CA2134443A1 - Inhibitory factor - Google Patents

Abstract

Novel inhibitory factors, oligonucleotides encoding the same, and methods of production are disclosed. Pharmaceutical compositions and methods of treating disorders are also disclosed.

Description

- W093/22449 ~ PCT~US93/03953 . . .

IN~IBITORY FACTOR

Field of the Invention The pre~ent invention relates to factors that mediate mammalian cell cycle arrest and maintain mammalian cell~ in a viable state, the use of such factor~, and to nucleic acid sequences encoding such factor~. In particular the invention relate~ to these factors, to fragment~ and polypeptide analogs thereof and to DNA sequence~ encoding the same.

Backaround of the Invention Both growth factors (stimulators of cell cycling) and growth inhibitory factors (inhibitory factor~ of cell cycling) play an important role in cell-cell interaction~
a~nd cell division. Despite the practical importance of growth inhibitory actors, very few have been isolated and purifi~ed~and, in most cases, there is little evidence that *he biologically àctive inhibitorY factors previously isolated are residents of the cell surace or act by binding cell surface receptors. The existence of cell urface;inhibitory factors would be con~istent with the models of regulation of cell division a~ a result of cell-cell con~act.

Compared to the numero~ls growth factors that have been de~cribèd, very few inhibitory factors of cell ~.
proliferatlon have been isolated and characterized. The ma~or inhibltory factors described includ~: 1) the 25 kDa homodimer transforming growth factor-~ (TGF-~) that ha~
mitogenic activity with a variety of fibroblasts and yet expre~se~ a potent lnhibitory activity wlth normal human epithelial prokeratinocytes cultured in serum-free medium (Roberts et al., Proc. Natl. Acad. Sci. U.S.A., 82:
119-123, 1985; Coffey et al., Cancer Re~. 48: 1596-1602, 1988) and the structurally-related protein isolated from ~. ~
-: .

~1~4 z PCT/US93/03953 African green monkey cells (BCS-l) conditioned medium (Tucker et al., Science 226: 705-707, 1984); 2) a 12-14 kDa protein isolated from mammary ti~ue (Bohmer et al., Exp.
Cell Re~. 150; 466-476, 1984; M~lller et al., J. Cell.
Physiol. 138: 415-423, 1989) that ha~ been identified in cell nuclei and ~hown to be structurally related to a fibroblast growth inhibitory factor i~olated from mou~e 3T3 cell medium ~Voss et al., Exp. Cell Re~ . 138 : 397-407 , l9B2; Bohmer et al, J. Cell. Biochem. 38: 199-204, 1988);
3) a 17 kDa acidic protein, oriqinally described as a glial maturation factor ~, that h~s been ~hown to have antiproliferative activity ( Lim, Proc . Natl . Acad. Sci .
U. S . A . 86 : 3901-3905 , 1989 ; Lim et al ., Cell Regulat . 1 :
741-746, 1990); 4) an oligosaccharide from human diploid fibroblast~ (Wieser et al., J. Cell Biol~ 2631-2692, 1990); 5) a tissue-specific growth inhibitory fac-tor ~mammo~tatin) isolated from cell cult~re medium (Ervin et al., Science 244: 1585-1587, 1989); and, 6) two classe~ of glycopeptide inhibitory factors structurally unrelated to the pre~ent invention (Kinder~ et al., Exp. Cell Res. 136:
31-41, 1981; Charp et al., J. Cell Biol. 97: 311-316, 1983).

Cell proliferation inhibitory factors that are membrane residentæ and that play a role in cell-cell 3ignaling, most likely have significant hydrophobic domain~, or are complexed with hydrophobic integral membrane components. Thi 8 featnre ha~ led to technical difficulties in their isol~tion, identification, and their presentation to target cell.q for meaningf-ll biological assays -- particularly when deterqents (necessary to maintain the element~ in aqueolt~ ~uspension or solution) are toxic solvents to living cells.
* * *
A hydrophilic and active fragment of a larger glycoprotein inhibitory factor was relea6ed from intact cells that allowed purification by biochemical procedures (Sharlfi et al., J. Chromat. 324: 173-180, 1985; Sharifi et al., Neurochem. 46: 461-469, 1986a).

W093t22449 ~ 3 PCT/US93tO3953 .

The bovine inhibitory glycopeptide is composed of a single polypeptide chain of a molecular weight of approximately 18,000 that focuses by isoelectric focu~ing at about 3.0 (Sharifi et al, Ne~lrochem. 46: 461-469, 1986;
and Sharlfi et al, J. Cell. Biochem. 31: 41-47, 1986). The glycopeptide inhibits cell~llar protein and DNA synthesis, and arrests cells in the mitotic cycle at what appears to be a single block point near the G1/S interphase (Fattaey et al., J. Cell. Physiol. 139: 269-274, 1989; and Fattaey et al, Exper. Cell Res. 19~: 62-68, 1991). The glycopeptide inhibitory factor requires only a cell surface interaction to mediate its biological inhibitory activity (Sharifi et al., Biochem. Biophys. Res. Comm. 134:
1350-1357, 1986c), and the binding kinetics are consistent with a specific and saturable cell surface receptor (Bascom et al., J. Cell Physiol. 128: 202-208, 1986; Sharifi and ~; Johnson, J. Biol. Chem. 262: 15752-15755, 1987).

;~ ~ Consistent with the hypothesis that cell division is controlled by the interaction of ligands at the cell surace with both positive and negative influences, the D~ glycopeptide has been identiied on the surface~ of 3T3 - ~ ; cells (Lakshmanarao et ~1., Exper. Cell Res. 195: 412-415, l991), and to be a potent alltagonist of the tumor promoter 12-0-tetradecanoylpho~bol-13- ~cetate (TPA) (Chou et al., Cancer Lett. 35: 119-128, 1987), epidermal growth factor (EGF) (Bascom et al., J. Ce].l. Biochem. 3~: 283-291, 1987) and bombesin (Johnson and Sharifi, Biochem. Biophys. Res.
Comm. 161: 468-474, 1989).

Although the glycopeptide W~8 isolated from bovine cerebral cortex cells, its inhibitory action i8 effective on wide range of target cells. Cells sensitive to its proliferative inhibitory action include vertebrate and , -invertebrate (insect) cellæ, fibroblast and epithelial-like cells, primary cells and established cell cultures, as well a~ a wide range of transformed cell lines (Fattaey et al., '~ .

:: , W093/22~9 PCT/US93/03953 ; ~

J. Cell. Physiol. 139: 269-274, 1989; and Fattaey et al, Exper. Cell Res. 194: 62-68, 1991).

With the exception of one cell line, human HL-60 leukemic cells, all cells which were inhibited were reversibly inhibited by the glycopeptide in a nontoxic manner (Ed~on et al, Life Sci. 48: 1813-1820, 1991). HL-60 cells, however, were arrested in an irrever~ible fashion although they remained viable or at least 84 h. The glycopeptide mediated a terminal cellular differentiation, even after its removal.

An interesting feature of the glycopeptide i~ that the biological inhibitory activity clearly is Ca2 dependent, and po~sibly related to cellular Ca2 fluxes and/~r intracellular Ca2 mobiliz~tion (Toole-Simms et al., J.
Cell. Phy~iol. 147: 292-297, 1991). The addition of the calci~m ionophore A23187, b~lt not the sodium ionophore monen~in, before or within minutes of the inhibitory factor, result~ in the abrogation of the inhibition of protein synthesis (Sharifi et al., Biochem. Biophys. Re~.
Comm. 136: ~76-982, 1986).

Prior to the ~ubject invention, a particularly disturbing feature o~ the puriied glycopeptide was that a protease activity, of unknown specificity, always was measurable in even the most p~lrified preparations (Sharifi et al., J. Cell. Biochem. 31: 41-47, 1986). Whether the protease was an integral activity of the glycopeptide molecule itself, or a trace contaminant in the purified preparation~ could not be determined. Although the protea~e activity remained even when the biological inhibitory activity of the glycopeptide was destroyed (Sobie~Xi et al., Life Sci. 38: 1883-1888, 1986), the protea~es presence was unavoidable and complicated the preparation of ~amples for stlldies of protein 8equencing.

W093/22~ 9 4 ~ PCT/US93/03953 O~ects of the Invention It i~ an object of the invention to provide a method !~
of purifying naturally occurring inhibitory factor to apparent homogeneity.

It is an object of the invention to provide DNA and RNA ~equences encoding inhibitory factvr.

- It is an object of the invention to provide recombinant polypeptide factor~ which inhibit cell division.

It i~ a further object of the invention to provide factors which can be used alone or in combination with other agents in the treatment of neopla~tic or proliferative states or states relating to cell prolieration in a variety of species.

It i~ a further object of the invention to provide factor3 which can slow growth, development or aging.

SummarY of the Inventio~

According to the present i.nvention, polypeptide factors, referred to herein as "inhibitory factor" having the ability to inhibit cell division or cell cycling, are provided. Such factors include purified naturally-occurring inhibitory factors. The invention al80 relate~ to non-naturally occurring polypeptides having amino acid ~eguence~ sufficiently duplicative of that of naturally-occurring inhibitory factor to allow possession of a biological activity of natl-rally occurring inhibitory fact9r 8uch as th8 ability to inhibit cell division.

The pre3ent invention also provides i~olated nucleic acid 8equence8 for use in securing expre~sion in ~/
procaryotic or eucaryotic host cell~ of polypeptide W093/22449 PCTJUS93/03953 ~
213Q~4~ 6 products having amino acid ~eq~ence~ sufficiently duplicative of that of naturally- occurring inhibitory factors to allow possessioll of a biological activity of naturally occurring inhibitory factor. Such DNA ~equences include:
a~ DNA ~equences encoding naturally occurring i~hibitory factor disclosed in Example VII or their complementary strands;
b) DNA ~e~uences whicll hybridize to the DNA sequences defined in a) or fr~gments thereof; and c) DNA sequences which, but for the degeneracy o the genetic code, would hybridize to the DNA ~e~uence~
defined in a~ and b).

The invention ~lso provides modified or substituted nucleic acid sequences (methyl phosphonate, thiolate, etc.) which bind to ~eguences either encoding inhibitory factor or complementary to those coding for inhibitory factor.

Al~o provided are vectors containing ~uch DNA
~e~uence~, and host cells tran~formed or tran~fected with such vectors. Also comprehended by the invention are methods of producing inhibitory factors by recombinant technique~, and methods of treating disorders.
Additionally, pharmaceutical compositions including inhibitory factors are provided. Antibodies specifically binding inhi~itory factors ~re also provided.

The invention also relates to a process for the efficient recouery of inhibitory fRctors from a material containing inhibitory factors.

Brief DescriDtion of the Drawings FIGURE 1 show~ an immunoblot analy~i 8 of components released from bovine brain cerebral cortex cell membrane.
Equal aliquots of plasma membrane were incubated for 30 min. at 4C with either isotonic buffer alone (0.154 M

- W093t22~9 ~1 3 ~ 1 3 PCT/~S93/03953 NaCl; 0.01 M potassium pho6phate, 1 ~g/~l each of phosphoramidon, pepstatin i;, leupeptin and aprotinin; pH
7.2), or with i~otonic b~lffer containing either 3 M NaCl or 3 M urea. After incubation the membranes were pelleted by centrifugation a~ described in the Material~ and Method~
and 100 ~1 of each supernatant fluid where tested for antigenicity as membrane released material (slots A, C and E). The membrane pellets were solubilized in 1%
octyl-B-D~glucopyranoside ~2.5 mg protein/ ml) and 100 ~1 were te~ted for antigenicity as membrane bound material ~slot~ B, D and F).

FIGURE 2 shows an SDS-PAGE analysi~ of bo~ine brain cerebral cortex cell membrane proteins during purification. Samples were separated by SDS-PAGE under reducing conditions, and tl~e gel~ were then silver stained. Original membrane preparation (100 ~g protein, lane A), 3 M NaCl relea~ed membrane proteins (50 ~g protein, lane B), preparative isoelectric focused pI 5.1 purified protein~ (10 ~g protein, lane C) and LPA affinity chromatography purified sample (~ ~g protein, lane D).

FIGURE 3 BhOW~ preparative isoelectric focusing analysis of 3 M NaCl released protein~ from membranes of bovine brain cerebra1 cortex cells. 1 mg membrane protein, relea~ed by 3 M NaCl, was isoelectrofocused as des~ribed in the Materials and Methods, and the pH of the twenty 2 ml fractions was measured. 50 ~lg protein from each fraction was analyzed by immunoblot using polyclonal antibody against the native bovine inhibitory factor. The relative amount o`f antigen in each fraction was ~uantlfied by de~itometer scanning.

FIGURE 4 ~hows RB protein immunoprecipitation of inhibitory factor arrested HSBP and SWi88 3T3 fibrobla~ts.
Spaxae HSBP and Swiss 3T3 cult~tre~ were treated for 24 hours with either, ~6 x 10 ~ M inhibitory factor in DMEM
with 10% newborn calf serum, or DMEM alone as a control.

W093/2~9 PCT/US93/03953 .~-2~34443 8 The cells were then radiol~belled for 3 1/2 hours with 135Slmethionine, immunoprecipitated with monoclonal mou~e anti-human RB IgG1, and the proteins separated by SDS-PAGE
as de~cribed in Example VIII. L~ne 1, control logarithmically growing HSBP cells; Lane 2, inhibitory factor arrested HSBP cells; Lane 3, control logarithmically growing Swiss 3T3 cells; and, Lane 4, inhibitory factor arrested Swi~s 3T3 cells.
.

FIGURE 5 ~hows RB protein immunoprecipitation of density-dependent quiescent HS~P and Swiss 3T3 fibroblasts. HSBP and Swiss 3T3 cells were plated and allowed to grow to confluence as described in Example VIII. After reaching confluency the cultures were incubated an additional 24 hour~, and the cells were then radiolabelled and immunoprecipitated. Another set of HSBP
and Swi 8 3T3 cultureæ were plated on the ~ame day at -1/3 the density. The~e cells were treated in the same fashion as the first set however, at the time of immunoprecipitation were ~tlbconfluent. Lane 1, subconfluent HSBP cultures; Lane 2, confluent HSBP
cultures; Lane 3, ~ubconfluellt Swiss 3T3 cultures; and, Lane 4, confluent Swis~ 3T3 cultures.

FIGURE 6 shows inhibitory factor cell proliferation inhibition assays carried out on the human osteosarcoma U20S (RB ) and SAOS-2 (RB ) cell lines. Osteosarcoma cells grown in DMEM and 10% fetal calf serum, and either 9 x 10 8M inhibitory factor (o) or an equal volume of PBS t) wa~ added at the time indicated by the arrows. Data are plotted as the average of d~lplicate well~.

FIGURE 7 shows inhibitory factor cell proliferation inhibition a88ay8 carried out on the human bladder carcinoma J82 (RB ) and human prostate carcinoma DU145 (RB ) cell lines. Carcinoma cells grown in DMEM and 10%
fetal calf serum, and either 9 x 10 8M inhibitory factor (o) or an equal volume of PBS () was added at the time W0~3~22~9 ;~1 3 ~ ~ 4 3 PCT/U~93/03953 indicated by the arrows. Data are plotted ~s th~ ~ver~e of duplicate wells.
FIGURE 8 shows inhibitory factor cell prolif~ration inhibition assays carried out on the human keratin~cyte HFK
(normal) and human papillomavirus transforme~ (13~ and NC0) cell lines. Cells were grown in KGM medium with appropriate growth factors (Clonetics, San Diego, CA), and either 9 x lO 8 M inhibitory fackor (o) or an eq~lal vo3ume of PBS (~) was added at the time indicated by the ~rrows.
Data are plotted as the average of duplicat~ well~.
FIGURE 9 shows inhibitory factor cell proliferation inhibition assays carried out on the adenovi~u~ t:r~nsformed human kidney epithelial cell line 293. Cells were grown in DMEM and 10% fetal calf serum, and either 9 x ~ M
inhibitory factor (o) or an e~ual volume of PBA (~) was added at the time indicated by the arrows. ~ata ~re plotted as the average of duplicate wells.
FI5URE lO shows inhibitory factor cell ~roliferatio i~hibition assays carried out on the murine fibrohlast Swiss 3T3 ~normal) and SV40 transformed (SVT-2 an~ F5~) cell lines. Fibroblasts were grown in DMEM ~n~ ]~/~ c~l~
serum, and either 9 x lO 8 M inhibitory factor (! ol ~n equal volume of PBA (-) were added at the time jn~icat:~-l hy the arrows. Data are plotted as the average o ~ t~
wells.
FIGURE ll shows inhibition o hy~ridoma cel~
proliferation Detailed De~criPtion of the In~ent_on The subject invention relates to purified ~atural].y I occurring factors and nov~l factors that inhi~it cell I growth, and to DNA se~uences encoding such f~ctorc... The invention also relates to the use of inhibitory factor as a research diagnostic or therapeutic agent. Additionally, the invention includes-method~ of purifying the inhibJ.tory factor of the invention.

i W~9~/22~g PCT~US93/03953 :10 2l<~4l43 - Inhibitory factor is an inhibitor ~f cell divi~ion (or cell cycling) of a wide variety of cells including those derived from various ti~ues of mice, monkey, human, avian and in~ect specie~. The factor inhibits cell divi~ion in a reversible and nontoxic fashion. It acts by binding a cell surface receptor and causing a variety of intracellular changes including alteration in Ca2 and phosphorylation of key cell regulatory protein.s, e.g., the retinoblastoma protein ~RB). The term "inhibitory factory factor" as u~ed herein refers to naturally-occurring inhibitory factors (e.g., natural human inhibitory factor~ a~ well as non-naturally occurring (i.e., different from naturally occurring) factors having amino acid se~uences and gly~osylation sufficiently duplicative of that of a n~turally-occurring inhibitory factor to allow possession of a biological activity of naturally occurring inhibitory factor.

