CA2328486A1 - Osteoporosis treatment - Google Patents

Abstract

There is disclosed a process of treating or alleviating the symptoms of pathological conditions in which bone density is decreased, which comprises inhibiting, in a mammalian patient suffering from such a condition, the formation in vivo of a tertiary complex of IL-11, its cell surface membrane receptor and the cell surface glycoprotein gp130. Examples of such substances are recombinant soluble IL-11 receptor mutants modified, as compared with native IL-11 receptor, at their gp130 binding site, and peptides which can interact with IL-11. The process of the invention not only inhibits bone resorption and hence bone loss, but also increases the process of bone formation to increase bone density.

Description

OSTEOPO~(,~SIS TREATMENT
FIELD OF THE INVENTION
This invention relates to medical treatments and to agents useful therein. More particularly, it relates to the prevention and treatment of pathological s conditions in which the underlying pathology is an increase in bone resorption leading to bone loss, for example postmenopausal osteoporosis. It also relates to therapeutic agents useful in treatment and prevention of such conditions.
BACKGROUND OF THE INVENTION
to The remodelling of bone depends on a balance between bone formation and bone resorption. Osteoblasts are responsible for the formation of new bone osteoid, composed mainly of nonmineralized type 1 collagen. Bone resorption is mediated by large multinucleated cells called osteoclasts. To resorb i s bone, osteoclasts first establish zones of close contact with the mineralized matrix.
This forms a protected compartment between the osteoclast and the bone matrix intertace in which an acidic microenvironment is formed. Within these zones bone is demineralized, and the collagen fibres resorbed by the action of secreted lysosomal hydrolases. A number of factors have bean shown to be potent 2 o stimulators of bone resorption both in vitro and in vivo. These include parathyroid hormone, 1,25-dihydroxyvitamin D3 prostaglandin-Ez or-Iz, IL_-1, TNF-a, TNF-~i and bone-derived growth factors. None of these factors directly affect osteoclastic function; all require the presence of osteoblasts.
25 Increased bone resorption is a hallmark of a variety of clinical conditions. Thus, it occurs not only in postmenopausal women but is also a frequent complication of metastatic bone cancer, myeloma, and Paget's disease of bone. Current treatment involves the use of agents that block bone resorption wo 99~s~os pcric~~roos~6 (such as bisphosphonates and calcitonin) or, in the case of postmenopausal osteoporosis, hormone replacement therapy with estrogens.
Interleukin-11 (IL-11 ) has a role, either alone or in combination with s other cytokines, in bone formationlresorption.
IL-11 belongs to a family of cytokines which includes interleukin-6 (IL-6), leukemia inhibitory factor (LIF), and oncostatin M(OSM). These cytokines have similar tertiary structures, share a common signal transducer (gp130) and io have overlapping biological activities. In order for these cytokines to elicit a biological response, a tertiary complex, comprising the cytokine, its specific receptor (alpha chain), and gp130 must be formed.
15 BRIEF REFERENCE TO THE PRIO~, ART
Van Leuven et al.. Genomics (19961 Jan 1; 31 (1 ):65-70 report cloning the human gene for the interleukin-11 receptor (IL-11 R) , analysing the structure of the gene, and determining the predicted protein sequence. No 2 o analysis of protein structure and function was reported.
iCerow et ~L. Biochem J. ~(1996~ 318: 489-495, report cloning the gene for IL-11 a receptor and elucidation of its amino acid sequence, report the production of a soluble form of murine interlsukin-11 receptor (IL-11 R) and z s demonstrate that it interacts with the IL-11 ligand with high affinity.
The affinity of IL-11 alone for gp130 is reported to be below the level of detection, but a complex of IL-11 and soluble IL-11 R interacts with gp130 with high affinity. The receptor is a transmembrane protein that exhibits sequence homology with other members of the haemopoietin receptor family. However, the location of the iL-11 and gp130 3 o binding sites on the IL-11 R was not disclosed.

WO 99/x9608 PCT/CA99100516 Teramura et al. Blood 1992, 79:327 and Musashi at al. Proc. Natl.
Aced. Sci. U.S.A. 1991, 88:765 report that in bone, IL-11 functions alone or in combination with other cytokines to support granulocyte/macrophage colony formation and to increase the number and ploidy of platelet forming cells, megakaryocytes.
Girasole et al. J. Clin. Invest. 1994, 93:1516 and Tamura et a1. Proc.
Natl. Aced. Sci.1993, 90:11924) report experimental workthat demonstrates a role for IL-11 as well as other members of this family of cytokines in the process of osteoclastogenesis.
U. S. Patent 5.215.895 Bennett et al., issued June 1,1993, discloses processes for production of IL-11, by culturing a cell transformed with a DNA
sequence coding for IL-11.
Canadian Patent Annlication 2.177.837 Ciliberto et al, discloses an IL-6 antagonist which has been mutated at its gp130 binding region.
SUMMARY OF THE INVENTION
ft is an object of the present invention to provide a novel procedure for treatment and for alleviation of the symptoms of clinical conditions, such as osteoporosis, in which increased bone resorption or decreased bone formation is the underlying pathology.
Zs It is a further object of the invention to provide novel therapeutic substances useful in the treatment of such clinical conditions andlor in the alleviation of symptoms thereof.
s o The present invention is based upon the elucidation of the role of the cytokine interleukin-11 (IL-11 ) in both the process of bone resorption and the process of bone formation, and the unexpectedly advantageous results to be obtained by inhibiting its actions. This cytokine has been found to be critical for osteoclast formation and activity and therefore bone resorption. Moreover, this cytokine has been found to act as an inhibitor of bone formation. The mode of action of IL-11 in its role in bone loss conditions involves the formation of a tertiary complex of IL-11, its cell surtace membrane receptor ( IL-11 R) and the cell surface glycopratein gp130 (gp130). if this tertiary complex is not formed, or is formed to only a lesser extent, bane resorption is not only inhibited, but in many cases the formation of new bone is promoted, so that effectively new bone is formed and i o bone density is increased.
Accordingly, the present invention from one broad aspect provides a process of treating or alleviating the symptoms of a pathological condition in which bone density is decreased, which comprises inhibiting, in a mammalian i s patient suffering from such a condition, the formation in vivo of a tertiary complex of IL-11, IL-11R and gp130. Such conditions include those involving increased bane resorption and lack of desirable bone formation. in addition to osteoporosis, these conditions include metastatic bone cancer, myeloma, Paget's disease of bone, and bone fracture healing, especially in the elderly human patient.
According to another aspect of the invention, there are provided therapeutic agents for administration to a mammalian patient to alleviate the symptoms of pathological conditions in which bone density is decreased, and comprising biologically acceptable peptide compounds, protein sequences, small 2 5 molecules and antibodies capable of interfering with the in lrivo formation of a tertiary complex of IL-11, its cell surface membrane receptor IL-11 R, and the cell surface glycoprotein gp130.
According to another aspect of the invention there is provided a 3 o novel use of the TRAP and bone nodule formation assays to allow the detection of IL-11 antagonists.

According to another aspect of the invention there are provided methods for the selective removal of IL-11 from a solution and methods for the purification or enrichment of IL-11 from solutions.
According to another aspect of the invention, there are provided therapeutic agents for administration to a mammalian patient to alleviate the symptoms of pathological conditions in which bone density is decreased, and comprising biologically acceptable IL-11 antagonists capable of intertering with the inin vivo formation of a tertiary complex of IL-11, its cell surface membrane receptor to IL-11R, and the cell surface glycoprotein gp130.
According to another aspect of the invention, there are provided therapeutic agents for administration to a mammalian patient to alleviate the symptoms of pathological conditions in which bone density is decreased, and comprising transcribable genetic materials including antisense nucleic acids capable of inhibiting the translation of a component necessary to the formation of the IL-11 I IL-11R I gp130 tertiary complex.
According to another aspect of the invention, there are provided 2 o therapeutic agents for administration to a mammalian patient to alleviate the symptoms of pathological conditions in which bone density is decreased, and comprising expressible genetic materials including transcribable genetic materials encoding amino acid sequences capable of inhibiting the formation of the IL-11 IL-11 R / gp130 tertiary complex.
BRIEF REFf RENCE TO THE DRAWINGS
Figure 1 A is a diagrammatic representation of native IL-11 receptor, showing the various regions thereof and its binding interactions with IL-11 and 3 o gp130;

Figure 1 B is a representation of cDNA sequence depicted in SEQ
ID NO. 3 and employed in Example 3, below.
Figure 2 is a detailed sequence of the gp130 binding region of native IL-11 R and indicating the specifically preferred mutation sites and mutations of the products of the present invention. The extensively mutated sequence disclosed is SEQ ID NO. 4.
Figure 3 is a representation of a portion of the IL-11 receptor peptide to sequence indicating in bold the region of interaction with IL-11.
Figure 4 is a representation of the sequence of peplide 1 (SEQ ID
NO. 1 ) and peptide 2 (SEQ ID NO. 2) used in Example 5 below, indicating the activity observed in that experiment.
Figure 5 is a graphical presentation of the results obtained according to Example 1 below.
Figures 6 A, B and C are graphical presentations of the results obtained according to Example 2 below.
Figures 7 A and B are graphical presentations of the results obtained according to Example 4 below.
z5 Figure 8 is a graphical presentation of the 'results obtained according to Example 5 below.
Figure 9 is a graphical presentations of the results obtained according to Example 6 below.

