CA2246578A1 - Novel human myeloid terminal differentiation response gene - Google Patents

Abstract

The present invention provides polynucleotide and amino acid sequences which encode and identify a novel human myeloid terminal differentiation response gene designated MYD118. The present invention also provides for MYD118 antisense molecules. The invention further provides genetically engineered expression vectors and host cells for the production of purified MYD118 polypeptide; antibodies, antagonists and inhibitors of MYD118 polypeptide; and pharmaceutical compositions and methods of treatment based on polynucleotide sequences encoding MYD118 polypeptide. The invention specifically provides for use of the MYD118 polynucleotide sequences as a diagnostic composition for the detection of myeloproliferative diseases and leukemias. The invention also relates to therapeutic methods and compositions based upon the nucleotide and amino acid sequences for MYD118. The invention further provides antibodies which specifically bind to MYD118.

Description

CA 02246~78 1998-08-14 WO 97/30157 PCTMS97/1~2458 NOVEL EIUMAN MYELOID TERMINAL DIFFERENTIATION RESPONSE GI~NE

RELATED APPLICATIONS
The present invention is a continl~tion in part application of United States Application s Serial Number 08/602,208 filed February 15, 1996, which is incorporated herein by reference in its entirety.

TECHNICAL FIELD
The present invention relates to nucleic acid and amino acid sequences of a novel human 0 myeloid terminal dirr~rellLiation response gene found in cDNA libraries made from human fetal liver/spleen tissue and non-adherent peripheral blood mononuclear cells and to the use of these sequences in the diagnosis, study and treatment of disease.

BACKGROUND ART
Cell numbers are regulated by a balance among proliferation, growth arrest and programmed cell death (apoptosis; Fornace AJ et al. 1992 Ann New York Acad Sci 663: 139-153). Genes induced by various growth arrest and apoptotic stimuli are the tumor ~L~p~ or gene p53, myeloid differentiation primary response genes (MyD genes), and growth arrest and DNA damage inducible genes (GADD genes, ~elvakumaran M et al. 1994 Mol Cell Biol20 14:2352-60).
Animal cells respond to differentiation signals which turn on or offthe appropriate genes resulting in conversion of proliferating, undifferentiated cells into nonproliferating, highly specialized, differentiated cells. An example of this process is the differentiation of myeloid precursor cells into mature granulocytes and macrophages. Blocks in the differentiation process 2s appear to be a major step in tumor progression, and lesions in genes involved in terrninal differentiation contribute to the development of m~ n~nt tumors (Liebermann MA et al. 1994 Stem Cells 12:352-69) .
A Lieberrnann et al. suggest that MyD genes function as positive regulators of terminal hematopoietic cell differentiation, which is associated with inhibition of cell growth and 30 apoptosis. Selvakumaran et al. supra provide evidence that MyD family member, murine MYD118, described as a termin~l differentiation response gene, is expressed in MlD+ myeloid precursor cells following induction of terrnin~l differentiation and growth arrest by I~6 and has SUBSTITUTE SHEET (RULE 26) CA 02246~78 1998-08-14 W O 97130157 PCT~US97/02458 -been shown to be a positive regulator of apoptosis ind~lce-l by TGF131. Additionally, leucine zipper transcription factors of the fos/jun family have been identified as MyD genes, specifically MyD21, MyD42 and MyD637 and function as positive regulators of hematopoietic cell differentiation, increasing the differentiation of myeloblastic leukemia cells in vitro and reducin~
5 the aggressiveness of the leukemic phenotype in nude mice. Liebermann et al. supra suggest that lesions in the MyD genes of the fos/jun family that affect expression or function of the genes contribute to development of lellkemi~
The cDNA sequence and cle~11lce~1 amino acid sequence of murine MYD118 is disclosed in Abdollahi A et al. (1991 Oncogene 6:165-17) who indicate that the cDNA nucleotide sequence 10 of murine MYD 118 predicts a protein of 160 amino acids, which does not contain protein secretory signals, tr~n~memhrane domains or known protein-DNA binding motifs, but does appear to contain a protein kinase phosphorylation site at position 204, two casein kinase II
phosphorylation sites at positions 215 and 231 and several AT3 motifs in its 3' untr~n~ted region. Abdollahi et al. observed detectable levels of MYD 118 RNA in myeloid precursor 15 enriched murine bone marrow, but not in several other non-myeloid murine tissues, such as liver or brain. Abdollahi et al. also observed that MYD118 expression was in~ ed in the absence of protein synthe~i~, following stimulation of MID+ cells by IL-1, LPS and le~lkcmi~ inhibitory factor (LIF).
The amino acid sequence for murine MYD118 is 75% similar (57% identical) to the 20 amino acid sequence for murine GADD gene, GADD45, which is regulated in part by the tumor suppressor gene pS3 (Zhan Q et al. (1994) Cancer Res 54: 2755-60, Carrier F et al. (1994) J Biol Chem 269:32672-32677). GADD and MYD118 are two separate but closely related genes and act synergistically to suppress growth of hematopoietic cell lineages.
Various portions of the nucleotide sequence encoding human MYD118 have been 2s disclosed in GenBank 92 in cDNA libraries made from tissue from hippocampus (M77995), retina (H84533 and H83991), olfactory epithelium (H71592), human fetal lung (D310470 and D31559), breast (H44355, R55161 and R82994), placenta (R24009, R63425, R21918 and R22497), human white blood cells (T33963), human brain (T35368), human pancreas (T29941), liver (T40088), prostate gland (T35225) and lung (T35563) . The complete nucleotide sequence 30 encoding human MYD118 has not been disclosed.
Myeloproliferative diseases and lellk~mi~ are neoplasms of the hematopoietic stem cell and include acute Iymphocytic leuk~mi~ (ALL), acute myelogenous leukemia (AML), chronic SUBSTITUTE SHEET (RULE 26) CA 02246~78 1998-08-14 WO 97/30157 PCT~US~7~0Z~8 lymphoc~tic leukemia (CLL), hairy cell lellkemi~ a~d chronic myelogenous leukemia (CML);
polycythemia vera (PV); agnogen;c myeloid metaplasia with myelofibrosis (AMM/MF); and essential thrombocytosis (ET). Research suggests that the myeloproliferative diseases arise as clonal expansions of a single transformed stem cell and that all of the myeloid cells of the blood s are derived fiom the neoplastic clone. In lellkemi~ leukemic cells proliferate primarily in the bone marrow and lymphoid tissues and are characterized according to the cell type involved (myeloid or lymphoid). Acute leukemia is characterized by proliferation of imm~h-re myeloid or lymphoid cells. CLL is a hematologic neoplasm characterized by the accumulation of mature-appearing Iymphocytes in the peripheral blood associated with infiltration of the bone marrow.
lo Hairy cell leukemia is characterized by peripheral blood cytopenias, splenomegaly and m~ n~nt cells in the blood and bone marrow. CML is characterized by marked splenomegaly and the production of increased numbers of granulocytes, particularly neutrophils, in the marrow and blood. PV is characterized by splenomegaly and an increased production of all myeloid element~, but is dominzltecl by an elevated hemoglobin concentration. AMM/MF is characterized 15 by the tendency of the neoplastic stem cells to lodge and grow in multiple sites outside the marrow, progressive splenomegaly, the gradual replacement of marrow element~ by fibrosis, and variable changes in the number of granulocytes and platelets. E~T is characterized by an elevated platelet count and represents the overproduction of platelets in the absence of a recognizable stimulus. In cultures of bone marrow cells from individuals subject to ET, colonies of 20 megakaryocytes from megakaryocyte progenitors for~n in the absence of added stimulus, whereas such colonies do not occur with marrow cell cultures from normal individuals. (Braunwald et al.
(Eds, 1 987) H~rrison's Principles of Internal Medicine. 11th ed. McGraw-Hill, New York NY).

DISCLOSURE OF THE INVENTION
2s The present invention relates to human MYD 1 18 whose nucleic acid sequence has been i~lentified among the polynucleotide sequences of cDNA libraries made from human fetal liver-spleen tissue and non-adherent peripheral blood mononuclear cells and to the use of the nucleic ~ acid and amino acid sequences of MYD1 18 in the study, diagnosis and treatment of disease states related to proliferation, specifically myeloprolir~ld~ive diseases and le~lk~
The nucleic acid sequence of mydl 18 (SEQ ID NO: 1 ) and the protein it encodes,MYDl 18 (SEQ ID NO:2) is disclosed herein in Figure 1. The amino acid homology among human MYD118, murine MYDl 18 (SEQ ID NO:3) and human GADD 45 (SEQ ID NO:4) is SUBSTITUTE SHEET (RULE 26) CA 02246~78 1998-08-14 shown in Figure 2.
The present invention is based in part on the amino acid homology that human MYD 1 l 8 shares with murine MYDl 18 and the ability of murine MYD1 18 and other MYD family members to stim~ te terminal differentiation of hematopoietic cells~ to arrest cell growth and to 5 modulate the leulcemic phenotype in vivo. The present invention is also based in part on the presence of nucleic acid sequences encoding MYD1 18 in a cDNA library made from fetal liver/spleen tissue and non-adherent peripheral blood mononuclear cells, where hematopoietic cells would be expected to be found. Nucleic acid sequences encoding MYD 1 18 are not detected in samples of c~NA libraries made from m~ n~nt hematopoietic cells sources where t;x~le~ion 10 of genes related to cell growth arrest and apoptosis may be absent, aberrant, deleted or expressed at low levels.
Therefore, expression of human MYD 1 18 may be altered, absent or at low levels in individuals subject to myeloproliferative disease and leukemias. Human MYDl 18, and nucleic acid sequences that encode it and oligonucleotides, peptide nucleic acid (PNA), fragments, 1S portions or ~n1i~n~e molecules thereof, provide the basis for diagnostic methods for the early and accurate detection and/or quantitation of MYDl 18 associated with abnormally proliferating hematopoietic cells such as, myeloproliferative diseases and lellkemi~, including but not limited to acute lymphocytic leukemia (ALL~, acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, chronic myelogenous leukemia (CML); polycythemia vera 20 (PV), agnogenic myeloid metaplasia with myelofibrosis (AMM/MF), and essentialthrombocytosis (ET). For example, the nucleotide sequence for mydl l8 disclosed herein, or fr~ment~ thereof, may be used in hybridization assays of biopsied cells or tissues or bodily fluids to diagnose abnormalities in mydl 18 gene expression in individuals subject to or at risk for a myeloproliferative disease or leukemia. Mydl 18 gene expression may be present at low levels, entirely absent or altered in such a disease states. Additionally, there may be a chromosomal aberration, such as a deletion or mutation, present in the region of the gene encoding MYD 1 18 in disease states related to proliferation of hematopoietic cells.
The nucleotide and arnino acid sequences for MYDl 18 may also be used in the diagnosis and treatment of other disease states related to abnormal proliferation of cells associated with 30 severe infl~mm~tion, such as rheumatoid arthritis, psoriasis (characterized by proliferation of epidermal cells) and lymrh~ m~toid granulomatosis (characterized as a lymphoproliferative disease) where it would be desirable to terrnin~lly di~lentiate proliferating cells and arrest their SUBSTITUTE SHEET (RULE 26) CA 02246~78 1998-08-14 WO 97/30157 PCT~US97~024S8 -growth.
According}y, the present invention provides diagnostic compositions and diagnostic tests for the detection of myd 1 18 nucleotide sequences in biological samples. Such a diagnostic test comprises the steps of combining the biological sample with a first nucleotide sequence which 5 comprises an mydl 18 nucleotide sequence, or a non-conserved fragment thereof, under ~ conditions suitable for the formation of a nucleic acid hybridization complex; detecting said hybridization complex, wherein the presence of said complex correlates with the presence of a second nucleotide sequence comprising mydl 18 nucleotide sequences in said biological sample, and comparing the amount of the second nucleotide sequence in said sample with a standard 10 thereby cletermining whether the amount of said second nucleotide sequence varies from said standard, wherein the presence of an abnormal level of said second nucleotide sequence correlates positively with a myeloproliferative disease. An abnorrnal level of nucleotide sequences encoding MYD118 in a biological sample may reflect a chromosomal aberration, such as a nucleic acid deletion or mutation. Accordingly, nucleotide sequences encoding MYD1 18 I S provide the basis for probes which can be used diagnostically to detect chromosomal aberrations such as deletions, mutations or chromosomal translocations in the gene encoding MYD118.
The present invention also provides a diagnostic test for the detection of mydl 18 nucleotide sequences in a biological sample, comprising the steps of combining the biological sample with polyrnerase chain reaction primers under conditions suitable for nucleic acid 20 amplification, wherein said primers comprise non-conserved fragments of the nucleotide sequence of SEQ ID NO:1, detecting amplified nucleotide sequences, and comparing the amount of amplified nucleotide sequences in said biological sample with a standard thereby cletermining whether the amount of said nucleotide sequence varies from said standard, wherein the presence of an abnorrnal level of said nucleotide sequence correlates positively with a myeloproliferative 25 disease.
Additionally, human MYD118 and the nucleic acid sequences that encode it will provide the basis for ph~ elltical compositions ~or the tresltment of myeloproliferative ~liee~ees, such as ALL, AML, CLL, hairy cell leukemia, CML, PV, AMM/MF and ET. For example, nucleotide sequences that encode MYD118 can be ~llmini~tered alone or in combination with 30 nucleotide sequences encoding tumor suppressor genes, such as p53 (known to regulate the expression of related family member GADD45), p 16 and p2 1, through gene therapy techniques to individuals with CML to induce t~rrnin~l differentiation of granulocytes, thereby arresting SUBSTITUTE SHEET (RULE 26) CA 02246~78 1998-08-14 W O 97/301S7 PCTAJSg7/02458 leukemic cell pro~iferation.
~ lternatively, mydl 18 nucleic acid antisense molecules or antagonists of MYDl 18 protein may be used to block the activity of human MYD l 18 in conditions where it would be preferable to block positive regulators of cell growth arrest. For example, MYD 1 18 may be used s alone or in combination with other agents in the ex vivo culturing of hematopoietic stem cells intended for autologous transplant to individuals lacking cells of the hematopoietic lineage such as, for example, individuals subject to HIV infection or individuals who have undergone chemotherapy or radiation therapy.
The present invention also relates, in part, to expression vectors and host cells comprising 0 polynucleotide sequences encoding MYD 1 18 for the in vivo or vitro production of MYD 1 18 protein.
Additionally, the present invention relates to the use of MYDl 18 polypeptides, or fragments or variants thereof, to produce anti-MYr)118 antibodies and to screen for antagonists or inhibitors of MYD 1 18 polypeptides which can be used diagnostically to detect and quantitate 15 MYDl 18 protein levels in disease states related to proliferation.
The present invention further relates to methods of treating individuals subject to myeloproliferative disease comprising :~1mini.ct~ring compositions comprising purified MYD118 polypeptides or variants thereof, to subjects at risk for or having myeloproliferative disease or leukemia.
The present invention also relates to pharm~ce~ltical compositions comprising effective amounts of MYD 1 18 protein or nucleic acid encoding MYD 1 18 for the treatment of myeloproliferative diseases or leukemias.
The present invention also encompasses the use of gene therapy methods for the introduction of nucleotide sequences encoding MYDl 18 into individuals having or at risk for 25 myeloproliferative ~ e~eçc7 or leukemias.
The invention ~urther provides diaignostic assays and kits for the detection of MYD118 in cells and tissues comprising purified MYD1 18 which may be used as a positive control, and anti-MYD 1 18 antibodies. Such antibodies may be used in solution-based, membrane-based, or tissue-based technologies to detect any disease state or condition related to the expression of 30 MYD118 protein or expression of deletions or variants thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

