WO2009109584A1

WO2009109584A1 - ANTIBODY BINDING ONLY TO IL6-sIL6R COMPLEX

Info

Publication number: WO2009109584A1
Application number: PCT/EP2009/052517
Authority: WO
Inventors: Ian Cottingham
Original assignee: Ferring International Center S. A.
Priority date: 2008-03-07
Filing date: 2009-03-03
Publication date: 2009-09-11

Abstract

An antibody that binds strongly to human IL-6/sIL-6R complex and thereby inhibits the transsignaling pathway and characterized by that the antibody essentially does not bind to human IL-6 alone (as such) and to human sIL-6R alone (as such) and thereby essentially does not affect the classical IL-6 signaling pathway.

Description

TITLE: Antibody binding only to IL6-slL6R complex

FIELD OF THE INVENTION:

The present invention relates to an antibody that binds strongly to human IL-6/slL- 6R complex and thereby inhibits the transsignaling pathway and characterized by that the antibody essentially does not bind to human IL-6 alone (as such) and to human slL-6R alone (as such) and thereby does not affect the classical IL-6 signaling pathway.

BACKGROUND

Figures 1 to 3 herein and below general text discussing these figures was essentially directly copied from the web-page of the company Conaris AG (www.conaris.de). It represents herein relevant general knowledge of the skilled person.

lnterleukin-6 (IL-6) is a pleiotropic cytokine which plays a crucial role in inflammation, immune regulation, hematopoiesis, and oncogenesis. It belongs to a family which comprises molecules such as interleukin-1 1 (IL-1 1 ), interleukin-27, leukemia inhibitory factor (LIF), oncostatin M (OSM), cardiotropin-1 (CT-1 ), ciliary neurotrophic factor (CNTF) and cardiotropin-like cytokine (CLC)).

All molecules need to bind to the glycoprotein 130 (gp130) to confer their biological function. The receptor complexes are depicted in figure 1.

The diversity of biological effects driven by IL-6 is surprising, because the specific IL-6 receptor (IL-6R) is only expressed by a small number of cells such as hepatocytes, monocytes, and inactive T- and B-lymphocytes. In contrast, gp130 is found on all body cells. Binding of IL-6 to the IL-6R leads to the recruitment and complexation of two gp130 molecules followed by the activation of certain intracellular processes, e.g. STAT1 and 3 phosphorylation by Janus kinases (JAKs). Neither IL-6 nor the IL-6R can bind to gp130 alone. This so-called classical IL-6 signaling way takes place during early immune responses and activates diverse acute phase proteins. The classical IL-6 signaling way is illustrated in figure 2. Besides the membrane-bound IL-6R, a soluble form (slL-6R) can be generated by proteolytic cleavage (TACE/ADAM17) or alternative splicing. The resulting IL-6/slL- 6R complex also binds to sgp130 and thereby activates intracellular signaling cascades. Interestingly, this also happens in cells which do not express an endogenous IL-6R. Consequently, cells which release the slL-6R protein render cells which only express gp130 responsive towards the cytokine IL-6. This mechanism has been termed transsiqnaling. Meanwhile, it has been shown that transsignaling plays a key role in the pathophysiology of chronic inflammatory disorders and most likely some cancer diseases. The transsignaling mechanism is illustrated in figure 3.

Both the classical IL-6 signaling way and the transsignaling mechanism have been known for years.

In the late eighties was cloned gp130 and in the early nineties the company Tosoh (in collaboration with the company Chugai, Japan) developed antibodies against gp130. In the early nineties Tosoh (Chugai) also started to develop antibodies against IL-6 and IL-6R.

Chugai with some collaboration with the company Roche has launched an antibody on the market (Japan and probably relatively soon also in Europe) named Atlizumab (with synonyms Actemra™ , MRA, Tocilizumab, Anti-IL-6, receptor Mab, Chugai R- 1569 Atlizumab). This commercial available Atlizumab antibody is a humanized anti-interleukin-6 receptor monoclonal antibody - i.e. it binds to IL-6R as such. It is being developed for the treatment of rheumatoid arthritis, Crohn's disease, multiple myeloma and the lymphoproliferative disorder giant lymph node hyperplasia (Castleman's disease).

Numerous prior art references describes antibodies that may be characterized as antibodies that bind directly to IL-6 or IL-6R as such (alone).

In other words, the prior art antibodies may be characterized as antibodies that can bind to IL-6 or IL-6R alone - i.e. before they are present in the IL-6/slL-6R complex.

Examples are EP783893A4 (Chugai - priority 1994) that describes antibodies that bind directly to IL-6 alone (in claim 1 termed "interleukin-6 antagonist) and EP983767A1 (Chugai -priority 1997) that describes antibodies that act via binding to IL-6R as such (alone) to inhibit the binding of IL-6 to IL-6R.

Recently published WO2007/104529A2 (Ablynx N. V. - publication date 20

September 2007) nicely summarized the numerous prior art publication relating to antibodies that bind directly to IL-6 or IL-6R as such (see e.g. page 3, lines 13 to 25).

In essence, WO2007/104529A2 relates to "special" types of antibodies (e.g. nanobodies) that do the same job as earlier described (e.g. by Chugai) antibodies - i.e. binding directly to IL-6 as such (alone).

Nanobodies are known - nanobodies are a type of antibodies derived from camels, and are much smaller than traditional antibodies. In claims 1 to 6 of WO2007/104529A2 is the antibody (e.g. nanobody) binding directly to IL-6 alone defined as "directed against IL-6". See also claim 7 referring to any of claims 1 to 6 and giving specific binding dissociation constants (Kd) to IL-6 as such (alone).

In claim 4 of WO2007/104529A2 is said that the e.g. nanobody direct binding to IL-6 as such may modulate the interaction between IL-6/IL-6R complex and gpl30. On page 37, lines 3 - 25 are elaborated further on this modulation of IL-6/IL-6R complex interaction. Relevant parts of page 37 reads (emphasis added):

"In a second aspect, the invention provides amino acid sequences comprising ... Nanobodies that can bind to IL-6 in such a way that they can modulate the interaction between IL-6/IL-6R complex and gpl30. In the context of the present invention "modulating the interaction between IL-6/IL-6R complex and gpl30" can for example mean: binding to IL-6 (i.e. as such or as present in the IL-6/IL-6R complex) in such a way that the formation of the I L-6/IL-6R complex is inhibited ...; or binding to IL-6 (i.e. as such or as present in the IL-6/IL-6R complex) in such a way that the formation of the IL-6/IL-6R complex essentially is not affected but that the binding of said complex to gpl30 is modulated (e.g. inhibited).... In this aspect, amino acid sequences or Nanobodies according to the invention preferably compete with gpl30 for binding to either the gbl30 interaction site Il of IL-6 (or of the IL-6/IL-6R complex) or the gpl30 interaction site III of IL-6 (or of the IL-6/IL-6R complex)."

In short and in directly agreement with the rest of the description of

WO2007/104529A2 is on page 37 described that the nanobodies shall bind directly to IL-6 as such ("alone"). In other words, in line with the prior art antibodies these nanobodies may be characterized as nanobodies that can bind to IL-6 alone - i.e. before IL-6 is present in the IL-6/slL-6R complex.