In addition to purified and isolated naturally-occurring inhibitory factors (i.e., purified from nature or manufactured ~uch that the primary, ~econdary and tertiary conformation, and the glycosylation pattern are identical to naturally-occurriIlg material), the ubject invention provides non-naturally occurring polypeptides having a primary structural conformation (i.e., continuous ~eguence o~ amino acid residues) and glycosylation ~ufficiently duplicative of that of naturally occurring inhibitory factor to allow pos.~ession of a biological activity of naturally occurring inhibitory factor. Such polypeptides include derivatives and analog~.

One embodiment of the invention i8 directed to an improved proc~dures to purify inhibitory factor, and the producta of ~uch purification. Although lt was thought that the bovine glycopeptide had been purified to homogeneity, ~ensitive silver-stained gels of the final product expo~ed the presence of low molecular weight protein contaminants. It w~s found that the use of : W093/22449 2 1 3 g ~ ~ 3 PCT/US~3/03953 ion-exchange column high performance liquid chromatography (HPLC) remove~ these contaminating protein specie~ and provide~ a glycopeptide purified to apparent homogeneity without protease activity.

The 3ubject invention includes a method to eliminate the protease activ~ty and to obtain the 18 kDa inhibitory factor in a homogenous form. Such method includes the following ~teps:

a) conducting mild proteolysis of intact cells or membrane~ u~ing a protease selected from the group including: pronase, trypsin, chymotrypsin, B
substilysin, ~erine proteases, thiolprotea3es, cathepsin D proteases, sulphydryl protea~e, metallo-protea~es, tryp~in like proteases, estra~e and carboxy prQteases, non-specific proteases and other ., specific protea~eiR;
b) conducting DEAE chromatography or preparative i~oelectric focussing;
c~ conducting lactin affinity chromatography;
d) HPLC size exclusion chromatography; and s e) conducting HPLC DEAE chromatography.
., The invention al80 includes a method of purifying the parental 66 kDa protein to apparent homogeneity. Such method include~ the ~tep~ of:

i .
a) eluting the protein from intact cell~ or membranes using salt ~uch a~ NaCl;
s b) conducting preparative i~oelectric focusing or j DEAE chromatography; and c) conducting lectin affinity chromatography.
-DEAE chromatography can be performed by batch me~hods or by using gravity fed or a variety of pressurized columns.

W093/22~9 PCT/US93/03953 ,-~

213~43 l~
Lectin affinity chrom~togr~hy can be done in batches or column~ using a variety of lectins to either bind the inhibitory fact~r (for example Limulu~ polyhemus agglutinin, LPA) or to bind cont~minants while leaving inhibitory factor unbound (for example Wheat Germ Agglutinin~ WGA). M~lltiple lectin affinity procedures optionally are ~ubstituted for tlle DEAE or i~oelectric focus~ing.

For purification of re~om~inant inhibitory factor, the following methods.can be ~Ised:

Method 1.
Affinity chromatography ~sing ~pecific labels or flags added to the inhibitory factor ~ a re~ult of the cloning proce3s, and DEAE chromatography Method 2.
Size aelection chromatography DEAE chromatography .
The ~teps listed in either of the~e procedures are u~ed alone or in combinatioll, depending on purity desired.
Lectins are not u~eful in isolating material grown in E.
coli but may be used in isnJ.atjng material~ from hosts capable of glycosylation.

According to another embodiment of the present invention, novel inhibitory factors and DNA and RNA
seguences coding for all or part of such inhibitory factor~
are provided. The pre~ent inv~ntion include~ DNA sequenceq which include: the incorporation of codon~ "preferred" for expression by selected nonmammalian ho~ts: the provision of 8ite8 ~or cleavage by restriction endonucleaæe enzymes; and the provi~ion of additiona]. initial, terminal or intermediate DNA sequences wllicl) facilitate construction of ~ 4 ~ ~
W093/2~9 PCT/US93/03953 readily-expres~ed vectors, or production or purification of inhibitory factor.

The present invention E~lso provide~ DNA sequence~
coding for polypeptide analogs or derivative~ of inhibitory factor which differ from nat~lr~lly-occurring form~ in terms of the identity or location of one or more amino acid residues (i.e., deletion analo~s containing less than all of the residue~ specified for inhibitory factor;
substitution analogs, wherein one or more residues specified are replaced by other residue~; and addition analogs wherein one or more ~mino acid residues is added to a ter~inal or medial portioll of the polypeptide) and which share some or all of the properties of naturally-occurring forms. The present invention ~pecifically provides DNA
sequence~ encoding the ful] length unprocessed amino acid sequence as well as DNA seq~lences encoding the proces~ed form of ~nhibitory factor.
: ' .
Novel DNA ~equences of the invention include ~equences . ,~, useful in ~ecuring expreQsi.on in procaryotic or eucaryotic host cells of polypeptide ~rod~l~t~ having at least a part of the structural conformation and ona or more of the biological properties of natllrally-occurring inhibitory factor. DNA ~equences of tl~e invention ~pecifically compri~e: (a) DNA sequence~ elt~oding inhibitory factor disclosed in Example VII o~ their complementary ~trands;
(b) DNA 3equences which ~lyh~.idiY.e (under the following hybridization conditions: ~.xSS~" 40% formamide, at 37C, 0.1% SDS, 5 x Denharts 801UtiOII, 0. 6 mg/ml yeast tRNA, lO
~g/ml ~heared herring sperm DNA, 5.0% polyethylene glycol and 20 mM tris pH 7.5, or more stringent conditions) to the DNA ~eguenceP diaclo~ed in F.xample VII or to fragments thereof; and tc~ DNA seq~ences which, but for the degenera~y of the geneti~ code, would hybridize to the DNA
sequences disclosed in Exampl.e VII. Specifically comprehended in parts (b) ~nd (c) are genomic DNA sequences encoding allelic variant forms of inhibitory factor and/or I

'~ 13 ~ 4 4~ PCT/USg3/039~3 ;~

encoding inhibitory factor from other mammalian species, and manufactured ~NA sequences encoding inhibitory factor, fragments of inhibitory factor, and analog~ of inhibitory factor. The DNA sequences may incorporate condons facilitating transcription and tran~lation of me~senger RNA
in microbial hosts. Such manuf~ctured sequences may readily be constructed according to the methods well known to tho~e skilled in the art.

According to another aspect of the present invention, the DNA sequences described llerein which encode polyp~ptide~ having inhibitory factor activity are valuable for ~he information which they provide concerning the amino acid sequence of the animal (including mammalian) proteins which have heretofore been ~mav~ilable. The DNA seq~ences are also valuable as products nseful in effecting the large ~cale synthesis of inhibitory factor by a variety of recombinant techniques. Put another way, DNA sequences provided by the invention are useful in generating new and useful viral and circular plasmid DNA vectors, new and u~eful transformed and transfected procaryotic and eucaryotic host cells (inclndillg bacterial and yeast cells and mammalian cells grown in cnlture), and new and useful methods for cultured growtl- of ~uch host cells capable of expre~sion of inhibitory facto~ and its related products.

DNA sequences of the invelltion are also suitable mater~als for use as labele~ probes in isolating human ~enomic DNA encoding inhibltory factor and other genes for related protein~ ~s well as cDNA and genomlc DNA sequences of other mammalian species. DN~ ~equence~ are also be useful in variou~ alternative methods of protein synthe~i 8 (e.g., in insect cells) or in genetic therapy in humanR and other mammals. DNA sequences o the lnvention are expected to be u~eful ~n developing transgenic mammalian species which may serve a~ eucaryotic "host~" for production of inhibitory factor and inhibitory factor products in WO 93/22449 2 1 ~ ~ 4 ~ 3 PC~tUS93/03~53 quantity. See, generally, Palm.i.ter et al., Science 222, 809-813 ( 1983 ) .

In an advantageous embodiment, inhibitory factor 1 characterized by being the product of procaryotic or eucaryotic host expression (e.g., by bacterial, yeast, higher plant, insect and mammalian cell~ in culture) of exogenous :DNA sequences obt~ined by genomic or cDNA cloning or by gene synthesis. That is, in an advantageous embodiment, inhibitory factor is "recombinant inhibitory factor." The products of expression in typical yeast (e.g., SaccharomYces cerevisiae~ or procaryote (e.g., E.
coli) host cells are free of association with any mammalian proteins. The products of expression in vertebrate le-g-, non-human mammalian (e.g., COS or CH0) and avianl cell3 are free of a sociation with ally hllman proteins. Depending upon the host employed, polypeptidea of the invention may be glycosylated with mammalian or other eucaryotic carbohydrates or may be non-glycosylated. Polypeptide~ of the invention optionally also i.nclude an initial methionine amino acid residue (at positio~

In addition to natural]y-occurring allelic forms of inhibitory factor, the present invention also embraces other inhibitory factor prod~lcts such as polypeptide analogs of inhibitory factor. S~lch analogs include fragments of inhibitory factol-. Following well known procedures, one can readily design and manufacture ~enes coding for microbial expresæion of polypeptides having primary conformation~ which differ from that herein speclfied for in terms of the identity or location of one or more re~idues (e.g., substitlltions, terminal and intermediate addition~ and deletion~). Alternately, modification~ of cDNA and ~enomic genes can be readily accomplished by well-known site-directed mutagene~is technigues and employed to generate analogs and derivatives of inhibitory factor. Sucll pro~ucts share at least one of the biological properties of inllibitory factor but may W093/22~9 PCT/US93/03953 ,~~
2~3 44 4~ 16 '`

differ in others. As examples, products of the invention include those which are foreshortened by e.g., deletions;
or those which are more stable to hydrolysi~ (and, therefore, may have more pronounced or longer-lasting e~fects than naturally-occurrillg); or which have been altered to delete or to add on~ or more potential ~ites or 0-glycosylation and/or N-glycosylation or which have one or more cysteine residues deleted or replaced by, e.g., alanine or serine residues and are potentially more ea~ily isolated in active form from microbial ~ystems; or which have one or more tyrosine residues replaced by phenylalanine and bind more or less readily to target protein~ or to receptors on target cells. Also comprehended are polypeptide fragments duplicating only a part of the continuous amino a~id sequence or seconclary conformations within inhibitory ~actor, which fragments may po8~e~8 one pr~perty of inhi.bitory factor, (e.g., receptor binding) and not others (e.g., cell inhibitory activity).
It iB noteworthy that activity iæ not neces~ary for any one or more of the products of the invention to have therapeutic utility l~ee, ~eiland et al., Blut, 44, 173-175 (1982~ or utility in other contexts, such as in assays of inhibitory factor antagoni~m. ~,ompetitive antagonists are useful in cases of overproA~lction of inhibitory factor or its receptor.

Of applicability to polypeptide analogs of the invention are reports of the immunological property of synthetic peptide~ which s~ t~ntially duplicate the amino acid 8e9uence extant in natura].ly-occurring proteins, glycoproteins and nucleoproteins. More ~pecifically, relatively low molecular weight. polypept~des have been shown to participate in imml-ne reaction~ which are similar in duration and extent to the immune reactions of physiologically-8ignificant proteins such as viral antigens, polypeptide hormones, and the llke. Included among the immune reactions of such polypeptides i~ the provocation of the formation of specific antibodies in W093/2~9 ~ PCT/U~93/03~53 immunologically-active animals See e.g., Lerner et al., Cell, 23, 309-310 (lg81) and Ross et al., Nature, 294, 654-656 (1981) See, also, Kaiser et al. IScience, 223, 249-255 (2984)] relating to biological and immunological properties o synthetic peptides which approximately share secondary structures of peptide hormones but may not share their primary ~tructural conformation.

The present invention also include~ that clas~ of polypeptides coded for by porti ons of the DNA complementary to the protein-coding strand of the human cDNA or genomic DNA ~eguences of inhibitory factor, i.e., "complementary inverted proteins" as described by Tramontano et al.
lNucleic Acid Res., 12, 5049-5059 (1984)].

Also comprehended by the invention are pharmaceutical c ~o~ition~ comprising therapeutically effective amounts o~ polypeptide products of the invention together with ~uitable diluents, preservatives, ~olubilizer~, emul~ifiers, adjuvants and/or carriers useful in inhibitory factor therapy.~ A "therap~tltically effective amount" as used herein refers to that ~mount which provides a therapeutic effect for a given condition and administration regimen. Such compositions ~re liquids, gel~, ointments, or lyophilized or otherwi~e dried formulations and include diluent~ of various buffer cont.ent (e.g., Tri~-HCl., acetate, phosphate), pH and iOlliC strength, additive~ such a~ albumin or gelatin to prevent ad~orption to surfaces, detergent~ (e.g., Tween 20, Tween 80, Pluronic F68, bile acid ~alts), solubilizing agent~ ~e.g., glycerol, polyethylene glycol), anti-oxidants (e.g., ascorbic acid~, sodium metabi~ulfite), preservatives (e.g., Thimerosal, . benzyl alcohol, parabens ?, b~llking sub~tances or tonicity modifers (e.g., lacto~e, mannitol), covalent attachment of polymer~ ~uch as polyethylene glycol to the protein, complexation With metal ions, or incorporation of the material into or onto particnlate preparations of polymeric compounds such as polylactic acid, poly~lycolic acid, W093/22~9 PCT/US93/039~3 l~-213~3 18 ~

hydrogel~, etc. or into l~posomes, microemulsion~, micelles, unilamellar or m~lltilamellar vesicle~, erythrocyte ghosts, spheroplasts, ~kin patches, or other known methods of releasing or packaging pharmaceuticals.
Such composition6 will infl~lence the phy~ical state, solubility, stability, rate of in vivo release, and rate of _ vivo clearance of inhibitory factor. The choice of composition will depend on the physical and chemical properties of the protein having inhibit~ry factor activity. For example, a prod-lct derivèd from a membrane-bound form of inhibitory fa~or may require a formulation containing detergent. Controlled or sustained release compositions incl~de formulation in lipophilic depots ~e.g., fatty acids, waxes, oil~). Also comprehended by the invention are particulate compo~tions coated with polymers (e.g., poloxamers or poloxamines) and inhibitory factor coupled to antibodies directed against tissue-specific receptors, ligand~ or antigens or coupled to ligand~ of tissue-specific receptors. Other embodiments of the compositions of the invention incorporate particulate forms protective coatings, protease inhibitory factors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal, topical (skin or mucosal) and oral.
.
The invention also comprises compositions including one or more additional factors such as chemotherapeutic agents, TNF, cytokines (e.g., interleukin~), antiproliferative drugs, 5FU, alkylating agent~, antimetabolites, and drugs which interfere with DNA
metabolism.

In another embodiment, inhibitory factory factor is admini~tered in conjunction with radiotherapy.

Polypeptide~ of the invention may be "labelled" by association with a detectable marker substance (e.g., radiolabeled with 125I, enzyme labelled, or W093/22449 2 ~ 3 4 ~I g 3 PCT/VS93/03g~3 biotinylated) to provide reagents u~eful in detection and guantification of inhibitory factor or it~ receptor bearing cells in ~olid tissue and fluid sample~ such as blood, urine, cerebral spinal fluid or culture media.

The subject invention al~o relates to antibodies specifically binding inhibitory factor. One embodiment i8 polyclonal antibodies which bin~ inhibitory factor but not any protea~es. A further ~mbodiment of the invention are stable hybridoma~, i.e., hybridomas capable of being passaged repeatedly and cryopreservation, ~uch hybridomas producing antibodies specifically binding inhibitory factor. In contrast to convelltional antibody (polyclonal) preparations which typic~lly incl~lde different antibodies directed against different determinant~ (epitopes), each monoclonal antibody is directed against a ~ingle d~terminant on the antigen. Monoclonal antibodie~ are useful to improve the selectivity and ~pecificity of diagnostic and analytical ~ssay methods using antigen-antibody binding. Also, both monoclonal and polyclonal antibodies are ~Ised to neutralize or remove inhibitory fa~tor from ser~lm or from culture media or other liquids. A ~econd advantage of monoclonal antibodies i~
that they can be synthesi7.ed by hybridoma cells in culture, uncontaminated by other imm~lnoglobulins. Monoclonal antibodie~ may be prepared from ~lpernatants of cultured hybridoma cells or from asci.te~ induced by intraperitoneal inoculation of hybridoma ce].ls into mice. The hybridoma technigue described oriqinally by Kohler and Mil~tein lEur.
J. Immunol. 6, 511-519 (19,76)1 has been widely applied to produce hybrid cell lines that secrete high level~ of monoclonal antibodies against many specific antiqens.