SUBSTITUTE SHEET (RULE 26) WO 97~0I57 PCTAUS97J~2458 -Figure 1 displays the polynucleotide (SEQ ID NO:l)and ~c~uce~l amino acid (SEQ ID
NO:2) sequence for MyD 118. Sequences shown in this Figure were produced using the multisequence alignment program of DNASTAR software (DNASTAR Inc, Madison WI). SEQ
ID NO: 1 also contains untrRn~l~tc~l S' and 3 ' regions of mydl 18.
Figure 2 displays the amino acid alignment of human MYD 118 (SEQ ID NO:2), murine MYD118 (SEQ ID NO:3) and human GADD45 (SEQ ID NO: 4). Boxed residues match the consensus sequence exactly.
Figure 3 displays an analysis of the hydrophobicity and other characteristics of MYD118 based on the predicted amino acid sequence (produced using DNASTAR software~.

MODES FOR CARRYING OUT THE INVENTION
The present invention relates to human MYD 118 and to the use of the nucleic acid and amino acid sequences of MYD 118 in the study, diagnosis and treatment of disease states, specifically myeloRroliferative diseases and le~kerniz~
The present invention is based in part on the amino acid homology that human MYD 118 shares with murine MYD 118 and the ability of murine MYD 118 and other M[YD family members to stimulate terminal differentiation of hematopoietic cells, to arrest cell growth and to reduce a leukemic phenotype in vivo. The present invention is also based in part on the presence of nucleic acid sequences encoding MYD118 in a cDNA library made from fetal liver/spleen 20 tissue and non-adherent peripheral blood mononuclear cells, where hematopoietic cells would be expected to be found. Nucleic acid sequences encoding MYD118 were not detected in randomly selected samples of about 500 to 5700 usable sequences in cDNA libraries made from THP-l cells, the human promonocyte line derived from the peripheral blood of an individual subject to acute monocytic leukemia (ATCC accession TIB 202; INCYTE libraries: THPlNOB01, 25 THPlPEB01, THPlPLB01 and THPlPLB02); U937 cells, made from m~ign~nt cells from the pleural effusion of an individual subject to histiocytic lymphoma (ATCC accession CRL 1593;
Sundstrom C and K Nilsson (1976) Int J Cancer 17:565-577; INCYTE library U937NOT01); T/B
lymphoblasts from a le-~ke~ source (Stratagene (La Jolla, CA), STR 937214; INCYTE library TBLYNOT01); peripheral blood white blood cells from an individual with myelogenous 30 leukemia (INCYTE library AMLBNOT01); and a human mast cell line from an individual subject to mast cell leukemia (INCYTE library HMClNOT01) where expression of genes related to cell growth arrest and apQptosis may be absent or aberrant. Nucleic acid sequences encoding Sl,~.a ~ JTE SHEET (RULE 26) CA 02246~78 1998-08-14 W O 97/30157 PCT~US97/0245 -MYD1 18 were not detected in a cDNA library from peripheral blood granulocytes (INCYTE
library NEUTFMT01); pooled bone marrow samples (INCYTE library BMARNOR02); ataxia telangiectasia fibroblast cell line ~INCYTE libraries: FIBRAGT01, F~BRAGT02 and FIBRANT01); or adult spleen (INCYTE library SPLNNOT0~). MYD118 related family 5 member, GADD45, is known to be absent in individuals subject to ataxia telangiectasia (Zhan et al. (199~) Mol and Cell Biol 14:2361-2371).
Therefore, expression of human MYD 1 18 may be altered, absent or not detected in individuals subject to myeloproliferative disease and leukemias. Additionally, the gene encoding MYDI 18 may be involved in a chromosomal aberration such as a deletion, mutation ie, a point or 10 int~ l mutation, translocation or may contain tri-nucleotide repeats known to be present in chromosomal abnormalities. Human MYDl 18, nucleic acid sequences that encode MYD1 18 and oligonucleotides, peptide nucleic acid (PNA), frzl~mçntc, portions or antisense molecules thereof, provide the basis for diagnostic methods for the early and accurate detection and/or ~uanLilaLion of MYD 1 18 associated with myeloproliferative diseases and leukemias, such as acute 15 lymphocytic leukemi~ (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, chronic myelogenous leukemia (CML); polycythemia vera (PV); agnogenic myeloid metaplasia with myelofibrosis (AMMtMF); and essential thrombocytosis (ET). For example, the nucleotide sequence for mydl 18 disclosed herein, or fragments thereof, may be used in hybridization assays of biopsied cells or tissues to diagnose 20 abnormalities in myd l 18 gene expression in individuals ~vith or at risk for a myeloproliferative disease or leukemias. Mydl 18 gene expression may be present at low levels, entirely absent or altered in such disease states.
Furtherrnore, the nucleic acid sequences disclosed herein may be used in the detection of aberrations, such as mutations and deletions, in the gene encoding MYD1 18. For exarnple, the 2s nucleotide sequences disclosed herein may be used to identify and isolate a genomic sequence for mydl 18. PCR primers can be designed from various portions of the introns and exons of the genomic mydl 18 that will allow detection of aberrations in the genomic sequence.
Additionally, human MYDl 18 and the nucleic acid sequences that encode it will provide the basis for pharmaceutical compositions for the treatment of myeloproliferative f1i~ç~es, such 30 as ALL, AML, CLL, hairy cell leul~emia, CML, PV, AMM/MF and ET. For example, nucleic acid sequences that encode MYDl 18 can be ~ mini~t~red to individuals subject to CML or AMM to induce tçrmin~l dirre~ iation of granulocytes, thereby arresting cell proliferation.

SUBSTITUTE SHEET (RULE 26) _ CA 02246~78 1998-08-14 W O97/30157 PCT~US97/024~8 ~lminictration of MYD118 or nucleic acid sequences that encode it may alleviate the symptoms associated with myeloproliferative diseases and leukemias, such as anemia, fatigue, splenomegaly, hyperrnetabolism and thrombohemorrhagic complications.
Alternatively, mydl 18 nucleic acid antisense molecules or antagonists of MYDl 18 s protein may be used to block the activity of human MYD118 in conditions where it would be ~ preferably to block positive regulators of cell growth arrest. For example, MYDl 18 may be used alone or in combination with other agents in the ex vivo culturing of hematopoietic stem cells intended for autologous transplant to HIV infected individuals or individuals who have undergone chemotherapy or radiation therapy.
o The present invention also relates, in part, to expression vectors and host cells comprising polynucleotide sequences encoding MYD 1 18 for the ~gy~yQ or in vitro production of MYD 1 18 protein.
"Nucleic acid sequence" as used herein refers to an oligonucleotide, nucleotide or polynucleotide sequence, and fra~ment~ or portions thereof, and to DNA or RNA of genomic or 15 synthetic origin which may be double-stranded or single-stranded whether reprcsçntin~ the sense or ~nti~n~e strand. As used herein "amino acid sequence" refers to peptide or protein sequences or portions thereof. As used herein, lower case mydl 18 refers to a nucleic acid sequence whereas upper case MYD 1 18 refers to a protein sequence. As used herein, peptide nucleic acid (PNA) refers to a class of informational molecules that have a neutral 'peptide like" backbone combined 20 with nucleotides that allow hybr;dization to complement~ry DNA or RNA with higher affinity and specificity than colle:j~ollding oligonucleotides (PerSeptive Biosystems, Marlborough MA).
As used herein, MYDl 18 refers to MYD1 18 from bovine, ovine, porcine, equine and preferably human, in naturally occurring or in variant form, or from any source, whether natural, synthetic, semi-synthetic or recombinant.
2s As used herein, "naturally occurring" refers to an MYDl 18 with an amino acid sequence found in nature, and "biologically active" refers to an MYDl 18 having structural, regulatory or biochemical functions ofthe naturally occurring MYDl 18. Likewise, ''immllnological activity"
~ is defined as the capability of the natural, recombinant or synthetic MYD 1 18 or any oligopeptide thereof, to induce a specific immllne response in a~p~ ate ~nim~l~ or cells and to bind with 30 specific antibodies.
The term "derivative" as used herein refers to the chemical modification of MYDl 18.
Illustrative of such modifications would be replacement of hydrogen by an alkyl, acyl, or amino SU..~ ~ ITE SHEET (RULE 263 CA 02246~78 l998-08-l4 W O 97/30157 PCT~US97/02458 group. An MYD~ 18 polypeptide derivative would encode a polypeptide which retains esst?ntizll biological characteristics of MYD118.
As used herein, the term "purified" refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment and isolated or separated from at 5 least one other component with which they are naturally associated.
The mydll8 Coding Sequences The nucleotide sequence of mydl 18 (SEQ ID NO: 1) is shown in Figure l . The entire coding region for human mydl 18 was initially identified within a cDNA library made from human fetal liver/spleen tissue where it was found 1 time in 2899 usable sequences. A BLAST
0 search (Basic Local Alignment Search Tool; Altschul SF (1993) J. Mol. Evol. 36: 290-300, Altschul SF et al (1990) J. Mol. Biol. 215:403-410) comparing the cDNAs ofthe human fetal liver/spleen library (SPLNFET01) against the primate ~t~h~e of GenBank 91 identified Incyte Clone 25214 as a non-exact match to murine mRNA for mydl 18 and related family member human gadd45 (NCBI GI number 53291), see Figure 2. The nucleotide sequence for mydl 18 s was identified within Incyte clone 25214 through a co~ ulel generated search for nucleotide sequence ~lignment~ The clone was resequenced, and the coding region rletermine~l Polynucleotide sequences encoding MYD118 were subsequently found in a cDNA library made from non-adherent peripheral blood mononuclear cells (PBMN) where it was found 1 time in 3941 usable sequences. As used herein term ~'usable sequences" refers to the total number of ~o clones in a library after the removal of vector, nucleotide repeats, cont~min~tion~ and mitochondrial DNA.
Various portions of the nucleotide sequence encoding MYD 118 have been found in GenBank from the following human tissue sources: hippocampus, retina, olfactory epithelium, fetal lung, breast, placenta, white blood cells, brain, pancreas, liver, prostate gland and lung.
25 Polynucleotide sequences encoding MYD118 have not been rlçtect-?~l however, in cDNA libraries made from malignant cells of hematopoietic lineage, including THP-1 cells; U937 cells; T/B
lymphoblasts from a leukemi~ source; peripheral blood white blood cells from an individual with myelogenous leukemia; and human mast cells from an individual with mast cell leukemia. The mydl 18 nucleotide sequence encodes an acidic protein of 160 amino acids having a predicted 30 isoelectric point of 4.4 and discrete hydrophobic and hydrophilic regions as shown in Figure 3.
Methods for DNA sequencing are well known in the art and employ such enzymes as the Klenow fragment of DNA polymerase I, Sequenase~D (US Biochemical Corp, Cleveland OH)), SUBSTITUTE SHEET (RI~LE 26) WO 97/3Q157 PCT~S97JO245~

