CA2006744A1 - Nucleid acids and polypeptides encoded thereby - Google Patents

Abstract

ABSTRACT
Amplified signals in both existing and non-detected restriction fragments were observed with 3 different enzymes (PVU2, HindIII and EcoRI) in two different AMLM2 cases out of 5. Most interestingly, the amplification of clone 14-hybridizable DNA was accompanied by amplification in BuChE positive sequences. The fact that different sizes of the restriction fragments hybridized with the 3' probe excluded the possibility of cross-hybridization, Several of the amplified bands could be observed, with lower intensities, in the presumptive polycytemia vera (Vaguez disease) case or in one or two of the other AML cases.
The adr. sample represents a patient, currently under remission following chemotherapy treatment, whose serum and erythrocyte ChE were apparently normal in levels and biochemical characteristics. ChE genes and clone 14 are both co-regulated and co-amplified under specific stimulants.

Description

This appllcation relates to growth factors. In p~rtlcular, it rel~tes to polypeptido3 whi h influonca the replicatio~ or differentiation of blood cells, especiQlly platelet progenitor ~lls.

In the bone marrow a pl-lripotent cell differentiates into meg~karyocytic, erythrocytic, nnd myelocytlc cell lines. There May be n llne of committed cells becween stem cells and megakaryocytes.
The earllest recognlz~ble member of the ~gakaryocyts ~meg) series, the megakaryoblast, is inltially 20 to 30 ~m ln dlameter with basophillc cycoplasm and a sl~ghtly irregular nucleus having loose, somewha~ reticular chromatln snd several nucleoli. Lnter megakaryoblas~s may contain up to 32 nuclei, bu~ the cytopl~sm remalns sparse and lmmature. As maturation proceeds, the nucleus becomes more lobul~ced and pyknotlc; the cy~oplasm lncre~ses in quantity and becomes more acidophllic and granular. The most mature cells may give the appearance oE relenslng pl~telets ~t thelr periphery. Normally, less than 10~ of megakaryocytes arc in the blast stage and more than 50~ are ~atur~. Arbltrary morphologic classifications eommonly applied to the megakaryocyte series are megakaryoblasc f~r the ~arllest form; promegakaryocyte ~o~

o~ basophillc me~akaryocy~¢ for ~hs lnee~medi~te for~; and ma~ure (acidophllic, granular, or placelet-producing) megak~ryocy~ for the lato forms. Tho m~ture meg~karyocyoo ~xcands fll~en~s of cy~opl~sm into slnusoidal spaces where ehey de~ach and fragment lnto lndlvidual pla~ele~s ~WLlllams es ~l., Hemacology, 1972).
.

Evldence 15 accumulacing that there is a dual level of regulat~on of megak~ryocy~opolesi~, Lnvolving more chan ons regulatory factor (WLlll~ns e ~1., Br. J. Hae~ol. ~:173 [1982~
a~d Wllliams et ~1., J. Ceil Physiol. ~ 101 [1982]). The early leval of megakaryocy~opoLesis is postulat~d as b~lne ~itotlc, co~c~rn~d with cell pr~ e~tlon and colony inltlatlon from CFU-meg but Ls not affectzd by pla~elet count tBursteln ee ~., J. Cell Physiol. lQ~:333 ~1981] ~nd Kimura et fll,, EXp. ~ematol. 1~:1048 lS [1985]). Tho later stago o~ maturatio~ is non-mitocic, involved w1th nuclear polyploldl~a~io~ and cyeoplasmlc m~cura~ion and i5 probably regulated ln a feedbaek mechanlsm by peripheral platelec number (Odell et fll., Blood ~8:765 [1976~ and Ebbe et nl., Blood 32:787 [1968~. Th~- existence of a distinct and specific megakaryocyce colnny-stimulatlng factor (meg-CSF) is still in dispute (Mazur, E., E~p. He~atol. 15:340-350 [1987]). Although meg-CSF's have been partly purified from experlmen~ally produced thxombocytopen~a ~ ., Exp. Hematol. 1~:752 tl986]) and human e~bryonlc kidney conditioned mediu~ ~CM~ (McDonald ç~ ~1., J.
Lab. Clin. Med. R5:59 [19753) ~nd in man fro~ aplastic anemla and idiopathic chrombocytopenlc purpura urinary extracts ~Kawakita e~
al , Blood 6:556 1l9B3]) and plasma (Hoffman et al., J. Clin.
Invest.' 75:1174 [1985]), their physiological function is as yet unks~own ~n most cases. The condi~ioned medium o pokeweed mit~en-activated spleen cells (PWM-SpCM) and WEHI-3 (WEHI-3CM) have been used as meg~karyocyte pot~nciator~. PWM-SpCM contains faccors enhancing CFU-meg grouth (Metcalf et al,. PNAS; USA X~:1744-1748 11975]; Quesenberry et al., Blood 65:214 [1985]; Bnd Iscove, N.N., in Hemat~poletic Cell Differentiation, ICN-UCLA Symposia o~
Molecular and Cellular Biology, Vol. 10, Golde et ~ ds. [New ~0~ 7~ ~

York, Academy Press] pp 37-52 119781), one o whlch is lnterleukin-3 (IL-3), a mul~illne~g~ colo~y stlmula~lng fact~r (multl-CSF
~Burscein, S.A., Blood Cells ll:469, 1986]). The other facrors ln this uedium have noe yet b~en iden~ifled and isolacod. UE~I-3 i~ a S murine myelomonocy~ic cull line ~ecretlng rela~lvely large a~oun~s of IL-3 and smaller ~o~m~s o~ G~-CS~. IL-3 has been recen~ly purlfled and clon~d (Ihl~ e~ al,, J. I~uno].. ~ 243L L1982]) and has been foun~ ~o pocen~late the growth of a wlde range oi hemopoletic ells (Ihle et ~0, J, Immunol. 13:282 ~1983]).
Besldes thls le can a1SG synergize wlth many of the known hemopoLetic hos~on0s or ~rowth fac~ors (Bartelmez et ~ . Cell Physiol. l2~:362-369 ~19B5~ and ~arren et al., Cell 46:667-674 [1~881). IL-3 has been found to synerglzc with both erythropoLetin (EPO) snd H-l (later known as in~erleukin-l or IL-l) in ~h~
induction of very early multipotential precursors and the ormation of very large mixed hemopoietic colonles.

Ocher sources of megakaryocyt~ potentia~ors have been found in the conditioned medla of muslne lung, bone, macrophage cell llnes, pesltoneal exudate cells and human embryonic kidney ~eLls.
Desplte cert~in conflictlng da~s ~Ma~ur, E., Exp. Hematol. 15:340-350 11987]), there is some evidence (Geissler et ~l-, Br. J.
Hae~Col. 60:233-238 ~1985~ that ~ctiYated T lymphocytes rather than monocy~ss pley an enh~ncing role in ~egakaryocy~opoiesis.
This may be suggestive o~ th~ ldea ~h~t ~ivs~ed T-ly~phocyte secre~ions such as interleukins may bc regula~ory ~ac~ori ln ~eg de~elopment tGeissler ee 1., Exp. Hematol. 15:845-853 ~19871). A
number of studies on megakaryocy~opoi~sis wi~h purified E~
(Vainchenker et al., Blood 54:940 ~979]; ~cLeod et al., Na~ure ~ 492-4 ~19761; and Willia~s ee al., ~xp. Hemato~ :734 [1984]~
indlc~te ehae ehis hormone h~ an ~nhanclng effect on meg colo~l-f ormatlo~. ~o~e recently this has baen demonstra~ed in bo~h seru~-fr~e and seru~-containing cultures and in the absence of accessory c~lls (W~lliams et 81., Exp. Hemaeol. l~-734 ~1984]~. EPO was pos~ulatDd ~0 be in~olved ~ore ln the single and two-c~ll s~e .

aspacts oi ~e~akaryocytGpoiesis is opposed to che effect of P'~-SpCM which ~a~ inYolved in the four-cell stnge of.~egakaryocyte develop~ant. The inceractlon of all these factors on bo~h aarly and la~e phases of megakaryocyta development remains to be elucidated.

Plat~lets ar~ critlcal ~or~ed alements of ehe blood clotting ~echanLsm. Depletlon of the circula~ing level of platelets, called th~ombocycopenls, occurs in varlous clinical condltions and dLsorders. In~reased rates of pla~elet destructlon characterize i~munologic or nonimmunologic acquired thrombocytopenla such lS that which is associacdd w~th infectlons, Moschcowl~z' Syndrome, Gasser's Syndrome, direc~ or drug-induced platelet toxlclty, post-transfuslon purpura and ~llerglc chro~bocytopenia ldiopnthic thrombocytopenic purpura; and lm,~unologic or nonimmunologlc congenit~l thrombocytopenia such as ery~hroblastosis fetalis, prematurity thrombocytopenia, infe~tious thrombocytopenia, thrombocytopenia with giant cavernous hemangioma, drug sensitivity, isoimmune neona~al thrombocy~openia, and thro~bocytopenia associa~ed with maternal idiopathic thrombocytopenic purpura. Diminished or defective platele~
production characterlzes Fanconi Syndrome, s~egakaryocy~ic thro~bocytop~nia, her~ditary thrombocytopenia, ~eonatal rubella, maternal ingestion of chiazide diuretics, aplastic ane~ia, ~allgnant infiltration o marrow, and the ~dm~nistr~ion of ionlzing radiatlon and ~yelosuppressi~e drugs.

Thrombocytopenia is clinically dangerous, exposlng patients with this condition to uncontrolled bleeding episodes. In the ordinary cours~ o events, th~ ti~ requlred for maturatlon of platelets from megaka~yocyt~s after an ~nsult is ~bout 4 to 5 days ln hu~ans. It ~ould be desirable to ldentify a~ agent Gap~ble of ~ccelerating platelet develop~ent and shortenin~ this perlod.

t7 Considerable efforts have been exp~nd~d on ldencifying su~h an agen~, commonly referred co as thrombopoeleln. Th~ actlvity of such an agent has been obsorved ~s early as 1959 (Rnk cc sl., ~ed.
Exp. n 1:12~) and a~empts to charac~eri_e ~nd purlfy ~hLa agenc S have conclnuad up co the pres&nt day. Soma have reporced par~ial purlficaclon of ~hrombopoeitln-acclve polypeptldes (see, for example, T~yrlen er al., "J. Biol. Chem." ~ 3262, 1987; and HoEEman ec al., "J. Clln. Invest." 7S:1174, 1~85) whlle others have pos~ula~ed cha~ thrombopoeitln is no~ a dlscrete en~i~y ln i~s own right but xather 1~ iimply th~ polyfunctlonal manlf~s~tion oi B
known hormone ~IL-3, Sparrow ee ~1., ^Psog. Clin. ~ioL. Re~
(~egakaryocyte DeY. Funct.):123, 19B6).

