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  • A61P19/00—Drugs for skeletal disorders
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  • A61P19/00—Drugs for skeletal disorders
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  • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
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  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
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  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61P5/00—Drugs for disorders of the endocrine system
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  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
• • C—CHEMISTRY; METALLURGY
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  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

• Anti-TNF alpha Antibodies which selectively inhibit TNF alpha signalling through the p55R
• the present invention relates to antibodies to TNF ⁇ .
• the present invention relates to antibodies which selectively inhibit TNF ⁇ signalling through the p55R relative to the p75R, for example by selectively inhibiting the binding of TNF ⁇ to the p55 receptor.
• Tumor necrosis factor alpha is a pro-inflammatory cytokine that is released by and interacts with cells of the immune system. TNF ⁇ has been shown to be upregulated in a number of human diseases, including chronic diseases such as rheumatoid arthritis, Crohn's disease, ulcerative colitis and multiple sclerosis.
• TNF- ⁇ Human TNF- ⁇ is a 17kDa protein and the active form exists as a homotrimer (Pennica et al, 1984, Nature, 312, 724-729; Davis et al, 1987, Biochemistry, 26, 1322- 1326; Jones et al, 1989, Nature, 338, 225-228). TNF ⁇ exerts its biological effects through interaction with two structurally related but functionally distinct cell surface receptors, p55R and p75R that are co-expressed on most cell types (Loetscher et al, 1990, Cell, 61, 351; Smith et al, 1990, Science, 248, 1019).
• the p55R is also known as p55TNFR; CD120a; TNFR I; TNFR 1 and TNFRSFIa.
• the p75R is also known as p75TNFR; CD120b; TNFR II; TNFR 2 and TNFRSFIb.
• Both receptors are also proteolytically released as soluble molecules capable of binding TNF ⁇ .
• the extracellular domains of the two receptors exhibit sequence similarity, consisting of four repeating cysteine-rich motifs containing four to six cysteines in conserved positions. In contrast their cytoplasmic signalling region sequences are unrelated, suggesting different modes of signalling and function.
• mice genetically deficient in one or both of the two receptors demonstrated that the p55R is responsible for the majority of TNF ⁇ -mediated inflammatory responses and the p75R may in some circumstances act to suppress TNF ⁇ -mediated inflammatory responses and that the two receptors can act as a balancing system for TNF ⁇ action.
• TNF ⁇ activity has been achieved by a number of different means using inhibitors such as antibodies and soluble receptors.
• inhibitors such as antibodies and soluble receptors.
• examples include etanercept, marketed by Immunex Corporation as EnbrelTM which is a recombinant fusion protein comprising two p75 soluble TNF-receptor domains linked to the Fc portion of a human immunoglobulin.
• EnbrelTM a recombinant fusion protein comprising two p75 soluble TNF-receptor domains linked to the Fc portion of a human immunoglobulin.
• Infliximab marketed by Centocor Corporation as RemicadeTM is a chimeric antibody having murine anti-TNF ⁇ variable domains and human IgG 1 constant domains.
• Adalimumab marketed by Abbott Laboratories as HumiraTM is a recombinant, fully human anti-TNF ⁇ antibody (Tussirot and Wendling, 2004, Expert Opin.Pharmacother, 5, 581-594).
• Other inhibitors include engineered TNF ⁇ molecules which form trimers with native TNF ⁇ and prevent receptor binding (Steed et al, 2003, Science, 301, 1895-1898; WO03033720; WO0164889). These current methods of inhibiting TNF ⁇ activity block binding of TNF ⁇ to both the p55 and p75 receptors (see for example Mease, 2005, Expert Opin. Biol. Therapy, 5, 11, 1491-1504).
• TNF ⁇ signalling through the p55R is necessary for the detrimental effects of TNF ⁇ during the acute phase of MS
• TNF ⁇ signalling through the p75R can lead to beneficial effects such as elimination of inflammatory infiltrates.
• This immunosuppressive role for TNF ⁇ has also been proposed in other autoimmune diseases (Cope, 1998, Current Opinion in Immunology, 10, 669-676). Indeed it has been suggested that p75R agonists could be used to treat allergic conditions such as allergic bronchial asthma (WO99/59632).
• both receptors can bind the same trimer at the same time (Barbara et al, 1994, EMBO, 13, 843-850). It has however, been possible to create TNF ⁇ mutants which selectively bind to either the p75 or the p55 receptor. TNF ⁇ mutants which do not bind to the p55R but do bind to the p75R have been demonstrated to retain antitumor activity but exhibit reduced proinflammatory activities (Barbara et al, 1994, EMBO J, 13, 843-850).
• the present invention provides an anti-TNF ⁇ antibody that selectively inhibits TNF ⁇ signalling through the p55R.
• the anti-TNF ⁇ antibody of the present invention selectively inhibits TNF ⁇ signalling through the p55R relative to the p75R.
• the antibodies of the present invention therefore have the advantageous property that they can selectively inhibit the effects of TNF ⁇ mediated by the p55R whilst retaining the beneficial effects of TNF ⁇ signalling through the p75R. Accordingly, the present invention also provides the use of an anti-TNF ⁇ antibody that selectively inhibits TNF ⁇ signalling through the p55R for the • manufacture of a medicament for the treatment and/or prophylaxis of an autoimmune or inflammatory disease. Also provided is a method for the treatment and/or prophylaxis of an autoimmune or inflammatory disease in a subject comprising administering to said subject a therapeutically effective amount of an antibody that selectively inhibits TNF ⁇ signalling through the p55R.
• the Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues.
• the actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure.
• CDR complementarity determining region
• the correct Kabat numbering of residues 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.
• CDR-Hl residues 31-35
• CDR-H2 residues 50-65
• CDR-H3 residues 95-102
• the loop equivalent to CDR-Hl extends from residue 26 to residue 32.
• 'CDR-Hl ' comprises 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 variable domain are located at residues 24-34 (CDR-
• the anti-TNF ⁇ antibodies of the present invention selectively bind to TNF ⁇ .
• Selectively binding means that the antibodies have a greater affinity for TNF ⁇ polypeptides than for other polypeptides.
• the TNF ⁇ polypeptide is human TNF ⁇ .
• TNF ⁇ polypeptide or cells expressing said polypeptide can be used to produce anti- TNF ⁇ antibodies which specifically recognise said polypeptide.
• the TNF ⁇ polypeptide may be a 'mature' polypeptide or a biologically active fragment or derivatives thereof which include the receptor binding site.
• the TNF ⁇ polypeptide is the mature polypeptide.
• TNF ⁇ 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.
• the term "polypeptides" includes peptides, polypeptides and proteins. These are used interchangeably unless otherwise specified.
• the TNF ⁇ polypeptide may in some instances be part of a larger protein such as a fusion protein for example fused to an affinity tag.
• Antibodies generated against these polypeptides 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 Experimental Immunology, D. M. Weir (ed.), VoI 4, Blackwell Scientific Publishers, Oxford, England, 1986).
• Many warm-blooded animals, such as rabbits, mice, rats, sheep, cows or pigs may be immunized. However, mice, rabbits, pigs and rats are generally preferred.
• Anti-TNF ⁇ 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, multi-valent, multi-specific, humanized or chimeric antibodies, single chain antibodies, Fab fragments, Fab' and F(ab') 2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
• Particular antibody fragments also include those described in International patent applications WO2005003169, WO2005003170 and WO2005003171 (all published on 13th January 2005).
• Antibody fragments and methods of producing them are well known in the art, see for example Verma et a!., 1998, Journal of Immunological Methods, 216, 165- 181.
• Antibodies for use in the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. molecules that contain an antigen binding site that specifically binds an antigen.
