may have been suffering from or experiencing pain for at least 12 months, thereby making it chronic pain. In some embodiments, the chronic pain experienced by the patient to be treated with the TNFR2 agonist, TNFR2 agonist polypeptide, TNFR2 agonist multimer, a polynucleotide, vector, or pharmaceutical composition of the present disclosure may be localised to particular joints, muscles or areas of the body.

[0262] In some embodiments, chronic pain may be defined as pain that persists for a period of time selected from the list consisting of: about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.

[0263] In some embodiments, chronic pain may be defined as pain that persists for a period of time selected from the list consisting of: more than 3 months, more than 4 months, more than 5 months, more than 6 months, more than 7 months, more than 8 months, more than 9 months, more than 10 months, more than 11 months, or more than 12 months.

[0264] In some embodiments, chronic pain may be defined as pain that persists for a period of time selected from the list consisting of: about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years.

[0265] In some embodiments, chronic pain may be defined as pain that persists for a period of time selected from the list consisting of: more than 1 year, more than 2 years, more than 3 years, more than 4 years, more than 5 years, more than 6 years, more than 7 years, more than 8 years, more than 9 years, or more than 10 years. In some embodiments, chronic pain may be defined as pain that persists for the life of a patient. In some embodiments, chronic pain may be defined as pain that persists for the rest of the life of a patient.

[0266] Suitably, neuropathic pain may be described as pain that affects nerves or the nervous system, which may be the result of neuronal damage or neuronal dysfunction. Neuropathic pain may be called nerve pain, neuropathy or neuralgia. Neuropathic pain may result from disorders of the peripheral nervous system or the central nervous system.

[0267] Suitably, multiple sclerosis may be described as a chronic, immune-mediated neurological disease that affects the brain and / or spinal cord, whereby damage to the nerve cells of the brain and / or spinal cord may cause a wide range of symptoms relating to vision, movement, sensation, cognition and balance.

[0268] The present disclosure provides a TNFR2 agonist polypeptide according to the disclosure, TNFR2 agonist multimer according to the disclosure, a polynucleotide according to the disclosure, a vector according to the disclosure, or a pharmaceutical composition according to the disclosure, for use in treating and / or preventing chronic pain (e.g., chronic neuropathic pain) or multiple sclerosis.

[0269] The present disclosure provides a TNFR2 agonist polypeptide, TNFR2 agonist multimer, a polynucleotide, vector, or pharmaceutical composition according to the disclosure for use in treating and / or preventing chronic pain (e.g., chronic neuropathic pain).

[0270] The present disclosure provides a TNFR2 agonist polypeptide, TNFR2 agonist multimer, a polynucleotide, vector according to the disclosure, or pharmaceutical composition according to the disclosure, for use in treating and / or preventing multiple sclerosis.

[0271] The present disclosure provides a method for treating and / or preventing chronic pain (e.g., chronic neuropathic pain) or multiple sclerosis comprising administering a TNFR2 agonist polypeptide according to the disclosure, TNFR2 agonist multimer according to the disclosure, a polynucleotide according to the disclosure, a vector according to the disclosure, or a pharmaceutical composition according to the disclosure, to a subject in need thereof.

[0272] The present disclosure provides a method for treating and / or preventing chronic pain (e.g., chronic neuropathic pain) comprising administering a TNFR2 agonist, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition according to the disclosure, to a subject in need thereof.

[0273] The present disclosure provides a method for treating and / or preventing multiple sclerosis comprising administering a TNFR2 agonist polypeptide, TNFR2 agonist, polynucleotide, vector, or pharmaceutical composition according to the disclosure, to a subject in need thereof.

[0274] The present disclosure provides a use of a TNFR2 agonist polypeptide according to the disclosure, TNFR2 agonist multimer according to the disclosure, a polynucleotide according to the disclosure, a vector according to the disclosure, or a pharmaceutical composition according to the disclosure, in the manufacture of a medicament for treating and / or preventing chronic pain (e.g., chronic neuropathic pain) or multiple sclerosis.

[0275] The present disclosure provides a use of a TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, a vector, or pharmaceutical composition according to the disclosure, in the manufacture of a medicament for treating and / or preventing chronic pain (e.g., chronic neuropathic pain).

[0276] The present disclosure provides a use of a TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition according to the disclosure, in the manufacture of a medicament for treating and / or preventing multiple sclerosis.

[0277] In some embodiments, the chronic pain may arise from osteoarthritis.

[0278] In some embodiments, the chronic pain may arise from rheumatoid arthritis.

[0279] In some embodiments, the chronic pain may arise from arthritis.

[0280] In some embodiments, the chronic pain is associated with osteoarthritis.

[0281] In some embodiments, the chronic pain is associated with rheumatoid arthritis.

[0282] In some embodiments, the chronic pain is associated with arthritis.

[0283] Suitably, the TNFR2 agonist may induce TNFR2 signalling in a cell. Suitably, the TNFR2 agonist may induce TNFR2 signalling in a nerve cell. Suitably, the TNFR2 agonist may induce TNFR2 signalling in neurons. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition, may induce TNFR2 signalling in a cell. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition, may induce TNFR2 signalling in a nerve cell. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition, may induce TNFR2 signalling in neurons.

[0284] Suitably, the neurons may be supraspinal neurons. Suitably, the supraspinal neurons may be Nex+ supraspinal neurons.

[0285] The present TNFR2 agonists (e.g. the TNFR2 agonist polypeptides or the TNFR2 agonist multimers) may have notable efficacy on disease-relevant pathways, including direct effects on neurons, such as supraspinal neurons. Without wishing to be bound by theory, the inventors consider that the direct effects on supraspinal neurons - distinct from previously reported effects on Treg cells - may facilitate long-term and preventative benefits.

[0286] Suitably, the TNFR2 agonist may be administered to treat chronic pain that may arise from, or is caused by, osteoarthritis. Suitably, the TNFR2 agonist may be administered to treat chronic pain that may arise from, or is caused by, rheumatoid arthritis. Suitably, the TNFR2 agonist may be administered to treat chronic pain that may arise from, or is caused by, arthritis.

[0287] Suitably, the TNFR2 agonist may be administered to treat chronic pain associated with osteoarthritis. Suitably, the TNFR2 agonist may be administered to treat chronic pain associated with rheumatoid arthritis. Suitably, the TNFR2 agonist may be administered to treat chronic pain associated with arthritis.

[0288] Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition, may be administered to treat chronic pain that may arise from, or is caused by, osteoarthritis. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition, may be administered to treat chronic pain that may arise from, or is caused by, rheumatoid arthritis. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition, may be administered to treat chronic pain that may arise from, or is caused by, arthritis. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition, may be administered to treat chronic pain associated with osteoarthritis. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition, may be administered to treat chronic pain associated with rheumatoid arthritis. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition, may be administered to treat chronic pain associated with arthritis.

[0289] Suitably, “chronic pain associated with osteoarthritis” may refer to treating the symptoms / manifestations of chronic pain in a subject suffering from osteoarthritis. Suitably, “chronic pain associated with osteoarthritis” may refer to chronic pain that is caused by osteoarthritis. In some instances, without being bound by theory, the TNFR2 agonist, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition of the disclosure is not treating or preventing the underlying condition (e.g., osteoarthritis), but rather is alleviating the pain associated with the underlying condition (e.g., osteoarthritis).

[0290] Suitably, “chronic pain associated with rheumatoid arthritis” or “chronic pain associated with arthritis” may refer to treating the symptoms / manifestations of chronic pain in a subject suffering from rheumatoid arthritis or arthritis, respectively. Suitably, “chronic pain associated with rheumatoid arthritis” or “chronic pain associated with arthritis” may refer to chronic pain that is caused by rheumatoid arthritis or arthritis, respectively. In some instances, without being bound by theory, the TNFR2 agonist, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition of the disclosure is not treating or preventing the underlying condition (e.g., rheumatoid arthritis or arthritis), but rather is alleviating the pain associated with the underlying condition (e.g., rheumatoid arthritis or arthritis).

[0291] Suitably, the TNFR2 agonist may be administered to a subject systemically. Suitably, the TNFR2 agonist may be administered orally, parenterally, intravenously (such as intravenous infusion), intramuscularly, subcutaneously, topically, by inhalation, rectally, sublingually, by a transdermal route, or vaginally. Suitably, the TNFR2 agonist may be injected into a patient.

[0292] Suitably, the TNFR2 agonist may be administered to a subject intravenously. Suitably, the TNFR2 agonist may be administered to a subject intravenously to treat chronic pain that may arise from osteoarthritis. Suitably, the TNFR2 agonist may be administered to a subject intravenously to treat chronic pain associated with osteoarthritis. Suitably, the TNFR2 agonist may be administered to a subject intravenously to treat chronic pain that may arise from rheumatoid arthritis. Suitably, the TNFR2 agonist may be administered to a subject intravenously to treat chronic pain associated with rheumatoid arthritis. Suitably, the TNFR2 agonist may be administered to a subject intravenously to treat chronic pain that may arise from arthritis. Suitably, the TNFR2 agonist may be administered to a subject intravenously to treat chronic pain associated with arthritis.

[0293] Suitably, the TNFR2 agonist may be administered to a subject intraparenterally. Suitably, the TNFR2 agonist may be administered to a subject intraparenterally to treat chronic pain that may arise from osteoarthritis. Suitably, the TNFR2 agonist may be administered to a subject intraparenterally to treat chronic pain associated with osteoarthritis. Suitably, the TNFR2 agonist may be administered to a subject intraparenterally to treat chronic pain that may arise from rheumatoid arthritis. Suitably, the TNFR2 agonist may be administered to a subject intraparenterally to treat chronic pain associated with rheumatoid arthritis. Suitably, the TNFR2 agonist may be administered to a subject intraparenterally to treat chronic pain that may arise from arthritis. Suitably, the TNFR2 agonist may be administered to a subject intraparenterally to treat chronic pain associated with arthritis.

