EP1161451A2 - Endokine alpha humain et methodes d'utilisation - Google Patents

Endokine alpha humain et methodes d'utilisation

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Publication number
EP1161451A2
EP1161451A2 EP00913602A EP00913602A EP1161451A2 EP 1161451 A2 EP1161451 A2 EP 1161451A2 EP 00913602 A EP00913602 A EP 00913602A EP 00913602 A EP00913602 A EP 00913602A EP 1161451 A2 EP1161451 A2 EP 1161451A2
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EP
European Patent Office
Prior art keywords
polypeptide
amino acid
ofthe
antibodies
seq
Prior art date
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Withdrawn
Application number
EP00913602A
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German (de)
English (en)
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EP1161451A4 (fr
Inventor
Guo-Liang Yu
Jian Ni
Craig A. Rosen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Human Genome Sciences Inc
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Human Genome Sciences Inc
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Priority to EP10179623A priority Critical patent/EP2357192A1/fr
Publication of EP1161451A2 publication Critical patent/EP1161451A2/fr
Publication of EP1161451A4 publication Critical patent/EP1161451A4/fr
Withdrawn legal-status Critical Current

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    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/525—Tumour necrosis factor [TNF]
    • C07K14/5255—Lymphotoxin [LT]
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Definitions

  • the present invention concerns a novel member of the tumor necrosis factor (TNF) family of cytokines.
  • TNF tumor necrosis factor
  • isolated nucleic acid molecules are provided encoding the endokine alpha protein.
  • Endokine alpha polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. Also provided are diagnostic and therapeutic methods concerning TNF family-related disorders.
  • TNF ⁇ tumor necrosis factor- ⁇
  • cachectin cachectin
  • TNF is a regulatory cytokine with pleiotropic biological activities. These activities include: inhibition of lipoprotein lipase synthesis ("cachectin” activity) (Beutler, B. et al, Nature 316:552 (1985)). activation of polymorphonuclear leukocytes (Klebanoff, S.J. et al, J. Immunol.
  • MHC major histocompatibility complex
  • TNF is noted for its pro-inflammatory actions which result in tissue injury, such as induction of procoagulant activity on vascular endothelial cells (Pober, J.S. et al, J. Immunol. 73(5:1680 (1986)), increased adherence of neutrophils and lymphocytes (Pober, J.S. et al, J. Immunol 735:3319 (1987)), and stimulation of the release of platelet activating factor from macrophages. neutrophils and vascular endothelial cells (Camussi, G. et al, J. Exp. Med. 166:1390 (1987)). Recent evidence implicates TNF in the pathogenesis of many infections
  • Cachexia includes progressive weight loss, anorexia, and persistent erosion of body mass in response to a malignant growth.
  • the cachectic state is thus associated with significant morbidity and is responsible for the majority of cancer mortality.
  • TNF is an important mediator ofthe cachexia in cancer, infectious pathology, and in other catabolic states.
  • TNF is thought to play a central role in the pathophysiological consequences of Gram-negative sepsis and endotoxic shock (Michie. H.R. el al, Br. J. Surg. 76:610-611 (1989); Debets, J.M.H. et al, Second Vienna Shock Forum, p.463-466 (1989); Simpson, S.Q. et ⁇ /., Crit. Care Clin. 5:27-47 (1989)), including fever, malaise, anorexia, and cachexia.
  • Endotoxin is a potent monocyte/macrophage activator which stimulates production and secretion of TNF (Kornbluth, S.K. et al, J. Immunol.
  • TNF could mimic many biological effects of endotoxin, it was concluded to be a central mediator responsible for the clinical manifestations of endotoxin-related illness. TNF and other monocyte-derived cytokines mediate the metabolic and neurohormonal responses to endotoxin (Michie, H.R. etal. N. Eng. J. Med. 375:1481-1486 (1988)). Endotoxin administration to human volunteers produces acute illness with flu-like symptoms including fever, tachycardia, increased metabolic rate and stress hormone release (Revhaug, A. et al, Arch. Surg. 723:162-170 (1988)).
  • Elevated levels of circulating TNF have also been found in patients suffering from Gram-negative sepsis (Waage, A. et al, Lancet 7:355-357 (1987); Hammerle, A.F. et al, Second Vienna Shock Forum p.
  • Passive immunotherapy directed at neutralizing TNF may have a beneficial effect in Gram-negative sepsis and endotoxemia, based on the increased TNF production and elevated TNF levels in these pathology states, as discussed above.
  • Antibodies to a "modulator" material which was characterized as cachectin
  • Kawasaki's pathology infantile acute febrile mucocutaneous lymph node syndrome; Kawasaki, T., Allergy 16:118 (1967); Kawasaki, T., Shonica (Pediatrics) 26:935 (1985)) were said to contain elevated TNF levels which were related to progress ofthe pathology (Yone et al, supra).
  • Other investigators have described mAbs specific for recombinant human
  • TNF neutralizing antibodies Some of these mAbs were used to map epitopes of human TNF and develop enzyme immunoassays (Fendly et al, supra; Hirai et al, supra; Moller et al, supra) and to assist in the purification of recombinant TNF (Bringman et al. , supra). However, these studies do not provide a basis for producing TNF neutralizing antibodies that can be used for in vivo diagnostic or therapeutic uses in humans, due to immunogenicity, lack of specificity and/or pharmaceutical suitability.
  • Neutralizing antisera or mAbs to TNF have been shown in mammals other than man to abrogate adverse physiological changes and prevent death after lethal challenge in experimental endotoxemia and bacteremia. This effect has been demonstrated, e.g., in rodent lethality assays and in primate pathology model systems (Mathison, J.C. et al. J. Clin. Invest. 57:1925-1937 (1988); Beutler, B. et al, Science 229:869-871 (1985); Tracey, K.J. et al, Nature 330:662-664 (1987); Shimamoto, Y. et al, Immunol. Lett. 77:31 1-318 (1988); Silva, A.T.
  • cytokine receptors sequence analysis of cytokine receptors has defined several subfamilies of membrane proteins (1) the Ig superfamily, (2) the hematopoietin (cytokine receptor superfamily and (3) the tumor necrosis factor (TNF)/nerve growth factor
  • NNF receptor superfamily (NGF) receptor superfamily (for review of TNF superfamily see, Gruss and
  • TNF/NGF receptor superfamily contains at least 10 different proteins. Gruss and Dower, supra. Ligands for these receptors have been identified and belong to at least two cytokine superfamilies.
  • cytokines similar to TNF that are involved in pathological conditions.
  • Such novel cytokines could be used to make novel antibodies or other antagonists that bind these TNF-like cytokines for therapy of TNF-like disorders.
  • the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding a cytokine that is similar to TNF and is believed to have similar biological effects and activities.
  • This cytokine is named endokine alpha, and includes endokine alpha polypeptides having at least a portion ofthe amino acid sequence in Figure 1 (SEQ ID NO:2) or an amino acid sequence encoded by the cDNA clone deposited in a bacterial host as ATCC Deposit Number 97640 on June 27, 1996.
  • the nucleotide sequence which was determined by sequencing the deposited endokine alpha cDNA clone, contains an open reading frame encoding a polypeptide of about 169 amino acid residues including an N-terminal methionine, an intracellular domain of about 17 amino acid residues, a transmembrane domain of about 26 amino acids, an extracellular domain of about 126 amino acids, and a deduced molecular weight for the complete protein of about 19 kDa.
  • 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 endokine alpha polypeptide having the complete amino acid sequence in SEQ ID NO:2; (b) a nucleotide sequence encoding the endokine alpha polypeptide having the complete amino acid sequence in SEQ ID NO:2 but minus the N-terminal methionine residue; (c) a nucleotide sequence encoding the endokine alpha polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in
  • nucleic acid molecules that comprise or, alternatively, consist of, a polynucleotide having a nucleotide sequence at least 80%, 85%, 90%, 92%, or 95% identical, and more preferably at least 96%, 97%, 98% or 99% identical, to any of the nucleotide sequences in (a), (b), (c), or (d), above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b), (c), or (d), 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 endokine alpha polypeptide having an amino acid sequence in (a), (b), (c), or (d), above.
  • nucleic acid fragments include nucleic acid molecules which encode: a polypeptide comprising the endokine alpha intracellular domain (amino acid residues from about 1 to about 17 in Figure 1 (SEQ ID NO:2)); a polypeptide comprising the endokine alpha transmembrane domain (amino acid residues from about 18 to about 43 in Figure 1 (SEQ ID NO:2)); and a polypeptide comprising the endokine alpha extracellular domain (amino acid residues from about 44 to about 169 in Figure 1 (SEQ ID NO:2)).
  • 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 endokine alpha polypeptides or peptides by recombinant techniques.
  • the invention further provides an isolated endokine alpha polypeptide having an amino acid sequence selected from the group consisting of: (a) the complete 169 amino acid sequence in SEQ ID NO:2; (b) the complete 169 amino acid sequence in SEQ ID NO:2 but minus the N-terminal methionine residue; (c) the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No.97640; and (d) the amino acid sequence of an epitope-bearing portion of any one ofthe polypeptides of (a), (b), or (c).
  • the polypeptides ofthe present invention also include polypeptides having an amino acid sequence at least 80%, 85%, 90%, 92%, or 95% identical, more preferably at least 96%, 97%, 98% or 99% identical to those above.
  • Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a endokine alpha polypeptide ofthe 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 ofthe invention described above also are included in the invention.
  • the invention provides an isolated antibody that binds specifically to an endokine alpha polypeptide having an amino acid sequence described in (a), (b), (c), or (d) above.
  • Preferred polypeptide fragments according to the present invention include a polypeptide comprising: the endokine alpha intracellular domain, the endokine alpha transmembrane domain, and the endokine alpha extracellular domain.
  • the invention further provides methods for isolating antibodies that bind specifically to an endokine alpha polypeptide having an amino acid sequence as described above. Such antibodies may be useful diagnostically or therapeutically as antagonists in the treatment of endokine alpha- and/or TNF-related disorders.
  • the invention also provides a diagnostic method for determining the presence of a TNF-related disorder.
  • Figure 1 shows the nucleotide (SEQ ID NO: l) and deduced amino acid (SEQ ID NO:2) sequences of the endokine alpha protein.
  • Amino acids 1 to 17 represent the intracellular domain, amino acids 18 to 43 the transmembrane domain (the underlined sequence), and amino acids 44 to 169 the extracellular domain (the remaining sequence).
  • Figure 2 shows the regions of similarity between the amino acid sequences ofthe endokine alpha protein (SEQ ID NO:2), tissue necrosis factor ⁇ (TNF- ⁇ ) (SEQ ID NO:3), and TNF- ⁇ (SEQ ID NO:4).
  • the J. Hein method was used with PAM 250 residue weight table. Shading with solid black indicates residues that match consensus exactly.
  • Figure 3 provides an analysis ofthe endokine alpha amino acid sequence.
  • Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.
  • amino acid residues 44-54, 57-68, 69-78, 94-105, 108-132 and 148-158 in Figure 1 correspond to the shown highly antigenic regions of the endokine alpha protein.
  • the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding an endokine alpha protein having an amino acid sequence shown in Figure 1 (SEQ ID NO:2), which was determined by sequencing a cloned cDNA.
  • Endokine alpha is a novel member of the tumor necrosis factor (TNF) ligand family and shares sequence homology with human TNF ⁇ and related TNF family members ( Figure 2).
  • the nucleotide sequence shown in Figure 1 (SEQ ID NO: l) was obtained by sequencing a cDNA clone, which was deposited on June 27, 1996, at the American Type Culture Collection, Patent Depository, 10801 University Boulevard, Manassas, VA 20110-2209, and given accession number 97640.
  • the deposited clone is contained in the pBluescript SK(-) plasmid (Stratagene, LaJolla, CA).
  • 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.
  • 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 99.99% identical to the actual nucleotide sequence ofthe sequenced DNA molecule.
  • the actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art.
  • nucleotide sequence 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 expected 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.
  • each "nucleotide sequence" set forth herein is presented as a sequence of deoxyribonucleotides (abbreviated A, G , C and T).
  • 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 deoxynucleotide (T) in the specified deoxynucleotide sequence in is replaced by the ribonucleotide uridine (U).
  • RNA molecule having the sequence of Figure 1 (SEQ ID NO: l ) set forth using deoxyribonucleotide abbreviations is intended to indicate an RNA molecule having a sequence in which each deoxynucleotide A, G or C of SEQ ID NO: 1 has been replaced by the corresponding ribonucleotide A, G or C, and each deoxynucleotide T has been replaced by a ribonucleotide U.
  • nucleic acid molecule ofthe present invention encoding an endokine alpha polypeptide can 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
  • SEQ ID NO: 1 the nucleic acid molecule described in Figure 1 was discovered in a cDNA library derived from human brain striatum. Expressed sequence tags corresponding to a portion ofthe endokine alpha cDNA were also found in several endothelial libraries and a fetal liver library.
  • the endokine alpha gene contains an open reading frame encoding a protein of about 169 amino acid residues, an intracellular domain of about 17 amino acids (amino acid residues from about 1 to about 17 in Figure 1 (SEQ ID NO:2)), a transmembrane domain of about 26 amino acids (amino acid residues from about 18 to about 43 in Figure 1 (SEQ ID NO:2)), an extracellular domain of about 126 amino acids (amino acid residues from about 44 to about 169 in Figure 1 (SEQ ID NO:2)); and a deduced molecular weight of about 19 kDa.
  • the endokine alpha protein shown in Figure 1 (SEQ ID NO: 2) is about 30% similar and about 22% identical to human TNF- ⁇ , which can be accessed on GenBank as Accession No. U42764.
  • the actual endokine alpha polypeptide encoded by the deposited cDNA comprises about 169 amino acids, but can be anywhere in the range of about 154-184 amino acids. It will also be appreciated by reasonable persons of skill in the art that, depending on the criteria used, the exact
  • the exact location ofthe endokine alpha intracellular, transmembrane and extracellular domains shown in Figure 1 may vary slightly (e.g. , the exact address may differ by about 1 to about 5 residues compared to that shown in Figure 1 ) depending on the criteria used to define the domain.
  • nucleic acid molecules ofthe 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 can be the coding strand, also known as the sense strand, or it can 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 ofthe 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 ofthe DNA molecules ofthe present invention.
  • Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
  • a nucleic acid contained in a clone that is a member of a library e.g., a genomic or cDNA library
  • a chromosome isolated or removed from a cell or a cell lysate e.g., a "chromosome spread," as in a karyotype
  • isolated for the purposes ofthe invention.
  • isolated nucleic acid molecules according to the present invention may be produced naturally, recombinantly, or synthetically.
  • Isolated nucleic acid molecules of the present invention include DNA molecules comprising the open reading frame (ORF) shown in Figure 1 (SEQ ID NO:
  • 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.
  • the invention provides isolated nucleic acid molecules encoding the endokine alpha polypeptide having an amino acid sequence encoded by the cDNA of the clone deposited as ATCC Deposit No. 97640 on June 27, 1996.
  • the invention further provides an isolated nucleic acid molecule having the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1 ) or the nucleotide sequence ofthe endokine alpha 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 are useful as probes for gene mapping by in situ hybridization with chromosomes and for detecting expression ofthe endokine alpha gene in human tissue, for instance, by Northern blot analysis. As described in detail below, detecting altered endokine alpha gene expression in certain tissues or bodily fluids is indicative of certain disorders.
  • the present invention is further directed to fragments of the isolated nucleic acid molecules described herein.
  • a fragment of an isolated nucleic acid molecule having the nucleotide sequence ofthe deposited cDNA or the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1) is intended fragments at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about
  • fragments 50, 100, 150, 200, 250, 300, 350, 400, 450, and 500 nt in length are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in Figure 1
  • fragments at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence ofthe deposited cDNA or the nucleotide sequence as shown in Figure 1 (SEQ ID NO: 1). Since the gene has been deposited and the nucleotide sequence shown in Figure 1 (SEQ ID NO 1) is provided, generating such DNA fragments would be routine to the skilled artisan. For example, restriction endonuclease cleavage or shearing by sonication could easily be used to generate fragments of various sizes. Alternatively, such fragments could be generated synthetically.
  • HEMCG04R SEQ ID NO: 11
  • BLAST analysis has 94% identity to nucleotides 26 to 482 of SEQ ID NO: 1.
  • Preferred nucleic acid fragments ofthe present invention include nucleic acid molecules encoding: apolypeptide comprising or, alternatively, consisting of, the endokine alpha intracellular domain (amino acid residues from about 1 to about 17 in Figure 1 (SEQ ID NO 2). or as encoded by the cDNA clone contained in ATCC Deposit No. 97640); a polypeptide comprising or, alternatively, consisting of, the endokine alpha transmembrane domain (amino acid residues from about 18 to about 43 in Figure 1 (SEQ ID NO 2), or as encoded by the cDNA clone contained in ATCC Deposit No.
  • nucleic acid fragments of the present invention include nucleic acid molecules encoding epitope-bearing portions ofthe endokine alpha protein.
  • nucleic acid fragments ofthe present invention include nucleic acid molecules encoding a polypeptide comprising or, alternatively, consisting of one, two, three or more of any ofthe following amino acid sequences and polynucleotides encoding these polypeptides: amino acid residues from about
  • Figure 1 SEQ ID NO:2; and amino acid residues from about 148 to about 158 in Figure 1 (SEQ ID NO:2).
  • the inventors have determined that the above polypeptide fragments are antigenic regions of the endokine alpha protein. Methods for determining other such epitope-bearing portions of the endokine alpha protein are described in detail below.
  • 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 complement of an endokine alpha polynucleotide fragment described herein, or the cDNA clone contained in ATCC
  • 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 ofthe reference polynucleotide e.g., the deposited cDNA clone
  • a portion 50-500 nt in length, or even to the entire length ofthe 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 Sambrook, J. et al. , eds., Molecular Cloning, A Laboratory Manual. 2nd. edition, Cold Spring Harbor Laboratory Press, Cold
  • an endokine alpha cDNA clone has been deposited and its nucleotide sequence is provided in Figure 1 (SEQ ID NO: l)
  • generating polynucleotides which hybridize to a portion of the endokine alpha cDNA molecule would be routine to the skilled artisan.
  • restriction endonuclease cleavage or shearing by sonication ofthe endokine alpha cDNA clone could easily be used to generate DNA portions of various sizes which are polynucleotides that hybridize to a portion ofthe endokine alpha cDNA molecule.
  • 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 endokine alpha cDNA shown in Figure 1 (SEQ ID NO:l)
  • a complementary stretch of T (or U) resides would not be included in a polynucleotide ofthe 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).
  • nucleic acid molecules ofthe present invention that encode an endokine alpha protein may include, but are not limited to, those encoding the amino acid sequence of the polypeptide. by itself; the coding sequence for the polypeptide and additional sequences, such as a pre-, or pro- or prepro- protein sequence; the coding sequence of the 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 ' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing - including splicing and polyadenylation signals, e.g..
  • the sequence encoding the polypeptide can be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification ofthe fused polypeptide.
  • 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 publicly and/or commercially available. As described in Gentz et al. Proc. Natl. Acad. Sci.
  • hexa-histidine provides for convenient purification ofthe fusion protein.
  • the "HA” tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin (HA) protein, which has been described by Wilson etal, Cell 37:161 (1984).
  • Other such fusion proteins include the endokine alpha protein fused to Fc at the N- or C-terminus.
  • the present invention further relates to variants of the nucleic acid molecules ofthe present invention, which encode portions, analogs or derivatives of the endokine alpha protein.
  • Variants can 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.
  • Non-naturally occurring variants can be produced, e.g. , using art-known mutagenesis techniques.
  • Non-naturally occurring variants may be produced using art-known mutagenesis techniques, which include, but are not limited to oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see e.g., Carter et al., Nucl. Acids Res. 73:4331 (1986); and Zoller et al., Nucl. Acids Res. 10:6481 (1982)). cassette mutagenesis (see e.g., Wells et al, Gene 34:315 (1985)), restriction selection mutagenesis (see e.g.. Wells et al, Philos. Trans. R. Soc. London SerA 377:415 (1986)).
  • variants include those produced by nucleotide substitutions, deletions or additions.
  • the substitutions, deletions or additions may involve one or more nucleotides.
  • the variants can be altered in coding or non-coding regions or both. Alterations in the coding regions can 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 endokine alpha protein or portions thereof. Also especially preferred in this regard are conservative substitutions.
  • nucleic acid molecules encoding the endokine alpha protein having the amino acid sequence shown in Figure 1 (SEQ ID NO:2) or the endokine alpha amino acid sequence encoded by the deposited cDNA clone.
  • nucleic acid molecules comprising or, alternatively, consisting of, a polynucleotide having a nucleotide sequence at least 80%, 85%, 90%, 92% or 95% identical, and more preferably at least 96%, 97%, 98%, or 99% identical to (a) a nucleotide sequence encoding the polypeptide having the amino acid sequence in SEQ ID NO:2; (b) a nucleotide sequence encoding the polypeptide having the amino acid sequence in SEQ ID NO:2, but lacking the N-terminal methionine; (c) a nucleotide sequence encoding the polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97640; or (d) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), or (c).
  • polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence encoding an endokine alpha 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 endokine alpha 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% ofthe total nucleotides in the reference sequence may be inserted into the reference sequence.
  • These mutations ofthe reference sequence may occur at the 5' or 3' terminal positions ofthe 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.