• the immunoglobulin molecules of the invention can be of any class ⁇ e.g. IgG, IgE, IgM, IgD and IgA) or subclass of immunoglobulin molecule.
• the constant region domains of the antibody molecule of the present invention may be selected having regard to the proposed function of the antibody molecule, and in particular the effector functions which may be required.
• the constant region domains may be human IgA, IgD, IgE, IgG or IgM domains.
• 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.
• IgG2 and IgG4 isotypes may be used when the antibody molecule is intended for therapeutic purposes and antibody effector functions are not required.
• constant region domains may also be used.
• IgG4 constant domain comprising this change.
• Monoclonal antibodies may be prepared by any method known in the art such as the hybridoma technique (Kohler & Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al, 1983, Immunology Today, 4:72) and the EBV-hybridoma 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 variable region cDNAs generated from single lymphocytes selected for the production of specific antibodies by for example the methods described 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.
• Humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule (see, e.g. US 5,585,089; WO91/09967).
• Chimeric antibodies are those antibodies encoded by immunoglobulin genes that have been genetically engineered so that the light and heavy chain genes are composed of immunoglobulin gene segments belonging to different species. These chimeric antibodies are likely to be less antigenic.
• Bivalent antibodies may be made by methods known in the art (Milstein et al., 1983, Nature 305:537-539; WO 93/08829, Traunecker et al, 1991, EMBO J. 10:3655-3659).
• Multi-valent antibodies may comprise multiple specificities or may be monospecific (see for example WO 92/22853).
• the antibodies for use in the present invention can also be generated using various phage display methods known in the art and include those disclosed by Brinkman 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.
• the present invention provides an anti-TNF ⁇ antibody which selectively inhibits TNF ⁇ signalling through the p55R, comprising a heavy chain, wherein the variable domain of the heavy chain comprises at least one of a CDR having the sequence given in SEQ ID NO:9 for CDR-Hl, a CDR having the sequence given in SEQ ID NO:10 or SEQ ID NO:21 for CDR-H2 and a CDR having the sequence given in SEQ ID NO: 11 for CDR-H3.
• an antibody of the present invention comprises a heavy chain wherein at least two of CDR-Hl, CDR-H2 and CDR-H3 of the variable domain of the heavy chain are selected from the following: the sequence given in SEQ ID NO:9 for CDR-Hl, the sequence given in SEQ ID NO:10 or SEQ ID NO:21 for CDR-H2 and the sequence given in SEQ ID NO:11 for CDR-H3.
• the antibody may comprise a heavy chain wherein CDR-Hl has the sequence given in SEQ ID NO: 9 and CDR-H2 has the sequence given in SEQ ID NO: 10.
• the antibody may comprise a heavy chain wherein CDR-Hl has the sequence given in SEQ ID NO: 9 and CDR-H3 has the sequence given in SEQ ID NO: 11, or the antibody may comprise a heavy chain wherein CDR-H2 has the sequence given in SEQ ID NO:21 and CDR-H3 has the sequence given in SEQ ID NO:11.
• CDR-Hl has the sequence given in SEQ ID NO: 9
• CDR-H3 has the sequence given in SEQ ID NO: 11
• the antibody may comprise a heavy chain wherein CDR-H2 has the sequence given in SEQ ID NO:21 and CDR-H3 has the sequence given in SEQ ID NO:11.
• an antibody according to the present invention comprises a heavy chain, wherein the variable domain comprises the sequence given in SEQ ID NO:9 for CDR- Hl, the sequence given in SEQ ID NO: 10 for CDR-H2 and the sequence given in SEQ ID NO: 11 for CDR-H3.
• an antibody according to the present invention comprises a heavy chain, wherein the variable domain comprises the sequence given in SEQ ID NO:9 for CDR- Hl, the sequence given in SEQ ID NO:21 for CDR-H2 and the sequence given in SEQ ID NO: 11 for CDR-H3.
• the antibody of the present invention comprises a heavy chain, wherein the variable domain of the heavy chain comprises the sequence given in SEQ ID NO:6.
• the antibody of the present invention comprises a heavy chain, wherein the variable domain of the heavy chain comprises the sequence given in SEQ ID NO:20.
• the antibody of the present invention comprises a heavy chain, wherein the variable domain of the heavy chain comprises a sequence having at least 60% identity or similarity to the sequence given in SEQ ID NO:6 or the sequence given in SEQ ID NO:20.
• the antibody of the present invention comprises a heavy chain, wherein the variable domain of the heavy chain comprises a sequence having at least 90%, 95% or 98% identity or similarity to the sequence given in SEQ ID NO:6 or the sequence given in SEQ ID NO:20.
• Identity indicates that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences.
• similarity indicates that, at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences.
• 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);
• the present invention also provides an anti-TNF ⁇ antibody which selectively inhibits TNF ⁇ signalling through the p55R, comprising a light chain, wherein the variable domain of the light chain comprises at least one of a CDR having the sequence given in SEQ ID NO: 12 for CDR-Ll, a CDR having the sequence given in SEQ ID NO: 13 for CDR-L2 and a CDR having the sequence given in SEQ ID NO: 14 for CDR-L3.
• the antibody of the present invention comprises 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: 12 for CDR-Ll, the sequence given in SEQ ID NO: 13 for CDR-L2 and the sequence given in SEQ ID NO: 14 for CDR-L3.
• the antibody may comprise a light chain wherein CDR-Ll has the sequence given in SEQ ID NO: 12 and CDR-L2 has the sequence given in SEQ ID NO:13.
• the antibody may comprise a light chain wherein CDR-Ll has the sequence given in SEQ ID NO: 12 and CDR-L3 has the sequence given in SEQ ID NO: 14, or the antibody may comprise a light chain wherein CDR-L2 has the sequence given in SEQ ID NO: 13 and CDR-L3 has the sequence given in SEQ ID NO: 14.
• CDR-Ll has the sequence given in SEQ ID NO: 12
• CDR-L3 has the sequence given in SEQ ID NO: 14
• the antibody may comprise a light chain wherein CDR-L2 has the sequence given in SEQ ID NO: 13 and CDR-L3 has the sequence given in SEQ ID NO: 14.
• the antibody of the present invention comprises a light chain, wherein the variable domain comprises the sequence given in SEQ ID NO: 12 for CDR-Ll, the sequence given in SEQ ID NO: 13 for CDR-L2 and the sequence given in SEQ ID NO: 14 for CDR-L3.
• the present invention comprises a light chain, wherein the variable domain of the light chain comprises the sequence given in SEQ ID NO:8.
• the antibody of the present invention comprises a light chain, wherein the variable domain of the light chain comprises a sequence having at least 60% identity or similarity to the sequence given in SEQ ID NO:8.
• the antibody of comprises a light chain, wherein the variable domain of the light chain comprises a sequence having at least 90%, 95% or 98% identity or similarity to the sequence given in SEQ ID NO:8.
• the antibody molecules of the present invention preferably comprise a complementary light chain or a complementary heavy chain, respectively.
• the antibody of the present invention comprises a heavy chain, wherein the variable domain of the heavy chain comprises the sequence given in SEQ ID NO:9 for CDR-Hl, the sequence given in SEQ ID NO: 10 or SEQ ID NO:21 for CDR-H2 and the sequence given in SEQ ID NO:11 for CDR-H3 and a light chain wherein the variable domain of the light chain comprises the sequence given in SEQ ID NO: 12 for CDR-Ll, the sequence given in SEQ ID NO:13 for CDR-L2 and the sequence given in SEQ ID NO:14 for CDR-L3.