[0294] Suitably, the TNFR2 agonist may be administered to a subject subcutaneously. Suitably, the TNFR2 agonist may be administered to a subject subcutaneously to treat chronic pain that may arise from osteoarthritis. Suitably, the TNFR2 agonist may be administered to a subject subcutaneously to treat chronic pain associated with osteoarthritis. Suitably, the TNFR2 agonist may be administered to a subject subcutaneously to treat chronic pain that may arise from rheumatoid arthritis. Suitably, the TNFR2 agonist may be administered to a subject subcutaneously to treat chronic pain associated with rheumatoid arthritis. Suitably, the TNFR2 agonist may be administered to a subject subcutaneously to treat chronic pain that may arise from arthritis. Suitably, the TNFR2 agonist may be administered to a subject subcutaneously to treat chronic pain associated with arthritis.

[0295] Suitably, the TNFR2 agonist may be administered to a subject intrathecally. Suitably, the TNFR2 agonist may be administered to a subject intrathecally to treat chronic pain that may arise from osteoarthritis. Suitably, the TNFR2 agonist may be administered to a subject intrathecally to treat chronic pain associated with osteoarthritis. Suitably, the TNFR2 agonist may be administered to a subject intrathecally to treat chronic pain that may arise from rheumatoid arthritis. Suitably, the TNFR2 agonist may be administered to a subject intrathecally to treat chronic pain associated with rheumatoid arthritis. Suitably, the TNFR2 agonist may be administered to a subject intrathecally to treat chronic pain that may arise from arthritis. Suitably, the TNFR2 agonist may be administered to a subject intrathecally to treat chronic pain associated with arthritis.

[0296] Suitably, the TNFR2 agonist may be administered to a subject intraarticularly. Suitably, the TNFR2 agonist may be administered to a subject intraarticularly to treat chronic pain that may arise from osteoarthritis. Suitably, the TNFR2 agonist may be administered to a subject intraarticularly to treat chronic pain associated with osteoarthritis. Suitably, the TNFR2 agonist may be administered to a subject intraarticularly to treat chronic pain that may arise from rheumatoid arthritis. Suitably, the TNFR2 agonist may be administered to a subject intraarticularly to treat chronic pain associated with rheumatoid arthritis. Suitably, the TNFR2 agonist may be administered to a subject intraarticularly to treat chronic pain that may arise from arthritis. Suitably, the TNFR2 agonist may be administered to a subject intraarticularly to treat chronic pain associated with arthritis.

[0297] Suitably, the TNFR2 agonist may be administered to a knee of a subject intraarticularly. Suitably, the TNFR2 agonist may be administered to a knee of a subject intraarticularly to treat chronic pain that may arise from osteoarthritis. Suitably, the TNFR2 agonist may be administered to a knee of a subject intraarticularly to treat chronic pain associated with osteoarthritis. Suitably, the TNFR2 agonist may be administered to a knee of a subject intraarticularly to treat chronic pain that may arise from rheumatoid arthritis. Suitably, the TNFR2 agonist may be administered to a knee of a subject intraarticularly to treat chronic pain associated with rheumatoid arthritis. Suitably, the TNFR2 agonist may be administered to a knee of a subject intraarticularly to treat chronic pain that may arise from arthritis. Suitably, the TNFR2 agonist may be administered to a knee of a subject intraarticularly to treat chronic pain associated with arthritis.

[0298] In some embodiments, the intraarticular administration is by intraarticular injection.

[0299] Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition, may be administered to a subject systemically.

[0300] Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition, may be administered orally, parenterally, intravenously (such as intravenous infusion), intramuscularly, subcutaneously, topically, by inhalation, rectally, sublingually, by a transdermal route, or vaginally. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition, may be injected into a patient.

[0301] Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject intravenously. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject intravenously to treat chronic pain that may arise from osteoarthritis. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject intravenously to treat chronic pain associated with osteoarthritis.

[0302] Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject intravenously to treat chronic pain that may arise from rheumatoid arthritis. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject intravenously to treat chronic pain associated with rheumatoid arthritis. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject intravenously to treat chronic pain that may arise from arthritis. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject intravenously to treat chronic pain associated with arthritis.

[0303] Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject intraparenterally. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject intraparenterally to treat chronic pain that may arise from osteoarthritis. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject intraparenterally to treat chronic pain associated with osteoarthritis.

[0304] Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject intraparenterally to treat chronic pain that may arise from rheumatoid arthritis. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject intraparenterally to treat chronic pain associated with rheumatoid arthritis. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject intraparenterally to treat chronic pain that may arise from arthritis. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject intraparenterally to treat chronic pain associated with arthritis.

[0305] Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject subcutaneously. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject subcutaneously to treat chronic pain that may arise from osteoarthritis. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject subcutaneously to treat chronic pain associated with osteoarthritis.

[0306] Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject subcutaneously to treat chronic pain that may arise from rheumatoid arthritis. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject subcutaneously to treat chronic pain associated with rheumatoid arthritis. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject subcutaneously to treat chronic pain that may arise from arthritis. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject subcutaneously to treat chronic pain associated with arthritis.

[0307] Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject intrathecally. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject intrathecally to treat chronic pain that may arise from osteoarthritis. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject intrathecally to treat chronic pain associated with osteoarthritis.

[0308] Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject intrathecally to treat chronic pain that may arise from rheumatoid arthritis. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject intrathecally to treat chronic pain associated with rheumatoid arthritis. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject intrathecally to treat chronic pain that may arise from arthritis. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject intrathecally to treat chronic pain associated with arthritis.

[0309] Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject intraarticularly. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject intraarticularly to treat chronic pain that may arise from osteoarthritis. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject intraarticularly to treat chronic pain associated with osteoarthritis.

[0310] Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject intraarticularly to treat chronic pain that may arise from rheumatoid arthritis. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject intraarticularly to treat chronic pain associated with rheumatoid arthritis. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject intraarticularly to treat chronic pain that may arise from arthritis. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject intraarticularly to treat chronic pain associated with arthritis.

[0311] Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a knee of a subject intraarticularly. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a knee of a subject intraarticularly to treat chronic pain that may arise from osteoarthritis. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a knee of a subject intraarticularly to treat chronic pain associated with osteoarthritis.

[0312] Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a knee of a subject intraarticularly to treat chronic pain that may arise from rheumatoid arthritis. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a knee of a subject intraarticularly to treat chronic pain associated with rheumatoid arthritis. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a knee of a subject intraarticularly to treat chronic pain that may arise from arthritis. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a knee of a subject intraarticularly to treat chronic pain associated with arthritis.

[0313] In some embodiments, the intraarticular administration is by intraarticular injection.

[0314] Suitably, the TNFR2 agonist may have direct effect in the central nervous system (CNS). Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition, may have direct effect in the central nervous system (CNS).

[0315] Suitably, the TNFR2 agonist may prevent the neuronal remodelling that causes chronic pain (e.g., chronic neuropathic pain). Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition, may prevent the neuronal remodelling that causes chronic pain (e.g., chronic neuropathic pain).

[0316] Suitably, the TNFR2 agonist may be administered to a patient over the life of the patient.

[0317] Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition, may be administered to a patient over the life of the patient.

[0318] 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. 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.

[0319] The terms “prevent”, “prevention” and “preventing” refers to preventing the onset of symptoms of a disease or condition, and as such encompasses prophylactic treatment.

[0320] Suitably, the TNFR2 agonist 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.

[0321] Suitably, the TNFR2 agonist may alleviate chronic pain faster, compared to other therapies that are known in the art. Suitably, the TNFR2 agonist may alleviate chronic pain that may arise from osteoarthritis faster, compared to other therapies that are known in the art. Suitably, the TNFR2 agonist may alleviate chronic pain associated with osteoarthritis faster, compared to other therapies that are known in the art.

[0322] Suitably, the TNFR2 agonist may alleviate chronic pain that may arise from rheumatoid arthritis faster, compared to other therapies that are known in the art. Suitably, the TNFR2 agonist may alleviate chronic pain associated with rheumatoid arthritis faster, compared to other therapies that are known in the art. Suitably, the TNFR2 agonist may alleviate chronic pain that may arise from arthritis faster, compared to other therapies that are known in the art. Suitably, the TNFR2 agonist may alleviate chronic pain associated with arthritis faster, compared to other therapies that are known in the art.

[0323] Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may result in a lowered incidence of disease or symptoms, compared to other therapies that are known in the art.

[0324] Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may result in a delayed onset of disease or symptoms, compared to other therapies that are known in the art.

[0325] Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may result in reduced severity of disease or symptoms, compared to other therapies that are known in the art. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition, may alleviate chronic pain faster, compared to other therapies that are known in the art. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may alleviate chronic pain that may arise from osteoarthritis faster, compared to other therapies that are known in the art. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may alleviate chronic pain associated with osteoarthritis faster, compared to other therapies that are known in the art.

[0326] Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may alleviate chronic pain that may arise from rheumatoid arthritis faster, compared to other therapies that are known in the art. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may alleviate chronic pain associated with rheumatoid arthritis faster, compared to other therapies that are known in the art. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may alleviate chronic pain that may arise from arthritis faster, compared to other therapies that are known in the art. Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may alleviate chronic pain associated with arthritis faster, compared to other therapies that are known in the art.

[0327] Without wishing to be bound by theory, the TNFR2 agonist, TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may reduce or obviate reliance on the use of opiates to treat chronic pain.

[0328] In some embodiments, the TNFR2 agonist, TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may reduce or obviate the need to treat chronic pain by administering opiates.

[0329] In some embodiments, the TNFR2 agonist, TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may reduce the dosage of opiates needed to treat chronic pain.

[0330] 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 chronic pain, multiple sclerosis, chemotherapeutic, immunomodulatory and / or radiotherapy treatments in the form of a combination therapy or treatment.

[0331] Suitably, treatments for chronic pain may include the administration of nonsteroidal antiinflammatory drugs (NSAIDs) and / or benzodiazepines.

[0332] Suitably, treatments for multiple sclerosis may include the administration of steroids (such as methylprednisolone, prednisone), amantadine, painkillers (such as gabapentin, amitriptyline, pregabalin, benzodiazepines), or disease modifying drugs (such as alemtuzumab, dimethyl fumarate, ofatumumab, ocrelizumab, natalizumab).

[0333] Suitably, the medical uses and methods of treatment described herein may be combined with one or more TNFR1 antagonist(s). In other words, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition, may be administered to a patient in combination with one or more TNFR1 antagonist(s). Illustrative TNFR1 antagonists 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.

