WO2015104322A1

WO2015104322A1 - Treatment of inflammatory diseases with non-competitive tnfr1 antagonists

Info

Publication number: WO2015104322A1
Application number: PCT/EP2015/050241
Authority: WO
Inventors: Adriaan Allart Stoop
Original assignee: Glaxosmithkline Intellectual Property Development Limited
Priority date: 2014-01-09
Filing date: 2015-01-08
Publication date: 2015-07-16

Abstract

The present invention is directed to anti-TNFα receptor type 1 (TNFR1; p55) inhibitors for use in the treatment of patients with TNF Receptor Associated Periodic Syndrome (TRAPS) as well as patients carrying low-penetrance TNFR1 mutations suffering from other inflammatory diseases.

Description

TREATMENT OF INFLAMMATORY DISEASES WITH

NON-COMPETITIVE TNFR1 ANTAGONISTS

FIELD OF THE INVENTION

The present invention is directed to anti-TNFa receptor type 1 (TNFR1; p55) inhibitors for use in the treatment of patients with TNF Receptor Associated Periodic Syndrome (TRAPS) as well as patients carrying low-penetrance TNFR1 mutations suffering from other inflammatory diseases.

BACKGROUND OF THE INVENTION

Tumour necrosis factor (TN F) receptor-associated periodic syndrome (TRAPS, OMIM 0142680) is an autosomal dominant autoinflammatory disorder linked to chromosome 12pl3 and more specifically to mutations within the TNFRSF1A gene encoding TNFR1 [ cDermott, 1999; Aganna, 2003;

Akesentijevich, 2001]. It's a very rare disease with an incidence of 5.6 cases per 10⁷ person-years, as determined in Germany over the 2003-2006 period [Lainka, 2009]. It belongs to the group of hereditary systemic autoinflammatory diseases, also known as hereditary periodic fever syndromes, which are diseases characterised by unprovoked recurrent attacks of systemic inflammation with lack of autoantibodies or auto-reactive T-cells [Masters, 2009]. In each of these syndromes a specific genetic defect has been identified involved in regulation of innate immunity.

The diagnosis of TRAPS relies on mutational analysis of the TNFRSF1A gene combined with the clinical symptoms. These symptoms are recurrent inflammatory episodes which occur either spontaneously or after minor triggers such as local injury, minor infection, stress, exercise and hormonal changes [Pettersson 2012]. In the most comprehensive and recent study of TRAPS phenotype, the median age at which symptoms began was 4.3 years, although 9.1% of patients presented after 30 years of age [Lachmann, 2013]. Attacks were recurrent in 88% of patients and the commonest features associated with the pathogenic variants were fever (88%), limb pain, abdominal pain, rash and eye manifestations, e.g. periorbital edema and conjunctivitis. At a median age of 43 years about 10% of TRAPS patients developed AA amyloidosis. Overall patients had a median of 70 symptomatic days a year with fever, limb and abdominal pain and rash being the commonest symptoms. There was little evidence of a significant effect of age or genotype on disease features at presentation.

Analysis of the TNFRSF1A gene identified mutations in the extracellular domains of TNFR1 to confer a dominantly inherited autoinflammatory syndrome [McDermott, 1999]. Since, a number of mutations in the TNFRISFIA gene have been identified and associated with TRAPS [Lachmann, 2013] (see Annex 1). The majority of mutations are restricted to the extracellular domain of the receptor with a striking absence of mutations that would result in loss of protein expression or truncation. Mutations disproportionally affect cysteine residues critical for folding of the extracellular domain and the majority of these are located in the first two N-terminal cysteine-rich domains CRD1 and CRD2. Most frequently these are cysteine mutations which lead to protein misfolding and to changes in levels of receptor shedding. In addition, these mutations have been shown to affect receptor trafficking and lead to intracellular receptor retention in the endoplasmatic reticulum, likely because of abnormal oligomerisation of mutant receptors through non-physiological disulfide bonds and protein misfolding [Lobito, 2006; Todd 2004, 2007]. These mutant receptors failed to interact with wild-type TNFR1, which is also present as patients are heterozygous for the mutation, and bind TNF [Lobito, 2006]. Because of the effect of these mutations on the protein structure and function of TNFR1, these mutations are often referred to as 'structural' mutations. Examples of structural mutations are; C30R, C33Y, C43G, C43Y, T50M, C52Y and C55Y. in addition to the structural mutations, a second class of mutations have been described which do not involve cysteine residues and do not impact receptor trafficking or shedding [Lobito, 2006]. These 'non-structural' mutations, e.g. P46L and R92Q, occur in 1-5% of the general population and show distinctive clinical features, are considered to be low-penetrance mutations and are associated with a milder form of disease [Hull 2002; Akesentijevich, 2001; Ravet 2006; Pelagatti, 2011]. The P46L mutation is found in unexpected high frequency in sub-Saharan west African populations [Tchernitchko, 2005]. Although a low penetrance mutation, the R92Q mutation is by far the most frequently found mutation in TRAPS patients, with 83% of patients harbouring this mutation in an epidemiology study [Lainka, 2009] and 34% of patients in a cross-European phenotype study [Lachmann, 2013].

Structural mutations in TNFR1 result in an enhanced and ligand-independent signalling of TNFR1, manifesting in the auto-inflammatory phenotype observed in patients [Simon 2010; Todd, 2004; Yousaf, 2005; IMedjai, 2008]. The structural mutations are thought to lead to intracellular accumulation of TNFR1 mutant protein which then activate JNK and p38 signalling. This activation sensitises cells to the effects of other innate immune stimuli, e.g. LPS, resulting in enhanced production of inflammatory cytokines and chemokines at low doses of such stimuli. In this situation, the enhanced subsequent rise in TNF, which then signals through wt TNFR1 also present on cells, leads to further increases of other inflammatory cytokines, e.g. IL-6 and IL-Ιβ [Simon, 2010]. The exaggerated clinical responses observed in TRAPS patients to trivial stimuli leading to clinical episodes of fever and other inflammatory symptoms would fit with this mechanism.

The non-structural mutations, and in particular R92Q, have also been shown to lead to ligand- independent signalling [Rebelo, 2006; Todd, 2007]. Although this mutant is expressed on the cell surface and is processed normally, cells expressing this mutant showed spontaneous upregulation of multiple proinflammatory genes, e.g. PTX3 and GM-CSF, relative to wild-type TNFR1 transfected cells. This enhanced signalling was observed at both the mRNA and protein level following incubation of transfected Sk-Hep-1 cells. Using mutants with point mutations in the death domain of TNFR1, it was established that this domain was implicated in the observed signalling [Rebelo, 2009]. A mechanistic explanation for the enhanced signalling has been provided by [Lewis et al., 2012] who suggested that signalling is associated with backbone conformational changes of receptor dimers consistent with overactivation of the R92Q mutant. They determined that R92Q dimerises with a lower Kd than wild-type receptor, suggesting that the mutant homodimerises with greater affinity [Lewis, 2012]. This observation may imply TNF receptor 1 homodimerisation as an element in activation of signalling on which the R92Q. mutation has an impact.

The R92Q. mutation has also been associated with clinical phenotypes other than TRAPS. These include: 1) rheumatoid arthritis in which a higher frequency of this mutation was observed [Hull, 2002]; 2) Behcet disease where a significantly higher proportion of patients were carriers of the R92Q polymorphism (6.8%) compared to control individuals without the disease. Furthermore, these R92Q carriers had an increased risk to subsequently develop extracranial deep vein thrombosis (30% of patients developing this thrombosis were R92Q carriers) [Amoura, 2005]. 3) In patients with premature myocardial infarction (Ml), the R92Q. allele was associated with a population-adjusted increased odds ratio of 2.15 (95% CI: 1.09-4.23) for developing Ml. The same study, further identified the R92Q allele to be positively associated with the presence of carotid plaques in smokers (OR 5.07; 95% CI: 1.64-15.63) and with increases in carotid intima-media thickness. Overall the study suggested that carriers of R92Q may be at increased risk of atherosclerosis [Poirier, 2004] . 4) An increased incidence of R92Q. (3 out of 30 patients) was also observed in a sma ll study of patients with recurring pericarditis who were refractory to colchicines treatment [Ca ntarini, 2009] and clustering of pericarditis in certain families might also be linked to TRAPS [Cantarini, 2012b, 2010]. 5) the R92Q allele has been associated by different groups with an increased risk of developing multiple sclerosis (MS), both in the context of TRAPS and as a stand-alone risk factor. The carrier frequency of the R92Q. mutation in a selected group of MS patients with additional TRAPS-like symptoms was found to be 13%, a frequency higher than those reported for other patient groups [Kumpfel, 2008]. A sepa rate study also identified an increased frequency of R92Q in MS patients, with 4 out of 5 carriers reporting autoinflammatory symptoms prior to MS diagnosis [Kauffman,

2011] . Furthermore, many additional studies and reviews have associated R92Q a nd/or TRAPS with m ultiple sclerosis and central nervous system disease [Caminero, 2011; Goris, 2011; Havla, 2013; Hoffma nn, 2008; Kumpfel, 2007, 2009; M inden, 2004]. Finally, la rge genome wide association studies (GWAS) have also identified R92Q as a risk factor for MS, odds ratio (OR) 1.6 [de Jager, 2009] . This was confirmed in a larger study in which a sub analysis (supplementary table 2) also implied an increased OR for R92Q carriers, as well as for a more frequently found polymorphism in the

TNFRSF1A gene polymorphism (rsl800693) [I MSGC, 2013] .

The ligand-independent mechanism underlying the signalling observed in TRAPS patients might also help explain why TNF blockade only results in partia l responses in TRAPS patients [Kimberley 2007, Hull 2002; Jacobelli 2007]. Unlike other autoinflammatory diseases in which a nti-TN F therapy is largely a successful treatment option, therapy with the anti-TNF drug Infliximab is often ineffective in patients with TRAPS. Moreover, in certain cases, Infliximab actually triggers severe episodes of inflammation [Nedjai, 2009] . Etanercept has been used more successfully, however it does not completely normalise symptoms or acute phase reactants a nd long-term ad herence to Etanercept is poor [Bulua, 2012] . Its efficacy may be more a reflection of generic a nti-inflammatory properties of Etanercept [Cantarini, 2012a] . Effective treatment of TRAPS is still a significant unmet medical need .

SUMMARY OF THE INVENTION

The present invention provides an anti-TN Fa receptor type 1 (TN FRl; p55) binding protein which is a non-competitive antagonist of TN FRl for use in the treatment of patients with TN F Receptor Associated Periodic Syndrome (TRAPS) as well as patients carrying low-penetra nce TNFRl mutations suffering from other inflammatory diseases.

In another aspect, the invention provides an anti-TN Fa receptor type 1 (TNFRl; p55) binding protein which is an antagonist of ligand-independent signalling of TN FRl in a huma n patient, for use in the treatment of patients with TN F Receptor Associated Periodic Syndrome (TRAPS) as well as patients carrying low-penetrance TN FRl m utations suffering from other inflammatory diseases.

In a nother aspect, the invention provides an anti-TNFa receptor type 1 (TN FRl; p55) binding protein which is an antagonist of TNFRl dimerisation, for use in the treatment of patients with TNF Receptor Associated Periodic Syndrome (TRAPS) as well as patients carrying low-penetrance TNFRl mutations suffering from other inflammatory diseases.

The inventors believe that a drug which specifica lly inhibits ligand-independent TN FRl signalling may address the cause of TRAPS mechanistica lly, and provide a benefit to TRAPS patients by inhibiting the spontaneous inflammation observed in these patients. BRIEF DESCRIPTION OF THE FIGURES

Figure 1, Anti-TNFRl fAb confirms TNFRl expression on HEK293 transfected with TNFRl full length (left) but not on cells transfected with truncated TNFR1-TR (right). Legend to traces and conditions used: (a) Isotype control IgGl-PE in presence doxycyline induction); (b) anti-TNFRl-PE in presence doxycline induction; (c) Isotype control -PE in absence Dox; (d) anti-TNFRl-PE in absence Dox.

Figure 2. Binding of D S5541 to HEK293 cells expressing full-length TNFRl wild type in the presence and absence of doxycyline induction, (a) for the streptavidin-AF 647 control; (b) for DMS5556 negative control; and (c) for DMS5541 anti-TNFRl.

Figure 3. Binding of DMS5541 to HEK293 cells expressing truncated TNFRl (TR) in the presence and absence of doxycyline induction, (a) for the streptavidin-AF 647 control; (b) for DMS5556 negative control; and (c) for DMS5541 anti-TNFRl.

