Combination comprising a tumor necrosis factor receptor 2 agonist

Abstract

The present invention relates to a combination comprising a Tumor necrosis factor (TNF) inhibitor and a Tumor necrosis factor receptor 2 (TNFR2) agonist.

Description

[0001] COMBINATION COMPRISING A TUMOR NECROSIS FACTOR RECEPTOR 2 AGONIST

[0002] FIELD

[0003] The present disclosure relates to approaches for modulating tumour necrosis factor (TNF) signalling by targeting multiple aspects of the TNF signalling pathway. In particular, provided herein is a combination comprising a TNF inhibitor and a Tumor necrosis factor receptor 2 (TNFR2) agonist.

[0004] BACKGROUND

[0005] The TNF superfamily is a family of structurally related cytokines with various functions.

[0006] TNF itself is a multifunctional cytokine with pleiotropic functions. It is a master regulator of the immune system and a key player in the initiation and orchestration of inflammation and immunity. TNF, like most ligands of the superfamily, is synthesized as a trimeric type 2 transmembrane protein (tmTNF) that can be proteolytically processed into soluble circulating TNF homotrimers (sTNF). Interestingly, sTNF and tmTNF differ in their capability to activate the two distinct TNF receptors (TNFRs): TNFR1 and TNFR2. Whereas TNFR1 is activated by both sTNF and tmTNF, TNFR2 is dependent on tmTNF to be robustly activated (Muhlenbeck et al, 2000, J. Biol., Chem. 275, 32208-32213; Wajant et al, 2001 , Oncogene 20, 4101-4106).

[0007] There remains a need for improved approaches for targeting TNF pathways.

[0008] SUMMARY

[0009] The present disclosure provides a surprising provision of a combination of a TNF inhibitor and a TNFR2 agonist.

[0010] In a particular embodiment, the combination comprises a TNF mutein, which acts as a TNFR2 agonist, and a TNF inhibitor which binds to both sTNF and tmTNF. In particular, the TNF inhibitor is capable of binding to endogenous (e.g. wild-type) sTNF and tmTNF but does not bind the TNF mutein. The TNF mutein is therefore capable of inducing signalling via TNFR2 in the presence of the TNF inhibitor.

[0011] As such, the TNF inhibitor is capable of binding sTNF in order to reduce, or abrogate, TNF signalling through TNFR1 . The TNF inhibitor may also bind to endogenous tmTNF and reduce or prevent its signalling via TNFR2. However, because the TNF inhibitor does not bind the TNF mutein (or other TNFR2 agonist), the TNF mutein (or other TNFR2 agonist) is able to induce signalling via TNFR2 and promote its associated activities (e.g. immune suppression, tissue homeostasis and regeneration). Without wishing to be bound by theory, the present combination may therefore provide the advantageous anti-inflammatory effects of a TNFR1 inhibition along with the regenerative effects of TNFR2-mediated signalling (e.g. immune suppression, tissue homeostasis and regeneration).

[0012] Accordingly, in a first aspect the present invention provides a combination comprising a Tumor necrosis factor (TNF) inhibitor and a Tumor necrosis factor receptor 2 (TNFR2) agonist.

[0013] In a further aspect there is provided a combination comprising a TNF inhibitor and a TNFR2 agonist, wherein the TNFR2 agonist is a TNF mutein and the TNF inhibitor is capable of binding to TNF but does not bind the TNF mutein.

[0014] Further provided is a composition comprising a TNF inhibitor and a TNFR2 agonist as defined in the first or further aspect.

[0015] Also provided is a polynucleotide encoding a TNF inhibitor and a TNFR2 agonist as defined in the first or further aspect.

[0016] Further provided is a composition comprising a first polynucleotide encoding a TNF inhibitor and a second polynucleotide encoding a TNFR2 agonist as defined in the first or further aspect.

[0017] Also provided is a pharmaceutical composition comprising a TNF inhibitor and a TNFR2 agonist as defined in the first or further aspect, a polynucleotide, or a first and second polynucleotide as defined herein.

[0018] Also provided is (i) a kit comprising a TNF inhibitor and a TNFR2 agonist as defined in the first or further aspect, (ii) a kit comprising a first polynucleotide encoding a TNF inhibitor and a second polynucleotide encoding a TNFR2 agonist as defined in the first or further aspect, or (iii) a kit comprising a composition comprising a TNF inhibitor and a composition comprising TNFR2 agonist as defined in the first or further aspect.

[0019] Also provided is a method of treatment comprising administering to a subject in need thereof (i) a TNF inhibitor, as defined herein, and (ii) a TNFR2 agonist, as defined herein.

[0020] Also provided is a method of treatment comprising administering to a subject in need thereof (i) a TNF inhibitor, as defined herein, and (ii) a TNFR2 agonist, as defined herein, wherein the TNFR2 agonist is a TNF mutein, and the TNF inhibitor is capable of binding to TNF but does not bind the TNF mutein. Also disclosed is a combination comprising a TNF inhibitor and a TNFR2 agonist, for use in treating or preventing a disease.

[0021] Also disclosed is a combination comprising a TNF inhibitor and a TNFR2 agonist, wherein the TNFR2 agonist is a TNF mutein and the TNF inhibitor is capable of binding to TNF but does not bind the TNF mutein for use in treating or preventing a disease.

[0022] Further disclosed is a combination comprising a TNF inhibitor and a TNFR2 agonist for use in treating acute pain.

[0023] Further disclosed is a combination comprising a TNF inhibitor and a TNFR2 agonist, wherein the TNFR2 agonist is a TNF mutein and the TNF inhibitor is capable of binding to TNF but does not bind the TNF mutein for use in treating acute pain.

[0024] BRIEF DESCRIPTION OF THE FIGURES

[0025] Figure 1 - Adalimumab does not interact with SCTNFR2-FC. Interaction of the TNF inhibitors Adalimumab, Infliximab and Golimumab with immobilized scTNFR2-Fc analyzed by enzyme- linked immunosorbent assay (ELISA). n=1 , mean±range of technical duplicates.

[0026] Figure 2 - Adalimumab does not neutralize SCTNFR2-FC. Functionality of Adalimumab, Infliximab, Golimumab and Certolizumab to neutralize either recombinant human TNF (rhTNF) (A, C) or SCTNFR2-FC (B, D) measured using a TNF reporter cell line. Luminescence was quantified in relative luminescence unit (RLU) 24 hours post stimulation using Bio-Gio reagent. n=1 , mean±range of technical duplicates.

[0027] Figure 3 - Mutations in the TNF region abrogate the interaction between Adalimumab and SCTNFR2-FC. Binding of Adalimumab and Infliximab to CHO-K1 cells expressing (A) non- cleavable wildtype TNF or TNFR2-selective TNFR2 mutein or (B) non-cleavable covalently stabilized trimeric wildtype scTNF or TNFR2-selective SCTNFR2, quantified by flow cytometry. Representative graph of three independent experiments, mean±range of technical duplicates.

[0028] Figure 4: Additional TN FR2 -selective mutations disrupt the Adalimumab-TNF interaction. Binding of Adalimumab to CHO-K1 cells expressing non-cleavable wildtype (WT) TNF or indicated TNFR2-selective TNF muteins, quantified by flow cytometry. Representative graph showing mean±range of technical duplicates.

[0029] Figure 5: Combination of Adalimumab and SCTNFR2-FC leads to superior antiinflammatory macrophage function. CD14+ monocyte-derived macrophages were differentiated into M1-or M2-macrophages in presence or absence of Adalimumab and / or SCTNFR2-FC. After 24 hours (A) ROS release was quantified, (B) phagocytotic activity was analyzed and normalized to number of nuclei (Draq5). n=6; mean±SEM, two-way ANOVA, Tukey's multiple comparison test, ** p<0.01 , *** p<0.001 , **** p<0.0001.

[0030] Figure 6: Combination of Adalimumab and SCTNFR2-FC leads to anti-inflammatory macrophage polarization. Primary CD14+ monocyte-derived macrophages were differentiated into M1- or M2-macrophages in the presence or absence of Adalimumab and / or SCTNFR2. After 24 hours, release of pro-inflammatory MIP-1 and IL-1 p by M1 macrophages and release of anti-inflammatory MDC by M2 macrophages was quantified using MSD multiplex assay. n=6, representative donor shown, ± range

[0031] Figure 7: Combination of adalimumab and SCTNFR2-FC leads to superior inhibition of pro-inflammatory cytokine release. IL-8 concentrations, quantified by ELISA, in harvested supernatant from HUVEC cells stimulated for 24 hours with a cytokine cocktail in the presence or absence of Adalimumab and / or SCTNFR2. n=3±SD, * p<0.05, ** p<0.01.

[0032] Figure 8: The combination of a TNF inhibitor and a TNFR2 agonist provides superior inhibition of ROS release by macrophages. The impact of the combination of an anti-TNF antibody with agonistic TNFR2-selective antibodies on release of ROS by M1 macrophages was analyzed. ROS release was quantified. n=3 donors; mean±SEM, two-way ANOVA, Tukey's multiple comparison test, ** p<0.01 , *** p<0.001 , **** p<0.0001.

[0033] Figure 9: TNFR2 agonist. TNFR2 agonistic activity of SCTNFR2-FC and TNFR2-binding antibody clones 80M2 and MR2-1 was analyzed using TNFR2-reporter cells. Luminescence was quantified in relative luminescence units 24 hours post-stimulation using the Bio-Gio reagent, and the fold change compared to the medium control was calculated. n=1 , mean ± range of technical triplicates.

[0034] DETAILED DESCRIPTION

[0035] TUMOR NECROSIS FACTOR (TNF)

[0036] The tumor necrosis factor (TNF) superfamily is a family of structurally related cytokines with various functions. The structural hallmark defining the TNF ligand family is the carboxyterminal TNF homology domain (THD) which is composed of two stacked b-pleated sheets that adopt a conserved jellyroll-like tertiary fold (Bodmer et al, 2000, Trends Biochem. Sci. 27, 19-26; Fesik, 2000, Cell 103, 273-282; Locksley et ak, 2001 , Cell 104, 487-501). This structural composition leads to the self-association of THD monomers into trimers and is necessary for receptor binding. Due to the carboxy-terminal localization of the THD, both the transmembrane form as well as soluble TNF ligands assemble into trimers. TNF is synthesized as a trimeric type 2 transmembrane protein (tmTNF) that can be proteolytically processed into soluble circulating TNF homotrimers (sTNF). sTNF and tmTNF differ in their capability to activate the two distinct TNF receptors (TNFRs): TNFR1 and TNFR2. Whereas TNFR1 is activated by both sTNF and tmTNF, TNFR2 is dependent on tmTNF to be robustly activated (Muhlenbeck et ak, 2000, J. Biol., Chem. 275, 32208-32213; Wajant et al, 2001 , Oncogene 20, 4101-4106). TNFR1 is constitutively expressed on most cell types, whereas TNFR2 is restricted primarily to endothelial, epithelial, and subsets of neurons and immune cells.

[0037] 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).

[0038] 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).

[0039] TNF inhibitors such as adalimumab, infliximab and golimumab bind both sTNF and tmTNF. This causes inhibition of both pro-inflammatory signalling via TNFR1 as well as inhibition of immune suppression, tissue homeostasis and regeneration via TNFR2. An illustrative TNF amino acid sequence is the human TNF sequence designated as UniProt P01375. An illustrative TNF amino acid sequence is shown as SEQ ID NO: 20.

[0040] SEQ ID NO: 20 - illustrative TNF amino acid sequence

[0041] MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEF PRDLSLISPLAQAVRSSSRTPSDKPVAHWANPQAEGQLQWLNRRANALLANGVELRDNQ LVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAE AKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL

[0042] As used herein, the terms TNF (or endogenous TNF or wild-type TNF) may refer to a polypeptide comprising SEQ ID NO: 20 or a variant thereof. Suitably, the variant may have at least at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 20.

[0043] Accordingly, TNF as referred to herein may comprise one or more mutations (e.g. addition, deletion or substitution) compared to SEQ ID NO: 20.

[0044] Suitably, the TNF may comprise or consist of SEQ ID NO: 20.

[0045] An illustrative TNFR1 amino acid sequence is the human TNFR1 sequence designated as UniProt P19438.

[0046] 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.

[0047] TNF INHIBITOR

[0048] The present disclosure relates to a combination comprising a TNF inhibitor and a TNFR2 agonist.

[0049] Without wishing to be bound by theory, the present combination of a TNF inhibitor and a TNFR2 agonist is surprisingly effective, as it would have been expected that the TNF inhibitor would inhibit / block all TNF signalling, including the desired TNFR2 signalling. As such, it was not necessarily expected that the TNFR2 agonist could induce effective signalling in the presence of a TNF inhibitor, particularly not to the extent demonstrated in the present Examples. The present inventors have surprisingly found that the combination of a TNFR2 agonist and a TNF inhibitor shows synergistic benefit that goes beyond the effects that may have been expected from the two individual components. Firstly, it would have been expected that the use of a TNF inhibitor would abrogate all TNF signalling, through both TNFR2 (positive) and TNFR1 (negative), thereby effectively reducing the signalling to background. Hence, at best, the effect of the combination might have been expected to equal the effect of the TNFR2 agonist alone. However, the inventors have surprisingly demonstrated that the combination shows a significant improvement compared with either individual component alone (see e.g., Examples 5-8). Furthermore, the data provided herein demonstrate that the combination results in dual reprogramming of macrophages, both destabilising and reducing the M1 phenotype, whilst simultaneously upregulating and enhancing the M2 phenotype resulting in superior anti-inflammatory macrophage function, i.e. release of ROS and increased phagocytotic activity (see e.g., Examples 5-8). Thereby, the combination promotes an actual repolarization of the macrophage population to the regenerative M2 phenotype. It was previously not predictable that the combination would have such an effect, e.g., that the TNFR2 agonist effects would outweigh the general TNF inhibition provided by the TNF inhibitor, and that both complementary effects could occur simultaneously and in the correct balance to provide additional synergistic therapeutic benefit.

[0050] Macrophages are pivotal immune cells that play a crucial role in various pathological and physiological indications. They show a high plasticity and are represented by a spectrum of different phenotypes ranging from the pro-inflammatory M1 phenotype to the antiinflammatory and reparative M2 phenotype, thus influencing disease progression and resolution (Chen et al., Signal Transduct. Target Ther. 2023; 8(1):207).

[0051] M1 macrophages are characterised by classical activation, in vitro mimicked typically by IFN- y or lipopolysaccharide (LPS), and release large quantities of proinflammatory cytokines to initiate and maintain immune response. M1 macrophages produce nitric oxide (NO) or reactive oxygen intermediates (ROI) to protect against bacteria and viruses. However, uncontrolled release of these mediators induces oxidative stress and promotes tissue damage.

[0052] M2 macrophages are alternatively activated by exposure to certain cytokines such as IL-4, IL- 10, or IL-13. M2 macrophages produce, for example, either polyamines to induce proliferation or proline to induce collagen production. These macrophages show increased phagocytotic activity to clear tissue debris and are associated with wound healing and tissue repair.

[0053] Suitably, the present combination may reduce the number or activity of M1 macrophages and increase the number or activity of M2 macrophages.

[0054] Without wishing to be bound by theory, the dual reprogramming of macrophages described herein is considered to provide particular therapeutic benefits, for example in respect of both reduced proinflammatory and increased regenerative effects. In a particular aspect, there is provided a combination comprising a TNF inhibitor and a TNFR2 agonist, wherein the TNFR2 agonist is a TNF mutein and the TNF inhibitor is capable of binding to TNF but does not bind the TNF mutein. In one embodiment, there is provided a combination comprising a TNF inhibitor and a TNFR2 agonist, wherein the TNFR2 agonist is a TNF mutein, wherein the TNF mutein comprises a polypeptide comprising a TNFR2 binding domain comprising three TNF homology domains (THD) that specifically bind to TNFR2, and the TNF inhibitor is capable of binding to human TNF but does not bind the TNF mutein.

[0055] A ‘TNF inhibitor’ as defined herein is capable of binding TNF (e.g. endogenous or wild-type TNF). For example, a TNF inhibitor may be capable of binding to human TNF, also called TNF-a or TNF-alpha. For example, a TNF inhibitor may be capable of binding to a polypeptide comprising SEQ ID NO: 20 or a variant thereof.

[0056] As described herein, TNF (e.g. as exemplified by SEQ ID NO: 20) may be present in the form of sTNF or tmTNF.

[0057] As used herein, a ‘TNF inhibitor’ is an agent that is capable of binding sTNF and tmTNF in order to inhibit their functions, for example signalling via TNFR1 . Suitably, a ‘TNF inhibitor’ as referred to herein is capable of binding sTNF and tmTNF to prevent their signalling via TNFR1.

[0058] A TNF inhibitor may decrease (e.g., inhibit) signalling through TNFR1 and TNFR2. In some embodiments, a ‘TNF inhibitor’ is capable of binding sTNF and tmTNF to prevent their signalling via TNFR1 and TNFR2. In some embodiments, a ‘TNF inhibitor’ is a non-selective TNF inhibitor that binds to and inhibits both sTNF and tmTNF. This is in contrast, for example, to selective inhibitors of soluble TNF (such as XPro1595 or SAR441566), which inhibit only TNFR1 signalling. Such selective inhibitors are typically understood to specifically bind soluble TNF (e.g., as compared to tmTNF).

[0059] A TNF inhibitor may decrease (e.g. inhibit) signalling through TNFR1.

[0060] A TNF inhibitor may decrease (e.g., inhibit) signalling through TNFR1 by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, as compared to a control (i.e. corresponding conditions without the TNF inhibitor present). It will also be understood that a TNF inhibitor may decrease (i.e., inhibit) signalling through TNFR1 by at least 2-fold, as compared to a control. Such signalling may be assessed using a reporter assay. Suitably, inhibitors of TNFR1 signalling may be identified using a cellular reporter system where an NFkB reporter gene is fused to a luciferase reporter and activation of the NFkB pathway by TNFR1 activation induces luciferase expression in reporter cells. Suitable reporter cell lines for identifying inhibitors of TNFR1 signalling are commercially available, e.g., from SBI System Biosciences (Catalog # TR860A-1 , NF-KB / 293 / GFP-LUC™ Transcriptional Reporter Cell Line). In such a reporter system, an inhibitor of TNFR1 signalling may decrease the luminescence signal by at least 2 fold as compared to a control.