Taq polymerase (Perk;n Elmer, Norwalk CT), thermostable T7 polymerase (~ mer.ch~n~, Chicago IL), or combinations of recombinant polymerases and proofreading exonucleases such as the ELONGASE Amplification System marketed by Gibco BRL (Gaithersburg MD). Methods to extend the DNA from an oligonucleotide primer annealed to the DNA template of interest have s been developed for both single-stranded and double-stranded templates. Chain termination reaction products were separated using electrophoresis and detected via their incorporated, labeled precursors. Recent improvements in mech~ni7P~ reaction ~lc~alalion, sequencing and analysis have permitted expansion in the number of sequences that can be deterrnined per day.
Preferably, the process is automated with machines such as the Hamilton Micro Lab 2200 0 (Harnilton, Reno NV), Peltier Therrnal Cycler (PTC200; MJ Research, Watertown MA) and the ABI Catalyst 800 and 377 and 373 DNA sequencers (Perkin Elmer).
The quality of any particular cDNA library from which polynucleotides encoding MYD 1 18 are found may be determined by performing a pilot scale analysis of the cDNAs and checking for percentages of clones cont~ining vector, lambda or E. ç~ DNA, mitochondrial or 1 s repetitive DNA, and clones with exact or homologous m~t~h~s to public ~lat~k~eels.
Extending mydll8 Polynucleotide Sequence The polynucleotide sequence of mydl 18 may be exten~ ltili7in~ the nucleotide sequences from SEQ ID NO: I in various methods known in the art to detect upstream sequences such as promoters and regulatory element~ Gobinda et al (1993; PCR Methods Applic 2:318-20 22) disclose '~restriction-site polymerase chain reaction (PCR)" as a direct method which uses universal primers to retrieve unknown sequence adjacent to a known locus. First, genomic DNA
is amplified in the presence of primer to a linker sequence and a primer specific to the known region. The amplified sequences are subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are 2s transcribed with an ~lupl;ate RNA polymerase and sequenced using reverse transcriptase.
Inverse PCR can be used to amplify or extend sequences using di~lg*llt primers based on a known region (Triglia T et al(l988) Nucleic Acids Res 16:8186). The primers may be designed using Oligo 4.0 (National Biosciences Inc, Plymouth MN), or another appropriate program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal 30 to the target sequence at t~ cldlUlCS about 68~-72~ C. The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.

S~IBSTITUTE SHEET (RULE 26) CA 02246~78 l998-08-l4 W O97/301S7 PCT~US97/02458 Capture PCR (Lagt;~ M et al (1991) PCR Methods Applic 1~ 19) is a method for PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artif~cial chromosome (YAC) DNA. Capture PCR also requires multiple restriction enzyme digestions and ligations to place an engineered double-stranded sequence into an unknown portion of the 5 DN~ molecule before PCR.
Parker JD et al (1991; Nucleic Acids Res 19:3055-60), teach walking PC~, a method for targeted gene walking which permits retrieval of unknown sequence. PromoterFinderTM is a new kit available from Clontech (Palo Alto CA) which uses PCR, nested primers and special libraries to "walk in" genomic DNA. This process avoids the need to screen libraries and is useful in o finding intron/exon junctions.
Another PCR method, "Tmproved Method for Obtaining Full Len~th cDNA Sequençes"
by Guegler et al, Patent Application Serial No 08/487,1 12, filed June 7, 1995 and hereby incorporated by reference, employs XL-PCRTM enzymes (Perkin-Elmer, Foster City CA) to amplify and/or extend nucleotide sequences.
Preferred libraries for screening for full length cDNAs are ones that have been size-selected to include larger cDNAs. Also, random primed libraries are preferred in that they will contain more sequences which contain the 5' and upstream regions of genes. A randomly primed library may be particularly useful if an oligo d(T) library does not yield a full-length cDNA.
Genomic libraries are useful for obtaining introns and extending 5' sequence.
A new method for analyzing either the size or confirrning the nucleotide sequence of sequencing or PCR products is capillary electrophoresis. Systems for rapid sequencing are available from Perkin Elmer, Beckrnan Instruments (Fullerton CA), and other companies.
Capillary sequencing employs flowable polymers for electrophoretic separation, four different fluorescent dyes (one i~or each nucleotide) which are laser activated, and detection of the emitted 2s wavelengths by a charge coupled devise carnera. Output/light intensity is converted to electrical signal using appropriate software (eg. GenotyperTM and Sequence NavigatorTM from Perkin Elmer) and the entire process from loading of samples to computer analysis and electronic data display is computer controlled. Capillary electrophoresis is particularly suited to the sequencing of small pieces of DN~ which might be present in limited amounts in a particular sample. The 30 reproducible sequencing of up to 350 bp of M 13 phage DNA in 30 min has been reported (Ruiz-Martinez MC et al (1993) Anal Chem 65:2851-8).
Expression of MYD118 SUBSTITUTE SHEET ~RULE 26) CA 02246~78 1998-08-14 WO 97/301~!;7 PCT/US97/024~;~

In accordance with the present invention, mydl 18 polynucleotide sequences whichencode MYD118 polypeptide sequences, fr~menl~, fusion proteins or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of mydl 18 in ~,op-;ate host cells. Due to the inherent degeneracy ofthe genetic code, DNA
5 sequences other than the polynucleotide sequences of SEQ ID NO: 1 which encode substantially ~ the same or a functionally equivalent amino acid sequence, may be used to clone and express mydl 18. As will be understood by those of skill in the art, it may be advantageous to produce MYD 1 1 8-encoding nucleotide sequences possessing non-naturally occurring codons. Codons preferred by a particular prokaryotic or eukaryotic host (Murray E et al ~1989) Nuc Acids Res o 17:) can be selected, for example, to increase the rate of mydl 18 expression or to produce recombinant ~NA transcripts having desirable properties, such as a longer half-life, than transcripts produced ~rom naturally occurring sequence.
Also included within the scope of the present invention are polynucleotide sequences that are capable of hybridizing to SEQ ID NO: 1 under conditions of intermediate to m~im~l 5 stringency as long as the polynucleotide sequence capable of hybridizing encodes a protein that retains a biological activity of the naturally occurring MYD l 18. Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Wahl GM et al. (1987, Methods Enzymol 152:399-407) incorporated herein by reference, and confer a defined "stringency" as explained below.
"Maximum stringency" typically occurs at about Tm-5 ~C (5 ~C below the Tm of theprobe); "high stringency" at about 5~C to 10~C below Tm; "int~rme~ te stringency" at about 10~C to 20~C below Tm; and "low stringency" at about 20~C to 25~C below Tm. As will be understood by those of skill in the art, a msl~imllm stringency hybridization can be used to identify or detect identical polynucleotide sequences while an intermediate (or low) stringency 25 hybridization can be used to identify or detect similar or related polynucleotide sequences.
The term "hybri~1i7:~tion" as used herein refers to "the process by which a strand of nucleic acid joins with a complementary strand through base pairing" (Coombs J (1994) Dictioîl~ry of - Biotechnology~ Stockton Press, New York NY). Amplification as carried out in polymerase chain reaction technologies is described in Dieffenbach CW and GS Dveksler (1995, PCR
30 Primer. a Laboratory Manual, Cold Spring Harbor Press, Plainview NY) and incorporated herein by reference.
As used herein a "deletion" is defined as a change in either nucleotide or amino acid S~J~S ~ ITE SHI~ET (RULE 26) CA 02246~78 1998-08-14 sequence in which one or more nucleotides or amino acid residues, respectively, are absent.
As used herein an "insertion" or "addition" is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring molecule.
s As used herein "substitution" results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.
Variant mydl 18 polynucleotide sequences may be used in accordance with the invention and include deletions, insertions or substitutions of dirre~ t nucleotide residues resulting in a polynucleotide that encodes the same or a functionally equivalent MYD118 polypeptide.
o Variant MYD 1 18 protein may also be used in accordance with the invention and may include deletions, insertions or substitutions of amino acid residues as long as the result is a functionally equivalent MYD 1 18. As used herein, the term functionally equivalent refers to a variant polynucleotide or variant polypeptide sequence that retains at least one of the biological activities of the naturally occurring sequence. Arnino acid substitutions may be made on the basis of 15 similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, andlor the arnphipathic nature of the residues as long as a biological activity of MYDl 18 is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include Iysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values are grouped as follows: leucine, isoleucine and valine; glycine and 20 alanine; asparagine and glutamine; and lastly, serine, threonine phenyl~l~nine, and tyrosine.
Included within the scope of the present invention are alleles of nlydl 18. As used herein, an "allele" or "allelic sequence" is an alternative form of mydl 18. Alleles result from a mutation, ie, a change in the nucleic acid sequence, and generally produce altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given gene may have 25 none, one or many allelic forms. Common mutational changes which give rise to alleles are generally ascribed to deletions, additions or substitutions of nucleic acids. Each of these types of changes may occur alone, or in combination with the others, and at the rate of one or more times in a given sequence.
The nucleotide sequences of the present invention may be engint?ered in order to alter an 30 mydl 18 coding sequence for a variety of reasons, including but not limited to, alterations which modify the cloning, processing and/or expression of the gene product. For example, mutations may be introduced using techniques which are well known in the art, eg, site-directed SUBSTITUTE SHEET (RULE 26) CA 02246~78 l998-08-l4 mutagenesis to insert new restriction sites, to alter glycosylation patterns, to change codon preference, etc.
In another embodiment of the invention, an mydl 18 natural, modified or recombinant sequence may be ligated to a heterologous sequence to encode a fusion protein. For example, for s screening of peptide libraries for inhibitors of MYD 1 18 activity, it may be useful to encode a chimeric MYD1 18 protein expressing a heterologous epitope that is recognized by a commercially available antibody. A fusion protein may also be engineered to contain a cleavage site located between an MYD1 18 sequence and the heterologous protein sequence, so that the MYD 1 18 may be cleaved and purified away from the heterologous moiety.
o In an alternate embodiment of the invention, the coding sequence of mydl 18 could be synth~?ci7~-1, whole or in part, using chemical methods well known in the art (See Caruthers MH
et al (1980) Nuc Acids Res Symp Ser 215-23, Horn T et al(1980) Nuc Acids Res Symp Ser 225-32, etc). ~Iternatively, the protein itself could be produced using chemical methods to syn~h~i7~
an MYD118 amino acid sequence, whole or in part. For example, peptides can be synthesized by S solid phase techniques, cleaved from the resin, and purified by ~ u~dtive high performance liquid chromatography (eg, Creighton (1983) Proteins Structures And Molecular Principles. WH
Freeman and Co, New York NY). The composition of the synthetic peptides may be confirrne~l by amino acid analysis or sequencing (eg, the Edman degradation procedure, Creighton, supra) Direct peptide synthesis can be performed using various solid-phase techniques (Roberge 2~ JY et al (1995) Science 269:202-204) and automated synthesis may be achieved, for example, using ABI 431 A Peptide Synth~ r (Perkin Elmer) in accordance with the instructions provided by the m~nnf~cturer. Additionally the amino acid sequence of MYD1 18, or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with other sequences to produce a variant po}ypeptide.
2s Expression Systems In order to express a biologically active MYD118, the nucleotide sequence coding for MYD118, or a functional equivalent thereof, is inserted into an appropriate ~ s~ion vector, ie, a vector which contains the nececs~ry elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art can be used 30 to construct expression vectors cont~ining an MYD118 coding sequence and appropl;ate transcriptional or translational controls. These methods include in vitro recombinant DNA
techniques, synthetic techniques and in vivo recombination or genetic recombination. Such SUBSTITUTE SHEET(RULE26~

