The present invention relates to antibody molecules having specificity for antigenic determinants of OX40 and compositions compπsing the same The present invention also relates to the therapeutic uses of the antibody molecules, compositions and methods for producing said antibody molecules

OX40 (also known as CDl 34, TNFRSF4, ACT35 or TXGPl L) is a member of the TNF receptor superfamily, which includes 4- IBB, CD27, CD30 and CD40 The extracellular hgand binding domain of OX40 is composed of 3 full cysteine-nch domains (CRDs) and a partial, fourth C-terminal CRD (Bodmer et al , 2002, Ti ends Biochem Sci, 27, 19-26)

The hgand for OX40 is OX40L and 3 copies of OX40 bind to the trimenc hgand to form the OX40-OX40L complex (Compaan and Hymowitz, 2006, Structure, 14, 1321-1330) OX40 is a membrane-bound receptor, however a soluble isoform has also been detected (Taylor and Schwarz, 2001 , J Immunol Methods, 255, 67-72) The functional significance of the soluble form is presently unknown OX40 is not expressed on resting T cells, but is transiently expressed on activated T cells after ligation of the T cell receptor (TCR) The hgand for OX40, OX40L, is a member of the TNF family and is expiessed on activated antigen presenting cells (APC), including B cells, macrophages, endothelial cells and dendritic cells (DC)

OX40 is a major costimulatory receptor with sequential engagement of CD28 and OX40 being required for optimal T cell proliferation and survival Ligation of OX40 on activated T cells leads to enhanced cytokine production and piohferation of both CD4+ and CD8+ T cells (Gramagha et al , 2000, J Immunol, 165, 3043-3050, Bansal-Pakala et al , 2004, J Immunol, 172, 4821-425) and can contribute to both ongoing ThI and Th2 responses (Gramagha et al , 1998, J Immuno , 161 , 6510-6517, Arestides e/ fl/ , 2002, Eui J Immunol 32, 2874-2880) OX40 costimulation piolongs T cell survival beyond the initial effector phase of the immune iesponse and increases the number of memory T cells through inhibition of effector T cell death

When immune activation is excessive or uncontrolled, pathological allergy, asthma, inflammation, autoimmune and othei related diseases may occur Because OX40 functions to enhance immune responses, it may exacerbate autoimmune and inflammatory diseases

The role of OX40/OX40L interactions in models of disease has been demonstrated in OX40 knockout mice In experimental allergic encephalomyelitis (EAE), a model of multiple sclerosis, less severe clinical signs of disease and reduced inflammatory infiltrate within the CNS was noted in OX40 knockout mice (Carboni et al , 2003, J Neuroimmunology, 145, 1-11) Also OX40 knockout mice pπmed and challenged with ovalbumin exhibit diminished lung inflammation (80 - 90% reduction in eosinophiha), reduced mucus production, and significantly attenuated airway hypei -reactivity (Jember et al , 2001 , J Exp Med , 193, 387- 392) Monoclonal antibodies to murine OX40 hgand have shown beneficial effects in the collagen-induced arthritis model of rheumatoid arthritis (Yoshioka et al , 2000, Eur J Immunol , 30, 2815-2823), EAE (Nohara e/ «/ , 2001 , J Immunol , 166, 2108-21 15), non-obese diabetic (NOD) mice (Pakala et al , 2004, Eur J Immunol , 34, 3039-3046), colitis in T cell restored mice (Malmstrom et al , 2001, J Immunol , 166, 6972-6981, Totsuka et al , 2003, Am J Physiol Gastrointest Liver Physiol , 284, G595-G603) and models of lung inflammation (Salek-Ardakaru e/ α/ , 2003, J Exp Med , 198, 315-324, Hoshino et al , 2003, Eur J Immunol, 33, 861-869) An antibody to human OX40L has been profiled in a model of lung inflammation in rhesus monkeys and resulted in reduced levels of IL-5, IL-13 and effector memory T cells in bronchiolar lavage fluid aftei allergen challenge (Seshasayee et al , 2007, J Clin Invest, 1 17, 3868-3878)

An increase in the expression of OX40 has been noted in several autoimmune and inflammatory diseases This includes an increase in OX40 expression on T cells isolated from the synovial fluid of rheumatoid arthritis patients (Brugnoni D et al , 1998, Br J Rheum , 37, 584-585, Yoshioka et al , 2000, Eur J Immunol , 30, 2815-2823, Giacomelh R et al , 2001, Clin Exp Rheumatol , 19, 317-320) Similarly an increase in OX40 expression has been noted in gastrointestinal tissue from patients with ulcerative colitis and Crohn's disease (Souza et al , 1999, Gut, 45, 856-863, Stuber et al , 2000, Eur J Clin Invest , 30, 594-599) and in active lesions of patients with multiple sclerosis (Carboni et al , 2003, J Neuroimmunology, 145, 1- 1 1 ) OX40L can also be detected on human airway smooth muscle (ASM) and asthma patients ASM cells show gi eater inflammatory responses to OX40L ligation than healthy donors, indicating a role for the OX40/OX40L pathway in asthma (Burgess et al , 2004, J Allergy Clin Immunol , 1 13, 683-689, Burgess et al , 2005, J Allergy Clin Immunol , 1 15, 302-308) It has also been reported that CD4+ T cells isolated from the penpheral blood of systemic lupus erythematosus (SLE) patients express elevated levels of OX40 which is associated with disease activity (Patschan et al , 2006, Clin Exp Immunol , 145, 235-242)

Given the role of OX40 in allergy, asthma and diseases associated with autoimmunity and inflammation, one approach to therapy in these diseases is to block OX40-OX40L signalling through the use of anti-OX40L antibodies or antagonistic anti-OX40 antibodies

Anti-OX40L antibodies have been descπbed, see for example WO2006/029879 Numerous agonistic anti-OX40 antibodies have been descπbed but very few antagonistic anti- OX40 antibodies are known A rabbit polyclonal anti-mouse OX40 antibody was produced by Stuber et al , 1996, J Exp Med, 183, 979-989 which blocks the interaction between OX40 and OX40L Mouse monoclonal antibodies, 131 and 315 which bind human OX40 were generated by Imura et al , 1996, J Exp Med, 2185-2195

Fully human antagonistic antibodies have been descπbed in WO2007/062245, the highest affinity of these antibodies had an affinity for cell surface expressed OX40 (activated T cells) of HnM Humanised antagonistic antibodies have been descnbed in WO2008/1061 16 and the antibody with the best affinity for OX40 had an affinity of 0 94nM

Other anti-OX40 antibodies have been descπbed, including muπne L 106 (US Patent numbei 6,277,962) and muπne ACT35, commercially available from eBioscience

Accordingly there is still a need in the art for an unproved anti-OX40 antibody suitable for ti eating patients We have now identified a high affinity antagonistic anti-OX40 antibody suitable for use in the treatment or prophylaxis of pathological disorders mediated by OX40 or associated with an increased level of OX40 Brief Description of the Drawings Figure 1 shows certain amino acid or DNA sequences relating to an antibody according to the disclosure Figure 2 shows a diagrammatic representation of an antibody of the A26 Fab'-PEG format

Figure 3 shows the cell-based affinity of the A26 Fab'-PEG antibody for cell surface OX40

Figure 4 shows percentage inhibition of OX40L binding to activated T cells by A26 Fab'-

PEG antibody Figure 5 shows peicentage inhibition of T cell proliferation by A26 Fab'-PEG antibody in the human MLR Figuie 6 shows A26 Fab'-PEG inhibition of proliferation of PBMC exposed to Tetanus

Toxoid Figuie 7 shows A26 Fab'-PEG peicentage inhibition of IL-13 production from PBMCs exposed to Dermatophagoides pteronyssinus allergenic extract

Figure 8 shows A26 Fab'-PEG peicentage inhibition of cytokine production from PBMCs exposed to Dermatophagoides pteronyssinus allergenic extract

Figure 9 shows A26 Fab'-PEG inhibits CD4+ and CD8+ T cell proliferation in a Hu-

SCID model Figure 10 shows inhibition of arthritis score (as area under the curve) by A26 Fab'-PEG in an in vivo model Figure 1 1 shows total histological scores in an in vivo model for arthritis

The onginal rat antibody from which the humanised antibodies are deπved is referred to herein as CA044_00026

Humanised CA044_00026 generally in the form of a Fab fragment or other fragments, is iefened to as A26 PEGylated antibody "A26" in the format shown in Figure 2, is referred to herein as

A26Fab'-PEG

The iesidues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al This system is set forth in Kabat et al , 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereafter "Kabat et al (supra)") This numbering system is used in the piesent specification except where otherwise indicated

The Kabat residue designations do not always correspond directly with the linear numbenng of the amino acid residues The actual linear amino acid sequence may contain fewei oi additional amino acids than in the strict Kabat numbenng corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic vaπable domain structure The correct Kabat numbering of iesidues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a "standard" Kabat numbered sequence

The CDRs of the heavy chain variable domain are located at residues 31-35 (CDR-Hl), residues 50-65 (CDR-H2) and residues 95-102 (CDR-H3) according to the Kabat numbering system However, according to Chothia (Chothia, C and Lesk, A M J MoI Biol , 196, 901- 917 ( 1987)), the loop equivalent to CDR-Hl extends from residue 26 to residue 32 Thus unless indicated otherwise 'CDR-Hl ' as employed herein is intended to refer to residues 26 to 35, as described by a combination of the Kabat numbering system and Chothia 's topological loop definition The CDRs of the light chain vaπable domain are located at residues 24-34 (CDR-Ll), residues 50-56 (CDR-L2) and residues 89-97 (CDR-L3) according to the Kabat numbering system

As used herein, the term 'antagonistic antibody' descπbes an antibody that is capable of inhibiting and/or neutralising the biological signalling activity of OX40, for example by blocking binding or substantially i educing bmding of OX40 to OX40 hgand and thus inhibiting the activation of OX40

Antibodies for use in the present invention may be obtained using any suitable method known in the art The OX40 polypeptide/protein including fusion pioteins, for example OX40- Fc fusions proteins or cells (lecombinantly or naturally) expressing the polypeptide (such as activated T cells) can be used to produce antibodies which specifically recognise OX40 The OX40 polypeptide may be the 'mature' polypeptide or a biologically active fragment or denvative thereof Suitably the OX40 polypeptide is the mature human polypeptide or the extracellular domain or fragment theieof The exhacellular domain typically compπses amino acids 29-214 of the OX40 protein (SWISS PROT entry P43489) OX40 polypeptides may be prepared by processes well known in the art from genetically engineered host cells comprising expression systems or they may be recovered from natural biological sources In the present application, the term "polypeptides" includes peptides, polypeptides and proteins These are used interchangeably unless otherwise specified The OX40 polypeptide may in some instances be part of a larger protein such as a fusion protein foi example fused to an affinity tag Antibodies generated against the OX40 polypeptide may be obtained, where immunisation of an animal is necessary, by administering the polypeptides to an animal, preferably a non-human animal, using well-known and routine protocols, see for example Handbook of Expeπmental Immunology, D M Weir (ed ), VoI 4, Blackwell Scientific Publishers, Oxford, England, 1986) Many warm-blooded animals, such as rabbits, mice, rats, sheep, cows, camels or pigs may be immunized However, mice, rabbits, pigs and rats are generally most suitable

Antibodies for use in the present invention include whole antibodies and functionally active fragments or derivatives thereof and may be, but are not limited to, monoclonal, humanised, fully human or chimeric antibodies

Monoclonal antibodies may be prepared by any method known in the art such as the hybπdoma technique (Kohler & Milstein, 1975, Nature, 256 495-497), the tπoma technique, the human B-cell hybπdoma technique (Kozbor et al , 1983, Immunology Today, 4 72) and the EBV-hybπdoma technique (Cole et al , Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan R Liss, Inc , 1985)

Antibodies for use in the invention may also be generated using single lymphocyte antibody methods by cloning and expressing immunoglobulin vaπable region cDNAs generated fiom single lymphocytes selected for the production of specific antibodies by, for example, the methods descnbed by Babcook, J et al , 1996, Proc Natl Acad Sci USA 93(15) 7843-78481,

WO92/02551, WO2004/051268 and International Patent Application number WO2004/106377

Screening for antibodies can be performed using assays to measure binding to human

OX40 and/or assays to measure the ability to block the binding of OX40 to its hgand, OX40L An example of a binding assay is an ELISA, in particular, using a fusion protein of human OX40 and human Fc, which is immobilized on plates, and employing a conjungated secondary antibody to detect anti-OX40 antibody bound to the fusion protein An example of a blocking assay is a flow cytometry based assay measuring the blocking of OX40 ligand fusion protein binding to OX40 on human CD4 cells A fluorescently labelled secondary antibody is used to detect the amount of OX40 ligand fusion protein binding to the cell This assay is looking for a reduction in signal as the antibody in the supernatant blocks the binding of ligand fusion protein to OX40 A further example of a blocking assay is an assay where the blocking of costimulation of naive human T cells mediated by OX40 ligand fusion protein coated to a plate is measured by measuπng tπtiated thymidine incorporation Humanised antibodies (which include CDR-grafted antibodies) are antibody molecules having one oi more complementarity determining regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule (see, e g US 5,585,089, WO91/09967) It will be appreciated that it may only be necessary to transfer the specificity determining residues of the CDRs rather than the entire CDR (see for example, Kashmiri et al , 2005, Methods, 36, 25-34) Humanised antibodies may optionally further comprise one or more framework residues derived from the non-human species from which the CDRs were derived

Chimeπc antibodies are composed of elements denved from two different species such that the elements retain the charactenstics of the species from which it is denved Generally a chimeric antibody will comprise a variable region from one species, for example a mouse, rat, iabbit or similar and constant region from another species such as a human