Ac¢ordlngly, it is nn ob~ect of ~hia invencion t~ obtaln a lS chrombopoeitln-ac~ive polypap~lde whlch is in a pharmaceu~ic~lly pure for~.

Ic is snooher obJec~ co provide ~ucleic acid encoding ~he polypap~ide and to use chis nuclelc acld co produce ~he polypepcide in racombinane caLl culture for diagnostic ~se or for therapeu~ic usa ln che treat~en~ of thrombocytopenia. I
1.
It is sclll further an object to cyneheslza variants of ~he polypeptide, including amino ac~d sequence and covalent d~ri~a~ives thereof.

Another object is to produce antibodies capable of binding to the polypeptlda.

~ urther ob~ect is to pro~ide dlagnos~lc methods or the determinatlon of the polypept~de, ~r nuclelc acid or ltS
anribodies, in tes~ samples whlch ~ethods are useful in the detectlon and classifica~lon o~ leukemias.

~nc~i7~

These and other ob~ects of the lnventlon will be apparent to the ordlnary artisan upon consldera~ion of the speciflcation as a whole.
, S-I~m~v of the In~entlon The,~ccomplLshment of these obJectives is made possible by ~he ideneificacLon of a gena ~ncoding a thro~bopoietin-acti~/e polypeptide (hereina~ter, me~akaryocytopoeitin or "MKP"). A D~A
sPquence encoding a butyryl-chollnes~erase deri~ative was fortuitously dlzcov~red during ~n i~veecigation of multiple cDNA
- librarias fr~m hu~an ~eeal ~iss~a3 wlth bueyryl-cholineste~as~ cDNA
pro~. Thl~ derivaelve concaincd the ~irst exon. ~rom the human butyryl-chollneseeras~ ~ene ~250 nucleotides of the 5' region of the bucyryl-cholinesterase codlng sequence), ~owever, the 3' end of this clone (herelnafter "clone 14a) concained ~n open seading frame encoding a polypepeida other than the remainder of butyryl-cholinesterase, The DNA ~t the 3' end of che butyryl-cholinest~rase derlvaei~e was further found to hybridlze to geno~lc DNA fr~gmencs obtained ln an scuce myelocy~ic laukemia.

Nucleic acid co~pris~ng tha clone 14 sequence is provided in order to prepar~ MXP by recomblnsnt cult~re. Nuclelc ~cid which is hybridizable co ehe clone 14 sequence under strin~0ne condi~ions (but which ~ay no~ encode ~he clone 14 polypeptide or any portlon thereof) is used to probe for sources o~ DNA encoding related polypep~id~s or ~or t~ss~es or cell l~nes which ar~ ~ranscrlbing this Bene or for prlmer exeension.
. .
Also within ~he scope o~ ~hls inven~ion are therapeu~lc prepara~ions of MKP obtained by conven~ional ~echnlques fro~ nati~e or recombinant sources of ~KP, an~lbodles agalns~ ~P ~nd diagnos~lc mechods for the de~ermlnation of MKP, les nucle~c acid or its sn~ibodics.

~rl~ mmarv_o ~h~ ~r~win Figs. la - le depLc~ th~ nucloocide and impu~ed amLn~ acid 6equences of clon~ 14. The upper readlng ~rame encoded by S nucleo~ides l to 56~r i~puces a polypepcide found within full-length MKP, probably represencln~ cha C-termlnus oE ~P, and ls referred ~o herein as.the sequence or polypepcide of Fi~. 1 pe~ai1ed Descr~Pcion of he Invenc;on ' ~CP is a hormonally-acti~e polypep~ide believed to have uc~lity in th~ modulatlon ~f hematopolesis and lmmune responses, and ln d~agnos~ic t~sting. MKP, it9 imm~nogens, lCs Jneibodle~ and nucleic acid whlch hybrldiz~s to nucleic acid cncoding MKP ~re dtagnostlcally ~seul in the subclassification oi rare leukemiai.
MKP polypzp~de is employed in stnndards for ~he'dececclon of ~KP
expresslon by leukemias. NucleLc acld which is capable of hyhridi~Ln~ to ~KP-encoding RNA finds u~llicy ln the decection of ~KP gene a~plificacion in thos~ sa~e leuk~mias. MKP lmmunogens are useful for the manufacture of antibodles to MKP, whlch in curn are useful in lmmunoass&ys for MKP as wlll be more fully descrlbed below.

ThQ hematopoleeic and lmmunomodulatory characteriscics of MKP nre resolved by conventional 6craening ~s~ays which ~asure the lnfluence of ~ ~ on the grow~h and/or diferenciaclon of blood cells and thelr progenitors, includlng erythrocy~es, megakaryocytes, st0m cells, monocy~es, ly~ph~cytes and ~he llke.
Based Jn preli~inary data, it is belleved that MKP potentiates ~he developmen~ or differentiatlon o~ me~akaryocy~es and has u~illcy tn ~herapeu~ic ccndltions where such an activlty is required.
HoweYer, 1t ~ill be understood tha~ MKP ls Llkely to be polyfunctional and the determi~a~lon of oeh r activi~les, and hence uses, for ~KP wlll be wl~hin the ordinary skill of rhs artlsan.

.

MKP i5 defln2d as a polypeptlda having ~he sequencs S2C
forth ln ~he d~signa~ad upp~r readLng frame of Fig. l~eo~e~har with ~ulno acid sequ~nr~ v~rla~ th~reof wherein one or mora ~mlno acld resldues hav~ be~n ln~rted, dela~od or ~ubs~ltuced and wh~reln the varinnts exhibi~ 6ubscsnelally th~ same qualltatlve blolo~lcal scclvicy ns the polypept~de of Fl~. 1 or lts fulL~leng~h, ma~ure ;
coun~erparc"

The blologlcal actlvlty of ~KP i8 defined ~s the abillty co modul~ce che growch or dlff~rentln~lon o~ blood cells or their progenitor ¢ells, or ehe abill~y to cro~a-react with an antibody raised a~ainse ehe polypeptidu of Fig. l. Preferably, the biologlcal ac~lvity of ~XP is dater~lned by thc modified Iscove' method described in the ~amplc below, or by other convenc~onal assays known eo those in the field.

~KP variants wlll have sequenc~s thac ordinarlly are greacer thnn sbout 80 percenc homolo~ous wlth the design~ed sequence of Fig. l, havin~ allgned the sequences to achieve maximu~
homology snd no~ consld~rlng ~s homologous any conaerva~ive substl~utlons. N- or C-terminal aubs~itu~lons 6hall noC be cons~rued as reduclng the homology of the v~riant to the Flg. 1 sequence. MKP varlan~s exclude ~ny polypep~lde heretofore ldentl~ied. Typical variants of the Flg. 1 sequ nce found in nature may lnclude the full-len th ~nalogue of MKP, coun~erpar~s from nnlmals such as porcine, non-human prlmate, ~quine, murine and ovine, and nlleles of the foregoing. O~her emino acid sequence variants are predetermined ~nd prepared by site-dlrected muragenesLs.
Amino ar~d s~quenc~ variants of MXP incl~d~ pr~dut~rmlned dele~isns from, os lnsertlons or subs~ieu~ions o ~esidues within ~he MKP seq~ence shown in Fig. 1 or l~s ~aeure homologu~. Amino scid se4uence dele~lons gener~lly range from,about l to lO residues and typically ar~ coneiguous. Coneiguous delations ordin~r$1y are . .
.. .

- s m~de in even numbers of resldua~, buc 6~ngle or odd numbers oE
d~letions ~ra wirhin eh~ 6cope h~r~of.

Insertions al80 ~re pr00rably msde ln ~ven numbers of S residu~s althou~h insertlons m~y rnnge from l to 5 residues in 6ener~1. Howev~r, ins~rtlons also include fuslons on~o one or bo~h of ~he amino or casboxyl terminl of MKP, ranging in len~h fro~ 1 ~sldue to polypepcldes containln~ one hundred or mor~ resldues. An ~xampla of a singla eermlnal ins~rtion ls ~a~ura ~XP having an N- j I0 te~mlnal methlonyl. Thl~ varlant ls an artifact of the direc~ I
expresslon of MK~ ln recombinnnc cell cul~ure, l.e., expression wlthout a signal sequence to direct the ~ecreclon of msture ~XP.
Ocher ex~mples of eermlnal la~sreions lncluda 1) fuslons of he~erologous signal sequences to the N-eermlnus of ~atuse ~KP in order co faclliente tha secreeion of mature ~XP from recombinant hosts, 2) fuslon~s of immunogenic polypeptide , e.g. bacterial polyp~ptidas such as betn-lsce~mase or an en~ym~ encodad by the ~.
trp locus ~nd 3~ N-termlnal fuslons with cholinesterases or N-~ termlnal fra~menes of cholin~sterases, especially butyryl-holinester~s~. The ~ntibody And MKP ar~ covalen~ly bonded, for exa~ple, by the method of EP ~70,697A, ~lthough other methods for linking proteins ~re conveneion~l and ~nown ~o the ar~is~n.
I~munogenic fusions nre usoful for preparing im~unogenic MKPs suitable ~s vlccines for prepnring a~ti-MXP an~lbodies.
The thl~d group of variants aro those ln which a~ leas~ one residue ln the MXP molecul~ has been removed ~nd a different residue inserted in lts place. Such ~ubstlcutions general?y are made in accordance wl~h following Table.

s 1 o -T~ble l $n~ Qs~dua _ F.xe~ rY subs~itu~loQ~
Ala gly; ser Arg lys Asn . gln; his Asp glu Cys. ser Gln A sn Cl~ ~sp Gly ala His asn; gln Il~ leu; v~
Leu il~; v~l Lys ~r~; ~,ln; ~,lu lS ~c met; leu; tyr Ser thr 11 Thr ser Trp ~yr Tyr trp; phe Val ile; leu .