[0061] A TNF inhibitor may decrease (e.g., inhibit) signalling through TNFR2 by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, as compared to a control (e.g. a corresponding assay without the TNF inhibitor present). It will also be understood that a TNF inhibitor may decrease signalling through TNFR2 by at least 2-fold as compared to a control (e.g. a corresponding assay without the TNF inhibitor present). Such signalling may be assessed using a reporter assay. Inhibitors of TNFR2 signalling may 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 TNF inhibitors (e.g., inhibitors of TNFR1 and TNFR2 signalling) are commercially available, e.g., from InvivoGen (HEK-Blue™ TNF-a cells). Alternatively, a reporter cell line for identifying TNF inhibitors 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 TNF inhibitor may decrease the luminescence signal by at least 2 fold as compared to a control.

[0062] A TNF inhibitor may decrease (e.g., inhibit) signalling through TNFR1 by at least 10% (e.g., at least 20% - as provided above), and decrease (i.e., inhibit) signalling through TNFR2 by at least 10% (e.g., at least 20% - as provided above), for example as tested using both of the reporter assay systems described above.

[0063] Suitably, the TNF inhibitor may be an antibody, a polypeptide, a nucleic acid or a small molecule.

[0064] The TNF inhibitor may be selected from the group consisting of adalimumab, infliximab, etanercept, golimumab, and certrolizumab. Suitably, certrolizumab may be in the form certrolizumab pegol.

[0065] The TNF inhibitor may be a monoclonal anti-TNF antibody. The TNF inhibitor may be a monoclonal antagonistic anti-TNF antibody. The TNF inhibitor may be a monoclonal TNF- blocking antibody. The TNF inhibitor may be selected from the group consisting of adalimumab, infliximab, golimumab, and certrolizumab. Suitably, certrolizumab may be in the form certrolizumab pegol. The TNF inhibitor may be adalimumab or infliximab. Illustrative CDR sequences for TNF inhibitor antibodies (or antibody-like molecules) are provided in Table 2. Suitably, the TNF inhibitor may be an antibody, or fragment thereof comprising the CDRs and / or VH / VL combinations shown in Table 2.

[0066] Table 2

[0067] Suitably, the TNF inhibitor may be an antibody which comprises HCDRs 1-3 which are present in SEQ ID NO: 16 and LCDRs 1-3 which are present in SEQ ID NO: 17.

[0068] Suitably, the TNF inhibitor may be an antibody which comprises the following CDRs:

[0069] HCDR1 : DYAMH (SEQ ID NO: 10) HCDR2: AITWNSGHIDYADSVEG (SEQ ID NO: 11)

[0070] HCDR3: VSYLSTASSLDY (SEQ ID NO: 12)

[0071] LCDR1 : RASQGIRNYLA (SEQ ID NO: 13)

[0072] LCDR2: AASTLQS (SEQ ID NO: 14)

[0073] LCDR3: QRYNRAPYT (SEQ ID NO: 15) The TNF inhibitor may be an antibody which comprises a variable heavy domain (VH) which comprises SEQ ID NO: 16 or a variant which has at least 90% identity to SEQ ID NO: 16; and a variable heavy domain (VL) which comprises SEQ ID NO: 17 or a sequence which has at least 90% identity to SEQ ID NO: 17.

[0074] Suitably, the variant of SEQ ID NO: 16 may have at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 16.

[0075] Suitably, the variant of SEQ ID NO: 17 may have at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 17.

[0076] Suitably, the TNF inhibitor may comprise heavy chain which comprises SEQ ID NO: 18 or a variant which has at least 90% identity to SEQ ID NO: 18; and a light chain which comprises SEQ ID NO: 19 or a variant which has at least 90% identity to SEQ ID NO: 19.

[0077] SEQ ID NO: 18

[0078] EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHID YADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP

[0079] SVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG N VFSCSVM H EALH N H YTQKSLSLSPG K

[0080] SEQ ID NO: 19

[0081] DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRF SGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0082] Suitably, the variant of SEQ ID NO: 18 may have at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 18.

[0083] Suitably, the variant of SEQ ID NO: 19 may have at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 19.

[0084] Suitably, the TNF inhibitor may be adalimumab.

[0085] Suitably, the TNF inhibitor may be an antibody which comprises HCDRs 1-3 which are present in SEQ ID NO: 41 and LCDRs 1-3 which are present in SEQ ID NO: 42.

[0086] Suitably, the TNF inhibitor may be an antibody which comprises the following CDRs:

[0087] HCDR1 : GFIFSNHW (SEQ ID NO: 43) HCDR2: IRSKSINSAT (SEQ ID NO: 44)

[0088] HCDR3: SRNYYGSTYDY (SEQ ID NO: 45)

[0089] LCDR1 : QFVGSS (SEQ ID NO: 46)

[0090] LCDR2: YAS (SEQ ID NO: 47)

[0091] LCDR3: QQSHSWPFT (SEQ ID NO: 48)

[0092] The TNF inhibitor may be an antibody which comprises a variable heavy domain (VH) which comprises SEQ ID NO: 41 or a variant which has at least 90% identity to SEQ ID NO: 41 ; and a variable heavy domain (VL) which comprises SEQ ID NO: 42 or a sequence which has at least 90% identity to SEQ ID NO: 42.

[0093] Suitably, the variant of SEQ ID NO: 41 may have at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 41.

[0094] Suitably, the variant of SEQ ID NO: 42 may have at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 42.

[0095] Suitably, the TNF inhibitor may comprise heavy chain which comprises SEQ ID NO: 65 or a variant which has at least 90% identity to SEQ ID NO: 65; and a light chain which comprises SEQ ID NO: 66 or a variant which has at least 90% identity to SEQ ID NO: 66.

[0096] SEQ ID NO: 65:

[0097] EVKLEESGGGLVQPGGSMKLSCVASGFIFSNHWMNWVRQSPEKGLEWVAEIRSKSINSAT HYAESVKGRFTISRDDSKSAVYLQMTDLRTEDTGVYYCSRNYYGSTYDYWGQGTTLTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS

[0098] VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY

[0099] RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDTAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK

[0100] SEQ ID NO: 66:

[0101] DI LLTQSPAI LSVSPGERVSFSCRASQFVGSSI H WYQQRTNGSPRLLI KYASESMSGI PSRF SGSGSGTDFTLSINTVESEDIADYYCQQSHSWPFTFGSGTNLEVKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0102] Suitably, the variant of SEQ ID NO: 65 may have at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 65. Suitably, the variant of SEQ ID NO: 66 may have at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 66.

[0103] Suitably, the TNF inhibitor may be infliximab.

[0104] TUMOUR NECROSIS FACTOR RECEPTOR 2 (TNFR2) AGONIST

[0105] A ‘TNFR2 agonist’ as used herein refers to an agent that is capable of inducing signalling via TNFR2.

[0106] Accordingly, a TNFR2 agonist is capable of activating the TNFR2 signalling pathway.

[0107] It will be understood that a TNFR2 agonist (e.g., a TNFR2 agonist protein or antibody) may increase signalling through TNFR2 by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, as compared to a control (e.g. a corresponding assay without the TNFR2 agonist present). It will also be understood that a TNFR2 agonist (e.g., a TNFR2 agonist protein or antibody) may increase signalling through TNFR2 by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, or at least 25-fold, at least 50-fold or at least 100-fold as compared to a control (e.g. a corresponding assay without the TNFR2 agonist present). Such signalling may be assessed using a reporter assay. TNFR2 agonists may 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.

[0108] Suitably, a TNFR2 agonist may increase a reporter signal (e.g. a luminescence reporter signal) in a reporter cell, for example a HEK:TNFR2 reporter cell as shown in Example 9. Suitably, a TNFR2 agonist may increase the reporter signal by at least 2 fold or at least 3-fold as compared to a control. Suitably, a TNFR2 agonist may increase a reporter signal, for example a luminescence signal, by at least 2-fold or at least 3-fold as compared to a control in a reporter cell line, for example a HEK:TNFR2 reporter cell as shown in Example 9. The TNFR2 agonist may be selected from a TNF mutein, an antibody, a polypeptide, a nucleic acid or a small molecule.

[0109] In some embodiments, the TNFR2 agonist is a polypeptide or an antibody. In some embodiments, the TNFR2 agonist is a polypeptide. In some embodiments, the TNFR2 agonist is a fusion protein. In some embodiments, the TNFR2 agonist comprises a TNF polypeptide 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).

[0110] 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. In some embodiments the TNFR2 agonist antibody may be an antibody fragment. For example, in some embodiments the TNFR2 agonist antibody may comprise or consist of a Fab, a scFv, or a VHH. In some embodiments the TNFR2 agonist antibody may comprise or consist of a Fab, a scFv, or a VHH 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). Further exemplary suitable TNFR2 agonist antibodies are described, for example: (i) in W02021141907A1 & WO2023281313A1 , for exampleHiFiBiO Therapeutics’ monoclonal TNFR2 agonist antibody HFB200301 ; (ii) in W02020089473A2, for eaxmple BioInvent’s, clinical monoclonal TNFR2 agonist antibody BI-1910; (iii) in WO2021231922A1 , W02020102739A1 , & WO2017040312A1 from the General Hospital Corporation; (iv) in WO2024151515A2, anfor example Odyssey Therapeutics monospecific tetravalent TNFR2 agonist V-body OD-00910, and (v) by NKTR-0165 from Nektar Therapeutics. In some embodiments, the TNFR2 agonist is HFB200301 , BI-1910, OD-00910, or NKTR-0165.

[0111] 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. TNF mutein

[0112] In some embodiments, the TNFR2 agonist is a TNF mutein.

[0113] As used herein, a ‘mutein’ refers to a mutant protein, with an altered amino acid sequence that differs from that of the original wild-type protein. Accordingly, the TNF mutein comprises an amino acid sequence which comprises at least one mutation compared to the wild-type TNF protein. An illustrative human TNF protein is provided by Uniprot accession number P01375 and shown herein as SEQ ID NO: 20.

[0114] In a particular embodiment, the combination comprises a TNF mutein, which acts as a TNFR2 agonist, and a TNF inhibitor which binds to sTNF and tmTNF. In particular, the TNF inhibitor is capable of binding to endogenous (e.g. wild-type) sTNF and tmTNF but does not bind the TNF mutein. The TNF mutein is therefore capable of inducing signalling via TNFR2 in the presence of the TNF inhibitor.

[0115] Suitably, binding of the TNF inhibitor to the TNF mutein is reduced or abrogated compared to binding of the TNF inhibitor to endogenous (e.g. wild-type) TNF.

[0116] As such, the TNF inhibitor may selectively bind endogenous (e.g. wild-type) TNF compared to the TNF mutein.

[0117] Suitable assays and techniques for measuring and / or quantifying the activity (e.g. binding capability and / or activity) of the TNF inhibitor to the TNF mutein may include, but are not limited to, ELISA, surface plasmon resonance (SPR), quartz crystal microbalance (QCM), bioluminescence assays and flow cytometry. Illustrative methods are provided in the present Examples.

[0118] In some embodiments, the TNFR2 agonist is a polypeptide comprising a TNFR2 binding domain comprising three TNF homology domains (THD) that preferentially, suitably specifically, bind to TNFR2.

[0119] In some embodiments, the TNFR2 agonist may induce TNFR2 signalling in a cell. In some embodiments, the TNFR2 agonist may induce TNFR2 signalling in a nerve cell. In some embodiments, the TNFR2 agonist may induce TNFR2 signalling in neurons. In some embodiments, the TNFR2 agonist may induce TNFR2 signalling in microglia. In some embodiments, the TNFR2 agonist may induce TNFR2 signalling in immune cells. TNF homology domain (THD)

[0120] The term THD as used herein refers to a protein domain shared by all tumor necrosis factor (TNF, formerly known as TNFa or TNF alpha) ligand family members. Homology implies evolutionary lineage from a common ancestor. A homology domain is a conserved part of a given protein sequence and (tertiary) structure that can evolve, function, and exist independently of the rest of the protein chain. It is a structural feature shared by all members of a certain protein family. Each domain forms a compact three-dimensional structure and often can be independently stable, folded and critical for biological activity.

[0121] In some embodiments, the TNFR2 agonist is a TNF mutein comprising a TNFR2 binding domain comprising three TNF homology domains (THD) that preferentially, suitably specifically, bind to TNFR2. In some embodiments, the TNFR2 agonist is a TNF mutein comprising a TNFR2 binding domain comprising at least three THDs, as further defined herein. In some embodiments, the TNFR2 agonist is a TNF mutein comprising a TNFR2 binding domain comprising three THDs, as further defined herein.

[0122] In some embodiments, the C-terminus of the first and second THD, respectively, which is in each case defined by the C-terminal consensus sequence

[0123] V-X1-F-G-X2-X3 (SEQ ID NO: 28), is linked to the N-terminus of the second and third THD, respectively, which is in each case defined by the N-terminal consensus sequence:

[0124] P-X4-A-H-X5 (SEQ ID NO: 29), through a peptide Xa, which is in each case independently selected and optionally has a length of 9 to 12 amino acids, preferably 9 to 11 , more preferably 9 to 10, wherein X1 is F or Y, wherein X2 is A or I, wherein X3 is a non-polar / hydrophobic or polar / neutral amino acid, preferably selected from the group consisting of F and I, wherein X4 is V or A, and wherein X5 is V or L.

[0125] 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.

[0126] In some embodiments, one or more THD may independently comprise or consist of the amino acid sequence according to SEQ ID NO: 2. 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.

[0127] In some embodiments, each THD may independently comprise or consist of the amino acid sequence according to SEQ ID NO: 2.

[0128] SEQ ID NO: 2 - THD sequence without mutations

[0129] SRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFK GQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLE KGDRLSAEINRPDYLDFAESGQVYFGIIAL

[0130] In some embodiments, the three THDs of the TNFR2 agonist are identical in their amino acid sequence.

[0131] 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).

[0132] In some embodiments, one or more, or each, THD according to the invention may independently comprise one or more mutations. Such a THD may be considered as a variant.

[0133] In some embodiments, one or more, or each, THD may independently comprise a contiguous amino acid sequence as outlined in WQ2020 / 260368 (incorporated herein by reference). WQ2020 / 260368 describes TNFR2 agonists with improved stability characteristics. In particular, WQ2020 / 260368 describes polypeptides comprising three TNF homology domains (THD) of TNF-ligand family member 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.

[0134] In some embodiments, the THD may comprise a contiguous amino acid sequence comprising SEQ ID NO: 9, optionally comprising at least one mutation or set of mutations selected from the group consisting of: D143Y, D143F, D143E, D143N, D143T, D143S, E146Q, E146H, E146K, A145R / S147T, Q88N / T89S / A145S / E146A / S147D, Q88N / A145I / E146G / S147D, A145H / E146S / S147D, A145H / S147D, L29V / A145D / E146D / S147D, A145N / E146D / S147D, A145T / E146S / S147D, A145Q / E146D / S147D, A145T / E146D / S147D, A145D / E146G / S147D, A145D / S147D, A145K / E146D / S147T, A145R / E146T / S147D, A145R / S147T, E146D / S147D, E146N / S147, S95C / G148C, K65A, K65W, Q67K, Q67T, Q67Y, L75H, L75W, D143W, D143V, D143V / F144L / A145S, D143N / A145R, D143V / A145S, L29V, L29T, L29S, L29A, L29G, R31 H, R31 I, R31 L, R32G, R32E, S147L, S147R, S147P S147T, S147A, Q149E, Q149N, E146D, E146N, E146S, E146G, A145R, A145S, A145T, A145H, A145K, A145F, A145D, A145G, A145N, A145P, A145Q, A145Y, A145V and A145W, preferably selected from D143N and A145R, wherein the above position numbering is in respect of the sequence of SEQ ID NO: 9.

[0135] Suitably, the THD may comprise one or more mutations in the amino acid sequence from D143 to Q149; wherein the position numbering is in respect of the sequence of SEQ ID NO: 9. The position numbering may alternatively be provided in respect of SEQ ID NO: 2 or 21 , as described herein. The amino acid sequence from D143 to Q149 wherein the position numbering is in respect of the sequence of SEQ ID NO: 9 may be referred to as the ‘G-H loop’. This is the loop connecting beta-strands G and H in the beta-sandwich structure of the TNF monomer. Suitably, the THD may comprise at least one, at least two, at least three or at least four mutations in the amino acid sequence from D143 to Q149; wherein the position numbering is in respect of the sequence of SEQ ID NO: 9. Suitably, the THD may comprise one, two, three or four mutations in the amino acid sequence from D143 to Q149; wherein the position numbering is in respect of the sequence of SEQ ID NO: 9.

[0136] The mutation may be any mutation (or set of mutations) in the amino acid sequence from D143 to Q149; wherein the position numbering is in respect of the sequence of SEQ ID NO: 9 as described herein.

[0137] Suitably, the THD may comprise a contiguous amino acid sequence comprising SEQ ID NO: 9; further comprising a D143Y and / or A145G mutation; wherein the position numbering is in respect of the sequence of SEQ ID NO: 9.

