CA2238885A1 - Nucleic acids encoding interferon gamma inducing factor-2 - Google Patents

Abstract

The present invention provides a polynucleotide (igif-2) which identifies and encodes a novel interferon gamma inducing factor-2 (IGIF-2) which was expressed in adenoid, brain, kidney, liver, lung, skin, synovium, and Tlymphocytes. The present invention also provides for antisense molecules. The invention further provides genetically engineered expression vectors and host cells for the production of purified IGIF-2; antibodies, antagonists and inhibitors; and pharmaceutical compositions and methods of treatment based on the polypeptide, its antibodies, antagonists and inhibitors. The invention specifically provides for use of the polypeptide as therapeutic for immunocompromised individuals and as a positive control in diagnostic assays for the detection of aberrant IGIF-2 expression or altered leukocyte or lymphocyte activity.

Description

CA 02238885 1998-0~-29 W O 97/24441 PCTrUS96/20432 NUCLEIC A~IDS ENCODING INTERFERON G~MMA lNDUCING FACTOR-2 TECHNICAL FIELD
The present invention relates to nucleic acid and amino acid sequences of a novel cytokine~ interferon gamma inducing factor-2 and to the use of these sequences in the diagnosis~ study~ prevention and tre~tment of disease.

I~ACKGROUND ART
Cytokines are active in cell proliferation, differentiation and movement at picomolar to nanomolar conc~ dlions and have effects on such activities as leukocyte migration and function, hematopoietic cell numbers. temperature regulation, acute response to infections. tissue remodeling and cell survival. Since cytokines are produced in groups and in patterns characteristic of the particular stimulus or disease, studies using antibodies or other drugs that modify the activity of a particular cytokine are beginning to elucidate the roles of individual cytokines in pathology and physiology. For purposes of example, two cytokines that are rapidly expressed in response to infl~mm~tion interferon gam~na (IF'N-y) and interleukin-12 (IL-12), will be described.
IFN-y is a pleiotropic cytokine involved in the regulation of immllne and infl~mm~t-~ry responses. It is produced by CD4+, CD8;, andThl lymphocytes and natural killer cells in response to antigens or mitogens. IFN-y is a homodimer formed by the antiparallel association of two subunits. each with six a helices held together bv short non-helical sequences. The overall structure of this 40-70 kD dimer is globular. and it contains two potential N-glycosylation sites. The single gene which encodes IFN-y is located on the long arm of chromosome 12, and its low homology with sequences from other species accounts for species specific activity. IFN-y participates in the activation, growth and differentiation of T, B, natural killer and endothelial cells, macrophages and fibroblasts. IF'N-y enables IgG2a production, poten~i~tes the antiviral and antiproliferative activity of IFN a/l~, increases ~x~lcs~ion of class II major histocompatibilitv complex molecules on B cells and macrophages. and inhibits IgGl and Ig~ production. Th2 cell proliferation. and the effects of IL-3. IL-4, granulocyte-monocyte colonv stim~ ting factor (GM-CSF) and 1 NF-a on bone marrow cells. In --I

:

CA 0223888~ 1998-05-29 mbdel studies of septic shock and cerebral malaria. IFN- y has exacerbated disease pathology by generating toxic levels of TNF-a.
IL-l 2 is composed of two disulfide linked chains of 35 and 40 kD which forrn a bioloQically active heterodimer. Neither chain is closely related to known proteins. The 40 kD chain. which shows 70% homology between man and mouse. appears to be speeies specific and contains sequence motifs present in the Ig Sl~I)çrf~mily and the hematopoietin family of receptors. The known functions of IL-12 include activation of cytotoxic lymphocytes, induction of IFN- y synthesis by T cells and natural killer eells, mediation of the Th-1 response to antigenic ~h~llen~e, growth faetor-like stim~ tion of activated CD4-T cells, CD81T eells, and natural killer cells, and acceleration of the lytic activity of natural killer and lymphocyte or Iymphokine activated killer cells.
IFN-~ intl~l~ing faetor (IGIF) is a recently discovered cytokine from mouse liver (Okamura H et al. (1995) Nature 378:88-91). The IGIF gene encodes a unique precursor protein of 192 amino acids and a mature protein of 157 amino acids which has no glycosylation sites. The mRNA for IGIF has been found in Kupffer cells and activated ac.ophages, and the recombinant protein is a stronger inducer of IFN-y than IL-12.
Invçstig~ti~ nc with IL-12 and IGIF suggest that these proteins may work synergistically to induce Th-1 production of IFN-y, however, antibody studies demonstrate that IGIF and I~-12 use different p~lhw~ys to carry out this induction. Additionally, Okamura ~
demonstrated that ~1minictr~tion of anti-IGIF antibody prevented liver damage in miee treated with bacteria and challenged with lipopolysaccharides to induce toxic shock.
Okamura et al. suggest that IGIF may decrease leukocyte initiated tissue destruction in the liver and that modulation of IGIF may have other applications in infl~mm~t~rv ~1iC~ce~
sueh as AIDS, plllmon~ry tubereulosis, and leprosy.
Current teehniques for the diagnosis of abnorm~litie~ in inflarned or ~licç~ced tissues mainly rely on clinical observations or serological analyses of body fluids or tissues for hormones, polypeptides or various metabolites. h/l~mm~lc, however. may not manifest clinical symptoms early in the in~l~mmsltion and disease process, and serological analyses do not always differentiate between invasive ~icç~cçc and genetic syndromes which have similar or overlapping ranges. Thus, development of new diagnostic methods for the detection of inflslmm~fion and disease states would be important in providing early and accurate diagnosis. undersf~n~ling molecular pathoeenesis. and developing effective therapies.
Cytokines are reviewed, inter alia. in Callard RE and AJH Gearing (1994) The ~ytokine Fact~book~ ~c~ .mic Press, New York NY? Guyton, AC (1991) Textbook of S Medical Physiology, WB Saunders Co. Philadelphia PA. and Paul WE (1993) F~lndamental ~mmunolo~y. Raven Press, New York NY.

DISCLOSURE OF THE INVENTION
The present invention relates to a novel cytokine, h~ r~,rol1 gamma intl~lcing factor-2 (IGIF-2), found in cDNA libraries made from human adenoid. brain. kidney, liver. lung, skin, synovium, and T-lymphocytes, and to the use of the nucleic acid and amino acid sequences of this novel cytokine in the study, diagnosis. prevention and treatment of infl~mm~tion and ~ e~e IGIF-2 disclosed herein was first iclentified in Incyte Clone 631796 through a computer generated search for amino acid sequence ~lignnl~ont~ The present invention has a length of 193 amino acids and ~ xilllately 60% amino acid similarity to the ~a~
musculus amino acid sequence of interferon garnrna in-lllring factor (IGIF; Okamura H et al. (1995) Nature 378:88-91; GI 1064823). The nucleic acid sequence (shown in lower ca~se, igif-2), SEQ ID NO: 1, and amino acid sequence (shown in upper case. IGIF-2), SEQ ID NO: 2, are disclosed herein.
Also disclosed herein is an IGIF-2 variant cont~ining a point mutation at nucleotide 622 of SEQ ID NO:1 r~slllting in an arnino acid substitution at residue 140.
Igif-2 from the T-lymphocyte cDNA library has a guanine (G) at nucleotide position 622 of SEQ ID NO: 1 which encodes an ~u~hlille while Igif-2 from the liver cDNA library has a thymine (rI ) at nucleotide position 622 which encodes an isoleucine at amino acid residue 140. Also disclosed herein is an IGIF-2 variant found in infl~mf ~l adenoid which is 42 amino acids in length and shares the N-terminal 30 amino acid residues of IGIF-2 (SEQ ID NO:2), the additional amino acid residues (in single letter code) being GKVEMNLFFFAN (SEQ ID NO:3). This Igif-2 variant has a poly A tail and may represent an ~It~ tively spliced Igif-2 transcript.
IGIF has been shown to induce IFN-y, a cytokine involved in regulating the CA 0223888~ 1998-0~-29 imrnune response and shown to exacerbate disease pathology by generating toxic levels of TNF-a. A-lminictration of anti-IGIF has been shown to prevent liver damage in mice treated with bacteria and challenged with lipopolysaccharides to induce toxic shock.
Therefore. nucleic acid and amino acid sequences of the present invention will provide the basis for the development of diagnostic and tre~tmçnt methods for the early and accurate detection and treatment of disease states and conditions associated with infl~mm~tion and/or the c~ ion of IGIF-2. Furtherrnore, the nucleic acid and amino acid sequences disclosed herein will provide the basis for ~ gnostic and therapeutic compositions for the detection and tre~fment of disease states and conditions associated with infl~mm~fion and/or the expression of IGIF-2.
The po~ynucleotide sequence disclosed herein which encodes IGIF-2. or variants thereo~ provides the basis for ~lç~igning oligonucleotide probes for the diagnosis of disease and conditions associated with infl~mm~tion andlor the expression of I~IF-2.
Such probes may be used to diagnose infl~mm~tion and tissue destruction in cells and tissues before the onset of severe clinical ~yllly~ollls~ The invention also provides for igif-2 ~nti~çn~e molecules which may be used to ~limini~h or elimin,.te expression of genomic igif-~ nucleotide sequences in individuals subject to an overactive or hla~
immllne response such as in allergies and asthrna. The present invention also relates, in part~ to e~ ssion vectors and host cells CUlll~liSillg igif-2 for in vitro or in ViVQ
production of IGIF-2.
The present invention also relates to the use of IGIF-2, or fr~ment~ or variantsthereof, to produce anti-IGIF-2 antibodies and to screen for antagonists or inhibitors of IGIF-2 which can be used therapeutically to prevent IGIF-2 in~ .tion of proliferation, dirf~ Liation. and maturation of leukocytes and lymphocytes. Such antagonists orinhibitors can be used to downregulate the immune response thereby preventing the secretion of proteolytic enzymes which may cause profound tissue damage.
The present invention further relates to ~nmini~tration of compositions comprising purified IGIF-2 or variants thereof, to imrnune-co",~loll,ised individuals. such as individuals subject to HIV, for the purpose of inducing endogenous antiviral molecules, such as IFN-y~ and for inducing proliferation~ differentiation and ,,I~Lu~dLion of leukocytes and lymphocytes.

CA 0223888~ 1998 - 0~ - 29 The present invention also relates to compositions comprisin_ anti-IGIF-2 antibodies, or other antagonists or inhibitors for the diagnosis~ prevention or tre~tnn~nt of inherited or acquired rli~e~ce~, involving the abnormal expression of igif-2 or altered leukocyte or lymphocyte activity. Such conditions would include such as viral (AIDS, hepatitis), bacterial (septic shock), fungal (histoplasmosis) or helminthi~. infections;
allergies or asthma; mechanical injury through exposure ~to asbestos, coal dust. etc) or trauma; arteriosclerosis. atherogenesis or collagen vascular t1ice~cr~ hereditarv tli~ç~ce-s such as autoimmnne hemo~ytic ~nemi~ biliary cirrhosis, juvenile diabetes mellitus, lupus erythematosus, multiple sclerosis, mya~theni~ gravis, or rh~llm~toid arthritis; leukemia, Iymphomas or carcinomas; Crohn's or other infl~mm~trlry bowel fii~ es: or other conditions which involve the abnormal activity of leukocytes or Iymphocytes.
The igif-2 polynucleotide sequences disclosed herein oligonucleotides. fr~gmentcportions or ~nti~çn~e molecules thereof, may be used in diagnostic assays to detect and quantify levels of igif-2 mRNA in cells and tissues. For example, the igif-2 polynucleotide sequence may be used to detect related or identical sequences in solution-based~ membrane-based, or tissue-based assays to ~ gn~se abnorm~litie~ in gene lession. The invention further provides diagnostic assays and kits for the detection of IGIF-2 in cells and tissues comprising purified IGIF-2, which may be used as a positive control, and anti-IGIF-2 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 abnormal expression of IGIF-2 or altered leukocyte or lymphocyte activity.
Igif-2 ~nti~çn~e molecules, anti-IGIF-2 antibodies, antagonists or inhibitors ofIGIF-2 may be used for therapeutic purposes, for example, in inhibiting or nP~ltr~ ing ovc;l~;x~ ion of IGIF-2 associated with infl~mm~tion, for example, in individuals 2~ subject to hepatitis or p~cle~liLis. The present invention provides ph~ celltic~l compositions for the tre~tm~nt of disease states associated with abnormal expression of igif-2 or altered leukocyte or Iymphocyte activity. Such ph~ re~-tical compositions will comprise effective amounts of ~nti~Pn~e molecules capable of inhibiting transcription and/or translation of genomic polynucleotide sequences. anti-IGIF-2 antibodies. or antagonists or inhibitors of IGIF-2. Alternatively, the present invention also provides pharrn~relltical compositions comprising effective amounts of IGIF-2 polypeptide. or W O 97/24441 PCTA~S96/20432 variants thereof. for the treatment of immune-compromised individuals~ such as individuals subject to HIV, for the purpose of inducing endogenous antiviral molecules, such as IFN-y, and for inducing proliferation, differentiation and maturation of leukocytes and lymphocytes.
The present invention also encomp~ses the use of gene therapy methods for the introduction of nucleotide sequences of the present invention into individuals subject to diseases or conditions associated with immllne response.

