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WO1998008969A1 - Soluble interleukin-1 receptor accessory molecule - Google Patents

Soluble interleukin-1 receptor accessory molecule Download PDF

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Publication number
WO1998008969A1
WO1998008969A1 PCT/US1996/013954 US9613954W WO9808969A1 WO 1998008969 A1 WO1998008969 A1 WO 1998008969A1 US 9613954 W US9613954 W US 9613954W WO 9808969 A1 WO9808969 A1 WO 9808969A1
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Prior art keywords
seq
acm
polypeptide
amino acid
sequence
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PCT/US1996/013954
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French (fr)
Inventor
Daniel P. Bednarik
Henrik S. Olsen
Craig A. Rosen
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Human Genome Sciences, Inc.
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Application filed by Human Genome Sciences, Inc. filed Critical Human Genome Sciences, Inc.
Priority to AU70116/96A priority Critical patent/AU7011696A/en
Priority to PCT/US1996/013954 priority patent/WO1998008969A1/en
Publication of WO1998008969A1 publication Critical patent/WO1998008969A1/en

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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a novel soluble Interleukin-1 receptor accessory molecule (IL-IR AcM).
  • IL-IR AcM is a member of the Ig superfamily by analysis of its putative extracellular domain and bears limited homology throughout the protein to both Type I and Type II IL-1 receptors. More specifically, isolated nucleic acid molecules are provided encoding a human microvascular endothelial-derived soluble IL-IR AcM. The IL-IR AcM polypeptides are also provided.
  • the present invention further relates to screening methods for putative agonists and antagonists of IL-1 signal transduction.
  • Interleukin 1 is a polypeptide cytokine with multiple diverse effects on immunological and inflammatory processes. While many of the roles of IL-1 in inflammation and the immune response have been well characterized, the molecular basis of these responses remains unclear (reviewed by Dinarello,
  • IL-1 is produced by a diversity of cell types and elicits a wide variety of physiological effects in he atopoetic and nonhematopoetic cells.
  • IL-1 has biological effects on hematopoietic cells, the digestive tract, bone, cartilage and connective tissue, vascular cells, the skin, the endocrine system, the gonads, and on neural tissue.
  • IL-1 is produced by malignant cells. (Pimentel, Handbook of Growth Factors: Volume III Hematopoietic Growth Factors and Cytokines, pp. 35-53, CRC Press, Boca Raton, FL 1994).
  • the IL-1 family of proteins comprises three members: IL-l ⁇ and IL-1 ⁇ (capable of inducing IL-1 biological responses) and IL-lra (a pure receptor antagonist). These ligands bind to two distinct and separate receptors: the Type I and Type II IL-1 receptors (IL-IRs).
  • the 80-kD Type I IL-IR is found mainly on T cells and fibroblasts (Sims, J.E., et al , Science 247:585-589 (1988); Chizzonite, R., et al , Proc Natl Acad Sci USA 86 8029-8033 (1989); Sims, J.E., etal., Proc. Natl.
  • Type I IL-1 R has a cytoplasmic tail of approximately 200 ammo acids, while the Type II
  • IL-IR cytoplasmic tail is only 29 amino acids
  • the agonists IL-l ⁇ and IL-l ⁇ bind to the extracellular domains of both receptors, although with different affinities (reviewed in Dower et al , Cellular and Molecular Mechanisms of Inflammation, pp. 137-172, Academic Press, Orlando FL )
  • the relative importance of the Type I and Type II IL-IRs in IL-1 signaling has been recently clarified.
  • a critical role for the Type I IL-IR in IL-1- induced activation of NF- ⁇ B, IL-6, and IL-8 secretion, and cell adhesion molecule expression has been demonstrated by several groups (Styhanou, E , et al , J Biol.
  • the Type II IL-IR appears to be dispensable for IL-1 signaling and may act as a decoy receptor (Stylianou, E., et al , J Biol Chem 267:15836- 15841 (1992); Colotta, F., et al , Science 261 472-475 (1993); Sims, J.E., et al , Proc Natl Acad Sci USA 90:6155-6159 (1993)) While it appears clear that the Type I IL-IR is necessary for IL-1 signal transduction, it is uncertain if it is the only cell-surface molecule involved in IL-1 signaling.
  • Type I IL-IR is a single chain receptor (Curtis, B.M., et al , Proc Natl Acad Sci USA, 56:3045-3049 (1989)).
  • affinity cross-linking of IL-1 to cells expressing natural IL-1 receptor has yielded complex patterns of cross-linked proteins (Dower, et al , Cellular and
  • IL-IR accessory proteins Only two IL-IR accessory proteins are have been identified. Studies initiated to identify components of a potential IL-1 receptor complex suggest that there is a cell-surface protein in close association with the IL-IR that may play a role in IL-1 receptor binding and signaling.
  • a murine IL-1 receptor accessory protein (mulL-lR AcP) has been cloned and expressed (Greenfeder et al. J.
  • GenBank GenBank.
  • the reported sequence for this partial cDNA is 396 bp long and represents one of 1600 cDNAs that were sequenced from a library made to contain only expressed sequence tags.
  • the regions to overlap with the mulL-lR AcP sequence is nucleotides 893-1286 of the muIL-R AcP, which include the transmembrane domain.
  • IL-IR accessory molecule has a number of implications for IL-1 receptor biology.
  • mulL-lR AcM may not bind IL-1 directly
  • the accessory molecule forms a complex with the muType I IL-IR allowing
  • IL-l ⁇ to bind with higher affinity than the muType I IL-IR alone (Greenfeder, et al. J. Biol Chem, 270: 13757-13765 (1995)).
  • the presence or absence of the accessory molecule in different cell lines determined whether the low or the higher affinity site was detected, suggesting that the low affinity site corresponds to the muType I IL-IR alone, while the higher affinity site represents a complex of the muType I IL-IR with the mulL-lR AcM (Greenfeder, et al J Biol Chem, 270: 13757-13765 (1995)).
  • the IL-I R AcM would be analogous to affinity conversion and signal transduction subunits such as gp 130 in the IL-6 system (Hibi, M., et al , Cell 63.1 149-1 157 (1990)).
  • affinity conversion and signal transduction subunits such as gp 130 in the IL-6 system (Hibi, M., et al , Cell 63.1 149-1 157 (1990)).
  • the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the soluble IL-IR AcM polypeptide having the amino acid sequence is shown in Figure 1 [SEQ ID NO:2] or the amino acid sequence encoded by the cDNA clone deposited in a bacterial host as ATCC Deposit
  • the nucleotide sequence determined by sequencing the deposited IL-IR AcM clone which is shown in Figure 1 [SEQ ID NO:l], contains an open reading frame encoding a polypeptide of 356 amino acid residues, including an initiation codon at positions 303-305, with a leader sequence of about 17 amino acid residues, and a predicted molecular weight of about 42 kDa.
  • the amino acid sequence of the mature IL-I R AcM protein is shown in Figure 1 , amino acid residues 18-356 fSEQ ID NO:2].
  • one aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of : (a) a nucleotide sequence encoding the soluble IL-
  • IR AcM polypeptide having the complete amino acid sequence in Figure 1 [SEQ ID NO:2];
  • (b) a nucleotide sequence encoding the mature soluble IL-IR AcM polypeptide having the amino acid sequence at positions 18-356 in Figure 1 [SEQ ID NO:2];
  • (c) a nucleotide sequence encoding the soluble IL-IR AcM polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97666;
  • (e) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c) or (d) above.
  • nucleic acid molecules that comprise a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical, to any of the nucleotide sequences in (a), (b), (c), (d) or (e), above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b), (c), (d) or (e), above.
  • This polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues.
  • An additional nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a IL-IR AcM polypeptide having an amino acid sequence in (a), (b), (c) or (d), above.
  • the present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of soluble IL-IR AcM polypeptides or peptides by recombinant techniques.
  • the invention further provides an isolated soluble IL-I R AcM polypeptide having amino acid sequence selected from the group consisting of: (a) the amino acid sequence of the soluble IL-IR AcM polypeptide having the complete 356 amino acid sequence, including the leader sequence shown in Figure 1 [SEQ ID NO:2]; (b) the amino acid sequence of the mature soluble IL- 1 R AcM polypeptide (without the leader) having the amino acid sequence at positions 18-356 in Figure 1 [SEQ ID NO:2]; (c) the amino acid sequence of the soluble IL-IR AcM polypeptide having the complete amino acid sequence, including the leader, encoded by the cDNA clone contained in ATCC Deposit No.
  • polypeptides of the present invention also include polypeptides having an amino acid sequence with at least 90% similarity, and more preferably at least 95% similarity to those described in (a), (b), (c) or (d) above, as well as polypeptides having an amino acid sequence at least 80% identical, more preferably at least 90% identical, and still more preferably 95%, 96%, 97%, 98% or 99% identical to those above.
  • An additional embodiment of this aspect of the invention relates to a peptide or polypeptide which has the amino acid sequence of an epitope-bearing portion of a soluble IL-IR AcM polypeptide having an amino acid sequence described in (a), (b), (c) or (d), above.
  • Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a soluble IL- 1 R AcM polypeptide of the invention include portions of such polypeptides with at least six or seven, preferably at least nine, and more preferably at least about 30 amino acids to about 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the invention described above also are included in the invention.
  • the invention provides an isolated antibody that binds specifically to a soluble IL-IR AcM polypeptide having an amino acid sequence described in (a), (b), (c) or (d) above.
  • the soluble IL-IR AcM may not bind IL-1 directly, however, the accessory molecule forms a complex with the Type 1 IL-IR that binds IL-l ⁇ with higher affinity than the Type I IL-IR alone.
  • the presence or absence of the accessory molecule in different cell lines determines whether the low or the higher affinity site is detected suggesting that the low affinity site corresponds to the Type I IL-IR alone, while the higher affinity site represents a complex of the Type I IL-IR with the IL-IR AcM.
  • the present invention further provides a screening method for identifying IL-1 receptor agonists and antagonists, which involves: (a) providing a polypeptide comprising a type I IL-1 receptor and a polypeptide comprising IL-IR AcM or IL-IR AcM fragment, wherein IL-IR and IL-IR AcM or IL-IR and the IL-IR AcM fragment form a complex; (b) providing a candidate compound; (c) providing a polypeptide conprising IL-1 or a functional II- 1 fragment; and (d) determining the binding affinity of said complex for IL-1 whereby an increased binding affinity of said complex for IL-1 in the presence of said compound is indicative that said compound is an agonist for IL-1 signal transduction and a decreased binding affinity of said complex for IL-1 in the presence of said compound is indicative that said compound is an antagonist of IL-1 signal transduction.
  • Figure 1 shows the nucleotide [SEQ ID NO: 1] and deduced amino acid [SEQ ID NO:2] sequences of soluble IL-IR AcM.
  • the protein has a leader sequence of about 17 amino acid residues (underlined) and a deduced molecular weight of about 42 kDa.
  • the predicted amino acid sequence of the mature soluble IL-IR AcM protein is also shown in Figure 1 [SEQ ID NO:2].
  • Figures 2A and B shows the regions of similarity between the amino acid sequences of the soluble IL-IR AcM protein (HMEEJ22) and mouse interleukin 1 receptor accessory protein [SEQ ID NO:3] and between the amino acid sequences of the soluble IL-IR AcM protein (HMEEJ22) and the partial cDNA isolated from human infant brain (Adams, M.D.. et al.. Nature Genet. 4:373-380 (1993)) [SEQ ID NO:4].
  • Figure 3 shows an analysis of the IL-I R AcM amino acid sequence.
  • Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.
  • the amino acid sequence of the IL-IR AcM protein is shown with the amino aicds that border each peak from the antigenic index plot displayed as underlined characters.
  • the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding soluble IL-IR AcM polypeptide, having the amino acid sequence shown in Figure 1 [SEQ ID NO:2], which was determined by sequencing a cloned cDNA.
  • the soluble IL-IR AcM protein of the present invention shares sequence homology with mouse interleukin 1 receptor accessory protein (Figure 2) [SEQ ID NO:3].
  • Figure 1 The nucleotide sequence shown in Figure 1 [SEQ ID NO: 1 ] was obtained by sequencing the HMEEJ22 clone, which was deposited on July 25, 1996 at the American Type Culture Collection, 12301 Park Lawn Drive, Rockville, Maryland 20852, and given accession number 97666.
  • isolated nucleic acid molecules which encode the soluble IL-IR AcM protein.
  • the IL-IR AcM is a novel member of the Ig superfamily.
  • nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art.
  • a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
  • nucleotide sequence set forth herein is presented as a sequence of deoxyribonucleotides (abbreviated A, G , C and T).
  • nucleic acid molecule or polynucleotide a sequence of deoxyribonucleotides
  • RNA molecule or polynucleotide the corresponding sequence of ribonucleotides (A, G, C and U), where each thymidine deoxyribonucleotide (T) in the specified deoxyribonucleotide sequence is replaced by the ribonucleotide uridine (U).
  • RNA molecule having the sequence of SEQ ID NO:l set forth using deoxyribonucleotide abbreviations is intended to indicate an RNA molecule having a sequence in which each deoxyribonucleotide A, G or C of SEQ ID NO: 1 has been replaced by the corresponding ribonucleotide A, G or C, and each deoxyribonucleotide T has been replaced by a ribonucleotide U.
  • nucleic acid molecule of the present invention encoding a soluble IL-IR AcM polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material.
  • nucleic acid molecule described in Figure 1 [SEQ ID NO: 1] was discovered in a cDNA library derived from human microvascular endothelial cells.
  • the determined nucleotide sequence of the soluble IL-IR AcM cDNA of Figure 1 [SEQ ID NO: l] contains an open reading frame encoding a protein of 356 amino acid residues, with an initiation codon at positions 303-306 of the nucleotide sequence in Figure 1 [SEQ ID NO:l], a predicted leader sequence of about 17 amino acid residues, and a deduced molecular weight of about 42 kDa.
  • the amino acid sequence of the predicted mature soluble IL-IR AcM is shown in Figure 1 [SEQ ID NO:2] from amino acid residue 18 to residue 356.
  • the soluble IL-IR AcM protein shown in Figure 1 [SEQ ID NO:2] is about 94% similar and 85% identical to mouse interleukin 1 accessory protein ( Figure 2A).
  • the nucleotides 1060 to 1353 of soluble IL-IR AcM protein shown in Figure 1 [SEQ ID NO:2] is about 99% similar and 98% identical to the first 294 nucleotides partial cDNA isolated from human infant brain by Adams, M.D., et al, Nature Genet. 4:373-380 (1993) [SEQ ID NO:4]( Figure 2B).
  • the partial cDNA isolated by Adams was 396 nucleotides in length.
  • the actual soluble IL-IR AcM polypeptide encoded by the deposited cDNA comprises about 356 amino acids, but may be anywhere in the range of 345-370 amino acids; and the actual leader sequence of this protein is about 17 amino acids, but may be anywhere in the range of about 10 to about 20 amino acids.
  • nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically.
  • the DNA may be double-stranded or single-stranded.
  • Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non- coding strand, also referred to as the anti-sense strand.
  • isolated nucleic acid molecule(s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment
  • recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention.
  • DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
  • Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention.
  • Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
  • Isolated nucleic acid molecules of the present invention include DNA molecules comprising an open reading frame (ORF) with an initiation codon at positions 303-306 of the nucleotide sequence shown in Figure 1 [SEQ ID NO:l]; DNA molecules comprising the coding sequence for the mature soluble IL-IR AcM protein shown in Figure 1 (last 339 amino acids) [SEQ ID NO:2]; and DNA molecules which comprise a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the soluble IL-IR AcM protein.
  • ORF open reading frame
  • SEQ ID NO:l DNA molecules comprising the coding sequence for the mature soluble IL-IR AcM protein shown in Figure 1 (last 339 amino acids) [SEQ ID NO:2]
  • the genetic code is well known in the art. Thus, it would be routine
  • the invention provides isolated nucleic acid molecules encoding the soluble IL-IR AcM polypeptide having an amino acid sequence encoded by the cDNA clone contained in the plasmid deposited as ATCC Deposit No. 97666 on July 25, 1996.
  • this nucleic acid molecule will encode the mature polypeptide encoded by the above-described deposited cDNA clone.
  • the invention further provides an isolated nucleic acid molecule having the nucleotide sequence shown in Figure 1 (SEQ ID NO: l ] or the nucleotide sequence of the soluble IL-IR AcM cDNA contained in the above-described deposited clone, or a nucleic acid molecule having a sequence complementary to one of the above sequences.
  • isolated molecules particularly DNA molecules, are useful as probes for gene mapping, by in situ hybridization with chromosomes, and for detecting expression of the soluble IL-I R AcM gene in human tissue, for instance, by Northern blot analysis.
  • the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, for instance, the cDNA clone contained in ATCC Deposit 97666.
  • stringent hybridization conditions is intended overnight incubation at 42 °C in a solution comprising: 50%> formamide, 5x SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x
  • a polynucleotide which hybridizes to a "portion" of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70 nt of the reference polynucleotide. These are useful as diagnostic probes and primers as discussed above and in more detail below.
  • polynucleotides hybridizing to a larger portion of the reference polynucleotide e.g., the deposited cDNA clone
  • a portion 50-750 nt in length, or even to the entire length of the reference polynucleotide are also useful as probes according to the present invention, as are polynucleotides corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in Figure 1 [SEQ ID NO:l].
  • such portions are useful diagnostically either as a probe according to conventional DNA hybridization techniques or as primers for amplification of a target sequence by the polymerase chain reaction (PCR), as described, for instance, in Molecular Cloning, A
  • the hybridizing polynucleotides of the present invention could be generated synthetically according to known techniques.
  • a polynucleotide which hybridizes only to a poly A sequence such as the 3' terminal poly(A) tract of the soluble IL-IR AcM cDNA shown in Figure 1 [SEQ ID NO.T]), or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide of the invention used to hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).
  • the invention further provides isolated nucleic acid molecules comprising a polynucleotide encoding an epitope-bearing portion of the soluble IL- 1 R AcM protein.
  • isolated nucleic acid molecules are provided encoding polypeptides comprising the following amino acid residues in Figure 1 (SEQ ID NO:2), which the present inventors have determined are antigenic regions of the soluble IL-IR AcM protein: a. S18-M32: SDASERCDDWGLDTM (SEQ ID NO:5) b. V37-I45: VFEDEPARI (SEQ ID NO:6) c. G65-Y71 : GLTLIWY (SEQ ID NO: 7) d.
  • R74-I83 RQDRDLEEPI (SEQ ID NO: 8) e. L87-K96: LPENRISKEK (SEQ ID NO:9) f. S123-L129: SKVAFPL (SEQ ID NOTO) g. V132-P141 : VQKDSCFNSP (SEQ ID NO:l 1 ) h. V146-E152: VHKLYIE (SEQ ID NO: 12) i. I175-N189: ITWYMGCYKIQNFNN (SEQ ID NO: 13) j. LI 97-1204: LSFLIALI (SEQ ID NO: 14) k. H224-K232: HLTRTLTVK (SEQ ID NO: 15)
  • P248-E261 PNDHVVYEKEPGEE (SEQ ID NO: 16) m. I264-F272: IPCTVYFSF (SEQ ID NO: 17) n. D285-T293: DGKKPDDIT (SEQ ID NO: 18) o. I302-R312: ISHSRTEDETR (SEQ ID NO: 19) p. K320-K327: KVTSEDLK (SEQ ID NO: 20) q. S336-V347: SAKEGEVAKAAKV (SEQ ID NO: 21) r. 350-Q356: KGNRCGQ (SEQ ID NO: 22) Methods for generating such epitope-bearing portions of soluble IL-IR AcM are described in detail below.
  • nucleic acid molecules of the present invention which encode a soluble IL-IR AcM polypeptide may include, but are not limited to those encoding the amino acid sequence of the mature polypeptide, by itself; the coding sequence for the mature polypeptide and additional sequences, such as those encoding the about 17 amino acid leader or secretory sequence, such as a pre-, or pro- or prepro- protein sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3 1 sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals, for example - ribosome binding and stability of mRNA; an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.
  • the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the fused polypeptide.
  • the marker amino acid sequence is a hexa-histidine peptide.
  • the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available.
  • hexa-histidine provides for convenient purification of the fusion protein.
  • the "HA" tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al, Cell 37: 767 (1984).
  • the present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of the soluble IL-IR AcM protein.
  • Variants may occur naturally, such as a natural allelic variant.
  • allelic variant is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally occurring variants may be produced using art-known mutagenesis techniques.
  • variants include those produced by nucleotide substitutions, deletions or additions.
  • the substitutions, deletions or additions may involve one or more nucleotides.
  • the variants may be altered in coding regoins, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the soluble IL-IR AcM protein or portions thereof. Also especially preferred in this regard are conservative substitutions.
  • nucleic acid molecules encoding the mature protein having the amino acid sequence shown in Figure 1 [SEQ ID NO:2] or the mature soluble IL-IR AcM amino acid sequence encoded by the deposited cDNA clone are highly preferred.
  • Further embodiments of the invention include isolated nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical to (a) a nucleotide sequence encoding the full-length soluble IL-IR AcM polypeptide having the complete amino acid sequence in Figure 1 [SEQ ID NO:2], including the predicted leader sequence; (b) a nucleotide sequence encoding the mature soluble IL-IR AcM polypeptide (full-length polypeptide with the leader removed) having the amino acid sequence at positions 18-356 in Figure 1 [SEQ ID NO:2]; (c) a nucleotide sequence encoding the full-length
  • nucleotide sequence encoding the mature soluble IL-IR AcM polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97666; or (e) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c) or (d).
  • a polynucleotide having a nucleotide sequence at least, for example,
  • nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the soluble IL-IR AcM polypeptide.
  • a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • nucleotide sequence shown in Figure 1 or to the nucleotides sequence of the deposited cDNA clone can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981), to find the best segment of homology between two sequences.
  • the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
  • the present application is directed to nucleic acid molecules at least 90%, 95%o, 96%», 97%, 98% or 99% identical to the nucleic acid sequence shown in
  • Figure 1 [SEQ ID NO:l] or to the nucleic acid sequence of the deposited cDNA, irrespective of whether they encode a polypeptide having soluble IL-IR AcM activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having soluble IL-IR AcM activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer.
  • PCR polymerase chain reaction
  • nucleic acid molecules of the present invention that do not encode a polypeptide having soluble IL-IR AcM activity include, inter alia, (1) isolating the soluble IL-IR AcM gene or allelic variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of the soluble IL-IR AcM gene, as described in
  • nucleic acid molecules having sequences at least 90%), 95%), 97%, 98% or 99% identical to the nucleic acid sequence shown in
  • Figure 1 [SEQ ID NO:l] or to the nucleic acid sequence of the deposited cDNA which do, in fact, encode a polypeptide having soluble IL-IR AcM protein activity.
  • a polypeptide having soluble IL-I R AcM activity is intended polypeptides exhibiting activity similar, but not necessarily identical, to an activity of the soluble IL-IR AcM protein of the invention (either the full-length protein or, preferably, the mature protein), as measured in a particular biological assay.
  • Assays of IL-1 R AcM protein activity are well-known to those in the art. These assays can be used to measure IL-IR AcM protein activity of partially purified or purified native or recombinant protein. For example, an equilibrium and competitive binding studies using CHO stable cell lines
  • a CHO-IR/AcM cell line is established by simultaneous cotransfection of two expression vectors such that both IL-1 R and IL-IR AcM or a candidate IL-IR AcMare expressed at about a 1 :10 ratio of molecules/cell.
  • control cell lines which express only IL-IR or IL-IR AcM are also established.
  • CHO-dhfr cells are maintained in DMEM with 10% fetal bovine serum, 25mM HEPES, pH 7.0, 0.1 mML glutamine, IX HT supplement (0.1 mM hypoxanthine, 0.016 mM thymidine) (Boehringer Mannheim), 50 ⁇ g/ml gentamicin, IX penicillin/streptomycin/fungizone (JRH Biosciences).
  • Cells are transfected with pSV2-dhfr (Subramani, et al Mol. Cell. Biol.
  • each of the above generated cell lines are analyzed by equilibrium binding with 125 I- labeled IL-1 ⁇ .
  • Equilibrium binding of 12S I-labeled IL-1 to the cells can performed as described by Mizel, et al. J. Immunol. 138: 2906-2912 (1987).
  • 25 I labeling of IL-l ⁇ can be performed by methods well known to those skilled in the art, for example, as described by Chizzonite, et al. J. Immunol. 147:1548-1556 (1991).
