CA2210724A1 - Human interleukin-1 receptor accessory protein - Google Patents

Abstract

This invention is directed to polynucleotides encoding human IL-1 receptor accessory protein, isolated IL-1 receptor accessory protein, and antibodies to IL-1 receptor accessory protein. This protein is particularly useful to prevent inflammation due to the action of IL-1.

Description

WO 96123067 PCT/J!;r~c~ool8 Human Interleukin-l Receptor accessory protein The present invention relates generally to cytokine receptors, and more specifically to accessory proteins of interleukin 1 receptors.
Interleukin 1 (IL- 1 ) is a polypeptide hormone that acts on a variety of cell types and has multiple biological properties (Dinarello, Blood 77: 1627, 1991). IL-l is a major mediator of inflammatory and immune responses. Therefore, regulation of IL- 1 activity provides a 10 means of controlling and modulating these responses.

Two species of IL- 1 have been characterized, interleukin 1 a (IL- l o~) and interleukin 1~ (IL-l~), both of which are referred to herein as IL- 1. The biological activities produced by IL- 1 are 15 mediated by binding to specific plasma membrane receptors, termed the Type I and Type II IL- 1 receptors. The IL- 1 receptors (IL- 1 R's) are transmembrane proteins with extracellular domains of about 300 amino acids, and are members of the immunoglobulin superfamily of molecules (Sims et al., Science 241: 585, 1988; Sims et al., Proc. Natl 20 Acad. Sci. USA 86: 8946, 1989; McMahan et al., EMBO J. 10: 2821, 1991). Both IL-l species bind to each of these receptors and compete completely with each other for binding.

It has been assumed that the Type I IL- 1 R encodes the entire ~5 functional IL- 1 receptor. Experiments with the cloned Type I IL- l R
indicated that when this receptor protein was transfected and expressed in Chinese hamster ovary cells, it was sufficient to bind IL- 1 and to transduce the IL- 1 signal (Curtis et al., Proc. Natl. Acad.
Sci. USA 86: 3045, 1989). The presence of an accessory protein 3 o endogenous to the hamster cells was not determined in these studies It had been suggested that the Type II IL- 1 R represented an accessory chain of the IL-lR (Solari, Cytokine 2: 21, 1990). However, more recent studies have shown that the Type II IL- 1 R is unlikely to function as a signal-transducing accessory protein, and that it acts W 096/23067 P~~ G~ 181 instead as a decoy receptor to bind excess IL- 1 and regulate its activity (Colotta et al., Science 261: 472, 1993).

Since IL-l binding to the IL-l receptor mediates the biological s effects of IL- 1, an understanding of the mechanism of receptor binding and activation is important for regulating IL- 1 's activities.
Affinity crosslinking and binding studies with labelled IL- 1 have shown that the IL- 1 receptor exists as a complex of multiple proteins that can bind IL- 1 with different affinities (Lowenthal and lO MacDonald, J. Exp. Med. 164: 1060, 1986; Ben~im~n et al., J. Immunol.
143:1168, 1989; McMahan et al., EMBO J. 10:2821, 1991). A murine monoclonal (mAb) 4C5 has been described that recognizes a 90 kDa protein on murine cells that is associated with IL- 1 R and is required for signal transduction and biological activity (Powers et al., AAI
15 meeting, Denver, CO, May 21-25, 1993). It was not known if an equivalent protein existed on human cells, or what biological function, if any, was associated with such a protein.

Prior to the present invention, efforts to identify a human IL- 1 R
20 accessory protein or to clone and express genes encoding this protein have been significantly impeded by lack of purified protein, lack of an antibody that recognizes this protein, and inability to identify cells that express large ~mounts of this protein and its mRNA. Even the murine accessory protein had not been obtained in sufficient amounts 25 to use in efforts to identify the corresponding human accessory protein. Murine cell lines known to express the accessory protein did so only in amounts (-1000 molecules/cell) too low to purify sufficient protein for obtaining unambiguous amino acid sequence information.
There was no mAb known to recognize a human homologue of the 4C5 3 o target protein (the murine accessory protein) . In addition, binding to IL- 1 was not known to be an effective screen for identifying a human accessory protein, since it is known that many accessory proteins do not bind ligand or bind with very low affinity (Hibi et al., Cell 63:
1149, 1990; Takeshita et al., Science 257: 379, 1992).
This invention makes available for the first time purified human IL- 1 receptor accessory protein which can be used to regulate the effects of IL- 1. The addition of soluble accessory protein inhibits the effect of IL-l on the cells. Hence, an aspect of the invention is the treatment of pathological conditions caused by excess activity of cells responding to IL-l by adding an amount of soluble human IL-lR
accessory protein (IL-lR AcP) sufficient to inhibit activation of cells s by IL- 1. This methodology can also be modified, and the soluble accessory protein can be used as a screening agent for pharmaceuticals .

Briefly, a pharmaceutical which works as an IL- 1 antagonist can 0 do so by blocking the interaction of IL-1 with the IL-lR AcP. The presence of IL-lR AcP in a cell membrane is necessary to permit IL-1 to interact effectively with the IL- 1 receptor complex (by effective interaction is meant binding to the receptor complex so as to initiate a biological response). The IL- 1 receptor complex includes the Type I or 15 Type II IL-l receptor in association with the IL-lR AcP (additional proteins may also be part of the complex). Adding soluble IL-lR AcP
inhibits this interaction by allowing IL- 1 or the IL- 1 receptor to interact with the soluble protein instead of IL-lR AcP on the cell surface, thus reducing the biological response caused by IL- 1.
20 Antibodies to the IL-lR AcP of this invention ~imil~rly inhibit the biological response of cells to IL-l. By binding to the IL-lR AcP, antibodies prevent IL-l from interacting effectively with the IL-l receptor. By blocking IL-lR AcP, these antibodies inhibit the binding of IL-l to the IL-l receptor complex, which depends on interaction 25 with IL-lR AcP. IL-lR AcP will inhibit IL-l interaction with the IL-l receptor, thus preventing activation of IL-l responsive cells and decreasing the infl~mm~tory response. One may also use the purified IL-lR AcP to screen a potential pharmaceutical. If the pharmaceutical blocks IL-l binding to the IL-lR AcP, it will be an effective IL-l 30 antagonist.

The present invention provides polynucleotides which encode IL-l receptor accessory proteins or active fragments thereof, preferably, the polynucleotides are selected from a group consisting 3 ~ of (a) polynucleotides, preferably cDNA clones, having essentially a nucleotide sequence derived from the coding region of a native IL-l R
AcP gene, such as shown in Figure 15 [SEQ ID NO. l]; (b) poly-nucleotides capable of hybridizing to the cDNA clones of (a) under W 096/23067 CA 02210724 1997-07-17 PCTnEP96/00181 moderately stringent conditions and which encode IL-lR AcP or fragments thereof; and (c) polynucleotides which are degenerate as a result of the genetic code to the DNA sequences defined in (a) and (b) and which encode IL-lR AcP molecules or fragments thereof.
s Particularly preferred compounds are the polynucleotides which encode human IL-l receptor accessory proteins, e. g. the polynucleotides encoding the amino acid sequence [SEQ ID NO:3] or an active fragment thereof, especially a polynucleotide having the sequence ~SEQ ID NO:l]. Especially preferred compounds encode 1 o soluble IL-1 receptor accessory proteins, e. g. human soluble IL- 1 receptor accessory proteins having for example the amino acid sequence [SEQ ID NO:9]. The polynucleotide [SEQ ID NO:7] codes for a human soluble IL- 1 receptor accessory protein. Also part of this invention are the antisense polynucleotides of the above compounds.
The present invention also provides vectors and suitable host cells, preferably expression vectors comprising the DNA sequences defined above, recombinant IL-lR AcP produced using the expression vectors, and a method for producing the recombinant accessory 20 protein molecules utilizing the expression vectors.

The present invention makes available IL- 1 receptor accessory proteins and active fragments thereof, encoded by polynucleotides as defined above. Preferred compounds are human IL- 1 receptor 2~ accessory proteins, preferably a protein having the amino acid sequence [SEQ ID NO:3]. Especially preferred are soluble human IL-l receptor accessory proteins, e. g. having the amino acid sequence [SEQ
ID NO:9]. Also part of this invention are IL-lR AcP proteins carrying one or more side groups which have been modified.
The present invention also provides antibodies to IL-lR AcP.
These antibodies bind specifically to the human IL- 1 receptor accessory protein and prevent activation of the IL- 1 receptor complex by IL- 1. The preferred antibodies have a binding affinity to the IL- 1 3s receptor accessory complex of from about KD 0.1 nM to about KD 10 nM and are for example monoclonal antibodies or derivatives thereof.

W 096/23067 PCT/~3Cl00181 Also part of this invention are pharmaceutical compositions which comprise an antisense polynucleotide, a IL- 1 receptor accessory protein or an antibody as described above. These pharmaceutical compositions may include one or more other cytokine 5 antagonists.

The invention also provides a process for the preparation of an IL- 1 receptor accessory protein comprising the steps of (a) expressing a polypeptide encoded by an above mentioned polynucleotide in a 10 suitable host, (b) isolating said IL- 1 receptor accessory protein, and (c) if desired, converting it in an analogue wherein one or more side groups are modified. Moreover, the invention includes a process for the preparation of an IL- 1 receptor accessory protein antibody comprising the steps of (a) preparation of a hybridoma cell line - 15 producing a monoclonal antibody which specifically binds to the IL- 1 receptor accessory protein and (b) production and isolation of the monoclonal antibody. Corresponding polyclonal antibodies may be produced using known methods.

The above mentioned compounds are useful as therapeutically active substances, e. g. for use in the treatment of infl~mm~tory or immune responses and/or for regulating and preventing infl~mm~tory or immunological activities of Interleukin- 1. Especially, these compounds are useful in the treatment of acute or chronic 25 diseases, preferably rheumatoid arthritis, infl~mm~tory bowel disease, septic shock, transplant rejection, psoriasis, asthma and Type I diabetes or, in the treatment of cancer, preferably acute and chronic myelogenous leukemia.

As used herein, IL-l includes both IL-loc and IL-l,B, and IL-l receptor includes Type I and Type II IL- 1 receptors, unless otherwise specifically indicated.

35 B~IEF DESCRIPTIQN OF DRAWlNGS

Figure 1. Equilibrium Binding Of [1 25I]-4C5 to Murine EL-4 Cells at Room Temperature. EL-4 cells (1.5 x 106 cells) were W 096t23067 CA 02210724 1997-07-17 PCT/~l3G~u~l8 incubated for 2 hrs at room temperature with increasing concentrations of [125I]-4C5 in the absence (o) or presence (V) of 100 nM unlabeled 4C5. Total (o) and non-specific (V) cell bound radioactivity were determined as described in Fx~mrle 1. Specific s binding of [125I]-4Cs (-) was calculated by subtracting non-specific binding from total binding. lA. Binding of EL-4 cells incubated with [125I]-4C5. lB. Analysis of the binding data according to the method of Scatchard (Scatchard, Ann. N.Y. Acad. Sci. 51: 660, 1949) as determined by Ligand (Munson and Rodbard, Anal. Biochem. 107:
o 220, 1980; McPherson, J. Pharmacol. Methods 14: 213, 1985) with a single-site model.

Figure 2. Equilibrium binding of [125I]-4C5 to Murine 70Z/3 Cells. 70Z/3 cells (1.5 x 106) were incubated for 2 hrs at room 15 temperature with increasing concentrations of [125I]-4C5 in the absence (o) or presence (V) of 100 nM unlabeled 4C5. Total (o) and non-specific (V ) cell bound radioactivity were determined as described in F.x~mple 1. Specific binding of [125I]-4C5 (-) was calculated by subtracting non-specific binding from total binding. 2A.
20 Binding of 70Z/3 cells incubated with [125I]-4C5. 2B. Analysis of the binding data according to the method of Scatchard (Scatchard, Ann.
N.Y. Acad. Sci. 51: 660, 1949) as determined by Ligand (Munson and Rodbard, Anal. Biochem. 107: 220, 1980; McPherson, J. Pharmacol.
Methods 14: 213, 1985) with a single-site model.
Figure 3. I~hibition of Human [125I]-IL-1 Binding to IL-1 Receptor on 70Z/3 Cells by Monoclonal Antibodies 4C5, 4E2 and 35F5.
Inhibition assays were performed as described in Fx~mple 1. The data are expressed as the percent inhibition of [125I]-IL-l binding in 30 the presence of the indicated concentrations of antibody when compared to the specific binding in the absence of antibody. Proteins are human IL- 1 a (H-alpha) and human IL- 1 ~ (H-beta) .

Figure 4. Inhibition of Human [125I]-IL-1 Binding to IL-1 3s Receptor on EL-4 Cells by Monoclonal Antibodies 4C5, 4E2 and 35F5.
Inhibition assays were performed as described in Fx~mple l. The data are expressed as the percent inhibition of [125I]-IL-l binding in the presence of the indicated concentrations of antibody when W O 96/23067 PCTi~G/OnlXl compared to the specific binding in the absence of antibody. Proteins are human IL- 1 a (H-alpha) and human IL- 1~ (H-beta) .

Figure 5. Isolation of Two Proteins of 90 and 50 kDa from a 5 Solubilized Extract of EL-4 Cells by 4C5 Affinity Chromatography.
Proteins were partially purified from a detergent extract of EL-4 cells by lentil lectin affinity chromatography followed by affinity chromatography on a matrix containing either an anti-Type I IL-lR
antibody (7E6), murine IL-la (Ma) or anti-accessory protein antibody 10 (4C5) as described in Fx~mple 1. Proteins in the detergent extract of EL-4 cells were also directly purified on a 4C5 affinity matrix (4C5) .
The proteins eluted from the columns were separated by SDS-PAGE, transferred to nitrocellulose and probed with [l25I]-4C5. The molecular sizes indicated in the margins were estimated from 15 molecular weight standards (Amersham Prestained Standards) run in parallel lanes. Exposure time was 1 day.

Figure 6. Inhibition of IL-l Induced Splenic B Cell Proliferation by Monoclonal Antibodies 4C5, 4E2 and 35F5. Inhibition 20 assays were performed as described in Lxample 1. The data are expressed as the incorporation of 3H-thymidine (CPM) by B cells in the presence of the indicated concentrations of antibody when compared to the incorporation in the absence of antibody. Proteins are: 6A. human IL-la (IL-la)) and 6B. human IL-1~ (IL-l~).
Figure 7. Inhibition of IL-1 Induced Proliferation of DlO.G4.1 Helper T-cells by Monoclonal Antibodies 4C5 and 35F5 and Human IL- lra. Inhibition assays were performed as described in Example 1.
The data are expressed as the incorporation of 3H-thymidine (CPM) 30 by D10 cells in the presence of the indicated concentrations of antibody and IL- lra when compared to the incorporation in the absence of antibody or IL-lra. Proteins are: 7A. human IL-loc~ 7B.
human IL- l ~.
.

Figure 8. Inhibition of IL-1 Induced Kappa Light Chain Expression by 70Z/3 Cells: Effect of Monoclonal Antibodies 4C5, 4E2 and 35F5. The induction of kappa light chain expression and inhibition with the antibodies was as described in Example 1. The W 096/23067 CA 02210724 1997-07-17 PCTi~l~G~'~G181 data are expressed as the percent of cells expressing kappa light chain in the presence of the indicated concentrations of antibody when compared to the percent of cells in the absence of antibody.
Proteins are human IL-la (IL-la) and human IL-l~ (IL-l~).
S
Figure 9. Inhibition of IL- 1 Induced Serum IL-6 in C57BL/6 Mice by Monoclonal Antibodies 4C5 and 35F5. Mice were pretreated with the monoclonal antibody at 4 hrs and 10 mins prior to subcutaneous injection of human IL-la (alpha) or human IL-l~
10 (beta) (0.03 ~lg). Two hours after the IL-l ~dmini~tration, the serum IL-6 concentration was determined as described in P,x ~mple 1. Mab X-7B2 is a control antibody.

Figure 10. Nucleotide Sequence and Deduced Amino Acid 1~ Sequence of Murine IL-lR AcP. 10A. The nucleotide sequence of the opening reading frame of murine IL-lR AcP cDNA clone E2-K is shown. The top strand is the coding sequence [SEQ ID NO:4]. 10B. The amino acid sequence of mllrine IL-lR AcP as deduced from the coding sequence shown in Figure 10A is shown [SEQ ID NO:6]. The 20 signal peptide cleavage site is predicted to occur after Ala -1, resulting in a 550 amino acid mature protein that extends from Ser 1 to Val 550. The cleavage site has been confirmed by NH2-terminal sequence analysis of purified natural muIL-lR AcP (F~x~mple 10). The predicted transmembrane domain extends from Leu 340 through Leu 25 363.

Figure 1 1. Immunoprecipitation of Recombinant MuIL- 1 R AcP
from Transfected COS cells with mAbs 4C5 and 2E6. COS cells were transfected by electroporation with either pEF-BOS/muIL-lR AcP or 30 pEF-BOS alone (mock). Transfected cells were metabolically labelled with [35S]Met as described (F,x~mple 8). Labelled transfectants were solubilized with RIPA buffer and immunoprecipitated with either mAb 4C5 or 2E6 (see Table 2) as described (F,x~mple 8). Both mAbs immunoprecipitated labelled protein from COS cells transfected with 35 pEF-BOS/muIL-lR AcP which migrated as a broad band between 70-90 kDa. No labelled protein was detected in this size range from mock transfected COS cells. A higher molecular weight species (>200 kDa) is present in both mock and muIL-lR AcP transfected COS cells.

W O 96123067 PcT/~lr~/~ol8 _ 9 _ Figure 12. Equilibrium Binding of [125I]-Labeled 4C5 and IL-l to Murine Recombinant IL-lR AcP Expressed in COS-7 Cells. Cells (4-8 x 104) transfected with an IL-lR AcP expression plasmid [COS(AcP)]
s or control plasmid [COS(PEF-BOS)] were incubated for 3 hrs at 4~C
with increasing concentrations of [125I]-4Cs or [125I]-IL-la in the absence (Total) or presence (Non-Specific) of 100 nM unlabeled 4C5 or 50 nM unlabeled IL- 1 oc . Total (Total) and non-specific (Non-Specific) cell bound radioactivity were determined as described in Example 1. Specific binding of [125I]-4C5 (Specific) and [125I]-IL~
(Specific) were calculated by subtracting non-specific binding from total binding. The binding of [125I]-IL-la to COS cells transfected with the control plasmid [COS(PEF-BOS)] showed that Cos-7 cells naturally express approxim~tely 600 high affinity binding sites for 15 IL- 1 a. The right hand panel shows analysis of the binding data according to the method of Scatchard (Scatchard, Ann. N.Y. Acad. Sci.
51: 660, 1949) as determined by Ligand (Munson and Rodbard, Anal.
Biochem. 107: 220, 1980; McPherson, J. Pharmacol. Methods 14: 213, 1985) with a single-site model. 12A. Binding of COS(AcP) cells 20 incubated with [125I]-4C5 12B. Scatchard plot of 12A data. 12C.
Binding of COS(AcP) cells incubated with [125I]-IL-la 12D. Scatchard plot of 12C data. 12E. Binding of [COS(P~F-BOS)] cells incubated with [125I]-IL-la. 12F. Scatchard plot of 12E data.

2s Figure 13. Equilibrium Binding of [1 25I]-Labeled 35F5 and IL-1 to Murine Recombinant Type I IL-lR Expressed in COS-7 Cells. Cells (4-8 x 104) transfected with an Type I IL-lR expression plasmid [COS(Mu-IL-lR)] were incubated for 3 hrs at 4~C with increasing concentrations of [125I]-35F5 or [125I]-IL-la and [125I]-IL-1~ in the absence (Total) or presence (Non-Specific) of 100 nM unlabeled 35F5 or 50 nM unlabeled IL- 1. Total (Total) and non-specific (Non-Specific) cell bound radioactivity were determined as described in Fx~mple 1.
Specific binding of [125I]-35F5 (Specific) and [125I]-IL-la or IL-1 (Specific) were calculated by subtracting non-specific binding from 3 s total binding. The right hand panel shows analysis of the binding data according to the method of Scatchard (Scatchard, Ann. N.Y. Acad. Sci.
51: 660, 1 949) as determined by Ligand (Munsan and Rodbard, Anal.
Biochem. 107: 220, 1980; McPherson, J. Pharmacol. Methods 14: 213, W 096/23067 CA 02210724 1997-07-17 PcT/~l~G~c~l8l 1985) with a single-site model. 13A. Billding of [COS(Mu-IL-lR)] cells incubated with [12~I]-35F5. 13B. Scatchard plot of 13A data. 13C.
Binding of [COS(Mu-IL-lR) cells incubated with [125I]-IL- 1,B 13D.
Scatchard plot of 13C data. 13E. Binding of [COS(Mu-IL-lR)] cells s incubated with [125I]-IL-la. 13F. Scatchard plot of 13E data.
Figure 14. Construction of Full-length cDNA Clone of Human IL-lR AcP. Schematic representations of the structures of the human IL-lR AcP cDNA inserts in clones #3 and #6 are shown in the upper 10 portion of the figure. Clone #3 contains 5' noncoding sequences, the initiating ATG codon, and a significant portion of the coding region.
Clone #6 overlaps with clone #3, containing most of the coding region, the TGA stop codon, and 3' noncoding sequences. The 846 bp XbaI/BstXI fragment from clone #3 and the - 2700 bp Bs~XI/XbaI
15 fragment from clone #6 were isolated and ligated into the expression vector pEF-BOS as described (F~x~mrles 12 and 13). A schematic representation of the resulting cDNA encoding full-length human IL-lR AcP is shown o~ the bottom line.

