WO2010081787A1

WO2010081787A1 - IMPROVED TNFα ANTAGONISM, PROPHYLAXIS & THERAPY WITH REDUCED ORGAN NECROSIS

Info

Publication number: WO2010081787A1
Application number: PCT/EP2010/050234
Authority: WO
Inventors: Neil Brewis; Steve Holmes
Original assignee: Domantis Limited
Priority date: 2009-01-14
Filing date: 2010-01-11
Publication date: 2010-07-22

Abstract

The invention relates to anti-TNFR1 polypeptides, binding moieties, antibody single variable domains (dAbs) and antagonists for treating and/or preventing a TNFα-mediated condition in a patient and substantially protecting against TNFα-mediated organ necrosis. The invention also relates to anti-TNFR1 polypeptides and antibody single variable domains (dAbs) and antagonists for antagonising TNFα in a patient and substantially protecting against TNFα-mediated organ necrosis. Methods and uses are also provided. Thus, liver, kidney, heart, pancreas, lung and/or other organs are substantially protected while TNFα is addressed using the approach of the invention.

Description

IMPROVED TNFα ANTAGONISM, PROPHYLAXIS & THERAPY WITH REDUCED ORGAN NECROSIS

The present invention relates to improved antagonism of TNFα and the treatment and/or prophylaxis of TNFα-mediated conditions. In particular for these purposes, the invention relates to anti-Tumor Necrosis Factor receptor 1 (TNFRl, p55, CD120a, P60, TNF receptor superfamily member IA, TNFRSFlA) immunoglobulin (antibody) single variable domains (dAbs), antagonists comprising these, methods and uses.

BACKGROUND OF THE INVENTION

TNFRl

TNFRl is a transmembrane receptor containing an extracellular region that binds ligand and an intracellular domain that lacks intrinsic signal transduction activity but can associate with signal transduction molecules. The complex of TNFRl with bound TNF contains three TNFRl chains and three TNF chains. (Banner et al., Cell, 73(3) 431-445 (1993).) The TNF ligand is present as a trimer, which is bound by three TNFRl chains. (Id.) The three TNFRl chains are clustered closely together in the receptor-ligand complex, and this clustering is a prerequisite to TNFRl -mediated signal transduction. In fact, multivalent agents that bind TNFRl, such as anti-TNFRl antibodies, can induce TNFRl clustering and signal transduction in the absence of TNF and are commonly used as TNFRl agonists. (See, e.g., Belka et al., EMBO, 14(6):\ 156-1165 (1995); Mandik-Nayak et al, J. Immunol, 167:1920-1928 (2001).) Accordingly, multivalent agents that bind TNFRl, are generally not effective antagonists of TNFRl even if they block the binding of TNFα to TNFRl .

SEQ ID numbers in this paragraph refer to the numbering used in WO2006038027. The extracellular region of TNFRl comprises a thirteen amino acid amino-terminal segment (amino acids 1-13 of SEQ ID NO: 603 (human); amino acids 1- 13 of SEQ ID NO:604 (mouse)), Domain 1 (amino acids 14-53 of SEQ ID NO:603 (human); amino acids 14-53 of SEQ ID NO:604 (mouse)), Domain 2 (amino acids 54- 97 of SEQ ID NO: 603 (human); amino acids 54-97 of SEQ ID NO:604 (mouse)), Domain 3 (amino acids 98-138 of SEQ ID NO: 603 (human); amino acid 98-138 of SEQ ID NO:604 (mouse)), and Domain 4 (amino acids 139-167 of SEQ ID NO:603 (human); amino acids 139-167 of SEQ ID NO:604 (mouse)) which is followed by a membrane-proximal region (amino acids 168-182 of SEQ ID NO:603_(human); amino acids 168-183 SEQ ID NO: 604 (mouse)). (See, Banner et al, Cell 75(3) 431-445 (1993) and Loetscher et al, Cell 61(2) 351-359 (1990).) Domains 2 and 3 make contact with bound ligand (TNFβ, TNFα). (Banner et al., Cell, 73(3) 431-445 (1993).) The extracellular region of TNFRl also contains a region referred to as the pre-ligand binding assembly domain or PLAD domain (amino acids 1-53 of SEQ ID NO:603_(human); amino acids 1-53 of SEQ ID NO:604 (mouse)) (The Government of the USA, WO 01/58953; Deng et al, Nature Medicine, doi: 10.1038/nml304 (2005)).

TNFRl is shed from the surface of cells in vivo through a process that includes proteolysis of TNFRl in Domain 4 or in the membrane-proximal region (amino acids 168-182 of SEQ ID NO:603; amino acids 168-183 of SEQ ID NO:604), to produce a soluble form of TNFRl . Soluble TNFRl retains the capacity to bind TNFα, and thereby functions as an endogenous inhibitor of the activity of TNFα.

WO2006038027, WO2008149144 and WO2008149148 disclose anti-TNFRl immunoglobulin single variable domains and antagonists comprising these. These documents also disclose the use of such domains and antagonists for the treatment and/or prevention of conditions mediated by TNFα. Anti- TNFα drugs such as infliximab and etanercept are used for addressing

TNFα-mediated conditions such as arthritis, Crohn's disease, psoriasis and ankylosing spondylitis. There are reports, however, that anti- TNFα drugs are associated with organ necrosis. This may be due to TNFα inhibition inducing or un-masking autoimmune disease leading to necrosis (the specific organ involved depending upon the autoimmune response involved). For example, see Thiefϊn et al, Joint Bone Spine, 75 (2008), 737-739; and Ozorio et al, MJA 2007, 187:524-526.

SUMMARY OF THE INVENTION

In one aspect, the invention provides an anti-TNFα receptor type 1 (TNFRl; p55) binding moiety for treating and/or preventing a TNFα-mediated condition in a patient and substantially protecting against TNFα-mediated organ necrosis.

In another aspect, the invention provides an anti-TNFα receptor type 1 (TNFRl; p55) binding moiety for antagonising TNFα in a patient and substantially protecting against TNFα-mediated organ necrosis.

In one aspect, the invention provides an anti-TNFα receptor type 1 (TNFRl; p55) antagonist comprising the binding moiety.

One aspect of the invention provides the use of an anti-TNFα receptor type 1 (TNFRl ; p55) binding moiety in the manufacture of a medicament for treating and/or preventing a TNFα-mediated condition and substantially protecting against TNFα- mediated organ necrosis.

One aspect of the invention provides the use of an anti-TNFα receptor type 1 (TNFRl; p55) binding moiety in the manufacture of a medicament for antagonising TNFα in a patient and substantially protecting against TNFα-mediated organ necrosis.

One aspect of the invention provides a method of treating and/or preventing a TNFα-mediated condition in a patient and substantially protecting against TNFα- mediated organ necrosis, wherein the method comprises administering to the patient a pharmaceutically effective dose of an anti-TNFα receptor type 1 (TNFRl; p55) binding moiety.