• the antibody comprises a heavy chain, wherein the variable domain of the heavy chain comprises the sequence given in SEQ ID NO: 6 and a light chain, wherein the variable domain of the light chain comprises the sequence given in SEQ ID NO:8.
• the antibody comprises a heavy chain, wherein the variable domain of the heavy chain comprises the sequence given in SEQ ID NO:20 and a light chain, wherein the variable domain of the light chain comprises the sequence given in SEQ rD NO:8.
• the antibody comprises a heavy chain and a light chain, wherein the variable domain of the heavy chain comprises a sequence having at least 60% identity or similarity to the sequence given in SEQ ID NO:6 and the variable domain of the light chain comprises a sequence having at least 60% identity or similarity to the sequence given in SEQ ID NO:8.
• the antibody comprises a heavy chain, wherein the variable domain of the light chain comprises a sequence having at least 90%, 95% or 98% identity or similarity to the sequence given in SEQ ID NO:6 and a light chain, wherein the variable domain of the light chain comprises a sequence having at least 90%, 95% or 98% identity or similarity to the sequence given in SEQ ID NO:8.
• the antibody comprises a heavy chain and a light chain, wherein the variable domain of the heavy chain comprises a sequence having at least 60% identity or similarity to the sequence given in SEQ ID NO:20 and the variable domain of the light chain comprises a sequence having at least 60% identity or similarity to the sequence given in SEQ ID NO: 8.
• the antibody comprises a heavy chain, wherein the variable domain of the light chain comprises a sequence having at least 90%, 95% or 98% identity or similarity to the sequence given in SEQ ID NO:20 and a light chain, wherein the variable domain of the light chain comprises a sequence having at least 90%, 95% or 98% identity or similarity to the sequence given in SEQ ID NO: 8.
• antibody '462' One antibody provided by the present invention is referred to herein as antibody '462'.
• the complete nucleotide and amino acid sequences of the heavy chain variable domain of rat antibody '462' are given in SEQ ID NOS: 5 and 6 and the complete nucleotide and amino acid sequences of the light chain variable domain of rat antibody '462' are given in SEQ ID NOS: 7 and 8.
• the nucleotide and amino acid sequences of the heavy chain variable region of this antibody including the rat leader sequence are given in SEQ ID NOs: 1 and 2 and the light chain variable regions are given in SEQ ID NOs:3 and 4.
• Another antibody provided by the present invention is referred to herein as antibody
• the complete nucleotide and amino acid sequences of the heavy chain variable domain of rat antibody '463' are given in SEQ ID NOS: 19 and 20 and the complete nucleotide and amino acid sequences of the light chain variable domain of rat antibody '463' are given in SEQ ID NOS: 7 and 8.
• the nucleotide and amino acid sequences of the heavy chain variable region of this antibody including the rat leader sequence are given in SEQ ID NOs: 15 and 16 and the light chain variable regions are given in SEQ ID NOs:17 and 18.
• CDR-grafted (or humanised) anti-TNF ⁇ antibody characterised in that the antibody selectively inhibits TNF ⁇ signalling through the p55R.
• one or more of the CDRs in the CDR-grafted antibody molecule have been obtained from either of the rat antibodies 462 or 463.
• the CDRs of rat antibody 462 are provided in SEQ ID NOS:9, 10, 11, 12, 13 and 14.
• the CDRs of rat antibody 463 are provided in SEQ ID NOS:9, 21, 11, 12, 13 and 14.
• the term 'CDR- grafted antibody molecule' refers 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 rat antibody such as antibody '462' or '463' as described herein) grafted into a heavy and/or light chain variable region framework of an acceptor antibody (e.g. a human antibody).
• a donor antibody e.g. a rat antibody such as antibody '462' or '463' as described herein
• acceptor antibody e.g. a human antibody
• any appropriate acceptor variable region framework sequence may be used having regard to the class/type of the donor antibody from which the CDRs are derived, including mouse, primate and human framework regions.
• the CDR-grafted antibody of the present invention has a variable domain comprising human acceptor framework regions as well as one or more of the CDRs derived from the donor antibody as referred to above.
• the variable domain comprises human acceptor framework regions and non-human, preferably rat, donor CDRs.
• human frameworks which can be used in the present invention are KOL,
• 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.
• human germline sequences may be used; these are available at: http://vbase.mrc-cpe.cam.ac.uk/
• the acceptor heavy and light chains do not necessarily need to be derived from the same antibody and may, if desired, comprise composite chains having framework regions derived from different chains.
• 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 regions 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 donor antibody.
• a protocol for selecting residues in the acceptor framework regions which may need to be changed is set forth in WO 91/09967.
• Donor residues are residues from the donor antibody, i.e. the antibody from which the CDRs were originally derived, which may in one embodiment of the present invention be either of the rat antibodies '462' or '463' as described herein.
• the antibody molecule of any aspect of the present invention preferably has a high binding affinity for TNF ⁇ , preferably picomolar.
• the antibody molecule of the present invention has a binding affinity of between about 1 and 50OpM.
• the antibody molecule of the present invention has a binding affinity of between about 10 and about 400 pM. 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 TNF ⁇ .
• affinity maturation protocols known in the art, such as mutating the CDRs (Yang et al, J. MoI. Biol., 254, 392- 403, 1995), chain shuffling (Marks et al, Bio/Technology, 10, 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.
• the anti-TNF ⁇ antibodies provided by the present invention selectively inhibit TNF ⁇ signalling through the p55R, for example by selectively inhibiting the binding of TNF ⁇ to the p55R i.e. they reduce the signalling through this receptor.
• the term 'selectively inhibit' means that the antibodies of the present invention inhibit TNF ⁇ signalling through the p55R to a greater extent than they inhibit TNF ⁇ signalling through the p75R.
• the invention provides an anti-TNF ⁇ antibody which selectively inhibits TNF ⁇ signalling through the p55R relative to the p75R.
• the antibody substantially reduces TNF ⁇ signalling through the p55R.
• the antibody of the present invention substantially reduces binding of TNF ⁇ to the p55R.
• the antibodies of the present invention inhibit binding of TNF ⁇ to the p55R by more than they inhibit binding of TNF ⁇ to the p75R.
• the term 'inhibit' as used herein includes total and partial inhibition. Hence the term includes total and partial inhibition of TNF ⁇ signalling through the p55R. It will be appreciated that the extent of inhibition may be affected by the concentration of antibody used.
• the anti-TNF ⁇ antibody inhibits TNF ⁇ signalling through the p55R by greater than 40%, preferably between 40 and 100%, even more preferably between 45 and 100%.
• the anti-TNF ⁇ antibody inhibits TNF ⁇ signalling through the p55R by 50% or greater.
• the anti-TNF ⁇ antibody inhibits TNF ⁇ signalling through the p55R by 60% or greater.
• the anti-TNF ⁇ antibody inhibits TNF ⁇ signalling through the p55R by 70% or greater.
• the anti- TNF ⁇ antibody inhibits TNF ⁇ signalling through the p55R by 80% or greater.
• the anti-TNF ⁇ antibody inhibits TNF ⁇ signalling through the p55R by 90% or greater.
• the anti-TNF ⁇ antibody of the present invention reduces the binding of TNF ⁇ to the p55R by greater than 40%, preferably between 40 and 100%, even more preferably between 45 and 100%.
• the anti-TNF ⁇ antibody of the present invention leaves TNF ⁇ signalling through the p75R largely unaffected.
• the anti-TNF ⁇ antibody of the present invention reduces TNF ⁇ signalling through the p75R by no more than around 50%, preferably by between 0 and 50%.
• the anti-TNF ⁇ antibody of the present invention reduces TNF ⁇ signalling through the p75R by no more than around 40%.
• the anti-TNF ⁇ antibody of the present invention reduces TNF ⁇ signalling through the p75R by no more than around 30%.