Substantial changes in functlon or lm~unological iden~i~y ~re made by select~ng substitutions ehae Are less conservatiYe than those in Table 1, i.a., s~lec~in~ residues that d~f~er mo~e signif$cantly in ~helr e~fece on maln~alnlng (a) th~ st~ucr~re of the polypep~ide backbone ln the area of the subs~i~ution, for exa~pls s a shee~ of hellcsl conformation, tb) ~he charga or hydrophobicity of the molecule at the ~ar~ee siee Gr (c) the bulk ol ~he side chain. The substStutions in genersl e~pec~ed to produce the greatest chan~es in MKP properties will be those in whlch (a~ a hydrophilie resldus, 2. g. seryl or threonyl, ls substltuted for (or by) a hydrophoblc residue, e.g. leucyl, lsoleucyl, phenylalanyl, valyl, or ~lanyl; (b) a cystaine or proline i5 substituted fo~ tor by) any other,residue; ~c) a residue havln~ an electroposielve side chaln, e.g., lysyl, arginyl, or ..;

histidyl, is subseieuted for (or by) ln electsonega~ivs residue, a.g., ~lu~amyl or sspartyl; or (d) ~ residue havlng a bulky side chain, ~.g., ph~nyl~l~nin~, is s~bs~ieu~ed for (or by) one no~
haYing such a si~e chaln, e.g. glycLne.
MKP nuclelc acld ls deflned as ~NA or DNA ~hich encod~s ~P
or which hybridlzes ~o such DNA snd rem~ins s~ably bound ~o i~
under strin~enc conditions snd is greacer than about 10 bsses in leng~h. ~tringent condlclons are deflned as low lonic s~rengch and high temperacure for washing, for example, 0.15 M NaCl/0.015 Sodlum cltrzce/0.1~ NaDodSo4 at 50-G, or al~ernaeively ~he presence of dena~urlng agents such as formamide, for example, 50~ (vol/Yol) formamldo with 0.1~ bovlne serum ~lbumLn/0.1~ Ficoll/0.1~
polyvinylpyrrolidone/50 m~ sodium phosphace buffer at p~ 6.5 wLth 750 mM NaCl, 75 ~ sodium citrata and 42-C for hybridization. DNA
en~oding MKP is obtalned from fetal tissue cDNA llbraries, ordinarily llvsr or dissecc~d braln reglons, from cDNAs or ~enom1c DNA o~ AML leukemic cells, or by in ~ synthesis. Hybridizing nucleic acid gen~rslly is obtainad by 1~ vit~o synthesis. The original discovery of ~ DNA sequence for MXP was forcuitous, but i~
wlll be understood tha~ one skilled ln the art would be sble ~o readily obeaLn M~P-ancoding nuclelc scid having been supplied wich th~ nucleotide sequence uncoding MKP. Thi~ mos~ convenlenely is acco~pllshed by prob~ng human cDNA or genomlc libr~ries by labelled oligonucleotide sequencas seleceed from Flg. 1 1~ accord wi~h known ~ ~ crlteria, ~mon~ which is thse the se~uence should be of sufflclen~
lengch and sufficien~ly un~mbiguous tha~ f~ls~ poqlti~as ~re mln~ized. Typlcally, ~ P32 labelled ollgonucleo~ide havlng ~boue 30 to 50 bAce~ ls 6uf~1ci~nt, p~rticularly if ~h~ ollgonucl~oclde cont~ins one or ~or~ codons for ~ethionlne or tryptophsne.

Of parcicular intexest i5 ~KP nuclele a~-id ~hat eneodes the ull lengch molecule, including buC not necessarlly its natlve signal sequence. This is ob~ained by screening cDNA or genomic llbraries usin~ ch~ full length elone depicted ln Flg. 1 and, if .

i'7~

nocessary, usLng conv~ntLonal primer ~xten~1on procedures ln orde~ ,, eo se ura DN~ whlch is co~plete at lrs S' codlng end.~ Such a clone ls readily ldantlEled by the presanca of a st~re codon ln readln~
~rama w~ch the Flg. 1 sequance, ~he full langeh clona most llkaly also wlll ancode a sl~nal s~quence of about 15 to 25 largely ;
hydrophobic rssld~les l~nediaealy followlng tha seart codon. The i full length.clon~ than is transfec~d lnto a ma~all~n hose cell under the conerol of appropria~e expre6sion concrol æe~uences, whereupon the precursor will be processed and sacreeed as ma~ure full len~th MKP. The N-terminus o thi3 molecule i6 dat~rmined by conv~n~lonal Ed~an degr~daCion or the like ~o elucidaee the m~eure ~mlno terminus. Thl9, tc6eth~r wlth an ~n~lysiY o~ ~he C-~ermlnal and of tha natlve signal sequenca, guldss the salec~ion of the pr~par N-~ermin~ slte for conatructing heterologous slgnal sequence fuslons usable, or example, ~n microblal host-~eceor syste~s.

The MKP-encodlng n~clelc acld i5 then ligated in~o raplicable veceor for further cloning or for axpressLon. Vec~ors ara useful fur parfor~ing two funceions in collaboracion wich co~pstible hose cells (A host-vactor system). One funceion is to facilieate the cloning of the nucleic acid that encodes the ~KP, i.e., ~o produce usable quaneitles of the nucleic acid. The ocher function is-to dir~ct tha exprassion of ~KP. One or bo~h of these functions are performed by the vector-host sysce~. The vectors will con~aln differ?~t co~pone~ts dap~nding upon the unc ion they are to perform ss well as ~he host cell tha~ is selected for cloning or expresslon.

Each vector will con~aLn nucleic ~cid th~t encodes MKP as describ~d above. Typlcally, thls will be DNA that encodes the MKP
ln its mature form linked at lts amLno terminus to a secretion signal. ~his secretion signal prefer~bly is the MXP presequence that normally directs the secre~lon of MKP ~ro~ human c~lls in ~ . However, suitable secretion signals ~lso ~nclude si~ ls .. ..

~3~ 7~ -~ -13- ~
, from oeher anim~l MKP, virsl aignals or sign~ls from secreted polypept~des of ehe same or related specles.

Expresslon and cioning veceors con~ain a nuclaie acid sequunco th~e en~bl~s the veceor to replicace ln one or more solected host cellQ. Cenerally, in clonlng veccors thls sequence ls one th~ enable~ tha veccor to replicate lndependently of ~he host chromosome-q, and lncludas ori~ins of raplicarion or autonomously replicating sequences. Such sequences ~re well-known for a variety of bactarla, ye~sC ~nd viruses. Tha origln of rcplicatlon from the w~ll-known plasmid pBR322 19 suitable for mosc gra~ nsgstiva bactaria, ~ha 2~ plasmld orlgin fo~ y~ast and Yarlou~
vlral orlglns ~SV40, polyoma, ~denovirus, VSV or BPV~ sra useful for eloning vectora in mammalian eells. Origins are noe needed for mamm~llan oxpression vectors (the SV40 origin is used in the Examples only beeause it contains th3 early promoter). ~ost expresslon vectors are ~'shuttle" Y~ctors, i.e. they are cspable of repllca~lon in at leas~ one clAss o or~anisma buc can be tr~nsfected into another org~nlsm for expression. For example, vector is cloned in E. col~ and chen che same vector is cransfec~ed ~nto yeast or ma~malian eells for expreqslon even thou~h i~ ls noc capablQ o replic~ing independently of ~h~ host cell chro~osome.

DNA also is eloned by lnsertion into cha host ~enome. This is re~dlly ~ccompllshed wi~h b~cillus specles, for e~mple, by Lneludin~ ln the vector ~ DNA sequence ~hac is eomplemencAry to a sequenee found 1~ bacillus geno~ic DNA. Transfection o ba illus with thls ~ec~or results ln ho~ologous recombination wich che g~no~ ~nd lns~r~lon of MKP DNA. HoweYer, th~ reco~ary of ganomlG
DNA eneoding MK~ is more complex chan tha~ of ~n exog~nously replieated ~eotor because restriction enzyme digestion is required to excise the MKP DNA.

Expression and eloning veetors should cont~in ~ seleetion 3~ gena, also car~d ~ salectabl~ marker. ~hi3 iS a gene tha~ encodes "

6~

.

a prot~ln necessary for ehe survival or gro~ch of a hos~ cell tr~nsformed wi~h ~he v~o~or. The presenc~ of this- gene ensures th~ ~ny host cell whlch dslet~ th~ Y~c~or wlll no~ obeAIn an advan;ag~ ln growch or ~production over trsnsformed hosts.
Typical selec~lon genea ~ncode proteln.~ th~t (a) confer resistance to antlblotlcs or other to~lns, e.g. ~mpicillin, neomycin, m~hotrexate or tetr~cycllne, ~b) comple~ent auxotrophic deflclencL~3, or ~c) supply c~ielcal nu~rl~n~s noc available rom complex media, e.g. ~hs genu ~ncodlng D-alanine racemase for b~cilli. I
~- I
A suitable selaction gene for U90 ln yeas~ is the trpl gene present in tha yeasc plasmid YRp7 (Stlnchcomb et al~, 1979, UNa~ure~ 39; Kin~sman ~ ~1., 1979, ~Gene" 7:141; or Tschemper et al , 1980, ~Cene~ 10:157) The ~pl gen0 provides a selectlon marker for a mutan~ strain of yeas~ lacklng the sbllley to grow ln tryptophan, for example ATCC No. 44076 or PEP4-1 (3Ones, 1977, UGene~ics'' 85:12) The presence of the trpl leslon in ~he yeast hosc cell genome then provides an effective environment for detectlng transfor~aclon by ~ro~h ln the absenca of ~rypeophan.
Similarly, L~2 defic~ent yeast strains ~ATCC 20,622 or 38,626) are comple~enead by known plasmids baarlng the Leu2 gene.

Examples of sulesbla selecc~bla markars for mammali~n cells nra dihydrGfolate reduceasa (DHFR) or ~hy~idlne kinase. Such m~rkers enable the ldenCification of cells whlch wer~ compa~ent to taka up the ~KP nucleic ~cld. The mammAlian Call transforman~s sre placed under selec~ion pressure which only ~he ~ransform~n~s are ~niquely adap~ed to su~vi~a by Yirtud of having taken up ~he marker. Selec~1On pressure ls imposed by cul~urlng the tr~nsformants under cond~tlons in which th~ conoentratlon of selection Agent in thP. ~edlum i3 success~vely chan~ed, thereby lead~ng to amplificatio~ of borh the selection gen~ ~nd the DNA
~ha~ encodes ~KP. Ampllflcation is the process by which gen2s in groa~er demand for the product$on of ~ protein crieical for growth ~o.P~3~

-1 5~

ar~ reiter~ted Ln t~ndem w~thln ch~ chromosomes of ~uccess~ve ~ener~ion3 o recombinsnt cells. ~ncr~ased qu~ntle~ei of MKP sre syntheslzed from th~ ~mplifiad DNA.