Figure 15. Nucleotide Sequence of Human IL-lR AcP. The nucleotide sequence of the open reading frame in the full-length human IL-lR AcP cDNA (Fx~mple 13, Figure 14) is shown. The top strand is the coding sequence [SEQ ID NO:1].

2s Figure 16. Amino Acid ~equence of Human IL-lR AcP. The amino acid se~uence of human IL-lR AcP as deduced from translation of the nucleotide sequence in Figure 15 is shown ~SEQ ID
NO:3~. The signal peptide cleavage site is predicted to occur after Ala-1, resulting in the production of a 550-amino acid mature protein that extends from Serl to ValS50. The predicted transmembrane domain extends from Leu340 to Leu363.

Figure 17. IL-1 Induction of IL-6 Production in MRC-5 Cells:
Inhibition by IL- 1 Receptor Antagonist and Anti-Type I IL- 1 3 s Receptor Antibody 4C 1. Human embryonic lung fibroblast MRC-5 cells (5 X 104cells; ATCC# CCL-171) were plated into 24-well cluster dishes (No. 3524; Costar) for 24 hrs at 37~C in a humidified incubator. After the 24 hr period, the cells were pretreated with increasing concentrations of either IL-1 receptor antagonist (IL-lRA; 10-2 to W O 96/23067 ~ PcT/~l~C~ 181 103pM), anti-Type I IL-l receptor antibody 4Cl (10-4 to lol ~g/ml) or nothing for 1 hr at 37~C. At the end of 1 hr, either 5 pM or 100 pM
human IL-l~ was added and the incubation continued for 24 hrs at 37~C. At the end of the incubation period, 100 ~Ll of cell supernatent - 5 was removed from each well and assayed for IL-6 concentration by the Quantikine Human IL-6 Assay Kit (R & D Systems). The data are 0 expressed as the concentration (pg/ml) of IL-6 secreted from the MRC-5 cells in prese~ce of either IL-1~ alone or in the presence of IL-1~ plus inhibitor. The effect of increasing concentrations of tumor o necrosis factor-a (TNFa) on the stimulation of IL-6 secretion from MRC-5 cells was also determinerl. TNFa was less potent (~500-fold) than IL-1~ in stimulating IL-6 secretion from these cells and appeared to be partially dependent on an autocrine secretion of IL- 1 by these cells. 17A shows data for IL-l~, TNFoc, and inhibition by IL-lra. 17B shows data for inhibition by mAb 4Cl.

Figure 18 . Nucleotide Sequence of the Soluble Human IL- 1 R
AcP. The nucleotide sequence of the soluble human IL-lR AcP cDNA
is shown. The top strand is the coding sequence [SEQ ID NO:7].
Figure 19. Amino Acid Sequence of the Soluble Human IL-lR
AcP. The amino acid sequence of soluble human IL-lR AcP as deduced from tr~n~l~tion of the nucleotide sequence in Figure 18 is shown [SEQ ID NO:9].
The present invention is directed to an isolated polynucleotide that encodes a IL-lR AcP (IL-lR AcP) or an active fragment of a IL-lR AcP (i.e. capable of inhibiting the ability of IL-1 to bind to or otherwise activate the IL-l receptor), in particular a human or 30 murine IL-lR AcP. Fx~m~les of such a polynucleotide are the DNA
polynucleotide having the sequence [SEQ ID NO: 1], and the DNA
polynucleotide encoding the human IL-lR AcP which has the amino acid sequence [SEQ ID NO: 3]. The polynucleotides of this invention may be used as intermediates to produce the protein IL-lR AcP as 35 described below. This protein is useful in treatment of conditions related to IL-l infl~mm~tory activity. The polynucleotides may themselves be used in treatment by known antisense modalities.

The invention is also directed to IL- 1 receptor accessory protein (IL-lR AcP) isolated free of other proteins, or an isolated active fragment of IL-lR AcP. The IL-lR AcP of this invention is a protein or active fragment which inhibits the ability of IL-1 to bind to or otherwise activate the IL- 1 receptor.

Part of this invention is a method of obtaining human IL-lR
AcP, which method uses as intermediates the following compounds:
polynucleotides encoding murine IL-lRAcP, murine IL-lR AcP, 10 antibodies to murine IL-lR AcP, and polynucleotides encoding human IL-lR AcP. From polynucleotides encoding human IL-lR AcP, soluble human IL- lR AcP and antibodies thereof can be obtained. The critical first intermediate for this invention is the isolation of mAbs for the murine IL- 1 R accessory protein. These mAbs are obtained by 15 immllni7~tion with a partially purified preparation of solubilized crosslinked IL-la/IL-lR complex from murine 70Z/2 pre-B cells (described in F~m~le 1). The use of the crosslinked ligand-receptor complex was uniquely suitable, since the accessory protein could only be purified as a result of its interaction in such a complex. One of 20 these mAbs (4C5) was then used to isolate a cDNA encoding the murine IL-lR AcP. This murine cDNA was used to obtain a partial genomic clone of the human homologue. A probe derived from the partial genomic clone was then used to isolate the full-length cDNA for human IL-lR AcP.
As used herein, "polynucleotide" refers to an isolated DNA or RNA polymer, in the form of a separate molecule or as a component of a larger DNA or RNA construct, which has been derived from DNA or RNA isolated at least once in substantially pure form, i.e., free of 30 cont~min~ting endogenous materials and in a quantity or concentration enabling identification, manipulation, and recovery of the sequence and its component nucleotide sequences by standard biochemical methods, for example, using a cloning vector. Such sequences are preferably provided in the form of an open reading 3 5 frame uninterrupted by internal nontranslated sequences, or introns, which are typically present in eukaryotic genes. However, it will be evident that genomic DNA containing the relevant sequences could also be used. Sequences of non-translated DNA may be present 5' or 3' from the open reading frame, where the same do not interfere with manipulation or expression of the coding regions.

These polynucleotides, e. g. DNA, include those containing one or 5 more of the above-identified DNA sequences and those sequences which hybridize under stringent hybridization conditions (see, T.
l~ni~tis et al., Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory (1982), pp. 387 to 389) to the DNA sequences. An example of one such stringent hybridization condition is hybridization lO at 4 x SSC at 65~C, followed by a washing in 0.1 x SSC at 65~C for an hour. Alternatively an exemplary stringent hybridization condition is in 50 % formamide, 4 x SSC at 42~C.

Polynucleotides which hybridize to the sequences for IL- lR AcP
15 under moderate hybridization conditions and which code on expression for IL-lR AcP peptides having IL-lR AcP biological properties also encode novel IL-ll~ AcP polypeptides. Examples of such non-stringent hybridization conditions are 4 x SSC at 50~C or hybridization with 30 - 40 % formamide at 42~C. Additional io hybridization conditions are mentioned in Fx~mple 11. For example, a DNA sequence which shares regions of significant homology, e. g. sites of glycosylation or disulfide linkages, with the sequences of IL-lR AcP
and encodes a protein having one or more IL-lR AcP biological properties clearly encodes a IL-lR AcP polypeptide even if such a 2s DNA sequence would not stringently hybridize to the IL- 1 R AcP
sequences.

Polynucleotides of this invention were obtained as described in Examples 7-13 by expressing murine cDNA in eucaryotic cells and 3 o screening cell-surface proteins using assays described in Example 7. A
murine cDNA clone was identified which results in the expression of a protein immunoreactive with mAb 4C5. This cDNA clone was used to obtain the homologous human genomic clone. Briefly, human genomic DNA was screened with the intermediate murine IL- 1 R AcP probe 3 s obtained from mouse cells in Example 7 . Clones were isolated and sequenced as described. The partial human genomic clones were then used as intermediates to screen a human cDNA library and clones W 096/23067 CA 022l0724 l997-07-l7 PcTl~lr~t were isolated and sequenced as described to obtain full-length polynucleotides of this invention encoding human IL- lR AcP.

A specific polynucleotide of this invention has the sequence [SEQ
s ID NO: 1]. Another polynucleotide of this invention encodes the human IL-lR AcP having the amino acid sequence [SEQ ID NO: 3]. Any polynucleotide capable of encoding the amino acid sequence of IL-lR
AcP, or specifically [SEQ ID NO: 3] is part of this invention. Another polynucleotide of invention has the sequence [SEQ ID NO: 4].
Also part of this invention is a polynucleotide encoding an active fragment of IL- 1 R AcP. Such polynucleotides are fragments of the polynucleotides provided above (fragmented by known methods such as restriction digestion or shearing) which, when expressed by 15 conventional methods, produce proteins that block IL- 1 activity in an IL-1 assay described below. A polynucleotide encoding a soluble IL-lR AcP is a preferred fragment of this invention. An example of such a polynucleotide has the sequence [SEQ ID NO:7].

Polynucleotides encoding the IL-lR AcP and its active fragments are useful as intermediates from which IL-lR AcP and its active fragments are obtained. In addition, these polynucleotides are useful as antisense therapeutics which block the production of IL-lR AcP.
Antisense therapeutics are used as described in Akhtar and Ivinson, 2s Nature Genetics 4:21~, 1993. RNA or DNA polynucleotides both have these utilities. Antisense polynucleotides which are complementary to [SEQ ID NO:l] or to a fragment of this sequence are part of this invention. Such polynucleotides may be obtained by known methods such as DNA or RNA synthesis to produce a complementary sequence.
3 o Thus, any sequence from the polynucleotides of this invention which is capable of hybridizing to DNA or RNA encoding IL-lR AcP under moderately stringent conditions known in the art and which when so hybridized prevents the synthesis of IL- 1 R AcP is also part of this invention .
3s This invention includes vectors which contain the poly-nucleotides described herein which encode IL- lR AcP or an active fragment. Any vector known in the art may be used in this capacity, CA 022l0724 l997-07-l7 W 096~3067 -~15 - P~li~l~G~'00181 such as plasmids, phagemids, viral vectors, cosmids and other vectors.
The polynucleotides are inserted in the vectors by methods well known in the art of recombinant DNA technology. Expression vectors are a particular example of vectors.
As used herein, "expression vector" refers to a vector such as plasmid comprising a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or 10 coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termin~tion sequences. Structural elements intended for use in various eukaryotic expression systems preferably include a signal sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, where recombinant protein is expressed without a signal or transport sequence, it may include an N-terminal methionine residue. This residue may optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.
Also part of this invention are host cells containing expression vectors containing polynucleotides of this invention, which express IL-lR AcP or active fragments. The polynucleotides are inserted into vectors containing transcriptional regulatory sequences to form 2s expression vectors. These expression vectors are then inserted into host cells by transfection, infection, electroporation, or other well-known methods. Such host cells are capable of producing protein from the expression vectors inserted therein. Other host cells, e.g. yeast, Chinese hamster ovary cells, bacterial cells, can be utilized with the 30 appropriate and suitable expression vectors.

As noted above, this invention is also directed to IL- 1 receptor accessory protein (IL-lR AcP) isolated free of other proteins, or an active fragment of IL-lR AcP. The IL-lR AcP of this invention is a 3 5 protein or active fragment which inhibits the ability of IL-l to bind to or otherwise activate the IL- 1 receptor, especially the Type I IL- 1 receptor. Inhibiting activation of the human IL-l receptor is accomplished by the human IL-lR AcP or active fragments, and has W 0 96123067 CA 02210724 1997-07-17 PCT~EP96/00181 - 16 -various effects, in particular reducing infl~mm~tion. Thus by means of the IL-lR AcP or active fragment, it is possible to inhibit IL-l activation of cells and thereby to reduce or alleviate the symptoms associated with infl~mm~tion.
Active fragments of IL- 1 R AcP may be obtained by conventional methods for obtaining protein fragments. For example, DNA of this invention may be fragmented by restriction digest or shearing and expressed in host cells by conventional methods to provide fragments 0 of IL-lR AcP. Fragments of the IL-lR AcP may also be obtained by proteolysis of the IL- lR AcP of this invention. Active fragments of this invention are determined by screening for activity using IL-l assays described below.

Soluble IL-lR AcP is an IL-lR AcP fragment of this invention in which deletions of the COOH-terminal sequences result in secretion of the protein into the culture medium. The soluble IL-lR AcP
corresponds to all or part of the extracellular region of the IL-lR AcP.
Methods for elucidating the COOH termin~l~ and extracellular regions 20 of proteins are well known. The resulting protein preferably retains its ability to interact with IL-l or the Type I and Type II IL-lR's.
Particularly preferred sequences include those in which the transmembrane region and intracellular domain of the IL-lR AcP are deleted or substituted to facilitate secretion of the accessory protein 2~ into the culture medium. The soluble IL-lR AcP may also include part of the transmembrane region, provided that the soluble IL-lR AcP is capable of being secreted from the cell. Soluble IL-lR AcP is obtained as described in Fx~mples 14 and 15. A specific soluble IL-lR AcP of this invention has the sequence [SEQ ID NO:9].
A preferred example of IL-lR AcP has the amino acid sequence [SEQ ID NO: 3]. The amino acid sequence of the IL-lR AcP as deduced from the cDNA sequence [SEQ ID NO: 1] is shown in Figure 16. Any IL- 1 R AcP which affects IL- 1 binding as described above, is included 3~ in this invention, such as an analogue having the sequence of [SEQ ID
NO: 3], in which one or more side groups have been modified in a known manner, by attachment of compounds such as polyethylene glycol, or by incorporation in a fusion protein (with other protein W 096123067 PcT/~ 181 sequences such as immunoglobulin sequences), for example, or proteins whose activity has otherwise been maintained or enhanced by any such modification. Also included are proteins which inhibit IL- 1 binding to the IL- 1 receptor and have essentially the sequence 5 ~SEQ ID NO:3] with one or more amino acids added, deleted, or substituted by known techniques such as site-directed mutagenesis.
,~ The change in amino acids is limited and conservative so as to maintain the identity of the protein as an IL-lR AcP with all or part of its activity as described, or enhanced activity. Means for 10 determining IL- 1 inhibiting activity are described in Examples 5, 6, 16 and include inhibition of IL- 1 binding to IL- 1 receptor, inhibition of lymphocyte proliferation or kappa light chain expression, and decrease of IL-1 induced IL-6 expression.

IL-lR AcP isolated free of other proteins may be obtained from the polynucleotides of this invention which encode IL- lR AcP. For example, IL-lR AcP may be obtained by conventional methods of expressing a polynucleotide provided herein encoding IL-lR AcP, preferably the DNA of [SEQ ID NO: 1] or [SEQ ID NO: 7] in a host cell, 20 and isolating the resulting protein. Once the IL-lR AcP is obtained, the protein can be isolated free of other proteins by conventional methods. These methods include but are not limited to purification or antibody affinity columns with the antibodies of this invention, chromatography on ion exchange or gel filtration columns, purification 25 by high performance liquid chromatography, and purification with an IL- 1 affinity column.

IL-lR AcP may be stabilized by attaching a polyalkylene glycol polymer by known methods. Polyalkylene glycol includes poly-3 0 ethylene glycol, and other polyalkylene polymers which may bebranched or unbranched. The polymers may be directly linked to the protein, or may be linked by means of linking groups connecting for example the COOH of the polymer to the NH2 of a lysine on the protein .
IL-lR AcP of this invention may be used directly in therapy to bind or scavenge IL-1, thereby providing a means for regulating and preventing the inflammatory or immunological activities of IL- 1. In W 0 ~6/23067 CA 02210724 1997-07-17 PCT/~l~lool8l its use to prevent or reverse pathologic responses, soluble IL-lR AcP
or antibodies to the IL-lR AcP can be combined with other cytokine antagonists such as antibodies to the IL-2 receptor, soluble TNF
receptor, the IL-l receptor antagonist, soluble IL- 1 receptor and the 5 like. In addition, isolated IL-lR AcP of this invention is useful in raising antibodies to IL-lR AcP which are themselves useful in therapy. R~i~in~: such antibodies is made feasible because this invention makes available IL- 1 R AcP in sufficient amounts for antibody production.
Thus, this invention is also directed to antibodies to human IL-lR AcP. Murine or rat monoclonal antibodies to human IL-lR AcP
are obtained as in Fx~mple 15. These antibodies are obtained by immnni7~tion with purified or partially purified amounts of human 15 IL-lR AcP, which is obtained after expression of the recombinant full-length or soluble human IL-lR AcP using the DNA's of this invention.
The human IL-lR A~cP cDNA's were isolated using the murine IL-lR
AcP DNA of this invention which was isolated with the unique mAb 4C5 described in F.x~mples 2 and 3. For the murine or rat mAbs to 20 human IL-lR AcP, hybridoma techniques well known in the art may then be used to obtain hybridomas to generate mAbs. Chimeric antibodies and hnm~ni7ed antibodies may be obtained from these rodent antibodies using known methods. (Brown et al., Proc. Natl.
Acad. Sci. USA 88: 2663, 1991; WO 90/7861, EP 620276) or by 25 producing heterodimeric bispecific antibodies (Kostelny et al., J.
Immunol. 148: 1547, 1992).

Antibodies to human IL-lR AcP of this invention bind specifically to human IL- 1 R AcP and prevent activation of the IL- 1 30 receptor complex by IL-1. This activity may be determined by assays as described herein. Specifically, biological assays include screens based on the ability of the antibody to inhibit the proliferation of IL- 1 -responsive cells or the IL- 1 -induced secretion of prostaglandin E 2 and IL-6. Such assays can be carried out by conventional methods 3s in cell biology. Suitable cells for these assays include splenic B cells, cell lines such as the human B cell line RPMI 1788 (Vandenabeele et al., J. Immunol. Meth. 135: 25, 1990), and human fibroblasts such as the human lung fibroblast line MRC-5 (Chin et al., J. Exp. Med. 165: 70, -W 096/23067 PcT/~5~ 8 1987). Methods for such assays using mouse cells are found in F.x~mrles 1, 2, 5, and 6. For example an in vivo assay may be used, which measures inhibition of IL- 1 induced IL-6 production in mice.
These assays may be performed using human cells to effectively s screen for the desired activity using the same techniques provided in the Fx~mples. A preferred antibody has a binding affinity to the IL-l receptor accessory complex of about KD 0.1 nM to about KD 10 nM, as determined by conventional methods (Scatchard, Ann. N.Y. Acad. Sci.
5 1: 660, 1949).
The antibodies of this invention may be ~lmini~tered by known methods to relieve conditions caused by the presence of IL-l. In particular, the antibodies of this invention are useful in reducing infl~mm~tion. These antibodies to the IL-lR AcP can be ~lmini~tered~
15 for example, for the purpose of suppressing infl~rnm~tory or immune responses in a human. A variety of diseases or conditions caused by inflammatory processes (e.g. rheumatoid arthritis, infl~mm~tory bowel disease, and septic shock) or by immune reactions (e.g. Type I
diabetes, transplant rejection, psoriasis, and asthma) are associated 20 with elevated levels of IL-l (Dinarello and Wolff, New Engl. J. Med.
328: 106, 1993). Treatment with antibodies that inhibit IL-1 interaction with the IL-lR AcP may therefore be used to effectively ~u~ress infl~mm~tory or immune responses in the clinical treatment of acute or chronic diseases such as rheumatoid arthritis, 25 infl~mm~tory bowel disease, and Type I diabetes. In addition, antibodies are useful in the treatment of certain cancers, such as acute and chronic myelogenous leukemia (Rambaldi et al., Blood 78: 3248, 1991; Estrov et al., Blood 78: 1476, 1991).

Included in this invention are antibodies to murine IL-lR AcP, specifically 4C5, 2B5, 3Fl, 4C4, 24C5, 4D4 (see Table 1) and lD2, 2D6, 2E6, lF6, 2D4, 2F6, 3F5, and 4Al (see Table 2). These antibodies are useful to obtain human IL- lR AcP, as described.

3 s As noted above, antibodies may be produced naturally by appropriate cells, or may be produced by recombinant expression vectors that modify the antibody proteins, e.g. by hllm~ni7ing the antibody (Brown et al., Proc. Natl. Acad. Sci. USA 88: 2663, 1991) or by producing heterodimeric bispecific antibodies (Kostelny et al., J.
Immunol. 148: 1547, 1992; WO 90/7861, EP 620276) that can recognize both the accessory protein and the Type I or Type II IL-lR.

The dose ranges for the ~lmini~tration of the IL-lR AcP and fragments thereof or of antibodies to the IL-lR AcP or antisense polynucleotides may be determined by those of ordinary skill in the art without undue experimentation. In general, appropriate dosages are those which are large enough to produce the desired effect, for l o example, blocking the activity of endogenous IL- 1 to cells responsive to IL-1. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of disease in the patient, counter-indications, if any, l 5 immune tolerance and other such variables, to be adjusted by the individual physician. The IL- lR AcP and fragments thereof or antibodies to this protein or antisense polynucleotides can be ~lministered parenterally by injection or by gradual perfusion over time. They can be ~-1mini~tered intravenously, intraperitoneally, io intramuscularly, or subcutaneously.

This invention includes pharmaceutical compositions comprising the proteins and/or antibodies of this invention in amounts effective to reduce infl~mm~tion, and a pharmaceutically acceptable carrier 2~ such as the preparations and vehicles described below. Such compositions may include other active compounds if desired. For the proteins, an example of an effective amount is in the range of about 4 to about 32 mg/meter2. For antibodies, an exarnple of an effective amount is in the range of about 0.1 to about 15 mg/kg body weight.
Preparations for parenteral Aflmini~tration include sterile or aqueous or non-aqueous solutions, suspensions, and emulsions.
Fx~mples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters 3 5 such as ethyl oleate. Aqueous carriers include water, alcoholic/
aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or W O 96/23067 PCT~EP96/00181 fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on ~inger's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, anti-microbials, anti-oxidants, chelating 5 agents, inert gases and the like. See, generally, Remington's Pharmaceutical Science, 16th Ed., Mack Eds., 1980.