As an overall summary of herein relevant related numerous antibody prior art, one may say that the prior art antibodies may be characterized as antibodies that can bind to IL-6 or IL-6R alone - i.e. before IL-6 or IL-6R are present in the IL- 6/slL-6R complex. In view of above, it is evident that such prior art antibodies will, in some way or the other, significantly affect the classical IL-6 signaling way as illustrated in figure 2.

EP1 148065A1 (Conaris AG - published 2001 ) describes a fusion molecule comprising two soluble gp130 (sgp130) molecules. In particular, an Fc fusion protein of soluble gp130 (sgp130Fc) was generated and shown in several cell culture models from Crohn's disease patients to specifically block IL-6 responses dependent on soluble IL-6R (transsignaling mechanism), whereas responses via the membrane bound IL-6R remain unaffected (classical IL-6 signaling).

At the filing date of the present application, the Conaris AG web-page essentially described sgp130Fc molecule as:

The fusion protein CR5/18 is a further development of the parental compound sgp130Fc with improved purifiability and enhanced biological activity. It is known to inhibit IL-6-induced transsignaling, whereas the classical IL-6-induced signal transduction is not affected. The molecule bears a significant therapeutic potential in experimental animal models of colitis, arthritis, colon cancer and asthma. The by Conaris described sgp130Fc related protein comprises two extracellular gp130 domains dimerized by an IgG-Fc molecule - see figure 4 for an illustration - figure 4 was downloaded from Conaris web-page.

Since sgp130Fc comprises the two extracellular soluble domains of gp130 the sgp130Fc may be said to be based on the part of the natural gp130 molecule that in vivo binds the IL-6/slL-6R complex.

SUMMARY OF THE INVENTION

The problem to be solved by the present invention is to provide an alternative antibody for treatment of e.g. chronic Inflammation.

The solution is based on a new antibody concept, wherein the antibody

(i): essentially only binds to the IL-6/slL-6R complex (thereby inhibiting the transsignaling mechanism as illustrated in figure 3); and

(ii) essentially does not bind to IL-6 or IL-6R alone - i.e. before IL-6 or IL-6R are present in the IL-6/slL-6R complex - thereby not affecting the classical IL-6 signaling way as illustrated in figure 2 in a clinical significant way.

As discussed above, for the last 20 years or more have been described numerous different herein relevant antibodies. As an overall summary of herein relevant related numerous antibody prior art, one may say that the prior art antibodies may be characterized as antibodies that can bind to IL-6 or IL-6R alone - i.e. before IL-6 or IL-6R are present in the IL-6/slL-6R complex. In view of above, it is evident that such prior art antibodies will, in some way or the other, significantly affect the classical IL-6 signaling way as illustrated in figure 2.

Accordingly for the last 20 years or more have there been developed numerous different antibodies with a functional characteristic (binds IL-6 or IL-6R alone), which are clearly different from the antibody of the present invention (does not bind IL-6 or IL-6R alone - binds only to the IL-6/slL-6R complex).

Without being limited to theory, the herein described new antibody concept is based on that once the IL-6/slL-6R complex is formed in vivo there will be created new epitope(s), which are not present in IL-6 or IL-6R alone - i.e. before IL-6 or IL-6R are present in the IL-6/slL-6R complex.

An example of such a new epitope may be an epitope created by a partial sequence of IL-6 and a partial sequence from IL-6R - i.e. the new epitope needs both IL-6 and slL-6R in order to be present - accordingly the epitope will not be present in IL-6 or SIL-6R alone.

Accordingly, a first aspect of the invention relates to a pharmaceutical composition comprising suitable pharmaceutical excipients and also comprising a human in vivo clinical effective amount of an antibody that under human physiological conditions (a): binds to human IL-6/slL-6R complex with a dissociation constant (Kd) of at least 10^"6 moles/liter; and

(b): inhibits the transsignaling pathway due to that when the antibody is bound to the IL-6/slL-6R complex the binding of the complex to human membrane bound gp130 is at least 10³ times less as compared to the binding of the same IL-6/slL-

6R complex without the antibody bound to the same gp130; and characterized by that the antibody essentially does not affect the classical IL-6 signaling pathway due to that the antibody:

(i) has an at least 10² times stronger binding dissociation constant (Kd) to the IL- 6/slL-6R complex as compared to the strongest binding dissociation constant

(Kd) to human IL-6 alone (as such) and to human slL-6R alone (as such).

The antibody as described herein must have an at least 10² times stronger binding dissociation constant (Kd) to the IL-6/slL-6R complex as compared to the strongest binding dissociation constant (Kd) to human IL-6 alone (as such) and human slL-6R alone (as such).

As understood by the skilled person this means that if for instance the antibody binding constant to the IL-6/slL-6R complex is e.g. 10^"8 the maximum strongest binding constant to any of human IL-6 alone and human slL-6R alone shall be less than 10^"6. Similarly, if for instance the antibody binding constant to the IL-6/slL-6R complex is e.g. 10^"10 the maximum strongest binding constant to any of human IL-6 alone and human slL-6R alone shall be less than 10^"8.

As evident to the skilled person the "logic" behind the functional definition of an antibody as described herein relates to the function of it and how skilled person can use it clinically. If for instance the antibody binding constant to the IL-6/slL-6R complex is very strong one only needs to administrate a relatively low dose to the patient and for such a relatively low dose one can accept some relatively "minor" binding to human IL-6 alone and human slL-6R alone without the antibody would clinically significantly affect the classical IL-6 signaling pathway.

Without being limited to theory it is believed that the fact that the novel antibody as described herein in vivo strongly binds to the human IL-6/slL-6R complex makes that it inhibits binding of the complex to human membrane bound gp130 due to e.g. steric hindrance. As can be seen on e.g. figure 3 the gp130 binding "pocket" is made to perfectly match the IL-6/slL-6R complex structure as such - accordingly that an antibody as described herein is also bound to the complex will make it too big to fit into the gp130 binding "pocket".

Alternatively the antibody as described herein may remove IL-6/slL-6R complex as such from the circulation.

Without being limited to theory it is believed that the relatively very low binding dissociation constants of the novel antibody as described herein to IL-6 and slL-6R as such (alone) will do that there is no in vivo clinical measurable effect of this low (virtually not existing) possible binding to IL-6 and slL-6R as such (alone) - in other words the antibody does not in a clinical measurable way affect the classical IL-6 signaling pathway.

As evident to the skilled person, if an antibody as described herein does not in a clinically measurable way bind to slL-6R this antibody will also not bind to membrane bound IL-6R - see e.g. figure 3 for an illustration.

In working example 1 herein is provided a detailed description of a method to screen for and thereby identify an antibody as described herein.

In working example 2 (ELISA) and example 3 (SPR) herein is provided a detailed description of how to measure the herein relevant antibody dissociation constants

(Kd).

In working example 4 herein is provided a detailed description of how to measure the inhibition of the transsignaling pathway due to significant less binding of the "antibody - IL-6/slL-6R complex" to human membrane bound gp130 - i.e. item (b) of first aspect.