5For ex~mple, c~lls tran~formad wich ehe DHFR selection ~ene nre i~st identlfL~d by culturlng ~11 of tha transorma~es ln a cultura m~diu~ whlch ll~cks hypoxanchine, ~lycine, and ehymidine.
An appropriace host c~ll ln thi~ cnsa iq ~he Chlnese h~mster ovary (CHO) c~ll line deficienc ~n DHFR sccivlty, prepared ~nd propagaced 10as descrlbad,by Urlaub and Chasin, 1980, "Proc. Na~'l. Acad. Sci.
USA~ 4216. A p~r~Lcularly useful DHFR is a mu~an~ DHFR that is .
highly resistant to ~TX (EP 117,060A~. This selactlon ~genc c~n be used wieh ~ny o~herwlse sultabl~ hose, e.g. ATCC No. CCL61 CHO-Kl, notwithstanding the presence of endogenous DHFR. The DHFR a~d ~XP-15encodlng DNA then is amplified by exposure to an a~ent (metho-trexa~e, or MTX) that inaccivaces th~ DHFR. One ensures that ~he oell requlres mora DHFR (and consequencly ampllfies ~11 axo~nous DNA) by seleccing only for cells thac can grow in successive rounds , of evar-~reacer MTX concencratlon.
Other me~hods, vectors and hsst cells suieable for adaptaCion co che synchasiQ of ths hybrid rscepCor in racombinanc ~ereebrat~ cell cultura are d2scribed ln M.J. Ge~hing Ç ~
"Natura" ~ 620-625 (1981) N. Manccl e~ ~1.. "Macur~ 40-46 ~1979); and A. Levlnson Q~ ~1...... EP 117,060A ~nd 117,058A. A
partlcularly useful starting plas~ld for m~mmalian ~11 culeura expresslon of MKP is pE342~HBV E400.D22 (also c~lled pE34~HBVE400D22, EP 117,058A):

30. Expressio~ vectors, unllka ~lonln~ vectors, should concain ~ promocer wh~ch is recognized by ~he host or~anis~ and is opsr~bly llnked eo th~ HKP nucleic ac~d. Promo~ers ~r~ un~r~nslated ~equences loca~ed ~pstrea~ fro~ che start codon of ~ s~ructural ene (generally wLchin abou~ 100 to 1000 bp~ that control the 35transcrlpeLon and transla~lon o~ nuclelc acid und~r their control.

7~ :

;.

They typically fall into tws classes, induclble and constituciYa.
Induclbla promotars a~e promot~rs ehac inltlnca lncr~sed lavels of transcrlp~lon from DNA under their con~rol ln responsa to some change ln Gultu~ condielona, o,g. tha presanc~ or sbaence of a S nutrient or ~ change in temp~ra~ure. At ehls time a l~rge nu~ber o~ promot~rs reco~nized by B variacy of potenclal hosc cells are wull known.. These p~omo~ars a~e operably llnked co MX~-encoding DNA by re~ovln~ che~ from chelr gene of orl~in by rescrlcclon enzy~e dlgeselon~ ollowed by inser~lon 5' eo the scart codon for ~KP. This is noc to aay ~hat the genomic MXP promocer is no~ ¦
usabla. Howe~er, heterologous promoterq gener~lly wlll reYulr ln ~reater cr3nscrip~ion and hl~her ylelds of expressed ~KP.

Nuclelc ~cid is operably llnked when ic is plac~d into a functlonal rela~ionship wlth another nucleic acid sequence, For ex~mple, DNA for ~ pr~sequenc~ or ~ecracory le~d~r is oparably llnked to DNA for a polypep~ide if it ls expressed as a prepro~ein whlch par~lcipsras ln the secretion of` ~he polypeptide; a promoter or enhancer is operably linked to a coding sequence if lt afects the transcriptlon of the sequence, or a riboso~e binding slce is operably llnkad to a coding sequence if it is positioned so as to facili~ate translation. Generally, operably linked m~ana tha~ ths DNA sequenc~s beIng llnked are conti~uous and, In the case of a secr~tory lead~r, con~guous ~nd ln readlng phass. Llnklng is accomplished by ligation at convsnlent restrlctlGn sites. I~ such sl~es do not ~xls~ than synthet~c ollgonuclsoride ~daptors or linkers are used in accord with conven~lonel prac~ice.

Promoters suitable f 4~ use wlth prokaryotlc hosts lncluda the ~-lactamase ~nd lactose promoter systems ~Chang e~ al., 1978, UNaeura" ~ 6}5; and ~oeddel ~ al., 1979, "Natura" ~1:544), alkallne phosphaease, ~ tryptophan (trp3 promotsr syste~ (~oeddel, 1980, "Nucleic Acids R~s." 8:4057 and EP0 Appln. ~ublo No. 36,776 and hybr~d promoters such as ~h~ tnc promoter (H. de Boer e~
1983, ~Proc. Nae'l. ~cnd. Sci. USA" ~Q:21-25~. How~vsr, ocher known b~ct~rL~ promocers ~r~ sult~ble. Their nucle~id~ sequencesh~v~ bQen publlshcd, ~he~by enabling a skllled worker operably to llga~s ~hem eo DNA encodlng M~P (Si~b~nlis~ e~ ~1.. 1980, ~C~
~Q:269~ using linkers or ad~ptor~ eo supply any r~qulred r~acria~lon sl~es. Promoc~rs for use ln bacterl~l syscema also wlll con~2in ~ Shlne-D~lgarrlo (S.D.) s~quen^e opQr~bly linked to ~he DNA encodin~ MKP.

Suleable promo~lng sequences for use wleh yease hosts lncluds the promoters for 3-phosphoglycera~s kinase (~ltzeman e~
pl., 1980, ~J. Blol. Chem." ~ 073) or o~her glycolytlc enzy~es (Hess et al., 1968, ~J. Adv. En~yme Rag.~ ~:149; ~nd Holland, 1978, "Biochemlsery~ l?:4900), such as enol~se, glyceraldehyde-3-phos-phate dehydrogenase, hexokin~ss, pyruv~ee decarboxylase, phospho-fructokinase, glucose-6-phosphata isomerase, 3-phosphoglycera~e muo~se, pyruvate klnass, trlosephospha~e isomerasa, phosphoglucose iso~erasa, and glucokinase.

Other yeast promoters, whlch ara lnduclble pro~oesrs h~ving ehe addi~ional advan~age of ~rnnscrlption con~rolled by growth condl~ons, are ehe promotsr reglons for alcohol dehydrogenase 2, isocytochrome C, ~c$d phosph~tsse, degradativ~ enzy~es sssocl~sd with nitrogen me~abollsm, metallothionein, glycer~ldshyde-3-phos-phate dehydro~enase, ~rld ~nzymes responslblQ f or maltose ~nd galactose u~illzation. Suitable vec~ors ~nd pro~oters for ~s~ in yeast exprQssion ~re fur~her deseribed in R. Hitz~man e~ ~l., EP
73,657A. Yeas~ enhancers also ~re ~dY~nta~eously used with ye~se promorers.

-30 MKP transcriptlon from Ye~tOrs ln m~mmallan hose cells is controlled by promoters obt~lned fro~ th9 genomes of vlruses such ss polyoma, cytomeg~lovirus, adenovirus, retro~lruse~, hepa~itls-~
I virus ~nd ~Ose preferably simi~n Vlrus 40 (SV40), or from ! heeerologous m = alisn promoeers, e.g. ~he ~celn promoter. Theearly ~nd l~ta promoters o~ the SV4~ ~lrus gre con~en~ently '7 -~a-obtaLned as ~n SV40 res~rlcelon fra&men whlch ~150 eontalns ehe SV40 vir~l orlgln of repllcatlon (Flers Q~ ~1., 1~78, a~acur3"
211:113). 0 course, promocers from tha hosc eell or ralated speeies also are usaful heruln.
Transcrlpclon of MKP-eneodlng DNA by h~ghar eukaryotes is lncreasud by illser~ing an enhancer suqusnee lnto ehe vec~or. An enhaneer i5 a nuclaocld0 aequence, usually abouc from 10-300 bp, thac acts on a promoc2r to lner~as2 leY trnnscrlption and does so ln a manner chac 13 relativ~ly orlenta~lon and posl~ion lndependenc. Many enhanear se~ueneo~ are now known from ma~mallan genes (globln, elastase, albu~in, ~-fe~oprot~ln ~nd insulin).
Typleally, however, onq wlll us~ ~n enhane~r from a eukaryoeic cell virus. Examples lnelude the SV40 ~nhanc~r on the late slde oi the repllcation orlgln (bp 100-270)~ the eytomegalovlrus early promo~er enhancer, the polyoma enhane~r on the la~e sld~ of the xepLlcation origin, and ndenovlral enhane~rs. The enhancer ~ay be spliced in~o the veetor ac a posiclon 5' or 3' to tha MKP-encodin~ sequance, bu~
ls preferably loca~ed at ~ slce 5' from the promo~er.
Exprassion vectors used ln eukaryotlc host cells tyeast, fungl, inseee, planc, animal, human or nucleated eells from o~her multieellular organisms) will also eontain sequenees neeessary for thc terminatlon o~ transcription and for seabili~ing ~ha ~RNA.
Sueh sequ~nees are eommonly available irom the 5' and, oeeasionally 3' untranslac~d regions of CU~dryU~iC or viral DNAs or eDNAs.
These regions contaln reglons that are ~ranscribed as polyadenylat~d segmenes ln the uncranslat~d por~ion of che mRNA
encoding MKP. The 3' untranslated reglons ~lso include transcrlption termlnation sltes.

Sult~ble host cells for cloning or ~xpresslng the ~eGtors herein are the prokaryote, yeas~ or high~r euk~ryote cells d~scrLbed above. Suleable p~okaryoees include gram negative or ! 35 gram posieive organlsms, for example ~ i or bacllli. A

~3~
. ~ ~

pr~farred eloning host Is ~. Ç~ll 294 (ATCC 31,446) al~hough orher F~rA~ noga~l~D ~r grwm positlv~ prokary~ees _urh as ~ oli B, ~.
~11 X1776 (ATCG 31,537), ~. Qll U3110 (ATCC 27,32S), pseud~monas speeies, or ~Q~ nreQq~n~ a~e suitabla.
.
In nddltion to prokaryotes, eukaryoclc ~lcrobas queh as fll~uontous.fungi or yeas~ ard suicabl~ hostq for MKP-eneodln~
veet~rs. ~S ~hQ~aDYs~L cerevtsiAe~ or common baker's yease, i6 ~h~
most commonly ~s~d among lower sukaryotic host mieroorg~nisms.
However, a number Df o~her ~enera, 6p~clss and stra~ns ar~ commonly ~allabl~ and useful herein.

Sui~able host e~lls for the ~xpresslon of MKP are derl~d from ~ultieellular organis~s. Such hosc cells ~re c~pable of eomplex proeessing and glyeosylation aetivitles. In prineiple, any .higher euk~ryotie eall eulture is workable, whe~her from ~er~ebra~e or inver~ebratd cul~ure, although eslls from mammals such as humans are preferrsd. Propaga~ion of such cells in eulture is ~X se well known. See T~$sue Çulture, Aeademic Press, Krus~ and Pa~terson, 20 editors ~1973). Examples of useful m~mmallan host cell lines are VERO &nd HeLa eells, Chinese hams~er ova~y eell llnes, ~he WI38, BHK, COS-7, MDCK eell lines and human embryon~e kidney cell line 293.
~1 Host cells ~re transform~d with ehe ~ove-descrlbe~
expression or cloning vectors and cul~ured ln con~entional nutrien~
medLa modii~d as $s appropriate for inducing pr~moters or s~lecting transformants co~talnlng ampli~ied genas. The cultur~
condi~ions, such ~ temper~ture, pH and che llke, sultably are ehose previously used with ~he hose ~ell select~d ~r oion$ng or expressisn, as the case may b~, a~d w~ll ba apparent to the ordinary artIsan.

