HK1193113B - Binding members for interleukin-6 - Google Patents

Description

Binding members for interleukin-6

This application is a divisional application entitled "binding members for interleukin-6" filed under 2007.11.28, CN 200780044214.7.

Technical Field

The present invention relates to binding members, in particular antibody molecules, that inhibit the biological effects of IL-6. The binding members are useful in the treatment of IL-6 related diseases, including inflammatory diseases and tumors.

Background

Interleukin 6(IL-6) is a 26kDa pleiotropic proinflammatory cytokine produced by a variety of cell types, including stimulated fibroblasts, monocytes, and endothelial cells, which constitute a major in vivo source of IL-6. Cells such as T cells, B cells, macrophages, keratinocytes, osteoblasts and several other cells can be stimulated to produce IL-6. IL-6 is also expressed by tumor cell lines and cells of tumors, such as lung cancer, prostate cancer, myeloma, adreno-like tumors and cardiac myxoma [1,2 ]. Under non-inflammatory conditions, IL-6 is secreted by adipose tissue [3 ].

The regulation of IL-6 expression depends on the cell that produces it. In multiple myeloma cells, IL-6 appears to act as a positive feedback loop-stimulating cell growth and producing more IL-6[4,5 ]. In other cell types, IL-6 appears to inhibit cell growth and activation, and may act as a negative regulator of some proinflammatory cytokines.

To initiate cell signaling, IL-6 binds with low affinity to transmembrane receptor IL-6 receptor alpha (also known as IL-6Ra, IL-6R, gp80, or CD126) to form an "IL-6: IL-6Ra" complex. This complex binds to gp130 signaling receptor; IL-6Ra and gp130 together form a high affinity IL-6 binding site and induce the formation of a hexamer consisting of two copies of each of IL-6, IL-6Ra and gp130 [6 ]. The transmembrane and cytoplasmic domains of IL-6Ra are not required for signal transduction, since soluble secreted forms of IL-6Ra also exist (sIL-6R or sIL-6 Ra). Soluble receptors are produced by differential splicing or proteolytic shedding of the IL-6Ra messenger. sIL-6R is capable of forming a ligand-receptor complex "IL-6: sIL-6Ra" with IL-6. This complex binds gp130 on cells, thereby initiating cell signaling in gp 130-positive cells, even if these cells do not express IL-6 Ra. Therefore, sIL-6R has the potential to broaden the spectrum of IL-6 responsive cells, which is thought to play an important role in IL-6-mediated inflammation [7 ].

The crystal structure of human IL-6 ligands has been elucidated [6 ]. The crystal structure of the extracellular domain of human IL-6Ra has also been obtained [8] and the hexamer structure of the IL-6/IL-6R/gp130 complex [9 ]. After combining these structures with mutagenesis studies, three sites were identified on the surface of IL-6 that are involved in the functional activity of IL-6 in forming complexes with various receptor components. The site 1 residue is involved in the interaction of IL-6 and IL-6 Ra. The site 2 residue is involved in the interaction of the IL-6 and gp130 cytokine binding domains. The site 3 residue of IL-6 is involved in the interaction with the Ig-like domain of the second gp130 in the hexamer complex. A fourth site on IL-6 has also been identified where IL-6 interacts with a second IL-6 molecule in the IL-6/IL-6R/gp130 hexamer complex [10 ].

A number of anti-IL-6 ligand monoclonal antibodies have been isolated. Mapping studies have been performed which show that they bind to different binding sites on the surface of human IL-6 as described above [11,12,13,14,15 ].

A number of anti-IL-6 Ra monoclonal antibodies have also been generated, mapping their binding sites on IL-6Ra [16,14,15,17 ].

IL-6 belongs to a family of cytokines that includes interleukin-11 (IL-11), ciliary neurotrophic factor (CNTF), oncostatin M (OsM), Leukemia Inhibitory Factor (LIF), cardiotrophin-like cytokine (CLC), and cardiotrophin 1 (CT-1). Each member of this family has its own specific receptor alpha subunit and forms a complex with the co-receptor subunit gp 130. For embryos, targeted disruption of the gp130 gene is lethal [18,19 ]. All IL-6 family members induce liver cells to express acute phase proteins.

IL-6 signaling involves tyrosine phosphorylation by JAK family kinases, followed by activation of two major intracellular signaling cascades, the SHP2/ERKMAPK and STAT1/3 pathways, leading to gene expression by NF-IL-6 and AP-1 [18,20 ].

IL-6 exhibits a broad spectrum of biological functions, including: hematopoiesis, induction of acute phase responses, T cell activation, stimulation of antibody secretion, host defense against infection, myeloma and osteoclast activation [21,22 ]. For a review of IL-6 effects see reference [23 ]. IL-6 was originally identified as a B cell differentiation factor produced by T cells [24], but was subsequently identified as a potent activator and growth promoting factor for many cell types. It induces the final maturation of B cells into antibody-producing cells, an essential cofactor for T cell activation and proliferation. Studies have shown that IL-6 is involved in the activation of autoreactive T lymphocytes and in the proliferation and differentiation of cytotoxic T cells. IL-6 has been shown to be involved in the hematopoietic process as a cofactor, causing the activation and differentiation of hematopoietic stem cells. The effect of IL-6 on acute phase responses was also documented [25 ]. IL-6 induces human hepatocytes to produce various acute phase proteins, including fibrinogen, alpha-anti-chymotrypsin, serum amyloid A, and C-reactive proteins. Acute phase proteins control immune responses and inflammation and have an effect on tissue remodeling. In various diseases, IL-6 serum levels and C reactive protein serum levels are well correlated, which suggests IL-6 in acute phase response in the etiological role. It has also been demonstrated that IL-6 is produced by osteoblasts and appears to be involved in osteoclast activation and bone resorption [26, 27, 28 ]. Paradoxically, it is suggested that IL-6 not only acts as a pro-inflammatory cytokine, but in certain cases and cell types, can attenuate the effects of other pro-inflammatory cytokines, resulting in a reduction in inflammation.

Since IL-6 has a variety of biological effects, elevated levels of IL-6 may be a key cytokine in various disease conditions. Elevated levels of circulating IL-6 have been demonstrated in a variety of diseases, such as rheumatoid arthritis, Castleman's disease, juvenile idiopathic arthritis and Crohn's disease [29 ]. Thus, IL-6 is involved in the pathology driving these inflammatory disorders. Moreover, IL-6 has been shown to stimulate a variety of tumor types, including melanoma, renal cell carcinoma, Kaposi's sarcoma, ovarian cancer, lymphoma, leukemia, multiple myeloma, and prostate cancer [30 ]. In addition, circulating levels of IL6 have been reported to be elevated in several cancers. In some cancer disorders, elevated IL-6 levels have been used as a prognostic indicator for the disease.

Because of the role of IL-6 in disease, a variety of murine and chimeric anti-human IL-6 monoclonal antibodies have been developed as potential therapeutics.

US5856135 describes reshaped human antibodies to IL-6 derived from the mouse monoclonal antibody "SK 2".

JP-10-66582 reports a chimeric antibody to IL-6, which recognizes the helical D region (site 1) of IL-6.

WO2004/020633(EP1536012) describes human scFv antibody molecules for IL-6 isolated by phage display technology. The affinity of the scFv was reported to be 13 nM.

The murine anti-IL-6 antibody, Esicimomab (also known as B-E8), has been used to treat patients with multiple myeloma [31, 32], renal cell carcinoma [33], and rheumatoid arthritis [34], with improvements in certain diagnostic markers observed in patients with all three diseases treated. BE-8 has also been used to treat HIV-positive immunoblastic or pleomorphic large cell lymphoma [35] patients with remission of systemic symptoms (i.e. fever, sweating, cachexia) and suppressed spontaneous growth of lymphoma in about 50% of patients.

However, the rapid clearance of this antibody and the possible induction of allergic reactions due to the production of human anti-mouse antibodies (HAMA) to imazamab limit its clinical utility [36 ].

Generally, clinical applications of murine monoclonal antibodies are limited because such antibodies often induce HAMA. HAMA is often produced on the Fc portion of mouse immunoglobulins, leading to rapid clearance and possible allergic reactions by anti-IL-6 mAbs [36 ]. It is also known that mouse antibodies have pharmacokinetic properties in humans that differ from human antibodies, with shorter half-lives and improved clearance rates.

To reduce the immunogenicity of murine antibodies in humans, chimeric antibodies with mouse variable regions and human constant regions were constructed. Patients with multiple myeloma have been treated with the chimeric human-mouse anti-IL-6 antibody clb8 (known as CNTO328) [5, 37], and disease stabilization has been observed in most patients.

However, although chimeric antibodies are less immunogenic than murine mabs, human anti-chimeric antibody (HACA) responses have been reported [38 ].

A mapping study was performed on cCLB8, which showed that it is a site I inhibitor of IL-6 activity. Brakenhoff et al [39] demonstrated that cCLB8 binds to the IL-6 amino-terminal deletion variants Pro46, Ser49, Glu51, Ile53, Asp54, and also to the deletion variants Asp62 and Met77 (although with reduced affinity). The same authors demonstrated that clb8 inhibited wild-type IL-6 but not C-terminal deletion 5 in a B9 cell proliferation assay, and that clb8 did not bind to IL-6delC-4 from which the last 4C-terminal amino acid residues were deleted. These data indicate that clb8 binds to an epitope comprising the C-terminal residue of IL-6.

Kalai et al [17] demonstrated that cCLB8 failed to recognize IL-6 mutants F106E, F102E/F106E, or R207E/R210E. However, this antibody was able to recognize the IL-6 mutants R207E and R207W. The binding performance of clb8 to mutants R207W and R207E was approximately 50% of that of wild type, indicating that residues F106 and R210 are involved in the clb8 binding epitope and that residue R207 is involved in binding but has less effect than residues F106 and R210. cCLB8 bound IL-6 site-I mutants R196M, K199N/Q203L and Q203L with 100% activity compared to the wild type. Brakenhoff et al [13] demonstrated that cCLB8 binds to the following IL-6 variants: Q182H, N183K, W185Q, W185G, W185R, T190P, Q182H/Q184P, W185R/S197N, Q187E/T190P, I164L/L186R/M189I, which is not surprising, since most of them are far apart from the 1 residue at position IL-6.

The advantageous effect of inhibiting IL-6 signaling in cancer and inflammatory diseases is further emphasized using the humanized anti-IL-6 Ra antibody tollizumab (also known as hPM-1, MRA and Actemra). This is the humanized murine anti-IL 6Ra antibody PM-1. Treatment of patients with such antibodies is effective against a variety of diseases including rheumatoid arthritis, juvenile idiopathic arthritis, Crohn's disease, myeloproliferative diseases, Karstmann's disease and systemic lupus erythematosus [40 ].

Disclosure of Invention

We have successfully isolated a high efficiency, high affinity binding member for IL-6. Due to the high affinity and potency of the binding members of the invention, and their performance in functional studies as described herein, the binding members of the invention are particularly useful in the therapeutic and diagnostic treatment of the human or animal body.

The binding members are useful for treating diseases associated with IL-6, as detailed elsewhere herein.

Human anti-IL-6 antibodies for the treatment of inflammatory diseases and cancer have significant advantages over existing methods. For example, human antibodies do not induce HAMA or HACA responses and have a longer half-life in vivo than non-human or chimeric antibodies.

We also recognize that IL-6 binding members have significant advantages over binding members for IL-6Ra, particularly with respect to in vivo administration and treatment, as described elsewhere herein.

As described in more detail in the examples section, we isolated a parent antibody molecule (CAN022D10) containing a set of CDR sequences as shown in table 7. By optimization, we generated a panel of antibody clones: antibodies 2, 3,4, 5,7, 8, 10, 14, 16, 17, 18,19, 21,22 and 23, the CDR sequences derived from the parent CDR sequences and containing substitutions at the positions indicated in table 7.

Thus, for example, it can be observed from Table 7 that antibody 2 has the parent HCDR1 sequence with Kabat residue 35 substituted with Thr (SEQ ID NO: 13). Antibodies 14 and 22 contain additional residues in HCDR3, i.e. amino acid insertions: ile is contained at Kabat residue 100D, which is not present in the parent HCDR3 sequence of SEQ ID NO: 5. Antibodies 7, 8, 10, 16-19, 21 and 23 do not contain Kabat residue 95 in LCDR3, while parent LCDR3(SEQ ID NO:10) contains Pro at Kabat residue 95. The parent HCDR3 and HCDR3 sequences of all antibodies 2, 3,4, 5,7, 8, 10, 14, 16, 17, 18,19, 21,22 and 23 contained Trp at Kabat residue 95 and Asp at residue 101, indicating that H95Trp and H101Asp may contribute to the binding member of the invention's binding to IL-6 and/or efficacy.

The VH, VL and CDR sequences of the parent antibody CAN022D10 and antibodies 2, 3,4, 5,7, 8, 10, 14, 16, 17, 18,19, 21,22 and 23 described herein are found in the accompanying sequence listing.

As described in detail below, the binding members of the invention are capable of neutralizing IL-6 with high efficiency. Neutralization indicates inhibition of the biological activity of IL-6. Binding members of the invention may neutralize one or more biological activities of IL-6. The biological activity that is inhibited is typically IL-6 in combination with one or more of its binding partners. For example, the biological activity that is inhibited can be the binding of IL-6 to transmembrane and/or soluble IL-6R α. This is demonstrated in the following experiments, which are briefly described here, and which are described in detail below: TF-1 assays demonstrate that binding members of the invention inhibit the binding of IL-6 to membrane IL-6Ra, since TF-1 cells do not appear to produce soluble IL-6 Ra. Likewise, binding members of the invention inhibit the binding of IL-6 to membrane receptors. In synovial fibroblast assays, binding members of the invention inhibit the binding of IL-6 to soluble IL-6Ra, as sIL-6Ra needs to be added to the assay to be effective. The added IL-1 β induces endogenous IL-6 production and when inhibited by a binding member of the invention prevents VEGF production.

According to the invention, binding of IL-6 to IL-6Ra in a human or non-human primate, such as a cynomolgus monkey, may be inhibited, e.g.a binding member may inhibit binding of mature human IL-6 to IL-6 Ra.

Biological activity may be partially or completely inhibited. A binding member can inhibit IL-6 biological activity by 100%, or by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% compared to the activity in the absence of the binding member.

The neutralizing potency of the binding member can be determined. Unless otherwise stated, potency is usually expressed as IC in nM₅₀The value is obtained. In functional testing, IC₅₀Is the concentration of binding member that reduces the biological response by 50% from its maximum value. In ligand binding studies, IC₅₀Is the concentration that reduces the level of ligand-receptor complex formation by 50% of the maximum level of specific binding. IC can be calculated by plotting% of highest biological response as a function of log (binding member concentration) using a software program such as Prism from graph pad company (GraphPad) or Origin from the Origin laboratory (Origin labs)₅₀To fit a sigmoid function to the data to generate an IC₅₀The value is obtained. Efficacy may be determined or detected using one or more assays known to those skilled in the art or described or depicted herein.

The ability of a binding member to bind and neutralize IL-6 activity in assays described herein, such as the TF-1 proliferation assay or other cell assays described below, indicates that the binding member is capable of binding and neutralizing IL-6. Other methods that can be used to determine binding of a binding member to IL-6 include ELISA, Western blotting, immunoprecipitation, affinity chromatography, and biochemical assays.

The binding members described herein are capable of binding to and neutralizing the biological effects of endogenous human IL-6 as demonstrated by the assays reported herein in examples 1.7 and 2.7 that inhibit the release of VEGF by human synovial fibroblasts in response to endogenous human IL-6. In this experiment, synovial fibroblasts from rheumatoid arthritis patients produced IL-6 in response to IL-1 β and soluble IL-6R α stimulation, resulting in IL-6-induced VEGF secretion. Thus, human synovial fibroblasts produce IL-6 representing endogenous human IL-6. Endogenous IL-6 is a molecular target for medical treatment of humans, and therefore neutralization of endogenous IL-6 is an important indicator of the therapeutic potential of a binding member. Since the assay was performed using synovial fibroblasts obtained from rheumatoid arthritis patients, the results were particularly relevant for the treatment of rheumatoid arthritis with the binding members. The neutralizing potency of the optimized antibody molecules detected in the VEGF release assay exceeded that of the known anti-IL-6 antibody CNTO-328.

IC of binding members of the invention in an assay for the inhibition of VEGF release from human synovial fibroblasts stimulated with 0.6pM human IL-1 β and 2.4nM soluble human IL-6R α₅₀May be less than 50nM, such as less than 5nM, for example less than 1 nM.

Endogenous IL-6 is known to be a mixture of glycosylated and non-glycosylated forms. Binding of a binding member of the invention to endogenous IL-6 is demonstrated in a synovial fibroblast assay, since this assay utilizes IL-6 produced by human synovial fibroblasts, i.e., endogenous IL-6.

Binding members of the invention inhibit IL-6-induced proliferation of TF-1 cells. TF-1 is a human promyelocytic cell line (premyelocellline) established from erythroleukemia patients (Kitamura et al, 1989). The survival and proliferation of TF-1 cell lines requires the presence of growth factors. TF-1 cells can respond to various growth factors including IL-6, GM-CSF and oncostatin M. IC of binding members of the invention in an assay to inhibit proliferation of TF-1 cells in response to 20pM human IL-6₅₀It may be less than 100nM, for example less than 20nM, 10nM or 1nM, for example less than 100pM, 70pM, 50pM, 40pM, 30pM, 20pM or 10 pM. As described herein (see example 1.5), the IC of the parent IgG "CAN022D10" in TF-1 proliferation experiments has been demonstrated₅₀At about 93nM, we subsequently generated optimized variants (IC) of CAN022D10 with significantly improved potency₅₀Typically less than 100pM) as shown in examples 2.2, 2.5 and 2.6 (tables 3,4 and 5 respectively). In particular some of the cloned ICs₅₀Values were detected as low as 5pM or less, e.g. germlined IgG antibody 7, antibody 17 and antibody 18, indicating that these antibodies are extremely high in neutralizing potency.

The binding members of the invention inhibit IL-6-induced proliferation of B9 cells. B9 cells were subclones of the murine B-cell hybridoma cell line B13.29 selected for specific response to IL-6. Survival and proliferation of B9 cells requires IL-6, responding to extremely low concentrations of IL-6. Thus, the proliferation of these cells in the presence of IL-6 antibodies can be assessed and the affinity of the antibodies determined. Example 2.10 described herein demonstrates that antibody 18 inhibits proliferation of B9 cells in response to IL-6 and shows high affinity in this experiment.

Autoantibodies produced in rheumatoid arthritis are mainly of the IgM type. SKW6.4 is a cloned human lymphoblast B cell line secreting IgM. These cells secrete IgM after stimulation with IL-6, and therefore this experiment is considered to be associated with rheumatoid arthritis. Binding members can be assayed for their efficacy in neutralizing IL-6 by measuring inhibition of secreted IgM in response to IL-6, using SKW6.4 cells. IC of binding members of the invention in an assay for SKW6.4 cells that inhibit IgM secretion in response to 100pM human IL-6₅₀May be less than 10pM, for example less than 5 pM. Antibody 18 has been shown to neutralize the effects of IL-6 in this experiment-see example 2.11 (Table 9).

The present invention provides high affinity binding members for human IL-6. Also show with short tail monkey IL-6 high affinity. Binding members of the invention may bind human IL-6 and/or cynomolgus IL-6, K thereof_DNot more than 1nM, e.g. not more than 100pM, 50pM, 30pM or 10 pM. By surface plasmon resonance, e.g.Determination of K_D. By usingThe method for determining affinity is described in example 2.9 herein. It is evident that the affinity of antibodies 7 and 18 was found to exceed that of the useMeasurement limits of the apparatus, thus indicating K_DValues below 10 pM.

As described elsewhere herein, surface plasmon resonance involves flowing an analyte in a liquid phase through a support-attached ligand and measuring the binding between the analyte and the ligand. For example, plasmon resonance can be effected by flowing IL-6 in a liquid phase through a support-attached binding member. Surface plasmon resonance data can be fitted to a monovalent analyte data model. The ratio of the rate constants, Kd/ka, can be determined by surface plasmon resonance using a monovalent analyte data model, and the affinity constant, Kd, can be calculated from the ratio of the rate constants.

Alternatively, the affinity of the binding member for IL-16 may be calculated by a Schild assay, for example based on experiments inhibiting the proliferation of TF-1 cells in response to different concentrations of human IL-6. Binding members of the invention may have an affinity of less than 10pM, for example less than 1pM, as calculated by schild analysis. As reported in example 2.10 herein, the affinity of antibody 18 to human IL-6, as calculated by the Schilder analysis, was 0.4 pM.

Optionally, a binding member of the invention may not cross-react with one or more, or all, of the following: leukemia Inhibitory Factor (LIF), ciliary neurotrophic factor (CNTF), IL-11, or oncostatin M.

Optionally, a binding member of the invention may not cross-react with rat IL-6, mouse IL-6 and/or canine IL-6.

Time-resolved fluorescence assays that can, for example, inhibit the binding of human IL-6 to a binding member immobilized on a support, as described in example 16Epitope competition experiments were performed to detect the cross-reactivity of binding members to other proteins or non-human IL-6. For example, in a time-resolved fluorescence assay that inhibits the binding of labeled human IL-6 to a binding member immobilized on a support, it is possible that none or all of LIF, CNTF, IL-11, Oncostatin M, rat IL-6, and mouse IL-6 exhibit inhibition, less than 50% inhibition, or IC₅₀Greater than 0.5mM or greater than 1 mM. For example, in detecting cross-reactivityIn fluorescence-resolved assays, it is possible that none or all of LIF, CNTF, IL-11, Oncostatin M, rat IL-6, and mouse IL-6 show inhibition, or IC₅₀At least 10-fold or 100-fold higher than unlabeled human IL-6. In this experiment, labeled mature wild-type human IL-6 was used at a final concentration equal to the Kd of the binding member interaction.

Binding members of the invention can cross-react with cynomolgus IL-6. In the time-resolved fluorescence assay described above, cross-reactivity is determined by inhibition of binding of labeled human IL-6 to the immobilized binding member on the support. For example, IC of cynomolgus IL-6 in this time-resolved fluorescence experiment₅₀May be less than 5nM, such as less than 2.5nM, such as about 1 nM. In this experiment, IC of IL-6 from cynomolgus monkey₅₀IC with unlabeled human IL-6₅₀May be less than a factor of 10, such as less than a factor of 5.

Materials and methods section provides detailed protocols for time-resolved fluorescence experiments to determine cross-reactivity. An example of the cross-reaction data obtained in this experiment is given in table 2 of example 1.6.

As reported in example 2.8, the binding members described herein have high cross-reactivity with cyno IL-6, but no or limited cross-reactivity with rat, mouse or canine IL-6.

Cross-reactivity data indicate that binding members described herein bind to IL-6 epitopes that are conserved between human and cyno IL-6 sequences, but that are different in mouse, rat and canine IL-6 sequences compared to human sequences.

It is believed that the binding members described herein are capable of binding to the "site 1" region of IL-6, which is the region that interacts with IL-6 Ra. Thus, the binding members of the invention competitively inhibit the binding of IL-6 to IL-6Ra, thereby neutralizing the IL-6 Ra-mediated biological effects of IL-6.

We investigated the ability of one of the antibodies described herein, antibody 18, to bind to mutant human IL-6 engineered to produce a mutation at residue 1. As described in example 3, we identified mutations in human IL-6 that result in a reduction in the binding capacity of antibody 18, indicating that these mutated residues are involved in the recognition of antibody 18 and may form part of the epitope of IL-6 that the antibody binds to.

For example, in a time-resolved fluorescence assay that inhibits the binding of labeled wild-type human IL-6 to antibody 18 immobilized on a support, no inhibition was observed with Arg207Glu mutant human IL-6(SEQ ID NO: 177), indicating that antibody 18 is capable of binding to residue Arg207 of human IL-6.

Since antibody 18 and antibodies 2, 3,4, 5,7, 8, 10, 14, 16, 17, 19, 21,22 and 23 are all derived from the parent antibody CAN22C10 and all have structurally related CDRs, it is expected that all of these antibody molecules will bind the same or very similar overlapping epitopes. Thus, it is also expected that the epitope mapping results obtained with antibody 18 CAN represent CAN22D10, another optimized antibody described herein.

Binding members of the invention can bind Phe102 and/or Ser204 of human IL-6. Binding members of the invention may also bind Arg207 of human IL-6. Optionally, in addition to binding Phe102 and/or Ser204, a binding member can also bind to a flanking residue or a structurally adjacent residue in the IL-6 molecule. By convention, the residue numbering corresponds to full-length human IL-6(SEQ ID NO: 161). However, binding can be determined using mature human IL-6. Binding to IL-6 residues was determined by site-directed mutagenesis as described below.

The person skilled in the art is well aware of the mutagenesis of single amino acids and multiple regions of proteins in order to relate structure to activity, and this method is used to determine the region of a protein that binds to an antibody [41 ]. Binding and/or neutralizing mutant human IL-6 can be used to assess whether a binding member binds Phe102, Ser204, and/or Arg 207. Failure to bind or neutralize, or a significant decrease in binding or neutralization, with mutant IL-6 as compared to wild type indicates that the binding member binds to this mutant residue.

Binding to an IL-6 residue can be determined using IL-6 mutated at the selected residue in a time-resolved fluorescence assay that inhibits binding of labeled wild-type human IL-6 to a binding member immobilized on a support, wherein the final concentration of labeled wild-type mature human IL-6 is equal to the Kd of its interaction with the binding member. Examples of such experiments and the competition data obtained are illustrated in example 3, the results of which are shown in Table 10. When the mutant IL-6 does not inhibit the binding of the labeled wild type IL-6 to the binding member, or when the mutant IL-6 has an IC50 greater (e.g., more than 10-fold or 100-fold) than the unlabeled wild type IL-6, it is indicative that the mutant residue binds to the binding member.