One aspect of the invention provides a method of antagonising TNFα in a patient and substantially protecting against TNFα-mediated organ necrosis, wherein the method comprises administering a pharmaceutically effective dose of an anti-TNFα receptor type 1 (TNFRl; p55) binding moiety. One aspect of the invention provides the use of an antagonist comprising an anti-TNFα receptor type 1 (TNFRl; p55) immunoglobulin single variable domain in the manufacture of a medicament for treating and/or preventing a TNFα-mediated condition in a patient and substantially protecting against TNFα-mediated organ necrosis, wherein the antagonist has a terminal half-life of at least 20 hours.

One aspect of the invention provides the use of an antagonist comprising an anti-TNFα receptor type 1 (TNFRl; p55) immunoglobulin single variable domain in the manufacture of a medicament for antagonising TNFα in a patient and substantially protecting against TNFα-mediated organ necrosis, wherein the antagonist has a terminal half-life of at least 20 hours.

One aspect of the invention provides a method of treating and/or preventing a TNFα-mediated condition in a patient and substantially protecting against TNFα- mediated organ necrosis, wherein the method comprises administering to the patient a pharmaceutically effective dose of an antagonist comprising an anti-TNFα receptor type 1 (TNFRl; p55) immunoglobulin single variable domain, wherein the antagonist has a terminal half-life of at least 20 hours.

One aspect of the invention provides a method of antagonisting TNFα in a patient and substantially protecting against TNFα-mediated organ necrosis, wherein the method comprises administering to the patient a pharmaceutically effective dose of an antagonist comprising an anti-TNFα receptor type 1 (TNFRl; p55) immunoglobulin single variable domain, wherein the antagonist has a terminal half-life of at least 20 hours.

In all aspects of the invention, the binding moiety can be an immunoglobulin single variable domain (ie, a dAb). In all aspects of the invention, the organ can be selected from the group consisting of liver, kidney, heart, pancreas, lung and skin. In one embodiment, the organ is liver. In one embodiment, the organ is kidney. In one embodiment, the organ is heart.

WO2006038027, WO2008149144 and WO2008149148 disclose anti-TNFRl binding moieties, immunoglobulin single variable domains and antagonists comprising these, as well as the sequences (amino acid and nucleotide) of the variable domains and antagonists. Selection of the variable domains (eg, from repertoires, eg, by phage display), formatting, expression and half-life extension are also disclosed. These documents also disclose the use of such domains and antagonists for antagonizing TNFα and the treatment and/or prevention of diseases and conditions mediated by TNFα, as well as applicable assays and models for use with anti -TNFRl binding domains, such as variable domains, and antagonists. All of these disclosures are incorporated herein by reference as though explicitly written herein with the express intention of providing disclosure for incorporation into claims herein and as examples of TNFRl binding moieties, variable domains, antagonists and TNFα-mediated conditions for application in the context of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : Protection of anti-TNFRl treatment of TNF-toxicity.

Mice in the six groups were scored for survival 48h after administration of TNFα. The figure plots the six treatment groups, 1) saline, 2) ENBREL™ (Immunex Corporation) 10 mg/kg, 3) 10 mg/kg DOMlm-21-23/DOM7m-16, 4) 1 mg/kg DOMlm-21-23/DOM7m-16, 5) 10 mg/kg DOMlm-21-23/PEG and 6) 1 mg/kg DOMlm-21-23/PEG against the percentage of surviving animals. The saline group had 0% survival whereas all animals in the different treatment groups survived. The amino acid and nucleic acid sequences of DOMl m-21-23 are found in WO2006038027 as

SEQ ID NOs: 173 and 186. DOMlm-21-23/DOM7m-16 is also known as TAR2m-21- 23 3U TAR7m-16. A nucleotide sequence encoding TAR2m-21-23 3U TAR7m-16 and the amino acid sequence of the dual specific ligand are presented in WO2006038027 as SEQ ID NO: 375 and SEQ ID NO:376, wherein "3U" is a (Gly₄Ser)₃ linker disclosed as SEQ ID NO: 199 in WO2006038027. These specific sequences and related disclosures in WO2006038027 are incorporated herein by reference as though explicitly written herein with the express intention of providing disclosure for incorporation into claims herein and as examples of variable domains and antagonists for application in the context of the present invention. "TAR2m" and "TARJm" notation in WO2006038027 are equivalent to "DOMIm" and "D0M7m" notation used herein.

Figure 2: Histopathological analysis on liver sections. Livers from eight mice per group were isolated 48h after TNFα stimulation and sectioned for histopathological analysis. The level of necrosis was scored on a scale from 0-4. The first group of the study (saline) is not shown as there were no surviving animals. The five surviving groups are shown in this figure, which are group 2) ENBREL™ 10 mg/kg, 3) 10 mg/kg DOMlm-21-23/DOM7m-16, 4) 1 mg/kg DOMIm- 21-23/DOM7m-16, 5) 10 mg/kg DOMlm-21-23/PEG, and 6) 1 mg/kg DOMlm-21- 23/PEG and are plotted against their liver necrosis score. The error-bars represent the statistical variation within each treatment group.

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) 4^th Ed, John Wiley & Sons, Inc. which are incorporated herein by reference) and chemical methods.

As used herein, the term "antagonist of Tumor Necrosis Factor Receptor 1 (TNFRl)" or "anti-TNFRl antagonist" or the like refers to an agent (e.g., a molecule, a compound) which binds TNFRl and can inhibit a (i.e., one or more) function of TNFRl . For example, an antagonist of TNFRl can inhibit the binding of TNF α to TNFRl and/or inhibit signal transduction mediated through TNFRl . Accordingly, TNFRl -mediated processes and cellular responses (e.g., TNFα-induced cell death in a standard L929 cytotoxicity assay) can be inhibited with an antagonist of TNFRl. As used herein, the term "antagonizing" TNFα refers to inhibiting a (i.e., one or more) function of TNFα. For example, inhibiting the binding of TNFα to TNFRl and/or inhibiting signal transduction mediated through TNFRl. Accordingly, TNFα - mediated processes and cellular responses can be inhibited in a patient as indicated by an improvement in a TNFα-mediated condition in the patient or a symptom thereof, for example as evident from an improvement in scoring or one or more standard indicia used for assessing the condition, as would be readily apparent and routine to the skilled person addressing the particular TNFα-mediated condition at hand.

A "patient" is any animal, eg, a mammal, eg, a mouse, human, rabbit, rat, dog, cat or pig. In one embodiment, the patient is a human. As used herein, "peptide" refers to about two to about 50 amino acids that are joined together via peptide bonds.