[0334] Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition, may be administered to a patient in combination with the TNFR1 antagonist Atrosab. The CDRH1 , CDRH2, and CDRH3 and CDRL1 , CDRL2, and CDRL3 sequences of Atrosab are presented below as SEQ ID NOs: 10-15 respectively. The VH and VL sequences of Atrosab are presented below as SEQ ID NO: 16 and SEQ ID NO: 17 respectively.

[0335] Suitably, the TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition, may be administered to a patient in combination with the TNFR1 antagonist Atrosimab. It will be understood that the CDRH1 , CDRH2, and CDRH3 and 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

[0336] Suitably, the TNFR1 antagonist may be 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;

[0337] [i] 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); or

[0338] [ii] wherein the VH comprises or consists of the sequence QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDFYINWVRQAPGQGLEWIGEIYPYSGHAYY NEKFKARVTITADKSTSTAYMELSSLRSEDTAVYYCARWDFLDYWGQGTTVTVSS (SEQ ID NO: 16); and the VL comprises or consists of the sequence DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGNTYLHWYLQKPGQSPQLLIYTVSNRFSG VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGGGTKVEIKR (SEQ ID NO: 17); or

[0339] [iii] 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); or

[0340] [iv] wherein the VH comprises or consists of the sequence HVQLVQSGAEVKKPGSSVKVSCKASGYTFTDFYINWVRQAPGQGLEWIGEIVPSQGEAKY NDKFKARVTITADKSTSTAYMELSSLRSEDTAVYYCARWDFLDYWGQGTTVTVSS (SEQ ID NO: 24); and the VL comprises or consists of the sequence DVQMTQSPSSLSASVGDRVTITCRSSQSLLHSNGNTYLHWYQQKPGKAPKLLIYTVSNRFS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCSQSTHVPYTFGGGTKVEIK (SEQ ID NO: 25).

[0341] The TNFR1 antagonist may be an antibody comprising or consisting of (i) a light chain of the sequence DVQMTQSPSSLSASVGDRVTITCRSSQSLLHSNGNTYLHWYQQKPGKAPKLLIYTVSNRFS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCSQSTHVPYTFGGGTKVEIKGTGGGSGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPSVFPLAPSSKSTSGGT AALGCLVKDYFPSDIAVEWESGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYSC

[0342] SVMHEALHNHYTQKSVEPKSC (SEQ ID NO: 26), and (ii) a heavy chain of the sequence HVQLVQSGAEVKKPGSSVKVSCKASGYTFTDFYINWVRQAPGQGLEWIGEIVPSQGEAKY NDKFKARVTITADKSTSTAYMELSSLRSEDTAVYYCARWDFLDYWGQGTTVTVSSGTGGG SGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPSVFIFPPSDEQ LKSGTASVVCLVNNFYPRDIAVEWEVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYSCSVMHEALHNHYTQKSFNRGEC (SEQ ID NO: 27).

[0343] Suitably, the TNFR1 antagonist described herein may have may comprise a variant of the amino acid sequence according to any one of SEQ ID NOs: 10-27 with at least 80% sequence identity thereto. Suitably, the TNFR1 antagonist described herein may have may comprise a variant of the amino acid sequence according to any one of SEQ ID NOs: 10-27 with at least 85% sequence identity thereto. Suitably, the TNFR1 antagonist described herein may have may comprise a variant of the amino acid sequence according to any one of SEQ ID NOs: IQ- 27 with at least 90% sequence identity thereto. Suitably, the TNFR1 antagonist described herein may have may comprise a variant of the amino acid sequence according to any one of SEQ ID NOs: 10-27 with at least 95% sequence identity thereto. Suitably, the TNFR1 antagonist described herein may have may comprise a variant of the amino acid sequence according to any one of SEQ ID NOs: 10-27 with at least 96% sequence identity thereto. Suitably, the TNFR1 antagonist described herein may have may comprise a variant of the amino acid sequence according to any one of SEQ ID NOs: 10-27 with at least 97% sequence identity thereto. Suitably, the TNFR1 antagonist described herein may have may comprise a variant of the amino acid sequence according to any one of SEQ ID NOs: 10-27 with at least 98% sequence identity thereto. Suitably, the TNFR1 antagonist described herein may have may comprise a variant of the amino acid sequence according to any one of SEQ ID NOs: IQ- 27 with at least 99% sequence identity thereto.

[0344] SUBJECT

[0345] As used herein, the terms “patient” and “subject” may be used interchangeably.

[0346] Suitably, the subject of the medical uses and methods of treatment according to the present disclosure may be a mammal.

[0347] In some embodiments, the subject is male. In some embodiments, the subject is female.

[0348] Suitably, the subject may be a human. Suitably, 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.

[0349] In some embodiments, the subject is human. In some embodiments, the subject is a human male. In some embodiments, the subject is a human female. ADMINISTRATION

[0350] Suitably, the form of administration of the TNFR2 agonist, TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or 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.

[0351] Suitably, the TNFR2 agonist, TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be injected into a subject.

[0352] In some embodiments, the TNFR2 agonist, TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be in a form suitable for intravenous infusion.

[0353] In some embodiments, the TNFR2 agonist, TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be in a form suitable for intravenous administration.

[0354] In some embodiments, the TNFR2 agonist, TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be in a form suitable for intraparenteral administration.

[0355] In some embodiments, the TNFR2 agonist, TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be in a form suitable for systemic administration.

[0356] In some embodiments, the TNFR2 agonist, TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be in a form suitable for subcutaneous administration.

[0357] In some embodiments, the TNFR2 agonist, TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be in a form suitable for intrathecal administration. In some embodiments, the TNFR2 agonist, TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be in a form suitable for intraarticular administration.

[0358] In some embodiments, the TNFR2 agonist, TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be in a form suitable for intraarticular administration to a knee.

[0359] In some embodiments, the intraarticular administration is by intraarticular injection.

[0360] Suitably, the TNFR2 agonist, TNFR2 agonist polypeptide, TNFR2 agonist multimer, polynucleotide, vector, or pharmaceutical composition may be administered to a subject once, or may be administered to a subject over a period of hours, days, weeks, months or years.

[0361] FURTHER EMBODIMENTS

[0362] The present disclosure provides a TNFR2 agonist for use in treating and / or preventing:

[0363] (i) chronic pain (e.g., chronic neuropathic pain);

[0364] (ii) multiple sclerosis; or

[0365] (iii) chronic pain associated with osteoarthritis.

[0366] The present disclosure provides a TNFR2 agonist for use in treating and / or preventing chronic pain.

[0367] The present disclosure provides a method of treating and / or preventing chronic pain, the method comprising administering to a subject a TNFR2 agonist.

[0368] The present disclosure provides a TNFR2 agonist in the manufacture of a medicament for treating and / or preventing chronic pain.

[0369] In some embodiments, the chronic pain may arise from osteoarthritis. In some embodiments, the chronic pain may arise from rheumatoid arthritis. In some embodiments, the chronic pain may arise from arthritis.

[0370] In some embodiments, the chronic pain is associated with osteoarthritis. In some embodiments, the chronic pain is associated with rheumatoid arthritis. In some embodiments, the chronic pain is associated with arthritis. The present disclosure provides a TNFR2 agonist for use in treating and / or preventing multiple sclerosis.

[0371] The present disclosure provides a method of treating and / or preventing multiple sclerosis, the method comprising administering to a subject a TNFR2 agonist.

[0372] The present disclosure provides a TNFR2 agonist in the manufacture of a medicament for treating and / or preventing multiple sclerosis.

[0373] It will be understood that a TNFR2 agonist is an agent that is capable of directly or indirectly activating the TNFR2 signalling pathway. As such, it will be understood that the TNFR2 agonist may be capable of inducing signalling via TNFR2.

[0374] In some embodiments, the TNFR2 agonist is any agent that is capable of activating the TNFR2 signalling pathway.

[0375] In some embodiments, the TNF signalling is TNF alpha and / or TNF beta signalling. In some embodiments, the TNF signalling is TNF alpha signalling.

[0376] 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).

[0377] 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.

[0378] In some embodiments, the TNFR2 agonist is any TNFR2 agonist as described herein. Suitable assays and techniques for measuring and / or quantifying the activity (e.g. binding capability and / or activity) of the TNFR2 agonist 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

[0379] It will be understood that a TNFR2 agonist may increase signalling through TNFR2 by at least 10%, as compared to a control. It will also be understood that a TNFR2 agonist may increase signalling through TNFR2 by at least 2-fold, as compared to a control.

[0380] 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.

[0381] NUMBERED PARAGRAPHS

[0382] The present disclosure may be described by way of the following numbered paragraphs:

[0383] 1. A tumour necrosis factor receptor 2 (TNFR2) agonist polypeptide comprising (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.

[0384] 2. The TNFR2 agonist polypeptide according to paragraph 1 wherein the Fc domain (i) does not comprise a proline at position 233 and / or (ii) comprises a glycine at position 236. 3. The TNFR2 agonist polypeptide according to paragraph 1 or paragraph 2 wherein the Fc domain comprises a sequence with at least 85% sequence identity to SEQ ID NO: 1.

[0385] 4. The TNFR2 agonist polypeptide according to any of paragraphs 1 to 3 wherein each THD comprises an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 2.

[0386] 5. The TNFR2 agonist polypeptide according to paragraph 4 wherein each THD independently comprises 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, Q84N / A141 I / E142G / S143D,

[0387] A141 H / E142S / S143D, A141 H / S143D, L25V / A141 D / E142D / S143D, A141 N / E142D / S143D, A141T / E142S / S143D, A141Q / E142D / S143D, A141T / E142D / S143D, A141 D / E142G / S143D, A141 D / S143D, 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, preferably selected from D139N and A141 R, in the corresponding position of the sequence of SEQ ID NO: 2.

[0388] 6. The TNFR2 agonist polypeptide according to any preceding paragraph 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.

[0389] 7. The TNFR2 agonist polypeptide according to any preceding paragraph 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.

[0390] 8. The TNFR2 agonist polypeptide according to any preceding paragraph wherein (i) the first and second THD and (ii) the second and third THD are directly linked without any intervening peptide linkers.

[0391] 9. The TNFR2 agonist polypeptide according to any preceding paragraph, wherein the TNFR2 binding domain comprises or consists of a sequence with at least 80% sequence identity to SEQ ID NO: 5. 10. The TNFR2 agonist polypeptide according to any preceding paragraph wherein the TNFR2 binding domain is joined to the Fc domain by a flexible linker consisting of 3-20 amino acids.