In time-resolved fluorescence experiments inhibiting the binding of labeled wild-type human IL-6 to the binding members of the invention immobilized on a support, Phe102Glu mutant human IL-6(SEQ ID NO: 175), Ser204Glu mutant human IL-6(SEQ ID NO: 176) and/or Arg207Glu mutant human IL-6(SEQ ID NO: 177) may show no inhibition, or its IC₅₀IC over wild-type human IL-6(SEQ ID NO: 165)₅₀100-fold higher, wherein the final concentration of labeled wild-type human IL-6 is equal to the Kd of its interaction with the binding member.

Optionally, a binding member of the invention may not bind and/or neutralize mutant human IL-6 that contains mutations at residues Phe102, Ser204, and/or Arg207, e.g., mutations Phe102Glu, Ser204Tyr, and/or Arg207 Glu. An example of a mutant human IL-6 sequence is SEQ ID NO: 175-177. Thus, a binding member of the invention may not inhibit the binding of one or more of these mutant IL-6 molecules to IL-6 Ra.

The binding member of the invention may comprise an antibody molecule, for example a human antibody molecule. The binding member typically comprises an antibody VH and/or VL domain. The VH and VL domains of the binding members are also provided as part of the invention. Within each VH and VL domain are complementarity determining regions ("CDRs") and framework regions ("FRs"). The VH domain comprises a set of HCDRs and the VL domain comprises a set of LCDRs. The antibody molecule may comprise an antibody VH domain comprising VHCDR1, CDR2 and CDR3 and framework regions. Or it may further comprise an antibody VL domain comprising VLCDRs 1, CDR2 and CDR3 and framework regions. The framework regions of the VH or VL domain comprise four framework regions FR1, FR2, FR3 and FR4 interspersed with CDRs and have the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。

examples of antibody VH and VL domains and CDRs of the present invention are set forth in the accompanying sequence Listing which forms a part hereof. Other CDRs are shown below and in table 7. All VH and VL sequences, CDR sequences, sets of CDRs and sets of HCDRs and sets of LCDRs disclosed herein represent aspects and embodiments of the present invention. The "set of CDRs" described herein includes CDR1, CDR2, and CDR 3. Thus, a set of HCDRs refers to HCDR1, HCDR2, and HCDR3, and a set of LCDRs refers to LCDR1, LCDR2, and LCDR 3. Unless otherwise stated, a "set of CDRs" includes HCDRs and LCDRs. The binding members of the invention are typically monoclonal antibodies.

Binding members of the invention may comprise an antigen binding site within a non-antibody molecule, typically provided by one or more CDRs, such as a set of CDRs, in a non-antibody protein scaffold, as will be discussed further below.

Described herein are binding members comprising the parent set of CDRs shown in table 7 of parent CAN022D10, wherein HCDR1 is seq id no:3(Kabat residues 31-35), HCDR2 is seq id no:4(Kabat residues 50-65), HCDR3 is seq id no:5(Kabat residues 95-102), LCDR1 is seq id no:8(Kabat residues 24-34), LCDR2 is seq id no:9(Kabat residues 50-56) and LCDR3 is seq id no:10(Kabat residues 89-97).

Binding members of the invention may comprise one or more CDRs as described herein, for example CDR3, and optionally also CDR1 and CDR2, thereby forming a set of CDRs. The CDR or set of CDRs may be a parent CDR or set of CDRs, or may be a CDR or set of CDRs of any of antibodies 2, 3,4, 5,7, 8, 10, 14, 16, 17, 18,19, 21,22 or 23, or may be variants thereof, as described herein.

For example, a binding member or VL domain of the invention may comprise a VL having the amino acid sequence of seq id no: LCDR3 of 120.

The binding member may comprise the H and/or LCDR panel of the parent antibody or any of antibodies 2, 3,4, 5,7, 8, 10, 14, 16, 17, 18,19, 21,22 or 23, which comprises one or more amino acid mutations. The amino acid mutation is a substitution, deletion or insertion of one amino acid. For example, there may be up to 20, such as up to 12, 11, 10, 9, 8, 7, 6,5, 4,3 or 2 mutations, such as substitutions, within the H and/or LCDR groups. For example, there may be up to 6,5, 4,3, or 2 mutations, e.g., substitutions, in HCDR3 and/or up to 6,5, 4,3, or 2 mutations, e.g., substitutions, in LCDR 3. Optionally, the HCDR3 and/or LCDR3 may contain an insertion or deletion of one amino acid compared to the H and/or LCDR groups. For example, the substitution can be a position of substitution in any of antibodies 2, 3,4, 5,7, 8, 10, 14, 16, 17, 18,19, 21,22, or 23, as shown in table 7. Thus, the substitution may optionally occur at Kabat numbering selected from the group consisting of:

kabat residue 35 in HCDR 1;

kabat residue 64 in HCDR 2;

kabat residues 96, 97, 98, 99, 100A, 100B, 100C, and/or 102 in HCDR 3;

kabat residue 34 in LCDR 1;

kabat residues 89, 90, 91, 92, 93, 94, 96 or 97 in LCDR 3.

The amino acid mutations can comprise mutations shown in table 7, such as the amino acid substitutions shown.

For example, a binding member or VH domain of the invention may comprise a parent HCDR1 having Kabat residue Ile35 substituted with Thr or Val.

The binding member or VH domain of the invention may comprise a parent HCDR2 with Kabat residues Lys64 substituted with Arg.

The binding member or VH domain may comprise a parent HCDR3 containing one or more of the following mutations:

kabat residue Ala96 substituted with Glu;

kabat residue Asp97 substituted with Glu or Asn;

kabat residue Asp98 substituted with Gly, Glu, or His;

kabat residue His99 substituted with Gly or Thr;

kabat residue Tyr100 is substituted with Pro, Asn, Arg, Trp, or Ala;

kabat residue Tyr100A substituted with Ala, Arg, Thr, Gly, Asn, Pro, or Ser;

kabat residue 100B by His, Trp, Gln, Pro or Thr;

kabat residue Ile100C substituted by Ala, Val, His, Tyr or Leu;

ile insertion into Kabat residue 100D;

kabat residue Val102 by Leu, His, Met or Ile substitution.

Thus, a binding member or VH domain of the invention may comprise an HCDR3 wherein Kabat residue 100D is Ile or wherein Kabat residue 100D is not present.

The binding member or VL domain of the invention may comprise a parent LCDR1 having Kabat residue Ala34 substituted with Thr.

Binding members of a VL domain of the invention may comprise a parent LCDR3 comprising one or more of the following mutations:

kabat residues Gln89 substituted by Met or Ala;

kabat residues Gln90 substituted with Asn, Ser, or Ala;

kabat residue Ser91 substituted with Asn, Gly, Ala, or His;

kabat residue Tyr92 substituted with Trp, Ser, Lys, or Phe;

kabat residue Ser93 substituted with Leu, Lys, Arg, or Ala;

kabat residue Thr94 substituted with Ala, Gly, or Pro;

kabat residue Pro95 deletion;

kabat residue Trp96 substituted with Gly;

kabat residue Thr97 was substituted with Ser.

Thus, a binding member or VL domain of the invention may comprise LCDR3 where Kabat residue 95 is Pro or Kabat residue 95 is not present.

The present invention provides an isolated human IL-6 binding member comprising a set of CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the set of CDRs contain 22 or fewer amino acid changes from a set of CDRs, e.g., at most 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,5, 4,3, 2,1 change or no change, wherein:

HCDR1 contains the amino acid sequence SEQ ID NO. 3;

HCDR2 contains the amino acid sequence SEQ ID NO. 4;

HCDR3 contains the amino acid sequence SEQ ID NO. 115;

LCDR1 contains amino acid sequence SEQIDNO 8;

LCDR2 contains amino acid sequence SEQIDNO 9; and

LCDR3 contains amino acid sequence SEQIDNO 120.

The amino acid change may be a substitution, insertion or deletion. Examples of Kabat positions that may be substituted and examples of residue substitutions are discussed below, with table 7 illustrating some substitutions.

As shown in table 7, the length of HCDR3 and LCDR3 varied between the different optimized antibodies described herein. Relative to the parent CDRs of CAN022D10, an insertion between Kabat residues 100 to 102 was observed in some antibodies (Kabat residues 100D as shown in table 7), and a deletion between Kabat residues 92 to 97 was observed in other antibodies. The deletion of Kabat residue 95 does not occur simultaneously with the insertion. Thus, it may be advantageous to combine the longer 12-residue HCDR3 sequence with the longer 9-residue LCDR3 sequence, and to combine the shorter 11-residue HCDR3 sequence with the shorter 8-residue LCDR3 sequence.

The residues of LCDR3 were numbered 89 to 97 according to the Kabat numbering system. The Kabat numbering system does not consider LCDR3 sequences shorter than 9 residues. In the present invention, the binding member may contain LCDR3 shorter than 9 residues, for example LCDR3 may be 8 residues in length, as shown in table 7. We numbered the 8 residues of LCDR3 as 89, 90, 91, 92, 93, 94, 96 and 97, respectively. Thus, in table 7, the deletion occurs at Kabat residue 95. It will be appreciated, however, that the effect of the deletion is to shorten the LCDR3 sequence, and in principle it is envisaged that the deletion may occur at any of residues 89 to 97, for example residues 92 to 97.

In HCDR3, the Kabat numbering system can accommodate CDR length changes by extending the numbering system between Kabat residues 100 and 101, for example suitably including HCDR3 where residue 100A is9 residues, plus HCDR3 where 100B is 10 residues, plus HCDR3 where 100C is 11 residues, plus HCDR3 where 100D is 12 residues. In table 7, the insertion of additional amino acids in HCDR3 of some optimized clones relative to the parent HCDR3 is shown at Kabat residue 100D. However, it is to be understood that in principle it is contemplated that such insertion may occur at any of Kabat residues 100 to 102.

As described herein, one or more insertions or deletions may occur in one or more CDRs of a binding member, e.g., HCDR3 and/or LCDR 3. For example, a binding member of the invention may comprise a set of CDRs of any of antibodies 2, 3,4, 5,7, 8, 10, 14, 16, 17, 18,19, 21,22 and 23, or variants thereof (described herein), wherein each CDR optionally comprises an insertion to extend the CDR by one residue, or a deletion of one residue to shorten the CDR by one residue. Insertions and/or deletions may be made in HCDRs and/or LCDRs, such as HCDR3 and/or LCDR 3.

For example, a binding member may comprise a set of CDRs comprising 20 or fewer amino acid substitutions, wherein:

HCDR1 contains the amino acid sequence SEQ ID NO. 3;

HCDR2 contains the amino acid sequence SEQ ID NO. 4;

HCDR3 contains the amino acid sequence SEQ ID NO. 115;

LCDR1 contains amino acid sequence SEQIDNO 8;

LCDR2 contains amino acid sequence SEQIDNO 9; and

LCDR3 contains amino acid sequence SEQIDNO 120;

wherein the binding member optionally contains an insertion of one residue to extend HCDR3 by one residue, or a deletion of one residue to shorten HCDR3 by one residue, and/or

Contains an insertion of one residue to extend LCDR3 by one residue, or contains a deletion of one residue to shorten LCDR3 by one residue.

Binding members of the invention may comprise an insertion of one residue in HCDR3SEQ ID NO:115 and/or an insertion of one residue in LCDR3SEQ ID NO: 120.

Insertions or deletions can be made at any site in the CDRs. For example, in HCDR3, the insertion or deletion can occur at any of Kabat residues 95-102, such as Kabat residue 100-102. For example, in LCDR3, the insertion or deletion may occur at any of Kabat residues 89 to 97, such as any of Kabat residues 92 to 97.

A binding member or VH domain of the invention may comprise HCDR1 where Kabat residue 35 is Ile, Thr or Val.

The binding member or VH domain of the invention may comprise HCDR2 with Kabat residue 64 being Lys or Arg.

A binding member or VH domain of the invention may comprise HCDR3 with Kabat residue 95 being Trp and/or Kabat residue 101 being Asp.

A binding member or VH domain of the invention may comprise HCDR3, wherein:

kabat residue 96 is Ala or Glu;

kabat residue 97 is Asp, Glu, or Asn;

kabat residue 98 is Asp, Gly, Glu, or His;

kabat residue 99 is His, Gly, or Thr;

kabat residue 100 is Pro, Tyr, Asn, Arg, Trp, or Ala;

kabat residue 100A is Pro, Tyr, Ala, Arg, Thr, Gly, Asn, Pro, or Ser;

kabat residue 100B is Trp, Tyr, His, Gln, Pro, or Thr;

kabat residue 100C is Ile, Ala, Val, His, Tyr or Leu; and

kabat residue 102 is Leu, Val, His, Met or Ile.

The binding member or VL domain of the invention may comprise LCDR1 where Kabat residue 34 is Ala or Thr.

A binding member or VL domain of the invention may comprise LCDR3, wherein:

kabat residue 89 is Gln, Met, or Ala;

kabat residue 90 is Gln, Asn, Ser, or Ala;

kabat residue 91 is Ser, Asn, Gly, Ala, or His;

kabat residue 92 is Trp, Tyr, Ser, Lys, or Phe;

kabat residue 93 is Leu, Ser, Lys, Arg, or Ala;

kabat residue 94 is Gly, Thr, Ala or Pro;

kabat residue 96 is Gly or Trp; and

kabat residue 97 is Ser or Thr.

The invention provides binding members comprising the HCDR1, HCDR2 and/or HCDR3 of any of the parent or antibodies 2, 3,4, 5,7, 8, 10, 14, 16, 17, 18,19, 21,22 and 23 and/or the LCDR1, LCDR2 and/or LCDR3 of any of the parent or antibodies 2, 3,4, 5,7, 8, 10, 14, 16, 17, 18,19, 21,22 and 23, for example any of the antibodies 2, 3,4, 5,7, 8, 10, 14, 16, 17, 18,19, 21,22 and 23 of the parent or antibodies shown in table 7, for example a set of CDRs of any of the parent or antibodies 2, 3,4, 5,7, 8, 10, 14, 16, 17, 18,19, 21,22 and 23.

For example, a binding member of the invention may comprise a set of CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein:

HCDR1 is seq id no: 113; HCDR2 is seq id no: 114, and a carrier; HCDR3 is seq id no:115, 115; LCDR1 is seq id no: 118; LCDR2 is seq id no: 119; and LCDR3 is seq id no:120, representing the CDRs of antibody 18.

The binding member may comprise a set of VHCDRs of one of these antibodies. It may also optionally comprise a set of VLCDRs of one of these antibodies, which may be from the same or different antibody as the VHCDRs.

The invention also provides a set of VH domains comprising HCDRs of any of the parent or antibodies 2, 3,4, 5,7, 8, 10, 14, 16, 17, 18,19, 21,22 and 23, and/or a set of VL domains comprising LCDRs of any of the parent or antibodies 2, 3,4, 5,7, 8, 10, 14, 16, 17, 18,19, 21,22 and 23.

The VH domain is typically paired with the VL domain to provide an antibody antigen-binding site, although as discussed further below, the VH or VL domains may be used alone to bind antigen. The antibody 2VH domain may be paired with the antibody 2VL domain, thereby forming an antibody antigen-binding site comprising both antibody 2VH and VL domains. Similar embodiments of the other VH and VL domains described herein are provided. In other embodiments, the antibody 2VH is paired with a VL domain other than the antibody VL. Light chain scrambling is well known in the art. The invention also provides similar embodiments of the other VH and VL domains described herein.

Thus, the VH of any of the parent or antibodies 2, 3,4, 5,7, 8, 10, 14, 16, 17, 18,19, 21,22, and 23 may be paired with the VL of any of the parent or antibodies 2, 3,4, 5,7, 8, 10, 14, 16, 17, 18,19, 21,22, and 23.

The binding member may comprise an antibody molecule having one or more CDRs, e.g. a set of CDRs, in the antibody framework. For example, one or more CDRs or a set of CDRs of an antibody can be grafted into a framework (e.g., a human framework) to provide an antibody molecule. The framework regions may be derived from human germline gene segment sequences. Thus, the framework can be germlined, whereby one or more residues within the framework are replaced to match the residues at equivalent positions in most similar human germline frameworks. The skilled artisan can select the germline segment of the antibody framework sequence whose sequence is closest to that prior to germlining and test the affinity or activity of the antibody in the assay described herein to confirm that germlining does not significantly reduce antigen binding or potency. The sequence of the human germline gene segment is known to those skilled in the art and can be accessed, for example, by Vbase compilation (software).

The binding members of the invention may be isolated human antibody molecules having a VH domain comprising a set of HCDRs in the human germline framework, e.g., Vh3_ DP-86_ (3-66). Thus, the VH domain framework regions FR1, FR2 and/or FR3 may comprise the framework regions of the human germline gene segment VH3_ DP-86_ (3-66), and/or may be germlined by mutating framework residues to match framework residues of such human germline gene segment. FR4 may comprise the framework region of human germline j section JH 2. The amino acid sequence of VHFR 1can be SEQ ID NO: 167. The amino acid sequence of VHFR2 may be seq id no: 168. the amino acid sequence of VHFR 3can be SEQ ID NO. 169. The amino acid sequence of VHFR 4can be SEQ ID NO: 170.

Binding members also typically have a VL domain that comprises, for example, human germline framework regions such as a set of LCDRs in Vk1_ L12. Thus, the VL domain framework region may comprise the framework regions FR1, FR2 and/or FR3 of the human germline gene segment Vk1_ L12 and/or may be germlined by mutating framework residues to match framework residues of such human germline gene segment. FR4 may comprise the framework region of human germline j segment JK 2. The amino acid sequence of VLFR 1can be SEQ ID NO: 171. The amino acid sequence of VLFR 2can be SEQ ID NO: 172. The amino acid sequence of VLFR 3can be SEQ ID NO: 173. The amino acid sequence of VLFR 4can be SEQ ID NO: 174.

A germlined VL domain may or may not be germlined at one or more trim residues (vernier residues), but typically is not germlined at one or more trim residues.

The antibody molecule or VH domain of the invention may comprise the following group of heavy chain framework regions:

FR1SEQIDNO:167；

FR2SEQIDNO:168；

FR3SEQIDNO:169；

FR4SEQIDNO:170；

alternatively, the set of heavy chain framework regions comprises 1,2, 3,4 or 5 amino acid changes, e.g. substitutions.

The antibody molecule or VL domain of the invention may comprise the following set of light chain framework regions:

FR1SEQIDNO:171；

FR2SEQIDNO:172；

FR3SEQIDNO:173；

FR4SEQIDNO:174；

alternatively, the set of light chain framework regions comprised may contain 1,2, 3,4 or 5 amino acid changes, e.g. substitutions.

The amino acid change may be a substitution, insertion or deletion.

For example, an antibody molecule of the invention may comprise a set of heavy and light chain framework regions, wherein:

heavy chain FR1 is seq id no: 167;

heavy chain FR2 is seq id no: 168;

heavy chain FR3 is seq id no: 169;

heavy chain FR4 is seq id no: 170;

light chain FR1 is seq id no: 171;

light chain FR2 is seq id no: 172;

light chain FR3 is seq id no: 173;

light chain FR4 is seq id no:174, and (b) a;

alternatively, the set of heavy and light chain framework regions comprises 10 or fewer, e.g., 5 or fewer, amino acid changes, e.g., substitutions. For example, there may be 1 or 2 amino acid substitutions in the set of heavy and light chain framework regions.

Compared to the germlined antibody molecule, the non-germlined antibody molecule has the same CDRs, but the framework is different. Among the antibody sequences of the sequence listing appended hereto, the sequences of antibodies No. 7, 10, 17 and 18 are germlined. Germlined antibodies 2-5, 8, 14, 16, 19 and 21-23 can be generated by germlining the framework regions of the VH and VL domain sequences shown herein in these antibodies.

The expressed scFv and IgG sequences of these antibodies comprise the 3' cgt codon and the corresponding arginine residue shown in the nucleotide and amino acid sequences of the kappa VL domains of antibodies 2, 3,4, 5,7, 8, 10, 14, 16, 17, 18,19, 21,22, and 23. The C-terminal arginine residue of the sequence corresponds to Kabat residue 108. The origin of this residue and its coding triplet cgt are explained below.

For expression of the light chain of IgG, a nucleotide sequence encoding the light chain of the antibody is provided comprising a first exon encoding the VL domain, a second exon encoding the CL domain, an intron separating the first exon from the second exon. Under normal circumstances, cellular mRNA processing machinery splices out introns, linking the 3 'end of a first exon to the 5' end of a second exon. Thus, when the DNA having the nucleotide sequence is expressed as RNA, the first and second exons are spliced together. Translation of the spliced RNA can produce a polypeptide comprising a VL domain and a CL region.

The choice of constant region is important because for the kappa light chain, the bridging amino acid is arginine formed by the cga codon, with the first cytosine encoded by exon 1 and guanine and adenine encoded by exon 2.

After splicing, for antibodies 2, 3,4, 5,7, 8, 10, 14, 16, 17, 18,19, 21,22 and 23, the Arg at Kabat residue 108 is encoded by the last base (c) of the VL domain framework 4 sequence and the first two bases (gt) of the CL region.

The arginine residue at Kabat residue 108 may be considered the C-terminal residue of the VL domain of the antibody molecule.

A binding member of the invention may be a binding member that competes for binding to IL-6 with any binding member that (i) binds IL-6 and (ii) comprises a binding member, VH and/or VL domain, CDR, e.g. HCDR3 and/or set of CDRs as disclosed herein.

Competition between binding members can be readily tested in vitro, for example by ELISA and/or by labelling one binding member with a specific reporter molecule (which can be detected in the presence of one or more other unlabelled binding members), to enable identification of binding members that bind the same epitope or overlapping epitopes. Such methods will be readily apparent to those of ordinary skill in the art, and are described in more detail herein (see the materials and methods section of the detailed description and examples for epitope competition experiments). Thus, in a further aspect, the invention provides a binding member comprising an antigen-binding site of a human antibody which competes for binding to IL-6 with an antibody molecule, e.g. an antibody molecule comprising a VH and/or VL domain, a CDR, e.g. HCDR3 or a set of CDRs of a parent antibody or any of antibodies 2, 3,4, 5,7, 8, 10, 14, 16, 17, 18,19, 21,22 and 23.

In other aspects, the invention provides isolated nucleic acids comprising a nucleic acid encoding a binding member, VH domain and/or VL domain of the invention, and methods of making a binding member, VH domain and/or VL domain of the invention, comprising expressing the nucleic acid under conditions such that the binding member, VH domain and/or VL domain is produced and recovering it.

In another aspect, the invention provides a nucleic acid encoding a VHCDR or VLCDR sequence disclosed herein, typically isolated.

Another aspect provides a host cell comprising or transformed with a nucleic acid of the invention.

Other aspects of the invention provide compositions comprising a binding member of the invention, and their use in methods of binding, inhibiting and/or neutralizing IL-6, including methods of treatment of the human or animal body.

The binding members of the invention may be used in a method of therapy or diagnosis, for example in a method of treatment (which may include prophylactic treatment) of a disease or condition of the human or animal body (e.g. a human patient) which method comprises administering to said patient an effective amount of a binding member of the invention. Disorders that can be treated according to the invention include any disorder in which IL-6 plays a role, as detailed elsewhere herein.

These and other aspects of the invention are described in further detail below.

Term(s) for

It is noted herein that where "and/or" is used herein, it is understood that each of the two recited features or components is specifically disclosed, with or without the other. For example, "a and/or B" should be understood to specifically disclose each of (i) a, (ii) B, and (iii) a and B, as if each were specifically set forth herein.

IL-6 and IL-6 receptors

IL-6 is interleukin 6. IL-6 may also be referred to herein as "antigen".

The full-length amino acid sequence of the human IL-6 is SEQ ID NO: 161. this sequence was cleaved in vivo to remove the N-terminal leader peptide, yielding mature IL-6. Mature human IL-6 has the amino acid sequence of SEQ ID NO: 165. The mature sequence represents circulating IL-6 in vivo, which is the target antigen for therapeutic and in vivo diagnostic applications described herein. Thus, IL-6 herein generally refers to mature human IL-6, unless otherwise indicated.

IL-6 may be conjugated to a detectable tag, such as HISFLAG, e.g., for use in the assays described herein. For example, a fusion protein comprising IL-6 coupled to the hislag sequence may be used. The sequence of the HISFLAG labeled human IL-6 is SEQ ID NO: 162.

IL-6 receptor a, IL-6Ra, is a receptor for interleukin 6. IL-6Ra is also known as IL-6R α, IL-6Ra, IL-6R and CD 126. IL-6Ra exists in vivo in transmembrane and soluble forms. IL-6Ra can be transmembrane IL-6Ra and/or soluble IL-6Ra, unless otherwise specified.

As used herein, the IL-6 receptor generally refers to the human IL-6 receptor, unless otherwise indicated. The amino acid sequence of human soluble IL-6Ra (sIL-6Ra, sIL-6R) is SEQ ID NO: 163. The amino acid sequence of human transmembrane IL-6Ra is SEQ ID NO: 164.

IL-6 binds to IL-6Ra to form the complex IL-6: IL-6 Ra. The complex may be soluble (with sIL-6Ra) or membrane-bound (with transmembrane IL-6 Ra). When IL-6Ra is in soluble form, the complex is referred to as IL-6: sIL-6 Ra. IL-6Ra can include IL-6 that forms a complex with transmembrane IL-6Ra or with soluble IL-6Ra, unless otherwise specified.

gp130

gp130 is the receptor for the IL-6: IL-6Ra complex. Methods for cloning and identifying gp130 are described in Hibi et al, Cell63:1149-1157 (1990). The sequence of human gp130 is shown in SEQ ID NO: 166.