Figure 4. Binding of DMS5541 to HEK293 cells expressing TRAPS mutant TNFRl R92Q. in the presence and absence of doxycyline induction, (a) for the streptavidin-AF 647 control; (b) for DMS5556 negative control; and (c) for D S5541 anti-TNFRl.

Figure 5. Bindin of DMS5541 to HEK293 cells expressing TRAPS mutant TNFRl P46L in the presence and absence of doxycyline induction, (a) for the streptavidin-AF 647 control; (b) for DMS5556 negative control; and (c) for DMS5541 anti-TNFRl.

Figure 6. Binding of DMS5541 to HEK293 ceils expressing TRAPS mutant TNFRl T50M in the presence and absence of doxycyline induction, (a) for the streptavidin-AF 647 control; (b) for DMS5556 negative control; and (c) for D S5541 anti-TNFRl.

Figure 7. Binding of D S5541 to HEK293 cells expressing TRAPS mutant TNFRl C33Y in the presence and absence of doxycyline induction, (a) for the streptavidin-AF 647 control; (b) for DMS5556 negative control; and (c) for DMS5541 anti-TNFRl.

Figure 8. Binding of DMS5541 to HEK293 cells expressing TRAPS mutant TNFRl C33Y R347A (with disabled death domain) in the presence and absence of doxycyline induction, (a) for the

streptavidin-AF 647 control; (b) for DMS5556 negative control; and (c) for D S5541 anti-TNFRl.

Figure 9. Effects of either DMS5541 (anti-TNFRl) or DMS5556 (negative control) on IL-8 secretion after T Fa (3 ng/ml) stimulation by different HEK293 cell lines over-expressing TNFRl variants associated with TRAPS. TR is truncated TNFRl providing background TNFRl levels in HEK293 cells; WT is over expressing full-length TNFRl and all other variants are full-length TNFRl with single amino-acid mutations.

Figure 10. Effects of either DMS5541 (anti-TNFRl) or DMS5556 (negative control) on IL-8 secretion after T Fa (3 ng/ml) stimulation by different HEK293 cell lines over-expressing TNFRl variants associated with TRAPS. The intracellular domain of these transfected TNFRl mutants has been disabled by introduction of the R347A mutation in the death domain. TR is truncated TNFRl providing background TNFRl levels in HEK293 cells (NT = not tested); WT is over expressing full- length TNFRl and all other variants are full-length TNFRl with indicated single amino-acid mutations in the extracellular domain and the R347A mutation in the death domain. Figure 11. Effects of either DMS5541 (anti-TNFRl, black square) or DMS5556 (negative control, open square) on G-CSF secretion after T Fa (1 ng/ml) stimulation by different SK-Hepl cell lines over- expressing TNFR1 variants associated with TRAPS.

Figure 12. Effects of either DMS5541 (anti-TNFRl, black square) or DMS5556 (negative control, open square) on CCL-2 secretion after TNFa (1 ng/ml) stimulation by different SK-Hepl cell lines over- expressing TNFR1 variants associated with TRAPS.

Figure 13. Effects of either DMS5541 (anti-TNFRl, black square) or DMS5556 (negative control, open square) on CCL-5 secretion after TNFa (1 ng/ml) stimulation by different SK-Hepl cell lines over- expressing TNFR1 variants associated with TRAPS. Figure 14. Effects of either DMS5541 (anti-TNFRl, black square) or DMS5556 (negative control, open square) on IL-6 secretion after TNFa (1 ng/ml) stimulation by different SK-Hepl cell lines over- expressing TNFR1 variants associated with TRAPS.

Figure 15. Effects of either DMS5541 (anti-TNFRl, black square) or DMS5556 (negative control, open square) on 1L-8 secretion after TNFa (1 ng/ml) stimulation by different SK-Hepl cell lines over- expressing TNFR1 variants associated with TRAPS.

Figure 16. Effects of either DMS5541 (anti-TNFRl, black square) or DMS5556 (negative control, open square) on PTX-3 secretion after TNFa (1 ng/ml) stimulation by different SK-Hepl cell lines over- expressing TNFR1 variants associated with TRAPS.

DETAILED DESCRIPTION OF THE INVENTION

Within this specification the invention has been described, with reference to embodiments, in a way which enables a clear and concise specification to be written. It is intended and should be appreciated that embodiments may be variously combined or separated without parting from the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons, Inc. which are incorporated herein by reference) and chemical methods.

As used herein, the phrase "low-penetrance" refers to an allele which will only sometimes produce the symptom or trait with which it has been associated at a detectable level.

As used herein, the term "TNFR1 binding protein" refers to antibodies and engineered protein constructs, such as DARPins (designed ankyrin repeat proteins), which are capable of binding to TNFR1. TNFR1 binding proteins may be antagonists of TNFR1. Antagonists of TNFR1 may be noncompetitive antagonists of TNFR1, in that the binding of TNFR1 binding protein does not antagonise the binding of TNFa ligand to the TNFR1. Non-competitive antagonists of TNFR1 are described, for example, in WO2005/035572, WO2011/006914, WO2011/051217 and WO2012/172070. The term "antibody" is used herein in the broadest sense to refer to molecules with an immunoglobulin-like domain and includes monoclonal, recom bina nt, polyclonal, chimeric, human, humanised, multispecific including bispecific antibodies, and heteroconjugate antibodies, antigen- binding fragments of any of the foregoing; a single variable domain (e.g. V_H, V_HH, V domain antibody (dAb™)), a ntigen binding fragments including Fa b , F(ab')2, Fv, disulphide linked Fv, scFv, d isulphide- linked scFv, diabody TAN DABS™, etc. a nd modified versions of any of the foregoing (for a summary of alternative "antibody" formats see Holliger and Hudson, Nature Biotechnology, 2005, Vol 23, No. 9, 1126-1136).

The phrase "single varia ble domain" refers to a folded polypeptide domain comprising sequences characteristic of a ntibody varia ble domains. It therefore includes complete antibody variable doma ins such as V_H» V_HH, V_L and modified antibody varia ble domains, for example, in which one or more loops have been replaced by sequences which a re not characteristic of antibody variable domains, or antibody variable domains which have been truncated or comprise N- or C- term inal extensions, as well as fragments of va riable domains which retain at least the binding activity and specificity of the full-length domain. A single variable domain is capable of binding an antigen or epitope independently of other variable regions or domains. A single variable domain may be a huma n single va riable domain, but a lso includes single variable domains from other species such as rodent (for example, as disclosed in WO 00/29004), nurse shark and Camelid V_HH dAbs™. Camelid V_HH a re immunoglobulin single variable domains that are derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies natura lly devoid of light chains. Such VHH domains may be humanised according to standard techniques available in the art, a nd such domains are considered to be "single variable domains" . As used herein V_H includes camelid V_HH domains.

A single variable domain can be present in a format (e.g., homo- or hetero-multimer) with other varia ble regions or va riable domains where the other regions or domains a re not required for antigen binding by the single variable domain (i.e., where the immunoglobulin single variable domain binds antigen independently of the additional variable domains). In one embodiment, in any aspect described herein, the TNFR1 binding protein is not an immunoglobulin single variable domain. A "domain" is a folded protein structure which has tertiary structure independent of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins, and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain.

As used herein, "functional" describes a polypeptide or peptide that has biological activity, such as specific binding activity. For example, the term "functional polypeptide" includes an antibody or antigen-binding fragment thereof that binds a target antigen through its antigen-binding site.

As used herein, "antibody format", "formatted" or similar refers to any suitable polypeptide structure in which one or more antibody variable domains can be incorporated so as to confer binding specificity for antigen on the structure. A variety of suitable antibody formats are known in the art, such as, chimeric antibodies, humanized antibodies, human antibodies, single chain antibodies, bispecific antibodies, antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy chains and/or light chains, antigen-binding fragments of any of the foregoing (e.g., a Fv fragment (e.g., single chain Fv (scFv), a disulfide bonded Fv), a Fab fragment, a Fab' fragment, a F(ab')₂ fragment), a single variable domain (e.g., a dAb, V_H, V_HH, V_Lj, and modified versions of any of the foregoing (e.g., modified by the covalent attachment of polyethylene glycol or other suitable polymer or a humanized V_HH).

An antigen binding fragment may be provided by means of arrangement of one or more CD s on non-antibody protein scaffolds such as a domain, The domain may be a domain antibody or may be a domain which is a derivative of a scaffold selected from the group consisting of DARPin, CTLA-4, lipocalin, SpA, an Affibody, an avimer, GroEl, transferrin, GroES and fibronectin/adnectin, which has been subjected to protein engineering in order to obtain binding to an antigen, such as TNFR1, other than the natural ligand.

An antigen binding fragment or an immunologically effective fragment may comprise partial heavy or light chain variable sequences. Fragments are at least 5, 6, 8 or 10 amino acids in length. Alternatively the fragments are at least 15, at least 20, at least 50, at least 75, or at least 100 amino acids in length.

The term "epitope" as used herein has its regular meaning in the art. Essentially, an epitope is a protein determinant capable of specific binding to an antigen binding protein, such as a TNFRl binding protein. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.

The term "binding" or "specific binding" used herein in the context of "binding to an epitope comprising residue X" is given its normal meaning in the art. Identifying the amino acid residues which make up an epitope on a target antigen - i.e. those residues involved in the "binding" interaction between binding protein a nd ta rget antigen is routine in the art. An epitope may be determined by, for example, competition assays with monoclonal antibodies (or other a ntigen binding proteins) of which the binding epitope is known, on e.g. Biacore, peptide mapping, site- directed mutagenesis (e.g. alanine scanning m utagenesis), hyd rogen-deuterium exchange mass- spectrometry, x-ray crystallography. For example, an epitope may be defined accurately by mapping those residues in the antigen which are determined by X-ray crysta llography to be within 4.0A (i.e. 4.0A or less than 4.0A) of a residue in the antigen binding protein.

As used herein, the term "antagonist of Tumour Necrosis Factor Receptor 1 (TN FRl)", "TN FR1 antagonist" or the like refers to an agent (e.g., a molecule, a compound) which binds TN FRl and can inhibit a (i.e., one or more) function of TN FRl. For example, an a ntagonist of TNFRl can inhibit signa l transduction mediated through TN FRl. Antagonists of TN FRl include those which partially, but not completely, inhibit a function of TNFRl (herein referred to as "partial antagonists" of TN FRl). For instance, the a ntagonists described herein may partially, but not completely, abrogate signal transduction mediated through TN FRl (e.g. may abrogate signal transduction substantially completely at a first concentration of TNFct, but only partially at a second, higher concentration).

Antagonists which partially inhibit TN FRl are described in WO2011/006914, the content of which is hereby incorporated in its entirety. Non-competitive TNFRl binding proteins have been observed to display a decreased level of inhibition at increasing TNFct concentrations (WO2011/006914), suggesting that they would be partial inhibitors of T Fa when high concentrations of TNFa are present. Consequently at high TNFa concentrations this class of inhibitors would leave residual TNFa signalling uninhibited. They offer potential advantages vis-a-vis complete inhibition of the effects of TNFa, as they do not completely inhibit all TNFa, but only the excess amount of TNFa found during chronic inflammation, e.g. in arthritis.

In one embodiment of any aspect of the invention, the TNFRl binding protein is a non-competitive antagonist which neutralizes TNFRl with an ND50 of (or about of) 5, 4, 3, 2 or 1 nM or less in a standard RC5 assay as determined by inhibition of TNF alpha-induced IL-8 secretion.

In one embodiment of any aspect of the invention, the antagonist also neutralizes (murine) TNFRl with an ND50 of 150, 100, 50, 40, 30 or 20 nM or less; or from (about) 150 to 10 nM; or from (about) 150 to 20 nM; or from (about) 110 to 10 nM; or from (about) 110 to 20 nM in a standard L929 assay as determined by inhibition of TNF alpha-induced cytotoxicity.

In one embodiment of any aspect of the invention, the antagonist also neutralizes (Cynomolgus monkey) TNFRl with an ND50 of 5, 4, 3, 2 or 1 nM or less; or (about) 5 to (about) 1 nM in a standard Cynomologus Kl assay as determined by inhibition of T F alpha-induced IL-8 secretion.

The TNFRl binding proteins of the present invention may be specific antagonists of TNFRl, in that they do not antagonize (inhibit signal transduction mediated through) TNFR2, and/or do not antagonize (inhibit signal transduction mediated through) other members of the TNF/NGF receptor superfamily.