The antibodies for use in the present invention can also be generated using vaπous phage display methods known in the art and include those disclosed by Bπnkman et al (in J Immunol Methods, 1995, 182 41-50), Ames et al (J Immunol Methods, 1995, 184 177-186), Kettleborough et al (Eur J Immunol 1994, 24 952-958), Persic et al (Gene, 1997 187 9-18), Burton et al (Advances in Immunology, 1994, 57 191-280) and WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/1 1236, WO 95/15982, WO 95/20401 , and US 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969, 108 Fully human antibodies are those antibodies in which the vanable regions and the constant regions (where piesent) of both the heavy and the light chains are all of human oπgin, or substantially identical to sequences of human oπgin, not necessaπly from the same antibody Examples of fully human antibodies may include antibodies produced, for example by the phage display methods descπbed above and antibodies produced by mice in which the murine immunoglobulin vaπable and optionally the constant region genes have been replaced by their human counterparts eg as descπbed in general terms in EP0546073 Bl, US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,661,016, US5,770,429, EP 0438474 and EP0463151

In one embodiment the present invention provides an antagonistic antibody having specificity for human OX40, comprising a heavy chain, wherein the vanable domain of the heavy chain comprises at least one CDR having the sequence given in Figure 1 (c) SEQ ID NO 1 for CDR-Hl , a CDR having the sequence given in Figure l (c) SEQ ID NO 2 or SEQ ID NO 20 for CDR-H2 and a CDR having the sequence given in Figure l(c) SEQ ID NO 3 for CDR-H3

In another embodiment the present invention provides an antagonistic antibody having specificity foi human OX40, compnsing a heavy chain, wheiein at least two of CDR-Hl , CDR- H2 and CDR-H3 of the vanable domain of the heavy chain are selected from the following the sequence given in SEQ ID NO 1 for CDR-Hl , the sequence given in SEQ ID NO 2 for CDR- H2 and the sequence given in SEQ ID NO 3 for CDR-H3 For example, the antibody may comprise a heavy chain wherein CDR-Hl has the sequence given in SEQ ID NO 1 and CDR- H2 has the sequence given in SEQ ID NO 2 Alternatively, the antibody may comprise a heavy chain wheiein CDR-Hl has the sequence given in SEQ ID NO 1 and CDR-H3 has the sequence given in SEQ ID NO 3, or the antibody may comprise a heavy chain wherein CDR-H2 has the sequence given in SEQ ID NO 2 and CDR-H3 has the sequence given in SEQ ID NO 3 For the avoidance of doubt, it is understood that all permutations are included In another embodiment the present invention provides an antagonistic antibody having specificity for human OX40, compnsing a heavy chain, wherein the vanable domain of the heavy chain compnses the sequence given in SEQ ID NO 1 for CDR-Hl , the sequence given in SEQ ID NO 2 for CDR-H2 and the sequence given in SEQ ID NO 3 for CDR-H3

In another embodiment the present invention provides an antagonistic antibody having specificity foi human OX40, comprising a heavy chain, wherein the vanable domain of the heavy chain compnses the sequence given in SEQ ID NO 1 for CDR-H l , the sequence given in SEQ ID NO 20 for CDR-H2 and the sequence given in SEQ ID NO 3 for CDR-H3

In one embodiment the present invention provides an antagonistic antibody having specificity for human OX40, compnsing a light chain, wherein the vanable domain of the light chain compnses at least one CDR having the sequence given in Figure 1 (c) SEQ ID NO 4 or

SEQ ID NO 21 for CDR-Ll , a CDR having the sequence given in Figure 1 (c) SEQ ID NO 5 for CDR-L2 and a CDR having the sequence given in Figure 1 (c) SEQ ID NO 6 for CDR-L3

In another embodiment the present invention provides an antagonistic antibody having specificity for human OX40, compnsing a light chain, wherein at least two of CDR-Ll, CDR- L2 and CDR-L3 of the variable domain of the light chain are selected from the following the sequence given in SEQ ID NO 4 for CDR-Ll, the sequence given in SEQ ID NO 5 for CDR-L2 and the sequence given in SEQ ID NO 6 for CDR-L3 For example, the antibody may comprise a light chain wherein CDR-Ll has the sequence given in SEQ ID NO 4 and CDR-L2 has the sequence given in SEQ ID NO 5 Alternatively, the antibody may compnse a light chain wherein CDR-Ll has the sequence given in SEQ ID NO 4 and CDR-L3 has the sequence given in SEQ ID NO 6, or the antibody may compnse a light chain wheiein CDR-L2 has the sequence given in SEQ ID NO 5 and CDR-L3 has the sequence given in SEQ ID NO 6 Foi the avoidance of doubt, it is understood that all permutations are included

In another embodiment the present invention provides an antagonistic antibody having specificity for human OX40, comprising a light chain, wherein the vanable domain compπses the sequence given in SEQ ID NO 4 for CDR-Ll , the sequence given in SEQ ID NO 5 for CDR-L2 and the sequence given in SEQ ID NO 6 for CDR-L3

In another embodiment the present invention provides an antagonistic antibody having specificity for human OX40, composing a light chain, wherein the vanable domain compnses the sequence given in SEQ ID NO 21 for CDR-Ll, the sequence given in SEQ ID NO 5 for CDR-L2 and the sequence given in SEQ ID NO 6 for CDR-L3

The antibody molecules of the present invention suitably compnse a complementary light chain oi a complementary heavy chain, respectively

Hence in one embodiment, an antibody accoiding to the present invention compnses a heavy chain, wherein the vanable domain of the heavy chain compnses the sequence given in SEQ ID NO 1 for CDR-H 1 , the sequence given in SEQ ID NO 2 or SEQ ID NO 20 for CDR- H2 and the sequence given in SEQ ID NO 3 for CDR-H3 and a light chain wherein the variable domain of the light chain compnses the sequence given in SEQ ID NO 4 or SEQ ID NO 21 foi CDR-Ll, the sequence given in SEQ ID NO 5 for CDR-L2 and the sequence given in SEQ ID NO 6 foi CDR-L3 It will be appreciated that one or more amino acid substitutions, additions and/or deletions may be made to the CDRs provided by the present invention without significantly altering the ability of the antibody to bind to OX40 and to neutralise OX40 activity The effect of any amino acid substitutions, additions and/or deletions can be readily tested by one skilled in the art, for example by using the methods descnbed herein, in particular m the Examples, to determine OX40 binding and inhibition of the OX40/OX40L interaction Accordingly, the present invention provides an antibody having specificity for human OX40 compnsing one or more CDRs selected fiom CDRH-I (SEQ ID NO 1), CDRH-2 (SEQ ID NO 2 or SEQ ID NO 20), CDRH-3 (SEQ ID NO 3), CDRL-I (SEQ ID NO 4 or SEQ ID NO 21), CDRL-2 (SEQ ID NO 5) and CDRL-3 (SEQ ID NO 6) in which one or more amino acids in one or more of the CDRs has been substituted with another amino acid, suitably a similar ammo acid as defined herein below In one embodiment, the present invention provides an antibody having specificity for human OX40 compnsing CDRH-I (SEQ ID NO 1), CDRH-2 (SEQ ID NO 2 or SEQ ID NO 20), CDRH-3 (SEQ ID NO 3), CDRL-I (SEQ ID NO 4 or SEQ ID NO 21 ), CDRL-2 (SEQ ID NO 5) and CDRL-3 (SEQ ID NO 6) as shown in Figure l(c), for example in which one oi more amino acids in one or more of the CDRs has been substituted with another amino acid, such as a similar amino acid as defined herein below In one embodiment, an antibody of the present invention compnses a heavy chain, wherein the vaπable domain of the heavy chain compnses three CDRs wherein the sequence of CDRH-I has at least 60% identity or similarity to the sequence given in SEQ ID NO 1, CDRH- 2 has at least 60% identity or similarity to the sequence given in SEQ ID NO 2 and/or CDRH-3 has at least 60% identity or similarity to the sequence given in SEQ ID NO 3 In another embodiment, an antibody of the present invention compnses a heavy chain, wherein the vanable domain of the heavy chain compnses three CDRs wherein the sequence of CDRH-I has at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence given in SEQ ID NO 1, CDRH-2 has at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence given in SEQ ID NO 2 and/or CDRH-3 has at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence given in SEQ ID NO 3

"Identity", as used herein, indicates that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences "Similanty", as used herein, indicates that, at any particular position in the aligned sequences, the amino acid iesidue is of a similar type between the sequences For example, leucine may be substituted for isoleucine or valine Other amino acids which can often be substituted for one another include but are not limited to

- phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains),

- lysine, arginine and histidine (amino acids having basic side chains), - aspartate and glutamate (amino acids having acidic side chains),

- asparagine and glutamine (amino acids havmg amide side chains), and

- cysteine and methionine (amino acids having sulphur-containing side chains) Degrees of identity and similanty can be readily calculated (Computational Molecular Biology, Lesk,

A M , ed , Oxford University Press, New York, 1988, Biocomputing Informatics and Genome Projects, Smith, D W , ed , Academic Press, New York, 1993, Computer Analysis of Sequence Data, Part 1, Gnffin, A M , and Gnffin, H G , eds , Humana Press, New Jersey, 1994, Sequence Analysis in Molecular Biology, von Heinje, G , Academic Press, 1987, Sequence Analysis Primer, Gnbskov, M and Devereux, J , eds , M Stockton Press, New York, 1991 , the BLAST™ software available from NCBI (Altschul, S F et a! , 1990, J MoI Biol 215 403-410, Gish, W & States, D J 1993, Nature Genet 3 266-272 Madden, T L et al , 1996, Meth Enzymol 266 131 -141 , Altschul, S F et al , 1997, Nucleic Acids Res 25 3389-3402, Zhang, J & Madden, T L 1997, Genome Res 7 649-656,)

In another embodiment, an antibody of the present invention compnses a light chain, wherein the vanable domain of the light cham comprises three CDRs wherein the sequence of CDRL-I has at least 60% identity or similanty to the sequence given in SEQ ID NO 4, CDRL- 2 has at least 60% identity or similanty to the sequence given in SEQ ID NO 5 and/or CDRL-3 has at least 60% identity or similanty to the sequence given in SEQ ID NO 6 In another embodiment, an antibody of the present invention compnses a light chain, wherein the vanable domain of the light chain compnses three CDRs wherein the sequence of CDRL-I has at least 70%, 80%, 90%, 95% or 98% identity or similanty to the sequence given in SEQ ID NO 4, CDRL-2 has at least 70%, 80%, 90%, 95% or 98% identity or similanty to the sequence given in SEQ ID NO 5 and/or CDRL-3 has at least 70%, 80%, 90%, 95% oi 98% identity or similarity to the sequence given in SEQ ID NO 6

In one embodiment the antibody provided herein is a monoclonal antibody In one embodiment the antibody provided by herein is a chimeπc antibody In one embodiment the antibody provided by the present invention is a CDR-grafted antibody molecule composing one or more of the CDRs provided in SEQ ID NOs 1, 2, 3, 4, 5, 6, 20 and/or 21 (Figure 1 (c)) or variants thereof As used herein, the term 'CDR-grafted antibody molecule' iefers to an antibody molecule wherein the heavy and/or light chain contains one or more CDRs (including, if desired, one or more modified CDRs) from a donor antibody (e g a muπne monoclonal antibody) grafted into a heavy and/or light chain variable region framework of an acceptor antibody (e g a human antibody) For a review, see Vaughan et al, Nature Biotechnology, 16, 535-539, 1998 In one embodiment rather than the entire CDR being transferred, only one or more of the specificity determining residues from any one of the CDRs descπbed herein above are transferred to the human antibody framework (see for example, Kashmni et al , 2005, Methods, 36, 25-34) In one embodiment only the specificity determining residues from one or more of the CDRs descnbed herein above are transferred to the human antibody framewoik In another embodiment only the specificity determining residues from each of the CDRs described herein above are transferred to the human antibody framework When the CDRs or specificity determining residues are grafted, any appropπate acceptor vanable legion framework sequence may be used having regard to the class/type of the donor antibody from which the CDRs are deπved, including mouse, pπmate and human framework regions Suitably, the CDR-grafted antibody according to the present invention has a vanable domain comprising human acceptor framework regions as well as one or more of the CDRs or specificity determining residues descnbed above Thus, provided in one embodiment is a neutralising CDR-grafted antibody wherein the vanable domain compnses human acceptor framework regions and non-human donor CDRs

Examples of human frameworks which can be used in the present invention are KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Kabat et al , supra) For example, KOL and NEWM can be used for the heavy chain, REI can be used for the light chain and EU, LAY and POM can be used for both the heavy chain and the light chain Alternatively, human germline sequences may be used, these are available at http //vbase mrc-cpe cam ac uk/

In a CDR-grafted antibody of the present invention, the acceptor heavy and light chains do not necessanly need to be denved from the same antibody and may, if desired, compnse composite chains having framework regions denved from different chains

The suitable framework region for the heavy chain of the CDR-grafted antibody of the present invention is denved from the human sub-group VH3 sequence 1-3 3-07 together with JH4 Accoidingly, provided is a neutiahsing CDR-grafted antibody comprising at least one non-human donor CDR wherein the heavy chain framewoik region is denved from the human subgroup VH3 sequence 1-3 3-07 together with JH4 The sequence of human JH4 is as follows (YFDY)WGQGTLVTVSS (Seq ID No 22) The YFDY motif is part of CDR-H3 and is not part of framework 4 (Ravetch, JV et al , 1981, Cell, 27, 583-591)

The suitable framework region for the light chain of the CDR-grafted antibody of the present invention is deπved from the human germhne sub-group VKl sequence 2-1 1-02 togethei with JK4 Accordingly, piovided is a neutralising CDR-grafted antibody comprising at least one non-human donor CDR wherein the light chain framework region is deπved from the human subgroup sequence 2-1 1-02 together with JK4 The JKl sequence is as follows (WT)FGQGTKVEIK (Seq ID No 23) The WT motif is part of CDR-L3 and is not part of framework 4 (Hieter, PA , et al , 1982, J Biol Chem , 257, 1516-1522) Also, in a CDR-grafted antibody of the present invention, the framework regions need not have exactly the same sequence as those of the acceptor antibody For instance, unusual residues may be changed to more frequently-occurring residues for that acceptor chain class or type Alternatively, selected residues in the acceptor framework legions may be changed so that they correspond to the residue found at the same position in the donor antibody (see Reichmann et al , 1998, Nature, 332, 323-324) Such changes should be kept to the minimum necessary to recover the affinity of the donoi antibody A protocol for selecting residues in the acceptor framework regions which may need to be changed is set forth in WO 91/09967