  • the activity of the accessory protein, IL-IR AcM can be examined in the binding of IL-l ⁇ to a CHO-IR/AcM cell line obtained above. It is desirable for this cell line to express an excess amount of IL-IR AcM protein relative to IL- IR. Cell lines bearing only IL-IR AcM protein do not bind IL-l ⁇ and cell lines bearing only IL-R will bind IL-l ⁇ with low affinity (i.e., approximately K n 1.0-
  • a CHO-IR/AcM cell line, bearing both the IL-IR and IL-IR AcM results in IL-IR having a higher affinity binding site (i.e., approximately K D 0.02- 0.8 nM).
  • IL-1 R AcM activity can be monitored by testing whether the putative accessory protein interacts with the IL-IR so as to generate a high affinity IL-l ⁇ binding site.
  • the IL-IR AcM protein of the present invention can serve as a reference for the assay for IL-IR AcM activity associated with the high affinity IL-IR binding state.
  • a candidate polypeptide has IL-IR AcM activity by performing simple binding kinetics can be measured to determine receptor affinity for the ligand (i.e., IL- 1 ⁇ ).
  • Binding kinetic analysis experiments are well known to those skilled in the art (Chizzonite et al, Proc Natl. Acad. Sci. 56:8029-8033 (1989); Mizel, et al, J. Immunol. 735:2906-2912 (1987)).
  • the IL-IR AcM protein of the present invention can serve as a reference for the assay for IL-IR AcM activity associated with the high affinity IL- 1 R binding state.
  • a polypeptide having soluble IL-I R AcM protein activity includes polypeptides that exhibit IL-IR AcM activity, in the above-described assay. Although the degree of activity need not be identical to that of the IL-IR
  • a polypeptide having IL-I R AcM protein activity will exhibit substantially similar activity as compared to the IL-IR AcM protein (i.e., the candidate polypeptide will exhibit greater activity or not more than about tenfold less and, preferably, not more than about ten- old less activity relative to the reference IL-IR AcM protein).
  • nucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of the deposited cDNA or the nucleic acid sequence shown in Figure 1 [SEQ ID NO:l] will encode a polypeptide "having soluble IL-IR AcM protein activity.”
  • degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay.
  • nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having soluble IL-IR AcM protein activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid).
  • the present invention also relates to vectors which include the isolated DNA molecules of the present invention, host cells which are genetically engineered with the recombinant vectors, and the production of soluble IL-IR AcM polypeptides or fragments thereof by recombinant techniques.
  • Recombinant constructs may be introduced into host cells using well known techniques such infection, transduction, transfection, transvection, electroporation and transformation.
  • the vector may be, for example, a phage, plasmid, viral or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
  • the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host.
  • a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
  • vectors comprising cis-acting control regions to the polynucleotide of interest.
  • Appropriate trans-acting factors may be supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.
  • the vectors provide for specific expression, which may be inducible and/or cell type-specific. Particularly preferred among such vectors are those inducible by environmental factors that are easy to manipulate, such as temperature and nutrient additives.
  • Expression vectors useful in the present invention include chromosomal-, episomal- and virus-derived vectors, e.g., vectors derived from bacterial plasmids, bacteriophage, yeast episomes, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adcnoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as cosmids and phagemids.
  • vectors derived from bacterial plasmids, bacteriophage, yeast episomes, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adcnoviruses, fowl pox viruses, pseudorabies viruses and retroviruses and vectors derived from combinations thereof, such as cosmids and phagemids.
  • the DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
  • an appropriate promoter such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
  • Other suitable promoters will be known to the skilled artisan.
  • the expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
  • the expression vectors will preferably include at least one selectable marker.
  • markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria.
  • Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli,
  • Streptomyces and Salmonella typhimurium cells
  • fungal cells such as yeast cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS and Bowes melanoma cells
  • plant cells Appropriate culture mediums and conditions for the above-described host cells are known in the art.
  • vectors preferred for use in bacteria include pA2, pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
  • preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
  • Other suitable vectors will be readily apparent to the skilled artisan.
  • bacterial promoters suitable for use in the present invention include the E. coli lacl and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter.
  • Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV), and metallothionein promoters, such as the mouse metallothionein-I promoter.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid- mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al, Basic Methods In Molecular Biology (1986).
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type.
  • enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • secretion signals may be inco ⁇ orated into the expressed polypeptide.
  • the signals may be endogenous to the polypeptide or they may be heterologous signals.
  • the polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.
  • a preferred fusion protein comprises a heterologous region from immunoglobulin that is useful to solubilize receptors.
  • EP-A-O 464 533 (Canadian counte ⁇ art 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another human protein or part thereof.
  • the Fc part in fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232262).
  • Fc portion proves to be a hindrance to use in therapy and diagnosis, for example when the fusion protein is to be used as antigen for immunizations.
  • human proteins such as, shIL5- has been fused with Fc portions for the piupose of high- throughput screening assays to identify antagonists of hIL-5. See, D. Bennett et al., Journal of Molecular Recognition, Vol. 8 52-58 (1995) and K. Johanson et al., The Journal of Biological Chemistry, Vol. 270, No. 16, pp 9459-9471 (1995).
  • the soluble IL-IR AcM protein can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification.
  • Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells.
  • polypeptides of the present invention may be glycosylated or may be non-glycosylated.
  • polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
  • the invention further provides an isolated soluble IL-IR AcM polypeptide having the amino acid sequence encoded by the deposited cDNA, or the amino acid sequence in Figure 1 [SEQ ID NO:2J, or a peptide or polypeptide comprising a portion of the above polypeptides.
  • peptide and oligopeptide are considered synonymous (as is commonly recognized) and each term can be used interchangeably as the context requires to indicate a chain of at least to amino acids coupled by peptidyl linkages.
  • polypeptide is used herein for chains containing more than ten amino acid residues. All oligopeptide and polypeptide formulas or sequences herein are written from left to right and in the direction from amino terminus to carboxy terminus.
  • the invention further includes variations of the soluble IL-IR AcM polypeptide which show substantial soluble IL-IR AcM polypeptide activity or which include regions of soluble IL-IR AcM protein such as the protein portions discussed below.
  • Such mutants include deletions, insertions, inversions, repeats, and type substitutions (for example, substituting one hydrophilic residue for another, but not strongly hydrophilic for strongly hydrophobic as a rule). Small changes or such "neutral" amino acid substitutions will generally have little effect on activity.
  • polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified.
  • a recombinantly produced version of the soluble IL-IR AcM polypeptide can be substantially purified by the method described by Bradley, M.K. "Overexpressio of Proteins in Eukaryotes” Methods in Enzymology, Vol. 182.
  • polypeptides of the present invention include the polypeptide encoded by the deposited cDNA including the leader, the mature polypeptide encoded by the deposited the cDNA minus the leader (i.e., the mature protein), the polypeptide of Figure 1 [SEQ ID NO:2] including the leader, the polypeptide of Figure 1 [SEQ ID NO:2] minus the leader, as well as polypeptides which have at least 90%) similarity, more preferably at least 95% similarity, and still more preferably at least 96%, 91%, 98% or 99% similarity to those described above.
  • polypeptides of the present invention include polypeptides at least 80% identical, more preferably at least 90% or 95% identical, still more preferably at least 96%), 97%, 98% or 99% identical to the polypeptide encoded by the deposited cDNA, to the polypeptide of Figure 1 [SEQ ID NO:2], and also include portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids.
  • % similarity for two polypeptides is intended a similarity score produced by comparing the amino acid sequences of the two polypeptides using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive,
  • polypeptide having an amino acid sequence at least, for example,
  • a reference amino acid sequence of a soluble IL-IR AcM polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of the soluble IL-1 R AcM polypeptide.
  • up to 5%> of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • any particular polypeptide is at least 90%, 95%, 96%), 97%, 98% or 99% identical to, for instance, the amino acid sequence shown in Figure 1 [SEQ ID NO:2J or to the amino acid sequence encoded by deposited cDNA clone can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 5371 1.
  • the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5%> of the total number of amino acid residues in the reference sequence are allowed.
  • polypeptide of the present invention could be used as a molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art.
  • the polypeptides of the present invention can also be used to raise polyclonal and monoclonal antibodies, which are useful in assays for detecting soluble IL-IR AcM protein expression as described below or as agonists and antagonists capable of enhancing or inhibiting soluble IL-IR AcM protein function.
  • polypeptides can be used in the yeast two- hybrid system to "capture" soluble IL-IR AcM protein binding proteins which are also candidate agonist and antagonist according to the present invention. The yeast two hybrid system is described in Fields and Song, Nature 340:245-246 (1989).
  • the invention provides a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention.
  • the epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention.
  • An "immunogenic epitope" is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen. These immunogenic epitopes are believed to be confined to a few loci on the molecule.
  • a region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope.”
  • the number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes. See, for instance, Geysen et al, Proc. Natl. Acad. Sci. USA 57:3998- 4002 (1983).
  • peptides or polypeptides bearing an antigenic epitope i.e., that contain a region of a protein molecule to which an antibody can bind
  • relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M., Green, N. and Learner, R.A. (1983) Antibodies that react with predetermined sites on proteins. Science 219:660-666.
  • Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals. Peptides that are extremely hydrophobic and those of six or fewer residues generally are ineffective at inducing antibodies that bind to the mimicked protein; longer, soluble peptides, especially those containing proline residues, usually are effective. Sutcliffe et al., supra, at 661.
  • 18 of 20 peptides designed according to these guidelines containing 8-39 residues covering 15% of the sequence of the influenza virus hemagglutinin HA1 polypeptide chain, induced antibodies that reacted with the HA1 protein or intact virus; and 12/12 peptides from the MuLV polymerase and 18/18 from the rabies glycoprotein induced antibodies that precipitated the respective proteins.
  • Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention.
  • a high proportion of hybridomas obtained by fusion of spleen cells from donors immunized with an antigen epitope-bearing peptide generally secrete antibody reactive with the native protein.
  • the antibodies raised by antigenic epitope-bearing peptides or polypeptides are useful to detect the mimicked protein, and antibodies to different peptides may be used for tracking the fate of various regions of a protein precursor which undergoes post-translational processing.
  • the peptides and anti-peptide antibodies may be used in a variety of qualitative or quantitative assays for the mimicked protein, for instance in competition assays since it has been shown that even short peptides (e.g., about 9 amino acids) can bind and displace the larger peptides in immunoprecipitation assays. See, for instance, Wilson et al. Cell 37:761-118 (1984) at 777.
  • the anti- peptide antibodies of the invention also are useful for purification of the mimicked protein, for instance, by adso ⁇ tion chromatography using methods well known in the art.
  • Antigenic epitope-bearing peptides and polypeptides of the invention designed according to the above guidelines preferably contain a sequence of at least seven, more preferably at least nine and most preferably between about 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention.
  • peptides or polypeptides comprising a larger portion of an amino acid sequence of a polypeptide of the invention, containing about 30 to about 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention also are considered epitope-bearing peptides or polypeptides of the invention and also are useful for inducing antibodies that react with the mimicked protein.
  • the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues and highly hydrophobic sequences are preferably avoided); and sequences containing proline residues are particularly preferred.
  • Non-limiting examples of antigenic polypeptides or peptides that can be used to generate soluble IL-IR AcM-specific antibodies include the following: a. S 18-M32: SDASERCDDWGLDTM (SEQ ID NO:5) b. V37-I45: VFEDEPARI (SEQ ID NO:6) c. G65-Y71 : GLTLIWY (SEQ ID NO:7) d. R74-I83: RQDRDLEEPI (SEQ ID NO: 8) e. L87-K96: LPENRISKEK (SEQ ID NO:9) f. S123-L129: SKVAFPL (SEQ ID NO: 10) g.
  • V132-P141 VQKDSCFNSP (SEQ ID NO:l 1)
  • V146-E152 VHKLYIE (SEQ ID NO: 12)
  • I175-N189 ITWYMGCYKIQNFNN
  • L197-I204 LSFLIALI
  • H224-K232 HLTRTLTVK (SEQ ID NO: 15)
  • P248-E261 PNDHVVYEKEPGEE (SEQ ID NO: 16) m. I264-F272: IPCTVYFSF (SEQ ID NO: 17) n. D285-T293: DGKKPDDIT (SEQ ID NO: 18) o. I302-R312: ISHSRTEDETR (SEQ ID NO: 19) p. K320-K327: KVTSEDLK (SEQ ID NO: 20) q. S336-V347: SAKEGEVAKAAKV (SEQ ID NO: 21) r. K350-Q356: KGNRCGQ (SEQ ID NO: 22)
  • the epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means for making peptides or polypeptides including recombinant means using nucleic acid molecules of the invention. For instance, a short epitope-bearing amino acid sequence may be fused to a larger polypeptide which acts as a carrier during recombinant production and purification, as well as during immunization to produce anti-peptide antibodies. Epitope-bearing peptides also may be synthesized using known methods of chemical synthesis.
  • Houghten has described a simple method for synthesis of large numbers of peptides, such as 10-20 mg of 248 different 13 residue peptides representing single amino acid variants of a segment of the HA1 polypeptide which were prepared and characte ⁇ zed (by ELISA-type binding studies) in less than four weeks.
  • Houghten, R A. (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids.
  • Epitope-bearing peptides and polypeptides of the invention are used to induce antibodies according to methods well known in the art See, for instance, Sutcliffe et al , supra; Wilson et al , supra. Chow, M et al , Proc Natl Acad Sci
  • animals may be immunized with free peptide, however, anti-peptide antibody titer may be boosted by coupling of the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid
  • KLH keyhole limpet hemacyanin
  • peptides containing cysteine may be coupled to carrier using a linker such as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carrier using a more general linking agent such as glutaraldehyde
  • Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intrape ⁇ toneal and/or intradermal injection of emulsions containing about 100 ⁇ g peptide or carrier protein and Freund'
  • the titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adso ⁇ tion to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
  • Immunogenic epitope-bearing peptides of the invention i.e., those parts of a protein that elicit an antibody response when the whole protein is the immunogen, are identified according to methods known in the art. For instance,
  • Geysen et al. discloses a procedure for rapid concurrent synthesis on solid supports of hundreds of peptides of sufficient purity to react in an enzyme-linked immunosorbent assay. Interaction of synthesized peptides with antibodies is then easily detected without removing them from the support. In this manner a peptide bearing an immunogenic epitope of a desired protein may be identified routinely by one of ordinary skill in the art. For instance, the immunologically important epitope in the coat protein of foot-and-mouth disease virus was located by Geysen et al. with a resolution of seven amino acids by synthesis of an overlapping set of all 208 possible hexapeptides covering the entire 213 amino acid sequence of the protein.
  • U.S. Patent No. 5,194,392 to Geysen (1990) describes a general method of detecting or determining the sequence of monomers (amino acids or other compounds) which is a topological equivalent of the epitope (i.e., a "mimotope") which is complementary to a particular paratope (antigen binding site) of an antibody of interest. More generally, U.S. Patent No. 4,433,092 to Geysen (1989) describes a method of detecting or determining a sequence of monomers which is a topographical equivalent of a ligand which is complementary to the ligand binding site of a particular receptor of interest. Similarly, U.S. Patent No. 5,480,971 to Houghten, R. A. et al. (1996) on
  • Peralkylated Oligopeptide Mixtures discloses linear C,-C 7 -alkyl peralkylated oligopeptides and sets and libraries of such peptides, as well as methods for using such oligopeptide sets and libraries for determining the sequence of a peralkylated oligopeptide that preferentially binds to an acceptor molecule of interest.
  • non-peptide analogs of the epitope-bearing peptides of the invention also can be made routinely by these methods.
  • the present inventors believe that soluble IL-IR AcM is involved in IL-1 activity and that it forms a complex with the Type I IL- 1 R allowing IL- 1 ⁇ to bind with higher affinity than to the Type I IL-IR alone.
  • the presence or absence of the accessory molecule in different cell lines may determine whether the low or the high affinity site in the Type I IL-IR is formed.
  • the low affinity site corresponds to the Type I IL-IR alone, while the higher affinity site represents a complex of the Type I IL-IR with the IL-IR AcM.
  • IL-IR AcM may to be necessary for IL-1 signal transduction events and may to be required for the formation of a high affinity IL- 1 R binding state.
  • Stable cell lines established by simultaneous cotransfection of two expression vectors such that both IL-IR and IL-IR AcM are expressed at a 1 :10 ratio of molecules/cell and control cell lines which express only IL-IR or IL-IR AcM could be used in screening assays to identify potential agonists and antagonists for IL- 1.
  • the present invention further provides a screening method of identifying IL-1 receptor agonists, which involves: (a) providing a host cell containing recombinant genes which express a polypeptide comprising a type I IL-1 receptor and a polypeptide comprising IL-I R AcM or a IL-IR AcM fragment, wherein IL-IR and IL-IR AcM or an IL-IR AcM fragment form a complex; (b) administering a candidate agonist to said cell; and (c) determining the binding affinity of said complex for said candidate agonist relative to the binding of said comples for IL-1.
  • each of the cell lines are analyzed by equilibrium binding with 125 I-labeled IL-l ⁇ .
  • Equilibrium binding of 125 I-labeled IL-l ⁇ to the cells can performed as described by Mizel, et al. J. Immunol. 138: 2906-2912 (1987) and Greenfeder et al, J. Biol. Chem. 270:13757- 13765 (1995).
  • the activity of the candidate agonist can be examined relative to the binding of IL-l ⁇ to a CHO-IR/AcM cell line (as described, supra). It is desirable for this cell line to express an excess amount of
  • IL-IR AcM protein relative to IL-IR.
  • Cell lines bearing only IL-IR AcM protein will not bind IL-l ⁇ and cell lines bearing only IL-R bind IL-l ⁇ with low affinity (i.e., on the order of K D 1.0-3.3nM).
  • a CHO-IR/AcM cell line, bearing both the IL-IR and IL-IR AcM results in the IL-IR having a higher affinity binding site (i.e., on the order of K D 0.02-0.8 nM).
  • K D 0.02-0.8 nM affinity binding site
  • the invention provides a screening method of identifying an IL-1 signal transduction antagonist.
  • the results of Greenfeder, et al. (J. Biol. Chem, 270: 13757-13765 (1995)) suggest that the antagonist IL-lra prevents or disrupts formation of a complex between mulL-lR and mulL-lR AcP.
  • the screening method for identifying other antagonist of signal transduction which involves: (a) providing a host cell containing recombinant genes which express a polypeptide comprising a type I IL-1 receptor and a polypeptide comprising IL-IR AcM or an IL-1 R AcM fragment, wherein IL-IR and IL-IR AcM or IL-IR and the IL-IR AcM fragment form a complex; (b) administering a candidate antagonist to said cell; and (c) determining the whether said candidate antagonist disrupts or prevents formation of a complex between IL-IR and IL-IR AcM or IL-IR and the IL-IR AcM fragment.
  • IL-IR complex between IL-IR and IL-IR AcM
  • IL-l ⁇ cross-linked to IL-l ⁇ , IL-lra or the candidate antagonist.
  • an anti-type I IL-IR mAb or an anti-IL-lR AcM mAb a protein complex of > 200 kDa range will be observed for the cross linking reaction when IL-1 ⁇ is used as a ligand indicating formation of an IL-1 binding complex.
  • these antibodies will immunoprecipitate labeled IL 1 ⁇ .
  • the anti-IL-l R AcM mAb will not immunoprecipitate the labeled agonist. Rather, the anti-IL- 1 R AcM antibody will only precipitate an IL- 1 ra or a candidate antagonist complex in the 100- 120 kDa range indicating that the IL-IR and IL-IR AcM have not formed a complex.
  • the present invention further provides a screening method for identifying IL-1 receptor agonists and antagonists, which involves: (a) providing a polypeptide comprising a type I IL-1 receptor and a polypeptide comprising IL-
  • IR AcM or IL-IR AcM fragment wherein IL-1 R and IL-1 R AcM or IL-I R and the IL-IR AcM fragment form a complex; (b) providing a candidate compound; (c) providing a polypeptide conprising IL-1 or a functional IL-1 fragment; and (d) determining the binding affinity of said complex for I -1 whereby an increased binding affinity of said complex for IL- 1 in the presence of said compound is indicative that said compound is an agonist for IL-1 signal transduction and a decreased binding affinity of said complex for IL-1 in the presence of said compound is indicative that said compound is an antagonist of IL-1 signal transduction.
  • Candidate antagonists and agonist according to the present invention includelde IL-lra, polypeptides and antibodies that either enhance or inhibit formation of the IL-IR IL-IR AcM complex.
  • an antibody that inhibits the murine IL-1R-IL-1R accessory protein complex is described in Greenfeder, et al. J. Biol Chem, 270: 13757-13765 (1995).
  • antibodies directed against murine type I IL-1 receptors which block IL-1 activity are described in Chizzonite et al. Proc. Natl Acad Sci USA 56:8029 (1989); Lewis et al. Eur. J. Immunol. 20:201 (1990); Dinarello, Int. J. Clin. Lab. Res. 24:61-79
  • IL- 1 ra converts IL- 1 ra from an antagonist to a partial agonist of IL-1 activity in Ju et al. Proc. Natl. Acad. Sci USA 55:2658- 2662 (1991). Others have developed recombinant IL-1 mutants with altered activities (Dinarello Blood 77:1627-1652; Gehrke et al. J. Biol Chem. 265:5922- 5925 (1990)). Thus, methods are known in the art for developing candidate IL-
  • Administration of the candidate agonist or antagonist can be exogenous or endogenous and the candidate agonist or antagonist can be obtained from natural or recombinant sources.
  • the screening method further provides for host cells containing recombinant genes expressing IL-IR and IL- IR AcM, as described above.
  • a host cell containing recombinant genes is intended host cells which one or more of the recombinant constructs described herein have been introduced or a progeny of such host cells.
  • the invention provides antibodies directed to this accessory molecule which inhibit the interaction of IL-IR with IL-IR AcM thereby modulating IL-1 response of the cells.
  • These antibodies could be useful for immunoprecipitating cross-linked complexes for the antagonist screening assay as well as showing that the stable cell lines are in fact expressing the type I IL- 1 R and IL-IR AcM proteins. Methods for obtaining these antibodies are set forth below.
  • Soluble IL-IR AcM protein-specific antibodies for use in the present invention can be raised against the intact soluble IL-IR AcM protein or an antigenic polypeptide portion thereof, which may presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse) or, if it is long enough (at least about 25 amino acids), without a carrier.
  • a carrier protein such as an albumin
  • antibody As used herein, the term "antibody” (Ab) or “monoclonal antibody” (Mab) is meant to include intact molecules as well as antibody portions (such as, for example, Fab and F(ab') 2 portions) which are capable of specifically binding to soluble IL-1 R AcM protein. Fab and F(ab') 2 portions lack the Fc portion of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al, J. Nucl. Med. 24:316-325 (1983)). Thus, these portions are preferred.
  • the antibodies of the present invention may be prepared by any of a variety of methods.
  • cells expressing the soluble IL-IR AcM protein or an antigenic portion thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies.
  • a preparation of soluble IL-1 R AcM protein is prepared and purified as described above to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.
  • the antibodies of the present invention are monoclonal antibodies (or soluble IL-IR AcM protein binding portions thereof). Such monoclonal antibodies can be prepared using hybridoma technology
  • Such procedures involve immunizing an animal (preferably a mouse) with a soluble IL-IR AcM protein antigen or, more preferably, with a soluble IL-IR AcM protein-expressing cell. Suitable cells can be recognized by their capacity to bind soluble IL-IR AcM protein antibody.
  • Such cells may be cultured in any suitable tissue culture medium; however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56°C), and supplemented with about 10 ⁇ g/l of nonessential amino acids, about 1 ,000 U/ml of penicillin, and about 100 ⁇ g/ml of streptomycin.
  • the splenocytes of such mice are extracted and fused with a suitable myeloma cell line.
  • Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP 2 O), available from the American Type Culture Collection, Rockville, Maryland.
  • the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gasiroenterology 50:225-232 (1981)). The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the soluble IL-IR AcM antigen.
  • additional antibodies capable of binding to the soluble IL- IR AcM protein antigen may be produced in a two-step procedure through the use of anti-idiotypic antibodies.
  • Such a method makes use of the fact that antibodies are themselves antigens, and therefore it is possible to obtain an antibody which binds to a second antibody.
  • soluble IL-IR AcM protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the soluble IL-IR AcM protein- specific antibody can be blocked by the soluble IL-I R AcM protein antigen.
  • Such antibodies comprise anti-idiotypic antibodies to the soluble IL-IR AcM protein-specific antibody and can be used to immunize an animal to induce formation of further soluble IL-IR AcM protein-specific antibodies.
  • Fab and F(ab') 2 and other portions of the antibodies of the present invention may be used according to the methods disclosed herein. Such portions are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab portions) or pepsin (to produce F(ab') 2 portions).
