Genentech, Inc.Genentech, Inc.

TrombopoetinThrombopoietin

Predloženi izum se nanaša na izolacijo, čiščenje in rekombinantno ali kemično sintezo proteinov, ki vplivajo na preživetje, proliferacijo, diferenciacijo ali zorenje hematopoetičnih celic, posebno trombocitnih predniških celic. Predloženi izum se posebno nanaša na kloniranje in ekspresijo nukleinskih kislin, ki kodirajo proteinski ligand, ki je sposoben vezanja in aktiviranja mpl, člana citokinske receptorske superdružine. Predloženi izum se nadalje nanaša na uporabo teh proteinov, samih ali v kombinaciji z drugimi citokini, za zdravljenje imunskih ali hematopoetičnih motenj, vključno trombocitopenije.The present invention relates to the isolation, purification and recombinant or chemical synthesis of proteins that affect the survival, proliferation, differentiation or maturation of hematopoietic cells, especially platelet progenitor cells. The present invention relates specifically to the cloning and expression of nucleic acids encoding a protein ligand capable of binding and activating mpl, a member of the cytokine receptor superfamily. The present invention further relates to the use of these proteins, alone or in combination with other cytokines, for the treatment of immune or hematopoietic disorders, including thrombocytopenia.

I. Hematopoetični sistemI. Hematopoietic system

Hematopoetični sistem ustvaijajo zrele visokospecializirane krvne celice, za katere je znano, da so potrebne za preživetje sesalcev. Te zrele celice vključujejo: eritrocite, specializirane za transport kisika in ogljikovega dioksida, limfocite T in B, odgovorne za celično in protitelesno posredovane imunske odzive, krvne ploščice ali trombocite, specializirane za tvorbo krvnih strdkov, in granulocite ter makrofage, specializirane kot odstranjevalce (scavengers) in kot akcesome celice, ki se borijo proti infekciji. Granulociti so nadalje razdeljeni v: nevtrofilce, eozinofilce, bazofilce in mastocite, specializirane celične tipe, ki imajo diskretne funkcije. Posebnost je v tem, da so vse specializirane zrele krvne celice izvedene samo iz enega, navadno primitivnega, celičnega tipa, imenovanega pluripotentna (ali totipotentna) matična celica, ki so jo prvotno ugotovili v kostnem mozgu (Dexter et al., Ann., Rev. Celi Biol., 3:423-441 [198η).The haematopoietic system enters mature, highly specialized blood cells that are known to be necessary for mammalian survival. These mature cells include: erythrocytes specialized in the transport of oxygen and carbon dioxide, T and B lymphocytes responsible for cell and antibody-mediated immune responses, blood platelets or platelets specialized in blood clot formation, and granulocytes and macrophages specialized as scavengers (scavengers) ) and as accessory cells that fight infection. Granulocytes are further subdivided into: neutrophils, eosinophils, basophils and mast cells, specialized cell types with discrete functions. The peculiarity is that all specialized mature blood cells are derived from only one, usually primitive, cell type called the pluripotent (or totipotent) stem cell, which was originally found in bone marrow (Dexter et al., Ann., Rev Celi Biol., 3: 423-441 [198η).

Zrele visokospecializirane krvne celice morajo nastajati v velikem številu kontinuirno skozi vse življenje sesalca. Precejšnji večini teh specializiranih krvnih celic je usojeno, da ostanejo funkcionalno aktivne le nekaj ur do nekaj tednov (Cronkite et al., Blood Celiš, 2:263-284 [1976]). Zaradi tega je potrebno kontinuirno obnavljanje zrelih krvnih celic, samih primitivnih matičnih celic, kot tudi vseh intermediatnih ali rodovno usmerjenih predniških celičnih linij, ki so med primitivnimi in zrelimi celicami, za vzdrževanje normalnih potreb po ustaljenem stanju krvnih celic sesalca.Mature, highly specialized blood cells must form in large numbers continuously throughout the mammalian life. Most of these specialized blood cells are destined to remain functionally active for only a few hours to a few weeks (Cronkite et al., Blood Cell, 2: 263-284 [1976]). This requires the continuous renewal of mature blood cells, the primitive stem cells themselves, as well as all intermediate or lineage progenitor cell lines that are between the primitive and mature cells, to maintain the normal steady-state needs of the mammalian blood cells.

V osredju hematopoetičnega sistema so pluripotentne matične celice. Teh celic je po številu relativno malo in so izpostavljene samoobnovitvi s proliferacijo, tako da nastanejo hčerinske matične celice ali pa se transformirajo v vrsti diferenciacijskih stopenj v vedno bolj zrele rodovno omejene predniške celice, ki končno tvorijo visokospecializirane zrele krvne celice.At the heart of the hematopoietic system are pluripotent stem cells. These cells are relatively small in number and undergo proliferative self-renewal to produce daughter stem cells or transform in a series of differentiation stages into increasingly mature, gene-restricted ancestral cells, ultimately forming highly specialized mature blood cells.

Nekatere multipotentne predniške celice, ki so npr. imenovane CFC-Mix, izvedene iz matičnih celic, so izpostavljene proliferaciji (samoobnovitvi) in razvoju, da nastanejo kolonije, ki vsebujejo vse različne mieloidne celice; eritrocite, nevtrofilce, megakariocite (predhodniki trombocitov), makrofage, bazofilce, eozinofilce in mastocite. Druge predniške celice limfoidnega rodu so izpostavljene proliferaciji in razvoju v celice T in B.Some multipotent ancestral cells, e.g. called stem cell-derived CFC-Mix are exposed to proliferation (self-renewal) and development to form colonies containing all different myeloid cells; erythrocytes, neutrophils, megakaryocytes (platelet precursors), macrophages, basophils, eosinophils and mast cells. Other ancestral cells of the lymphoid genus are exposed to proliferation and development into T and B cells.

Poleg tega je med predniškimi celicami CFC-Mix in mieloidnimi celicami druga vrsta predniških celic z intermediatno usmerjenostjo za njihovo potomstvo. Te rodovno omejene predniške celice so razvrščene na osnovi potomstva, ki ga ustvaijajo. Znani neposredni predhodniki mieloidnih celic so: eritroidno kolonijo tvoreče enote (CFU-E) za eritrocite, granulocitno/makrofagno kolonijo tvoreče celice (GM-CFC) za nevtrofilce in makrofage, megakariocitno kolonijo tvoreče celice (Meg-CFC) za megakariocite, eozinofilno kolonijo tvoreče celice (Eos-CFC) za eozinofilce in bazofilno kolonijo tvoreče celice (Bas-CFC) za mastocite. Druge intermediatne predhodniške celice med pluripotentnimi matičnimi celicami in zrelimi krvnimi celicami so znane (glej spodaj) ali pa bo verjetno dokazano, da imajo različne stopnje rodovne omejitve in sposobnosti za samoobnovitev.In addition, there is another type of ancestral cell with intermediate orientation for their progeny between CFC-Mix and myeloid cells. These gendered ancestral cells are sorted on the basis of the offspring they consume. Known direct myeloid cell precursors are: erythroid colony forming unit (CFU-E) for erythrocytes, granulocytic / macrophage colony forming cell (GM-CFC) for neutrophils and macrophages, megakaryocytic colony forming cell (Meg-CFC) for megakaryophyte cytochlorocytic cells (Eos-CFC) for eosinophils and basophilic colony-forming cells (Bas-CFC) for mast cells. Other intermediate precursor cells between pluripotent stem cells and mature blood cells are known (see below) or are likely to be shown to have varying degrees of gendered restriction and self-renewal capacity.

Osnovni princip normalnega hematopoetičnega celičnega sistema je zmanjšana sposobnost za samoobnovitev, ker se multipotentnost izgubi, pridobi pa se rodovna omejitev in zrelost. Torej je na eni strani hematopoetičnega celičnega spektra pluripotentna matična celica, ki ima sposobnost za samoobnovitev in diferenciacijo v vse vrste predniških celic, ki so rodovno specifično usmerjene. Ta sposobnost je osnova za terapijo transplantacije kostnega mozga, kjer primitivne matične celice repopulirajo celoten hematopoetični celični sistem. Na drugi strani spektra pa so visoko rodovno omejeni predniki in njihovo potomstvo, ki so izgubili sposobnost za samoobnovitev, pridobili pa zrelo funkcionalno aktivnost.The basic principle of a normal hematopoietic cellular system is a reduced capacity for self-renewal as multipotency is lost and gender restriction and maturity are acquired. So, on the one hand, the hematopoietic cell spectrum is a pluripotent stem cell that has the capacity for self-renewal and differentiation into all types of ancestral cells that are gender-specific. This ability is the basis for bone marrow transplantation therapy, where primitive stem cells repopulate the entire hematopoietic cell system. On the other side of the spectrum are highly ancestral restricted ancestors and their offspring, who have lost the ability to self-renew and gain mature functional activity.

Proliferacijo in razvoj matičnih celic in rodovno omejenih predniških celic skrbno nadzorujejo različni hematopoetični rastni faktorji ali citokini. Vloga teh rastnih faktorjev in vivo je kompleksna in ni popolnoma razumljiva. Nekateri rastni faktorji, kot npr. interlevkin-3 (IL-3), so sposobni, da stimulirajo tako multipotentne matične celice kot tudi usmerjene predniške celice različnih rodov, vključno npr. megakariocite. Za druge faktorje, kot je granulocitno/makrofagno kolonijo stimulirajoči faktor (GM-CSF), so prvotno mislili, da so omejeni v svojem delovanju na GM-CFC. Kasneje pa so ugotovili, da GM-CSF med drugim vpliva tudi na proliferacijo in razvoj megakariocitov. Za IL-3 in GM-CSF so torej ugotovili, da imata prekrivajoče biološke aktivnosti, čeprav z različno zmogljivostjo. Zadnje čase pa ugotavljajo, da tako interlevkin-6 (EL-6) kot tudi interievkin-11 (IL-11), čeprav sama nimata vidnega vpliva na tvorbo meg- kolonije, delujeta sinergistično z IL-3, tako da je zorenje megakariocitov stimulirano (Yonemura et al., Exp. Hematol., 20:10111016 [1992]).The proliferation and development of stem cells and progenitor-restricted progenitor cells is carefully controlled by various hematopoietic growth factors or cytokines. The role of these growth factors in vivo is complex and not completely understood. Some growth factors, such as interleukin-3 (IL-3), are capable of stimulating both multipotent stem cells and targeted ancestral cells of different genera, including e.g. megakaryocytes. Other factors, such as granulocyte / macrophage colony stimulating factor (GM-CSF), were originally thought to be limited in their action on GM-CFC. However, GM-CSF was later found to affect, among other things, the proliferation and development of megakaryocytes. IL-3 and GM-CSF were therefore found to have overlapping biological activities, albeit with different capacities. Recently, however, both interleukin-6 (EL-6) and interievkin-11 (IL-11), although they have no apparent effect on megacolonium formation, interact synergistically with IL-3, so that megakaryocyte maturation is stimulated (Yonemura et al., Exp. Hematol., 20: 10111016 [1992]).

Hematopoetični rastni faktorji torej lahko vplivajo na rast in diferenciacijo enega ali več rodov, lahko se prekrivajo z drugimi rastnimi faktorji pri vplivanju na posamezno predniško celično linijo, ali pa lahko delujejo sinergistično z drugimi faktorji.Hematopoietic growth factors may therefore affect the growth and differentiation of one or more genera, may overlap with other growth factors when affecting a single ancestral cell line, or may act synergistically with other factors.

Razvidno je tudi, da imajo hematopoetični rastni faktorji lahko vpliv na različnih stopnjah celičnega razvoja, od totipotentne matične celice preko raznih usmerjenih rodovno omejenih prednikov do zrele krvne celice. Za eritropoetin (epo) je npr. ugotovljeno, da pospešuje le proliferacijo zrelih eritroidnih predniških celic. Za IL-3 je ugotovljeno, da izraža svoj učinek prej, pri čemer vpliva na primitivne matične celice in intermediatne rodovno omejene predniške celice. Drugi rastni faktorji, kot npr. faktor matične celice (SCF), lahko vplivajo celo na razvoj bolj primitivnih celic.It has also been shown that hematopoietic growth factors can have an effect on various stages of cellular development, from the totipotent stem cell through various directed generically restricted ancestors to mature blood cells. For erythropoietin (epo), e.g. found to accelerate only the proliferation of mature erythroid progenitors. IL-3 is found to exert its effect earlier, affecting primitive stem cells and intermediate gendered restricted progenitors. Other growth factors such as stem cell factor (SCF) can even affect the development of more primitive cells.

Iz navedenega sledi, da bi bili novi hematopoetični rastni faktorji, ki vplivajo na preživetje, proliferacijo, diferenciacijo ali zorenje vseh krvnih celic ali njihovih predhodnikov, koristni, posebno kot pomoč pri ponovni vzpostavitvi oslabljenega hematopoetičnega sistema, povzročenega z boleznijo, ali po obsevanjih, ali kemoterapiji.It follows that novel hematopoietic growth factors affecting the survival, proliferation, differentiation or maturation of all blood cells or their precursors would be useful, especially in helping to restore the disease-induced or post-irradiated hematopoietic system, or chemotherapy.

II. Megakariocitopoeza - nastajanje trombocitovII. Megakaryocytopoiesis - platelet formation

Regulacija megakariocitopoeze in nastajanje trombocitov sta prikazana v Mazur, Exp. Hematol., 15:248 [1987] in Hoffman, Blood, 74:1196-1212 [1989]. Na kratko, pluripotentne matične celice kostnega mozga se diferencirajo v megakariocitne, eritrocitne in mielocitne celične linije. Verjetno obstaja hierarhija usmerjenih megakariocitnih predniških celic med matičnimi celicami in megakariociti. Vsaj trije razredi megakariocitnih predniških celic so identificirani, in sicer: izbruh tvoreča enota megakariocitov (BFU-MK), kolonijo tvoreča enota megakariocitov (CFUMK) in megakariocitne predniške celice z majhno gostoto (LD-CFU-MK). Samo megakariocitno zorenje je nadaljevanje razvoja, ki se loči v stopnje, ki temeljijo na standardnih morfoloških merilih. Najzgodnejši spoznani člani megakariocitne (MK ali meg) družine so megakarioblasti. Te celice imajo na začetku premer 20 do 30 μ-m, bazofilno citoplazmo in rahlo nepravilno jedro s prostim, nekoliko retikulamim kromatinom, in več nukleoli. Kasneje pa so megakarioblasti lahko vsebovali do 32 jeder (ploiploid), vendar pa je citoplazma ostala redka in nezrela. Pri nadaljnjem zorenju pa je postalo jedro bolj lobulamo in piknotično, citoplazma se je količinsko povečala in postala bolj acidofilna in granulama. Večina zrelih celic te družine lahko daje videz sproščanja trombocitov v njihovi periferiji. Normalno je manj kot 10 % megakariocitov v blastni stopnji, več kot 50 % pa je zrelih. Arbitrarne morfološke klasifikacije, ki se navadno uporabljajo za megakariocitno vrsto, so: mekarioblast za najzgodnejšo obliko; promegakariocit ali bazofilni megakariocit za intermediatno obliko; in zreli (acidofilni, granulami ali trombocite tvoreči) megakariocit za pozne oblike. Zreli megakariociti razširjajo filamente citoplazme v sinusoidne prostore, kjer se ločijo in fragmentirajo v individualne trombocite (Williams et al., Hematology, 1972).Regulation of megakaryocytopoiesis and platelet production are shown in Mazur, Exp. Hematol., 15: 248 [1987] and Hoffman, Blood, 74: 1196-1212 [1989]. Briefly, bone marrow pluripotent stem cells differentiate into megakaryocytic, erythrocyte, and myelocytic cell lines. There is probably a hierarchy of directed megakaryocyte progenitor cells between stem cells and megakaryocytes. At least three classes of megakaryocyte progenitor cells have been identified as follows: outbreak megakaryocyte forming unit (BFU-MK), colony forming megakaryocyte unit (CFUMK), and low density megakaryocyte progenitor cell (LD-CFU-MK). Megakaryocyte maturation alone is a continuation of development, which is separated into stages based on standard morphological criteria. The earliest known members of the megakaryocyte (MK or meg) family are megakaryoblasts. These cells initially have a diameter of 20 to 30 μ-m, a basophilic cytoplasm, and a slightly irregular nucleus with free, somewhat reticulum chromatin, and several nucleoli. Later, megakaryoblasts were able to contain up to 32 nuclei (ploiploid), but cytoplasm remained sparse and immature. On further maturation, however, the nucleus became more lobular and pycnotic, the cytoplasm increased quantitatively and became more acidophilic and granular. Most mature cells in this family can give the appearance of releasing platelets in their periphery. Normally, less than 10% of megakaryocytes are in the blast stage, and more than 50% are mature. The arbitrary morphological classifications commonly used for megakaryocytic species are: mecaryoblast for the earliest form; promegakaryocyte or basophilic megakaryocyte for intermediate form; and mature (acidophilic, granuloma, or platelet-forming) megakaryocytes for late forms. Mature megakaryocytes extend cytoplasmic filaments into sinusoidal spaces, where they separate and fragment into individual platelets (Williams et al., Hematology, 1972).

Za megakariocitopoezo domnevajo, da vključuje različne regulatome faktorje (Williams et al., Br. J. Haematol., 52:173 [1982] in Williams et al., J. Celi Physiol., 110:101 [1982]). Za zgodnjo stopnjo megakariocitopoeze domnevajo, da je mitotična in vpliva na celično proliferacijo in iniciacijo kolonije iz CFU-MK, vendar nanjo ne vpliva število trombocitov (Burstein et al., J. Celi Physiol., 109:333 [1981] in Kimura et al., Exp. Hematol., 13:1048 [1985]). Kasnejša stopnja zorenja je nemitotična in vključuje nuklearno poliploidizacijo in citoplazemsko zorenje in je verjetno uravnavana s povratnim mehanizmom s številom perifernih trombocitov (Odeli et al., Blood, 48:765 [1976] in Ebbe et al., Blood, 32:787 [1968]).Megakaryocytopoiesis is thought to involve various regulating factors (Williams et al., Br. J. Haematol., 52: 173 [1982] and Williams et al., J. Celi Physiol., 110: 101 [1982]). Early-stage megakaryocytopoiesis is thought to be mitotic and affect cell proliferation and colony initiation from CFU-MK, but is not affected by platelet counts (Burstein et al. J. Celi Physiol. 109: 333 [1981] and Kimura et al. ., Exp. Hematol., 13: 1048 [1985]). The later stage of maturation is non-mitotic and involves nuclear polyploidization and cytoplasmic maturation and is probably regulated by a feedback mechanism by peripheral platelet counts (Odeli et al., Blood, 48: 765 [1976] and Ebbe et al., Blood, 32: 787 [1968 ]).

O eksistenci jasnega in specifično megakariocitno kolonijo stimulirajočega faktorja (MK-CSF) obstajajo razprave (Mazur, Exp. Hematol., 15:340-350 [1987]). Večina avtorjev verjame, da naj bi postopek, ki je tako nujno potreben za preživetje, kot je nastajanje trombocitov, uravnavali citokini, izključno odgovorni za ta postopek. Hipoteza, da obstajajo megakariocitno/trombocitno specifični citokini, je dala osnovo za več kot 30-letne raziskave, vendar pa do sedaj še niso očistili, sekvencirali in določili s poskusi nobenega takega citokina, kot je edinstveni MK-CSF (TPO).The existence of a clear and specific megakaryocyte colony stimulating factor (MK-CSF) has been debated (Mazur, Exp. Hematol., 15: 340-350 [1987]). Most authors believe that a process as essential to survival as platelet production should be regulated by cytokines solely responsible for this process. The hypothesis that megakaryocyte / platelet-specific cytokines exist has provided the basis for more than 30 years of research, but so far they have not been purified, sequenced, and determined by testing any such cytokine such as the unique MK-CSF (TPO).

Čeprav je navedeno, da so bili MK-CSF delno očiščeni na osnovi eksperimentalno ustvarjene trombocitopenije (Hill et al., Exp. Hematol., 14:752 [1986]), iz kondicioniranega medija humanih embrionalnih ledvic [CM] (McDonald et al., J. Lab. Ciin. Med., 85:59 [1975]) ter v človeku z aplastično anemijo in idiopatično trombocitopenično purpuro urinamih ekstraktov (Kawakita et al., Blood, 6:556 [1983]) in plazme (Hoffman et al., J. Ciin. Invest., 75:1174 [1985]), pa je njihovo fiziološko delovanje še neznano v večini primerov.Although stated that MK-CSFs were partially purified based on experimentally generated thrombocytopenia (Hill et al., Exp. Hematol. 14: 752 [1986]), from the conditioned human embryonic kidney medium [CM] (McDonald et al. , J. Lab. Ciin. Med., 85:59 [1975]) and in humans with aplastic anemia and idiopathic thrombocytopenic purpura urine extracts (Kawakita et al., Blood, 6: 556 [1983]) and plasma (Hoffman et al. J. Ciin. Invest. 75: 1174 [1985], however, their physiological function is still unknown in most cases.

Kondicioniran medij vraničnih celic, aktiviranih s pokeweed mitogenom (PWMSpCM), in murino mielomonocitno celično linijo WEHI-3 (WEHI-3CM) so uporabili kot megakariocitne potenciatorje. PWM-SpCM vsebuje faktorje, ki povečujejo rast CFU-MK (Metcalf et al., Pro. Natl. Acad. Sci., ZDA, 72:1744-1748 [1975]; Quesenberry et al., Blood, 65:214 [1985]; in Iscove, N.N., v Hematopoietic Celi Differentiation, ICN-UCLA Symposia on Molecular and Cellular Biology, Vol. 10, Golde et al., izd. [New York, Academy Press] str. 37-52 [1978]), pri čemer je eden od teh interlevkin-3 (IL-3), večrodovno kolonijo stimulirajoči faktor (multi-CSF [Burstein, Blood Celiš, 11:469 [1986]). Drugi faktorji v tem mediju niso bili identificirani in izolirani. WEHI-3 je murina mielomonocitična celična linija, ki izloča relativno velike količine IL-3 in manjše količine GM-CSF. Za IL-3 so ugotovili, da omogoča rast širokega območja hematopoetičnih celic (Ihle et al., J. Immunol., 13:282 [1983]). Za IL-3 so tudi ugotovili, daje v sinergiji z mnogimi znanimi hematopoetičnimi hormoni ali rastnimi faktorji (Barteimez et al., J. Celi Physiol., 122:362-369 [1985] in Warren et al., Celi, 46:667-674 [1988]), ki vključujejo tako eritropoetin (EPO) kot tudi interlevkin-1 (IL-1), pri indukciji zelo zgodnjih multipotentnih predhodnikov in tvorbi zelo velikih mešanih hematopoetičnih kolonij.Conditioned medium of pokeweed mitogen-activated spleen cells (PWMSpCM) and murine myelomonocytic cell line WEHI-3 (WEHI-3CM) were used as megakaryocyte potentiators. PWM-SpCM contains CFU-MK growth enhancing factors (Metcalf et al., Pro. Natl. Acad. Sci., USA, 72: 1744-1748 [1975]; Quesenberry et al., Blood, 65: 214 [1985 ]; and Iscove, N.N., in Hematopoietic Whole Differentiation, ICN-UCLA Symposia on Molecular and Cellular Biology, Vol 10, Golde et al., ed. [New York, Academy Press] pp. 37-52 [1978]), one of these being interleukin-3 (IL-3), a multifamily colony stimulating factor (multi-CSF [Burstein, Blood Celish, 11: 469 [1986]). Other factors were not identified and isolated in this medium. WEHI-3 is a murine myelomonocytic cell line that secretes relatively large amounts of IL-3 and smaller amounts of GM-CSF. IL-3 has been found to allow the growth of a wide range of hematopoietic cells (Ihle et al. J. Immunol. 13: 282 [1983]). IL-3 has also been found to synergize with many known hematopoietic hormones or growth factors (Barteimez et al., J. Celi Physiol., 122: 362-369 [1985] and Warren et al., Celi, 46: 667 -674 [1988]), involving both erythropoietin (EPO) and interleukin-1 (IL-1), in the induction of very early multipotent precursors and the formation of very large mixed hematopoietic colonies.

Druge vire megakariocitnih potenciatorjev so ugotovili v kondicioniranih medijih murinih pljuč, kosti, makrofagnih celičnih linij, peritonealnih eksudatnih celic in humanih embrionalnih ledvičnih celic. Kljub nekaterim spornim podatkom (Mazur, Exp. HematoL, 15:340-350 [1987]) je nekaj dokazov (Geissler et al., Br. J. HaematoL, 60:233-238 [1985]), da imajo aktivirani limfociti T večjo vlogo v megakariocitopoezi kot pa monociti. Te ugotovitve navajajo k temu, da so lahko sekrecije aktiviranih limfocitov T, kot npr. interlevkinov, regulatorni faktorji pri razvoju MK (Geissler et al., Exp. Hematol., 15:845-853 [1987]). Iz številnih študij megakariocitopoeze z očiščenim eritropoetinom EPO (Vainchenker et al., Blood, 54:940 [1979]); McLeod et al., Nature, 261:492-4 [1976]); in Williams et al., Exp. Hematol., 12:734 [1984]) je razvidno, da ima ta hormon pospeševalni učinek na tvorbo kolonije MK. To je bilo tudi prikazano tako v kulturah brez sexuma kot tudi v tistih, ki ga vsebujejo, in v odsotnosti akcesomih celic (Williams et al., Exp. Hematol., 12:734 [1984]). Za EPO domnevajo, da je vključen bolj v eno- in dvocelični stadij megakariocitopoeze v nasprotju z učinki PWM-SpCM, ki je vključen v štiricelični stadij megakariocitnega razvoja. Interakcija vseh teh faktorjev tako v zgornji kot tudi pozni fazi megakariocitnega razvoja še vedno ostaja nepojasnjena.Other sources of megakaryocyte potentiators have been identified in conditioned media of murine lungs, bones, macrophage cell lines, peritoneal exudate cells, and human embryonic kidney cells. Despite some controversial data (Mazur, Exp. HematoL, 15: 340-350 [1987]), there is some evidence (Geissler et al., Br. J. HaematoL, 60: 233-238 [1985]) that activated T lymphocytes a greater role in megakaryocytopoiesis than monocytes. These findings suggest that secretions of activated T lymphocytes such as e.g. interleukins, regulatory factors in MK development (Geissler et al. Exp. Hematol. 15: 845-853 [1987]). From numerous studies of megakaryocytopoiesis with purified erythropoietin EPO (Vainchenker et al., Blood, 54: 940 [1979]); McLeod et al., Nature, 261: 492-4 [1976]; and Williams et al., Exp. Hematol., 12: 734 [1984]) shows that this hormone has an accelerating effect on MK colony formation. This has also been shown in cultures without sexum as well as in those containing it and in the absence of cell accesomes (Williams et al. Exp. Hematol. 12: 734 [1984]). The EPO is thought to be involved more in the one- and two-cell stage of megakaryocytopoiesis, in contrast to the effects of PWM-SpCM, which is involved in the four-cell stage of megakaryocytic development. The interaction of all these factors in both the upper and late stages of megakaryocytic development remains unclear.

Podatki iz različnih laboratorijev navajajo na to, da so edini večrodovni faktorji, ki imajo individualno MK-kolonijo stimulirajočo aktivnost: GM-CSF in IL-3 ter v manjši meri B-celični stimulirajoči faktor IL-6 (Ikebuchi et al., Proč. Natl. Acad. Sci. ZDA, 84:9035 [1987]). Nedavno so različni avtorji poročali, da IL-11 in levkemični inhibitomi faktor (LIF) delujeta sinergistično z IL-3 pri povečevanju megakariocitne velikosti in ploidije (Yonemura et al., British Journal of Hematology, 84:16-23 [1993]; Burstein et al., J. Celi. Physiol., 153:305-312 [1992]; Metcalf et al., Blood, 76:50-56 [1990]; Metcalf et al., Blood, 77:2150-2153 [1991]; Bruno et al., Exp. Hematol., 19:378-381 [1991]; in Yonemura et al., Exp. Hematol., 20:1011-1016 [1992]).Data from various laboratories indicate that the only multifamily factors that have individual MK colony stimulating activity are: GM-CSF and IL-3, and to a lesser extent B-cell stimulating factor IL-6 (Ikebuchi et al., Ref. Nat. Acad. Sci. USA 84: 9035 [1987]. Recently, various authors have reported that IL-11 and leukemic inhibitory factor (LIF) act synergistically with IL-3 in increasing megakaryocytic size and ploidy (Yonemura et al., British Journal of Hematology, 84: 16-23 [1993]; Burstein Physiol., 153: 305-312 [1992]; Metcalf et al., Blood, 76: 50-56 [1990]; Metcalf et al., Blood, 77: 2150-2153 [1991 ]; Bruno et al., Exp. Hematol., 19: 378-381 [1991]; and Yonemura et al., Exp. Hematol., 20: 1011-1016 [1992].

Drugi zanimivi dokumenti vključujejo: Eppstein et al., US Patent št. 4,962,091; Chong, US Patent št. 4,879,111; Femandes et al., US Patent št. 4,604,377; Wissler et al., US Patent št. 4,512,971; Gottlieb, US Patent št. 4,468,379; Bennett et al., US Patent št. 5,215,895; Kogan et al., US Patent št. 5,250,732; Kimura et al., Eur. J. Immunol. 20(9):1927-1931 [1990]; Secor et al., J. Of Immunol., 144(4):1484-1489 [1990]; Warren et al., J. of Immunol., 140(1):94-99 [1988]; Warren et al., Exp. Hematol., 17(11):1095-1099 [1989]; Bruno et al., Exp. Hematol., 17(10):1038-1043 [1989]; Tanikawa et al., Exp. Hematol., 17(8):883-888 [1989]; Koike et al., Blood, 75(12):2286-2291 [1990]; Lotem, Blood, 75(5):1545-1551 [1989]; Rennick et al., Blood, 73(7):1828-1835 [1989]; in Outterbuck et al., Blood, 73(6):1504-1512 [1989].Other interesting documents include: Eppstein et al., U.S. Pat. 4,962,091; Chong, US Pat. 4,879,111; Femandes et al., U.S. Pat. 4,604,377; Wissler et al., U.S. Pat. 4,512,971; Gottlieb, US Pat. 4,468,379; Bennett et al., U.S. Pat. 5,215,895; Kogan et al., U.S. Pat. 5,250,732; Kimura et al., Eur. J. Immunol. 20 (9): 1927-1931 [1990]; Secor et al., J. Of Immunol., 144 (4): 1484-1489 [1990]; Warren et al., J. of Immunol., 140 (1): 94-99 [1988]; Warren et al., Exp. Hematol., 17 (11): 1095-1099 [1989]; Bruno et al., Exp. Hematol., 17 (10): 1038-1043 [1989]; Tanikawa et al., Exp. Hematol., 17 (8): 883-888 [1989]; Koike et al., Blood, 75 (12): 2286-2291 [1990]; Lotem, Blood, 75 (5): 1545-1551 [1989]; Rennick et al., Blood, 73 (7): 1828-1835 [1989]; and Outterbuck et al., Blood, 73 (6): 1504-1512 [1989].

III. TrombocitopeniiaIII. Thrombocytopenia

Trombociti so kritični elementi mehanizma strjevanja krvi. Do deplecije krožečega nivoja trombocitov, imenovanega trombocitopenija, pride pri različnih kliničnih stanjih in motnjah. Trombocitopenija je navadno definirana kot število trombocitov, kije pod 150 x IO⁹/!. Glavne vzroke trombocitopenije lahko na široko razdelimo v tri kategorije na osnovi življenjske dobe trombocitov, in sicer: (1) poslabšano nastajanje trombocitov v kostnem mozgu, (2) sekvestracija trombocitov v vranici (splenomegalija) in (3) povečana destrukcija trombocitov v perifernem kroženju (npr. avtoimunska trombocitopenija ali kemijska in obsevalna terapija). Poleg tega se v pacientnih, ki prejemajo velike volumne krvnih produktov, revnih s trombociti, s hitrim dajanjem lahko razvije trombocitopenija zaradi razredčitve.Platelets are critical elements of the blood clotting mechanism. Depression of circulating platelet levels, called thrombocytopenia, occurs in various clinical conditions and disorders. Thrombocytopenia is usually defined as the number of platelets below 150 x IO ⁹ / !. The major causes of thrombocytopenia can be broadly divided into three categories based on platelet life, namely: (1) impaired bone marrow formation, (2) sequestration of platelets in the spleen (splenomegaly), and (3) increased platelet destruction in the peripheral circulation e.g., autoimmune thrombocytopenia or chemical and radiation therapy). In addition, thrombocytopenia due to dilution may develop rapidly in patients receiving large volumes of platelet-poor blood products.

Manifestacije kliničnih krvavitev pri trombocitopeniji so odvisne od resnosti trombocitopenije, njenih vzrokov in možnih združenih koagulacijskih defektov. Na splošno so pacienti, ki imajo število trombocitov med 20 in 100 x 10⁹/l, v nevarnosti za prekomerno posttravmatsko krvavenje, medtem ko lahko tisti, ki imajo število trombocitov pod 20 x 10⁹/l, krvavijo spontano. Ti, slednji pacienti so kandidati za transfuzijo trombocitov s spremljajočim imunskim in virusnim tveganjem. Za katerokoli povzročeno stopnjo trombocitopenije je tendenca krvavenja bolj resna, če je vzrok zmanjšano nastajanje trombocitov, kot pa če je vzrok zvečana destrukcije le-teh. V zadnji situaciji je posledica pospešenega preobrata trombocitov, kroženje mlajših, večjih in hemostatično bolj učinkovitih trombocitov. Trombocitopenija je lahko posledica raznih motenj, ki so na kratko opisane spodaj. Bolj podroben opis je v Schafner, A. I., Thrombocytopenia and Disorders of Platelet Function, Intemal Medicine, 3. izdaja, John J. Hutton et al., izd., Little Brown in Co., Boston/Toronto/London [1990].The manifestations of clinical haemorrhage in thrombocytopenia depend on the severity of thrombocytopenia, its causes, and the possible coagulation defects. In general, patients with platelet counts between 20 and 100 x 10 ⁹ / l are at risk for excessive post-traumatic bleeding, while those with platelets below 20 x 10 ⁹ / l may bleed spontaneously. These, the latter patients, are candidates for platelet transfusion with accompanying immune and viral risk. For any degree of thrombocytopenia caused, the bleeding tendency is more serious if the cause is reduced platelet production than if the cause is increased destruction. In the latter situation, accelerated platelet turnover results in the circulation of younger, larger and hemostatically more efficient platelets. Thrombocytopenia may be due to various disorders, which are briefly described below. A more detailed description is in Schafner, AI, Thrombocytopenia and Disorders of Platelet Function, Intemal Medicine, 3rd edition, John J. Hutton et al., Ed., Little Brown and Co., Boston / Toronto / London [1990].

(a) Trombocitopenija zaradi poslabšanega nastajanja trombocitov(a) Thrombocytopenia due to impaired platelet production

Vzroki za kongenitalno trombocitopenijo vključujejo kongenitalno aplastično anemijo (sindrom Fanconi) in prirojeno amegakariocitično trombocitopenijo, ki je lahko povezana s skeletno deformiranostjo. Pridobljene motnje za nastajanje trombocitov povzročajo bodisi hipoplazija megakariocitov ali neefektivna trombopoeza. Megakariocitična hipoplazija je lahko posledica različnih stanj, ki vključujejo aplazijo mozga (vključno idiopatične oblike mielosupresije zaradi kemoterapevtskih sredstev ali obsevalne terapije), mielofibrozo, levkemijo in invazijo kostnega mozga z metastatičnimi tumorji ali granulomi. V nekaterih primerih lahko toksini, infekcijska sredstva ali zdravila interferirajo s trombopoezo relativno selektivno; primeri vključujejo prehodne trombocitopenije, povzročene z alkoholom in določenimi virusnimi infekcijami, in blago trombocitopenijo, povezano z dajanjem tiazidnih diuretikov. Končno lahko neefektivna trombopoeza, sekudamo megaloblastičnim procesom (pomanjkanje folata ali B₁₂), tudi povzroči trombocitopenijo, navadno z soobstoječima anemijo in levkopenijo.Causes of congenital thrombocytopenia include congenital aplastic anemia (Fanconi syndrome) and congenital amegakaryocytic thrombocytopenia, which may be associated with skeletal deformity. Acquired platelet production disorders are caused by either megakaryocyte hypoplasia or ineffective thrombopoiesis. Megakaryocytic hypoplasia may be due to a variety of conditions involving brain aplasia (including idiopathic forms of myelosuppression due to chemotherapeutic agents or radiation therapy), myelofibrosis, leukemia, and bone marrow invasion by metastatic tumors or granulomas. In some cases, toxins, infectious agents, or drugs may interfere with thrombopoiesis relatively selectively; examples include transient alcohol-induced thrombocytopenia and certain viral infections and mild thrombocytopenia associated with the administration of thiazide diuretics. Finally, ineffective thrombopoiesis, sequestered by megaloblastic processes (folate deficiency or B ₁₂ ), can also lead to thrombocytopenia, usually with coexisting anemia and leukopenia.

Sedanje zdravljenje trombocitopenije zaradi zmanjšanega nastajanja trombocitov je odvisno od identifikacije in preobrata osnovnega vzroka napake kostnega mozga. Transfuzije trombocitov so navadno rezervirane za paciente z resnimi krvavitvenimi komplikacijami ali za kritje med kirurškimi postopki, ker izoimunizacija lahko vodi do refraktamosti za nadaljnjo transfuzijo trombocitov. Mukozne krvavitve, ki so posledica resne trombocitopenije, lahko izboljšajo z oralnim ali intravenoznim dajanjem antifibrinolitičnih sredstev. Seveda pa se lahko razvijejo trombotične komplikacije, če antifibrinolitična sredstva uporabimo pri pacientih z razširjeno intravaskulamo koagulacijo (DIC).Current treatment of thrombocytopenia due to reduced platelet production depends on the identification and reversal of the underlying cause of bone marrow defect. Platelet transfusions are usually reserved for patients with serious bleeding complications or for coverage during surgical procedures because isoimmunization can lead to refractivity for subsequent platelet transfusions. Mucosal haemorrhage resulting from severe thrombocytopenia may be improved by oral or intravenous administration of antifibrinolytic agents. Of course, thrombotic complications can develop if antifibrinolytic agents are used in patients with advanced intravascular coagulation (DIC).

(b) Trombocitopenija zaradi vranične sekvestracije(b) Thrombocytopenia due to splenic sequestration

Splenomegalija, zaradi kateregakoli vzroka, je lahko povezana z blago do zmerno trombocitopenijo. To je precej pasiven proces (hipersplenizem) sekvestracije vraničnih trombocitov v nasprotju z aktivno destrukcijo trombocitov v vranici v primerih imunsko posredovane trombocitopenije, obravnavane spodaj. Čeprav je najbolj običajen vzrok hipersplenizma kongestivna splenomegalija zaradi portalne hipertenzije pri alkoholni cirozi, pa so druge oblike kongestivne, infiltrativne ali limfoproliferativne splenomegalije prav tako povezane s trombocitopenijo. Število trombocitov na splošno ne pride pod 50 x 10⁹/l kot posledica hipersplenizma samega.Splenomegaly, for whatever reason, may be associated with mild to moderate thrombocytopenia. This is a rather passive process (hypersplenism) of the sequestration of spleen platelets, in contrast to the active destruction of platelets in the spleen in cases of immune-mediated thrombocytopenia discussed below. Although the most common cause of hypersplenism is congestive splenomegaly due to portal hypertension in alcoholic cirrhosis, other forms of congestive, infiltrative or lymphoproliferative splenomegaly are also associated with thrombocytopenia. Platelet counts generally do not come in below 50 x 10 ⁹ / l as a result of hypersplenism itself.

(c) Trombocitopenija zaradi neimunsko posredovanega nastajanja trombocitov(c) Thrombocytopenia due to non-immune mediated platelet production

Trombocitopenija je lahko posledica pospešene destrukcije trombocitov z raznimi neimunološkimi postopki. Motnje tega tipa vključujejo razširjeno intravaskulamo koagulacijo, prostetične intravaskulame naprave, ekstrakorporalni krvni obtok in trombotične mikroangiopatije, kot je trombotična trombocitična purpura. V vseh teh situacijah so krožeči trombociti, ki so izpostavljeni tako umetnim površinam kot tudi nenormalni vaskulami intimi, bodisi konzumirani na teh mestih ali poškodovani in nato prenaglo očiščeni z retikuloendotelijskim sistemom. Bolezenska stanja ali motnje, pri katerih lahko nastane razširjena intravaskulama koagulacija (DIC), so podrobno navedena v Braunwald et al. (izd.), Harrison’s Principles of Internal Medicine, 11. izdaja, str. 1478, McGraw Hill [1987]. Intravaskulame prostetične naprave, ki vključujejo srčne ventile in intraaortne balone, lahko povzročijo blago do zmerno destruktivno trombocitopenijo; prehodna trombocitopenija v pacientih, ki so izpostavljeni kardiopulmonamemu obvodu (bypass) ali hemodializi, pa je lahko posledica iztrošenja ali poškodbe trombocitov v ekstrakorporalnem obtoku.Thrombocytopenia may be the result of accelerated platelet destruction by various non-immunological procedures. Disorders of this type include widespread intravascular coagulation, prosthetic intravascular devices, extracorporeal blood circulation, and thrombotic microangiopathies such as thrombotic platelet purpura. In all these situations, circulating platelets exposed to both artificial surfaces and abnormal intima vasculature are either consumed at these sites or damaged and subsequently prematurely purified by the reticuloendothelial system. Diseases or disorders that can cause widespread intravascular coagulation (DIC) can be described in detail in Braunwald et al. (ed.), Harrison's Principles of Internal Medicine, 11th ed., p. 1478, McGraw Hill [1987]. Intravascular prosthetic devices involving cardiac valves and intra-aortic balloons may cause mild to moderate destructive thrombocytopenia; transient thrombocytopenia in patients undergoing cardiopulmonary bypass or hemodialysis may result from platelet depletion or damage in the extracorporeal circulation.

(d) Z zdravili inducirana imunska trombocitopenija(d) Drug-induced immune thrombocytopenia

Več kot sto zdravil je vključenih v imunološko posredovano trombocitopenijo. Vendar so dobro označeni le gvanidin, gvanin, zlato, sulfonamidi, cefalotin in heparin. Trombocitopenija, inducirana z zdravili, je pogosto zelo resna in do nje značilno pride pospešeno v dnevih, ko pacienti jemljejo senzibilizima zdravila.More than one hundred drugs are involved in immune-mediated thrombocytopenia. However, only guanidine, guanine, gold, sulfonamides, cephalothin and heparin are well characterized. Drug-induced thrombocytopenia is often very serious and is typically accelerated on days when patients are taking drug sensitization.

(e) Imunska (avtoimunska) trombocitopenična purpura (ΓΓΡ)(e) Immune (autoimmune) thrombocytopenic purpura (ΓΓΡ)

ΓΓΡ pri odraslih je kronična bolezen, označena z avtoimunsko destrukcijo trombocitov. Avtoprotitelo je navadno IgG, čeprav so navedeni tudi drugi imunoglobulini. Čeprav so za avtoprotitelo ΓΓΡ ugotovili, da je povezano s trombocitno membrano GPII_bIII_a, pa specifičnost trombocitnega antigena v večini primerov še ni identificirana. Do ekstravaskulame destrukcije senzibiliziranih trombocitov pride v retikuloendotelijskem sistemu vranice in jeter. Čeprav je več kot polovica vseh primerov ITP idiopatskih, pa ima mnogo pacientov osnovno revmatično ali avtoimunsko bolezen (npr. sistemski lupus eritematosus) ali limfoproliferativne motnje (npr. kronično limfocitno levkemijo).Odraslih In adults, it is a chronic disease characterized by autoimmune platelet destruction. The autoantibody is usually IgG, although other immunoglobulins are also indicated. Although the auto-antibody ΓΓΡ has been found to be related to the platelet membrane GPII _b III _a , the specificity of the platelet antigen in most cases has not yet been identified. Extravascular destruction of sensitized platelets occurs in the reticuloendothelial system of the spleen and liver. Although more than half of all cases of ITP are idiopathic, many patients have underlying rheumatic or autoimmune disease (eg systemic lupus erythematosus) or lymphoproliferative disorders (eg chronic lymphocytic leukemia).

(f) ΓΓΡ, inducirana s HIV(f) ΓΓΡ, induced by HIV

ΓΓΡ je vedno bolj običajna komplikacija infekcije s HIV (Morris et al., Ann. Intem. Med., 96:714-717 [1982]), do katere lahko pride v vsaki stopnji bolezenske progresije, tako pri pacientih z diagnozo za sindrom pridobljene imunske pomankljivosti (AIDS), pri tistih s kompleksom, sorodnim AIDS-u, in tistih z infekcijo s HIV, toda brez simptomov za AIDS. Infekcija s HIV je prenosljiva bolezen, končno označena s temeljitim pomanjkanjem celičnega imunskega delovanja kot tudi z navzočnostjo priložnostne infekcije in malignosti. Primarna imunološka nenormalnost, ki je posledica infekcije s HIV, je progresivna deplecija in funkcionalna oslabitev limfocitov T, ki eksprimirajo celični površinski glikoprotein CD4 (Lane et al., Ann. Rev. Immunol., 3:477 [1985]). Izguba CD4 pomočniške/induktorske T-celične funkcije je veijetno osnova za nedoumljive defekte v celični in humoralni imunosti, ki vodi do priložnostnih infekcij in malignosti, značilnih za AIDS (H. Lane, zgoraj).ΓΓΡ It is an increasingly common complication of HIV infection (Morris et al., Ann. Intem. Med., 96: 714-717 [1982]), which can occur at any stage of disease progression, both in patients diagnosed with acquired syndrome. immune deficiencies (AIDS) in those with a complex related to AIDS and those with HIV but without symptoms for AIDS. HIV infection is a transmissible disease, finally characterized by a thorough lack of cellular immune function as well as the presence of occasional infection and malignancy. The primary immunological abnormality resulting from HIV infection is progressive depletion and functional impairment of T lymphocytes expressing the cell surface glycoprotein CD4 (Lane et al., Ann. Rev. Immunol., 3: 477 [1985]). Loss of CD4 helper / inducer T-cell function is probably the basis for the incomprehensible defects in cellular and humoral immunity that lead to occasional AIDS-specific infections and malignancies (H. Lane, supra).

Čeprav je mehanizem ΓΓΡ, povezane s HIV, neznan, pa smatrajo, da je različen od mehanizma ΓΓΡ, ki ni povezana z infekcijo s HIV. (Walsh et al., N. Eng. J. Med., 311:635-639 [1984]; in Ratner, Am. J. Med., 86:194-198 [1989]).Although the mechanism HIV associated with HIV is unknown, they consider it to be different from the mechanism ni not associated with HIV infection. (Walsh et al., N. Eng. J. Med. 311: 635-639 [1984]; and Ratner, Am. J. Med. 86: 194-198 [1989]).

IV. Zdajšnja terapija trombocitopeniieIV. Current thrombocytopenia therapy

Terapevtski način zdravljenja pacientov s trombocitopenijo je določen z resnostjo in nujnostjo klinične situacije. Zdravljenje je podobno za trombocitopenijo, ki je v zvezi s HIV, in za tisto, ki ni v zvezi z njim, in čeprav uporabljajo številne različne terapevtske načine, je terapija še vedno sporna.The therapeutic modality for treating patients with thrombocytopenia is determined by the severity and urgency of the clinical situation. Treatment is similar for HIV-related and non-HIV-related thrombocytopenia, and although they use many different therapeutic modalities, therapy is still controversial.

Število trombocitov v pacientih z diagnozo za trombocitopenijo uspešno povečajo s terapijo z glukokortikoidi (kot je npr. prednisolon), vendar je pri večini pacientov odziv nepopolen ali pa pride do relapsa, če je doza glukokortikoidov zmanjšana, ali je dajanje prekinjeno. Na osnovi študija pacientov z ΓΓΡ, povezane s HIV, nekateri raziskovalci navajajo, da je lahko posledica terapije z glukokortikoidi predispozicija za AIDS. Glukokortikoide navadno dajejo, če število trombocitov pade pod 20 x 10^/1, ali če pride do spontane krvavitve.Platelet counts in patients diagnosed with thrombocytopenia are successfully increased with glucocorticoid therapy (such as prednisolone), but in most patients the response is incomplete or relapsed if glucocorticoid dose is discontinued or discontinued. Based on a study of patients with HIV-related ΓΓΡ, some researchers state that glucocorticoid therapy may be a predisposition to AIDS. Glucocorticoids are usually given if platelet counts fall below 20 x 10 ^ / 1, or if there is spontaneous bleeding.

Za paciente, ki so odporni proti glukokortikoidom, uspešno uporabijo spojino:For glucocorticoid-resistant patients, the compound is successfully used:

4-(2-klorfenil)-9-metil-2-[3-(4-morfolinil)’3-propanon-l-il]6H-tieno[3,2,f][l,2,4]triazolo[4,3,a][l,4]diazepin (WEB 2086), za zdravljenje resnega primera ITP, ki ni povezana s HIV. Paciente, ki imajo število trombocitov od 37000-58000/μ1, zdravijo z WEB 2086, in po 1-2 tednih zdravljenja število trombocitov naraste na 140000-190000/μ1 (EP 361,077 in Lohman et al., Lancet, 1147 [1988]).4- (2-chlorophenyl) -9-methyl-2- [3- (4-morpholinyl) 3-propanon-1-yl] 6H-thieno [3,2, f] [1,2,4] triazolo [ 4,3a] [l, 4] diazepine (WEB 2086) for the treatment of a serious case of non-HIV-related ITP. Patients with platelet counts of 37000-58000 / μ1 are treated with WEB 2086 and after 1-2 weeks of treatment platelet counts increase to 140000-190000 / μ1 (EP 361,077 and Lohman et al. Lancet, 1147 [1988]) .

Čeprav optimalno zdravljenje za pridobljeno amegakariocitično trombocitopenično purpuro (AATP) ni jasno, pa je za antitimocitni globulin (ATG), konjski antiserum za humano timusno tkivo, pokazano, da proizvede podaljšano kompletno remisijo (Trimble et al., Am. J. Hematol., 37:126-127 [1991]). V nedavnem poročilu je prikazano, da so hematopoetični učinki ATG pripisljivi timerosalu, kjer verjetno protein deluje kot nosilec živega srebra (Panella et al., Cancer Research, 50:44294435 [1990]).Although the optimal treatment for the acquired amegakaryocytic thrombocytopenic purpura (AATP) is not clear, antithymocytic globulin (ATG), an equine antiserum for human thymus tissue, has been shown to produce prolonged complete remission (Trimble et al., Am. J. Hematol., 37: 126-127 [1991]. A recent report shows that the hematopoietic effects of ATG are attributable to thimerosal, where the protein is likely to act as a carrier of mercury (Panella et al., Cancer Research, 50: 44294435 [1990]).

Dobri rezultati so navedeni za splenektomijo. Splenektomija odstrani glavno mesto destrukcije trombocitov in glavni vir proizvajanja avtoprotiteles pri mnogih pacientih. Ta postopek ima za posledico podaljšano zdravljenje brez remisij pri velikem številu pacientov. Ker se kirurškim postopkom na splošno izogibajo pri imunsko ogroženih pacientih, splenektomijo priporočajo le v resnih primerih trombocitopenije (npr. resna ΓΓΡ, povezana s HIV) pri pacientih, ki se ne odzovejo po 2-3 tednih zdravljenja z glukokortikoidi ali ne dosežejo zadržanega odziva po prekinitvi dajanja glukokortikoidov. Glede na sedanje znanstveno znanje ni jasno, ali splenektomija predisponira paciente za AIDS.Good results are reported for splenectomy. Splenectomy removes the major site of platelet destruction and the main source of autoantibody production in many patients. This procedure results in prolonged remission-free treatment in a large number of patients. Because surgical procedures are generally avoided in immunocompromised patients, splenectomy is recommended only in serious cases of thrombocytopenia (eg, severe HIV-related) in patients who do not respond after 2-3 weeks of glucocorticoid therapy or do not achieve a sustained response after discontinuation of glucocorticoids. Based on current scientific knowledge, it is unclear whether splenectomy predisposes patients to AIDS.

Poleg terapije s prednisolonom in splenektomije se kažejo obetavna tudi nekatera citotoksična sredstva, npr. vincristin in azidotimidin (AZT, zidovudin), pri zdravljenju ΓΓΡ, inducirane s HIV, vendar pa so rezultati preliminarni.In addition to prednisolone therapy and splenectomy, some cytotoxic agents are also promising, e.g. vincristine and azidothymidine (AZT, zidovudine) in the treatment of HIV-induced,, but the results are preliminary.

Iz predhodno navedenega sledi, da bi bil en način zdravljenja trombocitopenije takšen, da bi dobili sredstvo, ki bi bilo sposobno pospeševanja diferenciacije in zorenja megakariocitov ali njihovih predhodnikov v trombocite tvorečo obliko. Precejšnji trud je bil vložen v identificiranje takšnega sredstva, ki ga navadno imenujemo trombopoetin (TPO). Druga imena za TPO, ki jih navadno najdemo v literaturi, so: trombocitopoezo stimulirajoči faktor (TSF), megakariocitno kolonijo stimulirajoči faktor (MK-CSF), megakariocite stimulirajoči faktor in megakariocitni potenciator. Aktivnost TPO so opazili že leta 1959 (Rak et al., Med. Exp., 1:125), poskusi za označitev in očiščenje tega sredstva pa se nadaljujejo do danes. Čeprav obstajajo poročila o delnem čiščenju TPO-aktivnih polipeptidov (npr.: Tayrien et al., J. Biol. Chem., 262:3262 [1987] in Hoffman et al., J. Ciin. Invest. 75:1174 [1985]), pa nekateri navajajo, da TPO ni diskretna entiteta sam po sebi, ampak je prej enostavno polifunkcionalna manifestacija znanega hormona (IL-3, Sparrow et al., Prog. Ciin. Biol. Res., 215:123 [1986]). Molekula, ki bi imela trombopoetično aktivnost, bi imela precejšnjo terapevtsko vrednost, ne glede na njeno obliko ali izvor. Čeprav noben protein ni bil tako nedvoumno identificiran kot TPO, pa je precej zanimanja okrog sedanjega odkritja, da mpl, domnevni citokinski receptor, lahko transducira trombopoetični signal.It follows from the foregoing that one method of treating thrombocytopenia would be to obtain an agent capable of promoting the differentiation and maturation of megakaryocytes or their precursors into platelets. Considerable effort has been made to identify such an agent, commonly referred to as thrombopoietin (TPO). Other names for TPO commonly found in the literature are: thrombocytopoiesis stimulating factor (TSF), megakaryocyte colony stimulating factor (MK-CSF), megakaryocyte stimulating factor and megakaryocytic potentiator. TPO activity was observed as early as 1959 (Rak et al. Med. Exp. 1: 125), and attempts to label and purify this agent continue to this day. Although there are reports of partial purification of TPO-active polypeptides (e.g., Tayrien et al., J. Biol. Chem., 262: 3262 [1987] and Hoffman et al., J. Ciin. Invest. 75: 1174 [1985] ), however, some argue that TPO is not a discrete entity per se, but rather simply a polyfunctional manifestation of a known hormone (IL-3; Sparrow et al., Prog. Ciin. Biol. Res., 215: 123 [1986]). A molecule that has thrombopoietic activity would have considerable therapeutic value, regardless of its shape or origin. Although no protein has been as unambiguously identified as TPO, there is considerable interest around the current finding that mpl, a putative cytokine receptor, can transduce a thrombopoietic signal.

V. Mpl ie megakariocitopoetični citokinski receptorV. Mpl is a megakaryocytopoietic cytokine receptor

Verjetno je, da proliferacijo in zorenje hematopoetičnih celic močno uravnavajo faktorji, ki pozitivno ali negativno modulirajo proliferacijo pluripotentnih matičnih celic in večrodovno diferenciacijo. Ti učinki so posredovani z visoko afinitetno vezavo ekstraceličnih proteinskih faktorjev na specifične celične površinske receptorje. Ti celični površinski receptorji prispevajo precejšnjo homologijo in so na splošno klasificiram kot člani citokinske receptorske superdružine. Člani superdružine vključujejo receptorje za: IL-2 (β in γ verige) (Hatakeyama et al., Science, 244:551556 [1989]; Takeshita et al., Science, 257:379-382 [1991]), IL-3 (Itoh et al., Science, 247:324-328 [1990]; Gorman et al., Proč. Natl. Acad. Sci. ZDA, 87:54595463 [1990]; Kitamura et al., Celi, 66:1165-1174 [1991a]; Kitamura et al., Proč. Natl. Acad. Sci. ZDA, 88:5082-5086 [1991b]), IL-4 (Mosley et al., Celi, 59:335-348 [1989], IL-5 (Takaki et al., EMBO J., 9:4367-4374 [1990]; Tavemier et al., Celi, 66:1175-1184 [1991]), IL-6 (Yamasaki et al., Science, 241:825-828 [1988]; Hibi et al., Celi, 63:1149-1157 [1990]), IL-7 (Goodwin et al., Celi, 60:941-951 [1990]), IL-9 (Renault et al., Proč. Natl. Acad. Sci. ZDA, 89:5690-5694 [1992]), granulocitno makrofagno kolonijo stimulirajoči faktor (GM-CSF) (Gearing et al., EMBO J., 8:3667-3676 [1991]; Hayashida et al., Proč. Natl. Acad. Sci. ZDA, 244:9655-9659 [1990]), granulocitno kolonijo stimulirajoči faktor (G-CSF) (Fukunaga et al., Celi, 61:341-350 [1990a]; Fukunaga et al., Proč. Natl. Acad. Sci. ZSA, 87:8702-8706 [1990b]; Larsen et al., J. Exp. Med., 172:1559-1570 [1990]), EPO (D’Andrea et al. Celi, 57:277-285 [1989]; Jones et al., Blood, 76:31-35 [1990]), inhibitomi faktor levkemije (LIF) (Gearing et al., EMBO J., 10:2839-2848 [1991]), onkostatin M (OSM) (Rose et al., Proč. Natl. Acad. Sci. ZDA, 88:8641-8645 [1991]) in tudi receptorje za prolaktin (Boutin et al., Proč. Natl. Acad. Sci. ZDA, 88:7744-7748 [1988]; Edery et al., Proč. Natl. Acad. Sci. ZSAA, 86:2112-2116 [1989]), rastni hormon (GH) (Leung et al., Nature, 330:537-543 [1987]) in ciliami nevrotrofni faktor (CNTF) (Davis et al., Science, 253:59-63 [1991].The proliferation and maturation of hematopoietic cells is likely to be strongly regulated by factors that positively or negatively modulate the proliferation of pluripotent stem cells and multifamily differentiation. These effects are mediated by the high affinity binding of extracellular protein factors to specific cell surface receptors. These cell surface receptors contribute considerable homology and are generally classified as members of the cytokine receptor superfamily. Members of the superfamily include receptors for: IL-2 (β and γ chains) (Hatakeyama et al., Science, 244: 551556 [1989]; Takeshita et al., Science, 257: 379-382 [1991]), IL-3 (Itoh et al., Science, 247: 324-328 [1990]; Gorman et al., Proc. Natl. Acad. Sci. USA, 87: 54595463 [1990]; Kitamura et al., Celi, 66: 1165- 1174 [1991a]; Kitamura et al., Proc Natl. Acad. Sci. USA, 88: 5082-5086 [1991b], IL-4 (Mosley et al., Celi, 59: 335-348 [1989]. IL-5 (Takaki et al., EMBO J., 9: 4367-4374 [1990]; Tavemier et al., Celi, 66: 1175-1184 [1991]), IL-6 (Yamasaki et al., Science. 241: 825-828 [1988]; Hibi et al., Celi, 63: 1149-1157 [1990]), IL-7 (Goodwin et al., Celi, 60: 941-951 [1990]), IL-9 (Renault et al., Proc. Nat. Acad. Sci. USA, 89: 5690-5694 [1992]), granulocyte macrophage colony stimulating factor (GM-CSF) (Gearing et al., EMBO J., 8: 3667- 3676 [1991]; Hayashida et al., Et al., Acad. Sci. USA, 244: 9655-9659 [1990]), granulocyte colony stimulating factor (G-CSF) (Fukunaga et al., C eli, 61: 341-350 [1990a]; Fukunaga et al. Natl. Acad. Sci. ZSA, 87: 8702-8706 [1990b]; Larsen et al., J. Exp. Med., 172: 1559-1570 [1990], EPO (D'Andrea et al. Celi, 57: 277-285 [1989]; Jones et al., Blood, 76: 31-35 [1990]), inhibitomes leukemia factor (LIF) (Gearing et al., EMBO J., 10: 2839-2848 [1991]), Oncostatin M (OSM) (Rose et al., Proc. Natl. Acad. Sci. USA, 88: 8641- 8645 [1991]) as well as prolactin receptors (Boutin et al., Read. Natl. Acad. Sci. USA, 88: 7744-7748 [1988]; Edery et al., Away. Natl. Acad. Sci. ZSAA. 86: 2112-2116 [1989]), growth hormone (GH) (Leung et al., Nature, 330: 537-543 [1987]), and ciliami neurotrophic factor (CNTF) (Davis et al., Science, 253: 59 -63 [1991].

Člane citokinske receptorske superdružine lahko razdelimo v tri funkcionalne kategorije (za pregled glej: Nicola et al., Celi, 67:1-4 [1991]). Prvi razred obsega receptorje z enojno verigo, kot npr. eritropoetinski receptor (EPO-R) ali receptor za granulocitno kolonijo stimulirajoči faktor (G-CSF-R), ki veže ligand z visoko afiniteto preko ekstracelične domene in tudi ustvari intracelični signal. Drugi razred receptorjev, tako imenovane α-podenote, vključuje receptor interlevkina-6 (EL6-R), receptor granulocitno-makrofagno kolonijo stimulirajočega faktorja (GM-CSF-R), receptor interlevkina-3 (IL-3-Ra) in druge člane citokinske receptorske superdružine. Te α-podenote vežejo ligand z nizko afiniteto, vendar ne morejo transducirati intraceličnega signala. Visoko afinitetni receptor, ki je sposoben signaliziranja, ustvari heterodimer med α-podenoto in članom tretjega razreda citokinskih receptorjev, označenih j8-podenot, npr. 0_c, ki je običajna j8-podenota za tri α-podenote IL3-Ra: in GM-CSF-R.The members of the cytokine receptor superfamily can be divided into three functional categories (for review, see: Nicola et al., Celi, 67: 1-4 [1991]). The first class comprises single-chain receptors, e.g. an erythropoietin receptor (EPO-R) or granulocyte colony receptor stimulating factor (G-CSF-R) that binds a high-affinity ligand through the extracellular domain and also generates an intracellular signal. The second class of receptors, the so-called α-subunits, include the interleukin-6 receptor (EL6-R), the granulocyte-macrophage colony stimulating factor receptor (GM-CSF-R), the interleukin-3 receptor (IL-3-Ra) and other members cytokine receptor superfamilies. These α-subunits bind a low-affinity ligand but are unable to transduce the intracellular signal. The high-affinity signaling receptor generates a heterodimer between the α-subunit and a member of the third class of cytokine receptors labeled the j8-subunit, e.g. 0 _c , which is the usual j8-subunit for the three α-subunits of IL3-Ra: and GM-CSF-R.

Dokaz, da je mpl član citokinske receptorske superdružine, izhaja iz sekvenčne homologije (Gearing, EMBO J., 8:3667-3676 [1988]; Bazan, Proč. Natl. Acad. Sci. ZDA, 87:6834-6938 [1990]; Davis et al., Science, 253:59-63 [1991] in Vigon et al., Proč. Natl. Acad. Sci. ZDA, 89:5640-5644 [1992]) in njegove sposobnosti, da transducira proliferativne signale.Evidence that mpl is a member of the cytokine receptor superfamily comes from sequence homology (Gearing, EMBO J. 8: 3667-3676 [1988]; Bazan, Proc. Natl. Acad. Sci. USA, 87: 6834-6938 [1990] ; Davis et al., Science, 253: 59-63 [1991] and Vigon et al., Proc Natl. Acad. Sci. USA, 89: 5640-5644 [1992]) and its ability to transduce proliferative signals.

Proteinska sekvenca, izvedena z molekulskim kloniranjem murinega c-mpl, razkriva, da je protein homologen za druge citokinske receptorje. Ekstracelična domena vsebuje 465 aminokislinskih ostankov in je sestavljena iz dveh poddomen, vsaka s 4 visoko konzerviranimi cisteini in posebnim motivom v N-terminalni subdomeni in C-terminalni subdomeni. Za ligand vezavne ekstracelične domene domnevajo, da imajo podobno dvojno j8-sodčkasto (j8-barrel) zgubano strukturno geometrijo. Ta podvojena ekstracelična domena je visoko homologna za signal transducirajočo verigo, navadno za receptorje EL-3, IL-5 in GM-CSF kot tudi za nizko afinitetno vezavno domeno LIF (Vigon et al., Oncogene, 8:2607-2615 [1993]). Tako lahko mpl spada v nizko afinitetni razred citokinskih receptorjev, ki vežejo ligand.A protein sequence derived by molecular cloning of murine c-mpl reveals that the protein is homologous to other cytokine receptors. The extracellular domain contains 465 amino acid residues and consists of two subdomains, each with 4 highly conserved cysteines and a specific motif in the N-terminal subdomain and C-terminal subdomain. The ligand binding extracellular domains are thought to have a similar double j8-barrel (j8-barrel) folded structural geometry. This duplicate extracellular domain is highly homologous to the signal transducing chain, typically to the EL-3, IL-5 and GM-CSF receptors as well as to the low affinity binding domain of LIF (Vigon et al., Oncogene, 8: 2607-2615 [1993] ). Thus, mpl may belong to the low affinity class of ligand-binding cytokine receptors.

k primerjave med murinim mpl in zrelim humanim mpl P je razvidno, da imata ta dva proteina 81 % sekvenčno identičnost. Bolj specifično imata N-terminalna in C-terminalna ekstracelična subdomena 75 % oz. 80 % sekvenčno identičnost. Najbolj konzervirana regija mpl je citoplazemska domena, ki ima 91 % aminokislinsko identičnost s sekvenco 37 ostankov blizu transmembranske domene, ki je identična v obeh vrstah. V skladu s tem je za mpl navedeno, da je eden od najbolj konzerviranih članov citokinske receptorske superdružine (Vigon, zgoraj).Comparisons between murine mpl and mature human mpl P show that these two proteins have 81% sequence identity. More specifically, the N-terminal and C-terminal extracellular subdomains have 75% and. 80% sequential identity. The most conserved mpl region is the cytoplasmic domain, which has 91% amino acid identity with a sequence of 37 residues close to the transmembrane domain, which is identical in both species. Accordingly, mpl is said to be one of the most conserved members of the cytokine receptor superfamily (Vigon, supra).

Dokaz, da je mpl funkcionalen receptor, ki je sposoben transduciranja proliferativnega signala, izhaja iz konstrukcije kimemih receptorjev, ki vsebujejo ekstracelično domeno od citokinskega receptorja, ki ima visoko afiniteto za znan citokin s citoplazemsko domeno mpl. Ker ni naveden še noben znan ligand za mpl, je potrebno, da konstruiramo kimemo visoko afinitetno ligand vezavno ekstracelično domeno iz razreda citokinskih receptorjev, kot so IL-4R ali G-CSFR. Vigon et al., zgoraj, so spojili ekstracelično domeno G-CSFR tako s transmembransko kot tudi s citoplazemsko domeno c-mpl. IL-3 odvisno celično linijo BAF/B03 (Ba/F3) so transfektirali z G-CSFR/mp/ kimero skupaj z G-CSFR kontrolo s popolno dolžino. Celice, transfektirane s kimero, rastejo enako dobro bodisi v prisotnosti citokinskega IL-3 ali G-CSF. Podobno celice, transfektirane z G-CSFR, tudi dobro rastejo bodisi v IL-3 ali G-CSF. Vse celice odmrejo v odsotnosti rastnih faktorjev. Podoben eksperiment so izvedli Skoda et al., EMBO J., 12(7):2645-2653 [1993], v katerem so tako ekstracelično kot tudi transmembransko domeno humanega receptorja IL-4 (hIL-4R) spojili s citoplazemsko domeno murinega mpl in transfektirali v murino celično linijo Ba/F3, odvisno od IL-3. Celice Ba/F3, transfektirane s hIL-4-R divjega tipa, proliferirajo normalno v prisotnosti enega od obeh vrstno specifičnih IL-4 ah IL-3. Celice Ba/F3, transfektirane s hIL-4R/wp/, proliferirane normalno v prisotnosti hIL-4 (v prisotnosti ah odsotnosti IL-3), dokazujejo, da v celicah Ba/F3 citoplazemska domena mpl vsebuje vse elemente, ki so potrebni za transduciranje proliferativnega signala.Evidence that mpl is a functional receptor capable of transducing a proliferative signal originates from the construction of chimeric receptors containing the extracellular domain from a cytokine receptor having a high affinity for a known cytokine with the cytoplasmic domain of mpl. Since no known ligand for mpl is indicated, it is necessary to construct a high affinity ligand binding extracellular domain from a class of cytokine receptors such as IL-4R or G-CSFR. Vigon et al., Above, fused the extracellular domain of G-CSFR with both the transmembrane and cytoplasmic domain of c-mpl. The IL-3 dependent BAF / B03 cell line (Ba / F3) was transfected with G-CSFR / mp / chimera together with a full-length G-CSFR control. Chimera-transfected cells grow equally well either in the presence of cytokine IL-3 or G-CSF. Similarly, cells transfected with G-CSFR also grow well in either IL-3 or G-CSF. All cells die in the absence of growth factors. A similar experiment was performed by Skoda et al., EMBO J., 12 (7): 2645-2653 [1993], in which both the extracellular and transmembrane domains of the human IL-4 receptor (hIL-4R) were coupled to the cytoplasmic domain of murine mpl and transfected into murine IL-3-dependent Ba / F3 cell line. Ba / F3 cells transfected with wild-type hIL-4-R proliferate normally in the presence of one of both species-specific IL-4 ah IL-3. HIL-4R / wp / transfected Ba / F3 cells, proliferated normally in the presence of hIL-4 (in the presence of ah in the absence of IL-3), demonstrate that in the Ba / F3 cells, the mpl cytoplasmic domain contains all the elements necessary for transducing a proliferative signal.

Iz teh kimemih eksperimentov je razvidna sposobnost citoplazemske domene mpl, da signalizira proliferacijo, ni pa razvidno, da ekstracelična domena mpl lahko veže ligand. Ti rezultati so konsistentni z vsaj dvema možnostima, in sicer, daje mpl receptor z enojno verigo (razred 1), podoben EPO-R ali G-CSFR, ali je signal transducirajoča /3-podenota (razred tri), ki potrebuje α-podenoto, podobno kot IL-3 (Skoda et al. zgoraj).These kimem experiments show the ability of the cytoplasmic domain of mpl to signal proliferation, but it does not show that the extracellular domain of mpl can bind a ligand. These results are consistent with at least two options, namely that the single-chain mpl receptor (class 1) is similar to EPO-R or G-CSFR, or is the signal a transduction / 3-subunit (class three) that requires an α-subunit , similar to IL-3 (Skoda et al. above).

VI, Mpl ligand ie trombopoetin (TPO)VI, Mpl ligand ie thrombopoietin (TPO)

Kot je opisano zgoraj, vsebuje serum edini faktor, na katerega se včasih sklicujemo kot na trombopoetin (TPO), ki deluje sinergistično z raznimi drugimi citokini, tako da zvečuje rast in zorenje megakariocitov. Nobenega takega naravnega faktorja še niso izolirali iz seruma ali kakšnega drugega vira, čeprav so številne skupine v to vložile precej truda. Čeprav še ni znano, ali je mpl sposoben direktne vezave megakariocite stimulirajočega faktorja, pa je iz nedavnih eksperimentov razvidno, da je mpl vključen v transdukcijo proliferativnega signala iz faktorja ali faktorjev, ugotovljenih v serumu pacientov z aplastičnim kostnim mozgom (Methia et al., Blood, 82(5):1395-1401 (1993]).As described above, serum contains the only factor sometimes referred to as thrombopoietin (TPO), which acts synergistically with various other cytokines to increase the growth and maturation of megakaryocytes. No such natural factor has yet been isolated from serum or any other source, although many groups have put considerable effort into it. Although it is not yet known whether mpl is capable of directly binding the megakaryocyte stimulating factor, recent experiments show that mpl is involved in the transduction of proliferative signal from a factor or factors found in the serum of patients with aplastic bone marrow (Methia et al., Blood , 82 (5): 1395-1401 (1993]).

Dokaz, da edini serumsko kolonijo tvoreči faktor, različen od IL-Ία, IL-3, EL-4, IL-6, EL-11, SCF, EPO, G-CSF in GM-CSF, transducira proliferativni signal preko mpl, izhaja iz raziskave porazdelitve ekspresije c-mpl v primitivnih in usmerjenih hematopoetičnih celičnih linijah in iz antisens študij mpl v eni od teh celičnih linij.Evidence that the only serum colony forming factor other than IL-Ία, IL-3, EL-4, IL-6, EL-11, SCF, EPO, G-CSF, and GM-CSF transduces proliferative signal via mpl from a study of the distribution of c-mpl expression in primitive and targeted hematopoietic cell lines and from antisense studies of mpl in one of these cell lines.

Z uporabo reverzne transkriptaze (RT)-PCR v imunsko očiščenih humanih hematopoetičnih celicah so Methia et al., zgoraj, dokazali, da so močne signale mRNA mpl ugotovili le v očiščenih celicah CD34⁺, megakariocitih in trombocitih. Celice CD34⁺, očiščene iz kostnega mozga (BM), pomenijo približno 1 % vseh celic BM in so obogatene v primitivnih in usmerjenih prednikih vseh rodov (npr. eritroidnem, granulomakrofagnem in megakariocitnem).Using reverse transcriptase (RT) -PCR in immune-purified human hematopoietic cells, Methia et al., Above, demonstrated that potent mpl mpl signals were detected only in purified CD34 ⁺ cells, megakaryocytes, and platelets. Bone marrow (BM) purified CD34 ⁺ cells represent about 1% of all BM cells and are enriched in primitive and targeted ancestors of all genera (eg erythroid, granulomacrophage, and megakaryocytic).

Za mpl antisens oligodeoksinukleotide je prikazano, da preprečujejo tvorbo megakariocitične kolonije iz pluripotentnih celic CD34⁺, kultiviranih v serumu pacientov z aplastičnim mozgom (bogat vir megakariocitno kolonijo stimulirajoče aktivnosti [MK-CSA]). Ti, isti antisens oligodeoksinukleotidi nimajo vpliva na tvorbo eritroidne ali granulomakrofagne kolonije.Mpl antisense oligodeoxynucleotides have been shown to prevent the formation of megakaryocytic colonies from CD34 ⁺ pluripotent cells cultured in the serum of patients with aplastic brains (a rich source of megakaryocytic colony of stimulating activity [MK-CSA]). These, the same antisense oligodeoxynucleotides, have no effect on erythroid or granulomacrophage colony formation.

Ali mpl direktno veže ligand in ali serumski faktor, za katerega je znano, da povzroča megakariocitopoezo, delujeta preko mpl, je še vedno neznano. Vendar pa navajajo, da je, če mpl direktno veže ligand, njegova aminokislinska sekvenca verjetno visoko konzervirana in ima navzkrižno reaktivnost za vrste, zaradi znatne sekvenčne identičnosti med ekstraceličnimi domenami humanega in murinega mpl (Vigon et al., zgoraj [1993]).Whether mpl binds directly to the ligand and whether the serum factor known to cause megakaryocytopoiesis act via mpl is still unknown. However, they state that, if mpl directly binds a ligand, its amino acid sequence is likely to be highly conserved and has cross-reactivity for species due to its considerable sequence identity between the extracellular domains of the human and murine mpl (Vigon et al., Above [1993]).

VII. PredmetiVII. Objects

Glede na pred tem navedeno je razumljivo, da obstaja nenehna potreba v tehniki, da bi izolirali in identificirali molekule, ki bi bile sposobne stimulirati proliferacijo, diferenciacijo in zorenje hematopoetičnih celic, posebno megakariocitov ali njihovih predhodnikov, za terapevtsko uporabo pri zdravljenju trombocitopenije. Verjetno je takšna molekula mpl ligand in zato obstaja nadaljnja potreba, da bi izolirali takšen ligand(e), da bi ovrednotili njegovo vlogo (njihove vloge) v celični rasti in diferenciaciji.Given the foregoing, it is understood that there is an ongoing need in the art to isolate and identify molecules capable of stimulating the proliferation, differentiation and maturation of hematopoietic cells, especially megakaryocytes or their precursors, for therapeutic use in the treatment of thrombocytopenia. It is likely that such a mpl molecule is a ligand and therefore there is a further need to isolate such ligand (s) in order to evaluate its role (their roles) in cell growth and differentiation.

V skladu s tem je prvi predmet predloženega izuma ustvaritev farmacevtsko čiste molekule, sposobne stimuliranja proliferacije, diferenciacije in/ali zorenja megakariocitov v zrelo trombocite tvorečo obliko.Accordingly, the first object of the present invention is to provide a pharmaceutically pure molecule capable of stimulating the proliferation, differentiation and / or maturation of megakaryocytes into a mature platelet-forming form.

Nadaljnji predmet je, da zagotovimo molekulo v obliki za terapevtsko uporabo pri zdravljenju hematopoetičnih motenj, posebno trombocitopenije.It is a further object to provide a molecule in a form for therapeutic use in the treatment of hematopoietic disorders, especially thrombocytopenia.

Nadaljnji predmet predloženega izuma je, da izoliramo, očistimo in specifično identificiramo proteinske ligande, sposobne vezave receptorja citokinske superdružine, znanega kot mpl, in vivo, in transduciranja proliferativnega signala.It is a further object of the present invention to isolate, purify and specifically identify protein ligands capable of binding the cytokine superfamily receptor known as mpl in vivo and transducing a proliferative signal.

Nadaljnji predmet je, da zagotovimo molekule nukleinske kisline, ki kodirajo takšne proteinske ligande, in da uporabimo te molekule nukleinske kisline za izdelavo mpl vezivnih ligandov v rekombinantni celični kulturi za diagnostično in terapevtsko uporabo.It is a further object to provide nucleic acid molecules encoding such protein ligands and to use these nucleic acid molecules to produce mpl binding ligands in recombinant cell culture for diagnostic and therapeutic use.

Še nadaljnji predmet je, da zagotovimo derivate in modificirane oblike proteinskih ligandov, ki vključujejo variante aminokislinskih sekvenc, variante glikoproteinskih oblik in njihove kovalentne derivate.A further object is to provide derivatives and modified forms of protein ligands that include variants of amino acid sequences, variants of glycoprotein forms and their covalent derivatives.

Dodaten predmet je, da zagotovimo fuzijske polipeptidne oblike, ki združujejo mpl ligand in heterologni protein ter njegove kovalentne derivate.An additional object is to provide fusion polypeptide forms that combine the mpl ligand and the heterologous protein and its covalent derivatives.

Še dodatni predmet je, da zagotovimo variante polipeptidnih oblik, ki združujejo mpl ligand z aminokislinskimi adicijami in substitucijami iz sekvence EPO, da se tvori protein, sposoben reguliranja proliferacije in rasti tako trombocitnih prednikov kot tudi prednikov rdečih krvnih celic.Another object is to provide variants of polypeptide forms that combine the mpl ligand with amino acid additions and substitutions from the EPO sequence to form a protein capable of regulating the proliferation and growth of both platelet ancestors and red blood cell ancestors.

Še nadaljnji predmet je, da pripravimo imunogene za snovanje protiteles proti mpl ligandom ali njihovim fuzijskim oblikam, kot tudi da dobimo protitelesa, ki bi lahko vezala takšne ligande.A further object is to prepare immunogens for the production of antibodies against mpl ligands or their fusion forms, as well as to obtain antibodies that can bind such ligands.

Ti in drugi predmeti predloženega izuma bodo jasni strokovnjakom ob upoštevanju celotne specifikacije.These and other objects of the present invention will be apparent to those skilled in the art having regard to the full specification.

Predmete predloženega izuma dosežemo s tem, da zagotovimo izoliran protein sesalcev, ki pospešuje megakariocitopoetično proliferacijo in zorenje, imenovan mpl ligand (ML) ali trombopoetin (TPO), in je sposoben stimuliranja proliferacije, zorenja in/ali diferenciacije megakariocitov v obliko, tvorečo zrele trombocite.The objects of the present invention are achieved by providing an isolated mammalian protein that promotes megakaryocytopoietic proliferation and maturation, called mpl ligand (ML) or thrombopoietin (TPO), and is capable of stimulating the proliferation, maturation and / or differentiation of megakaryocytes in the form of mature platelets .

Ta, v bistvu homogeni protein lahko očistimo iz naravnega vira s postopkom, ki obsega: (1) kontaktiranje plazemskega vira, ki vsebuje molekule mpl liganda, ki jih je potrebno očistiti, z mobiliziranim receptorskim polipeptidom, posebno mpl ali mpl fuzijskim polipeptidom, mobiliziranim na nosilcu, pri pogojih, kjer molekule mpl liganda, ki jih je potrebno očistiti, selektivno adsorbiramo na mobiliziran receptorski polipeptid, (2) izpiranje mobiliziranega receptorskega polipeptida in njegovega nosilca, da odstranimo neadsorbirano snov, in (3) eluiranje molekul mpl liganda iz mobiliziranega receptorskega polipeptida, na katerem so adsorbirane, z eluimim pufrom. Prednostni naravni vir je plazma ali urin sesalca, ki vsebuje mpl ligand. V danem primeru je sesalec aplastičen in je mobiliziran receptor fuzija mplIgG.This essentially homogeneous protein can be purified from a natural source by a process comprising: (1) contacting a plasma source containing the mpl ligand molecules to be purified with a mobilized receptor polypeptide, especially a mpl or mpl fusion polypeptide mobilized to the carrier, under conditions where the mpl ligand molecules to be purified are selectively adsorbed onto the mobilized receptor polypeptide, (2) washing the mobilized receptor polypeptide and its carrier to remove the unabsorbed substance, and (3) eluting the mpl ligand molecules from the mobilized receptor of the adsorbed polypeptide with eluate buffer. A preferred natural source is mammalian plasma or urine containing the mpl ligand. In the present case, the mammal is aplastic and the mplIgG fusion receptor is mobilized.

V danem primeru je prednosten protein, ki pospešuje megakariocitopoetično proliferacijo in zorenje, izoliran v bistvu homogen mpl ligandski polipeptid, narejen s sintetičnimi ali rekombinantnimi postopki.In this case, a preferred protein that promotes megakaryocytopoietic proliferation and maturation is an isolated homogeneous mpl ligand polypeptide made by synthetic or recombinant methods.

Mpl ligandski polipeptid ali 'TPO v smislu izuma ima prednostno vsaj 70 % celotno sekvenčno identičnost z aminokislinsko sekvenco visoko očiščenega, v bistvu homogenega prašičjega mpl Ugandskega polipeptida, in vsaj 80 % sekvenčno identičnost z EPO-domeno prašičjega mpl Ugandskega poUpeptida. V danem primeru je mpl Ugand v smislu izuma zreU humani mpl Ugand (hML), ki ima zrelo aminokislinsko sekvenco, prikazano na sl. 1 (SEQ ID NO [identifikacijska številka sekvence]: 1) aU njegova varianta ali posttranskripcijsko modificirana oblika aU protein, ki ima približno 80 % sekvenčno identičnost z zrelim humanim mpl Ugandom. V danem primeru je varianta mpl Uganda fragment, posebno amino terminalni ali fragment EPO-domene zrelega humanega mpl Uganda (hML). Prednostno amino terminalni fragment ohrani v bistvu vso sekvenco humanega ML med prvim in četrtim cisteinskim ostankom, lahko pa vsebuje dodatne adicije, delecije ali substitucije zunaj te regije. V skladu s to izvedbo lahko polipeptidni fragment predstavimo s formulo:The mpl ligand polypeptide or 'TPO of the invention preferably has at least 70% overall sequence identity to the amino acid sequence of a highly purified, substantially homogeneous pig mpl Ugandan polypeptide, and at least 80% sequence identity to the EPO domain of the pig mpl Ugandan popeptide. In the present example, the Ugand mpl of the invention is a mature human Ugand mpl (hML) having a mature amino acid sequence shown in FIG. 1 (SEQ ID NO [Sequence Identification Number]: 1) aU a variant thereof or a post-transcriptionally modified form of aU protein having approximately 80% sequence identity to mature human mpl Uganda. In the present example, the Ugandan mpl variant is a fragment, especially the amino terminal or EPO domain fragment of mature human Ugandan mpl (hML). Preferably, the amino terminal fragment retains substantially the entire sequence of human ML between the first and fourth cysteine residues, but may contain additional additions, deletions or substitutions outside this region. According to this embodiment, the polypeptide fragment can be represented by the formula:

X-hML(7-151)-Y kjer pomeni hML(7-151) aminokislinsko sekvenco humanega TPO (hML), od Cys⁷ do vključno Cys¹⁵¹; X pomeni amino skupino Cys⁷ ali enega ali več aminoterminalnih aminokislinskih ostankov zrelega hML ali ekstenzije aminokislinskih ostankov le-tega, kot so npr. Met, Tyr, ali vodilne sekvence, ki vsebujejo npr. proteolitična cepišča (npr. faktor Xa ali trombin); in Y pomeni karboksi terminalno skupino Cys¹⁵¹ ali enega ali več karboksiterminalnih aminokislinskih ostankov zrelega hML ali ekstenzije le-teh.X-hML (7-151) -Y where hML (7-151) represents the amino acid sequence of human TPO (hML), from Cys ⁷ to Cys ¹⁵¹ inclusive; X stands for the amino group of Cys ⁷ or one or more amino-terminal amino acid residues of mature hML or an extension of amino acid residues thereof, such as e.g. Met, Tyr, or leader sequences containing e.g. proteolytic cleavages (eg factor Xa or thrombin); and Y represents the carboxy terminal group of Cys ¹⁵¹ or one or more carboxyterminal amino acid residues of mature hML or extensions thereof.

V danem primeru mpl Ugandski poUpeptid aU njegov fragment lahko spojimo s heterolognim poUpeptidom (kimera). Prednostni heterologni poUpeptid je citokin, kolonijo stimuUrajoči faktor, aU interlevkin aU njegov fragment, posebno kit-Ugand (KL), EL-1, EL-3, IL-6, IL-11, EPO, GM-CSF aU LIF. V danem primeru je prednostni heterologni poUpeptid imunoglobuUnska veriga, posebno humani IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgD, IgM aU njegov fragment, ki posebno obsega konstantno domeno težke verige IgG.In the present example, the mpl Ugandan poUpeptide aU can fragment its fragment with the heterologous poUpeptide (chimera). A preferred heterologous poUpeptide is a cytokine, a colony stimulating factor, aU interleukin aU fragment thereof, especially kit-Ugand (KL), EL-1, EL-3, IL-6, IL-11, EPO, GM-CSF aU LIF. In the present case, the preferred heterologous poUpeptide immunoglobulin chain, in particular human IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgD, IgM aU, is a fragment thereof that specifically comprises the constant domain of the heavy chain IgG.

Z nadaljnjega vidika predloženega izuma je zagotovljen sestavek, ki obsega izoUranega agonista mpl, ki je biološko aktiven in prednostno sposoben stimuliranja vgraditve označenih nukleotidov (npr. ³H-timidin) v DNA cehe Ba/F3, odvisnih od IL-3, transfektiranih s humanim mpl. N danem primeru je agonist mpl biološko aktiven mpl Ugand in je prednostno sposoben stimuliranja vgraditve ³⁵S v krožeče trombocite pri testu oživitve mišjih trombocitov. Prikladni agonisti mpl vključujejo hML₁₅₃, hML(R153A, R154A), hML2, hML3, hML4, mML, mML2, mML3, pML in pML2 aU njihove fragmente.In a further aspect of the present invention, a composition is provided comprising an isoL mon agonist that is biologically active and preferably capable of stimulating the incorporation of labeled nucleotides (e.g., ³ H-thymidine) into IL-3-dependent Ba / F3 guild DNA transfected with human mpl. In the present example, the mpl agonist is biologically active mpl Ugand and is preferably capable of stimulating the incorporation of ³⁵ S into circulating platelets in a mouse platelet resuscitation test. Suitable mpl agonists include hML ₁₅₃ , hML (R153A, R154A), hML2, hML3, hML4, mML, mML2, mML3, pML, and pML2 aU fragments thereof.

V drugi izvedbi zagotavlja predloženi izum izolirano protitelo, kije sposobno vezave na mpl Ugand. Izolirano protitelo, ki je sposobno vezave na mpl Ugand, lahko v danem primeru spojimo z drugim poUpeptidom in protitelo aU njegovo fuzijo uporabimo, da izoUramo in očistimo mpl Ugand iz vira, kot je opisano zgoraj za imobilizirani mpl. N nadaljnjem vidiku te izvedbe zagotavlja predloženi izum postopek za detektiranje mpl liganda in vitro ali in vivo, pri čemer obsega kontaktiranje protitelesa z vzorcem, posebno serumskim vzorcem, za katerega predvidevamo, da vsebuje Ugand, in detektiranje, če pride do vezave. V še nadaljnjih izvedbah zagotavlja predloženi izum izolirano molekulo nukleinske kisline, ki kodira mpl ligand ali njegove fragmente, pri čemer je lahko molekula nukleinske kisline v danem primeru označena z detektibilnim deležem, in molekulo nukleinske kisline, ki ima sekvenco, ki je komplementarna oz. se hibridizira pri zmernih do ostrih pogojih z molekulo nukleinske kisline, ki ima sekvenco, ki kodira mpl ligand. Prednostne molekule nukleinske kisline so tiste, ki kodirajo humani, prašičji in murini mpl ligand in vključujejo RNA in DNA, tako genomsko kot tudi cDNA. Nadaljnji vidik te izvedbe je molekula nukleinske kisline DNA, ki kodira mpl ligand in nadalje obsega replikabilni vektor, v katerem je DNA funkcionalno vezana na kontrolne sekvence, spoznane od gostitelja, transformiranega z vektorjem. DNA je v danem primeru cDNA s sekvenco, prikazano na sl. 1, 5’-3’ (SEQ ID NO: 2), 3’-5’ ali njen fragment. Ta vidik nadalje vključuje gostiteljske celice, prednostno celice CHO, transformirane z vektorjem, in postopek uporabe DNA, da tvorimo mpl ligand, ki prednostno obsega ekspresijo cDNA, ki kodira mpl ligand v kulturi transformiranih gostiteljskih celic, in rekuperiranje mpl liganda iz gostiteljskih celic ali gostiteljske celične kulture. Mpl ligand, pripravljen na ta način, je prednostno humani mpl ligand.In another embodiment, the present invention provides an isolated antibody capable of binding to the Ugand mpl. An isolated antibody capable of binding to mpl Ugand can optionally be fused to another poUpeptide and the antibody aU its fusion is used to isolate and purify mpl Ugand from the source as described above for immobilized mpl. In a further aspect of this embodiment, the present invention provides a method for detecting mpl ligands in vitro or in vivo, comprising contacting the antibody with a sample, especially a serum sample, which is intended to contain Ugandan, and detecting if binding occurs. In still further embodiments, the present invention provides an isolated nucleic acid molecule encoding an mpl ligand or fragments thereof, wherein the nucleic acid molecule may optionally be labeled with a detectable moiety, and a nucleic acid molecule having a sequence that is complementary to or. hybridizes under moderate to harsh conditions with a nucleic acid molecule having a sequence encoding the mpl ligand. Preferred nucleic acid molecules are those that encode human, porcine and murine mpl ligands and include RNA and DNA, both genomically and cDNA. A further aspect of this embodiment is a nucleic acid molecule of DNA encoding the mpl ligand and further comprising a replicable vector in which the DNA is functionally linked to control sequences recognized by the vector transformed host. The DNA is optionally cDNA with the sequence shown in FIG. 1, 5′-3 ′ (SEQ ID NO: 2), 3′-5 ′ or a fragment thereof. This aspect further includes host cells, preferably vector transformed CHO cells, and a process of using DNA to form an mpl ligand, preferably comprising the expression of a cDNA encoding the mpl ligand in the culture of the transformed host cells, and recovering the mpl ligand from the host cells or host cell cultures. The mpl ligand prepared in this manner is preferably a human mpl ligand.

Predloženi izum nadalje vključuje postopek za zdravljenje sesalcev s hematopoetično motnjo, posebno trombocitopenije, ki obsega dajanje terapevtsko učinkovite količine mpl liganda sesalcu. V danem primeru damo mpl ligand v kombinaciji s citokmom, posebno s kolonijo stimulirajočim faktorjem ali interlevkinom. Prednostni kolonijo stimulirajoči faktorji ali interlevkini vključujejo: kit-ligand (KL), LIF, G-CSF, GMCSF, M-CSF, EPO, IL-1, IL-3, IL-6 in IL-11.The present invention further includes a method for treating a mammal with a hematopoietic disorder, in particular thrombocytopenia, comprising administering a therapeutically effective amount of a mpl ligand to a mammal. If appropriate, the mpl ligand is administered in combination with a cytokine, especially a colony-stimulating factor or interleukin. Preferred colony-stimulating factors or interleukins include: kit-ligand (KL), LIF, G-CSF, GMCSF, M-CSF, EPO, IL-1, IL-3, IL-6 and IL-11.

Predloženi izum nadalje vključuje postopek za izoliranje in čiščenje TPO (ML) iz TPO izdelujočega mikroorganizma, ki obsega:The present invention further includes a method for isolating and purifying TPO (ML) from a TPO-producing microorganism comprising:

(1) razkrojevanje ali liziranje celic, ki vsebujejo TPO (2) v danem primeru ločevanje topne snovi od netopne, ki vsebuje TPO (3) solubiliziranje TPO v netopni snovi s solubilizimim pufrom (4) ločitev solubiliziranega TPO od drugih topnih in netopnih snovi (5) renaturiranje TPO v redoks pufru in (6) ločevanje pravilno zgubanega TPO od nepravilnega.(1) disintegration or lysis of cells containing TPO (2) optionally separating soluble substance from insoluble containing TPO (3) solubilizing TPO in insoluble substance with solubilizable buffer (4) separating solubilized TPO from other soluble and insoluble substances ( 5) renaturating the TPO in redox buffer and (6) separating the correctly folded TPO from the incorrect one.

Postopek zagotavlja solubiliziranje netopne snovi, ki vsebuje TPO, s kaotropičnim sredstvom, pri čemer le-to izberemo izmed soli gvanidina, natrijevega tiocianata ali sečnine. Postopek nadalje zagotavlja, da solubilizirani TPO ločimo od drugih topnih in netopnih snovi v eni ali več stopnjah, izbranih izmed centrifugiranja, gelske filtracije in kromatografije z reverzno fazo. Procesno stopnjo renaturiranja izvedemo z redoks pufrom, ki vsebuje tako oksidacijsko kot tudi redukcijsko sredstvo. Na splošno je oksidacijsko sredstvo kisik ali spojina, ki vsebuje vsaj eno disulfidno vez, redukcijsko sredstvo pa je spojina, ki vsebuje vsaj en prosti sulfhidril. Prednostno oksidacijsko sredstvo izberemo izmed oksidiranega glutationa (GSSG) in čistina, redukcijsko sredstvo pa izberemo izmed reduciranega glutationa (GSH) in cisterna. Najbolj prednostno oksidacijsko sredstvo je oksidirani glutation (GSSG) in redukcijsko sredstvo reducirani glutation (GSH). Prednostno sta oksidacijsko in redukcijsko sredstvo v enakem molskem razmerju oz. je oksidacijsko sredstvo v prebitku. Redoksni pufer dodatno vsebuje detergent, prednostno izbran izmed CHAPS in CHAPSO, v količini vsaj 1 %. Redoks pufer dodatno vsebuje NaC, prednostno v koncentracijskem območju približno 0,1 do 0,5 M, in glicerol, prednostno v koncentraciji, večji od 15 %. pH redoks pufra je prednostno v območju od približno 7,5 do 9,0, procesno stopnjo renaturiranja pa vodimo pri 4 °C 12-48 ur. V stopnji renaturiranja dobimo biološko aktiven TPO, v katerem se tvori disulfidna vez med Cys, kije najbližji amino-terminalu, in Cys, kije najbližji karboksi-terminalu domene EPO.The process ensures the solubilization of an insoluble substance containing TPO with a chaotropic agent, which is selected from the salts of guanidine, sodium thiocyanate or urea. The process further ensures that solubilized TPOs are separated from other soluble and insoluble substances in one or more stages selected from centrifugation, gel filtration and reversed-phase chromatography. The process of renaturation is carried out with redox buffer containing both oxidizing and reducing agents. In general, the oxidizing agent is oxygen or a compound containing at least one disulfide bond, and the reducing agent is a compound containing at least one free sulfhydryl. The preferred oxidizing agent is selected from oxidized glutathione (GSSG) and purity, and the reducing agent is selected from reduced glutathione (GSH) and cistern. The most preferred oxidizing agent is oxidized glutathione (GSSG) and the reducing agent reduced glutathione (GSH). Preferably, the oxidizing and reducing agents are in the same molar ratio, respectively. is an oxidizing agent in excess. The redox buffer additionally contains a detergent, preferably selected from CHAPS and CHAPSO, in an amount of at least 1%. Redox buffer additionally contains NaC, preferably in a concentration range of about 0.1 to 0.5 M, and glycerol, preferably in a concentration greater than 15%. The pH of the redox buffer is preferably in the range of about 7.5 to 9.0, and the process of renaturation is conducted at 4 ° C for 12-48 hours. In the renaturation step, a biologically active TPO is formed in which a disulfide bond is formed between the Cys closest to the amino terminal and the Cys closest to the carboxy terminal of the EPO domain.