  • the reference (query) sequence may be the entire endokine alpha encoding nucleotide sequence shown in Figure 1 (SEQ ID NO: l) or any endokine alpha polynucleotide fragment as described herein.
  • any particular nucleic acid molecule is at least 80%. 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to, for instance, the nucleotide sequence shown in Figure 1 or to the nucleotide 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 5371 1).
  • BESTFIT uses the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 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 ofthe 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 identity between a reference (query) sequence (a sequence of the present invention) and a subject sequence is determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
  • the percent identity is corrected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. A determination of whether a nucleotide is matched/aligned is determined by results ofthe FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of this embodiment.
  • the deletions occur at the 5' end of the subject sequence and therefore, the FASTDB alignment does not show a match/alignment ofthe first 10 bases at 5' end.
  • the 10 unpaired bases represent 10% ofthe sequence (number of bases at the 5' and 3' ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%.
  • a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5' or 3' ofthe subject sequence which are not matched/aligned with the query . In this case the percent identity calculated by FASTDB is not manually corrected.
  • nucleic acid molecules which are at least 80%. 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence described above irrespective of whether they encode a polypeptide having endokine alpha protein activity. This is because, even where a particular nucleic acid molecule does not encode a polypeptide having endokine alpha 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 ofthe present invention that do not encode a polypeptide having endokine alpha activity include, ter alia, (1) isolating the endokine alpha gene or allelic variants thereof from a cDNA library; (2) in situ hybridization (FISH) to metaphase chromosomal spreads to provide precise chromosomal location ofthe endokine alpha gene as described in Verma et al, Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988); and (3) Northern Blot analysis for detecting endokine alpha mRNA expression in specific tissues.
  • FISH in situ hybridization
  • nucleic acid molecules having sequences at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence described above which do, in fact, encode a polypeptide having endokine alpha protein activity.
  • a polypeptide having endokine alpha activity is intended polypeptides exhibiting similar, but not necessarily identical, activity as compared to the endokine alpha protein as measured in a particular biological assay. Endokine alpha activity can be assayed according to known methods. For example, a cytotoxicity assay or cell proliferation assay can be used where endokine alpha polypeptides are added to cells in culture and the effect of the endokine on the cells is determined by measuring the decrease or increase in cell numbers.
  • nucleic acid molecules having a sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence described above will encode a polypeptide "having endokine alpha protein activity.”
  • degenerate variants all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay.
  • a reasonable number will also encode a polypeptide having endokine alpha 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).
  • a polypeptide having endokine alpha functional activity is intended polypeptides exhibiting activity similar, but not necessarily identical, to an activity of the endokine alpha receptors of the present invention (either the full-length polypeptide, or the splice variants), as measured, for example, in a particular immunoassay or biological assay.
  • endokine alpha activity can be measured by determining the ability of an endokine alpha polypeptide to bind an endokine alpha ligand (e.g. , TR11 (GITR, AITR)).
  • Endokine alpha activity may also be measured by determining the ability of a polypeptide, such as cognate ligand which is free or expressed on a cell surface, to stimulate proliferation, differentiation or activation, or to stimulate TNF- ⁇ production, and/or to inhibit IL- 12 production in cells expressing the polypeptide, for example, B cells, T cells and monocytes.
  • a polypeptide such as cognate ligand which is free or expressed on a cell surface
  • the present invention is further directed to fragments of the isolated nucleic acid molecules (i.e. polynucleotides) described herein.
  • nucleic acid molecule having, for example, the nucleotide sequence of the deposited cDNA (clone 97640), a nucleotide sequence encoding the polypeptide sequence encoded by the deposited cDNA, a nucleotide sequence encoding the polypeptide sequence depicted in Figure 1 (SEQ ID NO:2).
  • nucleotide sequence shown in Figure 1 is intended fragments at least 15 nucleotides, and more preferably at least about 20 nucleotides, still more preferably at least 30 nucleotides, and even more preferably, at least about 40, 50, 100, 150, 200, 250, 300, 325, 350, 375, 400, 450, 500, 550, or 600 nucleotides in length.
  • "about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2 or 1 ) nucleotides.
  • fragments such as those of 501-1500 nucleotides in length are also useful according to the present invention as are fragments corresponding to most, if not all, ofthe nucleotide sequences ofthe deposited cDNA (clone 97640) or as shown in Figure 1 (SEQ ID NO: 1).
  • a fragment at least 20 nucleotides in length for example, is intended fragments which include 20 or more contiguous bases from, for example, the nucleotide sequence ofthe deposited cDNA, or the nucleotide sequence as shown in Figure 1 (SEQ ID NO: 1).
  • endokine alpha polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to 50, 51 to 100, 101 to 150, 151 to 200, 201 to 250, 251 to 300, 301 to 350, 351 to 400, 401 to 450, 451 to 500, 501 to 550, 551 to 600, 600 to 650, 651 to 700, 701 to 750, 751 to 800, 801 to
  • 850, 851 to 900, 901 to 950, 951 to 1000 1001 to 1050, 1051 to 1100, 1101 to 1150, 1 151 to 1200, 1201 to 1250, 1251 to 1300, 1301 to 1350, 1351 to 1400, 1401 to 1450, 1451 to 1500, 1501 to 1550, 1551 to 1600, 1601 to 1650, 1651 to 1700, 1701 to 1750, 1751 to 1800. and/or 1801 to 1840 of SEQ ID NO: 1, or the complementary strand thereto, or the cDNA contained in the deposited clone.
  • “about” includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
  • the polynucleotide fragments of the invention comprise, or alternatively, consist of, a sequence from nucleotide 961 to 1000, 1730 to 1770, 1770 to 1800. and/or 1800 to 1840, of SEQ ID NO:l. or the complementary strand thereto.
  • Polynucleotides that hybridize to these polynucleotide fragments are also encompassed by the invention.
  • the polynucleotide fragments of the invention encode a polypeptide which demonstrates an endokine alpha functional activity.
  • a polypeptide demonstrating "functional activity” is meant, a polypeptide capable of display ing one or more known functional activities associated with a full-length endokine alpha polypeptide.
  • Such functional activities include, but are not limited to, biological activity (e.g., stimulation of B cell proliferation, differentiation or activation; stimulation of T cell proliferation, differentiation or activation; stimulation of TNF- ⁇ production in monocytes; and/or inhibition of IL-12 production in monocytes), antigenicity (ability to bind (or compete with an endokine alpha polypeptide for binding) to an anti-endokine alpha antibody), immunogenicity (ability to generate antibody which binds to a endokine alpha polypeptide), ability to multimerize with native endokine alpha and ability to bind to a receptor or ligand for a endokine alpha polypeptide (e.g., TR11 ; see U.S.
  • biological activity e.g., stimulation of B cell proliferation, differentiation or activation; stimulation of T cell proliferation, differentiation or activation; stimulation of TNF- ⁇ production in monocytes; and/or inhibition of IL-12 production in monocytes
  • antigenicity ability to bind (or compete with an endokine alpha poly
  • endokine alpha polypeptides and fragments, variants, derivatives, and analogs thereof, can be assayed by various methods.
  • various immunoassays known in the art can be used, including, but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays,
  • agglutination assays e.g., gel agglutination assays, hemagglutination assays
  • complement fixation assays
  • antibody binding is detected by detecting a label on the primary antibody .
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
  • the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope ofthe present invention.
  • an endokine alpha ligand is identified (e. g. ,
  • binding can be assayed, e.g. , by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky, E., et al, Microbiol. Rev. 59:94-123 (1995).
  • physiological correlates (signal transduction) of endokine alpha binding to its substrates can be assayed.
  • assays described herein may routinely be applied to measure the ability of endokine alpha polypeptides and fragments, variants, derivatives and analogs thereof to elicit endokine alpha related biological activity (e.g., stimulation of B cell proliferation, differentiation or activation; stimulation of T cell proliferation, differentiation or activation; stimulation of TNF- ⁇ production in monocytes; and/or inhibition of IL-12 production in monocytes in vitro or in vivo).
  • endokine alpha related biological activity e.g., stimulation of B cell proliferation, differentiation or activation; stimulation of T cell proliferation, differentiation or activation; stimulation of TNF- ⁇ production in monocytes; and/or inhibition of IL-12 production in monocytes in vitro or in vivo.
  • Other methods will be known to the skilled artisan and are within the scope of the invention.
  • 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 endokine alpha polypeptides or portions thereof by recombinant techniques.
  • Recombinant constructs may be introduced into host cells using well known techniques such as 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.
  • the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
  • 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, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as cosmids and phagemids. See. e.g., Ausubel, infra; Sambrook, infra.
  • the DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E.
  • 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 include a translation initiating AUG at the beginning and a termination codon appropriately positioned at the end ofthe 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 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 media and conditions for the above-described host cells are known in the art.
  • vectors preferred for use in bacteria include pQ ⁇ 70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNHl ⁇ a, 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 lad and Z ⁇ cZ 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 ofthe 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 ofthe replication origin, and adenovirus enhancers.
  • secretion signals may be incorporated 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.
  • 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.
  • a region(s) also 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 0,464.533 also, Canadian counterpart 2.045,869 discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another human protein or part thereof.
  • the Fc part in the fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP A 0,232,262).
  • 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 hIL-5
  • Fc portions for the purpose of high-throughput screening assays to identify antagonists (for example, hIL-5). See, D. Bennett et al, Journal of Molecular Recognition 5:52-58 (1995) and K. Johanson et al, The Journal of Biological Chemistry 270(16):9459-941 ⁇ (1995).
  • the endokine alpha 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 ofthe 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 also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., endokine alpha coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with endokine alpha polynucleotides ofthe invention, and which activates, alters, and/or amplifies endogenous endokine alpha polynucleotides.
  • endogenous genetic material e.g., endokine alpha coding sequence
  • genetic material e.g., heterologous polynucleotide sequences
  • heterologous control regions e.g., promoter and/or enhancer
  • endogenous endokine alpha polynucleotide sequences via homologous recombination (see, e.g., U.S. Patent No. 5,641,670, issued June 24, 1997; International Publication No. WO 96/2941 1, published September 26, 1996; International Publication No. WO 94/12650, published
  • the invention further provides an isolated endokine alpha polypeptide having the amino acid sequence encoded by the deposited cDNA, or the amino acid sequence in Figure 1 (SEQ ID NO :2), or a peptide or polypeptide comprising a portion ofthe 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 two 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.
  • isolated polypeptide or protein is intended a polypeptide or protein removed from its native environment.
  • recombinantly produced polypeptides and proteins expressed in recombinant host cells are considered isolated for purposes ofthe invention as are native or recombinant polypeptides and proteins which have been substantially purified by any suitable technique such as, for example, the one-step method described in Smith and Johnson, Gene 7:31-40 (1988).
  • the invention further includes variations of the endokine alpha polypeptide which show substantial endokine alpha polypeptide activity or which include regions of endokine alpha protein such as the protein fragments 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.
  • conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and He; interchange ofthe 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.
  • the fragment, derivative or analog of the polypeptide of SEQ ID NO:2. or that encoded by the deposited cDNA. may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence.
  • a conserved or non-conserved amino acid residue preferably a conserved amino
  • the replacement of amino acids can also change the selectivity of binding to cell surface receptors. Ostade et al, Nature 361:266-268 (1993) describes certain mutations resulting in selective binding of TNF- ⁇ to only one ofthe two known types of TNF receptors.
  • the endokine alpha ofthe present invention may include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation.
  • changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table 1 ). TABLE 1. Conservative Amino Acid Substitutions.
  • Amino acids in the endokine alpha protein ofthe present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as receptor binding or in vitro, or in vitro proliferative activity. Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaff ⁇ nity labeling (Smith et al, J. Mol.
  • polypeptides ofthe present invention are preferably provided in an isolated form.
  • isolated polypeptide is intended a polypeptide removed from its native environment.
  • a polypeptide produced and/or contained within a recombinant host cell is considered isolated for purposes ofthe present invention.
  • polypeptides that have been purified, partially or substantially, from a recombinant host cell are polypeptides that have been purified, partially or substantially, from a recombinant host cell.
  • a recombinantly produced version of the endokine alpha polypeptide can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988).
  • polypeptides of the present invention include the polypeptides comprising or, alternatively, consisting of: (a) the complete amino acid sequence as shown in Figure 1 (SEQ ID NO:2); (b) the complete amino acid sequence as shown in Figure 1 (SEQ ID NO:2). but minus the N-terminal methionine residue; (c) the amino acid sequence ofthe endokine alpha polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No.
  • apolypeptide having an amino acid sequence at least, for example, 95% "identical" to a reference amino acid sequence of an endokine alpha 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 sequence of the endokine alpha 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% ofthe total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • These alterations of he reference sequence may occur at the amino or carboxy terminal positions ofthe 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 comprises or, alternatively, consists of, a sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence shown in Figure 1 (SEQ ID NO:2) or to the amino acid sequence encoded by deposited cDNA clone
  • BESTFIT program Wiconsin 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% ofthe total number of amino acid residues in the reference sequence are allowed.
  • the identity between a reference (query) sequence (a sequence of the present invention) and a subject sequence is determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
  • the percent identity is corrected by calculating the number of residues ofthe query sequence that are N- and C-terminal ofthe subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent ofthe total bases ofthe query sequence.
  • a determination of whether a residue is matched/aligned is determined by results ofthe FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of this embodiment. Only residues to the N- and C-termini ofthe subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues ofthe subject sequence. For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity.
  • the deletion occurs at the N-terminus ofthe subject sequence and therefore, the FASTDB alignment does not show a matching/alignment ofthe first 10 residues at the N-terminus.
  • the 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%.
  • a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini ofthe subject sequence which are not matched/aligned with the query.
  • the percent identity calculated by FASTDB is not manually corrected.
  • residue positions outside the N- and C-terminal ends ofthe subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are made for the purposes of this embodiment.
  • the present inventors have discovered that the endokine alpha protein is a 169 residue protein exhibiting three main structural domains.
  • the intracellular domain was identified within residues from about 1 to about 17 in Figure 1 (SEQ ID NO: 2).
  • the transmembrane domain was identified within residues from about 18 to about 43 in Figure 1 (SEQ ID NO:2).
  • the extracellular domain was identified within residues from about 44 to about 169 in Figure 1 (SEQ ID NO:2).
  • the invention further provides preferred endokine alpha protein fragments comprising a polypeptide selected from: the endokine alpha intracellular domain, the transmembrane domain and the endokine alpha extracellular domain.
  • the extracellular domain ofthe endokine alpha protein can be combined with parts ofthe constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides.
  • IgG immunoglobulins
  • These fusion proteins show an increased half-life in vivo. This has been shown, e.g. , for chimeric proteins consisting ofthe first two domains ofthe human CD4-polypeptide and various domains ofthe constant regions ofthe heavy or light chains of mammalian immunoglobulins (EP A 394,827; Traunecker et al, Nature 337:84-86 (1988)).
  • Fusion proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing the ligands than the monomeric extracellular domains alone (Fountoulakis et al, J. Biochem. 270:3958-3964 (1995)).
  • Polypeptide fragments of the present invention include polypeptides comprising or alternatively, consisting of, an amino acid sequence contained in
  • SEQ ID NO:2 encoded by the cDNA contained in the deposited clone, or encoded by nucleic acids which hybridize (e.g., under stringent hybridization conditions) to the nucleotide sequence contained in the deposited clone, or shown in Figure 1 (SEQ ID NO: l) or the complementary strand thereto.
  • Protein fragments may be "free-standing," or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region.
  • Representative examples of polypeptide fragments of the invention include, for example, fragments that comprise or alternatively, consist of from about amino acid residues: 1 to 50, 51 to 100, 101 to 150 and/or 151 to 169 of SEQ ID NO:2.
  • polypeptide fragments can be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150 or 168 amino acids in length.
  • Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • Polynucleotides that hybridize to the complement of these encoding polynucleotides are also encompassed by the invention, as are the polypeptides encoded by these hybridizing polynucleotides.
  • fragments ofthe invention are fragments characterized by structural or functional attributes of endokine alpha.
  • Such fragments include amino acid residues that comprise alpha-helix and alpha-helix-forming regions ("alpha-regions"), beta-sheet and beta-sheet-forming regions ("beta-regions"), turn and turn-forming regions ("turn-regions”), coil and coil-forming regions ("coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, surface forming regions, and high antigenic index regions (i.e., containing four or more contiguous amino acids having an antigenic index of greater than or equal to 1.5, as identified using the default parameters ofthe Jameson- Wolf program) of full-length endokine alpha (SEQ ID NO:2).
  • Certain preferred regions are those set out in Figure 3 and include, but are not limited to, regions ofthe aforementioned types identified by analysis of the amino acid sequence depicted in Figure 1 (SEQ ID NO:2), such preferred regions include; Garnier-Robson predicted alpha-regions, beta-regions, turn-regions, and coil-regions; Chou-Fasman predicted alpha-regions, beta- regions, turn-regions, and coil-regions; Kyte-Doolittle predicted hydrophilic and hydrophobic regions; Eisenberg alpha and beta amphipathic regions: Emini surface-forming regions: and Jameson-Wolf high antigenic index regions, as predicted using the default parameters of these computer programs. Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • the data presented in columns VIII, IX, XIII, and XIV of Table 2 can be used to determine regions of endokine alpha which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or IV by choosing values which represent regions ofthe polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
  • the above-mentioned preferred regions set out in Figure 3 and in Table 2 include, but are not limited to, regions ofthe aforementioned types identified by analysis ofthe amino acid sequence set out in Figure 3. As set out in Figure 3 and in Table 2, such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and coil-regions. Kyte-Doolittle hydrophilic regions and hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Emini surface-forming regions and Jameson- Wolf regions of high antigenic index.
  • Val 102 A B 090 051 * -034 062 Ala 103 A B 004 -024 * * 043 067 Pro 104 A B 040 000 * * -030 030
  • the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the endokine alpha amino acid sequence shown in Figure 1 (i.e., SEQ ID NO:2), up to the asparagine residue at position number 164 and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues n-l 69 of Figure 1 (SEQ ID NO:2), where n is an integer in the range of 2 to 164, and 165 is the position of the first residue from the N-terminus ofthe complete endokine alpha polypeptide believed to be required for at least immunogenic activity ofthe endokine alpha polypeptide.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of a member selected from the group consisting of: residues P-2 to S-169; L-3 to S-169; S-4 to S-169; H-5 to S-169; S-6 to S-169; R-7 to S-169; T-8 to S-169; Q-9 to S-169; G-10 to S-169; A-l l to S-169; Q-12 to S-169; R-13 to S-169; S-14 to S-169; S-15 to S-169; W-16 to S-169; K-17 to S-169; L-18 to S-169; W-19 to S-169: L-20 to S-169; F-21 to S-169; C-22 to S-169; S-23 to S-169; -44-
  • nucleic acid molecules comprising or, alternatively, consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%o identical to the polynucleotide sequences encoding the endokine alpha polypeptides described above.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence.
  • Polypeptides encoded by these polynucleotide sequences are also encompassed by the invention.
  • the ability ofthe shortened endokine alpha mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that an endokine alpha mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six endokine alpha amino acid residues may often evoke an immune response.
  • the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence ofthe endokine alpha polypeptide shown in Figure 1 (SEQ ID NO:2), up to the serine residue at position number 6, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues 1-m of Figure 1 (i.e.. SEQ ID NO:2), where m is an integer in the range of 6 to 169, and 6 is the position ofthe first residue from the C-terminus of the complete endokine alpha polypeptide -46-
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of a member selected from the group consisting of: residues M-1 to 1-168; M-1 to F-167; M-1 to Q-166; M-1 to P-165; M-1 to N-164; M-1 to A-163; M-1 to L-162; M-1 to L-161 ; M-1 to 1-160; M-1 to 1-159; M-1 to G-158; M-1 to W-157; M-1 to Y-156; M-1 to T-155; M-1 to N-154; M-1 to N-153; M-1 to K-152; M-1 to L-151 ; M-1 to V-150; M-1 to Q-149; M-1 to H-148; M-1 to E-147; M-1 to S-146; M-1 to N-145; M-1 to F-144; M-1 to 1-143; M-1 to L-142; M-1 to D-141 ;
  • the present invention is also directed to nucleic acid molecules comprising or, alternatively, consisting of, a polynucleotide sequence at least 80%>, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequences encoding the endokine alpha polypeptides described above.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotide sequences are also encompassed by the invention.
  • the invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of an endokine alpha polypeptide, which may be described generally as having residues n-m of Figure
  • n and m are integers as described above.
  • the endokine alpha polypeptides ofthe invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers). Accordingly, the present invention relates to monomers and multimers of the endokine alpha polypeptides of the invention, their preparation, and compositions (preferably, pharmaceutical compositions) containing them.
  • the polypeptides of the invention are monomers, dimers, trimers or tetramers.
  • the multimers ofthe invention are at least dimers, at least trimers, or at least tetramers. Multimers encompassed by the invention may be homomers or heteromers.
  • homomer refers to a multimer containing only endokine alpha polypeptides ofthe invention (including endokine alpha fragments, variants, and fusion proteins, as described herein). These homomers may contain endokine alpha polypeptides having identical or different amino acid sequences.
  • a homomer ofthe invention is a multimer containing only -48-
  • a homomer of the invention is a multimer containing endokine alpha polypeptides having different amino acid sequences.
