EP2091974A1

EP2091974A1 - Polypeptides specific for complexes involved in receptor-mediated signaling, such as the il-6/il-6 receptor complex

Info

Publication number: EP2091974A1
Application number: EP07857366A
Authority: EP
Inventors: Els Anna Alice Beirnaert; Joost Alexander Kolkman; Hendricus Renerus Jacobus Matteus Hoogenboom
Original assignee: Ablynx NV
Current assignee: Ablynx NV
Priority date: 2006-12-13
Filing date: 2007-12-11
Publication date: 2009-08-26
Also published as: WO2008071685A1; CA2672386A1; JP2010512734A; AU2007331596A1

Abstract

The present invention relates to polypeptides that can be used to prevent or treat diseases and disorders relating to and/or associated with interleukin-6 ('IL-6'), with the interleukin-6 receptor ('IL-6R') and/or with the IL-6/IL-6R complex.

Description

Polypeptides specific for complexes involved in receptor -mediated signaling, such as the IL-6/IL-6 receptor complex

The present invention relates to polypeptides that can be used to prevent or treat diseases and disorders relating to and/or associated with interleukin-6 ("IL-6"), with the interleukin-6 receptor ("IL-6R") and/or with the IL-6/IL-6R complex.

In particular, the present invention relates to polypeptides that can be used to modulate the signaling pathway(s) and/or the biological functions and responses in which interleukin-6 ("IL-6"), the interleukin-6 receptor ("IL-6R") and/or the IL-6/IL-6R complex are involved.

More in particular, the present invention relates to polypeptides that can be used to modulate the interaction between the IL-6/IL-6R complex and gpl30.

The invention also relates to nucleic acids encoding such polypeptides; to methods for preparing such polypeptides; to host cells expressing or capable of expressing such polypeptides; to compositions, and in particular to pharmaceutical compositions, that comprise such polypeptides, nucleic acids and/or host cells; and to uses of such polypeptides, nucleic acids, host cells and/or compositions, in particular for prophylactic, therapeutic or diagnostic purposes, such as the prophylactic, therapeutic or diagnostic purposes mentioned herein. The polypeptides of the present invention and pharmaceutical compositions comprising the same can be used in the prevention and/or treatment of diseases and disorders associated with interleukin-6 ("IL-6"), with the interleukin-6 receptor ("IL-6R"), with the IL- 6/IL-6R complex, and/or with the signaling pathway(s) and/or the biological functions and responses in which interleukin-6 ("IL-6"), the interleukin-6 receptor ("IL-6R") and/or the IL- 6/IL-6R complex are involved.

In particular, the polypeptides of the present invention and pharmaceutical compositions comprising the same can be used in the prevention and/or treatment of diseases and disorders which can benefit from modulating the signaling pathway(s) and/or the biological functions and responses in which interleukin-6 ("IL-6"), the interleukin-6 receptor ("IL-6R") and/or the IL-6/IL-6R complex are involved. Generally, these diseases and disorder will be characterized by abnormal, undesired, increased and/or reduced signaling associated with interleukin-6 ("IL-6"), the interleukin-6 receptor ("IL-6R") and/or the IL- 6/IL-6R complex. More in particular, the polypeptides of the present invention and pharmaceutical compositions comprising the same can be used in the prevention and/or treatment of diseases and disorders which can benefit from modulating the interaction between the IL-6/IL-6R complex and gp 130. Examples of the diseases and disorders referred to above (herein collectively: "IL-6 related disorders^'") will be clear to the skilled person, for example from the prior art, such as the background art as referred to herein below.

Other aspects, embodiments, advantages and applications of the invention will become clear from the further description herein. The interaction of IL-6, a protein originally identified as a B cell differentiation factor

(Hirano et al., 1985; EP0257406), with IL-6R (Yamasaki et al, 1988; EP0325474) results in the formation of the IL-6/IL-6R complex. This complex binds to gρ!30 (Taga et al., 1989; EP041 1946), a membrane protein on a target cell, which transmits various physiological actions of IL-6. For this, gρl30 forms a dimer with two molecules of the complex formed by IL-6 and the IL-6R, thus triggering IL-6 mediated signaling.

IL-6 is currently known to be involved in - amongst others - the regulation of the immune response, hematopoiesis, the acute phase response, bone metabolism, angiogenesis, and inflammation. Deregulation of IL-6 production is implicated in the pathology of several autoimmune and chronic inflammatory proliferative disease processes (Ishihara and Hirano, 2002). As a consequence, inhibitors of IL-6 induced signaling have attracted much attention in the past (Hirano et al., 1990). Polypeptides specifically binding to IL-6 (Klein et al., 1991; EP0312996), IL-6R (EP0409607) or gp!30 (Saito et al., 1993; EP0572U8) proved to exhibit an efficient inhibitory effect on IL-6 functioning.

IL-6 overproduction and signalling (and in particular so-called trans-signalling) are involved in various diseases disorders, such as sepsis (Starnes et al., 1999) and various forms of cancer such as multiple myeloma disease (MM), renal cell carcinoma (RCC), plasma cell leukaemia (Klein et al, 1991), lymphoma, B-lymphoproliferative disorder (BLPD) and prostate cancer. Non-limiting examples of other diseases caused by excessive IL-6 production or signalling include bone resorption (osteoporosis) (Roodman et al., 1992; Jilka et al., 1992), cachexia (Strassman et al., 1992), psoriasis, mesangial proliferative glomerulonephritis, Kaposi's sarcoma, AIDS-related lymphoma (Emilie et al., 1994), inflammatory diseases and disorder such as rheumatoid arthritis, systemic onset juvenile idiopathic arthritis, hypergammaglobulinemia (Grau et al., 1990); Crohn's disease, ulcerative colitis, systemic lupus erythematosus (SLE), multiple sclerosis, Castleman's disease, IgM gammopathy, cardiac myxoma, asthma (in particular allergic asthma) and autoimmune insulin-dependent diabetes mellitus (Campbell et al., 1991). Other IL-6 related disorders will be clear to the skilled person. As can for example be seen from the references above, the prior art describes antibodies and antibody fragments directed against human IL-6, against human IL-6R and against human gρl30 protein for the prevention and treatment of IL-6 relates disorders. Examples are Tocilizumab (see Woo P, et al. Arthritis Res Ther. (2005) 7: 1281-8, Nishimoto N et al. Blood. (2005) 106: 2627-32, Ito H et al. Gastroenterology. (2004) 126: 989-96, Choy EH et al. Arthritis Rheum. (2002) 46: 3143-50.), BE8 (see Bataille R et al. Blood (1995) 86:685-91, Emilie D et al. Blood (1994) 84:2472-9, Beck JT et al. N Engl J Med. (1994) 330:602-5, Wendling D et al. J Rheumatol. (1993) 20:259-62.) and CNTO-328 of Centocor (see Journal of Clinical Oncology, (2004) 22/14S: 2560; Journal of Clinical Oncology, (2004) 22/14S: 2608; Int J Cancer (2004) 1 11:592-5). Another active principle known in the art for the prevention and treatment of IL-6 related disorders is an Fc fusion of soluble gρl30 (see Becker C et al. Immunity. (2004) 21: 491-501 , Doganci A et al. J Clin Invest. (2005) 115:313-25, Nowell MA et al. J Immunol. (2003) 171: 3202-9., Atreya R et al. Nat Med. (2000) 6:583-8.)

Nevertheless, there is a need for alternative or improved active principles that can be used for the prevention or treatment of IL-6 related disorders.

The present invention solves this problem by providing polypeptides that are specific and/or selective for the IL-6/IL-6R complex, and/or that bind with higher avidity to the IL- 6/IL-6R-complex compared with binding to IL-6 alone and/or to IL-6R alone, respectively. The polypeptides and preparations of the present invention can generally be used to modulate, and in particular inhibit and/or prevent, binding of the IL-6/IL-6R complex to gpl30 protein, and thus to modulate, and in particular inhibit or prevent, the IL-6 mediated signalling (such as, without limitation, trans-signalling) and/or to modulate the biological responses and effects associated with such signalling. As such, the polypeptides and preparations of the present invention can be used for the prevention and treatment of IL-6 related disorders, and in particular for such diseases and disorders which are characterized by excessive and/or unwanted IL-6-mediated signalling.

Thus, without being limited thereto, the polypeptides of the invention can for example be used to prevent or treat all diseases and disorders that are currently being prevented or treated with active principles that can modulate IL-6 or IL-6R mediated signalling, such as antibodies or antibody fragments against IL-6, against IL-6 receptor or against gp 130, or the Fc fusion of soluble gρl30. It is also envisaged that the polypeptides of the invention can be used to prevent or treat all diseases and disorders for which treatment with such active principles is currently being developed, has been proposed, or will be proposed or developed in future. In addition, it is envisaged that - because of their unique properties as further described herein - the polypeptides of the present invention may be used for the prevention and treatment of other diseases and disorders than those for which these known active principles are being used or will be proposed or developed; and/or that the polypeptides of the present invention may provide new methods and regimens for treating the diseases and disorders described herein.

The invention in its broadest sense provides polypeptides that are can bind to the IL- 6/IL-6R complex, and by such binding can modulate the interaction between said complex and gρl30. In this context, such modulation can either result in an increase of the binding affinity of the complex for gp 130 (or visa versa) or in a decrease of the binding affinity of the complex for gp 130 (or visa versa), i.e. compared to the complex without the presence of the polypeptide of the present invention, As such, the polypeptides of the present invention can have an agonistic or an antagonistic effect on IL-6-mediated signaling and the biological functions and responses associated therewith. According to one preferred, but non-limiting embodiment, binding of the polypeptide of the present invention results in a decrease of the binding affinity of the complex for gp 130 (or visa versa) and/or results in an antagonistic effect on IL-6-mediated signaling and the biological functions and responses associated therewith. However, as will be clear from the description herein, the invention in its broadest sense is not limited thereto and for example also comprises polypeptides that have an agonistic effect.

Thus, generally, the invention provides a polypeptide that is directed against the IL- 6/IL-6R complex. Said polypeptide is preferably capable of inhibiting the interaction between IL-6/IL-6R complex and gpl30 protein, and more preferably capable of competitively inhibiting the interaction between IL-6/IL-6R complex and gpl30 protein, Said polypeptide preferably comprises or essentially consists of at least one binding unit, or a fragment thereof, directed against IL-6/IL-6R complex (as defined herein).

Alternatively, said polypeptide comprises or essentially consists of at least one binding unit, or a fragment thereof, directed against IL-6, and at least one binding unit, or a fragment thereof, directed against IL-6R. Preferably, such a polypeptide comprises at least one binding unit, or a fragment thereof, directed against IL-6, and at least one binding unit, or a fragment thereof, directed against IL-6R, such that the resulting polypeptide binds with higher avidity to the IL-6/TL-6R complex than to either IL-6 alone or 3L-6R alone. hi the polypeptides of the invention, said at least one binding unit is preferably an immunoglobulin or immunoglobulin fragment, more preferably an antibody or an antibody fragment, such as a Fab fragment, a F(ab') fragment, a F(ab'₂) fragment, an Fv fragment, or a scFv fragment. Even more preferably, said at least one binding unit is an immunoglobulin variable domain, such as a V_H domain or a V_L domain, and in particular a (single) domain antibody ((s)dAb). Most preferably, said at least one binding unit is a Nanobody^®.

The polypeptides of the invention (monospecific or bispecific) are preferably directed against mammalian IL-6/IL-6R complex, more preferably against human IL-6/IL-6R complex.

More specifically, the present invention provides polypeptides that are specific for the IL-6/IL-6R complex and/or that bind with higher avidity to the IL-6/IL-6R-complex compared with binding to IL-6 alone and/or to IL-6R alone, respectively, and which preferably inhibit - for example competitively - the binding of human IL-6/IL-6R complex to human gpl30 protein. According to one preferred, but non-limiting embodiment, the polypeptides of the invention are capable of (selectively) inhibiting so called "trans- signalling". However, it should be noted that generally, the polypeptides of the present invention are not limited by the biological mechanism by which they achieve a beneficial effect on the IL-6 related disorders. Generally, however, and without limitation, it may be said that some amino acid residues present in the polypeptides of the present invention will interact with amino acid residues that are present within the IL-6 part of the complex, and that other amino acid residues that some amino acid residues present in the polypeptides of the present invention will interact with amino acid residues that are present within the IL-6R part of the complex.

In the context of the present invention by "IL-6/IL-6R complex" is meant the complex that is formed upon association of IL-6 and IL-6R (e.g. either IL-6R in a membrane-bound form or sIL-6R, which comprises essentially the soluble extracellular domain of the membrane-bound form of IL-6R).

The present invention is in its broadest sense also not particularly limited to or defined by a specific antigenic determinant, epitope, part, domain, subunit or confirmation of IL-6/IL- 6R complex against which the polypeptides of the invention are directed. Thus, for example and as will be mentioned below and as schematically shown in Figure 1, the polypeptides of the invention may be directed against the IL-6/IL-6R interface. In the context of this embodiment, the polypeptide of the present invention is preferably specific for the IL-6/IL-6 receptor complex, in which by "specific for" is meant that the polypeptide interacts with the human IL-6/IL-6R complex in the presence of IL-6 and/or IL-6 receptor, for example with an affinity for the complex that is at least two times, such as at five times and preferably more than ten times higher than the affinity of the polypeptide for IL-6 and/or IL-6R individually, as determined using a suitable assay. For example, but without limitation, in this embodiment of the present invention, the polypeptides of the present invention comprise a single binding site that is directed to an epitope composed of amino acid residues from both IL-6 and IL-6R.

It is also within the scope of the invention that a polypeptide of the invention can bind to two or more antigenic determinants, epitopes, parts, domains, subunits or confirmations of

IL-6/IL-6R complex. In such a case, the antigenic determinants, epitopes, parts, domains or subunits of 3L-6/IL-6R complex to which the polypeptides of the invention bind may be the essentially same (for example, if IL-6/IL-6R complex contains repeated structural motifs or is present as a multimer, for example in complex with IL-6R and/or cell-surface linked gpl30 and/or soluble gpl30) or may be different (and in the latter case, the polypeptides of the invention may bind to such different antigenic determinants, epitopes, parts, domains, subunits of IL-6/IL-6R complex with an affinity and/or specificity which may be the same or different). Also, for example, when BL-6/IL-6R complex exists in an activated conformation and in an inactive conformation, the polypeptides of the invention may bind to either one of these conformations, or may bind to both these conformations (i.e. with an affinity and/or specificity which may be the same or different). Also, for example, the polypeptides of the invention may bind to a conformation of IL-6/IL-6R complex in which it is bound to a pertinent ligand (such as gp 130), may bind to a conformation of IL-6/IL-6R complex in which it not bound to a pertinent ligand (such as sp 130), and/or may bind to both such conformations (again with an affinity and/or specificity which may be the same or different).

Some preferred IL-6 binders for use as binding units in the context of the present invention are the amino acid sequences described in the US provisional application filed by ABLYNX N. V. US 60/782.243 (filed on March 13, 2006) and in the US provisional application filed by ABLYNX N. V. entitled: "Nanobodies against IL-6 and polypeptides comprising the same " (filed on December 1, 2006). Of these, the Nanobodies described in this application are particularly preferred. Of these Nanobodies, the following are especially preferred: SEQ ID's 195 to 322 (SEQ ID's are as listed in the US provisional application filed by ABLYNX N.V. US 60/782,243 (filed on March 13, 2006) and in the US provisional application filed by ABLYNX N. V. entitled: "Nanobodies against I.L-6 and polypeptides comprising the same" (filed on December 1, 2006)).

Some preferred IL-6R binders for use as binding units in the context of the present invention are the amino acid sequences described in the US provisional application filed by ABLYNX N. V. US 60/838,904 (filed on August 18, 2006) and in the US provisional application filed by ABLYNX N.V. entitled: "Amino acid sequences directed against IL-6R and polypeptides comprising the same for the treatment of diseases and disorders associated with IL-6-mediated signalling " (filed on December 5, 2006). Of these, the Nanobodies described in this application are particularly preferred. Of these Nanobodies, the following are especially preferred: SEQ ID's 399 to 471 (SEQ ID's are as listed in the US provisional application filed by ABLYNX N.V. US 60/838,904 (filed on August 18, 2006) and in the US provisional application filed by ABLYNX N.V. entitled: "Amino acid sequences directed against IL-6R and polypeptides comprising the same for the treatment of diseases and disorders associated with IL-6-mediated signalling" (filed on December 5, 2006).

Thus, as will be clear to the skilled person, the polypeptides according to the present invention can for example be polypeptides, and in particular multi specific polypeptides, as described in the patent applications as referred to above, containing at least one amino acid sequence directed against IL-6 and at least one amino acid sequence directed against IL-6R, and optionally one or more further amino acid sequences, again as described in the above patent applications.

The one or more further amino acid sequence may be any suitable and/or desired amino acid sequences. The further amino acid sequences may or may not change, alter or otherwise influence the (biological) properties of the polypeptide, and may or may not add further functionality to the polypeptide of the invention. Preferably, the further amino acid sequence is such that it confers one or more desired properties or functionalities to the polypeptide of the invention. For example, the further amino acid sequence may also provide a second binding site, which binding site may be directed against any desired protein, polypeptide, antigen, antigenic determinant or epitope (including but not limited to the same protein, polypeptide, antigen, antigenic determinant or epitope against which the polypeptide of the invention is directed, or a different protein, polypeptide, antigen, antigenic determinant or epitope).

Example of such amino acid sequences will be clear to the skilled person, and may generally comprise all amino acid sequences that are used in peptide fusions based on conventional antibodies and fragments thereof (including but not limited to ScFv' s and single domain antibodies). Reference is for example made to the review by Holliger and Hudson, Nature Biotechnology, 23, 9, 1 1.26-1 136 (2005),

For example, such an amino acid sequence may be an amino acid sequence that increases the half-life, the solubility, or the absorption, reduces the immunogenicity or the toxicity, eliminates or attenuates undesirable side effects, and/or confers other advantageous properties to and/or reduces the undesired properties of the polypeptides of the invention, compared to the polypeptide of the invention per se. Some non-limiting examples of such amino acid sequences are serum proteins, such as human serum albumin (see for example WO 00/27435) or haptenic molecules (for example haptens that are recognized by circulating antibodies, see for example WO 98/22141).

In particular, it has been described in the art that linking fragments of immunoglobulins (such as V_H domains) to serum albumin or to fragments thereof can be used to increase the half-life. Reference is for made to WO 00/27435 and WO 01/077137). According to the invention, the polypeptide of the invention is preferably either directly linked to serum albumin (or to a suitable fragment thereof) or via a suitable linker, and in particular via a suitable peptide linked so that the polypeptide of the invention can be expressed as a genetic fusion (protein). According to one specific aspect, the polypeptide of the invention may be linked to a fragment of serum albumin that at least comprises the domain III of serum albumin or part thereof. Reference is for example made to the US provisional application 60/788,256 of Ablynx N. V. entitled "Albumin derived amino acid sequence, use thereof for increasing the half-life of therapeutic proteins and of other therapeutic proteins and entities, and constructs comprising the same" filed on March 31, 2006.

Alternatively, the further amino acid sequence may provide a second binding site or binding unit that is directed against a serum protein (such as, for example, human serum albumin or another serum protein such as IgG), so as to provide increased half-life in serum. Such amino acid sequences for example include the Nanobodies described below, as well as the small peptides and binding proteins described in WO 91/01743, WO 01/45746 and WO 02/076489 and the dAb^'ε described in WO 03/002609 and WO 04/003019. Reference is also made to Harmsen et al., Vaccine, 23 (41); 4926-42, as well as to EP 0 368 684, as well as to the following the US provisional applications 60/843,349, 60/850,774, 60/850,775 by Ablynx N. V. mentioned herein. Such amino acid sequences may in particular be directed against serum albumin (and more in particular human serum albumin) and/or against IgG (and more in particular human IgG). For example, such amino acid sequences may be amino acid sequences that are directed against (human) serum albumin and that amino acid sequences that can bind to amino acid residues on (human) serum albumin that are not involved in binding of serum albumin to FcRn (see for example WO 06/0122787) and/or amino acid sequences that are capable of binding to amino acid residues on serum albumin that do not form part of domain III of serum albumin (see again see for example WO 06/0122787); amino acid sequences that have or can provide an increased half-life (see for example the US provisional application 60/843,349 by Ablynx N. V. entitled "Serum albumin binding proteins with long half-lives'^' filed on September 8, 2006); amino acid sequences against human serum albumin that are cross-reactive with serum albumin from at least one species of mammal, and in particular with at least one species of primate (such as, without limitation, monkeys from the genus Macaca (such as, and in particular, cynomologus monkeys {Macaca fascicularis) and/or rhesus monkeys (Macaca mulatto)) and baboon {Papio ursinus), reference is again made to the US provisional application 60/843,349); amino acid sequences that can bind to serum albumin in a pH independent manner (see for example the US provisional application 60/850,774 by Ablynx N. V. entitled "Amino acid sequences that bind to serum proteins in a manner that is essentially independent ofthepH, compounds comprising the same, and uses thereof, filed on October 11, 2006) and/or amino acid sequences that are conditional binders (see for example the US provisional application 60/850,775 by Ablynx N.V. entitled "Amino acid sequences that bind to a desired molecule in a conditional manner", filed on October 11, 2006).

Generally, the polypeptides of the invention with increased half-life preferably have a half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding amino acid sequence of the invention per se. For example, the compounds or polypeptides of the invention with increased half-life may have a half -life that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding amino acid sequence of the invention per se.

In a preferred, but non-limiting aspect of the invention, such polypeptides of the invention exhibit a serum half-life in human of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably a! least 72 hours or more. For example, compounds or polypeptides of the invention may have a half-life of at least 5 days (such as about 5 to 1.0 days), at preferably at least 9 days (such as about 9 to 14 days), more preferably at least about 10 days (such as about 10 to 15 days), or at least about 11 days (such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days).

According to another aspect, the one or more further amino acid sequences may comprise one or more parts, fragments or domains of conventional 4-chain antibodies (and in particular human antibodies) and/or of heavy chain antibodies. For example, although usually less preferred, a polypeptide of the invention may be linked to a conventional (preferably human) V_H or V_L domain domain or to a natural or synthetic analog of a V_H or V_L domain, again optionally via a linker sequence (including but not limited to other (single) domain antibodies, such as the dAb's described by Ward et al.).

The at least one polypeptide may also be linked to one or more (preferably human) C_RI , C_H2 and/or C_H3 domains, optionally via a linker sequence. For instance, a polypeptide linked to a suitable C_H1 domain could for example be used - together with suitable light chains - to generate antibody fragments/structures analogous to conventional Fab fragments or F(ab')₂ fragments, but in which one or (in case of an F(ab')₂ fragment) one or both of the conventional V_H domains have been replaced by a polypeptide of the invention. Also, two Nanobodies could be linked to a C_H3 domain (optionally via a linker) to provide a construct with increased half-life in vivo.

According to one specific aspect of a polypeptide of the invention, one or more Nanobodies of the invention may linked to one or more antibody parts, fragments or domains that confer one or more effector functions to the polypeptide of the invention and/or may confer the ability to bind to one or more Fc receptors. For example, for this purpose, and without being limited thereto, the one or more further amino acid sequences may comprise one or more C_H2 and/or C_H 3 domains of an antibody, such as from a heavy chain antibody (as described herein) and more preferably from a conventional human 4-chain antibody; and/or may form (part of) and Fc region, for example from IgG, from IgE or from another human Ig. For example, WO 94/04678 describes heavy chain antibodies comprising a Camelid V_HH domain or a humanized derivative thereof (i.e. a polypeptide), in which the Camelidae C_H2 and/or C_H3 domain have been replaced by human C_H2 and C_H3 domains, so as to provide an immunoglobulin that consists of 2 heavy chains each comprising a polypeptide and human C_H2 and C_R3 domains (but no C_HI domain), which immunoglobulin has the effector function provided by the C_H2 and C_H3 domains and which immunoglobulin can function without the presence of any light chains. Other amino acid sequences that can be suitably linked to the Nanobodies of the invention so as to provide an effector function will be clear to the skilled person, and may be chosen on the basis of the desired effector function(s). Reference is for example made to WO 04/058820, WO 99/42077 and WO

05/017148, as well as the review by Holliger and Hudson, supra. Coupling of a polypeptide of the invention to an Fc portion may also lead to an increased half-life, compared to the corresponding polypeptide of the invention. For some applications, the use of an Fc portion and/or of constant domains (i.e. C_H2 and/or C_H3 domains) that confer increased half-life without any biologically significant effector function may also be suitable or even preferred. Other suitable constructs comprising one or more Nanobodies and one or more constant domains with increased half-life in vivo will be clear to the skilled person, and may for example comprise two Nanobodies linked to a Cκ3 domain, optionally via a linker sequence. Generally, any fusion protein or derivatives with increased half-life will preferably have a molecular weight of more than 50 kD, the cut-off value for renal absorption.

The further amino acid sequences may also form a signal sequence or leader sequence that directs secretion of the polypeptide of the invention from a host cell upon synthesis (for example to provide a ρre-. pro- or prepro- form of the polypeptide of the invention, depending on the host cell used to express the polypeptide of the invention). The further amino acid sequence may also form a sequence or signal that allows the polypeptide of the invention to be directed towards and/or to penetrate or enter into specific organs, tissues, cells, or parts or compartments of cells, and/or that allows the polypeptide of the invention to penetrate or cross a biological barrier such as a cell membrane, a cell layer such as a layer of epithelial cells, a tumor including solid tumors, or the blood-brain-barrier. Suitable examples of such amino acid sequences will be clear to the skilled person, and for example include, but are not limited to, the "Peptrans" vectors mentioned above, the sequences described by Cardinale et al. and the amino acid sequences and antibody fragments known per se that can be used to express or produce the Nanobodies and polypeptides of the invention as so-called "intrabodies", for example as described in WO 94/02610, WO 95/22618, US-A-7004940, WO 03/014960, WO 99/07414; WO 05/01690; EP 1 512 696; and in Cattaneo, A. & Biocca, S. (1997) Intracellular Antibodies: Development and Applications. Landes and Springer- Verlag; and in Kontermann, Methods 34, (2004), 163-170, and the further references described therein.

As also described in the above applications, multispecific polypeptides according to the invention can contain one or more linkers linking the binding units in the polypeptide. These linkers can also be as described in the above mentioned two patent applications. Such multi-specific proteins can also contain further amino acid sequences or binding units, can be formatted and/or can have an increased half-life.

Some preferred but non-limiting examples of multispecific polypeptides useful in the invention are the constructs of SEQ ID's 42 to 61, listed in the present application.

Thus, for example and as further mentioned below and as schematically shown in Figure 1, a polypeptide of the invention can be a bispecific polypeptide with at least one binding site (for example a first domain antibody or Nanobody) directed against an epitope on the IL-6 part of the IL-6/IL-6R complex and with at least one binding site (for example a second domain antibody or Nanobody) against an epitope on the IL-6R part of the IL-6/IL-6R complex, such that the polypeptide can bind to the complex via both binding sites, resulting in a higher avidity to the IL-6/IL-6R-complex compared with binding to IL-6 alone and/or to IL-6R alone, respectively

In this embodiment, the first binding site (i.e. binding unit or Nanobody) is preferably specific for (as defined herein) for IL-6 and the second binding site (i.e. binding unit or Nanobody) is preferably specific for (as defined herein) for IL-6R.