  • soluble IL-IR AcM protein-binding portions can be produced through the application of recombinant DNA technology or through synthetic chemistry. Where in vivo imaging is used to detect enhanced levels of soluble IL-1 R
  • chimeric monoclonal antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric antibodies are known in the art. See, for review, Morrison, Science 229: 1202 ( 1985); Oi et al, BioTechniques
  • suitable enzyme labels include malate dehydrogenase, staphylococcal nucleasc, delta-5- steroid isomerase, yeast-alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholine esterase.
  • radioisotopic labels examples include 3 H, '"In, l25 I, 13 I I, 32 P,
  • m In is a preferred isotope where in vivo imaging is used since its avoids the problem of dehalogenation of the 125 I or l3 l I-labeled monoclonal antibody by the liver.
  • this radionucleotide has a more favorable gamma emission energy for imaging (Perkins et al, Eur. J. Nucl Med. 70:296-301 (1985); Carasquillo et al, J. Nucl Med.
  • suitable non-radioactive isotopic labels include ,57 Gd, 55 Mn, ,62 Dy, 52 Tr, and 56 Fe.
  • suitable fluorescent labels include an 152 Eu label, a fluorescein label, an isothiocyanate label, a rhodamine label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label, an o-phthaldehyde label, and a fluorescamine label.
  • Suitable toxin labels include diphtheria toxin, ricin, and cholera toxin.
  • chemiluminescent labels include a luminal label, an isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, and an aequorin label.
  • nuclear magnetic resonance contrasting agents include heavy metal nuclei such as Gd, Mn, and Fe.
  • Typical techniques for binding the above-described labels to antibodies are provided by Kennedy et al. (Clin. Chim. Ada 70: 1 -31 (1976)), and Schurs et al (Clin. Chim. Acta 57:1-40 (1977)). Coupling techniques mentioned in the latter are the glutaraldehyde method, the periodate method, the dimaleimide method, the m-maleimidobenzyl-N-hydroxy-succinimide ester method, all of which methods are inco ⁇ orated by reference herein.
  • the nucleic acid molecules of the present invention are also valuable for chromosome identification.
  • the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
  • mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
  • the cDNA herein disclosed is used to clone genomic DNA of a soluble IL-IR AcM protein gene.
  • genomic DNA then is used for in situ chromosome mapping using well known techniques for this pu ⁇ ose.
  • some trial and error may be necessary to identify a genomic probe that gives a good in situ hybridization signal.
  • sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3' untranslated region of the gene is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified portion. PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome.
  • mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow- sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
  • Fluorescence in situ hybridization of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step.
  • This technique can be used with probes from the cDNA as short as 50 or 60 bp.
  • Verma et al Human Chromosomes: A Manual Of Basic Techniques, Pergamon Press, New York (1988).
  • a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. This assumes 1 megabase mapping resolution and one gene per 20 kb.
  • IL-1 has an important role in modulating the proliferation, maturation, and functional activation of hematopoietic cells, including lymphoid and nonlymphoid cells. IL-1 may have an important function in the regulation liver metabolism and be responsible for some of the marked changes in hepatic protein synthesis that occur in the acute phase response to inflammation or tissue injury.
  • IL-1 is also involved in the regulation of bone remodulation, may be involved in the pathogenesis of chronic inflammatory joint diseases, such as rheumatoid arthritis, osteoarthritis, and may also play a role in the mechanisms of articular cartilage destruction hat occurs in degradative arthropathies.
  • Human IL-1 is capable of increasing collagen protein and mRNA levels in cultured normal human dermal fibroblasts, thus, IL-1 may have a role in the early stages of scleroderma and other fibrotic diseases.
  • IL-1 is capable of inducing a proliferative response in fibroblasts.
  • IL-1 may have important effects on vascular cells, including endothelial cells and vascular smooth muscle cells.
  • IL-1 may be involved in the pathogenesis of certain skin diseases, including chronic diseases such as psoriasis and epithelial fungus infections. IL-1 may have important effects on the functions of the hypothalamus-pituitary axis and thyroid gland. IL-1 has an important role in the regulation of insulin secretion by ⁇ cells in the pancreatic islets of Langerhans. Finally, IL-1 has important effects on the gonads and may play a role in the physiology of neural tissues. (Reviewed in Pimentel, Handbook of Growth Factors: Volume HI Hematopoietic Growth
  • IL-lra blocks the activity of exogenously administered IL-1 in a variety of animal models.
  • rabbits or baboons are injected with IL-1 they develope hypertension which is prevented by a prior injection of IL-lra (Ohlsson et al. Nature 348: 550 (1990); Fischer et al. Am. J. Physiol 261: R442 (1991)).
  • IL-IR blockade significantly reduces the severity of diseases, including those associated with infections, inflammation and metabolic disturbances (Arend, W.P. J. Clin. Invest 55:1445 (1991); Dinarello et al. Immunol Today 12: 404 (1991 )).
  • Table 1 of Dinarello, Int. J. Clin. Lab. Res. 24:61-79 (1994) (which is inco ⁇ orated herein by reference) different models are listed wherein a specific reduction in IL-IR activity has been employed to reduce the disease severity.
  • antibodies directed against IL-IR AcM are expected to behave as agonists or antagonist of IL-1 activity.
  • an antibody directed against the murine IL-I R accessory protein blocked the binding of IL-l ⁇ to murine type I IL-IR (Greenfeder et al. J. Biol. Chem, 270: 13757-13765 (1995)).
  • antibodies directed against the IL-IR AcM of the present invention that abrogate IL-1 activity can be used therapuetically to reduce the severity of diseases associated with IL-1.
  • the present invention is further directed antibody-based therapies which involve administering an anti-IL-lR AcM antibody to a mammalian, preferably human, patient for treating one or more of above-described disorders.
  • an anti-IL-lR AcM antibody to a mammalian, preferably human, patient for treating one or more of above-described disorders.
  • Methods for producing anti-IL-lR AcM polyclonal and monoclonal antibodies are described in detail above.
  • Such antibodies can may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
  • a summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding IL-IR AcM locally or systemically in the body or by direct cytotoxicity of the antibody, e.g., as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below.
  • compositions of the present invention may be administered by any means that achieve their intended pu ⁇ ose.
  • Amounts and regimens for the administration of antibodies, their fragments or derivatives can be determined readily by those with ordinary skill in the clinical art of treating colon cancer and related disease.
  • administration may be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes.
  • administration may be by the oral route.
  • the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concunent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • compositions within the scope of this invention include all compositions wherein the antibody, fragment or derivative is contained in an amount effective to achieve its intended pu ⁇ ose. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art.
  • the effective dose is a function of the individual chimeric or monoclonal antibody, the presence and nature of a conjugated therapeutic agent (see below), the patient and his clinical status, and can vary from about 10 ⁇ g/kg body weight to about 5000 mg/kg body weight.
  • the preferred dosages comprise 0.1 to 500 mg/kg body wt.
  • the new pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • the preparations contain from about 0.01 to 99 percent, preferably from about 20 to 75 percent of active compound(s), together with the excipient.
  • preparations of an IL-IR AcM antibody or fragment of the present invention for parenteral administration such as in detectably labeled form for imaging or in a free or conjugated form for therapy, include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oil 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 including sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, anti-oxidants. chelating agents, and inert gases and the like. See, generally, Remington's Pharmaceutical Science, 16th ed., Mack Publishing Co., Easton, PA, 1980.
  • the antibodies, fragments and derivatives of the present invention are useful for treating a subject having or developing IL-IR AcM related disorders as described herein.
  • Such treatment comprises parenterally administering a single or multiple doses of the antibody, fragment or derivative, or a conjugate thereof.
  • the antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hemopoietic growth factors, etc., which serve to increase the number or activity of effector cells which interact with the antibodies. Since it appear to be necessary to block nearly all IL-lR's to block IL-I activity, it is prefened to use high affinity and/or potent in vivo IL-IR AcM-inhibiting and/or neutralizing antibodies, fragments or regions thereof, for both IL-IR AcM immunoassays and therapy of IL-1 related disorders.
  • Such antibodies, fragments, or regions will preferably have an affinity for human IL- IR AcM, expressed as Ka, of at least 10 8 M" 1 , more preferably, at least 10 9 M' 1 , such as 5 X 10 8 M '1 , 8 X 10 8 M "1 , 2 X 10 9 M ', 4 X 10 9 M 1 , 6 X 10 9 M 1 , 8 X 10 9
  • Preferred for human therapeutic use are high affinity murine and murine/human or human/human chimeric antibodies, and fragments, regions and derivatives having potent in vivo IL-1 -inhibiting and/or neutralizing activity, according to the present invention, e.g., that block IL-1 -induced IL-6 secretion, and mitogenic activity, in vivo, in situ, and in vitro.
  • Example I Expression and Purification of IL-IR AcM in E. coli
  • the DNA sequence encoding the mature soluble IL-IR AcM protein in the deposited cDNA clone is amplified using PCR oligonucleotide primers specific to the amino acid carboxyl terminal sequence of the soluble IL-1 R AcM protein and to vector sequences 3' to the gene. Additional nucleotides containing restriction sites BamHI and Sail to facilitate cloning are added to the 5' and 3' sequences, respectively.
  • the 5' oligonucleotide primer has the sequence 5' GGATCCATGACACTTCTGTGGTGTG 3' (SEQ ID NO:23) containing the underlined BamHI restriction site, followed by 16 nucleotides complementary to bp 1834-1853 of the antisense strand of the IL-IR AcM protein coding sequence set out in Figure 1 (SEQ ID NO: 1).
  • the 3* primer has the sequence 5' GTCGACTCACTGACCGCATCT 3'
  • the restrictions sites are convenient to restriction enzyme sites in the bacterial expression vector pQE-9, which is used for bacterial expression in these examples. (Qiagen, Chatsworth, CA, 9131 1).
  • the amplified IL-IR AcM protein DNA and the vector pQE-9 are both digested with BamHI and Sail and the digested DNAs are subsequently ligated together. Insertion of the IL-IR AcM protein DNA into the pQE-9 restricted vector places the IL-IR AcM protein coding region downstream of and operably linked to the vector's promoter and in-frame with an initiating AUG appropriately positioned for translation of IL-1 R AcM protein.
  • E. coli strain M15/rep4 containing multiple copies of the plasmid pREP4, which expresses lac repressor and confers kanamycin resistance ("Kanr"), is used in carrying out the illustrative example described here.
  • Kanr lac repressor and confers kanamycin resistance
  • Transformants are identified by their ability to grow on LB plates in the presence of ampicillin. Plasmid DNA is isolated from resistant colonies and the identity of the cloned DNA is confirmed by restriction analysis.
  • Clones containing the desired constructs are grown overnight ("O/N") in liquid culture in LB media supplemented with both ampicillin ( 100 ⁇ g/ml) and kanamycin (25 ⁇ g/ml).
  • the O/N culture is used to inoculate a large culture, at a dilution of approximately 1 : 100 to 1 :250.
  • the cells are grown to an optical density at 600 nm ("OD600”) of between 0.4 and 0.6.
  • Isopropyl-B-D-thiogalactopyranoside (“IPTG”) are then added to a final concentration of 1 mM to induce transcription from lac repressor sensitive promoters, by inactivating the lad repressor. Cells subsequently are incubated further for 3 to 4 hours.
  • Inclusion bodies are purified from the disrupted cells using routine collection techniques, and protein are solubilized from the inclusion bodies into 8M urea.
  • the 8M urea solution containing the solubilized protein is passed over a PD-10 column in 2X phosphate buffered saline ("PBS"), thereby removing the urea, exchanging the buffer and refolding the protein.
  • PBS 2X phosphate buffered saline
  • the protein is purified by a further step of chromatography to remove endotoxin. Then, it is sterile filtered.
  • the sterile filtered protein preparation is stored in 2X PBS at a concentration of 95 micrograms per mL.
  • vectors used for the transient expression of the IL-R AcM protein gene sequence in mammalian cells should carry the SV40 origin of replication. This allows the replication of the vector to high copy numbers in cells (e.g., COS cells) which express the T antigen required for the initiation of viral DNA synthesis. Any other mammalian cell line can also be utilized for this pu ⁇ ose.
  • a typical mammalian expression vector contains the promoter element, which mediates the initiation of transcription of mRNA, the protein coding sequence, and signals required for the termination of trancription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).
  • LTRs long terminal repeats
  • cellular signals can also be used (e.g., human actin promoter).
  • Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109).
  • Mammalian host cells that could be used include, human Hela, 283, H9 and Jurkart cells, mouse NIH3T3 and Cl 27 cells, Cos 1 , Cos 7 and CV 1 , African green monkey cells, quail QC1-3 cells, mouse L cells and Chinese hamster ovary cells.
  • the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome.
  • a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transfected cells.
  • the transfected gene can also be amplified to express large amounts of the encoded protein.
  • the DHFR dihydrofolate reductase
  • GS glutamine synthase
  • Another useful selection marker is the enzyme glutamine synthase (GS) (Mwphy et al, Biochem J. 227.277-279 ( 1991); Bebbington et al, Bio/Technology 70:169-175 (1992)).
  • GS glutamine synthase
  • the mammalian cells are grown in selective medium and the cells with the highest resistance are selected.
  • These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) cells are often used for the production of proteins.
  • the expression vectors pCl and pC4 contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al. , Molecular and Cellular Biology, 438-447 (March, 1985)) plus a fragment of the CMV -enhancer (Boshart et al,
  • the vectors contain in addition the 3 ' intron, the polyadenylation and termination signal of the rat preproinsulin gene.
  • the expression plasmid, pIL-lR AcM HA is made by cloning a cDNA encoding IL-R AcM into the expression vector pcDNAI/Amp (which can be obtained from Invitrogen, Inc.).
  • the expression vector pcDNAI/amp contains: ( 1 ) an E.coli origin of replication effective for propagation in E. coli and other prokaryotic cells; (2) an ampicillin resistance gene for selection of plasmid-containing prokaryotic cells; (3) an SV40 origin of replication for propagation in eukaryotic cells; (4) a CMV promoter, a polylinker, an SV40 intron, and a polyadenylation signal arranged so that a cDNA conveniently can be placed under expression control of the CMV promoter and operably linked to the SV40 intron and the polyadenylation signal by means of restriction sites in the polylinker.
  • a DNA fragment encoding the IL- 1 R AcM protein and an HA tag fused in frame to its 3' end is cloned into the polylinker region of the vector so that recombinant protein expression is directed by the CMV promoter.
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein described by Wilson et al, Cell 37: 767 (1984). The fusion of the HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
  • the plasmid construction strategy is as follows
  • the IL-1 R AcM cDNA of the deposited clone is amplified using primers that contain convenient restriction sites, much as described above regarding the construction of expression vectors for expression of IL-R AcM in E. coli.
  • one of the primers contains a hemagglutinin tag ("HA tag") as described above.
  • HA tag hemagglutinin tag
  • Suitable primers include the following, which are used in this example.
  • the 5' primer, containing the underlined BamHI site, an AUG start codon and 5 codons of the 5' coding region has the following sequence:
  • the 3' primer containing the underlined Xbal site, a stop codon, 9 codons thereafter forming the hemagglutinin HA tag, and 12 bp of 3' coding sequence (at the 3' end) has the following sequence:
  • the PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digested with BamHI and Xbal and then ligated.
  • the ligation mixture is transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037), and the transformed culture is plated on ampicillin media plates which then are incubated to allow growth of ampicillin resistant colonies, Plasmid DNA is isolated from resistant colonies and examined by restriction analysis and gel sizing for the presence of the IL-IR AcM-encoding fragment.
  • COS cells are transfected with an expression vector, as described above, using DEAE-DEXTRAN, as described, for instance, in Sambrook et al, Molecular Cloning: a Laboratory
  • Cells are incubated under conditions for expression of IL- 1 R AcM by the vector.
  • IL-IR AcM HA fusion protein Expression of the IL-IR AcM HA fusion protein is detected by radiolabelling and immunoprecipitation, using methods described in, for example
  • NP-40 0.1% SDS, l%NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. cited above.
  • Proteins are precipitated from the cell lysate and from the culture media using an HA-specific monoclonal antibody. The precipitated proteins then are analyzed by SDS-PAGE gels and autoradiography. An expression product of the expected size is seen in the cell lysate, which is not seen in negative controls.
  • Plasmid pCl is a derivative of the plasmid pSV2-dhfr [ATCC Accession No. 37146]. Both plasmids contain the mouse DHFR gene under control of the SV40 early promoter. Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (alpha minus MEM, Life Technologies) supplemented with the chemotherapeutic agent methotrexate.
  • a selective medium alpha minus MEM, Life Technologies
  • MTX methotrexate
  • Plasmid pCl contains for the expression of the gene of interest a strong promoter of the long terminal repeat (LTR) of the Rouse Sarcoma Virus (Cullen, et al, Molecular and Cellular Biology, March 1985:438-4470) plus a fragment isolated from the enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart et al, Cell 47:521-530, 1985). Downstream of the promoter are the following single restriction enzyme cleavage sites that allow the integration of the genes: BamHI, Pvull, and Nrul.
  • LTR long terminal repeat
  • CMV cytomegalovirus
  • the plasmid contains translational stop codons in all three reading frames followed by the 3' intron and the polyadenylation site of the rat preproinsulin gene.
  • Other high efficient promoters can also be used for the expression, e.g., the human ⁇ -actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, e.g., HIV and HTLVI.
  • the polyadenylation of the mRNA other signals, e.g., from the human growth hormone or globin genes can be used as well.
  • Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co-transfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker in the beginning, e.g., G418 plus methotrexate.
  • the plasmid pCl is digested with the restriction enzyme BamHI and then dephosphorylated using calf intestinal phosphates by procedures known in the art.
  • the vector is then isolated from a 1% agarose gel.
  • the DNA sequence encoding IL-I R AcM, ATCC 97666, is amplified using PCR oligonucleotide primers corresponding to the 5 ' and 3 ' sequences of the gene: T h e 5 ' p r i m e r h a s t h e s e q u e n c e
  • the 3' primer has the sequence 5' Q ⁇ UCCTCACTGACCGCATCT 3' (SEQ ID NO:28) containing the EcoRI restriction followed by nucleotides complementary to the last 15 nucleotides of the IL-IR AcM coding sequence set out in Figure 1 (SEQ ID NO:l), including the stop codon.
  • amplified fragments are isolated from a 1% agarose gel as described above and then digested with the endonucleases BamHI and EcoRI and then purified again on a 1% agarose gel.
  • the isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase.
  • E. coli HBlOl cells are then transformed and bacteria identified that contained the plasmid pCl inserted in the correct orientation using the restriction enzyme BamHI. The sequence of the inserted gene is confirmed by DNA sequencing.
  • Chinese hamster ovary cells lacking an active DHFR enzyme are used for transfection.
  • 5 ⁇ g of the expression plasmid Cl are cotransfected with 0.5 ⁇ g of the plasmid pSVneo using the lipofecting method (Feigner et al, supra).
  • the plasmid pSV2-neo contains a dominant selectable marker, the gene neo from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418.
  • the cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418.
  • the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) and cultivated from 10-14 days. After this period, single clones are trypsinized and then seeded in 6-well petri dishes using different concentrations of methotrexate (25 nM, 50 nM, 100 nM, 200 nM, 400 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (500 nM, 1 ⁇ M, 2 ⁇ M, 5 ⁇ M). The same procedure is repeated until clones grow at a concentration of 100 ⁇ M.
  • Example 3 Cloning and expression of the Soluble IL-IR AcM protein in a baculovirus expression system
  • the cDNA sequence encoding the soluble IL-IR AcM protein in the deposited clone was amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene:
  • the 5' primer has the sequence 5 '
  • GACTGGATCCGCCATCATGACACTTCTGTGGTGTG 3' (SEQ ID NO:29) containing the underlined BamHI restriction enzyme site followed by 19 bases (bp 1834-1853) complementary to the antisense strand of the soluble IL-1 R AcM protein coding sequence of Figure 1 (SEQ ID NO: 1 ).
  • the 5' end of the amplified fragment encoding soluble IL-IR AcM protein receptor provides an efficient signal peptide.
  • An efficient signal for initiation of translation in eukaryotic cells as described by Kozak, M., J. Mol Biol. 796:947-950 (1987), may be located, as appropriate, in the vector portion of the construct.
  • the 3' primer has the full length sequence 5' GAC TGG TAG CCA TAG AAA TCA TGT GTA TAC C 3' (SEQ ID NO:30), containing the underlined Asp718 restriction followed by 25 nucleotides complementary to bp 2049-2070 of the sense strand of the soluble IL-1 R AcM protein set out in Figure 1 [SEQ ID NO: 1], and a stop codon.
  • the amplified fragment was isolated from a 1 % agarose gel using a commercially available kit ("Geneclean,” BIO 101 Inc., La Jolla, Ca.). The fragment then is digested with BamHI and Asp718 and again is purified on a 1% agarose gel. This fragment is designated herein "F2".
  • the vector pA2 is used to express the soluble IL-1 R AcM protein in the baculovirus expression system, using standard methods, such as those described in Summers et al, A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experimental Station Bulletin No. 1555 (1987).
  • This expression vector contains the strong polyhedrin promoter of the Autographa califomica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites.
  • AcMNPV Autographa califomica nuclear polyhedrosis virus
  • the beta- galactosidase gene from E. coli is inserted in the same orientation as the polyhedrin promoter and is followed by the polyadenylation signal of the polyhedrin gene.
  • the polyhedrin sequences are flanked at both sides by viral sequences for cellmediated homologous recombination with wild-type viral DNA to generate viable virus that express the cloned polynucleotide.
  • baculovirus vectors could be used in place of pA2, such as pAc373, pVL941 and pAcIMl provided, as those of skill readily will appreciate, that construction provides appropriately located signals for transcription, translation, trafficking and the like, such as an in-frame AUG and a signal peptide, as required.
  • Such vectors are described, for example, in Luckow et al, Virology 770:31-39 (1989). Suitable vectors will be readily apparent to the skilled artisan.
  • the plasmid was digested with the restriction enzymes BamHI and Asp718 and then was dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art.
  • the DNA was then isolated from a 1% agarose gel using a commercially available kit ("Gencclean" BIO 101 Inc., La Jolla, Ca.). This vector DNA is designated herein "V2".
  • Fragment ⁇ 2 and the dephosphorylated plasmid V2 were ligated together with T4 DNA ligase.
  • E. coli HB 101 cells were transformed with ligation mix and spread on culture plates.
  • Bacteria were identified that contain the plasmid with the human soluble IL-IR AcM protein gene by digesting DNA from individual colonies using BamHI and Asp718 and then analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment was confirmed by DNA sequencing. This plasmid is designated herein as pA2HG 16302.
  • plasmid pA2HG16302 was co-transfected with 1.0 ⁇ g of a commercially available linearized baculovirus DNA ("BaculoGoldTM baculovirus DNA", Pharmingen, San Diego, CA), using the lipofection method described by Feigner et al, Proc. Natl Acad. Sci. USA 54:7413-7417 (1987). 1 ⁇ g of a commercially available linearized baculovirus DNA ("BaculoGoldTM baculovirus DNA", Pharmingen, San Diego, CA), using the lipofection method described by Feigner et al, Proc. Natl Acad. Sci. USA 54:7413-7417 (1987). 1 ⁇ g of
  • BaculoGoldTM virus DNA and 5 ⁇ g of the plasmid pA2HG 16302 were mixed in a sterile well of a microliter plate containing 50 ⁇ l of serum free Grace's medium (Life Technologies Inc., Gaithersburg, MD). Afterwards, 10 ⁇ l Lipofectin plus 90 ⁇ l Grace's medium were added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture was added drop- wise to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with I ml Grace's medium without serum. The plate was rocked back and forth to mix the newly added solution. The plate is then incubated for 5 hours at 27 °C. After 5 hours the transfection solution was removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. The plate is put back into an incubator and cultivation is continued at 27 °C for four days.
  • serum free Grace's medium Life Technologies Inc.
  • plaque assay After four days the supernatant was collected and a plaque assay was performed, as described by Summers and Smith, supra.
  • An agarose gel with "Blue Gal” (Life Technologies Inc., Gaithersburg, MD) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques.
  • a detailed description of a "plaque assay” of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, MD, at pages 9-10.
  • Sf9 cells are grown in Grace's medium supplemented with 10% heat- inactivated FBS.
  • the cells are infected with the recombinant baculovirus A2HG16302 at a multiplicity of infection ("MOI") of about 2 (about 1 to about
  • Northern blot analysis is carried out to examine the levels of expression of the gene encoding the IL-IR AcM protein in human tissues, using methods described by, among others, Sambrook et al, cited above.
  • a cDNA probe containing the entire nucleotide sequence of the IL- 1 R AcM protein of the present invention (SEQ ID NO: 1 ) is labeled with "P using the redip ⁇ meTM DNA labeling system (Amersham Life Science), according to manufacturer's instructions. After labelling, the probe was purified using a CHROMA SPIN- 100TM column (Clontech Laboratories, Inc.), according to manufacturer's protocol number PT1200-1. The purified labelled probe is then used to examine various human tissues for the expression of the gene encoding the IL- 1 R AcM protein.