MKP pre~erably ls recovered from the culeure mediu~ ~s a secs~ced proeein, ~Lthough it ~lso m~y b0 recov0r~d fro~ hos~ cell ~3~

. .
lys~ees when dlrecely expr~ssad withou~ a aecr~eo~y slgnsl. As a f1LSC seep, th~ cultur~ medlum or lys~te ls c0n~rif~ged to r~move psre~cula~e ccll d~bris, MKP a180 ls purified ~ro~ conramlnan~
soluble proteins for exampls by fractlons~lon on lmmunoaffinley or ion ~xchange columns. Such processes ~s chroma~ography on alkyl ~-S~ph~rose, silica or an anion nr ca~ion exchange resln or gel elecerophores~s are used ~o separnte the MXP from contaminants.
MKP v~rl~ncs ln which residues havo been dele~ed, inserted or subs~ituted are recovcred ln the sRme ~ashion ~s native MKP, taking account of any substantial changes ln properties occasioned by the ~ari~ion. For example, preparation o~ an MKP fus~on with anorher proteln, ~.g. bac~erial ancigens, facilltates puriflcation be~.ause an immunoaffinicy column containlng ~n~ibody to the antlgen can be used to adsorb thc fusion. A proeeas~ inhibltor such as PMSF also may bc useful to lnhlblt proteslytic degradation during puriflcaeion, and antibio~ic~ may be lncluded to prevent the ~rowth of adven~i ious contaminants. One skilled ln the art will appreclate that puriicatlon methods suitable for na~i~e ~KP may req~lre modiflcacion co accounc for changes ln the charac~e~ of MKP
or its ~arian~s upon expression in ~ecombinanc cell culture.

Therapeutic ormul~eiona of MKP ~re prepared for stor~ge by mixing MXP ha~ing the desired degr~ of purity wi~h optional physlologically acceptsble carriera, excipien s or stabillzers in th~ or~ o lyophilized cake or nqueous solutions. A&ceptable carriers, excipienta or stabilizers are non~oxlc to reclpien~s at the dosages and concentracions employed, and include buffers su h a5 phosphate cierate and other organic acids; ~nclox1dan~s 1nG1Uding nscorb~c acld; low molecul~r wei~ht (less than about 10 - 30 resldues) polypeptides; protelns, such as ~erum ~lbumin, gelacin or immunoglobulins; hydrophillc poly~ers surh as polyvlnylpyrrolidone ~mlno ~cids such as glycina, glu~mic ~cld or ~spartlc acid;
monos~ccharides~ disaccharidss and other ca~bohydrates includln~
glucose, ~annos~, or de~trins; chela~ng ag~ts s~ch as EDTA; ~ugar alcohola such as mannitol o~ sorbi~ol; ~ale-form~ng countarions .

' , ~ g~ t~

such ~s sodlu~, ~nd~or nonlonlc sur~c~anta ~uch ag T~e~n, Pluronlca or PEC. MKE ~o b~ used f~r therapeu~ic ~dministra~Lon mus~ b~ ~eeril~. Thls ls r~adlly accompll~h~d by fll~r~lon through st~rll~ filcratlon membranes, prior to or foll~wing lyophlliz~tlon. ~KP ordinarlly ~ill be ~ored ln lyophllized form.
Th~ p~l of che MKP preparations eypically will be abouc from 6 ~o 8.

$h&rapeuclc MKP composltions generally ar~ placed ln~o a concalner havlng a sterlle access porc, for exa~ple, an lncravenous solucion bag or Ylal having a scopper plerceabl~ by a hypodermlc ln~ecelon needle.

MKP optionally is comblned wich or ~dministered in ~oncerc wlth other t~rombo- or arythro-potentlatlng ~gencs lncludlng lncerleukln-3, erythropoletin or ~ctlvin, and ls used wlch other convenclonal cherapies for thrombocy~openia.

Aneibodles to ~P ~r~ us~d w~th o~her co~sn~lonal ther~pies for thrombocyeemla, where they block ehe abnormPLly hl~h ~egakaryocy~opoiesis rates characterl~lng thLs typ~ of ~yndrome.

MKP preparations typically ~re admln~stered to animals exhlbicing chrombocytopenia.. 5he route ~f adminls~rat~on is in accord wlch known ~eehods, e.g. lncra~enous, ~ntraperlton~al, ln~ramuscular, intralesional infusion or in~ectlon, or by sus~ained release syscems ~s noted b~low. MKP ia ~d~ni~t~ed ~on~inuously by inusLon, ~lthough bDlus in~ ction ls ~ccep~ble.
.
S~i~able examples o~ sustained release preparatlons include sem~permeable polymer matrices in th~ form of shaped ~rticles, e.~.
~llms, or mLcrocapsules. Sustalned releas2 ~atrices lnclude polylactides ~U.S. Patent 3,773,919, EP 58,481), copolymers of L-gluta~$c acid and gamma ethyl-L-~l~tamate ~U. Sidman Q ~ 1983, i "B~opolymers~ 2~ 547-5563, poly ~2-hydroxy~thyl~ hacrylate) ~ 35 (R. Lang~r, Q~ ~l.. l9Bl, uJ. Bio~ed. Matsr. Res." 1~: 167~277 ~nd , R. L~n~r, 1982, Chem. T~ch.~ 98-105), echylenu vinyl ~ce~ate ~R. Lang~ ~1., Id.) or poly-D-(-)-3-~lydroxyb~t~ric acid (EP
133,988A~. Susealnad r~leas~ ~XP compos~ion~ also lnclud~
liposomally enernpped ~KP. Llpo¢omes containing MXP ar~ prepared .
by methods kno~n pe~ E 3,218,121A; Epstein e~ ~1., 1985, "Proc. ~ael. Acad. 5cl. USA~ 82: 368~-3692; ~wang ee ~., 1980, "Proc. Natl~ Acad. Scl. USA" 77: 4030-4034; EP 52322A; EP 36676A;
EP 88046A; EP 143949A; EP 142641A; Japanese patenc Appllcacion 83-118008; U.S. patants 4,485,045 and 4~544,545; and EP 102,324A.
Ordlnarlly th* liposomes ar~ of ~he ~mall (about 200~800 Angstroms) unllam~lar ~yp~ ln which tha lipld contanc ls greac~r than abo~t 30 mol. ~ oholastesol, ~ha s~l~cted proportlon being ad~u¢ted for the opclmal MKP therapy.

The ~ount o MXP to be employed therapeutically will depend, for example, upon che therape~tic ob~ectlves, the xoute of ad~iniscration, and the condlelon of the patient. Accordingly, it will b~ necessary for the tharapise to tlcer che dosage and modlfy tha rouce of administratlon as r~quirad to obcain th~ optimal therapeutic eEfect. Typically, th~ clinician will administer ~KP
~ntil a dosag~ i5 raached thae reestablishes a normal pLacela~
lavel of about 250 ~ 50 x 103/~1 o~ blood. The prograss o~ this therapy is ~asily monitored by convent'onal hematology assays or laboratory c~ll counes. Sultabl~ star~ing ther~peutic doses can he .
axtrapol~ted fro~ tha in Y~sEQ ~fficacy studia3 descrlbed abova.

Sui~able dlagnostic assays for ~P ~nd lts ~ntibodies ~re wall known P~X e. In addltion eo rh~ bioassay deseribed belo~, on~ may amploy competl~ve, sandwich and sterlc lnhibi~lon l~unoAsssy technique~. The compe~ltl~e ~nd sandwlch me~hods employ a phase separatlon step as sn in~egral part ~f ~h~ method ~hlls s~2ric lnhlbition sssays ~re conduct~d ln ~ 6ingla reac~lon mixture. Fundamenrally, the s~me procedures are used ~or th~ assay of MKP and or subs~ances th~t bind ~KP, ~lthough certsi~ ~ethods will b fa~ored dependlng upon the mol~cular weighc of the , . -23- t substanc~ b~ing ass~yed. Th~refora, tho substance to ba t~sted is refe~r~d to hereln as nn anslyt~, irr~speccive of its sta~us och~rwlsa as an nncigen or nnclbody, and protains whlch blnd co ehe analy~a are denomln~ted binding partners, whe~h~r chey be nnelbodi~s, cell surfaca receptors or ~ncigens.

Ar.alytlcal m~chods for ~KP or lcs anclbodl~s ~11 use one or more of the followLng ~eagents: labelled anilyra analogue, lmmobill~ad ~nalytu anslogua, labell~d blndLng partner, im~oblll7ed binding par~ner and steric conJu~atas. ~he labelled r~agencs ~lso aro known a~ ~tr~ersn.

The lsbel used i~ ~ny d~tectable functlonality which does not interfer~ with the binding of analyte and its binding p~s~ner.
Nu~erous labels ara known for use ln lmmunoassay, examples lncludlng enzymes 6uch as horser~dlsh peroxidase, radiolsotopes such as 14C and 131I, iluorophores such as rare earth chelates or fluorescein, stabla free radicals ~nd ehe like. Conventional method~ ar~ avallabla to covalently bind thase l~bels to proteins or polypepeidQs. Such bondlng me~hods ara su$~bla for usa with ~UCP Gr its antlbodi~s, all of which ara protelna~eous.

I~mobillzaelon of seag~nt~ i8 s~quired for certain assay methods. Immobilization eneall8 separating the binding partner fro~ any snalyte whlch remain~ fr~e i~ 601ution. Thi9 conventionally is ~ccomplished by either insolub~lizin~ the bind-lng partner or ~nalyt~ analogue before the sss~y procedure, as by adsorp~lQn ~o ~ er insoluble m~tsix os surface (Bennich t 1., U.S. 3,720,760), by covalent: Goupllng ~for exampl~ using glu~araldehyde cross-linklng), or by insolubill~ing ~he par~ner or - analogue lfterward, e.g., by l~munoprecipitation.

O~her ~ssay method~, knowQ BS competi~ve or sandwich assnys, are well establl5hed and widely used in th~ commerci~l d~agnostlcs industry. ~

.
,.

Comp~sltive assays rely on the abilley of a labelled analogu~ (th~ ~trae~r~) to eompece wlch th~ test sample ~nalyte for a limlted number of blnding 9iCQS on ~ eommon binding pnrtner. The blnding partnQr ~enerally l~ insolubilized befora or after the eompotitlon ~nd then the traeer and analyta bound to ehe bindlng partner ~re.separ~ted rom the unbound traeer and Bn~lyce. This separacion ls ~ccomplished by decanclng (where the bindlng par~ner w~s prelnsolubillzed) or by e~ntrlfugin~ (whar~ the blndlng par~ner was pr~elpitated ~frer th~ compatitive reactlon). Th~ amount of tast sampl~ analyt~ ia lnversQly proport~Dnal te ~he amoune of bound trac~r BS measured by eh~ amount o~ marker substsnee. Dose~
response eurves wlth known ~mD~nts of analyt~ are prepared ~nd eompared with the tasG results in ord~r to quantitatively determine the amount of analyta presenc in tha test s~mple, These assays ~ra called ELIS~ syste~s when enzymes are used as t~a detec~able markers.
.