Binding members

The term describes one member of a pair of molecules bound to each other. The members of the binding pair may be naturally occurring or wholly or partially synthetically produced. The members of a molecular pair have regions or cavities on their surface that can bind to, and thus complement, the specific steric and polar structure of the other member of the molecular pair. Examples of types of binding pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. The present invention relates to antigen-antibody type reactions.

Binding members generally include molecules having an antigen binding site. For example, the binding member may be an antibody molecule or a non-antibody protein containing an antigen-binding site.

Can be prepared by reacting a compound in the presence of, for example, fibronectinOr a non-antibody protein scaffold such as cytochrome B, and the like [42,43,44]Or by randomizing or mutating amino acid residues within the inner loop of the protein scaffold to provide an antigen binding site that can confer binding specificity to a desired target. Nygren et al [44]Scaffolds for engineering novel binding sites in proteins are summarized. Protein scaffolds for antibody mimetics are disclosed in WO/0034784, which is incorporated herein by reference in its entirety, wherein the inventors describe proteins comprising a fibronectin type III domain with at least one randomized loop (antibody mimetics). Suitable scaffolds to be grafted into one or more CDRs, e.g., a set of HCDRs, can be provided by any domain member of the immunoglobulin gene superfamily. The scaffold may be a human or non-human protein. One of the advantages of non-antibody protein scaffolds is that they can provide antigen binding sites in scaffold molecules that are at least smaller and/or easier to manipulate than some antibody molecules. The small size of the binding member can confer useful physiological properties, such as the ability to enter cells, penetrate deeper into tissues or react with targets within other structures, or bind to protein cavities of target antigens. Wess, 2004[45]The use of antigen binding sites in non-antibody protein scaffolds is summarized. Proteins typically have a stable backbone and one or more variable loops, wherein the amino acid sequence of the one or more loops is mutated specifically or randomly to create an antigen binding site that binds to a target antigen. These proteins include staphylococcus aureus (s. aureus) a protein, transferrin, tetranectin, fibronectin (e.g., the 10 th fibronectin type III domain), lipocalin, as well as gamma-crystallin and other affilins^TMIgG-binding domain of scaffold (silk ear protein (ScilProteins)). Examples of other methods include synthetic "microbodies" based on cyclic peptides (cyclotides) -small proteins containing intramolecular disulfide bonds, microbial proteins (Versabodies)^TMAmimazex (Amunix)) and ankyrin repeat (DARPins, molecular partners (molecular parttners)).

In addition to antibody sequences and/or antigen binding sites, binding members of the invention may comprise other amino acids, for example to form a peptide or polypeptide, such as a folding domain, or to confer another functional characteristic to the molecule in addition to its ability to bind antigen. The binding members of the invention may carry a detectable label or may be conjugated to a toxin or targeting moiety or enzyme (e.g. via a peptide bond or linker). For example, a binding member can comprise a catalytic site (e.g., in an enzyme domain) and an antigen binding site, wherein the antigen binding site binds to an antigen, thus targeting the catalytic site to the antigen. The catalytic site may, for example, inhibit the biological function of the antigen by cleavage.

Although it is known that non-antibody scaffolds may carry CDRs, the structures of the invention carrying a CDR or set of CDRs will typically be antibody heavy or light chain sequences or substantial portions thereof, wherein the CDR or set of CDRs are located at the corresponding positions of the CDR or set of CDRs of the naturally occurring VH and VL antibody variable regions encoded by the rearranged immunoglobulin genes. The structure and position of the variable regions of immunoglobulins can be determined by reference to Kabat, et al, 1987[46] and updates thereto. The database may be queried by a number of academic and business online resources. For example, the web address http:// www.bioinf.org.uk/abs/simkab. html is currently accessible, see reference [47] and related online resources.

The hypervariable regions of the heavy and light chains of an immunoglobulin are defined by CDR regions or CDRs which are intended to indicate Kabat et al, 1991[48] and subsequent versions. Antibodies typically contain 3 heavy chain CDRs and 3 light chain CDRs. The term CDR is used herein to denote (as the case may be) one or several or even all of these regions, which contain the majority of the amino acid residues responsible for binding by the affinity of the antibody to the antigen or epitope it recognizes.

Of the 6 short CDR sequences, the third CDR of the heavy chain (HCDR3) has greater variability in volume (primarily due to the gene rearrangement mechanism that produces it). It can be as short as 2 amino acids, although the longest volume known is 26. The CDR lengths may also vary according to the length that a particular potential framework region can provide. Functionally, HCDR3 plays a part in determining antibody specificity [ references 49, 50, 51, 52, 53, 54, 55, 56 ].

The HCDR 1can be 5 amino acids in length and consists of Kabat residues 31-35.

The HCDR 2can be 17 amino acids in length and consists of Kabat residues 50-65.

The HCDR 3can be 11 or 12 amino acids in length, consisting of Kabat residues 95-102, optionally including Kabat residue 100D.

LCDR1 may be 11 amino acids in length and consists of Kabat residues 24-34.

LCDR2 may be 7 amino acids long and consists of Kabat residues 50-56.

LCDR3 may be 8 or 9 amino acids in length, consisting of Kabat residues 89-97, optionally including Kabat residue 95.

Antibody molecules

The term describes immunoglobulins, whether produced naturally or partially or wholly synthetically. The term also encompasses any polypeptide or protein comprising an antibody antigen-binding site. It must be understood herein that the invention does not relate to antibodies in their natural form, i.e. they are not in their natural environment, but they can be isolated or obtained from natural sources by purification, or they can be obtained by genetic recombination or chemical synthesis, which may then contain unnatural amino acids, as will be further described below. Antibody fragments containing an antibody antigen-binding site include, but are not limited to: molecules such as Fab, Fab' -SH, scFv, Fv, dAb, Fd, etc. Various other antibody molecules have been engineered that contain one or more antibody antigen-binding sites, including, for example, Fab2, Fab3, diabodies, triabodies, tetrabodies, and minibodies. Antibody molecules and methods for their construction and use are described in [57 ].

It is possible to use monoclonal or other antibodies and use techniques such as recombinant DNA techniques to generate other antibodies or chimeric molecules that bind to the target antigen. These techniques may include the introduction of DNA encoding the immunoglobulin variable region, or CDRs of an antibody, or constant regions plus framework regions of different immunoglobulins. See, for example, EP-A-184187, GB2188638A or EP-A-239400, and ｃA number of subsequent documents. Genetic mutations or other changes may be made to the antibody-producing hybridoma or other cell, which may or may not alter the binding specificity of the produced antibody.

Since antibodies can be modified in a variety of ways, the term "antibody molecule" should be understood to encompass any binding member or substance having the desired specificity, having an antibody antigen-binding site, and/or capable of binding to an antigen. Thus, the term encompasses antibody fragments and derivatives, including any polypeptide comprising an antigen-binding site of an antibody, whether natural or wholly or partially synthetic. Thus included are chimeric molecules comprising an antibody antigen-binding site and fused to another polypeptide (e.g., derived from another species or belonging to another antibody type or subclass), or equivalents thereof. Cloning and expression of chimeric antibodies is described in EP-A-0120694 and EP-A-0125023, as well as in ｃA number of subsequent publications.

Other techniques available in the field of antibody engineering enable the isolation of human and humanized antibodies. For example, human hybridomas can be prepared as described by Kontermann and Dubel [58 ]. For example, the following numerous publications describe in detail the generation of another prior art binding member-phage display: kontermann and Dubel [58] as well as WO92/01047 (discussed further below) and US patents US5969108, US5565332, US5733743, US 58657, US5871907, US5872215, US5885793, US5962255, US6140471, US6172197, US6225447, US 1650, US6492160, US 6521404.

Human antibodies can be isolated using transgenic mice in which the mouse antibody genes are inactivated and functionally replaced with human antibody genes, but other components of the mouse immune system remain intact [59 ]. Humanized antibodies can be generated using techniques known in the art, e.g., as described in WO91/09967, US5,585,089, EP592106, US565,332, and WO 93/17105. In addition, WO2004/006955 describes a method for humanizing antibodies based on: the canonical CDR structure types of the non-human antibody variable region CDR sequences are compared to the canonical CDR structure types of the corresponding CDRs in a human antibody sequence, e.g., a library of germline antibody gene segments, to select variable region framework sequences in human antibody genes. Human antibody variable regions having a canonical CDR structure type similar to the non-human CDRs form a subset of human antibody sequence members from which human framework sequences are selected. Members of this subgroup can also be ordered by amino acid similarity between human and non-human CDR sequences. In the method of WO2004/006955, the top-ranked human sequences are selected to provide framework sequences such that chimeric antibodies are constructed using selected subset member human framework regions to functionally replace human CDR sequences with non-human CDR counterparts, thereby providing humanized antibodies of high affinity and low immunogenicity without the need to compare the framework sequences of non-human and human antibodies. Chimeric antibodies made according to this method are also disclosed.

Genes can be generated by means of synthetic oligonucleotides and assembled in suitable expression vectors for expression to produce synthetic antibody molecules, as described by Knappik et al [60] or Krebs et al [61 ].

Fragments of intact antibodies have been shown to perform the function of binding antigen. Examples of binding fragments are (i) a Fab fragment consisting of the VL, VH, CL and CH1 domains; (ii) an Fd fragment consisting of the VH and CH1 domains; (iii) (ii) an Fv fragment consisting of the VL and VH domains of a single antibody; (iv) dAb fragments consisting of VH or VL domains [62,63,64 ]; (v) an isolated CDR region; (vi) a F (ab')2 fragment which is a bivalent fragment comprising two linked Fab fragments; (vii) a single chain Fv molecule (scFv), wherein the VH domain and the VL domain are connected by a peptide linker, such that the two domains can be joined to form an antigen binding site [65,66 ]; (viii) bispecific single chain Fv dimers (PCT/US92/09965) and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO 94/13804; [67 ]). Fv, scFv or diabody molecules can be stabilized by the inclusion of a disulfide bond linking the VH and VL domains [68 ]. Small antibodies (minibodies) comprising scFv linked to a CH3 domain may also be prepared [69 ]. Other examples of binding fragments are Fab ' which differs from Fab fragments by the addition of a few residues at the carboxy terminus of the CH1 domain of the heavy chain, including one or more cysteines in the hinge region of the antibody, and Fab ' -SH, which is a Fab ' fragment in which the cysteine residues of the constant region carry a free thiol group.

Qui et al [70] describe antibody molecules containing only two CDRs connected by a framework region. The CDR3 of the VH or VL domain is linked to the CDR1 or CDR2 loops of the other domain. The C-terminus of the selected CDR1 or CDR2 is linked to the N-terminus of CDR3 by the FR region. Qui et al selected the FR region with the least hydrophobic region. The best combination of antibodies tested was found to be the VLCDR1 and VHCDR3(VHCDR1-VHFR2-VLCDR3) linked by VHFR 2. These antibody molecules have the advantage of improved tissue penetration compared to intact immunoglobulin molecules (about 150kDa) or scFv (about 28kDa) at a molecular weight of about 3 kDa.

The antibody fragments of the invention may be obtained starting from a parent antibody molecule or any of the antibodies in antibody molecules 2, 3,4, 5,7, 8, 10, 14, 16, 17, 18,19, 21,22 and 23 by cleavage of disulfide bridges, such as by enzymatic digestion, e.g. pepsin or papain digestion, and/or by chemical reduction. In another mode, the antibody fragment of the present invention can be obtained by genetic recombination or the like techniques well known to those skilled in the art, or by peptide synthesis or by nucleic acid synthesis and expression by means of, for example, an automated peptide synthesizer such as those provided by applied biosystems (applied biosystems) or the like.

Functional antibody fragments of the invention include any functional fragment that has increased half-life by chemical modification, particularly by pegylation, or by incorporation into liposomes.

dAbs (domain antibodies) are small monomeric antigen-binding fragments of antibodies, i.e., the variable regions of the heavy or light chains of antibodies [64]. Vhdabs are naturally produced in camelids (e.g. camels, llamas) and can be produced by immunising a camelid with a target antigen, isolating antigen-specific B cells and directly cloning the dAb gene for each B cell. dAbs can also be produced in cell culture. The small size, good solubility and temperature stability make them particularly physiologically useful and thus suitable for selection and affinity maturation. A therapeutic alpaca VHdAb under development is marketed under the name "Nanobody^TM". The binding members of the invention may be substantiallyA VH or VL domain as described herein or a dAb comprising a VH or VL domain of a set of CDRs substantially as described herein.

Bispecific or bifunctional antibodies constitute a second generation monoclonal antibody combining two different variable regions in the same molecule [71]. They have been shown to be useful in the diagnostic and therapeutic fields due to the ability to recruit several molecules that have new effector functions or target the surface of tumor cells. If bispecific antibodies are to be used, these antibodies can be prepared by conventional methods [72 ]]E.g., a conventional bispecific antibody prepared chemically or from a hybridoma, or can be any of the bispecific antibody fragments described above. These antibodies can be prepared by chemical methods [73,74 ]]Or somatic cell methods [75,76]Obtained, but preferably also by genetic engineering techniques to force heterodimerization, thus facilitating the purification process of the sought antibody [77]. Examples of bispecific antibodies include BiTE^TMThose antibodies which are technically obtainable in which the binding domains of two antibodies of different specificity are used and are directly linked by a short flexible peptide. The antibody combines two antibodies on a short single polypeptide chain. Diabodies and scFvs can be constructed without the Fc region using only the variable region, thereby potentially reducing the effect of anti-idiotypic reactions.

Bispecific antibodies can be constructed as intact IgG, bispecific Fab '2, Fab' PEG, diabodies, or bispecific scFv. In addition, two bispecific antibodies can be linked to form a tetravalent antibody using conventional methods known in the art.

Bispecific diabodies may also be particularly useful, as opposed to intact bispecific antibodies, because they are not difficult to construct and express in e. Selection of diabodies (and many other polypeptides, such as antibody fragments) with appropriate binding specificity from the library is readily accomplished using phage display (WO 94/13804). If one arm of a diabody is to be maintained constant, e.g., specific for IL-6, a library different from the other arm can be prepared and an antibody of appropriate specificity selected. Complete bispecific antibodies can be prepared by alternative engineering methods as described in Ridgeway1996[78 ].

Antibodies to IL-6 can be obtained by various methods in the art. These antibodies may be monoclonal antibodies, in particular of human, murine, chimeric or humanized origin, which may be obtained by standard methods well known to those skilled in the art.

For the preparation of monoclonal Antibodies or their functional fragments, in particular of murine origin, it is generally possible to indicate the manual "Antibodies" [79 ]]Specially adapted for the techniques orAnd Milstein [80 ]]Techniques for preparation from hybridomas.

Monoclonal antibodies can be obtained, for example, from B cells of animals immunized with IL-6 or one of the IL-6 fragments containing the epitope recognized by the monoclonal antibody. Suitable fragments and peptides or polypeptides comprising them are described herein, which can be used to immunize an animal to produce antibodies against IL-6. In particular, one of said IL-6 or fragments thereof can be prepared by peptide synthesis starting from an amino acid sequence contained in the peptide sequence of IL-6 and/or fragments thereof, by genetic recombination starting from a nucleic acid sequence containing a cDNA sequence coding for IL-6 or fragments thereof, according to conventional methods.

Monoclonal antibodies can be purified, for example, using an affinity column to which has been immobilized IL-6 or one of its fragments containing the epitope recognized by the monoclonal antibody. More specifically, monoclonal antibodies can be purified by protein A and/or protein G chromatography, with or without ion exchange chromatography to remove residual protein contaminants in themselves, as well as DNA and LPS, and with or without Sepharose exclusion chromatography to remove potential aggregates due to the presence of dimers or other multimers. In one embodiment, all of these techniques may be employed simultaneously or in succession.

Antigen binding sites

The term describes the portion of the molecule that binds to and is complementary to all or part of the target antigen. In an antibody molecule, the term refers to an antibody antigen-binding site, including the portion of an antibody that binds to and is complementary to all or part of a target antigen. If the antigen is large, the antibody may bind only a specific portion of the antigen, which portion is referred to as an epitope. The antibody antigen-binding site may be provided by one or more antibody variable regions. The antibody antigen-binding site may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

WO2006/072620 describes engineering an antigen binding site in a structural (non-CDR) loop extending between immunoglobulin domain β chains. The antigen binding site may be engineered in regions of the antibody molecule separate from the native positions of the CDRs, such as the VH or VL domain framework regions, or in antibody constant regions, such as CH1 and/or CH 3. The engineered antigen binding sites in the domains may be additional binding sites to or in place of those formed by the sets of CDRs of the VH and VL domains. When multiple antigen binding sites are present in an antibody molecule, they can bind to the same antigen (IL-6), thereby increasing the valency of the binding member. Alternatively, multiple antigen binding sites can bind different antigens (IL-6 and one or more other antigens), which can be used to add effector function, extend half-life, or improve in vivo delivery of antibody molecules.

Separated from each other

The term indicates that the binding members of the invention, or nucleic acids encoding such binding members, are generally in accordance with the invention. Thus, the binding members, VH and/or VL domains and encoding nucleic acid molecules and vectors of the invention provided may be, for example, isolated and/or purified from their natural environment, in substantially pure or homogeneous form, or in the case of nucleic acids, free or substantially free of nucleic acids or genes from sources other than the sequence encoding the polypeptide having the desired function. Isolated members and isolated nucleic acids are free or substantially free of materials with which they are naturally associated, such as other polypeptides or nucleic acids found in their natural environment or in the environment in which they are prepared (e.g., cell culture fluid) when recombinant DNA techniques are practiced in vitro or in vivo. The members and nucleic acids may be formulated with diluents or adjuvants, but are still separate for practical purposes-for example, if used to coat microtiter plates for immunoassays, the members will typically be mixed with gelatin or other carriers, or, when used in diagnosis or therapy, with pharmaceutically acceptable carriers or diluents. The binding members may be glycosylated, either naturally or by the system of heterologous eukaryotic cells (e.g., CHO or NS0(ECACC85110503)), or they may be non-glycosylated (e.g., if expressed in prokaryotic cells).

Heterogeneous preparations comprising anti-IL-6 antibody molecules also form part of the invention. For example, these preparations may be a mixture of antibodies having full-length heavy chains, heavy chains lacking C-terminal lysine, varying degrees of glycosylation, and/or having derivatized amino acids, such as N-terminal glutamic acid cyclized to form pyrrolyl-glutamic acid residues.

The phrase "substantially said" as used herein means that the relevant CDR characteristics of the VH or VL domain of the binding member described herein are the same or highly similar to particular regions of the sequences described herein. As used herein, the phrase "highly similar" with respect to a given region of one or more variable regions includes that 1 to about 5, e.g., 1 to 4, including 1 to3, or 1,2, 3, or 4 amino acid substitutions may be made in the CDR and/or VH or VL domains.

Brief description of the drawings

FIG. 1 shows the effect of administration of anti-IL-6 antibody (antibody 18) on the increase of human recombinant IL-6-induced haptoglobin in mice.

Detailed Description

As mentioned above, the binding members of the invention are capable of modulating and possibly neutralizing the biological activity of IL-6. The neutralizing potency of the IL-6 binding members of the invention may be optimized as described herein. Potency optimisation will generally involve mutating the sequence of the selected binding member (typically the variable region sequence of an antibody) to generate a library of binding members, then testing for potency and selecting for a more potent binding member. Thus, the selected "potency-optimised" binding members may be more potent than the binding members from which the library was generated. However, high potency binding members may also be obtained without optimisation, e.g. they may be obtained directly from an initial screening, e.g. biochemical neutralisation assay. A "potency-optimized" binding member refers to a binding member that has been optimized for its potency in neutralizing a particular activity or downstream function of IL-6. The tests and efficacy are described in more detail elsewhere herein. The invention provides potency-optimised and non-optimised binding members, and methods for potency optimisation of selected binding members. Thus, the invention enables the skilled person to generate highly efficient binding members.

In another aspect, the invention provides a method of obtaining one or more binding members capable of binding an antigen, the method comprising contacting a library of binding members of the invention with said antigen, and selecting the one or more binding members in the library that are capable of binding to said antigen.

The library may be displayed on particles or molecular complexes, e.g. replicable genetic packages, such as yeast, bacterial or bacteriophage (e.g. T7) particles, viruses, cells or covalent, ribosomal or other in vitro display systems, each particle or molecular complex containing nucleic acid encoding an antibody VH variable region and optionally a displayed VL domain, if present, displayed thereon. The following documents describe phage display: WO92/01047 and, for example, US patents US5969108, US5565332, US5733743, US5858657, US5871907, US5872215, US5885793, US5962255, US6140471, US6172197, US6225447, US 1650, US6492160 and US6521404, each of which is incorporated herein by reference in its entirety.

After selecting a binding member capable of binding to an antigen and displayed on a bacteriophage or other library particle or molecular complex, nucleic acid may be obtained from the bacteriophage or other particle or molecular complex displaying the selected binding member. Such nucleic acids may then be used to generate binding members or antibody VH or VL variable regions by expression of nucleic acid sequences comprising nucleic acids obtained from bacteriophage or other particles or molecular complexes displaying said selected binding members.

The antibody VH variable region comprising the amino acid sequence of the antibody VH variable region of the selected binding member may be provided in isolated form, for example a binding member comprising such a VH domain.

The ability to bind IL-6, and to compete with, for example, the parent or antibody molecule 2, 3,4, 5,7, 8, 10, 14, 16, 17, 18,19, 21,22 or 23 (e.g., scFv format and/or IgG format, e.g., IgG1) for binding to IL-6, can be further tested. The ability to neutralize IL-6 can be tested as described elsewhere herein.

The affinity of a binding member of the invention for binding to IL-6 may be that of a parent or other antibody molecule, such as an scFv, or one of antibodies 2, 3,4, 5,7, 8, 10, 14, 16, 17, 18,19, 21,22 and 23, such as IgG1, or better.

The potency of a binding member of the invention to neutralise the biological activity of IL-6 may be that of a parent or other antibody molecule, or one of antibodies 2, 3,4, 5,7, 8, 10, 14, 16, 17, 18,19, 21,22 and 23, such as scFv or IgG1, or better.

The binding affinity and neutralizing potency of different binding members can be compared under suitable conditions.

Variants of the VH and VL domains and CDRs of the invention, including those amino acid sequences set out herein and useful in binding members of the invention, may be obtained by sequence alteration or mutation, as well as methods of screening for antigen binding members having desired properties. Examples of desirable characteristics include, but are not limited to:

increased affinity for antigen binding relative to known antigen-specific antibodies

If an antigenic activity is known, the level of neutralization of that activity is increased relative to known antigen-specific antibodies.

Specific competition ability with known antibodies or ligands to the antigen at a specific molar ratio

Ability to immunoprecipitate complexes

The ability to bind to a particular epitope

Linear epitopes, e.g. peptide sequences identified using peptide binding scans as described herein, e.g. peptides using linear and/or defined conformations for screening

Conformational epitopes formed by non-contiguous residues

The ability to modulate the novel biological activity of IL-6, or a downstream molecule.

Such methods are also provided herein.

Variants of the antibody molecules disclosed herein may be prepared and used in the present invention. Multivariate data analysis techniques are applied to the structure/property-activity relationships [81] as directed by computational chemistry, and quantitative activity-property relationships of antibodies can be obtained using well-known mathematical techniques such as statistical regression, pattern recognition and classification [82,83,84,85,86,87 ]. The properties of an antibody can be obtained from empirical and theoretical models of antibody sequence, function and three-dimensional structure (e.g., analysis of potentially accessible residues or calculated physicochemical properties), and these properties can be considered individually or in combination.

The antigen-binding site of an antibody, consisting of a VH domain and a VL domain, is typically formed by 6 polypeptide loops: 3 from the light chain variable region (VL) and 3 from the heavy chain variable region (VH). Analysis of antibodies with known atomic structures elucidates the relationship between sequence and three-dimensional structure of antibody binding sites [88,89 ]. These relationships suggest that the binding site loop has a small number of backbone conformations, one of the classical structures, in addition to the third region (loop) in the VH domain. The formation of classical structures in specific loops is shown to be determined by their size and the presence of certain residues in key sites in the loop and framework regions [88,89 ].

Such sequence-structure relationship studies can be used to predict those residues of an antibody of known sequence but unknown three-dimensional structure that are critical for maintaining the three-dimensional structure of its CDR loops and thus binding specificity. These predictions can be supported by comparing them to the results of the lead optimization experiment. In the structural approach, a model [90] of the antibody molecule can be generated using any freely available or commercially available software package, such as WAM [91 ]. Possible substitutions at each position in the CDR can then be assessed using a protein visualization and analysis software package, such as InsightII or DeepView [92] from Acxelle Miller (Acceleys, Inc.). This information can then be used to make substitutions that may have minimal or beneficial effects on activity.

The techniques required to make substitutions within the sequences of the CDRs, antibody VH or VL domain and binding member are generally known in the art. Variant sequences may be prepared containing substitutions that may or may not be predicted to have minimal or beneficial effect on activity, tested for their ability to bind and/or neutralize IL-6, and/or any other desired property.

The present invention may utilize variable region amino acid sequence variants of any VH and VL domains, sequences as specifically disclosed herein, as described herein.

Variants of the VL domains of the invention and binding members or antibody molecules comprising them include VL domains in which no arginine is present at Kabat residue 108, e.g., Kabat residue 108 is a different residue or is deleted. For example, an antibody molecule, e.g., an antibody molecule lacking a constant region such as an scFv can comprise a VL domain having a VL domain sequence as described herein or a variant thereof, wherein Kabat residue 108 is an arginine, an amino acid residue other than an arginine, or a deletion.