CA 02246~78 1998-08-14 W O 97/30157 P~T~US97/~2458 techniques are described in Maniatis et al (1989) Molecular ~loni~ A Laboratory Manual, Cold Spring Harbor Press, Plainview NY and ~usubel FM et al. (1989) Current Protocols in ~olecular Biolo~y. John Wiley & Sons, New York NY.
Analysis of the functions of growtll inhibitory genes, such as mydl 18, by usings expression vectors can be technically difficult, since even low-level expression may block growth of the host cell. Stable transfectants that do grow out are often revertants that have lost e~ s~ion or have had other compensatory changes (Zhan et al. supra). Therefore, a preferred expression system for the expression of MYD1 18 in host cells is one that allows for expression of proteins toxic to the host cell. For expression in m~mm~ n cells, Zhan et al. ~supra, p. 2366), 0 describe a m~mm~ n t;x~ie~ion system for the expression of MYD1 18 related family members GADD45, which has been shown to be regulated by p53, and murine MYD118. This expression system employs the cytomegalovirus promoter and cotransfection with a selectable marker, pSV2neo. Expression studies were perforrned in human tumor lines with a null pS3 phenotype (H1299), in cells with a normal pS3 phenotype (RKO), and in cells cont~inin~ a viral protein, E6, 15 that interferes with pS3 function (HeLa).
For expression of MYD118 in ~. coli, Brown et al. (Gene 1993, 1~7:99-103) describe a method that was used for expression of the toxic POL3 gene of ~. cerevi~i~t? which involves the use of the vector pT7SC. Brown obtained up to 15 mg of protein from as little as 3 grams of cells and the expressed protein was in the forrn of inclusion bodies.
20 Identification of Transformants Containing mydll8 Although the presence/absence of marker gene expression suggests that the gene of interest is also present, its presence and expression should be confirmed. For example, if the mydl 18 is inserted within a marker gene sequence, recombinant cells contslinin~ mydl 18 can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in ~s tandem with an mydl 18 sequence under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of mydl 18 as well.

Alternatively, host cells which contain the coding sequence for mydl 18 and express mydl 18 may be identified by a variety of procedures known to those of skill in the art. These 30 procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridization and protein bioassay or immllnoassay techniques which include membrane-based, solution-based, or chip-based technologies for the detection and/or quantification of the nucleic acid or protein.

SUBSTITUTE SHEET (RULE 26) =
CA 02246~78 1998-08-14 WO 97/301~;7 PCT/US97~024~8 The presence o~the mydl 18 polynucleotide sequence can be detected by DNA-DNA orDNA-RNA hybr~dization or amplification using probes, portions or fragments of mydl 18 disclosed in SEQ ID NO: 1. Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the mydl 18 sequence to detect transforrnants cont~inin~
s mydl 18 DNA or RNA. As used herein "oligonucleotides" or "oligomers" refer to a nucleic acid sequence of at least about 1 Q nucleotides and as many as about 60 nucleotides, preferably about 15 to 30 nucleotides, and more preferably about 20-25 nucleotides which can be used as a probe or amplimer. Preferably, oligonucleotides are derived from the 3 ' region of the mydl 18 nucleotide sequence shown in Figure 1.
o A variety of protocols for detecting and measuring the expression of MYDI ~8 polypeptide, using either polyclonal or monoclonal antibodies specific for the protein are known in the art. Examples include enzyme-linked irnmunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent act;vated cell sorting (FACS). A two-site, monoclonal-based immunoassay ~7tili7.in~ monoclonal antibodies reactive to two non-i~llelr~ lg epitopes on 15 MYD 118 polypeptides is ~I.,fc~l~ d, but a competitive binding assay may be employed. These and other assays are described, among other places, in Hampton R et al (1990, Serolo~ical Me~hods. a T ~horatorv M~nl-~l APS Press, St Paul MN) and Maddox DE et al (1983, J Exp Med 158:1211).
A wide variety of labels and conjugation techni(lues are known by those skilled in the art 20 and can be used in various nucleic and arnino acid assays. Means for producing labeled hybridization or PCR probes for d~tectinE mydl 18 polynucleotide sequences include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide.
Alternatively, the mydl 18 sequence, or any portion of it, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, 25 and may be used to synthe~i7P RNA probes in vitro by addition of an a~,o{~iate RNA
polymerase such as T7, T3 or SP6 and labeled nucleotides.
A number of companies such as Pharmacia Biotech (Piscataway NJ), Promega (Madison WI), and US Biochemical Corp (Cleveland OH) supply commercial kits and protocols for these procedures. Suitable reporter molecules or labels include those radionuclides, enzymes, 30 fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like. Patents teSI~hing the use of such labels include US Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. Also, SUE3STITUTE SHEET ~RULE 26) , ~ CA 02246~78 1998-08-14 W O 97/301~7 PCT~US97/02458 recombinant immunoglobulins may be produced as shown in US Patent No. 4,816,567 and incorporated herein by reference.
Purification of MYD118 Host cells transformed with a mydl 18 nucleotide sequence may be cultured under 5 conditions suitable for the expression and recovery of the encoded protein from cell culture. The protein produced by a recombinant cell may be secreted or may be contained intracellularly depending on the se~uence and/or the vector used. As will be understood by those of skill in the art, e~ ion vectors co..~ ;"~ mydl 18 can be designed with signal sequences which direct secretion of MYDl 18 through a particular prokaryotic or eukaryotic cell membrane. Other I o recombinant constructions may join mydl 18 to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins (Kroll DJ et al (1993) DNA Cell Biol 12:441-53, see also above discussion of vectors co--l~ fusion proteins).
MYD I 18 may also be expressed as a recombinant protein with one or more additional polypeptide domains added to facilitate protein purification. Such purification facilit~tin~
5 domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals (Porath J ( 1992) Protein Expr Purif 3 :263-281), protein A domains that allow purification on imrnobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle WA). The inclusion of a cleavable linker sequences such as Factor XA or enterokinase (Invitrogen, San 20 Diego CA) between the purification domain and MYDl 18 is useful to facilitate purification.
Uses of MYDl18 MYD 1 18 appears to induce terminal differentiation of hematopoietic cells thereby arresting cell proliferation and inducing apoptosis. Polynucleotide sequences encoding MYD 1 18, or portions thereof, have been found in a variety of cDNA libraries from tissues 25 including fetal liver/spleen, PBMN cells, hippocampus, retina, olfactory epithel;um, human fetal lung, breast, placenta, human white blood cells, human brain, human pancreas, liver, prostate gland and lung. Polynucleotide sequences encoding MYD118 are not ~letected in cDNA libraries made from m~lign~nt cells of hematopoietic lineage, where genes related to cell growth arrest and apoptosis would be expected to be expressed at undetectable levels or not e~le~s~d at all.
30 Based upon 1 ) its characterization as an MYD farnily member, 2) the ability of MYD family members to terminally dirr~lellliate cells of a hematopoietic lineage and 3~ the ability of related MYD family members to reverse a leukemic phenotype in vivo, human MYD118 disclosed SUBSTITUTE SHEET (RULE 2 CA 02246~78 1998-08-14 WO 97/30157 PCTAUS97~02458 herein may be used as a diagnostic or th~ldpc;ulic agent in the detection or trç~tm~nt of proliferative diseases of hematopoietic cells, specifically in myeloproliferative diseases and lellkemiz,~, Accordingly, MYD 1 18 can be used to treat or ameliorate the symptoms of s myeloproli~erative diseases or leukemi~, such as acute Iymphocytic leukemia (ALL), acute - myelogenous leukemia (AML), chronic Iymphocytic leukemia (CLL), hairy cell leukemia, chronic myelogenous leukemia (C~L); polycythemia vera (PV); agnogenic myeloid metaplasia with myelofibrosis (AMM/MF); and essential thrombocytosis (ET). MYD118 may be used alone or in combination with other drugs or agents in the treatment of such ~1iee~ees For example, 10 ~lmini~tration of MYD118 to individuals subject to myeloproliferative disease, which is characterized by the production of increased numbers of i ~n l l li1 l l l l ~ cells in the peripheral blood, may induce terminal differentiation of the imm~ re cells thereby leading to the maturation of the cells and eventu~l apoptosis. ~A~1mini~tration of MYD1 18 may be used to al-gment chemotherapy, such as ~lminietration of bTlslllfz~n, radiation therapy and/or bone marrow 5 transplant. MYD118 may be used therapeutically to ameliorate the symptoms associated with myeloproliferative diseases and leukemias, such as, anemia, fatigue, splenomegaly hypermetabolism and thrombohemorrhagic complications.
MYD l 18 may also be used to treat disease states related to abnormal proliferation of cells associated with severe infl~mm~tion including, but not limited, to rheumatoid arthritis, psoriasis, 20 and lymphomatoid granulomatosis.
In another embodiment of the present invention, antibodies or antagonists of MYD1 18 may be used in the ex vivo culturing of cells from a hematopoietic cell linea~e intçnclç~l for autologous transplant where it would be desirable to elimin~te a cell growth arrest factor that might interfere with desirable cell proliferation.
25 MYD118 Antibodies Procedures well known in the art may be used for the production of antibodies toMYD118 polypeptides. Such antibodies include, but are not limited to, polyclonal, monoclonal, - chimeric, single chain, Fab fr~gment~ and fragments produced by a Fab expression library.
Neutralizing antibodies, ie, those which inhibit biological activity of MYD118 polypeptides, are 30 especially preferred for diagnostics and therapeutics.
For the production of antibodies, various hosts including goats, rabbits, rats, mice, etc may be immunized by injection with MYD 1 18 polypeptide or any portion, fragment or SUBSTITUTE SHEET (RULE 26) CA 02246~78 1998-08-14 W O 97/30157 PCT~US97/02458 oligopeptide which retains immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as Iysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet 5 hemocyanin, and dinitrophenol. BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are potentially useful human adjuvants which may be employed if purified MYD118 po}ypeptide is ~(lmini~tered to immunologically colllplvlllised individuals for the purpose of s1im~ tin~
systemic defense.
Monoclonal antibodies to MYD118 polypeptide may be prepared using any technique 0 which provides for the production of antibody molecules by continuous cell lines in culture.
These include but are not limited to the hybridoma technique originally described by Koehler and Milstein (1975 Nature 256:495-497), the human B-cell hybridoma technique (Kosbor et al (1983) Immunol Today 4:72; Cote et al (1983) Proc Natl Acad Sci 80:2026-2030) and the EBV-hybridoma techni~ue (Cole et al (1985) Monoclon~l Antibodies ~nd ~ncer Therapy~ Alan R Liss 15 Inc, pp 77-96). In addition, techniques developed for the production of "chimeric antibodies", the splicing of mouse antibody genes to human antibody genes to obtain a molecule with ~p~ iate antigen specificity and biological activity can be used (Morrison et al (1984) Proc Natl Acad Sci 81:6851-6855; Neuberger et al (1984) Nature 312:604-608, Takeda et al (1985) Nature 314:452-454). Alternatively, techniques described for the production of single chain antibodies (US
Patent No. 4,946,778) can be adapted to produce MYD118 specific single chain antibodies.
Antibodies may also be produced by inducing in vivo production in the Iymphocytepopulation or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi R et al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G and Milstein C (1991; Nature 349:293-299).
2s Antibody fr~ment~ which contain specific binding sites for MYD118 may also be generated. For example, such fragments include, but are not limited to, the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')~ fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fr~ment~ with the desired specificity (Huse WD et al (1989) Science 256:1275-1281).
MYD118-specific antibodies are useful for the diagnosis of conditions and rlice~e~
associated with expression of MYD 118 polypeptide. A variety of protocols for competitive SUBSTITUTE SHEET ~RULE 26) CA 02246~78 1998-08-14 WO 97~0IS7 PCT~US97~024S~

binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the formation of complexes between MYD1 18 polypeptides and its specific antibody (or similar MYD 1 1 8-binding molecule) and the measurement of complex formation. A~ two-site, 5 monoclonal-based immunoassay utili7in~ monoclonal antibodies reactive to two noninterfering - epitopes on a specific MYD 1 18 protein is preferred, but a competitive binding assay may also be employed. These assays are described in Maddox (supra) Diagnostic ~ssays Using MYD118 Specific Antibodies Anti-MYD 1 18 antibodies are useful for the diagnosis of myeloproliferative disease or lo leukemias or other conditions, disorders or diseases characterized by abnormal expression of MYDl 18. Diagnostic assays for MYDl 18 include methods lTtili7in~ the antibody and a label to detect MYr~118 polypeptide in human body fluids, cells, tissues or sections or extracts of such tissues. The polypeptides and antibodies of the present invention may be used with or without modification. Fre~uently, the polypeptides and antibodies will be labeled by joining them, either 15 covalently or noncovalently, with a reporter molecule. A wide variety of reporter molecules are known to those of skill in the art.
A variety of protocols for measuring MYDl 18 polypeptide, using either polyclonal or monoc~onal antibodies specific for the respective protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELI~;A), radioimmunoassay (RIA) and fluorescent 20 activated cell sorting (FACS). A two-site, monoclonal-based immlmoassay ~ltili7in~ monoclonal antibodies reactive to two non-interfering epitopes on MYDI 18 polypeptide is preferred, but a coll~eLilive binding assay may be employed. These assays are described, among other places, in Maddox (supra).
In order to provide a basis for the diagnosis of disease, normal or standard values for 25 MYD1 18 polypeptide expression must be established. This is accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with antibody to MYDl 18 polypeptide under conditions suitable for complex formation which are well known in - the art. The amount of standard complex formation may be ~uantified by comparing it with a dilution series of positive controls where a known amount of antibody is combined with known 30 concentrations of purified MYD 1 18 polypeptide. Then, standard values obtained from normal samples may be compared with values obtained from samples from subjects potentially affected by a disorder or disease related to MYDl 18 polypeptide ex~,e~sion. Deviation between standard SUBSTITUTE SHEET (RULE 26 CA 02246~78 1998-08-14 W O 97/30157 PCTrUS97/02458 -and subject values establishes the presence of the disease state.
Drug Screening MYDI 18 polypeptide, its immunogenic fragments or oligopeptides can be used for screening therapeutic compounds in any of a variety of drug screening techniques. The fragment 5 employed in such a test may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The abolition of activity or the formation of binding complexes, between MYDl 18 polypeptide and the agent being tested, may be measured. Accordingly, the present invention provides a method for screening a plurality of compounds for specific binding af~mity with MYD I 18, or a portion thereof, comprising providing a plurality of compounds;
o combining MYDl 18, or a portion thereof, with each of a plurality of compounds for a time sufficient to allow binding under suitable conditions; and detecting binding of MYDl 18, or portion thereof, to each of the plurality of compounds, thereby identifying the compounds which specifically bind MYDl 18.
Another technique for drug screening provides for high throughput screening of 15 compounds having suitable binding affinity to the MYD118 polypeptides and is described in detail in Geysen, European Patent Application 84/03564, published on September 13, 1984, incorporated herein by reference. ln surnmary, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with MYD118 fr~gmeT-t~ and washed. Bound MYDl 18 is 20 then detected by methods well known in the art. Purifled MYD1 18 can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding MYD118 specifically compete with a test compound 25 for binding MYD 1 18. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic detçrmin~nt~ with MYD118.
Uses of mydll8 Polynucleotide An mydl 18 polynucleotide, or any part thereof, may be used for diagnostic and/or therapeutic purposes. For diagnostic purposes, mydl 18 polynucleotide sequences may be used to 30 detect and quantitate gene expression in conditions, disorders or diseases in which mydl 18 activity may be implicated, for example in myeloproliferative diseases or lellkt?mi~c, where mydl 18 gene ~x~ s~ion appears to be absent, aberrant or not detected. For therapeutic purposes, SUBSTITUTE SHEET (RULE 26) CA 02246~78 1998-08-14 WO 97/30157 PC~AUS97~024~8 mydl 18 polynucleotide sequences may be ~lmini~tered to individuals with myeloid proliferative disease or leukemia to induce terminal differentiation of proliferating imm~t-lre hematopoietic cells origin, thereby arresting cell proliferation, arneliorating the symptoms of disease, and reversing the leukemic phenotype.
Included in the scope of the invention are oligonucleotide sequences, antisense RNA and - DNA molecules and ribozymes, which function to inhibit translation of an myd 1 18. Such nucleotide sequences may be used in conditions where is would be preferable to blocl~ positive regulators of cell growth arrest, for example, in ex vivo culturing of cells intended for autologous transplant to individuals lacking cells of the hematopoietic lineage, such as individuals who are 0 immllnoco~llp~ lised due to disease, such as HIV infection, or individuals subject to chemotherapy or radiation therapy which depletes certain cells of hematopoietic lineage.
Another aspect of the subject invention is to provide for nucleic acid hybridization or PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding MYDl 18 or closely related molecules, such as alleles. The specificity of 5 the probe, ie, whether it is derived from a highly conserved, conserved or non-conserved region or domain, and the stringency of the hybridization or amplification (high, intermediate or low) will ~leterrnine whether the probe identifies only naturally occurring mydl 18, or related sequences. Probes for the detection of related nucleic acid sequences are selected from conserved or highly conserved nucleotide regions of MYD farnily members and such probes may 20 be used in a pool of degenerate probes. For the detection of identical nucleic acid sequences, or where m~ximl-m specificity is desired, nucleic acid probes are selected from the non-conserved nucleotide regions or unique regions of mydl 18 polynucleotides. As used herein, the term "non-conserved nucleotide region" refers to a nucleotide region that is unique to mydl 18 and does not occur in related family members, such as the nucleotide sequence encoding GADD45.

Diagnostic Uses of mydll8 Polynuc~eotide ~ An mydl 18 encoding polynucleotide sequence may be used for the diagnosis of diseases resulting from abnormal expression of mydl 18 or other genes associated witll myeloproliferative 30 disease or lellk~mi~ For example, polynucleotide sequences encoding MYDl 18 may be used in hybridization or P(~R assays of tissues from biopsies or autopsies or biological fluids, such as serum, to detect abnormalities in mydl 18 ~ ession. The form of such qualitative or SlJts~ 111 ~JTE SHEET (RULE 26) CA 02246~78 1998-08-14 W O 97130157 PCTrUS97/02458 quantitative methods may include Southern or northern analysis, dot blot or other membrane-based technologies, PCR technologies, dip stick, pin or chip technologies; and ELISA or other multiple sample format technologies. All of these techniques are well known in the art, and are in fact the basis of many commercially available diagnostic kits.
Such assays may be tailored to evaluate the efficacy of a particular therapeutic treatment regime and may be used in animal studies, in clinical trials, or in monitoring the treatment of an individual patient. In order to provide a basis for the diagnosis of ~ e~e, a normal or standard profile for myd 118 expression must be established. This is accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with mydl 18 or a lo portion thereof, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by colllpallng the values obtained for normal subjects with a dilution series of positive controls run in the same experiment where a known amount of purified mydl 18 is used. Standard values obtained from normal samples may be compared with values obtained from samples from subJects potentially affected by a disorder or disease related to 15 mydl 18 expression. Deviation between standard and subject values establishes the presence of the disease state. If disease is established, an existing tn~ t;uL;c agent is ~-lmini~tered, and tre~tmçnt profile or values may be generated. Finally, the assay may be repeated on a regular basis to evaluate whether the values progress toward or return to the normal or standard pattern.
Successive tre~1ment profiles may be used to show the efficacy of treatment over a period of 20 several days or several months.
PCR as described in US Patent Nos. 4,683,195; 4,800,195; and 4,965,188 provides additional uses for oligonucleotides based upon the mydl 18 sequence. Such oligomers are generally chemically synthesized, but they may be generated enzymatically or produced from a recombinant source. Oligomers generally comprise two nucleotide sequences, one with sense 25 orientation (5'->3') and one with ~nti~çn~e (3'<-5') employed under optimized conditions for identification of a specific gene or condition. The same two oligomers, nested sets of oligomers, or even a degenerate pool of oligomers may be employed under less stringent conditions for detection and/or ql-~ntit~tion of closely related DNA or RNA sequences.
Additionally methods to ~u~ntit~te the expression of a particular molecule include 30 radiolabeling (Melby PC et al 1993 J Immunol Methods 159:235-443 or biotinylating (Duplaa C
et al 1993 Anal Biochem 229-36) nucleotides, coamplification of a control nucleic acid, and standard curves onto which the experimental results are interpolated. Quantitation of multiple SUBSTITUTE SHEET(RULE26) CA 02246~78 1998-08-14 WO 97/30~57 PCT~JS97/02458 samples may be speeded up by running the assay in an ELISA format where the an oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
Therapeutic Uses of an mydl 18 Polynucleotide An myd 1 18 polynucleotide sequence may provide the basis for treatment of various - abnorrnal conditions involving abnormally proliferating cells of hematopoietic lineage, including myeloproliferative diseases and le~ emi~s, such as acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic Iymphocytic leukemia (CLL), hairy cell lellkemi~, chronic myelogenous leukemia (C: ML); polycythemia vera (PV); agnogenic myeloid metaplasia o with myelofibrosis (AMM/MF); and essential thrombocytosis (ET), where it would be advantageous to induce terminal differentiation of the proliferating cells thereby arresting cell proliferation. Stable transformation of appropriate germ line cells, or a zygote, with a vector co~ polynucleotide sequences encoding MYDI 18 may produce a transgenic organism (US
Patent No. 4,736,866, 12 April 1988) producing enough copies of the polynucleotide sequence to 5 induce terrnin~1 differentiation of cells of hematopoietic lineage, thereby leading to cell growth arrest Such vectors and expression systems intenclel1 for in vivo or gene therapy use should be designed for expression of molecules toxic to the host cell as disclosed infra. A preferable expression vector would be one which drives expression of mydl 18 at levels comparable to its expression in normal non-proliferating cells.
Alternatively, an mydl 18 polynucleotide sequence may also provide the basis for design of antisense molecules that are able to inhibit transcription or translation of mydl 18 in conditions where it would be advantageous to block positive regulators of cell growth arrest, such as for example in ex vivo culturing of cells from a hematopoietic lineage intended for autologous transplant, where proliferation of cells of hematopoietic lineage would be desirable. The 25 introduction of vectors into stem cells taken from a patient and clonally prop~tecl for -autologous transplant is described in US Patent Nos. 5,399,493 and 5,437,994, disclosed herein by reference.
- Expression vectors derived from retroviruses, adenovirus, herpes or vaccinia viruses, or from various b~ct~ l plasmids, may be used for delivery of recombinant mydl 18, sense or 30 ~nti~ence molecules, to the targeted cell population. Methods which are well known to those skilled in the art can be used to construct recombinant vectors cont~inin~ mydll 18. See, for example, the techniques described in Maniatis et al (supra) and Ausubel et al(supra).