Based on the detailed descriptions/instructions in examples 1 to 4 and the common knowledge of the skilled person it is routine work for the skilled person to identify a specific antibody of interest according to the first aspect of the invention plus to determine if this specific antibody of interest complies with the criteria of the first aspect - i.e. the given antibody binding dissociation constants etc. For instance, in the working examples is explicitly described how to make e.g. the relevant ligands:

(1 ): human IL-6/slL-6R complex; (2): human IL-6 alone (as such); and (3): human slL-6R alone (as such).

Further is explicitly described how to measure the given antibody binding dissociation constants under human physiological conditions.

As said above, it has been shown that transsignaling plays a key role in the pathophysiology of a chronic inflammatory disorders and most likely some cancer diseases.

Accordingly, a second aspect of the invention relates to a clinical effective amount of the pharmaceutical composition comprising the antibody of the first aspect for use in a method for treatment of a chronic inflammatory disorder or a cancer disease in a human person.

This second aspect may alternatively be formulated as a method for treatment of a chronic inflammatory disorder or a cancer disease in a human person comprising administrating to a human person a clinical effective amount of the pharmaceutical composition comprising the antibody of the first aspect.

A third aspect relates to a method for preparing a pharmaceutical composition comprising a human in vivo clinical effective amount of an antibody of the first aspect comprising following steps:

(i): screening/selecting for an antibody that fulfills the functional characteristics of the antibody of first aspect to identify an antibody that fulfills said functional characteristics;

(ii): producing the identified antibody of step (i) in relevant quantities; and (iii): incorporating the relevant quantities of the identified antibody into a pharmaceutical composition comprising suitable pharmaceutical excipients to get the pharmaceutical composition comprising a human in vivo clinical effective amount of an antibody of the first aspect.

An advantage of that an antibody as described herein does not bind in a clinical measurable way to IL-6 or slL-6R alone and thereby essentially does not affect the classical IL-6 signaling pathway is that when used for e.g. treatment of chronic inflammatory disorder the natural "good/positive" in vivo effect of the classical IL-6 signaling pathway is maintained in the patient. Examples of normal "good/positive" in vivo effect of this classical IL-6 signaling pathway are the positive involvement in e.g. early immune responses. Accordingly, one may say that an antibody as described herein gives less negative side-effects as compared to the numerous prior art described antibodies that are specifically directed to bind IL-6 or IL-6R as such (alone).

An advantage of an antibody as described herein over e.g. the fusion molecule comprising two soluble gp130 (sgp130) molecules as described in e.g.

EP1 148065A1 (Conaris AG - published 2001 ) is that an antibody is quite a small molecule as compared to sgp130. The molecule sgp130 is quite "big" - see e.g. EP1 148065A1 and figure 4 herein. A smaller molecule is generally easier to produce and it is also believed that e.g. the in vivo stability half-life of an antibody will generally be bigger than for sgp130.

DEFINITIONS:

Prior to a discussion of the detailed embodiments of the invention is provided a definition of specific terms related to the main aspects of the invention.

In general, all specific technical terms used herein shall be understood as the skilled person would understand them in the present technical context.

The term a pharmaceutical composition comprising "a human in vivo clinical effective amount of an antibody" shall be understood as an amount that is sufficient high to give a wanted clinical effect in a human after administration of the pharmaceutical composition to the human - e.g. a wanted clinical effect in relation to treatment of a chronic inflammatory disorder or a cancer disease. In the present context the skilled person routinely is able to adjust the amount of antibody to be administrated in order to get a wanted clinical effect.

The term "under human physiological conditions" of first aspect should be understood as the skilled person would understand it in the present context. The antibody as described herein is used for treatment of a human person.

Accordingly, the antibody as described herein shall comply with the criteria of the first aspect under human physiological conditions. The skilled person knows what that is - e.g. temperature at 37°C etc.

Embodiments of the present invention are described below, by way of examples only.

DRAWINGS

Figure 1 : Illustrates the IL-6/gp130 Cytokine family.

Figure 2: Illustrates the classical IL-6 Signaling pathway.

Figure 3: Illustrates the IL-6 Transsignaling pathway.

Figure 4: Illustrates the sgp130Fc protein.

DETAILED DESCRIPTION OF THE INVENTION

Pharmaceutical composition comprising suitable pharmaceutical excipients

As known to the skilled person a pharmaceutical composition comprises pharmaceutical acceptable excipients and/or carriers. "Pharmaceutically acceptable" is meant to encompass any excipient and/or carrier, which does not interfere with the effectiveness of the biological activity of the active ingredient and that is not toxic to the host to which is administered.

In a preferred embodiment the pharmaceutical composition is a composition for subcutaneous administration, intramuscular administration or intravenous injection. This is examples of preferred administration routes for treatment of a relevant disorder/disease as described herein.

For treatment of a relevant disorder/disease as described herein it is preferred to do it by systemic administration.

The pharmaceutical composition may comprises other antibody/antibodies that an antibody as described herein.

Disorders/diseases

The term "chronic inflammatory disorder" is well known to the skilled person and e.g. a doctor can routinely identify if a person/patient suffers from a chronic inflammatory disorder.

Chronic inflammation disorder (also known as chronic systemic inflammation) is an inflammatory immune response of prolonged duration that eventually leads to tissue damage. Chronic inflammation is differentiated from acute inflammation by extended duration, lasting anywhere from a week to an indefinite time frame. The exact nature of chronic inflammation depends on the causative agent and the body's attempts to ameliorate it.

As known to the skilled person, chronic inflammation may develop as a progression from acute inflammation if the original stimulus persists or after repeated episodes of acute inflammation. Examples of diseases that can cause chronic inflammation include tuberculosis, chronic cholecystitis, bronchiectasis, rheumatoid arthritis, Hashimoto's thyroiditis, inflammatory bowel disease (ulcerative colitis and Crohn's disease), silicosis and other Pneumoconiosis and an implanted foreign body in a wound among many others. A disease of this list is a preferred disease to be treated with an antibody as described herein. From this list it is preferred to treat inflammatory bowel disease, Crohn's disease or rheumatoid arthritis.

Generally speaking, an antibody as described herein may be used for treatment of at least one disease as listed below - even though one of below listed disease may not directly be a chronic inflammation disorder related disease or a cancer disease. An example of a disease to be treated with an antibody as described herein is at least one disease selected from the group consisting of: sepsis, various forms of cancer, multiple myeloma disease (MM), renal cell carcinoma (RCC), plasma cell leukaemia, lymphoma, B-lymphoproliferative disorder (BLPD), prostate cancer, bone resorption, osteoporosis, cachexia, psoriasis, mesangial proliferative glomerulonephritis, Kaposi's sarcoma, AIDS-related lymphoma, inflammatory diseases, rheumatoid arthritis, systemic onset juvenile idiopathic arthritis, hypergammaglobulinemia, Crohn's disease, ulcerative colitis, systemic lupus erythematosus (SLE), multiple sclerosis, Castleman's disease, IgM gammopathy, cardiac myxoma, asthma, allergic asthma and autoimmune insulin-dependent diabetes mellitus.

Antibody

The term "antibody" is well known to the skilled person.