Another aspect of the invention is an antibody molecule comprising a VH domain having an amino acid sequence at least 60, 70, 80, 85, 90, 95, 98 or 99% identical to the VH domain of any of antibodies 2, 3,4, 5,7, 8, 10, 14, 16, 17, 18,19, 21,22 and 23 of the accompanying sequence listing and/or a VL domain having an amino acid sequence at least 60, 70, 80, 85, 90, 95, 98 or 99% identical to the VL domain of any of antibodies 2, 3,4, 5,7, 8, 10, 14, 16, 17, 18,19, 21,22 and 23 of the accompanying sequence listing. Algorithms that can be used to calculate percent identity between two amino acid sequences include, for example, BLAST [93], FASTA [94], or the Smith-Watermann algorithm (Smith-Watermanagorithm) [95], e.g., using default parameters.

A particular variant may comprise one or more amino acid sequence alterations (additions, deletions, substitutions and/or insertions of amino acid residues).

Changes may be made in one or more framework regions and/or one or more CDRs. The alteration does not generally result in loss of function, and thus a binding member comprising an amino acid sequence so altered may retain the ability to bind and/or neutralize IL-6. Which may retain the same quantitative binding and/or neutralising ability as an unaltered binding member, e.g. as measured in the assay described herein. Binding members comprising such altered amino acid sequences may have an increased ability to bind and/or neutralize IL-6.

Alterations may include substitutions of one or more amino acid residues with a non-naturally occurring or non-standard amino acid, modifications of one or more amino acid residues to a non-naturally occurring or non-standard form, or insertions of one or more non-naturally occurring or non-standard amino acids into a sequence. Exemplary numbers and positions of changes in the sequences of the invention are described elsewhere herein. Naturally occurring amino acids include the 20 "standard" L-amino acids identified by their standard one-letter code as G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, C, K, R, H, D, E. Non-standard amino acids include any other residue that may be incorporated into the polypeptide backbone or result from modification of an existing amino acid residue. The non-standard amino acids may be naturally occurring or non-naturally occurring. Several naturally occurring non-standard amino acids are known in the art, such as 4-hydroxyproline, 5-hydroxyproline, 3-methylhistidine, N-acetylserine, and the like [96 ]. Those amino acid residues derived at their N-alpha positions are located only at the N-terminus of the amino acid sequence. In the present invention, the amino acid is usually an L-amino acid, but may be a D-amino acid. Thus, the alteration includes modification of the L-amino acid to a D-amino acid, or substitution of the L-amino acid with a D-amino acid. Methylated, acetylated and/or phosphorylated forms of amino acids are also known, and in the present invention, amino acids may be subjected to such modifications.

The amino acid sequences of the antibody domains and binding members of the invention may comprise the non-natural or non-standard amino acids described above. Non-standard amino acids (e.g., D-amino acids) can be incorporated into the amino acid sequence during synthesis, or "original" standard amino acids can be modified or substituted after the amino acid sequence is synthesized.

The use of non-standard and/or non-naturally occurring amino acids can increase the structural and functional differences and thus increase the likelihood that a binding member of the invention will achieve the desired IL-6 binding and neutralizing properties. Furthermore, D-amino acids and analogues have shown different pharmacokinetic profiles compared to standard L-amino acids, since polypeptides with L-amino acids degrade in vivo after administration to animals, e.g.humans, which means that D-amino acids are advantageous in some in vivo applications.

Random mutagenesis of one or more selected VH and/or VL genes may be employed to generate mutations throughout the variable region, thereby generating novel VH or VL domains of the invention bearing CDR-derived sequences. Gram et al [97] describe this technique, which uses error-prone PCR. In some embodiments, one or two amino acid substitutions are made throughout the variable region or set of CDRs.

Another method that may be employed is site-directed mutagenesis of the CDR regions of the VH or VL genes. Barbas et al [98] and Schier et al [99] disclose these techniques.

All of the above techniques are known in the art and can be employed by the skilled person to provide binding members of the invention using methods conventional in the art.

In another aspect, the invention provides a method of obtaining an antibody antigen-binding site for IL-6, the method comprising providing a VH domain, which is an amino acid sequence variant of a VH domain as set out herein, by adding, deleting, substituting or inserting one or more amino acids into the amino acid sequence of the VH domain as set out herein, optionally combining the VH domain so provided with one or more VL domains, and testing the VH domain or one or more VH/VL combinations to identify a binding member for IL-6 or an antibody antigen-binding site, and optionally one or more desired properties, such as the ability to neutralise IL-6 activity. The amino acid sequence of the VL domain is substantially as shown herein. Similar methods may be employed wherein one or more sequence variants of a VL domain disclosed herein are combined with one or more VH domains.

As described above, the CDR amino acid sequences carried substantially as shown herein may be a CDR in a human antibody variable region or a substantial portion thereof. The HCDR3 sequences substantially as shown herein represent embodiments of the invention, and each of these sequences carried may be HCDR3 in the human heavy chain variable region or a substantial portion thereof.

The variable regions used in the present invention may be obtained or derived from any germline or rearranged human variable region, or may be synthetic variable regions based on consensus or actual sequences of known human variable regions. The variable regions may be derived from non-human antibodies. The CDR sequences of the invention (e.g., CDR3) can be introduced into a CDR-deficient (e.g., CDR3) variable region library (repotoreie) using recombinant DNA techniques. For example, Marks et al [100]]Methods of generating antibody variable region repertoires are described in which a consensus primer at or adjacent to the 5' end of the variable region is linked to a consensus primer of the third framework region of a human VH gene to provide a repertoire of VH variable regions lacking CDR 3. Marks et al also describe how to combine the library with the CDR3 of a particular antibody. Using similar techniques, CDR 3-derived sequences of the invention may be shuffled with a repertoire of VH or VL domains lacking CDR3, the shuffled complete VH and VL domains being combined with a homologous VL or VH domain to provide binding members of the invention. The library may then be displayed in a suitable host system, such as WO92/01047 or a number of subsequent documents including Kay, Winter and McCafferty [101 ], incorporated herein by reference in their entirety, so that suitable binding members may be selected]The phage display system. One library may be composed of 10⁴Of the above individual members, e.g. at least 10⁵At least 10⁶At least 10⁷At least 10⁸At least10⁹Or at least 10¹⁰One or more members. Other suitable host systems include, but are not limited to: yeast display, bacterial display, T4 display, viral display, cell display, ribosome display and covalent display.

There is provided a method of making a binding member for an IL-6 antigen, the method comprising:

(a) providing a starting repertoire of nucleic acids encoding a VH domain, which nucleic acids comprise CDR3 to be replaced or lack a CDR encoding region;

(b) combining the nucleic acid library with a donor nucleic acid encoding an amino acid sequence of VHCDR3 substantially as set out herein, thereby inserting the donor nucleic acid into the CDR3 region of the nucleic acid library, thereby providing a nucleic acid product library encoding a VH domain;

(c) expressing the nucleic acids of the product repertoire;

(d) selecting a binding member for IL-6; and

(e) recovering the binding member or nucleic acid encoding it.

An analogous method can also be used, wherein the VLCDR3 of the invention is combined with a library of nucleic acids encoding VL domains comprising the CDR3 to be replaced or lacking the CDR3 encoding region.

Similarly, one or more or all 3 CDRs may be grafted into a VH or VL domain repertoire, and the repertoire screened for one or more binding members for IL-6.

For example, one or more of the HCDR1, HCDR2 and HCDR3 of parent or antibody 2, 3,4, 5,7, 8, 10, 14, 16, 17, 18,19, 21,22 or 23, or the HCDR panel of parent or antibody 2, 3,4, 5,7, 8, 10, 14, 16, 17, 18,19, 21,22 or 23, and/or one or more of the LCDR1, LCDR2 and LCDR3 of parent or antibody 2, 3,4, 5,7, 8, 10, 14, 16, 17, 18,19, 21,22 or 23, or the LCDR panel of parent or antibody 2, 3,4, 5,7, 8, 10, 14, 16, 17, 18,19, 21,22 or 23 may be employed.

Similarly, other VH and VL domains, sets of CDRs and sets of HCDRs and/or sets of LCDRs disclosed herein may be utilized.

A substantial portion of the immunoglobulin variable region may comprise at least the three CDR regions, as well as their intervening framework regions. The portion may also comprise at least about 50% of the first or fourth framework regions, or both, said 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. Other residues at the N-terminus or C-terminus of a substantial portion of the variable region may be those not normally associated with a naturally occurring variable region. For example, construction of a binding member of the invention by recombinant DNA techniques may result in the introduction of N-or C-terminal residues encoded by linkers introduced to facilitate cloning or other manipulation steps. Other manipulation steps include the introduction of linkers to link the variable regions of the invention with other protein sequences comprising antibody constant regions, other variable regions (e.g., in the production of diabodies), or detectable/functional markers, as detailed elsewhere herein.

Although in some aspects of the invention the binding member comprises a pair of VH and VL domains, a single binding domain based on VH or VL domain sequences constitutes a further aspect of the invention. Single immunoglobulin domains, particularly VH domains, are known to bind target antigens in a specific manner. See, for example, the discussion of dAbs above.

In the case of each single binding domain, these domains can be used to screen for complementary domains that form a two-dimensional binding member capable of binding to IL-6. The so-called hierarchical double recombination scheme disclosed in WO92/01047, incorporated by reference in its entirety, can be employed by phage display screening methods in which each colony containing an H or L chain clone is used to infect a complete library of clones encoding the other chain (L or H), and the resulting double-stranded binding members are selected according to phage display techniques, such as those described in that reference. This technique is also described in Marks et al, supra [100 ].

The binding members of the invention may also comprise antibody constant regions or portions thereof, for example human antibody constant regions or portions thereof. For example, the VL domain can be linked at its C-terminus to an antibody light chain constant region comprising a human C.kappa.or C.lambda.chain. Similarly, a VH domain based binding member may be linked at its C-terminus to all or part of an immunoglobulin heavy chain (e.g. a CH1 domain) derived from any antibody isotype, for example IgG, IgA, IgE and IgM, and any isotype subclass, particularly IgG1 and IgG 4. IgG1 is preferred for its effector function and ease of handling. Any synthetic or other constant region variant that possesses these properties and stabilizes the variable region may also be used in the present invention.

Binding members of the invention may be labeled with a detectable or functional label. Thus, the binding member or antibody molecule may be present in the form of an immunoconjugate in order to obtain a detectable and/or quantifiable signal. The immunoconjugate may comprise an antibody molecule of the invention conjugated to a detectable or functional label. The label may be any molecule that is capable of producing or is induced to produce a signal, including but not limited to: fluorescers, radiolabels, enzymes, chemiluminescent agents or photosensitizers. Thus, binding can be detected and/or measured by detecting fluorescence or luminescence, radioactivity, enzymatic activity, or absorbance.

Suitable labels include, for example, but are not limited to:

enzymes, such as alkaline phosphatase, glucose-6-phosphate dehydrogenase ("G6PDH"), α -D-galactosidase, glucose magnesium oxide, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase and peroxidases, such as horseradish peroxidase;

-a dye;

fluorescent labels or fluorescers, such as fluorescein and its derivatives, fluorochromes, rhodamine compounds and derivatives, GFP (GFP means "green fluorescent protein"), dansyl, umbelliferone, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluoramine; fluorophores such as lanthanide cryptates and chelates, such as europium, etc. (PerkinElmer and Western biologies International),

chemiluminescent labels or chemiluminescent substances, such as isoaminobenzenedihydrazide, aminobenzenedihydrazide and dioxetane (dioxetane);

bioluminescent markers such as luciferase and luciferin;

-a photosensitizer;

-a coenzyme;

-an enzyme substrate;

-radioactive labels including but not limited to: bromine 77, carbon 14, cobalt 57, fluorine 8, gallium 67, gallium 68, hydrogen 3 (tritium), indium 111, indium 113m, iodine 123m, iodine 125, iodine 126, iodine 131, iodine 133, mercury 107, mercury 203, phosphorus 32, rhenium 99m, rhenium 101, rhenium 105, ruthenium 95, ruthenium 97, ruthenium 103, ruthenium 105, scandium 47, selenium 75, sulfur 35, technetium 99m, tellurium 121m, tellurium 122m, tellurium 125m, thulium 165, thulium 167, thulium 168, yttrium 199, and other radioactive labels described herein;

-particles, such as latex or carbon particles, which may be further labeled with dyes, catalysts or other detectable groups; gold sol; microcrystals; a liposome; cells, etc.;

-molecules such as biotin, digoxin or 5-bromodeoxyuridine;

-a toxin moiety, for example a toxin moiety selected from the group consisting of: pseudomonas exotoxin (PE or a cytotoxic fragment or mutant thereof), diphtheria toxin or a cytotoxic fragment or mutant thereof, botulinum toxin A, B, C, D, E or F, ricin or a cytotoxic fragment thereof such as ricin a, abrin or a cytotoxic fragment thereof, saporin or a cytotoxic fragment thereof, pokeweed antiviral toxin or a cytotoxic fragment thereof, and bryodin 1 or a cytotoxic fragment thereof.

Suitable enzymes and coenzymes are disclosed in Litman et al, U.S. Pat. No. 4,275,149, and Boguslazi et al, U.S. Pat. No. 4,318,980, each of which is incorporated herein by reference in its entirety. Suitable fluorescers and chemiluminescent agents are disclosed in Litman et al, U.S. patent No. 4,275,149, which is incorporated herein by reference in its entirety. Labels also include chemical moieties such as biotin which can be detected by binding to a specific relevant detectable moiety, such as labeled avidin or streptavidin. The detectable label may be attached to the antibody of the invention using conventional chemical methods known in the art.

Immunoconjugates or functional fragments thereof can be prepared by methods known to those skilled in the art. They may be conjugated to the enzyme or fluorescent label directly or mediated by a spacer or linking group, for example a polyaldehyde, such as glutaraldehyde, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DPTA), or in the presence of a coupling agent, for example those described above for the therapeutic conjugates. Conjugates comprising a fluorescein-based marker can be prepared by reaction with an isothiocyanate.

The person skilled in the art knows the existing methods for coupling therapeutic radioisotopes to antibodies, either directly or via chelating agents, such as the above-mentioned EDTA, DATA, which may make use of the radioelements used for diagnosis. It is also possible to label with Na125 by the chloramine T method [102], or with technetium 99m by the technique of Crockford et al (U.S. Pat. No. 4,424,200, incorporated herein by reference in its entirety) or by ligation via DPTA as described by Hnatowich (U.S. Pat. No. 4,479,930, incorporated herein by reference in its entirety).

The label can be caused to generate a signal by a number of means which can be detected by external means, for example by visual inspection, electromagnetic radiation, heat and chemical reagents. The label may also be bound to another binding member that binds to an antibody of the invention, or to a support.

The label can generate the signal directly, and therefore, no other components are required to generate the signal. Many organic molecules, such as fluorescers, absorb ultraviolet and visible light, and the absorbed light transfers energy to these molecules and raises them to an excited level. The absorbed energy is then dissipated by emitting light at a second wavelength. The radiation of the second wavelength is also capable of transferring energy to the labelled acceptor molecule, the resulting energy being dissipated from the acceptor molecule by emission of light, for example Fluorescence Resonance Energy Transfer (FRET). Other labels that directly generate a signal include radioisotopes and dyes.

Alternatively, the label may require additional components to generate a signal, and the signal producing system will include all components required to produce a detectable signal, including substrates, coenzymes, enhancers, other enzymes, substances that react with the enzyme product, catalysts, activators, cofactors, inhibitors, scavengers, metal ions, and specific binding substances required to bind the signal producing substance. Details regarding suitable signal generation systems are found in Ullman et al, U.S. Pat. No. 5,185,243, which is incorporated herein by reference in its entirety.

The methods provided herein comprise binding a binding member provided herein to IL-6. It is noted that such binding may occur in vivo, e.g., following administration of the binding member or nucleic acid encoding the binding member, or in vitro, e.g., ELISA, western blot, immunocytochemistry, immunoprecipitation, affinity chromatography, and biochemical or cellular assays, e.g., TF-1 cell proliferation assays.

The invention also enables the use of binding members of the invention, for example for direct detection of antigen levels in a biosensor system. For example, the invention includes a method of detecting and/or measuring binding to IL-6, comprising (i) contacting the binding member with IL-6 and (ii) detecting binding of the binding member to IL-6, wherein the binding is detected using any of the methods or detectable labels described herein. This method, and any other binding detection methods described herein, can be directly interpreted by the person carrying out the method, e.g., by visual observation of a detectable label. Alternatively, the method or any other combination of detection methods described herein can produce an autoradiogram, a photograph, a computer printout, a flow cytometry report, a picture, a chart, a tube or container or well containing the results, or any other visual or physical representation of the results of the method.

The amount of binding of the binding member to IL-6 can be determined. The quantitative determination may be related to the amount of the antigen of diagnostic interest in the test sample. Screening for IL-6 binding and/or its quantitative determination can be used, for example, to screen patients with the diseases or disorders described herein and/or any other diseases or disorders associated with abnormal IL-6 expression and/or activity.

The diagnostic methods of the invention may comprise (i) obtaining a tissue or fluid sample from a subject; (ii) contacting the tissue or fluid sample with one or more binding members of the invention; and (iii) detecting bound IL-6 in comparison to a control sample, wherein an increase in IL-6 binding compared to the control is indicative of an abnormal level of IL-6 expression or activity. The tissue or liquid sample to be tested comprises blood, serum, urine, biopsy material, tumor or any tissue suspected of containing an abnormal level of IL-6. Positive subjects tested for abnormal IL-6 levels or activity may also benefit from the treatment methods disclosed subsequently herein.

With the aid of the methods disclosed herein, the person skilled in the art can select a suitable way of determining the binding of a binding member to an antigen based on their preferences and general knowledge.

The reactivity of the binding member in the sample may be determined by any suitable means. Radioimmunoassay (RIA) is one possible. The radiolabeled antigen is mixed with unlabeled antigen (test sample) and allowed to bind to the binding member. The bound antigen is physically separated from the unbound antigen and the amount of radioactive antigen bound to the binding member is determined. The more antigen in the test sample, the less radioactive antigen is bound to the binding member. Competitive binding assays with nonradioactive antigens may also be used, using an antigen or analog linked to a reporter. The reporter may be a fluorescent dye, a phosphorous or a laser dye with spectrally separated absorption or emission characteristics. Suitable fluorescent dyes include fluorescein, rhodamine, phycoerythrin, texas red, and lanthanide chelators or cryptates. Suitable chromogenic dyes include diaminobenzidine.

Other reporter molecules include macromolecular colloidal particles or particulate materials such as colored, magnetic or paramagnetic latex beads and biologically or chemically active agents that directly or indirectly produce a detectable signal that can be visually observed, electronically detected or otherwise recorded. For example, these molecules may be enzymes that catalyze reactions to produce or change color or cause changes in electronic properties. They may be excitable molecules and thus electron transfer between energy levels may result in a characteristic absorption or emission spectrum. They may include chemical entities used in conjunction with biosensors. Biotin/avidin or biotin/streptavidin and alkaline phosphatase detection systems may be employed.

The signal generated by each binding member-reporter conjugate can be used to obtain quantifiable absolute or relative data relating to binding of the binding member in the sample (both normal and test).

In one aspect the invention also provides a kit comprising a binding member according to any aspect or embodiment of the invention. In the kit, the binding member may be labelled so as to allow its reactivity in the sample to be determined, for example as described below. The binding member may also be attached to a solid support, with or without. The components of the kit are typically sterile and placed in sealed vials or other containers. The kit may be used in diagnostic assays or other methods that utilize binding members. The kit may contain instructions for using the components in a method, e.g., a method of the invention. The kits of the invention may contain auxiliary substances to assist or carry out such methods. The auxiliary substance includes a second, different binding member that is capable of binding to the first binding member and is coupled to a detectable label (e.g., a fluorescent label, a radioisotope, or an enzyme). The antibody kit may also contain beads for performing immunoprecipitation. The components of the kit are typically contained in their own suitable containers. Thus, these kits typically comprise different containers for each binding member. In addition, these kits may contain instructions for performing the assays and methods for interpreting and analyzing the data obtained from performing the assays.

The invention also provides the use of a binding member as described above in a competition assay to detect the level of an antigen, i.e. a method of detecting the level of an antigen in a sample in a competition assay using a binding member as provided by the invention. This may not require physical separation of bound and unbound antigen. The reporter molecule is attached to the binding member such that a physical or optical change may occur upon binding. The reporter molecule can directly or indirectly generate a quantifiable detectable signal. The reporter molecule may be linked directly or indirectly, covalently, e.g. via a peptide bond or non-covalently. Linkage via a peptide bond may be the result of recombinant expression of a gene fusion encoding an antibody or reporter.

In various aspects and embodiments, the invention extends to a binding member that competes for binding to IL-6 with any binding member defined herein, e.g. a parent antibody or any of antibodies 2, 3,4, 5,7, 8, 10, 14, 16, 17, 18,19, 21,22 and 23, e.g. an antibody in the form of IgG 1. Competition between binding members can be readily tested in vitro, for example by labelling one binding member with a specific reporter molecule (which can be detected in the presence of other unlabelled binding members) to enable identification of binding members that bind the same epitope or overlapping epitopes. For example, competition can be determined by an ELISA in which IL-6 is immobilized on a plate to which a labeled or tagged first binding member is added along with one or more unlabeled or untagged other binding members. The presence of unlabelled binding member capable of competing with the labelled binding member is observed by a decrease in the signal emitted by the labelled binding member.

For example, the invention encompasses methods of identifying IL-6 binding compounds comprising (i) immobilizing IL-6 to a support, (ii) contacting said immobilized IL-6, simultaneously or in a stepwise manner, with at least one labeled binding member of the invention and one or more unlabeled test binding compounds, and (iii) identifying new IL-6 binding compounds by observing a decrease in the amount of binding tag of the labeled binding member. These methods can be performed in a high throughput manner using a multi-well or array format. Such experiments can be performed in solution. See, for example, U.S.5,814,468, which is incorporated herein by reference in its entirety. As mentioned above, the detection of binding may be directly interpreted by the person carrying out the method, for example by visual observation of the detectable label, or its presence may be reduced. Alternatively, a binding member of the invention may produce an autoradiogram, photograph, computer printout, flow cytometry report, picture, chart, test tube or container or well containing the results, or any other visual or physical representation of the results of the method.

Competition assays can also be used for epitope mapping. In one example, epitope mapping can be used to identify epitopes bound by IL-6 binding members, which may optionally have optimized neutralization and/or modulation characteristics. Such epitopes may be linear or conformational epitopes. Conformational epitopes can comprise at least two different fragments of IL-6, wherein the fragments are positioned adjacent to each other when IL-6 is folded into its tertiary or quaternary structure to form a conformational epitope recognized by an IL-6 inhibitor, e.g., an IL-6 binding member. In testing for competition, peptide fragments of the antigen, in particular peptides comprising or essentially consisting of the epitope of interest, may be utilized. Peptides having an epitope sequence with one or more amino acids added at each end can be used. Binding members of the invention may be such that their binding to the antigen is inhibited by a peptide having or comprising a given sequence.

The invention also provides isolated nucleic acids encoding binding members of the invention. The nucleic acid may comprise DNA and/or RNA. In one aspect, the invention provides a nucleic acid encoding a CDR or set of CDRs or a VH domain or a VL domain or an antibody antigen-binding site or an antibody molecule, e.g., scFv or IgG1, of the invention as described above.

The invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes comprising at least one polynucleotide as described above.

The invention also provides recombinant host cells comprising one or more of the above constructs. The nucleic acid encoding the CDR or set of CDRs or VH domain or VL domain or antibody antigen-binding site or antibody molecule, e.g. scFv or IgG1, itself forms an aspect of the invention, as does the method of producing the encoded product, which method comprises expressing the nucleic acid encoding it. Expression can be achieved without difficulty by culturing recombinant host cells containing the nucleic acid under suitable conditions. Following production by expression, the VH or VL domain or binding member may be isolated and/or purified using any suitable technique and then used as appropriate.

The nucleic acids of the invention may comprise DNA or RNA, which may be wholly or partially synthetic. Unless otherwise stated, reference to a nucleotide sequence as set forth herein includes a DNA molecule having the sequence set forth, and also includes an RNA molecule having the sequence set forth, wherein U replaces T.

In yet another aspect, there is provided a method of producing a VH variable region of an antibody, which method comprises expressing encoding nucleic acid. The method may comprise culturing the host cell under conditions to produce the antibody VH variable region.

Similar methods for producing a VL domain and a binding member comprising a VH and/or VL domain are further aspects of the invention.

The preparation method may comprise the step of isolating and/or purifying the product. The method of preparation may comprise formulating the product into a composition comprising at least one other component, such as a pharmaceutically acceptable excipient.

Systems for cloning and expressing polypeptides in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, plant cells, filamentous fungi, yeast and baculovirus systems, and transgenic plants and animals. The expression of antibodies and antibody fragments in prokaryotic cells is well known in the art. For a review see Pl ü ckthun [103 ]. A commonly used bacterial host is e.

As an alternative to the production of binding members, the skilled person may also use cultured eukaryotic cells for expression [104,105,106 ]. Mammalian cell lines useful in the art for the expression of heterologous polypeptides include Chinese Hamster Ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NS0 mouse melanoma cells, YB2/0 rat myeloma cells, human embryonic kidney cells, human embryonic retina cells, and many others.

Suitable vectors containing suitable regulatory sequences may be selected or constructed, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences, if desired. The vector may be a plasmid, such as a phagemid, or a virus, such as a phage (if desired) [107 ]. Ausubel [108] describes in detail a number of known techniques and methods for manipulating nucleic acids, such as preparing nucleic acid constructs, mutagenesis, sequencing, introducing DNA into cells and gene expression, and analyzing proteins.