As used herein, "polypeptide" refers to at least about 50 amino acids that are joined together by peptide bonds. Polypeptides generally comprise tertiary structure and fold into functional domains. As used herein an antibody refers to IgG, IgM, IgA, IgD or IgE or a fragment

(such as a Fab , F(ab')2, Fv, disulphide linked Fv, scFv, closed conformation multispecifϊc antibody, disulphide-linked scFv, diabody) whether derived from any species naturally producing an antibody, or created by recombinant DNA technology; whether isolated from serum, B-cells, hybridomas, transfectomas, yeast or bacteria. As used herein, "antibody format" 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, bispecifϊc 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 antibody variable domain (e.g., a dAb, V_H, V_HH, V_L), 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)-

The phrase "immunoglobulin single variable domain" refers to an antibody variable domain (V_H, V_HH, V_L) that specifically binds an antigen or epitope independently of other V regions or domains. An immunoglobulin single variable domain can be present in a format (e.g., homo- or hetero-multimer) with other variable regions or variable domains where the other regions or domains are not required for antigen binding by the single immunoglobulin variable domain (i.e., where the immunoglobulin single variable domain binds antigen independently of the additional variable domains). A "domain antibody" or "dAb" is the same as an "immunoglobulin single variable domain" as the term is used herein. A "single immunoglobulin variable domain" is the same as an "immunoglobulin single variable domain" as the term is used herein. A "single antibody variable domain" or an "antibody single variable domain" is the same as an "immunoglobulin single variable domain" as the term is used herein. An immunoglobulin single variable domain is in one embodiment a human antibody variable domain, but also includes single antibody variable domains from other species such as rodent (for example, as disclosed in WO 00/29004, the contents of which are incorporated herein by reference in their entirety), nurse shark and Cαmelid V_HH dAbs. Camelid V_HH are immunoglobulin single variable domain polypeptides that are derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies naturally devoid of light chains. The V_HH may be humanized. 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. A "single antibody variable domain" is a folded polypeptide domain comprising sequences characteristic of antibody variable domains. It therefore includes complete antibody variable domains and modified variable domains, for example, in which one or more loops have been replaced by sequences which are not characteristic of antibody variable domains, or antibody variable domains which have been truncated or comprise N- or C-terminal extensions, as well as folded fragments of variable domains which retain at least the binding activity and specificity of the full- length domain.

In the instant application, the term "prevention" and "preventing" involves administration of the protective composition prior to the induction of the disease or condition. "Treatment" and "treating" involves administration of the protective composition after disease or condition symptoms become manifest. "Suppression" or "suppressing" refers to administration of the composition after an inductive event, but prior to the clinical appearance of the disease or condition.

As used herein, the term "dose" refers to the quantity of ligand administered to a subject all at one time (unit dose), or in two or more administrations over a defined time interval. For example, dose can refer to the quantity of ligand (e.g., ligand comprising an immunoglobulin single variable domain that binds target antigen) administered to a subject over the course of one day (24 hours) (daily dose), two days, one week, two weeks, three weeks or one or more months (e.g. , by a single administration, or by two or more administrations). The interval between doses can be any desired amount of time. The term "pharmaceutically effective" when referring to a dose means sufficient amount of the antagonist, domain or pharmaceutically active agent to provide the desired effect. The amount that is "effective" will vary from subject to subject, depending on the age and general condition of the individual, the particular drug or pharmaceutically active agent and the like. Thus, it is not always possible to specify an exact "effective" amount applicable for all patients. However, an appropriate "effective" dose in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

The term "necrosis" when applied to organs is well understood in the art. This term is used to refer to the presence of dead cells in organ tissue, including cells that have died due to primary necrosis (type 1 necrosis; see, eg, www.copewithcytokines.de) or any other mechanism. Necrosis can be observed, as is conventional, using histophathology, for example employing microscopy, eg, light microscopy or electron microscopy. Alternatively, organ necrosis can be determined by non-invasive methods, as is conventional in the art, by determining organ function. For example, liver function can be assessed using standard techniques know to the skilled person, eg, by determining levels of liver enzymes and/or metabolites in the blood of the patient, and using this to determine whether or not these are indicative of organ necrosis, as well as the level of necrosis, if present. Similarly, lung, heart and kidney function tests can be used to determine whether or not lung, heart or kidney necrosis respectively is present, and to what degree, using conventional techniques and tests.

The term "substantially protecting against TNFα-mediated organ necrosis" means that organ necrosis is not observed (or not to a substantial degree) following administration of the anti-TNFRl binding moiety, single variable domain or antagonist. In one embodiment, the presence (or not) of organ necrosis is assessed once antagonism, treatment or prevention of a TNFα-mediated disease has been achieved. The assessment can be made using standard organ function tests as discussed above or using standard histopathology techniques, for example, conventionally an organ tissue biopsy is taken, tissue sample is fixed on a slide and visual inspection of the tissue sample (usually following staining with a standard stain, eg, H&E stain) is made under a microscope to determine whether or not dead cells are present (indicating necrosis). From this analysis, the skilled person can assess whether necrosis is present. In one embodiment, necrosis is determined when dead cells are prevalent in the tissue sample. For example, substantial protection against TNFα-mediated organ necrosis is determined when no more than about [Steve/Allart: are these ranges okay?] 10%, 5%, 4%, 3%, 2% or 1% of the cells in the sample (or as an average over more than one sample, eg, two, three, four, five, six, seven, eight, nine or 10 or more samples from the same organ) are determined to be dead by standard histopatho logical analysis. In one embodiment, determination of necrosis is made using standard techniques, eg using microscopy, eg, light microscopy and/or electron microscopy, for example judged using the following standard criteria. In a first embodiment, necrosis is detected when a cell has been identified as having a pale cytoplasm with a plurality of vacuoles and damaged cytoplasmic membrane with an intact (or fairly intact) nucleus. In a second embodiment, necrosis is detected when a cell has been identified as exhibiting loss of cytoplasm and a damaged and/or irregular nuclear membrane with an intact (or partially intact) nuclear structure. In a third embodiment, necrosis is detected when a cell has been identified as a disintegrated cell having a (very) hydropic cell sap and where the plasma membrane and membranes of the nucleus and organelles appear disrupted. In a fourth embodiment, necrosis is detected when a cell has been identified as showing swelling of the cytoplasm, rupture of the plasma membrane, chromatin aggregation and optionally loss of the nucleus. In a fifth embodiment, necrosis is detected when a cell has been identified as showing chromatin condensation with intact cytoplasmic and nuclear boundaries. In a sixth embodiment, necrosis is detected when a cell has been identified as exhibiting nuclear fragmentation into smaller nuclear bodies with an intact cytoplasm and/or cytoplasmic membrane. In a seventh embodiment, necrosis is detected when a cell has been identified as showing condensation and increased electron density in the cell sap with densely packed organelles, distended vacuoles and cisterns, optionally with some mitochondria showing crystal distension, the nucleus showing clumping and margination of the chromatin, the nuclear membrane appearing folded and the nuclear volume appearing decreased. In an embodiment, necrosis is detected for cells according to one or more of embodiments one, two, three and four, but not according to embodiments five to seven. In an embodiment, necrosis is detected for cells according to any of embodiments one to seven. See Fenech et al, Mutagenesis, VoI 14, No. 6, pp605-612, 1999. See also Solez et al, Kidney International, VoI 43, 1193, ppl058-1067.