According to this embodiment, this "bispecific" (or optionally multispecific or bispecific tri- or multivalent) polypeptide of the invention has a selective or preferential binding for the complex compared with the individual components. This can be measured as a higher overall binding affinity (or avidity) for the complex than for IL-6 or for the IL-6R separately, such as at least two times greater, for example five times greater or even ten times greater or more. The overall binding affinity towards the complex is determined by the affinity of interaction of the individual polypeptide building units for the components in the complex, and by the nature of the linkage and spacing between these two building units in the bispecific polypeptide. Thus, without limitation, by modulating the affinities of the individual building units for their respective antigens, the overall affinity (or avidity) level of the bispecific molecule, and hence selectively of binding towards the complex compared to individual components, can be modulated. The affinities of the individual building units for their respective antigens or epitopes can be chosen based on the average, measured or expected level of the individual antigen and of the complex in the compartment where the bispecific polypeptide is expected to have or require selective or preferential bind the complex. In such manner this avidity- based targeting can achieve selectivity (preferential binding) for the complex compared to the individual components. The most optimal overall affinity for the polypeptide to achieve selective (or preferential) binding to the complex may also or in addition be determined based on experimental approaches. Selective or preferential binding, the relative difference of binding activity due to the avid binding of the complex over binding of the bispecific to the individual components of the complex, can be measured by testing the binding interaction between the bispecific polypeptide and the complex in the presence of the individual components, e.g. in ELISA in which the IL-6/IL-6R complex is coated directly or via capture with a monoclonal antibody that recognizes an epitope far removed from the interface regions of the IL-6 and IL-6R, providing the competitors IL-6 and IL-6R in solution, and then measuring the binding activity of the bispecific polypeptide to the complex. Alternatively the relative binding activity and/or selectivity of the bispecific polypeptide for the complex or the individual components may be measured at different concentrations of the bispecific polypeptide. The binding affinities of the individual components can hence be adjusted to obtain a selective binding for the bispecific polypeptide at physiological relevant concentrations of both the polypeptide, the complex and/or the individual components. Using this experimental approach, all factors that could contribute to or counteract the selective binding of the bispecific polypeptide are taken into account, including those factors that are less readily predictable, not completely understood to too little biochemical data (e.g. the presence of dimers of the individual components of the complex such as IL-6R dimer, or the presence of precomplexed gpl30/TL-6/TL-6R, the presence of other serum proteins that could effect the equilibrium between the different components). A preferential binding of the polypeptide to the IL-6/IL-6R complex has the advantage that the individual components, JL- 6, cell-surface displayed IL- 6R and soluble IL-6R, are not bound as well as the complex. The affinities of the interactions may be chosen in such manner that in the presence of a physiological concentration of the polypeptide of the invention (maximally typically 10-50 micrograra/ml in humans), the non-complexed components may still have a certain level of interaction with their natural ligands. For example when the binding unit has a low affinity for IL-6, circulating of newly produced and secreted non-complexed IL-6 may under such conditions be able to bind 1L-6R displayed on liver cell surfaces and induce signal transduction. On the contrary with the binding unit has a high affinity for IL-6, it will be readily bound by the bispecific polypeptide of the invention and form trimolecular complexes with IL-6R. Further consequences on the biology of the IL-6 pathway will depend on whether such complexes can or cannot bind the soluble gpl30 or cell-surface linked gp!30 and can or cannot induce signaling. For this, the binding sites/units in the bispecific polypeptide of the invention are most preferably such that they can bind to epitopes on IL-6 and IL- 6R, respectively, that are accessible for binding by the polypeptide of the present invention when IL-6 and IL-6R are associated into a complex. Also, the polypeptide of the invention is most preferably such that each binding site/unit can bind to its respective epitope on IL-6 and IL-6R, respectively, when IL-6 and IL-6R are associated into a complex.

It is also expected that the polypeptides of the invention will generally bind to all naturally occurring or synthetic analogs, variants, mutants, alleles, parts and fragments of the IL-6/IL-6R complex (and/or of IL-6 and IL-6R in case of a bispecific molecule), or at least to those analogs, variants, mutants, alleles, parts and fragments of IL-6/IL-6R complex that contain one or more antigenic determinants or epitopes that are essentially the same as the antigenic determinant(s) or epitope(s) to which the polypeptides of the invention bind in IL- 6/IL-6R complex (e.g. in wild-type IL-6/IL-6R complex). It is also included within the scope of the invention that the polypeptides of the invention bind to some analogs, variants, mutants, alleles, parts and fragments of IL-6/IL-6R complex, but not to others. It is also within the scope of the invention to use parts, fragments, analogs, mutants, variants, alleles and/or derivatives of the polypeptides of the invention, and/or to use proteins or polypeptides comprising or essentially consisting of the same, as long as these are suitable for the uses envisaged herein. Such parts, fragments, analogs, mutants, variants, alleles, derivatives, proteins and/or polypeptides will be described in the further description herein. According to a non-limiting but preferred embodiment of the invention, the polypeptides of the invention are preferably such that they bind to IL-6/IL-6R complex with a dissociation constant (K_D) of 10^-5 to 10^{- 12} moles/liter or less, and preferably 10^-7 to 10^-12 moles/liter or less and more preferably 10- to 10^{- ~} moles/liter, and/or with a binding affinity (association constant (K_A) of at least 10⁷ M^-1 , preferably at least 10^s M^{- 1}, more preferably at least 10⁹ M^-1, such as at least 10¹² M^-1 and/or with an affinity (dissociation constant K_D) less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. The affinity of the polypeptide of the invention against IL-6/IL-6R complex can be determined in a manner known per se, for example using the assay described herein.

According to a non-limiting but preferred embodiment of the invention, the polypeptides of the invention that comprise multiple binding site to the IL-6 and IL-6R complex, are preferably such that they bind to IL-6/1L-6R complex with a higher avidity then the individual components separately. For such interactions, both both overall affinity or avidity values can be determined, with the caveat that it is well established in the literature that for measuring avid interactions (interactions with more than one binding site between two molecules such as antigen and IgG molecule), the assay conditions may influence the measured value. The measured overall dissociation constant for such avid interaction is preferably 10^-5 to 10^-12 moles/liter or less, and preferably 10^-7 to 10^-12 moles/liter or less and more preferably 10^-8 to 10^{- 12} moles/liter, and/or with a binding affinity of at least 10⁷ M^{- 1}, preferably at least 10⁸ M^*\ more preferably at least 10⁹ M^-1 , such as at least 10¹² M^-1 and/or with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. The increase in relative binding strength of the polypeptides of the invention towards the IL-6/IL-6R complex compared to the individual components of the complex is preferably 2-fold, 5-fold, 10-fold, 20-fold and 50-fold. This difference may be apparent from the comparison of the affinity values determined in a particular test, or by comparing directly the interactions between the polypeptides of the invention with the individual components of the complex and the complex itself in a binding test. Increase in relative binding or in avidity can be determined in a manner known in the art, e.g based on relative signals in three ELISAs with respectively immobilized IL-6/IL-6R complex, IL-6 and IL-6R, or based on BIAcore measurements in which there three components are injected onto a surface onto which the polypeptide of the invention is immobilized, or any other methods used for determining affinity and/or avidity constants, or using the assays described herein. Typically the binding affinity for the individual is moderate to low (10^-5 to 10^-8 moles/liter), and the combination of the two binding sites creates an avid effect that can be measured experimentally, e.g. by an effect on the dissociation phase in surface plasmon resonance (BIAcore), by stronger binding in flow cytometry, stronger signals in ELISA etc. Also, according to the invention, polypeptides that are directed against IL-6/IL-6R complex from a first species of warm-blooded animal may or may not show cross-reactivity with IL-6/IL-6R complex from one or more other species of warm-blooded animals. For example, polypeptides directed against human IL-6/IL-6R complex may or may not show cross-reactivity with IL-6/IL-6R complex from one or more other species of primates and/or with IL-6/3L-6R complex from one or more species of animals that are often used in animal models for diseases (for example mouse, rat, rabbit, pig or dog), and in particular in animal models for diseases and disorders associated with IL-6/IL-6R complex (such as the species and animal models mentioned herein). In this respect, it will be clear to the skilled person that such cross-reactivity, when present, may have advantages from a drug development point of view, since it allows the polypeptides against human IL-6/IL-6R complex to be tested in such disease models.

More generally, it is also encompassed within the scope of the invention that polypeptides directed against IL-6/IL-6R complex from one species of animal (such as polypeptides against human IL-6/IL-6R complex) are used in the treatment of another species of animal, as long as the use of the polypeptides provide the desired effects in the species to be treated.

The efficacy of the polypeptides of the invention, and of compositions comprising the same, can be tested using any suitable in vitro assay, cell-based assay, in vivo assay and/or animal model known per se, or any combination thereof, depending on the specific disease or disorder involved. Suitable assays and animal models will be clear to the skilled person, and for example include cell proliferation assays using IL-6-dependent cell lines including B9, XG1 and 7TD1, collagen induced arthritis model, transplant model of synovial tissue in SCID mice, xenograft models of various human cancers, including lymphoma, myeloma, prostate cancer and renal cell carcinoma, IBD models including TNBS, DSS and ILlO knockout models.

Hereinbelow, two preferred but non-limiting embodiments of the polypeptides of the invention will be described.

In a first embodiment, the polypeptides of the present invention comprise at least one binding unit that is specific for the human IL-6/IL-6R complex. Such a binding unit can for example be any suitable binding domain, such as an immunoglobulin sequence, an antibody, or an antibody fragment, such as, but not limited to, a Fab fragment, a F(ab') fragment, a F(ab'₂) fragment, an Fv fragment, or a scFv fragment. The binding unit preferably comprises at least one variable domain, such as V_H domain or a V_L domain. Even more preferably the binding unit comprises at least one (single) domain antibody. Still even more preferably the binding unit comprises at least one Nanobody^®. Nanobodies^® as well as methods for the preparation of the same are well known to the skilled person. In this embodiment, the polypeptide of the invention is preferably such that it binds to the IL-6/IL-6R complex interface (as schematically shown in Figure 1) and/or such that the polypeptide of the invention, upon binding to the IL-6/IL-6R complex, is capable of modulating, and in particular inhibiting association of the IL-6/IL-6R complex with gpl30.

Binding units suitable for use in this first embodiment of the invention can be generated in any suitable way. For example, immunoglobulin sequences against the IL-6/IL- 6R complex (or nucleic acids encoding the same) can be generated using suitable techniques known to the skilled person, such as the screening techniques reviewed by Hoogenboom, Nature Biotechnology, 23, 9, 1105-1116 (2005). Other techniques for generating immunoglobulins against a specified target include for example the Nanoclone technology (as for example described in the non-prepublished US provisional patent application 60/648,922), so-called SLAM technology (as for example described in the European patent application 0 542 810), the use of transgenic mice expressing human immunoglobulins or the well-known hybridoma techniques (see for example Larrick et al, Biotechnology, Vol.7, 1989, p. 934). All these techniques can be used to generate immunoglobulin sequences against the IL-6/IL-6R complex, for example starting from a naϊve library of human immunoglobulin sequences, from a library containing the immune repertoire from a mammal that has been suitably immunized with the IL-6/IL-6R complex, or from the blood or a sample of B-cells obtained from a mammal that has been suitably immunized with the IL- 6/IL-6R complex (i.e. so as to raise an immune response against the complex). For this purpose, the library may also be screened and/or the mammal immunized with a protein fusion of IL- 6 and IL-6R, a non-limiting example of which is the protein fusion known as "hyper IL-6" (see for example Fischer M et al., Nat Biotechnol (1997) 15: 142-145). Also, the mammal may be a transgenic mammal expressing a human(ized) antibody repertoire, such as the Xenomouse™. Thus, according to one embodiment of the invention, the polypeptide as described herein is a polypeptide that is directed against (as defined herein) a protein fusion of IL-6 and IL-6R, such as hyper IL-6 and/or that comprises at least one binding site (such as binding unit, for example a Nanobody) that is directed against (as defined herein) a protein fusion of IL-6 and IL-6R, such as hyper IL-6. Another embodiment of the invention is a polypeptide specific for (as defined herein) a protein fusion of IL-6 and IL-6R, such as hyper IL-6, and/or that comprises at least one binding site (such as binding unit, for example a Nanobody) that is specific for (as defined herein) a protein fusion of IL-6 and IL-6R, such as hyper IL-6. Another embodiment is a polypeptide, a binding unit or a polypeptide comprising at least one binding unit (all as further described herein) that is obtained by the general methods described herein for providing the polypeptides or binding units described herein, wherein said polypeptide or binding unit is directed against and/or specific for a protein fusion of IL-6 and IL-6R, such as hyper IL-6. For obtaining such proteins or polypeptides, the methods described herein can be performed in a manner known per se and/or as further described herein, for example starting from blood or B-celϊs of a mammal (preferably a Camelid) that has been suitably immunized with a protein fusion of IL-6 and IL-6R, such as hyper IL-6; and/or by screening (for example of an library of immunoglobulin sequences) with a protein fusion of IL-6 and IL-6R, such as hyper IL-6. After a suitable binding unit (or a nucleotide sequence encoding the same) has been identified and isolated, it may either be used as such as a polypeptide of the invention, or linked to one or more further amino acid sequences (or nucleotide sequences encoding the same) so as to provide a polypeptide of the invention (optionally after suitable expression).

All this may be performed essentially as described herein. In particular, a Camelid such as a llama may be immunized with the complex or with hyper-IL-6, after which Nanobody sequences directed against the complex may be generated and isolated, starting from B-cells obtained from the immunized animal, all as further described herein.

In a second embodiment the polypeptides of the present invention are polypeptides that bind with higher avidity to the IL-6/IL-6R-complex compared with binding to IL-6 alone and/or to IL-6R alone, respectively. Preferably, such polypeptides comprise at least one binding site directed against an epitope on the IL-6 part of the IL-6/IL-6R complex and at least one binding site against an epitope on the IL-6R part of the IL-6/IL-6R complex, such thai the first binding site can bind to an epitope on the IL-6 part of the IL-6/IL-6R complex and the second binding site can bind to an epitope on the IL-6R part of the IL-6/IL-6R complex. For example, such a bispecific molecule can comprise at least one binding unit that is directed to human IL-6, and at least one binding unit that is directed to human IL-6R, such that the polypeptides of the invention have a higher avidity or affinity for human IL-6/IL-6R complex then to the individual components, in which the term binding unit is as defined above.

Thus, for example, a bispecific molecule according to this embodiment of the invention can comprise a first (single) domain antibody or Nanobody directed against IL-6 and a second (single) domain antibody or Nanobody directed against IL-6R (optionally linked via a suitable linker or via another suitable amino acid sequence), in which the polypeptide is such that the first (single) domain antibody or Nanobody against IL-6 can bind to an epitope on the IL-6 part of the IL-6/IL-6R complex and the second (single) domain antibody or Nanobody directed against IL-6R can bind to an epitope on the IL-6R part of the IL-6/IL-6R complex.

To provide bispecific molecules according to this second embodiment, a binding unit directed against IL-6 (or a nucleotide sequence encoding the same) may be linked to a binding unit directed against IL-6R (or to a nucleotide sequence encoding the same), optionally via a suitable linker (or via a nucleotide sequence encoding the same), and optionally to and/or via one or further amino acid sequences (or nucleotide sequences encoding the same), such as a further binding unit that provides for increased half-life (as further described herein), so as to provide a polypeptide of the invention (or a nucleotide sequence encoding the same). The binding units against IL-6 and IL-6R (or the nucleotide sequences encoding the same) may be known per se or may be generated in any suitable manner, such as the techniques mentioned herein. In particular, a Camelid such as a llama may be immunized with the IL-6 and/or with IL-6R, respectively, after which Nanobody sequences directed against IL-6 and IL-6R, respectively, may be generated and isolated, starting from B -cells obtained from the immunized animal, all as further described herein.

The binding units directed against IL-6 and IL-6R, respectively, that are included in the bispecific molecules according to the invention preferably have a specificity for IL-6 and IL-6R, respectively, that is as defined herein. Some preferred, but non-limiting Nanobodies against IL-6 that can be used in the polypeptides of the present invention are described in the co-pending US provisional application by applicant entitled "Nanobodies® against IL-6 and polypeptides comprising the same", which has the same filing date as the present application. Generally, in the present description and claims, proteins or polypeptides that comprise or essentially consist of a single binding unit (such as a single binding unit of the invention) will be referred to herein as "monovalent" proteins or polypeptides or as "monovalent constructs". Proteins and polypeptides that comprise or essentially consist of two or more binding units (such as at least two binding units of the invention or at least one binding unit of the Invention and at least one other binding unit) will be referred to herein as "multivalent" proteins or polypeptides or as "multivalent constructs", and these may provide certain advantages compared to the corresponding monovalent binding units of the invention. Some non-limiting examples of such multivalent constructs will become clear from the further description herein.

According to another specific, but non-limiting embodiment, a polypeptide of the invention comprises or essentially consists of at least one binding unit of the invention (for example in case of a Nanobody directed against the IL-6/IL-6R complex) or of at least two binding units (e.g. in the case of a bispecific molecule directed against the IL-6-part and the IL-6 receptor part of the complex) and at least one other binding unit (i.e. directed against another epitope, antigen, target, protein or polypeptide). Such proteins or polypeptides are also referred to herein as "multispecific" proteins or polypeptides or as 'multi specific constructs", and these may provide certain advantages compared to the corresponding monovalent binding units of the invention. Again, some non-limiting examples of such multispecific constructs will become clear from the further description herein.

According to yet another specific, but non-limiting embodiment, a polypeptide of the invention comprises or essentially consists of at least one binding unit of the invention (for example in case of a Nanobody directed against the IL-6/IL-6R complex) or of at least two binding units (e.g. in the case of a bispecific molecule directed against the IL-6-part and the IL-6 receptor part of the complex) and at least one other amino acid sequence (such as a protein or polypeptide) that confers at least one desired property to the binding unit of the invention, and/or to the resulting fusion protein. Again, such fusion proteins may provide certain advantages compared to the corresponding monovalent or bispecific binding units of the invention. Some non -limiting examples of such amino acid sequences and of such fusion constructs will become clear from the further description herein.

In the above constructs, the one or more binding units and/or other amino acid sequences may be directly linked or linked via one or more linker sequences. Some suitable but non-limiting examples of such linkers will become clear from the further description herein.

Preferably, a polypeptide of the invention either comprises two or three binding units of the invention, optionally linked via one or two linkers, or is a multispecific polypeptide, comprising one or two, and preferably two, binding units of the invention and at least one binding unit that provides an increased half-life (such as a binding unit directed against a serum protein, and in particular against a human serum protein, such as against human serum albumin), in which said binding units again optionally linked via one or more linkers. hi another preferred embodiment of the invention, a polypeptide of the invention comprises one or more (such as two or preferably one) binding units of the invention linked (optionally via one or more suitable linker sequences) to one or more (such as two and preferably one) amino acid sequences that confer an increased half -life in vivo to the resulting polypeptide of the invention. In particular, said amino acid sequences that confer an increased half -life in vivo to the resulting polypeptide of the invention may be one or more (such as two and preferably one) binding units, and in particular binding units directed against a human serum protein, such as human serum albumin.

Generally, for pharmaceutical use, the polypeptides of the invention may be formulated as a pharmaceutical preparation comprising at least one polypeptide of the invention and at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more further pharmaceutically active polypeptides and/or compounds.

In another aspect, the invention relates to a nucleic acid that encodes a polypeptide of the invention. Such a nucleic acid will also be referred to herein as a "nucleic acid of the invention" and may for example be in the form of a genetic construct, as defined herein. In another aspect, the invention relates to host or host cell that expresses or that is capable of expressing a polypeptide of the invention and/or that contains a nucleic acid of the invention. Some preferred but non-limiting examples of such hosts or host cells will become clear from the further description herein.

The invention further relates to a product or composition containing or comprising at least one polypeptide of the invention and/or at least one nucleic acid of the invention, and optionally one or more further components of such compositions known per se, i.e. depending on the intended use of the composition. Such a product or composition may for example be a pharmaceutical composition (as described herein), a veterinary composition or a product or composition for diagnostic use (as also described herein). Some preferred but non-limiting examples of such products or compositions will become clear from the further description herein. The invention further relates to methods for preparing or generating the polypeptides, nucleic acids, host cells, products and compositions described herein. Some preferred but non-limiting examples of such methods will become clear from the further description herein.

The invention further relates to applications and uses of the polypeptides, nucleic acids, host cells, products and compositions described herein, as well as to methods for the prevention and/or treatment for diseases and disorders associated with IL-6/IL-6R complex.

Some preferred but non-limiting applications and uses will become clear from the further description herein.

Other aspects, embodiments, advantages and applications of the invention will also become clear from the further description hereinbelow.

The invention will be further described below using Nanobodies^® as a specific example of the binding unit(s). However, it will be clear to the skilled person from the disclosure herein that other binding units (including but not limited to those mentioned herein) may also be used in the present invention.

Detailed description of the invention

The above and other aspects, embodiments and advantages of the invention will become clear from the further description hereinbelow, in which: a) Unless indicated or defined otherwise, all terms used have their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks, such as Sambrook et al, "Molecular Cloning: A Laboratory Manual¹¹ ( 2nd.Ed.), VoIs, 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al, eds., "Current protocols in molecular biology'¹, Green Publishing and

Wiley Interscience, New York (1987); Lewin, "Genes IF', John Wiley & Sons, New York, N. Y-, (1985); Old et al., "Principles of Gene Manipulation: An Introduction to Genetic Engineering'^*, 2nd edition, University of California Press, Berkeley, CA (1981 ); Roitt et al., "Immunology" (6th. Ed.), Mosby/EIsevier, Edinburgh (2001); Roitt et al., Roitt's Essential Immunology, 1O⁰¹ Ed. Blackwell Publishing, UK (2001); and

J anew ay et al., "Immunobiology" (6th Ed.), Garland Science Publishing/Churchill Livingstone, New York (2005), as well as to the general background art cited herein; b) Unless indicated otherwise, the term "immunoglobulin sequence" - whether it used herein to refer to a heavy chain antibody or to a conventional 4-chain antibody - is used as a general term to include both the full-size antibody, the individual chains thereof, as well as all parts, domains or fragments thereof (including but not limited to antigen - binding domains or fragments such as V_HH domains or V_H/V_L domains, respectively).

In addition, the term "sequence'- as used herein (for example in terms like "immunoglobulin sequence", "antibody sequence", "variable domain sequence", "V_HH sequence" or "protein sequence"), should generally be understood to include both the relevant amino acid sequence as well as nucleic acid sequences or nucleotide sequences encoding the same, unless the context requires a more limited interpretation; c) Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to the standard handbooks and the general background art mentioned herein and to the further references cited therein; d) Amino acid residues will be indicated according to the standard three-letter or one- letter amino acid code, as mentioned in Table A-2;

Table A-2: one-letter and three-letter amino acid code

e) For the purposes of comparing two or more nucleotide sequences, the percentage of "sequence identity" between a first nucleotide sequence and a second nucleotide sequence may be calculated by dividing [the number of nucleotides in the first nucleotide sequence thai are identical to the nucleotides at the corresponding positions in the second nucleotide sequence] by [the total number of nucleotides in the first nucleotide sequence] and multiplying by [100%], in which each deletion, insertion, substitution or addition of a nucleotide in the second nucleotide sequence - compared to the first nucleotide sequence - is considered as a difference at a single nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences may be calculated using a known computer algorithm for sequence alignment such as NCBI Blast v2.0, using standard settings.

Some other techniques, computer algorithms and settings for determining the degree of sequence identity are for example described in WO 04/037999, EP 0 967 284, EP 1 085 089, WO 00/55318, WO 00/78972, WO 98/49185 and GB 2 357 768-A.

Usually, for the purpose of determining the percentage of "sequence identity" between two nucleotide sequences in accordance with the calculation method outlined hereinabove, the nucleotide sequence with the greatest number of nucleotides will be taken as the "first" nucleotide sequence, and the other nucleotide sequence will be taken as the "second" nucleotide sequence; f) For the purposes of comparing two or more amino acid sequences, the percentage of "sequence identity" between a first amino acid sequence and a second amino acid sequence may be calculated by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence] by [the total number of nucleotides in the first amino acid sequence] and multiplying by [700%], in which each deletion, insertion, substitution or addition of an amino acid residue in the second amino acid sequence - compared to the first amino acid sequence - is considered as a difference at a single amino acid residue (position), i.e. as an "amino acid difference" as defined herein.

Alternatively, the degree of sequence identity between two amino acid sequences may be calculated using a known computer algorithm, such as those mentioned above for determining the degree of sequence identity for nucleotide sequences, again using standard settings.

Usually, for the purpose of determining the percentage of "sequence identity" between two amino acid sequences in accordance with the calculation method outlined hereinabove. the amino acid sequence with the greatest number of amino acid residues will be taken as the "first" amino acid sequence, and the other amino acid sequence will be taken as the "second" amino acid sequence.

Also, in determining the degree of sequence identity between two amino acid sequences, the skilled person may take into account so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and which has little or essentially no influence on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example from WO 04/037999, GB-A-2 357 768, WO 98/49185, WO 00/46383 and WO 01/09300; and (preferred) types and/or combinations of such substitutions may be selected on the basis of the pertinent teachings from WO 04/037999 as well as WO 98/49185 and from the further references cited therein.

Such* conservative substitutions preferably are substitutions in which one amino acid within the following groups (a) - (e) is substituted by another amino acid residue within the same group: (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and GIy; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, GIu and GIn; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, VaI and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

Particularly preferred conservative substitutions are as follows: Ala into GIy or into Ser; Arg into Lys; Asn into GIn or into His; Asp into GIu; Cys into Ser; GIn into Asn; GIu into Asp; GIy into Ala or into Pro; His into Asn or into GIn; He into Leu or into VaI; Leu into lie or into VaI; Lys into Arg, into GIn or into GIu; Met into Leu, into Tyr or into lie; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into

Tyr; Tyr into Trp; and/or Phe into VaI₁ into De or into Leu.

Any amino acid substitutions applied to the polypeptides described herein may also be based on the analysis of the frequencies of amino acid variations between homologous proteins of different species developed by Schulz et al., Principles of Protein Structure, Springer-Verlag, 1978, on the analyses of structure forming potentials developed by Chou and Fasman, Biochemistry 13: 21 1, 1974 and Adv. EnzymoL, 47: 45-149, 1978, and on the analysis of hydrophobicity patterns in proteins developed by Eisenberg et al., Proc. Nad. Acad Sci. USA 81: 140-144, 1984; Kyte &

Doolittle; J Molec. Biol. 157: 105-132, 198 1, and Goldman et al., Ann. Rev. Biophys. Chem. 15: 321-353, 1986, all incorporated herein in their entirety by reference. Information on the primary, secondary and tertiary structure of Nanobodies given in the description herein and in the genera] background art cited above. Also, for this purpose, the crystal structure of a V_HH domain from a llama is for example given by Desmyter et al., Nature Structural Biology, Vol. 3, 9, 803 (1996); Spinelli et al., Natural Structural Biology (1996); 3, 752-757; and Decanniere et al., Structure, Vol. 7, 4, 361 (1999). Further information about some of the amino acid residues that in conventional V_H domains form the V_H/V_L interface and potential camelizing substitutions on these positions ; g) Amino acid sequences and nucleic acid sequences are said to be "exactly the same" if they have 100% sequence identity (as defined herein) over their entire length; h) When comparing two amino acid sequences, the term "amino acid difference'" refers to an insertion, deletion or substitution of a single amino acid residue on a position of the first sequence, compared to the second sequence; it being understood that two amino acid sequences can contain one, two or more such amino acid differences; i) A nucleic acid sequence or amino acid sequence is considered to be "(in) essentially isolated (form)" - for example, compared to its native biological source and/or the reaction medium or cultivation medium from which it has been obtained - when it has been separated from at least one other component with which it is usually associated in said source or medium, such as another nucleic acid, another protein/polypeptide, another biological component or macromolecule or at least one contaminant, impurity or minor component. In particular, a nucleic acid sequence or amino acid sequence is considered "essentially isolated" when it has been purified at least 2-fold, in particular at least 10-fold, more in particular at least 100-fold, and up to 1000-fold or more. A nucleic acid sequence or amino acid sequence that is "in essentially isolated form" is preferably essentially homogeneous, as determined using a suitable technique, such as a suitable chromatographic al technique, such as polyacrylamide-gelelectrophoresis; j) The term "domain" as used herein generally refers to a globular region of an antibody chain, and in particular to a globular region of a heavy chain antibody, or to a polypeptide that essentially consists of such a globular region. Usually, such a domain will comprise peptide loops (for example 3 or 4 peptide loops) stabilized, for example, as a sheet or by disulfide bonds. k) The term 'antigenic determinant' refers to the epitope on the antigen recognized by the antigen-binding molecule (such as a Nanobody^® or a polypeptide of the invention) and more in particular by the antigen-binding site of said molecule. The terms "antigenic determinant" and "epitope' may also be used interchangeably herein. 1) An amino acid sequence (such as a Nanobody^®, an antibody, a polypeptide of the invention, or generally an antigen binding protein or polypeptide or a fragment thereof) that can bind to, that has affinity for and/or that has specificity for a specific antigenic determinant, epitope, antigen or protein (or for at least one part, fragment or epitope thereof) is said to be "against" or "directed against" said antigenic determinant, epitope, antigen or protein. m) The term "specificity" refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding molecule or antigen-binding protein (such as a Nanobody^® or a polypeptide of the invention) molecule can bind. The specificity of an antigen-binding protein can be determined based on affinity and/or avidity. The affinity, represented by the equilibrium constant for the dissociation of an antigen with an antigen-binding protein (K_D), is a measure for the binding strength between an antigenic determinant and an antigen-binding site on the antigen-binding protein: the lesser the value of the K_D1 the stronger the binding strength between an antigenic determinant and the antigen-binding molecule (alternatively, the affinity can also be expressed as the affinity constant (K_A), which is 1/K_D). AS will be clear to the skilled person (for example on the basis of the further disclosure herein), affinity can be determined in a manner known per se, depending on the specific antigen of interest. Avidity is the measure of the strength of binding between an antigen-binding molecule (such as a Nanobody^® or polypeptide of the invention) and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antigen-binding molecule and the number of pertinent binding sites present on the antigen-binding molecule. Typically, antigen-binding proteins (such as the Nanobodies^® and/or polypeptides of the invention) will bind with a dissociation constant (K_D) of 10^-5 to 10^-12 moles/liter or less, and preferably 10^-7 to 10^-12 moles/liter or less and more preferably 10^-8 to 10^{- 12} moles/liter, and/or with a binding affinity of at least 10⁷ M^{- 1}, preferably at least 10⁸ M^-1, more preferably at least 10⁹ M^-1, such as at least 10¹² M^-1. Any K_D value greater than 10^-4 liters/mol is generally considered to indicate non-specific binding. Preferably, a Nanobody^® or polypeptide of the invention will bind to the desired antigen with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. Specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays

(EIA) and sandwich competition assays, and the different variants thereof known per se in the art. n) As further described herein, the amino acid sequence and structure of a Nanobody^® can be considered - without however being limited thereto - to be comprised of four framework regions or "FR's", which are referred to in the art and herein as "Framework region 1" or "FR1"; as "Framework region 2" or"FR2"; as "Framework region 3" or "FR3"; and as "Framework region 4" or "FR4", respectively; which framework regions are interrupted by three complementary determining regions or "CDR' s", which are referred to in the art as "Complementarity Determining Region l"or "CDR1"; as "Complementarity Determining Region 2" or "CDR2"; and as "Complementarity

Determining Region 3" or "CDR3", respectively; o) As also further described herein, the total number of amino acid residues in a Nanobody^® can be in the region of 110-120, is preferably 112-115, and is most preferably 113. It should however be noted that parts, fragments, analogs or derivatives (as further described herein) of a Nanobody^® are not particularly limited as to their length and/or size, as long as such parts, fragments, analogs or derivatives meet the further requirements outlined herein and are also preferably suitable for the purposes described herein; p) The amino acid residues of a Nanobody are numbered according to the general numbering for V_H domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91), as applied to V_HH domains from Camelids in the article of Riechmanrs and Muyldermans, referred to above (see for example Figure 2 of said reference). According to this numbering, FRl of a Nanobody^® comprises the amino acid residues at positions 1-30, CDRl of a Nanobody^® comprises the amino acid residues at positions 31-36, FR2 of a Nanobody^® comprises the amino acids at positions 36-49, CDR2 of a Nanobody^® comprises the amino acid residues at positions 50-65, FR3 of a Nanobody^® comprises the amino acid residues at positions 66-94, CDR3 of a Nanobody^® comprises the amino acid residues at positions 95-102, and FR4 of a Nanobody^® comprises the amino acid residues at positions 103-113. [In this respect, it should be noted that - as is well known in the art for V_H domains and for V_HH domains - the total number of amino acid residues in each of the CDR 's may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering). This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. Generally, however, it can be said that, according to the numbering of Kabat and irrespective of the number of amino acid residues in the CDR^' s, position 1 according to the Kabat numbering corresponds to the start of FRl and vice versa, position 36 according to the Kabat numbering corresponds to the start of FR2 and vice versa, position 66 according to the Kabat numbering corresponds to the start of FR3 and vice versa, and position 103 according to the Kabat numbering corresponds to the start of FR4 and vice versa.].