The following F.x~mples are provided to further describe the invention and are not intended to limit it in any way.

Example l Methods 15 Preparation, Screening and Purification of Hybridoma Antibodies Lewis Rats (Charles River Laboratories) were immunized by the intraperitoneal (i.p) route with detergent solubilized preparations of human IL-la (Gubler et al., J. Immunol. 136: 2492, 1986), affinity 20 cross-linked to IL-lR from murine 70Z/3 pre-B cells (ATCC #TIB 158).
For the primary immllni7~tion, the rats received solubilized IL-la/
IL-lR complex (0.4 ml) that was prepared and purified from 1 x 101 1 70Z/3 cells (Chizzonite et al., Proc. Natl. Acad. Sci. USA 86: 8029, 1989) and emulsified in Freund's Complete Adjuvant at a 1:2 ratio and 2s injected i.p. (described below). Six weeks later, the rats received solubilized IL-la/IL-lR complex (0.3 rnl) that was prepared and purified from 2.25 x 101 1 cells and emulsified in Freund's Complete Adjuvant at a ratio of 1:2 and injected in each hind foot pad and i.p.
Sera were collected from the rats at 2 and 6 weeks after the last 30 immllni7~tion and tested for activity that blocked [125I]-IL-l~
binding to IL-lR on 70Z/3 cells. Four months after the last immllni7~tion, one rat was immunized with the following amounts of solubilized IL-l~/IL-lR complex in preparation for splenocyte - isolation: 0.1 ml (prepared and purified from 8 x 101~ cells) 3s emulsified at a 1:4 ratio with Freund's Complete AdJuvant and injected in each hind foot pad and subcutaneous (s.c.) in each hind limb, and 0.9 ml (prepared and purified from 7.4 x 101 1 70Z/3 cells) injected intravenous (i.v.) and i.p. Two days later, the rat was immunized with solubilized IL-lo~/IL-lR complex (0.5 ml; prepared , and purified from 2 x 1011 70Z/3 cells) mixed with phosphate buffered saline (PBS), pH 7.4 (0.5 ml) and injected s.c. in each hind limb. Two days after this last immllni7~fion, spleen cells were isolated from the rat and fused with SP2/0 cells (ATCC CRL 1581) at a ratio of 5 1:1 (spleen cells:SP2/0 cells) with 35% polyethylene glycol (PEG 4000, E. Merck) according to a published procedure (Fazekas et al., J.
Immunol. Meth. 35: 1, 1980). The fused cells were plated at a density of 3 x 105 cells/well/ml in 48 well plates in IMDM supplemented with 15% FBS, glllt~mine (2 mM), beta-mercaptoethanol (0.1 mM), 10 gentamicin (50 llg/ml), HEPES (10 mM), 5% ORIGIN hybridoma cloning factor (IGEN, Inc.), 5% P388Dl supernatant (Nordon et al. J. Immunol.
13 9: 813, 1987) and 100 Units/ml recombinant human IL-6 (Genzyme).

Hybridoma supernatants were screened for inhibitory and non-inhibitory antibodies specific for an IL-lR AcP and the Type II IL-lR
in four assays: 1) iEor inhibitory antibodies: inhibition of [125I]-IL-l~
binding to 70Z/3 and EL-4 thymoma cells (described below), 2) for non-inhibitory antibodies: immunoprecipitation of solubilized complex 20 of [125I]-IL-1~ crosslinked to Type II IL-lR, 3) for inhibitory antibodies specific ~or IL-lR AcP or Type II IL-lR: inhibition of [125I]-IL-ll~ and [125I]-IL-la binding to cells expressing recombinant Type I and Type II IL-lRs, and 4) to elimin~te any antibodies specific for IL-l: immunoprecipitation of [1 25I]-IL- l a and 25 [125I]-IL-l~. Hybridoma cell lines secreting antibodies specific for Type II IL-lR and the IL-lR AcP were cloned by limiting dilution.
Antibodies were purified from large scale hybridoma cultures or ascites fluids by affinity chromatography on protein G bound to Sepharose 4B fast flow according to the manufacturer's protocol 30 (Pharmacia).

Cultured Cells and Biological Assays Mouse EL-4.IL-2 thymoma cells (TIB 181) and D10.G4.1 (TIB
3 5 224) cells were maintained as previously described (Kilian et al., J.
Immunol. 136: 1, 1986). Mouse 3T3L1 (CL 173) and 70Z/3 pre-B (TIB
158) cells were maintained in IMDM containing 5% fetal bovine serum W 096123067 P~~ 0181 in 600 cm2 dishes. The above cells were obtained from the American Type Culture Collection and the ATTC numbers are in parenthesis.

The biological activity of unlabeled IL-1 and [1 25I]-IL- 1 s proteins were evaluated in the murine D 10 proliferation assay (Kaye et al., J. Exp. Med. 158: 836, 1983).

Labeling of IL-l and Purified Monoclonal Antibodies with 1 25I

0 Recombinant murine IL-lo~, human IL-la and human IL-1~
were puriffed as previously described (Kilian et al., J. I~nmunol. 136:
1, 1986; Gubler et al., J. Immunol 136: 2492, 1986) except that murine IL-la was prepared in 25 mM Tris-HCl, 0.4 M NaCl. Protein determin~tions were performed by BCA protein assay (Pierce Chemical Co., Rockford, IL). Human IL-la human IL-1~, murine IL-la, murine IL-l~ and purified IgG were labeled with 125I by a modification of the Iodogen method (Pierce Chemical Co.). Iodogen was dissolved in chloroform and 0.05 mg dried in a 12 x 15 mm borosilicate glass tube. For radiolabeling, 1.0 mCi Na[l25I]
20 (Amersham, Chicago, IL) was added to an Iodogen-coated tube containing 0.05 ml of Tris-iodination buffer (25 mM Tris-HCl pH 7.5, 0.4 M NaCl, 1 mM EDTA) and incubated for 4 min at room temperature. The activated 125I solution was transferred to a tube containing 0.05 to 0.1 ml IL-l (5-13 ,ug) or IgG (100 ,ug) in Tris-25 iodination buffer and the reaction was incubated for 5-8 min at room temperature. At the end of the incubation, 0.05 ml of Iodogen stop buffer (10 mg/ml tyrosine, 10% glycerol in Dulbecco's PBS, pH 7.4) was added and reacted for 3 min. The mixture was then diluted with 1.0 ml Tris-iodination buffer, and applied to a Bio-Gel PlODG desalting 30 column (BioRad Laboratories) for chromatography. The column was eluted with Tris-iodination buffer, and fractions ( 1 ml) containing the peak amounts of labeled protein were combined and diluted to 1 x 108 cpm/ml with 1% BSA in Tris-iodination buffer. The TCA
precipitable radioactivity (10% TCA final concentration) was typically 35 in excess of 95% of the total radioactivity. The radiospecific activity was typically 2000 to 3500 cpm/fmol for purified antibodies and 3500 to 4500 cpm/fmole for IL- 1.

W 096/23067 CA 02210724 1997-07-17 PCT/~

Mouse IL- 1 Receptor Binding Assays Binding of radiolabeled IL- l to mouse cells grown in suspension culture was measured by a previously described method (Kilian et al., s J. Immunol. 136: 1, 1986). Briefly, cells were washed once in binding buffer (RPMI-1640, 5% FBS, 25 mM HEPES, pH 7.4), resuspended in binding buffer to a cell density of 1.5 x 107 cells/ml and incubated (1.5 x 106 cells) with various concentrations of [125I]-IL-1 (5-1000 pM) at 4~C for 3-4 hrs. Cell bound radioactivity was separated from 10 free [125I]-IL-1 by centrifugation of the assay mixture through 0.1 ml of an oil mixture (1:2 mixture of Thomas Silicone Fluid 6428-R15:
A.H. Thomas, and Silicone Oil AR 200: Gallard-Schlessinger) at 4~C for 90 sec at 10,000 x g. The tip containing the cell pellet was excised, and cell bound radioactivity was determined in a gamma counter. Non-15 specific binding was determined by inclusion of 50 nM unlabeled IL- 1 in the assay. Incubations were carried out in duplicate or triplicate.
Receptor binding data were analyzed by using the non-linear regression programs EBDA, LIGAND and Kinetic (Munson and Rodbard, Anal. Biochem 107: 220, 1980) as adapted for the IBM personal 20 computer by McPherson (McPherson, J. Pharmacol. Methods 14: 213, 1985) from Elsevier-BIOSOFT.

The binding of radioiodinated IL- 1 proteins to adherent cells was performed by incubating cells and ligands in a 24 or 12 well plate 2s at 4~C on a rocker platform for 4 hrs in binding buffer (24).
Monolayers were then rinsed 3 times with binding buffer at 4~C, solubilized with 0.5 ml 1% SDS and the released radioactivity courlted in a gamma counter. Non-specific binding was determined in the presence of 50 nM unlabeled IL- 1. Analysis of the binding data was 3 o performed as described above.

Equilibrium Binding of [ I]-labeled Monoclonal Antlbodies to Murine Cells 3 5 Murine cells were washed once in binding buffer (RPMI 1640, 5% FBS, 25 mM Hepes, pH 7.4) and resuspended in binding buffer to a cell density of 1.5 x 107 cells/ml. Cells (1.5 x 106) were incubated with various concentrations of [125I]-specific IgG (.005 to 2 nM) at WO 96~23067 PCT/EP96100181 - 2~ -room temperature for 1.5-2 hrs. Cell bound radioactivity was separated from free [1 25I]-labeled antibody by centrifugation of the assay mixture through 0.1 ml silicone oil at 4~C for 90 seconds at 10,000 x g. The tip cont~ining the cell pellet was exercised, and cell 5 bound radioactivity was determined in a gamma counter. Non-specific binding was determined by inclusion of 100 nM unlabeled antibody in the assay. Incubations were carried out in duplicate or triplicate.
Receptor binding data were analyzed as described above for IL- 1 binding to cells.

Antibody Mediated Inhibition of [125I]-IL-1 Binding to Murine Cells Bearing Type I or Type II IL-1 Receptors The ability of hybridoma supernatant solutions, purified IgG, or 5 antisera to inhibit the binding of Ll 25I]-IL-l proteins to murine cells bearing IL-1 receptor was measured as follows: serial dilutions of culture supernatants, purified IgG or antisera were mixed with cells (1-1.5 x 106 cells) in binding buffer (RPMI-1640, 5% FBS, 25 mM
Hepes, pH 7.4) and incubated on an orbital shaker for 1 hour at room 20 temperature. [125I]-IL-l (1 x 105 cpm; 25 pM) was added to each tube and incubated for 3-4 hours at 4~C. Non-specific binding was determined by inclusion of 50 nM unlabeled IL-1 in the assay.
Incubations were carried out in duplicate or triplicate. Cell bound radioactivity was separated from free [1 25I]-IL-1 by centrifugation of 2s the assay through 0.1 ml of an oil mixture as described above. The tip cont~inin~ the cell pellet was excised, and cell bound radioactivity was determined in a gamma counter.

Affinity Cross-linking and Purification of Solubilized [125I]-IL-la/
30 IL-lR Complexes Affinity cross-linking of radioiodinated IL- 1 proteins to cells was performed as described (Riske et al., J. Biol. Chem. 266: 11245, 1991) with minor modifications. Briefly, cells (1.5 x 107 cells/ml) 3 5 were incubated with radiolabeled IL- 1 (60-300 fmoles/ml) in the presence or absence of 50 nM unlabeled IL-l for 4 hrs at 4~C in binding buffer. The cells were then washed with ice cold PBS, pH 8.3 (25 mM sodium phosphate, pH 8.3, 0.15 M NaCl, 1 mM MgC12), resuspended at a concentration of 5 x 106 cells/ml in PBS, pH 8.3.
Disuccinimidyl suberate (DSS) or bis(sulfosuccinimidyl)suberate (BS3) (Pierce Chemical Co.) in dimethyl sulfoxide was added to a final concentration of 0.4 mM. Incubation was continued for 30-60 min at 5 4~C with constant agitation. The cells were washed with ice cold 25 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 5 mM EDTA and solubilized at 0.5-1 x 108 cells/ml in solubilization buffer (50 mM sodium phosphate, pH
7.5, cont~ining either 8 mM CHAPS or 1% Triton X-100, 0.25 M NaCl, 5 mM EDTA, 40 ~lg/ml phenylmethylsulfonyl fluoride, and 0.05% NaN3) 10 for 1 hr at 4~C. The detergent extract was centrifuged at 120,000 x g for 1 hr at 4~C to remove nuclei and other debris. The extracts were directly analyzed by SDS-PAGE on 8% pre-cast gels (NOVEX) followed by autoradiography. Alternatively, the extracts were immuno-precipitated with antibody bound to Gamma-Bind G Plus (Pharmacia).
l 5 The precipitated proteins were released by treatment with Laemmli sample buffer (Laemmli, Nature 22 7: 680, 1970), separated by SDS-PAGE and analyzed by autoradiography.

Preparation of the solubilized crosslinked complex of IL- 1 a/
20 IL-lR that was used as the immunogen was performed as described above with minor modifications. Briefly, 70Z/3 cells (0.5-1.0 x 108 cells/ml) were incubated with IL-la (0.5 to 1.0 nM) for 4 hrs at 4~C in binding assay buffer. The cells were then washed with ice cold PB S, pH 8.3, resuspended at a concentration of 5 x 107 cells/ml in PBS, pH
2s 8.3 and bis(sulfosuccinimidyl)suberate (BS3) (Pierce Chemical Co.) in dimethyl sulfoxide was added to a final concentration of 0.4 mM.
Incubation was continued for 30-60 min at 4~C with constant agitation. The quenching of the affinity crosslinking procedure and the detergent solubilization of the cells was as described above.
For purification of the solubilized IL-la/IL-lR complex that was used as the immunogen, the detergent extract of 70Z/3 cells was applied to an affinity column ( 10 ml) of goat anti-human IL- 1 a immobilized on crosslinked beaded agarose (Affi-Gel 10, BioRad 3 5 Laboratories). The goat anti-human IL- 1 a affinity column was prepared according to the manufacturer's instructions at a density of 1 mg of IgGlml of packed gel. After application of the detergent extract, the column was washed with 10 column volumes of W 096123067 - 27 - PCT~EP96100181 solubilization buffer without Chaps or Triton X- 100 or urltil the absorbance at 280nM was at baseline. The column was then eluted with 3 M potassium thiocyanate, 25 mM sodium phosphate, pH 7.5, 5 mM EDTA, 40 ,ug/ml phenylmethylsulfonyl fluoride, and 0.05% NaN3.
5 The proteins eluted from the affinity column were concentrated 10 to 100 fold and used for imm1lni7~tion.

Immunoblot Analysis of Proteins Solubilized from Murine Cells Murine 70Z/3 and EL-4 cells were washed 3 times with ice-cold PBS and solubilized at 0.5 - 1 x 108 cellsfml in solubilization buffer that contained either 8 mM CHAPS or 1% Triton X-100 and 1 mg/ml BSA for 1 hr at 4~C. The extracts were centrifuged at 120,000 x g for 45 min at 4~C to remove nuclei and other debris. The extracts were 15 incubated with either 4C5 (anti-IL-lR AcP obtained as described in Fx~mrle 2), 12A6 (anti-Type I IL-lR obtained as described in Chizzonite et al., Proc. Natl. Acad. Sci. USA 86:8029, 1989) or control antibody bound to protein-G immobilized on crosslinked agarose mm~ Bind G Plus, Pharmacia). The precipitated proteins were 20 released by treatment with 0.1 M glycine pH 2.3, neutralized with 3M
Tris, mixed with l/5 volume of 5X Laemmli sample buffer, and separated by SDS/PAGE on 8% pre-cast acrylamide gels (NOVEX). The separated proteins were transferred to nitrocellulose membrane (0.2 ~M) for 16 hours at 100 volts in 10 mM Tris-HCl pH 8.3, 76.8 mM
25 glycine, 20% methanol and 0.01% SDS. The nitrocellulose membrane was blocked with BLOTTO (50% w/v nonfat dry milk in PBS + .05%
Tween 20) and duplicate blots were probed with [125I]-4Cs IgG (1 x 106 cpm/ml in 8mM CHAPS, PBS, 0.25 M NaCl, 10% BSA and 5 mM
EDTA) with and without unlabeled 4C5 IgG (67nM).
Expression of Murine Recombinant Type I and Type II IL- 1 Receptors and IL-lR AcP in COS Cells and Deterrnin~tion of [125I]-labeled 4C5, 35F5 and IL- 1 Binding 3 5 COS cells (4-5 x 107 ) were transfected by electroporation with 25 ~Lg of plasmid DNA expressing recombinant murine IL-lR proteins or IL-lR AcP in a BioRad Ciene Pulser (250 ,uF, 250 volts) according to the manufacturer's protocol. The cells were plated in a 600 cm2 W 0 96/23067 CA 02210724 1997-07-17 P~~ 5~ ol8l culture plate, harvested after 72 hours by treatment with No-Zyme (JRH Biologics) and scraping, washed and resuspended in binding buffer. Transfected cells (4-8 x 104) were incubated with increasing concentrations of [125I]-labeled 4C5, 35F5 or IL-1 proteins at 4~C for s 3 hrs. Cell bound radioactivity was separated from free [1 25I]-labeled antibody or IL- 1 as described above.

Kappa Light Chain Expression by 70Z/3 Cells in Response to IL-l:
Inhibition by Monoclonal Antibodies 35F5, 4E2 and 4C5 70Z/3 cells (1 x 105/ml in RPMI 1640, supplemented with 10%
FB S, ~-mercaptoethanol and gentamicin) were incubated with and without 100 U/ml (0.19 nM) of human recombinant IL-l~x or IL-l~
for 24 hrs or 48 hrs. The cells were preincubated for one hour before 15 the addition of IL-l with 30 ~lg/ml of the indicated antibodies in a total volume of 0.5 ml. An additional 0.5 ml of medium cont~ining the IL- 1 or medium alone was added to the wells for a final concentration of 15 ,ug/ml (100 nM) antibodies. The cells were washed once after culture and stained with either a control rat antibody conjugated with 20 FITC or rat anti-mouse kappa light chain antibody conjugated with FITC (Tago, Burlingame, Ca). The cells were then analyzed for kappa light chain expression on a FACScan flow cytometer (Becton-Dickinson).

25 Proliferation of Murine Splenic B cells in Response to IL- 1: Inhibition by Monoclonal Antibodies 35F5, 4C5 and 4E2.

Splenic B cells were purified by treating splenocytes isolated from C57BL/6 mice with anti-Thyl.2 antibody and rabbit 3 o complement, followed by two sequential passages through a Sephadex G10 (Pharmacia) columns. B cells (5 x 105 cells) were treated with goat anti-mouse IgM (1 ~lg/ml) (ZYMED) and dibutyryl cAMP (10-3 M) in a final volume of 200 ~ll of RPMI 1640 media supplemented with 10% FBS, ~-mercaptoethanol and gentamicin. Splenic B cells were 3 5 treated with and without IL- 1 ( 100 U/ml) and with and without antibodies 35F5, 4C5 and 4E2. The cells were incubated for two days in the presence of the various reagents and then pulsed with 0.5 ,uC i tritiated thymidine, incubated for an additional 6 hrs and harvested.

Proliferation of Murine DlO.G4.1 Cells in Response to IL-l: Inhibition by Monoclonal Antibodies 4C5 and 35F5 and Human IL-lra DlO.G4.1 helper T cells were maintained as described (Kaye et al., J. Exp. Med. 158: 836, 1983; McIntyre et al., J. Exp. Med. 173: 931, 1991) and stimulated with IL-l as previously described (McIntyre et al., J. Exp. Med. 173: 931, 1991). Cells (1 x 105 in 200 ~11) were incubated with 0.2 pM IL-1 in RPMI 1640 containing 5% FBS, ~-mercaptoethanol (5 x 10-5 M), gentamicin (8 ~lg/ml), 2 mM
L-glutamine, 2.5 ,ug/ml concanavalin A and the indicated concentrations of antibodies or human IL- 1 receptor antagonist (IL- lra) . The cultures were incubated for two days, pulsed with 0.5 ~lCi tritiated thymidine and harvested 16 hrs later.
1~
In Vivo Induction of Serum IL-6 by IL- 1: Inhibition by Monoclonal Antibodies 3~F5 and 4C~

The induction of serum IL-6 by IL- 1 was performed as previously described (McIntyre et al., J. Exp. Med. 173: 931, 1991).
Briefly, C57BL/6 mice were pretreated (i.p) with 250 ~lg of antibody at 4 hrs and 10 min before ~tlmini~tration of IL-la or IL-l~ (0.3 g/mouse, s.c.). Sera were collected from the mice 2 hrs after ~lministration of IL-1 and analyzed for IL-6 concentration by a modification of the B9 hybridoma cell bioassay as described (Aarden et al., Eur. J. Immunol. 17: 1411, 1987).