[0392] 11. The TNFR2 agonist polypeptide according to paragraph 10 wherein the flexible linker is a Ser-Gly linker; preferably GGSGGGGSGG (SEQ ID NO: 6).

[0393] 12. The TNFR2 agonist polypeptide according to any preceding paragraph, wherein the TNFR2 agonist polypeptide comprises a sequence with at least 90% sequence identity to SEQ ID NO: 7.

[0394] 13. A TNFR2 agonist multimer wherein the TNFR2 agonist multimer is in the form of a dimer comprising two TNFR2 agonist polypeptides, wherein each TNFR2 agonist polypeptide is independently a TNFR2 agonist polypeptide according to any preceding paragraph.

[0395] 14. A TNFR2 agonist multimer according to paragraph 13, wherein each TNFR2 agonist polypeptide in the dimer is identical.

[0396] 15. A polynucleotide encoding a TNFR2 agonist polypeptide according to any of paragraphs 1 to 12.

[0397] 16. A vector comprising a polynucleotide according to paragraph 15.

[0398] 17. A pharmaceutical composition comprising a TNFR2 agonist polypeptide according to any of paragraphs 1 to 12, a TNFR2 agonist multimer according to paragraph 13 or paragraph 14, a polynucleotide according to paragraph 15 or a vector according to paragraph 16.

[0399] 18. A TNFR2 agonist comprising three TNF homology domains (THD) that specifically bind to TNFR2 and an Fc domain for use in treating and / or preventing chronic pain.

[0400] 19. A TNFR2 agonist comprising three TNF homology domains (THD) that specifically bind to TNFR2 and an Fc domain for use in treating and / or preventing multiple sclerosis.

[0401] 20. A method for treating and / or preventing chronic pain comprising administering a TNFR2 agonist to a subject in need thereof, wherein the TNFR2 agonist comprises three TNF homology domains (THD) that specifically bind to TNFR2 and an Fc domain. 21. A method for treating and / or preventing multiple sclerosis comprising administering a TNFR2 agonist to a subject in need thereof, wherein the TNFR2 agonist comprises three TNF homology domains (THD) that specifically bind to TNFR2 and an Fc domain.

[0402] 22. Use of a TNFR2 agonist comprising three TNF homology domains (THD) that specifically bind to TNFR2 and an Fc domain in the manufacture of a medicament for treating and / or preventing chronic pain.

[0403] 23. Use of a TNFR2 agonist comprising three TNF homology domains (THD) that specifically bind to TNFR2 and an Fc domain in the manufacture of a medicament for treating and / or preventing multiple sclerosis.

[0404] 24. The TNFR2 agonist for use according to paragraph 18 or paragraph 19, the method according to paragraph 20 or paragraph 21 , or the use according to paragraph 22 or paragraph 23 ; wherein the TNFR2 agonist is a TNFR2 agonist polypeptide according to any of paragraphs 1 to 12, a TNFR2 agonist multimer according to paragraph 13 or paragraph 14, a polynucleotide according to paragraph 15, a vector according to paragraph 16, or provided as a pharmaceutical composition according to paragraph 17.

[0405] 25. The TNFR2 agonist for use according to paragraph 18 or paragraph 24, the method according to paragraph 20 or paragraph 24, or the use according to paragraph 22 or paragraph 24; wherein the chronic pain may arise from osteoarthritis.

[0406] 26. The TNFR2 agonist for use according to paragraph 18 or paragraph 24, the method according to paragraph 20 or paragraph 24, or the use according to paragraph 22 or paragraph 24; wherein the chronic pain is associated with osteoarthritis.

[0407] 27. The TNFR2 agonist for use according to any of paragraphs 18, 19 or 24 to 26, the method according to any of paragraphs 20, 21 , or 24 to 26, or the use according to any of paragraphs 22 to 26 wherein the TNFR2 agonist induces TNFR2 signalling in neurons.

[0408] 28. The TNFR2 agonist for use, the method, or the use according to paragraph 27 wherein the neurons are supraspinal neurons, preferably Nex+ supraspinal neurons.

[0409] 29. The TNFR2 agonist for use according to any of paragraphs 18, 19 or 24 to 28, the method according to any of paragraphs 20, 21 or 24 to 28, or the use according to any of paragraphs 22 to 28 wherein the TNFR2 agonist is administered to a subject systemically. 30. The TNFR2 agonist for use according to any of paragraphs 18, 19 or 24 to 29, the method according to any of paragraphs 20, 21 or 24 to 29, or the use according to any of paragraphs 22 to 29 wherein the TNFR2 agonist is administered to a subject by a route selected from the list consisting of: systemical administration, intravenous administration, intraparenteral administration, subcutaneous administration, intrathecal administration, and intraarticular administration.

[0410] 31. The TNFR2 agonist, the method, or the use according to paragraph 30, wherein the TNFR2 agonist is administered to a subject systemically.

[0411] 32. The TNFR2 agonist, the method, or the use according to paragraph 30, wherein the TNFR2 agonist is administered to a subject subcutaneously.

[0412] 33. The TNFR2 agonist, the method, or the use according to paragraph 30, wherein the TNFR2 agonist is administered to a subject intraarticularly.

[0413] 34. The TNFR2 agonist for use according to any of paragraphs 18, 19 or 24 to 33, the method according to any of paragraphs 20, 21 or 24 to 33, or the use according to any of paragraphs 22 to 33, wherein the TNFR2 agonist is administered in combination with a TNFR1 antagonist.

[0414] 35. The TNFR2 agonist for use, the method, or the use according to paragraph 34, wherein the TNFR1 antagonist is (i) Atrosab, or (ii) Atrosimab, or (iii) 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:

[0415] (a) the CDRH1 sequence is SEQ ID NO: 10; the CDRH2 sequence is SEQ ID NO: 11 ; the CDRH3 sequence is SEQ ID NO: 12; the CDRL1 sequence is SEQ ID NO: 13; the CDRL2 sequence is SEQ ID NO: 14; and the CDRL3 sequence is SEQ ID NO: 15; or

[0416] (b) the VH comprises or consists of SEQ ID NO: 16; and the VL comprises or consists of SEQ ID NO: 17; or

[0417] (c) the CDRH1 sequence is SEQ ID NO: 18; the CDRH2 sequence is SEQ ID NO: 19; the CDRH3 sequence is SEQ ID NO: 20; the CDRL1 sequence is SEQ ID NO: 21 ; the CDRL2 sequence is SEQ ID NO: 22; and the CDRL3 sequence is SEQ ID NO: 23; or

[0418] (d) the VH comprises or consists of SEQ ID NO: 24; and the VL comprises or consists of SEQ ID NO: 25; or (e) the antibody comprises or consists of a light chain of SEQ ID NO: 26, and a heavy chain of SEQ ID NO: 27.

[0419] 36. A TNFR2 agonist for use in treating and / or preventing chronic pain.

[0420] 37. The TNFR2 agonist for use according to paragraph 36, wherein the chronic pain is associated with osteoarthritis

[0421] 38. A TNFR2 agonist for use in treating and / or preventing multiple sclerosis.

[0422] GENERAL TERMS AND DEFINITIONS

[0423] 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.

[0424] 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.

[0425] 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.

[0426] 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.

[0427] 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. 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.

[0428] 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.

[0429] 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'.

[0430] 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.

[0431] 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.

[0432] 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. 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.

[0433] The disclosure 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 disclosure and are not intended in any way to limit the scope of the disclosure.

[0434] EXAMPLES

[0435] As used in the present Examples, the TNFR2 agonist scTNFR2-Fc[LALA-Aa] is understood to comprise a TNFR2 agonist polypeptide according to the present disclosure, comprising (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. The scTNFR2-Fc[LALA-Aa] may be in the form of a dimeric protein consisting of two disulfide-bond (via Fc-hinge) stabilized SCTNFR2-FC chains, wherein each chain is a TNFR2 agonist polypeptide according to the present disclosure, i.e. comprising a TNFR2 binding domain (comprising three TNF homology domains (THD) that specifically bind to TNFR2 (SCTNFR2)) fused to a mutated Fc domain, as described herein. An example of the TNFR2 agonist polypeptide according to the present disclosure in dimeric form is provided in Figure 1A.

[0436] In particular, it will be understood that a Fc domain comprising the sequence of SEQ ID NO: 1 (i.e. with “A” at position 14, “A” at position 15, “G” at position 107, “S” at position 110 and “S” at position 111 , in line with the described L14A, L15A, A107G, A110S and P111S mutations described herein), may be defined as a “Fc[LALA-Aa]”.

[0437] It will also be understood that scTNFR2-Fc[Aab] as used herein refers to a molecule comprising a Fc domain that is not “Fc[LALA-Aa]”. In particular, the Fc domain of scTNFR2-Fc[Aab] may comprise the following mutations, which are not present in “Fc[LALA-Aa]”: E233P, L234V and AG236. As such, whereas “Fc[LALA-Aa]” comprises L234A, L235A, A327G, A330S, P331S, “Fc[Aab]” comprises E233P, L234V, L235A, AG236 A327G, A330S, P331S. Example 1 - scTNFR2-Fcn ALA-Aa1 characterization

[0438] The TNFR2 agonist scTNFR2-Fc[LALA-Aa] was analyzed in terms of quality and functionality. After production in CHO cells by transient transfection, scTNFR2-Fc[LALA-Aa] was purified by 2-step purification, i.e. by protein A affinity chromatography followed by preparative sizeexclusion chromatography (SEC) to remove remaining aggregates.

[0439] To determine purity and aggregate content, an analytical SEC analysis using a high- performance liquid chromatography (HPLC) instrument (Agilent 1260 Infinity) was performed. Here, 5 g protein were loaded onto a TSKgel G3000SWXL column and analyzed using a 0.3 M NaP buffer as a mobile phase. Here, a flow rate of 0.5 ml / min was used and the analysis was stopped after 40 min. The purity was then calculated using the area under the curve (AUC) of the measured signal in milli-Absorbance Units (mAU) in relation to the retention time in minutes. To determine the molecular weight of the proteins, a gel filtration standard from Bio-Rad containing a mixture of molecular weight markers (1.35-670 kD) was used (Figure 1A). The TNFR2 agonist eluted as single peak at a molecular weight (MW) of approx. 150 kD (calculated MW: 154.01 kD) and showed a purity of 99.04%.