Application~ of the Inhibi~ory Factor of the Invention . ~
The nontoxic and rever~ible nature of the cell cycling inhibition by the polypeptide of the invention perm~t~ many applications.

w093/22449 2 1 3 1 ~ ~3 PCT/US93/03953 -A) Synchronization of Cells The inhibitory factor of the subject invention i~
useful a~ a reagent to sync}lronize cell populations in culture for ~tudies includin~, b~lt not limited to, measuring specific biochemica~ events in specific stages of the cell cycle, receptor-ligand interactions that influence cell divisi~n, drug effects, effects of viru~es, effects of transforming agents, effects of mutagens, and effects on the ability of cells to fuse with other cells or react to environmental stimuli (heat, cold, etc.), and 8ignal transduction events that occur sub~equent to receptor-ligand interactio21. The inhibitory factor is useful in studies with cells derived from mammalian and non-mammalian species, primary c~llture3 and established cell line3 and nontransformed and suitable tumorigenic cell lines .

i) Exponentially Dividing Cell Cultures.

The inhibitory factor is useful for examining various 8tagç8 of the cell cycle. Adding, e.g., 1 to 10 x 10 8 M
of the inhibitory factor to exponentially dividing cell culture~, and incubating t}le cells for a period of time, allow~ all cells to come to arrest at one point in the cell cycle. Long incubation (over one generation time) provides the largest percentage of ~rrested cells. The incubation time varie~ according to the a~plication. The inhibitory factor-containing medium is then removed or inactivated (e.g., u~ing antibodies), (removing the media and replacing it will effect removal of inhibitory actor), and the cells are allowed to proceed thro~gh the cell cycle. By timing the period of ~xperimental intervention, vario-ls stages of the cell cycle can be examined for virtually all metabolic events of interest. For comparative reasons, several types of control cultures are used containing: 1) control cultures never exposed to inhibitory factor; 2) control culture~

W093/Z~9 ~ PCT/US93/039~3 with inhibitory factor not removed or deactivated; and 3) control cultures refed with media containing inhibitory factor. Synchronized cultures will provide greater magnitude effects in evaluation of a large number of i environmental, pharmocologic or other ~timuli.

ii) Cell Cycle Arrested Cultures.

The polypeptide of the subject invention i~ u~eful for studying metabolic events of cells and effect of growth stimulators on confluent cells (density inhibited cell~).
Mitogens are added to non-growing cells to stimulate division of the cells and inhibitory factor e.g., 1 to 10 x 10 8 ~ i~ added at the same time or at various time~
after the mitogens. Various metabolic events including, but not limited to, DNA synthesis, RNA ~ynthesi~, protein ~ynthesi~ and posttranscriptional and poQttran~lational modifications of macromolec~les can be studied as related to mitogen ~timulated cell cycling. In addition, thi~
method ~ffers a novel method to ~tudy the potential interactions between the lnllibitory factor and various mitogenic substances.

I iii) Other Cultures Primary explants or c~llture~ of tumors or ~ome other cultures which are not in exponential growth or stable (cvn~luent) state can be treated with inhibitory factor..

CYTOGENETIC APPLICATIONS

Unlike other methods of ~ynchronizing cells (such as mitotic shake off or drug treatments) inhibitory factor ~ynchronize~ the va~t majority of the cells in a culture.
Inhibitory factor treated cells do not divide until approximately ten hours after removal of inhibitory factor;
then, wi~hin one hour, greater than 95% enter and successfully complete mito~is, re~ulting in a striking W093/22~9 PCT/US93/03~3 ~
. 22 2i3444~
doubling of cell number. This contrast~ with the effect~
of currently u~ed mitogens to stimulate divi~ion, for example following the stimulation of lymphocytes with PHA
the number of mitosis gradually rises beginning at 40 ~
hours, reaches a maximum and levels off at approximately 3%
after 72 hours (Verma et al. Human Chromosome~, Pergamon Press, N.Y., N.Y. (1989~). Inhibitory factor reversibly inhibit~ over 90% of cultured human product~ of conception cells and cultures of many type~ of cancer cell~.

The time cour~e of inhibitory factor effect i~
consistent with knowledge of cell cycle tran~ition timen (Stubblefi21d, Methods Cell Phy~. 3,25-44, 1968): S last~
6-9 hr~, G2 la8t5 2 to 5 hr~ and M can take rom twelve to 60 minute~.

~ hile inhibitory factor appears to halt the cell~ at a single highly di~creet point in late Gl/GO (Fatteay et al., ~upra 1~91) most arresting agents cause cell~ to grind to a halt when they run out o DNA precursors ~ometime in S~ A
higher degr~e of synchrony wi 11 be achieved u~ing inhibitory factor. The experimental data shows a very sharp increa~e in cell numbe~s and pre~ervation of synchrony for more than one mitosisr a highly unusual property. In light of the nat~lral occurrence of the molecule in normal human tisæues, the natural occurrence of it~ receptor, and the reversibility of inhibition, synchronization by inhibitoxy factor i~ les~ toxic than currently available methods.

The u~e of inhibitory factor i8 expected to greatly increa~e the number of mito~e~ compared to untreated cells and ¢ven compared to cells sub~ect to a typical metaphase block of a few hours, or PHA. Optionally, thi8 method iB
u~ed in combination with traditional metaphase blocker~
(e.g., Colcemid). Thi8 i8 especially important in solid tumor~ where the cells have a variety of doubling time~.
The use of the inhibitory factor along with metaphase ~ 1 jl4~ ~ 5 W093/22~49 ~ PCT~U~93/03953 blocker~ allows the convenient simultaneous collection of cells with different doubling times.

Inhibitory factor is useful for obtaining different groups of mitotic figures that represent subpopulations of cells with different rates of growth. This occurs since the cells with the most rapid S phase enter mitoses before cells with a longer S phase. These cells are preferentially observed or isolated. Similarly cells with various length S phases can be isolated or observed. An easy way of observing such cells is with metaphase spreads and an easy way of isolating such cell~ is by shaking of the mitotic cells from the culture vessel.

If an additional generation is allowed to proceed before isolation or observation, differences in the length of M and especially G1 can also be noted.

B) Inducing Differentiation The inhibitory factor is useful to experimentally ' induce cellular differentiation and the subsequent morphological and biochemical alterations that accompany this proceæs. This includes cells obtained from solid and fluid tissues from mammaliall and non-mammalian species.

Various cells in culture are treated with, e.g., 1 to 10 x 10 8 M of the polypeptide inhibitory factor, and/or it~ peptide fra~ments and events associated with cellular differentiation, including but not limited to specific metabolic processes and morphological changes, are monitored during the culture period. In vivo differentiation is u~eful to treat various type~ of cancers and other diseases (see below).

C) Arresting the Cell Cycle The inhibitory factor provides cell~cycle arrest and 2 1 3 ~ ~ 4 PCT/US93~03953 cultures in "suspended animation" that subse~uently permits an investigator to store the cultures without routine and laborious refeeding or subculturing the cells on as frequent a schedule.

This application also provides a means to maintain cell cultures in "suspended animation" for purposes associated with shipping the cells over long distances, or maintaining the cultures outside of the culture facility for extended periods of time, without routine refeeding or exchanging cell culture medium. It can be especially difficult to refeed or perform maintenance or cells being prepared for transport to space or a large number of clones being analyzed for function. Embryos, fetu~es and adult organisms can similarly be caused to suspend division temporarily by use of inhibitory factor.

D) Treatment of Neoplastic Disease The first embodiment of the invention for treatment of neoplastic diseases (e.g., carcinomos, melanomas, sarco~as, lymphomas, adenomas) is the direct treatment to effect improved clinical state. Inhibitory factor may be used alone or in combination with drugs to directly slow or stop unwanted proliferations. Drugs most useful in combination with inhibitory factor to s~op cancer cells are those that work throughout the cell cycle such as alkylating agents which inhibit glycolysis and respiration as well as effecting DNA. Examples of these are Busulfan, Chlorambucil, Cyclophosphamide, Dacarbazine, Mechlorethahamine, Melphalan and Thiotepa. Certain antitumor antibiotics such as the anthracycline and chromycin~ (Dactinomycin, Daunorubicin, Doxorabicin, Placamyccin, Mitomycin C) and the nitroureas and cytokines which are cell cycle nonspecific may similarly be used in combination with inhibitory factor to cause direct toxicity.

~ t5 W093/2t~9 PCT/~S93J039~3 Cancers are particularly dangerous because the cancerous cells continue to proliferate and often metastasize (spread to and proliferate at di~tant sites).
In general, "undifferentiated" or "embryonic" or ~
"primitive" cells within the cancer are the most likely to proliferate and spread. Cancers that spontaneously regress often do so by undergoing differentiation. In addition, successful therapy often induces differentiation.
Worsening of the disease in contrast is associated with emergence of less differentiated cells. Pathologists routinely use greater degrees of differentiation as a good prognostic indicator and find more poorly differentiated tumors to the most aggressive. Thus, differentiation is good for the patient. Teratocarcinoma, ovarian carcinoma, thyroid carcinoma, neuroblastoma, glioma, melanoma, lymphomas, leukemias, prostrate cancer, colon cancer, breast cancer, lung cancer and other cancers all behave in this manner. In virtually all cancers the level of differentiation is an important factor, in the cases mentioned abo~e, it is a critical factor.

Animal cancers (including human cancers) are subject to inhibitory factor therapy by ca~sing differentiation.
An example of this is the differentiation and permanent irreversible inhibition of the h-lman leukemia line H6-60 by inhibitory factor.

~ nhibitory factor may be used in combination with known chemotherapy, as well as on its own to cause diffferentiation. Certain drugs are known to act by stimulation differentiation and slowing the growth of tumor cells. These includes androgens, estrogens, steroids and some cytokines. Inhibitory factor may be advantageously combined with drugs like Tamoxifin, Estradiol, Ethynl E~tradiol, Diethylstibesterol, Premarin, Medrooxy proge~terin, Megestrol, Hydroxyprogesterone, Testosterone, Floxymestrone, Methyl testosterone, Testolactone and other androgens, and corticosteroids including Predsinsone, W093J22~9 PCT/US93/039~3 ~-.
2,13~4~3 ~ ~

Hydroxycortisone and Dexamethasone to stimulate differentiation and slow tumor growth.

Even if the cancer is not forced to differentiate by inhibitory factor but is forced to remain in a nondi~iding state major clinical effect can occur due to the halting of disease progress and prevention of further metastasis. The body's natural immunity may act to destroy cancers that are no longer growing.

A second example of application in the class of direct t.herapy is treatment of any skin or squamous cancer or overproliferation of skin cells. It has been found that some cells -- human keratinocytes -- are especially sensitive to inhibitory factor. Basal Cell Epitheli.oma's (BCE), squamous carcinomas and a wlde variety of proliferative skin lesions including various icthyosis and p~oriasi~ are all treatable with inhibitory factor. Other proliferative diseases which are treated with inhibitory factor include ea~inophilia, benign reactive lymphocylic hyperplasia, lymphoproliferative diseases, adenomas and certain preneoplastic lesions like familiar polyposis.
j:
Inhibitory ~actor is useful in the treatment of human and animal leukemic disease (feline leukemia, HTLV virus, etc.).

The second embodiment of the invention to treat proliferative lessions (either concerns or benign proliferations) i~ in combination with other drugs. The above di~ea~es and other diseases may be treated thusly.
It has been demonstrated that _n vitro inhibitory factor act~ in a ~ynergistic manner with other cell modulators.
For example preliminary experiments (Woods, et al., FASEB
J, 5: 1463, 1991) have shown that inhibitory factor increases TNF cytotoxicity to certain tumor cells.

W093/22~9 PCT/US93/03953 ;- ~7 Other applications involve the inhibitory factor as adjuvant to increase the sensitivity of neoplastic cells to other agents. This permits the use of lower concentrations of anti-neoplastic agents to provide effective doses at less toxic levels.

Application is also be found in the use of the inhibitory factor in multiple-drug therapy for neoplastic disease. The inhibitory factor augments the efficacy of treatment by other compounds by a molecular mechanism that is separate but synergistic. This application is equally appropriate fsr both human and veterinary medicine.
Inhibitory factor can be used alone or with one or more additional factors such as TNF and cytokines in the treatment of disorders.

The administration of inhibitory factor with other agents such as one or more other factors, is temporally spaced or given together. The route of administration may be intravenous, intraperitoneal sub-cutaneous, intramuscular, topical, oral or nasal.

A third embodiment of the invention relating to the u~e of inhibitory factor as a chemoprotectant for normal cells in combination with chemotherapy agents. This combination decreases side effects. It is dependent on the cancer being resi~tant to inhibitory factor. Since inhibitory factor 1) prevents Rb phosphorylation;
2) underphosphorylated Rb maintains cells in a guiescent state; 3) certain cancer cells have oncogene producers which complex Rb; and 4) complexed Rb i8 not be effected by inhibitory factor. Certain tumors are insensitive to inhibitory factor. Cell lines transformed with SV40 laxge T antigen were assayed for inhibition by inhibitory factor. These cells were not inhibited by inhibitory factor. Simi~arly, human fibroblasts transformed with Adeno~irus are not inhibited. Control 3T3 cells used in these experiments were inhibited as in previous presented W093/22~9 2 ~ 3 4 ~ 4 3 PCT/US93/03953 experiments. A11 three of the cell linçs chosen because they contain Rb binding oncoproteins were found to resist inhibition, while none of the randomly chosen lines previously screened were resistant. Prostate cancer, bone cancer and bladder cancer are examples of cancer types insensitive to inhibitory factor. Similarly, in treatment of cancers derived from lung, breast, immune cells, blood cells, or other cells, inhibitory factor acts as a chemoprotectant. Other mechanisms of resistance are also possible.

In the presence of inhibitory factor, human cancer cells not inhibited by inhibitory factor, can be killed by a variety of treatment~ that destroy dividing cells while normal cells which are reversibly inhibited by i~hibitory factor would be protected from destruction. Thu~
inhibitory factor is very useful as a drug to decrease the side effects of chemotherapeutic agents. It is given along with or just prior to cytotoxic therapies. The normal cells would respond to inhibitory factor by stopping in a physiologically "safe" Gl resting phase while the cancer cells would continue to grow and be susceptible to killing by cytotoxic agents such as drugs or radiation.

Certain drugs are known to specifically efffect cells in M or G2 phase of the cell cycle. These include Zinostatin Bleomycin and some other anti-tumor antibiotics. In addition, the ~lkyloids such as vinbla~tin, vincristine, vindesine and others specifically act in M phase by blocking microtubule action. The Epipodophyllotoxins, etoposide and teniposide also specifically act in M phase with some effect in G2 and S.
The antimetabolites such as Fl~lorouracil, Floxurridine, Cytarabine, purine antagonist (mercaptopurlne, 6 thioguanine, azathioprine) and folate antagonist (methotrexate, dichloromethotrexate, triazinate), hydroxurea and hexamethylmelamine are also S sphase ~pecific. Inhibitory factor which will keep cells in Gl - W093/22449 ~ PCT/US93/03953 and specifically chemoprotect the normal against the toxicity of agents in these classes.

The specific cancers which may be best treated in the combination with certain drugs are evident from previous knowledge of the mode of action of these drugs and the cancers against which they are effective see, for example, The Washi.ngton Manual 1989, Dept. of Medicine, Washington University.

Certain dangerous DNA viruses are believed to interfere with cellular control mechanisms by producing molecules that interact with RB or with other cell regulatory molecules controlled by phosphorylation or by mechanisms affected by inhibitory factor (e.~., p53 or cyclins). It is believed that certain types of Human Papilloma Virus play a major role in causing cervical cancer. Carcinogenic types of Human Papilloma Virus (HPV
types 16 and 18) produce proteins inactivating RB (Dyson~
et al., 1989) (similarly to cells transformed with Adeno ElA or SV40 large T). This invention includes assays to determine if certain tumors have affected the RB mechanism, and the use of inhibitory factor as a protectant during therapy of these tumors.

In the case of preneoplastic (dysplastic) mucosal lesions or in situ carcinoma caused by HPV, a combination of inhibitory factor to protect adjacent normal tissue and a cytotoxic agent is useful for treatment. Such combinations can be locally applied. Current treatments are vari!ous surgical procedures which tend to leave some in situ cancer behind unless relatively large areas are removed.
.
Inhibitory factor without cytotoxic therapies are effective again~t the condyloma (wart) producing viruses ~hat do not produce oncogene products reactive with RB

W093/22~9 PCT/US93/03953 ~,'L3 4 4 ~3 (e.g., HPV Types 6/11, Dyson, et al., Science 243, 934-937, 1989~.

E) 5creening Antineoplastic Agents The inhibitory factor (which provides mitotic arrest) has application in screening of drugs. Many drugs act specifically in one region of the cell cycle (see above).
By comparing the putative anti-cancer drugs effect on parallel sets of cultures - one set proliferating and the other mitotically arrested with inhibitory factor - the relative action on stable versus multiplying populations is readily assessed. Since the mitotic arrest mediated by the polypeptide is totally reversible, future growth measured by colony formation as well as survival of inhibitory factor treated cells is easily assessed. Drugs which act at other specific stages of the cell cycle can also be advantageously sought and analyzed using ihihibitory factor. Inhibitory factor is used to place cells in a specific stage of the cell cycle as described in section A) above.