Suitably, in a CDR-grafted antibody molecule of the present invention, if the acceptor heavy chain has the human VH3 sequence 1-3 3-07 together with JH4, then the acceptor framework regions of the heavy chain comprise, in addition to one or more donor CDRs, a donor residue at at least one of positions 37, 73, 78 or 94 (according to Kabat et al , (supra)) Accordingly, provided is a CDR-grafted antibody, wherein at least the residues at positions 37, 73, 78 and 94 of the variable domain of the heavy chain are donor residues

Suitably, in a CDR-grafted antibody molecule according to the present invention, if the acceptor light chain has the human sub-group VKl sequence 2-1 1-02 together with JK4, then the acceptor framework regions of the light chain compπse, in addition to one or more donor CDRs, a donor residue at at least one of positions 64 or 71 Accordingly, provided is a CDR- grafted antibody, wherein at least the iesidues at positions 64 and 71 of the vaπable domain of the light chain are donor residues Donoi residues are residues from the donor antibody, i e the antibody from which the

CDRs were originally deπved

In one embodiment, an antibody of the present invention compπses a heavy chain, wherein the vanable domain of the heavy chain compπses the sequence given in Figure 1 (b) SEQ ID NO 9 It will be appreciated that one or more amino acid substitutions, additions and/or deletions may be made to the antibody vanable domains, provided by the present invention, without significantly alteπng the ability of the antibody to bind to OX40 and to neutralise OX40 activity The effect of any amino acid substitutions, additions and/or deletions can be readily tested by one skilled in the art, for example by using the methods descnbed herein, in particular the Examples, to deteπnine OX40 binding and hgand blocking In another embodiment, an antibody of the present invention comprises a heavy chain, wherein the variable domain of the heavy chain compnses a sequence having at least 60% identity or similarity to the sequence given in SEQ ID NO 9 In one embodiment, an antibody of the present invention compnses a heavy chain, wherein the vaπable domain of the heavy chain comprises a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence given in SEQ ID NO 9

In one embodiment, an antibody of the present invention composes a light chain, wherein the vanable domain of the light chain comprises the sequence given in Figure 1 (a) SEQ ID NO 7 In another embodiment, an antibody of the present invention compnses a light chain, wherein the variable domain of the light chain compnses a sequence having at least 60% identity or similanty to the sequence given in SEQ ID NO 7 In one embodiment the antibody of the present invention compnses a light chain, wheiein the vanable domain of the light chain comprises a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similanty to the sequence given in SEQ ID NO 7

In one embodiment an antibody of the present invention compnses a heavy chain, wherein the vanable domain of the heavy chain compnses the sequence given in SEQ ID NO 9 and a light chain, wherein the vanable domain of the light chain compnses the sequence given in SEQ ID NO 7 In another embodiment of the invention, the antibody compnses a heavy chain and a light chain, wherein the vanable domain of the heavy chain compnses a sequence having at least 60% identity or similanty to the sequence given in SEQ ID NO 9 and the vanable domain of the light chain compnses a sequence having at least 60% identity or similanty to the sequence given in SEQ ID NO 7 Suitably, the antibody comprises a heavy chain, wherein the vanable domain of the heavy chain compnses a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence given in SEQ ID NO 9 and a light chain, wherein the vanable domain of the light chain compnses a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similanty to the sequence given in SEQ ID NO 7

The antibody molecules of the present invention may compnse a complete antibody molecule having full length heavy and light chains or a fragment thereof and may be, but are not limited to Fab, modified Fab, Fab', modified Fab', F(ab') ₂ , Fv, single domain antibodies (e g VH or VL or VHH), scFv, bi, tn or tetra-valent antibodies, Bis-scFv, diabodies, tnabodies, tetrabodies and epitope-binding fragments of any of the above (see for example Holliger and Hudson, 2005, Nature Biotech 23(9) 1 126-1 136, Adair and Lawson, 2005, Drug Design Reviews - Online 2(3), 209-217) The methods for creating and manufactunng these antibody fragments are well known in the art (see for example Verma et al , 1998, Journal of Immunological Methods, 216, 165-181) Other antibody fragments for use in the present invention include the Fab and Fab' fragments descnbed in International patent applications WO2005/003169, WO2005/003170 and WO2005/003171 Multi-valent antibodies may compnse multiple specificities or may be monospecific (see for example WO 92/22853 and WO05/113605) In one embodiment the antibody according to the present disclosure is provided as an OX40 binding antibody fusion protein which comprises an immunoglobulin moiety, for example a Fab or Fab' fragment, and one or two single domain antibodies (dAb) linked directly or indirectly thereto, for example as descnbed in WO2009/040562 In one embodiment the fusion protein comprises two domain antibodies, for example as a vaπable heavy (VH) and variable light (VL) paiπng, optionally linked by a disulphide bond

In one embodiment the Fab of Fab' element of the fusion protein has the same or similar specificity to the single domain antibody or antibodies In one embodiment the Fab or Fab' has a different specificity to the single domain antibody or antibodies, that is to say the fusion protein is multivalent In one embodiment a multivalent fusion protein according to the present invention has an albumin binding site, for example a VH/VL pair therein provides an albumin binding site

The constant region domains of the antibody molecule of the piesent invention, if present, may be selected having regard to the proposed function of the antibody molecule, and in particular the effector functions which may be required For example, the constant region domains may be human IgA, IgD, IgE, IgG or IgM domains In particular, human IgG constant region domains may be used, especially of the IgGl and IgG3 isotypes when the antibody molecule is intended for therapeutic uses and antibody effector functions are required Alternatively, IgG2 and IgG4 isotypes may be used when the antibody molecule is intended foi therapeutic purposes and antibody effector functions are not required, e g for simply blocking OX40 activity It will be appreciated that sequence vanants of these constant region domains may also be used For example IgG4 molecules in which the seπne at position 241 has been changed to proline as descnbed in Angal et al , Molecular Immunology, 1993, 30 (1), 105-108 may be used It will also be understood by one skilled in the art that antibodies may undergo a vaπety of posttranslational modifications The type and extent of these modifications often depends on the host cell line used to express the antibody as well as the culture conditions Such modifications may include vaπations in glycosylation, methionine oxidation, diketopiperazine formation, aspartate lsomenzation and asparagine deamidation A frequent modification is the loss of a carboxy-terminal basic residue (such as lysine or arginine) due to the action of carboxypeptidases (as descnbed in Hams, RJ Journal of Chromatography

705 129-134, 1995) Accordingly, the C-terminal lysine of the antibody heavy chain given in Figure 1 (f), SEQ ID NO 15 may be absent

In one embodiment the antibody heavy chain compnses a CHl domain and the antibody light chain compnses a CL domain, either kappa or lambda In one embodiment the antibody provided by the piesent invention is an antagonistic antibody having specificity for human OX40 in which the heavy chain constant region compnses a modified hinge region Accordingly, the present invention provides an antibody in which the heavy chain comprises or consists of the sequence given in Figure 1 (f), SEQ ID NO 15 It will be appieciated that one or more amino acid substitutions, additions and/or deletions may be made to the antibody vanable and/or constant domains provided by the present invention without significantly altenng the ability of the antibody to bind to OX40 and to neutralise OX40 activity The effect of any amino acid substitutions, additions and/or deletions can be ieadily tested by one skilled in the art, for example by using the methods described herein, in particular in the Examples, to determine OX40 binding and blocking of the OX40/OX40L interaction

In one embodiment of the invention, the antibody comprises a heavy chain, wherein the heavy chain compnses a sequence having at least 60% identity or similarity to the sequence given in SEQ ID NO 15 Suitably, the antibody compnses a heavy chain, wherein the heavy chain compnses a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similanty to the sequence given in SEQ ID NO 15

In one embodiment an antibody molecule according to the present invention compnses a light chain compnsing the sequence given in Figure 1 (d), SEQ ID NO 1 1

In one embodiment of the invention, the antibody compnses a light chain, wherein the light chain compnses a sequence having at least 60% identity or similanty to the sequence given in SEQ ID NO 1 1 For example, the antibody compnses a light chain, wherein the light chain compnses a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence given in SEQ ID NO 1 1

In one embodiment the present invention provides an antibody in which the heavy chain comprises or consists of the sequence given in SEQ ID NO 15 and the light chain comprises oi consists of the sequence given in SEQ ID NO 1 1

In one embodiment of the invention, the antibody compnses a heavy chain and a light chain, wherein the heavy chain comprises a sequence having at least 60% identity or similanty to the sequence given in SEQ ID NO 15 and the light chain compnses a sequence having at least 60% identity or similarity to the sequence given in SEQ ID NO 1 1 Generally, the antibody comprises a heavy chain, wheiem the heavy chain compnses a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similanty to the sequence given in SEQ ID NO 15 and a light chain, wherein the light chain compnses a sequence having at least 70%, 80%, 90%, 95% or 98% identity or similanty to the sequence given in SEQ ID NO 1 1

Biological molecules, such as antibodies or fragments, contain acidic and/or basic functional groups, thereby giving the molecule a net positive or negative charge The amount of overall "observed" charge will depend on the absolute amino acid sequence of the entity, the local environment of the charged groups in the 3D structure and the environmental conditions of the molecule The isoelectric point (pi) is the pH at which a particular molecule or solvent accessible surface thereof carries no net electncal charge In one embodiment the antibody or fiagment according to the present disclosure has an isoelectnc point (pi) of at least 7 In one embodiment the antibody or fragment has an isoelectnc point of at least 8, such as 8 5, 8 6, 8 7, 8 8 or 9

The OX40 antibody and fragments of the invention have been engineered to have an appiopnate isoelectnc point This may lead to antibodies and/or fragments with more robust properties, in particular suitable solubility and/or stability profiles and/or improved punfication charactenstics Thus in one aspect the invention provides a humanised OX40 antibody engineered to have an isoelectric point different to that of the originally identified antibody C A044__00026 The antibody may, for example be engineered by replacing an amino acid residue such as replacing an acidic ammo acid residue with one or more basic amino acid residues Alternatively, basic amino acid residues may be introduced or acidic amino acid residues can be removed Alternatively, if the molecule has an unacceptably high pi value acidic residues may be introduced to lower the pi, as required The target pi of the engineeied antibody or fragment desirably may, for example be 8 or above, such 8 5 or 9 It is important that when manipulating the pi care must be taken to retain the desirable activity of the antibody or fragment Thus in one embodiment the engineered antibody or fragment has the same or substantially the same activity as the "unmodified" antibody or fragment

Programs such as ** ExPASY http //www expasy ch/tools/pi tool html, and http //www lut-arles up univ-mrs fr/w3bb/d abim/compo-p html, may be used to predict the isoelectric point of the antibody or fragment Also provided by the present invention is a specific region or epitope of human OX40 which is bound by an antibody provided by the present invention, m particular an antibody compnsing the heavy chain sequence gH2 (SEQ ID NO 9) and/or the light chain sequence gL8 (SEQ ID NO 7)

This specific region or epitope of the human OX40 polypeptide can be identified by any suitable epitope mapping method known in the art in combmation with any one of the antibodies provided by the present mvention Examples of such methods mclude screening peptides of varying lengths derived from OX40 for binding to the antibody of the present invention with the smallest fiagment that can specifically bind to the antibody containing the sequence of the epitope recognised by the antibody The OX40 peptides may be produced synthetically or by proteolytic digestion of the OX40 polypeptide Peptides that bind the antibody can be identified by, for example, mass spectrometπc analysis In another example, NMR spectroscopy or X-ray crystallography can be used to identify the epitope bound by an antibody of the present invention Once identified, the epitopic fragment which binds an antibody of the present mvention can be used, if required, as an immunogen to obtain additional antagonistic antibodies which bind the same epitope

Antibodies which cross-block the binding of an antibody according to the present invention in particular an antibody comprising the heavy chain sequence gH2 (SEQ ID NO 9) and the light chain sequence gL8 (SEQ ID NO 7) may be similarly useful in antagomsmg OX40 activity Accordmgly, the present mvention also provides an antagonistic antibody having specificity for human OX40, which cross-blocks the binding of any one of the antibodies descπbed above to human OX40 and/or is cross-blocked from binding OX40 by any one of those antibodies In one embodiment, such an antibody binds to the same epitope as an antibody descπbed herein above In another embodiment the cioss-blocking neutralising antibody binds to an epitope which borders and/or overlaps with the epitope bound by an antibody descπbed herein above In another embodiment the cross-blocking neutralising antibody of this aspect of the invention does not bind to the same epitope as an antibody of the present invention or an epitope that borders and/or overlaps with said epitope

Cross-blocking antibodies can be identified using any suitable method in the art, for example by using competition ELISA or BIAcore assays where binding of the cross blocking antibody to human OX40 prevents the binding of an antibody of the present invention or vice versa

In one embodiment there is provided an antagonistic antibody having specificity for human OX40, which cross-blocks the binding of an antibody whose heavy chain compnses the sequence gH2 (SEQ ID NO 9) and whose light chavn compnses the sequence gL8 (SEQ ID NO 7) to human OX40 In one embodiment the cross-blocking antibodies provided by the present invention inhibit the binding of an antibody comprising the heavy chain sequence gH2 (SEQ ID NO 9) and the light chain sequence gL8 (SEQ ID NO 7) by greater than 80%, for example by greatei than 85%, such as by greater than 90%, in particular by greatei than 95% Alternatively or in addition, antagonistic antibodies according to this aspect of the invention may be cross-blocked fiom binding to human OX40 by an antibody comprising the heavy chain sequence gH2 (SEQ ID NO 9) and the light chain sequence gL8 (SEQ ID NO 7) Also provided therefore is an antagonistic antibody molecule having specificity for human OX40 which is cross-blocked fiom binding human OX40 by an antibody comprising the heavy chain sequence gH2 (SEQ ID NO 9) and the light chain sequence gL8 (SEQ ID NO 7) In one embodiment the antagonistic antibodies provided by this aspect of the invention are inhibited from binding human OX40 by an antibody comprising the heavy cham sequence gH2 (SEQ ID NO 9) and the light chain sequence gL8 (SEQ ID NO 7) by greater than 80%, for example by greater than 85%, such as by greater than 90%, in particular by greater than 95%