Another aspect of the invention provides a host cell containing a nucleic acid disclosed herein. Such host cells may be in vitro and may be in culture. Such host cells may be in vivo. The presence of a host cell in vivo may bring about the intramolecular expression of a binding member of the invention as an "intrabody" or intrabody. Intrabodies are useful for gene therapy.

Yet another aspect provides a method comprising introducing a nucleic acid of the invention into a host cell. Such introduction may be carried out using any suitable technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, such as vaccinia virus, or in the case of insect cells, baculovirus. Introduction of nucleic acids into host cells, particularly eukaryotic cells, may utilize viral or plasmid systems. The plasmid system may be maintained episomally or may be incorporated into the host cell or into the artificial chromosome. Incorporation can be random or targeted integration of one or more copies at a single or multiple loci. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation, and transfection using bacteriophage.

The nucleic acid can be expressed after introduction, for example, by culturing the host cell under conditions in which the gene is expressed. The expressed product can be purified by methods known to those skilled in the art.

The nucleic acid of the invention may be integrated into the genome (e.g., chromosome) of the host cell. Integration may be facilitated by the inclusion of sequences that facilitate recombination with the genome, in accordance with standard techniques.

The invention also provides methods comprising using the constructs described above in an expression system to express the binding members or polypeptides described above.

There is evidence that IL-6 is involved in various diseases, as described elsewhere herein. Thus, the binding members of the invention are useful in methods of diagnosing or treating diseases associated with IL-6. Such a disease may be, for example, an inflammatory and/or autoimmune disease, for example, rheumatoid arthritis, osteoarthritis, cachexia, chronic obstructive pulmonary disease, juvenile idiopathic arthritis, asthma, systemic lupus erythematosus, inflammatory bowel disease, crohn's disease, or atherosclerosis. Binding members of the invention may also be used to treat diseases such as tumors and/or cancer.

Binding members of the invention may also be used in methods of diagnosing or treating at least one IL-6-associated disease in a patient, animal, organ, tissue or cell, including but not limited to:

- (respiratory tract) obstructive airway diseases including Chronic Obstructive Pulmonary Disease (COPD); asthma, such as bronchial, allergic, intrinsic, extrinsic and dust asthma, in particular chronic or obsessive-compulsive asthma (e.g. late asthma and airway hyperresponsiveness); bronchitis; acute, allergic, atrophic rhinitis and chronic rhinitis including rhinitis caseosa, hypertrophic rhinitis, rhinitis purulenta, rhinitis sicca and rhinitis medicamentosa; membranous rhinitis, including croupy, fibrinous, and pseudomembranous rhinitis, and adenopathic rhinitis; seasonal rhinitis including rhinitis nervosa (hay fever) and vasomotor rhinitis, sinusitis, Idiopathic Pulmonary Fibrosis (IPF); sarcoidosis, farmer's lung and related diseases, adult respiratory distress syndrome, hypersensitivity pneumonitis, fibrotic lung and idiopathic interstitial pneumonia;

(bone and joint) rheumatoid arthritis, juvenile chronic arthritis, juvenile systemic onset of arthritis, seronegative spondyloarthropathies (including ankylosing spondylitis, psoriatic arthritis and reiter's disease), behcet's disease, sjogren's syndrome and systemic sclerosis, gout, osteoporosis and osteoarthritis;

(skin) psoriasis, atopic dermatitis, contact dermatitis and other eczematous skin diseases, allergic contact dermatitis, seborrheic dermatitis, lichen planus, scleroderma, pemphigus, bullous pemphigoid, epidermolysis bullosa, urticaria, xeroderma (angiodermas), vasculitis, erythema, hypereosinophilia of the skin, uveitis, alopecia areata, allergic conjunctivitis and vernal conjunctivitis (vernalvamlconjunctitis);

(gastrointestinal tract) gastric ulcer, coeliac disease, proctitis, eosinophilic gastroenteritis, mastocytosis, inflammatory bowel disease, crohn's disease, ulcerative colitis, antiphospholipid syndrome)), food-related allergies that produce effects remote from the gut, such as migraine, rhinitis and eczema;

(other tissue and systemic diseases) cachexia, multiple sclerosis, atherosclerosis, acquired immunodeficiency syndrome (AIDS), mesangial proliferative glomerulonephritis, nephrotic syndrome, nephritis, glomerulonephritis, acute renal failure, hemodialysis, uremia, localized or discoid lupus erythematosus, systemic lupus erythematosus, Karman's disease, hashimoto's thyroiditis, myasthenia gravis, type I diabetes, type B insulin resistant diabetes, sickle cell anemia, iridocyclitis/uveitis/optic neuritis, nephritic syndrome, hypereosinophilic fasciitis, hyper IgE syndrome, systemic vasculitis/wegener's granulomatosis, orchitis/vasectomy reversal, leprosy, alcohol-induced hepatitis, sezary syndrome, and idiopathic thrombocytopenic purpura; post-operative adhesions, renal disease, systemic inflammatory response syndrome, sepsis syndrome, gram-positive sepsis, gram-negative sepsis, culture-negative sepsis, fungal sepsis, neutropenic fever, acute pancreatitis, urinary sepsis, Graves 'disease, Raynaud's disease, antibody-mediated cytotoxic types of cells, type III hypersensitivity, POEMS syndrome (polyneuropathy, orgasmic disease, endocrinopathy, monoclonal gammopathy, and dermopathic syndrome), mixed connective tissue disease, idiopathic addison's disease, diabetes, chronic active hepatitis, primary biliary cirrhosis, vitiligo, post (cardiotomy) syndrome, type IV hypersensitivity, intracellular-induced granuloma, Wilson's disease, hemochromatosis, alpha-I-antitrypsin deficiency, diabetic retinopathy, hashimoto thyroiditis, Graves's disease, Graves' disease, Gra, Hypothalamic-pituitary-adrenal axis assessment, thyroiditis, encephalomyelitis, chronic lung disease in neonates, familial lymphophagous lymphohistiocytosis (familial lymphoblastic lymphohistiocytosis), alopecia, radiation therapy (including for example but not limited to: weakness, anemia, cachexia, etc.), chronic salicylic acidosis, sleep apnea, obesity, heart failure, and meningococcemia;

(allograft rejection) acute and chronic rejection after kidney, heart, liver, lung, pancreas, bone marrow, bone, small intestine, skin, cartilage and cornea transplants; and chronic graft versus host disease;

(malignant disease) leukemia, Acute Lymphoblastic Leukemia (ALL), acute leukemia, T-cell, B-cell or FABALL, Chronic Myelogenous Leukemia (CML), Acute Myelogenous Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), lymphoma, hodgkin's disease, non-hodgkin's lymphoma, malignant lymphoma, burkitt's lymphoma, multiple myeloma, kaposi's sarcoma, renal cell carcinoma, colorectal cancer, prostate cancer, pancreatic cancer, nasopharyngeal cancer, malignant histiocytosis, tumor-associated syndrome/malignant hypercalcemia, solid tumors, adenocarcinoma, sarcoma, malignant melanoma, hemangioma, metastatic disease, cancer-related bone resorption, cancer-related bone pain; inhibition of cancer metastasis; improvement in cancer cachexia;

cystic fibrosis, stroke, reperfusion injury of the heart, brain, extremities (peripheralimbs) and other organs;

burns, trauma/hemorrhage, ionizing radiation exposure, chronic skin ulcers;

reproductive disorders (e.g., ovulation, menstruation and implantation disorders, premature labor, preeclampsia, endometriosis);

(infection) acute or chronic bacterial infection, acute and chronic parasitic or infectious processes, including bacterial, viral and fungal infections, HIV infection/HIV neuropathy, meningitis, hepatitis (type A, type B or type C, or other viral hepatitis, etc.), septic arthritis, peritonitis, pneumonia, epiglottitis, E.coli 0157: h7, hemolytic uremic syndrome/thrombotic thrombocytopenic purpura, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy, toxic shock syndrome, streptococcal myositis, gas gangrene, tuberculosis (Mycobacteria), avian-intracellular Mycobacteria, pneumocystis carinii (pneumocystis) pneumonia, pelvic inflammatory disease, orchitis/epididymitis, legionella, Lyme disease, influenza A, Epstein-Barr virus, and viral-associated hemophagocytic syndrome, Viral encephalitis/aseptic meningitis, and the like.

Accordingly, the present invention provides a method of treating an IL-6 related disease, said method comprising administering to a patient in need thereof an effective amount of one or more binding members of the invention, alone or in a combination regimen, in combination with another suitable agent known in the art or described herein.

Elsewhere herein is summarized the evidence of IL-6 involvement in certain diseases. In addition, the data provided herein also illustrate that the binding members of the invention may be used to treat such diseases, including prophylactic treatment and reduction of the severity of such diseases. Accordingly, the present invention provides a method of treating or reducing the severity of at least one symptom of any of the diseases described herein, comprising administering to a patient in need thereof an effective amount of one or more binding members of the invention, alone or in a combination treatment regimen, in combination with another suitable agent known in the art or described herein, thereby reducing the severity of at least one symptom of the disease.

Thus, the binding members of the invention are useful as therapeutic agents for the treatment of diseases or disorders associated with aberrant expression and/or activity, particularly expression/activity, of IL-6 and/or IL-6 Ra. A method of treatment may comprise administering to a patient in need thereof an effective amount of a binding member of the invention, wherein aberrant expression and/or activity of IL-6 and/or IL-6Ra is reduced. A method of treatment may comprise (i) identifying a patient demonstrating abnormal IL-6: IL-6Ra levels or activity, e.g. using a diagnostic method as described above, and (ii) administering to the patient an effective amount of a binding member of the invention, wherein abnormal expression and/or activity of IL-6Ra and/or IL-6 is reduced. According to the present invention, an effective amount is an amount that reduces the aberrant expression and/or activity of IL-6 and/or IL-6Ra, thereby reducing or alleviating at least one symptom of the disease or disorder being treated, but without curing the disease or disorder.

The invention also provides a method of antagonizing at least one effect of IL-6, the method comprising contacting or administering an effective amount of one or more binding members, such that the at least one effect of IL-6 is antagonized. The effects of IL-6 that can be antagonized by the methods of the invention include binding of IL-6 to gp130 and downstream effects resulting from such binding.

Thus, other aspects of the invention provide methods of treatment comprising administering a binding member provided, pharmaceutical compositions comprising such binding members, and the use of such binding members in the manufacture of a medicament for administration, for example a method of manufacture of a medicament or pharmaceutical composition, comprising formulating the binding member with a pharmaceutically acceptable excipient. A pharmaceutically acceptable excipient may be a compound or combination of compounds that can be incorporated into a pharmaceutical composition without causing secondary reactions and that can, for example, facilitate administration of the active compound, increase its in vivo lifespan and/or its efficacy, increase its solubility in solution, or prolong its shelf life. Such pharmaceutically acceptable carriers are well known and can be modified by those skilled in the art to suit the nature and mode of administration of the active compound selected.

The binding members of the invention are typically administered in the form of a pharmaceutical composition which may comprise at least one ingredient other than the binding member. Thus, for use in the present invention, the pharmaceutical compositions of the present invention may contain, in addition to the active ingredient, pharmaceutically acceptable excipients, carriers, buffers, stabilizers or other substances well known to those skilled in the art. These substances should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other substance depends on the route of administration, which may be oral, inhaled, intratracheal, topical, intracapsular or by injection, as described below.

The invention also contemplates pharmaceutical compositions for oral administration, such as single domain antibody molecules (e.g., nanobodies)^TM) And the like. Such oral formulations may be in the form of tablets, capsules, powders, liquids or semi-solids. Tablets may contain solid carriers such as gelatin or adjuvants. Liquid pharmaceutical compositions typically comprise a liquid carrier, such as water, petroleum, animal or vegetable oils, mineral oils, or synthetic oils. Physiological saline solution, dextrose or other sugar solution, or glycerol, such as ethylene glycol, propylene glycol or polyethylene glycol, may be included.

For intravenous injection, or injection at the site of the disease, the active ingredient may be in the form of a parenterally acceptable aqueous solution, which is pyrogen-free and has suitable pH, isotonicity and stability. Those skilled in the art will be able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, ringer's injection, and lactated ringer's injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be utilized if desired, including buffers such as phosphoric acid, citric acid and other organic acids; antioxidants, such as ascorbic acid and methionine; preservatives (for example octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, for example methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3' -pentanol; and m-cresol); a low molecular weight polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, e.g. sucrose, mannitol, seaweedSugar or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants, e.g. tweens^TMPluronic^TMOr polyethylene glycol (PEG).

Binding members of the invention may be formulated in liquid, semi-solid or solid form, depending on the physicochemical properties of the molecule and the route of delivery. The formulation may comprise, for example, the following excipients or combination of excipients: sugars, amino acids, and surfactants. Liquid formulations contain a wide range of antibody concentrations and pH. Solid formulations can be prepared, for example, by freeze drying, spray drying or drying by supercritical liquid techniques. The formulation of the binding member depends on the desired delivery route: for example, formulations for pulmonary delivery may consist of particles whose physical properties ensure penetration into the deep lung after inhalation; topical formulations (e.g., topical formulations for treating scars, such as skin scars) may contain viscosity modifiers which prolong the residence time of the drug at the site of action. The binding member may be prepared with carriers that prevent rapid release of the binding member, such as controlled release formulations, including implants, transdermal patches and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be utilized, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for preparing these formulations are known to those skilled in the art [109 ].

Orally (e.g., single domain antibody molecules (e.g., "nanobodies)^TM"), by injection (e.g., subcutaneously, intraarticularly, intravenously, intraperitoneally, intraarterially, or intramuscularly), by inhalation, intratracheally, intravesicularly (instillation into the bladder), or by topical (e.g., intraocular, intranasal, rectal, administration into a wound, or administration onto the skin). Treatment may be given by pulsed infusion, particularly with a dose-declining binding member. The route of administration may be determined by the physicochemical characteristics of the treatment, by specific considerations of the disease or the requirement to optimise efficacy or to minimise side effects. One particular route of administration is intravenous. Another route of administration of the pharmaceutical compositions of the present invention is subcutaneously. It is estimated that treatment will not be confined to the clinicThe application is as follows. Thus, subcutaneous injection using a needle-less device is preferred.

The compositions may be administered alone or in combination with other therapies, which may be simultaneous or sequential, depending on the condition to be treated.

The binding members of the invention may be used in combination with other pharmaceutical ingredients as part of a combination therapy. Combination therapy may be employed to provide significant synergy, particularly in combination with one or more other drugs. For the treatment of one or more of the diseases described herein, a binding member of the invention may be administered simultaneously or sequentially or as a combined preparation containing another therapeutic agent or agents.

The binding members of the invention may be used as chemosensitisers to enhance the therapeutic effect of the cytotoxic agent and may therefore be administered in combination with one or more cytotoxic agents, either simultaneously or sequentially. The binding member may also be used as a radiosensitizer to enhance the efficacy of radiotherapy and may therefore be administered simultaneously or sequentially with radiotherapy.

The binding members of the invention may be provided in combination or in addition with one or more of the following agents:

-cytokines or agonists or antagonists of cytokine function (e.g. agents acting on cytokine signalling pathways, such as modulators of the SOCS system), such as alpha-, beta-and/or gamma-interferon; insulin-like growth factor type I (IGF-1), its receptor and related binding proteins; an Interleukin (IL), such as one or more of IL-1-33, and/or an interleukin antagonist or inhibitor, such as anakinra; inhibitors of receptors for members of the interleukin family or inhibitors of specific subunits of these receptors; tumor necrosis factor alpha (TNF-alpha) inhibitors, such as anti-TNF monoclonal antibodies (e.g., infliximab; adalimumab and/or CDP-870), and/or TNF receptor antagonists, such as immunoglobulin molecules (e.g., etanercept) and/or low molecular weight agents, such as pentoxifylline (pentoxyfylline);

b cell modulators, such as monoclonal antibodies targeting B-lymphocytes (e.g. CD20 (rituximab) or MRA-aILl6R) or T-lymphocytes (e.g. CTLA4-Ig, HuMaxIl-15 or abercapt);

modulators that inhibit osteoclast activity, such as antibodies to RANKL;

modulators of chemokine or chemokine receptor function, such as antagonists of the following chemokines: CCR1, CCR2, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10 and CCR11 (for the C-C family); CXCR1, CXCR2, CXCR3, CXCR4 and CXCR5 and CXCR6 (for the C-X-C family) and C-X₃-Cfamily CX₃CR1；

-Matrix Metalloproteinases (MMPs), inhibitors of one or more of the following: stromelysin, collagenase and gelatinase and aggrecanase; in particular collagenase-1 (MMP-1), collagenase-2 (MMP-8), collagenase-3 (MMP-13), stromelysin-1 (MMP-3), stromelysin-2 (MMP-10) and/or stromelysin-3 (MMP-11) and/or MMP-9 and/or MMP-12, for example doxycycline;

-inhibitors of leukotriene biosynthesis, 5-lipoxygenase (5-LO) inhibitors or 5-lipoxygenase activating protein (FLAP) antagonists, such as: zileutong; ABT-761; fenton; teposalin; abbott-79175; abbott-85761; n- (5-substituted) -thiophen-2-alkylsulfonamide; 2, 6-di-tert-butylphenol hydrazone; methoxytetrahydropyrans, such as zenecar ZD-2138; compound SB-210661; pyridyl-substituted 2-cyanonaphthalene compounds, such as L-739,010; 2-cyanoquinoline compounds, such as L-746,530; indole and/or quinoline compounds, such as MK-591, MK-886 and/or BAYx 1005;

-receptor antagonists of Leukotriene (LT) B4, LTC4, LTD4 and LTE4 selected from: phenothiazine-3-1 s, such as L-651,392; amidino compounds, such as CGS-25019 c; aminobenzoxazoles (benzaxamines), such as e.g. ondansetron; benzamidines (benzamides), such as BIIL 284/260; and compounds such as zafirlukast, arlukast, montelukast, pranlukast, vilelukast (MK-679), RG-12525, Ro-245913, iralukast (CGP45715A), and BAYx 7195;

phosphodiesterase (PDE) inhibitors, for example methylxanthines (methylxanthines), such as theophylline and/or aminophylline; and/or a selective PDE isozyme inhibitor, e.g. a PDE4 inhibitor and/or an inhibitor of the isoform PDE4D, and/or a PDE5 inhibitor;

histamine receptor antagonists of type 1, such as cetirizine, loratadine, desloratadine, fexofenadine, avastin, terfenadine, astemizole, azelastine, levocabastine, chlorpheniramine, procaine, cyclizine and/or mizolastine (usually applied orally, topically or parenterally);

-proton pump inhibitors (e.g. omeprazole) or gastroprotective histamine type 2 receptor antagonists;

-a histamine type 4 receptor antagonist;

-alpha-1/alpha-2 adrenoceptor agonists, vasoconstrictors, sympathomimetics such as cyprodinyl, phenylephrine, phenylpropanolamine, ephedrine, pseudoephedrine hydrochloride, naphazoline hydrochloride, oxymetazoline hydrochloride, tetrahydrozoline hydrochloride, xylometazoline hydrochloride, tramazoline hydrochloride, and ethylnorepinephrine hydrochloride;

anticholinergics, such as muscarinic receptor (M1, M2 and M3) antagonists, such as atropine, scopolamine, glycopyrrolate (glycopyrrolate), ipratropium bromide, tiotropium bromide, oxitropium bromide, pirenzepine and telenzepine;

-beta-adrenoceptor agonists (including beta receptor subtypes 1-4), such as isoproterenol, salbutamol, formoterol, salmeterol, terbutaline, metaproterenol, albuterol mesylate and/or pirbuterol, for example their chiral enantiomers;

chromones, such as cromolyn sodium and/or nedocromil sodium;

glucocorticoids, such as flunisolide, prednisolone acetonide, beclomethasone dipropionate, budesonide, fluticasone propionate, ciclesonide and/or mometasone furoate;

agents that modulate nuclear hormone receptors, such as PPAR;

immunoglobulin (Ig) or an Ig preparation or an antagonist or antibody that modulates Ig function, such as anti-IgE (e.g., omalizumab (omalizumab));

other anti-inflammatory agents for systemic or topical application, such as thalidomide or its derivatives, retinoids, anthratriphenols and/or calcipotriol;

-a combination of aminosalicylate and sulfapyridine, such as sulfasalazine, mesalazine, balsalazide and olsalazine; immunomodulators, such as mercaptopurine (thioprines) and corticosteroids, such as budesonide;

antibacterial agents, such as penicillin derivatives, tetracycline, macrolides, β -lactams, fluoroquinolones, metronidazole and/or inhaled aminoglycosides; and/or antiviral agents, such as acyclovir, famciclovir, valacyclovir, ganciclovir, cidofovir; tricyclodecylamine, rimantadine; ribavirin; zanamivir and/or oseltamivir; protease inhibitors, such as indinavir, nelfinavir, ritonavir, and/or saquinavir; nucleoside reverse transcriptase inhibitors, such as didanosine, lamivudine, stavudine, zalcitabine, zidovudine; non-nucleoside reverse transcriptase inhibitors, such as nevirapine, efavirenz;

cardiovascular drugs, such as calcium channel blockers, beta-adrenoceptor blockers, Angiotensin Converting Enzyme (ACE) inhibitors, angiotensin-2 receptor antagonists; lipid lowering agents, such as statins and/or fibrates; modulators of blood cell morphology, such as pentoxifylline; thrombolytic and/or anticoagulant agents, such as blood cell aggregation inhibitors;

CNS drugs, such as antidepressants (e.g. sertraline), anti-parkinson drugs (e.g. celecoxib, levodopa, ropinirole, pramipexole, MAOB inhibitors, such as selegiline (selegine) and rasagiline, comP inhibitors, such as tolcapone, a-2 inhibitors, dopamine reuptake inhibitors, NMDA antagonists, nicotinic agonists, dopamine agonists and/or neuronal nitric oxide synthase inhibitors) and anti-alzheimer drugs, such as donepezil, rivastigmine, tacrine, COX-2 inhibitors, propentofylline or metrafonate;

-agents for the treatment of acute and chronic pain, such as centrally or peripherally acting analgesics, e.g. opiate analogues or derivatives, carbamazepine, phenytoin, sodium valproate, amitriptyline (amyltiline) or other antidepressants, acetaminophen or non-steroidal anti-inflammatory drugs;

parenteral or topical-application (including inhaled) local anaesthetics, such as lignocaine or its analogues;

anti-osteoporosis agents, for example hormonal drugs, such as raloxifene or bisphosphonates, such as alendronate;

- (i) trypsin inhibitors; (ii) platelet Activating Factor (PAF) antagonists; (iii) interleukin Converting Enzyme (ICE) inhibitors; (iv) an IMPDH inhibitor; (v) adhesion molecule inhibitors, including VLA-4 antagonists; (vi) (ii) a cathepsin; (vii) kinase inhibitors, such as tyrosine kinase inhibitors (e.g., Btk, Itk, examples of Jak3MAP inhibitors may include Gefitinib (Gefitinib), imatinib mesylate), serine/threonine kinases (e.g., MAP kinases such as p38, JNK, protein kinase A, B, and inhibitors of C and IKK), or kinases involved in cell cycle regulation (e.g., cyclin-dependent kinases); (viii) glucose-6 phosphate dehydrogenase inhibitors; (ix) kinin-B₁-and/or B₂Receptor antagonists, (x) anti-gout agents such as colchicine, (xi) xanthine oxidase inhibitors such as allopurinol, (xii) uricosuric agents such as probenecid, metrazolone and/or benzbromarone, (xiii) growth hormone secretagogues, (xiv) transforming growth factor (TGF β), (xv) platelet-derived growth factor (PDGF), (xvi) fibroblast growth factor, examples of which areSuch as basic fibroblast growth factor (bFGF); (xvii) Granulocyte macrophage colony stimulating factor (GM-CSF); (xviii) Capsaicin cream; (xix) Tachykinin NK₁And/or NK₃Receptor antagonists, such as NKP-608C, SB-233412 (talnetant) and/or D-4418, (xx) elastase inhibitors, such as UT-77 and/or ZD-0892, (xxi) TNF- α convertase inhibitors (TACE), (xxii) Inducible Nitric Oxide Synthase (iNOS) inhibitors or (xxiii) chemoattractant receptor-homologous molecules expressed on TH2 cells, (e.g., CRTH2 antagonists), (xxiv) inhibitors of P38, (xxv) agents that modulate the function of Toll-like receptors (TLRs) and (xxvi) agents that modulate purinergic receptor activity, such as P2X7, (xxvii) inhibitors of transcription factor activation, such as NFkB, API and/or STATS.

The inhibitor may be a specific inhibitor or may be a mixed type of inhibitor, e.g., an inhibitor that targets multiple molecules (e.g., receptors) or types of molecules as described above.

The binding members may also be co-administered or in the form of an immunoconjugate in combination with a chemotherapeutic agent or another tyrosine kinase inhibitor. The antibody non-fragments can also be used in bispecific antibodies obtained by recombinant mechanisms or biochemical couplings, capable of binding the specificity of the above antibodies to the specificity of other antibodies capable of recognizing other molecules involved in IL-6 related activities.

For the treatment of inflammatory diseases, a binding member of the invention may be used in combination with one or more agents, including, for example, non-steroidal anti-inflammatory drugs (hereinafter NSAIDs), including non-selective Cyclooxygenase (COX) -1/COX-2 inhibitors, whether for topical or systemic use (e.g., piroxicam, diclofenac, propionic acids such as naproxen, flurbiprofen, fenoprofen, ketoprofen and ibuprofen, fenamate, such as mefenamic acid, indomethacin, sulindac, apazone, pyrazolones, such as phenylbutazone, salicylates, such as aspirin); selective COX-2 inhibitors (e.g., meloxicam, celecoxib, rofecoxib, valdecoxib, ramicoxib (lumaroxib), parecoxib, and etoricoxib); cyclooxygenase enzymes (CINODs) that inhibit nitric oxide donors; glucocorticoids (whether by topical, oral, intramuscular, intravenous or intra-articular routes); methotrexate, leflunomide; hydroxychloroquine, d-penicillamine, auranofin or other parenteral or oral gold preparations; an analgesic; diacerein; intra-articular treatments, such as hyaluronic acid derivatives; and nutritional supplements such as glucosamine.