ABNORMAL PROLIFERATIVE STATES.
Inhibitory factor is also useful in the treatment of other diseases having abnormal proliferation such as psoriasis, other icthyosis, keloid or certain autoimmune diseases. Keratinocytes ar~ especially susceptible to inhibitory factor. Inhibitory factor is useful in the treatment of psoria~is, keloids and other proliferative skin diseases.

P~oriasi~

.
Psoriasis results from the exces~ divi~ion of skin cells ~as do other proliferative skin diseases called "icthyosiY"). In patients with psoriasis, skin cells divide ~even times faster than normal. This disorder is treatable with inhibitory factor.

.. . . . .. . . .. ... . . ... . . . . .. . .. . .. . ... . . .

;- W0~3/22449 2 I 3 ~ ~ 4 3 PCT~US93/03~53 Warts Warts (or "condyloma") also are the result of excess epthelial cell proliferation, as are several other~skin pathologies. Warts are caused by Human Papilloma Viru~es (HPV's~. Since it is known that keratinocytes (the type of epitheli~l cells that overproliferate in these lesions? are especially sensitive (approximately 30-50 fold more sensitive than most cells) to inhibitory factor these lesions can be treated efectively with inhibitory factor.

~eloid Keloid is a disease caused by overproliferation of scar tis~ue, thus it also can be advantageously treated with inhibitory factor as it is known that human fibroblasts are inhibited.

Athero~clerosi~

Atherosclerosis involves the overproliferation of cells lining the blood vessels. Inhibitory factor reversibly prevents proliferation of such cells including smooth muscle cells and endothelial cells.
Overproliferation leads to a variety of a~normalities including heart disease, strokes, renal disease and others. These diseases also can be prevented by decreasing atherosclerosis with inhibitory factor.

Proliferative Disease of the Eye Proliferative diseases of the eye including retinopathy are treatable with inhibitory factor to stop unwanted proliferation.

In1a~atory Di~oxders Unwanted inflammatory states, such as allergies and Wo93J22~9 2~ 4~ 32 PCT/US93/03g~3 autoimmune disease, even some types of arthritis involve the proliferation of certain cells that can be stopped with appropriate inhibitory factor therapy. Multiple sclerosis has been postulated to be either a viral or autoim~une (inflammatory disease). In either case TNF is known to be altered locally in M.S. an~ thus inhibitory factor can be used as therapy.

Aging It is believed that normal human cells have a limited capacity to divide. After a certain number of divisions this capacity is exhausted and the human body becomes unable to replenish itself. This is supported by evidence including the fact that cells from a young person will divide many times in tissue culture before "senescence"
(failure to divide) while cells from an older person have a much more limited capacity to divide before senescence.
The use of inhibitory factor early in life to prevent unnecessary divisions might allow some of the limited number of divisions to be saved for old age and thus to delay the onset of various organ degenerations seen in old age. This includes use as an ointment for skin aging as well as by other means to prevent deterioration of various internal tissues.

EUCARYOTIC CELL CLONING.
Temporary inhibition by inhibitory factory factor might also be extremely useful in situations where cell passageæ are difficult. For instance in eucaryotic cell cloning often a iarge number of clones are initially ~
obtained but only a few will be useful. Growth and passage of many clone~ during the evaluation period (for example while as~ays of the clone's ability to produce a biologic material, e.g., a monoclonal antibody, a biologic response modifier or an enzyme of interest) may be difficult.
Inhibitory factor can be u~ed to easily place cells in a ~afe but non-~ividing state. This method preæerves a much '1 ~ 4~
W093/2~9 PCT/US93/03953 larger number of important clones during the evaluation period with less effort and chance of loss or contamination.

Production of Cell Products.

Inhibitory factor reorients the protein synthetic mechanism of cells; initially it shuts off the synthesis of many proteins (total synthesis drops by 80%) however within hours the total synthesis is only 20% to 25% less than in exponentially growing cells. Thus, it is believed that certain structural proteins necessary for an increase in cell number are shut off but many proteins made in Gl are actually synthesized at a higher rate. ThuR increased production of certain biologically useful proteins (e~ g., monoclonal antibodies or other excreted proteins) is possible using inhibitory factor. Inhibitory factor increases production of monoclonal antibodies. Even if production per cell is not increased it may be very beneficial to have a metabolically active "bioreactor" with stable cell number in many instances. In theory, such bioreactors might even provide an entire metabolic pathway.

Diagnostic Te~ting Inhibitory factor is helpf~ll as an aid in karyotypic (chromosome) analysis. It is especially important in situations where low numbers of mitotic cells are present, such as solid cancers, or in situations where low numbers of cells are available for analysis (some difficult amniotic fluid taps or the isolation of sub populations of cells).

Isolation of Viru~es Additionally, the inhibitory factor is useful in isolating viruses. Viruses often require activitely divlding cells. The most frequent reason for in laboratory failure to isolate viruses from adequate clinical specimens W093/2~449 PCT/US93/03g53 2~3 ~ ~ ~3 34 in plating of the virus on cells that have become too dense or too confluent. Overgrown cells are also the major cause of delay in isolation of viruses clinical labs most frequentiy isolate. Inhibitory factor can be used~to hold cells at the ideal density for virus growth and then initiating exponential growth (which is most helpful for growing viruses such as herpes virus, cytomegulovirus and many other viruses which require activily growing cells) by removing inhibitory factor as described above.

Some viruses are difficult to culture using current methods. However, if cells were infected at the optimum point in the cell cycle, for example, during S phase or M
phase or G2 growth is much more reliable. The only methods currently available to achieve ~ultures with high amounts of S, M or G2 phase cells for this or any purpose is inhibitory factor.

Lastly is the use of inhibitory factor to allow growth of viruses which cannot currently be grown in the lab (e.g., HPV). It is believed that HPV and other viruses requires cells with certain differentiation properties.
Since inhibitory fa tor wil]. cause differentiation, it can facilitate growth of this class of viru~e~.

* * *

Nucleic acid products of the invention are useful when labeled with detectable markers (such as radiolabels and non-isotopic labels such as-biotin) and employed in ~, , , , ~
hybridization pxocesses to locate the human inhibitory factor gene position and/or the position of any related gene family in a chromosomal map. They are also useful for identifying human inhibitory factor gene disorders at the DNA level and used as gene markers for identifying neighboring genes and their disorders. The identification of the genes and defects in them are important in diagnosis and prognosis of proliferative diseases and cancers. The -- W093/22~9 ~1 3 4 ~ 4 3 PCT/US93/~3953 protein from these genes is assayed by use of monoclonal or polyclonal antibodies in various formats including western blots, dots blots and ELISAs. The detection of protein facilitates diagnosis and prognosis of various diseases involving altered levels of cell proliferation.

Typically, to affect cells inhibitory factor should be administer~d in a range of 1 nanomolar to 1 micromolar, advantageously the factor is administered at a concentration from 1 to 10 x 10-8 molar.

Other components of the media affect the optimal concentration of inhibitory factor: for example, media with low calcium concentration increases the sensitivity to inhibitory factor.

The following examples are offered to more fully illustrate the invention, but are not to be cvnstrued as limiting the scope thereof.
l~e E~CanlP1e~

EXAMPLE I

Isolation and Purification of the 18 kD Bovine Glycopeptide A suspension of bovine cerebral cortex cells was prepared in Dulbecco's minimal essential medium (DMEM) containing 25 mM HEPES buffer (pH 7.1). The cells were pelleted by centrifugation at 2000 x g for 5 min, the cell pellet was su pended in HKM buffer (10 mM HEPES, 120 mM
KCl, 5 mM MgC12, pH 7.1) and incubated with 0.02 units/ml of proteinase from S. grise~ls ("pronase") for 15 min at 37C with con~tant mixing.

Pre~ious reports suggested a single treatment with the protease was sufficient tSharifi et al., Neurochem. 46:
461-469, 1986a~, however, three subseguent protease treatments of.the bovine cerebr~l cortex cells essentially W093l22~9 ~ PCr/US93/03~3 - ~344~ 36 l~

triples the yield of the si ~loglycopeptide inhibitory factor released.

The cells were then removed by centrifugation~at 2000 g for 5 min. The supernatant fluids containing released molecules were then collected, the macromolecules were precipitated with ethanol overnight, and the resulting precipitate was collected by centrifugation, resuspended in 100 ml distilled water, extracted with chloroform/methanol (2:1, v/v), dialyzed against four liters of distilled water overnight, with at least six water changes, and the dialysate was then lyophilized to dryness.

The lyophilized material was resuspended in 2 ml of 0.05 M acetate buffer (pH S.0), clarified by three subsequent centrifugations ~t 1,000 g for two minutes and applied to a DEAE-agarose gel. Approximately 50 mg protein of the chloroform/methanol-extracted material was incubated with DEAE-agaro~e gel (10 ml bed volume) at 4C for 30 min with constant mixing. The gel was wa~hed three times with 3 ml of the acetate buffer and the biological inhibitory factor was eluted with 3 ml of 0.4 M NaCl in 0.05 M acetate buffer (pH 5.0~. The eluate was then dried in a Savant speed-vac apparatus.

The DEAE-agarose purified samples were then further purified with agarose-bound wheat germ agglutinin (WGA).
The WGA was previously equilibrated with phosphate buffered saline ~PBS, pH 7.1~, and the protein fraction was suspended in 1.5 ml of PBS and applied to a 1.0 ml WGA
column. After incubation at 4C for 30 minutes, the inhibitory factor-containing fraction that does not bind to the WGA column was removed by washing with 2 ml of PBS.

The WGA-eluted fraction was then further purified by applying the protein to a HPLC TSK-3000 size exclusion column. The elution buffer consisted of 0.1 M sodium phosphate (pH 6.8), and the flow rate was adjusted to 0.1 W093/2~9 2 1 3 1 4 4 3 PCT/US93/03953 . . .

ml/min. The eluate was monitored for absorption at A280, and the fractions associated with the major protein peak were pooled, dialyzed overnight at 4C against four liters of dilute PBS. The sample was then dried in a spee,~d-vac apparatus, resuspended in 0.5 ml of distilled water and the protein content and the biological inhibitory activity were measured.

EXAMPLE II
Measurement of the Biological Inhibitory Properties of the Inhibitory Factor i) Protein Synthesis Inhibition Protein synthesis was measured with cells from subconfluent cultures that were suspended in DMEM
containin~ 25 mM HEPES buffer, pH 7.1 (Sharifi et al., Neurochem. 46: 461-469, 1986a; Bascom et al., J. Cell Physiol. 12B: 202~208, 1986). Either HKM buffer alone (controls), or HKM buffer with various concentrations of the 18 k~ brain inhibitory factor (experimentals) were added to each reaction tube. The cells were incubated for 30 to 45 min at 37C to allow the cells to bind the inhibitory factor, 35S-methionine in HKM was then added to radiolabel cellular proteins and the cells were incu~ated at 37C for an additional 10 to 30 min. After this incubation period the macromolecules were precipitated with trichioroacetic acid (TCA~ and the amounts of intracellular acid-soluble and acid-insoluble radioactivity were determined by scintillation collnting. This assay is rapid and requires only nanograms of inhibitor, and one unit of biological activi,ty is set as the ~uantity that provides a 25% inhi~ition of mouse 3T3 cell protein ynthesis.

li) DNA Synthesis I~libition 3H-thymidine incorporation was measured with cultures in 24- or 48-well culture plates. For experiments on mitotic arrest and cell cycle kinetics, subconfluent cell monolayers were incubated with 0.2 ml of DMEM medium w093/2~449 ~3 ~ ~ ~3 38 PCT/U~91/~3953 containing 2.5% calf serum and H-thymidine (adjusted with non radioactive thymidine to a specific activity of 0.5 Ci/mmole) for 2 hr at 37~C. After incubation the media were removed and the cells were solubilized in 1 ml~of 0.2 N NaOH. Macromolecules were precipitated with 10% TCA, after the addition of 0.1 ml of 1% BSA as a carrier.
Radioactive thymidine in the intracellular acid-soluble pools and in the cell DNA was measured by scintillation counting (Chou et al., Cancer Lett. 35: 119-128, 1987;
Fattaey et al., J. Cell. Physiol. 139: 269-274, 1989).

Experiments where the sialoglycopeptide was studied as a potential antagonist to mitogens, that stimulate cell division, (e.g., EGF, TPA and bombesin) utilize confluent and quiescent cultures (See Bascom et al., J. Cel:L.
Biochem. 34: 283-291, 1987; Chou et al., Cancer Lett. 35:
119-128, 1987; Johnson and Sharifi, Biochem. Biophys. Res.
Comm. 161: 468-*74, 1989).

iii) Cell Growth Inhibition Cells were plated in 48-well tissue culture plates at a density of 2 to 5 x 103 cells per well and the cultures were incubated for ~4 hr prior to initiating growth-inhibition experiments. Cultures were refed with filter-sterilized medium at the start of the experiment, either with 0~5 ml of complete medium alone (controls) or with 0.5 ml of complete medium containing various concentrations of the glycopeptide inhibitory factor (experimentals) (Fattaey et al., J. Cell. Physiol. 139:
269-274, 1989; Fattaey et al, Exper. Cell Res. 194: 62-68,`
1991; Edson et al., Life Sci. 48: 1813-1820, 1991).

Triplicate samples were taken at least once every generation time (18 to 24 hr), and cells were harvested by trypsinization, washed, dissociated by gentle pipetting, and counted in a 1:20 dilution of Isoton II in a Coulter Counter.

W093/22449 2 1 ~ ~ 4 ~ 3 PCT/US93/039~3 EXAMPLE III
Improved Purification, and the Eliminati~n of the Protease Activity by the Addition of a Final HPLC Ion-Exchange Step Although the purification procedure described~in Example I appears to provide a 18 kDa glycopeptide product that was homogeneous, small molecular weight peptides contaminated the samples to various degrees from preparation to preparation. These contaminants were difficult to visualize when purified samples of the glycopeptide were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and stained by the Comassie Blue method of Sasse et al., in Current Protocols in Molecular Biology, (F.A. AuSabel, R. Brent, R.E. Kingston, D.D.
Moore, J.G. Saidman, J.A. Smith and K. Struhl, eds.), pp.
10.6.1-10.6.2 (1991). When the gels were stained by the silver-stain method of Merril et al., Methods Enzymol. 104:
441-447 (1984), the pre~ence of the contaminating peptides could be ~een as light and diffuse stained areas, often masked by the tracking dye used to time the electrophoresis run, and in the area of the SDS-PAGE sample representing contaminating molecules of 14 kDa or smaller. Careful washing and de5taining is necessary to reveal this smear.
Depending on the particular glycopeptide preparation being analyzed, the relative amouIlt o small molecular weight peptide contamination varied from 20% to 40% of the total glycopeptide product (determined by densitometric scanning of the silver-stained gels).

The presence of these small molecular weight peptides reduced the specific biological inhibitory activity (units per nanogram) and prevented meaningful studies concerning structural analysis and protein sequencing of the glycopeptide inhibitory factor.

A ~imple, but effective, procedure was developed to provide a homogeneous glycopeptide preparation. This procedure involves the use of a HPLC/DEAE ion-exchange step as the final step of bioseparation.

W093/22~9 4~ PCT/US93/03953 -~ 40 A Protein-Pak D~AE (Waters3 HP1C column was equilibrated with 20 mM Tris-HCl (pH 8.2) or 40 mM NH4HC03 (pH 8.0) and lO mM NaCl. The glycopeptide (20 to 60 micrograms) was added to the DEAE column and the glycoprotein was eluted by int~oducing over a 30 minute period a linear NaCl gradient that increased from lO to lO0 mM. The eluant was monitored at A280, and the purified glycopeptide that eluted from the HPLC column as a single and sharp peak, at approximately 50 mM NaCl (as determined by refractometry), and well-separated from the contaminating small molecular weight peptides, was collected manually.

The glycopeptide inhibitory factor was then lyophilized to dryness and resuspended in 2.0 ml of distilled water. The sample was then desalted by five serial centrifugations ~each with 2.0 ml of distilled water) in microconcentrators (Amicon) fitted with a membrane that retained molecules over lO kDa. The retentate, containing the 18 kDa glycopeptide was lyophilized and stored frozen at -70C.

Analysis of the final glycopeptide inhibitory factor pro~uct by silver-stained SDS-PAGE gels stained by the silver method revealed a single 18 kDa protein band that had been succe~fully resolved from the contaminating small molecular weight peptides.

The improved procedure for purification yielded a homogeneously purified glycopeptide that was free of any detectable protease activity, thereby providing preparation for structural analysis and protein sequencing. It also provide~ a product with a single mode of action due exclusively to inhibitory factor.

W093/22449 ~ 4 ~ PCT/U~93/039~3 EXAMPLE IV

Amino Acid Sequence Analysis Sequencing was attempted using the general procedure~
set forth in Lane et al, J. Protein Chem. 10 No. ~, 151-160 tl991~ .