  • MTN Multiple Tissue Northern
  • H human tissues
  • IM human immune system tissues
  • ADDRESSEE STERNE, KESSLER, GOLDSTEIN & FOX, P.L.L.C.
  • ACTAGAACAT CAGCAGGCCC TTAGAAGCCT CACTCTTGCC CCTCCCTTTA ATATCTCAAA 300
  • GCT GGC CTT ACT CTG ATC TGG TAT TGG ACT AAG CAG
  • GAC CGG GAC CTT 539 Ala Gly Leu Thr Leu lie Trp Tyr Trp Thr Lys Gin Asp Arg Asp Leu 50 55 60
  • TGT CCA AAT GTA GAT GGA TAT TTT CCT TCC AGT GTC AAA CCG ACT ATC 827 Cys Pro Asn Val Asp Gly Tyr Phe Pro Ser Ser Val Lys Pro Thr lie 145 150 155
  • AAATATTCAA AGCTACCAAA GATAGAAAAA ACTGGTAGAG CCACATATTG TTGGTGAATT 1710
  • Trp Tyr Met Gly Cys Tyr Lys He Gin Asn Phe Asn Asn Val He Pro 160 165 170 175
  • MOLECULE TYPE cDNA
  • the applicant hereby request that, until the application has been laid open to public inspection (by the Norwegian Patent Office), or has been finally decided upon by the Norwegian Patent Office without having been laid open to public inspection, the furnishing of a sample shall only be effected to an expert in the art.
  • the request to this effect shall be filed by the applicant with the Norwegian Patent Office not later than at the time when the application is made available to the pbulic under Sections 22 and 33(3) of the Norwegian Patents Act. If such a request has been filed by the applicant, any request made by a third party for the furnishing of a sample shall indicate the expert to be used. That expert may be any person entered on a list of recognized experts drawn up by the Norwegian Patent office or any person approved by the applicant in the individual case.

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Abstract

The present invention relates to a novel soluble IL-1 receptor accessory molecule (IL-1R AcM) protein which is a member of the Ig superfamily. In particular, isolated nucleic acid molecules are provided encoding the human IL-1R AcM protein. IL-1R AcM polypeptides are also provided as are vectors, host cells and recombinant methods for producing the same. Screening methods are further provided for identifying agonist and antagonists of IL-1 signal transduction. The invention further relates to methods for treating physiologic and pathologic disease conditions with IL-1R AcM antagonists.

Description

Soluble Interleukin-1 Receptor Accessory Molecule
Field of the Invention
The present invention relates to a novel soluble Interleukin-1 receptor accessory molecule (IL-IR AcM). IL-IR AcM is a member of the Ig superfamily by analysis of its putative extracellular domain and bears limited homology throughout the protein to both Type I and Type II IL-1 receptors. More specifically, isolated nucleic acid molecules are provided encoding a human microvascular endothelial-derived soluble IL-IR AcM. The IL-IR AcM polypeptides are also provided. The present invention further relates to screening methods for putative agonists and antagonists of IL-1 signal transduction.
Background of the Invention
Interleukin 1 (IL-1) is a polypeptide cytokine with multiple diverse effects on immunological and inflammatory processes. While many of the roles of IL-1 in inflammation and the immune response have been well characterized, the molecular basis of these responses remains unclear (reviewed by Dinarello,
Blood, 77: 1627-1652). IL-1 is produced by a diversity of cell types and elicits a wide variety of physiological effects in he atopoetic and nonhematopoetic cells. Thus, IL-1 has biological effects on hematopoietic cells, the digestive tract, bone, cartilage and connective tissue, vascular cells, the skin, the endocrine system, the gonads, and on neural tissue. In addition, IL-1 is produced by malignant cells. (Pimentel, Handbook of Growth Factors: Volume III Hematopoietic Growth Factors and Cytokines, pp. 35-53, CRC Press, Boca Raton, FL 1994).
The IL-1 family of proteins comprises three members: IL-lα and IL-1 β (capable of inducing IL-1 biological responses) and IL-lra (a pure receptor antagonist). These ligands bind to two distinct and separate receptors: the Type I and Type II IL-1 receptors (IL-IRs). The 80-kD Type I IL-IR is found mainly on T cells and fibroblasts (Sims, J.E., et al , Science 247:585-589 (1988); Chizzonite, R., et al , Proc Natl Acad Sci USA 86 8029-8033 (1989); Sims, J.E., etal., Proc. Natl. Acad, Sci USA 56:8946-8950 (1989)). The 68-kD Type II IL-IR is found predominantly on B cells and neutrophils (Chizzonite, R., et al , Proc Natl Acad Set USA 56:8029-8033 (1989); Sims, J.E., et al , Proc Natl
Acad, Sci USA 56:8946-8950 (1989); McMahan, C.J., et al , EMBOJ 70:2821- 2832 (1991)). Both receptor types contain a large cytoplasmic region, a single transmembrane domain, and three extracellular Ig- ke domains, a structural organization that classifies them as members of the Ig superfamily The Type I IL-1 R has a cytoplasmic tail of approximately 200 ammo acids, while the Type II
IL-IR cytoplasmic tail is only 29 amino acids The agonists IL-lα and IL-lβ bind to the extracellular domains of both receptors, although with different affinities (reviewed in Dower et al , Cellular and Molecular Mechanisms of Inflammation, pp. 137-172, Academic Press, Orlando FL ) The relative importance of the Type I and Type II IL-IRs in IL-1 signaling has been recently clarified. A critical role for the Type I IL-IR in IL-1- induced activation of NF-κB, IL-6, and IL-8 secretion, and cell adhesion molecule expression has been demonstrated by several groups (Styhanou, E , et al , J Biol. Chem 267:15836-15841 (1992), Colotta, F , et al , Science 261 472- 475 (1993); Sims, J.E., et al , Proc Natl Acad Sci USA 90:6155-6159 (1993))
In contrast, the Type II IL-IR appears to be dispensable for IL-1 signaling and may act as a decoy receptor (Stylianou, E., et al , J Biol Chem 267:15836- 15841 (1992); Colotta, F., et al , Science 261 472-475 (1993); Sims, J.E., et al , Proc Natl Acad Sci USA 90:6155-6159 (1993)) While it appears clear that the Type I IL-IR is necessary for IL-1 signal transduction, it is uncertain if it is the only cell-surface molecule involved in IL-1 signaling.
It has been assumed that the functional Type I IL-IR is a single chain receptor (Curtis, B.M., et al , Proc Natl Acad Sci USA, 56:3045-3049 (1989)). However, affinity cross-linking of IL-1 to cells expressing natural IL-1 receptor has yielded complex patterns of cross-linked proteins (Dower, et al , Cellular and
Molecular Mechanisms of Inflammation, pp 137-172, Academic Press, Orlando FL (1990); Dinarello, et al., Immunol. Today, 10: 49-51 (1989)). These cross- linking studies detect molecular mass complexes consistent with both the Type I and Type II IL-lRs cross-linked to IL-1. In addition, in some studies, higher molecular mass complexes (>200 kD) are apparent (Kupper, T.S., et al, J. Clin. Invest. 52:1787-1792 (1988); Dinarello, C.A., et al, Immunol. Today 70:49-51
(1989); Solari, R., Cytokine 2:21-28 (1990); Mancilla, J., et al, Lymph. Cytokine Res. llύ 97-205 ( 1992)). Some reports have inteφreted these higher molecular mass complexes to be dimmers of receptor-ligand complexes. Others have concluded that these high molecular mass complexes maybe indicative of a multi- subunit IL-1 receptor complex.
Only two IL-IR accessory proteins are have been identified. Studies initiated to identify components of a potential IL-1 receptor complex suggest that there is a cell-surface protein in close association with the IL-IR that may play a role in IL-1 receptor binding and signaling. A murine IL-1 receptor accessory protein (mulL-lR AcP) has been cloned and expressed (Greenfeder et al. J.
Biol.Chem, 270: 13757-13765 (1995)) . This protein was present in brain, lung, spleen, and thymus tissues. A search of the GenBank data base with the mulL- 1 R AcP cDNA sequence revealed significant homology (82%) to a cDNA isolated from human infant brain (accession no. T08277) (Adams, M.D., et al, Nature Genet. 4:373-380 (1993)). No other significant homologies were found in
GenBank. The reported sequence for this partial cDNA is 396 bp long and represents one of 1600 cDNAs that were sequenced from a library made to contain only expressed sequence tags. The regions to overlap with the mulL-lR AcP sequence is nucleotides 893-1286 of the muIL-R AcP, which include the transmembrane domain. Although Adams et al. (Adams, M.D., et al, Nature
Genet. 4:373-380 (1993)) assigned no function to this partial cDNA, it is likely that it encodes a portion of a human homologue of mulIL-1 R AcP. Using the mulIL-lR AcP cDNA has >95% homology to the partial sequence of Adams et al. and -90% homology to the mulL-lR AcP cDNA. This partial cDNA was isolated as an expressed gene in infant brain. This was consistent with Northern analysis results of Greenfeder et al. demonstrating that mulIL-lR AcP mRNA is constitutively expressed at high levels in mouse brain.
The discovery of IL-IR accessory molecule has a number of implications for IL-1 receptor biology. First, while mulL-lR AcM may not bind IL-1 directly, the accessory molecule forms a complex with the muType I IL-IR allowing
IL-lβ to bind with higher affinity than the muType I IL-IR alone (Greenfeder, et al. J. Biol Chem, 270: 13757-13765 (1995)). Thus, the presence or absence of the accessory molecule in different cell lines determined whether the low or the higher affinity site was detected, suggesting that the low affinity site corresponds to the muType I IL-IR alone, while the higher affinity site represents a complex of the muType I IL-IR with the mulL-lR AcM (Greenfeder, et al J Biol Chem, 270: 13757-13765 (1995)). In this respect, the IL-I R AcM would be analogous to affinity conversion and signal transduction subunits such as gp 130 in the IL-6 system (Hibi, M., et al , Cell 63.1 149-1 157 (1990)). the common β chain of the IL-3, granulocyte/macrophage colony-stimulating factor, and IL-5 receptors
(Kitamura, T., et al, Cell 66: 1 165-1 174 ( 1991 )), and the γc subunit first identified as part of the IL-2 receptor (reviewed in Mmami, et al , Annu Rev Immunol. 11 245-267 ( 1993)).
Second, the possible existence of a multi-subunit IL-1 receptor complex contradicts a previous hypothesis that the 1 ype I IL- 1 R is the entire functional receptor for IL-1 signaling (Dower, S.K. & Sims. J.L , Cellular and Molecular Mechanisms of Inflammation, Academic Press, Orlando, FL (1990), pp. 137-172; Curtis, B.M., et al, Proc Natl. Acad. Sci USA, 56.3045-3049 (1989)) This hypothesis was based on the observation that CHO cells expressing recombinant muπne Type I IL-IR were more sensitive than control CHO cells to low concentrations of IL-1, and that the increase in sensitivity was proportional to the number of murine Type I IL-lRs (Curtis, B.M., et al , Proc Natl Acad Sci USA, 56:3045-3049 (1989)). An alternative explanation for these results is that the endogenous hamster IL-IR, was able to form a functional receptor complex with the rau Type I IL-IR, thus enhancing IL-1 signaling in the transfected cells
(Greenfeder, et al J Biol Chem, 270: 13757-13765 (1995)) Third, the discovery of the accessory protein provides an intriguing explanation for the antagonist activity of IL-lra despite its high affinity binding to the Type I IL-IR. The inability of IL-lra to interact with the mulL-lR AcP, the putative signal transducing subunit of the IL-IR complex, would result in the absence of a biological response.
Summary of the Invention
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the soluble IL-IR AcM polypeptide having the amino acid sequence is shown in Figure 1 [SEQ ID NO:2] or the amino acid sequence encoded by the cDNA clone deposited in a bacterial host as ATCC Deposit
Number 97666 on July 25, 1996. The nucleotide sequence determined by sequencing the deposited IL-IR AcM clone, which is shown in Figure 1 [SEQ ID NO:l], contains an open reading frame encoding a polypeptide of 356 amino acid residues, including an initiation codon at positions 303-305, with a leader sequence of about 17 amino acid residues, and a predicted molecular weight of about 42 kDa. The amino acid sequence of the mature IL-I R AcM protein is shown in Figure 1 , amino acid residues 18-356 fSEQ ID NO:2].
Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of : (a) a nucleotide sequence encoding the soluble IL-
IR AcM polypeptide having the complete amino acid sequence in Figure 1 [SEQ ID NO:2]; (b) a nucleotide sequence encoding the mature soluble IL-IR AcM polypeptide having the amino acid sequence at positions 18-356 in Figure 1 [SEQ ID NO:2]; (c) a nucleotide sequence encoding the soluble IL-IR AcM polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97666; (d) a nucleotide sequence encoding the mature soluble IL-IR AcM polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97666; and (e) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c) or (d) above.
Further embodiments of the invention include isolated nucleic acid molecules that comprise a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical, to any of the nucleotide sequences in (a), (b), (c), (d) or (e), above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b), (c), (d) or (e), above. This polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues. An additional nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a IL-IR AcM polypeptide having an amino acid sequence in (a), (b), (c) or (d), above. The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of soluble IL-IR AcM polypeptides or peptides by recombinant techniques. The invention further provides an isolated soluble IL-I R AcM polypeptide having amino acid sequence selected from the group consisting of: (a) the amino acid sequence of the soluble IL-IR AcM polypeptide having the complete 356 amino acid sequence, including the leader sequence shown in Figure 1 [SEQ ID NO:2]; (b) the amino acid sequence of the mature soluble IL- 1 R AcM polypeptide (without the leader) having the amino acid sequence at positions 18-356 in Figure 1 [SEQ ID NO:2]; (c) the amino acid sequence of the soluble IL-IR AcM polypeptide having the complete amino acid sequence, including the leader, encoded by the cDNA clone contained in ATCC Deposit No. 97666; and (d) the amino acid sequence of the mature soluble IL-IR AcM polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97666. The polypeptides of the present invention also include polypeptides having an amino acid sequence with at least 90% similarity, and more preferably at least 95% similarity to those described in (a), (b), (c) or (d) above, as well as polypeptides having an amino acid sequence at least 80% identical, more preferably at least 90% identical, and still more preferably 95%, 96%, 97%, 98% or 99% identical to those above.
An additional embodiment of this aspect of the invention relates to a peptide or polypeptide which has the amino acid sequence of an epitope-bearing portion of a soluble IL-IR AcM polypeptide having an amino acid sequence described in (a), (b), (c) or (d), above. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a soluble IL- 1 R AcM polypeptide of the invention include portions of such polypeptides with at least six or seven, preferably at least nine, and more preferably at least about 30 amino acids to about 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the invention described above also are included in the invention. In another embodiment, the invention provides an isolated antibody that binds specifically to a soluble IL-IR AcM polypeptide having an amino acid sequence described in (a), (b), (c) or (d) above.
The soluble IL-IR AcM may not bind IL-1 directly, however, the accessory molecule forms a complex with the Type 1 IL-IR that binds IL-lβ with higher affinity than the Type I IL-IR alone. Thus, the presence or absence of the accessory molecule in different cell lines determines whether the low or the higher affinity site is detected suggesting that the low affinity site corresponds to the Type I IL-IR alone, while the higher affinity site represents a complex of the Type I IL-IR with the IL-IR AcM. The present invention further provides a screening method for identifying IL-1 receptor agonists and antagonists, which involves: (a) providing a polypeptide comprising a type I IL-1 receptor and a polypeptide comprising IL-IR AcM or IL-IR AcM fragment, wherein IL-IR and IL-IR AcM or IL-IR and the IL-IR AcM fragment form a complex; (b) providing a candidate compound; (c) providing a polypeptide conprising IL-1 or a functional II- 1 fragment; and (d) determining the binding affinity of said complex for IL-1 whereby an increased binding affinity of said complex for IL-1 in the presence of said compound is indicative that said compound is an agonist for IL-1 signal transduction and a decreased binding affinity of said complex for IL-1 in the presence of said compound is indicative that said compound is an antagonist of IL-1 signal transduction.
Brief Description of the Figures
Figure 1 shows the nucleotide [SEQ ID NO: 1] and deduced amino acid [SEQ ID NO:2] sequences of soluble IL-IR AcM. The protein has a leader sequence of about 17 amino acid residues (underlined) and a deduced molecular weight of about 42 kDa. The predicted amino acid sequence of the mature soluble IL-IR AcM protein is also shown in Figure 1 [SEQ ID NO:2].
Figures 2A and B shows the regions of similarity between the amino acid sequences of the soluble IL-IR AcM protein (HMEEJ22) and mouse interleukin 1 receptor accessory protein [SEQ ID NO:3] and between the amino acid sequences of the soluble IL-IR AcM protein (HMEEJ22) and the partial cDNA isolated from human infant brain (Adams, M.D.. et al.. Nature Genet. 4:373-380 (1993)) [SEQ ID NO:4].
Figure 3 shows an analysis of the IL-I R AcM amino acid sequence. Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown. In the "Antigenic Index - Jameson- Wolf ' graph (boxed graph), the amino acid sequence of the IL-IR AcM protein is shown with the amino aicds that border each peak from the antigenic index plot displayed as underlined characters. Detailed Description
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding soluble IL-IR AcM polypeptide, having the amino acid sequence shown in Figure 1 [SEQ ID NO:2], which was determined by sequencing a cloned cDNA. The soluble IL-IR AcM protein of the present invention shares sequence homology with mouse interleukin 1 receptor accessory protein (Figure 2) [SEQ ID NO:3]. The nucleotide sequence shown in Figure 1 [SEQ ID NO: 1 ] was obtained by sequencing the HMEEJ22 clone, which was deposited on July 25, 1996 at the American Type Culture Collection, 12301 Park Lawn Drive, Rockville, Maryland 20852, and given accession number 97666.
Accordingly, in one embodiment of the present invention, isolated nucleic acid molecules are provided which encode the soluble IL-IR AcM protein. The IL-IR AcM is a novel member of the Ig superfamily.
Nucleic Acid Molecules
Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
Unless otherwise indicated, each "nucleotide sequence" set forth herein is presented as a sequence of deoxyribonucleotides (abbreviated A, G , C and T). However, by "nucleotide sequence" of a nucleic acid molecule or polynucleotide is intended, for a DNA molecule or polynucleotide, a sequence of deoxyribonucleotides, and for an RNA molecule or polynucleotide, the corresponding sequence of ribonucleotides (A, G, C and U), where each thymidine deoxyribonucleotide (T) in the specified deoxyribonucleotide sequence is replaced by the ribonucleotide uridine (U). For instance, reference to an RNA molecule having the sequence of SEQ ID NO:l set forth using deoxyribonucleotide abbreviations is intended to indicate an RNA molecule having a sequence in which each deoxyribonucleotide A, G or C of SEQ ID NO: 1 has been replaced by the corresponding ribonucleotide A, G or C, and each deoxyribonucleotide T has been replaced by a ribonucleotide U. Using the information provided herein, such as the nucleotide sequence in Figure 1, a nucleic acid molecule of the present invention encoding a soluble IL-IR AcM polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material. Illustrative of the invention, the nucleic acid molecule described in Figure 1 [SEQ ID NO: 1] was discovered in a cDNA library derived from human microvascular endothelial cells. The determined nucleotide sequence of the soluble IL-IR AcM cDNA of Figure 1 [SEQ ID NO: l] contains an open reading frame encoding a protein of 356 amino acid residues, with an initiation codon at positions 303-306 of the nucleotide sequence in Figure 1 [SEQ ID NO:l], a predicted leader sequence of about 17 amino acid residues, and a deduced molecular weight of about 42 kDa. The amino acid sequence of the predicted mature soluble IL-IR AcM is shown in Figure 1 [SEQ ID NO:2] from amino acid residue 18 to residue 356. The soluble IL-IR AcM protein shown in Figure 1 [SEQ ID NO:2] is about 94% similar and 85% identical to mouse interleukin 1 accessory protein (Figure 2A). In addition, the nucleotides 1060 to 1353 of soluble IL-IR AcM protein shown in Figure 1 [SEQ ID NO:2] is about 99% similar and 98% identical to the first 294 nucleotides partial cDNA isolated from human infant brain by Adams, M.D., et al, Nature Genet. 4:373-380 (1993) [SEQ ID NO:4](Figure 2B). The partial cDNA isolated by Adams was 396 nucleotides in length.
As one of ordinary skill would appreciate, due to the possibilities of sequencing errors discussed above, as well as the variability of cleavage sites for leaders in different known proteins, the actual soluble IL-IR AcM polypeptide encoded by the deposited cDNA comprises about 356 amino acids, but may be anywhere in the range of 345-370 amino acids; and the actual leader sequence of this protein is about 17 amino acids, but may be anywhere in the range of about 10 to about 20 amino acids.
As indicated, nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically. The DNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non- coding strand, also referred to as the anti-sense strand.
By "isolated" nucleic acid molecule(s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated
DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically. Isolated nucleic acid molecules of the present invention include DNA molecules comprising an open reading frame (ORF) with an initiation codon at positions 303-306 of the nucleotide sequence shown in Figure 1 [SEQ ID NO:l]; DNA molecules comprising the coding sequence for the mature soluble IL-IR AcM protein shown in Figure 1 (last 339 amino acids) [SEQ ID NO:2]; and DNA molecules which comprise a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the soluble IL-IR AcM protein. Of course, the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate the degenerate variants described above.
In another aspect, the invention provides isolated nucleic acid molecules encoding the soluble IL-IR AcM polypeptide having an amino acid sequence encoded by the cDNA clone contained in the plasmid deposited as ATCC Deposit No. 97666 on July 25, 1996. Preferably, this nucleic acid molecule will encode the mature polypeptide encoded by the above-described deposited cDNA clone.
The invention further provides an isolated nucleic acid molecule having the nucleotide sequence shown in Figure 1 (SEQ ID NO: l ] or the nucleotide sequence of the soluble IL-IR AcM cDNA contained in the above-described deposited clone, or a nucleic acid molecule having a sequence complementary to one of the above sequences. Such isolated molecules, particularly DNA molecules, are useful as probes for gene mapping, by in situ hybridization with chromosomes, and for detecting expression of the soluble IL-I R AcM gene in human tissue, for instance, by Northern blot analysis.
In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, for instance, the cDNA clone contained in ATCC Deposit 97666. By "stringent hybridization conditions" is intended overnight incubation at 42 °C in a solution comprising: 50%> formamide, 5x SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x
Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 x SSC at about 65 °C. By a polynucleotide which hybridizes to a "portion" of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70 nt of the reference polynucleotide. These are useful as diagnostic probes and primers as discussed above and in more detail below.
Of course, polynucleotides hybridizing to a larger portion of the reference polynucleotide (e.g., the deposited cDNA clone), for instance, a portion 50-750 nt in length, or even to the entire length of the reference polynucleotide, are also useful as probes according to the present invention, as are polynucleotides corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in Figure 1 [SEQ ID NO:l]. By a portion of a polynucleotide of "at least 20 nt in length," for example, is intended 20 or more contiguous nucleotides from the nucleotide sequence of the reference polynucleotide (e.g., the deposited cDNA or the nucleotide sequence as shown in Figure 1 [SEQ ID NO:l]). As indicated, such portions are useful diagnostically either as a probe according to conventional DNA hybridization techniques or as primers for amplification of a target sequence by the polymerase chain reaction (PCR), as described, for instance, in Molecular Cloning, A
Laboratory Manual, 2nd. edition, edited by Sambrook, J., Fritsch, E. F. and Maniatis, T., (1989), Cold Spring Harbor Laboratory Press, the entire disclosure of which is hereby incoφorated herein by reference.
Since a soluble IL-IR AcM cDNA clone has been deposited and its determined nucleotide sequence is provided in Figure 1 [SEQ ID NO:lJ, generating polynucleotides which hybridize to a portion of the soluble IL-IR AcM cDNA molecule would be routine to the skilled artisan. For example, restriction endonuclease cleavage or shearing by sonication of the soluble IL-IR AcM cDNA clone could easily be used to generate DNA portions of various sizes which are polynucleotides that hybridize to a portion of the soluble IL- 1 R AcM cDNA molecule. Alternatively, the hybridizing polynucleotides of the present invention could be generated synthetically according to known techniques. Of course, a polynucleotide which hybridizes only to a poly A sequence (such as the 3' terminal poly(A) tract of the soluble IL-IR AcM cDNA shown in Figure 1 [SEQ ID NO.T]), or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide of the invention used to hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).
