DK176220B1 - New DNA segment - has gene encoding human tissue factor heavy chain protein and is useful for inhibiting coagulation - Google Patents

Abstract

New DNA segment (s) contg. no more than 1200 nucleotide base pairs includes a sequence defining a structural gene encoding human tissue factor heavy chain protein (huTFh) (I). The aminoacid sequence of (I) is defined. Also claimed is a recombinant DNA molecule comprising a vector operatively linked to (S). Human tissue factor binding site polypeptide analogues (II) with 50 aminoacid residues and including a sequence VNQVYTVQIST (a) or LYYWKSSSSBKKT (b) are also new. The following (II) are specifically claimed: H-EPKP-(a)-KSGDWKSKC-OH (IIa), H-VFGKDLIYTL-(b)-OH (IIG), H-SSSGKKTAKTNTNEFLIDVDKGENYCFSV-OH (IIc) H-SGTTNTVAAYNLTWKSTNFKTILEWEPKPV-OH (IId), H-TKSGDWKSKCFYTTDTECDLTDEIVKDVKQTY-OH (IIe) H-KSGDWKSKC-OH (IIf), H-LARVFSYPAGNVESTGSA BEPLYENSPEFTPYLC-OH (IIg), H-YENSPEFTPYLETNLBQ PTIQSFEQVGTKV-OH (IIh), and H-QAVIPSRTV NRKSTDSPVEC-OH (IIi) Also claimed are: (1) antibodies which immunoreact with (I), (IIa)-(IIi) but not with a polypeptide with sequence 204-226 of (I); (2) antibody secreting hybridomas TF8-5G9, TF9-10H10, TF9-6B4 and TF9-5B7; (3) various uses of the novel antibodies; and (4) diagnostic kits.

Description

i DK 176220 B1in DK 176220 B1

Den foreliggende opfindelse angår monoklonale antistoffer, som binder human vævsfaktor-protein, samt deres anvendelse.The present invention relates to monoclonal antibodies that bind human tissue factor protein, and their use.

Koagulering af blod involverer en seriekaskade af 5 enzym-, cofaktor-, proteolyse- og gelatineringsreaktioner, der medieres af en gruppe af cellulære proteiner og plasmaproteiner, der er kendte som koagulationsfaktorer. Initiering af denne kaskade kan forekomme, når den cellulære receptor, der er kendt som vævsfaktor {TF}, binder koagule-10 ringsfaktor VII eller dens derivat faktor Vila til dannelse af et katalytisk aktivt kompleks. I fraværelse af TF og uden fortsat binding til et kompleks initierer faktor VII/-VIla ikke koagulering. Den kemiske og biologiske karakterisering af TF er derfor klart af betydning for forståelsen 15 af koaguleringsmekanismen.Blood coagulation involves a series cascade of 5 enzyme, cofactor, proteolysis and gelatinization reactions mediated by a group of cellular proteins and plasma proteins known as coagulation factors. Initiation of this cascade may occur when the cellular receptor known as tissue factor {TF} binds coagulation factor VII or its derivative factor Vila to form a catalytically active complex. In the absence of TF and without continued binding to a complex, factor VII / -VIIa does not initiate coagulation. The chemical and biological characterization of TF is therefore clearly important for understanding the coagulation mechanism.

Vævsfaktor er et membranbundet glycoprotein, der ikke normalt findes opløst i kredsløbet eller tilgængeligt for plasmaproteiner omfattende faktor Vll/VIIa og de andre koagulationsfaktorer. Medens vævsfaktor ikke normalt ekspri-20 meres på overfladen af celler, som danner karvæv, kan dets ekspression af monocyter i karvævet fremkaldes af bestanddele af infektiøse agenser, såsom bakterielt lipopolysac-charid, lymphokiner afledt af nogle antigenstimulerede T--hjælperceller, direkte af nogle stimulerede T-hjælperceller 25 og immunkomplekser. Visse inflammatoriske mediatorer af monocyt/makrofag-oprindelse, f.eks. interleukin 1 og tumor--nekrose-faktor a samt bakterielt lipopolysaccharid, kan stimulere endotheliale celler, som forer den humorale overflade af blodkar til eksprimering af TF. Ekspression af TF 30 i kar-rummet medfører typisk spredt intravaskulær koagulering eller lokaliseret initiering af koagulering, dvs. throm-bogenese.Tissue factor is a membrane-bound glycoprotein that is not normally found in the circulation or available for plasma proteins comprising factor VIII / VIIa and the other coagulation factors. While tissue factor is not normally expressed on the surface of cells forming vascular tissue, its expression of monocytes in the vascular tissue can be elicited by constituents of infectious agents, such as bacterial lipopolysaccharide, lymphokines derived from some antigen-stimulated T helper cells, directly from some stimulated T helper cells 25 and immune complexes. Certain inflammatory mediators of monocyte / macrophage origin, e.g. Interleukin 1 and tumor necrosis factor α as well as bacterial lipopolysaccharide can stimulate endothelial cells lining the humoral surface of blood vessels to express TF. Expression of TF 30 in the vasculature typically results in disseminated intravascular coagulation or localized initiation of coagulation, ie. thrombin bogenese.

Vævsfaktor eksprimeres konstitutivt på overfladen af visse ekstravaskulære celler i kultur in vitro, herunder 35 fibroblaster, nogle endnu ikke identificerede typer af hjerneceller og visse epitheler, der er adskilt fra de cirkule- 2 DK 176220 B1 rende plasmaproteiner af basismembran-barrierer. Tilstedeværelsen af TF på disse celler medfører koage1-dannelse ved kontakt med blod som resultat af vævsbeskadigelse. TF er således grundlaget for initieringen af det hæmostatiske 5 system.Tissue factor is constitutively expressed on the surface of certain extravascular cells in culture in vitro, including 35 fibroblasts, some as yet unidentified types of brain cells, and certain epitheliums that are separate from the circulating plasma proteins of basement membrane barriers. The presence of TF on these cells causes coagulation1 formation upon contact with blood as a result of tissue damage. Thus, TF is the basis for the initiation of the hemostatic system.

Rapporten af Howell, Am. J. Physiol, 31:1 (1912) var den første til at foreslå, at et isoleret vævsproteinpræparat indeholdende TF kun kunne fremme koagulering, når det var til stede som et phospholipidprotein-kompleks (lipoprotein-10 -kompleks). Rekonstitution af den pro-koagulerende aktivitet af TF ved relipidering af det isolerede protein har været nødvendig, fordi isolering af det TF-holdige vævsprotein typisk medfører fjernelse af phospholipiderne, som normalt er knyttet til TF-proteinet, og en sådan rekonstitution er 15 blevet undersøgt af et antal forskere. Det er f.eks. blevet rapporteret, at rekonstitutionen af koagulerende aktivitet påvirkes af phospholipidtypen, forholdet mellem phospholipid og protein og detergentet og den ioniske sammensætning af rekonstitutionsblandingen, se Nemerson, J. Clin. Invest., 20 47-72 (1968); Nemerson, J. Clin. Invest., 48-322 (1969); ogThe report by Howell, Am. J. Physiol, 31: 1 (1912) was the first to suggest that an isolated tissue protein preparation containing TF could only promote coagulation when present as a phospholipid protein complex (lipoprotein-10 complex). Reconstitution of the pro-coagulant activity of TF by repopulating the isolated protein has been necessary because isolation of the TF-containing tissue protein typically results in removal of the phospholipids normally associated with the TF protein, and such reconstitution has been investigated. by a number of researchers. It is e.g. have been reported that the reconstitution of coagulant activity is influenced by the phospholipid type, the ratio of phospholipid to protein and the detergent and the ionic composition of the reconstitution mixture, see Nemerson, J. Clin. Invest., 20 47-72 (1968); Nemerson, J. Clin. Invest., 48-322 (1969); and

Carson et al., Science, 208:307 (1980).Carson et al., Science, 208: 307 (1980).

Både isolerede og relipiderede TF-holdige proteinpræ-parater er blevet fremstillet ved ekstraktion fra væv af forskellige arter. Historisk har de anvendte metoder været 25 vanskelige, tidskrævende og givet lave udbytter, fordi vævsfaktor kun er til stede i yderst små mængder i naturligt forekommende væv. Vedrørende en gennemgang af de klassiske metoder, se Nemerson et al., Prog. Hem. Thromb., 6:237-261 (1982).Both isolated and precipitated TF-containing protein preparations have been prepared by extraction from tissues of different species. Historically, the methods used have been 25 difficult, time-consuming and given low yields because tissue factor is present only in extremely small amounts in naturally occurring tissues. For a review of the classical methods, see Nemerson et al., Prog. Hem. Thromb., 6: 237-261 (1982).

30 Senere har Broze et al., J. Biol. Chem., 260:10917- 20 (1985), Bom et al., Thromb. Res., 42:635-643 (1986) ogLater, Broze et al., J. Biol. Chem., 260: 10917-20 (1985), Bom et al., Thromb. Res., 42: 635-643 (1986) and

Guha et al., Proc. Natl. Acad. Sci, USA, 83:299-302 (1986) rapporteret isolering af human vævsfaktor-protein (huTF--protein) ved anvendelse af en metode baseret på opdagelsen 35 af, at delipideret vævsfaktorprotein kan binde faktor VII/-Vlla, når proteinet er opløst i en vandig opløsning indehol- 3 DK 176220 B1 dende et ikke-ionisk detergent og calciumchlorid. Imidlertid er anvendeligheden af denne metode, hvorved der anvendes et faktor VII/VIla-affinitetssorptionsmiddel som middel til isolering af vævsfaktorprotein, ikke blot begrænset af van-5 skelighederne ved at opnås væsentlige mængder af isoleret faktor Vll/VIIa, men også af labiliteten af faktor Vll/VIIa.Guha et al., Proc. Natl. Acad. Sci, USA, 83: 299-302 (1986) reported isolation of human tissue factor protein (huTF protein) using a method based on the discovery that delipidized tissue factor protein can bind factor VII / -VIIa when the protein is dissolved in an aqueous solution containing a nonionic detergent and calcium chloride. However, the utility of this method using a factor VII / VIa affinity sorbent as agent for tissue factor protein isolation is not only limited by the difficulties of obtaining significant amounts of isolated factor VII / VIIa but also the lability of factor VII / VIIa.

Broze et al., supra, har foreslået, at udvikling af monoklonale antistoffer, som er specifikke for huTF, og deres anvendelse som immunoaffinitets-sorptionsmidler kan 10 omgå problemer forårsaget af den begrænsede tilgængelighed af faktor Vll/VIIa, Der er imidlertid ikke rapporteret nogen monoklonale anti-huTF-antistoffer i litteraturen. Endvidere immunoreagerer to monoklonale antistoffer fremkaldt mod bovin TF [Carson et al., Blood, 662-156 (1985)] ikke med 15 huTF (Guha et al., supra).Broze et al., Supra, have suggested that the development of monoclonal antibodies specific for huTF and their use as immunoaffinity sorbents may circumvent problems caused by the limited availability of factor Vll / VIIa, however, no reports have been reported. monoclonal anti-huTF antibodies in the literature. Furthermore, two monoclonal antibodies induced against bovine TF [Carson et al., Blood, 662-156 (1985)] do not immunoreact with 15 huTF (Guha et al., Supra).

Den foreliggende opfindelse angår følgende: - Et monoklonalt antistof, som binder human vævsfaktor-protein, til anvendelse ved forebyggelse af sepsis.The present invention relates to the following: - A monoclonal antibody that binds human tissue factor protein for use in the prevention of sepsis.

- Et monoklonalt antistof, som binder human vævsfak-20 tor-protein, til anvendelse ved behandling af thrombohæ- morrhagiske lidelser.- A monoclonal antibody that binds human tissue factor protein for use in the treatment of thrombohomorrhagic disorders.

- Et monoklonalt antistof, som binder human vævsfaktor-protein, til anvendelse ved inhibering af koagulation.A monoclonal antibody which binds human tissue factor protein for use in inhibiting coagulation.

Ifølge en udførelsesform reagerer et af disse mono-25 klonale antistoffer yderligere med en human vævsfaktor-bin-dingssted-polypeptidanalog; især med en sekvens valgt blandt følgende: E P KPVNQVYTVQISTKSGDWKS KC, VFGKDLIYTLYYWKS SS SGKKT, 3 0 SSSGKKTAKTNTNEFLIDVDKGENYCFSV, SGTTNTVAAYNLTWKSTNFKTILEWEPKPV, TKSGDWKSKCFYTTDTECDLTDEIVKDVKQTY, KSGDWKSKC, ECDLTDEIVKDVKQTY, 3 5 LARVFSYPAGNVESTGSAGEPLYENSPEFTPYLC, YENSPEFTPYLEMNIGQPTIQSFEQVGTKV, 4 DK 176220 B1 og QAVIPSRTVNRKSTDSPVEC; og immunoreagerer i det væsentlige ikke med et polypeptid med formlen vist i fig. 1 fra position 204 til position 226.In one embodiment, one of these monoclonal antibodies further reacts with a human tissue factor binding site polypeptide analog; in particular having a sequence selected from the following: E P KPVNQVYTVQISTKSGDWKS KC, VFGKDLIYTLYYWKS SS SGKKT, 3 0 SSSGKKTAKTNTNEFLIDVDKGENYCFSV, SGTTNTVAAYNLTWKSTNFKTILEWEPKPV, TKSGDWKSKCFYTTDTECDLTDEIVKDVKQTY, KSGDWKSKC, ECDLTDEIVKDVKQTY, 3 5 LARVFSYPAGNVESTGSAGEPLYENSPEFTPYLC, YENSPEFTPYLEMNIGQPTIQSFEQVGTKV, 4 DK 176220 B1 and QAVIPSRTVNRKSTDSPVEC; and do not substantially react with a polypeptide of the formula shown in FIG. 1 from position 204 to position 226.

5 Opfindelsen angår endvidere: - Anvendelse af et monoklonalt antistof, som binder human vævsfaktor-protein, ved fremstilling af et medikament til behandling af sepsis.The invention further relates to: Use of a monoclonal antibody which binds human tissue factor protein in the manufacture of a medicament for the treatment of sepsis.

- Anvendelse af et monoklonalt antistof, som binder 10 human vævsfaktor-protein, ved fremstilling af et medikament til behandling af thrombohæmorrhagiske lidelser.Use of a monoclonal antibody that binds 10 human tissue factor protein in the manufacture of a drug for the treatment of thrombohemorrhagic disorders.

- Anvendelse af et monoklonalt antistof, som binder human vævsfaktor-protein, ved fremstilling af et medikament til inhibering af koagulation.Use of a monoclonal antibody that binds human tissue factor protein in the preparation of a coagulation inhibiting drug.

15 Et DNA-segment, som er relateret til den foreliggende opfindelse, omfatter ikke mere end ca. 12.000 nukleotid-basepar og omfatter en sekvens, der definerer et strukturelt gen, som koder for et protein med tung kæde af human vævsfaktor (huTFh). Det strukturelle gen koder fortrinsvis for 20 et protein, der har en aminosyrerest-sekvens vist i fig. 1 fra ca. rest nr. 1 til ca. rest nr. 263. Mere foretrukket har det strukturelle gen en nuklotid-basesekvens vist i fig. 2 fra ca. base nr. 130 til ca. base nr. 918.A DNA segment related to the present invention encompasses no more than ca. 12,000 nucleotide base pairs and comprises a sequence defining a structural gene encoding a human tissue heavy chain factor (huTFh) protein. Preferably, the structural gene encodes a protein having an amino acid residue sequence shown in FIG. 1 from approx. residue # 1 to approx. residue # 263. More preferably, the structural gene has a nucleotide base sequence shown in FIG. 2 from approx. base # 130 to approx. base # 918.

Fortrinsvis omfatter DNA-segmentet også en anden 25 sekvens, der støder .op til 5'-terminussen af den første sekvens og koder for en aminosyrerest-ledersekvens bundet til aminoterminussen af huTFh-proteinet. Den første og den anden DNA-sekvens definerer sammen et sammensat strukturelt gen, der koder for et forstadium af et protein med tung 30 kæde af human vævsfaktor (pre-huTFh). Det sammensatte strukturelle gen koder fortrinsvis for et protein, der har en aminosyresekvens vist i fig. 1 fra ca. rest nr. -32 til ca. rest nr. 263 . Det sammensatte strukturelle gen har mere foretrukket en nukleotid-basesekvens vist i fig. 2 fra ca.Preferably, the DNA segment also comprises another sequence adjacent to the 5 'terminus of the first sequence and encoding an amino acid residue leader sequence bound to the amino terminus of the huTFh protein. The first and second DNA sequences together define a composite structural gene encoding a precursor of a human tissue heavy chain factor (pre-huTFh) protein. The composite structural gene preferably encodes a protein having an amino acid sequence shown in FIG. 1 from approx. residue # -32 to approx. residue # 263. More preferably, the composite structural gene has a nucleotide base sequence shown in FIG. 2 from approx.

35 base nr. 34 til ca. base nr. 918.35 base # 34 to approx. base # 918.

5 DK 176220 B15 DK 176220 B1

Et rekombinant DNA-molekyle, som er relateret til den foreliggende opfindelse, omfatter en vektor, der er operativt bundet til et først DNA-segment, som definerer et strukturelt gen, der koder for et protein med tung kæde af 5 human vævsfaktor. Det rekombinante DNA-molekyle omfatter yderligere fortrinsvis et andet DNA-segment, der støder op til 51-terminussen af det første segment og koder for en aminosyrerest-ledersekvens bundet til det nævnte protein.A recombinant DNA molecule related to the present invention comprises a vector operatively linked to a first DNA segment defining a structural gene encoding a heavy chain protein of human tissue factor. Preferably, the recombinant DNA molecule further comprises a second DNA segment adjacent to the 51-terminus of the first segment and encoding an amino acid residue leader sequence bound to said protein.

Det første og det andet DNA-segment definerer sammen et 10 sammensat strukturelt gen, som koder for en forstadie-form for det nævnte protein.The first and second DNA segments together define a composite structural gene encoding a precursor form of said protein.

En polypeptidanalog af bindingsstedet af human vævs-faktor, som er relateret til den foreliggende opfindelse, omfatter ikke mere end ca. 50 aminosyrerester og omfatter en 15 aminosyresekvens, der svarer til en sekvens med formlen: -VNQVYT- især en aminosyresekvens, der svarer til en sekvens med formlen: -VNQVYTVQIST-, eller 2 0 -LYYWKS S S SGKKT-.A polypeptide analog of the human tissue factor binding site related to the present invention encompasses no more than ca. 50 amino acid residues and comprises a 15 amino acid sequence corresponding to a sequence of formula: -VNQVYT- especially an amino acid sequence corresponding to a sequence of formula: -VNQVYTVQIST-, or 20 -LYYWKS S S SGKKT-.

De omhandlede antistoffer kan produceres af hybrido-maerne betegnet TF8-5G9, TF9-10H10, TF9-5B7 og TF9-6B4.The antibodies in question may be produced by the hybridomas designated TF8-5G9, TF9-10H10, TF9-5B7 and TF9-6B4.

Kort beskrivelse af tegningen.Brief description of the drawing.

25 Fig. 1 illustrerer den komplette aminosyresekvens af den modne form og forstadieformen af proteiner med tung kæde af human vævsfaktor (henholdsvis huTFh og pre-huTFh), der vist fra venstre til højre og i retning fra aminoter-minussen til carboxyterminussen ved anvendelse af etbogstavs-30 -aminosyrekoden. Aminosyresekvensen af det overvejende naturligt forekommende modne protein er nummereret 1 til 263. Sekvensen af den i mindre omfang forekommende modne form begynder ved aminosyrerest nr. 3 og ender ved rest nr. 263.FIG. 1 illustrates the complete amino acid sequence of the mature form and the precursor form of heavy chain human tissue factor proteins (huTFh and pre-huTFh, respectively), shown from left to right and in the direction from the amino terminus to the carboxy terminus using the one-letter amino acid code. The amino acid sequence of the predominantly naturally occurring mature protein is numbered 1 to 263. The sequence of the less abundant mature form begins at amino acid residue # 3 and ends at residue # 263.

Aminosyresekvensen svarende til ledersekvensen (for-35 stadiedelen) af pre-huTFh-proteinet, som fjernes under mod-ningsprocesen, er betegnet ved negative tal. Det ekstracel- 6 DK 176220 B1 lulære domæne og transmembran-forankringsregionen svarer til aminosyrerest nr. 1 til 219 henholdsvis 220 til 242.The amino acid sequence corresponding to the leader sequence (precursor portion) of the pre-huTFh protein which is removed during the maturation process is denoted by negative numbers. The extracellular domain and transmembrane anchoring region correspond to amino acid residues Nos. 1 to 219 and 220 to 242, respectively.

Fig. 2 illustrerer nukleotidsekvensen af et cDNA. der koder for pre-huTFh- og huTFh-proteinerne, vist fra 5 venstre til højre og i retning fra 5'-terminussen til 3'-terminussen ved anvendelse af etbogstavs-nukleotidbasekoden.FIG. 2 illustrates the nucleotide sequence of a cDNA. encoding the pre-huTFh and huTFh proteins, shown from 5 left to right and in the direction from the 5 'terminus to the 3' terminus using the one-letter nucleotide base code.

Det strukturelle gen for huTFh begynder ved base nr. 130 og ender ved base nr. 918.The structural gene for huTFh begins at base # 130 and ends at base # 918.

Læserammen er vist ved placering af den afledte ami-10 nosyresekvens over nukleotidsekvensen således, at bogstavet, der repræsenterer hver aminosyrerest, er placeret over den midterste base i det tilsvarende kodon.The reading frame is shown by placing the deduced amino acid sequence over the nucleotide sequence such that the letter representing each amino acid residue is positioned above the middle base of the corresponding codon.

Fig. 3 er en graf, der illustrerer koaguleringsbestemmelsen anvendt til måling af huTF-prokoaguleringsak-15 tiviteten som beskrevet i eksempel 2. Der er vist en dobbeltlogaritmisk afsætning af koaguleringstiden af humant citratplasma i sekunder mod koncentrationen af human vævsfaktor (huTF) i picogram (pg) pr. ml.FIG. Figure 3 is a graph illustrating the coagulation assay used to measure huTF procoagulation activity as described in Example 2. A double logarithmic plot of human citrate plasma coagulation time in seconds against human tissue factor (huTF) concentration in picogram (pg) is shown. per. ml.

Fig. 4 illustrerer et autofluorogram af faktor VII/-20 VIIa-affinitetsisoleret huTF, som er underkastet elektrofo-rese i en 10%'s polyacrylamidgel. Bane A viser ·*·^^Ι-mærket huTF, som er isoleret og reduceret med dithiothreitol (DTT) før elektroforese som beskrevet i eksempel 4. Bane B viser molekylvægtstandarder med tilsyneladende molekylvægte anført 25 i kilodalton (k).FIG. 4 illustrates an autofluorogram of factor VII / -20 VIIa affinity-insulated huTF, which is electrophoresed in a 10% polyacrylamide gel. Lane A shows · * · ^^ Ι labeled huTF isolated and reduced with dithiothreitol (DTT) before electrophoresis as described in Example 4. Lane B shows molecular weight standards with apparent molecular weights listed in kilodaltons (k).

Fig. 5 illustrerer et autofluorogram af faktor VII/-Vlla-affinitetsisoleret huTF underkastet elektroforese i 15%'s polyacrylgeler. Isoleringen, mærkningen med Qg elektroforesen af huTF gennemføres som beskrevet i eksempel 30 4. Bane A viser det isolerede huTF efter reduktion med DTT.FIG. Figure 5 illustrates an autofluorogram of factor VII / -VIIa affinity-insulated huTF subjected to electrophoresis in 15% polyacrylic gels. The isolation, labeling with Qg electrophoresis of huTF is performed as described in Example 30 4. Lane A shows the isolated huTF after reduction with DTT.

Bane B viser den samme prøve underkastet elektroforese uden reduktion med DTT. Det øvre og nedre bånd (mærket U og L) svarer til formerne med en størrelse på ca. 58 og 47 k af huTF. Efter autofluorografi udskæres de øvre og nedre bånd, 35 rehydreres i SDS-prøvepuffer indeholdende DTT, indsættes i prøvebrønden af en anden 15%'s polyacrylamidgel og underka- 7 DK 176220 B1 stes elektroforese. Bane C viser reelelektroforesen af det nedre bånd fra bane B. Bane D viser reelektroforesen af det øvre bånd fra bane B. Proteinerne med en tilsyneladende molekylægt på 12,5 og 47 kilodalton (k) er vist ved pile.Lane B shows the same sample subjected to electrophoresis without reduction with DTT. The upper and lower band (labeled U and L) correspond to the shapes with a size of approx. 58 and 47 k of huTF. After autofluorography, the upper and lower bands are cut, rehydrated in SDS sample buffer containing DTT, inserted into the sample well of another 15% polyacrylamide gel and subjected to electrophoresis. Lane C shows the real electrophoresis of the lower band from lane B. Lane D shows the real electrophoresis of the upper band from lane B. The proteins with an apparent molecular weight of 12.5 and 47 kilodaltons (k) are shown by arrows.

5 Fig. 6 illustrerer et autofluorogram af faktor VII/- VIIa-affinitetsisoleret huTF, som først er immunopræcipiteret med det huTF-specifikke monoklonale antistof TF8-5G9 og derefter er underkastet elektroforese i en 8-17%'s polya-crylamid-gradientgel som beskrevet i eksempel 4. Bane A 10 viser 125I-mærket huTF, som er underkastet elektroforese med reduktion med DTT. Bane B viser den samme prøve underkastet elektroforese uden reduktion.FIG. 6 illustrates an autofluorogram of factor VII / VIIa affinity isolated huTF which is first immunoprecipitated with the huTF specific monoclonal antibody TF8-5G9 and then subjected to electrophoresis in an 8-17% polyacrylamide gradient gel as described in Example 4. Lane A 10 shows the 125 I-labeled huTF, which is electrophoresed with DTT reduction. Lane B shows the same sample subjected to electrophoresis without reduction.

Fig. 7 illustrerer et autofluorogram af faktor VII/-Vlla-affinitetsisoleret huTF, som er underkastet elektrofo-15 rese i 15%1 s polyacrylamidgeler. Isoleringen, mærkningen med -*-251' reduktionen og deglycosyleringen gennemføres som beskrevet i eksempel 4. Bane 1 indeholder følgende proteinstandarder, der underkastes elektroforese som markører med de tilsyneladende molekylvægte (Mr) anført i kilodalton: 20 lysozym 14,3, carbonsyreanhydrase 30,0, ovalbumin 46,0, bovint serumalbumin 69,0, phosphorylase b 92,5 og myosin 200,0, alle fra Amersham, Arlington Heights, IL. 125I-huTF--holdige prøver underkastes elektroforese, enten med DTT (bane 2 og 3) eller uden DTT (bane 4 og 5) . Nogle af disse 25 125I-huTF-holdige prøver er deglycosylerede (bane 3 og 5), medens andre ikke er deglycosylerede (bane 2 og 4) før elek-troforese. De l^I-huTF-holdige prøver i bane 3 og 5 degly-cosyleres før elektroforese, medens prøverne i bane 2 og 4 ikke deglycosyleres.FIG. Figure 7 illustrates an autofluorogram of factor VII / -VIIa affinity-insulated huTF subjected to electrophoresis in 15% 1 s polyacrylamide gels. The isolation, labeling with - * - 251 'reduction and deglycosylation are carried out as described in Example 4. Lane 1 contains the following protein standards which are electrophoresed as markers with the apparent molecular weights (Mr) listed in kilodaltons: 20 lysozyme 14.3, carboxylic anhydrase 30, 0, ovalbumin 46.0, bovine serum albumin 69.0, phosphorylase b 92.5, and myosin 200.0, all from Amersham, Arlington Heights, IL. 125I-huTF - containing samples are subjected to electrophoresis, either with DTT (lanes 2 and 3) or without DTT (lanes 4 and 5). Some of these 25 125 I-huTF containing samples are deglycosylated (lanes 3 and 5), while others are not deglycosylated (lanes 2 and 4) prior to electrophoresis. The 11 H-huTF-containing samples in lanes 3 and 5 deglycosylated before electrophoresis, while the samples in lanes 2 and 4 were not deglycosylated.

30 Fig. 8 illustrerer et autofluorogram af immunoaf- finitetsisoleret huTF, som er underkastet elektroforese i 10%'s polyacrylamidgeler som beskrevet i eksempel 9. Bane 1 indeholder følgende proteinstandarder, der er underkastet elektrof orese som markører og med tilsyneladende molekylvægte 35 (Mr) anført i kg dalton: cytochrom c 12,4, lactoglobulin 18,4, carbonsyreanhydrase 29,0, lactat-dehydrogenase 36,0, 8 DK 176220 B1 ovalbumin 43,0, glutamat-dehydrogenase 55,0 og phosphorylase b 95,5, alle fra Diversified Biotech (Newton Centre, MA).FIG. Figure 8 illustrates an autofluorogram of immunoaffinity isolated huTF subjected to electrophoresis in 10% polyacrylamide gels as described in Example 9. Lane 1 contains the following protein standards subjected to electrophoresis as markers and with apparent molecular weights 35 (Mr) in kg dalton: cytochrome c 12.4, lactoglobulin 18.4, carboxylic anhydrase 29.0, lactate dehydrogenase 36.0, 8 DK 176220 B1 ovalbumin 43.0, glutamate dehydrogenase 55.0 and phosphorylase b 95.5, all from Diversified Biotech (Newton Center, MA).

Bane 2 indeholder ca. 2 0 μg protein, bestemt ved anvendelse af BCA-proteinbestemmelsesmetoden ifølge Smith et 5 al., Anal. Bioch., 150:76-85 (1985), og reduceret under anvendelse af DTT. Tung kæde af huTF (huTFh) er klart synlig ved ca. 47 Mr, og let kæde af huTF ses svagt ved ca. 12,5 Mr. Proteinet gøres synligt ved farvning med Coomassie-blåt som beskrevet af Laemmli, Nature, 227:680-685 (1970}.Lane 2 contains approx. 20 µg protein, determined using the BCA protein determination method of Smith et al., Anal. Bioch., 150: 76-85 (1985), and reduced using DTT. HuTF heavy chain (huTFh) is clearly visible at ca. 47 Mr, and light chain of huTF is weakly seen at ca. 12.5 Mr. The protein is made visible by staining with Coomassie Blue as described by Laemmli, Nature, 227: 680-685 (1970}.

10 Fig. 9 er en graf, der illustrerer dosis-reaktionskurven for inhibering af huTF-initieret coagulering ved hjælp af ikke-phospholipiderede (ikke-lipiderede) polypep-tidanaloge af huTFh. Den procentiske inhibering af koagulering ved forskellige koncentrationer af ikke-lipiderede 15 polypeptider måles som beskrevet i eksempel 12. De undersøgte polypeptider omfatter p26-49 (åben trekant, TF26.49), pl21--155 (åben cirkel, TF121.155), pl46-167 (udfyldt cirkel, TF146.167) og p204-226 (udfyldt firkant, TF204.226).FIG. Figure 9 is a graph illustrating the dose-response curve for inhibition of huTF-initiated coagulation by non-phospholipidated (non-lipidated) polypeptide analogs of huTFh. The percent inhibition of coagulation at various concentrations of non-lipidated polypeptides is measured as described in Example 12. The polypeptides examined include p26-49 (open triangle, TF26.49), p21--155 (open circle, TF121.155), pl46-167 (filled circle, TF146,167) and p204-226 (filled square, TF204,226).

Fig. 10 er en graf, der illustrer dosis-reaktionskur-20 ven for inhibering af huTF-initieret koagulering ved hjælp af phospholipiderede (lipiderede) polypeptidanaloge af huTFh.FIG. 10 is a graph illustrating the dose-response curve for inhibition of huTF-initiated coagulation by phospholipidated (lipidated) polypeptide analogs of huTFh.

De procentiske inhiberinger måles ved samme metode og for de samme analoge som beskrevet for fig. 9.The percent inhibitions are measured by the same method and for the same analogs as described in FIG. 9th

Fig. 11 illustrerer restriktionskortene for EcoRI-25 segment-indsatserne i rekombinant DNA-plasmiderne pCTF64, pCTF314 og pCTF403. Indsatserne (vist ved en bjælke) repræsenterer overlappende dele af nukleotidsekvenser, der sammen svarer til den komplette nukleotidsekvens af pre-huTFh-genet.FIG. 11 illustrates the restriction maps of the EcoRI-25 segment inserts in the recombinant DNA plasmids pCTF64, pCTF314 and pCTF403. The inserts (shown by a bar) represent overlapping portions of nucleotide sequences that together correspond to the complete nucleotide sequence of the pre-huTFh gene.

