HK1125113B - Antibody molecules having specificity for human il-6 - Google Patents

Description

Antibody molecules specific for human IL-6

The present invention relates to antibody molecules having specificity for IL-6 antigenic determinants. The invention also relates to the therapeutic use of the antibody molecule and to a method for the production of the antibody molecule.

IL-6 is a pleiotropic multifunctional cytokine produced by a variety of cell types. It was originally identified as a B-cell differentiation factor (BSF-2) that induces the final maturation of B-cells into antibody-producing cells (Hirano et al, 1986Nature 324, 73-76). IL-6 has been shown to play an important role in immune regulation, inflammation, hematopoiesis and tumorigenesis. Within the immune system, IL-6 induces B-cell antibody production that increases polyclonal immunoglobulin content. It also induces interleukin-2 (IL-2) receptor expression on T-cells (Nomo et al, 1987, Immunol. letters, 15, 3, 249-154253) and promotes IL-2 production in activated T-cells, thereby inducing the growth and differentiation of cytotoxic T-cells (Okada et al, 1988, J Immunol, 141, 5, 1543-1549). IL-6 is also known to determine monocyte differentiation into macrophages (Chomarat P et al, 2000, Nature Immunol., 6, 510-514).

The function of IL-6 is not limited to immune responses as it plays a role in hematopoiesis, thrombosis, osteoclastogenesis, and acute phase response elicitation of the liver, resulting in elevation of C-reactive protein (CRP) and serum amyloid a (saa). It is known to be a growth factor for epidermal keratinocytes, mesangial cells, myelomas and plasmacytomas (Grossman et al, 1989, ProtNatl Acad Sci.86, (16) 6367-. IL-6 is produced by a variety of cell types including monocytes/macrophages, fibroblasts, epidermal keratinocytes, vascular endothelial cells, mesangial cells, glial cells, chondrocytes, T and B-cells, and some tumor cells (Akira et al, 1990, FASEB J., 4, 11, 2860-. Normal cells do not express IL-6 except for tumor cells that constitutively produce IL-6, unless appropriately stimulated.

IL-6 is a glycoprotein, is a 184 amino acid molecule, according to post-translational modifications, with a molecular weight of 21 to 28 kD. Alternative splice variants are found in several cell types (Kishimoto et al, 1995, Blood, 86, 4, 1243-1254). The IL-6 receptor (IL-6R) complex is composed of two functionally distinct membrane proteins, an 80kD IL-6 specific binding chain (gp80) and a 130kD signal transduction chain (gp 130). Although IL-6 cannot directly bind gp130, but can bind IL-6R, produce IL-6/IL-6R/gp30 high affinity three-element complex. IL-6R binds IL-6 with low affinity, however, IL-6R does not have an intracellular signaling domain, and thus this linkage alone cannot lead to cell activation. Similarly, cell surface expression of IL-6R does not imply that the cells respond to IL-6 stimulation. Proteolytic cleavage results in the release of soluble IL-6R (sIL-6R; sgp80), which can bind circulating IL-6 and increase the half-life of IL-6. For cell activation, IL-6 first binds to cells that bind to IL-6R or sIL-6R; the heterodimeric IL-6/IL-6R complex then binds to the cell surface glycoprotein gp 130. The resulting ternary hybrid binds to another IL-6/IL-6R/gp130, followed by signal transduction (Bravo and Heath 2000, EMBO J., 19, (11), 2399-. IL-6 signaling through cell-bound IL-6R is referred to as cis (cis) signaling, while cellular activation through soluble IL-6R is described as trans (trans) signaling. Cells expressing gp130 but not IL-6R can be stimulated by IL-6 via sIL-6R.

Neutralizing murine antibodies to human IL-6 are known to interfere with the binding of human IL-6 to IL-6R (site 1) or to gp130 (sites 2 and 3) (Kalai et al, 1997, Eur. J. biochem.249, 690-charge 700; Brakenhoff et al, 1990, Journal of Immunology, 145, 561-charge 568; Wendling et al, 1993, Journal of Rheumatology, 29, 259-charge 262).

U.S. Pat. No. 4, 5,856,135 discloses the reconstitution of human IL-6 antibodies that will block IL-6 binding to IL-6R. These antibodies were derived from mouse monoclonal antibody SK2, in which Complementarity Determining Regions (CDRs) from the mouse antibody SK2 variable region were grafted to the variable region of a human antibody.

Neutralizing human autoantibodies to IL-6 are also known (Hansen et al, Eur. J. Immunol, 1995, 25, 348-354).

WO2004039826 describes site I chimeric mouse/human anti-IL-6 antibodies for use in therapy.

Humanized anti-human IL-6 receptor monoclonal antibodies are in phase III clinical trials for rheumatoid arthritis treatment (Kishimoto, 2005, Annu Rev Immunol.23: 1-21). The same antibodies were also reported to be effective in phase II studies of crohn's disease. Also in NZB/W F₁The efficacy of anti-IL-6 and anti-IL-6R antibodies was demonstrated in lupus-like disease in mice (Fink et al, 1994, J. Clin. invest.94, 585; Mihara et al, 1998, Clin. exp. Immunol.112, 397). Neutralizing antibodies to the murine IL-6 receptor inhibited colitis in an adoptive transfer model of the disease (Yamamoto et al, 2000, Journal of immunology, 164, 4878; Atreya et al, 2000, Nature Med 6, 583). The latter study also demonstrated the efficacy of anti-receptor antibodies in an IL-10 knockout mouse model of colitis and in a TNBS model of enteritis.

We have now identified a high affinity neutralizing anti-IL-6 antibody that is particularly effective in vivo, e.g., in the in vivo model described herein.

Residues in the variable region of an antibody are numbered conventionally according to a system designed by Kabat et al. This system is listed in Kabat et al, 1987, Sequences of Proteins of immunologicalcalest, US Department of Health and Human Services, NIH, USA (hereinafter "Kabat et al (supra)"). Unless otherwise indicated, this numbering system is used in this specification.

The Kabat residue numbering does not always directly correspond to the straight line numbering of amino acid residues. Actual linear amino acid sequences may contain fewer or more amino acids than correspond to a shortening of the structural component, or insertion into the structural component of strict Kabat numbering, whether framework or Complementarity Determining Regions (CDRs) of the basic variable domain structure. For a given antibody, the correct Kabat numbering of residues can be determined by alignment of antibody sequences to homologous residues of a "standard" Kabat numbered sequence.

The CDRs of the heavy chain variable domain are located at residues 31-35(CDR-H1), 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.mol.biol., 196, 901-917(1987)), the loop corresponding to CDR-H1 extends from residue 26 to residue 32. Thus, "CDR-H1" as used herein includes residues 26 to 35 as described in connection with the Kabat numbering system and the topological loop definition of Chothia.

The CDRs of the light chain variable domain are located at residues 24-34(CDR-L1), residues 50-56(CDR-L2) and residues 89-97(CDR-L3), according to the Kabat numbering system.

As used herein, the term "neutralizing antibody" describes an antibody that is capable of neutralizing IL-6 biological signaling activity, e.g., by blocking IL-6 binding to site 3 of the gp130 receptor.

Any suitable method known in the art may be used to obtain the antibodies used in the present invention. IL-6 polypeptides or cells expressing polypeptides can be used to produce antibodies that specifically recognize IL-6. The IL-6 polypeptide may be a "mature" polypeptide or a biologically active fragment or derivative thereof. Preferably, the IL-6 polypeptide is a mature polypeptide. IL-6 polypeptides may be prepared by methods well known in the art from genetically engineered host cells including expression systems or may be collected from natural biological sources. In the present application, the term "polypeptide" includes peptides, polypeptides and proteins. These may be used interchangeably unless otherwise indicated. In some cases, the IL-6 polypeptide can be a portion of a larger protein, such as a fusion protein, e.g., fused to an affinity tag. Antibodies raised against IL-6 polypeptides can be obtained, wherein immunization of an animal is necessary, by administering the polypeptide to the animal, preferably a non-human animal, using well known and conventional protocols, see, e.g., Handbook of experimental Immunology, d.m. weir (ed.), Vol 4, blackwell scientific Publishers, Oxford, England, 1986. Many warm-blooded animals, such as rabbits, mice, rats, sheep, cows or pigs, can be immunized. However, mice, rabbits, pigs and rats are generally preferred.

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

Monoclonal Antibodies can be prepared by any method known in the art, such as hybridoma technology (Kohler & Milstein, 1975, Nature, 256: 495-497), trioma technology, human B-cell hybridoma technology (Kozbor et al, 1983, Immunology Today, 4: 72) and EBV-hybridoma technology (Cole et al, Monoclonal Antibodies and cancer therapy, pp77-96, Alan R Liss, Inc., 1985).

Antibodies used in the present invention can also be produced using the single lymphocyte antibody method by cloning and expressing immunoglobulin variable region cDNA produced from a single lymphocyte selected for the production of a specific antibody by, for example, Babcook, j. et al, 1996, proc.natl.acad.sci.usa 93 (15): 7843-78481; WO 92/02551; the methods described in WO2004/051268 and International patent application No. WO 2004/106377.

Humanized antibodies, which include CDR-grafted antibodies, are antibody molecules from non-human species having one or more Complementarity Determining Regions (CDRs) from the non-human species and framework regions from human immunoglobulin molecules (see, e.g., U.S. Pat. No. 5,585,089; WO 91/09967). It will be appreciated that it may only be necessary to transfer the specificity determining residues of the CDRs, rather than the entire CDRs (see, e.g., Kashmiri et al, 2005, Methods, 36, 25-34). The humanized antibody may optionally further comprise one or more framework residues derived from a non-human species from which the CDRs are derived.

Chimeric antibodies are those antibodies encoded by genetically engineered immunoglobulin genes such that their light and heavy chain genes are composed of immunoglobulin gene segments belonging to different species.

Antibodies useful in the invention can also be generated using a variety of phage display methods known in the art, including Brinkman et al (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, 1879-18), Burton et al (Advances in Immunology, 1994, 57: 191-280) and WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401 and US5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753, respectively; 5,821,047, respectively; 5,571,698; 5,427,908; 5,516,637; 5,780,225, respectively; 5,658,727, respectively; 5,733,743 and 5,969,108.

Fully human antibodies are those in which the variable and constant regions of both heavy and light chains (if present) are all of human origin, or are substantially identical to, but not necessarily from the same sequence as, that of human origin. Examples of fully human antibodies include, for example, antibodies produced by the phage display method described above and antibodies produced by mice in which the murine immunoglobulin variable and constant part genes have been replaced by human counterparts, for example, as outlined in EP 0546073B 1, US5,545,806, US5,569,825, US5,625,126, US5,633,425, US5,661,016, US5,770,429, EP0438474B1 and EP 0463151B 1.

In one embodiment, the invention provides a neutralizing antibody specific for human IL-6 and comprising a heavy chain, wherein the variable domain of the heavy chain comprises a heavy chain having the sequence of SEQ ID NO: 5, having the sequence of CDR-H1 shown in SEQ ID NO: 6 and a CDR having the sequence of CDR-H2 shown in SEQ ID NO: 7 of the CDR-H3 sequence shown in fig.

In another embodiment, the invention provides a neutralizing antibody specific for human IL-6 and comprising a heavy chain, wherein at least two of the CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable domain are selected from the following: SEQ ID NO: 5, the CDR-H1 sequence shown in SEQ ID NO: 6, the CDR-H2 sequence shown in SEQ ID NO: 7, and a CDR-H3 sequence. For example, an antibody can include a CDR-H1 having the amino acid sequence of SEQ ID NO: 5 and CDR-H2 has the sequence shown in SEQ id no: 6, or a light chain of the sequence shown in figure 6. Alternatively, the antibody may comprise a CDR-H1 having the amino acid sequence of SEQ id no: 5 and CDR-H3 has the sequence shown in SEQ ID NO: 7, or the antibody can comprise a light chain wherein CDR-H2 has the sequence shown in SEQ ID NO: 6 and CDR-H3 has the sequence shown in SEQ ID NO: 7, or a light chain having the sequence shown in seq id no. For the avoidance of doubt, all permutations are understood to be included.