Figure10 is a graphical presentation of the results obtained according to Example 7 below.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The preferred process according to the invention comprises treating or alleviating the symptoms of a pathological condition in which bone density is decreased in a mammalian patient suffering from such a condition, by administering to the patient an effective amount of a substance which inhibits the 1 o in vivo formation of a tertiary complex of IL-11, its cell surface membrane receptor IL-11 R and the cell surface glycoprotein gp130. Examples of such substances include antibodies to IL-11, antibodies to IL-11R, antibodies to gp130, mutant forms of IL-11 receptor, small molecule antagonists of IL-11, and peptide compounds which include sequences which selectively interact with IL-11 in the region normally bound by IL-11R, so as to intertere with the normal interaction between IL-11 and IL-11 R.
One group of preferred compounds of the present invention are recombinant soluble IL-11 R mutants which are modified, as compared with native 2 o IL-11 R, so that they can participate in the IL-111 IL-11 R interaction but do not productively interact with gp130. As a result, the tertiary complex of IL-11, and gp130 is not formed, or is formed only to a lesser extent, so that there is no, or a lesser, biological response. In a preferred embodiment, amino acid substitutions in the gp130 binding region, in these preferred compounds, substantially or completely abolish IL-11 R interactions with gp'130, while having little or no effect on lL-11 binding. However, mutations in other regions of the lL-11 R protein can alter the characteristics of the gp130 binding site on IL-11 R and prevent or inhibit productive IL-11 R / gp130 interaction. Any and alt soluble receptor mutants which interfere with the formation of the IL-11 1 gp130 / IL-3o tertiary complex, are within the scope of the present invention. Although the WO 99/39fii1 PCTICA99/00516 specific examples relate to the use of human-derived IL-11 R sequences, the corresponding sequences from other mammals will also work.
The soluble IL-11 Rs of the present invention are preferably mutated s at one or more of positions 282, 283, 286, 288 and 291, as depicted in 5Et0 iD
NO. 4, all of which are within the gp130 binding site of native !L-11 R.
Specific preferred mutations are D282 to G, A283 to D, 6286 to D, H289 to Y, and V291 to L, independently or in combination of two or more such mutations. Amino acids are described herein with reference to their standard three letter and one letter i o codes. In particular, the symbols D, G, A, H, Y, V and L have the usual meanings in connection with individual amino acids, namely D represents aspartic acid, G
represents glycine, A represents alanine, H represents histidine, Y represents tyrosine, V represents valine and L represents leucine.
i5 Native IL-11 R is a known protein having a molecular mess of about 46 kd. Its amino acid sequence has been determined. It comprises several distinct functional regions, as generally illustrated in Figure 1A of the accompanying dn~nrings. It is normally bound to the cell surtace membrane. It has a region for binding to IL-11. Another of its regions, from about position 270-300, zo is ita gp130 binding site. These regions are indicated on Figure 1A wherein represents the amino-terminal region containing 4 positional conserved cysteine residues, 2 represents an IL-11 binding region, 3 represents a gp130 binding region, and 4 represents the transmembrane domain.
2 s The amino aad sequence of the gp130 binding site is shown in Figure 2. The present invention, in one of its preferred embodiments, provides IL-11 Rs which are mutated in the gp130 binding site illustrated in Figure 2, by replacement of one or more of the native amino acids in this region with other amino acids. Specific preferred products of the present invention are those which a o have mutations at one or more of positions 282, 283, 286, 289 and 290 as WO 99/69b0s PCT/CA99/OOS16 illustrated and as described above. These mutations effectively reduce or even eliminate binding to gp130, but do not materially affect binding to IL-11.
The mutant soluble IL-11 Rs (sIL-11 Rs) of the present invention can be prepared by known techniques. In a preferred process, eDNA encoding the IL-11 R is cloned by RT-PCR using tL-11 R specific primers and total RNA isolated from human osteosarcoma cells. The primers contain terminal restriction endonuclease sites for subsequent cloning into plasmid vectors. The sequence of a preferred cDNA sequence for insertion is depicted in Figure 1 B. After ligation io into a suitable vector, the IL-11R DNA may be expressed in mammalian cells.
Baby hamster kidney (BHK) cells constitute an example of a suitable host mammalian cell for this purpose, After extraction and purification, the sIL-DNA can be subjected to site-directed mutagenesis to modify the amino adds in the IL-11 receptor which mediate gp130 binding but do not significantly affect i5 binding, for example the amino acids identified above.
A second group of preferred compounds in accordance with the present invention are IL-11 binding peptides, which are peptide sequences which selectively interact with IL-11 and prevent the interaction between IL-11 and IL-2 0 11 R necessary for the formation of the IL-11 / IL-11 R / gp130 tertiary complex.
Surprisingly, it has been determined that small amino acid sequences are capable of binding to IL-11 and preventing the normal interaction between IL-11 and IL-11 R. In particular, a peptide with the amino acid sequence Arg Arg Leu Arg Ala Ser Trp Thr Tyr Pro Ala Ser Trp Pro Cys Gln Pro His Phe Leu (SEQ lD NO 1 ) has z5 been identified which binds IL-11 and prevents its productive interaction with IL-11 R. Even more surprisingly, it has been found that the peptide sequence Arg Arg Leu Arg Ala Ser Trp (SEA ID NO 5) contained in the amino terminus of SEQ ID
NO 1 is important to the ability of this peptide to prevent the productive interaction of IL-11 and IL-11 R. This short peptide (SEQ ID NO. 5) is also capable of 3 o inhibiting the productive interaction of IL-11 and IL-11 R. In addition, a third peptide (SEQ. ID. NO. 6) which is located near, but does not overlap, the s 4 .s sequences corresponding to SEQ. ID. N0. 1 or SEQ. ID. NO. 5 on the human IL-11 R has been found to inhibit the productive interaction between IL-11 and IL-11 R.
Surprisingly, the amino acid sequences of the murine and human IL-s 11 R differ somewhat in the regions corresponding to SEQ. ID. NO 5 and SEQ.
ID.
NO. 6 (which depict the human sequences). In particular, the human amino acid sequence described in SEQ. ID. NO. 5 is RRLRASW, whereas the murine sequence is RRLHASW (SEQ. ID. NO. 10). Thus, the presence of a basic amino acid residue in the position corresponding to position 4 in peptide 1 (SEQ.
ID.
1 o N0.5) is preserved between the human and murine sequences, although the actual amino acid in that position varies. Thus, the peptides corresponding to SEQ ID NO 5 and SEQ ID NO 6 are IL-11 binding peptides and peptides having the amino acid sequence RRLXASW, where X is a basic amino acid (SEQ. ID.
N0.7) are potential IL-11 binding peptides.
Additionally, SEQ. ID. NO. 6 depicts the amino acid sequence of an IL-11 binding region identified within the human IL-11 R, namely: Ser Ile Leu Arg Pro Asp Pro Pro Gln Gly Leu Arg Val Glu Ser Val Pro Gly Tyr Pro. The corresponding murine sequence is depicted in SEQ. ID. NO. 8 and is: Ser Ile Leu 2 o Arg Pro Asp Pro Pro Gln Gly Leu Arg Val Glu Ser Val Pro Ser Tyr Pro. These sequences differ in their eighteenth amino acid whereby the human peptide has Gly and the murine sequence has Ser. Gly and Ser are both relativety small amino acid residues, having volumes of 60.1 and 89.0 ~3 respectively, and accessible surface areas of 75 and 115 RZ respectively. This suggests that the relatively 2s small size of amino acid 18 in this peptide facilitates interactions with IL-11.
However, Gly and Ser differ in their hydrophilicity, suggesting that several factors may interact to govern the suitability of particular amino acid substitutions at position 18 in these peptides. Thus, although IL-11 binding peptides exist which have the amino acid sequence: Ser Ile Leu Arg Pro Asp Pro Pro Gln Gly Leu Arg Val 3 o Glu Ser Val Pro xxx Tyr Pro, where xxx is a suitable amino acid, it will be necessary to screen potential IL-11 binding peptides having this sequence using the TRAP