Alternative methods for numbering the amino acid residues of V_H domains, which methods can also be applied in an analogous manner to V_HH domains from Camelids and to Nanobodies^®, are the method described by Chothia et al. (Nature 342, 877-883 (1989)), the so-called "AbM definition" and the so-called "contact definition".

However, in the present description, claims and figures, the numbering according to

Kabat as applied to V_HH domains by Riechmann and Muyldermans will be followed, unless indicated otherwise; and The Figures, Sequence Listing and the Experimental Part/Examples are only given to further illustrate the invention and should not be interpreted or construed as limiting the scope of the invention and/or of the appended claims in any way, unless explicitly indicated otherwise herein. For a general description of heavy chain antibodies and the variable domains thereof, reference is inter alia made to the following references, which are mentioned as general background art: WO 94/04678, WO 95/04079 and WO 96/34103 of the Vrije Universiteit Brussel; WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO 02/48193 of Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of the Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 of Algonomics N.V. and applicant; WO 01/90190 by the National Research Council of Canada; WO 03/025020 (= EP 1 433 793) by the Institute of Antibodies; as well as WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863, WO 04/062551 by applicant and the further published patent applications by applicant;

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In accordance with the terminology used in the above references, the variable domains present in naturally occurring heavy chain antibodies will also be referred to as "V_HH domains", in order to distinguish them from the heavy chain variable domains that are present in conventional 4-chain antibodies (which will be referred to hereinbelow as "V_H domains") and from the light chain variable domains that are present in conventional 4-chain antibodies (which will be referred to hereinbelow as "V_L domains").

As mentioned in the prior art referred to above, V_HH domains have a number of unique structural characteristics and functional properties which make isolated V_HH domains (as well as Nanobodies^® based thereon, which share these structural characteristics and functional properties with the naturally occurring V_HH domains) and proteins containing the same highly advantageous for use as functional antigen-binding domains or proteins. In particular, and without being limited thereto, V_HH domains (which have been "designed" by nature to functionally bind to an antigen without the presence of, and without any interaction with, a light chain variable domain) and Nanobodies" can function as a single, relatively small, functional antigen -binding structural unit, domain or protein. This distinguishes the V_HH domains from the V_H and V_L domains of conventional 4-chain antibodies, which by themselves are generally not suited for practical application as single antigen -binding proteins or domains, but need to be combined in some form or another to provide a functional antigen-binding unit (as in for example conventional antibody fragments such as Fab fragments; in ScFv's fragments, which consist of a V_H domain covalentiy linked to a V_L domain).

Because of these unique properties, the use of V_HH domains and Nanobodies^® as single antigen-binding proteins or as antigen-binding domains (i.e. as part of a larger protein or polypeptide) offers a number of significant advantages over the use of conventional V_R and V_L domains, scFv's or conventional antibody fragments (such as Fab- or F(ab')₂- fragments): only a single domain is required to bind an antigen with high affinity and with high selectivity, so that there is no need to have two separate domains present, nor to assure that these two domains are present in the right spacial conformation and configuration (i.e. through the use of especially designed linkers, as with scFv's); V_HH domains and Nanobodies^® can be expressed from a single gene and require no post-translational folding or modifications; - V_HH domains, and Nanobodies^® can easily be engineered into multivalent and multispecific formats (as further discussed herein);

V_HH domains and Nanobodies^® are highly soluble and do not have a tendency to aggregate (as with the mouse-derived antigen-binding domains" described by Ward et al., Nature, Vol.341, 1989, p. 544); - V_HH domains and Nanobodies^® are highly stable to heat, pH, proteases and other denaturing agents or conditions (see for example Ewert et al, supra); V_HH domains and Nanobodies^® are easy and relatively cheap to prepare, even on a scale required for production. For example, V_HH domains, Nanobodies^® and proteins/polypeptides containing the same can be produced using microbial fermentation (e.g. as further described below) and do not require the use of mammalian expression systems, as with for example conventional antibody fragments; V_HH domains and Nanobodies^® are relatively small (approximately 15 kDa, or 10 times smaller than a conventional IgG) compared to conventional 4-chain antibodies and antigen-binding fragments thereof, and therefore show high(er) penetration into tissues (including but not limited to solid tumors and other dense tissues) than such conventional 4-chain antibodies and antigen-binding fragments thereof; V_HH domains and Nanobodies^® can show so-called cavity-binding properties (inter alia due to their extended CDR3 loop, compared to conventional V_H domains) and can therefore also access targets and epitopes not accessable to conventional 4-chain antibodies and antigen-binding fragments thereof. For example, it has been shown that

V_HH domains and Nanobodies^® can inhibit enzymes (see for example WO 97/49805: Transue et al., (1998), supra; Lauwereys et al., (1998), supra.

As mentioned above, the invention generally relates to Nanobodies^® directed against IL-6/IL-6R complex, as well as to polypeptides comprising or essentially consisting of one or more of such Nanobodies^®, that can be used for the prophylactic, therapeutic and/or diagnostic purposes described herein.

As also further described herein, the invention further relates to nucleic acids encoding such Nanobodies^® and polypeptides, to methods for preparing such Nanobodies^® and polypeptides, to host cells expressing or capable of expressing such Nanobodies^® or polypeptides, to compositions comprising such Nanobodies^®, polypeptides, nucleic acids or host cells, and to uses of such Nanobodies^®, polypeptides, nucleic acids, host cells or compositions. Generally, it should be noted that the term Nanobody^® as used herein in its broadest sense is not limited to a specific biological source or to a specific method of preparation. For example, as will be discussed in more detail below, the Nanobodies^® of the invention can generally be obtained: (1) by isolating the V_HH domain of a naturally occurring heavy chain antibody; (2) by expression of a nucleotide sequence encoding a naturally occurring V_HH domain; (3) by "humanization" (as described herein) of a naturally occurring V_HH domain or by expression of a nucleic acid encoding a such humanized V_HH domain; (4) by "camelization" (as described herein) of a naturally occurring V_H domain from any animal species, and in particular a from species of mammal, such as from a human being, or by expression of a nucleic acid encoding such a camelized V_H domain; (5) by "camelisation" of a "domain antibody" or "Dab" as described by Ward et al (supra), or by expression of a nucleic acid encoding such a camelized V_H domain; (6) by using synthetic or semi -synthetic techniques for preparing proteins, polypeptides or other amino acid sequences known per se; (7) by preparing a nucleic acid encoding a Nanobody^® using techniques for nucleic acid synthesis known per se, followed by expression of the nucleic acid thus obtained; and/or (8) by any combination of one or more of the foregoing. Suitable methods and techniques for performing the foregoing will be clear to the skilled person based on the disclosure herein and for example include the methods and techniques described in more detail herein.

One preferred class of Nanobodies^® corresponds to the V_HH domains of naturally occurring heavy chain antibodies directed against IL-6/IL-6R complex. As further described herein, such VH_H sequences can generally be generated or obtained by suitably immunizing a species of Camelid with IL-6/IL-6R complex (i.e. so as to raise an immune response and/or heavy chain antibodies directed against IL-6/IL-6R complex), by obtaining a suitable biological sample from said Camelid (such as a blood sample, serum sample or sample of B- cells), and by generating V_HH sequences directed against IL-6/IL-6R complex starting from said sample, using any suitable technique known per se. Such techniques will be clear to the skilled person and/or are further described herein.

Alternatively, such naturally occurring V_HH domains against IL-6/IL-6R complex can be obtained from naϊve libraries of Camelid V_HH sequences, for example by screening such a library using IL-6/IL-6R complex or at least one part, fragment, antigenic determinant or epitope thereof using one or more screening techniques known per se. Such libraries and techniques are for example described in WO 99/37681, WO 01/901.90, WO 03/025020 and WO 03/035694. Alternatively, improved synthetic or semi-synthetic libraries derived from naϊve V_HH libraries may be used, such as V_HH libraries obtained from naive V_HH libraries by techniques such as random mutagenesis and/or CDR shuffling, as for example described in WO 00/43507.

Yet another technique for obtaining V_HH sequences directed against IL-6/IL-6R complex involves suitably immunizing a transgenic mammal that is capable of expressing heavy chain antibodies (i.e. so as to raise an immune response and/or heavy chain antibodies directed against IL-6/IL-6R complex), obtaining a suitable biological sample from said transgenic mammal (such as a blood sample, serum sample or sample of B-cells), and then generating V_HH sequences directed against IL-6/IL-6R complex starting from said sample, using any suitable technique known per se. For example, for this purpose, the heavy chain antibody-expressing mice and the further methods and techniques described in WO 02/085945 and in WO 04/049794 can be used.

A particularly preferred class of Nanobodies^® of the invention comprises Nanobodies^® with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring V_HH domain, but that has been "humanized" , i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring V_HH sequence (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a V_H domain from a conventional A- chairs antibody from a human being (e.g. indicated above). This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the further description herein and the prior art on humanizalion referred to herein. Again, it should be noted that such humanized Nanobodies " of the invention can be obtained in any suitable manner known per se (i.e. as indicated under points (1) - (8) above) and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring V_HH domain as a starting material. Another particularly preferred class of Nanobodies^® of the invention comprises

Nanobodies^® with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring V_H domain, but that has been "camelized", i.e. by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring V_H domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a V_HH domain of a heavy chain antibody. This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the further description herein. Such "camelizing" substitutions are preferably inserted at amino acid positions that form and/or are present at the V_H-V_L interface, and/or at the so- called Camelidae hallmark residues, as defined herein (see for example WO 94/04678 and Davies and Riechmann (1994 and 1996), supra). Preferably, the V_H sequence that is used as a stalling material or starting point for generating or designing the camelized Nanobody^® is preferably a V_H sequence from a mammal, more preferably the V_H sequence of a human being, such as a V_H3 sequence. However, it should be noted that such camelized Nanobodies^® of the invention can be obtained in any suitable manner known per se (i.e. as indicated under points (1) - (8) above) and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring V_H domain as a starting material For example, again as further described herein, both "humanization" and

"camelization" can be performed by providing a nucleotide sequence that encodes a naturally occurring V_HH domain or V_H domain, respectively, and then changing, in a manner known per se, one or more codons in said nucleotide sequence in such a way that the new nucleotide sequence encodes a "humanized" or "camelized" Nanobody^® of the invention, respectively. This nucleic acid can then be expressed in a manner known per se, so as to provide the desired Nanobody^®of the invention. Alternatively, based on the amino acid sequence of a naturally occurring V_HH domain or V_H domain, respectively, the amino acid sequence of the desired humanized or canielized Nanobody^® of the invention, respectively, can be designed and then synthesized de novo using techniques for peptide synthesis known per se. Also, based on the amino acid sequence or nucleotide sequence of a naturally occurring V_HH domain or V_H domain, respectively, a nucleotide sequence encoding the desired humanized or camelized Nanobody^® of the invention, respectively, can be designed and then synthesized de novo using techniques for nucleic acid synthesis known per se, after which the nucleic acid thus obtained can be expressed in a manner known per se, so as to provide the desired Nanobody^® of the invention.

Other suitable methods and techniques for obtaining the Nanobodies^® of the invention and/or nucleic acids encoding the same, starting from naturally occurring V_H sequences or preferably V_HH sequences, will be clear from the skilled person, and may for example comprise combining one or more parts of one or more naturally occurring V_H sequences (such as one or more FR sequences and/or CDR sequences), one or more parts of one or more naturally occurring V_HH sequences (such as one or more FR sequences or CDR sequences), and/or one or more synthetic or semi-synthetic sequences, in a suitable manner, so as to provide a Nanobody^® of the invention or a nucleotide sequence or nucleic acid encoding the same.

According to one preferred, but non-limiting aspect of the aspect of the invention, a Nanobody^® in its broadest sense can be generally defined as a polypeptide comprising:

(a) an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which the amino acid residue at position 108 according to the Kabat numbering is Q; and/or:

(b) an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which the amino acid residue at position 45 according to the Kabat numbering is a charged amino acid (as defined herein) or a cysteine residue, and position 44 is preferably an E; and/or:

(c) an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S, and is in particular chosen from the group consisting of R and S.

Thus, in a first preferred, but non-limiting aspect, a Nanobody^® of the invention may have the structure

FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4

in which FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which (a) the amino acid residue at position 108 according to the Kabat numbering is Q; and/or in which: (b) the amino acid residue at position 45 according to the Kabat numbering is a charged amino acid or a cysteine and the amino acid residue at position 44 according to the

Kabat numbering is preferably E; and/or in which: (c) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S, and is in particular chosen from the group consisting of R and S,

In particular, a Nanobody in its broadest sense can be generally defined as a polypeptide comprising:

(a) an amino acid sequence that is comprised of four framework regions/sequences interrapted by three complementarity determining regions/sequences, in which the amino acid residue at position 108 according to the Kabat numbering is Q; and/or: (b) an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which the amino acid residue at position 44 according to the Kabat numbering is E and in which the amino acid residue at position 45 according to the Kabat numbering is an R; and/or: (c) an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S, and is in particular chosen from the group consisting of R and S. Thus, according to a preferred, but non-limiting aspect, a Nanobody^® of the invention may have the structure

FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4

in which FRl to FR4 refer to framework regions 1 to 4. respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which a) the amino acid residue at position 108 according to the Kabat numbering is Q; and/or in which: b) the amino acid residue at position 44 according to the Kabat numbering is E and in which the amino acid residue at position 45 according to the Kabat numbering is an R; and/or in which: c) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S, and is in particular chosen from the group consisting of R and S;

In particular, a Nanobody^® against IL-6/IL-6R complex according to the invention may have the structure:

FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4

in which FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3. respectively, and in. which (a) the amino acid residue at position 108 according to the Kabat numbering is Q; and/or in which: (b) the amino acid residue at position 44 according to the Kabat numbering is E and in which the amino acid residue at position 45 according to the Kabat numbering is an R; and/or in which: (c) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S, and is in particular chosen from the group consisting of R and S;

In particular, according to one preferred, but non-limiting aspect of the aspect of the invention, a Nanobody^® can generally be defined as a polypeptide comprising an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which;

(a-1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of A, G, E, D, G, Q, R, S, L; and is preferably chosen from the group consisting of G, E or Q; and (a-2) the amino acid residue at position 45 according to the Kabat numbering is chosen from the group consisting of L, R or C; and is preferably chosen from the group consisting of L or R; and (a-3) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of W, R or S; and is preferably W or R, and is most preferably W;

(a-4) the amino acid residue at position 108 according to the Kabat numbering is Q; or in which:

(b-1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of E and Q; and

(b-2) the amino acid residue at position 45 according to the Kabat numbering is R; and (b-3) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of W, R and S ; and is preferably W;

(b-4) the amino acid residue at position 108 according to the Kabat numbering is chosen from the group consisting of Q and L; and is preferably Q; or in which:

(c-1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of A, G, E, D, Q, R, S and L; and is preferably chosen from the group consisting of G, E and Q; and (c-2) the amino acid residue at position 45 according to the Kabat numbering is chosen from the group consisting of L, R and C; and is preferably chosen from the group consisting of L and R; and (c-3) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S; and is in particular chosen from the group consisting of R and S; and (c-4) the amino acid residue at position 108 according to the Kabat numbering is chosen from the group consisting of Q and L; is preferably Q; Thus, in another preferred, but non-limiting aspect, a Nanobody^® of the invention may have the structure

FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4

in which FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: (a) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of A, G, E, D, G, Q, R, S, L; and Is preferably chosen from the group consisting of G, E or Q; and in which: (b) the amino acid residue at position 45 according to the Kabat numbering is chosen from the group consisting of L, R or C: and is preferably chosen from the group consisting of

L or R; and in which:

(c) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of W, R or S; and is preferably W or R, and is most preferably W: and in which

(d) the amino acid residue at position 108 according to the Kabat numbering is Q;

In another preferred, but non-limiting aspect, a Nanobody^® of the invention may have the structure

FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4

in which FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which:

(a) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of E and Q; and in which:

(b) the amino acid residue at position 45 according to the Kabat numbering is R; and in which:

(c) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of W, R and S; and is preferably W; and in which:

(d) the amino acid residue at position 108 according to the Kabat numbering is Q; In another preferred, but non-limiting aspect, a Nanobody^® of the invention may have the structure

FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which:

(a) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of A, G, E, D, Q, R, S and L; and is preferably chosen from the group consisting of G, E and Q; and in which:

(b) the amino acid residue at position 45 according to the Kabat numbering is chosen from the group consisting of L, R and C; and is preferably chosen from the group consisting of L and R; and in which:

Cc) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S; and is in particular chosen from the group consisting of R and S; and in which:

(d) the amino acid residue at position 108 according to the Kabat numbering is chosen from the group consisting of Q and L; is preferably Q;

Two particularly preferred, but non -limiting groups of the Nanobodies^® of the invention are those according to a) above; according to (a-1) to (a-4) above; according to b) above; according to Cb-I) to (b-4) above; according to Cc) above; and/or according to (c-1) to Cc -4) above, in which; a) the amino acid residues at positions 44-47 according to the Kabat numbering form the sequence GLEW (or a GLEW-like sequence as defined herein) and the amino acid residue at position 108 is Q; or in which: b) the amino acid residues at positions 43-46 according to the Kabat numbering form the sequence KERE or KQRE Cor a KERE-like sequence) and the amino acid residue at position 108 is Q or L, and is preferably Q.

Thus, in another preferred, but non-limiting aspect, a Nanobody^® of the invention may have the structure

FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: (a) the amino acid residues at positions 44-47 according to the Kabat numbering form the sequence GLEW (or a GLEW-like sequence as defined herein) and the amino acid residue at position 108 is Q;

FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4

in which FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which the amino acid residues at positions 43-46 according to the Kabat numbering form the sequence KERE or KQRE (or a KERE-like sequence) and the amino acid residue at position 108 is Q or L, and is preferably Q;

In the Nanobodies^® of the invention in which the amino acid residues at positions 43- 46 according to the Kabat numbering form the sequence KERE or KQRE, the amino acid residue at position 37 is most preferably F. hi the Nanobodies^® of the invention in which the amino acid residues at positions 44-47 according to the Kabat numbering form the sequence GLEW, the amino acid residue at position 37 is chosen from the group consisting of Y, H, I, L, V or F, and is most preferably F.

Thus, without being limited hereto in any way, on the basis of the amino acid residues present on the positions mentioned above, the Nanobodies^® of the invention can generally be classified is on the basis of the following three groups: a) The "GLEW -group": Nanobodies^® with the amino acid sequence GLEW at positions 44-47 according to the Kabat numbering and Q at position 108 according to the Kabat numbering. As further described herein, Nanobodies^® within this group usually have a V at position 37, and can have a W, P, R or S at position 103. and preferably have a W at position 103. The GLEW group also comprises some GLEW-like sequences such as those mentioned in Table A-3 below; b) The "KERB- group": Nanobodies^® with the amino acid sequence KERE or KQRE or at positions 43-46 according to the Kabat numbering and Q or L at position 108 according to the Kabat numbering. As further described herein, Nanobodies^® within this group usually have a F at position 37, an L or F at position 47; and can have a W, P, R or S at position 103, and preferably have a W at position 103; c) The "103 P, R₁ S-group": Nanobodies^® with a P, R or S at position 103, These Nanobodies^® can have either the amino acid sequence GLEW at positions 44-47 of the Kabat numbering or the amino acid sequence KERE or KQRE at positions 43-46 according to the Kabat numbering, the latter most preferably in combination with an F at position 37 and an L or an F at position 47 (as defined for the KERE-group); and can have Q or L at position 108 according to the Kabat numbering, and preferably have Q.

Also, more generally and in addition to the 108Q, 43E/44R and 103P,R,S residues mentioned above, the Nanobodies^® of the invention can contain, at one or more positions that in a conventional V_H domain would form (part of) the -VVV_L interface, one or more amino acid residues that are more highly charged than the amino acid residues that naturally occur at the same position(s) in the corresponding naturally occurring V_H sequence, and in particular one or more charged amino acid residues (as mentioned in Table A-2). Such substitutions include, but are not limited to, the GLEW-like sequences mentioned in Table A- 3 below; as well as the substitutions that are described in the International Application WO 00/29004 for so-called "microbodies", e.g. so as to obtain a Nanobody^® with Q at position 108 in combination with KLEW at positions 44-47. Other possible substitutions at these positions will be clear to the skilled person based upon the disclosure herein. In one embodiment of the Nanobodies^® of the invention, the amino acid residue at position 83 is chosen from the group consisting of L, M, S, V and W; and is preferably L.

Also, in one embodiment of the Nanobodies^® of the invention, the amino acid residue at position 83 is chosen from the group consisting of R, K, N, E, G, I, T and Q; and is most preferably either K or E (for Nanobodies^® corresponding to naturally occurring V_HH domains) or R (for "humanized" Nanobodies ⁸, as described herein). The amino acid residue at position 84 is chosen from the group consisting of P, A, R, S, D T, and V in one embodiment, and is most preferably P (for Nanobodies^® corresponding to naturally occurring V_HH domains) or R (for "humanized" Nanobodies^®, as described herein).

Furthermore, in one embodiment of the Nanobodies^® of the invention, the amino acid residue at position 104 is chosen from the group consisting of G and D; and is most preferably G.

Collectively, the amino acid residues at positions 1 1, 37, 44, 45, 47, 83, 84, 103, 104 and 108, which in the Nanobodies^® are as mentioned above, will also be referred to herein as the "Hallmark Residues". The Hallmark Residues and the amino acid residues at the corresponding positions of the most closely related human V_H domain, V_H3, are summarized in Table A-3.

Some especially preferred but non-limiting combinations of these Hallmark Residues as occur in naturally occurring V_HH domains are mentioned in Table A-4. For comparison, the corresponding amino acid residues of the human V_H3 called DP-47 have been indicated in italics.

Table A-2: Hallmark Residues in Nanobodies

Table A-3: Some preferred but non-limiting combinations of Hallmark Residues in naturally occurring Nanobodies¹

For humanization of these combinations, reference is made to the specification.

In the Nanobodies^®, each amino acid residue at any other position than the Hallmark Residues can be any amino acid residue that naturally occurs at the corresponding position (according to the Kabat numbering) of a naturally occurring VHH domain. Such amino acid residues will be clear to the skilled person. Tables 5 - 8 mention some non-limiting residues that can be present at each position (according to the Kabat numbering) of the FRl, FR2, FR3 and FR4 of naturally occurring V_HH domains. For each position, the amino acid residue that most frequently occurs at each position of a naturally occurring V_HH domain (and which is the most preferred amino acid residue for said position in a Nanobody^®) is indicated in bold; and other preferred amino acid residues for each position have been underlined (note: the number of amino acid residues that are found at positions 26-30 of naturally occurring V_HH domains supports the hypothesis underlying the numbering Chothia (supra) that the residues at these positions already form part of CDRl.) In Tables 5 - 8, some of the non-limiting residues that can be present at each position of a human V_H3 domain have also been mentioned. Again, for each position, the amino acid residue that most frequently occurs at each position of a naturally occurring human V_H3 domain is indicated in bold; and other preferred amino acid residues have been underlined. For reference only, Table A-5 also contains data on the V_HH entropy {"V_HH

Ent ") and V_HH variability {"V_HH Van") at each amino acid position for a representative sample of 1118 V_HH sequences (data kindly provided by David Lutje Hulsing and Prof. Theo Verrips of Utrecht University). The values for the V_HH entropy and the V_HH variability provide a measure for the variability and degree of conservation of amino acid residues between the 1118 V_HH sequences analyzed: low values (i.e. <1, such as < 0.5) indicate that an amino acid residue is highly conserved between the V_HH sequences (i.e. little variability). For example, the G at position 8 and the G at position 9 have values for the V_HH entropy of 0.1 and 0 respectively, indicating that these residues are highly conserved and have vary little variability (and in case of position 9 is G in all 1118 sequences analysed), whereas for residues that form part of the CDR' s generally values of 1.5 or more are found (data not shown). Note that (1) the amino acid residues listed in the second column of Table A- 5 are based on a bigger sample than the 11 18 V_HH sequences that were analysed for determining the V_HH entropy and V_HH variability referred to in the last two columns; and (2) the data represented below supports the hypothesis that the amino acid residues at positions 27-30 and maybe even also at positions 93 and 94 already form part of the CDR' s (although the invention is not limited to any specific hypothesis or explanation, and as mentioned above, herein the numbering according to Kabat is used). For a general explanation of sequence entropy, sequence variability and the methodology for determining the same, see Oliveira et aJ., PROTEINS: Structure, Function and Genetics, 52: 544-552 (2003).