Sequencing Procedures Strategy A 12 microgram (600 pmoles) of inhibitory factor wa~
prepared as in Example I and further purified as in Example III. Since the protein is both N-terminally bloc~:ed and glycosylated, seguencing and associated ta~ks were extremely difficult.

Cyanogen bromide was obtained from Sigma, sequencing grad~ trypæin, chymotrypsin, endoproteinase Asp-N and Olu-C
from Boehringer Mannheim. Iodoacetic acid was purcha~ed from Sigma, dithiothreitol from Calbiochem. HPLC grade tri1uoroscetic acid was obtained from Applied Biosystems, Inc. (Foster City, California); HPLC-trade acetonitrile and water from Burdick ~ Jackson; 6N HCl from Pierce; and Vydac Hoer.c columns from the Nest Group (Southboro, Massachusetts). Automated sequencer and analyzer reagents were provided by the manufacturer. All other reagents were purchased from common commercial sources in the highe~t ~rade available.

Reduction and Alkylation of Inhibitory Factor : i Inhibitory factor destined for proteolytic cleavage wa~ reduced and S-carboxymethylated as described by Stone et al, Technique~ in Protein Chemistry (Hugll, ed.) Academic Pres~, San Diego, pp. 377-39~, (1989). 6.0 ~g (300 pmol) aliguot~ of bovine inhibitory factor were di~solved in 50 ~1 8 M urea/0.4 M NH4HC03 and reduced with 5 ~1 of 45 mM dithiothreitol at 50C for 15 min. Cysteine ~ 344~à 42 re6idue~ were alkylated by reaction with 5 ~ of lO0 mM
iodoacetic acid at room temperature for 15 min. Subsequent enzymic cleavage was carried out without further desalting or tran~fer as described below.

Proteolytic Cleavage of Inhibitory Factor Try~sin, chymotrypsin digestions: The above alkylation mixture containing S-carboxymethylated bovine inhibitory factor was diluted fourfold without further processing (Stone et al, supra 1989~ to a final buffer concentration of ~ M urea/0.1 M NH4HC0~. Enzyme was added to this solution to maintain a substrate to enzyme ratio of 25:1 (w/w), and the mixture was allowed to incubat:e at 37C
for 20 hr. The resultant peptide mixture was frozen at -20C until a separation by reverse-phase HPLC was performed.

Narrow-Bore Reverse-Phase HPLC Separation of Peptides Peptides were chromatographed on a Hewlett-Packard 1090 HPLC equipped with a 1040 diode array detector, using a Vydac 2.1 mm x 150 mm C18 column. The gradient employed was a modification of that previously described by Stone et al, æuPrat (1989). Briefly, where buffer A was 0.06%
trifluoroacetic acid/ H20 and buffer B was 0.055%
trifluoroacetic acid/acetonitrile, a gradient of 5%B at 0 min~ 33%B at 63 min, 60%B at 95 min, and ~0%B at 105 min with a flow rate of 150 ~1/min was used. Chromatographic data at 210 nm, 277 nm, 292 nm, and W ~pectra from 209-321 nm of each peak were ac~uired. While monitoring absorbance at 210 nm, fractions were manually collected into 1.5 ml microfuge tube~ and immediately stored without drying at -20C in preparation for peptide sequence analysis.

The inhibitory factor was unable to be analyzed and believed to have been refractory to those standard procedures; thus, alternative procedures were developed.

W093/2~ P~T/US93/03~3 Analytical Step to Show that the Bovine Inhibitory Factor Can be Proteolytically Cleaved Since inhibitory factor could not be cleaved with either trypsin or chymotrypsin, a series of analytical tests were run with bovine inhibitory factor, using 5 micrograms per assay, to determine if the 18 ~Da bovine inhibitor,y factor could be proteolytically cleaved.

The inhibitory factor was first solubilized in phosphate buffered saline (PBS, pH 7.0), heated to 95C for 5 min. to denature the polypeptide and then cooled to room temperature prior to the addition of the enzymes which were in 0.2 M ammonium bicarbonate (pH 8.0). The two enzymes used were bovine pancreatic trypsin (TPCK-treated) (Sigma Chem. Co., catalog ~ T-8642), and endoproteinase Asp-N
(Si~ma Chem. Co., catalog ~ P-3303, suitable for sequencing and peptide mapping). Other suitable enzymes may be chosen from the group including Ol~l-C and pronase, trypsin, chymotrypæin, B substilysin, serine proteases, thiolproteases, cathepsin D proteases, sulphydryl protease, metallo-protea~es, trypsin like protease, estrase and - carboxy proteases, non-specific protea~es and other specific proteases.

Five micrograms of the inhibitory factor, in PBS, were incubated for 1 hour at 37C, in a thermal cycler (reaction volume of 25 to 30 microliters), at an enzyme/substrate ratio of 1:10, 1:50 or 1:100; i.e., 0.5 microgram of enzyme, 0.1 mi~rogram of enzyme, O.05 microgram o enzyme.
Controls"were also run with the inhibitory factor incubated and handled in an identical manner, but without either the trypsin or endoproteinase Asp-N ~dded. Reactions were terminated by raising the temperature to 70C in the thermal cycler.

The resulting reactants were analyzed by SDS-PAGE
(Lakshmanarao et al, supra 1991) and silver-staining as described by Merril et al, Methods Enzymol. 104: 441-447 W093/2~9 ~3 ~ 4 43 PCT/U~93/039~3 t~-(1984). All three tr~psin concentrations completely hydrolyzed the 18 kDa inhibitory factor and little, if any remaining substrate could be vis~alized on the gels. Both of the higher concentrations of endoproteinase Asp~N almost completely hydolyzed the ;18 kDa substrate, while the lower concentration clearly was effective but some substrate (maybe one-third) remained as an 18 kDa band. In any event, the inhibitory factor clearly is sensitive to proteolytic hydrolysis. Using these new conditions following heat denaturation.

The resulting fragments are then separated and sequenced. For preparatory scale work the fragmentation of the purified 18 kDa bovine inhibitory factor (described in Example III), is carried O~lt essentially as described above but with approximately 25 to 50 ~g with an enzyme/protein ratio of at least 1:50. This provides adequate quantities of the fragments for separation of the fragments by HPLC
and subsequent sequencing by routine methods.

Amino Acid Analysis of the 18 kDa Bovine Inhibitory Factor Standard amino acid analysis was carried out on a sample of inhibitory factol. Based on the known mass of the inhibitory factor and the knowledge that it is a glycosyla~ed molecule with less than 10% of the mass composed of carbohydrates, the amino acid composition was çstimated. The total number of amino acids per bovine inhibitory factor molecule appears to be lS3, and the identity of the amino acids is shown below.

~ ~ v ' ~
WO 93/22449 PCI`/US93/03953 Amino Acid Compo~ition of the 18 kDa Bovine Inhibitory Eactor Number of Residue~
Amino Acid( s)* per Molecule Asp/Asn t D&N ) 15 Glu/Cln ~ E&Q ) 25 Ser (S) 17 Gly (G) 24 His (H) 3 Arg (R) 3 Thr ~T) 8 Ala (A) 12 Pro (P) 11 Tyr (Y) Val (V) 6 Met ~M) o Ilu (I) 3 Leu (L~ 6 Phe (F) 2 Lys (K) . 13 Total - 149 * Both the three- and one-letter abbreviations for the amino acids are listed This analysis is based on the most likely fit knowing that the bovine inhibitory factor is approximately 18 kDa had having carbohydrate residues th~t compose no more than 10%
of the mass.

The DNA seguence encoding inhibitory factor can be obtained using routine procedures for synthesizing - oligonucleotide probes using the amino acid seguence, and screening libraries. See Sambrook et al., Molecular Cloninq A LaboratorY Manual, 2d Edition, Cold Spring Harbor Laboratory Press, Chapter 11, 1989; Heller et al., Biotechniques 12, No. 1, p. 30-35 (1992); Itakura et al, Annual Rev. of Biochem, 53, 323-356 (1984); or Wood et al, PNA5, 82, 1585-1588 (1985).

W093/22~9 PCT/US93/03953 -3~3 4~
-E~AMPLE V

Antibody Production Rabbit polyclonal antibod~ àgainst the native (nondenatured) form of the inhibitory factor was prepared by subcutaneous injection of New Zealand white rabbit~ with 200 ~g of the 18 kDa bovine inhibitory factor purified as in Example 1 in àn equal volume of Freund's complete adjuvant or mixed with TltermaxlM adjuvant and as described by Lak~hmanar~o et al. Exptl. Cell Res. 195, 412-415, (1991~. Alternatively, the autoimmune offspring of a male Balb/c/J mouse and a female Balb/AJ mouse are used.
Further boosting once a month with antigen in complete Fruend~ adjacent has resulted in higher avidity antibody.
In order to detect the reduced and denatured antigen by Western blot analysi~ antibody aiso was prepared against the raduced and denatured inhibitory factor. The 18 kDa inhibitory factor band, recovered from Laemmli, SDS-PAGE
was excised, minced and passed through a syringe several times and blended with an equal volume of Freund's complete adjuvant. Rabbits were then îmmunized in the same manner as deæcribed above. Serum IgG, obtained with both the native and denatured forms of the bovine inhibitory factor was purified with a DEAE affigeJ. blue column and by ammonium sulfate precipitation (Lakshmanarao et al., Exptl.
Cell Res. 195, 412-415, 1991).

EXAMPLE VI

Purification of the Parentàl Inhibitory Factor Plas~a membrane pr~paration and NaCl release - Plasma membranes were obtained from cell suspensions of bovine cerebral coxtex ti~sue homogenized by 10 strokes in a Dounce homogenizer. The homogenate was centrifuged at 1,000 x g for 15 min, and the supernatant fluid was collected and recentrifuged at l,000 x g for l5 min. The W093~22~9 ~1 3 ~ 4 4 3 P~T/U~93/03~3 resulting supernatant fluid was centrifuged at 40,000 x g for 60 min to pellet the plasma membranes, and the membrane-associated proteins were released by resuspending the pellet in 10 vol of a b~lffered 3M NaCl solution~ (3M
NaCl, 0.1 M phosphate buffer, pH 7.2, containing l~g/~l each of phosphoramidon, pepstatin A, leupeptin and aprotinin). The membrane suspension was mixed for 30 min at 4C, centrifuged at 104,000 x g for 60 min and the supernatant fluid was collected and dialyzed overnight at 4C. The samples were first dialyzed against 1 M NaCl, followed by three changes of double-distilled water and after dialysis protein determinations were carried out by the method of Bradford, Anal. Biochem. 72, 248-254 (1976).

Preparative i~oelectric focu~ing - The NaCl-released membrane proteins were resuspended in 40 ml of double-distilled water, and electrofocu~ed at 12W for 4 h with 2% amphyli~es (pH 4-10, Pharmacia-LKB Biotechnology Inc., Gaithersburg, MD) in a BioRad Rotofor apparatus (Bio-Rad, Richmond, CA). The resulting 20 (2 ml) fractions were dialyzed against three changes of dilute PBS and concentrated to dryness in ~ S~vant Speedvac (Savant In~truments Inc., Hicksville, NY).

Lectin affinity chromatogr~hy - The electrofocused samples were solubilized in working buffer (50 mM Tris-HCl, 10 mM
CaC12, p~ 8.0) and then added to a column (1 ml bed-volume) of Limulus PolYhemus agglutinin (LPA) (EY Laboratories, San Mateo, CA) that previously was equilibrated with working buffer. The ~amples were incubated with constant mixing for 1 h;at room temperaturè, and the column was then washed with working buffer until no el-lting protein (A280) could be d~tected. The bound proteins were eluted with elution buffer (50 mM Tri~-HCl, ~ mM EDTA, pH 8.0), and both the bound and u~bound fractions were extensively dialyzed at 4C against dilute PBS and lyophilized to dryness. Equal volume samples of the dialysis fluids were also lyophilized W~93/22449 PCT/US93/03gS3 ~-2~34443 4~ ~, as controls for measurements of biologiçal inhibitory activity.

Antibody affinity chromatography - IgG (1 mg) prepared to the native inhibitory facto~`~as bound overnight at 4C to ~ ml of prewashed Affi-Ge~ HZ beads ~Bio-Rad, Richmond, CA) following the protocol provided by the commercial supplier. The protein fraction that isoelectric focused at pI 5.1 (~1 mg protein) was added to the column, incubated overnight at 4C and the column was then washed with column buffer until no eluting protein (A280) could be detected.
3 M MgC12 (pH 7.1~ was used to release the bound proteins, and the eluted protein fractions were collected, pooled, dialyzed overnight against dilute PBS at 4C and lyophilized to dryness.

We~tern analy~i~ and im~unoblots - Western analysis was carried out with a polyclonal antibody raised against the denatured bovine inhibitory factor essentially as described above. Immunoblots were carried out by transferring sample5 to nitrocellulose Ising a crosablot apparatus (Sebia, Paris, France), blots were then analyzed with antibody prepared to the native inhibitory fa~tor and the relative antigenicity of vario~ls protein bands was determined by densitometric scanning (Lakshmanarao et al., Exptl. Cell Res. 195, 412-415, 1991).

Protein 8ynthe~i8 inhibition a~ay - At various stages of purification the ability of samples to inhibit protein synthesis was tested with Swiss 3T3 cells essentially as described by Shari~i et al., J. Neurochem. 46, 461-469, (1986).

Cell proliferation inhibition assay - Cell proliferation inhibition wa~ measured with exponentially dividing culture~ of Swi8s 3T3 cells propagated in 48-well plates as described by Fattaey et al., J. Cell Physiol. 139, 269-274, (1989). The total medium volume of all cultures was 300 ~15~44~
W093~2~ PCT/US93tO3953 ~1, and one set of control cultures received 40 ~1 of PBS
while another received 40 ~11 of the lyophilized dialy~is fluids that were solubilized in 1 ml of sterile double-distilled water. Experimental cultures recçived complete culture medium with 40 ~1 containing various concentrations of LPA bound or unbound protein solubilized in 1 ml of sterile double-distilled water. At the start of the experiment and after vario~ls periods of incubation with thè additives cell numbers in each well were determined with a Coulter Counter, model ZM as described by Fattaey et al suPra. (1989). Comparisons of cell proliferation were determined by the formula [FeXp - AeXpl/l3cont Acont]
100 where A was the cell number (7.5 x 10 ) when the medium supplements were added! and F was the final cell num~er in the PBS (cont) and experimental (exp) wells at the end of the experiment.

* * *

As a preliminary assessment of the nature of the association o the parental inhibitory factor with the' bovine cerebral cortex membranes, 50 mg protein aliguots of membrane preparations were inc~lbated with either isotonic buffer, 3 M NaCl or 3 M urea at 4C for 30 min. and the membranes were then pelleted by centrifugation as described above. Immunoblot analysis of the membrane soluble extract and the pelleted membrane fractions, using polyclonal IgG
raised to the native form of the inhibitory factor, revealed that the antigenic material was not released from the membranes when they were incubated in isotonic buffer.
Incubation of membranes with 3 M NaCl, however, efficiently released the majority of the antigenic component suggesting that the parental inhibitory factor was not an integral membrane component but rather a membrane-associated element. Treatment of membranes with 3.0 M urea, another regent commonly used to release membrane-associated proteins, rendered both the soluble and the membrane fractions nonreactive to the polyclonal IgG against the W093/22~9 2~3 4 4 ~ ~ PCT~US93/03953 ~

native bovine inhibitory factor (Fig. 1). The loss of antigenicity of both the membrane and ~oluble fractions suggested that the incubation period with 3 M urea denatured the antigenic material and rendered it nonreactive. Since the denatured form of the parental bovine inhibitory factor most likely would not be biologically active, further use of 3 M urea to release the molecul~ from membranes was not pursued.

Since 3 M NaCl proved to efficiently release virtually all of the antigenically-reactive material from cell membrane preparations, we were led to utilize this reagent a~ our initial step in the parental inhibitory factor purification scheme. In order to purify a greater quantity of the parental inhibitory actor, 500 mg protein of the membrane preparation was subjected to 3 M NaCl treatment which yielded 85 mg of protein (17% of the total membrane protein3, and a six-fold purification o the parental inhibitory factor (Table 1).