BRIEF DESCRIPTION OF DRAWINGS
Figures 1 A and B displays the nucleic acid and amino acid sequences of i~ f~rungarnrna inducing factor-2. The ~lignment was produced using MacDNASIS PROTM
sofn~are (Hitachi Software F.ngineering Co., Ltd., San Bruno, CA).
Figure 2 shows the arnino acid sequence similarity between IGIF-2 (631796: SEQ
ID NO:2) and IGIF (GI 1064823; SEQ ID NO:4). Sequences shown were produced usingthe m~llti~e~uence alignm~nt program of DNASTARTM software (DNASTAR Inc, Madison WI) Figure 3 shows an image of the laboldL~J.y northern analysis of IGIF-2. The leftside of the image shows standard size markers, and the bottom shows the nurnbered lanes which contain nucleic acids extracted from: 1) spleen, 2) lymph node, 3) thymus, 4) appendix, 5) peripheral blood, 6) bone marrow, and 7) fetal liver. The IGIF-2 band appears in various lanes across the image at ~pl~oxilllately the sarne level as the 1.35 standard molecular weight marker.
Figure 4 shows the electronic northern analysis of IGIF-2 produced using Incyte clone 631796 and the LIFESEQlM d~ ha~e ancyte Ph~rm~el~fic~l~ Inc., Palo Alto, CA).
MODES FOR CARRYING OUT TIIE INVENTION
The present invention relates to a novel cytokine referred to herein as "inl.~f~gamrna in~ cing factor-2 " which was found expressed in cDNA libraries made fromhurnan kidney, liver, T lymphocytes and infl~m( ~l adenoid. As used herein. the abbreviation for the novel i~ .r~.un gamma inducing factor-2 in lower case (igif-2) refers to a nucleic acid sequence while the upper case (IGIF-2) refers to an arnino acid se~uence.
"Nucleic acid sequence" as used herein refers to an oligonucleotide. nucleotide or CA 0223888~ 1998 - 0~ - 29 polvnucleotide sequence. and fragments or portions thereof. and to DNA or RNA ofgenomic or synthetic origin which may be double-stranded or single-stranded whether representing the sense or ~nti~l~n~e strand. Similarly. "amino acid se~uence" as used herein refers to peptide or protein sequences or portions thereof.
As used herein. IGIF-2 refers to IGIF-2 from any species, including, 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. A preferred IGI~-2 variant is one having at least 80% amino acid sequence similarity, another r~ id IGIF-2 variant is one having at least 90% amino acid sequence simi}arity and another pl~rt;l.~,d IGIF-2 variant is one having at least 95% amino acid sequence similarity to the IGIF-2 amino acid sequence illustrated in Figure 1 (SEQ ID N(:):2). A
efel.ed IGIF-2 variant of the present invention is one having isoleucine at amino acid position 140 of SEQ ID NO:2. Another plcr~ d IGIF-2 variant is one having the first 30 amino acid residues of SEQ ID NO:2 with ~ tion~l amino acids GKVEMNLFFFAN
(SEQ ID N~:3) te~min~ting at amino acid residue 42.
IGIF-2 is a cytokine which is 60% homologous to IGIF at the amino acid level (GI1064823; Okamura H et al. (1995) Nature 378:88-91) as shown in the consensus sequences in Figure 2. Cytokines are involved in leukocyte and Iymphocyte cell proliferation, diff~lGllLiation, and movement, have effects on hematopoietic cell numbers, temperature regulation, acute response to infections, tissue remodeling and cell survival, and are known to be produced by damaged or stressed cells as well as cells of the immnnto system.
As used herein, "naturally occ-17rin~'' refers to an IGIF-2 with an mRNA sequence found in nature, and "biologically active" refers to an IGIF-2 having structural, regulatory or biochemical functions of the naturally occurring IGIF-2. ~ikewise, "imml~nological activity" is defined as the capability of the natural. recombinant or synthetic IGIF-2, or any oligopeptide thereof. to induce a specific immllne response in a~ul,~;ate animals or cells and to bind with specif1c antibodies.
The term "derivative" as used herein refers to the chemical modification of an igif-3~ 2 or the encoded IGIF-2. Illustrative of such modifications would be replacement of hydrogen by an alkyl, acyl. or amino group. An igif-2 derivative would encode a polvpeptide which retains f"::CPnti~l biological characteristics of IGIF-2 such as. for exarnple, the differentiation of monocytes.
As used herein, the terrn ';purified'' refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment and isolated or separated from at least one other component with which they are naturally associated.

The IGIF-2 Coding Sequences The nucleic acid (SEQ ID NO:I) and ~ eed amino acid sequences (SEQ ID
NO:2~ of IGIF-2 are shown in Figure 1. In accordance with the invention. any nucleotide sequence which encodes the amino acid sequence of IGIF-2 can be used to generaterecombinant molecules which express IGIF-2. Nucleotide sequences for igif-2 wereidentified through BLAST analysis of the N-te-m;n~l amino acid sequence derll~ce~l from Incyte Clone 631796 derived from the kidney cDNA library. The nucleic acid sequence of SEQ ID NO:l was assembled using Incyte Clones: 631796 from the kidney cDNA
library (-lç~ign~t~d KIDNNOT05); 450202 from the T- lymphocyte cDNA library n~t~-l TLYMNOT02); and 89908 from the liver cDNA library (deci~n~te(l LIVRNOT01). Incyte clone 159939 from the infl~m~d adenoid library (de~iEn~ted ADENINB01) is an exact match to Incyte Clone 631796 from nucleotides 30 to 294 and has a poly A+ tail. This clone appears to ~ ;S~ t an alternatively spliced IGIF-2 ~ sc.;p~. In IGIF-2 from the T-lymphocyte cDNA library, the nucleotide at position 622 of the nucleic acid sequence is G which would code for the arginine shown at position 140 in the amino acid sequence; however, at the same position in IGIF-2 from the liver cDNA library, the nucleotide at position 622 is T7 and the arnino acid would be isoleucine. Neither of these changes produce the lysine found at nucleotide position 622 in mouse and may r~ ,;.ent a polymorphic codon.
Methods for DNA sequencing are well known in the art and employ such enzymes as the Klenow fragment of DNA polymerase I, Sequenase~ (US Biochemical Corp, Cleveland OH)), Taq polymerase (Perkin Elmer, Foster City CA)~ thermostable T7 polymerase (Amersharn, Chicago IL), or combinations of recombinant polvmerases and 30~ proofreading exo~llcle~es such as the ELONGASETM Amplification system m~rkete~l by Gibco BRL (Gaithersburg MD) Methods to extend the DNA from an oli~onucleotide primer annealed to the DNA template of interest have been developed for both single-stranded and double-stranded templates. Chain termination reaction products were separated using electrophoresis and detected via their incorporated. Iabeled precursors.
Recent improvements in mecl~ l reaction ~,~pald~ion. sequencing and analysis have permitted expansion in the number of sequences that can be determined per day.
Preferably, the process is automated with m~t~hin~ s such as the Hamilton Micro Lab 2200 (Hamilton, ~eno NV), Peltier Thermal Cycler (PTC200; MJ Research, Watertown MA) and the Applied Biosystems (Foster City CA) Catalyst 800 and 377 and 373 DNA
sequencers.
The quality of any particular cDNA library may be ~lçt~rmin~d by p-,.r(~ g a pilot scale analysis of the cDNAs and ch.-~kin~ for p~lce~ ges of clones cont~ining vector. lambda or ~. ~Q1i DNA. mitochondrial or repetitive DNA, and clones with exact or homologous m~tr.htos to public t1~t~ees Extending igif-2 Polynucleotide S~ e The polynucleotide sequence of igif-2 may be extended lltili7ing partial nucleotide sequences from SEQ ID NO: 1 and various methods known in the art to procure U~ Usequences such as promoters and regulatory elen enf~ Sarkar, G. (1993; PCR Methods Applic. 2:318-322) ~ rloses "restriction-site polymerase chain reaction (PCR)" as a direct method which uses universal primers to retrieve unknown sequence ~ çnt 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 transcribed with an ~lu~l;ate RNA polymerase and sequenced using reverse transcriptase.
Inverse PCR can be used to arnplify or extend sequences using divergent primers based on a known region (Triglia T et al(1988) Nucleic Acids Res 16:8186). The primers may be designed using Oligo 4.0 (National Biosciences Inc, Plymouth MN). or another ~ ,iate program. to be 22-30 nucleotides in length, to have a GC content of 50~/O or more. and to anneal to the target sequence at te~ uc~ res 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.
Capture PCR (La~ Llu,ll M et al (1991) PCR Methods Applic 1~ 19) is a method for PCR amplification ûf DNA fragments ~ljacf~nt to a known sequence in human 5 _ and yeast artificial chromosome (YAC) DNA. Capture PCR also requires multiple restriction enzyme digestions and ligations to place an ~ngin.oPred double-stranded sequence into an unknown portion of the DNA molecule before PCR.
Parker JD et al (1991, Nucleic Acids Res 19:3055-60), teach walking PCR, a method for targeted gene walking which permits retrieval of unknown sequence. The PromoterFinderTMkit available from Clontech (Palo Alto CA) uses PCR, nested primers and PromoterFinder libraries to walk in genomic DNA. This process avoids the need to screen libraries and is useful in finding intron/exon ~unctions.
Another PCR method, ''Tn~roved Method for Obt~inin~ Full Ler~th cDNA
Sequen~f e" by Guegler et al, Patent Application Serial No 08/487,112, filed June 7, 1995 and hereby incorporated by l~ .ce, employs XL-PCRTM (Perkin-Elmer, Foster City CA) to amplify and/or extend nucleotide sequences.
PreferTed 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 u~all~ll 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 ~xtt n-iing 5' sequence.
A new method for analyzing either the size or confirrning the nucleotide sequence of seq~l~n~ing or PCR products is capillary electrophoresis. Systems for rapid sequencing are available from Perkin Elmer, Beckman Instruments (Fullerton CA), and other Cl Illp~l-ieS Capillary seqll~n~ing employs flowable polymers for electrophoretic separation, four dirrt .~n~ fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled devise camera.
Output/light hlL~ siLy is converted to electrical signal using al,plo~flate software (eg.
GenotyperTM and Sequence NavigatorTM from Perkin Elmer) and the entire process from loading of samples to computer analysis and electronic data displav is computer CA 02238885 l998-05-29 coMrolled. Capillary electrophoresis is particularly suited to the sequencing of small pieces of DNA which might be present in limited arnounts in a particular sarnple. The reproducible sequencing of up to 350 bp of M13 phage DNA in 30 min has been reported (Ruiz-Martinez MC et al (1993) Anal Chem 65:2851-8).