  • the multimer of the invention is a homodimer (e.g., containing endokine alpha polypeptides having identical or different amino acid sequences) or a homotrimer (e.g. , containing endokine alpha polypeptides having identical or different amino acid sequences).
  • the homomeric multimer ofthe invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.
  • heteromer refers to a multimer containing heterologous polypeptides (i.e., polypeptides of a different protein) in addition to the endokine alpha fragments and endokine alpha polypeptides ofthe invention.
  • the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer.
  • the heteromeric multimer ofthe invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.
  • Multimers ofthe invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation.
  • multimers ofthe invention such as, for example, homodimers or homotrimers
  • heteromultimers of the invention such as, for example, heterotrimers or heterotetramers, are formed when polypeptides ofthe invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution.
  • multimers ofthe invention are formed by covalent associations with and/or between the endokine alpha polypeptides ofthe invention.
  • Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g. , that recited in SEQ ID NO:2, or contained in the polypeptide encoded by the clone 97640).
  • association associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occurring) polypeptide.
  • the covalent associations are the consequence of chemical or recombinant manipulation.
  • such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in an endokine alpha fusion protein.
  • covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., U.S. Patent No. 5,478,925).
  • the covalent associations are between the heterologous sequence contained in a endokine alpha-Fc fusion protein of the invention (as described herein).
  • covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another TNF family ligand/receptor member that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (see, e.g., International Publication No. WO 98/49305, the contents of which are herein incorporated by reference in its entirety).
  • two or more endokine alpha polypeptides ofthe invention are joined through peptide linkers.
  • peptide linkers include those peptide linkers described in U.S. Patent No.5,073,627 (hereby incorporated by reference). Proteins comprising multiple endokine alpha polypeptides separated by peptide linkers may be produced using conventional recombinant DNA technology.
  • Leucine zipper and isoleucine zipper domains are polypeptides that promote multimerization ofthe proteins in which they are found.
  • Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al. , Science 240: 1759, (1988)), and have since been found in a variety of different proteins.
  • leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble multimeric endokine -50-
  • alpha proteins are those described in PCT application WO 94/10308, hereby incorporated by reference.
  • Recombinant fusion proteins comprising a soluble endokine alpha polypeptide fused to a peptide that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric endokine alpha is recovered from the culture supernatant using techniques known in the art.
  • trimeric endokine alpha may offer the advantage of enhanced biological activity.
  • Preferred leucine zipper moieties are those that preferentially form trimers.
  • One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344: 191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby incorporated by reference.
  • SPD lung surfactant protein D
  • Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric endokine alpha.
  • proteins ofthe invention are associated by interactions between Flag® polypeptide sequence contained in Flag®-endokine alpha fusion proteins ofthe invention.
  • associations proteins ofthe invention are associated by interactions between a heterologous polypeptide sequence contained in Flag®-endokine alpha fusion proteins ofthe invention and anti-Flag® antibody.
  • the multimers of the invention may be generated using chemical techniques known in the art.
  • polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art
  • multimers ofthe invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., U.S. Patent No. 5.478,925, -51-
  • polypeptides ofthe invention may be routinely modified by the addition of cysteine or biotin to the C terminus or N-terminus ofthe polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., U.S. Patent No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., U.S. Patent No. 5,478,925, which is herein incorporated by reference in its entirety).
  • multimers ofthe invention may be generated using genetic engineering techniques known in the art.
  • polypeptides contained in multimers ofthe invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g. , U.S. Patent No. 5,478,925, which is herein incorporated by reference in its entirety).
  • polynucleotides coding for a homodimer of the invention are generated by Hgating a polynucleotide sequence encoding a polypeptide ofthe invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N- terminus (lacking the leader sequence) (see, e.g., U.S. Patent No. 5,478,925, which is herein incorporated by reference in its entirety).
  • recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., U.S. Patent No. 5,478,925, which is herein incorporated by reference in its entirety).
  • polypeptides ofthe invention can be chemically synthesized using techniques known in the art (e.g. , see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N. Y., and Hunkapiller, M., et al. , 1984, Nature 310:105-11 1 ).
  • endokine alpha polypeptides ofthe invention can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the endokine alpha polynucleotide sequence.
  • Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4- diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3 -amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino
  • the invention encompasses endokine alpha polypeptides which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease,
  • Additional post-translational modifications encompassed by the invention include, for example, e.g. , N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends, attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression.
  • the polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
  • chemically modified derivatives of endokine alpha which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Patent No. 4,179,337).
  • the chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
  • the polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing.
  • Other sizes may be used, depending on the desired therapeutic profile (e. g. , the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects ofthe polyethylene glycol to a therapeutic protein or analog).
  • polyethylene glycol molecules should be attached to the protein with consideration of effects on functional or antigenic domains ofthe protein.
  • attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al, Exp. Hematol. 20: 1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride).
  • polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound.
  • amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid -54-
  • Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules.
  • Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
  • polyethylene glycol as an illustration ofthe present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (or peptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein.
  • the method of obtaining the N-terminally pegylated preparation i.e., separating this moiety from other monopegylated moieties if necessary
  • Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the
  • N-terminal available for derivatization in a particular protein.
  • substantially selective derivatization ofthe protein at the N-terminus with a carbonyl group containing polymer is achieved.
  • polypeptides ofthe present invention have uses which include, but are not limited to, as sources for generating antibodies that bind the polypeptides of the invention, and as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art.
  • proteins ofthe invention can be chemically synthesized using techniques known in the art (see, e.g., Creighton, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y. (1983), and Hunkapiller, M., et al. Nature 310: ⁇ 05A 1 1 (1984)).
  • a peptide corresponding to a fragment ofthe endokine-alpha polypeptides ofthe invention can be synthesized -55-
  • Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, alpha-amino isobutyric acid, 4-aminobutyric acid, Abu,
  • 2-amino butyric acid alpha- Abu, alpha- Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, alpha-alanine, fluoro-amino acids, designer amino acids such as alpha-methyl amino acids, Ca-methyl amino acids,
  • amino acids can be D (dextrorotary) or L (levorotary).
  • Non-naturally occurring variants may be produced using art-known mutagenesis techniques, which include, but are not limited to oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see, e.g., Carter et al. Nucl. Acids Res. 73:4331 (1986); and Zoller et al, Nucl. Acids Res. 10:6481 (1982)), cassette mutagenesis (see, e.g. , Wells et al, Gene 34:315 (1985)), and restriction selection mutagenesis (see, e.g.. Wells et al, Philos. Trans. R. Soc.
  • the invention additionally, encompasses endokine-alpha polypeptides which are differentially modified during or after translation, e.g. , by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH 4 . acetylation, formylation. oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.
  • Additional post-translational modifications encompassed by the invention include, for example. N-linked or O-linked carbohydrate chains, processing of
  • polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
  • chemically modified derivatives of endokine alpha which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Patent No. 4,179,337).
  • the chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
  • the polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing.
  • Other sizes may be used, depending on the desired therapeutic profile (e.g. , the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects ofthe polyethylene glycol to a therapeutic protein or analog).
  • the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000. 6500, 7000, 7500, 8000. 8500, 9000,
  • the polyethylene glycol may have a branched structure.
  • Branched polyethylene glycols are described, for example, in U.S. Patent No. -57-
  • polyethylene glycol molecules should be attached to the protein with consideration of effects on the functional or antigenic domains ofthe protein.
  • polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group.
  • Reactive groups are those to which an activated polyethylene glycol molecule may be bound.
  • the amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues, glutamic acid residues and the C-terminal amino acid residue.
  • Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
  • polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues.
  • polyethylene glycol can be linked to a protein via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues.
  • reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) ofthe protein or to more than one type of amino acid residue (e.g. , lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) ofthe protein.
  • specific amino acid residues e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine
  • amino acid residues e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof
  • polyethylene glycol As an illustration ofthe present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.). the proportion of polyethylene glycol molecules to protein (or -58-
  • the method of obtaining the N-terminally pegylated preparation may be by purification ofthe N-terminally pegylated material from a population of pegylated protein molecules.
  • Selective proteins chemically modified at the N-terminus may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization ofthe protein at the N-terminus with a carbonyl group containing polymer is achieved.
  • pegylation of the proteins of the invention may be accomplished by any number of means.
  • polyethylene glycol may be attached to the protein either directly or by an intervening linker.
  • Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al, Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al, Intern. J. of Hematol 68 ⁇ -X8 (1998); U.S. Patent No. 4,002,531; U.S. Patent No. 5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each of which are incorporated herein by reference.
  • One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride (ClSO 2 CH 2 CF 3 ).
  • MPEG monmethoxy polyethylene glycol
  • ClSO 2 CH 2 CF 3 tresylchloride
  • polyethylene glycol is directly attached to amine groups ofthe protein.
  • the invention includes protein-polyethylene glycol conjugates produced by reacting proteins ofthe invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.
  • Polyethylene glycol can also be attached to proteins using a number of different intervening linkers.
  • U.S. Patent No. 5,612,460 the entire disclosure of which is incorporated herein by reference, discloses urethane linkers for connecting polyethylene glycol to proteins.
  • conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG-succinimidylsuccinate, MPEG activated with f l '-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives.
  • compounds such as MPEG-succinimidylsuccinate, MPEG activated with f l '-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives.
  • a number additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in WO 98/32466, the entire disclosure of which is incorporated herein by reference. Pegylated protein products produced using the reaction chemistries set out herein are included within the scope
  • the number of polyethylene glycol moieties attached to each protein ofthe invention may also vary.
  • the pegylated proteins ofthe invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules.
  • the average degree of substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or
  • polypeptides ofthe present invention can be used to raise polyclonal and monoclonal antibodies, which are useful in diagnostic assays for detecting endokine alpha protein expression as described below or as agonists and antagonists capable of inhibiting endokine alpha protein function.
  • polypeptides can be used in the yeast two-hybrid system to "capture" endokine alpha 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.
  • epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide ofthe 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, H.M. et al, Proc. Natl. Acad. Sci. USA 57:3998-4002 (1984). As to the selection of peptides or polypeptides bearing an antigenic epitope
  • Antigenic epitope-bearing peptides and polypeptides ofthe invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention.
  • 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 posttranslational 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.
  • anti-peptide antibodies of the invention also are useful for purification of the mimicked protein, for instance, by adsorption 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 ofthe invention also are considered epitope-bearing peptides or polypeptides ofthe 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 that can be used to generate endokine-specific polyclonal and monoclonal antibodies include a polypeptide comprising or, alternatively, consisting of one, two, three or more of any ofthe following amino acid sequences and polynucleotides encoding these -62-
  • polypeptides amino acid residues from about 44 to about 158 in Figure 1 (SEQ ID NO:2); amino acid residues from about 44 to about 54 in Figure 1 (SEQ ID NO:2); amino acid residues from about 57 to about 68 in Figure 1 (SEQ ID NO:2); amino acid residues from about 69 to about 78 in Figure 1 (SEQ ID NO:2); amino acid residues from about 94 to about 105 in Figure 1 (SEQ ID NO:2)
  • 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 ofthe 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 ofthe HA1 polypeptide which were prepared and characterized (by ELISA-type binding studies) in less than four weeks. See, Houghten, R.A., Proc. Natl. Acad. Sci. USA 52:5131-5135 (1985). This "Simultaneous Multiple Peptide Synthesis (SMPS)" process is further described in U.S. Patent No. 4,631,21 1 to Houghten et al. (1986).
  • SMPS Simultaneous Multiple Peptide Synthesis
  • Epitope-bearing peptides and polypeptides of the invention are used to induce antibodies according to methods well known in the art. See, for instance,
  • mice 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 hemocyanin (KLH) or tetanus toxoid.
  • KLH keyhole limpet hemocyanin
  • 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.
  • a linker such as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)
  • MBS m-maleimidobenzoyl-N-hydroxysuccinimide ester
  • Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 ⁇ g 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 adsorption to the peptide on a solid support and elution ofthe selected antibodies according to methods well known in the art.
  • Immunogenic epitope-bearing peptides ofthe 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. (1984), 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.
  • 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 ofthe protein Then. -64-
  • Geysen (1987) further describes this method of identifying a peptide bearing an immunogenic epitope of a desired 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 ofthe 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. 5,194,392 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 ofthe epitope (i.e., a "mimotope") which is complementary to a particular paratope (antigen binding site)
  • the present invention further relates to antibodies and T-cell antigen receptors (TCR) which specifically bind the polypeptides ofthe present invention.
  • the antibodies ofthe present invention include IgG (including IgG 1 , IgG2, IgG3 , and IgG4), IgA (including IgAl and IgA2), IgD, IgE, or IgM, and IgY.
  • antibody is meant to include whole antibodies, including single-chain whole antibodies, and antigen-binding fragments thereof.
  • the antibodies are human antigen binding antibody fragments of the present invention which include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv). single-chain antibodies, disulfide-linked Fvs (sdFv) and -65-
  • the antibodies may be from any animal origin including birds and mammals.
  • the antibodies are human, murine, rabbit, goat, guinea pig, camel, horse, or chicken.
  • Antigen-binding antibody fragments may comprise the variable region(s) alone or in combination with the entire or partial ofthe following: hinge region, CHI, CH2, and CH3 domains. Also included in the invention are any combinations of variable region(s) and hinge region, CHI, CH2, and CH3 domains.
  • the present invention further includes monoclonal, polyclonal, chimeric, humanized, and human monoclonal and polyclonal antibodies which specifically bind the polypeptides of the present invention.
  • the present invention further includes antibodies which are anti-idiotypic to the antibodies of the present invention.
  • the antibodies ofthe present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide ofthe present invention or may be specific for both a polypeptide of the present invention as well as for heterologous compositions, such as a heterologous polypeptide or solid support material. See, e.g., WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, A. et al. (1991) J. Immunol 147:60-69: U.S. Patents Nos. 5,573,920, 4,474,893, 5,601,819, 4,714,681 , 4,925,648; Kostelny, S.A. et al. (1992) J. Immunol.
  • Antibodies ofthe present invention may be described or specified in terms ofthe epitope(s) or portion(s) of a polypeptide ofthe present invention which are recognized or specifically bound by the antibody.
  • the epitope(s) or polypeptide portion(s) may be specified as described herein, e.g. , by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or listed in the Tables and Figures.
  • Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides ofthe present invention, and allows for the exclusion ofthe same. -66-
  • Antibodies ofthe present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog ofthe polypeptides ofthe present invention are included. Antibodies that do not bind polypeptides with less than 95%, less than 90%>, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than
  • polypeptide ofthe present invention 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide ofthe present invention are also included in the present invention. Further included in the present invention are antibodies which only bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein). Antibodies ofthe present invention may also be described or specified in terms of their binding affinity.
  • Preferred binding affinities include those with a dissociation constant or Kd less than 5X10 " 6 M, 10 "6 M, 5X10- 7 M, 10 "7 M, 5X10 “8 M, 10 “8 M, 5X10 "9 M, 10 '9 M, 5X10- I0 M, 10 ' ,0 M, 5X10-"M, 10 ' "M, 5X10 ",2 M, 10 “,2 M, 5X10 " ' 3 M, 10 "13 M, 5X10-
  • Antibodies of the present invention have uses that include, but are not limited to, methods known in the art to purify, detect, and target the polypeptides of the present invention including both in vitro and in vivo diagnostic and therapeutic methods.
  • the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides ofthe present invention in biological samples. See, e.g. , Harlow et al. Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference in the entirety).
  • the antibodies of the present invention may be used either alone or in combination with other compositions.
  • the antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions.
  • antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, -67-
  • the antibodies ofthe present invention may be prepared by any suitable method known in the art.
  • a polypeptide ofthe present invention or an antigenic fragment thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies.
  • the term "monoclonal antibody” is not limited to antibodies produced through hybridoma technology.
  • the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not to the method by which it is produced.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant and phage display technology.
  • Hybridoma techniques include those known in the art and taught in Harlow et al. Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al, in: Monoclonal Antibodies and T-cell
  • Fab and F(ab')2 fragments may be produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
  • antibodies ofthe present invention can be produced through the application of recombinant DNA technology and phage display technology or through synthetic chemistry using methods known in the art.
  • the antibodies ofthe present invention can be prepared using various phage display methods known in the art.
  • phage display methods functional antibody domains are displayed on the surface of a phage particle which carries polynucleotide sequences encoding them.
  • Phage with a desired binding property are selected from a repertoire or combinatorial antibody library (e.g. human or murine) by selecting directly with antigen, typically antigen bound or captured to a solid surface or bead.
  • Phage used in these methods are typically filamentous phage including fd and Ml 3 with Fab, Fv or disulfide stabilized Fv antibody domains -68-
  • phage display methods that can be used to make the antibodies ofthe present invention include those disclosed in Brinkman U. et al. (1995) J. Immunol. Methods 182:41-50; Ames, R.S. et /. (1995) J. Immunol. Methods 184:177-186; Kettleborough, CA. et al. (1994) Eur. J. Immunol. 24:952-958; Persic, L. et al.
  • the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host including mammalian cells, insect cells, plant cells, yeast, and bacteria.
  • techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in WO 92/22324; Mullinax, R.L. et al. , BioTechniques 12(6):864-869 (1992); and Sawai, H. et al, AJRI 34:26-34 (1995); and Better, M. et al, Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties).
  • Antibodies can be humanized using a variety of techniques -69-
  • antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide ofthe present invention may be specific for antigens other than polypeptides of the present invention.
  • antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors.
  • Antibodies fused or conjugated to the polypeptides ofthe present invention may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g. , Harbor et al.
  • the present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions.
  • the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof
  • the antibody portion fused to a polypeptide ofthe present invention may comprise the hinge region, CHI domain. CH2 domain, and CH3 domain or any combination -70-
  • polypeptides ofthe present invention may be fused or conjugated to the above antibody portions to increase the in vivo half life ofthe polypeptides or for use in immunoassays using methods known in the art.
  • the polypeptides may also be fused or conjugated to the above antibody portions to form multimers.
  • Fc portions fused to the polypeptides ofthe present invention can form dimers through disulfide bonding between the Fc portions.
  • Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See e.g., U.S. Patents Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,112,946;
  • the invention further relates to antibodies which act as agonists or antagonists ofthe polypeptides ofthe present invention.
  • the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully.
  • receptor-specific antibodies include both receptor-specific antibodies and ligand-specific antibodies. Included are receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. Also include are receptor-specific antibodies which both prevent ligand binding and receptor activation. Likewise, included are neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor. Further included are antibodies which activate the receptor. These antibodies may act as agonists for either all or less than all of the biological activities affected by ligand-mediated receptor activation. The antibodies may be specified as agonists or antagonists for biological activities comprising specific activities disclosed herein. The above antibody agonists can be made using -71-
  • the present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO:2, or an epitope ofthe polypeptide sequence encoded by a polynucleotide sequence contained in deposited clone [Deposit information] or encoded by a polynucleotide that hybridizes to the complement ofthe sequence of SEQ ID NO: 1 or contained in the clone deposited as ATCC Deposit Number
  • the present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as. for example, the sequence disclosed in SEQ ID NO:l), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under -72-
  • epitopes refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human.
  • the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide.
  • An "immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al. , Proc.
  • antigenic epitope is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding, but does not necessarily exclude cross-reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic.
  • Fragments that function as epitopes may be produced by any conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 52:5131-5135 (1985), further described in U.S. Patent No. 4,631,211).
  • antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10. at least 15, at least 20, at least 25, and, most preferably, between about 15 to about 30 amino acids.
  • Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20. 25. 30, 35, 40, 45, 50, 55, 60, 65, 70,
  • Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope.
  • Antigenic epitopes can be used as the target molecules in immunoassays. (See, e.g., Wilson et al, Cell 37:161-118 (1984); Sutcliffe et al. Science 219:660-666 (1983)). -73-
  • immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. (See, e.g., Sutcliffe et al. , supra; Wilson et al. , supra; Chow et al. , Proc. Natl. Acad. Sci. USA 52:910-914; and Bittle et al. , J. Gen. Virol. 66:2341-2354 (1985).
  • a preferred immunogenic epitope includes the secreted protein.
  • the polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as, for example, rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier.
  • a carrier protein such as an albumin
  • immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to. at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).
  • Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al. , supra; Wilson et al. , supra, and Bittle et al. , J. Gen. Virol. 66:2341-2354 (1985).
  • animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid.
  • KLH keyhole limpet hemacyanin
  • peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde.
  • Animals such as, for example, rabbits, rats, and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 micrograms of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response.
  • booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody that can be detected by, for example, ELISA assay using free peptide adsorbed to a -74-
  • the titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution ofthe selected antibodies according to methods well known in the art.
  • the polypeptides of the present invention comprising an immunogenic or antigenic epitope can be fused to other polypeptide sequences.
  • polypeptides ofthe present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, and IgM), or portions thereof (CH 1 , CH2, CH3 , or any combination thereof and portions thereof) resulting in chimeric polypeptides.
  • immunoglobulins IgA, IgE, IgG, and IgM
  • portions thereof CH 1 , CH2, CH3 , or any combination thereof and portions thereof
  • chimeric polypeptides may facilitate purification and may increase half-life in vivo. This has been shown for chimeric proteins consisting ofthe first two domains ofthe human CD4-polypeptide and various domains ofthe constant regions ofthe heavy or light chains of mammalian immunoglobulins. See, e.g., EP 394,827; Traunecker et al. Nature 331:84-86 (1988).