[0138] In some embodiments, the THD may comprise a contiguous amino acid sequence comprising SEQ ID NO: 9, optionally comprising a mutation at one or more of the following positions: D143, A145, E146, S147, wherein the above position numbering is in respect of the sequence of SEQ ID NO: 9. In some embodiments, the THD may comprise a contiguous amino acid sequence comprising SEQ ID NO: 9, optionally comprising a mutation at D143 or A145 or E146 or S147, wherein the above position numbering is in respect of the sequence of SEQ ID NO: 9. In some embodiments, the THD may comprise a contiguous amino acid sequence comprising SEQ ID NO: 9, optionally comprising a mutation at D143 or A145. In some embodiments, the THD may comprise a contiguous amino acid sequence comprising SEQ ID NO: 9, optionally comprising a mutation at D143 and A145. In some embodiments, the THD may comprise a contiguous amino acid sequence comprising SEQ ID NO: 9, optionally comprising a set of mutations at the following positions: D143 / A145, D143 / E146, D143 / S147, A145 / E146, A145 / S147, E146 / S147, D143 / A145 / E146, D143 / A145 / S147, D143 / E146 / S147, A145 / E146 / S147, or D143 / A145 / E146 / S147, wherein the above position numbering is in respect of the sequence of SEQ ID NO: 9. In some embodiments, the THD may comprise a contiguous amino acid sequence comprising SEQ ID NO: 9, optionally comprising at least one mutation or set of mutations selected from the group consisting of: D143Y, D143F, D143E, D143N, D143T, D143S, E146Q, E146H, E146K, A145R / S147T, A145H / E146S / S147D, A145H / 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, D143W, D143V, D143V / F144L / A145S, D143N / A145R, D143V / A145S, S147R, S147P S147T, S147A, E146D, E146N, E146S, E146G, A145R, A145S, A145T, A145H, A145K, A145F, A145D, A145G, A145N, A145P, A145Q, A145Y, A145V, A145W, D143N, D143Y, A145R, A145K, wherein the above position numbering is in respect of the sequence of SEQ ID NO: 9. These mutations may be used in combination with the TNFR2 specific mutations described herein. In some embodiments, the THD may comprise a contiguous amino acid sequence comprising SEQ ID NO: 9, optionally comprising at least one mutation or set of mutations selected from the group consisting of: D143N, D143Y, A145R, A145K, D143N with A145R, D143Y with A145G, wherein the above position numbering is in respect of the sequence of SEQ ID NO: 9. These mutations may be used in combination with the TNFR2 specific mutations described herein.

[0139] 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: 9. Optionally, the one or more THD may comprise one or more mutations as described herein.

[0140] In some embodiments, the THD may comprise a contiguous amino acid sequence comprising SEQ ID NO: 21 , optionally comprising a mutation at one or more of the following positions: D132, A134, E135, S136, wherein the above position numbering is in respect of the sequence of SEQ ID NO: 21. In some embodiments, the THD may comprise a contiguous amino acid sequence comprising SEQ ID NO: 21 , optionally comprising a mutation at D132 or A134 or E135 or S136, wherein the above position numbering is in respect of the sequence of SEQ ID NO: 21. In some embodiments, the THD may comprise a contiguous amino acid sequence comprising SEQ ID NO: 21 , optionally comprising a mutation at D132 or A134. In some embodiments, the THD may comprise a contiguous amino acid sequence comprising SEQ ID NO: 21 , optionally comprising a mutation at D132 and A134. In some embodiments, the THD may comprise a contiguous amino acid sequence comprising SEQ ID NO: 21 , optionally comprising a set of mutations at the following positions: D132 / A134, D132 / E135, D132 / S136, A134 / E135, A134 / S136, E132 / S136, D132 / A134 / E135, D132 / A134 / S136, D132 / E135 / S136, A134 / E135 / S136, or D132 / A134 / E135 / S136, wherein the above position numbering is in respect of the sequence of SEQ ID NO: 21 . In some embodiments, the THD may comprise a contiguous amino acid sequence comprising SEQ ID NO: 21 , optionally comprising at least one mutation or set of mutations selected from the group consisting of: D132Y, D132F, D132E, D132N, D132T, D132S, E135Q, E135H, E135K, A134R / S136T, A134H / E135S / S136D, A134H / S136D, A134N / E135D / S136D, A134T / E135S / S136D, A134Q / E135D / S136D, A145T / E135D / S136D, A134D / E135G / S136D, A134D / S136D, A134K / E135D / S136T, A145R / E135T / S136D, A134R / S136T, E146D / S136D, E135N / S136, D132W, D143V, D132V / F144L / A145S, D132N / A134R, D132V / A145S, S136R, S136P S136T, S136A, E135D, E135N, E146S, E135G, A134R, A145S, A134T, A145H, A134K, A145F, A134D, A134G, A134N, A134P, A134Q, A134Y, A134V, A134W, D132N, D132Y, A134R, A134K, wherein the above position numbering is in respect of the sequence of SEQ ID NO: 21 . These mutations may be used in combination with the TNFR2 specific mutations described herein. In some embodiments, the THD may comprise a contiguous amino acid sequence comprising SEQ ID NO: 21 , optionally comprising at least one mutation or set of mutations selected from the group consisting of: D132N, D132Y, A134R, A134K, D132N with A134R, D132Y with A134G, wherein the above position numbering is in respect of the sequence of SEQ ID NO: 21. These mutations may be used in combination with the TNFR2 specific mutations described herein.

[0141] 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: 21. Optionally, the one or more THD may comprise one or more mutations as described herein. 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.

[0142] It will be understood that SEQ ID NO. 5 of W02020 / 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 W02020 / 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 W02020 / 260368 equates to position 1 in the above position numbering.

[0143] SEQ ID NO. 5 of W02020 / 260368 is presented below as SEQ ID NO: 8.

[0144] SEQ ID NO: 8 - SEQ ID NO. 5 of WQ2020 / 260368 (positions 77 to 233 are underlined)

[0145] MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEF PRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQ LVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAE AKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL

[0146] The amino acids at position 77 to 233 of SEQ ID NO: 8 (i.e. which is the same as SEQ ID NO. 5 of W02020 / 260368) are presented below as SEQ ID NO: 9, where position 77 of SEQ ID NO: 8 equates to position 1 of SEQ ID NO: 9.

[0147] SEQ ID NO: 9 - sequence of amino acids from position 77 to 233 of SEQ ID NO. 5 of WQ2020 / 260368 (which is SEQ ID NO: 8 of the present disclosure)

[0148] VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLWPSEGLYLIYS QVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGG VFQLEKGDRLSAEI N RPDYLDFAESGQVYFGI I AL

[0149] In some embodiments, the THD may comprise a contiguous amino acid sequence consisting of the amino acids sequence according to SEQ ID NO: 9, optionally comprising at least one mutation or set of mutations selected from the group consisting of: D143Y, D143F, D143E, D143N, D143T, D143S, E146Q, E146H, E146K, A145R / S147T,

[0150] Q88N / T89S / A145S / E146A / S147D, Q88N / A145I / E146G / S147D, A145H / E146S / S147D,

[0151] A145H / S147D, L29V / A145D / E146D / S147D, A145N / E146D / S147D, A145T / E146S / S147D, A145Q / E146D / S147D, A145T / E146D / S147D, A145D / E146G / S147D, A145D / S147D,

[0152] A145K / E146D / S147T, A145R / E146T / S147D, A145R / S147T, E146D / S147D, E146N / S147, S95C / G148C, K65A, K65W, Q67K, Q67T, Q67Y, L75H, L75W, D143W, D143V, D143V / F144L / A145S, D143N / A145R, D143V / A145S, L29V, L29T, L29S, L29A, L29G, R31 H, R31 I, R31 L, R32G, R32E, S147L, S147R, S147P S147T, S147A, Q149E, Q149N, E146D, E146N, E146S, E146G, A145R, A145S, A145T, A145H, A145K, A145F, A145D, A145G, A145N, A145P, A145Q, A145Y, A145V and A145W, preferably selected from D143N and A145R. It will be understood that a “set of mutations” as used above refers to a combination of mutations indicated by the use of 7”, where all of the mutations in the combination are selected, e.g. if “Q88N / T89S / A145S / E146A / S147D” as used above is selected, all of the indicated mutations will be present in the amino acid sequence.

[0153] In the context of the present invention, the mutations (e.g. for increasing specificity of binding to TNFR) may be described by reference to SEQ ID NO: 2.

[0154] In some embodiments, the THD may comprise at least one mutation or set of mutations selected from the group consisting of: D139Y, D139F, D139E, D139N, D139T, D139S, E142Q, E142H, E142K, A141 R / S143T, Q84N / T89S / A141S / E142A / S143D,

[0155] Q84N / A1411 / E142G / S143D, A141 H / E142S / S143D, A141 H / S143D,

[0156] L25V / A141 D / E142D / S143D, A141 N / E142D / S143D, A141T / E142S / S143D,

[0157] A141Q / E142D / S143D, A141T / E142D / S143D, A141 D / E142G / S143D, A141 D / S143D,

[0158] 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.

[0159] In some embodiments, one or more THD 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.

[0160] In some embodiments, each THD 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.

[0161] 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.

[0162] In some embodiments, one or more THD may independently comprise the amino acid sequence according to SEQ ID NO: 3 or a variant thereof with at least 80% sequence identity to SEQ ID NO: 3. In some embodiments, one or more THD may independently comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (optionally at least 90%, such as at least 95%) sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, one or more THD may independently consist of the amino acid sequence according to SEQ ID NO: 3. It will be understood that in these embodiments, regardless of the % sequence identity to SEQ ID NO: 3, the amino acid sequence of the one or more THD will include the D139N and A141 R mutations.

[0163] In some embodiments, each THD may independently comprise the amino acid sequence according to SEQ ID NO: 3 or a variant thereof with at least 80% sequence identity to SEQ ID NO: 3. In some embodiments, each THD may independently comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (optionally at least 90%, such as at least 95%) sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, each THD may consist of the amino acid sequence according to SEQ ID NO: 3. It will be understood that in these embodiments, regardless of the % sequence identity to SEQ ID NO: 3, the amino acid sequence of each THD will include the D139N and A141 R mutations.

[0164] SEQ ID NO: 3 - THD sequence with D139N and A141 R

[0165] SRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFK GQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLE KGDRLSAEINRPDYLNFRESGQVYFGIIAL

[0166] Without wishing to be bound by theory, the D139N and A141 R mutations may confer preferential TNFR2 binding, suitably TNFR2 selectivity.

[0167] Suitably, the TNF polypeptide may comprise an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 3 and one or both of the following mutations in the corresponding position of the sequence of SEQ ID NO: 2: D139N and / or A141 R.

[0168] In some embodiments, the three THDs of the TNFR2 binding domain are identical in their amino acid sequence. In some embodiments, each THD may independently comprise or consist of the amino acid sequence according to SEQ ID NO: 3 or a variant thereof.

[0169] In some embodiments, at least one of the THDs may comprise an additional “S” at the N terminus. In some embodiments, at least one of the THDs may comprise or consist of the amino acid sequence according to SEQ ID NO: 3 or a variant thereof, and may further comprise an additional “S” at the N terminus.

[0170] In some embodiments, only one of the THD (e.g. the first THD) may comprise an additional “S” at the N terminus. In some embodiments, the TNFR2 binding domain may comprise an additional “S” at the N terminus. In other words, the first THD of the TNFR2 binding domain may comprise an additional “S” at the N terminus.

[0171] In some embodiments, each THD of the TNFR2 binding domain according to the invention may independently comprise or consist of the amino acid sequence according to SEQ ID NO: 3 or a variant thereof, but the first THD of the TNFR2 binding domain may further comprise an additional “S” at the N terminus.

[0172] 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.

[0173] In some embodiments, a THD may comprise the amino acid sequence according to SEQ ID NO: 4 or a variant thereof with at least 80% sequence identity to SEQ ID NO: 4. In some embodiments, a THD may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (optionally at least 90%, such as at least 95%) sequence identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, a THD may consist of the amino acid sequence according to SEQ ID NO: 4. It will be understood that in these embodiments, regardless of the % sequence identity to SEQ ID NO: 4, the amino acid sequence of the THD will include the equivalent D139N and A141 R mutations.

[0174] Suitably, the THD may comprise an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 4 and one or both of the following mutations in the corresponding position of the sequence of SEQ ID NO: 2: D139N and / or A141 R.

[0175] SEQ ID NO: 4 - THD sequence with equivalent D139N and A141 R and additional S

[0176] SSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLF KGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQL EKGDRLSAEINRPDYLNFRESGQVYFGIIAL

[0177] In some embodiments, the first THD of the TNFR2 binding domain according to the invention may comprise or consist of the amino acid sequence according to SEQ ID NO: 4 or a variant thereof, and the second THD and third THD of the TNFR2 binding domain according to the invention may each comprise or consist of the amino acid sequence according to SEQ ID NO: 3 or a variant thereof.

[0178] In some embodiments, each THD may independently comprise the amino acid sequence according to SEQ ID NO: 23 or a variant thereof with at least 80% sequence identity to SEQ ID NO: 23. In some embodiments, each THD 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: 23. In some embodiments, each THD may independently comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 23. In some embodiments, each THD may consist of the amino acid sequence according to SEQ ID NO: 23. It will be understood that in these embodiments, regardless of the % sequence identity to SEQ ID NO: 23, the amino acid sequence of each THD will include the D132N and A134R mutations in the corresponding position of SEQ ID NO: 23 (which correspond to D139N and A141 R based on the corresponding position in SEQ ID NO: 2).

[0179] SEQ ID NO: 23 - THD sequence with D132N and A134R

[0180] PVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPS THVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSA EINRPDYLNFRESGQVYFGIIAL

[0181] 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.

[0182] In other embodiments, the first and second THD and / or second and third THD may be linked by intervening peptide linkers. In some embodiments, the intervening peptide linkers may be from 1 to 50, optionally from 1 to 20 amino acids in length. In some embodiments, the intervening peptide linkers may be from 8 to 12, optionally from 9 to 10 amino acids in length. The intervening peptide linkers may comprise any sequence, for example a flexible GS-rich sequence. In some embodiments, the intervening peptide linkers may be independently selected from a peptide which consists of XC-XL-XN; wherein:

[0183] Xc is selected from the group consisting of A, A-L, L, preferably A-L;

[0184] XL is absent or is an amino acid linker consisting of 1-11 amino acids,

[0185] XN is 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. In some embodiments, each intervening peptide linker may be A-L.

[0186] XL when present may comprise any amino acid sequence. XL when present may comprise a G / S linker sequence (i.e., 1-11 glycine and / or serine amino acids). SEQ ID NO: 5 is an amino acid sequence comprising (in sequential order) the amino acid sequence according to SEQ ID NO: 4 and two amino acid sequences according to SEQ ID NO: 3.

[0187] In some embodiments, the TNFR2 binding domain may comprise the amino acid sequence according to SEQ ID NO: 5 or a variant with at least 80% sequence identity to SEQ ID NO: 5. In some embodiments, the TNFR2 binding domain according to the invention may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (optionally at least 90%, such as at least 95%) sequence identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the TNFR2 binding domain according to the invention may consist of the amino acid sequence according to SEQ ID NO: 5. It will be understood that in these embodiments, regardless of the % sequence identity to SEQ ID NO: 5, the amino acid sequence of the TNFR2 binding domain will include the equivalent D139N and A141 R mutations for each THD.

[0188] SEQ ID NO: 5 - illustrative full trimeric THD TNFR2 binding domain

[0189] SSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLF KGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQL EKGDRLSAEINRPDYLNFRESGQVYFGIIALSRTPSDKPVAHVVANPQAEGQLQWLNRRAN ALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIK SPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLNFRESGQVYFGIIALSRT PSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQ GCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKG DRLSAEINRPDYLNFRESGQVYFGIIAL

[0190] Thus, in some embodiments, the TNFR2 agonist may be a TNF mutein comprising a THD as defined above. Suitably, binding of the TNF inhibitor to the TNF mutein is reduced or abrogated compared to binding of the TNF inhibitor to endogenous (e.g. wild-type) TNF due to the mutations present in the TNF mutein compared to endogenous (e.g. wild-type) TNF.

[0191] Suitably, the TNF inhibitor binds an epitope comprising one or more amino acids which are present in endogenous (e.g. wild-type) TNF but are not present in the TNF mutein.

[0192] Suitably, the TNF mutein may comprise one or mutations compared to SEQ ID NO: 20.

[0193] Suitably, the TNF mutein may comprise any mutation or set of mutations as described herein.

[0194] Suitably, the TNF mutein may comprise any mutation or set of mutations which provide TNFR2 selectivity; as described herein. For example, in some embodiments the TNF mutein may comprise three THDs, wherein each THD comprises a sequence of SEQ ID NO: 21 , and further comprising 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,

[0195] 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,

[0196] 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 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.

[0197] Suitably, the TNFR2 agonist may comprise any mutation or set of mutations which provide TNFR2 selectivity; as described herein. For example, in some embodiments the TNFR2 agonist may comprise three THDs, wherein each THD comprises a sequence of SEQ ID NO: 21 , and further comprising at least one mutation or set of mutations selected from the group consisting of: D132Y, D132F, D132E, D132N, D132T, D132S, E135Q, E135H, E135K, A134R / S136T, Q77N / T82S / A134S / E135A / S136D, Q77N / A134I / E135G / S136D,

[0198] A134H / E135S / S136D, A134H / S136D, L18V / A134D / E135D / S136D, A134N / E135D / S136D, A134T / E135S / S136D, A134Q / E135D / S136D, A134T / E135D / S136D, A134D / E135G / S136D, A134D / S136D, A134K / E135D / S136T, A134R / E135T / S136D, A134R / S136T, E135D / S136D, E135N / S136T, S84C / G137C, K54A, K54W, Q56K, Q56T, Q56Y, L64H, L64W, D132W, D132V, D132V / F133L / A134S, D132N / A134R, D132V / A134S, L18V, L18T, L18S, L18A, L18G, R20H, R20I, R20L, R21G, R21 E, S136L, S136R, S136P S136T, S136A, Q138E, Q138N, E135D, E135N, E135S, E135G, A134R, A134S, A134T, A134H, A134K, A134F, A134D, A134G, A134N, A134P, A134Q, A134Y, A134V and A134W, at the position corresponding to the sequence of SEQ ID NO: 21 , preferably selected from D132N and A134R at the positions corresponding to the sequence of SEQ ID NO: 21 . 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 “Q77N / T82S / A134S / E135A / S136D” as used above is selected, all of the indicated mutations will be present in the amino acid sequence.