[0440] Next, a sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed (Figure 1 B). Here, 2 pg protein was supplemented with Laemmli buffer and analyzed either under reducing (with DTT) or non-reducing (without DTT) conditions. The mixed samples were incubated at 95°C for 5 min to complete denaturation. The protein samples were then loaded onto a 4-12% Criterion TGX Stain-Free gel together with a protein standard (Precision Plus Protein Unstained Standards, 10-250 kD) and run at 200 V for approximately 40 min. Criterion TGX Stain-Free gels contain trihalo compounds that can react with tryptophan residues when activated with UV light thereby producing fluorescence. Therefore, the gel was activated for 45 seconds with UV light and the protein signals were visualized with the Bio-Rad ChemiDoc imaging system.

[0441] The SDS-PAGE analysis of scTNFR2-Fc[LALA-Aa] under reducing conditions showed a protein band at a MW of approximately 75 kDa, which corresponds to the calculated molecular weight of the SCTNFR2-FC monomers (77 kD each). Under non-reducing conditions the dimerized scTNFR2-Fc[LALA-Aa] was observed as a single band at approximately 150 kDa, which corresponds to the theoretical molecular weight of the correctly assembled protein (154.01 kD). Furthermore, binding of scTNFR2-Fc[LALA-Aa] to TNFR2 was analyzed by enzyme-linked immunosorbent assay (ELISA) (Figure 1C). Here, 0.5 pg / ml of human TNFR2-Fc diluted in 100 pl of PBS were immobilized on Flat bottom Nunc-lmmuno MaxiSorp 96-well plates overnight at 4°C. The next day, 200 pl of Assay Diluent were added to each well for 2 hours to block unspecific binding. Next, 100 pl of a 1 / sqrt(10) titration of the TNFR2 agonist or recombinant human TNF (rhTNF) in Assay Diluent were added and incubated for 1 hour. A start concentration of 10 nM was chosen. To detect bound fusion proteins, first 100 pl of a mouse anti-human TNF-alpha antibody (clone F6C5) diluted 1 :10,000 in Assay Diluent was added and incubated for 1 hour followed by a 1-hour incubation of 100 pl of an anti-mouse IgG antibody conjugated to a horseradish peroxidase (HRP) (dilution 1 :10,000 in Assay Diluent). Between each step the plate was washed 3 times with 200 pl of 0.05% Tween-20 diluted in PBS. Subsequently, 100 pl of TMB substrate solution were added per well. After 10 min the reaction was stopped with 50 pl of 1 M H2SO4. Finally, the absorbance at 450 nm was measured using the Tecan Infinite 200 PRO microplate reader. Binding was calculated in % by setting the absorbance of the highest concentration of scTNFR2-Fc[LALA-Aa] as 100% binding. scTNFR2-Fc[LALA-Aa] bound to TNFR2 in a dose dependent manner with an EC50 value of 0.19±0.06 nM. In contrast, rhTNF binding to TNFR2 resulted in a 10x higher EC50 value (1 ,93±0.26). These data support the concept that TNF ligand oligomerization by the SCTNFR2- Fc format results in superior binding to TNFR2.

[0442] Example 2 - Binding of SCTNFR2-FC muteins to FcyRlla

[0443] Binding of the SCTNFR2-FC muteins to FcyRlla was analyzed by ELISA. To determine binding to the high affinity FcyRlla (H131) (Figure 2A), 10 pg / ml of SCTNFR2-FC muteins diluted in 100 pl of PBS were immobilized on Flat bottom Nunc-lmmuno MaxiSorp 96-well plates and incubated at 4°C overnight. On the next day, 200 pl of Assay Diluent were added to each well and incubated for 2 hours to block unspecific binding. Next, 100 pl of a 1 / sqrt(10) titration of biotinylated FcyRlla (H131) diluted in Assay Diluent were added and samples were incubated for 1 hour. Here a start concentration of 10 pg / ml was chosen. To detect binding, 100 pl of Streptavidin-HRP diluted 1 :5,000 in Assay Diluent was added for 1 hour. Between each step, the plate was washed 3 times with 200 pl of 0.05% Tween-20 diluted in PBS. Subsequently, 100 pl of TMB substrate solution were added per well. The reaction was stopped after 30 min with 50 pl of 1 M H2SO4. Finally, the absorbance at 450 nm was measured using the CLARIOstarplusmicroplate reader. Binding was calculated in % by setting the absorbance of the highest concentration of the positive control, scTNFR2-Fc[WT], as 100% binding. FcyRlla (H131) bound to SCTNFR2-FC[WT] in a dose dependent manner, while at a concentration of 10 pg / ml of FcyRlla (H131) approx. 60% binding to scTNFR2-Fc[Aab] was observed. Surprisingly, no binding was detected for the scTNFR2-Fc[LALA-Aa] variant.

[0444] To detect binding to the low affinity receptor FcyRlla (R131) (Figure 2B), 1 pg / ml of Fey receptor diluted in 100 pl PBS were immobilized on Flat bottom Nunc-lmmuno MaxiSorp 96- well plates and incubated at 4°C overnight. On the next day, 200 pl of Assay Diluent were added to each well and incubated for 2 hours to block unspecific binding. Next, 100 pl of a 1 / sqrt(10) titration of the different muteins diluted in Assay Diluent were added and incubated for 1 hour. Here a start concentration of 100 pg / ml was chosen. To detect bound fusion protein 100 pl of an anti-human-Fc-HRP antibody diluted 1 :5,000 in Assay Diluent was added for 1 hour. Between each step, the plate was washed 3 times with 200 pl of 0.05% Tween-20 diluted in PBS. Subsequently, 100 pl of TMB substrate solution were added per well. The reaction was stopped after 1 hour with 50 pl of 1 M H2SO4. Finally, the absorbance at 450 nm was measured using the CLARIOstarplusmicroplate reader. Binding was calculated in % by setting the absorbance of the highest concentration of the positive control, SCTNFR2-FC[WT], as 100% binding.

[0445] SCTNFR2-FC[WT] bound to FcyRlla (R131) in a dose dependent manner, while at a concentration of 100 pg / ml of scTNFR2-Fc[Aab] approx. 20% binding to FcyRlla (R131) was observed. Surprisingly, no binding was detected for the scTNFR2-Fc[LALA-Aa] variant.

[0446] To quantify the binding kinetics of SCTNFR2-FC muteins to FcyRlla, SPR binding analysis was performed (Figure 3). To this end, recombinant FcyRlla (H131) receptor was captured on a C1 sensor chip. Serially two-fold diluted analytes (rituximab and scTNFR2-Fc[Aab]) in analysis buffer (10 mM HEPES pH 7.4, 150 mM NaCI, 3 mM EDTA, 0.05% Tween 20) were injected on FcyRlla (H131) capture surfaces and analysed in multi cycle kinetic mode at a temperature of 25°C using Biacore T200 (Figure 3A). The concentration range of analytes for FcyRlla analyses were 9.8 nM to 5 pM. Data processing (e.g. double referencing including blank run subtraction) and evaluation was performed by global fitting applying a Langmuir 1 :1 binding model including bulk refractive index (Rl) contribution using the Biacore T200 Evaluation Software version 3.2. For blank run subtraction the most appropriate approach was to use blank runs performed immediately before and after a concentration series. Statistical evaluation (mean, SD, CV) was conducted with Excel (Microsoft). Quantitative analyses of rituximab binding to FcyRIIA revealed transient kinetics showing fast association and fast dissociation. Equilibrium dissociation constants KD were calculated using a steady-state approach (KD = 2.93'07M). The Fc fusion protein scTNFR2-Fc[Aab] displayed complex and biphasic kinetics with binding signals well above the expected binding stoichiometry, suggesting multivalent heterogeneous binding and avidity. Evaluation was performed using a steady-state model (KD = 3.4T07M). Results need to be interpreted with caution due to not considered additional secondary binding (avidity) events.

[0447] Next, rituximab and scTNFR2-Fc[LALA-Aa] was captured on a C1 sensor chip (Figure 3B). Serially two-fold diluted analyte (recombinant FcyRlla (H131)) in analysis buffer (10 mM HEPES pH 7.4, 150 mM NaCI, 3 mM EDTA, 0.05% Tween 20) was injected on FcyRlla (H131) capture surfaces and analysed in multi cycle kinetic mode at a temperature of 25°C using Biacore T200. The concentration range of analytes for FcyRlla analyses were 9.8 nM to 5 pM. Data processing (e.g. double referencing including blank run subtraction) and evaluation was performed by global fitting applying a Langmuir 1 :1 binding model including bulk refractive index (Rl) contribution using the Biacore T200 Evaluation Software version 3.2. For blank run subtraction the most appropriate approach was to use blank runs performed immediately before and after a concentration series. Statistical evaluation (mean, SD, CV) was conducted with Excel (Microsoft).

[0448] Quantitative analyses of FcyRIIA binding to rituximab revealed transient kinetics showing fast association and fast dissociation. Equilibrium dissociation constants KD were calculated using a steady-state approach (KD = 3.57'07M). The Fc fusion protein scTNFR2-Fc[LALA-Aa] similarly displayed transient kinetics showing fast association and fast dissociation. Evaluation was performed using a steady-state model. A higher KD value (1.95’05M) was calculated indicating reducing affinity of scTNFR2-Fc[LALA-Aa] to FcyRIIA compared to rituximab and scTNFR2-Fc[Aab],

[0449] Example 3 - Activation of FcyRlla by scTNFR2-Fc muteins

[0450] The activation of FcyRlla by the TNFR2 agonists was quantified using reporter BW5147 cells expressing human FcyRIIA as a chimeric molecule providing the ectodomain of the FcyRIIA (haplotypes H131 (Figure 4A) and R131 (Figure 4B)) fused to the transmembrane domain and cytosolic tail of mouse CD3^. In response to FcyR activation, reporter cells secrete mlL-2 which was then quantified by ELISA. To quantify FcyRlla activation, TNFR2-expressing target cells (Colo205) were incubated with titrated amounts of the TNFR2 agonists in DM EM, supplemented with 10% FCS for 30 min at 37°C, 5% CO2. Cells were washed with RPMI and cocultured with BW5147-reporter cells (ratio E:T 20:1) for 16 hr at 37°C in a 5% CO2 atmosphere. Reporter cell mlL-2 secretion was quantified by subsequent anti mouse IL-2 sandwich ELISA. Each sample was analyzed in triplicates and data were analyzed using the software Microsoft Excel and GraphPad Prism (Figure 4A,B).