Hver for sig omfatter indsatserne nukleotidrester, der fra 30 venstre til højre og i retning fra 5' til 3' svarer til nukleotidsekvensen vist i fig. 2 fra base nr. 1 til 4 86 (indeholdt i pCTF64), fra base nr. 135 til 775 (indeholdt i pCTF314) og fra base nr. 776 til 1125 (indeholdt i pCTF403).Separately, the inserts comprise nucleotide residues corresponding from 30 left to right and in the direction of 5 'to 3' to the nucleotide sequence shown in FIG. 2 from base # 1 to 4 86 (contained in pCTF64), from base # 135 to 775 (contained in pCTF314), and from base # 776 to 1125 (contained in pCTF403).

Der er også vist den omtrentlige placering af restriktions-35 endonuklease-spaltningsstederne i indsatserne, der er anvendt til konstruktion af de forskellige rekombinante DNA-molekyler 9 DK 176220 B1 beskrevet i eksempel 16. Der er endvidere vist den omtrentlige placering af det tilsvarende pre-huTFh-protein med dets lederpeptid (prikket felt) og transmembran-forankrings-domæne (skraveret felt) vist intakte.Also, the approximate location of the restriction endonuclease cleavage sites in the inserts used to construct the various recombinant DNA molecules described in Example 16. is also shown. huTFh protein with its leader peptide (dotted field) and transmembrane anchoring domain (shaded field) shown intact.

5 Fig. 12 er en graf, der illustrerer dosis-reaktions- -kurven for inhibering af huTF-initieret koagulation ved hjælp af ikke-phospholipiderede (ikke-lipiderede) polypep-tidanaloge af huTFh. Den procentiske inhibering (%) for koagulering ved forskellige molære koncentrationer af ikke-10 lipiderede polypeptider måles som beskrevet i eksempel 12.FIG. 12 is a graph illustrating the dose-response curve for inhibition of huTF-initiated coagulation by non-phospholipidated (non-lipidated) polypeptide analogs of huTFh. The percent inhibition (%) of coagulation at various molar concentrations of non-lipid polypeptides is measured as described in Example 12.

De undersøgte polypeptider omfatter p24-35 (åben trekant), p26-49 (åben cirkel), pl52-169 (åben firkant) og peptiderne p40-71, p72-104, p94-123 og pl61-189, som alle i det væsentlige ikke giver nogen inhibering og i fælleskab er betegnet 15 ved en udfyldt cirkel.The polypeptides examined include p24-35 (open triangle), p26-49 (open circle), pl52-169 (open square) and peptides p40-71, p72-104, p94-123 and pl61-189, all of which are essentially does not provide any inhibition and is jointly designated 15 by a filled circle.

Fig. 13 er en graf, der illustrerer de kinetiske forhold for inhibering af koagulering ved hjælp af et TF8--5G9-antistofpræparat. Den procentiske inhibering (%) af koagulering er afsat over forskellige antistof-immunoreak-20 tionstider målt som beskrevet i eksempel 18.FIG. Figure 13 is a graph illustrating the kinetic conditions of inhibition of coagulation by a TF8 - 5G9 antibody preparation. The percent inhibition (%) of coagulation is plotted over various antibody immunoreaction times measured as described in Example 18.

Fig. 14 er en graf, der illustrerer dosis-reaktionen for inhibering af huTF-initieret koagulering ved hjælp af anti-huTF-antistoffer. Den procentiske inhibering (%) af koagulering med forskellige koncentrationer af det mono-25 klonale anti-huTF-antistof TF8-5G9 måles som beskrevet i eksempel 19.FIG. 14 is a graph illustrating the dose response for inhibition of huTF-initiated coagulation by anti-huTF antibodies. The percent inhibition (%) of coagulation with different concentrations of the monoclonal anti-huTF antibody TF8-5G9 is measured as described in Example 19.

Fig. 15 er en graf, der illustrerer dosis-reaktionskurven for inhibering af huTF-initieret koagulering med anti-huTF-antistoffer, hvor kilden til huTF er et humant 30 cellelysat af fibroblast-cellelinien GM1381. Den procentiske inhibering af koagulering ved forskellige koncentrationer af det monoklonale anti-huTF-antistof TF8-5G9 måles som beskrevet i eksempel 19. Åbne cirkler betegner TF8-5G9-an-tistof, og udfyldte cirkler betegner et irrelevant antistof.FIG. 15 is a graph illustrating the dose-response curve for inhibiting huTF-initiated coagulation with anti-huTF antibodies, the source of huTF being a human cell lysate of the fibroblast cell line GM1381. The percent inhibition of coagulation at various concentrations of the monoclonal anti-huTF antibody TF8-5G9 is measured as described in Example 19. Open circles represent TF8-5G9 antibody and filled circles represent an irrelevant antibody.

35 Fig. 16 illustrerer inhibering af den pro-koagulerende aktivitet af renset humant hjerne-TF med monoklonalt an- 10 DK 176220 B1 ti-TF-antistof TF8-5G9. Koaguleringsaktiviteten af renset humant hjerne-TF rekonstitueret i phospholipid-vesikler bestemmes efter forinkubering i 30 minutter ved 37°C med forskellige koncentrationer af renset IgG. Cirklerne betegner 5 anti-TF-antistof TF8-5G9, og trekanter betegner et irrelevant kontrol-antistof PAblOO. Resultaterne er anført som procen-tisk inhibering i forhold til den aktivitet, der observeres i fraværelse af tilsat antistof.FIG. 16 illustrates inhibition of the procoagulant activity of purified human brain TF by monoclonal antibody TF8-5G9 antibody. The coagulation activity of purified human brain TF reconstituted in phospholipid vesicles is determined after pre-incubation for 30 minutes at 37 ° C with various concentrations of purified IgG. The circles denote 5 anti-TF antibody TF8-5G9, and triangles denote an irrelevant control antibody PAblOO. The results are given as percent inhibition relative to the activity observed in the absence of added antibody.

Fig 17 illustrerer inhibering af faktor VII-binding 10 til og faktor Xa-dannelse af dyrkede J82-blærecarcinom-celler behandlet med rensede monoklonale anti-TF-antistoffer. Værdierne for inhibering af hastigheden af faktor Xa-dannelse er vist ved trekanter, og værdierne for inhibering af faktor VII-binding er vist ved cirkler. Resultaterne er anført som 15 procentisk inhibering i forhold til værdierne, der opnås for celler inkuberet uden tilsat antistof. Panel A viser effekten af antistof TF9-2C4, og panel B viser effekten af antistof TF9-5B7.Fig. 17 illustrates inhibition of factor VII binding to and factor Xa formation of cultured J82 bladder carcinoma cells treated with purified anti-TF monoclonal antibodies. The values for inhibiting the rate of factor Xa formation are shown by triangles, and the values for inhibiting factor VII binding are shown by circles. The results are stated as 15 percent inhibition relative to the values obtained for cells incubated without added antibody. Panel A shows the effect of antibody TF9-2C4, and panel B shows the effect of antibody TF9-5B7.

Fig. 18 illustrerer en Western-blot-analyse af im-20 munoaffinitetsisoleret huTF som beskrevet i eksempel 25.FIG. Figure 18 illustrates a Western blot analysis of immunoaffinity isolated huTF as described in Example 25.

15%'s polyacrylamidgeler lades på følgende måde: bane 1 indeholder molekylvægtsstandarder med tilsyneladende molekylvægte anført til venstre for panel A i kilodalton (k) , bane 2 indeholder 1 μg renset humant hæmoglobin reduceret 25 før elektroforese, bane 3 indeholder 0,5 μg isoleret huTF reduceret før elektroforese, og bane 4 indeholder 0,5 μg ikke-reduceret og isoleret huTF. Efter SDS-PAGE overføres de fremkomne proteinbånd elektroforetisk til nitrocellulose.15% polyacrylamide gels are charged as follows: lane 1 contains molecular weight standards with apparent molecular weights listed to the left of panel A in kilodalton (k), lane 2 contains 1 μg of purified human hemoglobin reduced before electrophoresis, lane 3 contains 0.5 μg isolated huTF reduced before electrophoresis, and lane 4 contains 0.5 μg of non-reduced and isolated huTF. Following SDS-PAGE, the resulting protein bands are electrophoretically transferred to nitrocellulose.

De således dannede Western-blots immunoreageres med 0,2 30 μg/ml affinitetsrenset kanin-anti-huTF-IgG {panel A) , 1 μg/ml kanin-antihæmoglobin-IgG (panel B) eller 1 μg/ml ikke-immun-kanin-IgG (panel C) . De tilsyneladende molekylvægte af immunofarvede bånd er anført i kDa til højre.The Western blots thus formed are immunoreacted with 0.2 µg / ml affinity purified rabbit anti-huTF IgG (panel A), 1 µg / ml rabbit anti-hemoglobin IgG (panel B) or 1 µg / ml non-immune rabbit IgG (panel C). The apparent molecular weights of immunostained bands are listed in kDa on the right.

35 11 DK 176220 B135 11 DK 176220 B1

Detaljeret beskrivelse af opfindelsen.Detailed description of the invention.

A. Definitioner.A. Definitions.

Aminosyrer: Alle de i den foreliggende beskrivelse anførte aminosyrerester har den naturlige L-konfiguration.Amino Acids: All of the amino acid residues listed in the present description have the natural L configuration.

5 I overensstemmelse med standard-polypeptidnomenklatur J.In accordance with standard polypeptide nomenclature J.

Biol. Chem., 243:3557-59, (1969), anvendes der forkortelser for aminosyreresterne som vist i den følgende tabel:Biol. Chem., 243: 3557-59, (1969), abbreviations for the amino acid residues are used as shown in the following table:

Symbol Aminosyre 10 - - 1-Bogstav 3-bogstav Y Tyr L-tyrosin G Gly glycin 15 F Phe L-phenylalanin M Met L-methionin A Ala L-alanin S Ser L-serin L Ile L-isoleucin 20 L Leu L-leucin T Thr L-threonin V Val L-valin P Pro L-prolin K Lys L-lysin 25 H His L-histidin Q Gin L-glutamin E Glu L-slutaminsyre W Trp L-tryptophan R Arg L-arginin 30 D Asp L-asparaginsyre N Asn L-asparagin C Cys L-cysteinSymbol Amino acid 10 - - 1-letter 3-letter Y Tyr L-tyrosine G Gly glycine 15 F Phe L-phenylalanine M Met L-methionine A Ala L-alanine S Ser L-serine L Ile L-isoleucine 20 L Leu L- leucine T Thr L-threonine V Val L-valine P Pro L-proline K Lys L-lysine 25 H His L-histidine Q Gin L-glutamine E Glu L-slutamic acid W Trp L-tryptophan R Arg L-arginine 30 D Asp L-aspartic acid N Asn L-aspartic C Cys L-cysteine

Det skal bemærkes, at alle aminosyresekvenser er vist i den 35 foreliggende beskrivelse ved formler, hvis orientering fra venstre til højre er i den konventionelle retning fra ami-noterminussen til carboxyterminussen. Endvidere skal det 12 DK 176220 B1 bemærkes, at en streg ved starten eller slutningen af en aminosyresekvens viser en binding til en gruppe, såsom H og OH (hydrogen og hydroxyl) ved henholdsvis amino- og carb-oxyterminussen eller en yderligere sekvens på en eller flere 5 aminosyrerester op til i alt ca. 50 rester i polypeptidkæden.It should be noted that all amino acid sequences are shown in the present specification by formulas whose orientation from left to right is in the conventional direction from the amine terminus to the carboxy terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid sequence shows a bond to a group such as H and OH (hydrogen and hydroxyl) at the amino and carb oxy terminus, respectively, or a further sequence at one or several 5 amino acid residues up to a total of approx. 50 residues in the polypeptide chain.

Polypeptid og peptid: Polypeptid og peptid er udtryk, der anvendes ensbetydende i den foreliggende beskrivelse til at betegne en lineær række af ikke mere end ca, 50 aminosyrerester forbundet med hinanden via peptidbindinger 10 mellem α-aminogruppen og carboxygruppen af tilstødende rester .Polypeptide and Peptide: Polypeptide and peptide are terms used in the present specification to denote a linear sequence of no more than about 50 amino acid residues linked to each other via peptide bonds 10 between the α-amino group and the carboxy group of adjacent residues.

Protein: Protein er et udtryk, der anvendes i den foreliggende beskrivelse til at betegne en lineær række af over 50 aminosyrerester, der er forbundet med hinanden som 15 i et polypeptid.Protein: Protein is a term used in the present specification to denote a linear sequence of over 50 amino acid residues linked to each other as 15 in a polypeptide.

Nukleotid: En monomer enhed af DNA eller RNA bestående af en sukkerdel (pentose), et phosphat og en nitrogenholdig heterocyclisk base. Basen er bundet til sukkerdelen via det glycosidiske carbonatom (11-carbonatomet af pentosen), og 20 denne kombination af base og sukkerart er et nukleosid. Når nukleosidet indeholder en phosphatgruppe bundet til 3'-eller 5'-positionen af pentosen, betegnes det et nukleotid.Nucleotide: A monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate and a nitrogen-containing heterocyclic base. The base is bound to the sugar moiety via the glycosidic carbon atom (the 11-carbon atom of the pentose), and this combination of base and sugar is a nucleoside. When the nucleoside contains a phosphate group attached to the 3 'or 5' position of the pentose, it is referred to as a nucleotide.

Basebar (bp) : En kombination af adenin (A) med thymin (T) eller af cytosin (C) med guanin (G) i et dobbeltstrenget 25 DNA-molekyle.Basebar (bp): A combination of adenine (A) with thymine (T) or of cytosine (C) with guanine (G) in a double-stranded DNA molecule.

B. DNA-segmenter.B. DNA segments.

I levende organismer er aminosyresekvensen af et protein eller polypeptid direkte relateret via den genetiske kode til deoxyribonukleinsyresekvensen (DNA-sekvensen) af 30 det strukturelle gen, der koder for proteinet. Et strukturelt gen kan således defineres som den aminosyresekvens, dvs. protein eller polypeptid, som det koder for.In living organisms, the amino acid sequence of a protein or polypeptide is directly related via the genetic code to the deoxyribonucleic acid sequence (DNA sequence) of the structural gene encoding the protein. Thus, a structural gene can be defined as the amino acid sequence, i.e. protein or polypeptide that it encodes.

Et vigtigt og velkendt træk ved den genetiske kode er dens redundans. Dette betyder, for de fleste af aminosy-35 rerne, der anvendes til at danne proteiner, at mere end én kodende nukleotidtriplet (kodon) kan kode for en bestemt 13 DK 176220 B1 aminosyrerest. Derfor kan et antal forskellige nukleotidse-kvenser kode for en bestemt aminosyresekvens. Sådanne nu-kleotidsekvenser betragtes som funktionelt ækvivalente, da de kan resultere i produktion af den samme aminosyresekvens 5 i alle organismer. Lejlighedsvis kan en methyleret variant af et purin eller pyrimidin være inkorporeret i en given nukleotidsekvens. Sådanne methyleringer påvirker imidlertid ikke kodningsforholdene på nogen måde.An important and well-known feature of the genetic code is its redundancy. This means, for most of the amino acids used to form proteins, that more than one coding nucleotide triplet (codon) can encode a particular amino acid residue. Therefore, a number of different nucleotide sequences can encode a particular amino acid sequence. Such nu-cleotide sequences are considered functionally equivalent as they can result in the production of the same amino acid sequence 5 in all organisms. Occasionally, a methylated variant of a purine or pyrimidine may be incorporated into a given nucleotide sequence. However, such methylations do not affect the coding conditions in any way.

DNA-segmenterne af den tidligere nævnte art er karak-10 teriseret ved at indeholde en DNA-sekvens, der koder for et protein med tung kæde af human vævsfaktor (huTFh). Fortrinsvis indeholder DNA-segmentet en DNA-sekvens, der koder for et forstadieprotein med tung kæde af human vævsfaktor (pre-huTFh). Dette betyder, at DNA-segmenterne er karakteriseret 15 ved tilstedeværelsen af et huTFh-strukturgen eller, mere foretrukket, et pre-huTFh-strukturgen. Yderligere foretrukne er DNA-segmenter, der indeholder en DNA-sekvens, der danner et strukturelt gen, som koder for et opløseligt huTFh- eller opløseligt pre-huTFh-protein. Genet er fortrinsvis til stede 20 som en ubrudt lineær række af codoner, hvor hvert codon koder for en aminosyrerest, som findes i huTFh- eller pre-huTFh-proteinet, dvs. et gen, som er frit for introner.The DNA segments of the aforementioned kind are characterized by containing a DNA sequence encoding a heavy chain human tissue factor (huTFh) protein. Preferably, the DNA segment contains a DNA sequence encoding a human tissue factor precursor heavy chain (pre-huTFh). This means that the DNA segments are characterized by the presence of a huTFh structural gene or, more preferably, a pre-huTFh structural gene. Further preferred are DNA segments that contain a DNA sequence forming a structural gene encoding a soluble huTFh or soluble pre-huTFh protein. The gene is preferably present as an unbroken linear array of codons, each codon encoding an amino acid residue found in the huTFh or pre-huTFh protein, i.e. a gene that is free of introns.

Homologe DNA- og RNA-sekvenser, der koder for de ovennævnte huTFh- og pre-huTFh-proteiner, inklusive deres 25 opløselige former, kommer også på tale, som ovenfor diskuteret .Homologous DNA and RNA sequences encoding the aforementioned huTFh and pre-huTFh proteins, including their 25 soluble forms, are also discussed, as discussed above.

DNA-segmenter, der koder for huTFh- og pre-huTFh-proteiner, kan let syntetiseres ved kemiske metoder, f.eks. ved phosphotriestermetoden ifølge Matteucci et al., J. Am.DNA segments encoding huTFh and pre-huTFh proteins can be readily synthesized by chemical methods, e.g. by the phosphotriester method of Matteucci et al., J. Am.

30 Chem. Soc., 103:3185 (1981). Ved kemisk syntese af den ko dende sekvens kan enhver ønsket modifikation naturligvis foretages ved blot at indføje de rette baser i stedet for de, der koder for den oprindelige aminosyresekvens. Imidlertid foretrækkes DNA-molekyler, der indeholder sekvenser, 35 som er nøjagtigt homologe med de, der er vist i fig. 2.Chem. Soc., 103: 3185 (1981). Of course, by chemical synthesis of the coding sequence, any desired modification can be made by simply inserting the appropriate bases instead of those encoding the original amino acid sequence. However, DNA molecules containing sequences that are exactly homologous to those shown in FIG. 2nd

14 DK 176220 B114 DK 176220 B1

Endvidere kan DNA-segmenter, der i det væsentlige består af strukturelle gener, som koder for huTFh- og pre--huTFh-proteinerne, fås ud fra rekombinante DNA-molekyler indeholdende disse gener. F.eks. indeholder de rekombinante 5 DNA-molekyler af plasmidtypen pCTF64, pCTF314 og pCTF403 hver især DNA-sekvenser, der koder for forskellige dele af huTFh- og pre-huTFh-proteinerne og sammen har hele den DNA- - sekvens, der er nødvendig for ekspression af hvert af proteinerne. Kulturer af Escherichia coli (E. coli) transforme- 10 ret med pCTF64, pCTF314 eller pCTF403 er deponeret i overensstemmelse med Budapest-traktaten hos American Type Culture Collection (ATCC) den 27. marts 1987 og har fået deponeringsnumrene 67.370, 67.368 henholdsvis 67.369.Furthermore, DNA segments consisting essentially of structural genes encoding the huTFh and pre-huTFh proteins can be obtained from recombinant DNA molecules containing these genes. Eg. the recombinant 5 DNA molecules of the plasmid type pCTF64, pCTF314 and pCTF403 each contain DNA sequences encoding different portions of the huTFh and pre-huTFh proteins and together have all the DNA sequence necessary for expression of each of the proteins. Cultures of Escherichia coli (E. coli) transformed with pCTF64, pCTF314 or pCTF403 have been deposited in accordance with the Budapest Treaty of the American Type Culture Collection (ATCC) on March 27, 1987, and have been assigned the deposit numbers 67,370, 67,368 and 67,369, respectively.

Et DNA-segment, der indeholder en DNA-sekvens, som 15 koder for huTFh eller pre-huTFh, kan fremstilles ved operativ binding (ligering) af passende restriktionsfragmenter fra hver af de ovenfor nævnte deponerede plasmider ved anvendelse af velkendte metoder. DNA-molekylerne produceret på denne måde har typisk klæbende ender, dvs. udragende enkeltstren-2 0 gede dele, som strækker sig ud over den dobbeltstrengede del af molekylet. Relateret til den foreliggende opfindelse er også ribonukleinsyreækvivalenter (RNA-ækvivalenter) af de ovenfor beskrevne DNA-segmenter.A DNA segment containing a DNA sequence encoding huTFh or pre-huTFh can be prepared by operative binding (ligation) of appropriate restriction fragments from each of the aforementioned deposited plasmids using well known methods. The DNA molecules produced in this way typically have adhesive ends, ie. protruding single strand-20 goat portions extending beyond the double stranded portion of the molecule. Related to the present invention are also ribonucleic acid equivalents (RNA equivalents) of the DNA segments described above.

C. Rekombinante DNA-molekvler.C. Recombinant DNA Molecules.

25 De rekombinante DNA-molekyler kan fremstilles ved operativ binding af en vektor til DNA-segment ifølge den foreliggende opfindelse.The recombinant DNA molecules can be prepared by operatively binding a vector to DNA segment of the present invention.

Som anvendt i den foreliggende beskrivelse refererer udtrykket "vektor" til et DNA-molekyle, der er i stand til 30 autonom replikation i en celle, og hvortil et andet DNA- - segment kan bindes operativt, således at der fås replikation af det tilføjede segment. Vektorer, der er i stand til at styre ekspressionen af huTFh- og pre-huTFh-gener, betegnes i den foreliggende beskrivelse "ekspressionsvektorer". Et 35 rekombinant DNA-molekyle (rDNA) er således et hybridt DNA- 15 DK 176220 B1 -molekyle, der omfatter mindst to nukleotidsekvenser, som ikke normalt findes sammen i naturen.As used in the present specification, the term "vector" refers to a DNA molecule capable of autonomous replication in a cell and to which another DNA segment can be operatively linked so that replication of the added segment is obtained. . Vectors capable of directing the expression of huTFh and pre-huTFh genes are referred to herein as "expression vectors". Thus, a recombinant DNA molecule (rDNA) is a hybrid DNA molecule comprising at least two nucleotide sequences not normally found together in nature.

Valget af vektor, hvortil et DNA-segment bindes operativt, afhænger direkte, således som det er velkendt, at de 5 ønskede funktionelle egenskaber, f.eks. proteinekspression, og værtscellen, der skal transformeres, idet disse er iboende begrænsninger ved konstruktion af rekombinante DNA-molekyler. Imidlertid er en vektor i det mindste i stand til at styre replikationen og fortrinsvis også ekspressionen af huTFh-10 eller pre-huTFh-strukturgenerne inkluderet i DNA-segmenter, hvortil den er operativt bundet.The choice of vector to which a DNA segment is operatively linked depends directly, as is well known, that the desired functional properties, e.g. protein expression, and the host cell to be transformed, as these are inherent limitations in constructing recombinant DNA molecules. However, at least one vector is capable of directing the replication and preferably also the expression of the huTFh-10 or pre-huTFh structural genes included in DNA segments to which it is operatively linked.

En vektor omfatter fortrinsvis et prokaryotisk repli-kon, dvs. en DNA-sekvens, der har evnen til direkte autonom replikation og ekstrakromosomal bibeholdelse af det rekom-15 binante DNA-molekyle i en prokaryotisk værtscelle, såsom en bakterie-værtscelle, der er transformeret dermed. Sådanne replikoner er velkendte. En sådan vektor, der omfatter et prokaryotisk replikon, omfatter også et gen, hvis ekspression giver lægemiddelresistens til en bakterievært, der er trans-20 formeret dermed. Typiske bakterielle lægemiddelresistens--gener, er sådanne der giver resistens mod ampicillin eller tetracyclin.A vector preferably comprises a prokaryotic replicon, i.e. a DNA sequence having the capability of direct autonomous replication and extrachromosomal retention of the recombinant DNA molecule in a prokaryotic host cell, such as a bacterial host cell transformed therewith. Such replicons are well known. Such a vector comprising a prokaryotic replicon also includes a gene whose expression confers drug resistance to a bacterial host transfected therewith. Typical bacterial drug resistance genes are those that confer resistance to ampicillin or tetracycline.

De vektorer, der omfatter et prokaryotisk replikon, kan også omfatte en prokaryotisk promotor, der er i stand 25 til at styre ekspressionen (transskriptionen og translationen) af huTFh- eller pre-huTFh-generne i en bakterie-værtscelle, såsom E. coli, der er transformeret dermed. En promotor er et ekspressionsregulerende element dannet af en DNA-sekvens, der muliggør binding af RNA-polymerase og til-30 lader transskriptionen at forekomme. Promotorsekvenser, som er forenelige med bakterielle værter, tilvejebringes typisk i plasmidvektorer indeholdende bekvemme restriktionssteder til indføjelse af et DNA-segment ifølge den foreliggende opfindelse. Typiske for sådanne vektorplasmider er pUC8, 35 pUC9, pBR322 og pBR329, der kan fås fra Biorad Laboratories, 16 DK 176220 B1 {Richmond, CA) og pPL og pKK223, der kan fås fra Pharmacia, Piscataway, N.J.The vectors comprising a prokaryotic replicon may also include a prokaryotic promoter capable of directing the expression (transcription and translation) of the huTFh or pre-huTFh genes in a bacterial host cell such as E. coli. that is transformed thereby. A promoter is an expression regulating element formed by a DNA sequence which allows binding of RNA polymerase and allows transcription to occur. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention. Typical of such vector plasmids are pUC8, 35 pUC9, pBR322 and pBR329 obtainable from Biorad Laboratories, 16 DK 176220 B1 (Richmond, CA) and pPL and pKK223 obtainable from Pharmacia, Piscataway, N.J.

Ekspressionsvektorer, der er forenelige med eukaryo-tiske celler, fortrinsvis sådanne, der er forenelige med 5 hvirveldyr-celler, kan også anvendes til dannelse af rekom-binante DNA-molekyler. Eukaryotiske celleekspressionsvektorer er velkendte og kan fås fra flere kommercielle kilder. Typisk fås sådanne vektorer med bekvemme restriktionssteder til indføjelse af det ønskede DNA-segment. Typiske for sådanne 10 vektorer er pSVL og pKSV-10 (Pharmacia), pBPV-l/pML2d (International Biotechnologies, Inc.), og pTDTl (ATCC31255).Expression vectors compatible with eukaryotic cells, preferably those compatible with 5 vertebrate cells, can also be used to form recombinant DNA molecules. Eukaryotic cell expression vectors are well known and can be obtained from several commercial sources. Typically, such vectors are provided with convenient restriction sites for insertion of the desired DNA segment. Typical of such 10 vectors are pSVL and pKSV-10 (Pharmacia), pBPV-1 / pML2d (International Biotechnologies, Inc.), and pTDT1 (ATCC31255).

Ekspressionsvektorerne af eukaryotiske celler, der anvendes til konstruktion de rekombinante DNA-molekyler, indeholder fortrinsvis en selektionsmarkør, der er effektiv 15 i en eukaryotisk celle, fortrinsvis en lægemiddelresistens-- selektionsmarkør. En foretrukken lægemiddelresistens-markør er genet, hvis ekspression resulterer i neomycinresistens, dvs. neomycin-phosphortransferase-genet (neo), jf. Southern et al., J. Mol. Appl. Genet., 1:327-341 (1982).The expression vectors of eukaryotic cells used to construct the recombinant DNA molecules preferably contain a selection marker that is effective in a eukaryotic cell, preferably a drug resistance selection marker. A preferred drug resistance marker is the gene whose expression results in neomycin resistance, ie. the neomycin phosphorus transferase gene (neo), cf. Southern et al., J. Mol. Appl. Genet., 1: 327-341 (1982).

20 Anvendelse af retrovirus-ekspressionsvektorer til dannelse af rDNA'er kommer også på tale. Som anvendt i den foreliggende beskrivelse refererer udtrykket "retrovirus-ekspressionsvektor" til et DNA-molekyle, der indeholder en promotorsekvens, som hidrører fra den lange terminale gen-25 tagelsesregion (LTR) af et retrovirus-genom.The use of retrovirus expression vectors to generate rDNAs is also contemplated. As used in the present specification, the term "retrovirus expression vector" refers to a DNA molecule containing a promoter sequence derived from the long terminal repeat region (LTR) of a retrovirus genome.

Ekspressionsvektoren er typisk en retrovirus-ekspressionsvektor, som fortrinsvis er replikations-inkompetent i eukaryotiske celler. Konstruktionen og anvendelsen af retro-virus-vektorer er beskrevet af Sorge et al.. Mol. Cell.The expression vector is typically a retrovirus expression vector which is preferably replication incompetent in eukaryotic cells. The construction and use of retroviral vectors is described by Sorge et al., Mol. Cell.

30 Biol., 4:1730-37 (1984).Biol., 4: 1730-37 (1984).

Forskellige metoder er blevet udviklet til operativ binding af DNA til vektorer via komplementære klæbende ender.Various methods have been developed for operative binding of DNA to vectors via complementary adhesive ends.

F.eks. kan komplementære homopolymere områder føjes til DNA-segmentet, der skal indføjes, og til vektor-DNA. Vektoren 35 og DNA-segmentet sammenføjes derefter ved hydrogenbinding 17 DK 176220 B1 mellem de komplementære homopolymere "haler" til dannelse af rekombinante DNA-molekyler.Eg. For example, complementary homopolymeric regions can be added to the DNA segment to be inserted and to vector DNA. The vector 35 and the DNA segment are then joined by hydrogen bonding between the complementary homopolymeric "tails" to form recombinant DNA molecules.

Syntetiske linkere, der indeholder et eller flere restriktionssteder, udgør en alternativ metode til indføjelse 5 af DNA-segmentet i vektorer. DNA-segmentet, dannet ved en-donuklease-restriktionsnedbrydning som tidligere beskrevet, behandles med bakteriofag T4-DNA-polymerase eller E. coli--DNA-polymerase I, som er enzymer, der fjerner fremspringende enkeltstrengede 3'-ender på grund af deres 31-51-eksonuk-10 leolytiske aktiviteter og udfylder tilbageliggende 31-ender på grund af deres polymeriserende aktiviteter. Kombinationen af disse aktiviteter danner derfor stumpendede DNA-segmenter.Synthetic linkers containing one or more restriction sites constitute an alternative method for inserting the DNA segment into vectors. The DNA segment formed by endonuclease restriction degradation as previously described is treated with bacteriophage T4 DNA polymerase or E. coli - DNA polymerase I, which are enzymes that remove protruding single stranded 3 'ends because of their 31-51 exonucleolytic activities and fills receding 31 ends due to their polymerizing activities. The combination of these activities therefore forms blunt-ended DNA segments.

De stumpendede segmenter inkuberes derefter med et stort molært overskud af linkermolekyler i nærværelse af et enzym, 15 som er i stand til at katalysere ligeringen af stumpendede DNA-molekyler, såsom bakteriofag T4-DNA-ligase. Produkterne af reaktionen er således DNA-segmenter, der bærer polymere linkersekvenser ved deres ender. Disse DNA-segmenter spaltes derefter med et passende restriktionsenzym og ligeres til 20 en ekspressionsvektor, der er blevet spaltet med et enzym, der producerer terminusser, som er forenelige med DNA-segmentets terminusser.The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme capable of catalyzing the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the products of the reaction are DNA segments bearing polymeric linker sequences at their ends. These DNA segments are then cleaved with an appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme producing terminuses compatible with the DNA segment terminals.

Syntetiske linkere, der indeholder forskellige re-striktionsendonukleasesteder, er kommercielt tilgængelige 25 fra et antal kilder, herunder International Biotechnologies,Synthetic linkers containing various restriction endonuclease sites are commercially available from a number of sources, including International Biotechnologies,

Inc., New Haven, CN.Inc., New Haven, CN.