Another speeies of eompetitive assay, called a ~homogeneous" assay, doe~ not requir~ a phase saparatlon. Her~, a eon~ugate of an ~nz~me with th~ analyte is prepared and used sueh that when ~n~i~Analyca binds to the ~nalyta tho presence of thQ
anti-analytQ modifi~s the ~nzyma aetiv~ty. In this oasQ, MXP or its i~unologleally aetiv~ frag~ants are eon~ugated with a bifunccion~l organic brid~s to an anzyme such a~ pe~oxldase.
Con~ugaees are selQcced for use wi~h anci-MKP so th~t blnding of the ancl-MKP lnhibi~s or potentiat~s ~he enzyme actlvity of the label. This me~hod ~ se ls widely practiced under the n~me of EMIT.
Steric con~u~ates axe used in sterlc hinder~nce ~eehods ~or homogeneous assay. Thasc con~ugAtes are synehesi~ed by cov21ently linking a low molecul~r wei~hc hap~en ~ B small snalyte so that ~ntibody to hapten subst~ntl~lly ls unable to blnd th~ con~u~a~u aC
th~ 5~m~ time ~s antl analyee. Under thi~ ~ssay procedure the ..

3~
-~5-analy~a prasene in tha e2sc s~mpl~ ~ill blnd anci-analyta, thereby allouing ancl-haptan ~o bind ~he con~ug~t~ resultlng ln B change in th~ character of the con~ugace hapt~n, e.g., a chan~e Ln fluorescence when thu hapcen ls ~ ~luorophore. Due to tha larg~
molacular welghc of MKP, homogen~ous or sterlc hinderance me~hods ~ro noe pr~erred for th~ de~ermlna~ion of t~P.

SandwLch ~ssays parcicularly are usefuL for che d~termlnatlon of MKP or ~KP ancibodies. In sequen~lal sandwLch assays ~n immobllized bindin~ pastner lq used to adsorb resc sampla analy~e, the test sample la ~moved ~9 by washlng, che bound analyc~ is u3ed to ~dsorb labell~d blndlng partn~r and bound material th~n separa~ed fro~ residual tracer. Th~ ~mounc of bo~nd trac~r 19 dirac ly proportional to test sa~ple analyte. In ~slmultaneous~ sandwich assays ehe ~e~c sample is noc ~epar2c~d befor~ addLng the labelled blnding partner. A s~quentlal ~andwlch assay using an anci-~KP monoclonal antibody as ono anclbody and a polyclonal anti-MKP anclbody as tha ocher ls useful in t~stln~
samples for MKP activity.
The for~goLng are merely exemplary dingnostic assays for MKP and antibodles. O~her me~hods now or hereafter developed for the d~Cermina~ion of these analytes ar~ included ~thin the ~copa hereof, including the bio~ssay dascrlbed lnfra.
: 25 Cova~en~ modifica~ons of the MKP ~olecule are lncluded wi~hln the scope hereof. Such ~odifi~ations are introduced intJ
ehe molecule by reacting targeted ~mino acid residues of ~he purified or crude proteln with an organic deriYa~izlng agen~ tha~
is cap~ble of reacting wlth selected slde c~ains or terminal residues. The resulting covalenc derl~atives are useful as immunogens or in progra~s directed at identifying residues i~por~ant for biologlcal actiYl q.

..
.' ' .

~ ~6 .

.. .
Cys~elnyl resldues mosc com~only are reacted with ~-haloacet~es (and corresponding amlnes), such as ch~oracetic acid or chloroace~amlds to gLve carboxymethyl or carboxamidomechyl derlvatives. Cysteinyl r~sidu~s also are derivac~z~d by reaceion S with bromotrifluoroace~on~, -bromo-~ ~S-imidozoyl) propionic acid, chloroac~tol phosphata, N-alkyl~al~imld~s, 3-nitro-2-pyrldyl disulflde, mathyl 2-pyrldyl disulfide, p-chloromercurlbenzoace, 2-chloro~rcuri-4-nicrophanol or chloro-7-ni~robenzo-2-oxa-1, 3-diazole.
Histidyl residues are derivaCized by reactlo~ with diethylpyrocarbonat~ ae pH 5.5 to 7.0; ~his ~gent is ~elatively speclfic for the hiseldyl ~ide ~hain. P~rabromoph~nacyl bro~ide ~lso is useful; ~he reaceion should be performed in O.lM sodium cacodylate ~t pH 6Ø

Lysinyl and amino termin~l residues are re~cted with succinlc or other carboxylic ~cid anhydrldes. Deriv~tization wi~h these agen~s has the effact of revarsin~ the charge of the lysinyl residues. Ocher uitAble re~genes for deriva~izing ~ ~mino-containing residuas lnclude imidoestars such as methyl picolinlmidate; pyridox~l phosphace; pyrldoxal; cl~roborohydrlde;
trlnitrobenz~nesulfonic acid; O-methyli~oure~; 2,4-pentanedione;
and trnns~minase-ca~lyzed re~ction with ~lyoxylaee.
Ar~lnyl residues are ~odified by reac~lon wlth one of sev~ral co~ven~ional rea~nts, ~mong them ph~nylglyoxal, 2,3-butanedione, 1,2-cyclohexanedlone, and ninhydrin. Deriva~Lzation of arginlne resldues requlres that the reac~ion be performed in ~0 alkaline conditions because of the hlgh pKa of the guanidine funceional group. Fusthqrmore, these ~e~gen~s may re~c~ wLth the groups oE lysine as well as the ~rglnlne - amino group.

The spec~fi~ ~odiflc~tion of eyrosyl resldues ~ se h~-~
been excen-sively s~udLed, wlch particular ~nteres~ in incrod~ring ,, .

~3~
. - ~7 ~pec~al labels lnto cyro~yl ~esldues by rascelon with aromaelc dlazonlum compounds or teeranltromethAn~. ~oae 'commonly, N-ac~eyli~ldizol and t~eranitromeehana ar~ u3ud to for~ 0-aceeyl tyrosyl specl4s and 3 nicro deriva~lve3, respecelv~ly. Tyrosyl S resldues are lodlna~ed using 12SI or 131I eo prupare labelled proceln~ for U52 ln radloimmunoasssy, che chlor.~mlne T method dascrlb~d abov~ bein~ suleabl~.

Carboxyl slda groups (aspartyl or glutamyl) ara selectively modlfled by re~ctlon wieh carbodli~ldcs (R'-N-C-N-R') such as 1-cyclohexyl-3-(2-morphollnyl-(4)-~thyl) carbodllmlde or 1-ethyl~3 (4-a~onia-4,4-dlmethylpantyl)-carbodiimid~. Furehermore, aspartyl and glutamyl r~sldue3 are ~on~er~ed to Asparaginyl and glueaminyl residues by renc~lon wieh smmonium lons.
Dsrivatization ~lth bifunctlonsl agenea ls useful ior prepar$~g intermolecular aggregates of tho prote$n with immunogenic polypeptides as well as fo~ cross-linking ehe proteln to a waeer insoluble support matrix or surface for use in the assay or ~fiinit~ purlficatlon of ~P antlbody. Commonly used cross-linklng agents lnclude l,l-bis(diazoacetyl)-2-phenyle~hane, Rlutaraldehyde, N-hydroxysurcini~lde es~rs, for ex~pl~ ~sters ~th 4-azidosalLcylLc acld, hom4bi~unctional imidoesters including disucclnimidyl esters such AS 3,3'-dithiobLs (sucrlnimldyl-proplon&ee), and blfunceionsl mal~imidea surh as bis-N-mal~i~ido-1,8-octane. Derlvaei~lng agenes such as methyl-3-[(p-azldo-phenyl)di~hlo) proplolmidac~ yield phoractiYa~abl~ inter~diates which are capable o~ formLng c~oss-links in the pr~sence of ligh~.
Alt0rns~1vely, r~activ~ w~t~r lnsolubl~ ~atrlces auch as cyanogen b~om$d~ ~c~lvated carbohydra~es and the reactiv~ substrates dsscribed ln ~.S. patents 3,969,287; 3,691,016; 4,195,128;

4,247,642; 4,229,537 ~nd 4,330,440 are employed for proteln lmmobilization.

3~
-2~- ~
, Clutamlnyl and s~paraglnyl r~sidues are fre~uen~ly d~smlda~ed ~o ~hs correspondlng gluc~myl and aspartyl resLdues.
Alternacively, thess resldu~s ar~ deamidarzd und~r mlldly acidic con~lcions. Ei~h~r form of chesa residu~s falls wiehin the scope of MKP.

Oeher modlXlcatlons include hydroxylacion of proline and lyslne, phosphorylacion of hydroxyl groups o~ seryl or threonyl res~dues, methyla~ion of the ~-amino groups of lysine, arglnine, and hystidlne side ch~ina (T.E. Cr~ighton, Pro~elns _Structure and , U.H. Freeman ~ Co., San Francisco pp 79-86 ~1983~), acetylacion of the N-termlnal ~mine snd, ln some ins~ances, amidation of the C~Carminal carboxyl.

Ancibod$es to MKP ~nerally ars ralsed in animals by .muleiplo 5c or ip lnjecelons of MXP and an adjuvant such as Freund's sd~uvant and/or tumor n~crosi6 ~actor. It may bs useful to con~ugats ~ha MKP to a protein ~h~ch ls lm~unogenlc ln ~he species to be lmmunized, e.g., keyhols llmpec hemocyanin, serum ~lbumln, bovin~ ehyroglobulLn, or soyb~an ~rypsln lnhibltor uslng a blfunctlonal or derivatizing agen~, or s~ample m~l~imldoben oyL
6ulfosuccinimlde ese~ (con~ugatlon throu~h cys~eine residues), N-hydsoxysuccinimlda (throuRh lysina r~sidue3), glu~araldehyde, succlnlc ~hydrid~, SOC12 or RlN C - NR. Alio, aggre~ating ~enrs such ~s alu~ may b~ used to enhance th~ immun9 response.