Table A-4: Non-limiting examples of amino acid residues in FR1 (for the footnotes, see the footnotes to Table A-3) Table A-4: Non-Iimiting examples of amino acid residues in FR1 (continued)

Table A-5: Non-limiting examples of amino acid residues in FR2 (for the footnotes, see the footnotes to Table A -3)

Table A -6: Non-limiting examples of amino acid residues in FR3 (for the footnotes, see the footnotes to Table A-3) Table A-6: Non-limiting examples of amino acid residues in FR3 (continued)

Table A-7: Non-limiting examples of amino acid residues in FR4 (for the footnotes, see the footnotes to Table A-3)

Thus, in another preferred, but not limiting aspect, a Nanobody^® of the invention can have the structure

FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4

in which FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which the Hallmark residues are as defined herein:

In another preferred, but not limiting aspect, a Nanobody^® of the invention can have the structure

FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4

in which FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3. respectively, and in which:

(a) FRl is chosen from the group consisting of the amino acid sequence:

[1] QVQLQESGGGXVQAGGSLRLSCAASG [26] [SEQ ED NO: 1]

or from the group consisting of amino acid sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with the above amino acid sequence; in which i) any amino acid substitution at any position other than a Hallmark position is preferably either a conservative amino acid substitution (as defined herein) and/or an amino acid substitution as defined in Table A- 5; and/or ii) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequence(s); and/or from the group consisting of amino acid sequences that have 3, 2 or only 1 "amino acid difference(s)" (as defined herein) with one of the above amino acid sequences, in which: i) any amino acid substitution at any position other than a Hallmark position is preferably either a conservative amino acid substitution (as defined herein) and/or an amino acid substitution as defined in Table A-5; and/or U) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequence(s); and in which:

(b) FR2 is chosen from the group consisting of the amino acid sequence:

[36] WXRQAPGKXXEXVA [49] [SEQ ID

NO: 2]

or from the group consisting of amino acid sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with the above amino acid sequence; in which i) any amino acid substitution at any position other than a Hallmark position is preferably either a conservative amino acid substitution (as defined herein) and/or an amino acid substitution, as defined in Table A-6; and/or ii) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequence(s); and/or from the group consisting of amino acid sequences that have 3, 2 or only 1 "amino acid difference(s)" (as defined herein) with one of the above amino acid sequences, in which: i) any amino acid substitution at any position other than a Hallmark position is preferably either a conservative amino acid substitution (as defined herein) and/or an amino acid substitution as defined in Table A-6; and/or ii) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequence(s); and in which: (c) FR3 is chosen from the group consisting of the amino acid sequence:

[66] RFTISRDNAKNTVYLQMNSLXXEDTAVYYCAA [94] [SEQ ID

NO: 3]

or from the group consisting of amino acid sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with the above amino acid sequence; in which i) any amino acid substitution at any position other than a Hallmark position is preferably either a conservative amino acid substitution (as defined herein) and/or an amino acid substitution as defined in Table A-7; and/or ii) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequence(s); and/or from the group consisting of amino acid sequences that have 3, 2 or only

1 "amino acid difference (s)" (as defined herein) with one of the above amino acid sequences, in which: i) any amino acid substitution at any position other than a Hallmark position is preferably either a conservative amino acid substitution (as defined herein) and/or an amino acid substitution as defined in Table A-7; and/or ii) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequence(s); and in which: (d) FR4 is chosen from the group consisting of the amino acid sequence:

[103] XXQGTXVTVSS [113] [SEQ ID NO: 4] or from the group consisting of amino acid sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with the above amino acid sequence; in which i) any amino acid substitution at any position other than a Hallmark position is preferably either a conservative amino acid substitution (as defined herein) and/or an amino acid substitution as defined in Table A-8; and/or ii) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequence(s); and/or from the group consisting of amino acid sequences that have 3, 2 or only 1 "amino acid difference(s)" (as defined herein) with one of the above amino acid sequences, in which: i) any amino acid substitution at any position other than a Hallmark position is preferably either a conservative amino acid substitution (as defined herein) and/or an amino acid substitution as defined in Table A-8; and/or ii) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequence(s); in which the Hallmark Residues are indicated by "X" and are as defined hereinabove and in which the numbers between brackets refer to the amino acid positions according to the Kabat numbering.

FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4

in which FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: and in which (a) FRl is chosen from the group consisting of the amino acid sequence: [1] QVQLQESGGGLVQAGGSLRLSCAASG [26] [SEQ ID NO: 5]

or from the group consisting of amino acid sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with the above amino acid sequence; in which i) any amino acid substitution at any position other than a Hallmark position is preferably either a conservative amino acid substitution (as defined herein) and/or an amino acid substitution as defined in Table A-5; and/or ii) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequence(s); and iii) the Hallmark residue at position is as indicated in the sequence above; and/or from the group consisting of amino acid sequences that have 3, 2 or only

1 "amino acid difference (s)" (as defined herein) with one of the above amino acid sequences, in which: i) any amino acid substitution at any position other than a Hallmark position is preferably either a conservative amino acid substitution (as defined herein) and/or an amino acid substitution as defined in Table A-5; and/or ii) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequence(s): and iii) the Hallmark residue at position is as indicated in the sequence above; and in which:

(b) FR2 is chosen from the group consisting of the amino acid sequences:

[36] WFRQAPGKERELVA [49] [SEQ ID

NO: 6] [36] WFRQAPGKEREFVA [49] [SEQ ID

NO: 7]

[36] WFRQAPGKEREGA [49] [SEQ ID NO: 8] [36] WFRQAPGKQRELVA [49] [SEQ ID

NO: 9]

[36] WFRQAPGKQREFVA [49] [SEQ ED

NO: 10] [36] WYRQAPGKGLEWA [49] [SEQ ID

NO: 11]

or from the group consisting of amino acid sequences that have at least 80%, preferably at least 90%, more preferably at least 95%. even more preferably at least 99% sequence identity (as defined, herein) with one of the above amino acid sequences; in which i) any amino acid substitution at any position other than a Hallmark position is preferably either a conservative amino acid substitution (as defined herein) and/or an amino acid substitution as defined in Table A-6; and/or ii) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequence(s); and iii) the Hallmark residues at positions 37, 44, 45 and 47 are as indicated in each of the sequences above; and/or from the group consisting of amino acid sequences that have 3, 2 or only

1 "amino acid difference(s)" (as defined herein) with one of the above amino acid sequences, in which: i) any amino acid substitution at any position other than a Hallmark position is preferably either a conservative amino acid substitution (as defined herein) and/or an amino acid substitution as defined in Table A-6; and/or ii) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequence(s); and iii) the Hallmark residues at positions 37, 44, 45 and 47 are as indicated in each of the sequences above; and in which: (c) FR3 is chosen from the group consisting of the amino acid sequence: [66] RFTISRDNAKNTVYLQMNSLKPEDT A VYYCAA [94] [SEQ ID

NO: 12]

or from the group consisting of amino acid sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with the above amino acid sequence; in which i) any amino acid substitution at any position other than a Hallmark position is preferably either a conservative amino acid substitution (as defined herein) and/or an amino acid substitution as defined in Table A-7; and/or ii) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequence(s); and iii) the Hallmark residues at positions 83 and 84 are as indicated in each of the sequences above; and/or from the group consisting of amino acid sequences that have 3, 2 or only 1 "amino acid difference(s)" (as defined herein) with one of the above amino acid sequences, in which: i) any amino acid substitution at any position other than a Hallmark position is preferably either a conservative amino acid substitution (as defined herein) and/or an amino acid substitution as defined in Table A-7; and/or ii) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequence(s); and iii) the Hallmark residues at positions 83 and 84 are as indicated in each of the sequences above; and in which: (d) FR4 is chosen from the group consisting of the amino acid sequences:

[103] WGQGTQVTVSS [113] [SEQ ID NO: 13]

[103] WGQGTLVTVSS [113] [SEQ ID NO: 14] or from the group consisting of amino acid sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with one of the above amino acid sequence; in which i) any amino acid substitution at any position other than a Hallmark position is preferably either a conservative amino acid substitution (as defined herein) and/or an amino acid substitution as defined in Table A-8; and/or ii) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequence(s); and iii) the Hallmark residues at positions 103, 104 and 108 are as indicated in each of the sequences above; and/or from the group consisting of amino acid sequences that have 3, 2 or only 1 "amino acid difference(s)" (as defined herein) with one of the above amino acid sequences, in which: i) any amino acid substitution at any position other than a Hallmark position is preferably either a conservative amino acid substitution (as defined herein) and/or an amino acid substitution as defined in Table A-8; and/or ii) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequence(s); and iii) the Hallmark residues at positions 103, 104 and 108 are as indicated in each of the sequences above;

FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4

and/or from the group consisting of amino acid sequences that have 3, 2 or only 1 "amino acid difference(s)" (as defined herein) with one of the above amino acid sequences, in which: i) any amino acid substitution at any position other than a Hallmark position is preferably either a conservative amino acid substitution (as defined herein) and/or an amino acid substitution as defined in Table A-5: and/or ii) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequence(s); and iii) the Hallmark residue at position is as indicated in the sequence above; and in which: (b) FR2 is chosen from the group consisting of the amino acid sequences:

[36] WFRQAPGKERELVA [49] [SEQ ID

NO: 6]

[36] WFRQAPGKEREFVA [49] [SEQ ID NO: 7]

[36] WFRQAPGKEREGA [49] [SEQ ID NO: 8]

[36] WFRQAPGKQRELVA [49] [SEQ ID

NO: 9]

[36] WFRQAPGKQREFVA [49] [SEQ ID NO: 10]

and/or from the group consisting of amino acid sequences that have 2 or only 1 "amino acid difference (s)" (as defined herein) with one of the above amino acid sequences, in which: i) any amino acid substitution at any position other than a Hallmark position is preferably either a conservative amino acid substitution (as defined herein) and/or an amino acid substitution as defined in Table A-6; and/or ii) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequence(s); and iii) the Hallmark residues at positions 37, 44, 45 and 47 are as indicated in each of the sequences above; and in which:

(c) FR3 is chosen from the group consisting of the amino acid sequence:

[66] RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA [94] [SEQ ID NO: 12]

and/or from the group consisting of amino acid sequences that have 3, 2 or only 1 "amino acid difference(s)" (as defined herein) with one of the above amino acid sequences, in which: i) any amino acid substitution at any position other than a Hallmark position is preferably either a conservative amino acid substitution (as defined herein) and/or an amino acid substitution as defined in Table A-7; and/or ii) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequence(s); and iii) the Hallmark residues at positions 83 and 84 are as indicated in each of the sequences above; and in which:

(d) FR4 is chosen from the group consisting of the amino acid sequences:

[103] WGQGTQVTVSS [1.13] [SEQ ID NO: 13]

[103] WGQGTLVTVSS [1 13] [SEQ ID NO: 14]

and/or from the group consisting of amino acid sequences that have 3, 2 or only 1 "amino acid difference(s)" (as defined herein) with one of the above amino acid sequences, in which: i) any amino acid substitution at any position other than a Hallmark position is preferably either a conservative amino acid substitution (as defined herein) and/or an amino acid substitution as defined in Table A-8; and/or ii) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequence(s); and iii) the Hallmark residues at positions 103, 104 and 108 are as indicated in each of the sequences above;

FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4

in which FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions I to 3, respectively, and in which: and in which

(a) FRl is chosen from the group consisting of the amino acid sequence:

[1] QVQLQESGGGLVQAGGSLRLSCAASG [26] [SEQ ED NO: 5]

and/or from the group consisting of amino acid sequences that have 3, 2 or only 1 "amino acid differences )" (as defined herein) with one of the above amino acid sequences, in which: i) any amino acid substitution at any position other than a Hallmark position is preferably either a conservative amino acid substitution (as defined herein) and/or an amino acid substitution as defined in Table A-5; and/or ii) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequence(s); and iii) the Hallmark residue at position is as indicated in the sequence above; and in which:

(b) FR2 is chosen from the group consisting of the amino acid sequence: [36] WYRQAPGKGLEWA [49] [SEQ ID

NO: U ]

and/or from the group consisting of amino acid sequences that have 2 or only 1

"amino acid difference(s)" (as defined herein) with one of the above amino acid sequences, in which: i) any amino acid substitution at any position other than a Hallmark position is preferably either a conservative amino acid substitution (as defined herein) and/or an amino acid substitution as defined in Table A-6; and/or ii) said amino acid sequence preferably oniy contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequence(s); and iii) the Hallmark residues at positions 37, 44, 45 and 47 are as indicated in each of the sequences above; and in which: (c) FR3 is chosen from the group consisting of the amino acid sequence:

[66] RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA [94] [SEQ ID NO: 12]

and/or from the group consisting of amino acid sequences that have 3, 2 or only 1 "amino acid difference(s)" (as defined herein) with one of the above amino acid sequences, in which: i) any amino acid substitution at any position other than a Hallmark position is preferably either a conservative amino acid substitution (as defined herein) and/or an amino acid substitution as defined in Table A-7; and/or ii) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequence(s); and iii) the Hallmark residues at positions 83 and 84 are as indicated in each of the sequences above; and in which: (d) FR4 is chosen from the group consisting of the amino acid sequence:

[103] WGQGTQVTVSS [113] [SEQ ED NO: 13]

and/or from the group consisting of amino acid sequences that have 3, 2 or only

1 "amino acid difference(s)" (as defined herein) with one of the above amino acid sequences, in which: i) any amino acid substitution at any position other than a Hallmark position is preferably either a conservative amino acid substitution (as defined herein) and/or an amino acid substitution as defined in Table A-8; and/or ii) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequence(s); and iii) the Hallmark residues at positions 103, 104 and 108 are as indicated in each of the sequences above;

Some other framework sequences that can be present in the Nanobodies^® of the invention can be found in the European patent EP 656 946 mentioned above (see for example also the granted US equivalent 5,759,808),

Preferably, the CDR sequences and FR sequences in the Nanobodies^® of the invention are such that the Nanobody^® of the invention binds to IL-6/IL-6R complex with a dissociation constant (Kn) of 10^-5 to 10^-12 moles/liter or less, and preferably 10-

⁷ to 10^{- 12} moles/liter or less and more preferably 10^-8 to 10^-12 moles/liter, and/or with a binding affinity of at least 10⁷ JVT¹, preferably at least 10⁸ M^{- 1}, more preferably at least 10⁹ M ¹, such as at least 10¹² M^-1 and/or with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. The affinity of the Nanobody^® of the invention against IL-6/IL-6R complex can be determined in a manner known per se, for example using the assay described herein.

As will be clear from the disclosure herein, it is also within the scope of the invention to use natural or synthetic analogs, mutants, variants, alleles, homologs and orthologs (herein collectively referred to as "analogs") of the Nanobodies^® of the invention as defined herein. Thus, according to one embodiment of the invention, the term "Nanobody^® of the invention" in its broadest sense also covers such analogs. Generally, in such analogs, one or more amino acid residues may have been replaced, deleted and/or added, compared to the Nanobodies^® of the invention as defined herein. Such substitutions, insertions or deletions may be made in one or more of the framework regions and/or in one or more of the CDR' s. When such substitutions, insertions or deletions are made in one or more of the framework regions, they may be made at one or more of the Hallmark residues and/or at one or more of the other positions in the framework residues, although substitutions, insertions or deletions at the Hallmark residues are generally less preferred (unless these are suitable humanizing substitutions as described herein). By means of non-limiting examples, a substitution may for example be a conservative substitution (as described herein) and/or an amino acid residue may be replaced by another amino acid residue that naturally occurs at the same position in another V_HH domain (see Tables 4-7 for some non -limiting examples of such substitutions), although the invention is generally not limited thereto. Thus, any one or more substitutions, deletions or insertions, or any combination thereof, that either improve the properties of the Nanobody^® of the invention or that at least do not detract too much from the desired properties or from the balance or combination of desired properties of the Nanobody^® of the invention (i.e. to the extent that the Nanobody^® is no longer suited for its intended use) are included within the scope of the invention. A skilled person will generally be able to determine and select suitable substitutions, deletions or insertions, or suitable combinations of thereof, based on the disclosure herein and optionally after a limited degree of routine experimentation, which may for example involve introducing a limited number of possible substitutions and determining their influence on the properties of the Nanobodies^® thus obtained.

For example, and depending on the host organism used to express the Nanobody^® or polypeptide of the invention, such deletions and/or substitutions may be designed in such a way that one or more sites for post-translational modification (such as one or more glycosylation sites) are removed, as will be within the ability of the person skilled in the art. Alternatively, substitutions or insertions may be designed so as to introduce one or more sites for attachment of functional groups (as described herein), for example to allow site-specific pegylation (again as described herein). As can be seen from the data on the V_HH entropy and V_HH variability given in Tables 4 to 7 above, some amino acid residues in the framework regions are more conserved than others. Generally, although the invention in its broadest sense is not limited thereto, any substitutions, deletions or insertions are preferably made at positions that are less conserved. Also, generally, amino acid substitutions are preferred over amino acid deletions or insertions.

The analogues are preferably such that they cac bind to IL-6/1L-6R complex with a dissociation constant (K_D) of IG^-5 to lCTⁱ² moles/liter or less, and preferably 10- ⁷ to 10^{- 12} moles/liter or less and more preferably 10^-8 to 10^-12 moles/liter, and/or with a binding affinity of at least 10⁷ M^{- 1}, preferably at least 10⁸ M^{- 1}, more preferably at least 10⁹ JvT¹, such as at least 10¹² M^{- 1} and/or with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. The affinity of the analogue against IL-6/IL-6R complex can be determined in a manner known per se, for example using the assay described herein. The analogues are preferably also such that they retain the favourable properties the Nanobodies^®, as described herein.

One preferred class of analogues of the Nanobodies^® of the invention comprise Nanobodies^® that have been humanized (i.e. compared to the sequence of a naturally occurring Nanobody^® of the invention). As mentioned in the background art cited herein, such humanization generally involves replacing one or more amino acid residues in the sequence of a naturally occurring V_HH with the amino acid residues that occur at the same position in a human V_H domain, such as a human V_H3 domain. Examples of possible humanizing substitutions or combinations of humanizing substitutions will be clear to the skilled person, for example from the Tables herein, from the possible humanizing substitutions mentioned in the background art cited herein, and/or from a comparison between the sequence of a Nanobody^® and the sequence of a naturally occurring human V_H domain.

The humanizing substitutions should be chosen such that the resulting humanized Nanobodies^® still retain the favourable properties of Nanobodies^® as defined herein, and more preferably such that they are as described for analogues in the preceding paragraphs. A skilled person will generally be able to determine and select suitable humanizing substitutions or suitable combinations of humanizing substitutions, based on the disclosure herein and optionally after a limited degree of routine experimentation, which may for example involve introducing a limited number of possible humanizing substitutions and determining their influence on the properties of the Nanobodies^® thus obtained.

Generally, as a result of humanization, the Nanobodies^® of the invention may become more "human-like", while still retaining the favourable properties of the Nanobodies^® of the invention as described herein. As a result, such humanized Nanobodies^® may have several advantages, such as a reduced immunogenicity, compared to the corresponding naturally occurring V_HH domains. Again, based on the disclosure herein and optionally after a limited degree of routine experimentation, the skilled person will be able to select humanizing substitutions or suitable combinations of humanizing substitutions which optimize or achieve a desired or suitable balance between the favourable properties provided by the humanizing substitutions on the one hand and the favourable properties of naturally occurring V_HH domains on the other hand. The humanized and other analogues, and nucleic acid sequences encoding the same, can be provided in any manner known per se. For example, the analogues can be obtained by providing a nucleic acid that encodes a naturally occurring V_HH domain, changing the codons for the one or more amino acid residues that are to be substituted into the codons for the corresponding desired amino acid residues (e.g. by site-directed mutagenesis or by PCR using suitable mismatch primers), expressing the nucleic acid/nucleotide sequence thus obtained in a suitable host or expression system; and optionally isolating and/or purifying the analogue thus obtained to provide said analogue in essentially isolated form (e.g. as further described herein). This can generally be performed using methods and techniques known per se, which will be clear to the skilled person, for example from the handbooks and references cited herein, the background art cited herein and/or from the further description herein. Alternatively, a nucleic acid encoding the desired analogue can be synthesized in a manner known per se (for example using an automated apparatus for synthesizing nucleic acid sequences with a predefined amino acid sequence) and can then be expressed as described herein. Yet another technique may involve combining one or more naturally occurring and/or synthetic nucleic acid sequences each encoding a part of the desired analogue, and then expressing the combined nucleic acid sequence as described herein. Also, the analogues can be provided using chemical synthesis of the pertinent amino acid sequence using techniques for peptide synthesis known per se, such as those mentioned herein.

In this respect, it will be also be clear to the skilled person that the Nanobodies^® of the invention (including their analogues) can be designed and/or prepared starting from human V_H sequences (i.e. amino acid sequences or the corresponding nucleotide sequences), such as for example from human V_H3 sequences such as DP-47, DP-51 or DP-29, i.e. by introducing one or more camelizing substitutions (i.e. changing one or more amino acid residues in the amino acid sequence of said human V_H domain into the amino acid residues that occur at the corresponding position in a V_HH domain), so as to provide the sequence of a Nanobody^® of the invention and/or so as to confer the favourable properties of a Nanobody^® to the sequence thus obtained. Again, this can generally be performed using the various methods and techniques referred to in the previous paragraph, using an amino acid sequence and/or nucleotide sequence for a human V_H domain as a starting point.

Some preferred, but non-limiting camelizing substitutions can be derived from Tables 4 to 7. It will also be clear that camelizing substitutions are one or more of the Hallmark residues will generally have a greater influence on the desired properties than substitutions at one or more of the other amino acid positions, although both and any suitable combination thereof are included within the scope of the invention. For example, it is possible to introduce one or more camelizing substitutions that already confer at least some the desired properties, and then to introduce further camelizing substitutions that either further improve said properties and/or confer additional favourable properties. Again, the skilled person will generally be able to determine and select suitable camelizing substitutions or suitable combinations of camelizing substitutions, based on the disclosure herein and optionally after a limited degree of routine experimentation, which may for example involve introducing a limited number of possible camelizing substitutions and determining whether the favourable properties of Nanobodies^® are obtained or improved (i.e. compared to the original V_H domain).

Generally, however, such camelizing substitutions are preferably such that the resulting an amino acid sequence at least contains (a) a Q at position 108; and/or (b) a charged amino acid or a cysteine residue at position 45 and preferably also an E at position, and more preferably E at position 44 and R at position 45; and/or (c) P, R or S at position 103; and optionally one or more further camelizing substitutions. More preferably, the camelizing substitutions are such that they result in a Nanobody^® of the invention and/or in an analogue thereof (as defined herein), such as in a humanized analogue and/or preferably in an analogue that is as defined in the preceding paragraphs.

As will also be clear from the disclosure herein, it is also within the scope of the invention to use parts or fragments, or combinations of two or more parts or fragments, of the Nanobodies^® of the invention as defined herein. Thus, according to one embodiment of the invention, the term "Nanobody^® of the invention" in its broadest sense also covers such parts or fragments.

Generally, such parts or fragments of the Nanobodies^® of the invention (including analogues thereof) have amino acid sequences in which, compared to the amino acid sequence of the corresponding full length Nanobody^® of the invention (or analogue thereof), one or more of the amino acid residues at the N-terminal end, one or more amino acid residues at the C-terminal end, one or more contiguous internal amino acid residues, or any combination thereof, have been deleted and/or removed.

The parts or fragments are preferably such that they can bind to IL-6/IL-6R complex with a dissociation constant (K_D) of 10^-5 to 10^-12 moles/liter or less, and preferably 10^-7to 10^{- 12} moles/liter or less and more preferably 10^-8 to 10^-ϊ2 moles/liter, and/or with a binding affinity of at least 10⁷ M^*1, preferably at least 10^s M^-1, more preferably at least 10⁹ M^{- 1}, such as at least 10¹² M^{- 1} and/or with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. The affinity of the analog against IL-6/IL-6R complex can be determined in a manner known per se, for example using the assay described herein.

Any part or fragment is preferably such that it comprises at least 10 contiguous amino acid residues, preferably at least 20 contiguous amino acid residues, more preferably at least 30 contiguous amino acid residues, such as at least 40 contiguous amino acid residues, of the amino acid sequence of the corresponding full length Nanobody^® of the invention.

Also, any part or fragment is such preferably that it comprises at least one of CDRl, CDR2 and/or CDR3 or at least part thereof (and in particular at least CDR3 or at least part thereof). More preferably, any part or fragment is such that it comprises at least one of the CDR' s (and preferably at least CDR3 or part thereof) and at least one other CDR (i.e. CDRl or CDR2) or at least part thereof, preferably connected by suitable framework sequence(s) or at least part thereof. More preferably, any part or fragment is such that it comprises at least one of the CDR' s (and preferably at least CDR3 or part thereof) and at least part of the two remaining CDR' s, again preferably connected by suitable framework sequence(s) or at least part thereof.

According to another particularly preferred, but non-limiting embodiment, such a part or fragment comprises at least CDR3, such as FR3, CDR3 and FR4 of the corresponding full length Nanobody^® of the invention, i.e. as for example described in the International application WO 03/050531 (Lasters et al).

As already mentioned above, it is also possible to combine two or more of such parts or fragments (i.e. from the same or different. Nanobodies^® of the invention), i.e. to provide an analogue (as defined herein) and/or to provide further parts or fragments (as defined herein) of a Nanobody^® of the invention. It is for example also possible to combine one or more parts or fragments of a Nanobody^® of the invention with one or more parts or fragments of a human V_H domain.

The parts and fragments, and nucleic acid sequences encoding the same, can be provided and optionally combined in any manner known per se. For example, such parts or fragments can be obtained by inserting a stop codon in a nucleic acid that encodes a full-sized Nanobody^® of the invention, and then expressing the nucleic acid thus obtained in a manner known per se (e.g. as described herein). Alternatively, nucleic acids encoding such parts or fragments can be obtained by suitably restricting a nucleic acid that encodes a full-sized Nanobody^® of the invention or by synthesizing such a nucleic acid in a manner known per se. Parts or fragments may also be provided using techniques for peptide synthesis known per se.

The invention in its broadest sense also comprises derivatives of the Nanobodies^® of the invention. Such derivatives can generally be obtained by modification, and in particular by chemical and/or biological (e.g enzymatical) modification, of the Nanobodies^® of the Invention and/or of one or more of the amino acid residues that form the Nanobodies^® of the invention.

Examples of such modifications, as well as examples of amino acid residues within the Nanobody^® sequence that can be modified in such a manner (i.e. either on the protein backbone but preferably on a side chain), methods and techniques that can be used to introduce such modifications and the potential uses and advantages of such modifications will be clear to the skilled person.

For example, such a modification may involve the introduction (e.g. by covalent linking or in an other suitable manner) of one or more functional groups. residues or moieties into or onto the Nanobody^® of the invention, and in particular of one or more functional groups, residues or moieties that confer one or more desired properties or functionalities to the Nanobody^® of the invention. Example of such functional groups will be clear to the skilled person.

For example, such modification may comprise the introduction (e.g. by covalent binding or in any other suitable manner) of one or more functional groups that that increase the half-life, the solubility and/or the absoiption of the Nanobody^® of the invention, that reduce the immunogenicity and/or the toxicity of the Nanobody^® of the invention, that eliminate or attenuate any undesirable side effects of the Nanobody^® of the invention, and/or that confer other advantageous properties to and/or reduce the undesired properties of the Nanobodies^® and/or polypeptides of the invention; or any combination of two or more of the foregoing. Examples of such functional groups and of techniques for introducing them will be clear to the skilled person, and can generally comprise all functional groups and techniques mentioned in the general background art cited hereiπabove as well as the functional groups and techniques known per se for the modification of pharmaceutical proteins, and in particular for the modification of antibodies or antibody fragments (including ScFv' s and single domain antibodies), for which reference is for example made to Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, PA (1980). Such functional groups may for example be linked directly (for example covalently) to a Nanobody " of the invention, or optionally via a suitable linker or spacer, as will again be clear to the skilled person.

One of the most widely used techniques for increasing the half-life and/or the reducing immunogenicity of pharmaceutical proteins comprises attachment of a suitable pharmacologically acceptable polymer, such as poly(ethyleneglycol) (PEG) or derivatives thereof (such as methoxypoly(ethyleneglycol) or mPEG). Generally, any suitable form of pegylation can be used, such as the pegylation used in the art for antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFv' s); reference is made to for example Chapman, Nat. Biotechnol., 54, 531-545 (2002); by Veronese and Harris, Adv. Drug Deliv. Rev. 54, 453-456 (2003), by Harris and Chess, Nat. Rev. Drug. Discov., 2, (2003) and in WO 04/060965. Various reagents for pegylation of proteins are also commercially available, for example from Nektar Therapeutics, USA. Preferably, site-directed pegylation is used, in particular via a cystine-residue

(see for example Yang et al., Protein Engineering, ] 6, 10, 761-770 (2003). For example, for this purpose, PEG may be attached to a cystine residue thai naturally occurs in a Nanobody^® of the invention., a Nanobody^® of the invention may be modified so as to suitably introduce one or more cystine residues for attachment of PEG, or an amino acid sequence comprising one or more cystine residues for attachment of PEG may be fused to the N- and/or C -terminus of a Nanobody^® of the invention, all using techniques of protein engineering known per se to the skilled person.

Preferably, for the Nanobodies^® and proteins of the invention, a PEG is used with a molecular weight of more than 5000, such as more than 10.000 and less than 200.000, such as less than 100.000; for example in the range of 20.000-80.000.

Another, usually less preferred modification comprises N-linked or O-linked glycosylation, usually as part of co-translational and/or post-translational modification, depending on the host cell used for expressing the Nanobody^® or polypeptide of the invention.