In one embodiment of any aspect of the invention, the TNFRl binding proteins of the present invention are non-competitive antagonists of TNFRl, in that the TNFRl binding protein binds to human TNFRl but does not compete with or inhibit T Fa for binding to TNFRl (e.g. in a standard receptor binding assay). In an embodiment, the TNFRl binding protein (e.g. an anti-TNFRl immunoglobulin variable domain) specifically binds to an epitope consisting of residues within domains 1, 2, 3 or 4 of TNFRl. More particularly, the TNR1 binding protein binds to an epitope consisting of residues in domain 4 and/or in Domain 3.

In one embodiment of any aspect of the invention, the TNFRl binding proteins of the present invention bind to an epitope on TNFRl (SEQ ID NO:4), wherein the epitope comprises or consists of one or more residues selected from: Q17, G18, K19, T31, K32, C33, H34, K35, G36, T37, G47, Q48, D49, E54, E64, V90, V91, H126, L127, Q.130, Q133, V136, T138 and L145 of SEQ ID NO:4. In a further embodiment, the epitope comprises or consists of one or more residues selected from: H126, L127, Q130, Q133, V136, T138 and L145 of SEQ ID NO:4.

Typically, the TNFRl binding proteins according to the invention are monovalent and contain one binding site that interacts with TNFRl. Monovalent binding proteins bind one TNFRl and may not induce cross-linking or clustering of TNFRl on the surface of cells which can lead to activation of the receptor and signal transduction. They can therefore be useful antagonists of TNFRl. In an embodiment, the monovalent antagonist binds to an epitope which spans more than one Domain of TNFRl. Multivalent TNFRl binding proteins may also have a first binding site for TNFRl and a second binding site for a separate antigen (for example human serum albumin). Multivalent TNFRl binding proteins which are capable of binding TNFRl and at least one different antigen may also be referred to herein as "multispecific ligands". In one aspect the invention provides an anti-TNFa receptor type 1 (TNFRl; p55) binding protein which is a non-competitive antagonist of TNFRl for use in the treatment of patients with TNF Receptor Associated Periodic Syndrome (TRAPS) as well as patients carrying low-penetrance TNFRl mutations suffering from other inflammatory diseases.

In another aspect, the invention provides an anti-T Fa receptor type 1 (TNFRl; p55) binding protein which is an antagonist of ligand-independent signalling of TNFRl in a human patient, for use in the treatment of patients with TNF Receptor Associated Periodic Syndrome (TRAPS) as well as patients carrying low-penetrance TNFRl mutations suffering from other inflammatory diseases.

In another aspect, the invention provides an anti-TNFa receptor type 1 (TNFRl; p55) binding protein which is an antagonist of TNFRl dimerisation, for use in the treatment of patients with TNF Receptor Associated Periodic Syndrome (TRAPS) as well as patients carrying low-penetrance TNFRl mutations suffering from other inflammatory diseases.

In an embodiment the binding protein is an immunoglobulin single variable domain. In a further embodiment the immunoglobulin single variable domain comprises an amino acid sequence that is at least 95, 96, 97, 98 or 99% identical (or is 100% identical) to the amino acid sequence of DOMlh- 574-208 (SEQ. ID NO.l). In a further embodiment the immunoglobulin single variable domain comprises an amino acid sequence that is identical to DOMlh-574-208 or has 1 or 2 amino acid differences compared to the amino acid sequence of DOMlh-574-208.

In one aspect, the invention provides a TNFRl binding protein as described herein for use in the treatment of patients with TNF Receptor Associated Periodic Syndrome (TRAPS) as well as patients carrying low-penetrance TNFRl mutations suffering from other inflammatory diseases, wherein the TNFRl binding protein comprises a second binding specificity for an antigen other than TNFRl. In an embodiment, the antigen other than TNFRl is human serum albumin.

In one aspect, the invention provides a multispecific ligand for use in the treatment of patients with TNF Receptor Associated Periodic Syndrome (TRAPS) as well as patients carrying low-penetrance TNFRl mutations suffering from other inflammatory diseases, comprising a TNFRl binding protein as described herein and a binding protein that specifically binds to an antigen other than TNFRl. In an embodiment, the antigen other than TNFRl is human serum albumin.

In one aspect the invention provides an anti-TNFa receptor type 1 (TNFRl; p55) immunoglobulin single variable domain which comprises an amino acid sequence that is at least 95, 96, 97, 98 or 99% identical (or is 100% identical) to the amino acid sequence of DOMlh-574-208 (SEQ ID NO.l) for use in the treatment of patients with TNF Receptor Associated Periodic Syndrome (TRAPS).

In one aspect, the invention provides an anti-TNFa receptor type 1 (TNFRl; p55) immunoglobulin single variable domain which comprises an amino acid sequence that is identical to DOMlh-574-208 or has 1 or 2 amino acid differences compared to the amino acid sequence of DOMlh-574-208 for use in the treatment of patients with T F Receptor Associated Periodic Syndrome (TRAPS). In another aspect, the invention provides an anti-TNFa receptor type 1 (TNFRl; p55) immunoglobulin single variable domain which comprises an amino acid sequence that is identical to DO lh-574-208 for use in the treatment of patients with TNF Receptor Associated Periodic Syndrome (TRAPS).

In one aspect, the invention provides a nucleic acid which comprises a nucleotide sequence that encodes an anti-TNFa receptor type 1 (TNFRl; p55) immunoglobulin single variable domain, wherein the nucleotide sequence is at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical (or is 100% identical) to the nucleotide sequence that encodes DOM lh-574-208 for use in the treatment of patients with TNF Receptor Associated Periodic Syndrome (TRAPS).

In one aspect, the invention provides a nucleic acid which comprises a nucleotide sequence that encodes an anti-TNFa receptor type 1 (TNFRl; p55) immunoglobulin single variable domain, wherein the variable domain comprises an amino acid sequence that is at least 95, 96, 97, 98 or 99% identical (or is 100% identical) to the amino acid sequence that encodes DOMlh-574-208 for use in the treatment of patients with TNF Receptor Associated Periodic Syndrome (TRAPS).

In another aspect the invention provides a multispecific ligand comprising (i) an anti-TNFa receptor type 1 (T FRl; p55) immunoglobulin single variable domain which comprises an amino acid sequence that is at least 95, 96, 97, 98 or 99% identical (or is 100% identical) to the amino acid sequence of DOMlh-574-208 (SEQ ID NO.l), and (ii) at least one anti-serum albumin (SA) immunoglobulin single variable domain that specifically binds SA for use in the treatment of patients with TNF Receptor Associated Periodic Syndrome (TRAPS).

In another aspect the invention provides a multispecific ligand comprising (i) an anti-TNFa receptor type 1 (TNFRl; p55) immunoglobulin single variable domain which comprises an amino acid sequence that is at least 95, 96, 97, 98 or 99% identical (or is 100% identical) to the amino acid sequence of DOMlh-574-208 (SEQ ID NO.l), and (ii) at least one anti-serum albumin (SA) immunoglobulin single variable domain that specifically binds SA, wherein the anti-SA single variable domain comprises an amino acid sequence that is at least 80% identical to the sequence of DOM7h- 11-3 (SEQ ID NO. 2) for use in the treatment of patients with TNF Receptor Associated Periodic Syndrome (TRAPS).

In another aspect the invention provides a multispecific ligand comprising (i) an anti-TNFa receptor type 1 (TNFRl; p55) immunoglobulin single variable domain which comprises an amino acid sequence that is at least 95, 96, 97, 98 or 99% identical (or is 100% identical) to the amino acid sequence of DOMlh-574-208 (SEQ ID NO.l), (ii) at least one anti-serum albumin (SA)

immunoglobulin single variable domain that specifically binds SA, wherein the anti-SA single variable domain comprises an amino acid sequence that is at least 80% identical to the sequence of DOM7h- 11-3 (SEQ ID NO. 2), and (iii) optionally wherein a linker is provided between the anti-TNFRl single variable domain and the anti-SA single variable domain for use in the treatment of patients with TNF

Receptor Associated Periodic Syndrome (TRAPS).

immunoglobulin single variable domain that specifically binds SA, wherein the anti-SA single variable domain comprises the amino acid sequence of DOM7h-ll-3 (SEQ ID NO. 2), and (iii) optionally wherein a linker is provided between the anti-TNFRl single variable domain and the anti-SA single variable domain for use in the treatment of patients with TNF Receptor Associated Periodic

Syndrome (TRAPS).

In another aspect the invention provides a multispecific ligand comprising (!) an anti-TNFa receptor type 1 (TNFRl; p55) immunoglobulin single variable domain which comprises an amino acid sequence that is at least 95, 96, 97, 98 or 99% identical (or is 100% identical) to the amino acid sequence of DOMlh-574-208 (SEQ ID NO.l), (ii) at least one anti-serum albumin (SA)

immunoglobulin single variable domain that specifically binds SA, wherein the anti-SA single variable domain comprises the amino acid sequence of DO 7h-ll-3 (SEQ ID NO. 2), and (iii) wherein a linker is provided between the anti-TNFRl single variable domain and the anti-SA single variable domain for use in the treatment of patients with TNF Receptor Associated Periodic Syndrome (TRAPS).

In one embodiment, the linker comprises the amino acid sequence AST, optionally ASTSGPS. In an embodiment the linker is AS(G,,S)_n, where n is 1, 2, 3 , 4, 5, 6, 7 or 8. In a further embodiment the linker is AS(G S)₃.

In another aspect the invention provides a multispecific ligand comprising the amino acid sequence of DMS5541 (SEQ ID NO. 3) for use in the treatment of patients with TNF Receptor Associated Periodic Syndrome (TRAPS).

In an embodiment the patient suffering from TRAPS carries a non-structural mutation. In a further embodiment the patient carries the R92Q or the P46L mutation.

In an embodiment the patient suffering from TRAPS carries a structural mutation. In a further embodiment the patient carries the C30R, C33Y, C43G, C43Y, T50M, C52Y or the C55Y mutation. In a further embodiment the patient carries the C33Y or the T50M mutation.

In one aspect the invention provides an anti-TNFa receptor type 1 (TNFRl; p55) immunoglobulin single variable domain which comprises an amino acid sequence that is at least 95, 96, 97, 98 or 99% identical (or is 100% identical) to the amino acid sequence of DOMlh-574-208 (SEQ ID NO.l) for use in the treatment of patients carrying low-penetrance TNFRl mutations suffering from inflammatory diseases.

In one aspect, the invention provides an anti-TNFa receptor type 1 (TNFRl; p55) immunoglobulin single variable domain which comprises an amino acid sequence that is identical to DOMlh-574-208 or has 1 or 2 amino acid differences compared to the amino acid sequence of DOMlh-574-208 for use in the treatment of patients carrying low-penetrance TNFRl mutations suffering from inflammatory diseases.

In another aspect, the invention provides an anti-TNFa receptor type 1 (TNFRl; p55)

immunoglobulin single variable domain which comprises an amino acid sequence that is identical to DOMlh-574-208 for use in the treatment of patients carrying low-penetrance TNFRl mutations suffering from inflammatory diseases.

In one aspect, the invention provides a nucleic acid which comprises a nucleotide sequence that encodes an anti-TNFa receptor type 1 (TNFRl; p55) immunoglobulin single variable domain, wherein the nucleotide sequence is at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical (or is 100% identical) to the nucleotide sequence that encodes DOMlh-574-208 for use in the treatment of patients carrying low-penetrance TNFRl mutations suffering from inflammatory diseases.

In one aspect, the invention provides a nucleic acid which comprises a nucleotide sequence that encodes an anti-TNFa receptor type 1 (TNFRl; p55) immunoglobulin single variable domain, wherein the variable domain comprises an amino acid sequence that is at least 95, 96, 97, 98 or 99% identical (or is 100% identical) to the amino acid sequence that encodes DOMlh-574-208 for use in the treatment of patients carrying low-penetrance TNFRl mutations suffering from inflammatory diseases.

In another aspect the invention provides a rnultispecific ligand comprising (i) an anti-TNFa receptor type 1 (TNFRl; p55) immunoglobulin single variable domain which comprises an amino acid sequence that is at least 95, 96, 97, 98 or 99% identical (or is 100% identical) to the amino acid sequence of DOMlh-574-208 (SEQ ID NO.l), and (ii) at least one anti-serum albumin (SA) immunoglobulin single variable domain that specifically binds SA for use in the treatment of patients carrying low-penetrance TNFRl mutations suffering from inflammatory diseases.