Predloženi izum nadalje vključuje postopek za čiščenje biološko aktivnega TPO iz mikroorganizma, ki obsega:The present invention further includes a method for purifying a biologically active TPO from a microorganism comprising:

(1) liziranje vsaj ekstracelične membrane mikroorganizma (2) obdelovanje lizata, ki vsebuje TPO, s kaotropičnim sredstvom (3) renaturiranje TPO in (4) ločevanje nečistot in nepravilno zgubanega TPO od pravilnega.(1) lysis of at least the extracellular membrane of the microorganism (2) treatment of the TPO-containing lysate with chaotropic agent (3) renaturation of the TPO and (4) separation of impurities and incorrectly folded TPO from the correct one.

Na sl. 1 je prikazana deducirana aminokislinska sekvenca (SEQ ID NO: 1) cDNA humanega mpl liganda (hML) in kodirna nukleotidna sekvenca (SEQ ID NO: 2). Nukleotidi so oštevilčeni na začetku vsake vrste. Netranslatirani regiji 5’ in 3’sta označeni z malimi črkami, aminokislinski ostanki so oštevilčeni nad sekvenco in se začnejo s Ser 1 proteinske sekvence zrelega mpl liganda (ML). Meje domnevnega eksona 3 so označene s puščicami, potencialna N-glikozilima mesta pa so v okvirčkih. Cisteinski ostanki so označeni s piko nad sekvenco. Podčrtana sekvenca ustreza N-terminalni sekvenci, določeni za mpl ligand, očiščen iz prašičje plazme.In FIG. 1 shows the deduced amino acid sequence (SEQ ID NO: 1) of the human mpl ligand (hML) cDNA and the coding nucleotide sequence (SEQ ID NO: 2). Nucleotides are numbered at the beginning of each species. The untranslated regions 5 'and 3′st are in lowercase, the amino acid residues are numbered above the sequence and begin with the Ser 1 protein sequence of the mature mpl ligand (ML). The boundaries of putative exon 3 are indicated by arrows, and the potential N-glycosyl sites are boxed. Cysteine residues are indicated by a dot above the sequence. The underlined sequence corresponds to the N-terminal sequence defined for the mpl ligand purified from porcine plasma.

SL 2 prikazuje postopek, uporabljen za test vgraditve ³H-timidina v mpl ligand. Da bi določili prisotnost mpl liganda iz različnih virov, celice Ba/F3 mpl P stradamo IL-3 24 ur v vlažnem inkubatoiju pri 37 °C, v mešanici 5 % CO₂ v zraku. Po končanem IL-3 stradanju celice zasadimo v posode za kulturo s 96 vdolbinicami z razredčenimi vzorci ali brez njih in kultiviramo 24 ur v inkubatorju za celično kulturo. V vsako vdolbinico dodamo 20 μΐ seruma brez medija RPMI, ki vsebuje 1 /xCi ³H-timidina, in pustimo 6-8 ur. Celice nato zberemo na filtrimih ploščah s 96 vdolbinicami in izperemo z vodo. Filtre nato preštejemo.SL 2 shows the procedure used to test the incorporation of ³ H-thymidine into the mpl ligand. To determine the presence of mpl ligand from different sources, Ba / F3 mpl P cells were starved of IL-3 for 24 hours in a humidified incubato at 37 ° C, in a mixture of 5% CO ₂ in air. After IL-3 starvation is complete, the cells are planted in 96-well culture dishes with or without diluted samples and cultured for 24 hours in a cell culture incubator. Add 20 μΐ serum free RPMI medium containing 1 / xCi ³ H-thymidine to each well and leave for 6-8 hours. The cells were then collected on 96-well filter plates and washed with water. The filters are then counted.

Sl. 3 prikazuje učinek pronaze, DTT in toplote na sposobnost APP za stimulacijo Ba/F3-mp/ celične proliferacije. Za pronazno digestijo APP pripojimo pronazo (Boehringer Mannheim) ali goveji serumski albumin na Affi-gellO (Biorad) in inkubiramo individualno z APP 18 ur pri 37 °C. Nato smole odstranimo s centrifugiranjem in testiramo supematante. APP segrejemo tudi za 4 minute na 80 °C ali naredimo 100 μΜ DTT, nato pa dializiramo proti PBS.FIG. 3 shows the effect of find, DTT, and heat on the ability of APP to stimulate Ba / F3-mp / cell proliferation. For the digestive digestion of APP, attach a find (Boehringer Mannheim) or bovine serum albumin to AffigelOl (Biorad) and incubate individually with APP for 18 hours at 37 ° C. The resins were then removed by centrifugation and the substrates tested. We also heat the APP for 4 minutes at 80 ° C or do 100 μΜ of DTT and then dialyze against PBS.

Sl. 4 prikazuje eluiranje aktivnosti mpl Uganda iz kolon: fenil-Toyopearl, bluesefaroze in ultralink-mp/. Frakcije 4-8 iz mpl afinitetnih kolon so tiste z najvišjo aktivnostjo, eluirane iz kolone.FIG. 4 shows the elution of mpl Uganda activity from columns: phenyl-Toyopearl, bluesepharose and ultralink-mp /. Fractions 4-8 from the mpl affinity columns are those with the highest activity eluted from the column.

Sl. 5 prikazuje SDS-PAGE eluiranih ultralink-mp/ frakcij. K 200 μΐ vsake frakcije 2-8 dodamo 1 ml acetona, ki vsebuje HCI (1 mM) pri -20 °C. Po 3 urah pri -20 °C vzorce centrifugiramo in nastale pelete izperemo 2-krat z acetonom pri -20 °C. Te pelete nato raztopimo v 30 μΐ SDS solubilizacijskega pufra, naredimo 100 μΜ DTT in segrevamo pri 90 °C 5 minut. Vzorce nato ločimo na SDS-poUakrilamidnem gelu (420 %) in proteine vizualiziramo z barvanjem s srebrom.FIG. 5 shows the SDS-PAGE of eluted ultralink-mp / fractions. To 200 μΐ of each fraction 2-8 was added 1 ml of acetone containing HCl (1 mM) at -20 ° C. After 3 hours at -20 ° C, the samples were centrifuged and the resulting pellets washed 2 times with acetone at -20 ° C. These pellets were then dissolved in 30 μΐ SDS solubilization buffer, made 100 μΜ DTT and heated at 90 ° C for 5 minutes. Samples were then separated on an SDS-poUacrylamide gel (420%) and proteins were visualized by silver staining.

Sl. 6 prikazuje eluiranje aktivnosti mpl liganda iz SDS-PAGE. Frakcijo 6 iz mpl afinitetne kolone ločimo na SDS-poliakrilamidnem gelu (4-20 %) pri nereduktivnih razmerah. Po elektroforezi razrežemo gel v 12 enakih regij in elektroeluiramo, kot je opisano v primerih. Elektroeluirane vzorce dializiramo v PBS in testiramo pri 1/20 razredčitvi. Standardi za Mr (molska masa), uporabljeni za kalibriranje gela, so standardi Novex Mark 12.FIG. 6 shows the elution of mpl ligand activity from SDS-PAGE. Separate the fraction 6 from the mpl affinity column on an SDS-polyacrylamide gel (4-20%) under non-reductive conditions. After electrophoresis, the gel was cut into 12 equal regions and electroeluted as described in the examples. Electro-eluted samples were dialyzed in PBS and tested at 1/20 dilution. The standards for Mr (molar mass) used to calibrate the gel are Novex Mark 12 standards.

Sl. 7 prikazuje učinek APP z odstranjenim mpl ligandom, na humano megakariocitopoezo. APP z odstranjenim mpl ligandom naredimo s prepuščanjem 1 ml preko 1 ml mpl afinitetne kolone (700 pg mpMgG/ml NHS-superoze, Pharmacia). Kulture humanih perifernih matičnih cehe naredimo 10 % z APP ali 10 % z APP z odstranjenim mpl ligandom in kultiviramo 12 dni. Megakariocitopoezo kvantitativno določimo, kot je opisano v primerih.FIG. 7 shows the effect of APP with the mpl ligand removed on human megakaryocytopoiesis. APP with the mpl ligand removed is made by passing 1 ml over 1 ml of the mpl affinity column (700 pg mpMgG / ml NHS superose, Pharmacia). Human peripheral mother guild cultures were made 10% with APP or 10% with APP with mpl ligand removed and cultured for 12 days. Megakaryocytopoiesis was quantified as described in the examples.

Sl. 8 prikazuje učinek mpZ-IgG na stimulacijo humane megakariocitopoeze z APP. Kulture humanih perifernih matičnih celic naredimo 10 % z APP in kultiviramo 12 dni. Na dneve 0,2 in 4 dodamo mpMgG (0,5 /xg) ali ANP-R-IgG (0,5 Mg)- Po 12 dneh kvantitativno določimo megakariocitopoezo, kot je opisano v Primerih. Grafično je prikazano povprečje paralelk vzorcev, dejanski podatki za paralelke pa so v oklepajih.FIG. 8 shows the effect of mpZ-IgG on the stimulation of human megakaryocytopoiesis by APP. Human peripheral stem cell cultures were made 10% with APP and cultured for 12 days. On days 0.2 and 4, mpMgG (0.5 / xg) or ANP-R-IgG (0.5 Mg) was added - After 12 days, megakaryocytopoiesis was quantified as described in the Examples. The average of the sample parallels is shown graphically, and the actual data for the parallels are in parentheses.

Sl. 9 prikazuje obe vijačnici fragmenta s 390 bp (bazni par) humane genomske DNA, ki kodira mpl ligand. Prikazani so: deducirana aminokislinska sekvenca eksona 3 (SEQ ED NO: 3), kodirna sekvenca (SEQ ID NO: 4) in njen komplement (SEQ ID NO:5).FIG. 9 shows both helixes of a 390 bp fragment (base pair) of human genomic DNA encoding the mpl ligand. Shown are: deduced amino acid sequence of exon 3 (SEQ ED NO: 3), coding sequence (SEQ ID NO: 4) and its complement (SEQ ID NO: 5).

Sl. 10 prikazuje deducirano aminokislinsko sekvenco zrelega humanega mpl liganda (hML) (SEQ ID NO: 6) in zrelega humanega eritropoetina (hEPO) (SEQ ID NO: 7). Napovedana aminokislinska sekvenca za humani mpl ligand je uvrščena s humano eritropoetinsko sekvenco. Identične amino kisline so v okvirčkih, praznine, uvedene za optimalno uvrstitev, pa so označene s pomišljaji. Potencialna N-glikozilacijska mesta so podčrtana s celo črto za hML in s prekinjeno za hEPO. Dva cisterna, pomembna za eritropoetinsko aktivnost, sta označena z veliko piko.FIG. 10 shows the deduced amino acid sequence of mature human mpl ligand (hML) (SEQ ID NO: 6) and mature human erythropoietin (hEPO) (SEQ ID NO: 7). The predicted amino acid sequence for the human mpl ligand is classified by the human erythropoietin sequence. Identical amino acids are boxed in, and gaps introduced for optimal ranking are dotted. Potential N-glycosylation sites are underlined by the entire line for hML and interrupted for hEPO. Two tanks important for erythropoietin activity are indicated by a large dot.

Sl. 11 prikazuje deducirano aminokislinsko sekvenco iz izo-oblik zrelega humanega mpl liganda hML (SEQ ID NO:6), hML2 (SEQ ID NO:8), hML3 (SEQ ED NO: 9) in hML4 (SEQ ID NO: 10). Identične amino kisline so v okvirjih, praznine pa so uvedene za optimalno uvrstitev in so označene s pomišljaji.FIG. 11 shows the deduced amino acid sequence from the iso-forms of the mature human mpl ligand hML (SEQ ID NO: 6), hML2 (SEQ ID NO: 8), hML3 (SEQ ED NO: 9), and hML4 (SEQ ID NO: 10). The identical amino acids are framed, and the gaps are introduced for optimal ranking and are marked with dashes.

Sl. 12A, 12B in 12C prikazujejo učinek humanega mpl liganda na Ba/F3-mp/ celično proliferacijo (A), humano megakariocitopoezo in vitro, kvantitativno določeno z uporabo radioaktivno označenega monoklonskega protitelesa murinega IgG, specifičnega za megakariocitni glikoprotein GPII_bIII_a (B), in murino trombopoezo, izmerjeno s testom oživitve trombocitov (C).FIG. 12A, 12B and 12C show the effect of human mpl ligand on Ba / F3-mp / cell proliferation (A), human megakaryocytopoiesis in vitro, quantified using radiolabeled murine IgG monoclonal antibody specific for megakaryocyte glycoprotein GPII _b III _a (B) , and murine thrombopoiesis as measured by a platelet recovery test (C).

293 celic transfektiramo s CaPO₄ postopkom (Gorman, C v DNA Cloning: A New Approach 2:143-190 [1985]) z vektorjem pRK5 samim, pRK5-hML ali s pRK5ML₁₅₃ preko noči (pRK5-ML₁₅₃ nastane z uvedbo stop kodona po ostanku 153 hML s PCR). Medij nato kondicioniramo 36 ur in testiramo za stimulacijo celične proliferacije Ba/F3-mp/, kot je opisano v Primeru 1 (A), ali humane megakariocitopoeze in vitro (B). Megakariocitopoezo kvantitativno določimo z uporabo ¹²⁵J radioaktivno označenega monoklonskega protitelesa (HP1-1D) murinega IgG za megakariocite specifičnega glikoproteina GPn_bffl_a, kot opisujejo (Grant et al., Blood 69:1334-1339 [1987]). Učinek delno očiščenega rekombinantnega ML (rML) na nastajanje trombocitov in vivo (C) določimo z uporabo testa oživitve trombocitoze, ki ga opisujejo McDonald, T.P. Proc.Soc.Exp. Biol. Med. 144:1006-1012 (1973). Delno očiščen rML pripravimo iz 200 ml kondicioniranega medija, ki vsebuje rekombinanten ML. Medij spustimo skozi 2 ml kolono bluesefaroze, uravnoteženo v PBS, in kolono izperemo s PBS ter eluiramo s PBS, ki vsebuje sečnino (2 M) in NaCl (2 M). Aktivno frakcijo dializiramo v PBS in naredimo 1 mg/ml z endotoksinom brez BSA. Vzorec vsebuje manj kot eno enoto endotoksina/ml. Mišim injiciramo bodisi 64000, 32000 ali 16000 enot rML ali samo polnilo. Vsako skupino sestavlja 6 miši. Prikazani sta srednja in standardna deviacija za vsako skupino, p vrednosti določimo z dvokončnim T-testom (2-tailed T-test) s primerjavo medianov.293 cells were transfected with the CaPO ₄ procedure (Gorman, C in DNA Cloning: A New Approach 2: 143-190 [1985]) with the pRK5 vector alone, pRK5-hML, or pRK5ML ₁₅₃ overnight (pRK5-ML _{153 was} generated by introducing a stop of codon by residue 153 hML with PCR). The medium was then conditioned for 36 hours and tested for stimulation of Ba / F3-mp / cell proliferation as described in Example 1 (A) or human megakaryocytopoiesis in vitro (B). Megakaryocytopoiesis was quantified using ¹²⁵ J radiolabeled monoclonal antibody (HP1-1D) murine IgG for megakaryocytes-specific glycoprotein GPn _b fl _a , as described (Grant et al., Blood 69: 1334-1339 [1987]). The effect of partially purified recombinant ML (rML) on platelet production in vivo (C) was determined using the thrombocytosis recovery assay described by McDonald, TP Proc.Soc.Exp. Biol. Med. 144: 1006-1012 (1973). Partially purified rML was prepared from 200 ml of conditioned medium containing recombinant ML. The medium was passed through a 2 ml column of bluesepharose balanced in PBS and the column was washed with PBS and eluted with PBS containing urea (2 M) and NaCl (2 M). The active fraction was dialyzed in PBS and made 1 mg / ml with endotoxin free of BSA. The sample contains less than one unit of endotoxin / ml. Mice were injected with either 64000, 32000, or 16000 rML units or filler alone. Each group consists of 6 mice. Shows the mean and standard deviation for each group, p values are determined by a two-tailed T-test comparing the medians.

Sl. 13 primerja učinek izo-oblik in variant humanega mpl liganda pri testu Ba/F3-znp/ celične proliferacije. hML, mock, hML2, hML3, hML(R153A, R154A) in hML153 testiramo pri raznih razredčitvah, kot je opisano v Primeru 1.FIG. 13 compares the effect of iso-forms and variants of the human mpl ligand on the Ba / F3-znp / cell proliferation assay. hML, mock, hML2, hML3, hML (R153A, R154A) and hML153 were tested at various dilutions as described in Example 1.

Sl. 14A, 14B in 14C prikazujejo deducirano aminokislinsko sekvenco (SEQ ID NO:1) humanega mpl liganda (hML) ali humanega TPO (hTPO) in kodirno sekvenco humane genomske DNA (SEQ ID NO: 11). Nukleotidni in aminokislinski ostanki so oštevilčeni na začetku vsake vrste.FIG. 14A, 14B and 14C show the deduced amino acid sequence (SEQ ID NO: 1) of human mpl ligand (hML) or human TPO (hTPO) and the human genomic DNA coding sequence (SEQ ID NO: 11). Nucleotide and amino acid residues are numbered at the beginning of each species.

Sl. 15 prikazuje SDS-PAGE očiščenih 293-rhML₃₃₂ in 293-rhML₁₅₃.FIG. 15 shows SDS-PAGE of purified 293-rhML ₃₃₂ and 293-rhML ₁₅₃ .

Sl. 16 prikazuje nukleotidno sekvenco: cDNA kodirno (SEQ ID NO: 12) in deducirano aminokislinsko sekvenco (SEQ ID NO: 13) odprtega bralnega okvirja izooblike murinega ML. Ta izo-oblika zrelega murinega mpl liganda vsebuje 331 amino kislinskih ostankov, 4 manj kot domnevni mML s popolno dolžino, in je zato označena kot mML2. Nukleotidi so oštevilčeni na začetku vsake vrste. Aminokislinski ostanki so oštevilčeni nad sekvenco in se začnejo s Ser 1. Potencialna N-glikozilacijska mesta so podčrtana. Cisteinski ostanki so označeni s piko nad sekvenco.FIG. 16 shows the nucleotide sequence: cDNA encoding (SEQ ID NO: 12) and deduced amino acid sequence (SEQ ID NO: 13) of the murine ML isoform open reading frame. This iso-form of the mature murine mpl ligand contains 331 amino acid residues, 4 less than the putative full-length mML, and is therefore designated mML2. Nucleotides are numbered at the beginning of each species. The amino acid residues are numbered above the sequence and begin with Ser 1. Potential N-glycosylation sites are underlined. Cysteine residues are indicated by a dot above the sequence.

Sl. 17 prikazuje sekvenco cDNA (SEQ ED NO: 14) in domnevno proteinsko sekvenco (SEQ ID NO: 15) izooblike tega murinega ML (mML). Nukleotidi so oštevilčeni na začetku vsake vrste. Aminokislinski ostanki so oštevilčeni nad sekvenco in se začnejo s Ser 1. Ta izo-oblika zrelega murinega mpl Uganda vsebuje 335 aminokislinskih ostankov in je verjetno mpl Ugand s popolno dolžino, označen kot mML. Signalna sekvenca je podčrtana s pomišljali, verjetna cepitvena točka pa je označena s puščico. Netranslatirani regiji 5’ in 3’ sta označeni z malimi črkami. Dve deleciji, ugotovljeni kot rezultat alternativnega spajanja (mML2 in mML3), sta podčrtani. Štirje cisteinski ostanki so označeni s piko. Sedem potencialnih N-glikozilacijskih mest je v okvirčkih.FIG. 17 shows the cDNA sequence (SEQ ED NO: 14) and the putative protein sequence (SEQ ID NO: 15) of the isoform of this murine ML (mML). Nucleotides are numbered at the beginning of each species. The amino acid residues are numbered above the sequence and begin with Ser 1. This iso-form of mature murine mpl Uganda contains 335 amino acid residues and is probably full length mpl Ugand designated mML. The signal sequence is underlined by dashes and the probable cleavage point is indicated by an arrow. Non-translated regions 5 'and 3' are case-sensitive. The two deletions identified as a result of alternative splicing (mML2 and mML3) are underlined. Four cysteine residues are indicated by a dot. Seven potential N-glycosylation sites are framed.

Na sl. 18 je primerjava sekvence deducirane amino kisline izo-oblike humanega ML (hML3) (SEQ ID NO:9) in izo-oblike murinega ML, označene kot mML3 (SEQ ED NO:16). Domnevna aminokislinska sekvenca za humani mpl Ugand je uvrščena s sekvenco murinega mpl Uganda. Identične amino kisline so v okvirčkih, praznine pa so uvedene za optimalno uvrstitev in označene s pomišljaji. Amino kisline so oštevilčene na začetku vsake vrste.In FIG. 18 is a comparison of the deduced amino acid sequence of the iso-form of human ML (hML3) (SEQ ID NO: 9) and the iso-form of murine ML, designated mML3 (SEQ ED NO: 16). The putative amino acid sequence for Ugandan human mpl is classified by the Ugandan mpl sequence. Identical amino acids are in the boxes and blanks are introduced for optimal ranking and dash-marked. The amino acids are numbered at the beginning of each species.

Na sl. 19 je primerjava domnevnih aminokislinskih sekvenc izo-oblik zrelega ML iz mišjega ML (SEQ ED NO:17), prašičjega ML (SEQ ED NO: 18) in humanega ML (SEQ ED NO: 6). Aminokislinske sekvence so uvrščene s prazninami, označenimi s pomišljaji, uvedenimi za optimalno uvrstitev. Amino kisline so oštevilčene na začetku vsake vrste z identičnimi ostanki v okvirčkih. Potencialna N-glikozilacijska mesta so označena z osenčenimi okvirčki, cisteinski ostanki pa so označeni s piko. Konzervirani dibazični aminokislinski motiv, ki je potencialno cepišče proteaze, je podčrtan. Delecija štirih amino kislin, do katere pride v vseh treh vrstah (ML2), je obkrožena s poudarjenim okvirčkom.In FIG. 19 is a comparison of putative amino acid sequences of iso-forms of mature ML from murine ML (SEQ ED NO: 17), porcine ML (SEQ ED NO: 18), and human ML (SEQ ED NO: 6). Amino acid sequences are ranked with blanks marked with dashes introduced for optimal ranking. Amino acids are numbered at the beginning of each species with identical residues in the frames. Potential N-glycosylation sites are indicated by shaded frames, and cysteine residues are indicated by a dot. The conserved dibasic amino acid motif, which is a potential protease cleavage site, is underlined. The deletion of the four amino acids occurring in all three species (ML2) is encircled by a prominent frame.

Sl. 20 prikazuje sekvenco cDNA (SEQ ID NO: 19) in domnevno sekvenco zrelega proteina (SEQ DD NO: 18) izo-obUke prašičjega ML (pML). Ta izo-oblika prašičjega mpl Uganda vsebuje 332 aminokislinskih ostankov in je verjetno mpl Ugand s popolno dolžino, označen kot pML. Nukleotidi so oštevilčeni na začetku vsake vrste. Aminokislinski ostanki so oštevilčeni nad sekvenco in se začnejo s Ser 1.FIG. 20 shows the cDNA sequence (SEQ ID NO: 19) and the putative sequence of the mature protein (SEQ DD NO: 18) of porcine ML (pML) iso-training. This iso-form of pig mpl Uganda contains 332 amino acid residues and is probably full-length mpl Ugand designated pML. Nucleotides are numbered at the beginning of each species. The amino acid residues are numbered above the sequence and begin with Ser 1.

Sl. 21 prikazuje sekvenco cDNA (SEQ ED NO: 20) in domnevno sekvenco zrelega proteina (SEQ ID NO: 21) izo-obUke prašičjega ML (pML2). Ta izo-oblika prašičjega mpl Uganda vsebuje 328 aminokisUnskih ostankov in je prašičji mpl Ugand s popolno dolžino v takšni obliki, ki ima delecijo štirih ostankov, označeni kot pML2.FIG. 21 shows the cDNA sequence (SEQ ED NO: 20) and the putative sequence of the mature protein (SEQ ID NO: 21) of porcine ML (pML2) iso-training. This iso-form of Uganda pig mpl contains 328 amino acid residues and is a full-length pig mpl Ugand in such a form that has a deletion of four residues designated pML2.

Nukleotidi so oštevilčeni na začetku vsake vrste. Amino kislinski ostanki so oštevilčeni nad sekvenco in se začnejo s Ser 1.Nucleotides are numbered at the beginning of each species. The amino acid residues are numbered above the sequence and begin with Ser 1.

Na sl. 22 je primerjava deducirane aminokislinske sekvence izo-oblike prašičjega ML (pML) s popolno dolžino (SEQ ID NO: 18) in izo-oblike prašičjega ML, označene kot pML2 (SEQ ID NO:21). Domnevna aminokislinska sekvenca za pML je uvrščena s sekvenco pML2. Identične amino kisline so v okvirčkih, praznine so uvedene za optimalno uvrstitev in so označene s pomišljaji. Amino kisline so oštevilčene na začetku vsake vrste.In FIG. 22 is a comparison of the deduced amino acid sequence of full-length porcine ML isoform (pML) (SEQ ID NO: 18) and porcine ML isoform, designated pML2 (SEQ ID NO: 21). The putative amino acid sequence for pML is classified by the pML2 sequence. Identical amino acids are in the boxes, blanks are introduced for optimum ranking and are marked with dashes. The amino acids are numbered at the beginning of each species.

Sl. 23 prikazuje pertinentne lastnosti plazmida pSVI5.ID.LL.MLORF (popolna dolžina ali TPO₃₃₂) , uporabljenega za transfekcijo gostiteljskih celic CHO-DP12 za izdelavo CHO-rhTPO₃₃₂.FIG. 23 shows the pertinent properties of the plasmid pSVI5.ID.LL.MLORF (full length or TPO ₃₃₂ ) used for transfection of CHO-DP12 host cells to produce CHO-rhTPO ₃₃₂ .

Sl. 24 prikazuje pertinentne lastnosti plazmida pSVI5.ID.LL.MLEPO-D (skrajšan ali TPO₁₅₃), uporabljenega za transfektiranje gostiteljskih celic CHO-DP12 za izdelavo CHO-rh-TPO₁₅₃.FIG. 24 shows the pertinent properties of the plasmid pSVI5.ID.LL.MLEPO-D (abbreviated or TPO ₁₅₃ ) used for transfection of CHO-DP12 host cells to produce CHO-rh-TPO ₁₅₃ .

Sl. 25A, 25B in 25C prikazujejo učinek E. coli-rhTPO(_Me ⁴ 153) na trombocite (A), eritrocite (B) in levkocite (C) v normalnih miših. Dvema skupinama po 6 samic miši C57 B6 injiciramo dnevno bodisi pufer PBS ali 0,3/xg E. coli-rhTPO(Me ⁴ 153) (100μΐ sc.). Na dan 0 in na dneve 3-7 vzamemo 40 μΐ krvi iz orbitalnega sinusa. To kri takoj razredčimo v 10 ml komercialnega razredčila, popolno štetje krvnih celic pa izvedemo na hematološkem analizatorju Serrono Baker Hematology Analyzer 9018. Podatki so predstavljeni kot povprečje ± standardna napaka povprečja.FIG. 25A, 25B and 25C show the effect of E. coli-rhTPO ( _Me ⁴ 153) on platelets (A), erythrocytes (B), and leukocytes (C) in normal mice. Two groups of 6 C57 B6 mice were injected daily with either PBS buffer or 0.3 / xg E. coli-rhTPO (Me ⁴ 153) (100μΐ sc.). On day 0 and days 3-7, 40 μΐ of blood is taken from the orbital sinus. Dilute this blood immediately to 10 ml of commercial diluent, and complete blood cell counts are performed on a Serrono Baker Hematology Analyzer 9018. Data are presented as mean ± standard error of the mean.

Sl. 26A, 26B in 26C prikazujejo učinek E. coli-rhTPO(_Met ⁴ 153) na trombocite (A), eritrocite (B) in levkocite (C) v subletalno obsevanih miših. Dve skupini po 10 samic miši C57 B6 izpostavimo subletalnemu obsevanju s 750 cGy gama radiacije iz vira ¹³⁷Cs in jim injiciramo dnevno bodisi pufer PBS ali 3,0 μξ E. coli-rhTPO(Met⁴ ₁₅₃) (100 μ\ sc.). Na dan 0 in pri kasnejših vmesnih časovnih točkah vzamemo 40 μΐ krvi iz orbitalnega sinusa. To kri takoj razredčimo v 10 ml komercialnega razredčila, popolno štetje krvnih celic pa izvedemo na hematološkem analizatorju Serrono Baker Hematology Analyzer 9018. Podatki so predstavljeni kot povprečje ± standardna napaka povprečja.FIG. 26A, 26B and 26C show the effect of E. coli-rhTPO ( _Met ⁴ 153) on platelets (A), erythrocytes (B) and leukocytes (C) in sublethally irradiated mice. Two groups of 10 C57 B6 mice were exposed to sublethal irradiation with 750 cGy gamma radiation from ¹³⁷ Cs and injected daily with either PBS buffer or 3.0 μξ E. coli-rhTPO (Met ⁴ ₁₅₃ ) (100 μ \ sc.). At day 0 and at later intermediate time points, 40 μΐ of blood is taken from the orbital sinus. Dilute this blood immediately to 10 ml of commercial diluent, and complete blood cell counts are performed on a Serrono Baker Hematology Analyzer 9018. Data are presented as mean ± standard error of the mean.

Sl. 27A, 27B in 27C prikazujejo učinek CHO-rhTPO₃₃₂ na trombocite (A), eritrocite (B) in levkocite (C) v normalnih miših. Dvema skupinama po 6 samic miši C67 B6 injiciramo dnevno bodisi pufer PBS ali 0,3 /tg CHO-rhTPO^ (100 /tl sc.). Na dan 0 in na dneve 3-7 vzamemo 40 /tl krvi iz orbitalnega sinusa. To kri takoj razredčimo v 10 ml komercialnega razredčila, popolno štetje krvnih celic pa izvedemo na hematološkem analizatorju Serrono Baker Hematology Analyzer 9018. Podatki so predstavljeni kot povprečje ± standardna napaka povprečja.FIG. 27A, 27B and 27C show the effect of CHO-rhTPO ₃₃₂ on platelets (A), erythrocytes (B) and leukocytes (C) in normal mice. Two groups of 6 C67 B6 mice were injected daily with either PBS buffer or 0.3 / tg CHO-rhTPO4 (100 / tl sc.). On day 0 and days 3-7, 40 / tl of blood is taken from the orbital sinus. Dilute this blood immediately to 10 ml of commercial diluent, and complete blood cell counts are performed on a Serrono Baker Hematology Analyzer 9018. Data are presented as mean ± standard error of the mean.

Sl. 28 prikazuje krivulje odziva na dozo za različne oblike rhTPO, dobljenega iz različnih celičnih linij. Krivulje odziva na dozo so konstruirane za rhTPO iz naslednjih celičnih linij: hTPO₃₃₂ iz CHO (popolna dolžina iz ovarijskih celic kitajskega hrčka); hTO_Met ⁴ ₁₅₃) (iz E. coli izvedena skrajšana oblika z N-terminalnim metioninom); hTPO₃₃₂ (TPO s popolno dolžino iz 293 humanih celic; 155 E-Coli brez Met (skrajšana oblika [rhTPO₁₅₅] brez terminalnega metionina iz E.coli). Skupinam po 6 samic miši C57 B6 injiciramo dnevno 7 dni rhTPO, odvisno od skupine. Vsak dan vzamemo 40 /tl krvi iz orbitalnega sinusa za popolno štetje krvi. Podatki, predstavljeni zgoraj, so maksimalni učinki, ki jih opazimo pri različnih obdelavah, z izjemo (met 153 E-Coli), in do katerih pride na sedmi dan obdelave. V zgoraj omenjeni skupini met 153 E-Coli opazimo petega dne maksimalni učinek. Podatki so predstavljeni kot povprečje ± standardna napaka povprečja.FIG. 28 shows dose response curves for different forms of rhTPO obtained from different cell lines. Dose response curves were constructed for rhTPO from the following cell lines: hTPO ₃₃₂ from CHO (full length from Chinese hamster ovary cells); hTO _Met ⁴ ₁₅₃ ) (abbreviated form with N-terminal methionine from E. coli); hTPO ₃₃₂ (full-length TPO from 293 human cells; 155 E-Coli without Met (shortened form [rhTPO ₁₅₅ ] without terminal methionine from E.coli). Groups of 6 C57 B6 mice were injected daily for 7 days with rhTPO depending on the group We take 40 / tl of blood from the orbital sinus every day for a complete blood count. The data presented above are the maximum effects observed with different treatments, with the exception of (met 153 E-Coli) occurring on the seventh day of treatment. In the met-153 E-Coli group mentioned above, the maximum effect is observed on day 5. Data are presented as mean ± standard error of the mean.

Sl. 29 prikazuje krivulje odziva na dozo, pri čemer primerjamo aktivnost nastalega rhTPO s popolno dolžino in pristriženega, nastalega v celicah CHO s skrajšano obliko iz E. coli. Skupinam po 6 samic miši C57 B6 injiciramo dnevno 0,3 /tg rhTPO različnih tipov. Na dneve 2-7 vzamemo 40 /tl krvi iz orbitalnega sinusa za popolno štetje krvi. Obdelovane skupine so: TPO₁₅₃, skrajšana oblika TPO iz E. coli; TPO₃₃₂ (mešana frakcija) TPO s popolno dolžino, ki vsebuje približno 80-90 % oblike s popolno dolžino in 10-20 % pristrižene oblike; TPO332(30K frakcija) = očiščena pristrižena frakcija iz originalnega mešanega pripravka; TPO332(70K frakcija) = očiščena frakcija TPO s popolno dolžino iz originalnega mešanega pripravka. Podatki so predstavljeni kot povprečje ± standardna napaka povprečja.FIG. 29 shows dose-response curves comparing the activity of full-length rhTPO produced and a haircut produced in shortened form E. coli CHO cells. Groups of 6 C57 B6 mice were injected daily with 0.3 / tg rhTPO of different types. On days 2-7, 40 / tl of blood is taken from the orbital sinus for complete blood counting. The treated groups are: TPO ₁₅₃ , abbreviated form of TPO from E. coli; TPO ₃₃₂ (mixed fraction) Full-length TPO containing about 80-90% of full-length shape and 10-20% of haircut shape; TPO332 (30K fraction) = purified clipped fraction from the original mixed preparation; TPO332 (70K fraction) = purified full-length TPO fraction from the original mixed preparation. Data are presented as mean ± standard error of the mean.

Na sl. 30 je shema, ki prikazuje test KIRA ELISA za merjenje TPO. Slika prikazuje kimero MPL/Rse.gD in ustrezne dele parentalnih receptorjev, končni konstrukti (desni del slike) in diagram poteka (levi del slike) pa prikazujejo ustrezne stopnje testa.In FIG. 30 is a diagram showing a KIRA ELISA test for measuring TPO. The figure shows the MPL / Rse.gD chimera and the corresponding parts of the parental receptors, and the final constructs (right part of the image) and the flowchart (left part of the image) show the corresponding test rates.

Sl. 31 je diagram poteka testa KIRA ELISA, ki prikazuje vsako stopnjo v postopku.FIG. 31 is a flowchart of the KIRA ELISA test showing each step in the process.

Sl. 32A-32L prikazujejo nukleotidno sekvenco (SEQ ID NO: 22) ekspresijskega vektorja pSVI17.ID.LL, uporabljenega za ekspresijo Rse.gD v primeru 17.FIG. 32A-32L show the nucleotide sequence (SEQ ID NO: 22) of the expression vector pSVI17.ID.LL used for the expression of Rse.gD in Example 17.

Sl. 33 shematično prikazuje pripravo plazmida pMPl.FIG. 33 schematically shows the preparation of the plasmid pMPl.

Sl. 34 shematično prikazuje pripravo plazmida pMP21.FIG. 34 schematically shows the preparation of plasmid pMP21.

Sl. 35 shematično prikazuje pripravo plazmida pMP151.FIG. 35 shows schematically the preparation of plasmid pMP151.

Sl. 36 shematično prikazuje pripravo plazmida pMP202.FIG. 36 schematically shows the preparation of plasmid pMP202.

Sl. 37 shematično prikazuje pripravo plazmida pMP172.FIG. 37 schematically shows the preparation of plasmid pMP172.

Sl. 38 shematično prikazuje pripravo plazmida pMP210.FIG. 38 schematically shows the preparation of plasmid pMP210.

Sl. 39 prikazuje tabelo petih klonov, ki najbolje izražajo TPO iz pMP210 plazmidne banke (SEQ ID NOS: 23,24,25,26,27 in 28).FIG. 39 shows a table of five clones that best express TPO from the pMP210 plasmid bank (SEQ ID NOS: 23,24,25,26,27 and 28).

Sl. 40 shematično prikazuje pripravo plazmida pMP41.FIG. 40 schematically shows the preparation of plasmid pMP41.

Sl. 41 shematično prikazuje pripravo plazmida pMP57.FIG. 41 schematically shows the preparation of plasmid pMP57.

Sl. 42 shematično prikazuje pripravo plazmida pMP251.FIG. 42 schematically shows the preparation of plasmid pMP251.

I. DefinicijeI. Definitions

Na splošno imajo naslednje besede ali fraze določeno definicijo, kadar jih uporabljamo v opisu, primerih in zahtevkih.Generally, the following words or phrases have a definite definition when used in descriptions, examples, and claims.

Kaotropično sredstvo se nanaša na spojino, ki v vodni raztopini in v prikladnih koncentracijah lahko povzroči spremembo v prostorski konfiguraciji ali konformaciji proteina z vsaj delno prekinitvijo sil, ki so odgovorne za vzdrževanje normalne sekundarne in terciarne strukture proteina. Take spojine vključujejo npr. sečnino, gvanidin.HCl in natrijev tiocianat. Visoke koncentracije, navadno 4-9M, teh spojin so normalno potrebne, da izrazijo konformacijske efekte na proteinu.A chaotropic agent refers to a compound which, in aqueous solution and at suitable concentrations, can cause a change in the spatial configuration or conformation of the protein by at least partially interrupting the forces responsible for maintaining the normal secondary and tertiary structure of the protein. Such compounds include e.g. urea, guanidine.HCl and sodium thiocyanate. High concentrations, typically 4-9M, of these compounds are normally required to express conformational effects on the protein.

Citokin je generični izraz za proteine, ki jih sprošča ena celična populacija in delujejo na drugo celico kot intercelični mediatoiji. Primeri takih citokinov so limfokini, monokini in tradicionalni polipeptidni hormoni. Med citokine so vključeni rastni hormoni, insulinu podobni rastni faktorji, humani rastni hormon, N-metionilni humani rastni hormon, goveji rastni hormon, paratiroidni hormon, tiroksin, insulin, proinsulin, relaksin, prorelaksin, glikoproteinski hormoni, kot npr. folikle stimulirajoči hormon (FSH), tiroid stimulirajoči hormon (TSH) in leutinizirajoči hormon (LH), hematopoetični rastni faktor, hepatični rastni faktor, fibroblastni rastni faktor, prolaktin, placentni laktogen, tumor nekrotizirajoči faktor - a (TNF-α in TNF-/3), mulerian inhibirajoča substanca, z mišjim gonadotropinom povezan peptid, inhibin, aktivin, vaksulami endotelijski rastni faktor, integrin, živčni rastni faktorji, kot npr. NGF-jS, trombocitni rastni faktor, faktorji za transformiranje rasti (TGF), kot npr. TGF-α in TGF-/3, insulinu podoben rastni faktor I in II, eritropoetin (EPO), osteoinduktivni faktorji, interferoni, kot interferon-α, β in γ, kolonije stimulirajoči faktorji (CSF), kot npr. makrofagni CSF (M-CSF), granulocitni makrofagni-CSF (GM-CSF) in granulocitni-CSF (G-CSF), interlevkini (IL), kot npr. IL-1, EL-Ια, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, BL-11, IL-12 in drugi polipeptidni faktorji, vključno LIF, SCF in kit-ligand. Predhodni izrazi, kot jih uporabljamo tukaj, so mišljeni, da vključujejo proteine iz naravnih virov ali iz rekombinatne celične kulture. Podobno so izrazi namenjeni, da vključujejo tudi biološko aktivne ekvivalente; npr. take, ki se razlikujejo v aminokislinski sekvenci po eni ali več amino kislin ali po tipu ali obsegu glikozilacije.Cytokine is a generic term for proteins released by one cell population and acting on another cell as intercellular mediatoids. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones, insulin-like growth factors, human growth hormone, N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, glycoprotein hormones, such as. follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH) and leutinizing hormone (LH), hematopoietic growth factor, hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrotizing factor - a (TNF-α and TNF- / 3), a mullerian inhibitory substance, a mouse gonadotropin-related peptide, inhibin, activin, endothelial growth factor, integrin, nerve growth factors, e.g. NGF-jS, platelet-derived growth factor, growth-transforming factors (TGF), e.g. TGF-α and TGF- / 3, insulin-like growth factor I and II, erythropoietin (EPO), osteoinductive factors, interferons, such as interferon-α, β and γ, colony stimulating factors (CSF), such as e.g. macrophage CSF (M-CSF), granulocyte macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF), interleukins (IL), such as e.g. IL-1, EL-Ια, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, BL-11, IL-12 and other polypeptides factors including LIF, SCF and kit ligand. The foregoing terms as used herein are intended to include proteins from natural sources or from recombinant cell culture. Similarly, the terms are intended to include biologically active equivalents; e.g. those which differ in amino acid sequence by one or more amino acids or by the type or extent of glycosylation.

mpl ligand, mpl ligandski polipeptid, ML, trombopoetin ali TPO so uporabljeni izmenljivo in obsegajo katerikoli peptid, ki ima lastnost za vezavo na mpl, člana citokinske receptorske superdružine, in ima biološko lastnost ML, definirano spodaj. Zgledna biološka lastnost je sposobnost, da stimulira vgraditev označenih nukleotidov (npr. ³H-timidin) v DNA od IL-3 odvisnih celic Ba/F3, transfektiranih s humanim mpl P. Druga zgledna biološka lastnost je sposobnost, da stimulira vgraditev ³⁵S v krožeče trombocite pri testu oživitve mišjih trombocitov. Ta definicija obsega polipeptid, izoliran iz vira mpl Uganda, kot npr. aplastične prašičje plazme, opisane tukaj, aU iz drugega vira, kot npr. druge živalske vrste, vključno ljudi, aU pripravljen z rekombinantnimi ali sintetičnimi postopki, vključuje pa tudi variantne obhke, vključno njihove funkcionalne derivate, fragmente, alele, izo-oblike in analoge.mpl ligand, mpl ligand polypeptide, ML, thrombopoietin or TPO are used interchangeably and comprise any peptide having binding properties to mpl, a member of the cytokine receptor superfamily, and having the ML biological property defined below. An exemplary biological property is the ability to stimulate the incorporation of labeled nucleotides (e.g., ³ H-thymidine) into DNA from IL-3 dependent Ba / F3 cells transfected with human mpl P. Another exemplary biological property is the ability to stimulate the incorporation of ³⁵ S into circulating platelets in a mouse platelet recovery test. This definition encompasses a polypeptide isolated from an Ugandan mpl source such as e.g. aplastic porcine plasma described herein, aU from another source, such as e.g. other animal species, including humans, aU prepared by recombinant or synthetic processes, and including variant arrays including their functional derivatives, fragments, alleles, iso-forms and analogues.

Fragment mpl Uganda ali fragment TPO je del sekvence naravnega zrelega mpl liganda ali TPO s popolno dolžino z delecijo enega ali več aminokislinskih ostankov ali enot ogljikohidratov. Do delecije aminokislinskih ostankov lahko pride kjerkoli v peptidu, vključno na bodisi N-terminalnem ali C-terminalnem koncu ali interno. Fragment ima vsaj eno biološko lastnost skupno z mpl ligandom. Fragment mpl liganda ima značilno zaporedno sekvenco vsaj 10,15,20,25,30 ali 40 aminokislinskih ostankov, ki so identični tistim v sekvenci mpl liganda, izoliranega iz sesalca, vključno liganda, izoliranega iz aplastične prašičje plazme ali humanega ali murinega liganda, posebno iz njegove EPO-domene. Reprezentativna primera N-terminalnih fragmentov sta hML₁₅₃ ali TP0(Met⁴ 1-153).The Ugand mpl fragment or TPO fragment is part of a sequence of natural mature mpl ligand or full-length TPO with the deletion of one or more amino acid residues or units of carbohydrates. Deletion of amino acid residues can occur anywhere in the peptide, including at either the N-terminal or C-terminal end or internally. The fragment has at least one biological property in common with the mpl ligand. The mpl ligand fragment has a typical sequential sequence of at least 10,15,20,25,30 or 40 amino acid residues identical to that in the mammalian mpl ligand sequence, including a ligand isolated from aplastic porcine plasma or human or murine ligand, in particular from his EPO domain. Representative examples of N-terminal fragments are hML ₁₅₃ or TP0 (Met ⁴ 1-153).

Variante mpl liganda ali sekvenčne variante mpl liganda, kot je definirano tukaj, pomenijo biološko aktiven mpl ligand, kot je definiran spodaj, ki ima manj kot 100 % sekvenčno identičnost z mpl ligandom, izoliranim iz rekombinantne celične kulture ali aplastične prašičje plazme ali humanim ligandom z deducirano sekvenco, prikazano na sl. 1 (SEQ ED NO: 1). Navadno ima varianta biološko aktivnega mpl liganda aminokislinsko sekvenco z vsaj približno 70 % aminokislinsko sekvenčno identičnostjo z mpl ligandom, izoliranim iz aplastične prašičje plazme ali zrelim murinom ali humanim ligandom ali njegovimi fragmenti (sl. 1 [SEQ ID NO: 1]), prednostno vsaj približno 75 %, bolj prednostno vsaj približno 80 %, še bolj prednostno vsaj približno 85 %, še celo bolj prednostno vsaj 90 % in najbolj prednostno vsaj približno 95 %.Mpl ligand variants or sequential mpl ligand variants as defined herein mean a biologically active mpl ligand, as defined below, that has less than 100% sequence identity with the mpl ligand isolated from recombinant cell culture or aplastic porcine plasma or human ligand with the deduced sequence shown in FIG. 1 (SEQ ED NO: 1). Typically, a variant of a biologically active mpl ligand has an amino acid sequence with at least about 70% amino acid sequence identity with the mpl ligand isolated from aplastic porcine plasma or mature murine or human ligand or fragments thereof (Fig. 1 [SEQ ID NO: 1]), preferably at least about 75%, more preferably at least about 80%, more preferably at least about 85%, even more preferably at least 90%, and most preferably at least about 95%.

Kimemi mpl ligand je polipeptid, ki obsega mpl ligand s popolno dolžino ali enega ali več njegovih fragmentov, ki so pripojeni ali vezani na drug heterologen polipeptid ali enega ali več njegovih fragmentov. Kimera ima vsaj eno biološko lastnost, skupno z mpl ligandom. Drugi polipeptid je značilno citokin, imunoglobulin ali njegov fragment.A kimemi mpl ligand is a polypeptide comprising a full length mpl ligand or one or more fragments thereof that are attached or bound to another heterologous polypeptide or one or more fragments thereof. The chimera has at least one biological property in common with the mpl ligand. The second polypeptide is typically a cytokine, an immunoglobulin, or a fragment thereof.

Izolirani mpl ligand, visoko očiščeni mpl ligand in v bistvu homogeni mpl ligand uporabljamo izmenljivo in pomeni mpl ligand, ki je očiščen iz vira mpl liganda ali je pripravljen z rekombinantnimi ali sintetičnimi postopki in je zadosti očiščen drugih peptidov ali proteinov, da (1) dobimo vsaj 15 in prednostno 20 aminokislinskih ostankov N-terminalne ali interne aminokislinske sekvence z uporabo sekvenatoija z vrtečo čašo ali najboljšega komercialno dosegljivega aminokislinskega sekvenatoija ali takega, ki je modificiran po objavljenih postopkih, npr. od datuma vložitve te prijave, ali (2) do homogenosti z SDS-PAGE pri nereduktivnih ali reduktivnih pogojih z uporabo barvila Coomassie blue ali prednostno srebrne barve.Isolated mpl ligand, highly purified mpl ligand and substantially homogeneous mpl ligand are used interchangeably and mean mpl ligand that is purified from the source of the mpl ligand or prepared by recombinant or synthetic processes and sufficiently purified by other peptides or proteins to (1) at least 15 and preferably 20 amino acid residues of the N-terminal or internal amino acid sequence using a rotary beaker sequenotope or the best commercially available amino acid sequence, or one modified by published methods, e.g. from the date of filing of this application, or (2) until homogeneous with SDS-PAGE under non-reductive or reductive conditions using Coomassie blue or preferably silver.

Homogenost tukaj pomeni manj kot približno 5 % kontaminacijo z drugimi proteinskimi viri.Homogeneity here means less than about 5% contamination with other protein sources.

Biološka lastnost, kadar jo uporabimo v zvezi z mpl ligandom ali izoliranim mpl ligandom, pomeni, da ima le-ta trombopoetično aktivnost ali efektorsko ali antigensko funkcijo ali aktivnost in vivo, ki je direktno ali indirektno povzročena ali izvedena z mpl ligandom (ali v njegovi naravni ali denaturirani konformaciji) ali njegovim fragmentom. Efektorske funkcije vključujejo vezavno aktivnost mpl in vse vezavne aktivnosti nosilca, agonizem ali antagonizem mpl, posebno transdukcijo proliferativnega signala, vključno replikacijo, regulatorno funkcijo DNA, modulacijo biološke ativnosti drugih citokinov, receptorsko (posebno citokinsko) aktivacijo, deaktivacijo, regulacijo navzgor ali navzdol, celično rast ali diferenciacijo ipd. Antigenska funkcija pomeni, da ima epitopsko ali antigensko mesto, ki je sposobno navzkrižne reakcije s protitelesi, vzgojenimi proti naravnemu mpl ligandu. Glavna antigenska funkcija mpl Ugandskega polipeptida je ta, da se veže z afiniteto vsaj 10⁶ 1/mol na protitelo, vzgojeno proti mpl ligandu, izoliranem iz aplastične prašičje plazme. Navadno se polipeptid veže z afiniteto vsaj 10⁷1/mol. Najbolj prednostno je antigensko aktiven mpl Ugandski polipeptid takšen, ki se veže na protitelo, vzgojeno proti mpl Ugandu, ki ima eno od zgoraj opisanih efektorskih funkcij. Protitelesa, uporabljena za to, da definirajo biološko aktivnost, so zajčja poliklonska protitelesa, ki jih vzgojimo s formuliranjem mpl Uganda, izoliranega iz rekombinantne ceUčne kulture ali aplastične prašičje plazme v Freundovem kompletnem adjuvansu, subkutanim injiciranjem formulacije, in poživitvijo imunskega odziva z intraperitonealno injekcijo formulacije do titra protitelesnega platoja mpl Uganda.A biological property when used in connection with an mpl ligand or an isolated mpl ligand means that it has a thrombopoietic activity or effector or antigenic function or activity in vivo that is directly or indirectly induced or derived by the mpl ligand (or in natural or denatured conformation) or a fragment thereof. Effector functions include mpl binding activity and all carrier binding activities, mpl agonism or antagonism, specific proliferation signal transduction, including replication, DNA regulatory function, modulation of biological activity of other cytokines, receptor (special cytokine) activation, deactivation, up or down regulation, cellular growth or differentiation, etc. Antigenic function means that it has an epitope or antigenic site capable of cross-reacting with antibodies raised against the natural mpl ligand. The main antigenic function of the mpl Ugandan polypeptide is that it binds with an affinity of at least 10 ⁶ 1 / mol to an antibody raised against the mpl ligand isolated from aplastic porcine plasma. Typically, the polypeptide binds with an affinity of at least 10 ⁷ 1 / mol. Most preferably, the antigenically active mpl Ugandan polypeptide is one that binds to an antibody grown against mpl Ugandan having one of the effector functions described above. The antibodies used to define biological activity are rabbit polyclonal antibodies raised by formulation of mpl Uganda isolated from recombinant tube culture or aplastic porcine plasma in Freund's complete adjuvant, subcutaneous injection of the formulation and boosting the immune response and formulation of the immune response to the titer of the antibody plateau mpl Uganda.

Izraz biološko aktiven, kadar ga uporabljamo v zvezi z mpl Ugandom aU izoliranim mpl Ugandom, pomeni mpl Ugand ali poUpeptid, ki ima trombopoetično aktivnost aU prispeva efektorsko funkcijo mpl Uganda, izoliranega iz aplastične prašičje plazme aU eksprimiranega v rekombinantni ceUčni kulturi, opisani tukaj. Glavna, tukaj znana efektorska funkcija mpl Uganda aU poUpeptida je vezanje na mpl in stimuliranje vgrajevanja označenih nukleotidov (³H-timidin) v DNA od IL-3 odvisnih cehe Ba/F3, transfektiranih s humanim mpl P. Druga, tukaj znana efektorska funkcija mpl Uganda ali poUpeptida je sposobnost, da stimulira vgraditev ³⁵S v krožeče trombocite pri izvajanju testa oživitve mišjih trombocitov. Še nadaljnja znana efektorska funkcija mpl liganda je sposobnost, da stimulira humano megakariocitopoezo in vitro, ki jo lahko kvantitativno določimo z uporabo radioaktivno označenega monoklonskega protitelesa, specifičnega za megakariocitni glikoprotein GPII_bIII_a.The term biologically active when used in reference to mpl Ugand aU isolated mpl Ugand means mpl Ugand or poUpeptide having thrombopoietic activity aU contributes to the effector function of mpl Ugand isolated from aplastic porcine plasma aU expressed in recombinant ceUc culture described herein. The main effector function known here, mpl Uganda aU poUpeptide, is binding to mpl and stimulating the incorporation of labeled nucleotides ( ³ H-thymidine) into DNA from IL-3 dependent guides Ba / F3 transfected with human mpl P. The second effector function known here is mpl Uganda or poUpeptide is the ability to stimulate the incorporation of ³⁵ S into circulating platelets when performing a mouse platelet recovery test. Another known effector function of the mpl ligand is the ability to stimulate human megakaryocytopoiesis in vitro, which can be quantified using a radiolabeled monoclonal antibody specific for the megakaryocyte glycoprotein GPII _b III _a .

Odstotek aminokislinske sekvenčne identičnosti glede na sekvenco mpl liganda je tukaj definiran kot odstotek aminokislinskih ostankov v kandidatni sekvenci, ki so identični ostankom v sekvenci mpl liganda, izoliranega iz aplastične prašičje plazme, ali murinega ali humanega liganda, ki ima deducirano aminokislinsko sekvenco, prikazano na sl. 1 (SEQ ED NO: 1), po uvrstitvi sekvenc in uvedbi praznin, če je potrebno, da dosežemo maksimalni odstotek sekvenčne identičnosti, ne da bi pri tem upoštevali katerokoli konzervativno substitucijo kot del sekvenčne identičnosti. Nobena od N-terminalnih, C-terminalnih ali internih ekstenzij, delecij ali insercij v sekvenci mpl liganda ni konstruirana tako, da bi vplivala na sekvenčno identičnost ali homologijo. Zgledni biološko aktivni mpl Ugandski polipeptidi, za katere je mišljeno, da imajo identične sekvence, vključujejo: prepro-mp/ Ugand, ρτο-mpl Ugand in zreU mpl Ugand.The percentage of amino acid sequence identity with respect to the mpl ligand sequence is defined herein as the percentage of amino acid residues in the candidate sequence identical to the residues in the mpl ligand sequence isolated from aplastic porcine plasma or murine or human ligand having a deduced amino acid, . 1 (SEQ ED NO: 1), after sequencing and introducing blanks, if necessary to achieve a maximum percentage of sequence identity without considering any conservative substitution as part of the sequence identity. None of the N-terminal, C-terminal, or internal extensions, deletions, or insertions in the mpl ligand sequence are constructed to affect sequence identity or homology. Exemplary biologically active mpl Ugandan polypeptides thought to have identical sequences include: prepro-mp / Ugand, ρτο-mpl Ugand, and zreU mpl Ugand.

Mikrosekvenciranje mpl Uganda lahko izvedemo s katerimkoU standardnim primernim postopkom, ki zagotavlja njegovo zadostno občutljivost. V enem takem postopku visoko očiščeni polipeptid, ki ga dobimo iz gelov SDS ali iz končne stopnje HPLC, sekvenciramo direktno z avtomatizirano Edmanovo (fenil izocianat) razgradnjo z uporabo sekvenatorja s plinsko fazo AppUed Biosystems model 470A, opremljenega s feniltiohidantionskim (ΡΊΉ) aminokislinskim analizatorjem 120A. Poleg tega lahko fragmente mpl Uganda, pripravljene s kemijsko (npr. CNBr, hidroksilamin, 2-nitro-5-tiocianobenzoat) ali encimatsko (npr. tripsin, klostripain, stafilokokna proteaza) digestijo in nato očiščene (npr. HPLC), podobno sekvenciramo. PTH amino kisline analiziramo z uporabo podatkovnega sistema ChromPerfect (Justice Innovations, Palo Alto, CA). Sekvenčno interpertacijo izvedemo na računalniku VAX 11/785 Digital Equipment Co., kot opisujejo Henzel et al., J. Chromatography, 404:41-52 [1987]. V danem primeru lahko alikvote frakcij HPLC izpostavimo elektroforezi na SDS-PAGE (5-20 %), elektrotransferiramo na membrano PVDF (ProBlott, AIB, Foster City, CA) in obarvamo z barvilom Coomassie BrilUant Blue (Matsurdiara, J. Biol. Chem., 262:10035-10038 [1987]. Specifični protein identificiramo po barvi, izrežemo iz popivka in N-terminalno sekvenciranje izvedemo s sekvenatorjem s plinsko fazo, opisanim zgoraj. Za interne proteinske sekvence frakcije HPLC posušimo v vakuumu (SpeedVac), resuspendiramo v ustreznih pufrih in izvedemo digestijo s cianogen bromidom, Lysspecifičnim encimom Lys-C (Wako Chemicals, Richmond, VA) ali Asp-N (Boehringer Mannheim, Indianapolis, IN). Po digestiji nastale peptide sekvenciramo kot zmes, ali po ločitvi s HPLC na koloni C4 razvijemo s propanolnim gradientom v TFA (0,1 %) pred sekvenciranjem v plinski fazi.The micro sequencing of Uganda mpl can be performed by any standard procedure that ensures its sensitivity. In one such process, the highly purified polypeptide obtained from SDS gels or from end-stage HPLC is sequenced directly by automated Edman (phenyl isocyanate) degradation using a gas-phase sequencer AppUed Biosystems model 470A equipped with a phenylthiohydantion (ΡΊΉ) amino acid analysis . In addition, Ugandan mpl fragments prepared by chemical (e.g. CNBr, hydroxylamine, 2-nitro-5-thiocyanobenzoate) or enzymatic (e.g. trypsin, clostripain, staphylococcal protease) digestion and subsequently purified (e.g. HPLC) can be similarly sequenced. PTH amino acids are analyzed using the ChromPerfect data system (Justice Innovations, Palo Alto, CA). Sequential interpertation was performed on a VAX computer 11/785 Digital Equipment Co., as described by Henzel et al., J. Chromatography, 404: 41-52 [1987]. In the present case, aliquots of the HPLC fractions can be subjected to SDS-PAGE electrophoresis (5-20%), electrotransfer to a PVDF membrane (ProBlott, AIB, Foster City, CA) and stained with Coomassie BrilUant Blue (Matsurdiara, J. Biol. Chem. , 262: 10035-10038 [1987] The specific protein is identified by color, excised from the batch, and N-terminal sequencing is performed with the gas phase sequencer described above.For internal protein sequences, the HPLC fractions are dried in vacuo (SpeedVac), resuspended in appropriate buffered and digested with cyanogen bromide, Lysspecific enzyme Lys-C (Wako Chemicals, Richmond, VA) or Asp-N (Boehringer Mannheim, Indianapolis, IN). After digestion, the peptides formed were sequenced as a mixture, or after HPLC separation on column C4 developed with a propanol gradient in TFA (0.1%) before sequencing in the gas phase.

Trombocitopenija je definirana kot število ploščic pod 150 χ IO⁹/! krvi.Thrombocytopenia is defined as the number of plaques below 150 χ IO ⁹ /! blood.

Trombopoetična aktivnost je definirana kot biološka aktivnost, ki je sestavljena iz pospeševanja proliferacije, diferenciacije in/ali zorenja megakariocitov ali megakariocitnih predhodnikov v trombocite tvorečo obliko teh celic. To aktivnost lahko izmerimo z različnimi testi, ki vključujejo test oživitve sinteze mišjih trombocitov in vivo, test indukcije celičnega površinskega antigena trombocitov, kot ga izmerimo z antitrombocitnim imunotestom (anti-GPII_bIII_a) za humano levkemično megakarioblastno celično linijo (CMK) in indukcijo poliploidizacije v megakarioblastni celični liniji (DAMI).Thrombopoietic activity is defined as biological activity, which consists of promoting the proliferation, differentiation and / or maturation of megakaryocytes or megakaryocyte precursors into platelets forming these cells. This activity may be measured by various tests, including test mouse platelet rebound synthesis and in vivo induction of platelet cell surface antigen, as measured by platelet immunoassay (anti-GPII _b III _a) for a human leukemia megakaryoblastic cell line (CMK), and induction of polyploidizations in a megakaryoblast cell line (DAMI).

'Trombopoetin (TPO) je definiran kot spojina s trombopoetično aktivnostjo ali takšna, ki je sposobna, da zveča število serumskih trombocitov v sesalcu. TPO je prednostno sposoben, da zveča število endogenih trombocitov za vsaj 10 %, bolj prednostno 50 % in najbolj prednostno, da zveča število trombocitov v človeku na več kot 150 x 10^/1 krvi.'Thrombopoietin (TPO) is defined as a compound with thrombopoietic activity or one capable of increasing the number of serum platelets in a mammal. TPO is preferably capable of increasing the number of endogenous platelets by at least 10%, more preferably 50% and most preferably increasing the number of platelets in humans to more than 150 x 10 ^ / 1 blood.

Izolirana nukleinska kislina mpl liganda je RNA ali DNA, ki vsebuje več kot 16 in prednostno 20 ali več zaporednih nukleotidnih baz, ki kodirajo biološko aktiven mpl ligand ali njegov fragment, je komplementarna RNA ali DNA, ali se hibridizira z RNA ali DNA in ostane stabilno vezana pri zmernih do ostrih pogojih. Ta RNA ali DNA je vsaj brez enega kontaminimega vira nukleinske kisline, s katerim je navadno skupaj v naravnem viru, in je prednostno v bistvu brez vsake druge RNA ali DNA sesalca. Fraza vsaj brez enega kontaminimega vira nukleinske kisline, s katerim je normalno skupaj, vključuje primer, kjer je nukleinska kislina prisotna v viru ali naravni celici, vendar je v različni kromosomski lokaciji ali je drugače bočno vezana na sekvence nukleinske kisline, ki se navadno ne nahaja v celičnem viru. Primer izolirane nukleinske kisline mpl liganda je RNA ali DNA, ki kodira biološko aktiven mpl ligand, ki ima vsaj 75 % sekvenčno identičnost, bolj prednostno vsaj 80 %, še bolj prednostno vsaj 85 %, še celo bolj prednostno 90 % in najbolj prednostno 95 % sekvenčno identičnost s humanim, murinim ali prašičjim mpl ligandom.Isolated mpl ligand nucleic acid is RNA or DNA containing more than 16 and preferably 20 or more consecutive nucleotide bases encoding a biologically active mpl ligand or fragment thereof, is complementary to RNA or DNA, or hybridizes with RNA or DNA and remains stable bound under moderate to harsh conditions. This RNA or DNA is at least free of one of the contaminating nucleic acid sources with which it is usually together in the natural source, and is preferably substantially free of any other mammalian RNA or DNA. The phrase at least without one of the contaminating nucleic acid sources with which it is normally co-located includes a case where a nucleic acid is present in a source or natural cell but is in a different chromosomal location or is otherwise laterally linked to nucleic acid sequences not normally found in the cell source. An example of an isolated nucleic acid mpl ligand is RNA or DNA encoding a biologically active mpl ligand having at least 75% sequence identity, more preferably at least 80%, more preferably at least 85%, even more preferably 90%, and most preferably 95% sequence identity with the human, murine or porcine mpl ligand.

Kontrolne sekvence, kadar se sklicujemo na ekspresijo, pomenijo sekvence DNA, potrebne za ekspresijo funkcionalno vezane kodirne sekvence v posebnem gos33 titeljskem organizmu. Kontrolne sekvence, ki so prikladne za prokariote vključujejo npr. promotor, v danem primeru operatersko sekvenco, ribosomsko vezišče, lahko pa tudi druge, še slabo razumljive sekvence. Evkariotske celice so znane, da uporabljajo promotorje, poliadenilacijske signale in spodbujevalce.Control sequences, when referring to expression, mean DNA sequences required for the expression of a functionally bound coding sequence in a specific gos33 titan organism. Control sequences that are suitable for prokaryotes include e.g. the promoter, optionally the operator sequence, the ribosomal binding site, as well as other, yet poorly understood sequences. Eukaryotic cells are known to use promoters, polyadenylation signals, and promoters.

Funkcionalno vezan, kadar se sklicujemo na nukleinske kisline, pomeni, da so nukleinske kisline nameščene v funkcionalni zvezi z drugo sekvenco nukleinske kisline. Npr. DNA je za presekvenco ali sekretomega voditelja funkcionalno vezana na DNA za polipeptid, če je ta eksprimiran kot preprotein, ki sodeluje pri sekreciji polipeptida; promotor ali spodbujevalec je funkcionalno vezan na kodirno sekvenco, če ta vpliva na transkripcijo sekvence; ali ribosomsko vezišče je funkcionalno vezano na kodirno sekvenco, če je ta v taki legi, da omogoča translacijo. Na splošno funkcionalno vezan pomeni, da so sekvence DNA, ki so vezane, sosednje, v primeru sekretomega voditelja sosednje in v fazi branja. Vendar pa za spodbujevalce ni potrebno, da so sosednji. Vezavo izvedemo z ligacijo ugodnih restrikcijskih mest. Če taka mesta ne obstajajo, uporabimo sintetične oligonukleotidne adapteije ali linkeije v skladu z običajno prakso.Functionally linked, when referring to nucleic acids, means that nucleic acids are located in a functional relationship with another nucleic acid sequence. E.g. The DNA is sequenced or secreted by the functionally linked DNA to the polypeptide if expressed as a preprotein involved in the secretion of the polypeptide; the promoter or promoter is functionally linked to the coding sequence if it affects the transcription of the sequence; or the ribosomal binding site is functionally linked to the coding sequence if it is in such a position as to permit translation. Generally functionally bound means that the DNA sequences that are bound are adjacent, in the case of a secret leader, adjacent and at the reading stage. However, pacemakers do not need to be adjacent. The binding is carried out by ligation of favorable restriction sites. If no such sites exist, use synthetic oligonucleotide adaptations or linkei according to normal practice.

Eksogen, kadar se nanaša na element, pomeni sekvenco nukleinske kisline, ki je tuja za celico ali homologna zanjo, vendar v položaju v nukleinski kislini gostiteljske celice, v kateri se element navadno ne nahaja.Exogen, when referring to an element, means a sequence of nucleic acid that is foreign to or homologous to the cell but in a position in the nucleic acid of the host cell in which the element is not normally located.

Celica, celična linija in celična kultura uporabljamo izmenljivo in te oznake vključujejo vse potomce celice ali celične linije. Tako npr. izrazi, kot so transformanti ali transformirane celice, vključujejo primarno subjektno celico in iz nje izvedene kulture ne glede na število prenosov. Razumljivo je, da ni potrebno, da so vsi potomci natančno identični po vsebnosti DNA zaradi namernih ali nenamernih mutacij. Vključeni so tudi mutantni potomci, ki imajo enako funkcijo ali biološko aktivnost, kot je selekcionirana za originalno transformirano celico. Kjer so predvidene razlikovalne oznake, je to razvidno iz teksta.Cell, cell line and cell culture are used interchangeably and these designations include all offspring of a cell or cell line. So e.g. terms such as transformants or transformed cells include the primary subject cell and the cultures derived therefrom regardless of the number of transmissions. It is understandable that not all offspring need to be exactly identical in DNA content due to intentional or unintentional mutations. Also included are mutant offspring having the same function or biological activity as selected for the originally transformed cell. Where distinctive markings are provided, this is evident in the text.

Plazmidi so avtonomno replikativne krožeče molekule DNA, ki imajo neodvisne izvore replikacije in so označeni tukaj z malo črko p, ki je pred velikimi črkami in/ali številkami in/ali ji le-te sledijo. Izhodni plazmidi so vsi komercialno in javno dosegljivi brez omejitev ali jih lahko konstruiramo iz takih, dosegljivih plazmidov v skladu z objavljenimi postopki. Poleg tega so v tehniki znani tudi drugi ekvivalentni plazmidi, ki so znani strokovnjakom.Plasmids are autonomously replicating circulating DNA molecules that have independent origin of replication and are identified here by the lowercase letter p, which is preceded by uppercase letters and / or numbers and / or sequences thereof. The starting plasmids are all commercially and publicly available without restriction, or can be constructed from such accessible plasmids in accordance with published procedures. In addition, other equivalent plasmids known to those skilled in the art are known in the art.

Restrikcijska encimska digestija, kadar je v zvezi z DNA, pomeni katalitično cepitev internih fosfodiesterskih vezi DNA z encimom, ki deluje le na določenih lokacijah ali mestih v sekvenci DNA Taki encimi se imenujejo restrikcijske endonukleaze. Vsaka restrikcijska endonukleaza spozna specifično sekvenco DNA, imenovano restrikcijsko mesto, ki ima dvojno simetrijo. Različni restrikcijski encimi, ki jih uporabljamo tukaj, so komercialno dosegljivi, pri tem pa upoštevamo njihove reakcijske pogoje, kofaktoije in druge zahteve, določene od dobavitelja. Restrikcijski encimi so navadno označeni z okrajšavami, sestavljenimi iz velike črke, kiji nato sledijo druge črke, ki pomenijo mikroorganizem, iz katerega je vsak restrikcijski encim originalno dobljen, in nato številka, ki označuje poseben encim. Na splošno uporabimo približno 1 μ-g plazmida ali fragmenta DNA s približno 1-2 enotama encima v približno 20 gl raztopine pufra. Ustrezne količine pufrov in substratov za posebne restrikcijske encime določi izdelovalec. Navadno uporabimo inkubacijo približno 1 uro pri 37 °C, vendar pa se lahko spreminja v skladu z navodili dobavitelja. Po inkubaciji protein ali polipeptid odstranimo z ekstrakcijo s fenolom in kloroformom, digerirano nukleinsko kislino pa rekuperiramo iz vodne frakcije z obaijanjem z etanolom. Digestiji z restrikcijskim encimom lahko sledi hidroliza terminalnih 5’ fosfatov z bakterijsko alkalno fosfatazo, da preprečimo, da bi dva restrikcijsko cepljena konca fragmenta DNA cirkularizirala oz. tvorila zaprto zanko, ki bi preprečila insercijo drugega fragmenta DNA na restrikcijskem mestu. Če ni drugače navedeno, digestiji plazmidov ne sledi 5’ terminalna defosforilacija. Postopki in reagenti za defosforilacijo so konvencionalni in so opisani v odstavkih 1.56-1.61 avtorja Sambrooka et al., Molecular Cloning: A Laboratory Manual [New York: Cold Spring Harbor Laboratory Press, 1989].Restriction enzyme digestion, when it comes to DNA, means the catalytic cleavage of DNA's internal phosphodiester bonds with an enzyme that acts only at specific sites or sites in the DNA sequence. Such enzymes are called restriction endonucleases. Each restriction endonuclease learns a specific DNA sequence called a restriction site that has double symmetry. The various restriction enzymes used herein are commercially available, taking into account their reaction conditions, cofactors and other requirements as specified by the supplier. Restriction enzymes are usually labeled with abbreviations consisting of a capital letter, followed by other letters representing the microorganism from which each restriction enzyme is originally derived, and then a number indicating a specific enzyme. In general, about 1 μ-g of plasmid or DNA fragment with about 1-2 units of enzyme in about 20 g of buffer solution is used. The appropriate amounts of buffers and substrates for specific restriction enzymes shall be specified by the manufacturer. Usually, incubation is used for about 1 hour at 37 ° C, but may vary according to the supplier's instructions. After incubation, the protein or polypeptide is removed by extraction with phenol and chloroform, and the digested nucleic acid is recovered from the aqueous fraction by ethanol staining. Digestion with a restriction enzyme may be followed by hydrolysis of the terminal 5 'phosphates by bacterial alkaline phosphatase to prevent the two restriction grafted ends of the DNA fragment from circulating or. forming a closed loop that would prevent the insertion of another DNA fragment at the restriction site. Unless otherwise stated, digestion of plasmids is not followed by 5 'terminal dephosphorylation. Dephosphorylation processes and reagents are conventional and are described in paragraphs 1.56-1.61 by Sambrook et al., Molecular Cloning: A Laboratory Manual [New York: Cold Spring Harbor Laboratory Press, 1989].

Rekuperacija ali izolacija nekega fragmenta DNA iz restrikcijskega digesta pomeni ločitev digesta na poliakrilamidnem ali agaroznem gelu z elektroforezo, identifikacijo fragmenta, ki nas zanima, s primerjanjem njegove mobilnosti s tisto od markiranih fragmentov DNA z znano molekulsko maso, odstranitev dela gela, ki vsebuje želen fragment in ločitev gela od DNA Ta postopek je splošno znan. Npr.: Lawn et al., Nucleic Acids Res., 9:6103-6114 [1981], in Goeddel et al., Nucleic Acids Res., 8:4057 [1980].Recovery or isolation of a DNA fragment from a restriction digest means separation of the digest on a polyacrylamide or agarose gel by electrophoresis, identification of the fragment of interest by comparing its mobility with that of a labeled DNA fragment of known molecular weight, removal of a portion of the gel containing the desired fragment and Separation of Gel from DNA This process is widely known. For example: Lawn et al., Nucleic Acids Res., 9: 6103-6114 [1981], and Goeddel et al., Nucleic Acids Res., 8: 4057 [1980].

Southern analiza ali Southern blotting je postopek, s katerim potrdimo prisotnost sekvenc DNA v restrikcijskem endonukleaznem digestu DNA ali DNA-vsebujočem sestavku s hibridizacijo z znanim, označenim oligonukleotidom ali fragmentom DNA.Southern analysis or Southern blotting is a process by which the presence of DNA sequences is confirmed in a restriction endonuclease DNA digest or DNA-containing composition by hybridization with a known, labeled oligonucleotide or DNA fragment.

Southern analiza značilno vključuje elektroforetsko ločitev digestov DNA na agaroznih gelih, denaturacijo DNA po elektroforetski ločitvi in prenos DNA na nitrocelulozo, najlon ali drugi primerni membranski nosilec za analizo z radioaktivno označeno, biotinilirano ali encimsko označeno sondo, kot je opisano v odstavkih 9.37-9.52, avtorja Sambrooka et al., zgoraj.Southern analysis typically includes electrophoretic separation of DNA digestions on agarose gels, denaturation of DNA after electrophoretic separation and transfer of DNA to nitrocellulose, nylon or other suitable membrane carrier for analysis with a radiolabelled, biotinylated or enzyme labeled probe as described in paragraphs 9.37-9.52, by Sambrook et al., supra.

Northern analiza ali Northern blotting je postopek, ki ga uporabimo za identificiranje sekvenc RNA, ki se hibridizirajo z znano sondo, kot je oligonukleotid, fragment DNA, cDNA ali njen fragment ali fragment RNA. Sondo označimo z radioaktivnim izotopom, kot je ³²P, ali z biotinilacijo ali z encimom. RNA, ki jo je treba analizirati, navadno ločimo elektroforetično na agarozi ali poliakrilamidnem gelu, prenesemo na nitrocelulozo, najlon ali drugo prikladno membrano in hibridiziramo s sondo s standardnimi tehnikami, dobro znanimi strokovnjakom, kot so npr. tiste, opisane v poglavjih 7.39-7.52, avtorja Sambrooka et al., zgoraj.Northern analysis or Northern blotting is a procedure used to identify RNA sequences that hybridize with a known probe, such as an oligonucleotide, a DNA fragment, a cDNA, or an RNA fragment or fragment thereof. The probe is labeled with a radioactive isotope, such as ³² P, or with biotinylation or an enzyme. The RNA to be analyzed is usually separated electrophoretically on an agarose or polyacrylamide gel, transferred to nitrocellulose, nylon or other suitable membrane and hybridized with a probe by standard techniques well known to those skilled in the art. those described in Chapters 7.39-7.52 by Sambrook et al., supra.

Ugacija je postopek tvorbe fosfodiestrskih vezi med dvema fragmentoma nukleinske kisline. Za ligacijo obeh fragmentov morata biti konca fragmentov kompatibilna, eden z drugim. V nekaterih primerih so konci direktno kompatibilni po endonukleazni digestiji. Vendar pa je lahko potrebno, da najprej konce, ki so nekompatibilno razporejeni in navadno nastanejo po endonukleazni digestiji, pretvorimo v odrezane (tope) konce, da jih naredimo kompatibilne za ligacijo. Zato, da odrežemo konce, DNA obdelujemo v prikladnem pufru vsaj 15 minut pri 15 °C s približno 10 enotami Klenowega fragmenta DNA polimeraze I ali T4 DNA polimeraze v prisotnosti štirih deoksiribonukleotidnih trifosfatov. DNA nato očistimo s fenolkloroformsko ekstrakcijo in etanolnim obarjanjem. Fragmente DNA, ki jih je potrebno ligirati skupaj, damo v raztopino v približno ekvimolamih količinah. Raztopina vsebuje tudi ATP, ligazni pufer in ligazo, kot npr. T4 DNA ligazo, v količini približno 10 enot na 0,5 μ-g DNA. Če je DNA potrebno ligirati v vektor le-tega, najprej lineariziramo z digestijo z ustreznimi restrikcijskimi endonukleazami. Lineariziran fragment nato obdelamo z bakterijsko alkalno fosfatazo ali telečjo intestinalno fosfatazo, da preprečimo samoligacijo med stopnjo ligiranja.Ugation is the process of forming phosphodiester bonds between two nucleic acid fragments. For the ligation of both fragments, the ends of the fragments must be compatible with each other. In some cases, the ends are directly compatible after endonuclease digestion. However, it may be necessary to first convert the incompatible ends, which are usually formed after endonuclease digestion, to the blunt ends to make them compatible for ligation. In order to cut the ends, the DNA is treated in suitable buffer for at least 15 minutes at 15 ° C with about 10 units of the Klenow fragment of DNA polymerase I or T4 DNA polymerase in the presence of four deoxyribonucleotide triphosphates. The DNA was then purified by phenol chloroform extraction and ethanol precipitation. The DNA fragments to be ligated together are added to the solution in approximately equimolar amounts. The solution also contains ATP, ligase buffer and ligase, such as e.g. T4 DNA ligase, in an amount of about 10 units per 0.5 μg of DNA. If DNA is to be ligated into a vector, it is first linearized by digestion with appropriate restriction endonucleases. The linearized fragment is then treated with bacterial alkaline phosphatase or calf intestinal phosphatase to prevent self-ligation during the ligation step.

Priprava DNA iz celic pomeni izoliranje plazmidne DNA iz kulture gostiteljskih celic. Navadno uporabljeni postopki za pripravo DNA so priprave plazmidov v velikem in majhnem obsegu, opisane v odstavkih 1.25-1.33, Sambrook et al., zgoraj. Po pripravi DNA le-to očistimo s postopki, dobro znanimi v tehniki, kot je opisano v odstavku 1.40, Sambrook et al., zgoraj.Preparation of DNA from cells means isolation of plasmid DNA from host cell culture. Commonly used DNA preparation methods are large and small scale plasmid preparation described in paragraphs 1.25-1.33, by Sambrook et al., Above. After DNA preparation, the DNA is purified by methods well known in the art, as described in paragraph 1.40, by Sambrook et al., Above.

Oligonukleotidi so kratki, enojno- ali dvojno-vijačni polideoksinukleotidi, ki jih kemijsko sintetiziramo z znanimi postopki (kot je kemija fosfotriestrov, fosfitov ali fosforamiditov, z uporabo tehnik v trdni fazi, kot je opisano v EP 266,032, obj. 4. maja 1988, ali preko deoksinukleozidnih H-fosfonatnih intermediatov, kot jih opisujejo Froehler et al., Nucl. Acids Res., 14:5399-5407 [1986]). Nadaljnji postopki vključujejo verižne polimerazne reakcije, definirane spodaj, in druge avtoprimerske metode in oligonukleotidne sinteze na trdnih nosilcih. Vse te postopke opisujejo Engels et al., Agnew. Chem. Int. ED. Engl. 28:716-734 (1989). Te postopke uporabimo, če je celotna sekvenca nukleinske kisline gena znana ali je sekvenca nukleinske kisline, ki je komplementarna s kodirno verigo, na voljo. Alternativno, če je ciljna aminokislinska sekvenca znana, lahko izvedemo potencialne sekvence nukleinske kisline z uporabo znanih in prednostnih kodirnih ostankov za vsak aminokislinski ostanek. Oligonukleotide nato očistimo na poliakrilamidnem gelu.Oligonucleotides are short, single- or double-stranded polydeoxynucleotides that are chemically synthesized by known methods (such as the chemistry of phosphotriesters, phosphites or phosphoramidites, using solid-phase techniques as described in EP 266,032, May 4, 1988, or via deoxynucleoside H-phosphonate intermediates as described by Froehler et al., Nucl. Acids Res., 14: 5399-5407 [1986]). Further methods include the polymerase chain reaction reactions defined below and other self-priming methods and oligonucleotide syntheses on solid carriers. All these procedures are described by Engels et al., Agnew. Chem. Int. ED. Engl. 28: 716-734 (1989). These procedures are used if the complete nucleic acid sequence of the gene is known or a nucleic acid sequence complementary to the coding chain is available. Alternatively, if the target amino acid sequence is known, potential nucleic acid sequences can be derived using known and preferred coding residues for each amino acid residue. The oligonucleotides are then purified on a polyacrylamide gel.

Verižna polimerazna reakcija ali PCR je postopek ali tehnika, pri kateri se zelo majhne količine specifičnega kosa nukleinske kisline, RNA in/ali DNA pomnožijo, kot je opisano v US patentu št. 4683195, izd. 28. julija 1987. Na splošno pa morajo biti na voljo sekvenčne informacije za konce regije, ki nas zanima, ali več kot le-to, tako da lahko oblikujemo oligonukleotidne primerje; ti primerji so identični ali podobni v sekvenci nasprotnim vijačnicam kalupa, ki ga je potrebno pomnožiti. 5’ terminalni nukleotidi obeh primeijev lahko sovpadajo s konci pomnožene snovi. PCR lahko uporabimo za pomnoževanje specifičnih sekvenc RNA, specifičnih sekvenc DNA iz celotne genomske DNA, in cDNA, transkribirane iz celotne celične RNA, bakteriofagne ali plazmidne sekvence itd. Na splošno to opisujejo Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51:263 [1987]; Erlich, izd. PCR Technology (Stockton Press, NY, 1989). Za PCR, kot uporabljamo tukaj, smatramo, da je eden vendar ne edini zgled postopka reakcije polimerazne nukleinske kisline za pomnoževanje nukleinske kisline testnega vzorca, pri čemer uporabimo znano nukleinsko kislino kot primer in polimerazno nukleinsko kislino, da pomnožimo ali naredimo specifičen kos nukleinske kisline.A polymerase chain reaction or PCR is a process or technique in which very small amounts of a specific strand of nucleic acid, RNA and / or DNA are amplified as described in U.S. Pat. 4683195, ed. July 28, 1987. In general, however, sequential information must be available for the ends of the region of interest or more than that in order to form oligonucleotide primers; these primers are identical or similar in sequence to the opposite helixes of the mold to be multiplied. The 5 'terminal nucleotides of both primers may coincide with the ends of the amplified substance. PCR can be used to amplify specific RNA sequences, specific DNA sequences from whole genomic DNA, and cDNA transcribed from whole cell RNA, bacteriophage or plasmid sequences, etc. This is generally described by Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51: 263 [1987]; Erlich, ed. PCR Technology (Stockton Press, NY, 1989). PCR as used herein is considered to be one but not the only example of a polymerase nucleic acid reaction process for nucleic acid amplification of a test sample, using known nucleic acid as an example and polymerase nucleic acid to amplify or make a specific nucleic acid piece.

Ostri pogoji so tisti (1), pri katerih uporabimo nizko ionsko jakost in visoko temperaturo za izpiranje, npr. NaCl (0,0015 M)/natrijev citrat (0,0015 M)/NaDodSO₄ (SDS) (0,1 %) pri 50 °C ali (2), pri katerih uporabimo med hibridizacijo denaturimo sredstvo, kot npr. formamid, npr. formamid 50 % (vol./vol.) z govejim serumskim albuminom (0,1 %)/Ficollom (0,1 %)/polivinilpirolidonom (0,1 %)/natrijim fosfatnim pufrom (50 mM) pri pH 6,5 z NaCl (750 mM) in natrijevim citratom (75 mM) pri 42 °C. Drugi zgled je uporaba formamida (50 %), 5-krat SSC [NaCl (0,75 M), natrijev citrat (0,075 M)], natrijev fosfat (50 mM, pH 6,8), natrijev pirofosfat (0,1 %), 5 x Denhardtova raztopina, sonificirana DNA lososove sperme (50/ig/ml), SDS (0,1 %) in dekstran sulfat (10 %) pri 42 °C z izpiranji pri 42 °C v 0,2 x SSC in SDS (0,1 %).The harsh conditions are those (1) where low ionic strength and high rinsing temperature are used, e.g. NaCl (0.0015 M) / Sodium citrate (0.0015 M) / NaDodSO ₄ (SDS) (0.1%) at 50 ° C or (2) using a denaturing agent such as e.g. formamide, e.g. formamide 50% (vol / vol) with bovine serum albumin (0.1%) / Ficoll (0.1%) / polyvinylpyrrolidone (0.1%) / sodium phosphate buffer (50 mM) at pH 6.5 z NaCl (750 mM) and sodium citrate (75 mM) at 42 ° C. Another example is the use of formamide (50%), 5 times SSC [NaCl (0.75 M), sodium citrate (0.075 M)], sodium phosphate (50 mM, pH 6.8), sodium pyrophosphate (0.1%) ), 5 x Denhardt solution, sonicated DNA of salmon sperm (50 / ig / ml), SDS (0.1%) and dextran sulfate (10%) at 42 ° C with rinses at 42 ° C in 0.2 x SSC and SDS (0.1%).

Zmerno ostri pogoji so opisani pri Sambrooku et al., zgoraj, in vključujejo uporabo izpiralne raztopine in hibridizacijske pogoje (npr. temperaturo, ionsko jakost in % SDS), manj ostre, kot so opisani zgoraj. Zgled zmerno ostrih pogojev je npr. inkubacija pri 37 °C preko noči v raztopini, ki vsebuje: formamid (20 %), 5 x SSC [NaCl (150 mM), trinatrijev citrat (15 mM)], natrijev fosfat (50 mM, pH 7,6), 5x Denhardtovo raztopino, dekstransulfat (10 %) in 20 μΐ/ml denaturirane strižno obdelane DNA lososove sperme, in nato izpiranje filtrov v 1 x SSC pri približno 37-50 °C. Strokovnjakom je jasno, kako naravnati temperaturo, ionsko jakost itd., kot je potrebno za prilagoditev faktorjev, kot je dolžina sonde ipd.Moderately harsh conditions are described in Sambrook et al., Above, and include the use of a rinse solution and hybridization conditions (e.g., temperature, ionic strength, and% SDS), less harsh than those described above. An example of moderately harsh conditions is e.g. incubation at 37 ° C overnight in a solution containing: formamide (20%), 5 x SSC [NaCl (150 mM), trisodium citrate (15 mM)], sodium phosphate (50 mM, pH 7.6), 5x Denhardt's solution, dextransulfate (10%) and 20 μΐ / ml denatured sheared DNA of salmon sperm, and then washing the filters in 1 x SSC at about 37-50 ° C. It will be apparent to those skilled in the art how to adjust the temperature, ionic strength, etc. as necessary to adjust factors such as probe length, etc.

Protitelesa (Ab) in imunoglobulini (Ig) so glikoproteini z enakimi strukturnimi značilnostmi. Medtem ko imajo protitelesa vezavno specifičnost za specifični antigen, imunoglobulini vključujejo tako protitelesa kot tudi druge protitelesom podobne molekule, ki nimajo antigenske specifičnosti. Polipeptide slednje vrste izdelujejo v nizkih nivojih limfni sistemi, v zvečanih pa mielomi.Antibodies (Ab) and immunoglobulins (Ig) are glycoproteins with the same structural characteristics. While antibodies have binding specificity for a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules that lack antigen specificity. The latter types of polypeptides are produced in low levels by the lymphatic systems and in the enlarged ones by myeloma.

Naravna protitelesa in imunoglobulini so navadno heterotetramemi glikoproteini s približno 150000 daltoni, sestavljeni iz dveh identičnih lahkih (L) verig in dveh identičnih težkih (H) verig. Vsaka lahka veriga je vezana na težko z eno kovalentno disulfidno vezjo, medtem ko število disulfidnih povezav variira med težkimi verigami različnih imunoglobulinskih izo-tipov. Vsaka težka in lahka veriga ima tudi regularno razmaknjene medverižne disulfidne mostičke. Vsaka težka veriga ima na enem koncu variabilno domeno (V_H), ki ji sledijo številne konstantne domene. Vsaka lahka veriga ima variabilno domeno na enem koncu ter (VJ in konstantno domeno na njenem drugem koncu; konstantna domena lahke verige se ujema s prvo konstantno domeno težke verige in variabilna domena lahke verige se ujema z variabilno domeno težke verige. Posebni aminokislinski ostanki verjetno tvorijo vmesno površino med variabilnimi domenami lahkih in težkih verig (Clothia et al., J. Mol. Biol., 186:651 - 663 [1985]; Novotny in Haber, Proč. Natl. Acad. Sci. ZDA, 82:4592-4596 [1985]).Natural antibodies and immunoglobulins are usually heterotetramic glycoproteins of approximately 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is bonded to the heavy by one covalent disulfide bond, while the number of disulfide bonds varies between the heavy chains of different immunoglobulin iso-types. Each heavy and light chain also has regularly spaced interchain disulfide bridges. Each heavy chain has a variable domain (V _H ) at one end, followed by a number of constant domains. Each light chain has a variable domain at one end and (VJ and a constant domain at its other end; the constant light chain domain matches the first constant heavy chain domain and the light chain variable domain matches the heavy chain variable domain. Special amino acid residues are likely to form amino acids. intermediate surface between variable domains of light and heavy chains (Clothia et al., J. Mol. Biol., 186: 651 - 663 [1985]; Novotny and Haber, Proc. Natl. Acad. Sci. USA, 82: 4592-4596 [1985]).

Izraz variabilen je v zvezi z dejstvom, da se nekateri deli variabilne domene znatno razlikujejo v sekvenci med protitelesi in se uporabljajo pri vezavi in specifičnosti vsakega posebnega protitelesa za njegov poseben antigen. Vendar pa variabilnost ni enakomerno porazdeljena v variabilnih domenah protiteles. Koncentrirana je v treh segmentih, imenovanih komplementarnost določujoče regije (CDR) ali hipervariabilne regije tako v variabilnih domenah lahke verige kot tudi težke. Bolj visoko konzervirani deli variabilnih domen so imenovani ogrodja (FR). Vsaka variabilna domena naravnih težkih in lahkih verig obsega štiri regije FR z močno privzeto /3-ravninsko konfiguracijo, povezane s tremi CDR, ki tvorijo povezavo zank in v nekaterih primerih del /3-ravninske strukture. CDR se v vsaki verigi držijo skupaj v neposredni bližini regij FR, in s CDR iz druge verige prispevajo k tvorbi antigenskih vezišč protiteles (Kabat et al., Sequences of Proteins of Immunological Interesi, National Institute of Health, Bethesda, MD [1987]). Konstantne domene niso direktno vključene pri vezavi protitelesa na antigen, vendar pa imajo razne efektorske funkcije, kot npr. sodelovanje protitelesa pri celični toksičnosti, odvisni od protitelesa.The term variable is related to the fact that some parts of the variable domain differ significantly in the sequence between antibodies and are used in the binding and specificity of each specific antibody to its particular antigen. However, variability is not evenly distributed across the variable domains of antibodies. It is concentrated in three segments, called complementarity determining regions (CDRs) or hypervariable regions, in both light chain and heavy chain variable domains. The more highly conserved portions of the variable domains are called frames (FRs). Each variable domain of natural heavy and light chains comprises four FR regions with a strong default / 3-plane configuration coupled to three CDRs that form a loop connection and in some cases part of a 3-plane structure. CDRs are held together in each chain in close proximity to FR regions, and contribute to the formation of antigenic antibody binding sites by CDRs from another chain (Kabat et al., Sequences of Proteins of Immunological Interests, National Institute of Health, Bethesda, MD [1987]) . The constant domains are not directly involved in the binding of the antibody to the antigen, but they have various effector functions, such as e.g. antibody involvement in antibody-dependent cellular toxicity.

S papainsko digestijo protiteles nastaneta dva identična fragmenta za vezavo antigena, imenovana fragmenta Fab, vsak z enojnim veziščem za antigen in residualnim fragmentom Fc, katerega ime izraža njegovo sposobnost, da z lahkoto kristalizira. Z obdelavo s pepsinom dobimo fragment F(ab’)₂, ki ima dve vezišči za antigen in je še vedno sposoben premreženja antigena.Papain antibody digestion produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site and a residual Fc fragment, whose name expresses its ability to crystallize easily. Treatment with pepsin yields the F (ab ') ₂ fragment, which has two antigen binding sites and is still capable of antigen cross-linking.

Fv je najmanjši fragment protitelesa, ki vsebuje kompletno antigensko spoznanje in vezišče. Ta regija je sestavljena iz dimera z eno težko in eno lahko verigo variabilne domene v tesni nekovalentni asociaciji. V tej konfiguraciji pride do interakcije treh CDR vsake variabilne domene, da definirajo vezišče za antigen na površini dimera V_H-V_L. Kolektivno šest CDR da specifičnost za vezavo antigena na protitelo. Vendar pa ima posamezna variabilna domena (ali polovica Fv, ki obsega samo tri CDR, specifične za antigen) sposobnost, da spozna in veže antigen, čeprav pri nižji afiniteti kot celotno vezišče.Fv is the smallest antibody fragment containing complete antigen recognition and binding. This region consists of a dimer with one heavy and one light chain variable domain in close noncovalent association. In this configuration, the interaction of three CDRs of each variable domain occurs to define an antigen binding site on the surface of the dimer V _H -V _L. Collectively, six CDRs give specificity for the binding of the antigen to the antibody. However, a single variable domain (or half Fv comprising only three antigen-specific CDRs) has the ability to recognize and bind the antigen, albeit at a lower affinity than the entire binding site.