The binding members of the invention may also be used in combination with existing therapeutic agents for the treatment of cancer. Suitable agents for use in combination include:

(i) antiproliferative/antineoplastic drugs and combinations thereof for medical oncology, such as gleevec (imatinib mesylate), alkylating agents (e.g., cisplatin, carboplatin, cyclophosphamide, mechlorethamine, melphalan, oncoclonine, busulfan, and nitrosoureas); antimetabolites (e.g., antifolates such as fluoropyrimidines such as 5-fluorouracil and tegafur, raltitrexed, methotrexate, cytarabine, hydroxyurea, gemcitabine and paclitaxel), antitumor antibiotics (e.g., anthracyclines such as doxorubicin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin), anti- (filaggregating) splitting agents (e.g., vinca alkaloids such as vincristine, vinblastine, vindesine and vinorelbine and taxanes such as taxol and taxotere), and topoisomerase inhibitors (e.g., etoposide such as etoposide and teniposide, amsacrine, topotecan and camptothecin);

(ii) cytostatic agents, such as antiestrogens (e.g., tamoxifen, toremifene, raloxifene, droloxifene, and indoxifene (iodoxyfene)), estrogen receptor downregulators (e.g., fulvestrant), antiandrogens (e.g., bicalutamide, flutamide, nilutamide, and cyproterone acetate), LHRH antagonists or LHRH agonists (e.g., goserelin, leuprolide, and buserelin), progestins (e.g., megestrol acetate), aromatase inhibitors (e.g., anastrozole, letrozole, vorozole (vorazole), and exemestane), and inhibitors of 5 α -reductase, such as finasteride;

(iii) agents that inhibit cancer cell invasion (e.g., metalloproteinase inhibitors, such as inhibitors of marimastat and urokinase plasminogen activator receptor function);

(iv) inhibitors of growth factor function, for example, such inhibitors include growth factor antibodies, growth factor receptor antibodies (e.g., anti-erbb 2 antibody, trastuzumab and anti-erbb 1 antibody cetuximab [ C225]), farnesyl transferase inhibitors, tyrosine kinase inhibitors and serine/threonine kinase inhibitors, for example, epidermal growth factor family inhibitors (e.g., EGFR family tyrosine kinase inhibitors such as N- (3-chloro-4-fluorophenyl) -7-methoxy-6- (3-morpholinopropoxy) quinazolin-4-amine (gefitinib, AZD1839), N- (3-ethynylphenyl) -6, 7-bis (2-methoxyethoxy) quinazolin-4-amine (erlotinib);), OSI-774) and 6-acrylamido-N- (3-chloro-4-fluorophenyl) -7- (3-morpholinopropoxy) quinazolin-4-amine (CI1033)), e.g., inhibitors of the platelet-derived growth factor family and e.g., inhibitors of the hepatocyte growth factor family;

(v) anti-angiogenic agents, such as those that inhibit the action of vascular endothelial growth factor, (e.g., the anti-vascular endothelial growth factor antibodies bevacizumab, the compounds disclosed in international patent applications WO97/22596, WO97/30035, WO97/32856, and WO98/13354, each of which is incorporated herein in its entirety) and compounds that act by other mechanisms (e.g., linoamine, inhibitors of integrin α v β 3 function, and angiostatin);

(vi) vascular disrupting agents such as combretastatin A4 and the compounds disclosed in International patent applications WO99/02166, WO00/40529, WO00/41669, WO01/92224, WO02/04434 and WO02/08213 (each of which is incorporated herein by reference in its entirety);

(vii) antisense therapies, e.g., those directed against the above targets, such as ISIS2503, anti-ras antisense;

(viii) gene therapy methods, including, for example, methods of replacing aberrant genes, such as aberrant p53 or aberrant BRCA1 or BRCA2, GDEPT (gene-directed enzyme prodrug therapy) methods, such as those utilizing cytosine deaminase, thymidine kinase, or bacterial nitroreductase, and methods of increasing a patient's resistance to chemotherapy or radiation therapy, such as multi-drug resistance gene therapy; and

(ix) immunotherapeutic approaches, including, for example, ex vivo and in vivo approaches to increase the immunogenicity of patient tumor cells, such as transfection with cytokines, such as interleukin 2, interleukin 4 or granulocyte macrophage colony stimulating factor, approaches to reduce T cell anergy, approaches using transfected immune cells, such as cytokine-transfected dendritic cells, approaches using cytokine-transfected tumor cell lines and approaches using anti-idiotypic antibodies.

The binding members of the invention and one or more of the other medical components described above may be used in the preparation of a medicament. The medicaments may be administered to the individual alone or in combination and may therefore comprise the binding member and other components, e.g. a combined preparation or separate preparations. Separate preparations may be used to facilitate separate and sequential or simultaneous administration and the components can be administered by different routes, e.g., oral and parenteral.

In accordance with the present invention, compositions are provided that can be administered to a mammal. Administration is generally a "therapeutically effective amount" sufficient to show benefit to the patient. Such benefit may at least alleviate at least one symptom. The actual amount and rate and course of administration will depend on the nature and severity of the condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the composition, the type of binding member, the method of administration, the schedule of administration and other factors known to medical practitioners. The ordinary practitioner and other doctors are responsible for the prescription of treatment, e.g., the determination of dosage, etc., depending on the severity and/or progression of the symptoms of the disease being treated. Suitable antibody dosages are well known in the art [110, 111 ]. Specific dosages of the herein described or Physician's desk reference (2003) appropriate for the type of drug being administered may be utilized. A therapeutically effective amount or suitable dose of a binding member of the invention may be determined by comparing its in vitro and in vivo activity in animal models. Methods are known to extrapolate effective doses in mice and other test animals to humans. The precise dosage will depend on a number of factors, including whether the antibody is to be used diagnostically, prophylactically, or therapeutically, the size and location of the treatment area, the precise nature of the antibody (e.g., whole antibody, fragment, or diabody), and the nature of any detectable label or other molecule attached to the antibody. For systemic administration, typical antibody doses are 100 μ g to 1g, for topical application 1 μ g to 1 mg. A higher loading dose may be administered first, followed by one or more lower doses. The antibody is typically a whole antibody, for example of the IgG1 isotype. This is the dose per treatment for adult patients, and can be adjusted appropriately for children and infants, and for other antibody formats can be adjusted proportionally to the molecular weight. Treatment may be repeated at daily, twice-weekly, or monthly intervals as prescribed. Treatment may be subcutaneous administration every 2-4 weeks and intravenous administration every 4-8 weeks. The treatment may be periodic, with a period of about two weeks or more between administrations, e.g., about 3 weeks or more, about 4 weeks or more, or about once per month. The treatment may be administered pre-and/or post-operatively, and/or may be administered or applied directly at the anatomical site of the surgical treatment.

The IL-6 binding members of the invention have advantages over antibodies to sIL-6Ra in terms of dosage and dosing requirements. As described elsewhere herein, circulating levels of IL-6 are significantly lower than circulating levels of sIL-6Ra in patients. Thus, the use of an IL-6 binding member, as opposed to an anti-IL-6R binding member, has significant advantages in that the amount of drug per dose administered to a patient that is produced may be lower. In addition, if the dose of anti-IL 6 treatment is lower, there may be a significant advantage because the lower dose facilitates subcutaneous and intravenous (i.v.) injections. It is well known to those skilled in the art that subcutaneous administration may be limited by the amount of binding member, e.g., antibody molecule, required per dose. This is because subcutaneous injections are limited to the injectable volume of a certain part of the skin. Subcutaneous injection volumes of 1.2ml or less are typically utilized. Because of the increased difficulty of formulating binding members for subcutaneous injection at concentrations above 50mg/ml, doses above 100mg administered by this route typically require multiple injections, which is even more uncomfortable for the patient.

Lower dose anti-IL-6 therapy may also require lower "loading" doses of antibody to inhibit all systemic IL-6, as compared to systemic sIL-6Ra, due to its higher concentration.

Other benefits may be associated with targeting IL-6 rather than the IL-6 receptor, which represents an additional advantage of the binding members of the invention over binding members for IL-6 Ra.

For example, circulating IL-6 levels in disease have been reported to be significantly lower than circulating sIL-6Ra [112, 113 ]. Since the level of sIL-6R is significantly higher than the level of IL-6, more anti-sIL-6R binding member may be required to neutralize sIL-6Ra than the amount of anti-IL-6 binding member required to neutralize IL-6. Thus, lower doses of anti-ligand binding member may be required than when an anti-receptor binding member is used.

Targeting IL-6 ligands rather than IL-6 receptors can reduce IL-6 levels in disease, but still allow IL-6 levels to rise during infection, where IL-6 is upregulated as part of the immune response.

Krebs et al [4] demonstrated that IL-6 is a potent growth factor and that myeloma cells freshly isolated from patients are capable of producing IL-6 and expressing its receptor. Furthermore, anti-IL-6 antibodies inhibit the growth of myeloma cells in vitro. This is direct evidence of autocrine loop work in human myeloma tumorigenesis. Following this study, VanZaanen et al [5] demonstrated decreased IL-6 production in multiple myeloma patients when treated with anti-IL-6 ligand antibodies.

A number of further studies have demonstrated that IL-6 is involved in the autocrine feedback loop of other cell types, such as Smooth Muscle Cells (SMC) [114], U373-MG astrocyte [115], 3T3 adipocytes [116], neurons [117], endothelial cells [118] and Kaposi's sarcoma cells [119 ]. Thus, inhibition of IL-6 with anti-IL 6 binding members in disease can lead to a decrease in the yield of IL-6 from the underlying disease.

In addition, anti-IL-6 binding members bind IL-6 in the systemic circulation, whereas IL-6 receptor binding members need to penetrate tissue to occupy receptors on the surface of cells involved in the pathology of the disease being treated.

In systemic circulation IL-6 binding members can form a balance with IL-6, creating a gradient across barriers such as the synovium, the net effect of which is to remove active IL-6 from the joint and form an inactive complex with the binding member. As a result, IL-6 binding members may have a faster onset of action, may have different dosage regimens, and may be readily optimized as compared to IL-6R binding members.

IL-6 signaling is mediated by IL-6 binding to IL-6R and the complex binding to gp 130. Given that the binding affinity of IL-6 to IL-6Ra is nanomolar (about 5nM) and that of the IL6: IL6R complex and gp130 are picomolar, binding members that target IL-6 are less competitive for binding to IL-6 and therefore may inhibit a higher proportion of IL-6 signaling. Although this may also be applicable to targeting soluble IL-6Ra and prevent IL-6: IL-6Ra complex formation of binding members, if IL-6Ra is membrane-bound, then due to space limitations, anti IL-6Ra may be more difficult to bind and inhibit IL-6Ra on the membrane.

Examples

Example 1 lead isolation

1.1 selection

Selection was performed using a naive human single chain fv (scFv) phage display library cloned into a phagemid vector according to filamentous phage M13 [120, 121 ]. anti-IL-6 specific scFv antibodies were isolated from phage display libraries using a series of selection cycles for recombinant human IL-6 essentially as described previously by Vaughan et al [120] and Hawkins et al [122 ]. Briefly, human IL-6 in PBS (Dulbecco's PBS, pH7.4) was adsorbed onto each well of a microtiter plate in a biopanning process at 4 ℃ overnight. The wells were washed with PBS and then blocked with PBS-Marvel (3% w/v) for 1 hour. Purified phage in PBS-Marvel (3% w/v) was added to each well and allowed to bind to the coated antigen for 1 hour. Multiple washes were performed with PBS-Tween (0.1% v/v) and PBS to remove unbound phage. The bound phage particles were eluted, infected into bacteria and rescued for the next round of selection [120 ].

1.2 inhibition of IL-6 binding to the IL-6 receptor by crude scFv

A representative number of each clone from the second round of selection was grown in 96-well plates. Expression of ScFv in the periplasm of bacteria(time-resolved homogeneous fluorescence (CIS BioInternational) human IL-6/human IL-6 receptor binding assays) were screened for their inhibitory activity. In this assay, samples compete with biotinylated IL-6R (Peprotech) for binding to crypt-labeled human IL-6 (R)&D systems Co Ltd (R)&DSystems)). A reference anti-IL-6 mAb (Biosource AHC0562) was included as a positive control in all efficacy tests. The detailed test methods are described in the materials and methods section.

1.3 scFv to IgG1

By mixing V_HAnd V_LThe domains were subcloned into vectors expressing the complete antibody heavy and light chains, respectively, to convert the clones from scFv to IgG format. Will V_HThe domains were cloned into a vector containing human heavy chain constant domains and regulatory elements (pEU15.1) to express the entire IgG heavy chain in mammalian cells. Similarly, the VL domain and regulatory elements were cloned into the vector peu3.4 to express the human kappa light chain or into peu4.4 to express the human lambda light chain constant region, thereby expressing the entire IgG light chain in mammalian cells. Reference [123 ]]The vectors expressing the heavy and light chains are first described. The vector of Cambridge antibody technology (Cambridge antibody technology) can be engineered simply by introducing OriP elements. To obtainIgG, heavy and light chain IgG expression vectors were transfected into EBNA-HEK293 mammalian cells. IgG is secreted into the culture medium upon expression. The collected materials are combined, filtered and purified. IgG was purified by protein a chromatography. The culture supernatant was applied to a protein A ceramic column (BioSepra) of appropriate size and washed with 50mM Tris-HClpH8.0, 250mM NaCl. Bound IgG was eluted from the column using 0.1M sodium citrate (pH3.0) and neutralized with Tris-HCl (pH 9.0). The buffer of the eluted material was replaced with PBS using Nap10 column (Amersham, 17-0854-02), and the concentration of IgG was measured spectrophotometrically using the extinction coefficient based on the amino acid sequence of IgG [124 ]]. Purified IgG was analyzed for aggregation or degradation by SEC-HPLC and SDS-PAGE.

1.4 inhibition of IL-6 binding to the IL-6 receptor by purified scFv and IgG

DNA sequencing of scFv that showed significant inhibition of IL-6A IL-6RA interaction (as crude periplasmic extract) [120,125 ]. The unique scFv was again expressed in bacteria and purified by affinity chromatography (as described by Bannister et al [126 ]). Purified IgG samples of these clones were also prepared as described in section 1.3. The efficacy of these samples was determined by competition with serial dilutions of purified preparations of anti-biotinylated sIL-6R for binding to hislag-labeled human IL-6 (internal e.coli-derived).

The results of cloning CAN022D10 as scFv and IgG with human heavy and kappa light chain constant regions are shown in table 1. The materials and methods section provides details.

Table 1: efficacy of CAN022D10scFv and IgG in receptor-ligand HTRF biochemical experiments

Cloning	IC50scFv(nM)	IC50IgG(nM)
			CAN022D10	45	0.31

1.5 inhibition of IL-6 induced TF-1 cell proliferation by purification of scFv and IgG

Neutralization efficacy of the purified scFv preparations against the biological activity of human and cynomolgus IL-6 was evaluated using the TF-1 cell proliferation assay. TF-1 is a human promyelocytic cell line established by erythroleukemia patients [134 ]. The survival and proliferation of TF-1 cell lines is dependent on certain factors. TF-1 cells were able to respond to human and cynomolgus IL-6 (internal E.coli-derived) and maintained in medium containing human GM-CSF (4ng/ml, R & D systems). Inhibition of IL-6 dependent proliferation was determined by detecting a decrease in incorporation of tritium-labeled thymidine into newly synthesized DNA of dividing cells. The details of this method are found in the materials and methods section.

At the highest concentrations tested, the purified scFv preparation of CAN022D10 was able to inhibit IL-6-induced TF-1 cell proliferation, but no complete inhibition was observed. Therefore, it is impossible to calculate an accurate IC from the obtained result₅₀Efficacy data. IC of CAN022D10 as purified IgG assay₅₀The calculation was 93 nM.

1.6 inAntibody selectivity and species cross-reactivity in epitope competition assays

By usingEpitope competition experiments, identified by measuring inhibition of binding of biotinylated HISFLAGIL-6 (internal E.coli-derived) to each immobilized anti-IL-6 antibodySpecies cross-reactivity and selectivity for IL-6 family member antibodies.

Purified Leukemia Inhibitory Factor (LIF) (Chemicon, Kemikang), ciliary neurotrophic factor (CNTF), IL-11, and oncostatin M (all from R) were tested in each assay&System D) to determine the efficacy of each structurally related protein, e.g., IC in the assay₅₀The values were measured.

Titers of various IL-6 substances including cynomolgus monkey (internal e.coli derived), human HISFLAGIL-6 (internal HEK-EBNA derived), rat and murine IL-6 (all from R & D systems) were tested in each assay to determine the species cross-reactivity of these antibodies. Exemplary results of this experiment are listed in table 2. The details of this method are found in the materials and methods section.

Table 2: potency of IL-6-related proteins and different IL-6 substances in CAN22D10 competition experiments

Numerical values are approximations of incomplete curves obtained from the sample

1.7 inhibition of endogenous IL-6 by purified IgG induces VEGF release by human synovial fibroblasts

Briefly, a titer of test IgG is added to cultured fibroblasts, which are then stimulated by the addition of human IL-1 β and soluble human IL-6R α to induce IL-6 expression, and cell signaling is initiated to induce VEGF expression.After 48 hours of incubation, the supernatant was removed and used with a commercially available kit (R)&System D) VEGF expression was detected by ELISA. Using these data to determine the IC of CAN022D10₅₀And calculated as 45 nM.

Example 2 antibody optimization

2.1 identification of amino acids that are likely to enhance binding of the lead antibody to IL-6

The protocol for identifying key residues in the parent antibody sequence that are likely to enhance binding to IL-6 was carried out by introducing random mutations throughout the CAN022D10scFv sequence. Using Diversify^TMPCR random mutagenesis kit (BD biosciences) two rounds of mutagenesis were performed according to the manufacturer's instructions to incorporate an average of 8.1 mutations per thousand bases of the nucleic acid sequence in each round of mutagenesis. The selection is carried out essentially as described previously (Hanes et al, 2000; methods enzymology.328.404-430). Briefly, a randomly mutagenized library of parental clones is transcribed into mRNA, and mRNA-ribosome-scFv complexes are formed using the hindered translation (stalledtranslation) method. These complexes were incubated with bio-huIL-6, and those bound to the antigen were then captured by streptavidin-coated magnetic beads. Non-specific ribosomal complexes were washed away, mRNA was isolated from the bound ribosomal complexes, reverse-transcribed into cDNA, and then subjected to PCR amplification. This DNA is used for the next round of selection and/or cloned for screening. This selection procedure was repeated in the presence of decreasing concentrations of bio-huIL-6 (the concentration of bio-huIL-6 was reduced from 100nM to 0.1nM in 4 rounds of screening). The ribosome display construct was digested with Nco1/Not1 restriction endonuclease (New England Biolabs) and ligated into Nco1/Not1 digested pCANTAB 6[ 127 ] with T4DNA ligase (New England Biolabs)]And the ScFv isolated by ribosome display was cloned into the phagemid vector pCANTAB 6. The ligated DNA was then transformed into chemically competent TG-1 cells and the crude scFv from each clone competed with CAN022D10IgG for binding to HIS/FLAGIL-6 detected in ligand-antibody biochemical experiments.

2.2 identification of improved clones Using antibody-ligand Biochemical experiments (Using CAN022D10IgG)

In CAN022D10 IgG-IL-6The crude scFv preparations of representative numbers of each clone were screened for inhibitory activity in binding experiments in the 3 rd and 4 th round results. In this experiment, cryptate-labeled anti-FLAG monoclonal antibody and streptavidin XL were used^ent！(TM) detection of binding of biotinylated antibody to FLAG-labeled IL-6. See materials and methods section for detailed assays.

ScFv that showed significant inhibition were sequenced and produced as a purified preparation as described in section 1.4. The IC of each scFv was then calculated from data obtained from purified samples of a series of test dilutions in HTRF antibody-ligand biochemical experiments and TF-1 proliferation experiments₅₀The value is obtained. As previously described, the most effective clones in TF-1 proliferation assays were converted to IgG containing the heavy chain constant region and kappa light chain constant region and retested in TF-1 proliferation assays. Exemplary potency data for purified scFv and IgG for each sample are shown in table 3.

Table 3: examples of clones with increased potency isolated from ribosome-display CAN022D10 random mutagenesis library in ligand-antibody biochemical experiments and TF-1 proliferation experiments

Protocol was varied to determine IgG potency, so scFv and IgG potency of individual clones should not be directly compared. For details of such changes, reference is made to materials and methods.

2.3 optimization of parental clones by Targeted mutagenesis

The leader antibody was optimized by directed mutagenesis using affinity phage display selection. In-situ targeted mutagenesis methodIn (1), standard molecular biology techniques are used [128 ]]Variable heavy chain (V) directed by oligonucleotides_H) And light chain (V)_L) Mutagenesis of complementarity determining region 3(CDR3) produced a large library of scFv-phages derived from the leader clone. These libraries were subjected to affinity-based phage display selection to select variants with higher affinity for IL-6. Thus, these variants should show an increased inhibitory activity on the binding of IL-6 to its receptor. Substantially as hereinbefore described [129 ]]Selection is performed. Briefly, scFv-phage particles were incubated with recombinant biotinylated human IL-6 in solution (bio-huIL-6, internal E.coli-derived and internal modified). Then streptavidin-coated paramagnetic beads according to the manufacturer's recommendations: (M280) capture scFv-phage that bind to antigen. Then as before [125 ]]The selected scFv-phage particles were rescued and the selection process was repeated in the presence of decreasing concentrations of bio-huIL-6 (reduced from 50nM to 0.1nM in 3 rounds).

Once 3 rounds of selection were completed, the VH and VL randomized libraries were pooled to form a single library, in which clones contained random pairings of individually randomized VH and VL sequences. Selection was then continued as described above in the presence of decreasing concentrations of bio-huIL-6 (again reduced from 0.1nM to 0.1pM in 4 rounds).

2.4 identification of clones improved by Targeted mutagenesis Using antibody-ligand Biochemical experiments (Using antibody 5IgG)

Crude scFv of clones isolated from targeted mutagenesis selection results were tested in antibody-ligand biochemical experiments essentially as described in section 2.2. In these results, the biochemical experiment was re-engineered to use the antibody 5 IgG. The antibody is an improved variant of CAN02210 with higher efficacy in TF-1 proliferation experiments. Incorporation of such more potent IgG resulted in the experiment being able to distinguish between more potent clones. The protocol of this modified assay was essentially the same as the original antibody-ligand biochemical assay using CAN022D10, but with the following changes. First, the HISFLAGIL-6 concentration used was reduced from 1nM to 0.5 nM.Second, anti-IL-6 antibody and streptavidin XL^ent！The concentration of (TM) increased from 1nM and 20nM to 16nM and 40nM, respectively. scFv that showed significant inhibition were sequenced and prepared as purified scFv and IgG, and then tested in TF-1 proliferation assay.

2.5. Inhibition of IL-6 induced TF-1 cell proliferation by optimization of cloned purified scFv and IgG

The efficacy of the optimized clones was determined using the IL-6 induced TF-1 proliferation assay as described previously. IgG detection clones as purified scFv preparations and variants. Examples of results for scFv and IgG are shown in Table 4.

Table 4: exemplary potencies of clones identified from a Targeted mutagenesis library when tested in TF-1 cell proliferation assay

Not determined N.D

Clones showed significant inhibition, but accurate IC₅₀Values could not be determined from serial dilutions of purified scFv.

2.6. Germlining

V to optimize anti-IL-6 antibodies_HAnd V_LAlignment of the amino acid sequence of the Domain with known human germline sequences in the VBASE database [130]The closest germline is identified by sequence similarity. For the VH domain of the CANDY022D10 antibody lineage, the closest germline v segment is Vh3_ DP-86_ (3-66) and the closest germline j segment is JH 2. For the VL domain, the closest germline v segment is Vk1_ L12 and the closest germline j segment is JK 2.

If the unaltered vernier residues [131] are not considered, three changes in the framework regions of the VH domain and 4 changes in the VL domain, they are all reverted to the closest germline sequence for a perfect match to the human antibody using standard site-directed mutagenesis techniques and appropriate mutagenesis primers.

A total of 5 trim residues were identified in the scFv sequence from germline mutated CAN022D 10. These residues are located at Kabat residues 29(I instead of V), 69(M instead of I), 73(I instead of N) and 78(V instead of L) in the heavy chain. A single Vernier mutation was also identified at Kabat residue 46(V instead of L) in the light chain sequence.

Then, the germlined IgG was again evaluated in IL-6 induced TF-1 proliferation experiments to confirm that the potency was not reduced. The potency of the Germlined (GL) antibodies is shown in table 5.

Table 5: exemplary potency data for germlined optimized clones when evaluated in IL-6 induced TF-1 cell proliferation experiments

Cloning	IC50(pM)
		Antibody 7(GL)	5
Antibody 10(GL)	71
		Antibody 17(GL)	1
Antibody 18(GL)	3
		CNTO-328	101

2.7. Inhibition of endogenous IL-6-induced VEGF release by optimized IgG in human synovial fibroblasts

Optimized IgG was detected in synovial fibroblast VEGF release experiments to assess efficacy against endogenously expressed IL-6. This method is described in detail in the materials and methods section, see section 1.7. Exemplary potencies of the detected IgG are shown in table 6 a. The mean potency data for the detected IgG are shown in table 6 b.