In another embodiment, the invention provides a neutralizing antibody specific for human IL-6 and comprising a heavy chain, wherein the variable region of the heavy chain comprises the amino acid sequence of SEQ ID NO: 5, the CDR-H1 sequence shown in SEQ ID NO: 6 and the sequence of CDR-H2 shown in SEQ ID NO: 7, and a CDR-H3 sequence.

In one embodiment, the invention provides a neutralizing antibody specific for human IL-6 and comprising a light chain, wherein the variable domain of the light chain comprises a heavy chain variable domain having the sequence of SEQ ID NO: 8, having the sequence of CDR-L1 shown in SEQ ID NO: 9 and a CDR having the sequence of CDR-L2 shown in SEQ ID NO: 10, at least one of the CDRs of the CDR-L3 sequence set forth in fig. 10.

In another embodiment, the invention provides a neutralizing antibody specific for human IL-6 and comprising a light chain, wherein at least two of the CDR-L1, CDR-L2 and CDR-L3 of the light chain variable domain are selected from the following: SEQ ID NO: 8, the CDR-L1 sequence shown in SEQ ID NO: 9, the CDR-L2 sequence shown in SEQ ID NO: 10, CDR-L3 sequence. For example, an antibody can include a polypeptide wherein CDR-L1 has the amino acid sequence of SEQ ID NO: 8 and CDR-L2 has the sequence shown in SEQ id no: 9, or a light chain of the sequence shown in seq id no. Alternatively, the antibody may comprise a CDR-L1 having the amino acid sequence of SEQ id no: 8 and CDR-L3 has the sequence shown in SEQ ID NO: 10, or an antibody can comprise a light chain wherein CDR-L2 has the sequence shown in SEQ ID NO: 9 and CDR-L3 has the sequence shown in SEQ id no: 10, or a light chain having the sequence shown in seq id No. 10. For the avoidance of doubt, all permutations are understood to be included.

In another embodiment, the invention provides a neutralizing antibody specific for human IL-6 and comprising a light chain, wherein the variable domain comprises the amino acid sequence of SEQ ID NO: 8, the CDR-L1 sequence shown in SEQ ID NO: 9 and the CDR-L2 sequence shown in SEQ ID NO: 10, CDR-L3 sequence.

It will be appreciated that one or more amino acid substitutions, additions and/or deletions may be made to the CDRs provided herein without significantly altering the ability of the antibody to bind IL-6 and neutralize IL-6 activity. The effect of any amino acid substitution, addition and/or deletion can be readily tested by one skilled in the art, for example, by determining IL-6 binding and neutralization using the methods described in the examples. Thus, in one embodiment, the invention provides an antibody specific for human IL-6 and comprising one or more CDRs selected from CDRH-1(SEQ ID NO: 5), CDRH-2(SEQ ID NO: 6), CDRH-3(SEQ ID NO: 7), CDRL-1(SEQ ID NO: 8), CDRL-2(SEQ ID NO: 9) and CDRL-3(SEQ ID NO: 10), wherein one or more amino acids in one or more CDRs are replaced by another amino acid.

Antibody molecules of the invention preferably comprise a complementary light chain or a complementary heavy chain, respectively.

Thus, in one embodiment, an antibody according to the invention comprises a heavy chain and a light chain, wherein the variable domain of the heavy chain comprises the amino acid sequence of SEQ ID NO: 5, SEQ id no: 6 and the sequence of CDR-H2 shown in SEQ ID NO: 7, and wherein the variable domain of the light chain comprises the CDR-H3 sequence set forth in SEQ ID NO: 8, the CDR-L1 sequence shown in SEQ ID NO: 9 and the CDR-L2 sequence shown in SEQ ID NO: 10, CDR-L3 sequence.

In one embodiment, the invention provides an antibody specific for human IL-6 and comprising a heavy chain and a light chain, wherein the variable domain of the heavy chain comprises SEQ ID NO: 5, the CDR-H1 sequence shown in SEQ ID NO: 6 and the sequence of CDR-H2 shown in SEQ ID NO: 7, and wherein the variable domain of the light chain comprises the CDR-H3 sequence set forth in SEQ ID NO: 8, the CDR-L1 sequence shown in SEQ ID NO: 9 and the CDR-L2 sequence shown in SEQ ID NO: 10, wherein one or more amino acids in one or more CDRs are replaced with another amino acid.

In one embodiment, the antibody of the invention comprises a heavy chain, wherein the variable domain of the heavy chain comprises SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

In another embodiment, the antibody of the invention comprises a heavy chain, wherein the variable domain of the heavy chain comprises a heavy chain having at least 60% of the amino acid sequence of SEQ ID NO: 2, or a sequence of identity or similarity to the sequence shown in figure 2. In one embodiment, the antibody of the invention comprises a heavy chain, wherein the variable domain of the heavy chain comprises a heavy chain having at least 70%, 80%, 90%, 95% or 98% of the sequence set forth in SEQ ID NO: 2, or a sequence of identity or similarity to the sequence shown in figure 2.

"identity" as used herein means that at any particular position in an aligned sequence, the amino acid residues between the sequences are identical. "similarity" as used herein means that at any particular position in the aligned sequences, the amino acid residues between the sequences are of a similar type. For example, leucine is used instead of isoleucine or valine. Other amino acids that may be substituted for one another in general include, but are not limited to:

phenylalanine, tyrosine and tryptophan (amino acids with aromatic side chains);

lysine, arginine and histidine (amino acids with basic side chains);

aspartic acid and glutamic acid (amino acids with acidic side chains);

asparagine and glutamine (amino acids with amide side chains); and

cysteine and methionine (amino acids with sulfur-containing side chains).

The degree of identity and similarity can be readily calculated (comparative Molecular Biology, Lesk, A.M. eds., Oxford university Press, New York, 1988; Biocomputating. information and genome project), Smith, D.W. eds., Academic Press, New York, 1993; Computer Analysis of Sequence Data, first part, Griff, A.M. and Griff, H.G. eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonjje, G.Ademen, Analysis, 1987; development Primer, Sequence Analysis, Primer J.1991).

In one embodiment, the antibody of the invention comprises a light chain, wherein the variable domain of the light chain comprises SEQ ID NO: 4, or a sequence shown in seq id no.

In another embodiment, an antibody of the invention comprises a light chain, wherein the variable domain of the light chain comprises a heavy chain variable region having at least 60% of the sequence set forth in SEQ ID NO: 4, or a sequence of identity or similarity to the sequence shown in figure 4. In one embodiment, the antibody of the invention comprises a light chain, wherein the variable domain of the light chain comprises a heavy chain variable region having at least 70%, 80%, 90%, 95% or 98% of the sequence set forth in SEQ ID NO: 4, or a sequence of identity or similarity to the sequence shown in figure 4.

In one embodiment, the invention comprises a heavy chain and a light chain, wherein the variable domain of the heavy chain comprises SEQ ID NO: 2 and wherein the variable domain of the light chain comprises SEQ ID NO: 4, or a sequence shown in seq id no.

In another embodiment of the invention, a heavy chain and a light chain are included, wherein the variable domain of the heavy chain comprises a heavy chain having at least 60% of the sequence set forth in SEQ ID NO: 2, and the variable domain of the light chain comprises a sequence having at least 60% of the sequence set forth in SEQ ID NO: 4, or a sequence of identity or similarity to the sequence shown in figure 4. Preferably, the antibody comprises a heavy chain and a light chain, wherein the variable domain of the heavy chain comprises a heavy chain having at least 70%, 80%, 90%, 95% or 98% of the sequence of seq id NO: 2, and the variable domain of the light chain comprises a sequence having at least 70%, 80%, 90%, 95%, or 98% of the sequence set forth in SEQ ID NO: 4, or a sequence of identity or similarity to the sequence shown in figure 4.

In one embodiment, the antibody of the invention is a rat antibody, wherein the variable domain of the heavy chain comprises the amino acid sequence of SEQ ID NO: 2, and the variable domain of the light chain comprises the sequence shown in SEQ ID NO: 4, or a sequence shown in seq id no. The rat antibody is referred to herein as the "132E 09" or "donor" antibody. SEQ ID NO: 1 and 2, the complete nucleotide sequence and amino acid sequence of the heavy chain variable domain of rat antibody 132E09 are provided, respectively. SEQ ID NO: 3 and 4, the complete nucleotide sequence and amino acid sequence of the light chain variable domain of rat antibody 132E09 are provided. SEQ ID NO: 5. the CDRs shown in 6, 7, 8, 9 and 10 were derived from rat antibody 132E 09.

In one embodiment, the antibody of the invention is a CDR-grafted antibody. As used herein, the term "CDR-grafted antibody" refers to an antibody in which 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) grafted into the heavy and/or light chain variable domain framework of a recipient antibody (e.g., a human antibody). For a review, see Vaughan et al, NatureBiotechnology,16,535-539, 1998. Preferably, one or more CDRs are obtained from murine antibody 132E09(SEQ ID NOS: 5,6, 7, 8, 9, and 10). In one embodiment, rather than transferring the entire CDR, only one or more specificity determining residues from any of the above CDRs are transferred to the human antibody framework (see, e.g., Kashmiri et al, 2005, Methods, 36, 25-34). In one embodiment, only specificity determining residues from one or more of the CDRs described above are transferred to the human antibody framework. In another embodiment, only specificity determining residues from each of the above CDRs are transferred to the human antibody framework.

When grafting the CDRs or specificity determining residues, any suitable acceptor variable region framework sequence may be used, taking into account the class/type of donor antibody that generates the CDRs. Preferably, the CDR-grafted antibodies of the present invention have at least one variable domain comprising a human acceptor framework region and one or more CDRs derived from the donor antibody described above. Accordingly, provided are neutralizing CDR-grafted antibodies, wherein the variable domains comprise human acceptor framework regions and non-human (preferably, rat) donor CDRs.

Examples of human frameworks that 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 nemw can be used for the heavy chain, REI can be used for the light chain, and EU, LAY and POM can be used for the heavy and light chains. Alternatively, human germline sequences may be used; these sequences are: http:// vbase. mrcpce.cam. ac. uk/available.

In the CDR-grafted antibody of the present invention, the acceptor heavy and light chains do not necessarily need to be derived from the same antibody, and may include a complex chain having framework regions derived from different chains, if desired.

Preferred framework regions for the heavy chains of CDR-grafted antibodies of the present invention are derived from the human subgroup VH3 sequences 1-43-72 and JH4 (shown in FIG. 2; SEQ ID NOS: 19 and 20). Accordingly, provided are neutralizing CDR-grafted antibodies comprising at least one non-human donor CDR, wherein the heavy chain framework regions are derived from human subgroup sequences 1-43-72 and JH 4. The sequence of human JH4 is as follows: (YFDY) WGQGTLVTVSS (SEQ ID NO: 20). The YFDY motif is part of CDR-H3 and is not part of framework 4 (Ravetch, JV. et al, 1981, Cell, 27, 583-. The donor sequence is the 132E09 VH sequence shown in FIG. 2(SEQ ID NO: 2), and the donor CDRs (SEQ ID NOS: 5,6 and 7) are underlined.

Preferred framework regions for the light chain of CDR-grafted antibodies of the present invention are derived from the human germline subgroup VK1 sequences 2-1- (1)012 and JK2, shown in FIG. 2(SEQ ID NOS: 21 and 22). Accordingly, a neutralizing CDR-grafted antibody comprising at least one non-human donor CDR is provided, wherein the light chain framework regions are derived from the human subgroup sequences VK 12-1- (1)012 and JK 2. The sequence of JK2 is as follows: (YT) FGQGTKLEIKR (SEQ ID NO: 22). The YT motif is part of CDR-L3 and is not part of framework 4 (Hieter, PA. et al, 1982, J.biol.chem., 257, 1516-1522). The donor sequence is the 132E09VL sequence shown in FIG. 2(SEQ ID NO: 4), and the donor CDRs (SEQ ID NO 8, 9, and 10) are underlined.