The invention further provides isolated nucleic acid molecules comprising a polynucleotide encoding an epitope-bearing portion of the soluble IL- 1 R AcM protein. In particular, isolated nucleic acid molecules are provided encoding polypeptides comprising the following amino acid residues in Figure 1 (SEQ ID NO:2), which the present inventors have determined are antigenic regions of the soluble IL-IR AcM protein: a. S18-M32: SDASERCDDWGLDTM (SEQ ID NO:5) b. V37-I45: VFEDEPARI (SEQ ID NO:6) c. G65-Y71 : GLTLIWY (SEQ ID NO: 7) d. R74-I83: RQDRDLEEPI (SEQ ID NO: 8) e. L87-K96: LPENRISKEK (SEQ ID NO:9) f. S123-L129: SKVAFPL (SEQ ID NOTO) g. V132-P141 : VQKDSCFNSP (SEQ ID NO:l 1 ) h. V146-E152: VHKLYIE (SEQ ID NO: 12) i. I175-N189: ITWYMGCYKIQNFNN (SEQ ID NO: 13) j. LI 97-1204: LSFLIALI (SEQ ID NO: 14) k. H224-K232: HLTRTLTVK (SEQ ID NO: 15)
1. P248-E261 : PNDHVVYEKEPGEE (SEQ ID NO: 16) m. I264-F272: IPCTVYFSF (SEQ ID NO: 17) n. D285-T293: DGKKPDDIT (SEQ ID NO: 18) o. I302-R312: ISHSRTEDETR (SEQ ID NO: 19) p. K320-K327: KVTSEDLK (SEQ ID NO: 20) q. S336-V347: SAKEGEVAKAAKV (SEQ ID NO: 21) r. 350-Q356: KGNRCGQ (SEQ ID NO: 22) Methods for generating such epitope-bearing portions of soluble IL-IR AcM are described in detail below.
As indicated, nucleic acid molecules of the present invention which encode a soluble IL-IR AcM polypeptide may include, but are not limited to those encoding the amino acid sequence of the mature polypeptide, by itself; the coding sequence for the mature polypeptide and additional sequences, such as those encoding the about 17 amino acid leader or secretory sequence, such as a pre-, or pro- or prepro- protein sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 31 sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals, for example - ribosome binding and stability of mRNA; an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities. Thus, the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the fused polypeptide. In certain preferred embodiments of this aspect of the invention, the marker amino acid sequence is a hexa-histidine peptide. such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al, Proc. Natl. Acad. Sci. USA 56:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. The "HA" tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al, Cell 37: 767 (1984).
The present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of the soluble IL-IR AcM protein. Variants may occur naturally, such as a natural allelic variant. By an "allelic variant" is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally occurring variants may be produced using art-known mutagenesis techniques.
Such variants include those produced by nucleotide substitutions, deletions or additions. The substitutions, deletions or additions may involve one or more nucleotides. The variants may be altered in coding regoins, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the soluble IL-IR AcM protein or portions thereof. Also especially preferred in this regard are conservative substitutions. Most highly preferred are nucleic acid molecules encoding the mature protein having the amino acid sequence shown in Figure 1 [SEQ ID NO:2] or the mature soluble IL-IR AcM amino acid sequence encoded by the deposited cDNA clone. Further embodiments of the invention include isolated nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical to (a) a nucleotide sequence encoding the full-length soluble IL-IR AcM polypeptide having the complete amino acid sequence in Figure 1 [SEQ ID NO:2], including the predicted leader sequence; (b) a nucleotide sequence encoding the mature soluble IL-IR AcM polypeptide (full-length polypeptide with the leader removed) having the amino acid sequence at positions 18-356 in Figure 1 [SEQ ID NO:2]; (c) a nucleotide sequence encoding the full-length soluble IL-IR AcM polypeptide having the complete amino acid sequence including the leader encoded by the cDNA clone contained in ATCC Deposit No.
97666; (d) a nucleotide sequence encoding the mature soluble IL-IR AcM polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97666; or (e) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c) or (d). By a polynucleotide having a nucleotide sequence at least, for example,
95%) "identical" to a reference nucleotide sequence encoding a soluble IL-IR AcM polypeptide is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the soluble IL-IR AcM polypeptide. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
As a practical matter, whether any particular nucleic acid molecule is at least 90%, 95%, 96%, 97%, 98%) or 99% identical to, for instance, the nucleotide sequence shown in Figure 1 or to the nucleotides sequence of the deposited cDNA clone can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981), to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
The present application is directed to nucleic acid molecules at least 90%, 95%o, 96%», 97%, 98% or 99% identical to the nucleic acid sequence shown in
Figure 1 [SEQ ID NO:l] or to the nucleic acid sequence of the deposited cDNA, irrespective of whether they encode a polypeptide having soluble IL-IR AcM activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having soluble IL-IR AcM activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer. Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having soluble IL-IR AcM activity include, inter alia, (1) isolating the soluble IL-IR AcM gene or allelic variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of the soluble IL-IR AcM gene, as described in
Verma et al, Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988); and Northern Blot analysis for detecting soluble IL-1 R AcM mRNA expression in specific tissues.
Preferred, however, are nucleic acid molecules having sequences at least 90%), 95%), 97%, 98% or 99% identical to the nucleic acid sequence shown in
Figure 1 [SEQ ID NO:l] or to the nucleic acid sequence of the deposited cDNA which do, in fact, encode a polypeptide having soluble IL-IR AcM protein activity. By "a polypeptide having soluble IL-I R AcM activity" is intended polypeptides exhibiting activity similar, but not necessarily identical, to an activity of the soluble IL-IR AcM protein of the invention (either the full-length protein or, preferably, the mature protein), as measured in a particular biological assay. Assays of IL-1 R AcM protein activity are well-known to those in the art. These assays can be used to measure IL-IR AcM protein activity of partially purified or purified native or recombinant protein. For example, an equilibrium and competitive binding studies using CHO stable cell lines
(Greenfeder et al, J. Biol. Chem. 270: 13757-13765 (1995)) can be performed to detect IL-IR AcM activity.
For this assay, a CHO-IR/AcM cell line is established by simultaneous cotransfection of two expression vectors such that both IL-1 R and IL-IR AcM or a candidate IL-IR AcMare expressed at about a 1 :10 ratio of molecules/cell. In addition, control cell lines which express only IL-IR or IL-IR AcM are also established. To establish the stable cell lines, CHO-dhfr cells are maintained in DMEM with 10% fetal bovine serum, 25mM HEPES, pH 7.0, 0.1 mML glutamine, IX HT supplement (0.1 mM hypoxanthine, 0.016 mM thymidine) (Boehringer Mannheim), 50 μg/ml gentamicin, IX penicillin/streptomycin/fungizone (JRH Biosciences). Cells are transfected with pSV2-dhfr (Subramani, et al Mol. Cell. Biol. 1: 845-864 (1981)) either alone or in combination with expression vectors containing IL-IR, IL-IR AcM protein and a candidate IL-IR AcM protein by the CaPO4 method following the manufacturer's directions (Stratagene). After three days, cells are transferred to medium lacking HT and allowed to grow an additional two weeks. Transfectants are then subjected to gene amplification by growth in increasing doses of methotrexate (0.1-1.0 μM). Clones are isolated by limiting dilution and screened by equilibrium binding with 125I-labeled IL-1, anti-IL-lR antibody or anti-IL-lR AcM antibody. To characterize the number and affinities of 1L- 1 β binding sites, each of the above generated cell lines are analyzed by equilibrium binding with 125I- labeled IL-1 β. Equilibrium binding of 12SI-labeled IL-1 to the cells can performed as described by Mizel, et al. J. Immunol. 138: 2906-2912 (1987). ,25I labeling of IL-lβ can be performed by methods well known to those skilled in the art, for example, as described by Chizzonite, et al. J. Immunol. 147:1548-1556 (1991).
The activity of the accessory protein, IL-IR AcM, can be examined in the binding of IL-lβ to a CHO-IR/AcM cell line obtained above. It is desirable for this cell line to express an excess amount of IL-IR AcM protein relative to IL- IR. Cell lines bearing only IL-IR AcM protein do not bind IL-lβ and cell lines bearing only IL-R will bind IL-lβ with low affinity (i.e., approximately Kn 1.0-
3.3nM). A CHO-IR/AcM cell line, bearing both the IL-IR and IL-IR AcM results in IL-IR having a higher affinity binding site (i.e., approximately KD 0.02- 0.8 nM). Thus, one can monitor the presence of IL-1 R AcM activity by testing whether the putative accessory protein interacts with the IL-IR so as to generate a high affinity IL-lβ binding site. The IL-IR AcM protein of the present invention can serve as a reference for the assay for IL-IR AcM activity associated with the high affinity IL-IR binding state.
In addition to the above described assay, one can evaluate whether a candidate polypeptide has IL-IR AcM activity by performing simple binding kinetics can be measured to determine receptor affinity for the ligand (i.e., IL- 1 β).
Binding kinetic analysis experiments are well known to those skilled in the art (Chizzonite et al, Proc Natl. Acad. Sci. 56:8029-8033 (1989); Mizel, et al, J. Immunol. 735:2906-2912 (1987)). One can monitor the presence of IL-IR AcM activity by testing whether the putative accessory protein interacts with the IL-IR so as to result in high affinity binding of IL- 1 β to the receptor. The IL-IR AcM protein of the present invention can serve as a reference for the assay for IL-IR AcM activity associated with the high affinity IL- 1 R binding state.
Thus, "a polypeptide having soluble IL-I R AcM protein activity" includes polypeptides that exhibit IL-IR AcM activity, in the above-described assay. Although the degree of activity need not be identical to that of the IL-IR
AcM protein, preferably, "a polypeptide having IL-I R AcM protein activity" will exhibit substantially similar activity as compared to the IL-IR AcM protein (i.e., the candidate polypeptide will exhibit greater activity or not more than about tenfold less and, preferably, not more than about ten- old less activity relative to the reference IL-IR AcM protein).
Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of the deposited cDNA or the nucleic acid sequence shown in Figure 1 [SEQ ID NO:l] will encode a polypeptide "having soluble IL-IR AcM protein activity." In fact, since degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay. It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having soluble IL-IR AcM protein activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid).
For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie, J. U. et al, "Deciphering the
Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247.T 306- 1310 (1990), wherein the authors indicate that there are two main approaches for studying the tolerance of an amino acid sequence to change. The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection. The second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selections or screens to identify sequences that maintain functionality. As the authors state, these studies have revealed that proteins are suφrisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at a certain position of the protein. For example, most buried amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Other such phenotypically silent substitutions are described in Bowie, J.U. et al, supra, and the references cited therein.
Vectors and Host Cells
The present invention also relates to vectors which include the isolated DNA molecules of the present invention, host cells which are genetically engineered with the recombinant vectors, and the production of soluble IL-IR AcM polypeptides or fragments thereof by recombinant techniques. Recombinant constructs may be introduced into host cells using well known techniques such infection, transduction, transfection, transvection, electroporation and transformation. The vector may be, for example, a phage, plasmid, viral or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
The polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
Preferred are vectors comprising cis-acting control regions to the polynucleotide of interest. Appropriate trans-acting factors may be supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.
In certain preferred embodiments in this regard, the vectors provide for specific expression, which may be inducible and/or cell type-specific. Particularly preferred among such vectors are those inducible by environmental factors that are easy to manipulate, such as temperature and nutrient additives.
Expression vectors useful in the present invention include chromosomal-, episomal- and virus-derived vectors, e.g., vectors derived from bacterial plasmids, bacteriophage, yeast episomes, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adcnoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as cosmids and phagemids.
The DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated. As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli,
Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art. Among vectors preferred for use in bacteria include pA2, pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.
Among known bacterial promoters suitable for use in the present invention include the E. coli lacl and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter.
Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV), and metallothionein promoters, such as the mouse metallothionein-I promoter. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid- mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al, Basic Methods In Molecular Biology (1986). Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incoφorated into the expressed polypeptide. The signals may be endogenous to the polypeptide or they may be heterologous signals.
The polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art. A preferred fusion protein comprises a heterologous region from immunoglobulin that is useful to solubilize receptors. For example, EP-A-O 464 533 (Canadian counteφart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another human protein or part thereof. In many cases, the Fc part in fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232262). On the other hand, for some uses it would be desirable to be able to delete the Fc pat after the fusion protein has been expressed, detected and purified in the advantageous manner described. This is the case when Fc portion proves to be a hindrance to use in therapy and diagnosis, for example when the fusion protein is to be used as antigen for immunizations. In drug discovery, for example, human proteins, such as, shIL5- has been fused with Fc portions for the piupose of high- throughput screening assays to identify antagonists of hIL-5. See, D. Bennett et al., Journal of Molecular Recognition, Vol. 8 52-58 (1995) and K. Johanson et al., The Journal of Biological Chemistry, Vol. 270, No. 16, pp 9459-9471 (1995). The soluble IL-IR AcM protein can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification. Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
IL-IR AcM Polypeptides and Fragments
The invention further provides an isolated soluble IL-IR AcM polypeptide having the amino acid sequence encoded by the deposited cDNA, or the amino acid sequence in Figure 1 [SEQ ID NO:2J, or a peptide or polypeptide comprising a portion of the above polypeptides. The terms "peptide" and "oligopeptide" are considered synonymous (as is commonly recognized) and each term can be used interchangeably as the context requires to indicate a chain of at least to amino acids coupled by peptidyl linkages. The word "polypeptide" is used herein for chains containing more than ten amino acid residues. All oligopeptide and polypeptide formulas or sequences herein are written from left to right and in the direction from amino terminus to carboxy terminus.
It will be recognized in the art that some amino acid sequences of the soluble IL-IR AcM polypeptide can be varied without significant effect of the structure or function of the protein. If such differences in sequence are contemplated, it should be remembered that there will be critical areas on the protein which determine activity. In general, it is possible to replace residues which form the tertiary structure, provided that residues performing a similar function are used. In other instances, the type of residue may be completely unimportant if the alteration occurs at a non-critical region of the protein.
Thus, the invention further includes variations of the soluble IL-IR AcM polypeptide which show substantial soluble IL-IR AcM polypeptide activity or which include regions of soluble IL-IR AcM protein such as the protein portions discussed below. Such mutants include deletions, insertions, inversions, repeats, and type substitutions (for example, substituting one hydrophilic residue for another, but not strongly hydrophilic for strongly hydrophobic as a rule). Small changes or such "neutral" amino acid substitutions will generally have little effect on activity. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and He; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gin, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr. As indicated in detail above, further guidance concerning which amino acid changes are likely to be phenotypically silent (i.e., are not likely to have a significant deleterious effect on a function) can be found in Bowie, J.U., et al, "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247. 1306-1310 (1990). The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of the soluble IL-IR AcM polypeptide can be substantially purified by the method described by Bradley, M.K. "Overexpressio of Proteins in Eukaryotes" Methods in Enzymology, Vol. 182.
The polypeptides of the present invention include the polypeptide encoded by the deposited cDNA including the leader, the mature polypeptide encoded by the deposited the cDNA minus the leader (i.e., the mature protein), the polypeptide of Figure 1 [SEQ ID NO:2] including the leader, the polypeptide of Figure 1 [SEQ ID NO:2] minus the leader, as well as polypeptides which have at least 90%) similarity, more preferably at least 95% similarity, and still more preferably at least 96%, 91%, 98% or 99% similarity to those described above.
Further polypeptides of the present invention include polypeptides at least 80% identical, more preferably at least 90% or 95% identical, still more preferably at least 96%), 97%, 98% or 99% identical to the polypeptide encoded by the deposited cDNA, to the polypeptide of Figure 1 [SEQ ID NO:2], and also include portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids.
By "% similarity" for two polypeptides is intended a similarity score produced by comparing the amino acid sequences of the two polypeptides using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive,
Madison, WI 53711) and the default settings for determining similarity. Bestfit uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2: 482-489, 1981) to find the best segment of similarity between two sequences. By a polypeptide having an amino acid sequence at least, for example,
95%> "identical" to a reference amino acid sequence of a soluble IL-IR AcM polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of the soluble IL-1 R AcM polypeptide. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5%> of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
As a practical matter, whether any particular polypeptide is at least 90%, 95%, 96%), 97%, 98% or 99% identical to, for instance, the amino acid sequence shown in Figure 1 [SEQ ID NO:2J or to the amino acid sequence encoded by deposited cDNA clone can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 5371 1. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5%> of the total number of amino acid residues in the reference sequence are allowed.
The polypeptide of the present invention could be used as a molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art. As described in detail below, the polypeptides of the present invention can also be used to raise polyclonal and monoclonal antibodies, which are useful in assays for detecting soluble IL-IR AcM protein expression as described below or as agonists and antagonists capable of enhancing or inhibiting soluble IL-IR AcM protein function. Further, such polypeptides can be used in the yeast two- hybrid system to "capture" soluble IL-IR AcM protein binding proteins which are also candidate agonist and antagonist according to the present invention. The yeast two hybrid system is described in Fields and Song, Nature 340:245-246 (1989).
In another aspect, the invention provides a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention. The epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention. An "immunogenic epitope" is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen. These immunogenic epitopes are believed to be confined to a few loci on the molecule. On the other hand, a region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope." The number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes. See, for instance, Geysen et al, Proc. Natl. Acad. Sci. USA 57:3998- 4002 (1983).
As to the selection of peptides or polypeptides bearing an antigenic epitope (i.e., that contain a region of a protein molecule to which an antibody can bind), it is well known in that art that relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M., Green, N. and Learner, R.A. (1983) Antibodies that react with predetermined sites on proteins. Science 219:660-666. Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals. Peptides that are extremely hydrophobic and those of six or fewer residues generally are ineffective at inducing antibodies that bind to the mimicked protein; longer, soluble peptides, especially those containing proline residues, usually are effective. Sutcliffe et al., supra, at 661. For instance, 18 of 20 peptides designed according to these guidelines, containing 8-39 residues covering 15% of the sequence of the influenza virus hemagglutinin HA1 polypeptide chain, induced antibodies that reacted with the HA1 protein or intact virus; and 12/12 peptides from the MuLV polymerase and 18/18 from the rabies glycoprotein induced antibodies that precipitated the respective proteins.
Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention. Thus, a high proportion of hybridomas obtained by fusion of spleen cells from donors immunized with an antigen epitope-bearing peptide generally secrete antibody reactive with the native protein. Sutcliffe et al., supra, at 663. The antibodies raised by antigenic epitope-bearing peptides or polypeptides are useful to detect the mimicked protein, and antibodies to different peptides may be used for tracking the fate of various regions of a protein precursor which undergoes post-translational processing. The peptides and anti-peptide antibodies may be used in a variety of qualitative or quantitative assays for the mimicked protein, for instance in competition assays since it has been shown that even short peptides (e.g., about 9 amino acids) can bind and displace the larger peptides in immunoprecipitation assays. See, for instance, Wilson et al. Cell 37:761-118 (1984) at 777. The anti- peptide antibodies of the invention also are useful for purification of the mimicked protein, for instance, by adsoφtion chromatography using methods well known in the art. Antigenic epitope-bearing peptides and polypeptides of the invention designed according to the above guidelines preferably contain a sequence of at least seven, more preferably at least nine and most preferably between about 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention. However, peptides or polypeptides comprising a larger portion of an amino acid sequence of a polypeptide of the invention, containing about 30 to about 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention, also are considered epitope-bearing peptides or polypeptides of the invention and also are useful for inducing antibodies that react with the mimicked protein. Preferably, the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues and highly hydrophobic sequences are preferably avoided); and sequences containing proline residues are particularly preferred.
Non-limiting examples of antigenic polypeptides or peptides that can be used to generate soluble IL-IR AcM-specific antibodies include the following: a. S 18-M32: SDASERCDDWGLDTM (SEQ ID NO:5) b. V37-I45: VFEDEPARI (SEQ ID NO:6) c. G65-Y71 : GLTLIWY (SEQ ID NO:7) d. R74-I83: RQDRDLEEPI (SEQ ID NO: 8) e. L87-K96: LPENRISKEK (SEQ ID NO:9) f. S123-L129: SKVAFPL (SEQ ID NO: 10) g. V132-P141 : VQKDSCFNSP (SEQ ID NO:l 1) h. V146-E152: VHKLYIE (SEQ ID NO: 12) i. I175-N189: ITWYMGCYKIQNFNN (SEQ ID NO: 13) j. L197-I204: LSFLIALI (SEQ ID NO: 14) k. H224-K232: HLTRTLTVK (SEQ ID NO: 15)
1. P248-E261 : PNDHVVYEKEPGEE (SEQ ID NO: 16) m. I264-F272: IPCTVYFSF (SEQ ID NO: 17) n. D285-T293: DGKKPDDIT (SEQ ID NO: 18) o. I302-R312: ISHSRTEDETR (SEQ ID NO: 19) p. K320-K327: KVTSEDLK (SEQ ID NO: 20) q. S336-V347: SAKEGEVAKAAKV (SEQ ID NO: 21) r. K350-Q356: KGNRCGQ (SEQ ID NO: 22)
The epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means for making peptides or polypeptides including recombinant means using nucleic acid molecules of the invention. For instance, a short epitope-bearing amino acid sequence may be fused to a larger polypeptide which acts as a carrier during recombinant production and purification, as well as during immunization to produce anti-peptide antibodies. Epitope-bearing peptides also may be synthesized using known methods of chemical synthesis. For instance, Houghten has described a simple method for synthesis of large numbers of peptides, such as 10-20 mg of 248 different 13 residue peptides representing single amino acid variants of a segment of the HA1 polypeptide which were prepared and characteπzed (by ELISA-type binding studies) in less than four weeks. Houghten, R A. (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc Natl
Acad Sci USA 52:5131-5135. This "Simultaneous Multiple Peptide Synthesis (SMPS)" process is further described in U S Patent No 4,631,21 1 to Houghten et al. (1986). In this procedure the individual resins for the solid-phase synthesis of various peptides are contained m separate solvent-permeable packets, enabling the optimal use of the many identical repetitive steps involved in solid-phase methods. A completely manual procedure allows 500-1000 or more syntheses to be conducted simultaneously. Houghten et al , supra, at 5134
Epitope-bearing peptides and polypeptides of the invention are used to induce antibodies according to methods well known in the art See, for instance, Sutcliffe et al , supra; Wilson et al , supra. Chow, M et al , Proc Natl Acad Sci
USA 52:910-914, and Bittle, F J. et al , Gen Vitol 66 2347-2354 (1985) Generally, animals may be immunized with free peptide, however, anti-peptide antibody titer may be boosted by coupling of the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid For instance, peptides containing cysteine may be coupled to carrier using a linker such as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carrier using a more general linking agent such as glutaraldehyde Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intrapeπtoneal and/or intradermal injection of emulsions containing about 100 μg peptide or carrier protein and Freund's adjuvant Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsoφtion to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
Immunogenic epitope-bearing peptides of the invention, i.e., those parts of a protein that elicit an antibody response when the whole protein is the immunogen, are identified according to methods known in the art. For instance,
Geysen et al. , supra, discloses a procedure for rapid concurrent synthesis on solid supports of hundreds of peptides of sufficient purity to react in an enzyme-linked immunosorbent assay. Interaction of synthesized peptides with antibodies is then easily detected without removing them from the support. In this manner a peptide bearing an immunogenic epitope of a desired protein may be identified routinely by one of ordinary skill in the art. For instance, the immunologically important epitope in the coat protein of foot-and-mouth disease virus was located by Geysen et al. with a resolution of seven amino acids by synthesis of an overlapping set of all 208 possible hexapeptides covering the entire 213 amino acid sequence of the protein. Then, a complete replacement set of peptides in which all 20 amino acids were substituted in turn at every position within the epitope were synthesized, and the particular amino acids conferring specificity for the reaction with antibody were determined. Thus, peptide analogs of the epitope-bearing peptides of the invention can be made routinely by this method. U.S. Patent No. 4,708,781 to Geysen (1987) further describes this method of identifying a peptide bearing an immunogenic epitope of a desired protein.
Further still, U.S. Patent No. 5,194,392 to Geysen (1990) describes a general method of detecting or determining the sequence of monomers (amino acids or other compounds) which is a topological equivalent of the epitope (i.e., a "mimotope") which is complementary to a particular paratope (antigen binding site) of an antibody of interest. More generally, U.S. Patent No. 4,433,092 to Geysen (1989) describes a method of detecting or determining a sequence of monomers which is a topographical equivalent of a ligand which is complementary to the ligand binding site of a particular receptor of interest. Similarly, U.S. Patent No. 5,480,971 to Houghten, R. A. et al. (1996) on
Peralkylated Oligopeptide Mixtures discloses linear C,-C7-alkyl peralkylated oligopeptides and sets and libraries of such peptides, as well as methods for using such oligopeptide sets and libraries for determining the sequence of a peralkylated oligopeptide that preferentially binds to an acceptor molecule of interest. Thus, non-peptide analogs of the epitope-bearing peptides of the invention also can be made routinely by these methods.