D. Transformerede celler og kulturer.D. Transformed cells and cultures.

En værtscelle transformeret med et rekombinant DNA-molekyle, fortrinsvis et rDNA, som er i stand til at ekspri-30 mere en opløselig form af huTFh eller pre-huTFh, kan enten være prokaryotisk eller eukaryotisk. Bakterieceller er foretrukne prokaryotiske værtsceller og er typisk en stamme af E. coli, f.eks. E. coli, stamme DH5, der kan fås fra Bethesda Research Laboratories, Inc., Bethesda, MD. Foretrukne eu-35 karyotiske værtsceller omfatter gærceller og pattedyrsceller, fortrinsvis hvirveldyr-celler, såsom celler fra en fibro- 18 DK 176220 B1 blast-cellelinie fra mus, rotte, abe eller menneske. Foretrukne eukaryotiske værtsceller omfatter ovarieceller af kinesisk hamster (CHO-celler) , der kan fås fra ATCC som CCL61, og NHI Swiss musefosterceller NHI/3T3, der kan fås 5 fra ATCC som CRL1658. Transformation af passende celleværter med et rekombinant DNA-molekyle gennemføres ved velkendte metoder, som typisk afhænger af typen af den anvendte vektor.A host cell transformed with a recombinant DNA molecule, preferably an rDNA capable of expressing a soluble form of huTFh or pre-huTFh, may be either prokaryotic or eukaryotic. Bacterial cells are preferred prokaryotic host cells and are typically a strain of E. coli, e.g. E. coli, strain DH5, available from Bethesda Research Laboratories, Inc., Bethesda, MD. Preferred eukaryotic host cells include yeast cells and mammalian cells, preferably vertebrate cells, such as cells from a mouse, rat, monkey or human fibroblast cell line. Preferred eukaryotic host cells include Chinese hamster ovary cells (CHO cells) obtainable from ATCC as CCL61, and NHI Swiss mouse fetal cells NHI / 3T3 obtainable from ATCC as CRL1658. Transformation of appropriate cell hosts with a recombinant DNA molecule is accomplished by well known methods, which typically depend on the type of vector used.

Med hensyn til transformation af prokaryotiske værtsceller kan der f.eks. henvises til Cohen et al., Proc. Natl. Acad.In terms of transformation of prokaryotic host cells, e.g. see Cohen et al., Proc. Natl. Acad.

10 Sci. USA, 69:2110 (1972}; og Maniatis et al., Molecular10 Sci. USA, 69: 2110 (1972); and Maniatis et al., Molecular

Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory,Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory,

Cold Spring Harbor, NY (1982) . Med hensyn til transformation af hvirveldyr-celler med retrovirus-vektorer indeholdende rDNA kan der f.eks. henvises til Sorge et al., Mol. Cell.Cold Spring Harbor, NY (1982). For transformation of vertebrate cells with retroviral vectors containing rDNA, e.g. see Sorge et al., Mol. Cell.

15 Biol., 4:1730-37 (1984); Graham et al., Virol., 52:456 (1973); og Wigler et al., Proc. Natl. Acad. Sci. USA, 76:1373-76 (1979) .Biol., 4: 1730-37 (1984); Graham et al., Virol., 52: 456 (1973); and Wigler et al., Proc. Natl. Acad. Sci. USA, 76: 1373-76 (1979).

Celler, som er transformeret med held, dvs. celler, der indeholder et rekombinant DNA-molekyle, kan identificeres 20 ved velkendte metoder. F.eks. kan celler, som hidrører fra indføring af rDNA, klones til dannelse af monoklonale kolonier. Celler fra disse kolonier kan høstes, lyseres, og deres DNA-indhold kan undersøges for tilstedeværelsen af rDNA ved anvendelse af en metode som f.eks. beskrevet af 25 Southern, J. Mol. Biol., 98:503 (1975) eller Berent et al., Biotech., 3:208 (1985).Successfully transformed cells, ie Cells containing a recombinant DNA molecule can be identified by well known methods. Eg. For example, cells resulting from introduction of rDNA can be cloned to form monoclonal colonies. Cells from these colonies can be harvested, lysed, and their DNA content examined for the presence of rDNA using a method such as described by Southern, J. Mol. Biol., 98: 503 (1975) or Berent et al., Biotech., 3: 208 (1985).

Foruden direkte bestemmelse af tilstedeværelsen af rDNA kan vellykket transformation bekræftes ved velkendte immunologiske metoder, når rDNA er i stand til at styre 30 ekspressionen af huTFh eller pre-huTFh. F.eks. producerer celler, som er vellykket transformeret med en ekspressionsvektor, proteiner, der udviser huTFh- eller pre-huTFh-anti-genitet. Prøver af celler, som formodes at være transformerede, høstes og undersøges for huTFh eller pre-huTFh ved 35 anvendelse af antistoffer, som er specifikke for disse antigener, såsom de, der produceres af et hybridoma.In addition to direct determination of the presence of rDNA, successful transformation can be confirmed by well-known immunological methods when rDNA is able to control the expression of huTFh or pre-huTFh. Eg. cells that are successfully transformed with an expression vector produce proteins exhibiting huTFh or pre-huTFh antigenicity. Samples of cells that are thought to be transformed are harvested and assayed for huTFh or pre-huTFh using antibodies specific for these antigens, such as those produced by a hybridoma.

19 DK 176220 B1 Næringsmedier, som er anvendelige til dyrkning af transformerede værtsceller, er velkendte og kan fås fra flere kommercielle kilder. I udførelsesformer, hvor værtscellen er en pattedyrscelle, anvendes der fortrinsvis et 5 "serumfrit" medium.19 DK 176220 B1 Nutrient media useful for growing transformed host cells are well known and are obtainable from several commercial sources. In embodiments where the host cell is a mammalian cell, a 5 "serum-free" medium is preferably used.

E. Metoder til fremstilling af huTFh- og pre-huTFh-proteiner.E. Methods for preparing huTFh and pre-huTFh proteins.

Proteiner, som udviser huTFh-antigenitet, er proteiner, som immunoreagerer med antistoffer fremkaldt af 10 naturlig vævsfaktor. Proteiner, som udviser huTFh-antigeni-tet, er anvendelige som antigener og til dannelse af antistoffer .Proteins exhibiting huTFh antigenicity are proteins that immunoreact with antibodies elicited by natural tissue factor. Proteins exhibiting the huTFh antigen are useful as antigens and for antibody formation.

Proteinerne fremstilles ved igangsætning af en kultur omfattende et næringsmedium indeholdende værtsceller, for-15 trinsvis humane celler, der er transformeret med et rekom-binant DNA-molekyle, der er i stand til at eksprimere et huTFh- eller pre-huTFh-protein, fortrinsvis et opløseligt huTFh- eller pre-huTFh-protein. Kulturen bibeholdes i et tilstrækkeligt tidsrum til, at de transformerede celler 20 eksprimerer et huTFh- eller pre-huTFh-protein. Det ekspri-merede protein udvindes derefter fra kulturen. huTFh-pro-teinerne produceret herved udviser fortrinsvis biologisk huTFh-aktivitet, dvs. evne til at binde faktor Vll/VIIa.The proteins are prepared by initiating a culture comprising a nutrient medium containing host cells, preferably human cells transformed with a recombinant DNA molecule capable of expressing a huTFh or pre-huTFh protein, preferably a soluble huTFh or pre-huTFh protein. The culture is maintained for a sufficient period of time for the transformed cells to express a huTFh or pre-huTFh protein. The expressed protein is then recovered from the culture. The huTFh proteins produced thereby preferably exhibit biological huTFh activity, ie. ability to bind factor Vll / VIIa.

Disse metoder omfatter dyrkning af pattedyr-værtsceller 25 transformeret med et rekombinant DNA-molekyle, der er i stand til at eksprimere pre-huTFh-genet i cellerne. Dyrkningen medfører ekspression af pre-huTFh-proteinet og efterfølgende intracellulær post-translations-modifikation af pre-huTFh til dannelse af et biologisk aktivt huTFh-protein.These methods include culturing mammalian host cells transformed with a recombinant DNA molecule capable of expressing the pre-huTFh gene in the cells. Culture involves expression of the pre-huTFh protein and subsequent intracellular post-translational modification of pre-huTFh to form a biologically active huTFh protein.

30 Metoder til udvinding af et eksprimeret protein fra en kultur er velkendte og omfatter fraktionering af den proteinholdige del af kulturen ved anvendelse af velkendte biokemiske metoder. F.eks. kan gelfiltrering, gelchromato-grafi, ultrafiltrering, elektroforese, ionbytning, affini-35 tetschromatografi og lignende, som er kendte til proteinfraktioneringer, anvendes til isolering af de eksprimerede 20 DK 176220 B1 proteiner, som findes i kulturen. Desuden kan immunokemiske metoder, såsom immunoaffinitet, immunoadsorption og lignende, gennemføres ved anvendelse af velkendte metoder.Methods for recovering an expressed protein from a culture are well known and include fractionation of the proteinaceous portion of the culture using well-known biochemical methods. Eg. For example, gel filtration, gel chromatography, ultrafiltration, electrophoresis, ion exchange, affinity chromatography and the like, known for protein fractionations, can be used to isolate the expressed proteins found in the culture. In addition, immunochemical methods such as immunoaffinity, immunoadsorption and the like can be carried out using well known methods.

F. huTFh- og pre-huTFh-proteinpræparater og 5 -ekspressionsprodukter.F. huTFh and pre-huTFh protein preparations and 5 expression products.

huTFh- og pre-huTFh-ekspressionsprodukter af rDNA'er har fortrinsvis en aminosyresekvens svarende til resterne 1 til 263 henholdsvis -32 til 263 som vist i fig. 1. Fortrinsvis har det eksprimerede protein en længde på mindst 90%, 10 mere foretrukket mindst 95% af længden af pre-huTFh- og huTFh-aminosyresekvensen vist i fig. 1, og er fortrinsvis en opløselig form af huTFh og pre-huTFh. Udtrykket "opløseligt" som anvendt i den foreliggende beskrivelse refererer til huTFh- og pre-huTFh-molekyler, som er karakteriseret 15 ved i det væsentlige at bestå af det ekstracellulære domæne af native huTFh- og pre-huTFh-molekyler, dvs. den del af huTFh- og pre-huTFh-molekylerne, der er aminoterminal til rest nr. 220 som vist i fig. 1. Opløseligt huTFh og opløseligt pre-huTFh indeholder derfor ikke nogen væsentlig del 20 af transmembran-forankringsregionen, som dannes i de native molekyler (resterne 220 til 242 som vist i fig. 1) . Det skal bemærkes, at udtrykkene "huTFh" og "pre-huTFh" som anvendt i den foreliggende beskrivelse omfatter de opløselige former af disse proteiner, med mindre andet er anført.The huTFh and pre-huTFh expression products of rDNAs preferably have an amino acid sequence corresponding to residues 1 to 263 and -32 to 263, respectively, as shown in FIG. 1. Preferably, the expressed protein has a length of at least 90%, more preferably at least 95% of the length of the pre-huTFh and huTFh amino acid sequence shown in FIG. 1, and is preferably a soluble form of huTFh and pre-huTFh. The term "soluble" as used in the present specification refers to huTFh and pre-huTFh molecules, which are characterized essentially by the extracellular domain of native huTFh and pre-huTFh molecules, i.e. the portion of the huTFh and pre-huTFh molecules that are amino terminal to residue # 220 as shown in FIG. Therefore, soluble huTFh and soluble pre-huTFh do not contain any substantial portion 20 of the transmembrane anchoring region formed in the native molecules (residues 220 to 242 as shown in Figure 1). It should be noted that the terms "huTFh" and "pre-huTFh" as used herein describe the soluble forms of these proteins, unless otherwise stated.

25 Da opløseligt huTFh og opløseligt pre-huTFh ikke indeholder en hydrofob transmembran-forankringsregion, ag-gregerer de ikke væsentligt i fysiologisk acceptable vandige opløsninger. Derfor er opløseligt huTFh og opløseligt pre-huTFh yderligere karakteriseret ved deres evne til at danne 30 en vandig opløsning ved anvendelse af et fysiologisk acceptabelt fortyndingsmiddel i en proteinkoncentration på ca.Since soluble huTFh and soluble pre-huTFh do not contain a hydrophobic transmembrane anchoring region, they do not aggregate substantially in physiologically acceptable aqueous solutions. Therefore, soluble huTFh and soluble pre-huTFh are further characterized by their ability to form an aqueous solution using a physiologically acceptable diluent at a protein concentration of ca.

0,1 pg/ml til ca. 100 ng/ml, hvor mindst ca. 70 vægt% fortrinsvis ca. 80 vægt% og mere foretrukket ca. 90 vægt% af huTFh- eller pre-huTFh-proteinet, som er til stede, er i 35 ikke-aggregeret (monomer) form. Metoder til bestemmelse af graden af aggregering i en proteinopløsning er velkendte og 21 DK 176220 B1 omfatter størrelsesfraktionering ved udelukkelses-søjlechro-matografi.0.1 µg / ml to approx. 100 ng / ml, with at least approx. 70% by weight, preferably approx. 80% by weight and more preferably approx. 90% by weight of the huTFh or pre-huTFh protein present is in non-aggregated (monomeric) form. Methods for determining the degree of aggregation in a protein solution are well known and include size fractionation by exclusion column chromatography.

huTFh- og pre-huTFh-ekspressionsprodukterne kan være ikke-glycosylerede, dvs. de er produceret i en prokaryotisk 5 celle transformeret med et rDNA. En ikke-glycosyleret form af huTFh og pre-huTFh er anvendelig som et immunogen og som et antigen.The huTFh and pre-huTFh expression products may be non-glycosylated, i.e. they are produced in a prokaryotic cell transformed with an rDNA. A non-glycosylated form of huTFh and pre-huTFh is useful as an immunogen and as an antigen.

Eukaryotisk produceret huTFh og pre-huTFh er typisk glycosyleret og biologisk aktivt foruden at være antigent 10 og immunogent. Som anvendt i den foreliggende beskrivelse referer udtrykket "biologisk aktivt" til et huTFh- eller pre-huTFh-protein eller -polypeptid, der har evne til at fremkalde faktor Vll/VIIa-afhængig koagulering.Eukaryotic produced huTFh and pre-huTFh are typically glycosylated and biologically active in addition to being antigenic and immunogenic. As used herein, the term "biologically active" refers to a huTFh or pre-huTFh protein or polypeptide capable of inducing factor VII / VIIa-dependent coagulation.

Et præparat omfattende en vandig opløsning indehol-15 dende biologisk aktivt huTFh er i det væsentlige er frit for protein med let kæde af human vævsfaktor. Præparatet er fortrinsvis også i det væsentlige frit for sådanne stoffer som ioniske detergenter, f.eks. natriumdodecylsulfat (SDS), polyacrylamid og proteiner hidrørende fra væv og med en 2 0 tilsyneladende molekylvægt på mindre end ca. 15.000 dalton som bestemt ved SDS-polyacrylamidgelelektroforese (SDS-PAGE) .A composition comprising an aqueous solution containing biologically active huTFh is substantially free of human tissue factor light chain protein. Preferably, the composition is also substantially free of such substances as ionic detergents, e.g. sodium dodecyl sulfate (SDS), polyacrylamide and proteins derived from tissues and having an apparent molecular weight of less than ca. 15,000 daltons as determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

De vandige huTFh-holdige opløsninger vil indeholde biologisk aktivt huTFh i en tilstrækkelig mængde til at bestemme koaguleringskompetencen af en karsystem-væskeprøve, 25 såsom blod eller produkter afledt af blod, såsom citratplasma. Udtrykket "koaguleringskompetence" refererer til evnen af karvæskeprøven til at koagulere i nærværelse af biologisk aktivt huTFh. Typiske huTFh-proteinkoncentrationer, som er tilstrækkelige til bestemmelse af koaguleringskom-30 petence, er ca. 0,1 pg/ml til ca. 100 ng/ml, fortrinsvis ca. 1 pg/ml til ca. 10 /xg/ml og mere foretrukket ca. 10 pg/ml til ca. 1 ng/ml, ved anvendelse af volumenforhold mellem prøve og huTFh, der ligner de i eksempel 2 anførte.The aqueous huTFh-containing solutions will contain biologically active huTFh in a sufficient amount to determine the coagulation competence of a vascular system fluid sample such as blood or blood derived products such as citrate plasma. The term "coagulation competence" refers to the ability of the vascular fluid sample to coagulate in the presence of biologically active huTFh. Typical huTFh protein concentrations sufficient to determine coagulation competence are approx. 0.1 µg / ml to approx. 100 ng / ml, preferably approx. 1 pg / ml to approx. 10 µg / ml and more preferably approx. 10 µg / ml to approx. 1 ng / ml, using sample to huTFh volume ratios similar to those of Example 2.

Der kan naturligvis også være tale om opløsninger, der inde- 3 5 holder huTFh i koncentrationer, som er højere end de, der 22 DK 176220 B1 er nødvendige til bestemmelse af koaguleringskompetence, men som kan fortyndes til en foretrukken koncentration.Of course, these may also be solutions containing huTFh at concentrations higher than those needed for coagulation competence determination, but which can be diluted to a preferred concentration.

De huTFh-holdige vandige opløsninger omfatter fortrinsvis huTFh dispergeret i et phospholipid eller ikke-5 ionisk detergent. Typiske vægtforhold mellem phospholipid og huTFh-protein er fra ca. 5:1 til 12.000:1, fortrinsvis ca. 50:1 til ca. 5.000:1 og mere foretrukket ca. 100:1 til 2.500:1.The huTFh-containing aqueous solutions preferably comprise huTFh dispersed in a phospholipid or nonionic detergent. Typical weight ratios of phospholipid to huTFh protein are from ca. 5: 1 to 12,000: 1, preferably approx. 50: 1 to approx. 5,000: 1 and more preferably approx. 100: 1 to 2.500: 1.

G. Polypeptider.G. Polypeptides.

10 Polypeptiderne nævnt ovenfor indeholder hver især ikke mere ca. 50 aminosyrerester, mere sædvanligt færre end ca. 35 aminosyrerester og fortrinsvis færre end ca. 25 aminosyrerester og indeholder mindst ca. 10 aminosyrerester.The polypeptides mentioned above each contain no more than ca. 50 amino acid residues, usually less than approx. 35 amino acid residues and preferably less than about 25 amino acid residues and contains at least about 10 amino acid residues.

Desuden er polypeptiderne karakteriseret ved deres aminosyre-15 sekvens og nye funktionelle egenskaber.In addition, the polypeptides are characterized by their amino acid sequence and novel functional properties.

Et sådant polypeptid kan således være en huTFh-bin-dingssted-polypeptidanalog, som til dels er karakteriseret ved dens evne til kompetitiv inhibering af bindingen af huTF til blodkoaguleringsfaktor Vll/VIIa. En bindingssted-20 analog binder fortrinsvis faktor Vll/VIIa uden at danne et aktiveret kompleks, dvs. uden at initiere koagulering.Thus, such a polypeptide may be a huTFh binding site polypeptide analogue, characterized in part by its ability to competitively inhibit the binding of huTF to blood coagulation factor VII / VIIa. Preferably, a binding site analog binds factor VII / VIIa without forming an activated complex, i.e. without initiating coagulation.

Ordet "kompleks" som anvendt i de foreliggende beskrivelse refererer til produktet af en specifik bindingsreaktion, såsom en antistof-antigen- eller receptor-ligand-25 -reaktion. Eksempler på komplekser er immunoreaktionsproduk-ter og vævsf aktor-faktor Vll/VIIa-bindingsreaktionsprodukter, der i den foreliggende beskrivelse betegnes TF:Vll/VIIa.The word "complex" as used in the present specification refers to the product of a specific binding reaction, such as an antibody-antigen or receptor-ligand reaction. Examples of complexes are immunoreaction products and tissue factor Factor VII / VIIa binding reaction products, herein referred to as TF: VII / VIIa.

En huTF-bindingssted-analog omfatter fortrinsvis i det mindste følgende aminosyresekvens: 30 -VNQVYT-, der repræsenterer aminosyreresterne 30-35 som vist i fig. 1.Preferably, a huTF binding site analog comprises at least the following amino acid sequence: -VNQVYT- representing amino acid residues 30-35 as shown in FIG. First

Mere foretrukket omfatter en huTFh-bindingssted-analog mindst én af følgende aminosyresekvenser: 23 DK 176220 B1 -VNQVYTVQIST-, og -LYYWKSSSSGKKT- .More preferably, a huTFh binding site analog comprises at least one of the following amino acid sequences: -VNQVYTVQIST-, and -LYYWKSSSSGKKT-.

Disse sekvenser repræsenterer huTFh-aminosyreresteme 30-40 henholdsvis 155-167 som vist i fig. 1.These sequences represent the huTFh amino acid residues 30-40 and 155-167, respectively, as shown in FIG. First

5 Yderligere mere foretrukket omfatter en huTFh-bin- dingssted-analog en aminosyresekvens valgt blandt -EPKPWQVYTVQISTKSGDWKSKC-, og -VFGKDLIYTLYYWKS S S SGKKT-, der repræsenterer aminosyreresterne 26-49 og 146-167 som 10 vist i fig. 1.Still more preferably, a huTFh binding site analog comprises an amino acid sequence selected from -EPKPWQVYTVQISTKSGDWKSKC-, and -VFGKDLIYTLYYWKS S S SGKKT- representing amino acid residues 26-49 and 146-167 as shown in Figs. First

Foretrukne huTFh-bindingssted-polypeptidanaloge omfatter sådanne, hvis aminosyresekvenser er vist i tabel 1.Preferred huTFh binding site polypeptide analogs include those whose amino acid sequences are shown in Table 1.

Tabel 1 15 Betegnelse3) AminosyresekvensTable 1 15 Designation3) Amino acid sequence

p2 6-4 9 H-EPKPVNQVYTVQISTKSGDWKSKC-OHp2 6-4 9 H-EPKPVNQVYTVQISTKSGDWKSKC-OH

pl46-167 H-VFGKDLIYTLYYWKS SSSGKKT-OHpl46-167 H-VFGKDLIYTLYYWKS SSSGKKT-OH

P161-189 H-S S SGKKTAKTNTNEFLIDVDKGENYCFSV-OHP161-189 H-S S SGKKTAKTNTNEFLIDVDKGENYCFSV-OH

20 a) Laboratoriebetegnelsen for hvert polypeptid repræsenterer den omfattede aminosyresekvens som vist i fig. 1.A) The laboratory designation for each polypeptide represents the amino acid sequence encompassed as shown in FIG. First

Polypeptiderne p26-49, pl46-167 og pl61-189 er også karakteriseret ved deres evne til at neutralisere (kompeti- 25 tivt inhibere) bindingen af anti-huTFh-antistofmolekyler til huTFh. Andre polypeptider, der har evne til at neutralisere bindingen af anti-huTFh-antistoffer til huTFh, omfatter de, der er anført i tabel 2.The polypeptides p26-49, pl46-167 and pl61-189 are also characterized by their ability to neutralize (competitively inhibit) the binding of anti-huTFh antibody molecules to huTFh. Other polypeptides that are capable of neutralizing the binding of anti-huTFh antibodies to huTFh include those listed in Table 2.

30 24 DK 176220 B130 24 DK 176220 B1

Tabel 2Table 2

Betegnelse5^ AminosvresekvensDesignation5 ^ Amino acid sequence

p1-3 0 H-SGTTNTVAAYNLTWKS TNFKT X LEWEPKPV-OHp1-3 0 H-SGTTNTVAAYNLTWKS TNFKT X LEWEPKPV-OH

p40-71 H-TKSGDWKSKCFYTTDTECDLTDEIVKDVKQTY-OHp40-71 H-TKSGDWKSKCFYTTDTECDLTDEIVKDVKQTY-OH

5 p41-49 H-KSGDWKSKC-OH5 p41-49 H-KSGDWKSKC-OH

p56-71 H-ECDLTDEIVKDVKQTY-OHp56-71 H-ECDLTDEIVKDVKQTY-OH

p72-104Ca) H - LAR V F S Y PAGNVESTGSAGEPLYENS PEFTPYLC - OHp72-104Ca) H - LAR V F S Y PAGNVESTGSAGEPLYENS PEFTPYLC - OH

p94-12 3 H-YENS PEFTPYLETNLGQPTIQS FEQVGIKY-OHp94-12 3 H-YENS PEFTPYLETNLGQPTIQS FEQVGIKY-OH

10 a) "C", som er føjet til laboratoriebetegnelsen, angiver, at en cysteinrest er føjet til den anførte sekvens som linker til proteinkonjugering.(A) "C", added to the laboratory designation, indicates that a cysteine residue is added to the indicated sequence linking to protein conjugation.

Det vil forstås, at et polypeptid ikke behøver at 15 være identisk i aminosyresekvens med huTFh, sålænge de omhandlede polypeptider er i stand til at konkurrere med nativ vævsfaktor om binding til faktor Vll/VIIa og/eller er i stand til kompetitiv inhibering af bindingen af anti-huTFh-antistofmolekyler til huTFh. Derfor kan et her omhandlet 20 polypeptid være genstand for forskellige ændringer, såsom indføjelser, udeladelser og substitutioner, enten konservative eller ikke-konservative, hvor sådanne ændringer giver visse fordele ved anvendelsen.It will be appreciated that a polypeptide need not be identical in amino acid sequence to huTFh as long as the polypeptides of the present invention are capable of competing with native tissue factor for binding to factor Vll / VIIa and / or capable of competitively inhibiting the binding of anti-huTFh antibody molecules to huTFh. Therefore, the subject polypeptide of this invention may be subject to various modifications, such as insertions, deletions and substitutions, either conservative or non-conservative, where such modifications confer certain advantages in use.

Konservative substitutioner er sådanne, hvor en ami-25 nosyrerest er erstattet med en anden, biologisk lignende rest. Eksempler på konservative substitutioner omfatter anvendelse af en hydrofob rest, såsom isoleucin, valin, leucin eller methionin i stedet for en anden, eller anvendelse af en polær rest i stedet for en anden, såsom erstat-30 ning af arginin med lysin, glutaminsyre med asparaginsyre eller glutamin med asparagin og omvendt og lignende. Udtrykket "konservativ substitution" omfatter også anvendelse af en substitueret aminosyre i stedet for en tilsvarende usub-stitueret aminosyre, forudsat at et sådant polypeptid også 35 udviser den nødvendige bindingsaktivitet.Conservative substitutions are those in which an amino acid residue is replaced by another biologically similar residue. Examples of conservative substitutions include the use of a hydrophobic residue such as isoleucine, valine, leucine or methionine in place of another, or use of a polar residue in place of another, such as replacing arginine with lysine, glutamic acid with aspartic acid. or glutamine with asparagine and vice versa and the like. The term "conservative substitution" also includes the use of a substituted amino acid instead of a corresponding unsubstituted amino acid, provided that such polypeptide also exhibits the necessary binding activity.

25 DK 176220 B1 Når et polypeptid har en sekvens, der ikke er identisk med sekvensen af nativt huTFh, fordi der er foretaget en eller flere konservative eller ikke-konservative substitutioner, er sædvanligvis ikke mere end 20% og mere sædvanligt 5 ikke mere end 10% af antallet af aminosyrerester substitueret, bortset fra når yderligere rester er tilføjet ved hver terminus med det formål at tilvejebringe en linker, ved hjælp af hvilken polypeptiderne på bekvem måde kan bindes til en mærkning eller en fast matriks eller bærer. Mærknin-10 ger, faste matrikser og bærere, der kan anvendes med polypeptiderne ifølge opfindelsen, beskrives nedenfor.When a polypeptide has a sequence that is not identical to the native huTFh sequence because one or more conservative or non-conservative substitutions have been made, usually no more than 20% and more usually no more than 10 % of the number of amino acid residues substituted, except when additional residues are added at each terminus for the purpose of providing a linker by which the polypeptides can conveniently be bound to a label or a solid matrix or carrier. Labels, solid matrices and carriers which can be used with the polypeptides of the invention are described below.

Aminosyrerest-linkere er sædvanligvis mindst én rest og kan være 40 eller flere rester, mere ofte 1-10 rester. Typiske aminosyrerester, som anvendes til binding, er tyro-15 sin, cystein, lysin, glutaminsyre og asparaginsyre eller lignende. Desuden kan en polypeptidsekvens adskille sig fra den naturlige sekvens ved, at sekvensen er modificeret ved acylering af endestillet NH2, f.eks. acetylering, eller thioglycolsyre-amidering, amidering af terminalt carboxyl, 20 f.eks. ammoniak, methylamin osv.Amino acid residue linkers are usually at least one residue and may be 40 or more residues, more often 1-10 residues. Typical amino acid residues used for binding are tyrosine, cysteine, lysine, glutamic acid and aspartic acid or the like. In addition, a polypeptide sequence may differ from the natural sequence in that the sequence is modified by acylation of terminal NH 2, e.g. acetylation, or thioglycolic acid amidation, amidation of terminal carboxyl, e.g. ammonia, methylamine, etc.

Når et polypeptid er koblet til en bærer via en linker til dannelse af, hvad der er kendt som et bærer-hapten-kon-jugat, er det i stand til at fremkalde antistoffer, der immunoreagerer med huTFh. På baggrund af det veletablerede 25 princip med immunologisk krydareaktivitet kan der derfor være tale om antigenrelaterede varianter af polypeptiderne vist i tabel 1 og 2. En "antigent relateret variant" er et polypeptid, der omfatter en sekvensdel på mindst seks aminosyrerester af et polypeptid fra tabel 1 eller tabel 2, og 30 som er i stand til at fremkalde antistofmolekyler, der immunoreagerer med et polypeptid fra tabel 1 eller 2 og huTFh.When a polypeptide is linked to a carrier via a linker to form what is known as a carrier-hapten conjugate, it is capable of inducing antibodies that immunoreact with huTFh. Therefore, based on the well-established principle of immunological spice reactivity, there may be antigen-related variants of the polypeptides shown in Tables 1 and 2. An "antigen-related variant" is a polypeptide comprising a sequence portion of at least six amino acid residues of a polypeptide from Table 1 or Table 2, and 30 capable of inducing antibody molecules that immunoreact with a polypeptide of Table 1 or 2 and huTFh.

I et præparat omfattende en vandig opløsning af en huTFh-bindingssted-polypeptidanalog, hvor polypeptidet er dispergeret i et phospholipid eller ikke-ionisk detergent, 35 er typiske vægtforhold mellem phospholipid og polypeptid-analog er fra ca. 5:1 til 2 00:1, fortrinsvis fra ca. 30:1 26 DK 176220 B1 til 80:1 og mere foretrukket ca. 45:1 til 55:1. Foretrukne huTFh-bindingssted-polypeptidanaloge, der er egnede til anvendelse dispergeret i et phospholipid, er de, der er anført i tabel 4, afsnit II.In a composition comprising an aqueous solution of a huTFh binding site polypeptide analog, wherein the polypeptide is dispersed in a phospholipid or nonionic detergent, typical weight ratios of phospholipid to polypeptide analog are from ca. 5: 1 to 200: 1, preferably from ca. 30: 1 26 DK 176220 B1 to 80: 1 and more preferably approx. 45: 1 to 55: 1. Preferred huTFh binding site polypeptide analogs suitable for use dispersed in a phospholipid are those listed in Table 4, Section II.

5 Et sådant polypeptid kan syntetiseres ved enhver metode, der er velkendt af en fagmand inden for polypeptidteknikken. En udmærket sammenfatning af de mange metoder, der står til rådighed, kan findes i J.M. Steward og J.D. Young, "Solid Phase Peptide Synthesis", W.H. Freeman Co., 10 San Francisco, 1969, og J. Meienhofer, "Hormonal Proteins and Peptides", bind 2, s. 46, Academic Press (New York), 1983, vedrørende fastfase-peptidsyntese, og E. Schroder og K. Kubke, "The Peptides", bind 1, Academic Press (New York), 1965, vedrørende klassisk syntese i opløsning.Such a polypeptide can be synthesized by any method well known to one of ordinary skill in the polypeptide art. An excellent summary of the many methods available can be found in J.M. Steward and J.D. Young, "Solid Phase Peptide Synthesis", W.H. Freeman Co., 10 San Francisco, 1969, and J. Meienhofer, "Hormonal Proteins and Peptides", Volume 2, p. 46, Academic Press (New York), 1983, on solid phase peptide synthesis, and E. Schroder and K. Kubke, "The Peptides," Volume 1, Academic Press (New York), 1965, on classical synthesis in solution.

15 H. Podematerialer.15 H. Inoculation Materials.

Et polypeptid af den nævnte art eller en antigent relateret variant deraf i et farmaceutisk acceptabelt vandigt fortyndingsmiddelpræparat kan anvendes til dannelse af et podemateriale, der ved indgivelse i en effektiv mængde er i 20 stand til at fremkalde antistoffer, der immunoreagerer med huTFh. Udtrykket "podemateriale" anvendes i den foreliggende beskrivelse til at betegne et præparat indeholdende et polypeptid som aktiv bestanddel anvendt til fremstilling af antistoffer mod huTFh. Når et polypeptid anvendes til at 25 fremkalde antistoffer, vil det forstås, at polypeptidet kan anvendes alene eller bundet til en bærer som et konjugat eller som en polypept idpolymer, men for nemheds skyld betegnes de forskellige polypeptidformer kollektivt ved udtrykket "polypeptid".A polypeptide of the aforementioned kind or an antigen-related variant thereof in a pharmaceutically acceptable aqueous diluent composition may be used to form a graft material capable of inducing, in administration in an effective amount, antibodies that immunoreact with huTFh. The term "graft material" is used in the present specification to designate a preparation containing a polypeptide as the active ingredient used to prepare antibodies against huTFh. When a polypeptide is used to induce antibodies, it will be understood that the polypeptide may be used alone or bound to a carrier as a conjugate or as a polypeptide polymer, but for convenience the various polypeptide forms are collectively referred to by the term "polypeptide".