[0199] In some embodiments the TNFR2 agonist may comprise three THDs, wherein each THD comprises a sequence having at least 90% (e.g., at least 95%) sequence identity to SEQ ID NO: 21 , and further comprising at least one mutation or set of mutations selected from the group consisting of: D132Y, D132F, D132E, D132N, D132T, D132S, E135Q, E135H, E135K, A134R / S136T, Q77N / T82S / A134S / E135A / S136D, Q77N / A134I / E135G / S136D,

[0200] A134H / E135S / S136D, A134H / S136D, L18V / A134D / E135D / S136D, A134N / E135D / S136D, A134T / E135S / S136D, A134Q / E135D / S136D, A134T / E135D / S136D, A134D / E135G / S136D, A134D / S136D, A134K / E135D / S136T, A134R / E135T / S136D, A134R / S136T, E135D / S136D, E135N / S136T, S84C / G137C, K54A, K54W, Q56K, Q56T, Q56Y, L64H, L64W, D132W, D132V, D132V / F133L / A134S, D132N / A134R, D132V / A134S, L18V, L18T, L18S, L18A, L18G, R20H, R20I, R20L, R21G, R21 E, S136L, S136R, S136P S136T, S136A, Q138E, Q138N, E135D, E135N, E135S, E135G, A134R, A134S, A134T, A134H, A134K, A134F, A134D, A134G, A134N, A134P, A134Q, A134Y, A134V and A134W. 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 “Q77N / T82S / A134S / E135A / S136D” as used above is selected, all of the indicated mutations will be present in the amino acid sequence. In some embodiments the TNFR2 agonist may comprise three THDs, wherein each THD comprises a sequence having at least 90% (e.g., at least 95%) sequence identity to SEQ ID NO: 21 , and further comprising mutations at positions D132 and A134. In some embodiments the TNFR2 agonist may comprise three THDs, wherein each THD comprises a sequence having at least 90% (e.g., at least 95%) sequence identity to SEQ ID NO: 21 , and further comprising mutations D132N and A134R. In some embodiments the TNFR2 agonist may comprise three THDs, wherein each THD comprises a sequence having at least 90% (e.g., at least 95%) sequence identity to SEQ ID NO: 21 , and further comprising D132Y and A134G.

[0201] SEQ ID NO: 21 - THD sequence

[0202] PVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPS THVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSA EINRPDYLDFAESGQVYFGIIAL

[0203] Preferably, the TNF mutein may comprise D139N and / or A141 R mutations corresponding to the positions shown in SEQ ID NO: 2. Most preferably, the TNF mutein may comprise D139N and A141 R mutations corresponding to the positions shown in SEQ ID NO: 2. Preferably, in embodiments wherein the TNFR2 agonist is a TNF mutein as described herein, most preferably wherein the TNF mutein comprises D139N and A141 R mutations corresponding to the position shown in SEQ ID NO: 2, the TNF inhibitor may be an antibody which comprises HCDRs 1-3 which are present in SEQ ID NO: 16 and LCDRs 1-3 which are present in SEQ ID NO: 17.

[0204] Preferably, in embodiments wherein the TNFR2 agonist is a TNF mutein as described herein, most preferably wherein the TNF mutein comprises D139N and A141 R mutations corresponding to the position shown in SEQ ID NO: 2, the TNF inhibitor may be an antibody which comprises the following CDRs:

[0205] HCDR1 : DYAMH (SEQ ID NO: 10)

[0206] HCDR2: AITWNSGHIDYADSVEG (SEQ ID NO: 11)

[0207] HCDR3: VSYLSTASSLDY (SEQ ID NO: 12)

[0208] LCDR1 : RASQGIRNYLA (SEQ ID NO: 13)

[0209] LCDR2: AASTLQS (SEQ ID NO: 14)

[0210] LCDR3: QRYNRAPYT (SEQ ID NO: 15)

[0211] Preferably, in embodiments wherein the TNFR2 agonist is a TNF mutein as described herein, most preferably wherein the TNF mutein comprises D139N and A141 R mutations corresponding to the position shown in SEQ ID NO: 2, the TNF inhibitor may be an antibody which comprises a variable heavy domain (VH) which comprises SEQ ID NO: 16 or a variant which has at least 90% identity to SEQ ID NO: 16; and a variable heavy domain (VL) which comprises SEQ ID NO: 17 or a sequence which has at least 90% identity to SEQ ID NO: 17.

[0212] The variant of SEQ ID NO: 16 may have at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 16. Suitably, the variant of SEQ ID NO: 17 may have at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 17.

[0213] Preferably, in embodiments wherein the TNFR2 agonist is a TNF mutein as described herein, most preferably wherein the TNF mutein comprises D139N and A141 R mutations corresponding to the position shown in SEQ ID NO: 2, the TNF inhibitor may be adalimumab.

[0214] In one embodiment, there is provided a combination comprising a TNF inhibitor and a TNFR2 agonist, wherein the TNFR2 agonist is a TNF mutein, wherein the TNF mutein comprises a polypeptide comprising a TNFR2 binding domain comprising three TNF homology domains (THD) that specifically bind to TNFR2, wherein each THD comprises D139N and A141 R mutations corresponding to the position shown in SEQ ID NO: 2, and the TNF inhibitor is capable of binding to human TNF but does not bind the TNF mutein. Fc domain

[0215] In some embodiments, the TNFR2 agonist, for example the TNF mutein, further comprises a multimerization domain. The multimerization domain may be selected from the group consisting of: an antibody, an antibody heavy chain, an immunoglobulin Fc domain, a heavy chain domain 2 (CH2) of IgM (MHD2), a heavy chain domain 2 (CH2) of IgE (EHD2), or the tetramerization domain of p53.

[0216] In some embodiments, the multimerization domain is a dimerization domain.

[0217] In some embodiments, the dimerization domain is an immunoglobulin Fc domain. The immunoglobulin Fc domain may be an lgG1 , lgG2 or lgG4 immunoglobulin Fc domain. The immunoglobulin Fc domain may be an lgG1 immunoglobulin Fc domain comprising an N297A mutation. In some embodiments, the dimerization domain is an immunoglobulin Fc domain mutant without FcR and / or C1 q binding, optionally wherein the Fc domain comprises one or more of the following mutations: FcAab, LALA, LALA-GP, lgG2, lgG2o, aglycosylated lgG1 , lgG1 (L234F / L235E / P331S), lgG2m4, lgG4 ProAlaAla.

[0218] 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.

[0219] In some embodiments, the TNFR2 agonist (e.g., TNF mutein) comprises an Fc domain, wherein the Fc domain comprises the following mutations L234A, L235A, A327G, A330S and P331S.

[0220] In some embodiments, the Fc domain does not comprise a proline at position 233.

[0221] In some embodiments, the Fc domain comprises a glycine at position 236.

[0222] In some embodiments, the Fc domain (i) does not comprise a proline at position 233 and (ii) comprises a glycine at position 236.

[0223] It will be understood that the amino acid numbering and residues for L234A, L235A, A327G, A330S, P331S, proline at position 233 and glycine at position 236, may be relative to the 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.

[0224] It will also be understood that the amino acid numbering and residues for L234A, L235A, A327G, A330S, P331S, proline at position 233 and glycine at position 236, may be relative to the IgG constant region residues and positions according to the Ell numbering system (found in Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S. and Foeller, C., Sequences of proteins of immunological interest. US Department of Health and Human services, NIH, Bethesda 1991).

[0225] It will also be understood that the positions of 233, 234, 235, 236, 327, 330, and 331 described above for the Fc domain may correspond to the corresponding positions in SEQ ID NO: 1 as outlined in Table 1 below.

[0226] Table 1 :

[0227] In some embodiments, where the TNFR2 agonist (e.g., TNF mutein) comprises an Fc domain, the Fc domain comprises the following mutations: L14A, L15A, A107G, A110S and P111S, wherein the amino acid numbering is relative to the sequence of SEQ ID NO: 1. It will be understood that SEQ ID NO: 1 already comprises “A” at position 14, “A” at position 15, “G” at position 107, “S” at position 110 and “S” at position 111.

[0228] It thus will be understood that L14A, L15A, A107G, A110S and P111S, wherein the amino acid numbering is relative to the sequence of SEQ ID NO: 1 , correspond to the L234A, L235A, A327G, A330S and P331S mutations and positions relative to the lgG1 positions and residues in the EU numbering system / as described in Armour et al.

[0229] It will be understood that an Fc domain comprising the L234A, L235A, A327G, A330S and P331S mutations, may be defined as a “Fc[LALA-Aa]”.

[0230] It will also be understood that an Fc domain 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]”.

[0231] In some embodiments, where the TNFR2 agonist (e.g., TNF mutein) comprises an Fc domain the Fc domain may comprise the amino acid sequence according to SEQ ID NO: 1 or a variant thereof with at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, an Fc domain may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (optionally at least 90%, such as at least 95%) sequence identity to the amino acid sequence of SEQ ID NO: 1 . In some embodiments, an Fc domain may consist of the amino acid sequence according to SEQ ID NO: 1. It will be understood that in these embodiments, regardless of the % sequence identity to SEQ ID NO: 1 , the amino acid sequence of the Fc domain will include the L14A, L15A, A107G, A110S and P111S mutations relative to the sequence of SEQ ID NO: 1 , or the equivalent mutations at corresponding positions (e.g. L234A, L235A, A327G, A330S and P331S).

[0232] SEQ ID NO: 1

[0233] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0234] (L14A, L15A, A107G, A110S and P111S are shown in bolded and underlined in the above sequence)

[0235] 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]”.

[0236] Without wishing to be bound by theory, it is considered that the Fc domain comprising the amino acid sequence of SEQ ID NO: 1 may have reduced, or essentially no, binding to Fc gamma receptors or C1q. In particular, the Fc domain (i.e. “Fc[LALA-Aa]”) of the TNFR2 agonist of the invention (e.g. an Fc domain comprising the sequence of SEQ ID NO: 1) may have reduced binding to FcyRlla. The ‘reduced’ binding may be in comparison to a corresponding Fc domain which does not comprise the L14A, L15A, A107G, A110S and P111S mutations. In some embodiments, the reduced binding to FcyRlla may be in comparison to the Fc[Aab] construct described by Armour et al., (Eur. J. Immunol. 1999. 29: 2613-2624).

[0237] 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.

[0238] Linker

[0239] In some embodiments, the TNFR2 agonist (e.g., TNF mutein) comprises a TNFR2 binding domain (as described herein) joined to a multimerization domain (e.g., an Fc domain) as described herein by a linker. Without wishing to be bound by theory, the linker may spatially separate the TNFR2 binding domain and the Fc domain.

[0240] In some embodiments, the linker may be a peptide linker. In some embodiments, the linker may be a flexible linker. Without wishing to be bound by theory, a flexible linker allows the TNFR2 binding domain and the Fc domain to orient in different directions to enable binding to their respective ligands / binding partners.

[0241] 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.

[0242] In some embodiments, the linker may be 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in length. In some embodiments, the linker may be more than 20 amino acids in length, such as 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 or more than 30 amino acids in length.

[0243] Suitable linkers will be known in the art and may be used in the context of the present invention.

[0244] In some embodiments, the flexible linker may be a Ser-Gly linker. In some embodiments, the linker may comprise a plurality of glycine and serine residues. The glycine (denoted as Gly or G) and serine (denoted as Ser or S) residues may be present in any combination. For example, the linker may comprise the format (Gly-Gly-Gly-Gly-Ser)n or (Gly-Ser)n, where n indicates the number of repeats. Suitable Ser-Gly linkers will be known in the art and may be used in the context of the present invention. In some embodiments, the flexible linker may be a Ser- Gly linker of 8-15 amino acids in length.

[0245] In some embodiments, the flexible linker may comprise or consist of the amino acid sequence: GGSGGGGSGG (SEQ ID NO: 6).

[0246] Full length TNFR2 agonist

[0247] In some embodiments, the TNFR2 agonist (in particular the TNF mutein) comprises a TNFR2 binding domain comprising three TNF homology domains (THD) that preferentially, suitably specifically, bind to TNFR2.

[0248] In some embodiments, the TNFR2 agonist (e.g., TNF mutein) comprises (i) a TNFR2 binding domain comprising three TNF homology domains (THD) that preferentially, suitably specifically, bind to TNFR2; and (ii) an Fc domain. In some embodiments, the TNFR2 agonist (e.g., TNF mutein) comprises (i) a TNFR2 binding domain comprising three TNF homology domains (THD) that preferentially, suitably specifically, bind to TNFR2; and (ii) an Fc domain, wherein the Fc domain comprises the following mutations L234A, L235A, A327G, A330S and P331S.

[0249] In some embodiments, it will be understood that the TNFR2 agonist (e.g., TNF mutein) may be considered as a fusion protein.

[0250] In some embodiments, the TNFR2 agonist (e.g., TNF mutein) has the following structure in a N-terminus to C-terminus orientation: TNFR2 binding domain - linker - Fc domain.

[0251] In some embodiments, the TNFR2 agonist (e.g., TNF mutein) comprises the following amino acid sequences in a N-terminus to C-terminus orientation: SEQ ID NO: 5 - SEQ ID NO: 6 - SEQ ID NO: 1

[0252] In some embodiments, variation within SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 1 may independently be selected from any of the variation permitted above.

[0253] 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.

[0254] In some embodiments, the TNFR2 agonist (e.g., TNF mutein) may comprise the amino acid sequence according to SEQ ID NO: 7 or a variant with at least 80% sequence identity to SEQ ID NO: 7. In some embodiments, the TNFR2 agonist according to the invention may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the TNFR2 agonist according to the invention may comprise an amino acid sequence having at least 90% (such as at least 95%) sequence identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the TNFR2 agonist according to the invention may comprise an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the TNFR2 agonist according to the invention may comprise the amino acid sequence according to SEQ ID NO: 7. In some embodiments, the TNFR2 agonist according to the invention may consist of the amino acid sequence according to SEQ ID NO: 7. It will be understood that in these embodiments, regardless of the % sequence identity to SEQ ID NO: 7, the amino acid sequence of each of the THDs will include the equivalent D139N and A141 R mutations, and the amino acid sequence of the Fc domain will include the equivalent L234A, L235A, A327G, A330S and P331S mutations. SEQ ID NO: 7 (full length molecule)

[0255] SSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLF KGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQL EKGDRLSAEINRPDYLNFRESGQVYFGIIALSRTPSDKPVAHVVANPQAEGQLQWLNRRAN ALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIK SPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLNFRESGQVYFGIIALSRT

[0256] PSDKPVAHWANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQ GCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKG DRLSAEINRPDYLNFRESGQVYFGIIALGGSGGGGSGGDKTHTCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRW SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

[0257] LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPG

[0258] TNFR2 agonist multimer

[0259] The TNFR2 agonist (in particular the TNF mutein) may combine with one or more other TNFR2 agonist(s) to form a multimeric molecule, for example through a multimerization domain.

[0260] It will be understood that the multimeric molecule may be referred to as a TNFR2 agonist multimer.

[0261] It will be understood that each individual TNFR2 agonist (e.g., TNF mutein) according to the invention can be considered as a TNFR2 agonist monomer.

[0262] 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.

[0263] In some embodiments, a TNFR2 agonist (e.g., a TNF mutein) may comprise a multimerization domain, which multimerization domain is configured to allow interaction with one or more other TNFR2 agonist(s) (e.g., TNF mutein(s)) according to the invention, to form a multimeric molecule. Illustrative multimerization domains include dimerization domains.

[0264] Illustrative dimerization domains include, but are not limited to, an antibody heavy chain, a Fc region, heavy chain domain 2 (CH2) of IgM (MHD2), heavy chain domain 2 (CH2) of IgE (EHD2), heavy chain domain 3 (CH3) of IgG, heavy chain domain 3 (CH3) of IgA, heavy chain domain 3 (CH3) of IgD, heavychain domain 4 (CH4) of IgM, heavy chain domain 4 (CH4) of IgE, Fab, Fab2, and the CHI and CL domain. A preferred dimerization domain from an antibody, is the Fc region, variants or fragments thereof. The Fc region usable as dimerization domain preferably originates from the following isotypes IgA, IgD, IgE, IgG, and IgM

[0265] Further dimerization domains include the immunoglobulin Fc region mutants without FcR and / or Clq binding. Examples of immunoglobulin Fc region mutants without FcR and / or Clq binding are FcAab, LALA, LALA-GP, lgG2, lgG2o, aglycosylated IgGI, IgGI (L234F / L235E / LP331 S), lgG2m4 and lgG4 ProAlaAla. Further examples of a Fc region mutant is FcAab which lacks Fey receptor I binding and Clq binding (Armour et al; Eur. J. Immunol. 1999, 29:2613-2624).

[0266] Other dimerization or multimerization domains include bamase-barstar, C4bp, CD59, peptides derived from collagen, leucine zipper motifs, miniantibodies, and ZIP miniantibodies, GST, the a and b subunits of inactive human chorionic gonadotropin, maltose-binding protein (MBP), p53 and fragments thereof, phosphatase, streptavidin, surfactant protein D, tenascin, tetranectin, dock-and-lock (DNL) motifs, and uteroglobin.

[0267] The multimerization domain may be a trimerization domain.

[0268] Suitable trimerization domains are tenascin C (TNC), the trimerization region of the C -terminal noncollagenous domain (NCI) of collagen XVIII, Fab3 like molecules, and TriBi- minibodies, more preferably TNC.

[0269] The multimerization domain may be a tetramerization domain.

[0270] Suitable tetramerization domains are the tetramerization domain of p53, the tetramerization domain of the general control protein 4 (GCN4), the tetramerization domain of VASP (vasodilator stimulated phosphoprotein), tandem diabodies, and di-diabodies.

[0271] In preferred embodiments, the TNFR2 agonist (e.g., TNF mutein) multimer may be in the form of a dimer.

[0272] In some embodiments, the TNFR2 agonist multimer may comprise six or more, optionally six, TNF homology domains (THD) that specifically bind to TNFR2.

[0273] In some embodiments, each TNFR2 agonist monomer may bind together through the interactions of each of the Fc domains to one another. For example, in a dimer, the Fc domain of one TNFR2 agonist may bind to the Fc domain of the other TNFR2 agonist. It will be understood that the Fc domain of each TNFR2 agonist monomer may interact with the Fc domain(s) of one or more other TNFR2 agonist monomer(s) through any interaction that is suitable to allow the Fc domains to bind together. As such, the binding of the Fc domains of the TNFR2 agonist monomers to each other is not limited to any one particular type of interaction.

[0274] Accordingly, in some embodiments, the TNFR2 agonist multimer is in the form of a dimer comprising two TNFR2 agonists, wherein each TNFR2 agonist is independently a TNFR2 agonist (e.g., TNF mutein) as disclosed herein.

[0275] In some embodiments, each TNFR2 agonist in the dimer may be identical (e.g. by having the same amino acid sequence).

[0276] Without wishing to be bound by theory, it is also considered that the TNFR2 agonists (in particular the TNF muteins) disclosed herein provide particular advantages 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. In particular, the dimer of the TNFR2 agonist is considered to unexpectedly provide improved efficacy in vivo, compared to known TNFR2 agonist molecules of the prior art comprising higher orders of THD domains, such as tetrameric TNFR2 agonist molecules comprising p53.