Fragment Fab prav tako vsebuje konstantno domeno lahke verige in prvo konstantno domeno (CH1) težke verige. Fragmenti Fab’ se razlikujejo od fragmentov Fab po dodatnih nekaj ostankih na karboksi terminalu težke verige CH1 domene, ki vključujejo enega ali več cisteinov iz gibljive regije protitelesa. Fab’-SH je tukaj oznaka za Fab’ v katerem cisteinski ostanki konstantne domene nosijo prosto tiolno skupino. Protitelesni fragmenti F(ab’)₂ nastanejo originalno kot pari fragmentov Fab’, ki imajo gibljive cisterne med seboj. Znane so tudi druge kemijske pripojitve protitelesnih fragmentov.The Fab fragment also contains the light chain constant domain and the first heavy chain constant domain (CH1). The Fab 'fragments differ from the Fab fragments by an additional few residues at the carboxy terminal of the CH1 domain of the heavy chain CH1 domain, which include one or more cysteines from the moving region of the antibody. Fab'-SH is here a designation for Fab 'in which cysteine residues of the constant domain carry a free thiol group. The F (ab ') ₂ antibody fragments originate originally as pairs of Fab' fragments having movable cisterns with each other. Other chemical couplings of antibody fragments are also known.

Lahke verige protiteles (imunoglobulinov) iz katerekoli vrste vretenčarjev lahko dodelimo enemu od dveh, jasno različnih tipov, imenovanih kapa in lambda (λ), na osnovi aminokislinskih sekvenc njihovih konstantnih domen.Light chains of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two, clearly different types, called kappa and lambda (λ), based on the amino acid sequences of their constant domains.

Imunoglobuline lahko, odvisno od aminokislinske sekvence konstantne domene njihovih težkih verig, razdelimo v različne razrede. Obstaja pet glavnih razredov imunoglobulinov: IgA, IgD, IgE, IgG in IgM in nekatere od teh lahko dalje razdelimo v podrazrede (izotipi), npr. IgG-1, IgG-2, IgG-3 in IgG-4; IgA-1 in IgA-2. Konstantne domene težke verige, ki ustrezajo različnim razredom imunoglobinov, so imenovane a, delta, epsilon, 7 oz. μ.. Strukture podenot in tridimenzionalnih konfiguracij različnih razredov imunoglobinov so dobro znane.Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be divided into different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further subdivided into subclasses (isotypes), e.g. IgG-1, IgG-2, IgG-3 and IgG-4; IgA-1 and IgA-2. The heavy chain constant domains corresponding to different classes of immunoglobins are termed a, delta, epsilon, 7 oz. The structures of subunits and three-dimensional configurations of different classes of immunoglobins are well known.

Izraz protitelo uporabljamo v najširšem smislu in specifično pokriva posamezna monoklonska protitelesa (vključno agonistična in antagonistična protitelesa), protitelesne sestavke s poliepitopično specifičnostjo, kot tudi fragmente protiteles (npr. Fab, F(ab’)₂ in Fv), toliko časa, dokler imajo želeno biološko aktivnost.The term antibody is used in the broadest sense and specifically covers individual monoclonal antibodies (including agonist and antagonist antibodies), antibody compositions with polyepitopic specificity, as well as antibody fragments (e.g., Fab, F (ab ') ₂ and Fv) for as long as they have desired biological activity.

Izraz monoklonsko protitelo, kot ga uporabljamo tukaj, je v zvezi s protitelesom, ki ga dobimo iz populacije v bistvu homogenih protiteles, t.j. individualna protitelesa, ki obsegajo populacijo, so identična, razen za možne naravne mutacije, ki so lahko prisotne v manjših količinah. Monoklonska protitelesa so visoko specifična, pri čemer so usmerjena proti posameznemu antigenskemu mestu. Poleg tega je v nasprotju z običajnimi (poliklonskimi) pripravki protiteles, ki značilno vključujejo različna protitelesa, usmerjena proti različnim determinantam (epitopi), vsako monoklonsko protitelo usmerjeno proti posamezni determinanti na antigenu. Poleg svoje specifičnosti pa so monoklonska protitelesa prednostna v tem, da jih sintetizira hibridomska kultura, nekontaminirana z drugimi imunoglobulini. Modificiran monoklonski pomeni značaj protitelesa, ki je dobljen iz v bistvu homogene populacije protiteles in ni konstruiran za potrebno tvorbo protitelesa po nekem posebnem postopku. Npr. monoklonska protitelesa za uporabo v skladu s predloženim izumom lahko naredimo s hibridomskim postopkom, ki so ga prvi opisali Kohler & Milstein, Nature, 256:495 (1975), ali ga lahko naredimo po postopku rekombinantne DNA (npr. US patent št. 4,816,567 [Cabilly et al.]).The term monoclonal antibody as used herein refers to an antibody derived from a population of substantially homogeneous antibodies, i.e. the individual antibodies that comprise the population are identical except for possible natural mutations that may be present in small quantities. Monoclonal antibodies are highly specific, targeting a single antigen site. In addition, unlike conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by a hybridoma culture uncontaminated with other immunoglobulins. Modified monoclonal means the character of an antibody derived from a substantially homogeneous antibody population and not designed for the required antibody formation by any particular procedure. E.g. monoclonal antibodies for use according to the present invention can be made by the hybridoma method first described by Kohler & Milstein, Nature, 256: 495 (1975), or can be made by recombinant DNA process (e.g., U.S. Patent No. 4,816,567 [ Cabilly et al.]).

Monoklonska protitelesa tukaj specifično vključujejo kimerna protitelesa (imunoglobuline), v katerih je del težke in/ali lahke verige identičen z ustreznimi sekvencami, ali homologen za te sekvence, v protitelesih, izvedenih iz posebne vrste ali pripadajočih k posebnemu razredu ali podrazredu protiteles, medtem ko je ostanek verige (verig) identičen ali homologen za ustrezne sekvence v protitelesih, izvedenih iz drugih vrst ali pripadajočih drugemu razredu ali podrazredu protiteles, kot tudi fragmente takih protiteles, toliko Časa, dokler imajo želeno biološko aktivnost (US patent št. 4,816,567 (Cabilly et al.); in Morrison et al., Proč. Natl. Acad. Sci. ZDA, 81:6851-6855 [1984]).Monoclonal antibodies herein specifically include chimeric antibodies (immunoglobulins) in which part of the heavy and / or light chain is identical to, or homologous to, those sequences, in antibodies derived from a particular species or belonging to a specific antibody class or subclass, while the chain (s) residue is identical or homologous to the corresponding sequences in antibodies derived from other species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, for as long as they have the desired biological activity (US Patent No. 4,816,567 (Cabilly et al.); and Morrison et al., Proc Natl. Acad. Sci. USA, 81: 6851-6855 [1984].

Humanizirane oblike ne-humanih (npr. murinih) protiteles so kirnemi imunoglobulini, imunoglobulinske verige ali njihovi fragmenti (kot npr. Fv, Fab, Fab’, F(ab’)₂ ali druge antigen-vezavne podsekvence protiteles), ki vsebujejo minimalno sekvenco, izvedeno iz nehumanega imunoglobulina. Večinoma so humanizirana protitelesa humani imunoglobulini (recipientno protitelo), v katerih so ostanki iz recipientne CDR nadomeščeni z ostanki CDR iz nehumanih vrst (donorsko protitelo), kot npr. miši, podgane ali zajca, ki imajo želeno specifičnost, afiniteto in kapaciteto. V nekaterih primerih so Fv ogrodni ostanki humanega imunoglobulina nadomeščeni z ustreznimi nehumanimi ostanki. Nadalje lahko humanizirano protitelo obsega ostanke, ki niso niti v protitelesnih recipientnih niti v uvoženih CDR ali ogrodnih sekvencah. Te modifikacije naredimo zato, da nadalje očistimo in optimiziramo izvedbo protitelesa. Na splošno, humanizirano protitelo obsega v bistvu v celoti vsaj eno in značilno dve variabilni domeni, v katerih vse ali v bistvu vse regije CDR ustrezajo nehumanim imunoglobulinskim in so vse ali v bistvu vse regije FR od humane imunoglobulinske konsenzne sekvence. Humanizirano protitelo optimalno obsega tudi vsaj del imunoglobulinske konstantne regije (Fc), značilno humane imunoglobulinske. Za nadaljnje podrobnosti glej: Jones et al., Nature, 321:522-525 [1986]; Reichmann et al., Nature, 332:323-329 [1988]; in Presta, Curr. Op. Struct. Biol., 2:593-596 [1992]).Humanized forms of non-human (eg, murine) antibodies are kyrgyz immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab ', F (ab') _2, or other antigen-binding antibody sequences) containing a minimal sequence derived from non-human immunoglobulin. Mostly, humanized antibodies are human immunoglobulins (recipient antibody), in which residues from recipient CDRs are replaced by CDR residues from non-human species (donor antibody), such as e.g. mice, rats or rabbits that have the desired specificity, affinity and capacity. In some cases, the Fv framework residues of human immunoglobulin are replaced by the corresponding non-human residues. Further, the humanized antibody may comprise residues that are neither in the antibody recipient nor in the imported CDRs or framework sequences. These modifications are made to further purify and optimize the performance of the antibody. In general, a humanized antibody comprises essentially at least one and typically two variable domains in which all or substantially all CDR regions correspond to non-human immunoglobulins and are all or substantially all FR regions of the human immunoglobulin consensus sequence. The humanized antibody also optimally comprises at least a portion of the immunoglobulin constant region (Fc), typically human immunoglobulin. For further details, see: Jones et al., Nature, 321: 522-525 [1986]; Reichmann et al., Nature, 332: 323-329 [1988]; and Presta, Curr. Op. Struct. Biol., 2: 593-596 [1992].

Ne-imunogenski v človeku pomeni, da po kontaktiranju polipeptida v farmacevtsko sprejemljivem nosilcu in v terapevtsko učinkoviti količini z ustreznim humanim tkivom, ni možno opaziti stanja občutljivosti ali rezistence na polipeptid po drugem dajanju polipeptida po ustrezni latentni periodi (npr. 8-14 dni).Non-immunogenic in humans means that after contacting the polypeptide in a pharmaceutically acceptable carrier and in a therapeutically effective amount with the appropriate human tissue, it is not possible to detect a state of sensitivity or resistance to the polypeptide after the second administration of the polypeptide after a suitable latency period (e.g., 8-14 days) .

II. Prednostne izvedbe izumaII. Preferred embodiments of the invention

Prednostni polipeptidi v smislu predloženega izuma so v bistvu homogeni polipeptidi, imenovani mpl Ugandi ah trombopoetini (TPO), ki imajo to lastnost, da se vežejo na mpl, člana receptorske citokinske superdružine in imajo biološko lastnost, da stimulirajo vgraditev označenih nukleotidov (³H-timidin) v DNA od IL-3 odvisnih ceUc Ba/F3, transfektiranih s humanim mpl P. Bolj prednostni mpl Ugandi so izolirani iz proteinov sesalcev, ki imajo hematopoetično, posebno megakariocitopoetično aU trombocitopoetično aktivnost, in sicer so sposobni stimuliranja proUferacije, zorenja in/aU diferenciacije nezrelih megakariocitov aU njihovih predhodnikov v zrelo trombocite tvorečo obliko. Najbolj prednostni poUpeptidi v smislu izuma so humani mpl Ugandi, vključno njihovi fragmenti, ki imajo hematopoetično, megakariocitopoetično aU trombopoetično aktivnost. V danem primeru pri teh humanih mpl Ugandih ni gUkozilacije. Drugi prednostni mpl Ugandi so EPO-domena od hML, imenovana hML₁₅₃ aU hTPO₁₅₃, skrajšana oblika hML, imenovana hML^ aU hTPO^, in zreU poUpeptid s popolno dolžino, ki ima aminokislinsko sekvenco, prikazano na sl. 1 (SEQ ID NO: 1), imenovan hML, hML₃₃₂ aU hTPO₃₃₂, in biološko aktivna substitucijska varianta hML(R153A, R154A).Preferred polypeptides of the present invention are essentially homogeneous polypeptides called mpl Ugand ah thrombopoietins (TPO), which have the property of binding to mpl, a member of the receptor cytokine superfamily, and have the biological property to stimulate the incorporation of labeled nucleotides ( ³ H- thymidine) in DNA from IL-3-dependent ceUc Ba / F3 transfected with human mpl P. More preferred mpl Ugandans are isolated from mammalian proteins having haematopoietic, especially megakaryocytopoietic aU thrombocytopoietic activity, and are capable of stimulating proliferation, maturation and / aU differentiation of immature megakaryocytes aU their precursors into mature platelet-forming form. The most preferred poUpeptides of the invention are Ugandan human mpl, including fragments thereof, which have hematopoietic, megakaryocytopoietic aU thrombopoietic activity. In the present case, there is no gucosylation in these humane mpl Ugandans. Other preferred mpl of Uganda are the EPO domain from hML called hML ₁₅₃ aU hTPO ₁₅₃ , a shortened form of hML called hML ^ aU hTPO ^, and a full length mature poUpeptide having the amino acid sequence shown in FIG. 1 (SEQ ID NO: 1) named hML, hML ₃₃₂ aU hTPO ₃₃₂ , and the biologically active hML substitution variant (R153A, R154A).

Fakultativno prednostni poUpeptidi v smislu izuma so biološko aU imunološko aktivne variante mpl Ugandov, izbrane izmed hML2, hML3, hML4, mML, mML2, mML3, pML in pML2.Optionally, the preferred polypeptides of the invention are biologically aU immunologically active variants of Ugandan mpl, selected from hML2, hML3, hML4, mML, mML2, mML3, pML and pML2.

Fakultativno prednostni poUpeptidi v smislu izuma so biološko aktivne variante mpl Uganda, ki imajo aminokisUnsko sekvenco, ki ima vsaj 70 % aminokislinsko sekvenčno identičnost s humanim mpl Ugandom (sl. 1 [SEQ ID NO: 1]), murinim mpl Ugandom (sl. 16 [SEQ ID NOS: 12 & 13]), rekombinantnim prašičjim mpl Ugandom (sl. 19 [SEQ ID NO: 18]) aU prašičjim mpl Ugandom, izoliranim iz aplastične prašičje plazme, prednostno vsaj 75 %, bolj prednostno vsaj 80 %, še bolj prednostno vsaj 85 %, celo bolj prednostno vsaj 90 % in najbolj prednostno vsaj 95 %.The optional polypeptides of the invention are biologically active Ugand mpl variants having an amino acid sequence having at least 70% amino acid sequence identity with human Ugl mpl (Fig. 1 [SEQ ID NO: 1]), murine Ugl mpl (Fig. 16 [SEQ ID NOS: 12 & 13]), recombinant pig mpl Uganda (Fig. 19 [SEQ ID NO: 18]) aIn pig mpl Uganda isolated from aplastic pig plasma, preferably at least 75%, more preferably at least 80%, further more preferably at least 85%, even more preferably at least 90% and most preferably at least 95%.

Mpl ligand, izoliran iz aplastične prašičje plazme, ima naslednje značilnosti:The mpl ligand isolated from aplastic porcine plasma has the following characteristics:

(1) delno očiščen Ugand se eluira iz kolone za gelsko filtracijo bodisi v(1) partially purified Ugand eluted from the gel filtration column either into

PBS, PBS, ki vsebuje SDS (0,1 %) aU PBS, ki vsebuje MgCl₂ (4M) z Mr 60000-70000;PBS, PBS containing SDS (0.1%) aU PBS containing MgCl ₂ (4M) with Mr 60000-70000;

(2) aktivnost Uganda uniči pronaza;(2) the activity of Uganda is destroyed by the find;

(3) Ugand je stabilen pri nizkem pH (2,5), SDS do 0,1 % in sečnini (2M);(3) Ugandan is stable at low pH (2.5), SDS up to 0.1% and urea (2M);

(4) ligand je glikoprotein, osnovan na njegovi vezavi v različnih lektinskih kolonah;(4) the ligand is a glycoprotein based on its binding in different lectin columns;

(5) visoko očiščeni ligand se eluira iz nereducirane SDS-PAGE z Mr 2500035000. Manjše količine aktivnosti se tudi eluirajo z Mr od približno 18000 do 22000 in 60000;(5) the highly purified ligand is eluted from non-reduced SDS-PAGE with Mr 2500035000. Smaller amounts of activity are also eluted with Mr from about 18000 to 22000 and 60000;

(6) visoko očiščeni ligand se loči na reducirani SDS-PAGE kot dublet z Mr 28000 in 31000;(6) the highly purified ligand is separated on reduced SDS-PAGE as a doublet with Mr 28000 and 31000;

(7) aminoterminalna sekvenca prog z Mr 18000-22000, 28000 in 31000 je enaka -SPAPPACDPRLLNKLLRDDHVLHGR (SEQ ID NO: 29); in (8) ligand se veže in eluira iz afinitetnih kolon, kot so:(7) the amino-terminal sequence of the lines with Mr 18000-22000, 28000 and 31000 is equal to -SPAPPACDPRLLNKLLRDDHVLHGR (SEQ ID NO: 29); and (8) the ligand binds and elutes from affinity columns such as:

blue-sefarozablue-sepharose

CM blue-sefarozaCM blue-Sepharose

MONO-QMONO-Q

MONO-S lektin(leča)-sefarozaMONO-S lectin (lens) -sepharose

WGA-sefarozaWGA-Sepharose

Con A(=konkanavalin A)-sefaroza eter 650 m Toyopearl butil 650 m Toyopearl fenil 650 m Toyopearl in fenil-sefaroza.Con A (= concanavalin A) -sepharose ether 650 m Toyopearl butyl 650 m Toyopearl phenyl 650 m Toyopearl and phenyl sepharose.

Bolj prednostni mpl Ugandski pohpeptidi so tisti, kijih kodira humana genomska ah cDNA, ki ima aminokislinsko sekvenco, prikazano na sl. 1 (SEQ ID NO: 1).More preferred mpl Ugandan popeptides are those encoded by the human genomic ah cDNA having the amino acid sequence shown in FIG. 1 (SEQ ID NO: 1).

Drugi prednostni naravni biološko aktivni mpl Ugandski poUpeptidi v smislu izuma vključujejo prepro-mp/ Ugand, ρτο-mpl Ugand, zreU mpl Ugand, fragmente mpl Uganda in njihove gUkozilacijske variante.Other preferred naturally occurring biologically active mpl Ugandan popeptides of the invention include prepro-mp / Ugand, ρτο-mpl Ugand, matured mpl Ugand, mpl Ugand fragments and their gcososylation variants.

Še drugi prednostni poUpeptidi v smislu izuma vključujejo variante sekvence mpl Uganda in kimere. Navadno so prednostne variante sekvence mpl Uganda in kimere biološko aktivne variante mpl Uganda, ki imajo aminokisUnsko sekvenco z vsaj 70 % aminokislinsko sekvenčno identičnostjo s humanim mpl ligandom ali mpl Ugandom, izoUranim iz aplastične prašičje plazme, prednostno vsaj 75 %, bolj prednostno vsaj 80 %, še bolj prednostno vsaj 85 %, še celo bolj prednostno vsaj 90 % in najboj prednostno vsaj 95 %. Zgledno prednostna varianta mpl Uganda je varianta N-terminalne domene hML (imenovana ΈΡΟ-domena zaradi njene sekvenčne homologije za eritropoetil). Prednostna ΕΡΟ-domena hML obsega približno prvih 153 aminokislinskihostankov zrelega hML in je označena hML₁₅₃. V danem primeru je prednostna varianta sekvence hML tista, v kateri je eden ali več bazičnih ali dibazičnih amino kislinskih ostankov v C-terminalni domeni substituiranih z ne-bazičnimi amino kislinskimi ostanki (npr. hidrofobni, nevtralni, kisli, aromatski, Gly, Pro ipd.). Prednostna varianta sekvence C-terminalne domene hML je tista, v kateri sta ostanka Arg 153 in 154 nadomeščena z ostanki Ala. Ta varianta je označena kot hML₃₃₂(R153A, R154A). Alternativno prednostna varianta hML obsega bodisi hML₃₃₂ ali hML₁₅₃, v katerem so amino ostanki 111-114 (QLPP ali LPPQ) deletirani ali nadomeščeni z drugačno tetrapeptidno sekvenco (npr. AGAG ipd.). Prej omenjeni delecijski mutanti so označeni kot A4hML₃₃₂ ali A4hML₁₅₃.Still other preferred poUpeptides of the invention include the Ugand mpl sequence variants and chimeras. Typically, mpl Uganda sequence variants and chimeras of biologically active mpl Uganda variants having an amino acid sequence with at least 70% amino acid sequence with a human mpl ligand or mpl Uganda isolated from aplastic porcine plasma, preferably at least 80%, more preferably at least 80%, are preferred. , more preferably at least 85%, even more preferably at least 90% and most preferably at least 95%. An exemplary preferred mpl variant of Uganda is the variant of the N-terminal hML domain (called the ΈΡΟ-domain due to its sequence homology to erythropoethyl). The preferred ΕΡΟ domain of hML comprises approximately the first 153 amino acid residues of mature hML and is designated hML ₁₅₃ . In the present case, a preferred variant of the hML sequence is one in which one or more basic or dibasic amino acid residues in the C-terminal domain are substituted by non-basic amino acid residues (e.g. hydrophobic, neutral, acidic, aromatic, Gly, Pro, etc.) .). The preferred sequence variant of the C-terminal hML domain is one in which residues Arg 153 and 154 are replaced by residues Ala. This variant is designated hML ₃₃₂ (R153A, R154A). Alternatively, the preferred hML variant comprises either hML ₃₃₂ or hML ₁₅₃ , in which the amino residues 111-114 (QLPP or LPPQ) are deleted or replaced by a different tetrapeptide sequence (e.g. AGAG, etc.). The aforementioned deletion mutants are designated A4hML ₃₃₂ or A4hML ₁₅₃ .

Prednostna kimera je fuzija med mpl ligandom ali njegovim fragmentom (definirano spodaj) in heterolognim polipeptidom ali njegovim fragmentom. Npr. hML₁₅₃ lahko spojimo s fragmentom IgG, da izboljšamo serumsko razpolovno dobo, ali z EL-3, G-CSF ali EPO, da nastane molekula s povečano trombopoetično ali kimemo hematopoetično aktivnostjo.The preferred chimera is a fusion between the mpl ligand or fragment thereof (defined below) and the heterologous polypeptide or fragment thereof. E.g. hML ₁₅₃ can be fused with an IgG fragment to improve serum half-life, or with EL-3, G-CSF or EPO to produce a molecule with increased thrombopoietic or nodding hematopoietic activity.

Alternativno prednostna kimera humanega mpl liganda je ML-EPO-domenska kimera, ki je sestavljena iz N-terminala 153 do 157 ostankov hML, substituiranih z enim ali več, toda ne vsemi, ostanki humanega EPO, približno uvrščenimi, kot je prikazano na sl. 10 (SEQ ED NO:7). V tej izvedbi naj bi imela kimera hML po dolžini približno 153-166 ostankov, kjer so individualni ostanki ali bloki le-teh iz sekvence humanega EPO dodani ali substituirani v sekvenci hML na položajih, ki ustrezajo uvrstitvi, prikazani na sl. 10 (SEQ ID NO: 6). Zgledni inserti sekvenčnih blokov v N-terminalnem delu hML naj bi vključevali eno ali več N-glikozilacijskih mest na položajih (EPO) 24-27, 38-40 in 83-85; enega ali več od Štirih napovedanih amfipatičnih α-vijačnih spletov na položajih (EPO) 9-22, 59-76, 90-107, 132-152; in druge visoko koncentrirane regije, ki vključujejo N-terminalne in C-terminalne regije in položaje ostankov (EPO) 44-52 (glej npr. Wen et al., Blood, 82:1507-1516 [1993] in Boissel et al., J. Biol. Chem., 268(21):15983-15993 [1993]). Po pričakovanju naj bi ta ML-EPO domenska kimera imela mešano trombopoetično-eritropoetično (TEPO) biološko aktivnost.An alternative preferred chimeric human mpl ligand is the ML-EPO domain chimera consisting of the N-terminal 153 to 157 hML residues substituted by one or more, but not all, human EPO residues approximately classified as shown in FIG. 10 (SEQ ED NO: 7). In this embodiment, the hML chimera should have about 153-166 residues in length, where individual residues or blocks thereof from the human EPO sequence are added or substituted in the hML sequence at the positions corresponding to the classification shown in FIG. 10 (SEQ ID NO: 6). Exemplary insertions of sequence blocks in the N-terminal portion of the hML should include one or more N-glycosylation sites at positions (EPO) 24-27, 38-40 and 83-85; one or more of the Four Predicted Amphipathic α-Screw Webs at Positions (EPO) 9-22, 59-76, 90-107, 132-152; and other highly concentrated regions involving the N-terminal and C-terminal regions and residue positions (EPOs) 44-52 (see, e.g., Wen et al., Blood, 82: 1507-1516 [1993] and Boissel et al., J. Biol. Chem., 268 (21): 15983-15993 [1993]. This ML-EPO domain chimera is expected to have mixed thrombopoietic-erythropoietic (TEPO) biological activity.

Drugi prednostni polipeptidi v smislu izuma vključujejo fragmente mpl liganda z zaporedno sekvenco vsaj 10, 15, 20, 25, 30 ali 40 aminokislinskih ostankov, ki so identični v sekvencah mpl liganda, izoliranega iz aplastične prašičje plazme, ali humanega mpl liganda, opisanega tukaj (tabela 14, Primer 24). Prednostni fragment mpl liganda je humani ML[1-X], kjer je X 153,164,191, 205, 207, 217, 229 ali 245 (sl. 1 [SEQ ID NO: 1] za sekvenco ostankov l-Χ). Drugi prednostni fragmenti mpl liganda vključujejo tiste, ki nastanejo kot rezultat kemijske ali encimatske hidrolize ali digestije očiščenega liganda.Other preferred polypeptides of the invention include fragments of the mpl ligand with a sequence of at least 10, 15, 20, 25, 30 or 40 amino acid residues identical in the sequences of the mpl ligand isolated from aplastic porcine plasma or the human mpl ligand described herein ( Table 14, Example 24). A preferred fragment of the mpl ligand is human ML [1-X], wherein X is 153,164,191, 205, 207, 217, 229 or 245 (Fig. 1 [SEQ ID NO: 1] for the sequence of residues l-Χ). Other preferred mpl ligand fragments include those resulting from the chemical or enzymatic hydrolysis or digestion of the purified ligand.

Nadaljnji prednostni vidik predloženega izuma je postopek za čiščenje molekul mpl liganda, ki obsega kontaktiranje vira mpl liganda, ki vsebuje molekule mpl liganda, z mobiliziranim receptorskim polipeptidom, posebno mpl ali mpl fuzijskim polipeptidom pri pogojih, kjer se molekule mpl liganda, ki jih je potrebno očistiti, selektivno adsorbirajo na imobilizirani receptorski polipeptid, izpiranje mobiliziranega nosilca, da odstranimo neadsorbirano snov, in eluiranje molekul, ki jih je potrebno očistiti, iz mobiliziranega receptorskega polipeptida z eluimim pufrom. Vir, ki vsebuje mpl ligand, je lahko plazma, kjer je mobiliziran receptor prednostno fuzija znpMgG.A further advantageous aspect of the present invention is a method for purifying mpl ligand molecules comprising contacting a mpl ligand source containing mpl ligand molecules with a mobilized receptor polypeptide, in particular mpl or mpl fusion polypeptide, under conditions where the mpl ligand molecules required are purified, adsorbed selectively on the immobilized receptor polypeptide, washing the mobilized carrier to remove the unabsorbed substance, and eluting the molecules to be purified from the mobilized receptor polypeptide with eluate buffer. The source containing the mpl ligand may be plasma, where the mobilized receptor is preferably a fusion of znpMgG.

Alternativno je vir, ki vsebuje mpl ligand, rekombinantna celična kultura, kjer je koncentracija mpl liganda bodisi v kulturnem mediju ali v celičnih lizatih na splošno višja kot v plazmi ali v drugih naravnih virih. V tem primeru zgoraj opisani imunoafinitetni postopek znpMgG, čeprav še vedno uporaben, navadno ni potreben in lahko uporabimo bolj tradicionalne postopke za čiščenje proteinov, znane v tehniki. Na kratko, prednostni postopek čiščenja, pri katerem dobimo v bistvu homogeni mpl ligand, obsega: odstranjevanje delcev debrisa, bodisi gostiteljskih celic ali liziranih fragmentov z npr. centrifugiranjem ali ultrafiltracijo; v danem primeru lahko protein koncentriramo s komercialno dosegljivim filtrom za koncentriranje proteinov; in nato ločevanje Uganda od drugih nečistot v eni ali več stopnjah, izbranih izmed: imunoafinitete, ionske izmenjave (npr. DEAE ali matrice, ki vsebujejo karboksimetilne ali sulfopropilne skupine), blue-sefaroze, CM blue-sefaroze, MONO-Q, MONO-S, lektin (leča)-sefaroze, WGA-sefaroze, Con A-sefaroze, eter Toyopearla, butil Toyopearla, fenil Toyopearla, protein A-sefaroze, SDS-PAGE, HPLC z reverzno fazo (npr. silikagel z dodanimi alifatskimi skupinami) ali molekularnih sit sefadeks ah velikostno izključitvene kromatografije in obaijanja z etanolom ali amonijevim sulfatom. Proteazni inhibitor, kot npr. metilsulfonilfluorid (PMSF), lahko vključimo v katerokoli od prej omenjenih stopenj za inhibiranje proteolize.Alternatively, the source containing the mpl ligand is a recombinant cell culture, where the concentration of the mpl ligand is either higher in the culture medium or in the cell lysates than in the plasma or in other natural sources. In this case, the znpMgG immunoaffinity procedure described above, although still useful, is usually not required and more conventional protein purification methods known in the art can be used. Briefly, a preferred purification process whereby a substantially homogeneous mpl ligand is obtained comprises: removing debris particles, whether host cells or lysed fragments with e.g. by centrifugation or ultrafiltration; optionally, the protein can be concentrated with a commercially available protein concentration filter; and then separating Uganda from other impurities in one or more stages, selected from: immunoaffinity, ion exchange (eg DEAE or matrix containing carboxymethyl or sulfopropyl groups), blue-sepharose, CM blue-sepharose, MONO-Q, MONO- S, lectin (lens) -sepharose, WGA-sepharose, Con A-sepharose, Toyopearl ether, butyl Toyopearl, phenyl Toyopearl, protein A-sepharose, SDS-PAGE, reversed-phase HPLC (eg silica gel with aliphatic groups added) or Sephadex ah molecular sieves size exclusion chromatography and ethanol or ammonium sulfate suspension. A protease inhibitor such as e.g. Methylsulfonylfluoride (PMSF) can be included in any of the aforementioned steps for inhibiting proteolysis.

V drugi prednostni izvedbi predloženi izum zagotavlja izolirano protitelo, ki je sposobno vezave na mpl ligand. Prednostno izolirano protitelo mpl liganda je monok45In another preferred embodiment, the present invention provides an isolated antibody capable of binding to the mpl ligand. The preferred isolated mpl ligand antibody is monoc45

Ionsko (Kohler and Milstein, Nature, 256:495-497 [1975]; Campbell, Laboratory Techniques in Biochemistry and Molecular Biology, Burdon et al., izd., Volume 13, Elsevier Science Publishers, Amsterdam [1985]; in Huse et al., Science, 246:12751281 [1989]). Prednostno izolirano protitelo mpl liganda je tisto, ki se veže na mpl ligand z afiniteto vsaj približno 10⁶1/mol. Bolj prednostno protitelo se veže z afiniteto vsaj približno 10⁷ 1/mol. Najbolj prednostno vzgojimo protitelo proti mpl ligandu, ki ima eno od zgoraj opisanih efektorskih funkcij. Izolirano protitelo, ki je sposobno vezave na mpl ligand, lahko v danem primeru spojimo z drugim polipeptidom, protitelo ali njegovo fuzijo pa lahko uporabimo, da izoliramo in očistimo mpl ligand iz vira, kot je opisano zgoraj za imobilizirani mpl polipeptid. V nadaljnjem prednostnem vidiku te izvedbe zagotavlja predloženi izum postopek za detektiranje mpl liganda in vitro ali in vivo, pri čemer obsega kontaktiranje protitelesa z vzorcem, posebno serumskim vzorcem, za katerega domnevamo, da vsebuje ligand, in detektiranje, če je prišlo do vezave.Ionian (Kohler and Milstein, Nature, 256: 495-497 [1975]; Campbell, Laboratory Techniques in Biochemistry and Molecular Biology, Burdon et al., Ed., Volume 13, Elsevier Science Publishers, Amsterdam [1985]; in Huse et al., Science, 246: 12751281 [1989]. Preferably, the isolated mpl ligand antibody is one that binds to the mpl ligand with an affinity of at least about 10 ⁶ 1 / mol. A more preferred antibody binds to an affinity of at least about 10 ⁷ 1 / mol. Most preferably, we raise an antibody to the mpl ligand that has one of the effector functions described above. An isolated antibody capable of binding to the mpl ligand can optionally be fused to another polypeptide, and the antibody or its fusion can be used to isolate and purify the mpl ligand from the source as described above for the immobilized mpl polypeptide. In a further preferred aspect of this embodiment, the present invention provides a method for detecting mpl ligands in vitro or in vivo, comprising contacting an antibody with a sample, especially a serum sample, which is believed to contain a ligand, and detecting if binding has occurred.

V še nadaljnji prednostni izvedbi zagotavlja predloženi izum izolirano molekulo nukleinske kisline, ki kodira mpl ligand ali njegove fragmente, pri čemer je molekula nukleinske kisline lahko označena ali neoznačena z detektibilnim deležem in ima molekula nukleinske kisline sekvenco, ki je komplementarna ali se hibridizira pro ostrih ali zmerno ostrih pogojih z molekulo nukleinske kisline, ki ima sekvenco, ki kodira mpl ligand. Prednostna nukleinska kislina mpl liganda je RNA ali DNA, ki kodira biološko aktiven mpl ligand in ima vsaj 75 % sekvenčno identičnost, bolj prednostno vsaj 80 %, še bolj prednostno vsaj 85 %, celo bolj prednostno 90 % in najbolj prednostno 95 % sekvenčno identičnost s humanim mpl ligandom. Bolj prednostne izolirane molekule nukleinske kisline so sekvence DNA, ki kodirajo biološko aktiven mpl ligand, izbrane izmed: (a) DNA, ki temelji na kodirni regiji gena mpl liganda sesalca (npr. DNA, ki obsega nukleotidno sekvenco, prikazano na sl. 1 (SEQ ID NO: 2) ali njeni fragmenti); (b) DNA, kije sposobna hibridiziranja z DNA od (a) pri vsaj zmerno ostrih pogojih; in (c) DNA, ki je degenerirana DNA, definirana pri (a) ali (b) in je rezultat degeneracije genetičnega koda. Ugotovili smo, da so lahko novi mpl Ugandi, opisani tukaj, člani družine Ugandov ah citokinov, ki imajo prikladno sekvenčno identičnost, da se njihova DNA lahko hibridizira z DNA s sl. 1 (SEQ ID NO: 2) (ali njenim komplementom ali fragmentom) pri manj do zmerno ostrih pogojih. Tako nadaljnji vidik predloženega izuma vključuje DNA, ki se hibridizira pri manj do zmerno ostrih pogojih z DNA, ki kodira mpl Ugandske poUpeptide.In a further preferred embodiment, the present invention provides an isolated nucleic acid molecule encoding an mpl ligand or fragments thereof, wherein the nucleic acid molecule may be labeled or unlabelled with a detectable moiety and the nucleic acid molecule has a sequence that is complementary or hybridizes pro-or moderately harsh conditions with a nucleic acid molecule having a sequence encoding the mpl ligand. Preferred mpl ligand nucleic acid is RNA or DNA encoding a biologically active mpl ligand and has at least 75% sequence identity, more preferably at least 80%, more preferably at least 85%, even more preferably 90%, and most preferably 95% sequence identity with human mpl ligand. More preferred isolated nucleic acid molecules are DNA sequences encoding a biologically active mpl ligand selected from: (a) DNA based on the coding region of the mammalian mpl ligand gene (e.g. DNA comprising the nucleotide sequence shown in Fig. 1 ( SEQ ID NO: 2) or fragments thereof); (b) DNA capable of hybridizing with DNA of (a) under at least moderately harsh conditions; and (c) DNA, which is degenerate DNA, defined in (a) or (b) and resulting from the degeneration of the genetic code. We have found that the new Ugandan mpl described herein may be members of the Ugand ah cytokine family having a convenient sequence identity so that their DNA can be hybridized to the DNA of FIG. 1 (SEQ ID NO: 2) (or a complement or fragment thereof) under less to moderately harsh conditions. Thus, a further aspect of the present invention includes DNA that hybridizes under less to moderately harsh conditions with DNA encoding mpl Ugandan popeptides.

V nadaljnji prednostni izvedbi predloženega izuma je molekula nukleinske kisline cDNA, ki kodira mpl ligand in nadalje obsega vektor, ki se da replicirati, v katerem je cDNA funkcionalno vezana na kontrolne sekvence, spoznane od gostitelja, transformiranega z vektorjem. Ta vidik nadalje vključuje gostiteljske celice, transformirane z vektorjem in postopek za uporabo cDNA, da vpliva na nastanek mpl liganda, ki obsega ekspresijo cDNA, ki kodira mpl ligand v kulturi transformiranih gostiteljskih celic, in rekuperiranje mpl liganda iz gostiteljske celične kulture. Mpl ligand, pripravljen na ta način, je prednostno v bistvu homogeni humani mpl ligand. Prednostne gostiteljske celice za tvorbo mpl liganda so ovarijske celice kitajskega hrčka (celice CHO).In a further preferred embodiment of the present invention, the nucleic acid molecule is a cDNA that encodes an mpl ligand and further comprises a replicable vector in which the cDNA is functionally linked to control sequences recognized by the host transformed with the vector. This aspect further includes vector transformed host cells and a process for using cDNA to influence mpl ligand formation comprising the expression of a cDNA encoding the mpl ligand in the transformed host cell culture and recovering the mpl ligand from the host cell culture. The mpl ligand prepared in this manner is preferably essentially a homogeneous human mpl ligand. Preferred host cells for mpl ligand formation are Chinese hamster ovary cells (CHO cells).

Predloženi izum nadalje vključuje prednosten postopek za zdravljenje sesalca z imunološkimi ali hematopoetičnimi motnjami, posebno trombocitopenije, ki obsega dajanje terapevtsko učinkovite količine mpl liganda temu sesalcu. V danem primeru damo mpl ligand v kombinaciji s dtokinom, posebno kolonijo stimulirajočim faktorjem ali interlevkinom. Prednosti kolonije stimulirajoči faktorji ali interlevkini vključujejo: kit-ligand, LIF, G-CSF, GM-CSF, M-CSF, EPO, IL-1, IL-2, LL-3, IL-5, IL-6, IL-7, IL-8, IL-9 ali IL-11.The present invention further includes a preferred method of treating a mammal with immunological or haematopoietic disorders, in particular thrombocytopenia, comprising administering a therapeutically effective amount of a mpl ligand to that mammal. If appropriate, the mpl ligand is administered in combination with dtokine, a particular colony-stimulating factor or interleukin. The benefits of colony-stimulating factors or interleukins include: kit-ligand, LIF, G-CSF, GM-CSF, M-CSF, EPO, IL-1, IL-2, LL-3, IL-5, IL-6, IL- 7, IL-8, IL-9, or IL-11.

III. Postopki izdelaveIII. Manufacturing procedures

Nekateri avtorji so dolgo mislili, da je nastajanje trombocitov kontrolirano z multiplimi rodovno specifičnimi humoralnimi faktorji. Domnevali so, da aktivnosti dveh različnih citokinov, imenovanih megakariocitno kolonijo stimulirajoči faktor (megCSF) in trombopoetin regulirata megakariocitopoezo in trombopoezo (Williams et al., J. Celi Physiol., 110:101-104 [1982]; Williams et al., Blood Celiš, 15:123-133 [1989]; in Gordon et al., Blood, 80:302-307 [1992]). V skladu s to hipotezo stimulira meg-CSF proliferacijo predniških megakariocitov, medtem ko trombopoetin primarno vpliva na zorenje bolj diferenciranih celic in na končno sproščanje trombocitov. Od leta 1960 naprej je dobro dokumentirana indukcija in nastanek aktivnosti tako za meg-CSF kot tudi za trombopoetin v plazmi, serumu in urinu živali ter ljudi po trombocitopoetičnih poteh (Odeli et al., Proč. Soc. Exp. Biol. Med., 108:428-431 [1961]; Nakeff et al., Acta Haematol., 54:340-344 [1975]; Specter, Proč. Soc. Exp. Biol., 108:146-149 [1961]; Schreiner et al., J. Ciin. Invest., 49:1709-1713 [1970]; Ebbe, Blood, 44:605-608 [1974]; Hoffman et al., N. Engl. J. Med., 305:533 [1981]; Straneva et al., Exp. Hematol., 17:1122-1127 [1988]; Mazur et al., Exp. Hematol., 13:1164 [1985]; Mazur et al., J. Ciin. Invest., 68:733-741 [1981]; Sheiner et al., Blood, 56:183-188 [1980]; Hill et al., Exp. Hematol.,Some authors have long thought that platelet production is controlled by multiple gender-specific humoral factors. The activities of two different cytokines called megakaryocyte colony stimulating factor (megCSF) and thrombopoietin have been hypothesized to regulate megakaryocytopoiesis and thrombopoiesis (Williams et al., J. Celi Physiol., 110: 101-104 [1982]; Williams et al., Blood Celiš, 15: 123-133 [1989]; and Gordon et al., Blood, 80: 302-307 [1992]. According to this hypothesis, it stimulates meg-CSF proliferation of ancestral megakaryocytes, whereas thrombopoietin primarily influences the maturation of more differentiated cells and the final release of platelets. Since 1960, the induction and emergence of activity for both meg-CSF and thrombopoietin in plasma, serum, and urine of animals and humans via thrombocytopoietic pathways has been well documented (Odeli et al., Soc Soc Exp. Biol. Med., 108 : 428-431 [1961]; Nakeff et al., Acta Haematol., 54: 340-344 [1975]; Specter, Proc Soc Soc Exp. Biol., 108: 146-149 [1961]; Schreiner et al. , J. Ciin Invest., 49: 1709-1713 [1970]; Ebbe, Blood, 44: 605-608 [1974]; Hoffman et al., N. Engl. J. Med., 305: 533 [1981] ; Straneva et al., Exp. Hematol., 17: 1122-1127 [1988]; Mazur et al., Exp. Hematol., 13: 1164 [1985]; Mazur et al., J. Ciin Invest., 68 : 733-741 [1981]; Sheiner et al., Blood, 56: 183-188 [1980]; Hill et al., Exp. Hematol.,

20:354-360 [1992]; in Hegyi et al., Int. J. Celi Cloning, 8:236-244 [1990]). Za te aktivnosti je navedeno, da so rodovno specifične in različne od znanih citokinov (Hill R.J. et al., Blood 80:346 (1992); Erickson-Miller C.L. et al., Brit. J. Haematol., 84:197-203 (1993); Straneva J.E. et al., Exp. Hematol. 20:4750 (1992); in Tsukada J. et al., Blood 81:866-867 [1993]). Prejšnji poskusi, da bi očistili meg-CSF ali trombopoetin iz trombocitopenične plazme ali urina, so bili neuspešni.20: 354-360 [1992]; and Hegyi et al., Int. J. Celi Cloning, 8: 236-244 [1990]. These activities are said to be gender specific and distinct from known cytokines (Hill RJ et al. Blood 80: 346 (1992); Erickson-Miller CL et al. Brit. J. Haematol. 84: 197-203 (1993); Straneva JE et al., Exp. Hematol. 20: 4750 (1992); and Tsukada J. et al., Blood 81: 866-867 [1993]. Previous attempts to purify meg-CSF or thrombopoietin from thrombocytopenic plasma or urine have been unsuccessful.

Skladno z zgornjimi opažanji, ki opisujejo trombocitopenično plazmo, smo ugotovili, da aplastična prašičja plazma (APP), dobljena iz obsevanih prašičev, stimulira humano megakariocitopoezo in vitro. Ugotovili smo, da se ta stimulatoma aktivnost odpravi s topno ekstracelično domeno c-mpl, kar potrjuje APP kot potencialni vir domnevnega mpl liganda (ML). Uspešno smo očistili mpl ligand iz APP, aminokislinsko sekvenčno informacijo pa smo uporabili za izolacijo cDNA murinega, prašičjega in humanega ML Ti ML imajo sekvenčno homologijo za eritropoetin in aktivnosti, podobne tako meg-CSF kot tudi trombopoetinu.Consistent with the above observations describing thrombocytopenic plasma, we found that aplastic porcine plasma (APP) obtained from irradiated pigs stimulates human megakaryocytopoiesis in vitro. We found that this stimulatory activity is abolished by the soluble extracellular c-mpl domain, confirming APP as a potential source of putative mpl ligand (ML). We successfully purified the mpl ligand from APP, and amino acid sequence information was used to isolate murine, porcine and human ML cDNAs. These MLs have sequence homology to erythropoietin and activities similar to both meg-CSF and thrombopoietin.

1. Čiščenje in identifikacija mp/ liganda iz plazmePlasma mp / ligand purification and identification

Kot je omenjeno zgoraj, je za aplastično plazmo iz različnih vrst navedeno, da vsebuje aktivnosti, ki stimulirajo hematopoezo in vitro, vendar pa pred tem ni bil naveden noben hematopoetični stimulatomi faktor, izoliran iz plazme. En vir aplastične plazme je tisti, ki ga dobimo iz obsevanih prašičev. Ta aplastična prašičja plazma (APP) stimulira humano hematopoezo in vitro. Da določimo, če APP vsebuje mpl ligand, testiramo njegov učinek z merjenjem vgraditve ³H-timidina v celice Ba/F3, transfektirane s humanim mpl P (Ba/F3-mp/) s postopkom, prikazanim na sl.As mentioned above, aplastic plasma from different species is said to contain activities that stimulate hematopoiesis in vitro, but no hematopoietic stimuli factor previously isolated from plasma has been reported previously. One source of aplastic plasma is that obtained from irradiated pigs. This aplastic porcine plasma (APP) stimulates human hematopoiesis in vitro. To determine if APP contains an mpl ligand, we test its effect by measuring the incorporation of ³ H-thymidine into Ba / F3 cells transfected with human mpl P (Ba / F3-mp /) by the procedure shown in FIG.

2. APP stimulira vgraditev ³H-timidina v celici Ba/F3-mp/, ne pa v kontrolne celice Ba/F3 (t.j. netransfektirane s humanim mpl P). Poleg tega nismo opazili nobene take aktivnosti v normalni prašičji plazmi. Iz teh rezultatov je razvidno, da APP vsebuje faktor ali faktorje, ki transducirajo proliferativni signal skozi mpl receptor in je zato naravni ligand za ta receptor. To je nadalje podprto z ugotovitvijo, da obdelava APP s topnim mpl-lgG blokira stimulatome učinke APP na celice Ba/F3-zzip/.2. APP stimulates the incorporation of ³ H-thymidine in the Ba / F3-mp / cell but not in the Ba / F3 control cells (ie, not transfected with human mpl P). In addition, no such activity was observed in normal porcine plasma. These results indicate that APP contains a factor or factors that transduce a proliferative signal through the mpl receptor and is therefore a natural ligand for this receptor. This is further supported by the finding that APP treatment with soluble mpl-1gG blocks the stimulatory effects of APP on Ba / F3-zzip / cells.

Videti je, da je aktivnost v APP proteinska, ker pronaza, DTT ali toplota uničijo aktivnost v APP (sl. 3). Aktivnosti se tudi ne da dializirati. Aktivnost pa je vsekakor stabilna pri nizkem pH (pH 2,5;2 uri) in se veže in eluira iz različnih lektinskih afinitetnih kolon, iz česar je razvidno, da je to glikoprotein. Za nadaljnjo osvetlitev strukture in identičnosti te aktivnosti le-to afinitetno očistimo iz APP z uporabo kimere mp/-IgG.Activity in APP appears to be protein because the find, DTT, or heat destroy the activity in APP (Fig. 3). Activities are also not dialysable. The activity is, however, stable at low pH (pH 2.5; 2 hours) and binds and elutes from different lectin affinity columns, indicating that it is a glycoprotein. To further illuminate the structure and identity of this activity, it is affinity purified from APP using the mp / -IgG chimera.

APP obdelamo v skladu s predpisi, navedenimi v Primerih 1 in 2. Na kratko, mpl ligand očistimo z uporabo hidrofobne interakcijske kromatografije (HIC), imobilizirane barvne kromatografije in mp/-afinitetne kromatografije. Rekuperiranje aktivnosti iz vsake stopnje je prikazano na sl. 4, večkratnost očiščenja pa v tabeli 1. Celotno rekuperiranje aktivnosti skozi mp/-afinitetno kolono je približno 10 %. Frakcija (F6) z maksimalno aktivnostjo iz mp/-afinitetne kolone ima določeno specifično aktivnost 9,8x1ο⁶ enot/mg. Celotno očiščenje iz 5 1 APP je približno 4x1ο⁶kratno (0,8 enot/mg do 3,3 χ 10⁶ enot/mg) s 83 x lO^kratno redukcijo proteina (250 g na 3 gg). Za specifično aktivnost liganda, eluiranega iz w/?/-afinitetne kolone, smo ugotovili, daje približno 3 χ 10⁶ enot/mg.The APP was treated according to the regulations given in Examples 1 and 2. Briefly, the mpl ligand was purified using hydrophobic interaction chromatography (HIC), immobilized color chromatography and mp / affinity chromatography. Recuperating activities from each stage is shown in Figs. 4, and the purification rates are shown in Table 1. The total recovery of activity through the mp / affinity column is about 10%. The fraction (F6) with maximum activity from the mp / affinity column has a specific activity of 9.8x1ο ⁶ units / mg. Total purification from 5 1 APP is approximately 4x1ο ⁶ times (0.8 units / mg to 3.3 χ 10 ⁶ units / mg) with 83 x lO ^ fold protein reduction (250 g to 3 gg). The specific activity of the ligand eluted from the w /? / - affinity column was found to be about 3 χ 10 ⁶ units / mg.

TABELA 1TABLE 1

Čiščenje mpl ligandaPurification of mpl ligand

vzorec sample volumen ml volume ml protein mg/ml protein mg / ml enote/ ml units / ml enote units specifič- na aktiv» nost^enote/mg specific- on activity ^ units / mg do- bi- tek % do- would- tek % n-kratno očiščenje n times purification APP APP 5000 5000 50 50 40 40 200,000 200,000 0.8 0.8 - - 1 1 fenil phenyl 4700 4700 0.8 0.8 40 40 200.000 200,000 50 50 94 94 62 62 blue-sef. blue-safe. 640 640 0.93 0.93 400 400 256,000 256,000 430 430 128 128 538 538 mpl Cul) ;št.fr. -5=n- mpl Cul); -5 = n- 12 12 5x10’⁴ 5x10 ' ⁴ 1666 1666 20,000 20,000 3.300,000 3.300,000 1 0 1 0 4,100,000 4,100,000

Protein določimo z Bradfordovim testom. Proteinsko koncentracijo mpl eluiranih frakcij 5-7 določimo na osnovi intenzitete barvanja gela SDS, obarvanega s srebrom. Ena enota je definirana tako, da povzroči 50 % maksimalne stimulacije Ba/F3-mp/ celične proliferacije.Protein was determined by Bradford assay. The protein concentration of the mpl eluted fractions 5-7 was determined on the basis of the staining intensity of the SDS gel stained with silver. One unit is defined to cause 50% of maximal stimulation of Ba / F3-mp / cell proliferation.

Iz analize eluiranih frakcij iz znpZ-afinitetne kolone z SDS-PAGE (4-20 %, Novex gel) pri redukcijskih pogojih je razvidna prisotnost različnih proteinov (sl. 5). Proteini, ki se obarvajo s srebrom z najmočnejšo intenziteto, se ločijo z jasnimi Mr: 66000,55000, 30000, 28000 in 18000-22000. Da določimo, kateri od teh proteinov stimulira proliferacijo Ba/F3-mpZ celičnih kultur, jih eluiramo iz gela, kot je opisano v PrimeruAnalysis of the eluted fractions from the ZnpZ affinity column by SDS-PAGE (4-20%, Novex gel) under reducing conditions shows the presence of different proteins (Fig. 5). Proteins that stain with the strongest intensity of silver are separated by clear Mr: 66000,55000, 30000, 28000 and 18000-22000. To determine which of these proteins stimulates the proliferation of Ba / F3-mpZ cell cultures, we elute them from the gel as described in Example

2.2.

Iz rezultatov tega eksperimenta je razvidno, da se večino aktivnosti eluira iz rezine gela, ki vključuje proteine z Mr 28000-32000, z manjšo aktivnostjo pa se eluirajo v delu gela z Mr 18000-22000 (sl. 6). Edini proteini, vidni v teh delih, imajo Mr 30000, 28000 in 18000-22000. Da identificiramo in dobimo proteinske sekvence za proteine, ki se ločijo v tem delu gela (t.j. proge pri 30, 28 in 18-22 kDa) te tri proteine elektro popivnamo na P VDF in sekvenciramo, kot je opisano v Primeru 3. Dobljene aminoterminalne sekvence so prikazane v tabeli 2.The results of this experiment show that most of the activity is eluted from a slice of gel that includes proteins with Mr 28000-32000, and with less activity, they are eluted in part of the gel with Mr 18000-22000 (Fig. 6). The only proteins seen in these parts have Mr 30000, 28000 and 18000-22000. To identify and obtain protein sequences for proteins that separate in this portion of the gel (i.e., lines at 30, 28, and 18-22 kDa), these three proteins were electroplated onto P VDF and sequenced as described in Example 3. The aminoterminal sequences obtained are shown in Table 2.

TABELA 2TABLE 2

Amino-terminalne sekvence mpl ligandaAmino-terminal sequences of mpl ligands

30 kDa 1 5 10 15 20 25 fS) P A P P A(C)D P R L L N K L L R D D (H/S) V L H (G) R L 30 kDa 1 5 10 15 20 25 fS) P A P P A (C) D P R L L N K L L R D D (H / S) V L H (G) R L (SEQ ID NO: 30) (SEQ ID NO: 30) 28 kDa 1 5 10 15 20 25 (S)PAPPAXDPRLLNKLLRDD(H)VL(H)GR 28 kDa 1 5 10 15 20 25 (S) PAPPAXDPRLLNKLLRDD (H) VL (H) GR (SEQ ID NO: 31) (SEQ ID NO: 31) 18-22 kDa 1 5 10 X P A P P A X D P R L X (N) (K) 18-22 kDa 1 5 10 X P A P P A X D P R L X (N) (K) (SEQ ID NO: 32) (SEQ ID NO: 32)

Računalniška analiza dokazuje, da so te aminokislinske sekvence nove. Ker so vse tri sekvence enake, je verjetno, da so proteini 30 kDa, 28 kDa in 18-22 kDa sorodni in so lahko različne oblike istega novega proteina. Nadalje so ti proteini možni kandidati za naravni mpl ligand, ker se aktivnost loči na SDS-PAGE v istem delu (28000-32000) gela (4-20 %). Poleg tega delno očiščeni ligand migrira v regijo z Mr 17000-30000, če ga izpostavimo gelski filtracijski kromatografiji z uporabo kolone superoze 12 (Pharmacia). Veijetno so oblike liganda z različnimi Mr rezultat proteoliznih ali glikozilacijskih razlik ali drugih po- ali pred-translacijskih modifikacij.Computational analysis proves that these amino acid sequences are novel. Because all three sequences are identical, it is likely that the 30 kDa, 28 kDa, and 18-22 kDa proteins are related and may be different forms of the same novel protein. Furthermore, these proteins are potential candidates for the natural mpl ligand because the activity is separated by SDS-PAGE in the same portion (28000-32000) of the gel (4-20%). In addition, the partially purified ligand migrates to the region with Mr 17000-30000 when subjected to gel filtration chromatography using a superose column 12 (Pharmacia). Ligand forms with different Mr are likely to be the result of proteolysis or glycosylation differences or other post- or pre-translational modifications.

Kot je opisano pred tem, antisens RNA humanega mpl odpravi megakariocitopoezo v kulturah humanega kostnega mozga, obogatenih s predniškimi celicami CD 34⁺, ne da bi vplivala na diferenciacijo drugih rodov hematopoetičnih celic (Methia et al., zgoraj). Ta rezultat navaja k temu, da ima lahko mpl receptor vlogo pri diferenciaciji in proliferaciji megakariocitov in vitro. Za nadaljnjo osvetlitev te vloge mpl liganda v megakariocitopoezi primerjamo učinke APP in APP z odstranjenim mpl ligandom na humano megakariocitopoezo in vitro. Učinek APP na humano megakariocitopoezo določimo z modifikacijo testa tekočinske suspenzijske megakariocitopoeze, opisanega v Primeru 4. Pri tem testu humane periferne matične celice (PSC) obdelamo z APP pred in po mp/-IgG afinitetni kromatografiji. GPIIbIIIa stimulacijo megakariocitopoeze kvantitativno določimo s ¹²⁵J-anti-IIbIII_a protitelesom (sl. 7). Kot je prikazano na sl. 7 10 % APP povzroči približno 3-kratno stimulacijo, medtem ko APP z odstranjenim mpl ligandom nima vpliva. Značilno je, da APP z odstranjenim mpl ligandom ne inducira proliferacije celic Ba/F3-mp/.As described previously, human mpl RNA antisense abolishes megakaryocytopoiesis in human bone marrow cultures enriched with CD 34 ⁺ ancestral cells without affecting differentiation of other hematopoietic cell lineages (Methia et al., Above). This result suggests that the mpl receptor may play a role in the differentiation and proliferation of megakaryocytes in vitro. To further illuminate this role of mpl ligand in megakaryocytopoiesis, we compare the effects of APP and APP with the removed mpl ligand on human megakaryocytopoiesis in vitro. The effect of APP on human megakaryocytopoiesis was determined by modifying the liquid suspension megakaryocytopoiesis test described in Example 4. In this test, human peripheral stem cells (PSCs) were treated with APP before and after mp / -IgG affinity chromatography. GPllbllla stimulation of megakaryocytopoiesis was quantitated with the ^{125I-anti-IIbIII} _a antibody (Fig. 7). As shown in FIG. 7 10% APP induces approximately 3-fold stimulation, whereas APP with mpl ligand removed has no effect. Typically, APP with the mpl ligand removed does not induce cell proliferation of Ba / F3-mp /.

Pri drugem eksperimentu topen humani mpl-lgG, dodan na dneve 0,2 in 4 kulturam, ki vsebujejo 10 % APP, nevtralizira stimulatorne učinke APP na humano megakariocitopoezo (sl. 8). Iz teh rezultatov je razvidno, da ima mpl ligand vlogo pri reguliranju humane megakariocitopoeze in je zato lahko koristen za zdravljenje trombocitopenije.In the second experiment, soluble human mpl-1gG added on days 0.2 and 4 to cultures containing 10% APP neutralized the stimulatory effects of APP on human megakaryocytopoiesis (Fig. 8). These results suggest that the mpl ligand plays a role in regulating human megakaryocytopoiesis and therefore may be useful for the treatment of thrombocytopenia.

2. Molekulsko kloniranje mpl liganda2. Molecular cloning of mpl ligand

Na osnovi amino-terminalne aminokislinske sekvence, dobljene iz 30 kDa, 28 kDa in 18-22 kDa proteinov (tabela 2, zgoraj), oblikujemo dva degenerirana oligonukleotidna primerska poola in uporabimo za pomnožitev prašičje genomske DNA s PCR. Utemeljeno je, da, če amino-terminalno aminokislinsko sekvenco kodira posamezen ekson, potem pričakujemo, da ima pravilen produkt PCR dolžino 69 bp. Fragment DNA te velikosti smo ugotovili in subklonirali v pGEMT. Sekvence oligonukleotidnih PCR primeijev in trije dobljeni kloni so prikazani v Primeru 5.Based on the amino-terminal amino acid sequence obtained from 30 kDa, 28 kDa, and 18-22 kDa proteins (Table 2, above), two degenerate oligonucleotide primer pools are formed and used to amplify porcine genomic DNA by PCR. It is justified that if the amino-terminal amino acid sequence encodes a single exon, then the correct PCR product is expected to have a length of 69 bp. A DNA fragment of this size was identified and subcloned into pGEMT. The sequences of the oligonucleotide PCR primers and the three clones obtained are shown in Example 5.

Aminokislinska sekvenca (PRLLNKLLR [SEQ ID NO: 33]) peptida, kodiranega med PCR primeiji, je identična tisti, ki jo dobimo pri sekvenciranju aminoterminalnega proteina prašičjega Uganda (ostanki 9-17 za sekvence prašičjega proteina z 28 in 30 kDa, zgoraj).The amino acid sequence (PRLLNKLLR [SEQ ID NO: 33]) of the peptide encoded during PCR primers is identical to that obtained from sequencing of the pig amino protein of Uganda (residues 9-17 for 28 and 30 kDa pig protein sequences, above).

Sintetični oligonukleotid, ki temelji na sekvenci fragmenta PCR uporabimo za selekcioniranje knjižnice humane genomske DNA. 45-memi oUgonukleid, označen s pR45, oblikujemo in sintetiziramo na osnovi sekvence fragmenta PCR. Ta oligonukleotid ima naslednjo sekvenco:A synthetic oligonucleotide based on the PCR fragment sequence is used to select a library of human genomic DNA. The pR45-tagged 45-membered oUgonucleide is formed and synthesized based on the PCR fragment sequence. This oligonucleotide has the following sequence:

5' GCC-GTG-AAG-GAC-GTG-GTC-GTC-ACG-AAG-CAG-TTT-ATT-TAG-GAG-TCG 3' (SEQ ID NO: 34)5 'GCC-GTG-AAG-GAC-GTG-GTC-GTC-ACG-AAG-CAG-TTT-ATT-TAG-GAG-TCG 3' (SEQ ID NO: 34)

Ta deoksioUgonukleotid uporabimo za selekcioniranje knjižnice humane genomske DNA v Xgeml2 pri manj ostrih pogojih hibridizacije in izpiranja v skladu s Primerom 6. Pozitivne klone zberemo, plake očistimo in analiziramo z restrikcijskim mapiranjem in Southern blotting-om. Fragment EcoRI-XbaI s 390 bp, ki se hibridizira s 45merom, subkloniramo v pBluescript SK-. Sekvenciranje DNA tega klona potrjuje, da smo izoliraU DNA, ki kodira humani homolog prašičjega mpl Uganda. Sekvenca humane DNA in sekvenca deducirane amino kisUne sta prikazani na sl. 9 (SEQ ED NO: 3 & 4). Napovedani položaji intronov v genomski sekvenci so tudi prikazani s puščicami in definirajo domnevni ekson (ekson 3).This deoxyUgonucleotide was used to select the human genomic DNA library in Xgeml2 under less harsh hybridization and washing conditions according to Example 6. Positive clones were collected, plaques were purified and analyzed by restriction mapping and Southern blotting. The 390 bp 39co bp EcoRI-XbaI fragment was subcloned into pBluescript SK-. DNA sequencing of this clone confirms that we have isolated DNA encoding a human homolog of pig mpl Uganda. The human DNA sequence and the deduced amino acid sequence are shown in FIG. 9 (SEQ ED NO: 3 & 4). The predicted positions of introns in the genomic sequence are also shown by arrows and define the putative exon (exon 3).

Na osnovi sekvence humanega eksona 3 (Primer 6) sintetiziramo oligonukleotide, ki ustrezajo 3’ in 5’ koncem sekvence eksona. Ta dva primerja uporabimo v reakcijah PCR, kjer uporabimo kot kalup cDNA, pripravljeno iz različnih humanih tkiv. Pričakovana velikost pravilnega produkta PCR je 140 bp. Po analizi produktov PCR na poliakrilamidnem gelu (12 %) detektiramo fragment DNA pričakovane velikosti v knjižnicah cDNA, pripravljene iz ledvic odraslega človeka, 293 celic fetalnih ledvic in cDNA, pripravljene iz humanih fetalnih jeter.Based on the human exon 3 sequence (Example 6), oligonucleotides corresponding to the 3 'and 5' ends of the exon sequence are synthesized. These two primers are used in PCR reactions where they are used as a cDNA mold prepared from different human tissues. The expected size of the correct PCR product is 140 bp. After analysis of PCR products on polyacrylamide gel (12%), a DNA fragment of the expected size was detected in cDNA libraries prepared from adult human kidneys, 293 fetal kidney cells, and human fetal liver cDNA cells.

Knjižnico cDNA fetalnih jeter (7x1ο⁶ klonov) v lambda DR2 nato selekcioniramo z istim 45-mernim oligonukleotidom, uporabljenim za selekcioniranje humane genomske knjižnice in knjižnice cDNA fetalnih jeter pri manj ostrih pogojih hibridizacije. Pozitivne klone zberemo, plake očistimo in določimo velikost inserta s PCR. En klon z insertom 1,8 kb izberemo za nadaljnjo analizo. Z uporabo postopkov, opisanih v Primeru 7, dobimo nukleotid in deducirano aminokislinsko sekvenco humanega mpl liganda (hML). Te sekvence so prikazane na sl. 1 (SEQ ID NO: 1 & 2).The fetal liver cDNA library (7x1ο ⁶ clones) in lambda DR2 is then selected with the same 45-oligonucleotide used to select the human genomic library and the fetal liver cDNA library under less harsh hybridization conditions. Positive clones were collected, plaques were cleaned, and the size of the insert was determined by PCR. One clone with a 1.8 kb insert is selected for further analysis. Using the procedures described in Example 7, the nucleotide and deduced amino acid sequence of the human mpl ligand (hML) are obtained. These sequences are shown in FIG. 1 (SEQ ID NO: 1 & 2).

3. Struktura humanega mpl liganda (hML)3. Structure of human mpl ligand (hML)

Sekvenca cDNA humanega mpl liganda (hML) (sl. 1 [SEQ ID NO: 2]) obsega 1774 nukleotidov, ki jim sledi poli(A) rep. Le ta vsebuje 215 nukleotidov 5’ netranslatirane sekvence in 3’ netranslatirano regijo 498 nukleotidov. Domnevni iniciacijski kodon na nukleotidnem položaju (216-218) je znotraj konsenzne sekvence, ugodne za evkariotsko translacijsko iniciacijo. Odprt bralni okvir ima dolžino 1059 nukleotidov in kodira polipeptid s 353 aminokislinskimi ostanki, začne pa se pri nukleotidnem položaju 220. N-terminal napovedane aminokislinske sekvence je visoko hidrofoben in vetjetno ustreza signalnemu peptidu. Iz računalniške analize predvidene aminokislinske sekvence (von Heijne et al., Eur. J. Biochem., 133:17-21 [1983]) je razvidno potencialno cepišče za signalno peptidazo med ostankoma 21 in 22. Cepitev v tem položaju bi dala zreli polipeptid s 332 aminokislinskimi ostanki, ki se začne z amino-terminalno sekvenco, dobljeno iz mpl liganda, očiščenega iz prašičje plazme. Napovedana molekulska masa neglikoziliranega liganda s 332 aminokislinskimi ostanki je približno 38 kDa. Ima 6 potencialnih N-glikozilacijskih mest in 4 cisteinske ostanke.The human mpl ligand (hML) cDNA sequence (Fig. 1 [SEQ ID NO: 2]) comprises 1774 nucleotides followed by a poly (A) tail. It contains 215 nucleotides of 5 'untranslated sequences and a 3' untranslated region of 498 nucleotides. The putative initiation codon at the nucleotide position (216-218) is within the consensus sequence favorable for eukaryotic translational initiation. The open reading frame has a length of 1059 nucleotides and encodes a polypeptide with 353 amino acid residues, starting at the nucleotide position 220. The N-terminal of the predicted amino acid sequence is highly hydrophobic and corresponds to the signal peptide. A computer analysis of the predicted amino acid sequence (von Heijne et al., Eur. J. Biochem., 133: 17-21 [1983]) reveals a potential cleavage site for signal peptidase between residues 21 and 22. Cleavage in this position would yield a mature polypeptide with 332 amino acid residues beginning with an amino-terminal sequence obtained from mpl of ligand purified from porcine plasma. The predicted molecular weight of the non-glycosylated ligand with 332 amino acid residues is approximately 38 kDa. It has 6 potential N-glycosylation sites and 4 cysteine residues.

Primerjava sekvence mpl liganda s sekvenco po podatkih Genbanke razkriva 23 % identičnost med 153 amino-terminalnimi ostanki zrelega humanega mpl liganda in humanega eritropoetina (sl. 10 [SEQ ID NOS: 6 & 7]). Če upoštevamo konzervativne substitucije, ima ta regija hML 50 % podobnost s humanim eritropoetinom (hEPO). Oba hEPO in hML vsebujeta štiri cisterne. Trije od štirih cisteinov so konzervirani v hML, vključno prvi in zadnji cistein. Iz eksperimentov za položajno usmerjeno mutagenezo je razvidno, da prvi in zadnji cistein eritropoetina tvorita disulfidno vez, ki je potrebna za delovanje (Wang, F.F. et al., Endocrinology 116:2286-2292 [1983]). Analogno prvi in zadnji cistein hML prav tako lahko tvorita kritično disulfidno vez. Nobeno glikozilacijsko mesto ni konzervirano v hML. Vsa potencialna N-vezana glikozilacijska mesta hML so locirana v karboksi-terminalni polovici hML polipeptida.Comparison of the mpl ligand sequence with the Genbank data reveals a 23% identity between 153 amino-terminal residues of the mature human mpl ligand and human erythropoietin (Fig. 10 [SEQ ID NOS: 6 & 7]). Considering conservative substitutions, this region of hML has 50% similarity to human erythropoietin (hEPO). Both hEPO and hML contain four tanks. Three of the four cysteines are conserved in hML, including the first and last cysteine. Experiments for position-directed mutagenesis indicate that the first and last cysteine of erythropoietin form the disulfide bond required for action (Wang, F.F. et al., Endocrinology 116: 2286-2292 [1983]). Analogously, the first and last cysteine hMLs can also form a critical disulfide bond. No glycosylation site is conserved in hML. All potential N-linked glycosylation sites of hML are located in the carboxy-terminal half of the hML polypeptide.

Podobno kot hEPO tudi hML mRNA ne vsebuje konsenzne poliadenilacijske sekvence AAUAAA niti regulatornega elementa AUUUA, ki je prisoten v 3’ netranslatiranih regijah mnogih citokinov in domnevno vpliva na stabilnost mRNA (Shawet et al., Celi, 46:659-667 [1986]). Analiza Northern blotting razkriva nizke nivoje posameznega 1,8 kb hML RNA transkripta tako v fetalnih kot tudi v odraslih jetrih. Po daljši izpostavitvi je možno detektirati slabšo progo enake velikosti v ledvicah odraslega. Za primerjavo humani eritropoetin eksprimirajo v fetalnih jetrih in za odziv na hipoksijo v ledvicah in jetrih odraslega človeka (Jacobs et al., Nature, 313:804-809 [1985] in Bondurant et al., Moleč. Celi. Biol., 6:2731-2733 [1986]).Similar to hEPO, hML mRNA does not contain the AAUAAA consensus polyadenylation sequence, nor the AUUUA regulatory element, which is present in the 3 'untranslated regions of many cytokines and supposedly affects mRNA stability (Shawet et al., Celi, 46: 659-667 [1986]). . Northern blotting analysis reveals low levels of single 1.8 kb hML RNA transcripts in both fetal and adult livers. After prolonged exposure, a poorer line of equal size in the adult kidney can be detected. In comparison, human erythropoietin is expressed in the fetal liver and in response to adult and adult kidney hypoxia (Jacobs et al., Nature, 313: 804-809 [1985] and Bondurant et al., Mol. Whole. Biol., 6: 2731-2733 [1986].

Pomembnost C-terminalne regije hML pa ostane nepojasnjena. Na osnovi prisotnosti šestih potencialnih mest za N-vezano glikozilacijo in sposobnosti liganda, da se veže v lektinskih afinitetnih kolonah, je ta regija hML verjetno glikozilirana. V nekaterih eksperimentih gelskega eluiranja opazimo aktivnost, ki se loči z Mr približno 60000, ki lahko pomeni glikozilirano molekulo s popolno dolžino. C-terminalna regija torej lahko deluje tako, da stabilizira in poveča razpolovno dobo krožečega hML. V primeru eritropoetina ima ne-glikozilirana oblika popolno biološko aktivnost in vitro, vendar ima značilno znižano plazemsko razpolovno dobo glede na glikoziran eritropoetin (Takeuchi et al., J. Biol. Chem., 265:12127-12130 [1990]; Narhi et al., J. Biol. Chem., 266:23022-23026 [1991] in Spivack et al., Blood, 7:90-99 [1989]). C-terminalna domena hML vsebuje dve dibazični aminokislinski sekvenci [Arg-Arg motiva na položajih 153-154 in 245-246], ki sta lahko potencialni procesimi mesti. Cepitev na teh mestih je lahko odgovorna za nastajanje oblik ML, izoliranega iz APP s 30, 28 in 18-22 kDa. Značilno je, da sekvenca Arg₁₅₃Arg₁₅₄ nastane takoj po domeni ML, podobni eritropoetinu. Iz teh opažanj je razvidno, da ML s popolno dolžino lahko pomeni predhodniški protein, ki je izpostavljen omejeni proteolizi, da tvori zreli ligand.The importance of the C-terminal region of hML, however, remains unclear. Based on the presence of six potential sites for N-linked glycosylation and the ability of the ligand to bind in lectin affinity columns, this hML region is likely glycosylated. In some gel elution experiments, an activity separating by Mr about 60000 is observed, which may represent a full-length glycosylated molecule. The C-terminal region can therefore function to stabilize and increase the half-life of circulating hML. In the case of erythropoietin, the non-glycosylated form has complete biological activity in vitro, but has a significantly reduced plasma half-life relative to glycosylated erythropoietin (Takeuchi et al. J. Biol. Chem. 265: 12127-12130 [1990]; Narhi et al ., J. Biol. Chem., 266: 23022-23026 [1991] and Spivack et al., Blood, 7: 90-99 [1989]. The C-terminal domain of hML contains two dibasic amino acid sequences [Arg-Arg motifs at positions 153-154 and 245-246], which may be potential process sites. The cleavage at these sites may be responsible for the generation of ML forms isolated from APPs of 30, 28, and 18-22 kDa. Typically, the Arg ₁₅₃ Arg ₁₅₄ sequence is generated immediately after the erythropoietin-like ML domain. These observations suggest that full-length ML may represent a precursor protein that undergoes limited proteolysis to form a mature ligand.

4. Izo-oblike in variante humanega mpl liganda4. Iso-forms and variants of human mpl ligand

Izo-oblike ali alternativno spojene oblike humanega mpl liganda detektiramo s PCR v jetrih odraslega človeka. Na kratko, primeije sintetiziramo ustrezno vsakemu koncu, prav tako pa izberemo tudi interne regije kodirne sekvence hML. Te primeije uporabimo v RT-PCR, da pomnožimo RNA jeter odraslega človeka, kot je opisano v Primeru 10. Poleg oblike s popolno dolžino, označene hML, opazujemo oz. deduciramo tudi tri druge oblike, označene hML2, hML3 in hML4. Zrele deducirane aminokislinske sekvence vseh štirih izo-oblik so prikazane na sl. 11 (SEQ ED NOS: 6, 8,9&10).Iso-forms or alternatively fused forms of the human mpl ligand are detected by PCR in the liver of an adult. Briefly, primes are synthesized at each end and the internal regions of the hML coding sequence are also selected. These primers were used in RT-PCR to amplify adult liver RNA as described in Example 10. In addition to the full-length, hML-labeled form, we observed or observed. we also inherit three other forms, designated hML2, hML3, and hML4. The mature deduced amino acid sequences of all four iso-forms are shown in Figs. 11 (SEQ ED NOS: 6, 8.9 & 10).

hML3 ima delecijo 116 nukleotidov na položaju 700, kar ima za posledico tako delecijo amino kisline kot tudi premik okvirja. cDNA sedaj kodira zreli polipeptid, ki ima dolžino 265 amino kislin in se razlikuje od sekvence hML pri aminokislinskem ostanku 139. Končno ima hML4 tako delecijo 12 nukleotidov po nukleotidnem položaju 618 (ugotovljeno tudi v mišjih in prašičjih sekvencah [glej spodaj]) in delecijo 116 bp, ugotovljeno v hML3. Čeprav niso bili izolirani nobeni kloni samo z delicijo 12 bp (po nukleotidu 619) v človeku (označeno hML2), ta oblika verjetno obstaja, ker je bila taka izo-oblika identificirana tako pri miši kot tudi pri prašiču (glej spodaj) in ker je bila identificirana v zvezi z delecijo 116 nukleotidov v hML4.hML3 has a deletion of 116 nucleotides at position 700, resulting in both amino acid deletion and frame shift. cDNA now encodes a mature polypeptide of 265 amino acids in length and different from the hML sequence at amino acid residue 139. Finally, hML4 thus has a deletion of 12 nucleotides at nucleotide position 618 (also found in mouse and porcine sequences [see below]) and deletion 116 bp found in hML3. Although no clones with only 12 bp deletion (after nucleotide 619) were isolated in man (labeled hML2), this form probably exists because such an iso-form has been identified in both mice and pigs (see below) and because was identified in relation to the deletion of 116 nucleotides in hML4.

Obe, substitucijsko varianto hML, v kateri je dibazična sekvenca Arg₁₅₃-Arg₁₅₄ nadomeščena z dvema alaninskima ostankoma, in skrajšano obliko 'ΈΡΟ-domene hML konstruiramo tako, da določimo, ali je ML s popolno dolžino potreben za biološko aktivnost. Substitucijsko varianto dibazične sekvence Arg₁₅₃-Arg₁₅₄, imenovano hML (R153A, R154A), konstruiramo z uporabo PCR, kot je opisano v Primeru 10. Naredimo tudi skrajšano obliko ΕΡΟ-domene, hML₁₅₃, z uporabo PCR z uvedbo stop kodona po Argl53.Both, the hML substitution variant in which the dibasic sequence Arg ₁₅₃ -Arg _{154 is} replaced by two alanine residues, and the truncated form of the ΈΡΟ-domain of hML are constructed to determine whether full-length ML is required for biological activity. A substitution variant of the Arg ₁₅₃ -Arg ₁₅₄ dibasic sequence, termed hML (R153A, R154A), was constructed using PCR as described in Example 10. We also make a truncated ΕΡΟ-domain form, hML ₁₅₃ , using PCR by introducing a stop codon according to Argl53 .

5. Ekspresija rekombinantnega humanega mpl liganda (rhML) v prehodno transfektiranih 293 celicah humanih embrionalnih ledvic5. Expression of recombinant human mpl ligand (rhML) in transiently transfected 293 human embryonic kidney cells

Da potrdimo, da klonirana humana cDNA kodira ligand za mpl, ligand eksprimiramo v 293 celicah sesalca ob kontroli citomegalovirusnega neposrednega zgodnjega promotoija z uporabo ekspresijskega vektorja pRK5-hML ali pRK5hML₁₅₃. Za supematante iz prehodno transfektiranih 293 celic humanih embrionalnih ledvic ugotovimo, da stimulirajo vgraditev ³H-timidina v celice Ba/F3-zMjpZ, ne pa v parentalne celice Ba/F3 (sl. 12A). Medij iz 293 celic, transfektiranih samo z vektorjem pRK, ne vsebuje te aktivnosti. Dodatek mplAgG k mediju uniči stimulacijo (podatki niso prikazani). Iz teh rezultatov je razvidno, da klonirana cDNA kodira funkcionalni humani ML (hML).To confirm that the cloned human cDNA encodes a ligand for mpl, the ligand is expressed in 293 mammalian cells while controlling the cytomegalovirus direct early promoter using the pRK5-hML or pRK5hML expression vector ₁₅₃ . Supematants from transiently transfected 293 human embryonic kidney cells were found to stimulate the incorporation of ³ H-thymidine into Ba / F3-zMjpZ cells but not into Ba / F3 parental cells (Fig. 12A). The medium from 293 cells transfected with the pRK vector alone does not contain this activity. Addition of mplAgG to the medium destroys stimulation (data not shown). These results suggest that cloned cDNA encodes functional human ML (hML).

Da določimo, ali samo EPO-domena lahko veže in aktivira mpl, skrajšano obliko hML, rhML₁₅₃, eksprimiramo v 293 celicah. Za supematante iz transfektiranih celic ugotovimo, da imajo aktivnost, podobno tisti, kije prisotna v supematantih iz celic, ki eksprimirajo hML s popolno dolžino (sl. 12A), iz česar je razvidno, da C-terminalna domena ML ni potrebna za vezavo in aktiviranje G-mpl.To determine whether only the EPO domain can bind to and activate the mpl, truncated hML form, rhML ₁₅₃ , was expressed in 293 cells. Transfected cell supernatants were found to have activity similar to that present in full-length hML cell supernatants (Fig. 12A), indicating that the C-terminal domain of ML is not required for binding and activation G-mpl.

6, mpl ligand stimulira megakariocitopoezo in trombopoezo6, mpl ligand stimulates megakaryocytopoiesis and thrombopoiesis

Tako oblika rhML s popolno dolžino kot tudi skrajšana oblika rhML₁₅₃ rekombinantnega hML stimulirata humano megakariocitopoezo in vitro (sl. 12B). Ta učinek opazujemo v odsotnosti drugih eksogeno dodanih hematopoetičnih rastnih faktorjev. Razen IL-3 je ML edini hematopoetični rastni faktor, kije testiran, da ima to aktivnost. IL-11, IL-6, IL-1, eritropoetin, G-CSF, IL-9, LIF, kit ligand (KL), M-CSF, OSM in GM-CSF nimajo vpliva na megakariocitopoezo, če jih v našem testu testiramo ločeno (podatki niso prikazani). Iz teh rezultatov je razvidno, da ima ML megakariocite stimulirajočo aktivnost in označuje vlogo ML pri reguliranju megakariocitopoeze.Both the full-length rhML form and the shortened rhML ₁₅₃ form of recombinant hML stimulate human megakaryocytopoiesis in vitro (Fig. 12B). This effect is observed in the absence of other exogenously added hematopoietic growth factors. Other than IL-3, ML is the only hematopoietic growth factor tested to have this activity. IL-11, IL-6, IL-1, erythropoietin, G-CSF, IL-9, LIF, kit ligand (KL), M-CSF, OSM and GM-CSF have no effect on megakaryocytopoiesis when tested in our test separately (data not shown). These results suggest that ML megakaryocytes have a stimulating activity and characterize the role of ML in regulating megakaryocytopoiesis.

Za trombopoetične aktivnosti, prisotne v plazmi trombocitopeničnih živali, je prikazano, da stimulirajo nastajanje trombocitov pri izvajanju testa oživitve mišje trombocitoze (McDonald, Proč. Soc. Exp. Biol. Med., 14:1006-1001 [1973] in McDonald et al., Scand. J. Haematol., 16:326-334 [1976]). V tem modelu naredimo miši akutno trombocitopenične z uporabo specifičnega antitrombocitnega seruma, kar ima za posledico predvidljivo ožitev trombocitoze. Take imunotrombocitemične miši so bolj odzivne na eksogene trombopoetinu podobne aktivnosti kot normalne miši (McDonald, Proč. Soc. Exp. Biol. Med., 14:1006-1001 [1973]), prav tako kot so ekshipoksične miši bolj občutljive za eritropoetin kot normalne miši (McDonald, et al., J. Lab. Ciin. Med., 77:134-143 [1971]). Da ugotovimo, ali rML stimulira nastajanje trombocitov in vivo, mišim z oživljeno trombocitozo injiciramo delno očiščen rhML. Kvantitativno določimo število trombocitov in vgraditev ³⁵S vanje. Injiciranje 64000 ali 32000 enot rML mišim znatno poveča nastajanje trombocitov, kar dokazuje ~20 % zvečanje števila trombocitov (p=0,0005 oz. 0,0001) in ~ 40 % zvečanje vgraditve ³⁵S vanje (p=0,003) v obdelanih miših v primerjavi s kontrolnimi, injiciranimi samo z nosilcem (sl. 12C). Ta nivo stimulacije je primerljiv s tistim, ki ga dobimo z IL-6 v tem modelu (podatki niso prikazani). Obdelava s 16000 enotami rML znatno ne stimulira nastajanja trombocitov. Ti rezultati dokazujejo, da ML stimulira nastajanje trombocitov na način, ki je odvisen od doze in ima zaradi tega trombopoetinu podobno aktivnost.Thrombopoietic activities present in plasma of thrombocytopenic animals have been shown to stimulate platelet production when performing a mouse thrombocytosis recovery test (McDonald, Proc. Soc. Exp. Biol. Med., 14: 1006-1001 [1973] and McDonald et al. , Scand. J. Haematol., 16: 326-334 [1976]. In this model, mice are acutely thrombocytopenic using a specific antiplatelet serum, resulting in a predictable reduction in thrombocytosis. Such immunotrombocythemic mice are more responsive to exogenous thrombopoietin-like activities than normal mice (McDonald, Proc. Soc. Exp. Biol. Med., 14: 1006-1001 [1973]), just as exhypoxic mice are more sensitive to erythropoietin than normal mice. mice (McDonald, et al., J. Lab. Ciin. Med., 77: 134-143 [1971]). To determine whether rML stimulates platelet formation in vivo, mice with resuscitated thrombocytosis were injected with partially purified rhML. Quantify the platelet count and incorporation of ³⁵ S into them. Injecting 64,000 or 32,000 rML units to mice significantly increased platelet production, as evidenced by a ~ 20% increase in platelet count (p = 0.0005 or 0.0001) and ~ 40% increase in ³⁵ S incorporation into them (p = 0.003) in treated mice in compared to controls injected with vehicle alone (Fig. 12C). This level of stimulation is comparable to that obtained with IL-6 in this model (data not shown). Treatment with 16,000 rML units did not significantly stimulate platelet production. These results demonstrate that ML stimulates platelet production in a dose-dependent manner and, as a result, has a similar activity to thrombopoietin.

293 celic transfektiramo tudi z drugimi konstrukti izo-oblike hML, opisanimi zgoraj, in supematante testiramo z uporabo testa proliferacije Ba/F3-znp/ (sl. 13). hML2 in hML3 nimata aktivnosti, ki bi sejo dalo detektirati v tem testu, vendar pa je aktivnost hML(R153A, R154A) podobna hML in hML₁₅₃, kar dokazuje, da procesiranje na dibazičnem mestu Arg₁₅₃-Arg₁₅₄ ni niti potrebno niti škodljivo za aktivnost.293 cells were also transfected with other hML isoform constructs described above, and the supernatants were tested using the Ba / F3-znp / proliferation assay (Fig. 13). hML2 and hML3 have no activity detectable by the session in this test, but the activity of hML (R153A, R154A) is similar to hML and hML ₁₅₃ , demonstrating that processing at the dibasic Arg ₁₅₃ -Arg _{154 site} is neither necessary nor harmful for activity.

7, Megakriocitopoeza in mpl ligand7, Megakriocytopoiesis and mpl ligand

Navedeno je, da megakariocitopoezo regulirajo multiple celične stopnje (Williaras et al., J.Cell Physiol., 110:101-104 [1982] in Williams et al., Blood Celiš, 15:123-133 [1989]). To temelji večinoma na opažanjih, da določeni hematopoetični rastni faktorji stimulirajo proliferacijo megakariocitnih prednikov, medtem ko drugi primarno vplivajo na zorenje. Rezultati, prikazani tukaj, navajajo k temu, da ML deluje kot proliferativni faktor in kot faktor zorenja. Da ML stimulira proliferacijo megakariocitnih predhodnikov, podpirajo številni dokazi. Kot prvo, APP stimulira tako proliferacijo, kot tudi zorenje humanih megakariocitov in vitro, to stimulacijo pa popolnoma inhibira mpl-lgG (sl. 7 in 8). Poleg tega inhibiranje tvorbe megakariocitne kolonije z antisens oligonukleotidi c-mpl (Methia et al., Blood, 82:1395-1401 [1993]) in ugotovitev, da c-mpl lahko transducira proliferativni signal v celicah, v katerih je transfektiran (Skoda et al., EMBO, 12:2645-2653 [1993] in Vigon et al., Oncogene, 8:2607-2615 [1993]), prav tako dokazuje, da ML stimulira proliferacijo. Očitna ekspresija c-mpl med vsemi stopnjami megakariocitne diferenciacije (Methia et al., Blood, 82:1395-1401 [1993]) in sposobnost rekombinantnega ML, da hitro stimulira nastajanje trombocitov in vivo, naznačuje, da ML vpliva tudi na zorenje. Rekombinantni ML omogoča natančno ocenitev njegove vloge pri reguliranju megakariocitopoeze in trombopoeze, kot tudi njegove zmogljivosti, da vpliva na druge hematopoetične rodove.Megakaryocytopoiesis has been reported to be regulated by multiple cell degrees (Williaras et al., J.Cell Physiol., 110: 101-104 [1982] and Williams et al., Blood Celish, 15: 123-133 [1989]). This is based largely on the observation that certain hematopoietic growth factors stimulate the proliferation of megakaryocytic ancestors, while others primarily influence maturation. The results presented here indicate that ML acts as a proliferative factor and as a maturation factor. That ML stimulates the proliferation of megakaryocyte precursors is supported by a wealth of evidence. First, APP stimulates both proliferation and maturation of human megakaryocytes in vitro, and this stimulation completely inhibits mpl-1gG (Figs. 7 and 8). In addition, inhibition of megakaryocyte colony formation by c-mpl antisense oligonucleotides (Methia et al., Blood, 82: 1395-1401 [1993]) and the finding that c-mpl can transduce proliferative signal in cells in which it is transfected (Skoda et al., EMBO, 12: 2645-2653 [1993] and Vigon et al., Oncogene, 8: 2607-2615 [1993]) also demonstrates that ML stimulates proliferation. The apparent expression of c-mpl during all stages of megakaryocyte differentiation (Methia et al., Blood, 82: 1395-1401 [1993]) and the ability of recombinant ML to rapidly stimulate platelet formation in vivo indicates that ML also affects maturation. Recombinant ML allows for an accurate assessment of its role in regulating megakaryocytopoiesis and thrombopoiesis, as well as its capacity to influence other hematopoietic genera.

8. Izolacija gena humanega mpl liganda (TPO)8. Isolation of the human mpl ligand (TPO) gene

Klone humane genomske DNA gena TPO izoliramo s selekcioniranjem humane genomske knjižnice v X-geml2 s pR45 pri manj ostrih pogojih ali pri zelo ostrih pogojih s fragmentom, ki ustreza 3’ polovici humane cDNA, ki kodira mpl ligand. Izoliramo dva prekrivajoča lambda klona z obsegom 35 kb. Dva prekrivajoča fragmenta (BamHl in EcoRI), ki vsebujeta celotni gen TPO, subkloniramo in sekvenciramo (sl. 14A, 14B in 14C).Clones of the human genomic DNA of the TPO gene are isolated by selection of the human genomic library in X-geml2 with pR45 under less harsh conditions or under very harsh conditions with a fragment corresponding to the 3 'half of the human cDNA encoding the mpl ligand. Two overlapping 35 kb lambda clones are isolated. Two overlapping fragments (BamH1 and EcoRI) containing the entire TPO gene were subcloned and sequenced (Figs. 14A, 14B and 14C).

Struktura humanega gena je sestavljena iz 6 eksonov znotraj 7 kb genomske DNA. Meje vseh eksonskih/intronskih povezav so konsistentne s konsenznim motivom, določenim za gene sesalcev (Shapiro, M.B., et al., Nucl. Acids Res. 15:7155 [1987]). Ekson 1 in ekson 2 vsebujeta 5’ netranslatirano sekvenco in začetne štiri amino kisline signalnega peptida. Ostanek sekietornega signala in prvih 26 amino kislin zrelega proteina so kodirani v eksonu 3. Celotna karboksilna domena in 3’ netranslatirana kot tudi približno 50 amino kislin eritropoetinu slične domene so kodirani v eksonu 6. Štiri amino kisline, vključene v delecijo, ki jo zaznamo v hML·! (hTPO-2), so kodirane na 5’ koncu eksona 6.The structure of the human gene consists of 6 exons within 7 kb of genomic DNA. The boundaries of all exon / intron junctions are consistent with the consensus motif established for mammalian genes (Shapiro, M.B., et al., Nucl. Acids Res. 15: 7155 [1987]). Exon 1 and exon 2 contain a 5 'untranslated sequence and the initial four amino acids of the signal peptide. The remainder of the secoether signal and the first 26 amino acids of the mature protein are encoded in exon 3. The entire carboxylic domain and the 3 'untranslated as well as about 50 amino acids of the erythropoietin-like domain are encoded in exon 6. The four amino acids involved in the deletion detected in hML ·! (hTPO-2) are encoded at the 5 'end of exon 6.

Analiza humane genomske DNA s Southern blottingom označuje, da je gen za TPO prisoten v enojni kopiji. Kromosomsko lokacijo gena določimo s fluorescentno hibridizacijo in situ (FISH), kije mapirana za kromosome 3q27-28.Analysis of human genomic DNA by Southern blotting indicates that the TPO gene is present in single copy. The chromosomal location of the gene is determined by fluorescence in situ hybridization (FISH) mapped for chromosomes 3q27-28.

9. Ekspresija in čiščenje TPO iz 293 celic9. Expression and purification of TPO from 293 cells

Priprava in čiščenje ML ali TPO iz 293 celic sta podrobno opisana v Primeru 19. Na kratko, cDNA, ki ustreza celotnemu odprtemu bralnemu okvirju TPO, dobimo s PCR z uporabo pRK5-h/np/I. Dobljeni PCR očistimo in kloniramo med restrikcijskima mestoma Clal in XBaI plazmida pRK5tkneo (vektor izveden iz pRK5, modificiran za ekspresijo gena, odpornega proti neomicinu ob kontroli zviševanja timidin kinaze), da dobimo vektor pRK5tkneo.ORF (vektor, ki kodira za celoten odprt bralni okvir).Preparation and purification of ML or TPO from 293 cells is described in detail in Example 19. Briefly, cDNA corresponding to the full open reading frame of TPO is obtained by PCR using pRK5-h / np / I. The resulting PCR was purified and cloned between the restriction sites Clal and XBaI of the plasmid pRK5tkneo (vector derived from pRK5 modified to express a neomycin-resistant gene while controlling the increase of thymidine kinase) to obtain the vector pRK5tkneo. ).

Drugi vektor, ki kodira za homologno domeno EPO, naredimo enako, le da uporabimo drugačne primeije PCR, da dobimo končni konstrukt, imenovan pRK5tkneoEPO-D.We do the second vector encoding for the homologous EPO domain, except to use different PCR primers to obtain a final construct called pRK5tkneoEPO-D.

Ta dva konstrukta transfektiramo v celice humanih embrionalnih ledvic s CaPO₄ postopkom ter klone, odporne proti neomicinu, selekcioniramo in pustimo, da rastejo do konfluence. Ekspresijo ML₁₅₃ ali ML₃₃₂ v kondicioniranem mediju iz teh klonov preizkusimo s testom proliferacije Ba/F3-mp/.These two constructs were transfected into human embryonic kidney cells by the CaPO ₄ procedure, and clones resistant to neomycin were selected and allowed to grow to confluence. The expression of ML ₁₅₃ or ML ₃₃₂ in the conditioned medium from these clones was tested by the Ba / F3-mp / proliferation assay.

