WO1996023067A1 - Proteine accessoire humaine du recepteur de l'interleukine-1 - Google Patents

Proteine accessoire humaine du recepteur de l'interleukine-1 Download PDF

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WO1996023067A1
WO1996023067A1 PCT/EP1996/000181 EP9600181W WO9623067A1 WO 1996023067 A1 WO1996023067 A1 WO 1996023067A1 EP 9600181 W EP9600181 W EP 9600181W WO 9623067 A1 WO9623067 A1 WO 9623067A1
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Prior art keywords
acp
cells
protein
human
polynucleotide
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PCT/EP1996/000181
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English (en)
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Richard Anthony Chizzonite
Grace Wong Ju
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F.Hoffmann-La Roche Ag
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Priority to BR9606837A priority Critical patent/BR9606837A/pt
Priority to EA199700265A priority patent/EA199700265A1/ru
Priority to AU45370/96A priority patent/AU4537096A/en
Priority to EP96901291A priority patent/EP0808365A1/fr
Priority to PL96321538A priority patent/PL321538A1/xx
Priority to CZ972081A priority patent/CZ208197A3/cs
Priority to MX9705501A priority patent/MX9705501A/es
Priority to JP8522598A priority patent/JPH10512453A/ja
Publication of WO1996023067A1 publication Critical patent/WO1996023067A1/fr
Priority to FI973089A priority patent/FI973089A/fi
Priority to NO973404A priority patent/NO973404D0/no

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to cyto ine receptors, and more specifically to accessory proteins of interleukin 1 receptors.
  • Interleukin 1 is a polypeptide hormone that acts on a variety of cell types and has multiple biological properties (Dinarello, Blood 77: 1627, 1991). IL-1 is a major mediator of inflammatory and immune responses. Therefore, regulation of IL-1 activity provides a means of controlling and modulating these responses.
  • IL-1 interleukin lot
  • IL-l ⁇ interleukin 16
  • the biological activities produced by IL- 1 are mediated by binding to specific plasma membrane receptors, termed the Type I and Type II IL-1 receptors.
  • the IL-1 receptors (IL-lR'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. Acad. Sci. USA 86: 8946, 1989; McMahan et al., EMBO J. 10: 2821,
  • Both IL-1 species bind to each of these receptors and compete completely with each other for binding.
  • Type I IL-I R encodes the entire functional IL-1 receptor.
  • Type II IL-IR is unlikely to function as a signal-transducing accessory protein, and that it acts instead as a decoy receptor to bind excess IL-1 and regulate its activity (Colotta et al., Science 261 : All, 1993).
  • IL-1 binding to the IL-1 receptor mediates the biological effects of IL-1
  • an understanding of the mechanism of receptor binding and activation is important for regulating IL- l'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 MacDonald, J. Exp. Med. 164: 1060, 1986; Bensiman et al., J. Immunol. 745: 1168, 1989; McMahan et al., EMBO J. 70:2821, 1991).
  • a murine monoclonal (mAb) 4C5 has been described that recognizes a 90 kDa protein on murine cells that is associated with IL- IR and is required for signal transduction and biological activity (Powers et al., AAI 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.
  • 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 huma 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-1 on the cells.
  • an aspect of the invention is the treatment of pathological conditions caused by excess activity of cells responding to IL-1 by adding an amount of soluble human IL-IR accessory protein (IL-IR AcP) sufficient to inhibit activation of cells by IL-1.
  • IL-IR AcP soluble human IL-IR accessory protein
  • This methodology can also be modified, and the soluble accessory protein can be used as a screening agent for pharmaceuticals.
  • a pharmaceutical which works as an IL-1 antagonist can do so by blocking the interaction of IL-1 with the IL-IR AcP.
  • the presence of IL-IR 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 Type II IL-1 receptor in association with the IL-IR AcP (additional proteins may also be part of the complex). Adding soluble IL-IR AcP inhibits this interaction by allowing IL-1 or the IL-1 receptor to interact with the soluble protein instead of IL-IR AcP on the cell surface, thus reducing the biological response caused by IL-1.
  • Antibodies to the IL-IR AcP of this invention similarly inhibit the biological response of cells to IL-1.
  • antibodies prevent IL-1 from interacting effectively with the IL-1 receptor.
  • blocking IL-IR AcP these antibodies inhibit the binding of IL-1 to the IL-1 receptor complex, which depends on interaction with IL-IR AcP.
  • IL-IR AcP will inhibit IL-1 interaction with the IL-1 receptor, thus preventing activation of IL-1 responsive cells and decreasing the inflammatory response.
  • the present invention provides polynucleotides which encode IL-1 receptor accessory proteins or active fragments thereof, preferably, the polynucleotides are selected from a group consisting of (a) polynucleotides, preferably cDNA clones, having essentially a nucleotide sequence derived from the coding region of a native IL-IR AcP gene, such as shown in Figure 15 [SEQ D NO.
  • Particularly preferred compounds are the polynucleotides which encode human IL-1 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].
  • soluble IL-1 receptor accessory proteins e. g. human soluble IL-1 receptor accessory proteins having for example the amino acid sequence [SEQ ED NO:9].
  • the polynucleotide [SEQ ID NO:7] codes for a human soluble IL-1 receptor accessory protein.
  • antisense polynucleotides of the above compounds are also part of this invention.
  • the present invention also provides vectors and suitable host cells, preferably expression vectors comprising the DNA sequences defined above, recombinant IL-IR AcP produced using the expression vectors, and a method for producing the recombinant accessory 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 accessory proteins, preferably a protein having the amino acid sequence [SEQ ED NO:3].
  • soluble human IL- 1 receptor accessory proteins e. g. having the amino acid sequence [SEQ ED NO:9].
  • IL-IR AcP proteins carrying one or more side groups which have been modified.
  • the present invention also provides antibodies to IL-IR 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 receptor accessory complex of from about KD 0.1 nM to about K 10 nM and are for example monoclonal antibodies or derivatives thereof.
  • 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 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 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.
  • 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 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 inflammatory or immune responses and/or for regulating and preventing inflammatory or immunological activities of Interleukin- 1.
  • these compounds are useful 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.
  • IL-1 includes both IL-l ⁇ and IL-l ⁇ , and IL-1 receptor includes Type I and Type II IL-1 receptors, unless otherwise specifically indicated.
  • FIG. 1 Equilibrium Binding of [ 1 25 I]-4C5 to Murine EL-4 Cells at Room Temperature.
  • EL-4 cells (1.5 x 10 ⁇ cells) were incubated for 2 hrs at room temperature with increasing concentrations of [ ⁇ 2 ⁇ I]-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 Example 1.
  • Specific binding of [ 125 I]-4C5 (•) was calculated by subtracting non-specific binding from total binding.
  • 1A Binding of EL-4 cells incubated with [125i]_4 5.
  • IB Analysis of the binding data according to the method of Scatchard (Scatchard, Ann. N.Y.
  • FIG. 3 Inhibition of Human [ 1 25 I]-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 Example 1. The data are expressed as the percent inhibition of [ ⁇ 2 ⁇ I]-IL-1 binding in the presence of the indicated concentrations of antibody when compared to the specific binding in the absence of antibody. Proteins are human IL-l ⁇ (H-alpha) and human IL-l ⁇ (H-beta).
  • FIG. 4 Inhibition of Human [ 1 2 ⁇ I]-IL-1 Binding to IL-1 Receptor on EL-4 Cells by Monoclonal Antibodies 4C5, 4E2 and 35F5. Inhibition assays were performed as described in Example 1. The data are expressed as the percent inhibition of [ ⁇ 2 ⁇ I]-IL-1 binding in the presence of the indicated concentrations of antibody when compared to the specific binding in the absence of antibody. Proteins are human IL-l ⁇ (H-alpha) and human IL-l ⁇ (H-beta).
  • FIG. 5 Isolation of Two Proteins of 90 and 50 kDa from a 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-IR antibody (7E6), murine IL-l ⁇ (Ma) or anti-accessory protein antibody (4C5) as described in Example 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 [ ⁇ 2 ⁇ I]-4C5. The molecular sizes indicated in the margins were estimated from molecular weight standards (Amersham Prestained Standards) run in parallel lanes. Exposure time was 1 day.
  • FIG. 6 Inhibition of IL-1 Induced Splenic B Cell Proliferation by Monoclonal Antibodies 4C5, 4E2 and 35F5. Inhibition assays were performed as described in Example 1. The data are expressed as the incorporation of ⁇ H-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-l ⁇ (IL-l ⁇ )) and 6B. human IL-l ⁇ (IL-l ⁇ ).
  • FIG. 7 Inhibition of IL-1 Induced Proliferation of D10.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 ⁇ H-thymidine (CPM) 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-l ⁇ , 7B. human IL-l ⁇ .
  • CPM ⁇ H-thymidine
  • FIG. 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-l ⁇ (alpha) or human IL-l ⁇ (beta) (0.03 ⁇ g). Two hours after the IL-1 administration, the serum IL-6 concentration was determined as described in Example 1. Mab X-7B2 is a control antibody.
  • FIG. 10 Nucleotide Sequence and Deduced Amino Acid Sequence of Murine IL-IR AcP.
  • 10 A The nucleotide sequence of the opening reading frame of murine IL-IR AcP cDNA clone E2-K is shown. The top strand is the coding sequence [SEQ ID NO:4].
  • 10B The amino acid sequence of murine IL-IR AcP as deduced from the coding sequence shown in Figure 10A is shown [SEQ ID NO:6].
  • the 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 (Example 10). The predicted transmembrane domain extends from Leu 340 through Leu 363.
  • FIG. 11 Immunoprecipitation of Recombinant MuIL-lR AcP from Transfected COS cells with mAbs 4C5 and 2E6.