In another aspect the invention provides a rnultispecific ligand comprising (i) an anti-TNFa receptor type 1 (TNFRl; p55) immunoglobulin single variable domain which comprises an amino acid sequence that is at least 95, 96, 97, 98 or 99% identical (or is 100% identical) to the amino acid sequence of DOMlh-574-208 (SEQ ID NO.l), and (ii) at least one anti-serum albumin (SA) immunoglobulin single variable domain that specifically binds SA, wherein the anti-SA single variable domain comprises an amino acid sequence that is at least 80% identical to the sequence of DOM7h- 11-3 (SEQ. ID NO. 2) for use in the treatment of patients carrying low-penetrance TNFRl mutations suffering from inflammatory diseases.

In another aspect the invention provides a rnultispecific ligand comprising (i) an anti-TNFa receptor type 1 (TNFRl; p55) immunoglobulin single variable domain which comprises an amino acid sequence that is at least 95, 96, 97, 98 or 99% identical (or is 100% identical) to the amino acid sequence of DOMlh-574-208 (SEQ ID NO.l), (ii) at least one anti-serum albumin (SA)

immunoglobulin single variable domain that specifically binds SA, wherein the anti-SA single variable domain comprises an amino acid sequence that is at least 80% identical to the sequence of DOM7h- 11-3 (SEQ ID NO. 2), and (iii) optionally wherein a linker is provided between the anti-TNFRl single variable domain and the anti-SA single variable domain for use in the treatment of patients carrying low-penetrance TNFRl mutations suffering from inflammatory diseases.

immunoglobulin single variable domain that specifically binds SA, wherein the anti-SA single variable domain comprises the amino acid sequence of DOM7h-ll-3 (SEQ ID NO. 2), and (iii) optionally wherein a linker is provided between the anti-TNFRl single variable domain and the anti-SA single variable domain for use in the treatment of patients carrying low-penetrance TNFRl mutations suffering from inflammatory diseases.

immunoglobulin single variable domain that specifically binds SA, wherein the anti-SA single variable domain comprises the amino acid sequence of DOM7h-ll-3 (SEQ ID NO. 2) and (iii) wherein a linker is provided between the anti-TNFRl single variable domain and the anti-SA single variable domain for use in the treatment of patients carrying low-penetrance TNFRl mutations suffering from inflammatory diseases.

In one embodiment, the linker comprises the amino acid sequence AST, optionally ASTSGPS. In an embodiment the linker is AS(G₄S)„, where n is 1, 2, 3 , 4, 5, 6, 7 or 8. In a further embodiment the linker is AS(G₄S)₃.

In another aspect the invention provides a multispecific ligand comprising the amino acid sequence of DMS5541 (SEQ ID NO. 3) for use in the treatment of patients carrying low-penetrance TNFRl mutations suffering from inflammatory diseases.

In one embodiment, the patients carrying low-penetrance mutations carry non-structural mutations. In a further embodiment the patients carrying low-penetrance TNFRl mutations carry the R92Q or the P46L mutation. In a further embodiment the patients carrying low-penetrance TNFRl mutations carry the R92Q mutation. In a still further embodiment the patients carrying low-penetrance TNFRl mutations carry the P46L mutation.

In one embodiment the patients carrying low-penetrance TNFRl mutations suffer from

inflammatory diseases selected from TRAPS, rheumatoid arthritis, Behcet disease, extracranial deep vein thrombosis, premature myocardial infarction (Ml), carotid plaques, carotid intima-media thickness, atherosclerosis, pericardits and multiple sclerosis (MS).

In a further embodiment the patients carrying low-penetrance TNFRl mutations suffer from inflammatory diseases selected from rheumatoid arthritis, Behcet disease, extracranial deep vein thrombosis, premature myocardial infarction (Ml), carotid plaques, carotid intima-media thickness, atherosclerosis, pericardits and multiple sclerosis (MS).

In one embodiment the patients carrying the R92Q mutation suffer from inflammatory diseases selected from TRAPS, rheumatoid arthritis, Behcet disease, extracranial deep vein thrombosis, premature myocardial infarction (Ml), carotid plaques, carotid intima-media thickness,

atherosclerosis, pericardits and multiple sclerosis (MS).

In a further embodiment the patients carrying the R92Q mutation suffer from inflammatory diseases selected from rheumatoid arthritis, Behcet disease, extracranial deep vein thrombosis, premature myocardial infarction (Ml), carotid plaques, carotid intima-media thickness, atherosclerosis, pericardits and multiple sclerosis (MS).

In one aspect the invention provides an anti-TNFa receptor type 1 (TNFRl; p55) binding protein which is a non-competitive antagonist of TNFRl for use in the treatment of patients suffering from TRAPS, rheumatoid arthritis, Behcet disease, extracranial deep vein thrombosis, premature myocardial infarction (Ml), carotid plaques, carotid intima-media thickness, atherosclerosis, pericardits and multiple sclerosis (MS) wherein the patient has the R92Q mutation in TNFRl.

In another aspect, the invention provides an anti-TNFa receptor type 1 (TNFRl; p55) binding protein which is an antagonist of ligand-independent signalling of TNFRl in a human patient, for use in the treatment of patients suffering from TRAPS, rheumatoid arthritis, Behcet disease, extracranial deep vein thrombosis, premature myocardial infarction (Ml), carotid plaques, carotid intima-media thickness, atherosclerosis, pericardits and multiple sclerosis (MS) wherein the patient has the R92Q mutation in TNFRl,

In another aspect, the invention provides an anti-TNFa receptor type 1 (TNFRl; p55) binding protein which is an antagonist of TNFRl dimerisation, for use in the treatment of patients suffering from TRAPS, rheumatoid arthritis, Behcet disease, extracranial deep vein thrombosis, premature myocardial infarction (Ml), carotid plaques, carotid intima-media thickness, atherosclerosis, pericardits and multiple sclerosis (MS) wherein the patient has the R92Q mutation in TNFRl.

In one aspect the invention provides an anti-TNFa receptor type 1 (TNFRl; p55) binding protein which is a non-competitive antagonist of TNFRl for use in the treatment of patients suffering from TRAPS, rheumatoid arthritis, Behcet disease, extracranial deep vein thrombosis, premature myocardial infarction (Ml), carotid plaques, carotid intima-media thickness, atherosclerosis, pericardits and multiple sclerosis (MS) wherein the patient has been characterised as having the R92Q mutation in TNFRl.

In another aspect, the invention provides an anti-TNFa receptor type 1 (TNFRl; p55) binding protein which is an antagonist of ligand-independent signalling of TNFRl in a human patient, for use in the treatment of patients suffering from TRAPS, rheumatoid arthritis, Behcet disease, extracranial deep vein thrombosis, premature myocardial infarction (Ml), carotid plaques, carotid intima-media thickness, atherosclerosis, pericardits and multiple sclerosis (MS) wherein the patient has been characterised as having the R92Q mutation in TNFRl.

In another aspect, the invention provides an anti-TNFa receptor type 1 (TNFRl; p55) binding protein which is an antagonist of TNFRl dimerisation, for use in the treatment of patients suffering from TRAPS, rheumatoid arthritis, Behcet disease, extracranial deep vein thrombosis, premature myocardial infarction (Ml), carotid plaques, carotid intima-media thickness, atherosclerosis, pericardits and multiple sclerosis (MS) wherein the patient has been characterised as having the R92Q mutation in TNFRl.

In one aspect the invention provides an anti-TNFa receptor type 1 (TNFRl; p55) binding protein which is a non-competitive antagonist of TNFRl for use in the treatment of patients suffering from TRAPS, rheumatoid arthritis, Behcet disease, extracranial deep vein thrombosis, premature myocardial infarction (Ml), carotid plaques, carotid intima-media thickness, atherosclerosis, pericardits and multiple sclerosis (MS) wherein the patient is characterised as having the R92Q mutation in TNFRl.

In another aspect, the invention provides an anti-TNFa receptor type 1 (TNFRl; p55) binding protein which is an antagonist of ligand-independent signalling of TNFRl in a human patient, for use in the treatment of patients suffering from TRAPS, rheumatoid arthritis, Behcet disease, extracranial deep vein thrombosis, premature myocardial infarction (Ml), carotid plaques, carotid intima-media thickness, atherosclerosis, pericardits and multiple sclerosis (MS) wherein the patient is characterised as having the R92Q mutation in TNFRl.

In another aspect, the invention provides an anti-TNFa receptor type 1 (TNFRl; p55) binding protein which is an antagonist of TNFRl dimerisation, for use in the treatment of patients suffering from TRAPS, rheumatoid arthritis, Behcet disease, extracranial deep vein thrombosis, premature myocardial infarction (Ml), carotid plaques, carotid intima-media thickness, atherosclerosis, pericardits and multiple sclerosis (MS) wherein the patient is characterised as having the R92Q. mutation in TNFR1.

In one aspect, the invention provides a method for treating, suppressing or preventing patients carrying low-penetrance TNFR1 mutations suffering from inflammatory diseases, comprising administering to a human in need thereof a therapeutically-effective dose or amount of a binding protein of TNFR1 according to any aspect of the invention.

In one aspect the invention provides the use of a binding protein according to any aspect of the invention for the manufacture of a medicament for the treatment of patients carrying low- penetrance TNFR1 mutations suffering from inflammatory diseases.

In one aspect, the invention provides a method for treating, suppressing or preventing T F Receptor Associated Periodic Syndrome (TRAPS), comprising administering to a human in need thereof a therapeutically-effective dose or amount of a binding protein of TNFR1 according to any aspect of the invention.

In one aspect the invention provides the use of a binding protein according to any aspect of the invention for the manufacture of a medicament for the treatment of patients with TNF Receptor Associated Periodic Syndrome (TRAPS).

Methods of Use

Generally, the binding proteins of the present invention will be utilised in purified form together with pharmacologically appropriate carriers. Typically, these carriers include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, any including saline and/or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's. Suitable physiologically-acceptable adjuvants, if necessary to keep a polypeptide complex in suspension, may be chosen from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.

Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack (1982) Remington's Pharmaceutical Sciences, 16th Edition). A variety of suitable formulations can be used, including extended release formulations.

The binding proteins of the present invention may be used as separately administered compositions or in conjunction with other agents. These can include various immunotherapeutic drugs, such as cylcosporine, methotrexate, adriamycin or cisplatinum, and immunotoxins. Pharmaceutical compositions can include "cocktails" of various cytotoxic or other agents in conjunction with the binding protein of the present invention, or even combinations of binding proteins according to the present invention having different specificities, such as binding proteins selected using different target antigens or epitopes, whether or not they are pooled prior to administration.

The route of administration of pharmaceutical compositions according to the invention may be any of those commonly known to those of ordinary skill in the art. For therapy, including without limitation immunotherapy, the selected binding proteins thereof of the invention can be administered to any patient in accordance with standard techniques. The administration can be by any appropriate mode, including parenterally, intravenously, intramuscularly, intraperitoneal^, subcutaneously, transdermal^, via the pulmonary route, or also, appropriately, by direct infusion with a catheter. The dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counterindications and other parameters to be taken into account by the clinician, Administration can be local (e.g., local delivery to the lung by pulmonary administration, e.g., intranasal

administration) or systemic as indicated.

The binding proteins of this invention can be lyophilised for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional

immunoglobulins and art-known lyophilisation and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and reconstitution can lead to varying degrees of antibody activity loss (e.g. with conventional immunoglobulins, IgM antibodies tend to have greater activity loss than IgG antibodies) and that use levels may have to be adjusted upward to compensate.

The compositions containing the present binding proteins or a cocktail thereof can be administered for prophylactic and/or therapeutic treatments. In certain therapeutic applications, an adequate amount to accomplish at least partial inhibition, suppression, modulation, killing, or some other measurable parameter, of a population of selected cells is defined as a "therapeutically-effective dose". Amounts needed to achieve this dosage will depend upon the severity of the disease and the general state of the patient's own immune system, but generally range from 0.005 to 10.0 mg of ligand per kilogram of body weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonly used. For prophylactic applications, compositions containing the present binding proteins or cocktails thereof may also be administered in similar or slightly lower dosages, to prevent, inhibit or delay onset of disease (e.g., to sustain remission or quiescence, or to prevent acute phase). The skilled clinician will be able to determine the appropriate dosing interval to treat, suppress or prevent disease. For example, it can be administered up to four times per day, twice weekly, once weekly, once every two weeks, once a month, or once every two months, at a dose off, for example, about 10 pg/kg to about 80 mg/kg, about 100 pg/kg to about 80 mg/kg, about 1 mg/kg to about 80 mg/kg, about 1 mg/kg to about 70 mg/kg, about 1 mg/kg to about 60 mg/kg, about 1 mg/kg to about 50 mg/kg, about 1 mg/kg to about 40 mg/kg, about 1 mg/kg to about 30 mg/kg, about 1 mg/kg to about 20 mg/kg , about 1 mg/kg to about 10 mg/kg, about 10pg/kg to about 10 mg/kg, about 10 pg/kg to about 5 mg/kg, about 10 pg/kg to about 2.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg or about 10 mg/kg.