The term "antibody" as used herein also comprises what in the art may be termed an antibody made of suitable antibody fragments - i.e. an antibody comprising or essentially consisting of an immunoglobulin variable domain (generally suitable light chain variable domain).

An example of an antibody may also be a nanobody.

Nanobodies are known -generally defined as nanobodies are a type of antibodies derived from camels, and are much smaller than traditional antibodies. See e.g. WO2007/104529A2 for further details to special suitable types of antibodies.

The antibody may be e.g. a rat or mouse antibody. However, since the antibody as described herein is used for treatment of a human it is preferred a humanized or human antibody. In a preferred embodiment the antibody is a monoclonal antibody (mAb), more preferably a humanized mAb and most preferably a human mAb.

Method to screen for and thereby identify an antibody as described herein

In this example 1 and in example 2 are in details provided all relevant technical details such as the relevant ligands - i.e.: (1 ): human IL-6/slL-6R complex; (2): human IL-6 alone (as such); and (3): human slL-6R alone (as such).

In the screening protocol of example 1 is screened for a mouse or rat antibody. However, the skilled person easily understands this is just an example of an antibody and exactly the same screening assay may be used to identify e.g. a human antibody with the characteristics as described herein.

In short, the screening assay is based on first identifying an antibody that binds to the human IL-6/slL-6R complex. Once a suitable pool of such antibodies have been identified then there is from this pool selected an antibody that essentially does not bind to human IL-6 alone (as such) and to human slL-6R alone (as such). As understood by the skilled person the strategy may also be "to the contrary" - i.e. first identify a pool of antibodies that essentially do not bind to human IL-6 alone (as such) and to human slL-6R alone (as such) and then from this pool identify an antibody that binds to the human IL-6/slL-6R complex.

Herein relevant antibody dissociation constants (Kd)

In working example 2 (ELISA) and example 3 (SPR) herein is provided a detailed description of how to measure the herein relevant antibody dissociation constants (Kd). Based on the detailed instructions of these examples and the common knowledge of the skilled person it is routine work for the skilled person to determine if a specific antibody of interest complies with the given antibody binding dissociation constants as described herein.

In the present context it is evident that it is preferred to have a strong binding to the human IL-6/slL-6R complex [item (a) of first aspect] and a very weak or no measurable binding to human IL-6 alone (as such) and to human slL-6R alone (as such) [item (i) of first aspects].

Accordingly, in a preferred embodiment the antibody

(a): binds to human IL-6/slL-6R complex with a dissociation constant (Kd) of at least 10^"8 moles/liter, more preferably the antibody

(a): binds to human IL-6/slL-6R complex with a dissociation constant (Kd) of at least 10^"10 to moles/liter, even more preferably the antibody

(a): binds to human IL-6/slL-6R complex with a dissociation constant (Kd) of at least 10^"12 to moles/liter.

Within being limited to theory it is believed that it would be difficult to make an antibody that binds to human IL-6/slL-6R complex with a dissociation constant (Kd) stronger than 10^"20 to moles/liter.

Similar, in a preferred embodiment the antibody (i) has an at least 10³ times stronger binding dissociation constant (Kd) to the IL-

6/slL-6R complex as compared to the strongest binding dissociation constant (Kd) to human IL-6 alone (as such) and to human slL-6R alone, more preferably the antibody

(i) has an at least 10⁵ times stronger binding dissociation constant (Kd) to the IL- 6/slL-6R complex as compared to the strongest binding dissociation constant

(Kd) to human IL-6 alone (as such) and to human slL-6R alone, even more preferably the antibody

(i) has an at least 10⁷ times stronger binding dissociation constant (Kd) to the IL- 6/slL-6R complex as compared to the strongest binding dissociation constant (Kd) to human IL-6 alone (as such) and to human slL-6R alone, most preferably

(i) has an at least 10⁹ times stronger binding dissociation constant (Kd) to the IL-

6/slL-6R complex as compared to the strongest binding dissociation constant

(Kd) to human IL-6 alone (as such) and to human slL-6R alone.

Similar, in a preferred embodiment the antibody

(ia) binds to human IL-6 alone (as such) with a dissociation constant (Kd) which is less than 10^"4 moles/liter and

(ib): binds to human slL-6R alone (as such) with a dissociation constant (Kd) which is less than 10^"4 moles/liter, more preferably the antibody

(ia) binds to human IL-6 alone (as such) with a dissociation constant (Kd) which is less than 10^"3 moles/liter and

(ib): binds to human slL-6R alone (as such) with a dissociation constant (Kd) which is less than 10^"3 moles/liter, even more preferably the antibody

(ia) binds to human IL-6 alone (as such) with a dissociation constant (Kd) which is less than 10^"2 moles/liter and

(ib): binds to human slL-6R alone (as such) with a dissociation constant (Kd) which is less than 10^"2 moles/liter, most preferably

(ia) binds to human IL-6 alone (as such) with a dissociation constant (Kd) which is less than 10^"1 moles/liter and

(ib): binds to human slL-6R alone (as such) with a dissociation constant (Kd) which is less than 10^"1 moles/liter.

Antibody as described herein - inhibition of the transsignaling pathway

Once a specific antibody of interest has been identified it is tested for its capacity to inhibit the transsignaling pathway.

Without being limited to theory it is believed that the fact that the novel antibody as described herein in vivo binds to the human IL-6/slL-6R complex makes that it inhibits binding of the complex to human membrane bound gp130 due to e.g. steric hindrance.

In the present context it is evident that it is preferred to have a strong inhibition of the transsignaling pathway.

Accordingly, in a preferred embodiment the antibody

(b): inhibits the transsignaling pathway due to that when the antibody is bound to the IL-6/slL-6R complex the binding of the complex to human membrane bound gp130 is at least 10⁵ times less as compared to the binding of the same IL-6/slL- 6R complex without the antibody bound to the same gp130; more preferably the antibody

(b): inhibits the transsignaling pathway due to that when the antibody is bound to the IL-6/slL-6R complex the binding of the complex to human membrane bound gp130 is at least 10⁷ times less as compared to the binding of the same IL-6/slL- 6R complex without the antibody bound to the same gp130.

EXAMPLES

Example 1 : Monoclonal production and screening

The methods described below are based on protocols describe in "Antibodies: A Laboratory Manual" by Harlow and Lane: ISBN 0-87969-314-2 1988 edition, referred to as Harlow and Lane with the page number in the text.

Antigen preparation

Recombinant IL-6 (Catalogue number 206-IL) and recombinant soluble IL-6 receptor (Catalogue number 227-SR) are available from R+D Systems (614 McKinley Place NE, Minneapolis, MN 55413) and other suppliers including Peprotech. Different forms of the antigen were prepared in order to optimize the production of antibodies in mice. In one example, IL-6 and soluble IL-6 receptor are separated from excipients by gel permeation chromatography and then mixed in an equimolar ratio in order to promote the spontaneous formation of a stoicheiometric complex due to the high binding affinity of IL-6 for its receptor. This material is made and used immediately for immunization. A second example is where the complex is made as described in phosphate buffered saline solution (PBS) and then cross-linked with gluteraldehyde using a 0.2% solution (Harlow and Lane; pg 78). Excess reagent is removed by dialysis overnight against PBS. A third approach is where IL-6 and soluble IL-6 receptor are recombinantly expressed as a covalently linked complex as described ('hyper IL-6'; ref 5). Alternatively, other cross-linking reagents can be used (Harlow and Lane; 130-131 ).