• the anti-TNF ⁇ antibody of the present invention reduces TNF ⁇ signalling through the p75R by no more than around 20%. In one example the anti-TNF ⁇ antibody of the present invention reduces TNF ⁇ signalling through the p75R by no more than around 10%.
• the anti-TNF ⁇ antibody of the present invention leaves the binding of TNF ⁇ to the p75R largely unaffected.
• the anti-TNF ⁇ antibody of the present invention reduces binding of TNF ⁇ to the p75R by no more than around 30%, preferably by between 0 and 30%, more preferably by between 0 and 20%, even more preferably by between 0 and 15%.
• TNF ⁇ signalling through the p75R is reduced by no more than 40%, generally by no more than 30%, usually by no more than 25%, typically by no more than 20%, ideally by no more than 10%.
• the anti-TNF ⁇ antibody of the present invention has an IC 5O for TNF ⁇ signalling through the p55R which is at least 5 fold lower, generally at least 10 fold lower, typically at least 15 fold lower, usually at least 20 fold lower, ideally at least 50 fold lower, preferably at least 100 fold lower than its IC 50 for TNF ⁇ signalling through the p75R.
• IC 5O for TNF ⁇ signalling through the p55R which is at least 5 fold lower, generally at least 10 fold lower, typically at least 15 fold lower, usually at least 20 fold lower, ideally at least 50 fold lower, preferably at least 100 fold lower than its IC 50 for TNF ⁇ signalling through the p75R.
• a lower IC 50 figure denotes a more active compound.
• antibodies with these properties are identified by first identifying antibodies that interact with TNF ⁇ and subsequently testing those antibodies to identify those that selectively inhibit TNF ⁇ signalling through the p55R.
• antibodies are identified by first identifying antibodies that interact with TNF ⁇ and subsequently testing those antibodies to identify those that selectively inhibit the binding of TNF ⁇ to the p55R and optionally further screening those antibodies for selective inhibition of signalling.
• antibodies may be screened directly to identify those that selectively inhibit TNF ⁇ signalling through the p55R relative to the p75R, for example by screening directly in signalling and/or binding assays.
• Antibodies that interact with TNF ⁇ may be identified using any suitable method, for example by using an assay system where the TNF ⁇ polypeptide is contacted with a candidate antibody and the ability of the candidate antibody to interact with the TNF ⁇ polypeptide is determined. Preferably, the ability of a candidate antibody to interact with a TNF ⁇ polypeptide is compared to a reference range or control. If desired, this assay may be used to screen a plurality of candidate antibodies using a plurality of TNF ⁇ polypeptide samples.
• a first and second sample comprising native or recombinant TNF ⁇ polypeptide are contacted with a candidate antibody or a control agent and the ability of the candidate antibody to interact with the TNF ⁇ polypeptide is determined by comparing the difference in interaction between the candidate antibody and the control agent.
• the TNF ⁇ polypeptide is first immobilized, by, for example, contacting the polypeptide with an immobilized antibody which specifically recognizes and binds it, or by contacting a purified preparation of TNF ⁇ polypeptide with a surface designed to bind proteins.
• the TNF ⁇ polypeptide may be partially or completely purified (e.g. partially or completely free of other polypeptides) or part of a cell lysate.
• polypeptide may be a fusion protein comprising the TNF ⁇ polypeptide or a biologically active portion thereof and a domain such as glutathionine-S-transferase or the Fc region of IgGl.
• polypeptide can be biotinylated using techniques well known to those of skill in the art (e.g. biotinylation kit, Pierce Chemicals; Rockford, IL).
• the TNF ⁇ polypeptide or the candidate antibody is labelled, for example with a radioactive label (such as 32 P, 35 S or 125 I) or a fluorescent label (such as fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde or fluorescamine) to enable detection of an interaction between the TNF ⁇ polypeptide and a candidate antibody.
• a radioactive label such as 32 P, 35 S or 125 I
• a fluorescent label such as fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde or fluorescamine
• FMAT fluorescent microvolume assay technology
• the antibodies of the present invention selectively inhibit TNF ⁇ signalling through the p55R by inhibiting binding of TNF ⁇ to the p55R.
• Antibodies which selectively inhibit the binding of TNF ⁇ to the p55R may be identified by any suitable method, for example by:
• Such assays can be used to screen candidate agents, in clinical monitoring and/or in drug development.
• TNF ⁇ receptor (p55R and p75R) binding inhibition assays have been described, see for example US 5,606,023 and Loetscher et ⁇ /., 1993, The Journal of Biological Chemistry, 268, 26350-26357. Further examples of suitable cell-free and cell- based assays are provided in the Examples.
• the ability of a candidate antibody to selectively inhibit the binding of TNF ⁇ to the p55R is compared to a reference range or control.
• this assay may be used to screen a plurality of candidate antibodies using a plurality of receptor binding inhibition assays.
• a first and second sample comprising native or recombinant TNF ⁇ polypeptide are contacted with a candidate antibody or a control agent and the ability of the candidate antibody to inhibit the binding of the TNF ⁇ polypeptide to either the p55R or p75R is determined by comparing the difference in binding of TNF ⁇ to each receptor in the presence of the candidate antibody and a control agent.
• the extracellular domain of the receptor polypeptide is first immobilized, by, for example, contacting the extracellular domain of the appropriate receptor with an immobilized antibody which specifically recognizes and binds it, or by contacting a purified preparation of the receptor polypeptide with a surface designed to bind proteins.
• the receptor polypeptide may be partially or completely purified (e.g. partially or completely free of other polypeptides) or part of a cell lysate.
• the receptor polypeptide may be a fusion protein comprising the extracellular domain of the receptor or a biologically active portion thereof and a domain such as glutathionine-S-transferase or the Fc portion of IgGl.
• the receptor polypeptide can be biotinylated using techniques well known to those of skill in the art (e.g. biotinylation kit, Pierce Chemicals; Rockford, IL).
• biotinylation kit Pierce Chemicals; Rockford, IL
• the ability of the candidate antibody to inhibit the binding of TNF ⁇ to the immobilised p55 or p75 receptors can be determined by methods known to those of skill in the art, for example, ELISA, BIAcoreTM, Flow cytometry or fluorescent microvolume assay technology (FMAT).
• the TNF ⁇ polypeptide is first immobilized, by, for example, contacting the polypeptide with an immobilized antibody which specifically recognizes and binds it, or by contacting a purified preparation of TNF ⁇ polypeptide with a surface designed to bind proteins.
• the ability of a candidate antibody to selectively inhibit the binding of TNF ⁇ to the p55R or p75R can be determined by incubating the candidate antibody with the immobilised TNF ⁇ polypeptide, contacting the TNF ⁇ polypeptide with either the p55R or the p75R polypeptide and detecting whether the receptor has bound to the TNF ⁇ polypeptide.
• the p55R and p75R polypeptides may each be a fusion protein comprising the extracellular domain of the receptor or a biologically active portion thereof and a domain such as the Fc portion of IgGl .
• Receptor binding may be detected by using for example anti-IgG Fc antibodies which bind to the Fc portion of the receptor fusion protein conjugated to a reporter group such as peroxidase. The presence or absence of receptor binding can be used to determine whether the candidate antibody has selectively blocked the binding of TNF ⁇ to the p55R.
• a population of cells expressing either the p55R or p75R is contacted with TNF ⁇ and a candidate antibody and the ability of the candidate antibody to inhibit the binding of TNF ⁇ to the receptor is determined.
• the ability of a candidate antibody to inhibit TNF ⁇ binding is compared to a reference range or control.