Treatment or therapy performed using the compositions described herein is considered "effective" if one or more symptoms are reduced (e.g., by at least 10% or at least one point on a clinical assessment scale), relative to such symptoms present before treatment, or relative to such symptoms in an individual (human or model animal) not treated with such composition or other suitable control. Symptoms will obviously vary depending upon the disease or disorder targeted, but can be measured by an ordinarily skilled clinician or technician. Such symptoms can be measured, for example, by monitoring the level of one or more biochemical indicators of the disease or disorder (e.g., levels of an enzyme or metabolite correlated with the disease, affected cell numbers, etc.), by monitoring physical manifestations (e.g., inflammation)or by an accepted clinical assessment scale, for example, the Expanded Disability Status Scale (for multiple sclerosis), systemic symptoms, social function and emotional status - score ranges from 32 to 224, with higher scores indicating a better quality of life), the Quality of Life Rheumatoid Arthritis Scale, or other accepted clinical assessment scale as known in the field. A sustained (e.g., one day or more, or longer) reduction in disease or disorder symptoms by at least 10% or by one or more points on a given clinical scale is indicative of "effective" treatment. Similarly, prophylaxis performed using a composition as described herein is "effective" if the onset or severity of one or more symptoms is delayed, reduced or abolished relative to such symptoms in a similar individual (human or animal model) not treated with the composition.

The binding proteins can be administered and or formulated together with one or more additional therapeutic or active agents. When a binding protein is administered with an additional therapeutic agent, the binding protein can be administered before, simultaneously with or subsequent to administration of the additional agent. Generally, the binding protein and additional agent are administered in a manner that provides an overlap of therapeutic effect.

In a further aspect of the invention, there is provided a composition comprising a binding protein according to the invention and a pharmaceutically acceptable carrier, diluent or excipient.

In other embodiments, any of the binding proteins described herein further comprises a half-life extending moiety, such as a polyalkylene glycol moiety, serum albumin or a fragment thereof, transferrin receptor or a transferrin-binding portion thereof, or a moiety comprising a binding site for a polypeptide that enhance half-life in vivo. In some embodiments, the half-life extending moiety is a moiety comprising a binding site for a polypeptide that enhances half-life in vivo selected from the group consisting of an affibody, a SpA domain, an LDL receptor class A domain, an EGF domain, and an avimer.

In other embodiments, the half-life extending moiety is a polyethylene glycol moiety. In one embodiment, the binding protein of the invention comprises a single variable domain linked to a polyethylene glycol moiety (optionally, wherein the moiety has a size of about 20 to about 50 kDa, optionally about 40 kDa linear or branched PEG). Reference is made to WO04081026 for more detail on PEGylation of dAbs and binding moieties.

In other embodiments, the half-life extending moiety is an antibody or antibody fragment (e.g, an immunoglobulin single variable domain) comprising a binding site for serum albumin or neonatal Fc receptor.

DATA

Selection and characterization of a ligand-independent inhibitory dAb of TNFR1 signaling

The isolation of non-competitive anti-TNFRl inhibitors has been described in detail in

WO2010/094720 and WO2011/051217. A targeted selection strategy was designed for de novo selections of dAbs from a large synthetic phage-display library. The selection approach included competition with TNF-a to enrich for TNFRl-binding dAbs which recognize antigen epitopes outside of the TNF-a binding site. Extensive screening of dAbs isolated after these early selections, identified dAbs with the desired epitope profile. However, these dAbs required further improvements in both affinity and stability, which were achieved through sequential rounds of mutagenesis and selection using a combination of phage-display and DNA-display technologies. These selections yielded an anti-TNFRl dAb, a single, heavy-chain variable domain (VH) referred to as DOMlh-574-208. To confirm that DOMlh-574-208 bound sTNFRl with high affinity, its binding kinetics for sTNFRl were measured on Biacore™ and determined to be 270 pM. A conformational difference could exist between recombinant sTNFRl used for in vitro experiments and TNFRl expressed when anchored on a cell membrane. Therefore, it was determined that incubation of a TNFRl-expressing monocyte cell line (U937) with DOMlh-574-208 resulted in a shift by flow cytometry comparable to that seen for an anti-TNFRl mAb, while being distinct from an isotype control dAb or cells only. To confirm binding of DOMlh-574-208 to TNFRl translated to inhibition of signaling, DOMlh-574-208 was assayed for inhibition of TNF-cx induced IL-8 secretion by U937 cells. In contrast to an isotype control dAb, DOM lh-574-208 demonstrated a dose-dependent inhibition of TNF-ci induced IL-8 secretion in this monocyte cell line. To verify that this inhibition is not limited to cell lines, DOMlh-574-208 demonstrated the ability to inhibit TNF-a-induced, cell-surface expression of vascular cell adhesion molecule-1 (VCAM-1) in primary HUVE cells. Hence, in two different cell types incubation with DOM lh-574-208 resulted in a dose-dependent inhibition of NF-κΒ mediated signaling products after TNF-cx stimulation.

The hypothesis and selection pressure were designed to generate TNFRl specific dAbs which do not compete with T F-ci for receptor binding. To confirm that DOMlh-574-208 binds independently of TNF-cx characterization of binding of TNF-a to sTNFRl in the absence and presence of saturating amounts of DOMlh-574-208 was performed. Injection of TNF-cx over a sTNFRl surface in a Biacore™ resulted in a clear binding signal with a peak of 64 Response Units (RUs). After saturation of the sTNFRl surface with DOMlh-574-208 the same amount of TNF-cx was re-injected and a very similar response was observed, 59 RUs. This would imply that binding of TNF-cx and DOMlh-574-208 to sTNFRl are two independent interactions and do not impact each other. Thus, DOMlh-574-208 binds sTNFRl with high affinity and inhibits TNFRl-mediated signaling but does not interfere in the binding of ligand to the receptor.

Crystallographic analysis of TNF- a/TNFRl/dA b ternary complex

This unique mechanism of action would suggest that a complex could be generated consisting of TNF-α, TNFRl and DOMlh-574-208. Therefore, we incubated these three proteins at a 1:1:1 ratio and subjected the mixture to a crystallization screen. X-Ray diffraction of these crystals yielded a 2.9A resolution structure with an asymmetric unit containing two copies of each protein organized as two ternary complexes paired through protein-protein contacts between the DOM lh-574-208. Surprisingly, DOMlh-574-208 bound to an epitope in the membrane-proximal cysteine rich domain 4 (CRD4) of TNFRl which is positioned at approximately a 150° angle of rotation around TNFRl from the TNF-cx binding site. The DOMlh-574-208 did not interfere in the TN FRl-TNF-a interaction as the three-fold symmetry of the structure remains compatible with iigand-mediated trimerization of the receptor through contacts made via the binding sites located in inter-subunit grooves of CRD2 and CRD3 of TNFRl. Furthermore, binding of DOMlh-574-208 did not induce any major conformational changes in either TNF-a (RMSD=0,7 A compared to lTNF.pdb [Eck, 1989] or TNFRl (RMSD= 1.4 A compared to INCF.pdb [Naismith, 1995] and ITNR.pdb [Banner, 1993], suggesting that its inhibitory activity does not rely on inducing structural changes in either TNFRl or its ligand. Interestingly, the binding epitope overlaps significantly with surface area involved in receptor homodimer formation [Naismith, 1996], suggesting a possible role for DOMlh-574-208 in breaking this interaction by sterical hindrance. We have demonstrated a novel mechanism to inhibit TNFRl signaling without interfering with ligand binding to the receptor. Our data indicate that in addition to TNF-oc binding to TNFRl, further receptor dimerization, or possibly oligomerization, of receptor is a prerequisite for efficient signaling.

Consequently, non-competitive inhibition of TNF signaling by blocking receptor homodimerisation offers a unique and novel approach to inhibit ligand-independent TNFRl signalling which drives e.g. the symptoms of TRAPS patients. Furthermore detailed analysis of a R92Q mutation was reported to decrease the dissociation constant (Kd) and correspondingly increase the affinity of TNFRl for dimer formation when compared to wild-type receptor [Lewis, 2012]. It is hypothesized that higher receptor dimerisation affinities would lead to increased dimer levels on cells and consequently an increased sensitivity to receptor clustering. This clustering would drive spontaneous signaling and contribute to the clinical manifestation of the disease with which this mutation is associated. In the case of individuals with increased levels of TNFRl homodimerisation, as can be expected in the carriers of the R92Q polymorphism, the use of a binding protein according to the invention as described herein could offer relief by inhibiting spontaneous and/or enhanced inflammation in TRAPS. In addition, the R92Q. mutation has also been implicated as a risk factor in other disease indications with an inflammatory component e.g. early arthritis [Hull, 2002], MS [de Jager, 2009; Kumpfel, 2008; IMSGC, 2013], ACS and atherosclerosis [Poirier, 2004], and Behcet disease [Amoura, 2005]. Surprisingly, carriers of the R92Q. polymorphism in these divergent diseases could benefit from the use of a non-competitive anti-TNFRl binding protein by reducing ligand-independent signaling.

Materials and Methods

Generation of DOMlh-574-208

The process used to identify and affinity mature DOMlh-574-208 is described in WO2010/094720 and WO2011/051217. Naive phage selections using the proprietary 4G and 6G Domantis' libraries were performed using biotinylated TNFRl as the pull-down reagent in the 1^st round. In the 2^nd and 3^rd rounds, the phage were incubated with unlabeled sTNFRl which was followed by addition of biotinylated TNF-ct as pull-down reagent. dAbs were re-cloned into an E. coli expression vector and the protein screened by Biacore™ for binding to TNFRl, inhibition of T F-ct signaling in a cell-based assay and lack of inhibition of the TNFRl/TNF-ct interaction. On selected dAbs, affinity maturation was carried out in sequential steps. Firstly, a phage-display library was made by error-prone PCR and subjected to sequential rounds of selection for improved binders. Secondly, dAbs identified were further improved by targeted diversification of CDR regions and selections using DNA-display technology (described in patent filing: WO2006/018650). Finally, improvements to biophysical properties were made by generating novel error-prone PCR, phage-displayed libraries of the most potent dAbs and subjecting these to specific selection pressures known to enrich for more stable molecules, e.g. trypsin digestion and heat incubation. Screening of these outputs identified DOMlh- 574-208.

Protein expression and purification for assays and crystallography

DOMlh-574-208 and isotype control dAb were expressed from a T7 expression vector in E. coli BL21(D£3) strain derivatives. All were purified from culture supernatants by Protein-A (GE

Healthcare) affinity capture followed by cation exchange chromatography and polished using size exclusion chromatography when necessary. Final material was concentrated and buffer exchanged into PBS and was determined to be >95% purity (SDS-PAG E and SEC) with low endotoxin content. TNFR1 and T F-a used for crystallography were expressed in E. coli.

Flow cytometry assays

Flow cytometry was determined using a Beckman Dickinson FACS Canto II flow cytometer. U937 cells were incubated with biotinylated DOM lh-574-208, relevant isotype control, PE conjugated anti-TNFRl mAb ( R&D systems, FAB225P) or PE conjugated IgGl istoype control (R&D systems, IC002P). Samples were incubated for 1 hour on ice before washing and incubation with Streptavidin- Alexa ^® Fluor 647 (Molecular Probes S21374) for 1 hour on ice to detect biotinylated molecules. Cells were washed and resuspended in PBS containing Sytox^® Blue dead cell exclusion dye

(Molecular Probes S34857).

Cell-based assays

U937 (ATCC CCL-171), a nd pooled HUVE (Promocell C-12203) cells were used in cell-based assays. U937 cells (ATCC CCL-171) were seeded in growth media (RPM I, 10% FBS, 2mM glutamine) in 96 well microplates (lxlO⁵ cells ). Cells were incubated with a dose range of test molecules or a nti-TNFRl mAb (R&D systems MAB225) at concentrations up to approximately ΙΟμΜ for 1 hour at 37 °C, 5%CC½. For the inhibition assays, human TNF-a ( Peprotech 300-1A) was added to the cells (lng/m l for U937 cells). All plates were incubated for a further 15 hours and levels of IL-8 in the supernatants were quantified by Duoset ELISA (R&D Systems) .