Čiščenje rhML₃₃₂ izvedemo, kot je opisano v Primeru 19. Na kratko, kondicioniran medij 293-rhML₃₃₂ nanesemo na kolono blue-sefaroze (Pharmacia), ki jo nato izperemo s pufrom, ki vsebuje sečnino (2M). Kolono eluiramo s pufrom, ki vsebuje sečnino (2M) in NaCl (IM). Elucijski pool blue-sefaroze nato direktno nanesemo na kolono WGA-sefaroze, izperemo z 10 kolonskimi volumni pufra, ki vsebuje sečnino (2M) in NaQ (IM) in eluiramo z istim pufrom, ki vsebuje N-acetil-D-glukozamin (0,5M). WGA-sefarozni eluat nanesemo na kolono C4-HPLC (Synchrom, Inc.) in eluiramo z diskontinuimim propanolnim gradientom. Pri SDS-PAGE migrira očiščeni 293-rhML₃₃₂ kot široka proga v področju gela 68-80 kDa (sl. 15).Purification of rhML _{332 was} performed as described in Example 19. Briefly, conditioned medium 293-rhML _{332 was} applied to a blue-Sepharose column (Pharmacia), which was then washed with urea-containing buffer (2M). The column was eluted with buffer containing urea (2M) and NaCl (IM). The blue-Sepharose elution pool was then directly applied to the WGA Sepharose column, washed with 10 column volumes of urea-containing (2M) and NaQ (IM) buffer and eluted with the same buffer containing N-acetyl-D-glucosamine (0, 5M). The WGA-Sepharose eluate was applied to a C4-HPLC column (Synchrom, Inc.) and eluted with a discontinuous propanol gradient. In SDS-PAGE, the purified 293-rhML ₃₃₂ migrates as a wide line in the gel region of 68-80 kDa (Fig. 15).

Čiščenje rhML₁₅₃ prav tako izvedemo, kot je opisano v Primeru 19. Na kratko, kondicioniran medij 293-rhML₁₅₃ ločimo na blue-sefarozi, kot je opisano za rhML₃₃₂. Eluat blue-sefaroze nanesemo direktno na /np/-afinentno kolono, kot je opisano zgoraj. rhML₁₅₃, ki se eluira iz mp/-afinitetne kolone, očistimo do homogenosti, pri čemer uporabimo kolono C4-HPLC pri enakih pogojih kot za rhML₃₃₂. Z SDSPAGE se očiščeni rhML₁₅₃ loči v dve večji in dve manjši progi z Mr ~ 18000-22000 (sl. 15).Purification of rhML ₁₅₃ was also performed as described in Example 19. Briefly, conditioned medium 293-rhML _{153 was} separated on blue-Sepharose as described for rhML ₃₃₂ . Apply blue-Sepharose eluate directly to the / np / -affinity column as described above. rhML ₁₅₃ eluted from the mp / affinity column was purified to homogeneity using the C4-HPLC column under the same conditions as for rhML ₃₃₂ . With SDSPAGE, purified rhML _{153 is} separated into two larger and two smaller lines with Mr ~ 18000-22000 (Fig. 15).

10. Murini mpl ligand10. Murini mpl ligand

Fragment DNA, ki ustreza kodirni regiji humanega mpl liganda, dobimo s PCR, gelsko očistimo in označimo v prisotnosti ³²P-dATP in ³²P-dCTP. To sondo uporabimo za selekcioniranje 10⁶ klonov knjižnice cDNA mišjih jeter v \GT10. Murini klon (sl. 16 [SEQ ID NOS: 12 & 13]), ki vsebuje insert 1443 baznih parov, izoliramo in sekvenciramo. Predvideni iniciacijski kodon je pri nukleotidnem položaju 138-141 v konsenzni sekvenci, ugodni za evkariotično translacijsko iniciacijo (Kozak, M. J.Cell Biol., 108:229-241 [1989]). Ta sekvenca definira odprt bralni okvir 1056 nukleotidov, kar napoveduje primarno translatiran produkt s 352 amino kislinami. Bočno k temu odprtemu bralnemu okvirju je 137 nukleotidov od 5’ in 247 nukleotidov od 3’ netranslatirane sekvence. Ni pa nobenega poli(A) repa po 3’ netranslatirani regiji, ki bi označeval, da klon verjetno ni kompleten. N-terminal predvidene aminokislinske sekvence je visoko hidrofoben in predstavlja signalni peptid. Računalniška analiza (von Heijne, G. Eur. J. Biochem. 133:17-21 [1983]) naznačuje potencialno cepišče za signalno peptidazo med ostankoma 21 in 22. Cepitev v tem položaju bi dala zreli polipeptid s 331 amino kislinami (35 kDa), identificiran kot mML₃₃₁ (ali mML2 zaradi razlogov, opisanih spodaj). Sekvenca vsebuje štiri cisteine, vse konzervirane v humani sekvenci, in sedem potencialnih N-glikozilacijskih mest, od katerih je pet konzerviranih v humani sekvenci. Ponovno, kot pri hML, je vseh sedem potencialnih N-glikozilacijskih mest lociranih v C-terminalni polovici proteina.The DNA fragment corresponding to the coding region of the human mpl ligand is obtained by PCR, gel purified and labeled in the presence of ³² P-dATP and ³² P-dCTP. We use this probe to select 10 ⁶ clones of the mouse liver cDNA library in \ GT10. A murine clone (Fig. 16 [SEQ ID NOS: 12 & 13]) containing insert 1443 base pairs was isolated and sequenced. The predicted initiation codon is at the nucleotide position 138-141 in a consensus sequence favorable for eukaryotic translational initiation (Kozak, MJCell Biol., 108: 229-241 [1989]). This sequence defines an open reading frame of 1056 nucleotides, predicting a primarily translated product with 352 amino acids. Lateral to this open reading frame are 137 nucleotides of 5 'and 247 nucleotides of 3' of the untranslated sequence. However, there is no poly (A) tail in the 3 'untranslated region to indicate that the clone is probably incomplete. The N-terminal of the predicted amino acid sequence is highly hydrophobic and is a signal peptide. Computer analysis (von Heijne, G. Eur. J. Biochem. 133: 17-21 [1983]) indicates a potential cleavage site for signal peptidase between residues 21 and 22. Cleavage in this position would yield a mature polypeptide with 331 amino acids (35 kDa ) identified as mML ₃₃₁ (or mML2 for the reasons described below). The sequence contains four cysteines, all conserved in the human sequence, and seven potential N-glycosylation sites, five of which are conserved in the human sequence. Again, as with hML, all seven potential N-glycosylation sites are located in the C-terminal half of the protein.

Če primerjamo s humanim ML, opazimo precejšnjo identičnost tako za nukleotidne kot tudi za deducirane aminokislinske sekvence v EPO-domenah teh ML. Vendar pa, če uvrstimo deducirane aminokislinske sekvence humanega ML in mišjega ML, kaže, da ima mišja sekvenca tetrapeptidno delecijo med ostanki 111-114, ki ustreza deleciji 12 nukleotidov po nukleotidnem položaju 618, razvidno tako v humani cDNA (glej zgoraj) kot tudi prašičji (glej spodaj). V skladu s tem dodatne klone raziščemo, da odkrijemo možne izo-oblike murinega ML En klon kodira deducirano sekvenco polipeptida s 335 amino kislinami, ki vsebuje manjkajoči (missing) tetrapeptid LPLQ. Za to obliko domnevamo, da je murini ML s popolno dolžino in je imenovana mML ali mML^. Nukleotid in deducirana aminokislinska sekvenca za mML sta prikazana na sl. 17 (SEQ ED NOS: 14 & 15). Ta klon cDNA je sestavljen iz 1443 baznih parov, ki jim sledi poli(A) rep. Ima odprt bralni okvir iz 1068 bp, ki imajo bočno 134 baz od 5’ in 241 baz od 3’ netranslatirane sekvence. Domnevni inidacijski kodon je pri nukleotidnih položajih 138-140. Odprt bralni okvir kodira napovedani protein s 356 amino kislinami, pri čemer je prvih 21 zelo hidrofobnih in veijetno delujejo kot sekrecijski signal.Compared to human ML, considerable identity is observed for both nucleotide and deduced amino acid sequences in the EPO domains of these MLs. However, when we classify deduced amino acid sequences of human ML and murine ML, the mouse sequence shows a tetrapeptide deletion between residues 111-114 corresponding to the deletion of 12 nucleotides at nucleotide position 618, seen in both human cDNA (see above) and pigs (see below). Accordingly, additional clones are being investigated to discover possible isoforms of murine ML. One clone encodes a deduced 335 amino acid polypeptide sequence containing the missing tetrapeptide LPLQ. For this form, we assume that the murini is a full-length ML and is called mML or mML ^. The nucleotide and deduced amino acid sequence for mML are shown in FIG. 17 (SEQ ED NOS: 14 & 15). This cDNA clone consists of 1443 base pairs followed by a poly (A) tail. It has an open reading frame of 1068 bp having 134 bases of 5 'laterally and 241 bases of 3' untranslated sequences. The putative inidation codon is at nucleotide positions 138-140. The open reading frame encodes a predicted protein with 356 amino acids, the first 21 being highly hydrophobic and probably acting as a secretion signal.

Končno izoliramo tretji murini klon, sekvenciramo in ugotovimo, da vsebuje delecijo 116 nukleotidov, ki ustreza hML3. Ta murina izo-oblika je zato označena mML3. Primerjava deducimih aminokislinskih sekvenc teh dveh izo-oblik je prikazana na sl. 18 (SEQ ID NOS: 9 & 16).Finally, the third murine clone was isolated, sequenced, and found to contain a 116 nucleotide deletion corresponding to hML3. This murine isoform is therefore designated mML3. A comparison of the deduced amino acid sequences of these two iso-forms is shown in Figs. 18 (SEQ ID NOS: 9 & 16).

Celotna aminokislinska sekvenčna identičnost med humanim in mišjim ML (sl. 19 [SEQ ED NOS: 6 & 17]) je 72 %, vendar pa ta homologija ni pravilno porazdeljena. Regija, ki je definirana kot ΕΡΟ-domena (amino kisline 1-153 za humano sekvenco in 1-149 za mišjo) je bolje konzervirana (86 % homologija) kot karboksiterminalna regija proteina (62 % homologija). To lahko nadalje pomeni, da je samo ΕΡΟ-domena pomembna za biološko aktivnost proteina. Zanimivo je, da je od dveh dibazičnih aminokislinskih motivov, ugotovljenih v hML, le dibazični motiv, kije neposredno takoj za EPO-domeno (položaj ostankov 153-154) v humani sekvenci, prisoten v murini sekvenci. To je konsistentno z možnostjo, da ML s popolno dolžino lahko predstavlja predhodniški protein, ki je izpostavljen omejeni proteolizi, da tvori zreli ligand. Alternativno lahko proteoliza med Arg₁₅₃-Arg₁₅₄ olajša odstranitev hML.The overall amino acid sequence identity between human and mouse ML (Fig. 19 [SEQ ED NOS: 6 & 17]) is 72%, but this homology is not correctly distributed. The region defined as the ΕΡΟ domain (amino acids 1-153 for the human sequence and 1-149 for the mouse) is better conserved (86% homology) than the carboxyterminal region of the protein (62% homology). This may further imply that only the Ε dom domain is important for the biological activity of the protein. Interestingly, of the two dibasic amino acid motifs identified in hML, only the dibasic motif, which is immediately behind the EPO domain (position of residues 153-154) in the human sequence, is present in the murine sequence. This is consistent with the possibility that full-length ML may represent a precursor protein that undergoes limited proteolysis to form a mature ligand. Alternatively, proteolysis during Arg ₁₅₃ -Arg _{154 may} facilitate the removal of hML.

Ekspresijski vektor, ki vsebuje celotno kodirno sekvenco mML, predhodno transfektiramo v 293 celic, kot je opisano v Primeru 1. Kondicioniran medij iz teh celic stimulira vgraditev ³H-timidina v celice Ba/F3, ki eksprimirajo tako murini kot humani mpl, nima pa vpliva na parentalno (brez mpl) celično linijo. Iz tega je raz60 vidno, da klonirana cDNA murinega ML kodira funkcionalni ligand, ki je sposoben da aktivira tako receptor murinega kot tudi humanega ML (mpl).An expression vector containing the entire mML coding sequence was pre-transfected into 293 cells as described in Example 1. The conditioned medium from these cells stimulates the incorporation of ³ H-thymidine into Ba / F3 cells expressing both murine and human mpl but lacking affects the parental (no mpl) cell line. From this, it can be seen that the cloned murine ML cDNA encodes a functional ligand capable of activating both murine and human ML (mpl) receptors.

11. Prašičji mpl ligand cDNA prašičjega ML (pML) izoliramo z RACE PCR, kot je opisano v Primeru 13. PCR cDNA produkt s 1342 bp ugotovimo v ledvicah in subkloniramo. Različne klone sekvenciramo in ugotovimo, da kodirajo prašičji mpl ligand s 332 aminokislinskimi ostanki, imenovan pML (ah pML₃₃₂), ki ima nukleotidno in deducirano aminokislinsko sekvenco, ki sta prikazani na sl. 20 (SEQ ID NOS: 18 & 19).11. The porcine mpl ligand of porcine ML cDNA (pML) was isolated by RACE PCR as described in Example 13. A 1342 bp PCR cDNA product was detected in the kidney and subcloned. Different clones are sequenced and found to encode a pig mpl ligand with 332 amino acid residues called pML (ah pML ₃₃₂ ), which has the nucleotide and deduced amino acid sequence shown in FIG. 20 (SEQ ID NOS: 18 & 19).

Ponovno identificiramo drugo obliko, označeno pML2, ki kodira protein z delecijo štirih aminokislinskih ostankov (228 aminokislinskih ostankov) (sl. 21 [SEQ ID NO: 21]). Iz primeijave aminokislinskih sekvenc pML in pML2 je razvidno, daje slednja oblika identična, razen da ima delecijo tetrapeptida QLPP, ki ustreza ostankom od 111 do vključno 114 (sl. 22 [SEQ ID NOS: 18 & 21]). Do delecije štirih amino kislin, ki jo zaznamo tako v cDNA murinega kot tudi prašičjega ML, pride na točno enakem položaju v napovedanih proteinih.We again identify another form, designated pML2, that encodes a protein with a deletion of four amino acid residues (228 amino acid residues) (Fig. 21 [SEQ ID NO: 21]). The primium amino acid sequences of pML and pML2 show that the latter form is identical except that it has a deletion of the tetrapeptide QLPP corresponding to residues 111 to 114 inclusive (Fig. 22 [SEQ ID NOS: 18 & 21]). The deletion of four amino acids, which is detectable in both murine and porcine ML cDNAs, occurs at exactly the same position in the predicted proteins.

Iz primeijave napovedanih aminokislinskih sekvenc zrelega ML človeka, miši in prašiča (sl. 19 [SEQ ID NOS: 6, 17 & 18]) je razvidno, da je celotna sekvenčna identičnost 72 %, če primeijamo mišjo in humano, 68 % če primerjamo mišjo in prašičjo ter 73 % če primeijamo prašičjo in humano. Homologija je bistveno večja v amino-terminalni polovici ML (EPO homologna domena). Ta domena ima 80-84 % identičnost, če primeijamo katerikoli dve vrsti, pri čemer je karboksiterminalna polovica (ogljikohidratna domena) samo 57-67 % identična. Dibazični aminokislinski motiv, ki bi lahko pomenil proteazno cepišče, je prisoten na karboksilnem koncu eritropoetinske homologne domene. Ta motiv je konzerviran med tremi vrstami (species) na tem položaju (sl. 19 [SEQ ID NOS: 6, 17 & 18]). Drugo dibazično mesto, prisotno na položajih 245 in 246 v humani sekvenci, ni prisotno v mišjih ali prašičjih sekvencah. Sekvenca murinega in prašičjega ML vsebuje 4 cisterne, vse konzervirane v humani sekvenci. Obstaja 7 potencialnih N-glikozilacijskih mest v mišjem Ugandu in 6 v prašičjem ML, od katerih je 5 konzerviranih v humani sekvenci. Ponovno so vsa potencialna N-glikozilacijska mesta locirana v C-terminalni polovici proteina.The primium predicted amino acid sequences of mature ML of man, mouse and pig (Fig. 19 [SEQ ID NOS: 6, 17 & 18]) show that the overall sequence identity is 72% when compared to mouse and human, 68% when compared to mouse and pig and 73% if pig and human are compared. Homology is significantly greater in the amino-terminal half of ML (EPO homologous domain). This domain has 80-84% identity when comparing any two species, with the carboxyterminal half (carbohydrate domain) only 57-67% identical. A dibasic amino acid motif that could represent a protease cleavage is present at the carboxyl end of the erythropoietin homologous domain. This motif is conserved among the three species in this position (Fig. 19 [SEQ ID NOS: 6, 17 & 18]). Another dibasic site present at positions 245 and 246 in the human sequence is not present in mouse or porcine sequences. The Mura and Pig Sequence ML contains 4 tanks, all conserved in human sequence. There are 7 potential N-glycosylation sites in murine Uganda and 6 in porcine ML, 5 of which are conserved in the human sequence. Again, all potential N-glycosylation sites are located in the C-terminal half of the protein.

12. Ekspresija in čiščenje TPO iz ovariiskih celic kitajskega hrčka (celice CHO)12. Expression and purification of TPO from Chinese hamster ovary cells (CHO cells)

Ekspresijski vektorji, uporabljeni za transfektiranje celic CHO so označeni: pSVI5.ID.LL.MLORF (popolna dolžina ali TPO^), in pSVI5.ID.LL.MLEPO-D (skrajšan ali TPO₁₅₃). Pertinentne lastnosti teh plazmidov so prikazane na sl. 23 in 24.Expression vectors used for transfection of CHO cells are denoted: pSVI5.ID.LL.MLORF (full length or TPO ^), and pSVI5.ID.LL.MLEPO-D (truncated or TPO ₁₅₃ ). The pertinent properties of these plasmids are shown in FIG. 23 and 24.

Transfekcijski postopki so opisani v Primeru 20. Na kratko, cDNA, ki ustreza celotnemu odprtemu bralnemu okviru TPO, dobimo s PCR. Produkt PCR očistimo in kloniramo med dve restrikcijski mesti (Clal in Šali) plazmida pSVI5.ID.LL, da dobimo vektor pSVI5.ID.LL.MLORF. Drugi konstrukt, ki ustreza homologni domeni EPO, izdelamo na enak način, le da uporabimo drugačen, reverzni primer (EPOD.SaI). Končni konstrukt za vektor, ki kodira za EPO homologno domeno TPO, je imenovan pSVI5.ID.LL.MLEPO-D.Transfection procedures are described in Example 20. Briefly, cDNA corresponding to the full open reading frame of TPO is obtained by PCR. The PCR product was purified and cloned between two restriction sites (Clal and Shali) of plasmid pSVI5.ID.LL to obtain the vector pSVI5.ID.LL.MLORF. The second construct corresponding to the homologous domain of the EPO is made in the same way except by using a different, reverse primer (EPOD.SaI). The final construct for the vector encoding the TPO homologous domain for the EPO is named pSVI5.ID.LL.MLEPO-D.

Ta dva konstrukta Imeariziramo z Noti in transfektiramo v ovarijske celice kitajskega hrčka (celice CHO-DP12, EP 307,247, objavljeno 15. marca 1989) z elektroporacijo. 10⁷ celic elektroporiramo v napravi za elektroporacijo BRL (350 voltov, 330 mF, nizka kapacitanca) v prisotnosti 10, 25 ali 50 mg DNA, kot opisujejo (Andreason, G.L J. Tissue Cult. Meth. 15,56 [1993]). Dan po transfekciji celice cepimo v selektivni medij DHFR (visoko glukozni DMEM-F12 50:50, brez glicina, glutamin (2 mM), dializiran fetalni telečji serum (2-5 %)). 10 do 15 dni kasneje prenesemo individualne kolonije na plošče s 96 vdolbinicami in pustimo, da rastejo do klonfluence. Ekspresijo ML₁₅₃ ali ML₃₃₂ v kondicioniranem mediju iz teh klonov ugotovimo s testom proliferacije Ba/F3-mpl (opisano v Primeru 1).These two constructs were imaged with Noti and transfected into Chinese hamster ovary cells (CHO-DP12 cells, EP 307,247, published March 15, 1989) by electroporation. 10 ⁷ cells were electroporated in a BRL electroporation device (350 volts, 330 mF, low capacitance) in the presence of 10, 25, or 50 mg of DNA as described (Andreason, GL J. Tissue Cult. Meth. 15.56 [1993]). The day after transfection, the cells were vaccinated in selective DHFR medium (high glucose DMEM-F12 50:50, glycine-free, glutamine (2 mM), fetal calf serum dialysed (2-5%)). 10 to 15 days later, transfer individual colonies to 96-well plates and allow them to grow to clonfluence. The expression of ML ₁₅₃ or ML ₃₃₂ in the conditioned medium from these clones was determined by the Ba / F3-mpl proliferation assay (described in Example 1).

Postopek čiščenj in izoliranj TPO iz zbrane tekočine celične kulture CHO je opisan v primeru 20. Na kratko, zbrano tekočino celične kulture (HCCF) nanesemo na kolono blu-sefaroze (Pharmacia) pri razmerju približno 100 1 HCCF na liter smole. Kolono nato izperemo s 3 do 5 kolonskimi volumni pufra, nato s 3 do 5 kolonskimi volumni pufra, ki vsebuje sečnino (2,0 M). TPO nato eluiramo s 3 do 5 kolonskimi volumni pufra, ki vsebuje sečnino (2,0M) in NaG (l,0M).The procedure for the purification and isolation of TPO from the collected CHO cell culture fluid is described in Example 20. Briefly, the collected cell culture fluid (HCCF) is applied to a blu-Sepharose column (Pharmacia) at a ratio of about 100 1 HCCF per liter of resin. The column was then washed with 3 to 5 column volumes of buffer followed by 3 to 5 column volumes of buffer containing urea (2.0 M). The TPO was then eluted with 3 to 5 column volumes of buffer containing urea (2.0M) and NaG (1.0M).

Eluatni pool iz blue-sefaroze, ki vsebuje TPO, nato nanesemo na kolono lektin (pšenični kalčki)-sefaroze (Pharmacia), uravnotežno v eluimem pufru blue-sefaroze pri razmerju od 8 do 16 ml eluata blue-sefaroze na ml smole. Kolono nato izperemo z 2 do 3 kolonskimi volumni uravnoteževalnega pufra. TPO nato eluiramo z 2 do 5 kolonskimi volumni pufra, ki vsebuje sečnino (2,0M) in N-acetil-D-glukozamin (0,5Μ).The TPO-containing blue-Sepharose eluate pool is then applied to a lectin (wheat germ) -sepharose (Pharmacia) column equilibrated in blue-Sepharose eluate buffer at a ratio of 8 to 16 ml of Blue-Sepharose eluate per ml of resin. The column was then washed with 2 to 3 column volumes of equilibration buffer. The TPO was then eluted with 2 to 5 column volumes of buffer containing urea (2.0M) and N-acetyl-D-glucosamine (0.5Μ).

Eluat lektina pšeničnih kalčkov, ki vsebuje TPO, nato nakisamo in dodamo C₁₂E_g do končne koncentracije 0,04 %. Dobljeni pool nanesemo na kolono z reverzno fazo C4, uravnoteženo v TFA (0,1 %), C₁₂E_g (0,04 %), s polnitvijo približno 0,2 do 0,5 mg proteina na ml smole.The wheat germ lectin eluate containing TPO was then acidified and C ₁₂ E _{g was added} to a final concentration of 0.04%. The resulting pool was applied to a TFA reversed phase column (0.1%), C ₁₂ E _g (0.04%), loading about 0.2 to 0.5 mg of protein per ml of resin.

Protein se eluira v dvofaznem linearnem gradientu acetonitrila, ki vsebuje TFA (0,1 %) in C₁₂E_g (0,04 %), pool pa naredimo na osnovi SDS-PAGE.The protein was eluted in a two-phase linear gradient of acetonitrile containing TFA (0.1%) and C ₁₂ E _g (0.04%), and the pool was made on the basis of SDS-PAGE.

Pool C4 nato razredčimo in diafiltriramo proti približno 6 volumnom pufra na ultrafiltracijski membrani Amicon YM ali podobni z mejnimi (cut-off) molekulskimi masami od 10000 do 30000 Da. Dobljeni diafiltrat nato direktno predelamo ali dalje koncentriramo z ultrafiltracijo. Diafiltratu/koncentratu navadno naravnamo končno koncentracijo 0,01 % Tweena-80.Pool C4 is then diluted and diafiltered against about 6 volumes of buffer on an Amicon YM ultrafiltration membrane or similar with cut-off molecular weights of 10,000 to 30000 Da. The resulting diafiltrate is then directly processed or further concentrated by ultrafiltration. The diafiltrate / concentrate is usually adjusted to a final concentration of 0.01% Tweena-80.

Ves diafiltrat/koncentrat ali le del, ekvivalenten 2 do 5 % izračunanega kolonskega volumna, nato nanesemo na kolono sefakril S-300 HR (Pharmacia), uravnoteženo v pufru, ki vsebuje Tween-80 (0,01 %), in kromatografiramo. Frakcije, ki vsebujejo TPO, ki so brez agregata in produkte proteolitične degradacije nato združimo v pool na osnovi SDS-PAGE. Dobljeni pool filtriramo in shranimo pri 2-8 °C.All diafiltrate / concentrate or only a portion equivalent to 2 to 5% of the calculated column volume was then applied to a Sephacryl S-300 HR column (Pharmacia) balanced in Tween-80 containing buffer (0.01%) and chromatographed. The TPO-containing fractions, which are aggregate-free and proteolytic degradation products, are then pooled in an SDS-PAGE pool. The resulting pool was filtered and stored at 2-8 ° C.

13. Postopki za transformiranje in induciranje sinteze TPO v mikroorganizmu in izoliranje, čiščenje in renaturiranie TPO, narejenega v njem13. Methods for transforming and inducing the synthesis of TPO in a micro-organism and for isolating, purifying and renaturing TPO made therein

Konstrukcija ekspresijskih vektorjev E. coli TPO je podrobno opisana v Primeru 21. Na kratko, plazmidi pMP21, pMP151, pMP141, pMP57 in pMP202 so vsi oblikovani tako, da eksprimirajo prvih 155 amino kislin TPO navzdol od majhnega voditelja, ki variira med različnimi konstrukti. Voditelji (leaders) primarno zagotavljajo visok nivo translacijske iniciacije in hitro čiščenje. Plazmidi pMP210-l, -T8, -21, -22, -24, -25 so oblikovani za ekspresijo prvih 153 amino kislin TPO navzdol od iniciacijskega metionina in se razlikujejo le v uporabi kodona za prvih 6 amino kislin TPO, plazmid pMP251 pa je derivat pMP210-l v katerem je karboksi-terminalni konec TPO razširjen z dvema amino kislinama. Vsi od zgornjih plazmidov dajo visoke nivoje intracelične ekspresije TPO v E. coli po indukciji triptofanskega promotorja (Yansura, D. G. et al. Methods in Enzymology (Goeddel, D.V., izd.) 185:54-60, Academic Press, San Diego [1990]). Plazmida pMPl in pMP172 sta intermediata v konstrukciji zgornjih intraceličnih ekspresijskih plazmidov TPO.The construction of E. coli TPO expression vectors is detailed in Example 21. Briefly, plasmids pMP21, pMP151, pMP141, pMP57, and pMP202 are all designed to express the first 155 amino acids of TPO downstream of a small leader that varies between different constructs. Leaders primarily provide a high level of translational initiation and rapid purification. Plasmids pMP210-l, -T8, -21, -22, -24, -25 are designed to express the first 153 amino acids of TPO downstream of the initiation methionine and differ only in the use of the codon for the first 6 amino acids of TPO, and plasmid pMP251 is pMP210-l derivative in which the carboxy-terminal end of TPO is extended by two amino acids. All of the above plasmids give high levels of intracellular TPO expression in E. coli after induction of the tryptophan promoter (Yansura, DG et al. Methods and Enzymology (Goeddel, DV, ed.) 185: 54-60, Academic Press, San Diego [1990] ). Plasmids pMP1 and pMP172 are intermediates in the construction of the upper intracellular TPO expression plasmids.

Zgornje ekspresijske plazmide TPO uporabimo za transformiranje E.coli z uporabo postopka toplotnega šoka s CaCl₂ (Mandel, M. et al., J. Mol. Biol., 53:159-162, [1970]) in drugih postopkov, opisanih v Primeru 21. Na kratko, transformirane celice najprej rastejo pri 37 °C, dokler optična gostota (600 nm) kulture ne doseže približno 2-3. Kulturo nato razredčimo in po rasti ob aeraciji dodamo kislino. Kulturo nato pustimo, da kontinuirno raste ob aeraciji nadaljnjih 15 ur, nakar celice zberemo s centrifugiranjem.The TPO expression plasmids above are used to transform E. coli using a heat shock procedure with CaCl ₂ (Mandel, M. et al., J. Mol. Biol., 53: 159-162, [1970]) and other methods described in Example 21. Briefly, transformed cells first grow at 37 ° C until the optical density (600 nm) of the culture reaches about 2-3. The culture was then diluted and acid was added after growth under aeration. The culture was then allowed to grow continuously with aeration for a further 15 hours, after which cells were collected by centrifugation.

Postopki izoliranja, čiščenja in renaturiranja, navedeni spodaj, za izdelavo biološko aktivnega renaturiranega humanega TPO ali njegovih fragmentov, so opisani v Primerih 22 in 23 in jih lahko uporabimo za rekuperiranje katerekoli variante TPO, vključno N- in C-terminalo razširjenih oblik. Drugi postopki, prikladni za renaturiranje rekombinantnega ali sintetičnega TPO, so v naslednjih patentih: Builder et al., U.S. Patent 4,511,502; Jones et al., U.S. Patent 4,512,922; Olson U.S. Patent 4,518,526 in Builder et al., U.S. Patent 4,620,948; za splošen opis postopkov rekuperiranja in renaturiranja za različne rekombinantne proteine, eksprimirane v netopni obliki v E. coli.The isolation, purification and renaturation procedures listed below for the production of biologically active renatured human TPO or fragments thereof are described in Examples 22 and 23 and can be used to recover any variant of TPO, including the N- and C-terminal of extended forms. Other processes suitable for the renaturation of recombinant or synthetic TPO are in the following patents: Builder et al., U.S. Pat. Patent 4,511,502; Jones et al., U.S. Pat. Patent 4,512,922; Olson U.S. Patent 4,518,526 and Builder et al., U.S. Pat. Patent 4,620,948; for a general description of the recovery and renaturation processes for various recombinant proteins expressed in insoluble form in E. coli.

A. Rekuperiranje netopnega TPOA. Recovery of insoluble TPO

Mikroorganizem, kot npr. E. coli, ki eksprimira TPO, kodiran s katerimkoli prikladnim plazmidom, fermentiramo pri pogojih, pri katerih se TPO deponira v netopna refraktilna telesa. V danem primeru celice najprej izperemo v pufru za razkroj celic. Značilno je, da približno 100 g celic resuspendiramo v približno 10 volumnih pufra za razkroj celic (npr. tris (10 mM), EDTA (5 mM), pH 8) npr. s homogenizatoijem Polytron in celice centrifugiramo pri 5000 g 30 minut. Celice nato liziramo s katerokoli konvencionalno tehniko, kot so npr. tonični šok, sonifikacija, tlačno cikliranje, kemijski ali encimatski postopki. Npr., zgornji izprani celični pelet lahko resuspendiramo v drugih 10 volumnih pufra za razkroj celic s homogenizatorjem in celično suspenzijo spustimo skozi napravo za razkroj celic (LH Celi Disrupter; LH Inceltech, Inc.) ali skozi mikrofluidizator (Microfluidizer; Microfluidics International) po navodilih izdelovalca. Delce snovi, ki vsebujejo TPO, nato ločimo od tekoče faze in v danem primeru izperemo s prikladno tekočino. Npr. suspenzijo celičnega lizata lahko centrifugiramo pri 5000 g 30 minut, resuspendiramo in v danem primeru drugič centrifugiramo, da naredimo izprani pelet refraktilnih teles.A microorganism, such as E. coli expressing TPO encoded by any suitable plasmid is fermented under conditions under which TPO is deposited in insoluble refractile bodies. In a given case, the cells are first washed in cell decomposition buffer. Typically, about 100 g of cells are resuspended in about 10 volumes of cell decomposition buffer (e.g., tris (10 mM), EDTA (5 mM), pH 8) e.g. using a Polytron homogenizer and the cells were centrifuged at 5000 g for 30 minutes. The cells are then lysed by any conventional technique such as e.g. tonic shock, sonication, pressure cycling, chemical or enzymatic processes. For example, the above washed cell pellet can be resuspended in another 10 volumes of cell decomposition buffer with a homogenizer and the cell suspension is lowered through a cell decomposition device (LH Whole Disrupter; LH Inceltech, Inc.) or through a microfluidizer (Microfluidizer; Microfluidics International) as instructed. of the manufacturer. The particulate matter containing the TPO is then separated from the liquid phase and, if appropriate, washed with a suitable liquid. E.g. the cell lysate suspension can be centrifuged at 5000 g for 30 minutes, resuspended and optionally centrifuged a second time to make the washed pellet of refractive bodies.

Izprani pelet lahko uporabimo takoj ali v danem primeru zamrznjenega shranimo (pri približno -70 °C).The washed pellet can be used immediately or, if appropriate, frozen (at about -70 ° C).

B. Solubiliziranje in čiščenje monomemega TPOB. Solubilizing and purifying monomemic TPO

Netopen TPO v peletu refraktilnih teles nato solubiliziramo s solubilizimim pufrom. Solubilizimi pufer vsebuje kaotropično sredstvo in ima navadno s pufrom naravnan bazični pH ter vsebuje redukcijsko sredstvo za izboljšanje dobitka monomemega TPO. Ustrezna kaotropična sredstva vključujejo sečnino, gvanidin.HCl in natrijev tiocianat. Prednostno kaotropično sredstvo je gvanidin.HCl. Koncentracija kaotropičnega sredstva je navadno 4-9M, prednostno 6-8M. pH solubilizimega pufra vzdržujemo s katerimkoli prikladnim pufrom v območju od približno 7,5 do 9,5, prednostno 8,0-9,0 in najbolj prednostno 8,0. Prednostno solubilizimi pufer vsebuje tudi redukcijska sredstva zaradi lažje tvorbe monomeme oblike TPO. Prikladna redukcijska sredstva vključujejo organske spojine, ki vsebujejo prosti tiol (RSH). Reprezentativna redukcijska sredstva vključujejo ditiotreitol (DTT), ditioeritritol (DTE), merkaptoetanol, glutation (GSH), cisteamin in cistein. Prednostno redukcijsko sredstvo je ditiotreitol (DTT). V danem primem lahko solubilizimi pufer vsebuje blago oksidacijsko sredstvo (npr. molekulami kisik) in sulfitno sol, da nastane monomemi TPO preko sulfitolize. V tej izvedbi nastali TPO-S-sulfonat kasneje renaturiramo v prisotnosti redoks pufra (npr. GSH/GSSG), da dobimo pravilno zguban TPO.Insoluble TPO in the pellet of refractile bodies is then solubilized with solubilizable buffer. The solubilizers buffer contains a chaotropic agent and generally has a basic pH-adjusted buffer and contains a reducing agent to improve the yield of monomemic TPO. Suitable chaotropic agents include urea, guanidine.HCl and sodium thiocyanate. A preferred chaotropic agent is guanidine.HCl. The concentration of the chaotropic agent is usually 4-9M, preferably 6-8M. The pH of the solubilizable buffer is maintained with any suitable buffer in the range of about 7.5 to 9.5, preferably 8.0-9.0 and most preferably 8.0. Preferably, solubilizers also contain reducing agents to facilitate the formation of a monomemic form of TPO. Suitable reducing agents include free thiol-containing organic compounds (RSH). Representative reducing agents include dithiothreitol (DTT), dithioerythritol (DTE), mercaptoethanol, glutathione (GSH), cysteamine and cysteine. A preferred reducing agent is dithiothreitol (DTT). In a given example, solubilizers may contain a mild oxidizing agent (eg oxygen molecules) and a sulfite salt to form TPO monomemes via sulfitolysis. In this embodiment, the resulting TPO-S-sulfonate is subsequently renatured in the presence of redox buffer (e.g. GSH / GSSG) to obtain a properly folded TPO.

Protein TPO navadno nadalje očistimo z uporabo npr. centrifugiranja, gelske filtracijske kromatografije in kolonske kromatografije z reverzno fazo.The TPO protein is usually further purified using e.g. centrifugation, gel filtration chromatography and reverse phase column chromatography.

Za ilustracijo navajamo naslednji postopek, s katerim smo dobili ugodne dobitke monomemega TPO. Pelet refraktilnih teles resuspendiramo v približno 5 masnih volumnih solubilizimega pufra (tris (20 mM), pH 8, z gvanidinom (6-8 M) in DTT (25 mM)) ter mešamo 1-3 ure ali preko noči pri 4 °C, da povzročimo solubilizacijo proteina TPO. Visoke koncentracije sečnine (6-8 M) so tudi uporabne, vendar dajo na splošno nekoliko nižje dobitke, če jih primerjamo z gvanidinom. Po solubilizaciji raztopino centrifugiramo pri 30000 g 30 minut, da dobimo bister supematant, ki vsebuje denaturiran monomeren protein TPO. Supematant nato kromatografiramo na gelski filtracijski koloni superdeks 200 (Pharmacia, 2,6 x 60 cm) pri hitrosti pretoka 2 ml/min in protein eluiramo z Na-fosfatom (20 mM), pH 6,0 z DTT (10 mM). Frakcije, ki vsebujejo monomemi denaturirani protein TPO, ki se eluirajo medFor illustration, the following procedure is used to obtain a favorable gain for a monomemic TPO. The pellets of the refractile bodies were resuspended in about 5 mass volumes of solubilizable buffer (tris (20 mM), pH 8, guanidine (6-8 M) and DTT (25 mM)) and stirred for 1-3 hours or overnight at 4 ° C. to solubilize the TPO protein. High concentrations of urea (6-8 M) are also useful, but generally yield slightly lower yields when compared to guanidine. After solubilization, the solution was centrifuged at 30000 g for 30 minutes to obtain a clear supernatant containing denatured monomeric protein TPO. The supernatant was then chromatographed on a superdex 200 gel filtration column (Pharmacia, 2.6 x 60 cm) at a flow rate of 2 ml / min and the protein was eluted with Na-phosphate (20 mM), pH 6.0 with DTT (10 mM). Fractions containing monomem denatured TPO protein eluted between

160 in 200 ml, zberemo v pool. Protein TPO nadalje očistimo na semipreparativni koloni z reverzno fazo C4 (2 x 20 cm VYDAC). Vzorec nanesemo s 5 ml/min na kolono, uravnoteženo v TFA (trifluoroocetna kislina (0,1 %)) z acetonitrilom (30 %). Protein eluiramo z linearnim gradientom acetonitrila (30-60 % v 60 min). Očiščeni reducirani protein se eluira približno pri 50 % acetonitrilu. To snov uporabimo za renaturiranje, da dobimo biološko aktivno varianto TPO.Collect 160 and 200 ml into the pool. The TPO protein was further purified on a C4 reverse phase semipreparative column (2 x 20 cm VYDAC). The sample was applied at 5 ml / min to a column equilibrated in TFA (trifluoroacetic acid (0.1%)) with acetonitrile (30%). The protein was eluted with a linear gradient of acetonitrile (30-60% in 60 min). The purified reduced protein was eluted at about 50% acetonitrile. This substance is used for renaturation to obtain the biologically active variant of TPO.

C. Renaturiranje TPO, da dobimo biološko aktivno oblikoC. Renaturation of TPO to obtain biologically active form

Po solubilizaciji in nadaljnjem čiščenju TPO dobimo biološko aktivno obliko z renaturiranjem denaturiranega monomemega TPO v redoks pufru. Zaradi visoke zmogljivosti TPO (polovica maksimalne stimulacije v testu Ba/F3 je dosežena pri približno 3 pg/ml) je možno, da dobimo biološko aktivno snov z uporabo različnih številnih pufrov, detergentov in redoks pogojev. Vendar pa pri večini pogojev dobimo le majhno količino pravilno zgubane snovi (<10 %). Za komercialne proizvodne postopke je želeno, da imajo vsaj 10 % dobitke renaturiranja, bolj prednostno 30-50 % in najbolj prednostno >50 %. Za mnoge, različne detergente, ki vključujejo Triton Χ-100, dodecil-beta-maltozid, CHAPS, CHAPSO, SDS, sarkozil, Tween 20 in Tween 80, Zwittergent 3-14 in druge, smo ugotovili, da so prikladni za izdelovanje vsaj nekoliko pravilno zgubane snovi. Od teh so najbolj prednostni detergenti iz družine CHAPS (CHAPS in CHAPSO), za katere smo ugotovili, da delujejo najbolje pri reakciji renaturiranja in da omejijo agregacijo proteina in nepravilno bisulfidno tvorbo. Nivoji CHAPS, ki so večji kot približno 1 %, so najbolj prednostni. Natrijev klorid je potreben za najboljše dobitke z optimalnimi nivoji med 0,1 M in 0,5 M. Prisotnost EDTA (1-5 mM) v redoks pufru je prednostna, da omeji oksidacijo, katalizirano s kovino (in agregacijo), zaznano pri nekaterih pripravkih. Koncentracije glicerola, večje od 15 %, ustvarijo optimalne pogoje za renaturiranje. Za maksimalne dobitke je bistveno, da imamo redoks par v redoks pufru, ki je sestavljen tako iz oksidiranega kot tudi iz reduciranega organskega tiola (RSH). Prikladni redoks pari vključujejo merkaptoetanol, glutation (GSH), cisteamin, cistein in njihove ustrezne oksidirane oblike. Prednostni redoks pari so glutation (GSH): oksidirani glutation (GSSG) ali cistein:cistin. Najbolj prednostni redoks parje glutation (GSH):oksidirani glutation (GSSG). Na splošno dobimo visoke dobitke, če sta oksidirani in reducirani član redoks para v enakem molskem razmerju oz. je oksidirani član v prebitku. pH vrednosti med 7,5 in približno 9 so optimalne za renaturiranje teh variant TPO. Organska topila (npr. etanol, acetonitril, metanol) toleriramo pri koncentracijah od 10 do 15 % ali nižjih. Višji nivoji organskih topil zvečajo količino nepravilno zgubanih oblik. Tris in fosfatni pufri so na splošno uporabni. Inkubacija pri 4 °C prav tako daje visoke nivoje pravilno zgubanega TPO.After solubilization and further purification of TPO, a biologically active form is obtained by renaturing denatured monomemic TPO in redox buffer. Due to the high TPO capacity (half of the maximum stimulation in the Ba / F3 assay is achieved at about 3 pg / ml), it is possible to obtain a biologically active substance using a variety of many buffers, detergents and redox conditions. However, under most conditions only a small amount of properly wrinkled matter (<10%) is obtained. Commercial manufacturing processes are desired to have at least 10% renaturation gains, more preferably 30-50% and most preferably> 50%. Many, many detergents, including Triton Χ-100, dodecyl beta-maltoside, CHAPS, CHAPSO, SDS, sarcosyl, Tween 20 and Tween 80, Zwittergent 3-14 and others, have been found to be suitable for at least a little properly wrinkled matter. Of these, the most preferred are the detergents from the CHAPS family (CHAPS and CHAPSO), which have been found to work best in the renaturation reaction and to limit protein aggregation and irregular bisulfide formation. CHAPS levels greater than about 1% are the most preferred. Sodium chloride is required for best yields with optimum levels between 0.1 M and 0.5 M. The presence of EDTA (1-5 mM) in redox buffer is preferred to limit the metal-catalyzed (and aggregation) oxidation detected in some preparations. Glycerol concentrations greater than 15% create optimal conditions for renaturation. For maximum yields, it is essential to have a redox pair in redox buffer consisting of both oxidized and reduced organic thiol (RSH). Suitable redox pairs include mercaptoethanol, glutathione (GSH), cysteamine, cysteine and their corresponding oxidized forms. Preferred redox pairs are glutathione (GSH): oxidized glutathione (GSSG) or cysteine: cystine. The most preferred redox pair is glutathione (GSH): oxidized glutathione (GSSG). Generally, high yields are obtained if the oxidized and reduced members of the redox pair are in the same molar ratio. is an oxidized member in excess. pH values between 7.5 and about 9 are optimal for the renaturation of these TPO variants. Organic solvents (eg ethanol, acetonitrile, methanol) are tolerated at concentrations of 10 to 15% or lower. Higher levels of organic solvents increase the amount of misfolded forms. Tris and phosphate buffers are generally useful. Incubation at 4 ° C also yields high levels of properly folded TPO.

40-60 % dobitki renaturiranja (glede na količino reduciranega in denaturiranega TPO, uporabljenega v reakciji renaturiranja) so značilni za pripravke TPO, ki jih očistimo v prvi stopnji C4. Aktivno snov lahko dobimo, če so pripravki manj čisti (npr. direktno po koloni superdeks 200 ali po začetni ekstrakciji refraktilnih teles), čeprav so dobitki manjši zaradi obsežne precipitacije in interference ne-TPOproteinov med postopkom renaturiranja TPO.40-60% of the renaturation gains (relative to the amount of reduced and denatured TPO used in the renaturation reaction) are typical of the TPO preparations purified in the first stage of C4. The active substance can be obtained if the preparations are less pure (eg directly after the superdex 200 column or after the initial extraction of refractory bodies), although the yields are lower due to the extensive precipitation and interference of non-TPOproteins during the TPO renaturation process.

Ker TPO vsebuje 4 cisteinske ostanke, je možno, da naredimo 3 različne disulfidne verzije proteina:Since TPO contains 4 cysteine residues, it is possible to make 3 different disulfide versions of the protein:

verzija 1: disulfidi med cisteinskimi ostanki 1-4 in 2-3 verzija 2: disulfidi med cisteinskimi ostanki 1-2 in 3-4 verzija 3: disulfidi med cisteinskimi ostanki 1-3 in 2-4.version 1: disulfides between cysteine residues 1-4 and 2-3 version 2: disulfides between cysteine residues 1-2 and 3-4 version 3: disulfides between cysteine residues 1-3 and 2-4.

Med začetnim raziskovanjem določevanja pogojev renaturiranja smo ločili številne različne vrhove, ki vsebujejo protein TPO, s kromatografijo z reverzno fazo C4. Le eden od teh vrhov ima značilno biološko aktivnost, ugotovljeno s testom Ba/F3. Kasneje smo pogoje renaturiranja optimizirali, da smo dobili to prednostno verzijo. Pri teh pogojih je nepravilno zgubanih verzij manj od 10-20 % celotnega monomemega TPO, dobljenega v stopnji solubiliziranja.During the initial exploration of the renaturation conditions, we separated a number of different peaks containing the TPO protein by C4 reversed phase chromatography. Only one of these peaks has the characteristic biological activity identified by the Ba / F3 assay. Later, the renaturation conditions were optimized to obtain this preferred version. Under these conditions, misfolded versions are less than 10-20% of the total monomemic TPO obtained in the solubilization step.

Za disulfidni model biološko aktivnega TPO je bilo ugotovljeno z masno spektrometrijo in proteinskim sekvenciranjem, da je verzija z disulfidi med cisteinskimi ostanki 1-4 in 2-3, pri čemer so cisteini oštevilčeni zaporedno od aminoterminala. Ta model cisteinskega premreževanja je konsistenten z znanim modelom disulfidnih vezav sorodne molekule eritropoetina.The disulfide model of biologically active TPO was found by mass spectrometry and protein sequencing to be the version with disulfides between cysteine residues 1-4 and 2-3, with cysteines numbered sequentially from the aminoterminals. This model of cysteine cross-linking is consistent with the known model of disulfide bonds of the erythropoietin related molecule.

D. Biološka aktivnost rekombinantnega renaturiranega TPOD. Biological activity of recombinant renatured TPO

Renaturirani in očiščeni TPO ima aktivnost tako pri testu in vitro kot tudi in vivo. Npr. pri testu Ba/F3 dosežemo polovično maksimalno stimulacijo vgraditve timidina v celice Ba/F3 za TPO (Met'¹1-153) pri 3,3 pg/ml (0,3 pM). V ELISI, ki temelji na receptorju mpl, pride do polovične maksimalne aktivnosti pri 1,9 ng/ml (120 pM). V normalnih živalih in takih z mielosupresijo, dobljenih s skoraj letalnim obsevanjem z rentgenskimi žarki, je renaturirani TPO (Met¹ 1-153) visoko zmogljiv (aktivnost opazna pri nizkih dozah, kot npr. 30 ng/miš) za stimulacijo nastajanja novih trombocitov. Podobno biološko aktivnost opazimo tudi pri drugih oblikah renaturiranega TPO v skladu z zgoraj opisanimi postopki (sl. 25,26 in 28).Renated and purified TPO has activity both in vitro and in vivo. E.g. in the Ba / F3 assay, half-maximal stimulation of thymidine incorporation into Ba / F3 cells for TPO (Met ' ¹ 1-153) was achieved at 3.3 pg / ml (0.3 pM). In mpl receptor-based ELISA, half maximal activity occurs at 1.9 ng / ml (120 pM). In normal myelosuppression animals obtained by near-air X-ray irradiation, renatured TPO (Met ¹ 1-153) is highly potent (activity observed at low doses, such as 30 ng / mouse) to stimulate new platelet production. Similar biological activity was observed in other forms of renatured TPO according to the procedures described above (Figs. 25, 26 and 28).

14. Postopki za merjenje trombopoetične aktivnosti14. Procedures for measuring thrombopoietic activity

Trombopoetično aktivnost lahko izmerimo z različnimi testi, ki vključujejo test mpl liganda Ba/F3 (opisano v Primeru 1), test oživitve sinteze mišjih trombocitov in vivo, test indukcije celičnega površinskega antigena trombocitov, kot ga izmerimo z antitrombocitnim imunotestom (anti-GPII_bIII_a) za humano levkemično megakarioblastično celično linijo (CMK) (Sato et al., Brit. J. Heamatol., 72:184-190 [1989]) (glej tudi test tekočinske suspenzijske megakariocitopoeze; opisano v Primeru 4), in indukcijo poliploidizacije v megakarioblastni celični liniji (DAMI) (Ogura et al., Blood, 72(1):49-60 [1988]). Zorenje megakariocitov iz nezrelih, pretežno DNA nesintetizimih celic, v megakariocite, ki se dajo morfološko identificirati, obsega postopek, ki vključuje nastanek citoplazemskih organelov, akvizicijo membranskih antigenov (GPII_bIII_a), endoreplikacijo in sproščanje trombocitov, kot je opisano v ozadju. Za rodovno specifični promotor (t.j. mpl ligand) megakariocitnega zorenja bi pričakovali, da inducira vsaj nekaj od teh sprememb v nezrelih megakariocitih, odločujočih za sproščanje trombocitov in zmanjšanje trombocitopenije. Testi so torej oblikovani za megenje dejanske pojavnosti teh parametrov v nezreli megakariocitni celični liniji, t.j. celicah CMK in DAMI. Test CMK (Primer 4) meri nastanek specifičnega trombocitnega markeg'a, GPII_bIII_a in izgubljanje trombocitov. Test DAMI (Primer 15) meri endoreplikacijo, ker so porasti v ploidiji pristni znaki megakariocitov. Spoznavni megakariociti imajo ploidne vrednosti 2N, 4N, 8N, 16N, 32N itd.. Končno je test oživitve mišjih trombocitov in vivo (Primer 16) koristen za to, da prikažemo, da je posledica dajanja testne spojine (tukaj mpl ligand) zvečanje števila trombocitov.Thrombopoietic activity can be measured by various assays, including the Ba / F3 mpl ligand assay (described in Example 1), the in vivo synthesis of murine platelet synthesis, the platelet cell surface antigen induction assay, as measured by anti-GPII _b III _a ) for human leukemic megakaryoblastic cell line (CMK) (Sato et al., Brit. J. Heamatol., 72: 184-190 [1989]) (see also liquid suspension megakaryocytopoiesis test; described in Example 4), and induction of polyploidization in a megakaryoblast cell line (DAMI) (Ogura et al. Blood, 72 (1): 49-60 [1988]). The maturation of megakaryocytes from immature, predominantly non-synthetic DNA cells, into morphologically identifiable megakaryocytes, involves a process involving the formation of cytoplasmic organelles, the acquisition of membrane antigens (GPII _b III _a ), endoreplication and platelet release, as described. The genus-specific promoter (ie mpl ligand) of megakaryocyte maturation would be expected to induce at least some of these changes in immature megakaryocytes, which are crucial for platelet release and reduction of thrombocytopenia. The tests are therefore designed to counteract the actual occurrence of these parameters in an immature megakaryocytic cell line, ie CMK and DAMI cells. The CMK test (Example 4) measures the formation of a specific platelet markg ', GPII _b III _a and the loss of platelets. The DAMI test (Example 15) measures endoreplication because the growths in the ploidy are genuine signs of megakaryocytes. Recognizable megakaryocytes have ploidy values of 2N, 4N, 8N, 16N, 32N, etc. Finally, the in vivo mouse platelet recovery test (Example 16) is useful in demonstrating that platelet count increase is caused by administration of the test compound (here mpl ligand) .

Dva dodatna testa in vitro sta razvita za megenje aktivnosti TPO. Prvi je analiza, ki temelji na aktivaciji kinaznega receptorja (KIRA) ELISA, v katerem celice CHO transfektiramo s kimemim mp/-Rse, tirozinsko fosforiliziranje Rse pa izmerimo s testom ELISA po izpostavitvi kimemega mpl dela mpl ligandu (Primer 17). Drugi pa je ELISA, ki temelji na receptogu, pri čemer zajčji antihumani IgG, razmazan na plošči za test ELISA, zajame (kaptira) humani kimemi receptor mp/-IgG, ki veže mpl ligand, ki ga testiramo. Biotinilirano zajčje poliklonsko protitelo za mpl ligand (TPO₁₅₅) uporabimo, da detektiramo vezani mpl ligand, ki ga izmerimo z uporabo streptavidin-peroksidaze, kot je opisano v Primeru 18.Two additional in vitro assays have been developed for fogging TPO activity. The first is an kinase receptor (KIRA) ELISA assay in which CHO cells are transfected with chimem mp / -Rse, and tyrosine phosphorylation of Rse is measured by ELISA assay after exposure to chimem mpl portion of mpl ligand (Example 17). The other is a recipe-based ELISA, wherein the rabbit anti-human IgG smeared on the ELISA panel captures the human chimeric mp / -IgG receptor that binds the mpl ligand being tested. The biotinylated rabbit polyclonal antibody for mpl ligand (TPO ₁₅₅ ) was used to detect the bound mpl ligand, which was measured using streptavidin peroxidase as described in Example 18.

15. Biološki odziv in vivo normalnih in subletalno obsevanih miši, obdelanih s TPO15. Biological response of in vivo normal and sublethally irradiated TPO-treated mice

Tako normalne kot tudi subletalno obsevane miši obdelamo s skrajšanim TPO in s takim s popolno dolžino, izoliranim iz ovarijskih celic kitajskega hrčka (celice CHO),Both normal and sublethal irradiated mice were treated with shortened TPO and full-length mice isolated from Chinese hamster ovary cells (CHO cells),

E. coli, in 293 celic humanih embrionalnih ledvic. Obe obliki TPO, nastali v teh treh gostiteljih, stimulirata nastajanje trombocitov v miših, vendar pa TPO s popolno dolžino, izoliran iz CHO, ustvari največji odziv in vivo. Iz teh rezultatov je razvidno, da je pravilna glikozilacija karboksi-terminalne domene verjetno potrebna za optimalno aktivnost in vivo.E. coli, and 293 human embryonic kidney cells. Both forms of TPO generated in these three hosts stimulate platelet production in mice, but full-length TPO isolated from CHO produces the largest response in vivo. These results suggest that proper glycosylation of the carboxy-terminal domain is likely required for optimal activity in vivo.

(aJE-coli-rh-TTO^·¹,^(aJE-coli-rh-TTO ^ · ¹ , ^

Obliko Met EPO-domene (Met v položaju -1 plus prvih 153 ostankov humanega TPO), nastalo v E. coli (Primer 23), injiciramo dnevno v normalne samice miši C57 B6, kot je opisano v legendah na sl. 25A, 25B in 25C. Te slike prikazujejo, da je neglikozilirana skrajšana oblika TPO, nastala v E.coli, in renaturirana, kot je opisano zgoraj, sposobna, da stimulira približno 2-kratno zvečanje nastajanja ploščic v normalnih miših, ne da bi vplivala na populacijo eritrocitov ali levkocitov.The meth form of the EPO domain (Met at position -1 plus the first 153 residues of human TPO) formed in E. coli (Example 23) was injected daily into normal female C57 B6 mice, as described in the legends of FIG. 25A, 25B and 25C. These images show that the non-glycosylated truncated form of TPO generated in E.coli and renatured as described above is capable of stimulating an approximately 2-fold increase in plaque formation in normal mice without affecting the erythrocyte or leukocyte population.

Ista molekula, injicirana dnevno v subletalno obsevane (¹³⁷Cs) samice miši C57 B6, kot je opisano v legendah na sl. 26A, 26B in 26C, stimulira rekuperirane trombocitov in vpliva na najnižjo točko, vendar pa ne vpliva na eritrocite ali levkocite.The same molecule injected daily into the sublethally irradiated ( ¹³⁷ Cs) females of C57 B6 mice as described in the legends of FIG. 26A, 26B and 26C, stimulates the recovered platelets and affects the lowest point, but does not affect erythrocytes or leukocytes.

(b) CHO-rhTPO₃₃₂ (b) CHO-rhTPO ₃₃₂

Oblika TPO s popolno dolžino, izdelana v CHO in injicirana dnevno v normalne samice miši C57 B6, kot je opisano v legendah na sl. 27A, 27B in 27C, ustvari približno 5-kratno zvečanje nastajanja trombocitov v normalnih miših, ne da bi vplivala na populacijo eritrocitov ali levkocitov.A full-length TPO formulation made in CHO and injected daily into normal C57 B6 female mice as described in the legends of FIG. 27A, 27B, and 27C, produce an approximately 5-fold increase in platelet production in normal mice without affecting the erythrocyte or leukocyte population.

(c) CHO-rhTPO₃₃₂; E. coli-rhTPO^'¹153); 293-rhTPO₃₃₂; in E. coli-rhTPO₁₅₅.(c) CHO-rhTPO ₃₃₂ ; E. coli-rhTPO ^ ' ¹ 153); 293-rhTPO ₃₃₂ ; and E. coli-rhTPO ₁₅₅ .

Krivulje odziva na dozo konstruiramo za obdelovanje normalnih miši z rhTPO iz različnih celičnih linij (CHO-rhTPO₃₃₂; E. coli-rhTPO^'¹153); 293-rhTPO₃₃₂ inDose response curves were designed to treat normal mice with rhTPO from different cell lines (CHO-rhTPO ₃₃₂ ; E. coli-rhTPO ^ ' ¹ 153); 293-rhTPO ₃₃₂ in

E. coli-rhTPO₁₅₅), kot je opisano v legendi na sl. 28. Ta slika prikazuje, da vse testirane oblike molekule stimulirajo nastajanje trombocitov, vendar pa ima oblika s popolno dolžino, nastala v CHO, največjo aktivnost in vivo.E. coli-rhTPO ₁₅₅ ), as described in the legend in FIG. 28. This figure shows that all tested forms of the molecule stimulate platelet formation, but the full-length form produced in CHO has the highest activity in vivo.

(d) CHO-rhTPO₁₅₃, CHO-rhTPOpristrižen in CHO-rhTPO₃₃₂ (d) CHO-rhTPO ₁₅₃ , CHO-rhTPO attached and CHO-rhTPO ₃₃₂

Konstruiramo tudi krivuljo odziva na dozo za obdelovanje normalnih miši z različnimi oblikami rhTPO, narejenega v CHO (CHO-rhTPO₁₅₃, CHOrhTPOpristrižen in CHO-rhTPO^), kot je opisano v legendi na sl. 29. Ta slika prikazuje, da vse testirane oblike molekule CHO stimulirajo nastajanje trombocitov, vendar pa ima oblika s popolno dolžino 70 kDa največjo aktivnost in vivo.We also construct a dose response curve for treating normal mice with various forms of rhTPO made in CHO (CHO-rhTPO ₁₅₃ , CHOrhTPO attached and CHO-rhTPO ^), as described in the legend in FIG. 29. This figure shows that all tested forms of the CHO molecule stimulate platelet formation, but the 70 kDa full-length form has the highest activity in vivo.

16. Splošna rekombinantna priprava mpl liganda in variant16. General recombinant preparation of mpl ligand and variants

Prednostno mpl ligand pripravimo s standardnimi rekombinantnimi postopki, ki vključujejo izdelovanje mpl Ugandskega poUpeptida s kultiviranjem cehe, transfektiranih za ekspresijo nukleinske kisline mpl Uganda (značilno s transformiranjem celic z ekspresijskim vektorjem) in rekuperiranje poUpeptida iz ceUc. UgotoviU smo, da v danem primeru mpl Ugand lahko izdelamo s homologno rekombinacijo aU rekombinantnimi postopki z uporabo kontrolnih elementov, uvedenih v ceUce, ki že vsebujejo DNA, ki kodira mpl Ugand. Npr., močan element promotor/spodbujevalec, supresor aU eksogeni transkripcijski modulatomi element lahko inseriramo v genom predvidene gostiteljske ceUce v zadovoljivi bližini in orientaciji, da vpUva na transkripcijo DNA, ki kodira želeni mpl Ugandski poUpeptid. Kontrolni element ne kodira mpl Uganda, ampak je prej DNA indigena za genom gostiteljske ceUce. Naslednji pa selekcionira ceUce za izdelavo receptorskega poUpeptida v smislu izuma aU za zvečanje aU zmanjšanje nivojev ekspresije, če je potrebno.Preferably, the mpl ligand is prepared by standard recombinant procedures involving the production of mpl Ugandan poUpeptide by culturing a guild transfected for the expression of the nucleic acid mpl Uganda (typically transforming cells with an expression vector) and recovering poUpeptide from ceUc. We have found that, in the present case, mpl Ugand can be produced by homologous recombination of aU recombinant processes using controls introduced into ceUce that already contain DNA encoding mpl Ugand. For example, a potent promoter / promoter element, a suppressor of the aU exogenous transcriptional modulus element, can be inserted into the genome of the predicted host ceUce in sufficient proximity and orientation to induce transcription of the DNA encoding the desired mpl Ugandan popeptide. The control element does not encode mpl Uganda but is formerly the DNA of the indigenous gene for the host ceUce genome. The following selects ceUce to produce the receptor poUpeptide of the invention aU for increasing aU reducing expression levels, if necessary.

V smislu izuma torej preučujemo postopek za izdelavo mpl Uganda, ki obsega insercijo v genom ceUce, ki vsebuje molekulo nukleinske kisline mpl Uganda, transkripcijskega modulatornega elementa v zadovoljivi bližini in orientaciji glede na molekulo nukleinske kisline, da vpUva na njeno transkripcijo, in v danem primeru nadaljnjo stopnjo, ki obsega kultiviranje ceUce, ki vsebuje transkripcijski modulatomi element in molekulo nukleinske kisline. Predloženi izum prav tako preučuje gostiteljsko celico, ki vsebuje indigeno molekulo nukleinske kisline mpl Uganda, funkcionalno vezano na eksogene kontrolne sekvence, spoznane od gostiteljske ceUce.According to the invention, therefore, we are examining a method for making Ugandan mpl comprising insertion into the ceUce genome containing the mpl Uganda nucleic acid molecule, a transcriptional modulator element in a satisfactory proximity and orientation with respect to the nucleic acid molecule to uptake its transcription, and, as the case may be, a further step comprising the cultivation of a ceUce containing a transcriptional modulus element and a nucleic acid molecule. The present invention also examines a host cell containing an indigenous mpl Uganda nucleic acid molecule functionally linked to exogenous control sequences recognized by the host ceUce.

A. Izolacija DNA, ki kodira mpl Ugandski poUpeptidA. Isolation of DNA encoding mpl Ugandan poUpeptide

DNA, ki kodira mpl Ugandski poUpeptid, lahko dobimo iz katerekoU knjižnice cDNA, pripravljene iz tkiva, za katerega verjamemo, da ima mRNA mpl Uganda in da le-to eksprimira na nivoju, ki se da detektirati. Gen mpl Uganda prav tako lahko dobimo iz knjižnice genomske DNA aU z oUgonukleotidno sintezo in vitro iz kompletne nukleotidne ali aminokislinske sekvence.DNA encoding the Ugandan poUpeptide mpl can be obtained from any tissue cDNA library believed to have Ugandan mpl mRNA and expressing it at a detectable level. The Ugand mpl gene can also be obtained from the aU genomic DNA library by oUgonucleotide synthesis in vitro from a complete nucleotide or amino acid sequence.

Knjižnice selekcioniramo s sondami, oblikovanimi tako, da identificirajo gen, ki nas zanima, aU protein, ki ga le-ta kodira. Za ekspresijske knjižnice cDNA prikladne sonde vključujejo monoklonska aU poliklonska protitelesa, ki spoznajo mpl Ugand in se specifično vežejo nanj. Za knjižnice cDNA prikladne sonde vključujejo oUgonukleotide s približno 20-80 bazami v dolžini, ki kodirajo znane aU domnevne dele cDNA mpl Uganda iz enakih aU razUčnih vrst in/aU komplementarne aU homologne cDNA aU njihove fragmente, ki kodirajo enak ali podoben gen. Ustrezne sonde za selekcioniranje knjižnic genomske DNA vključujejo, vendar ne omejujoče, oUgonukleotide, cDNA aU njihove fragmente, ki kodirajo enak aU podoben gen in/aU homologne genomske DNA ali njihove fragmente. Selekcioniranje cDNA ali genomske knjižnice z izbrano sondo lahko izvedemo z uporabo standardnih postopkov, kot je opisano v poglavjih 10-12 avtorja Sambrooka et al., zgoraj.Libraries are selected using probes designed to identify the gene of interest and the aU protein that encodes it. For cDNA expression libraries, suitable probes include monoclonal aU polyclonal antibodies that recognize and specifically bind to Ugand mpl. For cDNA libraries, suitable probes include oUgonucleotides of about 20-80 bases in length encoding known aU putative cDNA mpl portions of Uganda from identical aU assay species and / aU complementary aU homologous cDNA aU fragments encoding the same or similar gene. Suitable probes for selecting genomic DNA libraries include, but are not limited to, oUgonucleotides, cDNA aU fragments thereof encoding the same aU-like gene and / aU homologous genomic DNA or fragments thereof. Selection of a cDNA or genomic library with a selected probe can be performed using standard procedures as described in Chapters 10-12 by Sambrook et al., Above.

Alternativni način, da izoUramo gen, ki kodira mpl Ugand je, da uporabimo metodologijo PCR, kot je opisana v odstavku 14 avtorja Sambrooka et al., zgoraj. Ta postopek zahteva uporabo oUgonukleotidnih sond, ki se hibridizirajo z DNA, ki kodira mpl Ugand. Strategije za selekcijo oligonukleotidov so opisane spodaj.An alternative way to isolate the gene encoding Ugandan mpl is to use the PCR methodology as described in paragraph 14 by Sambrook et al., Above. This process requires the use of oUgonucleotide probes that hybridize with the DNA encoding the Ugand mpl. The strategies for oligonucleotide selection are described below.

V prednostnem postopku v smislu izuma uporabimo natančno izbrane oUgonukleotidne sekvence, da selekcioniramo knjižnice cDNA iz razUčnih tkiv, prednostno ceUčne Unije humanih ali prašičjih ledvic (odraslih aU fetalnih) aU jeter. Npr. knjižnice cDNA ceUčne Unije humanih fetalnih jeter selekcioniramo z oUgonukleotidnimi sondami. Alternativno lahko humane genomske knjižnice selekcioniramo z oUgonukleotidnimi sondami.In the preferred method of the invention, precisely selected oUgonucleotide sequences are used to select cDNA libraries from tissue tissues, preferably the entire Union of human or porcine (adult aU fetal) aU livers. E.g. Human fetal liver cDNA libraries are selected by oUgonucleotide probes. Alternatively, human genomic libraries can be selected by oUgonucleotide probes.

OUgonukleotidne sekvence, izbrane kot sonde, morajo biti dovolj dolge in dovolj nedvoumne, da so napačni pozitivi minimalni. Dejanska nukleotidna sekvenca je navadno oblikovana na osnovi regij mpl liganda, ki imajo najmanjšo kodonsko izobilje. Oligonukleotidi so lahko degenerirani na enem ali več položajih. Uporaba degeniranih oligonukleotidov je posebno pomembna, kadar je knjižnica selekcionirana iz vrste, v kateri preferenčna uporaba kodona ni znana.The oUgonucleotide sequences selected as probes should be long enough and unambiguous enough to minimize false positives. The actual nucleotide sequence is typically formed based on mpl ligand regions that have the least codon abundance. Oligonucleotides can be degenerated in one or more positions. The use of degenerate oligonucleotides is particularly important when the library is selected from a species in which codon preferential use is unknown.

Oligonukleotid mora biti označen tako, da ga lahko detektiramo po hibridizaciji z DNA v knjižnici, ki jo selekcioniramo. Prednosten postopek označevanja je uporaba ATP (npr. -γ³²Ρ) in polinukleotidne kinaze, da radioaktivno označimo 5’ konec oligonukleotida. Vsekakor pa so uporabni tudi drugi postopki za označevanje oligonukleotidov, vključno, vendar ne omejujoče, biotiniliranje ali encimsko označevanje.The oligonucleotide must be labeled so that it can be detected after hybridization with DNA in the library to be selected. A preferred labeling process is the use of ATP (e.g., -γ ³² Ρ) and polynucleotide kinase to radiolabel the 5 'end of the oligonucleotide. However, other methods for labeling oligonucleotides, including, but not limited to, biotinylation or enzymatic labeling are useful.

Posebno zanimiva je nukleinska kislina mpl liganda, ki kodira mpl Ugandski polipeptid s popolno dolžino. V nekaterih prednostnih izvedbah sekvenca nukleinske kisline vključuje signalno sekvenco naravnega mpl liganda. Nukleinsko kislino, ki ima vso proteinsko kodirno sekvenco, dobimo s selekcioniranjem izbrane cDNA ali genomskih knjižnic z uporabo deducirane aminokislinske sekvence.Of particular interest is the nucleic acid of the mpl ligand encoding the mpl full-length Ugandan polypeptide. In some preferred embodiments, the nucleic acid sequence includes the signal sequence of the natural mpl ligand. A nucleic acid having all the protein coding sequence is obtained by selecting the selected cDNA or genomic libraries using a deduced amino acid sequence.

B. Variante aminokislinske sekvence naravnega mpl UgandaB. Amino acid sequence variants of natural mpl Uganda

Variante aminokislinske sekvence mpl Uganda pripravimo z uvedbo ustreznih nukleotidih sprememb v DNA mpl Uganda aU s sintezo in vitro želenega mpl Ugandskega polipeptida. Take variante vključujejo npr. delecije aU insercije aU substitucije ostankov v aminokisUnski sekvenci za prašičji mpl Ugand. Npr. karboksi-terminalne dele zrelega mpl Uganda s popolno dolžino lahko odstranimo s proteoUtično cepitvijo bodisi in vivo aU in vitro ali s kloniranjem aU eksprimiranjem fragmenta aU DNA, ki kodira mpl Ugand s popolno dolžino, da dobimo biološko aktivno varianto. Vse kombinacije delecij, insercij in substitucij naredimo zato, da pridemo do končnega konstrukta, pod pogojem, da ima končni konstrukt želeno biološko aktivnost. AminokisUnske spremembe lahko spremenijo post translacijske postopke mpl Uganda, kot je npr. sprememba števila ali položajev mest za glikozilacijo. Za modeliranje variant aminokisUnske sekvence mpl Uganda sta lokacija mesta mutacije in narava mutacije odvisni od značilnosti mpl liganda, ki ga modificiramo. Mesta za mutacijo lahko modificiramo individualno aU v seriji, npr. (1) s substitucijo najprej s konzervativnimi izbirami amino kislin in nato z bolj radikalnimi selekcijami, odvisno od doseženih rezultatov, (2) z delecijo ciljnega ostanka ali (3) z insercijo ostankov enakega ali razUčnega razreda zraven lociranega mesta ali s kombinacijami opcij 1-3.Variants of the amino acid sequence of mpl Uganda are prepared by introducing appropriate nucleotide changes into the DNA of mpl Uganda aU by in vitro synthesis of the desired mpl Uganda polypeptide. Such variants include e.g. deletions of aU insertion aU substitution of residues in the amino acid sequence for pig mpl Ugand. E.g. carboxy-terminal portions of mature full length Ugandan mpl can be removed by proteoUtic cleavage either in vivo aU in vitro or by cloning aU by expressing a full length aU DNA encoding a full length Ugandan mpl to obtain a biologically active variant. All combinations of deletions, insertions, and substitutions are made to arrive at the final construct, provided that the final construct has the desired biological activity. Amino acid changes can change the post translational procedures of mpl Uganda, such as. change in the number or positions of glycosylation sites. To model variants of the amino acid sequence of the mpl Uganda, the location of the mutation site and the nature of the mutation depend on the characteristics of the mpl ligand being modified. Mutation sites can be modified individually by aU in a series, e.g. (1) by substitution, first by conservative choices of amino acids and then by more radical selections, depending on the results obtained, (2) by deletion of the target residue, or (3) by insertion of residues of the same or study class next to the located site, or by combinations of options 1- 3.

Uporaben postopek za identifikacijo določenih ostankov ali regij mpl Ugandskega poUpeptida, ki so prednostne lokacije za mutagenezo, je imenovan alaninska skenirna mutageneza, ki jo opisujejo Cunningham in WeUs, Science, 244:1081-1085 [1989]. Pri tem ostanek aU skupino ciljnih ostankov identificiramo (npr. nabiti ostanki, kot npr. arg, asp, his, lys in glu) in nadomestimo s katerokoli, prednostno pa z nevtralno ali negativno nabito amino kislino (najbolj prednostno alanin ali poUalanin), da vpUvamo na interakcijo amino kislin z obdajajočim vodnim okoljem v ceUci ali zunaj nje. Tiste domene, ki imajo funkcionalno občutljivost za substitucije, nato očistimo z uvedbo nadaljnjih aU drugih variant na aU za mesta za substitucijo. Medtem ko je mesto za uvedbo variante aminokislinske sekvence predhodno določeno, pa za naravo same mutacije ni potrebno, daje predhodno določena. Npr., da optimiziramo izvedbo mutacije na določenem mestu, vodimo ala-skeniranje ali naključno mutagenezo na ciljnem kodonu aU regiji, eksprimirane variante mpl Uganda pa selekcioniramo za optimalno kombinacijo želene aktivnosti.A useful procedure for identifying particular mpl residues or regions of Ugandan poUpeptide that are preferred sites for mutagenesis is called alanine scanning mutagenesis, described by Cunningham and WeUs, Science, 244: 1081-1085 [1989]. In doing so, the aU residue is identified by a group of target residues (e.g., charged residues, such as arg, asp, his, lys, and glu) and replaced by any, preferably neutral or negatively charged amino acid (most preferably alanine or poUalanin), we are introducing the interaction of amino acids with the surrounding aqueous environment inside or outside the ceUci. Those domains that have functional sensitivity to substitutions are then purified by introducing further aU other variants to aU for the substitution sites. While the site for introducing the amino acid sequence variant is predetermined, it is not necessary for the nature of the mutation itself to be predetermined. For example, to optimize the execution of a mutation at a particular site, we conduct ala-scanning or random mutagenesis at the target codon of the aU region, and express the mpl Uganda variants selected for the optimal combination of the desired activity.

V konstrukciji variante aminokislinske sekvence sta dve načelni spremenljivki: lokacija mesta mutacije in narava mutacije. Npr. variante mpl Ugandskega poUpeptida vključujejo variante sekvence mpl Uganda in lahko pomenijo naravne alele (za katere ni potrebna manipulacija DNA mpl Uganda), aU predhodno določene mutantne oblike, narejene z mutacijo DNA, da dobimo alelo aU varianto, ki se ne nahaja v naravi. Na splošno je izbira lokacije in narave mutacije odvisna od značilnosti mpl Uganda, ki ga modificiramo.There are two principle variables in the construction of an amino acid sequence variant: the location of the mutation site and the nature of the mutation. E.g. mpl variants of Ugandan poUpeptide include Ugandan mpl sequence variants and may represent natural alleles (which do not require the manipulation of mpl Uganda DNA), aU pre-defined mutant forms made by DNA mutation to produce the allele aU variant not found in nature. Generally, the choice of location and nature of the mutation depends on the characteristics of the mpl Uganda we are modifying.

Delecije aminokislinske sekvence obsegajo na splošno približno 1 do 30 ostankov, bolj prednostno približno 1 do 10 ostankov, in so značilno sosednje. Alternativno lahko delecije aminokisUnske sekvence za mpl Ugand vključujejo del karboksiterminalne glikoproteinske domene aU celotno. Delecije aminokisUnske sekvence lahko vključujejo tudi enega ali več od prvih 6 amino-terminalnih ostankov zrelega proteina. Opcijske delecije aminokisUnske sekvence obsegajo enega aU več ostankov v eni aU več regijah zank, ki obstajajo med spleti vijačnic. Sosednje delcije navadno naredimo tako, da obsegajo parno število ostankov, vendar pa so v obsegu predloženega izuma tudi delecije posameznih in neparnega števila ostankov. Delecije lahko uvedemo v regije z nizko homologijo med mpl Ugandi, ki prispevajo največ sekvenčne identičnosti, da modificiramo aktivnost mpl Uganda. Delecije pa lahko uvedemo tudi v regije z nizko homologijo med humanim mpl Ugandom in drugimi mpl Ugandskimi poUpetidi sesalca, ki prispevajo največ sekvenčne identičnosti za humani mpl ligand. Bolj verjetno je, da delecije mpl Ugandskega poUpeptida sesalca na področjih bistvene homologije z drugimi mpl Ugandi sesalca bolj značilno modificirajo biološko aktivnost mpl Uganda. Število sosednjih delecij izberemo tako, da ohranimo terciarno strukturo mpl Ugandov v prizadeti domeni, npr. beta nagubana ravnina aU alfa vijačnica.The amino acid deletions generally comprise about 1 to 30 residues, more preferably about 1 to 10 residues, and are typically adjacent. Alternatively, amino acid deletions of the Ug mpl sequences may include a portion of the carboxyterminal glycoprotein domain aU throughout. Amino acid deletions may also include one or more of the first 6 amino-terminal residues of a mature protein. Optional amino acid deletion sequences comprise one aU multiple residues in one aU multiple loop regions that exist between helix webs. Adjacent deletions are usually made to comprise an even number of residues, but within the scope of the present invention are also deletions of single and odd number of residues. Deletions can be introduced into regions with low homology among Ugandan mpls that contribute the most sequence identity to modify Ugandan mpl activity. However, deletions can also be introduced into regions with low homology between human mpl Ugand and other mpl Ugandan mammalian uPetides that contribute the most sequence identity to the human mpl ligand. It is more likely that deletions of the mammalian mpl Ugandan poUpeptide in areas of essential homology with other mammalian Ugandan mpl more significantly modify the biological activity of the Ugandan mpl. The number of adjacent deletions is selected by maintaining the tertiary structure of the Ugandan mpl in the affected domain, e.g. beta pleated plane aU alpha helix.

Insercije aminokislinske sekvence vključujejo amino- in/aU karboksilne-terminalne fuzije z dolžinami, ki obsegajo en ostanek, do poUpetidov s 100 aU več ostanki, kot tudi intrasekvenčne insercije posameznih aU multiplih aminokislinskih ostankov. Intrasekvenčne insercije (t.j. insercije v sekvenci zrelega mpl Uganda) lahko obsegajo na splošno približno 1 do 10 ostankov, bolj prednostno 1 do 5, še bolj prednostno 1 do 3. Zgledno prednostna je fuzija mpl Uganda aU njegovega fragmenta in drugega citokina aU njegovega fragmenta. Primeri terminalnih insercij vključujejo zreU mpl Ugand z N-terminalnim metionilnim ostankom, artefakt direktne ekspresije zrelega mpl Uganda v rekombinantni ceUčni kulturi in fuzijo heterologne N-terminalne signalne sekvence z N-terminalom molekule zrelega mpl Uganda, da se pospeši sekrecija zrelega mpl Uganda iz rekombinantnih gostiteljev. Take signalne sekvence - in na ta način tudi homologne - dobimo na splošno iz predvidenih vrst gostiteljskih ceUc. Prikladne sekvence vključujejo STII ali Ipp za E. coU, alfa faktor za kvasovke in virusne signale, kot npr. herpes gD za celice sesalcev.Amino acid sequence insertions include amino and / aU carboxyl-terminal fusions with lengths ranging from one residue to poUpetides with 100 aU multiple residues, as well as intrassequent insertions of single aU multiple amino acid residues. Intraspeak insertions (i.e., insertions in the sequence of a mature Ugandan mpl) may generally comprise about 1 to 10 residues, more preferably 1 to 5, more preferably 1 to 3. Fusion of the mpl Uganda aU of its fragment and another cytokine aU of its fragment is exemplary preferred. Examples of terminal insertions include mature mpl Ugand with N-terminal methionyl residue, artifact of direct expression of mature Ugandan mpl in recombinant tube culture, and fusion of heterologous N-terminal signal sequence with the N-terminal of the mature Ugandan mpl molecule to enhance the secretion of mature mpl Ugandans hosts. Such signal sequences - and thus homologous ones - are generally obtained from the intended host ceUc species. Suitable sequences include STII or Ipp for E. coU, an alpha factor for yeast and viral signals, such as e.g. herpes gD for mammalian cells.

Druge insercijske variante molekule mpl liganda vključujejo fuzijo z N- ali C-terminalom mpl Uganda imunogenih poUpeptidov (t.j., niso endogeni za gostitelja, na katerem izvajamo fuzijo), kot so npr.: bakterijski poUpeptidi, kot je beta laktamaza aU encim, kodiran z lokusom E. coU trp, aU protein kvasovk in C-terminalne fuzije s proteini, ki imajo dolgo razpolovno dobo, kot npr. imunoglobulinske konstantne regije (aU druge imunoglobulinske regije), albumin aU feritin, kot je opisano v WO 89/02922 objavljeno 6. aprila 1989.Other insertion variants of the mpl ligand molecule include fusion with the N- or C-terminal of the mpl Uganda immunogenic poUpeptides (i.e., they are not endogenous to the host on which the fusion is performed), such as: bacterial poUpeptides such as beta lactamase aU enzyme encoded by locus of E. coU trp, aU yeast protein, and C-terminal fusion with proteins with a long half-life, such as e.g. immunoglobulin constant regions (aU other immunoglobulin regions), albumin aU ferritin as described in WO 89/02922 published April 6, 1989.

Tretja skupina variant so aminokislinske substitucije. Te variante imajo odstranjen vsaj en aminokislinski ostanek v molekuU mpl Uganda odstranjen, na njegovo mesto pa je vstavljen drugačen ostanek. Mesta, ki so najbolj zanimiva za substitucijsko mutagenezo, vključujejo tista, ki so identificirana kot aktivna mesta mpl Uganda in tista, kjer so amino kisline, ki so ugotovljene v drugih analogih, v bistvu drugačne glede obsega stranske verige, naboja ah hidrofobnosti, vendar pa obstaja tudi visoka stopnja sekvenčne identičnosti na izbranem mestu med razUčnimi vrstami mpl Uganda in/ali v razUčnih živalskih analogih enega člana mpl Uganda.The third group of variants are amino acid substitutions. These variants have at least one amino acid residue in the mpl Uganda molecule removed and a different residue inserted in its place. The sites most interesting for substitution mutagenesis include those identified as the active sites of the Ugandan mpl and those where the amino acids found in the other analogues are substantially different in terms of side chain extent, ah hydrophobicity, but there is also a high degree of sequential identity at the selected site between the mpl Uganda species and / or in the animal analogues of one mpl Uganda member.

Druga zanimiva mesta so tista, kjer so posebni ostanki mpl liganda, dobljenega iz članov različnih družin in/ali živalskih vrst, identični v enem članu. Ta mesta, posebno tista, ki so znotraj sekvence vsaj treh drugih identično konzerviranih mest, so substituirana na relativno konzervativen način. Takšne konzervativne substitucije so prikazane v tabeli 3 pod naslovom prednostne substitucije. Če je posledica teh substitucij sprememba biološke aktivnosti, potem uvedemo več bistvenih sprememb, ki so v tabeli 3 imenovane zgledne substitucije, ali kot je dalje spodaj opisano pri navajanju aminokislinskih razredov, in produkte selekcioniramo.Other interesting sites are those where the specific residues of the mpl ligand obtained from members of different families and / or animal species are identical in one member. These sites, especially those within the sequence of at least three other identically conserved sites, are substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 3 under the heading of preferred substitutions. If these substitutions result in a change in biological activity, then a number of significant changes are introduced, which are called exemplary substitutions in Table 3, or as described below in the listing of amino acid classes, and the products are selected.

TABELA 3TABLE 3

Originalni Original Zgledne Exemplary Prednostne Preferred ostanek the residue substitucije substitutions substitucije substitutions Ala (A) Ala (A) Val; Leu; De Val; Leu; De Val Val Arg(R) Arg (R) Lys; Gin; Asn Lys; Gin; Asn Lys Lys Asn(N) Asn (N) Gin; His; Lys; Arg Gin; His; Lys; Arg Gin Gin Asp(D) Asp (D) Glu Glu Glu Glu Cys(C) Cys (C) Ser Sir Ser Sir Gln(Q) Gln (Q) Asn Asn Asn Asn Glu (E) Glu (E) Asp Asp Asp Asp Gly(G) Gly (G) Pro Pro Pro Pro His (H) His (H) Asn; Gin; Lys; Arg Asn; Gin; Lys; Arg Arg Arg Ile(I) Ile (I) Leu; Val; Met; Ala; Phe; Leu; Val; Met; Ala; Phe; norleucin norleucine Leu Leu Leu (L) Leu (L) norleucin; De; Val; Met; Ala; Phe norleucine; De; Val; Met; Ala; Phe De De Lys (K) Lys (K) Arg; Gin; Asn Arg; Gin; Asn Arg Arg Met (M) Met (M) Leu; Phe; De Leu; Phe; De Leu Leu Phe (F) Phe (F) Leu; Val; De; Ala Leu; Val; De; Ala Leu Leu Pro (P) Pro (P) Gly Gly Gly Gly Ser(S) Ser (S) Thr Thr Thr Thr Thr(T) Thr (T) Ser Sir Ser Sir Trp(W) Trp (W) Tyr Tyr Tyr Tyr Tyr(Y) Tyr (Y) Trp; Phe; Thr; Ser Trp; Phe; Thr; Sir Phe Phe Val (V) Val (V) De; Leu; Met; Phe; De; Leu; Met; Phe;

Ala; norlevcin LeuAla; norleucine leu

Bistvene modifikacije v funkciji ali imunološki identičnosti mpl liganda izvedemo z izbiranjem substitucij, ki se značilno razlikujejo po njihovem učinku na vzdrževanje (а) strukture polipeptidnega ogrodja na področju substitucij, npr. ravninska ali vijačna konformacija, (b) naboja ali hidrofobnosti molekule na ciljnem mestu ali (c) obsežnosti stranske verige. Naravni ostanki so razdeljeni v skupine, ki temeljijo na splošnih lastnostih stranske verige:Substantial modifications in the function or immunological identity of the mpl ligand are made by selecting substitutions that differ significantly in their effect on the maintenance of (a) the structure of the polypeptide framework in the field of substitutions, e.g. plane or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the extent of the side chain. Natural residues are divided into groups based on the general properties of the side chain:

(1) hidrofoben: norlevcin, Met, Ala, Val, Leu, Ile (2) nevtralno hidrofilen: Cys, Ser, Thr (3) kisel: Asp, Glu (4) bazičen: Asn, Gin, His, Lys, Arg (5) ostanki, ki vplivajo na orientacijo verige: Gly, Pro in (б) aromatski: Trp, Tyr, Phe.(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile (2) neutral hydrophilic: Cys, Ser, Thr (3) acidic: Asp, Glu (4) basic: Asn, Gin, His, Lys, Arg ( 5) residues affecting chain orientation: Gly, Pro and (b) aromatic: Trp, Tyr, Phe.

Nekonzervativne substitucije sprožijo zamenjavo enega člana teh razredov z drugim. Take substituirane ostanke lahko uvedemo tudi v konzervativna substitucijska mesta ali bolj prednostno v preostala (nekonzervirana) mesta.Non-conservative substitutions trigger the replacement of one member of these classes by another. Such substituted residues can also be introduced into conservative substitution sites, or more preferably into remaining (non-conserved) sites.

V eni izvedbi v smislu izuma je želeno, da inaktiviramo eno ali več proteaznih cepišč, ki so v molekuli. Ta mesta so identificirana z nadzorom kodirane aminokislinske sekvence, v primeru tripsina, npr. za arginilm ali lizililni ostanek. Ko identificiramo proteazna cepišča, jih naredimo neaktivna za proteolitično cepitev s substituiranjem ciljnega ostanka z drugim ostankom, prednostno bazičnim ostankom, kot npr. glutaminom ali hidrofobnim ostankom, kot je serin z delecijo ostanka ali z insercijo prolilnega ostanka, takoj po ostanku.In one embodiment of the invention, it is desirable to inactivate one or more protease cleavages present in the molecule. These sites are identified by controlling the encoded amino acid sequence, in the case of trypsin, e.g. for arginylm or lysilyl residue. Once the protease cleavages are identified, they are rendered inactive for proteolytic cleavage by substituting the target residue with another residue, preferably a basic residue, such as e.g. glutamine or a hydrophobic residue, such as serine, by deletion of the residue or by insertion of a shedding residue, immediately after the residue.

V drugi izvedbi substituiramo katerikoli metionilni ostanek, drugačen od začetnega metionilnega ostanka signalne sekvence, ali katerikoli ostanek, lociran znotraj približno treh ostankov N- ali C-terminala za vsak tak metionilni ostanek, z drugim ostankom (prednostno v skladu s tabelo 3), ali deletiramo. Alternativno lahko inseriramo približno 1-3 ostanke zraven takšnih mest.In another embodiment, any methionyl residue other than the initial methionyl residue of the signal sequence is substituted, or any residue located within about three N- or C-terminal residues for each such methionyl residue, with another residue (preferably in accordance with Table 3), or deletes. Alternatively, about 1-3 residues can be inserted next to such sites.

Vsak cisteinski ostanek, ki ni vključen v vzdrževanje pravilne konformacije mpl liganda, prav tako lahko substituiramo na splošno s serinom, da izboljšamo oksidativno stabilnost molekule in preprečimo zmotno premreženje. Ugotovili smo, da sta prvi in četrti cistein v EPO domeni, oštevilčeno od amino-terminala, potrebna za vzdrževanje pravilne konformacije, drugi in tretji pa ne. Zato lahko drugi in tretji cistein v EPO domeni substituiramo.Any cysteine residue that is not involved in maintaining the correct conformation of the mpl ligand can also be substituted in general with serine to improve the oxidative stability of the molecule and to prevent mistaken cross-linking. We found that the first and fourth cysteines in the EPO domain, numbered from the amino terminal, are required to maintain the correct conformation, and the second and third are not. Therefore, the second and third cysteines in the EPO domain can be substituted.

Molekule nukleinske kisline, ki kodirajo variante amino kislinske sekvence mpl liganda, pripravimo z raznimi postopki, znanimi v tehniki. Ti postopki vključujejo, vendar ne omejujoče, izolacijo iz naravnega vira (v primeru variant naravne aminokislinske sekvence) ali pripravo z oligonukleotidno posredovano (ali položajno usmerjeno) mutagenezo, mutagenezo PCR in kasetno mutagenezo predhodno pripravljene variante ali ne-variante mpl Ugandskega polipeptida.Nucleic acid molecules encoding variants of the amino acid sequence of the mpl ligand are prepared by various methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of natural amino acid sequence variants) or preparation by oligonucleotide-mediated (or position-directed) mutagenesis, PCR mutagenesis and cassette mutagenesis of a previously prepared or non-mpl variant of the Ugandan polypeptide.

OUgonukleotidno posredovana mutageneza je prednostni postopek za pripravo substitucijskih, delecijskih in insercijskih variant DNA mpl Uganda. Ta tehnika je dobro znana in jo opisujejo Adelman et al., DNA, 2:183 [1983]. Na kratko, DNA mpl Uganda spremenimo s hibridiziranjem oUgonukleotida, ki kodira želeno mutacijo za kalup DNA, kjer je kalup enojnovijačna oblika plazmida aU bakteriofaga, ki vsebuje nespremenjeno ali naravno sekvenco DNA mpl Uganda. Po hibridizaciji uporabimo polimerazo DNA za sintezo celotne druge komplementarne vijačnice kalupa, ki na ta način vgradi oUgonukleotidni primer in kodira za izbrano spremembo v DNA mpl Uganda.OUgonucleotide-mediated mutagenesis is the preferred process for the preparation of substitution, deletion and insertion variants of Ugandan DNA mpl. This technique is well known and is described by Adelman et al., DNA, 2: 183 [1983]. Briefly, DNA mpl of Uganda is altered by hybridizing an oUgonucleotide that encodes a desired mutation for a DNA mold, where the mold is a single-stranded form of the aU bacteriophage plasmid containing an unmodified or natural sequence of the Uganda DNA mpl. After hybridization, DNA polymerase is used to synthesize the entire second complementary helix of the mold, which thus embeds the oUgonucleotide primer and encodes for the selected change in the DNA mpl of Uganda.

Na splošno uporabimo oUgonukleotide z dolžino vsaj 25 nukleotidov. Optimalni oUgonukleotid ima 12 do 15 nukleotidov, ki so popolnoma komplementarni s kalupom na obeh straneh nukleotidov, ki kodirajo za mutacijo. To zagotavlja, da se oligonukleotid pravilno hibridizira s kalupom enojnovijačne molekule DNA. OUgonukleotide enostavno sintetiziramo z uporabo tehnik, znanih strokovnjakom, kot jih opisujejo Crea et al., Proč. Natl. Acad. Sci. ZDA, 75:5765 [1978].Generally, oUgonucleotides of at least 25 nucleotides in length are used. The optimal oUgonucleotide has 12 to 15 nucleotides that are completely complementary to the mold on either side of the nucleotides encoding for the mutation. This ensures that the oligonucleotide hybridizes properly with the mold of the single-stranded DNA molecule. OUgonucleotides are easily synthesized using techniques known to those skilled in the art as described by Crea et al., Et al. Natl. Acad. Sci. U.S.A. 75: 5765 [1978].

Kalup DNA lahko naredimo s tistimi vektoiji, ki so bodisi izvedeni iz vektorjev bakteriofaga M13 (komercialna dosegljiva vektorja M13mpl8 in M13mpl9 sta primerna), ali takih, ki vsebujejo fagni-enojnovijačni izvor replikacije, kot to opisujejo Viera et al., Meth. Enzymol., 153:3 [1987]. Na ta način DNA, ki jo je potrebno mutirati, lahko inseriramo v enega od teh vektorjev, da naredimo enojnovijačni kalup. Izdelava enojnovijačnega kalupa je opisana v odstavkih 4.21-4.41 avtorja Sambrooka et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, NY1989).DNA molding can be done with those vectors that are either derived from bacteriophage M13 vectors (commercially available vectors M13mpl8 and M13mpl9 are suitable), or those containing a phage-single-stranded replication source, as described by Viera et al., Meth. Enzymol., 153: 3 [1987]. In this way, the DNA to be mutated can be inserted into one of these vectors to make a single-stranded mold. The production of single-screw mold is described in paragraphs 4.21-4.41 by Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, NY1989).

Alternativno lahko naredimo kalup enojnovijačne DNA z denaturiranjem dvojnovijačnega plazmida (ali drugega) DNA z uporabo standardnih tehnik.Alternatively, a single-stranded DNA mold can be made by denaturing the double-stranded plasmid (or other) DNA using standard techniques.