I ;xpression of igif-2 In accordance with the present invention, igif-2 polynucleotide sequences which encode IGIF-2~ fr~gment~, fusion proteins or functional equivalents thereof. may be used to ~ d~ recombinant DNA molecules that direct the e~les~ion of IGIF-2 in a~ ,pliate host cells. Due to the inherent degeneracy ofthe genetic code, DNA
sequences other than the nucleotide sequences of SEQ ID NO: 1 which encode sllhst~nti~lly the same or a functionally equivalent amino acid sequence~ may be used to clone and express IGIF-2. As will be understood by those of skill in the art~ it may be advantageous to produce IGIF-2-encoding nucleotide sequences pos~escing non-naturally occurring codons. Codons l~lc;r~led by a particular prokaryotic or eukaryotic host (Murray E et al (1989) Nuc Acids Res 17:) can be selecte~l, for ~mple, to increase the rate of IGIF-2 ~ ,on or to produce recombinant RNA l~ s~ having desirable properties, such as a longer half-life, than L~dlrs~ Ls produced from naturally oCcUrring sequence.
Also included within the scope of the present invention are polynucleotide sequences that are capable of hybridizing to the nucleotide sequence of Figure 1 under conditions of intermediate to m~im~l stringency. Hybridization conditions are based on the melting tel~lpc.c~ e (Tm) of the nucleic acid binding complex, as taught in Wahl GM
et al. (1987, Methods Enzymol 152:399-407) incol~olaled herein by reference. and confer a defined "stringency" as explained below.
~;tX;IIIII-~I stringency" typically occurs at about Tm-5~C (5~C below the Tm of the probe); "high stringency" at about 5~C to 10~C below Tm, "in~rm~Ai~t~ stringency"
at about 10~C to 20~C below Tm; and "low stringency" at about 20~C to 25~C belowTm. As will be understood by those of skill in the art, a maximurn stringencv hybridization can be used to identify or detect identical polynucleotide sequences while an interrn~ te (or low) strinPen~y hybridization can be used to identify or detect similar CA 0223888~ 1998-0~-29 or related polynucleotide sequences.
The terrn "hybridization" 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) ~iction~ry of Biotechnologv. Stocl~ton Press, New York NY). Amplification as carried out in polymerase chain reaction technolo~ies is described in Dieffenbach CW and GS
Dveksler (1995, PCR 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 aminoacid sequence in which one or more nucleotides or amino acid residues, respectively, are 1 0 absent.
As used herein an "insertion" or "addition" is that change in a nucleotide or arnino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as co-~ ~ed to the naturally occl-rring molecules.
As used herein "substitution" results from the repl~ em~nt of one or more nucleotides or amino acids by di~,~n~ nucleotides or amino acids. lc:a~ecLi~ly.
Variant igif-2 polynucleotide sequences may be used in accordance with the invention and include deletions, insertions or substitutions of different nucleotide residues resulting in a polynucleotide that encodes the sarne or a functionally equivalent IGIF-2.
Variant IGIF-2 protein may also be used in accordance with the invention and mayinclude deletions, insertions or substitutions of arnino acid residues as long as the result is a functionally equivalent IGIF-2.
Arnino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the ~mphir~thic nature of the residues as long as the biological activity of IGIF-2 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 include leucine, isoleucine, valine, glycine. alanine;
asparagine, ghlt~min(; serine. threonine phenyl~l~nine and tyrosine.
Included within the scope of the present invention are alleles of igi~-2. As used herein, an "allele" or "allelic sequence" is an alternative form of igif-2. Alleles result from a mutation ie, a change in the nucleic acid sequence. and generally produce altered CA 0223888~ 1998 - 0~ - 29 WO 97/24441 PCT/US96l20432 mRN.4.s or polypeptides whose structure or function may or may not be altered. Any given gene may have 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, one or more times in a given sequence.
The nucleotide sequences of the present invention may be Pn~in~ered in order to alter an igif-2 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, e~,? site-directed mutagenesis to insert new restriction sites, to alter glycosylation patterns. to change codon ~Icr~,~ence, etc.
In another embodiment of the invention. an igif-2 natural. modified or recombinant sequence may be 1igated to a heterologous sequence to encode a fusion protein. For example, for s-;-c~.ling of peptide libraries for inhibitors of IGIF-2 activity, it may be useful to encode a chimeric IGIF-2 protein ~Ay~;, .ing a heterologous epitope that is recognized by a conl~llel~;ially available antibody. A fusion protein may also be ~ngin.-Pred to contain a cleavage site located between an IGIF-2 sequence and the heterologous protein sequence, so that the IGIF-2 may be cleaved and purified away from the heterologous moiety.
In an alternate embodiment of the invention, the coding sequence of igif-2 couldbe svnthP~i7P~l, whole or in part, using chemical methods well known in the art (See Caruthers et al (1980) Nuc Acids Res Symp Ser 7:215-233; Crea and Horn (1980) Nuc Acids ~es 9:2331; ~f~ ci and Caruthers (1980) Tetrahedron Lett 21:719; and Chow and Kempe (1981) Nuc Acids Res 9:2807-2817). ~It~ tively, the protein itself could be produced using ~.hemics~l methods to synthPci7e an IGIF-2 amino acid sequence. whole or in part. For example, peptides can be synthP~i7Pd by solid phase techniques? cleaved from the resin. and purified by ~ aldLi~e high pe~ ."~ e liquid chromatography (eg, Crei~hton T (1983~ Proteins Structures And Molecul:~r Principles. WH Freeman and Co, New York NY). The composition of the synthetic peptides may be confirmed by arnino acid analysis or sequencing (eg, the Edman degradation procedure: Creighton. supra) Direct peptide synthesis can be performed using various solid-phase techniques (Roberge JY et al (1995) Science 269:202-204) and automated synthesis may be achieved. for example, using Applied Biosystems 431A Peptide Synth~si7Pr in accordance with the instructions provided by the m~nlif~ctllrer. Additionally the amino acid sequence of IGIF-2. or any part thereof. may be altered during direct synthesis and/or combined using chemical methods with other cytokine sequences. or any part thereof, to produce a variant polypeptide.