assay and/or the bone nodule formation assay in order to determine if they are IL-11 binding peptides.
In addition to the amino acid substitutions discussed above, amino s acid sequences from other mammals which correspond to 1L-11 binding peptides identified from mammalian materials will also be potential IL-11 binding peptides.
Thus, where there is variation between mammalian sequences, having identified an IL-11 binding peptide sequence in one mammalian species, it is possible to identify other IL-11 binding peptides with reference to the corresponding amino to acid sequences in other mammals, and the pattern of conserved residues observed.
In light of the disclosure of the present application, it is within the capacity of a competent technician to identify and produce peptides capable of is interacting with IL-11 and inhibiting the productive interaction between IL-11 and IL-11 R, thereby inhibiting or preventing the formation of the IL-11 I IL-11 R
I gp130 complex. It will be obvious to one skilled in the art that peptide sequences containing amino acid substitutions or modifications which preserve the features essential for binding between the peptide and IL-11 are possible and are within the z o scope of the invention. Such peptides may comprise all or a portion of the amino acid sequence of SEQ ID NO 1, SEQ ID NO 5, SEQ. ID. NO. 6, SEA. ID. N0.7, SEQ. ID. NO 8, or SEQ. ID. NO. 10. Alternatively, these peptides may have a substantially different amino acid sequence from these sequences, but may have functional attributes permitting specific interactions between these peptides and z s IL-11. Peptides which are IL-11 binding peptides may be readily identified using one or both of the TRAP assay and the bone nodule formation assay, discussed herein.
Protein and peptide sequences which selectively interact with IL-11 3 o are valuable in vitro as well as in vivo. The present invention teaches a peptide sequence which selectively binds IL-11. It is therefore well within the capacity of i t. , WO 99/59608 PG?/CA99/OOSI6 a technician of ordinary skill to attach this peptide to a suitable substrate by way of an appropriate linking moiety. Once attached, the immobilized peptide sequence may be used to remove IL-11 from solutions. In particular, immobilized peptides can be used to deplete solutions of IL-11. Alternatively, immobilized s peptide can be used to bind IL-11 in one solution, and then release IL-11 in the presence of a second solution, thereby allowing the production of a solution enriched in IL-11 or with a reduced number or quantity of components other than IL-11.
1 o Thus, another preferred embodiment of the present invention is a process of selectively removing IL-11 from solutions using immobilized peptides of the invention having an affinity for IL-11.
In this application, the term "small molecule" refers to a compound 15 having a molecular weight of no more than 30 kd, and the term °IL-11 antagonist"
refers to compounds which inhibit or prevent the productive interaction between IL-11 and IL-11 R and which are less effective than IL-11 at promoting the productive interaction of IL-11 R and gp130.
2 o Particular IL-11 antagonists useful in the treatment of IL-11 related bone density disorders may be identified using the TRAP assay and the bone nodule formation assay, discussed herein. The TRAP assay is known in the art and involves co-culturing murine calvaria (osteoblasts) and bone marrow cells ~
vitro and then quantifying osteoclast formation by counting the number of tartrate-2 s resistant acid phosphatase positive (TRAP+) multinucleated dells. In light of the disclosure herein, and particularly the information pertaining to IL-11 binding peptides, including their sequences and the pattern of conserved residues, it is within the capacity of a competent technician to design potential IL-11 antagonists which are not peptides but which have comparable binding specificities. These 3o potential IL-11 antagonists may be screened to determine if they are IL-11 antagonists by examining their activity in the TRAP assay and the bone nodule formation assay.
Other preferred embodiments of the present invention are the use s of the TRAP assay and the bone nodule formation assay, individually or in combination, to screen samples for the presence of IL-11 antagonists, and to identify IL-11 antagonists.
A third group of preferred compounds are peptides which bind to IL-i o 11 R and prevent its productive interaction with IL-11. Candidate peptides which bind to IL-11 R can be designed by molecular modelling of the IL-11 ! IL-11 R
binding site in light of the disclosure in the present application.
Alternatively, such peptides can be identified by screening potential peptides. IL-11 R binding peptides may be identified from potential peptides by the ability of IL-11 R
binding ~5 peptides to reduce the formation of TRAP MNC's in the TRAP assay andlor the ability of IL-11 binding peptides to reduce the inhibitory effect of IL-11 on bone nodule formation in vitro, using the bone nodule formation assay.
A fourth type of preferred compound for use in the present invention 2 o for inhibiting or preventing the formation of the IL-11 /IL-11 R/gp130 tertiary complex comprises antibodies which selectively bind to one of the components of the complex to interfere with the interaction between components to form the tertiary complex. Some anti-IL-11 antibodies are commercially available. Alternatively, antibodies may be prepared using standard techniques following the injection of 2s a compound comprising the peptide of interest into a suitable animal.
Useful antibodies include anti-IL-11 antibodies, anti gp130 antibodies, anti-IL-11 R
antibodies, end humanized monoclonal antibodies specific for a site of interest on one of the components of the tertiary complex.
3 o The current invention teaches the sequence of the human IL-11 R
protein, the sequence of several IL-11 binding peptides, the sequence of IL-11 WO 99/x9608 PCT/CA99/OOS16 binding regions on the human and murine IL-11 Rs, and the sequence of the gp130 binding region on IL-11 R, as well as the means for the production of soluble IL-11 Rs. It is, therefore, within the capability of a competent technician to produce and screen antibodies which specifically bind either the IL-11 binding region or the s ~ gp130 binding region on the IL-11R. These specific antibodies may be used to specifically inhibit the binding of IL-11 to the IL-11 R through this binding region, thereby inhibiting formation of the aforementioned complex in vivo without substantially affecting the levels of IL-11 in solution. This may be particularly useful in situations where it is desired to inhibit the formation of the !L-11 / gp130 complex, but it is still desired to maintain free IL-11 and functional gp130 to achieve some other biological effect.
A fifth type of preferred compound for use in the present invention for inhibiting or preventing the formation of the IL-11 / IL-11 R I gp130 tertiary complex comprises small molecules capable of interfering with the IL-11 R / IL-interaction by specific binding to either IL-11 or IL-11 R in the IL-11 I IL-binding region. Small molecule antagonists of IL-11 and IL-11 R may be identified using the TRAP assay and the bone nodule formation assay. Both IL-11 antagonists and IL-11 R antagonists may be identified andlor synthesized in light of the features of the binding region of the compounds of the present invention.
Features such as size, shape, hydrophilicity and charge which control molecular binding affinity are well understood, and it is well within the capacity of a competent technician to identifyr candidate small molecules having the potential to bind either IL-11 R or IL-11 using molecular modelling in light of the peptides and 2s proteins of the present invention. It is also within the capacity of a competent technician to screen the candidate small molecules for the ability to inhibit or prevent the formation of the IL-11 / IL-11 R I gp130 complex using the TRAP
assay and the bone nodule formation assay, known in the prior art and described below.
3 o In light of the teachings of the present invention, it is also within the capability of a competent technician to produce transcribable genetic material WO 99/5%08 PCT/CA99/OOS16 capable of inhibiting the formation of the IL-11 / IL-11 R / gp130 tertiary complex.
One variety of transcribable genetic material which can be used is DNA encoding antisense RNA complementary to the mRNA encoding a s component necessary to the formation of the IL-11 / IL-11 R / gp130 tertiary complex (including IL-11, IL-11R, gp130, or portions thereof), and capable of inhibiting or preventing the translation of this mRNA. The mRNA sequence of IL-11 has been previously reported and sequence listings may be founds in the GENBANK database (Accession No. s M57766, M37007 (M. Fascicularis), 1 o Accession Nos. M81890, M57765, M37006 (human)). The mRNA sequence of gp130 has been previously reported and sequence listings may be founds the GENBANK database (Accession No.s M83336, MX62646 ). The mRNA sequence of the IL-11 R alpha chain has been reported and a sequence listing may be found in the GENBANK database (Accession No. U32324). In light of this sequence is information and the disclosure in the present patent application it is within the capacity of a competent technician to produce .transcribable genetic material capable of inhibiting the formation of the IL-11 I IL-11 R / gp130 tertiary complex.
The capacity of a particular antisense mRNA to inhibit the translation of a component of the IL-11 I IL-11 R I gp130 complex may be assessed using standard 2 o methods.
Antisense sequences may be used to inhibit the translation of the corresponding protein of interest (including IL-11, IL-11 R, and gp130), thereby effecting a reduction in levels of that protein in the treated cells. This reduction of 2s protein levels will reduce the protein available for binding in the IL-11 I

gp130 tertiary complex, thereby reducing the formation of the tertiary complex.
Transcribable genetic material encoding antisense nucleic acid sequences may be introduced into subjects by standard techniques, including gene therapy.
3 o A second type of transcribable genetic material which may be used to inhibit the formation of the IL-11 / IL-11 R / gp130 tertiary complex is transccibable genetic material encoding amino acid sequences capable of inhibiting the formation of the tertiary complex and containing amino acid sequences targeting these sequences to the appropriate location upon translation.
Examples of such amino acid sequences are soluble mutant IL-11 Rs, IL-11 binding peptides, and IL-11 R binding peptides of the present invention with suitable amino acid residues added which target these sequences for secretion. Protein targeting and selective cleavage sequences are known in the art and it is well within the capacity of a competent technician to produce transcribable genetic material encoding the amino acid sequence of interest and targeted for secretion.
to In addition to the amino acid sequences discussed above, it is well within the capacity of a competent technician to identify other amino acid sequences which will inhibit or prevent the formation of the IL-11 / IL-11 R /
gp130 tertiary complex using the TRAP' assay, and/or the bone nodule formation assay.
i5 Having identified a peptide or protein which is capable of inhibiting the formation of the tertiary complex, it is possible to determine the amino acid sequence of that peptide or protein using standard methods. It is therefore well within the capacity of a competent technician to devise an expressible genetic element encoding the amino acid sequence of the peptide or protein of interest and bearing the desired 2o post-translational modification and targeting information.
Transcriptional regulation elements may be selected so that the transcribable genetic material of the present invention is constitutively transcribed.
Alternatively, the level of transcription may be regulated by transcriptional control 2s elements which are sensitive to the level of an inducing compound. Suitable inducing compounds include substances naturally produced by tissues in the patient's body, the level of which may vary with the disease state or other factors.
Alternately, the inducing compound may be a substance which is not normally present in levels sufficient to allow transcription of the transcribable genetic 3 o material. In such a case, the inducing compound may be introduced into the A

subject at the time and dosage necessary to stimulate the desired level of transcription.
Post menopausal osteoporosis is characterized by a general reduction in bone mass, resulting from an imbalance between osteoblast-mediated bone formation and bone resorption by the osteoclast. While the osteoblast is responsible for the formation of new bone or osteoid, it also appears to control the activation and/or number of osteoclasts by releasing cytokines such as 1L-6 or IL-11. This process can be examined using a "TRAP assay" which involves co-1 o culturing marine calvaria (vsteoblasts) and bone marrow cells in vitro and then quantifying osteoclast formation by counting the number of tartrate-resistant acid phosphatase positive (TRAP+) multinucleated cells. Ovariectomized (OVX) rats or mice provide a satisfactory animal model for postmenopausal women in studies of osteoporosis.
Initial experiments comparing the ability of marrow cells isolated from sham-operated, OVX, and OVX mice treated with IL-11 neutralizing antibody, to form osteoclasts in vi demonstrated that IL-11 Ab treatment reduces osteoclast levels below those obtained from sham-operated animals. Accordingly, since bone 2 o density is determined by balancing bone formation with bone resorption, inhibitors of IL-11 R binding to gp130 reverse bone loss in post-menopausal patients.
Using cocultures of marine calvaria and bone marrow cells, it has been shown that IL-11 is a potent simulator of osteoclast formation in vitro.
Moreover, it has been demonstrated that IL-11 inhibits bone formation when marine calvaria cells (primary osteoblasts) are cult~xsd in the presence of ascorbic acid and 10mM p-glycerol phosphate (the "bone nodule formation assay"). Thus, by targeting IL-11 one can not only inhibit the process of osteoclastogenesis and therefore pathological bone loss, but one can also restore 3 o previously lost bone by stimulating the process of bone formation.