Immunisation

Mice and rats are immunized using a number of different protocols as described by Harlow and Dale (Chapter 5). In one protocol mice are immunized by intraperitoneal injection with adjuvant (Harlow and Dale; pg 158 onwards). An alternative is to inject subcutaneously (Harlow and Dale; pg 164). The injection schedule comprises an initial injection, using complete Freund's adjuvant, followed by a booster injection, in incomplete Freund's adjuvant, after two weeks. The concentration of antibodies to the target compound are then measured after ten days, by drawing a blood sample, and if the antibody response is not strong enough a further booster injection of antigen is given. This is repeated for up to three booster injections until a strong antibody response is observed. Animals showing a strong response are then given a final booster, by intravenous injection, and then three days later the spleen taken for hybridoma production. Harlow and Dale (pg 151 ) describes a typical immunization schedule using intraperitonal injection but subcutaneous injection is more appropriate since the amounts of antigen used are small (5 to 20micrograms antigen). A number of different studies are run using mice or rats, soluble complex, cross-linked complex or recombinant fusion protein as antigen. At least five animals are used in each experiment in order to increase the chances of obtaining a high affinity antibody with specificity for the complex. Experiments are repeated until a suitable antibody is obtained from a hybridoma. Screening of animals for antibody production

Animals making the required antibody, with higher affinity for the IL-6/soluble IL-6 receptor complex compared to the individual components, are identified by enzyme- linked immuno-absorbant assays (ELISA). The basic format for the ELISA screening assays are the same. A 96 well plate is coated with a solution of target antigen at an alkaline pH and then excess antigen is washed off. Next further binding sites on the ELISA plate are blocked with excess of an unrelated protein, for example albumin or fish gelatin, or by including 1 to 5% of non-ionic detergent in all solutions. The plate is then exposed to a range of dilutions of the test material; animal serum if screening for antibody formation or cell culture supernatant if screening hybridomas made from the spleen cells of immunized animals. After further washing is a suitable buffer, containing non-ionic detergent, the level of bound antibody is measured using a detection reagent comprising a rabbit antibody to mouse IgG which has been fused with an enzyme, for example horse raddish peroxidase. The plate is then washed again and the quantity of bound antibody visualized using a chromogenic substrate such as tetramethylbenzidine. Harlow and Dale describe the principles and practice of antibody-capture ELISA assays from pg 565 onwards.

In order to optimize the screening process a number of different ELISA formats are developed. Animals immunized with the IL-6/soluble IL-6 receptor complex and cross-linked complex are screened against soluble complex, cross-linked complex or fusion protein. Animals screened with the fusion protein are screened against IL-6/ soluble IL-6 receptor complex and cross-linked complex but not fusion protein since the response to the linker protein can predominate over the desired response to the complex itself.

In a second format plates are coated with IL-6 in order to eliminate animals that produce a high response to IL-6 but not to the complex. Likewise in a third format plates are coated with soluble IL-6 receptor only in order to eliminate animals that produce a high response to the receptor but not the complex.

The screening process can be summarized as follows based on the observation that antibodies with a high binding affinity to the target protein will give a signal at higher dilution compared to antibodies with a lower binding affinity.

Hybridoma production

The spleens are removed from animals showing the production of antibodies to IL-6/ soluble IL-6 receptor complex. Lymphocytes are extracted mechanically and fused to myeloma cells by spinning through 30% PEG (Harlow and Lane; pg 212). Pools of hybridomas are screened using the three ELISAs previously described for screening for antibody production in animals. However, in this case it is necessary to select clones that bind only IL-6/soluble receptor complex at higher dilution. In order not to miss any appropriate clones pools of clones were selected based on reactivity to the complex at a ten-fold higher dilution compared to IL-6 alone or soluble IL-6 receptor.

Selected pools are then subjected to limiting dilution and a further round of screening with ELISA in order to select single clones expressing antibodies to IL-6/ soluble IL-6 receptor (Harlow and Lane: pg 222-223).

A selection of clones is made based on a higher affinity (binding at higher dilution or with a lower dissociation constant) to the IL-6/soluble receptor complex, compared to either the IL-6 alone or the soluble IL-6 receptor alone. These clones are expanded to provide cells for banking (Harlow and Lane; pg 258) and supernatant to allow further characterisation of the individual antibodies.

Conclusions

As understood by the skilled person, by performing the screening protocol of this example one can routinely identify an antibody that binds to the human IL-6/slL-6R complex but essentially does not bind human IL-6 alone (as such) and human slL- 6R alone (as such).

Example 2: Screening by ELISA of hybridoma supernatants and purified monoclonals for binding to IL-6/ soluble IL-6 receptor complex, IL-6 and IL-6 soluble receptor alone.

1.0 Enzyme-linked immunosorbant assay (ELISA) for characterization of monoclonal antibodies against IL-6R, IL-6 and IL-6R-IL-6 complexes. The ELISA entails the following steps:

IL-6R-IL-6 complexes

1. Coating of the wells of a micro titer plate with purified, IL-6R (capture antigen).

2. Blocking of non-specific sites; 3. Binding of IL-6 to the IL-6R;

4. Removal of non-bound proteins by washing of the wells;

5. Distribution of mAb's test samples and controls into the micro titer plate wells;

6. Binding of rabbit anti-mouse antibody-horseradish-peroxidase conjugate to the IL-6R-IL-6-mAb antigen complexes;

7. Removal of non-bound proteins by washing of the wells;

8. Addition of substrates hydrogen peroxide and ABTS.

9. Incubation of the titer plate and measurement of the specific absorbance of oxidized ABTS, 10. Data processing and determination of test sample binding degree based on the positive control and the background.

IL-6R 1. Coating of the wells of a micro titer plate with purified, IL-6R

(capture antigen).

2. Blocking of non-specific sites;

3. Removal of non-bound proteins by washing of the wells;

4. Distribution of mAb's test samples and controls into the micro titer plate wells;

5. Binding of rabbit anti-mouse antibody-horseradish-peroxidase conjugate to the IL-6R-mAb antigen complexes;

6. Removal of non-bound proteins by washing of the wells;

7. Addition of substrates hydrogen peroxide and ABTS. 8. Incubation of the titer plate and measurement of the specific absorbance of oxidized ABTS,

9. Data processing and determination of test sample binding degree based on the positive control and the background.

IL-6

1. Coating of the wells of a micro titer plate with purified, IL-6 (capture antigen).

2. Blocking of non-specific sites;

3. Removal of non-bound proteins by washing of the wells; 4. Distribution of mAb's test samples and controls into the micro titer plate wells;

5. Binding of rabbit anti-mouse antibody-horseradish-peroxidase conjugate to the IL-6-mAb antigen complexes;

6. Removal of non-bound proteins by washing of the wells; 7. Addition of substrates hydrogen peroxide and ABTS.

8. Incubation of the titer plate and measurement of the specific absorbance of oxidized ABTS,

9. Data processing and determination of test sample binding degree based on the positive control and the background. 2.0 MATERIALS AND REAGENTS

The reagent stock solutions required for the ELISA are detailed in SOP 04-67-180 and equivalents may be substituted accordingly to availability. In addition, the following reagents are specifically required for the detection of IL-6R-IL-6 complexes.