Expression Systems In order to express a biologically active lGIF-2, the nucleotide sequence codingfor IGIF-2, or a functional equivalent, is inserted into an ~p~u~ L~ e~l,res~ion vector, ie.
a vector which contains the n-oCPcc~ry el~m~ntc for the transcription and translation of the inserted coding sequence.
Methods which are well known to those skilled in the art can be used to construct expression vectors co~ ;"il~g an igif-2 coding sequence and ~lu~,.iate l~ls~-~;ylional or tr~ncl~tiorl~l controls. These methods include in yitro recombinant DNA techniques, synthetic techniques and in vivo recombination or genetic recombination. Such techniques are described in Sambrook, J. et al. (1989) Molec~ r Clo~ir~ A T ~horatory M~nn~l Cold Spring Harbor Press, Plainview, NY, and Ausubel, F.M. et al. (1989) Gurrent Protocols in Moll-rnl~r Biology. John Wiley & Sons, New York, NY.
A variety of expression vector/host systems may be utilized to contain and express an igif-2 coding sequence. These include but are not limited to bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA ex~re;,~ion vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus e~ aion vectors (eg, baculovirus), plant cell systems transfected with virus expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or kansforrned with bacterial e~ ession vectors (eg, Ti or pBR322 pl~cmi~l3; or animal cell systems.
The "control elements" or "regulatory sequences" of these systems vary in their skength and specificities and are those nonkr~ncl~ted regions of the vector~ enh~nc~rs, promoters, and 3' unk~ncl~ted regions~ which interact with host cellular prûteins to carry out ~ s.,.i~lion and kanslation~ Depending on the vector system and host utilized~ any CA 02238885 l998-0~-29 W O 97/24441 PCT~US96/2~432 number of suitable transcription and translation elements~ including constitutive and inducible promoters, may be used. For exarnple, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the Bluescript~ phagemid(Stratagene, LaJolla CA) and ptrp-lac hybrids and the like may be used. The baculovirus polyhedrin promoter may be used in insect cells. Promoters or çnh~n~ers derived from the genomes of plant cells (eg, heat shock, RUBISCO; and storage protein genes) or from plant viruses (eg, viral promoters or leader sequences) may be cloned into the vector. In m~mm~ n cell svstems, promoters from the mz-mm~ n genes or from m~rnm~ n viruses are most ~pL~ ;ate. If it is n~cesci~ y to ~ lleld~t~ a cell line that contains multiple copies of igif-2, vectors based on SV40 or EBV may be used with an ~ropliate selectable marker.
In bacterial systems, a number of ~x~l~ssion vectors may be selected depending upon the use inten-l~cl for IGIF-2. For exarnple, when large quantities of IGIF-2 are needed for the induction of antibodies, vectors which direct high level ~ s~.ion of fusion proteins that are readily purified may be desirable. Such vectors include, but are not limited to, the ~. ~21i cloning and ~A~ s~ion vector Bluescript~ (Stratagene), in which the igif-2 coding sequence rnay be ligated into the vector in frarne with sequences for the amino-te~,Tnin~l Met and the subsequent 7 residues of 13-galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke & Schuster (1989) J Biol Chem 264:5503-5509); and the like. pGEX vectors (P,.~lllega, Madison WI) may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems are ~ ign~d to include heparin, thrombin or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
In the yeast, Sacch~olnyces cerev;~iae. a nurnber of vectors co..~
constitutive or inducible promoters such as alpha factor. alcohol oxidase and PGH may be used. ror reviews. see Ausubel et al (supra) and Grant et al (1987) Methods Enzymol 153:516-544.
In cases where plant ~ ssion vectors are used, the expression of an IGIF-2 -WO 97/2'1441 PCT/US96/20432 coding sequence may be driven by any of a number of promoters. For example~ viral promoters such as the 35S and 19S promoters of CaMV (Brisson et al (1984) Nature310:511 -514) may be used alone or in combination with the omega leader sequence from TMV (T~k~m~t~u et al (1987) EMBO J 6:307-311). Alternatively, plant promoters such as the small subunit of RUBISCO (Coruzi et al (1984) EMBO J 3:1671-1680: Broglie et al (1984) Science 224:838-843); or heat shock promoters (Winter J and Sinibaldi RM
(1991) Results Probl Cell Differ 17:85-105) may be used. These constructs can beintroduced into plant cells by direct DNA transformation or pathogen-me~ ted transfection. For reviews of such techniques, see Hobbs S or Murry LE in McGraw Yearbook of Science and Technology (1992~ McGraw Hill New York NY, pp 191-196 orWei~sb~eh and Weissbach (1988) Methods for Plant Molecular Biology, Academic Press, Neu York NY, pp 421 -463.
An altern:~tive expression system which could be used to express igif-2 is an insect system. In one such system, Auto~rapha californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera fr Igiperda cells or in Trichoplusia larvae. The igif-2 coding seqllpnre may be cloned into a non-e~serltiz~
region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of igif-2 will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses are then used to infect ~. fi~iperda cells or Trichoplusia larvae in which IGIF-2 is ~ ,ssed (Smith et al (1983) J Virol 46:584; Engelhard EK et al (1994) Proc Nat Acad Sci 91 :3224-7).
In m~mm~ n host cells, a number of viral-based ~ ssion systems may be utilized. In cases where an adenovirus is used as an ~cyre5~ion vector. an igif-2 coding sequence may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader se4uence. Insertion in a non-~ssenti~l E 1 or E3 region of the viral genome will result in a viable virus capable of c~lGs~hlg IGIF-2 in infected host cells. (Logan and Shenk (1984) Proc Natl Acad Sci 81:3655-3659). In addition, L~ sc~ ion enh~ncers, such as the rous sarcoma virus (RSV) ~nh~n~er may be used to increase expression in m~mm~ n host cells.
Specific initiation signals may also be required for efficient trarlslation of an CA 0223888~ 1998-OS-29 WO 97/24441 PCTrUS96/20432 inserted igif-2 coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where igif-2. its initiation codon and upstream sequences are inserted into the ~yylu~liate expression vector, no additional translational control signals may be needed. However, in cases where only coding sequence. or a portion thereof. is inserted~ exogenous transcriptional control signals, including the ATG
~ initiation codon. must be provided. Furthermore, the initiation codon must be in the correct reading frarlle to ens,.re trzE~.scr;ptior. cf the entire insert.. Exogenous transcriptional elements and initiation codons can be of various origins, both natura} and synthetic. The efficiency of ~ es~ion may be enh~nre~l by the inclusion of enh~n~ers ~ylo~liate to the cell system (Scharf et al (1994) Results Probl Cell Differ 20: 125-62;
Bittner et al (1987) Methods Enzymol 153:516-544).
In addition. a host cell strain mav be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. ~uch mor~ tions of the polypeptide in-~lu~le7 but are not limited to~
acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing which cleaves a precursor form of the protein may also be hllyol ~anL for correct insertion, folding and/or function. Different host cells such as CHO, HeLa, MDC~K, 293, WI38, etc have specific cellular machinery and characteristic m~h~nicmc for such post-translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign protein.
For long-term, high-yield production of recombinant proteins. stable expression is yle~ll~d. For example, cell lines which stably express igif-2 may be transformed using expression vectors which contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media, before they are switched to selective media. The selectable marker confers recict~n~e to selection and allows identification of cells which have stably integrated the introduced sequences into their DNA. Reci~t~nt clumps of cells can be proliferated using tissue culture techniques ~yyl~yllate to the cell type.
Any number of selection svstems may be used to recover transformed cell line.
These include. but are not limited to, the herpes simplex virus thyrnidine kinase (Wigler WO 97t24441 PCT/US96/20432 et al (1977) Cell 11:223) and ~(lçnine phosphoribosyltransferase (Lowy et al (1980) Cell 22:817) genes which can be employed in tk- or aprt~ cells~ respectively. Also, antimetabolite antibiotic or herbicide resistance can be used as the basis of seleclion, for example, dhfr confers rç~i~t~nee to methotrexate (Wigler et al (1980) Natl Acad Sci 77:3567); npt, which confers re~i~t~nce to the aminoglycosides, neomycin and G~18 (Colberre-Garapin et al (1981) J Mol Biol 150: 1), and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase (Murry, supra). Additional selectable genes have been described, for exarnple, trpB, which allows cells to utilize indole in place of tr~toph~ll, or hisD, which allows cells to utilize histinol in place of hi~tiriin.o (Hartman and Mulligan (1988) Proc Natl Acad Sci 85:8047). Recently, the use of visible markers has gained popularity with such markers as 13 glucurnni~ e anthocvanin~ and luciferin being widely used not only to identify transformants. but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes CA et al. (1995) Methods Mol Biol 55:121-31).
Identifi~tion of Transformallts Co~~t~nirlg igi~-2 Although the presence/absence of marker gene ~ s~ion ~ugge~l~ that the gene of interest is also present, its presence and exl~.ei,~ion should be confirmed. For exarnple, if the igif-2 is ills~"ed within a marker gene sequence, recombinant cells co~ g igif-2 can be identified by the absence of rnarker gene function. Al~ Liv~ly, a marker gene can be placed in tandem with an igif-2 sequence under the control of a single promoter.
Expression of the marker gene in response to induction or selection usually inr~ic~tes expression of igif-2 as well.
~lt~-rn~tively, host cells which contain the coding sequence for igif-2 and express IGIF-2 may be identified by a variety of procedures known to those of skill in the art.
These procedures include, but are not lirnited to, DNA-DNA or DNA-RNA hybridization and protein bioassay or imml-nc-a~ay techniques which include mennhr~nl -based, solu~ion-based~ or chip-based technologies for the detection andlor quantification of the nucleic acid or protein.
The presence of the igif-2 polynucleotide sequence can be detected by DNA-DNA
or DNA-RNA hybridization or amplification using probes, portions or fr~gmentc of igif-CA 0223888~ 1998 - 0~ - 29 WO 97t24441 PCTIUS96/20432 2. ~ucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the igif-2 sequence to detect transformants cont~inin~ igif-2 DNA or RNA. As used herein "oligonucleotides' or "oligomers" refer to a nucleic acid se~uence of at least about 10 nucleotides and as many as about 60 nucleotides7 preferablv about 15 S to 30 nucleotides. and more preferably about 20-25 nucleotides which can be used as a probe or amplimer.
The t~ ;,sion of an IGIF-2 protein product can be ~scecsef~ biologically in a chemotaxis or Ca~~ mobilization assay or immunologically in Western blot, enzyme-linked imm~1n-~cs~ys (ELISA) and the like.
Falk WR et al (1980, J Tmmllnl~l Methods 33:239) first described the ~Ccescment of chemotactic activity using 48-well microchemotaxis chambers. In this assay, the expressed cy+ol~ine is placed in media on one side of a polycarbonate filter and a particular population of cells is suspended in the same media on the opposite side of the filter. Sufficient in~l-h~tion time allows the cells to traverse the filter in response to the cytol~ine col1c~ lld~ion gradient. Filters are recovered from each well, and the cells a&ering to the side of the filter facing the cytokine are typed and quantified.
Populations of cells used in such assays may include blood cells obtained from venipuncture or enriched populations of neutrophils, peripheral blood mononuclear cells, monocytes and lymphocytes obtained by density gr~1ient centrifugation and/or negative selection using antibodies specific for surface molecules of the non-desired population.
For example. inc~lb~ting a population of T cells with CD4+ and s~;~dLi~lg out CD4 bound T cells may result in a CD8+ enriched T-cell population.
To assay non-chemotactic activity of neutrophils and monocytes, testing may involve measurement of actin polymerization, increase in respiratory burst activity, degranulation of the ~ulo~l~ilic granule or mobilization of Ca++ and comparison of the results with standard measurements. The assay for mobilization of Ca++ as part of the signal tr~nc~h~ction pathway requires preloading neutrophils with a fluorescent probe whose emission characteristics have been altered by Ca++ binding. When the ~ells are exposed to an activating sfim~ c, Ca++ flux is cl~t( rmined by observation of the cells in a fluorometer. The measurement of Ca+~ mobilization has been described in G}~nkievicz G
et al (1985) J Biol Chem 260:3440, and McColl S et al (1993) J Immnnnl 150:4550-4555, _ CA 0223888~ l998-0~-29 WO 97/24441 PCT/US96t20432 incorporated herein by reference.
Degr~n~ tion and respiratory burst responses are similarly measured in monocytes (Zachariae COC et al. (1990) J Exp Med 171: 2177-82). Further measures of monocyte activation are regulation of adhesion molecule c~ cssion in lymphocytes(Jiang Y et al (1992) J Immunol 148: 2423-8; Taub D et al (1993) Science 260: 355-358).
A variety of protocols for detecting and measuring the expression of IGIF-2. using either polyclonal or monoclonal antibodies specific for the protein are known in the art.
Examples include enzyme-linked imml-n~corbent assay (EL}SA). radioirnmunoassay (RIA) and fluorescent activated cell sorting (FACS). A two-site. monoclonal-based imm~lnn~Cc~y litili7in~ monoclonal antibodies reactive to two non-illlclr~ g epitopes on IGIF-2 is ~.efc--cd, but a colll~cliti~e binding assay may be employed. These and other assays are described. among other places! in Hampton R et al (1990. Serolo~ical Methods a I~aboratory M~n~ APS Press, St Paul MN) and Maddox DE et al (1983, J ~xp Med 158:1211).
A wide variety of labels and conJugation techniques are known by those skilled in the art and can be used in various nucleic and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to igif-2 include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide. Alternatively, the igif-2 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. and may be used to synthrci7P RNA probes in vitro by addition of an ~l~lu~iate RNA polymerase such as T7, T3 or SP6 and labeled nucleotides.
A number of comp~nies such as Ph~rrnzlri~ Biotech (Piscataway NJ), Promega (Madison WI~, and US Biochçmicz-l Corp (Cleveland OH) supply comrnercial kits and protocols for these procedures. Suitable lc~clLel molecules or labels include those radionuclides. enzymes, fluorescent, chemilllminPscPnt, or chromo~enic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like. Patents te~chin~ the use of such labels include US Patents 3,817.837; 3,850,752; 3,939,350; 3,996,345: 4.277,437;

4,275rl49 and 4r366,241. Also, recombinant imrnunoglobulins may be produced as shown in US Patent No. 4.816,567 incol~ol~lcd herein by ~crclcllcc.

CA 0223888~ 1998-0~-29 Purification of IGIF-2 Host cells transformed with an igif-2 nucleotide sequence may be cultured under 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 sequence and/or the vector used. As will be understood by those of skill in the art~ ession vectors CO~ ling igif-2 can be ~1~cign~d with signal sequences which direct secretion of IGIF-2 through a particular prokaryotic or eukaryotic cell membrane. Other recombinant constructions may join igif-2 to nucleotide sequence encoding a polypeptide domain w'nich will f~cili1~te purification of soluble proteins (Kroll DJ et al (1993) DNA Cell Biol 12:441-53; see also above discussion of vectors cont~inin~ fusion proteins).
IGIF-2 may also be expressed as a recombinant protein with one or more additional polypeptide domains added to facilitate protein purification. Such purification facilitating domains include, but are not limited to, metal che1~ting peptides such as hictirl;n~ Lc,phan modules that allow purification on immobilized metals (Porath J
( 1992) Protein Expr Purif 3 :263-281), protein A domains that allow purification on immobilized immnnoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Tmmlln--~ Corp, Seattle WA). The inclusion of a cleavable linker se~uences such as Factor XA or enterokinase (Invitrogen, San Diego CA) between the purification domain and IGIF-2 is useful to facilitate pnrific~tion.

Uses of IC~IF-2 Based on its amino acid sequence similarity to IGIF and its ~ es~ion in Iymphocytes, leukocytes and inil~nn~d or cancerous tissues, IGIF-2 would appear to play a role in systemic defense. Therefore, IGIF-2, a~ti-IGIF-2 antibodies or other small molecules, biological or organic, that modulate the activity of IGIF-2 directly or indirectly can be used therapeutically in the treatment of disease states resulting from abnormal expression of IGIF-2; altered leukocyte or Iymphocyte activity; or altered immlme response.
A therapeutic composition comprising IGIF-2 may have application in the prevention and tre~tment of individuals subject to diseases or conditions which CA 0223888~ 1998-0~-29 compromise the immune system, eg, for example, HIV infection where it would be useful to induce endogenous antiviral molecules, eg, IFN-r. Given the homology between IGIF
(Okamura supra) and IGIF-2, IGIF-2 is expected to stim~ te production of IFN~. and to influence the development of Thl cells and to favorably modulate the response of Thl CD4-T and CD8-T cells in HIV infected individuals. Likewise, the modulation. i.e., down regulation! of i~ opliate Th2 responses or the induction of I~Ny mav be useful in treating allergies, particularly in hy~ci~ onsive individuals.
In another embodiment of the present invention, anti-IGIF-2 antibodies capable of neutralizing the activity of IGIF-2 may be used to prevent or treat conditions or disease states such as asthma or septic shock in which tissue destruction results from IGIF-2 ,es~ion in combination with the ex~lession of other cytokines. The ability of antibodies or ligands to modulate the effects of IGIF-2 may be measured using the microchemotaxis, Ca~ flux or other assays described infra.
Procedures well known in the art may be used for the production of antibodies toIGIF-~. Such antibodies include, but are not limited to, polyclonal, monoclonal,chimeric, single chain, Fab fragmt?ntc and fr~m~ntc produced by a Fab t~ .,s~ionlibrary. Neutra}izing antibodies, ie, those which inhibit biological activity of IGIF-2, are t~cpeci~lly pLc~llcd for diagnostics and therapellti~s For the production of antibodies, various hosts including goats, rabbits. rats, mice, etc may be immllni7lod by injection with IGIF-2 or any portion, fragment or oligopeptide which retains immunogenic properties. Depending on the host species, various adjuvants may be used to increase immlmnlogical response. Such adjuvants include~ but are not limited to, Freund's, mineral gels such as al-lminllm hydroxide, and surface active sllkst~nrec such as Iysolecithin, pluronic polyols, polyanions, peptides~ oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (bacilli Calmette-Guerin) and Coryneba~ ,.iull- parvum are potentially useful human adjuvants which may be employed if purified IGIF-2 is ~mini~red to immlln(~logically co~ ,olnised individuals for the purpose of stimulating systemic defense.
Monoclonal antibodies to IGIF-2 may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture.
These include but are not limited to the EBV hybridoma technique originallv described by CA 0223888~ 1998 - 0~ - 29 (Kohler G et al (1975) Nature 256:495-497: Kozbor D et al (1985) J Immunol Methods 81:31-42; Cote RJ et al (1983) Proc Natl Acad Sci 80:2026-2030: Cole SP et al (1984) Mol Cell Biol 62: 109-120). 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 appropriate 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 IGIF-2 specific single chain antibodies.
Antibodies may also be produced by in~ cing in vivo production in the Iymphocyte population 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).
Antibody fr~gm~nt~ which contain specific binding sites for IGIF-2 may also be ~ clalt:d. For example, such fr~gment~ include, but are not limited to, the F(ab')l ~gmPnt~ which can be produced by pepsin digestion of the antibody molecule and the Fab fr~gm~nt~ which can be gen~-r~t~cl by reducing the disulfide bridges of the F(ab')2 ~m~ntc ~ltPm~tively, Fab eA3Jl~ion libraries may be constructed to allow rapid and easy identification of monoclonal Fab fr~m~ntc with the desired specificity (Huse WD
et al (1989) Science 256:1275-1281).
IGIF-2-specific antibodies are useful for the diagnosis of conditions and diseases associated with e:Aplc:~sion of IGIF-2. A variety of protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such imml-no~ ys typically involve the formation of complexes between IGIF-2 and its specific antibody (or similar IGIF-2-binding molecule) and the measurement of complex formation. A two-site, monoclonal-based immunoassay utili7ing monoclonal antibodies reactive to two nonhllelr~li"gepitopes on a specific IGIF-2 protein is ~c~l~d, but a competitive binding assay may also be employed. These assays are described in Maddox DE et al (1983. J Exp Med 158:1211).