-2s-SUBSTITUTE StlEET (RULE 26) CA 02246~78 1998-08-14 W O 97130157 PCT~US97/02458 Alternatively, recombinant mydl 18 can be delivered to target cells in liposomes.
The full length cDNA sequence and/or its regulatory elements enable researchers to use mydl 18 as a tool in sense (Youssoufian H and E~F Lodish 1993 Mol Cell Biol 13:98-104) or antisense (Eguchi et al (1991) Annu Rev Biochem 60:631-652) investigations of gene function.
5 Oligonucleotides, designed from the cDNA or control sequences obtained from the genomic DNA can be used in vitro or in vivo to inhibit expression. Such technology is now well known in the art, and sense or antisense oligonucleotides or larger fr~gment~ can be designed from various locations along the coding or control regions.
Additionally, mydl 18 ~ ssion can be modulated by transfecting a cell or tissue with o expression vectors which express high levels of an mydl 18 fragment in conditions where it would be preferably to block the activity of positive regulator of cell growth arrest, such as in bone marrow transplant therapy and ex vivo culturing of cells intended for autologous transplant.
Such constructs can flood cells with untr~n~ ble sense or ~nti~.?nce sequences. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules 5 until all copies of the vector are disabled by endogenous nucleases. Such transient expression may last for a month or more with a non-replicating vector (Mettler I, personal co~ lication) and even longer if a~ro~iate replication elements are part of the vector system.Modifications of gene ~ ssion can be obtained by cleci~ning antisense sequences to the conkol regions of the mydl 18 gene, such as the promoters, enhancers, and introns.
20 Oligonucleotides derived from the transcription initiation site, eg, between -lO and +10 regions of the leader sequence, are preferred. Antisense RNA and DNA molecules may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes. Similarly, inhibition can be achieved using Hogeboom base-pairing methodology, also known as "triple helix" base pairing. Triple helix pairing compromises the ability of the double helix to open 25 sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The meçh~ni~m of ribozyme action invo}ves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by a endonucleolytic cleavage.
Within the scope of the invention are engineered h~mmerhead motif ribozyme molecules that 30 specifically and efficiently catalyze endonucleolytic cleavage of mydl 18 RNA sequences.
Specific ribozyme cleavage sites within any potential RNA target are initially identified by sc~nnin~ the target molecule for ribozyme cleavage sites which include the following SUtJ;~ 1 1 1 UTE SHEET (RULE 26) CA 02246~78 1998-08-14 WO 97/30157 rCT~US97/024~
sequences, GUA, GUIJ and GU~. ~nce identified, short RNA sequences of between 15 and 20 ~ibonucleotides corresponding to the region of the target gene cont~ining the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide se~uence inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to s hybridization with complelnent~ry oligonucleotides using ribonuclease protection assays.
Both antisense RNA and DNA molecules and ribozymes of the invention may be prepared by any method kno~,vn in the art for the synthesis of RNA molecules. These include techniques for chemically synth~o~i7in~ oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro or i~ ViVQ
lo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. ~ltern~tively, antisense cDNA constructs that synthe~ ? antisense RNA
constitutively or inducibly can be introduced into cell lines, cells or tissues.DNA molecules may be modified to increase intracellular stability and half-life. Possible 15 modifications include, but are not limited to, the addition of fl~nkin~ sequences of the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
Methods for introducing vectors into cells or tissue include those methods ~ c~lc~e~
In addition, several of these transformation or transfection methods are equally suitable for the ex 20 vivo therapy, Furtherrnore, the myd 1 18 polynucleotide sequences disclosed herein may be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleotide sequences that are curre~tly known, including but not limited to such properties as the triplet genetic code and specific base pair interactions.
2s Detection and Mapping of Polynucleotide Sequences Related to mydll~
The nucleic acid sequence for myd 1 18 can also be used to generate hybridization probes as previously described, ~or mapping the endogenous genomic sequence. The sequence may be mapped to a particular chromosome or to a specific region of the chromosome using well known techniques. These include ~ ~ hybridization to chromosomal spreads (Verma et al (198g) 30 Hl-m~n Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York City), flow-sorted chromosomal ~alc-Lions, or artificial chromosome constructions such as YACs, bacterial artificial chromosomes (BACs), bacterial Pl constructions or single chromosome cDNA libraries.

SlJe~ JTE SHEET (RULE 26) CA 02246~78 1998-08-14 W O 97/301S7 PCT~US97/02458 hybridization of chromosomal preparations and physical mapping techniques such as linkage analysis using established ehromosomal markers are invaluable in exten~lin~ genetic maps. Examples of genetic maps ean be found in Science (1995; 270:410fand 1994; 265:1981f).
Often the placement of a gene on the ehromosome of another m~mmz~ n species may reveal s associated markers even if the number or arm of a partieular human ehromosome is not known.
New sequenees ean be assigned to chromosomal arms, or parts thereof, by physieal mapping.
This provides valuable information to investigators searching for disease genes using positional eloning or other gene diseovery teehniques. Once a disease or syndrome, such as ataxia telangiectasia (AT), has been crudely localized by genetie linkage to a particular genomic region, for example, AT to I lq22-23 (Gatti et al (1988) Nature 336:577-580), any sequences mapping to that area may represent associated or regulatory genes for further investigation. The nueleotide sequenee of the subject invention may also be used to detect differences in the chromosomal loeation due to transloeation, inversion, ete between normal, earrier or affected individuals.
Pharmaceutical Compositions The present invention relates to ph~rm~ce~ltical compositions which may eomprise all or portions of mydl 18 polynueleotide sequenees, MYD118 polypeptides, inhibitors or antagonists o~MYD118 bioactivity, including antibodies, alone or in combination with at least one other agent, such as stabilizing compound, and may be sl~lmini~tçred in any sterile, bioeompatible ph~rm~-~eutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
Mydl 18 nucleotide and MYl~118 amino acid sequenees can be ~-~minictered to a patient alone, or in combination with other nucleotide sequences, sueh as nucleotide sequenees encoding tumor suppressor genes, sueh as pS3, p16 and p21, drugs or hormones or in pharmaceutieal eompositions where it is mixed with excipient(s) or other pharrn~çeutically acceptable carriers.
~n one embodiment of the present invention, the ph~ eeutieally aeeeptable earrier is 25 pharm~eutieally inert. A ~le~ll~,d route of ~dmini~tration for treatment of myeloproliferative diseases or leukemias would be intravenous delivery whereas a preferred route of ~dmini~tration for tre~tment of eonditions related to infl~mm~tion would be loeal arlmini~tration at the site of infl~mm~tion, such as the joint affected in rheum~toid arthritis.
Mydl 18 polynueleotide sequenees or MYI~118 amino aeid sequenees may be 30 ~-lmini~tered alone to individuals subjeet to myeloproliferative rli~ç~ces or lenkemi~ or in eombination with other types of agents or therapy including ehemotherapy, radiation therapy or stem cell transplant therapy.

Sl/~S ~ ITE SHEET (RULE 26) CA 02246~78 l998-08-l4 WO 97/30157 PCT~US97/024~8 -Depending on the condition being treated, these pharrnaceutical coml~ositions may be formnl~te~l and ~imini~tered systemically or locally. Techniques for formulation and ~1ministration may be found in the latest edition of "Remington's Pharmaceutical Sciences"
(Mack Publishing Co, Easton PA). Suitable routes may, for example, include oral or s tr~n~mllcosal ~(lmini~tration; parenteral delivery, including intramuscular, subcutaneous, - inkarnedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal ~mini ~tration.
For injection, the ph~ el-tical compositions of the invention may be form~ te~l in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, 0 Ringer's solution, or physiologically buffered saline. For tissue or cellular ~lmini~tration, penetrants dl,~rupl;ate to the particular barrier to be perme~te~l are used in the forrnulation. Such penetrants are generally known in the art.
The pharmaceutical compositions can be formulated using ph~rn ~ eutically acceptable carriers well known in the art in dosages suitable for oral ~lmini~tration. Such carriers enable the 5 pharrnaceutical compositions to be form~ tecl as tablets, pills, capsules, liquids, gels, syrups, slurries, sllcp~?ncions and the like, for oral or nasal ingestion by a patient to be treated.
Phz~rm~ceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. For example, an effective amount of MYD 1 18 may be that amount that 20 ameliorates the symptoms of ~nemi~, fatigue, splenomegaly, hyperrnetabolism and thrombohemorrhagic complications. Deterrnination of effective amounts is well within the capability of those skilled in the art, especially in light of the disclosure provided below.
In addition to the active ingredients these pharmaceutical compositions may contain suitable ph~rrn~f~eutically acceptable carriers comprising excipients and auxiliaries which 25 facilitate processing of the active compounds into preparations which can be used ph~rm~eutically. The pl~l)aldlions formulated for oral ~-lmini~tration may be in the form of tablets, dragees, capsules, or solutions.
The ph~rm:~elltical compositions of the present invention may be m~nllf~rtured in a manner that is itself known, eg, by means of conventional mixing, dissolving, gr~n~ ting, 30 dragee-m~king, levigating, emulsifying, encapsulating, e,~ L,pillg or Iyophili~in~ processes.
Ph~rm~cel~tical formulations for p~cllleldl ~imini~tration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds SUBSTITUTE SHEET (RULE 26) CA 02246~78 1998-08-14 W O 97130157 PCT~US97/02458 -may be ~ d as ~ iate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or 5 dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the ~le~ualdlion of highly concentrated solutions.
Ph~ eutical ~cpaldLions for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the 10 mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
Suitable excipients are carbohydrate or protein fillers such as sugars, including lactose, sucrose, m~nnitol, or sorbitol; starch from corn, wheat, rice, potato, etc; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and gums including arabic and tr~ nth~ and proteins such as gelatin and collagen. If desired, 15 disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid or a salt thereof such as sodium z~ n~te~
Dragee cores are provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent 20 mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, ie, dosage.
Ph~ ceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients mixed with a filler or binders such as 25 lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid ~a~ , or liquid polyethylene glycol with or without stabilizers.
Compositions comprising a compound of the invention formulated in a ph~ eutical acceptable carrier may be prepared, placed in an appropriate container, and labeled for treatment 30 of an indicated condition. For polynucleotide or amino acid sequences of MYDl 18, conditions indicated on the label may include treatment of myeloproliferative ~lice~cs or leukemias.
The pharmaceutical composition may be provided as a salt and can be formed with many SU~ 111 ~JTE SHEET (RULE 26) CA 02246~78 1998-08-14 WO 97/301~i7 PCT~JS97/024~;8 acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forrns. In other cases, the preferred preparation may be a Iyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH range of 4.5 to 5.5 s that is combined with buffer prior to use.
- For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. Then, preferably, dosage can be formnl~ted in animal models (particularly murine models) to ach;eve a desirable circulating concentration range that adjusts MYDl 18 levels.
o A therapeutically effective dose refers to that amount of MYDl 18 which ameliorates symptoms of the disease state. Toxicity and therapeutic efficacy of such compounds can be rlet~rn~inçd by standard ph~rm~(~eutical procedures in cell cultures or experimental :~nim~l.c, eg, for det~rrnining the LD50 (the dose lethal to 50% of the population~ and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio I,D50/ED50. Compounds which exhibit large therapeutic indices are ~ler~ d. The data obtained from these cell culture assays and additional animal studies can be used in formulating a range of dosage for human use.
The dosage of such compounds lies preferably within a range of circ~ ting concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon 20 the dosage form employed, sensitivity of the patient, and the route of ~rlmini~tration~
The exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and ~mini~tration are adjusted to provide sufficient levels of the active moiety or to m~int~;n the desired effect. Additional factors which may be taken into account include the severity of the disease state; age, weight, and gender of the patient; diet, time and frequency of 25 ~lmini~tration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.
Long acting ph~rm~c~e~ltical compositions might be ~lmini~tered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.
- Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of ~flmini~tration. Guidance as to particular dosages and 30 methods of delivery is provided in the literature. See US Patent No. 4,657,760, 5,206,344; or 5,225,212. Those skilled in the art will employ dirrerellL formulations for MYD118 than for the inhibitors of MYDl 18. A~imini~tration to the bone marrow may necessitate delivery in a manner .. SlJI:s~~ JTE SHEET (RULE 26~

CA 02246~78 1998-08-14 W O 97/301S7 PCTrUS97/024~8 different from intravenous injections.
These examples are provided by way of illustration and are not included for the purpose of limiting the invention.