AnimaLs, ordinarily rabblts or mlr~, are 1mmuniæed ~gainst th0 immunogenic con~u~ates ~or derlvatlves, prefesably a glutaraldehyde or s~cclnlc anhydrlde con~ugate to soybean tryp5in inhibicor, by coLblnlng 1 m~ or 1 ~g of ~on3ugs~ ~for rabblts or mic~, reqpectlvely) wlth 3 volumes ~f Freund' 5 G~mplete ~djuvane - and lnj ect~ ng he solu~lon in~rader~lly se muleiple s~es with human TNF-~ ~e ~g/K~. Ona month l~er ~h~ ~nim~ls are boose~d with 1/5 to 1~10 the 4rlginal ~mount of conjug8t~ i~ Freund's ~ompl~te ad~uv~ne by subcut~n~ou~ in~ec~ion ~t muleiple Sit~S. 7 i'7~ :

"

to 14 daya l~cer animal~ ~re bl~d and ~he ser~ ssayed for an~i-MKP ti~r. ~nl~als ~re boosted uncll ~e ~lter pla~eaus.
Pr~f~rably, the ~nimal is boosead ~ich th~ con~ug~ce of rhe s~me MKP polyp~p~lde, but conJugated to a dl fe~enc proc~in and/or through 8 different cross~linking agent.

~onoclonal an~ibodies are prepared by recovering spleen cells from approprlate immunlzed animsls and lmmorcallzing the cells in conven~ional fashion, e.g. by fuslon with myeloma cells sr 13 by EB virus transfDrmation and screéning for clones expressing the dasLrsd ancibody.

4u~ntlflcaticn of ~plified MKP ganes in peripharal blood cells oi' leukemla pscients ls diagnosed by DNA dot blo~
lS hybridizaeion. For this purpose, se~isl dLlutions of dena~ured genomic DNa purif~ed by stsndard procedur~a from blood s~mple of tested patlenCs ar~ spotted onto nylon filter6 using appropria~e applicators. Puriflad MKP DNA ls bainB spotted ln parAllel for ~alibraelon. All samples also contaln denac~red carrier DNA sucn as salmon sper~ DNA to yi~ld ~ total constant ~moun~ of DNA per spot (usually, 2 ~g). Filcers are incubated for hybrldiza~ion with P32 MK~ DNA or ~lth biotinylated MKP DNA and washed under atringen~
conditions. Quantltles of genomic MKP DNA that hybrldize ~ith ~h~
~sbeled probe ln each pat$ent are than deter~ined ln values equlvalent to pg of ~KP DNA by optical densicomecry of exposed X-ray film (in the case of P32-labeled probe) or of con~ugated second fluoroprobe ~in the case of bioclnyla~ed MKP D~A). In a typiral diagnos~ic ~est, samples of up to 2 ~g of genomic DNA are : hybrldized for 16 hr. and washed ~5 deeailed above~
The principle o~ this ~ssay is the da~ection of putati~e pe~turba~ions a~ the level of the MKP ~ene and not ~h~ pro~ein produot. SL~ce ~oleoul~r changes at the level of genes and ohromoso~s appea~ to be link~d to the occu~rQnce ~ f congen$tal x~

syndro~es ~s well ~5 to reflact ~ha ~ev~rRness of csrtaLn diseaaes, tpis eyp~ of measursment can ndd novel ~linlcal lnfor~elon.

Exampl~ 1 A modificacion of Iscove's mechod tln Hematopoie~Lc Cell i DifEarencla~ion, ICN-UCLA SymposLa on Molecular and Cellular Biology, ~ol. 10, Colde ec ~1.. eds. [N~w YorX, Academy Press], pp 37~52) for growlng hamopoleslo stem cells ln methyl cellulose oulEure was ~sed comblned wlth I~D~ (Iscov~'s modiflcatlon of Dulbecco's ~edlu~) medlum. Bon~ marrow (BM) cells from 8-12 week-old C3H/HeJ (endo~oxin-resistant) mice were e~ected by syringe and grown ln the above m~diu~ ac a concsntratlon of 105 cellsJml ac 5 C2 and 37-C.

5~ PWM-Sp-C~ + 10~ ~EHI-CM constituted ths positiv~ control while 10~ WE~I-CM alone was the negativ~ control for ~11 subse~uent expariments. In a typlc~l ~xperime~t the number of ~egakaryocycic oolonies scored ~t seven days ln the posltive con~rol was 60~ml/pl~te whLle ~0/ml/plats were noted in the nag~tive ooncrol.

I.
A novel cDNA sequence was ldqntlfied in reve~se transcrlp~s of feeal gan~lio61d~ mRNA by ~ho use of BuCh~ probes. ~his consis~ed o~ thQ exact flrse 249 nuclaotides of BuCh~ cDNA ~Prody et ~l., 1987~ followed by tha -~eq~enca $ncludad ln ~igur~ l. The connectlng site ~herefore w~s ... TTT CTT GGA A~T CGC GGC ...
Phe Leu Gly Ila Arg Cly BuChE cDNA Clone 14 ~DNA
Note, however, that lt i~ posslbl~ th~t the ~T trlplet was coneribuCed ~lso from BuChE cDNA and is not part of clone 14.

Xenopus oocytes were ln~ected wlth ~ith~r clone 14 nonsense mRNA, clone 14 sense ~lA, Butyrylchollneseerssa (BuChE) ~RNA or ~arch medl~ (ehe m~diu~ used for growing the ooc~tes).-Th~ supernAtancs w~r~ harvested ~Eter 20-22 hours, dlluted with I~DM to 1:20, filter-steril~zed and diluted once more co 1:100 wich IMDM. Each o th~ supernatants was lntroduced at two conc~ntraclons, 1:20 and 1:100, ln~o culture medla concaining only WE~ C~. In each cas~ ~huy constltuccd 5~ of the total volume.
M~g~kAryocy~lc coloni~s wer~ scor~d aftex seven dsys of lncubation. J
Fac~ors Added No. of ~egaka~yoc~slc .
Colonl~s/mla Poslti~e Ne ati~ Inhlbltory ~b Resulcs 1 PWM ~ WEHI 63+4 17 2 WEHI 2~+3 R
3. 14 nonsense~ 1:20 54 4 4 ~ 100 36~8 2 4. 14 sense, 1:20 68~1 3 " , 1:100 64~9 4 5. BuCh~, 1:20 11~5 2 ~ 1:100 16~4 2 6. Barth, 1:20 91+4 4 ~ 1:100 6~8 4 a. Resulcs e~p~essed ln mesn +S~M.
b. N ~ No. of plat~s scored.

.
~0 Fro~ ~his ~xperi~en~ lt was e~lden~ that both th~ 14 sense clone and th~ Barth ~dlum produced resul~s slmllar to or superl3r to ~he synerg~scic ~ffect o~ PWM-SpCM ~nd U~ C~ while ~uChE was dis~incrly lnhlbltory o~ me~akaryocy~ic c~lnny grow~h. Thls latter ~lnding is i~ keeplng wlth the earlier results o~ Burstein ee ~.
(J. Cell Physiol. lQ~:201-208 tl980]) whlch demonst~sted ~hat lnhibitor to aceeylcholines~erase (AChE) scimulated megakaryocylic colony grow~h. The 14 nonsense clone gave intermedll~e va~ lles which were d~ficult to interpret. At this point, we coul~ oc distinguish betwzen the background e~fect of the endogensus o~ e ,.

D~

._ ' sQcr~tions in thc 3~rth medium ~nd ~hosa indu~ed by th~ 14 sense clon~ when in~ec~ad lnro the oOcyees. IC should bs noeed in thls respee~ ~hat mRNA mlcroin~ction r~ducas the endogenous level of proc~ln secretlon from oocytes (~. Soreq, unpublished). It is henee possibla that the clon~ 14 nonsense group ls the correc~
negn~ive control for the 14 sense clone.

In orde~ to d~ter~ine whethsr ehe ~urrounding cells of rhe oocy~es wer~ producLng factors ~c~iva in poten~iating megakaryocytic developmene the ~bove experimene w~s repea~ad using whol~ oocyte~ as be~or~ and in addition ~ocyt~s depleted of surrounding eells by ooll~genas~ tr~ cnc ~eollagsnaso Type I, Sigma). Both types of oocyc~s were ini~cced with 14 sens~ clone.
lS The results corroborated tho3e o~ Experi~en~ I which were repea~ed her~, including the 14 sensq and antlsense elonea, BuChE, and ehe ~areh-in~ected oocyt~ CM.
F~ctors added Positive Results N
14 sense, collagenase-tre~ted, 1:20 96+18 2 , 1:100 38 2 2 The number of meg~karyocytie colonies produced fro~ the C~
of the collagenase-trested oocyts~ ln;ected with 14 scns~ clone ~r~ equiv~lent to thoss producad fro~ the B~th medl~m and sllgh~ly hi~her than thosc ormcd ~rom the untr~tsd 14 fiens~
clone-ooeyces and the PWM ~ ~E~I controls. It w~s ~herefore ~enta~ively eoncluded ~h~t th~ surroundln~ cells of the oocy~es wer~ not con~r~buting any ~ddi~lonal grow~h f~G~ors to those produced by the B~rth-lnJ~eted oocy~e~ and/o~ the 14 sensa clone mRNA.

III.
In anothex at~e~pt to dlstinguish b~ween the possible ~ff~cts of native secreCl~ns o th~ ~oocyts and ~hose of the 14 ~ense clone, chc dilut~d supern~tant~ of un~rea~ed oocyces and ~ ' . ' - .

Bar~h-ln~acc~d oocyt~s wer~ treaced Xor 3' And 15' ~t 4~-C. The ~aclonalu here belng tha~ ~phlbian protei~s ar~ far Iore sensi~ive to cemperaturea such ~9 45-C than are ~m~slian prot~Lns and would posslbly be lnactlvaced. Care was taken to dilute the supernatanes before heA~ing so that concentrae~on affects would not ~ask those due to heaclng. The results ar~ aa tabulae~d:
Factors Added No. o Mggakaryocy~ic Colonle~/~l N
1. PUM ~ WEHI 63~3 17 2. ~II 28~3 8 3. Untres~ed Oocy~e S~cr~tlon 1:~0, 3' lncuba~ion ~2~4 2 1:20, 15~ ~ncubation 20~4 4 1:100, 15' ~ncubatlon 26~7 4. Barth-treat~d Oocyes ~0cretion 1:100, 3' incubation 36~4 2 1:20, 15' incubation 27~5 4 1:100, 15' incuba~ion 42~B . 4 It was concluded fro~ the above experi~ene that heating oocyte superna~an~s for eYen 3' causes a si~niflcant r~duc~ion ln megakaryocy~e~potentiating acti~lty. Both Bar~h-inJected and untreated oocyt~ ~ecretions coneain his actlvity in si~ilar ~mounts, de~onstrating ~hat ~he native oocyte factor is not a result o~ the in~ection i~self.

IY.
The e~fact of CM's from oocyte~ ln~ected with 14 S~nSB
clone or Bar~h medlum before and after colla~enase ereat~ent on ~he stlmulation of early mul~lpoten~ial hemopoietic precursors was tested. Cul~ures containing erythropoietin (EP0) and WE~I-3-C~
w~re used as ~h~ ~a~s of comparison for all experi~en~al - 35 co~bLna~ions. ~hls co~blnation of factors is synergistlc and isknown to produce a fair number of lar~ ~ixed colonies, l.e., - clon&l colonies con~a~ning seversl typec of hamopoietic c~lls. Inthis case, the predominant cell typ~ is erythroid, with smaller numbers of macropha~es, granulocytes and me~akaryocy~s.
. ~0 . , -- .