Yet another modification may comprise the introduction of one or more detectable labels or other signal-generating groups or moieties, depending on the intended use of the labelled Nanobody^®. Suitable labels and techniques for attaching, using and delecting them will be clear to the skilled person, and for example include, but are not limited to, fluorescent labels (such as fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine and fluorescent metals such as ¹⁵²Eu or others metals from the lanthanide series), phosphorescent labels, chemilumine scent labels or bio luminescent labels (such as luminal, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, oxalate ester, dioxetane or GFP and its analogs ), radio-isotopes (such as ³H, ¹²⁵I, ³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ³⁶Cl, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, and ⁷⁵Se), metals, metals chelates or metallic cations (for example metallic cations such as ^99IΏTC, ¹²³I, ^{11 1}In, ¹³¹I, ⁹⁷Ru, ⁶⁷Cu, ⁶⁷Ga, and ⁶⁸Ga or other metals or metallic cations that are particularly suited for use in in vivo, in vitro or in situ diagnosis and imaging, such as (¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr, and ⁵⁶Fe), as well as chromophores and enzymes (such as malate dehydrogenase, staphylococcal nuclease, delta- V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, biotinavidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose- VI- phosphate dehydrogenase, glucoamylase and acetylcholine esterase). Other suitable labels will be clear to the skilled person, and for example include moieties that can be detected using NMR or ESR spectroscopy. Such labelled Nanobodies^® and polypeptides of the invention may for example be used for in vitro, in vivo or in situ assays (including immunoassays known per se such as ELISA, RIA, EIA and other "sandwich assays", etc.) as well as in vivo diagnostic and imaging purposes, depending on the choice of the specific label. As will be clear to the skilled person, another modification may involve the introduction of a chelating group, for example to chelate one of the metals or metallic cations referred to above. Suitable chelating groups for example include, without limitation, diethyl-enetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Yet another modification may comprise the introduction of a functional group that is one part of a specific binding pair, such as the biotin-(strept)avidin binding pair. Such a functional group may be used to link the Nanobody^® of the invention to another protein, polypeptide or chemical compound that is bound to the other half of the binding pair, i.e. through formation of the binding pair. For example, a Nanobody^® of the invention may be conjugated to biotin, and linked to another protein, polypeptide, compound or carrier conjugated to avidin or streptavidin. For example, such a conjugated Nanobody^® may be used as a reporter, for example in a diagnostic system where a detectable signal-producing agent is conjugated to avidin or streptavidin. Such binding pairs may for example also be used to bind the Nanobody^® of the invention to a carrier, including carriers suitable for pharmaceutical purposes. One non-limiting example are the liposomal formulations described by Cao and Suresh, Journal of Drug Targetting, 8, 4, 257 (2000). Such binding pairs may also be used to link a therapeutically active agent to the Nanobody^® of the invention.

For some applications, in particular for those applications in which it is intended to kill a cell that expresses the target against which the Nanobodies^® of the invention are directed (e.g. in the treatment of cancer), or to reduce or slow the growth and/or proliferation such a cell, the Nanobodies^® of the invention may also be linked to a toxin or to a toxic residue or moiety. Examples of toxic moieties, compounds or residues which can be linked to a Nanobody^® of the invention to provide - for example - a cytotoxic compound will be clear to the skilled person and can for example be found in the prior art cited above and/or in the further description herein. One example is the so-called ADEPT™ technology WO 03/055527.

Other potential chemical and enzymatical modifications will be clear to the skilled person. Such modifications may also be introduced for research purposes (e.g. to study function-activity relationships). Reference is for example made to Lundblad and Bradshaw, Biotechnol. Appl. Biochem., 26, 143-151 (1997).

Preferably, the derivatives are such that they bind to IL-6/IL-6R complex with an dissociation constant (K_D) of 10^-5 to 10^-12 moles/liter or less, and preferably 10^-7 to 10^-12 moles/liter or less and more preferably 10^-8 to 10^-12 moles/liter, and/or with a binding affinity of at least 10⁷ M^'1, preferably at least 10⁸ M^-1, more preferably at least 10⁹ M^-1, such as at least 10¹² M^-1 and/or with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. The affinity of a derivative of a Nanobody^® of the invention against IL-6/IL-6R complex can be determined in a manner known per se, for example using the assay described herein, As mentioned above, the invention also relates to proteins or polypeptides that essentially consist of or comprise at least one Nanobody^® of the invention. By "essentially consist of is meant that the amino acid sequence of the polypeptide of the invention either is exactly the same as the amino acid sequence of a Nanobody^® of the invention or corresponds to the amino acid sequence of a Nanobody^® of the invention which has a limited number of amino acid residues, such as 1-20 amino acid residues, for example 1-10 amino acid residues and preferably 1-6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 amino acid residues, added at the amino terminal end, at the carboxy terminal end, or at both the amino terminal end and the carboxy terminal end of the amino acid sequence of the Nanobody^®.

Said amino acid residues may or may not change, alter or otherwise influence the (biological) properties of the Nanobody^® and may or may not add further functionality to the Nanobody \ For example, such amino acid residues: a) can comprise an N-terminal Met residue, for example as result of expression in a heterologous host cell or host organism. b) may form a signal sequence or leader sequence that directs secretion of the Nanobody^® from a host cell upon synthesis. Suitable secretory leader peptides will be clear to the skilled person, and may be as further described herein.

Usually, such a leader sequence will be linked to the N-terminus of the Nanobody^®, although the invention in its broadest sense is not limited thereto; c) may form a sequence or signal that allows the NaDobody^® to be directed towards and/or to penetrate or enter into specific organs, tissues, cells, or parts or compartments of cells, and/or that allows the Nanobody^® to penetrate or cross a biological barrier such as a cell membrane, a cell layer such as a layer of epithelial cells, a tumor including solid tumors, or the blood-brain-barrier. Examples of such amino acid sequences will be clear to the skilled person. Some non-limiting examples are the small peptide vectors ("Pep-trans vectors") described in WO 03/026700 and in Temsamani et al., Expert Opin. Biol. Ther.,

1, 773 (2001); Temsamani and Vidal, Drug Discov. Today, 9, 1012 (004) and Rousselle, J. Pharmacol. Exp. Ther., 296, 124-131 (2001), and the membrane translocator sequence described by Zhao et al., Apoptosis, 8, 631-637 (2003). C-terminal and N-terminal amino acid sequences for intracellular targeting of antibody fragments are for example described by Cardinale et al., Methods, 34,

171 (2004). Other suitable techniques for intracellular targeting involve the expression and/or use of so-called "intrabodies" comprising a Nanobody^® of the invention, as mentioned below; d) may form a "tag", for example an amino acid sequence or residue that allows or facilitates the purification of the Nanobody^®, for example using affinity techniques directed against said sequence or residue. Thereafter, said sequence or residue may be removed (e.g. by chemical or enzymatical cleavage) to provide the Nanobody^® sequence (for this purpose, the tag may optionally be linked to the Nanobody" sequence via a cleavable linker sequence or contain a cleavable motif). Some preferred, but non-limiting examples of such residues are multiple histidine residues, glutatione residues and a myc-tag such as AAAEQKLISEEDLNGAA [SEQ ID NO:31]; e) may be one or more amino acid residues that have been functionalized and/or that can serve as a site for attachment of functional groups. Suitable amino acid residues and functional groups will be clear to the skilled person and include, but are not limited to, the amino acid residues and functional groups mentioned herein for the derivatives of the Nanobodies^® of the invention. According to another embodiment, a polypeptide of the invention comprises a

Nanobody^® of the invention, which is fused at its amino terminal end, at its carboxy terminal end, or both at its amino terminal end and at its carboxy terminal end to at least one further amino acid sequence, i.e. so as to provide a fusion protein comprising said Nanobody^® of the invention and the one or more further amino acid sequences. Such a fusion will also be referred to herein as a "Nanobody^® fusion".

The one or more further amino acid sequence may be any suitable and/or desired amino acid sequences. The further amino acid sequences may or may not change, alter or otherwise influence the (biological) properties of the Nanobody^®, and may or may not add further functionality to the Nanobody^® or the polypeptide of the invention. Preferably, the further amino acid sequence is such that it confers one or more desired properties or functionalities to the Nanobody^® or the polypeptide of the invention.

Example of such amino acid sequences will be clear to the skilled person, and may generally comprise all amino acid sequences that are used in peptide fusions based on conventional antibodies and fragments thereof (including but not limited to

ScFv's and single domain antibodies). Reference is for example made to the review by Holliger and Hudson, Nature Biotechnology, 23, 9, 11.264136 (2005),

For example, such an amino acid sequence may be an amino acid sequence that increases the half-life, the solubility, or the absorption, reduces the immunogenicity or the toxicity, eliminates or attenuates undesirable side effects, and/or confers other advantageous properties to and/or reduces the undesired properties of the polypeptides of the invention, compared to the Nanobody^® of the invention per se. Some non-limiting examples of such amino acid sequences are serum proteins, such as human serum albumin (see for example WO 00/27435) or haptenic molecules (for example haptens that are recognized by circulating antibodies, see for example WO 98/22141).

The further amino acid sequence may also provide a second binding site, which binding site may be directed against any desired protein, polypeptide, antigen, antigenic determinant or epitope (including but not limited to the same protein, polypeptide, antigen, antigenic determinant or epitope against which the Nanobody^® of the invention is directed, or a different protein, polypeptide, antigen, antigenic determinant or epitope). For example, the further amino acid sequence may provide a second binding site that is directed against a serum protein (such as, for example, human serum albumin or another serum protein such as IgG), so as to provide increased half-life in serum. Reference is for example made to EP 0 368 684, WO 91/01743, WO 01/45746 and WO 04/003019 (in which various serum proteins are mentioned), the International application by applicant entitled "Nanobodies® against amyloid-beta and polypeptides comprising the same for the treatment of degenerative neural diseases such as Alzheimer's disease" (in which various other proteins are mentioned), as well as to Harmsen et al., Vaccine, 23 (41); 4926-42.

According to another embodiment, the one or more further amino acid sequences may comprises one or more parts, fragments or domains of conventional 4- chain antibodies (and in particular human antibodies) and/or of heavy chain antibodies. For example, although usually less preferred, a Nanobody^® of the invention may be linked to a conventional (preferably human) V_H or V_L domain domain or to a natural or synthetic analogue of a V_H or V_L domain, again optionally via a linker sequence (including but not limited to other (single) domain antibodies, such as the dAb's described by Ward et al.).

The at least one Nanobody^® may also be linked to one or more (preferably human) CHj, CHa and/or CH₃ domains, optionally via a linker sequence. For instance, a Nanobody^® linked to a suitable CH₁ domain could for example be used - together with suitable light chains - to generate antibody fragments/structures analogous to conventional Fab fragments or F(ab')2 fragments, but in which one or (in case of an F(ab')2 fragment) one or both of the conventional V_H domains have been replaced by a Nanobody^® of the invention. Also, two Nanobodies^® could be linked to a CH3 domain (optionally via a linker) to provide a construct with increased half-life in vivo.

According to one specific embodiment of a polypeptide of the invention, one or more Nanobodies^® of the invention may linked to one or more antibody parts, fragments or domains that confer one or more effector functions to the polypeptide of the invention and/or may confer the ability Io bind to one or more Fc receptors. For example, for this purpose, and without being limited thereto, the one or more further amino acid sequences may comprise one or more CH₂ and/or CH₃ domains of an antibody, such as from a heavy chain antibody (as described herein) and more preferably from a conventional human 4-chain antibody; and/or may form (part of) and Fc region, for example from IgG, from IgE or from another human Ig. For example, WO 94/04678 describes heavy chain antibodies comprising a Camelid V_HH domain or a humanized derivative thereof (i.e. a Nanobody^®), in which the Camelidae CH₂ and/or CH₃ domain have been replaced by human CH₂ and CH₃ domains, so as to provide an immunoglobulin that consists of 2 heavy chains each comprising a Nanobody^® aad human CH2 and CH3 domains (but no CHl domain), which immunoglobulin has the effector function provided by the CH2 and CH3 domains and which immunoglobulin can function without the presence of any light chains. Other amino acid sequences that can be suitably linked to the Nanobodies^® of the invention so as to provide an effector function will be clear to the skilled person, and may be chosen on the basis of the desired effector function(s). Reference is for example made to WO 04/058820, WO 99/42077 and WO 05/017148, as well as the review by Holliger and Hudson, supra. Coupling of a Nanobody of the invention to an Fc portion may also lead to an increased half-life, compared to the corresponding Nanobody^® of the invention. For some applications, the use of an Fc portion and/or of constant domains (i.e. CH₂ and/or CH₃ domains) that confer increased half-life without any biologically significant effector function may also be suitable or even preferred. Other suitable constructs comprising one or more Nanobodies^® and one or more constant domains with increased half-life in vivo will be clear to the skilled person, and may for example comprise two Nanobodies^® linked to a CH3 domain, optionally via a linker sequence. Generally, any fusion protein or derivatives with increased half-life will preferably have a molecular weigth of more than 50 kD, the cut-off value for renal absorption. The further amino acid sequences may also form a signal sequence or leader sequence that directs secretion of the Nanobody^® or the polypeptide of the invention from a host cell upon synthesis (for example to provide a pre-, pro- or prepro- form of the polypeptide of the invention, depending on the host cell used to express the polypeptide of the invention).

The further amino acid sequence may also form a sequence or signal that allows the Nanobody^® or polypeptide of the invention to be directed towards and/or to penetrate or enter into specific organs, tissues, cells, or parts or compartments of cells, and/or that allows the Nanobody^® or polypeptide of the invention to penetrate or cross a biological barrier such as a cell membrane, a cell layer such as a layer of epithelial cells, a tumor including solid tumors, or the blood-brain-barrier. Suitable examples of such amino acid sequences will be clear to the skilled person, and for example include, but are not limited to, the "Peptrans" vectors mentioned above, the sequences described by Cardinale et al. and the amino acid sequences and antibody fragments known per se that, can be used to express or produce the Nanobodies^® and polypeptides of the invention as so-called "intrabodies", for example as described in WO 94/02610, WO 95/22618, US-A-6004940, WO 03/014960, WO 99/07414; WO 05/01690; EP 1 512 696; and in Cattaneo, A. & Biocca, S. (1997) Intracellular Antibodies: Development and Applications. Landes and Springer- Verlag; and in Kontermann, Methods 34, (2004), 163-170, and the further references described therein.

For some applications, in particular for those applications in which it is intended to kill a cell that expresses the target against which the Nanobodies^® of the invention are directed (e.g. in the treatment of cancer), or to reduce or slow the growth and/or proliferation such a cell, the Nanobodies^® of the invention may also be linked to a (cyto)toxic protein or polypeptide. Examples of such toxic proteins and polypeptides which can be linked to a Nanobody^® of the invention to provide - for example - a cytotoxic polypeptide of the invention will be clear to the skilled person and can for example be found in the prior art cited above and/or in the further description herein. One example is the so-called ADEPT™ technology WO 03/055527.

According to one preferred, but non-limiting embodiment, said one or more further amino acid sequences comprise at least one further Nanobody^®, so as to provide a polypeptide of the invention that comprises at least two, such as three, four, five or more Nanobodies^®, in which said Nanobodies^® may optionally be linked via one or more linker sequences (as defined herein). Polypeptides of the invention that comprise two or more Nanobodies^®, of which at least one is a Nanobody^® of the invention, will also be referred to herein as "multivalent" polypeptides of the invention, and the Nanobodies^® present in such polypeptides will also be referred to herein as being in a "multivalent format". For example a "bivalent" polypeptide of the invention comprises two Nanobodies^®, optionally linked via a linker sequence, whereas a "trivalent" polypeptide of the invention comprises three Nanobodies^®, optionally linked via two linker sequences; etc.; in which at least one of the Nanobodies^® present in the polypeptide, and up to all of the Nanobodies^® present in the polypeptide, is/are a Nanobody^® of the invention.

In a multivalent polypeptide of the invention, the two or more Nanobodies^® may be the same or different, and may be directed against the same antigen or antigenic determinant (for example against the same part(s) or epitope(s) or against different parts or epitopes) or may alternatively be directed against different antigens or antigenic determinants; or any suitable combination thereof. For example, a bivalent polypeptide of the invention may comprise (a) two identical Nanobodies^®; (b) a first Nanobody^® directed against a first antigenic determinant of a protein or antigen and a second Nanobody directed against the same antigenic determinant of said protein or antigen which is different from the first Nanobody^®; (c) a first Nanobody^® directed against a first antigenic determinant of a protein or antigen and a second Nanobody^® directed against another antigenic determinant of said protein or antigen; or (d) a first Nanobody^® directed against a first protein or antigen and a second Nanobody^® directed against a second protein or antigen (i.e. different from said first antigen). Similarly, a trivalent polypeptide of the invention may, for example and without being limited thereto, comprise (a) three identical Nanobodies^®; (b) two identical Nanobody^® against a first antigenic determinant of an antigen and a third Nanobody^® directed against a different antigenic determinant of the same antigen; (c) two identical Nanobody^® against a first antigenic determinant of an antigen and a third Nanobody^® directed against a second antigen different from said first antigen; (d) a first Nanobody^® directed against a first antigenic determinant of a first antigen, a second Nanobody^® directed against a second antigenic determinant of said first antigen and a third Nanobody^® directed against a second antigen different from said first antigen; or (e) a first Nanobody^® directed against a first antigen, a second Nanobody directed against a second antigen different from said first antigen, and a third Nanobody^® directed against a third antigen different from said first and second antigen.

Polypeptides of the invention that contain at least two Nanobodies^®, in which at least one Nanobody^® is directed against a first antigen (i.e. against IL-6/IL-6R complex) and at least one Nanobody^® is directed against a second antigen (i.e. different from IL-6/IL-6R complex), will also be referred to as "multispecific" polypeptides of the invention, and the Nanobodies^® present in such polypeptides will also be referred to herein as being in a "multivalent format". Thus, for example, a "bispecific" polypeptide of the invention is a polypeptide that comprises at least one Nanobody^® directed against a first antigen (i.e. IL-6/IL-6R complex) and at least one further Nanobody^® directed against a second antigen (i.e. different from IL-6/IL-6R complex), whereas a "trispecific" polypeptide of the invention is a polypeptide that comprises at least one Nanobody^® directed against a first antigen (i.e. IL-6/IL-6R complex), at least one further Nanobody^® directed against a second antigen (i.e. different from IL-6/IL-6R complex) and at least one further Nanobody^® directed against a third antigen (i.e. different from both IL-6/IL-6R complex and the second antigen); etc.

Accordingly, in its simplest form, a bispecific polypeptide of the invention is a bivalent polypeptide of the invention (as defined herein), comprising a first Nanobody^® directed against IL-6/IL-6R complex and a second Nanobody^® directed against a second antigen, in which said first and second Nanobody^® may optionally be linked via a linker sequence (as defined herein); whereas a trispecific polypeptide of the invention in its simplest form is a trivalent polypeptide of the invention (as defined herein), comprising a first Nanobody^® directed against LL-6/IL-6R complex, a second Nanobody^® directed against a second antigen and a third Nanobody^® directed against a third antigen, in which said first, second and third Nanobody^® may optionally be linked via one or more, and in particular one and more in particular two, linker sequences.

However, as will be clear from the description hereinabove, the invention is not limited thereto, in the sense that a multispecific polypeptide of the invention may comprise at least one Nanobody^® against IL-6/IL-6R complex and any number of Nanobodies^® directed against one or more antigens different from IL-6/IL-6R complex.

Furthermore, although it is encompassed within the scope of the invention that the specific order or arrangement of the various Nanobodies ° in the polypeptides of the invention may have some influence on the properties of the final polypeptide of the invention (including but not limited to the affinity, specificity or avidity for IL- 6/IL-6R complex or against the one or more other antigens), said order or arrangement is usually not critical and may be suitably chosen by the skilled person, optionally after on some limited routine experiments based on the disclosure herein. Thus, when reference is made to a specific multivalent or multispecific polypeptide of the invention, it should be noted that this encompasses any order or arrangements of the relevant Nanobodies^®, unless explicitly indicated otherwise.

Finally, it is also within the scope of the invention that the polypeptides of the invention contain two or more Nanobodies^® and one or more further amino acid sequences (as mentioned herein).

For multivalent and multispecific polypeptides containing one or more V_HH domains and their preparation, reference is also made to Conrath et al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001, as well as to for example WO 96/34103 and WO 99/23221. Some other examples of some specific multispecific and/or multivalent polypeptide of the invention can be found in the applications by applicant referred to herein.

One preferred, but non-limiting example of a multispecific polypeptide of the invention comprises at least one Nanobody^® of the invention and at least one Nanobody^® that provides for an increased half-life. Some preferred, but non-limiting examples of such Nanobodies^® include Nanobodies^® directed against serum proteins, such as human serum albumin, thyroxine-binding protein, (human) transferrin, fibrinogen, an immunoglobulin such as IgG, IgE or IgM,.or one of the other serum proteins listed in WO 04/003019. For example, for experiments in mice, Nanobodies^® against mouse serum albumin (MSA) can be used, whereas for pharmaceutical use, Nanobodies^® against human serum albumin can be used. Another embodiment of the present invention is a polypeptide construct as described above wherein said at least one (human) serum protein is any of (human) serum albumin, (human) serum immunoglobulins, (human)

According to a specific, but non-limiting aspect of the invention, the polypeptides of the invention contain, besides the one or more Nanobodies of the invention, at least one Nanobody against human serum albumin. Although these Nanobodies against human serum albumin may be as generally described in the applications by applicant cited above (see for example W04/062551), according to a particularly preferred, but non-limiting embodiment, said Nanobody against human serum albumin consists of 4 framework regions (FRl to FR4 respectively) and 3 complementarity determining regions (CDRl to CDR3 respectively), in which: i) CDRl is an amino acid sequence chosen from the group consisting of:

SFGMS [SEQ ID NO: 15]

LNLMG [SEQ ID NO: 16] INLLG [SEQ ID NO: 17]

NYWMY; [SEQ ID NO: 18] and/or from the group consisting of amino acid sequences that have 2 or only 1 "amino acid difference(s)" (as defined herein) with one of the above amino acid sequences, in which: (1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or

(2) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequences; and in which: ii) CDR2 is an amino acid sequence chosen from the group consisting of:

SISGSGSDTLYADSVKG [SEQ ID NO: 19]

TITVGDSTNYADSVKG [SEQ ID NO: 20]

TITVGDSTSYADSVKG [SEQ ID NO: 21] SINGRGDDTRYADSVKG [SEQ ID NO: 22]

AISADSSTKNYADSVKG [SEQ ED NO: 23]

AISADSSDKRYADSVKG [SEQ ED NO: 24]

RISTGGGYSYYADSVKG [SEQ ID NO: 25] or from the group consisting of amino acid sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with one of the above amino acid sequences; in which (1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or

(2) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequences; and/or from the group consisting of amino acid sequences that have 3, 2 or only 1 "amino acid difference(s)" (as defined herein) with one of the above amino acid sequences, in which:

(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or (2) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequences; and in which: iii) CDR3 is an amino acid sequence chosen from the group consisting of: DREAQVDTLDFDY [SEQ ID NO: 26] or from the group consisting of amino acid sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with one of the above amino acid sequences; in which (1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or

(2) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequences; and/or from the group consisting of amino acid sequences that have 3, 2 or only 1 "amino acid difference(s)" (as defined herein) with one of the above amino acid sequences, in which: (1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or

(2) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequences; or from the group consisting of:

GGSLSR [SEQ ID NO: 27]

RRTWHSEL [SEQ DD NO: 28]

GRSVSRS [SEQ ID NO: 29] GRGSP [SEQ K) NO: 30] and/or from the group consisting of amino acid sequences that have 3, 2 or only 1 "amino acid difference(s)" (as defined herein) with one of the above amino acid sequences, in which:

(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or

(2) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequences.

In another aspect, the invention relates to a Nanobody against human serum albumin, which consist of 4 framework regions (FRl to FR4 respectively) and 3 complementarity determining regions (CDRl to CDR3 respectively), which is chosen from the group consisting of Nanobodies with the one of the following combinations of CDRl, CDR2 and CDR3, respectively:

- CDRl: SFGMS; CDR2: SISGSGSDTLYADSVKG; CDR3: GGSLSR; - CDRl: LNLMG; CDR2: TITVGDSTNYADSVKG; CDR3: RRTWHSEL;

- CDRl: INLLG; CDR2: T1TVGDSTSYADSVKG; CDR3: RRTWHSEL;

- CDRl: SFGMS; CDR2: SLNGRGDDTRYADSVKG; CDR3: GRSVSRS;

- CDRl: SFGMS; CDR2: AISADSSDKRYADSVKG; CDR3: GRGSP;

- CDRl: SFGMS; CDR2: AISADSSDKRYADSVKG; CDR3: GRGSP; - CDRl: NYWMY; CDR2: RISTGGGYS YYADSVKG; CDR3:

DREAQVDTLDFDY.

In the Nanobodies of the invention that comprise the combinations of CDR' s mentioned above, each CDR can be replaced by a CDR chosen from the group consisting of amino acid sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with the mentioned CDR' s; in which

(2) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequences; and/or chosen from the group consisting of amino acid sequences that have 3, 2 or only 1 (as indicated in the preceding paragraph) "amino acid difference(s)" (as defined herein) with the mentioned CDR(s) one of the above amino acid sequences. in which:

(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and/or (2) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequences.

However, of the Nanobodies of the invention that comprise the combinations of CDR' s mentioned above, Nanobodies comprising one or more of the CDR' s listed above are particularly preferred; Nanobodies comprising two or more of the CDR' s listed above are more particularly preferred; and Nanobodies comprising three of the

CDR' s listed above are most particularly preferred.

In these Nanobodies against human serum albumin, the Framework regions

FRl to FR4 are preferably as defined hereinabove for the Nanobodies of the invention.

Some preferred, but non-limiting examples of Nanobodies directed against human serum albumin that can be used in the polypeptides of the invention are listed in Table A-9 below. ALB-8 is a humanized version of ALB-I.

Table A-9: Preferred, but non-limiting examples of albumin -binding Nanobodies

<Name, SEQ ID #; PRT (protein) ; ->

Sequence

Generally, any derivatives and/or polypeptides of the invention with increased half-life (for example pegylated Nanobodies or polypeptides of the invention, multispecific Nanobodies directed against Xxxx, and (human) serum albumin, or Nanobodies fused to an Fc portion, all as described herein) have a half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, the half-life of the corresponding Nanobody of the invention. Also, any derivatives or polypeptides of the invention with an increase half- life preferably have a half-life of more than 1 hour, preferably more than 2 hours, more preferably of more than 6 hours, such as of more than 12 hours, and for example of about one day. two days, one week, two weeks or three weeks, and preferably no more than 2 months, although the latter may be less critical. Half-life can generally be defined as the time taken for the serum concentration of the polypeptide to be reduce by 50%, in vivo, for example due to degradation of the ligand and/or clearance or sequestration of the ligand by natural mechanisms. Methods for pharmacokinetic analysis and determination of half-life are familiar to those skilled in the art. Details may be found in Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and in Peters et al, Pharmacokinete analysis: A Practical Approach (1996). Reference is also made to "Pharmacokinetics", M Gibaldi & D Perron, published by Marcel Dekker, 2 nd Rev. ex edition (1982).

According to one aspect of the invention the polypeptides are capable of binding to one or more molecules which can increase the half-life of the polypeptide in vivo.

The polypeptides of the invention are stabilised in vivo and their half-life increased by binding to molecules which resist degradation and/or clearance or sequestration. Typically, such molecules are naturally occurring proteins which themselves have a long half-life in vivo.

Another preferred, but non-limiting example of a multispecific polypeptide of the invention comprises at least one Nanobody of the invention and at least one Nanobody that directs the polypeptide of the invention towards, and/or that allows the polypeptide of the invention to penetrate or to enter into specific organs, tissues, cells, or parts or compartments of cells, and/or that allows the Nanobody to penetrate or cross a biological barrier such as a cell membrane, a cell layer such as a layer of epithelial cells, a tumor including solid tumors, or the blood-brain-barrier. Examples of such Nanobodies include Nanobodies that are directed towards specific cell- surface proteins, markers or epitopes of the desired organ, tissue or cell (for example cell-surface markers associated with tumor cells), and the single-domain brain targeting antibody fragments described in WO 02/057445, of which FC44 (SEQ ID NO 35) and FC5 (SEQ ID NO: 36) are preferred examples.

Table A-10: Sequence listing of FC44 and FC5

In the polypeptides of the invention, the one or more Nanobodies and the one or more polypeptides may be directly linked to each other (as for example described in WO 99/23221) and/or may be linked to each other via one or more suitable spacers or linkers, or any combination thereof.

Suitable spacers or linkers for use in multivalent and multispecific polypeptides will be clear to the skilled person, and may generally be any linker or spacer used in the art to link amino acid sequences. Preferably, said linker or spacer is suitable for use in constructing proteins or polypeptides that are intended for pharmaceutical use. Some particularly preferred spacers include the spacers and linkers that are used in the art to link antibody fragments or antibody domains. These include the linkers mentioned in the general background art cited above, as well as for example linkers that are used in the art to construct diabodies or ScFv fragments (in this respect, however, its should be noted that, whereas in diabodies and in ScFv fragments, the linker sequence used should have a length, a degree of flexibility and other properties that allow the pertinent V_H and V_L domains to come together to form the complete antigen-binding site, there is no particular limitation on the length or the flexibility of the linker used in the polypeptide of the invention, since each Nanobody by itself forms a complete antigen-binding site).

For example, a linker may be a suitable amino acid sequence, and in particular amino acid sequences of between 1 and 50, preferably between 1 and 30, such as between 1 and 10 amino acid residues. Some preferred examples of such amino acid sequences include gly-ser linkers, for example of the type (gly_λser_y)_z, such as (for example (gly₄ser)₃ or (gly₃ser₂)₃, as described in WO 99/42077, hinge-like regions such as the hinge regions of naturally occurring heavy chain antibodies or similar sequences (such as described in WO 94/04678 ).