[0277] Fusion protein

[0278] A ‘fusion protein’ comprising a TNF inhibitor and a TNFR2 agonist (e.g., TNF mutein) as used herein may refer to a polypeptide comprising at least two functional domains (i.e. a TNF inhibitor and a TNFR2 agonist). Suitably, a ‘fusion protein’ may be defined as a protein comprising at least two domains encoded by separate coding regions that originally coded for separate proteins which have been joined so that they are transcribed and translated as a single unit, producing a single polypeptide.

[0279] The at least two domains may be connected by a linker region. Suitably, the linker may be a flexible polypeptide linker. For example, the linker may be a serine-glycine linker.

[0280] POLYNUCLEOTIDE

[0281] The present disclosure provides a polynucleotide encoding a TNF inhibitor as described herein and a TNFR2 agonist as described herein.

[0282] Also provided is one or more polynucleotide(s) encoding a TNF inhibitor as described herein and a TNFR2 agonist as described herein. The present disclosure also provides a composition comprising a first polynucleotide encoding a TNF inhibitor as described herein and a second polynucleotide encoding a TNFR2 agonist as described herein. The present disclosure also provides a composition comprising a first polynucleotide encoding a TNF inhibitor as described herein and one or more further polynucleotides encoding a TNFR2 agonist as described herein.

[0283] Suitably, the TNFR2 agonist may be a TNF mutein as described herein.

[0284] As used herein, the terms “polynucleotide”, “nucleotide”, and “nucleic acid” are intended to be synonymous with each other.

[0285] Due to the redundancy of the genetic code, variations in nucleic acid sequences are possible that encode for the same polypeptide. These sequences are encompassed by the present invention. Therefore multiple polynucleotides are envisaged, each with a different nucleic acid sequence but which encodes a polypeptide as described herein or a further polypeptide as described herein. It is possible to design and produce such nucleic acid sequences without difficulty.

[0286] 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.

[0287] The polynucleotide may be codon optimised for production in the host cell of choice.

[0288] In some embodiments, the polynucleotide encoding the TNF inhibitor and / or TNFR2 agonist is a DNA polynucleotide. In some embodiments, the polynucleotide encoding the TNF inhibitor and / or TNFR2 agonist is an RNA polynucleotide. In some embodiments, the polynucleotide encoding the TNF inhibitor and / or TNFR2 agonist is an mRNA polynucleotide. In some embodiments, the polynucleotide encoding the TNF inhibitor and / or TNFR2 agonist is a cDNA polynucleotide.

[0289] In some embodiments, the polynucleotide encoding the TNF inhibitor is a DNA polynucleotide. In some embodiments, the polynucleotide encoding the TNF inhibitor is an RNA polynucleotide. In some embodiments, the polynucleotide encoding the TNF inhibitor is an mRNA polynucleotide. In some embodiments, the polynucleotide encoding the TNF inhibitor is a cDNA polynucleotide.

[0290] In some embodiments, the polynucleotide encoding the TNFR2 agonist is a DNA polynucleotide. In some embodiments, the polynucleotide encoding the TNFR2 agonist is an RNA polynucleotide. In some embodiments, the polynucleotide encoding the TNFR2 agonist is an mRNA polynucleotide. In some embodiments, the polynucleotide encoding TNFR2 agonist is a cDNA polynucleotide.

[0291] In some embodiments, the polynucleotide encoding the TNF inhibitor and TNFR2 agonist is a DNA polynucleotide. In some embodiments, the polynucleotide encoding the TNF inhibitor and TNFR2 agonist is an RNA polynucleotide. In some embodiments, the polynucleotide encoding the TNF inhibitor and TNFR2 agonist is an mRNA polynucleotide. In some embodiments, the polynucleotide encoding the TNF inhibitor and TNFR2 agonist is a cDNA polynucleotide.

[0292] In some embodiments, the nucleic acid sequence may be operably linked to a heterologous sequence, such as a promoter or regulatory sequence, forming an expression cassette.

[0293] The expression cassette may comprise one or more control sequences. Control sequences are sequences that control and regulate transcription and, where appropriate, translation, and include promoter sequences, transcriptional regulators encoding sequences, ribosome binding sequences (RBS) and / or transcription terminating sequences. The expression cassette may additionally include an enhancer, which may be adjacent to or distant from the promoter sequence and can function to increase transcription from the same. The expression control sequence may be functional in prokaryotic cells or in eukaryotic cells and organisms, such as mammalian cells. The expression cassette may comprise a promoter. Any promoter may be used in this methodology. In general, it is advantageous to employ a strong promoter functional in eukaryotic cells. The strong promoter may be, but not limited to, the immediate early cytomegalovirus promoter (CMV-IE) of human or murine origin, or optionally having another origin such as the rat or guinea pig.

[0294] In more general terms, the promoter has either a viral, or a cellular origin. A strong viral promoter other than CMV-IE that may be usefully employed in the practice of the invention is the early / late promoter of the SV40 virus or the LTR promoter of the Rous sarcoma virus. A strong cellular promoter that may be usefully employed in the practice of the invention is the promoter of a gene of the cytoskeleton, such as e.g. the desmin promoter (Kwissa et al., 2000), or the actin promoter (Miyazaki et al., 1989).

[0295] The promoter may be a constitutive promoter. The promoter may be a tissue specific promoter.

[0296] VECTOR

[0297] In some embodiments, the one or more polynucleotide(s) may be comprised within one or more vector(s). Such a vector may be used to introduce the nucleic acid sequence into a cell so that the cell expresses and / or produces the TNF inhibitor and / or TNFR2 agonist as described herein.

[0298] 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.

[0299] The vector may be any agent capable of delivering a polynucleotide to a cell and / or maintaining a polynucleotide in a cell. In some embodiments, the vector may be selected from the list consisting of: viral vectors, plasmids, naked nucleic acids, transposon-based vectors, nucleic acids complexed with polypeptide or other molecules, and nucleic acids immobilised onto solid phase particles.

[0300] In some embodiments, the vector may be a plasmid or a viral vector. In some embodiments, the vector may be a retroviral vector or a lentiviral vector.

[0301] The vector may be capable of transfecting or transducing a cell.

[0302] In some embodiments, a cell may comprise a polynucleotide or a vector as described herein. The cell may be capable of producing and / or expressing TNF inhibitor and / or TNFR2 agonist as described herein. 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.

[0303] In some embodiments, the polynucleotide or vector may, for example, be introduced into a cell by transduction or transfection in vitro or ex vivo. The cell is then capable of expressing and / or producing the TNF inhibitor and / or TNFR2 agonist, when the cell is cultured under suitable conditions. The TNF inhibitor and / or TNFR2 agonist can be harvested from the cell or supernatant of the cell.

[0304] Accordingly, described herein is a method of making a TNF inhibitor and / or TNFR2 agonist as described herein, which comprises the steps of: (i) introducing a polynucleotide or a vector as described above; (ii) culturing the cell under suitable conditions to express and / or produce the TNF inhibitor and / or TNFR2 agonist; and (iii) harvesting the TNF inhibitor and / or TNFR2 agonist. PHARMACEUTICAL COMPOSITION

[0305] Also disclosed herein is a pharmaceutical composition comprising a TNF inhibitor and / or TNFR2 agonist as described herein; a fusion protein as described herein; a polynucleotide or combination of a first and second polynucleotide as described herein; or a vector as described herein.

[0306] 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.

[0307] In some embodiments, the pharmaceutical composition may comprise one or more additional compounds, components and / or active agents.

[0308] In some embodiments, a first pharmaceutical composition comprising a TNF inhibitor as described herein may be administered in combination with a second pharmaceutical composition comprising a TNFR2 agonist as described herein.

[0309] In some embodiments, a first pharmaceutical composition comprising a TNFR2 agonist as described herein may be administered in combination with a second pharmaceutical composition comprising a TNF inhibitor as described herein.

[0310] Pharmaceutical compositions typically should be sterile and stable under the conditions of manufacture and storage. The pharmaceutical composition according to the invention may be produced using current good manufacturing practices (CGMP).

[0311] 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.

[0312] Pharmaceutical compositions comprise solid, semisolid, liquid, transdermal therapeutic systems (TTS). Solid compositions are selected from the group consisting of tablets, coated tablets, powder, granulate, pellets, capsules, effervescent tablets or transdermal therapeutic systems. Also comprised are liquid compositions, selected from the group consisting of solutions, syrups, infusions, extracts, solutions for intravenous application, solutions for infusion or solutions of the carrier systems of the present invention. Semisolid compositions that can be used in the context of the invention comprise emulsion, suspension, creams, lotions, gels, globules, buccal tablets and suppositories.

[0313] 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.

[0314] A sterile saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously.

[0315] 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.

[0316] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. The compounds of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[0317] In some embodiments, the salt may comprise a metal cation, such as a sodium salt or a potassium salt. In some embodiments, the pharmaceutical composition may comprise an aqueous diluent or solvent. In some embodiments, the aqueous diluent or solvent may be a phosphate buffered saline solution, such as a sterile phosphate buffered saline solution.

[0318] In some embodiments, the pharmaceutical composition may be, for example, in a form suitable for oral, parenteral administration, intravenous (such as intravenous infusion), intramuscular, subcutaneous, topical, inhalative, rectal, sublingual, transdermal, or vaginal administration.

[0319] KIT

[0320] Also provided herein is a kit comprising a TNF inhibitor as described herein and a TNFR2 agonist as described herein.

[0321] Further provided is a kit comprising a first polynucleotide encoding a TNF inhibitor as described herein and a second polynucleotide encoding TNFR2 agonist as described herein.

[0322] Also provided is a kit comprising a first pharmaceutical composition encoding a TNF inhibitor as described herein and a second pharmaceutical composition encoding TNFR2 agonist as described herein.

[0323] In each aspect relating to a kit as described herein, the TNF inhibitor may be a TNF inhibitor as described in previous aspects of the present disclosure.

[0324] In each aspect relating to a kit as described herein, the TNFR2 agonist may be a TNFR2 agonist as described in previous aspects of the present disclosure. Suitably, the TNFR2 agonist may be a TNFR2 mutein.

[0325] MEDICAL USE

[0326] In one aspect, provided herein is a combination comprising a TNF inhibitor and a TNFR2 agonist for use in treating or preventing a disease.

[0327] Also described is a method of treating a disease comprising administering a combination of a TNF inhibitor and a TNFR2 agonist to a subject in need thereof.

[0328] 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.

[0329] 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.

[0330] Suitably, the combination of a TNF inhibitor and a TNFR2 agonist, as provided in any format described herein, may treat a disease a described herein.

[0331] The terms “prevent”, “prevention” and “preventing” refers to preventing the onset of symptoms of a disease or condition, and as such encompasses prophylactic treatment.

[0332] Suitably, the combination of a TNF inhibitor and a TNFR2 agonist, as provided in any format described herein, may prevent a disease a described herein.

[0333] In some embodiments, the combination of a TNF inhibitor and a 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.

[0334] The present disclosure provides a TNFR2 agonist, preferably a TNF mutein as described herein, for use in treating and / or preventing a disease, wherein the TNFR2 agonist is administered in combination with TNF inhibitor.

[0335] The present disclosure provides a TNF inhibitor for use in treating and / or preventing a disease, wherein the TNF inhibitor is administered in combination with a TNFR2 agonist. Preferably the TNFR2 agonist is a TNF mutein as described herein.

[0336] The present disclosure provides a method for treating and / or preventing a disease comprising administering a TNFR2 agonist to a subject in need thereof, preferably wherein the TNFR2 agonist is a TNF mutein as described herein, wherein the TNFR2 agonist is administered in combination with a TNF inhibitor. The present disclosure provides a method for treating and / or preventing a disease comprising administering a TNF inhibitor to a subject in need thereof, wherein the TNF inhibitor is administered in combination with a TNFR2 agonist. Preferably the TNFR2 agonist is a TNF mutein as described herein.

[0337] In some embodiments, the TNFR2 agonist and the TNF inhibitor are administered concurrently.

[0338] In some embodiments, the TNFR2 agonist and the TNF inhibitor are administered sequentially.

[0339] Suitably, TNFR2 agonist is comprised within a pharmaceutical composition as described herein. Suitably, the TNFR2 agonist is a TNF mutein as described herein.

[0340] Suitably, the TNF inhibitor is comprised within a pharmaceutical composition as described herein.

[0341] Suitably, the disease or indication to be treated and / or prevented may be selected from inflammatory disorders, cancers, hyperproliferative disorders, autoimmune disorders, metabolic diseases, cardiovascular diseases, neuropathic diseases and neurological insults. Suitably, the disease or indication to be treated and / or prevented is an inflammatory disorder. Suitably, the disease or indication to be treated and / or prevented is cancer. Suitably, the disease or indication to be treated and / or prevented is pain, such as neuropathic pain. Suitably, the disease or indication to be treated and / or prevented is arthritis or multiple sclerosis.

[0342] ACUTE PAIN

[0343] Suitably, the disease or indication to be treated and / or prevented may be acute pain.

[0344] In some embodiments, the acute pain may be associated with any disease, disorder, or condition associated with and / or involving TNF, such as aberrant TNF signalling.

[0345] Acute pain is typically understood to be distinct from chronic pain, in that it is usually sudden in onset and usually caused by something specific (e.g., an injury, surgery or particular diseases). Acute pain is typically sharp or intense in quality, at least at the onset. Acute pain typically lasts for a short period of time (e.g., from a few minutes to less than six months) and may disappear when the underlying cause is treated / healed. Acute and chronic pain are different clinical entities and typically require different approaches to treatment. In some instances, acute pain may be understood to be pain that is not chronic pain. Both chronic and acute pain are well-defined in the art and have been acknowledged as distinct conditions, see e.g., Bonezzi C, et al., Pain Ther. 2020; 9(Suppl 1):1-15; Grichnik KP, Ferrante FM. Mt Sinai J Med. 1991 ; 58(3):217-20; International Association for the Study of Pain; Acute Pain; 2023 (https: / / www.iasp-pain.org / resources / topics / acute-pain / ).

[0346] In some embodiments, the acute pain may arise from injury or trauma.

[0347] In some embodiments, the acute pain may arise from surgery and / or health treatments. In some embodiments, the acute pain may be post-operative pain.

[0348] In some embodiments, the acute pain may arise from osteoarthritis and related diseases.

[0349] In some embodiments, the acute pain is associated with osteoarthritis.

[0350] In some embodiments, the acute pain is post-operative acute pain.

[0351] Acute pain includes, but is not limited to, acute herpes zoster pain, acute inflammatory pain, acute intermittent pain, acute musculoskeletal pain, acute obstetric pain, acute tendonitis pain, acute visceral pain, post-traumatic pain, burns, myocardial infarction, acute pancreatitis, cancer-associated acute pain, acute pain associated with tumor-related pain such as bone pain, headache and facial pain, visceral pain, or cancer treatment, such as post-chemotherapy symptoms, acute pain that may be attributed to a herniated or ruptured intervertabral disc.

[0352] In some embodiments, acute pain may be defined as pain that persists for less than 6 months.

[0353] In some embodiments, acute pain may be defined as pain that persists for a period of time selected from the list consisting of: about 5 months, about 4 months, about 3 months, about 2 months, or about 1 month.

[0354] In some embodiments, acute pain may be defined as pain that persists for a period of time selected from the list consisting of: less than 5 months, less than 4 months, less than 3 months, less than 2 months, or less than 1 month.

[0355] In some embodiments, acute pain may be defined as pain that persists for about 6 weeks. In some embodiments, acute pain may be defined as pain that persists for less than 6 weeks.

[0356] In some embodiments, acute pain may be defined as pain that persists for a period of time selected from the list consisting of: about 5 weeks, about 4 weeks, about 3 weeks, about 2 weeks, or about 1 week. In some embodiments, acute pain may be defined as pain that persists for a period of time selected from the list consisting of: less than 5 weeks, less than 4 weeks, less than 3 weeks, less than 2 weeks, or less than 1 week.

[0357] In some embodiments, acute pain may be defined as pain that persists for about 7 days. In some embodiments, acute pain may be defined as pain that persists for less than 7 days.

[0358] In some embodiments, acute pain may be defined as pain that persists for a period of time selected from the list consisting of: about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day.

[0359] In some embodiments, acute pain may be defined as pain that persists for a period of time selected from the list consisting of: less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days, or less than 1 day.

[0360] In some embodiments, acute pain may be defined as pain that persists for about 24 hours. In some embodiments, acute pain may be defined as pain that persists for less than 24 hours.

[0361] In some embodiments, acute pain may be defined as pain that persists for a period of time selected from the list consisting of: about 23 hours, about 22 hours, about 21 hours, about 20 hours, about 19 hours, about 18 hours, about 17 hours, about 16 hours, about 15 hours, about 14 hours, about 13 hours, about 12 hours, about 11 hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, or about 1 hour.

[0362] In some embodiments, acute pain may be defined as pain that persists for a period of time selected from the list consisting of: less than 23 hours, less than 22 hours, less than 21 hours, less than 20 hours, less than 19 hours, less than 18 hours, less than 17 hours, less than 16 hours, less than 15 hours, less than 14 hours, less than 13 hours, less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, or less than 1 hour.

[0363] In some embodiments, acute pain may be defined as pain that persists for up to 12 weeks.

[0364] In some embodiments, acute pain may be defined as pain that persists for (i.e., lasts for) less than 3 months. In some embodiments, the pharmaceutical compositions described herein may be useful in the treatment of acute pain that has persisted (or is expected to persist, e.g., where surgery is planned) for more than an hour but less than 3 months. In some embodiments, the pharmaceutical compositions described herein may be useful in the treatment of acute pain that has persisted (or is expected to persist) for more than 24 hours but less than 3 months. In some embodiments, the pharmaceutical compositions described herein may be useful in the treatment of acute pain that has persisted (or is expected to persist) for more than 1 week but less than 3 months.

[0365] In some embodiments, acute pain may be defined as pain that persists for about 6 weeks to about 3 months.

[0366] It will be understood that the TNFR2 agonist and the TNF inhibitor may alleviate acute pain faster than known therapies. It will also be understood that the one or more modulator(s) of TNF signalling according to the invention may promote improved recovery after surgery, compared to known therapies.

[0367] MYOCARDIAL INFARCTION

[0368] Suitably, the disease or indication to be treating and / or prevented may be myocardial infarction.