~', ' . .

~. , Two unlts of EPO/ml were used ln rhes~ experi~enes and ths ~ncubatlon perlod was for 8~10 dayY a~ 5~ C02 and 37-~.
Factors Addad ~ix~d C-M
Colonle3~1 Colonles/ml N
EPO ~ WEHI 19~1 22 2 EP0 ~ 14 sens~ 20 0 0 2 UEHI ~ 14 sense, 1:20 0 llL 2 10EPO ~ llEHI ~ 14 sensa 1:20 30~2 n.c. 2 1:100 17~3 n.c. 2 EPO + WEHI f L4 sens~
-collagenasa-ereated 1:20 34~3 n.c. 2 ~PO ~ UE~I ~ B~rth .-l:lOO 3~0.5 n. 2 It was concluded from thls sXper$mBnt tha~ C~'s fsom Bar~h and 14 sense clone contain ~ hamopoietic growth factor, that in the presence of both EPO and ~ C~ enhanc~s synergistically ~he produc~ion of mixed colsnies containing predominantly cells oE the erychroid lineage. Th2 effact W35 most pronounc~d a~ the 1:20 dilucion. ~era, as ln ths megakaryocyti~ experimanes, ~here was no diffsrence beeween oocy~ leh or wlthou~ surrounding c~lls. Once a~ain, i~ was not possibl~ to dlstlnguish between th~ ~ffec~ of the natlv~ oocyte secretlon ~nd that lnduced by the l~ct~on of thH 14 ~nse clons. From ~ha few Dbssrvat1 ons ~ad~, ic would sePm th~t eh~ 14 aens~ clona also had ~ pronounGcd sffect on tho ~umb~r of gr~nulocyt~^macrophag~ colon$es produced.

Morpholo~ical Seudies Cytosp$n preparationa were mlde of colonles app~arin~ in cultures p~oduced under varylng conditions, using a Shandon, ~odel 2 cytocentr$fuge and ~ay-Crunwald-Giemsa stain. Coloni~s wer~
~ found ~o contaln ~ large proportio~ ~up ~o 80~) of me~akaryocytes, - both early nnd ~acure, and a smaller proport~on of granulocyt~s, mac~ophages and plasmacyt~s. Early m~akaryocyt~s, cha~scterizsd 7~ ~
^35 by ~lngle, lar~2, lobed or kldney-shApsd nuclel, wsre much more ab~ndan~ than thc m~cure forms.

Using d1fferentlal lnter0rence contrast, ~n inceres~ing group of phenomena was obsarved, Ae glven seages of developmenc of the cultures (~t 6^10 days), tremendou~ amoaboid^like forms were seen to move^flow through the medLum, frequen~ly coverlng the whole ield of viqion. Th~sa free^form m~akaryocy~s, probably th~
~esul~ of fusion, ~ould wlthin minuces b~gin to pinch off lneo ~wo smaLler mega~aryocyts~. Ae tl~es, one could even observ~
si~ultaneoua pinching-of ~ two sap~ra~ poin~s, r~sultln~ in ~he for~aclon of ~hreo smaller megakaryocytes. Miniatu~o d~nsi~y gr~dients wera fcrm~d ln the cultu~s where ~umerous cell-like for~s in chu sam2 slze range ~3 plat~le~s (O.S - 1 mlcron) wer~
found at the bo~tom of th~ Petr1 dishes. A~ Lntermediate levels, sm~ll 3nd medium-sl~d m~gakaryocy~es would be concentra~ed and a~
the top floated the tremendous free-form megakaryocy~es.
Fraquen~ly, clusters of grsnulocytes would be a~tached to tha sura~e of thc in~ermediate-~zed me~akaryocy~es. Ic is possible tha~ th~ pLnching-off of smaller megakaryocytes from th~ larger free-forms may be par~ of the non-~ltotic, cytopl~smic matura~ion of megakaryQpoiesls, Conclusions 1. The synergist~c co~blna~lon of PUM-SpC~ ~ ~E~I-3-CM and W~I-3-CM alone can be ~sed ~s meg3karyocytP po~ensi2tors in ~
methyl cellulose-IMDM clonal assay and serYe as ~ b2sis of co~parLson of ~egakaryocytopo~eelc activity o unknown grow~h factors, The sourc~s of ~hes~ growth actors ~re ehe supernas~nts of X~nopus oocy~es induced by mRNAs of differe~t clones dev~loped from a cDNA library of hu~an newborn br~in 5tem.

2. Subsci~ution o~ ~h~ PWM-SpCM by ~he v~rious supern~tants ~f th~ sense elon 14 ~nd the Barth ~ediu~ used ~or oocy~ growth ~sulted in ~ simllar synergis~ic poteneiation 9f CFU-megJk.~ cyt~

i'7~

., .
gro~h. Thl5 lef~ in doubt, however, the sourca of ~ha synergiscic factors, 3. BuChE mRNA, ~h~n eranslaeed into protein by Xenopus .
oocyt~s, inhiblted the growth of CFU-m~gakaryocyt~s, beha~ing ln this fashlon behavin~ ln this fRshion inverseley eo lnhlbitors of AChE, a resul~ expacted in vlew of inhibitor studies and the cDnsiderable sequence homology bec~een hu~an AChE and BuCh.

4. Oocyt~ surrounding cells wer~ no~ r~sponsible ior the synerglstic acr~viry noted, as their removal by ~olla~enase had, if ~ny, ~n increascd effec~ on m~gakaryocyt~c colony formaeion.

S H~acing of eh~ supern~t~nts of untre~ted or Barth-in~ecced oocyees fo~ 3-15~ at 45-C caused a sign$ficant reduc~ion in megakaryocytic synergis~lc actlvl~y, lndicating thae a~ least some of the synergIs~ic sctivity may have been due to temperature-sensitive amphibian proteins.

6. Tha 6~me sup~rn~tln~s of ~he 14 clona and the Barth-in~ected oocy~es proved to hava 8 substantial 6ynerglstic ~ction with EPO ~nd UHI-CM on mlxed co~ony for~tion and CFU-C~
forma~lon. Thls activlty, li ~ound to be Associ~ted wich the s~e molecule as ~h~G produGing th~ syn~rgi6~1c ~ctlon of CFU-megakaryocyelc ~oloni~s, ~sy ba suggestlv0 of th~ ~ulti-CSF nature of ~ha ~ctlvl~y. In this ooneext, EPO msy h~ve ~n effect on ~ha develop~ent of the CFU-meglkaryocytlc colonles.

7. On the basii of prevlous i~ves~igations wlth WE~I-3-C~, lt c~n be ~ssumed that the action o~ ~EHI in the ~bove experi~ents ~s caused by the prese~ce of IL-3 in th~s CM.

~3~;7~At Exampl~ 2 , The study ln thi~ was und~rt~ken to inv~s~ig~. the _ ~ssump~ion that clone 14 and the human cholln~3t~r~s~ g~nei ~r~
physic~lly r~latQd and do not mer~ly repres~t an artifactu~l r~combLn~lon ~van~, ~nd th~t alt~red clone 14 gen~ ~rearr3nged, a~pllfled o~ mut~genlzsd ln ~ny oth~r w~y) are in~olved wi~h ~he hl~h plat~le~ coun~s ln ~ou~ my~lold leukemia pntlsllts.

17 cases o~ l~ukeml~ wer~ ~herefor~ looked ~t, using butyryl cholLna~ter~s~ (BuChE) cDNA and clone 14 cDNA as probes.
Th~s9 lnclud~d 9 ~s~s of ~ML, 5 c~s~s of AML~2 (char~t~r~zed by b~d pro~nosis and r~pid progress), on~ cas~ of A~OL ~cute monocytic leuk~ia), ona c~s~ of AMCL ~acue~ meg~karyocytic leukemia) ~nd one ~lth ~he potenti~l diagnosis of polycyc~mia ~ra, a semL-malignant syndrome implicated with abnormal ery~hropoiesis.
Mlnor modifications in the DNA restriction patrerns could be obse~ved ~n sever~l of these, but were difficul~ ~o interpret.
However, cwo of che AMLM2 blood DNA samples displayed ~onside~able ~mpllflcacion as foLlows:
An~pLif~,ed 1~
S~mpL~ No. 1070(AMLM2) Adr.(-L2)~AMLM2) HeaLChy ________________ Concro~
LAbeled oDNA Probe ~S
Clo~ 14 3.~ Kb++(P~U
2.4 Kb++~PW 2) 1.8 Kb+~(PVU
3.0 Kb~t~ n~III) 2.0 Kb+f(~_RI) 2.0 Kb~++(HindIII) 1.8 Kb~t(~indIII~

BuCh~cDNA 4.4 Kbt~(PW 2) ~ 3.0 Kb~t~W 2~ 3-0 xb~(PW 2) + 2.5 Kb+t~(PW 2) 2.5 Kb+(PW 2) 3.0 Kb++~ n~III) 3.0 Kb~t*~ç_RI) - - 49 4.2 Xb+SEc~RX) 3.9 Kb+(~QRI) . ,.

Claims (19)

1. A method for producing a megakaryocytopoeitin which comprises culturing oocytes and recovering the factor from the culture medium of the oocytes.

2. The factor produced by the method of claim 1.

3. A composition comprising call-free MKP.

4. A composition comprising human MKP free of human plasma or serum.

5. An isolate of MKP protein.

6. MKP free of polypeptides with which it is ordinarily associated.

7. An MKP variant having the sequence set forth in Fig. 1 wherein a predetermined amino acid is substituted, inserted or deleted, provided, however, that the variant is not an animal analogue of the Fig. 1 polypeptide or a naturally-occurring allele thereof.

8. An isolate of nucleic acid encoding MKP.

9. The isolate of claim 8 which is DNA free of 5' or 3' flanking

10. Nucleic acid having greater than about 10 bp and which is capable of hybridizing with the nucleic acid of claim 8 under stringent conditions.

11. A replicable vector comprising nucleic acid encoding MKP.

12. A host cell transformed with nucleic acid encoding MKP.
LC8x147.mdh

13. The host of claim 12 which is a mammalian cell.

14. A method for making MKP comprising culturing the host cell of claim 12 and recovering MKP from the culture modium of the host cell.

15. A method for diagnosis of leukemia comprising assaying a leukemia cell for transcription or translation of the MKP gene or for detection of structural changes such as deletions, rearrangements or amplification of such gene.

16. An antibody capable of binding MKP.

17. An MKP derivative labeled with a detectable moiety.

18. The tracer of Claim 17 where in the moiety is a radioisotope or enzyme.

19. A composition suitable for administration to a patient comprising a therapeutically effective amount of antibodies to MKP, capable of blocking its physiological activity, and an acceptable carrier.