The rat anti-mouse IL- 1 accessory protein monoclonal antibody 4C5 was prepared, characterized and generated as follows:
Example 2 Preparation, Characterization and Identification of Monoclonal Antibodies Specific for IL-lR AcP and Type II IL-lR
In the course of preparing antibodies to the Type II IL- 1 receptor, antibodies to an unexpected, novel component of the IL- 1 receptor complex were detected. Since murine 70Z/3 cells express almost exclusively the Type II IL-lR, immllni~tion of rats with the purified crosslinked IL-la/IL-lR complex solubilized from these cells was the initial strategy pursued to develop monoclonal anti-Type II
IL-lR antibodies. Rats immunized with this solubilized IL-la/IL- 1 R
s complex developed serum antibodies that blocked [125I]-IL-l~
binding to 70Z/3, indicating the presence of blocking antibodies specific for the Type II IL-lR. The serum samples also contained antibodies that imnnuno-precipitated the [125I]-IL-l~/IL-lR complex solubilized from 70Z/3 cells, indicating the presence of non-blocking 0 anti-Type II IL-lR antibodies. [125I]-IL-~ was used for the IL-lR
binding and immunoprecipitation assays to elimin~te identification of antibodies specific for IL- 1 a instead of the Type II receptor.

Hybridomas resulting from the fusion of splenocytes isolated 15 from the immunized rat were screened for antibodies that blocked IL-l~ binding to both 70Z/3 (Type II receptor bearing) and EL-4 (Type I receptor bearing) cells. Antibodies that block binding only to 70Z/3 cells were identified and elimin~ted from further analysis because they are antibodies to Type II IL-lR, and antibodies that 20 blocked binding only to EL-4 cells were identified and elimin~ted from further analysis because they are antibodies to Type I IL- lR.
Antibodies that blocked IL- 1 binding to both cell types are specific for the IL-lR AcP.

2s From the initial fusion, seven antibodies were identified that blocked IL-l~ binding to 70Z/3 cells (Table 1). Six of these antibodies (2BS, 4C5, 3Fl, 4C4, 24CS, and 4D4) blocked IL-l~ binding to both 70Z/3 and EL-4 cells. These antibodies did not block IL-l~ binding to CHO cells expressing murine recombinant Type I IL- 1 R, and were 30 therefore specific for an IL-lR AcP. One antibody, 4E2, only blocked IL- 1~ binding to 70Z/3 cells, indicating that it was specific for the Type II IL-lR.

The initial fusion was also screened for non-blocking antibodies 35 that were specific for either the IL-lR AcP or the Type II IL-lR. Eight antibodies (lD2, 2D6, 2E6, lF6, 2D4, 2F6, 3F5 and 4Al) immuno-precipitated the IL-l~/IL-lR complex solubilized from 70Z/3 cells (Table 2). These antibodies also immunoprecipitated the IL-l~/IL-lR

W 096123067 - 31 - PCT~P96/00181 complexes solubilized from two other Type II IL- 1 R bearing murine cell lines, AMJ2Cl 1 and P388D1. Seven of these antibodies also immunoprecipitated the IL-l~/IL-lR complex solubilized from EL-4 cells, demonstrating that they recognized an IL- 1 R AcP. One antibody, s lF6, did not bind to the IL-lB/IL-lR complex solubilized from EL-4 W 0 96123067 CA 02210724 1997-07-17 PCT/~

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W 096/23067 CA 02210724 1997-07-17 PCT~EPg6100181 cells, indicating it was a non-blocking Type II IL-lR antibody. To confirm that these antibodies did not bind to the Type I IL-lR, they were tested in immunoprecipitation assays with murine soluble Type IL- lR (Table 2). None of these antibodies immunoprecipitated the 5 complex of [125I]-IL-l~ crosslinked to recombinant soluble Type I
IL-lR ([125I]-sMsR[bv]). They also did not immunoprecipitate [125I]
labeled soluble Type I receptor produced either in a baculovirus/
insect cell expression system or in a COS cell expression system (Table 2).

Since the rats were immunized with the solubilized IL-la/IL-l R
complex, antibodies in the rat serum were also detected that recognized IL- l a . Each monoclonal antibody was tested in immunoprecipitation assays with [1 25I]-labeled murine and human 15 IL-l proteins to co:nfirm that they did not bind to IL-l. All 15 antibodies (Tables 1 and 2) were negative in these assays.

Example 3 20 Characterization of Murine IL- 1 Rs and IL-l R AcP by Reactivity with Anti-Type I (35F5), Type II (4E2) and Accessory Protein (4C5) Monoclonal Antibodies Following the initial identification and characterization of the 25 antibodies described above, 4C5, a putative blocking IL- l R AcP (IL- 1 R
AcP) antibody, and 4E2, a blocking Type II IL-lR antibody, were chosen as probes for the further study of the IL- lR AcP. A previously identified and characterized anti-Type I IL-lR antibody, 35F5, was also included in this study (Chizzonite et al., Proc. Natl. Acad. Sci. USA
30 86: 8029, 1989), McIntyre et al., J. Exp. Med. 173: 931, 1991).

These three antibodies were used to identify the presence of Type I and Type II IL-lR's and IL-lR AcP on various murine cells.
Equilibrium binding assays with [125I]-labeled mAb 4C5 35 demonstrated the presence of IL-lR AcP on murine cells bearing predomin~tely Type I (EL-4 cells) or Type II (70Z/3 cells) receptors (Figures 1 and 2). Other cells bearing predomin~tely Type I (3T3Ll cells) or Type II (P388Dl cells) receptors also expressed IL-lR AcP

W 096/23067 PCTi~ 181 (Table 3). Cells (S49.1) that do not express either Type I or Type II
IL-lR AcP did not express IL-lR AcP, indicating a link between expression of IL-lR and IL-lR AcP. During its initial characterization, mAb 4C5 blocked [12~I]-human IL-lB binding to 5 both EL-4 and 70Z/3 cells. Further studies established W 096/23067 CA 02210724 1997-07-17 PCT/~l~6~G~

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that mAb 4C5 also inhibited the binding of radiolabeled human IL- 1 a (Fig. 3), murine IL-la and IL-l~ to 70Z/3 cells (Table 4). Similar to its inhibition of [125I]-human IL-1~ binding to EL-4 cells, 4C5 also blocked [125I]-murine IL-l~ binding to these cells (Table 4).
5 However, 4C5 did not block either radiolabeled human IL-la (Fig. 4) or murine IL- 1 a (Table 4) binding to EL-4 cells. Moreover, 4C5 did not block the binding of [125I]-labeled IL-l proteins to CHO or COS cells expressing murine recombinant Type I or Type II receptors. The anti-Type I receptor antibody, 35F5, and the anti-Type II receptor lo antibody, 4E2, inhibited both IL-1~ and IL-1~ binding to their respective IL- 1 receptors, regardless of whether the receptors were the natural or recombinant forms (Table 4). The ICsos for 4C5-mediated inhibition of IL- 1 binding to EL-4 and 70Z/3 cells were at least 1000-fold lower than ICsos for inhibition of binding to cells l s expressing recombinant Type I or Type II receptors (Table 5). These ICso data suggested two conclusions: 1) mAb 4C5 did not crossreact to any significant extent with Type I or Type II IL-lR's, and 2) the difference in the ability of 4C5 to block IL-1~, but not IL-la, binding to natural IL-lR's was unrelated to the affinity of the antibody.~0 l~xample 4 Determination of the Size of the IL-lR AcP Recognized by Monoclonal Antibody 4C5 The approxim~te molecular size of the cell surface protein recognized by mAb 4C5 on EL-4 cells was determined by affinity chromotography and immunoblotting to be approxim~tely 90 kDa (Fig. 5). Detergent extracts prepared from EL-4 cells were purified on a lentil lectin 30 affinity m~triX followed by affinity chromatography on either an anti-Type I receptor antibody (7E6), murine IL-la (Ma) or 4C5 affinity gel.
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> ~ ' ~ ~ V oC" P~ _ --W 096/23067 CA 02210724 1997-07-17 PCT~P96tO0181 by autoradiography. A major protein of ~90 kDa and a minor protein of 5~ kDa were immunoreactive with radiolabeled 4C5. These two proteins were also identified on the immllnoblot if the EL-4 extract was directly purified on a 4C5 affinity matrix. These data indicated 5 that the apparent molecular weight of the natural, glycosylated IL- 1 R
AcP is ~90 kDa and that proteolytic processing may reduce its size to ~55 kDa.

Example S
Neutralization of IL-l~ Biologic Activity by Monoclonal Antibody 4C5 The ability of mAb 4C5 to neutralize IL-l~ biologic activity in a dose-dependent manner was demonstrated in three biologic assays: 1) 15 IL- l induced proliferation of murine splenic B cells, 2) IL-1 induced proliferation of DlO.G4.1 helper T cells, and 3) IL-1 induced kappa light chain expression in 70Z/3 cells. MAb 4C5 demonstrated a dose-dependent inhibition of IL-1~, but not IL-la, induced proliferation of the splenic B cells (Fig. 6). In contrast to mAb 4C5, the anti-Type I
20 receptor antibody 35FS blocked both IL-la and IL-1~ induced proliferation of B cells. The anti-Type II IL-lR antibody 4E2 did not inhibit proliferation induced by either IL-la or IL-l~. In a ~jmil~r fashion, mAb 4C5 inhibited IL-la, but not IL-l~, induced proliferation of DlO.G4.1 T cells (Fig. 7). Both mAb 35F5 and human IL-lra blocked 2s IL-la and IL-l~ induced proliferation of the DlO.G4.1 cells. MAb 4C5 also blocked IL-l~, but not IL-la, induced expression of kappa light chain on 70Z/3 cells (Fig. 8). Antibody 35F5 blocked both IL- 1 a and IL-l~ induced effects in this assay, whereas mAb 4E2, which recognizes the Type II IL-lR, was inactive . For these assays, 30 neutralization of IL-l activity by the antibodies or by IL-lra is detected as a dose-dependent decrease in the biological response. The block in response may be 100% inhibition (i.e. equal to no IL-l added) or to a lower level depending on the potency of the antibody.

W Og6/t3067 PCT/~ 00181 Example 6 Inhibition of IL-l~ Biologic Activity In Vivo by Monoclonal Antibody Mice ~(1mini~tered IL-1 show a rapid and dramatic increase in the concentration of IL-6 in their serum. The magnitude of the increase in serum IL-6 is dependent on the IL- 1 dose and can be blocked by factors that interfere with IL-l binding to Type I IL-lR.
10 When tested in this IL-l biological model, 4C5 blocked by approxim~te.ly 90% the IL-l~, but not IL-la, induced increase in serum IL-6 (Fig. 9). The anti-Type I IL-lR antibody 35F5 blocked both IL-la and IL-l~ induced increase in serum IL-6. A control mAb X-7B2 had no inhibitory effect.
1~
Fx~mI~le 7 Expression cloning of Mouse (Murine) IL- 1 R AcP using Mab 4C~

20 F~xtraction of RNA
3T3-LI cells were harvested and total RNA was extracted using guanidinium isothiocyanate/phenol as described (P. Chomczynski and N. Sacchi, Anal. Biochem. 162:156, 1987). Poly A+ RNA was isolated from total RNA by one batch adsorption to oligo dT latex beads as 25 described (K. Kuribayashi et al., Nucl. Acids Res. Symposium Series 19:
61, 1988). The mass yield of poly A+ RNA from this purification was approximately 6 Yo. The integrity of the RNA preparations was analyzed by fractionating in 1.0% agarose gels under denaturing conditions in the presence of 2.2M formaldehyde (Molecular Cloning, 30 A Laboratory Manual, second edition, J. Sambrook, E.F. Pritsch, T.
Maniatis, Cold Spring Harbor Laboratory Press, 1989).

3T3-L1 cDNA library construction From the above poly A+ RNA, a cDNA library was established in the 35 m~mm~ n expression vector pEF-BOS (Mizushima and Nagata, Nucl.
Acids Res. 18: 5322, 1990). 10 ~Lg of poly A+ RNA were reverse transcribed using RNaseH~ reverse transcriptase (GIBCO BRL Life Technologies Inc., Gaithersburg, MD). The resulting mRNA-cDNA

WO 96/23067 CA 02210724 1997 - O7 - 17 P~ lool8 hybrids were converted into blunt ended doublestranded cDNAs by established procedures (Gubler and Chua, in: Essential Molecular Biology, Volume II, T.A. Brown, editor, pp. 39-56, IRL Press 1991).
BstXI linkers (Aruffo and Seed, Proc. Natl. Acad. Sci (USA) 84:8573, 5 1987) were ligated to the resulting cDNAs and molecules >1000 base pairs (bp) were selected by passage over a Sephacryl SF500 column.
The Sephacryl SF500 column (0.8 x 29 cm) was packed by gravity in lOmM Tris-HCl pH 7.8/lmM EDTA/lOOmM NaAcetate. BstXI linker-treated cDNA was applied to the column and 0.5 ml fractions were l o collected. A small aliquot of each fraction was fractionated in a 1.0%
agarose gel. The gel was dried down by vacuum and the size distribution of the radioactive cDNA was visualized by exposure of the gel to X-ray film. Fractions cont~ining cDNA molecules >lOOO bp were selected and pooled. The cDNA was concentrated by ethanol l 5 precipitation and ligated to the cloning vector. The cloning vector was the plasmid pEF-BOS that had been digested with BstXI restriction enzyme and purified over two consecutive agarose gels. 375 ng of plasmid DNA were ligated to 18.75 ng of size selected cDNA from above in 150 ,ul of ligation buffer (50 mM Tris-HCl pH 7.8/lOmM
20 MgCl2/lOmM DTT/1 mM rATP/25 mg/ml bovine serum albumin) at 15~C overnight. The following day the ligation reaction was extracted with phenol/chloroform/isoamyl alcohol (25:24:1). The nucleic acids were ethanol precipitated in the presence of 5 ~g of oyster glycogen.
The precipitate was dissolved in water and ethanol precipitated again, 2s followed by washing with 70% ethanol. The final pellet was dissolved in 14 ~11 of water and 1 ~l aliquots were electroporated into E. coli strain DH-lOB (GIBCO-BRL). By this method, a library of approximately 4x 106 recombinants was generated.

30 Screenin~ for murine IL-l Receptor Accessory Protein (muIL-lR AcP~
cDNAs by pannin~ with monoclonal antibody 4C5 The panning method has been described previously (Aruffo and Seed, Proc. Natl. Acad. Sci. (USA) 84: 8573, 1987). Ten aliquots from 3 s the 3T3-LI library each representing approximately 5x 104 clones were plated on LB agar plates containing lOO ~lg/ml ampicillin (amp) and grown overnight at 37~C~. The next day, the colonies from each pool were scraped from the plates into separate 50 ml aliquots of LB +

amp and cultures grown at 37~C for another 2-3 hrs. Plasmid DNA was subsequently extracted using QIAGEN plasmid kits (Qiagen Inc., Chatsworth, CA). The ten separate DNA pools were then used to transfect COS-7 cells by the DEAE dextran technique (5 ~lg DNA/2x 1 o6 5 cells/9 cm diameter dish) (Molecular Cloning, A Laboratory Manual, second edition, J. Sambrook, E.F. Fritsch, T. l~ni7.ti~, Cold Spring Harbor Laboratory Press 1989). 72 hrs after transfection, the COS cells were detached from the plates using 0.5 mM EDTA/0.02% Na-azide in phosphate buffered saline (PBS). A single cell suspension was made of 10 each pool. The anti-muIL-lR AcP mAb 4C5 was bound to the cells for 1 hr on ice [(10 ~g/ml 4C5 mAb in 3 ml PBS/05 mM EDTA/0.02% Na azide/ 5.0% Fetal Calf Serum (FCS)]. The 3 ml of cell-mAb suspension was centrifuged through 6 ml of 2% Ficoll in the above buffer (~300 x g, 5 minutes) to remove unbound mAb. The cells were gently l 5 resuspended in the above buffer. The cells from each pool were subsequently added to a single bacterial plate (9 cm diameter) that had been coated with polyclonal goat anti-rat IgG (20 ,ug/ml in 50 mM
Tris-HCl pH 9.5, room temperature, 1.5 hrs) and blocked overnight with PBS/1% BSA at room temperature. COS cells were left on the 20 bacterial plates for 2-3 hrs at room temperature with gentle rocking.
Nonadherent cells were gently removed by washing with PBS. The rem~inin,~ cells were lysed by the addition of 0.8 ml of Hirt lysis solution (0.6% SDS/10 mM EDTA). The lysates were transferred to 1.5 ml Eppendorf tubes and made 1 M NaCl, incubated overnight on ice 2~ and spun at 15,000 xg for 15 min at 4~C. The supernatants were extracted with phenol/chloroform/isoamyl alcohol (25:24:1) one time, lO~ug of oyster glycogen was added and the DNA precipitated twice by addition of 0.5 volumes of 7.5 M NH40Ac and 2.5 volumes of ethanol. The pellet was washed with 70% ethanol, dried and 30 resuspended in 1 ,ul of H20. Each panned pool of DNA was then electroporated into E. coli strain DH- 1 OB . After electroporation, 5x 104 colonies of each pool were grown as above and plasmid DNA was isolated as above. This DNA represents one round of panning enrichment of the library. A total of three panning rounds were 3 5 completed keeping each of the ten library pools separate throughout.

After the third round of panning, each of the ten pools was used to transfect COS cells by the DEAE dextran method (1 ,ug DNA/2x105 cells/well of a 6-well Costar dish). 72 hrs post transfection, the COS
cells were screened for pools that expressed muIL- 1 R AcP by rosetting with secondary antibody coated polystyrene beads (Dynal Inc., Great Neck, NY). 4C5 mAb was bound to transfected COS cells in 5 PBS/2% FCS (2 ,ug Ab/well) for 15 hrs at room temperature with gentle rocking. Antibody was removed and cells were washed with PBS/2% FCS. 1 ml PBS/2% FCS/1 ,ul of sheep anti-rat IgG coated polystyrene beads (~4x 105 Dynabeads M-450) was added and incubated 1.5 hrs at room temperature with gentle rocking. The beads 10 were removed and the cells washed 5-10 times with PBS. Cells were then fixed by incubation in 95% ethanol/5% acetic acid and examined microscopically for rosetting. One of the ten pools (p~nning pool #2) was found positive for surface expression of muIL-lR AcP.

15 To identify positive clone(s), 100 ,ul of LB + amp was placed in the wells of two 96-well microtiter plates. Each well was then inoculated with 4 individual colonies from p~nning pool #2. The bacterial cells were allowed to grow for 5-6 hrs at 37~C. Pools were then made by combining 10 ~1 aliquots from each well in the 8 rows and 12 columns 20 of each plate, keeping each row and column separate. These pools were each used to inoculate a separate 5 ml culture in LB + amp and grown overnight at 37~C. The next day plasmid DNA was isolated using QIAGEN plasmid kits. Each DNA preparation represented pools of either 48 (rows) or 32 (columns) individual isolates from panning 2s pool #2. Each microtiter pool was used to transfect COS cells in 6-well plates as above and 72 hrs after transfection the cells were screened for Dynabead rosetting as above. Two positive pools were found from one of the microtiter plates, one from row E and one from column 2. A
10 ml aliquot was taken from the well at the intersection of the 30 column and row (well E2) and plated onto LB agar + amp. After overnight incubation, 40 individual colonies were used to each inoculate a 5 ml LB + amp culture. Plasmid DNA was isolated from these cultures using QIAGEN plasmid kits. Each plasmid isolate was digested with XbaI restriction enzyme, to release the cDNA insert, and 3 s fractionated on a 1.0% agarose gel. This analysis revealed that only three sizes of cDNA inserts were represented in the positive microtiter pool. A single representative of each of the three plasmids was used to transfect COS cells in a 6-well plate as above and screened by W O 96123067 P~ Cl~181 rosetting with Dynabeads. In this way a single cDNA clone (E2-K) was identified that encoded the 4C5-reactive muIL- 1 R AcP.

Characterization of muIL-lR AcP cDNA's The cDNA clone E2-K (pEF-BOS/muIL-lR AcP) was initially characterized by restriction enzyme mapping. Digestion of this clone with XbaI released a 3.2 kilobasepair (kb) cDNA insert. The 3.2 kb XbaI fragment was gel-purified and the DNA sequence of both strands 0 was determined by using an ABI automated DNA sequencer along with thermostable DNA polymerase and dye-labeled dideoxy-nucleotides as termin~tors. The DNA sequence revealed an open re~-lin~ frame (ORF) in the 5-prime half of the clone (see below).
Restriction enzyme mapping using Intelligenetics computer software 15 indicated a 1.4 kb PstI restriction fragment within the ORF. This 1.4 kb fragment was gel isolated and used as a probe to identify additional muIL-lR AcP cDNA clones. Approxim~tely 6 x 10~
additional clones from the 3T3-LI cDNA library described previously were plated as above. Colony lifts were performed (Molecular Cloning, 20 A Laboratory Manual, second edition, J. Sambrook, E.F. Fritsch, T.
~ni~ti~, Cold Spring Harbor Laboratory Press 1989) and the lifts were probed with the 1.4 kb PstI restriction fragment labelled with [3 2P]-dCTP by random-priming using the Multiprime DNA labelling system (Amersham Co., Arlington Heights, IL). In this way two 2~ additional homologous cDNA clones were isolated. One contained a 1.0 kb insert and the other a 4.3 kb insert as determined by Xb a I
digestion. The DNA sequence of the 4.3 kb insert was determined as above to confirm the sequence of the muIL-lR AcP ORF.