Some other particularly preferred linkers are poly-alanine (such as AAA), as well as the linkers mentioned in Table A-11, of which AAA, GS-7 and GS-9 are particularly preferred.

Table A-11: Sequence listing of linkers

Other suitable linkers generally comprise organic compounds or polymers, in particular those suitable for use in proteins for pharmaceutical use. For instance, poly(ethyleneglycol) moieties have been used to link antibody domains, see for example WO 04/081026.

It is encompassed within the scope of the invention that the length, the degree of flexibility and/or other properties of the linker(s) used (although not critical, as it usually is for linkers used in ScFv fragments) may have some influence on the properties of the final polypeptide of the invention, including but not limited to the affinity, specificity or avidity for Xxxx, or against the one or more of the other antigens. Based on the disclosure herein, the skilled person will be able to determine the optimal linker(s) for use in a specific polypeptide of the invention, optionally after on some limited routine experiments.

For example, in multivalent polypeptides of the invention that comprise Nanobodies directed against a multimeric antigen (such as a multimeric receptor or other protein), the length and flexibility of the linker are preferably such that it allows each Nanobody of the invention present in the polypeptide to bind to the antigenic determinant on each of the subunits of the multimer. Similarly, in a multispecific polypeptide of the invention that comprises Nanobodies directed against two or more different antigenic determinants on the same antigen (for example against different epitopes of an antigen and/or against different subunits of a multimeric receptor, channel or protein), the length and flexibility of the linker are preferably such that it allows each Nanobody to bind to its intended antigenic determinant. Again, based on the disclosure herein, the skilled person will be able to determine the optimal Hnker(s) for use in a specific polypeptide of the invention, optionally after on some limited routine experiments.

It is also within the scope of the invention that the linker(s) used confer one or more other favourable properties or functionality to the polypeptides of the invention, and/or provide one or more sites for the formation of derivatives and/or for the attachment of functional groups (e.g. as described herein for the derivatives of the Nanobodies of the invention). For example, linkers containing one or more charged amino acid residues (see Table A-2 above) can provide improved hydrophilic properties, whereas linkers that form or contain small epitopes or tags can be used for the purposes of detection, identification and/or purification. Again, based on the disclosure herein, the skilled person will be able to determine the optimal linkers for use in a specific polypeptide of the invention, optionally after on some limited routine experiments.

Finally, when two or more linkers are used in the polypeptides of the invention, these linkers may be the same or different. Again, based on the disclosure herein, the skilled person will be able to determine the optimal linkers for use in a specific polypeptide of the invention, optionally after on some limited routine experiments.

Usually, for easy of expression and production, a polypeptide of the invention will be a linear polypeptide. However, the invention in its broadest sense is not limited thererto. For example, when a polypeptide of the invention comprises three of more Nanobodies, it is possible to link them use a linker with three or more "arms", which each "arm" being linked to a Nanobody, so as to provide a "star-shaped" construct. It is also possible, although usually less preferred, to use circular constructs. The invention also comprises derivatives of the polypeptides of the invention, which may be essentially analogous to the derivatives of the Nanobodies^® of the invention, i.e. as described herein.

The invention also comprises proteins or polypeptides that "essentially consist" of a polypeptide of the invention (in which the wording "essentially consist of" has essentially the same meaning as indicated hereinabove).

According to one embodiment of the invention, the polypeptide of the invention is in essentially isolated from, as defined herein.

The Nanobodies^®, polypeptides and nucleic acids of the invention can be prepared in a manner known per se, as will be clear to the skilled person from the further description herein. For example, the Nanobodies^® and polypeptides of the invention can be prepared in any manner known per se for the preparation of antibodies and in particular for the preparation of antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments). Some preferred, but non-limiting methods for preparing the Nanobodies^®, polypeptides and nucleic acids include the methods and techniques described herein.

As will be clear to the skilled person, one particularly useful method for preparing a Nanobody^® and/or a polypeptide of the invention generally comprises the steps of: the expression, in a suitable host cell or host organism (also referred to herein as a "host of the invention") or in another suitable expression system of a nucleic acid that encodes said Nanobody^® or polypeptide of the invention (also referred to herein as a "nucleic acid of the invention"), optionally followed by: - isolating and/or purifying the Nanobody^® or polypeptide of the invention thus obtained,

In particular, such a method may comprise the steps of: cultivating and/or maintaining a host of the invention under conditions that are such that said host of the invention expresses and/or produces at least one Nanobody^® and/or polypeptide of the invention; optionally followed by: isolating and/or purifying the Nanobody^® or polypeptide of the invention thus obtained.

A nucleic acid of the invention can be in the form of single or double stranded

DNA or RNA, and is preferably in the form of double stranded DNA. For example, the nucleotide sequences of the invention may be genomic DNA, cDNA or synthetic

DNA (such as DNA with a codon usage that has been specifically adapted for expression in the intended host cell or host organism).

According to one embodiment of the invention, the nucleic acid of the invention is in essentially isolated from, as defined herein. The nucleic acid of the invention may also be in the form of, be present in and/or be part of a vector, such as for example a plasmid, cosmid or YAC, which again may be in essentially isolated form.

The nucleic acids of the invention can be prepared or obtained in a manner known per se, based on the information on the amino acid sequences for the polypeptides of the invention given herein, and/or can be isolated from a suitable natural source. To provide analogues, nucleotide sequences encoding naturally occurring V_HH domains can for example be subjected to site-directed mutagenesis, so at to provide a nucleic acid of the invention encoding said analogue. Also, as will be clear to the skilled person, to prepare a nucleic acid of the invention, also several nucleotide sequences, such as at least one nucleotide sequence encoding a

Nanobody^® and for example nucleic acids encoding one or more linkers can be linked together in a suitable manner. Techniques for generating the nucleic acids of the invention will be clear to the skilled person and may for instance include, but are not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and/or synthetic sequences (or two or more parts thereof), introduction of mutations that lead to the expression of a truncated expression product; introduction of one or more restriction sites (e.g. to create cassettes and/or regions that may easily be digested and/or ligated using suitable restriction enzymes), and/or the introduction of mutations by means of a PCR reaction using one or more "mismatched" primers, using for example a sequence of a naturally occurring GPCR as a template. These and other techniques will be clear to the skilled person, and reference is again made to the standard handbooks, such as Sambrook et al. and Ausubel et al., mentioned above, as well as the Examples below.

The nucleic acid of the invention may also be in the form of, be present in and/or be part of a genetic construct, as will be clear to the person skilled in the art. Such genetic constructs generally comprise at least one nucleic acid of the invention that is optionally linked to one or more elements of genetic constructs known per se, such as for example one or more suitable regulatory elements (such as a suitable promoter(s), enhancer(s), terminator(s), etc) and the further elements of genetic constructs referred to herein. Such genetic constructs comprising at least one nucleic acid of the invention will also be referred to herein as "genetic constructs of the invention".

The genetic constructs of the invention may be DNA or RNA, and are preferably double- stranded DNA. The genetic constructs of the invention may also be in a form suitable for transformation of the intended host cell or host organism, in a form suitable for integration into the genomic DNA of the intended host cell or in a form suitable independent replication, maintenance and/or inheritance in the intended host organism. For instance, the genetic constructs of the invention may be in the form of a vector, such as for example a plasmid, cosmid, YAC, a viral vector or transposon. In particular, the vector may be an expression vector, i.e. a vector that can provide for expression in vitro and/or in vivo (e.g. in a suitable host cell, host organism and/or expression system).

In a preferred but non-limiting embodiment, a genetic construct of the invention comprises a) at least one nucleic acid of the invention; operably connected to b) one or more regulatory elements, such as a promoter and optionally a suitable terminator; and optionally also c) one or more further elements of genetic constructs known per se; in which the terms "regulatory element", "promoter", "terminator" and "operably connected" have their usual meaning in the art (as further described herein); and in which said "further elements" present in the genetic constructs may for example be 3'- or 5'-UTR sequences, leader sequences, selection markers, expression markers/reporter genes, and/or elements that may facilitate or increase (the efficiency of) transformation or integration. These and other suitable elements for such genetic constructs will be clear to the skilled person, and may for instance depend upon the type of construct used, the intended host cell or host organism; the manner in which the nucleotide sequences of the invention of interest are to be expressed (e.g. via constitutive, transient or inducible expression); and/or the transformation technique to be used. For example, regulatory sequences, promoters and terminators known per se for the expression and production of antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments) may be used in an essentially analogous manner. Preferably, in the genetic constructs of the invention, said at least one nucleic acid of the invention and said regulatory elements, and optionally said one or more further elements, are "operably linked" to each other, by which is generally meant that they are in a functional relationship with each other. For instance, a promoter is considered "operably linked" to a coding sequence if said promoter is able to initiate or otherwise control/regulate the transcription and/or the expression of a coding sequence (in which said coding sequence should be understood as being "under the control of said promotor). Generally, when two nucleotide sequences are operably linked, they will be in the same orientation and usually also in the same reading frame. They will usually also be essentially contiguous, although this may also not be required.

Preferably, the regulatory and further elements of the genetic constructs of the invention are such that they are capable of providing their intended biological function in the intended host cell or host organism. For instance, a promoter, enhancer or terminator should be "operable" in the intended host cell or host organism, by which is meant that (for example) said promoter should be capable of initiating or otherwise controlling/regulating the transcription and/or the expression of a nucleotide sequence - e.g. a coding sequence - to which it is operably linked (as defined herein).

Some particularly preferred promoters include, but are not limited to, promoters known per se for the expression in the host cells mentioned herein; and in particular promoters for the expression in the bacterial cells, such as those mentioned herein and/or those used in the Examples. A selection marker should be such that it allows - i.e. under appropriate selection conditions - host cells and/or host organisms that have been (successfully) transformed with the nucleotide sequence of the invention to be distinguished from host cells/organisms that have not been (successfully) transformed. Some preferred, but non-limiting examples of such markers are genes that provide resistance against antibiotics (such as kanamycin or ampicillin), genes that provide for temperature resistance, or genes that allow the host cell or host organism to be maintained in the absence of certain factors, compounds and/or (food) components in the medium that are essential for survival of the non-transformed cells or organisms.

A leader sequence should be such that - in the intended host cell or host organism - it allows for the desired post-translational modifications and/or such that it directs the transcribed mRNA to a desired part or organelle of a cell, A leader sequence may also allow for secretion of the expression product from said cell. As such, the leader sequence may be any pro-, pre-, or prepro-sequence operable in the host cell or host organism. Leader sequences may not be required for expression in a bacterial cell. For example, leader sequences known per se for the expression and production of antibodies and antibody fragments (including but not limited to single domain antibodies and ScFv fragments) may be used in an essentially analogous manner.

An expression marker or reporter gene should be such that - in the host cell or host organism - it allows for detection of the expression of (a gene or nucleotide sequence present on) the genetic construct. An expression marker may optionally also allow for the localisation of the expressed product, e.g. in a specific part or organelle of a cell and/or in (a) specific cell(s), tissue(s), organ(s) or part(s) of a multicellular organism. Such reporter genes may also be expressed as a protein fusion with the amino acid sequence of the invention. Some preferred, but non-limiting examples include fluorescent proteins such as GFP.

Some preferred, but non-limiting examples of suitable promoters, terminator and further elements include those that can be used for the expression in the host cells mentioned herein; and in particular those that are suitable for expression bacterial cells, such as those mentioned herein and/or those used in the Examples below. For some (further) non-limiting examples of the promoters, selection markers, leader sequences, expression markers and further elements that may be present/used in the genetic constructs of the invention - such as terminators, transcriptional and/or translational enhancers and/or integration factors - reference is made to the general handbooks such as Sambrook et al. and Ausubel et al, mentioned above, as well as to the examples that are given in WO 95/07463, WO 96/23810, WO 95/07463, WO

95/21191 , WO 97/11094, WO 97/42320, WO 98/06737, WO 98/21355, US-A- 6,207,410, US-A- 5,693,492 and EP 1 085 089. Other examples will be clear to the skilled person. Reference is also made to the general background art cited above and the further references cited herein.

The genetic constructs of the invention may generally be provided by suitably linking the nucleotide sequence(s) of the invention to the one or more further elements described above, for example using the techniques described in the general handbooks such as Sambrook et al. and Ausubel et al., mentioned above.

Often, the genetic constructs of the invention will be obtained by inserting a nucleotide sequence of the invention in a suitable (expression) vector known per se. Some preferred, but non-limiting examples of suitable expression vectors are those used in the Examples below, as well as those mentioned herein.

The nucleic acids of the invention and/or the genetic constructs of the invention may be used to transform a host cell or host organism, i.e. for expression and/or production of the Nanobody^® or polypeptide of the invention. Suitable hosts or host cells will be clear to the skilled person, and may for example be any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism, for example: a bacteria] strain, including but not limited to gram-negative strains such as strains of Escherichia coli; of Proteus, for example of Proteus mirabilis; of Pseuάomonas, for example of Pseudomonas fluorescens; and gram-positive strains such as strains of Bacillus, for example of Bacillus subtilis or of Bacillus brevis; of Streptomyces, for example of Streptomyces lividans; of Staphylococcus, for example of Staphylococcus carnosus; and of Lactococcus, for example of Lactococcus lactis; a fungal cell, including but not limited to cells from species of Trichoderma, for example from Trichoderma reesei; of Neurospora, for example from Neurospora crassa; of Sordaήa, for example from Sordaria macrospora; of Aspergillus, for example from Aspergillus niger or from Aspergillus sojae; or from other filamentous fungi; a yeast cell, including but not limited to cells from species of Saccharomyces, for example of Saccharomyces cerevisiae; of Schizosaccharomyces, for example of Schizosaccharomyces pombe; of Pichia, for example of Pichia pastoris or of Pichia methanolica; of Hansenula, for example of Hansenula polymorpha; of Kluyveromyces, for example of Kluyveromyces lactis; of

Arxula, for example of Arxula adeninivorans; of Yarrowia, for example of Yarrowia lipolytica; an amphibian cell or cell line, such as Xenopus oocytes; an insect-derived cell or cell line, such as cells/cell lines derived from lepidoptera, including but not limited to Spodopiera SF9 and Sf21 cells or cells/cell lines derived from Drosophila, such as Schneider and Kc cells; a plant or plant cell, for example in tobacco plants; and/or a mammalian cell or cell line, for example derived a cell or cell line derived from a human, from the mammals including but not limited to CHO -cells, BHK-cells (for example BHK-21 cells) and human cells or cell lines such as

HeLa, COS (for example COS-7) and PER.C6 cells; as well as all other hosts or host cells known per se for the expression and production of antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments), which will be clear to the skilled person. Reference is also made to the general background art cited hereinabove, as well as to for example WO 94/29457; WO 96/34103; WO 99/42077; Frenken et al, (1998), supra; Riechmann and Muyldermans, (1999), supra; van der Linden, (2000), supra; Thomassen et al., (2002), supra; Joosten et al., (2003), supra; Joosten et al., (2005), supra; and the further references cited herein.

The Nanobodies^® and polypeptides of the invention can also be introduced and expressed in one or more cells, tissues or organs of a multicellular organism, for example for prophylactic and/or therapeutic purposes (e.g. as a gene therapy). For this purpose, the nucleotide sequences of the invention may be introduced into the cells or tissues in any suitable way, for example as such (e.g. using liposomes) or after they have been inserted into a suitable gene therapy vector (for example derived from retroviruses such as adenovirus, or parvoviruses such as adeno-associated virus). As will also be clear to the skilled person, such gene therapy may be performed in vivo and/or in situ in the body of a patent by administering a nucleic acid of the invention or a suitable gene therapy vector encoding the same to the patient or to specific cells or a specific tissue or organ of the patient; or suitable cells (often taken from the body of the patient to be treated, such as explanted lymphocytes, bone marrow aspirates or tissue biopsies) may be treated in vitro with a nucleotide sequence of the invention and then be suitably (re-)introduced into the body of the patient. All this can be performed using gene therapy vectors, techniques and delivery systems which are well known to the skilled person, for Culver, K. W., "Gene Therapy'¹, 1994, p. xii, Mary Ann Lϊebert, Inc., Publishers, New York, N.Y). Giordano, Nature F Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911- 919; Anderson, Science 256 (1992),808-813; Verma, Nature 389 (1994),239; Isner, Lancet 348 (1996),370-374; Muhlhauser, Circ. Res. 77 (1995),1077-1086; Onodera, Blood 91; (1998),30- 36; Verma, Gene Ther. 5 (1998),692-699; Nabel, Ann. N.Y. Acad. Sci. : 811 (1997), 289-292; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-51; Wang, Nature Medicine 2 (1996),714-716; WO 94/29469; WO 97/00957, US 5,580,859; 1 US 5,5895466; or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640. For example, in situ expression of ScFv fragments (Afanasieva et al., Gene Ther., 10, 1850-1859 (2003)) and of diabodies (Blanco et al., J. Immunol, 171, 1070- 1077 (2003)) has been described in the art. For expression of the Nanobodies^® in a cell, they may also be expressed as so- called or as so-called "intrabodies", as for example described in WO 94/02610, WO 95/22618 and US-A-6004940; WO 03/014960; in Cattaneo, A. & Biocca, S. (1997) Intracellular Antibodies: Development and Applications. Landes and Springer- Verlag; and in Kontermann, Methods 34, (2004), 163-170.

For production, the Nanobodies ^R and polypeptides of the invention can for example also be produced in the milk of transgenic mammals, for example in the milk of rabbits, cows, goats or sheep (see for example US-A-5,741,957, US-A- 5,304,489 and US-A-5, 849.992 for general techniques for introducing traαsgenes into mammals), in plants or parts of plants including but not limited to their leaves, flowers, fruits, seed, roots or turbers (for example in tobacco, maize, soybean or alfalfa) or in for example pupae of the silkworm Bombix mori. Furthermore, the Nanobodies^® and polypeptides of the invention can also be expressed and/or produced in cell-free expression systems, and suitable examples of such systems will be clear to the skilled person. Some preferred, but non-limiting examples include expression in the wheat germ system; in rabbit reticulocyte lysates; or in the E. coli Zubay system. As mentioned above, one of the advantages of the use of Nanobodies^® is that the polypeptides based thereon can be prepared through expression in a suitable bacteria] system, and suitable bacterial expression systems, vectors, host cells, regulatory elements, etc., will be clear to the skilled person, for example from the references cited above. It should however be noted that the invention in its broadest sense is not limited to expression in bacterial systems.

Preferably, in the invention, an (in vivo or in vitro) expression system, such as a bacterial expression system, is used that provides the polypeptides of the invention in a form that is suitable for pharmaceutical use, and such expression systems will again be clear to the skilled person. As also will be clear to the skilled person, Polypeptides of the invention suitable for pharmaceutical use can be prepared using techniques for peptide synthesis.

For production on industrial scale, preferred heterologous hosts for the (industrial) production of Nanobodies^® or Nanobody^®-containing protein therapeutics include strains of E. coli, Pichia pastoris, S. cerevisiae that are suitable for large scale expression/production/fermentation, and in particular for large scale pharmaceutical expression/production/fermentation. Suitable examples of such strains will be clear to the skilled person. Such strains and production/expression systems are also made available by companies such as Biovitrum (Uppsala, Sweden). Alternatively, mammalian cell lines, in particular Chinese hamster ovary

(CHO) cells, can be used for large scale expression/production/fermentation, and in particular for large scale pharmaceutical expression/production/fermentation. Again, such expression/production systems are also made available by some of the companies mentioned above.

The choice of the specific expression system would depend in part on the requirement for certain post-translational modifications, more specifically glycosylation. The production of a Nanobody^®-containing recombinant protein for which glycosylation is desired or required would necessitate the use of mammalian expression hosts that have the ability to glycosylate the expressed protein. In this respect, it will be clear to the skilled person that the glycosylation pattern obtained (i.e. the kind, number and position of residues attached) will depend on the cell or cell line that is used for the expression. Preferably, either a human cell or cell line is used (i.e. leading to a protein that essentially has a human glycosylation pattern) or another mammalian cell line is used that can provide a glycosylation pattern that is essentially and/or functionally the same as human glycosylation or at least mimics human glycosylation. Generally, prokaryotic hosts such as E. coli do not have the ability to glycosylate proteins, and the use of lower eukaryotes such as yeast are usually leads to a glycosylation pattern that differs from human glycosylation. Nevertheless, it should be understood that all the foregoing host cells and expression systems can be used in the invention, depending on the desired Nanobody^® or protein to be obtained.

Thus, according to one non-limiting embodiment of the invention, the Nanobody^® or polypeptide of the invention is glycosylated. According to another non-limiting embodiment of the invention, the Nanobody^® or polypeptide of the invention is non-glycosylated.

According to one preferred, but non-limiting embodiment of the invention, the Nanobody^® or polypeptide of the invention is produced in a bacterial cell, in particular a bacterial cell suitable for large scale pharmaceutical production, such as cells of the strains mentioned above.

According to another preferred, but non-limiting embodiment of the invention, the Nanobody^® or polypeptide of the invention is produced in a yeast cell, in particular a yeast cell suitable for large scale pharmaceutical production, such as cells of the species mentioned above.

According to yet another preferred, but non-limiting embodiment of the invention, the Nanobody^® or polypeptide of the invention is produced in a mammalian cell, in particular in a human cell or in a cell of a human cell line, and more in particular in a human cell or in a cell of a human cell line that is suitable for large scale pharmaceutical production, such as the cell lines mentioned hereinabove.

When expression in a host cell is used to produce the Nanobodies^® and the proteins of the invention, the Nanobodies^® and proteins of the invention can be produced either intracellullarly (e.g. in the cytosol, in the periplasms or in inclusion bodies) and then isolated from the host cells and optionally further purified; or can be produced extracellularly (e.g. in the medium in which the host cells are cultured) and then isolated from the culture medium and optionally further purified. When eukaryotic hosts cells are used, extracellular production is usually preferred since this considerably facilitates the further isolation and downstream processing of the Nanobodies^® and proteins obtained. Bacterial cells such as the strains of E. coli mentioned above normally do not secrete proteins extracellularly, except for a few classes of proteins such as toxins and hemolysin, and secretory production in E. coli refers to the translocation of proteins across the inner membrane to the periplasmic space. Periplasmic production provides several advantages over cytosolic production. For example, the N-terminal amino acid sequence of the secreted product can be identical to the natural gene product after cleavage of the secretion signal sequence by a specific signal peptidase. Also, there appears to be much less protease activity in the periplasm than in the cytoplasm. In addition, protein purification is simpler due to fewer contaminating proteins in the periplasm. Another advantage is that correct disulfide bonds may form because the periplasm provides a more oxidative environment than the cytoplasm. Proteins overexpressed in E. coli are often found in insoluble aggregates, so-called inclusion bodies. These inclusion bodies may be located in the cytosol or in the periplasm; the recovery of biologically active proteins from these inclusion bodies requires a denaturation/refolding process. Many recombinant proteins, including therapeutic proteins, are recovered from inclusion bodies. Alternatively, as will be clear to the skilled person, recombinant strains of bacteria that have been genetically modified so as to secrete a desired protein, and in particular a Nanobody^® or a polypeptide of the invention, can be used.

Thus, according to one non-limiting embodiment of the invention, the

Nanobody^® or polypeptide of the invention is a Nanobody^® or polypeptide that has been produced intracellularly and that has been isolated from the host cell, and in particular from a bacterial cell or from an inclusion body in a bacterial cell.

According to another non-limiting embodiment of the invention, the Nanobody^® or polypeptide of the invention is a Nanobody^® or polypeptide that has been produced extracellularly, and that has been isolated from the medium in which the host cell is cultivated.

Some preferred, but non-limiting promoters for use with these host cells include, for expression in E. coli: lac promoter (and derivatives thereof such as the lacUV5 promoter); arabinose promoter; left- (PL) and rightward (PR) promoter of phage lambda; promoter of the tip operon; hybrid lac/trp promoters (tac and trc); T7-promoter (more specifically that of T7-phage gene J O) and other T- phage promoters; promoter of the TnIO tetracycline resistance gene; engineered variants of the above promoters that include one or more copies of an extraneous regulatory operator sequence; - for expression in S. cerevisiae: constitutive: ADHl (alcohol dehydrogenase 1), ENO (enolase), CYCl (cytochrome c iso-1), GAPDH (glyceraldehydes-3- phosphate dehydrogenase); PGKl (phosphoglycerate kinase), PYKl (pyruvate kinase); regulated: GALl, 10,7 (galactose metabolic enzymes), ADH2 (alcohol dehydrogenase 2), PHO5 (acid phosphatase), CUPl (copper metallothionein); heterologous: CaMV (cauliflower mosaic virus 35S promoter); for expression in Pichia pastoris: the AOXl promoter (alcohol oxidase I) for expression in mammalian cells: human cytomegalovirus (hCMV) immediate early enhancer/promoter; human cytomegalovirus (hCMV) immediate early promoter variant that contains two tetracycline operator sequences such that the promoter can be regulated by the Tet repressor; Herpes

Simplex Virus thymidine kinase (TK) promoter; Rous Sarcoma Virus long terminal repeat (RSV LTR) enhancer/promoter; elongation factor lα (hEF-lα) promoter from human, chimpanzee, mouse or rat; the SV40 early promoter; HIV-I long terminal repeat promoter; β-actin promoter; Some preferred, but non-limiting vectors for use with these host cells include: vectors for expression in mammalian cells: pMAMneo (Clontech), pcDNA3 (Invitrogen), pMClneo (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo

(ATCC 37593), pBPV-1 (8-2) (ATCC 37110), pdBPV-MMTneo (342-12) (ATCC 37224). pRSVgpl (ATCC37199), pRSVneo (ATCC37198). _pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460) and 1ZD35 (ATCC 37565), as well as viral -based expression systems, such as those based on adenovirus; - vectors for expression in bacterials cells: pET vectors (Novagen) and pQE vectors (Qiagen); vectors for expression in yeast or other fungal cells: pYES2 (Invitrogen) and Pichia expression vectors (Invitrogen); vectors for expression in insect cells: pBlueBacII (Invitrogen) and other baculo virus vectors vectors for expression in plants or plant cells: for example vectors based on cauliflower mosaic virus or tobacco mosaic virus, suitable strains of Agrobacterium. or Ti-plasmid based vectors.

Some preferred, but non-limiting secretory sequences for use with these host cells include: for use in bacterial cells such as E. coli: PeIB, BIa, OmpA, OmpC, OmpF, OmpT, StII, PhoA, PhoE, MaIE, Lpp, LamB, and the like; TAT signal peptide, hemolysin C -terminal secretion signal for use in yeast: α-mating factor prepro-sequence, phosphatase (phol), invertase (Sue), etc.; for use in mammalian cells: indigenous signal in case the target protein is of eukaryotic origin; murine Ig κ-chain V-J2-C signal peptide; etc.

Suitable techniques for transforming a host or host cell of the invention will be clear to the skilled person and may depend on the intended host cell/host organism and the genetic construct to be used. Reference is again made to the handbooks and patent applications mentioned above.

After transformation, a step for detecting and selecting those host cells or host organisms that have been successfully transformed with the nucleotide sequence/genetic construct of the invention may be performed. This may for instance be a selection step based on a selectable marker present in the genetic construct of the invention or a step involving the detection of the amino acid sequence of the invention, e.g. using specific antibodies. The transformed host cell (which may be in the form or a stable cell line) or host organisms (which may be in the form of a stable mutant line or strain) form further aspects of the present invention.

Preferably, these host cells or host organisms are such that they express, or are (at least) capable of expressing (e.g. under suitable conditions), an amino acid sequence of the invention (and in case of a host organism: in at least one cell, part, tissue or organ thereof). The invention also includes further generations, progeny and/or offspring of the host cell or host organism of the invention that may for instance be obtained by cell division or by sexual or asexual reproduction.

To produce/obtain expression of the amino acid sequences of the invention, the transformed host cell or transformed host organism may generally be kept, maintained and/or cultured under conditions such that the (desired) amino acid sequence of the invention is expressed/produced. Suitable conditions will be clear to the skilled person and will usually depend upon the host cell/host organism used, as well as on the regulatory elements that control the expression of the (relevant) nucleotide sequence of the invention. Again, reference is made to the handbooks and patent applications mentioned above in the paragraphs on the genetic constructs of the invention.

Generally, suitable conditions may include the use of a suitable medium, the presence of a suitable source of food and/or suitable nutrients, the use of a suitable temperature, and optionally the presence of a suitable inducing factor or compound (e.g. when the nucleotide sequences of the invention are under the control of an inducible promoter); all of which may be selected by the skilled person. Again, under such conditions, the amino acid sequences of the invention may be expressed in a constitutive manner, in a transient manner, or only when suitably induced. It will also be clear to the skilled person that the amino acid sequence of the invention may (first) be generated in an immature form (as mentioned above), which may then be subjected to post-translational modification, depending on the host cell/host organism used. Also, the amino acid sequence of the invention may be glycosylated, again depending on the host cell/host organism used.