  • COS cells were transfected by electroporation with either pEF-BOS/muIL-lR AcP or pEF-BOS alone (mock).
  • Transfected cells were metabolically labelled with [3 ⁇ S]Met a s described (Example 8). Labelled transfectants were solubilized with RIPA buffer and immunoprecipitated with either mAb 4C5 or 2E6 (see Table 2) as described (Example 8).
  • Cells (4-8 x 10 4 ) transfected with an IL-IR AcP expression plasmid [COS (AcP)] or control plasmid [COS(PEF-BOS)] were incubated for 3 hrs at 4°C with increasing concentrations of [ ⁇ 2 ⁇ I]-4C5 or [ ⁇ 2 ⁇ I]-IL- l ⁇ in the absence (Total) or presence (Non-Specific) of 100 nM unlabeled 4C5 or 50 nM unlabeled IL-l ⁇ .
  • Total (Total) and non-specific (Non- Specific) cell bound radioactivity were determined as described in Example 1.
  • Figure 14 Construction of Full-length cDNA Clone of Human IL-IR AcP. Schematic representations of the structures of the human IL-IR AcP cDNA inserts in clones #3 and #6 are shown in the upper 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.
  • FIG. 15 Nucleotide Sequence of Human IL-IR AcP.
  • the nucleotide sequence of the open reading frame in the full-length human IL-IR AcP cDNA (Example 13, Figure 14) is shown.
  • the top strand is the coding sequence [SEQ ED NO: l].
  • FIG. 16 Amino Acid Sequence of Human IL-IR AcP.
  • the amino acid sequence of human IL-IR 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 Nal550.
  • the predicted transmembrane domain extends from Leu340 to Leu363.
  • FIG. 17 IL-1 Induction of IL-6 Production in MRC-5 Cells: Inhibition by IL-1 Receptor Antagonist and Anti-Type I IL-1 Receptor Antibody 4C1.
  • Human embryonic lung fibroblast MRC-5 cells (5 X lC ⁇ cells; 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-1RA; 10" 2 to 10 3 pM), anti-Type I IL-1 receptor antibody 4C1 (10" 4 to 10 1 ⁇ g/ml) or nothing for 1 hr at 37° C.
  • IL-1 receptor antagonist IL-1RA
  • anti-Type I IL-1 receptor antibody 4C1 10" 4 to 10 1 ⁇ g/ml
  • TNF ⁇ was less potent ( ⁇ 500-fold) than IL-l ⁇ 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 ⁇ , TNF ⁇ , and inhibition by IL- Ira.
  • 17B shows data for inhibition by mAb 4C1.
  • FIG. 18 Nucleotide Sequence of the Soluble Human IL-IR AcP.
  • the nucleotide sequence of the soluble human IL-IR AcP cDNA is shown.
  • the top strand is the coding sequence [SEQ ED NO:7].
  • FIG. 19 Amino Acid Sequence of the Soluble Human IL-IR AcP.
  • the amino acid sequence of soluble human IL-IR AcP as deduced from translation 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-IR AcP (IL-IR AcP) or an active fragment of a IL-IR AcP (i.e. capable of inhibiting the ability of IL-1 to bind to or otherwise activate the IL-1 receptor), in particular a human or murine IL-IR AcP.
  • IL-IR AcP IL-IR AcP
  • active fragment of a IL-IR AcP i.e. capable of inhibiting the ability of IL-1 to bind to or otherwise activate the IL-1 receptor
  • human or murine IL-IR AcP examples of such a polynucleotide are the DNA polynucleotide having the sequence [SEQ ED NO: 1], and the DNA polynucleotide encoding the human IL-IR AcP which has the amino acid sequence [SEQ ED NO: 3].
  • the polynucleotides of this invention may be used as intermediates to produce the protein IL-IR AcP
  • This protein is useful in treatment of conditions related to IL-1 inflammatory 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-IR AcP) isolated free of other proteins, or an isolated active fragment of IL-IR AcP.
  • IL-IR 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-IR AcP, which method uses as intermediates the following compounds: polynucleotides encoding murine IL-lRAcP, murine IL-IR AcP, antibodies to murine IL-IR AcP, and polynucleotides encoding human IL-IR AcP. From polynucleotides encoding human IL-IR AcP, soluble human IL-IR AcP and antibodies thereof can be obtained.
  • the critical first intermediate for this invention is the isolation of mAbs for the murine IL-IR accessory protein.
  • mAbs are obtained by immunization with a partially purified preparation of solubilized crosslinked IL-l ⁇ /IL-lR complex from murine 70Z/2 pre-B cells (described in Example 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 these mAbs (4C5) was then used to isolate a cDNA encoding the murine IL-IR 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-IR AcP.
  • 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 contaminating 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.
  • sequences are preferably provided in the form of an open reading 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.
  • polynucleotides include those containing one or more of the above-identified DNA sequences and those sequences which hybridize under stringent hybridization conditions (see, T. Maniatis 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 at 4 x SSC at 65°C, followed by a washing in 0.1 x SSC at 65°C for an hour.
  • an exemplary stringent hybridization condition is in 50 % formamide, 4 x SSC at 42°C.
  • Polynucleotides which hybridize to the sequences for IL-IR AcP under moderate hybridization conditions and which code on expression for IL-IR AcP peptides having IL-IR AcP biological properties also encode novel IL-IR AcP polypeptides.
  • non-stringent hybridization conditions are 4 x SSC at 50°C or hybridization with 30 - 40 % formamide at 42°C. Additional hybridization conditions are mentioned in Example 11.
  • a DNA sequence which shares regions of significant homology e. g.
  • IL-IR AcP sequences of IL-IR AcP and encodes a protein having one or more IL-IR AcP biological properties clearly encodes a IL-IR AcP polypeptide even if such a DNA sequence would not stringently hybridize to the IL-IR AcP sequences.
  • Polynucleotides of this invention were obtained as described in Examples 7-13 by expressing murine cDNA in eucaryotic cells and 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-IR AcP probe 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 were isolated and sequenced as described to obtain full-length polynucleotides of this invention encoding human IL-IR AcP.
  • a specific polynucleotide of this invention has the sequence [SEQ ID NO: 1].
  • Another polynucleotide of this invention encodes the human IL-IR AcP having the amino acid sequence [SEQ ED NO: 3]. Any polynucleotide capable of encoding the amino acid sequence of IL-IR AcP, or specifically [SEQ ED NO: 3] is part of this invention.
  • Another polynucleotide of invention has the sequence [SEQ ED NO: A].
  • polynucleotide encoding an active fragment of IL-IR AcP.
  • Such polynucleotides are fragments of the polynucleotides provided above (fragmented by known methods such as restriction digestion or shearing) which, when expressed by conventional methods, produce proteins that block IL-1 activity in an IL-1 assay described below.
  • a polynucleotide encoding a soluble IL-IR 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- IR AcP and its active fragments are useful as intermediates from which IL-IR AcP and its active fragments are obtained.
  • these polynucleotides are useful as antisense therapeutics which block the production of IL-IR AcP.
  • Antisense therapeutics are used as described in Akhtar and Ivinson, Nature Genetics 4:215, 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.
  • any sequence from the polynucleotides of this invention which is capable of hybridizing to DNA or RNA encoding IL-IR AcP under moderately stringent conditions known in the art and which when so hybridized prevents the synthesis of IL-IR AcP is also part of this invention.
  • This invention includes vectors which contain the poly ⁇ nucleotides described herein which encode IL-IR AcP or an active fragment. Any vector known in the art may be used in this capacity, 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.
  • 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 coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination 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.
  • recombinant protein 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-IR AcP or active fragments.
  • the polynucleotides are inserted into vectors containing transcriptional regulatory sequences to form expression vectors.
  • These expression vectors are then inserted into host cells by transfection, infection, electroporation, or other well- known methods.
  • 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 appropriate and suitable expression vectors.
  • this invention is also directed to IL-1 receptor accessory protein (IL-IR AcP) isolated free of other proteins, or an active fragment of IL-IR AcP.
  • IL-IR AcP IL-1 receptor accessory protein
  • the IL-IR 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, especially the Type I IL-1 receptor. Inhibiting activation of the human IL-1 receptor is accomplished by the human IL-IR AcP or active fragments, and has various effects, in particular reducing inflammation. Thus by means of the IL-IR AcP or active fragment, it is possible to inhibit IL-1 activation of cells and thereby to reduce or alleviate the symptoms associated with inflammation.
  • Active fragments of IL-IR AcP may be obtained by conventional methods for obtaining protein fragments.
  • DNA of this invention may be fragmented by restriction digest or shearing and expressed in host cells by conventional methods to provide fragments of IL-IR AcP.
  • Fragments of the IL-IR AcP may also be obtained by proteolysis of the IL-IR AcP of this invention. Active fragments of this invention are determined by screening for activity using IL-1 assays described below.
  • Soluble IL-IR AcP is an IL-IR 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-IR AcP corresponds to all or part of the extracellular region of the IL-IR AcP. Methods for elucidating the COOH terminals and extracellular regions of proteins are well known.
  • the resulting protein preferably retains its ability to interact with IL-1 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-IR AcP are deleted or substituted to facilitate secretion of the accessory protein into the culture medium.
  • the soluble IL-IR AcP may also include part of the transmembrane region, provided that the soluble IL-IR AcP is capable of being secreted from the cell. Soluble IL-IR AcP is obtained as described in Examples 14 and 15. A specific soluble IL-IR AcP of this invention has the sequence [SEQ ED NO:9].
  • IL-IR AcP has the amino acid sequence [SEQ ED NO: 3].