3 o Sequence analysis of muIL- 1 R AcP cDNA clone The nucleotide sequence of the open reading frame in the muIL- 1 R
AcP cDNA insert is shown in Figure 10A. [SEQ ID NO:4] This open re~-lin~ frame (ORF) consists of 1710 bp which encodes a protein of 35 570 amino acids. The amino acid sequence, shown in Figure 10B [SEQ
ID NO:6], predicts a 20 amino acid NH2-terminal signal peptide with cleavage after Ala-1, an extracellular domain from Serl-Glu339, a hydrophobic transmembrane domain from Leu340-Leu363 and a W 096/23067 CA 02210724 1997-07-17 PCTAE~96/00181 cytoplasmic tail from Glu364 to the COOH-terminus. Seven potential N-linked glycosylation sites are all contained within the extracellular domain .

Database searches with the protein sequence using the Intelligenetics computer program indicate that muIL- 1 R AcP has significant homology to both IL- 1 Type I and IL- l Type II receptors from mouse, human, chicken and rat. The homology to each of these proteins is approxim~tely 25% and is uniformly distributed o throughout the protein sequence. Further analysis of the amino acid sequence of muIL-lR AcP shows it to be a member of the immunoglobulin superfamily. The three pairs of cysteine residues, conserved in the extracellular domain of all of the IL- 1 receptors and responsible for formation of three IgG-like domains, are perfectly 15 conserved in muIL- 1 R AcP.

l~xample 8 Mab 4C5 binding to Murine Recombinant IL-lR AcP Expressed in COS
20 cells To confirm that the cDNA for muIL-lR AcP encodes a protein reactive with mAb 4C5, recombinant muIL-lR AcP was expressed on transfected COS cells and examined for direct binding of [125I]-4C5.
25 COS cells were electroporated, by standard methods, with pEF-BOS/muIL-lR AcP. After electroporation, cells were seeded onto a 6 well tissue culture plate at 2-3 x 105 cells/well. After 48-72 hrs growth medium was removed and 1 ml of binding buffer (RPMI/5%FCS) containing 1 x 106 cpm of [125I]-4Cs was added per 30 well either alone (total binding) or in the presence of 2 ,ug unlabelled 4C5 as cold inhibitor (non-specific binding). Both total and non-specific binding were carried out in duplicate. After 3 hrs incubation at 4~ C, binding buffer was removed and the cells were washed 3 times with PBS. The cells were then lysed by addition of 0.75 ml of 35 0.5% SDS. The lysates were harvested and bound counts were determined. Specific binding was calculated by subtracting non-specific counts from total counts. Specific counts were approxim~tely 30,000 cpm/ well with a non-specific background of 8% indicating CA 022l0724 l997-07-l7 W O 96/23067 PCTi~l~ DlXl that pEF-BOS/muIL-lR AcP directs the expression of 4C5 immunoreactive protein in COS cells.

The size of recombinant muIL-lR AcP expressed in COS cells was determined by metabolic labelling of transfected COS cells with [3 5 S ] -methionine and immunoprecipitation of labelled muIL- 1 R AcP with the mAbs 4C5 or 2E6 (Table 2). 36 hrs after electroporation with pEF-BOS/muIL-lR AcP, medium was removed and COS cells were washed 1 time with methionine-free medium [DMEM(high glucose, without 10 methionine-GIBCO-BRL)/10% FBS/l mM L-glutamine/ 1 mM Na pyruvate)]. Fresh methionine-free medium was added and after 5-8 hrs incubation at 37~ C, 50-100 ~LCi of 35S-methionine was added per ml of medium and incubation continued for 24 hrs. Medium was then rernoved and the cells washed 2 times with cold PBS. Cells wère 15 solubilized by the addition of RIPA buffer (0.5% NP-40, 0.5% Tween-20, 0.5% Deoxycholate, 420mM NaCl, 10mM KCl, 20mM Tris pH 7.5, lmM EDTA) and incubation on ice for 15 min. The lysate was transferred to tubes and spun at 15,000 x g for 15 min. Lysates were precleared by the addition of 40 ,ul of G~mm~Rind G Sepharose (50%
20 v/v in RIPA buffer) (Pharmacia Biotech Inc., Piscataway, NJ) to 500 ,ul of lysate and incubation overnight at 4~ C. The next day the precleared lysates were spun 30 sec in a microfuge and lysates were transferred to clean tubes. Another 40 ~11 of G~mm~Rind G Sepharose was added along with 20 ~g mAb 4C5 or 2E6 (Table 2) and the 25 immunoprecipitations were incubated for 3 hrs at 4~ C with rotation.
The Sepharose-Ab complexes were spun down and washed 1 X with RIPA buffer, lX with 50mM HEPES pH 7.91200mM NaCl/lmM
EDTA/0.~% NP-40 and lX with 25mM Tris pH 7.5/lOOmM NaCl/0.5%
Deoxycholate/1.0% Triton X-10010.1% SDS. Protein was released from 30 the beads by addition of 20 ~Ll of 2X Laemmli sample buffer (Laemmli, Nature 227:680, 1970). The proteins were separated by electro-phoresis in Tris-Glycine PAGE and visualized by autoradiography. As shown in Figure 11 , recombinant muIL- l R AcP immunoprecipitated with mAb 4C5 or 2E6 from transfected COS cells migrates as a broad 35 band from 70-90 kDa. No protein was precipitated from mock transfected COS cells.

W 096/23067 CA 02210724 1997-07-17 P~~ r~/ool8l l~xample 9 Expression of Recombinant IL-lR AcP in COS Cells: Reactivity with [125]-I-Labeled IL-l Proteins and Monoclonal Antibodies The binding characteristics of the recombinant IL-lR AcP for [125I]-labeled IL-1, 4C5 and 4E2 were determined (Fig. 12). The data showed high level expression of recombinant IL-lR AcP ~Cos(4C5)] as determined by [125 I]-4C5 binding, but no increase in [125 I] -human l o IL- 1 a binding when compared to control transfected COS cells [Cos(PEF-BOS)]. For comparison, the high level expression of murine recombinant Type I receptor in COS cells [COS (Mu-IL-lR)] as determined by [125I]-35F5 binding was accompanied by a corresponding increase in radiolabeled human IL- 1~ and IL- 1 a 15 binding (Fig. 13).

Fx~nlple 1 0 Purification of Natural Murine IL-1 Receptor Accessory Protein (IL-lR
20 AcP) from EL-4 Cells Murine EL-4 cells (100 gm) were solubilized in 1 liter of PBS
containing 8 mM CHAPS, 5 mM EDTA and the protease inhibitors pepstatin (10 ,ug/ml), leupeptin (10 ~lg/ml), benzamidine (1 mM), 25 aprotinin ( 1 ~lg/ml) and PMSF (0.2 mM). After centrifugation at 100,000 x g to remove insoluble material, the supernatant was loaded onto a 50 ml wheat germ agglutinin (WGA) agarose column (Vector Laboratories, Inc.) at 0.8 ml/min. The column was washed with equilibration buffer (PBS, 8 mM CHAPS, 5 mM EDTA) followed by 30 equilibration buffer cont~ining 0.5 M NaCl, and bound protein was eluted with PBS cont~ining 8 mM CHAPS and 0.3 M N-acetyl-D-glucosamine.

The sugar-eluted fractions from three WGA agarose column runs 3 5 were pooled and loaded onto a 5 ml immunoaffinity column ~mAb 4C5 antibody cross-linked to Protein G Sepharose via dimethyl-pimelimidate (Stern, A.S. and Podlaski, F.J., in: Techniques in Protein Chemistry IV, R.H. Angeletti, ed., pp. 353-360, Academic Press, NY, W 096123067 P~1I~1~CJ~181 1993)] equilibrated with PBS containing 8 mM CHAPS at 1 ml/min.
The column was washed with equilibration buffer followed by equilibration buffer containing 1 M NaCl. Bound protein was eluted with 50 mM diethylamine buffer, pH 11.5, containing 8 mM CHAPS.
s The fractions containing IL-lR AcP were dialyzed against PBS
containing 4 mM CHAPS and concentrated.

All column fractions were monitored for the presence of IL- 1 R
AcP by SDS-PAGL/immunoblot analysis with mAb 4C5. SDS-PAGE was 10 performed on 8-16% gradient gels (Novex), and proteins were transferred to nitrocellulose as described (Towbin et al., Proc. Natl.
Acad. Sci. USA 76: 4350, 1979). After blocking the nitrocellulose with 2.5% casein in 50 mM Tris cont~ining 150 mM NaC12 and 0.01%
thimerosal (pH 7.5), blots were incubated with mAb 4C5 (5 ~lg/ml) 15 followed by incubation with HRP-conjugated goat (Fab)2 anti-rat antibody (Tago Immunologicals). Blots were developed with the ECL
System (Amersham Life Science).

The amino acid composition (Hollfelder et al., J. Protein Chem.
20 12: 435, 1993) of the final protein preparation is shown in Table 6; it is ~imi1~r to the composition predicted from the deduced protein sequence [SEQ ID NO: 3] from the cDNA clone [SEQ ID NO:l] (Figure 16).
The remainder of the sample was subjected to SDS-PAGE, transferred to a PVDF membrane (Matsudaira, J. Biol. Chem. 262: 10035, 1987) 2s and stained with Coomassie blue R-250. The protein-stained band at 80 kDa, which was immunoreactive with 4C5 antibody, was analyzed by NH2-termin~l sequence analysis (Hollfelder et al., J. Protein Chem.
12: 435, 1993). Two sequences were obtained (1-3 pmoles of each amino acid per cycle), one of which matched residues 1-12 3 o (SERXDDWXLDTM) of the deduced protein sequence obtained from expression cloning of murine IL-lR AcP (Figure lOB).

Although IL-lR AcP solubilized from EL-4 cells has a Mr = 80 kDa as determined by immunoblot analysis with the 4C5 antibody, the 3 5 predicted molecular weight of the protein from the cDNA sequence is 66 kDa. This apparent difference is likely due to glycosylation of the accessory protein. To address this issue, the affinity purified IL-lR
AcP was subjected to SDS-PAGE, and the Coomassie blue-stained band corresponding to the 80 kDa, 4C5-immunoreactive protein was eluted from the gel and chemically deglycosylated with trifluoromethane sulfonic acid (Edge et al., Anal. Biochem. 118: 131, 1981). The deglycosylated protein migrates witn a Mr = 63-64 kDa in SDS-PAGE, s a value in good agreement with the predicted molecular weight from the cDNA sequence.

Example 1 1 10 Isolation of Genomic Clones of Human IL- 1 Receptor Accessory Protein Screening bv cross-hvbridization Attempts were made to identify and isolate a cDNA coding for l 5 the human homologue of IL- lR AcP by screening human cDNA
libraries by Table 6 Amino Acid Composition of Natural Murine IL- 1 Receptor Accessc . ~ Protein from EL-4 Cells amino acid mole %
Cys n.d.
Asx 10.5 Thr 53 Ser 5 1 Glx 13.1 Pro n.d.
Gly 8.5 Ala 8.9 Val 7.4 Met 2.7 I le 6.9 Leu 10.6 Thr 3 3 Phe 4 5 His 2.1 Lys 5 7 Trp n.d.
Arg 5 4 n.d. = not dctennined 20 cross-hybridization with sequences from murine IL- 1 R AcP. Human cDNA libraries prepared from mRNA isolated from RAJ1 cells or NC37 cells were probed with the murine IL-lR AcP cDNA, but initial W O 96123067 PCT/~G~ 181 - ~5 -attempts were unsuccessful, possibly due to very low expression of the human homologue in these cells (see Fx~mple 12). We decided to screen a human genomic library to isolate specific sequences that could be used to subsequently screen a human cDNA library.

The murine IL-lR AcP cDNA clone [3.2 kb XbaI fragment] and restriction fragments of the murine IL-lR AcP cDNA clone [1.4 kb PstI
fragment and 843 basepair (bp) Bam HT/SalI fragment] were used as probes to perform low-stringency Southern blot analysis of human lO genomic DNA (Clontech, Palo Alto, CA). This analysis was performed to determine optimal hybridization and washing conditions under which the murine probe could detect homologous sequences present in the human genome. Hybridization with the murine IL-lR AcP cDNA
probes were carried out at 37~C overnight in hybridization buffer A
(2X SSC, 20% formamide, 2X Denhardt's, 100 ~g/ml yeast RNA, 0.1%
SDS). Probes were labelled with [3 2P]-dCTP using the Prime-It II
Random Primer Labeling Kit (Stratagene, La Jolla, CA). The blots were washed with 2X SSC and 0.01% SDS at various temperature points beginning at 37~C. The optimal conditions were determined to be the 20 use of the [32P]-843 bp BamHI/SalI fragment, hybridizing at 37~C
overnight in hybridization buffer A, washing in 2X SSC, 0.01% SDS at ~5~C. These conditions yielded the lowest background and were used to screen a commercially available human genomic library.

2~ To identify human genomic clones of IL-lR AcP, a human lung fibroblast library in Lambda FIX #944201 (Stratagene, La Jolla, CA) was screened. 4.8 x 105 plaques were screened by standard plaque hybridization techniques (Molecular Cloning, A Laboratory Manual, second edition, J. Sambrook, E.F. Fritsch, T. ~qni~ti~, Cold Spring 30 Harbor Laboratory Press, 1989) using the conditions described above.
Six hybridization positive phage clones were purified by successive plaque hybridization. Two phage clones were further characterized (#1 and #7).
.

3 5 Characterization of human genomic clones The human IL-lR AcP genomic clones were initially characterized by restriction enzyme mapping. Bacteriophage lambda W 096/23067 CA 02210724 1997-07-17 PCT/~l~c~cA

DNA was isolated iFrom clones #1 and #7 using LambdaSorb phage adsorbent (Promega, Madison, WI). The phage DNAs were digested with SacI to release the inserts, and the fragments were then separated by electrophoresis on 1% agarose gels. Inserts for both s clones 3Yl and #7 were ~17 kb in size. Further mapping of clones #l and $~7 was performed using XbaI and EcoRI. The digested DNAs were.
separated by electrophoresis on 1% agarose, transferred to a nylon membrane (ICN, Irvine, CA) and crosslinked for Southern blot analysis. The membrane was hybridized with the 843 bp 0 (BamHIlSalI) fragment of murine IL-lR AcP previously described.
The probe was labelled with [32P]-dCTP using Prime-It II Random Primer Labeling Kit (Stratagene, La Jolla, CA). The blots were hybridized and washed using the low stringency hybridization conditions previously described.
A 4.5 kb fragment from the EcoRI digest and a 2.6 kb fragment from the XbaI digest were identified as positive for hybridization to the murine IL- 1 R AcP sequences . The 4.5 kb fragment and the 2.6 kb fragment were isolated from 0.8% Seaplaque agarose (FMC, Rockland, 20 ME) and purified with Qiaex (Qiagen, Chatsworth, CA). The fragments were subcloned into the vector pBluescript II SK+ (Stratagene, La Jolla, CA) to facilitate characterization. Plasmid DNA was prepared using the Qiagen plasmid kit (Qiagen, Chatsworth, CA).

Southern blot analysis was performed to determine which fragment would be more suitable to detect homologous sequences in the human genome. The 4.5 kb and 2.6 kb fragments were used as probes. Low stringency hybridization conditions were used as follows:
5X SSC, 50% formamide, 5X Denhardt's, 100 ,ug/ml yeast RNA, 0.1%
SDS, 37~C, overnight hybridization. Probes were labelled with [32P]-d CTP using Prime-It II Random Primer Labeling Kit (Stratagene, La Jolla, CA). The membranes were washed using high stringency conditions (0.1 X SSC, 0.01% SDS) at various temperature points beginning at 37~C. Optimal conditions were determined to insure 3 5 selecting a probe that would be specific for huIL- 1 R AcP when screening a human cDNA library. These optimal conditions are described in Fx~mple 12.

W 096/23067 PCTi~ 100181 Sequence analysis of human genomic clone The pBluescript II SK+/2.6 kb human genomic IL-lR AcP
plasmid DNA was sequenced using an ABI automated DNA sequencer s along with thermostable DNA polymerase and dye-labeled dideoxynucleotides as termin~tors. Prelimin~ry DNA sequence analysis showed that this DNA contained a 150-nucleotide region with 90% homology to a sequence coding for the intracellular domain of the murine IL-lR AcP.
Fxample 1 2 Isolation of cDNA Clones of Human IL-lR AcP

15 YT cell cDNA library construction The mAb 2E6 (Fx~mple 2, Table 2) was originally characterized by its reactivity with the murine IL-lR AcP. Prelimin~ry data indicated that mAb 2E6 detects the IL-lR AcP on human cells. A
20 number of human cell lines were screened with [1 25I]-2E6 and it was determined that the YT cell line (Yodoi et al., J. Immunol. 134: 1623~
1985) expressed relatively high numbers of 2E6 reactive sites per cell compared to other hllm~n cell lines, e.g. RAJI. The YT cell line was therefore chosen as the source of RNA for cDNA library construction.
Total RNA was extracted from YT cells and cDNA was made from this RNA as described herein (F.x~mple 7: 3T3-LI cDNA library construction). EcoRI adapters (Stratagene, La Jolla, CA) were ligated to the resulting cDNAs and molecules > 1000 bp were selected by passage 30 over a Sephacryl SF500 column as described herein (EXAMPLE 7: 3T3-LI cDNA library construction). The cDNA was concentrated by ethanol precipitation and ligated to the cloning vector. The cloning vector was Lambda ZAP II phage (Stratagene) that had been digested with EcoR I
restriction enzyme and dephosphorylated (as provided by the 35 supplier). 10 aliquots of lOOng of size selected cDNA from above were each ligated to 1 ~g of Lambda ZAP II arms (EcoRI digested and dephosphorylated) in S ~11 of ligation buffer (66 mM Tris-HCl pH
7.5/SmM MgCl2/lmM DTE/lmM rATP) at 15 C overnight. The W O 96/23067 CA 02210724 1997-07-17 PCTAEP96tO0181 following day the ligations were pooled and packaged into Lambda phage in twelve 4- LLl aliquots using Gigapack II packaging extracts and following the manufacturer's instructions (Stratagene). Packaged phage were titered by plating in bacterial strain XL 1 -Blue-MRF' 5 (Stratagene) in the presence of 5 mM Isopropyl-~-D-thiogalacto-pyranoside (IPTG) (Boehringer Mannheim Co., Indianapolis, IN) and 4 mg/ml 5-bromo-4-chloro-3-indolyl-~-D-galactopyranoside(X-Gal) (Boerhinger-Mannheim) to distinguish non-recombinant phage.
Plaque counts the following day indicated that a library of 3.55 x l o6 10 recombinants was obtained with a non-recombinant background of <0.1%.

Screenin~ of human cDNA library by hybridization with human genomic clone fragments of IL- 1 R AcP
The 2.6 kb XbaI restriction fragment which was previously described as being a specific probe for the huIL-lR AcP was used at low stringency hybridization (SX SSC, 50% formamide, 5X Denhardt's, 100 ~lg/ml yeast RNA, 0.1% SDS, 37~C overnight), high stringency wash 20 conditions (O.lX SSC, 0.01% SDS, 40~C) to screen the YT cDNA library.

4.8 x 105 plaques were screened by standard plaque hybridization techniques (Molecular Cloning, A Laboratory Manual, second edition, J.
Sambrook, E.I. Fritsch, T. M~ni~tis, Cold Spring Harbor Laboratory Press, 1989). Three hybridization positive phage clones (#3, ~5, and 25 #6) were identified and purified by successive plaque hybridization.
Excision of pBluescript SK (-) phagemids containing insert DNA from the Lambda Zap II vector was performed according to manufacturer's protocol.

3 0 Characterization of human cDNA clones The human IL-lR AcP cDNA inserts #3, #5, and #6 in pBluescript SK (-) were further characterized by restriction enzyme mapping. Initially, miniprep plasmid DNA was prepared by the rapid 35 boil method (Holmes and Quigley, Anal. Biochem. 114: 193, 1981).
Subsequently, plasmid DNA was prepared with the Qiagen plasmid kit.
The plasmid DNAs were digested with EcoRI to release the inserts, and the inserts were separated by electrophoresis on 1% agarose. Clone #3 W O 96/23067 PCT/~l~5~ 8 _ 59 _ contained a 2.3 kb insert, clone ~5 contained a 1.4 kb insert, and clone #6 contained a 2.7 kb insert. Further restriction mapping indicates a single PvuII site present in all three clones.

5 Sequence analvsis of human IL- 1 R AcP cDNA clones Plasmid DNA from clones #3, #5 and #6 were sequenced using an ABI automated DNA sequencer along with thermostable DNA
polymerase and dye-labeled dideoxynucleotides as terminators.
10 Prelimin~ry sequence analysis indicated that only clones #3 and #6 had inserts that were homologous to the murine IL-lR AcP cDNA.
Therefore, clones #3 and #6 inserts were sequenced completely.
Sequence analysis indicates that clones #3 and #6 are overlapping clones. Schematic representations of clones #3 and #6 are shown in 5 Figure 14. Clone #3 contains the ATG initiation codon and the 5' portion of the coding region. Clone #6 contains the 3' portion of the cDNA and the TGA stop codon. These two overlapping clones were used to construct a full length huIL-lR AcP cDNA.

Example 13 Construction of Full Length Human IL-lR AcP cDNA

Restriction endonuclease mapping and prelimin~ry sequence analysis indicated that there was a single BstXI site present in clone 2s #3 and clone #6. Shown in Figure 14 is a schematic representation of overlapping clones #3 and #6. Clones #3 and #6 were digested with the restriction enzymes BstXI and XbaI. Fragments of approxim~tely 846 bp and approxim~tely 2700 bp were prepared from clone #3 and clone #6, respectively, by electrophoresis in 0.7% Seaplaque agarose 30 (FMC, Rockland, ME) and purified with Qiaex (Qiagen, Chatsworth, CA).