3 0 I. Antistoffer og antistofpræparater.3 0 I. Antibodies and antibody preparations.

Udtrykket "antistof" anvendes i den foreliggende beskrivelse til at betegne immunoglobulinmolekyler og immunologisk aktive dele af immunoglobulinmolekyler, dvs. molekyler, der indeholder et antistofkombinerende sted eller 35 paratop. Eksempler på antistofmolekyler er intakte immunoglobulinmolekyler, i det væsentlige intakte immunoglobulin- 27 DK 176220 B1 molekyler og de dele af et immunoglobulinmolekyle, der indeholder paratopen, herunder de dele, der er kendte som Fab, Fab', F(ab')2 °9 F (v) .The term "antibody" is used in the present specification to denote immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. molecules containing an antibody combining site or paratope. Examples of antibody molecules are intact immunoglobulin molecules, essentially intact immunoglobulin molecules and those parts of an immunoglobulin molecule containing the paratope, including those known as Fab, Fab ', F (ab') 2 ° 9. F (v).

Et antistofpræparat er således, er et anti-peptid-5 antistof, der er karakteriseret ved at indeholde antistof-molekyler, der immunoreagerer med huTfh og mindst ét specifikt polypeptid af ovennævnte art.Thus, an antibody preparation is an anti-peptide antibody characterized by containing antibody molecules that immunoreact with huTfh and at least one specific polypeptide of the above kind.

F.eks. er et antistofpræparat, der indeholder antistofmolekyler, som immunoreagerer med huTFh og en polypep-10 tidanalog af vævsfaktor-bindingsstedet, men ikke immunoreagerer væsentligt med p2 04-22 6, i stand til at neutralisere evnen af vævsfaktor til at binde faktor VII/VIIa. Foretrukne antistofpræparater er således sådanne, der indeholder antistofmolekyler, som immunoreagerer med huTFh og p26-49 eller 15 pl46-167 og i det væsentlige er frie for immunoreaktion med p2 04 -2 2 6.Eg. is an antibody preparation containing antibody molecules that immunoreact with huTFh and a polypeptide analog of the tissue factor binding site, but do not substantially immunoreact with p2 04-22 6, capable of neutralizing the ability of tissue factor to bind factor VII / VIIa. Thus, preferred antibody preparations are those containing antibody molecules that immunoreact with huTFh and p26-49 or 15 pl46-167 and are essentially free of immunoreaction with p2 04-226.

Det skal bemærkes, at polyklonale antisera, som er dannet med huTFh, indeholder antistoffer, der immunoreagerer med p2 04-22 6. Det hovedsagelige fravær af anti-p204-226-20 immunoreaktivitet er således et træk, der adskiller de nævnte antistofpræparater fra præparaterne beskrevet ifølge kendt teknik.It should be noted that polyclonal antisera formed with huTFh contain antibodies that immunoreact with p2 04-22 6. The major absence of anti-p204-226-20 immunoreactivity is thus a feature that separates said antibody preparations from the compositions. described in the prior art.

Et antistofpræparat produceres typisk ved at immunisere et pattedyr med et podemateriale ifølge opfindelsen og 25 derved fremkalde antistofmolekyler i pattedyret, der har den rette polypeptid-immunospecificitet. Antistofmolekyleme udvindes derefter fra pattedyret og isoleres i ønsket omfang ved velkendte metoder, f.eks. ved immunoaffinitetschromato-grafi. Det således fremstillede antistofpræparat kan bl.a.An antibody preparation is typically produced by immunizing a mammal with a graft material of the invention, thereby eliciting antibody molecules in the mammal having the proper polypeptide immunospecificity. The antibody molecules are then recovered from the mammal and isolated to the desired extent by well known methods, e.g. by immunoaffinity chromatography. The antibody preparation thus prepared can i.a.

30 anvendes ved diagnostiske metoder og systemer til påvisning af huTFh i en kropsprøve.30 is used in diagnostic methods and systems for detecting huTFh in a body specimen.

Et præparat af monoklonalt antistof indeholder inden for påvisningsgrænserne kun én art af antistofkombinerings-sted, som er i stand til effektivt at binde huTFh. Et præ-35 parat af monoklonalt antistof udviser således typisk en enkelt bindingsaffinitet til huTFh, selv om det kan indeholde 28 DK 176220 B1 antistoffer, der er i stand til at binde andre proteiner end huTFh. Opfindelsen omfatter et monoklonalt antistof, der immunoreagerer med huTFh og en polypeptidanalog af vævs-faktor-bindingsstedet, fortrinsvis p26-49 eller pl46-l67.A monoclonal antibody preparation contains within the detection limits only one species of antibody combining site capable of efficiently binding huTFh. Thus, a monoclonal antibody preparation typically exhibits a single binding affinity for huTFh, although it may contain antibodies capable of binding proteins other than huTFh. The invention comprises a monoclonal antibody which immunoreacts with huTFh and a polypeptide analog of the tissue factor binding site, preferably p26-49 or p146-167.

5 Opfindelsen omfatter også et antikoagulerende (neu traliserende) monoklonalt antistof (MoAb) indeholdende antistofmolekyler, der immunoreagerer med huTFh og inhiberer huTFh-initieret koagulering.' Et foretrukket monoklonalt antistof, der inhiberer koagulering, er yderligere karak-10 teriseret ved at immunoreagere med en polypeptid af den ovennævnte art, fortrinsvis en huTFh-bindingssted-polypep-tidanalog og mere foretrukket et polypeptid som vist i tabel 1 .The invention also includes an anticoagulant (neutralizing) monoclonal antibody (MoAb) containing antibody molecules that immunoreact with huTFh and inhibit huTFh-initiated coagulation. A preferred monoclonal antibody that inhibits coagulation is further characterized by immunoreacting with a polypeptide of the aforementioned kind, preferably a huTFh binding site polypeptide analog and more preferably a polypeptide as shown in Table 1.

Ifølge en anden udførelsesform omfatter et antikoa-15 gulerende monoklonalt antistof antistofmolekyler, der immunoreagerer med huTFh og et huTFh: faktor Vll/VIIa-kompleks og inhiberer (neutraliserer) huTFh-initieret koagulering.In another embodiment, an anticoagulant monoclonal antibody comprises antibody molecules that immunoreact with huTFh and a huTFh: factor V11 / VIIa complex and inhibit (neutralize) huTFh-initiated clotting.

Et foretrukket antikoagulerende monoklonalt antistof er yderligere karakteriseret ved at immunoreagere med huTFh-20 -polypeptiderne pl-30 eller p26-49 og immunoreagerer fortrinsvis ikke med huTFh-polypeptid p56-71.A preferred anticoagulant monoclonal antibody is further characterized by immunoreacting with the huTFh-20 polypeptides p1-30 or p26-49, and preferably does not immunoreact with huTFh polypeptide p56-71.

Der kan også være tale om et præparat af ikke-neutral i serende monoklonalt antistof indeholdende antistof-molekyler, der ikke neutraliserer evnen af vævsfaktor til 25 at initiere koagulering. Et sådant præparat indeholder fortrinsvis antistofmolekyler, der immunoreagerer med huTFh og polypeptidet pl-30 og produceres (udskilles) af hybridoma TF9-1OHIO.It may also be a preparation of neutral in serous monoclonal antibody containing antibody molecules which does not neutralize the ability of tissue factor to initiate coagulation. Such a preparation preferably contains antibody molecules that immunoreact with huTFh and the polypeptide p1-30 and are produced (secreted) by hybridoma TF9-1OH10.

Et præparat af monoklonalt antistof kan produceres 30 ved at igangsætte en kultur af monoklonalt hybridoma omfattende et næringsmedium indeholdende et hybridoma, der udskiller antistofmolekyler med den rette polypeptid-specificitet .A preparation of monoclonal antibody can be produced by initiating a culture of monoclonal hybridoma comprising a nutrient medium containing a hybridoma that secretes antibody molecules with the proper polypeptide specificity.

Kulturen holdes under betingelser og i et tidsrum, 35 der er tilstrækkeligt til, at hybridomaet udskiller antistofmolekylerne i mediet. Det antistofholdige medium opsamles 29 DK 176220 B1 derefter. Antistofmolekylerne kan derpå isoleres yderligere ved velkendte metoder.The culture is maintained under conditions and for a time sufficient for the hybridoma to secrete the antibody molecules in the medium. The antibody-containing medium is then collected. The antibody molecules can then be further isolated by well known methods.

Medier, som er anvendelige til fremstilling af disse præparater, er både velkendte og kommercielt tilgængelige og 5 omfatter syntetiske kulturmedier, indavlede mus og lignende.Media useful in the preparation of these compositions are both well known and commercially available and include synthetic culture media, inbred mice and the like.

Et eksempel på et syntetisk medium er Dulbecco's minimale essentielle medium (DMEM; Dulbecco et al., Virol. 8:396 (1959) suppleret med 4,5 g/liter glucose, 20 mM glutamin og 20% kalvefosterserum. Et eksempel på en indavlet musestamme 10 er Balb/c. Præparaterne af monoklonalt antistof, der er fremstillet ved den ovenfor anførte metode, kan f.eks. anvendes diagnostisk og terapeutisk, hvor dannelsen af et huTFh-holdigt immunoreaktionsprodukt ønskes.An example of a synthetic medium is Dulbecco's Minimum Essential Medium (DMEM; Dulbecco et al., Virol. 8: 396 (1959) supplemented with 4.5 g / liter glucose, 20 mM glutamine and 20% fetal calf serum. An example of an inbred The mouse strain 10 is Balb / c. The monoclonal antibody preparations prepared by the above method can be used, for example, diagnostically and therapeutically where the formation of a huTFh-containing immunoreaction product is desired.

J. Hybridomaer.J. Hybridomas.

15 Hybridomaer kan anvendes til at producere antistof molekyler, der immunoreagerer med huTFh. Et foretrukket hybridoma er yderligere karakteriseret ved at producere antistofmolekyler, der inhiberer huTFh-initieret koagulering og fortrinsvis immunoreagerer med et polypeptid af den oven* 20 nævnte art, fortrinsvis en huTFh-bindingssted-polypeptid-analog og mere foretrukket et polypeptid som vist i tabel 1. Ved yderligere foretrukne udførelsesformer immunoreagerer et antikoagulerende monoklonalt antistof med ikke-humant primat-Tf.Hybridomas can be used to produce antibody molecules that immunoreact with huTFh. A preferred hybridoma is further characterized by producing antibody molecules that inhibit huTFh-initiated coagulation and preferably immunoreact with a polypeptide of the above-mentioned species, preferably a huTFh binding site polypeptide analog and more preferably a polypeptide as shown in Table 1. In further preferred embodiments, an anticoagulant monoclonal antibody with non-human primate Tf immunoreacts.

2 5 Et hybridoma kan også fortrinsvis producere antistof - molekyler, der immunoreagerer med huTFh og et huTFh: faktor Vll/VIIa-kompleks og neutraliserer huTFh-initieret koagulering. Fortrinsvis er et hybridoma, der producerer antistoffer, som immunoreagerer med et huTFh: faktor VII/VIla-kompleks 30 yderligere karakteriseret ved evnen af antistofmolekylerne til at immunoreagere med huTFh-polypeptiderne pl-30 eller p26-49, fortrinsvis begge, idet det er mere foretrukket, at de nævnte antistofmolekyler ikke immunoreagerer med poly-huTFh-polypeptid p56-71.Preferably, a hybridoma can also produce antibody molecules that immunoreact with huTFh and a huTFh: factor V11 / VIIa complex and neutralize huTFh-initiated coagulation. Preferably, a hybridoma producing antibodies that immunoreact with a huTFh: factor VII / VIa complex 30 is further characterized by the ability of the antibody molecules to immunoreact with the huTFh polypeptides p1-30 or p26-49, preferably both, being more abundant. preferably, said antibody molecules do not immunoreact with poly-huTFh polypeptide p56-71.

35 Metoder til fremstilling af hybridomaer, der produ cerer (udskiller) antistofmolekyler, der har en ønsket im- 30 DK 176220 B1 munospecificitet, dvs. har evnen til at immunoreagere med et bestemt protein og/eller polypeptid, er velkendte. Særlig anvendelig er hybridoma-teknologien beskrevet af Niman et al., Proc. Natl. Acad. Sci. USA, 80:4949-4953 (1983). Fore- 5 trukne hybridomaer er de, der er anført i tabel 5 i eksempel 13 .Methods for preparing hybridomas producing (secreting) antibody molecules having a desired immunospecificity, ie. having the ability to immunoreact with a particular protein and / or polypeptide are well known. Particularly useful is the hybridoma technology described by Niman et al., Proc. Natl. Acad. Sci. USA, 80: 4949-4953 (1983). Preferred hybridomas are those listed in Table 5 of Example 13.

Hybridomakulturerne TF8-5G9, TF9-6B4 og TF9-10H10 er deponeret i overensstemmelse med Budapest-traktaten hos ATCC den 27. marts 1987 og har fået deponeringsnumrene 10 HB9382, HB9381, henholdsvis HB9383.The hybridoma cultures TF8-5G9, TF9-6B4 and TF9-10H10 have been deposited in accordance with the Budapest Treaty with the ATCC on March 27, 1987 and have been given deposit numbers 10 HB9382, HB9381 and HB9383 respectively.

En yderligere hybridomakultur TF9-5B7 er deponeret i overensstemmelse med Budapest-traktaten hos ATTC den 9. marts 1988 og har fået deponeringsnummeret HB9658.A further hybridoma culture TF9-5B7 has been deposited in accordance with the Budapest Treaty with the ATTC on March 9, 1988 and has been given the deposit number HB9658.

K. Terapeutiske metoder og præparater.K. Therapeutic Methods and Preparations.

15 huTFh- f aktor-VIl/VlIa-bindingssted-polypeptidanalo- gene, antistofpræparaterne, præparaterne af monoklonalt antistof og de antikoagulerende monoklonale antistoffer kan hver især anvendes til at modulere bindingen af faktor Vll/VIIa af vævsfaktor in vivo.The 15 huTFh-factor VI1 / VIIa binding site polypeptide analogues, the antibody preparations, the monoclonal antibody preparations and the anticoagulant monoclonal antibodies can each be used to modulate the factor VII / VIIa binding of tissue factor in vivo.

20 F.eks. kan en huTFh-faktor Vll/VIIa-bindingssted-po- lypeptidanalog anvendes i et farmaceutisk acceptabelt præparat, der ved indgivelse til en person i en effektiv mængde er i stand til kompetitiv inhibering af bindingen af faktor Vll/VIIa til vævsfaktor. Denne inhibering antages at bevirke 25 en nedsat hastighed af dannelsen af vævsfaktor-faktor VII/-VIla-kompleks. Således kan indgivelse in vivo af en huTFh--faktor VII/VIla-bindingssted-polypeptidanalog anvendes til at modulere enhver fysiologisk reaktion, der initieres af binding af vævsfaktor til faktor Vll/VIIa, såsom koagulering 30 og nogle inflammatoriske reaktioner. Polypeptidet indgives fortrinsvis dispergeret i et phospholipid som ovenfor beskrevet .For example, For example, a huTFh factor VII / VIIa binding site polypeptide analog can be used in a pharmaceutically acceptable preparation capable of competitively inhibiting the binding of factor VII / VIIa to tissue factor upon administration to a subject. This inhibition is thought to cause a decreased rate of tissue factor-factor VII / VIIa complex formation. Thus, in vivo administration of a huTFh factor VII / VIa1 binding site polypeptide analogue can be used to modulate any physiological response initiated by the binding of tissue factor to factor VIII / VIIa such as coagulation 30 and some inflammatory responses. The polypeptide is preferably administered dispersed in a phospholipid as described above.

En anden metode til modulering af bindingen af faktor Vll/VIIa af vævsfaktor in vivo er intravenøs indgivelse af 35 en effektiv mængde af et antistofpræparat (anti-peptid-an-tistof) eller et antikoagulerende monoklonalt antistof ifølge 31 DK 176220 B1 opfindelsen. Fortrinsvis er antistofmolekylerne sådanne, der indeholder denne paratopiske region og er frie for Fc--regionen, såsom immunoglobulinfragmenterne F (ab')2/ Fab °9 lignende. Terapeutisk effektive mængder af et antikoagule-5 rende monoklonalt antistof ligger i området fra 15 iig/kg legemsvægt til 5 mg/kg legemsvægt, fortrinsvis i området fra ca. 100 /zg/kg legemsvægt til ca. 1 mg/kg legemsvægt og mere foretrukket i området fra ca. 150 Mg/kg legemsvægt til ca. 500 Mg/kg legemsvægt.Another method for modulating the binding of factor VII / VIIa by tissue factor in vivo is intravenous administration of an effective amount of an antibody preparation (anti-peptide antibody) or an anticoagulant monoclonal antibody of the invention. Preferably, the antibody molecules are those containing this paratopic region and are free of the Fc region, such as the immunoglobulin fragments F (ab ') 2 / Fab ° 9 similar. Therapeutically effective amounts of an anticoagulant monoclonal antibody range from 15 µg / kg body weight to 5 mg / kg body weight, preferably in the range of from 100 µg / kg body weight to approx. 1 mg / kg body weight and more preferably in the range of about 150 Mg / kg body weight to approx. 500 Mg / kg body weight.

10 Antistofmolekylerne af et monoklonalt antistof, anti- koagulerende monoklonalt antistof eller ikke-neutraliserende monoklonalt antistof ifølge opfindelsen kan også bindes til et antitumormiddel til dannelse af et antitumorvirksomt terapeutisk præparat. En effektiv mængde antitumorvirksomt 15 terapeutisk præparat dannet på denne måde kan indgives til en person, der har tumorceller, som eksprimerer vævsfaktor på deres overflade. Eksempler på sådanne tumorceller er brystkarcinom og lungekarcinom.The antibody molecules of a monoclonal antibody, anticoagulant monoclonal antibody or neutralizing monoclonal antibody of the invention may also be bound to an antitumor agent to form an antitumor therapeutic preparation. An effective amount of antitumor therapeutic preparation thus formed can be administered to a person having tumor cells expressing tissue factor on their surface. Examples of such tumor cells are breast carcinoma and lung carcinoma.

Typiske for sådanne antitumormidler er radionuklider, 131 188 212 20 såsom I, Re, Bi og lignende. Metoder til fremstilling af terapeutiske præparater af radionuklid-konjugeret monoklonalt antistof og deres anvendelse er beskrevet i Kozak et al·., Trends In Biotech., 4:259-264 (1986).Typical of such antitumor agents are radionuclides such as I, Re, Bi and the like. Methods for preparing therapeutic compositions of radionuclide-conjugated monoclonal antibody and their use are described in Kozak et al., Trends In Biotech., 4: 259-264 (1986).

De indgivne polypeptid- eller antistofmolekyleholdige 25 præparater har form af opløsninger eller suspensioner, men polypeptiderne kan også have form af tabletter, piller, kapsler, formuleringer med langvarig frigivelse eller pulvere. I alle tilfælde indeholder præparaterne 0,10-95% aktiv bestanddel, fortrinsvis 25-70%.The administered polypeptide or antibody molecule-containing preparations take the form of solutions or suspensions, but the polypeptides may also take the form of tablets, pills, capsules, sustained-release formulations or powders. In all cases, the compositions contain 0.10-95% of active ingredient, preferably 25-70%.

30 Fremstillingen af terapeutiske præparater, der in deholder polypeptider eller antistofmolekyler som aktive bestanddele, er velkendt. Typisk fremstilles sådanne præparater som injektionspræparater, enten som væskeopløsninger eller suspensioner, men faste former, der er egnet til op-35 løsning eller suspension i væske før injektion, kan også fremstilles. Præparatet kan også være emulgeret. Den aktive 32 DK 176220 B1 terapeutiske bestanddel blandes ofte med ekscipienser, der er farmaceutisk acceptable og forenelige med den aktive bestanddel. Egnede ekscipienser er f.eks. vand, saltvand, dextrose, glycerol, ethanol eller lignende og kombinationer 5 deraf. Desuden kan præparatet om ønsket indeholde mindre mængder af hjælpestoffer, såsom befugtningsmidler eller emulgatorer, pH-puffermidler, som forøger effektiviteten af den aktive bestanddel.The preparation of therapeutic compositions containing polypeptides or antibody molecules as active ingredients is well known. Typically, such compositions are prepared as injection preparations, either as liquid solutions or suspensions, but solid forms suitable for solution or suspension in liquid prior to injection may also be prepared. The composition may also be emulsified. The active ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are e.g. water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. In addition, if desired, the composition may contain minor amounts of excipients such as wetting agents or emulsifiers, pH buffering agents which enhance the effectiveness of the active ingredient.

Et præparat af polypeptid eller antistof kan i det 10 terapeutiske præparat formuleres som neutraliserede, farmaceutisk acceptable saltformer. Farmaceutisk acceptable salte omfatter syreadditionssalte (dannet med de frie amino-grupper af polypeptidet eller antistoffet) og dannes med uorganiske syrer, f.eks. saltsyre eller phosphorsyre, eller 15 med sådanne organiske syrer som eddikesyre, oxalsyre, vinsyre, mandelsyre og lignende. Salte dannet med de frie carb-oxylgrupper kan også fås ud fra uorganiske baser, f.eks. natrium-, kalium-, ammonium-, calcium- eller ferrihydroxid, og sådanne organiske baser som isopropylamin, trimethylamin, 20 2-ethylaminoethanol, histidin, procain og lignende.A polypeptide or antibody preparation may be formulated in the therapeutic composition as neutralized, pharmaceutically acceptable salt forms. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the polypeptide or antibody) and formed with inorganic acids, e.g. hydrochloric or phosphoric acid, or with such organic acids as acetic, oxalic, tartaric, mandelic and the like. Salts formed with the free carb oxyl groups can also be obtained from inorganic bases, e.g. sodium, potassium, ammonium, calcium or ferric hydroxide, and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine and the like.

De terapeutiske polypeptid- eller antistofholdige præparater indgives sædvanligvis topisk eller intravenøst, f.eks. ved injektion af en enhedsdosis. Udtrykket "enhedsdosis" refererer i sammenhæng med et terapeutisk præparat 25 ifølge den foreliggende opfindelse til fysisk særskilte enheder, der er egnede som enhedsdoser til mennesker, idet hver enhed indeholder en forud bestemt mængde aktivt materiale, der er beregnet til at give den ønskede terapeutiske effekt i kombination med det nødvendige fortyndingsmiddel, 30 dvs. bærer eller bærestof.The therapeutic polypeptide or antibody-containing preparations are usually administered topically or intravenously, e.g. by injection of a unit dose. The term "unit dose" in the context of a therapeutic composition 25 of the present invention refers to physically separate units suitable as unit doses for humans, each unit containing a predetermined amount of active material intended to provide the desired therapeutic effect. in combination with the necessary diluent, i.e. carrier or carrier.

Præparaterne indgives på en måde, der er forenelig med dosisformuleringen, og i en terapeutisk effektiv mængde. Mængden, der skal indgives, afhænger af patienten, der skal behandles, evnen af patientens blodkoaguleringssystem til 3 5 at udnytte den aktive bestanddel og den ønskede grad af inhibering eller neutralisering af vævsfaktor-bindingskapa- 33 DK 176220 B1 citet. De nøjagtige mængder af aktiv bestanddel, der skal indgives, afhænger af lægens vurdering og er specielle for hvert individ. Imidlertid er egnede polypeptid-dosisområder af størrelsesordenen et til flere mg aktiv bestanddel pr. in-5 divid pr. dag og afhænger af indgivelsesmåden. Egnede planer for begyndelsesindgivelse og booster-indgivelser er også variable, men udgøres typisk af en begyndelsesindgivelse efterfulgt af gentagne doser med mellemrum på en eller flere timer, som indgives ved injektion eller på anden måde. Al-10 ternativt kan der foretages en kontinuerlig intravenøs infusion, der er tilstrækkelig til at opretholde koncentrationer på 10 nM til 10 mM i blodet.The compositions are administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The amount to be administered depends on the patient to be treated, the ability of the patient's blood coagulation system to utilize the active ingredient and the desired degree of inhibition or neutralization of tissue factor binding capacity. The exact amounts of active ingredient to be administered depend on the physician's assessment and are specific to each individual. However, suitable polypeptide dose ranges are on the order of one to several mg of active ingredient per day. in-5 dividends per share. day and depends on the mode of administration. Suitable initial and booster administration plans are also variable, but typically constitute an initial administration followed by repeated doses at intervals of one or more hours administered by injection or otherwise. Alternatively, a continuous intravenous infusion sufficient to maintain concentrations of 10 nM to 10 mM in the blood may be performed.

Eksempler 15 De følgende eksempler tjener til at illustrere, men ikke begrænse, den foreliggende opfindelse.Examples 15 The following examples serve to illustrate, but not limit, the present invention.

1. Fremstilling af vævsfaktorholdig human hjerneekstrakt1. Preparation of Tissue Factor-Containing Human Brain Extract

Normale hjerner tilvejebragt ved autopsi forarbejdes 20 enten inden for 12 timer eller opbevares nedfrosset ved -80°C. Hjernehinderne og cerebellum fjernes, og de tilbageværende hjernedele homogeniseres i et lige så stort volumen kold (0°C) acetone ved anvendelse af en Polytron-homogeni-seret {Brinkman Instruments, Co., Westbury, NY). Det frem-25 komne homogenisat blandes med yderligere tre volumener kold acetone, og fraktionen af fast væv fraskilles ved filtrering under anvendelse af en sintret glastragt. Acetoneopløseligt materiale ekstraheres fra det tilbageholdte faste stof syv yderligere gange, idet der hver gang blandes med to volumener 30 kold acetone og derefter filtreres. Efter den sidste filtrering får tilbageværende acetone lov at fordampe ved atmosfæretryk fra det tilbageværende faste stof natten over ved ca. 2 0 °C.Normal brains provided by autopsy are processed either within 12 hours or stored frozen at -80 ° C. The meninges and cerebellum are removed and the remaining brain sections are homogenized in an equal volume of cold (0 ° C) acetone using a Polytron homogenized {Brinkman Instruments, Co., Westbury, NY). The resulting homogenate is mixed with an additional three volumes of cold acetone and the solid tissue fraction is separated by filtration using a sintered glass funnel. Acetone-soluble material is extracted from the retained solid seven additional times, each mixing with two volumes of 30 cold acetone and then filtering. After the last filtration, the remaining acetone is allowed to evaporate at atmospheric pressure from the residual solid overnight at ca. 20 ° C.

Det tilbageværende faste stof af hjernevæv underkastes 35 derefter fem ekstraktioner, der hver især gennemføres ved at blande det faste stof med en heptan/butanol-opløsning i 34 DK 176220 B1 forholdet 2:1 i et forhold på 1 g fast stof pr. 25 ml hep-tan/butanol efterfulgt af filtrering til fraskillelse af det faste stof. Efter den sidste filtrering tørres det tilbageværende faste stof natten over ved ca. 20°C under atmos-5 færetryk, hvorved der fås delipideret hjernevæv-pulver, som opbevares ved -80°C indtil anvendelsen.The remaining brain tissue solid is then subjected to five extractions, each of which is accomplished by mixing the solid with a heptane / butanol solution in a 2: 1 ratio at a ratio of 1 g of solid per ml. 25 ml of hepatane / butanol followed by filtration to separate the solid. After the last filtration, the residual solid is dried overnight at ca. 20 ° C under atmospheric pressure, yielding delipidated brain tissue powder which is stored at -80 ° C until use.

25 g af hjernevæv-pulveret blandes derefter med 500 ml TS/EDTA-puffer (100 mM natriumchlorid, 50 mM tris-HCl (pH-værdi 7,5), 0,02% natriumazid, 5 mM ethylendiamintetra-10 eddikesyre (EDTA), 0,1 volumenprocent "Triton X-100" (poly-arylethylen-9-octyl-phenylether)) og omrøres natten over ved 4°C. Blandingen centrifugeres derefter ved 15.300 g i 1 time. Den fremkomne pellet resuspenderes i 500 ml puffer A (100 mM natriumchlorid, 50 mM tris-HCl (pH-værdi 7,5), 0,02% 15 natriumazid, 2% "Triton X-100") til dannelse af en opslæmning. Efter omrøring i 1 time ved stuetemperatur centrifugeres opslæmningen som ovenfor beskrevet. Den fremkomne ovenstående væske fraskilles, frysetørres og opløseliggøres derefter i 100 ml puffer A til dannelse af en huTF-holdig 20 hjerneekstraktopløsning.25 g of the brain tissue powder is then mixed with 500 ml of TS / EDTA buffer (100 mM sodium chloride, 50 mM tris-HCl (pH 7.5), 0.02% sodium azide, 5 mM ethylenediaminetetraacetic acid (EDTA) , 0.1 volume percent "Triton X-100" (poly-arylethylene-9-octyl-phenyl ether)) and stirred overnight at 4 ° C. The mixture is then centrifuged at 15,300 g for 1 hour. The resulting pellet is resuspended in 500 ml of buffer A (100 mM sodium chloride, 50 mM tris-HCl (pH 7.5), 0.02% sodium azide, 2% "Triton X-100") to form a slurry. After stirring for 1 hour at room temperature, the slurry is centrifuged as described above. The resulting liquid above is separated, freeze-dried and then solubilized in 100 ml of buffer A to form a huTF-containing 20 brain extract solution.

2. Koagulerinosbestemmelse til måling af huTF-pro-koagulerende aktivitet. huTF-pro-koagulerende aktivitet måles ved en ettrins koaguleringsbestemmelse gennemført med alle reagenser og 25 blandinger holdt ved 37°C. En pool af normalt humant plasma citratbehandles ved blanding af et volumen plasma med et volumen af en opløsning indeholdende 20 mM natriumcitrat-di-hydrat og 140 mM natriumchlorid, pH-værdi 7,4. 100 μΐ af en prøve indeholdende huTF fortyndet i TBS/BSA-opløsning (150 30 mM natriumchlorid, 50 mM tris-HCl, pH-værdi 7,5, 0,1% bovint serumalbumin) blandes med 100 μΐ af det citratbehandlede plasma. 100 μΐ af en 25 mM calciumchloridopløsning iblandes derefter til dannelse af en koaguleringsblanding, der vugges forsigtigt, indtil koaguleringen sker. Tidsrummet mellem 35 tilsætningen af calciumchlorid og dannelsen af koagel måles.2. Coagulerin assay for measuring huTF pro-coagulant activity. huTF pro-coagulant activity is measured by a one-step coagulation assay performed with all reagents and mixtures maintained at 37 ° C. A pool of normal human plasma citrate is treated by mixing a volume of plasma with a volume of a solution containing 20 mM sodium citrate dihydrate and 140 mM sodium chloride, pH 7.4. 100 μΐ of a sample containing huTF diluted in TBS / BSA solution (150 30 mM sodium chloride, 50 mM tris-HCl, pH 7.5, 0.1% bovine serum albumin) is mixed with 100 μΐ of the citrate-treated plasma. 100 μΐ of a 25 mM calcium chloride solution is then mixed to form a coagulation mixture which is gently rocked until coagulation occurs. The time between the addition of calcium chloride and the formation of clot is measured.

En standardkurve for huTF-aktivitet konstrueres derefter 35 DK 176220 B1 ved at afsætte fortyndingen mod koaguleringst iden i sekunder, et eksempel på en standardkurve er vist i fig. 3.A standard curve for huTF activity is then constructed by plotting the dilution against the coagulation time in seconds. An example of a standard curve is shown in FIG. Third

3. Fremstilling af en faktor Vll-holdig fast bærer til affinitetsisolering af huTF.3. Preparation of a factor VII-containing solid support for affinity isolation of huTF.