In one embodiment the cross-blocking antibodies provided by the present invention are fully human In one embodiment the cross-blocking antibodies provided by the present invention are humanised In one embodiment the cross-blocking antibodies provided by the present invention have an affinity for human OX40 of lOOpM or better In one embodiment the cross-blocking antibodies provided by the present invention have an affinity for human OX40 of 5OpM or better In one embodiment the cross-blocking antibody has an isoelectric point of at least 7, for example at least 8, such as 8 5, 8 6, 8 7, 8 8, 8 9 or 9 0

The antibody molecules of the present invention suitably have a high binding affinity, in particular picomolar Affinity may be measured using any suitable method known in the art, including BIAcore, as descnbed in the Examples herein, using isolated natural or recombinant OX40 or a suitable fusion protein/polypeptide In one example affinity is measured using recombinant human OX40 extracellular domain as described in the Examples herein In one example the recombinant human OX40 extracellular domain used is a dimer, for example an Fc fusion dimer Suitably the antibody molecules of the present invention have a binding affinity for isolated human OX40 of about 20OpM or better In one embodiment the antibody molecule of the piesent invention has a binding affinity of about 100 pM or better In one embodiment the antibody molecule of the present invention has a binding affinity of about 5OpM or better In one embodiment the antibody molecule of the present invention has a binding affinity of about 4OpM or better In one embodiment the antibody molecule of the present invention has a binding affinity of about 3OpM or better In one embodiment the antibody molecule of the present invention is fully human or humanised and has a binding affinity of about lOOpM or bettei

The antibody molecules of the present invention suitably have a high binding affinity for human OX40 expressed on the surface of activated T cells, for example nanomolar or picomolar affinity Affinity may be measured usmg any suitable method known in the art, including the method as described in the Examples herein using activated CD4 OX40 ⁺ human T cells In particular the antibody molecules of the present invention have a binding affinity for cell surface expressed human OX40 of about 2nM or better In one example the antibody molecules of the present invention have a binding affinity for cell surface expressed human OX40 of about 1 5 nM or better In another example the antibody molecules of the present invention have a binding affinity for cell surface expressed human OX40 of about 1 2 nM or better In one embodiment there is provided a fully human or humamsed antibody molecule which has a binding affinity of about 2nM or better for human cell surface expressed OX40 It will be appreciated that the affinity of antibodies provided by the present invention may be altered using any suitable method known in the art The present invention therefore also relates to variants of the antibody molecules of the present invention, which have an improved affinity for OX40 Such variants can be obtained by a number of affinity maturation protocols including mutating the CDRs (Yang et al , J MoI Biol , 254, 392-403, 1995), chain shuffling (Marks et al , Bio/Technology, K), 779-783, 1992), use of mutator strains of E coli (Low et al , J MoI Biol , 250, 359-368, 1996), DNA shuffling (Patten et al , Curr Opin Biotechnol , 8, 724-733, 1997), phage display (Thompson et al , J MoI Biol , 256, 77-88, 1996) and sexual PCR (Crameπ et al , Nature, 391, 288-291, 1998) Vaughan et al {supra) discusses these methods of affinity maturation

In one embodiment the antibody molecules of the present invention block the interaction between OX40 and OX40L Numerous assays suitable for determining the ability of an antibody to block this interaction are descπbed in the examples herein In one embodiment the present invention provides a neutralising antibody having specificity for human OX40 which is capable of inhibiting the binding of human OX40L (tested at a final concentiation of 2μg/ml) to activated human CD4+OX40+ T cells by 50% at a concentration of less than 5nM In one embodiment the human OX40L used in the assay is natural human OX40 In one embodiment the human OX40 used in the assay is recombinant human OX40 In one embodiment the neutralising antibody is a humanised oi fully human antibody

If desired an antibody for use in the present invention may be conjugated to one or more effector molecule(s) It will be appreciated that the effector molecule may comprise a single effector molecule or two or more such molecules so linked as to form a single moiety that can be attached to the antibodies of the piesent invention Where it is desired to obtain an antibody fragment linked to an effector molecule, this may be prepared by standard chemical or recombinant DNA procedures in which the antibody fragment is linked either directly or via a coupling agent to the effector molecule Techniques for conjugating such effector molecules to antibodies are well known in the art (see, Hellstrom et al , Controlled Drug Delivery, 2nd Ed , Robinson et al , eds , 1987, pp 623-53, Thorpe et al , 1982 , Immunol Rev , 62 119-58 and Dubowchik et al , 1999, Pharmacology and Therapeutics, 83, 67-123) Particular chemical procedures include, for example, those descπbed in WO 93/06231, WO 92/22583, WO 89/00195, WO 89/01476 and WO03031581 Alternatively, where the effector molecule is a protein oi polypeptide the linkage may be achieved using recombinant DNA procedures, for example as descπbed in WO 86/01533 and EP0392745

The term effector molecule as used herein includes, for example, antineoplastic agents, drugs, toxins, biologically active proteins, for example enzymes, other antibody oi antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof e g DNA, RNA and fragments thereof, radionuclides, particularly radioiodide, radioisotopes, chelated metals, nanoparticles and reporter groups such as fluorescent compounds or compounds which may be detected by NMR or ESR spectroscopy Examples of effector molecules may include cytotoxins or cytotoxic agents including any agent that is detπmental to (e g kills) cells Examples include combrestatins, dolastatins, epothilones, staurospoπn, maytansinoids, spongistatins, rhizoxin, hahchondπns, rondins, hemiasterhns, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof

Effector molecules also include, but are not limited to, antimetabolites (e g methotrexate, 6-mercaptopuπne, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e g mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracychnes (e g daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e g dactinomycin (formerly actinomycin), bleomycin, mithramycin, anthramycin (AMC), calicheamicins or duocarmycins), and anti-mitotic agents (e g vincristine and vinblastine)

Other effector molecules may include chelated radionuclides such as " ¹ In and ⁹⁰ Y, Lu ¹⁷⁷ , Bismuth ²¹³ , Californium ²⁵² , Indium ¹⁹² and Tungsten ¹⁸⁸ /Rhenium ¹⁸⁸ , or drugs such as but not limited to, alkylphosphocholines, topoisomerase I inhibitors, taxoids and suramin Other effector molecules include pioteins, peptides and enzymes Enzymes of interest include, but are not limited to, proteolytic enzymes, hydrolases, lyases, isomerases, transferases

Proteins, polypeptides and peptides of interest include, but are not limited to, immunoglobulins, toxins such as abπn, πcin A, pseudomonas exotoxin, or diphtheria toxin, a protein such as insulin, tumour necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet derived growth factoi or tissue plasminogen activatoi, a thrombotic agent or an anti-angiogenic agent, e g angiostatin or endostatin, or, a biological response modifier such as a lymphokine, interleukin-1 (IL-I ), interleukin-2 (IL-2), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), nerve growth factor (NGF) or other giowth factoi and immunoglobulins

Other effector molecules may include detectable substances useful for example in diagnosis Examples of detectable substances include vaπous enzymes, prosthetic groups, fluorescent materials, luminescent mateπals, bioluminescent materials, radioactive nuclides, positron emitting metals (for use in positron emission tomography), and nonradioactive paramagnetic metal ions See generally U S Patent No 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase, suitable prosthetic groups include streptavidin, avidin and biotin, suitable fluorescent mateπals include umbelhferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotπazinylamine fluorescein, dansyl chloπde and phycoerythnn, suitable luminescent materials include luminol, suitable bioluminescent mateπals include luciferase, lucifenn, and aequoπn, and suitable radioactive nuclides include ¹²⁵ I, ¹³¹ I, In and Tc In another example the effector molecule may increase the half-life of the antibody in vivo, and/or reduce immunogenicity of the antibody and/or enhance the delivery of an antibody across an epithelial barrier to the immune system Examples of suitable effector molecules of this type include polymers, albumin, albumin binding proteins oi albumin binding compounds such as those descπbed in WO05/117984 Wheie the effector molecule is a polymer it may, in general, be a synthetic or a natuially occurring polymer, foi example an optionally substituted straight or branched chain polyalkylene, polyalkenylene or polyoxyalkylene polymer or a branched or unbranched polysacchaπde, e g a homo- or hetero- polysacchaπde

Specific optional substituents which may be present on the above-mentioned synthetic polymers include one or more hydroxy, methyl or methoxy groups

Specific examples of synthetic polymers include optionally substituted straight or branched chain poly(ethyleneglycol), poly(propyleneglycol) poly(vinylalcohol) or deπvatives thereof, especially optionally substituted poly(ethyleneglycol) such as methoxypoly(ethyleneglycol) or derivatives thereof Specific natuially occurπng polymers include lactose, amylose, dextran, glycogen or derivatives thereof

"Derivatives" as used herein is intended to include reactive deπvatives, for example thiol-selective reactive groups such as maleimides and the like The reactive group may be linked directly or through a linker segment to the polymer It will be appreciated that the iesidue of such a group will in some instances form part of the product as the linking group between the antibody fragment and the polymer

The size of the polymer may be vaπed as desired, but will generally be in an average molecular weight range from 500Da to 50000Da, for example from 5000 to 40000Da such as from 20000 to 40000Da The polymer size may in particular be selected on the basis of the intended use of the pioduct for example ability to localize to certain tissues such as tumors or extend circulating half-life (for review see Chapman, 2002, Advanced Drug Delivery Reviews, 54, 531-545) Thus, for example, where the product is intended to leave the circulation and penetrate tissue, for example for use in the treatment of a tumour, it may be advantageous to use a small molecular weight polymer, for example with a molecular weight of around 5000Da For applications where the product remains in the circulation, it may be advantageous to use a highei molecular weight polymer, for example having a molecular weight in the range from 20000Da to 40000Da

Suitable polymers include a polyalkylene polymer, such as a poly(ethyleneglycol) or, especially, a methoxypoly(ethyleneglycol) or a denvative thereof, and especially with a molecular weight in the range from about 15000Da to about 40000Da In one example antibodies for use in the piesent invention are attached to poly(ethyleneglycol) (PEG) moieties In one particular example the antibody is an antibody fragment and the PEG molecules may be attached through any available amino acid side-chain or terminal amino acid functional group located in the antibody fragment, for example any fiee amino, lnnno, thiol, hydroxyl or carboxyl group Such amino acids may occur naturally in the antibody fragment or may be engineered into the fragment using recombinant DNA methods (see for example US 5,219,996, US 5,667,425, WO98/25971) In one example the antibody molecule of the present invention is a modified Fab fragment wherein the modification is the addition to the C-terminal end of its heavy chain one or more amino acids to allow the attachment of an effector molecule Suitably, the additional amino acids form a modified hinge region containing one or more cysteine residues to which the effector molecule may be attached Multiple sites can be used to attach two or more PEG molecules

Suitably PEG molecules are covalently linked through a thiol group of at least one cysteine residue located in the antibody fragment Each polymer molecule attached to the modified antibody fragment may be covalently linked to the sulphur atom of a cysteine residue located in the fragment The covalent linkage will generally be a disulphide bond or, in particular, a sulphur-carbon bond Where a thiol group is used as the point of attachment appropπately activated effector molecules, for example thiol selective deπvatives such as maleimides and cysteine denvatives may be used An activated polymer may be used as the starting matenal in the preparation of polymer-modified antibody fragments as descnbed above The activated polymer may be any polymer containing a thiol reactive group such as an α- halocarboxyhc acid or ester, e g iodoacetamide, an lmide, e g maleimide, a vinyl sulphone or a disulphide Such starting materials may be obtained commercially (for example from Nektar, formerly Shearwater Polymers Inc , Huntsville, AL, USA) or may be prepared from commercially available starting materials using conventional chemical procedures Particulai PEG molecules include 2OK methoxy-PEG-amine (obtainable from Nektar, formerly

Shearwater, Rapp Polymere, and SunBio) and M-PEG-SPA (obtainable from Nektar, formerly Shearwater)

In one embodiment, the antibody is a modified Fab fragment or diFab which is PEGylated, i e has PEG (poly(ethyleneglycol)) covalently attached thereto, e g according to the method disclosed in EP 0948544 or EP 1090037 [see also "Poly(ethyleneglycol) Chemistry, Biotechnical and Biomedical Applications", 1992, J Milton Hams (ed), Plenum Press, New York, "Poly(ethyleneglycol) Chemistry and Biological Applications", 1997, J Milton Harris and S Zahpsky (eds), American Chemical Society, Washington DC and "Bioconjugation Protein Coupling Techniques for the Biomedical Sciences", 1998, M Aslam and A Dent, Grove Publishers, New York, Chapman, A 2002, Advanced Drug Delivery Reviews 2002, 54 531 -545] In one example PEG is attached to a cysteine in the hinge region In one example, a PEG modified Fab fragment has a maleimide group covalently linked to a single thiol group in a modified hinge region A lysine residue may be covalently linked to the maleimide group and to each of the amine groups on the lysine residue may be attached a methoxypoly(ethyleneglycol) polymer having a molecular weight of approximately 20,000Da The total molecular weight of the PEG attached to the Fab fragment may therefore be approximately 40,000Da

In one embodiment, the present invention provides an antagonistic antibody molecule having specificity for human OX40, which is a modified Fab fragment having a heavy chain compπsing the sequence given in SEQ ID NO 9 and a light chain compnsing the sequence given in SEQ ID NO 7 and having at the C-terminal end of its heavy chain a modified hinge region contaimng at least one cysteine residue to which an effector molecule is attached Suitably the effector molecule is PEG and is attached using the methods descnbed in (WO98/25971 and WO20040721 16 or in WO2007/003898) Suitably the effector molecule is attached in such as way that a lysyl-maleimide group is attached to the cysteine residue at the C-terminal end of the heavy chain, and each amino group of the lysyl residue has covalently linked to it a methoxypoly(ethyleneglycol) residue having a molecular weight of about 20,000 Da The total molecular weight of the PEG attached to the antibody is therefore appioximately 40,000Da Particular PEG molecules include 2-[3-(N-rnaleimido)propionamido]ethyl amide of N,N'-bis(methoxypoly(ethylene glycol) MW 20,000) modified lysine, also known as PEG2MAL40K (obtainable from Nektar, formerly Shearwater)