Table 6a: exemplary efficacy data for optimization of clones in evaluating efficacy against endogenous IL-6 in IL-6-induced synovial fibroblast VEGF release experiments

Cloning (GL germlined clone)	IC50(nM)
		Antibody 2	0.59
Antibody 3	0.38
		Antibody 4	0.52
Antibody 5	0.70
		Antibody 7(GL)	0.75
Antibody 10(GL)	0.55
		Antibody 17(GL)	0.57
Antibody 18(GL)	0.93
		CNTO-328	1.31

Table 6 b: average efficacy data for optimized clones when assessing efficacy against endogenous IL-6 in IL-6 induced synovial fibroblast VEGF release experiments

Cloning (GL germlined clone)	IC50(nM)(95％CI)	n
			Antibody 7(GL)	0.78(0.54-1.11)	3
Antibody 17(GL)	0.57(0.51-0.64)	3
			Antibody 18(GL)	0.67(0.20-2.25)	4
CNTO-328	1.02(0.39-2.63)	4

2.8. In thatOptimizing antibody selectivity and species cross-reactivity in epitope competition assays

Again using as described previously (see section 1.6 and materials and methods)Epitope competition experiments evaluated a series of clones for selectivity and species cross-reactivity. Human and cynomolgus IL-6 produced overlapping inhibition curves, and thus IC of all IgG detected₅₀The value is uncertain. No inhibition of the panel of antibodies by murine, rat or canine IL-6 or any related human protein detected was observed. These data indicate that a panel of clones tested cross-reacted with cyno IL-6, but did not bind murine, rat, or canine IL-6, or the human protein most closely related to human IL-6.

2.9 calculation of affinity data for optimized clones Using BIAcore

The binding affinity of purified IgG samples of representative antibodies 7 and 18 to human and cynomolgus IL-6 was determined by surface plasmon resonance using a BIAcore2000 biosensor (BIAcore corporation (BIAcore ab)) essentially as described in reference [132 ]. Briefly, purified antibodies were coupled to the surface of a CM5 sensor chip using an amine coupling kit (BIAcore corporation) to provide a surface density of 220-225 Ru. Human and cynomolgus IL-6 were passed over the sensor chip surface at a range of concentrations between 200nM and 0.2nM in HBS-EP buffer. The resulting sensorgrams were evaluated using BIA evaluation 3.1 software to provide relative binding data.

BIAcore2000^TMAffinity assay for biosensorsThe lower limit of the enclosure is about 10pM (BIAcore2000 Equipment handbook). From the data obtained, it can be seen that the affinity of the antibodies to human and cyno IL-6 is below the detection limit of 10pM, i.e. the antibodies are more potent than the measurable values. Therefore, no accurate affinity measurements were calculated. Using this method, it is believed that the affinity of the two antibodies to the two IL-6 substances is less than 10 pM.

2.10 calculation of affinity data for optimized clones Using TF-1 cell proliferation in vitro experiments

The affinity of antibody 18 was calculated by a Schilder analysis using TF-1 experiments. Standard curve for IL-6 (7.7X 10)^-15M to 3x10^-9M) and a range of IgG concentrations (2.67X 10)^-13M to 8.3x10^-10M) mixing and repeating twice. The affinity of IgG was determined by plotting Log10 (antibody concentration) against Log10 (dose ratio). Using this method, the affinity of antibody 18(GL) to human IL-6 was calculated to be 0.40pM (95% ci0.12pm-0.69 pM, n ═ 6).

2.11 determination of antagonist efficacy of human recombinant IL-6 Using IL-6 mediated in vitro proliferation of B9 cells

IL-6-induced B9 cell proliferation was evaluated in the presence of antibody 18 and an isotype control antibody. Each antibody was evaluated at a range of concentrations (1X 10)^-13M to 1x10^-9M) vs IL-6 Standard Curve (concentration Range 1X 10)^-14M to 1x10^-9M). Data points are in duplicate. B9 proliferation was determined after 4 days of incubation by reduction (fluorescence) of alamarblue.

Antibody 18 was shown to inhibit IL-6-induced B9 proliferation. Isotype control had no inhibitory effect. The average data are shown in Table 8.

Table 8: average Kb values for inhibition of IL-6 induced B9 proliferation

	Average Kb pM(95％CI)	n
			Antibody 18(GL)	0.3(0.1–0.5)	6

2.12 determination of the Effect of antagonists on human recombinant IL-6 Using IL-6-mediated IgM release from SKW6.4 cells in vitro

IL-6 induces the secretion of IgM by the human B lymphoblast line SKW 6.4. SKW6.4 cells and a range of IL-6 concentrations (1X 10)^-13M to 3x10^-8.5M) incubation with the resulting average for IgM secretion [ A ]]50 was 77pM (n-3). By observing different antibody concentrations in the presence of 100pMIL-6 (1X 10)^-12.5M to 1x10^-8M) evaluation of the effect of anti-human IL-6 antibodies 7, 17 and 18 and of an isotype control antibody on IL-6-induced IgM secretion. After 4 days, IgM secretion was measured using anti-human IgMELISA. Data points are in duplicate.

Antibodies 7, 17 and 18 inhibit IL-6-induced IgM secretion. Isotype controls had no inhibitory effect in these experiments. The average data are shown in Table 9.

Table 9: average inhibition of IgM secretion by SKW6.4 cells

	Average IC50pM	n
			Antibody 7(GL)	2.64	3
Antibody 17(GL)	3.21	3
			Antibody 18(GL)	2.63	3

Example 3 epitope mapping

3.1 comparison of epitopes of anti-IL-6 antibodies with known anti-human IL-6 antibodies

The epitope of antibody 18(GL) was compared to the epitopes of two anti-human IL-6 antibodies, B-E8 and cCLB 8. These two antibodies are known to inhibit the binding of IL-6 to IL-6Ra and have been studied as potential therapeutic agents [5,31,34,37,133 ]. To compare the epitopes of these three antibodies, a set of IL-6 mutants each containing an amino acid mutation compared to the wild-type (wt) sequence was constructed. These mutants were then evaluated for binding to different antibodies using biochemical competition experiments. These experiments were based on the biochemical competition experiments described in example 1.6, with the concentrations of antibody and IL-6 variant being varied as required. Briefly, PBS was formulated at 2nM (antibody 18) or 4nM (B-E8 and cCLB8) antibody coated onto the surface of a 96-well NM immunoassay (NuncMaxisorpimmunassay) plate and incubated overnight at 4 ℃. After blocking the well surface with 3% (w/v) BSA in PBS, a dilution of inhibitor in the range of 200nM to 10pM mixed with biotinylated human IL-6 at a final concentration of 0.15nM was added to the antibody-coated wells to allow binding. Binding of biotinylated IL-6 to the antibody was determined using europium-labeled streptavidin.

By comparing the IC of the mutant with that of unlabeled wild-type human IL-6₅₀Value, the potency ratio of each mutant can be established. Then, by comparing these ratios of different antibodies, the effect of each mutation on the binding of the antibody to the IL-6 molecule can be assessed. Typical results of these experiments are shown in Table 10, and the experiments were repeated two more times.

Table 10: IC of a panel of IL-6 mutants for anti-human IL-6 antibody 18, B-E8 and cCLB8₅₀And ratio of potency

The residue numbers in Table 10 represent the amino acid sequence of full-length human IL-6(SEQ ID NO: 161).

The results indicate that the three antibodies have different binding properties to the panel of IL-6 mutants and therefore bind to different epitopes on the surface of the cytokine. It was previously observed by Kalai et al (1997) that cCLB8 did not recognize the IL-6 mutant F106E. This was confirmed in our experiments because it did not inhibit the binding of biotinylated IL-6 to the antibody. In contrast, IL-6 mutant F106E was only 5-fold less potent than wtIL-6 in competition experiments with antibody 18, indicating that it binds strongly to the antibody. Similar results were observed with mutant Q211A, where the potency ratio for antibody 18 was 1.5 and 158 for clb 8. In contrast, mutants F102E, R207E, R207L and S204E were potent inhibitors in the clb8 experiment, but were observed to be significantly less potent than wtIL-6 in the antibody 18 experiment.

Differences in binding of antibody 18 and B-E8 were observed with mutants R58E and S204E. The ratio of potency of R58E to antibody 18 was 2.009 and B-E8 was 79.083, indicating that this mutation reduced the binding of B-E8 to IL-6. Of the three antibodies tested, the effect of the mutation S204E appeared to be specific to antibody 18. As with cCLB8, this mutation had little effect on the potency of IL-6 binding to B-E8, whereas for antibody 18 the mutant was more than 100-fold less potent than wild-type IL-6 in biochemical experiments.

Example 4 in vivo administration of anti-IL-6 antibodies

4.1 Effect of administration of anti-IL-6 antibodies on human recombinant IL-6-induced increases in neutrophils and haptoglobin in mice

Systemic administration of IL-6 is known to cause a systemic increase in neutrophil and acute phase protein concentrations. Intraperitoneal injection of human IL-6 into male C57/B/6/J mice generated an in vivo model, and neutrophil and acute phase response protein haptoglobin concentrations were determined. The ability of antibody 18(GL) administered by subcutaneous injection to inhibit the response was determined.

4.2 haptoglobin assay

Intra-peritoneal injection of human IL-6(5.2nmol/kg, equivalent to 12mg/kg twice daily) resulted in a significant increase in plasma haptoglobin levels from 0.02 + -0.01 mg/mL (vehicle control) to 1.19 + -0.27 mg/mL (IL-6 treated group) over the 7 day period (T-test, P < 0.01). Although the IgG1 isotype control had no effect, antibody 18 dose-dependently inhibited the response, with significant inhibition levels observed at doses of 10.6nmol/kg (156mg/kg) and higher (ANOVA, P <0.01 compared to IL-6 alone) (figure 1).

4.3 neutrophil assay

Intra-peritoneal injection of human IL-6(5.2nmol/kg, equivalent to 12mg/kg twice daily) resulted in neutrophil counts from 1.1. + -. 0.44X10 over 7 days⁹Individual cells/liter (vehicle control) increased significantly to 2.47. + -. 0.12X10⁹Individual cells/liter (IL-6 treated group) (T test, P<0.01). Although the IgG1 isotype control had no effect, antibody 18 dose-dependently inhibited the response, with significant inhibition levels observed at doses of 1.5nmol/kg (23mg/kg) and higher (ANOVA, P compared to IL-6 alone<0.01)。

These results confirmed, anti IL-6 antibody in vivo inhibition of IL-6 systemic effects ability.

Materials and methods

Inhibition of IL-6 binding to IL-6 receptor by crude scFv

With receptor-ligand binding(time resolved homogeneous fluorescence) assay format selection of the resulting inhibitory pocket Compound-labeled human IL-6 (R)&D systems 206-IL) or HISFLAG-labeled human IL-6 (internal E.coli-derived) with biotinylated IL-6R (Papp company 200-06R).

The results during lead isolation were screened as undiluted crude scFv containing periplasmic extracts formulated in 200mM hepes buffer pH7.4, 0.5mM EDTA and 0.5M sucrose. With 8nM streptavidin XL^ent！(TM) (CIS Biol International 611SAXLA) 8nM biotinylated human IL-6R was preincubated with light at room temperature for 30 minutes. All dilutions were performed in Phosphate Buffered Saline (PBS) containing 0.4M potassium fluoride and 0.1% BSA (assay buffer).

After reagent pre-incubation, 10 μ l of the crude scFv sample was added to a 384 well low volume assay plate (Costar 3676). Then 5. mu.l of pre-incubated biotinylated receptor and streptavidin XL were added^ent！(TM) mixture followed by 5. mu.l of 11.2nM cryptate-labeled human IL-6.

The assay plates were then centrifuged at 1000rpm for 1 minute at room temperature, incubated for 2 hours at room temperature, and the time-resolved fluorescence at the emission wavelengths of 620nm and 665nm was read using an EnVision plate reader (parkin elmer).

Inhibition of IL-6 binding to IL-6 receptor by purified scFv and IgG

In thatPurified scFv and IgG from positive clones identified by the screen were tested in the assay for inhibition of binding of HISFLAG-labeled human IL-6 to biotinylated IL-6R. With 8nM streptAvidin XL^ent！(TM) 8nM biotinylated human IL-6R30 min was preincubated at room temperature with exclusion of light. All dilutions were performed in Phosphate Buffered Saline (PBS) containing 0.4M potassium fluoride and 0.1% BSA (assay buffer).

Purification of samples by titration to determine IC from assay₅₀The cloning efficiency was evaluated. After reagent pre-incubation, 10 μ l of the titrate of the purified scFv sample was added to a 384-well low volume assay plate (Costar 3676). Then 5. mu.l of pre-incubated biotinylated receptor and streptavidin XL were added^ent！(TM) mixture. After mixing 2 nMHISFLG-labeled human IL-6 with 1.732nM of crypt-labeled anti-flag IgG (CIS biosciences International 61FG2KLB), 5. mu.l of the mixture was immediately added to the assay plate.

The assay plates were then centrifuged at 1000rpm for 1 minute at room temperature, incubated for 2 hours at room temperature, and the time-resolved fluorescence at the emission wavelengths of 620nm and 665nm was read using an EnVision plate reader (parkin elmer).

Data processing

Analysis of the samples from above by the following methodMeasured data.

The data were analyzed by calculating the% af value for each sample. Δ F was determined according to equation 1.

Equation 1:

the% specific binding was then calculated using the% Δ F values as described in equation 2.

Equation 2:

IC was determined by curve fitting using the 4-parameter logistic equation (equation 3) using Mat corporation Prism software₅₀The value is obtained.

Equation 3:

bottom + (top-bottom)/(1 +10^ ((LogEC50-X) } HillSlope))

X is the logarithm of the concentration. Y is a specific binding.

Y starts at the "bottom" and rises in an S-shape to the "top".

All assays contained a reference anti-IL-6 mAb (Biosource) AHC0562) as a positive control.

Inhibition of IL-6 induced TF-1 cell proliferation by purification of scFv and IgG

TF-1 cells are composed of R&The D system company gives away and cultures according to the provided scheme. Assay media included RPMI-1640 with GLUTAMAXI (Invitrogen), 5% fetal bovine serum (JRH), and 1% sodium pyruvate (Sigma). Before each assay, the TF-1 cells were pelleted by centrifugation at 300Xg for 5 minutes, the medium was aspirated, and the cells were resuspended in assay medium. By using at 5x10⁵The process was repeated twice for cells resuspended in assay medium at a final concentration of individual cells/ml. Cells were seeded in 96-well assay plates at a density of 100 μ l/well. At 37 ℃ and 5% CO₂The plates were incubated for 24 hours and GM-CSF removed to starve the cells. Test solutions of purified scFv or IgG (in duplicate) were diluted to the desired concentration with assay medium. Irrelevant antibodies not directed against IL-6 were used as a negative control. Recombinant bacteria-derived human (R) was added to the mixture in a total volume of 100. mu.l/well mixed with the appropriate test antibody&D) And cynomolgus (internal) IL-6 to a final concentration of 20pM (human IL-6) or 100pM (cynomolgus), respectively. The concentration of IL-6 used in the assay was selected as the dose that produced about 80% of the maximal proliferative response at the final assay concentration. All samples were incubated at room temperature for 30 minutes. Then 100 microliter IL-6 and antibody mixture is added to 100 microliter cells, making the total volume of 200 microliterLiter/well. At 37 ℃ and 5% CO₂Plates were incubated for 24 hours. Then, 20. mu.l tritiated thymidine (5. mu. Ci/ml) was added at each assay point, and the plate was returned to the incubator and incubated for another 24 hours. Cells were harvested on glass fiber filter plates (PerkinElmer) using a cell harvester. Thymidine incorporation was determined using a Packotron (PackardTopcount) microplate liquid scintillation counter. Then, the data was analyzed using Prism software of graphic pad company.

Determination of inhibition of binding of biotinylated human IL-6 to immobilized anti-IL-6 antibody by time-resolved fluorescence assay Method of producing a composite material

The particular method used for this determination and which results are provided in example 2.6 was adoptedReagents, as indicated above. The method is described more generally below and is suitable for use as an assay for determining and/or quantifying the binding of other IL-6 forms and related proteins to anti-IL-6 mabs.

In this assay, an anti-IL-6 monoclonal antibody is bound to a solid support, for example via Fc attachment to the support. Polystyrene high protein binding plates, such as NM plates (NuncMaxisorbplate) can be used as suitable supports.

anti-IL-6 Mab in PBS was coated on plates at 50. mu.l/well and overnight at 4 ℃.

All subsequent steps are carried out at room temperature.

The plates were washed 3 times with PBS containing 0.05% Tween 20(PBST, currently available from Sigma, P1379) and then blocked with 300. mu.l/well of PBS containing 3% (w/v) BSA (currently available from Roche diagnostics, 70129138) for 1 hour.

Wash the plate three times with PBST.

Inhibitor titrations were prepared with PBS containing 3% (w/v) BSA and added to 'dilute' plates (40 μ l/well) followed by 40 μ l/well of biotinylated IL-6, the final concentration of biotinylated IL-6 being equal to the KD of the protein with the antibody. Transfer 50. mu.l of sample from dilution plate to corresponding well of assay plate

Incubate the plates for 1 hour.

-washing the plate three times with PBST, then adding 50 μ l/well 0.1 μ g/ml europium-labeled streptavidin formulated with 50mm tris-HCl containing 0.9% NaCl, 0.5% purified BSA, 0.1% tween 20, and 20 μ MEDTA, ph7.5 to each well, followed by incubation for 1 hour.

Wash the plates seven times with wash buffer (25 ℃) consisting of 0.05MTris buffered saline (0.138m nacl, 0.0027m kcl), 0.05% (v/v) tween 20, ph 8.0.

Add 50. mu.l of enhancing solution to each well and acidify with acetic acid containing Triton X-100 and the chelating agents β NTA and TOPO. The resulting change in pH from basic to acidic results in rapid separation of europium ions from the streptavidin conjugate. The free europium ions then form fluorescent chelates with available chelators. By providing TOPO to remove water, the chelating agent is made to form micelles, prolonging the fluorescence of the chelating agent.

Incubation for 5 minutes, followed by measurement of time-resolved fluorescence at emission wavelength 620 nm. Fluorescence data was converted to% specific binding according to equation 1. Total binding was determined in control wells containing biotinylated huIL-6 but no competitor. Non-specific binding was determined in wells containing biotinylated huIL-6 and 100-fold excess of huIL-6. Fitting the resulting data to a sigmoidal curve to calculate IC according to equation 2₅₀The value is obtained.

Antibody coating and biotinylated huIL-6 concentrations for determination of biochemical epitope competition experiments

The concentration of antibody used for coating and the concentration of biotinylated huIL-6 used for epitope competition experiments were dependent on the affinity of the interaction of the two reagents and the efficiency of antibody immobilization. Therefore, the standard antibody coating concentration and the desired concentration of biotinylated huIL-6 for each antibody detected must be determined.

The general principle is that the final concentration of biotinylated huIL-6 used in each assay is equal to the KD of the ligand to the corresponding antibody as determined by saturation analysis. The concentration of antibody used for coating should be such that the lowest signal to background ratio measured under competitive assay conditions when biotinylated huIL-6 was added with KD is 10: 1.

In that Antibody selectivity and species cross-reactivity in epitope competition assays

Purified IgG was adsorbed to 96-well Maxisorp microtiter plates (Nunc) in PBS at a concentration that gave a clear signal when biotinylated human IL-6 was added approximately with the estimated Kd for that particular IgG. Excess IgG was washed off with PBS-Tween (0.1% v/v) and each well was blocked with PBS-Marvel (3% w/v) for 1 hour. Starting from approximately 200-fold concentrations of the KD for the interaction of biotinylated human IL-6 and corresponding IgG, serial dilutions of each of the following competitors were prepared in PBS: human IL-6, macaque IL-6, rat IL-6 (R)&D systems 506-RL/CF), murine IL-6 (R)&D systems Inc. 406-ML/CF), human CNTF (R)&257-NT/CF, D systems Inc.), LIF (LIF 1010, Kalimekang), IL-11 (R)&D systems 518-IL/CF), human Oncostatin M (R)&D systems company 295-OM/CF). Non-biotinylated human IL-6 was used as a positive control. To this serial dilution, an equal volume of biotinylated recombinant human IL-6 was added at a concentration of about twice the Kd (serial dilutions were obtained with initial ratio of competitive antigen: biotinylated human IL-6 of about 100: 1). These mixtures were then transferred to blocked IgG and equilibrated for 1.5 hours. Unbound antigen was removed by washing with PBS-Tween (0.1% v/v) and streptavidin-europium 3+ conjugate (M: (M))Detection, Parkinelmer (Perkinelmer)) detected the remaining biotinylated human IL-6. The 620nm time-resolved fluorescence was detected using an EnVision plate reader (parkin elmer). Fluorescence data were converted to%Specific binding (100% from containing biotinylated human IL-6 but no competitor control hole determination, 0% from containing biotinylated human IL-6 and more than 100 times of non-biotinylated human IL-6 hole determination). The resulting data were analyzed using the Prism curve fitting software of Mat corporation to determine IC according to equation 3₅₀The value is obtained.

Determination of inhibition of binding of biotinylated human IL-6 to immobilized anti-IL-6 antibody by time-resolved fluorescence assay Method of producing a composite material

The particular method used for this determination and which results are provided in example 2.8 was adoptedReagents, as indicated above. The method is described more generally below and is suitable for use as an assay for determining and/or quantifying the binding of other IL-6 forms and related proteins to anti-IL-6 mabs.

In this assay, an anti-IL-6 monoclonal antibody is bound to a solid support, for example via Fc attachment to the support. Polystyrene high protein binding plates, such as NM plates (NuncMaxisorbplate) can be used as suitable supports.

anti-IL-6 Mab in PBS was coated on plates at 50. mu.l/well and overnight at 4 ℃.

All subsequent steps are carried out at room temperature.

The plates were washed 3 times with PBS containing 0.05% Tween 20(PBST, currently available from Sigma, P1379) and then blocked with 300. mu.l/well of PBS containing 3% (w/v) BSA (currently available from Roche diagnostics, 70129138) for 1 hour.

Wash the plate three times with PBST.

Inhibitor titrations were prepared with PBS containing 3% (w/v) BSA and added to 'dilute' plates (40 μ l/well) followed by 40 μ l/well of biotinylated IL-6, the final concentration of biotinylated IL-6 being equal to the KD of the protein with the antibody. Transfer 50. mu.l of sample from dilution plate to corresponding well of assay plate

Incubate the plates for 1 hour.

-washing the plate three times with PBST, then adding 50 μ l/well 0.1 μ g/ml europium-labeled streptavidin formulated with 50mm tris-HCl containing 0.9% NaCl, 0.5% purified BSA, 0.1% tween 20, and 20 μ MEDTA, ph7.5 to each well, followed by incubation for 1 hour.

Wash the plates seven times with wash buffer (25 ℃) consisting of 0.05MTris buffered saline (0.138m nacl, 0.0027m kcl), 0.05% (v/v) tween 20, ph 8.0.

Add 50. mu.l of enhancing solution to each well and acidify with acetic acid containing Triton X-100 and the chelating agents β NTA and TOPO. The resulting change in pH from basic to acidic results in rapid separation of europium ions from the streptavidin conjugate. The free europium ions then form fluorescent chelates with available chelators. By providing TOPO to remove water, the chelating agent is made to form micelles, prolonging the fluorescence of the chelating agent.

Incubation for 5 minutes, followed by measurement of time-resolved fluorescence at emission wavelength 620 nm. Fluorescence data was converted to% specific binding according to equation 1. Total binding was determined in control wells containing biotinylated huIL-6 but no competitor. Non-specific binding was determined in wells containing biotinylated huIL-6 and 100-fold excess of huIL-6. Fitting the resulting data to a sigmoidal curve to calculate IC according to equation 2₅₀The value is obtained.

Antibody coating and biotinylated huIL-6 concentrations for determination of biochemical epitope competition experiments

The concentration of antibody used for coating and the concentration of biotinylated huIL-6 used for epitope competition experiments were dependent on the affinity of the interaction of the two reagents and the efficiency of antibody immobilization. Therefore, the standard antibody coating concentration and the desired concentration of biotinylated huIL-6 for each antibody detected must be determined.

The general principle is that the final concentration of biotinylated huIL-6 used in each assay is equal to the KD of the ligand to the corresponding antibody as determined by saturation analysis. The concentration of antibody used for coating should be such that the lowest signal to background ratio measured under competitive assay conditions when biotinylated huIL-6 was added with KD is 10: 1.

Identification of improved clones Using antibody-ligand Biochemical experiments

Competition at epitopeSelection of leads in the assay format the inhibitory capacity of the selection products obtained by optimization of the leads on the binding of hislag-labelled human IL-6 (internal e.coli-derived) to biotinylated anti-IL-6 antibody (internal IgG produced by separation of the leads, CAN022D10) was determined.

The product during lead optimization was screened as undiluted crude scFv containing periplasmic extracts formulated in 50nMMOPS buffer ph7.4, 0.5mMEDTA and 0.5M sorbitol. 1nM human HISFLAGIL-630 min was preincubated with 1.732nM crypt-labeled anti-flag IgG (CIS organism International 61FG2KLB) protected from light at room temperature. All dilutions were performed in assay buffer. In parallel, 20nM streptavidin XL^ent！(TM) (CIS Biol. International 611SAXLB) 1nM biotinylated anti-IL-6 IgG (to which the test binding member to be detected competes) was pre-incubated for 30 min at room temperature in the absence of light.

After pre-incubation of reagents, 10 μ l of crude scFv sample was added to a black 384-well optical plate (pamoermer catalog No. 6007279). Then 10. mu.l of assay buffer was added to the whole plate. Then 10. mu.l of pre-incubated biotinylated anti-IL-6 IgG and streptavidin XL were added^ent！(TM) mixture, and 10. mu.l of pre-incubated human IL-6 anti-flag cryptate mixture with HISFLAG marker.