[0369] A myocardial infarction occurs when blood flow decreases or stops in one of the coronary arteries of the heart, causing infarction (tissue death) to the heart muscle.

[0370] A myocardial infarction, may be defined by elevated cardiac biomarkers with a rising or falling trend and at least one of the following: symptoms relating to ischemia, changes on an electrocardiogram (ECG), such as ST segment changes, new left bundle branch block, or pathologic Q waves, changes in the motion of the heart wall on imaging and / or demonstration of a thrombus on angiogram.

[0371] COMBINATION THERAPY

[0372] The medical uses and methods of treatment described herein may be used in combination with additional treatments and / or medicaments. For example, the medical uses and methods of treatment described herein may be combined with known treatments for acute pain, known chemotherapeutic treatments, known immunomodulatory treatments and / or known radiotherapy treatments in the form of a combination therapy or treatment.

[0373] In some embodiments, treatments for acute pain may include the administration of nonsteroidal anti-inflammatory drugs (NSAIDs) and / or benzodiazepines.

[0374] It will be understood that the combination of a TNFR2 agonist and TNF inhibitor may alleviate acute pain faster than known therapies and / or may promote improved recovery after surgery, compared to known therapies. It will be understood that the combination of a TNFR2 agonist and TNF inhibitor may also reduce or obviate reliance on the use of opiates to treat acute pain.

[0375] In some embodiments, the combination of a TNFR2 agonist and TNF inhibitor may alleviate acute pain faster, compared to other therapies that are known in the art.

[0376] In some embodiments, the combination of a TNFR2 agonist and TNF inhibitor may reduce or obviate the need to treat acute pain by administering opiates.

[0377] In some embodiments, the combination of a TNFR2 agonist and TNF inhibitor may reduce the dosage of opiates needed to treat acute pain.

[0378] SUBJECT

[0379] As used herein, the terms “patient” and “subject” may be used interchangeably.

[0380] In some embodiments, the subject of the medical uses and methods of treatment according to the present invention may be a mammal.

[0381] In some embodiments, the subject is human. In some embodiments, the subject may alternatively be a non-human mammal, including for example, a dog, a cat, a horse, a cow, a sheep or a pig.

[0382] In some embodiments, the subject is male. In some embodiments, the subject is female.

[0383] In some embodiments, the subject is a human male. In some embodiments, the subject is a human female.

[0384] ADMINISTRATION

[0385] In some embodiments, the TNFR2 agonist and / or TNF inhibitor is administered orally, parenterally, intravenously (such as intravenous infusion), locally, intrathecally, intraarticularly, intramuscularly, subcutaneously, topically, by inhalation, rectally, sublingually, by a transdermal route, or vaginally.

[0386] In some embodiments, the TNFR2 agonist is administered orally, parenterally, intravenously (such as intravenous infusion), locally, intrathecally, intra-articularly, intramuscularly, subcutaneously, topically, by inhalation, rectally, sublingually, by a transdermal route, or vaginally.

[0387] In some embodiments, the TNF inhibitor is administered orally, parenterally, intravenously (such as intravenous infusion), locally, intrathecally, intra-articularly, intramuscularly, subcutaneously, topically, by inhalation, rectally, sublingually, by a transdermal route, or vaginally.

[0388] In some embodiments, the TNFR2 agonist and / or TNF inhibitor is administered to a subject systemically.

[0389] In some embodiments, the TNFR2 agonist and / or TNF inhibitor may be administered one or more (e.g., multiple) times to a subject. In some embodiments, the pharmaceutical composition may be administered locally and systemically to a subject.

[0390] In some embodiments, the TNFR2 agonist and / or TNF inhibitor is injected into a subject. In some embodiments, the TNFR2 agonist is injected into a subject. In some embodiments, the TNF inhibitor is injected into a subject.

[0391] In some embodiments, the TNFR2 agonist and / or TNF inhibitor is administered to a subject once. In some embodiments, the TNFR2 agonist is administered to a subject once. In some embodiments, the TNF inhibitor is administered to a subject once.

[0392] In some embodiments, the TNFR2 agonist and / or TNF inhibitor is administered to a subject over a period of hours, days, weeks, months or years. In some embodiments, the TNFR2 agonist is administered to a subject over a period of hours, days, weeks, months or years. In some embodiments, the TNF inhibitor is administered to a subject over a period of hours, days, weeks, months or years.

[0393] In some embodiments, the TNFR2 agonist and / or TNF inhibitor treats and / or prevents acute pain within 2 hours after the pharmaceutical composition is administered to a subject (e.g., where the pharmaceutical composition is administered intravenously).

[0394] In some embodiments, the TNFR2 agonist and / or TNF inhibitor treats and / or prevents acute pain within 3 hours after the pharmaceutical composition is administered to a subject intravenously.

[0395] In some embodiments, the TNFR2 agonist and / or TNF inhibitor treats and / or prevents acute pain within 6 hours after the pharmaceutical composition is administered to a subject subcutaneously.

[0396] FURTHER ASEPCTS

[0397] The present invention may be described by way of the following numbered paragraphs (para): 1. A combination comprising a Tumor necrosis factor (TNF) inhibitor and a Tumor necrosis factor receptor 2 (TNFR2) agonist, wherein the TNFR2 agonist is a TNF mutein and the TNF inhibitor is capable of binding to TNF but does not bind the TNF mutein.

[0398] 2. The combination according to para 1 wherein the TNF inhibitor comprises an antibody, a polypeptide, a nucleic acid or a small molecule.

[0399] 3. The combination according to para 2 wherein the TNF inhibitor comprises an antibody.

[0400] 4. The combination according to any preceding para wherein the TNF inhibitor binds an epitope comprising one or more amino acids which are present in TNF but are not present in the TNF mutein.

[0401] 5. The combination according to para 4, wherein the TNF mutein comprises one or more amino acids 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 / A1411 / E142G / S143D, A141 H / E142S / S143D, A141 H / S143D,

[0402] L25V / A141 D / E142D / S143D, A141 N / E142D / S143D, A141T / E142S / S143D,

[0403] A141Q / E142D / S143D, A141T / E142D / S143D, A141 D / E142G / S143D, A141 D / S143D,

[0404] 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.

[0405] 6. The combination according to para 5, wherein the one or more amino acid mutations present in the TNF mutein comprise D139N and / or A141 R mutations corresponding to the position shown in SEQ ID NO: 2.

[0406] 7. The combination according to any of paras 3 to 6 wherein the antibody comprises HCDRs 1-3 which are present in SEQ ID NO: 16 and LCDRs 1-3 which are present in SEQ ID NO: 17.

[0407] 8. The combination according to para 7 wherein the antibody comprises the following CDRs:

[0408] HCDR1 : DYAMH (SEQ ID NO: 10)

[0409] HCDR2: AITWNSGHIDYADSVEG (SEQ ID NO: 11)

[0410] HCDR3: VSYLSTASSLDY (SEQ ID NO: 12) LCDR1 : RASQGIRNYLA (SEQ ID NO: 13)

[0411] LCDR2: AASTLQS (SEQ ID NO: 14)

[0412] LCDR3: QRYNRAPYT (SEQ ID NO: 15)

[0413] 9. The combination according to para 7 or 8 wherein the antibody comprises a variable heavy domain (VH) which comprises SEQ ID NO: 16 or a variant which has at least 90% identity to SEQ ID NO: 16; and a variable heavy domain (VL) which comprises SEQ ID NO: 17 or a sequence which has at least 90% identity to SEQ ID NO: 17.

[0414] 10. The combination according to any of paras 7 to 9 wherein the antibody comprises a heavy chain which comprises SEQ ID NO: 18 or a variant which has at least 90% identity to SEQ ID NO: 18; and a light chain which comprises SEQ ID NO: 18 or a variant which has at least 90% identity to SEQ ID NO: 19.

[0415] 11. The combination according to para 10 wherein the antibody is adalimumab.

[0416] 12. The combination according to any preceding para wherein the TNF mutein comprises a polypeptide comprising a TNFR2 binding domain comprising three TNF homology domains (THD) that specifically bind to TNFR2.

[0417] 13. The combination according to para 12 wherein at least one, preferably each, of the THD domains comprises a D139N and / or A141R mutation compared to SEQ ID NO: 2.

[0418] 14. The combination according to para 12 or 13, wherein the C-terminus of the first and second THD, respectively, which is in each case defined by the C-terminal consensus sequence:

[0419] V-X1-F-G-X2-X3 (SEQ ID NO: 28); is linked to the N-terminus of the second and third THD, respectively, which is in each case defined by the N-terminal consensus sequence:

[0420] P-X4-A-H-X5 (SEQ ID NO: 29); through a peptide Xa, which is in each case independently selected and has a length of 9 to 12 amino acids, preferably 9 to 11 , more preferably 9 to 10, wherein X1 is F or Y, wherein X2 is A or I, wherein X3 is a non-polar / hydrophobic or polar / neutral amino acid, preferably selected from the group consisting of F and I, wherein X4 is V or A, and wherein X5 is V or L. 15. The combination according to any of paras 12 to 14, wherein each THD comprises an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 2

[0421] 16. The combination according to any of paras 12 to 15, 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 and the D139N and A141 R mutations at the positions corresponding to SEQ ID NO: 2.

[0422] 17. The combination according to any of paras 12 to 16, 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 and the D139N and A141 R mutations at the positions corresponding to SEQ ID NO: 2.

[0423] 18. The combination according to any of paras 12 to 17, wherein the TNFR2 binding domain comprises or consists of a sequence with at least 80% sequence identity to SEQ ID NO: 5.

[0424] 19. The combination according to any of paras 12 to 18, wherein the polypeptide further comprises a multimerization domain.

[0425] 20. The combination according to para 19, wherein the multimerization domain is a dimerization domain, optionally wherein the dimerization domain is an immunoglobulin Fc domain.

[0426] 21. The combination according to any of paras 12 to 20, wherein the TNF mutein 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.

[0427] 22. The combination according to para 20 or 21 , wherein:

[0428] (a) the Fc domain: (i) does not comprise a proline at position 233 and / or (ii) comprises a glycine at position 236; and / or

[0429] (b) the Fc domain comprises a sequence with at least 85% sequence identity to SEQ ID NO: 1.

[0430] 23. The combination according to any of paras 12 to 22, wherein the TNFR2 agonist is a polypeptide comprising a sequence with at least 80% sequence identity to SEQ ID NO: 7.

[0431] 24. The combination according to paras 12 to 23, wherein the TNFR2 agonist is a polypeptide comprising the sequence of SEQ ID NO: 7. 25. The combination according to any preceding para wherein the TNF inhibitor comprises CDRs as defined in para 7 or 8 and the TNFR2 agonist is a polypeptide comprising a sequence with at least 80% sequence identity to SEQ ID NO: 7.

[0432] 26. The combination according to para 25 wherein the TNF inhibitor comprises a heavy chain which comprises SEQ ID NO: 18, or a variant which has at least 90% identity to SEQ ID NO: 18; and a light chain which comprises SEQ ID NO: 18, or a variant which has at least 90% identity to SEQ ID NO: 19; and the TNFR2 agonist is a polypeptide comprising a sequence with at least 80% sequence identity to SEQ ID NO: 7.

[0433] 27. A composition comprising a TNF inhibitor and a TNFR2 agonist according to any preceding para.

[0434] 28. A fusion protein comprising a TNF inhibitor and a TNFR2 agonist according to any preceding para, preferably wherein the TNF inhibitor is an antibody or a polypeptide.

[0435] 29. A polynucleotide encoding a TNF inhibitor and a TNFR2 agonist according to any preceding para.

[0436] 30. A composition comprising a first polynucleotide encoding a TNF inhibitor according to any preceding para and a second polynucleotide encoding a TNFR2 agonist according to any preceding para.

[0437] 31. A pharmaceutical composition comprising a TNF inhibitor and a TNFR2 agonist according to any of paras 1 to 26, a fusion protein according to para 28, a polynucleotide according to para 29, or a first and second polynucleotide according to para 30.

[0438] 32. A kit comprising a TNF inhibitor and a TNFR2 agonist according to any of paras 1 to 26, a first and second polynucleotide as defined in para 30 or a composition comprising a TNF inhibitor and a composition comprising TNFR2 agonist.

[0439] 33. A combination comprising a TNF inhibitor and a TNFR2 agonist, wherein the TNFR2 agonist is a TNF mutein and the TNF inhibitor is capable of binding to TNF but does not bind the TNF mutein for use in treating or preventing a disease.

[0440] 34. A combination comprising a TNF inhibitor and a TNFR2 agonist, wherein the TNFR2 agonist is a TNF mutein and the TNF inhibitor is capable of binding to TNF but does not bind the TNF mutein for use in treating acute pain.

[0441] 35. A TNF inhibitor for use in treating acute pain, wherein the TNF inhibitor is administered in combination with a TNFR2 agonist, wherein the TNFR2 agonist is a TNF mutein and the TNF inhibitor is capable of binding to TNF but does not bind the TNF mutein. 36. A TNFR2 agonist for use in treating acute pain, wherein the TNFR2 agonist is administered in combination with a TNF inhibitor, wherein the TNFR2 agonist is a TNF mutein and the TNF inhibitor is capable of binding to TNF but does not bind the TNF mutein.

[0442] 37. A pharmaceutical composition according to para 31 for use in treating acute pain.

[0443] 38. The combination for use according to para 34, the TNF inhibitor for use according to para 35, the TNFR2 agonist for use according to para 36 or the pharmaceutical composition for use according to para 37, wherein the acute pain has persisted for less than 3 months, for less than 2 months, or for less than 1 month.

[0444] 39. The combination for use according to para 34, the TNF inhibitor for use according to para 35, the TNFR2 agonist for use according to para 36 or the pharmaceutical composition for use according to para 37, wherein the acute pain has persisted, for less than 6 weeks, for less than 5 weeks, for less than 4 weeks, for less than 3 weeks, for less than 2 weeks, or for less than 1 week.

[0445] 40. The combination for use according to para 34, the TNF inhibitor for use according to para 35, the TNFR2 agonist for use according to para 36 or the pharmaceutical composition for use according to para 37, wherein the acute pain has persisted, for less than 7 days, for less than 6 days, for less than 5 days, for less than 4 days, for less than 3 days, for less than 2 days, or for less than 1 day.

[0446] 41 . The combination, TNF inhibitor, TNFR2 agonist or pharmaceutical composition for use according to any of paras 34 to 40, wherein the acute pain arises from injury or trauma.

[0447] 42. The combination, TNF inhibitor, TNFR2 agonist or pharmaceutical composition for use according to any of paras 34 to 40, wherein the acute pain arises from surgery and / or health treatment.

[0448] 43. The combination, TNF inhibitor, TNFR2 agonist or pharmaceutical composition for use according to any of paras 34 to 40, wherein the acute pain is associated with osteoarthritis.

[0449] 44. The combination, TNF inhibitor, TNFR2 agonist or pharmaceutical composition for use according to any of paras 34 to 40, wherein the acute pain is post-operative acute pain. 45. The combination, TNF inhibitor, TNFR2 agonist or pharmaceutical composition for use according to any of paras 34 to 44 wherein the TNF inhibitor is a TNF inhibitor as defined in any of paras 1 to 26.

[0450] 46. The combination, TNF inhibitor, TNFR2 agonist or pharmaceutical composition for use according to any of paras 34 to 45 wherein the TNFR2 agonist is a TNFR2 agonist as defined in any of paras 1 to 26.

[0451] 47. A method of treating or preventing a disease in a subject, comprising administering to the subject a therapeutically effective amount of a TNF inhibitor and a TNFR2 agonist, wherein the TNFR2 agonist is a TNF mutein and the TNF inhibitor is capable of binding to TNF but does not bind the TNF mutein.

[0452] 48. Use of a TNF inhibitor and a TNFR2 agonist in the manufacture of a medicament for treating or preventing a disease, wherein the TNFR2 agonist is a TNF mutein and the TNF inhibitor is capable of binding to TNF but does not bind the TNF mutein.

[0453] 49. A method according to para 47 or a use according to para 48 wherein the disease is acute pain.

[0454] ADDITIONAL ASEPCTS

[0455] Additional aspects of the present invention are described by way of the following numbered clauses:

[0456] 1. A combination comprising a Tumor necrosis factor (TNF) inhibitor and a Tumor necrosis factor receptor 2 (TNFR2) agonist.

[0457] 2. A combination according to clause 1 wherein the TNF inhibitor comprises an antibody, a polypeptide, a nucleic acid or a small molecule.

[0458] 3. The combination according to clause 2 wherein the TNF inhibitor comprises an antibody.

[0459] 4. The combination according to clause 3 wherein the antibody is selected from adalimumab, infliximab, golimumab, and certrolizumab.

[0460] 5. The combination according to any preceding clause wherein the TNFR2 agonist is selected from an antibody, a polypeptide, a nucleic acid or a small molecule.

[0461] 6. The combination according to clause 5 wherein the TNFR2 agonist is an antibody selected from 80M2 or MR2-1 . 7. The combination according to any preceding clause wherein the TNFR2 agonist is a TNF mutein.

[0462] 8. The combination according to any preceding clause wherein the TNF inhibitor is capable of binding to TNF but does not bind the TNFR2 agonist.

[0463] 9. The combination according to clause 8 wherein the TNF mutein comprises a polypeptide comprising a TNFR2 binding domain comprising three TNF homology domains (THD) that specifically bind to TNFR2.

[0464] 10. The combination according to clause 9 wherein at least one, preferably each, of the THD domains comprises a D139N and / or A141 R mutation as compared to SEQ ID NO: 2.

[0465] 11. The combination according to clause 9 or 10, wherein the C-terminus of the first and second THD, respectively, which is in each case defined by the C-terminal consensus sequence:

[0466] V-X1-F-G-X2-X3 (SEQ ID NO: 28); is linked to the N-terminus of the second and third THD, respectively, which is in each case defined by the N-terminal consensus sequence:

[0467] P-X4-A-H-X5 (SEQ ID NO: 29); through a peptide Xa, which is in each case independently selected and has a length of 9 to 12 amino acids, preferably 9 to 11 , more preferably 9 to 10, wherein X1 is F or Y, wherein X2 is A or I, wherein X3 is a non-polar / hydrophobic or polar / neutral amino acid, preferably selected from the group consisting of F and I, wherein X4 is V or A, and wherein X5 is V or L.