Alternative sources of PEG linkeis include NOF who supply GL2-400MA2 (wherein m in the structure below is 5) and GL2-400MA (where m is 2) and n is approximately 450

That is to say each PEG is about 20,000Da Further alternative PEG effector molecules of the following type are available from Dr Reddy, NOF and Jenkem

In one embodiment there is provided an antibody which is PEGylated (for example with a PEG described herein), attached through a cysteme amino acid residue at or about amino acid 226 in the chain, for example amino acid 226 of the heavy chain (by sequential numbering) In one embodiment, the present invention provides an antagonistic antibody molecule having specificity foi human OX40, which is a modified Fab fragment having a heavy chain compnsing or consisting of the sequence given in SEQ ID NO 15 and a light chain compπsing or consisting of the sequence given in SEQ ID NO 1 1 and having an effector molecule attached to the cysteine at position 226 of the heavy chain (linear numbering from SEQ ID NO 15) Suitably the effector molecule is PEG and is attached using the methods descπbed in (WO98/25971 and WO20040721 16 or WO2007/003898) and a lysyl-maleimide group is attached to the cysteine residue at position 226 of the heavy chain (SEQ ID NO 15), and each amino group of the lysyl residue has covalently linked to it a methoxypoly(ethyleneglycol) residue having a molecular weight of about 20,000Da The total molecular weight of the PEG attached to the antibody is therefore approximately 40,000Da Particular PEG molecules include 2-[3-(N-maleimido)propionamido]ethyl amide of N,N'-bis(methoxypoly(ethylene glycol) MW 20,000) modified lysine, also known as PEG2MAL40K (obtainable from Nektar, formerly Shearwater) Suitably, the antibody molecule of the present invention is a PEGylated modified Fab' fragment as shown in Figure 2 This PEGylated molecule is referred to herein as A26Fab'-PEG In another example effector molecules may be attached to antibody fiagments using the methods descπbed in applications WO2005/003169, WO2005/003170 & WO2005/003171 The present invention also provides an isolated DNA sequence encoding the heavy and/oi light chain(s) of an antibody molecule of the present invention Sutiably, the DNA sequence encodes the heavy or the light chain of an antibody molecule of the present invention The DNA sequence of the present invention may compπse synthetic DNA, for instance produced by chemical processing, cDNA, genomic DNA or any combination thereof DNA sequences which encode an antibody molecule of the present invention can be obtained by methods well known to those skilled in the art For example, DNA sequences coding for part or all of the antibody heavy and light chains may be synthesised as desired from the determined DNA sequences or on the basis of the corresponding amino acid sequences DNA coding foi acceptor framework sequences is widely available to those skilled in the art and can be readily synthesised on the basis of their known amino acid sequences

Standard techniques of molecular biology may be used to prepare DNA sequences coding for the antibody molecule of the present invention Desired DNA sequences may be synthesised completely or in part using oligonucleotide synthesis techniques Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be used as appropπate

Examples of suitable sequences are provided in Figure 1 (h) SEQ ID NO 8, Figure 1 (i) SEQ ID NO 10, Figure 1 0) SEQ ID NO 13, Figure 1 (k) SEQ ID NO 14, Figure 1 (1) SEQ ID NO 17 and Figure 1 (m) SEQ ID NO 18 Nucleotides 1-63 in SEQ ID NO 18 and 1-63 in SEQ ID NO 14 encode the signal peptide sequence OmpA which is cleaved to give an antagonistic antibody molecule of the present invention (the signal peptide corresponds to amino acid residues 1-21 in Figure 1 (g) SEQ ID NO 16 and 1-21 in Figure 1 (e) SEQ ID NO 12 lespectively) The present invention also provides an isolated DNA sequence encoding the heavy chain of an antibody of the present invention which compπses SEQ ID NO 17 or SEQ ID NO 18 The present invention also provides an isolated DNA sequence encoding the light chain of an antibody of the present invention which compπses SEQ ID NO 13 or SEQ ID NO 14

The present invention also i elates to a cloning or expression vector compπsing one or more DNA sequences of the present invention Accordingly, provided is a cloning or expression vector compnsing one or more DNA sequences encoding an antibody of the present invention Suitably, the cloning or expression vector compπses two DNA sequences, encoding the light chain and the heavy chain of the antibody molecule of the present invention, respectively Suitably, a vector according to the present invention compπses the sequences given in SEQ ID NO 14 and SEQ ID NO 18 Nucleotides 1-63 in SEQ ID NO 18 and 1-63 in SEQ ID NO 14 encode the signal peptide sequence from OmpA (residues 1-21 in SEQ ID NO 16 and 1-21 in SEQ ID NO 12 respectively) which is most suitably cleaved to give a neutralising antibody molecule of the present invention In one example the vector compπses an intergenic sequence between the heavy and the light chains, such as IGS2 (see WO03/048208) Accordingly in one embodiment the vector of the present invention comprises the sequence given in Figure 1 (n) (SEQ ID NO 19) General methods by which the vectors may be constructed, transfection methods and culture methods are well known to those skilled in the art In this respect, ieference is made to "Current Protocols in Molecular Biology", 1999, F M Ausubel (ed), Wiley Interscience, New York and the Maniatis Manual produced by Cold Spring Harbor Publishing

Also provided is a host cell compnsing one or more cloning or expression vectors compnsing one or more DNA sequences encoding an antibody of the present invention Any suitable host cell/vectoi system may be used for expression of the DNA sequences encoding the antibody molecule of the present invention Bactenal, for example E coh, and other microbial systems may be used or eukaryotic, for example mammalian, host cell expression systems may also be used Suitable mammalian host cells include CHO, myeloma or hybπdoma cells

The present invention also provides a process for the production of an antibody molecule accoiding to the present invention comprising cultuπng a host cell containing a vector of the present invention undei conditions suitable for leading to expression of protein from DNA encoding the antibody molecule of the present invention, and isolating the antibody molecule

The antibody molecule may compπse only a heavy or light chain polypeptide, in which case only a heavy chain or light chain polypeptide coding sequence needs to be used to transfect the host cells For production of products comprising both heavy and light chains, the cell line may be transfected with two vectois, a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide Alternatively, a single vector may be used, the vector including sequences encoding light chain and heavy chain polypeptides The antibodies and fragments accoiding to the present disclosure are expressed at good levels from host cells Thus the properties of the antibodies and/or fragments are optimised and condusive to commercial processing

As the antibodies of the present invention are useful in the treatment and/or piophylaxis of a pathological condition, the present invention also provides a pharmaceutical or diagnostic composition compπsing an antibody molecule of the present invention in combination with one or more of a pharmaceutically acceptable excipient, diluent or earner Accordingly, provided is the use of an antibody of the invention for the manufacture of a medicament The composition will usually be supplied as part of a steπle, pharmaceutical composition that will normally include a pharmaceutically acceptable earner A pharmaceutical composition of the piesent invention may additionally compnse a pharmaceutically-acceptable adjuvant

The present invention also provides a process for preparation of a pharmaceutical or diagnostic composition compnsing adding and mixing the antibody molecule of the present invention together with one or more of a pharmaceutically acceptable excipient, diluent or carnei The antibody molecule may be the sole active ingredient in the pharmaceutical or diagnostic composition or may be accompanied by other active ingredients including other antibody ingredients, for example anti-TNF, anti- IL- 1/3, anti-T cell, anti-IFNγ or anti-LPS antibodies, or non-antibody ingiedients such as xanthines Other suitable active ingredients include antibodies capable of inducing tolerance, for example, anti-CD3 or anti-CD4 antibodies

In a further embodiment the antibody, fragment or composition according to the disclosure is employed in combination with a further pharmaceutically active agent, for example a corticosteroid (such as fluticasonoe propionate) and/or a beta -2 -agonist (such as salbutamol, salmeterol or formoterol) or inhibitors of cell growth and proliferation (such as rapamycin, cyclophosphmide, methotrexate) or alternative a CD28 and /or CD40 inhibitor In one embodiment the inhitor is a small molecule In another embodiment the inhibitor is an antibody specific to the target

The pharmaceutical compositions suitably comprise a therapeutically effective amount of the antibody of the invention The term "therapeutically effective amount" as used herein refers to an amount of a therapeutic agent needed to treat, ameliorate or prevent a targeted disease or condition, or to exhibit a detectable therapeutic or preventative effect For any antibody, the therapeutically effective amount can be estimated initially either in cell culture assays or in animal models, usually in rodents, rabbits, dogs, pigs or pπmates The animal model may also be used to determine the appropriate concentration range and route of administration Such information can then be used to determine useful doses and routes for administration in humans

The precise theiapeutically effective amount for a human subject will depend upon the seventy of the disease state, the general health of the subject, the age, weight and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities and tolerance/response to therapy This amount can be determined by routme experimentation and is within the judgement of the clinician Generally, a therapeutically effective amount will be from 0 01 mg/kg to 50 mg/kg, for example 0 1 mg/kg to 20 mg/kg Pharmaceutical compositions may be conveniently presented in unit dose forms containing a predetermined amount of an active agent of the invention per dose Compositions may be administered individually to a patient or may be administered in combination (e g simultaneously, sequentially or sepaiately) with other agents, drugs oi hormones

The dose at which the antibody molecule of the present invention is administered depends on the nature of the condition to be treated, the extent of the inflammation present and on whethei the antibody molecule is being used prophylactically or to treat an existing condition

The frequency of dose will depend on the half-life of the antibody molecule and the duration of its effect If the antibody molecule has a short hall-life (e g 2 to 10 hours) it may be necessary to give one or more doses per day Alternatively, if the antibody molecule has a long half life (e g 2 to 15 days) it may only be necessary to give a dosage once per day, once per week or even once every 1 or 2 months

The pharmaceutically acceptable earner should not itself induce the production of antibodies harmful to the individual receiving the composition and should not be toxic Suitable earners may be large, slowly metabolised macromolecules such as proteins, polypeptides, liposomes, polysacchandes, polylactic acids, polyglycolic acids, polymenc amino acids, amino acid copolymers and inactive virus particles

Pharmaceutically acceptable salts can be used, for example mineral acid salts, such as hydrochlondes, hydrobromides, phosphates and sulphates, or salts of organic acids, such as acetates, propionates, malonates and benzoates Pharmaceutically acceptable earners in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol Additionally, auxiliary substances, such as wetting or emulsifying agents or pH buffeπng substances, may be present in such compositions Such earners enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by the patient Suitable forms for administration include forms suitable for parenteral administration, e g by injection or infusion, for example by bolus injection or continuous infusion Where the product is for injection or infusion, it may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulatory agents, such as suspending, preservative, stabilising and/or dispersing agents Alternatively, the antibody molecule may be in dry form, for reconstitution before use with an appropriate steπle liquid

Once formulated, the compositions of the invention can be administered directly to the subject The subjects to be treated can be animals However, in one or more embodiments the compositions are adapted for administration to human subjects

Suitably in formulations according to the present disclosure, the pH of the final formulation is not similar to the value of the isoelectric point of the antibody or fragment, for example if the pH of the formulation is 7 then a pi of from 8-9 or above may be appropπate Whilst not wishing to be bound by theory it is thought that this may ultimately provide a final formulation with improved stability, for example the antibody or fragment remains in solution In one embodiment the pharmaceutical formulation at a pH in the range of 4 0 to 7 0 comprises 1 to 200mg/mL of an antibody according to the present disclosure, 1 to 10OmM of a buffer, 0 001 to 1% of a surfactant, a) 10 to 50OmM of a stabiliser, b) 10 to 50OmM of a stabiliser and 5 to 500 niM of a tonicity agent, or c) 5 to 500 mM of a tonicity agent

For example the formulation at approximately pH6 may compπse 1 to 50mg/mL of antibody, 2OmM L-histadine HCl, 240 mM trehalose and 0 02% polysorbate 20 Alternatively a formulation at approximately pH 5 5 may comprise 1 to 50mg/mL of antibody, 2OmM citrate buffer, 24OmM sucrose, 2OmM arginine, and 0 02% polysorbate 20

The pharmaceutical compositions of this invention may be administered by any number of ioutes including, but not limited to, oral, intravenous, intramuscular, intra-arteπal, intramedullary, intrathecal, intraventricular, transdermal, transcutaneous (for example, see WO98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal routes Hyposprays may also be used to administer the pharmaceutical compositions of the invention Typically, the therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions Solid forms suitable for solution in, or suspension in, liquid vehicles pπor to injection may also be prepared Direct delivery of the compositions will geneially be accomplished by injection, subcutaneously, intrapentoneally, intravenously or intramuscularly, or delivered to the inteistitial space of a tissue The compositions can also be administered into a lesion Dosage treatment may be a single dose schedule or a multiple dose schedule

It will be appreciated that the active ingredient in the composition will be an antibody molecule As such, it will be susceptible to degradation in the gastrointestinal tract Thus, if the composition is to be administered by a route using the gastrointestinal tract, the composition will need to contain agents which protect the antibody from degradation but which release the antibody once it has been absorbed from the gastrointestinal tract

A thorough discussion of pharmaceutically acceptable earners is available in Remington's Pharmaceutical Sciences (Mack Publishing Company, N J 1991) In one embodiment the formulation is provided as a formulation for topical administrations including inhalation

Suitable inhalable preparations include inhalable powders, meteπng aerosols containing propellant gases or inhalable solutions free from propellant gases Inhalable powders according to the disclosure containing the active substance may consist solely of the abovementioned active substances or of a mixture of the abovementioned active substances with physiologically acceptable excipient

These inhalable powders may include monosacchaπdes (e g glucose or arabinose), disacchaπdes (e g lactose, saccharose, maltose), ohgo- and polysacchaπdes (e g dextranes), polyalcohols (e g sorbitol, mannitol, xyhtol), salts (e g sodium chloπde, calcium carbonate) or mixtuies of these with one another Mono- or disacchaπdes are suitably used, the use of lactose oi glucose, particularly but not exclusively in the form of their hydrates