  • the amino acid sequence of the IL-IR AcP as deduced from the cDNA sequence [SEQ ID NO: 1] is shown in Figure 16.
  • Any IL-IR AcP which affects IL-1 binding as described above, is included 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 sequences such as immunoglobulin sequences), for example, or proteins whose activity has otherwise been maintained or enhanced by any such modification.
  • proteins which inhibit IL-1 binding to the IL-1 receptor have essentially the sequence [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-IR AcP with all or part of its activity as described, or enhanced activity.
  • Means for 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-IR AcP isolated free of other proteins may be obtained from the polynucleotides of this invention which encode IL-IR AcP.
  • IL-IR AcP may be obtained by conventional methods of expressing a polynucleotide provided herein encoding IL-IR AcP, preferably the DNA of [SEQ ID NO: 1] or [SEQ ID NO: 7] in a host cell, and isolating the resulting protein.
  • 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 by high performance liquid chromatography, and purification with an IL-1 affinity column.
  • IL-IR AcP may be stabilized by attaching a poly alky lene glycol polymer by known methods.
  • Poly alky lene glycol includes poly- ethylene glycol, and other polyalkylene polymers which may be branched 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-IR 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.
  • soluble IL-IR AcP or antibodies to the IL-IR AcP can be combined with other cytokine antagonists such as antibodies to the IL-2 receptor, soluble TNF receptor, the IL-1 receptor antagonist, soluble IL-1 receptor and the like.
  • isolated IL-IR AcP of this invention is useful in raising antibodies to IL-IR AcP which are themselves useful in therapy. Raising such antibodies is made feasible because this invention makes available IL-IR AcP in sufficient amounts for antibody production.
  • this invention is also directed to antibodies to human IL-IR AcP.
  • Murine or rat monoclonal antibodies to human IL-IR AcP are obtained as in Example 15. These antibodies are obtained by immunization with purified or partially purified amounts of human IL-IR AcP, which is obtained after expression of the recombinant full- length or soluble human IL-IR AcP using the DNA's of this invention.
  • the human IL-IR AcP cDNA's were isolated using the murine IL-IR AcP DNA of this invention which was isolated with the unique mAb 4C5 described in Examples 2 and 3.
  • hybridoma techniques well known in the art may then be used to obtain hybridomas to generate mAbs.
  • Chimeric antibodies and humanized 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 producing heterodimeric bispecific antibodies (Kostelny et al., J. Immunol. 148: 1547, 1992).
  • Antibodies to human IL-IR AcP of this invention bind specifically to human IL-IR AcP and prevent activation of the IL-1 receptor complex by IL-1.
  • This activity may be determined by assays as described herein.
  • 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 E2 and IL-6.
  • Such assays can be carried out by conventional methods 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.
  • human fibroblasts such as the human lung fibroblast line MRC-5 (Chin et al., J. Exp. Med. 165: 70, 1987).
  • Methods for such assays using mouse cells are found in Examples 1, 2, 5, and 6.
  • 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 screen for the desired activity using the same techniques provided in the Examples.
  • a preferred antibody has a binding affinity to the IL-1 receptor accessory complex of about K 0.1 nM to about K 10 nM, as determined by conventional methods (Scatchard, Ann. N.Y. Acad. Sci. LL: 660, 1949).
  • the antibodies of this invention may be administered by known methods to relieve conditions caused by the presence of IL-1.
  • the antibodies of this invention are useful in reducing inflammation.
  • These antibodies to the IL-IR AcP can be administered, for example, for the purpose of suppressing inflammatory or immune responses in a human.
  • a variety of diseases or conditions caused by inflammatory processes e.g. rheumatoid arthritis, inflammatory bowel disease, and septic shock
  • immune reactions e.g. Type I diabetes, transplant rejection, psoriasis, and asthma
  • IL-1 Dishid arthritis
  • psoriasis e.g. Type I diabetes, transplant rejection, psoriasis, and asthma
  • Treatment with antibodies that inhibit IL-1 interaction with the IL-IR AcP may therefore be used to effectively suppress inflammatory or immune responses in the clinical treatment of acute or chronic diseases such as rheumatoid arthritis, inflammatory bowel disease, and Type I diabetes.
  • 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).
  • IL-IR AcP antibodies to murine IL-IR AcP, specifically 4C5, 2B5, 3F1, 4C4, 24C5, 4D4 (see Table 1) and 1D2, 2D6, 2E6, 1F6, 2D4, 2F6, 3F5, and 4A1 (see Table 2). These antibodies are useful to obtain human IL-IR AcP, as described.
  • antibodies may be produced naturally by appropriate cells, or may be produced by recombinant expression vectors that modify the antibody proteins, e.g. by humanizing 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-IR.
  • the dose ranges for the administration of the IL-IR AcP and fragments thereof or of antibodies to the IL-IR AcP or antisense polynucleotides may be determined by those of ordinary skill in the art without undue experimentation.
  • appropriate dosages are those which are large enough to produce the desired effect, for 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.
  • the dosage will vary with the age, condition, sex and extent of disease in the patient, counter-indications, if any, immune tolerance and other such variables, to be adjusted by the individual physician.
  • the IL-IR AcP and fragments thereof or antibodies to this protein or antisense polynucleotides can be administered parenterally by injection or by gradual perfusion over time. They can be administered intravenously, intraperitoneally, intramuscularly, or subcutaneously.
  • compositions comprising the proteins and/or antibodies of this invention in amounts effective to reduce inflammation, and a pharmaceutically acceptable carrier such as the preparations and vehicles described below.
  • a pharmaceutically acceptable carrier such as the preparations and vehicles described below.
  • Such compositions may include other active compounds if desired.
  • an effective amount is in the range of about 4 to about 32 mg/meter 2 .
  • an example of an effective amount is in the range of about 0.1 to about 15 mg/kg body weight.
  • Preparations for parenteral administration include sterile or aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters 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 fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, anti-microbials, anti-oxidants, chelating agents, inert gases and the like. See, generally, Remington's Pharmaceutical Science, 16th Ed., Mack Eds., 1980.
  • Lewis Rats (Charles River Laboratories) were immunized by the intraperitoneal (i.p) route with detergent solubilized preparations of human IL-l ⁇ (Gubler et al., J. Immunol. 136: 2492, 1986), affinity cross-linked to IL-IR from murine 70Z/3 pre-B cells (ATCC #TIB 158).
  • the rats received solubilized IL-l ⁇ / IL-IR complex (0.4 ml) that was prepared and purified from 1 x 10* 70Z 3 cells (Chizzonite et al., Proc. Natl. Acad. Sci.
  • IL-l ⁇ /IL-lR complex in preparation for splenocyte isolation: 0.1 ml (prepared and purified from 8 x 10* " cells) 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 10 1 1 70Z/3 cells) injected intravenous (i.v.) and i.p.
  • spleen cells were isolated from the rat and fused with SP2/0 cells (ATCC CRL 1581) at a ratio of 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.
  • the fused cells were plated at a density of 3 x 10 ⁇ cells/well/ml in 48 well plates in IMDM supplemented with 15% FBS, glutamine (2 mM), beta-mercaptoethanol (0.1 mM), gentamicin (50 ⁇ g/ml), HEPES (10 mM), 5% ORIGIN hybridoma cloning factor (IGEN, Inc.), 5% P388D1 supernatant (Nordon et al. J. Immunol. 759: 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-IR AcP and the Type II IL- I R in four assays: 1 ) for inhibitory antibodies: inhibition of [ ⁇ 2 ⁇ I]-IL- l ⁇ binding to 70Z/3 and EL-4 thymoma cells (described below), 2) for non-inhibitory antibodies: immunoprecipitation of solubilized complex of [ 1 25 I]-IL-l ⁇ crosslinked to Type II IL-IR, 3) for inhibitory antibodies specific for IL-IR AcP or Type II IL-IR: inhibition of [125j-j_iL_i ⁇ an( j [125 -j_jL_ j ⁇ binding to cells expressing recombinant Type I and Type II IL-IRs, and 4) to eliminate any antibodies specific for IL-1 : immunoprecipitation of [ 1 ⁇ I]-IL- l ⁇ and [ ⁇ ⁇ I]-IL-l ⁇ .
  • Hybridoma cell lines secreting antibodies specific for Type II IL-IR and the IL-IR 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 (Pharmacia).
  • Mouse EL-4.IL-2 thymoma cells (TIB 181 ) and D10.G4.1 (TIB 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 in 600 cm 2 dishes. The above cells were obtained from the American Type Culture Collection and the ATTC numbers are in parenthesis.
  • murine IL-l ⁇ , human IL-l ⁇ and human IL-l ⁇ were purified as previously described (Kilian et al., J. Immunol. 756: 1, 1986; Gubler et al., J. Immunol 136: 2492, 1986) except that murine IL-l ⁇ was prepared in 25 mM Tris-HCl, 0.4 M NaCl. Protein determinations were performed by BCA protein assay (Pierce Chemical Co., Rockford, IL).
  • Human IL-l ⁇ human IL-l ⁇ , murine IL-l ⁇ , murine IL-l ⁇ and purified IgG were labeled with ⁇ 2 ⁇ I 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.
  • 1.0 mCi Na[ ⁇ ⁇ I] (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 l 2 5j solution was transferred to a tube containing 0.05 to 0.1 ml EL-1 (5-13 ⁇ g) or IgG (100 ⁇ g) in Tris- iodination buffer and the reaction was incubated for 5-8 min at room temperature.
  • 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 P10DG desalting 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 10 8 cpm/ml with 1% BSA in Tris-iodination buffer.
  • the TCA precipitable radioactivity (10% TCA final concentration) was typically 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.