WO 93/2~449 ~ 1 3 4 4 1 ~ PCl/US93~039~3 U~
o ~ I o W ~ "

~ I ~ '~D O
O 10 O ~ N
.,, ~ E~ ,~ In .q ~

H ~ ~
C~ ~ ~I ~ ~ O
~- e~ ~ ~
~D
~D
~, _ O O CO U~
~, ~a ~ ~`
~1 ~ _ o ~1 u'Jo' ~ o D O o rj S3 0 1~ ~ O
U~

~'O
Q~ ~ O
o ~
E~
~: ~c u~ ~ ~
w WO 93/22449 ~ l 3 ,~ ~ 43 P~/US93/03953 ;^ -SDS-PAGE analysis of this initial extract revealed numerous protein bands indicating the necessity for further purification ~Fig. 2, lane B).~

The NaCl-released membrane proteins were next su~jected to preparative isoelectric focusing utilizing a BioRad Rotofor as described in the Materials and Methods.
85 mg of the NaCl-released proteins were introduced to the Rotofor unit, the material was focused for 4 h and 20 fractions (2 ml) were collected across a pH gradient from 4.0 to 12Ø The proteins were relatively egually distribu~ed across the gradient with each fraction having æomewhere between 3.5 to 4.0 mg of protein. Immunoblot analysis of each fraction revealed that the antigenically-reactive material primarily was associated with two fractionso a major reactive peak was found to be focu~ed at a pI of 5.1 (fraction number 4); and, a minor reactive peak was focused at a pI of 7.2 (fraction number 10) (Fig. 3). Approximately 90% of the reactive antigen was focused at pH 5.1, and the amount of protein recovered constituted 0.8% of the original membrane protein. No' antigenically reactive materials could be found by immunoblot analysi~ in the remaining 18 fractions (Fig.
3). At this stage of purification the parental inhibitory factor was purified approximately 123-fold (Table 1~.
Eowever, there still wa~ a heterogenous array of protein bands when the isolectric focused fraction at pH 5.1 was analyzed by SDS-PAGE and developed by silver staining (Fig.
2, lane C). Although there were three distinct major bands, an additional 12 minor bands were detected at this stage of the purification.

Because previous information had shown that the inhibitory factor contained sialic acid residues (Sharifi et al., J. Neurochem. 46, 461-469, 1986), we took advantage of Limulus polyhemus agglutinin (LPA? lectin affinity chromatography to further purify the parental inhibitory factor. l mg protein of the immunoreactive pI 5.1 W~93/2~9 ~ PCT/U~93/03953 fraction, obtained by preparative isoelectric focusing, was loaded on a LPA column as described above, the column was extensively wa~hed with working buffer and the bound material was then released (48 ~g protein) with an elution buffer containing 2 mM EDTA. SDS PAGE analysis of the LPA
bound and released protein preparation provided a single band at approximately 66 kDa (Fig. 2, lane D). A visual comparison of the LPA and Rotofor purified fractions clearly showed that the 66 kDa band was a relatively minor component of the isoelectric focused material. Since the SDS-PAGE gel was run with the LPA fractioned protein, reduced just prior to gel analysis, and no other bands were evident by silver staining, we concluded that the parental inhibitory factor most likely was a single polypeptide without subunit structure. At this stage of the purification the parental inhibitory factor appeared homogeneous, and enriched 2,520-fold over the original membrane protein (Table 1).

Affinity columns with immobilized polyclonal IgG, raised to the native inhibitory factor, also were used in the purification protocol. A 66 kDa molecule was bound and eluted from the column, and the small amount of recovered protein (approximately 1 to 2 ~g) was insufficient to test biological inhibitory activity. Within the resolution of SDS-PAGE gels stained with silver stain, the IgG affinity puri~ied parental inhibitory factor appeared similar to that obtained by LPA affinity chromatography.

To provide assurance that the 66 kDa protein, obtained by LPA affinity chromatography was antigenically related to the inhibitory factor, dot blot analyses were conducted with polyclonal IgG rai~ed against the native inhibitory factor. 200 ng protein of both the LPA bound and eluted, and the LPA unbound fractions were blotted and probed with the anti-inhibitory fa~tor IgG. The bound and eluted ~raction wa8 strongly antigenic while the unbound material ~howed only slight reactivity that most likely reflected a W093/Z2~9 3 ~ ~ 43 PCT/U~93/03953 t~

slight overloading of the affinity column. Western analyses, carried out with the IgG raised to the denatured inhibitory factor, were consistent with the dot-blot analy~es and showed that only a 66 kDa band of LPA~bound and eluted fraction reacted Wit}l the IgG, while the proteins in the unbound fraction essentially was nonreactive.

Both the purified 66 kDa membrane protein and the LPA
unbound protein fraction were tested for biological - inhibitory activity with exponentially dividing mouse Swiss 3T3 fibroblast cells. The lyophilized 66 kDa protein was resuspended in 1 ml of distilled water, and 40 ~1 containing 1, 5 or 10 ~lg of protein were added to culture medium to provide a total volume of 300 ~1, resulting in final concentrations of the parental inhibitory factor of 5 x 10 ~ M, 2.5 x 10 7 M and 5 x 10 7 M, respectively. The LPA unbound proteins were added at the same concentrations, and other ~ets of cultures received 40 ~1 of the dialysis fluids, previously lyophilized and resuspended in 1 ml of di~tilled water. The addition of the 66 kDa parental inhibitory factor clearly showed a marked inhibition of 3T3 cell division when compared to cultures that received the dialysis fluid or PBS (Tab~.e 1). The measured inhibition appeared at least semi-quantit~tive since the cultures receiving 10 ~g (5 x 10 7 M) of protein attained only 7% of the ~rowth compared to the control and dialysis fluid-treated cultures, while the cultures receiving 5 ~g (2.5 x 10 7 M) and 1 ~g (5 x 10 8 M), attained 26% and ~3%, respectively. In contrast, the cultures receiving the LPA unbound protein continued proliferating as those that received reconstituted dialysis fluid (Table 2).

WO 93/22449 ~ PCr/US93/03953 Tab1e 2. Inh1bitiOn O~ C~11 D1YiSiOn by th~ 66 kDa Inhibito2.~ ~actor .

~n~l Growth Additions to Proteln C~ll Compar-d to Culture Medium Added Number ~ Control ~in 40 ~1) (Yg) (X 104) ~%) PBS (control) 2.9 100 Dlalysi~ Fluid - 2.~ 100 66 kDa Prot~n 1 1.9 53 66 kDa ~rotein 5 1.3 26 66.XDa Protein 10 0.9 7 P~S (control) ~ 2.5 100 Dialysis Fluid - 2.8 117 LPA-Unbound Protein 1 2.5 100 LPA-Unbo~nd Protein 5 2.8 117 ~-~A-Unbound Prote~n 10 2.4 94 Each data point represents th~ mean o~ duplicate cultures ~nd three independent ~easurements of cell number in each well.

*~ Additions wsr~ ~ade when the total cell number per culture was 7.5 x 103, and cell proliferation was compared to those cul-tures recelv~ng 40 ~1 o~ PBS
. ~ .

W09~/22~9 PCT/US93/03953 ~
~34~3 56 : EXAMPLE VII
Cloning of the Genes for Mouse, Bovine and Human Inhibitory Factor Immunoscreening of cDNA libraries with the pol~yclonal - antibody prepared to the~denatured inhibitory factor was carried out with commercially avaiLable cCNA libraries prepared from bovine cerebr~l cortex, human fetal brain and mouse k.dney.

The preparation of the lambda bacteriophages, the immunological screening and identification of positive clones were essentially carried out by the proceclures described in Molecular Cloni_g_ A Laboratory Manual (J.
Sambrook, E.F. Frisch and T. Manlatis, 2nd Edition, Cold Spring Harbor Laboratory press, 1989).
i Preparation of the Ho~t Bacteria (modified from Part 1 of the Molecular Çlonina: A Laboratory Manual cited above).

50 ml of sterile rich medium (LB) supplemented with 0.2% maltoæe and 10 mM magne~ium sulfate was placed in a sterile 250-ml flask and illoculated with a single bacterial colony. The culture was grown overnight at 37C with moderate agitation (250 cyc].es/minute in a rotary shaker~.
When XL1-Blue cells were to be ~1sed as the host as in the case of the HFB and BCC Libraries ~see below) 5 micrograms of tetracycline was also added to the medium.
i ~; The cells were then centrifuged at 4000 x g for 10 minutes at room temperature and resuspended in 5 ml to 10 ml of LB medium supplemented as described above.

Immunological Screening of Expre~sion Libraries (from Part 2 of the Molecular Cloninq: A LaboratorY Manual cited above).

Screening Expression Libraries Constructed In Bacteriophage ~ Vectors CJ
,- WOg3/27~9 PCT~US~3/03953 Using a single colony of the appropriate strain of E.
coli as inoculum, prepare a plating cult~re as described in Chapter 2 of Molecular Clon~g _A Laborator~ Manual.

E. coli strain Y109OhsdR, which is commonly used as the host for immunological screening of expression li~raries const~ucted in ~gtll as was the case for the MK
library (see below), carries a plasmid (pMC9) that codes for the lac repressor and prevents synthesis of potentially toxic fusion proteins from the ~-galactosidase promoter.
This plasmid also carries a selectable marker (ampr). To ensure against loss of the plasmid, E. coli strain YlO~OhsdR was grown in media containing 50 ~g/ml ampi~illin.

~ . coli strains BB4 and XL1-Blue, which were used for immunological screening of libraries constructed in ~ZAP, carry a ~acI9 gene and a tet marker on an F' factor.
These strains were therefore grown in media containing 12.5 ~g/ml tetracycline.

Twenty plates were typically used. A set of sterile tubes (13 mm x lOO mm~ were arranged in a rack; a fresh tube was used for each plate infected. In each tube, 0.1 ml of the plating bacteria was mixed with 0.1 ml of sodium magnesium media (manniatis, supra) containing 3 x 104 pfu (90-mm plates) or 105 pfu (150 mm plates) of the bacteriophage ~ expression library. The infected bacteria was incubated for 20 minutes at 37C.

To each tube was added 4.0 ml (90-mm plate) or 7.5 ml (150-mm plate) of molten top agarose, and the mixture was immediately poured onto an LB agar plate. The infected plates were incubated for 3.5 hours at 42C.

Nitrocellùlose filters were numbered. The filters were handled with gloved hands. The filters were soaked in a solution of isopropylthio-~-n-galactoside (IPTG) (10 mM
in distilled water) for a few minutes. One set of plates W093/22~9 '~ ~ 4 ~ ~ ~ P~T/US93~03953 -:~

were done without IPTG ~ treated nitrocellulose as a control. Using blunt-ended forceps (e.g;, Millipore forceps), the filters were removed from the solution, and allowed to dry at room temperat~lre on a pad of Kimwipe~.

The plates were removed from the incubator, and the agar quickly overlayed with the IPTG-impregnated nitrocellulose filters.

The lids were left off the plates and the incubation continued for a further 20 minutes at 37C.

The plates were moved in small batche~ to room temperature. Each filter was marked in at least three asymmetric locations by stabbing through it and into the agar underneath with an 18-guage needle attached to a syringe containing waterproof black ink.

Using blunt-ended forceps, ths filters were peeled off the plates and immediately imm~rsed in a large volume of TNT. Any small remnants of agarose was rinsed away by gently agitating the filters in the buffer. The TNT was agitated to prevent the filters from sticking to one another.

TNT
10 mM Tris Cl (pH 8.0 150 mM NaCl 0.05% Tween 20 The plates were wrapped in Saran Wrap, and stored at 4C until the re~ults of the imm~lnological screening were available~

When all of the filters are removed and rinsed, they are transferred one at a time to a fresh batch of TNT.
When all of the filters have been tran~ferred, the buffer W093/22449 PCTfUS93/039~3 is agitated gently for a further 30 minutes at room temperature.

Using blunt-ended forceps, the filters were transferred individually to glass trays or petri dishes containing blocking buffer (7.5 ml for each 82-mm filter;
15 ml or each 138-mm filter). When all of the filters had been submerged, the buffer was agitated slowly on a rotary platform for 30 minutes at room temperature.

Blocking Buffer 2% nonfat dry milk iIl TNT

The blocking buffer W~5 stored at 4C and reused several times. Sodium azide was added to a final concentration of 0.05% to inhibit the growth of microorganisms.

Using blunt-ended forceps, the filters were transferred to fresh glass trays or petri dishes containing the primary antibody diluted in blocking ~uffer (7.5 ml for each 82-mm filter; 15 ml for each 138-mm filter). The highest dilution of antibody W~8 used that gives acceptable background yet still allows detection of 50-100 pg of denatured antigen. When all of the filters had been submerged, the solutions were ~gitated gently on a rotary platform overnight at room temperature.

The antibody solution was stored at 4C and reused several times. Sodium azide waæ added to a final concentration of 0.05% to inhibit the growth of microorganism~.

The filtèrs were washed for 10 minutes in each of the buffers-below in the order given. The filters were transferred individually from one buffer to the next. 7.5 w093/2~9 ~3 ~ 4 43 PCT/US93~03953 i -:
6~

ml of each buffer was used for each 82-mm filter and 15 ml for each 138-mm filter.

TNT ~ 2% nonfat dry milk TNT + 2% nonfat dry milk-~ O.l ~ Nonidet P-40 TNT + 2% nonfat dry milk.

The antigen-antibody complexes were detected with the radiochemical reagents.

Approximately 1 ~Ci of 1~ I-labeled protein A
~the preferred reagent) or anti-immunoglobulin were used per filter. Radiolabeled protein A
is available from commercial sources (sp. act.
30 mCi/mg). Radioiodinated second antibody is prepared according to well known techniques.
Radiolabeled ligands were diluted in blocking buffer (7.5 ml for each 82-mm filter; 15 ml for each 138-mm filter). The filters were incubated 2 hrs. at room temperature, and then wa~hed several times in TNT before autoradiographs were establi shed .

The locations of positive pla~les were identified.

a. A sheet of Saran Wrap was layed over the filters.

b. On the surface of the Saran Wrap, the locations of th¢ holes in the filter and the locations of antigen-positive clone~ were marked with different colored waterproof markers. The Saran Wrap was labeled to identify the plates from which the filters were derived.

c. The ~heet of Saran Wrap was placed on a light box, and the plates were aligned containing the original bacteriophage ~ plaques on top of it.

wo g3/22~9 2 1 3 4 '1 ~ 3 PCT/US93/03953 - d. The area containing the positive plaque is identified, and a plug of agar from this area is removed using the large end of a pasteur pipette. The plug wa~ transferred to l~ml of SM containing 2 drops of chloroform.

- e. The sheet of Saran Wrap, which provides a permanent record of the locations of the ; positive plaques is retained.
-The bacteriophage particles were allowed to elute fromthe ~gar plug for several hours at 4C. The titer o~ the bacteriopha~es in the eluate was determined, and then replated so as to obtain approximately 3000 plaques per gO-~m plate. The plaques were rescreened as described above, and the process of screening and platin~ was repeated until a homogeneous population of immunopositive recombinant bacteriophages was obtained. The clonal isolates were subcloned at least three time~ to provide a ho~ogeneous positive population, and each time the plaques were tested with the polyclonal antibody probe to provide assurance of continued anti~en product expression and the homogeneity of the final isolate.

The results were as follows:

Bovine Cerebral Cortex Library (BCC), lambda ZAPII
phage/XL-l-Blue E. coli hos~ ,OOO,OOO plaques ~creened, five po~itive clones; the five postiive clones were pooled and labelled "B" and deported ~t the ATCC on April 27, 1992.
.
Human Fetal Brain Library (HFB), lambda ZAPII
phage/XL-1-Blue E. coli host, 4,000,000 plaque~ screened, three positive clones; the three positive clone~ were pooled and labelled "M" and deposited at the ATCC on April 27, 1992, and, W0~3/22449 PC~/US93/03g~3 ~
3 4~ ~3 Mouse Kidney Library (MK3, lambda gtll phage/Y1090 E.
coli host, 5,000,000 plaques screened, two positive clones;
the two po~itive clones were pooled and-labelled "H" and deposited at the ATCC on April 27, 1992.

Lambda DNA Isolation Three separate protocols were used in order to isolate lambda DNA but only one way Lëd to successfully isolating sufficient DNA for sequencing. All three methods used the ~ame general protocol to grow the host and phage.

Two 20-ml starter cultures of bacteria were grown overnight in LB medium supplemented with maltose and magnesium sulfate as described above. A lambda virus preparation with a titer of 101 plaque forming units per ml was mixed with 200 microliters of the host cell starter culture, and the preparation was incubated at 37C for 15 to 30 minutes. The preparation was then added to the 40 ml host culture and incubated at 37C, with constant mixing, until lysis occurred (7 to 8 hours~.

The three methods used for isolation of DNA were:

1) Promega Technical Rulletin No. 142, "Purification of Lamda DNA with Magicl~Lamda Preps DNA Purification System", p. 3, "Liquid Culture Method," (steps 1 through 5); p. 4, "Removal of Lambd~ Phage Coat," (all steps~; page 5 ignore; and, p. 6, "Lambda DNA Purification Without a Vacuum Maniford," (all steps). Using this technique, only 2 to 4 micrograms of lambda DNA were recovered.

2) Pharmacia P-L Biochemicals Bulletin, SephaglasT~
PhagePrep Kit (1991), pp. 9-12. U~ing this techni~ue, only 2 to 4 microgram~ of lambda DNA were recovered.

3) Modified techniques for isolation of lambda DNA

~ l ~ 4l~ ~
W093t2~9 ~ PCT/US93/03953 After host cell lysis, 200 microliters of pure chloroform was added and the preparations were shaken at 37C for 5 minutes. The lysates were centrifuged at 1000 x g for 15 minutes to pellet debris. The supernatant 1uids were centrifuged at 40,0Q0 x g at 4~C for 1.5 hours to pellet the bacteriophage particles. The supernatant fluids - were discarded, the phage pellets were drained and resuspended in a total of 300 microliters of 50 mM Tris-HCl (pH 7.5). The preparations were treated with DNase and RNase (1 microgram per m/l each) for 30 minutes at 37C.
The DNA was then extracted with l volume of TE buffer ~10 mM Tris-HCl and 1 mM EDTA, pH 7.4) saturated with phenol plus chloroform and isoamylalcohol ~50:48:2). The tubes were rocked gently for l minute, then centrifuged at 12,000 x g at 4C for 5 minutes, and the initial extrarction was repeated.