Pooled H UVE cells (4xl0⁴ cells) were seeded overnight in gelatine coated 96 well microplates (VWR #734-0403) in Endothelial Cell Growth Media with supplements (Promocell # C22210 & supplements #C39210). Cells were then incubated with a dose ra nge of test molecules at concentrations up to approximately 10μΜ or anti-TNFRl mAb (R&D systems MAB225) for 1 hour at 37 °C, 5%C0₂. For the inhibition assays, huma n TN F-a (Peprotech 300-1A) was added to the cells at a concentration of lng/ml. All plates were returned to the incubator for a further 15 hours. Following the incubation the cells were lysed with lysis buffer (20mM Tris, 13.7m M NaCI, 1% Triton-X-100, 10% Glycerol, 50m M NaF, Im M Na₃V0₄ & 1 x mini-complete protease inhibitor tablet (Roche #11836153001) during incubation on ice for ISmins. VCAM-1 levels, an established marker of TNF-a signaling in HUVE cells [Mackay, 1993], in the cell lysates were determined by ELISA with mouse anti-human VCAM-1 mAb (R&D Systems MAB809), biotinylated sheep anti-human VCAM-1 pAb (R&D Systems BAF809) (0.4ug/ml in 0.1% BSA in PBS/0.05% Tween-20) and streptavidin-H RP ( R&D Systems DY998).

For all assays, individua l data points were determined in triplicate wells. All assay data was analysed using GraphPad Prism (version 4.03, GraphPad Software Inc.). Data was transformed using x=log x transformation & fitted using a 4 parameter sigmoidal dose response model or a bell shaped dose response model where appropriate. If fitting criteria were not met, individ ual data points are shown and no line has been plotted.

Biacore™ experiments

Biacore™ experiments were performed on a Biacore'" T200 instrument at 20μΙ/ίτιίη flow rates in H BS-EP+ buffer (G E hea lthcare) at 25°C. Biacore™ CM5 chip (Series S Sensor Chip certified GE

Healthcare Bio-Sciences AB, Uppsala, Sweden, BR-1005-30) was coated with recombinant human TNFRl (rhTNFRl) using Amine Coupling Kit according to manufacturer's instructions (Amine Coupling Kit, GE Healthcare Bio-Sciences AB, Uppsala, Sweden, BR- 1000-50). ΙμΜ recombinant human TNF-a (rhTNF-a, Peprotech 300-1A) was injected over the chip surface (120s). Chip surface was then regenerated back to baseline using regeneration buffer (lOmM Glycine, pH 2.0), Next, ΙμΜ 208-dAb was injected and immediately followed by rhTNF-a (dual injection) or running buffer (H BS-EP+, lOmM HEPES, 150m NaCI, 3m M EDTA, 0.05% Surfactant P20, pH 7.4) injection. Injection time was 100s for DOMlh-574-208. Biacore™ traces were double referenced and binding responses (in RUs) of rhTNF-a to rhTNFRl in the absence or presence of DOM lh-574-208 were compared. Experiments determining interaction competition were done at a 20 μΐ/min flow rate; kinetic measurements were performed at 50 μΙ/min and 25°C. Data collection rate was 10Hz. Association a nd dissociation parameters were fit using 1:1 binding model in T200 BIAeva luation software.

Crystallography

TN F-a ( 10.5 mg), TN FRl (11.6 mg) and DOM lh-574-208 (9 mg) were mixed at 1:1:1 stoichimetric ratio in a total volume of 8mL and incubated on ice for 30 minutes. After centrifugation the protein com plex was purified by gel filtration chromatography on a Superdex 200 (26/60) colum n (GE Lifesciences) running in 20m M Hepes/NaOH pH7.5, 150m M NaCI (buffer A) at a flow rate of 2ml/min. Fractions containing the ternary complex were collected, pooled and concentrated to 18mg/m L (tota l of 20mg).

The terna ry complex was diluted to 7mg/ml with buffer A and subjected to a broad crystallisation screen. Crystals were grown from 37% pentaerythritol propoxylate, 200mM KCI and 50mM

Hepes/NaOH pH7.5 at 4°C and further improved by microseeding. Cube-shaped crystals grew within 2-4 days.

For cryoprotection a crystal was mounted on the Free Mounting System™ and PEG550 was added to a final concentration of 30% using the Picod ropper Technology™ [Kiefersauer, 2000] . During this proced ure the relative humidity was decreased from 95% to 86%. Crystals were frozen by transfer into liquid nitrogen.

Diffraction data were collected at the Swiss Light Source (Villigen, Switzerland) on beam line X06SA under cryogenic conditions at a wavelength of 1.0 A. Beamline and detector details are given in Table SI below. Data were integrated a nd scaled with XDS and SCALA (Kabsch, 2010] . The structure was solved by molecular replacement using MOLREP using the TNF-J3/TNFR1 (lTNR.pdb) complex as a search model. The model was completed using COOT and refined with REFMAC5 [Collaborative Com putational Project, 1994] .

Example 1 - Confirmation that anti-TNFRl dAb (DMS5541) binds TNFRl-expressing cells and retains binding to TRAPS-associated variants of TNFRl. For an anti-TN FRl dAb to inhibit TN FRl signa lling, a first requirement will be that the dAb binds wild- type TN FRl and retains this binding to the TN FRl m utant variants associated with TRAPS. This bind ing could be esta blished using for example H EK293 cells expressing on their cell surface either wild-type TN FRl or TNFRl variants associated with TRAPS (Todd, 2004). By incubating these cells with a control anti-TN FRl FAb, an anti-TN FRl dAb or a negative control dAb, which are directly or indirectly labelled fluorescently, the specific binding of the antibody for TNFRl variants can be monitored by flow cytometry. As an extra control, the expression of TN FRl can be ind uced using doxycycline, which would increase the surface expression levels of TNFRl and facilitate discrimination from background levels. As over-expression of active TNFRl often leads to cell death, transfected variants can be included lacking a function death domain for the overexpressing TNFRl variant. This can be accomplished by introducing the R347A mutation in the death domain of TNFRl. Therefore, this example describes experiments with both full-length TNFRl versions with functional death domain and second versions in which the activity of the death domain had been removed by introducing the R347A mutation. The variants included in the example are: TNFRl wild-type and the TRAPS associated variants TNFRl R92Q, TNFRl P46L, TNFRl T50M and TNFRl C33Y. In addition, a truncated TNFRl was also used which served as a negative control for no TNFRl over expression, but would continue to show the background levels of endogenous TNFRl expression in HEK293 cells.

The TNFRl mutant variants chosen to exemplify binding of the anti-TNFRl dAb cover both structural and non-structural mutations found in TRAPS patients. The R92Q. and P46L are mutations with normal surface expression and are considered low-penetrance mutations which are non structural. The C33Y mutation is a cysteine mutation described to lead to misfolding with reduced surface expression levels, while the T50M is also considered a structural mutation although it doesn't concern a cysteine.

Methods:

The HEK293 cells expressing TNFRl were cultured as described in Todd et al. (2004). To induce expression levels of TNFRl further, cells were exposed to Doxycycline and ZVAD.FMK (Caspase 1 Inhibitor) at final concentrations of ^g/ml and 10μΜ respectively, overnight (20h). Controls were also included which were not induced with Doxycycline.

Cells were harvested and washed with 1% FBS/PBS and incubated for lh at 4°C with:

Phycoerythrin (PE)-labelled anti-TNFRl Fab (anti-TNFRl-PE, from R&D Systems, cat no. FAB225P)

- Isotype Control Fab-PE labelled,

nothing (secondary Control only)

10p.g/ml DMS5541-biotin (anti-TNFRl dAb genetically fused with an albumin-binding dAb),

or lC^g/ml DMS5556-biotin (a negative control dAb with no specific antigen binding (Vh-dummy) genetically fused with an albumin-binding dAb).

Anti-TNFRl-PE and Isotype Control-PE were washed with PBS and resuspended in PBS. Secondary control, DMS5541-biotin and DMS5556-biotin tubes were washed with 1% FBS/PBS and then incubated with Ipg/ml Streptavidin-Alexa Fluor 647 for lh at 4°C. Cells were washed and then resuspended in PBS before data was acquired on a flow cytometer. Prior to data acquisition, Sytox Green was added to all tubes to a final concentration of ΙΟηΜ to discriminate live from dead cells.

Biotinylation of DMS5541 and DMS5556

For detection of binding of the dAbs to the cells, the dAbs were biotinylated which enables subsequent detection with fluorescently-labelled streptavidin, e.g. Streptavidin-Alexa Fluor 647. An aliquot of each protein was retrieved from -80°C storage and biotinylated according to their reported concentrations of lmg/ml and MWT of 25kDa. Biotinylation was performed with EZ-Link Sulfo-NHS-LS-Biotin (Thermo Scientific, cat no, 21327) and the samples buffer exchanged using Zeba Spin Desalting columns (Thermo Scientific, cat no, 89889) to remove non-conjugated biotin. Protein product was quantified by BCA protein assay.

Biotinylation was confirmed to be positive in an EL!SA based system.

Freshly biotinylated DMS5541-biotin and DMS5556-biotin were tested along with two positive controls for biotinylation: previously biotinylated D S5541-Biotin (positive control) and a known IgG positive control (Vector Labs Anti-Human IgG-Biotin). A known negative control (R&D Systems

AB625) was also included. Proteins were coated in carbonate/bicarbonate buffer onto Nunc

Maxisorp F96 plates o/n at room temperature (RT°). Wells were washed, and then blocked with 1% BSA for lh at RT°. Wells were washed again then incubated with Streptavidin-HRP for 20mins at RT°» After a final wash, TMB liquid substrate was added, the colour allowed to develop and the reaction stopped with 2N H₂S0₄. Data was acquired at 450nm on Labsystems Multiskan EX Plate Reader. Results:

Confirmation of biotinylation of D S5541 and DMS5556. Samples of biotinylated samples pre- and post desalting were analysed in an ELISA as described above. The values obtained below

demonstrate that post-desalting, when non-conjugated biotin has been removed, a specific biotin signal remains for DMS5541 and DMS5556 and not for the negative control (MAB625). The signals observed for DMS5541 (post desalting) and DMS5556 (post desalting) are within the range provided by the two positive controls (DMS5541-Biotin) and Vector Labs IgG Biotin, suggesting biotinylation has been successful.

Confirmation of TNFR1 expression on HEK293 cells transfected with full length TNFR1 and comparison to cells transfected with truncated TNFR1 (TR). Experiments were performed in the presence and absence of doxycycline induction of TNFR1 expression. The four different traces show the shifts as determined by flow cytometry for: 1) Isotype control IgGl-PE in presence doxycycline induction (trace indicated with an 'a'); 2) anti-TNFRl-PE in presence doxycycline induction (b); 3) Isotype control -PE in absence Dox (c); 4) anti-TNFRl-PE in absence Dox (d). Figure 1 shows the results for both TNFR1 wildtype and TNFR1 (TR). As can be seen in figure 1, the cells display TNFR1 full-length and upon induction by doxycycline the signal (line 'b') shifts further to the right, indicating higher expression levels. In contrast, for the truncated version (TNFR1-TR), all the curves overlay each other indicating no significant expression and no induction upon doxycycline incubation.

The next step was to test the same TNFRl full-length and TR cell lines with DMS5541-biotin (anti- TNFRl dAb) and the negative control DMS5556-biotin and confirm a similar result is obtained as shown when using the commercial anti-TNFRl-PE, Figures 2 and 3 shows the traces for this experiment and the trace code used is: (a) for the streptavidin-AF 647 control; (b) for DMS5556- biotin (negative control); and (c) for DMS5541-biotin (anti-TNFRl dAb). The results mirror those seen with the anti-TNFRl-PE used in figure 1 and confirm that DMS5541 specifically binds TNFRl, which is induced by doxycycline incubation, and that DMS5556 is a negative control. Furthermore, if a truncated version of TNFRl (TR) is expressed only a very small shift is seen. upon incubation with DMS5541-biotin and this shift does not increase with doxycycline incubation, reflecting binding to endogenously expressed TNFRl present on the HEK293 cells

The ability of either DMS5541-biotin or DMS5556-biotin to bind cells expressing TNFRl variants associated with TRAPS: R92Q, P46L, T50M and C33Y was tested. To increase TNFRl expression levels and have a more distinctive shift by flow, the cells were also incubated in the presence of doxycycline. The results are shown in figures 4-7, all having the same trace coding as in figure 2. The figures indicate that D S5541-biotin binds specifically to the TRAPS-associated TNFRl mutant expressing cell lines in the absence of doxycycline. Induction of TNFRl expression by doxycyline resulted in increased levels of TNFRl expression for the wild-type and the non-structural TRAPS mutants (R92Q and P46L), which was observed to a slightly lesser extent for the structural mutation T50M. These increased shifts upon doxycycline induction confirmed that binding is specific for the expressed mutant form of TNFRl. For the C33Y cell-line, no significant shift is seen upon induction with doxycycline, suggesting that binding is possibly predominantly to endogenous TNFRl. However, by using the death domain disabled variant of C33Y, the C33Y R347A, a shift is observed after doxycycline induction (figure 8) confirming that DMS5541 does bind the extracellular region of C33Y as well.