2.1 Human IL-6R

Human IL-6R from Peprotech catalogue No. 200-06R or equivalent.

2.2 Human IL-6

Human IL-6 from Peprotech catalogue No. 200-06 or equivalent.

2.3 Rabbit-Anti-Mouse-lgG/M HRP conjugated Antibodies From Jackson catalogue No. 315-035-048 or equivalent.

2.4 Mouse anti Human IL-6 mAb

Mouse anti Human IL-6 from Peprotech catalogue No 500-M06.

2.5 Mouse anti Human IL-6R mAb

Mouse anti Human IL-6R from Serotec catalogue No MCA822.

3.0 ELISA PROCEDURE IL-6R-IL-6 COMPLEXES The ELISA is performed according to the general ELISA procedure detailed in SOP 04-67-180 version 4, with the additions/changes noted below:

3.1 Well Coating with IL-6R

3.1.1 Dilute the I L-6R (1 mg/ml) to a concentration of 10 g/ml in carbonate- bicarbonate buffer (pH 9.6). Dispense 100 I per well, shake for 1 h at 37⁰C and Incubate 1 h at 4°C.

3.2 Plate Blocking with BSA in PBS 3.2.1 Discard the fluid from the wells and wash the wells four times with 300 I solution containing PBS and 0.05% Tween-20 (PBST). Invert the plate at each step and blot dry on paper towels. Alternatively, wash the plate with an automatic plate washer.

3.2.2 Dispense 200 I PBS containing 3% BSA into each well.

3.2.3 Seal the plate with plastic lid and incubate with gentle shaking for 1 h at 37⁰C.

3.2.4 Wash the plate as in 3.2.1.

3.3 IL-6R-IL-6 Complex

3.3.1 Dilute the I L-6 (1 mg/ml) to a concentration of 10 g/ml in PBS-1 % BSA. Dispense 100 I per well, shake for 1 h at 37⁰C and incubate 1 h at 4°C.

3.3.2 Wash the plate as in 3.2.1.

3.4 Monoclonal antibody test samples and controls

Dilute the samples 1 :10, 1 :100 and 1 :1000 by adding 50 I of sample to 450 I of PBS-1 % BSA respectively.

3.4.1 Dilute the commercial mouse anti-IL-6 mAb 1 :10, 1 :100 and 1 :1000 by adding 50 I of sample to 450 I of PBS-1 % BSA respectively.

3.4.2 Incubate the plates for 2 hours at 37°C with shaking and 1 h at 4°C.

3.4.3 Wash the plate four times with PBST.

3.5 Rabbit Anti-Mouse HRP-Conjugated Antibody

3.5.1 Dilute the rabbit anti-Mouse-HRP conjugated antibody 1 :2000 with PBST- 1 % BSA. 3.5.2 Dispense 100 I per well.

3.5.3 Incubate the plate for 1 hour at 37°C, with shaking.

3.5.4 Wash the plate four times with PBST.

3.6 Activation of HRP Conjugate

3.6.1 Dispense 100 μl ABTS substrate solution per well.

3.6.2 Incubate the plate for 0.5 hour at 37°C and monitor the optical density (OD) at 405 nm using the ELISA plate reader. When the OD of the highest positive control concentration is between 1.0 to 1.5, stop the reaction by adding 100 I per well of HRP stop solution. Measure the absorbance of the wells using the current Multicalc protocol (4PL transformation) for the detection of monoclonal antibodies against IL-6R-IL-6 complex samples.

4.0 ELISA PROCEDURE IL-6R The ELISA is performed according to the general ELISA procedure detailed in SOP 04-67-180 version 4, with the additions/changes noted below:

4.1 Well Coating with IL-6R

4.1.1 Dilute the I L-6R (1 mg/ml) to a concentration of 10 g/ml in carbonate- bicarbonate buffer (pH 9.6). Dispense 100 I per well, shake for 1 h at 37⁰C and Incubate 1 h at 4°C.

4.2 Plate Blocking with BSA in PBS

4.2.1 Discard the fluid from the wells and wash the wells four times with 300 I solution containing PBS and 0.05% Tween-20 (PBST). Invert the plate at each step and blot dry on paper towels. Alternatively, wash the plate with an automatic plate washer. 4.2.2 Dispense 200 I PBS containing 3% BSA into each well.

4.2.3 Seal the plate with plastic lid and incubate with gentle shaking for 1 h at 37⁰C.

4.2.4 Wash the plate as in 4.2.1.

4.3 Monoclonal antibody test samples and controls Dilute the samples 1 :10, 1 :100 and 1 :1000 by adding 50 I of sample to

450 I of PBS-1 % BSA respectively.

4.3.1 Dilute the commercial mouse anti-IL-6R mAb 1 :10, 1 :100 and 1 :1000 by adding 50 I of sample to 450 I of PBS-1 % BSA respectively.

4.3.2 Incubate the plates for 2 hours at 37°C with shaking and 1 h at 4°C.

4.3.3 Wash the plate four times with PBST.

4.4 Rabbit Anti-Mouse HRP-Conjugated Antibody

4.4.1 Dilute the rabbit anti-Mouse-HRP conjugated antibody 1 :2000 with PBST- 1 % BSA.

4.4.2 Dispense 100 I per well.

4.4.3 Incubate the plate for 1 hour at 37°C, with shaking.

4.4.4 Wash the plate four times with PBST.

4.5 Activation of HRP Conjugate

4.5.1 Dispense 100 μl ABTS substrate solution per well. 4.5.2 Incubate the plate for 0.5 hour at 37°C and monitor the optical density (OD) at 405 nm using the ELISA plate reader. When the OD of the highest positive control concentration is between 1.0 to 1.5, stop the reaction by adding 100 I per well of HRP stop solution. Measure the absorbance of the wells using the current Multicalc protocol (4PL transformation) for the detection of monoclonal antibodies against IL-6R samples.

5.0 ELISA PROCEDURE IL-6 The ELISA is performed according to the general ELISA procedure detailed in SOP 04-67-180 version 4, with the additions/changes noted below:

5.1 Well Coating with IL-6R

5.1.1 Dilute the IL-6 (1 mg/ml) to a concentration of 10 g/ml in carbonate- bicarbonate buffer (pH 9.6). Dispense 100 I per well, shake for 1 h at 37⁰C and Incubate 1 h at 4°C.

5.2 Plate Blocking with BSA in PBS

5.2.1 Discard the fluid from the wells and wash the wells four times with 300 I solution containing PBS and 0.05% Tween-20 (PBST). Invert the plate at each step and blot dry on paper towels. Alternatively, wash the plate with an automatic plate washer.