In an example, substantial protection against TNFα-mediated organ necrosis is determined according to standard organ tissue necrosis scoring. Standard scoring systems are known to the person skilled in histopathology, and these may vary according to the particular organ being assessed. In an embodiment, substantial protection against TNFα-mediated organ necrosis is determined when the scoring system comprised multiple levels of score (eg, 0, 1, 2, 3, and 4) and when the score for a tissue sample is determined to be the lowest score (the least amount of dead cells) or no necrosis at all, but not a higher score according to that scoring system which would indicate necrosis above said "least amount" score. For example, where the scoring system has levels 0-4, with 0 being no necrosis at all, 1 being the lowest level (minimal necrosis) and 4 being the most necrosis, substantial protection against TNFα-mediated organ necrosis is determined when a score of 0 or 1 only is obtained for a sample or as an average over a plurality of samples, eg, from the same organ, (eg, as a numerical mean score over 2, 3, 4, 5, 6, 7, 8, 9, 10 or more samples). In an embodiment, a control sample is used, wherein the sample is from the organ of a donor that has received a TNFα antagonist, eg, an anti- TNFα antibody, eg, ENBREL™ (Immunex Corp), HUMIRA™ (Abbott Laboratories) or REMICADE™ (Johnson & Johnson) or equivalent generic antagonists etanercept, adalimumab or infliximab. The score(s) for the control sample(s) are compared with the score(s) for the test samples (from a donor having received an anti-TNFRl binding moiety, variable domain or antagonist according to the invention). From the comparison of scores, the skilled person can readily determine if the score for the test sample (or average score for the test samples) is better than the score for the control sample (or average score for the control samples). If the difference is significant, then this is indicative of substantial protection against TNFα-mediated organ necrosis. Conventional statistical analysis can be used to determine significance, eg, by using a standard p test, a p value of <0.001 being indicative of significance when a test score (or average score) is compared with a control score (or average score). In this example, a p value of >0.05 would not be indicative of significance when a test score (or average score) is compared with a control score (or average score).

In one embodiment, therefore, "substantial protection against TNFα-mediated organ necrosis " is indicated by significantly lower organ necrosis using the anti- TNFRl binding moiety, variable domain or antagonist according to the invention, as compared with using a binding moiety or antagonist that binds TNFα, eg an anti-TNFα antibody, eg, ENBREL™ (Immunex Corp), HUMIRA™ (Abbott Laboratories) or REMICADE™ (Johnson & Johnson), etanercept, adalimumab or infliximab.

In one embodiment, standard histopathology scoring is applied and the level of necrosis is determined, as is conventional, from the scoring. For example, for assessing necrosis (eg, kidney necrosis), one can apply scoring (here called Jablonski Scoring) on a scale of 1 to 4 as follows (see also Jablonski et al, Translplantation, VoI 35, No. 3, 1983, ppl98-204):- 1. Mitoses and necrosis of individual cells;

2. Necrosis of all cells in proximal convoluted tubules, with survival of surrounding tubules; 3. Necrosis confined to the distal third of the proximal convoluted tubule with a band of necrosis extending across the inner cortex; and 4. Necrosis affecting all three segments of the proximal convoluted tubule.

In one embodiment, the organ is kidney and "substantially protecting against TNFα-mediated organ necrosis" means that a Jablonski Score of 1 or no necrosis at all is obtained. In one embodiment, the organ is kidney and "substantially protecting against TNFα-mediated organ necrosis" means that a Jablonski Score of 2 or less is obtained. See also Lee et al, J Am Soc Nephrol 15: 102-111, 2004, in particular "Histologic Examinations to Detect Necrosis" on page 103, where Jablonski Scoring was also used. See also Calder et al, Br Med J, 1971, 4, 818, "The comparative nephrotoxicity of asprin and phenacetin derivatives" for further discussion on determining toxicity and necrosis in the kidney. See also Rafϊa et al, Laboratory Investigation, VoI 81, No 11, pp 1503 -1515 for general tissue fixing and staining methodology and microscopy that can be applied in the context of the present invention and for any organ.

For example, where the organ is liver, scoring may be carried out using the SimplePCI software (C Imaging, Inc.). Digital images can be acquired with a SPOT Jr camera mounted on a Nikon E400 microscope using a 1Ox objective. The score can be determined by dividing the measured necrotic area by the total area of the field (see, eg, Carmen & Godolfi, Toxicology, VoI 185, Issues 1-2, 14 March 2003, pp 79-87).

In another example, where the organ is liver, scoring may be carried out using the scoring system disclosed in Soheila et al₃ Toxico logical Sciences, 47, 1999, ppl 10- 117, which discloses a scoring system (herein called the Soheila Scoring system) for necrosis of 1 to 4, as follows:- 1. Occasional (<1%) necrotic hepatocytes;

2. Frequent (5-10%) necrotic hepatocytes; 3. Small foci of necrosis (clusters greater than 10 necrotic hepatocytes); and

4. Extensive areas of necrosis (over 25% of the lobular unit).

In one embodiment of this example, "substantially protecting against TNFα-mediated organ necrosis" means that a score of 1 or no necrosis at all is seen, according to the Soheila Scoring system. In this embodiment, in an example, the score is an average score over a plurality of samples, eg, from the same liver, (eg, as a numerical mean score over 2, 3, 4, 5, 6, 7, 8, 9, 10 or more samples).

In one embodiment of any aspect of the invention, the TNFRl binding moiety is an immunoglobulin single variable domain. In one embodiment, the binding moiety, eg, immunoglobulin single variable domain, is a non-competitive inhibitor of TNFRl. In one example, the binding moiety, eg, single variable domain, is specific for domain 1 of TNFRl. In one example, the binding moiety, eg, single variable domain, is specific for PLAD domain of TNFRl . In another embodiment, the binding moiety, eg single variable domain, competes with TNFα for binding to TNFRl . In another embodiment, the binding moiety, eg, single variable domain, inhibits TNFα binding to TNFRl . Competitive and non-competitive binding moieties are disclosed in WO2006038027 as are the stest and assays for determining whether such moieties are competitive or noncompetitive with TNFα for binding to TNFRl, and this specific disclosure is incorporated herein by reference.