  • Binding of radiolabeled IL-1 to mouse cells grown in suspension culture was measured by a previously described method (Kilian et al., J. Immunol. 756: 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 10 ⁇ cells/ml and incubated (1.5 x 10" cells) with various concentrations of [ ⁇ ⁇ I]-IL-1 (5-1000 pM) at 4°C for 3-4 hrs.
  • binding buffer RPMI-1640, 5% FBS, 25 mM HEPES, pH 7.4
  • Cell bound radioactivity was separated from free [1 2 ⁇ I]-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- 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 707: 220, 1980) as adapted for the IBM personal computer by McPherson (McPherson, J. Pharmacol. Methods 14: 213, 1985) from Elsevier-BIOSOFT.
  • radioiodinated IL-1 proteins was performed by incubating cells and ligands in a 24 or 12 well plate 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 counted in a gamma counter. Non-specific binding was determined in the presence of 50 nM unlabeled IL-1. Analysis of the binding data was performed as described above.
  • hybridoma supernatant solutions, purified IgG, or antisera to inhibit the binding of 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 10 6 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 temperature. [ ⁇ 2 ⁇ I]-IL-1 (1 x 10 ⁇ 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.
  • 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 10 7 cells/ml) were incubated with radiolabeled IL-1 (60-300 fmoles/ml) in the presence or absence of 50 nM unlabeled IL-1 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 MgCl 2 ), resuspended at a concentration of 5 x 10 ⁇ cells/ml in PBS, pH 8.3.
  • DSS Disuccinimidyl suberate
  • BS3 bis(sulfosuccinimidyl)suberate
  • Incubation was continued for 30-60 min at 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 10° cells/ml in solubilization buffer (50 mM sodium phosphate, pH 7.5, containing either 8 mM CHAPS or 1% Triton X-100, 0.25 M NaCl, 5 mM EDTA, 40 ⁇ g/ml phenylmethylsulfonyl fluoride, and 0.05% NaN3) 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.
  • the extracts were immuno ⁇ precipitated with antibody bound to Gamma-Bind G Plus (Pharmacia).
  • the precipitated proteins were released by treatment with Laemmli sample buffer (Laemmli, Nature 227: 680, 1970), separated by SDS- PAGE and analyzed by autoradiography.
  • Preparation of the solubilized crosslinked complex of IL-l ⁇ / IL-IR that was used as the immunogen was performed as described above with minor modifications. Briefly, 70Z/3 cells (0.5-1.0 x 10 ⁇ cells/ml) were incubated with IL-l ⁇ (0.5 to 1.0 nM) for 4 hrs at 4°C in binding assay buffer. The cells were then washed with ice cold PBS, pH 8.3, resuspended at a concentration of 5 x 10 ⁇ cells/ml in PBS, pH 8.3 and bis(sulfosuccinimidyl)suberate (BS3) (Pierce Chemical Co.) in dimethyl sulf oxide 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.
  • the detergent extract of 70Z/3 cells was applied to an affinity column (10 ml) of goat anti-human IL-l ⁇ immobilized on crosslinked beaded agarose (Affi-Gel 10, BioRad Laboratories).
  • the goat anti-human IL-l ⁇ affinity column was prepared according to the manufacturer's instructions at a density of 1 mg of IgG/ml of packed gel.
  • the column was washed with 10 column volumes of solubilization buffer without Chaps or Triton X-100 or until 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 ⁇ g/ml phenylmethylsulfonyl fluoride, and 0.05% NaN 3 .
  • the proteins eluted from the affinity column were concentrated 10 to 100 fold and used for immunization.
  • Murine 70Z/3 and EL-4 cells were washed 3 times with ice-cold
  • nitrocellulose membrane was blocked with BLOTTO (50% w/v nonfat dry milk in PBS + .05% Tween 20) and duplicate blots were probed with [ 1 25 I]-4C5 IgG (1 x 10 6 cpm ml in 8mM CHAPS, PBS, 0.25 M NaCl, 10% BSA and 5 mM EDTA) with and without unlabeled 4C5 IgG (67nM).
  • COS cells (4-5 x 10 ⁇ ) were transfected by electroporation with
  • 70Z/3 cells (1 x 10 5 /ml in RPMI 1640, supplemented with 10% FBS, ⁇ -mercaptoethanol and gentamicin) were incubated with and without 100 U/ml (0.19 nM) of human recombinant IL-l ⁇ or IL-l ⁇ for 24 hrs or 48 hrs.
  • the cells were preincubated for one hour before the addition of IL-1 with 30 ⁇ g/ml of the indicated antibodies in a total volume of 0.5 ml.
  • An additional 0.5 ml of medium containing the IL-1 or medium alone was added to the wells for a final concentration of 15 ⁇ g/ml (100 nM) antibodies.
  • the cells were washed once after culture and stained with either a control rat antibody conjugated with 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).
  • Splenic B cells were purified by treating splenocytes isolated from C57BL/6 mice with anti-Thyl.2 antibody and rabbit complement, followed by two sequential passages through a Sephadex G10 (Pharmacia) columns.
  • B cells (5 x 10 ⁇ cells) were treated with goat anti-mouse IgM (1 ⁇ g/ml) (ZYMED) and dibutyryl cAMP (10 -3 M) in a final volume of 200 ⁇ l of RPMI 1640 media supplemented with 10% FBS, ⁇ -mercaptoethanol and gentamicin.
  • Splenic B cells were treated with and without IL-1 (100 U/ml) and with and without antibodies 35F5, 4C5 and 4E2.
  • D10.G4.1 helper T cells were maintained as described (Kaye et al., J. Exp. Med. 158: 836, 1983; Mclntyre et al., J. Exp. Med. 775: 931, 1991) and stimulated with IL-1 as previously described (Mclntyre et al., J. Exp. Med. 775: 931, 1991).
  • Cells (1 x 10 5 in 200 ⁇ l) were incubated with 0.2 pM IL-1 in RPMI 1640 containing 5% FBS, ⁇ -mercaptoethanol (5 x 10 "5 M), gentamicin (8 ⁇ g/ml), 2 mM L-glutamine, 2.5 ⁇ g/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 ⁇ Ci tritiated thymidine and harvested 16 hrs later.
  • the rat anti-mouse IL-1 accessory protein monoclonal antibody 4C5 was prepared, characterized and generated as follows:
  • the serum samples also contained antibodies that immuno-precipitated the [ 1 2 ⁇ I]-IL-l ⁇ /IL-lR complex solubilized from 70Z/3 cells, indicating the presence of non-blocking anti-Type II IL-IR antibodies.
  • [ 125 I]-IL- ⁇ was used for the IL-IR binding and immunoprecipitation assays to eliminate identification of antibodies specific for IL-l ⁇ instead of the Type II receptor.
  • Hybridomas resulting from the fusion of splenocytes isolated 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 eliminated from further analysis because they are antibodies to Type II IL-IR, and antibodies that blocked binding only to EL-4 cells were identified and eliminated from further analysis because they are antibodies to Type I IL-IR.
  • Antibodies that blocked IL-1 binding to both cell types are specific for the IL-IR AcP.
  • the initial fusion was also screened for non-blocking antibodies that were specific for either the IL-IR AcP or the Type II IL-IR.
  • These antibodies also immunoprecipitated the IL-l ⁇ /IL-lR complexes solubilized from two other Type II IL-IR bearing murine cell lines, AMJ2C11 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-IR AcP.
  • One antibody, 1F6, did not bind to the IL-l ⁇ /IL-lR complex solubilized from EL-4
  • rHuIL-l ⁇ human recombinant IL-l ⁇ .
  • rHuIL-l ⁇ human recombinant IL-l ⁇ .
  • rMuIL-l ⁇ murine recombinant IL- l ⁇ .
  • IL- IR AcP putative blocking IL-IR AcP
  • 4E2 a blocking Type II IL-IR antibody
  • a previously identified and characterized anti-Type I IL-IR antibody, 35F5 was also included in this study (Chizzonite et al., Proc. Natl. Acad. Sci. USA 86: 8029, 1989), Mclntyre et al., J. Exp. Med. 775 : 931, 1991).
  • mAb 4C5 also inhibited the binding of radiolabeled human IL-l ⁇ (Fig. 3), murine IL-l ⁇ and IL-l ⁇ to 70Z/3 cells (Table 4). Similar to its inhibition of [ 1 25 I]-human IL-l ⁇ binding to EL-4 cells, 4C5 also blocked [ 1 25 I]-murine IL-l ⁇ binding to these cells (Table 4). However, 4C5 did not block either radiolabeled human IL-l ⁇ (Fig. 4) or murine IL-l ⁇ (Table 4) binding to EL-4 cells. Moreover, 4C5 did not block the binding of [125I]-labeled IL-1 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 antibody, 4E2 inhibited both IL-l ⁇ and IL-l ⁇ binding to their respective IL-1 receptors, regardless of whether the receptors were the natural or recombinant forms (Table 4).
  • the IC50S for 4C5- mediated inhibition of IL-1 binding to EL-4 and 70Z/3 cells were at least 1000-fold lower than IC50S for inhibition of binding to cells expressing recombinant Type I or Type II receptors (Table 5).
  • the approximate molecular size of the cell surface protein recognized by mAb 4C5 on EL-4 cells was determined by affinity chromotography and immunoblotting to be approximately 90 kDa (Fig. 5).
  • Detergent extracts prepared from EL-4 cells were purified on a lentil lectin affinity matrix followed by affinity chromatography on either an anti- Type I receptor antibody (7E6), murine IL-l ⁇ (Ma) or 4C5 affinity gel.
  • the proteins eluted from each affinity column were treated with Laemmli sample buffer, separated by SDS-PAGE on 8% gels and transferred to nitrocellulose membrane.