The full-length human IL-lR AcP was prepared by subcloning ~ into the m~mm~ n expression vector pEF-BOS (Mizllshim~ and Nagato, Nuc. Acids Res. 18: 5322, 1990). pEF-BOS plasmid DNA was 35 digested with XbaI, treated with calf intestinal phosphatase (Boehringer Mannheim, Indianapolis, IN), separated by electro-phoresis on a 0.7% Seaplaque agarose gel, and purified with Qiaex (Qiagen, Chatsworth, CA). The ~46 bp and approxim~tely 2700 bp W 096123067 CA 02210724 1997-07-17 PCT~P96/00181 BstXIlXbaI fragments described above were ligated into the XbaI-cleaved pEF-BOS expression vector, and the ligation products were transformed into MC 1061 competent cells. The transformed cells were plated onto LB agar plates containing 100 ~lg/ml ampicillin and grown 5 overnight at 37 C. The next day, 12 individual colonies were picked, inoculated into LB and ampicillin (100 llg/ml) and incubated overnight at 37 C. Miniprep plasmid DNA was prepared from each inoculated colony by the rapid boil method (Holmes and Quigley, Anal.
Biochem. 114: 193, 1981). Restriction endonuclease analysis 10 confirmed that 10 clones contained the appropriate insert in the proper orientation relative to the promoter region in pEF-BOS.

Plasmid DNA was isolated from two positive clones ~1 and #9 by the Qiagen method (Qiagen, Chatsworth, CA). The nucleotide 15 sequence of both strands of both plasmids was determined as described in F.x~mple 7. The sequence of the 1710 bp open reading frame (ORF) contained within the full-length huIL- lR AcP cDNA is shown in Figure 15. [SEQ ID NO:1] The deduced amino acid sequence, shown in Figure 16 [SEQ ID NO:3], would encode a protein of 570 20 residues consisting of a 20 arnino acid signal peptide (Met~20-Ala~ l ), a putative extracellular domain (Serl-Glu339), a hydrophobic transmembrane domain (Leu340-Leu363), and a cytoplasmic tail (Glu364-ValS50). Seven potential N-linked glycosylation sites are all contained within the extracellular domain. All seven sites are 25 conserved between murine and human IL-lR AcP.

Example 1 4 Expression of Soluble Human IL-lR AcP
To express the huIL-lR AcP, a soluble form of the protein was engineered for expression in the baculoviral expression system. This system is useful for overproducing recombinant proteins in eukaryotic cells (Luckow and Summers, Bio/Technology 6: 47, 1988).
3 5 Using the polymerase chain reaction (PCR) method (Innis M.A., et al., PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego~, an amplicon was produced that encoded a soluble form of the extracellular domain of huIL-lR AcP. Briefly, two oligonucleotide WO 96/23067 CA 0 2 2 l O 7 2 4 l 9 9 7 - O 7 - 17 PcT/~ ' ' I 3181 primers were synthesized on an Applied Biosystems synthesizer. The forward primer contained the BamHI site and the codons for the first 11 amino acids of the signal peptide: (~')GGCC GGA TCC ATG ACA CTT
CTG TGG TGT GTA GTG AGT CTC TAC (3') [SEQ ID NO:10]. The reverse 5 primer sequence coded for the 11 amino acids just before the transmembrane domain, an Ala spacer, and a Glu-Glu-Phe tag, followed by the terrnin~tion codon TAG and a KpnI site: (5') CGCGCG
GGT ACC CI A GAA CI'C TTC AGC TTC CAC TGT GTA TCI TGG AGC TG&
CAC TTT CTGC(3') [SEQ ID NO:ll]. The Glu-Glu-Phe tripeptide tag at the l 0 COOH-terminus was engineered to provide an epitope for antibody detection of the recombinant protein. This tripeptide tag is recognized by a commercially available monoclonal antibody to a-tubulin (Skinner et al., J. Biol. Chem. 266: 14163, 1991).

The forward and reverse primers were used to amplify the extracellular domain of the huIL-lR AcP, using clone #3 (Figure 14) as template. The resulting approxim~tely 800 bp PCR amplicon was digested with BamHI and KpnI. The digested fragment was subjected to electrophoresis through 0.7% Seaplaque agarose and purified with 20 Qiaex (Qiagen, Chatsworth, CA). The soluble human IL-lR AcP
extracellular domain was then subcloned into pNRl, a derivative of the baculovirus transfer vector pVL941 (PharMingen, San Diego, CA).
pNRl was prepared from pVL941 by removal of the EcoRI site at position 7196 (cleavage with EcoRI and filling in of sticky ends with 25 Klenow DNA polymerase). The DNA was then subjected to religation, then cleavage with BamHI and Asp718 (KpnI isoschizomer) and insertion of the following oligonucleotides which contain BamHI, EcoRI, and Asp718 recognition sequences:
30 (5') GATCCAGAATTCATAATAG (3') [SEQ ID NO:12]
(3') GTCTTAAGTAI-rATCCATG (5')[SEQ ID NO:13]
The BamHI, EcoRI, and Asp718 restriction sites are unique in pNRl.

3s pNRl plasmid DNA was digested with BamHI and KpnI and purified from a 0.7% Seaplaque agarose gel with Qiaex (Qiagen, Chatsworth, CA). The Bam HI/KpnI approximately 800 bp huIL-lR
AcP PCR amplicon fragment was ligated into the BamHI/KpnI cleaved pNR1 expression vector. The ligation products were transformed into W 096123067 CA 02210724 1997-07-17 PCT/~ ol8l MC 1061 competent cells, which were then plated onto LB agar containing ampicillin (100 ~lg/ml) and grown overnight at 37~C. The next day, 36 independent colonies were picked and inoculated into LB
and ampicillin (100 ,ug/ml). Miniprep DNA was prepared by the rapid s boil method (Holmes and Quigley, Anal. Biochem. 114: 193, 1981). The DNA was analyzed by restriction endonuclease rnapping. Thirty plasmid clones were shown to contain the correct insert. Plasmid DNA
was prepared from two positive clones (~ 25) by the Qiagen method (Qiagen, Chatsworth, CA). These clones were verified by o sequence analysis.

The pNRl/soluble human IL- lR AcP DNA (clone #25) was co-transfected with linearized AcRP23.1ac Z baculovirus DNA
(PharMingen, San Diego, CA) into Sf9 (Spodoptera frugiperda) cells 15 using the BaculoGold Transfection Kit (PharMingen, San Diego, CA).
Following transfection, recombinant baculovirus were isolated and plaque purified according to a protocol described in the BaculoGold Transfection Kit (PharMingen). Plaques were visualized by staining with MTT as described (Shanafelt, Biotechniques 11: 330, 1991).
20 Twelve individual viral plaques were isolated and the virus particles were eluted from the agarose into 0.5 mls of SF-9 media (IPL-41 +
10% FBS - JRH Biosciences, Lenexa, KS) by incubating overnight at 4 C
on a rotator. Each recombinant virus was analyzed for the presence of insert by PCR analysis and for the expression of recombinant human 2s IL-lR AcP by immunoblot analysis. For PCR amplification, viral DNA
was extracted, incubated with Taq DNA polymerase and the appropriate pNRl forward and reverse primers (relative to the BamHI/Asp718 cloning sites), and amplified using standard PCR
methods (Innis et al., PCR Protocols, Academic Press, San Diego 1990).
30 Each amplicon was analyzed by electrophoresis on 1.5% agarose. The results confirmed that 10 out of the 11 plaques tested contained an insert of ~ 1 kb, corresponding to the proper insert size.

For immunoblot analysis, human IL- 1 R AcP + tag (from the 3 5 supernatant of Sf9 cells infected with recombinant virus) was isolated by reacting with biotinylated anti-tubulin antibody (YLl/2) (Harlan Bioproducts) immobilized on streptavidin-agarose (Pierce, Rockford, IL). Proteins were eluted from the anti-tubulin antibody matrix with WO 96/23067 CA 02210724 1997 - O7 - 17 PCTI~;~,5.'~0181 0.2M glycine pH 2.7, and the fractions neutralized with 3M Tris base.
Eluted proteins were treated with Laemmli sample buffer without ~-mercaptoethanol, separated on 8% acrylamide (Novex) slab gel and transferred to 0.2~L nitrocellulose membrane (Schleicher & Schuell, 5 Keene, NH). The immobilized proteins were probed with the YL1/2 antibody (10 ,ug/ml), and peroxidase-conjugated goat-anti-rat antibody ( 1:10,000 dilution) (Boehringer Mannheim Biochemicals).
Immunoreactive bands were visualized by ECL (Amersham) according to the manufacturer's protocol. This analysis identified a protein of o >200 kDa, that was expressed by recombinant virus containing the human IL-lR AcP ~ tag insert.

Recombinant virus from plaques #2 and #12 (identified by immunoblot analysis as expressing human IL-lR AcP + tag )were 5 amplified to obtain virus stocks which were used in the large-scale production of human IL-lR AcP + tag for immllni7~tion purposes. Sf9 cells were cultured in logarithmic growth (1 x 106 cells/ml) in EX-CELL 401 with 1% Fetal Bovine Serum (JRH Biosciences, Lenexa, KS) at 27 ~C, infected with recombinant baculovirus as described (O'Reilly e t 20 al., Baculovirus Expression Vectors, a Laboratory Manual, Oxford Univ.
Press, 1994) and spent culture media were harvested at 3-5 days post-infection. The cells were removed from the spent culture media by centrifugation and the soluble human IL-lR AcP + tag was purified over an affinity matrix composed of immobilized YL1/2 antibody as 2s described in Fx~mple 15 below. The purified human IL-lR AcP + ta~
was used to immunize mice.

Fxample 1 5 30 Preparation and Screening for Monoclonal Antibodies Specific for Human IL-l Receptor Accessory Protein (huIL-lR AcP) Three methods are employed to develop antibodies specific for the huIL-lR AcP.
3~

W 0 96/23067 CA 02210724 1997-07-17 PCT/~00181 Tmmuni7~tion of mice and rats with COS cells expressing human recombinant IL- 1 R AcP

COS cells (4 X 107) are transfected by electroporation with the 5 full-length huIL-lR AcP expression plasmid (20 ~lg, described in F.x~mple 13) in a BioRad Gene Pulser at 250 ~lF and 350 volts as per the manufacturer's protocol. The transfected cells are plated into a 250 mm x 250 mm Nunc tissue culture tray and harvested after 72 hrs growth. The transfected cells are released from the tissue culture tray 10 by treatment with NO-zyme (JRH Biosciences) for 10 min at 37~C. The cells are harvested, washed in PBS, pH 7.4 and used for immllni7~tions.
Mice and rats are immunized by the intraperitoneal (i.p.) route with COS cells expressing huIL-lR AcP (1 X 107cells/~nimAl) on Days 0, 7, 14 and 28. On day 40, the ~nim~ls are bled to determine the titer of 15 the antibody response against huIL-lR AcP (see below for specific assays). ~nim~l~ are given booster immllni7~tions (1 X 107 cells, i.p.) at 2-4 week intervals after day 40. Serum antibody titers specific for huIL- 1 R AcP are deterrnined at 10-12 days after each booster immllni7~tion. When the ~nim~l~ develop a sufficient serum antibody 20 titer (e.g., 1/1000 dilution of the serum immunoprecipitates at least 50% of a given ~mount of the complex of [125I]-IL-l~ crosslinked to IL-lR AcP solubilized from human YT and RAJI cells), they are given booster immnni7~tions in preparation to isolating their spleen cells.
These final booster immllni7.~tions are composed of 1 X 107 cells given 25 both i.v.and i.p. on two consecutive days. Three days after the last immlmi7~tion, spleen cells are isolated from the ~nim~l and hybridoma cells are produced as described previously. Hybridoma cells secreting antibodies specific for huIL- lR AcP are identified by the assays described below. Hybridoma cells are cloned as described previously 30 in Example 1.

Tmmunization of mice and rats with purified human recombinant soluble IL-lR AcP

a. Preparation of human recombinant soluble IL-lR AcP in COS
cell and baculovirus expression systems. As described above, COS cells are transfected with plasmid DNA expressing the extracellular domain of huIL-lR AcP that has a tag (Glu, Glu, Phe) (Skinner et al., J. Biol.

W O 96123067 ~ /0~18 Chem. 266: 14163, 1991) inserted at the C-terminus (soluble IL-lR
AcP, amino acids 1-339 ~ Ala ~ Glu + Glu + Phe). The tag encodes the sequence for recognition by the anti-tubulin antibody YLl/2 (Harlan Bioproducts). The medium is harvested from the cells 72 hrs after 5 transfection and soluble IL- 1 R AcP+tag is detected and purified as described below.
r Standard methods (Gruenwald and Heitz, Baculovirus Expression Vector System: Procedures and Methods Manual, Second l~dition, 1993, lo PharMingen, San Diego, CA) are employed to generate a pure recombinant baculovirus expressing the soluble IL-lR AcP protein.
Briefly, plasmid DNA coding for the soluble extracellular domain of human IL-lR AcP~tag is inserted into the transfer vector pNRl as described in Fx~mrle 14. The recombinant transfer vector is purified 5 and co-transfected with linearized ACVVVl.lacZ DNA (PharMingen) into Sf9 (Spodoptera frugiperda) cells. Recombinant baculovirus are isolated and plaque-purified. SF-9 cells (2 X 106 cells/ml) are cultured to logarithmic glo~vLh phase in TMH-FH medium (PharMingen) at 27~C, infected with recombinant baculovirus, and spent culture media 20 harvested after 3-5 days. The cells are removed from the spent culture media by centrifugation and the soluble IL-lR AcP+tag protein is detected and purified as described below.

b. Preparation of an affinity matrix composed of immobilized 25 YLl/2 antibody. Many methods can be utilized to immobilize the YL1/2 antibody to an affinity m~tri~ including covalent crosslinking to either an activated agarose gel such as Affi-Gel 10 (BioRad Laboratories) or to an agarose gel cont~ining immobilized Protein G
(Stern and Podlaski, in: Techniques in Protein Chemistry IV, R.H.
30 ~ngelletti, ed., pp. 353-360, Academic Press, NY, 1993). However, for the purification of soluble IL-lR AcP, the YL1/2 antibody is covalently modified with NHS-LC-biotin (Pierce Chemical Co.) and immobilized on a streptavidin-agarose gel (Pierce Chemical Co.). YL1/2 antibody (3 mg/ml) is dialyzed against 0.1 M borate buffer, pH 8.~ followed by 35 reaction with NHS-LC-biotin at a molar ratio of 40:1 (LC-biotin:YL1/2 antibody) for 2 hrs at room temperature. The unreacted LC-biotin is quenched with 1 M glycine/0. 1 M borate buffer, pH 8.4. The unreacted and quenched NHS-LC-biotin is removed by centrifugation at 1000 xg W 0 96/23067 CA 02210724 1997-07-17 PCT/~l~G1~181 for 15-30 min using a Centricon-30 microconcentrator (Amicon). After centrifugation, the biotinylated YLl/2 antibody is diluted with 0.1 M
sodium phosphate, pH 7.0 and the process repeated two more times.
Biotinylated-YLl/2 antibody (6 mg in 0.1 M sodium phosphate, pH
5 7.0) is reacted with streptavidin-agarose (6 ml of a 50% suspension) for 2 hrs at room temperature. The streptavidin agarose with the immobilized biotinylated YLl/2 antibody is placed into a column and washed with 10 column volumes of PBS, pH 7.4.

o c. Purification of soluble IL-lR AcP. Media from either COS cells or Sf9 cells containing soluble IL-lR AcP are passed through the YLl/2 affinity column at a flow rate of 3 ml/min. The column is washed sequentially with 2 column volumes of PBS, pH 7.4, 5 column volumes of 50 mM sodium phosphate, pH 7.5, 0.5 M NaCl, 0.2 % Tween 20, 0.05% NaN3 and 2 column volumes of PBS, pH 7.4. The soluble IL-lR
AcP + tag is eluted with 0.1 M glycine-HCL, pH 2.8 and the fractions (1 ml) are neutralized with 3 M Tris base (0.015 ml per 1 ml fraction).
The protein eluted from the column (purified soluble IL- 1 R AcP + tag) is characterized by reducing and non-reducing SDS-PAGE on 12%
20 acrylamide slab gels followed by silver st~inin~ to visualize the protein bands. The soluble IL-lR AcP + tag present in the conditioned media from the COS cell and baculovirus expression systems and in the purified preparations can also be identified by western blotting procedures. Proteins in the conditioned media (~.04 ml) and purified 25 soluble IL-lR AcP + tag (0.1 to 1 ~g) are treated with Laemmli sample buffer without ~-mercaptoethanol, separated by SDS-PAGE on 12% gels and transferred to nitrocellulose membrane (0.2 ~M) as described above in F.x~mrle 1. The proteins irnmobilized on the nitrocellulose are probed with YL1/2 antibody (5 ~g/ml) and peroxidase-conjugated goat 30 anti-murine or -rat IgG antibody (1/1000 dilution) (Boehringer Mannheim Biochemicals). The immunoreactive bands are identified by ECL technique (Amersham Inc.) according to the manufacturer's protocol. The soluble IL-lR AcPs that are purified from COS cell and baculovirus expression systems should migrate as proteins of 3 ~ approxim~tely 65-67 kDa and 45-47 kDa, respectively.

d. Immunization of mice and rats with soluble IL-lR AcP + ta~.
Mice and rats are immunized by the i.p. and foot pad routes on days 0, CA 022l0724 l997-07-l7 W 0~6123067 PCT/~ 'OCl81 14 and 28 with 10-100 ~lg of soluble IL-lR AcP + tag. The protein is prepared as described in F~mrles 1 and 2 in Freund's complete adjuvant for the primary immllniz~tion and in Freund's incomplete adjuvant for the day 14 and 28 booster immllni7~tions. Serum is ~ 5 collected from the ~nim~l~ on day 40 and tested for antibody reactivity (see assays below). The ~nimAl~ are given booster t immllni7:1tionS (i.p., 10-25 llg of protein prepared in Freund's incomplete adjuvant) at 4 week intervals and the titer of serum antibodies determined two weeks after each immunization. When the 10 ~nim~l~ develop a potent serum antibody titer (e.g., 1/104 dilution of the serum gives a 50% response in the EIA), they are given booster immllni7~tions (i.v. and i.p.) of 10-100 ~lg of soluble IL-lR AcP + tag on two consecutive days. Three days later, spleen cells are isolated from the ~nim~l and fused with SP210 cells as described in Fx~m~le 1 for 15 the development of the anti-murine IL- 1 R AcP antibodies . Hybridoma supernatants are screened for inhibitory and non-inhibitory antibodies by the assays described below. Hybridoma cell lines secreting anti-huIL-lR AcP antibodies are cloned by limiting dilution. Anti-huIL-lR
AcP antibodies are purified as described in Fx~mple 1.
e. J4ssays to detect antibodies specific for human II~-lR ~cP. The presence of anti-IL- lR AcP antibodies in the serum is initially determined by enzyme immunoassay (EIA) with soluble IL-lR AcP +
tag immobilized on a 96 well plate. Briefly, soluble IL-lR AcP + tag (1 25 ~lg/ml) is diluted with 50 mM sodium carbonate buffer, pH 9.0, 0.15 M
NaCl (BC saline) and passively adsorbed (100 ~11, 100 ng) to the wells of a Nunc Maxisorb plate for 16 hrs at room temperature. After washing, the plates are reacted with PBS, pH 7.4, 1% bovine serum albumin (BSA) for 1 hr at 37~C. Serial dilutions [1/100 to 1/106 in 50 mM
30 sodium phosphate, pH 7.5, 0.5 M NaCL, 0.1% Tween-20, 1% BSA and 0.05% NaN3 (antibody binding buffer)] of the serum samples are incubated with the immobilized soluble IL-lR AcP for 2 hrs at room temperature. After washing the plate with PBS, pH 7.4, 0.05% Tween-20, the bound antibody is detected with peroxidase-conjugated goat 35 anti-murine or -rat IgG antibody (Boerhringer-Mannheim Inc.) and visualized with TMB (tetramethylbenzidine) substrate. The color intensity in the individual wells is measured at 450 nm in a multi-W 096123067 CA 02210724 1997-07-17 P~~ 5~ool8 channel photometer and is proportional to the concentration of anti-IL-lR AcP antibody in the serum.