s INDUSTRIAL APPLICABILITY
SPLNFET01 cDNA Library Construction and Isolation of cDNA Clones The human spleen cell cDNA library (SPLNFET01) was custom constructed by Stratagene. The tissue was obtained from fetal spleens pooled from different sources and contained many different types of cells. Poly(A+) RNA (mRNA) was purified, and cDNA was o synthesized from the mRNA. Synthetic adaptor oligonucleotides were ligated onto cDNA ends enabling its insertion into Uni-ZAPTM vector system (Stratagene), allowing high efficiency unidirectional (sense orientation) lambda library construction and the convenience of a plasmid system with blue/white color selection to detect clones with cDNA insertions. Alternative unidirectional vectors are pcDNAl (Invitrogen, San Diego CA~ and pSHlox-l (Novagen, 15 Madison WI).
The custom-constructed library phage particles were transfected into E~. coli host strain XL 1 -Blue~) (Stratagene), which has a high transformation efficiency, increasing the probability of obtaining rare, under-represented clones in the cDNA library.
The phagemid forms of individual cDNA clones were obtained by the in vivo excision 20 process, in which the host bacterial strain was coinfected with both the lambda library phage and an fl helper phage. Proteins derived from both the library-cont~ining phage and the helper phage nicked the lambda DNA, initiated new DNA synthesis from defined sequences on the lambda target DNA and created a smaller~ single stranded circular phagemid DNA molecule that included all DNA sequences of the pBluescript(~) plasmid and the cDNA insert. The phagemid 25 DNA was secreted from the cells and purified, then used to re-infect fresh host cells, where the double stranded phagemid DNA was produced. Because the phagemid carries the gene ~or 13-t~m~e, the newly-transformed bacteria were selected on medium cont~inin~ ampicillin.
Phagemid DNA was purified using the QIAwell-8 Plasmid, QIAwell P~US, or QIAwell ULTRA DNA~ purification system (QIAGEN Inc Chatsworth CA). This product line provides a 30 convenient, rapid and reliable high-throughput method to lyse bacterial cells and isolate highly purified phagemid DNA using QIAGEN anion-~-~eh~n~e resin particles with EMPORE~Mmembrane technology (3M, Minneapolis MN) in a multiwell format. The DNA was eluted from SU~ 1~1 LITE SHEET (RULE 26) W~ 97/30157 PCT~US97J~24~8 the purification resin already prepared for DNA sequencing and other analytical manipulations.
The cDNAs were sequenced by the method of Sanger F and A3~ Coulson (1975; J Mol Biol 94:441f), using the Catalyst 800 and 373 DNA Sequencing System (Perkin Elmer).
II Homology Searching of cDNA Clones and Their Deduced Proteins s Each cDNA was compared to sequences in GenBank using a search algo,ilhll, d - incorporated into the ABI INHERITTM 670 Sequence Analysis System (Perkin Elmer3. In this algorithm, Pattern Specification Language (TRW Inc, Los Angeles CA) was used to lletermine regions of homology. The three parameters that determine how the sequence comparisons run were window size, window offset, and error tolerance. Using a combination of these three o parameters, the DNA database was searched for sequences cont~inin~ regions of homology to the ~uery se~uence, and the appropriate sequences were scored with an initial value. Subsequently, these homologous regions were examined using dot matrix homology plots to distinguish regions of homology from chance matches. Smith-W~terrn~n alignments were used to display the results of the homology search.
BLAST, which stands for Basic Local ~lignment Search Tool (Altschul SF (1993) J Mol Evol 36:290-3û0; Altschul, SF et al (1990) J Mol Biol 215:403-10), was used to search for local sequence alignments . BLAST produces alignments of both nucleotide and amino acid sequences to determine sequence similarity. Because of the local nature of the ~ nments, BLAST is especially useful in determining exact matches or in identifying homologs. 13LAST is useful for 20 m~tf~hes which do not contain gaps. The filn~l~ment~l unit of BLAST algorithm output is the High-scoring Segment Pair (HSP).
An HSP consists of two sequence fragments of arbitrary but equal lengths whose alignment is locally maximal and for which the ~ nment score meets or exceeds a threshold or cutoff score set by the user. The BLAST approach is to look for HSPs between a query sequence 25 and a fl~t~bS-ce sequence, to evaluate the statistical significance of any m~t~hec found, and to report only those matches which satisfy the user-selected threshold of significance. The parameter E establishes the statistically significant threshold for reporting ~ t~qk~e sequence - matches. E is interpreted as the upper bound of the expected frequency of chance occurrence of an HSP (or set of HSPs) within the context of the entire ~l~t~k~e search. Any ~l~t~b~e sequence 30 whose match satisfies E is reported in the program output.
M YD118 was identified using the ABI INHERITTM DNA Analysis System (Perkin Elmer) software which identified clone 25214 as being related to human GADD45 and murine SU~ JTE SHEET (RULE 26 CA 02246~78 1998-08-14 W O 97/301S7 PCT~US97/~2458 MYD 1 18. PCR extension analysis was performed to determine the 5' coding region. The nucleotide sequence for clone 25214 was subjected to a confirm~tory DNA sequence analysis.
The polynucleotide (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO:2) of MYDl 18 are , disclosed herein in Figure 1.
5 III Determination of Re~ling Frame of cDNA Clone The reading frame of individual cDNA~ clones obtained from the SPLNFET0 1 library was obtained by analyzing the polynucleotide sequences for the presence of start (ATG, GTG, etc.) and stop codons (TGA, TAA, TAG). Typically, one frame will continue throughout the major portion of all of a cDNA sequence and the other two pending frames tend to contain numerous o stop codons. Algorithms for dete~rrnining reading frame have been developed which analyze the occurrence of individual nuc}eotide bases of each putative codon triplet (eg, Fickett, ~W ( 1982) Nucleic Acids Res 10:5303-18). Coding DNA tends to contain predomin~ntly certainnucleotides within certain triplet periodicities, such as a significant ~ felellce for pyrimidines in the third codon position. These algorithms have been incorporated into widely available software 15 and can be easily used to determine coding potential (and frarne) of a given stretch of DNA. This algorithm-derived inforrnation, combined with start/stop codon inforrnation, was used to determine proper frame of individual clones within the SPLNFET0 1 library with a high degree of c~ ly, thus permitting the correct reading frame alignment with ~I.ropl ;ate expression vehicles.
20 IV F.~t~n~i~n of mydll8 to Recover Regulato~y Elements The nucleic acid sequence of mydl 18 may be used to design oligonucleotide primers for obtaining full length sequences from genomic libraries. One primer is synthesized to initiate extension in the antisense direction (XLR) and the other is synthesi7~d to extend sequence in the sense direction (XLF). The primers allow the known mydl 18 sequence to be extended ~5 "outward" generat;ng arnplicons con~ining new, unknown nucleotide sequence for the control region of interest. The initial primers are designed from the cl~lA using Oligo 4.0 (National Biosciences Inc, Plymouth MN), or another al~plvpl;ate program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at t~ eldl lres about 68~-72~ C. Any stretch of nucleotides which would result in hairpin structures and 30 primer-primer dimerizations is avoided.
A human genomic library is used to extend and amplify 5' upstream sequence. ~f necessary, a second set of primers is designed to further extend the known region. By following SUBSTITUTESHEET(RULE26) CA 02246~78 l998-08-l4 the instructions for the XL-PCR kit (Perkin Elmer) and thoroughly mixing the enzyme and reaction mix, high fidelity amplification is obtained. Beginning with 40 pmol of each primer and the recommended concentrations of all other components of the kit, PCR is performed using the Peltier Therrnal Cycler (PTC200; MJ Research, Watertown MA) and the following parameters:
5 Step 1 94~ C for 1 min (initial denaturation) Step 2 65~ C for 1 min Step 3 68~ C for 6 min Step 4 94~ C for 15 sec Step 5 65 ~ C for 1 min 1~ Step 6 68~ C for 7 min Step 7 Repeat step 4-6 for 15 additional cycles Step 8 94~ C for 15 sec Step 9 65~ C for 1 min Step 10 68~ C for 7:15 min Step 11 Repeat step 8-10 for 12 cycles Step 12 72~ C for 8 min Step 13 4~ C (and holding) A 5-10 ,ul aliquot of the reaction mixture is analyzed by electrophoresis on a low 2~ concentration ~about 0.6-0.8%) agarose mini-gel to determine which reactions were successful in extending the sequence. The largest products or bands were selected and cut out of the gel.
Further purification involves using a commercial gel extraction method such as QIAQuickTM
(QIAGEN Inc). After recovery of the DNA, Klenow enzyme was used to trim single-stranded, nucleotide overhangs creating blunt ends which facilitate religation and cloning.
After ethanol precipitation, the products are redissolved in 13 ,ul of ligation buffer, 1,ul T4-DNA ligase (15 units) and l,ul T4 polynucleotide kinase are added, and the mixture is incubated at room temperature for 2-3 hours or overnight at 16 ~ C. Competent E. ~Qli cells (in 40 ,ul of appropriate media) are transformed with 3 ,ul of ligation mixture and cultured in 80 ,ul of SOC m.~ m (Sarnbrook J et al, supra~. After incubation for one hour at 37~ C, the whole 30 transformation mixture is plated on Luria Bertani (LB)-agar (Sambrook J et al, supra) Con~ining 2x Carb. The following day, several colonies are randomly picked from each plate and cultured in 150 ,ul of liquid LB/2xCarb mediurn placed in an individual well of an appropriate, commercially-available, sterile 96-well microtiter plate. The following day, 5 ,ul of each overnight culture is transferred into a non-sterile 96-well plate and after dilution 1:10 with water, 35 5 ,ul of each sample is transferred into a PCR array.
For PCR amplification, 18 ,ul of concentrated PCR reaction mix (3.3x) cont~inin~ 4 units of rTth DNA polymerase, a vector primer and one or both of the gene specific primers used for SUBSTITUTE SHEET (RULE 26) CA 02246~78 1998-08-14 W O 97/301S7 PCT~US971~2458 -the extension reaction are added to each well. Amplification is performed using the following conditions:
Step 1 94~ C for 60 sec - ~Step 2 94~ C for 20 sec s Step 3 55 ~ C for 30 sec Step 4 72~ C for 90 sec Step 5 Repeat steps 2-4 for an additional 29 cycles Step 6 72 ~ C for 180 sec Step 7 4~ C (and holding) lo Aliquots of the PCR reactions are run on agarose gels together with molecular weight markers. The sizes of the PCR products are compared to the original partial cDNAs, and applo~liate clones are selected, }igated into plasmid and sequenced.
V Labeling of Hybridization Probes Hybridization probes derived from SEQ ID NO: 1 may be employed to screen cDNAs, 15 mRNAs or genomic DNAs. Although the labeling of oligonucleotides, consisting of about 20 base-pairs, is specifically described, essentially the same procedure may be used with larger cDNA fragments. Oligonucleotides are labeled by combining 50 pmol of each oligomer and 250 m~i of [g 32p] adenosine triphosphate (Arnersham, Chicago IL) and T4 polynucleotide kinase (DuPont NEN'19, Boston MA). The labeled oligonucleotides are purified with Sephadex G-25 20 super fine resin column (Pharmacia). A portion cont~ining 107 counts per minute of each is used in a typical membrane based hybridization analysis of hurnan genomic DNA digested with one of the following endonucleases (Ase I, Bgl II, EcoR I, Pst I, Xba 1, or Pvu II; DuPont NEN~).
The DNA from each digest is fractionated on a 0.7 percent agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is carried out 2s for 16 hours at 40~C. To remove nonspecific signals, blots are sequentially washed at room temperature under increasingly stringent conditions up to 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate. After XOMAT ARTM film (Kodak, Rochester NY) is exposed to the blots in a Phosphoimager cassette (Molecular Dyn~micc, Sunnyvale CA) for several hours, hybridization patterns are compared visually.
30 VI ~ntisense Molecules The mydl 18 sequence, or any part thereof, may be used to inhibit in viw or vitro expression of endogenous mydl 18. Although use of antisense oligonucleotides, consisting of about 20 base-pairs, is specifically described, essentially the same procedure may be used with larger cDNA fi~f~mentc An oligonucleotide based on the coding sequence of mydl 18 may be SUBSTITUTE SHEET(RULE2~) -WO 97/30157 PCT~US97~02458 used to inhibit t;x~les::iion of endogenous mydl 18. Using Oligo 40, the compleme~ntPry oligonucleotide can be designed from the conserved 5' sequence and used either to inhibit transcription by preventing promoter binding to the upstream nontranslated sequence or translation of an mydl 18 transcript by preventing the ribosome from binding to the mRNA.
5 VII Production of MYD118 Specific Antibodies For production of polyclonal antibodies, the cleclllce~i amino acid sequence of MYDl 18 is analyzed using DNASTAR software (DNASTAR Inc) to deterrnine regions of high immunogenicity and a corresponding oligopeptide is synthesized and used to raise antibodies in rabbits. Analysis to select ~plu~liate epitopes, such as those near the C-terrnin~-~ or in adjacent l o hydrophilic regions is described by Ausubel FM et al (supra). An oligopeptide of about 15 residues in length is syntl~i7~cl using an ABI Peptide Synthf~si7Pr Model 431A (Perkin Elmer, Norwalk, CN) using fmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma) by reaction with M-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel FM et al, supra). Rabbits are imrnunized with the oligopeptide-KLH complex in complete Freund's 15 adjuvant. The resulting antisera are tested for antipeptide activity, for example, by binding the peptide to plastic, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radioio~lin~te-l goat anti-rabbit IgG.
VIII Purification of MYDl 18 Using Specific Antibodies Endogenous or recombinant MYDl 18 can be purified by immunoaffinity 20 chromatography using antibodies specific for MYDl 18. An immllno~ffinity column is constructed by covalently coupling MYDl 18 antibody to an activated chromatographic resin such as CnBr-activated Sepharose (Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
Media cont~ininp MYD 1 18 is passed over the irnmunoaffinity column, and the column is 25 washed under conditions that allow the preferential absorbance of MYD 1 18 ~eg, high ionic strength buffers in the presence of d~tc.gellL). The column is eluted under conditions that disrupt antibody/MYDl 18 binding (eg, a buffer of pH 2-3 or a high concentration of a chaotrope such as - urea or thiocyanate ion), and MYDl 18 is collected.
IX Identification of Molecules Which Interact with MYDl 18 MYDl 18, or biologically active ~rnent~ thereof, are labeled with '25I Bolton-Hunter reagent (Bolton, AE and Hunter, WM (1973) Biochem J 133: 529). Candidate small molecules previously arrayed in the wells of a 96 well plate are incubated with the labe}ed MYDl 18, SUBSTITUTE SHEET (RULE 26) CA 02246~78 1998-08-14 W O 97/30157 PCTrJS97/02458 washed and any wells with labeled MYD118 complex are assayed. Data obtained using ~lirrelellL
concentrations of MYD 1 18 are used to calculate values for the number, affinity, and association of MYD 1 18 with the candidate molecules.