As an alternative to an immunoglobulin single variable domain, the binding moiety is an antibody or antigen-binding fragment thereof, such as a monovalent antigen-binding fragment (e.g., scFv, Fab, Fab', dAb) that has binding specificity for TNFRl . Other suitable binding moieties are ligands described in WO2006038027 that bind TNFRl . The ligands can comprise an immunoglobulin single variable domain or domain antibody (dAb) that has binding specificity for TNFRl, or the complementarity determining regions of such a dAb in a suitable format. In some embodiments, the binding moiety is a dAb monomer that consists essentially of, or consists of, an immunoglobulin single variable domain or dAb that has binding specificity for TNFRl. In other embodiments, the binding moiety is a polypeptide that comprises a dAb (or the CDRs of a dAb) in a suitable format, such as an antibody format, as disclosed in WO2006038027.

In one aspect, the invention provides an anti-TNFα receptor type 1 (TNFRl; p55) antagonist comprising the binding moiety, eg where the binding moiety is an immunoglobulin single variable domain.

In one embodiment, the antagonist comprises the binding moiety (eg, immunoglobulin single variable domain) linked to a polyalkylene glycol moiety, optionally a polyethylene glycol moiety. Reference is made to WO04081026 for suitable PEGs and ways of conjugating PEG to single variable domains and associated tests and assays.

In one embodiment, the antagonist is a multispecific ligand comprising the binding moiety, eg, immunoglobulin single variable domain, and an immunoglobulin single variable domain that specifically binds serum albumin (SA). Reference is made to WO2008149148 for suitable anti-SA single variable domains, ways of linking these to binding moieties and associated tests and assays. The sequences of all anti-SA single variable domains disclosed in WO2008149148 are incorporated herein by reference as though explicitly written herein with the express intention of providing disclosure for incorporation into claims herein and as examples of anti-SA single variable domains to be included in antagonists of the present invention. One aspect of the invention provides the use of an anti-TNFα receptor type 1

(TNFRl; p55) binding moiety (eg, immunoglobulin single variable domain) as herein described in the manufacture of a medicament for treating and/or preventing a TNF α- mediated condition and substantially protecting against TNFα-mediated organ necrosis. One aspect of the invention provides the use of an anti-TNFα receptor type 1 (TNFRl; p55) binding moiety (eg, immunoglobulin single variable domain) as herein described in the manufacture of a medicament for antagonising TNFα in a patient and substantially protecting against TNFα-mediated organ necrosis. One aspect of the invention provides a method of treating and/or preventing a

TNFα-mediated condition in a patient and substantially protecting against TNFα- mediated organ necrosis, wherein the method comprises administering to the patient a pharmaceutically effective dose of an anti-TNFα receptor type 1 (TNFRl; p55) binding moiety (eg, immunoglobulin single variable domain) as herein described. One aspect of the invention provides a method of antagonising TNFα in a patient and substantially protecting against TNFα-mediated organ necrosis, wherein the method comprises administering a pharmaceutically effective dose of an anti-TNFα receptor type 1 (TNFRl; p55) binding moiety (eg, immunoglobulin single variable domain) as herein described. In one example of the use, method, binding moiety (eg, single variable domain) according to the present invention, the binding moiety is provided as part of an antagonist as herein described.

In one embodiment, the organ is selected from the group consisting of liver, kidney, heart, pancreas, lung and skin. In one embodiment, the organ is liver. In one embodiment, the organ is kidney. In one embodiment, the organ is heart.

One aspect of the invention provides the use of an antagonist comprising an anti- TNFα receptor type 1 (TNFRl; p55) immunoglobulin single variable domain in the manufacture of a medicament for treating and/or preventing a TNFα-mediated condition in a patient and substantially protecting against TNFα-mediated organ necrosis, wherein the antagonist has a terminal half- life of at least 12 hours. Optionally, the antagonist can have an alternative terminal half-life, for example any of the terminal half-lives described below.

One aspect of the invention provides the use of an antagonist comprising an anti- TNFα receptor type 1 (TNFRl; p55) immunoglobulin single variable domain in the manufacture of a medicament for antagonising TNFα in a patient and substantially protecting against TNFα-mediated organ necrosis, wherein the antagonist has a terminal half-life of at least 12 hours. Optionally, the antagonist can have an alternative terminal half-life, for example any of the terminal half- lives described below.

It may not, in some instances, be necessary, expedient or ethical to make a determination that the binding moiety or antagonist substantially protects against TNFα -mediated organ necrosis in the patient in which the TNFα-mediated condition is treated or prevented, or in which TNFα is antagonised. For example, trials to determine if a particular binding moiety or antagonist substantially protects against TNFα - mediated organ necrosis as well as antagonises, treats and/or protects against a TNFα- mediated condition may be conducted in a model system, such as in a mouse, volunteer human or non-human primate (eg, Cynomolgus monkey, rhesus, marmoset or baboon). Thereafter, once protection has been determined in the model, the binding moiety or antagonist may be subsequently administered to a patient (eg, a human) that is different from the animal(s) used in the trials, in order to antagonise TNFα or treat and/or prevent against a TNFα -mediated condition in that patient . In some instances, the earlier determination of necrosis is made from tissue obtained from a volunteer human to which the binding moiety or antagonist has been administered. For example, this may be in clinical trials. Once the binding moiety or antagonist has been shown to antagonise TNFα or treat and/or prevent a TNFα-mediated condition in the trial human(s) and substantially protect against organ necrosis in those human(s), it is contemplated by the present invention that the binding moiety or antagonist can be used to antagonise TNFα or treat and/or prevent a TNFα-mediated condition in a patient (eg, a human patient) for the purpose of such antagonism, treatment and/or prevention and with the expectation of substantially protecting against organ necrosis, without the need to determine from that patient's organs whether or not there is substantial protection against organ necrosis. Thus, in one example of the use, method, binding moiety (eg, single variable domain) or antagonist according to the present invention, "substantially protecting against TNFα-mediated organ necrosis" means that substantial protection has been previously observed in an animal (eg, a mammal, eg, a mouse, volunteer human or non-human primate (eg, Cynomolgus monkey, rhesus, marmoset or baboon)) that is different from the patient. In one embodiment, the animal is a non-human primate, and the patient is a human. In one embodiment, the animal is a Cynomolgus monkey, rhesus, marmoset or baboon, and the patient is a human. In one embodiment, the animal is a Cynomolgus monkey, and the patient is a human. In one embodiment, the animal is a mouse, and the patient is a human. In one embodiment, the animal is a human, and the patient is a different human. The invention relates to isolated and/or recombinant nucleic acids encoding immunoglobulin single variable domains and antagonists described herein.

Nucleic acids referred to herein as "isolated" are nucleic acids which have been separated away from other material (e.g., other nucleic acids such as genomic DNA, cDNA and/or RNA) in its original environment (e.g., in cells or in a mixture of nucleic acids such as a library). An isolated nucleic acid can be isolated as part of a vector (e.g., a plasmid).