  • the proteins immobilized on the nitrocellulose were probed with [ 1 ⁇ I]-4C5 and the immuno- reactive bands identified
  • mAb 4C5 The ability of mAb 4C5 to neutralize IL-l ⁇ biologic activity in a dose-dependent manner was demonstrated in three biologic assays: 1) IL-1 induced proliferation of murine splenic B cells, 2) IL-1 induced proliferation of D10.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-l ⁇ , but not IL-l ⁇ , induced proliferation of the splenic B cells (Fig. 6).
  • the anti-Type I receptor antibody 35F5 blocked both IL-l ⁇ and IL-l ⁇ induced proliferation of B cells.
  • the anti-Type II IL-IR antibody 4E2 did not inhibit proliferation induced by either IL-l ⁇ or IL-l ⁇ .
  • mAb 4C5 inhibited IL-l ⁇ , but not IL-l ⁇ , induced proliferation of D10.G4.1 T cells (Fig. 7).
  • Both mAb 35F5 and human IL-lra blocked IL- l ⁇ and IL-l ⁇ induced proliferation of the D10.G4.1 cells.
  • MAb 4C5 also blocked IL-l ⁇ , but not IL-l ⁇ , induced expression of kappa light chain on 70Z/3 cells (Fig. 8).
  • Antibody 35F5 blocked both IL-l ⁇ and IL-l ⁇ induced effects in this assay, whereas mAb 4E2, which recognizes the Type II IL-IR, was inactive.
  • neutralization of IL-1 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-1 added) or to a lower level depending on the potency of the antibody.
  • mice administered 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-1 binding to Type I IL-IR.
  • 4C5 blocked by approximately 90% the IL-l ⁇ , but not IL-l ⁇ , induced increase in serum IL-6 (Fig. 9).
  • the anti-Type I IL-IR antibody 35F5 blocked both IL-l ⁇ and IL-l ⁇ induced increase in serum IL-6.
  • a control mAb X-7B2 had no inhibitory effect.
  • 3T3-LI cells were harvested and total RNA was extracted using guanidinium isothiocyanate/phenol as described (P. Chomczynski and N. Sacchi, Anal. Biochem. 762:156, 1987).
  • Poly A + RNA was isolated from total RNA by one batch adsorption to oligo dT latex beads as described (K. Kuribayashi et al., Nucl. Acids Res. Symposium Series 79: 61, 1988). The mass yield of poly A + RNA from this purification was approximately 6%.
  • 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, A Laboratory Manual, second edition, J. Sambrook, E.F. Fritsch, T. Maniatis, Cold Spring Harbor Laboratory Press, 1989).
  • a cDNA library was established in the mammalian expression vector pEF-BOS (Mizushima and Nagata, Nucl. Acids Res. 75: 5322, 1990). 10 ⁇ g of poly A + RNA were reverse transcribed using RNaseH " reverse transcriptase (GIBCO BRL Life Technologies Inc., Gaithersburg, MD). The resulting mRNA-cDNA 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.
  • the cDNA was concentrated by ethanol precipitation and ligated to the cloning vector.
  • the cloning vector was the plasmid pEF-BOS that had been digested with BstXl 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 ⁇ l of ligation buffer (50 mM Tris-HCl pH 7.8/1 OmM MgCl 2 /10mM DTT/1 mM rATP/25 mg/ml bovine serum albumin) at
  • the ten separate DNA pools were then used to transfect COS-7 cells by the DEAE dextran technique (5 ⁇ g DNA/2xl0 6 cells/9 cm diameter dish) (Molecular Cloning, A Laboratory Manual, second edition, J. Sambrook, E.F. Fritsch, T. Maniatis, 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 each pool.
  • PBS phosphate buffered saline
  • 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/0.5 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 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 ⁇ g/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 bacterial plates for 2-3 hrs at room temperature with gentle rocking. Nonadherent cells were gently removed by washing with PBS. The remaining 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 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, 10 ⁇ g of oyster glycogen was added and the DNA precipitated twice by addition of 0.5 volumes of 7.5 M NH4OAC and 2.5 volumes of ethanol.
  • the pellet was washed with 70% ethanol, dried and resuspended in 1 ⁇ l of H2O.
  • Each panned pool of DNA was then electroporated into E. coli strain DH-10B. After electroporation, 5x10 ⁇ 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 completed keeping each of the ten library pools separate throughout.
  • each of the ten pools was used to transfect COS cells by the DEAE dextran method (1 ⁇ g DNA/2xl0 5 cells/well of a 6-well Costar dish).
  • the COS cells were screened for pools that expressed muIL-lR AcP by rosetting with secondary antibody coated polystyrene beads (Dynal Inc., Great Neck, NY).
  • 4C5 mAb was bound to transfected COS cells in PBS/2% FCS (2 ⁇ g Ab/well) for 1.5 hrs at room temperature with gentle rocking. Antibody was removed and cells were washed with PBS/2% FCS.
  • LB + amp 100 ⁇ l was placed in the wells of two 96- well microtiter plates. Each well was then inoculated with 4 individual colonies from panning pool #2. The bacterial cells were allowed to grow for 5-6 hrs at 37°C. Pools were then made by combining 10 ⁇ l aliquots from each well in the 8 rows and 12 columns 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 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 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 Xbal restriction enzyme, to release the cDNA insert, and fractionated on a 1.0% agarose gel.
  • the cDNA clone E2-K (pEF-BOS/muIL-lR AcP) was initially characterized by restriction enzyme mapping. Digestion of this clone with Xbal released a 3.2 kilobasepair (kb) cDNA insert. The 3.2 kb Xbal fragment was gel-purified and the DNA sequence of both strands was determined by using an ABI automated DNA sequencer along with thermostable DNA polymerase and dye-labeled dideoxy- nucleotides as terminators. The DNA sequence revealed an open reading frame (ORF) in the 5-prime half of the clone (see below). Restriction enzyme mapping using Intelligenetics computer software indicated a 1.4 kb Pstl restriction fragment within the ORF.
  • ORF open reading frame
  • This 1.4 kb fragment was gel isolated and used as a probe to identify additional muIL-lR AcP cDNA clones. Approximately 6 x 10 ⁇ additional clones from the 3T3-LI cDNA library described previously were plated as above. Colony lifts were performed (Molecular Cloning, A Laboratory Manual, second edition, J. Sambrook, E.F. Fritsch, T. Maniatis, Cold Spring Harbor Laboratory Press 1989) and the lifts were probed with the 1.4 kb Pstl restriction fragment labelled with [32pj. ( jcTP by random-priming using the Multiprime DNA labelling system (Amersham Co., Arlington Heights, IL). In this way two additional homologous cDNA clones were isolated. One contained a 1.0 kb insert and the other a 4.3 kb insert as determined by Xbal digestion. The DNA sequence of the 4.3 kb insert was determined as above to confirm the sequence of the muIL-lR AcP ORF.
  • FIG. 10A The nucleotide sequence of the open reading frame in the muIL-lR AcP cDNA insert is shown in Figure 10A.
  • This open reading frame (ORF) consists of 1710 bp which encodes a protein of 570 amino acids.
  • the amino acid sequence shown in Figure 10B [SEQ ED NO: 6], predicts a 20 amino acid NH 2 -terminal signal peptide with cleavage after Ala-1, an extracellular domain from Serl-Glu339, a hydrophobic transmembrane domain from Leu340-Leu363 and a cytoplasmic tail from Glu364 to the COOH- terminus. Seven potential N-linked glycosylation sites are all contained within the extracellular domain.
  • muIL-lR AcP has significant homology to both IL-1 Type I and IL-1 Type II receptors from mouse, human, chicken and rat. The homology to each of these proteins is approximately 25% and is uniformly distributed 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 conserved in muIL-lR AcP.
  • 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 [ ⁇ 2 ⁇ I]-4C5.
  • 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 10 ⁇ cells/well. After 48-72 hrs growth medium was removed and 1 ml of binding buffer (RPMI/5%FCS) containing 1 x 10 6 cpm of [ 1 25 I]-4C5 was added per well either alone (total binding) or in the presence of 2 ⁇ g 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 0.5% SDS.
  • binding buffer RPMI/5%FCS
  • the ly sates were harvested and bound counts were determined. Specific binding was calculated by subtracting non ⁇ specific counts from total counts. Specific counts were approximately 30,000 cpm/ well with a non-specific background of 8% indicating 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 [ ⁇ S]- methionine and immunoprecipitation of labelled muIL-lR 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 methionine-GIBCO-BRL)/10% FBS/1 mM L-glutamine/ 1 mM Na pyruvate)].
  • Fresh methionine-free medium was added and after 5-8 hrs incubation at 37° C, 50-100 ⁇ Ci of ⁇ S-methionine was added per ml of medium and incubation continued for 24 hrs. Medium was then removed and the cells washed 2 times with cold PBS. Cells were solubilized by the addition of RIPA buffer (0.5% NP-40, 0.5% Tween- 20, 0.5% Deoxycholate, 420mM NaCl, lOmM KC1, 20mM Tris pH 7.5, ImM EDTA) and incubation on ice for 15 min. The lysate was transferred to tubes and spun at 15,000 x g for 15 min.
  • RIPA buffer 0.5% NP-40, 0.5% Tween- 20, 0.5% Deoxycholate, 420mM NaCl, lOmM KC1, 20mM Tris pH 7.5, ImM EDTA
  • Lysates were precleared by the addition of 40 ⁇ l of GammaBind G Sepharose (50% v/v in RIPA buffer) (Pharmacia Biotech Inc., Piscataway, NJ) to 500 ⁇ l 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 ⁇ l of GammaBind G Sepharose was added along with 20 ⁇ g mAb 4C5 or 2E6 (Table 2) and the immunoprecipitations were incubated for 3 hrs at 4° C with rotation.