Za spremembo sekvence naravne DNA (za izdelavo variant aminokislinske sekvence) oligonukleotid hibridiziramo z enojnovijačnim kalupom pri prikladnih pogojih za hibridizacijo. Nato dodamo DNA polimerizimi encim, navadno Klenow fragment DNA polimeraze I, da sintetiziramo komplementarno vijačnico kalupa z uporabo oligonukleotida kot primerja za sintezo. Na ta način se tvori heterodupleksna molekula, tako da ena vijačnica DNA kodira mutirano obliko mpl Uganda, druga vijačnica (originalni kalup) pa kodira naravno nespremenjeno sekvenco mpl Uganda. To heterodupleksno molekulo nato transformiramo v prikladno gostiteljsko ceUco, ki je navadno prokariot, kot npr. E. coli JM101. Potem ko ceUce zrastejo, jih zasadimo na agarozne plošče in selekcioniramo z uporabo oUgonukleotidnega primerja, radioktivno označenega z 32-fosfatom, da identificiramo bakterijske kolonije, ki vsebujejo mutirano DNA. Mutirano regijo nato odstranimo in damo v ustrezen vektor za izdelavo proteina, na splošno ekspresijski vektor tipa, ki se značilno uporablja za transformacijo ustreznega gostitelja.To alter the sequence of natural DNA (to produce amino acid sequence variants), the oligonucleotide is hybridized with a single-stranded mold under suitable hybridization conditions. An DNA polymerase enzyme, usually the Klenow DNA polymerase I fragment, is then added to synthesize the complementary helix of the mold using oligonucleotide as a primer for synthesis. In this way, a heteroduplex molecule is formed so that one helix of DNA encodes a mutated form of mpl Uganda, and another helix (original mold) encodes a naturally unchanged sequence of mpl Uganda. This heteroduplex molecule is then transformed into a suitable host ceUco, which is usually a prokaryote, such as e.g. E. coli JM101. After the ceUce have grown, they are seeded onto agarose plates and selected using an oUgonucleotide primer radiolabeled with 32-phosphate to identify bacterial colonies containing the mutated DNA. The mutated region is then removed and placed in a suitable vector for protein production, generally an expression vector of the type typically used to transform the corresponding host.

Postopek, opisan tik pred tem zgoraj, lahko modificiramo tako, da oblikujemo homodupleksno molekulo, v kateri obe vijačnici plazmida vsebujeta mutacije. Modifikacije so naslednje: enojnovijačni oUgonukleotid aneliramo na enojnovijačni kalup, kot je opisano zgoraj. Zmes treh deoksiribonukleotidov, deoksiriboadenozina (dATP), deoksiribogvanozina (dGTP) in deoksiribotimidina (dlTP) kombiniramo z modificiranim tiodeoksiribocitozinom, imenovanim dCTP-(aS) (ki ga lahko dobimo pri Amersham Corporation). To zmes dodamo k kalupnemu oUgonukleotidnemu kompleksu. Po dodatku polimeraze DNA k tej zmesi nastane vijačnica DNA, identična kalupu, razen za mutirane baze. Poleg tega ta nova vijačnica DNA vsebuje dCTP-(aS) namesto dCTP, ki jo varuje pred restrikcijsko endonukleazno digestijo.The procedure described immediately above can be modified to form a homoduplex molecule in which both plasmid helixes contain mutations. The modifications are as follows: The single-stranded oUgonucleotide is anelinated onto the single-stranded mold as described above. A mixture of three deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP) and deoxyribotimidine (dlTP) is combined with a modified thiodeoxyribocytosine called dCTP- (aS) (obtainable from Amersham Corporation). This mixture was added to the molded oUgonucleotide complex. Adding DNA polymerase to this mixture produces a DNA helix identical to the mold except for the mutated bases. In addition, this new DNA helix contains dCTP- (aS) instead of dCTP, which protects it from restriction endonuclease digestion.

Potem ko vijačnico kalupa dvojnovijačnega heterodupleksa prelomimo z ustreznim restrikcijskim encimom, lahko vijačnico kalupa digeriramo z nukleazo ΕχοΙΙΙ ali drugo ustrezno nukleazo zunaj regije, ki vsebuje mesta za mutagenezo. Reakcijo nato ustavimo, da ostane molekula, ki je le delno enojnovijačna. Popolni homodupleks dvojnovijačne DNA nato tvorimo z uporabo polimeraze DNA v prisotnosti vseh štirih deoksiribonukleotidnih trifosfatov ATP in ligaze DNA. To molekulo homodupleksa lahko nato transformiramo v prikladno gostiteljsko celico, kot je E. coli JM101, kot je opisano zgoraj.After breaking the helix of the double-stranded heteroduplex mold with an appropriate restriction enzyme, the helix of the mold can be digested with the ΕχοΙΙΙ nuclease or other appropriate nuclease outside the region containing the mutagenesis sites. The reaction is then stopped to remain a single-stranded molecule. The complete double-stranded DNA homoduplex is then formed using DNA polymerase in the presence of all four deoxyribonucleotide triphosphates ATP and DNA ligase. This homoduplex molecule can then be transformed into a suitable host cell, such as E. coli JM101, as described above.

DNA, ki kodira mutante mpl liganda, v katerih je potrebno substituirati več kot eno amino kislino, lahko izdelamo na različne načine. Če so amino kisline locirane blizu skupaj v polipeptidni verigi lahko mutirajo simultano z uporabo tistega oligonukleotida, ki kodira za vse želene aminokislinske substitucije. Če pa so amino kisline locirane v neki razdalji ena od druge (ločene z več kot približno 10 amino kislinami), je bolj težko, da naredimo posamezen oligonukleotid, ki kodira vse želene spremembe. Namesto tega lahko uporabimo enega od dveh alternativnih postopkov.DNA encoding mpl ligand mutants in which more than one amino acid needs to be substituted can be produced in various ways. If the amino acids are located close together in the polypeptide chain, they can mutate simultaneously using the oligonucleotide that codes for all the desired amino acid substitutions. However, if the amino acids are located at a distance from each other (separated by more than about 10 amino acids), it is more difficult to make a single oligonucleotide that encodes any desired changes. Instead, one of two alternative methods can be used.

V prvem postopku naredimo ločen oligonukleotid za vsako amino kislino, ki jo je treba substituirati. Oligonukleotide nato aneliramo simultano na enojnovijačni kalup DNA, druga vijačnica DNA, ki jo sintetiziramo iz kalupa, pa kodira vse želene aminokislinske substitucije.In the first process, a separate oligonucleotide is made for each amino acid to be substituted. The oligonucleotides are then anelinated simultaneously to a single-stranded DNA strand, and another strand of DNA synthesized from the strand encodes all the desired amino acid substitutions.

Alterantivni postopek vključuje dva ali več krogov mutageneze, da nastane želen mutant. Prvi krog je tak, kot je opisano za posamezne mutante: DNA divjega tipa uporabimo za kalup, oligonukleotid, ki kodira prvo želeno aminokislinsko substitucijo, aneliramo na ta kalup in nato nastane heterodupleksna molekula DNA. V drugem krogu mutageneze uporabimo mutirano DNA, narejeno v prvem krogu mutageneze kot kalup. Ta kalup že vsebuje eno ali več mutacij. Oligonukleotid, ki kodira dodatne želene aminokislinske substitucije nato aneliramo na ta kalup in tako sedaj nastala vijačnica DNA kodira mutacije, tako iz prvega kot tudi drugega kroga mutageneze. To nastalo DNA lahko uporabimo kot kalup v tretjem krogu mutageneze itd.An alternative procedure involves two or more rounds of mutagenesis to produce the desired mutant. The first round is as described for the individual mutants: wild-type DNA is used to mold, an oligonucleotide encoding the first desired amino acid substitution, anelated to that mold, and then a heteroduplex DNA molecule is formed. In the second round of mutagenesis, we use mutant DNA made in the first round of mutagenesis as a mold. This mold already contains one or more mutations. An oligonucleotide encoding the additional desired amino acid substitutions is then anelated to this mold and the resulting helix of DNA encodes mutations from both the first and second rounds of mutagenesis. This resulting DNA can be used as a mold in the third round of mutagenesis, etc.

Mutageneza PCR je tudi prikladna za izdelavo aminokislinskih variant mpl Ugandskega polipeptida. Čeprav je naslednja obravnava v zvezi z DNA, je razumljivo, da je tehnika uporabna tudi za RNA- Tehnika PCR je na splošno v zvezi z naslednjim postopkom (Erlich, zgoraj, poglavje od R. Higuchi, str. 61-70): kadar uporabimo majhne količine kalupne DNA kot izhodno snov v PCR, lahko uporabimo primeije, ki se v sekvenci malo razlikujejo od ustrezajoče regije v kalupni DNA, da izdelamo relativno velike količine fragmenta specifične DNA, ki se razlikuje od sekvence kalupa le v položajih, kjer se primeiji razlikujejo od kalupa. Za uvedbo mutacije v plazmidno DNA oblikujemo enega od primerjev tako, da prekriva položaj mutacije in vsebuje mutacijo; sekvenca drugega primerja mora biti identična odseku sekvence nasprotne vijačnice plazmida, vendar pa je ta sekvenca lahko locirana kjerkoli vzdolž plazmidne DNA. Prednostno je, da je sekvenca drugega primeija locirana znotraj 200 nukleotidov prvega primerja, tako da na koncu celotno pomnoženo regijo DNA, vezano s primeiji, z lahkoto sekvenciramo. Pomnoževanje PCR z uporabo para primeijev, takega kot je bil pravkar opisan, ima za posledico populacijo fragmentov DNA, ki je različna v položaju mutacije, ki jo specificira primer in možno na drugih položajih, kjer je kopiranje kalupa nekako nagnjeno k napakam.Mutagenesis of PCR is also suitable for the production of amino acid variants of the mpl Ugandan polypeptide. Although the following treatment is related to DNA, it is understood that the technique is also applicable to RNA- The PCR technique is generally related to the following procedure (Erlich, supra, chapter from R. Higuchi, pp. 61-70): when used small amounts of template DNA as the starting material in PCR, primers that differ slightly in sequence from the corresponding region in the template DNA can be used to produce relatively large amounts of a specific DNA fragment that differ from the mold sequence only in positions where primes differ of mold. To introduce a mutation into plasmid DNA, one of the primers is designed to cover the position of the mutation and to contain the mutation; the sequence of the second primer must be identical to the sequence sequence of the opposite helix of the plasmid, but this sequence may be located anywhere along the plasmid DNA. Preferably, the second primer sequence is located within 200 nucleotides of the first primer, so that the entire primer region-bound DNA region can be easily sequenced. Multiplication of PCR using a primer pair, such as that just described, results in a population of DNA fragments that is different in the mutation position specified by the case and possibly in other positions where the mold copying is somehow prone to error.

Če je razmeije kalupa proti izdelani snovi ekstremno nizko, velika večina fragmentov izdelane DNA vgradi želene mutacije. To izdelano snov uporabimo, da nadomestimo ustrezno regijo v plazmidu, ki rabi kot kalup PCR, z uporabo standardne tehnologije DNA. Mutacije na ločenih položajih lahko uvedemo simultano, bodisi z uporabo mutantnega drugega primeija ali izvajanjem druge PCR z različnimi mutantnimi primeiji in ligacijo obeh nastalih fragmentov PCR, simultano za vektorski fragment vIf the mold spacing against the manufactured substance is extremely low, the vast majority of fragments of manufactured DNA incorporate the desired mutations. This manufactured substance is used to replace the corresponding region in the plasmid used as a PCR mold using standard DNA technology. Mutations at separate positions can be introduced simultaneously, either by using a mutant second primer or by performing a second PCR with different mutant primers and ligation of both resulting PCR fragments simultaneously for the vector fragment in the

3- (ali več) delni ligaciji.3- (or more) partial ligation.

V specifičnem primeru mutageneze PCR, lineariziramo kalupno plazmidno DNA (1 jug) z digestijsko restrikcijsko endonukleazo, ki ima edinstveno spoznavno mesto v plazmidni DNA zunaj regije, ki jo je treba pomnožiti. Od te snovi dodamo 100 ng k zmesi PCR, ki vsebuje pufer PCR, ki vsebuje 4 deoksinukleotidne trifosfate in je vključen v kompletu GeneAmp® (dobavitelj Perkm-Elmer Cetus, Norwalk, CT in Emeryville, CA), in 25 pmol vsakega oligonukleotidnega primeija do končnega volumna 50 /ul. Reakcijsko zmes prekrijemo s 35 jul mineralnega olja. Reakcijsko zmes denaturiramo 5 minut pri 100 °C, damo za kratek čas na led in nato dodamo 1 jul Thermus aquaticus (Taq) polimeraze DNA (5 enot/jul, dobavitelj Perkin-Elmer Cetus) pod plast mineralnega olja.In the specific case of PCR mutagenesis, we linearize the plasmid plasmid DNA (1 south) with digestive restriction endonuclease, which has a unique recognition site in plasmid DNA outside the region to be amplified. From this substance, 100 ng was added to a PCR mixture containing PCR buffer containing 4 deoxynucleotide triphosphates and included in the GeneAmp® kit (supplier Perkm-Elmer Cetus, Norwalk, CT and Emeryville, CA), and 25 pmol of each oligonucleotide primer to of a final volume of 50 / ul. Cover the reaction mixture with 35 Jul mineral oil. The reaction mixture was denatured for 5 minutes at 100 ° C, placed on ice for a short time, and then Thermus aquaticus (Taq) DNA polymerase (5 units / July, supplier Perkin-Elmer Cetus) was added under a layer of mineral oil.

Reakcijsko zmes nato vložimo v napravo za toplotno termostatiranje DNA Thermal Cycler (dobavitelj Perkin-Elmer Cetus), programirano takole:The reaction mixture is then fed to a DNA Thermal Cycler (Perkin-Elmer Cetus supplier), programmed as follows:

min., 55 °C s, 72 °C, nato 19 ciklov nasalednje:min., 55 ° C with, 72 ° C, followed by 19 cycles of inheritance:

s, 94 °C s, 55 °C in s, 72 °C.s, 94 ° C s, 55 ° C and s, 72 ° C.

Na koncu programa reakcijsko fiolo odstranimo iz naprave za toplotno termostatiranje in vodno fazo prenesemo v novo fiolo, ekstrahiramo s fenolom/kloroformom (50:50 vol.), oborimo z etanolom in DNA rekuperiramo s standardnimi postopki. To snov nato izpostavimo ustreznim obdelavam za insercijo v vektor.At the end of the program, the reaction vial is removed from the thermal thermostat and the aqueous phase is transferred to a new vial, extracted with phenol / chloroform (50:50 vol.), Precipitated with ethanol and DNA recovered by standard procedures. This substance is then subjected to appropriate treatments for vector insertion.

Drugi postopek za pripravo variant kasetne mutageneze temelji na tehniki, ki jo opisujejo Wells et al., Gene, 34:315 [1985]. Izhodna snov je plazmid (ali drugi vektor), ki obsega DNA mpl liganda, ki jo je treba mutirati. Kodoni v DNA mpl liganda, ki jih je potrebno mutirati, so identificirani. Na vsaki strani indentificiranega mesta za mutacijo mora biti edinstveno restrikcijsko endonukleazno mesto. Če ni takih restrikcijskih mest, jih lahko naredimo z uporabo zgoraj opisane oligonukleotidno posredovane mutageneze, da jih uvedemo na primerna mesta v DNA mpl liganda. Po uvedbi restrikcijskih mest v plazmid, le-tega prerežemo na teh mestih, da ga lineariziramo. Dvojnovijačni oligonukleotid, ki kodira sekvenco DNA med restrikcijskima mestoma, vsebuje pa želene mutacije, sintetiziramo z uporabo standardnih postopkov. Obe vijačnici sintetiziramo ločeno in nato hibridiziramo skupaj z uporabo standardnih tehnik. Ta dvojnovijačni oligonukleotid imenujemo kaseta. Ta kaseta je oblikovana tako, da ima 3’ in 5’ konca, ki sta kompatibilna s konci lineariziranega plazmida, tako da jo lahko direktno legiramo na plazmid. Plazmid tako vsebuje mutirano sekvenco DNA mpl liganda.A second process for preparing variants of cluster mutagenesis is based on a technique described by Wells et al., Gene, 34: 315 [1985]. The starting material is a plasmid (or other vector) that comprises the DNA of the mpl ligand to be mutated. The codons in the DNA of the mpl ligand to be mutated are identified. There must be a unique restriction endonuclease site on each side of the identified mutation site. In the absence of such restriction sites, they can be made using the oligonucleotide-mediated mutagenesis described above to introduce them into suitable sites in the mpl ligand DNA. After introducing restriction sites into the plasmid, it is cut at these sites to linearize it. A double-stranded oligonucleotide encoding the DNA sequence between restriction sites and containing the desired mutations was synthesized using standard procedures. The two screws are synthesized separately and then hybridized together using standard techniques. This double-stranded oligonucleotide is called a cassette. This cassette is designed to have 3 'and 5' ends that are compatible with the ends of the linearized plasmid so that it can be directly alloyed to the plasmid. The plasmid thus contains a mutated DNA sequence of the mpl ligand.

C. Insercija nukleinske kisline v replikabilni vektorC. Nucleic acid insertion into a replicable vector

Nukleinsko kislino (npr. cDNA ali genomska DNA), ki kodira naravni mpl Ugandski polipeptid ali njegovo varianto, inseriramo v replikabilni vektor za nadaljnje kloniranje (pomnoževanje DNA) aU ekspresijo. Mnogi vektoiji so na voljo in izbira ustreznega vektoija je odvisna od tega, (1) aU ga bomo uporabiU za pomnoževanje DNA aU za ekspresijo DNA (2) od velikosti nukleinske kisline, ki bo inserirana v novi vektor in (3) od gostiteljske ceUce, ki bo transformirana z vektorjem. Vsak vektor vsebuje različne komponente, odvisne od njegove funkcije (pomnoževanje DNA ali ekspresija DNA) in gostiteljske celice, s katero je kompatibilen. Vektorske komponente na splošno vključujejo, vendar ne omejujoče, eno ali več od naslednjih: signalno sekvenco, izvor repUkacije, enega ali več markerskih genov, spodbujevalni element, promotor in transkripcijsko terminacijsko sekvenco.A nucleic acid (eg, cDNA or genomic DNA) encoding a natural mpl Ugandan polypeptide or variant thereof is inserted into a replicable vector for further cloning (DNA amplification) aU expression. Many vectors are available and the choice of a suitable vector depends on (1) aU to use it to amplify the DNA aU for DNA expression (2) from the size of the nucleic acid to be inserted into the new vector and (3) from the host ceUce. which will be transformed by a vector. Each vector contains different components depending on its function (DNA amplification or DNA expression) and the host cell with which it is compatible. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, a origin of repUction, one or more marker genes, a stimulus element, a promoter, and a transcriptional termination sequence.

(i) Komponenta signalna sekvenca(i) Signal sequence component

Mpl Ugand v smislu izuma lahko eksprimiramo, ne samo direktno, ampak tudi kot fuzijo s heterolognim poUpeptidom, prednostno s signalno sekvenco aU drugim poUpeptidom, ki ima specifično cepišče na N-terminalu zrelega proteina ali poUpep81 tida. Na splošno je signalna sekvenca lahko komponenta vektorja, ali je lahko del DNA mpl liganda, inseriranega v ta vektor. Izbrana heterologna signalna sekvenca naj bi bila tista, ki je spoznana in procesirana (t.j. cepljena s signalno peptidazo) od gostiteljske celice. Za prokariotske gostiteljske celice, ki ne spoznajo in ne procesirajo signalne sekvence mpl liganda, substituiramo signalno sekvenco s prokariotsko signalno sekvenco, izbrano npr. iz skupine alkalne fosfataze, penicilinaze, Ipp ali voditeljev toplotno stabilnih enteretoksinov II. Za izločanje kvasovk lahko naravno signalno sekvenco substituiramo npr. z invertazo kvasovk, alfa faktorjem ali kislinskimi fosfataznimi voditelji, C. albicans glukoamilaznim voditeljem (EP 362,179, obj. 4. aprila 1990) ali signalom, opisanim v WO 90/13646, obj. 15. novembra 1990. Pri ekspresiji celice sesalca je zadovoljiva naravna signalna sekvenca (t.j. presekvenca mpl liganda, ki normalno usmerja sekrecijo mpl liganda iz njegovih naravnih celic sesalca in vivo), čeprav so prikladne tudi druge signalne sekvence sesalca, kot npr. signalne sekvence iz drugih mpl Ugandskih poUpeptidov aU iz enakega mpl Uganda iz razUčnih živalskih vrst, signalne sekvence iz mpl Uganda in signalne sekvence iz izločenih poUpeptidov, enakih aU sorodnih vrst, kot tudi virusni sekretomi voditelji, npr. herpes simpleks gD signal.Mpl Ugand of the invention can be expressed not only directly but also as a fusion with a heterologous poUpeptide, preferably with the signal sequence aU another poUpeptide having a specific cleavage at the N-terminal of the mature protein or poUpep81 tide. In general, the signal sequence may be a component of the vector, or may be part of the DNA of the mpl ligand inserted into that vector. The heterologous signal sequence selected is intended to be one that is recognized and processed (i.e., vaccinated by signal peptidase) from the host cell. For prokaryotic host cells that do not recognize and process the mpl ligand signal sequence, we substitute the signal sequence with a prokaryotic signal sequence selected e.g. from the alkaline phosphatase group, penicillinase, Ipp or leaders of heat stable enteretoxins II. For yeast secretion, the natural signal sequence can be substituted e.g. by yeast invertase, alpha factor or acid phosphatase leaders, C. albicans glucoamylase leader (EP 362,179, obj. 4 April 1990), or the signal described in WO 90/13646, obj. November 15, 1990. Natural mammalian signal sequence (i.e., mpl ligand sequence that normally directs secretion of mpl ligand from its natural mammalian cells in vivo) is satisfactory for mammalian cell expression, although other mammalian signal sequences, such as e.g. signal sequences from other mpl Ugandan aUpeptides aU from the same mpl Uganda from wildlife species, signal sequences from mpl Uganda and signal sequences from secreted poUpeptides of the same aU related species, as well as viral secretome leaders, e.g. herpes simplex gD signal.

(n) Komponenta izvor replikacije(n) Replication source component

Tako ekspresijski kot tudi klonimi vektorji vsebujejo sekvenco nukleinske kisline, ki omogoča replikacijo vektorja v eno aU več izbranih gostiteljskih ceUc. Na splošno je v klonimih vektorjih ta sekvenca tista, ki omogoča replikacijo vektorja, neodvisno od gostiteljske kromosomske DNA, in vključuje izvore replikacije aU avtonomno replikativne sekvence. Take sekvence so dobro znane za različne vrste bakterij, kvasovk in virusov. Izvor replikacije iz plazmida pBR322 je prikladen za večino gram negativnih bakterij, izvor 2 μια plazmid je prikladen za kvasovke, razni virusni izvori (SV40, polioma, adenovirus, VSV ali BPV) pa so koristni za kloniranje vektorjev v ceUcah sesalcev. Na splošno komponenta - izvor replikacije - ni potrebna za ekspresijske vektorje sesalcev (izvor SV40 lahko značilno uporabimo le zato, ker vsebuje zgodnji promotor).Both expression and clone vectors contain a nucleic acid sequence that allows replication of the vector into one aU of multiple selected ceUc hosts. Generally, in clone vectors, this sequence is the one that enables the replication of the vector independently of the host chromosomal DNA and includes the sources of replication of the aU autonomously replicative sequence. Such sequences are well known for various types of bacteria, yeasts and viruses. The replication source from plasmid pBR322 is suitable for most gram-negative bacteria, the 2 μια plasmid source is suitable for yeast, and various viral sources (SV40, polio, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian ceUc. In general, the component - the origin of replication - is not required for mammalian expression vectors (the SV40 source can typically be used only because it contains an early promoter).

Večinoma so ekspresijski vektorji dvojni (shuttle), to je, sposobni so repUkacije vsaj enega razreda organizmov, vendar pa se lahko transfektirajo v drug organizem za ekspresijo. Npr. vektor je kloniran v E. coli in nato je isti vektor transfektiran v ceUcah kvasovk aU sesalca za ekspresijo, čeprav ni sposoben repUkacije, neodvisno od kromosoma gostiteljske celice.In most cases, expression vectors are double (shuttle), that is, capable of repuction of at least one class of organisms, but can be transfected into another organism for expression. E.g. the vector is cloned into E. coli and then the same vector is transfected into the yeast aU of the mammalian aU for expression, although it is incapable of repUction independent of the host cell chromosome.

DNA prav tako lahko pomnožimo z insercijo v gostiteljski genom. To z lahkoto izvedemo z uporabo vrste Baccilus kot gostitelja npr. tako, da vključimo v vektor sekvenco DNA, kije komplementarna sekvenci, ugotovljeni v genomski DNA Bacillusa. Transfekcija Bacillusa z drugim vektorjem ima za posledico homologno rekombinacijo z genomom in insercijo DNA mpl liganda. Vendar pa je rekuperiranje genomske DNA, ki kodira mpl ligand, bolj kompleksno kot tisto od eksogeno repliciranega vektorja, ker je potrebna restrikcijska encimska digestija, da izrežemo DNA mpl liganda.DNA can also be amplified by insertion into the host genome. This is easily accomplished using a Baccilus species as a host e.g. by incorporating into the vector a DNA sequence that is complementary to the sequences found in the genomic DNA of Bacillus. Transfection of Bacillus with another vector results in homologous recombination with the genome and insertion of the DNA mpl ligand. However, recovering genomic DNA encoding an mpl ligand is more complex than that of an exogenously replicated vector because restriction enzyme digestion is required to cut out the mpl ligand DNA.

(iii) Komponenta selekcijski gen(iii) Component selection gene

Ekspresijski in klonimi vektorji naj bi vsebovali selekcijski gen, tudi označen kot selektibilni marker. Ta gen kodira protein, potreben za preživetje ali rast transformiranih gostiteljskih celic, zraslih v selektivnem kulturnem mediju. Gostiteljske celice, ki niso transformirane z vektorjem, ki vsebuje selekcijski gen, ne preživijo v kulturnem mediju. Značilni selekcijski geni kodirajo proteine, ki (a) dajo odpornost proti antibiotikom ali drugim toksinom, npr. ampicilinu, neomicinu, metotreksatu ali tetraciklinu (b) dopolnijo avksotrofne pomankljivosti ali (c) oskrbijo kritična hranila, ki niso na voljo v kompleksnem mediju, kot je npr. gen kodirna D-alanin racemaza za Bacille.Expression and clone vectors are expected to contain a selection gene, also designated as a selectable marker. This gene encodes a protein required for the survival or growth of transformed host cells grown in a selective culture medium. Host cells that have not been transformed with the vector containing the selection gene do not survive in the culture medium. Characteristic selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline (b) supplement auxotrophic deficiencies, or (c) supply critical nutrients not available in complex media such as e.g. gene encoding D-alanine racemase for Bacille.

En primer selekcijske sheme uporablja zdravilo, da ustavi rast gostiteljske celice. Tiste celice, ki so uspešno transformirane s heterolognim genom, eksprimirajo protein, ki daje odpornost proti zdravilom, in na ta način preživijo selekcijski režim. Zgledi take dominantne selekcije uporabljajo zdravila: neomicin (Southern et al., J. Moleč. Appl. Genet., 1:327 [1982]), mikofenolno kislino (Mulligan et al., Science, 209:1422 [1980]) ali higromicin (Sugden et al., Mol. Celi. Biol., 5:410-413 [1985]). Trije zgledi, navedeni zgoraj, uporabljajo bakterijske gene ob evkariotski kontroli, da posredujejo odpornost za ustrezno zdravilo G418 ali neomicin (geneticin), xgpt (mikofenolna kislina) oz. higromicin.One example of a selection scheme uses a drug to stop the growth of the host cell. Those cells that are successfully transformed with the heterologous gene express a drug that confer resistance to the drug and thus survive the selection regimen. Examples of such dominant selection utilize drugs: neomycin (Southern et al., J. Molec. Appl. Genet., 1: 327 [1982]), mycophenolic acid (Mulligan et al., Science, 209: 1422 [1980]), or hygromycin. (Sugden et al., Mol. Celi. Biol., 5: 410-413 [1985]). The three examples listed above use bacterial genes under eukaryotic control to mediate resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or. hygromycin.

Zgledi drugih prikladnih selektibilnih marketjev za celice sesalcev so tisti, ki omogočajo identifikacijo celic, kompetentnih, da navzamejo nukleinsko kislino mpl liganda, kot je npr. dehidrofolat reduktaza (DHFR) ali timidin kinaza. Transformante celic sesalcev damo pod selekcijski pritisk, tako da so transformantni edini, ki so adaptirani za preživetje zaradi tega, ker so navzeli marker. Selekcijski pritisk uvedemo s kultiviranjem transformantov pri pogojih, pri katerih se koncentracija selekcijskega sredstva v mediju zaporedno spreminja, kar vodi do pomnožitve tako selekcijskega gena kot tudi DNA, ki kodira mpl Ugandski polipeptid. Pomnožitev je postopek, pri katerem se geni v večji zahtevi za izdelovanje proteina, kritični za rast, ponavljajo v tandemu v kromosomih zaporednih generacij rekombinantnih ceUc. Povečane koUčine mpl Uganda sintetiziramo iz pomnožene DNA.Examples of other suitable selectable mammalian cell markets are those that enable the identification of cells competent to harbor the mpl ligand nucleic acid, such as e.g. dehydrofolate reductase (DHFR) or thymidine kinase. Transformants of mammalian cells are put under selection pressure, so that transformants are the only ones that are adapted to survive because they are a marker. Selection pressure is introduced by culturing the transformants under conditions in which the concentration of the selection agent in the medium changes successively, leading to amplification of both the selection gene and the DNA encoding the mpl Ugandan polypeptide. Multiplication is a process in which genes in a major requirement for the production of a protein critical for growth are replicated in tandem in the chromosomes of successive generations of recombinant ceUc. Increased amounts of mpl Uganda are synthesized from DNA amplified.

Npr. ceUce, transformirane s selekcijskim genom DHFR, najprej identificiramo s kultiviranjem vseh transformantov v mediju kulture, ki vsebuje metotreksat (Mtx), konkurenčni antagonist DHFR. Ustrezna gostiteljska ceUca, kadar uporabimo DHFR divjega tipa, je ovarijska ceUčna linija kitajskega hrčka (celična Unija CHO) s pomanjkanjem aktivnosti DHFR, pripravljena in propargirana, kot opisujeta Urlaub in Chasin, Proč. Natl. Acad. Sci. ZDA, 77:4216 [1980]. Transformirane ceUce nato eksponiramo povečanim nivojem Mtx. To vodi do sinteze multiplih kopij gena DHFR in istočasno multiplih kopij druge DNA, ki obsega ekspresijske vektorje, kot npr. DNA, ki kodira mpl Ugand. To tehniko pomnoževanja lahko uporabimo z vsakim, sicer prikladnim gostiteljem, npr. ATCC št. CCL61 CHO-K1, kljub prisotnosti endogenega DHFR, če npr. uporabimo mutantni gen DHFR, ki je zelo odporen proti Mtx (EP 117.060). Alternativno lahko gostiteljske ceUce [posebno gostiteljske ceUce divjega tipa, ki vsebujejo endogeni DHFR] transformirane aU sotransformirane s sekvencami DNA, ki kodirajo mpl Ugand, protein DHFR divjega tipa in drug selektibilen marker, kot npr. aminoglikozid 3’ fosfotransferazo (APH), selekcioniramo s celično rastjo v mediju, ki vsebuje selekcijsko sredstvo za selektibilni marker, kot je amino gUkozidni antibiotiki, npr. kanamicin, neomicin aU G418. Glej US patent št. 4,965,199.E.g. ceUce transformed with the DHFR selection gene is first identified by culturing all the transformants in a culture medium containing methotrexate (Mtx), a competitive DHFR antagonist. A suitable host ceUca, when wild-type DHFR is used, is a Chinese hamster ovary tubular (CHO cellular) ovarian tube with a lack of DHFR activity, prepared and parparagized as described by Urlaub and Chasin, Off. Natl. Acad. Sci. U.S.A. 77: 4216 [1980]. The transformed ceUce is then exposed to increased levels of Mtx. This leads to the synthesis of multiple copies of the DHFR gene and at the same time multiple copies of another DNA comprising expression vectors, such as e.g. DNA encoding Ugandan mpl. This multiplication technique can be used with any otherwise convenient host, e.g. ATCC no. CCL61 CHO-K1, despite the presence of endogenous DHFR, if e.g. we use the mutant DHFR gene, which is highly resistant to Mtx (EP 117.060). Alternatively, host ceUce [especially wild-type host ceUce containing endogenous DHFR] may be transformed aU co-transformed with DNA sequences encoding Ugand mpl, wild-type DHFR protein and other selectable marker, such as e.g. aminoglycoside 3 'phosphotransferase (APH), is selected by cell growth in a medium containing a selectable marker agent, such as amino glycosidic antibiotics, e.g. kanamycin, neomycin aU G418. See U.S. Pat. No. 4,965,199.

Prikladen selekcijski gen za uporabo pri kvasovkah je gen trpi, prisoten v plazmidu kvasovk YRp7 (Stinchcomb et al., Nature, 282:39 [1979]; Kingsman et al., Gene, 7:141 [1979]; aU Tschemper et al., Gene, 10:157 [1980]). Gen trpi zagotavlja selekcijski marker za mutantni sev kvasovk, ki niso sposobne, da rastejo v triptofanu, npr. ATCC št. 44076 ali PEP4-1 (Jones, Genetics, 85:12 [1977]). Prisotnost lezije trpi v genomu gostiteljskih kvasovk nato zagotovi efektivno okolje za detektiranje transformacije z rastjo v odsotnosti triptofana. Podobno komplementiramo seve kvasovk s pomanjkanjem Leu2 (ATCC št. 20,622 ali 38,626) z znanimi plazmidi, ki nosijo gen Leu2.A suitable selection gene for use in yeast is the suffering gene present in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282: 39 [1979]; Kingsman et al., Gene, 7: 141 [1979]; aU Tschemper et al. , Gene, 10: 157 [1980]. The sufferer gene provides a selection marker for a mutant yeast strain unable to grow in tryptophan, e.g. ATCC no. 44076 or PEP4-1 (Jones, Genetics, 85:12 [1977]). The presence of the lesion suffers in the host yeast genome then provides an effective environment for detecting growth transformation in the absence of tryptophan. Similarly, we complement Leu2-deficient yeast strains (ATCC No 20,622 or 38,626) with known plasmids carrying the Leu2 gene.

(iv) Komponenta promotor(iv) Promoter component

Ekspresijski in klonimi vektorji navadno vsebujejo promotor, ki ga spozna gostiteljski organizem in je funkcionalno vezan na nukleinsko kislino mpl liganda. Promotoiji so netranslatirane sekvence, locirane navzgor (5’) od začetnega kodona strukturnega gena (na splošno znotraj približno 100 do 1000 bp), ki kontrolirajo transkripcijo in translacijo posebne sekvence nukleinske kisline, kot npr. sekvenco nukleinske kisline mpl liganda, na katero so funkcionalno vezani. Taki promotoiji značilno spadajo v dva razreda, inducibilnega in konstitutivnega. Inducibilni promotoiji so tisti, ki inicirajo povečane nivoje transkripcije iz DNA ob njihovi kontroli odziva na nekatere spremembe v pogojih kulture, npr. prisotnost ali odsotnost hranila ali spremembe temperature. Sedaj je znanih veliko število promotoijev, ki spoznajo različne potencialne gostiteljske celice. Te promotorje funkcionalno vežemo na DNA, ki kodira mpl ligand, z odstranitvijo promotoija iz vira DNA z restrikcijsko encimsko digestijo in inseriranjem izolirane promotorske sekvence v vektor. Tako promotorsko sekvenco naravnega mpl liganda kot tudi mnoge heterologne promotorje lahko uporabimo za direktno pomnožitev in/ali ekspresijo DNA mpl liganda. Vendar so prednostni heterologni promotoiji, ker na splošno dopuščajo večjo transkripcijo in višje dobitke eksprimiranega mpl liganda v primerjavi s promotoijem naravnega mpl liganda.Expression and clone vectors typically contain a promoter that is recognized by the host organism and is functionally bound to the nucleic acid of the mpl ligand. Promotions are untranslated sequences located upstream (5 ') from the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription and translation of a specific nucleic acid sequence, such as. the nucleic acid sequence of the mpl ligand to which they are functionally bound. Such promotions typically fall into two classes, inducible and constitutive. Inducible promotions are those that initiate increased levels of transcription from DNA while controlling their response to some changes in culture conditions, e.g. presence or absence of nutrient or temperature change. A large number of promoters are now known to learn about different potential host cells. These promoters bind functionally to the DNA encoding the mpl ligand by removing the promoter from the DNA source by restriction enzyme digestion and inserting the isolated promoter sequence into the vector. Both the promoter sequence of the natural mpl ligand and many heterologous promoters can be used to directly amplify and / or express the DNA of the mpl ligand. However, heterologous promotions are preferred because they generally allow for greater transcription and higher yields of the expressed mpl ligand compared to the promoter of the natural mpl ligand.

Promotoiji, prikladni za uporabo v prokariotskih gostiteljih, vključujejo betalaktamazo in laktozne promotorske sisteme (Chang et al., Nature, 275:615 [1978]; in Goeddel et al., Nature, 281:544 [1979]), alkalno fosfatazo, triptofanski (trp) promotorski sistem (Goeddel, Nucleic Acids Res., 8:4057 [1980] in EP 36,776) in hibridne promotorje, kot npr. tac promotor (deBoer et al., Proč. Natl. Acad. Sci. ZDA, 80:21-25 [1983]). Primerni pa so tudi drugi bakterijski promotoiji. Njihove nukleotidne sekvence so objavljene, kar omogoča strokovnjakom, da jih funkcionalno ligirajo na DNA, ki kodira mpl ligand (Siebenlist et al., Celi, 20:269 [1980]), z uporabo linkerjev ah adapteijev za uvedbo katerihkoli potrebnih restrikcijskih mest. Promotoiji za uporabo v bakterijskih sistemih vsebujejo tudi sekvenco Shine-Dalgamo (S.D.), funkcionalno vezano na DNA, ki kodira mpl ligandski polipeptid.Promotions suitable for use in prokaryotic hosts include beta-lactamase and lactose promoter systems (Chang et al., Nature, 275: 615 [1978]; and Goeddel et al., Nature, 281: 544 [1979]), alkaline phosphatase, tryptophan (trp) promoter system (Goeddel, Nucleic Acids Res. 8: 4057 [1980] and EP 36,776) and hybrid promoters, such as e.g. tac promoter (deBoer et al., read. Natl. Acad. Sci. USA, 80: 21-25 [1983]). Other bacterial promotions are also suitable. Their nucleotide sequences are published, allowing experts to functionally ligate them to DNA encoding the mpl ligand (Siebenlist et al., Celi, 20: 269 [1980]), using linkers ah adaptations to introduce any necessary restriction sites. Promotions for use in bacterial systems also contain a Shine-Dalgamo (S.D.) sequence functionally linked to DNA encoding the mpl ligand polypeptide.

Promotorske sekvence so znane za evkariote. Dejansko imajo vsi evkariotski geni regijo, bogato z AT, locirano približno 25 do 30 baz navzgor od mesta, kjer se začne transkripcija. Druga sekvenca, ugotovljena 70 do 80 baz navzgor od začetka transkripcije mnogih genov, je regija CXCAAT, kjer je lahko X katerikoli nukleotid. Na 3’ koncu večine evkariotskih genov je sekvenca AATAAA, ki je lahko signal za dodatek poli A repa na 3’ koncu kodirne sekvence. Vse te sekvence so prikladno inserirane v evkariotske ekspresijske vektorje.The promoter sequences are known for eukaryotes. In fact, all eukaryotic genes have an AT-rich region located about 25 to 30 bases upstream of where transcription begins. Another sequence identified 70 to 80 bases upstream of the start of transcription of many genes is the CXCAAT region, where X can be any nucleotide. At the 3 'end of most eukaryotic genes is the AATAAA sequence, which may be a signal for the addition of a poly A tail at the 3' end of the coding sequence. All these sequences are conveniently inserted into eukaryotic expression vectors.

Primeri prikladnih promovimih sekvenc za uporabo z gostitelji kvasovk vključujejo promotorje za 3-fosfoglicerat kinazo (Hitzeman et al., J. Biol. Chem., 255:2073 [1980]) ali druge glikolitične encime (Hess et al., J. Adv. Enzyme Reg., 7:149 [1968]); in Holland, Biochemistry, 17:4900 [1978]), kot npr. enolazo, gliceraldehid-3-fosfat dehidrogenazo, heksokinazo, piruvatno dekarboksilazo, fosfofruktokinazo, glukoza-6-fosfat izomerazo, 3-fosfoglicerat mutazo, piruvat kinazo, triosefosfat izomerazo, fosfoglukoza izomerazo in glukokinazo.Examples of suitable promoter sequences for use with yeast hosts include promoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem., 255: 2073 [1980]) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg., 7: 149 [1968]); and Holland, Biochemistry, 17: 4900 [1978]), e.g. enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, isomerase glucose phosphomer isomerase

Drugi promotoiji kvasovk, ki so inducibilni promotorji z dodatno prednostjo za transkripcijo, kontrolirano z rastnimi pogoji, so promotorske regije za alkohol dehidrogenazo 2, izocitokrom C, kislinsko fosfatazo, razgradne encime, povezane z metabolizmom dušika, metalotionein, gliceraldehid-3-fosfat dehidrogenazo in encime, odgovorne za uporabo maltoze in galaktoze. Prikladne vektorje in promotorje za uporabo v ekspresiji kvasovk opisujejo Hitzeman et al., EP 73,657A Spodbujevalce kvasovk prav tako prednostno uporabimo s promotorji kvasovk.Other yeast promoters, which are inducible promoters with an added advantage for growth-controlled transcription, are promoter regions for alcohol dehydrogenase 2, isocytocrom C, acid phosphatase, degradation enzymes related to nitrogen metabolism, metalothionein, glyceraldehyde-3-phosphate dehydrogenase and enzymes responsible for the use of maltose and galactose. Suitable vectors and promoters for use in yeast expression are described by Hitzeman et al., EP 73,657A Yeast promoters are also preferably used with yeast promoters.

Transkripcija mpl liganda iz vektorjev gostiteljskih celic sesalcev je kontrolirana npr. s promotorji, dobljenimi iz genomov virusov, kot so npr.: polioma virus, ptičji poxvirus, (VB 2,211,504, obj. 5. julija 1989), adenovirus (kot npr. Adenovirus 2), goveji papiloma virus, avian virus sarkoma, citomegalovirus, retrovirus, virus hepatitis-B in najbolj prednostno Simian Virus 40 (SV40) iz heterolognih promotorjev sesalcev, kot je npr. aktinski promotor ah imunoglobulinski promotor, iz promotorjev toplotnega šoka in promotorjev, ki so normalno povezani s sekvenco mpl liganda, pod pogojem, da so le-ti kompatibilni s sistemi gostiteljskih celic.The transcription of mpl ligand from mammalian host cell vectors is controlled e.g. with promoters derived from genome viruses such as: polio virus, avian poxvirus, (UK 2,211,504, obj. July 5, 1989), adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis-B virus, and most preferably Simian Virus 40 (SV40) from heterologous mammalian promoters such as e.g. actin promoter ah immunoglobulin promoter, from heat shock promoters and promoters normally associated with the mpl ligand sequence, provided that they are compatible with host cell systems.

Zgodnje in pozne promotorje virusa SV40 ugodno dobimo kot restrikcijski fragment SV40, ki tudi vsebuje virusni (SV40) izvor replikacije. Fiers et al., Nature, 273:113 [1978]; Muligan in Berg, Science, 209:1422-1427 [1980]; Pavlakis et al., Proč. Natl. Acad. Sci. ZDA, 78:7398-7402 [1981]. Neposredni zgodnji promotor humanega citomegalovirusa ugodno dobimo kot restrikcijski fragment Hindlll E Greenaway et al., Gene, 18:355-360 [1982]. Sistem za ekspresijo DNA v gostiteljih sesalcih z uporabo govejega virusa papiloma kot vektorja je prikazan v US patentu št. 4,419,446. Modifikacija tega sistema je opisana v US patentu št. 4,601,978. Glej tudi Gray et al., Nature, 295:503-508 [1982] za ekspresijo cDNA, ki kodira imuni inter86 feron v opičjih celicah; Reyes et al., Nature, 297:598-601[1982] za ekspresijo cDNA humanega jS-interferona v mišjih celicah ob kontroli promotorja timidin kinaze iz virusa herpes simpleks; Canaani in Berg, Proč. Natl. Acad. Sci. ZDA, 79:5166-5170 [1982] za ekspresijo gena humanega interferona βΐ v kultiviranih celicah miši in zajcev; in Gorman et al., Proč. Natl. Acad. Sci. ZDA, 79:6777-6781 [1982] za ekspresijo sekvenc bakterijskih CAT v opičjih ledvičnih celicah CV-1, piščančjih embrionalnih fibroblastih, ovarijskih celicah kitajskega hrčka, celicah HeLa in mišjih celicah NIH-3T3 z uporabo Rousovega virusa sarkoma - dolgo terminalno ponavljanje - kot promotorja.Early and late promoters of the SV40 virus are advantageously obtained as a restriction fragment of the SV40, which also contains a viral (SV40) origin of replication. Fiers et al., Nature, 273: 113 [1978]; Muligan and Berg, Science 209: 1422-1427 [1980]; Pavlakis et al., Proc. Natl. Acad. Sci. U.S.A. 78: 7398–7402 [1981]. The immediate early promoter of human cytomegalovirus is advantageously obtained as a restriction fragment by Hindlll E Greenaway et al., Gene, 18: 355-360 [1982]. A system for expressing DNA in mammalian hosts using bovine papilloma virus as a vector is shown in U.S. Pat. No. 4,419,446. A modification of this system is described in U.S. Pat. No. 4,601,978. See also Gray et al., Nature, 295: 503-508 [1982] for expression of cDNA encoding immune inter86 ferrons in monkey cells; Reyes et al., Nature, 297: 598-601 [1982] for expression of human jS-interferon cDNA in mouse cells upon control of the herpes simplex virus thymidine kinase promoter; Canaani and Berg, Away. Natl. Acad. Sci. USA, 79: 5166-5170 [1982] for human interferon βΐ gene expression in cultured mouse and rabbit cells; and Gorman et al., ch. Natl. Acad. Sci. USA, 79: 6777-6781 [1982] for expression of bacterial CAT sequences in CV-1 monkey kidney cells, chicken embryonic fibroblasts, Chinese hamster ovary cells, HeLa cells and NIH-3T3 mouse cells using Rous's sarcoma virus - long terminal recurrence - as a promoter.

(v) Komponenta spodbujevalni element(v) Incentive component

Transkripcijo DNA, ki kodira mpl ligand v smislu izuma, z višjimi evkarioti pogosto povečamo z inseriranjem spodbujevalne sekvence v vektor. Spodbujevalci so cis delujoči elementi DNA, ki imajo navadno približno 10 do 300 bp, ki delujejo na promotor tako, da povečajo njegovo transkripcijo. Spodbujevalce, relativno neodvisne od orientacije in položaja, so ugotovili 5’ (Laimins et al., Proč. Natl. Acad. Sci. ZDA, 78:993 [1981]) in 3’ (Lusky et al., Mol. Celi Bio., 3:1108 [1983]) glede na transkripcijsko enoto v intronu (Banerji et al., Celi, 33:729 [1983]) kot tudi v sami kodirni sekvenci (Osborne et al., Mol. Celi Bio., 4:1293 [1984]). Mnoge sekvence spodbujevalcev so znane iz genov sesalcev (globin, elastaza, albumin, a-fetoprotein in insulin). Značilno lahko uporabimo spodbujevalce iz evkariotskega celičnega virusa. Primeri vključujejo spodbujevalec SV40 na pozni strani replikacijskega izvora (bp 100-270), spodbujevalec citomegalovirusnega zgodnjega promotorja, spodbujevalec polioma na pozni strani replikacijskega izvora in adenovirusne spodbujevalce. Glej tudi Yaniv, Nature, 297:17-18 [1982] za spodbujevalne elemente za aktivacijo evkariotskih promotoijev. Spodbujevalec lahko spojimo v vektor na položaju 5’ ali 3’ sekvence, ki kodira mpl ligand, vendar pa je prednostno lociran na mestu 5’ od promotorja.The transcription of DNA encoding the mpl ligand of the invention is often increased by higher eukaryotes by inserting the stimulus sequence into the vector. The promoters are cis-acting DNA elements, typically having about 10 to 300 bp, that act on the promoter by enhancing its transcription. Promoters relatively independent of orientation and position were identified 5 '(Laimins et al., Off. Natl. Acad. Sci. USA, 78: 993 [1981]) and 3' (Lusky et al., Mol. Whole Bio. , 3: 1108 [1983]) with respect to the transcriptional unit in the intron (Banerji et al., Celi, 33: 729 [1983]) as well as in the coding sequence itself (Osborne et al., Mol. Celi Bio., 4: 1293 [1984]. Many enhancer sequences are known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Typically, eukaryotic cell virus promoters may be used. Examples include the SV40 promoter on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter promoter, the polio promoter on the late side of the replication origin, and adenoviral promoters. See also Yaniv, Nature, 297: 17-18 [1982] for stimulus elements for activation of eukaryotic promoters. The promoter can be fused into a vector at the position 5 'or 3' of the sequence encoding the mpl ligand, but is preferably located at position 5 'from the promoter.

(vi) Komponenta transkripcijska terminacija(vi) Transcriptional termination component

Ekspresijski vektorji, uporabljeni v evkariotskih gostiteljskih celicah (kvasovke, glive, insekti, rastline, živali, ljudje ali nukleirane celice iz drugih multiceličnih organizmov), tudi vsebujejo sekvence, potrebne za terminacijo transkripcije in za stabiliziranje mRNA. Take sekvence so navadno dosegljive od 5’ in priložnostno 3’ netranslatiranih regij evkariotskih ali virusnih DNA ali cDNA. Te regije vsebujejo nukleotidne segmente, transkribirane kot poliadenilirane fragmente v netranslatiranem delu mRNA, ki kodira mpl ligand.Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms) also contain the sequences required for transcription termination and mRNA stabilization. Such sequences are typically reachable from 5 'and occasionally 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mpl encoding the mpl ligand.

(vii) Konstrukcija in analiza vektorjev(vii) Construction and analysis of vectors

Za konstrukcijo prikladnih vektorjev, ki vsebujejo eno ali več zgoraj navedenih komponent, uporabljamo standardne tehnike ligacije. Izolirane plazmide ali fragmente DNA cepimo, ukrojimo in ponovno ligiramo v želeno obliko, da tvorimo potrebne plazmide.Standard ligation techniques are used to construct suitable vectors containing one or more of the above components. Isolated plasmids or DNA fragments are cleaved, cut and re-ligated to the desired form to form the required plasmids.

Za analizo potrditve pravilnih sekvenc v konstruiranih plazmidih uporabljamo ligacijske zmesi, da transformiramo E. coli K12 sev 294 (ATCC št. 31,446) in izberemo uspešne transformante glede na ampicilinsko ali tetraciklinsko odpornost, kjer je potrebno. Plazmide iz transformantov pripravimo in analiziramo z restrikcijsko endonukleazno digestijo ali sekvenciramo po postopku od Messinga et al., Nucleic Acids Res., 9:309 [1981] ali po postopku od Maxama et al., Methods in Enzymology, 65:499 [1980].Ligation mixtures are used to analyze the validation of the correct sequences in engineered plasmids to transform E. coli K12 strain 294 (ATCC No. 31,446) and select successful transformants according to ampicillin or tetracycline resistance where necessary. Plasmids from transformants are prepared and analyzed by restriction endonuclease digestion or sequenced according to the procedure from Messing et al., Nucleic Acids Res., 9: 309 [1981] or following the procedure from Maxama et al., Methods in Enzymology, 65: 499 [1980] .

(viii) Vektorji za prehodno ekspresijo(viii) Transient expression vectors

Posebno uporabni pri izvajanju predloženega izuma so ekspresijski vektorji, ki zagotovijo prehodno ekspresijo DNA, ki kodira mpl Ugandski poUpeptid v ceUcah sesalca. Na splošno prehodna ekspresija vključuje uporabo ekspresijskega vektoija, ki je sposoben, da se učinkovito replicira v gostiteljski ceUci, tako da gostiteljska ceUca akumulira mnogo kopij ekspresijskega vektoija in v nadaljevanju sintetizira visoke nivoje želenega poUpeptida, kodiranega z ekspresijskim vektorjem. Sambrook et al., zgoraj, str. 16.17-16.22. Prehodni ekspresijski sistemi, ki obsegajo prikladen ekspresijski vektor in gostiteljsko celico, dopuščajo prikladno pozitivno identifikacijo poUpeptidov, kodiranih s kloniranimi DNA, kot tudi hitro selekcioniranje takih poUpeptidov za želene biološke aU fiziološke lastnosti. Tako so prehodni ekspresijski sistemi posebno uporabni v smislu izuma za namene identificiranja analogov in variant mpl Ugandskega poUpeptida, ki ima biološko aktivnost mpl Ugandskega poUpeptida.Particularly useful in the implementation of the present invention are expression vectors that provide transient expression of DNA encoding the mpl Ugandan poUpeptide in the ceUc of a mammal. Generally, transient expression involves the use of an expression vector that is capable of being effectively replicated in the host ceUci, such that the host ceUca accumulates many copies of the expression vector and subsequently synthesizes high levels of the desired poUpeptide encoded by the expression vector. Sambrook et al., Supra, p. 16.17-16.22. Transient expression systems, comprising a suitable expression vector and a host cell, allow convenient positive identification of cloned DNA-encoded poUpeptides as well as rapid selection of such poUpeptides for desired biological aU physiological properties. Thus, transient expression systems are particularly useful in the invention for the purpose of identifying analogs and variants of mpl Ugandan poUpeptide having the biological activity of mpl Ugandan poUpeptide.

(ix) Prikladni zgledni vektoiji celic vretenčarjev(ix) Appropriate exemplary vertebrate cell vectors

Druge postopke, vektoije in gostiteljske ceUce, prikladne za adaptacijo za sintezo mpl liganda v rekombinantni celični kulturi vretenčarjev, opisujejo Gething et al., Nature, 293:620-625 [1981]; Mantei et al., Nature, 281:40-46 [1979]; Levinson et al.;Other methods, vectors, and host ceUce suitable for adaptation for mpl ligand synthesis in recombinant vertebrate cell culture are described by Gething et al., Nature, 293: 620-625 [1981]; Mantei et al., Nature, 281: 40-46 [1979]; Levinson et al .;

EP 117,060; in EP 117,058. Posebno koristen plazmid za ekspresijo mpl liganda celične kulture sesalca je pRK5 (EP 307,247, US patent št. 5,258,287) ali pSVI6B (PCT št. objave WO 91/08291).EP 117,060; and EP 117,058. A particularly useful plasmid for expression of the mammalian cell culture ligand mpl is pRK5 (EP 307,247, U.S. Patent No. 5,258,287) or pSVI6B (PCT Publication No. WO 91/08291).

D. Selekcija in transformacija gostiteljskih celicD. Selection and transformation of host cells

Prikladne gostiteljske celice za kloniranje ali ekspresijo vektorjev so tukaj prokarioti, kvasovke ali višje evkariotske celice, opisane zgoraj. Prikladni prokarioti vključujejo bakterije, kot gram-negativne ali gram-pozitivne organizme, npr. E. coli, Bacille, kot npr. B. subtilis, vrsto Pseudomonas, kot npr. P. aeruginosa, Salmonella typhimurium, ali Serratia marcescans. Prednostni E. coli klonimi gostitelj je E. coli 294 (ATCC št. 31,446), čeprav so primerni tudi drugi sevi, kot npr. E. coli B, E. coli Χ1776 (ATCC št. 31,537) in E. coli W3110 (ATCC št. 27,325). Ti zgledi so bolj ilustrativni kot pa omejujoči. Prednostno naj bi gostiteljska celica izločala minimalne količine proteolitičnih encimov. Alternativno so prikladni postopki kloniranja in vitro npr. PCR ali druge reakcije polimeraznih nukleinskih kislin.Suitable host cells for cloning or expression of vectors are herein the prokaryotes, yeasts or higher eukaryotic cells described above. Suitable prokaryotes include bacteria such as gram-negative or gram-positive organisms, e.g. E. coli, Bacille, e.g. B. subtilis, a species of Pseudomonas, as e.g. P. aeruginosa, Salmonella typhimurium, or Serratia marcescans. The preferred E. coli clone host is E. coli 294 (ATCC No. 31,446), although other strains such as e.g. E. coli B, E. coli Χ1776 (ATCC No. 31,537), and E. coli W3110 (ATCC No. 27,325). These examples are more illustrative than restrictive. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. Alternatively, in vitro cloning methods, e.g. PCR or other polymerase nucleic acid reactions.

Poleg prokariotov so evkariotski mikrobi, kot npr. filamentne glive ali kvasovke, tudi prikladni gostitelji za vektorje, ki kodirajo mpl ligand. Saccharomyces cerevisiae ali splošni pekovski kvas je najbolj splošno uporabljen med nižjimi evkariotskimi gostiteljskimi mikroorganizmi. Vendar pa v smislu izuma lahko uporabimo tudi druge rodove, vrste in seve, ki so splošno dosegljivi in uporabni, kot so npr. Schizosaccharomyces pombe (Beach in Nurse, Nature, 290:140 [1981]; EP 139,383, obj. 2. maja 1985), gostitelji Kluyveromyces (US patent št. 4,943,529), kot npr. K. lactis (Louvencourt et al., J. Bacteriol., 737 [1983]), K. fragilis, K. bulgaricus, K. thermotolerans in K. maraanus, yarrowia [EP 402,226], Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278 [1988]), Candida, Trichoderma reesia (EP 244,234), Neurospora crassa (Čase et al., Proč. Natl. Acad. Sci. ZDA, 76:5259-5263 [1979]), in filamentne glive, kot npr. Neurospora, Penicillium, Tolypocladium (WO 91/00357, obj. 10. januarja 1991), in gostitelji Aspergillus, kot npr. A. nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 112:284-289 [1983]; Tilbum et al., Gene, 26:205-221 [1983]; Yelton et al., Proč. Natl. Acad. Sci. ZDA, 81:1470-1474 [1984]) in A. niger (Kelly in Hynes, EMBO J., 4:475-479 [1985]).In addition to prokaryotes, there are eukaryotic microbes, such as. filamentous fungi or yeasts, also suitable hosts for vectors encoding the mpl ligand. Saccharomyces cerevisiae or general baker's yeast is most commonly used among lower eukaryotic host microorganisms. However, other genera, species and strains that are generally available and useful, such as e.g. Schizosaccharomyces pombe (Beach in Nurse, Nature, 290: 140 [1981]; EP 139,383, published May 2, 1985), hosts Kluyveromyces (U.S. Patent No. 4,943,529), such as e.g. K. lactis (Louvencourt et al., J. Bacteriol., 737 [1983]), K. fragilis, K. bulgaricus, K. thermotolerans and K. maraanus, yarrowia [EP 402,226], Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28: 265-278 [1988], Candida, Trichoderma reesia (EP 244,234), Neurospora crassa (Chase et al., Natl. Acad. Sci. USA, 76: 5259- 5263 [1979]), and filamentous fungi such as e.g. Neurospora, Penicillium, Tolypocladium (WO 91/00357, obj. 10 January 1991), and Aspergillus hosts, such as e.g. A. nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 112: 284-289 [1983]; Tilbum et al., Gene, 26: 205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81: 1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4: 475-479 [1985]).

Prikladne gostiteljske celice za ekspresijo glikoziliranega mpl liganda so izvedene iz multiceličnih organizmov. Take gostiteljske celice so sposobne kompleksnega procesiranja in glikozilacijskih aktivnosti. Na splošno so uporabne vse višje prokariotske celične kulture, bodisi iz kulture vretenčaijev ali nevretenčaijev. Primeri celic nevretenčaijev vključujejo rastlinske celice in celice insektov. Številni bakulovirusni sevi in variante ter ustrezne dopustne gostiteljske celice insektov iz gostiteljev, kot npr. Spodoptera frugiperda (gosenica), Aedes aegypti (komar), Aedes albopictus (komar), Drosophila melanogaster (vinska mušica), in Bombyx mori so identificirani. Glej npr. Luckow et al., Bio/Technology, 6:47-55 [1988]; Miller et al., Genetic Engineering, Setlow et al., izd. vol. 8 (Plenum Publishing, 1986), str. 277-279; in Maeda et al., Nature, 315:592-594 [1985]. Številni virusni sevi za transfekcijo so javno dosegljivi, npr.: L-l varianta od Autographa californica NPV in sev Bm-5 od Bombyx mori NPV, in jih lahko uporabimo kot viruse v smislu predloženega izuma, posebno za transfekcijo celic Spodoptera frugiperda.Suitable host cells for the expression of glycosylated mpl ligand are derived from multicellular organisms. Such host cells are capable of complex processing and glycosylation activities. Generally, all higher prokaryotic cell cultures, whether from vertebrate or invertebrate cultures, are useful. Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains and variants, and suitable tolerant host insect cells from hosts, such as e.g. Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (wine fly), and Bombyx seas have been identified. See, e.g. Luckow et al., Bio / Technology, 6: 47-55 [1988]; Miller et al., Genetic Engineering, Setlow et al., Ed. vol. 8 (Plenum Publishing, 1986), p. 277-279; and Maeda et al., Nature, 315: 592-594 [1985]. Many transfection virus strains are publicly available, for example: the L-l variant from Autograph californica NPV and the Bm-5 strain from Bombyx mori NPV, and can be used as viruses of the present invention, especially for transfection of Spodoptera frugiperda cells.

Kulture rastlinskih celic bombaža, koruze, krompiija, soje, petunij, paradižnika in tobaka lahko uporabimo kot gostitelje. Značilno rastlinske celice transfektiramo z inkubacijo z določenimi sevi bakterij Agrobacterium tumefaciens, ki jih predhodno obdelamo, da vsebujejo DNAznp/ liganda. Med inkubacijo rastlinskih celičnih kultur z A. tumefaciens se DNA, ki kodira mpl ligand, transferira v rastlinsko gostiteljsko celico, tako da je le-ta transfektirana in pri ustreznih pogojih eksprimira DNA mpl liganda. Poleg tega so regulatome in signalne sekvence, kompatibilne z rastlinskimi celilcami, dosegljive, kot npr. promotor nopalin sintaza in poliadenilacijske signalne sekvence. Depicker et al., J. Mol. Appl. Gen., 1:561 [1982]. Poleg tega so segmenti DNA, izolirani iz navzgomje regije T-DNA gena 780, sposobni aktiviranja ali zvečevanja transkripcijskih nivojev rastlinsko ekspresibilnih genov v rekombinantni DNA, ki jo vsebujejo rastlinska tkiva. EP 321,196, obj. 21. junija 1989.Plant cultures of cotton, maize, potato, soybean, petunia, tomato, and tobacco plant cells can be used as hosts. Typically, plant cells are transfected by incubation with certain strains of Agrobacterium tumefaciens pre-treated to contain DNAznp / ligand. During incubation of plant cell cultures with A. tumefaciens, DNA encoding the mpl ligand is transferred to the plant host cell so that it is transfected and under the appropriate conditions expressed the mpl ligand DNA. In addition, plant-healing helper-compatible regulators and signal sequences are available, such as e.g. promoter of nopalin synthase and polyadenylation signal sequences. Depicker et al., J. Mol. Appl. Gen. 1: 561 [1982]. In addition, DNA segments isolated from the upstream region of the T-DNA gene 780 are capable of activating or enhancing the transcriptional levels of plant-expressing genes in recombinant DNA contained in plant tissues. EP 321,196, obj. June 21, 1989

Seveda pa so najbolj zanimive celice vretenčaijev, tako da je propagacija celic vretenčaijev v kulturi (tkivna kultura) postala rutinski postopek v zadnjih letih (Tissue Culture, Academic Press, Kruse in Patterson, izd. [1973]). Primeri uporabnih gostiteljskih celičnih linij sesalca so opičja ledvična linija CV1, transformirana z SV40 (COS-7, ATCC CRL 1651); humana embrionalna ledvična linija (293) ali 293 celic, subkloniranih za rast v suspenzijski kulturi, Graham et al., J. Gen Virol., 36:59 [1977]); ledvične celice mladiča hrčka (BHK, ATCC CCL 10);ovarijske celice kitajskega hrčka/DHFR (CHO, Urlaub in Chasin, Proč. Natl. Acad. Sci. ZDA, 77:4216 [1980]); mišje celice sertoli (TM4, Mather, Biol. Reprod., 23:243-251 [1980]); opičje ledvične celice (CV1ATCC CCL 70); ledvične celice afriške zelene opice (VERO-76, ATCC CRL-1587); celice humanega cervikalnega karcinoma (HELA, ATCC CCL 2); pasje ledvične celice (MDCK, ATCC CCL 34); jetrne celice volovske podgane (buffalo rat liver celiš) (BRL 3A, ATCC CRL 1442); humane pljučne celice (W138, ATCC CCL 75); humane jetrne celice (HEP G2, HB 8065); mišji mamami tumor (MMT 060562, ATCC CCL51); celice TRI (Mather et al., Annals Ν.Υ. Acad. Sci., 383:44-68 [1982]); celice MRC 5; celice FS4; in humana hepatomna linija (Hep G2).Of course, vertebrate cells are the most interesting, so propagation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years (Tissue Culture, Academic Press, Kruse, & Patterson, ed. [1973]). Examples of useful mammalian host cell lines are the CV40 monkey renal line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293) or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 [1977]); hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216 [1980]); mouse sertoli cells (TM4; Mather, Biol. Reprod., 23: 243-251 [1980]); monkey kidney cells (CV1ATCC CCL 70); African Green Monkey Kidney Cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine renal cells (MDCK, ATCC CCL 34); buffalo rat liver cellulose liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (HEP G2, HB 8065); mouse mamami tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals Ν.Υ. Acad. Sci., 383: 44-68 [1982]); MRC 5 cells; FS4 cells; and human hepatoma line (Hep G2).

Gostiteljske celice transfektiramo in prednostno transformiramo z zgoraj opisanimi ekspresijskimi ali klonimimi vektorji v smislu izuma in kultiviramo v konvencionalnih hranilnih medijih, modificiranih kot je potrebno za induciranje promotoijev, selekcioniranje transfomantov ali pomnoževanje genov, ki kodirajo želene sekvence.The host cells are transfected and preferably transformed with the expression or clone vectors of the invention described above and cultured in conventional nutrient media modified as necessary to induce promoters, select transfomants, or amplify genes encoding the desired sequences.

Transfekcija se nanaša na navzemanje ekspresijskega vektorja z gostiteljsko celico, bodisi, da je dejansko eksprimirana kakršnakoli kodirna sekvenca ali ne. Številni postopki za transfekcijo so znani strokovnjakom, npr. CaPO₄ in elektroporadja. Uspešno transfekcijo na splošno spoznamo po katerikoli indikaciji delovanja vektorja v gostiteljski celici.Transfection refers to the uptake of an expression vector by a host cell, whether or not any coding sequence is actually expressed. Many transfection procedures are known to those skilled in the art, e.g. CaPO ₄ and power generation. Successful transfection is generally recognized by any indication of vector activity in the host cell.

Transformacija pomeni uvedbo DNA v organizem tako, da je DNA replikabilna, bodisi kot ekstrakromosomski element ali s kromosomskim integrantom. Odvisno od uporabljene gostiteljske celice naredimo transformacijo z uporabo standardnih tehnik, ustreznih za take celice. Obdelava s kalcijem z uporabo kalcijevega klorida, kot je opisano v poglavju 1.82 avtorja Sambrooka et al., zgoraj, se na splošno uporablja za prokariote ali druge celice, ki vsebujejo bistvene celične stenske bariere. Infekcija z Agrobacterium tumefaciens se uporablja za transformacijo določenih rastlinskih celic, kot to opisujejo Shaw et al., Gene, 23:315 [1983] in WO 89/05859, obj. 29. junija 1989. Poleg tega lahko rastline transfektiramo z uporabo ultrazvoka, kot je opisano v WO 91/00358, obj. 10. januarja 1991. Za celice sesalcev brez takih celičnih sten je prednosten obarjalni postopek s kalcijevim fosfatom, ki ga opisujejo Graham in van der Eb, Virology, 52:456-457 [1978]. Splošne vidike za transformacije sistema gostiteljskih celic sesalcev opisuje Axel v US patentu št. 4,399,216, izd. 16. avgusta 1983. Transformacije v kvasovke značilno izvedemo v skladu s postopkom od Van Solingena et al., J. Bact., 130:946 [1977] in Hsiao et al., Proč. Natl. Acad. Sci. (ZDA, 76:3829 [1979]. Prav tako lahko uporabimo tudi druge postopke za uvedbo DNA v celice, kot npr. nuklearno injiciranje, elektroporacijo ali protoplastno fuzijo.Transformation involves the introduction of DNA into an organism in such a way that DNA is replicable, either as an extrachromosomal element or with a chromosomal integrant. Depending on the host cell used, a transformation is made using standard techniques appropriate for such cells. Treatment with calcium using calcium chloride, as described in Section 1.82 by Sambrook et al., Above, is generally used for prokaryotes or other cells containing essential cell wall barriers. Infection with Agrobacterium tumefaciens is used to transform certain plant cells, as described by Shaw et al., Gene, 23: 315 [1983] and WO 89/05859, obj. June 29, 1989. In addition, plants can be transfected using ultrasound as described in WO 91/00358, obj. January 10, 1991. For mammalian cells without such cell walls, a calcium phosphate precipitation process described by Graham and van der Eb, Virology, 52: 456-457 [1978] is preferred. General aspects of mammalian host cell system transformations are described by Axel in U.S. Pat. No. 4,399,216, ed. August 16, 1983. Transformations into yeasts are typically performed according to the procedure of Van Solingen et al., J. Bact., 130: 946 [1977] and Hsiao et al. Natl. Acad. Sci. (U.S.A. 76: 3829 [1979]. Other methods for introducing DNA into cells, such as nuclear injection, electroporation, or protoplast fusion, may also be used.

E. Kultiviranje gostiteljskih celicE. Culturing host cells

Prokariotske celice, uporabljene za izdelavo mpl Ugandskega poUpeptida v smislu izuma kultiviramo v prikladnih medijih, kot to na splošno opisujejo Sambrook et al., zgoraj.The prokaryotic cells used to make the mpl of the Ugandan poUpeptide of the invention are cultured in suitable media, as generally described by Sambrook et al., Supra.

Gostiteljske celice sesalcev, uporabljene za izdelavo mpl liganda v smislu predloženega izuma, lahko kultiviramo v različnih medijih. Komercialno dosegljivi mediji, kot npr.: Ham’s F10 (Sigma), minimalno esencialni medij ([ΜΕΜ], Sigma), RPMI-1640 (Sigma), in Eaglov medij, modificiran po Dulbecco-u ([DMEM], Sigma), so prikladni za kultiviranje gostiteljskih ceUc. Poleg tega še kateregakoU od medijev, ki jih opisujejo: Ham in WaUace, Meth. Enz., 58:44 [1979], Bames in Sato. Anal. Biochem., 102:255 [1980], US patent št. 4,767,704; 4,657,866; 4,927,762; aU 4,560,655; WO 90/03430; WO 87/00195; US patent Re. 30,985; aU istočasno v postopku US serij. št. 07/592,107 aU 07/592,141, oba vložena 3. oktobra 1990, katerih opisi so vključeni tukaj z referencami, lahko uporabimo kot kulturni medij za gostiteljske ceUce. Vse te medije lahko dopolnimo, če je potrebno s hormoni in/aU drugimi rastnimi faktorji (kot so inzulin, transferin aU epidermalni rastni faktor), solmi (kot so natrijev klorid, kalcij, magnezij in fosfat), pufri (kot je HEPES), nukleozidi (kot sta adenozin in timidin), antibiotiki (kot je Gentamycin™ zdravilo), elementi v sledovih (definirani kot anorganske spojine, ki so navadno prisotne v končnih koncentracijah v mikromolskih koUčinah) in glukozo aU ekvivalentnim virom energije. Vse druge potrebne dodatke tudi lahko vključimo v ustreznih koncentracijah, znanih strokovnjakom. Pogoji za kulturo, kot npr. temepratura, pH ipd., so tisti, ki se prednostno uporabljajo za gostiteljsko celico, izbrano za ekspresijo, in so strokovnjakom znani.The mammalian host cells used to make the mpl ligand of the present invention can be cultured in different media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Media ([ΜΕΜ], Sigma), RPMI-1640 (Sigma), and Eagle's Dulbecco Modified Media ([DMEM], Sigma) are suitable for cultivating host ceUc. In addition, any of the media described by Ham and WaUace, Meth. Enz., 58:44 [1979], Bames and Sato. Anal. Biochem., 102: 255 [1980], U.S. Pat. 4,767,704; 4,657,866; 4,927,762; aU 4,560,655; WO 90/03430; WO 87/00195; U.S. Pat. 30,985; aU simultaneously in the process of US series. no. 07 / 592,107 aU 07 / 592,141, both filed October 3, 1990, the descriptions of which are incorporated herein by reference, may be used as a cultural medium for host ceUce. All these media can be supplemented if necessary with hormones and / aU other growth factors (such as insulin, transferrin aU epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as Gentamycin ™), trace elements (defined as inorganic compounds that are usually present at final concentrations in micromole quantities), and glucose aU equivalent energy sources. All other necessary additives can also be incorporated into appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, etc. are those which are preferably used for the host cell selected for expression and are known to those skilled in the art.

Gostiteljske celice, na katere se sklicujemo v temu opisu, obsegajo celice v kulturi in vitro kot tudi celice, ki so v gostiteljski živali.The host cells referred to in this specification include cells in culture in vitro as well as cells that are in the host animal.

F. Detektiranje pomnožitve/ekspresije genaF. Detection of gene amplification / expression

Pomnožitev in/ali ekspresijo gena lahko izmerimo v vzorcu direktno, npr. s konvencionalnimi tehnikami, kot so: Southern blotting, Northern blotting, da kvantitativno določimo transkripcijo mRNA (Thomas, Proč. Natl. Acad. Sci. ZDA 77:5201-5205 [1980]), s kapljičnim nanosom (dot blotting) (DNA analiza) ali s hibridizacijo in situ, z uporabo ustreznih označenih sond, ki temeljijo na sekvencah, zagotovljenih tukaj. Uporabimo lahko različna sredstva za označevanje, najbolj običajno radioaktivne izotope, posebno ³²P. Uporabimo pa lahko tudi druge tehnike, kot npr. tisto z uporabo nukleotidov, modificiranih z biotinom, za uvedbo v polinukleotid. Biotin nato rabi kot mesto za vezavo na avidin ali protitelesa, ki so lahko označena z različnimi označevalnimi sredstvi, kot npr. radionuklidi, fluorescentnimi sredstvi, encimi ipd. Alternativno lahko uporabimo protitelesa, ki lahko spoznajo specifične duplekse, vključno duplekse DNA duplekse RNA in hibridne duplekse DNA-RNA ali duplekse DNA-protein. Protitelesa lahko nato označimo in izvedemo test, kjer se dupleks veže na površino tako, da po tvorbi dupleksa na površini lahko detektiramo prisotnost protitelesa, vezanega na dupleks.Gene amplification and / or expression can be measured directly in a sample, e.g. using conventional techniques such as: Southern blotting, Northern blotting, to quantify mRNA transcription (Thomas, Proc. Natl. Acad. Sci. USA 77: 5201-5205 [1980]), dot blotting (DNA analysis ) or by in situ hybridization, using appropriate labeled probes based on the sequences provided herein. Different labeling agents, most commonly radioactive isotopes, in particular ³² P. may be used, but other techniques may be used, such as e.g. one using biotin-modified nucleotides for introduction into a polynucleotide. Biotin is then used as a binding site for avidin or antibodies that may be labeled with various labeling agents, such as. radionuclides, fluorescent agents, enzymes and the like. Alternatively, antibodies that can recognize specific duplexes, including DNA RNA duplexes and DNA-RNA hybrid duplexes or DNA-protein duplexes, may be used. The antibodies can then be labeled and a test is performed where the duplex binds to the surface so that after the formation of the duplex on the surface, the presence of the antibody bound to the duplex can be detected.

Gensko ekspresijo lahko alternativno izmerimo z imunološkimi postopki, kot npr. z imunohistokemijskim barvanjem tkivnih predelov in testiranjem celične kulture ali telesnih tekočin, da direktno kvantitativno določimo ekspresijo nastalega gena. Z imunohistokemijskimi tehnikami barvanja pripravimo celični vzorec, značilno z dehidracijo in fiksiranjem, čemur sledi reakcija z označenimi protitelesi, specifičnimi za pripojeni nastali gen, pri čemer so oznake navadno vizualno detektibilne, kot npr. encimatske oznake, fluorescentne, luminiscentne in ipd. Posebno občutljivo tehniko barvanja, prikladno za uporabo v predloženem izumu, opisujejo Hsu et al., Am. J. Ciin. Path., 75:734-738 [1980].Alternatively, gene expression can be measured by immunological methods, such as e.g. by immunohistochemical staining of tissue sections and testing of cell culture or body fluids to directly quantify the expression of the resulting gene. Immunohistochemical staining techniques produce a cell pattern characterized by dehydration and fixation, followed by reaction with labeled antibodies specific for the annexed resulting gene, the labels being typically visually detectable, such as e.g. enzymatic labels, fluorescent, luminescent and the like. A particularly sensitive staining technique suitable for use in the present invention is described by Hsu et al., Am. J. Ciin. Path., 75: 734-738 [1980].

Protitelesa, uporabna za imunohistokemijsko barvanje in/ali testiranje vzorčnih tekočin, so lahko bodisi monoklonska ali poliklonska in jih lahko pripravimo v kateremkoli sesalcu. Prikladno lahko pripravimo protitelesa proti naravnemu mpl Ugandskemu poUpeptidu ali protisintetičnemu peptidu, ki temelji na sekvencah DNA zagotovljenih tukaj, kot je opisano nadalje spodaj.Antibodies useful for immunohistochemical staining and / or testing of sample fluids can be either monoclonal or polyclonal and can be prepared in any mammal. Conveniently, antibodies can be prepared against native mpl Ugandan poUpeptide or antisynthetic peptide based on the DNA sequences provided herein, as described below.

G. Čiščenje mpl Ugandskega poUpeptidaG. Purification of mpl Ugandan poUpeptide

Mpl Ugand prednostno rekuperiramo iz medija kulture kot izločen poUpeptid, čeprav ga lahko rekuperiramo tudi iz lizatov gostiteljske celice, kadar je direktno eksprimiran brez sekretomega signala.Mpl Ugand is preferably recovered from the culture medium as secreted poUpeptide, although it can also be recovered from host cell lysates when directly expressed without a secretome signal.

Če je mpl Ugand eksprimiran v rekombinantni ceUci, ki ni humanega izvora, je mpl ligand popolnoma brez proteinov ali polipeptidov humanega izvora. Vendar je Še vedno navadno potrebno, da očistimo mpl ligand drugih rekombinantnih celičnih proteinov ali polipeptidov, da dobimo pripravke, ki so v bistvu homogeni glede na mpl ligand sam po sebi. Kot prvo stopnjo medij kulture ali lizat centrifugiramo, da odstranimo delčke celičnega debrisa. Membrano in topne proteinske frakcije nato ločimo. Alternativno lahko uporabimo komercialno dosegljiv proteinski koncentracijski filter (npr. ultrafiltracijske enote Amicon ali Millipore Pellicon). Mpl ligand lahko nato očistimo iz topne proteinske frakcije in iz membranske frakcije kulturnega lizata, če se mpl ligand veže na membrano. Mpl ligand nato očistimo kontiminantnih topnih proteinov in polipeptidov z izsoljevanjem in izmenjavo ali kromatografskimi postopki z uporabo različnih gelskih matric. Te matrice vključujejo: akrilamid, agarozo, dekstran, celulozo in druge, običajne za čiščenje proteinov. Zgledni kromatografski postopki, prikladni za čiščenje proteinov vključujejo: imunoafinitetno kromatografijo (npr. anti-hmp/ ligand Mab), receptorsko afiniteto (npr. znpZ-IgG ali protein A-sefarozo), hidrofobno interakcijsko kromatografijo (HIC) (npr.: eter, butil ali fenil Toyopearl), lektinsko kromatografijo (npr.: Con A-sefaroza, lektin (leča)-sefaroza), velikostno izključitveno kromtografijo (npr.: sefadeks G-75), kationsko in anionsko izmenjalno kolonsko kromatografijo (npr.: DEAE ali karboksimetil- in sulfopropil-celuloza) in HPLC z reverzno fazo (RP-HPLC) (glej npr. Urdal et al., J. Chromatog., 296:171 [1984], kjer uporabimo dve zaporedni stopnji RP-HPLC, da očistimo rekombinantni humani IL-2). Druge stopnje čiščenja v danem primeru vključujejo: etanolno obaijanje; obaijanje z amonijevim sulfatom, kromatofokusiranje, prepartivno SDS-PAGE ipd.If mpl Ugand is expressed in non-human recombinant ceUci, the mpl ligand is completely free of human origin proteins or polypeptides. However, it is still generally necessary to purify the mpl ligand of other recombinant cellular proteins or polypeptides to obtain preparations that are substantially homogeneous with respect to the mpl ligand per se. As a first step, the culture medium or lysate is centrifuged to remove cell debris particles. The membrane and soluble protein fractions are then separated. Alternatively, a commercially available protein concentration filter (eg, Amicon ultrafiltration units or Millipore Pellicon) may be used. The mpl ligand can then be purified from the soluble protein fraction and from the membrane fraction of the culture lysate if the mpl ligand binds to the membrane. The Mpl ligand is then purified of contaminant soluble proteins and polypeptides by salting and exchange or chromatographic procedures using different gel matrices. These matrices include: acrylamide, agarose, dextran, cellulose and other common protein purification agents. Exemplary chromatographic processes suitable for protein purification include: immunoaffinity chromatography (e.g. anti-hmp / Mab ligand), receptor affinity (e.g. znpZ-IgG or protein A-sepharose), hydrophobic interaction chromatography (HIC) (e.g., ether, butyl or phenyl Toyopearl), lectin chromatography (eg: Con A-Sepharose, lectin (lens) -sepharose), size exclusion chromatography (eg: Sephadex G-75), cation and anion exchange column chromatography (eg: DEAE or carboxymethyl- and sulfopropyl-cellulose) and reversed-phase HPLC (RP-HPLC) (see, e.g., Urdal et al., J. Chromatog., 296: 171 [1984], where two consecutive steps of RP-HPLC are used to purify recombinant human IL-2). Other cleaning steps in this case include: ethanol staining; ammonium sulfate dyeing, chromatofocusing, pre-partition SDS-PAGE and the like.

Variante mpl liganda, v katerih so ostanki deletirani, inserirani ali substituirani, rekuperiramo na enak način kot naravni mpl ligand, pri čemer upoštevamo vse bistvene spremembe v lastnostih, povzročenih z variiranjem. Npr., pripravek fuzije mpl liganda z drugim proteinom ali polipeptidom, npr. bakterijskim ali virusnim antigenom, olajša čiščenje; imunoafinitetno kolono, ki vsebuje protitelo za antigen, lahko uporabimo za adsorbiranje fuzijskega polipeptida. Imunoafinitetne kolone, kot npr. kolono za zajčji poliklonski anti-mp/ ligand, lahko uporabimo za absorbiranje variante mpl liganda z njeno vezavo na vsaj en preostali imuni epitop. Alternativno lahko mpl ligand očistimo z afinitetno kromatografijo z uporabo očiščenega /np/-IgG, pripojenega na (prednostno) imobilizirano smolo, kot npr. Affi-Gel 10 (Bio-Rad, Richmond, CA) ipd. na način, dobro znan v tehniki. Proteazni inhibitor, kot npr. fenil metil sulfonil fluorid (PMSF) tudi lahko uporabimo za inhibiranje proteolitične degradacije med čiščenjem, antibiotike pa lahko vključimo, da preprečimo rast naključnih kontaminantov. Strokovnjakom je jasno, da je postopke čiščenja, prikladne za naravni mpl ligand, morda potrebno modificirati zaradi sprememb v značaju mpl liganda ali njegovih variant po ekspresiji v rekombinantni celični kulturi.Variants of the mpl ligand in which the residues are deleted, inserted or substituted are recovered in the same manner as the natural mpl ligand, taking into account any significant changes in the properties caused by the variation. For example, the preparation of mpl ligand fusion with another protein or polypeptide, e.g. bacterial or viral antigens, facilitates purification; an immunoaffinity column containing an antibody for the antigen can be used to adsorb the fusion polypeptide. Immunoaffinity columns, e.g. the rabbit polyclonal anti-mp / ligand column can be used to absorb the mpl ligand variant by binding it to at least one remaining immune epitope. Alternatively, the mpl ligand can be purified by affinity chromatography using purified / np / -IgG attached to a (preferably) immobilized resin, such as e.g. Affi-Gel 10 (Bio-Rad, Richmond, CA), etc. in a manner well known in the art. A protease inhibitor such as e.g. Phenyl methyl sulfonyl fluoride (PMSF) can also be used to inhibit proteolytic degradation during cleansing, and antibiotics can be included to prevent the growth of accidental contaminants. It will be appreciated by those skilled in the art that purification procedures suitable for the natural mpl ligand may need to be modified due to changes in the character of the mpl ligand or variants thereof after expression in recombinant cell culture.

H. Kovalentne modifikacije mpl Ugandskega poUpeptidaH. Covalent modifications of mpl Ugandan poUpeptide

Kovalentne modifikacije mpl Ugandskega poUpeptida so vključene v obseg predloženega izuma. Tako naravni mpl Ugand kot tudi variante aminokislinske sekvence mpl Uganda lahko kovalentno modificiramo. En tip kovalentne modifikacije, vključen v obseg predloženega izuma, je fragment mpl Uganda. Varianto fragmenta mpl Uganda, ki ima do približno 40 aminokisUnskih ostankov, lahko prikladno pripravimo s kemijsko sintezo aU z encimatskim ali kemijskim cepljenjem mpl Ugandskega poUpeptida s popolno dolžino ali njegove variante. Druge tipe kovalentnih modifikacij mpl Uganda aU njegovih fragmentov uvedemo v molekulo z reakcijo ciljnih aminokislinskih ostankov mpl Uganda aU njegovih fragmentov z organskim derivatizimim sredstvom, ki je sposobno reagirati z izbranimi stranskimi verigami ali N- aU C-terminalnimi ostanki.Covalent modifications of the mpl of the Ugandan poUpeptide are included within the scope of the present invention. Both natural mpl Ugand and variants of the amino acid sequence of mpl Uganda can be covalently modified. One type of covalent modification included in the scope of the present invention is the Ugand mpl fragment. A variant of a Ugandan mpl fragment having up to about 40 amino acid residues can be conveniently prepared by chemical synthesis of aU by enzymatic or chemical cleavage of full-length Ugandan poUpeptide mpl or variants thereof. Other types of covalent modifications of the Ugand aU mpl mpl fragments of it are introduced into a molecule by reacting the target amino acid residues of the mpl Uganda aU fragments thereof with an organic derivative capable of reacting with selected side chains or N-aU C-terminal residues.

Cisteinilni ostanki najbolj običajno reagirajo z α-haloacetati (in ustreznimi amini), kot npr. klorocetno kislino aU kloracetamidom, da nastanejo karboksimetil aU karboksiamidometil derivati. Cisteinilne ostanke prav tako derivatiziramo z reakcijo z bromotrifluoroacetonom, a-bromo-/?-(5-imidozoil)propionsko kislino, kloroacetil fosfatom, N-aUrilmaleimidi, 3-nitro-2-piridil disulfidom, metil 2-piridil disulfidom, p-kloromerkuribenzoatom, 2-kloromerkuri-4-nitrofenolom, aU kloro-7-nitrobenzo2-oksa-l,3-diazolom.Cysteinyl residues are most commonly reacted with α-haloacetates (and corresponding amines), such as e.g. chloroacetic acid aU with chloroacetamide to form carboxymethyl aU carboxyamidomethyl derivatives. Cysteinyl residues are also derivatized by reaction with bromotrifluoroacetone, α-bromo - N - (5-imidozoyl) propionic acid, chloroacetyl phosphate, N-aurylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, methyl pyridyl disulfide , 2-chloromercuri-4-nitrophenol, aU chloro-7-nitrobenzo2-oxa-1,3-diazole.

Histidilne ostanke derivatiziramo z reakcijo z dietilpirokarbonatom pri pH 5,5-7,0, ker je to sredstvo relativno specifično za histidilno stransko verigo. Parabromofenacil bromid je prav tako uporaben; reakcijo prednostno izvedemo v natrijevem kakodilatu (O,1M) pri pH 6,0.Histidyl residues are derivatized by reaction with diethyl pyrocarbonate at pH 5.5-7.0 because this agent is relatively specific to the histidyl side chain. Parabromophenacyl bromide is also useful; the reaction is preferably carried out in sodium cacodylate (O, 1M) at pH 6.0.

Lizinil in amino-terminalni ostanki reagirajo z anhidridi jantarne aU drugih karboksilnih kislin. Derivatizacija s temi sredstvi vpliva na spremembo naboja Uzinilnih ostankov. Drugi prikladni reagenti za derivatiziranje ostankov, ki vsebujejo amino ostanke, vključujejo imidoestre, kot so : metil pikolinimidat; piridoksal fosfat; piridoksal; kloroborohidrid; trinitrobenzensulfonska kislina; O-metilizosečnina, 2,4pentandion; in reakcijo z glioksilatom, katalizirano z transaminazo.Lysinyl and amino-terminal residues react with succinic aU anhydrides of other carboxylic acids. Derivatization by these means influences the change in charge of the Uzinil residues. Other suitable reagents for derivatizing residues containing amino residues include imidoesters such as: methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea, 2,4pentanedione; and transaminase-catalyzed glyoxylate reaction.

Arginilne ostanke modificiramo z reakcijo z enim ali več konvencionalnimi reagenti, med katerimi so: fenilglioksal, 2,3-butandion, 1,2-cikloheksandion in ninhidrin. Derivatizacija argininskih ostankov mora potekati v alkalnih pogojih zaradi visoke pK_a gvanidinske funkcionalne skupine. Nadalje lahko ti reagenti reagirajo s skupinami lizina kot tudi z argininsko epsilon-amino skupino.The arginyl residues are modified by reaction with one or more conventional reagents, including: phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione and ninhydrin. The derivatization of arginine residues must take place under alkaline conditions due to the high pK _{a of the} guanidine functional group. Furthermore, these reagents can react with the lysine groups as well as with the arginine epsilon-amino group.

Specifično modifikacijo tirozilnih ostankov lahko naredimo posebno zanimivo z uvedbo spektralnih označb v tirozilne ostanke z reakcijo z aromatskimi diazonijevimi in spojinami ali tetranitrometanom. Najbolj običajno uporabimo N-acetilimidazol in tetranitrometan, da tvorimo O-acetil tirozilno vrsto oz. 3-nitro derivate. Tirozilne ostanke jodiramo z uporabo ¹²⁵J ali¹³¹ J, da pripravimo označene proteine za uporabo v radioimunskem testu, pri čemer je prikladen postopek s kloraminom T, opisan zgoraj.The specific modification of tyrosyl residues can be made particularly interesting by introducing spectral designations into tyrosyl residues by reaction with aromatic diazonium and compounds or tetranitromethane. Most commonly, N-acetylimidazole and tetranitromethane are used to form the O-acetyl tyrosyl species, respectively. 3-nitro derivatives. Tyrosyl residues are iodized using ¹²⁵ J or ¹³¹ J to prepare labeled proteins for use in radioimmunoassay, the chloramine T procedure described above being suitable.

Karboksilne stranske skupine (aspartil ali glutamil) selektivno modificiramo z reakcijo s karbodiimidi (R-N=C=N-R’), kjer sta R in R’ različni alkilni skupini, kot npr. l-cikloheksil-3-(2-morfolinil-4-etil)karbodiimid ali l-etil-3-(4-azonia-4,4dimetilpentil)karbodfimid. Nadalje aspartilne in glutamilne ostanke pretvorimo v asparaginilne in glutaminilne ostanke z reakcijo z amonijevimi ioni.The carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R-N = C = N-R '), where R and R' are different alkyl groups, such as e.g. 1-Cyclohexyl-3- (2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3- (4-azonia-4,4 dimethylpentyl) carbodimide. Furthermore, the aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

Derivatizacija z bifunkcionalnimi sredstvi je uporabna za premreževanje mpl liganda na nosilni matrici ali površini, ki ni topna v vodi, za uporabo v postopku za čiščenje anti-mp/ Ugandskih protiteles in obratno. Navadno uporabljena premreževalna sredstva vključujejo npr. l,l-bis(diazoacetil)-2-feniletan, glutaraldehid, N-hidroksisukcinimid estre, npr. estre s 4-azidosaUcilno kislino, homobifunkcionalne imidoestre, vključno disukcinimidil estre, kot npr. 3,3’ditiobis(sukcinimidilpropionat), in bifunkcionalne maleimide, kot npr. bis-Nmaleimido-l,8-oktan. Derivatizima sredstva, kot je metil3-[(p-azidofenil)ditio]propioimidat, dajo fotoaktivime intermediate, ki so sposobni, da tvorijo premreženja v prisotnosti svetlobe. Alternativno, reaktivne v vodi netopne matrice, kot so ogljikovi hidrati, aktivirani s cianogenbromidom in reaktivne substrate, opisane v US patentih št. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; in 4,330,440, uporabimo za proteinsko imobilizacijo.Derivatization with bifunctional agents is useful for crosslinking the mpl ligand on a carrier matrix or a water-insoluble surface for use in an anti-mp / Ugandan antibody purification process and vice versa. Commonly used cross-linking agents include e.g. 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, e.g. 4-azidosa estersUnic acid, homobifunctional imidoesters, including disuccinimidyl esters such as e.g. 3,3'ditiobis (succinimidylpropionate), and bifunctional maleimides such as e.g. bis-Nmaleimido-1,8-octane. Derivatives agents such as methyl3 - [(p-azidophenyl) dithio] propioimidate give photoactivities intermediates that are capable of forming crosslinks in the presence of light. Alternatively, water-insoluble matrices such as cyanogen bromide-activated carbohydrates and reactive substrates described in U.S. Pat. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440, used for protein immobilization.

Glutaminilne in asparaginilne ostanke pogosto deamidiramo v ustrezne glutamilne oz. aspartilne ostanke. Ti ostanki se deamidirajo pri nevtralnih ali bazičnih pogojih.Glutaminyl and asparaginyl residues are often deamidated to the corresponding glutamyl or. aspartyl residues. These residues are deamidated under neutral or basic conditions.

Deamidirane oblike teh ostankov so tudi v obsegu predloženega izuma.The deamidated forms of these residues are also within the scope of the present invention.

Druge modifikacije vključujejo hidroksiliranje prolina in lizina, fosforiliranje hidroksilnih skupin serilnih ali treonilnih ostankov, metiliranje α-amino skupin lizina, arginina in histidinskih stranskih verig (T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, str. 79-86 [1983]), acetiliranje N-terminalnega amina in amidiranje katerekoli C-terminalne karboksilne skupine.Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of α-amino groups of lysine, arginine and histidine side chains (TE Creighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, p. 79-86 [1983]), acetylation of the N-terminal amine and amidation of any C-terminal carboxyl group.

Drugi tip kovalentne modifikacije mpl Ugandskega poUpeptida, kije vključen v obseg predloženega izuma, obsega spreminjanje modelov naravne glikozilacije poUpeptida. S spreminjanjem je mišljena delecija enega ali več ogljikohidratnih deležev, ugotovljenih v naravnem mpl Ugandu, in/ali dodajanje enega aU več glikozilacijskih mest, ki niso prisotna v naravnem mpl Ugandu.Another type of covalent modification of the mpl of Ugandan poUpeptide, which is included in the scope of the present invention, involves modifying the natural glycosylation patterns of poUpeptide. Alteration means deletion of one or more carbohydrate moieties found in natural mpl Ugand and / or addition of one aU more glycosylation sites that are not present in natural mpl Ugand.

Glikozilacija poUpeptidov je značilno bodisi N-vezana aU O-vezana. N-vezana se nanaša na povezanost ogljikohidratnega deleža s stransko verigo asparaginskega ostanka. Tripeptidne sekvence asparagin-X-serin in asparagin-X-treonm, kjer je X katerakoU amino kislina razen prolina, so spoznavne sekvence za encimatsko povezanost ogljikohidratnega deleža z asparaginsko stransko verigo. Na ta način prisotnost katerekoU od teh tripeptidnih sekvenc v poUpeptidu ustvari potencialno mesto za glikozilacijo. O-vezana gUkozilacija se nanaša na povezanost enega od sladkorjev,kot so N-acetilgalaktozamin, galaktoza aU ksiloza s hidroksiamino kislino, najbolj običajno serinom ali treoninom, čeprav lahko uporabimo tudiThe glycosylation of poUpeptides is typically either N-linked aU O-linked. N-linked refers to the association of the carbohydrate moiety with the side chain of the asparagine residue. The tripeptide sequences of asparagine-X-serine and asparagine-X-threonine, where X is any amino acid other than proline, are recognizable sequences for the enzymatic linkage of the carbohydrate moiety to the asparagine side chain. In this way, the presence of any of these tripeptide sequences in the poUpeptide creates a potential glycosylation site. O-linked gucosylation refers to the association of one of the sugars, such as N-acetylgalactosamine, galactose aU xylose, with hydroxyamic acid, most commonly serine or threonine, although we may also use

5-hidroksiprolin ali 5-hidroksiUzin.5-hydroxyproline or 5-hydroxyUzine.