Furthermore, in the CDR-grafted antibody of the present invention, the framework region need not have exactly the same sequence as the acceptor antibody. For example, the rare residues may be changed to residues that occur more frequently for the class or type of receptor chain. Alternatively, selected residues in the acceptor framework regions may be altered so that they correspond to residues 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 restore donor antibody affinity. The protocol for selecting residues in the acceptor framework region that may need to be altered is set forth in WO 91/09967.

Preferably, in a CDR-grafted antibody molecule of the present invention, if the acceptor heavy chain has the human VH3 sequences 1-43-72 and JH4, the acceptor framework region of the heavy chain includes a donor residue at least at position 49 in addition to one or more CDRs (according to Kabat et al, (supra)). Accordingly, a CDR-grafted antibody is provided wherein at least the residue at position 49 of the heavy chain variable domain is a donor residue.

Preferably, in a CDR-grafted antibody molecule according to the invention, if the acceptor light chain has the human subgroup VK1 sequences 2-1- (1)012 and JK2, then no donor residues are used in the acceptor framework regions of the light chain and only one or more CDRs are transferred.

The donor residues are residues from the donor antibody, i.e., the antibody from which the CDRs were originally generated, in the present case rat antibody 132E 09.

Accordingly, the invention provides antibodies in which the heavy chain variable region comprises the sequence of gH13 (FIG. 2; SEQ ID NO: 11).

In one embodiment of the invention, the antibody comprises a heavy chain, wherein the variable domain of the heavy chain comprises a heavy chain having at least 60% of the amino acid sequence of SEQ ID NO: 11, or a sequence of identity or similarity to the sequence shown in figure 11. Preferably, the antibody comprises a heavy chain, wherein the variable domain of the heavy chain comprises a heavy chain having at least 70%, 80%, 90%, 95%, or 98% of the amino acid sequence of SEQ ID NO: 11, or a sequence of identity or similarity to the sequence shown in figure 11.

In addition, the invention provides antibodies in which the light chain variable region comprises gL10 (FIG. 2; SEQ ID NO: 13).

In one embodiment of the invention, the antibody comprises a light chain, wherein the variable domain of the light chain comprises a heavy chain variable region having at least 60% of the sequence set forth in SEQ ID NO: 13, or a sequence of identity or similarity to the sequence shown in figure 13. Preferably, the antibody comprises a light chain, wherein the variable domain of the light chain comprises a heavy chain variable domain having at least 70%, 80%, 90%, 95%, or 98% of the sequence set forth in SEQ ID NO: 13, or a sequence of identity or similarity to the sequence shown in figure 13.

Preferably, the CDR-grafted antibody according to the present invention comprises a heavy chain comprising the sequence of gH13(SEQ ID NO: 11) and a light chain comprising the sequence of gL10(SEQ ID NO: 13).

In one embodiment of the invention, an antibody comprises a heavy chain and a light chain, wherein the variable domain of the heavy chain comprises a heavy chain having at least 60% of the sequence set forth in SEQ ID NO: 11, and the variable domain of the light chain comprises a sequence having at least 60% of the sequence set forth in SEQ id no: 13, or a sequence of identity or similarity to the sequence shown in figure 13. Preferably, the antibody comprises a heavy chain and a light chain, wherein the variable domain of the heavy chain comprises a heavy chain having at least 70%, 80%, 90%, 95% or 98% of the sequence set forth in SEQ ID NO: 11, wherein the variable domain of the light chain comprises a sequence having at least 70%, 80%, 90%, 95%, or 98% of the sequence set forth in SEQ ID NO: 13, or a sequence of identity or similarity to the sequence shown in figure 13.

The antibody molecules of the invention may include intact antibody molecules having full-length heavy and light chains or fragments thereof, and may be, but are not limited to, Fab, modified Fab, Fab ', F (ab')₂Fv, single domain antibodies, scFv, bivalent, trivalent or tetravalent antibodies, Bis-scFv, diabody, triabody, tetrabody and epitope-binding fragments of any of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech.23 (9): 1126-. Methods for forming and making these antibody fragments are known in the art (see, e.g., Verma et al, 1998, Journal of Immunological Methods, 216, 165-181). Other antibody fragments for use in the present invention include Fab and Fab' fragments as described in International patent applications WO2005/003169, WO2005/003170 and WO 2005/003171. Multivalent antibodies may include multiple specificities or may be monospecific (see, e.g., WO92/22853 and WO 05/113605).

The constant region domains of the antibody molecules of the invention, if present, may be selected taking into account the proposed function of the antibody molecule, particularly the effector functions which may be required. For example, the constant region domain may be a human IgA, IgD, IgE, IgG or IgM domain. In particular, where the antibody molecule is intended for therapeutic use and an antibody effector molecule is desired, human IgG constant region domains, particularly of the IgG1 and IgG3 isotype, may be used. Alternatively, where the antibody molecule is intended for therapeutic purposes but antibody effector functions are not required, for example for simple blockade of IL-6 activity, IgG2 and IgG4 isotypes may be used. It will be appreciated that sequence variants of these constant region domains may also be used. For example, IgG molecules that change serine at position 241 to proline as described in Angal et al, Molecular Immunology, 1993, 30(1), 105-108 can be used. It is particularly preferred to include such altered IgG4 constant domains. One skilled in the art will also appreciate that antibodies can be subjected to various post-translational modifications. The type and extent of these modifications typically depends on the host cell line used to express the antibody and the culture conditions. Such modifications may include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization, and asparagine deamidation. Common modifications are the loss of the carboxy-terminal basic residue (e.g., lysine or arginine) by carboxypeptidase action (e.g., Harris, RJ, Journal of chroma tography 705: 129-134, 1995). Thus, SEQ ID NO: 16 may be absent.

In a preferred embodiment, the antibodies provided herein are neutralizing antibodies specific for human IL-6, wherein the heavy chain constant region comprises a human IgG4 constant region wherein the serine at position 241 has been replaced with a proline, as described above by Angal et al. Accordingly, the invention provides a polypeptide wherein the heavy chain comprises SEQ ID NO: 16 or a sequence represented by SEQ ID NO: 16. Preferably, the light chain constant region is ck.

In one embodiment, the invention provides wherein the heavy chain comprises SEQ ID NO: 16 or a sequence represented by SEQ ID NO: 16 and the light chain comprises SEQ ID NO: 18 or a sequence represented by SEQ ID NO: 18, or a pharmaceutically acceptable salt thereof.

In one embodiment of the invention, the antibody comprises a heavy chain, wherein the heavy chain comprises a heavy chain having at least 60% of the amino acid sequence of SEQ ID NO: 16, or a sequence of identity or similarity to the sequence shown in figure 16. Preferably, the antibody comprises a heavy chain, wherein the heavy chain comprises a heavy chain having at least 70%, 80%, 90%, 95%, or 98% of seq id NO: 16, or a sequence of identity or similarity to the sequence shown in figure 16.

In one embodiment of the invention, the antibody comprises a light chain, wherein the light chain comprises a heavy chain variable region having at least 60% of SEQ ID NO: 18, or a sequence of identity or similarity to the sequence shown in figure 18. Preferably, the antibody comprises a light chain, wherein the light chain comprises a heavy chain variable region having at least 70%, 80%, 90%, 95%, or 98% of seq id NO: 18, or a sequence of identity or similarity to the sequence shown in figure 18.

In one embodiment of the invention, an antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain having at least 60% of the sequence set forth in SEQ ID NO: 16, and the light chain comprises a sequence having at least 60% identity or similarity to the sequence set forth in SEQ ID NO: 18, or a sequence of identity or similarity to the sequence shown in figure 18. Preferably, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain having at least 70%, 80%, 90%, 95%, or 98% of the sequence set forth in SEQ ID NO: 16, and the light chain comprises a sequence having at least 70%, 80%, 90%, 95%, or 98% of the sequence set forth in SEQ ID NO: 18, or a sequence of identity or similarity to the sequence shown in figure 18.

The invention also provides specific regions or epitopes of human IL-6 to which an antibody according to the invention binds, in particular an antibody comprising any one of CDR-H1(SEQ ID NO: 5), CDR-H2(SEQ ID NO: 6), CDR-H3(SEQ ID NO: 7), CDR-L1(SEQ ID NO: 8), CDR-L2(SEQ ID NO: 9) or CDR-L3(SEQ ID NO: 10), for example antibody 132E09, or an antibody comprising heavy chain variable region sequence gH13(SEQ ID NO: 11) and/or light chain variable region sequence gL10(SEQ ID NO: 13).

The specific region or epitope of such a human IL-6 polypeptide can be identified by any suitable epitope mapping method known in the art in conjunction with any of the antibodies provided by the present invention. Examples of such methods include screening for peptides of varying lengths derived from IL-6 that bind to the antibodies of the invention, using the smallest fragment that can specifically bind to an antibody containing the epitope sequence recognized by the antibody. IL-6 peptides may be produced synthetically or by proteolytic digestion of IL-6 polypeptides. For example, peptides that bind to antibodies can be identified by mass spectrometry. In another example, NMR spectroscopy can be used to identify the epitope to which the antibody of the invention binds, as described in the examples herein. It will be appreciated that an epitope fragment which binds to an antibody of the invention may be used, if desired, as an immunogen to obtain additional neutralizing antibodies which bind to the same epitope.

In one embodiment, the epitope of human IL-6 to which the antibody of the invention binds includes at least amino acid residues S47, C50, E93, R104, F105, E106, T149, K150, A153, Q156, Q159 and S169 of human IL-6 (according to the residue numbering of Boulanger et al, Science, 300, 2101-. In one embodiment, the epitope of human IL-6 to which an antibody of the invention binds comprises amino acid residues S47, C50, E93, R104, F105, E106, T149, K150, a153, Q156, Q159 and S169 and one or more residues selected from C44, S53, a58, V96, Q152, Q154, N155, W157, T163, L165 and E172.

In one embodiment, the epitope of human IL-6 to which an antibody of the invention binds includes amino acid residues C44, S47, C50, S53, a58, E93, V96, R104, F105, E106, T149, K150, Q152, a153, Q154, N155, Q156, W157, Q159, T163, L165, S169, and E172.

In one embodiment, the invention provides a neutralizing antibody specific for human IL-6 that binds to an epitope of mature human IL-6 that includes amino acid residues S47, C50, E93, R104, F105, E106, T149, K150, A153, Q156, Q159, and S169.

In one embodiment, the invention provides neutralizing antibodies specific for human IL-6 that bind to an epitope of mature human IL-6 comprising amino acid residues S47, C50, E93, R104, F105, E106, T149, K150, A153, Q156, Q159, and S169 and one or more residues selected from the group consisting of C44, S53, A58, V96, Q152, Q154, N155, W157, T163, L165, and E172.

In one embodiment, the invention provides neutralizing antibodies specific for human IL-6 that bind to an epitope of mature human IL-6 that includes amino acid residues C44, S47, C50, S53, A58, E93, V96, R104, F105, E106, T149, K150, Q152, A153, Q154, N155, Q156, W157, Q159, T163, L165, S169, and E172.

It will be appreciated that the numbering of the above residues may also be based on the amino acid numbering of the unprocessed IL-6 precursor (Swiss Prot accession number P05231). Using this numbering, residues numbered as described above, such as C44, S47, C50, S53, a58, E93, V96, R104, F105, E106, T149, K150, Q152, a153, Q154, N155, Q156, W157, Q159, T163, L165, S169, and E172, according to Boulanger et al (supra), become C72, S75, C78, S81, a86, E121, V124, R132, F133, E134, T177, K178, Q180, a181, Q182, N183, Q184, W185, Q187, T191, L193, S197, and E200, respectively.

Preferably, the antibodies of the invention block gp130 receptor binding to site 3 of human IL-6.