Particles for deposition in the lung requne a particle size less than 10 microns, such as 1-9 microns for example from 0 1 to 5 μm, in particular from 1 to 5 μm The particle size of the active ingredient (such as the antibody or fragment) is of primary importance The propellent gases which can be used to prepare the inhalable aerosols are known in the art Suitable propellent gases are selected from among hydrocarbons such as n-propane, n- butane or isobutane and halohydrocarbons such as chlonnated and/or fluoπnated deπvatives of methane, ethane, propane, butane, cyclopropane or cyclobutane The abovementioned propellent gases may be used on their own oi in mixtures thereof Particularly suitable propellent gases are halogenated alkane deπvatives selected from among TG 1 1, TG 12, TG 134a and TG227 Of the abovementioned halogenated hydrocarbons, TG 134a (1 ,1 , 1,2-tetrafluoroethane) and TG227 (1 ,1,1 ,2,3,3,3-heptafluoropropane) and mixtures thereof are particularly suitable

The propellent-gas-containing inhalable aerosols may also contain other ingredients such as cosolvents, stabilisers, surface-active agents (surfactants), antioxidants, lubπcants and means for adjusting the pH All these ingredients are known in the art

The propellant-gas-containing inhalable aerosols according to the invention may contain up to 5 % by weight of active substance Aerosols according to the invention contain, foi example, 0 002 to 5 % by weight, 0 01 to 3 % by weight, 0 015 to 2 % by weight, 0 1 to 2 % by weight, 0 5 to 2 % by weight or 0 5 to 1 % by weight of active ingredient

Alternatively topical administrations to the lung may also be by administration of a liquid solution or suspension formulation, for example employing a device such as a nebulizer, for example, a nebulizer connected to a compressor (e g , the Pan LC-Jet Plus(R) nebulizer connected to a Pan Master(R) compressor manufactured by Pan Respiratory Equipment, Inc , Richmond, Va ) The antibody of the invention can be delivered dispeised in a solvent, e g , in the form of a solution or a suspension It can be suspended in an appropπate physiological solution, e g , saline or other pharmacologically acceptable solvent or a buffered solution Buffered solutions known in the art may contain 0 05 mg to 0 15 mg disodium edetate, 8 0 mg to 9 0 mg NaCl, 0 15 mg to 0 25 mg polysorbate, 0 25 mg to 0 30 mg anhydrous citnc acid, and 0 45 mg to 0 55 mg sodium citrate per 1 ml of water so as to achieve a pH of about 4 0 to 5 0 A suspension can employ, for example, lyophihsed antibody

The therapeutic suspensions or solution formulations can also contain one or more excipients Excipients are well known in the art and include buffers (e g , citrate buffer, phosphate buffer, acetate buffer and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (e g , serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol Solutions oi suspensions can be encapsulated in liposomes or biodegradable microspheres The formulation will generally be provided in a substantially sterile form employing steπle manufacture processes This may include production and sterilization by filtration of the buffered solvent/solution used for the formulation, aseptic suspension of the antibody in the stenle buffered solvent solution, and dispensing of the formulation into sterile receptacles by methods familiar to those of ordinary skill in the art

Nebuhzable formulation according to the present disclosure may be provided, for example, as single dose units (e g , sealed plastic containers or vials) packed in foil envelopes Each vial contains a unit dose in a volume, e g , 2 mL, of solvent/solutionbuffer

The antibodies disclosed herein may to be suitable for devehvery via nebuhsation

It is also envisaged that the antibody of the present invention may be admimstered by use of gene therapy In order to achieve this, DNA sequences encoding the heavy and light chains of the antibody molecule under the control of appropπate DNA components are introduced into a patient such that the antibody chains are expressed from the DNA sequences and assembled in situ

The present invention also provides an antibody molecule (or compositions comprising same) for use in the control of inflammatory diseases, for example acute or chronic inflammatory disease Suitably, the antibody molecule (or compositions comprising same) can be used to reduce the inflammatory process oi to prevent the inflammatory process In one embodiment there is provided an in vivo reduction of activated T cells, in particular those involved in inappropπate inflammatory immune responses, for example recruited to the vicinity/location of such a response Reduction of activated T cells, as employed herein, may be a reduction, 10, 20, 30, 40,

50, 60, 70, 80, 90 or more percent in comparison to before treatment or without tieatment Advantageously, treatment with an antibody, fiagment or composition according to the present invention, may allow the reduction in the level of activated T cells, without reducing the patients general level of T cells (unactivated T cells) This may result in fewer side effects, and possibly prevent T cell depletion in the patient The present invention also provides the antibody molecule of the present invention for use in the treatment or prophylaxis of a pathological disorder that is mediated by OX40 or associated with an increased level of OX40 The pathological condition, may, for example be selected from the group consisting of infections (viral, bactenal, fungal and parasitic), endotoxic shock associated with infection, arthritis, rheumatoid arthritis, asthma, COPD, pelvic inflammatory disease, Alzheimer's Disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peyronie's Disease, coehac disease, gallbladder disease, Pilonidal disease, peritonitis, psoπasis, vasculitis, surgical adhesions, stroke, Type I Diabetes, lyme disease, arthritis, meningoencephalitis, autoimmune uveitis, immune mediated inflammatory disoiders of the central and peripheral nervous system such as multiple sclerosis, lupus (such as systemic lupus erythematosus) and Guillain-Barr syndrome, Atopic dermatitis, autoimmune hepatitis, fibrosing alveolitis, Grave's disease, IgA nephropathy, idiopathic thrombocytopenic purpura, Meniere's disease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma, Wegener's granulomatosis, other autoimmune disorders, pancreatitis, trauma (surgery), graft-versus-host disease, transplant rejection, heart disease including ischaemic diseases such as myocardial infarction as well as atherosclerosis, intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis and hypochlorhydia

In one embodiment the antibody according to the invention is employed in the treatment of allery, COPD, autoimmune disease or rheumatoid arthritis The present invention also provides an antibody molecule according to the present invention for use in the treatment or prophylaxis of pain, particularly pain associated with inflammation

The present invention further provides the use of an antibody molecule, fragment or composition according to the present invention in the manufacture of a medicament for the treatment or prophylaxis of a pathological disorder that is mediated by OX40 or associated with an increased level of OX40, in particulai the pathological disorder is rheumatoid arthritis, asthma or COPD

The present invention further provides the use of an antibody molecule, fragment or composition according to the piesent invention in the manufacture of a medicament for the treatment or prophylaxis of one or more medical indications descπbed herein

An antibody molecule, fragment or composition of the present invention may be utilised in any therapy where it is desired to reduce the effects of OX40 in the human or animal body OX40 may be circulating in the body or may be present in an undesirably high level localised at a particular site in the body, for example a site of inflammation In one embodiment the antibody molecule of the present invention or a composition comprising the same is used for the control of inflammatory disease, e g as descπbed herein

The present invention also provides a method of treating human or animal subjects suffeπng from or at risk of a disorder mediated by OX40, the method comprising administering to the subject an effective amount of the antibody molecule of the present invention, or a composition compnsing the same In one embodiment there is provided a process for puπfiying an antibody (in particular an antibody or fragment according to the invention) comprising the steps performing anion exchange chromatography in non-binding mode such that the impurities are retained on the column and the antibody is eluted Suitable ion echange resins for use in the process include Q FF iesin (supplied by GE-

Healthcare) The step may, for example be performed at a pH about 8

The process may further comprise an intial capture step employing cation exchange chromatography, performed for example at a pH of about 4 to 5, such as 4 5 The cation exchange chromatography may, for example employ a resin such as CaptoS resin or SP sepharose FF (supplied by GE-Healthcare) The antibody or fragment can then be eluted from the resin employing an ionic salt solution such as sodium chloπde, for example at a concentration of 20OmM

Thus the chromatograph step or steps may include one or more washing steps, as appropnate The purification process may also compπse one or moie filteration steps, such as a dia filtration step

A pi above 8, such as 8 5, 8 6, 8 7, 8 8 or 9 0 of the antibody or fragment is thought to assist the purification to provide the antibody or fragment "free" or "substantially free" from impurities, such as endotoxin, DNA and host cell proteins Thus in one embodiment there is provided a puπfied OX40 antibody or fragment, for example a humanised antibody or fiagment, in particular an antibody or fragment according to the invention, in substantially purified fiom, in particular free or substantially free of endotoxin and/oi host cell protein or DNA

Puπfied form as used supra is intended to refer to at least 90% purity, such as 91 , 92, 93, 94, 95, 96, 97, 98, 99% w/w or more pure

Substantially free of endotoxin is generally intended to refer to an endotoxin content of 1 EU per mg antibody product or less such as 0 5 or 0 1 EU per mg product

Substantially free of host cell protein or DNA is generally intended to refer to host cell piotein and/or DNA content 400μg per mg of antibody product oi less such as lOOμg per mg or less, in particular 20μg per mg, as appropnate

The antibody molecule of the present invention may also be used in diagnosis, for example in the in vivo diagnosis and imaging of disease states involving OX40

Compnsing in the context of the present specification is intended to meaning including

Where technically appropnate embodiments of the invention may be combined Embodiments are descnbed herein as compnsing certain features/elements The disclosure also extends to separate embodiments consisting or consisting essentially of said features/elements

The piesent invention is further descnbed by way of illustration only in the following examples, which refer to the accompanying Figures, in which EXAMPLES

Figure 1 in detail: a) Light chain V region of antibody A26 (SEQ ID NO 7) b) Heavy chain V region of antibody A26 (SEQ ID NO 9) c) CDRHl (SEQ ID NO 1), CDRH2 (SEQ ID NO 2), CDRH3 (SEQ ID NO 3), CDRLl (SEQ ID NO 4), CDRL2 (SEQ ID NO 5), CDRL3 (SEQ ID NO 6) of antibody A26) and CDRH2 (SEQ ID NO 20) and CDRL 1 (SEQ ID NO 21 ) of antibody C A044_00026 d) Light chain of antibody A26 (SEQ ID NO 1 1) e) Light chain of antibody A26 including signal sequence (underlined) (SEQ ID NO 12) f) Heavy chain of antibody A26 (SEQ ID NO 15) g) Heavy chain of antibody A26 including signal sequence (underlined) (SEQ ID NO 16) h) DNA encoding light chain vanable region of antibody A26 (SEQ ID NO 8) i) DNA encoding heavy chain variable region of antibody A26 (SEQ ID NO 10) j) DNA encoding light chain of antibody A26 (SEQ ID NO 13) k) DNA encoding light chain of antibody A26 including signal sequence (SEQ ID NO 14)

1) DNA encoding heavy chain of antibody A26 (SEQ ID NO 17) m) DNA encoding heavy chain of antibody A26 including signal sequence (SEQ ID NO 18) n) DNA encoding heavy and light chain of antibody A26 including signal sequences and intergenic sequence IGS2 (SEQ ID NO 19) DNA manipulations and general methods E coli strain INV of' (Invitrogen) was used for transformation and routine culture growth DNA restriction and modification enzymes were obtained from Roche Diagnostics Ltd and New England Biolabs Plasmid preparations were performed using Maxi Plasmid purification kits (QIAGEN, catalogue No 12165) DNA sequencing ieactions were performed using the ABI Pπsm Big Dye terminator sequencing kit (catalogue No 4304149) and run on an ABI 3100 automated sequencer (Applied Biosystems) Data was analysed using the program AutoAssembler (Applied Biosystems) Oligonucleotides were obtained from Invitrogen Genes encoding initial V-region sequences were designed and constructed by an automated synthesis approach by Entelechon GmbH, and modified to generate the grafted versions by oligonucleotide directed mutagenesis The concentration of Fab' was determined using Fab' assembly ELISA Example 1 Production and humanisation of a neutralising anti-OX40 antibody A26

Female Sprague Dawly rats were immunised with recombinant fusion protein of human OX40 and mFC Antibody CA044 00026 which binds human OX-40 was isolated using the methods descπbed in WO92/02551 Genes for the heavy chain vanable domain (VH) and light chain variable domain (VL) of antibody CA044_00026 were isolated and sequenced following cloning via reverse transcnption PCR

A series of humanised VL and VH regions were designed using human V-region acceptor frameworks and by varying the number of donor residues in the framework regions Two grafted VH regions (gHl and 2) and 8 grafted VL regions (gLl-8) were designed and genes were built by oligonucleotide assembly and PCR mutagenesis Antibody Fab' fragments were constructed for each graft using the genes encoding the humanised vanable domains which were sub-cloned into E coli expression vector pTTOD, which contains DNA encoding the human Cγl heavy chain CHl domain (G ImI 7 allotype) and the human C kappa light chain constant domain (Klm3 allotype) (as previously described in WO03/048208) The hinge region is truncated and modified, consisting of the sequence change from Cys-Pro-Pro-Cys to Cys-Ala-Ala to generate a hinge region with a single cysteine residue available foi site specific attachment of a PEG moiety (see Example 2)

Sequences encoding the OmpA signal peptide were attached to the 5' end of the genes encoding both the heavy and light chains On expression, the signal sequences target the transport of each polypeptide to the bacteπal penplasm Following translocation through the cell membrane the signal sequence is cleaved off, leaving the mature Fab' heavy & light chains The pTTOD vector containing each graft was transformed into the host strain E coli

Kl 2 W31 10 and the antibody Fab' fragments produced in E coli by high cell density cultivation using standard methods Antibodies were puπfied using cation exchange followed by amon exchange chromatography using standard methods (Humphreys et al , 2002, Protein Expression and Purification, 26, 309-320) The various Fab' fragments produced were tested in the binding and blocking described hereinbelow and each was evaluated in terms of their expression in E coli, their potency relative to the paient antibody, and their suitability for purification and downstream processing This lead to the selection of giaft gL8gH2 which was named A26 The V region sequences of this graft are shown in Figure 1 (a) and (b) and in SEQ ID NOs 7 and 9 for the light chain (gL2) and heavy chains (gH2) respectively