• the cell for example, can be of eukaryotic origin (e.g. yeast or mammalian) and can express the p55R or p75R endogenously or be genetically engineered to express the polypeptide.
• the TNF ⁇ polypeptide is labelled, for example with a radioactive label (such as 32 P, 35 S or 125 I) or a fluorescent label (such as fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde or fluorescamine) to enable detection of an interaction between the TNF ⁇ polypeptide and the receptor.
• a radioactive label such as 32 P, 35 S or 125 I
• a fluorescent label such as fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde or fluorescamine
• Alternative methods such as ELISA, flow cytometry and FMAT may also be used.
• Antibodies which selectively inhibit TNF ⁇ signalling through the p55R for example by selectively inhibiting binding of TNF ⁇ to the p55R may be identified using cell
• L929 cells (a mouse fibroblast cell line) which express the mouse p55R but not the p75R are used to determine whether a candidate antibody blocks TNF ⁇ signalling through the p55R e.g. by inhibiting binding to the p55R.
• These cells are killed by human TNF ⁇ if sensitised with a protein synthesis inhibitor such as actinomycin D hence, for example, if a candidate antibody blocks binding of TNF ⁇ to the p55R it protects the cells from TNF ⁇ mediated cytotoxicity.
• Blocking antibodies can therefore be detected by determining cell viability at the end of the assay.
• the assay is described in detail in the Examples provided herein and in WO92/11383.
• the binding of TNF ⁇ to one of its receptors and the resulting receptor signalling can be detected using a cell based reporter gene assay using reporter genes such as, for example, a luciferase, ⁇ -galactosidase, alkaline phosphatase, or green fluorescent protein linked to at least the extracellular region (or a TNF ⁇ binding portion thereof) of TNF ⁇ receptors p55 or p75 to detect downstream gene expression following TNF ⁇ binding. Details of examples of such assays are provided in the Examples.
• a reduction in reporter gene expression is indicative of a candidate antibody blocking TNF ⁇ signalling through the receptor, for example by inhibiting binding to the receptor.
• the present invention therefore provides a method of obtaining an anti-TNF ⁇ antibody that selectively inhibits the binding of TNF ⁇ to the p55R comprising: a) obtaining at least one anti-TNF ⁇ antibody b) screening the antibody obtained in step (a) to determine whether the antibody selectively inhibits TNF ⁇ signalling through the p55R, for example by selectively inhibiting the binding of TNF ⁇ to the p55R and where necessary, repeating steps (a) and (b) until at least one selective antibody is found.
• the antibody identified in step (b) of the method selectively inhibits the binding of TNF ⁇ to the p55R by greater than 45% and inhibits the binding of TNF ⁇ to the p75R by no more than 30%.
• the antibodies obtained in step (a) of the method are obtained from an immunised animal, preferably using the methods described in for example, Babcook, J. et al, 1996, Proc. Natl. Acad. Sci. USA 93(15):7843-7848; WO92/02551; WO2004/051268 and International Patent Application number WO2004/106377.
• Anti-TNF ⁇ antibodies that selectively inhibit TNF ⁇ signalling through the p55R, for example through inhibiting the binding of TNF ⁇ to the p55R may be identified or further tested, for example to determine therapeutically effective amounts in one or more animal models.
• suitable animals include, but are not limited to, mice, rats, rabbits, monkeys, guinea pigs, dogs and cats.
• the animal used represents a model of an autoimmune or inflammatory disease, such as MS, diabetes, SLE, rheumatoid arthritis, autoimmune haemolytic anemia, myasthenia gravis, Grave's disease, idiopathic thrombocytopenic purpura, autoimmune thyroiditis, Behcets disease, Wegener's granulomatosis, psoriasis, psoriatic arthritis, ankylosing spondylitis or inflammatory bowel disease, including Crohn's disease and Ulcerative colitis.
• an autoimmune or inflammatory disease such as MS, diabetes, SLE, rheumatoid arthritis, autoimmune haemolytic anemia, myasthenia gravis, Grave's disease, idiopathic thrombocytopenic purpura, autoimmune thyroiditis, Behcets disease, Wegener's granulomatosis, psoriasis, psoriatic arthritis, ankylosing spondy
• the selective inhibition of TNF ⁇ signalling through the p55R can be determined by monitoring an amelioration or improvement in disease symptoms, a delayed onset or slow progression of the disease, for example but without limitation, a reduction in clinical score.
• Techniques known to physicians familiar with autoimmune disease can be used to determine whether a candidate agent has altered one or more symptoms associated with the disease.
• autoimmune disease A number of different models of autoimmune disease are known in the art, for example there are a number of disease models for MS ('t Hart and Amor 2003, Current Opinion in Neurology 16:375-83).
• EAE experimental autoimmune encephalomyelitis
• the present invention also provides a specific region of the TNF ⁇ polypeptide wherein binding of an antibody to that region selectively inhibits TNF ⁇ signalling through the p55R, for example by inhibiting the binding of TNF ⁇ to the p55R relative to the p75R.
• This specific region or epitope of the TNF ⁇ polypeptide can be identified by any suitable epitope mapping method known in the art in combination with the antibody provided by the present invention. Examples of such methods include screening peptides of varying lengths derived from TNF ⁇ for binding to the antibody of the present invention with the smallest fragment that can specifically bind to the antibody containing the sequence of the epitope recognised by the antibody.
• the TNF ⁇ peptides may be produced synthetically or by proteolytic digestion of the TNF ⁇ polypeptide.
• Peptides that bind the antibody can be identified by mass spectrometric analysis. In another example, NMR spectroscopy can be used to identify the epitope of the present invention. Once identified, the epitopic fragment which binds an antibody of the present invention can be used, if required, to obtain additional antibodies which bind the same epitope.
• a specific region or epitope of human TNF ⁇ wherein binding of antibody '462' or '463' or antibodies comprising one or more CDRs given in SEQ ID NOs 9, 10, 11, 12, 13, 14 and 21 to that region selectively inhibits TNF ⁇ signalling through the p55R.
• TNF ⁇ may be similarly useful in selectively inhibiting TNF ⁇ signalling through the p55R.
• an antibody having specificity for human TNF ⁇ which cross-blocks the binding of antibody '462' or antibody '463' or any antibody comprising one or more of the CDRs provided in SEQ ID NOs 9, 10, 11, 12, 13, 14 and 21 to human TNF ⁇ and/or is cross-blocked from binding to human TNF ⁇ by any one of those antibodies.
• an antibody according to this aspect of the invention binds to the same epitope as antibody '462' or antibody '463' or any antibody comprising one or more of the CDRs provided in SEQ ID NOs 9, 10, 11, 12, 13, 14 and 21.
• the antibody according to this aspect of the invention binds to an epitope which borders and/or overlaps with the epitope bound by antibody '462' or antibody '463' or any antibody comprising one or more of the CDRs provided in SEQ ID NOs 9, 10, 11, 12, 13, 14 and 21.
• the antibody according to this aspect of the invention does not bind to the same epitope as antibody '462' or antibody '463' or any antibody comprising one or more of the CDRs provided in SEQ ED NOs 9, 10, 11, 12, 13, 14 and 21 or an epitope that borders and/or overlaps with said epitope.
• Cross-blocking antibodies according to this aspect of the present invention can be identified using any suitable method in the art, for example by using competition ELISA or BIAcore where binding of the cross blocking antibody to human TNF ⁇ prevents the binding of antibody '462' or antibody '463' or any antibody comprising one or more of the CDRs provided in SEQ ID NOs 9, 10, 11, 12, 13, 14 and 21 or vice versa.