Conclusion:

D S5541, the anti-TNFRl dAb/AlbudAb fusion, binds full-length TNFRl expressed on HEK293 cells and this binding is maintained for all TRAPS-associated TNFRl mutants tested. Example 2 - Confirmation that anti-TNFRl dAb (D S5541) inhibits TNF-ct induced secretion of IL-8 by HE 293 cells expressing TRAPS-associated mutant versions of TNFRl.

HEK293 cells were incubated for 24h and 72h in the presence of 3 ng/ml of TNFa and either the anti- TNFRl dAb (DMS5541) or the control dAb (DMS5556) at 10 jig/ml. After the incubation, the supernatant was removed and assayed for IL-8 levels to determine the effect of the dAbs on TNF-a mediated secretion of IL-8.

Methods:

Measurement of IL-8 in supernatants from stimulated HEK293 TNFRl mutants

IL-8 was measured using an R&D Systems CXCL8/IL-8 Duoset match pair antibody ELISA development kit. Nunc 96-well Maxisorp plates were coated with 4μ§/ηιΙ of mouse anti-human IL-8 then incubated o/n at RT°. Wells were washed with 0.05% Tween 20 in PBS, then blocked with 1% BSA in PBS for 1 hour at RT°. Wells were washed again, and samples added to the wells in duplicate, diluted 1 in 10 in TBS (0.1% BSA, 0.05% Tween 20). Supernatant from only one replicate was used. A standard curve of rhlL-8 was produced with a range of 2000pg/ml to 31.3pg/ml and was added to each plate. A negative was also included. Plates were incubated 2h at RT°. Wells were washed, and 20ng/ml of biotinylated goat anti human IL-8 was added to the wells and incubated for 2h at RT°. Wells were again washed, then Streptavidin-HRP added to each well for 20min at RT°. Cells were washed a final time, then TMB liquid substrate was added to each well. Substrate development reaction was stopped after 5min with 2N H₂S0₄.

Absorbance was measured at 450nm with 570nm correction in a Fluorostar Omega plate reader. Optical densities were analysed using Graphpad Prism 6.

Cell culture

HEK293 cells were cultured in 96-well flat-bottomed tissue culture plates, with cell densities dependent on the length of planned incubation. For 24h incubations 1.8xl0⁴ cells per well were used and SxlO³ for 72h incubations with final volume per well of 200 μΙ, Overall culture conditions were as described previously for these cells in Todd et al. (2004, 2007). The HEK293 cells covering the following mutations were used: WT FL, WT R347A, R92Q FL, R92Q A347R, P46L FL, P46L R347A, T50 FL, T50M R347A, C33Y FL, C33Y R347A and TR. Each of these cell lines was incubated in triplicate at four different conditions:

1) 3ng/ml rhTNF-a, 10μΜ ZVAD.F K, 10μ§/ηιΙ DMS5541

2) lOng/ml DMS5541

3) 3ng/ml rhTNF-a, 10μΜ ZVAD.FMK, 10μ§/πιΙ DMS5556

From each well 120 μΙ supernatant was harvested at the appropriate time point and stored at -2Q°C for subsequent analysis using the IL-8 R&D Systems Duoset.

Results:

The levels of IL-8 secretion after 72h by the six different full-length cell lines after incubation with 3ng/ml of TNF-a in the presence of either an anti-TNFRl or a negative control dAb are shown in figure 9. Addition of the anti-TNFRl dAb inhibits IL-8 secretion in all cell lines tested. A striking observation is that even with the truncated TNFR1 cell line still a significant amount of IL-8 is produced. This is most likely due to the endogenous levels of T FR1 present on these cells which induce IL-8 secretion after incubation with TNF-a. Clearly, the anti-TNFRl dAb is able to effectively inhibit induction of IL-8 secretion by the different TNFR1 variants associated with TRAPS.

A second set of experiments is shown in figure 10. TRAPS variants were tested in which the death domain activity had been disabled by introducing the R347A mutation. In these variants, the IL-8 secretion after TNF-a incubation will be due to heterozygous pairing of the mutant TNFR1 version with the endogenous wild-type TNFR1 copy, as only the endogenous TNFR1 will have a death domain capable of signalling. In all these variants the anti-TNFRl is able to inhibit IL-8 secretion. Interestingly, the TRAPS associated mutations with the most pronounced clinical effects (T50 and C33Y) in this set-up show the highest levels of IL-8 secretion after TNF-a stimulation in the presence of the negative control dAb (D S5556).

Conclusion:

The anti-TNFRl dAb not only binds TNFRl variants associated with TRAPS patients, but is also able to effectively inhibit IL-8 secretion induced by these TNFRl variants after incubation with TNF-a.

Example 3 - anti-TNFRl dAb (DMS5541) inhibits T F-a induced secretion of G-CSF, IL-6, IL-8, CCL-2, CCL-5 and PTX-3 by SK Hep-1 cells expressing TRAPS associated mutant versions of TNFRl.

To confirm and broaden the observations made in the HEK293 cells expressing TRAPS associated mutants, a more extensive experiment was done using SK Hep-1 cells. This is an endothelial cell line (Heffelfinger, 1992), which had previously been stably transfected with different TRAPS associated TNFRl mutants (Rebelo, 2006, 2009). This cell line might be physiologically more relevant than HEK293 cells as endothelial cells are an important site of TNFRl expression and function in inflammation (Pober, 2007), and endothelial cell activation is likely to contribute to the monocytic fasciitis (Hull, 2002) and dermal rashes (Toro, 2000) that are characteristic of TRAPS. Hence, we investigated cells transfected with TRAPS mutants C33Y, T50M, R92Q, P46L and wt for the potential of DMS5541 to inhibit TNF-a induced effects on G-CSF, IL-6, IL-8, CCL-2 (MCP-1), CCL-5 (RANTES) and PTX-3. For these experiments, the levels of receptor expression were not enhanced by the addition of doxycycline, as we would consider this less representative of the physiological situation.

Methods

Cell culture

SK-Hepl cells were cultured as described in Rebelo 2009, with the modification that no doxycycline was added. Cells stably transfected with either one of these mutations WT FL, R92Q. FL, P46L FL, T50M FL or C33Y FL as well as a TR version, containing only the extracellular domain and no intracellular signalling domain, were grown in 96-well flat-bottomed tissue culture plates. Cells were seeded at 5xl0³ cells per well in a final volume of 200μΙ and for each of the biological replicates, wells were stimulated as follows:

1) 3ng/ml rhTNF-c , 10μΜ ZVAD.FMK, ^g/ml DMS5541

2) 3ng/ml rhTNF-a, 10μΜ ZVAD.FMK, H^g/ml DMS5556

3) lng/ml rhTNF-a, 10μΜ ZVAD.FMK, lOng/ml DMS5541

4) lng/ml rhTNF-a, 10μΜ ZVAD.FMK, 10|ag/ml DMS5556

After 72h incubation, the supernatant was harvested and stored at -20°C for protein microarray analysis of G-CSF, IL-6, IL-8, CCL-2, CCL-5, and PTX-3.

Micro-array analysis

Protein micro array analysis was basically performed as described in Salva rajah (2014).

Specific reagents used were:

- Printing Buffer: PBS, 50mM Trehalose

- Blocking Buffer: 0.2% l-Block in PBS with 0.1% Tween 20 - Washing Buffer: PBS with 0.1% Tween-20

- Reagent Diluent (R&D Systems DY995 10% BSA) to be diluted 1:10 with PBS

Normal Goat Serum (NGS) (R&D Systems DY005) 2.5μΙ per ΙΟΟμΙ diluted antibody in reagent diluents

- 3%BSA Buffer: dilute 30% BSA in 1XPBS

Tissue Culture Supernatants

For detection the following R&D Systems Duosets were used: for G-CSF (DY214), IL-6 (DY206), IL-8 (DY208), CCL2 MCP-1 (DY279)_; CCL5 RANTES (DY278), and Pentraxin-3 (PTX-3) (DY1826)

Method for microarray:

Capture antibodies were printed at lOOng/ml, diluted in printing buffer onto Superchip

Poly-L-Lysine Slides (16-pad) using a TAS Microgrid II High Density Arrayer. Each of the six antibodies was printed 6 times in each of the 16 pads.

Pads were blocked with ΙΟΟμΙ l-Block and incubated on a shaker at RT° for lh.

A cytokine Standard master mix containing the six cytokines was prepared in Reagent

Diluent. Each cytokine had a top concentration of lOOOpg/ml with the exception of

Pentraxin-3 at 7000pg/ml. From this, a 7-point, 1 in 2 serial dilution was prepared plus a blank.

Tissue culture supernatants were diluted appropriately in Reagent diluent.

Each slide was washed for 3 min x 3 times with wash buffer on a shaker.

To each pad was added ΙΟΟμΙ of Cytokine Standard or diluted sample, as appropriate.

Each slide was incubated on the shaker for lh at RT°.

- Detection Antibodies for each pad were made lh prior to use. A Detection Antibody master mix was prepared in Reagent diluent with 2% Normal Goat Serum, at the working concentrations shown above.

Slides were washed as before, then each pad incubated with ΙΟΟμΙ of diluted Detection antibody master mix on a shaker for lh at RT°.

- Slides were washed as before.

To each pad was added ΙΟΟμΙ of Streptavidin-Cy5 (stock diluted 1 in 1000 in 3% BSA), and incubated on the shaker for 15min, in the dark, at RT°.

Slides were washed as before, then washed again once in PBS, and once with ultra pure water. Slides were dried by centrifugation 5min at 1200 rpm at RT°. Each slide was scaned at 635nm with an Axon Genepix 4200AL Scanner, with the PMT set to 450V. Scanned data was analysed using Genepix Pro, R Statistical Computing software (http://www.r-project.org/) and Graphpad Prism. Results

The effects of DMS5541 incubation, compared to negative control incubation (DMS5556), were investigated for each of the TNFR1 mutant cell lines in the absence and presence of T F-a (1 and 3 ng/ml) stimulation. The data for the inhibitory effect of DMS5541 on cytokine secretion by cells stimulated with 1 ng/ml TNF-a are summarised in Figures 11-16. The 3ng/ml T F concentration results were very similar whilst the no TNF provided background cytokine/chemokine levels with no major difference between TNFR1 mutants or response to DMS5541 addition. The cytokines studied were: G-CSF, IL-6, IL-8, CCL-2, CCL-5, and PTX-3. For each cytokine/chemokine studied, the level of activation varies for each mutant cell line. Overall, the introduction of mutant TNFR1 variants translate to increased responsiveness to TNF-a stimulation, which is most clear for R92Q and T50M mutants when looking at G-CSF, CCL-2 and I L-8. Incubation of cells with DMS5541 clearly reduces the secretion levels of these cytokines/chemokines in these mutant cell lines when compared to the level seen for the negative control DMS5556. The P46L mutation specifically seems to result in a more pronounced increase of CCL-5, for which a smaller increase is seen with the other TRAPS variants. Also this increase is inhibited by incubation with DMS5541. For C33Y, only small increases in sensitivity to T F-a are observed for CCL-2 and IL-8. Both of these small increases are inhibited by DMS5541.

Conclusions

Confirming the data generated with the HEK-293 cells, the non-competitive TNFR1 antagonist DMS5541 also inhibited the increase in cytokine and chemokine secretion observed in the endothelial cell line SK-Hepl in response to low-level stimulation with TNF-a. The relative levels of inhibition and stimulation were dependent on the TRAPS mutation and the cytokine/chemokine studied. For the R92Q, P46L, C33Y and T50M mutations the level of cytokine/chemokine increase were reduced when incubated with DMS5541, suggesting that the dAb could be able to reduce inflammatory responses in patients carrying these mutations and responding strongly to low-level TNF-a stimulation.