[0451] The activation of FcyRlla by the TNFR2 agonists was also quantified using FcyRlla-H reporter cells (Promega Cat.# G9901) expressing the human FcyRIIA-H haplotype (H131) (Figure 4C). Upon FcyRlla activation, reporter cells lead to NFAT-response element-mediated luciferase activity. To quantify FcyRlla activation, TNFR2-high-expressing target cells (HeLa-TNFR2) were incubated with titrated amounts of scTNFR2-Fc muteins in RPMI, supplemented with 4% low-IgG serum and cocultured with FcyRlla-H effector cells (ratio E:T 2:1) for 6 hours at 37°C in a 5% CO2atmosphere. Reporter cell luciferase activity was quantified by the addition of Bio-Gio™ Reagent for 10 min and subsequent detection using the CLARIOstarplusmicroplate reader. Each sample was analyzed in duplicates and data were analyzed using the software Microsoft Excel and GraphPad Prism (Figure 4C).

[0452] Whereas SCTNFR2-FC[WT] and scTNFR2-Fc[Aab] dose-dependently induced activation of FcyRIIA(H131) (Figure 4A,C) and FcyRIIA(R131) (Figure 4B), no response was observed upon stimulation with scTNFR2-Fc[LALA-Aa], confirming that scTNFR2-Fc[LALA-Aa] in contrast to scTNFR2-Fc[Aab] does not activate FcyRIIA.

[0453] Example 4 - Alleviation of disease in EAE mice

[0454] To evaluate the therapeutic potential of the TNFR2 agonist scTNFR2-Fc mutein format (i.e. the scTNFR2-Fc[LALA-Aa] as referred to in Examples 1-3 above), the impact of a mouse TNFR2- specific sc-mTNFR2-Fc mutein treatment was evaluated, compared to the mouse TNFR2 agonist prototype p53-sc-mTNFR2 using the EAE model.

[0455] It will be understood that the TNFR2 agonist scTNFR2-Fc mutein may be in the form of a dimer of two monomers of sc-mTNFR2, where each sc-mTNFR2 itself comprises three THDs (i.e. each sc-mTNFR2 is trivalent). Thus, the dimeric TNFR2 agonist scTNFR2-Fc mutein may be considered as hexavalent sc-mTNFR2-Fc mutein.

[0456] It will be understood that p53-sc-mTNFR2 is in the form of a tetramer of four monomers of sc- mTNFR2, where each monomeric sc-mTNFR2 itself comprises three THDs (i.e. the sc-mTNFR2 is trivalent). Thus, a p53-sc-mTNFR2may be considered as a dodecavalent binding molecule (i.e. a dodecavalent TN FR2 agonist p53-sc-mTNFR2). To this end, female or male C57BL / 6J mice were immunized using subcutaneous injections containing an emulsion of 100 g of MOG35-55 peptide and complete Freund's adjuvant (CFA; InvivoGen, San Diego, California) supplemented with 200 pg of heat-inactivated Mycobacterium tuberculosis H37Ra in CFA. These injections were administered 50 pL each to the posterior right and left flanks. All mice received a booster MOG / CFA injection 7 days after the first injection. Mice were either treated with the, regarding the TNF subunits, dodecavalent TNFR2 agonist p53-sc-mTNFR2 (Figure 5A) or the hexavalent sc-mTNFR2-Fc mutein (Figure 5B) on days 10, 13 and 16 post immunization. Mice were evaluated daily for both body weight changes and signs of motor deficits. Motor scores were assigned based on the following: 0 - no motor deficits, 1 - loss of tail tone, 2 - fully flaccid tail, 3 - hind-limb paralysis, 4 - forelimb paralysis, 5 - moribund, 6 - death.

[0457] Both p53-sc-mTNFR2 and sc-mTNFR2-Fc alleviated motor disease in female mice, but surprisingly, only sc-mTNFR2-Fc promoted disease alleviation in male animals (Table 2). This therapeutic effect in males was in part dependent on the impact of sc-mTNFR2-Fc on supressing the development of motor disease, i.e. inducing the incident of disease development (Figure 6).

[0458] Table 2 - Motor disease area under the curve (AUC)

[0459] Mechanical allodynia was assessed using the Von Frey test. Therefore, Von Frey hair monofilaments were used to assess sensitivity to a mechanical stimulus administered to the hind paw of each mouse. Mice were placed in individual clear plexiglass chambers elevated on a mesh-wire platform and allowed to acclimate for 30 min. The von Frey hair filaments were applied to the plantar surface of each hind paw using the up-down method of analysis. If no reaction is seen to the administered monofilament, a monofilament with a slightly greater diameter is used. This is done for a series of 5 trials once the first positive pain response is noted. The paw withdrawal threshold is then measured in grams (g) of force, with thresholds typically ranging from 0.0 to 2.0 g. Hind paw withdrawal was only considered a pain response if paired with at least one or more of the following actions: looking at the hind paw, stimulus avoidance behaviour, tucking the tail under stimulated hind paw, and / or licking the stimulated hind paw, which are indicative of cognitive awareness of the stimulus as being painful. Baseline testing was done a week prior to EAE induction and then pain was assessed on a weekly basis.

[0460] Both p53-sc-mTNFR2 and sc-mTNFR2-Fc alleviated mechanical allodynia in female and male mice (Figure 7). In contrast to p53-sc-mTNFR2, sc-mTNFR2-Fc promoted faster recovery of males and females, highlighted by the stronger increase in the withdrawal threshold on day 14 post immunization.

[0461] Example 5 - Alleviation of neuropathic pain after spinal cord injury

[0462] Adult female C57BL / 6J mice (3 months old) purchased from Jackson Laboratory were acclimated for at least 1 week before undergoing surgery. To this end, following anaesthesia with ketamine (100 mg / kg) and xylazine (10 mg / kg), a laminectomy was performed at thoracic level T9 and the exposed spinal cord was contused using the Infinite Horizon impactor (Precision Systems and Instrumentation, LLC) with a desired force of 65 kDynes. Following injury, muscles of the back, as well as the skin, were sutured. After injury, lactated Ringer's solution was administered subcutaneously twice daily for at least 7 days to prevent dehydration, buprenorphine (0.1 mg / kg) twice a day for 2 days for pain relief and gentamicin (80 mg / kg) once a day for 7 days as a preventive measure against bladder infection. Bladders were expressed twice daily until normal function was recovered. Mice were either treated with the TNFR2 agonist EHD2-sc-mTNFR2 (Figure 8A) or sc-mTNFR2-Fc (Figure 8B) on days 7, 10 and 13 post injury.

[0463] Mechanical allodynia was assessed using the Von Frey test. Therefore, Von Frey hair monofilaments were used to assess sensitivity to a mechanical stimulus administered to the hind paw of each mouse. Mice were placed in individual clear plexiglass chambers elevated on a mesh-wire platform and allowed to acclimate for 30 min. The von Frey hair filaments were applied to the plantar surface of each hind paw using the up-down method of analysis. If no reaction is seen to the administered monofilament, a monofilament with a slightly greater diameter is used. This is done for a series of 5 trials once the first positive pain response is noted. The paw withdrawal threshold is then measured in grams (g) of force, with thresholds typically ranging from 0.0 to 2.0 g. Hind paw withdrawal was only considered a pain response if paired with at least one or more of the following actions: looking at the hind paw, stimulus avoidance behaviour, tucking the tail under stimulated hind paw, and / or licking the stimulated hind paw, which are indicative of cognitive awareness of the stimulus as being painful. Baseline testing was done a week prior to surgery. Then pain was assessed on a weekly basis starting two weeks after injury.

[0464] Both EHD2-sc-mTNFR2 and sc-mTNFR2-Fc alleviated mechanical allodynia after spinal cord injury (Figure 8). However, in contrast to EHD2-sc-mTNFR2, sc-mTNFR2-Fc promoted faster recovery observable 2 weeks after injury, where pain sensitivity of sc-mTNFR2-Fc-treated animals was back at baseline, whereas EHD2-sc-mTNFR2-treated mice did not show improved pain tolerance at this point in time.

[0465] Non-reflexive pain testing was performed using the mechanical conflict avoidance test. Therefore, a conflict avoidance system (Noldus) is used. Rodents are placed on one side of a height-adjustable array of nociceptive probes and given the opportunity to cross the array to escape from an aversive lighted area to a preferred dark area. The array consists of blunt tapered probes that are painful but not sharp enough to cause any tissue damage when walked on by the rodent. The Mechanical Conflict-Avoidance System contains an aversive (lit) area, an attractive (dark) area, and a walkway in between with blunt tapered probes. This system is used to determine the level of pain felt by an animal in correlation with their desire to return to the dark side of the testing chamber. Therefore, a mouse is placed in chamber for 20 minutes with spikes set to 0.5 mm. This will allow the mouse to learn the chamber and understand where there are spikes and are not. Both spike wheels on the left and right side were adjusted to the same level. After 20 minutes, the door with the mouse on one side of the apparatus was closed. The spike height was adjusted to 1 mm, which served as the first height for testing. An industrial bright light was placed over the closed chamber containing the mouse for 20 seconds. Then, the door was opened and the time it took the mouse to travel from light to dark area was recorded. The time was started, when all 4 paws had left the light and stopped, when all 4 have reached the dark. When the mouse reached the dark side, the door was closed behind them, and the mice were kept in the for 1 minute as a reward. Then, the industrial light was placed back over the chamber with the mouse and the described steps were repeated while gradually increasing the spike height.