The entire disclosure of each document cited in this section on "Polypeptides and Peptides" is hereby incoφorated herein by reference.
Screening Applications of Soluble IL-IR AcM
The present inventors believe that soluble IL-IR AcM is involved in IL-1 activity and that it forms a complex with the Type I IL- 1 R allowing IL- 1 β to bind with higher affinity than to the Type I IL-IR alone. Thus, the presence or absence of the accessory molecule in different cell lines may determine whether the low or the high affinity site in the Type I IL-IR is formed. The low affinity site corresponds to the Type I IL-IR alone, while the higher affinity site represents a complex of the Type I IL-IR with the IL-IR AcM.
Given that IL-1 has many diverse effects on immunologic and inflammatory process and that IL-1 elicits a wide variety of effects in hematic and nonhematic cells, it would be of interest to identify agonist and antagonist for IL-1 activity. IL-IR AcM may to be necessary for IL-1 signal transduction events and may to be required for the formation of a high affinity IL- 1 R binding state. Stable cell lines established by simultaneous cotransfection of two expression vectors such that both IL-IR and IL-IR AcM are expressed at a 1 :10 ratio of molecules/cell and control cell lines which express only IL-IR or IL-IR AcM could be used in screening assays to identify potential agonists and antagonists for IL- 1. Thus, the present invention further provides a screening method of identifying IL-1 receptor agonists, which involves: (a) providing a host cell containing recombinant genes which express a polypeptide comprising a type I IL-1 receptor and a polypeptide comprising IL-I R AcM or a IL-IR AcM fragment, wherein IL-IR and IL-IR AcM or an IL-IR AcM fragment form a complex; (b) administering a candidate agonist to said cell; and (c) determining the binding affinity of said complex for said candidate agonist relative to the binding of said comples for IL-1.
To characterize candidate IL-IR agonist binding to IL-IR, each of the cell lines are analyzed by equilibrium binding with 125I-labeled IL-lβ.
Equilibrium binding of 125I-labeled IL-lβ to the cells can performed as described by Mizel, et al. J. Immunol. 138: 2906-2912 (1987) and Greenfeder et al, J. Biol. Chem. 270:13757- 13765 (1995). The activity of the candidate agonist, can be examined relative to the binding of IL-lβ to a CHO-IR/AcM cell line (as described, supra). It is desirable for this cell line to express an excess amount of
IL-IR AcM protein relative to IL-IR. Cell lines bearing only IL-IR AcM protein will not bind IL-lβ and cell lines bearing only IL-R bind IL-lβ with low affinity (i.e., on the order of KD 1.0-3.3nM). A CHO-IR/AcM cell line, bearing both the IL-IR and IL-IR AcM results in the IL-IR having a higher affinity binding site (i.e., on the order of KD 0.02-0.8 nM). Thus, one can monitor the relative binding affinity of the candidate agonist relative to the IL-lβ. The presence of the IL-IR AcM protein of the present invention in the cell line used for agonist screening ensures that the IL1-R is in a high affinity binding state.
In a further aspect, the invention provides a screening method of identifying an IL-1 signal transduction antagonist. The results of Greenfeder, et al. (J. Biol. Chem, 270: 13757-13765 (1995)) suggest that the antagonist IL-lra prevents or disrupts formation of a complex between mulL-lR and mulL-lR AcP. Thus, the screening method for identifying other antagonist of signal transduction which involves: (a) providing a host cell containing recombinant genes which express a polypeptide comprising a type I IL-1 receptor and a polypeptide comprising IL-IR AcM or an IL-1 R AcM fragment, wherein IL-IR and IL-IR AcM or IL-IR and the IL-IR AcM fragment form a complex; (b) administering a candidate antagonist to said cell; and (c) determining the whether said candidate antagonist disrupts or prevents formation of a complex between IL-IR and IL-IR AcM or IL-IR and the IL-IR AcM fragment. In this antagonist screening assay, one can measure the formation of complex between IL-IR and IL-IR AcM by immunoprecipitating labeled protein complexes cross-linked to IL-lβ, IL-lra or the candidate antagonist. Using an anti-type I IL-IR mAb or an anti-IL-lR AcM mAb, a protein complex of > 200 kDa range will be observed for the cross linking reaction when IL-1 β is used as a ligand indicating formation of an IL-1 binding complex. In addition, these antibodies will immunoprecipitate labeled IL 1 β. However, when labeled IL- 1 ra or a candidate antagonist is used as ligand, the anti-IL-l R AcM mAb will not immunoprecipitate the labeled agonist. Rather, the anti-IL- 1 R AcM antibody will only precipitate an IL- 1 ra or a candidate antagonist complex in the 100- 120 kDa range indicating that the IL-IR and IL-IR AcM have not formed a complex.
Alternative screening assay:
The present invention further provides a screening method for identifying IL-1 receptor agonists and antagonists, which involves: (a) providing a polypeptide comprising a type I IL-1 receptor and a polypeptide comprising IL-
IR AcM or IL-IR AcM fragment, wherein IL-1 R and IL-1 R AcM or IL-I R and the IL-IR AcM fragment form a complex; (b) providing a candidate compound; (c) providing a polypeptide conprising IL-1 or a functional IL-1 fragment; and (d) determining the binding affinity of said complex for I -1 whereby an increased binding affinity of said complex for IL- 1 in the presence of said compound is indicative that said compound is an agonist for IL-1 signal transduction and a decreased binding affinity of said complex for IL-1 in the presence of said compound is indicative that said compound is an antagonist of IL-1 signal transduction.
Candidate Agonists and Antagonists
Candidate antagonists and agonist according to the present invention inclulde IL-lra, polypeptides and antibodies that either enhance or inhibit formation of the IL-IR IL-IR AcM complex. For example, an antibody that inhibits the murine IL-1R-IL-1R accessory protein complex is described in Greenfeder, et al. J. Biol Chem, 270: 13757-13765 (1995). In addition, antibodies directed against murine type I IL-1 receptors which block IL-1 activity are described in Chizzonite et al. Proc. Natl Acad Sci USA 56:8029 (1989); Lewis et al. Eur. J. Immunol. 20:201 (1990); Dinarello, Int. J. Clin. Lab. Res. 24:61-79
( 1994). In addition, a point mutation in IL- 1 ra converts IL- 1 ra from an antagonist to a partial agonist of IL-1 activity in Ju et al. Proc. Natl. Acad. Sci USA 55:2658- 2662 (1991). Others have developed recombinant IL-1 mutants with altered activities (Dinarello Blood 77:1627-1652; Gehrke et al. J. Biol Chem. 265:5922- 5925 (1990)). Thus, methods are known in the art for developing candidate IL-
IR agonists and antagonists for screening in the present invention.
Methods for determining agonist or antagonist activity are known in the art (Ju et al Proc. Nat Acad. Sci USA 55:2658-2662 (1991); Dinarello Blood 77:1627-1652; Gehrke et al. J. Biol. Chem. 265:5922-5925 (1990)). Methods for determining whether a candidate agonist or antagonist enhances or interferes with the formation of the IL-1 receptor complex between IL-IR and IL-I R AcM is disclosed in Greenfeder et al, J. Biol. Chem. 270 13757-13765 (1995).
Administration of the candidate agonist or antagonist can be exogenous or endogenous and the candidate agonist or antagonist can be obtained from natural or recombinant sources. In addition, the screening method further provides for host cells containing recombinant genes expressing IL-IR and IL- IR AcM, as described above.
By "a host cell containing recombinant genes" is intended host cells which one or more of the recombinant constructs described herein have been introduced or a progeny of such host cells.
In addition, the invention provides antibodies directed to this accessory molecule which inhibit the interaction of IL-IR with IL-IR AcM thereby modulating IL-1 response of the cells. These antibodies could be useful for immunoprecipitating cross-linked complexes for the antagonist screening assay as well as showing that the stable cell lines are in fact expressing the type I IL- 1 R and IL-IR AcM proteins. Methods for obtaining these antibodies are set forth below.
Soluble IL-IR AcM protein-specific antibodies for use in the present invention can be raised against the intact soluble IL-IR AcM protein or an antigenic polypeptide portion thereof, which may presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse) or, if it is long enough (at least about 25 amino acids), without a carrier.
As used herein, the term "antibody" (Ab) or "monoclonal antibody" (Mab) is meant to include intact molecules as well as antibody portions (such as, for example, Fab and F(ab')2 portions) which are capable of specifically binding to soluble IL-1 R AcM protein. Fab and F(ab')2 portions lack the Fc portion of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al, J. Nucl. Med. 24:316-325 (1983)). Thus, these portions are preferred. The antibodies of the present invention may be prepared by any of a variety of methods. For example, cells expressing the soluble IL-IR AcM protein or an antigenic portion thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies. In a preferred method, a preparation of soluble IL-1 R AcM protein is prepared and purified as described above to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.
In the most preferred method, the antibodies of the present invention are monoclonal antibodies (or soluble IL-IR AcM protein binding portions thereof). Such monoclonal antibodies can be prepared using hybridoma technology
(Kohler et al, Nature 256:495 (1975); Kohler et al, Eur. J. Immunol. 6:51 1 (1976); Kohler et al, Eur. J. Immunol. 6:292 (1976); Hammerling et al, In: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)). In general, such procedures involve immunizing an animal (preferably a mouse) with a soluble IL-IR AcM protein antigen or, more preferably, with a soluble IL-IR AcM protein-expressing cell. Suitable cells can be recognized by their capacity to bind soluble IL-IR AcM protein antibody. Such cells may be cultured in any suitable tissue culture medium; however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56°C), and supplemented with about 10 μg/l of nonessential amino acids, about 1 ,000 U/ml of penicillin, and about 100 μg/ml of streptomycin. The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP2O), available from the American Type Culture Collection, Rockville, Maryland. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gasiroenterology 50:225-232 (1981)). The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the soluble IL-IR AcM antigen.
Alternatively, additional antibodies capable of binding to the soluble IL- IR AcM protein antigen may be produced in a two-step procedure through the use of anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, and therefore it is possible to obtain an antibody which binds to a second antibody. In accordance with this method, soluble IL-IR AcM protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the soluble IL-IR AcM protein- specific antibody can be blocked by the soluble IL-I R AcM protein antigen.
Such antibodies comprise anti-idiotypic antibodies to the soluble IL-IR AcM protein-specific antibody and can be used to immunize an animal to induce formation of further soluble IL-IR AcM protein-specific antibodies.
It will be appreciated that Fab and F(ab')2 and other portions of the antibodies of the present invention may be used according to the methods disclosed herein. Such portions are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab portions) or pepsin (to produce F(ab')2 portions). Alternatively, soluble IL-IR AcM protein-binding portions can be produced through the application of recombinant DNA technology or through synthetic chemistry. Where in vivo imaging is used to detect enhanced levels of soluble IL-1 R
AcM protein for diagnosis in humans, it may be preferable to use "humanized" chimeric monoclonal antibodies. Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric antibodies are known in the art. See, for review, Morrison, Science 229: 1202 ( 1985); Oi et al, BioTechniques
4:214 (1986); Cabilly et al, U.S. Patent No. 4,816,567; Taniguchi et al, EP 171496; Morrison et al, EP 173494; Neuberger et al, WO 8601 33; Robinson et al, WO 8702671; Boulianne et al, Nature 372:643 (1984); Neuberger et al, Nature 374:268 (1985). Further suitable labels for the soluble IL-I R AcM protein-specific antibodies of the present invention are provided below. Examples of suitable enzyme labels include malate dehydrogenase, staphylococcal nucleasc, delta-5- steroid isomerase, yeast-alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholine esterase.
Examples of suitable radioisotopic labels include 3H, '"In, l25I, 13 II, 32P,
35S, 14C, 51Cr, "To, 58Co, 59Fe, 75Se, ,52Eu, 90Y, 67Cu, 2 l7Ci, 2"At, 212Pb, 47Sc, 109Pd, etc. mIn is a preferred isotope where in vivo imaging is used since its avoids the problem of dehalogenation of the 125I or l3 lI-labeled monoclonal antibody by the liver. In addition, this radionucleotide has a more favorable gamma emission energy for imaging (Perkins et al, Eur. J. Nucl Med. 70:296-301 (1985); Carasquillo et al, J. Nucl Med. 25:281-287 (1987)). For example, '"In coupled to monoclonal antibodies with l-(P-isothiocyanatobenzyl)-DPTA has shown little uptake in non-tumorous tissues, particularly the liver, and therefore enhances specificity of tumor localization (Esteban et al, J. Nucl. Med. 25:861-870 (1987)).
Examples of suitable non-radioactive isotopic labels include ,57Gd, 55Mn, ,62Dy, 52Tr, and 56Fe. Examples of suitable fluorescent labels include an 152Eu label, a fluorescein label, an isothiocyanate label, a rhodamine label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label, an o-phthaldehyde label, and a fluorescamine label.
Examples of suitable toxin labels include diphtheria toxin, ricin, and cholera toxin.
Examples of chemiluminescent labels include a luminal label, an isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, and an aequorin label. Examples of nuclear magnetic resonance contrasting agents include heavy metal nuclei such as Gd, Mn, and Fe.
Typical techniques for binding the above-described labels to antibodies are provided by Kennedy et al. (Clin. Chim. Ada 70: 1 -31 (1976)), and Schurs et al (Clin. Chim. Acta 57:1-40 (1977)). Coupling techniques mentioned in the latter are the glutaraldehyde method, the periodate method, the dimaleimide method, the m-maleimidobenzyl-N-hydroxy-succinimide ester method, all of which methods are incoφorated by reference herein.
Chromosome Assays
The nucleic acid molecules of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data
(repeat polymoφhisms) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
In certain preferred embodiments in this regard, the cDNA herein disclosed is used to clone genomic DNA of a soluble IL-IR AcM protein gene.
This can be accomplished using a variety of well known techniques and libraries, which generally are available commercially. The genomic DNA then is used for in situ chromosome mapping using well known techniques for this puφose. Typically, in accordance with routine procedures for chromosome mapping, some trial and error may be necessary to identify a genomic probe that gives a good in situ hybridization signal.
In addition, in some cases, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3' untranslated region of the gene is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified portion. PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of portions from specific chromosomes or pools of large genomic clones in an analogous manner. Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow- sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
Fluorescence in situ hybridization ("FISH") of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with probes from the cDNA as short as 50 or 60 bp. For a review of this technique, see Verma et al, Human Chromosomes: A Manual Of Basic Techniques, Pergamon Press, New York (1988).
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian
Inheritance In Man, available on-line through Johns Hopkins University, Welch Medical Library. The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes). Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. This assumes 1 megabase mapping resolution and one gene per 20 kb.
Therapeutic Uses of Soluble IL-IR AcM
IL-1 has an important role in modulating the proliferation, maturation, and functional activation of hematopoietic cells, including lymphoid and nonlymphoid cells. IL-1 may have an important function in the regulation liver metabolism and be responsible for some of the marked changes in hepatic protein synthesis that occur in the acute phase response to inflammation or tissue injury.
IL-1 is also involved in the regulation of bone remodulation, may be involved in the pathogenesis of chronic inflammatory joint diseases, such as rheumatoid arthritis, osteoarthritis, and may also play a role in the mechanisms of articular cartilage destruction hat occurs in degradative arthropathies. Human IL-1 is capable of increasing collagen protein and mRNA levels in cultured normal human dermal fibroblasts, thus, IL-1 may have a role in the early stages of scleroderma and other fibrotic diseases. IL-1 is capable of inducing a proliferative response in fibroblasts. In addition, IL-1 may have important effects on vascular cells, including endothelial cells and vascular smooth muscle cells. IL-1 may be involved in the pathogenesis of certain skin diseases, including chronic diseases such as psoriasis and epithelial fungus infections. IL-1 may have important effects on the functions of the hypothalamus-pituitary axis and thyroid gland. IL-1 has an important role in the regulation of insulin secretion by β cells in the pancreatic islets of Langerhans. Finally, IL-1 has important effects on the gonads and may play a role in the physiology of neural tissues. (Reviewed in Pimentel, Handbook of Growth Factors: Volume HI Hematopoietic Growth
Factors and Cytokines, pp. 35-53, CRC Press. Boca Raton, FL 1994).
Antibody Therapy
In view of the wide range of roles that IL- 1 plays in physiologic and pathologic processes, regulating the action of IL- 1 by abrogating signal transduction from the IL-1 binding complex is expected to be useful for therapeutic puφoses. For example, recombinant IL-lra blocks the activity of exogenously administered IL-1 in a variety of animal models. When rabbits or baboons are injected with IL-1 they develope hypertension which is prevented by a prior injection of IL-lra (Ohlsson et al. Nature 348: 550 (1990); Fischer et al. Am. J. Physiol 261: R442 (1991)). In animal studies, IL-IR blockade significantly reduces the severity of diseases, including those associated with infections, inflammation and metabolic disturbances (Arend, W.P. J. Clin. Invest 55:1445 (1991); Dinarello et al. Immunol Today 12: 404 (1991 )). In Table 1 of Dinarello, Int. J. Clin. Lab. Res. 24:61-79 (1994) (which is incoφorated herein by reference) different models are listed wherein a specific reduction in IL-IR activity has been employed to reduce the disease severity.
In addition, studies with human subjects have also demonstrated that blockade of IL-1 R is effective where the severity of disease is high. For example, dramatic results have been seen in patients with septic shock. In clinical trials there was a statistically significant reduction in mortality. (Dinarello, Int. J. Clin. Lab. Res. 24:61-79 (1994))
In another clinical trial, rheumatoid arthritis patients treated with IL-lra, in addition to other non-steroidal anti-inflammatory drugs, had a significant reduction in the number and severity of painful and swollen joints. These results demonstrate an improvement in the clincal disease of these patients. (Dinarello,
Int. J. Clin. Lab. Res. 24:61-79 (1994))
Similarly, antibodies produced to the murine type I IL-1 R have been used to block IL-1 effects in vitro and in vivo. (Chizzonite et al. Proc. Natl Acad Sci
USA 56:8029 (1989); Lewis et al. Eur. J. Immunol. 20:201 (1990)). For example, in animal models of infection and inflammation, administration of anti- IL-1R antibodies have reduced disease severity (Dinarello, Int. J. Clin. Lab. Res. 24:61-79 (1994)). The advantage of the anti-IL-lR antibodies is that they block IL-1 effects for several hours to days, whereas IL-l ra blood levels need to be sustained at sufficiently high levels to block IL- 1 effects (Dinarello, Int. J. Clin. Lab. Res. 24:61-79 (1994)).
Given that IL-IR AcM is involved in IL- 1 signal transduction, antibodies directed against IL-IR AcM are expected to behave as agonists or antagonist of IL-1 activity. For example, an antibody directed against the murine IL-I R accessory protein blocked the binding of IL-l β to murine type I IL-IR (Greenfeder et al. J. Biol. Chem, 270: 13757-13765 (1995)). Thus, antibodies directed against the IL-IR AcM of the present invention that abrogate IL-1 activity can be used therapuetically to reduce the severity of diseases associated with IL-1.
Thus, the present invention is further directed antibody-based therapies which involve administering an anti-IL-lR AcM antibody to a mammalian, preferably human, patient for treating one or more of above-described disorders. Methods for producing anti-IL-lR AcM polyclonal and monoclonal antibodies are described in detail above. Such antibodies can may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding IL-IR AcM locally or systemically in the body or by direct cytotoxicity of the antibody, e.g., as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic puφoses without undue experimentation.
The pharmaceutical compositions of the present invention may be administered by any means that achieve their intended puφose. Amounts and regimens for the administration of antibodies, their fragments or derivatives can be determined readily by those with ordinary skill in the clinical art of treating colon cancer and related disease.
For example, administration may be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concunent treatment, if any, frequency of treatment, and the nature of the effect desired.
Compositions within the scope of this invention include all compositions wherein the antibody, fragment or derivative is contained in an amount effective to achieve its intended puφose. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. The effective dose is a function of the individual chimeric or monoclonal antibody, the presence and nature of a conjugated therapeutic agent (see below), the patient and his clinical status, and can vary from about 10 μg/kg body weight to about 5000 mg/kg body weight. The preferred dosages comprise 0.1 to 500 mg/kg body wt. In addition to the pharmacologically active compounds, the new pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Preferably, the preparations, contain from about 0.01 to 99 percent, preferably from about 20 to 75 percent of active compound(s), together with the excipient. Similarly, preparations of an IL-IR AcM antibody or fragment of the present invention for parenteral administration, such as in detectably labeled form for imaging or in a free or conjugated form for therapy, include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil 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 including sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
Intravenous vehicles include fluid and nutrient replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, anti-oxidants. chelating agents, and inert gases and the like. See, generally, Remington's Pharmaceutical Science, 16th ed., Mack Publishing Co., Easton, PA, 1980.
In particular, the antibodies, fragments and derivatives of the present invention are useful for treating a subject having or developing IL-IR AcM related disorders as described herein. Such treatment comprises parenterally administering a single or multiple doses of the antibody, fragment or derivative, or a conjugate thereof.
The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hemopoietic growth factors, etc., which serve to increase the number or activity of effector cells which interact with the antibodies. Since it appear to be necessary to block nearly all IL-lR's to block IL-I activity, it is prefened to use high affinity and/or potent in vivo IL-IR AcM-inhibiting and/or neutralizing antibodies, fragments or regions thereof, for both IL-IR AcM immunoassays and therapy of IL-1 related disorders. Such antibodies, fragments, or regions, will preferably have an affinity for human IL- IR AcM, expressed as Ka, of at least 108 M"1, more preferably, at least 109 M'1, such as 5 X 108 M'1, 8 X 108 M"1, 2 X 109 M ', 4 X 109 M 1, 6 X 109 M 1, 8 X 109
MX
Preferred for human therapeutic use are high affinity murine and murine/human or human/human chimeric antibodies, and fragments, regions and derivatives having potent in vivo IL-1 -inhibiting and/or neutralizing activity, according to the present invention, e.g., that block IL-1 -induced IL-6 secretion, and mitogenic activity, in vivo, in situ, and in vitro.
Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.
Examples
Example I: Expression and Purification of IL-IR AcM in E. coli
The DNA sequence encoding the mature soluble IL-IR AcM protein in the deposited cDNA clone is amplified using PCR oligonucleotide primers specific to the amino acid carboxyl terminal sequence of the soluble IL-1 R AcM protein and to vector sequences 3' to the gene. Additional nucleotides containing restriction sites BamHI and Sail to facilitate cloning are added to the 5' and 3' sequences, respectively.
The 5' oligonucleotide primer has the sequence 5' GGATCCATGACACTTCTGTGGTGTG 3' (SEQ ID NO:23) containing the underlined BamHI restriction site, followed by 16 nucleotides complementary to bp 1834-1853 of the antisense strand of the IL-IR AcM protein coding sequence set out in Figure 1 (SEQ ID NO: 1).
The 3* primer has the sequence 5' GTCGACTCACTGACCGCATCT 3'
(SEQ ID NO:24) containing the underlined Sail restriction site, followed by 15 nucleotides complementary to bp 1056-1071 of the sense strand of the IL-IR AcM protein coding sequence set out in Figure 1 (SEQ ID NO: l), and a stop codon.
The restrictions sites are convenient to restriction enzyme sites in the bacterial expression vector pQE-9, which is used for bacterial expression in these examples. (Qiagen, Chatsworth, CA, 9131 1). The amplified IL-IR AcM protein DNA and the vector pQE-9 are both digested with BamHI and Sail and the digested DNAs are subsequently ligated together. Insertion of the IL-IR AcM protein DNA into the pQE-9 restricted vector places the IL-IR AcM protein coding region downstream of and operably linked to the vector's promoter and in-frame with an initiating AUG appropriately positioned for translation of IL-1 R AcM protein.
The ligation mixture is transformed into competent E. coli cells using standard procedures. Such procedures are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strain M15/rep4, containing multiple copies of the plasmid pREP4, which expresses lac repressor and confers kanamycin resistance ("Kanr"), is used in carrying out the illustrative example described here. This strain, which is only one of many that are suitable for expressing IL-IR AcM protein, is available commercially from Qiagen.
Transformants are identified by their ability to grow on LB plates in the presence of ampicillin. Plasmid DNA is isolated from resistant colonies and the identity of the cloned DNA is confirmed by restriction analysis.