  • Sepharose- Ab complexes were spun down and washed IX with RIPA buffer, IX with 50mM HEPES pH 7.9/200mM NaCl/lmM EDTA/0.5% NP-40 and IX with 25mM Tris pH 7.5/1 OOmM NaCl/0.5% Deoxycholate/1.0% Triton X- 100/0.1% SDS. Protein was released from the beads by addition of 20 ⁇ l 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.
  • the binding characteristics of the recombinant IL-IR AcP for [ 1 25 I]-labeled IL-1, 4C5 and 4E2 were determined (Fig. 12).
  • the data showed high level expression of recombinant IL-IR AcP [Cos(4C5)] as determined by [ ⁇ ⁇ I]-4C5 binding, but no increase in [ ⁇ 2 ⁇ I]-human IL- l ⁇ binding when compared to control transfected COS cells
  • IL-IR AcP Natural Murine IL-1 Receptor Accessory Protein
  • 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 ⁇ g/ml), leupeptin (10 ⁇ g/ml), benzamidine (1 mM), aprotinin (1 ⁇ g/ml) and PMSF (0.2 mM). After centrifugation at a cell sorting at the cell sorting of the cell sorting of the cell sorting of the cell sorting of the cell sorting of the cells.
  • pepstatin 10 ⁇ g/ml
  • leupeptin 10 ⁇ g/ml
  • benzamidine (1 mM
  • aprotinin (1 ⁇ g/ml)
  • PMSF 0.2 mM
  • the amino acid composition (Hollfelder et al., J. Protein Chem. 72: 435, 1993) of the final protein preparation is shown in Table 6; it is similar to the composition predicted from the deduced protein sequence [SEQ ED 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) and stained with Coomassie blue R-250.
  • the affinity purified IL-IR 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. 775: 131, 1981).
  • the murine IL-IR AcP cDNA clone [3.2 kb Xbal fragment] and restriction fragments of the murine IL-IR AcP cDNA clone [1.4 kb Pstl fragment and 843 basepair (bp) Bam Hl/Sall fragment] were used as probes to perform low-stringency Southern blot analysis of human 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-IR 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 [ 32 P]-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 use of the [ 32 P]-843 bp BamRllSall fragment, hybridizing at 37°C overnight in hybridization buffer A, washing in 2X SSC, 0.01% SDS at 55 °C. These conditions yielded the lowest background and were used to screen a commercially available human genomic library.
  • a human lung fibroblast library in Lambda FIX #944201 (Stratagene, La Jolla, CA) was screened.
  • 4.8 x 10 ⁇ plaques were screened by standard plaque hybridization techniques (Molecular Cloning, A Laboratory Manual, second edition, J. Sambrook, E.F. Fritsch, T. Maniatis, Cold Spring 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).
  • the human IL-IR AcP genomic clones were initially characterized by restriction enzyme mapping. Bacteriophage lambda DNA was isolated from clones #1 and #7 using LambdaSorb phage adsorbent (Promega, Madison, WI). The phage DNAs were digested with Sac I to release the inserts, and the fragments were then separated by electrophoresis on 1% agarose gels. Inserts for both clones #1 and #7 were -17 kb in size. Further mapping of clones #1 and #7 was performed using Xbal and fcoRI. The digested DNAs were .
  • the membrane was hybridized with the 843 bp (B ⁇ mHIIS ⁇ ll) fragment of murine IL-IR AcP previously described.
  • the probe was labelled with [ 3 P]-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 £c ⁇ RI digest and a 2.6 kb fragment from the Xb ⁇ l digest were identified as positive for hybridization to the murine IL-IR AcP sequences.
  • the 4.5 kb fragment and the 2.6 kb fragment were isolated from 0.8% Seaplaque agarose (FMC, Rockland, 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).
  • the pBluescript II SK+/2.6 kb human genomic IL-IR AcP plasmid DNA was sequenced using an ABI automated DNA sequencer along with thermostable DNA polymerase and dye-labeled dideoxynucleotides as terminators. Preliminary 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-IR AcP.
  • the mAb 2E6 (Example 2, Table 2) was originally characterized by its reactivity with the murine IL-IR AcP. Preliminary data indicated that mAb 2E6 detects the IL-IR AcP on human cells. A number of human cell lines were screened with [ ⁇ ⁇ I]-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 human cell lines, e.g. RAJI. The YT cell line was therefore chosen as the source of RNA for cDNA library construction.
  • Eco ⁇ Xl adapters (Stratagene, La Jolla, CA) were ligated to the resulting cDNAs and molecules >1000 bp were selected by passage over a Sephacryl SF500 column as described herein (EXAMPLE 7: 3T3- LI cDNA library construction).
  • the cloning vector was Lambda ZAP II phage (Stratagene) that had been digested with EcoRl restriction enzyme and dephosphorylated (as provided by the supplier).
  • phage were titered by plating in bacterial strain XLl-Blue-MRF' (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 10 ⁇ recombinants was obtained with a non-recombinant background of ⁇ 0.1%.
  • the 2.6 kb Xbal restriction fragment which was previously described as being a specific probe for the huIL-lR AcP was used at low stringency hybridization (5X SSC, 50% formamide, 5X Denhardt's, 100 ⁇ g/ml yeast RNA, 0.1% SDS, 37°C overnight), high stringency wash conditions (0.1X SSC, 0.01 % SDS, 40°C) to screen the YT cDNA library.
  • 4.8 x 10 ⁇ plaques were screened by standard plaque hybridization techniques (Molecular Cloning, A Laboratory Manual, second edition, J. Sambrook, E.I. Fritsch, T. Maniatis, Cold Spring Harbor Laboratory Press, 1989).
  • Three hybridization positive phage clones (#3, #5, and #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.
  • the human IL-IR 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 boil method (Holmes and Quigley, Anal. Biochem. 114: 193, 1981).
  • plasmid DNA was prepared with the Qiagen plasmid kit.
  • the plasmid DNAs were digested with TfcoRI to release the inserts, and the inserts were separated by electrophoresis on 1 % agarose.
  • Clone #3 contained a 2.3 kb insert
  • clone #5 contained a 1.4 kb insert
  • clone #6 contained a 2.7 kb insert.
  • Further restriction mapping indicates a single Pvu ⁇ l site present in all three 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. Preliminary sequence analysis indicated that only clones #3 and #6 had inserts that were homologous to the murine IL-IR 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 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 huEL-lR AcP cDNA.
  • FIG. 14 Shown in Figure 14 is a schematic representation of overlapping clones #3 and #6. Clones #3 and #6 were digested with the restriction enzymes BstXl and Xbal. Fragments of approximately 846 bp and approximately 2700 bp were prepared from clone #3 and clone #6, respectively, by electrophoresis in 0.7% Seaplaque agarose (FMC, Rockland, ME) and purified with Qiaex (Qiagen, Chatsworth, CA).
  • the full-length human IL-IR AcP was prepared by subcloning into the mammalian expression vector pEF-BOS (Mizushima and Nagato, Nuc. Acids Res. 75: 5322, 1990).
  • pEF-BOS plasmid DNA was digested with Xbal, 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 846 bp and approximately 2700 bp BstXIIXbal fragments described above were ligated into the Xbal- cleaved pEF-BOS expression vector, and the ligation products were transformed into MCI 061 competent cells.
  • the transformed cells were plated onto LB agar plates containing 100 ⁇ g/ml ampicillin and grown o overnight at 37 C. The next day, 12 individual colonies were picked, inoculated into LB and ampicillin (100 ⁇ g/ml) and incubated o 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 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 sequence of both strands of both plasmids was determined as described in Example 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:l] The deduced amino acid sequence, shown in Figure 16 [SEQ ED NO:3], would encode a protein of 570 residues consisting of a 20 amino acid signal peptide (Met " 2 ⁇ -Ala " ⁇ ), a putative extracellular domain (Serl-Glu339), a hydrophobic transmembrane domain (Leu340-Leu363), and a cytoplasmic tail (Glu364-Val550). Seven potential N-linked glycosylation sites are all contained within the extracellular domain. All seven sites are conserved between murine and human IL-IR 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).
  • PCR polymerase chain reaction
  • an amplicon was produced that encoded a soluble form of the extracellular domain of huIL-lR AcP.
  • two oligonucleotide primers were synthesized on an Applied Biosystems synthesizer.
  • the forward primer contained the B am HI site and the codons for the first 11 amino acids of the signal peptide: (5')GGCC GGA TCC ATG ACA CTT CTG TGG TGT GTA GTG AGT CTC TAC (3') [SEQ ID NO: 10].
  • the Glu-Glu-Phe tripeptide tag at the 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 ⁇ -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 approximately 800 bp PCR amplicon was digested with Bam l and Kpnl.
  • the digested fragment was subjected to electrophoresis through 0.7% Seaplaque agarose and purified with Qiaex (Qiagen, Chatsworth, CA).
  • Qiaex Qiagen, Chatsworth, CA
  • the soluble human IL-IR 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 EcoRl site at position 7196 (cleavage with EcoRl and filling in of sticky ends with Klenow DNA polymerase). The DNA was then subjected to religation, then cleavage with BamHl and Aspll ' (Kpnl isoschizomer) and insertion of the following oligonucleotides which contain _5 ⁇ mHI, EcoRl, and Aspll ' recognition sequences:
  • pNRl plasmid DNA was digested with BamHl and Kpnl and purified from a 0.7% Seaplaque agarose gel with Qiaex (Qiagen, Chatsworth, CA).