The amino acid sequence of the invention may then be isolated from the host cell/host organism and/or from the medium in which said host cell or host organism was cultivated, using protein isolation and/or purification techniques known per se, such as (preparative) chromatography and/or electrophoresis techniques, differential precipitation techniques, affinity techniques (e.g. using a specific, cleavable amino acid sequence fused with the amino acid sequence of the invention) and/or preparative immunological techniques (i.e. using antibodies against the amino acid sequence to be isolated).

Generally, for pharmaceutical use, the polypeptides of the invention may be formulated as a pharmaceutical preparation comprising at least one polypeptide of the invention and at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more further pharmaceutically active polypeptides and/or compounds. By means of non-limiting examples, such a formulation may be in a form suitable for oral administration, for parenteral administration (such as by intravenous, intramuscular or subcutaneous injection or intravenous infusion), for topical administration, for administration by inhalation, by a skin patch, by an implant, by a suppository, etc.. Such suitable administration forms - which may be solid, semi-solid or liquid, depending on the manner of administration - as well as methods and carriers for use in the preparation thereof, will be clear to the skilled person, and are further described herein.

Thus, in a further aspect, the invention relates to a pharmaceutical composition that contains at least one Nanobody^® of the invention or at least one polypeptide of the invention and at least one suitable carrier, diluent or excipient (i.e. suitable for pharmaceutical use), and optionally one or more further active substances.

Generally, the Nanobodies^® and polypeptides of the invention can be formulated and administered in any suitable manner known per se, for which reference is for example made to the general background art cited above (and in particular to WO 04/041862, WO 04/041863, WO 04/041865 and WO 04/041867) as well as to the standard handbooks, such as Remington's Pharmaceutical Sciences, 18¹¹¹ Ed., Mack Publishing Company, USA (1990) or Remington, the Science and Practice of Pharmacy, 21th Edition, Lippincott Williams and Wilkins (2005).

For example, the Nanobodies^® and polypeptides of the inventions may be formulated and administered in any manner known per se for conventional antibodies and antibody fragments (including ScFv' ε and diabodies) and other pharmaceutically active proteins. Such formulations and methods for preparing the same will be clear to the skilled person, and for example include preparations suitable for parenteral administration (for example intravenous, intraperitoneal, subcutaneous, intramuscular, intraluminal, intra-arterial or intrathecal administration) or for topical (i.e. transdermal or intradermal) administration.

Preparations for parenteral administration may for example be sterile solutions, suspensions, dispersions or emulsions that are suitable for infusion or injection. Suitable carriers or diluents for such preparations for example include, without limitation, sterile water and aqueous buffers and solutions such as physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution; water oils; glycerol; ethanol; glycols such as propylene glycol or as well as mineral oils, animal oils and vegetable oils, for example peanut oil, soybean oil, as well as suitable mixtures thereof. Usually, aqueous solutions or suspensions will be preferred. The Nanobodies^® and polypeptides of the present invention can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient or a domestic animal in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, intranassally by inhalation, intravenous, intramuscular, topical or subcutaneous routes. A pharmaceutical composition of the present invention may comprise a Nanobody^® or polypeptide of the present invention and a suitable pharmaceutical vehicle as listed below.

The Nanobodies^® and polypeptides of the present invention can also be administered using gene therapy methods of delivery. See, e.g., U.S. Patent No. 5.399,346, which is incorporated by reference in its entirety. Using a gene therapy method of delivery, primary cells transfected with the gene for the a Nanobody^® or polypeptide of the present invention can additionally be transfected with tissue specific promoters to target specific organs, tissue, grafts, tumors, or cells and can additionally be transfected with signal and stabilization sequences for subcellularly localized expression.

Thus, the Nanobodies^® and polypeptides of the present invention may be systemic ally administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the Nanobodies^® and polypeptides of the present invention may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of a Nanobody^® or polypeptide of the present invention. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of Nanobody^® or polypeptide of the present invention in such therapeutically useful compositions is such that an effective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum iragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of winter green, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain a Nanobody^® or polypeptide of the present invention, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, a Nanobody^® or polypeptide of the present invention may be incoiporated into sustained-release preparations and devices. The Nanobodies^® and polypeptides of the present invention may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the Nanobodies^® and polypeptides of the present invention can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form must be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, εthanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the Nanobodies^® and polypeptides of the present invention in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions. For topical administration, the Nanobodies^® and polypeptides of the present invention may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, hydroxyalkyls or glycols or water- ale ohol/glycol blends, in which the Nanobodies^® and polypeptides of the present invention can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers. Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid earners to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

Examples of useful dermatological compositions which can be used to deliver pharmaceutically active compounds and proteins to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).

Useful dosages of the Nanobodies^® and polypeptides of the present invention can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.

Generally, the concentration of the Nanobodies^® and polypeptides of the present invention in a liquid composition, such as a lotion, will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%. The concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%. The amount of the Nanobodies ^R and polypeptides of the present invention required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician. Also the dosage of the Nanobodies^® and polypeptides of the present invention will vary depending on the target cell, tumor, tissue, graft, or organ.

The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.

An administration regimen could include long-term, daily treatment. By "long-term" is meant at least two weeks and preferably, several weeks, months, or years of duration. Necessary modifications in this dosage range may be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. See Remington's Pharmaceutical Sciences (Martin, E. W., ed. 4), Mack Publishing Co., Easton, PA. The dosage can also be adjusted by the individual physician in the event of any complication. Generally, for pharmaceutical use, the polypeptides of the invention may be formulated as a pharmaceutical preparation comprising at least one polypeptide of the invention and at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more further pharmaceutically active polypeptides and/or compounds. By means of non-limiting examples, such a formulation may be in a form suitable for oral administration, for parenteral administration (such as by intravenous, intramuscular or subcutaneous injection or intravenous infusion), for topical administration, for administration by inhalation, by a skin patch, by an implant, by a suppository, etc.. Such suitable administration forms - which may be solid, semi-solid or liquid, depending on the manner of administration - as well as methods and carriers for use in the preparation thereof, will be clear to the skilled person, and are further described herein.

Thus, in a further aspect, the invention relates to a pharmaceutical composition that contains at least one Nanobody of the invention or at least one polypeptide of the invention and at least one suitable carrier, diluent or excipient (i.e. suitable for pharmaceutical use), and optionally one or more further active substances.

Generally, the Nanobodies and polypeptides of the invention can be formulated and administered in any suitable manner known per se, for which reference is for example made to the general background art cited above (and in particular to WO 04/041862, WO 04/041863, WO 04/041865 and WO 04/041867) as well as to the standard handbooks, such as Remington's Pharmaceutical Sciences, 18^th Ed., Mack Publishing Company, USA (1990) or Remington, the Science and Practice of Pharmacy, 21th Edition, Lippincott Williams and Wilkins (2005).

For example, the Nanobodies and polypeptides of the invention may be formulated and administered in any manner known per se for conventional antibodies and antibody fragments (including ScFv' s and diabodies) and other pharmaceutically active proteins. Such formulations and methods for preparing the same will be clear to the skilled person, and for example include preparations suitable for parenteral administration (for example intravenous, intraperitoneal, subcutaneous, intramuscular, intraluminal, intra-arterial or intrathecal administration) or for topical (i.e. transdermal or intradermal) administration.

Preparations for parenteral administration may for example be sterile solutions, suspensions, dispersions or emulsions that are suitable for infusion or injection. Suitable carriers or diluents for such preparations for example include, without limitation, sterile water and aqueous buffers and solutions such as physiological phosphate -buffered saline, Ringer's solutions, dextrose solution, and Hank's solution; water oils; glycerol; ethanol; glycols such as propylene glycol or as well as mineral oils, animal oils and vegetable oils, for example peanut oil, soybean oil, as well as suitable mixtures thereof. Usually, aqueous solutions or suspensions will be preferred.

The Nanobodies and polypeptides of the invention can also be administered using gene therapy methods of delivery. See, e.g., U.S. Patent No. 5,399,346, which is incoiporated by reference in its entirety. Using a gene therapy method of delivery, primary cells transfected with the gene encoding a Nanobody or polypeptide of the invention can additionally be transfected with tissue specific promoters to target specific organs, tissue, grafts, tumors, or cells and can additionally be transfected with signal and stabilization sequences for subcellularly localized expression.

Thus, the Nanobodies and polypeptides of the invention may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the Nanobodies and polypeptides of the invention may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of the Nanobody or polypeptide of the invention. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of the Nanobody or polypeptide of the invention in such therapeutically useful compositions is such that an effective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the Nanobodies and polypeptides of the invention, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the Nanobodies and polypeptides of the invention may be incorporated into sustained-release preparations and devices. The Nanobodies and polypeptides of the invention may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the Nanobodies and polypeptides of the invention or their salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form must be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thiraerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the Nanobodies and polypeptides of the invention in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions. For topical administration, the Nanobodies and polypeptides of the invention may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, hydroxyalkyls or glycols or water-alcohol/glycol blends, in which the Nanobodies and polypeptides of the invention can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers. Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

Examples of useful dermatological compositions which can be used to deliver the Nanobodies and polypeptides of the invention to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820.508).

Useful dosages of the Nanobodies and polypeptides of the invention can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humaDS are known to the art; for example, see U.S. Pat. No. 4,938,949.

Generally, the concentration of the Nanobodies and polypeptides of the invention in a liquid composition, such as a lotion, will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%. The concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%. The amount of the Nanobodies and polypeptides of the invention required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician. Also the dosage of the Nanobodies and polypeptides of the invention varies depending on the target cell, tumor, tissue, graft, or organ.

An administration regimen could include long-term, daily treatment. By "long-term" is meant at least two weeks and preferably, several weeks, months, or years of duration. Necessary modifications in this dosage range may be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. See Remington's Pharmaceutical Sciences (Martin, E.W., ed. 4), Mack Publishing Co., Easton, PA. The dosage can also be adjusted by the individual physician in the event of any complication.

In another aspect, the invention relates to a method for the prevention and/or treatment of at least one IL-6 related disorders, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of a Nanobody of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same.

In the context of the present invention, the term "prevention and/or treatment" not only comprises preventing and/or treating the disease, but also generally comprises preventing the onset of the disease, slowing or reversing the progress of disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing and/or alleviating one or more symptoms associated with the disease, reducing the severity and/or the duration of the disease and/or of any symptoms associated therewith and/or preventing a further increase in the severity of the disease and/or of any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by the disease, and generally any pharmacological action that is beneficial to the patient being treated. The subject to be treated may be any warm-blooded animal, but is in particular a mammal, and more in particular a human being. As will be clear to the skilled person, the subject to be treated will in particular be a person suffering from, or at risk from, the diseases and disorders mentioned herein. The invention also relates to a method for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering a Nanobody or polypeptide of the invention to a patient, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of a Nanobody of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same.

More in particular, the invention relates to a method for the prevention and/or treatment of at least one disease or disorder chosen from the group consisting of the diseases and disorders listed herein, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of a Nanobody of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same.

In another embodiment, the invention relates to a method for immunotherapy, and in particular for passive immunotherapy, which method comprises administering, to a subject suffering from or at risk of the diseases and disorders mentioned herein, a pharmaceutically active amount of a Nanobody of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same.

In the above methods, the Nanobodies and/or polypeptides of the invention and/or the compositions comprising the same can be administered in any suitable manner, depending on the specific pharmaceutical formulation or composition to be used. Thus, the Nanobodies and/or polypeptides of the invention and/or the compositions comprising the same can for example be administered orally, intraperitoneally (e.g. intravenously, subcutaneously, intramuscularly, or via any other route of administration that circumvents the gastrointestinal tract), intranasally, transdermally. topically, by means of a suppository, by inhalation, again depending on the specific pharmaceutical formulation or composition to be used. The clinician will be able to select a suitable route of administration and a suitable pharmaceutical formulation or composition to be used in such administration, depending on the disease or disorder to be prevented or treated and other factorse well known to the clinician.

The Nanobodies and/or polypeptides of the invention and/or the compositions comprising the same are administered according to a regime of treatment that is suitable for preventing and/or treating the disease or disorder to be prevented or treated. The clinician will generally be able to determine a suitable treatment regimen, depending on factors such as the disease or disorder to be prevented or treated, the severity of the disease to be treated and/or the severity of the symptoms thereof, the specific Nanobody or polypeptide of the invention to be used, the specific route of administration and farmaceutical formulation or composition to be used, the age, gender, weight, diet, general condition of the patient, and similar factors well known to the clinician.

Generally, the treatment regimen will comprise the administration of one or more Nanobodies and/or polypeptides of the invention, or of one or more compositions comprising the same, in one or more pharmaceutically effective amounts or doses. The specific amount(s) or doses to administered can be determined by the clinician, again based on the factors cited above.

Generally, for the prevention and/or treatment of the diseases and disorders mentioned herein and depending on the specific disease or disorder to be treated, the potency of the specific Nanobody and polypeptide of the invention to be used, the specific route of administration and the specific pharmaceutical formulation or composition used, the Nanobodies and polypeptides of the invention will generally be administered in an amount between 1 gram and 0.01 microgram per kg body weight per day, preferably between 0.1 gram and 0.1 microgram per kg body weight per day, such as about 1, 10, 100 or 1000 microgram per kg body weight per day, either continuously (e.g. by infusion), as a single daily dose or as multiple divided doses during the day. The clinician will generally be able to determine a suitable daily dose, depending on the factors mentioned herein. It will also be clear that in specific cases, the clinician may choose to deviate from these amounts, for example on the basis of the factors cited above and his expert judgment. Generally, some guidance on the amounts to be administered can be obtained from the amounts usually administered for comparable conventional antibodies or antibody fragments against the same target administered via essentially the same route, taking into account however differences in affinity/avidity, efficacy, biodistribution, half-life and similar factors well known to the skilled person.

Usually, in the above method, a single Nanobody or polypeptide of the invention will be used. It is however within the scope of the invention to use two or more Nanobodies and/or polypeptides of the invention in combination.

The Nanobodies and polypeptides of the invention may also be used in combination with one or more further pharmaceutically active compounds or principles, i.e. as a combined treatment regimen, which may or may not lead to a synergistic effect. Again, the clinician will be able to select such further compounds or principles, as well as a suitable combined treatment regimen, based on the factors cited above and his expert judgement.

In particular, the Nanobodies and polypeptides of the invention may be used in combination with other pharmaceutically active compounds or principles that are or can be used for the prevention and/or treatment of the diseases and disorders cited herein, as a result of which a synergistic effect may or may not be obtained. Examples of such compounds and principles, as well as routes, methods and pharmaceutical formulations or compositions for administering them will be clear to the clinician.

When two or more substances or principles are to be used as part of a combined treatment regimen, they can be administered via the same route of administration or via different routes of administration, at essentially the same time or at different times (e.g. essentially simultaneously, consecutively, or according to an alternating regime). When the substances or principles are administered to be simultaneously via the same route of administration, they may be administered as different pharmaceutical formulations or compositions or part of a combined pharmaceutical formulation or composition, as will be clear to the skilled person.

Also, when two or more active substances or principles are to be used as part of a combined treatment regimen, each of the substances or principles may be administered in the same amount and according to the same regimen as used when the compound or principle is used on its own, and such combined use may or may not lead to a synergistic effect. However, when the combined use of the two or more active substances or principles leads to a synergistic effect, it may also be possible to reduce the amount of one, more or all of the substances or principles to be administered, while still achieving the desired therapeutic action. This may for example be useful for avoiding, limiting or reducing any unwanted side-effects that are associated with the use of one or more of the substances or principles when they are used in their usual amounts, while still obtaining the desired pharmaceutical or therapeutic effect.

The effectiveness of the treatment regimen used according to the invention may be determined and/or followed in any manner known per se for the disease or disorder involved, as will be clear to the clinician. The clinician will also be able, where appropriate and or a case-by-case basis, to change or modify a particular treatment regimen, so as to achieve the desired therapeutic effect, to avoid, limit or reduce unwanted side-effects, and/or to achieve an appropriate balance between achieving the desired therapeutic effect on the one hand and avoiding, limiting or reducing undesired side effects on the other hand.

Generally, the treatment regimen will be followed until the desired therapeutic effect is achieved and/or for as long as the desired therapeutic effect is to be maintained. Again, this can be determined by the clinician.

In another aspect, the invention relates to the use of a Nanobody or polypeptide of the invention in the preparation of a pharmaceutical composition for prevention and/or treatment of at least one IL-6 related disorders. The subject to be treated may be any warm-blooded animal, but is in particular a mammal, and more in particular a human being. As will be clear to the skilled person, the subject to be treated will in particular be a person suffering from, or at risk from, the diseases and disorders mentioned herein.

The invention also relates to the use of a Nanobody or polypeptide of the invention in the preparation of a pharmaceutical composition for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering a Nanobody or polypeptide of the invention to a patient.

More in particular, the invention relates to the use of a Nanobody or polypeptide of the invention in the preparation of a pharmaceutical composition for the prevention and/or treatment of IL-6 related disorders, and in particular for the prevention and treatment of one or more of the diseases and disorders listed herein. Again, in such a pharmaceutical composition, the one or more Nanobodies or polypeptides of the invention may also be suitably combined with one or more other active principles, such as those mentioned herein.

Finally, although the invention is described here with reference to the ΪL-6/JL- 6 receptor complex and its interaction with gpl30, it is envisaged that the concepts and embodiments of the present invention can also be applied to other signalling pathways in which two proteins (e.g. both in circulation or one in circulation and one on a cell surface) first form a complex and then interact with a third protein (i.e. on a cell surface) to activate a signalling pathway. Examples of such proteins are: - IL-I I and IL-J 1 receptor-alpha, which form a complex that interacts with gp!30, which triggers the IL-11 signalling cascade

- LIF (leukaemia inhibiting factor) and LIF-receptor-beta, which form a complex that interacts with gpl30, which triggers the LIF signalling cascade

- TGF-beta, which first binds to type II receptor (membrane bound), and then recruits type I receptor (see for example Wrana JL, Miner Electrolyte Metab.

1998;24(2-3): 120-30).

Thus, in one embodiment, the invention relates to a polypeptide (as described herein), and in particular a polypeptide comprising or essentially consisting of at least one Nanobody, that is directed against, and preferably specific for (as defined herein) a complex formed by a first and a second protein, wherein said complex interacts with a third protein to trigger downstream signalling events.

In another embodiment, the invention relates to a bispecific polypeptide comprising a first binding site binding to a first protein and a second binding site binding to a second protein, in which the first protein and the second protein form a complex which interacts with a third protein to trigger a signalling cascade or event.

Thus, for example, such a polypeptide can be a bispecific polypeptide with at least one binding site (for example a first domain antibody or Nanobody) directed against an epitope on a part of the complex that is formed by the first protein and with at least one binding site (for example a second domain antibody or Nanobody) against an epitope on a part of the complex that is formed by the second protein, such that the polypeptide can bind to the complex via both binding sites

In this embodiment, the first binding site (i.e. binding unit or Nanobody) is preferably specific for (as defined herein) for the first protein and the second binding site (i.e. binding unit or Nanobody) is preferably specific for (as defined herein) for the second protein.

According to this embodiment the avidity (as defined below) of the bi specific polypeptide of the invention for the complex is greater than the avidity of the polypeptide for the first and second protein, respectively, such as at least two times greater, for example five times greater or even ten times greater or more.

For this, the binding sites/units in the bispecific polypeptide of the invention are most preferably such that they can bind to epitopes on the first and second protein, respectively, that are accessible for binding by the polypeptide of the present invention when the first and second protein are associated into a complex. Also, the polypeptide of the invention is most preferably such that each binding site/unit can bind to its respective epitope on the first and second protein, respectively, when the first and second protein are associated into a complex.

The above proteins bind to the complex of the first and second protein, and by such binding can modulate the interaction between said complex and the third protein. In this context, such modulation can either result in an increase of the binding affinity of the complex for the third protein (or visa versa) or in a decrease of the binding affinity of the complex for third protein (or visa versa), i.e. compared to the complex without the presence of the polypeptide of the present invention. As such, the polypeptides of the present invention can have an agonistic or an antagonistic effect on the signaling and the biological functions and responses associated with the first protein, the second protein, the complex of the first and second protein and/or the third protein.

According to one preferred, but non-limiting embodiment, binding of the polypeptide of the present invention results in a decrease of the binding affinity of the complex for the third protein (or visa versa) and/or results in an antagonistic effect on the signaling and the biological functions and responses associated with associated with first protein, the second protein, the complex of the first and second protein and/or the third protein. However, as will be clear from the description herein, the invention in its broadest sense is not limited thereto and for example also comprises polypeptides that have an agonistic effect.

According to one preferred, but non-limiting embodiment, the first protein is a cytokine. The second protein may be membrane-bound or may be soluble or both.

The third protein is preferably membrane-bound, and may for example be, without limitation, a membrane-bound protein to receptor that is dimerized by the interaction with the complex formed by the first and second protein (e.g. by interaction with two molecules of the complex), thus triggering downstream signaling events. A preferred, but non-limiting example of the third protein is gpl30.

Thus, generally, the invention provides a polypeptide that is directed against the complex formed by the first and second protein, which is preferably capable of inhibiting the interaction between said complex and the third protein, and more preferably capable of competitively inhibiting the interaction between the complex and the third protein.

The invention also relates to nucleic acids encoding such polypeptides; to methods for preparing such polypeptides; to host cells expressing or capable of expressing such polypeptides; to compositions, and in particular to pharmaceutical compositions, that comprise such polypeptides, nucleic acids and/or host cells; and to uses of such polypeptides, nucleic acids, host cells and/or compositions, in particular for prophylactic, therapeutic or diagnostic purposes, such as the prophylactic, therapeutic or diagnostic purposes mentioned herein. The aforementioned may all be a generally defined herein. Said polypeptides of the present invention and pharmaceutical compositions comprising the same can be used in the prevention and/or treatment of diseases and disorders associated with the first protein, the second protein, the complex of the first and second protein, the third protein, and/or with the signaling pathway(s) and/or the biological functions and responses in which with the first protein, the second protein, the complex of the first and second protein, the third protein are involved.

With advantage, for the above embodiment, the methods of the invention can be used to raise polypeptides or binding units (also) against human proteins (i.e. by suitably immunizing Camelids).

It is also envisaged that the concepts and embodiments of the present invention can be applied to signalling pathways in which a protein (e.g. in circulation or on a cell surface) first forms a complex with a factor, compound, ligand or other molecule or entity, whereupon said complex than interacts with a second protein (i.e. on a cell surface) to activate a signalling pathway. Examples of such proteins, factors, compounds, Iigands or other molecules or entities, of the complexes that they form, of the proteins with which these complexes interact, and of the signalling pathways in which the foregoing are involved, will be clear to the skilled person.

Thus, in another embodiment, the invention relates to a polypeptide (as described herein), and in particular a polypeptide comprising or essentially consisting of at least one Nanobody, that is directed against, and preferably specific for (as defined herein) a complex formed by a first protein and a factor, compound, ligand or other molecule or entity, wherein said complex interacts with a second protein to trigger downstream signalling events. In another embodiment, the invention relates to a bispecific polypeptide comprising a first binding site binding to a first protein and a second binding site binding to a factor, compound, ligand or other molecule or entity, in which the first protein and the factor, compound, ligand or other molecule or entity form a complex which interacts with a second protein to trigger a signalling cascade or event. Thus, for example, such a polypeptide can be a bispecific polypeptide with at least one binding site (for example a first domain antibody or Nanobody) directed against an epitope on a part of the complex that is formed by the first protein and with at least one binding site (for example a second domain antibody or Nanobody) against an epitope on a part of the complex that is formed by the factor, compound, ligand or other molecule or entity, such that the polypeptide can bind to the complex via both binding sites

In this embodiment, the first binding site (i.e. binding unit or Nanobody) is preferably specific for (as defined herein) for the first protein and the second binding site (i.e. binding unit or Nanobody) is preferably specific for (as defined herein) for the factor, compound, ligand or other molecule or entity.

According to this embodiment the avidity (as defined below) of the bispecific polypeptide of the invention for the complex is greater than the avidity of the polypeptide for the first protein and the factor, compound, ligand or other molecule or entity, respectively, such as at least two times greater, for example five times greater or even ten times greater or more.

For this, the binding sites/units in the bispecific polypeptide of the invention are most preferably such that they can bind to epitopes on the first and second protein, respectively, that are accessible for binding by the polypeptide of the present invention when the first protein and the factor, compound, ligand or other molecule or entity are associated into a complex. Also, the polypeptide of the invention is most preferably such that each binding site/unit can bind to its respective epitope on the first protein and the factor, compound, ligand or other molecule or entity, when they are associated into a complex.

The above proteins bind to the complex of the first protein and the factor, compound, ligand or other molecule or entity, and by such binding can modulate the interaction between said complex and the second protein. In this context, such modulation can either result in an increase of the binding affinity of the complex for the second protein (or visa versa) or in a decrease of the binding affinity of the complex for second protein (or visa versa), i.e. compared to the complex without the presence of the polypeptide of the present invention. As such, the polypeptides of the present invention can have an agonistic or an antagonistic effect on the signaling and the biological functions and responses associated with the first protein, the factor, compound, ligand or other molecule or entity and/or the second protein.

According to one preferred, but non-limiting embodiment, binding of the polypeptide of the present invention results in a decrease of the binding affinity of the complex for the second protein (or visa versa) and/or results in an antagonistic effect on the signaling and the biological functions and responses associated with associated with first protein, the factor, compound, ligand or other molecule and/or the second protein. However, as will be clear from the description herein, the invention in its broadest sense is not limited thereto and for example also comprises polypeptides that have an agonistic effect.

In this embodiment, the factor, compound, ligand or other molecule may be membrane-bound or may be soluble (or both), and may for example be an agonist or antagonist of the first protein and/or of the relevant signaling pathway. Preferably, but without limitation, the factor, compound, ligand or other molecule is a factor, compound, ligand or other molecule that naturally interacts with the first protein so as to form a complex which interacts with the second protein, and so modulates the signalling cascade (i.e. as an agonist or an antagonist). Thus, for example and without limitation, the bi specific Nanobody may bind both the ligand as well as its receptor, and functions to enhance ligand-mediated effects on the receptor. Cross-linking a ligand to a receptor may have different outcomes depending on the exact nature of the interaction between the 3 components of the interaction, on the nature of the receptor and ligand, and on the epitopes recognized by the Nanobodies, their affinity of interaction and the flexibility between the two Nanobody binding sites. Once the free ligand is bound by this bispecific Nanobody, the complex can obtain a higher overall binding activity for the receptor. The Nanobody can also bind the receptor and the complex can then bind the ligand with overall higher affinity or specificity. By indirectly coupling ligand and receptor, the ligand once bound to the receptor will be maintained in the vicinity of the receptor even when the interaction with the receptor is lost. It can then rebind with high efficiency and mediate its biological effect. An overall higher affinity of interaction may yield a functional gain in ligand- induced receptor triggering, but it may also lead to a more efficient receptor desensitization.

It will also be clear to the skilled person that when the factor, compound, ligand or other molecule is a protein or polypeptide, this embodiment of the invention will be essentially the same as the embodiment that was described just above.

Thus, generally, the invention provides a polypeptide that is directed against the complex formed by the first protein and a factor, compound, ligand or other molecule, which is preferably capable of inhibiting the interaction between said complex and the second protein, and more preferably capable of competitively inhibiting the interaction between the complex and the second protein.

The invention also relates to nucleic acids encoding such polypeptides; to methods for preparing such polypeptides; to host cells expressing or capable of expressing such polypeptides; to compositions, and in particular to pharmaceutical compositions, that comprise such polypeptides, nucleic acids and/or host cells; and to uses of such polypeptides, nucleic acids, host cells and/or compositions, in particular for prophylactic, therapeutic or diagnostic purposes, such as the prophylactic, therapeutic or diagnostic purposes mentioned herein. The aforementioned may all be a generally defined herein.

Said polypeptides of the present invention and pharmaceutical compositions comprising the same can be used in the prevention and/or treatment of diseases and disorders associated with the first protein, the factor, compound, ligand or other molecule, the complex of the first protein and the factor, compound, ligand or other molecule, the second protein, and/or with the signaling pathway(s) and/or the biological functions and responses in which with the first protein, the factor, compound, ligancl or other molecule, the complex of the first protein and the factor, compound, ligand or other molecule, and/or the second protein are involved.

With advantage, for the above embodiment, the methods of the invention can be used to raise polypeptides or binding units (also) against human factors, compounds, ligands or other molecules (i.e. by suitably immunizing Camelids).

The invention will now be further described by means of the following non- limiting examples and figures, in which the Figures show:

- Figure 1 : schematic view of Nanobodies according to two embodiments of the invention, i.e. Nanobody binding to the IL-6/IL-6R interface, and bispecific Nanobody binding to the IL-6-part and the IL-6R-part of the complex, respectively.