WO 99/x9608 PCT/CA99/00516 The in vitro TRAP assay also provides a convenient means to screen the effectiveness of soluble mutant IL-11 Rs, IL-11-binding peptides, IL-11 R
binding peptides, and small molecules prior to use in vivo. In particular, when small molecules, IL-11 binding peptides, IL-11 R binding peptides or mutant IL-s 11 Rs ere produced, their effectiveness at inhibiting the formation of a functional tertiary complex may be assessed in vitro using the TRAP assay. Those compounds causing a significant reduction in TRAP+ MNC's in the presence of exogenous IL-11 under assay conditions are deemed to be effective at inhibiting the formation of the tertiary complex. Where a potential compound of interest is i o contained within a mixture of compounds, it may be desirable to separate these compounds by standard means prior to examination using the TRAP assay. Such separation will allow the removal of potentially undesirable compounds, as well as reducing the number of possible compounds producing the results observed.
15 In addition to allowing the identification of carnpounds useful to inhibit the formation of the tertiary complex, the TRAP assay allows an assessment of the relative effectiveness of various compounds of the present invention.
The terms small molecule, IL-11 binding peptide, IL-11 R binding peptide and mutant IL-11 R when used herein refer to small molecules, peptides or proteins which are 20 effective at inhibiting the formation of the IL-1111L-i 1 R/gp130 tertiary complex in vitro when tested using the TRAP+ assay. The terms anti IL-11 antibody, and anti IL-11 R antibody when used herein refer to antibodies or portions or functional equivalents thereof which are effective at inhibiting the formation of the IL-11 R I gp 130 tertiary complex in vitro when tested using the TRAP assay.
The bone nodule formation assay provides a second convenient method to screen the effectiveness of soluble IL-11 R's, IL-11 bonding peptides, IL-11 R binding peptides, and small molecules at inhibiting the formation of the IL-11 / IL-11 R / gp130 tertiary complex. A compound to be assessed may be added 3 o to the bone nodule formation assay system, and the effect of the compound on bone nodule formation may be assessed. As with the TRAP assay, in some _ ~g _ circumstances it may be desirable to separate various compounds contained within a sample prior to examination using the bone nodule formation assay.
Those compounds causing a significant reduction in the inhibition of bone nodule formation by IL-11 under assay conditions are deemed to be effective at inhibiting the formation of the tertiary complex. In addition to allowing the identification of compounds useful in inhibiting the formation of the tertiary complex, this assay allows an assessment of the relative effectiveness of various compounds of the present invention.
i o Compounds of the present invention may be administered systemically or locally, by various modes of administration, and in various forms.
One form in which the proteins, peptides, and small molecules of the invention may be administered is in liquid form, as solutions or suspensions in appropriate, biologically acceptable carriers. A preferred means of delivering such liquid compounds is by injection. Alternately, the proteins, peptides, and small molecules of the present invention may be encapsulated for oral administration.
The encapsulation material may be selected to allow the release of the encapsulated compounds at an optimal stage in the digestive process to allow maximal biological effect.
Where localized treatment is desired, the cxxnpounds of the present invention may be included in a suitable matrix for implantation near the desired treatment site. Such matrices may be permanent or biodegradable, depending on the clinical needs of the patient. One particularly effective means of localized 2s administration is the inclusion of compounds of the present invention in implanted pins used in the immobilization of bone fractures. Another preferred form of local administration is by injection into the region of the mammalian patient's body proximate to the site affected by the disorder under treatment.
3 o The transcribable genetic material of the present invention may be administered systemically or locally, by various modes of administration.
Where WO 99lS9608 PCT/CA99100516 _ 20 _ it is desired to use antisense RNA sequences to inhibit translation of a cellular protein, the DNA encoding the antisense RNA may introduced into the nuclei of target cells by standard means. Where it is desired to introduce DNA sequences encoding protein or peptide products capable of inhibiting the formation of the lL-11 / IL-11 R / gp130 tertiary complex, the DNA may be introduced into the nuclei of cells already present in the patient's body by standard means.
Alternatively, the DNA may be introduced into the nuclei of MHC-compatible cells by standard means in vitro, and the resulting cells expressing the introduced DNA may be administered to the patient systemically or locally by standard means such as io intravenous injection, or local injection near sites of concern.
The desired dosage of active compound will vary, depending on the mode of administration, the condition to be treated, the overall condition of the subject, and the compound administered. It is well within the capability of a competent technician to determine the appropriate dosage for a particular patient in light of these factors. It is anticipated that where the systemic administration of the solubilized mutant IL-11 R of the present invention by injection is desired, the appropriate dosage will be between 1 mg to 20 mg of the soluble mutant IL-11 R
per kg body weight. Depending on the subject and the condition to be treated, 2o dosages will be more preferably between 1 to 10 mg per kg body weight for subjects whose existing bone density is not extremely low and between 10 mg to mg per kg body weight for subjects whose bone density is extremely low. It is anticipated that in many pathological conditions the appropriate systemic dosage of solubilized mutant IL-11 R will be similar to the appropriate dosage of 2 s parathyroid hormone for the treatment of a comparable patient.
Where localized administration of the compounds of the present invention is desired, the appropriate localized dosage can be determined with reference to the level of compound desired in the treatment area. It is anticipated 3o that the total dosage required for localized treatment will be lower than that level required for systemic treatment, and in many cases the appropriate localized wo 99is~os rcric~~roos~s dosage will be ten to one-hundred fold lower than the amount of compound required for systemic treatment.
Where it is desired to use a small molecule, IL-11 binding peptide, IL-11 R binding peptide or a specific antibody instead of, or in addition to, solubilized mutant IL-11 R, the appropriate dosage may be easily determined based on the appropriate dosage of IL-11 R. The small molecule, peptide or antibody dosage will be a function of the solubilized mutant IL-11 R dosage, adjusted to provide a comparable level of effective target binding based on the to abundance of the target, the relative molecular mass and binding affinity of the small molecule, peptide or antibody for its target relative to the solubilized mutant It.-11 R, its relative effectiveness at blocking tertiary complex formation in vitro, and its relative half-fife in vnro. Target abundance, molecular mass, binding affinity and in vivo half life of a particular small molecule, antibody or peptide may be i s determined by standard methods. The effectiveness of the compound at blocking the formation of the tertiary complex in vitro may be assessed using the TRAP
assay and/or the bone nodule formation assay, as previously discussed. It is anticipated that the appropriate dosage of the IL-11-binding peptides of the present invention when administered by local injection will frequently be between 2 0 0.1 to 10 mg per kg body weight. The dosage of a particular IL-11 binding peptide needed for a particular patient may be easily determined with reference to the factors discussed above, the patient's overall condition, and the disorder to be treated.
2 5 In some instances it will be desirable to enhance the activity and/or in vivo half-life of the peptides of the present invention by the chemical modification of these peptides to increase activity or to inhibit in vivo degradation.
For example, chemical moieties may be covalently attached to speck amino acids to interfere with the action of degradative enzymes. Alternatively or 3 o additionally, specific amino acids in the peptide sequence may be chemically modified to increase the overall peptide half life without greatly reducing specific binding between the peptide and IL-11. Additionally, it will be desirable in some instances to employ one or more D-isomer amino acid residues in the formation of these peptides. In some instances it will be desirable to cyclize the peptides.
For example, cyclization can be used to stabilize a particular peptide conformation in order to obtain a desired level of binding. Methods of modifying peptide sequences by the addition of chemical moieties to amino acids, the chemical modification of specific amino acids, the cyclization of peptides, and the incorporation of D-isomers of amino acids are known in the art and it is within the capacity of a competent technician in light of this disclosure to determine what to modifications are appropriate and how to effect these modifications.
Where the compound to be administered comprises antisense genetic material to be expressed within the patient's cells, the appropriate dose will also depend on the level of transcription of the antisense material, and its i5 stability and binding affinity for its complementary !L-11 R RNA in the cell.
Alternatively or additionally, expressible genetic material encoding a secreted form of a protein or peptide sequence of interest may be introduced into suitable MHC-compatible cells ex vivo by standard methods and these cells may 2o be introduced into the body of the patient by standard methods. These cells may be administered locally in the region where increased bone deposition or decreased bone resorption is needed, or they may be administered generally.
The expressible genetic element may be constitutively active, or it may be inducible.
The appropriate dosage of expressible genetic elements will depend on the mode 25 of administration, the condition to be treated, the patient's condition, the stability of the coding RNA and its protein product, the level of protein expression, and other factors influencing the level of effective target binding, as discussed in relation to IL-11 binding peptides, IL-11 R binding peptides, small molecules and specific antibodies. In general, it is anticipated that the appropriate dosage of 3 o expressible genetic elements will be the dosage which leads to a level of secreted protein or peptide in the vicinity of the target cells which is generally similar to the level of protein or peptide present in the vicinity of the target cells when the appropriate level of that peptide is administered directly to the patient.
Where the compound to be administered comprises an antibody s which specifically binds to IL-11, IL-11R, or gp130 and prevents or inhibits the formation of the tertiary complex, either systemic or localized administratior7 of the compound may be possible. Localized administration may be accomplished by various means, including the injection of a solution containing the antibody of interest into the region proximate to the tissue to be treated. Alternatively or to additionally, MHC-compatible cells secreting the antibody of interest may be introduced into the subject's body in either a systemic or a localized manner.
The invention is further described, for illustrative purposes, in the following specific examples.
is EXAMPLE 1 - IL-11 INH~ITS BONE NODULE FORMATION
The effect of IL-11 on bone formation was examined using the bone nodule formation assay. Murine calvaria cells (primary osteoblasts) were cultured 2 o in the presence of 250 IrM ascorbic acid and 10 mM ~i-glycerol phosphate, and in the presence of various amounts of interleukin-11. After culturing, the culture media were plated out and the number of bone nodules per plate was counted.
The results are shown on the accompanying Figure 5, where number of bone nodules per plate is plotted as the vertical axis, with the different concentrations 2 s of interleukin on the horizontal axis, not to scale. Each bar represents the result on experiments containing a different concentration of interleukin-11, as noted on Figure 5.
It is clearly seen that the experiment conducted in the absence of 3 o interleukin-11 led to the highest number of bone nodules per plate, and that the number of bone nodules per plate decreased as the interleukin-11 concentration increased. This demonstrates that IL-11 inhibits bone formation.