5 Human faktor Vll/VIIa isoleres som beskrevet af Fair,Human factor Vll / VIIa is isolated as described by Fair,

Blood, 62:784-91 (1983). Denne isolerede faktor Vll/VIIa aktiveres til kobling til en fast agarosematriks ved at dialysere 5 mg mod 0,1 M 2-(N-morpholino)-ethansulfonsyre (MES) (pH-værdi 6,5) natten over ved 4°C. Calciumchlorid 10 tilsættes til en slutkoncentration på 1 mM. Faktor Vll/VIIa blandes derefter med 4 ml "AffiGel-15"-aktiverede agaroseper-ler (Biorad Laboratories, Richmond, CA) , og den resulterende koblingsreaktionsblanding roteres i 4 timer ved 4°C ifølge producentens anbefalinger (Biorad).Blood, 62: 784-91 (1983). This isolated factor Vll / VIIa is activated for coupling to a solid agarose matrix by dialyzing 5 mg against 0.1 M 2- (N-morpholino) ethanesulfonic acid (MES) (pH 6.5) overnight at 4 ° C. Calcium chloride 10 is added to a final concentration of 1 mM. Factor VIII / VIIa is then mixed with 4 ml of AffiGel-15-activated agarose beads (Biorad Laboratories, Richmond, CA) and the resulting coupling reaction mixture is rotated for 4 hours at 4 ° C according to the manufacturer's recommendations (Biorad).

15 Overskydende proteinbindingssteder på den faste bærer blokeres ved forsigtig omrøring af den faste bærer i 0,1 N glycin-ethylester i 1 time ved stuetemperatur. Derefter vaskes den faste bærer successivt på en sintret glastragt medens ca. 20 ml af hver af følgende: 1) puffer A, 2) puffer 20 A indeholdende 1 M natriumchlorid, 3) puffer A indeholdende 5 mM EDTA og 4) puffer A indeholdende 1 mM calciumchlorid. Overskydende væske fjernes ved hjælp af vakuum til dannelse af en halvtør partikelmasse (kage).Excess protein binding sites on the solid support are blocked by gently stirring the solid support in 0.1 N glycine ethyl ester for 1 hour at room temperature. Then the solid support is washed successively on a sintered glass funnel while approx. 20 ml of each of the following: 1) buffer A, 2) buffer 20 A containing 1 M sodium chloride, 3) buffer A containing 5 mM EDTA, and 4) buffer A containing 1 mM calcium chloride. Excess liquid is removed by vacuum to form a semi-dry particle mass (cake).

4. Faktor VII/Vila-affinitetsisolering 25 af huTF.4. Factor VII / Vila affinity isolation of huTF.

20 ml af en opløsning indeholdende 0,1 M glycinethyl-ester og 0,1 M MES, pH-værdi 6,5, blandes med 22,5 ml "AffiGel-15"-agaroseperler (Biorad) til dannelse af en koblingsreaktionsblanding. Koblingsreaktionsblandingen holdes 30 ved stuetemperatur i 1 time. Det fremkomne konjugat vaskes på en sintret glastragt 4 gange med 10 volumener puffer 1 og filtreres under vakuum til dannelse af en glycinethyl-ester-agarose-kage.20 ml of a solution containing 0.1 M glycinethyl ester and 0.1 M MES, pH 6.5, are mixed with 22.5 ml of AffiGel-15 agarose beads (Biorad) to form a coupling reaction mixture. The coupling reaction mixture is kept at room temperature for 1 hour. The resulting conjugate is washed on a sintered glass funnel 4 times with 10 volumes of buffer 1 and filtered under vacuum to form a glycine ethyl ester agarose cake.

30 ml hjerneekstraktopløsning fremstillet i eksempel 35 1 dialyseres natten over ved 4°C mod 6 liter puffer A in deholdende 1 mM calciumchlorid. Dialyseret hjerneekstrakt 36 DK 176220 B1 blandes med glycinethylester-agarose-kage til dannelse af en fastfase-væskefase-reaktionsblanding. Efter at være blevet holdt i 2 timer ved stuetemperatur under rotering adskilles den faste fase og den flydende fase ved filtrering under 5 anvendelse af en sintret glastragt. Væskefasen fraskilles og blandes med "Trasylol" (aprotinin; Sigma Chemical Co.30 ml of brain extract solution prepared in Example 35 1 is dialyzed overnight at 4 ° C against 6 liters of buffer A containing 1 mM calcium chloride. Dialyzed brain extract 36 is mixed with glycine ethyl ester agarose cake to form a solid-phase liquid-phase reaction mixture. After being kept for 2 hours at room temperature while rotating, the solid phase and the liquid phase are filtered by filtration using a sintered glass funnel. The liquid phase is separated and mixed with "Trasylol" (aprotinin; Sigma Chemical Co.

St. Louis, MO) til en slutkoncentration på 10 enheder/ml.St. Louis, MO) to a final concentration of 10 units / ml.

Den fraskilte væskefase blandes med faktor Vll/VIIa-agarose--kagen fremstillet i eksempel 3 til dannelse af en anden 10 fastfase/væskefase-blanding.The separated liquid phase is mixed with the factor II / VIIa agarose cake prepared in Example 3 to form another 10 solid / liquid phase mixture.

Denne blanding holdes natten over ved 4°C under rotation for at muliggøre dannelse af et huTF-faktor Vll/VIIa--holdigt fastfase-produkt. Den faste fase og den flydende fase adskilles derefter ved filtrering som ovenfor beskrevet.This mixture is kept overnight at 4 ° C under rotation to allow the formation of a huTF factor Vll / VIIa - containing solid phase product. The solid phase and the liquid phase are then separated by filtration as described above.

15 Den faste fase, som tilbageholdes på den sintrede glastragt, vaskes med 25 ml puffer A indeholdende 1 mM calciumchlorid.The solid phase retained on the sintered glass funnel is washed with 25 ml of buffer A containing 1 mM calcium chloride.

Den faste fase overføres derefter til en chromatografisøj le af sintret glas (0,5 x 15 cm, Biorad) og vaskes med 6 ml af den samme vaskepuffer. huTF, som er bundet til den faste 20 bærer efter de ovennævnte vaskninger, frigøres (elueres) derefter ved vaskning af den faste bærer med puffer A indeholdende 5 mM EDTA, medens den tilbageholdes på søjlen af sintret glas. Elueret materiale opsamles i fraktioner på 1 ml, og hver fraktion undersøges for tilstedeværelse af huTF 25 som beskrevet i eksempel 2. huTF-holdige fraktioner pooles og dialyseres natten over mod 6 liter TBS (150 mM natrium-chlorid, 50 mM tris-HCl, pH-værdi 7,5) indeholdende 1% "Triton X-100" (TBS/Triton) ved 4°C.The solid phase is then transferred to a sintered glass chromatography column (0.5 x 15 cm, Biorad) and washed with 6 ml of the same wash buffer. huTF, which is bonded to the solid support after the above washings, is then released (eluted) by washing the solid support with buffer A containing 5 mM EDTA while retained on the sintered glass column. Eluted material is collected in 1 ml fractions and each fraction assayed for the presence of huTF 25 as described in Example 2. huTF containing fractions are pooled and dialyzed overnight against 6 liters of TBS (150 mM sodium chloride, 50 mM tris-HCl, pH 7.5) containing 1% "Triton X-100" (TBS / Triton) at 4 ° C.

Det således dannede dialysat blandes derefter med 30 fire volumener kold acetone til udfældning af huTF-proteinet. Bundfaldet opsamles ved centrifugering ved 5.000 g i 30 minutter ved ca. -10°C. Den fremkomne pellet tørres under nitrogen. Typiske udbytter er 2 μg huTF pr. g (tør vægt) af delipideret hjernevæv-pulver.The dialysate thus formed is then mixed with 30 four volumes of cold acetone to precipitate the huTF protein. The precipitate is collected by centrifugation at 5,000 g for 30 minutes at ca. -10 ° C. The resulting pellet is dried under nitrogen. Typical yields are 2 μg huTF per ml. g (dry weight) of delipidized brain tissue powder.

35 En prøve af det således isolerede huTF suspenderes i TBS/Triton og mærkes derefter med Na-^^I (16 μCi/μg, 37 DK 176220 B135 A sample of the thus isolated huTF is suspended in TBS / Triton and then labeled with Na - ^^ I (16 µCi / µg, 37 DK 176220 B1

Amersham, Arlington Heights, IL) ved anvendelse af iodogen ifølge producentens anvisninger (Pierce Chemical Co.,Amersham, Arlington Heights, IL) using iodogen according to the manufacturer's instructions (Pierce Chemical Co.,

Rockford, IL) . Efter mærkningen fraskilles overskydende 12 5 uomsat I fra det mærkede huTF ved afsaltningschromatografi 5 på "Sephadex G25" (Pharmacia, Inc., Piscataway, NJ) under anvendelse af TBX/Triton.Rockford, IL). Following the labeling, excess 125 of unreacted I is separated from the labeled huTF by desalting chromatography 5 on "Sephadex G25" (Pharmacia, Inc., Piscataway, NJ) using TBX / Triton.

125I-mærkede huTF-holdige prøver vurderes ved natri -umdodecylsulf at -polyacrylamidgelelektroforese (SDS-PAGE) ifølge Laemmli, Nature, 227:680-685 (1970). Dithiotreitol 10 (DTT, Sigma) inkluderes i prøvepufferen i en koncentration på 100 mM for de prøver, der vurderes under reducerende betingelser. Immunopræcipitationerne gennemføres ved inku-bering natten over ved 4°C af 12^I-huTF i 1% "Triton X-100", 50 mM tris-HCl (pH-værdi 7,4), 150 mM natriumchlorid med 15 l/10 volumen TF8-5G9 eller PAb 100 (ATCC TIB 115, et hybri-doma, der producerer et SV40 stort T-antigenspecifikt antistof, som her anvendes som negativ kontrol) hybridomakultur-ovenstående væske. Gede-anti-muse-IgG immubiliseret på aga-roseperler (Sigma Chemical Co., St. Louis, MO) anvendes 20 derefter til at adsorbere de primære immunoreaktionsprodukter. Perlerne vaskes grundigt med den samme puffer, og det bundne 125I-huTF elueres ved kogning i 5 minutter i prøvepuffer med eller uden DTT. Proteinbåndene gøres synlige efter SDS-PAGE ved autofluorografi.125 I-labeled huTF-containing samples are evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli, Nature, 227: 680-685 (1970). Dithiotreitol 10 (DTT, Sigma) is included in the sample buffer at a concentration of 100 mM for the samples evaluated under reducing conditions. The immunoprecipitations are performed by overnight incubation at 4 ° C of 12 µl-huTF in 1% Triton X-100, 50 mM Tris-HCl (pH 7.4), 150 mM sodium chloride at 15 L / 10 volume TF8-5G9 or PAb 100 (ATCC TIB 115, a hybridoma producing a SV40 large T antigen-specific antibody used here as a negative control) hybridoma culture supernatant. Goat anti-mouse IgG immobilized on agarose beads (Sigma Chemical Co., St. Louis, MO) is then used to adsorb the primary immunoreaction products. The beads are washed thoroughly with the same buffer and the bound 125 I-huTF is eluted by boiling for 5 minutes in sample buffer with or without DTT. The protein bands are made visible after SDS-PAGE by autofluorography.

25 Når isoleret huTF er radioioderet, reduceret med DTT25 When isolated huTF is radioiodinated, reduced by DTT

og analyseret ved SDS-PAGE på 10%'s acrylamidgeler, observeres der et enkelt større bånd med en tilsyneladende molekylvægt på 47 kDa (fig. 4). Når ikke-reduceret huTF analyseres på lignende måde, observeres der imidlertid to bånd 30 på ca. 58 og 47 kDa i relativt lige store mængder (fig. 5, bane B), hvilket tyder på mindst to forskellige størrelsesformer .and analyzed by SDS-PAGE on 10% acrylamide gels, a single larger band with an apparent molecular weight of 47 kDa is observed (Fig. 4). However, when non-reduced huTF is similarly analyzed, two bands 30 of ca. 58 and 47 kDa in relatively equal amounts (Fig. 5, lane B), suggesting at least two different size shapes.

Mulige forklaringer af de to bånd, der observeres i fravær af reduktion, er, at det større, dvs. langsommere 35 vandrende bånd kan være kraftigere glycosyleret, kan have yderligere uforarbejdet protein eller kan være knyttet til 38 DK 176220 B1 yderligere disulfidbindings-bundne polypeptider. Tilstedeværelsen af et enkelt bånd efter reduktion stemmer ikke overens med de to første forslag. Den sidste mulighed forekommer mest sandsynlig, men på grund af den ringe størrel-5 sesforskel vil yderligere polypeptidkæder formentlig være små nok til at vandre ved eller nær farvefronten og ikke blive opløst ved elektroforese på 10%'s acrylamidgeler efter reduktion. Elektroforese af reduceret og ikke-reduceret huTF på 15%'s polyacrylamidgeler viser ingen enkelt særskilt 10 let kæde, selv om flere mindre, hurtigt vandrende bånd observeres (fig. 5, bane A og B) . Disse små polypeptider i mindre mængde kan repræsentere urenheder, som tidligere er blevet bemærket (Broze et al., J. Biol. Chem., 260:10917- 20 (1985) og Guha et al., Proc. Natl. Acad. Sci. USA 83:299-15 3 02 (1986) ) . For klart at løse problemet udskæres båndene på 47 og 58 kDa fra den ikke-reducerede gel, og hvert bånd reduceres med dithiothreitol og underkastes særskilt SDS-PAGE på en 15%'s acrylamidgel (fig. 5, bane C og D). 58 kDa-pro-teinet opløses i en let kæde på 12,5 kDa og en tung kæde på 20 47 kDa. Når 47 kDa-proteinet undersøges, observeres der kun en tung kæde med samme molekylvægt. Således har begge former tunge kæder med lignende opførsel ved SDS-PAGE.Possible explanations of the two bands observed in the absence of reduction are that the larger, i.e. slower 35 migratory bands may be more strongly glycosylated, may have additional unprocessed protein, or may be associated with additional disulfide bond-bound polypeptides. The presence of a single band after reduction does not agree with the first two proposals. The latter option seems most likely, but due to the small size difference, additional polypeptide chains will probably be small enough to migrate at or near the color front and not be resolved by electrophoresis on 10% acrylamide gels after reduction. Electrophoresis of reduced and non-reduced huTF on 15% polyacrylamide gels shows no single distinct light chain, although several smaller, rapidly migrating bands are observed (Fig. 5, lanes A and B). These small polypeptides in smaller amounts may represent impurities previously noted (Broze et al., J. Biol. Chem., 260: 10917-20 (1985) and Guha et al., Proc. Natl. Acad. Sci. USA 83: 299-1532 (1986)). To clearly solve the problem, the 47 and 58 kDa bands are excised from the non-reduced gel and each band reduced with dithiothreitol and subjected to SDS-PAGE separately on a 15% acrylamide gel (Figure 5, lanes C and D). The 58 kDa protein is dissolved in a light chain of 12.5 kDa and a heavy chain of 20 47 kDa. When the 47 kDa protein is examined, only a heavy chain of the same molecular weight is observed. Thus, both forms have heavy chains of similar behavior at SDS-PAGE.

For at påvise tilstedeværelsen af den lette kæde direkte immupræcipiteres -huTF med det huTF-specifikke 25 monoklonale antistof TF8-5G9 og underkastes elektroforese i nærværelse af reduktionsmiddel. Det større 47 kDa-bånd observeres sammen med et særskilt bånd på ca. 12,5 kDa (fig.In order to detect the presence of the light chain directly, the huTF is immunoprecipitated with the huTF-specific monoclonal antibody TF8-5G9 and subjected to electrophoresis in the presence of reducing agent. The larger 47 kDa band is observed together with a separate band of approx. 12.5 kDa (fig.

6, bane A). Elektroforese af prøven uden forudgående reduktion giver bånd på ca. 47 og 58 kDa, men ikke noget polypep-30 tid med lav molekylvægt (fig. 6, bane B). Elektroforese af ikke-reduceret huTF giver også mindre mængder af et 90 kDa-protein, hvilket stemmer overens med en dimer af den tunge kæde af huTF, der er blevet foreslået af Broze et al., J.6, lane A). Electrophoresis of the sample without prior reduction gives bands of approx. 47 and 58 kDa, but no low molecular weight polypeptide (Fig. 6, lane B). Electrophoresis of non-reduced huTF also yields smaller amounts of a 90 kDa protein, which is consistent with a dimer of the huTF heavy chain suggested by Broze et al., J.

Biol. Chem., 260:10917-20 (1985).Biol. Chem., 260: 10917-20 (1985).

3 5 For at undersøge muligheden for, at den lette kæde af huTF kan være afledt proteolytisk fra den tunge kæde, 39 DK 176220 B1 underkastes de lette og tunge kæder, der er isoleret ved SDS-PAGE, en N-terminal aminosyresekvensanalyse.To examine the possibility that the light chain of huTF may be proteolytically derived from the heavy chain, the light and heavy chains isolated by SDS-PAGE are subjected to an N-terminal amino acid sequence analysis.

De tunge og lette kæder adskilles ved SDS-PAGE og elektroblottes på aktiverede, aminoderivatiserede glasfiber-5 filtre ved anvendelse af høj-pH-værdimetoden ifølge Abersold et al., J. Biol. Chem., 261:4229-4238 (1986). Proteinbåndene gøres synlige på blottene ved fluorescensfarvning ifølge samme reference, udskæres og sekvensbestemmes, stadig bundet til glasfibermaterialet, i et Applied Biosystems 470A-pro- 10 teinsekvensbestemmelsesapparat med on-line-HPLC-analyse af PTH-derivater. Alternativt gøres proteinbåndene synlige på gelen ved farvning med Coomassie-blåt og elektroelueres til sekvensbestemmelse. Begge metoder giver ækvivalente resultater .The heavy and light chains are separated by SDS-PAGE and electroblotted onto activated, amino derivatized glass fiber filters using the high pH method of Abersold et al., J. Biol. Chem., 261: 4229-4238 (1986). The protein bands are made visible on the blots by fluorescence staining according to the same reference, cut and sequenced, still bound to the fiberglass material, in an Applied Biosystems 470A protein sequencing apparatus with on-line HPLC analysis of PTH derivatives. Alternatively, the protein bands are made visible on the gel by staining with Coomassie blue and electroeluted for sequencing. Both methods give equivalent results.

15 Mikrosekvensbestemmelse af de tunge kæder af huTFMicrosequencing of the heavy chains of huTF

giver konsekvent to samtidige aminosyresekvenser i tilnærmelsesvis ækvimolære mængder, I næsten alle tilfælde viser hver aminosyrerest sig to gange med to cyclers adskillelse.consistently gives two simultaneous amino acid sequences in approximately equimolar amounts. In almost all cases, each amino acid residue appears twice with two cycles of separation.

Dette er et tegn på forskudte N-terminusser af to varianter 2 0 af tung kæde af huTF, der adskiller sig med to rester i længde ved N-terminussen. N-terminussen af den større variant udledes at være Ser-Gly-X-X-Asn-Thr-Val-Ala-Ala-Tyr-X-Leu-Thr-Trp-Lys-Ser, hvor X betyder en uspecificeret aminosyrerest .This is a sign of staggered N-terminals of two variants 20 of the heavy chain of huTF differing by two residues in length at the N-terminus. The N-terminus of the larger variant is deduced to be Ser-Gly-X-X-Asn-Thr-Val-Ala-Ala-Tyr-X-Leu-Thr-Trp-Lys-Ser, where X represents an unspecified amino acid residue.

25 Flere forsøg på at sekvensbestemme den lette kæde har ikke givet nogen sekvensinformation, hvilket stemmer overens med en blokeret N-terminus. Imidlertid er den tunge og lette kæde af huTF antigent forskellig, idet to kanin--anti-huTF-antisera og 28 monoklonale muse-antistoffer frem- 3 0 kaldt mod isoleret huTFh alle viser sig at binde til den tunge kæde alene. Derfor er det usandsynligt, at den lette kæde er et proteolytisk fragment af den tunge kæde. Desuden reagerer den lette kæde ikke med antisera mod E2-mikroglo-bulin, 35 Betydningen af den 12,5 kDa lette kæde af huTF er i øjeblikket ukendt. Det er usandsynligt at den er blevet 40 DK 176220 B1 tilvejebragt kunstigt under isoleringen ved tilfældig disul-fidudveksling, da den er en enkelt særskilt molekylart. Når affinitetsisoleret huTF underkastes SDS-PAGE uden reduktion, elueres huTF-aktivitet fra gelerne svarende til både 58 kDa 5 og 47 kDa mo1eky1vægtsformerne. huTF-aktiviteten svarende til disse to molekylvægtsformer påvises også, når rå hjerne eller delvis isoleret placentaekstrakt underkastes elektro-forese på SDS-geler (resultater ikke vist). I alle tilfælde er aktiviteten faktor Vll-afhængig, hvilket viser huTF-spe-10 cifik aktivitet. Disse resultater viser, at huTFh alene kan aktivere faktor VII, og at den lette kæde ikke er nødvendig til denne funktion.Several attempts to sequence the light chain have yielded no sequence information, which is consistent with a blocked N-terminus. However, the heavy and light chain of huTF antigen is different in that two rabbits - anti-huTF antisera and 28 monoclonal mouse antibodies raised against isolated huTFh all appear to bind to the heavy chain alone. Therefore, the light chain is unlikely to be a proteolytic fragment of the heavy chain. Furthermore, the light chain does not respond with antisera to E2 microglobulin, 35 The significance of the 12.5 kDa light chain of huTF is currently unknown. It is unlikely to have been artificially provided during the isolation by random disulphide exchange, since it is a single distinct molecular species. When affinity-insulated huTF is subjected to SDS-PAGE without reduction, huTF activity is eluted from the gels corresponding to both the 58 kDa 5 and 47 kDa molecular weight forms. The huTF activity corresponding to these two molecular weight forms is also detected when raw brain or partially isolated placental extract is subjected to electrophoresis on SDS gels (results not shown). In all cases, the activity is factor VII-dependent, showing huTF-specific activity. These results show that huTFh alone can activate factor VII and that the light chain is not required for this function.

Det er af interesse, at den lette kæde kun er disul-fidbundet til ca. halvdelen af de tunge kæder af huTF. Den 15 er enten fraværende in vivo fra en væsentlig andel af huTF eller er til stede, men er tilknyttet via ikke-kovalente vekselvirkninger, der kan brydes med detergenter. Den lette kæde af huTF kan have været ubemærket ved tidligere undersøgelser, fordi den ringe størrelse vil bevirke vandring 20 ved farvestoffronten ved SDS-PAGE, og fordi publicerede analyser af huTF er blevet gennemført efter reduktion. De begrænsede mængder, der kan isoleres ved anvendelse af gængse affinitetsmetoder, gør det vanskeligt at påvise en associeret lille polypeptidkæde ved proteinfarvning.It is of interest that the light chain is only disulfide bonded to approx. half of the heavy chains of huTF. It is either absent in vivo from a substantial proportion of huTF or is present, but is associated through non-covalent interactions that can be broken by detergents. The light chain of huTF may have been unnoticed in previous studies because the small size will cause migration 20 at the dye front by SDS-PAGE and because published analyzes of huTF have been performed after reduction. The limited amounts that can be isolated using conventional affinity methods make it difficult to detect an associated small polypeptide chain in protein staining.

25 Selv om monomert huTF vil initiere koagulering in vitro, forekommer der fysiologisk initiering af koagulering ved hjælp af huTF på celleoverfladen. Man kan forestille sig, at den lette kæde kan spille mere subtile roller ved huTF-funktionen eller -organisationen, end man kan påvise 30 ved en sædvanlig koaguleringsbestemmelse. F.eks. kan den lette kæde være involveret i samlingen af receptoren af to underenheder for faktor VII, hvilket er fremsat som hypotese for at forklare den tilsyneladende positive samvirken af binding af faktor VII/VIIa til vævsfaktor. Alternativt kan 35 organisationen af huTF i strukturelle domæner på celleover- 41 DK 176220 B1 fladen og regulering af huTF-aktivitet på celleoverflader være medieret af den lette kæde af huTF,Although monomeric huTF will initiate coagulation in vitro, physiological initiation of coagulation by means of huTF occurs on the cell surface. It is envisaged that the light chain may play more subtle roles in the huTF function or organization than can be detected in a conventional coagulation assay. Eg. For example, the light chain may be involved in the assembly of the receptor of two factor VII subunits, which is hypothesized to explain the apparently positive interaction of factor VII / VIIa binding to tissue factor. Alternatively, the organization of huTF in structural domains on the cell surface and regulation of huTF activity on cell surfaces may be mediated by the light chain of huTF.

Rollen af N-bundne oligosaccharider undersøges ved at deglycosylere en prøve af 12^I-huTF. Ca. 12,74 ng afThe role of N-linked oligosaccharides is investigated by deglycosylating a sample of 12 µl-huTF. Ca. 12.74 ng of

5 mærket huTF indeholdende ca. 3,6 x 10^ tællinger pr. minut (cpm) blandes med 2 0 μΐ af en opløsning indeholdende 0,4 enheder glycopeptidase F (Boehringer-Mannheim Biochemicals, Indianapolis, IN), 20 mM tris-HCL (pH-værdi 7,5), 20 mM5 labeled huTF containing approx. 3.6 x 10 5 counts per per minute (cpm) is mixed with 20 µΐ of a solution containing 0.4 units of glycopeptidase F (Boehringer-Mannheim Biochemicals, Indianapolis, IN), 20 mM Tris-HCL (pH 7.5), 20 mM

EDTA og 1% "Triton X-100" og holdes derefter i 16 timer ved 10 37°C. De deglycosylerede produkter analyseres derefter ved SDS-PAGE som ovenfor beskrevet.EDTA and 1% "Triton X-100" and then kept for 16 hours at 10 37 ° C. The deglycosylated products are then analyzed by SDS-PAGE as described above.

Resultaterne af deglycosyleringsundersøgelserne, de er vist i fig. 7, bane 4 og 5, viser, at 58 kDa-formen af huTF udviser en højere relativ molekylær end 47 kDa-formen 15 på grund af tilstedeværelsen af yderligere proteindele, dvs. den lette kæde.The results of the deglycosylation studies shown in FIG. 7, lanes 4 and 5, show that the 58 kDa form of huTF exhibits a higher relative molecular weight than the 47 kDa form 15 due to the presence of additional protein moieties, i. the light chain.

Det således isolerede huTF relipideres til rekonstituering af dets prokoagulerende aktivitet. Det nødvendige forhold mellem vævsfaktor og lipid til tilvejebringelse af 20 et relipideret vævsfaktorprodukt, der har maksimal aktivitet, bestemmes empirisk ved at opløse det isolerede huTF, der er fremstillet ovenfor, i forskellige koncentrationer i HBS--pufferopløsning (20 mM Hepes, pH-værdi 6,0, 140 mM natrium-chlorid, 0,01% natriumazid) indeholdende 0,1% BSA. De for-25 skellige huTF-fortyndinger relipideres derefter som beskrevet nedenfor, og det forhold, der giver den højeste rekonstituerede aktivitet som bestemt ved koaguleringsbestemmelsen beskrevet i eksempel 2, benyttes til senere anvendelse.The huTF thus isolated is relipidated to reconstitute its procoagulant activity. The necessary ratio of tissue factor to lipid to provide a maximized activity of a reciprocated tissue factor product is empirically determined by dissolving the isolated huTF prepared above at various concentrations in HBS buffer solution (20 mM Hepes, pH 6.0, 140 mM sodium chloride, 0.01% sodium azide) containing 0.1% BSA. The various huTF dilutions are then precipitated as described below and the ratio giving the highest reconstituted activity as determined by the coagulation assay described in Example 2 is used for later use.

Lipider til relipidering af huTF fremstilles ved 3 0 ekstraktion fra kaninhjerne/acetone-pulver fra Sigma Chemical Co., St. Louis, MO. Pulveret blandes med heptan/butanol i volumenforholdet 2 .*1 i et forhold på 25 ml heptan/butanol pr. g pulver, og det faste stof indeholdt deri genvindes ved filtrering ved anvendelse af sintret glastragt. Denne 35 ekstraktionsproces gentages seks gange på det tilbageværende faste stof. Det tilbageværende faste stof tørres derefter i 42 DK 176220 B1 en rotationsfordamper, opløses i chloroform og opbevares ved -80°C. Efter behov tørres portioner af det chloroformopløste faste stof under nitrogen og opløses til en koncentration på 4 mg/ml i en opløsning af frisk fremstillet 0,25%'s na-5 triumdeoxypolat til dannelse af en kaninhjerne-phospholipid--opløsning (RBPL).Lipids for releasing huTF are prepared by extraction from rabbit brain / acetone powder from Sigma Chemical Co., St. Louis, MO. The powder is mixed with heptane / butanol in a volume ratio of 2. * 1 in a ratio of 25 ml of heptane / butanol per liter. g of powder and the solid contained therein is recovered by filtration using sintered glass funnel. This extraction process is repeated six times on the remaining solid. The residual solid is then dried in a rotary evaporator, dissolved in chloroform and stored at -80 ° C. As needed, portions of the chloroform-dissolved solid are dried under nitrogen and dissolved to a concentration of 4 mg / ml in a solution of freshly prepared 0.25% sodium deoxypolate to form a rabbit brain phospholipid solution (RBPL) .

Til relipidering blandes 100 μΐ af hver huTF-fortyn-ding med 100 μΐ RBPL-opløsning, 0,76 ml HBS-opløsning indeholdende 0,1% bovint serumalbumin (HBS/BSA) og 40 μΐ af en 10 100 mM cadmiumchloridopløsning. Denne blanding holdes ved 37°C i 2 dage, og aktiviteten af huTF deri bestemmes ved koaguleringsbestemmelsen beskrevet i eksempel 2.For precipitation, mix 100 μΐ of each huTF dilution with 100 μΐ of RBPL solution, 0.76 ml of HBS solution containing 0.1% bovine serum albumin (HBS / BSA) and 40 μΐ of a 10 100 mM cadmium chloride solution. This mixture is maintained at 37 ° C for 2 days and the activity of huTF therein is determined by the coagulation assay described in Example 2.

5. Fremstilling af hvbridomaer og monoklonale antistoffer.5. Preparation of hybridomas and monoclonal antibodies.

15 Alle hybridomaer fremstilles ved anvendelse af milt- celler fra Balb/c-hunmus med en alder på 6-8 uger, der fås fra Scripps Clinic and Research Institute's vivarium.All hybridomas are prepared using spleen cells from Balb / c female mice of 6-8 weeks age obtained from the Scripps Clinic and Research Institute's vivarium.

a. Immunisering af TF8-mus.a. Immunization of TF8 mice.

5 μg affinitetsisoleret huTF fremstillet i eksempel 20 4 opløses i normalt saltvand i en koncentration på 100 μg/ml, og kombineres og emulgeres derefter i volumenforholdet 1:1 med R-700-adjuvans fra Ribi Immunochem Research, Inc. , Hamilton, MO. Emulsionen injiceres derefter subcutant (s.c.) i TF8-mus.5 µg affinity-insulated huTF prepared in Example 20 4 is dissolved in normal saline at a concentration of 100 µg / ml and then combined and emulsified in 1: 1 volume ratio with R-700 adjuvant from Ribi Immunochem Research, Inc. , Hamilton, MO. The emulsion is then injected subcutaneously (s.c.) into TF8 mice.

25 TF8-mus podes på lignende måde ca. 2 uger senere ved anvendelse af en emulsion indeholdende denatureret huTF og R-700-adjuvans. Denatureret huTF fremstilles ved kogning i 5 minutter af TBS (150 mM calciumchlorid, 50 mM tris-HCl (pH-værdi 7,5)) indeholdende 0,09% "Triton X-100", 0,93% 30 SDS, 0,2 N 2-mercaptoethanol og huTF i en mængde på 270 /ig/ml. Derefter blandes det denaturerede huTF med et lige så stort volumen normalt saltvand indeholdende 0,6 mg/ml museserumalbumin. Derefter blandes 4 volumener acetone med opløsningen af denatureret huTF, og den fremkomne blanding 35 holdes natten over ved -20°C. Det fremkomne bundfald opsamles ved centrifugering ved ca. 13.000 g i 10 minutter, vaskes én 43 DK 176220 B1 gang med en blanding af acetone og vand i volumenforholdet 4:1 og suspenderes derefter i 200 μΐ normalt saltvand i en koncentration på 0,1 mg/ml.Similarly, 25 TF8 mice are seeded approx. 2 weeks later using an emulsion containing denatured huTF and R-700 adjuvant. Denatured huTF is prepared by boiling for 5 minutes by TBS (150 mM calcium chloride, 50 mM tris-HCl (pH 7.5)) containing 0.09% "Triton X-100", 0.93% SDS, 0. 2 N 2-mercaptoethanol and huTF in an amount of 270 µg / ml. Then, the denatured huTF is mixed with an equal volume of normal saline containing 0.6 mg / ml mouse serum albumin. Then, 4 volumes of acetone are mixed with the solution of denatured huTF and the resulting mixture is kept overnight at -20 ° C. The resulting precipitate is collected by centrifugation at ca. 13,000 g for 10 minutes, washed once with a mixture of acetone and water in a volume ratio of 4: 1 and then suspended in 200 μΐ normal saline at a concentration of 0.1 mg / ml.