The a~ueous phase was then removed an~ extracted once with chloroorm/isoamylalcohol (24:1) with gentle rocking for 1 minute. The mixture was centrifuged at 12,000 x g at 4C for 5 minutes and the aqueous phase removed. An equal volume of isopropanol was added, the tubes rocked gently, and the mixture was left at -70C for at least 20 minutes.
The mixture was again centrifuged at 12,000 x g at 4C for 10 minutes and ~he supernatant fluid wa~ removed. The pellet was rinsed by adding 1 ml-of 70% ethanol, followed by immediate centrifugation at 12,000 x g at 4C for 10 minutes. The resulting pellet was air-dried and resuspended in 50 microliters of 50 mM Tris-HCl (pH 7.5).

This method provided 35 micrograms of lambda DNA.

The preparation of this example was an isolate with a 1.3 kilobase pair insert, obtained from the human fetal brain cDNA library.

The DNA inserts removed by restriction enzymes or pcr were then subcloned into t~le vector p-Flag system, WO93/2244g PCT/US93/039S3 i~' 2~34~43 64 available commercially from International Biotechnologies, Inc. (New Haven, CT), in either XLl-Blue or JM101 E. coli hosts. Each subclone was tested for inducibility, fusion proteins isolated from the microbial periplasm and ~ -identified by Western analysis. The purified fusion proteins are tested for biologi~al inhibitory activity by assays de.scribed above.

EXAMPLE VIII

Effects of Inhibitory Factor on the Post-Translational Regulation of the Retinoblastoma Protein Inhibitory factor medi~ted cell cycle arrest of both human diploid fibroblasts (HSBP) and.mouse fibroblasts (Swis~ 3T3) results in the maintenance of the RB protein in the hypophosphorylated state, consistent with a late GI
arrest site. Although their normal nontransformed counterparts are sensitive to cell cycle arrest mediated by inhibitory factor, cell lines lacking a functional RB
protein, through either genetic mutation or DNA tumor virus oncoprotein interaction, are refractory.

Inhibitory factor purification. The sialoglycopeptide inhibitory factor inhibitor was released from intact bovine cerebral cortex cells by mi.ld proteolysis and purified to apparent homogeneity as descri~ed above. Briefly, bovine cerebral cortex cells were treated with dilute protease, the released molecules precipitated with ethanol, the precipitates were extracted Wit]l chloroform/methanol (2:2), and inhibitory factor was p~lrified by DEAE ion-exchange chromato~raphy, lectin affinity chromatography and HPLC
with a TSK-3000 size exclusion column. The samples were then dialyzed against distilled water, lyophilized and re3uspended in phosphate buffered saline (PBS; 145 mM NaCl, 5 mM potas~ium phosphate, pH 7.2). Protein determinations were carried out by the method of Bradford, et al, Anal.
Biochem. 72:248-254 (1976) using bovine serum albumin as a W093/22449 ~ 4 3 PC~/US93/03~3 protein standard, and the puriied inhibitory factor preparations were stored at -70C.

Cell culture. Cultures were grown as monolayers in a humidified incubator with a 5% C02/95% air atmosphere Fattaey, et al, J. Cell. Physiol. 139:~69-274 (1989).
Mouse Swiss 3T3 celLs, from the American Type Culture Collectivn, and the SV40 transformed 3T3 cell lines (SVT2 and F5B), were grown in Dulbecco's modified Eagle's medium (DMEM) (GIBC0/BRL, Grand Island, NY) with 10% calf serum.
Human diploid foreskin fibroblasts (HSBP), human osteosarcoma cells (U20S and SAOS-2), human bladder carcinoma cells (J82), human prostate carcinoma cells (DU145), and adenovirus transformed human epithelial cells ( 293 ? and grown in DMEM with 10% etal calf ~erum. Human fibroblast keratinocytes (HFK), and HFK cells transformed with papillomaviruses (28-NC0 and 1321) Pietenpol, et al, Cell 61:777-785 (1990); Romanczuk, et al, J. Virol.
65:2739-2744 (1991) and grown in KGM media with growth factors (Clonetics, San Diego, CA).

Protein æYnthesi~ inhibition assaY. Protein syntheæis inhibition was tested essent.ial~y as described by Sharifi et al, J, Neurochem. 46:46~.-469 (1986). Various concentrations of the purifi.ed inhibitory factor were added to 5 x 105 cells in 100 ~l of methionine-free minimal Eagle'æ medium (MEM/HEPES). The cells were preincubated with the inhibitor for 30 mi.n at 37C to allow inhibitory factor to bind to the cell s~lrface receptor, and then 2.0 ~Ci of [35S]methionine, in 10 ~ll of methionine-free MEM/HEPES were added, and the cells were incubated for an additional 15 min. The cell proteins were precipitated with trichloroacetic acid (TCA), the precipitates were washed several time~ with 5% TCA, and the amount of radioactivity incorporated into acid-insoluble protein was measured in a liquid scintillation system Sharifi et al, J.
Neurochem. 4~:461-469 (1986).

W093/22~9 PCT/USg3/03953 ;~
'~34i~43 66 -Cell proliferation assay. Cells were plated in 48-well culture plates (Costar, Cambridge, MA) and allowed to attached for at least 4 h. Then _6-9 x 10 8 M inhibitory factor, diluted in the appropriate culture medium, or medium alone, was added and the cell number determined at various times with a Coulter counter, model Z~ Edson, et al, Life Sci. 48:1813-1820 (1991) and Fattaey, et al, J.
Cell. Physiol. 139:269-274 (1989).

RB protein immunoprec~pitation and SDS-PAGE. Cell . _ , . . .
cultures, incubated with and without inhibitory factor for 24 hJ were radiolabelled for 3.5 h with 300 ~Ci/ml of [35Slmethionine (TRANS35SLABEL, ICN, Irvine, CA) :in methio~ine-free DMEM, ~nd immunoprecipitated from cell ly~ates for 12 h as described by Harlow and Lane Harlow, et al, Antibodies: a laborarory manual, Cold Spring Harbor Press, New York (1988), using monoclonal anti-human IgGl(PMG3-245, Pharminigen, San Diego, CA). Due to the lower reactivity between the mouse RB product and the PMG3-245 antibody, Swiss 3T3 lysates were incubated with the antibody for 24 h. The immunoprecipitates were boiled for 5 min in sample buffer Laemmli~ V.K., Nature ~27:680-685 (1970) and separated on a 7.5% SDS-PAGE at 15 mA for _3 h (samples were equalized with regard to the amount of radiolabelled protein loaded). After electrophoresis the protein.s were electroblotted to a PVDF
membrane (Millipore, Bedford, MA), prepared for fluorography (EN HANCE spray, NEN/DuPont, Willimington, DE) and expo8ed to X-ray film for 24 h at -70C.

InhibitorY factor bindinq as~y. Inhibitory factor was radioiodinated, and the binding studies were carried out as described by Ba~com et al, J. Cell. Physiol. 12B:202-208 (1986~. Briefly, radioiodination was by the chloramine T
method (Sigma Chem. C0., St. Louis, M0) that resulted in a biologically active inhibitory factor with a specific radioactivity of -1 x 104 cpm/ng protein. Cultures were grown in 24-well plates and various concentrations of the ~ -~ v -o ~
W093/22~9 PCT/US93/03953 125I-labelled inhibitory factor ~in 300 ~l of culture medium), with or without a 30-fold excess of nonradioactive inhibitory factor to measure nonspecific binding, were added to duplicate subconfluent cell culture~ ~-1.5~x 105 cells/well). The cells were incubated with the radiolabelled inhibitor preparations at 37C or 30 min and then quiGkly washed three times with PBS. The cells were then lysed by the addition of 300 ~l of distilled water containing lOO ~l of l M NaOH. The samples were collected and the bound radiolabelled inhibitory factor was determined with a gamma co~lnter Bascom et al, J. (,ell.
Physiol. 128:202-208 (1986).

Exponentially growing human diploid fibroblasts (HSBP) and Swiss 3T3 cells were used to study the potent.ial effect of inhibitory factor mediated cell cycle arrest on the phosphorylation states of the RB gene product. The cultur~s were incubated with or without inhibitcry factor for 24 h~ radiolabelled for 3.5 h with 135S~methionine, and the RB protein was immunoprecipitated with the monoclonal anti-human RB IgG as described above. Both exponentially growing cell cultures exhibited newly synthesized RB
protein in both the hypo- and hyperphosphorylated states (Fig. 4, lanes l & 3), while cells arrested by the inhibitory factor inhibitor contained only RB protein in the hypophosphorylated state (Fig. 4, lanes ~ & 4). These observations were consistent with the proposed Gl re~ulatory state of the RB protein and the site of cell cycle arrest mediated by the inhibitory factor inhibitor.

.
To further examine the potential role of posttranslational modification of the RB product in the biological inhibitory action of inhibitory factor, both HSBP and Swiss 3T3 cells were plated and allowed to grow to confluence, and when the cultures reached confluence they were incubated for an additional 24 h to ensure that the majority of the cells were density-dependent arrested. A
second set of cultures were plated at the same time at _l/3 W09c~ 3 PCT/US93/039~3 the density, and at the time of immunoprecipitation these cultures remained subconfluent. The results clearly showed that both HSBP and 3T3 density-dependent growth arrested cultures solely displayed the RBUnPhos protein (Fig~ 5, lanes 2 & 4). Exponentially dividing HSBP and 3T3 cells, however, again expressed the expected hyper- and hypophosphorylated form~ of~the RB product (Fig. ~, lanes 1 & 3). The results from the~e experiments indicated that cell cycle arre~t, mediated by inhibitory factor, was consistent with a block at or near the Gl arrest ~ite since the state of phosphorylation of the RB protein, under inhibitory factor mediated cell cycle arrest and density-dependent arrest, was indistinguishable.

These observations, however, did not necessarily establish a direct relationship between the RB protein and signal transductîon events associated with the inhibitory factor inhibitor. If the maintenance of the RB protein in the R ~ nphos state is necessary for inhibitory factor mediated inhibition, cell lines either lacking a functional RB product or having the RB protein sequestered by a viral oncoprotein lead to an insensitivity to the cell cycle regul.atory activity of the sialoglycopeptide.

In order to investigat~ thi.~, the ~ensitivity of two human osteosarcoma cell lines, tJ20S(RB ) and SAOS-2(RB ), to inhibitory factor were compAred. When 9 x 10 8 M
inhibitory factor was added to the culture medium the U20S
cells were efficiently inhibited within 20 h, while the SAOS-2 cells were refractory to inhibition throughout the incubation period (Fig 6). H~lman bladder càrcinoma cell lina J82 (RB ) and prostate carcinoma cell line DU145 (RB ) also were resi~tant to the inhibitory action of inhibitory factor (Fig. 7). Even higher concentration~ of the inhibitor also were ineffective in blocking cell cycling in RB cell lines.

W093/22~ 2 1 3 4 '1 ~ 3 PCT/US93/~39~3 Since cells that express a normal RB product can phenotypically act like RB cell lines when transformed with certain DNA tumor antigens, the pos~ibility that these cell lines might also be resistant to the inhibitory influence of inhibitory factor was examined. While normal human keratinocytes were readily arrested by inhibitory factor, the 1321 and NCO papillomavirus E6/E7 protein transformed cell lines were totally refractory to the action of the inhibitor (Fig. 8). The adenovirus ElA
protein transformed human epithelial cell line 293 also was resistant to the cell cycle arrest mediated by the inhibitory factor inhibitor ~Fig. 9).

Swiss 3T3 cells were found to be sensitive to the inhibitory action of inhibitory factor, while consistent with the observations of papillomavirus and adenovirus transformed human cell lines, the proliferation of both the SV40 large T antigen transformed cell lines SVT2 and F~B
- were not inhibited by the sialoglycopeptide (Fig. 10).
Clearly, the transformation of both human and mouse cells by the transforming antigens of several DNA oncogenic viruses, that are known to sequestered the nuclear RB
product, re~ulted in a refr~ctory phenotype with regard to inhibitory factor action.

Since it has been shown th~t cells resistant to the i~hibitory action of TGF-B can be a reflection of a decrease in the surface receptor population for the ligand, the number of receptors and the Kd of the inhibitor-receptor interaction w~s measured with the refractory human SAOS-2 and mouse SVT2 cell lines, and compared to the sensitive human U20S cells. The number of inhibitory factor receptors per cell, and their Kd, were remarkably similar.

W093/22449 P~r/lJS93/039~3 i-;
2134~ 3 7~ ~

Table 3. . CeReS-18 Receptors on Sensitive and Insenstive Cell Lines a ~ . . . . . . .. ~ .
Rcceptors CeReS-18 Cell Line Cell Type per Cell Kd Mediated Arrest (nM) .
SV~I~ mouse fibroblast 2.6 x 104 8.5 Insel~sitive U20S human osteosarcoma 2.9 x 104 9.7 Sensitive SAOS-2 human osteosarcoma 23 x 104 6.1 ~n~ensitive .

a Specific cell roceptors for C~ReS-18, and their Kd~ were determined with l25I-radiola~elled inhibitor as descnbed in the Materials and Metbods - ~ W0~3/22~9 PCT~US93/03953 The reason for the refractory nature of RB and viral transformed cell lines to inhibitory factor cell cycle arrest clearly could not be attributed to a change in cell surface receptors. Consistent with this observation, all cell lines were sensitive to the transient inhibitory factor inhibition of protein synthesis, which require6 occupancy of the inhibitory factor receptor to inhibit translational events, whether or not the more enduring cell cycle arrest was effected.

The cell lines used in this study also provided an examination of the potential role of a second tumor suppressor gen~ product, p53, with regard to the inhibitory action of inhibitory factor. The human carcinoma cell lines J82 and DU145 are p53 while being resistant to the inhibitory action of inhibitory factor.

w0 ~3/22449 72 Pcr/u~s3/o3s53 ~34~43 Table 4 . CeReS-18 Inhibition of Cell Proliferation Tumor Suppressor CeReS-18 Cell Iine Cell Type Product Mediated Inhibition a (RB) (p53) Swiss 3T3 mouse fibroblast + ~ +
HSBP human ~broblast + + +
HFK human keratinocte ~ ~ ~
U20S human osteosarcoma + ~ +
SAOS-2 buman osteosarcoma - - -DU145 human prostatc carcinoma - +
J82 human bladder carcinoma - ~ -1321 huma~ l~eratinocyte ~ b ~ -NCO human kcratino~ytc v ~ -293 huma~ lddncy cpithelial ~v v F5B mouse ~broblast ~ v - -SVI~ mouse fibroblast v v .

a The cell lines were exami~ed for sensitivi~r of cell proliferation with 9 x 10 8 M of the ;
CcReS-18 inhibitor.
b v - Denotes prcse~ce of ~riral oncoproteins capable of sequestering RB and pS3.

W093/2~9 ~ 3 PCT/US93/03953 HL-60 cells, however, are RB and p53 and are sensitive target cells to the inhibitor. These observations delineate that the RB protein, and not p53 product, appears to play a central role in the ability of inhibitory~factor to mediate arrest in the Gl phase of the cell cycle.
* * *

Cell cycle arrest of exponentially dividing human and mouse fibroblasts results in cells primarily having the tumor suppressor protein in the RBUnPhos state (Fig. 4).
For all practical purposes it appears that cell cycle arrest, mediated by the cell surface sialoglycopeptide, is equivalent to cells that naturally become arrestecl by density-dependent growth inhibition (Fig. 5~.

Inhibitory factor, derived from a parental cell surface component of bovine cerebral cortex cells, has an unusually broad target cell range. It has the ability to mediate cell cycle arrest of cells obtained from mouse, human, rat, avian and insect species, all of which necessarily have specific cell surface receptors for the inhibitor. In addition, many tumorigenic cell lines, derived by mutation or retroviruses, have been shown to be highly sensitive to the proliferation inhi~itor. For the most part, reversal experiments also have shown that this broad array of cells primarily are arrested in the G1 phase of the cell cycle.
Studies with mouse and hum~ti cells confirm a Gl phase block by the presence of solely t~e underphosphorylated form of the RB in the inhibited cells. The kinetics of reversal of DNA synthesis, cell doubling and the state of the RB
protein are all consistent with the restriction (R) point,~
near the Gl/S interphase. The one exception at the present time to this generality is the inhibitory factor mediated arrest of HL-60 cells. Unlike most others that appear to be synchronously released from cell cycle arrest when the inhibitor is removed, HL-60 cells are irreversibly arrested by the sialoglycopeptide, aJId even after inhibitory factor is removed the cells progres~ through differentiation.