SEQUENCE INFORMATION SEQ ID NO.l - Polypeptide sequence of DOMlh-576-208

EVQLLESGGGLVQPGGSLRLSCAASGFTFDKYS GWVRQAPGKGLEWVSQISDTADRTYYAHAVKGRFTISRDNS KNTLYLQMNSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVTVSS

SEQ ID N0.2 - Polypeptide sequence of DOM7h-ll-3

DIQMTQSPSSLSASVGDRVTITCRASRPIGTTLSWYQQKPGKAPKLLILWNSRLQSGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCAQAGTHPTTFGQGTKVEIKR

SEQ ID N0.3 - Polypeptide Sequence of DMS5541 EVQLLESGGGLVQPGGSLRLSCAASGFTFDKYSMGWVRQAPGKGLEWVSQISDTADRTYYAHAVKGRFTISRDNS

KNTLYLQ NSLRAEDTAVYYCAIYTGRWVPFEYWGQGTLVTVSSASTDIQMTQSPSSLSASVGDRVTITCRASRPI

GTTLSWYQQKPG APKLLILWNSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQAGTHPTTFGQGTKVEI

KR SEQ ID N0. - Poly eptide sequence of human TIMFR1 (extracellular region)

LVPHLGDREKRDSVCPQG KYIHPQ NSICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKC

RKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCTCHAGFFLRENECV

SCSNCKKSLECTKLCLPOJENVKGTEDSGTT

SEQ ID NO.5 - Polypeptide Sequence of D S5556 EVQLLESGGGLVQPGGSLRLSCAASGVNVSHDSMTWVRQAPGKGLEWVSAIRGPNGSTYYADSVKGRFTISRDN SKNTLYLQMNSLRAEDTAVYYCASGARHADTERPPSQQTMPFWGQGTLVTVSSASTDIQMTQSPSSLSASVGDR VTITCRASRPIGTTLSWYQQKPGKAPKLLILWNSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQAGTHPT TFGQGTKVEIKR

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Annex 1

The TNFRSF1A variants in 158 TRAPS patients [Lachmann, 2013]

Variant (protein variant)

D12E (p.Asp41Glu)

H22Q (p.His51Gln)

H22R (p.His51Arg)

C29F (p.Cys58Phe)

C29Y (p.Cys58Tyr)

C30F (p.Cys59Phe)

C30R (p.Cys59Arg)

C30Y (p.Cys59Tyr)

C33G (p.Cys62Gly)

C33Y (p.Cys62Tyr)

c.l93-14G>A lntron 2

C.194-150T

T37I (p.Thr66lle)

Y38S (c.200A>C)

L39F (p.Leu68Phe)

D42DEL (p.Asp71del)

C43G (c214T>G)

C43R (p.Cys72Arg)

C43S (p.Cys72Ser)

C43Y (p.Cys72Tyr)

P46L (p.Pro75Leu)

T50K (p.Thr79Lys)

T50M (p.Thr79Met)

C52Y (p.Cys81Tyr)

C55Y (p.Cys84Tyr) S59P (p.Ser88Pro)

F60L (p.Phe89Leu)

T61N (p.Thr90Asn)

N65I (p.Asn94lle)

H66L (p.His95Leu)

L67P (p.Leu96Pro)

H69fs (p.His98_Cys99delinsArg)

C73R (p.Cysl02Arg)

C73W (p.Cysl02Trp)

C88Y (p.Cysll7Tyr)

R92P (p.Argl21Pro)

R92Q (p.Argl21Gln)

V95M (p.Vall24Met)

C96Y (p.Cysl25Tyr)

Y103_R104DEL (p.Tyrl32_Argl33del)

E109A (p.Glul38Ala)

C114W (p.Cysl43Trp

N116S (p.Asnl45Ser)

C.472+60T Intron 4

L167_G175del (c.586_612dell27 Exon 6

Intronic substitution c626-32G>T Intron 6

Claims

I. An anti-TNFa receptor type 1 (TNFR1; p55) binding protein which is a non-competitive antagonist of TNFR1 for use in the treatment of patients with TNF Receptor Associated Periodic Syndrome (TRAPS).

2. The anti-TNFa receptor type 1 (TNFR1; p55) binding protein for use according to claim 1 which is an immunoglobulin single variable domain.

3. The anti-TNFa receptor type 1 (TNFR1; p55) binding protein for use according to claim 1 or 2 which is an immunoglobulin single variable domain which comprises an amino acid sequence that is at least 95, 96, 97, 98 or 99% identical (or is 100% identical) to the amino acid sequence of DOMlh- 574-208 (SEQ ID NO.l).

4. The anti-TNFa receptor type 1 (TNFR1; p55) binding protein for use according to any preceding claim which is an immunoglobulin single variable domain comprising an amino acid sequence that is identical to DOMlh-574-208 (SEQ ID NO. 1) or has 1 or 2 amino acid differences compared to the amino acid sequence of DOMlh-574-208.

5. The anti-TNFa receptor type 1 (TNFR1; p55) binding protein for use according to any preceding claim which is an immunoglobulin single variable domain comprising an amino acid sequence that is identical to DOMlh-574-208 (SEQ ID NO. 1).

6. The anti-TNFa receptor type 1 (TNFR1; p55) binding protein for use according to any preceding claim which comprises a multispecific ligand comprising (i) an anti-TNFa receptor type 1 (TNFR1; p55) binding protein as defined in any preceding claim and (ii) at least one anti-serum albumin (SA) immunoglobulin single variable domain that specifically binds SA.

7. The anti-TNFa receptor type 1 (TNFR1; p55) binding protein for use according to any preceding claim which comprises a multispecific ligand comprising (i) an anti-T Fa receptor type 1 (TNFR1; p55) binding protein as defined in any preceding claim and (ii) at least one anti-serum albumin (SA) immunoglobulin single variable domain that specifically binds SA, wherein the anti-SA single variable domain comprises an amino acid sequence that is at least 80% identical to the sequence of DOM7h-ll-3 (SEQ ID NO. 2).

8. The anti-TNFa receptor type 1 (TNFR1; p55) binding protein for use according to any preceding claim which comprises a multispecific ligand comprising (i) an anti-TNFa receptor type 1 (TNFR1; p55) binding protein as defined in any preceding claim and (ii) at least one anti-serum albumin (SA) immunoglobulin single variable domain that specifically binds SA, wherein the anti-SA single variable domain comprises an amino acid sequence that is at least 80% identical to the sequence of DOM7h-ll-3 (SEQ ID NO. 2), and (Hi) optionally wherein a linker is provided between the anti-TNFRl binding protein and the anti-SA single variable domain.

9. The anti-TNFa receptor type 1 (TNFR1; p55) binding protein for use according to claim 8 wherein the linker comprises the amino acid sequence AST, optionally ASTSG S.

10. The anti-TNFa receptor type 1 (TNFR1; p55) binding protein for use according to claim 8 or 9 wherein the linker is AS(G₄S)_n, where n is 1, 2, 3 , 4, 5, 6, 7 or 8.

II. The anti-TNFa receptor type 1 (TNFR1; p55) binding protein for use according to claims 8-10 wherein the linker is AS(G₄S)₃.

12. The anti-TNF receptor type 1 (TNFR1; p55) binding protein for use according to any preceding claim which comprises a multispecific ligand comprising the amino acid sequence of DMS5541 (SEQ ID NO. 3).

13. The anti-TNFa receptor type 1 (TNFR1; p55) binding protein for use according to any preceding claim wherein the patient carries a non-structural or structural mutation.

14. An anti-TNFa receptor type 1 (TNFR1; p55) binding protein which is a non-competitive antagonist of TNFR1 for use in the treatment of patients carrying low-penetrance TNFR1 mutations suffering from inflammatory diseases.

15. The anti-TNFa receptor type 1 (TNFR1; p55) binding protein for use according to claim 14 which is an immunoglobulin single variable domain.

16. The anti-TNFa receptor type 1 (TNFR1; p55) binding protein for use according to claim 14 or 15 which is an immunoglobulin single variable domain which comprises an amino acid sequence that is at least 95, 96, 97, 98 or 99% identical (or is 100% identical) to the amino acid sequence of DOMlh-574-208 (SEQ ID NO.l).

17. The anti-TNFa receptor type 1 (TNFR1; p55) binding protein for use according to claim 14-16 which is an immunoglobulin single variable domain which comprises an amino acid sequence that is identical to DOMlh-574-208 (SEQ ID NO. 1) or has 1 or 2 amino acid differences compared to the amino acid sequence of DOMlh-574-208.

18. The anti-TNFa receptor type 1 (TNFR1; p55) binding protein for use according to claim 14-17 which is an immunoglobulin single variable domain comprising an amino acid sequence that is identical to DOM lh-574-208 (SEQ ID NO. 1).

19. The anti-TNFa receptor type 1 (TNFR1; p55) binding protein for use according to claim 14-18 which comprises a multispecific ligand comprising (i) an anti-TNFa receptor type 1 (TNFR1; p55) binding protein as defined in any preceding claim and (ii) at least one anti-serum albumin (SA) immunoglobulin single variable domain that specifically binds SA.

20. The anti-TNFa receptor type 1 (TNFR1; p55) binding protein for use according to claim 14-19 which comprises a multispecific ligand comprising (i) an anti-TNFa receptor type 1 (TNFR1; p55) binding protein as defined in any preceding claim and (ii) at least one anti-serum albumin (SA) immunoglobulin single variable domain that specifically binds SA, wherein the anti-SA single variable domain comprises an amino acid sequence that is at least 80% identical to the sequence of DOM7h- 11-3 (SEQ ID NO. 2).

21. The anti-TNFa receptor type 1 (TNFR1; p55) binding protein for use according to claims 14- 20 which comprises a multispecific ligand comprising (i) an anti-TNFa receptor type 1 (TNFR1; p55) binding protein as defined in any preceding claim and (ii) at least one anti-serum albumin (SA) immunoglobulin single variable domain that specifically binds SA, wherein the anti-SA single variable domain comprises an amino acid sequence that is at least 80% identical to the sequence of DOM7h- 11-3 (SEQ ID NO. 2), and (iii) optionally wherein a linker is provided between the anti-TNFRl binding protein and the anti-SA single variable domain.

22. The anti-TNFa receptor type 1 (TNFR1; p55) binding protein for use according to claim 21 wherein the linker comprises the amino acid sequence AST, optionally ASTSGPS. WO 2015/104322 PCT/EP2015/050241

23, The anti-TNFa receptor type 1 (TNFR1; p55) binding protein for use according to claim 21 or

22 wherein the linker is AS(G₄S)_n, where n is 1, 2, 3 , 4, 5, 6, 7 or 8.

24. The anti-TNFa receptor type 1 (TNFR1; p55) binding protein for use according to claims 21-

23 wherein the linker is AS(G₄S)₃.

25. The anti-TNFa receptor type 1 (TNFR1; p55) binding protein for use according to claim 14-24 which comprises a multispecific ligand comprising the amino acid sequence of DMS5541 (SEQ. ID NO.

3),

26. The anti-TNFa receptor type 1 (TNFR1; p55) binding protein for use according to according to claim 14-25 wherein the patients carrying low-penetrance TNFR1 mutations carry the R92Q mutation.

27. The anti-TNFa receptor type 1 (TNFR1; p55) binding protein for use according to claims 14- 25 wherein the patients carrying low-penetrance TNFR1 mutations suffer from inflammatory diseases selected from TRAPS, rheumatoid arthritis, Behget disease, extracranial deep vein thrombosis, premature myocardial infarction (Ml), carotid plaques, carotid intima-media thickness, atherosclerosis, pericardits and multiple sclerosis (MS).

28. The anti-TNFa receptor type 1 (TNFR1; p55) binding protein for use according to claims 14- 25 wherein the patients carrying low-penetrance TNFR1 mutations suffer from inflammatory diseases selected from rheumatoid arthritis, Behfet disease, extracranial deep vein thrombosis, premature myocardial infarction (Ml), carotid plaques, carotid intima-media thickness,

atherosclerosis, pericardits and multiple sclerosis (MS).

29. The anti-TNFa receptor type 1 (TNFR1; p55) binding protein for use according to claims 26 wherein the patients carrying the R92Q. mutation suffer from inflammatory diseases selected from TRAPS, rheumatoid arthritis, Behcet disease, extracranial deep vein thrombosis, premature myocardial infarction (Ml), carotid plaques, carotid intima-media thickness, atherosclerosis, pericardits and multiple sclerosis (MS).

30. The anti-TNFa receptor type 1 (TNFR1; p55) binding protein for use according to claim 26wherein the patients carrying the R92Q mutation suffer from inflammatory diseases selected from rheumatoid arthritis, Behcet disease, extracranial deep vein thrombosis, premature myocardial infarction (Ml), carotid plaques, carotid intima-media thickness, atherosclerosis, pericardits and multiple sclerosis (MS).