CA 0223888~ 1998-0~-29 Diagnostic Assays Using IGIF-2 Specific Antibodies IGIF-2 antibodies are useful for the diagnosis of conditions disorders or ~lic~se~
characterized bv abnormal expression of IGIF-2. Diagnostic assays for IGIF-2 include methods ~ltili7ing the antibody and a label to detect IGIF-2 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. Frequently, the polypeptides and antibodies will be labe}ed by joining them, either covalently or noncovalently. with a reporter molecule. A wide variety of reporter molecules are known, several of which were described above.
A variety of protocols for mf~Sllring IGIF-27 using either polyclonal or monoclonal antibodies specific for the r~eclive protein are known in the art. Examples include enzyme-linked immlm~sorbent assay (ELISA), radioimmlmc-assav (RIA) and fluorescent activated cell sorting (FA~S). A two-site, monoclonal-based immllno~ ,y lltili7ing monoclonal antibodies reactive to two non-interfering epitopes on IGIF-2 is preferred, but a cv.~ e binding assay may be employed. These assays are described, arnong other places. in Maddox, DE et al (1983, J Exp Med 158:1211).
In order to provide a basis for the diagnosis of disease, normal or standard values for IGIF-2 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 IGIF-2 under conditions suitable for complex formation which are well known in the art. The amount of standard complex forrnation may be qll~n1ifiefl by comp~ring it with a dilution series of positive controls where a known amount of antibody is combined with known concentrations of purified IGIF-2 . Then7 standard values obtained from norrnal samples may be compared with values obtained from samples from subjects affected by a immllnological disorder or disease related to IGIF-2 expression. Deviation between standard and subject values establishes the presence of the disease state.

Drug Screening IGIF-2. ilS immun-)genic fr~gment~ or oligopeptides can be used for ~c~eel hlg therapeutic compounds in any of a variety of drug screening techniques. The fra_ment employed in such a test may be free in solution. affixed to a solid support. borne on a cell CA 0223888~ 1998-05-29 W O 97/24441 PCTrUS96/20432 surface. or located intracellularly. The abolition of catalytic activitv or the formation of bindin_ complexes. between I&IF-2 and the agent being tested~ may be measured.
Another technique for drug screening provides for high throughput screening of - compounds having suitable binding affinity to the IGIF-2 polypeptides and is described in detail in Guysen~ European Patent Application 84/03564, published on September 13, 1984. incorporated herein by reference. In summary, large numbers of different small peptide test compounds are synthPsi7~d on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with IGIF-2 fragment and washed.
Bound IGIF-2 is then detPcted by methods well known in the art. Purified IGIF-2 can also be coated directly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobili~ it on a solid support.
This invention also contemplates the use of col~lp~LiLive drug sclet;llillg assays in which neutralizing antibodies capable of binding IGIF-2 speçific~lly compete with a test compound for binding IGIF-2. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more ~ntigt?niC dete. ~ with IGIF-2.
Uses of igif-2 Polyn~le~tide An igif-2 polynucleotide, or any part thereof, may be used for ~ gn-lstjc and/ortherapeutic purposes. For diagnostic purposes. the igif-2 of this invention may be used to detect and quantitate abnormal gene expression in conditions, disorders or ~i~e~C~oc in which igif-2 activity may be implicated. These specifically include, but are not limited to, conditions with simi}ar biochemical or imml-n~logical properties such as viral (AIDS, hepatitis), bacterial (septic shock), fungal (histopl~mc-sic) or helminthic infections;
allergies or asthma; mechanical injury through exposure (to asbestos, coal dust, etc) or trauma; arteriosclerosis, atherogenesis or collagen vascular dic~ces, hereditarv tiice~c~?c such as autoimmlln~ hemolytic anemia, biliary cirrhosis, juvenile diabetes mellitus. lupus erythem~tosllc, multiple sclerosis, my~theni~ gravis. or rheumatoid arthritis; leukemia, lymphomas or carcinomas; Crohn's or other infl~mm~tory bowel cii~ CeS: or other ~ 30 conditions which involve the abnormal activity of leukocytes or lymphocytes.
Included in the scope of the invention are oligonucleotide sequences. ~ntis~nce CA 0223888~ 1998-0~-29 RNA and DNA molecules and ribozymes. which function to inhibit translation of an igif-2. Such nucleic acid sequences may be used in the tre~tment of individuals sub~ect to an ~lice~ces or conditions associated wtih infl~mm~tion. Another aspect of the subject invention is to provide for hybridization or PCR probes which are capable of detecting S polynucleotide sequences. including genomic sequences, encoding IGIF-2 or closely related molecules. The specificity of the probe, ie, whether it is derived from a highly conserved, conserved or non-conserved region or domain, and the stringencv of the hybridization or amplification (high, interme~ te or low) will rletermin~ whether the probe idçntifies only naturally occ-lrring igif-2, related igif sequences? or other cytokine molecules. Probes for the detection of related nucleic acid sequences are selected from conserved or highly conserved igif-2 sequence~ and such probes may be used in a pool of degenerate probes. For the detection of identical nucleic acid se~uences. or where maximum specificity is desired, nucleic acid probes are selected from non-conserved nucleotide regions or unique regions of igif-2. As used herein, the term "non-conserved nucleotide region" refers to a nucleotide region that is unique to igif-2 and does not occur in IGIF or other cytokines.

Diagnostic Uses of igif-2 Polynurl~oti~le An IGIF-2 encoding polynucleotide sequence may be used for the ~ gno~i~ of ~i~e~es reslllting from abnormal ~:x~ ssion of igif-2. For example, polynucleotide sequences encoding IGIF-2 may be used in hybridization or PCR assays of tissues from biopsies or autopsies to detect abnormalities in igif-2 ~ lcs~ion. The form of such qualitative or 4..~ e methods may include Southern or northern analysis. dot blot or other membr~nto-based technologies; P{~R technologies; dip stick, pin or chip technologies; and ELISA or other multiple salnple format technologies. All of these techniques are well known in the art, and are in fact the basis of many co~ elcially available diagnostic kits.
Such assays may be tailored to evaluate the efficacy of a particular therapeutictre~tment regime and may be used in animal studies, in clinical trials. or in moniloring the treatment of an individual patient. In order to provide a basis for the diagnosis of disease.
a normal or standard profile for igif-2 expression must be established. This is -2~-.

CA 02238885 1998-0~-29 W O 97t24441 PCTrUS9~nO432 accomplished bv combining body fluids or cell extracts taken from normal subjects. either animal or human. with igif-2 or a portion thereof~ under conditions suitable forhybridization or amplification. Standard hybridization may be qu~ntified by cull.palil1g the values ûbtained for normal subjects with a dilution series of positive controls run in S the same experiment where a known amount of purified igif-2 is used. Standard values obtained from nor}nal samples may be colllpal cd with values obtained from sampies from subjects potentially affected by a disorder or disease related to igif-2 G~rG~ion.
De~iation between standard and subject values establishes the presence of the disease state.
If disease is established. an e~i~ting thGId~GuLic agent is ~-lmini~t~red, and treatment 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 ll~~ profiles may be used to show the efficacy of treatment over a period of several days or several months.
PCR as described in US Patent Nos. 4,6~3,195; 4,800,195; and 4,965,188 provides additional uses for oligonucleotides based upon the igif-2 sequence. Such oligomers are generally chemically synthesized, but they may be generated Gl~ylll~Lically or produced from a recombinant source. Oligomers generally comprise two nucleotide sequences, one with sense orientation (5'->3') and one with ~nti~n~e (3'~-5'~ employed under oL,Lhl~i~ed 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 quantitation of closely related DNA or RNA sequences.
Additionally methods to ql~ntit~t~ the G~lG~ion of a particular molecule includeradiolabeling (Melby PC et al 1993 J ~mmllnol Methods 159:235-44) 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 sarnples may be spee~ed up by running the assay in an ELISA
format where the oligomer-of-interest is presented in various dilutions and a ~e-;~lo~l1otometric or colorimetric response gives rapid qll~ntit~tion~ For example, upregulation of igif-2 may result in an infl~mm~tc ry response, resulting in swelling and CA 0223888~ l998-0~-29 WO 97/24441 PCT/US96t20432 discomfort. In like manner. undt:lc~ession of igif-2 may result in an insufficient immunological response. In either case. a definitive diagnosis may allow health professionals to treat the patient and prevent further worsening of the condition.
Similarly, assays known to those of skill in the art can be used to monitor the progress of a patient displaying an igif-2 associated disease state during therapy.

Therapeutic Uses of an igif-2 Polynucleotide An igif-2 sequence may be useful in the tre~tm~nt of various abnormal conditions, including ~ ces or conditions wherein subjects are immllnc~collly~olllised, eg, HIV
infection, where it would be desirable to induce endogenous antiviral molecules. The introduction of the igif-2 sequence into cells can be used to induce IFN-y thereby s~im~ ting T cell populations. In such inct~nc~P~ the sequence encoding an IGIF-2 is int~ndf-d to supplement the activity of endogenous cytokines.
Igif-2 ~nti~n~e constructs may be useful in the tre?~tmtont of various ~hn~rrn~lconditions characterized by o~ res:~ion of igif-2 or other molecules of the imml-n~
system. The s~lccec~ful delivery and ~ c~ion of such sequences to individuals subject to such l1ice~cP~ will reduce or inhibit the transcription of igif-2 mRNA thereby rcd~lcin tissue damage resulting from infl~mm~tion.
Expression vectors derived from retroviruses, adenovirus, herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of recombinant igif-2~ sense or ~nti~Pnce molecules. to the targeted cell population. Methods which are well known to those skilled in the art can be used to construct recombinant vectors co~ g igif-2. See, for example, the techniques described in Sambrook et al (supra) and Ausubel et al (supra). Alternatively, recombinant igif-2 can be delivered to target cells in liposomes.
The full length cDNA sequence and/or its regulatory elements enable researchers to use igif-2 as a tool in sense (Youssoufian H and HF Lodish 1993 Mol Cell Biol 13 :98-104) or ~nti~.on~e (Eguchi et al (1991) Annu Rev Biochem 60:631-652) investi~ations of gene function. Oligonucleotides, decignl-d from the cDNA or control sequences obtained from the genomic DNA can be used in vitro or in vivo to inhibit expression. Suchtechnology is now well known in the art, and sense or ~nti~çn~e oligonucleotides or larger CA 0223888~ 1998-0~-29 fragments can be designed from various locations along the coding or control regions.
Additionally, igif-2 expression can be modulated by transfecting a cell or tissue with expression vectors which express high levels of an igif-2 fr~gn~nt Such constructs can flood cells with untr~n~l~t~hle sense or antisense sequences. Even in the absence of inte_ration into the DNA, such vectors may continue to transcribe RNA molecules until ~ all copies of the vector are disabled by endogenous nucleases. Such transient expression mav last for a month or more with a non-replicating vector (Mettler I, personal comm~mic~tion) and even longer if ~ .;ate replication elements are part of the vector system.
On the other hand, stable kansformation of ~I.lo~l;ate germ line cells, or preferably a zygote, with a vector co~ g the igif-2 fr~gmentc may produce a transgenic organism (US Patent No. 4,736,866, 12 April ~ 988), which produces enough copies of the sense or ~nfi~.once sequence to signific~ntly c~ lvlllise or entirely çlimin~tto activity of the endogenous igif-2 gene. Frequently, disruption of such genes can be asce.~illed by observing behaviors such as reduced infl~mm~tc-ry response or reduced leukocyte proliferation.
As mentioned previous}y, modifications of gene ~ression can be obtained by de~isnin~ ~nti~enSe sequences to the control regions of the igif-2 gene--the promoters, enhancers, and introns. Oligonuc}eotides derived from the transcription initiation site, eg, between -10 and +10 regions of the leader sequence. are preferred. ~nti~Pnce RNA and DNA molecules may also be ~1e~ignf~ to block translation of mRNA by preventing the sclipl from binding to ribosomes. Similarly, inhibition can be achieved using Hogeboom base-pairing methodology, also known as "kiple helix" base pairing. Triple helix pairing conl~.lolllises the ability of the double helix to open sufficiently for the binding of polymerases, kanscription factors, or regulatory molecules.
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The ml ~h~nicm of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complement~ry target RNA, followed by a ~ndon~lcleolytic cleavage. Within the scope of the invention are engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of igif-2 RNA sequences.