5.2.2 Dispense 200 I PBS containing 3% BSA into each well.

5.2.3 Seal the plate with plastic lid and incubate with gentle shaking for 1 h at 37⁰C.

5.2.4 Wash the plate as in 5.2.1.

5.3 Monoclonal Antibody Test Samples and Controls Dilute the samples 1 :10, 1 :100 and 1 :1000 by adding 50 I of sample to 450 I of PBS-1 % BSA respectively.

5.3.1 Dilute the commercial mouse anti-IL-6 mAb 1 :10, 1 :100 and 1 :1000 by adding 50 I of sample to 450 I of PBS-1 % BSA respectively.

5.3.2 Incubate the plates for 2 hours at 37°C with shaking and 1 h at 4°C.

5.3.3 Wash the plate four times with PBST.

5.4 Rabbit Anti-Mouse HRP-Conjugated Antibody

5.4.1 Dilute the rabbit anti-Mouse-HRP conjugated antibody 1 :2000 with PBST- 1 % BSA.

5.4.2 Dispense 100 I per well.

5.4.3 Incubate the plate for 1 hour at 37°C, with shaking.

5.4.4 Wash the plate four times with PBST.

5.5 Activation of HRP Conjugate

5.5.1 Dispense 100 μl ABTS substrate solution per well.

5.5.2 Incubate the plate for 0.5 hour at 37°C and monitor the optical density (OD) at 405 nm using the ELISA plate reader. When the OD of the highest positive control concentration is between 1.0 to 1.5, stop the reaction by adding 100 I per well of HRP stop solution. Measure the absorbance of the wells using the current Multicalc protocol (4PL transformation) for the detection of monoclonal antibodies against IL-6 samples.

6.0 ACCEPTANCE CRITERIA FOR THE TEST RESULTS 6.1 Positive reaction (in a dose response manner) of the commercial mAb's positive controls to the specific antigens.

Example 3: Measurement of the affinity of selected monoclonal antibodies by surface plasmon residence (SPR).

A selection of clones is further characterized in terms of their relative binding affinities for the IL-6/soluble IL-6 receptor complex compared to IL-6 alone and IL-6 soluble receptor alone.

The relevant ligands - i.e.:

(1 ): human IL-6/slL-6R complex;

(2): human IL-6 alone (as such); and (3): human slL-6R alone (as such). are the same as used in examples above.

Such characterisation is performed by ELISA (see Example 2 above) or using surface plasmon resonance (SPR). SPR is also capable of measuring the binding and dissociation rates of the antibody and antigen complex and the effects of pH and buffer concentration. These parameters are all used in identifying the most suitable candidates for therapeutic antibodies.

SPR is the basis for detecting binding of proteins with other surface-bound proteins in the Biocore instrument (www.Biacore.com). The principles of operation are well described in Biocore Technical Notes, Biocore methods and in the scientific literature.

Step 1. It is first necessary to immobilize a capture antibody to the surface of a detector chip. A variety of chemistries are used for this most typically coupling via amide groups to a carboxy-methyl-derivatised surface. A goat polyclonal antibody prepared to mouse IgG is used or other antibodies, such as monoclonal antibodies, with similar binding characteristics. Once the capture antibody is immobilized excess binding groups are blocked and excess reagents removed by washing. Step 2. A candidate monoclonal is then flowed over the capture antibody-modified chip either directly from the hybridoma supernatant or after cleaning up by protein A capture and elution. The binding until saturation of the candidate monoclonal is followed by the change in signal which increases as the monoclonal is progressively captured and peaks as all binding sites are filled. The chip is then washed with buffer until a stable signal is achieved. At this stage, if desired, the bound monoclonal can be chemically-cross-linked to the capture antibody to facilitate repetitive cycles of antigen binding and release. Excess reagents are then washed away to prepare the chip for the start of binding affinity measurements. Step 3. Binding for IL-6 alone is measured by flowing a dilute solution of IL-6 in buffer over sensor chip. Binding occurs up to a maximum signal determined by the concentration of reagents bound to the chip. The rate at which the maximum signal is reached is a measure of the binding affinity, known as the 'on-rate'. Subsequently the flow of antigen solution is replaced by buffer alone. Now the rate of decrease of the signal, as the antigen is washed away, is a measure of the unbinding of the antigen, known as the 'off-rate'. The dissociation constant for an individual monoclonal is determined by the ratio of the off-rate divided by the 'on-rate'. Step 4. The binding of the soluble IL-6 receptor alone is determined on the same chip or using a similar chip made under identical conditions. Again the on and off rates and the dissociation constant are measured and determined. Step 5. The binding characteristics of the same monoclonal for the IL-6/soluble IL-6 receptor are determined by flowing a dilute solution of pre-formed IL-6/soluble IL-6 receptor complex over the antibody or with a solution of the recombinant covalently- linked IL-6/soluble IL-6 receptor complex.

The selection of monoclonal candidates for further development is based on a higher binding affinity (smaller dissociation constant) for the IL-6/soluble IL-6 receptor complex compared to either IL-6 or soluble IL-6 receptor alone. In general a selected candidate monoclonal has a 100 to 1000 higher affinity for the complex over the individual components. In general existing therapeutic monoclonal antibodies have dissociation constants in the range 10^"7 to 10^"12. The final choice of monoclonal also depends on the relative values of the 'on' and 'off' rates for the complex compared to the individual components.

Conclusions Based on the detailed instructions in this example 3 [surface plasmon resonance (SPR)] and example 2 above (ELISA) and the common knowledge of the skilled person it is routine work for the skilled person to determine if a specific antibody of interest complies with the given antibody binding dissociation constants as described herein.

Example 4: Assay of IL-6/soluble IL-6 receptor binding activity in vitro activity

The biological activity of monoclonal antibodies with selected affinities for the IL- 6/soluble IL-6 receptor complex are screened in vitro against a BAF 3 cell line transfected with the natural binding receptor for the complex know variously as the interleukin 6 signal transducer and gp130 (Genebank accession number: M57230). The BAF3 cell line is normally dependent on an external supplementation of IL-3 for growth. Once transformed with the gp130 receptor growth can be supported by the IL-6/soluble IL-6 receptor complex usually in the form of the convalently-linked 'hyper IL-6' (ref 5).

Construction of a BAF3 cell line expressing the gp130 receptor protein

Molecular Biology

The complementary DNA (cDNA) encoding the full open reading frame, complete with initiation and stop codons for gp130 is either cloned by standard molecular biology techniques (reverse transcription of messenger RNA and Polymerase Chain Reaction (PCR)) or obtained from a commercial source (Invitrogen: www.invitrogen.com). Alternatively the gene was constructed synthetically according to the accession number M57230 by a contractor (for example NextGen: nextgensciences.com).

Restriction enzyme sites were added to the cDNA by PCR and sub-cloned into a multiple cloning site of an expression vector by standard molecular biology techniques. For example the vector can contain a cytomegalovirus (CMV) promoter and bovine polyadenylation as with the pcDNA series (Invitrogen). The vector also carries selection markers for cell line selection (neomycin, hygromycin, zeocin).

Vectors containing the insert are selected and amplified in preparation for transfection in a modified K12 E. CoIi strain. Vector DNA is purified to transfection quality using a plasmid purification system which also removes endotoxin (UltraMobius kit from Merck biosciences: merckbiosciences.com)Merck Biosciences).