Adicijo mest za glikoziliranje mpl Ugandskega poUpeptida ugodno izvedemo s spreminjanjem aminokislinske sekvence, tako da le-ta vsebuje eno aU več zgoraj opisanih tripeptidnih sekvenc (za N-vezana mesta glikozilacije). Spremembe lahko naredimo tudi tako, da izvedemo adicijo aU substitucijo enega ali več serinskih aU treoninskih ostankov v sekvenci naravnega mpl Uganda (za O-vezana mesta gUkozilacije). Ugodno aminokisUnsko sekvenco mpl Uganda prednostno spremenimo s spremembami na nivoju DNA, posebno z mutiranjem DNA, ki kodira mpl Ugandski poUpeptid na predhodno izbranih bazah, tako da nastanejo kodoni, ki se translatirajo v želene amino kisline. Mutacije DNA lahko naredimo s postopki, opisanimi zgoraj pod naslovom Variante aminokislinske sekvence mpl Uganda.The addition of the glycosylation sites of the mpl of the Ugandan poUpeptide is advantageously accomplished by altering the amino acid sequence such that it contains one aU of the tripeptide sequences described above (for N-linked glycosylation sites). Changes can also be made by adding an aU substitution of one or more serine aU threonine residues in the natural mpl Uganda sequence (for O-linked gcososylation sites). The advantageous amino acid sequence of mpl Uganda is advantageously altered by changes at the DNA level, in particular by mutating the DNA encoding the mpl Ugandan poUpeptide at previously selected bases to produce codons that translate into the desired amino acids. DNA mutations can be made using the procedures described above under the title Variants of the amino acid sequence of mpl Uganda.

Drug način za povečevanje števila ogljikohidratnih deležev mpl liganda je kemijsko ali encimatsko pripajanje glikozidov na polipeptid. Ti postopki so prednostni v tem, da ni potrebno izdelati polipeptida v gostiteljski celici s sposobnostjo glikoziliranja za N- ali O-vezano glikozilacijo. Odvisno od uporabljenega načina pripajanja se sladkorji lahko vežejo na (a) arginin in histidin, (b) prosto karboksilno skupino, (c) proste sulfhidrilne skupine, kot so tiste od cisterna, (d) proste hidroksilne skupine, kot so tiste od serina, treonina ali hidroksiprolina in (e) aromatske ostanke, kot so tisti od fenilanalina, tirozina ali triptofana ali (f) na amidno skupino glutamina. Ti postopki so opisani v WO 87/05330, obj. 11. septembra 1987, in v Aplin in Wriston, CRC Crit. Rev. Biochem., str. 259-306 [1981].Another way to increase the carbohydrate content of the mpl ligand is to chemically or enzymatically attach glycosides to the polypeptide. These methods are advantageous in that it is not necessary to produce a polypeptide in a host cell capable of glycosylation for N- or O-linked glycosylation. Depending on the coupling method used, sugars may bind to (a) arginine and histidine, (b) a free carboxyl group, (c) free sulfhydryl groups such as those from the cisterna, (d) free hydroxyl groups such as those from serine, threonine or hydroxyproline; and (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the glutamine amide group. These procedures are described in WO 87/05330, obj. September 11, 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., P. 259-306 [1981].

Odstranitev ogljikohidratnih deležev, prisotnih na mpl Ugandskem poUpeptidu, lahko izvedemo kemijsko aU encimatsko. Kemijska degUkozilacija zahteva izpostavitev poUpeptidov trifluorometansulfonski kislini aU ekvivalentni spojini. Ta obdelava povzroči cepitev večine ali vseh sladkorjev razen vezivnega sladkorja (Nacetilglukozamin ali N-acetilgalaktozamin), medtem ko ostane polipeptid intakten. Kemijsko degUkozilacijo opisujejo Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 [1987] in Edge et al., Anal. Biochem., 118:131 [1981]. Encimatsko cepitev ogljikohidratnih deležev na poUpeptidih lahko dosežemo z uporabo razUčnih endoin ekso-glikozidaz, kot opisujejo Thotakura et al., Meth. Enzymol., 138:350 [1987].Removal of the carbohydrate moieties present on the mpl Ugandan poUpeptide can be carried out chemically aU enzymatically. Chemical degucosylation requires the exposure of popeptides to trifluoromethanesulfonic acid aU equivalent compound. This treatment results in the cleavage of most or all sugars except the binder sugar (Nacetylglucosamine or N-acetylgalactosamine), while the polypeptide remains intact. Chemical degucosylation is described by Hakimuddin, et al., Arch. Biochem. Biophys., 259: 52 [1987] and Edge et al., Anal. Biochem., 118: 131 [1981]. Enzymatic cleavage of carbohydrate moieties on poUpeptides can be achieved by using endogenous exo-glycosidases as described by Thotakura et al., Meth. Enzymol., 138: 350 [1987].

Glikozilacijo na potencialnih mestih za glikozilacijo lahko preprečimo z uporabo spojine tunicamicina, kot opisujejo Duskin et al., J. Biol. Chem., 257:3105 [1982]. Tunicamicin blokira tvorbo protein-N-glikozidnih povezav.Glycosylation at potential glycosylation sites can be prevented using the compound of tunicamycin as described by Duskin et al., J. Biol. Chem., 257: 3105 [1982]. Tunicamycin blocks the formation of protein-N-glycosidic linkages.

Drugi tip kovalentne modifikacije mpl liganda obsega vezavo mpl Ugandskega polipeptida na enega od raznih neproteinskih polimerov, kot so npr. poUetilen glikol, polipropilen gUkol aU polioksialkileni, na način, opisan v US patentih št. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 ali 4,179,337. Mpl Ugandski poUpeptidi, kovalentno vezani na pred tem navedene polimere, so tukaj imenovani pegilirani mpl Ugandski poUpeptidi.Another type of covalent modification of the mpl ligand involves binding of the mpl Ugandan polypeptide to one of various nonprotein polymers, such as e.g. polyethylene glycol, polypropylene glycol aU polyoxyalkylene, as described in U.S. Pat. 4,640,835; 4,496,689; 4,301,144; 4,670,417; No. 4,791,192 or 4,179,337. Mpl Ugandan poUpeptides covalently linked to the polymers previously mentioned are referred to herein as pegylated mpl Ugandan poUpeptides.

Upoštevati je treba, da je potrebno selekcioniranje rekuperirane variante mpl Uganda, da izberemo optimalno varianto za vezavo na mpl, ki bo imela imunološko in/ali biološko aktivnost, definirano zgoraj. Selekcioniramo lahko glede na stabilnost v rekombinantni celični kulturi ali v plazmi (npr. proti proteolitični cepitvi), visoko afiniteto za člana mpl, oksidativno stabilnost, sposobnost, da se izloči v večjih dobit98 kih ipd. Spremembe imunološkega značaja mpl Ugandskega polipeptida, kot je afiniteta za dano protitelo, izmerimo z imunskim testom kompetitivnega tipa. Druge potencialne modifikacije proteina ali polipeptidnih lastnosti, kot je redoks ali termalna stabilnost, hidrofobnost ali občutljivost za proteoUtično degradacijo, preizkusimo s postopki, dobro znanimi v tehniki.It should be borne in mind that selection of the recovered mpl Uganda variant is required to select the optimal mpl binding variant that will have the immune and / or biological activity defined above. Selected according to stability in recombinant cell culture or in plasma (eg against proteolytic cleavage), high affinity for mpl member, oxidative stability, ability to excrete in higher yields, etc. Changes in the immunological character of mpl Ugandan polypeptide, such as affinity for a given antibody, are measured by a competitive-type immunoassay. Other potential modifications of the protein or polypeptide properties, such as redox or thermal stability, hydrophobicity or sensitivity to proteoUtic degradation, are tested by methods well known in the art.

17. Splošni postopki za pripravo protiteles za mpl Ugand17. General antibody preparation procedures for Ugandan mpl

Priprava protiteles (i) Poliklonska protitelesaAntibody preparation (s) Polyclonal antibodies

Poliklonska protitelesa za mpl Ugandske poUpeptide aU fragmente na splošno vzgojimo v živalih z multipUmi subkutanimi (sc) aU intraperitonealnimi (ip) injekcijami mpl Uganda in adjuvansa. Koristno je, da konjugiramo mpl Ugand aU fragment, ki vsebuje ciljno aminokisUnsko sekvenco, s proteinom, ki je imunogen v vrstah, ki jih je potrebno imunizirati, kot so npr. hemocianin morskega polža, serumski albumin, goveji tiroglobulin ali inhibitor sojin tripsin z uporabo bifunkcionalnega ali derivatizimega sredstva, npr. maleimidobenzoil sulfosukcinimid estra (konjugacija skozi cisteinske ostanke), N-hidroksisukcinimida (skozi lizinske ostanke), glutaraldehida, anhidrida jantarne kisUne, SOC^ aU R¹N=C=NR, kjer sta R in R¹ različni alkilni skupini.Polyclonal antibodies for mpl Ugandan poUpeptide aU fragments are generally raised in animals by multipU subcutaneous (sc) aU intraperitoneal (ip) injections of mpl Uganda and adjuvant. It is advantageous to conjugate the mpl Ugand aU fragment containing the target amino acid sequence with a protein that is immunogenic in the species to be immunized, such as e.g. sea snail hemocyanin, serum albumin, bovine thyroglobulin or a soybean trypsin inhibitor using a bifunctional or derivatizable agent, e.g. maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOC ^ aU R ¹ N = C = NR, where R and R ^{1 are} different alkyl groups.

ŽivaU imuniziramo proti mpl Ugandskemu polipeptidu aU fragmentu, imunogenim konjugatom aU derivatom s kombiniranjem 1 mg od 1 /ig peptida aU konjugata (za zajce oz. miši) s 3 volumni Freundovega kompletnega adjuvansa in injiciramo raztopino intradermalno na multipUh mestih. En mesec kasneje ŽivaU poživimo z 1/5 do 1/10 originalne koUčine peptida aU konjugata v Freundovem kompletnem adjuvansu s subkutano injekcijo na multipUh mestih. 7 do 14 dni kasneje živalim odvzamemo kri in serum testiramo za mpl Ugandski protiteiesni titer. Živalim dajemo poživilne injekcije, dokler ne dosežemo končne točke (plato) titra. Prednostno ŽivaU poživimo s konjugatom istega mpl Uganda, vendar konjugiranega z razUčnim proteinom in/ali skozi razUčen premreževalni reagent. Konjugate lahko naredimo tudi v rekombinantni celični kulturi kot proteinske fuzije. Agregacijska sredstva, kot je alum prav tako lahko uporabimo, da povečamo imunski odziv.Mercury was immunized against the mpl Ugandan polypeptide aU fragment, immunogenic conjugates of aU derivatives by combining 1 mg of 1 / ig peptide aU conjugate (rabbits or mice) with 3 volumes of Freund's complete adjuvant and injected the solution intradermally at multipU sites. One month later, Mercury was resuscitated with 1/5 to 1/10 of the original peptide weight of the aU conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. 7 to 14 days later, the blood is collected from the animals and serum tested for mpl Ugandan anti-body titer. Animals are given booster injections until the end point (plateau) of the titre is reached. Preferably, mercury is resuscitated with a conjugate of the same Ugandan mpl but conjugated with the digest protein and / or through the studied cross-linking reagent. Conjugates can also be made in recombinant cell culture as protein fusions. Aggregation agents such as alum can also be used to increase the immune response.

(u) Monoklonska protitelesa(u) Monoclonal antibodies

Monoklonska protitelesa dobimo iz populacije v bistvu homogenih protiteles, t.j. in99 dividualna protitelesa, ki obsegajo populacijo, so identična, razen možnih naravnih mutacij, ki so lahko prisotne v manjših količinah. Modificiran izraz monoklonski označuje značaj protitelesa, ki ni zmes diskretnih protiteles.Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e. and99 the individual antibodies that comprise the population are identical except for the possible natural mutations that may be present in small quantities. The modified term monoclonal indicates the character of an antibody other than a mixture of discrete antibodies.

Npr. monoklonska protitelesa mpl liganda v smislu izuma lahko naredimo z uporabo hibridomskega postopka, ki so ga prvi opisali Kohler & Milstein, Nature, 256:495 [1975] (US patent št. 4,816,567 [Cabilly et al.]).E.g. mpl ligand monoclonal antibodies of the invention can be made using the hybridoma method first described by Kohler & Milstein, Nature, 256: 495 [1975] (U.S. Patent No. 4,816,567 [Cabilly et al.]).

V hibridomskem postopku miš ali drugo ustrezno gostiteljsko žival, kot je hrček, imuniziramo , kot je opisano pred tem zgoraj, da izzovemo limfocite, ki tvorijo ali so sposobni tvorbe protiteles, da se specifično vežejo na protein, uporabljen za imunizacijo. Alternativno lahko limfocite imuniziramo in vitro. Limfocite nato fuzioniramo z mielomskimi celicami z uporabo prikladnega fuzijskega sredstva, kot je polietilen glikol, da nastane hibridomska celica (Goding, Monoclonal Antibodies: Principles and Practice, str. 59-103 [Academic Press, 1986]).In a hybridoma process, a mouse or other suitable host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies to bind specifically to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent such as polyethylene glycol to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 [Academic Press, 1986]).

Tako pripravljene hibridomske celice zasejemo in pustimo, da rastejo v prikladnem kulturnem mediju, ki prednostno vsebuje eno ali več substanc, ki inhibirajo rast ali preživetje nespojenih parentalnih mielomskih celic. Če npr. parentalne mielomske celice nimajo encima hipoksantin gvanin fosforibozil transferaze (HGPRT ali HPRT), kulturni medij za hibridome značilno vključuje hipoksantin, aminopterin in timidin (medij HAT), pri čemer substance preprečujejo rast celic s pomanjkanjem HGPRT.The hybridoma cells thus prepared are seeded and allowed to grow in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of unconnected parental myeloma cells. If e.g. parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for hybridomas typically includes hypoxanthine, aminopterin and thymidine (HAT medium), with the substances preventing the growth of HGPRT-deficient cells.

Prednostne mielomske celice so tiste, ki se fuzionirajo učinkovito, vzdržujejo stabilen visok nivo ekspresije protitelesa z izbranimi protitelesa tvorečimi celicami in so občutljive za medij, kot je HAT. Med prednostnimi mielomskimi celičnimi linijami so murine mielomske linije, kot tiste, izvedene iz mišjih tumorjev MOPC-21 in MPC-11, dosegljive pri Salk Institute Celi Distribution Center, San Diego, Califomia ZDA, in celice SP-2, dosegljive pri American Type Culture Collection Rockville, Maryland ZDA. Humane mielomske in mišje-humane heteromielomske celične linije so prav tako opisane za izdelavo humanih monoklonskih protiteles (Kozbor, J. Immunol., 133:3001 [1984]; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, str. 51-63, Marcel Dekker, Inc., New York, 1987).Preferred myeloma cells are those that fuse efficiently, maintain a stable high level of antibody expression with selected antibody-forming cells, and are sensitive to a medium such as HAT. Among the preferred myeloma cell lines are murine myeloma lines, such as those derived from mouse tumors MOPC-21 and MPC-11, available from the Salk Institute Whole Distribution Center, San Diego, Califomia USA, and SP-2 cells available from American Type Culture Collection Rockville, Maryland USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 [1984]; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 , Marcel Dekker, Inc., New York, 1987).

Kulturni medij, v katerem rastejo hibridomske celice, testiramo za izdelovanje monoklonskih protiteles, naravnanih proti mpl ligandu. Prednostno določimoThe culture medium in which the hybridoma cells grow is tested for the production of monoclonal antibodies directed against the mpl ligand. We prioritize

100 vezavno specifičnost monoklonskih protiteles, izdelanih od hibridomskih celic, z imunoprecipitacijo ali s testom vezave in vitro, kot je radioimunski test (RIA) ali z encimsko imunskim testom (ELISA).100 binding specificity of monoclonal antibodies made from hybridoma cells by immunoprecipitation or by an in vitro binding assay such as a radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

Vezavno afiniteto monoklonskih protiteles npr. določimo s Scatchardovo analizo od Munsona & Pollarda, Anai. Biochem., 107:220 [1980].The binding affinity of monoclonal antibodies e.g. determined by Scatchard analysis from Munson & Pollard, Anai. Biochem., 107: 220 [1980].

Potem ko hibridomske celice identificiramo, da izdelujejo protitelesa želene specifičnosti, afinitite in/ali aktivnosti, lahko klone subkloniramo z limitiranjem razredčevalnih postopkov in rastjo po standardnih postopkih (Goding, zgoraj). Prikladni kulturni mediji za te namene vključujejo npr. Eaglov medij, modificiran po Dulbecco-u, ali medij RPMI-1640. Poleg tega lahko hibridomske celice rastejo kot ascitesni tumorji v živalih in vivo.After hybridoma cells have been identified to produce antibodies of the desired specificity, affinity and / or activity, the clones can be subcloned by limiting dilution procedures and growing according to standard procedures (Goding, supra). Suitable cultural media for this purpose include e.g. Eagle medium modified by Dulbecco but RPMI-1640 medium. In addition, hybridoma cells can grow as ascites tumors in animals in vivo.

Monoklonska protitelesa, izločena od subklonov, prikladno ločimo iz kulturnega medija, ascitesne tekočine ali seruma s konvencionalnimi imunoglobulinskimi postopki čiščenja, kot so npr. protein-A-sefaroza, hidroksilapatitna kromatografija, gelska elektroforeza, dializa ali afinitetna kromatografija.Monoclonal antibodies secreted from subclones are conveniently separated from the culture medium, ascites fluid or serum by conventional immunoglobulin purification procedures, such as e.g. protein-A-sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

DNA, ki kodira monoklonska protitelesa v smislu izuma, z lahkoto izoliramo in sekvenciramo z uporabo konvencionalnih postopkov (npr. z uporabo oligonukleotidnih sond, ki so sposobne specifičnega vezanja na gene, ki kodirajo težke in lahke verige murinih protiteles). Hibridomske celice v smislu izuma so prednosten vir take DNA. Ko DNA izoliramo, jo lahko plasiramo v ekspresijske vektoije, ki jih nato transfektiramo v gostiteljske celice, kot simian COS, ovarijske celice kitajskega hrčka (celice CHO) ali mielomske celice, ki drugače ne tvorijo imunoglobulinskih proteinov, da pride do sinteze monoklonskih protiteles v rekombinantnih gostiteljskih celicah. DNA lahko modificiramo npr. tudi s substitucijo kodirne sekvence za težke in lahke verige konstantnih humanih domen namesto homolognih murinih sekvenc (Cabily et al., zgoraj; Morrison, et al., Proč. Nat. Acad. Sci., 81:6851 [1984]) ali s kovalentnim povezovanjem z imunoglobulinsko kodirno sekvenco celotne ali dela kodirne sekvence za ne-imunoglobulinski polipeptid.The DNA encoding the monoclonal antibodies of the invention can be easily isolated and sequenced using conventional methods (e.g., using oligonucleotide probes capable of specific binding to genes encoding murine heavy and light chains). The hybridoma cells of the invention are a preferred source of such DNA. Once isolated, DNA can be placed into expression vectors, which are then transfected into host cells, such as simian COS, Chinese hamster ovary cells (CHO cells), or myeloma cells that do not otherwise form immunoglobulin proteins to produce monoclonal antibody synthesis in recombinant host cells. DNA can be modified e.g. also by substituting the coding sequence for heavy and light chains of constant human domains instead of homologous murine sequences (Cabily et al., supra; Morrison, et al., read. Nat. Acad. Sci., 81: 6851 [1984]) or by covalent linking to an immunoglobulin coding sequence of all or part of the coding sequence for a non-immunoglobulin polypeptide.

Značilno take ne-imunoglobulinske polipeptide substituiramo za konstantne domene protitelesa v smislu izuma ali jih substituiramo za variabilno domeno enega antigenvezavnega mesta protitelesa v smislu izuma, da kreiramo kimemo divalentno protitelo, ki obsega eno antigen vezavno mesto s specifičnostjo za mpl ligand in drugoTypically, such non-immunoglobulin polypeptides are substituted for the constant domains of the antibodies of the invention or substituted for the variable domain of one antigen binding site of the invention to create a chimeric divalent antibody comprising one antigen binding site specific for mpl ligand and another

101 antigen vezavno mesto s specifičnostjo za drugačen antigen.101 antigen binding site with specificity for a different antigen.

Kimerna ali hibridna protitelesa lahko pripravimo tudi in vitro z uporabo znanih postopkov v kemiji sintetičnih proteinov, vključno tistih, ki vključujejo premreževalna sredstva. Npr. imunotoksine lahko konstruiramo z reakcijo izmenjave disulfidov ali s tvorbo tioetrske vezi. Primeri prikladnih reagentov za ta namen vključujejo imunotiolat in metil-4-merkaptobutirimidat.Chimeric or hybrid antibodies can also be prepared in vitro using known methods in the chemistry of synthetic proteins, including those involving cross-linking agents. E.g. immunotoxins can be constructed by a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include immunothiolate and methyl-4-mercaptobutyrimidate.

Za diagnostično uporabo značilno označimo protitelesa v smislu izuma z detektibilnim deležem. Detektibilni delež mora biti takšen, da je sposoben tvoriti bodisi direktno ali indirektno detektibilen signal. Detektibilni delež je lahko radioaktiven izotop, kot je ³H, ¹⁴C, ’Ψ, ³⁵S ali¹²⁵J, fluorescentna in kemiluminiscentna spojina, kot je fluorescein, izotiocianat, rodamin ali luciferin; radioaktivno izotopsko označevalno sredstvo, kot je npr. ¹²⁵J, ³²P, ¹⁴C ali ³H, ali encim, kot je alkana fosfataza, betagalaktozidaza ali hrenova peroksidaza.For diagnostic use, the antibodies of the invention are typically labeled with detectable fraction. The detectable fraction must be such that it is capable of producing either a directly or indirectly detectable signal. The detectable fraction may be a radioactive isotope such as ³ H, ¹⁴ C, ^'35 , ³⁵ S or ¹²⁵ J, a fluorescent and chemiluminescent compound such as fluorescein, isothiocyanate, rhodamine or luciferin; a radioactive isotope labeling agent such as e.g. ¹²⁵ J, ³² P, ¹⁴ C or ³ H, or an enzyme such as alkane phosphatase, beta-galactosidase or horseradish peroxidase.

Katerikoli postopek, znan v tehniki, za ločeno konjugiranje protitelesa z detektibilnim deležem, lahko uporabimo, vključno tiste postopke, kijih opisujejo Hunter et al., Nature, 144:945 [1962]; David, et al., Biochemistry, 13:1014 [1974]; Pain, et al., J. Immunol. Meth., 40:219 [1981]; in Nygren, J. Histochem. and Cytochem., 30:407 [1982].Any method known in the art for the separate conjugation of an antibody with a detectable fraction can be used, including those procedures described by Hunter et al., Nature, 144: 945 [1962]; David, et al., Biochemistry, 13: 1014 [1974]; Pain, et al., J. Immunol. Meth., 40: 219 [1981]; and Nygren, J. Histochem. and Cytochem., 30: 407 [1982].

Protelesa v smislu predloženega izuma lahko uporabimo v kateremkoli znanem testu, kot npr. v kompetitivnih vezavnih testih, direktnih in indirektnih sendvič testih in imunoprecipitacijskih testih. Zola, Monoclonal Antibodies: A Manual of Techniques, str. 147-158 (CRC Press. Inc., 1987).The antibodies of the present invention can be used in any known assay, such as e.g. in competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation tests. Zola, Monoclonal Antibodies: A Manual of Techniques, p. 147-158 (CRC Press. Inc., 1987).

Kompetitivni vezavni testi se nanašajo na sposobnost označenega standarda (ki je lahko mpl ligand ali njegov imunološko reaktivni del) za tekmovanje s testnim vzorcem analita (mpl ligand) za vezavo z omejeno količino protitelesa. Količina mpl liganda v testnem vzorcu je obratno proporcionalna količini standarda, ki se veže na protitelesa. Da olajšamo določevanje količine standarda, ki se veže, protitelesa navadno insolubiliziramo pred tekmovanjem ali po njem, tako da standard in analit, ki se vežeta na protitelesa, lahko prikladno ločimo od standarda in analita, ki ostaneta nevezana.Competitive binding assays refer to the ability of a labeled standard (which may be an mpl ligand or its immune-reactive moiety) to compete with an analyte test sample (mpl ligand) for binding with a limited amount of antibody. The amount of mpl ligand in the test sample is inversely proportional to the amount of antibody-binding standard. To facilitate determination of the amount of binding standard, antibodies are usually insolubilized before or after the race, so that the antibody-binding standard and analyte can be conveniently separated from the standard and analyte that remain unbound.

Sendvič testi vključujejo uporabo dveh protiteles, pri čemer je vsako sposobnoSandwich tests involve the use of two antibodies, each capable

102 vezave na različen imunogenski del ali epitop proteina (mpl ligand), ki ga detektiramo. V sendvič testu se testni vzorec analita veže najprej s prvim protitelesom, ki je imobilizirano na trdnem nosilcu, nato pa se drugo protitelo veže na analit in na tak način tvori netopen tridelni kompleks (David & Greene, US patent št. 4,376,110). Drugo protitelo je lahko samo označeno z detektibilnim deležem (direktni sendvič testi), ali pa ga lahko izmerimo z uporabo antiimunoglobulinskega protitelesa, ki je označeno z detektibilnim deležem (indirektni sendvič testi). Npr. en tip sendvič testa je test ELISA, pri čemer je detektibilni delež encim (npr. hrenova peroksidaza).102 binding to a different immunogenic portion or epitope of a protein (mpl ligand) that is detected. In a sandwich assay, the test sample of the analyte first binds to the first antibody immobilized on a solid support, and then the second antibody binds to the analyte, thus forming an insoluble three-part complex (David & Greene, US Patent No. 4,376,110). The second antibody can only be labeled with detectable fraction (direct sandwich tests), or can be measured using an anti-immunoglobulin antibody labeled with detectable fraction (indirect sandwich tests). E.g. one type of sandwich test is the ELISA test, with the detectable fraction being an enzyme (eg horseradish peroxidase).

(iii) Humanizirana in humana protitelesa(iii) Humanized and human antibodies

Postopki za humaniziranje nehumanih protiteles so dobro znani v tehniki. Na splošno ima humanizirano protitelo enega ali več aminokislinskih ostankov, ki so vanj uvedeni iz nehumanega vira. Ti nehumani aminokislinski ostanki so pogosto imenovani uvoženi ostanki, ki so značilno vzeti iz uvožene variabilne domene. Humanizacijo lahko v bistvu izvedemo po postopku Winteqa in sodelavcev (Jones et al., Nature, 321:522-525 [1986]; Riechmann et al., Nature, 332:323-327 [1988]; Verhoeyen et al., Science, 239:1534-1536 [1988]), tako da s CDR ali sekvencami CDR glodalcev substituiramo ustrezne sekvence humanega protitelesa. V skladu s tem so humanizirana protitelesa kimema protitelesa (Cabilly et al., zgoraj), v katerih je bistveno manj kot intaktna humana variabilna domena substituirana z ustrezno sekvenco iz nehumane vrste. V praksi so humanizirana protitelesa, značilno humana protitelesa v katerih je nekaj ostankov CDR in možno nekaj ostankov FR, substituiranih z ostanki iz analognih mest v protitelesih glodavcev.Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as imported residues, which are typically taken from the imported variable domain. Humanization can essentially be performed by the procedure of Winteq et al. (Jones et al., Nature, 321: 522-525 [1986]; Riechmann et al., Nature, 332: 323-327 [1988]; Verhoeyen et al., Science. 239: 1534-1536 [1988]) by substituting the corresponding human antibody sequences by CDR or rodent CDR sequences. Accordingly, humanized chimeric antibodies are antibodies (Cabilly et al., Supra) in which substantially less than the intact human variable domain is substituted by a corresponding sequence from a nonhuman species. In practice, humanized antibodies are typically human antibodies in which there are some CDR residues and possibly some FR residues substituted by residues from analogous sites in rodent antibodies.

Izbira humanih variabilnih domen, tako lahkih kot težkih, za izdelavo humaniziranih protiteles je zelo pomembna, da znižamo antigenost. V skladu s tki. najboljše prilagojenim postopkom sekvenco variabilne domene protitelesa glodalca selekcioniramo za celotno knjižnico znanih humanih sekvenc variabilne domene. Humana sekvenca, ki je najbližja glodalski, je nato sprejeta kot humano ogrodje (FR) za humanizirano protitelo (Sims et al., J. Immunol., 151:2296 [1993]; Chothia in Lesk,The choice of human variable domains, both light and heavy, for the production of humanized antibodies is very important to reduce antigenicity. According to tki. the rodent antibody variable domain sequence is best selected for the whole library of known human variable domain sequence sequences. The human sequence closest to the murine one is then adopted as a human framework (FR) for the humanized antibody (Sims et al. J. Immunol. 151: 2296 [1993]; Chothia and Lesk.

J. Mol. Biol., 196:901 [1987]). Drugi postopek uporablja posebno ogrodje, izvedeno iz konsenzne sekvence posebne podskupine lahkih ali težkih verig popolnoma humanih protiteles. Enako ogrodje lahko uporabimo za številna različna humanizirana protitelesa (Carter et al., Proč. Natl. Acad. Sci. ZDA, 89:4285 [1992]; Presta et al., J. Immnol., 151:2623 [1993]).J. Mol. Biol., 196: 901 [1987]. The second process uses a special framework derived from the consensus sequence of a specific subset of light or heavy chains of fully human antibodies. The same framework can be used for many different humanized antibodies (Carter et al., Proc. Nat. Acad. Sci. USA, 89: 4285 [1992]; Presta et al., J. Immnol., 151: 2623 [1993]).

103103

Nadalje je pomembno, da protitelesa humaniziramo z retencijo visoke afinitete za antigen in druge ugodne biološke lastnosti. Da dosežemo ta cilj v skladu s prednostnim postopkom, pripravimo humanizirana protitelesa s postopkom analiziranja parentalnih sekvenc in različnih konceptualno humaniziranih produktov z uporabo tridimenzionalnih modelov parentalnih in humaniziranih sekvenc. Tridimenzionalni imunoglobulinski modeli so navadno dosegljivi in strokovnjakom znani. Na voljo so računalniški programi, ki ilustrirajo in prikazujejo verjetne tridimenzionalne konformacijske strukture izbranih kandidatnih imunoglobulinskih sekvenc. Natančen pregled teh ponazoritev omogoča analiziranje verjetne vloge ostankov v delovanju kandidatne imunoglobulinske sekvence, tj. analizo ostankov, ki vplivajo na sposobnost kandidatnega imunoglobulina, da veže svoj antigen. Na ta način lahko ostanke FR izberemo in kombiniramo iz konsenzne in uvožene sekvence, tako da dosežemo želene značilnosti protitelesa, da povečamo afiniteto za ciljne antigene. Na splošno ostanki CDR direktno in najbolj bistveno vplivajo na vezavo antigena. Za nadaljnje podrobnosti glej US prijavo ser. št. 07/934,373, vloženo 21. avgusta 1992, ki je delno nadaljevanje prijave s ser. št. 07/715,272, vložene 14. junija 1991.Furthermore, it is important to humanize the antibodies by retaining high affinity for the antigen and other beneficial biological properties. To achieve this objective according to the preferred method, humanized antibodies are prepared by the method of analyzing parental sequences and various conceptually humanized products using three-dimensional models of parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and known to those skilled in the art. Computer programs are available to illustrate and demonstrate the probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. A close examination of these illustrations allows us to analyze the likely role of residues in the function of the candidate immunoglobulin sequence, i. analysis of residues that affect the candidate immunoglobulin's ability to bind its antigen. In this way, FR residues can be selected and combined from the consensus and imported sequences to achieve the desired antibody characteristics to increase the affinity for the target antigens. In general, CDR residues directly and most significantly affect antigen binding. For further details, see US application ser. no. No. 07 / 934,373, filed Aug. 21, 1992, which is, in part, a continuation of the application by Ser. no. No. 07 / 715,272, filed June 14, 1991.

Alternativno je sedaj možno, da vzgojimo transgenske živali (npr. miši), ki so sposobne, da po imunizaciji izdelajo popoln repertoar humanih protiteles v odsotnosti endogene imunoglobulinske produkcije. Opisano je, da je posledica homozigotne delecije težke verige protitelesa, ki povezuje regijo (J_H) gena v kimernih in mutantnih miših germinalne linije, popolna inhibicija izdelovanja endogenih protiteles. Transfer humane germinalno linijske imunoglobulinske genske vrste v take germinalno linijske mutantne miši ima za posledico izdelovanje humanih protiteles po antigenskem izzivu. Glej npr. Jakobovits et al., Proč. Natl. Acad. Sci. ZDA, 90:2551-255 [1993]; Jakobovits et al., Nature, 362:255-258 [1993]; Bruggermann et al., Year in Immuno., 7:33 [1993]. Humana protitelesa prav tako lahko nastajajo v knjižnicah za prikazovanje fagov (Hoogenboom in Winter, J. Mol. Biol. 227,381 [1991]; Marks et al., J. Mol. Biol. 222,581 [1991]).Alternatively, it is now possible to raise transgenic animals (eg mice) capable of producing a complete repertoire of human antibodies after immunization in the absence of endogenous immunoglobulin production. Homozygous deletion of an antibody heavy chain binding the region (J _H ) of a gene in chimeric and mutant germline mice has been described as a complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene species to such germline mutant mice results in the production of human antibodies after the antigen challenge. See, e.g. Jakobovits et al., Proc. Natl. Acad. Sci. U.S.A. 90: 2551-255 [1993]; Jakobovits et al., Nature, 362: 255-258 [1993]; Bruggermann et al., Year in Immuno., 7:33 [1993]. Human antibodies can also be generated in phage display libraries (Hoogenboom and Winter, J. Mol. Biol. 227,381 [1991]; Marks et al., J. Mol. Biol. 222,581 [1991]).

(iv) Bispecifična protitelesa(iv) Bispecific antibodies

Bispecifična protitelesa so monoklonska, prednostno humana ali humanizirana protitelesa, ki imajo vezavne specifičnosti za vsaj dva različna antigena. Postopki za izdelovanje bispecifičnih protiteles so znani v tehniki.Bispecific antibodies are monoclonal, preferably human or humanized antibodies that have binding specificities for at least two different antigens. Methods for making bispecific antibodies are known in the art.

Tradicionalno temelji rekombinantno izdelovanje bispecifičnih protiteles naRecombinant bispecific antibody production has traditionally been based on

104 koekspresiji dveh imunoglobulinskih parov težka veriga-lahka veriga, kjer imata obe težki verigi različne specifičnosti (Millstein in Cuello, Nature, 305:537-539 [1983]). Zaradi naključne izbire imunoglobulinskih težkih in lahkih verig ti hibridomi (kvadromi) izdelujejo potencialno zmes 10 različnih protitelesnih molekul, od katerih ima samo ena pravilno bispecifično strukturo. Čiščenje pravilne molekule, ki navadno poteka s stopnjami afinitetne kromatografije, je precej neugodno, dobitki produkta pa so nizki. Podobni postopki so prikazani v PCT, obj. št. WO 93/08829 (obj. 13. maja 1993) in v Traunecker et al., EMBO, 10:3655-3659 [1991].104 coexpression of two immunoglobulin heavy chain-light chain pairs, where both heavy chains have different specificities (Millstein and Cuello, Nature, 305: 537-539 [1983]). Due to the random selection of immunoglobulin heavy and light chains, these hybridomas (squares) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule, which is usually carried out with affinity chromatography steps, is rather disadvantageous and the product yields are low. Similar procedures are shown in PCT, obj. no. WO 93/08829 (published May 13, 1993) and in Traunecker et al., EMBO, 10: 3655-3659 [1991].

V skladu z različnim in bolj prednostnim pristopom protitelesa variabilne domene z želenimi vezavnimi specifičnostmi (protitelo-antigen vezišča) spojimo z imunoglobulinskimi sekvencami konstantnih domen. Fuzija je prednostno s konstantno domeno imunoglobulinske težke verige, ki obsega vsaj del gibljive, CH2 in CH3 regije. Prednostno je, da je konstantna regija prve težke verige (CHl), ki vsebuje mesto, potrebno za vezavo lahke verige, prisotna v vsaj eni od fuzij. DNA ki kodirajo fuzije imunoglobulinskih težkih verig, in če je potrebno imunoglobulinske lahke verige, inseriramo v ločene ekspresijske vektorje in kotransfektiramo v prikladni gostiteljski organizem. To zagotavlja veliko fleksibilnost pri prilagajanju skupnih razmerij treh polipeptidnih fragmentov v izvedbah, kjer neenaka razmerja treh polipeptidnih verig, uporabljenih pri konstrukciji, zagotavljajo optimalne dobitke. Možna je tudi insercija kodirnih sekvenc za dve ali tri polipeptidne verige v enem ekspresijskem vektorju, če ekspresija vsaj dveh polipeptidnih verig v enakih razmerjih daje visoke dobitke ali, če razmerja niso posebno pomembna. V prednostni izvedbi tega načina so bispecifična protitelesa sestavljena iz hibridne imunoglobulinske težke verige s prvo vezavno specifičnostjo v enem kraku in hibridnega imunoglobulinskega para težka veriga-lahka veriga (zagotovljena druga vezavna specifičnost) v drugem kraku. Ugotovili smo, da ta asimetrična struktura pospešuje ločitev želenih bispecifičnih spojin iz neželene kombinacije imunoglobulinske verige, ker prisotnost imunoglobulinske lahke verige samo v eni polovici bispecifične molekule zagotavlja lažjo pot ločitve. Ta način je prikazan v prijavi, ki je istočasno v postopku s ser. št. 07/931,811, vloženi 17. avgusta 1992.According to a different and more preferred approach, variable domain antibodies with desired binding specificities (antibody-binding antigen) are coupled to immunoglobulin constant domain sequences. Fusion is preferably with a constant immunoglobulin heavy chain domain comprising at least part of the moving, CH2 and CH3 region. Preferably, a constant region of the first heavy chain (CH1) containing the site required for light chain binding is present in at least one of the fusions. DNAs encoding fusion immunoglobulin heavy chains, and if necessary immunoglobulin light chains, are inserted into separate expression vectors and cotransfected into a suitable host organism. This provides great flexibility in adjusting the joint ratios of the three polypeptide fragments in embodiments where the unequal ratios of the three polypeptide chains used in the construction provide optimal yields. It is also possible to insert coding sequences for two or three polypeptide chains in one expression vector if the expression of at least two polypeptide chains in equal proportions yields high yields or if the ratios are not particularly significant. In a preferred embodiment of this method, the bispecific antibodies consist of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin pair heavy chain-light chain (guaranteed second binding specificity) in the second arm. We found that this asymmetric structure accelerates the separation of the desired bispecific compounds from the unwanted immunoglobulin chain combination, since the presence of the immunoglobulin light chain in only one half of the bispecific molecule provides an easier separation path. This mode is shown in the application, which is currently under procedure with Ser. no. No. 07 / 931,811, filed Aug. 17, 1992.

Nadaljnje podrobnosti za izdelovanje bispecifičnih protiteles opisujejo npr. Suresh et al., Methods in Enzymology, 121:210 [1986].Further details for the production of bispecific antibodies describe e.g. Suresh et al., Methods in Enzymology, 121: 210 [1986].

(v) Heterokonjugirana protitelesa(v) Heteroconjugated antibodies

105105

Heterokonjugirana protitelesa tudi spadajo v obseg predloženega izuma. Heterokonjugirana protitelesa so sestavljena iz dveh kovalentno povezanih protiteles. Taka protitelesa so predlagana za ciljne celice imunskega sistema za neželene celice (US patent št. 4,676,980) in za zdravljenje infekcij s HIV (PCT obj. št. WO 91/00360 in WO 92/00373; EP 03089). Heterokonjugirana protitelesa lahko naredimo z uporabo kateregakoli prikladnega premreževalnega postopka. Prikladna premreževalna sredstva so dobro znana v tehniki in so prikazana v US patentu št. 4,676,980 skupaj s številnimi premreževalnimi tehnikami.Heteroconjugated antibodies are also within the scope of the present invention. Heteroconjugated antibodies consist of two covalently linked antibodies. Such antibodies have been proposed for target cells of the immune system for unwanted cells (US Patent No. 4,676,980) and for the treatment of HIV infection (PCT Publication Nos. WO 91/00360 and WO 92/00373; EP 03089). Heteroconjugated antibodies can be made using any suitable cross-linking procedure. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980 together with a number of cross-linking techniques.

IV. Terapevtska uporaba megakariocitopoetičnega proteinskega mpl ligandaIV. Therapeutic use of megakaryocytopoietic protein mpl ligand

Biološko aktivni mpl ligand s hematopoetičnim efektorskem delovanjem, imenovan tukaj megakariocitopoetični ali trombocitopoetični protein (TPO), lahko uporabimo v sterilnem farmacevtskem pripravku ali formulaciji, da stimuliramo megakariocitopoetično ali trombopoetično aktivnost pri pacientih, ki trpijo zaradi trombocitopenije, zaradi poslabšanega nastajanja, sekvestracije ali povečane destrukcije trombocitov. Hipoplazijo kostnega mozga, povezano s trombocitopenijo (npr. aplastična anemija po kemoterapiji ali transplantaciji kostnega mozga) lahko učinkovito zdravimo s spojinami v smislu izuma, kot tudi motnje, kot je razširjena intravaskulama koagulacija (DIC), imuna trombocitopenija (ki vključuje ΓΓΡ inducirano s HIV in ΓΓΡ neinducirano s HIV), kronična idiopatična trombocitopenija, kongenitalna trombocitopenija, mielodisplazija in trombotična trombocitopenija. Poleg tega so ti megakariocitopoetični proteini lahko koristni pri zdravljenju mieloproliferativnih trombocitotičnih bolezni kot tudi trombocitoze zaradi vnetnih pogojev in pomankanja železa.A biologically active mpl ligand with hematopoietic effector activity, hereinafter referred to as megakaryocytopoietic or thrombocytopoietic protein (TPO), can be used in a sterile pharmaceutical composition or formulation to stimulate megakaryocytopoietic or thrombopoietic activity in patients suffering from thrombosis, thrombosis, platelets. Thrombocytopenia-related bone marrow hypoplasia (eg, aplastic anemia after chemotherapy or bone marrow transplantation) can be effectively treated with the compounds of the invention, as well as disorders such as widespread intravascular coagulation (DIC), immune thrombocytopenia (which includes HIV induced by HIV and ΓΓΡ non-HIV-induced), chronic idiopathic thrombocytopenia, congenital thrombocytopenia, myelodysplasia, and thrombotic thrombocytopenia. In addition, these megakaryocytopoietic proteins may be useful in the treatment of myeloproliferative thrombocytotic diseases as well as thrombocytosis due to inflammatory conditions and iron deficiency.

Prednostne uporabe megakariocitopoetičnega ali trombocitopoetičnega proteina (TPO) v smislu izuma so v: mielotoksični kemoterapiji za zdravljenje levkemije ali trdih tumoijev, mieloablativni kemoterapiji za avtologne ali alogene transplantacije kostnega mozga, mielodisplaziji, idiopatični aplastični anemiji, kongenitalni trombocitopeniji in imuni trombocitopeniji.Advantageous uses of megakaryocytopoietic or thrombocytopoietic protein (TPO) of the invention are in: myelotoxic chemotherapy for the treatment of leukemia or solid tumors, myeloablative chemotherapy for autologous or allogeneic bone marrow transplants, myelodysplasia, idiopathic aplopenic thrombocytopenia.

Nadaljnje motnje, ki jih uspešno zdravimo z megakariocitopoetičnimi proteini v smislu izuma, vključujejo defekte ali poškodbe trombocitov, nastale zaradi zdravil, zastrupitev ali aktivacije na umetnih površinah. V teh primerih lahko uporabimo predložene spojine, da stimuliramo prelivanje novih, nepoškodovanih trombocitov. Bolj popoln seznam koristnih aplikacij je naveden pod naslovom ozadje,Further disorders successfully treated with megakaryocytopoietic proteins of the invention include platelet defects or damage resulting from drugs, poisoning or activation on artificial surfaces. In these cases, the compounds provided may be used to stimulate the spillover of new, intact platelets. A more complete list of useful applications is provided under the heading Background,

106 zgoraj, posebno sekcije (a)-(f) in v tam citiranih referencah.106 above, especially sections (a) - (f) and references cited therein.

Megakariocitopoetične proteine v smislu predloženega izuma lahko uporabimo same ali v kombinaciji z drugimi citokini, hematopoetini, interlevkini, rastnimi faktorji ali protitelesi pri zdravljenju zgoraj identificiranih motenj ali stanj. Tako lahko predložene spojine uporabimo v kombinaciji z drugimi proteini ali peptidi, ki imajo trombopoetično aktivnost in vključujejo: G-CSF, GM-CSF, LIF, M-CSF, IL-1, IL-3, eritropoetin (EPO), kit ligand, IL-6 in IL-11.The megakaryocytopoietic proteins of the present invention can be used alone or in combination with other cytokines, hematopoietins, interleukins, growth factors or antibodies in the treatment of the disorders or conditions identified above. Thus, the present compounds can be used in combination with other proteins or peptides having thrombopoietic activity and include: G-CSF, GM-CSF, LIF, M-CSF, IL-1, IL-3, erythropoietin (EPO), kit ligand, IL-6 and IL-11.

Megakariocitopoetične proteine v smislu izuma pripravimo v zmesi s farmacevtsko sprejemljivim nosilcem. Ta terapevtski sestavek lahko damo intravenozno ali skozi nos ali pljuča. Sestavek lahko po želji damo parenteralno ali subkutano. Pri sistematičnem dajanju terapevtski sestavek ne sme biti pirogen, mora pa biti v parenteralno sprejemljivi raztopini, ki je glede na pH izotonična in stabilna. Ti pogoji so znani strokovnjakom. Na kratko, dozirne formulacije spojin v smislu izuma pripravimo za shranjevanje ali dajanje, z mešanjem spojine, ki ima želeno stopnjo čistote, s fiziološko sprejemljivimi nosilci, polnili ali stabilizatorji. Taki materiali so netoksični za recipiente v uporabljenih dozah in koncentracijah in vključujejo pufre, kot so fosfat, citrat, acetat in druge soli organskih kislin; antioksidante kot je askorbinska kislina; peptide z nizko molekulsko maso (manj od približno 10 ostankov), kot je poliargenin, proteine, kot je serumski albumin, želatino ali imunoglobuline; hidrofilne polimere, kot je polivinilpirolidinon; amino kisline, kot so glicin, glutaminska kislina, asparaginska kislina ali arginin; monosaharide, disaharide in druge ogljikohidrate, vključno celulozo ali njene derivate, glukozo, manozo ali dekstrine; kelima sredstva kot je EDTA; sladkorne alkohole, kot manitol ali sorbitol; protiione, kot je natrij in/ali neionska površinska sredstva, kot je Tween Pluronic ali polietilenglikol.The megakaryocytopoietic proteins of the invention are prepared in admixture with a pharmaceutically acceptable carrier. This therapeutic composition can be administered intravenously or through the nose or lungs. The composition may optionally be administered parenterally or subcutaneously. When administered systemically, the therapeutic composition must not be pyrogenic but must be in a parenterally acceptable solution that is isotonic and stable with respect to pH. These conditions are known to those skilled in the art. Briefly, the dosage formulations of the compounds of the invention are prepared for storage or administration, by mixing a compound having the desired purity level with physiologically acceptable carriers, fillers or stabilizers. Such materials are non-toxic to recipients at the doses and concentrations used and include buffers such as phosphate, citrate, acetate and other salts of organic acids; antioxidants such as ascorbic acid; low molecular weight peptides (less than about 10 residues) such as polyarginine, proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidinone; amino acids such as glycine, glutamic acid, aspartic acid or arginine; monosaccharides, disaccharides and other carbohydrates, including cellulose or derivatives thereof, glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as mannitol or sorbitol; counterions such as sodium and / or non-ionic surfactants such as Tween Pluronic or polyethylene glycol.

Približno 0,5 do 500 mg spojine ali zmesi megakariocitopoetičnega proteina v obliki proste kisline ali baze ali farmacevtsko sprejemljive soli združimo s fiziološko sprejemljivim nosilcem, polnilom, vezivom, prezervativom, stabilizatorjem, aromo itd., kot zahteva sprejeta farmacevtska praksa. Količina aktivne sestavine v teh sestavkih je taka, da dobimo prikladno doziranje v označenem območju.Approximately 0.5 to 500 mg of a compound or mixture of megakaryocytopoietic protein in the form of a free acid or base or a pharmaceutically acceptable salt is combined with a physiologically acceptable carrier, filler, binder, condom, stabilizer, flavor, etc., as required by accepted pharmaceutical practice. The amount of active ingredient in these compositions is such that suitable dosage is obtained within the indicated range.

Sterilne sestavke za injekcijo lahko formuliramo v skladu s konvencionalno farmacevtsko prakso. Npr. raztopine ali suspenzije aktivne spojine v nosilcih, kot je voda ali naravno rastlinsko olje, kot je sezamovo ali arašidno ali olje bombažnihSterile injectable compositions may be formulated in accordance with conventional pharmaceutical practice. E.g. solutions or suspensions of the active compound in carriers such as water or natural vegetable oil such as sesame or peanut or cottonseed oil

107 semen ali sintetična maščoba, kot etil oleat ipd., so lahko potrebne. Pufre, prezervative, antioksidante ipd. lahko vgradimo v skladu s sprejeto farmacevtsko prakso.107 seeds or synthetic fat, such as ethyl oleate, etc., may be required. Buffers, condoms, antioxidants, etc. can be incorporated in accordance with accepted pharmaceutical practice.

Prikladni primeri pripravkov za zadržano sproščanje vključujejo semipermeabilne matrice trdnih hidrofobnih polimerov, ki vsebujejo polipeptid, pri čemer so matrice v obliki izdelkov, kot so npr. tanke plasti ali mikrokapsule. Primeri matric za zadržano sproščanje vključujejo poliestre, hidrogele [npr. poli(2-hidroksietil-metakrilat), kot opisujejo Langer et al., J. Biomed. Mater. Res., 15:167-277 [1981] in Langer, Chem. Tech., 12:98-105 [1982], ali poli(vinilalkohol)], polilaktide (US patent št. 3,773,919, EP 58,481), sopolimere L-glutaminske kisline in gama etil-L-glutamata (Sidman et al., Biopolymers, 22:547-556 [1983]), nerazgradljivi etilen-vinil acetat (Langer et al., zgoraj), razgradljive sopolimere mlečne kisline-glikolne kisline, kot je Lupron Depot™ (injicime mikrokroglice sestavljene iz sopolimera mlečne kisline-glikolne kisline in levprolid acetat) in poli-D-(-)-3-hidroksimasleno kislino (EP 133,988).Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing a polypeptide, the matrices being in the form of products such as e.g. thin layers or microcapsules. Examples of sustained release matrices include polyesters, hydrogels [e.g. poly (2-hydroxyethyl methacrylate) as described by Langer et al., J. Biomed. Mater. Res., 15: 167-277 [1981] and Langer, Chem. Tech., 12: 98-105 [1982], or poly (vinyl alcohol)], polylactides (U.S. Patent No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers , 22: 547-556 [1983]), non-degradable ethylene-vinyl acetate (Langer et al., Supra), degradable lactic acid-glycolic acid copolymers such as Lupron Depot ™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D - (-) - 3-hydroxybutyric acid (EP 133,988).

Medtem ko polimeri, kot sta etilen-vinilni acetat in mlečna kislina-glikolna kislina omogočajo sproščanje molekul nad 100 dni, pa določeni hidrogeli sproščajo proteine krajši čas. Če zakapsulirani proteini ostanejo v telesu daljši čas, se lahko denaturirajo ali agregirajo kot posledica izpostavitve vlagi pri 37 °C, kar ima za posledico izgubo biološke aktivnosti in možne spremembe v imunogenosti. Racionalne spremembe si lahko zamislimo za proteinsko stabilizacijo, odvisno od vključenega mehanizma. Če odkrijemo, da je mehanizem agregacije tvorba intermolekulske S-S vezi skozi disulfidne izmenjave, lahko dosežemo stabilizacijo z modificiranjem sulfhidrilnih ostankov, liofiliziranjem iz kislinskih raztopin, kontroliranjem vsebnosti vlage, z uporabo ustreznih aditivov in z razvijanjem specifičnih polimernih matričnih sestavkov. Megakariocitopoetični proteinski sestavki za zadržano sproščanje vključujejo tudi liposomsko vgrajene megakariocitopoetične proteine. Uposome, ki vsebujejo megakariocitopoetični protein, pripravimo po postopkih, znanih per se: DE 3,218,121; Epstein et al., Proč. Natl. Acad. Sci. ZDA, 82:3688:3692 [1985]; Hwang et al., Proč. Natl. Acad. Sci. ZDA, 77:4030-4034 [1980]; EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; japonska patentna prijava 83-118008; US patenta št. 4,485,045 in 4,544,545; in EP 102,324. Navadno so liposomi majhni (približno 20-80 nm), enolamelnega tipa, v njih pa je vsebnost lipidov večja od približno 30 mol.% holesterola, pri čemer izbrano razmerje prilagodimo za optimalno megakariocitopoetično proteinsko terapijo. Doziranje določi spremljajoči zdravnik ob upoštevanju raznih faktorjev, znanih, da modificirajo delovanje zdravil, kot so resnost in tip bolezni, telesna masa, spol, dieta, čas in način dajanja, druga zdravila inWhile polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow the release of molecules beyond 100 days, certain hydrogels release proteins for a shorter time. If the encapsulated proteins remain in the body for an extended period, they can be denatured or aggregated as a result of exposure to humidity at 37 ° C, resulting in a loss of biological activity and possible changes in immunogenicity. We can think of rational changes for protein stabilization, depending on the mechanism involved. If the aggregation mechanism is found to be the formation of an intermolecular S-S bond through disulfide exchanges, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling the moisture content, using appropriate additives, and developing specific polymer matrix compositions. Megakaryocytopoietic protein compositions for sustained release also include liposomally incorporated megakaryocytopoietic proteins. Assumptions containing megakaryocytopoietic protein are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. U.S.A. 82: 3688: 3692 [1985]; Hwang et al., Off. Natl. Acad. Sci. U.S.A. 77: 4030-4034 [1980]; EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patent application 83-118008; U.S. Pat. No. 4,485,045 and 4,544,545; and EP 102,324. Typically, liposomes are small (about 20-80 nm), single-lamellar, and have a lipid content greater than about 30 mol% of cholesterol, adjusting the ratio selected for optimal megakaryocytopoietic protein therapy. The dosage is determined by the attending physician, taking into account the various factors known to modify the action of the drugs, such as the severity and type of the disease, weight, sex, diet, timing and route of administration, other medicines and

108 drugi relevantni klinični faktorji. Značilno je dnevni režim v območju od 0,1-100 /ig/kg telesne mase, prednostno je doziranje v območju od 0,1-50 μ-g/kg telesne mase. Bolj prednostno je začetno doziranje v območju od 1 do 5 jtig/kg/dan. V danem primeru je dozirno območje enako kot za druge citokine, posebno G-CSF, GM-CSF in EPO. Terapevtsko učinkovite doze lahko določimo s postopki in vitro ali in vivo.108 other relevant clinical factors. Typically, the daily regimen is in the range of 0.1-100 / g / kg body weight, preferably dosing in the range of 0.1-50 μ-g / kg body weight. More preferred is an initial dosage in the range of 1 to 5 µg / kg / day. In the given case, the dosage range is the same as for other cytokines, in particular G-CSF, GM-CSF and EPO. Therapeutically effective doses can be determined by in vitro or in vivo procedures.

PRIMERIEXAMPLES

Verjamemo, da lahko strokovnjaki brez nadaljnjega opisa, ob uporabi predhodnega opisa in ilustrativnih Primerov, naredijo in uporabijo predloženi izum v popolnem obsegu. Naslednji delovni Primeri tako specifično ponazarjajo prednostne izvedbe predloženega izuma in niso narejeni tako, da bi kakorkoli omejevali preostali opis.It is believed that, without further description, using the foregoing description and the illustrative Examples, it will be appreciated that those skilled in the art can make and use the present invention to the fullest extent. The following working Examples thus specifically illustrate preferred embodiments of the present invention and are not designed to limit in any way the rest of the description.

PRIMERIEXAMPLES

Delno čiščenje prašičjega mpl ligandaPartial purification of pig mpl ligand

Trombocitno revno plazmo zberemo v normalnih ali aplastično anemičnih prašičih. Prašiče naredimo aplastične z obsevanjem z 900 cGy celotnega obsevanja telesa z uporabo linearnega pospeševalnika 4meV. Obsevane prašiče vzdržujemo 6-8 dni z intramuskularnimi injekcijami cefazolina. Nato celoten volumen njihove krvi odstranimo pod splošno anestezijo, hepariniziramo in centrifugiramo pri 1800 g 30 minut, da naredimo trombocitno revno plazmo. Vrh megakariocitne stimulime aktivnosti ugotovimo 6 dni po obsevanju.Platelet poor plasma is collected in normal or aplastically anemic pigs. Pigs are made aplastic by irradiating 900 cGy of total body irradiation using a linear 4meV accelerator. Irradiated pigs were maintained for 6-8 days by intramuscular injection of cefazolin. The whole volume of their blood is then removed under general anesthesia, heparinized and centrifuged at 1800 g for 30 minutes to make platelet-poor plasma. The peak of megakaryocyte stimulus activity was detected 6 days after irradiation.

Aplastično prašičjo plazmo, ki jo dobimo iz obsevanih prašičev, naredimo 4M z NaCl in mešamo 30 minut pri sobni temperaturi. Nastalo oborino odstranimo s centrifugiranjem s 3800 obr./min v Sorvallu RC3B in supernatant napolnimo na kolono fenil-Toyopearl (220 ml) uravnoteženo v NaPO₄ (10 mM), ki vsebuje NaG (4M). Kolono izpiramo s tem pufrom, dokler ni A^ <0,05 in eluiramo z dH^O. Eluirani proteinski vrh razredčimo z dH₂O do konduktivnosti 15mS in s tem polnimo kolono blue-sefaroze, uravnoteženo v PBS (240 ml). Nato kolono izperemo s 5 kolonskimi volumni vsakega PBS in NaPO₄ (10 mM) (pH 7,4), ki vsebuje sečnino (2M). Proteine eluiramo iz kolone z NaPO₄ (10 mM) (pH 7,4), ki vsebuje sečnino (2M) in NaG (IM). Iz eluiranega proteinskega vrha naredimo 0,01 % oktil glukozid(n-oktil /3-D-glukopiranozid) in 1 mM, v obeh primerih z EDTA in Pefabloc-om (Boehinger Mannheim), in le-to polnimo direktno na tandemsko vezaniAplastic pig plasma obtained from irradiated pigs was made 4M with NaCl and stirred for 30 minutes at room temperature. The resulting precipitate was removed by centrifugation at 3800 rpm in Sorvall RC3B and the supernatant was loaded onto a phenyl-Toyopearl column (220 ml) equilibrated in NaPO ₄ (10 mM) containing NaG (4M). Wash the column with this buffer until A ^ <0.05 and elute with dH ^ O. The eluted protein peak was diluted with dH ₂ O to a conductivity of 15 mS, thereby filling a blue-Sepharose column equilibrated in PBS (240 ml). The column was then washed with 5 column volumes of each PBS and NaPO ₄ (10 mM) (pH 7.4) containing urea (2M). Proteins were eluted from the column with NaPO ₄ (10 mM) (pH 7.4) containing urea (2M) and NaG (IM). 0.01% octyl glucoside (n-octyl / 3-D-glucopyranoside) and 1 mM are made from the eluted protein peak and in both cases EDTA and Pefabloc (Boehinger Mannheim) charged and charged directly to the tandem bound

109 koloni CD4-IgG (Capon, D.J. et al. Nature 337:525-531 [1989]) in mp/-IgG Ultralink (Pierce) (glej spodaj). Kolono CD4-IgG (2 ml) odstranimo, potem ko vzorec napolnimo, kolono mp/-IgG (4 ml) pa izperemo z lO-kolonskimi volumni vsakega PBS in PBS, ki vsebuje NaCl (2 M), in eluiramo z glicin-HCl (0,1 M), pH 2,25. Frakcije zberemo v 1/10 volumna tris-HQ (1 M) (pH 8,0).109 columns of CD4-IgG (Capon, D.J. et al. Nature 337: 525-531 [1989]) and mp / -IgG Ultralink (Pierce) (see below). The CD4-IgG column (2 ml) was removed after filling the sample and the mp / -IgG column (4 ml) was washed with 10 column volumes of each PBS and PBS containing NaCl (2 M) and eluted with glycine-HCl (0.1 M), pH 2.25. The fractions were collected in 1/10 volume of tris-HQ (1 M) (pH 8.0).

Iz analize frakcij, eluiranih iz znp/-afinitetne kolone z SDS-PAGE (4-20 %, Novex gel), ki poteka pri redukcijskih pogojih, je razvidna prisotnost različnih proteinov (sl. 5). Proteini, ki se obarvajo s srebrom z najmočnejšo intenziteto, se ločijo z jasno Mr 66000, 55000, 30000, 28000 in 14000. Da določimo, kateri od teh proteinov stimulira proliferacijo BafF3-mpl celičnih kultur, te proteine eluiramo iz gela, kot je opisano v Primeru 2 spodaj.The analysis of the fractions eluted from the Znp / affinity column by SDS-PAGE (4-20%, Novex gel) under reducing conditions shows the presence of different proteins (Fig. 5). The proteins that stain with the strongest intensity of silver are separated by clear Mr 66000, 55000, 30000, 28000 and 14000. To determine which of these proteins stimulates the proliferation of BafF3-mpl cell cultures, these proteins are eluted from the gel as described in Example 2 below.

Afinitetne kolone UltralinkUltralink affinity columns

10-20 mg mpl-lgG ali CD4-IgG v PBS pripojimo na 0,5 g smole Ultralink (Pierce) po navodilih izdelovalca.10-20 mg of mpl-IgG or CD4-IgG in PBS were attached to 0.5 g of Ultralink resin (Pierce) according to the manufacturer's instructions.

Konstrukcija in ekspresija /npMgGConstruction and expression / npMgG

Kimemo molekulo, ki obsega celotno ekstracelično domeno humanega mpl (amino kisline 1-491) in regijo Fc humane molekule IgGl eksprimiramo v 293 celic. Fragment cDNA, ki kodira amino kisline 1-491 humanega mpl, dobimo s PCR iz humane megakariocitične CMK celične cDNA knjižnice in sekvenciramo. Mesto Clal inseriramo na 5’ koncu in mesto BstEII na 3’ koncu. Ta fragment kloniramo navzgor od kodirne regije IgGI Fc v vektor Bluescript med mesti Clal in BstEII po delni digestiji produkta PCR z BstEII zaradi dveh drugih mest BstEII, prisotnih v DNA, ki kodira ekstracelično domeno mpl. Mesto BstEII, uvedeno na 3’ koncu produkta PCR mpl je označeno, da ima regijo Fc v okviru z mpl ekstra celično domeno. Konstrukt subkloniramo v vektor pRK5-tkneo med mesti Clal in Xbal in transfektiramo v 293 humanih embrionalnih ledvičjih celic po postopku s kalcijevim fosfatom. Celice zberemo v 0,4 mg/ml G418 in izoliramo idividualne klone. Ekspresijo mpl-lgG iz izoliranih klonov določimo z uporabo ELISE, specifične za humani Fc. Najboljši ekspresijski klon ima ekspresijski nivo 1-2 mg/ml tnpl-lgG.A molecule comprising the entire extracellular domain of human mpl (amino acids 1-491) is knocked out and the Fc region of the human IgG1 molecule is expressed in 293 cells. A cDNA fragment encoding the amino acids 1-491 of human mpl is obtained by PCR from a human megakaryocytic CMK cell cDNA library and sequenced. Insert the Clal site at the 5 'end and the BstEII site at the 3' end. This fragment is cloned upstream of the IgGI Fc coding region into the Bluescript vector between Clal and BstEII sites after partial digestion of the PCR product with BstEII due to two other BstEII sites present in the DNA encoding the extracellular domain of mpl. The BstEII site introduced at the 3 'end of the PCR mpl product is indicated to have an Fc region within the mpl extra cellular domain. The construct was subcloned into the pRK5-vector vector between Clal and Xbal sites and transfected into 293 human embryonic kidney cells by calcium phosphate procedure. Cells were collected in 0.4 mg / ml G418 and isolated clones were isolated. The expression of mpl-lgG from isolated clones was determined using ELISA specific for human Fc. The best expression clone has an expression level of 1-2 mg / ml tnpl-1gG.

Ba/F3 mpl P eksprimime celiceBa / F3 mpl P express cell

110 cDNA, ki ustreza celotni kodirni regiji humanega mpl P kloniramo v pRK5-tkneo, ki ga nato lineariziramo z Noti in transfektiramo v celično linijo Ba/F3, odvisno od IL-3, z elektroporacijo (lxl0⁷ celic, 9605F, 250V). Tri dni kasneje začnemo selekcijo v prisotnosti 2mg/ml G418. Celice zberemo kot poole ali dobimo individualne klone z omejeno razredčitvijo na ploščah s 96 vdolbinicami. Izbrane celice vzdržujemo v RPMI, ki vsebuje FBS (15 %), 1 mg/ml G418, glutamin (20 mM), HEPES (10 mM) in 100 μg/ml Pen-Strepa. Ekspresijo mpl P v zbranih klonih določimo z analizo FACS z uporabo zajčjih poliklonskih protiteles anti-mp/ P.110 cDNA corresponding to the entire coding region of human mpl P was cloned into pRK5-tkna, which was then linearized with Noti and transfected into IL-3-dependent Ba / F3 cell line by electroporation (1x10 ⁷ cells, 9605F, 250V). Three days later, selection is initiated in the presence of 2mg / ml G418. Cells were harvested as pooles or individual clones were diluted with limited dilution on 96-well plates. The selected cells were maintained in RPMI containing FBS (15%), 1 mg / ml G418, glutamine (20 mM), HEPES (10 mM) and 100 μg / ml Pen-Strep. The expression of mpl P in the clones collected was determined by FACS analysis using rabbit anti-mp / P rabbit polyclonal antibodies.

Ba/F3 mpl ligandski test mpl ligandski test vodimo, kot je prikazano na sl. 2. Da določimo prisotnost mpl liganda iz različnih virov celice Ba/F3 mpl P stradamo IL-3 24 ur pri celični gostoti 5x1ο⁵ celic/ml v ovlaženem inkubatorju pri 37 °C v mešanici zraka in 5 % CO₂. Po končanem stradanju celice zasadimo v posode za kulturo s 96 vdolbinicami z gostoto 50000 celic v 200 μΐ medija z razredčenimi vzorci ali brez in kultiviramo 24 ur v inkubatorju za celično kulturo. 20 μΐ medija RPMI brez seruma, ki vsebuje 1 μ-Ci ³Htimidina, dodamo v vsako vdolbinico za 6-8 ur. Celice nato zberemo na filtrimih ploščah GF/C s 96 vdolbinicami in izperemo 5-krat z vodo. Filtre preštejemo v prisotnosti 40 μΐ scintilacijske tekočine (Microscint 20) v napravi za štetje Packard Top Count.Ba / F3 mpl ligand assay The mpl ligand assay is run as shown in FIG. 2. To determine the presence of the mpl ligand from different Ba / F3 mpl P cell sources, IL-3 is starved for 24 hours at a cell density of 5x1ο ⁵ cells / ml in a humidified incubator at 37 ° C in a mixture of air and 5% CO ₂ . After the starvation is complete, the cells are planted in 96-well culture dishes with a density of 50000 cells in 200 μΐ medium with or without diluted samples and cultured for 24 hours in a cell culture incubator. 20 μΐ serum-free RPMI medium containing 1 μ-Ci ³ Hymimidine was added to each well for 6-8 hours. The cells were then collected on 96-well GF / C filter plates and washed 5 times with water. Filters are counted in the presence of 40 μΐ scintillation fluid (Microscint 20) in a Packard Top Count counter.

PRIMER 2EXAMPLE 2

Visoko očiščeni prašičji mpl ligandHighly purified pig mpl ligand

Predpis gelske elucijeGel elution prescription

Enake količine afinitetno očiščenega mpl liganda (frakcija 6, eluirana iz kolone mplIgG) in vzorčnega pufra 2X Laemmli zmešamo pri sobni temperaturi brez redukcijskega sredstva in damo na poliakrilamidni gel Novex 4-20 %, kolikor hitro je mogoče. Vzorec se ne segreje. Kot kontrolo vzdržujemo vzorčni pufer brez liganda v sosednji liniji. Gel vzdržujemo pri 4-6 °C pri 135 voltih približno 2 1/4 ure. Pufer vzdržujemo na začetku pri sobni temperaturi. Gel nato odstranimo iz predela za gel in ploščo na eni strani gela odstranimo.Equal amounts of affinity-purified mpl ligand (fraction 6 eluted from the mplIgG column) and sample buffer 2X Laemmli were mixed at room temperature without reducing agent and applied to Novex polyacrylamide gel 4-20% as fast as possible. The sample does not heat up. As a control, sample ligand-free buffer in the adjacent line was maintained. The gel was maintained at 4-6 ° C at 135 volts for about 2 1/4 hours. The buffer was initially maintained at room temperature. The gel is then removed from the gel compartment and the plate on one side of the gel is removed.

111111

Repliko gela naredimo na nitrocelulozi, kot sledi: kos nitroceluloze omočimo z destilirano vodo in previdno položimo na vrh eksponirane površine gela, tako da izključimo zračne mehurčke. Izhodiščne (fiducial) oznake damo na nitrocelulozo in gelsko ploščo, tako da lahko repliko natančno repozicioniramo po barvanju. Po približno 2 minutah nitrocelulozo previdno odstranimo in gel ovijemo v ovitek iz umetne snovi in damo v hladilnik. Nitrocelulozo obarvamo z Bioradovim zlatim barvilom, ki obarva celotne proteine (Biorad’s gold total protein stain), tako da jo najprej mešamo v 3x10 ml Tweena 20 (0,1 %) + NaG (0,5 M) + tris-HG (0,1 M) (pH 7,5) približno 45 minut, nato pa s 3x10 ml očiščene vode 5 minut. Nato dodamo zlato barvilo in pustimo, da se razvija, dokler proge v standardih niso vidne. Repliko nato speremo z vodo, položimo čez ovitek iz umetne snovi v modelu in previdno poravnamo z izhodiščnimi oznakami. Pozicije standardov Novex označimo na gelski plošči in narišemo linije, da označimo mesta za prerez. Nitrocelulozo in ovitek iz umetne snovi nato odstranimo in gel prerežemo vzdolž označenih linij z ostrim rezilom britve. Prerezi se razširijo čez vzorčno linijo, tako da jih lahko uporabimo, da določimo pozicije za rezine, ko obarvamo gel. Ko rezine odstranimo, preostali gel obarvamo s srebrom in izmerimo pozicije standardov in oznake prerezov. Molekulske mase, ki ustrezajo pozicijam prerezov, določimo s standardi Novex.The gel replica is made on nitrocellulose as follows: wet a piece of nitrocellulose with distilled water and carefully place it on top of the exposed gel surface by excluding air bubbles. The fiducial markings are placed on the nitrocellulose and the gel plate so that the replica can be accurately repositioned after staining. After about 2 minutes, carefully remove the nitrocellulose and wrap the gel in a plastic wrap and place in the refrigerator. The nitrocellulose was stained with Biorad's gold colorant, which stained the whole proteins (Biorad's gold total protein stain), by first mixing it in 3x10 ml of Tween 20 (0.1%) + NaG (0.5 M) + tris-HG (0. 1 M) (pH 7.5) for about 45 minutes, then with 3x10 ml of purified water for 5 minutes. Then add the gold dye and allow it to develop until the lines are visible in the standards. The replica is then rinsed with water, placed over the plastic wrap in the model and carefully aligned with the starting markings. Mark the positions of the Novex standards on the gel plate and draw the lines to mark the sections for the section. The nitrocellulose and plastic wrap are then removed and the gel cut along the marked lines with a sharp razor blade. The sections extend beyond the sample line so that they can be used to determine the positions for the slices as the gel is stained. When the slices are removed, the remaining gel is stained with silver and the positions of the standards and section marks are measured. The molecular weights corresponding to the cross-sectional positions are determined by Novex standards.

rezin gela damo v celice v dveh napravah za elektroeluiranje Biorad model 422. Za celice uporabimo membranske kape z mejno vrednostjo za molekulsko maso 1214K. Eluimi pufer je amonijev bikarbonat (50 mM) + SDS (0,05 %) (pH pribl. 7,8). 11 pufra hladimo približno 1 uro pred uporabo v hladni sobi pri 4-6 °C. Rezine gela eluiramo pri 10 ma/celico (40 v začetno) v hladni sobi pri 4-6 °C. Eluiranje poteka približno 4 ure. Celice nato previdno odstranimo, tekočino nad fritom pa odstranimo s pipeto. Eluimo komoro odstranimo, kakršnokoli tekočino nad membransko kapo pa odstranimo s pipeto. Tekočino v membranski kapi odstranimo s pipeto (Pipetman) in shranimo. 50 /tl alikvote očiščene vode damo nato v kapo, mešamo in odstranimo, ko se vsi kristali SDS raztopijo. Te izpiralne tekočine združimo s shranjeno zgornjo tekočino. Celotni eluimi volumen vzorca je 300-500 /tl na rezino gela. Vzorce damo v 10 mm dializne cevke Spectrapor 4 z mejno vrednostjo za molekulsko maso 12-14K, ki smo jih namakali nekaj ur v očiščeni vodi. Vzorce dializiramo preko noči pri 4-6 °C proti 600 ml fiziološke raztopine s fosfatnim pufrom (PBS je približno 4 mM v kaliju) na 6 vzorcev. Pufer zamenjamo naslednje jutro in dializo nadaljujemo 2,5 ure. Vzorce nato odstranimo iz dializnih vrečk in damo v cevke za mikrofugiranje. Cevke damo za eno uro na led, mikrofugiramo pri 14K obr./min 3 minute in supematante previdno odstranimo iz oborjenega SDS. Super112 natante damo nato na led še za približno 1 uro in mikrofugiramo ponovno 4 minute. Supernatante razredčimo v fiziološki raztopini s fosfatnim pufrom in izpostavimo testu za aktivnost. Preostale vzorce zamrznemo pri -70 °C.The slice of the gel was placed into the cells in two Biorad model 422 electroelution devices. For the cells, membrane caps with a molecular weight of 1214K were used. The elution buffer was ammonium bicarbonate (50 mM) + SDS (0.05%) (pH ca. 7.8). The 11 buffers were cooled for about 1 hour before being used in a cold room at 4-6 ° C. Gel slices were eluted at 10 ma / cell (40 initially) in a cold room at 4-6 ° C. Elution takes about 4 hours. The cells are then carefully removed and the liquid over the frit removed with a pipette. The elution chamber is removed and any liquid above the membrane cap is removed by pipette. The liquid in the membrane stroke was removed with a pipette (Pipetman) and stored. 50 / tl aliquots of purified water are then added to the cap, stirred and removed when all SDS crystals have dissolved. These rinsing liquids are combined with the upper fluid stored. The total eluimi sample volume is 300-500 / tl per slice of gel. Samples were placed in 10 mm Spectrapor 4 dialysis tubes with a limit value for the molecular weight of 12-14K, which were soaked for several hours in purified water. Samples were dialyzed overnight at 4-6 ° C against 600 ml of phosphate buffered saline (PBS is approximately 4 mM in potassium) per 6 samples. The buffer was replaced the next morning and the dialysis continued for 2.5 hours. Samples were then removed from the dialysis bags and placed in microfuge tubes. The tubes were placed on ice for one hour, microfuged at 14K rpm for 3 minutes, and the substrates carefully removed from precipitated SDS. The Super112 was then placed on ice for about 1 hour and microfugged for 4 minutes. The supernatants were diluted in saline with phosphate buffer and subjected to an activity test. The remaining samples were frozen at -70 ° C.

PRIMER 3EXAMPLE 3

Mikrosekvenciranie prašičjega mpl ligandaMicrosequencing of pig mpl ligand

Frakcijo 6 (2,6 ml) iz afinitetne kolone mpl-lgG koncentriramo na napravi Microcon-10 (Amicon). Zato, da preprečimo absorbiranje mpl liganda na Microcon, membrano izperemo z SDS (1 %) in dodamo 5 μΐ SDS (10 %) k frakciji 6. Vzorčni pufer 2X (20 μΐ) dodamo k frakciji št. 6 po koncentriranju (20 μ.1) na Microconu in celotni volumen (40 μΐ) napolnimo na eno linijo gradientnega akrilamidnega gela (420 %, Novex). Gel vzdržujemo po predpisu Novexa. Gel nato uravnotežujemo 5 minut pred elektropopivnanjem (electroblotting) v pufru 3-(cikloheksilamino)-lpropansulfonske kisline (CAPS) (10 mM), pH 11,0, ki vsebuje metanol (10 %). Elektropopivnanje na membrane Immobilon-PSQ (Millipore) izvedemo v 45 minutah pri konstantnem toku 250 mA v napravi za transfer celic BioRad Trans-Blot (32). Membrane PVDF barvamo z barvilom Coomassie blue R-250 (0,1 %) v metanolu (40 %), ocetni kislini (0,1 %) 1 minuto in razbarvamo v 2-3 minutah z ocetno kislino (10 %) v metanolu (50 %). Edini proteini, ki so vidni v področju z Mr 18000-35000, imajo Mr 30000,28000 in 22000.Fraction 6 (2.6 ml) from the mpl-lgG affinity column was concentrated on a Microcon-10 device (Amicon). To prevent the mpl ligand from being absorbed onto Microcon, the membrane was washed with SDS (1%) and 5 μΐ SDS (10%) was added to fraction 6. Sample buffer 2X (20 μΐ) was added to fraction no. 6 after concentration (20 μ.1) on Microcon and the whole volume (40 μΐ) was filled to one line of gradient acrylamide gel (420%, Novex). The gel is maintained according to Novex regulations. The gel was then equilibrated 5 minutes before electroblotting in 3- (cyclohexylamino) -1propanesulfonic acid (CAPS) buffer (10 mM), pH 11.0 containing methanol (10%). Immobilization onto Immobilon-PSQ membranes (Millipore) is performed within 45 minutes at a constant current of 250 mA in a BioRad Trans-Blot cell transfer device (32). PVDF membranes were stained with Coomassie blue R-250 dye (0.1%) in methanol (40%), acetic acid (0.1%) for 1 minute and discolored for 2-3 minutes with acetic acid (10%) in methanol ( 50%). The only proteins visible in the region with Mr 18000-35000 have Mr 30000,28000 and 22000.

Proge pri 30,28 in 22 kDa izpostavimo sekvenciranju proteinov. Avtomatizirano sekvenciranje proteina izvedemo na sekvenatoiju Applied Biosystem model 470A, opremljenim z on-line analizatoijem PTH. Sekvenator modificiramo tako, da injiciramo 80-90 % vzorca (Rodriguez, J. Chromatogr., 350:217-225 [1985]). Aceton (~ 12 μ]/ϊ) dodamo k topilu A, da uravnotežimo UV absorbanco. Elektropopivnane proteine sekvenciramo v patrone za popivnanje (Blott cartridge). Vrhove integriramo z računalniškim programom Jostice Innovation z uporabo vmesnikov Nelson Analytical 970. Interpretacijo sekvenc izvedemo na VAX 5900 (Henzel et al., J. Chromatogr., 404:41-52 [1987]). N-terminalne sekvence (ob uporabi kode z eno črko z nedoločenimi ostanki v oklepajih) in količina dobljenega materiala (v oklepajih) so predstavljeni v tabeli 2’.The lines at 30.28 and 22 kDa are exposed to protein sequencing. Automated protein sequencing is performed on an Applied Biosystem model 470A sequencer equipped with an on-line PTH analyzer. The sequencer is modified by injecting 80-90% of the sample (Rodriguez, J. Chromatogr., 350: 217-225 [1985]). Acetone (~ 12 μ] / ϊ) was added to solvent A to balance UV absorbance. The electro-absorbent proteins are sequenced into blot cartridges. The peaks are integrated with the Jostice Innovation computer program using Nelson Analytical 970 interfaces. Sequence interpretation is performed on VAX 5900 (Henzel et al. J. Chromatogr. 404: 41-52 [1987]). The N-terminal sequences (using one-letter code with indefinite residuals in parentheses) and the amount of material obtained (in parentheses) are presented in Table 2 '.

113113

TABELA 2’TABLE 2 '

Amino-terminalne sekvence mpl ligandaAmino-terminal sequences of mpl ligands

30 kDa [1.8 pmol] 1 5 10 15 20 25 (S) P A P P A(C)D PRLLNKLLRDD (H/S) V L H (G) R L 30 kDa [1.8 pmol] 1 5 10 15 20 25 (S) P A P P A (C) D PRLLNKLLRDD (H / S) V L H (G) R L (SEQ ID NO: 30) (SEQ ID NO: 30) 28 kDa [0.5 pmol] 1 5 10 15 20 25 (S)PAPPAXDPRLLNKLLRDD(H)VL(H)GR 28 kDa [0.5 pmol] 1 5 10 15 20 25 (S) PAPPAXDPRLLNKLLRDD (H) VL (H) GR (SEQ ID NO: 31) (SEQ ID NO: 31) 18-22 kDa [0.5 pmol] 1 5 10 XPA,PPAX,DPR,LX(N)(K) 18-22 kDa [0.5 pmol] 1 5 10 XPA, PPAX, DPR, LX (N) (K) (SEQ ID NO: 32) (SEQ ID NO: 32)

PRIMER 4EXAMPLE 4

Test tekočinske suspenzijske megakriocitopoezeLiquid suspension megacryocytopoiesis test

Humane periferne matične celice (PSC) (dobljene od pacientov z njihovo privolitvijo) razredčimo 5-krat z medijem IMDM (Gibco) in centrifugiramo pri 800 g 15 minut pri sobni temperaturi. Celične pelete resuspendiramo v IMDM in damo na Percoll (60 %), gostota 1,077 g/ml (Pharmacia) in centrifugiramo pri 800 g 30 minut. Mononukleame celice z majhno (light) gostoto aspiriramo na vmesni površini in izperemo 2-krat z IMDM in zasadimo 1-2 χ 10⁶ celic/ml v IMDM, ki vsebuje FBS (30 %) (končni volumen 1 ml), v 24 vdolbinic s skupki tkivne kulture (Costar). APP ali APP z odstranjenim mpl ligandom dodamo do 10 % in pustimo, da kulture rastejo 12-14 dni v ovlaženem inkubatorju pri 37 °C v mešanici zraka in 5 % CO_y Kulture rastejo tudi v prisotnosti APP (10 %) z 0,5 gg znp/-IgG, dodanega na dneve 0, 2 in 4. APP odstranimo mpl ligand s prepuščanjem APP skozi mplAgG afinitetno kolono.Human peripheral stem cells (PSCs) (obtained from patients with their consent) were diluted 5 times with IMDM medium (Gibco) and centrifuged at 800 g for 15 minutes at room temperature. Cell pellets were resuspended in IMDM and placed on Percoll (60%), density 1.077 g / ml (Pharmacia) and centrifuged at 800 g for 30 minutes. Small-density mononuclear cells were aspirated on the intermediate surface and washed twice with IMDM and planted 1-2 χ 10 ⁶ cells / ml in FBS-containing IMDM (30%) (final volume 1 ml) in 24 wells with tissue culture assemblies (Costar). Add APP or APP with mpl ligand removed up to 10% and allow the cultures to grow for 12-14 days in a humidified incubator at 37 ° C in air and 5% CO _y Cultures also grow in the presence of APP (10%) with 0.5 gg znp / -IgG added on days 0, 2 and 4. APP removes the mpl ligand by passing APP through the mplAgG affinity column.

114114

Za kvantitavno določitev megakariocitopoeze v teh tekočinskih suspenzijskih kulturah uporabimo modifikacijo Solberga et al. in radioaktivno označeno monoklonsko protitelo murinega IgG (HP1-1D) za GPIIbIIIa (dobavitelj Dr. Nichols, Mayo Clinic). 100 μ-g HP1-1D (glej Grant, B. et al., Blood 69:1334-1339 [1987]), radioaktivno označimo z lmCi Na¹²⁵J z uporabo Enzymobeads (Biorad, Richmond, CA) po navodilih izdelovalca. Radioaktivno označen HP1-1D shranimo pri -70 °C v PBS, ki vsebuje oktilglukozid (0,01 %). Značilne specifične aktivnosti so 1-2 χ 10⁶ cpm/p-g (>95 % se obori s trikloroocetno kislino (12,5 %)).To quantify megakaryocytopoiesis in these liquid suspension cultures, we use the modification of Solberg et al. and radiolabeled murine IgG monoclonal antibody (HP1-1D) for GPIIbIIIa (supplied by Dr. Nichols, Mayo Clinic). 100 μ-g HP1-1D (see Grant, B. et al., Blood 69: 1334-1339 [1987]) was radiolabeled with lmCi Na ¹²⁵ J using Enzymobeads (Biorad, Richmond, CA) according to the manufacturer's instructions. The radiolabeled HP1-1D was stored at -70 ° C in PBS containing octylglucoside (0.01%). Typical specific activities are 1-2 χ 10 ⁶ cpm / pg (> 95% precipitated with trichloroacetic acid (12.5%)).

Tekočinske suspenzijske kulture pripravimo v trajniku za vsako eksperimentalno točko. Po 12-14 dneh kultiviranja po 1 ml kultur prenesemo v 1,5 ml eppendorfske cevke in centrifugiramo pri 800 g 10 minut pri sobni temperaturi in nastale celične pelete resuspendiramo v 100 μλ PBS, ki vsebuje EDTA (0,02 %) in goveji telečji serum (20 %). 10 ng ¹²⁵J-HP1-1D v 50 μΐ testnega pufra dodamo k resuspendiranim kulturam in inkubiramo 60 minut pri sobni temepraturi (RT) z občasnim stresanjem. Nato celice zberemo s centrifugiranjem pri 800 g 10 minut pri RT in izperemo 2-krat s testnim pufrom. Pelete štejemo 1 minuto v števcu gama (Packard). Nespecifično vezavo določimo z dodajanjem 1 /xg neoznačenega HP1-1D 60 minut pred dodatkom označenega HP1-1D. Specifično vezavo določimo kot celotno vezavo ¹²⁵J-HP1-1D, minus vezava v prisotnosti prebitka neoznačenega HP1-1D.Liquid suspension cultures were prepared in perennial for each experimental point. After 12-14 days of cultivation, transfer 1 ml of cultures into 1.5 ml Eppendorf tubes and centrifuge at 800 g for 10 minutes at room temperature and resuspend the resulting cell pellets in 100 μλ PBS containing EDTA (0.02%) and beef veal serum (20%). 10 ng of ¹²⁵ J-HP1-1D in 50 μΐ assay buffer was added to the resuspended cultures and incubated for 60 minutes at room temperature (RT) with occasional shaking. Cells were then collected by centrifugation at 800 g for 10 minutes at RT and washed twice with assay buffer. Count the pellets for 1 minute in a Gamma (Packard) counter. Non-specific binding is determined by adding 1 / xg of unlabeled HP1-1D 60 minutes before the addition of labeled HP1-1D. Specific binding is defined as total binding ¹²⁵ J-HP1-1D, minus binding in the presence of excess unlabeled HP1-1D.

PRIMER 5EXAMPLE 5

Oligonukleotidni PCR primeriiOligonucleotide PCR primers

Na osnovi amino-terminalne aminokislinske sekvence, dobljene iz proteinov s 30 kDa, 28 kDa in 18-22 kDa, oblikujemo degenerirane oligonukleotide, da jih uporabimo kot primeije verižne polimerazne reakcije (PCR) (glej tabelo 4). Sintetiziramo dva primerska poola, pozitivni-sens 20-memi pool, ki kodira aminokislinske ostanke 2-8 (mpl 1) in anti-sens 21-memi pool, komplementaren sekvencam, ki kodirajo amino kisline 18-24 (mpl 2).On the basis of amino-terminal amino acid sequences obtained from proteins of 30 kDa, 28 kDa, and 18-22 kDa, degenerate oligonucleotides are formed to be used as primers of the polymerase chain reaction (PCR) (see Table 4). We synthesize two primer pools, a positive-sens 20-memi pool encoding amino acid residues 2-8 (mpl 1) and an anti-sens 21-memi pool, complementary to sequences encoding amino acids 18-24 (mpl 2).

115115

TABELA4TABLE4

Primerski pooli degeneriranih olieonukleotidovPrimer pools of degenerate oleonucleotides

mpl 1:5' CCN GCN CCN CCN GCN TGY GA 3' (2,048-krat decmpri ran) mpl 1: 5 'CCN GCN CCN CCN GCN TGY GA 3' (2,048 times dec) (SEQ ID NO: 35) (SEQ ID NO: 35) mpl 2:5’ NCC RTG NAR NAC RTG RTC RTC 3’ (2,048-krat cfeomprirm) mpl 2: 5 'NCC RTG NAR NAC RTG RTC RTC 3' (2,048 times cfeomprirm) (SEQ ID NO: 36) (SEQ ID NO: 36)

Prašičjo genomsko DNA, izolirano iz prašičjih perifernih krvnih limfocitov, uporabimo kot kalup za PCR. 50 /tl reakcijske zmesi vsebuje: 0,8 /xg prašičje genomske DNA v tris-HCl (10 mM) (pH 8,3), KC1 (50 mM), MgC^ (3 mM), 100 /ig/ml BSA, dNTP (400 μΜ), 1 μΜ vsakega primerskega poola in 2,5 enot Taq polimeraze. Začetno kalupno denaturiranje je pri 94 °C 8 minut, čemur sledi 35 ciklov po 45 sekund pri 94 °C, 1 minuto pri 55 °C in 1 minuto pri 72 °C. Končni cikel pustimo, da traja 10 minut pri 72 °C. Produkte PCR ločimo z elektroforezo na poliakrilamidnem gelu (12 %) in vizualiziramo z barvanjem z etidijevim bromidom. Utemljeno je, da če amino-terminalno aminokislinsko sekvenco kodira posamezen ekson, potem pričakujemo, da ima pravilen produkt PCR 69 bp. Fragment DNA te velikosti eluiramo iz gela in subkloniramo v pGEMT (Promega). Sekvence treh klonov so prikazane spodaj v tabeli 5.Pig genomic DNA isolated from porcine peripheral blood lymphocytes is used as a PCR mold. 50 / tl of the reaction mixture contains: 0.8 / xg of porcine genomic DNA in tris-HCl (10 mM) (pH 8.3), KC1 (50 mM), MgC ^ (3 mM), 100 / ig / ml BSA, dNTP (400 μΜ), 1 μΜ of each primer pool and 2.5 units of Taq polymerase. Initial mold denaturation is at 94 ° C for 8 minutes, followed by 35 cycles of 45 seconds at 94 ° C, 1 minute at 55 ° C, and 1 minute at 72 ° C. Allow the final cycle to stand at 72 ° C for 10 minutes. The PCR products were separated by polyacrylamide gel electrophoresis (12%) and visualized by ethidium bromide staining. It is argued that if the amino-terminal amino acid sequence encodes a single exon, then the correct PCR product is expected to have 69 bp. A DNA fragment of this size is eluted from the gel and subcloned into pGEMT (Promega). The sequences of the three clones are shown below in Table 5.

TABELA 5TABLE 5

Fragmenti prašičje genomske DNA z 69 bp gemT3Pig genomic DNA fragments with 69 bp gemT3

5'CCAGCGCCGC CAGCCTGTGA CCCCCGACTC CTAAATAAAC TGCCTCGTGA 3'GGTCGCGGCG GTCGGACACT GGGGGCTGAG GATTTATTTG ACGGAGCACI5'CCAGCGCCGC CAGCCTGTGA CCCCCGACTC CTAAATAAAC TGCCTCGTGA 3'GGTCGCGGCG GTCGGACACT GGGGGCTGAG GATTTATTTG ACGGAGCACI

TGACCACGTT CAGCACGGC [69 bp] (SEQ ID NO: 37)TGACCACGTT CAGCACGGC [69 bp] (SEQ ID NO: 37)

ACTGGTGCAA GTCGTGCCG (SEP ID NO: 38)ACTGGTGCAA GTCGTGCCG (SEP ID NO: 38)

116 gemT7116 gemT7

5'CCAGCACCTC CGGCATGTGA CCCCCGACTC CTAAATAAAC TGCTTCGTGA 3'GGTCGTGGAG GCCGTACACT GGGGGCTGAG GATTTATTTG ACGAAGCA£I5'CCAGCACCTC CGGCATGTGA CCCCCGACTC CTAAATAAAC TGCTTCGTGA 3'GGTCGTGGAG GCCGTACACT GGGGGCTGAG GATTTATTTG ACGAAGCA £ I

CGACCACGTC CATCACGGC [69 bp] (SEQ ID NO: 39)CGACCACGTC CATCACGGC [69 bp] (SEQ ID NO: 39)

GCTGGTGCAG GTAGTGCCG(SEQ ID NO: 40) gemT9GCTGGTGCAG GTAGTGCCG (SEQ ID NO: 40) gemT9

P R L L N K L LR (SEQ IDP R L L N K L LR (SEQ ID

NO: 32)NO: 32)

S» pCAGCACCGCCGGCATGTGACCCCCGACTCCTAAATAAACTGCTTCGTGACG 3* GGTCGTGGCGGCCGTACACTGGGGGCTGAGGATTTATTTGACGAAGCA£Iia£S »pCAGCACCGCCGGCATGTGACCCCCGACTCCTAAATAAACTGCTTCGTGACG 3 * GGTCGTGGCGGCCGTACACTGGGGGCTGAGGATTTATTTGACGAAGCA £ Iia £

ATCATGTCTATCACGGT 3' (SEQ ID NO: 41)ATCATGTCTATCACGGT 3 '(SEQ ID NO: 41)

TAGTACAGATAGTGCCA 5' (SEP ID NO: 42)TAGTACAGATAGTGCCA 5 '(SEP ID NO: 42)

Položaj primerjev za PCR je označen s podčrtanimi bazami. Ti rezultati potrjujejo N-terminalno sekvenco, dobljeno za amino kisline 9-17 za proteine s 30 kDa, 28 kDa in 18-22 kDa in označujejo, da to sekvenco kodira posamezen ekson prašičje DNAThe position of the primers for PCR is indicated by the underlined bases. These results confirm the N-terminal sequence obtained for amino acids 9-17 for proteins of 30 kDa, 28 kDa, and 18-22 kDa and indicate that this sequence encodes a single exon of porcine DNA

PRIMER 6EXAMPLE 6

Gen humanega mpl ligandaHuman mpl ligand gene

Na osnovi rezultatov iz Primera 5 oblikujemo 45-merni deoksioligonukleotid, imenovan pR45, in ga sintetiziramo za selekcioniranje genomske knjižnice. 45-mer ima naslednjo sekvenco:Based on the results of Example 5, a 45-mer deoxyoligonucleotide, called pR45, is formulated and synthesized for selection in the genomic library. The 45-mer has the following sequence:

5' GCC-GTG-AAG-GAC-GTG-GTC-GTC-ACG-AAG-CAG-TTT-ATT-TAG-GAG-TCG 3' (SEQ ID NO: 28)5 'GCC-GTG-AAG-GAC-GTG-GTC-GTC-ACG-AAG-CAG-TTT-ATT-TAG-GAG-TCG 3' (SEQ ID NO: 28)

Ta oligonukleotid je ³²P-označen z (γ³²Ρ)-ΑΤΡ in kinazo T4 in ga uporabimo zaThis oligonucleotide is ³² P-labeled with (γ ³² Ρ) -ΑΤΡ and kinase T4 and used for

117 selekcioniranje humane genomske DNA knjižnice v Xgeml2 pri manj ostrih pogojih za hibridizacijo in izpiranje (glej Primer 7). Pozitivne klone zberemo, plake očistimo in analiziramo z restrikcijskim mapiranjem in Southern blottingom. Klon št. 4 izberemo za dodatno analizo.117 selection of the human genomic DNA library in Xgeml2 under less harsh hybridization and washing conditions (see Example 7). Positive clones were collected, plaques were cleaned and analyzed by restriction mapping and Southern blotting. Clone no. 4 is selected for additional analysis.