CA 0223888~ 1998-0~-29 WO 97t24441 PCT/US96/20432 Specific ribozyme cleavage sites within any potential RNA target are initially identified by ~c~nning the target molecule for ribozyme cleavage sites which include the following sequences. GUA~ GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target geneS cont~ining the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide seguence inoperable. The suitability of candidate targets may also be evaluated by testing ~cceccihility to hybridization with complementarv oligonucleotides using ribonuclease protection assays.
Both ~ntic~ nce R~A and DNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synth~ci7ing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated bv in vitro or ~n vivo transcription of DNA sequences encoding the ~ntic~-nce RNA molecule.
Such DNA seq~l~n~ toc may be inco.~oldl~d into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. ~AItern~tively, ~nticpnce cDNA constructs that synth~ci7~ ~nticence RNA con~LiluLiv~ly or inducibly can be introduced into cell lines, cells or tissues.
DNA molecules may be mo~1ifiecl to increase intr~t~tollular stability and half-life.
Possible modifications include, but are not limited to, the addition of fl~nking 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 ~iiccll~cecl in Section IV of the Exarnples. In addition, several of these transformation or transfection methods are equally suitable for the ex vivo therapy, the introduction of vectors into stem cells taken from the patient and clonally propagated for autologous transplant as described in US Patent Nos. 5,399,493 and 5,437,994, disclosed herein by ce.
~urthermore, the igif-2 polynucleotide sequences disclosed herein may be used inmolecular biology techniques that have not yet been developed, provided the new techniques rely on pl~pel Lies of nucleotide sequences that are currently known. including but not limited to such properties as the triplet genetic code and specific base pair CA 0223888~ 1998-0~-29 interactions.

Detection and Mapping o~ Polynucleotide Sequences Related to igif-2 The nucleic acid sequence for igif-2 can also be used to generate hybridization probes as previously described, for mapping the endogenous genomic sequence. Thesequence may be mapped to a particular chromosome or to a specific region of thechromosome using well l~nown techniques. These include in ~i~ hybridization to chromosomal spreads (Verma et al (1988) Hllmz~n Chromosomes: A Manual of Basic T~?rhnTques. Pergamon Press, New York City), flow-sorted chromosomal ~ ions, or artificial chromosome constructions such as YACs, bacterial artificial chromosomes (BACs), bacterial P 1 constructions or single chromosome cDNA libraries.
~ ~ hybridization of chromosomal ~ ~dLions and physical mapping techniques such as linkage analysis using established chromosomal markers are invaluable in e~ct~ lin~ genetic maps. Exarnples of genetic maps can be found in Science (1995; 270:410f and 1994; 265:1981f). Often the pl~ m~nt of a gene on the chromosome of another m~mms~ n species may reveal associated markers even if thenumber or arm of a particular human chromosome is not known. New sequences can be assigned to chronlc som~l arms, or parts thereof, by physical mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once a disease or syndrome, such as ataxia telangiectasia (AT), has been crudely localized by genetic linkage to a particular genomic region~ for example, AT to 1 lq22-23 (Gatti et al (1988) Nature 336:577-580). any sequences mapping to that area may represent associated or regulatory genes ~or further investigation. The nucleotide sequence of the subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion. etc bet~,veen normal. carrier or affected individuals.

Phar.~ r~ l Compositions The active compositions of the invention, which may comprise all or portions of IGIF-2 or inhibitors or antagonists, including antibodies. alone or in combination with at least one other agent, such as stabilizing compound, may be ~-1mini~tered in anv sterile, CA 0223888~ 1998-0~-29 WO 97/24441 PCT/US96t20432 biocompatible ph~n~elltical carrier. including. but not limited to. saline. buffered saline. dextrose. and water.
IGIF-2 can be ~lminictered to a patient alone. or in combination with other cytokines, agents. drugs or hormones or in pharmaceutical compositions where it is mixed with e~ccipient(s) or ph~rm~ eutically acceptable carriers. In one embodirnent of the present invention, the pharm~ eutically acceptable carrier is ph~mzl~e~ltir~lly inert.
Depending on the condition, disorder or disease being treated, these ph~rrn~rellti~ ~l compositions may be formulated and ~lminictered systemically or locally.
Techniques for formulation and ~rimini~tration may be found in the latest edition of "Remington's Ph~rrn~elltical Sciences" (Mack Publishing Co, Easton PA). Suitab}eroutes may, for example, include oral, transvaginal, or transmucosal ~(1mini~tration;
PLr~ e1d1 delivery. including intramuscular, subcutaneous, intr~m~d~ ry, intrathecal, intraventricular, intravenous, hlLldp~ oneaL or intranasal a~imini~tration. The p~ef~ c;d route for IGIF-2 or its inhibitors is intravenous ~imini~tration.
For injection. the rh~rrn~reutical compositions ofthe invention may be formni~t~d in aqueous solutions, preferably in physiologically compatible buffers such as ~anks's solution, Ringer's solution, or physiologically buffered saline. For tissue or cellular ~t1mini~tration, penetrants a~lo~l.dLe to the particular barrier to be p??~m~t~cl are used in the forrn~ ti- n. Such penetrants are generally known in the art.
The ph~rm~entical compositions can be forml-l~t~ using ph~rrn~reutically acceptable carriers well known in the art in dosages suitable for oral ~imini~tration. Such carriers enable the pharm~e~ltical compositions to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral or nasal ingestion by a patient to be treated.
Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the inten~d purpose. Det~rrnin~tion 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 ph~ eutical compositions may contain suitable pharrn~eutir~lly acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into ~,~pd-~Lions which CA 0223888~ 1998 - 0~ - 29 can be used ph~rrn~ceuticallv. The ~ ,a.dLions form~ ttocl for oral a-lminictration may be in the form of tablets. dragees, capsules. or solutions.
The pharmaceutical compositions of the present invention may be m~nllf~ctured ina manner that is itself known, eg, by means of conventional mixing, dissolving, gr~n~ ting, dragee-m~king, levigating, emulsifying, enr~ps~ ting ~n~ hlg or lyorhili7ing processes.
Ph~rrn~eutical formulations for l~e~lL~l~l administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as ~pp.O~.;ate 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 dextran. Optionally, the suspension may also contain suitable stabilizers or agents which illclease the solubility of the compounds to allow for the plc~dLion of highly concc;llLldLed solutions.
phs3rn~ eutical pre~Lions for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable ~llxili~riec~ if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, etc;
cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose. or sodium carboxymethylcellulose; and gums including arabic and trs~g~nth; and proteins such as gelatin and collagen. If desired, ~licintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid or a salt thereof such as sodium ~lginslt~.
Dragee cores are provided with suitable cozltingc such as concentrated sugar solutions, which may also contain gurn arabic. talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol. and/or LiL~lliull. dioxide. lacquer solutions, and suitable organic solvents or solvent 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.