[0466] Next to alleviating pain in stimulus-dependent pain testing, both EHD2-sc-mTNFR2 and sc- mTNFR2-Fc alleviated non-stimulus-dependent pain, i.e. decreased the latency time in the conflict avoidance test. However, a stronger effect was observed with sc-mTNFR2-Fc. (Figure 9). Example 6 - Microqlia-TNFR2 promotes pain recovery

[0467] To study the mechanisms of TNFR2-mediated pain recovery, CX3CR1cre:TNFR2fl / flmice were used where TNFR2 on can be deleted on microglial cells. Recombination was induced by 5 subsequent daily injections of tamoxifen. This led to deletion of TNFR2 in peripheral myeloid cells and central microglial cells. 4 weeks later, peripheral monocytes were replenished from the bone marrow, while deletion in self-renewing microglia was permanent. TNFR2fl / flmice injected with tamoxifen were used as controls. Then neuropathic pain was induced by chronic constriction injury (CCI). Therefore, mice were anesthetized via intraperitoneal (i.p.) injection of a ketamine (100 mg / kg) and xylazine (10 mg / kg) cocktail. Then, the right sciatic nerve was exposed at the level of the mid-thigh and 3 loose silk sutures (MV-711 , Oasis, Mettawa, IL) were tied around the sciatic nerve with a 1.0-1.5 mm interval between each ligature. The wound was closed with 6-0 Ethilon monofilament nylon. Sham operated animals underwent a similar procedure with exposure of the sciatic nerve without ligation. Mice then were randomly divided into treatment groups. Treatments with saline or sc- mTNFR2-Fc (10 mg / kg) were performed i.p. on days 7, 10, and 13 post injury. Upon sacrifice, an incision was applied at the level of the right thigh and the right sciatic nerve was exposed to verify that the ligatures were still intact. All behavioural analyses were performed by experimenters blinded to the surgery and the genotype or treatment. Mice were handled daily for at least 1 week prior to any behavioural analysis.

[0468] Mechanical allodynia was assessed using the Von Frey test. Therefore, Von Frey hair monofilaments were used to assess sensitivity to a mechanical stimulus administered to the hind paw of each mouse. Mice were placed in individual clear plexiglass chambers elevated on a mesh-wire platform and allowed to acclimate for 30 min. The von Frey hair filaments were applied to the plantar surface of each hind paw using the up-down method of analysis. If no reaction is seen to the administered monofilament, a monofilament with a slightly greater diameter is used. This is done for a series of 5 trials once the first positive pain response is noted. The paw withdrawal threshold is then measured in grams (g) of force, with thresholds typically ranging from 0.0 to 2.0 g. Hind paw withdrawal was only considered a pain response if paired with at least one or more of the following actions: looking at the hind paw, stimulus avoidance behaviour, tucking the tail under stimulated hind paw, and / or licking the stimulated hind paw, which are indicative of cognitive awareness of the stimulus as being painful. Baseline testing was done a week prior to EAE induction and then pain was assessed on a weekly basis. TNFR2fl / fltreated with sc-mTNFR2-Fc showed recovery starting 2 weeks post injury showing full recovery week 3 post injury. In contrast, CX3CR1cre:TNFR2fl / flmice did not fully recover, suggesting that microglia-TNFR2 is important for the therapeutic effect mediated by systemically administered sc-mTNFR2-Fc (Figure 10). It will be understood that sc-mTNFR2-Fc used herein comprises the “Fc[LALA-Aa]” described above.

[0469] Example 7 - Neuronal-TNFR2 promotes pain recovery

[0470] To study the mechanisms of TNFR2-mediated pain recovery, NEXcre:TNFR2fl / flmice were used where TNFR2 on can be deleted on neuronal cells. Recombination was induced by 5 subsequent daily injections of tamoxifen. TNFR2fl / flmice injected with tamoxifen were used as controls. 4 weeks post tamoxifen treatment, neuropathic pain was induced by chronic constriction injury (CCI). Therefore, mice were anesthetized via intraperitoneal (i.p.) injection of a ketamine (100 mg / kg) and xylazine (10 mg / kg) cocktail. Then, the right sciatic nerve was exposed at the level of the mid-thigh and 3 loose silk sutures (MV-711 , Oasis, Mettawa, IL) were tied around the sciatic nerve with a 1.0-1.5 mm interval between each ligature. The wound was closed with 6-0 Ethilon monofilament nylon. Sham operated animals underwent a similar procedure with exposure of the sciatic nerve without ligation. Mice then were randomly divided into treatment groups. Treatments with saline or sc-mTNFR2-Fc (10 mg / kg) were performed i.p. on days 7, 10, and 13 post injury. Upon sacrifice, an incision was applied at the level of the right thigh and the right sciatic nerve was exposed to verify that the ligatures were still intact. All behavioural analyses were performed by experimenters blinded to the surgery and the genotype or treatment. Mice were handled daily for at least 1 week prior to any behavioural analysis.

[0471] Mechanical allodynia was assessed using the Von Frey test. Therefore, Von Frey hair monofilaments were used to assess sensitivity to a mechanical stimulus administered to the hind paw of each mouse. Mice were placed in individual clear plexiglass chambers elevated on a mesh-wire platform and allowed to acclimate for 30 min. The von Frey hair filaments were applied to the plantar surface of each hind paw using the up-down method of analysis. If no reaction is seen to the administered monofilament, a monofilament with a slightly greater diameter is used. This is done for a series of 5 trials once the first positive pain response is noted. The paw withdrawal threshold is then measured in grams (g) of force, with thresholds typically ranging from 0.0 to 2.0 g. Hind paw withdrawal was only considered a pain response if paired with at least one or more of the following actions: looking at the hind paw, stimulus avoidance behaviour, tucking the tail under stimulated hind paw, and / or licking the stimulated hind paw, which are indicative of cognitive awareness of the stimulus as being painful. Baseline testing was done a week prior to EAE induction and then pain was assessed on a weekly basis.

[0472] TNFR2fl / fltreated with sc-mTNFR2-Fc showed recovery starting 2 weeks post injury. In contrast, NEXcre:TNFR2fl / flmice did not show recovery 5 weeks post injury, highlighting that neuronal- TNFR2 is important for the therapeutic effect mediated by systemically administered sc- mTNFR2-Fc (Figure 11). It will be understood that sc-mTNFR2-Fc used herein comprises the “Fc[LALA-Aa]” described above.

[0473] Example 8 - Lack of FcyRllla activation by SCTNFR2-FC muteins

[0474] The activation of FcyRllla by the TNFR2 agonists was quantified using FcyRllla reporter cells (Promega Cat.# G7015). Upon FcyRllla activation, reporter cells lead to NFAT-response element-mediated luciferase activity. To quantify FcyRllla activation, TNFR2-high-expressing target cells (HeLa-TNFR2, Figure 12A) were incubated with titrated amounts of SCTNFR2-FC muteins in RPMI, supplemented with 4% low-IgG serum and co-cultured with FcyRllla effector cells (ratio E:T 1.5:1) for 6 hours at 37°C in a 5% CO2 atmosphere. Reporter cell luciferase activity was quantified by the addition of Bio-Gio™ Reagent for 5-30 min and subsequent detection using the CLARIOstarplusmicroplate reader (Figure 12B). Each sample was analyzed in duplicates and data were analyzed using the software Microsoft Excel and GraphPad Prism.

[0475] Whereas SCTNFR2-FC[WT] dose-dependently induced activation of FcyRIIIA (Figure 12B), no response was observed upon stimulation with either scTNFR2-Fc[Aab] or SCTNFR2-FC[LALA- Aa], confirming that scTNFR2-Fc[LALA-Aa] does not activate FcyRIIIA.

[0476] Example 9 - Alleviation of chronic pain in a chemically induced OA model

[0477] To demonstrate the therapeutic relevance of TNFR2 agonism for chronic pain associated with osteoarthritis (OA), we assessed the impact of sc-mTNFR2-Fc administration in the monosodium iodoacetate (MIA) model of OA pain. It will be understood that sc-mTNFR2-Fc used herein comprises the “Fc[LALA-Aa]” described above. Intra-articular (i.a.) administration of MIA in the ipsilateral knee of Sprague Dawley (SD) rats leads to development of robust and long-lasting hyperalgesia and allodynia associated with an inflammatory response. The development of these signs in this animal model are believed to be clinically relevant; reflecting the symptoms displayed by patients presenting with chronic inflammatory pain associated with underlying conditions such as osteoarthritis (OA) or rheumatoid arthritis. The pain in this model follows a bi-phasic mechanism, with an early, predominantly inflammatory, component. This early inflammatory phase gives way to a more chronic neuropathic-type pain phenotype.

[0478] Chronic pain associated with OA was induced via intra-articular injection of 2 mg MIA (25 pl of 80 mg / mL) into the left knee. Sham animals underwent the same procedure with 25pl saline instead of MIA. On day 0, following the final baseline reading, animals were anaesthetised using isoflurane and oxygen mixed 3:1 in sterile conditions. The left knee area was shaved and cleaned with a dilute Hibi scrub solution. OA was induced via injection of MIA as described above. Animals were allowed to recover in a warmed environment, before being returned to their home cage. After MIA injection, animals develop an inflammatory response and may guard and lick the affected area. Therefore, rats were carefully monitored for unexpected signs of distress or severe pain, so that any animal displaying such signs could be culled immediately. Animals were weighed daily for 3 days post injection.

[0479] Animals were weighed at the start of the study, pre- and post-MIA to track any changes in weight. Animals were then weighed on all test days and before the administration of compounds to precisely adjust the dose. Animals experiencing weight loss >15% of the weight prior to MIA induction were culled immediately by a schedule 1 method.

[0480] Measurement of static weight bearing (WB) (calculated as % ratio ipsilateral reading and contralateral reading) through each hind limb was achieved using sensor pads on a rat incapacitance tester (Linton Instruments, UK). Naive rats distribute their body weight equally between the two hind paws. However, when the injected (left) hind knee is inflamed and / or painful, the weight is re-distributed so that less weight is put on the affected limb (decrease in WB on injured limb). Naive rats were acclimatized to the procedure room for at least 1 hour in their home cages, with food and water available ad libitum. Animals were typically habituated to the apparatus over 3 sessions. Rats were placed in the incapacitance tester with hind paws on the separate sensors and the average force exerted by both hind limbs (ipsi and contra) was recorded over 2 seconds.