Antibodies that cross-block the binding of the antibodies of the invention to IL-6 can similarly be used in neutralizing IL-6 activity. Thus, the invention also provides neutralizing antibodies specific for human IL-6 that cross-block the binding of any of the above antibodies to human IL-6 and/or are cross-blocked from binding to IL-6 by any of those antibodies. In one embodiment, such an antibody binds to the same epitope as the antibody described above. In another embodiment, the cross-blocking neutralizing antibody binds to an epitope that is contiguous with and/or overlapping with the epitope to which the above-described antibody binds. In another embodiment, the cross-blocking neutralizing antibody of this aspect of the invention does not bind to the same epitope as the antibody of the invention or to an epitope linked to and/or overlapping with said epitope.

Cross-blocking antibodies can be identified using any suitable method in the art, for example, using a competition ELISA or BIAcore, wherein binding of the cross-blocking antibody to human IL-6 prevents binding of the antibody of the invention, or vice versa.

In one embodiment, neutralizing antibodies specific for human IL-6 are provided which cross-block antibody 132E09 or an antibody whose heavy chain comprises the sequence gH13(SEQ ID NO: 11) or an antibody whose light chain comprises the sequence gL10(SEQ ID NO: 13) or an antibody comprising any of CDR-H1(SEQ ID NO: 5), CDR-H2(SEQ ID NO: 6), CDR-H3(SEQ ID NO: 7), CDR-L1(SEQ ID NO: 8), CDR-L2(SEQ ID NO: 9) or CDR-L3(SEQ ID NO: 10) for binding to human IL-6. In one embodiment, the cross-blocking antibody provided herein inhibits binding of human IL-6 by 80% or more, 85% or more, 90% or more, or 95% or more of 132E09 or an antibody whose heavy chain comprises the sequence gH13(SEQ ID NO: 11) or an antibody whose light chain comprises the sequence gL10(SEQ ID NO: 13) or an antibody which comprises any one of CDR-H1(SEQ ID NO: 5), CDR-H2(SEQ ID NO: 6), CDR-H3(SEQ ID NO: 7), CDR-L1(SEQ ID NO: 8), CDR-L2(SEQ ID NO: 9), or CDR-L3(SEQ ID NO: 10).

Alternatively or additionally, the neutralizing antibody according to this aspect of the invention may be cross-blocked by any of the antibodies of the invention from binding to human IL-6. Thus, neutralizing antibody molecules specific for human IL-6 are also provided that are cross-blocked from binding human IL-6 by antibody 132E09 or an antibody whose heavy chain comprises the sequence gH13(SEQ ID NO: 11) or an antibody whose light chain comprises the sequence gL10(SEQ ID NO: 13) or an antibody that comprises any one of CDR-H1(SEQ ID NO: 5), CDR-H2(SEQ ID NO: 6), CDR-H3(SEQ ID NO: 7), CDR-L1(SEQ ID NO: 8), CDR-L2(SEQ ID NO: 9), or CDR-L3(SEQ ID NO: 10). In one embodiment, the neutralizing antibody provided by this aspect of the invention is inhibited from binding to human IL-6 by 80% or more, 85% or more, 90% or more, or 95% or more by 132E09 or an antibody whose heavy chain comprises the sequence gH13(SEQ ID NO: 11) or an antibody whose light chain comprises the sequence gL10(SEQ ID NO: 13) or an antibody which comprises any one of CDR-H1(SEQ ID NO: 5), CDR-H2(SEQ ID NO: 6), CDR-H3(SEQ ID NO: 7), CDR-L1(SEQ ID NO: 8), CDR-L2(SEQ ID NO: 9) or CDR-L3(SEQ ID NO: 10).

The antibody molecules of the invention preferably have a high binding affinity for human IL-6, preferably in picomolar amounts. Affinity can be measured using any suitable method known in the art, including BIAcore as described in the examples herein. Preferably, the use of this embodiment described in recombinant human IL-6 to measure affinity. Preferably, the antibody molecule according to the invention has an affinity for human IL-6 of less than 500 pM. Preferably, the antibody molecule according to the invention has an affinity for human IL-6 of less than 50 pM. Thus, in one embodiment, the antibody molecule of the invention has an affinity of from about 1 to about 500 pM. In one embodiment, the antibody molecule of the invention has an affinity of about 1 to about 50 pM. Preferably, the antibody molecules of the invention have an affinity for human IL-6 of from about 1 to about 20 pM. In one embodiment, the antibodies of the invention have an affinity for human IL-6 of 8 to 12 pM. It will be appreciated that any suitable method known in the art may be used to alter the affinity of the antibodies provided by the present invention. Thus, the invention also relates to variants of the antibody molecules of the invention having improved affinity for human IL-6. Such variants can be obtained by various affinity maturation protocols, including mutating the CDRs (Yang et al, j.mol.biol.,254392-,10779-783, 1992), mutator strains using E.coli (Low et al, J.mol.biol.250359-368, 1996), DNA shuffling (Pattern et al, curr. Opin. Biotechnol,8724-733, 1997), phage display (Thompson et al, J.mol.biol.,25677-88, 1996) and sexual PCR (Crameri et al, Nature,391,288-291, 1998). Vaughan et al (supra) discuss these methods of affinity maturation.

The antibody molecules of the invention preferably neutralize IL-6 activity, for example, in vitro and in vivo assays as described in the examples. In one embodiment, these antibodies bind to site 3 of human IL-6.

In one embodiment, the invention provides neutralizing antibodies specific for human IL-6, capable of inhibiting the activity of 0.038nM human IL-650% at concentrations below 100pM, as measured on the basis of IL-6-induced T1165 cell proliferation. In one embodiment, the concentration of antibody that inhibits 50% IL-6 is less than 50pM, more preferably less than 20 pM. Preferably, the assay used in the IL-6 is human recombinant IL-6. In one embodiment, the neutralizing antibody is a humanized or fully human antibody.

In another embodiment, the invention provides neutralizing antibodies specific for human IL-6 that inhibit the activity of 3.84nM human IL-650% at concentrations below 1nM, as measured by HUVEC in response to MCP-1 production by human IL-6 and sIL-6R. Preferably, the assay used in the IL-6 is human recombinant IL-6. In one embodiment, the neutralizing antibody is a humanized or fully human antibody.

If desired, the antibodies according to the invention may be conjugated with one or more effector molecules. It will be appreciated that in one embodiment, the effector molecule may comprise a single effector molecule or two or more such molecules, such that the linkage forms a single moiety that may be linked to an antibody of the invention. Where it is desired to obtain antibody fragments linked to effector molecules, they may be prepared by standard chemical or recombinant DNA methods in which the antibody fragments are linked to the effector molecules either directly or via a coupling agent. Techniques for conjugating such effector molecules to antibodies are well known in the art (see Hellstrom et al, Controlled Drug Delivery, 2 nd edition, Robinson et al, eds., 1987, pp.623-53; Thorpe et al, 1982, Immunol.Rev., 62: 119-58 and Dubowchik et al, 1999, Pharmacology and spheres, 83, 67-123). Specific chemical methods include, for example, those described in WO93/06231, WO 92/22583, WO 89/00195, WO 89/01476, and WO 03031581. Alternatively, where the effector molecule is a protein or polypeptide, the linkage may be achieved using recombinant DNA methods, for example as described in WO86/01533 and EP 0392745.

The term effector molecule as used herein includes, for example, antineoplastic agents, drugs, toxins, biologically active proteins, e.g., enzymes, other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof, e.g., DNA, RNA and fragments thereof, radionuclides, particularly radioiodinates, radioisotopes, chelated metals, nanoparticles and receptor groups, such as fluorescent compounds or compounds that can be detected by NMR or ESR spectroscopy.

Examples of effector molecules may include cytotoxins or cytotoxic agents, including any agent that is harmful (e.g., kills) on cells. Examples include combretastatin, dolastatin, epothilones, oncogenes, inhibitors of maytansinoids, rhizobians, halichondrins, sclerostins, hamiltrin, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthrax dione, mitoxantrone, plicamycin, actinomycin D, 1-dehydrotestosterone, adrenocorticoids, 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 dacarbazine), alkylating agents (e.g., dichloromethyldiethylamine, thioepa chromabuucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cycloothoramide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (previously referred to as daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (previously referred to as actinomycin), bleomycin, mithramycin, Anthranilomycin (AMC), calicheamicin, or duocarmycin), and anti-mitotic cutting agents (e.g., vincristine and vinblastine).

Other effector molecules may include chelated radionuclides, such as¹¹¹In and⁹⁰Y、Lu¹⁷⁷bismuth, bismuth²¹³Californium²⁵²Iridium (III)¹⁹²And tungsten¹⁸⁸Rhenium¹⁸⁸(ii) a Or drugs such as, but not limited to, alkylphosphocholines, topoisomerase I inhibitors, taxoid, and suramin.

Other effector molecules include proteins, peptides and enzymes. Target enzymes 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 abrin, ricin a, pseudomonas exotoxin or diphtheria toxin, proteins, such as insulin, tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet-derived growth factor or tissue plasminogen activator, a thrombogenic agent or an anti-angiogenic agent, for example, angiostatin or endostatin, or biological response modifiers such as lymphokines, interleukin-1 (IL-1), 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 factors and immunoglobulins.

Other effector molecules may include, for example, detectable substances that may be used in diagnostics. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radionuclides, positron emitting metals (used in positron emission tomography), and nonradioactive paramagnetic metal ions. For metal ions that can be conjugated to antibodies for use as diagnostic agents, see generally U.S. Pat. No. 4,741,900. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin and biotin; suitable fluorescent substances include umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin;suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin and aequorin; suitable radionuclides include¹²⁵I、¹³¹I、¹¹¹In and⁹⁹Tc。

in another embodiment, the effector molecule may increase the half-life of the antibody in vivo, and/or decrease the immunogenicity of the antibody and/or facilitate delivery of the antibody to the immune system across the endothelial barrier. Examples of suitable effector molecules of this type include polymers, albumin binding proteins or albumin binding compounds, as described in WO05/117984 (published 12/15.05).

Where the effector molecule is a polymer, it may typically be a synthetic or naturally occurring polymer, for example, an optionally substituted linear or branched polyalkylene, polyalkenylene or polyoxyalkylene polymer or a branched or linear polysaccharide, for example, a homo-or hetero-polysaccharide.

Particular optional substituents that may be present on the above-described synthetic polymers include one or more hydroxyl, methyl, or methoxy groups.

Specific examples of synthetic polymers include optionally substituted linear or branched poly (ethylene glycol), poly (propylene glycol), poly (vinyl alcohol) or derivatives thereof, especially optionally substituted poly (ethylene glycol) such as methoxy poly (ethylene glycol) or derivatives thereof.

Specific naturally occurring polymers include lactose, amylose, dextran, glycogen or derivatives thereof.

As used herein, "derivative" is intended to include reactive derivatives, for example, thiol-selective reactive groups, such as maleimides and the like. The reactive group may be attached to the polymer directly or through a linker segment. It will be appreciated that in some cases the residue of such a group forms part of the product, as a linking group between the antibody fragment and the polymer.

The size of the polymer may vary as desired, but is generally in the molecular weight range of 500Da to 50000Da, preferably 5000 to 40000Da, more preferably 20000 to 40000 Da. In particular, the polymer size may be selected based on the intended use of the product, e.g., 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 tumours, it may be advantageous to use a polymer of small molecular weight, for example having a molecular weight of about 5000 Da. For applications where the product remains in the circulation, higher molecular weight polymers may be advantageously used, for example, having a molecular weight of 20000Da to 40000 Da.

Particularly preferred polymers include polyalkylene polymers such as poly (ethylene glycol), or especially methoxy poly (ethylene glycol) or derivatives thereof, and especially have a molecular weight of from about 15000Da to about 40000 Da.