NEUTRALIZING ANTIBODIES TO HALT AND TO REVERSE BONE t-OS$ IN
OVARIECTOMIZ~Q.A~JIMALS
Twenty four laboratory mice were divided into four equal groups of six. Three of the groups were ovariectomized (OVX) while the fourth group was io sham-operated (SHAM), to act as a control. One week after ovariectomization, treatment of one group of ovariectomized animals (OVX + Anti Il_-11 Ab) with a daily dose of 160 Nglmouse of anti-IL-11 Ab (an affinity-purified sheep anti-murine IL-11 polyclonal neutralizing antibody) commenced. At the same time, treatment of another group of ovariectomized animals (OVX + NSIgG) with the same daily i5 dosage of normal sheep immunoglobulin (NSIgG) was commenced. Treatments were delivered once daily by intraperitoneal injection. Plasma analysis demonstrated that IgG entered the circulation of NSIgG group animals. Animals in the remaining two groups received no treatment.
2 o On the day on which treatment was commenced, the sham-operated animals (Sham-Baseline) and an equal number of the untreated ovariectomized animals (OVX-Baseline) were sacrificed to obtain their right femurs for histomorphometric analysis, so that baseline values could be established.
25 On day 21 after the commencement of the treatment, the remaining animals were similarly killed and their right femurs removed for bone histomorphometry. For this, the undecalcified distal third of the right femur of each mouse was embedded in glycolmethacrylate (JB-4 embedding medium;
Analychem, Toronto, Ontario, Canada). Histvlogic sections of 6 to 8 Nm were ~ o obtained using a Riechert Jung microtome (model K4; Riechert Jung Canada, Toronto, Ontario), mounted, and then stained with either 1 % toluidine blue or hematoxyline and eosin (H 8 E) before being subjected to morphometric analysis.
In each case, a region 800 pm below the epiphyseal growth plate that included the entire metaphysis was subjected to light microscopy using a Merz grid (Carl Zeiss Canada, Don Mills, Ontario). Sections examined in this fashion encompassed a s total tissue area of 5-8mm~. The following parameters were determined: (1 ) cancellous bone volume, (2) osteoblast surface, (3) osteoid surface, and (4) osteoclast surface. For each section, cancellous bone volume was calculated from a total of >1,600 point measurements (45 fields; 400x magnification),which were selected at random using the Merz grid. The percent osteoblast, osteoid, or to osteoclast surface was calculated under oil immersion (1,000x) by recording the presence or absence of each where the hemispherical grid of the Merz radical crossed cancellous bone. Osteoblasts were identified morphologically as distinct cuboidal-shaped cells lining the cancellous bone surface, whereas osteoclasts were identified morphologically as large multinucleated cells in close proximity to i s the cancel lous bone surface, which stained for tartrate-resistant acid phosphatase (Sigma Chemical Co., St. Louis, MO; Procedure No. 386).
The results of measurements of cancellous bone volume are presented graphically in Figure 6A. Clearly the animals of the OVX + Anti IL-2o Ab group, treated with IL-11 antibody, had a much larger volume of cancellous bone than the untreated OVX group and the negative control OVX NSIgG, IgG
treated group. Measurements on the femurs from sham and OVX animals sacrificed on the day on which treatment was commenced establish baseline values for the bone volume increases. It will be noted that the test animals of the 25 OVX + Anti IL-11 Ab group showed a significant gain in canceltous bone volume as compared to OVX baseline, indicating that cancellous bone loss was not only prevented but that it was reversed by the inhibition of biological activity of IL-11.
Figure 6 B of the accompanying drawings presents the results of 3 0 osteoid surface measurements, and indicates that the OVX + Anti IL-11 Ab animals exhibited higher rates of bone formation than the comparative groups, demonstrating that by inhibiting IL-11 biological activity one can promote the formation of new bone, reverse bone loss, and increase bone density in OVX
mice.
Figure 6 C of the accompanying drawings similarly presents the results of osteoclast surface measurements, and shows a notable reduction in the case of the OVX + Anti IL-11 Ab animals treated with the IL-11 antibodies.
This again indicates that the OVX + Anti IL-11 Ab animals exhibited much less bone resorption than the comparative groups, in further demonstration of the fact that inhibition of biological activity of IL-11 prevents and even reverses bone resorption i o in OVX mice.
EXAMPLE 3 - PREPARATION OF SOLUBLE INTERLEUKIN-11 R~,CEPTOR

cDNA encoding the IL-11 receptor (minus the transmembrane and cytoplasmicdomains) was cloned by RT-PCR using IL-11 receptor specificprimers and total RNA isolated from the human osteosarcoma cell tine SAGS-2. Primer sequences were based on the DNA sequence for the human IL-11 receptor a-2o chain. The forward primer contains a Kozak consensus sequence preceding the start ATG codon, while the reverse primer contains, in addition to a termination codon, bases encoding a histidine tag. Both primers contain terminal restriction endonuclease sites for subsequent cloning into plasrnid vectors. Authenticity of the cDNA insert encoding the soluble IL-11 receptor (cDNA depicted in SEQ ID NO
2 5 3) is confirmed by restriction endonuclease analysis and by double-stranded DNA
sequencing using a modified T7 DNA polymerase system (Sequenase, Amersham).
Far stable expression in mammalian cells, the IL-11 R cDNA is gel-3 o purified and ligated into the pcDNA3.1 vector (Invitrogen, Carlsbad, CA) which encodes a neomycin gene and allows for selection under high concentrations of 6418. The cDNA is inserted upstream of the human cytomegalovirus (CMV) immediate-early promoter/enhancer (this allows for high-level expression in a variety of mammalian cell lines) and downstream of the bovine growth hormone (BGH) polyadenylation signal (which allows for efficient transcript stabilization and s termination). The cDNA sequence to be inserted was formed from the cDNA
sequence corresponding to nuGeotides 62 to 1156 on the IL-11 R cDNA as identified by Van Leuven et al., with an additional 39 nucleotides added to the 3' end of this sequence to provide a thrombin cleavage site, a histidine tag, and a stop codon. The cDNA sequence inserted (SEQ ID NO. 3) is depicted in Figure io 1B.
Proper orientation of the inserted IL-11 R cDNA is confirmed by using restriction endonuclease analysis and DNA sequence analysis. After approximately 10-12 days of selection in medium containing neomycin, drug-i s resistant colonies are isolated and the highest secreting clones are seeded into roller bottles and the medium collected every 2-3 days. Soluble IL-11 R is purled from the collected medium using a Ni2'-IDA column and subsequently eluted using EDTA. Antigen levels are determined by ELISA using antibodies to both the histidine tag as well as the IL-11 receptor.
To assure that the sIL-11R does not associate with gp130, site-directed mutagenesis is used to modify the amino acids in the IL-11 receptor which mediate gp130 binding but do not affect IL-11 binding. Specifically, the process mutates 0282 to G, A283 to D, 6286 to D, H289 to Y, and V290 to L, 2 s independently or in combination. The relevant portion of the IL-11 R, and the preferred mutations, are depicted in Figure 2. Site-directed mutagenesis is pertormed as described previously (Austin, Richard C. et al. "FEBS Letters", Vol.
280, No. 2, 254:258 (March 1991 ) Federation of European Biochemical Societies) using mutant oligodeoxynucleotide primers synthesized at the Central Facility of 3 o the Institute for Molecular Biology and Biotechnology, McMaster University. Prior to expression in BHK cells, the resultant sIL-11 R mutant cDNAs are then inserted ...._.~... __....~."",.~.. .-_,»,~...._.

wo mss pc~ricAmoos~6 into pcDNA3.1, and the sequences of all the sIL-11 R mutant cDNAs are confirmed by sequencing as described above.
EXAMPLE 4 - DETERMINATION OF THE EFFECT OF A sIL-11 R ANTAGONIST

The effect of IL-11 on osteoclast development in cocultures of murine bone marrow and calvaria cells was examined using standard techniques.
Briefly, bone marrow cultures were established by removing femurs from mice, 1 o dissecting away soft tissue, and removing the distal and proximal ends of the femur. The marrow was then flushed with 5 ml a MEM and 1.0 °~
penicillin-streptomycin using a 25 gauge needle. The bone marrow cells were then suspended to a concentration of 5 000 000 cells per ml in a MEM containing 15 °~ fetal calf serum (charcoal treated) to remove cells adherent to plastic. The non-adherent cells were then co-cultured for an additional 9 days with murine calvaria cells prior to being fixed and stained for TRAPase activity (stains were obtained from Sigma Chemical Co., St. Louis, MO). TRAP+ MNC's have the ability to form resorption pits in smooth cortical bone slices and are therefore considered to be of osteoclast origin.

(i) IL-11 dose-dependency of TRAP+ cell formation The effect of IL-11 on osteoclast development in cocultures of murine bone marrow and calvaria cells was examined by maintaining these cultures in the presence of 2 s various specific concentrations of IL-11 for 9 days. After 9 days of culture, the cells were stained for TRAPase activity and the number of multinucleated TRAP+
cells were determined. The results of this experiment are depicted in Table I
Data are expressed as mean +/- SEM.