The serum antibodies are also tested for reactivity by FACS
5 (fluorescence activated cell sorting) on 1) natural huIL-lR AcP
expressed on the human cell lines YT, NC-37 and RAJI and 2) recombinant huIL-lR AcP expressed on COS cells. Cells (1 X 106) are incubated with serurn dilutions (1/100 to 1/104) in PBS, pH 7.4 (100 ,ul) for 1 hr at 4~C. After washing the cells with PBS, pH 7.4, to remove 10 unbound antibody, the cells are incubated with fluorescein-conjugated goat-anti-mouse or -rat IgG antibody (Tago Laboratories) for 30 min at 4~C. The cells are washed with PBS, pH 7.4, and the quantity of antibody bound to the cell surface is determined by the increase in fluorescence intensity in a FACSort (Becton-Dickinson Co.).
The anti-murine IL-lR AcP antibodies 4C5 and 2E6 (Table 2) demonstrated inhibitory and non-inhibitory activity, respectively, against IL- lR AcP expressed on murine cells. To determine if sera from ~nim~l~ immunized with human IL-lR AcP contain both 20 inhibitory and non-inhibitory antibodies, two types of assays are performed: 1) inhibition of [125I]-IL-1,13 binding to human cells and 2) immunoprecipitation of the solubilized complex of [125I]-IL-l~
crosslinked to cell surface proteins from human cells. For the inhibition assays, serial dilutions of the sera are incubated with YT, NC-37 and 25 RAJI cells (1-2 x 106) in binding buffer for 1 hr at room temperature.
[125I]-IL-1,~ (25-250 pM) is added to each tube, incubated for 3 hrs at 4~C and cell bound radioactivity determined as previously described in Fx~mple 1. The titer of inhibitory antibodies is determined by the serum dilution that results in a 50% decrease in cell-bound 3 o radioactivity. For the immunoprecipitation assays, dilutions of serum are incubated for 16 hr at 4~C with the solubilized complexes of [125I]IL-1~ crosslinked to huIL-lR AcP and in the presence of protein-G-Plus immobilized on agarose beads. Fach serum sample is tested for reactivity with solubilized complexes prepared from human 3s cell lines YT, NC-37 and RAJI. After centrifuging and washing the protein-G-Plus agarose beads, the immunoprecipitated proteins are analyzed by SDS-PAGE and autoradiography as described in the F.x~mple 1 for the murine IL-lR AcP antibodies.

WQ 96123067 PCT/~ ,'u~181 Tmmllni7~tion of mice and rats with huIL-lR AcP peptides conjugated to keyhole limpet hemocyanin (KLH) s Peptides corresponding to sequences 1-10, 54-64, 68-77, 265-273, 285-294, 490-499 and 505-515 of the full-length huIL-lR AcP
were synthesized by standard solid phase techniques (Marglin and Merrifield, Ann. Rev. Biochem. 39: 841, 1970). The sequence of each peptide had a cysteine added to the C-terminus for the purpose of 10 covalent coupling to KLH by the MBS technique. Briefly, KLH (1.5 mg in PBS, pH 7.4) is reacted with 0.32 mg of 3-malemidobenzoyl-N-hydroxy-succinimide ester (MBS; Boehringer Mannheim Biochemicals) for 1 hr at 4~C. The reaction mix is applied to a prepacked BioGel P10 column (10 ml) (BioRad Laboratories) and chromatographed with PBS, 5 pH 7.4. The fractions cont~ining the KLH-MBS conjugate are pooled (2 ml) and reacted with peptide (2 mg) for 1 hr at 4~C. The KLH-peptide conjugate is concentrated in a Centricon 10 microconcentrator (Amicon) and used for imml1ni7~tions. Mice and rats are immunized by the i.p. and foot pad routes on day 0, 7, 14 and 28 with 200-500 ,ug of 20 KLH-peptide conjugate. The conjugate is prepared in Freund's complete adjuvant for the primary immunization and Freund's incomplete adjuvant for the booster immllni7~tions. Sera are collected from the ~nim~ls on day 40 and tested for antibody reactivity in the soluble IL-lR AcP EIA. The ~nim~l~ are given booster imm~lni7~tions (i.p., lO0 ~lg 2~ of KLH-peptide conjugate prepared in Freund's incomplete adjuvant) at 4 week intervals and the titer of serum antibodies determined two weeks after each immllni7~tion. When the ~nim~l~ develop a potent serum antibody titer (1/104 dilution gives a 50% response in the EIA), they are given booster immllni7~tions with free peptide (100 '~lg, i.v.
30 route) and KLH-peptide conjugate (500 ,ug, i.p. route) on two consecutive days. Three days later, spleen cells are isolated from the ~nim~l and hybridoma cells secreting huIL-lR AcP antibodies are produced and identified as described above.

WO 96/23067 CA o 2 2 l o 7 2 4 19 9 7 - o 7 - 17 PCT/EP96/00 181 Example 1 6 Neutralization of IL-l~ Biologic Activity by Anti-Human IL-lR AcP
Antibodies and Active Fragments of IL-lR AcP
The ability of anti-human IL-lR AcP antibodies to neutralize IL-1 biologic activity in a dose-dependent manner can be determined in the IL- 1 -induced IL-6 assay with human embryonic lung fibroblast MRC-5 cells (ATCC # CCL-171). MRC-5 cells are plated in 96-well 10 cluster dishes and pretreated for l hr with either increasing concentrations of anti-human IL-lR AcP or active fragment of IL-l R
AcP. Following the pretreatment, the cells are stimulated with either S
pM human IL-la or IL-l~ for 24 hrs. The amount of IL-6 secreted by the cells in response to IL- 1 is measured by a commercially available 15 IL-6 EIA (Qll~ntikine Assay for Human IL-6, R & D Systems, Minneapolis, MN). The inhibitory effects of the antibodies and active fragments of IL-lR AcP are calculated by determining the decrease in IL-6 secretion in the presence and absence of inhibitors. For example, S p M and 100 p M IL-l~ stimulated the secretion of approxim~ely 20 8100 and 9800 pg/ml of IL-6, respectively, from MRC-5 cells (Fig.
17). IL-l receptor antagonist (IL-lRA) and anti-human Type I IL-lR
antibody 4Cl blocked this IL-6 secretion in response to IL-l~ (Fig.
17). For IL-lRA and 4Cl, the ICso's for blocking 5 pM IL-lB were 200 pM and 0.025 ~g/ml, respectively (Fig. 17). The inhibition by IL-lRA
25 and 4Cl can be overridden by increasing the concentration of IL-l~ to 100 pM. With 100 pM IL-l~, the ICso's for IL-lRA and 4Cl inhibition were > 1 nM and 10 ,ug/ml, respectively. These data demonstrated that the IL- 1 -induced IL-6 response from the MRC-5 cells was specific for IL-l and a Type I IL-lR-dependent response, in the same 3 o way that IL- 1 -dependent responses in murine cells are also Type receptor-dependent (Figs. 6, 7 and 8). These IL- 1 biologic assays with murine cells led to the identification of neutalizing anti-murine IL- lR
AcP antibodies. Simil~rily, the IL-l biologic assay with MRC-5 cells can be used to identify neutralizing anti-human IL-lR AcP antibodies 3 5 and active fragments of IL- 1 R AcP.

CA 022l0724 l997-07-l7 W 096l23067 PCTi~l~C,'~~181 SEQUENC~ LISTING

(1) GENERAL INFORMATION
s (i) APPLICANT
(A) NAME F HOFFNANN-LA ROCHE AG
(B) STREET Grenzacherstrasse 124 (C) CITY Basle (D) STATE BS
(E) COUN'1'KY: Switzerland (F) POSTAL CODE (ZIP) CH-4010 (G) TE~EPHOh-E 061-6885108 (H) TELEFAX 061-6881395 (I) TELEX 962292/965542 hlr ch (ii) TITLE OF lNv~llON HUMAN ACCESSORY PROTEIN FOR INTE~T~TN-1 ~ '1'0~
(iii) NUMBER OF ~Qu~S 13 (iv) COMPUTER ~n~RT~ FORM
(A) MEDIUM TYPE Floppy disk (B) COMPUTER IBM PC compatible (C) OPERATING SYSTEN PC-DOS/MS-DOS
(D) SOFTWARE PatentIn Release #1 0, Version #1 30 (EPO) (v) CURRENT APPLICATION DATA

(2) INFORNATION FOR SEQ ID NO 1 (i) ~QU~N~ CHARACTERISTICS
(A) LENGTH 1713 base pairs (B) TYPE nucleic acid (C) STRAN~N~SS single (D) TOPOLOGY linear (ii) MOLECULE TYPE cDNA
(iii) HYPOTHETICAL NO
(iv) ANTI-SENSE NO

(xi) SEQUENCE DESCRIPTION SEQ ID NO 1 ATGACACTTC l~lG~~ AGTGAGTCTC TACTTTTATG GAATCCTGCA AAGTGATGCC 60 llCCGGCCCA ~l~-lC-lCAA TGACACTGGC AACTATACCT GCATGTTAAG GAACACTACA 360 TATTGCAGCA AAGTTGCATT ~lCC~ GGAA ~ll~llCAAA AAGACAGCTG TTTCAATTCC 420 W 096/23067 CA 022l0724 l997-07-l7 PCT/~

CCAAATGTAG ATGGATATTT lC~llCCAGT GTCAAACCGA CTATCACTTG GTATATGGGC 540 TGTTATAAAA TACAGAATTT TAATAATGTA ATACCCGAAG GTATGAACTT GA~lllC~l~C 600 ATTGCCTTAA TTTCAAATAA TGGAAATTAC ACAl~l~llG TTA~ATATCC AGAAAATGGA 660 GTGCCCCCTG TGATCCATTC ACCTAATGAT CAl~lG~l~l ATGAGAAAGA ACCAGGAGAG 780 GAGCTACTCA ~ CCCl~lAC GGTCTATTTT A~llll~l~A TGGATTCTCG CAATGAGGTT 840 lG~l~GACCA TTGATGGAAA AAAACCTGAT GACATCACTA TTGATGTCAC CATTAACGAA 90O
AGTATAAGTC ATAGTAGAAC AGAAGATGAA A~AA~AACTC AGATTTTGAG CATCAAGAAA 960 GTTACCTCTG AGGATCTCAA GCGCAGCTAT ~l~l~lCATG CTAGAAGTGC CAAAGGCGAA 1020 GTTGCCAAAG CAGCCAAGGT GACGCAGAAA GTGCCAGCTC rA~ATA~Ar AGTGGAACTG 1080 G~ll~l~ll~ TTGGAGCCAC A~lC~l~lA GTGGTGATTC TCAll~ll~l TTACCATGTT 1140 TACTGGCTAG AGATGGTCCT ATTTTACCGG GCTCATTTTG ~-AA~A~.ATGA AACCATTTTA 1200 GATGGAAAAG AGTATGATAT TTATGTATCC TATGCAAGGA ATGCGGAAGA A~AA~AATTT 1260 GTTTTACTGA CCCTCCGTGG A~llllGGAG AATGAATTTG G~TA~AAGCT GTGCATCTTT 1320 GACCGAGACA ~l~lGC~lGG GGGAATTGTC ACAGATGAGA CTTTGAGCTT CATTCAGAAA 1380 AGCAGACGCC TCClG~ll~l~ TCTAAGCCCC AACTACGTGC TCCAGGGAAC CCAAGCCCTC 1440 CTGGAGCTCA AGGCTGGCCT A~AAAATATG GG~ CGGG GCAACATCAA CGTCATTTTA 1500 GTACAGTACA AAGCTGTGAA GGAAAC~-AAG GTGAAAGAGC TGAAGAGGGC TAAGACGGTG 1560 CTCACGGTCA TTAAATGGAA AGGGGAAAAA TCCAAGTATC CACAGGGCAG ~ll~lGGAAG 1620 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1713 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES

(xi) ~Q~N~ DESCRIPTION: SEQ ID NO:2:

CA 022l0724 l997-07-l7 W 096123067 P~ 100181 A~l~'ll'GCGA CGCTACTGAC CCCTGATCTG TGGTACTCCG TTTAGGTTCA CAAACTTCTA 120 ~lCG~lC~AG CGTAGTTCAC GGGTGAGAAA ~ll~l~AAGA ACTTTAAGTT GA'l'~'l'C~l'~l' 180 CGGGTAAGTC GACCGGAATG AGACTAGACC ATAACCTGAT CC~lC'~lGGC CCTGGAACTC 240 CTCGGTTAAT TGAAGGCGGA GGGGCTCTTG GCGTAATCAT 'l'C~l~lll~l ACACGACACC 300 AAGGCCGGGT r-A~A~AGTT ACTGTGACCG TTGATATGGA CGTACAATTC CTTGTGATGT 360 ATAACGTCGT TTCAACGTAA AGGGAACCTT CAACAAGTTT ll~l~lC~AC AA~AGTTAAGG 420 ~ AA~A~TTT Al~l~llAAA ATTATTACAT TATGGGCTTC CATACTTGAA CTCAAAGGAG 600 TAACGGAATT AAAGTTTATT ACCTTTAATG TGTAcAcAAc AATGTATAGG 'l~'llllACCT 660 GCATGCAAAG TAGAGTGGTC CTGAGACTGA CATTTCCATC ATCCGAGAGG TTTTTTAcGT 720 CACGGGGGAC ACTAGGTAAG TGGATTACTA GTACACCAGA TA~l~lll~l TGGTCCTCTC 780 CTCGATGAGT AAGGGACATG CrA~A~A~AA TCAAAAGACT ACCTAAGAGC GTTACTCCAA 840 ACCACCTGGT AACTACCTTT llll~ ACTA CTGTAGTGAT AACTACAGTG GTAATTGCTT 900 TCATATTCAG TATCATCTTG l~llClACTT l~ll~ll~AG TCTAAAACTC GTA~ll~lll 960 CAACG~lll'C ~lC~llCCA CTGC~l~"l"l"l' CACGGTCGAG GTTCTATGTG TCACCTTGAC 1080 ATGACCGATC TCTACCAGGA TAAAATGGCC CGAGTAAAAC ~ll~l~lACT TTGGTAAAAT 1200 CTACCTTTTC TCATACTATA AATACATAGG ATACGTTCCT TACGCCTTCT l~ll~llAAA 1260 ~lCGl~lGCGG AGGACCAACA AGATTCGGGG TTGATGCACG AGGTCCCTTG G~llCGGGAG 1440 GACCTCGAGT TCCGACCGGA l~llllATAC CC~-A~-AGCCC C~ll~lAGTT GCAGTAAAAT 1500 CATGTCATGT TTCGACACTT C'~lllG~llC CA~"l"l"l'~'l'CG ACTTCTCCCG ATTCTGCCAC 1560 5~ GTCGACGTCC ACCGGTACGG TCA~ll~lll TCAGGGTCCG CCAGATCGTC ACTACTCGTC 1680 CCGGAGAGCA TAAGTAGAAA ~lllllACAT ACT 1713 W 096/23067 CA 022l0724 l997-07-l7 PCT/~lJG

(2) INFORMATION FOR SEQ ID NO:3:
(i) ~Q~ CHARACTERISTICS:
(A) LENGTH: 570 amino acids (B) TYPE: amino acid (C) STR~NDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Met Thr Leu Leu Trp Cys Val Val Ser Leu Tyr Phe Tyr Gly Ile Leu Gln Ser Asp Ala Ser Glu Arg Cys Asp Asp Trp Gly Leu Asp Thr Met Arg Gln Ile Gln Val Phe Glu Asp Glu Pro Ala Arg Ile Lys Cys Pro Leu Phe Glu His Phe Leu Lys Phe Asn Tyr Ser Thr Ala His Ser Ala Gly Leu Thr Leu Ile Trp Tyr Trp Thr Arg Gln Asp Arg Asp Leu Glu Glu Pro Ile Asn Phe Arg Leu Pro Glu Asn Arg Ile Ser Lys Glu Lys Asp Val Leu Trp Phe Arg Pro Thr Leu Leu Asn Asp Thr Gly Asn Tyr Thr Cys Met Leu Arg Asn Thr Thr Tyr Cys Ser Lys Val Ala Phe Pro Leu Glu Val Val Gln Lys Asp Ser Cys Phe Asn Ser Pro Met Lys Leu Pro Val His Lys Leu Tyr Ile Glu Tyr Gly Ile Gln Arg Ile Thr Cys Pro Asn Val Asp Gly Tyr Phe Pro Ser Ser Val Lys Pro Thr Ile Thr Trp Tyr Met Gly Cys Tyr Lys Ile Gln Asn Phe Asn Asn Val Ile Pro Glu Gly Met Asn Leu Ser Phe Leu Ile Ala Leu Ile Ser Asn Asn Gly Asn Tyr Thr Cys Val Val Thr Tyr Pro Glu Asn Gly Arg Thr Phe His Leu Thr Arg Thr Leu Thr Val Lys Val Val Gly Ser Pro Lys Asn Ala 225 230 ' 235 240 CA 022l0724 l997-07-l7 ''t~18l W 0 96l23067 P~11~15~ _ Val Pro Pro Val Ile His Ser Pro Asn Asp His Val Val Tyr Glu Lys Glu Pro Gly Glu Glu Leu Leu Ile Pro Cys Thr Val Tyr Phe Ser Phe Leu Met Asp Ser Arg Asn Glu Val Trp Trp Thr Ile Asp Gly Lys Lys - 275 ~80 285 Pro Asp Asp Ile Thr Ile Asp Val Thr Ile Asn Glu Ser Ile Ser His Ser Arg Thr Glu Asp Glu Thr Arg Thr Gln Ile Leu Ser Ile Lys Lys Val Thr Ser Glu Asp Leu Lys Arg Ser Tyr Val Cys His Ala Arg Ser Ala Lys Gly Glu Val Ala Lys Ala Ala Lys Val Thr Gln Lys Val Pro Ala Pro Arg Tyr Thr Val Glu Leu Ala Cys Gly Phe Gly Ala Thr Val Leu Leu Val Val Ile Leu Ile Val Val Tyr His Val Tyr Trp Leu Glu Met Val Leu Phe Tyr Arg Ala His Phe Gly Thr Asp Glu Thr Ile Leu Asp Gly Lys Glu Tyr Asp Ile Tyr Val Ser Tyr Ala Arg Asn Ala Glu Glu Glu Glu Phe Val Leu Leu Thr Leu Arg Gly Val Leu Glu Asn Glu Phe Gly Tyr Lys Leu Cys Ile Phe Asp Arg Asp Ser Leu Pro Gly Gly Ile Val Thr Asp Glu Thr Leu Ser Phe Ile Gln Lys Ser Arg Arg Leu Leu Val Val Leu Ser Pro Asn Tyr Val Leu Gln Gly Thr Gln Ala Leu Leu Glu Leu Lys Ala Gly Leu Glu Asn Met Gly Ser Arg Gly Asn Ile Asn Val Ile Leu Val Gln Tyr Lys Ala Val Lys Glu Thr Lys Val Lys Glu Leu Lys Arg Ala Lys Thr Val Leu Thr Val Ile Lys Trp Lys Gly Glu Lys Ser Lys Tyr Pro Gln Gly Arg Phe Trp Lys Gln Leu Gln Val Ala Met Pro Val Lys Lys Ser Pro Arg Arg Ser Ser Ser Asp Glu Gln Gly Leu Ser Tyr Ser Ser Leu Lys Asn Val WO 96123067 CA 022l0724 l997-07-l7 PCTIEP96/0018l (2) INFORMATION FOR SEQ ID NO:4:
r:5,1u~ : CHARACTERISTICS:
(A) LENGTH: 1713 }~ase pairs (B) TYPE: nucleic acid (C) STRANnF.nNF~S: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(xi) ~ QU~ DESCRIPTION: SEQ ID NO:4:

GAGCCGGCTC GAATCAAGTG CCCC~ lll GAACACTTCC TGAAGTACAA CTACAGCACT 180 25 GCCCATTCCT ~:TGGC~:llAC CCTGATCTGG TACTGGACCA GGCAAGACCG GGACCTGGAG 240 GAGCCCATTA ACTTCCGCCT CCt'A~'A~'-AA'r CGCATCAGTA AGGAGAAAGA l~lG~ lGG 300 TTCCGGCCCA CC~ C~ AA TGACACGGGC AATTACACCT GCATGTTGAG ~'.AA~'A~'AAC'T 360 TACTGCAGCA AAGTTGCATT TCCCCTGGAA ~'l"l'~ CAGA AGGACAGCTG TTTCAATTCT 420 35 CCAAATGTAG ACGGATACTT lC~:llCCAGT GTCAAACCAT CGGTCACTTG GTATAAGGGT 540 TGTACTGAAA TAGTGGACTT TCATAATGTA CTACCCGAGG GCATGAACTT GAG~ lllllC 600 ATCCCCTTGG TTTCAAATAA CGGAAATTAC ACAl~l~lGG TTACATATCC TGAAAACGGA 660 C~l~ lllC ACCTCACCAG GACTGTGACT GTAAAGGTGG TGGGCTCACC AAAGGATGCA 720 TTGCCACCCC AGATCTATTC TCCAAATGAC C:~i'l'~i'l"l'~'l'~"l' ATGAGAAAGA ACCAGGAGAG 780 GTCACCCCGG AGGATCTCAG GCGCAACTAT ~ll:l'~lCATG CTC~AAATAr CAAAGGGGAA 1020 GCTGAGCAGG CTGCCAAGGT GAAAt'A~.'AA~ GTCATACCAC CAAGGTACAC AGTAGAACTC 1080 55 GC~:l~lGc~ll TTGGAGCCAC G~ lG GTA~lG~llC TCAll~lG~l TTACCATGTT 1140 TACTGGCTGG AGAl~ l' CTTTTACCGA GCTCACTTTG t'AA('At'ATGA AACAATTCTT 1200 GATGGAAAGG AGTATGATAT TTAl'~'l"l"lCC TATGCAAGAA ATGTGGAAGA AGAGGAATTT 1260 GTGCTGCTGA CGCTGCGTGG A~l"ll"l'~AG AATGAGTTTG ~'-ATA~-AAGCT GTGCATCTTC 1320 GACAGAGACA GC~:lGC~lGG GGGAATTGTC ACAGATGAGA CCCTGAGCTT CATTCAGAAA 1380 CA 022l0724 l997-07-l7 W 096l23067 PcT/~ 181 AGrA~AC~AC 'l'C'~"l'G~'l"l'~'l' CCTAAGTCCC AACTACGTGC TCCAGGGAAC ACAAGCCCTC 1440 CTGGAGCTCA AGC~lGGCCT AGAAAATATG GCCTCCCGGG GCAACATCAA CGTCATTTTA 1500 s CTCACGGTCA TTAAATGGAA AG~A~A~AAA TCCAAGTATC CTCAGGGCAG ~'l"l~l~GAAG 1620 G~ C~l~ ACTCATCCCT ~AAAAA-cGTA TGA 1713 (2) INFORMATION FOR SEQ ID NO 5 (i) ~u~ CHARACTERISTICS
(A) LENGTH 1713 base pairs (B) TYPE nucleic acid ( C ) STRA N ~ N ~ S: s ingle (D) TOPOLOGY linear (ii) MOLECULE TYPE cDNA
(iii) HYPOTHETICAL NO
(i~) ANTI-SENSE YES