In one embodiment, the antibodies used in the present invention are linked to a poly (ethylene glycol) (PEG) moiety. In a particular embodiment, the antibody is an antibody fragment, and the PEG molecule may be attached through any available amino acid side chain or terminal amino acid functional group located in the antibody fragment, e.g., any free amino, imino, sulfhydryl, hydroxyl, or carboxyl group. Such amino acids may occur naturally in antibody fragments or may be engineered into fragments using recombinant DNA methods (see, e.g., U.S. Pat. No. 5,219,996; U.S. Pat. No. 5,667,425; WO 98/25971). In one embodiment, the antibody molecule of the invention is a modified Fab fragment, wherein the modification is the addition of one or more amino acids at the C-terminus of its heavy chain, such that an effector molecule may be attached. Preferably, the additional amino acids form a modified hinge region to which effector molecules containing one or more cysteines may be attached. Multiple sites can be used to link two or more PEG molecules.

Preferably, the PEG molecule is covalently attached through a thiol group of at least one cysteine located in the antibody fragment. Each polymer molecule attached to a modified antibody fragment may be covalently linked to the sulfur atom of a cysteine residue located in that fragment. The covalent linkage is typically a disulfide bond, or, in particular, a sulfur-carbon bond. Where a thiol group is used as the point of attachment, appropriately activated effector molecules may be used, for example, thiol-selective derivatives such as maleimides and cysteine derivatives. The activated polymer may be used as a starting material in the preparation of polymer-modified antibody fragments as described above. The activated polymer may be any polymer containing a thiol-reactive group, such as an alpha-halocarboxylic acid or ester, e.g., iodoacetamide, an imide, e.g., maleimide, vinyl sulfone, or a disulfide. Such feedstocks are commercially available (e.g., Nektar, formerly shearwater polymers inc., Huntsville, AL, USA) or can be prepared from commercially available feedstocks using conventional chemical methods. Specific PEG molecules include 20K methoxy-PEG-amine (available from Nektar, formerly Shearwater; Rapp Polymer; and SunBio) and M-PEG-SPA (available from Nektar, formerly Shearwater).

In one embodiment, the antibody is a modified Fab fragment or a di-Fab which is pegylated, i.e., has PEG (poly (ethylene glycol)) covalently attached thereto, e.g., according to the methods described in EP0948544 or EP1090037 [ see also "poly (ethylene glycol) Chemistry, Biotechnical and biomedical Applications" (poly (ethylene glycol) Chemistry, biotechnology and biomedical Applications), 1992, j.milton Harris (ed.), Plenum Press, New York, "poly (ethylene glycol) Chemistry and Biological Applications", 1997, j.milris and s.zallipsky (ed.), american Society, Washington DC and "Biological Coupling", scientific Chemical and Biological conjugation, technical, scientific, technical and Biological conjugation, 1998; chapman, a.2002, Advanced Drug Delivery Reviews 2002, 54: 531-545]. In one embodiment, the PEG is linked to cysteine of the hinge region. In one embodiment, the PEG-modified Fab fragment has a maleimide group covalently linked to a single thiol group in the modified hinge region. A lysine residue can be covalently linked to a maleimide group, and each amine group on the lysine residue can be linked to a methoxy poly (ethylene glycol) polymer having a molecular weight of about 20,000 Da. Thus, the total molecular weight of the PEG attached to the Fab fragment may be about 40,000 Da.

In one embodiment, the invention provides neutralizing antibody molecules specific for human IL-6, which are modified Fab fragments having an amino acid sequence comprising SEQ ID NO: 11 and a light chain comprising the sequence shown in SEQ ID NO: 13 and having at least one modified hinge region containing at least one cysteine residue at the C-terminus of its heavy chain to which an effector molecule may be attached. Preferably, the effector molecule is PEG and is attached using the methods described in (WO98/25971 and WO2004072116), whereby the lysyl-maleimide group is attached to the cysteine residue at the C-terminus of the heavy chain and each amino group of the lysyl residue is covalently attached to a methoxy poly (ethylene glycol) residue having a molecular weight of about 20,000 Da. Thus, the total molecular weight of the PEG linkage to the antibody is about 40,000 Da.

In another example, effector molecules can be attached to antibody fragments using the methods described in International patent applications WO2005/003169, WO2005/003170 and WO 2005/003171.

The invention also provides isolated DNA sequences encoding the heavy and/or light chains of the antibody molecules of the invention. Preferably, the DNA sequence encodes the heavy or light chain of an antibody molecule of the invention. The DNA sequences of the present invention may include synthetic DNA, e.g., produced by chemical means, cDNA, genomic DNA, or any combination thereof.

The DNA sequence encoding the antibody molecule of the invention is obtained by methods well known to those skilled in the art. For example, if desired, DNA sequences encoding a portion or all of the heavy and light chains of an antibody can be synthesized from the determined DNA sequences or based on the corresponding amino acid sequences.

DNA encoding acceptor framework sequences is widely available to those skilled in the art and can be readily synthesized based on known amino acid sequences.

Standard techniques of molecular biology may be used to prepare DNA sequences encoding the antibody molecules of the invention. Oligonucleotide synthesis techniques can be used to synthesize the desired DNA sequence, in whole or in part. Site-directed mutagenesis and Polymerase Chain Reaction (PCR) may be used, if appropriate.

Examples of suitable DNA sequences are provided in SEQ ID NO: 1; SEQ ID NO: 3; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 17. SEQ ID NO: 15 and nucleotides 1-57 of SEQ ID NO: nucleotides 1-60 in 17 encode a signal peptide sequence from murine antibody B72.3 (Whittle et al, 1987, Protein Eng.1(6)499-505), which is cleaved to obtain the neutralizing antibody molecule of the invention. Accordingly, the invention also provides an isolated DNA sequence encoding the heavy chain of an antibody of the invention, comprising SEQ ID NO: nucleotide 58-2008 of 15. The invention also provides an isolated DNA sequence encoding the light chain of an antibody of the invention, comprising SEQ ID NO: 17 from nucleotide 61 to 705.

The invention also relates to cloning or expression vectors comprising one or more DNA sequences of the invention. Accordingly, cloning or expression vectors comprising one or more DNA sequences encoding the antibodies of the invention are provided. Preferably, the cloning or expression vector comprises two DNA sequences encoding the light and heavy chains, respectively, of an antibody molecule of the invention. Preferably, the vector according to the invention comprises SEQ id no: 15 and 17. SEQ ID NO: 15 and nucleotides 1-57 of SEQ ID NO: nucleotides 1-60 of 17 encode a signal peptide sequence from murine antibody B72.3, which is most preferably cleaved to obtain the neutralizing antibody molecule of the invention.

General methods, transfection methods and culture methods that can be used to construct the vector are well known to those skilled in the art. In this respect, reference is made to "Current Protocols in molecular biology" (general Experimental protocol in molecular biology), 1999, Maniatis Manual, manufactured by F.M. Ausubel (eds.), Wiley Interscience, New York and Cold Spring Harbor publishing.

Also provided are host cells comprising one or more cloning or expression vectors comprising one or more DNA sequences encoding the antibodies of the invention. Any suitable host cell/vector system may be used to express the DNA coding sequence of the antibody molecule of the invention. Bacterial, e.g., E.coli and other microbial systems may be used, or eukaryotic, e.g., mammalian, host cell expression systems may also be used, see Verma et al, 1998, Journal of immunological Methods, 216, 165-181. Suitable mammalian host cells include CHO, NSO, myeloma or hybridoma cells. Examples of suitable expression systems include the glutamine synthase expression system described in WO 87/04462.

The invention also provides a method for producing an antibody molecule according to the invention, comprising culturing a host cell containing a vector of the invention under conditions suitable for expression of the protein from the DNA encoding the antibody molecule of the invention, and isolating the antibody molecule.

An antibody molecule may comprise only a heavy or light chain polypeptide, in which case only the heavy or light chain polypeptide coding sequence need be used to transfect the host cell. For the production of products comprising both heavy and light chains, the cell line may be transfected with two vectors, a first vector encoding the light chain polypeptide and a second vector encoding the heavy chain polypeptide. Alternatively, a single vector may be used which includes sequences encoding the light and heavy chain polypeptides.

Because the antibodies of the invention are useful for treating and/or preventing pathological conditions, the invention also provides pharmaceutical or diagnostic compositions comprising the antibody molecules of the invention and one or more pharmaceutically acceptable excipients, diluents or carriers. Thus, there is provided the use of an antibody of the invention for the manufacture of a medicament. The compositions are typically provided as part of a sterile pharmaceutical composition, which typically includes a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention may further comprise pharmaceutically acceptable auxiliaries.

The invention also provides a method of preparing a pharmaceutical or diagnostic composition comprising adding and mixing an antibody molecule of the invention and one or more pharmaceutically acceptable excipients, diluents or carriers.

The antibody molecule may be the sole active ingredient in a pharmaceutical or diagnostic composition or may be accompanied by other active ingredients, including other antibody ingredients, e.g., anti-TNF, anti-IL-1 β, anti-T cell, anti-IFN γ or anti-LPS antibodies, or non-antibody ingredients such as xanthines.

The pharmaceutical composition preferably comprises a therapeutically effective amount of an antibody of the invention. The term "therapeutically effective amount" as used herein refers to the amount of a therapeutic agent required to treat, ameliorate, or prevent a target disease or condition, or the amount of a therapeutic agent required to exhibit a detectable therapeutic or prophylactic effect. For any antibody, the therapeutically effective amount may be estimated initially in a cell culture assay or in an animal model, typically a rodent, rabbit, dog, pig or primate. Animal models can also be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine useful dosages and routes of administration in humans.

The exact therapeutically effective amount for a human patient will depend upon the severity of the condition, the general health of the subject, the age, weight and sex of the subject, the diet, the time and frequency of administration, the drug combination, the response sensitivity and the tolerance/response to treatment. The amount can be determined by routine experimentation and is within the judgment of the clinician. Generally, a therapeutically effective amount is from 0.01mg/kg to 50mg/kg, preferably from 0.1mg/kg to 20 mg/kg. The pharmaceutical compositions may generally be presented in unit dosage form containing a predetermined amount of the active agent/agents of the invention.

The compositions may be administered to the patient alone or may be administered in combination (e.g., simultaneously, sequentially or separately) with other active agents, drugs or hormones.

The dosage of the antibody molecule of the invention to be administered depends on the nature of the condition to be treated, the degree of inflammation present and whether the antibody molecule is to be used prophylactically or to treat an existing condition.

The frequency of administration will depend on the half-life of the antibody molecule and the duration of its effect. If the antibody molecule has a short half-life (e.g., 2 to 10 hours), one or more doses may need to be administered per day. Alternatively, if the antibody molecule has a long half-life (e.g., 2 to 15 days or 2 to 30 days), it may only be necessary to administer once per day, once per week or even once every 1 month or 2 months.

The pharmaceutically acceptable carrier should not itself induce the production of antibodies harmful to the individual receiving the composition and should be non-toxic. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polyamines, amino acid copolymers and inactive virus particles.

Pharmaceutically acceptable salts may be used, for example, inorganic acid salts such as hydrochloride, hydrobromide, phosphate and sulfate, or organic acid salts such as acetate, propionate, malonate and benzoate.

The pharmaceutically acceptable carrier in the therapeutic composition may additionally contain liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances, such as wetting or emulsifying agents or pH buffering substances, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by the patient.

Preferred forms of administration include those suitable for parenteral administration, for example, by injection or infusion, for example, by bolus injection or continuous infusion. In the case of products for injection or infusion, they may take the form of suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, preserving, stabilizing and/or dispersing agents. Alternatively, the antibody molecule may be in dry form for reconstitution with a suitable sterile liquid prior to use.

Once formulated, the compositions of the present invention can be administered directly to a subject. The patient to be treated may be an animal. However, it is preferred that the composition is suitable for administration to a human subject.

The pharmaceutical compositions of the present invention may be administered by a variety of routes of administration, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intraspinal, intrathecal, intraventricular, transdermal (see, e.g., WO98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal, or rectal routes. Needleless injectors may also be used to administer the pharmaceutical compositions of the present invention. Generally, the therapeutic compositions can be prepared as injectables, such as liquid solutions or suspensions. Solid forms suitable for dissolution or suspension in a liquid carrier prior to injection may also be prepared.

Direct delivery of the composition is typically achieved by subcutaneous, intraperitoneal, intravenous or intramuscular injection, or delivery to the intercellular spaces of the tissue. The composition may also be administered into the lesion. The dose treatment may be a single dose plan or a multiple dose plan.