[0468] 12. The combination according to any of clauses 9 to 11 , wherein each THD comprises an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 2

[0469] 13. The combination according to any of clauses 9 to 12, 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.

[0470] 14. The combination according to any of clauses 9 to 13, 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. 15. The combination according to any of clauses 9 to 14, wherein the TNFR2 binding domain comprises or consists of a sequence with at least 80% sequence identity to SEQ ID NO: 5.

[0471] 16. The combination according to any of clauses 9 to 15, wherein the polypeptide further comprises a multimerization domain.

[0472] 17. The combination according to clause 16, wherein the multimerization domain is a dimerization domain, optionally wherein the dimerization domain is an immunoglobulin Fc domain.

[0473] 18. The combination according to any of clauses 9 to 17, wherein the TNF mutein 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.

[0474] 19. The combination according to clause 17 or 18, wherein:

[0475] (a) the Fc domain: (i) does not comprise a proline at position 233 and / or (ii) comprises a glycine at position 236; and / or

[0476] (b) the Fc domain comprises a sequence with at least 85% sequence identity to SEQ ID NO: 1.

[0477] 20. The combination according to any of clauses 9 to 19, wherein the TNFR2 agonist is a polypeptide comprising a sequence with at least 80% sequence identity to SEQ ID NO: 7.

[0478] 21. The combination according to clauses 9 to 20, wherein the TNFR2 agonist is a polypeptide comprising the sequence of SEQ ID NO: 7.

[0479] 22. A composition comprising a TNF inhibitor and a TNFR2 agonist according to any preceding clause.

[0480] 23. A fusion protein comprising a TNF inhibitor and a TNFR2 agonist according to any preceding clause, preferably wherein each of the TNF inhibitor and TNFR2 agonist is independently selected from an antibody or a polypeptide.

[0481] 24. A polynucleotide encoding a TNF inhibitor according to any preceding clause and a TNFR2 agonist according to any preceding clause.

[0482] 25. A composition comprising a first polynucleotide encoding a TNF inhibitor according to any preceding clause and a second polynucleotide encoding a TNFR2 agonist according to any preceding clause. 26. A pharmaceutical composition comprising a TNF inhibitor and a TNFR2 agonist according to any of clauses 1 to 21 , a fusion protein according to clause 23, a polynucleotide according to clause 24, or a first and second polynucleotide according to clause 25.

[0483] 27. A kit comprising a TNF inhibitor and a TNFR2 agonist according to any of clauses 1 to 21 , a polynucleotide encoding a TNF inhibitor and a polynucleotide encoding a TNFR2 as defined in claim 24 or a first composition comprising a TNF inhibitor and a second composition comprising TNFR2 agonist.

[0484] 28. A combination comprising a TNF inhibitor and a TNFR2 agonist for use in treating or preventing a disease.

[0485] 29. A combination comprising a TNF inhibitor and a TNFR2 agonist for use in treating acute pain.

[0486] 30. A TNF inhibitor for use in treating acute pain, wherein the TNF inhibitor is administered in combination with a TNFR2 agonist.

[0487] 31. A TNFR2 agonist for use in treating acute pain, wherein the TNFR2 agonist is administered in combination with a TNF inhibitor.

[0488] 32. A pharmaceutical composition according to clause 26 for use in treating acute pain.

[0489] 33. The combination for use according to clause 29, the TNF inhibitor for use according to clause 30, the TNFR2 agonist for use according to clause 31 or the pharmaceutical composition for use according to clause 32, wherein the acute pain has persisted for less than 3 months, for less than 2 months, or for less than 1 month.

[0490] 34. The combination for use according to clause 29, the TNF inhibitor for use according to clause 30, the TNFR2 agonist for use according to clause 31 or the pharmaceutical composition for use according to clause 32, wherein the acute pain has persisted, for less than 6 weeks, for less than 5 weeks, for less than 4 weeks, for less than 3 weeks, for less than 2 weeks, or for less than 1 week.

[0491] 35. The combination for use according to clause 29, the TNF inhibitor for use according to clause 30, the TNFR2 agonist for use according to clause 31 or the pharmaceutical composition for use according to clause 32, wherein the acute pain has persisted, for less than 7 days, for less than 6 days, for less than 5 days, for less than 4 days, for less than 3 days, for less than 2 days, or for less than 1 day.

[0492] 36. The combination, TNF inhibitor, TNFR2 agonist or pharmaceutical composition for use according to any of clauses 29 to 35, wherein the acute pain arises from injury or trauma. 37. The combination, TNF inhibitor, TNFR2 agonist or pharmaceutical composition for use according to any of clauses 29 to 35, wherein the acute pain arises from surgery and / or health treatment.

[0493] 38. The combination, TNF inhibitor, TNFR2 agonist or pharmaceutical composition for use according to any of clauses 29 to 35, wherein the acute pain is associated with osteoarthritis.

[0494] 39. The combination, TNF inhibitor, TNFR2 agonist or pharmaceutical composition for use according to any of clauses 29 to 35, wherein the acute pain is post-operative acute pain.

[0495] 40. The combination, TNF inhibitor, TNFR2 agonist or pharmaceutical composition for use according to any of clauses 29 to 35 wherein the TNF inhibitor is a TNF inhibitor as defined in any of clauses 1 to 21.

[0496] 41 . The combination, TNF inhibitor, TNFR2 agonist or pharmaceutical composition for use according to any of clauses 29 to 35 wherein the TNFR2 agonist is a TNFR2 agonist as defined in any of clauses 1 to 21.

[0497] 42. A method of treating or preventing a disease in a subject, comprising administering to the subject a therapeutically effective amount of a TNF inhibitor and a TNFR2 agonist.

[0498] 43. Use of a TNF inhibitor and a TNFR2 agonist in the manufacture of a medicament for treating or preventing a disease.

[0499] 44. A method according to clause 42 or a use according to clause 43 wherein the disease is acute pain.

[0500] GENERAL TERMS AND DEFINITIONS

[0501] 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.

[0502] 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.

[0503] 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.

[0504] 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.

[0505] As used herein, “antibody” may refer to a protein or polypeptide having an antigen binding site or antigen-binding domain which comprises at least one complementarity determining region (CDR). The term “antibody” may therefore encompass antibody fragments such as a variable immunoglobulin domain; an antigen binding site of an antibody; a single-chain variable fragment (scFv); a Fab; an F(ab)’2; or an Fv. The term ‘antibody’ may also encompass dingle domain antibodies such as VHH. Suitably, the antibody may comprise 6 CDRs and have an antigen binding site which is equivalent to that of an antibody comprising a VH and VL domain (including antibody fragments such as scFv). “Heavy chain variable region” or “VH” refers to the fragment of the heavy chain of an antigen-binding domain or antibody that contains three CDRs interposed between flanking stretches known as framework regions, which are more highly conserved than the CDRs and form a scaffold to support the CDRs. “Light chain variable region” or “VL” refers to the fragment of the light chain of an antigen-binding domain or antibody that contains three CDRs interposed between framework regions. “Complementarity determining region” or “CDR” with regard to an antigen-binding domain or antibody or antigenbinding fragment thereof refers to a highly variable loop in the variable region of the heavy chain of the light chain of an antibody. CDRs can interact with the antigen conformation and largely determine binding to the antigen (although some framework regions are known to be involved in binding). The heavy chain variable region and the light chain variable region each contain 3 CDRs (heavy chain CDRs 1 , 2 and 3 and light chain CDRs 1 , 2 and 3, numbered from the amino to the carboxy terminus).

[0506] A number of definitions of the CDRs are commonly in use. The Kabat definition is based on sequence variability and is the most commonly used (see http: / / www.bioinf.org.uk / abs / ). The ImMunoGeneTics information system (IMGT) (see http: / / www.imgt.org) can also be used. According to this system, a complementarity determining region (CDR-IMGT) is a loop region of a variable domain, delimited according to the IMGT unique numbering for V domain. There are three CDR-IMGT in a variable domain: CDR1-IMGT (loop BC), CDR2-IMGT (loop C'C"), and CDR3-IMGT (loop FG). Other definitions of the CDRs have also been developed, such as the Chothia, the AbM and the contact definitions (see http: / / www.imgt.org). The CDRs of the antibodies described here may be defined using any suitable system, such as any suitable system known in the art. "Humanised antibody" may refer to a genetically engineered non-human antibody, which contains human antibody constant domains and non-human variable domains modified to contain a high level of sequence homology to human variable domains. This can be achieved by grafting of the non-human antibody complementarity-determining regions (CDRs), which together form the antigen binding site, onto a homologous human acceptor framework region (FR). Non-limiting examples of antibody humanisation methods include CDR grafting, and resurfacing (i.e. replacing surface residues to obtain a “more human” surface. For example, in order to fully reconstitute the binding affinity and specificity of the parental antibody, the substitution of framework residues from the parental antibody (i.e. the non-human antibody) into the human framework regions (back-mutations) may be required. Structural homology modelling may help to identify the amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanised antibody may comprise non-human CDR sequences, primarily human framework regions optionally comprising one or more amino acid back-mutations to the non-human amino acid sequence, and, optionally, fully human constant regions. Optionally, additional amino acid modifications, which are not necessarily back-mutations, may be introduced to obtain a humanized antibody with preferred characteristics, such as affinity and biochemical properties. Humanisation of non-human therapeutic antibodies is performed to minimise its immunogenicity in man while such humanised antibodies at the same time maintain the specificity and binding affinity of the antibody of non-human origin.

[0507] 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.

[0508] 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. 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.

[0509] 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'.

[0510] 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.

[0511] 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.

[0512] 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.

[0513] 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.

[0514] The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention. EXAMPLES

[0515] In all Examples, “SCTNFR2-FC” corresponds to a protein having the sequence of SEQ ID NO: 7.

[0516] Example 1 - Adalimumab does not interact with SCTNFR2-FC

[0517] The interaction of the anti-TNF inhibitors Adalimumab, Infliximab and Golimumab with the TNF mutein (SCTNFR2-FC) was analyzed by enzyme-linked immunosorbent assay (ELISA) (Figure 1).

[0518] 5 pg / ml of human SCTNFR2-FC diluted in 100 pl of PBS was immobilized on Flat bottom Nunc- Immuno MaxiSorp 96-well plates overnight at 4°C. The next day, 200 pl of Assay Diluent was added to each well for 2 hours to block unspecific binding. Next, 100 pl of a 1 / sqrt(10) titration of the anti-TNF inhibitors in Assay Diluent was added and incubated for 1 hour. A start concentration of 0.948 pg / ml was chosen. To detect bound anti-TNF inhibitors, 100 pl of an anti-human Fab antibody conjugated to a horseradish peroxidase (HRP) diluted 1 :40,000 in Assay Diluent was added and incubated for 1 h. 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 CLARIOstar Plus microplate reader and data were analyzed using Microsoft Excel and GraphPad Prism.

[0519] Surprisingly, Adalimumab did not bind to SCTNFR2-FC, while Infliximab and Golimumab bound to SCTNFR2-FC in a dose dependent manner with EC50 values of 28.9 ng / ml and 157.3 ng / ml; respectively. These data support the combinatorial use of Adalimumab and a TNF-based (i.e. a TNF mutein) TNFR2 agonist.

[0520] Example 2 - Adalimumab does not neutralize SCTNFR2-FC

[0521] To verify if the surprising lack of binding of Adalimumab to SCTNFR2-FC would also result in the lack of neutralizing capability a bioassay was performed.

[0522] The functionality of anti-TNF inhibitors to neutralize either recombinant human TNF (rhTNF) or SCTNFR2-FC was measured using a TNF reporter cell line. To measure TNFR2-induced luminescence, the Bio-Gio Luciferase assay reagent from Promega was used. First, 104reporter cells in 200 pl DM EM + 10% FBS medium were seeded in a Flat bottom Cell Culture- Treated white 96-well plate. The next day, a 10-step 1 / sqrt(10) dilution of either Adalimumab, Infliximab, Golimumab (Figure 2 A,B) or Certolizumab-pegol (Figure 2 C,D) was prepared. Here, a start concentration of 2 pg / ml was chosen. The prepared stimulation was incubated with a constant concentration of 20 ng / ml recombinant human TNF (rhTNF) or SCTNFR2-FC for 30 min, resulting in a 1 :1 dilution of the final concentration. Next, the medium of the previously seeded cells was aspirated and 75 pl of the prepared stimulation was added to the adherent cells and cultivated under normal conditions 24 hours. At the end of the incubation period, the plates were removed from the 37°C incubator and equilibrated to room temperature. After 5- 15 minutes, 75 pl of Bio-Gio reagent were added to each well and cells were incubated for further 10 min in the dark. Finally, the luminescence was measured using the CLARIOstar Plus multiplate reader.

[0523] As expected, Adalimumab, Infliximab, Golimumab and Certolizumab-pegol neutralized TNF induced luminescence dose dependently (Figure 2 A,C). While Infliximab, Golimumab and Certolizumab-pegol neutralized the TNF based TNFR2 agonist SCTNFR2-FC dose dependently, Adalimumab did not neutralize scTNFR2-Fc-induced TNFR2 activation. This confirms the surprising finding that Adalimumab does not bind (Figure 1) or inhibit (Figure 2) SCTNFR2-FC.

[0524] Example 3 - Mutations in the TNF region abrogate the interaction between Adalimumab and SCTNFR2-FC

[0525] To determine the reason why Adalimumab does not interact with SCTNFR2-FC, a cellular binding assay was performed. CHO-K1 cells were transfected with 0.1 pg of either non- cleavable wildtype TNF or TNFR2 bearing the mutations D143N and A145R (Figure 3A) or non-cleavable covalently stabilized trimeric single-chain TNF (scTNF) or TNFR2-selective SCTNFR2 (Figure 3B) encoding mRNA. Here, the commercially available MessengerMax Kit was used for transfection. The cells were incubated at 37°C and 5% CO2 overnight. All subsequent steps were performed at 4°C using precooled reagents. First, cells were harvested and collected in FACS buffer (DPBS containing 2% FBS and 2mM EDTA). Next, 100 pl of a (1 / sqrt(10)) titration of Adalimumab or 1 pg / ml Infliximab as a positive control was added and incubated for 1 hour at 4°C. Then 100 pl of FACS buffer was added and cells were centrifuged for 3 min at 300 g. Bound anti-TNF therapeutics were detected using an anti-Fc PE-labeled antibody (1 :100 dilution in 100 pl FACS buffer) for 30 min. After washing with 200 pl of FACS buffer, the cells were centrifuged for 3 min at 300 g and the supernatant was aspirated. Then cells were fixed using 200 pl of fixation solution and incubated for 30 min at 4°C. Lastly, the cells were centrifuged (3 min at 300 g), collected in 100 pl of FACS buffer and analyzed using the BD FACS Celesta. The Mean Fluorescent Intensity (MFI) was determined using FlowJo.

[0526] As expected, Adalimumab bound to wildtype TNF in a dose-dependent manner (Figure 3A). Furthermore, Adalimumab was able to bind the covalently stabilized scTNF in a dose- dependent manner (Figure 3B). Surprisingly, once the two point mutations D143N and A145R (which correspond to D139N and A141 R of SEQ ID NO: 2) were present in TNF or the TNFR2- selective TNF mutein, any binding to Adalimumab was abrogated (Figure 3 A, B). In contrast, Infliximab efficiently bound to all tested TNF muteins, highlighting the unique binding site of Adalimumab and the possibility of combining Adalimumab with a TNF-based TNFR2 agonist.

[0527] Example 4 - Investigating TNF mutations to disrupt Adalimumab-TNF interactions, while maintaining TNFR2 activation.

[0528] As described in Example 3, it was surprisingly identified that the TNF inhibitor Adalimumab does not interact with SCTNFR2-FC due to the point mutations D143N and A145R, which confer TNFR2 selectivity on the TNF mutein. This finding enables the prediction of additional mutations that might disrupt the interaction between Adalimumab and other TNFR2 agonist fusion proteins of the SCTNFR2-FC format.

[0529] To this end, a cellular binding assay to analyze the interaction of Adalimumab with different TNF muteins was performed. CHO-K1 cells were transfected with DNA expression constructs coding for either non-cleavable wildtype (WT) TNF (positive control) or TNF muteins bearing mutations listed in Table 3. TNF mutein 1 corresponds to SCTNFR2-FC, and TNF muteins 2-6 had the sequence of SEQ ID NO: 7, but with each THD having the mutations as indicated in Table 3, rather than the D143N, A145R mutations.

[0530] Table 3: Tested single-chain TNF muteins

[0531] Lipofectamine LTX (Thermo Fisher Scientific) was used for transfection according to the manufacturer’s protocol. The cells were incubated at 37°C and 5% CO2 overnight. All subsequent steps were performed at 4°C using precooled reagents. First, cells were harvested and collected in FACS buffer (DPBS containing 2% FBS and 2mM EDTA). Next, Adalimumab (1 pg / ml) was added and incubated for 1 hour at 4°C. Then 100 pl of FACS buffer was added and cells were centrifuged for 3 min at 300 g. Bound Adalimumab was detected using an anti-Fc PE-labeled antibody (1 :100 dilution in 100 pl FACS buffer) for 30 min. After washing with 200 pl of FACS buffer, the cells were centrifuged for 3 min at 300 g and the supernatant was aspirated. Then cells were fixed using 200 pl of fixation solution and incubated for 30 min at 4°C. Lastly, the cells were centrifuged (3 min at 300 g), collected in 100 pl of FACS buffer and analyzed using the BD FACS Celesta. The frequency of the PE- positive cells was quantified using FlowJo.

[0532] Interestingly, mutations in either the site D143 or A145 resulted in a loss of binding (Figure 4), indicating the relevance of both sites for loss of Adalimumab binding to TNF. The combined mutations D143N / A145R resulted in complete loss of Adalimumab binding. tion of Adalimumab and SCTNFR2-FC anti- function.