(ii) Impact of mutant IL-11 receptor on IL-11 induced osteoclast formation In order to assess the effect of a gp130 binding region mutant IL-11R on IL-11 induced osteoclast formation, the procedure described in (i) above was repeated using 20 nglml IL-11 and 10, 100, or 1000 nglml of either (A) the H2$9-~Y289 mutant solubilized IL-11 receptor described in Example 3, or (B) a corresponding solubilized native IL-11 receptor. The results of this experiment, depicted in Figures 7 A and B, demonstrate that a mutant IL-11 receptor is capable of inhibiting IL-11-induced osteoclast formation, whereas the native IL-11 receptor io is not.

is It is desirable to have a means of selectively inhibiting the interaction of IL-11 with the IL-11 R, without adding exogenous antibodies or other large proteins. Surprisingly, it was determined that a short peptide sequence could be created which is capable of inhibiting the interaction between IL-11 and the IL-2 0 11 R. A peptide with the amino acid sequence Arg Arg Leu Arg Ala Ser Trp Thr Tyr Pro Ala Ser Trp Pro Cys Gln Pro His Phe Leu ("peptide 1 ", SEQ ID NO. 1 ) was synthesized which is homologous to a region in the IL-11 receptor which appears to bind IL-11.
2s To determine if peptide 1 could inhibit IL-11-induced MNC formation, the procedure of Experiment 4 was repeated using peptide 1 in place of the gp130 binding region mutant !L-11 receptor protein, and using an overlapping peptide ("peptide 2", SEA ID NO. 2) in place of the solubilized native IL-11 receptor.
Peptide 2 has the amino acid sequence Thr Tyr Pro Ala Ser Trp Pro Cys Gln Pro 3o His Phe Leu Leu Lys Phe Arg Leu Gln Tyr (SEQ ID NO 2) and represents a portion of the amino acid sequence occurring in the native IL-11 receptor, and overlapping in part with the sequence of peptide 1. Peptide 2 lacks the N
terminal Arg Arg Leu Arg Ala Ser Trp sequence (SEGl ID N0. 5) which is contained in peptide 1. The sequence of these peptides is depicted in Figure 4.
s Peptide 1 inhibits IL-11 induced osteoclast formation, whereas peptide 2 does not. The results of this experiment pertaining to peptide 1 are depicted in Figure 8. This indicates that a peptide sequence comprising Arg Arg Leu Arg Ala Ser Trp is capable of interacting with IL-11 and acting as an antagonist to IL-11 mediated activation of the osteoclast formation. The ability of 1 o peptide 1, and particularly the peptide sequence Arg Arg Leu Arg Ala Ser Trp to inhibit osteoclast formation indicates that this peptide is interacting with IL-11.
Thus, peptide 1 is an example of an IL-11 binding peptide.
i 5 EXAMPLE B - DETERMINATION OF THE EFFECT OF IL-11 ANTAGONISTS ON
THE ABILITY OF IL-11 TO INHIBIT BONE NOQULE F ATIrZN IN VITRO
It was desired to determine if the IL-11 antagonists of the invention could control bone nodule formation, and in particular, if these antagonists could reduce the 2 o inhibitory effect of IL-11 on bone nodule formation.
Bone nodule formation was measured using standard techniques. Briefly, calvaria cell cultures were established as follows: Calvaria cells were obtained from 2 day old fetal mouse calavariae by collagenase digestion. The cells were then cultured 2s for 21 days in the presence of 0.5 mM ascorbic acid 'and 10 mM p-glycerophosphate. Where indicated in the experimental descriptions below, !L-and the mutant IL-11 receptor of experiment 4 were added to the culture at day and every 3-4 days thereafter until the removal of the medium for analysis.
Bone nodule formation was quantified by counting alizarin red stain nodules under a 3 0 light microscope.

Exogenous IL-11 alone added to murine calvaria cell culture can inhibit bone nodule formation, as depicted in Figure 5. The effect of a gp130 binding region mutant soluble IL-11 receptor of type described in Experiment 4 and produced by the process of Experiment 3 on bone nodule formation in the s presence of 20 nglml of exogenous IL-11 was assessed, and the results are depicted in Figure 9. These results indicate that very low levels of mutant solubilized IL-11 receptor can reverse the effect of exogenous IL-11.
Moreover, the addition of 10 nglml of the mutant receptor used in this experiment was capable of allowing enhanced bone nodule formation relative to the level observed to in the absence of exogenous IL-11. Thus, IL-11 antagonists such as mutant IL-11 Rs can enhance bone nodule formation.
EXAMPLE 7 - DETERMINATION OF TI'iE EFFECT OF AN IL-11 ANTAGONIST

~ 5 IN VITRO
It was desired to determine if bone nodule formation could be modulated in a manner similar to that reported in Example 6 using an IL-11 antagonist peptide such as the one used in Example 5. To achieve this, the 2 o procedure of Example 6 was repeated, with the substitution of peptide 1 for the solubilized mutant IL-11 receptor. The results of this experiment are depicted in Figure 10. Peptide 1 was able to reduce the inhibitory effects of 20 ng/ml exogenous IL-11 at very low concentrations, and at concentrations of 50 ng/ml, peptide 1 allowed bone nodule formation in excess of that seen in the control 2 5 samples. Peptide 2 was unable to reduce the inhibition of nodule formation by IL-11. Thus, peptide 1 (SEQ ID N0.1 ), and particularly the peptide sequence Arg Arg Leu Arg Ala Ser Trp (SEG1 ID N0.5), is capable of allowing enhanced bone nodule formation.

SEQUENCE LISTINGS
( 1 ) GENERAL INFORMATION
(i) APPLICANT:
SHAUGNESSEY, Stephen 72 Leaside Drive St. Catharines, Ontario CANADA
AUSTIN, Richard Carl 68 Rosemary Lane Ancaster, Ontario CANADA
(ii) TITLE OF INVENTION: OSTEOPOROSIS TREATMENT
(iii) NUMBER OF SEQUENCES: 10 (iv) CORRESPONDENCE ADDRESS:
RIDOUT & MAYBES
150 Metcalfe St.
Ottawa, Ontario K2P OJ7 CANADA
(v) COMPUTER READABLE FORM
(a) COMPUTER: IBM Compatible (b) OPERATING SYSTEM: WINDOWS 95 (c) SOFTWARE: WORDPERFECT 8 (vi) CURRENT APPLICATION DATA
(a) APPLICAT10N NUMBER: 2,237,915 (b) FILING DATE: 1998/05/19 (c) CLASSIFICATION:

(vii)PRIOR APPLICATION DATA

(a) APPLICATION NUMBER:
NONE

(b) FILING DATE:

(c) CLASSIFICATION:

(viii) PATENT AGENT INFORMATION
(a) NAME: Robert G. Hirons, RIDOUT & MAYBEE
(b) REFERENCE NUMBER: 29210-0011 (2) INFORMATION FOR SEQ ID NO: 1 (i) SEQUENCE CHARACTORISTICS
(a) LENGTH: 20 (b) TYPE: amino acid (c) STRANDEDNESS:
(d) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: no (iv) ANTISENSE: no (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: human interleukin-11 receptor protein sequence (vii) IMMEDIATE SOURCE: synthetic (viii) POSITION IN GENOME:

i WO 99159608 PCTICA99/OOSIb (a) CHROMOSOME1SEGMENT; 9p13 (b) MAP POSITION:
(c) UNITS:
(ix) FEATURE
(a) NAME/KEY: IL-11 binding region (b) LOCATION: amino acids 1-8 (c) IDENT1FICATION METHOD: experiment (d) OTHER INFORMATION: IL-11 receptor antagonist (x) PUBLICATION INFORMATION
(a) AUTHOR(S):
(b) TITLE:
(c) JOURNAL:
(d) VOLUME:
(e) ISSUE:
(~ PAGES:
(g) DATE:
(b) DOCUMENT NUMBER:
(i) FILING DATE:
(j) PUBLICATION DATE:
(k) RELEVANT RESIDUES IN SEQUENCE ID NO:
(xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO:1 WO 99/59608 PCTlCA99/0~516 Arg Arg Leu Arg Ala Ser Trp Thr Tyr Pro Ala Ser Trp Pro Cys Gln Pro His Phe Leu (3) INFORMATION FOR SEQ ID N0:2 (i) SEQUENCE CHARACTORISTICS
(a) LENGTH:20 (b) TYPE: amino acid (c) STRANDEDNESS:
(d) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: no (iv) ANTISENSE: no (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: human interleukin-11 receptor protein sequence (vii) IMMEDIATE SOURCE: synthetic (viii) POSITION IN GENOME:
(a) CHROMOSOME/SEGMENT: 9p13 (b) MAP POSITION:
(c) UNITS:
(ix) FEATURE
(a) NAME/KEY:
(b) LOCATION:
(c) IDENTIFICATION METHOD:

(d) OTHER INFORMAT10N:
(x) PUBLICATION INFORMATION
(a) AUTHOR(S):
(b) TITLE:
(c) JOURNAL:
(d) VOLUME:

(e) ISSUE:

(fj PAGES:

(g) DATE:

(h) DOCUMENT NUMBER:

(i) FILING DATE:

(j) PUBLICATION DATE:

(k) RELEVANT RESIDUES IN SEQUENCE ID NO:

(xi) SEQtJENCEDESCRIPTION: SEQUENCE ID N0:2 Thr Tyr Pro Ala Ser Trp Pro Cys Gln Pro His Phe Leu Leu Lys :~ Phe Arg Leu Gln Tyr (4) INFORMATION FOR SEQ ID NO; 3 (i) SEQUENCE CHARACTORISTICS
(a) LENGTH: 1140 bsscs (b) TYPE: nucleotide and amino acid (c) STRANDEDNESS: double stranded (d) TOPOLOGY: linear 6izz (ii) MOLECULE TYPE: cDNA, protein (iii) HYPOTHETICAL: no (iv) ANTISENSE: no (v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE: murine interleukin-11 receptor sequence (vii) IMMEDIATE SOURCE: mouse bone marrow stromal cells (viii) POSITION IN GENOME:
(a) CHROMOSOMEISEGMENT:
(b) MAP POSITION:
(c) iINITS:
(ix) FEATURE
(a) NAME/KEY:

(b) LOCATION:

(c) IDENTIFICATION METHOD: experiment (d) OTHER INFORMATION: soluble form of IL-11 receptor, sequence additionally modified from wild type for cloning and histidine purification purposes (x) PUBLICATION
INFORMATION

(a) AUTHOR(S):

(b) TITLE:

(c) JOURNAL:

(d) VOLUME:

WO 99/39608 PCT/CA99100s16 (e) ISSUE:
PAGES:
(g) DATE:
(h) DOCUMENT NUMBER:
(i) FILING DATE:
(j) PUBLICATION DATE: .
(k) RELEVANT RESIDUES IN SEQUENCE ID NO: 3 (xi) SEQUENCE DESCRIPTION: SEQUENCE ID N0:3 S' ATG AGC AGC AGC TGC TCA GGG CTG ACC AGG GTC CTG GTG GCC GTG GCT
ACG GCC
Met Ser Ser Ser Cys Ser Gly Leu Thr Arg Val Leu Val Ala Val Ala Thr Ala CTG GTG TCT TCC TCC TCC CCC TGC CCC CAA GCT TGG GGT CCT CCA GGG
GTC CAG
Leu Val Ser Ser Ser Ser Pro Cys Pro Gln Ala Trp Gly Pro Pro Gly Val Gln TAT GGA CAA CCT GGC AGG CCC GTG ATG CTG TGC TGC CCC GGA GTG AGT
CiCT GGG
Tyr Gly Gln Pro Gly Arg Pro Val Met Leu Cys Cys Pro Gly Val Ser Ala Gly ACT CCA GTG TCC TGG TTT CGG GAT GGA GAT TCA AGG CTG CTC CAG GGA