  • IgG fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion desulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al, J. Biochem. 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin ("HA”) tag or flag tag) to aid in detection and purification ofthe expressed polypeptide.
  • an epitope tag e.g., the hemagglutinin ("HA") tag or flag tag
  • a system described by Janknecht etal allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al, Proc. Natl. Acad. Sci. USA 55:8972- 897 (1991)).
  • the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame ofthe gene is translationally fused to an amino-terminal tag consisting of six histidine residues.
  • the tag serves as a matrix-binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni 2' nitriloacetic -75-
  • acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.
  • DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. See, generally, U.S. Patent Nos. 5,605,793; 5,81 1,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al, Curr. Opinion Biotechnol 5:724-33 (1997); Harayama, Trends Biotechnol
  • alteration of polynucleotides corresponding to SEQ ID NO: 1 and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling.
  • DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence.
  • polynucleotides of the invention may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination.
  • one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide coding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
  • the present invention further relates to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a polypeptide, preferably an -76-
  • Antibodies ofthe invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies ofthe invention), and epitope-binding fragments of any of the above.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen.
  • the immunoglobulin molecules ofthe invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3. IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
  • the antibodies are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain.
  • Antigen-binding antibody fragments, including single-chain antibodies may comprise the variable region(s) alone or in combination with the entirety or a portion ofthe following: hinge region, CHI, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CHI, CH2, and CH3 domains.
  • the antibodies of the invention may be from any animal origin including birds and mammals.
  • the antibodies are human, murine, donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.
  • "human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Patent No. 5.939.598 by Kucherlapati et al -77-
  • the antibodies ofthe present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide ofthe present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g.,
  • the epitope(s) or polypeptide portion(s) may be specified as described herein, e.g. , by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or listed in the Tables and Figures.
  • Antibodies that specifically bind any epitope or polypeptide ofthe present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.
  • Antibodies ofthe present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present invention are included. Antibodies that bind polypeptides with at least 95%, at least 90%>, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide ofthe present invention are also included in the present invention. Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%.
  • binding affinities include those with a dissociation constant or Kd less than 5X10 "2 M, 10 "2 M, 5X10 "3 M, 10 "3 M, 5X10 "4 M,
  • the invention also provides antibodies that competitively inhibit binding of an antibody to an epitope ofthe invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein.
  • the antibody competitively inhibits binding to the epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%o.
  • Antibodies ofthe present invention may act as agonists or antagonists of the polypeptides of the present invention.
  • the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides ofthe invention either partially or fully.
  • the invention features both receptor-specific antibodies and ligand-specific antibodies.
  • the invention also features receptor-specific antibodies which do not prevent ligand binding, but prevent receptor activation.
  • Receptor activation may be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra).
  • phosphorylation e.g., tyrosine or serine/threonine
  • antibodies are provided that inhibit ligand or receptor activity by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%) ofthe activity in absence ofthe antibody.
  • the invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the -79-
  • receptor-ligand complex and, preferably, do not specifically recognize the unbound receptor or the unbound ligand.
  • neutralizing antibodies which bind the ligand and prevent binding ofthe ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor.
  • antibodies which activate the receptor may act as receptor agonists, i.e., potentiate or activate either all or a subset ofthe biological activities ofthe ligand-mediated receptor activation.
  • the antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities ofthe peptides of the invention disclosed herein.
  • the above antibody agonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281 ; U.S. Patent No. 5,81 1,097; Deng et al, Blood 92(6):X98X-X988 (1998); Chen, et al, Cancer Res.
  • Antibodies of the present invention may be used, for example, but not limited to, to purify, detect, and target the polypeptides ofthe present invention, including both in vitro and in vivo diagnostic and therapeutic methods.
  • the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al. Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference herein in its entirety). -80-
  • the antibodies ofthe present invention may be used either alone or in combination with other compositions.
  • the antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions.
  • antibodies ofthe present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; and WO 89/12624; U.S. PatentNo. 5.314,995; and EP 396,387.
  • the antibodies ofthe invention include derivatives that are modified, e.g. , by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response.
  • the antibody derivatives include antibodies that have been modified, e.g.. by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
  • the antibodies ofthe present invention may be generated by any suitable method known in the art.
  • Polyclonal antibodies to an antigen-of-interest can be produced by various procedures well known in the art.
  • a polypeptide ofthe invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen.
  • Various adjuvants may be used to increase the immunological response, depending on the host species, and include, but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols. polyanions. peptides. oil emulsions, keyhole limpet hemocyanins. dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al , Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al, in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)
  • the term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
  • the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
  • mice can be immunized with a polypeptide ofthe invention or a cell expressing such peptide.
  • an immune response e.g.. antibodies specific for the antigen are detected in the mouse serum
  • the mouse spleen is harvested and splenocytes isolated.
  • the splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC.
  • Hybridomas are selected and cloned by limited dilution.
  • hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide ofthe invention.
  • Ascites fluid which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
  • the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a -82-
  • mice immunized with an antigen ofthe invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide ofthe invention.
  • Antibody fragments that recognize specific epitopes may be generated by known techniques.
  • Fab and F(ab')2 fragments ofthe invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
  • F(ab')2 fragments contain the variable region, the light chain constant region and the CHI domain ofthe heavy chain.
  • the antibodies ofthe present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
  • such phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine).
  • Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
  • Phage used in these methods are typically filamentous phage including fd and Ml 3 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein.
  • the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below.
  • techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax etal.
  • a chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art.
  • Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well -84-
  • Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Patent Nos.
  • Human antibodies are particularly desirable for therapeutic treatment of human patients.
  • Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Patent Nos. 4,444,887 and 4,716,1 1 1 ; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety. Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes.
  • the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells.
  • the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes.
  • the mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination.
  • homozygous deletion ofthe JH region prevents endogenous antibody production.
  • the modified embryonic stem cells are expanded and microinjected into blastocysts to produce -85-
  • chimeric mice The chimeric mice are then bred to produce homozygous offspring that express human antibodies.
  • the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology .
  • the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation.
  • IgG, IgA, IgM and IgE antibodies For an overview of this technology for producing human antibodies, see Lonberg and Huszar (Int. Rev.
  • antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide ofthe invention to a ligand can be used to generate anti-idiotypes that "mimic" the -86-
  • polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand.
  • neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand.
  • anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligands/receptors, and thereby block its biological activity.
  • the invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof.
  • the invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide ofthe invention, preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO:2.
  • the polynucleotides may be obtained, and the nucleotide sequence ofthe polynucleotides determined, by any method known in the art.
  • a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g. , as described in Kutmeier et al, BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions ofthe sequence encoding the antibody, annealing and ligation of those oligonucleotides, and then amplification ofthe ligated oligonucleotides by PCR.
  • chemically synthesized oligonucleotides e.g. , as described in Kutmeier et al, BioTechniques 17:242 (1994)
  • a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence ofthe antibody molecule is known, a nucleic acid encoding the immunoglobulin may be obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated therefrom, or nucleic acid, preferably poly A+ RNA, isolated therefrom, or any tissue or cells expressing the antibody, such as hybridoma cells selected to -87-
  • a suitable source e.g., an antibody cDNA library, or a cDNA library generated therefrom, or nucleic acid, preferably poly A+ RNA, isolated therefrom, or any tissue or cells expressing the antibody, such as hybridoma cells selected to -87-
  • an antibody ofthe invention by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody.
  • Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.
  • nucleotide sequence and corresponding amino acid sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (See, for example, the techniques described in Sambrook et ⁇ l.
  • the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability.
  • CDRs complementarity determining regions
  • one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra.
  • the framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al, J. Mol Biol.
  • the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention.
  • one or more amino acid substitutions may be made within the -88-
  • amino acid substitutions improve binding ofthe antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.
  • Single chain antibodies are formed by linking the heavy and light chain fragments ofthe Fv region via an amino acid bridge, resulting in a single chain polypeptide.
  • Techniques for the assembly of functional Fv fragments inE. coli may also be used (Skerra et al. Science 242:1038- 1041 (1988)).
  • the antibodies ofthe invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques. -89-
  • an antibody of the invention or fragment, derivative or analog thereof, e.g., a heavy or light chain of an antibody of the invention, requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof
  • the vector for the production ofthe antibody molecule may be produced by recombinant DNA technology using techniques well known in the art.
  • methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein.
  • the invention provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule ofthe invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter.
  • Such vectors may include the nucleotide sequence encoding the constant region ofthe antibody molecule (see, e.g.
  • variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
  • the expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody ofthe invention.
  • the invention includes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, operably linked to a heterologous promoter.
  • vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below. -90-
  • host-expression vector systems may be utilized to express the antibody molecules of the invention.
  • Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ.
  • These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g. , baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g.
  • plasmid expression vectors e.g., Ti plasmid
  • mammalian cell systems e.g., COS, CHO, BHK, 293, 3T3 cells harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
  • bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule.
  • mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al, Gene 45:101 (1986); Cockett et al, Bio/Technology 5:2 (1990)).
  • a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an -91-
  • vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al. , EMBO J. 2: 1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 75:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like.
  • pG ⁇ X vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the pG ⁇ X vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • AcNPV is used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • the antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) ofthe virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g.. the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non- essential region ofthe viral genome (e.g. , region ⁇ l or ⁇ 3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts, (e.g. , see Logan & Shenk. Proc. Natl Acad. Sci. USA 57:355-359 (1984)).
  • Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent -92-
  • initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert.
  • exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic.
  • the efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al. , Methods in Enzymol. 153 : 5 X -544 (1987)).
  • a host cell strain may be chosen which modulates the expression ofthe inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function ofthe protein.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing ofthe primary transcript, glycosylation, and phosphorylation ofthe gene product may be used.
  • Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS. MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.
  • cell lines which stably express the antibody molecule may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1 -2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the -93-
  • This method may advantageously be used to engineer cell lines which express the antibody molecule.
  • Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al, Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase
  • genes can be employed in tk-, hgprt- or aprt- cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al , 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al, Proc. Natl. Acad. Sci. USA 78: X 527 (1981 )); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci.
  • the host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
  • the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
  • a single vector may be used which encodes both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2X97
  • the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
  • an antibody molecule of the invention may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
  • centrifugation e.g., centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • the present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20 or 50 amino acids of the polypeptide) of the present invention to generate fusion proteins.
  • the fusion does not necessarily need to be direct, but may occur through linker -95-
  • the antibodies may be specific for antigens other than polypeptides (or portion thereof, preferably at least 10, 20 or 50 amino acids of the polypeptide) of the present invention.
  • antibodies may be used to target the polypeptides ofthe present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides ofthe present invention to antibodies specific for particular cell surface receptors.
  • Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art.
  • the present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions.
  • the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof.
  • the antibody portion fused to a polypeptide ofthe present invention may comprise the constant region, hinge region, CHI domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof.
  • the polypeptides may also be fused or conjugated to the above antibody portions to form multimers.
  • Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions.
  • Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides ofthe present invention to antibody portions are known in the art. See, e.g., U.S. Patent Nos. 5,336.603;
  • polypeptides of the present invention may be fused or conjugated to the above antibody portions to increase the in vivo half life ofthe polypeptides or for use in immunoassays using methods known in the art. Further, the polypeptides ofthe present invention may be fused or conjugated to the above antibody portions to facilitate purification.
  • One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains ofthe constant regions ofthe heavy or light chains of mammalian immunoglobulins. (EP 394,827; Traunecker et al. , Nature 55 : 84-86 (1988)).
  • polypeptides ofthe present invention fused or conjugated to an antibody having disulfide-linked dimeric structures may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone.
  • the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties.
  • EP A 232,262 Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired.
  • the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
  • human proteins, such as hIL-5 have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5.
  • the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitates their purification.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, C A, 9131 1), among others, many of which are commercially available.
  • a pQE vector QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, C A, 9131 1
  • hexa-histidine provides for convenient purification of the fusion protein.
  • Other peptide tags useful for purification include, but are not limited to. the "HA" tag. which corresponds to an epitope derived from the -97-
  • influenza hemagglutinin protein (Wilson et al , Cell 37: 767 ( 1984)) and the "flag" tag.
  • the present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent.
  • the antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g. , determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. See, for example, U.S. Patent No.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fiuorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin, and aequorin;
  • suitable radioactive material include 125 I, l31 I, ' "In or 99 Tc.
  • an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D. ethidium bromide, emetine, mitomycin. etoposide, tenoposide, vincristine. vinblastine, colchicin.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g. , methotrexate, -98-
  • alkylating agents e.g., mechlore
  • the conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, ⁇ -interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, a thrombotic agent or an anti- angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin- 1 ("IL-1 "), interleukin-2 ("IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin
  • a protein such as tumor necrosis factor,
  • Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification ofthe target antigen.
  • solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g. , Arnon et al , "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. eds., pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al, "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
  • Monoclonal Antibodies '84 Biological And Clinical Applications, Pinchera et al. eds., pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. eds., pp. 303-16 (Academic Press 1985), and Thorpe et al, "The Preparation And Cytotoxic
  • an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.
  • An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.
  • the antibodies ofthe invention may be assayed for immunospecific binding by any method known in the art.
  • the immunoassays which can be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few.
  • Such assays are routine and well known in the art (see, e.g. , Ausubel et al. , eds, Current
  • Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIP A buffer ( 1 % NP-40 or Triton X- 100, 1 % sodium deoxycholate, 0.1% SDS. 0.15 M NaCl.0.01 M sodium phosphate at pH 7.2. 1% -100-
  • a lysis buffer such as RIP A buffer ( 1 % NP-40 or Triton X- 100, 1 % sodium deoxycholate, 0.1% SDS. 0.15 M NaCl.0.01 M sodium phosphate at pH 7.2. 1% -100-
  • Trasylol supplemented with protein phosphatase and/or protease inhibitors (e.g. , EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g. , X -4 hours) at 4° C, adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C, washing the beads in lysis buffer and resuspending the beads in
  • protein phosphatase and/or protease inhibitors e.g. , EDTA, PMSF, aprotinin, sodium vanadate
  • the ability ofthe antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g. , western blot analysis.
  • One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background ( e -g- - > pre-clearing the cell lysate with sepharose beads).
  • immunoprecipitation protocols see, e.g., Ausubel (et al, eds. Current Protocols in Molecular Biology, Vol. 1 , John Wiley & Sons, Inc., New York (19914) at 10.16.1.
  • Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%- 20%
  • a membrane such as nitrocellulose, PVDF or nylon
  • blocking the membrane in blocking solution e.g. , PBS with 3%o BSA or non-fat milk
  • washing buffer e.g. , PBS-Tween 20
  • primary antibody the antibody of interest
  • secondary antibody which recognizes the primary antibody, e.g.. an anti-human antibody
  • conjugated to an enzymatic substrate e.g., horseradish peroxidase or alkaline phosphatase
  • radioactive molecule e.g.
  • ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g. , horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence ofthe antigen.
  • a detectable compound such as an enzymatic substrate (e.g. , horseradish peroxidase or alkaline phosphatase)
  • a detectable compound such as an enzymatic substrate (e.g. , horseradish peroxidase or alkaline phosphatase)
  • a second antibody conjugated to a detectable compound may be added following the addition ofthe antigen of interest to the coated well.
  • ELISAs see, e.g., Ausubel, et al, eds, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York (1994) at 11.2.1.
  • the binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays.
  • a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g. , 3H or 1251) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen.
  • the affinity ofthe antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis.
  • Competition with a second antibody can also be determined using radioimmunoassays.
  • the antigen is incubated with antibody of interest is conjugated to a labeled compound (e.g. , 3H or 1251) in the presence of increasing amounts of an unlabeled second antibody.
  • a labeled compound e.g. , 3H or 1251
  • Endokine alpha is a new member of the TNF family of cytokines.
  • tissue or other cells or bodily fluids e.g., sera, plasma, urine, synovial fluid or spinal fluid
  • a "standard" endokine alpha gene expression level that is, the endokine alpha expression level in tissue or bodily fluids from an individual not having the disorder.
  • the invention provides a diagnostic method useful during diagnosis of an endokine alpha-related disorder, which involves measuring the expression level ofthe gene encoding the endokine alpha protein in tissue or other cells or body fluid from an individual and comparing the measured gene expression level with a standard endokine alpha gene expression level, whereby an increase or decrease in the gene expression level compared to the standard is indicative of an endokine alpha related disorder.
  • measuring the expression level ofthe gene encoding the endokine alpha protein is intended qualitatively or quantitatively measuring or estimating the level ofthe endokine alpha protein or the level of the mRNA encoding the endokine alpha protein in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g. , by comparing to the endokine alpha protein level or mRNA level in a second biological sample).
  • the endokine alpha protein level or mRNA level in the first biological sample is measured or estimated and compared to a standard endokine alpha protein level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having a disorder involving endokine alpha.
  • a standard endokine alpha protein level or mRNA level is known, it can be used repeatedly as a standard for comparison.
  • biological sample any biological sample obtained from an individual, body fluid, cell line, tissue culture, or other source which contains endokine alpha protein or mRNA.
  • biological samples include body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) which contain secreted mature endokine alpha protein, or tissue sources found to express endokine alpha. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.
  • the present invention is useful for diagnosis of various endokine alpha -related disorders in mammals, preferably humans, as similar to TNF-like disorders known in the art or as presented herein. These include disorders associated with immunomodulation and inflammation, cell proliferation, angiogenesis, tumor metastases, apoptosis, sepsis and endotoxemia.
  • Total cellular RNA can be isolated from a biological sample using any suitable technique such as the single-step-guanidinium-thiocyanate -phenol- chloroform method described in Chomczynski and Sacchi, Anal. Biochem. 762:156-159 (1987). Levels of mRNA encoding an endokine alpha polypeptide are then assayed using any appropriate method. These include Northern blot analysis, SI nuclease mapping, the polymerase chain reaction (PCR), reverse transcription in combination with the polymerase chain reaction (RT-PCR), and reverse transcription in combination with the ligase chain reaction (RT-LCR).
  • PCR polymerase chain reaction
  • RT-PCR reverse transcription in combination with the polymerase chain reaction
  • RT-LCR reverse transcription in combination with the ligase chain reaction
  • RNA is prepared from a biological sample as described above.
  • an appropriate buffer such as glyoxal/dimethyl sulfoxide/sodium phosphate buffer
  • the filter is prehybridized in a solution containing formamide, SSC, Denhardt's solution, denatured salmon sperm, SDS, and sodium phosphate buffer.
  • Endokine alpha protein cDNA labeled according to any appropriate method (such as the 32 P-multiprimed DNA labeling -104-
  • cDNA for use as probe according to the present invention is described in the sections above and will preferably at least 15 bp in length. SI mapping can be performed as described in Fujita et al, Cell
  • probe DNA for use in SI mapping
  • the sense strand of above-described cDNA is used as a template to synthesize labeled antisense DNA.
  • the antisense DNA can then be digested using an appropriate restriction endonuclease to generate further DNA probes of a desired length.
  • Such antisense probes are useful for visualizing protected bands corresponding to the target mRNA (i.e., mRNA encoding the endokine alpha protein).
  • Northern blot analysis can be performed as described above.
  • levels of mRNA encoding the endokine alpha protein are assayed using the RT-PCR method described in Makino et al, Technique 2:295-301 (1990).
  • the radioactivities ofthe "amplicons" in the polyacrylamide gel bands are linearly related to the initial concentration of the target mRNA.
  • this method involves adding total RNA isolated from a biological sample in a reaction mixture containing a RT primer and appropriate buffer. After incubating for primer annealing, the mixture can be supplemented with a RT buffer, dNTPs, DTT, RNase inhibitor and reverse transcriptase.
  • RNA After incubation to achieve reverse transcription ofthe RNA, the RT products are then subject to PCR using labeled primers.
  • a labeled dNTP can be included in the PCR reaction mixture.
  • PCR amplification can be performed in a DNA thermal cycler according to conventional techniques. After a suitable number of rounds to achieve amplification, the PCR reaction mixture is electrophoresed on a polyacrylamide gel. After drying the gel, the radioactivity ofthe appropriate bands (corresponding to the mRNA encoding the endokine alpha protein) is quantified using an imaging analyzer.
  • RT and PCR reaction ingredients and conditions, reagent and gel concentrations, and labeling methods are well known in the art. Variations on the RT-PCR method will be apparent to the skilled artisan.
  • any set of oligonucleotide primers which will amplify reverse transcribed target mRNA can be used and can be designed as described in the sections above.
  • Assaying endokine alpha protein levels in a biological sample can occur using any art-known method.
  • Preferred for assaying endokine alpha protein levels in a biological sample are antibody-based techniques.
  • endokine alpha protein expression in tissues can be studied with classical immunohistological methods. In these, the specific recognition is provided by the primary antibody (polyclonal or monoclonal), but the secondary detection system can utilize fluorescent, enzyme, or other conjugated secondary antibodies.
  • an immunohistological staining of tissue section for pathological examination is obtained. Tissues can also be extracted, e.g.
  • endokine alpha protein for Western-blot or dot/slot assay (Jalkanen, M., etal, J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., etal, J. Cell . Biol. 105:3087-3096 (1987)).