[0533] To demonstrate the functional superiority of the adalimumab and SCTNFR2-FC combination, we analyzed its impact on macrophage function. Macrophages are pivotal innate immune cells that play a crucial role in a multitude of pathological and physiological indications. They play a key role in development and maintenance of almost every inflammatory disease. Macrophages are heterogeneous and their phenotype and functions are regulated by the surrounding micro-environment. Next to the tissue-resident macrophages, they can originate from blood monocytes by stimulus-dependent differentiation. Upon injury or disease, monocytes migrate to the damaged tissue and exert both inflammatory and protective functions. This macrophage plasticity is simplified by the M1 / M2 paradigm. Classically activated or M1 macrophages produce pro-inflammatory cytokines and contribute to oxidative stress responses by the release of radical oxygen species (ROS). In contrast, alternatively activated or M2 macrophages produce anti-inflammatory cytokines, and contribute to clearance of damaged tissue, to repair inflammation-associated injuries. ,. The impact of treatment with the adalimumab and SCTNFR2-FC combination on macrophage repolarization and key functions of polarized M1 and M2 macrophages, i.e. release of ROS (M1) and phagocytotic activity (M2) was assessed.

[0534] Primary CD14+(cluster of differentiation 14) monocytes were isolated from human PBMCs (peripheral blood mononuclear cells), donated from healthy donors, using Miltenyi's CD14 MACS (magnetic activated cell sorting) isolation kit. The isolated cells were then cultured with 10 ng / ml human M-CSF (macrophage colony-stimulating factor) for 5 days to induce differentiation into the M0 macrophage phenotype, under incubation conditions of 37°C and 5% CO2. After this differentiation period, cells were labeled with DCFDA (Abeam), a cell- permeable fluorescent probe used to detect reactive oxygen species (ROS) according to manufacturer’s instructions. After ROS labeling, macrophages were further treated with either 100 ng / ml LPS (lipopolysaccharide) and 20 ng / ml IFN-y (interferon gamma) to induce a pro- inflammatory M1 phenotype, or 20 ng / ml IL-4 (interleukin 4) and 20 ng / ml IL-13 to promote polarization into an anti-inflammatory M2 phenotype. In addition, cells were either left untreated or were stimulated with 100 ng / mL Adalimumab and / or SCTNFR2-FC. The cells were then incubated at 37°C and 5% CO2 overnight. After 24 hours, the fluorescence was measured using a multiplate reader (TECAN).

[0535] As expected, only the M1 macrophages showed increased release of ROS compared to control MO macrophages (Figure 5A). Both incubation with Adalimumab or SCTNFR2-FC resulted in a slight reduction of the ROS release. Interestingly, the combination of Adalimumab and SCTNFR2-FC significantly reduced ROS release compared to single treatments in all M1 stimulation paradigms (Figure 5A). In contrast, none of the conditions impacted ROS release in M2 macrophages. These data demonstrate that the combination of the anti-TNF Adalimumab with the TNFR2 agonist SCTNFR2-FC is superior in reducing pro-inflammatory release of ROS by M1 macrophages over the single treatments.

[0536] To investigate the impact of the TNF pathway modulation on phagocytotic activity, primary CD14+monocytes were isolated from human PBMCs (peripheral blood mononuclear cells), donated from healthy donors, using Miltenyi's CD14 MACS (magnetic activated cell sorting) isolation kit. The isolated cells were then cultured with 10 ng / ml human M-CSF (macrophage colony-stimulating factor) for 5 days to induce differentiation into the M0 macrophage phenotype, under incubation conditions of 37°C and 5% CO2. After this differentiation period, the cells were further treated with either 20 ng / ml IFN-y (interferon gamma) and 100 ng / ml LPS (lipopolysaccharide) [M1] or 20 ng / ml IL-4 (interleukin 4) and 20 ng / ml IL-13 [M2] to promote polarization into either pro-inflammatory M1 or anti-inflammatory M2 phenotypes, respectively. In addition, cells were either left untreated or exposed to 100 ng / ml SCTNFR2-FC or Adalimumab, or a combination of SCTNFR2-FC and Adalimumab, followed by a 24-hour incubation at 37°C and 5% CO2. Then, the cells were incubated with pHrodo-labeled E.coli particles (1 :50, Thermo Fisher Scientific) for 24h. Following this incubation period, the cells were labeled with PhenoVue DRAQ5 Total Cell Nuclear Stain from Revvity (1 :1000) and fluorescence signals were analzed using the Incucyte system (Sartorius). The analysis was performed by normalizing the total integrated intensity ratio of the phagocytozed E.coli particles over the total integrated intensity of the nuclear marker, allowing for accurate quantification of phagocytic activity.

[0537] Here, as expected, M1 macrophages showed reduced and M2 macrophages showed increased phagocytotic activity compared to M0 macrophages (Figure 5B). Interestingly, only the addition of the combination of SCTNFR2-FC and adalimumab resulted in improved phagocytotic activity in all 3 phenotypes. Whereas SCTNFR2-FC or adalimumab alone resulted in a slight upregulation of the phagocytotic activity of the M1 phenotype, only the combination of both TNF modulators strongly increased the phagocytotic activity to a level comparable with M2 macrophages (Figure 5B).

[0538] Altogether, these data sets demonstrate that only the combination of the TNF inhibitor Adalimumab together with the TNFR2 agonist SCTNFR2-FC results in superior antiinflammatory and reparative function of macrophages, whereas the stimulation with either adalimumab or SCTNFR2-FC only partially impacts the functional changes of macrophages.

[0539] Example 6 - Combination of adalimumab and SCTNFR2-FC promotes anti-inflammatory polarization of macrophages.

[0540] Next to the functional changes of macrophages, the impact of TNF modulation by Adalimumab and SCTNFR2-FC on macrophage polarization was analyzed. To this end, the release of pro- and anti-inflammatory mediators characteristic for the M1 or M2 macrophage phenotype was quantified. To quantify the M1 phenotype, the release of the chemoattractant MIP-1 and the cytokine IL-1 p was measured. The Th2-attracting chemokine MDC / CCL22 was quantified to represent the M2 phenotype.

[0541] Primary CD14+(cluster of differentiation 14) monocytes were isolated from human PBMCs (peripheral blood mononuclear cells), donated from healthy donors, using Miltenyi's CD14 MACS (magnetic activated cell sorting) isolation kit. The isolated cells were then cultured with 10 ng / ml human M-CSF (macrophage colony-stimulating factor) for 5 days to induce differentiation into the MO macrophage phenotype, under incubation conditions of 37°C and 5% CO2. After this differentiation period, the cells were further treated with either 20 ng / ml IFN-y (interferon gamma) and 100 ng / ml LPS (lipopolysaccharide) [M1] or 20 ng / ml IL-4 (interleukin 4) and 20 ng / ml IL-13 [M2] to promote polarization into either pro-inflammatory M1 or anti-inflammatory M2 phenotypes, respectively. In addition, cells were either left untreated or exposed to 100 ng / ml SCTN FR2-FC, 1 pg / ml Adalimumab, or a combination of SCTN FR2-FC and Adalimumab, followed by a 24-hour incubation at 37°C and 5% CO2. Then, the supernatants were collected and cytokine / chemokine levels were quantified using a multiplex assay from Meso Scale Discovery (MSD).

[0542] While adalimumab inhibited the release of MIP-1 and I L-1 p and thereby the differentiation of the pro-inflammatory M1 macrophage phenotype, SCTNFR2-FC did not affect the secretion of these pro-inflammatory cytokines by M1 macrophages (Figure 6A, B). In contrast, SCTNFR2- Fc promoted the release of MDC by anti-inflammatory M2 macrophages (Figure 6C). Interestingly, only the combination of adalimumab and SCTN FR2-FC retains both mechanisms of action, inhibiting the release of pro-inflammatory mediators by M1 macrophages while promoting the M2 macrophage phenotype (Figure 6A, B, C). This suggests that compared to single treatments, the combination therapy results in superior anti-inflammatory polarization of macrophages. Due to the high plasticity of macrophages this indicates superior performance of the combination therapy under physiological conditions, where a spectrum of phenotypes is present. The combined effect of inhibiting the M1 phenotype, while promoting the M2 phenotype leads to a superior polarization of the macrophage spectrum into an antiinflammatory and reparative M2 phenotype.

[0543] Example 7 - Combination of adalimumab and SCTNFR2-FC promotes superior inhibition of pro- inflammatory cytokine release.

[0544] Endothelial cells play an important role in regulating vasoreactivity, angiogenesis, immunomodulation and nutrient exchange and uptake; therefore maintaining their homeostasis is crucial for physiological functions (Pasut, A., et al., 2025, Nat Rev Cardiol., 22, 923-943). Pro-inflammatory cytokines such as TNF, and IL-1 beta (interleukin 1 beta) can induce endothelial activation (Amersfoort, J., et al., 2022, Nat Rev Immunol., 22, 576-588). This can lead to an increased expression of adhesion markers and other pro-inflammatory cytokines such as IL-8 (interleukin 8), which serves as a chemoattractant for neutrophils and was used as a diagnostic marker in several inflammatory diseases (Li, M., et al., 2018, Front Pharmacol., 9, 233). Human Umbillical Vein Endothelial Cells (HUVEC) cells were cultured in Endothelial Cell Basal Medium 2. Prior to the cell seeding, the 96-well flat-bottom plate was incubated with 50 pl per well of human fibronectin (concentration) for minimum 30 minutes. After that, the fibronectin was removed and 20,000 HUVEC cells in 200 pl ECBM2 (endothelial cell basal medium) medium were seeded. After the HUVEC cells reached confluency, a cytokine mixture that contained 10 ng / mL TNF, 10 ng / mL I L-1 and 10 ng / mL IFN-y (Interferon gamma) was prepared. The cells were then treated with the cytokine mixture alone or in combination with either 10 pg / mL anti-TNF adalimumab, 100 ng / mL TNFR2 agonist SCTNFR2- Fc, or 10 pg / mL adalimumab with 100 ng / mL SCTNFR2-FC. Cells treated only with the medium were used as the control group. After 24 hours of the treatments, supernatants were collected and IL-8 secretion was quantified using the Biolegend ELISA MAX™ Standard Set Human IL- 8 ELISA kit according to the manufacturer’s instruction. The results were interpolated using the GraphPad Prism non-linear fit, one-site binding (hyperbola) equation.

[0545] Stimulation with the cytokine mixture led to a significant increase in the IL-8 production of the HUVEC cells. While the treatments with only adalimumab or SCTNFR2-FC showed limited effects on IL-8 secretion, the combination of both adalimumab and SCTNFR2-FC demonstrated significant reduction in the IL-8 release induced by the cytokine mixture treatment (Figure 7). This indicates the superiority of combining the TNF antagonist with TNFR2 agonist on the induction of pro-inflammatory responses. Example 8 - Combination of anti-TNF antibodies and TNFR2 agonistic antibodies promotes superior effect in reducing induced ROS release.

[0546] It was further investigated whether superior effects were also observable for combinations of TNF inhibitors with different TNFR2 agonists. The release of pro-inflammatory ROS by M1 macrophages, a process often implicated in the pathogenesis of numerous inflammatory and autoimmune diseases (Manful et al. Int J Mol Sci. 2025 Aug 4;26(15):7520), was used as a read-out.

[0547] Primary CD14+(cluster of differentiation 14) monocytes were isolated from human PBMCs (peripheral blood mononuclear cells), donated from healthy donors, using Miltenyi's CD14 MACS (magnetic activated cell sorting) isolation kit. The isolated cells were then cultured with 10 ng / ml human M-CSF (macrophage colony-stimulating factor) for 5 days to induce differentiation into the MO macrophage phenotype, under incubation conditions of 37°C and 5% CO2. After this differentiation period, cells were labeled with DCFDA (Abeam), a cell- permeable fluorescent probe used to detect reactive oxygen species (ROS) according to manufacturer’s instructions. After ROS labeling, macrophages were further treated with either 100 ng / ml LPS (lipopolysaccharide) and 20 ng / ml IFN-y (interferon gamma) to induce a pro- inflammatory M1 phenotype. In addition, cells were either left untreated or were stimulated (i) with 100 ng / mL of the anti-TNF monoclonal antibodies adalimumab or infliximab, (ii) with 1 pg / mL of the commercially available TNFR2 agonist antibody MR2-1 or 80M2, or (iii) with a combination of each anti-TNF with each TNFR2 agonist antibody. The cells were then incubated at 37°C and 5% CO2 overnight. After 24 hours, the fluorescence was measured using a multiplate reader (TECAN).

[0548] Data were again normalized to the ROS release by M0 macrophages and the inhibition of Mi- induced ROS-release by the TNF modulator antibodies was quantified (Figure 8). As expected, either anti-TNFs or TNFR2 agonist antibodies alone induced a slight decrease in ROS-release. However, the combination of 80M2 or MR2-1 with the anti-TNFs resulted in the strongest decrease in ROS release (Figure 8). This confirmed the superiority of the combination of a TNF inhibitor with a TNFR2 agonist. These findings demonstrate that a combinatorial approach using inhibitory anti-TNF antibodies together with TNFR2 agonistic antibodies is significantly more potent than the monotherapies.

[0549] Example 9 - Definition of a TNFR2 agonist.

[0550] TNFR2 agonists were tested using a reporter cell line with high TNFR2 expression. This HEK cell line incorporated a luciferase response element downstream of the NF-kB transcription promoter. To measure TNFR2-induced luminescence, the Bio-Gio Luciferase assay reagent from Promega was used. First, 104HEK reporter cells in 200 pl DMEM + 10% FBS medium were seeded in a Flat bottom Cell Culture-Treated white 96-well plate. The next day, the medium of the previously seeded cells was aspirated and 100 pl of SCTNFR2-FC (100 ng / ml), 80M2 (2.5 pg / ml), MR2-1 (2.5 pg / ml) or medium was added to the adherent cells and cultivated under normal conditions for 24 hours. At the end of the incubation period, the plates were removed from the 37°C incubator and equilibrated to room temperature. After 5-15 minutes, 100 pl of Bio-Gio reagent were added to each well and cells were incubated for further 10 min in the dark. Finally, the luminescence was measured using the CLARIOstar Plus multiplate reader.

[0551] A TNFR2 agonist was characterized by its ability to induce a luminescence signal that exceeded the baseline activity of the cells by more than two-fold. As expected, SCTNFR2-FC and two commercially available TNFR2 agonistic antibody clones, 80M2 and MR2-1 , exceeded this threshold and elicited more than a three-fold increase in luminescence signal (Figure 9).

[0552] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Claims

CLAIMS1. A combination comprising a Tumor necrosis factor (TNF) inhibitor and a Tumor necrosis factor receptor 2 (TNFR2) agonist.

2. The combination according to claim 1 wherein (i) the TNFR2 agonist is selected from an antibody, a polypeptide, a nucleic acid or a small molecule and / or (ii) the TNF inhibitor is selected from an antibody, a polypeptide, a nucleic acid or a small molecule.

3. The combination according to any preceding claim wherein the TNFR2 agonist is a TNF mutein or a TNFR2 agonist antibody.

4. The combination according to any preceding claim wherein the TNF inhibitor is an anti-TNF antibody; preferably wherein the antibody is selected from adalimumab, infliximab, golimumab, and certrolizumab.

5. The combination according to any preceding claim, wherein the TNFR2 agonist is a TNF mutein and the TNF inhibitor is capable of binding to TNF but does not bind the TNF mutein.

6. The combination according to any of claims 3-5 wherein the TNF inhibitor binds an epitope comprising one or more amino acids which are present in TNF but are not present in the TNF mutein.

7. The combination according to any of claims 3-6, wherein the TNF mutein comprises a mutation at one or more of D139, A141 , E142, or S143 mutations corresponding to the position shown in SEQ ID NO: 2, optionally wherein the TNF mutein comprises a mutation at positions D139 and A141.

8. The combination according to any of claims 3-7, wherein the TNF mutein comprises D139N and / or A141 R mutations corresponding to the position shown in SEQ ID NO: 2.

9. The combination according to any of claims 4 to 8 wherein the TNF inhibitor is an antibody which comprises the following CDRs:HCDR1 : DYAMH (SEQ ID NO: 10)HCDR2: AITWNSGHIDYADSVEG (SEQ ID NO: 11)HCDR3: VSYLSTASSLDY (SEQ ID NO: 12)LCDR1 : RASQGIRNYLA (SEQ ID NO: 13)LCDR2: AASTLQS (SEQ ID NO: 14)LCDR3: QRYNRAPYT (SEQ ID NO: 15)10. The combination according to any of claims 3 to 9 wherein the TNF mutein comprises a polypeptide comprising a TNFR2 binding domain comprising three TNF homology domains (THD) that specifically bind to TNFR2.

11. The combination according to claim 10, wherein each THD comprises SEQ ID NO: 21 or an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 21 and comprising mutations at one, two, three or four of the following positions: D132, A134, E135, S136, optionally at the positions D132 and / or A134, further optionally comprising the mutations D132N and A134R.

12. The combination according to claim 11 , 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 and D139N and A141 R mutations at the positions corresponding to SEQ ID NO: 2.

13. The combination according to claim 12, wherein the TNFR2 agonist is a polypeptide comprising the sequence of SEQ ID NO: 7.

14. The combination according to any preceding claim wherein the TNF inhibitor comprises CDRs as defined in claim 9 and the TNFR2 agonist is a polypeptide comprising a sequence with at least 80% sequence identity to SEQ ID NO: 7 and a mutation at one or more of D139, A141 , E142, or S143 corresponding to the position shown in SEQ ID NO: 2, optionally wherein the TNFR2 agonist comprises a mutation at positions D139 and A141 ; preferably wherein the mutations are D139N and A141 R at the positions corresponding to SEQ ID NO: 2.

15. A composition comprising a TNF inhibitor and a TNFR2 agonist according to any preceding claim.

16. A composition comprising: (i) a polynucleotide encoding a TNF inhibitor and a TNFR2 agonist according to any preceding claim; or (ii) a first polynucleotide encoding a TNF inhibitor any preceding claim and a second polynucleotide encoding a TNFR2 agonist according to any preceding claim.

17. A pharmaceutical composition comprising a TNF inhibitor and a TNFR2 agonist according to any of claims 1 to 14, or a polynucleotide or a first and a second polynucleotide according to claim 16.

18. A kit comprising a TNF inhibitor and a TNFR2 agonist according to any of claims 1 to 14, a first and a second polynucleotide according to claim 16 or a composition comprising a TNF inhibitor and a composition comprising TNFR2 agonist.

19. A combination comprising a TNF inhibitor and a TNFR2 agonist, for use in treating a disease.

20. A combination comprising a TNF inhibitor and a TNFR2 agonist for use in treating acute pain.

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