The assay plates were then centrifuged at 1000rpm for 1 minute at room temperature, incubated for 2 hours at room temperature, and the time-resolved fluorescence at the emission wavelengths of 620nm and 665nm was read using an EnVision plate reader (parkin elmer). The% af and% specific binding were calculated as described previously to analyze the data.

After identification of improved leads from random mutagenesis libraries, competition with improved epitopesAn assay screening undiluted CDR3 targeted mutagenesis selected crude scFv harvest, comprising the following improvements: anti-flag IgG (CIS Bio International 61FG2KLB) labelled with 1.732nM crypt was preincubated with 0.5nM human HISFLAGIL-630 min at room temperature in the dark. In parallel, with 40nM streptavidin XL^ent！(TM) (CIS Biol. International 611SAXLB) 16nM biotinylated anti-IL-6 IgG (antibody 5, internal IgG identified by CAN022D10 random mutagenesis selection) was pre-incubated for 30 min at room temperature in the absence of light. All other conditions were identical to the CAN022D10 epitope competition assay. The% af and% specific binding were calculated as described previously to analyze the data.

Inhibition of endogenous IL-6-induced VEGF release by human synovial fibroblasts by purified IgG

Rheumatoid arthritis knee samples from total joint replacement were placed in DMEM containing antibiotics. Synovium was isolated from the joints in a media bath and finely minced. Synovial tissue was washed with medium containing 10% FCS. At 37 ℃ in CO₂The cell suspension was incubated with collagenase solution in an incubator for 2 hours. The digested synovial cell suspension was separated by repeated pipetting through a 10ml pipette and centrifuged at 400g for 5 minutes at room temperature to collect the cells. Cells were resuspended in DMEM containing 10% FCS and cell density adjusted to 1X10 by cell Filter (purifier)⁶Individual cells/ml, 37 ℃ in CO₂225-cm in incubator²Cell culture flasks (3001, costarcorning inc.). After adherence, most of the culture medium is discarded, the fresh culture medium is replaced, and the culture medium is put back into the incubator for long-term culture. Cells were examined weekly and passaged at confluency by trypsinization at one-third of the passage rate.

Each flask was treated with 10mL of a 0.1% trypsin-EDTA solution (25300-To separate confluent fibroblasts from the flask (P3-5). An equal volume of DMEM medium containing 10% FCS was added to the cells, and then the cells were pelleted by centrifugation at 330g for 5 minutes at room temperature. After washing the cells once with DMEM medium containing 10% FCS, the cell suspension (1X 10)⁵Individual cells/ml) were added (150 μ l/well) to wells of sterile 96-well cell culture colony flat-bottomed polystyrene plates (3598, kaskening) at a density of 1.5x10⁴Individual cells/well. DMEM medium (100. mu.l/well) containing 10% FCS was also added to each well to give a total volume of 250. mu.l/well. Cells were incubated overnight at 37 ℃ to allow attachment and dormancy (quiescence).

The 96-well plate is examined to ensure that the cells are confluent and in good condition (e.g., no contamination). Then, the medium in the wells was aspirated, and 100. mu.L of DMEM medium containing 10% FCS was immediately added. To this end, 50 μ L of DMEM medium containing sample IgG with 10% FCS or medium alone was added to each well (1:5 dilution into the experiment).

Then 50. mu.L of DMEM medium containing 10% FCS containing recombinant human soluble (rhs) IL-6R α (500 ng/mL; 12nM) and rhIL-1 β (50 pg/mL; 2.95pM, 1:5 diluted into the experiment) was added to each well.

To different wells, 50. mu.L of DMEM medium containing 10% FCS containing rh-IL-6(0, 100 ng/mL; 21.5nM), sIL-6Ra (500 ng/mL; 12nM), rhIL-1. beta. (50 pg/mL; 2.95pM) or medium alone (1:5 diluted into the experiment) was added. The final volume of each well was 250. mu.L.

The plates were incubated at 37 ℃ for 48 hours. Incubations were performed in duplicate or triplicate wells as described for the plate format. The plates were centrifuged at 330g for 5 minutes at room temperature, the culture supernatant was removed and stored in flat-bottomed microtiter plates (611F96, Sterilin) at-40 ℃.

ELISA was performed according to the manufacturer's instructions (DY293B, R)&System D) to determine VEGF. Briefly, ELISA plates were coated with mouse anti-human VEGF antibody overnight at 4 ℃ and blocked with 1% BSA/PBS. The plates were washed with 0.05% Tween 20/PBS and cultured with human synovial membrane-derived fibroblastsThe supernatant and biotinylated goat anti-human VEGF antibody were incubated overnight at room temperature. After washing, VEGF was detected using streptavidin horseradish peroxidase. Using 1:1H₂O₂Tetramethylbenzidine imparts color to the plate. With 2MH₂SO₄The reaction was terminated, the calibration wavelength was set to 540nm, and the optical density was measured at 450 nm.

BIAcore assay

Using BIAcore2000^TMA BIAcore study was performed. The antibody was coupled to the surface of the CM-5 sensor chip using an amine coupling kit to provide a surface density of 220-225 Ru. A series of concentrations of human IL-6 between 200nM and 0.2nM in HBS-EP buffer was passed over the sensor chip surface. The resulting sensorgrams were evaluated using BIA evaluation 3.1 software to calculate k for the detected antibodies_{Bonding of}、k_DissociationAnd K_DThe value is obtained.

IL-6 mediated B9 cell proliferation assay

B9 cells were subclones of the murine B-cell hybridoma cell line B13.29 selected for specific response to IL-6. Survival and proliferation of B9 cells requires IL-6, responding to extremely low concentrations of IL-6.

IL-6-induced proliferation of B9 cells was assessed in the presence of antibody 18 and an isotype control (CAT-002). Each antibody was evaluated at a range of concentrations (1X 10)^-13M to 1x10^-9M) vs IL-6 Standard Curve (concentration Range 1X 10)^-14M to 1x10^-9M). Data points are in duplicate. B9 proliferation was measured after 4 days of incubation by reduction of alamar blue (fluorescence method).

B9 cells were cultured in RPMI-1640 containing 5% FCS, 2 mML-glutamine and 50. mu.M 2-mercaptoethanol. Cells were passaged every 2-4 days to a cell density of 0.05X10⁶mL^-1To 0.1x10⁶mL^-1Supplement 5x10^-13M human IL-6. The cells used in the experiment were cultured for at least 48 hours before the experiment without IL-6 supplementation, but within 96 hours of the experiment with IL-6 supplementation. The cells used in the experiment are obtained from cells with a density not exceeding 0.8x10⁶mL^-1The mother liquor culture bottle of (1).

In assay medium (RPMI + 5% FCS, 2 mML-glutamine, 50. mu.M 2-mercaptoethanol, penicillin 100 UmL)^-1And streptomycin 100mgmL^-1) In order to dilute each antibody from the stock solution to 10 times the maximum desired assay concentration. Then diluted 10-fold with culture medium to obtain the desired concentration of each antibody.

The lyophilized powder was reconstituted to 1 × 10 by adding a suitable volume of sterile PBS + 0.1% BSA^-5IL-6 solution of M. Further dilution to 1x10 with media^-8M。1x10^-8M equal portions were frozen and stored for later use. On the day of assay, 1X10 was diluted as required^-8M aliquots to obtain a series of solutions ten times the desired final assay concentration.

The desired volume of cells was removed from the flask and centrifuged at 300g for 8 minutes. The supernatant was removed and the cells were resuspended in the appropriate volume of medium to obtain 0.5x10⁶mL^-1The cell density of (a).

Assays were performed in tissue culture treated polystyrene 96-well flat bottom plates. The final assay volume was 200. mu.L. mu.L of 10x antibody (antibody 18 or CAT-002) solution or medium was added to the appropriate wells of each plate followed by 140. mu.L of medium and 20. mu.L of IL-6 or medium at the appropriate concentration.

Place the plate in wet 5% CO₂And 37 ℃ incubator for 2 hours. Then 20 μ L of cells were added to each well. The final number of cells per well was 10000. Then, the plate was returned to the incubator and allowed to stand for 4 days. Cell proliferation was assessed by alamar blue incorporation. Add 10% v/v Arama blue to each well and place the plate back in the incubator for 6 hours. The plate was then read at 590nm after excitation at 544nm on a spectrofluorimeter.

Raw data were normalized to the curve of the control IL-6 on each plate to determine the maximum fluorescence as 100% and the base fluorescence as 0%. By means of a non-linear regression, sigmoidal dose reaction from prism4.01, Inc. (variable slope)Rate) fitting program fitted to the normalized data. Using control pEC₅₀Values and pEC in the Presence of antibody at various concentrations₅₀Values determine the Dose Ratio (DR). Determination of K at the lowest concentration of antibody causing a 3-fold or more shift in the IL-6 concentration response curve using the following chemical antagonism equation_bThe value:

K_b＝([Ab]/(DR-1))

(KenakinTP. journal of pharmacological analysis of drug receptor interactions (Pharmacological Analyzis of drugs-receptors interactions), 1 st edition, New York, Levens Press (ravenPress), 1987, pages 205-24).

Determination of IL-6-mediated IgM Release from SKW6.4 cells

IL-6 is involved in the final maturation of B cells into antibody-producing cells (B-lymphocyte differentiation process). SKW cells have been used to study B cell responses (Nawata et al, Ann. N. Y. Acad. Sci.557: 230-238.1989). Autoantibodies produced in rheumatoid arthritis are mainly of the IgM type. SKW6.4 is a cloned human lymphoblast B cell line secreting IgM. Cells were from ATCC, reference # TIB 215. These cells secrete IgM when stimulated with IL-6, and therefore this experiment is considered to be associated with rheumatoid arthritis.

IL-6 was evaluated in the presence of CAT6001 and CAT-002 (isotype control) for the induction of IgM secretion by SKW6.4 cells. Each antibody was evaluated at a range of concentrations in the presence of 100pMIL-6 (1X 10)^-12.5M to 1x10^-8M). Data points are in duplicate. After 4 days of incubation, IgM secretion in the cell supernatants was determined using the anti-human IgMELISA assay.

At 37 ℃, 95% relative humidity and 95/5% (v/v) air/CO₂Next, SKW6.4 cells were cultured in RPMI1640 containing 2 mML-glutamine and 10% (v/v) fetal bovine serum. Cell density was maintained between 0.4 and 2x106 cells/ml. In routine cell passaging, cells were harvested by centrifugation at 300Xg for 5 minutes at room temperature, old medium was removed, and cells were resuspended in the desired volume of fresh medium.

Appropriate dilutions were made in assay medium (RPMI + 10% FCS, 2 ml-glutamine) to dilute each antibody from the stock solution to 50-fold the maximum required assay concentration. Then diluted 10-fold with culture medium to obtain the desired concentration of each antibody.

Assays were performed in tissue culture treated polystyrene 96-well flat bottom plates. SKW6.4 cell stock was diluted with fresh medium to a cell density of 0.3X106ml^-1And plated at a density of 100 μ l/well (30,000 cells/well). To each well was added 2. mu.l of antibody at the final concentration indicated, followed by 2. mu.l of IL-6 at a final concentration of 100 pM.

The plates were then returned to 37 ℃ with 5% CO₂The incubator of (1). After 4 days of incubation, cell-free supernatants were harvested by centrifugation and then assayed by IgMELISA or by cryopreservation at-20 ℃ on the day of harvest.

The ELISA was generated with a pair of antibodies from sarotack (Serotec). The coating antibody was mouse anti-human IgM (MCA1662) and the detection antibody was HRP-linked goat anti-human IgM (STAR 98P). The assay was optimized for good signal-to-noise ratio by standard methods using 1:2000 dilution of the coating antibody (5. mu.g/ml) and 1:3500 dilution of the detection antibody (200 ng/ml).

IgM standard solutions (catalog number PHP003 human M κ purified protein) were purchased from Selotatch for generating standard curves.

The IgM standard curve data were polynomial fitted using a standard fitting procedure to analyze the data. The percent inhibition for each antibody sample was calculated relative to the control IgM production in the absence of antibody and IC was generated₅₀The value is obtained.

Generation of IL-6 and IL-6 muteins for epitope mapping

Cloning of human and cynomolgus IL-6 cDNAs

The sequences for human and cynomolgus IL-6 were obtained from Embl (accession numbers for human and cynomolgus monkey are BC015511 and AB000554, respectively). These sequences were used to design oligonucleotide primers to amplify cDNAs encoding human and cynomolgus IL-6. For human and cynomolgus monkeys, the N-terminal primers were hIL6_5' NdeI and macIL6_5' NdeI, respectively, and macIL6_3' NheI were used as the C-terminal primers for both (see Table 11 for oligonucleotide sequences).

Table 11: primer sequences

A PCVR reaction was performed to amplify both cdnas. The template for each PCR reaction was 10ng cdna obtained from human liver and cynomolgus monkey liver, respectively. The cDNA amplified from each reaction was purified and cloned into pCR4blunttopo (Invitrogen) using a topoisomerase ligation reaction according to the manufacturer's instructions.

Positive clones were identified and sequenced. The resulting cDNA was subcloned into various E.coli T7-promoter expression vectors using standard techniques to fuse the N-terminus of cDNAencoding mature human or cynomolgus IL-6 to the N-terminal HIS6-FLAG tag just upstream of the N-terminal valine of mature IL-6.

Generation of mutants

Site-directed mutagenesis was performed using the quick Change (Quikchange) XL kit from Schachatagene (Stratagene) according to the manufacturer's protocol. Design of the mutagenic primers was performed according to the manufacturer's protocol. Mutagenesis was performed according to the protocol using plasmid pT7flagHISIL-6 as a template. Subsequent DpnI digestions were then performed and transformed into chemically competent Top10 cells, which were cultured overnight at 37 ℃ on agar plates containing the appropriate antibiotics for selection. For each individual mutagenesis reaction, several clones were sequenced and the plasmid DNA of one correct clone per reaction was retained for use.

Expression of IL-6 and IL-6 muteins

The IL-6 expression plasmid was transformed into chemically competent BL21(DE3) astrocytes (starcell) (Invitrogen) using the manufacturer's protocol. The 1L terrific broth culture was inoculated with the transformed cells and incubated at 37 ℃ on an orbital incubator until A600 reached 0.5. IPTG was then added to 0.25mM and incubation continued overnight at 22 ℃. Cells were harvested by centrifugation and the cell pellet stored at-80 ℃.

Purification of IL-6 and IL-6 muteins

The cell pellet was thawed and each cell pellet was resuspended in 50ml of a solution containing 50mM potassium phosphate, pH7.4, 10mM imidazole, 0.3M NaCl, 5 mM. beta. -mercaptoethanol, 10% glycerol (buffer A) + complete protease inhibitor without EDTA (Roche). Cells were sonicated for 3x30 seconds on ice. The lysate was centrifuged at 100,000g and 4 ℃ for 30 minutes and the supernatant was subjected to NiNTA affinity chromatography. A5 ml Ni-NTA Superflow (Superflow) column (Qiagen) was equilibrated with buffer A at 3 ml/min. IL-6 samples were loaded and the column was washed with 10 column volumes of 15mM imidazole in buffer A. The column was then washed with 10 column volumes of 30mM imidazole in buffer A. IL-6 was eluted from the column by an upward wash with 0.3M imidazole wash prepared in 5 column volumes of buffer A. 10ml fractions were collected in the washing step and 5ml fractions were collected in the elution step. The column was operated at 4 ℃ with AKTA Explorer100Air (Explorer100 Air). Fractions containing purified IL-6 protein were pooled and dialyzed against 5LPBS4 deg.C overnight.

The dialyzed IL-6 protein was further purified by gel filtration chromatography. In each purification, 100,000g and 4 degrees C centrifugal dialysis of IL-6 protein for 20 minutes. Up to 13ml were applied to 318ml Superdex20026/60 column (GE healthcare) equilibrated with PBS at 2.5 ml/min. The column was operated at 4 ℃ with AKTA purified gas (Purifier). Fractions containing the monomeric IL-6 protein peak were collected for further analysis.

The purity of each protein was checked by standard SDS chromatography, the protein concentration was determined, and the protein mass was determined by Q-ToF mass spectrometry. Purified IL-6 was frozen in liquid nitrogen and stored at-80 ℃.

Materials and methods for in vivo studies

Animals were randomly assigned to test groups. Mice from each test group were then treated daily with a set subcutaneous dose (10ml/kg) of vehicle control (0.05% BSA in PBS) or 467 μ g/kg IgG1 isotype control or antibody 18 (ranging from 467 μ g/kg to 8 μ g/kg). At the same time, mice (10ml/kg) were given vehicle control (0.05% BSA in PBS) or 12. mu.g/kg human recombinant IL-6 by intraperitoneal injection twice daily.

On day 7, mice were sacrificed 2 hours after the last dose of IL-6 at 09:00h and final blood samples were obtained. The blood was transferred to a LabTek1ml EDTA blood tube and placed on a roller for 5 minutes. The samples were then kept on ice until use. Differential cell counts were performed using a Sesimimes (Sysmex) cell counter. The remaining samples were transferred to a microcentrifuge tube, centrifuged (300g, 5 min) to obtain plasma, which was again aliquoted and stored at-20 ℃ until haptoglobin levels were analyzed.

According to the PHASE of Biognosis Inc. (Biognosis, Hailsham, UK) of Haishan, UK^TMHaptoglobin assay was performed according to the instructions provided in the RANGETRIDeltaFormat kit (catalog number TP-801).

All results are expressed as mean ± SEM. Data analysis was performed by unpaired T-test or one-way ANOVA followed by Dunnett' test (GI corporation (GraphPadInstat)).

Sequence of

The VH domain, VL domain and CDR sequences of the binding members are shown in the accompanying sequence listing, wherein seq id nos correspond to the meanings given below:

1CAN022D10VH nucleotide

2CAN022D10VH amino acid

3CAN022D10VHCDR1 amino acid

4CAN022D10VHCDR2 amino acid

5CAN022D10VHCDR3 amino acid

6CAN022D10VL nucleotide

7CAN022D10VL amino acid

8CAN022D10VLCDR1 amino acid

9CAN022D10VLCDR2 amino acid

10CAN022D10VLCDR3 amino acid

11 antibody 2VH nucleotides

12 antibody 2VH amino acids

13 antibody 2VHCDR1 amino acid

14 antibody 2VHCDR2 amino acid

15 antibody 2VHCDR3 amino acid

16 antibody 2VL nucleotides

17 antibody 2VL amino acids

18 antibody 2VLCDR1 amino acid

19 antibody 2VLCDR2 amino acid

20 antibody 2VLCDR3 amino acids

21 antibody 3VH nucleotides

22 antibody 3VH amino acids

23 antibody 3VHCDR1 amino acid

24 antibody 3VHCDR2 amino acid

25 antibody 3VHCDR3 amino acid

26 antibody 3VL nucleotides

27 antibody 3VL amino acids

28 antibody 3VLCDR1 amino acids

29 antibody 3VLCDR2 amino acid

30 antibody 3VLCDR3 amino acids

31 antibody 4VH nucleotides

32 antibody 4VH amino acids

33 antibody 4VHCDR1 amino acid

34 antibody 4VHCDR2 amino acid

35 antibody 4VHCDR3 amino acid

36 antibody 4VL nucleotides

37 antibody 4VL amino acids

38 antibody 4VLCDR1 amino acid

39 antibody 4VLCDR2 amino acid

Amino acid 4VLCDR3 of antibody 40

41 antibody 5VH nucleotides

42 antibody 5VH amino acids

43 antibody 5VHCDR1 amino acid

44 antibody 5VHCDR2 amino acid

45 antibody 5VHCDR3 amino acid

46 antibody 5VL nucleotides

47 antibody 5VL amino acids

48 antibody 5VLCDR1 amino acids

49 antibody 5VLCDR2 amino acid

Amino acid of 5VLCDR3 of antibody 50

51 antibody 7VH nucleotides

52 antibody 7VH amino acids

53 antibody 7VHCDR1 amino acid

54 antibody 7VHCDR2 amino acid

Antibody 557 VHCDR3 amino acid

56 antibody 7VL nucleotides

57 antibody 7VL amino acids

Amino acid of antibody 58, 7VLCDR1

Antibody 59 7VLCDR2 amino acids

Amino acid of antibody 60 7VLCDR3

61 antibody 8VH nucleotides

62 antibody 8VH amino acids

63 antibody 8VHCDR1 amino acid

64 antibody 8VHCDR2 amino acid

65 antibody 8VHCDR3 amino acid

66 antibody 8VL nucleotides

67 antibody 8VL amino acids

68 antibody 8VLCDR1 amino acid

69 antibody 8VLCDR2 amino acid

70 antibody 8VLCDR3 amino acid

71 antibody 10VH nucleotides

72 antibody 10VH amino acids

73 antibody 10VHCDR1 amino acid

Antibody 74 10VHCDR2 amino acid

75 antibody 10VHCDR3 amino acid

76 antibody 10VL nucleotides

77 antibody 10VL amino acids

Antibody 78 amino acid 10VLCDR1

79 antibody 10VLCDR2 amino acids

80 antibody 10VLCDR3 amino acid

81 antibody 14VH nucleotides

82 antibody 14VH amino acids

83 antibody 14VHCDR1 amino acids

84 antibody 14VHCDR2 amino acid

85 antibody 14VHCDR3 amino acid

86 antibody 14VL nucleotides

87 antibody 14VL amino acid

88 antibody 14VLCDR1 amino acid

89 antibody 14VLCDR2 amino acid

90 antibody 14VLCDR3 amino acid

91 antibody 16VH nucleotides

92 antibody 16VH amino acids

93 antibody 16VHCDR1 amino acids

94 antibody 16VHCDR2 amino acid

95 antibody 16VHCDR3 amino acid

96 antibody 16VL nucleotides

97 antibody 16VL amino acids

98 antibody 16VLCDR1 amino acid

99 antibody 16VLCDR2 amino acids

100 antibody 16VLCDR3 amino acids

101 antibody 17VH nucleotides

102 antibody 17VH amino acids

103 antibody 17VHCDR1 amino acid

Antibody 17VHCDR2 amino acid

105 antibody 17VHCDR3 amino acid

106 antibody 17VL nucleotides

17VL amino acid of 107 antibody

108 antibody 17VLCDR1 amino acid

Antibody 109 17VLCDR2 amino acids

Amino acid of antibody 17VLCDR3

111 antibody 18VH nucleotides

112 antibody 18VH amino acids

113 antibody 18VHCDR1 amino acid

114 antibody 18VHCDR2 amino acid

115 antibody 18VHCDR3 amino acid

116 antibody 18VL nucleotides

117 antibody 18VL amino acid

118 antibody 18VLCDR1 amino acid

119 antibody 18VLCDR2 amino acid

Amino acid of 18VLCDR3 of antibody 120

121 antibody 19VH nucleotides

122 antibody 19VH amino acids

123 antibody 19VHCDR1 amino acid

Antibody 19VHCDR2 amino acid of 124

125 antibody 19VHCDR3 amino acid

126 antibody 19VL nucleotides

127 antibody 19VL amino acid

Antibody 128 19VLCDR1 amino acid

129 antibody 19VLCDR2 amino acid

130 antibody 19VLCDR3 amino acid

131 antibody 21VH nucleotides

132 antibody 21VH amino acids

Amino acid of 21VHCDR1 of antibody 133

134 antibody 21VHCDR2 amino acid

Amino acid of 21VHCDR3 of antibody 135

136 antibody 21VL nucleotides

137 antibody 21VL amino acid

138 antibody 21VLCDR1 amino acid

139 antibody 21VLCDR2 amino acids

140 antibody 21VLCDR3 amino acid

141 antibody 22VH nucleotides

142 antibody 22VH amino acids

143 antibody 22VHCDR1 amino acid

144 antibody 22VHCDR2 amino acid

145 antibody 22VHCDR3 amino acid

146 antibody 22VL nucleotides

147 antibody 22VL amino acid

148 antibody 22VLCDR1 amino acid

149 antibody 22VLCDR2 amino acid

Amino acid of 150 antibody 22VLCDR3

151 antibody 23VH nucleotides

152 antibody 23VH amino acids

153 antibody 23VHCDR1 amino acid

154 antibody 23VHCDR2 amino acid

155 antibody 23VHCDR3 amino acid

156 antibody 23VL nucleotides

157 antibody 23VL amino acid

158 antibody 23VLCDR1 amino acid

159 antibody 23VLCDR2 amino acid

Amino acid of 23VLCDR3 of antibody 160

161 full-Length human IL-6 amino acid

162 hislag tagged human IL-6

163 soluble IL-6Ra (human)

164 transmembrane IL-6Ra (human)

165 mature human IL-6 amino acid

166 human gp130

167-germlined VHFR1

168 germlined VHFR2

169 germlined VHFR3

170 germlined VHFR4

171 germlined VLFR1

172 germlined VLFR1

173 germlined VLFR1

174-germlined VLFR1

175F102E mutant IL-6

176S204E mutant IL-6

177R207E mutant IL-6

178F106E mutant IL-6

179Q211A mutant IL-6

180R58E mutant IL-6

181E200W mutant IL-6

182R207L mutant IL-6

183 primer macIL6_5' NdeI

184 primer macIL6_3' NheI

185 primer hIL6_5' NdeI

The sequences of antibodies 7, 10, 17 and 18 were germlined.

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Claims (4)

1. An isolated human IL-6 antibody, comprising in combination a heavy chain variable region sequence of SEQ ID NO 52 and a light chain variable region sequence of SEQ ID NO 57.

2. The antibody of claim 1, which is an IgG.

3. The antibody of claim 2, wherein the IgG is IgG 1.

4. A composition comprising the antibody of claim 1 and a pharmaceutically acceptable excipient.