  • quantitation of endokine alpha protein can be accomplished using isolated endokine alpha protein as a standard. This technique can also be applied to body fluids.
  • endokine alpha protein With these samples, a molar concentration of endokine alpha protein will aid to set standard values of endokine alpha protein content for different body fluids, like serum, plasma, urine, synovial fluid, spinal fluid, etc.
  • body fluids like serum, plasma, urine, synovial fluid, spinal fluid, etc.
  • the normal appearance of endokine alpha protein amounts can then be set using values from healthy individuals, which can be compared to those obtained from a test subject.
  • antibody-based methods useful for detecting endokine alpha protein levels include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • endokine alpha protein-specific monoclonal antibodies can be used both as an immunoadsorbent and as an enzyme-labeled probe to detect and quantify the endokine alpha protein.
  • the amount of endokine alpha protein present in the sample can be calculated by reference to the amount present in a standard preparation using a linear regression computer algorithm.
  • Such an ELISA for detecting a tumor antigen is described in Iacobelli et al. Breast Cancer Research and Treatment 77:19-30 (1988). In -106-
  • two distinct specific monoclonal antibodies can be used to detect endokine alpha protein in a body fluid.
  • one ofthe antibodies is used as the immunoadsorbent and the other as the enzyme-labeled probe.
  • the above techniques may be conducted essentially as a "one-step” or “two-step” assay.
  • the "one-step” assay involves contacting endokine alpha protein with immobilized antibody and, without washing, contacting the mixture with the labeled antibody.
  • the "two-step” assay involves washing before contacting the mixture with the labeled antibody.
  • Other conventional methods may also be employed as suitable. It is usually desirable to immobilize one component of the assay system on a support, thereby allowing other components of the system to be brought into contact with the component and readily removed from the sample.
  • Suitable enzyme labels include, for example, those from the oxidase group, which catalyze the production of hydrogen peroxide by reacting with substrate.
  • Glucose oxidase is particularly preferred as it has good stability and its substrate
  • oxidase label is readily available. Activity of an oxidase label may be assayed by measuring the concentration of hydrogen peroxide formed by the enzyme-labeled antibody/substrate reaction.
  • suitable labels include radioisotopes, such as iodine ( 125 I, 12I I). carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium (' 12 In), and technetium ( 99m Tc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
  • endokine alpha protein can also be detected in vivo by imaging.
  • Antibody labels or markers for in vivo imaging of endokine alpha protein include those detectable by X-radiography, NMR or ESR.
  • suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject.
  • suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.
  • a endokine alpha protein-specific antibody or antibody portion which has been labeled with an appropriate detectable imaging moiety such as a radioisotope (for example, 131 I, ' ' 'In, 99m Tc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the mammal to be examined for a disorder.
  • a radioisotope for example, 131 I, ' ' 'In, 99m Tc
  • a radio-opaque substance for example, a radio-opaque substance, or a material detectable by nuclear magnetic resonance
  • the size of the subject and the imaging system used will determine the quantity of imaging moieties needed to produce diagnostic images.
  • the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99m Tc.
  • the labeled antibody or antibody portion will then preferentially accumulate at the location of cells which contain endokine alpha protein.
  • In vivo tumor imaging is described in S. W. Burchiel et al, "Immunopharmacokinetics of Radiolabeled Antibodies and Their Portions" (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, Burchiel, S.W. and Rhodes, B.A. eds., Masson Publishing Inc. (1982)).
  • Endokine alpha-protein specific antibodies for use in the present invention can be raised against the intact endokine alpha 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 endokine alpha 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. 24:3 X6-325 (1983)). Thus, these portions are preferred.
  • the antibodies ofthe present invention may be prepared by any of a variety of methods.
  • cells expressing the endokine alpha protein or an antigenic portion thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies.
  • the antibodies ofthe present invention are monoclonal antibodies (or endokine alpha protein binding portions thereof).
  • Such monoclonal antibodies can be prepared using hybridoma technology (see, e.g., Colligan, Current Protocols in Immunology, Wiley Interscience, New York (1990-1996); Harlow & Lane, Antibodies: A Laboratory Manual, Chs. 6-9, Cold Spring Harbor Press, Cold Spring Harbor, N. Y. (1988); Ausubel, infra, at Chapter
  • Such procedures involve immunizing an animal (preferably a mouse) with an endokine alpha polypeptide antigen or with an endokine alpha polypeptide-expressing cell.
  • Suitable cells can be recognized by their capacity to bind anti-endokine alpha protein antibody.
  • Such cells may be cultured in any suitable tissue culture medium (e.g., Earle's modified Eagle's medium supplemented with 10%) fetal bovine serum (inactivated at about 56°C), supplemented with about 10 ⁇ g/1 of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 ⁇ g/ml of streptomycin).
  • tissue culture medium e.g., Earle's modified Eagle's medium supplemented with 10%
  • fetal bovine serum inactivated at about 56°C
  • 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 (e.g., parent myeloma cell line (SP 2 O), available from the American Type Culture Collection (ATCC) (Manassas, VA, USA)).
  • SP 2 O parent myeloma cell line
  • ATCC American Type Culture Collection
  • VA Manassas, VA, USA
  • the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. , Gaslroenterology 50:225-232 (1981); Harlow & Lane, infra, Chapter 7.
  • the hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the endokine alpha antigen.
  • additional antibodies capable of binding to the endokine alpha protein antigen may be produced in a two-step procedure through the use of anti-idiotypic antibodies.
  • Such a method makes use ofthe fact that antibodies -109-
  • endokine alpha 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 endokine alpha protein-specific antibody can be blocked by the endokine alpha protein antigen.
  • Such antibodies comprise anti-idiotypic antibodies to the endokine alpha protein-specific antibody and can be used to immunize an animal to induce formation of further endokine alpha 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). Alternatively, endokine alpha protein-binding portions can be produced through the application of recombinant DNA technology or through synthetic chemistry.
  • 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 4:2X4 (1986); Cabilly et al, U.S. Patent No. 4,816,567; Taniguchi et al, EP 171496; Morrison etal, EP 173494; Neubergeret ⁇ /., WO 8601533; Robinson etal, WO
  • suitable labels for the endokine alpha protein-specific antibodies ofthe present invention are provided below.
  • suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast-alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose -1 10-
  • 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 H, ' "In, l25 I, 1 1 1, 32 P, 35 S, ,4 C, 51 Cr, 57 To, 58 Co, 59 Fe, 75 Se, ,52 Eu, 90 Y, 67 Cu, 217 Ci, 2, , At, 212 Pb, 47 Sc, ,09 Pd, etc. ' "In and 99m Tc are preferred isotopes where in vivo imaging is used since they avoid the problem of dehalogenation of the 125 I or l31 I-labeled monoclonal antibody by the liver.
  • these radionucleotides have 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)).
  • fluorescent labels examples 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.
  • chemi luminescent 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 examples include heavy metal nuclei such as Gd, Mn, and Fe.
  • the present invention further relates to antibodies and T-cell antigen receptors (TCR) which specifically bind the polypeptides ofthe present invention.
  • TCR T-cell antigen receptors
  • the antibodies ofthe present invention include IgG (including IgGl , IgG2, IgG3, and IgG4), IgA (including IgAl and IgA2), IgD, IgE, IgM, and IgY.
  • antibody is meant to include whole antibodies, including single-chain whole antibodies, and antigen-binding fragments thereof.
  • the antibodies are human antigen binding antibody fragments of the present invention include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single- chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain.
  • the antibodies may be from any animal origin including birds and mammals.
  • the antibodies are human, murine, rabbit, goat, guinea pig, camel, horse, or chicken.
  • Antigen-binding antibody fragments may comprise the variable region(s) alone or in combination with the entire or partial ofthe following: hinge region, CHI , CH2, and CH3 domains. Also included in the invention are any combinations of variable region(s) and hinge region, CHI, CH2, and CH3 domains.
  • the present invention further includes chimeric, humanized, and human monoclonal and polyclonal antibodies which specifically bind the polypeptides of the present invention.
  • the present invention further includes antibodies which are anti-idiotypic to the antibodies of the present invention.
  • the antibodies ofthe present invention may be monospecific, bispecific, trispecific or of greater multispecificity.
  • Multispecific antibodies may be specific for different epitopes of a polypeptide ofthe present invention or may be specific for both a polypeptide of the present invention as well as for heterologous compositions, such as a heterologous polypeptide or solid support material. See, e.g.. WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, A. et al ,
  • Antibodies ofthe present invention may be described or specified in terms ofthe epitope(s) or portion(s) of a polypeptide ofthe present invention which are recognized or specifically bound by the antibody.
  • the epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or listed in the Tables and Figures.
  • Antibodies which specifically bind any epitope or polypeptide ofthe present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides ofthe present invention, and allows for the exclusion ofthe same.
  • Antibodies ofthe present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog ofthe polypeptides ofthe present invention are included. Antibodies that do not bind polypeptides with less than 95%, less than 90%>. less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than
  • polypeptide ofthe present invention 60%, less than 55%, and less than 50%> identity (as calculated using methods known in the art and described herein) to a polypeptide ofthe present invention are also included in the present invention. Further included in the present invention are antibodies which only bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein). Antibodies ofthe present invention may also be described or specified in terms of their binding affinity.
  • Preferred binding affinities include those with a dissociation constant or Kd less than 5X10 " °M, 10- 6 M, 5X10 "7 M, 10 “7 M, 5X10 “8 M, 10 “8 M, 5X10 "9 M, 10 “9 M, 5X10-'°M, 10 " l0 M, 5X10-"M, 10-"M, 5X10 ",2 M, 10 ',2 M, 5X10 "13 M, 10 ",3 M, 5X10 ",4 M, 10 ",4 M,
  • Antibodies of the present invention have uses that include, but are not limited to, methods known in the art to purify, detect, and target the polypeptides of the present invention including both in vitro and in vivo diagnostic and therapeutic methods.
  • the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides ofthe present -113-
  • the antibodies of the present invention may be used either alone or in combination with other compositions.
  • the antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions.
  • antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, or toxins. See, e.g., WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No. 5,314,995; and EP 0 396 387.
  • the antibodies ofthe present invention may be prepared by any suitable method known in the art.
  • a polypeptide ofthe present invention or an antigenic fragment thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies.
  • Monoclonal antibodies can be prepared using a wide of techniques known in the art including the use of hybridoma and recombinant technology. See, e.g., Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al, in: Monoclonal Antibodies and T-cell Hybridomas, pp.
  • Fab and F(ab')2 fragments may be produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
  • antibodies ofthe present invention can be produced through the application of recombinant DNA technology or through synthetic chemistry using methods known in the art.
  • the antibodies of the present invention can be prepared using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of a phage particle which carries polynucleotide sequences encoding them. -1 14-
  • Phage with a desired binding property are selected from a repertoire or combinatorial antibody library (e.g. human or murine) by selecting directly with antigen, typically antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and Ml 3 with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies ofthe present invention include those disclosed in Brinkman U. et al, J. Immunol. Methods 752:41-50 (1995); Ames, R.S. etal, J. Immunol. Methods 754:177-186 (1995); Kettleborough, CA. et al, Eur. J. Immunol. 24:952-958 (1994); Persic, L. et al, Gene 757:9-18 (1997); Burton,
  • the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host including mammalian cells, insect cells, plant cells, yeast, and bacteria.
  • techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in WO 92/22324; Mullinax, R.L. et al, BioTechniques 12 (6) : 864-869 (1992); and Sawai, H. et al, AJRI 54:26-34 (1995); and Better, M. et al. Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties).
  • chimeric, humanized, or human antibodies it may be preferable to use chimeric, humanized, or human antibodies.
  • Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al, BioTechniques 4:2X4 (1986); Gillies, S.D. et al, J. Immunol. Methods 725: 191-202 (1989); and U.S. Patent No. 5,807,715.
  • Antibodies can be humanized using a variety of techniques including CDR-grafting (EP 0 239 400; WO 91/09967; U.S. Patent No.
  • Human antibodies can be made by a variety of methods known in the art including phage display methods described above. See also, U.S. Patents Nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; and WO 98/46645 (said references incorporated by reference in their entireties). Further included in the present invention are antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide of the present invention. The antibodies may be specific for antigens other than polypeptides of the present invention.
  • antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors.
  • Antibodies fused or conjugated to the polypeptides ofthe present invention may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g. , Harbor et al. supra and WO 93/21232; EP 0 439 095; Naramura, M. et al, Immunol Lett.
  • the present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions.
  • the invention may be fused or conjugated to an antibody Fc region, or portion thereof.
  • the antibody portion fused to a polypeptide ofthe present invention may comprise the hinge region, CHI domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof.
  • the polypeptides ofthe present invention may be fused or conjugated to the above antibody portions to increase the in vivo half life ofthe polypeptides or for use in immunoassays using methods known in the art.
  • the polypeptides may also be fused or conjugated to the above antibody portions to form multimers.
  • Fc portions fused to the polypeptides ofthe present invention can form dimers through disulfide bonding between the Fc portions.
  • the invention further relates to antibodies which act as agonists or antagonists ofthe polypeptides ofthe present invention.
  • the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully. Included are both receptor-specific antibodies and ligand-specific antibodies. Included are receptor-specific antibodies which do not prevent ligand binding, but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. Also include are receptor-specific antibodies which both prevent ligand binding and receptor activation.
  • neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor.
  • antibodies which activate the receptor are included. These antibodies may act as agonists for either all or less than all of the biological -i n ⁇
  • the antibodies may be specified as agonists or antagonists for biological activities comprising specific activities disclosed herein.
  • the above antibody agonists can be made using methods known in the art. see e.g., WO 96/40281; U.S. Patent No. 5,811,097; Deng, B. et al, Blood 92 ⁇ :1981-1988 (1998); Chen, Z. et al, Cancer Res.
  • polypeptides of the invention can also be expressed in transgenic animals.
  • Animals of any species including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate transgenic animals.
  • techniques described herein or otherwise known in the art are used to express polypeptides ofthe invention in humans, as part of a gene therapy protocol.
  • transgene i.e., polynucleotides ofthe invention
  • transgene i.e., polynucleotides ofthe invention
  • Such techniques include, but are not limited to, pronuclear microinjection (Paterson etal, Appl Microbiol. Biotechnol. 40:691-698 (1994); Carver et al. Biotechnology (NY) 77:1263-1270 (1993); Wright et al, Biotechnology (NY) 9:830-834 (1991); and Hoppe etal, U.S. Pat. No. 4,873.191 (1989)); retrovirus mediated gene transfer into germ lines (Van der Putten et al , -1 18-
  • transgenic clones containing polynucleotides of the invention for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (Campell et al, Nature 550:64-66 (1996); Wilmut et al, Nature
  • the present invention provides for transgenic animals that carry the transgene in all their cells, as well as animals which carry the transgene in some. but not all their cells, i.e., mosaic animals or chimeric animals.
  • the transgene may be integrated as a single transgene or as multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems.
  • the transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko et al. , Proc. Natl. Acad. Sci. USA 59:6232-6236 (1992)).
  • the transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type, by following, for example, the teaching of Gu et al. (Gu et al. , Science 265: 103- 106 ( 1994)).
  • the regulatory sequences required for such a cell- type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. The contents of each ofthe documents recited in this paragraph is herein incorporated by reference in its entirety.
  • the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration ofthe transgene has taken place. The level of mRNA expression ofthe transgene in the tissues ofthe transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenic gene- expressing tissue may also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.
  • founder animals may be bred, inbred, outbred, or crossbred to produce colonies ofthe particular animal.
  • breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the transgene on a distinct background that is appropriate for an experimental model of interest.
  • Transgenic and "knock-out" animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of endokine alpha polypeptides, studying conditions and/or disorders associated with aberrant endokine alpha expression, and in screening for compounds effective in ameliorating such conditions and/or disorders.
  • cells that are genetically engineered to express the polypeptides ofthe invention, or alternatively, that are genetically engineered not to express the polypeptides of the invention are administered to a patient in vivo.
  • Such cells may be obtained from the patient (i.e., animal, including human) or an MHC compatible donor and can include, but are not limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells, etc.
  • the cells are genetically engineered in vitro using recombinant DNA techniques to introduce the coding sequence of polypeptides of the invention into the cells, or alternatively, to disrupt the coding sequence and/or endogenous regulatory sequence associated with the polypeptides ofthe invention, e.g. , by transduction (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc.
  • the coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, ofthe polypeptides ofthe invention.
  • the engineered cells which express and preferably secrete the polypeptides ofthe invention can be introduced into the patient systemically, e.g. , in the circulation, or intraperitoneally .
  • the cells can be incorporated into a matrix and implanted in the body, e.g. , genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft.
  • U.S. Patent No. 5,631,153 Capecchi, et al, Cells and Non-Human Organisms Containing Predetermined Genomic Modifications and Positive-Negative Selection Methods and Vectors for Making Same
  • U.S. Patent No. 4,736,866 Leder, et al, Transgenic Non-Human Animals
  • U.S. Patent No. 4,873,191 Wider, et al, Genetic Transformation of Zygotes
  • the cells to be administered are non-autologous or non-MHC compatible cells, they can be administered using well known techniques which prevent the development of a host immune response against the introduced cells.
  • the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.
  • antagonists according to the present invention are nucleic acids corresponding to the sequences contained in SEQ ID NO:l, or the complementary strand thereof, and/or to nucleotide sequences contained in the deposited clone 97640.
  • antisense sequence is generated internally by the organism, in another embodiment, the antisense sequence is separately administered (see, for example, O'Connor, J., Neurochem. 5 ⁇ " :560
  • the methods are based on binding of a polynucleotide to a complementary DNA or RNA.
  • the 5' coding portion of a polynucleotide that encodes the mature polypeptide ofthe present invention may be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
  • a DNA oligonucleotide is designed to be complementary to a region ofthe gene involved in transcription thereby preventing transcription and the production of the receptor.
  • the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into receptor polypeptide.
  • the endokine alpha antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence.
  • RNA antisense nucleic acid
  • a vector or a portion thereof is transcribed, producing an antisense nucleic acid (RNA) of the invention.
  • a vector would contain a sequence encoding the endokine alpha antisense nucleic acid.
  • Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
  • Such vectors can be constructed by recombinant DNA technology methods standard in the art.
  • Vectors can be plasmid, viral, or others know in the art, used for replication and expression in vertebrate cells. Expression of the sequence encoding endokine alpha, or fragments thereof, can be by any promoter known in the art to act in vertebrate, preferably human cells.
  • Such promoters can be inducible or constitutive.
  • Such promoters include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, Nature 29:304-3 X 0 ( 1981 ), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al. , Cell 22:181-191 (1980), the herpes thymidine promoter (Wagner et al, Proc. Natl. Acad. Sci.
  • the antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of an endokine alpha gene.
  • absolute complementarity although preferred, is not required.
  • RNA means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double stranded endokine alpha antisense nucleic acids, a single strand ofthe duplex DNA may thus be tested, or triplex formation may be assayed.
  • the ability to hybridize will depend on both the degree of complementarity and the length ofthe antisense nucleic acid Generally, the larger the hybridizing nucleic acid, the more base mismatches with an endokine alpha RNA it may contain and still form a stable duplex (or triplex as the case may be).
  • One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point ofthe hybridized complex.
  • Oligonucleotides that are complementary to the 5' end ofthe message should work most efficiently at inhibiting translation.
  • sequences complementary to the 3' untranslated sequences of mRNAs have been shown to be effective at inhibiting translation of mRNAs as well. See generally, Wagner, R., 1994, Nature 372:333-335.
  • 5'- or 3'- non-translated, non-coding regions ofthe nucleotide sequence shown in Figure 1 could be used in an antisense approach to inhibit translation of endogenous endokine alpha mRNA.
  • Oligonucleotides complementary to the 5' untranslated region ofthe mRNA should include the complement ofthe AUG start codon.
  • Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention.
  • antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides. at least 25 nucleotides or at least 50 nucleotides.
  • the polynucleotides of the invention can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double- stranded.
  • the oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, -124-
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al, Proc. Natl Acad. Sci. U.S.A. 5(5:6553-6556 (1989); Lemaitre et al, Proc Natl. Acad. Sci. 54:648-652 (1987); PCTPublicationNo. WO88/09810, publishedDecember 15,
  • the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross- linking agent, transport agent, hybridization-triggered cleavage agent, etc.
  • the antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
  • 5-carboxymethylaminomethyl-2-thiouridine 5-carboxymethylaminomethyluracil, dihydrouracil, a-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1 -methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine.
  • 2-thiouracil beta-D-mannosylqueosine, 5 '-methoxy carboxy methyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil.
  • the antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
  • the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group including, but not -125-
  • a phosphorothioate a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
  • the antisense oligonucleotide is an ⁇ -anomeric oligonucleotide.
  • An ⁇ -anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gautier et al, Nucl Acids Res. 75:6625-6641 (1987)).
  • the oligonucleotide is a 2'-0-methylribonucleotide (Inoue et al. , Nucl. Acids Res. 75:6131-6148 (1987)), or a chimeric RNA-DNA analogue (Inoue et al, FEBS Lett. 215:321-330 (1987)).
  • Polynucleotides ofthe invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.).
  • an automated DNA synthesizer such as are commercially available from Biosearch, Applied Biosystems, etc.