  • the Bam HVKpnl approximately 800 bp huIL-lR AcP PCR amplicon fragment was ligated into the BamHUKpnl cleaved pNRl expression vector.
  • the ligation products were transformed into MCI 061 competent cells, which were then plated onto LB agar containing ampicillin (100 ⁇ g/ml) and grown overnight at 37°C. The next day, 36 independent colonies were picked and inoculated into LB and ampicillin (100 ⁇ g/ml).
  • Miniprep DNA was prepared by the rapid boil method (Holmes and Quigley, Anal. Biochem. 114: 193, 1981). The DNA was analyzed by restriction endonuclease mapping. Thirty plasmid clones were shown to contain the correct insert. Plasmid DNA was prepared from two positive clones (#1 1 , #25) by the Qiagen method (Qiagen, Chatsworth, CA). These clones were verified by sequence analysis.
  • the pNRl/soluble human IL-IR AcP DNA (clone #25) was co- transfected with linearized AcRP23.1ac Z baculovirus DNA (PharMingen, San Diego, CA) into Sf9 (Spodoptera frugiperda) cells 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 77 : 330, 1991 ).
  • PCR amplification viral DNA was extracted, incubated with Taq DNA polymerase and the appropriate pNRl forward and reverse primers (relative to the BamHll Aspll ' cloning sites), and amplified using standard PCR methods (Innis et al., PCR Protocols, Academic Press, San Diego 1990). 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.
  • human IL- IR AcP + tag (from the supernatant of Sf9 cells infected with recombinant virus) was isolated by reacting with biotinylated anti-tubulin antibody (YL1/2) (Harlan Bioproducts) immobilized on streptavidin-agarose (Pierce, Rockford, IL). Proteins were eluted from the anti-tubulin antibody matrix with 0.2M glycine pH 2.7, and the fractions neutralised 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 ⁇ nitrocellulose membrane (Schleicher & Schuell, Keene, NH).
  • the immobilized proteins were probed with the YL1/2 antibody (10 ⁇ g/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 >200 kDa, that was expressed by recombinant virus containing the human IL-IR AcP + tag insert.
  • Recombinant virus from plaques #2 and #12 (identified by immunoblot analysis as expressing human IL-IR AcP + tag )were amplified to obtain virus stocks which were used in the large-scale production of human IL-IR AcP + tag for immunization purposes.
  • Sf9 cells were cultured in logarithmic growth (1 x 10 ⁇ 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 et 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-IR AcP + tag was purified over an affinity matrix composed of immobilized YL1/2 antibody as described in Example 15 below.
  • the purified human IL-IR AcP + tag was used to immunize mice.
  • COS cells (4 X 10 ' ) are transfected by electroporation with the full-length huIL-lR AcP expression plasmid (20 ⁇ g, described in Example 13) in a BioRad Gene Pulser at 250 ⁇ F 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 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 immunizations.
  • mice and rats are immunized by the intraperitoneal (i.p.) route with COS cells expressing huIL-lR AcP (1 X 10 7 cells/animal) on Days 0, 7, 14 and 28.
  • the animals are bled to determine the titer of the antibody response against huIL-lR AcP (see below for specific assays).
  • Animals are given booster immunizations (1 X 10 ⁇ cells, i.p.) at 2-4 week intervals after day 40.
  • Serum antibody titers specific for huIL-lR AcP are determined at 10-12 days after each booster immunization.
  • the animals develop a sufficient serum antibody titer (e.g., 1/1000 dilution of the serum immunoprecipitates at least 50% of a given amount of the complex of [l 2 5l]-IL- l ⁇ crosslinked to IL-IR AcP solubilized from human YT and RAJI cells), they are given booster immunizations in preparation to isolating their spleen cells. These final booster immunizations are composed of 1 X 10 ⁇ cells given both i.v.and i.p. on two consecutive days. Three days after the last immunization, spleen cells are isolated from the animal 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 in Example 1.
  • a sufficient serum antibody titer e.g., 1/1000 dilution of the serum immunoprecipitates at least 50% of
  • a Preparation of human recombinant soluble IL-IR AcP in COS cell and baculovirus expression systems.
  • 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. Chem. 266: 14163, 1991) inserted at the C-terminus (soluble IL-IR AcP, amino acids 1-339 + Ala + Glu + Glu + Phe).
  • the tag encodes the sequence for recognition by the anti-tubulin antibody YL1/2 (Harlan Bioproducts).
  • the medium is harvested from the cells 72 hrs after transfection and soluble IL-IR AcP+tag is detected and purified as described below.
  • Standard methods (Gruenwald and Heitz, Baculovirus Expression Vector System: Procedures and Methods Manual, Second Edition, 1993, PharMingen, San Diego, CA) are employed to generate a pure recombinant baculovirus expressing the soluble IL-IR AcP protein.
  • plasmid DNA coding for the soluble extracellular domain of human IL-IR AcP+tag is inserted into the transfer vector pNRl as described in Example 14.
  • the recombinant transfer vector is purified and co-transfected with linearized ACVWl.lacZ DNA (PharMingen) into Sf9 (Spodoptera frugiperda) cells.
  • Recombinant baculovirus are isolated and plaque-purified.
  • SF-9 cells (2 X 10 ⁇ cells/ml) are cultured to logarithmic growth phase in TMH-FH medium (PharMingen) at 27°C , infected with recombinant baculovirus, and spent culture media harvested after 3-5 days. The cells are removed from the spent culture media by centrifugation and the soluble IL-IR AcP+tag protein is detected and purified as described below.
  • YL1/2 antibody to an affinity matrix including covalent crosslinking to either an activated agarose gel such as Affi-Gel 10 (BioRad Laboratories) or to an agarose gel containing immobilized Protein G (Stern and Podlaski, in: Techniques in Protein Chemistry IV, R.H. Angelletti, ed., pp. 353-360, Academic Press, NY, 1993).
  • an activated agarose gel such as Affi-Gel 10 (BioRad Laboratories) or to an agarose gel containing immobilized Protein G (Stern and Podlaski, in: Techniques in Protein Chemistry IV, R.H. Angelletti, ed., pp. 353-360, Academic Press, NY, 1993).
  • 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 8.5 followed by reaction with NHS-LC-biotin at a molar ratio of 40:1 (LC-biotin:YLl/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 for 15-30 min using a Centricon-30 microconcentrator (Amicon). After centrifugation, the biotinylated YL1/2 antibody is diluted with 0.1 M sodium phosphate, pH 7.0 and the process repeated two more times.
  • Biotinylated- YL1/2 antibody (6 mg in 0.1 M sodium phosphate, pH 7.0) is reacted with streptavidin-agarose (6 ml of a 50% suspension) for 2 hrs at room temperature.
  • streptavidin agarose with the immobilized biotinylated YL1/2 antibody is placed into a column and washed with 10 column volumes of PBS, pH 7.4.
  • 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-IR AcP + tag) is characterized by reducing and non-reducing SDS-PAGE on 12% acrylamide slab gels followed by silver staining to visualize the protein bands.
  • the soluble IL-IR 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 (0.04 ml) and purified soluble IL-IR 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 Example 1.
  • the proteins immobilized on the nitrocellulose are probed with YL1/2 antibody (5 ⁇ g/ml) and peroxidase-conjugated goat anti-murine or -rat IgG antibody (1/1000 dilution) (Boehringer
  • the immunoreactive bands are identified by ECL technique (Amersham Inc.) according to the manufacturer's protocol.
  • the soluble IL-IR AcPs that are purified from COS cell and baculovirus expression systems should migrate as proteins of approximately 65-67 kDa and 45-47 kDa, respectively.
  • mice and rats are immunized by the i.p. and foot pad routes on days 0, 14 and 28 with 10-100 ⁇ g of soluble IL-IR AcP + tag.
  • the protein is prepared as described in Examples 1 and 2 in Freund's complete adjuvant for the primary immunization and in Freund's incomplete adjuvant for the day 14 and 28 booster immunizations. Serum is collected from the animals on day 40 and tested for antibody reactivity (see assays below).
  • the animals are given booster immunizations (i.p., 10-25 ⁇ g of protein prepared in Freund's incomplete adjuvant) at 4 week intervals and the titer of serum antibodies determined two weeks after each immunization.
  • booster immunizations i.p., 10-25 ⁇ g of protein prepared in Freund's incomplete adjuvant
  • they develop a potent serum antibody titer (e.g., 1/10 ⁇ dilution of the serum gives a 50% response in the EIA)
  • booster immunizations i.v. and i.p.
  • 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 Example 1.
  • Assays to detect antibodies specific for human IL-IR AcP The presence of anti-IL-lR AcP antibodies in the serum is initially determined by enzyme immunoassay (EIA) with soluble IL-IR AcP + tag immobilized on a 96 well plate. Briefly, soluble IL-IR AcP + tag (1 ⁇ g/ml) is diluted with 50 mM sodium carbonate buffer, pH 9.0, 0.15 M NaCl (BC saline) and passively adsorbed (100 ⁇ l, 100 ng) to the wells of a Nunc Maxisorb plate for 16 hrs at room temperature.
  • EIA enzyme immunoassay
  • 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 1/10 6 in 50 mM sodium phosphate, pH 7.5, 0.5 M NaCL, 0.1% Tween-20, 1% BSA and 0.05% NaN 3 (antibody binding buffer)] of the serum samples are incubated with the immobilized soluble IL-IR AcP for 2 hrs at room temperature.
  • the bound antibody is detected with peroxidase-conjugated goat anti-murine or -rat IgG antibody (Boerhringer-Mannheim Inc.) and visualized with TMB (tetramethylbenzidine) substrate.