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be ~ar~ to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embo-liment~, it should be understood that the invention as claimed should not be unduly limited 0 to such specific embo~liment~ Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are int~ncle~l to be within the scope of the following claims.

SUBSTITUTE SHEET (RULE 26) CA 02246~78 l998-08-l4 W O 97/301S7 PCT~US97~02458 SEQUENCE LISTING
(l) GENERAL INFORMATION
- - (i) APPLICANT: INCYTE PHARMACEUTICALS, INC.
(ii) TITLE OF THE INVENTION: NOVEL HUMAN MYELOID TERMINAL
DIFFERENTIATION RESPONSE GENE
- (iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Incyte Pharmaceuticals, Inc.
(B) STREET: 3174 Porter Drive (C) CITY: Palo Alto (D) STATE: CA
(E) COUNTRY: U.S.
(F) ZIP: 94303 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ Version 1.5 (vi) CURRENT APPLICATION DATA:
(A) PCT APPLICATION NUMBER: To Be Assigned (B) FILING DATE: Filed Herewith (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION SERIAL NUMBER: US 08/221,531 (B) FILING DATE: 02-FEB-1994 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION SERIAL NUMBER: US 08/602,208 (B) FILING DATE: 015-FEB-1996 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Billings, Lucy J.
(B) REGISTRATION NUMBER: 36,749 (C) REFERENCE/DOCKET NUMBER: PF-0054 PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 415-855-0555 (B) TELEFAX: 415-845-4166 (C) TELEX:

(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 745 base pairs (B) TYPE: nucleic acid - (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(vii) IMMEDIATE SOURCE:
(A) LIBRARY: Myeloid Terminal CA 02246~78 1998-08-14 W O 97/30157 PCT~US97/02458 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 160 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vii) IMMEDIATE SOURCE:
(A) LIBRARY: Myeloid Terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Thr Leu Glu Glu Leu Val Ala Cys Asp Asn Ala Ala Gln Lys Met 1 5 10 15~ln Thr Val Thr Ala Ala Val Glu Glu Leu Leu Val Ala Ala Gln.Arg Gln Asp Arg Leu Thr Val Gly Val Tyr Glu Ser Ala Lys Leu Met Asn Val Asp Pro Asp Ser Val Val Leu Cys Leu Leu Ala Ile Asn Glu Glu Glu Glu Asp Asp Ile Ala Leu Gln Ile His Phe Thr Leu Ile Gln Ser . 70 75 80~he Ser Cys Asn Asn Asp Ile Asn Ile Val Arg Val Ser Gly Met Gln 95~rg Leu Ala Gln Leu Leu Gly Glu Pro Ala Glu Thr Gln Gly Thr Thr Glu Ala Arg Asp Leu His Cys Leu Leu Val Thr Asn Pro His Thr Asp Ala Arg Lys Ser His Gly Leu Val Glu Val Ala Ser Tyr Cys Glu Glu Ser Arg Gly Asn Asn Gln Trp Val Pro Tyr Ile Ser Leu Gln Glu Arg 1~5 150 155 160 (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 165 amino acids (B) TYPE: amino acid ~C) STRANDEDNESS: single -(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide . .

CA 02246~78 l998-08-l4 WO 97/30157 PCT~US97J02453 (vii) IMMEDIATE SOURCE:
(A) LIBRARY: Myeloid Terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 Met Thr Leu Glu Glu Phe Ser Ala Gly Glu Gln Lys Thr Glu Arg Met 1 5 10 15~sp Lys Val Gly Asp Ala Leu Glu Glu Val Leu Ser ~ys Ala Leu Ser 30~ln Arg Thr Ile Thr Val Gly Val Tyr Glu Ala Ala Lys Leu Leu Asn Val Asp Pro Asp Asn Val Val Leu Cys Leu Leu Ala Ala Asp Glu Asp Asp Asp Arg Asp Val Ala Leu Gln Ile His Phe Thr Leu Ile Gln Ala 80~he Cys Cys Glu Asn Asp Ile Asn Ile Leu Arg Val Ser Asn Pro Gly 95~rg Leu Ala Glu Leu Leu Leu Leu Glu Thr Asp Ala Gly Pro Ala Ala 100 105 110~er Glu Gly Ala Glu Gln Pro Pro Asp Leu His Cys Val Leu Val Thr 115 lZ0 125 Asn Pro His Ser Ser Gln Trp Lys Asp Pro Ala Leu Ser Gln Leu Ile Cys Phe Cys Arg Glu Ser Arg Tyr Met Asp Gln Trp Val Pro Val Ile Asn Leu Pro Glu Arg (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 160 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vii) IMMEDIATE SOURCE:
(A) LIBRARY: Myeloid Terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Thr Leu Glu Glu Leu Val Ala Ser Asp Asn Ala Val Gln Lys Met 1 5 10 15~ln Ala Val Thr Ala Ala Val Glu Gln Leu Leu Val Ala Ala Gln Arg Gln Asp Arg Leu Thr Val Gly Val Tyr Glu Ala Ala Lys Leu Met Asn -Val Asp Pro Asp Ser Val Val Leu Cys Leu Leu Ala Ile Asp Glu Glu Glu Glu Asp Asp Ile Ala Leu Gln Ile His Phe Thr Leu Ile Gln Ser 80~he Cys Cys Asp Asn Asp Ile Asp Ile Val Arg Val Ser Gly Met Gln 95~rg Leu Ala Gln Leu Leu Gly Glu Pro Ala Glu Thr Leu Gly Thr Thr 100 105 110~lu Ala Arg Asp Leu His Cys Leu Leu Val Thr Asn Cys His Thr Asp 115 120 125~er Trp Lys Ser Gln Gly Leu Val Glu Val Ala Ser Tyr Cys Glu Glu CA 02246578 l998-08-l4 W O 97/30157 PCTrUS97/02458 130 135 .~ 140 Ser Arg Gly Asn Asn Gln Trp Val Pro Tyr Ile Ser Leu Glu Glu Arg

Claims (20)

1. A purified polynucleotide comprising a nucleic acid sequence encoding the polypeptide having the sequence shown in SEQ ID NO: 2.

2. The purified polynucleotide of claim 1 wherein the nucleic acid sequence comprises the sequence shown in SEQ ID NO:1.

3 . An antisense molecule comprising the complement of the polynucleotide of claim 1 or a portion thereof.

4. An expression vector comprising the polynucleotide of claim 1.

5. A host cell transformed with the expression vector of claim 4.

6. A method for producing the polypeptide having the amino acid sequence for MYD118 (SEQ ID NO:2), said method comprising the steps of:
a) culturing the host cell of claim 5 under conditions suitable for the expression of said polypeptide, and b) recovering said polypeptide from the host cell culture.

7. A purified polypeptide comprising the amino acid sequence for MYD118 as shown in SEQ ID NO:2.

8. A diagnostic composition for the detection of myd118 polynucleotide sequences comprising the polynucleotide of claim 1.

9. A diagnostic test for the detection of myd118 polynucleotide sequences in a biological sample, comprising the steps of:
a) combining the biological sample with a first nucleotide sequence of SEQ ID NO:
1, or a non-conserved fragment thereof, under conditions suitable for the formation of a nucleic acid hybridization complex, b) detecting said hybridization complex, wherein the presence of said complex correlates with the presence of a second nucleotide sequence comprising myd118 polynucleotide sequences in said biological sample, and c) comparing the amount of the second nucleotide sequence in said sample with a standard thereby determining whether the amount of said second nucleotide sequence varies from said standard, wherein the presence of an abnormal level of said second nucleotide sequence correlates positively with a myeloproliferative disease.

10. A diagnostic test for the detection of myd118 polynucleotide sequences in a biological sample, comprising the steps of:
a) combining the biological sample with polymerase chain reaction primer under conditions suitable for nucleic acid amplification, wherein said primers comprise non-conserved fragments of the nucleotide sequence of SEQ ID NO: 1, b) detecting amplified nucleotide sequences, and c) comparing the amount of amplified nucleotide sequences in said biological sample with a standard thereby determining whether the amount of said nucleotidesequence varies from said standard, wherein the presence of an abnormal level of said nucleotide sequence correlates positively with a myeloproliferative disease.

11. A method of screening a plurality of compounds for specific binding affinity with the polypeptide of claim 7 or a portion thereof comprising the steps of:
a) providing a plurality of compounds;
b) combining the polypeptide of claim 7 with each of a plurality of compounds for a time sufficient to allow binding under suitable conditions; and c) detecting binding of said polypeptide of claim 7 to each of the plurality of compounds, thereby identifying the compounds which specifically bind said polypeptide of claim 7.

12. A method for inducing terminal differentiation of human proliferating myeloid cells comprising administering an effective amount of the polynucleotide of claim 1 to said myeloid cells under conditions suitable for expression of MYD118 (SEQ ID NO:2).

13. A method for inducing terminal differentiation of human proliferating myeloid cells comprising administering an effective amount of the polypeptide of claim 7 to said myeloid cells.

14. A method for inhibiting the expression of MYD118 in human hematopoietic cells comprising administering an effective amount of the antisense molecule of claim 3 to said cells.

15. The method of claim 14 wherein said hematopoietic cells are in vitro.

16. A pharmaceutical composition for treating an individual having a myeloproliferative disease comprising an effective amount of the polynucleotide of claim 1, or a biologically active fragment thereof.

17. A method of treating an individual having a myeloproliferative disease comprising administering an effective amount of the pharmaceutical composition of claim 16 to said individual.

18. A pharmaceutical composition for treating an individual having a myeloproliferative disease comprising an effective amount of the polypeptide of claim 7, or a biologically active fragment thereof.

19. A method of treating an individual having a myeloproliferative disease comprising administering an effective amount of the pharmaceutical composition of claim 18 to said individual.

20. An antibody specific for the polypeptide of claim 7.