This DNA is sequenced to publication quality to verify the insert prior to transfection.

Cell line generation

The BAF3 parent cell line is available from DSMZ (ACC 300: Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH: www.dsmz.de) are grown in appropriate media to 50-60% confluence depending on the cell line in a T75 flask

(BD Falcon: bdbiosciences.com) at 37⁰C, 5% CO₂ in a humidified incubator. Cells are transfected with purified vector DNA (1 - 5 μg,) according to manufacturers' instructions. Typical reagents include liposome based such as Lipofectamine (Invitrogen), Effectene (Qiagen: www.qiagen.com) or a protein based carrier,

Genejuice (Merck Biosciences). At the same time a sham transfection is carried out as a control for the selection process.

The transfected cells are incubated for 2 days at 37⁰C, 5% CO₂ then the selection marker added. The working range is calculated a kill curve using cells transfected with the parent vector lacking the insert. At least three parallel transfections are carried out and a range of concentrations of selection marker used according to the kill curve. For example with G418 the concentrations typically used are 100 μg/ml, 200 μg/ml and 400 μg/ml.

When the cells reach confluence they are detached from the flask using 5 ml Versene (Invitrogen) and a dilution made using cell media (containing the appropriate concentration of selection marker), for example 1 in 50 - 100100 μg/ml. This dilution (500 μl) is aliquoted into a 24 well plate (BD Falcon) and incubated at 37⁰C, 5% CO₂ for 2 - 5 days. When colonies were visible (1-2 mm in diameter), single colonies are passaged into 6 well plates. When these wells are confluent they are detached with 1 ml Versene and transferred to T75 flasks and grown to confluence. These cells are expanded for testing and cryopreservation.

Ce// line analysis

Expression of the receptor is first tested at the confluent 6 well plate stage. A sample of the cells (100 μl) is taken when detached for transfer to the T75. The cells are lysed by boiling for 5 minutes in 1x LDS sample buffer (141 mM Tris base, 2% LDS, 10% Glycerol,0.51 mM EDTA,0.22 mM SERVA Blue G250, 0.175 mM phenol Red, pH 8.5). A pre-cast SDS PAGE gel is run and transferred to supported nitrocellulose according to the manufacturers' instructions. The receptor was then visualised using a selective antibody (goat anti-gp130: Santa Cruz Biotechnology: www.scbt.com) and compared to the parent line for protein expression. Cell lines are deselected on the basis of no expression. Cells that express some protein are continued.

Alternatively the cells are tested at this stage using reverse transcription PCR (RT PCR). At the same timepoint RNA may be prepared from the cells using RNeasy columns (Qiagen), transcribed to cDNA and receptor message amplified using primers designed to a small region of the receptor (200-300 bp) and standard molecular biology techniques.

Functional assays are carried out at the expanded T75 timepoint. Cell lines are selected by comparison of the lines, looking for a fully efficacious dose response to the native ligand, with a hill slope near or equal to one over the established time frame of the assay.

Biological assay for monoclonal antibody functionality.

The biological assay for a selection of monoclonal antibodies measures the concentration needed to fully sequester IL-6/soluble IL-6 receptor and thus withdraw the growth stimulus to the gp130 transfected BAF3 cells. The cells must be grown in the presence of IL-6 premixed with soluble IL-6 receptor. Fixed numbers of cells are exposed to a range of concentrations of a selected monoclonal antibody and after a period of growth the number of viable cells, compared to cells which are not exposed to the same monoclonal, are measured by a cell proliferation assay (example: ViaLight Plus from Lonza: www.lonza.com. The method is based on the publication by Jostock (ref 6).

The biological assay reflects the affinity of the monoclonal antibody as determined by ELISA and SPR (BiaCore). Antibodies with a high affinity for the complex, compared to IL-6 alone or soluble IL-6 receptor alone, will inhibit gp130/BAF3 cells at lower concentration in the media compared to low affinity monoclonal antibodies.

Conclusions

Based on the detailed instructions in this example and the common knowledge of the skilled person it is routine work for the skilled person to determine if a specific antibody of interest complies with the inhibition of the transsignaling pathway, as described herein, due to significant less binding of the "antibody - IL-6/slL-6R complex" to human membrane bound gp130 - i.e. item (b) of first aspect.

REFERENCE LIST

1 : EP783893A4 (Chugai - priority 1994) 2: EP983767A1 (Chugai -priority 1997) 3: WO2007/104529A2 (Ablynx N. V) 4: EP1148065A1 (Conaris AG - published 2001 )

Claims

1 : A pharmaceutical composition comprising suitable pharmaceutical excipients and also comprising a human in vivo clinical effective amount of an antibody that under human physiological conditions

(a): binds to human IL-6/slL-6R complex with a dissociation constant (Kd) of at least 10^"6 moles/liter; and

(Kd) to human IL-6 alone (as such) and to human slL-6R alone (as such).

2. The pharmaceutical composition of claim 1 , wherein it is a composition for subcutaneous administration, intramuscular administration or intravenous injection.

3. The pharmaceutical composition of claims 1 or 2, wherein the antibody is a nanobody or a monoclonal antibody (mAb).

4. The pharmaceutical composition of claim 3, wherein the monoclonal antibody is a human monoclonal antibody (mAb).

5. The pharmaceutical composition of any of the preceding claims, wherein the antibody

(i) has an at least 10⁷ times stronger binding dissociation constant (Kd) to the IL- 6/slL-6R complex as compared to the strongest binding dissociation constant

(Kd) to human IL-6 alone (as such) and to human slL-6R alone.

6. The pharmaceutical composition of claim 5, wherein the antibody

(a): binds to human IL-6/slL-6R complex with a dissociation constant (Kd) of at least 10^"10 to moles/liter and

(ib): binds to human slL-6R alone (as such) with a dissociation constant (Kd) which is less than 10^"2 moles/liter.

7. The pharmaceutical composition of any of the preceding claims, wherein the antibody

8. A clinical effective amount of the pharmaceutical composition comprising the antibody of any of claims 1 to 7 for use in a method for treatment of a chronic inflammatory disorder or a cancer disease in a human person.

9. The clinical effective amount of the pharmaceutical composition 8, wherein it is used for treatment of a chronic inflammatory disorder related disease selected from the group of diseases consisting of: tuberculosis, chronic cholecystitis, bronchiectasis, rheumatoid arthritis, Hashimoto's thyroiditis, inflammatory bowel disease, ulcerative colitis, Crohn's disease and silicosis.

10. A method for preparing a pharmaceutical composition comprising a human in vivo clinical effective amount of an antibody of any of claims 1 to 7 comprising following steps:

(i): screening/selecting for an antibody that fulfills the functional characteristics of the antibody of claim 1 to identify an antibody that fulfills said functional characteristics;

(ii): producing the identified antibody of step (i) in relevant quantities; and (iii): incorporating the relevant quantities of the identified antibody into a pharmaceutical composition comprising suitable pharmaceutical excipients to get the pharmaceutical composition comprising a human in vivo clinical effective amount of an antibody of any of claims 1 to 7.