CCT GAC
Thr Pro Val Ser Trp Phe Arg Asp Gly Asp Ser Arg I,eu Leu Gln Gly Pro Asp TCT GGG TTA GGA CAC AAA CTG GTC TTG GCC CAG GTG GAC AGC CCT GAT
GAA GGC
Ser Gly Leu Gly His Lys Leu Val Leu Ala Gln Val Asp Ser Pro Asp Glu Gly ACT TAT GTC TGC CAG ACC CTG GAT GGT GTA 1'CA GGG GGC ATG GTG ACC
CTG AAG
Thr Tyr Val Cys Gln Thr Leu Asp Gly Val Ser Gly Gly Met Val Thr Leu Lys CTG GGC TTT CCC CCA GCA CGT CCT GAA GTC TCC TGC CAA GCG GTA GAC
TAT GAA
Leu Gly Phe Pro Pro Ala Arg Pro Glu Val Ser Cys Gln Ala Val Asp Tyr Glu AAC TTC TCC TGT ACT TGG AGT CCA GGC CAG GTC AGC GGT TTG CCC ACC
CGC TAC
Asn Phe Ser Cys Thr Trp Ser Pro Gly Gln Val Ser Gly Leu Pro Thr Arg Tyr CTT ACT TCC TAC AGG AAG AAG ACG CTG CCA GGA GCT GAG AGT CAG AGG
GAA AGT
Leu Thr Ser Tyr Arg Lys Lys Thr Leu Pro Gly Ata Glu Ser Gln Arg Glu Ser CCA TCC ACC GGG CCT TGG CCG TGT CCA CAG GAC CCT CTG GAG GCC TCC
CGA TGT
Pro Ser Thr Gly Pro Trp Pro Cys Pro Gln Asp Pro Leu Glu Ala Ser Arg Cys GTG GTC CAT GGG GCA GAG TTC TGG AGT GAG TAC CGG ATC AAT GTG ACC
GAG GTG
Val Val His Gly Ala Glu Pb~ Trp Ser Glu Tyr Arg Ile Asn Val Thr Glu Vat AAC CCA CTG GGT GCC AGC ACG TGC CTA CTG GAT GTG AGA TTA CAG AGC
ATC TTG
Asn Pro l:.eu Gly Ala Ser Thr Cys Leu Leu Asp Val Arg L,eu Gln Ser Ile L.eu CGT CCT GAT CCA CCC CAA GGA CTG CGG GTG GAA TCC GTA CCT AGT TAC
CCG AGA
Arg Pro Asp Pro Pro Gln Gly Leu Arg Val Glu Ser Val Pro Ser Tyr Pro Arg CGC CTG CAT GCC AGC TGG ACA TAC CCT GCC TCC TGG CGT CGC CAA CCC
CAC TTT
Arg Leu His Ala Ser Trp Thr Tyr Pro Ala Ser Trp Arg Arg Gln Pro His Phe CTG CTC AAG TTC CGG TTG CAA TAC CGA CCA GCA CAG CAT CCA GCG TGG
TCC ACG

Leu Leu Lys Phe Arg Leu Gln Tyr Arg Pro Ala Gln His Pro Ala Trp Ser Thr GTG GAG CCC ATT GGC TTG GAG GAA GTG ATA ACA GAT GCT GTG GCT GGG
CTG CCA
Val Glu Pro Ile Gly Leu Glu Glu Val Ile Thr Asp Ala Val Ala Gly Leu Pro CAC GCG GTA CGA GTC AGT GCC AGG GAC TTT CTG GAT GCT GGC ACC TGG
AGC GCC
His Ala Val Arg Val Ser Ala Arg Asp Phe Leu Asp Ala Gly Thr Trp Ser Ala TGG AGC CCA GAG GCC TGG GGT ACT CCT AGC ACT GGT CCC CTG CAG GAT
GAG ATA
Trp Ser Pro Glu Ala Trp Gly Thr Pro Ser T6r Gly Pro Leu Gln Asp Glu Ile CCT GAT TGG AGC CAG GGA CAC GGA CAG CAG CTA GAG GCA GTA GTA GCT
CAG GAG
Pro Asp Trp Ser Gln Gly His Gly Gln Gln Leu Glu A1a Val Val Ala Gin Glu GAC AGC CCG GCT CCT GCA AGG CCT TCC TTG CAG CCG GAC CCA AGG CCA
CTT GAT
Asp Ser Pro Ala Pro Ala Arg Pro Ser Leu Gln Pro Asp Pro Arg Pro Leu Asp wo ms~os pcric~~roos~6 CAC AGG GAT CCC TTG GAG CAA CTG GTG CCA CGC GGT TCT CAC CAC CAC
CAC CAC
His Arg Asp Pro Leu G1u Gln Ixu Val Pro Arg Gly Scr His His His His His CAC TGA 3' His ***
(5) INFORMATION FOR SEQ ID N0:4 (1) SEQUENCE CHARACTORISTICS
( 1 ) LENGTH : 10 (2) TYPE: amino acid (3) STRANDEDNESS:
(4) TOPOLOGY: linear (2) MOLECULE TYPE: peptide (3) HYPOTHETICAL: yes (4) ANTISENSE: no (5) FRAGMENT TYPE: internal (6) ORIGINAL SOURCE: human interleukin-11 receptor protein sequetxe (7) IMMEDIATE SOURCE: synthetic (8) POSITION IN GENOME:
(a) CHROMOSOME/SEGMENT: 9p13 (b) MAP POSITION:
(c) UNITS:

WO 99/59608 PC1'/CA99/00516 (ix) FEATURE
(a) NAME/KEY: ineffectual gp130 binding region (b) LOCATION: entire sequence (c) IDENTIFICATION METHOD: experimcnt (d) OTHER INFORMATION:
(x) PUBLICATION INFORMATION
(a) AUTHOR(S):
(b) TITLE:
(c) JOURNAL:
(d) VOLUME:
(e) ISSUE:
(f) PAGES:
(g) DATE:
(h) DOCUMENT NUMBER:
(i) FILING DATE:
(j) PUBLICATION DATE:
(k) RELEVANT RESIDUES IN SEQUENCE ID NO:
(xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO: 4 Gly Asp Val Ala Asp Leu Pro Tyr Ala Leu (6) INFORMAT10N FOR SEQ ID NO: 5 (i) SEQUENCE CHARACTORISTICS

(a) LENGTH: 7 (b) TYPE: amino acid (c) STRANDEDNESS:
(d) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: no (iv) ANTISENSE: no (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: human intcrleukin-11 receptor protein sequence (vii) IMMEDIATE SOURCE: synthetic (viii) POSITION IN GENOME:
(a) CHROMOSOME/SEGMENT: 9p 13 (b) MAP POSITION:
(c) UNITS:
(ix) FEATURE
(a) NAMFJICEY: IL-11 binding region (b) LOCATION: entire sequence (c) IDENTIFICATION METHOD: experiment (d) OTHER INFORMATION: IL-11 receptor antagonist (x) PUBLICATION INFORMATION
(a) AUTHOR(S):

wo ms~at rc~ric~~oos~6 (b) TITLE:
(c) JOURNAL:
(d) VOLUME:
(e) ISSUE:
(t) PAGES:
(g) DATE:
(h) DOCUMENT NUMBER:
(i) FILING DATE:
(j) PUBLICATION DATE:
(k) RELEVANT RESIDUES IN SEQUENCE ID NO:
(xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO:S
Arg Arg Leu Arg Ala Ser Trp (7) INFORMATION FOR SEQ ID NO: 6 (i) SEQUENCE CHARACTORISTICS
(a) LENGTH: 20 (b) TYPE: amino acid (c) STRANDEDNBSS:
(d) TOPOLOGY: Iit~ear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: no (iv) ANTISENSE: no (v) FRAGMENT TYPE: internal WO 99/s9608 PCT/CA99/00516 (vi) ORIGINAL SOURCE: human interleukin-11 receptor protein sequence (vii) IMMEDIATE SOURCE: synthetic (viii) POSITION IN GENOME:
(a) CHROMOSOME/SEGMENT:9p13 (b) MAP POSITION:
(c) UNITS:
(ix) FEATURE
(a) NAMEIKEY: IL-11 binding region (b) LOCATION:
(c) IDENTIFICATION METHOD: experiment (d) OTHER INFORMATION: IL-11 receptor antagonist (x) PUBLICATION INFORMATION
(a) AUTHOR(S):
(b) TITLE:
(c)JOURNAL:

(d)VOLUME:

(e)ISSUE:

(f)PAGES:

(g)DATE:

(h)DOCUMENT NUMBER:

(i)FILING DATE:

WO 99/59608 PCT~A99I00S16 16r22 (j) PUBLICATION DATE:
(k) RELEVANT RESIDUES IN SEQUENCE ID NO:
(xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO: 6 Ser Ile Leu Arg Pro Asp Pro Pro Gln Gly Leu Arg Val Glu Ser Val Pro Gly Tyr Pro (8) INFORMATION FOR SEQ ID NO: 7 (i) SEQUENCE CHARACTORISTICS
(a) LENGTH: 7 (b) TYPE: amino acid (c) STRANDEDNESS:
(d) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL. yes (iv) ANTISENSE: no (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:
(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(a) CHROMOSOME/SEGMENT:
(b) MAP POSITION: _ (c) UNITS:
(ix) FEATURE

(a) NAMEJKEY: IL-11 binding region (b) LOCATION:
(c) IDENTIFICATION METHOD: experiment, homology (d) OTHER INFORMATION: IL-11 receptor antagonist (x) PUBLICATION INFORMATION
(a) AUTHOR(S):
(b) TI'fL E:
(c) IOURNAL:

(d) VOLUME:

(e) ISSUE:

(~ PAGES

(g) DATE:

(h) DOCUMENT NUMBER:

(i) FILING DATE:

(j) PUBLICATION DATE:

(k) RELEVANT RESIDUES IN SEQUENCE ID
NO: 7 (xi) SEQUENCE
DESCRIPTION:
SEQUENCE ID
NO: 7 Arg Arg Leu X Ala Ser Trp where X is a basic amino acid (9) INFORMATION FOR SEQ ID NO: 8 (i) SEQUENCE CHARACTORISTICS
(a) LENGTH:20 WO ~~~ PCT/CA99/00516 (b) TYPE: amino acid (c) STRANDEDNESS:
(d) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: no (iv) ANTISENSE: no (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: marine interleukin-11 receptor (vii) IMMEDIAT>r 5UUIZLI;:
(viii) POSITION IN GENOME:
(a) CHROMOSOME/SEGMENT:
(b) MAP POSITION:
(c) UNITS:
(ix) FEATURE
(a) NAMEJKEY: IL-11 binding region (b) LOCATION:
(c) IDENTIFICATION METHOD: experiment, homology (d) OTHER INFORMATION: IL-11 receptor antagonist (x) PUBLICATION INFORMATION
(a) AUTHOR(S):
(b) TITLE:

WO 99/5960$ PCT/CA9910051b (c) JOURNAL:
(d) VOLUME:
(e) ISSUE:
(t) PAGES:
(g) DATE:
(h) DOCUMENT NUMBER:
(i) FILING DATE:
(j) PUBLICAT10N DATE:
(k) RELEVANT RESIDUES IN SEQUENCE ID NO: 8 (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO: 8 Ser Ile Leu Arg Pro Asp Pro Pro Gln Gly Leu Arg Val Glu Ser Val Pro Ser Tyr Pro (10) INFORMATION FOR SEQ ID NO: 9 (i) SEQUENCE CHARACTORISTICS
(a) LENGTH: 20 (b) TYPE: amino acid (c) STRANDEDNESS:
(d) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: yes (iv) ANTISENSE: no (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:
(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(a) CHROMOSOME/SEGMENT:
(b) MAP POSITION:
(c) UNITS:
(ix) FEATURE
(a) NAMEJKEY:
(b) LOCATION:
(c) IDENTIFICATION METHOD:
(d) OTHER INFORMATION:
(x) PUBLICATION INFORMATION
(a) AUTHOR(S):
(b)TIZ?.E:

(c)JOURNAL:

(d)VOLUME:

(e)ISSUE:

(f)PAGES:

(g)DATE:

(h)DOCUMENT NUMBER:

(i)FILING DATE:

Q) PUBLICATION DATE:
(k) RELEVANT RESIDUES IN SEQUENCE ID NO: 9 (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO: 9 Ser Ile Leu Arg Pro Asp Pro Pm Gln Gly ieu Arg Val Glu Ser Val Pro xxx Tyr Pro where xxx is a suitable amino acid (11) INFORMATION FOR SEQ ID NO: 10 (i) SEQUENCE CHARACTORISTICS
(a) LENGTH: 7 (b) TYPE: amino acid (c) STRANDEDNESS:
(d) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: no (iv) ANTISENSE: no (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: marine IL-11 receptor (vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(a) CHROMOSOME/SfiGMENT:
(b) MAP POSITION:
(c) UNITS:
(ix) FEATURE

WO 99/59608 PCTlCA99/00516 (a) NAMEIKEY: IL-11 binding region (b) LOCATION: entire sequence (c) IDENTIFICATION METHOD: experiment and homology (d) OTHER INFORMATION: IL-11 receptor antagonist (x) PUBLICATION INFORMATION
(a) AUTHOR(S):

(b) TITLE:

(c) JOURNAL:

(d) VOLUME:

(e) ISSUE:

(f) PAGES:

(g) DATE:

(h) DOCUMENT NUMBER:

(i) FILING DATE:
(j) PUBLICATION DATE:
(k) RELEVANT RESIDUES IN SEQUENCE ID NO: 10 (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO: 10 Arg Arg Leu His Ala Ser Trp

Claims (47)

WHAT IS CLAIMED IS:

1. A process of treating or alleviating the symptoms of a pathological condition in which bone density is decreased which comprises inhibiting, in a mammalian patient suffering from such a condition, the formation of a tertiary complex of IL-11, IL-11R, and gp130.

2. The process of claim 1 which comprises administering to the patient an effective amount of a substance which inhibits, in vivo, the formation of a tertiary complex of IL-11, IL-11R, and gp130.

3. The process of claim 2 wherein the pathological condition is postmenopausal bone loss.

4. The process of claim 2 wherein the substance is a mutant IL-11R.

5. The process of claim 4 wherein the substance is a mutant IL-11R
with at least one mutation in its gp130 binding region.

6. The process of claim 5 wherein the substance is a mutant IL-11R
having at least one of the following mutations: D282.fwdarw.G282, A283.fwdarw.D283, G286.fwdarw.D286, H289.fwdarw.Y289, and V291.fwdarw.L291.

7. The process of claim 6 wherein the substance is a mutant IL-11R
having the mutation H289.fwdarw.Y289.

8. The process of claim 4, 5, 6, or 7 wherein the substance is a soluble mutant IL-11R.

9. The process of claim 8 wherein the mutant IL-11R is a human IL-11R.

10. The process of claim 2 wherein the substance is an anti IL-11 antibody.

11. The process of claim 2 wherein the substance is an IL-11 binding peptide.

12. The process of claim 11 wherein the substance is an IL-11 binding peptide having an amino acid sequence which specifically binds IL-11 in the region normally bound by IL-11R.

13. The process of claim 12 wherein the substance is a peptide comprising the sequence Arg Arg Leu Arg Ala Ser Trp.

14. The process of claim 2 wherein the substance is a small molecule.

15. The process of claim 2 wherein the substance is an IL-11 antagonist.

16. The process of claim 2 wherein the substance is an IL-11R binding peptide.

17. The process of claim 2 wherein the substance is an anti IL-11R
antibody which inhibits interactions between IL-11 and the IL-11R.

18. The process of claim 2 wherein the substance is an anti IL-11R
antibody which inhibits interactions between IL-11R and gp130.

19. The process of claim 1 which comprises administering to a patient an effective amount of transcribable genetic material which causes inhibition of the formation of the tertiary complex of IL-11, IL-11R, and gp130.

20. The process of claim 19 wherein transcribable genetic material encodes an RNA sequence capable of inhibiting the translation of a component necessary to the formation of the IL-11 / IL-11R /
gp130 tertiary complex.

21. The process of claim 20 wherein the transcribable genetic material comprises DNA encoding an RNA sequence complementary to IL-11 mRNA.

22. The process of claim 20 wherein the transcribable genetic material comprises DNA encoding an RNA sequence complementary to IL-11R mRNA.

23. The process of claim 20 wherein the transcribable genetic material comprises DNA encoding an RNA sequence complementary to gp130 mRNA.

24. The process of claim 19 wherein the transcribable genetic material comprises DNA encoding an amino acid sequence capable of inhibiting the formation of the IL-11 / IL-11R / gp130 tertiary complex.

25.The process of claim 24 wherein the transcribable genetic material encodes an IL-11R mutated to inhibit binding to gp130.

26. The process of claim 24 wherein the transcribable genetic material encodes an IL-11 binding peptide.

27. The process of any of claims 19 to 26 wherein the level of transcription of the transcribable genetic material is dependant on the concentration of an inducing compound.

28. The process of any preceding claim in which the patient is a human.

29. A composition of matter comprising a mutant IL-11R.

30. The composition of claim 29 in which the mutant IL-11R has at least one of the following mutations: D282.fwdarw.G282, A283.fwdarw.D283, G286.fwdarw. D286, H289.fwdarw.Y289, and V291.fwdarw.L291.

31. The composition of claim 30 in which the mutant IL-11R has the H289.fwdarw.Y289 mutation.

32. The composition of claim 29, 30, or 31 wherein the mutant IL-11R
is soluble.

33. The composition of claim 29, 30, 31, or 32 wherein the mutant IL-11R is a human IL-11R.

34. A composition of matter comprising an IL-11 binding peptide.

35. The composition of claim 34 wherein the IL-11 binding peptide comprises the sequence Arg Arg Leu Arg Ala Ser Trp.

36. The composition of claim 34 wherein the IL-11 binding peptide comprises the sequence Arg Arg Leu His Ala Ser Trp.

37. The composition of claim 34 wherein the IL-11 binding peptide comprises the sequence Arg Arg Leu X Ala Ser Trp, and X is a basic amino acid.

38. The composition of claim 34 wherein the IL-11 binding peptide comprises the sequence Ser Ile Leu Arg Pro Asp Pro Pro Gln Gly Leu Arg Val Glu Ser Vat Pro Gly Tyr Pro.

39. The composition of claim 34 wherein the IL-11 binding peptide comprises the sequence Ser Ile Leu Arg Pro Asp Pro Pro Gln Gly Leu Arg Val Glu Ser Val Pro Ser Tyr Pro.

40. Use of the peptide of any of claims 34 to 39 in the purification of 1L-11.

41. Use of the peptide of any of claims 34 to 39 in the depletion of IL-11 from a solution.

42. A composition of matter for the selective binding of IL-11 comprising the peptide of any of claims 34 to 39 suitably immobilized on an appropriate substrate.

43. A composition of matter comprising an IL-11 R binding peptide.

44. A composition of matter useful in inhibiting IL-11 / IL-11R
interactions comprising an antibody which specifically binds the IL-11R and blocks interactions between IL-11 and IL-11R.

45. A composition of matter useful in inhibiting IL-11R / gp130 interactions via the gp130 binding site on IL-11R comprising an antibody which specifically binds the IL-11R and blocks interactions between gp130 and IL-11R.

46. The use of the TRAP assay in identifying IL-11 antagonists.

47. The use of the bone marrow formation assay in identifying IL-11 antagonists.