2,8 kb fragment BamHI-XbaI, ki se hibridizira s 45-merom subkloniramo v pBluescript SK-. Delno sekvenciranje DNA tega klona izvedemo tako, da kot primeije uporabimo oligonukleotide, specifične za sekvenco DNA prašičjega mpl liganda. Dobljena sekvenca potrjuje, da smo izolirali DNA, ki kodira humani homolog prašičjega mpl liganda. Restrikcijo mesto EcoRI detektiramo v sekvenci, kar nam omogoča, da izoliramo fragment EcoRI-XbaI s 390 bp iz 2,8 kb BamHIXbal in ga subkloniramo v pBluescript SK-.A 2.8 kb 45-mer hybridized BamHI-XbaI fragment was subcloned into pBluescript SK-. Partial DNA sequencing of this clone is performed by using oligonucleotides specific for the DNA sequence of the pig mpl ligand as primers. The resulting sequence confirms that we isolated DNA encoding a human homolog of the porcine mpl ligand. The restriction of the EcoRI site is detected in sequence, allowing us to isolate the 390 bp EcoRI-XbaI fragment from 2.8 kb BamHIXbal and subcloning it into pBluescript SK-.

Obe vijačnici tega fragmenta sekvenciramo. Sekvenca humane DNA in deducirana aminokislinska sekvenca sta prikazani na sl. 9 (SEQ ID NOS: 3 & 4). Napovedani položaji intronov v genomski sekvenci so prikazani s puščicami, definiran pa je tudi domnevni ekson (ekson 3).Both helixes of this fragment are sequenced. The human DNA sequence and deduced amino acid sequence are shown in FIG. 9 (SEQ ID NOS: 3 & 4). The predicted positions of introns in the genomic sequence are indicated by arrows, and the putative exon (exon 3) is defined.

Raziskovanje napovedane aminokislinske sekvence potrjuje, da je serinski ostanek prva amino kislina zrelega mpl liganda, kot določimo z direktno analizo aminokislinske sekvence. Takoj navzgor od tega kodona napovedana aminokislinska sekvenca zelo spominja na signalno sekvenco, vključeno v izločanje zrelega mpl liganda. Ta kodirna regija sedanje sekvence je verjetno prekinjena pri nukleotidnem položaju 68 z intronom.Investigation of the predicted amino acid sequence confirms that the serine residue is the first amino acid of the mature mpl ligand as determined by direct analysis of the amino acid sequence. Immediately upstream of this codon, the predicted amino acid sequence is very reminiscent of the signal sequence involved in secretion of the mature mpl ligand. This coding region of the present sequence is probably interrupted at nucleotide position 68 by an intron.

V 3’ smeri kaže, da se ekson konča pri nukleotidu 196. Ta ekson zato kodira sekvenco 42 amino kislin, od katerih jih je 16 verjetno del signalne sekvence in 26 del zrelega humanega mpl liganda.In the 3 'direction, the exon ends at nucleotide 196. This exon therefore encodes a sequence of 42 amino acids, 16 of which are probably part of the signal sequence and 26 of the mature human mpl ligand.

PRIMER 7 cDNA humanega mpl liganda s popolno dolžinoEXAMPLE 7 full length human mpl ligand cDNA

Na osnovi humane sekvence eksona 3 (Primer 6) sintetiziramo dva nedegenerirana oligonukleotida, ki ustrezata 3’ in 5’ koncema sekvence eksona 3 (tabela 6).Based on the human exon 3 sequence (Example 6), two non-degenerate oligonucleotides corresponding to the 3 'and 5' ends of the exon 3 sequence are synthesized (Table 6).

118118

TABELA 6TABLE 6

Humana cDNA nedegeneriranih oligonukleotidnih primeriev PCRHuman cDNA of non-generated oligonucleotide primers PCR

(prednji 5' GCT AGC TCT AGA AAT TGC TCC TCG TGG TCA TGC TTC T 3' primer) (front 5 'GCT AGC TCT AGA AAT TGC TCC TCG TGG TCA TGC TTC T 3' example) (SEOIDNO: 43) (SEOID: 43) (reverzni ₅· _{CAG TCT} GCCGTG AAG GAC ATG G 3’ primer)(reverse ₅ · _{CAG TCT} GCCGTG AAG GAC ATG G 3 'example) (SEOIDNO: 44) (SEOID: 44)

Ta dva primerja uporabimo v reakcijah PCR, pri čemer uporabimo kot kalup DNA iz različnih knjižnic humane cDNA ali 1 ng cDNA Quick Clone (Clonetech) iz različnih tkiv ob pogojih, opisanih v primeru 5. Pričakovana velikost pravilnega produkta PCR je 140 bp. Po analizi produktov PCR na poliakrilamidnem gelu (12 %) detektiramo fragment DNA pričakovane velikosti v knjižnicah cDNA, pripravljenih iz odraslih ledvic, 293 fetalnih ledvičnih celic in cDNA pripravljene iz humanih fetalnih jeter (Clonetech kat. št. 7171-1).These two primers are used in PCR reactions, using as a DNA mold from different human cDNA libraries or 1 ng Quick Clone cDNA (Clonetech) from different tissues under the conditions described in Example 5. The expected size of the correct PCR product is 140 bp. After analysis of PCR products on polyacrylamide gel (12%), a DNA fragment of the expected size was detected in cDNA libraries prepared from adult kidneys, 293 fetal kidney cells and cDNA prepared from human fetal livers (Clonetech cat. No. 7171-1).

tt

Knjižnico cDNA fetalnih jeter v λ DR2 (Clonetech kat. št. HL115lx) selekcioniramo z istim 45-memim oligonukleotidom, ki smo ga uporabili za selekcioniranje humane genomske knjižnice. Oligonukleotid označimo z (γ³²Ρ)-ΑΤΡ z uporabo polinukleotidne kinaze T4. Knjižnico selekcioniramo pri manj ostrih pogojih hibridizacije. Filtre predhibridiziramo 2 uri in nato hibridiziramo s sondo preko noči 16 h pri 42 °C v formamidu (20 %), 5xSSC, 10xDenhardtovem reagentu, natrijevem fosfatu (0,05 M) (pH 6,5), natrijevem pirofosfatu (0,1 %), 50 Mg/ml sonificirane DNA lososove sperme. Filtre nato splaknemo v 2xSSC in nato izperemo enkrat v 0,5xSSC, SDS (0,1 %) pri 42 °C. Filtre izpostavimo preko noči kodakovemu rentgenskemu filmu. Pozitivne klone zberemo, plake očistimo, velikost inserta pa določimo s PCR z uporabo oligonukleotidov, ki so bočno k BamHI-XbaI, ki je kloniran v XDR2 (Conetch kat. št. 6475-1). 5 μΐ izhodnega faga uporabimo kot kalupni vir. Začetna denaturacija poteka 7 minut pri 94 °C, nato pa sledi 30 pomnoževalnih ciklov (1 min. pri 94 °C, 1 min. pri 52 °C in 1,5 min. pri 72 °C). Končna razširitev je 15 min pri 72 °C.The fetal liver cDNA library in λ DR2 (Clonetech cat. No. HL115lx) was selected using the same 45-membered oligonucleotide that was used to select the human genomic library. The oligonucleotide was labeled with (γ ³² Ρ) -ΑΤΡ using T4 polynucleotide kinase. We select the library under less harsh hybridization conditions. The filters were pre-hybridized for 2 hours and then hybridized with the probe overnight for 16 h at 42 ° C in formamide (20%), 5xSSC, 10xDenhardt reagent, sodium phosphate (0.05 M) (pH 6.5), sodium pyrophosphate (0.1 %), 50 Mg / ml sonicated sperm DNA. The filters were then rinsed in 2xSSC and then washed once in 0.5xSSC, SDS (0.1%) at 42 ° C. The filters are exposed overnight to a Kodak X-ray film. Positive clones were collected, plaques were cleaned, and the size of the insert was determined by PCR using oligonucleotides lateral to BamHI-XbaI cloned in XDR2 (Conetch cat. No. 6475-1). Use 5 μΐ of the output phage as a mold source. Initial denaturation takes 7 minutes at 94 ° C, followed by 30 multiplication cycles (1 min at 94 ° C, 1 min at 52 ° C and 1.5 min at 72 ° C). The final extension is 15 min at 72 ° C.

119119

Klon št. FL2b ima 1,8 kb insert in ga izberemo za nadaljnjo analizo.Clone no. FL2b has a 1.8 kb insert and is selected for further analysis.

Plazmid pDR2 (Clonetech, \DR2 & pDR2 cloning and Expression System Library Protocol Handbook, str. 42), ki se nahaja v krakih faga \DR2, osvobodimo po navodilih izdelovalca (Clonetech, XDR2 & pDR2 cloning and Expression System Library Protocol Handbook, str. 29-30). Iz restrikcijske analize plazmida pDR2-FL2b z BamHI in Xbal je razvidna prisotnost internega restrikcijskega mesta BamHI v insertu približno na položaju 650. Digestija plazmida BamHI-XbaI prereže insert na dva fragmenta, enega z 0,65 kb in drugega z 1,15 kb. Sekvenco DNA določimo s tremi različnimi razredi kalupov, ki so izvedeni iz plazmida pDR2-FL2b. Sekvenciranje dvojnovijačne plazmidne DNA izvedemo z avtomatskim fluorescentnim sekvenatoijem DNA ABI373 (Applied Biosystems, Foster City, Kalifornija), pri čemer uporabimo predpise za barvno označene dideoksi nukleozid trifosfatne terminatorje (barvni terminatorji) in po naročilih sintetizirane sprehajajoče (walking) primeije (Sanger et al., Proč. Natl. Acad. Sci. ZDA, 74:5463-5467 [1977]; Smith et al., Nature, 321:674-679 [1986]). Direktno sekvenciranje fragmentov, pomnoženih z verižno polimerazno reakcijo iz plazmida, naredimo s sekvenatorjem ABI373 z uporabo narejenih primerjev in reakcij z barvnimi terminatorji. Enojnovijačni kalup izdelamo z Janusovim vektorjem M13 (DNASTAR, Inc., Madison, Wisconsin) (Burland et al., Nucl. Acids Res., 21:3385-3390 [1993]). Fragmenta BamHI-XbaI (1,15 kb) in BamHI (0,65 kb) izoliramo iz plazmida pDR2-FL2b, konce izpolnimo (zacelimo) z DNA polimerazo T4 v prisotnosti deoksinukleotidov in nato subkloniramo v mesto Smal Janusovega vektorja M13. Sekvenciranje izvedemo po standardnih predpisih za barvno označene univerzalne M13 in reverzne primeije ali sprehajajoče primeije in barvne terminatorje. Reakcije manualnega sekvenciranja izvedemo na enojno vijačni DNA M13 z uporabo sprehajajočih primerjev in standardne dideoksi-terminatorske kemije (Sanger et al., Proč. Natl. Acad. Sci. ZDA, 74:5463-5467 [1977]), ³³P-označene α-dATP in Sequenase (United States Biochemical Corp., Cleveland, Ohio). Sestavljanje sekvence DNA izvedemo na napravi Sequencher V2.1bl2 (Gene Codes Corporation, Ann Arbor, Michigan). Nukleotidne in deducirane sekvence hML so prikazane na sl. 1 (SEQ ID NO:1).The plasmid pDR2 (Clonetech, \ DR2 & pDR2 cloning and Expression System Library Protocol Handbook, p. 42), which is located in the phage \ DR2 arms, is released according to the manufacturer's instructions (Clonetech, XDR2 & pDR2 cloning and Expression System Library Protocol Handbook, p. 29-30). The restriction analysis of the pDR2-FL2b plasmid with BamHI and Xbal shows the presence of an internal BamHI restriction site in the insert at approximately position 650. The digestion of the BamHI-XbaI plasmid cuts the insert into two fragments, one with 0.65 kb and the other with 1.15 kb. DNA sequence is determined by three different classes of molds derived from plasmid pDR2-FL2b. Sequencing of double-stranded plasmid DNA was performed with ABI373 automated fluorescence DNA sequencer (Applied Biosystems, Foster City, CA), using color-coded dideoxy nucleoside triphosphate terminators and custom-made walking walking synthesized. Acad. Sci. USA, 74: 5463-5467 [1977]; Smith et al., Nature, 321: 674-679 [1986]. The direct sequencing of fragments amplified by a polymerase chain reaction from a plasmid is done using the ABI373 sequencer using primers and reactions with color terminators. The single-stranded mold is made with a Janus vector M13 (DNASTAR, Inc., Madison, Wisconsin) (Burland et al., Nucl. Acids Res., 21: 3385-3390 [1993]). BamHI-XbaI fragments (1.15 kb) and BamHI (0.65 kb) were isolated from plasmid pDR2-FL2b, finally filled (healed) with T4 DNA polymerase in the presence of deoxynucleotides and then subcloned into the Smal Janus vector M13 site. Sequencing is performed according to standard regulations for color coded universal M13 and reverse primers or walking primers and color terminators. Manual sequencing reactions are performed on single-stranded M13 DNA using walking primers and standard dideoxy-terminator chemistry (Sanger et al., Natl. Acad. Sci. USA, 74: 5463-5467 [1977]), ³³ P-labeled α -dATP and Sequenase (United States Biochemical Corp., Cleveland, Ohio). DNA sequence assembly was performed on a Sequencher V2.1bl2 device (Gene Codes Corporation, Ann Arbor, Michigan). The nucleotide and deduced hML sequences are shown in FIG. 1 (SEQ ID NO: 1).

PRIMER 8EXAMPLE 8

Izolacija gena humanega mpl liganda (TPO)Isolation of the human mpl ligand (TPO) gene

Klone humane genomske DNA gena TPO izoliramo s selekcioniranjem humaneHuman genomic DNA clones of the TPO gene are isolated by human selection

120 genomske knjižnice v X-geml2 s pR45, predhodno opisano oligonukleotidno sondo, pri manj ostrih pogojih (Primer 7), pri zelo ostrih pogojih pa s fragmentom, ki ustreza 3’ polovici humane cDNA, ki kodira mpl ligand (od mesta BamHI do 3’ konca). Izoliramo dva prekrivajoča λ klona, ki obsegata 35 kb. Dva prekrivajoča fragmenta (BamHI in EcoRI), ki vsebujeta celoten gen TPO, subkloniramo in sekvenciramo. Struktura humanega gena je sestavljena iz šestih eksonov znotraj 7 kb genomske DNA (sl. 14A, B in C). Meje vseh eksonskih/intronskih povezav so konsistentne s konsenznim motivom, določenim za gene sesalcev (Shapiro, M. B., et al., Nucl. Acids Res. 15:7155 [1987]). Ekson 1 in ekson 2 vsebujeta 5’ netranslatirano sekvenco in začetne 4 amino kisline signalnega peptida. Ostanek sekretomega signala in prvih 26 amino kislin zrelega proteina so kodirani v eksonu 3. Celotna karboksilna domena in 3’ netranslatirana kot tudi približno 50 aminokislinska eritropoetinu podobna domena so kodirani v eksonu 6. Štiri amino kisline, vključene v delecije, opažene v hML-2 (hTPO-2), so kodirane na 5’ koncu eksona 6.120 genomic libraries in X-geml2 with pR45, oligonucleotide probe described previously, under less stringent conditions (Example 7), and under very harsh conditions with a fragment corresponding to the 3 'half of the human cpl DNA encoding the mpl ligand (from the BamHI site to 3 'ends). We isolate two overlapping λ clones spanning 35 kb. Two overlapping fragments (BamHI and EcoRI) containing the entire TPO gene were subcloned and sequenced. The structure of the human gene is composed of six exons within 7 kb of genomic DNA (Fig. 14A, B and C). The boundaries of all exon / intron linkages are consistent with the consensus motif established for mammalian genes (Shapiro, M. B., et al., Nucl. Acids Res. 15: 7155 [1987]). Exon 1 and exon 2 contain a 5 'untranslated sequence and the initial 4 amino acids of the signal peptide. The remainder of the secretome signal and the first 26 amino acids of the mature protein are encoded in exon 3. The entire carboxyl domain and the 3 'untranslated as well as about 50 amino acid erythropoietin-like domain are encoded in exon 6. The four amino acids involved in the deletions observed in hML-2 (hTPO-2) are encoded at the 5 'end of exon 6.

PRIMER 9EXAMPLE 9

Prehodna ekspresija humanega mpl liganda (hML)Transient expression of human mpl ligand (hML)

Zato da subkloniramo insert s popolno dolžino, vsebovan v pDR2-FL2b, plazmid popolnoma digeriramo z Xbal, nato pa delno z BamHI. Fragment DNA, ki ustreza insertu z 1,8 kb, gelsko očistimo in subkloniramo v pRK5 (pRK5-hmp/1) (US patent št. 5,258,287 za konstrukcijo pRK5) ob kontroli citomegalovirusnega neposrednega zgodnjega promotoija. DNA iz konstrukta pRK5-hmp/I pripravimo z metodo PEG in transfektiramo v 293 humanih embrionalnih ledvičnih celic, vzdrževanih v Eaglovem mediju, modificiranem po Dulbecco-u (DMEM), dopolnjenem s hranilno zmesjo F-12, Hepesom (20 mM) (pH 7,4) in fetalnim govejim serumom (10 %). Celice transfektiramo po kalcijevem fosfatnem postopku, kot opisujejo Gorman, C. [1985] v DNA Cloning: A Practical Approach (Glover, D.M., ed) Vol. II, str. 143190, IRL Press, Washington, D.C.. 36 ur po transfekciji supernatant transfektiranih celic testiramo za aktivnost v proliferacijskem testu (Primer 1). Supernatant 293 celic, transfektiranih samo z vektorjem pRK, ne stimulira celic Ba/F3 ali Ba/F3-mp/ (sl. 12A). Supernatant celic, transfektiranih s pRK5-hmp/I ne vpliva na celice Ba/F3, pač pa znatno stimulira proliferacijo celic Ba/F3-mp/ (sl. 12A), iz česar je razvidno, da cDNA kodira funkcionalno aktivni humani mpl ligand.In order to subclone the full length insert contained in pDR2-FL2b, the plasmid was completely digested with Xbal and then partially with BamHI. A 1.8 kb DNA fragment corresponding to the 1.8 kb insert was gel purified and subcloned into pRK5 (pRK5-hmp / 1) (US Patent No. 5,258,287 for the pRK5 construct) while controlling the cytomegalovirus immediate early promoter. DNA from the pRK5-hmp / I construct was prepared by the PEG method and transfected in 293 human embryonic kidney cells maintained in Eagle's Dulbecco modified medium (DMEM) supplemented with nutrient F-12, Hepes (20 mM) (pH 7.4) and fetal bovine serum (10%). Cells are transfected by the calcium phosphate process as described by Gorman, C. [1985] in DNA Cloning: A Practical Approach (Glover, D.M., ed) Vol. II, p. 143190, IRL Press, Washington, D.C .. 36 hours after transfection, supernatants of transfected cells were tested for proliferation assay activity (Example 1). The supernatant of 293 cells transfected with the pRK vector alone did not stimulate Ba / F3 or Ba / F3-mp / cells (Fig. 12A). The supernatant of cells transfected with pRK5-hmp / I did not affect Ba / F3 cells but significantly stimulated the proliferation of Ba / F3-mp / cells (Fig. 12A), indicating that the cDNA encodes a functionally active human mpl ligand.

121121

PRIMER 10EXAMPLE 10

Izo-oblike humanega mpl liganda hML2. hML3 in hML4Iso-forms of the human mpl ligand hML2. hML3 and hML4

Zato da idenficiramo alternativno spojene oblike hML, primerje sintetiziramo ustrezno vsakemu koncu kodirne sekvence hML. Te primeije uporabimo v RT-PCR, da pomnožimo RNA humanih odraslih jeter. Dodatno konstruiramo interne primeije, ki so bočno k izbranim regijam, ki nas zanimajo (glej spodaj), in jih podobno uporabimo. Direktno sekvenciranje koncev produkta PCR dokazuje enojno sekvenco, ki natančno ustreza sekvenci cDNA, izolirani iz knjižnice humanih fetalnih jeter (sl. 1 [SEQ ID NO: 1]). Vendar pa ima regija poleg C-terminala EPOdomene (v sredini produkta PCR) vzorec kompleksne sekvence, kar navaja k obstoju možnih spojenih variant v tej regiji. Da izoliramo te spojene variante, uporabimo primeije, prikazane v tabeli 7, ki so bočno k tej regiji v PCR, kot kalupe cDNA humanih odraslih jeter.In order to identify alternatively fused hML forms, the primer is synthesized accordingly at each end of the hML coding sequence. These primers are used in RT-PCR to amplify human adult liver RNA. We additionally construct internal primes that are lateral to the selected regions of interest (see below) and use them similarly. Direct sequencing of PCR product ends demonstrates a single sequence that closely matches the cDNA sequence isolated from the human fetal liver library (Fig. 1 [SEQ ID NO: 1]). However, in addition to the C-terminal of the EPOdomain (in the middle of the PCR product), the region has a pattern of complex sequence, indicating the existence of possible fused variants in that region. To isolate these spliced variants, we use the primers shown in Table 7, which are lateral to this region in PCR, as human adult liver liver cDNA molds.

TABELA 7TABLE 7

Primerii PCR izo-oblike humanega MLExamples of PCR isoforms of human ML

phmpllcdna.3e1: phmpllcdna.3e1: 5'TGTGGACTTTAGCTTGGGAGAATG3' 5'TGTGGACTTTAGCTTGGGAGAATG3 ' (SEQ ID NO: 45) (SEQ ID NO: 45) pbx4.f2: pbx4.f2: 5’GGTCCAGGGACCTGGAGGTTTG3’ 5'GGTCCAGGGACCTGGAGGTTTG3 ' (SEQ ID NO: 46) (SEQ ID NO: 46)

Produkte PCR subkloniramo topo (blunt) v M13. Iz sekvenciranja individualnih subklonov je razvidna eksistenca vsaj 3 izo-oblik ML. Ena od njih, hML (imenovana tudi hML₃₃₂), je najdaljša oblika in natančno ustreza sekvenci, izolirani iz knjižnice fetalnih jeter. Sekvence štirih izo-oblik humanega mpl liganda so navedene od najdaljše (hML) do najkrajše (hML-4) in so prikazane na sl. 11 [SEQ ID NOS: 6,8,9PCR products are subcloned blunt in M13. Sequencing of individual subclones reveals the existence of at least 3 ML isoforms. One of them, hML (also called hML ₃₃₂ ), is the longest in shape and closely matches the sequence isolated from the fetal liver library. The sequences of the four isoforms of the human mpl ligand are listed from longest (hML) to shortest (hML-4) and are shown in Figs. 11 [SEQ ID NOS: 6,8,9

122 & 10].122 & 10].

PRIMER 11EXAMPLE 11

Konstrukcija in prehodna ekspresija izo-oblik humanega mpl liganda in nadomestnih variant hML2, hML3 in hML(R153A, R154A1Construction and transient expression of iso-forms of the human mpl ligand and the alternative variants hML2, hML3 and hML (R153A, R154A1

Izo-oblike hML2 in hML3 in nadomestne variante hML(Rl53A, R154A) rekonstruiramo iz hML z uporabo rekombinantnih tehnik PCR, kot opisujejo Russell Higuchi v predpisih PCR, A Guide to Methods and Applications, Acad. Press, M.A.Innis, D.H. Gelfand, J.J. Sninsky & T.J. White, izdajatelji.The isoforms of hML2 and hML3 and the alternate variants of hML (Rl53A, R154A) are reconstructed from hML using recombinant PCR techniques as described by Russell Higuchi in PCR, A Guide to Methods and Applications, Acad. Press, M.A.Innis, D.H. Gelfand, J.J. Sninsky & T.J. White, the traitors.

V vseh konstruktih uporabimo zunanje primerje, ki so prikazani v tabeli 8, in prekrivajoče primerje, ki so prikazani v tabeli 9.In all constructs, we use the external primers shown in Table 8 and the overlapping primers shown in Table 9.

TABELA 8TABLE 8

Zunanji primerilExternal primer

Cla.FL.F2: 5'ATC GAT ATC GAT AGC CAG ACA CCC CGG CCA G3' Cla.FL.F2: 5'ATC GAT ATC GAT AGC CAG ACA CCC CGG CCA G3 ' (SEQ ID NO: 47) (SEQ ID NO: 47) HMPLL-R: 5'GCT AGC TCT AGA CAG.GGA AGG GAG CTG TAC ATG AGA3' HMPLL-R: 5'GCT AGC TCT AGA CAG.GGA AGG GAG CTG TAC ATG AGA3 ' (SEQ ID NO: 48) (SEQ ID NO: 48)

TABELA 9TABLE 9

Prekrivajoči primerilOverlapping example

hML-2: hML-2: 5'CTC CTT GGA ACC CAG GGC AGG ACC 3‘ 5'CTC CTT GGA ACC CAG GGC AGG ACC 3 ' (SEQ ID NO: 49) (SEQ ID NO: 49) MLA4.F: MLA4.F: MLA4.R MLA4.R 5'GGT CCT GCC CTG GGT TCC AAG GAG 3' 5'GGT CCT GCC CTG GGT TCC AAG GAG 3 ' (SEQ ID NO: 50) (SEQ ID NO: 50)

123123

hML:!; hMLA116+: 5'CTG CTC CGA GGA AAG GAC TTC TGG ATT 3* h M LA 116 -: 5ΆΑΤ CCA GAA GTC CTT TCC TCG GAG CAG 3' hML:!; hMLA116 +: 5'CTG CTC CGA GGA AAG GAC TTC TGG ATT 3 * h M LA 116 -: 5ΆΑΤ CCA GAA GTC CTT TCC TCG GAG CAG 3 ' (SEQ ID NO: 51) (SEQ ID NO: 52) (SEQ ID NO: 51) (SEQ ID NO: 52) hML(R15.3Au... R354A): RR-KO-F: 5'CCC TCT GCG TCG CGG CGG CCC CAC CCA C 3' RR-KO-R: 5'GTG GGT GGG GCC GCC GCG ACG CAG AGG G 3’ hML (R15.3Au ... R354A): RR-KO-F: 5'CCC TCT GCG TCG CGG CGG CCC CAC CCA C 3 ' RR-KO-R: 5'GTG GGT GGG GCC GCC GCG ACG CAG AGG G 3 ' (SEQ ID NO: 53) (SEQ ID NO: 54) (SEQ ID NO: 53) (SEQ ID NO: 54)

Vse pomnožitve PCR izvedemo s klonirano Pfu DNA polimerazo (Stratagene), pri čemer uporabimo naslednje pogoje: začetno kalupno denaturiranje je pri 94 °C 7 minut, čemur sledi 30 ciklov po 1 min. pri 94 °C, 1 min. pri 55 °C in 1,5 min. pri 72 °C. Končni cikel pustimo, da traja 10 min. pri 72 °C. Končni produkt PCR digeriramo s Clal-Xbal, gelsko očistimo in kloniramo v pRK5tkneo. 293 celic transfektiramo z različnimi konstrukti, kot je opisano zgoraj, supernatant pa testiramo, pri čemer uporabimo test profileracije Ba/F3-mp/. Za hML-2 in hML-3 ni razvidna detektibilna aktivnost v tem testu, vendar pa je aktivnost hML(R153A, R154A) podobna hML, iz česar je razvidno, da procesiranje na tem dibazičnem mestu ni potrebno za aktivnost (sl. 13).All PCR amplifications were performed using cloned Pfu DNA polymerase (Stratagene) using the following conditions: initial mold denaturation was at 94 ° C for 7 minutes, followed by 30 cycles of 1 min. at 94 ° C, 1 min. at 55 ° C and 1.5 min. at 72 ° C. Allow the final cycle to last 10 min. at 72 ° C. The final PCR product was digested with Clal-Xbal, gel purified and cloned into pRK5. 293 cells were transfected with different constructs as described above, and the supernatant was tested using the Ba / F3-mp / profiling assay. For hML-2 and hML-3, no detectable activity was seen in this assay, but hML activity (R153A, R154A) is similar to hML, indicating that processing at this dibasic site is not required for activity (Fig. 13).

PRIMER 12 cDNA murinega mpl liganda mML, mML-2 in mML-3EXAMPLE 12 murine mpl ligand cDNA mML, mML-2 and mML-3

Izolacija cDNA mMLMML cDNA isolation

Fragment DNA, ki ustreza celotni kodirni regiji humanega mpl liganda, dobimo s PCR, gelsko očistimo in označimo z naključnim iniciranjem v prisotnosti ³²P-dATP in ³²P-dCTP. To sondo uporabimo, da selekcioniramo 10⁶ klonov knjižnice cDNA mišjih jeter v \GT10 (Clontech kat. št. ML3001a). Filtre v paralelki hibridiziramo v formamidu (35 %), 5xSSC, 10xDenhardtovem reagentu, SDS (0,1 %), natrijevem fosfatu (0,05M) (pH 6,5), natrijevem pirofosfatu (0,1 %), 100 μ-g/ml sonificirane DNA lososove sperme preko noči v prisotnosti sonde. Filtre splaknemo v 2xSSC in nato izperemo enkrat v 0,5xSSC, SDS (0,1 %) pri 42 °C. Hibridizime fage očistimo plakov in inserte cDNA subkloniramo v mesto EcoRI plazmida Bluescript SK-. KlonThe DNA fragment corresponding to the entire coding region of the human mpl ligand is obtained by PCR, gel purified and randomly labeled in the presence of ³² P-dATP and ³² P-dCTP. This probe is used to select 10 ⁶ clones of the mouse liver cDNA library in \ GT10 (Clontech cat. No. ML3001a). Filters in parallel were hybridized in formamide (35%), 5xSSC, 10xDenchardt reagent, SDS (0.1%), sodium phosphate (0.05M) (pH 6.5), sodium pyrophosphate (0.1%), 100 μ- g / ml sonicated DNA of salmon sperm overnight in the presence of a probe. The filters were rinsed in 2xSSC and then washed once in 0.5xSSC, SDS (0.1%) at 42 ° C. Phage hybrids are purified by plaque and the cDNA inserts are subcloned into the EcoRI site of the Bluescript SK- plasmid. Clone

124124

LD z 1,5 kb insertom izberemo za nadaljnjo analizo in obe vijačnici sekvenciramo, kot je opisano zgoraj za cDNA humanega ML. Nukleotid in deducirane aminokislinske sekvence iz klona LD so prikazane na sl. 14 (SEQ ID NOS:1 & 11). Deducirana sekvenca zrelega ML iz tega klona ima dolžino 331 aminokislinskih ostankov in je identificirana kot mML₃₃₁ (ali mML-2 zaradi razlogov opisanih spodaj). Znatno identičnost tako za nukleotid kot tudi za deducirane aminokislinske sekvence opazimo v domenah, podobnih EPO teh ML. Vendar pa je pri razvrstitvi deduciranih aminokislinskih sekvenc humanih in mišjih ML razvidno, da ima mišja sekvenca tetrapeptidno delecijo med humanimi ostanki 111-114, ki ustreza deleciji 12 nukleotidov po nukleotidnem položaju 618, vidni tako v humani (glej zgoraj) kot tudi prašičji (glej spodaj) cDNA. V skladu s tem raziščemo tudi dodatne klone, da detektiramo možne izo-oblike murinega ML. En klon L7 ima 1,4 kb insert z deducirano sekvenco 335 amino kislin, ki vsebuje manjkajoči tetrapeptid LPLQ. Ta oblika je verjetno murini ML s popolno dolžino, imenovana mML ali mML^. Nukleotid in deducirana aminokislinska sekvenca za mML sta prikazana na sl. 16 (SEQ ID NOS: 12 & 13). Končno klon L2 izoliramo in sekvenciramo. Ta klon ima delecijo 116 nukleotidov, ki ustreza hML3 in je zato označen mML-3. Primerjava deduciranih aminokislinskih sekvenc teh dveh izo-oblik je prikazana na sl. 16.A LD with a 1.5 kb insert was selected for further analysis, and both helices were sequenced as described above for human ML cDNA. The nucleotide and deduced amino acid sequences from the LD clone are shown in Figs. 14 (SEQ ID NOS: 1 & 11). The deduced sequence of mature ML from this clone has a length of 331 amino acid residues and is identified as mML ₃₃₁ (or mML-2 for the reasons described below). Considerable identity for both nucleotide and deduced amino acid sequences is observed in the EPO-like domains of these MLs. However, the classification of deduced amino acid sequences of human and murine MLs shows that the mouse sequence has a tetrapeptide deletion between human residues 111-114 corresponding to a deletion of 12 nucleotides at nucleotide position 618, visible in both human (see above) and pig (see below) cDNA. Accordingly, additional clones are also investigated to detect possible isoforms of murine ML. One L7 clone has a 1.4 kb insert with a deduced 335 amino acid sequence containing the missing tetrapeptide LPLQ. This format is probably full-length murines called mML or mML ^. The nucleotide and deduced amino acid sequence for mML are shown in FIG. 16 (SEQ ID NOS: 12 & 13). Finally, clone L2 is isolated and sequenced. This clone has a 116 nucleotide deletion corresponding to hML3 and is therefore designated mML-3. A comparison of the deduced amino acid sequences of these two iso-forms is shown in Figs. 16.

Ekspresija rekombinantnega mMLExpression of recombinant mML

Ekspresijske vektorje za murine ML pripravimo v bistvu tako, kot je opisano v primeru 8. Klone, ki kodirajo mML in mML-2, subkloniramo v pRK5tkneo, ekspresijski vektor sesalca, ki zagotavlja ekspresijo ob kontroli promotorja CMV in poliadenilacijskega signala SV40. Nastale ekspresijske vektorje mMLpRKtkneo in mML2pRKtkneo prehodno transfektiramo v 293 celic s kalcijevim fosfatnim postopkom. Po prehodni transfekciji medij kondicioniramo 5 dni. Celice vzdržujemo v zelo glukoznem mediju DMEM, dopolnjenim s fetalnim telečjim serumom (10 %).Expression vectors for murine ML were prepared essentially as described in Example 8. Clones encoding mML and mML-2 were subcloned into pRK5tkneo, a mammalian expression vector providing expression upon control of the CMV promoter and the SV40 polyadenylation signal. The resulting expression vectors mMLpRKtkneo and mML2pRKtkneo were transiently transfected into 293 cells by the calcium phosphate process. After transient transfection, the medium was conditioned for 5 days. Cells were maintained in high glucose DMEM supplemented with fetal calf serum (10%).

Ekspresija murinega mpl (mmpl) v celicah Ba/F3Expression of mura mpl (mmpl) in Ba / F3 cells

Stabilne celične linije, ki eksprimirajo c-mpl, dobimo s transfekcijo mmpl pRKtkneo v bistvu tako, kot je opisano za humani mpl v Primeru 1. Na kratko, ekspresijski vektor (20 μ-g; lineariziran), ki vsebuje celotno kodirno sekvenco murinega mpl (Skoda, R. C., et al., EMBO J. 12:2645-2653 [1993]), transfektiramo v celice Ba/F3 z elektroporacijo (5x1ο⁶ celic, 250 voltov, 960 μ-F), nato pa sledi selekcija za neomicinsko odpornost z 2 mg/ml G418. Ekspresijo mpl testiramo s pretočnoStable cell lines expressing c-mpl were obtained by transfection of mmpl pRKtkneo essentially as described for human mpl in Example 1. Briefly, an expression vector (20 μ-g; linearized) containing the entire coding sequence of murine mpl (Skoda, RC, et al., EMBO J. 12: 2645-2653 [1993]), transfected into Ba / F3 cells by electroporation (5x1ο ⁶ cells, 250 volts, 960 μ-F), followed by selection for neomycin resistance with 2 mg / ml G418. The mpl expression was tested by flow

125 citometrično analizo z uporabo zajčjega antimurinega znp/-IgG anti-seruma. Celice Ba/F3 vzdržujemo v mediju RPMI 1640 iz celic WEHI-3B kot vir IL-3. Supematante 293 celic, prehodno transfektirane tako z mML kot tudi z mML-2, testiramo v celicah Ba/F3, transfektiranih tako z mmpl kot tudi hmpl, kot je opisano v Primeru 1.125 cytometric analysis using rabbit antimurine znp / -IgG anti-serum. Ba / F3 cells were maintained in RPMI 1640 medium from WEHI-3B cells as a source of IL-3. Supematants of 293 cells transiently transfected with both mML and mML-2 were tested in Ba / F3 cells transfected with both mmpl and hmpl as described in Example 1.

PRIMER 13 cDNA prašičjega mpl liganda pML in pML-2 cDNA prašičjega ML (pML) izoliramo z RACE PCR. Na kratko, primer oligo dT in 2 specifična primerja oblikujemo na osnovi sekvence eksona gena prašičjega ML, ki kodira amino terminal ML, očiščenega iz aplastičnega prašičjega seruma. Dobimo cDNA, pripravljeno iz raznih aplastičnih prašičjih tkiv, in pomnožimo. Produkt cDNA PCR s 1342 bp ugotovimo v ledvicah in subkloniramo. Različne klone sekvenciramo in ugotovimo, da kodirajo zreli prašičji mpl ligand (ni vključen popolen sekrecijski signal). Za cDNA ugotovimo, da kodira zreli protein s 332 amino kislinami (pMLj₃₂), ki ima sekvenco, prikazano na sl. 18 (SEQ ID NOS: 9 & 16).EXAMPLE 13 pig mpl ligand pML and pML-2 cDNA pig ML (pML) was isolated by RACE PCR. Briefly, the oligo dT primer and 2 specific primers are formed based on the sequence of an exon of a porcine ML gene encoding an amino terminal of ML purified from aplastic porcine serum. A cDNA prepared from various aplastic swine tissues was obtained and amplified. 1342 bp PCR cDNA product was detected in the kidney and subcloned. The different clones are sequenced and found to encode a mature porcine mpl ligand (complete secretion signal not included). The cDNA was found to encode a mature 332 amino acid protein (pMLj ₃₂ ) having the sequence shown in FIG. 18 (SEQ ID NOS: 9 & 16).

POSTOPEK:PROCESS:

Izolacija gena pML in cDNA. Genomske klone gena prašičjega ML izoliramo s selekcioniranjem prašičje genomske knjižnice v EMBL3 (Clontech Inc.) s pR45. Knjižnico selekcioniramo v bistvu tako, kot je opisano v Primeru 7. Različne klone izoliramo in ekson, ki kodira aminokislinsko sekvenco, identično tisti, ki jo dobimo iz očiščenega ML, sekvenciramo. cDNA prašičjega ML dobimo z modifikacijo predpisa RACE-PCR. Dva specifična primerja ML oblikujemo na osnovi sekvence gena prašičjega ML. Poliadenilirano mRNA izoliramo iz ledvic aplastičnih prašičev v bistvu tako, kot je opisano pred tem. cDNA pripravimo z reverzno transkripcijo s primeijem BamdT, (BamdT: 5’ GACTCGAGGATCCATCGAI1111111IIIIITITT 3') (SEQ ID NO: 55) usmerjenim proti poliadenozinskemu repu mRNA. Začetni cikel pomnoževanja PCR (28 ciklov pri 95 °C 60 sekund, pri 58 °C 60 sekund in 72 °C 90 sekund) iz126 vedemo tako, da uporabimo h-prednji (forward)-l primer, specifičen za ML (h-forward-1: 5' GCTAGCTCTAGAAATTGCTCCTCGTGGTCATGCTTCT 3¹) (SEQ ID NO: 43) in primer BAMAD (BAMAD: 5‘ GACTCGAGGATCCATCG 3') (SEQ ID NO: 56) v 100 ml reakcijske zmesi KC1 (50 mM), MgCl (1,5 mM), tris (10 mM) pH 8,0 dNTP (0,2 mM) z 0,05 E/ml Amplitaq polimeraze [Perkin Elmer Inc.]). Produkt PCR nato digeriramo s Clal, ekstrahiramo s fenolom-kloroformom (1:1), oborimo z etanolom in legiramo z 0,1 mg vektoija Bluescript SK- (Stratagene Inc.), ki je prerezan s Clal in Kpnl. Po dveumi inkubaciji pri sobni temperaturi eno četrtino ligacijske zmesi dodamo direktno k drugemu ciklu PCR (22 ciklov, kot je opisano zgoraj), pri čemer uporabimo drugi prednji-1 primer, specifičen za ML (forward-1: 5' GCTAGCTCTAGAAGCCCGGCTCCTCCTGCCTG 3') (SEQ ID NO: 57) in T3-21 (oligonukleotid, ki se veže na sekvenco zraven multiple klonime regije v vektorju Bluescript SK-):Isolation of the pML and cDNA gene. Genomic clones of the porcine ML gene were isolated by selecting the porcine genomic library in EMBL3 (Clontech Inc.) with pR45. The library was selected essentially as described in Example 7. Different clones were isolated and an exon encoding an amino acid sequence identical to that obtained from purified ML was sequenced. pig ML ML cDNA is obtained by modifying the RACE-PCR regulation. Two specific primers of ML are formed based on the sequence of the porcine ML gene. The polyadenylated mRNA is isolated from the kidneys of aplastic pigs essentially as described previously. cDNA was prepared by reverse transcription with BamdT primer (BamdT: 5 'GACTCGAGGATCCATCGAI1111111IIIIITITT 3') (SEQ ID NO: 55) directed to the polyadenosine tail of the mRNA. The initial PCR amplification cycle (28 cycles at 95 ° C for 60 seconds, at 58 ° C for 60 seconds and 72 ° C for 90 seconds) is calculated from126 using the ML-specific h-forward h-forward. -1: 5 'GCTAGCTCTAGAAATTGCTCCTCGTGGTCATGCTTCT 3 ¹ ) (SEQ ID NO: 43) and BAMAD (BAMAD: 5' GACTCGAGGATCCATCG 3 ') (SEQ ID NO: 56) in 100 ml of KC1 (50 mM), MgCl (1, 1, 5). 5 mM), tris (10 mM) pH 8.0 dNTP (0.2 mM) with 0.05 E / ml Amplitaq polymerase [Perkin Elmer Inc.]). The PCR product was then digested with Clal, extracted with phenol-chloroform (1: 1), precipitated with ethanol, and alloyed with 0.1 mg of Bluescript SK- vector (Stratagene Inc.) cut with Clal and Kpnl. After two incubations at room temperature, one quarter of the ligation mixture was added directly to another PCR cycle (22 cycles as described above) using a second ML-specific forward-1 primer (forward-1: 5 'GCTAGCTCTAGAAGCCCGGCTCCTCCTGCCTG 3') ( SEQ ID NO: 57) and T3-21 (an oligonucleotide that binds to a sequence adjacent to the multiple clonime region in the Bluescript vector SK-):

(5' CGAAATTAACCCTCACTAAAG 3’) (SEQ ID NO: 58).(5 'CGAAATTAACCCTCACTAAAG 3') (SEQ ID NO: 58).

Nastali produkt PCR digeriramo z Xbal in Clal in subkloniramo v Bluescript SK-. Sekvenciramo različne klone iz neodvisnih reakcij PCR.The resulting PCR product was digested with Xbal and Clal and subcloned into Bluescript SK-. We sequenced different clones from independent PCR reactions.

Ponovno identificiramo drugo obliko, označeno pML-2, ki kodira protein z delecijo 4 aminokislinskih ostankov (328 aminokislinskih ostankov) (sl. 21 [SEQ ID NO: 21]). Iz primerjave aminokislinskih sekvenc za pML in pML-2 je razvidno, da je slednja oblika identična, razen da ima delecijo tetrapeptida QLPP, ki ustreza ostankom od 111 do vključno 114 (sl. 22 [SEQ ID NOS: 18 & 21]). Do delecije štirih amino kislin,We again identify another form, designated pML-2, that encodes a protein with a deletion of 4 amino acid residues (328 amino acid residues) (Fig. 21 [SEQ ID NO: 21]). Comparison of the amino acid sequences for pML and pML-2 shows that the latter form is identical except that it has a deletion of the tetrapeptide QLPP corresponding to residues 111 to 114 inclusive (Fig. 22 [SEQ ID NOS: 18 & 21]). Up to a deletion of four amino acids,

127 ki jo zaznamo v cDNA murinega, humanega in prašičjega ML, pride na natančno enakem položaju v napovedanih proteinih.127, which is detected in murine, human and porcine ML cDNAs, occurs at exactly the same position in the predicted proteins.

PRIMER 14EXAMPLE 14

Test CMK za trombopoetinsko (TPO) indukcijo ekspresije trombocitnega antigenaCMK assay for thrombopoietin (TPO) induction of platelet antigen expression

GPU. IIIGPU. III

-—2-—2

Celice CMK vzdržujemo v mediju RMPI 1640 (Sigma), dopolnjenem s fetalnim govejim serumom (10 %) in glutaminom (10 mM). Pri pripravi za test celice zberemo, izperemo in resuspendiramo ΙΟχΙΟ⁵ celic/ml v mediju GIF brez seruma, dopolnjenem s 5 mg/1 govejega inzulina, 10 mg/1 apo-transferina, 1 X elementi v sledovih. V vsako vdolbinico plošče z ravnim dnom in s 96 vdolbinicami damo standard TPO ali eksperimentalne vzorce pri ustreznih razredčitvah v 100 μΐ volumnih. 100 μΐ celične suspenzije CMK dodamo v vsako vdolbinico in plošče inkubiramo pri 37 °C v inkubatorju s 5 % CO₂ 48 ur. Po inkubaciji plošče vrtimo s 1000 obr./min. pri 4 °C 5 minut. Supematante zavržemo, v vsako vdolbinico pa dodamo 100 μΐ FITCkonjugiranih GPII_bIII_a monoklonskih 2D2 protiteles. Po inkubaciji pri 4 °C 1 uro plošče ponovno vrtimo s 1000 obr./min. 5 minut. Supematante, ki vsebujejo nevezana protitelesa zavržemo in v vsako vdolbinico dodamo 200 μΐ BSA-PBS izpiralne raztopine (0,1 %). Stopnjo izpiranja z BSA-PBS (0,1 %) ponovimo trikrat. Celice nato analiziramo na FASCAN z uporabo standardne eno-parametrske analize, pri čemer merimo relativno fluorescentno intenziteto.CMK cells were maintained in RMPI 1640 medium (Sigma) supplemented with fetal bovine serum (10%) and glutamine (10 mM). When preparing for the test, cells were collected, washed and resuspended ΙΟχΙΟ ⁵ cells / ml in serum-free GIF supplemented with 5 mg / 1 bovine insulin, 10 mg / 1 apo-transferrin, 1 X trace elements. A standard TPO or experimental samples are applied to each well of the 96-well flat-bottomed plate at appropriate dilutions in 100 μΐ volumes. 100 μΐ of CMK cell suspension was added to each well and the plates were incubated at 37 ° C in an incubator with 5% CO _{2 for} 48 hours. After incubation, the plates were rotated at 1000 rpm. at 4 ° C for 5 minutes. The supernatants were discarded, and 100 μΐ FITC conjugated GPII _b III _a monoclonal 2D2 antibodies were added to each well. After incubation at 4 ° C, the plates are again rotated at 1000 rpm for 1 hour. 5 minutes. Discard the antibodies containing unbound antibodies and add 200 μΐ of BSA-PBS wash solution (0.1%) to each well. The rinsing rate with BSA-PBS (0.1%) was repeated three times. Cells were then analyzed on FASCAN using standard one-parameter analysis, measuring relative fluorescence intensity.

PRIMER 15EXAMPLE 15

Test DAMI za trombopoetin (TPO) z merjenjem endomitotične aktivnosti celicDAMI assay for thrombopoietin (TPO) measuring endomitotic cell activity

DAMI na mikrotiterskih ploščah s 96 vdolbinicamiDAMI on 96 well microtiter plates

Celice DAMI vzdržujemo v IMDM + konjskem serumu (10 %) (Gibco), dopolnjenem z glutaminom (10 mM), 100 ng/ml Penicillina G in 50 μ-g/ml streptomicina. Pri pripravi za ta test celice zberemo, izperemo in resuspendiramo ΙχΙΟ⁶ celic/ml v IMDM + konjskem serumu (1 %). V ploščo z okroglim dnom s 96 vdolbinicami dodamo 100 μΐ standarda TPO ali eksperimentalnih vzorcev k celični suspenziji DAMI. Celice nato inkubiramo 48 ur pri 37 °C v inkubatorju s 5 % CO₂. Po inkubaciji plošče vrtimo v centrifugi Sorvall 6000B s 1000 obr./min 5 minut pri 4 °C. Supematante zavržemo in ponovimo izpiralno stopnjo z 200 μΐ PBS-BSA (0,1 %).DAMI cells were maintained in IMDM + horse serum (10%) (Gibco) supplemented with glutamine (10 mM), 100 ng / ml Penicillin G, and 50 μg / ml streptomycin. In preparation for this assay, cells were collected, washed, and resuspended ΙχΙΟ ⁶ cells / ml in IMDM + horse serum (1%). Add 100 μ d of TPO standard or experimental samples to the DAMI cell suspension in a 96-well round bottom plate. The cells were then incubated for 48 hours at 37 ° C in an incubator with 5% CO ₂ . After incubation, the plates were rotated in a Sorvall 6000B centrifuge at 1000 rpm for 5 minutes at 4 ° C. The supernatants were discarded and the rinsing step repeated with 200 μΐ PBS-BSA (0.1%).

128128

Celice fiksiramo z dodatkom 200 gl ledeno-hladnega 70% etanola - PBS in resuspendiramo z aspiracijo. Po inkubaciji pri 4 °C 15 minut ploščo vrtimo z 2000 obr./min 5 minut in dodamo v vsako vdolbinico 150 gl od 1 mg/ml RNA-ze, ki vsebuje 0,1 mg/ml propidijevega jodida in Tween-20 (0,05 %). Po 1 uri inkubacije pri 37 °C izmerimo spremembe v vsebnosti DNA s pretočno citometrijo. Poliploidijo izmerimo in kvantitativno določimo, kot sledi:The cells were fixed with the addition of 200 g of ice-cold 70% ethanol - PBS and resuspended by aspiration. After incubation at 4 ° C for 15 minutes, the plate was rotated at 2000 rpm for 5 minutes and 150 ml of 1 mg / ml RNA-ze containing 0.1 mg / ml propidium iodide and Tween-20 (0 were added to each well) , 05%). After 1 hour of incubation at 37 ° C, changes in DNA content were measured by flow cytometry. Polyploidy is measured and quantified as follows:

Normalizirano poliploidno razmerje (NPR) = (%celic v >G2+M/%celic v <G2+M) s TPO (%celic v >G2+M/%celic v <G2+M) v kontroliNormalized polyploid ratio (NPR) = (% of cells in> G2 + M /% of cells in <G2 + M) with TPO (% of cells in> G2 + M /% of cells in <G2 + M) in control

PRIMER 16EXAMPLE 16

Test trombopoetina (TPO) in vivo (Test oživitve mišjih trombocitov)Thrombopoietin (TPO) test in vivo (Mouse platelet recovery test)

Test nastajanja trombocitov z določitvijo ^S, in vivoPlatelet production assay by determination of ^ S, in vivo

Mišim C57BL6 (dobljene pri Charles Riveiju) injiciramo intraperitonealno (IP) 1 ml kozjega protimišjega trombocitnega seruma (6 ampul) na dan 1, da ustvarimo trombocitopenijo. Na dneva 5 in 6 damo mišim dve injekciji faktorja IP ali PBS kot kontrolo. Na dan 7 injiciramo intravenozno 30 gCi Na₂ ³⁵SO₄ v 0,1 ml fiziološke raztopine soli in izmerimo odstotek vgraditve ³⁵S injicirane doze v krožeče trombocite v krvnih vzorcih, ki jih dobimo iz obdelanih in kontrolnih miši. Število trombocitov in levkocitov določimo istočasno v krvi, ki jo dobimo iz retro-orbitalnega sinusa.C57BL6 mice (obtained from Charles Rivei) were injected intraperitoneally (IP) 1 ml goat anti-mouse platelet serum (6 ampoules) on day 1 to generate thrombocytopenia. On days 5 and 6, two injections of IP factor or PBS were administered to the mice as a control. On day 7, 30 gCi Na ₂ ³⁵ SO _{4 was} injected intravenously in 0.1 ml saline and the percentage of incorporation of ³⁵ S injected dose into circulating platelets was measured in blood samples from treated and control mice. Platelet and leukocyte counts are determined simultaneously in blood obtained from the retro-orbital sinus.

PRIMER 17EXAMPLE 17

KIRA ELISA za trombopoetin (TPO) z merjenjem fosforilaciie kimemega receptorja mpJ-Rse.gDKIRA ELISA for Thrombopoietin (TPO) by Measurement of Phosphorylation of Chimeric Receptor mpJ-Rse.gD

Receptor humanega mpl so opisali Vigon et al., PNAS, ZDA 89:5640-5644 (1992). Kimemi receptor, ki obsega ekstracelično domeno (ECD) mpl receptorja in transmembrano (TM) in intracelično domeno (ICD) Rse (Mark et al., J. of Biol. Chem. 269 (14):10720-10728 [1994]) s karboksi-terminalnim polipeptidom z repom (flag polipeptidom) (t.j. Rse.gD) naredimo za uporabo v KIRA ELISA, opisano tukaj. Ta test je prikazan z diagrami na sl. 30 in 31.The human mpl receptor has been described by Vigon et al., PNAS, USA 89: 5640-5644 (1992). Kimemi receptor comprising the extracellular domain (ECD) of the mpl receptor and the transmembrane (TM) and intracellular domain (ICD) of Rse (Mark et al., J. of Biol. Chem. 269 (14): 10720-10728 [1994]) The carboxy-terminal tail polypeptide (ie flag polypeptide) (ie Rse.gD) is made for use in the KIRA ELISA described herein. This test is illustrated by the diagrams in FIG. 30 and 31.

(a) Pripravek kaptažnega sredstva(a) Preparation of a drip agent

129129

Monoklonski anti-gD (klon 5B6) izdelamo za peptid iz glikoproteina virusa Herpes simpleks (Paborsky et al., Protein Engineering 3(6):547-553 [1990]). Očiščeni izhodni pripravek naravnamo na 3,0 mg/ml v fiziološki raztopini soli s fosfatnim pufrom (PBS), pH 7,4 in 1,0 ml alikvote shranimo pri -20 °C.A monoclonal anti-gD (clone 5B6) is made for a peptide from the Herpes simplex virus glycoprotein (Paborsky et al., Protein Engineering 3 (6): 547-553 [1990]). The purified starting material was adjusted to 3.0 mg / ml in phosphate buffered saline (PBS), pH 7.4 and 1.0 ml aliquots were stored at -20 ° C.

(b) Pripravek anti-fosfotirozinskega protitelesa(b) Preparation of anti-phosphotyrosine antibody

Monoklonski anti-fosfotirozin, klon 4G10, nabavimo pri UBI (Lake Placid, NY) in biotiniliramo z dolgokrakim biotin-N-hidroksisukcinamidom (Biotin-X-NHS, Research Organics, Cleveland, OH).Monoclonal anti-phosphotyrosine, clone 4G10, was purchased from UBI (Lake Placid, NY) and biotinylated with long-acting biotin-N-hydroxysuccinamide (Biotin-X-NHS, Research Organics, Cleveland, OH).

(c) Ligand(c) Ligand

Mpl ligand pripravimo z rekombinantnimi tehnikami, opisanimi tukaj. Očiščeni mpl ligand shranimo pri 4 °C kot izhodno osnovno raztopino.The Mpl ligand is prepared by the recombinant techniques described herein. The purified mpl ligand was stored at 4 ° C as the stock stock solution.

(d) Priprava nukleinske kisline Rse.gD(d) Nucleic acid preparation Rse.gD

Sintetične dvojnovijačne oligonukleotide uporabimo za rekonstituiranje kodirne sekvence za C-terminal 10 amino kislin (880-890) humanega Rse in dodamo dodatnih 21 amino kislin, ki vsebujejo epitop za protitelo 5B6 in stop kodon. V tabeli 10 je prikazana končna sekvenca sintetičnega dela fuzijskega gena.Synthetic double-stranded oligonucleotides are used to reconstitute the coding sequence for the C-terminal 10 amino acids (880-890) of human Rse and add an additional 21 amino acids containing an epitope for the 5B6 antibody and stop codon. Table 10 shows the final sequence of the synthetic part of the fusion gene.

TABELA 10TABLE 10

Sintetični dvoinovijačni del fuzijskega gena humanega RseSynthetic double-stranded part of the human Rse fusion gene

kodirna vijačnica: 5'-TGCAGCAAGGGCTACTGCCACACTCGAGCTGCGCAGATGCTAGCCTCAAGA TGGCTG ATCCAAATCGATTCCGCGGCAAAGATCTTCCGGTCCTGTAGAAGCT-3' coding helix: 5'-TGCAGCAAGGGCTACTGCCACACTCGAGCTGCGCAGATGCTAGCCTCAAGA TGGCTG ATCCAAATCGATTCCGCGGCAAAGATCTTCCGGTCCTGTAGAAGCT-3 ' (SEQ ID NO: 59) (SEQ ID NO: 59)

130 nekodima (anti-sens) vijačnica:130 anti-sens screwdriver:

5'-AGCTTCTACAGGACCGGAAGATCTTTGCCGCGGAATCGATTTGGATCAGCCA TCTTG AGGCTAGCATCTGCGCAGCTCGAGTGTGGCAGTAGCCCTTGCTGCA-3* (SEQ ID5'-AGCTTCTACAGGACCGGAAGATCTTTGCCGCGGAATCGATTTGGATCAGCCA TCTTG AGGCTAGCATCTGCGCAGCTCGAGTGTGGCAGTAGCCCTTGCTGCA-3 * (SEQ ID

Sintetično DNA ligiramo s cDNA, ki kodira amino kisline 1-880 humanega Rse, na mestu Pstl z začetkom pri nukleotidu 2644 objavljene sekvence cDNA humanega Rse (Mark et al., Journal of Biological Chemistiy 269(14):10720-10728 [1994]) in mestih Hindlll v poli linkeiju ekspresijskega vektorja pSVI7.ID.LL (sl. 32 A-L: SEQ ID NO: 22), da ustvarimo ekspresijski plazmid pSV.ID.Rse.gD. Na kratko, ekspresijski plazmid obsega dicistronski primarni transkript, ki vsebuje sekvenco ki kodira DHFR, vezano z intronskimi spajalnimi mesti 5’ spajalnega donoija in 3’ spajalnega akceptoija, čemur sledi sekvenca, ki kodira Rse.gD. Sporočilo s popolno dolžino (nespojeno) vsebuje DHFR kot prvi odprti bralni okvir in zato ustvari protein DHFR, da omogoči selekcijo stabilnih transformatnov.Synthetic DNA is ligated with the cDNA encoding amino acids 1-880 of human Rse at the Pst1 site starting at nucleotide 2644 of the published human Rse cDNA sequence (Mark et al., Journal of Biological Chemistry 269 (14): 10720-10728 [1994] ) and HindIII sites in the poly linkei of the expression vector pSVI7.ID.LL (Fig. 32 AL: SEQ ID NO: 22) to generate the expression plasmid pSV.ID.Rse.gD. Briefly, the expression plasmid comprises a dicistronic primary transcript containing a DHFR encoding sequence coupled to the intron junctions of the 5 ′ splice dono and the 3 ′ splice acceptor, followed by the sequence encoding Rse.gD. The full-length message (unconnected) contains DHFR as the first open reading frame and therefore creates a DHFR protein to allow selection of stable transformants.

(e) Priprava nukleinske kisline mp/-Rse.gD(e) Nucleic acid preparation mp / -Rse.gD

Ekspresijski plazmid pSV.ID.Rse.gD, izdelan kot je opisano zgoraj, modificiramo, da izdelamo plazmid pSV.ID.M.tmRd6, ki vsebuje kodirne sekvence od ECD humanega mpl (amino kisline 1-491), spojene s transmembransko domeno in intracelično domeno Rse.gD (amino kisline 429-911). Sintetične oligonukleotide uporabimo, da povežemo kodirno sekvenco dela ekstracelične domene humanega mpl z delom kodirne sekvence Rse v dvostopenjski klonimi reakciji PCR, kot opisujejo Mark et al., J. Biol. Chem. 267:26166-26171 (1992). Primeija, ki ju uporabimo za prvo reakcijo PCR, sta Ml (5'-TCTCGCTACCGTTTACAG-3') (SEQ ID NO: 61) inM2The expression plasmid pSV.ID.Rse.gD, prepared as described above, is modified to produce a plasmid pSV.ID.M.tmRd6 containing the coding sequences from ECD of human mpl (amino acids 1-491) fused to the transmembrane domain and the intracellular domain of Rse.gD (amino acids 429-911). Synthetic oligonucleotides are used to link the coding sequence of a portion of the human mpl extracellular domain to a portion of the Rse coding sequence in a two-step PCR clone reaction as described by Mark et al., J. Biol. Chem. 267: 26166-26171 (1992). The primers used for the first PCR reaction are Ml (5'-TCTCGCTACCGTTTACAG-3 ') (SEQ ID NO: 61) and M2

131 (5*-CAGGTACCCACCAGGCGGTCTCGGT-3’) (SEQ ID NO: 62) s kalupno cDNA mpl in RI (5'-GGGCCATGACACTGTCAA-3‘) (SEQ ID NO: 63) in R2 (5'-GACCGCCACCGAGACCGCCTGGTGGGTACCTGTGGTCCTT-3') (SEQ ID NO: 64) s kapulno cDNA Rse. Delež PvuII-Smal te fuzijske povezave uporabimo za konstrukcijo kimernega receptorja s popolno dolžino.131 (5 * -CAGGTACCCACCAGGCGGTCTCGGT-3 ') (SEQ ID NO: 62) with cDNA mpl and RI template (5'-GGGCCATGACACTGTCAA-3') (SEQ ID NO: 63) and R2 (5'-GACCGCCACCGAGACCGCCTGGTGTTGGTTGGTTGCTGCTGCTGGTTGCTGCTGCTGCTGCTGGTTGCTG (SEQ ID NO: 64) with the capsule cDNA Rse. The PvuII-Smal fraction is used to construct this fusion receptor to construct a full-length chimeric receptor.

(f) Celična transformacija(f) Cell transformation

Celice DP12.CHO (EP 307,247, obj. 15. marca 1989) elektroporiramo s pSV.ID.M.tmRd6, ki ga lineariziramo kot edino mesto Noti v plazmidnem ogrodju. DNA oborimo z etanolom po ekstrakciji s fenolom/kloroformom in resuspendiramo v 20 μλ 1/10 tris EDTA Nato 10 /zg DNA inkubiramo z 10⁷ celic DP12 CHO v 1 ml PBS na ledu 10 minut pred elektroporiranjem pri 400 voltih in 330 /zF. Celice damo na led za 10 minut, predno jih zasadimo v neselektivni medij. Po 24 urah celice nahranimo z medijem, ki ne vsebuje nukleozidov, da jih izberemo za stabilen DHFR+klone.DP12.CHO cells (EP 307,247, published March 15, 1989) were electroporated with pSV.ID.M.tmRd6, which was linearized as the only Noti site in the plasmid framework. DNA was precipitated with ethanol after extraction with phenol / chloroform and resuspended in 20 μλ 1/10 tris EDTA Then 10 / g DNA was incubated with 10 ⁷ DP12 CHO cells in 1 ml PBS on ice for 10 minutes before electroporation at 400 volts and 330 / zF. Put the cells on ice for 10 minutes before planting them in a non-selective medium. After 24 hours, the cells were fed with nucleoside-free medium to be selected for stable DHFR + clones.

(g) Selekcija transformiranih celic za uporabo v KIRA ELISA(g) Selection of transformed cells for use in KIRA ELISA

Klone, ki eksprimirajo MPL/Rse.gD indentificiramo z westem-blotting-om celotnih celičnih lizatov po frakcionaciji z SDS-PAGE z uporabo protitelesa 5B6, ki detektira epitopni tag gD.Clones expressing MPL / Rse.gD were identified by westem-blotting of whole cell lysates after fractionation with SDS-PAGE using 5B6 antibody, which detects the gD epitope tag.

(h) Medij(h) The medium

Celice zrastejo v F12/DMEM 50:50 (Gibco/BRL, Life Technologies, Grand Island, NY). Medij dopolnimo z diafiltriranim FBS (10 %) (HyClone, Logan, Utah), HEPES-om (25 mM) in L-glutaminom (2 mM).Cells were grown in F12 / DMEM 50:50 (Gibco / BRL, Life Technologies, Grand Island, NY). The medium was supplemented with diafiltered FBS (10%) (HyClone, Logan, Utah), HEPES (25 mM) and L-glutamine (2 mM).

132 (i)KIRA ELISA132 (i) KIRA ELISA

Celice DP12.CHO, transformirane z mpZ-Rse.gD, zasejemo (3x1ο⁴ na vdolbinco) v vdolbinice plošče za kulturo z ravnim dnom in s 96 vdolbinicami v 100 μΐ medija in kultiviramo preko noči pri 37 °C v 5 % CO₂. Naslednjo jutro v vdolbinicah supernatante dekantiramo in plošče rahlo popivnamo na papirnate brisače. Nato dodamo v vsako vdolbinico 50 μΐ medija, ki vsebuje bodisi eksperimentalne vzorce ali 200,50, 12,5, 3,12, 0,78, 0,19, 0,048 ali 0 ng/ml mpl liganda. Celice stimuliramo pri 37 °C 30 minut, supematante v vdolbinicah dekantiramo in plošče ponovno enkrat rahlo popivnamo na papirnato brisačo. Za lizo celic in solubiliziranje kimemih receptoijev dodamo v vsako vdolbinico 100 μΐ liznega pufra. Lizni pufer je sestavljen iz NaCl (150 mM), ki vsebuje HEPES (50 mM) (Gibco), Triton-X 100 (0,5 %) (Gibco), timerosal (0,01 %), 30 KIE/ml aprotina (ICN Biochemicals, Aurora, OH), 4-(2aminoetil)-benzensulfonil fluorid hidroklorid (1 mM), (AEBSF: ICN Biochemicals), levpeptin (50 μΜ) (ICN Biochemicals) in natrijev ortovanadat (2 mM) (Na₃VO₄: Sigma Chemical Co, St. Louis. MO), pH 7,5. Plošče nato rahlo tresemo na ploščnem tresalniku (Belico Instruments, Vineland, NJ) 60 minut pri sobni temperaturi.DP12.CHO cells transformed with mpZ-Rse.gD were seeded (3x1ο ⁴ per well) into the wells of a flat bottom culture plate and 96 wells in 100 μΐ of medium and cultured overnight at 37 ° C in 5% CO ₂ . The following morning, the supernatants were decanted in the wells and the plates were lightly soaked on paper towels. Then, 50 μΐ of medium containing either experimental samples or 200,50, 12,5, 3,12, 0,78, 0,19, 0,048 or 0 ng / ml mpl ligand were then added to each well. The cells were stimulated at 37 ° C for 30 minutes, the supernatants in the wells were decanted and the plates were again lightly touched once on a paper towel. For cell lysis and solubilization of the chimeric receptors, 100 μΐ of lysis buffer was added to each well. Lysis buffer consisted of NaCl (150 mM) containing HEPES (50 mM) (Gibco), Triton-X 100 (0.5%) (Gibco), thimerosal (0.01%), 30 KIE / ml aprotin ( ICN Biochemicals, Aurora, OH), 4- (2aminoethyl) -benzenesulfonyl fluoride hydrochloride (1 mM), (AEBSF: ICN Biochemicals), levpeptin (50 μΜ) (ICN Biochemicals), and sodium orthovanadate (2 mM) (Na ₃ VO ₄ : Sigma Chemical Co., St. Louis. MO), pH 7.5. The plates were then shaken gently on a plate shaker (Belico Instruments, Vineland, NJ) for 60 minutes at room temperature.

Medtem ko se celice solubilizirajo, mikrotitrsko ploščo ELISA (Nune Maxisorp, Inter Med, Danska), ki je preko noči premazana z monoklonskim anti-gD protitelesom 5B6 pri 4 °C (5,0 μg/ml v karbonatnem pufru (50 mM), pH 9,6, 100 μΙ/vdolbinico), dekantiramo, popivnamo na papirnato brisačo in blokiramo s 150 μΙ/vdolbinico pufra za blokiranje [PBS, ki vsebuje BSA (0,5 %) (Intergen Company, Purchase, NY) in timerosal (0,01 %)] 60 minut pri sobni temperaturi ob rahlem tresenju. Po 60 minutah ploščo, premazano z anti-gD 5B6, izperemo 6-krat z izpiralnim pufrom (PBS, ki vsebuje Tween-20 (0,05 %) in timerosal (0,01 %)) z avtomatiziranim ploščnim izpiralnikom (ScanWasher 300, Skatron Instruments, Inc. Sterling, VA).While the cells were solubilizing, a microtiter plate ELISA (Nune Maxisorp, Inter Med, Denmark) overnight coated with monoclonal anti-gD 5B6 at 4 ° C (5.0 μg / ml in carbonate buffer (50 mM)). pH 9.6, 100 μΙ / well), decanted, refilled on a paper towel, and blocked with 150 μΙ / well of blocking buffer [PBS containing BSA (0.5%) (Intergen Company, Purchase, NY) and thimerosal ( 0.01%)] 60 minutes at room temperature with gentle shaking. After 60 minutes, the anti-gD 5B6-coated plate was washed 6 times with wash buffer (PBS containing Tween-20 (0.05%) and thimerosal (0.01%)) with an automated plate washer (ScanWasher 300, Skatron Instruments, Inc. Sterling, VA).

Lizat, ki vsebuje solubiliziran mp//Rse.gD, iz mikrotitrske vdolbinice s celično kulturo prenesemo v vdolbinico ELISE (85 μΙ/vdolbinico), premazano z anti-gD 5B6 in blokirano, ter nato inkubiramo 2 uri pri sobni temperaturi ob rahlem tresenju. Nevezani mpl Rse.gD odstranimo z izpiranjem z izpiralnim pufrom in v vsako vdolbinico dodamo 100 μΐ biotiniliranega 4G10 (anti-fosfotirozin) razredčenega 1:18000 v razrečevalnem pufru (PBS, ki vsebuje BSA (0,05 %), Tween-20 (0,05 %), EDTA (5Lysate containing solubilized mp // Rse.gD was transferred from a microtiter well with cell culture to an ELISA well (85 μΙ / well) coated with anti-gD 5B6 and blocked and then incubated for 2 hours at room temperature with gentle shaking. Unbound mpl of Rse.gD was removed by washing with wash buffer and 100 μΐ of biotinylated 4G10 (anti-phosphotyrosine) diluted 1: 18000 in dilution buffer (PBS containing BSA (0.05%), Tween-20 (0 was added to each well) , 05%), EDTA (5

133 mM) in timerosal (0,01 %), t.j. 56 ng/ml. Po inkubaciji 2 uri pri sobni temperaturi ploščo izperemo in v vsako vdolbinico dodamo 100 μΐ streptavidina, konjugiranega s hrenovo peroksidazo (HRPO) (Zymed Laboratories, S. San Francisco, CA) in razredčenega 1:60000 v razredčevalnem pufru. Ploščo inkubiramo 30 minut pri sobni temperaturi ob rahlem tresenju. Nevezani avidinski konjugat odstranimo s spiranjem in v vsako vdolbinico dodamo 100 μΐ sveže pripravljene substratne raztopine (tetrametil benzidin [TMB]; 2-komponentni substratni kit; Kirkegaard in Peny, Gaithersburg, MD). Pustimo, da reakcija poteka 10 minut, in nato ustavimo razvijanje barve z dodatkom 100 μλ H₃PO₄ (1,0 M)/vdolbinico. Absorbanco pri 450 nm odčitamo z referenčno valovno dolžino 650 nm (ABS_450/650) z uporabo ploščnega čitalnika vmax (Molecular Devices, Palo Alto, CA), nadzorovanega z Macintosh Centris 650 (Apple Computers, Cupertino, CA) in programske opreme Delta Soft (BioMetallics, Inc, Princeton, NJ).133 mM) and thimerosal (0.01%), ie 56 ng / ml. After incubation for 2 hours at room temperature, the plate was washed and 100 μΐ of horseradish peroxidase-conjugated (HRPO) conjugated streptavidin (Zymed Laboratories, S. San Francisco, CA) was added to each well and diluted 1: 60000 in dilution buffer. The plate was incubated for 30 minutes at room temperature with gentle shaking. The unbound avidin conjugate was removed by washing and 100 μΐ of freshly prepared substrate solution (tetramethyl benzidine [TMB]; 2-component substrate kit; Kirkegaard and Peny, Gaithersburg, MD) was added to each well. Allow the reaction to proceed for 10 minutes and then stop the development of the dye by the addition of 100 μλ H ₃ PO ₄ (1.0 M) / well. Absorbance at 450 nm is read at a reference wavelength of 650 nm (ABS _450/650 ) using a vmax plate reader (Molecular Devices, Palo Alto, CA) controlled by Macintosh Centris 650 (Apple Computers, Cupertino, CA) and Delta Soft (BioMetallics, Inc., Princeton, NJ).

Standardno krivuljo naredimo s stimuliranjem dpl2.trkA,B ali celic C.gD z 200, 50, 12,5, 3,12, 0,78, 0,19, 0,048 ali 0 ng/ml mpl liganda in prikažemo kot TPO v ng/ml proti povprečju ABS_450/650 ± sd (standardna deviacija) z uporabo programa DeltaSoft. Vzorčne koncentracije dobimo z interpolacijo njihovih absorbanc na standardni krivulji in jih izrazimo kot aktivnost TPO v ng/ml.The standard curve is made by stimulating dpl2.trkA, B, or C.gD cells with 200, 50, 12.5, 3.12, 0.78, 0.19, 0.048, or 0 ng / ml mpl ligand and plotted as TPO in ng / ml vs. ABS average _450/650 ± sd (standard deviation) using DeltaSoft. The sample concentrations are obtained by interpolating their absorbances on a standard curve and expressing them as TPO activity in ng / ml.

Za mpl ligand ugotovimo, da je sposoben da aktivira kimemi receptor mpl-Rse.gD na koncentracijsko odvisen in Ugandsko specifičen način. Nadalje smo ugotovfli, da je test mpl-Rse.gD s KERA-ELISO toleranten za humani serum do 100 % (prikazano) aU plazmo do 100 % (ni prikazano), kar dopušča, da test lahko uporabimo za enostavno selekcioniranje pacienta in vzorcev pK.The mpl ligand is found to be capable of activating the mpl-Rse.gD kimemia receptor in a concentration-dependent and Ugandan-specific manner. We further found that the mpl-Rse.gD assay with KERA-ELISA was tolerant for human serum up to 100% (shown) aU plasma up to 100% (not shown), allowing the assay to be used to easily select patient and pK samples .

134134

Standardna krivulja za TPO^, izdelan v 293 celicahStandard curve for TPO ^ made in 293 cells

(ng/ml)(ng / ml)

Povzetek EC50 vrednosti TPOSummary of EC50 TPO values

Oblika TPO (celice) TPO format (cells) EC50 (mas./vol.) EC50 (w / v) EC50 (molamost) EC50 (please) Hu TPO 332 (293) Hu TPO 332 (293) 2.56 ng/ml 2.56 ng / ml 67.4 pM 67.4 pM Mu TPO 332 (293) Mu TPO 332 (293) 3.69 ng/ml 3.69 ng / ml 97.1 pM 97.1 pM Hu TPO 153 (293) Hu TPO 153 (293) -41 ng/ml -41 ng / ml -1.08 nM -1.08 nM Hu TPO 155 (£ coli) Hu TPO 155 (£ inch) 0.44 ng/ml 0.44 ng / ml 11.6 pM 11.6 pM Hu TPO 153met (E. coli) Hu TPO 153met (E. coli) 0.829 ng/ml 0.829 ng / ml 21.8 pM 21.8 pM

135135

PRIMER 18EXAMPLE 18

Receptorsko osnovana ELISA za trombopoetin (TPO)Receptor Based Thrombopoietin ELISA (TPO)

Plošče za ELISO prevlečemo z zajčjim F(Ab’)₂ anti-humanim IgG (Fc) v karbonatnem pufru s pH 9,6 in vzdržujemo preko noči pri 4 °C. Plošče blokiramo z govejim serumskim albuminom (0,5 %) v PBS pri sobni temperaturi 1 uro. Produkt fermentacije, ki vsebuje kimemi receptor, mp/-IgG, dodamo na plošče in inkubiramo 2 uri. Nato dodamo na plošče dvojne serijske razredčitve (0,39 -25 mg/ml) standarda (TPO₃₃₂, izdelan v 293 celicah s koncentracijo, določeno s kvantitativno aminokislinsko analizo) in serijsko razredčene vzorce v govejem serumskem albuminu (0,5 %), Tween-u-20 (0,05 %) in inkubiramo 2 uri. Vezan TPO detektiramo s proteinom A, očiščenimi biotiniliranimi zajčjimi protitelesi za TPO₁₅₅, ki je izdelan v E. coli (inkubacija 1 uro), in nato s streptavidin-peroksidazo (inkubacija 30 minut) in 3,3’,5,5’-tetrametil benzidinom kot substratom. Absorbanco odčitamo pri 450 nm. Plošče med stopnjami izpiramo. Za analizne podatke umerimo standardno krivuljo z uporabo štiriparametrskega programa za umerjanje krivulj od Kaleidagrapha. Koncentracijo vzorcev izračunamo iz standardne krivulje.ELISA plates were coated with rabbit F (Ab ') ₂ anti-human IgG (Fc) in carbonate buffer at pH 9.6 and maintained overnight at 4 ° C. The plates were blocked with bovine serum albumin (0.5%) in PBS at room temperature for 1 hour. The fermentation product containing the kimemic receptor, mp / -IgG, was added to the plates and incubated for 2 hours. Subsequently, double standard dilution plates (0.39-25 mg / ml) of the standard (TPO ₃₃₂ produced in 293 cells at the concentration determined by quantitative amino acid analysis) and serial diluted samples in bovine serum albumin (0.5%) were added. Tween-u-20 (0.05%) and incubated for 2 hours. Bound TPO was detected with protein A, purified biotinylated rabbit antibodies for TPO ₁₅₅ produced in E. coli (incubation for 1 hour) and then streptavidin peroxidase (incubation for 30 minutes) and 3.3 ', 5.5'- tetramethyl benzidine as a substrate. Absorbance was read at 450 nm. Wash the plates between stages. We calibrate the standard curve for the analytical data using a four-parameter curve calibration program from Kaleidagraph. The concentration of the samples is calculated from the standard curve.

PRIMER 19EXAMPLE 19

Ekspresija in čiščenje TPO iz 293 celicExpression and purification of TPO from 293 cells

1. Priprava ekpresijskih vektorjev 293 celic cDNA, ki ustreza celotnemu odprtemu bralnemu okviiju TPO, dobimo s PCR z uporabo naslednjih oligonukleotidov kot primeijev:1. Preparation of the expression vectors of 293 cDNA cells corresponding to the full open reading frame of TPO is obtained by PCR using the following oligonucleotides as primers:

TABELAHTABELAH

Primerji PCR 293 celicPCR primers of 293 cells

Cla.FL.F: 5' ATC GAT ATC GAT CAG CCA GAC ACC CCG GCC AG 3‘ Cla.FL.F: 5 'ATC GAT ATC GAT CAG CCA GAC ACC CCG GCC AG 3' (SEOIDNO: 65) (SEOID: 65) hmpll-R: 5' GCT AGC TCT AGA CAG GGA AGG GAG CTG TAC ATG AGA 3' hmpll-R: 5 'GCT AGC TCT AGA CAG GGA AGG GAG CTG TAC ATG AGA 3' (SEOIDNO: 48) (SEOID: 48)

136136

PRK5-hmp/1 (opisan v Primeru 9) uporabimo kot kalup za reakcijo v prisotnosti pfu DNA polimeraze (Stratagene). Začetno denaturiranje je 7 minut pri 94 °C, čemur sledi 25 ciklov pomnoževanja (1 min. pri 94 °C, 1 min. pri 55 °C in 1 min. pri 72 °C). Končna razširitev je 15 min. pri 72 °C. Produkt PCR očistimo in kloniramo med restrikcijski mesti Clal in Xbal plazmida pRK5tkneo, vektorja, izvedenega iz pRK5, modificiranega za ekspresijo gena, rezistentnega proti neomicinu, ob kontroli timidin kinaznega promotorja, da dobimo vektor pRK5tkneo.ORF. Drugi konstrukt, ki ustreza homologni domeni EPO, izdelamo na enak način, le z uporabo Cla.FLF kot prednjega (forward)-primeija in naslednjega reverznega primerja:PRK5-hmp / 1 (described in Example 9) was used as a reaction mold in the presence of pfu DNA polymerase (Stratagene). The initial denaturation is 7 minutes at 94 ° C, followed by 25 cycles of amplification (1 min at 94 ° C, 1 min at 55 ° C and 1 min at 72 ° C). Final extension is 15 min. at 72 ° C. The PCR product was purified and cloned between the restriction sites Clal and Xbal of the plasmid pRK5tkneo, a vector derived from pRK5 modified to express the neomycin-resistant gene under control of a thymidine kinase promoter to obtain the pRK5tkneo.ORF vector. The second construct corresponding to the homologous domain of EPO is made in the same way, only using Cla.FLF as a forward (forward) and the following reverse primer:

Arg.STOP.Xba: 5' TCT AGA TCT AGA TCA CCT GAC GCA GAG GGT GGA CC 3' (SEQ ID NO: 66)Arg.STOP.Xba: 5 'TCT AGA TCT AGA TCA CCT GAC GCA GAG GGT GGA CC 3' (SEQ ID NO: 66)

Končni konstrukt je imenovan pRK5.tkneoEPO-D. Sekvenci obeh konstruktov potrdimo, kot je opisano v Primeru 7.The final construct is named pRK5.tkneoEPO-D. The sequences of both constructs were confirmed as described in Example 7.

2. Transfekcije humanih embrionalnih ledvičnih celic2. Transfection of human embryonic kidney cells

Ta dva konstrukta transfektiramo v humane embrionalne ledvične celice s CaPO₄ postopkom, kot je opisano v Primeru 9. 24 ur po transfekciji začnemo selekcijo klonov, rezistentnih proti neomicinu, v prisotnosti 0,4 mg/ml G418.10 do 15 dni kasneje individulane kolonije prenesemo na plošče s 96 vdolbinicami in pustimo, da rastejo do konfluence. Ekspresijo ML₁₅₃ ali MLj₃₂ v kondicioniranem mediju določimo iz teh klonov s testom proliferacije Ba/F3-mp/ (opisano v Primeru 1).These two constructs were transfected into human embryonic kidney cells by the CaPO ₄ procedure as described in Example 9. 24 hours after transfection, selection of neomycin resistant clones was initiated in the presence of 0.4 mg / ml G418.10 15 days later individually colonized transfer to 96-well plates and allow to grow to confluence. The expression of ML ₁₅₃ or MLj ₃₂ in conditioned medium was determined from these clones by the Ba / F3-mp / proliferation assay (described in Example 1).

3. Čiščenje rhML₃₃₂ 3. Purification of rhML ₃₃₂

Kondicioniran medij 293-rhML₃₃₂ nanesemo na kolono blue-sefaroze (Pharmacia), uravnoteženo v natrijevem fosfatu (10 mM), pH 7,4 (pufer A). Kolono nato izperemo z 10 kolonskimi volumni tako pufra A kot tudi pufra A, ki vsebuje sečnino (2M). Kolono nato eluiramo s pufrom A, ki vsebuje sečnino (2M) in NaCI (IM). Elucijski pool blue-sefaroze nato direktno nanesemo na kolono WGA-sefaroze, uravnotežene v pufru A Kolono WGA-safaroze nato izperemo z 10 kolonskimi volumni pufra A ki vsebuje sečnino (2M) in NaCI (IM) in eluiramo z enakim pufrom, ki vsebuje N-acetil-D-glukozamin (0,5 M). Eluat WGA-sefaroze nanesemoConditioned medium 293-rhML _{332 was} applied to a blue-Sepharose (Pharmacia) column equilibrated in sodium phosphate (10 mM), pH 7.4 (buffer A). The column was then washed with 10 column volumes of both buffer A and buffer A containing urea (2M). The column was then eluted with buffer A containing urea (2M) and NaCl (IM). The blue-Sepharose elution pool is then applied directly to the WGA-Sepharose column equilibrated in buffer A The WGA-safarose column is then washed with 10 column volumes of buffer A containing urea (2M) and NaCI (IM) and eluted with the same buffer containing N -acetyl-D-glucosamine (0.5 M). WGA Sepharose eluate was applied

137 na kolono C4-HPLC (Synchrom, Inc.), uravnoteženo v TFA (0,1 %). Kolono C4HPLC eluiramo z diskontinuimim propanolnim gradientom (0-25 %, 25-35 %, 35-70 %). rhMLj^ se eluira v 28-30 % propanolnem področju gradienta. Pri SDS-PAGE migrira očiščeni rHML^ kot široka proga v 68-80 kDa področju gela (sl. 15).137 per column C4-HPLC (Synchrom, Inc.) balanced in TFA (0.1%). The C4HPLC column was eluted with a discontinuous propanol gradient (0-25%, 25-35%, 35-70%). rhMLj ^ is eluted in a 28-30% propanol gradient range. In SDS-PAGE, the purified rHML ^ migrates as a wide line in the 68-80 kDa region of the gel (Fig. 15).

4. Čiščenje rhML₁₅₃ 4. Purification of rhML ₁₅₃

Kondicioniran medij 293-rhML₁₅₃ ločimo na blue-sefarozi, kot je opisano za rhML^. Eluat blue-sefaroze nanesemo direktno na mp/-afinitetno kolono, kot je opisano zgoraj. rhML₁₅₃, eluiran iz znp/-afinentne kolone, očistimo do homogenosti v koloni C4-HPLC pri enakih pogojih, kot so opisani za rhML^. Z SDS-PAGE očiščeni rhML₁₅₃ ločimo v 2 glavni in 2 manjši progi z Mr približno 18000-21000 (sl. 15).Conditioned medium 293-rhML _{153 was} separated on blue-Sepharose as described for rhML ^. Blue-Sepharose eluate was applied directly to the mp / affinity column as described above. rhML ₁₅₃ eluted from the znp / -affinity column was purified to homogeneity in the C4-HPLC column under the same conditions as described for rhML ^. With SDS-PAGE, purified rhML _{153 was} separated into 2 major and 2 minor lines with Mr approximately 18000-21000 (Fig. 15).

PRIMER 20EXAMPLE 20

Ekspresija in čiščenje TPO iz CHOExpression and purification of TPO from CHO

1. Opis ekspresijskih vektorjev CHO1. Description of CHO expression vectors

Ekspresijski vektorji, uporabljeni v predpisani elektroporaciji, opisani spodaj, so označeni:The expression vectors used in the prescribed electroporation described below are indicated:

pSVI5.ID.LL.MLORF (popolna dolžina ali hTPO₃₃₂) in pSVI5.ID.LL.MLEPO-D (skrajšan ali hTPO₁₅₃).pSVI5.ID.LL.MLORF (full length or hTPO ₃₃₂ ) and pSVI5.ID.LL.MLEPO-D (abbreviated or hTPO ₁₅₃ ).

Pertinentne lastnosti teh plazmidov so prikazane na sl. 23 in 24.The pertinent properties of these plasmids are shown in FIG. 23 and 24.

2. Priprava ekspresijskih vektorjev CHO cDNA, ki ustreza celotnemu odprtemu bralnemu okvirju hTPO, dobimo s PCR z uporabo oligonukleotidnih primeijev iz tabele 12.2. Preparation of CHO cDNA expression vectors corresponding to the full open reading frame of hTPO was obtained by PCR using the oligonucleotide primers of Table 12.

138138

TABELA 12TABLE 12

Primeril PCR ekspresijskih vektorjev CHOCompared the PCR of CHO expression vectors

Cla.FL.F2 Cla.FL.F2 5’ ATC GAT ATC GAT AGC CAG ACA CCC CGG CČA G 3' 5 'ATC GAT ATC GAT AGC CAG ACA CCC CGG CCA G 3' (SEOIDNO: 47) (SEOID: 47) ORF. Sal ORF. Sal 5' AGT CGA CGT CGA CGT CGG CAG TGT CTG AGA ACC 3' 5 'AGT CGA CGT CGA CGT CGG CAG TGT CTG AGA ACC 3' (SEQ ID NO: 67) (SEQ ID NO: 67)

PRK5-hmp/1 (opisan v Primerih 7 in 9) uporabimo kot kalup za reakcijo v prisotnosti pfu DNA polimeraze (Stratagene). Začetna denaturacija je 7 min. pri 94 °C, čemur sledi 25 ciklov pomnoževanja (1 min. pri 24 °C, 1 min. pri 55 °C in 1 min. pri 72 °C). Končna razširitev je 15 min. pri 72 °C. Produkt PCR očistimo in kloniramo med restrikcijski mesti Clal in Šali plazmida pSVI5.ID.LL, da dobimo vektor pSVI5.ID.LL.MLORF. Drugi konstrukt, ki ustreza homologni domeni EPO, izdelamo na enak način, le z uporabo CIa.FL.F2 kot prednjega primerja in naslednjega reverznega primerja:PRK5-hmp / 1 (described in Examples 7 and 9) was used as a reaction mold in the presence of pfu DNA polymerase (Stratagene). The initial denaturation is 7 min. at 94 ° C, followed by 25 multiplication cycles (1 min at 24 ° C, 1 min at 55 ° C and 1 min at 72 ° C). Final extension is 15 min. at 72 ° C. The PCR product was purified and cloned between the restriction sites Clal and Shali of plasmid pSVI5.ID.LL to obtain the vector pSVI5.ID.LL.MLORF. The second construct corresponding to the homologous domain of the EPO is made in the same way, only using CIa.FL.F2 as the forward primer and the following reverse primer:

EPOD.SaI 5' AGT CGA CGT CGA CTC ACC TGA CGC AGA GGG TGG ACC 3’ (SEQ ID NO: 68)EPOD.SaI 5 'AGT CGA CGT CGA CTC ACC TGA CGC AGA GGG TGG ACC 3' (SEQ ID NO: 68)

Končni produkt je imenovan pSVI5.ID.LL.MLEPO-D. Sekvence obeh konstruktov potrdimo, kot je opisano v Primerih 7 in 9.The final product is named pSVI5.ID.LL.MLEPO-D. The sequences of both constructs were confirmed as described in Examples 7 and 9.

Kodirne sekvence za ligand s popolno dolžino in skrajšani ligand v bistvu uvedemo v multiplo klonimo mesto CHO ekspresijskega vektorja pSVI5.ID.LL. Ta vektor vsebuje zgodnjo promotorsko/spodbujevalno regijo SV40, modificirano spojeno enoto, ki vsebuje cDNA mišjega DHFR, multiplo klonimo mesto za uvedbo iskanega gena (v tem primeru opisane sekvence TPO), poliadenilacijski signal SV40 in izvor replikacije ter beta-laktamazni gen za plazmidno selekcijo in pomnožitev v bakterijah.The coding sequences for the full-length ligand and the truncated ligand are essentially introduced into the multiple cloned site of the CHO expression vector pSVI5.ID.LL. This vector contains the early SV40 promoter / promoter region, a modified spliced unit containing the mouse DHFR cDNA, the multiple clone insertion site (TPO sequences described in this case), the SV40 polyadenylation signal and the origin of replication, and the beta-lactamase gene for plasmid selection and amplification in bacteria.

139139

3. Metodologija za vzpostavljenje stabilnih celičnih linij CHO, ki eksprimirajo rekombinantni humani TPO₃₃₂ in TPO₁₅₃ 3. Methodology for the establishment of stable CHO cell lines expressing recombinant human TPO ₃₃₂ and TPO ₁₅₃

a. Opis parentalne celične linije CHOa. Description of parental CHO cell line

Gostiteljska celična linija CHO, uporabljena za ekspresijo molekul TPO, opisana tukaj, je znana kot CHO-DP12 (EP 307,247, obj. 15. marca 1989). To celično linijo sesalcev klonalno izberemo od transfekcije parentalne linije (CHO-K1 DUXBll(DHFR-), ki jo dobimo pri Dr. Franku Lee-ju iz Stanford University z dovoljenjem dr. L Chasina) z vektorjem, ki eksprimira preproinsulin, da dobimo klone z znižanimi insulinskimi zahtevami. Te celice so tudi DHFR minus, klone pa izberemo za prisotnost vektorskih sekvenc cDNA DHFR z rastjo v mediju brez nukleozidnih dodatkov (glicin, hipoksantin in timidin). Ta selekcijski sistem se navadno uporablja za stabilno ekspresijo celične linije CHO.The CHO host cell line used for the expression of TPO molecules described herein is known as CHO-DP12 (EP 307,247, published March 15, 1989). This mammalian cell line is selected clonally from a parental line transfection (CHO-K1 DUXBll (DHFR-) obtained from Stanford University's Dr. Frank Lee with permission from Dr. L Chasin) with a preproinsulin-expressing vector to produce clones with reduced insulin requirements. These cells are also DHFR minus, and clones are selected for the presence of DHFR cDNA vector sequences with growth in medium free of nucleoside additives (glycine, hypoxanthine and thymidine). This selection system is commonly used for stable expression of the CHO cell line.

b. Transfekcijski postopek (elektroporacija)b. Transfection procedure (electroporation)

TPO₃₃₂ in TPO₁₅₃, ki eksprimirata celične linije, izdelamo s transfektiranjem celic DP12 z elektroporacijo (glej npr. Andreason, G.L. J. Tiss. Cult. Meth., 15,56 [1993]) z lineariziranim plazmidom pSVI5.ID.LL.MLORF oz. pSVI5.ID.LL.MLEPO-D. Tri reakcijske zmesi restrikcijskih encimov nastavimo za prerezovanje vsakega plazmida; 10 /ig, 25 /ig in 50 /ig vektorja z encimom Noti s standardnimi postopki molekularne biologije. To restrikcijsko mesto ugotovimo samo enkrat v vektorju v linearizacijski regiji 3’ in zunaj transkripcijskih enot TPO liganda (sl. 23). 100 μϊ reakcijske zmesi nastavimo za inkubacijo preko noči pri 37 °C. Naslednji dan zmesi ekstrahiramo s fenolom-kloroformom-izoamil alkoholom (50:49:1), enkrat, in oborimo z etanolom na suhem ledu v približno 1 uri. Oborino nato zberemo s 15 minutnim mikrocentrifugiranjem in posušimo. Linearizirano DNA resuspendiramo v 50 μϊ Hamovega medija DMEM-F12 1:1, dopolnjenega s standardnimi antibiotiki in glutaminom (2mM).TPO ₃₃₂ and TPO ₁₅₃ expressing cell lines are made by transfection of DP12 cells by electroporation (see, e.g., Andreason, GLJ Tiss. Cult. Meth., 15.56 [1993]) with a linearized plasmid pSVI5.ID.LL.MLORF or . pSVI5.ID.LL.MLEPO-D. Three reaction mixtures of restriction enzymes were set up to cut each plasmid; 10 / ig, 25 / ig and 50 / ig vectors with Noti enzyme using standard molecular biology procedures. We find this restriction site only once in the vector in the linearization region 3 'and outside the transcription units of the TPO ligand (Fig. 23). Adjust 100 μϊ of the reaction mixture overnight for incubation at 37 ° C. The next day, the mixture was extracted with phenol-chloroform-isoamyl alcohol (50: 49: 1), once, and precipitated with ethanol on dry ice for about 1 hour. The precipitate was then collected by micro-centrifugation for 15 minutes and dried. Linearized DNA was resuspended in 50 μϊ of Ham DMM-F12 1: 1 supplemented with standard antibiotics and glutamine (2mM).

V suspenziji rastoče celice DP12 zberemo, enkrat izperemo v mediju, opisanem za resuspendiranje DNA, in končno resuspendiramo v enakem mediju pri koncentraciji 10⁷ celic/750 /d. Alikvote celic (750 μΐ) in vsako linearizirano mešanico DNA inkubiramo skupaj pri sobni temperaturi 1 uro in nato prenesemo v komoro za elektroporacijo BRL Vsako reakcijsko mešanico nato elektroporiramo v standardni napravi za elektroporacijo BRL pri 350 voltih, naravnano pri 330 /iF in nizkiIn suspension, growing DP12 cells were collected, washed once in the medium described for resuspending DNA, and finally resuspended in the same medium at a concentration of 10 ⁷ cells / 750 / d. Aliquots of cells (750 μΐ) and each linearized DNA mixture were incubated together at room temperature for 1 hour and then transferred to a BRL electroporation chamber. Each reaction mixture was then electroporated in a standard 350 volt BRL electroporation apparatus, set at 330 / iF and low.