• an anti-TNF ⁇ antibody which selectively inhibits TNF ⁇ signalling through the p55R, which cross-blocks the binding of antibody '462' or antibody '463' or an antibody comprising one or more of the CDRs provided in SEQ ID NOs 9, 10, 11, 12, 13, 14 and 21 to human TNF ⁇ .
• the cross-blocking antibodies provided by this aspect of the invention inhibit the binding of antibody '462' or antibody '463' or an antibody comprising one or more of the CDRs provided in SEQ ID NOs 9, 10, 11, 12, 13, 14 and 21 to human TNF ⁇ by 80% or greater, preferably by 85% or greater, more preferably by 90% or greater, even more preferably by 95% or greater.
• antibodies according to this aspect of the invention may be cross-blocked from binding to human TNF ⁇ by any one of antibody '462' or antibody '463' or an antibody comprising one or more of the CDRs provided in SEQ ID NOs 9, 10, 11, 12, 13, 14 and 21.
• an anti-TNF ⁇ antibody which selectively inhibits TNF ⁇ signalling through the p55R which is cross-blocked from binding human TNF ⁇ by antibody '462' or antibody '463' or an antibody comprising one or more of the CDRs provided in SEQ ID NOs 9, 10, 11, 12, 13, 14 and 21.
• the cross- blocking antibodies provided by this aspect of the invention are inhibited from binding human TNF ⁇ by antibody '462' or antibody '463' or an antibody comprising one or more of the CDRs provided in SEQ ID NOs 9, 10, 11, 12, 13, 14 and 21 by 80% or greater, preferably by 85% or greater, more preferably by 90% or greater, even more preferably by 95% or greater.
• an antibody for use in the present invention may be conjugated to an effector molecule.
• effector molecule as used herein includes, for example, antineoplastic agents, drugs, toxins, biologically active proteins, for example enzymes, other antibody or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof e.g.
• anti-TNF ⁇ antibodies can be conjugated to an effector molecule, such as a cytotoxic agent, a radionuclide or drug moiety to modify a given biological response.
• the therapeutic agent may be a drug moiety which may be a protein or polypeptide possessing a desired biological activity.
• Such moieties may include, for example and without limitation, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin, a protein such as tumour necrosis factor, ⁇ -interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor or tissue plasminogen activator, a thrombotic agent or an anti- angiogenic agent, e.g.
• a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin
• a protein such as tumour necrosis factor, ⁇ -interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor or tissue plasminogen activator, 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), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G- CSF), nerve growth factor (NGF) or other growth factor.
• IL-I interleukin-1
• IL-2 interleukin-2
• IL-6 interleukin-6
• GM-CSF granulocyte macrophage colony stimulating factor
• G- CSF granulocyte colony stimulating factor
• NGF nerve growth factor
• the effector molecules may be cytotoxins or cytotoxic agents including any agent that is detrimental to (e.g. kills) cells.
• cytotoxins include 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- mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g. mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g.
• antimetabolites e.g. methotrexate, 6- mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine
• alkylating agents e.g. mechlorethamine, thioepa chlor
• daunorubicin (formerly daunomycin) and doxorubicin
• antibiotics e.g. dactinomycin (formerly actinomycin), bleomycin, mithramycin, anthramycin (AMC), calicheamicins or duocarmycins
• anti-mitotic agents e.g. vincristine and vinblastine.
• Other effector molecules may include radionuclides such as ⁇ 1 In and 90 Y, Lu 177 ,
• Bismuth 213 Californium 252 , Iridium 192 and Tungsten 188 /Rhenium 188 ; or drugs such as but not limited to, alkylphosphocholines, topoisomerase I inhibitors, taxoids and suramin.
• the antibody or fragment thereof is fused via a covalent bond (e.g. a peptide bond), at optionally the N-terminus or the C-terminus, to an amino acid sequence of another protein (or portion thereof; preferably at least a 10, 20 or 50 amino acid portion of the protein).
• a covalent bond e.g. a peptide bond
• the antibody, or fragment thereof is linked to the other protein at the N-terminus of the constant domain of the antibody.
• Recombinant DNA procedures may be used to create such fusions, for example as described in WO 86/01533 and EP 0392745.
• the effector molecule may increase half-life in vivo, and/or enhance the delivery of an antibody across an epithelial barrier to the immune system.
• suitable effector molecules of this type include polymers, albumin, albumin binding proteins or albumin binding compounds such as those described in PCT/GB2005/002084.
• antibodies of the present invention may be attached to poly(ethyleneglycol) (PEG) moieties.
• 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 free amino, imino, thiol, hydroxyl or carboxyl group.
• 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.
• Multiple sites can be used to attach two or more PEG molecules.
• PEG molecules are covalently linked through a thiol group of at least one cysteine residue located in the antibody fragment. Where a thiol group is used as the point of attachment appropriately activated effector molecules, for example thiol selective derivatives such as maleimides and cysteine derivatives may be used.
• the antibody is a modified Fab' fragment which is PEGylated, i.e. has PEG (poly(ethyleneglycol)) covalently attached thereto, e.g. according to the method disclosed in EP 0948544 [see also "Poly(ethyleneglycol) Chemistry, Biotechnical and Biomedical Applications", 1992, J. Milton Harris (ed), Plenum Press, New York, “Poly(ethyleneglycol) Chemistry and Biological Applications", 1997, J. Milton Harris and S. Zalipsky (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.
• PEG is attached to a cysteine in the hinge region.
• 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 20,000 Da.
• the total molecular weight of the PEG attached to the Fab' fragment may therefore be approximately 40,000 Da.
• Particular PEGylated antibody fragments also include those described in International patent applications WO2005003169, WO2005003170 and WO2005003171.
• the present invention also provides an isolated DNA sequence encoding the heavy and/or light chain(s) of an antibody molecule of the present invention.
• 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 comprise 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 for 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 appropriate.
• PCR polymerase chain reaction
• SEQ ID NO:1 SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19.
• the present invention also relates to a cloning or expression vector comprising one or more DNA sequences of the present invention.
• a cloning or expression vector comprising one or more DNA sequences encoding an antibody of the present invention.
• the cloning or expression vector comprises two DNA sequences, encoding the light chain and the heavy chain of the antibody molecule of the present invention, respectively.
• 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, reference 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.
• a host cell comprising one or more cloning or expression vectors comprising one or more DNA sequences encoding an antibody of the present invention.
• Any suitable host cell/vector system may be used for expression of the DNA sequences encoding the antibody molecule of the present invention.
• Bacterial, for example E. coli, 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 hybridoma cells.
• the present invention also provides a process for the production of an antibody molecule according to the present invention comprising culturing a host cell containing a vector of the present invention under 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 comprise 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.
• the cell line may be transfected with two vectors, a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide.
• a single vector may be used, the vector including sequences encoding light chain and heavy chain polypeptides.
• the present invention also provides a method for the treatment and/or prophylaxis of an autoimmune or inflammatory disease comprising administering a therapeutically effective amount of an anti-TNF ⁇ antibody that selectively inhibits TNF ⁇ signalling through the p55R, for example by selectively inhibiting the binding of TNF ⁇ to the p55R.
• the invention also provides the use of an anti-TNF ⁇ antibody that selectively inhibits TNF ⁇ signalling through the p55R, for example by selectively inhibiting the binding of TNF ⁇ to the p55R for the manufacture of a medicament for the treatment and/or prophylaxis of autoimmune or inflammatory disease.
• the term 'treatment' includes either therapeutic or prophylactic therapy.
• Antibodies which selectively inhibit TNF ⁇ signalling through the p55R, for example by inhibiting the binding of TNF ⁇ to the p55R can be used in the manufacture of a medicament for the treatment of any disease resulting from p55R mediated signalling, in particular autoimmune and inflammatory diseases.