Clones containing the desired constructs are grown overnight ("O/N") in liquid culture in LB media supplemented with both ampicillin ( 100 μg/ml) and kanamycin (25 μg/ml). The O/N culture is used to inoculate a large culture, at a dilution of approximately 1 : 100 to 1 :250. The cells are grown to an optical density at 600 nm ("OD600") of between 0.4 and 0.6. Isopropyl-B-D-thiogalactopyranoside ("IPTG") are then added to a final concentration of 1 mM to induce transcription from lac repressor sensitive promoters, by inactivating the lad repressor. Cells subsequently are incubated further for 3 to 4 hours. Cells are then harvested by centrifugation and disrupted, by standard methods. Inclusion bodies are purified from the disrupted cells using routine collection techniques, and protein are solubilized from the inclusion bodies into 8M urea. The 8M urea solution containing the solubilized protein is passed over a PD-10 column in 2X phosphate buffered saline ("PBS"), thereby removing the urea, exchanging the buffer and refolding the protein. The protein is purified by a further step of chromatography to remove endotoxin. Then, it is sterile filtered. The sterile filtered protein preparation is stored in 2X PBS at a concentration of 95 micrograms per mL.
Analysis of the preparation by standard methods of polyacrylamide gel electrophoresis reveals that the preparation contains about 95% monomer IL-1 R AcM protein having the expected molecular weight of approximately 42 kDa.
Example 2: Cloning and Expression in Mammalian Cells
Most of the vectors used for the transient expression of the IL-R AcM protein gene sequence in mammalian cells should carry the SV40 origin of replication. This allows the replication of the vector to high copy numbers in cells (e.g., COS cells) which express the T antigen required for the initiation of viral DNA synthesis. Any other mammalian cell line can also be utilized for this puφose.
A typical mammalian expression vector contains the promoter element, which mediates the initiation of transcription of mRNA, the protein coding sequence, and signals required for the termination of trancription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).
However, cellular signals can also be used (e.g., human actin promoter). Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be used include, human Hela, 283, H9 and Jurkart cells, mouse NIH3T3 and Cl 27 cells, Cos 1 , Cos 7 and CV 1 , African green monkey cells, quail QC1-3 cells, mouse L cells and Chinese hamster ovary cells.
Alternatively, the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome. The co-transfection with a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transfected cells.
The transfected gene can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) is a useful marker to develop cell lines that carry several hundred or even several thousand copies of the gene of interest. Another useful selection marker is the enzyme glutamine synthase (GS) (Mwphy et al, Biochem J. 227.277-279 ( 1991); Bebbington et al, Bio/Technology 70:169-175 (1992)). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) cells are often used for the production of proteins.
The expression vectors pCl and pC4 contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al. , Molecular and Cellular Biology, 438-447 (March, 1985)) plus a fragment of the CMV -enhancer (Boshart et al,
Cell 47:521-530 (1985)). Multiple cloning sites, e.g., with the restriction enzyme cleavage sites BamHI, Xbal and Asp718, facilitate the cloning of the gene of interest. The vectors contain in addition the 3 ' intron, the polyadenylation and termination signal of the rat preproinsulin gene.
Example 2(a): Cloning and Expression in COS Cells
The expression plasmid, pIL-lR AcM HA, is made by cloning a cDNA encoding IL-R AcM into the expression vector pcDNAI/Amp (which can be obtained from Invitrogen, Inc.).
The expression vector pcDNAI/amp contains: ( 1 ) an E.coli origin of replication effective for propagation in E. coli and other prokaryotic cells; (2) an ampicillin resistance gene for selection of plasmid-containing prokaryotic cells; (3) an SV40 origin of replication for propagation in eukaryotic cells; (4) a CMV promoter, a polylinker, an SV40 intron, and a polyadenylation signal arranged so that a cDNA conveniently can be placed under expression control of the CMV promoter and operably linked to the SV40 intron and the polyadenylation signal by means of restriction sites in the polylinker.
A DNA fragment encoding the IL- 1 R AcM protein and an HA tag fused in frame to its 3' end is cloned into the polylinker region of the vector so that recombinant protein expression is directed by the CMV promoter. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein described by Wilson et al, Cell 37: 767 (1984). The fusion of the HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
The plasmid construction strategy is as follows The IL-1 R AcM cDNA of the deposited clone is amplified using primers that contain convenient restriction sites, much as described above regarding the construction of expression vectors for expression of IL-R AcM in E. coli. To facilitate detection, purification and characterization of the expressed IL-1 R AcM, one of the primers contains a hemagglutinin tag ("HA tag") as described above. Suitable primers include the following, which are used in this example. The 5' primer, containing the underlined BamHI site, an AUG start codon and 5 codons of the 5' coding region has the following sequence:
5 'GGATCCATCCGCCATCATGACACTTCTGTGGTGTG 3 ' (SEQ ID NO:25).
The 3' primer, containing the underlined Xbal site, a stop codon, 9 codons thereafter forming the hemagglutinin HA tag, and 12 bp of 3' coding sequence (at the 3' end) has the following sequence:
5 CTAGAAAAGCGTAGTCTGGGACGTCGTATGGGTAC TGACCGCATCT 3' (SEQ IDNO:26).
The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digested with BamHI and Xbal and then ligated. The ligation mixture is transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037), and the transformed culture is plated on ampicillin media plates which then are incubated to allow growth of ampicillin resistant colonies, Plasmid DNA is isolated from resistant colonies and examined by restriction analysis and gel sizing for the presence of the IL-IR AcM-encoding fragment.
For expression of recombinant IL-I R AcM, COS cells are transfected with an expression vector, as described above, using DEAE-DEXTRAN, as described, for instance, in Sambrook et al, Molecular Cloning: a Laboratory
Manual, Cold Spring Laboratory Press, Cold Spring Harbor, New York (1989).
Cells are incubated under conditions for expression of IL- 1 R AcM by the vector.
Expression of the IL-IR AcM HA fusion protein is detected by radiolabelling and immunoprecipitation, using methods described in, for example
Harlow et al, Antibodies: A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988). To this end, two days after transfection, the cells are labeled by incubation in media containing 5S- cysteine for 8 hours. The cells and the media are collected, and the cells are washed and the lysed with detergent-containing RIPA buffer: 150 mM NaCI, 1 %
NP-40, 0.1% SDS, l%NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. cited above. Proteins are precipitated from the cell lysate and from the culture media using an HA-specific monoclonal antibody. The precipitated proteins then are analyzed by SDS-PAGE gels and autoradiography. An expression product of the expected size is seen in the cell lysate, which is not seen in negative controls.
Example 2(b): Cloning and Expression in CHO Cells
The vector pC 1 is used for the expression of IL- 1 R AcM protein. Plasmid pCl is a derivative of the plasmid pSV2-dhfr [ATCC Accession No. 37146]. Both plasmids contain the mouse DHFR gene under control of the SV40 early promoter. Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (alpha minus MEM, Life Technologies) supplemented with the chemotherapeutic agent methotrexate. The amplification of the DHFR genes in cells resistant to methotrexate (MTX) has been well documented (see, e.g., Alt, F.W., Kellems, R.M., Bertino, J.R., and Schimke, R.T., 1978, J. Biol. Chem.
253:1357-1370, Hamlin, J.L. and Ma, C. 1990, Biochem. et Biophys. Acta, 1097:107-143, Page, M.J. and Sydenham, M.A. 1991 , Biotechnology Vol. 9:64- 68). Cells grown in increasing concentrations of MTX develop resistance to the drug by oveφroducing the target enzyme, DHFR, as a result of amplification of the DHFR gene. If a second gene is linked to the DHFR gene it is usually co- amplified and over-expressed. It is state of the art to develop cell lines carrying more than 1,000 copies of the genes. Subsequently, when the methotrexate is withdrawn, cell lines contain the amplified gene integrated into the chromosome(s). Plasmid pCl contains for the expression of the gene of interest a strong promoter of the long terminal repeat (LTR) of the Rouse Sarcoma Virus (Cullen, et al, Molecular and Cellular Biology, March 1985:438-4470) plus a fragment isolated from the enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart et al, Cell 47:521-530, 1985). Downstream of the promoter are the following single restriction enzyme cleavage sites that allow the integration of the genes: BamHI, Pvull, and Nrul. Behind these cloning sites the plasmid contains translational stop codons in all three reading frames followed by the 3' intron and the polyadenylation site of the rat preproinsulin gene. Other high efficient promoters can also be used for the expression, e.g., the human β-actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, e.g., HIV and HTLVI. For the polyadenylation of the mRNA other signals, e.g., from the human growth hormone or globin genes can be used as well. Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co-transfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker in the beginning, e.g., G418 plus methotrexate.
The plasmid pCl is digested with the restriction enzyme BamHI and then dephosphorylated using calf intestinal phosphates by procedures known in the art.
The vector is then isolated from a 1% agarose gel.
The DNA sequence encoding IL-I R AcM, ATCC 97666, is amplified using PCR oligonucleotide primers corresponding to the 5 ' and 3 ' sequences of the gene: T h e 5 ' p r i m e r h a s t h e s e q u e n c e
5'GACTflQΔICCGCCATCATGACACTTCTGTGGTGTG 3' (SEQ ID NO:27) containing the underlined BamHI restriction enzyme site followed by 19 bases of the sequence of IL-IR AcM of Figure 1 (SEQ ID NO: 1 ). Inserted into an expression vector, as described below, the 5' end of the amplified fragment encoding human IL-IR AcM provides an efficient signal peptide. An efficient signal for initiation of translation in eukaryotic cells, as described by Kozak, M., J. Mol. Biol. 196:947-950 (1987) is appropriately located in the vector portion of the construct.
The 3' primer has the sequence 5' QΔΔUCCTCACTGACCGCATCT 3' (SEQ ID NO:28) containing the EcoRI restriction followed by nucleotides complementary to the last 15 nucleotides of the IL-IR AcM coding sequence set out in Figure 1 (SEQ ID NO:l), including the stop codon.
The amplified fragments are isolated from a 1% agarose gel as described above and then digested with the endonucleases BamHI and EcoRI and then purified again on a 1% agarose gel.
The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HBlOl cells are then transformed and bacteria identified that contained the plasmid pCl inserted in the correct orientation using the restriction enzyme BamHI. The sequence of the inserted gene is confirmed by DNA sequencing.
Transfection of CHO-DHFR-cells
Chinese hamster ovary cells lacking an active DHFR enzyme are used for transfection. 5 μg of the expression plasmid Cl are cotransfected with 0.5 μg of the plasmid pSVneo using the lipofecting method (Feigner et al, supra). The plasmid pSV2-neo contains a dominant selectable marker, the gene neo from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418. The cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) and cultivated from 10-14 days. After this period, single clones are trypsinized and then seeded in 6-well petri dishes using different concentrations of methotrexate (25 nM, 50 nM, 100 nM, 200 nM, 400 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (500 nM, 1 μM, 2 μM, 5 μM). The same procedure is repeated until clones grow at a concentration of 100 μM.
The expression of the desired gene product is analyzed by Western blot analysis and SDS-PAGE. Example 3: Cloning and expression of the Soluble IL-IR AcM protein in a baculovirus expression system
The cDNA sequence encoding the soluble IL-IR AcM protein in the deposited clone was amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene:
The 5' primer has the sequence 5 '
GACTGGATCCGCCATCATGACACTTCTGTGGTGTG 3' (SEQ ID NO:29) containing the underlined BamHI restriction enzyme site followed by 19 bases (bp 1834-1853) complementary to the antisense strand of the soluble IL-1 R AcM protein coding sequence of Figure 1 (SEQ ID NO: 1 ). Inserted into an expression vector, as described below, the 5' end of the amplified fragment encoding soluble IL-IR AcM protein receptor provides an efficient signal peptide. An efficient signal for initiation of translation in eukaryotic cells, as described by Kozak, M., J. Mol Biol. 796:947-950 (1987), may be located, as appropriate, in the vector portion of the construct.
For the full length gene, the 3' primer has the full length sequence 5' GAC TGG TAG CCA TAG AAA TCA TGT GTA TAC C 3' (SEQ ID NO:30), containing the underlined Asp718 restriction followed by 25 nucleotides complementary to bp 2049-2070 of the sense strand of the soluble IL-1 R AcM protein set out in Figure 1 [SEQ ID NO: 1], and a stop codon.
The amplified fragment was isolated from a 1 % agarose gel using a commercially available kit ("Geneclean," BIO 101 Inc., La Jolla, Ca.). The fragment then is digested with BamHI and Asp718 and again is purified on a 1% agarose gel. This fragment is designated herein "F2". The vector pA2 is used to express the soluble IL-1 R AcM protein in the baculovirus expression system, using standard methods, such as those described in Summers et al, A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experimental Station Bulletin No. 1555 (1987). This expression vector contains the strong polyhedrin promoter of the Autographa califomica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites. For an easy selection of recombinant virus the beta- galactosidase gene from E. coli is inserted in the same orientation as the polyhedrin promoter and is followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequences are flanked at both sides by viral sequences for cellmediated homologous recombination with wild-type viral DNA to generate viable virus that express the cloned polynucleotide.
Many other baculovirus vectors could be used in place of pA2, such as pAc373, pVL941 and pAcIMl provided, as those of skill readily will appreciate, that construction provides appropriately located signals for transcription, translation, trafficking and the like, such as an in-frame AUG and a signal peptide, as required. Such vectors are described, for example, in Luckow et al, Virology 770:31-39 (1989). Suitable vectors will be readily apparent to the skilled artisan.
The plasmid was digested with the restriction enzymes BamHI and Asp718 and then was dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art. The DNA was then isolated from a 1% agarose gel using a commercially available kit ("Gencclean" BIO 101 Inc., La Jolla, Ca.). This vector DNA is designated herein "V2".
Fragment ^2 and the dephosphorylated plasmid V2 were ligated together with T4 DNA ligase. E. coli HB 101 cells were transformed with ligation mix and spread on culture plates. Bacteria were identified that contain the plasmid with the human soluble IL-IR AcM protein gene by digesting DNA from individual colonies using BamHI and Asp718 and then analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment was confirmed by DNA sequencing. This plasmid is designated herein as pA2HG 16302.
5 μg of plasmid pA2HG16302 was co-transfected with 1.0 μg of a commercially available linearized baculovirus DNA ("BaculoGold™ baculovirus DNA", Pharmingen, San Diego, CA), using the lipofection method described by Feigner et al, Proc. Natl Acad. Sci. USA 54:7413-7417 (1987). 1 μg of
BaculoGold™ virus DNA and 5 μg of the plasmid pA2HG 16302 were mixed in a sterile well of a microliter plate containing 50 μl of serum free Grace's medium (Life Technologies Inc., Gaithersburg, MD). Afterwards, 10 μl Lipofectin plus 90 μl Grace's medium were added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture was added drop- wise to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with I ml Grace's medium without serum. The plate was rocked back and forth to mix the newly added solution. The plate is then incubated for 5 hours at 27 °C. After 5 hours the transfection solution was removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. The plate is put back into an incubator and cultivation is continued at 27 °C for four days.
After four days the supernatant was collected and a plaque assay was performed, as described by Summers and Smith, supra. An agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg, MD) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques. A detailed description of a "plaque assay" of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, MD, at pages 9-10.
Four days after serial dilution, the virus was added to the cells. After appropriate incubation, blue stained plaques are picked with the tip of an Eppendorf pipette. The agar containing the recombinant viruses was then resuspended in an Eppendorf tube containing 200 μ l of Grace's medium. The agar is removed by a brief centrifugation and the supernatant containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then they were stored at 4°C. A clone containing properly inserted soluble IL-IR AcM cDNA was identified by DNA analysis including restriction mapping and sequencing. This clone is designated herein as pA2HG 16302.
Sf9 cells are grown in Grace's medium supplemented with 10% heat- inactivated FBS. The cells are infected with the recombinant baculovirus A2HG16302 at a multiplicity of infection ("MOI") of about 2 (about 1 to about
3). Six hours later the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc., Gaithersburg, MD). 42 hours later, 5 μCi of 35S methionine and 5 MCi ^ cysteine (available from Amersham) are added. The cells are further incubated for 16 hours and then they are harvested by centrifugation, lysed and the labeled proteins are visualized by SDS-PAGE and autoradiography.
Example 4: Tissue distribution of Soluble IL-IR AcM protein expression
Northern blot analysis is carried out to examine the levels of expression of the gene encoding the IL-IR AcM protein in human tissues, using methods described by, among others, Sambrook et al, cited above. A cDNA probe containing the entire nucleotide sequence of the IL- 1 R AcM protein of the present invention (SEQ ID NO: 1 ) is labeled with "P using the redipήme™ DNA labeling system (Amersham Life Science), according to manufacturer's instructions. After labelling, the probe was purified using a CHROMA SPIN- 100™ column (Clontech Laboratories, Inc.), according to manufacturer's protocol number PT1200-1. The purified labelled probe is then used to examine various human tissues for the expression of the gene encoding the IL- 1 R AcM protein.
Multiple Tissue Northern (MTN) blots containing various human tissues (H) or human immune system tissues (IM) are obtained from Clontech and were examined with labelled probe using ExpressHyb™ Hybridization Solution (Clontech) according to manufacturer's protocol number PT 1 190-1. Following hybridization and washing, the blots are mounted and exposed to film at -70 °C overnight, and films developed according to standard procedures.
It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims. The entire disclosure of each document cited in this application is hereby incoφorated herein by reference.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Human Genome Sciences, Inc. 9410 Key West Avenue Rockville, MD 20850 United States of America APP ICANTS/INVENTORS : Bednarik, Daniel P.
Olsen, Henrik S. Rosen, Craig A.
(ii) TITLE OF INVENTION: Soluble Interleukin-1 Receptor Accessory
Molecule
(iii) NUMBER OF SEQUENCES: 30
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: STERNE, KESSLER, GOLDSTEIN & FOX, P.L.L.C.
(B) STREET: 1100 NEW YORK AVE . , N.W., SUITE 600
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(D) SOFTWARE: Patentin Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: To be assigned
(B) FILING DATE: 26-AUG-1996
(C) CLASSIFICATION:
(viii) ATTORNE /AGENT INFORMATION:
(A) NAME: Goldstein, Jorge A.