Ca. 4 uger efter den første injektion blandes 33 μg 5 affinitetsisoleret (ikke-denatureret) huTF i 0,1 ml normalt saltvand med 0,1 ml komplet Freund's adjuvans (CFA) til dannelse af en emulsion. Denne emulsion injiceres derefter intraperitonealt (i.p.) i TF8-mus.Ca. 4 weeks after the first injection, 33 µg of 5 affinity-insulated (non-denatured) huTF is mixed in 0.1 ml of normal saline with 0.1 ml of complete Freund's adjuvant (CFA) to form an emulsion. This emulsion is then injected intraperitoneally (i.p.) in TF8 mice.

Ca. 8 uger efter den første podning injiceres 15 μg 10 affinitetsisoleret huTF i phosphatpufret saltvand (PBS) intravenøst (i.v.), og en identisk huTF/PBS-podning gives intravenøst 24 timer senere. TF8-musenes miltceller høstes til fusion 3 dage senere.Ca. At 8 weeks after the first inoculation, 15 μg of 10 affinity-insulated huTF is injected into phosphate-buffered saline (PBS) intravenously (i.v.) and an identical huTF / PBS inoculation is given intravenously 24 hours later. Spleen cells of the TF8 mice are harvested for fusion 3 days later.

b. Immunisering af TF9-mus.b. Immunization of TF9 mice.

15 TF9-mus underkastes den samme podningsplan som TF8- -mus, bortset fra, at der ved begge Ribi-adjuvansinjektioner anvendes huTF, der er denatureret før emulgering. Desuden indgives det første PBS-podemateriale intraperitonealt og 4 1/2 måned efter det CFA-holdige podemateriale.15 TF9 mice are subjected to the same grafting schedule as TF8 mice, except that in both Ribi adjuvant injections, huTF denatured prior to emulsification is used. In addition, the first PBS graft material is administered intraperitoneally and 4 1/2 months after the CFA-containing graft material.

20 c. Hvbridoma-dannelse.20 c. Hybridoma formation.

Der anvendes samme fusionsprocedure for både TF8- og TF9-afledte miltceller. Ca. 1 x 10® miltceller fra hver mus blandes med 2 x ΙΟ7 P3X63 Ag8.653.1-myelomaceller i 200 μΐ af et fusionsmedium omfattende 30% (vægt/volumen) polyethy- 25 lenglycol (PEG 4000, ATCC 25322-68-3) . Efter cellefusion udsås de resulterende hybridomaer i 96-brønds plader, dyrkes i HAT-medium (hypoxanthin, aminopterin og thymidin) og screenes derefter for evnen til at producere antistofmolekyler, der reagerer med huTF.The same fusion procedure is used for both TF8 and TF9 derived spleen cells. Ca. 1 x 10 6 spleen cells from each mouse are mixed with 2 x 7 P3X63 Ag8.653.1 myeloma cells in 200 μΐ of a fusion medium comprising 30% (w / v) polyethylene glycol (PEG 4000, ATCC 25322-68-3). After cell fusion, the resulting hybridomas were seeded into 96-well plates, grown in HAT medium (hypoxanthine, aminopterin and thymidine) and then screened for the ability to produce huTF antibody molecules.

30 Både TF8- og TF9-muse-miltcelle-fusionerne resulterer i HAT-medium-resistente hybridoma-cellekloner. TF8-fusionen giver 907 HAT-resistente hybridomaer, medens TF9-fusionen giver 348 HAT-resistente hybridomaer.Both the TF8 and TF9 mouse spleen cell fusions result in HAT medium-resistant hybridoma cell clones. The TF8 fusion produces 907 HAT-resistant hybridomas, while the TF9 fusion yields 348 HAT-resistant hybridomas.

44 DK 176220 B1 6 * Screening af hybridomaer for produktion af anti-huTF-antistofmolekyler.44 DK 176220 B1 6 * Screening of hybridomas for production of anti-huTF antibody molecules.

a. Fastfase-RIA.a. Solid phase RIA.

100 μΐ gede-anti-muse-IgG (Boehringer-Mannheim Bio-5 chemicals, Indianapolis, IN) fortyndet til 20 /zg/ml i TBS blandes i brøndene af "Immulon" 96-brønds fleksible vinyl--mikrotiterplader (Dynatech Laboratories, Alexandria, VA) . Pladerne holdes derefter i 1 time ved 37°C, således at IgG får lov at adsorbere til væggene af brøndene. Efter vaskning 10 tre gange med TBS blandes 100 μΐ TBS/"Triton" indeholdende 3% ovalbumin i hver brønd til blokering af overskydende proteinbindingssteder.100 µg goat anti-mouse IgG (Boehringer-Mannheim Bio-5 chemicals, Indianapolis, IN) diluted to 20 µg / ml in TBS is mixed in the wells of "Immulon" 96-well flexible vinyl microtiter plates (Dynatech Laboratories, Alexandria, VA). The plates are then kept for 1 hour at 37 ° C so that IgG is allowed to adsorb to the walls of the wells. After washing 10 three times with TBS, 100 μΐ TBS / "Triton" containing 3% ovalbumin in each well is mixed to block excess protein binding sites.

Brøndene holdes i 1 time ved ca. 20°C, og derefter fjernes blokeringsopløsningen ved opsugning. 50 μΐ ovenstå-15 ende væske fra hybridomakultur blandes i hver brønd. Den resulterende fast fase/væskefase - i mmunoreakt ionsblanding holdes ved 37°C i 1 time. Brøndene skylles derefter tre gange med TBS, og overskud af væske fjernes ved opsugning.The wells are kept for 1 hour at approx. 20 ° C, and then the blocking solution is removed by aspiration. 50 μΐ of supernatant from hybridoma culture is mixed in each well. The resulting solid phase / liquid phase - in mmunoreaction mixture is maintained at 37 ° C for 1 hour. The wells are then rinsed three times with TBS and excess fluid is removed by aspiration.

50 μΐ 12^I-mærket huTF fremstillet i eksempel 4 og 20 indeholdende ca. 1 ng huTF og ca. 5 x 105 cpm i TBS/"Triton" blandes i hver brønd til dannelse af en anden fastfase/væ-skefase-immunoreaktionsblanding. Brøndene holdes i 2 timer ved 37°C og skylles derefter tre gange med TBS/"Triton" til isolering af de fastfasebundne 125I-huTF-holdige immunoreak-25 tionsprodukter. Overskydende væske fjernes ved opsugning, og brøndene får lov at tørre. De enkelte brønde skæres fra hinanden, og 125I indeholdt i hver brønd bestemmes med en -Y -tæller.50 µl of 12 µl labeled huTF prepared in Examples 4 and 20 containing ca. 1 ng huTF and approx. 5 x 10 5 cpm in TBS / "Triton" are mixed in each well to form a different solid-phase / liquid-phase immunoreaction mixture. The wells are maintained for 2 hours at 37 ° C and then rinsed three times with TBS / "Triton" to isolate the solid phase-bound 125 I-huTF-containing immunoreaction products. Excess liquid is removed by aspiration and the wells are allowed to dry. The individual wells are cut apart and 125 I contained in each well is determined with a -Y counter.

Baggrundsradioaktiviteten (ingen reaktion af huTF 30 med antistof) er i gennemsnit ca. 200-300 cpm pr. brønd, medens positive reaktioner af huTF med antistof giver 10.000 cpm pr. brønd. Hybridomaer, der bestemmes som positive med hensyn til produktion af anti-huTF-antistoffer, udvælges og udgør hybridomaer ifølge opfindelsen. Derefter screenes 35 disse hybridomaer ved den nedenfor beskrevne dot-blot-bestemmelse .The background radioactivity (no reaction of huTF 30 with antibody) averages approx. 200-300 cpm. well, while positive reactions of huTF with antibody yield 10,000 cpm. well. Hybridomas determined as positive for production of anti-huTF antibodies are selected and constitute hybridomas of the invention. Then, these hybridomas are screened by the dot-blot assay described below.

45 DK 176220 B1 b. Dot-blot-ELISA.45 DK 176220 B1 b. Dot-blot ELISA.

Acetoneudfældet huTF fremstillet i eksempel 4 ekstra-heres to gange med en blanding af acetone og vand i volumenforholdet 4:1. Det tilbageværende bundfald resuspenderes 5 i en mængde på 20 rø/ml i TBS. 20 ng (1 jul) af denne huTF-op-løsning afsættes som en plet på BA83-nitrocellulosepapir (Schleicher and Schuell, Keene, NH) ved siden af et tal skrevet på papiret med mærkeblæk. huTF afsat som en plet lufttørres, og de enkelte pletter udskæres derefter til 10 cirkulære papirskiver ved anvendelse af en perforator. De enkelte papirstykker nedsænkes i enkeltbrønde af en bakke med flere brønde indeholdende "BLOTTO" (Johnson et al.,Acetone precipitated huTF prepared in Example 4 is extracted twice with a mixture of acetone and water in the 4: 1 volume ratio. The remaining precipitate is resuspended in an amount of 20 tubes / ml in TBS. 20 ng (July 1) of this huTF solution is deposited as a spot on BA83 nitrocellulose paper (Schleicher and Schuell, Keene, NH) next to a number written on the paper with ink. huTF deposited as a stain is air dried and the individual stains are then cut into 10 circular paper slices using a perforator. The individual pieces of paper are immersed in single wells by a multi-well tray containing "BLOTTO" (Johnson et al.,

Gene. Anal. Tech., 1:3 (1984)) og holdes ved 37°C i ca. 1 time.Gene. Anal. Tech., 1: 3 (1984)) and maintained at 37 ° C for approx. 1 hour.

15. "BLOTTO" fjernes fra brøndene ved opsugning, og der sættes 200 μΐ ovenstående væske fra hybridomakultur til hver brønd. Brøndene holdes derefter ved 37°C i 2 timer. Papirstykkerne skylles to gange med TBS, fjernes fra brøndene og samles i enkelt større beholder til yderligere skylning 20 i TBS. Overskud af væske fjernes derefter fra beholderen.15. "BLOTTO" is removed from the wells by aspiration and 200 µΐ of the above liquid from hybridoma culture is added to each well. The wells are then kept at 37 ° C for 2 hours. The pieces of paper are rinsed twice with TBS, removed from the wells and collected in a single larger container for further rinsing 20 in TBS. Excess liquid is then removed from the container.

Alkalisk phosphatase-konjugeret anti-muse-IgG i pro-toblot-reagenssættet (Promega Biotech, Ann Arbor, MI) fortyndes 1:5700 i BLOTTO og kontakteres med papirstykkerne. Protoblot-opløsningen holdes i kontakt i 30 minutter ved 25 37°C. Papirstykkerne skylles derefter tre gange i TBS. Bundet alkalisk phosphatase påvises på papirstykkerne ved anvendelse af chromogene substrater, der leveres med protoblot-sættet ifølge producentens anvisninger.Alkaline phosphatase-conjugated anti-mouse IgG in the pro-toblot reagent kit (Promega Biotech, Ann Arbor, MI) is diluted 1: 5700 in BLOTTO and contacted with the pieces of paper. The protoblot solution is kept in contact for 30 minutes at 37 ° C. The pieces of paper are then rinsed three times in TBS. Bound alkaline phosphatase is detected on the pieces of paper using chromogenic substrates supplied with the protoblot kit according to the manufacturer's instructions.

c. Western blot-bestemmelse.c. Western blot determination.

30 Til Western-blot-bestemmelser opløses ca. 10 μg huTF30 For Western blot determinations, approx. 10 µg huTF

isoleret som beskrevet i eksempel 4 i prøvepuffer (2% SDS, 50 mM dithiothreitol, 10% glycerol, 125 mM tris-HCl, pH-værdi 6,8) og koges i 5 minutter. Det underkastes derefter SDS-polyacrylamidgel-elektroforese på en præparativ pladegel 35 som beskrevet af Laemmli, Nature, 226:680 (1970) i en bred bane, der på begge sider er flankeret af smalle baner in- 46 DK 176220 B1 deholdende for-farvede molekylvægtstandarder (Diversified Biotech, Newton Centre, MA) . Efter elektroforese og elektro-blotting på nitrocellulose som beskrevet af Towbin et al.,isolated as described in Example 4 in sample buffer (2% SDS, 50 mM dithiothreitol, 10% glycerol, 125 mM tris-HCl, pH 6.8) and boiled for 5 minutes. It is then subjected to SDS-polyacrylamide gel electrophoresis on a preparative plate gel 35 as described by Laemmli, Nature, 226: 680 (1970) in a wide web flanked on both sides by narrow webs containing pre-colored molecular weight standards (Diversified Biotech, Newton Center, MA). Following electrophoresis and electroblotting on nitrocellulose as described by Towbin et al.,

Proc. Natl. Acad. Sci. USA, 76:4350 (1979) blokeres blottet 5 med en opløsning af 5% fedtfrit mælkepulver i TBS og fastgøres i en manifold (Miniblotter: Immunetics, Cambridge, MA). Pools af otte ovenstående væsker fra hybridomacellekultur anbringes i hver manifold-spalte og inkuberes i 1 time ved 37°C, hvorefter blottet fjernes og skylles med TBA (TBS 10 indeholdende 0,02% natriumazid). Baner, som har bundet antistof, gøres synlige ved anvendelse af et alkalisk phospha-tase-konjugeret andet antistof fremkaldt med et chromogent substrat (Protoblot; Promega Biotech, Madison, WI) ifølge producentens foreslåede metoder. Ovenstående væsker fra 15 kultur fra positive pools testes igen enkeltvis ved en fortynding på 1:8 i 5% fedtfrit mælkepulver/TBA til identificering af individuelle hybridoma-kloner, der producerer anti-TF-antistoffer.Proc. Natl. Acad. Sci. United States, 76: 4350 (1979), bare 5 is blocked with a solution of 5% fat-free milk powder in TBS and fixed in a manifold (Mini Blotter: Immunetics, Cambridge, MA). Pools of eight supernatants from hybridoma cell culture are placed in each manifold slot and incubated for 1 hour at 37 ° C, then the blot is removed and rinsed with TBA (TBS 10 containing 0.02% sodium azide). Pathways that have bound antibody are made visible using an alkaline phosphatase-conjugated second antibody developed with a chromogenic substrate (Protoblot; Promega Biotech, Madison, WI) according to the manufacturer's suggested methods. The above 15 culture fluids from positive pools are again tested individually at a 1: 8 dilution in 5% fat-free milk powder / TBA to identify individual hybridoma clones producing anti-TF antibodies.

Hybridomaer, der bestemmes at være positive med hensyn 20 til produktion af anti-huTF-antistoffer, udvælges til yderligere karakterisering. F.eks. karakteriseres hybridomaer fra den ovennævnte TF8-fusion som anti-huTF-antistofproducerende hybridomakulturer, hvis de ovenstående væsker fra hybridomakulturen udviser immunoreaktion med huTF ved dot-25 -blot-bestemmelsen beskrevet i eksempel 6b og fastfase-RIA beskrevet i eksempel 6a. Denne karakterisering giver fire TF8-hybridoma-cellelinier som vist i tabel 5 i eksempel 13.Hybridomas which are determined to be positive for production of anti-huTF antibodies are selected for further characterization. Eg. hybridomas from the aforementioned TF8 fusion are characterized as anti-huTF antibody-producing hybridoma cultures if the aforementioned liquids from the hybridoma culture exhibit immunoreaction with huTF by the dot-25 blot assay described in Example 6b and the solid-phase RIA described in Example 6a. This characterization yields four TF8 hybridoma cell lines as shown in Table 5 of Example 13.

Hybridomaer fra TF9-fusionen karakteriseres som anti--huTF-antistofproducerende hybridomakulturer, hvis de oven-30 stående væsker fra hybridomakulturen udviser immunoreaktion med huTF ved fastfase-RIA beskrevet i eksempel 6a og ved Western blot-bestemmelsen beskrevet i eksempel 6c. Denne karakterisering giver 24 TF9-hybridoma-cellelinier, hvoraf de fleste er vist i tabel 5 i eksempel 13.Hybridomas from the TF9 fusion are characterized as anti-huTF antibody-producing hybridoma cultures if the supernatants of the hybridoma culture exhibit immunoreaction with huTF by the solid-phase RIA described in Example 6a and by the Western blot assay described in Example 6c. This characterization yields 24 TF9 hybridoma cell lines, most of which are shown in Table 5 of Example 13.

35 Antistofmolekyler produceret af et bestemt hybridoma, der er udvalgt ved de ovennævnte screeningsmetoder, betegnes 47 DK 176220 B1 i den foreliggende beskrivelse ved tegn, der viser 1) den immuniserede mus (dvs. TF8 eller TF9), der giver miltceller til en bestemt fusion, og 2) 96-brønds kulturpladen, rækkenummeret og brøndnummeret, som den særlige HAT-resistente 5 hybridomacelle er isoleret fra (dvs. 5B7, 11D12 osv.).Antibody molecules produced by a particular hybridoma selected by the aforementioned screening methods are designated in the present specification by characters showing 1) the immunized mouse (i.e., TF8 or TF9) that gives spleen cells for a particular fusion and 2) the 96-well culture plate, the row number and the well number from which the particular HAT-resistant hybridoma cell is isolated (i.e. 5B7, 11D12, etc.).

7. Isolering af immunoglobulin IqG7. Isolation of immunoglobulin IqG

Immunoglobulin IgG isoleres fra ascitesvæsken af en mus indeholdende musehybridoma-cellelinien TF8-5G9 (ATCC nr. HB9382) ved anvendelse af et Biorad Laboratories MAPS 10 II system ifølge producentens anvisninger. Proteinkoncentrationen af det isolerede IgG bestemmes ved anvendelse af "BCA Protein Assay Reagent" (Pierce Chemical Co.) ifølge producentens anvisninger.Immunoglobulin IgG is isolated from the ascites fluid of a mouse containing the mouse hybridoma cell line TF8-5G9 (ATCC No. HB9382) using a Biorad Laboratories MAPS 10 II system according to the manufacturer's instructions. The protein concentration of the isolated IgG is determined using the "BCA Protein Assay Reagent" (Pierce Chemical Co.) according to the manufacturer's instructions.

8. Fremstilling af en anti-huTF-holdig fast bærer8. Preparation of an anti-huTF containing solid carrier

15 til immunoaffinitetsisolering af huTF15 for immunoaffinity isolation of huTF

Anti-huTF-antistoffer aktiveres til kobling til en fast matriks af agarose ved dialysering af 10 mg MAPS-iso-leret monoklonalt TF8-5G9-antistof, fremstillet som beskrevet i eksempel 7, mod 500 ml af en dialysepuffer bestående af 2 0 0,1 M MES, pH-værdi 6,5, i 16 timer ved 4°C med mindst én udskiftning af dialysepufferen. De aktiverede TF8-5G9-anti-stoffer blandes derefter med 2 ml "AffiGel-10"-agaroseperler (Biorad), og den fremkomne koblingsreaktionsblanding forarbejdes ifølge producentens anvisninger til dannelse af en 25 fast bærer af TF8-5G9/agarose.Anti-huTF antibodies are activated for coupling to a solid matrix of agarose by dialyzing 10 mg MAPS-isolated monoclonal TF8-5G9 antibody prepared as described in Example 7 against 500 ml of a dialysis buffer consisting of 20 1 M MES, pH 6.5, for 16 hours at 4 ° C with at least one replacement of the dialysis buffer. The activated TF8-5G9 antibodies are then mixed with 2 ml of "AffiGel-10" agarose beads (Biorad), and the resulting coupling reaction mixture is processed according to the manufacturer's instructions to form a solid carrier of TF8-5G9 / agarose.

Overskydende proteinbindingssteder på den faste bærer blokeres derefter, og der vaskes og vakuumfiltreres som beskrevet i eksempel 3 til dannelse af en TF8-5G9/agarose--kage.Excess protein binding sites on the solid support are then blocked and washed and vacuum filtered as described in Example 3 to form a TF8-5G9 / agarose cake.

30 9. Immunoaffinitetsisolering af huTF9. Immunoaffinity Isolation of huTF

Hjerneekstraktopløsning svarende til ca. l/2 menne skehjerne, dvs. ca. 100 ml, og fremstillet som i eksempel 1 dialyseres i 3 dage med to udskiftninger mod i alt 6 liter puffer A ved 4°C. Den dialyserede hjerneekstrakt centrifu-35 geres derefter ved 10.000 g i 1,5 timer. Den fremkomne ovenstående væske blandes med glycinethylester-agarose-kagen 48 DK 176220 B1 fremstillet i eksempel 4 til dannelse af en fastfase/væ-skefase-reaktionsblanding. Efter bibeholdelse ved stuetemperatur i 2 timer under rotering adskilles den faste fase og væskefasen ved filtrering under anvendelse af en sintret 5 glastragt. Den huTF-holdige væskefase fraskilles og blandes med TF8-5G9/agarose-kagen fremstillet i eksempel 8 til dannelse af en fastfase/væskefase-immunoreaktionsblanding.Brain extract solution corresponding to approx. 1/2 men spoon brain, ie ca. 100 ml and prepared as in Example 1 are dialyzed for 3 days with two replacements against a total of 6 liters of buffer A at 4 ° C. The dialyzed brain extract is then centrifuged at 10,000 g for 1.5 hours. The resultant supernatant is mixed with the glycine ethyl ester agarose cake 48 in Example 4 to form a solid phase / liquid phase reaction mixture. After maintaining at room temperature for 2 hours while rotating, the solid phase and the liquid phase are filtered off using a sintered glass funnel. The huTF-containing liquid phase is separated and mixed with the TF8-5G9 / agarose cake prepared in Example 8 to form a solid phase / liquid phase immunoreaction mixture.

Immunoreaktionsblandingen holdes nattes over ved 4°C under rotering for at muliggøre dannelse af et vævsfaktor-10 holdigt fastfase-immunoreaktionsprodukt. Den faste fase og væskefasen adskilles derefter ved filtrering som ovenfor beskrevet. Den faste fase beholdes og vaskes derefter med 10 volumener puffer A. Den faste fase overføres derefter til en glaschromatografisøj le og vaskes successivt med 1) 15 to volumener 1 M natriumchlorid indeholdende 1% "Triton X -10011 og 2) to volumener 0,1 M glycin, pH-værdi 4,0, indeholdende 1% "Triton X-100".The immunoreaction mixture is kept overnight at 4 ° C while rotating to allow formation of a tissue factor-containing solid phase immunoreaction product. The solid phase and the liquid phase are then separated by filtration as described above. The solid phase is then retained and washed with 10 volumes of buffer A. The solid phase is then transferred to a glass chromatography column and washed successively with 1) two volumes of 1 M sodium chloride containing 1% Triton X -10011 and 2) two volumes of 0.1 M glycine, pH 4.0, containing 1% "Triton X-100".

huTF, som er bundet immunogisk til den faste bærer efter de ovennævnte vaskninger, frigøres (elueres) derefter 20 ved vaskning af den faste bærer, medens den tilbageholdes på en sintret glastragt, med 20 ml 0,1 M glycin, pH-værdi 2,5, og 1% "Triton X-100". Elueret materiale opsamles, undersøges for huTF, pooles og dialyseres, alt som beskrevet i eksempel 4.huTF which is immunologically bound to the solid support after the above washes is then released (eluted) 20 by washing the solid support while retained on a sintered glass funnel, with 20 ml of 0.1 M glycine, pH 2, 5, and 1% "Triton X-100". Eluted material is collected, examined for huTF, pooled and dialyzed, as described in Example 4.

25 Dialysatet blandes derefter med fire volumener kold acetone til udfældning af huTF-proteinet. Bundfaldet opsamles ved centrifugering ved 5.000 g i 30 minutter ved ca. -10°C.The dialysate is then mixed with four volumes of cold acetone to precipitate the huTF protein. The precipitate is collected by centrifugation at 5,000 g for 30 minutes at ca. -10 ° C.

Den fremkomne pellet tørres under nitrogen, og en portion af pelleten analyseres ved SDS-polyacrylamidgelelektroforese 30 (SDS-PAGE) under denaturerende betingelser.The resulting pellet is dried under nitrogen and a portion of the pellet is analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions.

Resultaterne af denne analyse, der er vist i fig. 8, viser, at huTFh kan immunoaffinitetsisoleres med et udbytte på 33 mg huTFh pr. g delipideret hjernepulver.The results of this analysis shown in Figs. 8, shows that huTFh can be immunoaffinity isolated with a yield of 33 mg huTFh per g delipidated brain powder.

49 DK 176220 B1 10 . Inhibering af koagulering med an ti - hul'F-an ti stoffer .49 DK 176220 B1 10. Inhibition of coagulation by an ti - hole'F-ten substances.

10 μΐ af ovenstående væske af en hybridomakultur blandes med 90 μΐ HBS/BSA indeholdende ca. 2 ng af det re-5 lipiderede huTF fremstillet i eksempel 4. Immunoreaktions-blandingerne dannet på denne måde holdes ved 37°C i 30 minutter for at gøre det muligt for anti-huTF-antistofmoleky-lerne at binde huTF immunologisk og danne et immunoreak-tionsprodukt. Immunoreaktionsblandingerne undersøges derefter 10 for huTF-prokoaguleringsaktivitet som beskrevet i eksempel 2. Et irrelevant IgG-præparat anvendes i stedet for anti-huTF-antistof som negativ kontrol.Mix 10 μΐ of the above liquid of a hybridoma culture with 90 μΐ HBS / BSA containing approx. 2 ng of the re-lipidated huTF prepared in Example 4. The immunoreaction mixtures thus formed are maintained at 37 ° C for 30 minutes to allow the anti-huTF antibody molecules to bind huTF immunologically and to form an immunoreaction. -tionsprodukt. The immunoreaction mixtures are then assayed for huTF procoagulation activity as described in Example 2. An irrelevant IgG preparation is used in place of anti-huTF antibody as a negative control.

En effektiv huTF-koncentration ekstrapoleres ud fra standardkurven dannet som i eksempel 2 ved anvendelse af 15 den målte koaguleringstid i nærværelse af inhibitor. In-hiberingen udtrykkes som det procentiske forhold mellem den effktive huTF-koncentration og den faktisk anvendte huTF--koncentration. Præparater af monoklonalle antistoffer, der giver mindst 50% inhibering, udvælges som neutraliserende 20 antistofpræparater ifølge opfindelsen.An effective huTF concentration is extrapolated from the standard curve formed as in Example 2 using the measured coagulation time in the presence of inhibitor. Inhibition is expressed as the percentage ratio between the effective huTF concentration and the huTF concentration actually used. Monoclonal antibody preparations which provide at least 50% inhibition are selected as neutralizing antibody preparations of the invention.

Adskillige ovenstående væsker fra kulturerne af hy-bridomaer dannet mod isoleret huTF som beskrevet i eksempel 5 måles ved den ovennævnte procedure for deres evne til at inhibere initiering af koagulering. Hybridomaer, der viser 25 sig at give en væsentlig inhibering af initieringen af koagulering, er anført i tabel V.Several of the above liquids from the cultures of hybridomas formed against isolated huTF as described in Example 5 are measured by the above procedure for their ability to inhibit coagulation initiation. Hybridomas found to give a substantial inhibition of the initiation of coagulation are listed in Table V.

Inhibering af koagulering med anti-huTF-antistoffer er også blevet opnået ved anvendelse af forud dannede huTF-- faktor VII-komplekser. 10 μΐ indeholdende ca. 1 ng relipi-30 deret huTF fremstillet i eksempel 4 blandes med 70 μΐ HBS/-BSA, 10 /il 20 mM calciumchlorid og, hvor det er anført, 10 μΐ indeholdende ca. 25 ng faktor VII fremstillet som beskrevet i eksempel 3. Denne blanding holdes ved 37°C i 15 minutter for at gøre det muligt for huTF at danne et kompleks 35 med eventuel faktor VII, som foreligger i blandingen. Derefter iblandes der yderligere 10 μΐ af en opløsning indehol- 50 DK 176220 B1 dende ca. 10 ng MAPS-isoleret monoklonalt antistof fremstillet som beskrevet i eksempel 7, og denne anden blanding holdes ved 3 7°C i 30 minutter. Inhibering af koagulering måles derefter i den resulterende blanding ved først at 5 tilsætte 100 μΐ 20 mM calciumchlorid og derefter 100 μΐ af enten humant citratplasma eller faktor VII-frit plasma fremstillet som beskrevet i eksempel 12 og observere koaguleringstiden i sekunder. Den procentiske inhibering udtrykkes som beskrevet i eksempel 10, og resultaterne af disse in-10 hiberinger med forud dannet huTF-faktor VII-kompleks er vist i tabel 6a.Inhibition of coagulation with anti-huTF antibodies has also been achieved using pre-formed huTF factor VII complexes. 10 μΐ containing approx. 1 ng of the reliably huTF prepared in Example 4 is mixed with 70 μΐ HBS / -BSA, 10 μl 20 mM calcium chloride and, where indicated, 10 μΐ containing approx. 25 ng of factor VII prepared as described in Example 3. This mixture is kept at 37 ° C for 15 minutes to allow huTF to form a complex 35 with any factor VII present in the mixture. Then add another 10 μΐ of a solution containing approx. 10 ng of MAPS-isolated monoclonal antibody prepared as described in Example 7, and this second mixture is maintained at 37 ° C for 30 minutes. Inhibition of coagulation is then measured in the resulting mixture by first adding 100 μΐ of 20 mM calcium chloride and then 100 μΐ of either human citrate plasma or factor VII free plasma prepared as described in Example 12 and observing the coagulation time in seconds. The percent inhibition is expressed as described in Example 10, and the results of these inhibitions with pre-formed huTF Factor VII complex are shown in Table 6a.

15 20 25 30 35 51 DK 176220 B115 20 25 30 35 51 DK 176220 B1

Tabel 6Table 6

Inhibering af huTF-faktor VII-initieret koagulering med anti-huTF-antistoffer 5 I. Koagulering med citratbehandlet humant plasmaInhibition of huTF Factor VII Initiated Coagulation with Anti-huTF Antibodies 5 I. Coagulation with Citrate-Treated Human Plasma

Antistof Faktor VI1^·) % InhiberingAntibody Factor VI1 *)% Inhibition

Blinda) + 0 TF8-5G9b) + 58%Blind) + 0 TF8-5G9b) + 58%

Kontrol0) + 0 10 TF8-5G9 - 83%Control 0 + 0 10 TF8-5G9 - 83%

Kontrol - 0 II. Koagulering med faktor VII-frit humant plasma Antistof Faktor VII % InhibitorControl - 0 II. Coagulation with Factor VII-Free Human Plasma Antibody Factor VII% Inhibitor

Blind + 0 15 TF8-5G9 + 58%Blind + 0 15 TF8-5G9 + 58%

Kontrol + 0 a) "Blind" betegner, at der ikke anvendes monoklonalt antistof ved bestemmelsen.Control + 0 (a) "Blind" means that no monoclonal antibody is used in the assay.

20 b) "TF8-5G9" betegner, at det monoklonale antistof isoleret fra hybridoma TF8-5G9 er antistoffet anvendt ved bestemmelsen .B) "TF8-5G9" means that the monoclonal antibody isolated from hybridoma TF8-5G9 is the antibody used in the assay.

c) "Kontrol" betegner, at et irrelevant monoklonalt antistof er antistoffet anvendt ved bestemmelsen.c) "Control" means that an irrelevant monoclonal antibody is the antibody used in the assay.

25 ^) "+" betegner, at faktor VII tilsættes og får lov at danne et kompleks med renset huTF, før antistof sættes til blandingen.25 +) means that factor VII is added and allowed to form a purified huTF complex before antibody is added to the mixture.

Yderligere undersøgelser af inhibering af koagulering 30 med anti-huTF-antistoffer er gennemført under betingelser, hvorved man sammenligner inhiberingen før og efter at TF er associeret til faktor Vll/VIIa til dannelse af et TF:faktor VII/VIla-kompleks.Further studies on inhibition of coagulation 30 with anti-huTF antibodies have been conducted under conditions comparing the inhibition before and after TF is associated with factor VII / VIIa to form a TF: factor VII / VIa complex.