(xi) ~QU~N~'~ DESCRIPTION SEQ ID NO 5 CTCGGCCGAG CTTAGTTCAC GGGGGAGAAA ~ GAAGG ACTTCATGTT GAl~lC~lGA 180 CGGGTAAGGA r-~CCGr-~ATG GGACTAGACC ATGACCTGGT CC~l~ l~GC CCTGGACCTC 240 CTCGGGTAAT TGAAGGCGGA GG~l~l~llA GCGTAGTCAT ~l~C~l~l"l"l~l ACACGAGACC 300 AAGGCCGGGT GGGAGGAGTT A~l~lGCCCG TTAATGTGGA CGTACAACTC ~ll~l'~llGA 360 ATGACGTCGT TTCAACGTAA AGGr,rACCTT CAACAAGTCT TC~l~lCGAC AAAGTTAAGA 420 GGTTTACATC TGCCTATGAA AGGAAGGTCA CA~lllG~lA GCCAGTGAAC CATATTCCCA 540 ACATGACTTT ATCACCTGAA AGTATTACAT GAlGGG~lCC CGTACTTGAA CTCGAAAAAG 600 TAGGGGAACC AAAGTTTATT GCCTTTAATG TGTArArACC AATGTATAGG A~llllGC~l 660 AACGGTGGGG TCTAGATAAG AGGTTTACTG GCACAACAGA TA~l~lll~l TGGTCCTCTC 780 CTTGACCAAT AAGGGACGTT T~A~-~AAAG TCAAAGTAAT ACCTGAGGGT GTTACTCCAG 840 TCACATTCAA TAAGAAGTTG CCTTCTACTT TGTTCCTGAG TCTAAAACTC GTA~ll~lll 960 W 096/23067 CA 022l0724 l997-07-l7 PCTAEP96/00181 CA~l~GGGCC TCCTAGAGTC C'GC~ll~ATA rA~A~AGTAC GAGCTTTATG GTTTCCCCTT 1020 CGA~lC~lCC GACGGTTCCA ~ CAGTATGGTG GTTCCATGTG TCATCTTGAG 1080 CGGACACCAA AAC~lCG~lG C~A~AAA~AC CATCACCAAG AGTAACACCA AATGGTACAA 1140 ATGACCGACC TCTACCAGGA GAAAATGGCT CGAGTGAAAC ~ l~lACT TTGTTAAGAA 1200 CTACCTTTCC TCATACTATA AATACAAAGG ATAC~ll~ll TACACCTTCT ~l~lC~llAAA 1260 CACGACGACT GCGACGCACC TCAAA~ACCTC TTACTCAAAC CTAl~llC~A CACGTAGAAG 1320 15 lC~l~lG~lG AGG,ACCAACA GGATTCAGGG TTGATGCACG AG~lCC~ll'G l~llCGGGAG 1440 GACCTCGAGT TCCGACCGGA ~ lATAC CGGAGGGCCC CGTTGTAGTT GCAGTAAAAT 1500 CACGTCATGT TTCGACACTT CCTGTACTTC CA~lll~lCG ACTTCGCCCG ATTCTGCCAC 1560 GAGTGCCAGT AATTTACCTT ~l~C~l~l~lll AGGTTCATAG GAGTCCCGTC CAAGACCTTC 1620 GTCAACGTCC ACCGGTACGG TCA~ll~llC TCAGGGTCCA CCAGATCGTT A~l~llC~lC 1680 25 CCAGAGAGGA TGAGTAGGGA ~lllllGCAT ACT 1713 (2) INFORMATION FOR SEQ ID NO:6:
(i) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: 570 amino acids (B) TYPE: amino acid (C) STR-A~n~n~F~s: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) ~Y~O~ CAL: NO
(iv) ANTI-SENSE: NO

(xi) ~Qu~ DESCRIPTION: SEQ ID NO:6:
Met Gly Leu Leu Trp Tyr Leu Met Ser Leu Ser Phe Tyr Gly Ile Leu Gln Ser His Ala Ser Glu Arg Cys Asp Asp Trp Gly Leu Asp Thr Met Arg Gln Ile Gln Val Phe Glu Asp Glu Pro Ala Arg Ile Lys Cys Pro 35 ~0 45 Leu Phe Glu His Phe Leu Lys Tyr Asn Tyr Ser Thr Ala His Ser Ser Gly Leu Thr Leu Ile Trp Tyr Trp Thr Arg Gln Asp Arg Asp Leu Glu Glu Pro Ile Asn Phe Arg Leu Pro Glu Asn Arg Ile Ser Lys Glu Lys W Og6123067 PCTAEP96/00181 Asp Val Leu Trp Phe Arg Pro Thr Leu Leu Asn Asp Thr Gly Asn Tyr Thr Cys Met Leu Arg Asn Thr Thr Tyr Cys Ser Lys Val Ala Phe Pro ~i 115 120 125 Leu Glu Val Val Gln Lys Asp Ser Cys Phe Asn Ser Ala Met Arg Phe Pro Val His Lys Met Tyr Ile Glu His Gly Ile His Lys Ile Thr Cys Pro Asn Val Asp Gly Tyr Phe Pro Ser Ser Val Lys Pro Ser Val Thr Trp Tyr Lys Gly Cys Thr Glu Ile Val Asp Phe His Asn Val Leu Pro Glu Gly Met Asn Leu Ser Phe Phe Ile Pro Leu Val Ser Asn Asn Gly Asn Tyr Thr Cys Val Val Thr Tyr Pro Glu Asn Gly Arg Leu Phe His Leu Thr Arg Thr Val Thr Val Lys Val Val Gly Ser Pro Lys Asp Ala Leu Pro Pro Gln Ile Tyr Ser Pro Asn Asp Arg Val Val Tyr Glu Lys Glu Pro Gly Glu Glu Leu Val Ile Pro Cys Lys Val Tyr Phe Ser Phe Ile Met Asp Ser His Asn Glu Val Trp Trp Thr Ile Asp Gly Lys Lys Pro Asp Asp Val Thr Val Asp Ile Thr Ile Asn Glu Ser Val Ser Tyr Ser Ser Thr Glu Asp Glu Thr Arg Thr Gln Ile Leu Ser Ile Lys Lys Val Thr Pro Glu Asp Leu Arg Arg Asn Tyr Val Cys His Ala Arg Asn Thr Lys Gly Glu Ala Glu Gln Ala Ala Lys Val Lys Gln Lys Val Ile Pro Pro Arg Tyr Thr Val Glu Leu Ala Cys Gly Phe Gly Ala Thr Val Phe Leu Val Val Val Leu Ile Val Val Tyr His Val Tyr Trp Leu Glu Met Val Leu Phe Tyr Arg Ala His Phe Gly Thr Asp Glu Thr Ile Leu Asp Gly Lys Glu Tyr Asp Ile Tyr Val Ser Tyr Ala Arg Asn Val Glu Glu Glu Glu Phe Val Leu Leu Thr Leu Arg Gly Val Leu Glu Asn Glu W 096l23067 CA 022l0724 l997-07-l7 PCT~EP96/00181 Phe Gly Tyr Lys Leu Cys Ile Phe Asp Arg Asp Ser Leu Pro Gly Gly Ile Val Thr Asp Glu Thr Leu Ser Phe Ile Gln Lys Ser Arg Ary Leu Leu Val Val Leu Ser Pro Asn Tyr Val Leu Gln Gly Thr Gln Ala Leu Leu Glu Leu Lys Ala Gly Leu Glu Asn Met Ala Ser Arg Gly Asn Ile Asn Val Ile Leu Val Gln Tyr Lys Ala Val Lys Asp Met Lys Val Lys Glu Leu Lys Arg Ala Lys Thr Val Leu Thr Val Ile Lys Trp Lys Gly Glu Lys Ser Lys Tyr Pro Gln Gly Arg Phe Trp Lys Gln Leu Gln Val Ala Met Pro Val Lys Lys Ser Pro Arg Trp Ser Ser Asn Asp Lys Gln Gly Leu Ser Tyr Ser Ser Leu Lys Asn Val (2) INFORMATION FOR SEQ ID NO:7:
(i) ~QU~N~ CHARACTERISTICS:
tA) LENGTH: 1077 base pairs (B) TYPE: nucleic acid (C) STRANnFnM~S: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(xi) ~Qu~ DESCRIPTION: SEQ ID NO:7:
ATGACACTTC ~ AGTGAGTCTC TACTTTTATG GAATCCTGCA AAGTGATGCC 60 T~.A~CGCT GCGATGACTG GGGACTAGAC ACCATGAGGC AAATCCAAGT GTTTGAAGAT 120 GAGCCAATTA ACTTCCGCCT CCCC~-A~-A~C CGCATTAGTA AGGAGAAAGA l~lG~l~l~GG 300 TATTGCAGCA AAGTTGCATT lCC~llGGAA ~ lCAA~A AA~A~AGCTG TTTCAATTCC 420 CA 022l0724 l997-07-l7 W 096l23067 P~ 3G~00181 TGTTATAAAA TA~A~AATTT TAATAATGTA ATACCCGAAG GTATGAACTT GAGTTTCCTC 600 AllGC~~ AA TTTCA~ATAA TGGAAATTAC ACAl~l~llG TTACATATCC AGAAAATGGA 660 GAGCTACTCA llCC~l~lAC GGTCTATTTT A~llllClGA TGGATTCTCG CAATGAGGTT 840 AGTATAAGTC ATAGTAGAAC A.~AA~ATGAA ACAAGAACTC AGATTTTGAG CAT~APA~AA~ 960 GTTACCTCTG AGGATCTCAA GCGCAGCTAT ~l~l~lCATG CTAGAAGTGC CAAAGGCGAA l020 GTTGCCAAAG ~GC~AAGGT GACGCAGAAA GTGCCAGCTC ~AA~A~CAC AGTGGAA 1077 (2) INFORMATION FOR SEQ ID NO:8:
(i) ~Q~N~ CHARACTERISTICS:
(A) LENGTH: 1077 base pairs tB) TYPE: nucleic acid (C) STRA~n~n~S: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES

3~ (xi) ~Q~N~'~ DESCRIPTION: SEQ ID NO:8:
TACTGTGAAG ACACCACACA TCACTCAGAG ATr-AAAATA~ CTTAGGACGT TTCACTACGG 60 A~l~ll~CGA CGCTACTGAC CCCTGATCTG TGGTACTCCG TTTAGGTTCA CAAACTTCTA 120 ~ lC w lCGAG CGTAGTTCAC GGGTGAGAAA CTTGTGAAGA ACTTTAAGTT GAl~lC~l~l l80 ~l~GGllAAT TGAAGGCGGA GGGG~l~llG GCGTAATCAT ~l~C~l~lll~l A~A~-A~CC 300 AAGGCCGGGT GAGAGGAGTT ACTGTGACCG TTGATATGGA CGTACAATTC ~ll~lGATGT 360 ATAACGTCGT TTCAACGTAA AGGGAACCTT CAACAAGTTT 'l"l'~'l'~'l'C~AC AAAGTTAAGG 420 GGTTTACATC TACCTATAAA AGGAAGGTCA CA~lll~G~l GATAGTGAAC CATATACCCG 540 ACAATATTTT Al~l~llAAA ATTATTACAT TATGGGCTTC CATACTTGAA CTCAAAGGAG 600 TAACGGAATT AAAGTTTATT ACCTTTAATG TGTACACAAC AATGTATAGG ~ lACCT 660 GCATGCAAAG TAGAGTGGTC CTGAGACTGA CATTTCCATC ATCCGAGAGG ~ lllACGT 720 CAC'GG~G~AC ACTAGGTAAG TGGATTACTA GTACACCAGA TA~~ W~l~C~l~lC 780 W 096/23067 CA 022l0724 l997-07-l7 PCTI~/00181 ACCACCTGGT AACTACCTTT TTTTGGACTA CTGTAGTGAT AACTACAGTG GTAAllG~ll 900 TCATATTCAG TATCATCTTG '~ lAcTT l~ll~ll~AG TCTAAAACTC GTA~ll~lll 960 s CAACGGTTTC GTCGGTTCCA ~ GC~ T CACGGTCGAG GTTCTATGTG TCACCTT 1077 (2) INFORMATION FOR SEQ ID NO:9:
Qu~ CHARACTERISTICS:
(A) LENGTH: 359 amino acids (B) TYP~: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(xi) ~u~ DESCRIPTION: SEQ ID NO:9:
Met Thr Leu Leu Trp Cys Val Val Ser Leu Tyr Phe Tyr Gly Ile Leu Gln Ser Asp Ala Ser Glu Arg Cys Asp Asp Trp Gly Leu Asp Thr Net Arg Gln Ile Gln Val Phe Glu Asp Glu Pro Ala Arg Ile Lys Cys Pro Leu Phe Glu His Phe Leu Lys Phe Asn Tyr Ser Thr Ala Hls Ser Ala Gly Leu Thr Leu Ile Trp Tyr Trp Thr Arg Gln Asp Arg Asp Leu Glu Glu Pro Ile Asn Phe Arg Leu Pro Glu Asn Arg Ile Ser Lys Glu Lys Asp Val Leu Trp Phe Arg Pro Thr Leu Leu Asn Asp Thr Gly Asn Tyr Thr Cys Met Leu Arg Asn Thr Thr Tyr Cys Ser Lys Val Ala Phe Pro Leu Glu Val Val Gln Lys Asp Ser Cys Phe Asn Ser Pro Met Lys Leu Pro Val His Lys Leu Tyr Ile Glu Tyr Gly Ile Gln Arg Ile Thr Cys Pro Asn Val Asp Gly Tyr Phe Pro Ser Ser Val Lys Pro Thr Ile Thr Trp Tyr Met Gly Cys Tyr Lys Ile Gln Asn Phe Asn Asn Val Ile Pro CA 022l0724 l997-07-l7 WO 96/23067 P~ 00181 Glu Gly Met Asn Leu Ser Phe Leu Ile Ala Leu Ile Ser Asn Asn Gly Asn Tyr Thr Cys Val Val Thr Tyr Pro Glu Asn Gly Arg Thr Phe His Leu Thr Arg Thr Leu Thr Val Lys Val Val Gly Ser Pro Lys Asn Ala Val Pro Pro Val Ile His Ser Pro Asn Asp His Val Val Tyr Glu Lys 2~5 250 255 Glu Pro Gly Glu Glu Leu Leu Ile Pro Cys Thr Val Tyr Phe Ser Phe Leu Met Asp Ser Arg Asn G1U Val Trp Trp Thr Ile Asp Gly Lys Lys Pro Asp Asp Ile Thr Ile Asp Val Thr Ile Asn Glu Ser Ile Ser His Ser Arg Thr Glu Asp Glu Thr Arg Thr Gln Ile Leu Ser Ile Lys Lys Val Thr Ser Glu Asp Leu Lys Arg Ser Tyr Val Cys His Ala Arg Ser Ala Lys Gly Glu Val Ala Lys Ala Ala Lys Val Thr Gln Lys Val Pro Ala Pro Arg Tyr Thr Val Glu (2) INFORMATION FOR SEQ ID NO:10:
(i) ~Qu~ CHARACTERISTICS:
(A) LENGTH: 43 base pairs (B) TYPE: nucleic acid (C) STR~N.~SS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAh: NO
(iv) ANTI-SENSE: NO

(xi) ~Qu N~ DESCRIPTION: SEQ ID NO:10:

; (2) INFORMATION FOR SEQ ID NO:11:
(i) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STR~N~ l)N~:~S: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

W 096/23067 CA 02210724 1997-07-17 P~li~l~C~00181 (iii) HYPOTHETICAL: NO
(iv) AWTI-SENSE: NO
s (xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
CGCGCGGGTA CCCTAGAACT CTTCAGCTTC CA~l~l~lAT CTTGGAGCTG GCA~lll~l~G 60 (2) INFORMATION FOR SEQ ID NO:12:
(i) ~Qu~N~: CHARACTERISTICS:
(A) LENGTH: l9 base pairs (B) TYPE: nucleic acid (C) sTRANn~n~s single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(xi) ~yu~ DESCRIPTION: SEQ ID NO:12:
GATccAGAAT TC~TAATAr~ l9 (2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: l9 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(xi) ~QU~N~ DESCRIPTION: SEQ ID NO:13:
GTCTTAAGTA TTATCCATG l9

Claims (35)

Claims

1. A polynucleotide which encodes an IL-1 receptor accessory protein or an active fragment thereof.

2. A polynucleotide of claim 1 comprising a DNA sequence selected from (a) a polynucleotide having essentially the sequence [SEQ ID NO:1]; or (b) a polynucleotide which hybridizes to the DNA of (a) under moderately stringent conditions; or (c) a polynucleotide which differs in codon sequence due to the degeneracy of the genetic code.

3. A polynucleotide of claim 1 or claim 2 which encodes a human IL-1 receptor accessory protein.

4. A polynucleotide of claim 3 which encodes the human IL-1 receptor protein having the amino acid sequence [SEQ ID NO:3] or an active fragment thereof.

5. A polynucleotide of claim 4 having the sequence [SEQ ID
NO:1]

6. A polynucleotide of claim 1 or claim 2 which encodes a soluble IL-1 receptor accessory protein.

7. A polynucleotide of claim 6 which encodes a human soluble IL-1 receptor accessory protein.

8. A polynucleotide of claim 7 which encodes the human soluble IL-1 receptor protein having the amino acid sequence [SEQ ID
NO:9] or an active fragment thereof.

9. A polynucleotide of claim 8 having the sequence [SEQ ID
NO:7].

10. A polynucleotide of claim 1 or claim 2 which is an antisense polynucleotide.

11. A vector which comprises a polynucleotide according to any of claims 1 to 10.

12. A vector of claim 11 which is an expression vector.

13. A host cell which comprises a vector of claim 11 or claim 12.

14. The IL-1 receptor accessory protein or an active fragment thereof.

15 . A protein of claim 14 encoded by a polynucleotide as defined in claim 2.

16. A protein according to claim 14 or claim 15 which is the human IL- 1 receptor accessory protein.

17. A protein of claim 16 which has the amino acid sequence [SEQ ID NO:3].

18. A protein according to claim 14 or claim 15 which is a soluble human IL-1 receptor accessory protein.

19. A protein of claim 18 having the amino acid sequence [SEQ
ID NO:9].

20. A protein according to any of claims 14 to 19 carrying one or more side groups which have been modified.

21 . An antibody which binds specifically to the human IL- 1 receptor accessory protein and prevents activation of the IL- 1 receptor complex by IL- 1.

22. An antibody of claim 21 which is a monoclonal antibody.

23. An antibody according to claim 21 or claim 22 having a binding affinity to the IL-1 receptor accessory complex of from about KD 0.1 nM to about KD 10 nM.

24. A pharmaceutical composition which comprises a compound according to any of claims 10 and 14 to 23 and a pharmaceutically acceptable carrier.

25. A pharmaceutical composition according to claim 24 in combination with one or more other cytokine antagonists.

26. A process for the preparation of an IL- 1 receptor accessory protein comprising the steps of:
(a) expressing a polypeptide encoded by a DNA according to any of claims 1 to 10 in a suitable host, (b) isolating said IL- 1 receptor accessory protein, and (c) if desired, converting it in an analogue wherein one or more side groups are modified.

27. A process for the preparation of an IL- 1 receptor accessory protein antibody comprising the steps of:
(a) preparation of a hybridoma cell line producing a monoclonal antibody which specifically binds to the IL- 1 receptor accessory protein and (b) production and isolation of the monoclonal antibody.

28. A compound as claimed in any one of claims 14 to 23 prepared by a process as claimed in claim 26 or claim 27.

29. A compound according to any of claims 10 and 14 to 23 for use as therapeutically active substance.

30. A compound according to any of claims 10 and 14 to 23 for use in the treatment of inflammatory or immune responses and/or for regulating and preventing inflammatory or immunological activities of Interleukin-1.

31. A compound according to any of claims 10 and 14 to 23 in the treatment of acute or chronic diseases, preferably rheumatoid arthritis, inflammatory bowel disease, septic shock, transplant rejection, psoriasis, asthma and Type I diabetes or in the treatment of cancer, preferably acute and chronic myelogenous leukemia.

32. The use of a compound according to any of claims 10 and 14 to 23 for the manufacture of a medicament for the control or prevention of illness.

33. The use of a compound of claim 10 and 14 to 23 for the manufacture of a medicament for the treatment of inflammatory or immune responses and/or for regulating and preventing inflammatory or immunological activities of Interleukin-1.

34. The use of a compound of claims 10 and 14 to 23 for the manufacture of a medicament for the treatment or prophylaxis of rheumatoid arthritis, inflammatory bowel disease, septic shock, transplant rejection, psoriasis, asthma and Type I diabetes or for the treatment or prophylaxis of cancer, preferably acute and chronic myelogenous leukemia.

35. The novel compounds, compositions, processes and uses thereof substantially as described herein.