; WOg3/22449 PCT/US93/03953 2~3~4~ 74 This i~ of particular relevance to the pre~ent study since the HL-60 cells are p53 and RB . The human osteosarcoma SAOS-2 cell line is p53 and RB but it appear~ that the RB
protein is the salient gene product with regard to ~
inhibitory factor inhibition of cell cycling. The central role of the RB product in inhibitory factor action was confirmed by the insensitivity of the human bladder J82 (RB and p53 ) and human prostate DU145 (RB and p53 ) carcinoma cell lines to the inhibitor (Fig. 7).

The insensitivity of RB mutants and DNA tumor virus transformed cell lines was not associated with neither a reduced level of receptors nor the measured binding affinities of inhibitory factor to U20S, SAOS-2 and SVT2 cells (Table 3). In fact, the number of inhibitory factor receptors per cell was quite comparable whether or not the cells were growth arrested by the sialoglycopeptide inhibitor, and consistent with earlier measurements of 2 x 104 receptors per Swiss 3T3 cell that ~erve as the standard cell line for many of the inhibitory factor studies.

It i5 clear that the RB product is more than a casual player in the series of metabolic events that mediate cell cycle arrest by the inhibitor. Inhibitory factor arrests cell~ at a site where the RBUnPhos state is the dominant form of the tumor suppressor protein. Further, RB cell lines are refractory to cell cycle inhibition by a sialoglycopeptide. Either its absence as a functional protein by mutation, or its being sequestered by transforming antigens of certain DNA oncoviruses, led to an insensitivity of cell cycle arrest by the sialoglycopeptide inhibitor (Table 4). The maintenance of the RB product in the hypophosphorylated state alone, although readily ~een in growth arrest cells (Fig. 4), is not the sole reason for the refractory nature of these cells. Consistent with khe information that the RB protein regulates progression through the cell cycle, there is a re~uirement for a W093/22449 ~1 3 9 '~ I 3 PCT/US93/039s3 functional RB protein in order for the cell surface inhibitor to mediate cell cycle arrest.

Inhibitory factor is one of the few naturally occurring potential growth regulators that abrogates the phosphorylation of the RB protein. The inhibitor is a cell surface component that infl~lences cell cycling of a wide variety of cell types. Further, there is a similarity at a molecular level between the inhibitory factor arrested cells and those that naturally reach confluency and quiescence, and the reversibility of its inhibitory action. Inhibitory factor represents a wide class of cell growth re~ulators that play a fundamental role in density-dependent growth inhibition. In this regard, the inhibitor is a valuable agent for studies of cell cycling, provides a controlled and synchronous population of cells in their progression through the cell cycle, and delineates the genetic and molecular events associated with the posttranslational modifications of the RB product that regulate cell proliferation.
EXAMPLE IX
The Effect of Inhibitory Factor on Hybridoma Cell Proliferation and Monoclonal Antibody Production Hybridoma cells (3G-lOG-5) were plated into 96 well plates at 1 x 10 cells/well (100 ~1 medium). Hybridoma line, 3G-lOG-5, produces molloclonal antibody to the budgerigar fledgling disease virus (BFDV) major capsid protein (VPl). Inhibitory factor treated (1.2 inhibitory units) experimental cultures (Fig. 11, open circles) had the inhibitor present at the time of plating (Fig. 11, arrow #1). Control cultures received medium without the inhibitor (Fig. 11, closed circles). Fresh medium was not added until seven days of culture. At the times indicated, the cells were pelleted by centrifugation, resuspended and counted, and the media were saved and used to quantitate the monoclonal antibody by ELISA.

; W093/22~9 PCT/~S93tO3~3 i 213~3 76 On day seven, cells were centrifuged, media were saved, and fresh medium (without inhibitory actor~ was added to both control and inhibitor-treated cultures (Fig. ll, arrow #2). On day 11 cells were counted and ELISA carrie~d out.
On day 14 cells were counted and tested for viability by Trypan Blue exclusion (Fig. 11, arrow #3).

Trypan Blue exclusion indicated 35%-50% of the cells in the inhibitory factor treated and reversed cultures were viable, while control cultures had ~5% viability.

ELISAs were performed with culture media from dayæ 2, 7 and ll, in 96 well plates containing 90 ng/well of BFDV
protein.

The addition of fresh medium is the reason for the ~econd set of lower points for the seventh day.

Hybridoma cells were effectively inhibited by i~hibitory factor, and the inhibition i8 reversible.
Antlbody synthecis continued while cells were arrested by inhibitory factor and after reversal. INote that the cell number was almost 30-times greater in control versus inhibitory factor treated c~lltures.]

WO93/2~449 i~ PCI'/US~3/03953 Table 5. Monoclonal Andibody Production by Hybrldoma Cell~ Incubated with Inhlbitory EaGtOr _ ~ ====_ =========_===========_====_=========_================
Period of Monoclonal Incubation Antibody*
(Day8) .
Control Inhibitor~Factor Total Per 104 Cells Total Per 104 Cells 2 ~).10 0.09 0.05 ~).07 7 (before media 0 . 33 0 . 02 0 . lB 0 . 45 chang~ ) 7 (after media 0.05 - 0.05 change ) 11 1.00 0.01 0.60 1.25 ________ ~ _ _____ _=======___==__====__=_===___ ====__==
}Iybridoma cells were incubated for seven days before medla were changed and the lnhibitory factor remo~ed from the lnhibited culture~ .
* Monoclonal antlbody concentratlon~ were messured by Eli sa a~ (A450) W093/22449 78 PCT~US93/03953 ), c~3~443 * * *

While the present invention has been described in terms of preferred embodiments, it is understood that variations and modifications will occur to those skilled in ; the art. Therefore, it is intended that the appended claims cover all such equivalent variations which come within the scope of the invention as claimed.

The features disclosed in the foregoing description, in the following claims and/or in the accompanying drawings may, both separately and in any combination thereof, be material for reali7.ing the invention in diverse forms ther--o f .

Claims (80)

WHAT IS CLAIMED IS:

1. A non-naturally-occurring polypeptide having an amino acid sequence sufficiently duplicative of that of naturally-occurring inhibitory factor to allow possession of a biological activity of naturally occurring inhibitory factor.

2. A purified polypeptide comprising naturally-occurring inhibitory factor.

3. A polypeptide according to Claim 1 or 2 wherein said polypeptide is the product of procaryotic or eucaryotic expression of an exogenous DNA sequence.

4. A polypeptide according to Claim 3 wherein said polypeptide is the product of CHO cell expression.

5. A polypeptide according to Claim 3 wherein the exogenous DNA sequence is a cDNA sequence.

6. A polypeptide according to Claim 1 or 2 wherein said inhibitory factor is human inhibitory factor.

7. A polypeptide according to Claim 3 wherein the exogenous DNA sequence is a genomic DNA sequence.

8. A polypeptide according to Claim 3 wherein the exogenous DNA sequence is carried on an autonomously replicating DNA plasmid or viral vectors.

9. A polypeptide according to Claim 1 which has an in vivo biological activity of naturally occurring inhibitory factor.

10. A polypeptide according to Claim 1 which has an in vitro biological activity of naturally occurring inhibitory factor.

11. A polypeptide according to Claim 1 further characterized by being covalently associated with a detectable label substance.

12. A polypeptide according to Claim 2 further characterized by being covalently associated with a detectable label substance.

13. An isolated DNA sequence for use in securing expression in a procaryotic or eucaryotic host cell of a polypeptide product having an amino acid sequence sufficiently duplicative of that of naturally occurring inhibitory factor to allow possession of a biological activity of naturally occurring inhibitory factor, said DNA
sequence selected from among:
(a) DNA sequences disclosed in Example VII;
(b) DNA sequences which hybridize to the DNA sequences defined in (a) or fragments thereof; and (c) DNA sequences which, but for the degeneracy of the genetic code, would hybridize to the DNA sequences defined in (a) and (b).

14. A procaryotic or eucaryotic host cell transformed or transfected with a DNA sequence according to Claim 13 in a manner allowing the host cell to express said polypeptide product.

15. A polypeptide product of the expression of a DNA
sequence of Claim 13 in a procaryotic or eucaryotic host cell.

16. An isolated DNA sequence coding for procaryotic or eucaryotic host expression of a polypeptide having an amino acid sequence sufficiently duplicative of that of naturally occurring inhibitory factor to allow possession of a biological activity of naturally-occurring inhibitory factor.

17. A cDNA sequence according to Claim 16.

18. A genomic DNA sequence according to Claim 16.

19. A DNA sequence according to Claim 16 wherein said DNA sequence codes for human inhibitory factor.

20. A DNA sequence according to Claim 19 and including one or more codons preferred for expression in E.
coli cells.

21. A DNA sequence according to Claim 16 and including one or more codons preferred for expression in yeast cells.

22. A DNA sequence according to Claim 16 covalently associated with a detectable label substance.

23. A DNA sequence coding for a polypeptide fragment or polypeptide analog of naturally-occurring inhibitory factor.

24. A DNA sequence as in Claim 23 coding for methionyl inhibitory factor.

25. A biologically functional plasmid or viral DNA
vector including a DNA sequence according to Claim 16.

26. A procaryotic or eucaryotic host cell stably transformed or transfected with a DNA vector according to Claim 25.

27. A polypeptide product of the expression in a procaryotic or eucaryotic host cell of a DNA sequence according to Claim 26.

28. A polypeptide having one or more of the in vitro biological activities of naturally-occurring inhibitory factor.

29. A polypeptide having part or all of the secondary conformation of naturally-occurring inhibitory factor and having a biological property of naturally-occurring human inhibitory factor.

30. A process for the production of inhibitory factor comprising:
growing, under suitable nutrient conditions, procaryotic or eucaryotic host cells transformed or transfected with a DNA according to Claim 13, and isolating desired polypeptide products of the expression of DNA sequences in said vector.

31. A composition comprising a purified and isolated human inhibitory factor free of association with any other human protein.

32. A pharmaceutical composition comprising an effective amount of a polypeptide according to Claim 1 and a pharmaceutically-acceptable diluent, adjuvant or carrier.

33. A DNA sequence coding for an analog of human inhibitory factor selected from the group consisting of:
a) [Met-1] inhibitory factor, and b) inhibitory factor wherein one or more cysteines are replaced by alanine or serine.

34. A polypeptide product of the expression in a procaryotic or eucaryotic host cell of a DNA sequence according to Claim 33.

35. A pharmaceutical composition comprising recombinant inhibitory factor having the human amino acid sequence, and a pharmaceutically acceptable diluent, adjuvant or carrier.

36. Polyclonal antibodies specifically binding inhibitory factor but not protease.

37. Antibodies as in Claim 36 wherein said antibodies bind the parental protein.

38. A non-naturally occurring polypeptide having the biological activity of naturally occurring inhibitory factor, said polypeptide having an amino acid sequence of naturally occurring inhibitory factor or any allelic variants, derivatives, deletion analogs, substitution analogs, or addition analogs thereof, and characterized by being the product of procaryotic or eucaryotic expression of an exogenous DNA sequence.

39. A biologically active composition comprising the polypeptide of Claim 1 covalently attached to a water-soluble polymer.

40. A composition as in Claim 39 wherein said polymer is selected from the group consisting of polyethylene glycol or copolymers of polyethylene glycol and polypropylene glycol, and said polymer is unsubstituted or substituted at one end with an alkyl group.

41. The composition of Claim 39 wherein said polymer has an average molecular weight of about 1,000 to 100,000 daltons.

42. The composition of Claim 39 wherein said polymer has an average molecular weight of about 4,000 to 40,000 daltons.

43. The composition of Claim 39 wherein said polymer is an unsubstituted polyethylene glycol or a monomethoxy polyethylene glycol.

44. The composition of Claim 39 wherein said polymer is attached to said polypeptide via reaction with an active ester of a carboxylic acid or carbonate derivative or aldehyde derivative of said polymer.

45. The composition of Claim 39 wherein one or more amino groups of said protein are conjugated to said polymer by reaction with a N-hydroxysuccinimide, p-nitrophenol or 1-hydroxy-2-nitro-benzene-4-sulfonate ester of the polymer.

46. The composition of Claim 39 wherein one or more free cysteine sulfhydryl groups are conjugated to said polymer via reaction with a maleimido or haloacetyl derivative of the polymer.

47. The composition of Claim 39 wherein the polypeptide is glycosylated and the polymer is attached by reaction of an amino, hydrazine or hydrazide derivative of the polymer with one or more aldehyde groups generated by oxidation of the carbohydrate moieties.

48. A method for preparing a biologically active polymer-polypeptide adduct which comprises reacting the polypeptide of Claim 39 with a water soluble polymer under conditions permitting the covalent attachment of the polymer to said polypeptide, and recovering the adduct so produced.

49. A method of providing growth-arrest to cells or embryos or tissues or tumors comprising the steps of administering an inhibitory factor to said cells embryos, tissues or tumors.

50. A method as in claim 49 wherein the cells or embryos are cells or embryos selected from the group consisting of mammalian cells, avian cells and invertebrate species cells.

51. A method of synchronizing cell populations in the mitotic cycle comprising the steps of:
administering an inhibitory factor to said cells population;
culturing said cells; and removing or inactivating said inhibitory factor.

52. A method of measuring specific metabolic events or drug or other agent effects in cells in specific stages of the cell cycle comprising administering an inhibitory factor to said cells.

53. A method of culturing viruses comprising administering inhibitory factor to cells infected with viruses or to cells in preparation for viral infection.

54. A method of performing karyotypic analysis comprising administering inhibitory factor to cells prior to making chromosome spreads.

55. A method of measuring signal transduction events in cells mediated by the interaction of an inhibitory factor, with its cell surface receptor, comprising administering an inhibitory factor to said cells.

56. A method of studying deoxyribonucleic acid synthesis associated with, but not limited to, specific stages of the cell cycle in cells, comprising administering an inhibitory factor to said cells.

57. A method of studying protein synthesis and posttranslational modifications of proteins associated with, but not limited to, specific stages of the cell cycle in cells, comprising administering an inhibitory factor to said cells.

58. A method of studying gene expression and ribonucleic acid metabolism, associated with, but not limited to, specific stages of the cell cycle in cells, comprising administering an inhibitory factor to said cells.

59. A method of inducing cellular differentiation in cells and subsequent morphological and biochemical processes that accompany cellular differentiation comprising the step of administering to said cells an inhibitory factor.

60. A method of maintaining cell cultures in suspended animation comprising the step of administering to said cell cultures an inhibitory factor.

61. A method of treating proliferative, neoplastic or preneoplastic diseases in an animal where cell cycle arrest and/or selective cytotoxicity is desired, comprising administering to aid animal a therapeutically effective amount of an inhibitory factor.

62. A method of treating proliferative, neoplastic, or preneoplastic diseases in an animal comprising the step of administering to said animal a therapeutically effective amount of a chemotherapeutic agent and a therapeutically effective amount of an inhibitory factor.

63. A method of treating proliferative, neoplastic or preneoplastic diseases in an animal comprising the step of administering to said animal a therapeutically effective amount of an inhibitory factor.

64. A method as in Claim 63, further comprising the step of conducting radiotherapy, surgical therapy, chemotherapy or immunotherapy.

65. A method for treatment of a carcinoma, melanoma, sarcoma or lymphoma in a mammal comprising administering a therapeutically effective amount of the polypeptide according to Claim 1.

66. A method for treatment of a lymphoma in a mammal comprising administering a therapeutically effective amount of the polypeptide according to Claim 1.

67. A method for treating multiple sclerosis in a mammal comprising administering a therapeutically effective amount of the polypeptide according to Claim 1.

68. A method for treating psoriasis in a mammal comprising administering a therapeutically effective amount of the polypeptide according to Claim 1.

69. A method of treating atherosclerosis in a mammal which comprises administering a therapeutically effective dose of inhibitory factor.

70. A method of treating viral diseases or viral caused lesions in a mammal comprising administering an inhibitory factor.

71. A method of delaying effects of aging or development in an animal comprising administering an inhibitory factor.

72. A method of purifying inhibitory factor comprising the steps of:
a) conducting mild proteolysis of intact cells or membranes using a protease, b) conducting DEAE chromatography or preparative isoelectric focussing, c) conducting lectin affinity chromatography, and d) conducting HPLC DEAE chromatography.

73. A method of purifying inhibitory factor comprising the steps of:
a) eluting the protein from intact cells or membranes using a salt, b) conducting preparative isoelectric focusing or DEAE chromatography, and c) conducting lectin affinity chromatography.

74. A method for treating lesions associated with HPV
comprising administering to a patient a therapeutically effective amount of the polyperptide according to Claim 1.

75. A kit comprising:
a) inhibitory factor, b) culture media, c) slides or substrates for cell preparation.

76. A kit as in Claim 75 further comprising cholchine or another mitotic arrest agent.

77. A kit comprising:
a) inhibitory factor, b) cells for viral infection, c) media for culturing said cells.

78. A cell culture media comprising inhibitory factor and nutrients for maintaining cells.

79. A method for protecting an animal from damage due to radiation or chemical exposure comprising administrating inhibitory factor to said animal prior to said radiation or chemical exposure.

80. A method of maintaining a hybridoma cell culture in growth arrest or increasing monoclonal antibody production from such culture comprising the step of administering to said culture inhibitory factor.