  • TMB tetramethylbenzidine
  • the serum antibodies are also tested for reactivity by FACS (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 10 6 ) are incubated with serum dilutions (1/100 to 1/10 4 ) in PBS, pH 7.4 (100 ⁇ l) for 1 hr at 4°C. After washing the cells with PBS, pH 7.4, to remove 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-IR AcP antibodies 4C5 and 2E6 demonstrated inhibitory and non-inhibitory activity, respectively, against IL-IR AcP expressed on murine cells.
  • two types of assays are performed: 1) inhibition of t ⁇ 2 ⁇ I]-IL-l ⁇ binding to human cells and 2) immunoprecipitation of the solubilized complex of [ ⁇ ⁇ I]-IL-l ⁇ crosslinked to cell surface proteins from human cells.
  • serial dilutions of the sera are incubated with YT, NC-37 and RAJI cells (1-2 x 10") in binding buffer for 1 hr at room temperature.
  • [ 1 25 I]-IL- l ⁇ (25-250 pM) is added to each tube, incubated for 3 hrs at 4°C and cell bound radioactivity determined as previously described in Example 1.
  • the titer of inhibitory antibodies is determined by the serum dilution that results in a 50% decrease in cell-bound radioactivity.
  • dilutions of serum are incubated for 16 hr at 4°C with the solubilized complexes of [_.
  • the reaction mix is applied to a prepacked BioGel P10 column (10 ml) (BioRad Laboratories) and chromatographed with PBS, pH 7.4.
  • the fractions containing 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 immunizations. Mice and rats are immunized by the i.p. and foot pad routes on day 0, 7, 14 and 28 with 200-500 ⁇ g of KLH-peptide conjugate.
  • the conjugate is prepared in Freund's complete adjuvant for the primary immunization and Freund's incomplete adjuvant for the booster immunizations.
  • Sera are collected from the animals on day 40 and tested for antibody reactivity in the soluble IL- IR AcP EIA.
  • the animals are given booster immunizations (i.p., 100 ⁇ g 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 immunization.
  • booster immunizations i.p., 100 ⁇ g of KLH-peptide conjugate prepared in Freund's incomplete adjuvant
  • the titer of serum antibodies determined two weeks after each immunization.
  • 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 cluster dishes and pretreated for 1 hr with either increasing concentrations of anti-human IL-IR AcP or active fragment of IL-IR AcP. Following the pretreatment, the cells are stimulated with either 5 pM human IL-l ⁇ 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 IL-6 EIA (Quantikine Assay for Human IL-6, R & D Systems,
  • the inhibitory effects of the antibodies and active fragments of IL-IR AcP are calculated by determining the decrease in IL-6 secretion in the presence and absence of inhibitors. For example, 5 pM and 100 pM IL-l ⁇ stimulated the secretion of approximately 8100 and 9800 pg/ml of IL-6, respectively, from MRC-5 cells (Fig. 17). IL-1 receptor antagonist (IL-1RA) and anti-human Type I IL-IR antibody 4C1 blocked this IL-6 secretion in response to IL-l ⁇ (Fig. 17). For IL-1RA and 4C1, the ICSQ'S for blocking 5 pM IL-l ⁇ were 200 pM and 0.025 ⁇ g/ml, respectively (Fig. 17).
  • IL-1RA and 4C1 can be overridden by increasing the concentration of IL-l ⁇ to 100 pM. With 100 pM IL-l ⁇ , the ICso's for IL-1RA and 4C1 inhibition were >1 nM and 10 ⁇ g/ml, respectively.
  • IL-1 -induced IL-6 response from the MRC-5 cells was specific for IL-1 and a Type I IL-lR-dependent response, in the same way that IL-1 -dependent responses in murine cells are also Type I receptor-dependent (Figs. 6, 7 and 8).
  • IL-1 biologic assays with murine cells led to the identification of neutalizing anti-murine IL-IR AcP antibodies. Similarily, the IL-1 biologic assay with MRC-5 cells can be used to identify neutralizing anti-human IL-IR AcP antibodies and active fragments of IL-IR AcP.
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • GAGCCAGCTC GCATCAAGTG CCCACTCTTT GAACACTTCT TGAAATTCAA CTACAGCACA 180 GCCCATTCAG CTGGCCTTAC TCTGATCTGG TATTGGACTA GGCAGGACCG GGACCTTGAG 240
  • GAGCTACTCA TTCCCTGTAC
  • GGTCTATTTT AGTTTTCTGA TGGATTCTCG
  • CTCACGGTCA TTAAATGGAA AGGGGAAAAA TCCAAGTATC CACAGGGCAG GTTCTGGAAG 1620
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • CAATGGAGAC TCCTAGAGTT CGCGTCGATA CAGACAGTAC GATCTTCACG GTTTCCGCTT 1020
  • CAACGGTTTC GTCGGTTCCA CTGCGTCTTT CACGGTCGAG GTTCTATGTG TCACCTTGAC 1080
  • TCGTCTGCGG AGGACCAACA AGATTCGGGG TTGATGCACG AGGTCCCTTG GGTTCGGGAG 1440
  • GAGTGCCAGT AATTTACCTT TCCCCTTTTT AGGTTCATAG GTGTCCCGTC CAAGACCTTC 1620 GTCGACGTCC ACCGGTACGG TCACTTCTTT TCAGGGTCCG CCAGATCGTC ACTACTCGTC 1680
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • Trp Tyr Met Gly Cys Tyr Lys He Gin Asn Phe Asn Asn Val He Pro 180 185 190 Glu Gly Met Asn Leu Ser Phe Leu He Ala Leu He Ser Asn Asn Gly 195 200 205
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • GAGCCGGCTC GAATCAAGTG CCCCCTCTTT GAACACTTCC TGAAGTACAA CTACAGCACT 180 GCCCATTCCT CTGGCCTTAC CCTGATCTGG TACTGGACCA GGCAAGACCG GGACCTGGAG 240
  • GAGCCCATTA ACTTCCGCCT CCCAGAGAAT CGCATCAGTA AGGAGAAAGA TGTGCTCTGG 300
  • CTACCTTTCC TCATACTATA AATACAAAGG ATACGTTCTT TACACCTTCT TCTCCTTAAA 1260
  • CTGTCTCTGT CGGACGGACC CCCTTAACAG TGTCTACTCT GGGACTCGAA GTAAGTCTTT 1380 TCGTCTGCTG AGGACCAACA GGATTCAGGG TTGATGCACG AGGTCCCTTG TGTTCGGGAG 1440
  • GAGTGCCAGT AATTTACCTT TCCTCTCTTT AGGTTCATAG GAGTCCCGTC CAAGACCTTC 1620
  • GAGCTACTCA TTCCCTGTAC
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

Abstract

L'invention porte sur des polynucléotides codant la protéine accessoire humaine du récepteur de l'IL-1, sur ladite protéine isolée, et sur des anticorps contre ladite protéine accessoire du récepteur d'IL-1. Ladite protéine s'avère particulièrement utile pour prévenir les inflammations dues à l'action de l'IL-1.
PCT/EP1996/000181 1995-01-23 1996-01-17 Proteine accessoire humaine du recepteur de l'interleukine-1 WO1996023067A1 (fr)

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BR9606837A BR9606837A (pt) 1995-01-23 1996-01-17 Proteína acessória receptora de interleucina-1 humana
EA199700265A EA199700265A1 (ru) 1995-01-23 1996-01-17 Добавочный протеин рецептора интерлейкина-1 человека
AU45370/96A AU4537096A (en) 1995-01-23 1996-01-17 Human interleukin-1 receptor accessory protein
EP96901291A EP0808365A1 (fr) 1995-01-23 1996-01-17 Proteine accessoire humaine du recepteur de l'interleukine-1
PL96321538A PL321538A1 (en) 1995-01-23 1996-01-17 Additional protein of a human interleukin-1 receptor
CZ972081A CZ208197A3 (en) 1995-01-23 1996-01-17 Side protein of human receptor for interleukin 1
MX9705501A MX9705501A (es) 1995-01-23 1996-01-17 Proteinas accesorias de los receptores de la interleucina 1 humana.
JP8522598A JPH10512453A (ja) 1995-01-23 1996-01-17 ヒト インターロイキン−１ レセプター アクセサリータンパク質
FI973089A FI973089A (fi) 1995-01-23 1997-07-22 Ihmisen interleukiini-1-reseptorin apuproteiini
NO973404A NO973404D0 (no) 1995-01-23 1997-07-23 Humant interleukin-1-reseptor aksessorisk protein

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US37626895A 1995-01-23 1995-01-23
US08/376,268 1995-01-23

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AR (1) AR003919A1 (fr)
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BR (1) BR9606837A (fr)
CA (1) CA2210724A1 (fr)
CO (1) CO4480033A1 (fr)
CZ (1) CZ208197A3 (fr)
EA (1) EA199700265A1 (fr)
FI (1) FI973089A (fr)
HU (1) HUP9702458A2 (fr)
IL (1) IL116796A0 (fr)
MX (1) MX9705501A (fr)
NO (1) NO973404D0 (fr)
PE (1) PE64396A1 (fr)
PL (1) PL321538A1 (fr)
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ZA96333B (en) 1996-07-23
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AU4537096A (en) 1996-08-14
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NO973404L (no) 1997-07-23
FI973089A0 (fi) 1997-07-22
PL321538A1 (en) 1997-12-08
PE64396A1 (es) 1997-01-28
BR9606837A (pt) 1998-05-26
JPH10512453A (ja) 1998-12-02
FI973089A (fi) 1997-07-22
AR003919A1 (es) 1998-09-30
IL116796A0 (en) 1996-05-14
TR199700652T1 (xx) 1998-02-21
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NO973404D0 (no) 1997-07-23
EA199700265A1 (ru) 1998-04-30

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