  • phosphorothioate oligonucleotides may be synthesized by the method of Stein et al, Nucl. Acids Res. 16:3209 (1988)
  • methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al, Proc. Natl Acad. Sci. U.S.A. 55:7448-7451 (1988)), etc.
  • antisense nucleotides complementary to the endokine alpha coding region sequence could be used, those complementary to the transcribed untranslated region are most preferred.
  • Potential antagonists according to the invention also include catalytic RNA, or a ribozyme (see, e.g., PCT International Publication WO 90/11364, published October 4, 1990; Sarver et al, Science 247:1222-1225 (1990). While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy endokine alpha mRNAs, the use of hammerhead ribozymes is preferred.
  • Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3'.
  • the construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, Nature 554:585-591 (1988).
  • the ribozyme is engineered so that the cleavage recognition site is located near the 5' end ofthe endokine alpha mRNA; i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.
  • the ribozymes of the invention can be composed of modified oligonucleotides (e.g. for improved stability, targeting, etc.) and should be delivered to cells which express endokine alpha in vivo. DNA constructs encoding the ribozyme may be introduced into the cell in the same manner as described above for the introduction of antisense encoding DNA.
  • a preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive promoter, such as, for example, pol III or pol II promoter, so that transfected cells will produce sufficient quantities ofthe ribozyme to destroy endogenous endokine alpha messages and inhibit translation. Since ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.
  • a strong constitutive promoter such as, for example, pol III or pol II promoter
  • Endogenous gene expression can also be reduced by inactivating or "knocking out” the endokine alpha gene and/or its promoter using targeted homologous recombination, (e.g., see Smithies et al, Nature 317:230-234 (1985); Thomas & Capecchi, Cell 51 :503-512 (1987); Thompson et al, Cell 5:313-321 (1989); each of which is incorporated by reference herein in its entirety).
  • targeted homologous recombination e.g., see Smithies et al, Nature 317:230-234 (1985); Thomas & Capecchi, Cell 51 :503-512 (1987); Thompson et al, Cell 5:313-321 (1989); each of which is incorporated by reference herein in its entirety).
  • a mutant, non-functional polynucleotide ofthe invention flanked by DNA homologous to the endogenous polynucleotide sequence (either the coding regions or regulatory regions of the gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express polypeptides of the invention in vivo.
  • techniques known in the art are used to generate knockouts in cells that contain, but do not express the gene of interest. Insertion ofthe DNA construct, via targeted homologous recombination, results in inactivation of the targeted gene.
  • Such approaches are particularly suited in research and agricultural fields where modifications to embryonic stem cells can be used to generate animal offspring with an inactive targeted gene (e.g., see -127-
  • antagonists according to the present invention include soluble forms of endokine alpha (e.g., fragments ofthe endokine alpha polypeptide shown in Figure 1 that include the ligand binding domain from the extracellular region ofthe full length receptor).
  • soluble forms of endokine alpha which may be naturally occurring or synthetic, antagonize endokine alpha mediated signaling by competing with the cell surface bound forms ofthe receptor for binding to TNF-family ligands.
  • Antagonists of the present invention also include antibodies specific for TNF-family ligands and endokine alpha-Fc fusion proteins.
  • TNF-family ligand is intended naturally occurring, recombinant, and synthetic ligands that are capable of binding to a member of the TNF receptor family and inducing and/or blocking the ligand/receptor signaling pathway.
  • TNF ligand family include, but are not limited to, TNF-a, lymphotoxin-a (LT-a, also known as TNF-b), LT-b (found in complex heterotrimer LT-a2-b), FasL, CD40L, CD27L, CD30L, 4-lBBL, OX40L and nerve growth factor (NGF).
  • TNF- ⁇ has been shown to protect mice from infection with herpes simplex virus type l (HSV-1). Rossol-Voth et j/., J .Gen. Virol. 72:143-147 (1991).
  • the mechanism ofthe protective effect of TNF- ⁇ is unknown but appears to involve neither interferons nor NK cell killing.
  • One member ofthe TNFR family has been shown to mediate HSV-1 entry into cells. Montgomery et al , Eur. Cytokine Newt. 7:159 (1996).
  • antibodies specific for the extracellular domain of this TNFR block HSV- 1 entry into cells.
  • endokine alpha antagonists ofthe present invention include both endokine alpha amino acid sequences and antibodies capable of preventing TNFR mediated viral entry into cells. Such -128-
  • sequences and antibodies can function by either competing with cell surface localized TNFR for binding to virus or by directly blocking binding of virus to cell surface receptors.
  • Antibodies according to the present invention may be prepared by any of a variety of standard methods using endokine alpha receptor immunogens ofthe present invention.
  • endokine alpha receptor immunogens include the endokine alpha receptor protein shown in FIGURE 1 (SEQ ID NO: 2) (which may or may not include a leader sequence) and polypeptide fragments ofthe receptor comprising the ligand binding, extracellular, transmembrane, the intracellular domains of the endokine alpha receptors, or any combination thereof.
  • Polyclonal and monoclonal antibody agonists or antagonists according to the present invention can be raised according to the methods disclosed herein and/or known in the art, such as, for example, those methods described in Tartagliaand Goeddel, J. Biol. Chem. 267(7):4304-4307(1992)); Tartaglia et al. , Cell 73:2X3-2X6 (1993)), and PCT Application WO 94/09137 (the contents of each of these three applications are herein incorporated by reference in their entireties), and are preferably specific to polypeptides ofthe invention having the amino acid sequence of SEQ ID NO:2.
  • endokine alpha polypeptides of the present invention and the epitope-bearing fragments thereof described above can be combined with heterologous polypeptide sequences.
  • the polypeptides ofthe present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, and IgM) or portions thereof (CHI , CH2, CH3, and any combination thereof, including both entire domains and portions thereof), resulting in chimeric polypeptides.
  • immunoglobulins IgA, IgE, IgG, and IgM
  • CHI constant domain of immunoglobulins
  • CH2, CH3 any combination thereof, including both entire domains and portions thereof
  • DNA shuffling The techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as "DNA shuffling") may be employed to modulate the activities of endokine alpha thereby effectively generating agonists and antagonists of endokine alpha. See generally, U.S. Patent Nos. 5,605,793,
  • alteration of endokine alpha polynucleotides and corresponding polypeptides may be achieved by DNA shuffling.
  • DNA shuffling involves the assembly of two or more DNA segments into a desired endokine alpha molecule by homologous, or site-specific, recombination.
  • endokine alpha polynucleotides and corresponding polypeptides may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination.
  • one or more components, motifs, sections, parts, domains, fragments, etc., of endokine alpha may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
  • the heterologous molecules are, for example, TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-lBBL, DcR3, OX40L, TNF-gamma (International Publication No. WO 96/14328).
  • AIM-I International Publication No. WO 97/33899
  • AIM-II International Publication No. WO 97/34911
  • APRIL J. Exp. Med.
  • endokine-alpha International Publication Nos. WO 98/07880 and WO 98/18921
  • OPG OPG
  • OX40 nerve growth factor
  • NGF nerve growth factor
  • soluble forms of Fas CD30, CD27, CD40 and 4-IBB
  • DR3 International Publication No. WO 97/33904
  • DR4 International Publication No. WO 98/32856
  • TR5 International Publication No. WO 98/30693
  • TR6 International Publication No. WO
  • heterologous molecules are any member of the TNF family.
  • the nucleic acid molecules ofthe 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.
  • Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) 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.
  • the cDNA herein disclosed is used to clone genomic DNA of an endokine alpha 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 purpose. 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.
  • sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis ofthe 3' untranslated region ofthe 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. -131-
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome.
  • 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 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).
  • the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V.
  • 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).
  • TNF Tumor Necrosis Factor
  • TNF-family ligands induce such various cellular responses by binding to TNF-family receptors.
  • Endokine alpha polynucleotides, polypeptides, agonists or antagonists of the invention may be used in developing treatments for any disorder mediated (directly or indirectly) by defective, or insufficient amounts of endokine alpha.
  • Endokine alpha polypeptides, agonists or antagonists may be administered to a patient (e.g., mammal, preferably human) afflicted with such a disorder.
  • endokine alpha nucleotide sequences permits the detection of defective endokine alpha genes, and the replacement thereof with normal endokine alpha-encoding genes.
  • Defective genes may be detected in in vitro diagnostic assays, and by comparision ofthe endokine alpha nucleotide sequence disclosed herein with that of a endokine alpha gene derived from a patient suspected of harboring a defect in this gene.
  • polypeptides ofthe present invention are used as a research tool for studying the biological effects that result from inhibiting TRl 1/endokine alpha interactions on different cell types
  • endokine alpha polypeptides also may be employed in in vitro assays for detecting TRl 1 or endokine alpha or the interactions thereof.
  • TNF is noted for its pro-inflammatory actions which result in tissue injury, such as induction of procoagulant activity on vascular endothelial cells (Pober. J.S. et al. J. Immunol 756:1680 (1986)). increased -133-
  • TNF is an important mediator ofthe cachexia in cancer, infectious pathology, and in other catabolic states.
  • the endokine alpha protein ofthe present invention can be used for tumor targeting, preferably, after conjugation with radioisotypes or cytostatic drugs (Gruss and Dower, Blood 85(12) .3318-3404 (1995)).
  • Endokine alpha can be used in patients with melanoma and sarcoma for tumor regression and extension of patient life span through a local injection or used in isolated limb perfusion (Aggarwal and Natarajan, Eur. Cytokine Netw. 7(2):92-X24 (1996)).
  • the endokine alpha ofthe present invention can also have a therapeutic role in specific situations, for example, activity against viral, bacterial, yeast, fungal, and other infections (including toxoplasma gondii, schistosoma mansoni, listeria monocytogens and BCG). These effects of endokine alpha can be indirect and thus preferably, mediated through activation of macrophages, eosinophils, fibroblasts, or neutrophils.
  • TNF is also thought to play a central role in the pathophysiological consequences of Gram-negative sepsis and endotoxic shock (Michie, H.R. et al, Br. J. Surg. 76:670-671 (1989); Debets. J.M.H. et al. Second Vienna Shock
  • Endotoxin is a potent monocyte/macrophage activator which stimulates production and secretion of TNF (Kornblufh, S.K. et al, J. Immunol. 757:2585-2591 (1986)) and other cytokines. Elevated levels of circulating TNF have also been found in patients suffering from Gram-negative sepsis (Waage. A. et al. Lancet 7:355-357 (1987); -134-
  • Neutralizing antisera or mAbs to TNF have been shown in mammals other than man to abrogate adverse phay siological changes and prevent death after lethal challenge in experimental endotoxemia and bacteremia. This effect has been demonstrated, e.g., in rodent lethality assays and in primate pathology model systems (Mathison, J.C. et al, J. Clin. Invest. 57:1925-1937 (1988); Beufler, B. et al, Science 229:869-871 (1985); Tracey, K.J. et al, Nature 330:662-664 (1987); Shimamoto, Y. et al, Immunol. Lett.
  • the present invention is further directed to antibody-based therapies which involve administering an anti-endokine alpha antibody to a mammalian, preferably human, patient for treating one or more of the above-described disorders.
  • antibody-based therapies which involve administering an anti-endokine alpha antibody to a mammalian, preferably human, patient for treating one or more of the above-described disorders.
  • Methods for producing anti-endokine alpha polyclonal and monoclonal antibodies are described in detail above.
  • Such antibodies may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
  • Polynucleotides and/or polypeptides of the invention, and/or agonists and/or antagonists thereof, are useful in the diagnosis and treatment or prevention of a wide range of diseases and/or conditions.
  • diseases and conditions include, but are not limited to, cancer (e.g., immune cell related cancers, breast cancer, prostate cancer, ovarian cancer, follicular lymphoma, gliobalstoma. cancer -135-
  • lymphoproliferative disorders e.g., lymphadenopathy and lymphomas (e.g., EBVinduced lymphoproliferations and Hodgkin's disease), microbial (e.g.
  • viral, bacterial, etc. infection e.g., HIV-1 infection, HIV-2 infection, herpesvirus infection (including, but not limited to, HSV-1, HSV-2, CMV, VZV, HHV-6, HHV-7, EBV), adenovirus infection, poxvirus infection, human papilloma virus infection, hepatitis infection (e.g., HAV, HBV, HCV, etc.), Helicobacter pylori infection, invasive Staphylococcia, etc.), parasitic infection, nephritis, bone disease
  • herpesvirus infection including, but not limited to, HSV-1, HSV-2, CMV, VZV, HHV-6, HHV-7, EBV
  • adenovirus infection e.g., poxvirus infection, human papilloma virus infection, hepatitis infection (e.g., HAV, HBV, HCV, etc.), Helicobacter pylori infection, invasive Sta
  • cardiovascular disorders e.g., neovascularization, hypovascularization or reduced circulation (e.g., ischemic disease (e.g., myocardial infarction, stroke, etc.)
  • ischemic disease e.g., myocardial infarction, stroke, etc.
  • AIDS allergy, inflammation
  • neurodegenerative disease e.g., Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, pigmentary retinitis, cerebellar degeneration, etc.
  • graft rejection acute and chronic
  • graft vs graft vs.
  • osteomyelodysplasia e.g., aplastic anemia, etc.
  • joint tissue destruction in rheumatism liver disease (e.g., acute and chronic hepatitis, liver injury, and cirrhosis)
  • autoimmune disease e.g., multiple sclerosis, myasthenia gravis, rheumatoid arthritis, systemic lupus erythematosus, immune complex glomerulonephritis, autoimmune diabetes, autoimmune thrombocytopenic purpura.
  • Grave's disease Hashimoto's thyroiditis.
  • cardiomyopathy e.g., dilated cardiomyopathy
  • diabetes e.g., diabetic complications
  • influenza e.g., diabetic nephropathy, diabetic neuropathy, diabetic retinopathy
  • asthma e.g., psoriasis, glomerulonephritis, septic shock, and ulcerative colitis.
  • Polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are useful in promoting angiogenesis, wound healing (e.g., wounds, burns, and bone fractures), and regulating bone formation and treating osteoporosis. -136-
  • Polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are also useful as an adjuvant to enhance immune responsiveness to specific antigen and/or anti-viral immune responses.
  • polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are useful in regulating (i.e.. elevating or reducing) immune response.
  • polynucleotides and/or polypeptides ofthe invention may be useful in preparation or recovery from surgery, trauma, radiation therapy, chemotherapy, and transplantation, or may be used to boost immune response and/or recovery in the elderly and immunocompromised individuals.
  • polynucleotides and/or polypeptides ofthe invention and/or agonists and/or antagonists thereof are useful as immunosuppressive agents, for example in the treatment or prevention of autoimmune disorders or in the prevention of transplant rejection.
  • polynucleotides and/or polypeptides of the invention are used to treat or prevent chronic inflammatory, allergic or autoimmune conditions, such as those described herein or are otherwise known in the art.
  • a summary ofthe ways in which the antibodies ofthe present invention may be used therapeutically includes binding endokine alpha 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.
  • CDC complement
  • ADCC effector cells
  • the pharmaceutical compositions of the present invention may be administered by any means that achieve their intended purpose. 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 TNF-related disease. For example, administration may be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. -137-
  • 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 purpose. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill ofthe 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 ofthe 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 endokine alpha antibody or fragment ofthe 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 -138-
  • the antibodies, fragments and derivatives of the present invention are useful for treating a subject having or developing endokine alpha related disorders as described herein.
  • Such treatment comprises parenterally administering single or multiple doses ofthe antibody, a 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.
  • endokine alpha (like TNF) tend to be extremely low, in the range of about 10 pg/ml in non-septic individuals, and reaching about 50 pg/ml in septic patients and above 100 pg/ml in the sepsis syndrome for TNF (Hammerle, A.F. et al, 1989, supra) or may be only be detectable at sites of endokine alpha-related disorders, it is preferred to use high affinity and/or potent in vivo endokine alpha-inhibiting and/or neutralizing antibodies, fragments or regions thereof, for both endokine alpha immunoassays and therapy of endokine related disorders.
  • Such antibodies, fragments, or regions will preferably have an affinity for human endokine alpha, 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 “ ',
  • 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 endokine-inhibiting and/or neutralizing activity, according to the present invention, e.g.. that block endokine-induced IL-1.
  • Additional preferred embodiments of the invention include, but are not limited to, the use of endokine- ⁇ polypeptides and functional agonists in the following applications: -139-
  • a vaccine adjuvant that enhances immune responsiveness to specific antigen.
  • An adjuvant to enhance anti- viral immune responses As a stimulator of B cell responsiveness to pathogens.
  • TH2 a humoral response
  • TH1 a TH1 cellular response
  • B cell and other ligand expressing cell e.g. , endothelial cells
  • specific binding protein to which specific activators or inhibitors of cell growth may be attached. The result would be to focus the activity of such activators or inhibitors onto normal, diseased, or neoplastic B cell populations.
  • B-lineage cells and/or ligand expressing cells e.g. , endothelial cells
  • This application may require labeling the protein with biotin or other agents to afford a means of detection.
  • B cell selection device As part of a B cell selection device the function of which is to isolate B cells as well as other ligand expressing cells (e.g.. endothelial cells) from a heterogenous mixture of cell types. Endokine alpha could be coupled to a solid support to which B cells would then specifically bind. Unbound cells would be -140-
  • This technique would allow purging of tumor cells from, for example, bone marrow or peripheral blood prior to transplant.
  • Endokine alpha-induced inhibition of IL-12 production might be helpful in controlling TH1 -associated conditions, such as autoimmune diseases, inflammation, acute allograft rejection, fetal reabsorption.
  • Products ofthe oxidative burst such as H2O2 are used by monocytes for the killing of phagocytosed pathogens or for the extracellular destruction of cells.
  • Antagonists of endokine alpha include binding and/or inhibitory antibodies, antisense nucleic acids, ribozymes or soluble forms of the endokine alpha receptor(s). These would be expected to reverse many of the activities of the ligand described above as well as find clinical or practical application as: -141-
  • endokine alpha may, like CD40 and its ligand, be regulated by the status ofthe immune system and the microenvironment in which the cell is located.
  • a therapy for preventing the B and/or T cell proliferation and Ig secretion associated with autoimmune diseases such as idiopathic thrombocytopenic purpura, systemic lupus erythramatosus and MS.
  • An inhibitor of B and or T cell migration in endothelial cells disrupts tissue architecture or cognate responses and is useful, for example, in disrupting immune responses, and blocking sepsis.
  • a therapy for B cell and/or T cell malignancies such as ALL, Hodgkins disease, non-Hodgkins lymphoma. Chronic lymphocyte leukemia, plasmacytomas, multiple myeloma, Burkitt's lymphoma, and EBV-transformed diseases.
  • a therapy for chronic hypergammaglobulinemeia evident in such diseases as monoclonalgammopathy of undetermined significance (MGUS), Waldenstrom's disease, and related idiopathic monoclonalgammopathies.
  • An immunosuppressive agent(s) is an immunosuppressive agent(s).
  • the agonists and antagonists may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as described above.
  • the antagonists may be employed for instance to inhibit endokine alpha chemotaxis and activation of macrophages and their precursors, and of -142-
  • neutrophils neutrophils, basophils, B lymphocytes and some T-cell subsets, e.g. , activated and CD8 cytotoxic T cells and natural killer cells, in certain auto-immune and chronic inflammatory and infective diseases.
  • auto-immune diseases include multiple sclerosis, and insulin-dependent diabetes.
  • the antagonists may also be employed to treat infectious diseases including silicosis, sarcoidosis, idiopathic pulmonary fibrosis by preventing the recruitment and activation of mononuclear phagocytes. They may also be employed to treat idiopathic hyper-eosinophilic syndrome by preventing eosinophil production and migration.
  • Endotoxic shock may also be treated by the antagonists by preventing the migration of macrophages and their production of the endokine alpha polypeptides of the present invention.
  • the antagonists may also be employed for treating atherosclerosis, by preventing monocyte infiltration in the artery wall.
  • the antagonists may also be employed to treat histamine-mediated allergic reactions and immunological disorders including late phase allergic reactions, chronic urticaria, and atopic dermatitis by inhibiting chemokine-induced mast cell and basophil degranulation and release of histamine.
  • IgE-mediated allergic reactions such as allergic asthma, rhinitis, and eczema may also be treated.
  • the antagonists may also be employed to treat chronic and acute inflammation by preventing the attraction of monocytes to a wound area. They may also be employed to regulate normal pulmonary macrophage populations, since chronic and acute inflammatory pulmonary diseases are associated with sequestration of mononuclear phagocytes in the lung. Antagonists may also be employed to treat rheumatoid arthritis by preventing the attraction of monocytes into synovial fluid in the joints of patients. Monocyte influx and activation plays a significant role in the pathogenesis of both degenerative and inflammatory arthropathies.
  • the antagonists may be employed to interfere with the deleterious cascades attributed primarily to IL- 1 and TNF, which prevents the biosynthesis of other inflammatory cytokines. In this way, the antagonists may be employed to prevent inflammation.
  • the antagonists may also be employed to inhibit prostaglandin-independent fever induced by endokine alpha.
  • the antagonists ma ⁇ ' -143-
  • the antagonists may also be employed to treat cases of bone marrow failure, for example, aplastic anemia and myelodysplastic syndrome.
  • the antagonists may also be employed to treat asthma and allergy by preventing eosinophil accumulation in the lung.