Experimental part

1 ) Immunization

With approval of the Ethical Committee of the Faculty of Veterinary Medicine (University Ghent, Belgium), llamas are immunized with human IL-6/ΓL- 6R complex according to all current animal welfare regulations. For immunization, the complex is formulated as an emulsion with an appropriate, animal-friendly adjuvant (Specoll, CEDI Diagnostics B. V.). The antigen is administered by double- spot injections intramuscularly in the neck. The animal receives 2 injections of the emulsion, containing 100 μg of IL-6/TL-6R complex and 4 subsequent injections containing 50 ug of antigen at weekly intervals. At different time points during immunization, 10-ml blood samples is collected from the animal and sera are prepared. The induction of an antigen specific humoral immune response is verified using the serum samples in an ELISA experiment with IL-6/DL-6R complex. Five days after the last immunization, a blood sample of 150 ml is collected. Peripheral blood lymphocytes (PBLs), as the genetic source of the llama heavy chain immunoglobulins (HcAbs), are isolated from the 150-ml blood sample using a Ficoll- Paque gradient (Amersham Biosciences) yielding 5xlO⁸ PBLs. The maximal diversity of antibodies is expected to be equal to the number of sampled B- lymphocytes, which is about 10 % of the number of PBLs (5xlO⁷). The fraction of heavy-chain antibodies in llama is up to 20 % of the number of B -lymphocytes. Therefore, the maximal diversity of HcAbs in the 150 ml blood sample is calculated as 10⁷ different molecules.

2) Cloning of a repertoire of Nanobodies^® recognizing specifically IL-6/IL-6R complex

Cloning of a repertoire of Nanobodies^® binding specifically to IL-6/IL-6R complex is carried out using the method described below.

Alternatively, the Nanoclone® method described in the co-pending application PCT/EP2005/011819 (filing date November 4, 2005) may be used.

"Repertoire cloning" and "phage display" techniques can be used for the cloning of immunoglobulin sequences, as for example described in EP 0 589 S77, US

5,969,108, US 6,248,516 and Reiter et aL, 1999. Generally, the selection and cloning of immunoglobulin sequences (also referred to below as "binders") by means of these techniques involves the steps of: a) providing "total" mRNA from a cell using a method described by Chomczynski and Sacchi (1987), wherein said cell can express the entire immune "repertoire" from a animal (such as B-cell) and wherein said mRNA contains the entire immune repertoire of said animal; b) synthesizing cDNA out of said mRNA with MMLV Reverse Transcriptase

(Superscript III, Invitrogen) using oligo d(T) oligonucleotides (de Haard et aL, 1999). c) selectively amplifying the nucleotide sequences that encode the immune repertoire using specific primers (EP 0 368 684; WO03/054016); in a first PCR, the repertoire of both conventional (1.6 kb) and heavy chain (1.3 kb) antibody gene segments is amplified using a leader specific primer and an oligo d(T) primer. The resulting DNA fragments are separated by agarose gel electrophoresis. The amplified 1.3 kb fragment, encoding heavy-chain antibody segments is purified from the agarose gel and used as template in a nested PCR using a FRl specific primer containing a Sfil restriction site and an oligo d(T) primer. The PCR products are subsequently digested with SfH and B stEll (naturally occurring in FR4); d) preparing phage particles that express the binders encoded by said amplified sequences on their surface; using a suitable micro-organism, such as E. coli: following gel electrophoresis, a DNA fragment of approximately 400 basepairs is purified from gel and 330 ng of amplified VHH repertoire is ligated into the corresponding restriction sites of one microgram of phagemid vector to obtain a library after electroporation of Escherichia coli TGl. The phagemid vector allows for production of phage particles, expressing the individual VHHs as a fusion protein with the geneIII product; e) selecting phage particles that express binder sequences that can bind to IL- 6/IL-6R complex: Various concentrations between 0 and 1 nM of biotinylated

IL-6/IL-6R complex are incubated with 10 ul phage in PBS containing 0.1% casein and 0.1 % Tween-20. After 1 hour incubation at RT, the samples are transferred to microtiter plate wells which are coated with 5 ug/ml streptavidin and subsequently blocked with PBS containing 1% casein for 3 hours at room temperature. After 5 min incubation, the wells are washed 10 times with PBS-Tween and 10 times with PBS. Phage are eluted by addition of 1 mg/ml trypsin followed by a 30 min incubation at 37⁰C or by addition of 100 μl of 100 μg/ml gpl30-Fc and overnight incubation at 4⁰C. Eluted phage are allowed to infect exponential] y growing TGl cells, and are then plated on LB agar plates containing 100 μg/ml ampicϋlin and 2% glucose. f) recloning and expression of the binder-encoding sequences from the phage particles selected in step e): DNA encoding binders to IL-6/IL-6R complex are recloned into a suitable expression vector and subsequently transformed into electrocompetent E. coli cells. A single colony is used to start an overnight culture in LB containing 2% glucose and 100 μg/ml ampicillin.

This overnight culture is diluted 100-fold in in TB medium containing 100 ug/ml ampicillin, and incubated at until ODeoo = 0.5. 1 mM IPTG is added and the culture is incubated for 3 more hours at 37°C or overnight at 28⁰C. The culture is centrifuged for 20 min at 10000 rpm at 4°C. The obtained pellet is frozen overnight or for 1 hour at -20^QC. Next, the pellet is thawn at room temperature, re-suspended in PBS at 1/10 of the original culture volume. The periplasmic fraction is isolated by centrifugation for 20 minutes at 4⁰C at 20000 rpm. The supernatant containing the VHH is used directly or is loaded on Ni-NTA and purified to homogeneity. The yield of VHH is calculated according to the extinction coefficient.

3) Functional characterization of Nanobodies^® recognizine specifically IL-6/IL-6R

3a) Binding of Nanobodies^® to JL-6, IL-6R or IL-6/IL-6R complex in ELISA Nanobodies specific for the IL-6/IL-6R complex is tested for binding to IL-6, 1L-6R or IL-6/IL-6R complex in ELISA. Therefore, a microtiterplate (Nunc, Maxisorb) is coated with human IL-6, human IL-6R or human IL-6/IL-6R complex at a concentration of 1 ug/ml. The plate is coated overnight at 4⁰C. The plate is then washed with PBS-Tween and blocked for two hours at room temperature with PBS- 1% casein. After washing, the samples are applied starting at a concentration of 10 μg/ml and 3-fold dilutions are made in PBS. After a two hours incubation period, the plates are washed and mouse monoclonal anti-myc antibody at a 1000-fold dilution is applied for 1 hour at room temperature. The plates are washed and polyclonal anti- mouse-HRP (DAKO) is applied at a 1000-fold dilution for one hour at room temperature. The plates are washed and ABTS/H₂O₂ substrate is applied. The OD 405 nm is measured.

3b) Inhibition of binding of human gpl 30 protein to IL-6/IL-6R complex

Nanobodies capable of inhibiting the interaction between human gpl 30 and the IL-6/IL-6R complex is identified by Alphascreen. In this assay, periplasmic extracts prepared from bispecific Nanobody expressing E. coli cells is incubated with biotinylated human IL-6/IL-6R complex in a 384-wells plate for 15 rnin. Subsequently a mixture of gpl 30-Fc chimera (R&D Systems) and protein A acceptor beads (20 ug/ml) coated is added and incubated for 30 min. Finally, streptavidin coated donor beads (20 ug/ml) is added. After 1 hour of incubation the plates can be read on the Envision Alphascreen reader (PerkinElmer).

3c) Analysis of Nanobody^® binding to IL-6/IL-6R complex on BIACORE

On and off-rates of Nanobodies binding to IL-6/IL-6R complex are determined by surface plasmon resonance on a Biacore 3000 instrument. For this analysis, IL-6/IL-6R complex is covalently bound to a CM5 sensor chip via amine coupling. The surface of the chip is activated by the injection of EDC/NHS (1: 1 mix of 0,4 M l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 0.1 M N- hydroxysuccinimide in water). IL-6/IL-6R complex is sent over the activated chip until an increase of 500 response units is obtained. The excess of reactive groups is inactivated with 1 M Ethanolamine-HCl (pH 8,5). Different concentrations of Nanobody are injected for 5 min to allow for binding to immobilized IL-6/TL-6R complex on the chip. Buffer without Nanobody is sent over the chip for 30 min to allow for spontaneous dissociation of bound Nanobody. The association and dissociation phases at different Nanobody concentrations are used to calculate the k_kn and k_off values, respectively for each individual Nanobody,

3d) Cross-reactivity of the Nanobodies^® to IL-6/IL-6R complex from other species Nanobodies^® are tested for cross-reactivity to IL-6/IL-6R complexes from different species (e.g. mouse, rat, dog, pig, rhesus monkey, baboon and cynomolgus monkey) by ELISA. Therefore, a microtiterplate (Nunc, Maxisorb) is coated with IL-6/IL-6R complex from the species of interest at a concentration of 1 ug/ml. The plate is coated overnight at 4⁰C. The plate is then washed with PBS-Tween and blocked for two hours at room temperature with PBS-1 % casein. After washing, the Nanobody samples are applied starting at a concentration of 10 μg/ml and 3-fold dilutions are made in PBS-Tween. After a two hours incubation period, the plates are washed and mouse monoclonal anti-myc antibody at a 1000-fold dilution is applied for 1 hour at room temperature. The plates are washed and polyclonal anti-mouse-HRP (DAKO) is applied at a 1000- fold dilution for one hour at room temperature. The plates are washed and ABTS/H₂O₂ substrate is applied. The OD 405 nm is measured.

4) Immunization

With approval of the Ethical Committee of the Faculty of Veterinary Medicine (University Ghent, Belgium), llamas is immunized with either human 1L-6, human IL-6R, or human IL-6/IL-6R complex according to all current animal welfare regulations. For immunization, the cytokine or cytokine receptor is formulated as an emulsion with an appropriate, animal -friendly adjuvant (Specoll, CEDI Diagnostics B. V.). The antigen cocktail is administered by double-spot injections intramuscularly in the neck. The animal receives 6 injections of the emulsion, containing 100 μg of IL-6, IL-6R, or IL-6/IL-6R complex at weekly intervals. At different time points during immunization, 10-ml blood samples is collected from the animal and sera is prepared. The induction of an antigen specific humoral immune response is verified using the serum samples in an ELISA experiment with IL-6, IL-6R, or IL-6/IL-6R complex. Five days after the last immunization, a blood sample of 150 ml is collected. From this sample, conventional and heavy-chain antibodies (HcAbs) are fractionated (Lauwereys et al. 1998) and used in an ELISA, which can show that the HcAbs are responsible for the antigen specific humoral immune response. Peripheral blood lymphocytes (PBLs), as the genetic source of the llama heavy chain immunoglobulins (HcAbs), are isolated from the 150-ml blood sample using a Ficoll- Paque gradient (Amersham Biosciences) yielding 5xlO⁸ PBLs. The maximal diversity of antibodies is expected to be equal to the number of sampled B- lymphocytes, which is about 10 % of the number of PBLs (5xlO⁷). The fraction of heavy-chain antibodies in llama is up to 20 % of the number of B -lymphocytes. Therefore, the maximal diversity of HcAbs in the 150 ml blood sample is calculated as 10⁷ different molecules.

5) Cloning of a repertoire of Nanobodies^* recognizing specifically IL-6 or IL-6R Cloning of a repertoire of Nanobodies^® binding either specifically to IL-6, to IL-6R or to the IL-6/IL-6R complex is carried out using one of the two hereinbelow described methods:

5a) Repertoire cloning combined with phage display "Repertoire cloning" and "phage display" techniques can be used for the cloning of immunoglobulin sequences, as for example described in EP 0 589 877, US 5,969,108, US 6,248,516 and Reiter et al., 1999. Generally, the selection and cloning of immunoglobulin sequences (also referred to below as "binders") by means of these techniques involves the steps of: a) providing "total" mRNA from a cell using a method described by

Chomczynski and Sacchi (1987), wherein said cell can express the entire immune "repertoire" from a animal (such as B-cell) and wherein said mRNA contains the entire immune repertoire of said animal; b) synthesizing cDNA out of said mRNA with MMLV Reverse Transcriptase (Invitrogen) using oligo d(T) oligonucleotides (tie Haard et al., 1999). c) selectively amplifying the nucleotide sequences that encode the immune repertoire using specific primers (EP 0 368 684; WO03/054016); in a first PCR, the repertoire of both conventional (1.6 kb) and heavy chain (1.3 Kb) antibody gene segments is amplified using a leader specific primer (ABL002) and an oligo d(T) primer (ABLOlO). The resulting DNA fragments are separated by agarose gel electrophoresis. The amplified 1.3 kb fragment, encoding heavy-chain antibody segments is purified from the agarose gel and used as template in a nested PCR using a mixture of FRl primers (ABL037-

ABL043) and ABLOlO. The PCR products are subsequently digested with SfH (introduced in the FRl primer) and BstEH (naturally occurring in FR4); d) preparing phage particles that express the binders encoded by said amplified sequences on their surface; using a suitable micro-organism, such as E. colt following gel electrophoresis, a DNA fragment of approximately 400 basepairs is purified from gel and 330 ng of amplified VHH repertoire is ligated into the corresponding restriction sites of one microgram of phagemid pAX004 to obtain a library after electroporation of Escherichia coli TGl. pAX004 allows the production of phage particles, expressing the individual VHHs as a fusion protein with the geneIII product; e) selecting phage particles that express binder sequences that can bind to either IL-6, 3L-6R, or IL-6/IL-6R complex: a well in a microtiterplate is coated with either 2 μg/ml IL-6, 2 μg/ml IL-6R, or 2 μg/ml IL-6/IL-6R complex; another well is coated with PBS containing 1% casein. After overnight incubation at 4°C, the wells are blocked with PBS containing 1% casein, for 3 hours at room temperature. 200 μl phages are added to the wells. After 2 hours incubation at room temperature, the wells are washed 10 times with PBS- Tween and 10 times with PBS. Elutions are performed overnight at room temperature. Eluted phages are allowed to infect exponentially growing TGl cells, and are then plated on LB agar plates containing 100 μg/ml ampicillin and 2% glucose. This experiment is repeated for a second round of panning, under the same conditions as described above. f) isolation/cloning of the binder-encoding sequences from the phage particles selected in step e): a plasmid is prepared for binders for IL-6, IL-6R, or IL- 6/IL-6R complex and is transformed into WK6 electrocompetent cells, A single colony is used to start an overnight culture in LB containing 2% glucose and 100 g/ml ampicillin. This overnight culture is diluted 100-fold in

300 ml TB medium containing 100 μg/ml ampicillin, and incubated at 37⁰C until OD600nm= 0.5. 1 mM IPTG is added and the culture is incubated for 3 more hours at 37⁰C or overnight at 28"C.The culture is centrifuged for 20 minutes at 10000 rpm at 4° C. The obtained pellet is frozen overnight or for 1 hour al -2O⁰C. Next, the pellet is thawed at room temperature for 40 minutes, re-suspended in 20 ml PBS and shaken on ice for 1 hour. The periplasmic fraction is isolated by centrifugation for 20 minutes at 4°C at 20000 rpm. The supernatant containing the VHH is loaded on Ni-NTA and purified to homogeneity. The yield of VHH is calculated according to the extinction coefficient.

5b) Repertoire cloning using Nanoclone® technology

This method exploits the fact that a sample or population of cells that is enriched in cells expressing, or capable of expressing, a heavy chain antibody against a desired antigen is readily available as a starting material for generating the desired immunoglobulin sequences, said method comprising the steps of: a) obtaining B-lymphocytes from at least one animal immunized with either IL- 6, IL-6R, or IL-6/IL-6R complex, b) selecting from said B-lymphocytes at least one B-3ymphocyte with specificity against IL-6, IL-6R, or IL-6/JL-6R complex: IL-6-, IL-6R-, or IL-6/IL-6R complex- specific B-lymphocytes can be obtained by panning B-lymphocytes in tubes, flasks, plates or magnetic microbeads (e.g. Dynal beads or MACS) coated with IL-6, IL-6R, or IL-6/IL-6R complex. Secreted immunoglobulin is captured on the cell membrane of the originating B -cell via an affinity matrix, consisting of a B -cell binding moiety, such as anti-CD19 or anti-CD45 and an immunoglobulin binding moiety, such as anti -llama immunoglobulin Fc. c) obtaining a nucleic acid from at least one B-lymphocyte selected in step (b), wherein said nucleic acid encodes said immunoglobulin against IL-6, IL-6R, or IL-6/TL-6R complex or encodes at least one part of said immunoglobulin directed against IL-6, IL-6R, or IL-6/IL-6R complex: RNA is isolated from

IL-6-, IL-6R-. or IL-6/IL-6R complex specific B-lymphocytes; methods to isolate RNA are known in the ait, and include TRIzol reagent (Invitrogen) and the Gough method (Gough, 1988); subsequently single stranded cDNA is synthesized from the isolated mRNA and double stranded DNA is prepared from the single stranded cDNA. Methods for the preparation of cDNA and double stranded DNA are known in the art. d) amplifying and/or cloning said nucleic acid so as to obtain an amplified and/or cloned nucleic acid which encodes said immunoglobulin against IL-6,

IL-6R, or IL-6/IL-6R complex or which encodes at least one part of said immunoglobulin directed against IL-6, IL-6R, or IL-6/IL-6R complex: amplification is performed using a universal 3'end primer having a sequence oligo-dT and a framework-specific 5' end primer (FRl). A unique restriction site BstEII is found to be generally present close to the 3'-end of the FR4 region of antibodies from human or other mammals. Such restriction site permits cloning of the amplified Nanobody gene without the need for a restriction site in the 3 '-end primer. Once obtained, the amplified DNA may be cloned into a vector not capable of expression in order to obtain a stable collection of antigen- specific Nanobody genes, preferably for storage. Such non-expression vectors are well known in the art. When expression of a sibgle Nanobody^® gene is required, a single, non-expression vector comprising a Nanobody^® gene is separated from the above mentioned collection, and the Nanobody^® gene therein is transferred to an expression vector. Means to perform such steps and vectors therefor are well known the art.

6) Construction of bispecific immunoglobulin sequences recognizing the human IL- 6/IL-6R complex, comprising a first Nanobody^® recognizing human IL-6 and a second Nanobody^® recognizing human IL-6R Recombinase systems such as non-expression / expression vectors systems allow the transfer of individual Nanobody^® encoding genes to be performed in bispecific format. For a detailed description of the preparation of bispecific immunoglobulin sequences, further reference is made to Conrath et al. (2001), as well as to for example WO96/34103 and WO99/23221.

7) Functional characterization of bispecific immunoglobulin sequences recognizing the human IL-6/IL-6R complex, comprising a first Nanobody^® recognizing human IL- 6 and a second Nanobod^ recoenizins human IL-6R

7a) Binding of bispecific immunoglobulin sequences to IL-6, IL-6R or IL-6/IL-6R complex in ELISA Bispecific immunoglobulin sequences is tested for binding to IL-6, IL-6R or IL-6/lL- 6R complex in ELISA. Therefore, a microtiterplate (Nunc. Maxisorb) is coated with human IL-6, human IL-6R or human IL-6/IL-6R complex at a 200-fold dilution and pre-warmed for 15 minutes at 37⁰C. The plate is coated overnight at 4°C. The plate is then washed with PBS-Tween and blocked for two hours at room temperature with PBS-I % casein. After washing, the samples are applied starting at a concentration of 10 μg/ml and 3-fold dilutions are made in PBS. After a two hours incubation period, the plates are washed and mouse monoclonal anti-myc antibody at a 1000-fold dilution is applied for 1 hour at room temperature. The plates are washed and polyclonal anti-mouse-HRP (DAKO) is applied at a 1000-fold dilution for one hour at room temperature. The plates are washed and ABTS/H₂O₂ substrate is applied. The OD 405 nm is measured. Sequences of polypeptides directed against the IL-6/IL-6R complex are shown in Table B-I.

7b) Inhibition of binding of human gp!30 protein to IL-6/IL-6R complex Thermanox coverslips (Nunc) are soaked overnight in 80 % ethanol, rinsed thoroughly with distilled water and air-dried. Human IL-6/IL-6R complex is solubilized in 0.05 mol/1 acetic acid and sprayed on the coverslips at a final density of

3Oμg/cm² with a retouching airbrush. After spraying the coverslips are blocked with

1% human albumin solution in PBS for at least 1 hour at room temperature. Gp 130 protein with or without addition of 2 μg/ml nanobody is then incubated for five minutes. The coverslip is subsequently rinsed in Hepes buffered saline (10 mM

Hepes, 150 mM NaCL, ph 7.4), fixed in 0.5% glutaraldehyde in PBS, dehydrated in methanol and stained with May-Grϋnwald and Giemsa (Riedel de Haen). Binding of gpl30 protein to IL-6/IL-6 receptor complex was measured as described above.

7c) Analysis in BIACORE for binding of homologous Nanobodies^® to IL-6/IL-6R complex

Homologue sequences of Nanobodies^® inhibiting the binding of gp!30 protein to IL- 6/IL-6R complex is obtained from a llama comprising certain amino acid differences. IL-6/IL-6R complex is covalently bound to the sensor chip surface via amine coupling. The CM5 surface of the chip is activated by the injection of EDC/NHS (1 : 1 mix of 0,4 M l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 0,1 M N- hydroxysuccinimide in water) for 7 minutes. Upon activation IL-6/IL-6R complex is injected until an increase of 6000 response units was detected. The excess of reactive groups is deactivated with 1 M Ethanolamine-HCl (pH 8,5) for 7 minutes. The flowrate is kept constant during the immobilization procedure at 5 ul/min. The eluent buffer is 0,01 M HEPES (pH 7,4) with 0,15 M NaCl, 3 mM EDTA and 0,005 % Surfactant P20. For the treatment of acute events, the fast inhibition of IL-6/IL-6R complex is very important, and thus a fast K-on rate is preferred. The K-on rate determines how fast a nanobody binds its target (1L-6/IL-6R complex) when injected into human or animals.

7d) Stability of nanobodies at elevated temperatures

A stock solution of nanobodies at a concentration of 200 μg/ml in PBS is prepared and divided into several tubes. Each tube containing nanobody is then incubated at different temperatures for 1 hour, cooled at room temperature for 2 hours and put at 4°C overnight. The next day, the samples are centrifuged for 30 minutes at 13000 rprn, and the supernatant is tested for OD280 nm. The concentration of supernatants are measured spectrophotometrically and expressed as a percentage of the concentration at room temperature. The supernatants are also tested in ELISA for binding to IL-6/1L-6R complex as described above in paragraph 4a.

7e) Cross-reactivity of the nanobodies with IL-6/IL-6R complex from other species A microtiterplate is coated with mouse anti-myc at 1/1000 overnight at 4°C. The plate is then washed with PB S -T ween and blocked for two hours at room temperature with PBS- 1% casein. After washing, the nanobodies are applied at a concentration of 10 μg/ml in PBS. After a one hour incubation period, the plates are washed and IL- 6/IL-6R complex (from dog, pig, human, baboon and cynomologues monkey) is applied at a suitable concentration. The plates are incubated for 1 hour at room temperature. The plates are washed and polyclonal anti-IL-6/IL-6R complex-HRP (DAKO) is applied at a 2000-fold dilution for one hour at room temperature. The plates are washed and ABTS/H₂O₂ substrate is applied. The OD 405 nm is measured.

7f) Humanization of nanobodies specific for IL-6/IL-6R complex Humanization of nanobodies specifically binding to IL-6/IL-6R complex can be achieved by substituting certain amino acid residues specific for variable domains of heavy chain antibodies of camelid species to amino acid residues specific for variable domains of conventional human antibodies. Methods for humanization are well known in the art.

Claims

C L A I M S

1. Polypeptide directed against the IL-6/IL-6R complex.

2. Polypeptide according to claim 1 that is specific for the IL-6/IL-6R complex and/or that binds with higher avidity to the IL-6/IL-6R-complex compared with binding to 1L-6 alone and/or to IL-6R alone, respectively.

3. Polypeptide according to claim 1 or 2, comprising or essentially consisting of at least one binding unit, or a fragment thereof, directed against the IL-6/IL-6R complex, and preferably specific for IL-6/IL-6R complex .

4. Polypeptide according to claim 1 or 2, comprising (a) at least one binding unit, or a fragment thereof, directed against IL-6, and preferably specific for IL-6; and (b) at least one binding unit, or a fragment thereof, directed against IL-6R, and preferably specific for IL-6R.

5. Polypeptide according to claim 4, comprising at least one binding unit, or a fragment thereof, directed against IL-6 (and preferably specific for IL-6), and at least one binding unit, or a fragment thereof, directed against IL-6R (and preferably specific for IL-6R), such that the resulting polypeptide binds with higher avidity to the IL-6/IL-6R-complex compared with binding to IL-6 alone and/or to IL-6R alone, respectively.

6. Polypeptide according to any of the preceding claims, which is capable of inhibiting the interaction between IL-6/IL-6R complex and gpl30 protein.

7. Polypeptide according to claim 6, which is capable of competitively inhibiting the interaction between IL-6/IL-6R complex and gpl30 protein.

8. Polypeptide according to any of the preceding claims, wherein said at least one binding unit is an immunoglobulin or immunoglobulin fragment.

9. Polypeptide according to any of the preceding claims, wherein said at least one binding unit is an antibody.

10. Polypeptide according to any of the preceding claims, wherein said at least one binding unit is an antibody fragment, such as a Fab fragment, a F(ab') fragment, a F(ab'₂) fragment, an Fv fragment, or a scFv fragment.

11. Polypeptide according to any of the preceding claims, wherein said at least one binding unit is an immunoglobulin variable domain, such as a V_H domain or a VL domain.

12. Polypeptide according to any of the preceding claims, wherein said at least one binding unit is a (single) domain antibody ((s)dAb).

13. Polypeptide according to any of the preceding claims, wherein said at least one binding unit is a Nanobody^®.

14. Polypeptide according to claim 3, wherein said at least one binding unit, or a fragment thereof, is directed against mammalian IL-6/IL-6R complex.

15. Polypeptide according to claim 3, wherein said at least one binding unit, or a fragment thereof, is directed against human IL-6/IL-6R complex.

16. Polypeptide according to claim 4, wherein

(a) said at least one binding unit, or a fragment thereof, is directed against mammalian IL- 6, and wherein (b) said at least one binding unit, or a fragment thereof, is direcled against mammalian IL-6R.

17. Polypeptide according to claim 4, wherein

(a) said at least one binding unit, or a fragment thereof, is directed against human IL-6, and wherein

(b) said at least one binding unit, or a fragment thereof, is directed against human IL-6R.

18. Polypeptide according to claim 13, wherein said Nanobody^® consists of 4 framework regions (FRl to FR4, respectively) and 3 complementary determining regions (CDRl to CDR3, respectively).

19. Polypeptide according to claims 13 or 18, wherein at least one Nanobody^®, or a fragment thereof, is a humanized Nanobody^®, or a fragment thereof.

20. Polypeptide according to claims 13, 18 or 19, wherein one or more amino acids of at least one Nanobody^®, or a fragment thereof, have been substituted without substantially altering the antigen binding capacity.

21. Polypeptide according to any of claims 13 or 18 to 19, wherein the number of Nanobodies^®, or a fragments thereof, is at least two.

22. Polypeptide according to any of the preceding claims, further comprising at least one polypeptide, directed to improving the half-life of the polypeptide in vivo.

23. Polypeptide according to claim 22, wherein said at least one polypeptide, directed to improving the half-life of the polypeptide in vivo is a polypeptide directed against a serum protein.

24. Polypeptide according to claim 23, wherein said at least one polypeptide, directed to improving the half-life of the polypeptide in vivo is a polypeptide directed against serum albumin, serum immunoglobulins, thyroxin binding protein, transferrin, or fibrinogen.

25. Polypeptide according to any of the preceding claims, which is pegylated.

26. Nucleic acid encoding a polypeptide according to any of claims 1 to 25.

27. Composition comprising a polypeptide according to any of claims 1 to 25 and/or nucleic acid according to claim 26.

28. Composition according to claim 27, which is a pharmaceutical composition, optionally comprising at least one pharmaceutically acceptable carrier.

29. Polypeptide according to any of claims 1 to 25, or the nucleic acid according to claim 24, or the composition according to claims 27 or 28, for the prevention and/or treatment of disorders or diseases relating to IL-6 and/or IL- 6R.

30. Use of the polypeptide according to any of claims 1 to 25, or the nucleic acid according to claim 26, for the preparation of a medicament for the prevention and/or treatment of disorders or diseases relating to IL-6 and/or IL-6R.

31. A method of producing a polypeptide according to any of claims 1 to 25 comⁿrisin &'.

(b) culturing host cells comprising a nucleic acid encoding a polypeptide according to any of claims 1 to 25 or capable of expressing a polypeptide according to claims 1 to 25 under conditions allowing the expression of the polypeptide, and,

(c) recovering the produced polypeptide from the culture; and (d) optionally pegylaling said polypeptide.

32. A method according to claim 31, wherein said host cells are bacterial cells or yeast cells.