140 kapacitanci. Po elektroporaciji celice pustimo 5 minut, da se ustalijo v napravi, in nato na ledu inkubiramo dodatnih 10 minut. Elektroporirane celice prenesemo v 60 mm posode za celično kulturo, ki vsebujejo 5 ml standarda, kompletni rastni medij za celice CHO (visoko glukozni DMEM-F12 50:50 brez glicina, dopolnjen z IX GHT, glutaminom (2 mM) in fetalnim telečjim serumom (5 %)) in pustimo, da rastejo preko noči v inkubatorju za celično kulturo s 5 % CO₂.140 capacitors. After electroporation, the cells were allowed to settle in the apparatus for 5 minutes and then incubated on ice for an additional 10 minutes. Electroporated cells were transferred to 60 mm cell culture dishes containing 5 ml of standard, complete growth medium for CHO cells (high glucose DMEM-F12 50:50 without glycine supplemented with IX GHT, glutamine (2 mM) and fetal calf serum ( 5%)) and allowed to grow overnight in a 5% CO ₂ cell culture incubator.

c. Selekcija in selekcioniral postopekc. Selection and selection process

Naslednji dan celice tripsiniziramo iz plošč s standardnimi postopki in jih prenesemo v 150 mm posode za tkivne kulture, ki vsebujejo selektivni medij DHFR (Hamov DMEM-F12 1:1, opisan zgoraj , dopolnjen z 2 % ali 5 % dializiranega fetalnega telečjega seruma, vendar brez glicina, hipoksantina in timidina; t.j. standardni selekcijski medij DHFR, ki ga mi uporabljamo). Celice iz vsake 60 mm posode nato ponovno zasadimo v 5/150 mm posode. Celice nato inkubiramo 10 do 15 dni (z eno zamenjavo medija) pri 37 °C/5 % CO₂, dokler ne nastanejo kloni in dosežejo velikost, primemo za prenos v posode s 96 vdolbinicami. Po 4 do 5 dnevih celične linije prenesemo v posode s 96 vdolbinicami s pipeto s sterilnimi rumenimi nastavki (pipettman), naravnanimi na 50 ml. Celice pustimo, da rastejo do konfluence (navadno 3-5 dni) in nato pladnje tripsiniziramo in dve kopiji originalnega pladnja reproduciramo. Dve od teh kopij za kratek čas shranimo v zmrzovalniku s celicami v vsaki vdolbinici, razredčenimi v 50 /tl FCS (10 %) v DMSO. Vzorce medija brez seruma, kondicionirane 5 dni, testiramo iz konfluentnih vdolbinic v tretjem pladnju za ekspresijo TPO s testom aktivnosti na osnovi celic Ba/F. Klone z najvišjo ekspresijo na osnovi tega testa oživimo iz zaloge in odmerimo za 2 konfluenta 150 mm T-stekleničk (scaled up to 2 confluent 150 mm T-flasks) za prenos v skupino celične kulture za suspenzijsko adaptacijo, ponovni test in shranjevanje v banki.The next day, the cells were trypsinized from standard procedure plates and transferred to 150 mm tissue culture dishes containing selective DHFR medium (Ham's DMEM-F12 1: 1 described above supplemented with 2% or 5% dialysed fetal calf serum, but without glycine, hypoxanthine and thymidine; ie the standard selection medium DHFR we use). Cells from each 60 mm container are then replanted in 5/150 mm containers. The cells were then incubated for 10 to 15 days (with one medium change) at 37 ° C / 5% CO ₂ until clones formed and reached a size, to be transferred to 96-well containers. After 4 to 5 days, transfer the cell lines to 96-well vessels with a 50 ml sterile yellow pipette pipette. The cells were allowed to grow to confluence (usually 3-5 days) and then the plates were trypsinized and two copies of the original tray were reproduced. Two of these copies were briefly stored in a freezer with cells in each well diluted in 50 / tl FCS (10%) in DMSO. Serum samples of serum-free medium conditioned for 5 days were tested from confluent wells in a third TPO expression tray with a Ba / F cell-based activity assay. Clones with the highest expression based on this test are resuscitated from stock and weighed for 2 confluents of 150 mm T-flasks (scaled up to 2 confluent 150 mm T-flasks) for transfer to the cell culture group for suspension adaptation, retest and storage in a bank.

d. Predpis pomnoževanjad. Multiplication regulation

Več celičnih linij z najvišjim titrom iz selekcije, opisane zgoraj, nato izpostavimo standardnemu pomnoževalnemu režimu z metotreksatom, da izdelamo visoko titme klone. Klone celic CHO ekspandiramo in zasadimo v posode s premerom 10 cm pri 4 koncentracijah metotreksata (t.j. 50 nM, 100 nM, 200 nM in 400 nM) pri dveh ali treh številih celic (105, 5x105 in 106 celic na posodo). Te kulture nato inkubiramo pri 37 °C/5 % CO₂, dokler niso kloni vzpostavljeni in primerni za transfer v posode s 96 vdolbinicami za nadaljnji test. Več visokotitmih klonov iz te selekcije ponovno izpos141 tavimo večjim koncentracijam metotreksata (t.j. 600 nM, 800 nM, 1000 nM in 1200 nM) in kot pred tem rezistentne klone pustimo, da se vzpostavijo in jih nato prenesemo v posode s 96 vdolbinicami in testiramo.Several highest-titre cell lines from the selection described above are then exposed to a standard methotrexate multiplication regimen to produce high-titer clones. CHO cell clones are expanded and planted in 10 cm diameter containers at 4 concentrations of methotrexate (i.e. 50 nM, 100 nM, 200 nM, and 400 nM) at two or three cell numbers (105, 5x105, and 106 cells per dish). These cultures were then incubated at 37 ° C / 5% CO ₂ until the clones were established and fit for transfer to 96-well containers for further assay. Several high-titre clones from this selection were re-emptied at higher concentrations of methotrexate (ie 600 nM, 800 nM, 1000 nM, and 1200 nM) and allowed the resistant clones to settle and then transferred to 96-well containers and tested.

4. Kultiviranje stabilne celične linije CHO, ki eksprimira rekombinantni humani TPO^z in TPO₁₅₃ 4. Cultivation of a stable CHO cell line expressing recombinant human TPO ^ z and TPO ₁₅₃

Celice, shranjene v banki, odtajamo in celično populacijo ekspandiramo s standamimi postopki za rast celic, bodisi v medij, ki ne vsebuje seruma ali takega, ki ga vsebuje. Po ekspanziji do zadostne celične gostote celice izperemo, da odstranimo izrabljen medij celične kulture. Celice nato kultiviramo s katerimkoli standardnim postopkom, vključno šaržnim, polnilnim šaržnim ali kontinuiranim kultiviranjem pri 2540 °C, nevtralnem pH, z vsebnostjo raztopljenega O₂ vsaj 5 %, da se akumulira konstitutivno izločen TPO. Tekočino celične kulture nato ločimo od celic mehanično, npr. s centrifugiranjem.Cells stored in a bank are thawed and the cell population expanded by standard cell growth procedures, either in serum-free or serum-containing medium. After expansion to sufficient cell density, the cells are washed to remove the spent cell culture medium. The cells were then cultured by any standard procedure, including batch, charge batch or continuous cultivation at 2540 ° C, neutral pH, with dissolved O ₂ content of at least 5% to accumulate constitutively secreted TPO. The cell culture fluid is then mechanically separated from the cells, e.g. with centrifugation.

5. Čiščenje rekombinantnega humanega TPO iz tekočin kulture CHO5. Purification of recombinant human TPO from CHO culture fluids

Zbrane tekočine celične kulture (HCCF) direktno nanesemo na hitro pretočno kolono blue-sefaroze 6 (Pharmacia), uravnoteženo v natrijevem fosfatu (0,01M), pH (7,4), NaCl (0,15 M), v razmerju približno 1001HCCF/1 smole in pri linearni pretočni hitrosti približno 300 ml/h/cm². Kolono nato izperemo s 3 do 5 kolonskimi volumni uravnoteževalnega pufra in nato s 3 do 5 kolonskimi volumni Na-fosfata (0,01M), pH 7,4, sečnine (2,0 M). TPO nato eluiramo s 3 do 5 kolonskimi volumni Na-fosfata (0,01 M), pH (7,4), sečnine (2,0 M), NaCl (1,0 M).The collected cell culture fluids (HCCF) were directly applied to a blue-Sepharose 6 rapid flow column (Pharmacia) equilibrated in sodium phosphate (0.01M), pH (7.4), NaCl (0.15 M), in a ratio of approximately 1001HCCF / 1 resin and at a linear flow rate of approximately 300 ml / h / cm ² . The column was then washed with 3 to 5 column volumes of equilibration buffer and then with 3 to 5 column volumes of Na-phosphate (0.01M), pH 7.4, urea (2.0 M). The TPO was then eluted with 3 to 5 column volumes of Na-phosphate (0.01 M), pH (7.4), urea (2.0 M), NaCl (1.0 M).

Pool blue-sefaroze, ki vsebuje TPO, nato nanesemo na kolono lektin(pščenični kalčki)-sefaroze 6MB (Pharmacia), uravnoteženo v Na-fosfatu (0,01M), pH (7,4), sečnini (2,0M) in NaCl (1,0 M), v razmerju od 8 do 16 ml poola blue-sefaroze/ml smole pri pretočni hitrosti približno 50 ml/h/cm². Kolono nato izperemo z 2 do 3 kolonskimi volumni uravnoteževalnega pufra. TPO nato eluiramo z 2 do 5 kolonskimi volumni Na-fosfata (0,01M), pH (7,4), sečnine (2,0M), N-acetil-Dglukozamina (0,5M).A pool of blue-Sepharose containing TPO was then applied to a column of lectin (wheat germ) -sepharose 6MB (Pharmacia) balanced in Na-phosphate (0.01M), pH (7.4), urea (2.0M) and NaCl (1.0 M), in a ratio of 8 to 16 ml of blue sepharose / ml resin at a flow rate of about 50 ml / h / cm ² . The column was then washed with 2 to 3 column volumes of equilibration buffer. The TPO was then eluted with 2 to 5 column volumes of Na-phosphate (0.01M), pH (7.4), urea (2.0M), N-acetyl-Dglucosamine (0.5M).

Pool lektina pšeničnih kalčkov nato naravnamo na končno koncentracijo C₁₂E₈ (0,04 %) in trifluorocetne kisline (TFA) (0,1 %). Nastali pool nanesemo na kolono z reverzno fazo C4 (Vydac 214TP1022), uravnoteženo v TFA (0,1 %), C₁₂E_g (0,04 %),The wheat germ lectin pool was then adjusted to a final concentration of C ₁₂ E ₈ (0.04%) and trifluoroacetic acid (TFA) (0.1%). The resulting pool was applied to a C4 reversed phase column (Vydac 214TP1022) balanced in TFA (0.1%), C ₁₂ E _g (0.04%),

142 pri čemer polnimo približno 0,2 do 0,5 mg proteina/ml smole s pretočno hitrostjo 157 ml/h/cm².142 filling about 0.2 to 0.5 mg of protein / ml of resin at a flow rate of 157 ml / h / cm ² .

Protein se eluira v dvofaznem linearnem gradientu acetonitrila, ki vsebuje TFA (0,1 %), C₁₂E₈ (0,04 %). Prva faza je sestavljena iz linearnega gradienta acetonitrila (0-30 %), 15 minut, druga faza pa je sestavljena iz linearnega gradienta acetonitrila (30-60 %), 60 minut. TPO se eluira pri približno 50 % acetonitrilu. Pool naredimo na osnovi SDS-PAGE.The protein was eluted in a two-phase linear gradient of acetonitrile containing TFA (0.1%), C ₁₂ E ₈ (0.04%). The first phase consists of a linear gradient of acetonitrile (0-30%), 15 minutes, and the second phase consists of a linear gradient of acetonitrile (30-60%), 60 minutes. TPO eluted at about 50% acetonitrile. The pool is made on the basis of SDS-PAGE.

Pool C4 nato razredčimo z 2 volumnoma Na-fosfata (0,01 M), pH (7,4), NaCl (0,15 M) in diafiltriramo proti približno 6 volumnom Na-fosfata (0,01 M), pH (7,4), NaCl (0,15 M) na ultrafiltracijski membrani Amicon YM ali podobni, ki ima mejne molekulske mase 10000 do 30000 Daltonov. Nastali diafiltrat lahko direktno procesiramo ali nadalje koncentriramo z ultrafiltradjo. Diafiltrat/koncentrat naravnamo na končno koncentracijo 0,01 % Tweena-80. Ves diafiltrat/koncentrat ali le del, ekvivalenten 2 do 5 % izračunanega kolonskega volumna, nanesemo na kolono sefakril S-300 HR (Pharmacia), uravnoteženo v Na-fosfatu (0,01 M), pH (7,4), NaCl (0,15M), Tweenu-80 (0,01 %) in kromatografiramo pri pretočni hitrosti približno 17 ml/h/cm². TPO, ki vsebuje frakcije, ki so brez agregatov in proteolitičnih degradacijskih produktov, zberemo v poole na osnovi SDS-PAGE. Dobljeni pool filtriramo na 0,22 μτη filtru, Millex-GV ali podobnem in shranimo pri 2-8 °C.Pool C4 is then diluted with 2 volumes of Na-phosphate (0.01 M), pH (7.4), NaCl (0.15 M), and diafiltered against approximately 6 volumes of Na-phosphate (0.01 M), pH (7 , 4), NaCl (0.15 M) on an Amicon YM ultrafiltration membrane or similar having a molecular weight limit of 10,000 to 30000 Daltons. The resulting diafiltrate can be directly processed or further concentrated by ultrafiltration. Adjust the diafiltrate / concentrate to a final concentration of 0.01% Tweena-80. All diafiltrate / concentrate, or only a portion equivalent to 2 to 5% of the calculated column volume, was applied to a column of sefacryl S-300 HR (Pharmacia) balanced in Na-phosphate (0.01 M), pH (7.4), NaCl ( 0.15M), Tweenu-80 (0.01%) and chromatographed at a flow rate of about 17 ml / h / cm ² . TPO containing fractions that are devoid of aggregates and proteolytic degradation products are collected into SDS-PAGE-based pooles. The resulting pool is filtered on a 0.22 μτη filter, Millex-GV or the like and stored at 2-8 ° C.

PRIMER 21EXAMPLE 21

Transformacija in indukcija sinteze proteina TPO v E.coliTransformation and induction of TPO protein synthesis in E.coli

1. Konstrukcija ekspresijskih vektoijev E.coli TPO1. Construction of E.coli TPO expression vectors

Plazmidi pMP21, pMP151, pMP41, pMP57 in pMP202 so vsi oblikovani za ekspresijo prvih 155 amino kislin TPO navzdol od majhnega voditelja, ki variira med različnimi konstrukti. Voditelji zagotavljajo primarno visok nivo translacijske iniciacije in hitro čiščenje. Plazmidi pMP210-l, -T8, -21, -22, -24, -25 so oblikovani za ekspresijo prvih 153 amino kislin TPO navzdol od iniciacijskega metionina in se razlikujejo le v kodonski uporabi za prvih 6 amino kislin TPO, medtem ko je plazmid pMP251 derivat pMP210-l, v katerem je karboksi terminalni konec TPO razširjen z dvema amino kislinama. Vsi zgornji plazmidi proizvedejo visoke nivoje intracelične ekspresije TPO v E.coli po indukciji triptofanskega promotoija (Yansura, D.G. et al., Methods in Enzymology (Goeddel, D.V., izd.) 185:54-60, Academic Press, San DiegoPlasmids pMP21, pMP151, pMP41, pMP57 and pMP202 are all designed to express the first 155 amino acids of TPO downstream of a small leader that varies between different constructs. Leaders provide a primarily high level of translational initiation and rapid purification. Plasmids pMP210-l, -T8, -21, -22, -24, -25 are designed to express the first 153 amino acids of TPO downstream of the initiating methionine and differ only in codon usage for the first 6 amino acids of TPO, while the plasmid is pMP251 is a pMP210-l derivative in which the carboxy terminal end of TPO is extended by two amino acids. All of the above plasmids produce high levels of intracellular TPO expression in E. coli after induction of tryptophan promoter (Yansura, D.G. et al., Methods and Enzymology (Goeddel, D.V. ed.) 185: 54-60, Academic Press, San Diego

143 [1990]). Plazmida pMPl in pMP172 sta intermediata v konstrukciji zgornjih intraceličnih ekspresijskih plazmidov TPO.143 [1990]. Plasmids pMP1 and pMP172 are intermediates in the construction of the upper intracellular TPO expression plasmids.

(a) Plazmid pMPl(a) Plasmid pMPl

Plazmid pMPl je sekrecijski vektor za prvih 155 amino kislin TPO in ga konstruiramo z ligiranjem skupaj 5 fragmentov DNA, kot je prikazano na sl. 33. Prvi od teh je vektor pPho21, v katerem je odstranjen majhen fragment MIuI-BamHI. pPho21 je derivat phGHl (Chang, C.N. et al., Gene 55:189-196 [1987]), v katerem je gen humanega rastnega hormona nadomeščen z genom phoA E.coli in je restrikcijsko mesto MIuI konstruirano v kodirni sekvenci za signalno sekvenco STII pri amino kislinah 20-21.Plasmid pMPl is the secretion vector for the first 155 amino acids of TPO and is constructed by ligation of a total of 5 DNA fragments as shown in FIG. 33. The first of these is the pPho21 vector, in which a small fragment of MIuI-BamHI is removed. pPho21 is a derivative of phGH1 (Chang, CN et al., Gene 55: 189-196 [1987]), in which the human growth hormone gene is replaced by the E. coli phoA gene and the restriction site of MIuI is constructed in the coding sequence for the STII signal sequence at amino acids 20-21.

Naslednja dva fragmenta, košček DNA z 258 bp Hinfl-Pstl iz pRK5-hznp/I (Primer 9), ki kodira amino kisline 19-103 TPO in naslednjo sintetično DNA, ki kodira amino kisline 1-18The next two fragments, a piece of DNA with 258 bp Hinfl-Pstl from pRK5-hznp / I (Example 9) encoding amino acids 19-103 TPO and the next synthetic DNA encoding amino acids 1-18

5'-CGCGTATGCCAGCCCGGCTCCTCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCG5'-CGCGTATGCCAGCCCGGCTCCTCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCG

TGTG

ATACGGTCGGGCCGAGGAGGACGAACACTGGAGGCTCAGGAGTCATTTGACGAAGCATACGGTCGGGCCGAGGAGGACGAACACTGGAGGCTCAGGAGTCATTTGACGAAGC

ACTGA-5' (SEQ ID NO: 69) (SEQ ID NO: 70) predhodno ligiramo z ligazo T4-DNA, drugega pa prerežemo s Pstl. Četrti je fragment s 152 bp Pstl-Haelll od pRK5hmpII, ki kodira amino kisline 104-155 TPO. Zadnji je fragment s 412 bp StuI-BamHI iz pdhl08, ki vsebuje lambdo za transkripcijski terminator, kot so pred tem opisali (Scholtissek, S. et al., NAR 15:3185 [1987]).ACTGA-5 '(SEQ ID NO: 69) (SEQ ID NO: 70) was previously ligated with T4-DNA ligase and the second was cut with Pstl. The fourth is a 152 bp Pstl-Haelll fragment of pRK5hmpII encoding amino acids 104-155 TPO. The last is a 412 bp StuI-BamHI fragment from pdhl08 containing a lambda for the transcription terminator as previously described (Scholtissek, S. et al., NAR 15: 3185 [1987]).

(b) Plazmid pMP21(b) Plasmid pMP21

Plazmid pMP21 oblikujemo za ekspresijo prvih 155 amino kislin TPO z voditeljem s 13 amino kislinami, ki obsega del signalne sekvence STIL Konstruiramo ga z ligiranjem skupaj treh framgnetov DNA, kot je prikazano na sl. 34, pri čemer je prvi vektor pVEG31, v katerem je majhen fragment Xbal-Sphl odstranjen. Vektor pVEG31 jePlasmid pMP21 is designed to express the first 155 amino acids of TPO with a 13 amino acid leader comprising part of the STIL signal sequence. It is constructed by ligation of a total of three DNA framgnets, as shown in FIG. 34, the first vector being pVEG31 in which a small fragment of Xbal-Sphl is removed. The vector is pVEG31

144 derivat pHGH207-l (de Boer, H.A. et al., v Promoter Strucutre and Function (Rodriguez, R.L. in Chamberlain, M.J. izd.), 462, Praeger, New York [1982]), v katerem je gen humanega rastnega hormona nadomeščen z genom vaskulamega endotelijskega rastnega faktorja (ta identični vektorski fragment lahko dobimo iz slednjega plazmida).144 derivative of pHGH207-l (de Boer, HA et al., In Strucutre and Function Promoter (Rodriguez, RL and Chamberlain, MJ eds.), 462, Praeger, New York [1982]), in which the human growth hormone gene is replaced with the vascular endothelial growth factor gene (this identical vector fragment can be obtained from the latter plasmid).

Drugi del v ligaciji je sintetični dupleks DNA z naslednjo sekvencoThe second part in the ligation is a synthetic DNA duplex with the following sequence

5'-CTAGAATTATGAAAAAGAATATCGCATTTCTTCTTAA5'-CTAGAATTATGAAAAAGAATATCGCATTTCTTCTTAA

TTAATACTmTCTTATAGCGTAAAGAAGAATTGCGC-5· (SEQ ID NO: 71) (SEQ ID NO: 72)TTAATACTmTCTTATAGCGTAAAGAAGAATTGCGC-5 · (SEQ ID NO: 71) (SEQ ID NO: 72)

Zadnji košček je fragment MluI-SphI z 1072 bp iz pMPl, ki kodira 155 amino kislin TPO.The last bit is a 1072 bp MluI-SphI fragment from pMP1 encoding 155 amino acids of TPO.

(c) Plazmid pMP151(c) Plasmid pMP151

Plazmid pMP151 je oblikovan za ekspresijo prvih 155 amino kislin TPO navzdol od voditelja, ki obsega 7 amino kislin signalne sekvence STII, 8 histidinov in cepišče faktorja Xa. k sl. 35 je razvidno, daje pMP151 konstruiran z ligiranjem skupaj treh fragmentov DNA, pri čemer je prvi, predhodno opisani vektor, pVEG31, iz katerega je odstranjen majhen fragment XbaI-SphL Drugi je sintetični dupleks DNA z naslednjo sekvenco:Plasmid pMP151 is designed to express the first 155 amino acids of TPO downstream of a leader comprising 7 amino acids of the STII signal sequence, 8 histidines, and factor Xa cleavage. k fig. 35 shows that pMP151 is constructed by ligation of a total of three DNA fragments, the first vector previously described being pVEG31 from which a small XbaI-SphL fragment is removed The second is a synthetic DNA duplex with the following sequence:

5'-CTAGAATTATGAAAAAGAATATCGCATTTCATCACCATCACCATCACCATCACATCGAAG5'-CTAGAATTATGAAAAAGAATATCGCATTTCATCACCATCACCATCACCATCACATCGAAG

GTCGTAGCCGTCGTAGCC

TTAATACTTTTTCTTATAGCGTAAAGTAGTGGTAGTGGTAGTGGTAGTGTAGCTTCTTAATACTTTTTCTTATAGCGTAAAGTAGTGGTAGTGGTAGTGGTAGTGTAGCTTC

CAGCAT-5' (SEQ ID NO: 73) (SEQ ID NO: 74)CAGCAT-5 '(SEQ ID NO: 73) (SEQ ID NO: 74)

Zadnji je fragment Bgll-SphI s 1064 bp iz pMPll, ki kodira 154 amino kislin TPO. Plazmid pMPll je identičen pMPl, razen nekaj kodonskih sprememb v signalni sekvenci STII (ta fragment lahko dobimo iz pMPl).The last is a 1064 bp Bgll-SphI fragment from pMPll encoding 154 amino acids of TPO. The plasmid pMPll is identical to pMPl except for some codon changes in the STII signal sequence (this fragment can be obtained from pMPl).

145 (d) Plazmid pMP202145 (d) Plasmid pMP202

Plazmid pMP202 je zelo podoben ekspresijskemu vektorju pMP151, razen da je cepišče faktorja Xa v voditelju nadomeščeno s trombinskim cepiščem. Iz sl. 36 je razvidno, da pMP202 konstruiramo z ligiranjem skupaj treh fragmentov DNA. Prvi od teh je predhodno opisani pVEG31, v katerem je majhen fragment Xbal-Sphl odstranjen. Drugi je sintetični dupleks DNA z naslednjo sekvenco:Plasmid pMP202 is very similar to the expression vector pMP151 except that the factor Xa cleavage site in the leader is replaced by a thrombin cleavage site. From FIG. 36 shows that pMP202 is constructed by ligation of a total of three DNA fragments. The first of these is the previously described pVEG31, in which a small fragment of Xbal-Sphl is removed. The second is a synthetic DNA duplex with the following sequence:

5’-CTAGAATTATGAAAAAGAATATCGCATTTCATCACCATCACCATCACCATCACATCGAA5'-CTAGAATTATGAAAAAGAATATCGCATTTCATCACCATCACCATCACCATCACATCGAA

CCACGTAGCCCCACGTAGCC

TTAATACPTTTCTTATAGCGTAAAGTAGTGGTAGTGGTAGTGGTAGTGTAGCTTTTAATACPTTTCTTATAGCGTAAAGTAGTGGTAGTGGTAGTGGTAGTGTAGCTT

GGTGCAT-5' (SEQ ID NO: 75) (SEQ ID NO: 76)GGTGCAT-5 '(SEQ ID NO: 75) (SEQ ID NO: 76)

Zadnji košček je fragment Bgll-SphI s 1064 bp iz predhodno opisanega plazmida pMPll.The last bit is a 1064 bp Bgll-SphI fragment from the previously described plasmid pMPll.

(e) Plazmid pMP172(e) Plasmid pMP172

Plazmid pMP172 je sekrecijski vektor za prvih 153 amino kislin TPO in je intermediat za konstrukcijo pMP210. Iz sl. 37 je razvidno, da pMP172 pripravimo z ligiranjem skupaj treh fragmentov DNA, pri čemer je prvi vektor pLS32IamB, v katerem je majhen predel EcoRI-Hindlll odstranjen. Drugi je framgnet EcoRI-Hgal s 946 bp iz predhodno opisanega plazmida pMPll. Zadnji košček je sintetični dupleks DNA z naslednjo sekvenco:Plasmid pMP172 is the secretion vector for the first 153 amino acids of TPO and is an intermediate for the construction of pMP210. From FIG. 37 shows that pMP172 is prepared by ligation of three DNA fragments together, the first vector being pLS32IamB, in which a small EcoRI-HindIII region is removed. The other is the 946 bp EcoRI-Hgal framgnet from the previously described plasmid pMPll. The last bit is a synthetic DNA duplex with the following sequence:

5'-TCCACCCTCTGCGTCAGGT5'-TCCACCCTCTGCGTCAGGT

GGAGACGCAGTCCATCGA-5' (SEQ ID NO: 77) (SEQ ID NO: 78) (f) Plazmid pMP210GGAGACGCAGTCCATCGA-5 '(SEQ ID NO: 77) (SEQ ID NO: 78) (f) Plasmid pMP210

146146

Plazmid pMP210 oblikujemo za ekspresijo prvih 153 amino kislin TPO po translacijskem iniciacijskem metioninu. Ta plazmid dejansko naredimo kot banko plazmidov, v katerih je prvih 6 kodonov TPO naključnih v tretjem položaju vsakega kodona, in ga konstruiramo, kot je prikazano na sl. 38, z ligacijo treh fragmentov DNA. Prvi od teh je predhodno opisani vektor pVEG31, v katerem je majhen fragment Xbal-Sphl odstranjen. Drugi je sintetični dupleks DNA, prikazan spodaj, obdelan najprej z DNA polimerazo I (Klenow) in nato digeriran z Xbal in Hinfl, in kodira iniciacijski metionin in naključnih prvih 6 kodonov TPO.Plasmid pMP210 is formulated to express the first 153 amino acids of TPO after translational initiation methionine. We actually make this plasmid as a bank of plasmids in which the first 6 TPO codons are random in the third position of each codon, and construct it as shown in FIG. 38, by ligation of three DNA fragments. The first of these is the previously described vector pVEG31, in which a small fragment of Xbal-Sphl is removed. The second is the synthetic DNA duplex shown below, first treated with DNA polymerase I (Klenow) and then digested with Xbal and Hinfl, and encodes the initiating methionine and random first 6 TPO codons.

5'-GCAGCAGTTCTAGAATTATGTCNCCNGCNCCNCCNGCNTGTGACCTCCGA5'-GCAGCAGTTCTAGAATTATGTCNCCNGCNCCNCCNGCNTGTGACCTCCGA

ACACTGGAGGCTACACTGGAGGCT

GTTCTCAGTAAA (SEQ ID NO: 79)GTTCTCAGTAAA (SEQ ID NO: 79)

CAAGAGTCATTTGACGAAGCACTGAGGGTACAGGAAG-5’ (SEQ ID NO: 80)CAAGAGTCATTTGACGAAGCACTGAGGGTACAGGAAG-5 '(SEQ ID NO: 80)

Tretji je fragment Hinfl-Sphl z 890 bp iz pMP172, ki kodira amino kisline 19-153 TPO.The third is an 890 bp Hinfl-Sphl fragment from pMP172 encoding amino acids 19-153 TPO.

Plazmidno banko pMP210 s približno 3700 kloni retransformiramo na visoko tretraciklinske (50 /ig/ml) plošče LB, da izberemo visoko translacijsko iniciacijske klone (Yansura, D.G. et al., Methods: A Companion to Methods in Enzymology 4:151-158 [1992]). Od 8 kolonij, ki nastanejo na visoko tetraciklinskih ploščah, jih pet najboljših glede na ekspresijo TPO izpostavimo sekvenciranju DNA, rezultati pa so prikazani na sl. 39 (SEQ ID NOS: 23,24,25,26,27 in 28).The plasmid bank pMP210 with about 3700 clones was retransformed onto high Tretricycline (50 / ig / ml) LB plates to select high translational initiation clones (Yansura, DG et al., Methods: A Companion to Methods in Enzymology 4: 151-158 [1992 ]). Of the 8 colonies formed on highly tetracycline plates, the five best exposed to DNA sequencing in terms of TPO expression, and the results are shown in Figs. 39 (SEQ ID NOS: 23,24,25,26,27 and 28).

(g) Plazmid pMP41(g) Plasmid pMP41

Plazmid pMP41 oblikujemo za ekspresijo prvih 155 amino kislin TPO, spojenega z voditeljem, ki je sestavljen iz 7 amino kislin signalne sekvence STII, čemur sledi cepišče faktoija Xa. Plazmid konstruiramo, kot je prikazano na sl. 40, z ligiranjem skupaj treh koščkov DNA, pri čemer je prvi predhodno opisani vektor pVEG31, v katerem je majhen fragment Xbal-Sphl odstranjen. Drugi je naslednji sintetični dupleks DNA:Plasmid pMP41 is formulated to express the first 155 amino acids of TPO fused to a leader consisting of 7 amino acids of the STII signal sequence, followed by the Xa facto cleavage site. The plasmid is constructed as shown in FIG. 40, ligating a total of three pieces of DNA, the first being the previously described vector pVEG31, in which a small Xbal-Sphl fragment is removed. The second is the following synthetic DNA duplex:

147147

5'-CTAGAATTATGAAAAAGAATATCGCATTTATCGAAGGTCGTAGCC (SEQ ID NO: 81)5'-CTAGAATTATGAAAAAGAATATCGCATTTATCGAAGGTCGTAGCC (SEQ ID NO: 81)

TTAATACTnTTCTTATAGCGTAAATAGCTTCCAGCAT-5' (SEQ ID NO: 82)TTAATACTnTTCTTATAGCGTAAATAGCTTCCAGCAT-5 '(SEQ ID NO: 82)

Zadnji košček ligacije je fragment Bgll-SphI s 1064 bp iz predhodno opisanega plazmida pMPll.The last piece of ligation is a 1064 bp Bgll-SphI fragment from the previously described plasmid pMPll.

(h) Plazmid pMP57(h) Plasmid pMP57

Plazmid pMP57 eksprimira prvih 155 amino kislin TPO navzdol od voditelja, ki je sestavljen iz 9 amino kislin signalne sekvence STII in dibazičnega mesta Lys-Arg. To dibazično mesto zagotavlja sredstvo za odstranitev voditelja s proteazo ArgC. Plazmid konstruiramo, kot je prikazano na sl. 41, z ligiranjem skupaj treh koščkov DNA. Prvi od teh je predhodno opisani vektor pVEG31, v katerem je majhen fragment Xbal-Sphl odstranjen. Drugi je naslednji sintetični dupleks DNA:Plasmid pMP57 expresses the first 155 amino acids of TPO downstream of a leader consisting of 9 amino acids of the STII signal sequence and the dysbasic Lys-Arg site. This dibasic site provides a means of removing the leader with the ArgC protease. The plasmid is constructed as shown in FIG. 41, by ligation of a total of three pieces of DNA. The first of these is the previously described vector pVEG31, in which a small fragment of Xbal-Sphl is removed. The second is the following synthetic DNA duplex:

5'-CTAGAATTATGAAAAAGAATATCGCATTTCTTCTTAAACGTAGCC (SEQ ID NO: 83)5'-CTAGAATTATGAAAAAGAATATCGCATTTCTTCTTAAACGTAGCC (SEQ ID NO: 83)

TTAATACTTTTTCTTATAGCGTAAAGAAGAATTTGCAT-5' (SEQ ID NO: 84)TTAATACTTTTTCTTATAGCGTAAAGAAGAATTTGCAT-5 '(SEQ ID NO: 84)

Zadnji del ligacije je fragment Bgll-SphI s 1064 bp iz predhodno opisanega plazmida pMPll.The last part of the ligation is a 1064 bp Bgll-SphI fragment from the previously described plasmid pMPll.

(i) Plazmid pMP251(i) Plasmid pMP251

Plazmid pMP251 je derivat pMP210-l, v katerega sta vljučeni dve dodatni amino kislini TPO na karboksi terminalnem koncu. Iz sl. 42 je razvidno, da ta plazmid konstruiramo z ligiranjem skupaj dveh koščkov DNA pri čemer je prvi od teh predhodno opisani pMP21, v katerem je majhen fragment Xbal-Apal odstranjen. Drugi del ligacije je fragment s 316 bp Xbal-Apal iz pMP210-l.Plasmid pMP251 is a pMP210-l derivative that includes two additional amino acids TPO at the carboxy terminal end. From FIG. 42 shows that this plasmid is constructed by ligation of two pieces of DNA together, the first of these being the previously described pMP21, in which a small Xbal-Apal fragment is removed. Another part of the ligation is a 316 bp Xbal-Apal fragment from pMP210-l.

2. Transformacija in indukcija E.coli z ekspresijskimi vektoiji TPO2. Transformation and induction of E.coli by TPO expression vectors

Zgornje ekspresijske plazmide TPO uporabimo za transformiranje E.coli seva 44C6 (w3110 ίοηΑ_Δ rpoH_u Ιοη_Δ clpP_A galE), pri Čemer uporabimo postopek toplotnega šoka s CaClj (Mandel, M. et al., J. Mol. Biol. 53:159-162, [1970]). Transformirane celice rastejo najprej pri 37 °C v mediju LB, ki vsebuje 50 Mg/ml karbenicilina, dokler optična gostota (600 nm) kulture ne doseže 2-3. Kulturo LB nato razredčimo 20-kratThe TPO expression plasmids above were used to transform E.coli strain 44C6 (w3110 ίοηΑ _Δ rpoH _u Ιοη _Δ clpP _A galE), using a thermal shock procedure with CaClj (Mandel, M. et al., J. Mol. Biol. 53: 159-162, [1970]. The transformed cells were first grown at 37 ° C in LB medium containing 50 Mg / ml carbenicillin until the optical density (600 nm) of the culture reached 2-3. The LB culture was then diluted 20 times

148 v medij M9, ki vsebuje kazamino kisline (0,49 mas./vol.%) in 50 /ig/ml karbenicilina. Po enourni rasti z aeracijo pri 30 °C dodamo indol-3-akrilno kislino do končne koncentracije 50/xg/ml. Kulturo nato pustimo, da raste pri 30 °C ob aeraciji nadaljnjih 15 ur, nakar celice zberemo s centrifugiranjem.148 to medium M9 containing casino acid (0.49 w / v%) and 50 / g / ml carbenicillin. After one hour of growth by aeration at 30 ° C, indole-3-acrylic acid was added to a final concentration of 50 / xg / ml. The culture was then allowed to grow at 30 ° C with aeration for a further 15 hours, then the cells were collected by centrifugation.

PRIMER 22EXAMPLE 22

Izdelovanje biološko aktivnega TPO (Met-1-153) v E. coliProduction of biologically active TPO (Met-1-153) in E. coli

Postopke, navedene spodaj, za izdelovanje biološko aktivnega renaturiranega TPO (Met⁴ 1-153) lahko analogno uporabimo za rekuperiranje drugih variant TPO, ki vključujejo N- in C-terminalno razširjene oblike (Primer 23).The procedures below for the production of biologically active renatured TPO (Met ⁴ 1-153) can be analogously used to recover other variants of TPO that include N- and C-terminally extended forms (Example 23).

A. Rekuperiranje netopnega TPO (Met⁴ 1-153)A. Recovery of Insoluble TPO (Met ⁴ 1-153)

Celice E. coli, ki eksprimirajo TPO (Met⁴ 1-153), ki je kodiran s plazmidom pMP210-l fermentiramo, kot je opisano zgoraj. Značilno približno 100 g celic resuspendiramo v 11 (10 volumnov) pufra za razkroj celic (tris (10 mM), EDTA (5 mM), pH 8) s homogenizatorjem Polytron in celice centrifugiramo pri 5000 g 30 minut. Izprani celični pelet ponovno resuspendiramo v 11 pufra za razkroj celic s homogenizatorjem Polytron in celično suspenzijo spustimo skozi napravo za razkroj celic LH Celi Dissrupter (LH Inceltech, Inc.) ali skozi mikrofluidizator (Microfluidizer, Microfluidics International) po navodilih izdelovalca. Suspenzijo centrifugiramo pri 5000 g 30 minut in resuspendiramo ter centrifugiramo drugič, da naredimo izprani pelet refraktilnih teles. Izprani pelet takoj uporabimo ali zmrznjenega shranimo pri -70 °C.E. coli cells expressing TPO (Met ⁴ 1-153) encoded by plasmid pMP210-l were fermented as described above. Typically, about 100 g of cells were resuspended in 11 (10 volumes) cell digestion buffer (tris (10 mM), EDTA (5 mM), pH 8) with a Polytron homogenizer, and the cells were centrifuged at 5000 g for 30 minutes. The washed cell pellet was resuspended in 11 cell dissolution buffers with a Polytron homogenizer and the cell suspension was lowered through a LH Whole Dissrupter cell decomposition device (LH Inceltech, Inc.) or through a microfluidizer (Microfluidizer, Microfluidics International) according to the manufacturer's instructions. The suspension was centrifuged at 5000 g for 30 minutes and resuspended and centrifuged a second time to produce the washed pellet of refractile bodies. The washed pellet should be used immediately or stored frozen at -70 ° C.

B. Solubilizacija in čiščenje monomemega TPO (Met⁴ 1-153)B. Solubilization and purification of monomemic TPO (Met ⁴ 1-153)

Zgornji pelet resuspendiramo v 5 volumnih (masnih) tris-a (20 mM), pH 8 z gvanidinom (6-8 M) in DTT (ditiotreitol) (25 mM) in mešamo 1-3 ure ali preko noči pri 4 “C, da povzročimo solubilizacijo proteina TPO. Visoke koncentracije sečnine (6-8 M) prav tako lahko uporabimo, vendar pa so navadno dobitki nižji, če jih primerjamo z gvanidinom. Po solubilizaciji raztopino centrifugiramo pri 30000 g (30 minut), da dobimo bister supernatant, ki vsebuje denaturirani monomemi protein TPO. Supernatant nato kromatografiramo na gelski filtracijski koloni superdeks 200 (Pharmacia, 2,6 x 60 cm) pri pretočni hitrosti 2 ml/min in protein eluiramo z Na149 fosfatom (20 ml), pH 6,0 in zberemo poole frakcij DTT (10 mM), ki vsebujejo monomemi denaturirani protein TPO, ki se eluira med 160 in 200 ml. Protein TPO nadalje očistimo na semipreparativni koloni z reverzno fazo C4 (2 x 20 cm VYDAC). Vzorec nanesemo s 5 ml/min. na kolono, uravnoteženo v TFA (trifluoroocetna kislina) (0,1 %) z acetonitrilom (30 %). Protein se eluira z linearnim gradientom acetonitrila (30-60 % v 60 min). Očiščeni redukcijski protein se eluira pri približno 50 % acetonitrilu. To snov uporabimo za renaturiranje, da dobimo biološko aktivno varianto TPO.The above pellet was resuspended in 5 volumes of tris (20 mM), pH 8 with guanidine (6-8 M) and DTT (dithiothreitol) (25 mM) and stirred for 1-3 hours or overnight at 4 ° C. to solubilize the TPO protein. High concentrations of urea (6-8 M) can also be used, but the yields are usually lower when compared to guanidine. After solubilization, the solution was centrifuged at 30000 g (30 minutes) to obtain a clear supernatant containing the denatured monomeric TPO protein. The supernatant was then chromatographed on a SuperDex 200 gel filtration column (Pharmacia, 2.6 x 60 cm) at a flow rate of 2 ml / min and the protein was eluted with Na149 phosphate (20 ml), pH 6.0, and pools of DTT fractions (10 mM) were collected. containing monomyme denatured TPO protein eluting between 160 and 200 ml. The TPO protein was further purified on a C4 reverse phase semipreparative column (2 x 20 cm VYDAC). The sample was applied at 5 ml / min. per column balanced in TFA (trifluoroacetic acid) (0.1%) with acetonitrile (30%). The protein was eluted with a linear gradient of acetonitrile (30-60% in 60 min). The purified reducing protein was eluted with about 50% acetonitrile. This substance is used for renaturation to obtain the biologically active variant of TPO.

C. Izdelava biološko aktivnega TPO (Met¹1-153)C. Production of Bioactive TPO (Met ¹ 1-153)

Približno 20 mg monomemega reduciranega in denaturiranega proteina TPO v 40 ml TFA (0,1 %)/acetonitril (50 %) razredčimo v 360 ml renaturimega pufra, ki vsebuje optimalno naslednje reagente:Approximately 20 mg of the monomemic reduced and denatured TPO protein in 40 ml of TFA (0.1%) / acetonitrile (50%) is diluted in 360 ml of renaturim buffer containing optimally the following reagents:

mM trismM tris

0,3 M NaCl0.3 M NaCl

5mMEDTA % detergent CHAPS % glicerol mM oksidirani glutation mM reducirani glutation pH naravnan na 8,3.5mMEDTA% detergent CHAPS% glycerol mM oxidized glutathione mM reduced glutathione pH adjusted to 8.3.

Po zmešanju komponent renaturimi pufer blago mešamo pri 4 °C 12-48 ur, da povzročimo maksimalne dobitke renaturiranja pravilno disulfidno vezane oblike TPO (glej spodaj). Raztopino nato nakisamo s TFA do končne koncentracije 0,2 %, filtriramo skozi 0,45 ali 0,22 μτη filter in dodamo 1/10 volumna acetonitrila. To raztopino nato črpamo direktno na kolono z reverzno fazo C4 in očiščeni, renaturirani TPO (Met¹ 1-153) eluiramo z enakim gradientnim programom kot zgoraj. Renaturirani biološko aktivni TPO se eluira pri približno 45 % acetonitrilu pri enakih pogojih. Nepravilne disulfidno vezane verzije TPO se eluirajo prej.After mixing the components, the renaturimi buffer was gently stirred at 4 ° C for 12-48 hours to produce maximum renaturation gains of the properly disulfide-bound form of TPO (see below). The solution was then acidified with TFA to a final concentration of 0.2%, filtered through a 0.45 or 0.22 μτη filter, and 1/10 volume of acetonitrile was added. This solution was then pumped directly onto a C4 reversed column and the purified, renatured TPO (Met ¹ 1-153) was eluted with the same gradient program as above. The renatured biologically active TPO was eluted with about 45% acetonitrile under the same conditions. Irregular disulfide-bound versions of TPO are eluted earlier.

Končni očiščeni TPO (Met¹ 1-153) ima čistoto, ki je višja od 95 %, kot določimo z SDS geli in analitično kromatografijo z reverzno fazo C4. Za živalske študije snov, očiščeno na C4, dializiramo v fiziološko kompatibilnih pufrih. Uporabimo izotonične pufre (Na-acetat (10 mM), pH 5,5, Na-sukcinat (10 mM), pH 5,5 ali Na-fosfat (10The final purified TPO (Met ¹ 1-153) has a purity higher than 95% as determined by SDS gels and reversed phase C4 analytical chromatography. For animal studies, the substance purified on C4 is dialyzed in physiologically compatible buffers. Isotonic buffers (Na-acetate (10 mM), pH 5.5, Na-succinate (10 mM), pH 5.5 or Na-phosphate (10) are used.

150 mM), pH 7,4), ki vsebujejo NaCl (150 mM) in Tween 80 (0,01 %).150 mM), pH 7.4) containing NaCl (150 mM) and Tween 80 (0.01%).

Zaradi visoke zmogljivosti TPO v testu Ba/F3 (polovična maksimalna stimulacija, dosežena pri približno 3 pg/ml) je možno, da dobimo biološko aktivno snov z uporabo mnogih različnih pufrov, detergentov in redoks pogojev. Vendar pa pri večini pogojev dobimo le majhno količino pravilno zgubane snovi (<10 %). Za komercialne izdelovalne postopke so želeni dobitki renaturiranja vsaj 10 %, bolj prednostno 30-50 % in najbolj prednostno >50 %. Mnogo različnih detergentov (Triton Χ-100, dodecil-beta-maltozid, CHAPS, CHAPSO, SDS, sarkozil, Tween 20 in Tween 80, Zwittergent 3-14 in drugi) smo preizkusili glede njihove učinkovitosti, da prispevajo k visokim dobitkom renaturiranja. Od teh detergentov smo ugotovili, da je le družina CHAPS (CHAPS in CHAPSO) na splošno uporabna v reakcijah renaturiranja, da omejimo proteinsko agregacijo in nepravilno disulfidno tvorbo. Nivoji CHAPS, večji od 1 %, so najbolj uporabni. Natrijev klorid je potreben za boljše dobitke z optimalnimi nivoji med 0,1 M in 0,5 M. Prisotnost EDTA (1-5 mM) omeji oksidacijo, katalizirano s kovino (in agregacijo), ki jo opazimo pri nekaterih pripravkih. Koncentracije glicerola, večje od 15 %, ustvarijo optimalne pogoje za renaturiranje. Za maksimalne dobitke je bistveno, da imamo tako oksidiran kot tudi reduciran glutation ali oksidiran in reduciran cistein kot redoks reagentni par. Na splošno dobimo višje dobitke, če sta oksidirani in reducirani reagent v redoks paru v enakem molskem razmerju oz. je oksidirani reagent v prebitku. pH vrednosti med 7,5 in približno 9 so optimalne za renaturiranje teh variant TPO. Organska topila (npr. etanol, acetonitril, metanol) so dopustna pri koncentracijah od 10 do 15 % ali nižjih. Višji nivoji organskih topil povečajo količino nepravilno zgubanih oblik. Na splošno uporabimo tris in fosfatne pufre. Inkubacija pri 4 °C prav tako ustvari višje nivoje pravilno zgubanega TPO.Due to the high TPO capacity in the Ba / F3 assay (half maximal stimulation achieved at about 3 pg / ml), it is possible to obtain a biologically active substance using many different buffers, detergents and redox conditions. However, under most conditions only a small amount of properly wrinkled matter (<10%) is obtained. For commercial manufacturing processes, the desired renaturation gains are at least 10%, more preferably 30-50% and most preferably> 50%. Many different detergents (Triton Χ-100, dodecyl beta-maltoside, CHAPS, CHAPSO, SDS, sarcosyl, Tween 20 and Tween 80, Zwittergent 3-14 and others) have been tested for their effectiveness to contribute to high renaturation gains. Of these detergents, we found that only the CHAPS family (CHAPS and CHAPSO) is generally useful in renaturation reactions to limit protein aggregation and irregular disulfide formation. CHAPS levels greater than 1% are most useful. Sodium chloride is required for better yields with optimum levels between 0.1 M and 0.5 M. The presence of EDTA (1-5 mM) limits the metal-catalyzed (and aggregation) oxidation observed in some preparations. Glycerol concentrations greater than 15% create optimal conditions for renaturation. For maximum yields, it is essential to have both oxidized and reduced glutathione or oxidized and reduced cysteine as a redox reagent pair. Generally higher yields are obtained if the oxidized and the reduced reagent are in the same molar ratio or in the redox pair. is the excess oxidized reagent. pH values between 7.5 and about 9 are optimal for the renaturation of these TPO variants. Organic solvents (eg ethanol, acetonitrile, methanol) are acceptable at concentrations of 10 to 15% or lower. Higher levels of organic solvents increase the amount of misfolded forms. We generally use tris and phosphate buffers. Incubation at 4 ° C also creates higher levels of properly folded TPO.

Renaturirni dobitki od 40 do 60 % (na osnovi količine reduciranega in denaturiranega TPO, uporabljenega v reakciji renaturiranja) so značilni za pripravke TPO, ki so očiščeni s prvo stopnjo C4. Aktivno snov dobimo tudi, če so pripravki manj čisti (npr. direktno po koloni superdeks 200 ali po začetni ekstrakciji refraktilnih teles), čeprav so dobitki manjši, zaradi obširne precipitacije in interference neTPO proteinov med postopkom renaturiranja TPO.Renaturation gains of 40 to 60% (based on the amount of reduced and denatured TPO used in the renaturation reaction) are typical of TPO preparations purified by first stage C4. The active substance is also obtained if the preparations are less pure (eg, directly after the superdex 200 column or after the initial extraction of the refractive bodies), although the yields are smaller, due to the extensive precipitation and interference of neTPO proteins during the TPO renaturation process.

Ker TPO (Met¹ 1-153) vsebuje 4 cisteinske ostanke, je možno, da izdelamo tri različne disulfidne verzije tega proteina:Since TPO (Met ¹ 1-153) contains 4 cysteine residues, it is possible to produce three different disulfide versions of this protein:

151 verzija 1: disulfidi med cisteinskimi ostanki 1-4 in 2-3 verzija 2: disulfidi med cisteinskimi ostanki 1-2 in 3-4 verzija 3: disulfidi med cisteinskimi ostanki 1-3 in 2-4151 version 1: disulfides between cysteine residues 1-4 and 2-3 version 2: disulfides between cysteine residues 1-2 and 3-4 version 3: disulfides between cysteine residues 1-3 and 2-4

Med začetnim raziskovanjem določevanja pogojev renaturiranja smo številne različne vrhove, ki so vsebovali protein TPO, ločili s kromatografijo z reverzno fazo C4. Samo eden od teh vrhov je imel značilno biološko aktivnost, določeno s testom Ba/F3. Nato smo pogoje renaturiranja optimizirali, da smo dobili to prednostno verzijo. Pri teh pogojih je nepravilno zgubane verzije manj od 10-20 % celotnega dobljenega monomera TPO.During the initial exploration of renaturation conditions, many different peaks containing TPO protein were separated by C4 reversed phase chromatography. Only one of these peaks had the characteristic biological activity determined by the Ba / F3 assay. We then optimized the renaturation conditions to obtain this preferred version. Under these conditions, the misfolded versions are less than 10-20% of the total TPO monomer obtained.

Za disulfidni vzorec biološko aktivnega TPO smo določili z masno spektrometrijo in proteinskim sekvenciranjem, da je 1-4 in 2-3 (t.j. verzija 1). Alikvote različnih vrhov, ločenih na C4 (5-10 mmol) smo digerirali s tripsinom (molsko razmerje tripsina:proteinu 1:25). Digestijsko zmes smo analizirali z lasersko desorpcijsko masno spektrometrijo z matrico pred redukcijo z DTT in po njej. Po redukciji smo detektirali mase, ki ustrezajo večini od večjih triptičnih peptidov TPO. V nereduciranih vzorcih so nekatere od teh mas manjkale, opazili pa smo nove. Masa novih vrhov je ustrezala v bistvu vsoti individualnih triptičnih peptidov, vključenih v disulfidni par. Tako je bilo možno, da smo nedvoumno določili za sulfidni vzorec renaturiranega, rekombinantnega biološko aktivnega TPO, da je 1-4 in 2-3. To je konstistentno z znanim disulfidnim vzorcem sorodne molekule eritropoetina.The disulfide pattern of biologically active TPO was determined by mass spectrometry and protein sequencing to be 1-4 and 2-3 (i.e., version 1). Aliquots of different peaks separated on C4 (5-10 mmol) were digested with trypsin (molar trypsin: protein ratio 1:25). The digestion mixture was analyzed by matrix matrix laser desorption mass spectrometry before and after reduction with DTT. After reduction, masses corresponding to most of the larger tryptic peptides of TPO were detected. Some of these masses were missing in the untreated samples, but new ones were observed. The mass of the new peaks corresponded essentially to the sum of the individual tryptic peptides included in the disulfide pair. Thus, it was possible that we determined unequivocally for the sulfide sample of renatured, recombinant biologically active TPO to be 1-4 and 2-3. This is consistent with the known disulfide pattern of the related erythropoietin molecule.

D. Biološka aktivnost rekombinantnega renaturiranega TPO (Met'¹1-153)D. Biological activity of recombinant renatured TPO (Met ' ¹ 1-153)

Renaturirani in očiščeni TPO (Met¹ 1-153) ima aktivnost tako v testih in vitro kot tudi in vivo. V testu Ba/F3 dosežemo polovično maksimalno stimulacijo timidinske vgraditve v celice Ba/F3 pri 3,3 pg/ml (0,3 pM). V ELISI na osnovi mpl receptorja pride do polovične maksimalne aktivnosti pri 1,9 ng/ml (120 pM). V normalnih in mielosuprimiranih živalih, ki jih dobimo s skoraj letalnim obsevanjem z rentgenskimi žarki, je TPO (Met¹ 1-153) visoko potenten (aktivnost vidna pri tako nizkih dozah, kot npr. 30 ng/miš) za stimuliranje nastajanje novih trombocitov.Renated and purified TPO (Met ¹ 1-153) has activity in both in vitro and in vivo assays. In the Ba / F3 assay, half-maximal stimulation of thymidine incorporation into Ba / F3 cells was achieved at 3.3 pg / ml (0.3 pM). In the mpl receptor ELISA, half maximal activity occurs at 1.9 ng / ml (120 pM). In normal and myelosuppressed animals obtained by near-air X-ray irradiation, TPO (Met ¹ 1-153) is highly potent (activity seen at as low doses as 30 ng / mouse) to stimulate new platelet production.

PRIMER 23EXAMPLE 23

Izdelovanje drugih biološko aktivnih variant TPO v E. coliProduction of other biologically active TPO variants in E. coli

152152

Tri različne variante TPO, izdelane v E. coli, očiščene in renaturirane v biološko aktivnih oblikah, so navedene spodaj.Three different TPO variants made in E. coli purified and renatured in biologically active forms are listed below.

(1) MLF - 13 ostankov iz bakterijsko izvedene signalne sekvence STII spojimo z N-terminalno domeno TPO (ostanki 1-155). Nastala sekvenca je:(1) MLF-13 residues from the bacterially derived STII signal sequence are coupled to the N-terminal domain of TPO (residues 1-155). The resulting sequence is:

MKKNIAFLLNAYASPAPPAC.....CVRRA (SEQ ID NO: 85) kjer je vodilna sekvenca podčrtana in C....C pomeni Cys⁷ do Cys¹⁵¹. To varianto konstruiramo, da dobimo tirozin za radioaktivno jodiranje TPO za receptorske in biološke študije.MKKNIAFLLNAYASPAPPAC ..... CVRRA (SEQ ID NO: 85) wherein the leader sequence is underlined and C .... C stands for Cys ⁷ to Cys ¹⁵¹ . We construct this variant to obtain tyrosine for radioactive iodination of TPO for receptor and biological studies.

(2) H8MLF - 7 ostankov iz sekvence STII, 8 histidinskih ostankov in s faktorjem Xa encimatsko cepljivo sekvenco IEGR spojimo z N-terminalno domeno (ostanki 1-155) TPO. Sekvenca je:(2) H8MLF - 7 residues from the STII sequence, 8 histidine residues and an XEG enzymatic cleavage sequence of IEGR are coupled to the N-terminal domain (residues 1-155) of TPO. The sequence is:

M.KKNIAFHHHHHHHHIFARSPAPPAC.....CVRRA (SEQ ID NO: 86) kjer je vodilna sekvenca podčrtana, in C....C pomeni Cys⁷ do Cys¹⁵¹. To varianto, ko je očiščena in renaturirana, lahko obdelamo z encimom faktorjem Xa, ki cepi po argininskem ostanku sekvence IEGR, pri čemer nastane varianta TPO z dolžino 155 ostankov z naravno serinsko N-terminalno amino kislino.M.KKNIAFHHHHHHHHIFARSPAPPAC ..... CVRRA (SEQ ID NO: 86) wherein the leader sequence is underlined, and C .... C is Cys ⁷ to Cys ¹⁵¹ . When purified and renatured, this variant can be treated with the enzyme factor Xa, which cleaves the arginine residue of the IEGR sequence, producing a 155-residue TPO variant with the natural serine N-terminal amino acid.

(3) T-H8MLF pripravimo, kot je opisano zgoraj, za varianto (2), razen da trombinsko senzitivno sekvenco IEPR spojimo z N-terminalno domeno TPO. Nastala sekvenca je:(3) T-H8MLF is prepared as described above for variant (2) except that the thrombin-sensitive IEPR sequence is coupled to the N-terminal domain of TPO. The resulting sequence is:

MKKNIAFHHHHHHHHIEPRSPAPPAC.....CVRRA (SEQ ID NO: 87) kjer je vodilna sekvenca podčrtana, in C....C pomeni Cys⁷ do Cys¹⁵¹. To varianto lahko po čiščenju in renaturiranju obdelamo z encimom trombinom, da naredimo naravno N-terminalno varianto TPO z dolžino 155 ostankov.MKKNIAFHHHHHHHHIEPRSPAPPAC ..... CVRRA (SEQ ID NO: 87) wherein the leader sequence is underlined, and C .... C is Cys ⁷ to Cys ¹⁵¹ . After purification and renaturation, this variant can be treated with the enzyme thrombin to produce a natural N-terminal TPO variant with a length of 155 residues.

153153

A. Rekuperiranje, solubilizacija in čiščenje monomemih biološko aktivnih variant TPO (1),(2) in (3).A. Recovery, solubilization and purification of monomem biologically active variants of TPO (1), (2) and (3).

Vse te variante eksprimiramo v E. coli. Večino variant ugotovimo v reffaktilnih telesih, kot je razvidno v Primeru 22 za TPO (Met⁴ 1-153). Izvedemo identične postopke za rekuperiranje, solubilizacijo in čiščenje monomemih variant TPO, kot je opisano v primem 22. Uporabimo identične pogoje za renaturiranje kot za TPO (Met⁴ 1-153) s celotnimi dobitki 30-50 %. Po renaturiranju variante TPO očistimo s kromatografijo z reverzno fazo C4 v TFA (0,1 %) z uporabo acetonitrilnega gradienta, kot je že opisano pred tem. Vse variante TPO (v njihovih neproteoliziranih oblikah) imajo biološko aktivnost določeno s testom Ba/F3 s polovičnimi maksimalnimi aktivnostmi 2-5 pM.All these variants are expressed in E. coli. Most variants are found in refactile bodies, as shown in Example 22 for TPO (Met ⁴ 1-153). Perform identical procedures for recovering, solubilizing and purifying monomemic variants of TPO as described in Example 22. Use identical conditions for renaturation as for TPO (Met ⁴ 1-153) with total yields of 30-50%. After renaturation, the TPO variant was purified by reverse phase C4 chromatography in TFA (0.1%) using an acetonitrile gradient as described previously. All TPO variants (in their unproteolized forms) have biological activity determined by the Ba / F3 assay with half maximal activities of 2-5 pM.

B. Proteolitično procesiranje variant (2) in (3), da izdelamo avtentični N-terminalni TPO (1-155)B. Proteolytic processing of variants (2) and (3) to produce an authentic N-terminal TPO (1-155)

Variante TPO (2) in (3), zgoraj, oblikujemo z encimatsko cepljivim vodilnim peptidom pred normalnim N-terminalnim in aminokislinskim ostankom TPO. Po renaturiranju in čiščenju variant (2) in (3), kot je opisano zgoraj, vsako izpostavimo digestiji z ustreznim encimom. Za vsako varianto acetonitril iz stopnje kromatografije z reverzno fazo C4 odstranimo z vpihovanjem blagega toka dušika v raztopino. Nato obe varianti obdelamo bodisi s faktorjem Xa ali s trombinom, kot je opisano spodaj.Variants of TPO (2) and (3), above, are formulated with an enzymatically cleavable leader peptide against the normal N-terminal and amino acid residues of TPO. After renaturation and purification of variants (2) and (3) as described above, each is digested with the appropriate enzyme. For each variant, acetonitrile is removed from the reversed phase C4 chromatography step by blowing a gentle stream of nitrogen into the solution. Then, both variants are treated with either factor Xa or thrombin as described below.

Za varianto TPO (2), dodamo tris pufer (1 M), pH 8, k raztopini, ki ne vsebuje acetonitrila, do končne koncentracije 50 mM in pH naravnamo na 8, če je potrebno. Dodamo NaG in CaC^ do 0,1 M oz. 2 mM. Faktor Xa (New England Biolabs) dodamo, da dosežemo približno molsko razmerje encima proti varianti od 1:25 do 1:100. Vzorec inkubiramo pri sobni temperaturi 1-2 uri, da dosežemo maksimalno cepitev, ki jo določimo s spremembo v migraciji na gelih SDS, kar pomeni izgubo vodilne sekvence. Nato rekacijsko zmes očistimo s kromatografijo z reverzno fazo C4 z uporabo enakega gradienta in pogojev, kot je opisano zgoraj za čiščenje pravilno zgubanih variant. Necepljeno varianto B ločimo od cepljene variante (2) pri teh pogojih. Za N-terminalne amino kisline je razvidno, da so SP APP, kar pomeni, da je odstranitev N-terminalne vodilne sekvence uspešna. Faktor Xa prav tako naredi variabilne količine interne cepitve v domeni TPO; cepitev opazimo po argininskem ostanku na položaju št. 118, zaradi česar nastane dodatna N-terminalna sekvenca TTAHKDP (SEQ ID NO: 88). Na nereducimih gelih SDS, opazimo eno progo priFor TPO variant (2), add tris buffer (1 M), pH 8, to the acetonitrile-free solution to a final concentration of 50 mM and adjust the pH to 8 if necessary. Add NaG and CaC ^ to 0.1 M oz. 2 mM. Factor Xa (New England Biolabs) is added to achieve an approximate molar ratio of enzyme to variant from 1:25 to 1: 100. The sample is incubated at room temperature for 1-2 hours to achieve maximum cleavage, which is determined by the change in migration on SDS gels, resulting in loss of the leader sequence. The reaction mixture was then purified by reverse phase C4 chromatography using the same gradient and conditions as described above to purify the properly folded variants. Unvaccinated variant B is separated from vaccinated variant (2) under these conditions. The N-terminal amino acids are shown to be SP APPs, which means that the removal of the N-terminal leader sequence is successful. Factor Xa also makes variable amounts of internal cleavage in the TPO domain; cleavage is observed after arginine residue at position no. 118, resulting in an additional N-terminal TTAHKDP sequence (SEQ ID NO: 88). On irreducible SDS gels, one line at

154 približno 17000 Da, za varianto, cepljeno s faktorjem Xa; na reducirnih gelih opazimo dve progi z molekulskimi masami približno 12000 in 5000 Da, konsistentni s cepitvijo na argininu 118. To zaznavanje potrjuje, da se dva dela molekule držita skupaj z disulfidno vezjo med prvim in četrtim cisteinskim ostankom, kar izvedemo s triptičnimi digestijskimi eksperimenti, opisanimi zgoraj. V biološkem testu Ba/F3 ima očiščena varianta TPO (1-155), po odstranitvi N-terminalne vodilne sekvence in interni cepitvi, polovično maksimalno aktivnost 0,2 do 0,3 pmol. Intaktna varianta z vodilno sekvenco ima polovično maksimalno aktivnost 2-4 pmol.154 approximately 17000 Yes, for the variant vaccinated with factor Xa; two lines with molecular weights of about 12000 and 5000 Da, consistent with cleavage on arginine 118. are observed on reducing gels. This detection confirms that two parts of the molecule are held together by the disulfide bond between the first and fourth cysteine residues, which is carried out by tryptic digestion experiments. described above. In the Ba / F3 bioassay, the purified TPO variant (1-155), after removal of the N-terminal leader sequence and internal cleavage, has a half maximum activity of 0.2 to 0.3 pmol. The leading sequence intact variant has a half maximal activity of 2-4 pmol.

Za varianto (3) je digestijski pufer sestavljen iz trisa (50 mM), pH 8, CHAPS-a (2 %), NaCl (0,3 M), EDTA (5 mM) in humanega ali govejega trombina (Calbiochem) pri masnem razmerju encima proti proteinu variante TPO od 1:25 do 1:50. Digestijo vodimo pri sobni temperaturi 2-6 ur. Napredek digestije določimo z geli SDS, kot je opisano zgoraj za cepitveno reakcijo faktorja Xa. Na splošno dosežemo več kot 90 % cepitev vodilne sekvence v tem času. Nastali TPO očistimo na kolonah z reverzno fazo C4, kot je opisano zgoraj, z aminokislinskim sekvenciranjem pa določimo, da ima želeni N-terminal. Dobimo le zelo majhne (<5 %) količine interne cepitve na isti arginin-treoninski vezi, ki smo jih zaznali zgoraj s faktorjem Xa. Nastali protein TPO ima visoko biološko aktivnost s polovičnimi maksimalnimi odzivi pri testu Ba/F3 pri 0,2-0,4 pmol proteinu. V testu ELISA na osnovi receptorja mpl ima ta protein polovični maksimalni odziv pri 2-4 ng/ml očiščenega proteina (120-240 pmol), medtem ko je intaktna varianta, ki vsebuje vodilno sekvenco, 5-10 krat manj zmogljiva v obeh testih. Za živalske študije s HPLC očiščen cepljen protein dializiramo v fiziološko sprejemljive pufre z NaCl (150 mM), Tweenom 80 (0,1 %) in natrijevim sukcinatom (10 mM), pH 5,5 ali natrijevim acetatom (10 mM), pH 5,5 ali natrijevim fosfatom (10 mM), pH 7,4. S HPLC in geli SDS očiščen protein je stabilen več tednov, če ga shranimo pri 4 °C. Pri normalnih in mielosuprimiranih miših je ta očiščeni TPO z avtentično N-terminalno sekvenco visoko aktiven in stimulira nastajanje trombocitov pri nizkih dozah, kot npr. 30 ng/miš.For variant (3), digestion buffer consists of tris (50 mM), pH 8, CHAPS (2%), NaCl (0.3 M), EDTA (5 mM) and human or bovine thrombin (Calbiochem) at wt. enzyme to protein ratio of TPO variant from 1:25 to 1:50. Digestion is conducted at room temperature for 2-6 hours. Digestion progress was determined with SDS gels as described above for the factor Xa cleavage reaction. In general, more than 90% of the leader sequence cleavages are achieved during this time. The resulting TPO was purified on the reversed phase C4 columns as described above, and determined by amino acid sequencing to have the desired N-terminal. Only very small (<5%) amounts of internal cleavage are obtained on the same arginine-threonine bond detected above by factor Xa. The resulting TPO protein has high biological activity with half maximal responses in the Ba / F3 assay at 0.2-0.4 pmol protein. In the mpl receptor ELISA assay, this protein has a half maximal response at 2-4 ng / ml of purified protein (120-240 pmol), whereas the intact variant containing the leader sequence is 5-10 times less potent in both assays. For animal studies by HPLC, the purified cleaved protein was dialyzed into physiologically acceptable buffers with NaCl (150 mM), Tween 80 (0.1%), and sodium succinate (10 mM), pH 5.5, or sodium acetate (10 mM), pH 5. , 5 or sodium phosphate (10 mM), pH 7.4. The HPLC and gels SDS purified protein is stable for several weeks when stored at 4 ° C. In normal and myelosuppressed mice, this purified TPO with an authentic N-terminal sequence is highly active and stimulates platelet formation at low doses, such as e.g. 30 ng / mouse.

PRIMER 24EXAMPLE 24

Sintetični mpl ligandSynthetic mpl ligand

Čeprav humani mpl ligand (hML) navadno naredimo z uporabo rekombinantnih postopkov, pa ga prav tako lahko sintetiziramo z encimatsko ligacijo sintetičnih peptidnih fragmentov z uporabo postopkov, opisanih spodaj. Sintetična izdelava hMLAlthough human mpl ligand (hML) is usually made using recombinant methods, it can also be synthesized by enzymatic ligation of synthetic peptide fragments using the procedures described below. Synthetic hML production

155 dopušča vgraditev nenaravnih amino kislin ali sintetičnih funkcionalnosti, kot je polietilenglikol. Pred tem so mutant serin proteaza subtilizin BPN, subtiligazo (S221C/P225A), konstruirali z učinkovito ligacijo peptidnih estrov v vodni raztopini (Abrahmsen et al., Biochem., 30:4151-4159 [1991]). Sedaj pa je razvidno, da lahko sintetične peptide encimatsko ligiramo v zaporedju, da dobimo encimatsko aktivne dolge peptide in proteine, kot je ribonukleaza A (Jackson et al., Science, [1994]). Ta tehnologija, opisana bolj podrobno spodaj, omogoča kemijsko sintetiziranje dolgih proteinov, ki so jih pred tem lahko naredili le s tehnologijo rekombinantne DNA.155 allows the incorporation of unnatural amino acids or synthetic functionalities such as polyethylene glycol. Previously, the mutant serine protease subtilisin BPN, subtiligase (S221C / P225A), was constructed by efficient ligation of peptide esters in aqueous solution (Abrahmsen et al., Biochem. 30: 4151-4159 [1991]). It is now apparent, however, that synthetic peptides can be enzymatically ligated in sequence to produce enzymatically active long peptides and proteins such as ribonuclease A (Jackson et al., Science, [1994]). This technology, described in more detail below, allows the chemical synthesis of long proteins, which previously could only be made using recombinant DNA technology.

Splošna strategija sinteze hML₁₅₃ z uporabo subtiligaze je prikazana na shemi 1. Pričnemo s popolnoma deprotektiranim peptidom, ki ustreza C-terminalnemu fragmentu proteina, ki mu dodamo N-terminalno zaščiten C-terminalno aktiviran estrski peptid skupaj s subtiligazo. Ko je reakcija končana, produkt izoliramo s HPLC z reverzno fazo in zaščitno skupino odstranimo od N-terminala. Ligiramo naslednji peptidni fragment, deprotektiramo in postopek ponovimo z uporabo zaporednih peptidov, dokler ne dobimo popolne dolžine proteina. Ta postopek je podoben metodologiji v trdni fazi v tem, da N-terminalno zaščiten C-terminalno aktiviran peptid ligiramo na N-terminalu predhodnega peptida in protein sintetiziramo v smeri C-*N. Ker je posledica vsake pripojitve dodatnih 50 ostankov in ker produkte izoliramo po vsaki ligaciji, lahko sintetiziramo mnogo daljše visoko čiste proteine v primernih dobitkih.A general strategy for the synthesis of hML ₁₅₃ using subtiligase is shown in Scheme 1. We begin with a fully deprotected peptide corresponding to the C-terminal fragment of the protein, to which is added an N-terminally protected C-terminally activated ester peptide together with subtiligase. When the reaction is complete, the product is isolated by reversed-phase HPLC and the protecting group is removed from the N-terminal. The next peptide fragment was ligated, deprotected and the procedure repeated using consecutive peptides until the full length of the protein was obtained. This process is similar to the solid-phase methodology in that the N-terminally protected C-terminally activated peptide is ligated at the N-terminal of the previous peptide and the protein is synthesized in the C- * N direction. Since each addition results in an additional 50 residues and since the products are isolated after each ligation, much longer highly pure proteins can be synthesized in suitable yields.

156156

Shema 1. Strategija za sintezo hML z uporabo subtiligazeScheme 1. Strategy for hML synthesis using subtiligase

R-NH-Peptid₂ -CO-R' + HjN-Peptid, -CO₂ R-NH-Peptide ₂ -CO-R '+ HjN-Peptide, -CO ₂

1¹⁾ subtiligaza1 ¹⁾ subtiligase

R-NH-Peptid₂ -CO-NH-Peptid, -co₂ I 2) Zn/CH₃CO₂HR-NH-Peptide ₂ -CO-NH-Peptide, -co ₂ I 2) Zn / CH ₃ CO ₂ H

H₂N-Peptid₂ -CO-NH-Peptid, -CO₂ H ₂ N-Peptide ₂ -CO-NH-Peptide, -CO ₂

3) ponovitev 1+23) 1 + 2 repetition

H₂N-Peptid ₃ -CO-NH-Peptid ₂ -CO-NH-Peptid! -CO₂ H ₂ N-Peptide ₃ -CO-NH-Peptide ₂ -CO-NH-Peptide! -CO ₂

Glede na naše poznavanje sekvenčne specifičnosti subtiligaze kot tudi aminokislinske sekvence biološko aktivne EPO-domene hML, razdelimo hML₁₅₃ v sedem fragmentov z dolžino 18-25 ostankov. Testne ligacijske tetrapeptide sintetiziramo, da določimo primerne ligacijske povezave za 18-25-mere. Iz tabele 13 so razvidni rezultati teh testnih ligacij.Given our knowledge of the sequence specificity of subtiligase as well as the amino acid sequence of the biologically active hML EPO domain, we divide hML ₁₅₃ into seven fragments with a length of 18-25 residues. Test ligation tetrapeptides are synthesized to determine suitable ligation links for 18-25-mer. Table 13 shows the results of these test ligation.

157157

TABELA 13TABLE 13

Testne ligaciie hML. Donor in nukleofilne peptide raztopimo, da dobimo koncentracijo 10 mM, v tricinu (100 mM), pH 7,8 pri 22 °C. Ligazo dodamo do končne koncentracije 10 μΜ iz izhodne raztopine 1,6 mg/ml (~70 μΜ) in pustimo, da ligacija poteka preko noči. Dobitki so osnovani na % ligacije proti hidrolizi donorskih peptidov.HML test ligations. The donor and nucleophilic peptides were dissolved to give a concentration of 10 mM, in tricin (100 mM), pH 7.8 at 22 ° C. Ligase was added to a final concentration of 10 μΜ from a stock solution of 1.6 mg / ml (~ 70 μΜ) and allowed to ligation overnight. The gains are based on% ligation against hydrolysis of donor peptides.

Mesto City Donor (gle-K-NH2) Donor (see-K-NH2) Nukleofil-ML, Nucleophil-ML, % hidrolize % hydrolysis fe ligaci jel fe ligaci jel 1 (23/24) 1 (23/24) HVLH (SEQ ID NO: 89) HVLH (SEQ ID NO: 89) SRLS (SEQ ID NO: 90) SRLS (SEQ ID NO: 90) 92 92 08 08 (22/23) (22/23) SHVL (SEQ ID NO: 91) SHVL (SEQ ID NO: 91) HSRL (SEQ ID NO: 92) HSRL (SEQ ID NO: 92) 48 48 52 52 2 (46/47) 2 (46/47) AVDF (SEQ ID NO: 93) AVDF (SEQ ID NO: 93) SLGE (SEQ ID NO: 94) SLGE (SEQ ID NO: 94) 22 22 78 78 3 (69/70) 3 (69/70) AVTL (SEQ ID NO: 95) AVTL (SEQ ID NO: 95) LLEG (SEQ ID NO: 96) LLEG (SEQ ID NO: 96) 53 53 47 47 4 (89/90) 4 (89/90) LSSL (SEQ ID NO: 97) LSSL (SEQ ID NO: 97) LGQL (SEQ ID NO: 98) LGQL (SEQ ID NO: 98) 95 95 05 05 (88/89) (88/89) C(acm)LSS (SEQ ID NO: 99) C (acm) LSS (SEQ ID NO: 99) LLGQ (SEQ ID NO: 100) LLGQ (SEQ ID NO: 100) 00 00 00 00 (90/91) (90/91) SSLL (SEOID NO: 101) SSLL (SEOID NO: 101) GQLS (SEQ ID NO: 102) GQLS (SEQ ID NO: 102) 45 45 55 55 (88/89) (88/89) CLSS (SEQ ID NO: 103) CLSS (SEQ ID NO: 103) LLGQ (SEQ ID NO: 100) LLGQ (SEQ ID NO: 100) 90 90 10 10 5(107/108) 5 (107/108) LQSL (SEQ ID NO: 104) LQSL (SEQ ID NO: 104) LGTQ (SEQ ID NO: 105) LGTQ (SEQ ID NO: 105) 99 . 99. 01 01 (106/107) (106/107) ALQS (SEQ ID NO: 106) ALQS (SEQ ID NO: 106) LLGT (SEQ ID NO: 107) LLGT (SEQ ID NO: 107) 70 70 30 30 6(128/129) 6 (128/129) NAIF (SEQ ID NO: 108) NAIF (SEQ ID NO: 108) LSFQ (SEQ ID NO: 109) LSFQ (SEQ ID NO: 109) 60 60 40 40

j·,·.r.kv:ntnc '<<,< •Λΐ'ίΰνιχ.* ij ·, · .r.kv: ntnc '<<, <• Λΐ'ίΰνιχ. * i

158158

Na osnovi teh eksperimentov se ligacijski peptidi, prikazani v tabeli 14, učinkovito ligirajo s subtiligazo. Prikladna zaščitna skupina za N-terminal vsakega donorskega estrskega peptida je potrebna, da preprečimo samoligacijo. Izbrali smo izonikotinilno (iNOC) zaščitno skupino (Veber et al., J. Org. Chem., 42:3286-3289 [1977]), ker je vodotopna in jo lahko vgradimo v zadnji stopnji sinteze peptidov v trdni fazi in je stabilna v brezvodnem HF, uporabljenem za deprotekcijo in cepitev peptidov od smolne trdne faze. Poleg tega jo lahko odstranimo od peptida po vsaki ligaciji pri milih redukcijskih pogojih (Zn/CHjCO^), da dobimo prosti N-terminal za kasnejše ligacije. Glikolat-lizil-amid (glc-K-NH^) ester uporabimo za C-terminalno aktivacijo, osnovano na predhodnih eksperimentih, iz katerih je razvidno, da se le-ta učinkovito acilira z subtiligazo (Abrahmsen et al., Biochem., 30:4151-4159 [1991]). iNOC zaščitene, glc-K-amidno aktivirane peptide lahko sintetiziramo z uporabo standardnih postopkov v trdni fazi, kot je prikazano na shemi 2. Peptide nato zaporedno ligiramo, dokler ne izdelamo popolnega proteina in končni produkt renaturiramo in vitro. Na osnovi homologije z EPO domnevamo, da se disulfidni pari tvorijo med ostankoma 1 in 51 in med 28 in 85. Oksidacijo disulfidov lahko izvedemo z enostavnim mešanjem reducirane snovi nekaj ur pod atmosfero kisika. Renaturirano snov lahko nato čistimo s HPLC in zberemo poole frakcij, ki vsebujejo aktivni protein in liofiliziramo. Alternativno lahko disulfide različno zaščitimo za nadzorovanje zaporedne oksidacije med specifičnimi disulfidnimi pari. Zaščita cisteinov 7 in 151 z acetamidometilnimi (acm) skupinami zagotavlja oksidacijo 28 in 85. Skupine acm lahko nato odstranimo in ostanka 7 in 151 oksidiramo. Obratno lahko ostanka 28 in 85 zaščitimo z acm in oksidiramo v primeru, da je potrebna zaporedna oksidacija za pravilno zgubanje. V danem primeru lahko cisterna 28 in 85 substituiramo z drugimi naravnimi ali nenaravnimi ostanki, drugačnimi od Cys, da zagotovimo pravilno oksidacijo cisteinov 7 in 151.Based on these experiments, the ligation peptides shown in Table 14 are effectively ligated by subtiligase. A suitable N-terminal protecting group of each donor ester peptide is required to prevent self-ligation. An isonicotinyl (iNOC) protecting group was selected (Veber et al., J. Org. Chem., 42: 3286-3289 [1977]) because it is water-soluble and can be incorporated in the final stage of solid phase peptide synthesis and is stable in anhydrous HF used for deprotection and cleavage of peptides from resin solid phase. In addition, it can be removed from the peptide after each ligation under mild reducing conditions (Zn / CH2CO2) to give a free N-terminal for subsequent ligation. The glycolate-lysyl-amide (glc-K-NH ^) ester is used for C-terminal activation, based on previous experiments, which show that it is effectively acylated by subtiligase (Abrahmsen et al., Biochem., 30 : 4151-4159 [1991]. iNOC protected, glc-K-amide activated peptides can be synthesized using standard solid phase procedures as shown in Scheme 2. The peptides are then sequentially ligated until complete protein is produced and the final product is renatured in vitro. Based on homology with EPO, it is assumed that the disulfide vapor forms between residues 1 and 51 and between 28 and 85. The oxidation of the disulfides can be carried out simply by stirring the reduced substance for several hours under an oxygen atmosphere. The recovered substance can then be purified by HPLC and pools of fractions containing the active protein are collected and lyophilized. Alternatively, disulfides can be variously protected to control sequential oxidation between specific disulfide pairs. Protection of cysteines 7 and 151 by acetamidomethyl (acm) groups provides oxidation of 28 and 85. The acm groups can then be removed and residues 7 and 151 oxidized. Conversely, residues 28 and 85 can be protected with acm and oxidized in the event that sequential oxidation is required for proper folding. In the present case, the cisterns 28 and 85 may be substituted with other natural or unnatural residues other than Cys to ensure proper oxidation of cysteines 7 and 151.

159159

TABELAMTABELAM

Peptidni fragmenti, uporabljeni za celokupno sintezo h-ML z uporabo subtiligazePeptide fragments used for whole h-ML synthesis using subtiligase

FragmentFragment

Sekvenca (SEQ ID NO: 110) iNOC-HN-SPAPPACDLRVLSKLLRDSHVL-glc-K-NH2 (1 -22) (SEQ ID NO: 111)Sequence (SEQ ID NO: 110) iNOC-HN-SPAPPACDLRVLSKLLRDSHVL-glc-K-NH2 (1 -22) (SEQ ID NO: 111)

INOC-HN-HSRLSQCPEVHPLPTPVLLPAVDF-glc-K-NH2 (23-46) (SEQ ID NO: 112) iNOC-HN-SLGEWKTQMEETKAQDILGAVTL-glc-K-NK2 (47-69) (SEQ ID NO: 113) iNOC-HN-LLEGVMAARGQLGPTCLSSLL-glc-K-NH2 (70-90) (SEQ ID NO: 114) iNOC-HN-GQLSGQVRLLLGALQS-glc-K-NH2 (90-106) (SEQ ID NO: 115) iNOC-HN-LLGTQLPPQGRTTAHKDPNAIF-glc-K-NH2 (107-128) (SEQ ID NO: 116)INOC-HN-HSRLSQCPEVHPLPTPVLLPAVDF-glc-K-NH2 (23-46) (SEQ ID NO: 112) iNOC-HN-SLGEWKTQMEETKAQDILGAVTL-glc-K-NK2 (47-69) (SEQ ID NO: 113) iNOC-HN- LLEGVMAARGQLGPTCLSSLL-glc-K-NH2 (70-90) (SEQ ID NO: 114) iNOC-HN-GQLSGQVRLLLGALQS-glc-K-NH2 (90-106) (SEQ ID NO: 115) iNOC-HN-LLGTQLPPQIFT-glCDP- K-NH2 (107-128) (SEQ ID NO: 116)

H2N-LSFOHLLRGKVRFLMLVGGSTLCVR-CO2 (129-153)H2N-LSFOHLLRGKVRFLMLVGGSTLCVR-CO2 (129-153)

Peptidne ligacije izvedemo pri 25 °C v tricinu (100 mM), pH 8 (sveže pripravljen in razplinjen z vakuumsko filtracijo skozi 5 μΜ filter). Značilno C-terminalni fragmentPeptide ligation was performed at 25 ° C in tricin (100 mM), pH 8 (freshly prepared and degassed by vacuum filtration through a 5 μΜ filter). Typically a C-terminal fragment

160 raztopimo v pufru (2-5 mM peptid) in dodamo 10x izhodno osnovno raztopino subtiligaze (1 mg/ml v tricinu (100 mM), pH 8), da dosežemo končno encimsko koncentracijo približno 5 gM. Nato dodamo 3-5 molov prebitka donorskega peptida, aktiviranega z glc-K-Nl·^, kot trdno snov, raztopimo in zmes pustimo, da stoji pri 25 °C. Ligacije nadzorujemo z analitično HPLC z reverzno fazo 08 (CH^CN/F^O gradient s TFA (0,1 %)). Produkte ligacije očistimo s preparativno HPLC in liofiliziramo. Izonikotinilno (iNOC) deprotekcijo izvedemo z mešanjem cinkovega prahu, aktiviranega s HC1, z zaščitenim peptidom v ocetni kislini. Cinkov prah odstranimo s filtracijo in ocetno kislino uparimo v vakuumu. Nastali peptid lahko uporabimo direktno v naslednji ligaciji in postopek ponovimo. Sintetični hML₁₅₃ lahko ligiramo po postopkih, analognih tistim, opisanim zgoraj, za sintetični ali rekombinantni hML₁₅₄₃₃₂, da naredimo sintetični ali semisintetični hML popolne dolžine.160 was dissolved in buffer (2-5 mM peptide) and a 10x stock subtiligase stock solution (1 mg / ml in tricin (100 mM), pH 8) was added to achieve a final enzyme concentration of about 5 gM. Then, 3-5 moles of excess donor peptide activated with glc-K-Nl · ^ are added as a solid, dissolved, and the mixture is allowed to stand at 25 ° C. Ligations were monitored by reverse phase analytical HPLC 08 (CH ^ CN / F ^ O gradient with TFA (0.1%)). The ligation products were purified by preparative HPLC and lyophilized. Isonicotinyl (iNOC) deprotection is performed by mixing HCl-activated zinc powder with a protected peptide in acetic acid. The zinc dust was removed by filtration and the acetic acid was evaporated in vacuo. The resulting peptide can be used directly in the next ligation and repeated. Synthetic hML ₁₅₃ can be ligated by procedures analogous to those described above for synthetic or recombinant hML ₁₅₄₃₃₂ to make full-length synthetic or semisynthetic hML.

Sintetični hML ima mnogo prednosti pred rekombinantnim. Nenaravne stranske verige lahko uvedemo zato, da izboljšamo zmogljivost ali specifičnost. Polimerne funkcionalnosti, kot je polietilenglikol, lahko vgradimo, da izboljšamo trajanje delovanja. Polietilenglikol lahko npr. vežemo na lizinske ostanke individualnih fragmentov (tabela 14) pred ali po eni ali več izvedenih ligacijskih stopenj. Proteazno občutljive peptidne vezi lahko odstranimo ali spremenimo, da izboljšamo stabilnost in vivo. Poleg tega lahko sintetiziramo derivate težkih atomov za pomoč pri določevanju strukture.Synthetic hML has many advantages over recombinant. Unnatural side chains can be introduced to improve performance or specificity. Polymer functionalities such as polyethylene glycol can be incorporated to improve the duration of operation. Polyethylene glycol may e.g. bind to lysine residues of individual fragments (Table 14) before or after one or more ligation steps performed. Protease-sensitive peptide bonds can be removed or altered to improve stability in vivo. In addition, derivatives of heavy atoms can be synthesized to help determine the structure.

161161

Shema 2. Sinteza v trdni fazi peptidnih fragmentov za segmentno ligaciioScheme 2. Solid phase synthesis of peptide fragments for segmental ligation

H,N'H, N '

O .(Peptid K nhO. (Peptide K nh

e,f (cepitev)»e, f (cleavage) »

izonikotinil (iNOC) glikolat-lizil-amid (glc-K-NHp)isonicotinyl (iNOC) glycolate-lysyl-amide (glc-K-NHp)

a) Lizil-parametilbenzhidrilaminsko (MBHA) smolo 1 (0,63 mekv./g, Advanced ChemTech) mešamo z bromoocetno kislino in diizopropilkarbodiimidom (5 ekv.) eno uro pri 25 °C v dimetilacetamidu (DMA), da dobimo bromoacetilni derivat 2. b) Smolo temeljito izperemo z DMA in individualne Boc-zaščitene amino kisline (3 ekv., Bachem) esterificiramo z mešanjem z natrijevim bikarbonatom (6 ekv.) v dimetilformamidu (DMF) 24 ur pri 50 °C, da dobimo ustrezno glikolat-fenilalanilamidno smolo 3. Amino acetilirano smolo 3 izperemo z DMF (3x) in dik162 lorometanom (CHjClj) (3x) in lahko shranimo pri sobni temperaturi za več mesecev. Smolo 3 lahko nato napolnimo v avtomatiziran peptidni sintetizator (Applied Biosystems 430A) in peptide podaljšamo z uporabo standardnih postopkov v trdni fazi (5). c) Ν-α-Boc-skupino odstranimo z raztopino 45 % trifluoroocetne kisline v CF^C^. d) Boc-zaščitene amino kisline (5 ekv.) nato predaktiviramo z uporabo benzotriazol-1il-oksi-tris-(dimetilamino) fosfonijevega heksafluorofosfata (BOP, 4 ekv.) in N-metilmorfolina (NMM, 10 ekv.) v DMA in pripojimo v 1-2 urah. e) Končno N-aBoc skupino odstranimo (TFA/CF^Cl^, da dobimo 4, in izonikotinilno (iNOC) zaščitno skupino uvedemo, kot je opisano pred tem, (4), z mešanjem s 4-izonikotinil2-4-dinitrofenil karbonatom (3 ekv.) in NMM (6 ekv.) v DMA pri 25 °C 24 ur. f) S cepitvijo in deprotekcijo peptida z obdelavo z brezvodnim HF (5 % anizol/5 % etilmetil sulfid) pri 0 °C 1 uro dobimo iNOC-zaščiten, glikolat-lys-amidno aktiviran peptid 5, ki ga očistimo s HPLC z reverzno fazo 08 (CH^CN/H^O gradient, TFA (0,1 %)). Identičnost vseh substratov potrdimo z masno spektrometrijo.a) Lysyl-parameterbenzhydrylamine (MBHA) resin 1 (0.63 mEq / g, Advanced ChemTech) was mixed with bromoacetic acid and diisopropylcarbodiimide (5 eq) for one hour at 25 ° C in dimethylacetamide (DMA) to give the bromoacetyl derivative 2 b) The resin is washed thoroughly with DMA and the individual Boc-protected amino acids (3 eq, Bachem) are esterified by mixing with sodium bicarbonate (6 eq) in dimethylformamide (DMF) for 24 hours at 50 ° C to give the corresponding glycolate- phenylalanilamide resin 3. The amine acetylated resin 3 is washed with DMF (3x) and dik162 loromethan (CH2Cl2) (3x) and can be stored at room temperature for several months. The resin 3 can then be filled into an automated peptide synthesizer (Applied Biosystems 430A) and the peptides extended using standard solid phase procedures (5). c) The Ν-α-Boc group is removed with a solution of 45% trifluoroacetic acid in CF ^C C ^. d) Boc-protected amino acids (5 eq) are then inactivated using benzotriazol-1-yl-oxy-tris- (dimethylamino) phosphonium hexafluorophosphate (BOP, 4 eq) and N-methylmorpholine (NMM, 10 eq) in DMA and attach in 1-2 hours. e) The final N-aBoc group is removed (TFA / CF2 Cl2 to give 4, and the isonicotinyl (iNOC) protecting group is introduced as previously described (4) by mixing with 4-isonicotinyl 2-4-dinitrophenyl carbonate (3 eq.) And NMM (6 eq.) In DMA at 25 ° C for 24 h. F) Cleavage and deprotection of the peptide by treatment with anhydrous HF (5% anisole / 5% ethylmethyl sulfide) at 0 ° C for 1 hour iNOC-protected, glycolate-lys-amide-activated peptide 5, which was purified by reverse phase HPLC 08 (CH2 CN / H2O gradient, TFA (0.1%)). The identity of all substrates is confirmed by mass spectrometry.

DODATNE INFORMACIJEMORE INFORMATION

Predloženi izum je omogočen na osnovi zgornjega opisa in z lahkoto dosegljivih referenc in izhodnih materialov. Prijavitelji so deponirali pri American Type Culture Collection, Rockville, Md., ZDA (ATCC) naslednje celične linije:The present invention is made possible on the basis of the foregoing description and readily available references and starting materials. Applicants have deposited the following cell lines with the American Type Culture Collection, Rockville, Md., USA (ATCC):

Escherichia coli, DHlOB-pBSK-hrap/ I 1,8, ATCC dostopna št. CRL 69575, deponirano 24. februarja 1994;Escherichia coli, DHlOB-pBSK-hrap / I 1.8, ATCC accession no. CRL 69575, deposited February 24, 1994;

plazmid, pSVI5.ID.LLML0RF, ATCC dostopna št. CRL 75958; deponirano 2. decembra 1994; in celice CHO DP-12, ML 1/50 MCB (označeno št. 1594), ATCC dostopna št. CRL 11770; deponirano 6. decembra 1994.plasmid, pSVI5.ID.LLML0RF, ATCC accession no. CRL 75958; deposited December 2, 1994; and CHO DP-12 cells, ML 1/50 MCB (designated no. 1594), ATCC access no. CRL 11770; deposited on 6 December 1994.

Le to deponiramo po predpisih budimpeštanske pogodbe za mednarodno priznanje deponiranih mikroorganizmov za namene patentnih postopkov in temu ustreznih pravil (Budapest Treaty). To zagotavlja vzdrževanje kultur, sposobnih za življenje 30 let od dneva depozita. Organizmi so dosegljivi od ATCC po določbah Budapest Treaty in izpostavljeni dogovoru med prijavitelji in ATCC, ki zagotavlja neomejeno dosegljivost po izdaji pristojnega US patenta. Dosegljivost deponiranega seva si ni možno razlagati kot licenco za izvajanje predloženega izuma v nasprotju s pravicami, podeljenimi pod avtoriteto katerekoli vlade v skladu z njenimi patentnimi zakoni.This is deposited in accordance with the provisions of the Budapest Treaty for the International Recognition of Deposited Microorganisms for the Purposes of Patent Procedures and the relevant Budapest Treaty. This ensures that cultures capable of living for 30 years from the date of deposit are maintained. The organisms are available from the ATCC under the provisions of the Budapest Treaty and are subject to agreement between the applicants and the ATCC, which guarantees unlimited availability after the grant of a competent US patent. The reach of a deposited strain cannot be interpreted as a license to carry out the present invention in violation of the rights conferred under the authority of any government under its patent laws.

163163

Čeprav je predloženi izum nujno opisan v povezavi s prednostnimi izvedbami in specifičnimi delovnimi primeri, pa strokovnjak na osnovi predhodnega opisa lahko iz vede številne spremembe ter substitucije ekvivalentov in preoblikuje predložene predmete, navedene tukaj, ne da bi se oddaljil od njegovega namena in obsega. Torej lahko izum izvajamo na načine, ki so drugačni od tistih, specifično navedenih tukaj. Zato je mišljeno, da naj bi zaščita, podeljena s patentno listino, omejevala le z predložene zahtevke in njihove ekvivalente.Although the present invention is necessarily described in connection with preferred embodiments and specific working examples, the person skilled in the art may, from the foregoing description, make many changes and substitutions of equivalents and transform the objects presented herein without departing from its purpose and scope. Thus, the invention may be carried out in ways other than those specifically stated herein. It is therefore intended that the protection conferred by the patent should be restricted to the applications and their equivalents only.

Vse reference, navedene tukaj, so jasno vključene z referenco.All references cited here are clearly incorporated by reference.