It will be appreciated that the active ingredient in the composition is an antibody molecule. Thus, it will be susceptible to degradation in the gastrointestinal tract. Thus, if the composition is to be administered using the gastrointestinal route, the composition will need to contain ingredients that protect the antibody from degradation, but release the antibody once absorbed from the gastrointestinal tract.

A thorough discussion of pharmaceutically acceptable carriers is available in Remington's pharmaceutical Sciences (Mark Publishing Company, N.J.1991).

It is also contemplated that the antibodies of the invention may be administered by use of gene therapy. To achieve this goal, DNA sequences encoding the heavy and light chains of an antibody molecule under the control of appropriate DNA components are introduced into a patient such that the antibody chains are expressed from the DNA sequences and assembled in situ.

The invention also provides antibody molecules for use in the control of inflammatory diseases. Preferably, the antibody molecule may be used to reduce an inflammatory process or to prevent an inflammatory process.

Antibody molecules according to the invention are also provided for the treatment and/or prevention of pathological disorders mediated by IL-6 or associated with elevated IL-6 levels. The invention further provides the use of an antibody molecule according to the invention in the manufacture of a medicament for the treatment and/or prevention of a pathological disorder mediated by IL-6 or associated with elevated IL-6 levels. Preferably, the pathological condition is selected from the group consisting of infections (viral, bacterial, fungal and parasitic), endotoxic shock associated with infections, arthritis, rheumatoid arthritis, psoriatic arthritis, systemic onset juvenile specific arthritis (JIA), Systemic Lupus Erythematosus (SLE), asthma, pelvic inflammatory disease, alzheimer's disease, crohn's disease, ulcerative colitis, irritable bowel syndrome, castleman's disease, ankylosing spondylitis, dermatomyositis, uveitis, spongitis, celiac disease, cystic disease, pilathies, peritonitis, psoriasis, vasculitis, surgical adhesions, stroke, type I diabetes, lyme arthritis, meningitis, immune-mediated inflammatory diseases of the central and peripheral nervous system (such as multiple sclerosis and guillain-barre syndrome), other autoimmune diseases, mumps, trauma (surgery), and surgery, Graft versus host disease, graft rejection, cancer (solid tumors such as melanoma, hepatoblastoma, sarcoma, squamous cell carcinoma, transitional cell carcinoma, ovarian cancer and hematologic malignancies, particularly acute myeloid leukemia, chronic myelogenous leukemia, gastric cancer and colon cancer), heart disease (including ischemic diseases such as myocardial infarction and atherosclerosis), intravascular coagulation, bone resorption, burn patients, osteoporosis, periodontal disease and gastric hyperacidity.

Preferably, the pathological condition is rheumatoid arthritis or Systemic Lupus Erythematosus (SLE).

The invention also provides an antibody molecule according to the invention for use in the treatment or prevention of pain.

The invention further provides the use of an antibody molecule according to the invention in the manufacture of a medicament for the treatment or prevention of pain.

The antibody molecules of the invention may be used in any treatment where it is desirable to reduce the effect of IL-6 in a human or animal body. IL-6 can circulate in the body or can be present at undesirably high levels at specific sites in the body, such as sites of inflammation.

Preferably, the antibody molecules of the invention are used for the control of inflammatory diseases.

The invention also provides a method of treating a human or animal subject having or at risk of a disease mediated by IL-6, which method comprises administering to the subject a therapeutically effective amount of an antibody molecule of the invention.

The antibody molecules of the invention may also be used in diagnostics, for example, in vivo diagnostics, and in imaging of disease states involving IL-6.

The invention will be further described by the following examples, which are intended to be illustrative only, and which will be described with reference to the accompanying drawings, in which:

FIG. 1 shows a design of the grafting of the sequences of the gl heavy chain (FIG. 2 a; SEQ ID NO: 11) and light chain (FIG. 2 b; SEQ ID NO: 13). The symbol (|) highlights the donor: receptor: differences between the grafted framework sequences. CDRs are single underlined. These are defined by Kabat, except for CDR-H1, which includes Kabat and Chothia definitions. The double underlined sequence is the donor framework residue remaining in the graft.

Figure 2a shows the translated sequence of the heavy chain of gl IgG4, showing the intron/exon boundaries.

Fig. 2b shows the translated sequence of the gl light chain, showing the intron/exon boundaries.

Figure 3a shows the inhibition of T1165 cell proliferation induced by gl human recombinant, human mammal derived, rhesus monkey, cynomolgus monkey and murine I-6.

FIG. 3b shows the inhibition of human recombinant IL-6 and sIL-6R-induced MCP-1 production in HUVEC by gl antibody 240.

FIG. 3c shows the inhibition of IL-17 induced endogenous IL-6 and sIL-6R induced MCP-1 production in HUVEC by antibody 240. gl.

Figure 4. hll-6 induced SAA in mice was neutralized in vivo by administration of CA030-240.gl (site 3 antibody), n-7-8/group, except PBS, n-6. Statistical analysis was performed by ANOVA using the Bonferroni post test, compared to IL-6 alone,^**P＜0.01。

DNA manipulation and general methods

Coli strain INV α F' (Invitrogen) was used for transformation and conventional culture growth. DNA restriction and modification enzymes were obtained from Roche Diagnostics Ltd. and New England Biolabs. Plasmid preparation was performed using Maxi Plasmid purification kit (QIAGEN, cat # 12165). DNA sequencing reactions were performed using the ABI Prism Big Dye stop sequencing kit (catalog No. 4304149) and run on an ABI 3100 automated sequencer (applied biosystems). Data were analyzed using the AutoAssembler (applied biosystems) program. Oligonucleotides were obtained from INVITROGEN. A synthetic gene was constructed in Entelechon. Fab and IgG concentrations were determined using assembly ELISA.

Example 1: separation of 132E09

Rats were immunized by subcutaneous injections of recombinant human IL-6(Peprotech) at two week intervals, initially with Freund's complete adjuvant, followed by Freund's incomplete adjuvant. Spleens were collected one to two weeks after the last immunization and single cell suspensions were prepared. Lymphocytes from immunized rats were cultured in 96-well microtiter plates for one week in the presence of irradiated murine thymoma EL4 cells and rabbit T cell conditioned medium. Supernatants were screened in an ELISA for the presence of human IL-6 specific antibodies. The DS1 cell line assay was further screened for the ability to positively neutralize the biological effects of human IL-6 (Bock et al, 1993, Cytokine, 5, 480-489).

Individual B cells secreting antibodies with appropriate binding characteristics were isolated from positive microtiter plate wells according to the lymphocyte antibody selection method (Babcook et al, 1996, Proc. Natl. Acad. Sci. USA 93, 7843-7848; WO92/02551) and the heavy and light chain variable region genes were cloned from individual rat B cells by reverse transcription PCR. The variable regions were expressed as recombinant IgG to confirm binding and 132E09 was selected for humanization and further studies. The rat variable region sequence was registered as CA030_00240.

The V-region sequence is shown in SEQ ID NO: 1 to 4.

Example 2: CDR-grafting of 132E09

A series of humanized VL and VH regions were designed in which CDR hypervariable regions plus varying numbers of framework residues from 132E09 were grafted onto a human V-region acceptor framework.

Ten grafted VL regions (gL1-10) were designed and genes were constructed by oligonucleotide assembly and PCR mutagenesis. A total of 13 grafted VH regions (gH1-13) were also constructed using two different framework regions, VH 31-43-72 and VH 31-33-21. The light chain grafted sequence was subcloned into the human light chain expression vector pkh10.1, which contained DNA encoding the human C- κ constant region (Km3 isoform). The heavy chain grafted sequence was subcloned into the human γ -4 expression vector pVhg4P FL, which contained DNA encoding the human γ -4 constant region containing the hinge stabilizing mutation S241P (Angal et al, supra). Plasmids were co-transfected into CHO cells and screened for activity of the antibodies produced in IL-6 binding and in vivo assays. Using Lipofectamine^TMProcedure 2000 transfection of CHO cells was carried out according to the manufacturer's instructions (InVitrogen, Cat. No. 11668).

Of the 13 heavy chain grafts generated, two contained only a single framework donor residue (Ala) at position 49, and two different heavy chain frameworks were used to generate these. VH 31-33-21 was poorly expressed in CHO cells and showed reduced affinity for IL-6. In contrast, grafting with the VH 31-43-72 framework was well expressed and retained the affinity of the donor antibody. This heavy chain graft, comprising only a single donor framework residue, was selected in conjunction with the CDR-only light chain graft gL 10.

Figure 1 shows the alignment between the donor rat sequence 132E09 and the acceptor human framework. The heavy chain acceptor framework is the human germline sequence VH 31-33-72 with framework 4 from this position in the human JH-region germline JH 4. The light chain acceptor framework is the human germline sequence VK 12-1- (1)012, with framework 4 from this position in the human JK-region germline JK 2. The grafted sequences of gH13 and gL10 are shown in FIG. 1(SEQ ID NOS: 11, 12, 13, and 14). This grafted antibody was designated CA030 — 00240. gl. This was generated as intact IgG4 comprising a serine to proline substitution at position 241 as described above. The complete translated heavy and light chain sequences are shown in fig. 2a and 2b, respectively. SEQ ID NO: 16, and SEQ ID NO: 18 is a light chain. SEQ ID NO: 15 and 17 provide the DNA coding sequences for the heavy and light chains, respectively. SEQ ID NO: 15 and nucleotides 1-57 in figure 2a encode the signal peptide sequence from murine antibody B72.3VH, while SEQ ID NO: nucleotides 1-60 in FIG. 17 and FIG. 2b encode the signal peptide sequence from murine antibody B72.3VL.

Murine antibody B72.3 is described in Whittle et al, 1987, Protein Eng.1(6) 499-505.

Example 3: binding affinity

CA030_00240.gl binding affinity measurement

The BIAcore technology monitors binding between biomolecules in real time without the need for labeling. One of the interactors, called the ligand, is immobilized or captured directly on the immobilized surface, while the other, called the analyte, flows in solution over the captured surface. The sensor detects a change in the substance on the sensor surface as the analyte binds to the ligand to form a complex on the surface. This corresponds to the aggregation process. The dissociation process is monitored as the buffer is substituted for the analyte. In the affinity BIAcore test, the ligand is CA 030-00240. gl intact IgG4, and the analyte is human IL-6.

Instrument for measuring the position of a moving object

Biacore3000，Biacore AB，Uppsala，Sweden

Sensor chip

CM5 (research grade), catalog number: BR-1001-14, Biacore AB, Uppsala, Sweden.

The chip was stored at 4 ℃.

BIA standardized solution

70% (w/w) glycerol. Part of biamidantence kit catalog No.: BR-1002-51, Biacore AB, Uppsala, Sweden. The biamaintence kit was stored at 4 ℃.

Amine coupling kit

Catalog number: BR-1000-50, Biacore AB, Uppsala, Sweden.

Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Made up to 75mg/mL in distilled water and stored in 200. mu.L aliquots at-70 ℃.

N-hydroxysuccinimide (NHS). Made up to 11.5mg/mL in distilled water and stored in 200. mu.L aliquots at-70 ℃.

1M ethanolamine hydrochloride-NaOH pH 8.5. Stored in 200. mu.L aliquots at-70 ℃.

Buffer solution

Running a gel buffer solution: HBS-EP (0.01M HEPES pH7.4, 0.15M NaCl, 3mM EDTA, 0.005% surfactant P20). Catalog number: BR-1001-88, Biacore AB, Uppsala, Sweden. The buffer was stored at 4 ℃.

Fixing buffer solution: acetate 5.0(10mM sodium acetate ph 5.0). Catalog number: BR-1003-51, Biacore AB, Uppsala, Sweden. The buffer was stored at 4 ℃.

Ligand capture

Affinipure F(ab’)₂Fragment goat anti-human IgG, Fc fragment specific. Jackson immunoresearch Inc (Pennsylvania, USA), catalog No.: 109-006-098. The reagents were stored at 4 ℃.