Nucleic acids referred to herein as "recombinant" are nucleic acids which have been produced by recombinant DNA methodology, including methods which rely upon artificial recombination, such as cloning into a vector or chromosome using, for example, restriction enzymes, homologous recombination, viruses and the like, and nucleic acids prepared using the polymerase chain reaction (PCR).

The section entitled "NUCLEIC ACIDS, HOST CELLS AND METHODS FOR PRODUCING PROTEASE RESISTANT POLYPEPTIDES" in WO2008149148 is expressly incorporated herein by reference to provide disclosure applicable to nucleotide sequences, nucleic acids, hosts and vectors of the invention and encoding variable domains and antagonists of the invention and methods and uses of these. Generally, the present ligands (e.g., variable domains, antagonists) 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 ligands (e.g., antagonits) 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 ligands of the present invention, or even combinations of ligands according to the present invention having different specificities, such as ligands 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 ligands 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, intraperitoneally, transdermally, 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 ligands 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 ligands (e.g., antagonists) 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 included in the definition of 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 5.0 mg of ligand, e.g. dAb or antagonist 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 ligands 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. When an ligand of TNFRl (e.g., antagonist) is administered to treat, suppress or prevent a chronic inflammatory disease, 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 μg/kg to about 80 mg/kg, about 100 μg/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 10 μg/kg to about 10 mg/kg, about 10 μg/kg to about 5 mg/kg, about 10 μg/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. In particular embodiments, the ligand of TNFRl (e.g., antagonist) is administered to treat, suppress or prevent a chronic inflammatory disease once every two weeks or once a month at a dose of about 10 μg/kg to about 10 mg/kg (e.g., about 10 μg/kg, about 100 μg/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 to have taken place 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, tumor size, etc.), or by an accepted clinical assessment scale, for example, the Expanded Disability Status Scale (for multiple sclerosis), the Irvine Inflammatory Bowel Disease Questionnaire (32 point assessment evaluates quality of life with respect to bowel function, 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 treatment. Similarly, prophylaxis (prevention) performed using a composition as described herein is achieved 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.

A composition containing a ligand (e.g., antagonist) or cocktail thereof according to the present invention may be utilised in prophylactic and therapeutic settings to aid in the alteration, inactivation, killing or removal of a select target cell population in a mammal. In addition, the selected repertoires of polypeptides described herein may be used extracorporeally or in vitro selectively to kill, deplete or otherwise effectively remove a target cell population from a heterogeneous collection of cells. Blood from a mammal may be combined extracorporeally with the ligands whereby the undesired cells are killed or otherwise removed from the blood for return to the mammal in accordance with standard techniques. A composition containing a ligand (e.g., antagonist) according to the present invention may be utilised in prophylactic and therapeutic settings to aid in the alteration, inactivation, killing or removal of a select target cell population in a mammal.

The ligands (e.g., anti -TNFRl antagonists, dAb monomers) can be administered and or formulated together with one or more additional therapeutic or active agents. When a ligand (eg, a dAb) is administered with an additional therapeutic agent, the ligand can be administered before, simultaneously with or subsequent to administration of the additional agent. Generally, the ligand and additional agent are administered in a manner that provides an overlap of therapeutic effect.

In one embodiment, the "TNFα-mediated condition" is a chronic inflammatory disease, such that the purpose (eg, of the method or use) of the invention is for treating, suppressing or preventing a chronic inflammatory disease and substantially protecting against TNFα-mediated organ necrosis.

In one embodiment, the "TNFα-mediated condition" is a arthritis, such that the purpose (eg, of the method or use) of the invention is for treating, suppressing or preventing arthritis (e.g., rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis) and substantially protecting against TNFα-mediated organ necrosis.

In one embodiment, the "TNFα-mediated condition" is a psoriasis, such that the purpose (eg, of the method or use) of the invention is for treating, suppressing or preventing psoriasis and substantially protecting against TNFα-mediated organ necrosis.

In one embodiment, the "TNFα-mediated condition" is inflammatory bowel disease, such that the purpose (eg, of the method or use) of the invention is for treating, suppressing or preventing inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis) and substantially protecting against TNFα-mediated organ necrosis.

In one embodiment, the "TNFα-mediated condition" is chronic obstructive pulmonary disease, such that the purpose (eg, of the method or use) of the invention is for treating, suppressing or preventing chronic obstructive pulmonary disease (e.g., chronic bronchitis, chronic obstructive bronchitis, emphysema) and substantially protecting against TNFα-mediated organ necrosis. In one embodiment, the "TNFα-mediated condition" is pneumonia, such that the purpose (eg, of the method or use) of the invention is for treating, suppressing or preventing pneumonia (e.g., bacterial pneumonia, such as Staphylococcal pneumonia) and substantially protecting against TNFα-mediated organ necrosis.

In other embodiments, the purpose of the invention is for treating, suppressing or preventing other pulmonary diseases in addition to chronic obstructive pulmonary disease, and pneumonia, and substantially protecting against TNFα-mediated organ necrosis. Other pulmonary diseases that can be treated, suppressed or prevented in accordance with the invention include, for example, cystic fibrosis and asthma (e.g., steroid resistant asthma). Thus, in another embodiment, the "TNFα-mediated condition" is a pulmonary disease (e.g., cystic fibrosis, asthma), such that the purpose (eg, of the method or use) of the invention is for treating, suppressing or preventing a pulmonary disease (e.g., cystic fibrosis, asthma) and substantially protecting against TNFα-mediated organ necrosis.

In particular embodiments, an antagonist of TNFRl is administered via pulmonary delivery, such as by inhalation (e.g. , intrabronchial, intranasal or oral inhalation, intranasal drops) or by systemic delivery (e.g., parenteral, intravenous, intramuscular, intraperitoneal, subcutaneous). In one embodiment, the "TNFα-mediated condition" is septic shock, such that the purpose (eg, of the method or use) of the invention is for treating, suppressing or preventing septic shock and substantially protecting against TNFα-mediated organ necrosis.