CA 02238885 l998-05-29 Pharm~entical ~lepa.~Lions which can be used orally include push-fit capsules made of gelatin. as well as soft, sealed capsules made of gelatin and a coatin~ such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients mixed with a filler or binders such as }actose or starches, lubricants such as talc or m~gn~cium stearate~
and~ optionally. stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids~ such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabiii~rs.
Compositions coln~ ing a compound of the invention form~ te~i in a ph~rm~/~e~ltic~l acceptable carrier may be prepared, placed in an ~lv~liate container, and labeled for tre~tment of an indicated condition. For IGIF-2 inhibitors, conditions indicated on the label may include trP~tnnent of infl~mm~tinn.
The ph~rm~elltical composition may be provided as a salt and can be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic. Iactic, tartaric.
malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the c- l.e~onding free base forms. In other cases, the plef~ ,d ~r~pa-~Lion may be a lyophili7rd powder in lmM-50mM hi~ticlinP~ 0.1%-2% sucrose, 2%-7% mRnnitol at a pH range of 4.5 to 5.5 that is combined with buffer prior to use.
For any compound used in the method of the invention, the therapeutically effective dose can be e~l;,n~l~cl initially from cell culture assays. Then, preferably, dosage can be formnl~t~ i in animal models to achieve a desirable circulating conrentr~tion range that adjusts IGIF-2 levels. Such inforrnation can be used todetermine useful doses in hnm~nc. Examples of animal models useful for studying therapeutic applications of IGIF-2 or its inhibitors include those c~esrrihed in Hutz (1989) Biol Reproduction 40:709-713; Hutz et al. (1990) J Med Primatol 19:553-571; Kitzman et al. (1992) Cell Tissue Res 268:191-196; and Quandt et al. (1993) Biol Reprod 48:1088-1094.
A th.,.,.p~ 11 ;c~lly effective dose refers to that arnount of IGIF-2 or its inhibitor which ameliorates symptoms which may mean stim~ tinn of the immnn~ svstem and T
lymphocytes or the reduction of mfl~mm~tion and pain. Toxicity and therapeutic efficacy of such compounds can be iPt~rmin~?d by standard ph~ eutical procedures in cell cultures or e~ illlental ~nim~l~, eg, for determining the LD50 (the dose lethal to 50% of CA 0223888~ 1998 - 0~ - 29 the population) and the ED50 (the dose therapentiç~ily 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 LD50/ED50. Compounds which exhibit large therapeutic indices are ~}lef~ llcd. The data obtained from these cell culture assays and additional animal studies can be used in forrnulating a range of dosage for human use.
The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient. and the route of ~iminictration.
The exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and ~flminictration are adjusted to provide sufficient levels of the active moie~v or to m~int~in 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 ~-lmini~tration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long acting pharm~elltic~l compositions might be ~lminictered every 3 to 4 days, every week, or once every two weeks depending onhalf-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 ~lminicfration Guidance as to particular 2Q dosages and 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 .lirr~
formulations for IGIF-2 than for the inhibitors of IGIF-2. ~lminictration to the lungs may nec~ceit~te delivery in a manner different from that to the kidney, or stom~r~
Disease states or conditions associated with the imm-lne system which may be treated with nucleic acid or amino acid sequences disclosed herein include, but are not lilnited to, viral (AIDS, hepatitis), bacterial (septic shock), fungal (histopl~crnocic) or h~lminthic infections; allergies or ~cthm~ merh~nic~ iniury through exposure (toasbestos, coal dust, etc) or trauma; arteriosclerosis. atherogenesis or collagen vascular tlice~cçs, hereditary ~1ice~ces such as autoimml-ne hemolytic anemia, biliary cirrhosis, juvenile diabetes mellitus, lupus erythematosus. multiple sclerosis, myasthenia gravis, or rheurnatoid arthritis; leukemia, lymphomas or carcinomas; Crohn s or other infl~mm:~tory bowel (lice~ce~ or other conditions which involve the abnormal activity of leukocytes or lymphocytes and which may be specificallv diagnosed by the assays previously discussed.
These examples are provided by wav of illustration and are not included for the purpose of limiting the invention.
j l[NDUSTE~IAL APPLICABILITY
DNA Libraly Construction For purposes of discussion, ~ ,ald~ion of the kidney cDNA library is described.
cDNA libraries from other tissues in which IGIF-2 or its variants are found were ple~ d by similar methods well known to those of skill in the art.
The kidney cDNA library was constructed from 1.8 micrograms of mRNA made from kidney tissue of a two day old hispanic female (Lot #95-04-0274; International Tn~~ t~ for Advanced Medicine, Exton PA). The tissue was Iysed in buffer cnnt~ining gll~ni~linium isothiocyanate and the lysate was centrifuged over a 5.7 M CsCl cushion using an Beckman SW28 rotor in a Beckman L8-70M Ultracentrifuge (Beckman Instruments) for 18 hours at 25,000 rpm at ambient Lt;lllp~ Lul ~. The RNA was extracted once with acid phenol pH 4.0, plecipil~ted using 0.3 M sodium acetate and ~.5 volumes of ethanol, resuspended in water and DNase treated for 15 min at 37~C. The poly A' RNA was isolated using the Qiagen Oligotex kit (QIAGEN Inc, Chatsworth CA) The poly A+ RNA was handled according to the recomm~n~le~l protocols in the SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning (Catalog ~ 18248-013; Gibco/BRL). First strand cDNA synthesis was accomplished using oli_o d(T) priming and second strand synthesis was p~,ro""ed using a combination of DNA
polymerase I, ~. çs~ ligase and RNase H. The cDNA was blunted with T4 polymerase, and a Sal I linker was added to the blunt ended cDNA. The Sal I adapted, double-stranded cDNAs were the digested with Not I and fractionated on a Sepharose CL4B column (Catalog ;~275105, ph~ ). Those cDNAs e~ceerling 400 bp were ligated into pSportI which was subsequently transforrned into DH5aTM competent cells (Catalog #18258-012. Gibco/BRL).
II Isolation and Sequencing of cDNA Clones CA 02238885 l998-05-29 Plasmid DNA was released from the cells and purified using the Miniprep Kit (Cat~ 77468: Advanced Genetic Technologies Corporation~ Gaithersburg MD). This kit consists of a 96 well block with reagents for 960 purifications. The recommen~led protocol was employed except for the following changes: l ) the 96 wells were each filled with only 1 ml of sterile Terrific Broth (Cat~ 22711, LIFE TECHNOLOGIESTM, Gaithersburg MD) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2~ the bacteria were cultured for 24 hours after the wells were inoculated and then Iysed with 60 ,ul of Iysis buffer; 3) a centrifugation step employing the Ber~m~n GS-6R ~2900 rpm for 5 min was performed before the contents of the block were added to the primary filter plate, and ~ the optional step of adding is~.~,opal~ol to TRIS buffer was not routinelyperformed. After the last step in the protocol, samples were transferred to a Beckman 96-well block for storage.
The cDNAs were sequenced by the method of Sanger F and AR Coulson (1975; J
Mol Biol 94:441f), using a Hamilton Micro Lab 2200 (Hamilton, Reno NV) in combination with four Peltier Therrnal Cyclers (PTC200 from MJ Research, Watertown MA) and Applied Biosystems 377 or 373 DNA Sequencing Systems (Perkin Elmer~, andthe reading frame was ~letlorrninerl III Homology Searching of cDNA Clones and Their Deduced Proteins Each cDNA was compared to sequences in GenBank using a search algorithrn developed by Applied Biosystems and incorporated into the INHERITTM 670 SequenceAnalysis System. In this algorithm, Pattern Specification Language ~TRW Inc, LosAngeles CA) was used to determine regions of homology. The three parameters thatdetermine how the sequence comp~isons run were window size, window offset. and error tolerance. Using a combination of these three p~r~meters, the DNA rl~t~h~e was searched for sequences ~o~ g regions of homology to the query sequence. and the a~ iate sequences were scored with an initial value. Subsequently, these homologous regions were ex~min~cl using dot makix homology plots to distinguish regions of homology from chance m~t~hes Smith-W~ft-rm~n :~lignment~ were used to display the results of the homology search.
Peptide and protein sequence homologies were ascertained using the rNH~RITTM

WO 97t24441 PCT/US96/20432 670 Sequence Analysis System in a way similar to that used in DNA sequence homologies. Pattern Specification Language and pararneter windows were used to search protein ~l~t~haces for sequences cont~ining regions of homology which were scored with an initial value. Dot-matrix homology plots were ex~minPcl to distinguish re~ions of ci~nific~nt homology from chance m~trhPs BLAST~ which stands for Basic ~ocal ~lignmerlt Search Tool (Altschul SF
(19g3) J Mol Evol 36:290-300; Altschul, SF et al (199û) J Mol Biol 215:403-10), was used to search for local sequence ~lignm~nt~ . BLAST produces ~lignment~ of bothnucleotide and amino acid sequences to ~et~rrnin~- sequence similarity. Because of the local nature ofthe ~lig,."~ ;, BLAST is especially useful in clet~rmining exact m~t~h~s or in identifying homologs. BLAST is useful for m~trh~s which do not contain gaps. The fi~ ment~l unit of BLAST algorithm output is the High-scoring Segm~nt Pair (HSP).
An HSP consists of two sequence fr~gmçntc of al uiLId~ y but equal lengths whosenment is locally m~xim~I and for which the ~ nm~nt score meets or exceeds a threshold or cutoffscore set by the user. The BLAST approach is to look for HSPsbetween a query sequence and a tl~t~h~ce sequence. to evaluate the st~ti~tic~l significance of any m~trh~s found, and to report only those m~t~h~?s which satisfy the user-selected threshold of cj~nific~nce The p~r~m~t~r E establishes the statistically ~ignifir~nt threshold for reporting (l~t~h~ce sequence ~ cl-loc E is i-lLe,~l~Led as the upper bound of the e~rect~d frequency of chance oc~;ul.~nce of an HSP (or set of HSPs) within the context of the entire t1~t~h~ce search. Any ~l~t~hzlce sequence whose match satisfies E is reported in the program output.

IV Northern Analysis Northern analysis (Sambrook et al., supra) was used to detect the presence of anigif-2 L~ s~;L;~L among the mRNAs of imml-n~logical tissues. Fr~gm~nt~ of the igif-2 sequence (SEQ ID NO: 1) were randomly primed with radioactive label (see Exarnple and hybridized to a Multiple Tissue Northern blot (MTN; Clontech). Figure 3 shows the image of the resulting igif-2 northern. Expression of igif-2 is clearly shown as a band aligned with standard protein molecular weight marker 1.35 in the lanes cont~ining RNAs from appendix and peripheral blood as well as the lymphoid tissues--spleen. Iymph node, thvmus. and bone marrow). The igif-2 trar script was not ~l.ot.?ctecl in fetal liver RNA at the stringency used in this experiment.
Analogous electronic northern analysis used BLAST (Altschul, S.F. 1993 and 1990. supra) to search for identical or related molecules in the LIFESEQTM ~i~t~h~e S (Inc te ph~rm~ceuticals, Inc.).The sensitivity of the co~ ul~1 search was set at a product score of 50. Product score is defined as:
% seq-~nce itl~ntity x % rn~ximum BLAST score The product score takes into account both the degree of similarity between two sequences l 0 and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1-2% error; and at 70, the match will be exact. E~omologous molecules are usually identified by selecting those which show product scores between 15 and 40. although lower scores may identify related molecules.
Figure 4 shows the results of electronic northern analysis reported as a list oflibraries in which highly homologous molecules or tldllSelipl~ encoding IGIF occurred.
Abundance and percent abllntl~nre are also reported. Ab--ncl~n~e directly reflects the nurnber of times a particular transcript is represented in a cDNA library, and percent abl-n-i~nce is abun-l~n~e divided by the total number of sequences e~r~minP(l in the cDNA
library.
V F.~t~ncjon of igif-2 to Recover Regulatory Elements The nucleic acid sequence of full length igif-2 (SEQ ID NO:1) is used to design oligonucleotide primers for obtair~ing 5' sequences from genomic libraries. One primer is synth~-ci7~d to initiate extension in the ~nti~nce direction (XLR3 and the other is synth~si7~ A to extend sequence in the sense direction (XLF). The primers allowed the known igif-2 sequence to be extended "outward" g~ g amplicons cont~ining new, unknown nucleotide sequence for the control region of interest. The initial primers are de~i_n~l from the cDNA using Oligo 4.06 (National Biosciences Inc, Plymouth MN), or another a~ ;a~e 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 te~ dtllres about 68~-72~ C. Any stretch of nucleotides which would result in hairpin structures and primer-primer CA 0223888~ 1998-0~-29 dimerizations is avoided.
A human genomic library is used IO extend and amplify 5' ul,sllealll sequence. If necessary, a second set of primers is designed to further extend the known region. By following the instructions for the XL-PCR kit (Perkin Elmer) and thoroughly mixing the S enzvme and reaction mix, high fidelity amplification is obtained. Beginning with 40 pmol of each primer and the recommenllPd concentrations of all other components of the kit. PCR is ~ olllled using the Peltier Thermal Cycler (PTC200; MJ Research.
Watertown MA) and the following parameters:
Step 1 94~ C for l 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 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 lO 68~ C for 7:15 min Stepll Repeatstep8-10 forl2cycles 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 concellL-d~ion (about 0.6-0.8%) agarose mini-gel to ~terrninf~ which reactions were successful in ~t~n-ling 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 ~lecipi~Lion, the products are redissolved in 13 ~l of ligation buffer, l,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 coli cells (in 40 ,ul of ~ opliate media) are transformed with 3 ,ul of ligation mixture and cultured in 80 ,ul of SOC mediurn (Sambrook J et al, supra). After incubation for one hour at 37~ C~ the whole transforrnation mixture is plated on Luria Bertani (BB)-agar (Sambrook J et al. supra) cont~ining 2x Carb. The following day, several colonies are CA 02238885 1998-0~-29 W O 97/24441 PCTrUS96/20432 randomly picked from each plate and cultured in 150 ul of liquid LB/2xCarb medium placed in an individual well of an ~p~ iate, 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: l 0 with water, 5 ,ul of each sample is transferred into a PCR array.
For PC~ amplification, 18 ,ul of concentrated PCR reaction mix (3.3x) cont~ining4 units of rTth DNA polymerase, a vector primer and one or both of the gene specific primers used for the extension reaction are added to each well. Amplification isp~ ncd using the following conditions:
Step 1 94~ C for 60 sec Step 2 94~ C for 20 sec 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) Aliquots of the PCR reactions are run on agarose gels together with molecular weight m~rker.~. The sizes of the PCR products are compared to the original partial cDNAs, and a~.o~liate clones are selected, ligated into plasmid and sequenced.
VI T 9h-~ ~ of Hybridization Probes Hybridization probes derived from SEQ ID NO: 1 are employed to screen cDNAs, mRNAs or genomic DNAs. Although the labeling of oligonucleotides, consisting of about 20 base-pairs, is specifically described, e~el nti~lly the sarne procedure is used with larger cDNA fr~gm~ntc Oligonucleotides are labeled by combining 50 pmol of each oligomer and 250 mCi of [y-3''P] adenosine triphosphate (Amersham, Chicago IL) and T4 polynucleotide kinase (DuPont NEN~, Boston MA). The labeled oligonucleotides arepurified with Sephadex G-25 super fine resin column (Pha~.lla~;ia). A portion cont~ining 107 counts per minute of each is used in a typical membrane based hybridization analysis 3 0 of human genomic DNA digested with one of the following endonucleases (Ase I, Bgl II, EcoR I? Pst I, Xba 1, or PYU 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).

CA 02238885 l998-05-29 Hybridization is carried out for 16 hours at 40~C. To remove nonspecific signals. blots are sequentially washed at room telllpc:lalure 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 Dynamics, Sunnyvale CA) for several hours, hybridization patterns are compared visually.