[0481] Animals underwent a pre-MIA (baseline) assessment, performed from day -3 to -1 for WB to check absence of alteration of values. An inflammatory baseline post-M IA was measured (predosing) for all animals injected with MIA (n=29 group B-D) and saline (n=6 group A; Vehicle Sham,) on day 3. Animals were ranked and randomized to treatment groups (n=6 group A; n=10 per groups B and C; n=9 group D) according to their MIA window (based on a Latin square design). Further WB measurements were taken at 2 hours post compound dosing on days 7, 14, 21 , 28 & 34. WB (g) readings were taken for both right and left hind paws and the difference in weight distribution (ratio) was calculated as follows. Data are expressed as % ratio ipsilateral / contralateral ((WB left / WB right)*1OO) (mean ± SEM).

[0482] Treatment was started on day 7 post MIA-injection and repeated twice weekly during the duration of the study (Figure 13A). Whereas sc-mTNFR2-Fc was administered using a dose of 5 mg / kg via s.c. administration, Pregabalin was applied p.o using a dose of 30 mg / kg. On each day of administration animals were weighed to precisely adjust the daily dose. The volume administered was 5 mL / kg and was administered subcutaneously (s.c.) or per orally (p.o.) via a plastic gavage. Syringe and gavage tube were filled immediately prior to dosing and formulations were under constant stirring throughout.

[0483] Both sc-mTNFR2-Fc and Pregabalin alleviated pain hypersensitivity in the MIA model of chronic pain associated with osteoarthritis (Figure 13B), indicating the suitability of TNFR2 agonists to treat chronic pain is associated with osteoarthritis.

[0484] Example 10 - Alleviation of chronic pain in a post-traumatic model of chronic pain associated with osteoarthritis

[0485] The impact of the route of administration on the therapeutic effect of sc-mTNFR2-Fc on chronic pain associated with osteoarthritis was evaluated using the DMM model of post-traumatic OA. It will be understood that sc-mTNFR2-Fc used herein comprises the “Fc[LALA-Aa]” described above.

[0486] OA was induced by DMM surgery of the right leg leading to progressive development of pain hypersensitivity. To this end, animals were anesthetized using 4-5% isoflurane and maintained with 1.5-2% isoflurane during the procedure. After shaving the knee with razors, the surgical site was sterilized with PVP iodine wipes and alcohol (3x) and the mouse eyes were covered with ointment. Then, a 1 cm longitudinal medial para-patellar incision to expose the knee joint was made, and the knee joint was opened gently through the lateral patella and patellar ligament dislocation. The medial meniscotibial ligament, which anchors the medial meniscus to the tibial plateau was cut, while avoid injuring the cartilage beneath the medial meniscus. After the procedure, the skin was closed with a 6-0 absorbable suture.

[0487] Treatment with the TNFR2 agonist was started 4 weeks post-surgery, when animals had developed knee pain, by either systemic intraperitoneal (Figure 15A) or local intra-articular injection (Figure 14A). The local intra-articular injections of sc-mTNFR2-Fc were applied once a week. All intra-articular injections were applied under 1.5-2% isoflurane anesthesia, withlO pL, Neuros Syringe, Model 1701 RN, 33 gauge, Point Style 4. Similarly, systemic intraperitoneal application was repeated once weekly.

[0488] Pain was assessed by Von Frey test for mechanical allodynia at multiple time points postsurgery. Using a predetermined set of 8 von Frey (vF) monofilaments (0.008-6 g, Stoelting) with the up-down method, hind paw 50% mechanical withdrawal thresholds was measured (Chaplan SR, et al., J Neurosci Methods. 1994 Jul;53(1):55-63.). Mice were acclimated for at least 30 minutes in an opaque acrylic box on an elevated wire mesh platform. The vF hair was applied perpendicular to the proximal region of the glabrous skin on the plantar surface of the hind paw adjacent to the incision site. Each trial commenced with applying an intermediate filament (0.16 g) for 3 s, causing slight filament bending. The next smallest filament was tested in the event of a positive response (i.e., rapid withdrawal or licking of the paw within 3 s of filament removal). If a negative response was observed, 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 (Dixon WJ, J Am Stat Assoc. 1965;60:967-978.).

[0489] Both local intra-articular (Figure 14B) and systemic intraperitoneal (Figure 15B) application of sc-mTNFR2-Fc resulted in alleviated pain in the treated animals, indicating the suitability of TNFR2 agonists to treat chronic pain associated with osteoarthritis using different administration routes.

[0490] Example 11 - Alleviation of disease in EAE mice

[0491] To evaluate the therapeutic potential of the human TNFR2 agonist SCTNFR2-FC mutein the EAE model of multiple sclerosis was used and the impact of systemic SCTNFR2-FC administration on motor disease and pain hypersensitivity was assessed. It will be understood that sc-mTNFR2-Fc used herein comprises the “Fc[LALA-Aa]” described above.

[0492] To this end, huTNFR2-k / i C57BL / 6J mice were immunized using subcutaneous injections containing an emulsion of 100 pg of MOG35-55 peptide and complete Freund's adjuvant (CFA; InvivoGen, San Diego, California) supplemented with 200 pg of heat-inactivated Mycobacterium tuberculosis H37Ra in CFA. These injections were administered 50 pL each to the posterior right and left flanks. All mice received a booster MOG / CFA injection 7 days after the first injection. Mice were treated with the sc-mTNFR2-Fc mutein (Figure 16A) on days 10, 13 and 16 post immunization. Mice were evaluated daily for both body weight changes and signs of motor deficits. Motor scores were assigned based on the following: 0 - no motor deficits, 1 - loss of tail tone, 2 - fully flaccid tail, 3 - hind-limb paralysis, 4 - forelimb paralysis, 5 - moribund, 6 - death.

[0493] Mechanical allodynia was assessed using the Von Frey test. Therefore, Von Frey hair monofilaments were used to assess sensitivity to a mechanical stimulus administered to the hind paw of each mouse. Mice were placed in individual clear plexiglass chambers elevated on a mesh-wire platform and allowed to acclimate for 30 min. The von Frey hair filaments were applied to the plantar surface of each hind paw using the up-down method of analysis. If no reaction is seen to the administered monofilament, a monofilament with a slightly greater diameter is used. This is done for a series of 5 trials once the first positive pain response is noted. The paw withdrawal threshold is then measured in grams (g) of force, with thresholds typically ranging from 0.0 to 2.0 g. Hind paw withdrawal was only considered a pain response if paired with at least one or more of the following actions: looking at the hind paw, stimulus avoidance behaviour, tucking the tail under stimulated hind paw, and / or licking the stimulated hind paw, which are indicative of cognitive awareness of the stimulus as being painful. Baseline testing was done a week prior to EAE induction and then pain was assessed on a weekly basis.

[0494] Importantly, SCTNFR2-FC alleviated both motor disease (Figure 16B), as well as mechanical allodynia (Figure 16C) in female and male mice. This demonstrates the suitability of TNFR2 agonists to treat multiple sclerosis.

[0495] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific preferred embodiments, it should be understood that the disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure 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 tumour necrosis factor receptor 2 (TNFR2) agonist polypeptide comprising (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.

2. The TNFR2 agonist polypeptide according to claim 1 wherein (a) the Fc domain (i) does not comprise a proline at position 233 and / or (ii) comprises a glycine at position 236; and / or (b) the Fc domain comprises a sequence with at least 85% sequence identity to SEQ ID NO: 1.

3. The TNFR2 agonist polypeptide according to claim 1 or claim 2 wherein each THD comprises an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 2; optionally wherein each THD independently comprises 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, Q84N / A141 I / E142G / S143D, A141 H / E142S / S143D, A141 H / S143D,L25V / A141 D / E142D / S143D, A141 N / E142D / S143D, A141T / E142S / S143D,A141Q / E142D / S143D, A141T / E142D / S143D, A141 D / E142G / S143D, A141 D / S143D,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, preferably selected from D139N and A141 R, in the corresponding position of the sequence of SEQ ID NO: 2.

4. The TNFR2 agonist polypeptide according to any preceding claim wherein (a) 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; and / or (b) 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.

5. The TNFR2 agonist polypeptide according to any preceding claim, wherein the TNFR2 binding domain comprises or consists of a sequence with at least 80% sequence identity to SEQ ID NO: 5.

6. The TNFR2 agonist polypeptide according to any preceding claim wherein the TNFR2 binding domain is joined to the Fc domain by a flexible linker consisting of 3-20 amino acids; optionally wherein the flexible linker is a Ser-Gly linker; preferably GGSGGGGSGG (SEQ ID NO: 6).

7. The TNFR2 agonist polypeptide according to any preceding claim, wherein the TNFR2 agonist polypeptide comprises a sequence with at least 90% sequence identity to SEQ ID NO:7.

8. A TNFR2 agonist multimer wherein the TNFR2 agonist multimer is in the form of a dimer comprising two TNFR2 agonist polypeptides, wherein each TNFR2 agonist polypeptide is independently a TNFR2 agonist polypeptide according to any preceding claim; optionally wherein each TNFR2 agonist polypeptide in the dimer is identical.

9. A polynucleotide encoding a TNFR2 agonist polypeptide according to any of claims 1 to 7.

10. A vector comprising a polynucleotide according to claim 9.

11. A pharmaceutical composition comprising a TNFR2 agonist polypeptide according to any of claims 1 to 7, a TNFR2 agonist multimer according to claim 8, a polynucleotide according to claim 9 or a vector according to claim 10.

12. A TNFR2 agonist comprising three TNF homology domains (THD) that specifically bind to TNFR2 and an Fc domain for use in treating and / or preventing chronic pain or multiple sclerosis.

13. The TNFR2 agonist for use according to claim 12, wherein the TNFR2 agonist is a TNFR2 agonist polypeptide according to any of claims 1 to 7, a TNFR2 agonist multimer according to claim 8, a polynucleotide according to claim 9, a vector according to claim 10, or provided as a pharmaceutical composition according to claim 11.

14. The TNFR2 agonist for use according to claim 12 or claim 13, wherein the TNFR2 agonist induces TNFR2 signalling in neurons; optionally wherein the neurons are supraspinal neurons, preferably Nex+ supraspinal neurons.

15. The TNFR2 agonist for use according to any of claims 12 to 14, wherein the TNFR2 agonist is administered to a subject, wherein the administration is selected from the list consisting of: systemic administration, intravenous administration, intraparenteraladministration, subcutaneous administration, intrathecal administration, and intraarticular administration.

16. The TNFR2 agonist for use according to any of claims 11 to 15, wherein the chronic pain is associated with osteoarthritis.

Images