• autoimmune and inflammatory diseases include demyelinating autoimmune diseases of the CNS, multiple sclerosis (MS), diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis, autoimmune haemolytic anemia, myasthenia gravis, Grave's disease, idiopathic thrombocytopenic purpura, autoimmune thyroiditis, Behcets disease, Wegener's granulomatosis, psoriasis, psoriatic arthritis, ankylosing spondylitis, inflammatory bowel disease, including Crohn's disease and Ulcerative colitis.
• MS multiple sclerosis
• SLE systemic lupus erythematosus
• rheumatoid arthritis autoimmune haemolytic anemia
• myasthenia gravis Grave's disease
• idiopathic thrombocytopenic purpura autoimmune thyroiditis
• Behcets disease Wegener's gran
• anti-TNF ⁇ antibodies which selectively inhibit TNF ⁇ signalling through the p55R for example by inhibiting the binding of TNF ⁇ to the p55R can be used in the treatment and/or prophylaxis of autoimmune and inflammatory diseases.
• the agents will generally be administered in the form of a pharmaceutical composition.
• composition comprising an anti-TNF ⁇ antibody which selectively inhibits TNF ⁇ signalling through the p55R and a pharmaceutically acceptable carrier.
• the composition will usually be supplied as part of a sterile, pharmaceutical composition that will normally include a pharmaceutically acceptable carrier.
• This composition may be in any suitable form (depending upon the desired method of administering it to a patient).
• the antibodies of the invention are preferably administered to a subject by a variety of other routes such as orally, transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intrathecally and intracerebroventricularly. The most suitable route for administration in any given case will depend on the particular antibody, the subject, and the nature and severity of the disease and the physical condition of the subject.
• the antibodies of use in the invention may be administered in combination, e.g.
• compositions may be conveniently presented in unit dose forms containing a predetermined amount of an active agent of the invention per dose.
• Such a unit may contain for example but without limitation, 750mg/kg to 0.1mg/kg depending on the condition being treated, the route of administration and the age, weight and condition of the subject.
• Pharmaceutically acceptable carriers for use in the invention may take a wide variety of forms depending, e.g. on the route of administration.
• compositions for oral administration may be liquid or solid.
• Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use.
• Oral liquid preparations may contain suspending agents as known in the art.
• oral solid preparations such as powders, capsules and tablets
• carriers such as starches, sugars, microcrystalline cellulose, granulating agents, lubricants, binders, disintegrating agents, and the like may be included. Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are generally employed.
• active agents of the invention may also be administered by controlled release means and/or delivery devices.
• Tablets and capsules may comprise conventional carriers or excipients such as binding agents for example, syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tableting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch; or acceptable wetting agents such as sodium lauryl sulphate.
• the tablets may be coated by standard aqueous or non-aqueous techniques according to methods well known in normal pharmaceutical practice.
• compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active agent, as a powder or granules, or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water- in-oil liquid emulsion.
• Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association the active agent with the carrier, which constitutes one or more necessary ingredients.
• the compositions are prepared by uniformly and intimately admixing the active agent with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.
• a tablet may be prepared by compression or moulding, optionally with one or more accessory ingredients.
• Pharmaceutical compositions suitable for parenteral administration may be prepared as solutions or suspensions of the active agents of the invention in water suitably mixed with a surfactant such as hydroxypropylcellulose.
• Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
• the pharmaceutical forms suitable for injectable use include aqueous or non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the composition isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
• aqueous or non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the composition isotonic with the blood of the intended recipient
• aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
• Extemporaneous injection solutions, dispersions and suspensions may be prepared from sterile powders, granules and tablets.
• compositions can be administered with medical devices known in the art.
• a pharmaceutical composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in US 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556.
• Examples of well-known implants and modules useful in the present invention include: US 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; US 4,486,194, which discloses a therapeutic device for administering medicaments through the skin; US 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; US 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; US 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and US 4,475,196, which discloses an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known to those skilled in the art.
• compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, impregnated dressings, sprays, aerosols or oils, transdermal devices, dusting powders, and the like.
• These compositions may be prepared via conventional methods containing the active agent.
• they may also comprise compatible conventional carriers and additives, such as preservatives, solvents to assist drug penetration, emollients in creams or ointments and ethanol or oleyl alcohol for lotions.
• Such carriers may be present as from about 1% up to about 98% of the composition. More usually they will form up to about 80% of the composition.
• a cream or ointment is prepared by mixing sufficient quantities of hydrophilic material and water, containing from about 5-10% by weight of the compound, in sufficient quantities to produce a cream or ointment having the desired consistency.
• compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.
• the active agent may be delivered from the patch by iontophoresis.
• compositions are preferably applied as a topical ointment or cream.
• the active agent may be employed with either a paraffinic or a water-miscible ointment base.
• the active agent may be formulated in a cream with an oil-in-water cream base or a water-in-oil base.
• compositions adapted for topical administration in the mouth include lozenges, pastilles and mouth washes.
• Pharmaceutical compositions adapted for topical administration to the eye include eye drops wherein the active agent is dissolved or suspended in a suitable carrier, especially an aqueous solvent. They also include topical ointments or creams as above.
• compositions suitable for rectal administration wherein the carrier is a solid are most preferably presented as unit dose suppositories.
• Suitable carriers include cocoa butter or other glyceride or materials commonly used in the art, and the suppositories may be conveniently formed by admixture of the combination with the softened or melted carrier(s) followed by chilling and shaping moulds. They may also be administered as enemas.
• the dosage to be administered of an anti-TNF ⁇ antibody which selectively inhibits TNF ⁇ signalling through the p55R will vary according to the particular antibody, the type of autoimmune or inflammatory disease, the subject, and the nature and severity of the disease and the physical condition of the subject, and the selected route of administration; the appropriate dosage can be readily determined by a person skilled in the art.
• pharmaceutical compositions comprising antibodies can be administered to patients (e.g., human subjects) at therapeutically or prophylactically effective dosages (e.g. dosages which result in inhibition of an autoimmune or inflammatory disease and/or relief of autoimmune or inflammatory disease symptoms) using any suitable route of administration, such as injection and other routes of administration known in the art for clinical products, such as antibody-based clinical products.
• compositions may contain from 0.1% by weight, preferably from 10-60%, or more, by weight, of the inhibitor of the invention, depending on the method of administration.
• Figure 1 p55TNFR and p75TNFR binding inhibition assay showing the effect of different anti-TNF ⁇ antibodies on the binding of TNF ⁇ to the p55R and the p75R.
• Figure 2 shows the expression cassette cloned into the large Notl and Xhol restricted fragment of pBluescript® II SK(+) to generate the bioassay receptor shuttle vector.
• Figure 3 shows a titration of the TNF ⁇ induced luciferase response from the p75/CD28-
• TCR zeta bioassay receptor TCR zeta bioassay receptor
• Figure 4 shows the effect of antibody '462', infliximab and adalimumab on p55R signalling.
• Figure 5 shows the effect of antibody '463' on p55R signalling.
• Figure 6 shows the effect of antibody '462', infliximab and adalimumab on p75R signalling.
• Figure 7 shows the effect of antibody '463' on p75R signalling. Examples Example 1. Isolation of a panel of anti-TNF ⁇ antibodies
• Rats were immunised with soluble human recombinant TNF ⁇ . 4x 5ug at 3-4 week intervals initially in complete Freund's adjuvant by the sub-cutaneous route.
• Spleen cells from one rat were then seeded into 40 microtitre plates at a cell density that ensures that any detected TNF ⁇ binding antibody is clonal.
• the cells were then cultured in T cell conditioned media (3%) and EL-4 cells (5xlO 4 /well) for seven days.