VII ~lr~t5~~ Mol(~c~
The complement of the igif-2 sequence, or any part thereof, is used to inhibit vivo or in vitro expression of endogenous igif-2. Although use of ~ntie~n~e oligonucleotides. consisting of about 20 base-pairs, is specifically described. e~c~ontisllly the same procedure is used with larger cDNA fragments. An oligonucleotide comp!eml~n~ry to the coding sequence of igi~-2 is used to inhibit expression of endogenous igif-2. Using Oligo 4.06, the complementary oligonucleotide is ~ ci~n~d from the conserved 5' sequence and used to inhibit l-alls~ Lion or from 3' sequence and used to prevent the ribosome from tr~ncl~ting the mRNA.

VIII Cloning and Expression of IGIF-2 Nucleotides for a Kozak sequence were t?npinPered uluaLlt~ of the initi~ting~TG,and nucleotides ~n~orling a six residue hictic1ine tag were added to the 3' end of SEQ ID
NO:''. The o~LillliGed nucleotide sequence encoding IGIF-2 was cloned into the pCEP
vector (Invitrogen). Lipofect~min~ (Gibco BRL) was used to transform pCEP into 293 cells which had been Ll~lar~med previously with EBNA (Invitrogen). Stable transfomants were selecte~l using 300 ug/ml hygromycin. The native signal sequence of IGIF provides for secretion of the protein into the growth media which is used in the test for IGIF-2 activity.

IX IGIF-2 Activity Cytokine chemotactic activity is usually measured in 48-well microchemotaxis chambers. In each welL two colll~Lments are separated by a filter that allows the passage of cells from one COlll~ ent into the other in response to a chemical gradient.

CA 0223888~ 1998 - 0~ - 29 Cell culture medium into which IGIF-2 has been secreted is placed on one side of a polvcarbonate filter, and peripheral blood cells are suspended in the same media opposite side of the filter. Sufficient incubation time is allowed for the cells to traverse the filter in response to diffusion and resulting concentration gradient of IGIF-2. Filters are recovered from each well and specific cell types, eg, monocytes, ~hering to the side of the filter facing the cytokine are identified and counted.
Specificity of the chemoattraction is ~etermin~1 by performing the assay on fractionated populations of cells such as enriched populations of monocytes or lymphocytes obtained by density gradient centrifugation. Specific T cell populations are further purified using CD8+ and CD4+ specific antibodies for negative selection.
X Production of IGIF-2 Specific Antibodies Although IGIF-2 purified using PAGE electrophoresis (Sambrook et al, supra~
may be used to imml-ni7~ rabbits using standard protocols, a monoclonal a~pluacll is more commonly employed. The amino acid sequence tr~n~l~tecl from igif-2 is analyzed using DNASTAR software (DNASTAR Inc) to ~1eterrnin~ regions of high immunc-genicity and a corresponding oligopeptide is synth~ei7~d and used to raise antibodies by means known to those of skill in the art. Analysis to select ~ u~,ial~
epitopes, such as those near the C-te ~ or in ad3acent hydrophilic regions is described by Ausubel FM et al (supra).
Typically, the oligopeptides are 15 residues in length, synth~si7~A using an Applied Biosystems Peptide Synth~-si7~r Model 431A using fmoc-ch~ try, and coupled to keyhole limpet hemocyanin (KLH, Sigma) by reaction with M-maleimidobenzoyl-N-hydroxys-~cci~ e ester (MBS; Ausubel FM et al, supra). Rabbits are immllni7~i with the oligopeptide-KLH complex in comp}ete Freund's 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 radioiot1in7~te~;, goat anti-rabbit IgG.

XI Purification of IGIF-2 Using Specific Antibodies Tmmllnnaffinity chlullldLography is used to purify endogenous or recombinant IGlF-~ using antibodies specific for IGIF-2. An immunoaffinity colurnn is constructed by covalently coupling IGIF-2 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 m~mlf~ct~lrer's instructions.
S Media cont~ining IGIF-2 is passed over the immlln~-affinity column. and the column is washed under conditions that allow the ~l~f~.e.lLial absorbance of IGIF-2 ~eg, high ionic strength buffers in the presence of deLc.g~llL). The column is eluted under conditions that disrupt antibody/IGIF-2 binding (eg, a buffer of pH 2-3 or a high c~"lce..L,dLion of a chaotrope such as urea or thiocyanate ion), and IGIF-2 is collected.
XII Identifil~tion of Mo1e: l~c Which Interact with IGIF-2 IGIF-2, or biologically active fr~gmf nt~ thereof. are labeled with 1''51 Bolton-Hunter reagent (Bolton~ AE and Hunter, WM (1973) Biochem J 133: 529).
~nr~ tt? small molecules previously arrayed in the wells of a 96 well plate are inrubat~cl with the labeled IGIF-2, washed and any wells with labeled IGIF-2 complex are assayed. Data obtained using different concentrations of IGIF-2 are used to r~lcul~te values for the nurnber, affinity, and association of IGIF-2 with the r~ntlit1~t~ molecules.
All publications and patents mentioned in the above specification are herein inco,lJoldLt:d by reference. Various modifications and variations ofthe described method and system of the invention will be d~pdl~::llL 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~limPnt~, it should be understood that the invention as claimed should not be unduly limited to such specific embo-lim~ntc 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~nflecl to be within the scope of the following claims.

CA 02238885 l998-0~-29 W O 97/24441 PCTrUS96/20432 SEQVENCE LISTING
;1) GENERAL INFORMATION
(i) APPLICANT: INCYTE PHARMACEUTICALS, INC.
~ ii) TITLE OF THE INVENTION: NUCLEIC ACIDS ENCODING INTERFERON GAMMA

(iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Incyte Pharmaceuticals, Inc.
(~) STREET: 3174 Porter Drive (C) CITY: Palo Alto (D) STATE: CA
(E) COUNTRY: USA
(~) ZIP: 94304 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette (B) COMPUTER: IBM Compati~le (C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ ~or Windows Version 2.0 (vi) CURRENT APPLICATION DATA:
(A) PCT APPLICATION NUMBER: To Be Assigned (B) FILING DATE: Herewith (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION SERIAL NUMBER: US 08/580,667 (B) FILING DATE: 29-DEC-1995 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Billings, Lucy J.
(B) REGISTRATION NUMBER: 36,749 (C) REFERENCE/DOCKET NUMBER: PF-0051 PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 415-855-0555 (B) TELEFAX: 415-845-4166 (C) TELEX:

(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1101 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~vii) IMMEDIATE SOURCE:
(A) LIBRARY: Kidney (B) CLONE: 631796 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

~5 CA 0223888~ l998-0~-29 WO 97/24441 PCTrUS96/20432 GTCGACCCA- GCGTCCGCTG GCGACTGCCT GGACAGTCAG CAAGGAATTG TCTXCAGTG 60CATTTTGCG~ TCCTGGCTGC CAACGCTGGC TGCTAAAGTG GCTGCCACCT GCTGCAGTCT i20 TTGTGGC~.T GAAATTTATT GACAATACGC TTTACTTTAT AGCTGAAGAT GATGAAAACC 300 ACTGTAGAGA TAATGCACCC CGGACCATAT TTATTATAAG TATGTATAAA GATAGCCAGC g80 AGAACAAA~T TATTTCCTTT AAGGAAATGA ATCCTCCTGA TAACATCAAG GA.ACAAAAA 600 CTCCAAAAAA ~P~AP~ ~AA A 1101 (2) INFORMATION FO~ SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 193 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (vii) IMMEDIATE SOURCE:
(A) LIBRARY: Kidney (B) CLONE: 631796 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Ala Ala Glu Pro Val Glu Asp Asn Cys Ile Asn Phe Val Ala Met 1 5 10 15~ys Phe Ile Asp Asn Thr Leu Tyr Phe Ile Ala Glu Asp Asp Glu Asn Leu Glu Ser Asp Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg 80~hr Ile Phe Ile Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Met g0 95~la Val Thr Ile Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys Ser Asp Ile Ile Phe Phe Gln Arg Ser Val Pro Gly His Asp Asn Lys Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Ty- Phe Leu Ala Cys Glu Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lvs Lys 165 170 17~~lu Asp Glu Leu Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp _ CA 0223888~ 1998-0~-29 ;2) INFORMATION FOR SEQ ID NO:3:
(-' SEQUENCE C~ARACTERISTICS:
~A) LENGTH: 12 amino acids ~3) TYPE: amino acid _) STRANDEDNESS: single ~) TOPOLOG'~: linear (v~i) IMMEDIATE SOURCE:
(A) LIBRARY: adenoid (B) CLONE: 159939 (x~) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Gly Lys Val Glu Met Asn Leu Phe Phe Phe Ala Asn (2) INEORMATION FOR SEQ ID NO:4:
(-) SEQUENC_ CHARACTERISTICS:
'.~) LENGTX: 192 amino acids :3) TYPE: amino acid 'C) STRANDEDNESS: single ~3) TOPOLOGY: linear (vii) IMMEDIATE SOURCE:
(A) LIBRARY: GenBank (B) CLONE: 1064823 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Ala ~la Met Ser Glu Asp Ser Cys Val Asn Phe Lys Glu Met Met 1 5 10 15~he Ile Asp Asn Thr Leu Tyr Phe Ile Pro Glu Glu Asn Gly Asp Leu Glu Ser Asp Asn Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn Asp Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Glu Asp Met Thr ASD Ile Asp Gln Ser Ala Ser Glu Pro Gln Thr Arg 80~eu Ile _le Tyr Met Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val 95~hr Leu Ser Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile I1e Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser Asp Leu Ile Phe Phe Gln Lys Arg Val Pro Gly His Asn 130 135 lg0 Lys Met Glu Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys 145 150 155 160~ln Lys Glu Asp ASD Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu 165 170 175~sn Gly Asp Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

Claims (20)

1. A purified polynucleotide comprising a nucleic acid sequence encoding interferon gamma inducing factor-2 (IGIF-2) of SEQ ID NO:2.

2. The polynucleotide of claim 1 wherein the polynucleotide sequence consists ofSEQ ID NO: 1.

3. The purified polynucleotide of claim 1 wherein said polypeptide has isoleucine at residue 140.

4. The polynucleotide of claim 3 wherein the polynucleotide sequence of SEQ ID
NO: 1 has thymine at position 622.

5. A purified polynucleotide comprising a nucleic acid sequence encoding the polypeptide having amino acid residues 1-30 of SEQ ID NO:2 followed by the amino acid residues of SEQ ID NO:3.

6. An antisense molecule comprising the complement of the polynucleotide of claim 2 or a portion thereof.

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

8. A host cell transformed with the expression vector of claim 7.

9. A purified polypeptide comprising the amino acid sequence of SEQ ID NO:2.

10. The purified polypeptide of claim 9 having isoleucine at amino acid residue 140.

11. A purified polypeptide comprising amino acid residues 1-30 of claim 9 followed by the amino acid residues of SEQ ID NO:3.

12. A pharmaceutical composition comprising an effective amount of the antisensemolecule of claim 6 and a pharmaceutically acceptable excipient.

13. A method of treating a subject having a condition associated with altered igif-2 expression comprising administering an effective amount of the pharmaceutical composition of claim 12 to the subject.

14. A diagnostic composition comprising an oligonucleotide probe of the polynucleotide of claim 2.

15. A diagnostic test for the detection of nucleotide sequences encoding IGIF-2 in a biological sample, comprising the steps of:
a) combining the biological sample with a first nucleotide sequence which comprises the nucleotide sequence of claim 2, or a 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 encoding IGIF-2 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 condition associated with inflammation or aberrant expression of IGIF-2.

16. A method of screening a plurality of compounds for specific binding affinity with the IGIF-2 or a portion thereof comprising the steps of:
a) providing a plurality of compounds;
b) combining the polypeptide of claim 9 with each of a plurality of compounds for a time sufficient to allow binding under suitable conditions; and c) detecting binding of IGIF-2 to each of the plurality of compounds, thereby identifying the compounds which specifically bind IGIF-2.

17. A method for producing IGIF-2, said method comprising the steps of:
a) culturing the host cell of claim 8 under conditions suitable for the expression of said polypeptide, and b) recovering said polypeptide from the host cell culture.

18. An antagonist specifically binding the polypeptide of claim 9 or a portion thereof.

19. A pharmaceutical composition comprising the antagonist of claim 18 and a pharmaceutically acceptable excipient.

20. A method of treating a subject with a condition associated with altered IGIF-2 expression comprising administering an effective amount of the pharmaceutical composition of claim 19 to the subject.