Analyte

Recombinant human IL-6(R & D Systems Europe Ltd, Abingdon, Oxon. catalog number 206-IL-050, batch A131402A), stored at-70 ℃ and thawed once for each experiment.

Recombinant cynomolgus IL-6 and recombinant rhesus IL-6 were produced by transient transfection of CHO cells. The material was used as a non-purified and non-quantitative cell culture supernatant.

Regeneration solution

40mM HCl was prepared from 11.6M stock (BDH, Poole, catalog # 101254H) by dilution with distilled water.

5mM NaOH was prepared by dilution from a 50mM stock solution with distilled water.

Catalog number: BR-1003-58, Biacore AB, Uppsala, Sweden.

Test method

BIA (biomolecular interaction analysis) was performed using BIAcore 3000(BIAcore AB). Affinipure F (ab')₂Fragment goat anti-human IgG, Fc fragment specific (Jackson ImmunoResearch) was immobilized on CM5 sensor chip to a capture level of ≈ 5000 Response Units (RU). HBS-EP buffer (10mM HEPES pH7.4, 0.15M NaCl, 3mM EDTA, 0.005% surfactant P20, BIAcoreAB) was used as running buffer with a flow rate of 10. mu.l/min. Mu.l of 4. mu.g/mL CA030-240.gl injection was used for capture via immobilized anti-human IgG-Fc. Human IL-6 was titrated with different concentrations of CA030-240.gl at a flow rate of 30. mu.l/min. The surface was regenerated by 10. mu.L of 40mM HCl injection followed by 5. mu.L of 5mM NaOH injection at a flow rate of 10. mu.L/min.

Background substrate binding curves were analyzed using BIA evaluation software (version 3.2) according to standard methods. Kinetic parameters were determined from the fitting algorithm.

A batch of CA030_240.gl was used in this study. Affinity was measured at human IL-6 concentrations of 20nM or less than 20 nM. The affinity values determined for CA030-240.gl were in the range of 9.02-10.50pM, with an average of 9.76. + -. 0.74pM (Table 1.1). The use of immobilized on BIAcore chip on human IL-6 is impossible to measure affinity, because the fixed IL-6 caused the loss of native conformation.

Since it was not possible to quantify cynomolgus or rhesus IL-6, it was also not possible to determine the real kinetic parameters of their interaction with CA030_00240. gl. However, visual inspection of the binding sensorgrams revealed that CA 030-00240. gl binds IL-6 in these non-human primates with similar affinity to human IL-6.

Table 1.1: affinity of CA030-240.gl for human IL-6

Reference to	ka(M-1s-1)	kd(s-1)	Kd(M)	Kd pM
10017474/39-51	7.31E+05	7.68E-06	1.05E-11	10.5
10017474/39-51	8.52E+05	7.68E-06	9.02E-12	9.02

Example 5: in vitro neutralization assay

The efficacy of the antibody CA030_00240.gl (hereinafter abbreviated as 240.gl) was determined using two different assays. The first uses a murine IL-6 dependent cell line, designated T1165, which proliferates in response to murine, rhesus, cynomolgus and human IL-6. This direct signaling of IL-6 to the cell surface IL-6 receptor, along with the signaling receptor subunit known as gp130, is known as cis-signaling. The second assay uses Human Umbilical Vein Endothelial Cells (HUVEC) stimulated with human IL-6 plus a soluble IL-6 receptor (IL-6R), and reads the production of monocyte chemoattractant protein-1 (MCP-1). In this assay, IL-6 is produced by exogenous addition or by stimulation of HUVEC with the cytokine interleukin-17 (IL-17). These two assay variants are called trans-signaling because HUVECs do not express IL-6R and respond to the production of MCP-1 only when both IL-6 and soluble IL-6R are added exogenously.

Using these different assays, ND50 (50% neutralization dose) values for gl against human (recombinant and natural), rhesus, cynomolgus and murine IL-6 can be generated.

Material

Culture medium

T1165 medium-RPMI 1640, supplemented with 10% fetal bovine serum, penicillin (100 units/ml), streptomycin (50. mu.g/ml), glutamine (2mM) and 10ng/ml human recombinant IL-6, R & D systems, UK.

HUVEC medium-macrovascular endothelial cell basal medium (LVECBM) TCSCellworks, UK, macrovascular endothelial cell growth supplements TCS Cellworks, UK and antibiotic supplements TCS Cellworks, UK.

CA 03000240.gl was generated internally at a concentration of 6.83mg/ml in PBS.

Human IL-6R & D systems, UK, human mammal derived IL-6 (derived internally using CHO transfection of human IL-610017108/67), rhesus IL-6 (derived internally using CHO transfection of rhesus IL-610017108/67), cynomolgus IL-6 (derived internally using CHO transfection of cynomolgus IL-610017108/67), murine IL-6R & D systems, UK. Anti-human MCP-1 capture antibody (555055) and anti-human MCP-1 detection antibody (554664) and human recombinant MCP-1(890225), BD Biosciences, CA. streptavidin-HRP (AMDEX) Amersham bioscience, UK. Thrombin Merck Biosciences, Darmstadt, Germany. sIL-6R R & D systems, UK. Human IL-17R & D systems, UK.

T1165 test-CellTiter 96AQueous Promega，CA。

TM Blue(Serologicals，GA)。

Measurement of 240.gl Activity Using IL-6 dependent proliferation of T1165 cells

T1165 cells were thawed 4 days prior to use and cultured in RPMI1640 supplemented with 10% FCS, antibiotics, glutamine, and 10ng/ml human IL-6. Trypan blue exclusion was used to monitor cell viability, only cells considered viable at least 90% were used. Prior to use, cells were washed twice in RPMI1640 in the absence of human IL-6. The cells were then counted and counted at 5X 10⁴Cell/well density was distributed into 96-well plates. Serially diluted 240.gl were incubated in separate plates in the presence of a fixed concentration of 1ng/ml (0.038nM) of human recombinant, human mammal derived (also known as human CHOIL-6), rhesus monkey, cynomolgus monkey or murine IL-6. The pre-mixed mixture of 240.gl and IL-6 was then transferred to wells containing T1165 cells, which were in humidified 5% CO₂Incubate at 37 ℃ for 48 hours in the atmosphere. During the last six hours of incubation, 20ml of CellTiter 96 was addedAQueous to determine the number of proliferating cells. Inhibition of IL-6 dependent proliferation of T1165 cells by gl was expressed as the percentage inhibition of wells treated with IL-6 alone minus control wells containing cells but no IL-6.

The activity of gl against the human recombinant, human mammal derived, rhesus monkey, cynomolgus monkey and murine IL-6 induced proliferation of cell line T1165 can be seen in figure 3 a. Gl effectively inhibits human recombinant, human mammal derived, rhesus monkey, cynomolgus IL-6 activity, but not murine IL-6 activity.

Human recombinant IL-6 has ND50 of 1.1 + -0.5 ng/ml (7.26 + -3.3 pM). Human mammal-derived IL-6 has ND50 of 3.6 + -2.4 ng/ml (23.76 + -15.84 pM). Rhesus IL-6 had an ND50 of 2.2. + -. 1.1ng/ml (14.52+7.26 pM). Mimosan IL-6 ND50 was 5.4. + -. 1.1ng/ml (35.64. + -. 7.26 pM).

Measurement of 240.gl Activity Using IL-6 and soluble IL-6 receptor to induce MCP-1 production in HUVEC

HUVECs (TCS Cellworks, UK) were grown in macrovascular endothelial cell basal medium (LVECBM) and passaged no more than five times in culture. Cells were grown until 75% confluency prior to use. Cells were detached using trypsin/EDTA, resuspended in fresh LVECBM and washed once. The cells were then counted and counted at 2X 10⁴Cell/well density was distributed into 96-well flat-bottom plates. The cells were then incubated in 5% CO in humidity₂The culture was carried out overnight at 37 ℃ in the atmosphere. The following day, cells were washed in fresh medium supplemented with biotinylated thrombin (3U/ml) and set aside. Serial dilutions of 240.gl were incubated in separate plates in the presence of human recombinant IL-6(50 ng/ml; 3.84nM) and a fixed concentration of 500ng/ml (10.15nM) of sIL-6R. In addition, 240.gl was incubated with human recombinant IL-17(25 ng/ml; 1.18nM) that stimulates HUVEC to produce IL-6 and a fixed concentration of 500ng/ml (10.15nM) of sIL-6R. The pre-mixed 240.gl and IL-6/sIL-6R or IL-17/sIL-6R complexes were then transferred to HUVEC-containing wells, which were incubated in a humidified 5% CO₂Incubate at 37 ℃ for 24 hours in the atmosphere. After the incubation period, cell-free supernatants were collected and human MCP-1 levels were determined by sandwich ELISA (experimental protocol given below). The inhibition of IL-6/sIL-6R or IL-17/sIL-6R induced MCP-1 production by gl 240 is expressed as the percentage inhibition of wells treated with IL-6/sIL-6R or IL-17/sIL-6R minus control wells containing cells but no stimulus. In addition, added control to show the presence of sIL-6R cells to add human IL-6 response.

MCP-1 ELISA

Nunc Maxisorp plates were covered with anti-MCP-1 capture antibody at a concentration of 2. mu.g/ml. The plates were incubated overnight at +4 ℃ and then washed twice in PBS plus 0.1% Tween 20 (wash buffer). The plates were blocked in PBS plus 5% bovine serum albumin for 1 hour. The plate was then washed four times with wash buffer and standards and samples were added. The plates were incubated at room temperature for two hours. The plates were then washed and biotinylated anti-MCP-1 antibody was added at a concentration of 1 mg/ml. The plates were incubated for a further 2 hours and then washed four times. A1: 5000 dilution of streptavidin-HRP was then added and the plates were incubated for 30 min. Plates were washed four final times and TMB substrate was added. Colorimetric readings were taken at 630nm and background readings were taken at 492 nm. MCP-1 concentrations were generated from standard curves using four parameter logistic curve fitting on Genesis II software.

The activity of 240.gl in HUVEC against human recombinant IL-6 and sIL-6R-induced trans-signaling can be seen in FIG. 3 b. Gl effectively inhibited human recombinant IL-6 and sIL-6R-induced MCP-1 production by HUVEC. ND50 was 64. + -. 62ng/ml (422. + -.409 pM).

The activity of 240.gl in HUVEC against IL-17 induced cis-signaling induced by endogenous IL-6 and sIL-6R can be seen in FIG. 3 c. Gl effectively inhibited IL-17-induced MCP-1 production of endogenous IL-6 and sIL-6R-induced HUVEC. ND50 was 93. + -. 70ng/ml (614. + -. 462 pM).

Conclusion

Gl is capable of neutralizing the biological activity of human recombinant, human mammal derived, rhesus and cynomolgus IL-6 in an IL-6 cis-signaling assay, but not murine IL-6. In addition, the gl can neutralize recombinant or endogenous IL-6 induced IL-6 trans-signaling.

Example 6: in vivo Activity

IL-6 is known to induce acute phase proteins. In mice, the most prominent acute phase protein is blood \ amyloid a (saa). Human IL-6 is capable of acting on murine receptors, so human IL-6 can be injected into mice and SAA production measured in serum.

Balb/c mice were s.c. injected with site-specific anti-hIL-6 antibody, CA030-240.gl intact IgG 4. After 24 hours, the mice were injected intraperitoneally with 30. mu.g/kg of hIL-6(Peprotech Cat No. 200-06, batch No. 0203B 16). After 20 hours, blood was collected by cardiac puncture and serum was collected for determination of serum amyloid a (saa) by ELISA (delta, lot 22KT 022). As depicted in fig. 4, CA030 — 240.gl inhibited SAA induction of hIL-6, noting a statistically significant reduction in SAA at doses of 0.3, 0.1, and 0.03 mg/kg.

n-7-8/group except PBS n-6. Statistical analysis was performed by ANOVA using the Bonferroni post test, compared to IL-6 alone.^**P＜0.01。

Two further experiments demonstrated that CA030-240.gl at doses of 0.3 and 0.1mg/kg significantly inhibited IL-6 (30. mu.g/kg) induced SAA.