  • the antagonists may also be employed to treat subepithelial basement membrane fibrosis which is a prominent feature ofthe asthmatic lung.
  • Antibodies against endokine alpha may be employed to bind to and endokine alpha activity to treat ARDS, by preventing infiltration of neutrophils into the lung after injury.
  • the antagonists and antagonists ofthe instant may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as described hereinafter.
  • Agonists and antagonists of the invention also have uses in stimulating wound and tissue repair, stimulating angiogenesis, stimulating the repair of vascular or lymphatic diseases or disorders. Additionally, agonists and antagonists ofthe invention may be used to stimulate the regeneration of mucosal surfaces.
  • the compositions of the invention may be administered alone or in combination with other therapeutic agents.
  • Therapeutic agents that may be administered in combination with the compositions ofthe invention include but not limited to, other members of the TNF family, chemotherapeutic agents, antibiotics, steroidal and non-steroidal anti-inflammatories, conventional immunotherapeutic agents, cytokines and/or growth factors. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially.
  • Administration "in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.
  • compositions ofthe invention are administered in combination with other members ofthe TNF family.
  • TNF TNF-related or TNF- like molecules that may be administered with the compositions of the invention -144-
  • TNF-alpha soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT- alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-lBBL, DcR3, OX40L, TNF-gamma (International Publication No. WO 96/14328), AIM-I (International Publication No. WO 97/33899),, endokine-alpha (International Publication No.
  • WO 98/07880 International Publication No. WO 98/30694
  • OPG International Publication No. WO 98/30694
  • neutrokine-alpha International Publication No. WO 98/18921 , OX40, and nerve growth factor (NGF)
  • soluble forms of Fas CD30, CD27, CD40 and 4-IBB
  • TR2 International Publication No. WO 96/34095
  • DR3 International Publication No. WO 97/33904
  • DR4 International Publication No. WO
  • TR5 International Publication No. WO 98/30693
  • TR6 International Publication No. WO 98/30694
  • TR7 International Publication No. WO 98/41629
  • TRANK International Publication No. WO 98/56892
  • TR10 International Publication No. WO 98/54202
  • 312C2 International Publication No. WO 98/06842
  • TR12 and soluble forms CD154, CD70, and CD153.
  • Nonspecific immunosuppressive agents that may be administered in combination with the compositions ofthe invention include, but are not limited to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide methylprednisone, prednisone, azathioprine, FK-506, 15-deoxyspergualin, and other immunosuppressive agents that act by suppressing the function of responding T cells.
  • compositions of the invention are administered in combination with an antibiotic agent.
  • Antibiotic agents that may be administered with the compositions ofthe invention include, but are not limited to. tetracycline, metronidazole, amoxicillin, beta-lactamases, aminoglycosides, macrolides. quinolones. fluoroquinolones, cephalosporins, erythromycin, ciprofloxacin, and streptomycin.
  • compositions of the invention are administered alone or in combination with an anti-inflammatory agent.
  • Anti- inflammatory agents that may be administered with the compositions of the invention include, but are not limited to, ⁇ lucocorticoids and the nonsteroidal anti- -145-
  • inflammatories aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e- acetamidocaproic acid, S-adenosylmethionine, 3 -amino-4-hydroxy butyric acid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, and tenidap.
  • compositions ofthe invention are administered in combination with a chemotherapeutic agent.
  • Chemotherapeutic agents that may be administered with the compositions ofthe invention include, but are not limited to, antibiotic derivatives (e.g.. doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g. , tamoxifen); antimetabolites (e.g.
  • cytotoxic agents e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cis-platin, and vincristine sulfate
  • hormones e.g., medroxyprogesterone.
  • estramustine phosphate sodium ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene. and testolactone
  • nitrogen mustard derivatives e.g. , mephalen, chorambucil, mechlorethamine (nitrogen mustard) and thiotepa
  • steroids and combinations e.g., bethamethasone sodium phosphate
  • others e.g., dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).
  • compositions of the invention are administered in combination with cytokines.
  • Cytokines that may be administered with the compositions of the invention include, but are not limited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-gamma and TNF-alpha.
  • compositions of the invention are administered in combination with angiogenic proteins.
  • Angiogenic proteins that may be administered with the compositions ofthe invention include, but are not -146-
  • GDGF Glioma Derived Growth Factor
  • EP-399816 Glioma Derived Growth Factor
  • PDGF-A Platelet Derived Growth Factor-A
  • PDGF-B Platelet Derived Growth Factor-B
  • PIGF Placental Growth Factor
  • VEGF Vascular Endothelial Growth Factor
  • P1GF-2 Placental Growth Factor-2
  • VEGF Vascular Endothelial Growth Factor
  • VEGF-A Vascular Endothelial Growth Factor-A
  • VEGF-2 Vascular Endothelial Growth Factor-2
  • VEGF-B186 Vascular Endothelial Growth Factor B-186
  • VEGF-D Vascular Endothelial Growth Factor-D
  • VEGF-D Vascular Endothelial Growth Factor-D
  • VEGF-D Vascular Endothelial Growth Factor-D
  • VEGF-D Vascular Endothelial Growth Factor-D
  • VEGF-D Vascular Endothelial Growth Factor-D
  • VEGF-D Vascular Endothelial Growth Factor-D
  • VEGF-D Vascular Endothelial Growth Factor-D
  • VEGF-D Vascular Endothelial Growth Factor-D
  • WO 98/07832 and Vascular Endothelial Growth Factor-E (VEGF-E), as disclosed in German Patent Number DEI 9639601. The above mentioned references are incorporated herein by reference.
  • compositions of the invention are administered in combination with Fibroblast Growth Factors. Fibroblast Growth Factors. Fibroblast Growth
  • Factors tha may be administered with the compositions ofthe invention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8. FGF-9, FGF-10. FGF-1 1, FGF-12, FGF-13, FGF-14, and FGF-15.
  • compositions of the invention are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.
  • Endokine alpha compositions of the invention are also suitably administered by sustained-release systems.
  • sustained-release compositions include suitable polymeric materials (such as. for example, semi-permeable polymer matrices in the form of shaped articles, e.g.. films, or mirocapules), suitable hydrophobic materials (for example, as an emulsion in an -147-
  • Sustained-release matrices include polylactides (U.S. Patent No.
  • Sustained-release compositions also include liposomally entrapped compositions of the invention (see generally, Langer, Science 249:1527-1533
  • Liposomes containing Endokine alpha polypeptide may be prepared by methods known per se: DE 3,218,121 ; Epstein et al, Proc. Natl.
  • the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the
  • compositions of the invention include crystal formulations known in the art.
  • the compositions ofthe invention are delivered by way of a pump (see Langer. supra; Sefton. CRC Crit. Refi Biomed.
  • compositions of the invention may be administered alone or in combination with other adjuvants.
  • adjuvants that may be administered with the -148-
  • compositions of the invention include, but are not limited to, alum, alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21 (Genentech, Inc.), BCG, and MPL.
  • compositions of the invention are administered in combination with alum.
  • compositions of the invention are administered in combination with QS-21.
  • compositions ofthe invention include, but are not limited to, monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21 , QS-18, CRL 1005, aluminum salts, MF-59, and Virosomal adjuvant technology.
  • Vaccines that may be administered with the compositions of the invention include, but are not limited to, vaccines directed toward protection against MMR (measles, mumps, rubella), polio, varicella, tetanus/diptheria, hepatitis A, hepatitis B, Haemophilus influenzae B, whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus, cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies, typhoid fever, and pertussis, and/or PNEUMOVAX-23TM.
  • MMR measles, mumps, rubella
  • polio varicella
  • tetanus/diptheria hepatitis A
  • hepatitis B Haemophilus influenzae B
  • cholera yellow fever
  • Japanese encephalitis poliomyelitis
  • rabies typhoid fever
  • pertussis and/
  • Combinations may be administered either concomitantly, e.g., as an admixture, separately, but simultaneously or concurrently; or sequentially.
  • Administration "in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.
  • compositions ofthe invention are used in combination with PNEUMOVAX-23TM to treat, prevent, and/or diagnose infection and/or any disease, disorder, and/or condition associated therewith.
  • compositions of the invention are used in combination with PNEUMOVAX-23TM to treat, prevent, and/or diagnose any Gram positive bacterial infection and/or any disease, disorder, and/or condition associated therewith.
  • compositions of the invention are used in combination with PNEUMOVAX-23TM to treat, prevent, and/or diagnose infection and/or anv disease, disorder, and/or condition associated with one or -149-
  • compositions ofthe invention are used in any combination with PNEUMOVAX-23TM to treat, prevent, and/or diagnose infection and/or any disease, disorder, and/or condition associated with one or more members ofthe Group B streptococci.
  • compositions ofthe invention are used in combination with PNEUMOVAX-23TM to treat, prevent, and/or diagnose infection and/or any disease, disorder, and/or condition associated with Streptococcus pneumoniae.
  • compositions of the invention may be administered alone or in combination with other therapeutic agents, including, but not limited to, chemotherapeutic agents, antibiotics, antivirals, steroidal and non-steroidal anti-inflammatories, conventional immunotherapeutic agents and cytokines.
  • Combinations may be administered either concomitantly, e.g., as an admixture, separately, but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately, but simultaneously, e.g. , as through separate intravenous lines into the same individual.
  • Administration "in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.
  • compositions ofthe invention are administered in combination with other members of the TNF family.
  • TNF, TNF-related or TNF-like molecules that may be administered with the compositions of the invention include, but are not limited to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-lBBL, DcR3, OX40L.
  • TNF-gamma International Publication No. WO 96/14328
  • AIM-I International Publication No.
  • NGF nerve growth factor
  • soluble forms of Fas CD30, CD27, CD40 and 4-IBB
  • TR2 International Publication No. WO 96/34095
  • DR3 International Publication No. WO 97/33904
  • DR4 International Publication No. WO 98/32856
  • TR5 International Publication No. WO 98/30693
  • TR6 International Publication No. WO 98/30694
  • TR7 International Publication No.
  • WO 98/41629 discloses WO 98/41629
  • TR9 International Publication No. WO 98/56892
  • TRIO International Publication No. WO 98/54202
  • 312C2 International Publication No. WO 98/06842
  • compositions of the invention are administered alone or in combination with CD40 ligand (CD40L), a soluble form of CD40L (e.g. , AVREND), biologically active fragments, variants, or derivatives of CD40L, anti-CD40L antibodies (e.g., agonistic or antagonistic antibodies), and/or anti-CD40 antibodies (e.g., agonistic or antagonistic antibodies).
  • CD40L CD40 ligand
  • AVREND soluble form of CD40L
  • biologically active fragments, variants, or derivatives of CD40L e.g., anti-CD40L antibodies (e.g., agonistic or antagonistic antibodies), and/or anti-CD40 antibodies (e.g., agonistic or antagonistic antibodies).
  • compositions ofthe invention are administered in combination with antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors.
  • Nucleoside reverse transcriptase inhibitors that may be administered in combination with the compositions ofthe invention, include, but are not limited to, RETROVIRTM (zidovudine/AZT), VIDEXTM (didanosine/ddl), HIVIDTM (zalcitabine/ddC), ZERITTM (stavudine/d4T), EPIVIRTM (lamivudine/3TC), and
  • Non-nucleoside reverse transcriptase inhibitors that may be administered in combination with the compositions ofthe invention, include, but are not limited to, VIRAMUNETM (nevirapine), RESCRIPTORTM (delavirdine), and SUSTIVATM (efavirenz).
  • Protease inhibitors that may be administered in combination with the compositions ofthe invention, include, but are not limited to, CRIXIVANTM (indinavir), NORVIRTM (ritonavir), INVIRASETM (saquinavir), and VIRACEPTTM (nelfinavir).
  • antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors may be -151-
  • compositions ofthe invention may be administered in combination with anti-opportunistic infection agents.
  • Anti-opportunistic agents that may be administered in combination with the compositions ofthe invention, include, but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLETM, DAPSONETM, PENTAMIDINETM, ATOVAQUONETM, ISONIAZIDTM, RIFAMPINTM, PYRAZINAMIDETM, ETHAMBUTOLTM, RIFABUTINTM, CLARITHROMYCINTM, AZITHROMYCINTM, GANCICLOVIRTM, FOSCARNETTM, CIDOFOVIRTM, FLUCONAZOLETM, ITRACONAZOLETM,
  • compositions of the invention are used in any combination with TRIMETHOPRIM- SULFAMETHOXAZOLETM, DAP S ONETM,
  • compositions of the invention are used in any combination with ISONIAZIDTM, RIFAMPINTM, PYRAZINAMIDETM, and/or ETHAMBUTOLTM to prophylactically treat, prevent, and/or diagnose an opportunistic Mycobacterium avium complex infection.
  • compositions of the invention are used in any combination with RIFABUTINTM, CLARITHROMYCINTM, and/or AZITHROMYCINTM to prophylactically treat, prevent, and/or diagnose an opportunistic Mycobacterium tuberculosis infection.
  • compositions of the invention are used in any combination with GANCICLOVIRTM, FOSCARNETTM, and/or CIDOFOVIRTM to prophylactically treat, prevent, and/or diagnose an opportunistic cytomegalovirus infection.
  • compositions of the invention are used in any combination with FLUCONAZOLETM. ITRACONAZOLETM, and/or KETOCONAZOLETM to -152-
  • compositions ofthe invention are used in any combination with AC YCLOVIRTM and/or FAMCICOLVIRTM to prophylactically treat, prevent, and/or diagnose an opportunistic herpes simplex virus type I and/or type II infection.
  • compositions ofthe invention are used in any combination with PYRIMETHAMINETM and/or LEUCOVORINTM to prophylactically treat, prevent, and/or diagnose an opportunistic Toxoplasma gondii infection.
  • compositions of the invention are used in any combination with LEUCOVORINTM and/or NEUPOGENTM to prophylactically treat, prevent, and/or diagnose an opportunistic bacterial infection.
  • compositions of the invention are administered in combination with an antiviral agent.
  • Antiviral agents that may be administered with the compositions ofthe invention include, but are not limited to, acyclovir, ribavirin, amantadine, and remantidine.
  • compositions of the invention are administered in combination with an antibiotic agent.
  • Antibiotic agents that may be administered with the compositions ofthe invention include, but are not limited to, amoxicillin, aminoglycosides, beta-lactam (glycopeptide), beta-lactamases, Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin, ciprofloxacin, erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins, quinolones, rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim, trimethoprim-sulfamthoxazole, and vancomycin.
  • nonspecific immunosuppressive agents that may be administered in combination with the compositions ofthe invention include, but are not limited to, steroids, cyclosporine, cyclosporine analogs cyclophosphamide, cyclophosphamide IV, methylprednisolone, prednisolone, azathioprine, FK-506, 15-deoxyspergualin, and other immunosuppressive agents that act by suppressing the function of responding T cells.
  • steroids cyclosporine
  • cyclosporine analogs cyclophosphamide cyclophosphamide IV
  • methylprednisolone prednisolone
  • azathioprine FK-506, 15-deoxyspergualin
  • other immunosuppressive agents that act by suppressing the function of responding T cells.
  • compositions ofthe invention are administered in combination with immunosuppressants.
  • Immunosuppressants preparations that may be administered with the compositions ofthe invention include, but are not l i m i t e d t o , O R T H O C L O N E TM ( O K T 3 ) , SANDIMMUNETM/NEORALTM/SANGDYATM (cyclosporin), PROGRAFTM
  • immunosuppressants may be used to prevent rejection of organ or bone marrow transplantation.
  • compositions of the invention are administered in combination with steroid therapy.
  • Steroids that may be administered in combination with the compositions ofthe invention include, but are not limited to, oral corticosteroids, prednisone, and methylprednisolone (e.g. , IV methylprednisolone).
  • compositions ofthe invention are administered in combination with prednisone.
  • the compositions ofthe invention are administered in combination with prednisone and an immunosuppressive agent.
  • Immunosuppressive agents that may be administered with the compositions ofthe invention and prednisone are those described herein, and include, but are not limited to, azathioprine, cylophosphamide, and cyclophosphamide IV.
  • compositions of the invention are administered in combination with methylprednisolone.
  • the compositions ofthe invention are administered in combination with methylprednisolone and an immunosuppressive agent.
  • Immunosuppressive agents that may be administered with the compositions of the invention and methylprednisolone are those described herein, and include, but are not limited to, azathioprine, cylophosphamide. and cyclophosphamide IV.
  • compositions of the invention are administered in combination with an antimalarial.
  • Antimalarials that may be administered with the compositions ofthe invention include, but are not limited to, hydroxychloroquine, chloroquine, and/or quinacrine. -154-
  • compositions of the invention are administered in combination with an NSAID.
  • compositions of the invention are administered in combination with one, two, three, four, five, ten, or more ofthe following drugs: NRD-101 (Hoechst Marion Roussel), diclofenac (Dimethaid), oxaprozin potassium (Monsanto), mecasermin (Chiron), T-614 (Toyama), pemetrexed disodium (Eli Lilly), atreleuton (Abbott), valdecoxib (Monsanto), kornac (Byk Gulden), campath, AGM-1470 (Takeda), CDP-571 (Celltech Chiroscience), CM-101 (CarboMed), ML-3000 (Merckle), CB-2431 (KS Biomedix), CBF-BS2 (KS Biomedix), IL-lRa gene therapy (Valentis), JTE-522
  • compositions of the invention are administered in combination with one, two. three, four, five or more of the following drugs: methotrexate, sulfasalazine, sodium aurothiomalate, auranofin, cyclosporine, penicillamine, azathioprine, an antimalarial drug (e.g. , as described herein), cyclophosphamide, chlorambucil, gold, ENBRELTM (Etanercept), anti-TNF antibody, and prednisolone.
  • drugs methotrexate, sulfasalazine, sodium aurothiomalate, auranofin, cyclosporine, penicillamine, azathioprine, an antimalarial drug (e.g. , as described herein), cyclophosphamide, chlorambucil, gold, ENBRELTM (Etanercept), anti-TNF antibody, and prednisolone.
  • compositions of the invention are administered in combination with an antimalarial, methotrexate, anti-TNF antibody, ENBRELTM and/or suflasalazine.
  • the compositions ofthe invention are administered in combination with methotrexate.
  • the compositions ofthe invention are administered in combination with anti-TNF antibody.
  • the compositions of the invention are administered in combination with methotrexate and anti-TNF antibody.
  • the compositions of the invention are -155-
  • compositions ofthe invention are administered in combination with suflasalazine.
  • the compositions ofthe invention are administered in combination with methotrexate, anti-TNF antibody, and suflasalazine.
  • the compositions of the invention are administered in combination ENBRELTM.
  • the compositions ofthe invention are administered in combination with ENBRELTM and methotrexate.
  • the compositions of the invention are administered in combination with ENBRELTM, methotrexate and suflasalazine.
  • compositions of the invention are administered in combination with ENBRELTM, methotrexate and suflasalazine.
  • one or more antimalarials is combined with one of the above-recited combinations.
  • compositions ofthe invention are administered in combination with an antimalarial (e.g., hydroxychloroquine), ENBRELTM, methotrexate and suflasalazine.
  • an antimalarial e.g., hydroxychloroquine
  • sulfasalazine e.g., sulfasalazine
  • anti-TNF antibody e.g., sulfasalazine
  • methotrexate e.g., hydroxychloroquine
  • compositions of the invention are administered alone or in combination with one or more intravenous immune globulin preparations.
  • Intravenous immune globulin preparations that may be administered with the compositions ofthe invention include, but are not limited to, GAMMARTM, IVEEGAMTM, SANDOGLOBULINTM, GAMMAGARD S/DTM, and GAMIMUNETM.
  • compositions of the invention are administered in combination with intravenous immune globulin preparations in transplantation therapy (e.g., bone marrow transplant).
  • transplantation therapy e.g., bone marrow transplant.
  • the compositions of the invention are administered alone or in combination with an anti-inflammatory agent.
  • Anti-inflammatory agents that may be administered with the compositions ofthe invention include, but are not limited to, glucocorticoids and the nonsteroidal anti-inflammatories, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid -156-
  • compositions ofthe invention are administered in combination with a chemotherapeutic agent.
  • Chemotherapeutic agents that may be administered with the compositions ofthe invention include, but are not limited to, antibiotic derivatives (e.g. , doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g. , tamoxifen); antimetabolites (e.g.
  • cytotoxic agents e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cis-platin, and vincristine sulfate
  • hormones e.g.
  • medroxyprogesterone estramustine phosphate sodium, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and testolactone
  • nitrogen mustard derivatives e.g., mephalen, chorambucil, mechlorethamine (nitrogen mustard) and thiotepa
  • steroids and combinations e. g.
  • bethamethasone sodium phosphate e.g., bethamethasone sodium phosphate
  • others e.g., dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide.
  • compositions ofthe invention are administered in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or any combination of the components of CHOP.
  • CHOP cyclophosphamide, doxorubicin, vincristine, and prednisone
  • compositions ofthe invention are administered in combination with Rituximab.
  • compositions of the invention are administered with Rituxmab and CHOP, or Rituxmab and any combination ofthe components of CHOP.
  • the compositions of the invention are administered in combination with cytokines. Cytokines that may be administered -157-
  • compositions ofthe invention include, but are not limited to, GM-CSF, G-CSF, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha, and TNF-beta.