(B) REGISTRATION NUMBER: 29,021
(C) REFERENCE/DOCKET NUMBER: 1488.045PC00
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 202-371-2600
(B) TELEFAX: 202-371-2540
(2) INFORMATION FOR SEQ ID NO : 1 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2155 base pairs
(B) TYPE: nucleic acid
( C) STRANDEDNESS : doub1e
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 303..1370
(ix) FEATURE:
(A) NAME/KEY: sig_peptide
(B) LOCATION: 303..353
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 354..1370
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :
CGGTGGCGCC CGTTCTAGAA CTAGTGGATC CCCCGGGATG CAGGAATTCG GCACGAGAAA 60
GTGCGGCGGA AAGTAAGAGG CTCACTGGGG AAGACTGCCG GGATCCAGGT CTCCGGGGTC 120
CGCTTTGGCC AGAGGCGCGG AAGGAAGCAG TGCCCGGCGA CACTGCACCC ATCCCGGCTG 180
CTTTTGCTGC GCCCTCTCAG CTTCCCAAGA AAGGCATCGT CATGTGATCA TCACCTAAGA 240
ACTAGAACAT CAGCAGGCCC TTAGAAGCCT CACTCTTGCC CCTCCCTTTA ATATCTCAAA 300
GG ATG ACA CTT CTG TGG TGT GTA GTG AGT CTC TAC TTT TAT GGA ATC 347 Met T r Leu Leu Trp Cys Val Val Ser Leu Tyr P e Tyr Gly lie -17 -15 -10 -5
CTG CAA AGT GAT GCC TCA GAA CGC TGC GAT GAC TGG GGA CTA GAC ACC 395 Leu Gin Ser Asp Ala Ser Glu Arg Cys Asp Asp Trp Gly Leu Asp Thr
1 5 10
ATG AGG CAA ATC CAA GTG TTT GAA GAT GAG CCA GCT CGC ATC AAG TGC 443 Met Arg Gin lie Gin Val Phe Glu Asp Glu Pro Ala Arg lie Lys Cys 15 20 25 30
CCA CTC TTT GAA CAC TTC TTG AAA TTC AAC TAC AGC ACA GCC CAT TCA 491 Pro Leu Phe Glu His Phe Leu Lys Phe Asn Tyr Ser Thr Ala His Ser
35 40 45
GCT GGC CTT ACT CTG ATC TGG TAT TGG ACT AAG CAG GAC CGG GAC CTT 539 Ala Gly Leu Thr Leu lie Trp Tyr Trp Thr Lys Gin Asp Arg Asp Leu 50 55 60
GAG GAG CCA ATT AAC TTC CGC CTC CCC GAG AAC CGC ATT AGT AAG GAG 587 Glu Glu Pro lie Asn Phe Arg Leu Pro Glu Asn Arg lie Ser Lys Glu 65 70 75
AAA GAT GTG CTG TGG TTC CGG CCC ACT CTC CTC AAT GAC ACT GGC AAC 635 Lys Asp Val Leu Trp Phe Arg Pro Thr Leu Leu Asn Asp Thr Gly Asn 80 85 90 TAT ACC TGC ATG TTA AGG AAC ACT ACA TAT TGC AGC AAA GTT GCA TTT 683 Tyr Thr Cys Met Leu Arg Asn Thr Thr Tyr Cys Ser Lys Val Ala Phe 95 100 105 110
CCC TTG GAA GTT GTT CAA AAA GAC AGC TGT TTC AAT TCC CCC ATG AAA 731 Pro Leu Glu Val Val Gin Lys Asp Ser Cys Phe Asn Ser Pro Met Lys 115 120 125
CTC CCA GTG CAT AAA CTG TAT ATA GAA TAT GGC ATT CAG AGG ATC ACT 779 Leu Pro Val His Lys Leu Tyr lie Glu Tyr Gly lie Gin Arg lie Thr 130 135 140
TGT CCA AAT GTA GAT GGA TAT TTT CCT TCC AGT GTC AAA CCG ACT ATC 827 Cys Pro Asn Val Asp Gly Tyr Phe Pro Ser Ser Val Lys Pro Thr lie 145 150 155
ACT TGG TAT ATG GGC TGT TAT AAA ATA CAG AAT TTT AAT AAT GTA ATA 875 Thr Trp Tyr Met Gly Cys Tyr Lys lie Gin Asn Phe Asn Asn Val lie 160 165 170
CCC GAA GGT ATG AAC TTG AGT TTC CTC ATT GCC TTA ATT TCA AAT AAT 923 Pro Glu Gly Met Asn Leu Ser Phe Leu lie Ala Leu lie Ser Asn Asn 175 180 185 190
GGA AAT TAC ACA TGT GTT GTT ACA TAT CCA GAA AAT GGA CGT ACG TTT 971 Gly Asn Tyr Thr Cys Val Val Thr Tyr Pro Glu Asn Gly Arg Thr Phe 195 200 205
CAT CTC ACC AGG ACT CTG ACT GTA AAG GTA GTA GGC TCT CCA AAA AAT 1019 His Leu Thr Arg Thr Leu Thr Val Lys Val Val Gly Ser Pro Lys Asn 210 215 220
GCA GTG CCC CCT GTG ATC CAT TCA CCT AAT GAT CAT GTG GTC TAT GAG 1067 Ala Val Pro Pro Val lie His Ser Pro Asn Asp His Val Val Tyr Glu 225 230 235
AAA GAA CCA GGA GAG GAG CTA CTC ATT CCC TGT ACG GTC TAT TTT AGT 1115 Lys Glu Pro Gly Glu Glu Leu Leu He Pro Cys Thr Val Tyr Phe Ser 240 245 250
TTT CTG ATG GAT TCT CGC AAT GAG GTT TGG TGG ACC ATT GAT GGA AAA 1163 Phe Leu Met Asp Ser Arg Asn Glu Val Trp Trp Thr He Asp Gly Lys 255 260 265 270
AAA CCT GAT GAC ATC ACT ATT GAT GTC ACC ATT AAC GAA AGT ATA AGT 1211 Lys Pro Asp Asp He Thr He Asp Val Thr He Asn Glu Ser He Ser 275 280 285
CAT AGT AGA ACA GAA GAT GAA ACT AGA ACT CAG ATT TTG AGC ATC AAG 1259 His Ser Arg Thr Glu Asp Glu Thr Arg Thr Gin He Leu Ser He Lys 290 295 300
AAA GTT ACC TCT GAG GAT CTC AAG CGC AGC TAT GTC TGT CAT GCT AGA 1307 Lys Val Thr Ser Glu Asp Leu Lys Arg Ser Tyr Val Cys His Ala Arg 305 310 315 AGT GCC AAA GGC GAA GTT GCC AAA GCA GCC AAG GTG AAG CAG AAA GGT 1355 Ser Ala Lys Gly Glu Val Ala Lys Ala Ala Lys Val Lys Gin Lys Gly 320 325 330
AAT AGA TGC GGT CAG TGATGAATCT CTCAGCTCCA AATTAACATT GTGGTGAATA 1410
Asn Arg Cys Gly Gin
335
AGGACAAAAG GAGAGATTGA GAACAAGAGA GCTCCAGCAC CTAGCCTGAC GGCATCTAAC 1470
CCATAGTAAT GAATCAAACT TAAATGAAAA ATATGAAAGT TTTCATCTAT GTAAGATACT 1530
CAAAATATTG TTTCTGATAT TGTTAGTACC GTAATGCCCA AATGTAGCTA AAAAAATCGA 1590
CGTGAGTACA GTGAGACACA ATTTTGTGTC TGTACAATTA TGAAAAATTA AAAACAAAGA 1650
AAATATTCAA AGCTACCAAA GATAGAAAAA ACTGGTAGAG CCACATATTG TTGGTGAATT 1710
ATTAAGACCC TTTTAAAAAT CATTCATGGT AGAGTTTAAG AGTCATAAAA AAGATTGCAT 1770
CATCTGACCT AAGACTTTCG GAATTTTTCC TGAACAAATA ACAGAAAGGG AATTATATAC 1830
CTTTTAATAT TATTAGAAGC ATTATCTGTA GTTGTAAAAC ATTATTAATA GCAGCCATCC 1890
AATTGTATGC AACTAATTAA GGTATTGAAT GTTTATTTTC CAAAAATGCA TAATTATAAT 1950
ATTATTTTAA ACACTATGTA TCAATATTTA AGCAGGTTTA TAATATACCA GCAGCCACAA 2010
TTGCTAAAAT GAAAATCATT TAAATTATGA TTTTAAATGG TATACACATG ATTTCTATGT 2070
TGATAGTACT ATATTATTCT ACAATAAATG GAAATTATAA AGCCTTCTTG TCAGAAGTGC 2130
TGCTCCTAAA AAAAAAAAAA AAAAA 2155
(2) INFORMATION FOR SEQ ID NO : 2 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 356 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :
Met Thr Leu Leu Trp Cys Val Val Ser Leu Tyr Phe Tyr Gly He Leu -17 -15 -10 -5
Gin Ser Asp Ala Ser Glu Arg Cys Asp Asp Trp Gly Leu Asp Thr Met
1 5 10 15
Arg Gin He Gin Val Phe Glu Asp Glu Pro Ala Arg He Lys Cys Pro 20 25 30 Leu Phe Glu His Phe Leu Lys Phe Asn Tyr Ser Thr Ala His Ser Ala 35 40 45
Gly Leu Thr Leu He Trp Tyr Trp Thr Lys Gin Asp Arg Asp Leu Glu 50 55 60
Glu Pro He Asn Phe Arg Leu Pro Glu Asn Arg He Ser Lys Glu Lys 65 70 75
Asp Val Leu Trp Phe Arg Pro Thr Leu Leu Asn Asp Thr Gly Asn Tyr 80 85 90 95
Thr Cys Met Leu Arg Asn Thr Thr Tyr Cys Ser Lys Val Ala Phe Pro 100 105 110
Leu Glu Val Val Gin Lys Asp Ser Cys Phe Asn Ser Pro Met Lys Leu 115 120 125
Pro Val His Lys Leu Tyr He Glu Tyr Gly He Gin Arg He Thr Cys 130 135 140
Pro Asn Val Asp Gly Tyr Phe Pro Ser Ser Val Lys Pro Thr He Thr 145 150 155
Trp Tyr Met Gly Cys Tyr Lys He Gin Asn Phe Asn Asn Val He Pro 160 165 170 175
Glu Gly Met Asn Leu Ser Phe Leu He Ala Leu He Ser Asn Asn Gly 180 185 190
Asn Tyr Thr Cys Val Val Thr Tyr Pro Glu Asn Gly Arg Thr Phe His 195 200 205
Leu Thr Arg Thr Leu Thr Val Lys Val Val Gly Ser Pro Lys Asn Ala 210 215 220
Val Pro Pro Val He His Ser Pro Asn Asp His Val Val Tyr Glu Lys 225 230 235
Glu Pro Gly Glu Glu Leu Leu He Pro Cys Thr Val Tyr Phe Ser Phe 240 245 250 255
Leu Met Asp Ser Arg Asn Glu Val Trp Trp Thr He Asp Gly Lys Lys 260 265 270
Pro Asp Asp He Thr He Asp Val Thr He Asn Glu Ser He Ser His 275 280 285
Ser Arg Thr Glu Asp Glu Thr Arg Thr Gin He Leu Ser He Lys Lys 290 295 300
Val Thr Ser Glu Asp Leu Lys Arg Ser Tyr Val Cys His Ala Arg Ser 305 310 315
Ala Lys Gly Glu Val Ala Lys Ala Ala Lys Val Lys Gin Lys Gly Asn 320 325 330 335
Arg Cys Gly Gin
(2) INFORMATION FOR SEQ ID NO : 3 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 350 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS : not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :
Met Gly Leu Leu Trp Tyr Leu Met Ser Leu Ser Phe Tyr Gly He Leu 1 5 10 15
Gin Ser His Ala Ser Glu Arg Cys Asp Asp Trp Gly Leu Asp Thr Met 20 25 30
Arg Gin He Gin Val Phe Glu Asp Glu Pro Ala Arg He Lys Cys Pro 35 40 45
Leu Phe Glu His Phe Leu Lys Tyr Asn Tyr Ser Thr Ala His Ser Ser 50 55 60
Gly Leu Thr Leu He Trp Tyr Trp Thr Arg Gin Asp Arg Asp Leu Glu 65 70 75 80
Glu Pro He Asn Phe Arg Leu Pro Glu Asn Arg He Ser Lys Glu Lys 85 90 95
Asp Val Leu Trp Phe Arg Pro Thr Leu Leu Asn Asp Thr Gly Asn Tyr 100 105 110
Thr Cys Met Leu Arg Asn Thr Thr Tyr Cys Ser Lys Val Ala Phe Pro 115 120 125
Leu Glu Val Val Gin Lys Asp Ser Cys Phe Asn Ser Ala Met Arg Phe 130 135 140
Pro Val His Lys Met Tyr He Glu His Gly He His Lys He Thr Cys 145 150 155 160
Pro Asn Val Asp Gly Tyr Phe Pro Ser Ser Val Lys Pro Ser Val Thr 165 170 175
Trp Tyr Lys Gly Cys Thr Glu He Val Asp Phe His Asn Val Leu Pro 180 185 190 Glu Gly Met Asn Leu Ser Phe Phe He Pro Leu Val Ser Asn Asn Gly 195 200 205
Asn Tyr Thr Cys Val Val Thr Tyr Pro Glu Asn Gly Arg Leu Phe His 210 215 220
Leu Thr Arg Thr Val Thr Val Lys Val Val Gly Ser Pro Lys Asp Ala 225 230 235 240
Leu Pro Pro Gin He Tyr Ser Pro Asn Asp Arg Val Val Tyr Glu Lys 245 250 255
Glu Pro Gly Glu Glu Leu Val He Pro Cys Lys Val Tyr Phe Ser Phe 260 265 270
He Met Asp Ser His Asn Glu Val Trp Trp Thr He Asp Gly Lys Lys 275 280 285
Pro Asp Asp Val Thr Val Asp He Thr He Asn Glu Ser Val Ser Tyr 290 295 300
Ser Ser Thr Glu Asp Glu Thr Arg Thr Gin He Leu Ser He Lys Lys 305 310 315 320
Val Thr Pro Glu Asp Leu Arg Arg Asn Tyr Val Cys His Ala Arg Asn 325 330 335
Thr Lys Gly Glu Ala Glu Gin Ala Ala Lys Vάϊ Lys Gin Lys 340 345 350
(2) INFORMATION FOR SEQ ID NO : 4 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 294 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : :
TCTATGAGAA AGAACAAGGA GAGGAGCTAC TCATTCCCTG TACGGTCTAT TTTAGTTTTC 60
TGATGGATTC TCGCAATGAG GTTTGGTGGA CCATTGATGG AAAAAAACCT GATGACATCA 120
CTATTGATGT CACCATTAAC GAAAGTATAA GTCATAGTAG AACAGAAGAT GAAACAAGAA 180
CTCAGATTTT GAGCATCAAG AAAGTTACCT CTGAGGATCT CAAGCGCANT ANTGTCTGTC 240
ATGCTAGAAG TGCCAAAGGC GAAGTTGCCA AAGCAGCCAA GGTGAAGCAG ΛAAG 294 (2) INFORMATION FOR SEQ ID NO • 5 :
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH 15 amino acids
(B) TYPE: ammo acid
(C) STRANDEDNESS not relevant
(D) TOPOLOGY: not relevant
(n) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION SEQ ID NO 5
Ser Asp Ala Ser Glu Arg Cys Asp Asp Trp Gly Leu Asp Thr Met
1 5 10 15
(2) INFORMATION FOR SEQ ID NO 6
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH 9 ammo acids
(B) TYPE amino ac d
(C) STRANDEDNESS not relevant
(D) TOPOLOGY- not relevant
(11) MOLECULE TYPE peptide
(xi) SEQUENCE DESCRIPTION SEQ ID NO 6
Val Phe Glu Asp Glu Pro Ala Arg He 1 5
(2) INFORMATION FOR SEQ ID NO , 7
(l) SEQUENCE CHARACTERISTICS-
(A) LENGTH 7 ammo acids
(B) TYPE: ammo ac d
(C) STRANDEDNESS not relevant
(D) TOPOLOGY: not relevant
(ll) MOLECULE TYPE- peptide
(xi) SEQUENCE DESCRIPTION- SEQ ID NO 7
Gly Leu Thr Leu He Trp Tyr 1 5
(2) INFORMATION FOR SEQ ID NO 8 (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 ammo acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
( n ) MOLECULE TYPE peptide
(xi ) SEQUENCE DESCRIPTION - SEQ ID NO 8
Arg Gin Asp Arg Asp Leu Glu Glu Pro He
1 5 10
(2) INFORMATION FOR SEQ ID NO : 9
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH- 10 ammo acids
Figure imgf000072_0001
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(li) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION. SEQ ID NO 9
Leu Pro Glu Asn Arg He Ser Lys Glu Lys 1 5 10
(2) INFORMATION FOR SEQ ID NO: 10
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH: 7 ammo acids
(B) TYPE: ammo acid
(C) STRANDEDNESS. not relevant
(D) TOPOLOGY, not relevant
(ii) MOLECULE TYPE- peptide
(xi) SEQUENCE DESCRIPTION SEQ ID NO.10
Ser Lys Val Ala Phe Pro Leu 1 5
(2) INFORMATION FOR SEQ ID NO: 11
(l) SEQUENCE CHARACTERISTICS (A) LENGTH: 10 ammo acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(n) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
Val Gin Lys Asp Ser Cys Phe Asn Ser Pro
1 5 10
(2) INFORMATION FOR SEQ ID NO : 12 :
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 ammo acids
Figure imgf000073_0001
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(n) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO i;
Val His Lys Leu Tyr He Glu 1 5
(2) INFORMATION FOR SEQ ID NO:13:
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH: 15 ammo acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
( i ) MOLECULE TYPE : peptide
(x ) SEQUENCE DESCRIPTION . SEQ ID NO . 13
He Thr Trp Tyr Met Gly Cys Tyr Lys He Gin Asn Phe Asn Asn 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 ammo acids (B) TYPE: ammo acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14
Leu Ser Phe Leu He Ala Leu He
1 5
(2) INFORMATION FOR SEQ ID NO: 15:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 ammo acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(i ) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION- SEQ ID NO 15
His Leu Thr Arg Thr Leu Thr Val Lys 1 5
(2) INFORMATION FOR SEQ ID NO: 16.
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(n) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION- SEQ ID NO 16
Pro Asn Asp His Val Val Tyr Glu Lys Glu Pro Gly Glu Glu 1 5 10
(2) INFORMATION FOR SEQ ID NO -17
(l) SEQUENCE CHARACTERISTICS-
(A) LENGTH: 9 amino acids
(B) TYPE: ammo acid (C) STRANDEDNESS- not relevant
(D) TOPOLOGY not relevant
(11) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO -17
He Pro Cys Thr Val Tyr Phe Ser Phe 1 5
(2) INFORMATION FOR SEQ ID NO: 18
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH 9 ammo acids
(B) TYPE amino acid
(C) STRANDEDNESS- not relevant
(D) TOPOLOGY not relevant
(n) MOLECULE TYPE peptide
(Xl) SEQUENCE DESCRIPTION- SEQ ID NO 18
Asp Gly Lys Lys Pro Asp Asp He Thr 1 5
(2) INFORMATION FOR SEQ ID NO 19
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH. 11 ammo acids
Figure imgf000075_0001
(C) STRANDEDNESS- not relevant
(D) TOPOLOGY- not relevant
(ii) MOLECULE TYPE- peptide
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO 19
He Ser His Ser Arg Thr Glu Asp Glu Thr Arg 1 5 10
(2) INFORMATION FOR SEQ ID NO: 20.
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 ammo acids
Figure imgf000075_0002
(C) STRANDEDNESS not relevant (D) TOPOLOGY, not relevant (ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO -20.
Lys Val Thr Ser Glu Asp Leu Lys
1 5
(2) INFORMATION FOR SEQ ID NO: 21
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH: 13 ammo acids
Figure imgf000076_0001
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION. SEQ ID NO 21
Ser Ala Lys Glu Gly Glu Val Ala Lys Ala Ala Lys Val 1 5 10
(2) INFORMATION FOR SEQ ID NO .22
(l) SEQUENCE CHARACTERISTICS.
(A) LENGTH: 7 ammo acids
Figure imgf000076_0002
(C) STRANDEDNESS- not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO 22
Lys Gly Asn Arg Cys Gly Gin 1 5
(2) INFORMATION FOR SEQ ID NO.23.
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY, linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: GGATCCATGA CACTTCTGTG GTGTG 25
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: GTCGACTCAC TGACCGCATC T 21
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: CDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: GGATCCATCC GCCATCATGA CACTTCTGTG GTGTG 35
(2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 47 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single '
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: TCTAGAAAAG CGTAGTCTGG GACGTCGTAT GGGTACTGAC CGCATCT 47
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: GACTGGATCC GCCATCATGA CACTTCTGTG GTGTG 35
(2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: GAATTCCTCA CTGACCGCAT CT 22
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: GACTGGATCC GCCATCATGA CACTTCTGTG GTGTG 35 (2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: GACTGGTACC CATAGAAATC ATGTGTATAC C 31
Applicant's or agent's file International application No. Reference number 1488 . 045PC00 IBA
I NDICATIONS RELATING- TO A DEPOSITED MICROORGANISM
(PCT Rule \3bis)
Figure imgf000080_0001
For International Bureau use only
I j This sheet was received by the International Bureau on:
Λulhoπ/.cd officer
Figure imgf000080_0002
Form I'CI /UO/I VI Jul 1<W2 -78/1-
DNA PLASMID 1450621 ATTCC 97666
SINGAPORE
The applicant hereby request that the furnishing of a sample of a microorganism shall only be made available to an expert. The request to this effect must be filed by the applicant with the International Bureau before the completion of the technical preparations for international publication of the application.
NORWAY
The applicant hereby request that, until the application has been laid open to public inspection (by the Norwegian Patent Office), or has been finally decided upon by the Norwegian Patent Office without having been laid open to public inspection, the furnishing of a sample shall only be effected to an expert in the art. The request to this effect shall be filed by the applicant with the Norwegian Patent Office not later than at the time when the application is made available to the pbulic under Sections 22 and 33(3) of the Norwegian Patents Act. If such a request has been filed by the applicant, any request made by a third party for the furnishing of a sample shall indicate the expert to be used. That expert may be any person entered on a list of recognized experts drawn up by the Norwegian Patent office or any person approved by the applicant in the individual case.
AUSTRALIA
The applicant hereby gives notice that the furnishing of a sample of a microorganism shall only be effected prior to the grant of a patent, or prior to the lapsing, refusal or withdrawal of the application, to a person who is a skilled addressee without an interest in the invention (Regulation 3.25(3) of the Australian Patents Regulations).
FINLAND
The applicant hereby requests that, until the application has been laid open to public inspection (by the National Board of Patents and Registration), or has been finally decided upon by the National Board of Patents and Registration without having been laid open to public inspection, the furnishing of a sample shall only be effected to an expert in the art.

Claims

-79-What Is Claimed Is:
1. An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of: (a) a nucleotide sequence encoding the human EL- 1 receptor accessory molecule polypeptide having the complete amino acid sequence in Figure 1 [SEQ ID NO:2];
(b) a nucleotide sequence encoding the human IL-1 receptor accessory molecule polypeptide having the amino acid sequence at positions 18- 356 in Figure 1 [SEQ ID NO:2];
(c) a nucleotide sequence encoding the human IL- 1 receptor accessory molecule polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97666;
(d) a nucleotide sequence encoding the mature human IL-1 receptor accessory molecule polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No 97666; and
(e) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c) or (d)
2. The nucleic acid molecule of claim 1 wherein said polynucleotide has the complete nucleotide sequence in Figure 1 [SEQ ID NO:l]
3. The nucleic acid molecule of claim 1 wherein said polynucleotide has the nucleotide sequence in Figure 1 [SEQ ID NO.1 ] encoding the human IL- 1 receptor accessory molecule polypeptide having the complete amino acid sequence in Figure 1 [SEQ ID NO:2].
4. The nucleic acid molecule of claim 1 wherein said polynucleotide has the nucleotide sequence in Figure 1 [SEQ ED NO: l] encoding the mature -80-
human IL-1 receptor accessory molecule polypeptide having the amino acid sequence in Figure 1 [SEQ ID NO:2].
5. The nucleic acid molecule of claim 1 wherein said polynucleotide has the complete nucleotide sequence of the cDNA clone contained in ATCC
Deposit No. 97666.
6. The nucleic acid molecule of claim 1 wherein said polynucleotide has the nucleotide sequence encoding the human IL-1 receptor accessory molecule polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97666.
7. The nucleic acid molecule of claim 1 wherein said polynucleotide has the nucleotide sequence encoding the mature human EL-l receptor accessory molecule polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97666.
8. An isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide having a nucleotide sequence identical to a nucleotide sequence in (a), (b), (c), (d) or (e) of claim 1 wherein said polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues.
9. An isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a human IL- 1 receptor accessory molecule polypeptide having an amino acid sequence in (a), (b), (c) or (d) of claim 1. -81-
10 The isolated nucleic acid molecule of claim 9, which encodes an epitope-bearing portion of a human IL-1 receptor accessory molecule polypeptide selected from the group consisting of a S18-M32 SDASERCDDWGLDTM (SEQ ID NO 5), b V37-145 VFEDEPARI (SEQ ID NO 6), c G65-Y71 GLTLIWY (SEQ ID NO 7), d R74-I83 RQDRDLEEPI (SEQ ID NO 8), e L87-K96 LPENRISKEK (SEQ ID NO 9), f S123-L129 SKVAFPL (SEQ ID NO 10), g V132-P141 VQKDSCFNSP (SEQ ID NO 11), h V146-E152 VHKLYIE (SEQ ED NO 12), i I175-N189 ITWYMGCYKIQNFNN (SEQ ID NO 13), j L197-I204 LSFLIALI (SEQ ID NO 14), k H224-K232 HLTRTLTVK (SEQ ID NO 15), 1 P248-E261 PNDHWYEKEPGEE (SEQ ID NO 16), m I264-F272 IPCTVYFSF (SEQ ID NO 17), n D285-T293 DGKKPDDIT (SEQ ID NO 18), o I302-R312 ISHSRTEDETR (SEQ ID NO 19), p K320-K327 KVTSEDLK (SEQ ID NO 20), q S336-V347 SAKEGEVAKAAKV (SEQ ID NO 21), and r K350-Q356 KGNRCGQ (SEQ ID NO 22)
1 1 A method for making a recombinant vector comprising inserting an isolated nucleic acid molecule of claim 1 into a vector
12 A recombinant vector produced by the method of claim 11
13 A method of making a recombinant host cell comprising introducing the recombinant vector of claim 12 into a host cell
14 A recombinant host cell produced by the method of claim 13 -82-
15. A recombinant method for producing a human IL-1 receptor accessory molecule polypeptide, comprising culturing the recombinant host cell of claim 14 under conditions such that said polypeptide is expressed and recovering said polypeptide.
16. An isolated human IL-1 receptor accessory molecule polypeptide having an amino acid sequence at least 95% identical to a sequence selected from the group consisting of:
(a) the amino acid sequence of the human IL- receptor accessory molecule polypeptide having the complete amino acid sequence in
Figure 1 [SEQ ID NO:2];
(b) the amino acid sequence of the mature human IL-1 receptor accessory molecule polypeptide having the amino acid sequence at positions 18- 356 in Figure 1 [SEQ ID NO:2]; (c) the amino acid sequence of the human IL-1 receptor accessory molecule polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97666;
(d) the amino acid sequence of the mature human IL-1 receptor accessory molecule polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97666; and
(e) the amino acid sequence of an epitope-bearing portion of any one of the polypeptides of (a), (b), (c), or (d).
17. An isolated polypeptide comprising an epitope-bearing portion of the human EL-l receptor accessory molecule protein, wherein said portion is selected from the group consisting of: a. S18-M32: SDASERCDD GLDTM (SEQ ID NO:5); b. V37-I45: VFEDEPARI (SEQ ID NO:6); c. G65-Y71: GLTLIWY (SEQ ID NO:7); d. R74-I83: RQDRDLEEPI (SEQ ID NO: 8); -83-
e. L87-K96: LPENRISKEK (SEQ ID NO:9); f. S123-L129: SKVAFPL (SEQ ID NO: 10); g. V132-P141 : VQKDSCFNSP (SEQ ID NO: 11); h. V146-E152: VHKLYIE (SEQ ID NO: 12); i. I175-N189: ITWYMGCYKIQNFNN (SEQ ID NO: 13), j. L197-I204: LSFLIALI (SEQ ID NO 14), k. H224-K232: HLTRTLTVK (SEQ ID NO: 15), 1. P248-E261: PNDHWYEKEPGEE (SEQ ID NOT 6), m. I264-F272: IPCTVYFSF (SEQ ID NO. 17), n. D285-T293: DGKKPDDIT (SEQ ID NO 18), o. I302-R312: ISHSRTEDETR (SEQ ID NO 19), p. K320-K327: KVTSEDLK (SEQ ID NO. 20), q. S336-V347: SAKEGEVAKAAKV (SEQ ID NO. 21), and r. K350-Q356: KGNRCGQ (SEQ ID NO 22)
18 An isolated antibody that binds specifically to a human IL-1 receptor accessory molecule polypeptide of claim 16
19 A method for identifying a type 1 IL-1 receptor agonist, comprising: (a) providing a host cell containing recombinant genes which express a polypeptide comprising a type I IL-1 receptor and a polypeptide comprising IL-IR AcM or a IL-IR AcM fragment, wherein said type I IL-IR and IL-IR AcM or said type I IL-IR and IL-IR AcM fragment form a complex, (b) administering a candidate agonist to said cell, and (c) determining the binding affinity of said complex for said candidate agonist relative to the binding affinity of said complex for IL-1.
20. A method for identifying a type I IL-1 receptor antagonist comprising' -84-
(a) providing a host cell containing recombinant genes which express a polypeptide comprising a type I IL-1 receptor and a polypeptide comprising IL-IR AcM or a IL-IR AcM fragment, wherein said type I IL-IR and IL-IR AcM or said type I EL-l R and EL-IR AcM fragment form a complex;
(b) administering a candidate antagonist to said cell; and
(c) determining the whether said candidate agonist disrupts or prevents formation of a complex between type I IL-IR and IL-IR AcM.
PCT/US1996/013954 1996-08-26 1996-08-26 Soluble interleukin-1 receptor accessory molecule WO1998008969A1 (en)

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Cited By (9)

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US6733753B2 (en) 1997-02-10 2004-05-11 Amgen Inc. Composition and method for treating inflammatory diseases
EP1229047A3 (en) * 1998-09-25 2002-10-02 Regeneron Pharmaceuticals, Inc. IL-1 Receptor fusion proteins used as antagonists and methods of making and using
GB2375604A (en) * 2001-05-18 2002-11-20 Warner Lambert Co Methods for screening using interleukin soluble trimolecular complex
WO2002095414A1 (en) * 2001-05-18 2002-11-28 Warner-Lambert Company Llc Methods for screening using interleukin soluble trimolecular complex
US7619066B2 (en) 2004-04-02 2009-11-17 Amgen Inc. IL-1ra variants
US10765747B2 (en) 2004-04-02 2020-09-08 Swedish Orphan Biovitrum Ab (Publ) Methods of reducing aggregation of IL-1ra
WO2014100772A1 (en) * 2012-12-21 2014-06-26 Cellerant Therapeutics, Inc. Antibodies that bind membrane-bound il1rap
US11168140B2 (en) 2018-08-17 2021-11-09 23Andme, Inc. Anti-IL1RAP antibodies
US12304959B2 (en) 2018-08-17 2025-05-20 23Andme, Inc. Method of treating asthma with anti-IL1RAP antibodies

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