Example 7: CA 030-00240. gl epitope mapping

The epitopes recognized by CA 030-00240. gl on human IL-6 were mapped using NMR techniques, using 240.gl as Fab' fragments. This requires the expression of human IL-6 in E.coli, and use¹⁵N/¹³C/²H-stable isotopes are labeled uniformly. For free IL-6, the complete sequence-specific backbone resonance assignments were obtained and changes in these signal positions induced by binding of the Fab' fragment of CA030 — 00240.gl were detected using 3D TROSY HNCO spectroscopy.

Expression and purification of recombinant IL-6:

human IL-6 was prepared from an E.coli expression vector (pET3d) containing the coding sequence for the mature form of the protein. Expression of proteins in Tuner (DE3) pLysS cells to obtain high yieldsInsoluble product. Preparation from cells grown on appropriately labeled enrichment Medium (Celtone)¹⁵N、¹⁵N/¹³C and¹⁵N/¹³C/²h labeled IL-6 samples.

IL-6 was purified from transformed E.coli cells using well known procedures. Initially, cells harvested from 1 liter of medium were resuspended in 40mL of buffer A [100mM KC1, 2mM DTT, 10mM Tris-HCl pH8.5, 25% (w/v) sucrose, solubilized protease inhibitor tablet (Boehringer) ]. 10mL of buffer B [300mM Tris-HCl pH8.5, 100mM EDTA, 4mg/mL lysozyme ] was added and the suspension was incubated on ice for 10-30 minutes with occasional stirring. Then 50ml of buffer C [1M LiCl, 20mM EDTA, 0.5% (v/v) NP-40] was added, and the suspension was passed twice through a 20,000psi French Press (French Press). The homogenate was then centrifuged at 16,000g rpm for 15 minutes at 4 ℃ and the pellet was retained. The pellet was resuspended in 40ml buffer D [10mM Tris-HCl pH8.5, 0.1mM EDTA, 0.5M LiCl, 0.5% (v/v) NP-40, 1mM DTT, solubilized protease pellet ], and passed through a French press and centrifugation as before. The process is repeated. The pellet was then resuspended in 40ml buffer E [10mM Tris-HCl pH8.5, 0.1mM EDTA, 0.5% (v/v) NP-40, 1mM DTT, solubilized protease pellet ], and passed through a French press and centrifuged as before. The process is repeated. The final pellet was dissolved in 6mL of 6M GuHCl, 50mM Tris-HCl pH8.0 and centrifuged at 48,000g for 30 min at 4 ℃. The supernatant was retained and dissolved IL-6 was quantified spectrophotometrically.

Dissolved IL-6 was diluted in 2.5mg/mL 5M GuHCl, 50mM NaCl, 50mM Tris-HCl, 2mM GSH, 0.2mM GSSG, 1mM EDTA, pH8.0 and incubated at 25 ℃ for 1 hour. The sample was then further diluted in a dropwise fashion to 250. mu.g/mL of 50mM NaCl, 50mM Tris-HCl, 2mM GSH, 0.2mM GSSG, 1mM EDTA, pH8.0 and incubated at 25 ℃ for 3 hours. Any precipitate formed in this stage was removed by centrifugation at 30,000g for 30 minutes at 4 ℃. The clarified material was dialyzed against 50mM Tris-HCl, 10% (v/v) glycerol, pH9.0, at 4 ℃ for a minimum of 16 hours, and the buffer was changed twice. After dialysis, by dissolving in 30,000g was centrifuged at 4 ℃ for 30 minutes to clarify the solution. The supernatant was retained and loaded onto an 8mL monoQ (Amersham Biosciences) ion exchange column and eluted with a linear gradient of sodium chloride (0-1.0M). Fractions containing refolded IL-6 were identified by SDS-PAGE and then pooled and diluted in 25mM sodium phosphate, 100mM NaCl, 0.01% (w/v) NaN₃pH 6.5.

Fab' fragments of CA 030-00240. gl were generated, purified and formulated in 25mM sodium phosphate, 100mM NaCl, 0.01% (w/v) NaN₃pH 6.5. A1: 1 complex of IL-6 and a fragment of CA 030-00240. glFab' was prepared for NMR analysis by mixing equimolar concentrations of protein ranging from 0.2 to 0.6 mM.

NMR spectra

For 0.35ml of the protein sample, 25mM sodium phosphate, 100mM sodium chloride and 0.01% (w/v) sodium azide buffer (95% H) at pH6.5₂O and 5% D₂O) was subjected to NMR experiments. Preparation of¹⁵N/¹³C/²A1: 1 complex of H-labeled IL-6 and unlabeled CA 030-00240. gl Fab' fragments, was mixed with equimolar amounts of protein to obtain a final concentration of 0.1mM for NMR analysis. For free IL-6 and for IL-6: a complex of a fragment of CA 030-00240. glFab' when assembled with a triple resonance (¹⁵N/¹³C/¹H) NMR data were acquired on a 800MHz Bruker Avance spectrometer on a cryoprobe. Standard HNCACB, CBCA (CO) NH and HNCO spectra (Wittekind, M. and Mueller, L. (1993) J Magn Reson, series B101 (2), 201; Grzesek, S. and Bax, A. (1993) J Biomol NMR3(2), 185. sub.204; Muhandiram, D.R., and Kay, L.E. (1994) J MagnReson, series B103 (3), 203; Grzesek, S. and Bax, A. (1992) J MagnReson 96(2), 432) were used to prepare the complete sequence-specific backbone resonance assignments for free IL-6 (Wittekind, M. and Mueller, L. (1993) J Magn Reson, S., and Bax¹⁵N，¹³C and¹H) using 0.9mM¹⁵N/¹³C homogeneously labeled sample.

The change in position of the IL-6 backbone signal induced by the binding of the CA 030-00240. gl Fab' fragment was detected using 3D TROSYHNCO spectroscopy (Salzmann, M. et al (1998) Proc Natl Acad Sci USA 95(23), 13585-. Typical acquisition parameters for all 3d nmr experiments are provided in table 1.

All spectra were processed using NMRPipe (Delaglio, F., et al, (1995) J BiomolNMR 6(3), 277- & 293), and the 3D data was analyzed using linear prediction¹⁵The effective acquisition time in the N dimension is extended to about 30 ms. With moderate resolution enhancement in all dimensions, a displacement sine-squared function is used. Spectral analysis was performed using spark (Goddard, t.d. and Kneller, d.g. Sparky 3.In., university of california, san francisco).

Analysis of Fab binding data:

the change in the position of the IL-6NMR signal caused by the binding of the CA 030-00240. gl Fab' fragment was determined using the minimum Displacement method (Farmer, B.T. et al, (1996) Nat Struct mol Biol 3(12), 995; Muskett, F.W. et al, (1998) J Biol Chem 273(34), 21736-. Initially, all peaks in the free IL-63DHNCO spectrum and the IL-63D TROSYHNCO spectrum of the bound g132E09Fab were picked at their center. Correction for temperature differences between complex and free protein spectra¹⁵N and¹h chemical shift value (for)¹⁵N is not-0.8 ppm, with respect to¹H was-0.05 ppm) (Baxter, N.J., and Williamson, M.P, (1997) J Biomol NMR 9(4), 359-. Calculation by Using Microsoft Excel¹⁵N、¹³C and¹chemical shift differences in H binding to obtain minimal changes in peak positions between free and Fab bound IL-6 for comparing each specified peak in the HNCO spectrum of the free protein with all peaks observed in the TROSY-HNCO spectrum of the Fab complex. The difference in chemical shift (Δ δ) of bound amide protons, nitrogen and carbon is defined according to the following formula (formula 1), where Δ δ_HN、Δδ_NAnd delta_CBetween pairs of peaks corresponding to compared HNCO¹H、¹⁵N and¹³difference in C displacement, and α_NAnd alpha_CAre scaling factors of 0.2 and 0.33 required to account for differences in chemical shift ranges of amide protons, amide nitrogen, and carbon. For each individual HNCO peak, according to the lowest possibleShift values for binding (Δ δ) to obtain the minimum shift induced by Fab binding.

(formula 1)

To identify Fab binding sites (epitopes) on IL-6, a histogram of the minimum shift in binding versus protein sequence was used to reveal IL-6 fragments containing significant perturbation signals. If the combined chemical shift change for a single amino acid exceeds the mean limit value of the combined chemical shift changes for all amino acids plus one standard deviation of the mean, these residues are selected for further evaluation as potentially touching residues in the Fab binding site. Finally, the positions of candidate binding site residues were detected on a high resolution IL-6 structure (Xu, G.Y., et al (1997) J Mol Biol 268(2), 468) -481 and only residues located on the protein surface were considered available for Fab binding.

Two different limits were used to identify Fab-bound residues, mean minimum shift +1SD (0.143) and mean minimum shift +2SD (0.213). The binding site of the antibody was found to include the critical site 3 signal residue of Trp157 (Boulanger et al, 2003, Science, 300, 2101-2104). Using the amino acid numbering used by Boulanger et al, supra, it was found that antibody 240gl binds to at least the following residues (average +2SD (0.213)) S47, C50, E93, R104, F105, E106, T149, K150, a153, Q156, Q159, and S169. The antibody may bind all of residues (average +1SD (0.143)) C44, S47, C50, S53, a58, E93, V96, R104, F105, E106, T149, K150, Q152, a153, Q154, N155, Q156, W157, Q159, T163, L165, S169, and E172.

It will be appreciated that the same residues may also be numbered based on the amino acid numbering of the unprocessed IL-6 precursor (Swiss Prot accession number P05231). Using this numbering, antibody 240g.l binds to at least residues S75, C78, E121, R132, F133, E134, T177, K178, a181, Q182, Q187 and S197 below. The antibody may bind all of residues C72, S75, C78, S81, a86, E121, V124, R132, F133, E134, T177, K178, Q180, a181, Q182, N183, Q184, W185, Q187, T191, L193, S197, and E200.

Table 1: basic parameters of NMR experiments

Direct acquisition using a scan width of 14ppm and an acquisition time of 85ms¹H (F3 or F2).

It will of course be understood that the present invention has been described by way of example only and is not intended to be limiting in any way, and that variations in detail may be made within the scope of the claims that follow. The preferred features of each embodiment of the invention are equally applicable, mutatis mutandis, to the other respective embodiments. 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.

Claims (10)

1. Has specificity for human IL-6 and has an amino acid sequence comprising the sequence gH13 or SEQ ID NO: 11 and a light chain comprising the sequence gL10 or SEQ ID NO: 13, neutralizing antibody to the light chain.

2. Has specificity for human IL-6 and has the amino acid sequence of SEQ ID NO: 16 and the heavy chain of the sequence shown in SEQ ID NO: 18, or a neutralizing antibody to the light chain of the sequence shown in 18.

3. Isolated DNA sequences encoding the heavy and light chains of an antibody according to claim 1 or 2.

4. The isolated DNA sequence according to claim 3, wherein the DNA sequence encoding the heavy chain comprises the amino acid sequence of SEQ ID NO: 12, and the DNA sequence encoding the light chain comprises SEQ ID NO: 14, or a sequence shown in fig. 14.

5. A cloning or expression vector comprising one or more DNA sequences according to claim 3 or 4.

6. A host cell comprising one or more cloning or expression vectors according to claim 5.

7. A method of producing an antibody having binding specificity for human IL-6 comprising culturing the host cell of claim 6 and isolating the antibody.

8. A pharmaceutical composition comprising an antibody of claim 1 or 2, in combination with one or more pharmaceutically acceptable excipients, diluents or carriers.

9. The pharmaceutical composition according to claim 8, additionally comprising other active ingredients.

10. An antibody according to claim 1 or 2 or a pharmaceutical composition according to claim 8 or claim 9 for use in the treatment or prevention of a pathological disorder mediated by IL-6 or associated with elevated IL-6 levels.