Ved disse undersøgelser gennemføres inhiberingen af 35 koagulering med anti-huTFh-antistoffer ved anvendelse af forud dannede TF:Vll/Vlla-komplekser i det væsentlige som beskrevet ovenfor i eksempel 10, bortset fra at de anvendte 10 μΙ^θΓ opløsning indeholdende monoklonalt antistof er 52 DK 176220 B1 ovenstående væske fra hybridomakultur i stedet for en opløsning indeholdende MAPS-isoleret monoklonalt antistof. Til sammenligning vurderes inhiberingen af koagulering med anti-huTF-antistoffer ved dannelse af immunokomplekser mellem 5 disse antistoffer og relipideret huTF før blanding med citratbehandlet plasma indeholdende faktor VII/VIIa som beskrevet ovenfor i eksempel 10.In these studies, the inhibition of coagulation with anti-huTFh antibodies is carried out using pre-formed TF: Vll / Vllla complexes essentially as described above in Example 10, except that the 10 µΙΙ θΓ solution containing monoclonal antibody is used. 52 DK 176220 B1 the above liquid from hybridoma culture instead of a solution containing MAPS-isolated monoclonal antibody. In comparison, the inhibition of coagulation by anti-huTF antibodies is assessed by forming immunocomplexes between these antibodies and precipitated huTF before mixing with citrate-treated plasma containing factor VII / VIIa as described above in Example 10.

Selv om alle de her beskrevne antistoffer undersøges ved denne sammenlignende inhiberingsbestemmelse, anses kun 10 de antistoffer, der udviser en mere end ca. 60%'s inhibering, for at have en væsentlig evne til at inhibere koagulering initieret af et huTF:VII/VIIa-kompleks. Disse monoklonale antistoffer er TF9-1B8, TF9-5B7, TF8-5C4, TF8-11D12 og TF8-21F2 .Although all of the antibodies described herein are assayed by this comparative inhibition assay, only 10 of the antibodies exhibiting a greater than ca. 60% inhibition, to have a substantial ability to inhibit coagulation initiated by a huTF: VII / VIIa complex. These monoclonal antibodies are TF9-1B8, TF9-5B7, TF8-5C4, TF8-11D12 and TF8-21F2.

15 11. Polvpeptidsvntese11. Polypeptide Synthesis

Polypeptiderne svarende til de forskellige huTFh-regioner, som anvendes her, syntetiseres kemisk på en Applied Biosystems Model 43OA Peptide Synthesizer ved anvendelse af 20 den symmetriske anhydrid-metode ifølge Hagenmaier, et al., Hoppe-Seyler's Z. Physiol. Chem., 353:1973 (1982). Ud over polypeptiderne anført i tabel 1 og 2 syntetiseres også po-lypeptiderne anført i tabel 3 nedenfor og udgør polypeptider ifølge den foreliggende opfindelse, der er anvendelige til 25 produktion af anti-polypeptid-antistoffer, der kan reagere med huTFh.The polypeptides corresponding to the various huTFh regions used herein are chemically synthesized on an Applied Biosystems Model 43OA Peptide Synthesizer using the symmetric anhydride method of Hagenmaier, et al., Hoppe-Seyler's Z. Physiol. Chem., 353: 1973 (1982). In addition to the polypeptides listed in Tables 1 and 2, the polypeptides listed in Table 3 below are also synthesized and constitute polypeptides of the present invention useful for the production of anti-polypeptide antibodies that can react with huTFh.

Tabel 3Table 3

Antigene polypeptiderAntigenic polypeptides

30 p121-155 H-TKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTL-OH30 p121-155 H-TKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTL-OH

p2 04 -2 2 6 H-DSPVECMGQEKGEFREIFYIIGA-OHp2 04 -2 2 6 H-DSPVECMGQEKGEGEFREIFYIIGA-OH

p2 2 5-2 44 H-GAWFWIILVIILAISLHK-OHp2 2 5-2 44 H-GAWFWIILVIILAISLHK-OH

p245-263 H-CRKAGVGQSWKENS PLNVS-OHp245-263 H-CRKAGVGQSWKENS PLNVS-OH

35 53 DK 176220 B1 12. Inhibering af koagulering med polypeptider35 53 DK 176220 B1 12. Inhibition of coagulation with polypeptides

Evnen af polypeptiderne ifølge opfindelsen til at inhibere huTF-initieret koagulering bestemmes ved først at inkubere polypeptiderne i nærværelse af faktor Vll/VIIa og 5 calciumioner og derefter sætte denne blanding til faktor VIi/VIla-frit plasma og vurdere koaguleringstiderne.The ability of the polypeptides of the invention to inhibit huTF-initiated coagulation is determined by first incubating the polypeptides in the presence of factor VIIa / VIIa and 5 calcium ions and then adding this mixture to factor VI1 / VIa1 free plasma and assessing the coagulation times.

Human faktor Vll/VIIa isoleres som beskrevet i eksempel 3. 10 //liter af en opløsning af 200 ng af denne isolerede faktor Vll/VIIa pr. ml HBS/BSA sættes til en opløsning om-10 fattende 100 //liter HBS, 20 μΐίίοΐ 25 mM calciumchlorid og 100 μΙ^θΓ TBS/"Triton" indeholdende syntetisk polypeptid.Human factor Vll / VIIa is isolated as described in Example 3. 10 // liter of a solution of 200 ng of this isolated factor Vll / VIIa per ml of HBS / BSA is added to a solution of about 100 µL of HBS, 20 μΐίίοΐ 25 mM calcium chloride and 100 μΙ ^ θΓ TBS / "Triton" containing synthetic polypeptide.

Der fremstilles på denne måde adskillige blandinger, der indeholder forskellige koncentrationer af polypeptidet, og de holdes derefter ved 37°C i 15 minutter. Relipideret vævs-15 faktor fremstillet som beskrevet i eksempel 4 fortyndes i HBS/BSA, således at 10 μΙ^βΓ vil give en koaguleringstid på ca. 45 sekunder ved undersøgelse ved koaguleringsbestemmelsen beskrevet i eksempel 2. Den ovenfor nævnte blanding blandes yderligere med denne 10 μΐΐΐθτθ fortynding af reli-20 pideret huTF, med 100 μliter 25 mM calciumchlorid og med 100 μΙίΙθΓ faktor Vll/VIIa-frit plasma (George King Bio-Medical, Inc., Overland Park, KA) fortyndet i forholdet 1 del plasma til 1,5 dele HBS. Koaguleringstiden bestemmes derpå og afsættes som beskrevet i eksempel 2. En forlængelse 25 af koaguleringstiden viser inhibering af koagulering med det syntetiske polypeptid. Den procentiske inhibering beregnes som beskrevet i eksempel 10. Polypeptider, der giver mindst 30% inhibering af koagulering, betragtes som huTFh-bindingssted-polypeptidanaloge, dvs. polypeptiderne p26-49, 30 pl46-167 og pl61-189 som vist i afsnit I i tabel 4.In this way, several mixtures containing different concentrations of the polypeptide are prepared and then kept at 37 ° C for 15 minutes. Released tissue-15 factor prepared as described in Example 4 is diluted in HBS / BSA so that 10 μΙΙβΓ will give a coagulation time of about 45 seconds when examined by the coagulation assay described in Example 2. The above mixture is further mixed with this 10 μΐΐΐθτθ dilution of reliably huTF, with 100 μl 25 mM calcium chloride and with 100 μΙίΙθΓ factor Vll / VIIa free plasma (George King Bio -Medical, Inc., Overland Park, KA) diluted in 1 part plasma to 1.5 parts HBS. The coagulation time is then determined and plotted as described in Example 2. An extension 25 of the coagulation time shows inhibition of coagulation with the synthetic polypeptide. The percent inhibition is calculated as described in Example 10. Polypeptides giving at least 30% inhibition of coagulation are considered huTFh binding site polypeptide analogs, i.e. polypeptides p26-49, 30 pl46-167 and pl61-189 as shown in Section I of Table 4.

Alternativt er der ved den ovenfor beskrevne inhibe-ringsbestemmelse blevet anvendt faktor VII/Vila-frit plasma fremstillet ud fra plasma, der er befriet for faktor Vll/VIIa ved immunoaffinitetsadsorption med monoklonale antistoffer.Alternatively, in the inhibition assay described above, factor VII / Vila-free plasma prepared from plasma released from factor VII / VIIa by immunoaffinity adsorption with monoclonal antibodies has been used.

35 Et monoklonalt antistof mod human faktor Vll/VIIa fremstilles i det væsentlige som beskrevet i eksempel 5, bortset fra at 54 DK 176220 B1 faktor VII/VIIa isoleret som beskrevet i eksempel 3 anvendes som immunogen i stedet for huTF. De resulterende hybridomaer vurderes ved ELISA til identificering af et hybridoma, som ikke reagerer med de humane blodproteiner protein S, faktor 5 IX, faktor X og faktor II, der kan fås fra Enzyme Research Laboratories, Inc., South Bend, IN. Et sådant hybridoma FV11.F1.2H3-3.2 kan fås fra Dr. T.S. Edgington (Scripps Clinic and Research Foundation, La Jolla, CA) . Immunoglobulin IgG isoleres fra ascites fra en mus indeholdende hybridoma 10 FV11 F1.2H3-3.2, og det isolerede IgG konjugeres til en fast bærer som beskrevet i eksempel 8. Den fremkomne faste bærer indeholdende monoklonalt faktor Vll/VIIa-antistof anvende til at fjerne faktor VII/VIIa fra poolet normalt citratbehandlet plasma ved anvendelse af immunoaffinitets-15 proceduren beskrevet i eksempel IX, bortset fra, at væskefasen indeholdende plasma opsamles og beholdes.A human factor Vll / VIIa monoclonal antibody is prepared essentially as described in Example 5, except that Factor VII / VIIa isolated as described in Example 3 is used as immunogen instead of huTF. The resulting hybridomas are evaluated by ELISA to identify a hybridoma that does not react with the human blood proteins protein S, Factor 5 IX, Factor X and Factor II available from Enzyme Research Laboratories, Inc., South Bend, IN. Such a hybridoma FV11.F1.2H3-3.2 can be obtained from Dr. T. S. Edgington (Scripps Clinic and Research Foundation, La Jolla, CA). Immunoglobulin IgG is isolated from ascites from a mouse containing hybridoma 10 FV11 F1.2H3-3.2 and the isolated IgG is conjugated to a solid support as described in Example 8. The resulting solid carrier containing monoclonal factor VII / VIIa antibody used to remove factor VII / VIIa from the pool normally citrate-treated plasma using the immunoaffinity procedure described in Example IX except that the liquid phase containing plasma is collected and retained.

Evnen af nogle af polypeptiderne til kompetitivt at inhibere koagulering ved anvendelse i lipideret form vurderes ved den ovennævnte bestemmelse ved at anvende 100 /eliter 20 lipideret syntetisk polypeptid i stedet for de 100 ^liter opløsning af syntetisk polypeptid.The ability of some of the polypeptides to competitively inhibit coagulation by use in lipidized form is assessed by the above determination by using 100 µl of 20 lipid synthetic polypeptide instead of the 100 µl solution of synthetic polypeptide.

Lipiderede syntetiske peptider fremstilles som beskrevet i eksempel 4 for relipidering af isoleret huTF, bortset fra at der anvendes syntetisk polypeptid i stedet 25 for isoleret huTF. Der anvendes rutinemæssigt vægtforholdet 52:1 mellem lipid og polypeptid. Lipiderede polypeptider, der giver mindst 30% inhibering af koagulering, betragtes som huTFh-bindingssted-polypeptidanaloge, når de foreligger i lipideret form, dvs. polypeptiderne anført i afsnit II i 30 tabel 4.Lipidized synthetic peptides are prepared as described in Example 4 for the isolation of isolated huTF, except that synthetic polypeptide is used instead of isolated huTF. A 52: 1 weight ratio of lipid to polypeptide is routinely used. Lipidated polypeptides that provide at least 30% inhibition of coagulation are considered huTFh binding site polypeptide analogs when in lipid form, i.e. the polypeptides listed in Section II of Table 4.

35 55 DK 176220 B135 55 DK 176220 B1

Tabel 4Table 4

Inhibering af huTF-initieret koagulering med polypeptidanaloge af huTFh 5 Peptid Inhibering3 Koncentration I. Ikke-phospholipiderede peptider pi-30 25,0 10 μΜ p26-49 88,8 10 μΜ p41-71 25,0 10 μΜ 10 p40-49 25,0 10 μΜ p56-71 25,0 10 μΜ p72-104 25,0 10 μΜ p94-123 20,0 10 μΜ pl21-155 10,0 10 μΜ 15 pl46-167 87,5 10 μΜ plSl-189 32,5 10 μΜ pl90-209 20,0 10 μΜ p204-226 20,0 10 μΜ 0 20 II. Phospholipiderede peptider pi-30 81,0 10 μΜ p26-4 0 83,0 10 μΜ p40-71 65,0 10 μΜ 25 p50-71 73,3 30 μΜ p94-123 93,7 10 μΜ pl21-155 55,0 10 μΜ pl46-167 80,0 10 μΜ pl61-189 94,0 10 μΜ 3 0__ a De procentiske inhiberinger bestemmes som beskrevet i eksempel 12.Inhibition of huTF-initiated clotting with polypeptide analogs of huTFh 5 Peptide Inhibition3 Concentration I. Non-phospholipidated peptides pi-30 25.0 10 μΜ p26-49 88.8 10 μΜ p41-71 25.0 10 μΜ 10 p40-49 25, 0 10 μΜ p56-71 25.0 10 μΜ p72-104 25.0 10 μΜ p94-123 20.0 10 μΜ pl21-155 10.0 10 μΜ 15 pl46-167 87.5 10 μΜ plSl-189 32.5 10 μΜ pl90-209 20.0 10 μΜ p204-226 20.0 10 μΜ 0 20 II. Phospholipidated peptides pi-30 81.0 10 μΜ p26-4 0 83.0 10 μΜ p40-71 65.0 10 μΜ 25 p50-71 73.3 30 μΜ p94-123 93.7 10 μΜ pl21-155 55.0 10 μΜ pl46-167 80.0 10 μΜ pl61-189 94.0 10 μΜ 3 0__ a The percent inhibitions are determined as described in Example 12.

Eksempler på dosis/reaktions-kurver opnået ved gen-35 nemførelse af de ovennævnte polypeptid-inhiberingsundersø-gelser er vist i fig. 9 og 10 56 DK 176220 B1 13. Inhibering af antistof-huTF- -immunoreaktion med polypeptiderExamples of dose / response curves obtained by performing the above polypeptide inhibition studies are shown in FIG. 9 and 10 56 DK 176220 B1 13. Inhibition of antibody huTF immunoreaction with polypeptides

Brøndene af "Immulon" plader med 96 brønde (U-bund) fremstillet af fleksibelt vinyl (Dynatech) overtrækkes med 5 gede-anti-muse-IgG (Boehringer-Mannheim) som beskrevet i eksempel 6, bortset fra at blokeringen af overskydende proteinbindingssteder gennemføres i 20 minutter ved 37°C.The wells of 96-well (U-bottom) Immulon plates made of flexible vinyl (Dynatech) are coated with 5 goat anti-mouse IgG (Boehringer-Mannheim) as described in Example 6, except the blocking of excess protein binding sites is accomplished. for 20 minutes at 37 ° C.

50 filter af ovenstående væske fra hybridomakultur anbringes i hver brønd og holdes i 1 time ved 37°C. Brøndene 10 skylles derefter tre gange med TBS, og væskeoverskud fjernes ved opsugning.50 filters of the above liquid from hybridoma culture are placed in each well and kept for 1 hour at 37 ° C. The wells 10 are then rinsed three times with TBS and liquid excess is removed by aspiration.

Isoleret huTF fremstilles på immunoaffinitetssøjler som beskrevet i eksempel 9. Det fremkomne acetonebundfald indeholdende isoleret huTF opløses i TBS/"Triton", og pro-15 teinkoncentrationen bestemmes ved anvendelse af "BCA Protein Assay Reagent" (Pierce) ifølge producentens specifikationer. Kulhydrat~sidegrupper på huTF biotinyleres ved anvendelse af biotin-hydrazid (ICN Biomedicals Inc., Plainview, NY) ved metoden beskrevet af O'Shannessy et al., Immunol. Let-20 ters, 8:273-277 (1984) til dannelse af en opløsning af bio- tinyleret huTF.Isolated huTF is prepared on immunoaffinity columns as described in Example 9. The resulting acetone precipitate containing isolated huTF is dissolved in TBS / "Triton" and the protein concentration is determined using "BCA Protein Assay Reagent" (Pierce) according to the manufacturer's specifications. Carbohydrate ~ side groups on huTF are biotinylated using biotin hydrazide (ICN Biomedicals Inc., Plainview, NY) by the method described by O'Shannessy et al., Immunol. Latters, 8: 273-277 (1984) to form a solution of biotinylated huTF.

50 /eliter af en opløsning af biotinyleret huTF fremstillet til 60 ng/ml af TBS/"Triton" anbringes derefter i hver brønd sammen med 5 μΜ syntetisk polypeptid og holdes i 25 en time ved 37°C. Brøndene skylles derefter tre gange med TBS/"Triton".50 µl of a solution of biotinylated huTF prepared for 60 ng / ml of TBS / "Triton" is then placed in each well with 5 µΜ of synthetic polypeptide and held for 25 hours at 37 ° C. The wells are then rinsed three times with TBS / "Triton".

100 μ1±ίΒτ streptavidin-konjugeret alkalisk phosphatase (Detek I-alk, Enzo Biochem Inc., New York, NY) fortyndet 1:100 i TBS indeholdende 5 mM EDTA, 0,5% "Triton X-100" og 3 0 1% BSA anbringes i hver brønd og holdes i 3 0 minutter ved 37°C. Brøndene skylles derefter fire gange med en opløsning indeholdende 10 mM kaliumphosphat (pH-værdi 6,5), 2% BSA, 0,5% "Triton X-100", 0,5 M natriumchlorid og 1 mM EDTA, efterfulgt af en enkelt skylning med detektionspuffer (0,1 35 M Tris-HCl (pH-værdi 8,8), 0,1 M natriumchlorid, 5 mM mag- nesiumchlorid).100 μ1 ± ίΒτ streptavidin-conjugated alkaline phosphatase (Detek I-alk, Enzo Biochem Inc., New York, NY) diluted 1: 100 in TBS containing 5 mM EDTA, 0.5% Triton X-100, and 3 0 1 % BSA is placed in each well and maintained for 30 minutes at 37 ° C. The wells are then rinsed four times with a solution containing 10 mM potassium phosphate (pH 6.5), 2% BSA, 0.5% Triton X-100, 0.5 M sodium chloride and 1 mM EDTA, followed by a single rinse with detection buffer (0.1 M Tris-HCl (pH 8.8), 0.1 M sodium chloride, 5 mM magnesium chloride).

57 DK 176220 B1 100 /xliter af en opløsning indeholdende 2 mM p-nitro-phenylphosphat i detektionspuffer sættes derefter til hver brønd og holdes i 1 time ved 3 7°C. Den optiske absorption ved 405 nm måles derefter for hver brønd ved anvendelse af 5 en Bio-Tek-mikropladelæser (Bio-Tek Instruments, Winooski, VT) .57 GB 176220 B1 100 µl of a solution containing 2 mM p-nitro-phenylphosphate in detection buffer is then added to each well and maintained for 1 hour at 37 ° C. The optical absorption at 405 nm is then measured for each well using a Bio-Tek microplate reader (Bio-Tek Instruments, Winooski, VT).

Resultaterne af undersøgelsen af kompetitiv inhibering er vist i tabel 5.The results of the competitive inhibition study are presented in Table 5.

58 DK 176220 B158 DK 176220 B1

Tabel 5Table 5

Oversigt over peptidvekselvirkninger med monoklonale antistoffer 5Overview of peptide interactions with monoclonal antibodies 5

Maba> ABCDEFGHIJKMaba> ABCDEFGHIJK

TF85G9 + TF811D12 + TF85C4 + 10 TF821F2 + TF91D5 + + + TF92C4 + + + + TF92F6 + + TF95C7 + + + + 15 TF96B4 + + TF99C3 + + + + TF910C2 + + TF91F1 + TF91E7 + + + 20 TF91B8 + + + TF91B9 + TF94D11 + + + TF95G4 + + TF95B7 + + 25 TF96G4 + TF97E10 + + TF98E8 + + + TF99E1 + + + TF99B4 + + 30 TF96C8b> + + + TF910H5b) + + TF99D6b) + + TF910 H10b> + + + 35 A = pi-30, B = p2 6-49, C = p40-71, D = p41-49, E = p56-71, F = p72-104, G = p94-12 3, Η = pl21-155, I = pl46-167, J = pl61-189, K = pl90-209 59 DK 176220 B1 a Hvert monoklonalt antistof (Mab) produceres af et hybri-doma med samme betegnelse. Alle monoklonale antistoffer screenes ved anvendelse af ovenstående væsker fra hybri-domakultur som beskrevet i eksempel 13.TF85G9 + TF811D12 + TF85C4 + 10 TF821F2 + TF91D5 + + + TF92C4 + + + + TF92F6 + + TF95C7 + + + + 15 TF96B4 + + TF99C3 + + + + TF910C2 + + TF91F1 + TF91E7 +8 + TF91E7 + + TF94D11 + + + TF95G4 + + TF95B7 + + 25 TF96G4 + TF97E10 + + TF98E8 + + + TF99E1 + + + TF99B4 + + 30 TF96C8b> + + + TF910H5b) + + TF99D6b) + + TF910 = pi-30, B = p2 6-49, C = p40-71, D = p41-49, E = p56-71, F = p72-104, G = p94-12 3, Η = pl21-155, I = pl46-167, J = pl61-189, K = pl90-209 59 DK 176220 B1 a Each monoclonal antibody (Mab) is produced by a hybrid domain of the same designation. All monoclonal antibodies are screened using the above hybroma culture fluids as described in Example 13.

5 ^ Disse antistoffer betragtes som ikke-neutraliserende ifølge resultaterne i eksempel 10. Alle andre antistoffer betragtes som neutraliserende ifølge disse samme resultater .These antibodies are considered neutralizing according to the results of Example 10. All other antibodies are considered neutralizing according to these same results.

10 Inhibering betragtes som signifikant, hvis den målte absorptionsværdi i nærværelse af polypeptid er mere end en standardafvigelse fra middelværdien opnået for et givet antistof i fraværelse af polypeptid.Inhibition is considered significant if the measured absorption value in the presence of polypeptide is more than a standard deviation from the mean obtained for a given antibody in the absence of polypeptide.

15 14. Påvisning af huTF i en kroosprøve ved to-steds ELISA15 14. Detection of huTF in a cross-sample by two-site ELISA

huTF kan påvises i en kropsprøve, såsom blod, plasma, spyt, urin osv., ved anvendelse af to monoklonale antistoffer, der sideløbende kan binde det samme huTF-molekyle.huTF can be detected in a body sample, such as blood, plasma, saliva, urine, etc., using two monoclonal antibodies that can simultaneously bind the same huTF molecule.

"Immulon"-polystyrenplader med 96 brønde med U-bund 20 (Dynatech) overtrækkes med gede-anti-muse-IgG (Boehringer-Mannheim) ved, at der i hver brønd først anbringes 100 //liter af IgG fortyndet til 10 //g/ml i TBS, og IgG-opløsningen derefter holdes i kontakt med brønden natten over ved 4°C. Brøndene skylles tre gange med TBS, og 100 //liter TBS/"Tri-25 ton" indeholdende 3% BSA sættes til hver brønd. Brøndene holdes derefter i 1 time ved 37°C, skylles tre gange med TBS, og overskydende væske fjernes ved opsugning.Immunon 96-well polystyrene plates with U-bottom 20 (Dynatech) are coated with goat anti-mouse IgG (Boehringer-Mannheim) by first placing 100 µl of IgG diluted to 10 // in each well. g / ml in TBS and the IgG solution is then kept in contact with the well overnight at 4 ° C. The wells are rinsed three times with TBS, and 100 µL of TBS / "Tri-25 ton" containing 3% BSA is added to each well. The wells are then kept for 1 hour at 37 ° C, rinsed three times with TBS and excess liquid removed by aspiration.

100 //liter af en anti-huTF-antistofmolekylholdig ovenstående væske fra en kultur af et første hybridoma, 30 TF9-6B4, blandes i hver brønd og holdes i 1 time ved 37°C.100 µl of an anti-huTF antibody molecule-containing supernatant from a culture of a first hybridoma, TF9-6B4, is mixed in each well and maintained for 1 hour at 37 ° C.

Brøndene skylles derefter tre gange med TBS, og overskydende væske fjernes ved opsugning.The wells are then rinsed three times with TBS and excess liquid is removed by aspiration.

Immunoaffinitetsisoleret og acetoneudfældet huTF fremstillet som i eksempel 9 opløses i TBS/"Triton". Fortyn-35 dinger af huTF-opløsningen fremstilles i området fra 5 /zg/ml til 0,5 ng/ml af TBS/"Triton", og 100 //liter af en fortynding 60 DK 176220 B1 anbringes i en brønd af "Immulon"-pladen. Fortyndingerne af huTF holdes i kontakt raed det første antistof i 1 time ved 37°C. Fortyndingerne fjernes derefter, og brøndene skylles tre gange med TBS/"Triton". Overskydende væske fjernes ved 5 opsugning.Immunoaffinity isolated and acetone precipitated huTF prepared as in Example 9 are dissolved in TBS / "Triton". Dilutions of the huTF solution are prepared in the range of 5 µg / ml to 0.5 ng / ml of TBS / "Triton", and 100 µl of a dilution 60 is applied to a well of "Immulon". "plate. The dilutions of huTF are kept in contact with the first antibody for 1 hour at 37 ° C. The dilutions are then removed and the wells are rinsed three times with TBS / "Triton". Excess fluid is removed by aspirating.

Anti-huTF-antistoffer MAPS-isoleres fra ascites af et andet hybridoma, TF9-10H10, ved metoderne beskrevet i eksempel 7. Den fremkomne antistofopløsning måles for protein og mærkes derefter ved biotinylering som beskrevet i eksempel 10 13.Anti-huTF antibodies are MAPS isolated from ascites of another hybridoma, TF9-10H10, by the methods described in Example 7. The resulting antibody solution is measured for protein and then labeled by biotinylation as described in Example 10 13.

Det biotinylerede anti-huTF-antistof fortyndes til 60 mg/ml af TBS/"Triton", og 100 /uliter af denne opløsning blandes i hver brønd. Brøndene holdes ved 37°C i 1 time og skylles derefter tre gange i TBS/"Triton".The biotinylated anti-huTF antibody is diluted to 60 mg / ml of TBS / "Triton" and 100 µl of this solution is mixed in each well. The wells are kept at 37 ° C for 1 hour and then rinsed three times in TBS / "Triton".

15 Det bundne biotinylerede anti-huTF-antistof påvises derefter ved anvendelse af Detek I-alk systemet beskrevet i eksempel 13. De monoklonale antistoffer, der anvendes som det første og andet antistof ved denne bestemmelse, kan varieres, så længe de har evnen til at binde sideløbende 20 til huTF. Når TF9-6B4 er anvendt som det første antistof, kan f.eks. TF9-11D12 anvendes som det andet antistof i stedet for TF9-10H10. Opfindelsen omfatter således enhver kombination af antistoffer, der kan binde sideløbende ved denne bestemmelse.The bound biotinylated anti-huTF antibody is then detected using the Detek I-alk system described in Example 13. The monoclonal antibodies used as the first and second antibodies in this assay can be varied as long as they have the ability to concurrently bind 20 to huTF. When TF9-6B4 is used as the first antibody, e.g. TF9-11D12 is used as the second antibody in place of TF9-10H10. The invention thus encompasses any combination of antibodies that can bind concurrently with this assay.

25 15. Konstruktion af et DNA-segment indeholdende hele den pre-huTFh-kodende sekvensConstruction of a DNA segment containing the entire pre-huTFh coding sequence

Et DNA-segment indeholdende hele den pre-huTFh-kodende sekvens kan konstrueres på følgende måde ved anvendelse af 30 de rekombinante plasmider pCTF64, pCTF403 og pCTF314, hvis restriktionskort er vist i fig. 11, og procedurer, der er velkendte, se f. eks. Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY (1983).A DNA segment containing the entire pre-huTFh coding sequence can be constructed as follows using the recombinant plasmids pCTF64, pCTF403 and pCTF314, whose restriction map is shown in FIG. 11, and procedures well known, see, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY (1983).

35 Indsats-segmenterne indeholdt i de rekombinante DNA- plasmider vist i fig. 11 har EcoRI-linkeren 5'-GGAATTCC-3' 61 DK 176220 B1 (Collaborative Research Lexington, MA.) ved hver terminus for at lette kloningsprocessen. Disse linkersekvenser er ikke til stede i nukleotidsekvensen vist i fig. 2, idet de ikke er en del af den naturligt forekommende huTFh-DNA-ko-5 dende sekvens. For at de følgende beskrivelser af konstruktion af rekombinante DNA-molekyler kan være klare med hensyn til de involverede huTFh-DNA-sekvenser, vil segmenter dannet ved nedbrydninger, der omfatter EcoRI-terminusser og således kan indeholde disse yderligere linkersekvenser, blive beteg-10 net ved nukleotidbasenummeret vist i fig. 2. Det vil forstås, at segmenterne kan indeholde disse yderligere sekvenser ved deres terminusser.The insert segments contained in the recombinant DNA plasmids shown in FIG. 11, the EcoRI linker has 5'-GGAATTCC-3 '61 Collaborative Research Lexington, MA. At each terminus to facilitate the cloning process. These linker sequences are not present in the nucleotide sequence shown in FIG. 2, as they are not part of the naturally occurring huTFh DNA coding sequence. In order for the following descriptions of the construction of recombinant DNA molecules to be clear with respect to the huTFh DNA sequences involved, segments formed by degradations comprising EcoRI terminals and thus containing these additional linker sequences will be referred to at the nucleotide base number shown in FIG. 2. It will be understood that the segments may contain these additional sequences at their terminals.

Plasmid pCTF64 nedbrydes med restriktionsendonuk-leaserne EcoRI og Dralll til dannelse af et DNA-segment 15 omfattende en nukleotidsekvens svarende til sekvensen vist i fig. 2 fra baserest nr. 1 til rest nr. 296. Segmentet på 302 nukleotid-basepar (bp) produceret på denne måde isoleres ved størrelses fraktionering under anvendelse af en agarosegel og dephosphoryleres derefter ved behandling med alkalisk 20 phosphatase.Plasmid pCTF64 is digested with the restriction endonucleases EcoRI and Dralll to form a DNA segment 15 comprising a nucleotide sequence similar to the sequence shown in FIG. 2 from base residue # 1 to residue # 296. The 302 nucleotide base pair (bp) segment produced in this way is isolated by size fractionation using an agarose gel and then dephosphorylated by treatment with alkaline phosphatase.

Plasmid pCTF403 nedbrydes med restriktionsendonu-kleasen EcoRI til dannelse af et DNA-segment omfattende en nukleotidsekvens svarende til sekvensen vist i fig. 2 fra rest 776 til rest nr. 1125. Det resulterende 352 bp segment 25 isoleres ved størrelsesfraktionering under anvendelse af en agarosegel.Plasmid pCTF403 is digested with the restriction endonuclease EcoRI to form a DNA segment comprising a nucleotide sequence similar to the sequence shown in FIG. 2 from residue 776 to residue # 1125. The resulting 352 bp segment 25 is isolated by size fractionation using an agarose gel.

Plasmid pCTF314 nedbrydes med restriktionsendonu-kleasen EcoRI, og det resulterende 647 bp segment isoleres ved størrelsesfraktionering. Dette segment omfatter en nu-30 kleotidsekvens, der svarer til sekvensen vist i fig. 2 fra rest nr. 135 til rest nr. 775. 647 bp segmentet isoleres ved størrelsesfraktionering og dephosphoryleres ved alkalisk phosphatase.Plasmid pCTF314 is digested with the restriction endonuclease EcoRI, and the resulting 647 bp segment is isolated by size fractionation. This segment comprises a nucleotide sequence corresponding to the sequence shown in FIG. 2 from residue # 135 to residue # 775. The 647 bp segment is isolated by size fractionation and dephosphorylated by alkaline phosphatase.

352 bp segmentet og det dephosphorylerede 647 bp 35 segment forbindes derefter operativt (ligeres) ved reaktion med T4-DNA-ligase, hvorved der dannes et 999 bp segment, 62 DK 176220 B1 der har en nukleotidsekvens svarende til sekvensen vist i fig. 2 fra rest nr. 135 til rest nr. 1125. 999bp segmentet nedbrydes derefter med restriktionsendonukleasen Drain, der spalter 999 bp segmentet ved en position mellem baseres-5 terne 2 96 og 297 vist i fig. 2, hvorved der dannes et 168 bp segment og et 831 bp segment. Det dephosphorylerede 302 bp segment og 831 bp segmentet forbindes derefter operativt ved anvendelse af T4-DNA-ligase til dannelse af et 1125 bp segment omfattende en nukleotidsekvens svarende til sekvensen 10 vist i fig. 2 fra rest nr. 1 til rest nr. 1125.The 352 bp segment and the dephosphorylated 647 bp 35 segment are then operatively linked (ligated) by reaction with T4 DNA ligase, thereby forming a 999 bp segment, 62 having a nucleotide sequence similar to the sequence shown in FIG. 2 from residue # 135 to residue # 1125. The 999bp segment is then degraded by the restriction endonuclease Drain, which cleaves the 999bp segment at a position between base sites 2 96 and 297 shown in FIG. 2, thereby forming a 168 bp segment and an 831 bp segment. The dephosphorylated 302 bp segment and the 831 bp segment are then operatively linked using T4 DNA ligase to form a 1125 bp segment comprising a nucleotide sequence corresponding to the sequence 10 shown in FIG. 2 from residue # 1 to residue # 1125.