• L929 Assay L929 cells (a mouse fibroblast cell line) that express the mouse p55TNF ⁇ receptor but not the p75TNF ⁇ receptor were used to assay for anti-TNF ⁇ antibodies that block binding of TNF ⁇ to this receptor. These cells are killed by human TNF ⁇ if sensitised with a protein synthesis inhibitor.
• Standard ELISA plates were coated with a sheep anti-human TNF ⁇ polyclonal antibody diluted 1/10,000. The plates were then blocked with PBS+1% BSA. Human TNF ⁇ was then added to each well at 25-50ng/ml. After 1 hour unbound TNF ⁇ was washed off.
• Supernatants containing anti-TNF ⁇ antibodies were then added to replicate wells. In addition to one well of each replicate was added either human p55TNFR- Human Fc fusion protein or Human p75TNFR- Human Fc fusion protein. These were incubated for lhour and then washed to remove unbound receptor. Following this step an anti-Human IgG Fc peroxidase conjugated polyclonal antibody (Stratech Scientific) was added at 1/2000 dilution. The plates were left for lhr and then washed to remove unbound conjugate. TMB substrate was then added to each well, and the colour allowed to develop. Wells where the anti- TNF ⁇ antibodies have blocked binding of the receptor(s) can therefore be visualised.
• Figure 1 shows the percentage inhibition of TNF ⁇ binding to the p55TNFR and p75TNFR by four different anti-TNF antibodies.
• Antibody '3D6' inhibited binding of TNF ⁇ to the p55TNFR by 49.3% but only inhibited binding of TNF ⁇ to the p75TNFR by 14.6%.
• antibody 22H3 for example inhibited binding of TNF ⁇ to the p55R and the p75R by 78.9 and 71.9% respectively.
• Antibody 3D6 therefore selectively blocks binding of TNF ⁇ to the p55R.
• Example 1 Using the same rat population as Example 1 cultured B cells were screened to identify TNF ⁇ selective antibodies.
• TNF ⁇ Human TNF ⁇ (Strathman Biotech GmbH) was biotinylated with a 10 fold molar excess of Sulfo-NHS-LC-LC-biotin (Pierce) for 1 hour at room temperature following the manufacturers' protocol. 5 ⁇ g of biotinylated TNF ⁇ was mixed with 50 ⁇ l of 9.95 micron superavidin coated microspheres (Bangs Beads) for 1 hour at room temperature in a volume of 500 ⁇ l (mix for 1 x 384-well plate). Beads were then washed 5 times in PEG block (1% PEG/0.1% tween/PBS) to remove unbound TNF ⁇ . TNF ⁇ -coated beads were then resuspended in approx.
• Variable regions were expressed in recombinant IgG format to confirm binding and activity in signalling assays by sub-cloning into expression vectors containing the human antibody constant region genes (human kappa light chain and gamma-4 heavy chain in which the serine at position 241 has been changed to proline as described in Angal et al., Molecular Immunology, 1993, 30 (1), 105-108) and a rat/human chimeric antibody expressed transiently in CHO cells.
• human antibody constant region genes human kappa light chain and gamma-4 heavy chain in which the serine at position 241 has been changed to proline as described in Angal et al., Molecular Immunology, 1993, 30 (1), 105-108
• V-region sequences of '462' are given in SEQ ID NOS: 1, 2, 3 and 4.
• the variable region sequences without the leader sequences are provided in SEQ ID NOS: 5, 6, 7 and 8.
• the V-region sequences of 463' are given in SEQ ID NOS: 15, 16, 17 and 18.
• the variable region sequences without the leader sequences are provided in SEQ ID NOS: 19, 20, 7 and 8.
• A549-ES-Luc cells were used for this reporter gene assay.
• A549 cells are an epithelial lung cell carcinoma that express the p55 TNF receptor and have been stably transfected with a vector comprising the E-selectin promoter (contains 3 x NFkB binding sites) linked to the luciferase gene and a selectable marker for stable cell line generation.
• A549-ES-Luc were grown in the following media:
• Jurkat cells that have been stably transfected with a vector containing a cassette coding for the p75R extra-cellular domain linked to the intra-cellular signalling regions of CD28 and TCR zeta was used to assay for p75 signalling.
• a vector containing a cassette coding for the p75R extra-cellular domain linked to the intra-cellular signalling regions of CD28 and TCR zeta was used to assay for p75 signalling.
• Within the same vector there are 5 binding sites for NFKB with a minimal E-selectin promoter region, this drives expression of the reporter gene luciferase, and a selectable marker for stable cell line generation.
• Stimulation of the p75 bioassay receptor with its ligand, human TNF ⁇ leads via the CD28/zeta regions of the bioassay receptor, to the initiation of a signalling cascade within the cell.
• the signalling cascade induces NFKB activation and allows transcription of the luciferase reporter gene. Activation levels can then be measured in a luciferase assay. Antibodies that can block this activation will prevent expression of luciferase.
• This vector includes the cloning cassette devised in pBluescript SK+ (Stratagene) described previously (Finney et al., J.Immunol. 2004 172: 104). 5' to this cloning cassette is the HCMV promoter, and the SV40 polyadenylation signal is 3' to this cloning cassette.
• the cloning cassette consists of an extracellular domain (ECD) binding component, a transmembrane component and a signalling region component, and facilitates easy exchange of each individual component. Combining the following DNA fragments generated the shuttle vector:
• a fragment comprising residues 135 to 202 of human CD28 transmembrane and signalling region and residues 31 to 142 of human TCR zeta intracellular region was digested from a plasmid previously described (Finney et al., J.Immunol. 2004 172: 104) with restriction enzymes Narl and EcoRI. Construction of Bioassay receptor reporter gene vectors
• the full length expression cassette for the Bioassay receptor was generated by combining the binding, transmembrane and signalling components described above in the shuttle vector described above. This was then subcloned into the reporter gene vector pNifty2-Luc(Invivogen). This vector contains a Luciferase reporter gene under control of a NF-kB inducible promoter and the selectable marker ZeocinTM for selection in both E.coli and mammalian cells. The Bioassay receptor expression cassette was removed from the shuttle vector on a Notl to Notl fragment and cloned into the Notl site of pNifty2-Luc.
• TNF ⁇ Luclite assay kit
• the TNF ⁇ induced Luciferase response from the p75/CD28-TCR zeta Bioassay receptor is shown in figure 3.
• a concentration of TNF ⁇ was selected from this titration and used to assess the ability of an anti-TNF ⁇ antibody to block Luciferase production via the p75/CD28-TCR zeta Bioassay receptor.
• protease inhibitor Jurkat cells were plated out into white opaque 96-well plates using a cell suspension of 2x10 6 cells/ml. Antibodies were then added to the plate in the desired titration scale. The plate was incubated for 30 minutes at 37 0 C and lO ⁇ l of human TNF ⁇ ligand added to each well at a concentration of 30 ng/ml to give a final concentration of 3ng/ml human TNF ⁇ in each well. The plate was incubated for 4 hours at 37 0 C. Luciferase expression was then assayed using a luciferase reporter gene assay kit (Luclite 1000 kit, Perkin-Elmer).
• Adalimumab and Infliximab on Luciferase production in the p75R signalling assay is shown in Figure 6. It is clear that only Adalimumab and Infliximab inhibit TNF ⁇ signalling through the p75R while antibody '462' leaves TNF ⁇ signalling through the p75R largely unaffected. Figure 7 shows that antibody '463' also leaves TNF ⁇ signalling through the p75R largely unaffected. Both antibodies '462' and '463' were significantly less potent in the p75R signalling assay than in the p55R signalling assay. Antibodies '462' and '463' therefore selectively inhibit TNF ⁇ signalling through the p55R.

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