  • compositions ofthe invention may be administered with any interleukin, including, but not limited to, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4,
  • compositions ofthe invention are administered in combination with IL4 and IL 10.
  • the compositions of the invention are administered with a chemokine.
  • the compositions ofthe invention are administered with chemokine beta-8, chemokine beta-1 , and/or macrophage inflammatory protein-4.
  • the compositions ofthe invention are administered with chemokine beta-8.
  • compositions of the invention are administered in combination with an IL-4 antagonist.
  • IL-4 antagonists that may be administered with the compositions ofthe invention include, but are not limited to: soluble IL-4 receptor polypeptides. multimeric forms of soluble IL-4 receptor polypeptides; anti-IL-4 receptor antibodies that bind the IL-4 receptor without transducing the biological signal elicited by IL-4, anti-IL4 antibodies that block binding of IL-4 to one or more IL-4 receptors, and muteins of IL-4 that bind IL-4 receptors but do not transduce the biological signal elicited by IL-4.
  • the antibodies employed according to this method are monoclonal antibodies
  • compositions of the invention are administered in combination with hematopoietic growth factors.
  • Hematopoietic growth factors that may be administered with the compositions ofthe invention include, but are not limited to, LEUKINETM (SARGRAMOSTIMTM) and NEUPOGENTM (FILGRASTIMTM).
  • compositions of the invention are administered in combination with fibroblast growth factors.
  • Fibroblast growth factors that may be administered with the compositions ofthe invention include, -158-
  • FGF-1 FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-1 1, FGF-12, FGF-13, FGF-14, and FGF-15.
  • compositions ofthe invention may be administered alone or in combination with other therapeutic regimens, including, but not limited to, radiation therapy.
  • Such combinatorial therapy may be administered sequentially and/or concomitantly.
  • the present invention is further directed to antibody-based therapies which involve administering antibodies ofthe invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more ofthe described disorders.
  • Therapeutic compounds ofthe invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof as described herein).
  • the antibodies ofthe invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide ofthe invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein such as, for example autoimmune diseases, disorders, or conditions associated with such diseases or disorders (including, but not limited to, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (e.g,, IgA nephropathy), Multiple Sclerosis, Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Purpura (e.g.,
  • Goodpasture's syndrome Pemphigus, Receptor autoimmunities such as, for example, Graves' Disease, Myasthenia Gravis, and insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, rheumatoid arthritis, schleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis/dermatomyositis, pernicious anemia, idiopathic Addison's disease, infertility, glomerulonephritis such as primary glomerulonephritis and IgA nephropathy, bullous pemphigoid, Sjogren's syndrome, diabetes millitus, and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis), chronic active hepatitis, primary biliary cirrhosis, other endocrine gland failure, vitiligo, vasculitis, post-MI, cardiotomy syndrome,
  • antibodies ofthe invention are used to treat, inhibit, prognose, diagnose or prevent systemic lupus erythematosis.
  • the treatment and/or prevention of diseases and disorders associated with aberrant expression and/or activity of a polypeptide ofthe invention includes, but is not limited to, alleviating symptoms associated with those diseases and disorders.
  • Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
  • a summary ofthe ways in which the antibodies ofthe present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention 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 purposes without undue experimentation. -160-
  • the antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g. , IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.
  • lymphokines or hematopoietic growth factors such as, e.g. , IL-2, IL-3 and IL-7
  • the antibodies of the invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that ofthe patient is preferred.
  • human antibodies, fragments derivatives, analogs, or nucleic acids are administered to a human patient for therapy or prophylaxis.
  • polypeptides or polynucleotides of the present invention It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragments thereof of the present invention.
  • Such antibodies, fragments, or regions will preferably have an affinity for polynucleotides or polypeptides, including fragments thereof.
  • Preferred binding affinities include those with a dissociation constant or Kd less than 5 X 10 "6 M, 10 "6 M, 5 X 10 "7 M, 10 “7 M, 5 X 10 "8 M, 10 “8 M, 5 X 10 "8 M, 10 "9 M, 5 X lO "10 M, lO "10 M, 5 X 10 " M, 10 ' “ M, 5 X 10 '12 M, 10 "12 M, 5 X 10 13 M, 10 " ' 3 M, 5 X 10 14 M, 10 "14 M, 5 X lO "15 M, and 10 "15 M.
  • the invention provides a method of delivering compositions containing the polypeptides of the invention (e.g., compositions containing Endokine alpha polypeptides or anti-Endokine alpha antibodies associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs) to targeted cells, expressing the membrane-bound form of Endokine alpha on their surface, or intratley, an Endokine alpha receptor (e.g.. TRl 1 ) on their surface.
  • heterologous polypeptides may be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions.
  • the invention provides a method for the specific delivery of compositions ofthe invention to cells by administering polypeptides ofthe invention (e.g. , Endokine alpha or anti-Endokine alpha antibodies) that are associated with heterologous polypeptides or nucleic acids.
  • polypeptides ofthe invention e.g. , Endokine alpha or anti-Endokine alpha antibodies
  • the invention provides a method for delivering a therapeutic protein into the targeted cell.
  • the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.
  • a single stranded nucleic acid e.g., antisense or ribozymes
  • double stranded nucleic acid e.g., DNA that can integrate into the cell'
  • the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides ofthe invention (e.g. , Endokine alpha polypeptides or anti-Endokine alpha antibodies) in association with toxins or cytotoxic prodrugs.
  • polypeptides ofthe invention e.g. , Endokine alpha polypeptides or anti-Endokine alpha antibodies
  • the invention provides a method for the specific destruction of cells expressing TRl 1 on their surface (e.g., activated T cells, and/or T cell and/or B cell related leukemias or lymphomas) by administering Endokine alpha polypeptides in association with toxins or cytotoxic prodrugs.
  • the invention provides a method for the specific destruction of cells expressing the membrane-bound form of Endokine alpha on their surface (e.g., endothelial cells) by administering anti-Endokine alpha antibodies in association with toxins or cytotoxic prodrugs.
  • toxin is meant compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, cytotoxins (cytotoxic agents), or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death.
  • Toxins that may be used according to the methods of the invention include, but are not limited to. radioisotopes known in the art, compounds such as. for example, antibodies (or complement fixing containing -162-
  • Toxin also includes a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e. g.
  • alpha-emitters such as, for example, 2l3 Bi, or other radioisotopes such as, for example, ,03 Pd, ,33 Xe, ,3 T, 68 Ge, 57 Co, 65 Zn, 85 Sr, 32 P, 35 S, 90 Y, ,53 Sm, 153 Gd, 169 Yb, 5 l Cr, 54 Mn, 75 Se, " 3 Sn, 90 Yttrium, 117 Tin, , 86 Rhenium, ,66 Holmium, and 188 Rhenium; luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
  • radioisotopes such as, for example, ,03 Pd, ,33 Xe, ,3 T, 68 Ge, 57 Co, 65 Zn, 85 Sr, 32 P, 35 S, 90 Y, ,53 Sm, 153 Gd, 169 Yb, 5
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.
  • Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine. vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione. mitoxantrone, mithramycin, actinomycin D,
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil. melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide.
  • antimetabolites e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine
  • alkylating agents e.g., mechlorethamine, thioepa chlorambucil. melphalan, carmustine (BSNU) and lomustine (CCNU)
  • BSNU carmustine
  • CCNU lomustine
  • busulfan dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti -mito tic agents (e.g. , vincristine and vinblastine).
  • II dichlorodiamine platinum
  • DDP cisplatin
  • anthracyclines e.g., daunorubicin (formerly daunomycin) and doxorubicin
  • antibiotics e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)
  • cytotoxic prodrug is meant a non-toxic compound that is converted by an enzyme, normally present in the cell, into a cytotoxic compound.
  • Cytotoxic prodrugs that may be used according to the methods ofthe invention include, but are not limited to, glutamyl derivatives of benzoic acid mustard alkylating agent, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubisin, and phenoxyacetamide derivatives of doxorubicin.
  • compositions of the invention may be administered to an animal (including, but not limited to, those listed above, and also including transgenic animals) incapable of producing functional endogenous antibody molecules or having an otherwise compromised endogenous immune system, but which is capable of producing human immunoglobulin molecules by means of a reconstituted or partially reconstituted immune system from another animal (see, e.g., published PCT Application Nos. WO 98/24893, WO 96/34096, WO 96/33735, and WO 91/10741).
  • Compositions ofthe invention include, but are not limited to, endokine-alpha polypeptides and polynucleotides and agonists and antagonists thereof, antibodies, anti-antibodies, etc.
  • compositions described herein may be used as a vaccine adjuvant that enhances immune responsiveness to specific antigen.
  • the vaccine adjuvant is an endokine alpha polypeptide described herein.
  • the vaccine adjuvant is an endokine alpha polynucleotide described herein (i.e., the endokine alpha polynucleotide is a genetic vaccine adjuvant).
  • endokine alpha polynucleotides may be administered using techniques known in the art, including but not limited to, liposomal delivery, recombinant vector delivery, injection of naked DNA, and gene gun delivery.
  • compositions described herein may also be an adjuvant used to enhance tumor-specific immune responses.
  • Anti-viral immune responses that may be enhanced using the compositions of the invention as an adjuvant, include, but are not limited to, virus and virus associated diseases or symptoms described herein or otherwise known in the art.
  • compositions of the invention are used as an -164-
  • compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: HIV/AIDS,
  • compositions ofthe invention are used as an adjuvant to enhance an immune response to the HIV gpl20 antigen.
  • compositions ofthe invention include bacteria or fungus and bacteria or fungus associated diseases or symptoms described herein or otherwise known in the art.
  • the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: tetanus, Diphtheria, botulism, and meningitis type B.
  • the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: Vibrio cholerae, Mycobacterium leprae, Salmonella typhi,
  • compositions of the invention include parasite and parasite associated diseases or symptoms described herein or otherwise known in the art.
  • the compositions of the invention are used as an adjuvant to enhance an immune response to a parasite.
  • the compositions ofthe invention are used as an adjuvant to enhance an immune response to Plasmodium (malaria). -165-
  • compositions ofthe invention may be used as a stimulator of B or T cell responsiveness to pathogens.
  • compositions ofthe invention may be used as an agent that elevates the immune status of an individual prior to their receipt of immunosuppressive therapies; as an agent to induce higher affinity antibodies; as an agent to increase serum immunoglobulin concentrations; as an agent to accelerate recovery of immunocompromised individuals; as an agent to boost immunoresponsiveness among aged populations; and as an immune system enhancer prior to, during, or after bone marrow transplant and/or other transplants (e.g., allogeneic or xenogeneic organ transplantation).
  • compositions of the invention may be administered prior to, concomitant with, and/or after transplantation.
  • compositions ofthe invention are administered after transplantation, prior to the beginning of recovery of T-cell populations.
  • compositions of the invention are first administered after transplantation after the beginning of recovery of T cell populations, but prior to full recovery of B cell populations.
  • compositions of the invention may be used as an agent to boost immunoresponsiveness among B cell and/or T cell immunodeficient individuals, such as, for example, an individual who has undergone a partial or complete splenectomy.
  • B cell immunodeficiencies that may be ameliorated or treated by administering the endokine alpha polypeptides or polynucleotides ofthe invention, or agonists thereof, include, but are not limited to, severe combined immunodeficiency (SCID)-X linked, SCID-autosomal, adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton's disease, congenital agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia
  • Wiskott-Aldrich Syndrome WAS
  • non X-linked immunodeficiency with hyper IgM selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), antibody deficiency with normal or elevated Igs, immunodeficiency with thymoma, Ig heavy chain deletions, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), selective IgM immunodeficiency, recessive agammaglobulinemia (Swiss type), reticular dysgenesis, neonatal neutropenia, severe congenital leukopenia, thymic alymphoplasia-aplasia or dysplasia with immunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezelof syndrome-combined immunodeficiency with Igs, purine nucleoside phosphorylase deficiency (PNP), M
  • T cell immunodeficiencies that may be ameliorated or treated by administering the endokine alpha polypeptides or polynucleotides ofthe invention, or agonists thereof, include, but are not limited to, DiGeorge anomaly, thymic hypoplasia. chronic mucocutaneous candidiasis, natural killer cell deficiency, idiopathic CD4+ T-lymphocytopenia, and immunodeficiency with predominant T-cell defect, graft versus host disease, graft rejections and inflammation associated with an immuno-deficiency.
  • Additional conditions resulting in an acquired loss of B or T cell function that may be ameliorated or treated by administering the endokine alpha polypeptides or polynucleotides ofthe invention, or agonists thereof, include, but are not limited to, HIV Infection, AIDS, bone marrow transplant, and B cell chronic lymphocytic leukemia (CLL).
  • HIV Infection HIV Infection
  • AIDS bone marrow transplant
  • CLL B cell chronic lymphocytic leukemia
  • compositions of the invention may also be used as an agent to boost immunoresponsiveness among individuals having a temporary immune deficiency.
  • Conditions resulting in a temporary immune deficiency that may be ameliorated or treated by administering the endokine alpha polypeptides or polynucleotides ofthe invention, or agonists thereof, include, but are not limited to, recovery from viral infections (e.g.. influenza), conditions associated with malnutrition, recovery from infectious mononucleosis, or conditions associated with stress, recovery from measles, recovery from blood transfusion, and recovery from surgery.
  • viral infections e.g. influenza
  • endokine alpha polynucleotides, polypeptides, and/or agonists and/or antagonists thereof are used to treat, prevent, and/or diagnose diseases or disorders affecting or conditions associated with any one or more ofthe various mucous membranes ofthe body.
  • Such diseases or disorders include, but are not limited to, for example, mucositis, mucoclasis, mucocolitis, mucocutaneous leishmaniasis (such as, for example, American leishmaniasis, leishmaniasis americana, nasopharyngeal leishmaniasis, and New World leishmaniasis), mucocutaneous lymph node syndrome (for example, Kawasaki disease), mucoenteritis, mucoepidermoid carcinoma, mucoepidermoid tumor, mucoepithelial dysplasia, mucoid adenocarcinoma, mucoid degeneration, myxoid degeneration, myxomatous degeneration, myxomatosis, mucoid medial degeneration (for example, cystic medial necrosis), mucolipidosis (including, for example, mucolipidosis I, mucolipidosis II, mucolipidosis III, and mucolipidosis IV), mucolysis disorders, mucomembranous
  • Hurler's syndrome type IS mucopolysaccharidosis (i.e., Scheie's syndrome or type V mucopolysaccharidosis), type II mucopolysaccharidosis (i.e., Hunter's syndrome), type III mucopolysaccharidosis (i.e., Sanfilippo's syndrome), type IV mucopolysaccharidosis (i.e., Morquio's syndrome), type VI mucopolysaccharidosis (i.e., Maroteaux-Lamy syndrome), type VII mucopolysaccharidosis (i.e.
  • mucopolysaccharidosis due to beta-glucuronidase deficiency), and mucosulfatidosis mucopolysacchariduria, mucopurulent conjunctivitis, mucopus, mucormycosis (i.e., zygomycosis), mucosal disease (i.e., bovine virus diarrhea), mucous colitis (such as, for example, mucocolitis and myxomembranous colitis), and mucoviscidosis (such as, for example, cystic fibrosis, cystic fibrosis of the pancreas, Clarke-Hadfield syndrome, fibrocystic disease of the pancreas, mucoviscidosis, and viscidosis).
  • endokine alpha polynucleotides, polypeptides, and/or agonists and/or antagonists thereof are used to treat, prevent, and/or diagnose mucositis, especially as associated with chemotherapy.
  • Endokine alpha polynucleotides or polypeptides of the invention may be used to diagnose, prognose, treat or prevent one or more of the following diseases or disorders, or conditions associated therewith: primary immuodeficiencies, immune-mediated thrombocytopenia, Kawasaki syndrome, bone marrow transplant (e.g., recent bone marrow transplant in adults or children), chronic B-cell lymphocytic leukemia, HIV infection (e.g., adult or pediatric HIV infection), chronic inflammatory demyelinating polyneuropathy, and post-transfusion purpura.
  • diseases or disorders, or conditions associated therewith include: primary immuodeficiencies, immune-mediated thrombocytopenia, Kawasaki syndrome, bone marrow transplant (e.g., recent bone marrow transplant in adults or children), chronic B-cell lymphocytic leukemia, HIV infection (e.g., adult or pediatric HIV infection), chronic inflammatory demyelinating polyneuropathy, and post-transfusion purpura.
  • Endokine alpha polynucleotides or polypeptides of the invention may be used to diagnose, prognose, treat or prevent one or more of the following diseases, disorders, or conditions associated therewith, Guillain-Barre syndrome, anemia (e.g..
  • anemia associated with parvovirus B 19 patients with stable mutliple myeloma who are at high risk for infection (e.g., recurrent infection), autoimmune hemolytic anemia (e.g., warm-type autoimmune hemolytic anemia), thrombocytopenia (e.g, neonatal thrombocytopenia), and immune-mediated neutropenia, transplantation (e.g, cytamegalovirus (CMV)-negative recipients of CMV-positive organs), hypogammaglobulinemia (e.g. , hypogammaglobulinemic neonates with risk factor for infection or morbidity), epilepsy (e.g. intractable epilepsy), systemic vasculitic syndromes, myasthenia gravis (e.g, decompensation in myasthenia gravis), dermatomyositis, and polymyositis.
  • CMV cytamegalovirus
  • hypogammaglobulinemia e.g.
  • Endokine alpha polynucleotides or polypeptides ofthe invention and/or agonists and/or antagonists thereof may be used to treat, prevent, and/or diagnose various immune system-related disorders and/or conditions associated with these disorders, in mammals, preferably humans. Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of endokine alpha polynucleotides or polypeptides ofthe invention and/or agonists and/or antagonists thereof that can inhibit an immune response, particularly the proliferation of B cells and/or the production of immunoglobulins, may be an -169-
  • endokine alpha antagonists of the invention are used to treat, prevent, and/or diagnose an autoimmune disorder.
  • Autoimmune disorders and conditions associated with these disorders that may be treated, prevented, and/or diagnosed with the endokine alpha polynucleotides, polypeptides, and/or antagonists of the invention (e.g,.
  • anti-endokine alpha antibodies include, but are not limited to, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (e.g, IgA nephropathy), Multiple Sclerosis, Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura), Reiter's Disease, Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation,
  • autoimmune hemolytic anemia include, but are not limited to, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, autoi
  • autoimmune disorders that are highly probable
  • autoimmune thyroiditis i.e., IL-12, IL-12
  • Hashimoto's thyroiditis (often characterized, e.g. , by cell-mediated and humoral thyroid cytotoxicity), systemic lupus erhythematosus (often characterized, e.g., by circulating and locally generated immune complexes), Goodpasture's syndrome (often characterized, e.g., by anti-basement membrane antibodies), Pemphigus (often characterized, e.g., by epidermal acantholytic antibodies), Receptor autoimmunities such as, for example, Graves' Disease (often characterized, e.g.
  • TSH receptor antibodies Myasthenia Gravis (often characterized, e.g., by acetylcholine receptor antibodies), and insulin resistance (often characterized, e.g. , by insulin receptor antibodies), autoimmune hemolytic anemia (often characterized, e.g., by phagocytosis of antibody-sensitized RBCs). and -170-
  • autoimmune thrombocytopenic purpura (often characterized, e.g., by phagocytosis of antibody-sensitized platelets.
  • autoimmune disorders that may be treated, prevented, and/or diagnosed with the compositions ofthe invention include, but are not limited to, rheumatoid arthritis (often characterized, e.g. , by immune complexes in joints), schleroderma with anti-collagen antibodies (often characterized, e.g. , by nucleolar and other nuclear antibodies), mixed connective tissue disease (often characterized, e.g., by antibodies to extractable nuclear antigens (e.g., ribonucleoprotein)), polymyositis/dermatomyositis (often characterized, e.g.
  • pernicious anemia often characterized, e.g., by antiparietal cell, microsomes, and intrinsic factor antibodies
  • idiopathic Addison's disease often characterized, e.g., by humoral and cell-mediated adrenal cytotoxicity
  • infertility often characterized, e.g. , by antispermatozoal antibodies
  • glomerulonephritis often characterized, e.g.
  • glomerular basement membrane antibodies or immune complexes such as primary glomerulonephritis and IgA nephropathy, bullous pemphigoid (often characterized, e.g., by IgG and complement in basement membrane), Sjogren's syndrome (often characterized, e.g., by multiple tissue antibodies, and/or a specific nonhistone ANA (SS-B)), diabetes millitus (often characterized, e.g. , by cell -mediated and humoral islet cell antibodies), and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis) (often characterized, e.g., by beta-adrenergic receptor antibodies).
  • SS-B specific nonhistone ANA
  • diabetes millitus often characterized, e.g. , by cell -mediated and humoral islet cell antibodies
  • adrenergic drug resistance including adrenergic drug resistance with asthma or cystic fibrosis

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Abstract

L'invention concerne un nouveau membre de la famille de cytokines de facteur de nécrose tumorale (TNF). Cette invention concerne notamment des molécules d'acides nucléiques isolées codant la protéine alpha endokine, ainsi que des polypeptides endokine alpha, des vecteurs, des cellules hôtes et des méthodes de recombinaison permettant de les produire. Ladite invention concerne aussi des méthodes diagnostiques et thérapeutiques relatives aux troubles liés à la famille TNF.
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EP2357192A1 (fr) 2011-08-17
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