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

Moreover, the present invention provides a method for the treatment of disease using a polypeptide, dAb or antagonist of TNFRl or a composition according to the present invention. In an embodiment the disease is cancer or an inflammatory disease, eg rheumatoid arthritis, asthma or Crohn's disease. In one embodiment, the "TNFα-mediated condition" is a infarction, such that the purpose (eg, of the method or use) of the invention is for treating, suppressing or preventing infarction (eg, myocardial infarction) and substantially protecting against TNFα-mediated organ necrosis. Methods for pharmacokinetic analysis and determination of ligand (eg, single variable domain or antagonist) half-life will be familiar to those skilled in the art. Details may be found in Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and in Peters et al, Pharmacokinetc analysis: A Practical Approach (1996). Reference is also made to "Pharmacokinetics", M Gibaldi & D Perron, published by Marcel Dekker, 2^nd Rev. ex edition (1982), which describes pharmacokinetic parameters such as t alpha and t beta half lives and area under the curve (AUC). Optionally, all pharmacokinetic parameters and values quoted herein are to be read as being values in a human. Optionally, all pharmacokinetic parameters and values quoted herein are to be read as being values in a mouse. Half lives (t/4 alpha and Wi beta) and AUC can be determined from a curve of serum concentration of ligand against time. The WinNonlin analysis package, eg version 5.1 (available from Pharsight Corp., Mountain View, CA94040, USA) can be used, for example, to model the curve. When two-compartment modeling is used, in a first phase (the alpha phase) the ligand is undergoing mainly distribution in the patient, with some elimination. A second phase (beta phase) is the phase when the ligand has been distributed and the serum concentration is decreasing as the ligand is cleared from the patient. The t alpha half life is the half life of the first phase and the t beta half life is the half life of the second phase. Thus, in one embodiment, in the context of the present invention, the variable domain or antagonist has a tα half-life in the range of (or of about) 15 minutes or more. In one embodiment, the lower end of the range is (or is about) 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 11 hours or 12 hours. In addition, or alternatively, the variable domain or antagonist according to the invention will have a tα half life in the range of up to and including 12 hours (or about 12 hours). In one embodiment, the upper end of the range is (or is about) 11, 10, 9, 8, 7, 6 or 5 hours. An example of a suitable range is (or is about) 1 to 6 hours, 2 to 5 hours or 3 to 4 hours.

In one embodiment, the present invention provides the variable domain or antagonist according to the invention has a tβ half-life in the range of (or of about) 2.5 hours or more. In one embodiment, the lower end of the range is (or is about) 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours , 11 hours, or 12 hours. In addition, or alternatively, the tβ half-life is (or is about) up to and including 21 or 25 days. In one embodiment, the upper end of the range is (or is about) 12 hours, 24 hours, 2 days, 3 days, 5 days, 10 days, 15 days, 19 days 20 days, 21 days or 22 days. For example, the variable domain or antagonist according to the invention will have a tβ half life in the range 12 to 60 hours (or about 12 to 60 hours). In a further embodiment, it will be in the range 12 to 48 hours (or about 12 to 48 hours). In a further embodiment still, it will be in the range 12 to 26 hours (or about 12 to 26 hours).

As an alternative to using two-compartment modeling, the skilled person will be familiar with the use of non-compartmental modeling, which can be used to determine terminal half-lives (in this respect, the term "terminal half-life" as used herein means a terminal half-life determined using non-compartmental modeling). The WinNonlin analysis package, eg version 5.1 (available from Pharsight Corp., Mountain View, CA94040, USA) can be used, for example, to model the curve in this way. In this instance, in one embodiment the single variable domain or antagonist has a terminal half life of at least (or at least about) 8 hours, 10 hours, 12 hours, 15 hours, 28 hours, 20 hours, 1 day, 2 days, 3 days, 7 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days or 25 days. In one embodiment, the upper end of this range is (or is about) 24 hours, 48 hours, 60 hours or 72 hours or 120 hours. For example, the terminal half-life is (or is about) from 8 hours to 60 hours, or 8 hours to 48 hours or 12 to 120 hours, eg, in man.

In addition, or alternatively to the above criteria, the variable domain or antagonist according to the invention has an AUC value (area under the curve) in the range of (or of about) 1 mg.min/ml or more. In one embodiment, the lower end of the range is (or is about) 5, 10, 15, 20, 30, 100, 200 or 300 mg.min/ml. In addition, or alternatively, the variable domain or antagonist according to the invention has an AUC in the range of (or of about) up to 600 mg.min/ml. In one embodiment, the upper end of the range is (or is about) 500, 400, 300, 200, 150, 100, 75 or 50 mg.min/ml. Advantageously the variable domain or antagonist will have a AUC in (or about in) the range selected from the group consisting of the following: 15 to 150 mg.min/ml, 15 to 100 mg.min/ml, 15 to 75 mg.min/ml, and 15 to 50mg.min/ml.

One or more of the t alpha, t beta and terminal half- lives as well as the AUCs quoted herein can be obtained in a human and/or mouse by providing one or more anti- TNFRl single variable domains (or other binding moieties defined herein) linked to either a PEG or a single variable domain (or binding moiety) that specifically binds to serum albumin, eg mouse and/or human serum albumin (SA). The PEG size can be (or be about) at least 20 kDa, for example, 30, 40, 50, 60, 70 or 80 kDa. In one embodiment, the PEG is 40 kDa, eg 2x20kDa PEG. In one embodiment, to obtain a t alpha, t beta and terminal half-lives or an AUC quoted herein, there is provide an antagonist comprising an anti-TNFRl immunoglobulin single variable domain linked to an anti-SA immunoglobulin single variable domain. In one embodiment, the PEG is 40 kDa, eg 2x20kDa PEG. For example, the antagonist comprises only one such anti- TNFRl variable domains, for example one such domain linked to only one anti-SA variable domains. In one embodiment, to obtain a t alpha, t beta and terminal half-lives or a AUC quoted herein, there is provide an antagonist comprising an anti-TNFRl immunoglobulin single variable domain linked to PEG, eg, 40-80 kDa PEG, eg, 40 kDa PEG. For example, the antagonist comprises only one such anti-TNFRl variable domains, for example one such domain linked to 40 kDa PEG.

EXEMPLIFICATION

EXAMPLE: Effect of anti-TNFRl immunoglobulin single variable domain treatment on TNF in vivo toxicity Administration of mouse TNFα to mice results in systemic toxicity and mortality. Mouse TNFα binds both TNFRl and TNFR2 and inhibition of either TNFα or one of the receptors might have a beneficial effect in inhibiting the mortality induced by TNFα. Therefore, we set out to determine if inhibition of TNFα, by treating mice with murine TNFα -binding ENBREL™ or with a dAb specific for mouse TNFRl would be able to block the detrimental effects of TNFα in mice. To increase the sensitivity of the analysis we not only looked at mortality and animal well-being, but also more specifically at the level of organ necrosis which would be indicative of the protective effect of TNFRl inhibition on organ necrosis. For this study, we chose liver as a representative organ.

Description of protocol:

Six groups of 8 animals each received either saline (group 1), 10 mg/kg ENBREL™ (group 2), 10 mg/kg DOM lm-21 -23/DOM7m- 16 (group 3), 1 mg/kg DOM lm-21 - 23/DOM7m-16 (group 4), 10 mg/kg DOM lm-21-23/PEG (group 5) and 1 mg/kg DOMlm-21-23/PEG (group 6). Test compounds were administered twice, at 24 hours and 4 hours before intravenous challenge with mouse TNFα (10 μg per 25 grams body weight). Survival was monitored over a period of 48 hours. Serum and liver tissue were collected from animals that did not succumb to TNFα toxicity. The PEG used was 2x20 kDa PEG (Nektar Therapeutics, San Carlos, CA, USA).