The heavy chain acceptor framework is the human germline sequence VH3 1-3 3-07 with framework 4 coming from this portion of the human JH-region germline JH4 The light chain acceptor framework is the human germline sequence VKl 2-1 1-02, with framework 4 coming from this portion of the human JK-region germline JK4 The amino acids at positions 37, 73, 78 and 94 (Kabat numbenng) in the heavy chain of SEQ ID NO 9 are donor residues (fiom the parent antibody) which were found to be essential for retention of full potency Residue 64 within CDRH2 was converted from the donor glutamate to the acceptor lysine (E64K) to create a molecule with a higher pi, more favourable for ion exchange purification The amino acids at positions 64 and 71 (Kabat numbenng) in the light chain of SEQ ID NO 7 are donor residues which were found to be essential for retention of full potency Residues 27 and 28 within CDR-Ll were converted from the donor glutamate and aspartate to the acceptor glutamine (E27Q) and seπne (D28S) to create a molecule with a higher pi, more favourable for ion exchange purification

The CDRs of this antibody are shown in Figure l(c) as are the oπginal CDRH2 (SEQ ID NO 20) and CDRLl (SEQ ID NO 21) which are unmodified The full-length light and heavy chains are shown in Figures l(d) and (f) respectively

The gL8 and gH2 genes were iedesigned at the DNA level containing codons for both vanable and constant regions optimized for expression in E coli and expressed in the pTTOD(Fab') vector as descnbed above The DNA sequences encoding the light and heavy chains are shown in Figures l(k), SEQ ID NO 14 and (m) SEQ ID NO 18 respectively The protein sequence of this Fab' (including the constant regions) is provided in SEQ ID NOS 1 1 and 12 (light chain without and with OmpA signal peptide) and SEQ ID NOS 15 and 16 (heavy chain without and with OmpA signal peptide) The pTTOD (A26 IGS2) dicistromc expression vector includes the sequence provided in Figure 1 (n) and SEQ ID NO 19 The sequence contains an intergenic sequence, IGS2, between the light and heavy chain genes (See WO03/048208) and the OmpA leader sequence at the start of both the light and heavy chain genes Example 2: Production of A26Fab'-PEG The Fab' fragment A26 produced in E coli and puπfied as descnbed in Example 1 was PEGylated according to the methods descnbed in or WO2007/003898

The PEG was attached to the hinge cysteine at position 226 (linear numbering) of the heavy chain (SEQ ID NO 15) such that a lysyl-maleimide group was attached to the cysteine residue at position 226 of the heavy chain (SEQ ID NO 15), and each amino group of the lysyl residue has covalently linked to it a methoxypoly(ethyleneglycol) residue having a molecular weight of about 20,000 Da The total molecular weight of the PEG attached to the antibody was therefore approximately 40,000Da, as shown in Figure 2

Example 3: Assessment of the affinity of A26 and A26Fab'-PEG for OX40 The BIAcore technology monitors the binding between biomolecules in real time and without the requirement for labelling One of the interactants, termed the hgand, is either immobilised directly or captured on the immobilised surface while the othei, termed the analyte, flows in solution over the captured surface The sensoi detects the change in mass on the sensor surface as the analyte binds to the hgand to form a complex on the surface This corresponds to the association process The dissociation of the analyte from the hgand is monitored when the analyte is replaced by buffei In the affinity BIAcore assay, the hgand is the antibody being tested and the analyte is human OX40

Instrument Biacore ® 3000, Biacore AB, Uppsala, Sweden

Sensor chip CM5 (research grade) Catalogue Number BR- 1001 -14, Biacore AB, Uppsala,

Sweden Chips were stored at 4 ⁰ C

Amine Coupling Kit Catalogue Number BR-1000-50, Biacoie AB, Uppsala, Sweden Ethyl-3-(3-dimethylaminopropyl) carbodπmide hydrochloπde (EDC) Made up to 75 mg/mL in distilled water and stored in 200 μL ahquots at -70 ⁰ C

N-Hydioxysuccinimide (NHS) Made up to 11 5 mg/mL in distilled water and stored in 200 μL ahquots at -70 ⁰ C 1 M Ethanolamine hydrochloπde-NaOH pH 8 5 Stored in 200 μL ahquots at -70 ⁰ C Buffers Running buffer HBS-EP (being 0 01 M HEPES pH 7 4, 0 15 M NaCl, 3 mM EDTA, 0 005 % Surfactant P20) Catalogue Number BR- 1001-88, Biacore AB, Uppsala, Sweden Buffer stored at 4 ⁰ C

Immobilisation buffer Acetate 5 0 (being 10 mM sodium acetate pH 5 0) Catalogue number BR- 1003-51, Biacore AB, Uppsala, Sweden Buffer stored at 4 ⁰ C Ligand capture Affinφure F(ab') ₂ fragment goat anti-human IgG, F(ab')2 fragment specific

Jackson ImmunoResearch Inc (Pennsylvania, USA) Catalogue number 109-006-097 Reagent stored at 4 ⁰ C

Ligand Antibodies A26 and A26Fab'-PEG, stored at 4 ⁰ C

Analyte Human OX40 extracellular (185 aa) domain fused to the murine IgG2a Fc (232 aa)

(0 5mg/ml, Ancell No 513-020 lot 142805), stored at 4 ⁰ C

Regeneration Solution 40 mM HCl prepared by dilution with distilled water from an 11 6 M stock solution (BDH, Poole, England Catalogue number 101254H)

5 mM NaOH prepared by dilution with distilled water from a 50 mM stock solution Catalogue number BR-1003-58, Biacore AB, Uppsala, Sweden

Assay Method The assay format was capture of the antibody by immobilised anti-human

F(ab')? then titration of the human extracellulai domain OX40 over the captured surface

An example of the procedure is given below

BIA (Biamolecular Interaction Analysis) was performed using a BIAcore 3000 (BIAcore AB) Affinipure F(ab') ₂ Fragment goat anti-human IgG, F(ab') ₂ fragment specific (Jackson ImmunoResearch) was immobilised on a CM5 Sensor Chip via amine coupling chemistry to a capture level of »4000 response units (RUs) HBS-EP buffer (1OmM HEPES pH 7 4, 0 15 M NaCl, 3 mM EDTA, 0 005 % Surfactant P20, BIAcore AB) was used as the running buffer with a flow rate of 10 μl/min A 10 μl injection of Fab' at 0 5μg/mL or Fab'- PEG at 50μg/mL was used for capture by the immobilised anti-human IgG-F(ab') ₂ Human OX40 was titrated over the captured antibody at vaπous concentrations (25nM to 0 78nM) at a flow rate of 30 μL/min The surface was regenerated by a 10 μL injection of 40 mM HCl, followed by a 5 μL injection of 5 mM NaOH at a flowrate of lOμL/min

Background subtraction binding curves were analysed using the BIAevaluation software (version 3 2) following standaid procedures Kinetic parameters were determined from the fitting algorithm

The affinity value determined for A26 was in the range 19-45 4pM and A26Fab'-PEG was in the range 13 7-50 3pM

The following table shows replicate data for unPEGylated humanised Fab fragment A26 (fab*) and A26 Fab'-PEG Fab fragment (Fab-PEG**), binding human OX40 Table 1

Sample ka(l/Ms) Kd(l/s) KD(M) KD(pM)

Fab* 4 86 + 1 6 E+05 1 29 ± 0 07 E-05 2 96E- 11 29 6 Fab-PEG** 4 76 ± 2 1 E+05 1 30 + 0 46 E-05 3 13E-1 1 31 3

* average of 5 determinations, ** average of 4 determinations

Example 3a: Cell-based affinity and ligand-blocking capacity of A26Fab'-PEG Cell-based affinity To determine the affinity of A26Fab'-PEG for cell surface expressed antigen, saturation binding experiments were performed using activated CD4 ⁺ OX40 ⁺ T cells, and FITC labeled antibody Specific binding of antibody to receptor at equilibrium across a range of hgand concentrations was used to determine K _D , assuming that only a very small fraction of antibody was bound to receptor at any point on the binding curve

Equilibrium binding is described using the following equation kon

Receptor free + Antibody free < Receptoi -Antibody The rate of association of antibody with receptor = Ic _0n x [Receptor ^e] x [Antibody _free ] The rate of dissociation of receptor-antibody complex = k _off X [Receptor-Antibody] At equilibrium, the association and dissociation rates are equal and an equation can be derived which descπbes the binding isotherm, on a semi-log plot the binding is sigmoidal The K _D IS defined by k _ott / Ic _0n and can be calculated from the binding curve as the concentration at which half-maximal binding occurs

Binding of FITC labelled A26Fab'-PEG to activated human CD4 ⁺ OX40 ⁺ T cells was measured by flow cytometry across a 4-log concentration range A representative binding curve foi A26Fab'-PEG is shown in Figure 3 Kp values obtained on activated cells from 3 different donors were 1 193nM, 1 07InM and 1 055nM The cell-based K ₀ of A26Fab'-PEG (mean 1 106nM) is significantly weaker than the binding to iecombinant OX40 measured by BIAcoie (31 3pM) This could be due to a number of factors A26Fab'-PEG may have higher affinity for recombinant OX40 expressed as a dimeric Fc fusion protein, which has a different tertiary and quaternary structure than native cell surface expressed OX40, predicted to associate as a non-covalent trimer in the cell membrane (Chan et al , 2000) Furthermore, the affinity may be altered by differential glycosylation of recombinant versus native OX40 The 3-dimensional environment of the cell membrane such as membrane convolutions and co-localised proteins may also provide steric hindrance, limiting the accessibility of OX40 to A26Fab'-PEG Consequently, the cell-based affinity probably represents a closer measurement of the true drug affinity in vivo Methods: A26Fab'-PEG Binding to human activated CD4 ⁺ OX40 ⁺ T Cells.

PBMC weie isolated by separation on a Ficoll gradient and activated with l μg/mL PHA-L for 3 days at 37 ⁰ C, 5% CO ₂ , 100% humidity CD4 ⁺ T cells were isolated by negative selection using magnetic beads (CD4 ⁺ T cell Isolation Kit II for Human, Miltenyi Biotec) Approximately 1 2 x 10 ³ cells weie incubated in the presence of antibody (final concentration range lOμg/mL - 0 0006μg/mL (1 1 InM - 0 0068nM)) for 2 hours on ice The cells were washed pπor to analysis by flow cytometry using a FACScalibur (Becton Dickinson) Two titration curves were produced, one with A26Fab'-PEG and a second with gA33 Fab'-PEG as a non-specific binding control Lineal regression analysis was used to subtract non-specific binding and the specific binding curve thus generated was analysed by non-linear regression (Graphpad Prism®) to determine K _D Example 3b: Ligand-blocking capacity

The capacity of A26Fab ^Λ -PEG to block the interaction between cell-surface expressed OX40 and recombinant OX40L was measured using a flow cytometry-based hgand blocking assay Briefly, activated human CD4 ⁺ OX40 ⁺ T cells were pre-incubated with a titration of A26Fab'- PEG Recombinant OX40L was subsequently added to the cells and allowed to bind in the presence of A26 Fab'-PEG The proportion of OX40L bound was then detected by flow cytometry using a labelled secondary reagent Figure 4 shows a representative inhibition curve and demonstrates that A26Fab'-PEG is capable of completely blocking OX40L binding The mean IC ₅₀ for inhibition of recombinant OX40L binding was 4 InM (n = 2 donors) Methods: Inhibition of OX40L binding to human activated CD4 ⁺ OX40 ⁺ T Cells by A26Fab'-PEG. PBMC were isolated by separation on a Ficoll gradient and activated with l μg/mL PHA-L for 3 days at 37 ⁰ C, 5% CO ₂ , 100% humidity 2 5 x 10 ⁵ cells were incubated in the piesence of antibody (final concentration range 20μg/mL - 0 0003μg/mL (229 nM - 0 OO35nM)) for 10 minutes on ice OX40L (biotinylated CD252 muCD8, Ancell) was added at a final concentration of 2 μg/ml and incubated for a further 30 minutes on ice Cells were washed and OX40L binding detected by incubation with PE-labelled streptavadin (Jackson Immunoresearch) prior to analysis by flow cytometry using a FACScalibur (Becton Dickinson) gA33 Fab'-PEG was used as a non-specific control The inhibition curve was analysed by non- hneai regression (Graphpad Prism®) to determine the IC ₅₀ The data shown is from one representative donor of two

Example 4: Potency of A26Fab'-PEG in human functional assays

To assess its potency in blocking endogenous OX40-OX40L binding dunng cellulai interactions, A26 Fab'-PEG was tested in a range of antigen-driven human T cell responses Example 4a: Mixed lymphocyte reaction Fust developed in 1964 (Bach et al 1964, Science 143, 813-814) the allogeneic mixed lymphocyte reaction (MLR) is an in vitro model of alloreactive T cell activation and proliferation (O'Flaherty et al , 2000, Immunology, 100, 289-299), using whole peπpheral blood mononuclear cells (PBMCs) from two unrelated donors Donor T cells are activated through recognition of allogeneic major histocompatibility complex (MHC) antigens on unrelated donor stimulator PBMCs, resulting in cellular proliferation and cytokine production (Lukacs et al , 1993, Am J Pathology, 143, 1179-1 188) T lymphocyte alloreaction has been shown to be dπven by both the allogeneic MHC antigen and bound peptide (Sherman et al , 1993, Annu Rev Immunol, 1 1, 385-402), suggesting an MLR response may be against both stimulator allogeneic MHC antigens and bound peptides The magnitude of an MLR response correlates with the degree of MHC mis-matching between the responder-stimulator pair (Forrester et al , 2004, Corneal Transplantation An Immunological Guide to the Clinical Problem, Imperial College Press, 66-67) An MLR response results in the proliferation of cells fiom the responding donor and the pioduction of both T _H I (IL-2, IFN-γ and TNF-α) and T _H 2 (IL-4, IL-5, IL-10 and IL- 13) T cell deπved cytokines The exact cytokine profile in an MLR is thought to be specific to the responder-stimulator painng (Jordan et al , 2002, J Immunol Methods, 260, 1-14) MLR assays have been used widely in research to study T cell activation pathways and screen immunosuppressive drugs, and in clinical settings to assess immune function in acquired immune deficiency syndrome (AIDS) patients and predict possible donor organ rejection in transplant recipients (Bromelow et al , 2001 , J Immunol Methods, 247, 1-8)

The effect of A26Fab'-PEG on in vitro human alloreactive T cell activation and proliferation was investigated using an MLR assay essentially as described by O'Flaherty et al ,