Toxicity results:

All mice pretreated with either ENBREL™ or the anti-TNFRl dAb formats were protected from the deleterious effects of systemic TNFα administration, in contrast to the saline treated group where all mice died. Notably, all mice treated with ENBREL™ developed a clinical phenotype, malaise and rough fur, whereas mice treated with the anti-TNFRl dAbs remained clinically healthy throughout the protocol (figure 1). Histopatho logical analysis on liver sections:

The livers of the surviving mice were collected and analysed microscopically for the presence of necrotic cells. The level of necrosis was scored for each individual animal and the level of variability between the animals is indicated by the error-bars in Figure 2.

Mice receiving either of the two anti-TNFRl dAb formats and at each dose were significantly protected from the deleterious effect of TNF α toxicity on liver tissue and protected from necrosis as compared to the ENBREL™ treated group, where all mice developed liver necrosis .

The statistical analysis of the histopatho logical data demonstrated that the inhibitory effect of anti-TNFRl treatment was significant compared to ENBREL™ (p<0.001), whilst the differences between the different dAb formats, i.e. PEGylated or fused to a serum-albumin binding dAb, or dAb dose (1 vs 10 mg/kg) were not significant (p>0.05) (Table 1)

Table 1. Statistical analysis of the liver necrosis scores between the different treatment groups to determine significance of observed effects. The different treatment groups are: 2) ENBREL™10 mg/kg, 3) 10 mg/kg DOMlm-21-23-(G₄S)₃-DOM7m-16, 4) 1 mg/kg DOMlm-21-23-(G₄S)₃-DOM7rn-16, 5) 10 mg/kg DOMlm-21-23/(2x20K)PEG and 6) 1 mg/kg DOMlm-21-23/(2x20K)PEG. Standard p-test used.

Comparison groups Liver necrosis score

Group 2 vs Group 4 P<0.001

Group 2 vs Group 5 P<0.01 Group 2 vs Group 6 PO.001 Group 3 vs Group 4 P>0.05 Group 3 vs Group 5 P>0.05 Group 3 vs Group 6 P>0.05 Group 4 vs Group 5 P>0.05 Group 4 vs Group 6 P>0.05 Group 5 vs Group 6 P>0.05

The inventors believe that targeting of TNFRl with an immunoglobulin single variable domain (eg, a variable domain as disclosed herein or as provides as part of an antagonist disclosed herein) is beneficial when antagonizing TNFα-mediated diseases, eg to treat or prevent such diseases, since this can be achieved while protecting against organ necrosis.

Claims

1. An anti-TNFα receptor type 1 (TNFRl; p55) immunoglobulin single variable domain for treating and/or preventing a TNFα-mediated condition in a patient and substantially protecting against TNFα-mediated organ necrosis.

2. An anti-TNFα receptor type 1 (TNFRl; p55) immunoglobulin single variable domain for antagonizing TNFα in a patient and substantially protecting against TNFα-mediated organ necrosis.

3. The immunoglobulin single variable domain of claim 1 or 2, wherein the variable domain is a non-competitive inhibitor of TNFRl.

4. An anti-TNFα receptor type 1 (TNFRl; p55) antagonist comprising an immunoglobulin single variable domain according to any preceding claim.

5. The antagonist of claim 4, wherein the antagonist has a terminal half-life of at least 12 hours.

6. The antagonist of claim 5, wherein the immunoglobulin single variable domain is linked to a polyalkylene glycol moiety, optionally a polyethylene glycol moiety.

7. The antagonist of claim 5, wherein the antagonist is a multispecific ligand comprising an immunoglobulin single variable domain according to claim 1 or 2 and an immunoglobulin single variable domain that specifically binds serum albumin (SA).

8. Use of an anti-TNFα receptor type 1 (TNFRl; p55) immunoglobulin single variable domain in the manufacture of a medicament for treating and/or preventing a TNFα- mediated condition in a patient and substantially protecting against TNFα-mediated organ necrosis.

9. Use of an anti-TNFα receptor type 1 (TNFRl; p55) immunoglobulin single variable domain in the manufacture of a medicament for antagonising TNFα in a patient and substantially protecting against TNFα-mediated organ necrosis.

10. Use of an antagonist comprising an anti-TNFα receptor type 1 (TNFRl; p55) immunoglobulin single variable domain in the manufacture of a medicament for treating and/or preventing a TNFα-mediated condition in a patient and substantially protecting against TNFα-mediated organ necrosis, wherein the antagonist has a terminal half-life of at least 12 hours.

11. Use of an antagonist comprising an anti-TNFα receptor type 1 (TNFRl; p55) immunoglobulin single variable domain in the manufacture of a medicament for antagonising TNFα in a patient and substantially protecting against TNFα-mediated organ necrosis, wherein the antagonist has a terminal half-life of at least 12 hours.

12. Method of treating and/or preventing a TNFα-mediated condition in a patient and substantially protecting against TNFα-mediated organ necrosis, wherein the method comprises administering to the patient a pharmaceutically effective dose of an anti- TNFα receptor type 1 (TNFRl; p55) immunoglobulin single variable domain.

13. Method of antagonising TNFα in a patient and substantially protecting against TNFα-mediated organ necrosis, wherein the method comprises administering to the patient a pharmaceutically effective dose of an anti-TNFα receptor type 1 (TNFRl; p55) immunoglobulin single variable domain.

14. Method of treating and/or preventing a TNFα-mediated condition in a patient and substantially protecting against TNFα-mediated organ necrosis, wherein the method comprises administering to the patient a pharmaceutically effective dose of an antagonist comprising an anti-TNFα receptor type 1 (TNFRl; p55) immunoglobulin single variable domain, wherein the antagonist has a terminal half- life of at least 12 hours.

15. Method of antagonisting TNFα in a patient and substantially protecting against

TNFα-mediated organ necrosis, wherein the method comprises administering to the patient a pharmaceutically effective dose of an antagonist comprising an anti-TNFα receptor type 1 (TNFRl; p55) immunoglobulin single variable domain, wherein the antagonist has a terminal half-life of at least 12 hours.

16. The use of any one of claims 8 to 11, or the method of any one of claims 12 to 15, wherein the single variable domain is according to claim 3 or is provided as part of an antagonist according to any one of claims 4 to 7.

17. The variable domain, antagonist, use or method of any preceding claim, wherein the organ is selected from the group consisting of liver, kidney, heart, pancreas, lung and skin.