2000 PBMCs from two uni elated donors were co-cultured in the presence and absence of

A26Fab'-PEG and cellular proliferation measured by ³ H-thymdine incorporation As shown in figure 5, A26 Fab'-PEG inhibited T cell proliferation in a dose dependent manner with an IC ₅ 0 value of 2 149nM (0 1877μg/mL) and a maximal inhibition of 57% Supernatents from the human MLR were analysed in a Meso Scale Discovery (MSD) human cytokine assay to investigate the effect of A26 Fab'-PEG on cytokine pioduction A26Fab'-PEGylated partially inhibited production of IFN-γ (55% inhibition), IL- 13 (50% inhibition) and IL-5 (80% inhibition) in the MLR (data not shown) Method: Inhibition of the human allogeneic one-way whole PBMC MLR proliferative response by A26Fab'-PEG. Human PBMCs from two unrelated donors were isolated from whole blood Cells from one donor were inactivated by γ-irradiation to generate the stimulator population Cells from the remaining donor formed the responder population Stimulator and responder populations were mixed at a 1 1 ratio (IxIO ⁵ cells/donor) and cultured in the presence A26Fab'-PEG (ing-lOOμg/mL) for 6 days A33 Fab'-PEG (in-house reagent) was utilized as a contiol reagent Cellular proliferation was measured at day 6 by ³ H-thymidine incorporation (0 5μCi/well) Data is displayed as peicentage inhibition relative to the responder plus stimulator iesponse in the absence of biologic reagent, and is the combined data from 10 different donor painngs (mean ± SEM) IC50 values were calculated using Graphpad Pπsm® software The results are shown in Figure 5 Example 4b: Tetanus toxoid response

Tetanus toxoid (TT) induces strong T cell specific immune responses in vaccinated individuals In vitro antigen-specific recall responses to TT challenge can be detected by monitoring proliferation and cytokine production (T _H 1 & T _H 2) from PBMC (Bishop et al 2005) A26Fab'- PEG inhibited proliferation and IL-5, IL-13, IFN-γ and TNF-α production (data not shown) in a dose-dependent manner, with maximal inhibition of proliferation reaching 38% IC ₅₀ values for inhibition of proliferation, calculated for 2 donors, were 0 58nM (0 051μg/mL) and 1 HnM (0 097μg/mL) Fig 6 shows the A26Fab'-PEG proliferation inhibition curve for 1 donor Method A26 Fab'-PEG inhibits proliferation of PBMC exposed to Tetanus Toxoid. PBMC were isolated by separation on a Ficoll gradient and exposed to lμg/mL Tetanus Toxoid (Calbiochem) in the presence of A26Fab'-PEG (concentration range 5μg/mL to 0 OOlμg/mL) in a final volume of 200μL per well in a 96-well round-bottomed plate After 5 days incubation at 37 ⁰ C, 5% COJ, 100% humidity, cell proliferation was measured by incorporation of ³ H thymidine (0 5μCi/well) into actively dividing cells Results from a single lepresentative donor are presented IC ₅ O values were calculated using Graphpad Pπsm® software Example 4c: House dust mite response Severe acute asthma can be tπggered by inhaled antigens such as house dust mite (Tillie- Leblond et a! , 2005, Allergy, 60, (1), 23-29), including species from the genus Dermatophagoides pteronyssinus Such allergens induce proliferative responses by penpheral blood cells and TH2 polarised cytokine production, in atopic but not non-atopic patients (Ling et al , 2004, Lancet, 363, 608-615) An in vitro assay was set up to determine the effect of OX40 blockade on production of the T _H 2 cytokine IL- 13 in response to antigen challenge PBMCs were taken from atopic people with an allergen-specific IgE (RAST) score between 3 and 5 (scale 0 to 6) and stimulated with Dermatophagoides pteronyssinus antigen in the presence of A26Fab'-PEG or control antibody A26Fab'-PEG inhibited IL-13 production to a maximum of 60% with an IC ₅₀ value of 1 23nM (figure 7) Furthermore, A26 Fab'-PEG also potently inhibited production of the cytokines IL-4, IL-5 and TNF-α in this assay whilst enhancing levels of the regulatory cytokine IL-10 (figure 8)

Method Figure 7: A26Fab'-PEG inhibits IL-13 production from PBMC exposed to Dermatophagoides pteronyssinus allergenic extract. PBMC were isolated from allergic volunteers by separation on a Ficoll gradient Purified PBMC were exposed to 25μg/mL Dermatophagoides pteronyssinus allergenic extract (Greer) in the presence of test antibody (concentration range lOμg/mL to 0 0005μg/mL) in a final volume of 200μL per well in a 96- well round-bottomed plate Aftei 6 days incubation at 37 ⁰ C, 5% CO ₂ , 100% humidity, supernatants were harvested and assayed for IL-13 content by ELISA (Biosource) The giaph iepresents pooled data from three donors (mean ± SEM) IC50 values were calculated using Graphpad Prism® software

Method Figure 8: A26Fab'-PEG modulates cytokine production from PBMC exposed to Dermatophagoides pteronyssinus allergenic extract. PBMC were isolated from allergic volunteeis by separation on a Ficoll gradient Purified PBMC were exposed to 25μg/mL Dermatophagoides pteronyssinus allergenic extiact (Greei) in the presence of lOμg/mL A26Fab'-PEG (1 14nM) or control (TN3 Fab'-PEG) in a final volume of 200μL per well in a 96-well round-bottomed plate After 6 days incubation at 37 ⁰ C, 5% CO?, 100% humidity, supernatants were harvested and assayed for cytokine content using a multi-spot assay (MSD) The graphs represent pooled data from three donors (mean ± SEM) Summary

The IC ₅₀ values for A26Fab'-PEG in human functional assays are summarised in Table 2. The potency of A26Fab-PEG is similar across all three assays and correlates well with the cell- based affinity measurement of 1 106nM In these assays, either cellular proliferation and/or production of multiple inflammatory cytokines was significantly suppressed, demonstrating that A26Fab'-PEGy profoundly inhibits T cell activation The Tetanus Toxoid and House Dust Mite assays both measure recall responses by memory T cells, signifying that A26Fab'-PEG is capable of inhibiting established T cell responses to a variety of antigens

Table 2 Mean IC5 ₀ values for A26Fab'-PEG in human functional in vitro assa s

The atopic memory T _H 2 response to House Dust Mite antigen provides a relevant in vitro assay for allergic asthma and the data suggests that A26Fab'-PEG may be an effective therapy in this indication OX40 co-stimulation has previously been linked to lung inflammation where it is suggested to play a critical role in both the differentiation of allergen-specific naive CD4 ⁺ T cells into inflammatory T _H 2 cells and the recall iesponses of memory T _H 2 cells (Wang & Lm, 2007, J Clin Invest, 117 (12), 3655-3657) Dunng allergic inflammation, the innate cytokine thymic stromal lyphopoietin (TSLP) produced by stressed epithelial cells dπves maturation of human dendntic cells and induces expression of OX40L OX40L functions to promote TH2 polarisation of CD4 ⁺ T cells with an inflammatory phenotype of enhanced TNF-α but no IL-10 production (Ito et al, 2005, J Exp Med, 202 (9), 1213-1223) In the HDM response, A26 Fab'- PEG potently inhibited the classic TH2 cytokines IL- 13, IL-5 and IL-4 as well as TNF -a Furthermore, in two out of four allergic donors A26Fab'-PEG enhanced IL-10 production Thus, A26Fab'-PEG may have the capacity not only to inhibit allergic responses but also to modulate them towards a regulatory phenotype Example 5:

A26Fab'-PEG inhibits CD4+ & CD8+ T cell proliferation in a Hu-SCID model. The Hu-SCID model involves reconstitution of SCID mice with human PBMCs which then elicit a strong xenogeneic response against the host mouse This response is tracked by the proliferation of human T cells in the mouse Using experimentally determined data on the PK of A26Fab'-PEG a dosing regime was designed which resulted in steady state plasma concentrations of 8, 23 and 34μg/ml A26Fab'-PEG The data in Figure 9 demonstrates that CD4 ⁺ and CD8 ⁺ T cells are profoundly inhibited by maintaining steady state plasma levels of A26Fab'-PEG at 8, 23 and 34 μg/ml Method: A26Fab'-PEG inhibits CD4+ and CD8+ T cell proliferation in a Hu-SCID model. Mice were given a s c loading dose of 0 825, 2 475 or 8 25 mg/kg on day -2 and then daily s c maintenance doses of 0 25, 0 75 or 2 5 mg/kg respectively Mice are depleted of NK cells by dosing with TMβ 1 one day pπor to transfer of eight million human PBMCs into the peritoneal cavity on day 0 The experiment is then terminated on day 14 and blood, pentoneal lavage fluid and spleen homogenate are analysed for CD4 ⁺ & CD8 ⁺ cells Day 14 mice were killed by cervical dislocation & bled by cardiac puncture The number of human CD4 ⁺ & CD8 ⁺ cells was then determined by FACS analysis Data (n=10) is expressed as means ± SEM The decrease in CD4 ⁺ & CD8 ⁺ cells in the blood after administration of A26Fab'-PEG is shown in Figure 9 Example 6: Cross-reactivity of A26Fab'-PEG with non-human primate QX40 To validate use of A26Fab'-PEG in non-human primate (NHP) disease models and pre-clinical toxicology, its relative affinity and functional potency were compared on human & NHP cells

Cell-based Affinity on NHP Cells

Cynomolgus or rhesus CD4 ⁺ T cells were isolated from peπpheral blood and activated to express high levels of OX4O The affinity of A26Fab'-PEG was measured by non-linear regression analysis of equilibrium binding curves as shown in Figure 3 A26Fab'-PEG showed a less than 2-fold drop off in affinity for cynomolgus or rhesus CD4 ⁺ T cells as compared to human, indicating it is highly cross-reactive (Table 3)

Table 3 Cell-based affinit com arison of A26Fab'-PEG on human and NHP cells

NHP PBMC were separated on a Lympholyte (VH Bio) gradient, activated with 1 μg/mL PHA- L for 3 days at 37 ⁰ C, 5% CO ₂ , 100% humidity and CD4 ⁺ T cells were isolated by negative selection using magnetic beads (CD4 ⁺ T cell Isolation Kit II for non-human pπmate, MiltenyiBiotec) Affinities were measured as descπbed in Example 3a (Figure 3) Example 7: Efficacy study in the Cynomolgus monkey CIA model Rationale for study and study design

Cynomolgus collagen-induced arthritis is a standard model used to profile potential anti- arthritic drugs pπor to human expenmentation In our hands, this model responds to treatments directed against TNFα and IL-6 These data are consistent with the clinical RA findings with equivalent anti-human therapeutics

The induction of arthritis in cynomolgus monkeys requires two immunisation steps with collagen II separated by a peπod of 3 weeks Arthritis symptoms (swelling and tenderness of one or more joints) can be manifest at any time after the second immunisation and were assessed weekly using an arthritis score The experiment was run for 1 1 weeks in total OX40 is a co-stimulation molecule and so interference with function would be expected to have effects on the immunisation phases of the model Three dosing regimes with A26 Fab'-PEG were evaluated One group leceived A26Fab'-PEG (100mg/kg) once only on the day before first immunisation A second group received A26 Fab'-PEG (100mg/kg) once only on the day before the second immunisation and the third group received A26 Fab'-PEG (100mg/kg) one day prior first and second immunisations A control group of animals received an acetate buffer vehicle Disease onset in the vehicle treated group was characteπsed by serum elevations in the acute phase proteins C-reactive protein (CRP) and haptoglobin, (biomarkers that are measured clinically in RA tπals) Joint integrity was assessed by x-ray and by histological examination Results and conclusion

In animals treated with A26Fab'-PEG on the day before first immunisation, arthritis seventy was generally lower than in the vehicle treated group These differences in arthritis score were statistically significant on days 49, 63 and 76 Figure 10 shows an overall summary of the data for individual animals expressed as area under curve for clinical scores X ray assessment of bone erosion to joints was also ieduced (Table 4) as were histopathological changes (Figure 1 1) Concentrations of CRP and haptoglobin tended to be lower than in the control group Similar results were obtained for the group of animals dosed with A26Fab'-PEG one day pnor to first immunisation and one day prior to second immunisation However, there was no convincing anti arthritic effect observed in animals receiving A26Fab'-PEG once only on the day before second immunisation These data show an anti arthritic effect of anti OX40 treatment in cynomolgus CIA and demonstrate the importance of OX40 to the initiation of the pathogenic immune response

Figure 10 Inhibition of arthritis score by A26 Fab'-PEG in cynomolgus CIA. Data shows individual animal area under the curve (AUC) for cluneal score data for control animals receiving acetate buffer pnor to first and second immunisations (Ac Ac), animals receiving pnor to first immunisation (A26 Ac), animals receiving A26 Fab'-PEG pnor to second immunisation (Ac A26) and anunals receiving A26 Fab'-PEG pnor to first and second immunisations (A26 A26) Bars are medians

Table 4 Effects of A26 Fab'-PEG treatment on x-ra scores of bone erosion

Ac Ac animals received acetate buffer vehicle, A26 Ac animals received A26Fab'-PEG pnoi to first immunisation, Ac A26 animals leceived A26 Fab'-PEG pnor to second immunisation and A26 A26 animals received A26 Fab'-PEG pnor to first and second immunisations Means ± s e m , *p<0 05, **p<0 01 Wilcoxin's test

Method Figure 11: Reduction in total histological score in cynomolgus CIA by A26Fab'- PEG. Data shows total histological scores (incorporating degeneration of cartilage and bone, fibrosis, granulation tissue and hyperplasia) for individual animals at termination of the study Ac Ac animals received acetate buffer vehicle, A26 Ac animals received A26 Fab'-PEG pnor to first immunisation, Ac A26 animals received A26Fab'-PEG prior to 2nd immunisation & A26A26 animals received A26 Fab'-PEG pnor to 1 ^st & 2 ^nd immunisations Bars are medians It will of course be understood that the present invention has been descnbed by way of example only, is in no way meant to be limiting, and that modifications of detail can be made within the scope of the claims hereinafter Preferred features of each embodiment of the invention are as for each of the other embodiments mutatis mutandis All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.