Kloningsplasmidvektoren pUC8 lineariseres ved nedbrydning med EcoRI. Det ovenfor fremstillende 1133 bp segment og denne EcoRI-nedbrudte vektor forbindes operativt ved anvendelse af T4-DNA-ligase til dannelse af det cirkulære 15 rekombinante DNA-molekyle pUC-pre-huTFh.The cloning plasmid vector pUC8 is linearized by digestion with EcoRI. The 1133 bp segment prepared above and this EcoRI degraded vector are operably linked using T4 DNA ligase to form the circular 15 recombinant DNA molecule pUC pre-huTFh.

E. coli stamme RR1 (Bethesda Research Laboratories, Gaithersburg, MD) transformeres med pUC-pre-huTFh, og vellykkede transformanter udvælges på basis af ampicillinresi-stens. De udvalgte transformanter klones derefter og screenes 20 for tilstedeværelse af et rekombinant DNA-molekyle, der har pre-huTFh-strukturgenet.E. coli strain RR1 (Bethesda Research Laboratories, Gaithersburg, MD) is transformed with pUC pre-huTFh and successful transformants are selected on the basis of ampicillin resistance. The selected transformants are then cloned and screened for the presence of a recombinant DNA molecule having the pre-huTFh structural gene.

Screeningen for tilstedeværelse af rekombinante DNA-molekyler, der har pre-huTFh-strukturgenet, gennemføres ved at nedbryde rDNA fra hver udvalgt transformant med EcoRI.The screening for the presence of recombinant DNA molecules having the pre-huTFh structural gene is performed by degrading the rDNA of each selected transformant with EcoRI.

25 De resulterende EcoRI-fragmenter opløses i et mønster efter størrelse på en agarosegel. Rekombinante DNA-molekyler, som giver et mønster af tre bånd svarende til DNA-segmenter på 352, 781 og 2682 bp, indeholder pre-huTFh-strukturgenet. E. coli RR1-transformanter, som har rDNA, der giver det ovenfor 30 beskrevne EcoRI-nedbrydningsmønster, indeholder et rekombinant DNA-molekyle ifølge den foreliggende opfindelse og udvælges (beholdes).The resulting EcoRI fragments are dissolved in a pattern by size of an agarose gel. Recombinant DNA molecules, which give a pattern of three bands corresponding to DNA segments of 352, 781 and 2682 bp, contain the pre-huTFh structural gene. E. coli RR1 transformants having rDNA giving the EcoRI degradation pattern described above contain a recombinant DNA molecule of the present invention and are selected (retained).

Et DNA-segment, der indeholder en væsentlig del af den pre-huTFh-kodende sekvens omfattende den ekstracellulære 35 forankringsregion, men mangler transmembran-forankringsre- 63 DK 176220 B1 gionen ved carboxyterminussen og derved koder for et opløseligt huTFh-protein, konstrueres på følgende måde.A DNA segment containing a substantial portion of the pre-huTFh coding sequence comprising the extracellular anchoring region but lacking the transmembrane anchoring region at the carboxy terminus, thereby encoding a soluble huTFh protein, is constructed as follows. manner.

Plasmid pCTF64 nedbrydes med restriktionsendonukleasen EcoRI til dannelse af et DNA-segment omfattende en nukleo-5 tidsekvens svarende til sekvensen vist i fig. 2 fra rest nr. 1 til rest nr. 486. Segmentet på 486 nukleotid-basepar <bp) fremstillet på denne måde isoleres ved størrelsesfraktionering under anvendelse af en agarosegel og dephosphory-leres derefter ved behandling med alkalisk phosphatase. Det 10 dephosphorylerede 486 bp segment nedbrydes derefter med restriktionsendonukleasen Dralll, der spalter 486 bp segmentet ved en position mellem base nr. 296 og 297 vist i fig.Plasmid pCTF64 is digested with the restriction endonuclease EcoRI to form a DNA segment comprising a nucleotide sequence similar to the sequence shown in FIG. 2 from residue # 1 to residue # 486. The 486 nucleotide base pair (bp) segment prepared in this way is isolated by size fractionation using an agarose gel and then dephosphorylated by treatment with alkaline phosphatase. The 10 dephosphorylated 486 bp segment is then degraded by the restriction endonuclease Dralll, which cleaves the 486 bp segment at a position between base 296 and 297 shown in FIG.

2, hvorved der dannes et 296 bp segment og 190 bp segment.2, thereby forming a 296 bp segment and 190 bp segment.

296 bp segmentet isoleres ved størrelsesfraktionering under 15 anvendelse af en agarosegel.The 296 bp segment is isolated by size fractionation using an agarose gel.

Plasmidet pCTF314 nedbrydes med restriktionsendonukleasen EcoRI til dannelse af et DNA-segment omfattende en nukleotidsekvens svarende til sekvensen vist i fig. 2 fra rest nr. 135 til rest nr. 775. Det resulterende 641 bp seg-20 ment isoleres ved størrelsesfraktionering under anvendelse af en agarosegel og dephosphoryleres derefter ved behandling med alkalisk phosphatase. Det dephosphorylerede 641 bp segment nedbrydes derefter med Dralll, der spalter 641 bp segmentet ved en position mellem baserest nr. 296 og 297 vist 25 i fig. 2, hvorved der dannes et 162 bp segment og et 479 bp segment. 479 bp segmentet isoleres ved størrelsesfraktionering under anvendelse af en agarosegel.The plasmid pCTF314 is digested with the restriction endonuclease EcoRI to form a DNA segment comprising a nucleotide sequence similar to the sequence shown in FIG. 2 from residue # 135 to residue # 775. The resulting 641 bp segment is isolated by size fractionation using an agarose gel and then dephosphorylated by treatment with alkaline phosphatase. The dephosphorylated 641 bp segment is then degraded with Dralll, which cleaves the 641 bp segment at a position between base residue Nos. 296 and 297 shown in FIG. 2, thereby forming a 162 bp segment and a 479 bp segment. The 479 bp segment is isolated by size fractionation using an agarose gel.

De ovenfor fremstillede segmenter på 296 og 479 bp forbindes derefter operativt {ligeres) ved reaktion med T4-3 0 DNA-ligase, hvorved der dannes et 775 bp segment, der har en nukleotid-adapter-sekvens svarende til sekvensen vist i fig. 2 fra rest nr. 1 til rest 775.The 296 and 479 bp segments prepared above are then operatively linked (ligated) by reaction with T4-3 DNA ligase to form a 775 bp segment having a nucleotide adapter sequence similar to the sequence shown in FIG. 2 from residue # 1 to residue 775.

Kloningsplasmidvektor pUC18 lineariseres ved nedbrydning med EcoRI. Det ovenfor fremstillede 775 bp segment og 35 den EcoRI-nedbrudte vektor forbindes operativt ved anvendelse 64 DK 176220 B1 af T4-DNA-ligase til dannelse af det cirkulære rekombinante DNA-molekyle pUC-pre-huTFh-T.Cloning plasmid vector pUC18 is linearized by degradation with EcoRI. The 775 bp segment prepared above and the EcoRI degraded vector are operably linked using 64 T17 DNA ligase to form the circular recombinant DNA molecule pUC-pre-huTFh-T.

E. coli RRl transformeres med pUC-pre-huTFh-T, og ampicillinresistente transformanter, dvs. kloner indeholdende 5 pUC-pre-huTFh-T, udvælges.E. coli RR1 is transformed with pUC pre-huTFh-T and ampicillin resistant transformants, ie. clones containing 5 pUC-pre-huTFh-T are selected.

Det rekombinante DNA-molekyle pUC-pre-huTFh-T nedbrydes med EcoRI, og det resulterende 775 bp segment isoleres ved størrelsesfraktionering.The recombinant DNA molecule pUC pre-huTFh-T is digested with Eco RI and the resulting 775 bp segment is isolated by size fractionation.

Syntetiske oligonukleotid-adaptersegmenter med se-10 kvenserne: 5'-AATTTAGAGAATAAGAATTCGGG-3’ og 31-ATCTCTTATTCTTAAGCCC-51 fremstilles ved metoden ifølge Caruthers et al., J. Am.Synthetic oligonucleotide adapter segments having the sequences: 5'-AATTTAGAGAATAAGAATTCGGG-3 'and 31-ATCTCTTATTCTTAAGCCC-51 are prepared by the method of Caruthers et al., J. Am.

Chem. Soc. , 103:3185 (1981), og Gait et al., Cold Spring 15 Harbor Symp. Quant. Biol., 47:393 (1983), bortset fra, at de således fremstillede oligonukleotider ikke phosphoryleres med polynukleotid-kinase for at forhindre operativ binding (ligering) af disse oligonukleotider til hinanden. Oligonu-kleotiderne kondenseres til dannelse af et dobbeltstrenget 20 DNA-linker-segment indeholdende en klæbende EcoRI-terminus og en stump terminus ved metoderne ifølge Rotherstein et al., Methods in Enzymol., 68:98 (1979). Dette linker-segment forbindes derefter operativt til 775 bp segmentet fremstillet ud fra pUC-pre-huTFh-T til dannelse af et 817 bp segment 25 indeholdende et kondenseret segment ved hver ende af 775 bp segmentet. Det resulterende 817 bp segment nedbrydes derefter med EcoRI til omdannelse af hver terminus af 817 bp segmentet fra en stump ende til en EcoRI-klæbende ende, hvorved der dannes et 805 bp segment. Det resulterende 805 bp segment 30 isoleres ved størrelsesfraktionering under anvendelse af en agarosegel.Chem. Soc. , 103: 3185 (1981), and Gait et al., Cold Spring 15 Harbor Symp. Quant. Biol., 47: 393 (1983), except that the oligonucleotides thus produced are not phosphorylated with polynucleotide kinase to prevent operative binding (ligation) of these oligonucleotides to each other. The oligonucleotides are condensed to form a double-stranded 20 DNA linker segment containing an adhesive EcoRI terminus and a blunt terminus by the methods of Rotherstein et al., Methods in Enzymol., 68:98 (1979). This linker segment is then operatively connected to the 775 bp segment made from pUC pre-huTFh-T to form an 817 bp segment 25 containing a condensed segment at each end of the 775 bp segment. The resulting 817 bp segment is then decomposed with Eco RI to convert each terminus of the 817 bp segment from a blunt end to an Eco RI adhesive, thereby forming an 805 bp segment. The resulting 805 bp segment 30 is isolated by size fractionation using an agarose gel.

Kloningsplasmidvektoren pUC18 lineariseres ved nedbrydning med EcoRI. Det ovenfor fremstillede 805 bp segment og den EcoRI-nedbrudte vektor forbindes operativt ved anven-35 delse af T4-DNA-ligase til dannelse af det cirkulære rekombinante DNA-molekyle pUC-pre-huTFh-TR.The cloning plasmid vector pUC18 is linearized by degradation with EcoRI. The 805 bp segment prepared above and the Eco RI degraded vector are operably linked using T4 DNA ligase to form the circular recombinant DNA molecule pUC-pre-huTFh-TR.

65 DK 176220 B1 E. coli RR1 transformeres med pUC-pre-huTFh-TR, og ampicillinresistente transformanter, dvs. kloner indeholdende pUC-pre-huTFh-TR, udvælges.65 DK 176220 B1 E. coli RR1 is transformed with pUC pre-huTFh-TR and ampicillin resistant transformants, ie. clones containing pUC-pre-huTFh-TR are selected.

5 16. Fremstilling af huTFh ved ekspression af rekombinante huTFh-kodende sekvenser5 16. Preparation of huTFh by expression of recombinant huTFh coding sequences

Ekspressionen af rekombinant huTFh fra rekombinante DNA-molekyler kan gennemføres i forskellige ekspressionsme-dier, omfattende prokaryotiske bakterieceller, eukaryotiske 10 celler af hvirvelløse dyr og eukaryotiske celler af hvirveldyr. Eksempler på sådanne ekspressionsmedier er E. coli, S. cerevisiae og ovarieceller af kinesisk hamster (CHO-celler).The expression of recombinant huTFh from recombinant DNA molecules can be performed in various expression media, including prokaryotic bacterial cells, eukaryotic cells of invertebrates and eukaryotic cells of vertebrates. Examples of such expression media are E. coli, S. cerevisiae and Chinese hamster ovary cells (CHO cells).

a. Ekspression af pre-huTFh i E. coli 15 Et rekombinant DNA-molekyle, der kan eksprimere pre- huTFh-strukturgenet i E. coli-celler, kan konstrueres ved at isolere et pre-huTFh-gen-holdigt DNA-segment fra det rekombinante pUC-pre-huTFh-DNA-molekyle fremstillet i eksempel 15 og forbinde dette segment operativt med en prokaryo-20 tisk ekspressionsvektor.a. Expression of pre-huTFh in E. coli 15 A recombinant DNA molecule capable of expressing the pre-huTFh structural gene in E. coli cells can be constructed by isolating a pre-huTFh gene-containing DNA segment from the recombinant pUC pre-huTFh DNA molecule prepared in Example 15 and operatively linking this segment with a prokaryotic expression vector.

Det rekombinante DNA-molekyle pUC-pre-huTFh nedbrydes med EcoRI under sådanne betingelser, at nogle, men ikke alle EcoRI-stederne i plasmidet spaltes. Denne partielle nedbrydningsprocedure er beskrevet mere detaljeret i Maniatis 2 5 et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratories, Cold Spring Harbor, NY (1982). Et 1133 bp segment omfattende en nukleotidsekvens svarende til sekvensen vist i fig. 2 fra rest nr. 1 til rest nr. 1125 isoleres fra produkterne af den delvise EcoRI-nedbrydning ved 30 størrelsesfraktionering.The recombinant DNA molecule pUC pre-huTFh is digested with EcoRI under conditions such that some, but not all of the EcoRI sites in the plasmid are cleaved. This partial degradation procedure is described in more detail in Maniatis 25 et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratories, Cold Spring Harbor, NY (1982). An 1133 bp segment comprising a nucleotide sequence corresponding to the sequence shown in FIG. 2 from residue # 1 to residue # 1125 are isolated from the products of partial EcoRI degradation by 30 size fractionation.

Den prokaryotiske ekspressionsvektor pKK223-3 (Pharmacia Fine Chemicals, Pisctaway, NJ) lineariseres ved nedbrydning med EcoRI. Den nedbrudte vektor og 1133 bp segmentet indeholdende pre-huTFh-strukturgenet forbindes operativt 35 ved anvendelse af T4-DNA-ligase til dannelse af det cirkulære rekombinante DNA-molekyle pKK-pre-huTFh.The prokaryotic expression vector pKK223-3 (Pharmacia Fine Chemicals, Pisctaway, NJ) is linearized by degradation with EcoRI. The degraded vector and the 1133 bp segment containing the pre-huTFh structural gene are operatively linked using T4 DNA ligase to form the circular recombinant DNA molecule pKK pre-huTFh.

66 DK 176220 B1 E. coli RR1 transformeres med pKK-pre-huTFh, og am-picillinresistente transformanter, dvs. kloner indholdende pKK-pre-huTFh, udvælges.66 DK 176220 B1 E. coli RR1 is transformed with pKK pre-huTFh and am-picillin resistant transformants, ie. clones containing pKK pre-huTFh are selected.

5 b. Ekspression af huTFh i E. coli5 b. Expression of huTFh in E. coli

Et rekombinant DNA-molekyle, der kan eksprimere huTF-genet i E. coli, konstrueres ved manipulering af 1133 bp segmentet fremstillet i eksempel 16a. Dette segment dephos-phoryleres først med alkalisk phosphatase og nedbrydes der-10 efter med restriktionsendonukleasen Bbvl. Det resulterende 964 bp segment omfatter en nukleotidsekvens svarende til sekvensen vist i fig. 2 fra rest nr. 164 til rest nr. 1125 og isoleres ved størrelsesfraktionering.A recombinant DNA molecule capable of expressing the huTF gene in E. coli is constructed by manipulating the 1133 bp segment prepared in Example 16a. This segment is first dephosphorylated with alkaline phosphatase and then degraded by the restriction endonuclease Bbvl. The resulting 964 bp segment comprises a nucleotide sequence similar to the sequence shown in FIG. 2 from residue # 164 to residue # 1125 and isolated by size fractionation.

Syntetiske oligonukleotid-adapter-segmenter med se-15 kvenserne: 5’-AATTGACATGTCAGGCACTACAAATACTGTGGCAGCATATAATT-3' og 3'-CTGTACAGTCCGTGATGTTTATGACACCGTCGTATATTAAATTG-5' fremstilles som ovenfor beskrevet og kondenseres til dannelse 20 af et dobbeltstrenget DNA-linker-segment indeholdende klæbende EcoRI- og Bbvl-ender ved metoden ifølge Rotherstein et al., Methods in Enzymol. , 68:98 (1979). Linkeren forbindes først operativt til 964 bp segmentet til dannelse af 1008 bp segment. 1008 bp segmentet forbindes derefter operativt 25 til den EcoRI-nedbrudte vektor pKK223-3 ved anvendelse af T4-DNA-ligase til dannelse af det cirkulære rekombinante DNA-molekyle pKK-huTFh.Synthetic oligonucleotide adapter segments with the sequences: 5'-AATTGACATGTCAGGCACTACAAATACTGTGGCAGCATATAATT-3 'and 3'-CTGTACAGTCCGTGATGTTTATGACAT using the method of Rotherstein et al., Methods in Enzymol. , 68:98 (1979). The linker is first operatively connected to the 964 bp segment to form the 1008 bp segment. The 1008 bp segment is then operatively linked to the EcoRI degraded vector pKK223-3 using T4 DNA ligase to form the circular recombinant DNA molecule pKK-huTFh.

Det rekombinante DNA-molekyle pKK-huTFh adskiller sig kun fra pKK-pre-huTFh ved, at 1) et segment fra rest nr.The recombinant DNA molecule pKK-huTFh differs from pKK pre-huTFh only in that 1) a segment from residue no.

30 1 til rest nr. 129 er udeladt, og 2) et nyt methionin-kodon er operativt forbundet før rest nr. 130, således at proteinekspressionen (translationen) begynder ved det indføjede methionin-kodon.30 1 to residue # 129 is omitted and 2) a new methionine codon is operatively linked before residue # 130, so that protein expression (translation) begins at the inserted methionine codon.

De rekombinante DNA-molekyler pKK-pre-huTFh og pKK-35 huTFh indføres i et prokaryotisk værtsmedium foreneligt med ekspression af huTFh- eller pre-huTFh-proteinet, som kodes 67 DK 176220 B1 af det strukturelle gen indeholdt deri. Eksempler på værtsceller indeholdende sådant medium er E. coli stamme RR1. Værten transformeres med det rekombinante DNA-molekyle og dyrkes under betingelser, der er forenelige med cellevækst 5 og ekspression af det rekombinante DNA, og det eksprimerede protein høstes ved velkendte metoder.The recombinant DNA molecules pKK pre-huTFh and pKK-35 huTFh are introduced into a prokaryotic host medium compatible with expression of the huTFh or pre-huTFh protein encoded therein by the structural gene. Examples of host cells containing such medium are E. coli strain RR1. The host is transformed with the recombinant DNA molecule and cultured under conditions compatible with cell growth 5 and expression of the recombinant DNA, and the expressed protein is harvested by well known methods.

c. Ekspression af pr-huTFh i CHO-cellerc. Expression of pr-huTFh in CHO cells

Et rekombinant-DNA-molekyle, der er i stand til at 10 eksprimere pre-huTFh-genet i hvirveldyr-celler, konstrueres ved anvendelse af 1133 bp segmentet fremstillet i eksempel 16a.A recombinant DNA molecule capable of expressing the pre-huTFh gene in vertebrate cells is constructed using the 1133 bp segment prepared in Example 16a.

Syntetiske oligonukleotid-adapter-segmenter med sekvenserne : 15 5'-AATTCCCGGG-3' og 5'-GATCCCCGGG-3’ fremstilles ved metoden ifølge Caruthers et al., supra og Gait et al., supra. Oligonukleotid-adaptersegmenterne forbindes derefter til hver terminus af 1133 bp segmentet ved 20 anvendelse af metoderne beskrevet af Rotherstein et al., Methods in Enzymol., 68:98 (1979). De klæbende EcoRl-ender, som oprindelig er til stede på 1133 bp segmentet, omdannes derved til klæbende Bglll-ender.Synthetic oligonucleotide adapter segments having the sequences: 5'-AATTCCCGGG-3 'and 5'-GATCCCCGGG-3' are prepared by the method of Caruthers et al., Supra and Gait et al., Supra. The oligonucleotide adapter segments are then connected to each terminus of the 1133 bp segment using the methods described by Rotherstein et al., Methods in Enzymol., 68:98 (1979). The adhesive EcoR1 ends, which are initially present on the 1133 bp segment, are thereby converted to adhesive BglII ends.

Den eukaryotiske abevirus-(SV40-)-baserede ekspres-25 sionsvektor pKSV-10 (Pharmacia Fine Chemicals, Piscataway, NJ) lineariseres ved nedbrydning med restriktionsendonu-kleasen Bglll . Det Bglll-adapterede 1133 bp segment og den BgllI-nedbrudte vektor forbindes operativt ved anvendelse af T4-DNA-ligase til dannelse af det cirkulære rekombinante 30 DNA-molekyle pSV-pre-huTFh.The eukaryotic monkey virus (SV40 -) based expression vector pKSV-10 (Pharmacia Fine Chemicals, Piscataway, NJ) is linearized by digestion with the restriction endonuclease BglII. The BglII-adapted 1133 bp segment and the BglII-degraded vector are operatively linked using T4 DNA ligase to form the circular recombinant DNA molecule pSV pre-huTFh.

E. coli RR1 transformeres med pSV-pre-huTFh, og vellykkede transformanter udvælges på basis af ampicillinresi-stens og klones. De udvalgte transformanter klones derefter og screenes for tilstedeværelse af pSV-pre-huTFh ved under-35 søgelse af hver klon for tilstedeværelsen af eksprimeret 68 DK 176220 B1 pre-huTFh-protein ved anvendelse af det monoklonalle antistof TF8-5G9.E. coli RR1 is transformed with pSV pre-huTFh and successful transformants are selected on the basis of ampicillin resistance and cloned. The selected transformants are then cloned and screened for the presence of pSV pre-huTFh by examining each clone for the presence of expressed 68 DK 176220 B1 pre-huTFh protein using the monoclonal antibody TF8-5G9.

d. Ekspression af huTFh i CHO-celler 5 Et rekombinant DNA-molekyle, der er i stand til at eksprimere huTFh-genet i pattedyrceller, konstrueres ved at nedbryde pSV-pre-huTFh fra eksempel 16c med restriktionsen-donuklease Bglll. Det resulterende 1153 bp segment isoleres ved størrelsesfraktionering og nedbrydes derefter med re-10 striktionsendonukleasen Bbvl. Det resulterende 974 bp segment omfatter en nukleotid-adapter-sekvens svarende til sekvensen vist i fig. 2 fra rest nr. 164 til rest nr. 1125 og isoleres ved størrelsesfraktionering.d. Expression of huTFh in CHO Cells 5 A recombinant DNA molecule capable of expressing the huTFh gene in mammalian cells is constructed by degrading pSV pre-huTFh of Example 16c with restriction endonuclease BglII. The resulting 1153 bp segment is isolated by size fractionation and then degraded by the restriction endonuclease Bbvl. The resulting 974 bp segment comprises a nucleotide adapter sequence similar to the sequence shown in FIG. 2 from residue # 164 to residue # 1125 and isolated by size fractionation.

Syntetiske oligonukleotid-adapter-segmenter med se-15 kvenserne: 5'-GATCGACATGTCAGGCACTACAAATACTGTGGCAGCATATAATT-31 og 3'-CTGTACAGTCCGTGATGTTTATGACACCGTCGTATATTAAATTG-51 fremstilles som ovenfor beskrevet og kondenseres til dannelse 20 af et dobbeltstrenget DNA-linker-segment indeholdende klæbende Bglll-ender og klæbende Bbvl-ender. Linkeren forbindes derefter operativt til 974 bp segmentet ved anvendelse af T4-DNA-ligase til dannelse af 1018 bp segment omfattende en nukleotidsekvens svarende til sekvensen vist i fig. 2 fra 25 rest nr. 130 til rest nr. 1125.Synthetic Oligonucleotide Adapter Segments with Sequences: 5'-GATCGACATGTCAGGCACTACAAATACTGTGGCAGCATATAATT-31 -ends. The linker is then operatively linked to the 974 bp segment using T4 DNA ligase to generate 1018 bp segment comprising a nucleotide sequence similar to the sequence shown in FIG. 2 from 25 residue # 130 to residue # 1125.

Plasmidekspressionsvektoren pKSV-10 lineariseres ved nedbrydning med Bglll og forbindes derefter operativt til 1018 bp segmentet ved anvendelse af T4-DNA-ligase til dannelse af det cirkulære rekombinante DNA-molekyle pSV-huTFh.The plasmid expression vector pKSV-10 is linearized by degradation with BglII and then operatively linked to the 1018 bp segment using T4 DNA ligase to form the circular recombinant DNA molecule pSV-huTFh.

30 De rekombinante DNA-molekyler pSV-pre-huTFh og pSV~ huTFh indføres i et eukaryotisk værtsmedium foreneligt med ekspression af huTFh- eller pre-huTFh-proteinet kodet af det strukturelle gen indeholdt deri. Eksempler på værtsceller indeholdende et sådant medium er CHO-celler.The recombinant DNA molecules pSV pre-huTFh and pSV ~ huTFh are introduced into a eukaryotic host medium compatible with expression of the huTFh or pre-huTFh protein encoded by the structural gene contained therein. Examples of host cells containing such a medium are CHO cells.

35 Værten transficeres med det rekombinante DNA-molekyle, og stabile transformanter udvælges ved velkendte metoder, 69 DK 176220 B1 se f.eks. Graham et al., Virol., 52:456 (1973); og Southern et al., J. Mol. Appl. Genet., 1:327-341 (1982). Transformerede værtsceller dyrkes under betingelser, der er forenelige med cellevækst og ekspression af det rekombinante DNA, og 5 det eksprimerede protein høstes ved velkendte metoder.The host is transfected with the recombinant DNA molecule and stable transformants are selected by methods well known in the art. Graham et al., Virol., 52: 456 (1973); and Southern et al., J. Mol. Appl. Genet., 1: 327-341 (1982). Transformed host cells are grown under conditions compatible with cell growth and expression of the recombinant DNA, and the expressed protein is harvested by well known methods.

e. Ekspression af pre-huTFh i gære. Expression of pre-huTFh in yeast

Et rekombinant DNA-molekyle, der er i stand til at eksprimere pre-huTFh-genet i S, cerevisiae, konstrueres ved 10 at fremstille oligonukleotid-adapter-segmenter med sekvensen: 5'-AATTCCCGGG-3' og 5'-CGCCCGGG-31 og forbinde dem til terminusserne af 1133 bp segmentet fra eksempel 16a som ovenfor beskrevet. Det adapterede segment 15 dannet på danne måde har klæbende Clal-ender.A recombinant DNA molecule capable of expressing the pre-huTFh gene in S, cerevisiae is constructed by producing oligonucleotide adapter segments having the sequence: 5'-AATTCCCGGG-3 'and 5'-CGCCCGGG-31 and connecting them to the terminals of the 1133 bp segment of Example 16a as described above. The adapted segment 15 formed in this manner has adhesive Clal ends.

Gærekspressionsvektoren pTDTl (ATCC 31255) lineari-seres ved nedbrydning med restriktionsendonukleasen Clal.The yeast expression vector pTDT1 (ATCC 31255) is linearized by digestion with the restriction endonuclease Clal.

Det ovennævnte Clal-adapterede 1133 bp segment og den Clal-nedbrudte vektor forbindes operativt ved anvendelse af T4-20 DNA-ligase til dannelse af det cirkulære rekombinante DNA-molekyle pY-pre-huTFh.The aforementioned Clal-adapted 1133 bp segment and the Clal degraded vector are operably linked using T4-20 DNA ligase to form the circular recombinant DNA molecule pY-pre-huTFh.

E. coli RR1 transformeres med pre-huTFh, og transfor-manter, der eksprimerer pre-huTFh-strukturgenet, identificeres og udvælges som beskrevet i eksempel 16c.E. coli RR1 is transformed with pre-huTFh, and transformants expressing the pre-huTFh structural gene are identified and selected as described in Example 16c.

25 f. Ekspression af huTFh i gær25 f. Expression of huTFh in yeast

Et rekombinant DNA-molekyle, der er i stand til at eksprimere huTFh-strukturgenet i S. cerevisiae, konstrueres ved at nedbryde pY-pre-huTFh med Clal til dannelse af et 30 1151 bp segment omfattende en nukleotidsekvens svarende til sekvensen vist i fig. 2 fra rest nr. 1 til rest nr. 1125.A recombinant DNA molecule capable of expressing the huTFh structural gene in S. cerevisiae is constructed by degrading pY pre-huTFh with ClaI to form a 30,111 bp segment comprising a nucleotide sequence similar to the sequence shown in FIG. 2 from residue # 1 to residue # 1125.

Efter isolering ved størrelsesfraktionering nedbrydes 1151 bp segmentet med Bbvl til dannelse af et 97 8 bp segment omfattende en nukleotidsekvens svarende til sekvensen vist 35 i fig. 2 f ra rest nr. 164 til rest nr. 1125. 978 bp segmentet isoleres derefter ved størrelsesfraktionering.After isolation by size fractionation, the 1151 bp segment is broken down with Bbv1 to form a 97 8 bp segment comprising a nucleotide sequence similar to the sequence shown in FIG. 2 from residue # 164 to residue # 1125. The 978 bp segment is then isolated by size fractionation.

70 DK 176220 B170 DK 176220 B1

Syntetiske oligonukleotid-adapter-segmenter med sekvenserne : 5'-CGGACATGTCAGGCACTACAAATACTGTGGCAGCATATAATT- 3' og 5 3'-CTGTACAGTCCGTGATGTTTATGACACCGTCGTATATTAAATTG-5' fremstilles og kondenseres som ovenfor beskrevet til dannelse af DNA-adapter-segmenter, der har klæbende Clal- og BbvI-ender. Adaptersegmentet forbindes først operativt til 978 bp segmentet til dannelse af 1020 bp segment. 1020 bp seg-10 mentet forbindes derefter til den Clal-nedbrudte pTDTl-vek-tor, fremstillet som beskrevet i eksempel 16e, ved anvendelse af T4-DNA-ligase til dannelse af det cirkulære rekombinante DNA-molekyle pY-huTFh,Synthetic oligonucleotide adapter segments having the sequences: The adapter segment is first operatively connected to the 978 bp segment to form the 1020 bp segment. The 1020 bp segment is then connected to the ClaI digested pTDT1 vector, prepared as described in Example 16e, using T4 DNA ligase to generate the circular recombinant DNA molecule pY-huTFh.

De rekombinante DNA-molekyler pY-pre-huTFh og pY-huTFh 15 indføres i et gær-værtsmedium foreneligt med ekspression af huTFh- eller pre-huTFh-proteinet kodet af strukturgenet indeholdt deri. Eksempler på værtsceller omfattende et sådant medium er S. cerevisiae-celler.The recombinant DNA molecules pY-pre-huTFh and pY-huTFh are introduced into a yeast host medium compatible with expression of the huTFh or pre-huTFh protein encoded by the structural gene contained therein. Examples of host cells comprising such a medium are S. cerevisiae cells.

Værten transformeres med det rekombinante DNA-molekyle 20 og dyrkes i selektionsmedium til isolering af vellykket transformerede celler ved velkendte metoder, se f.eks. Hinnen et al., Proc. Natl. Acad. Sci. USA, 75:1929 {1978); og Mi- yajima et al., Mol. Cell. Biol., 4:407 (1984). Transformerede celler dyrkes under betingelser, der er forenelige med cel-25 levækst og ekspression af det rekombinante DNA, og det eks-primerede protein høstes ved velkendte metoder.The host is transformed with the recombinant DNA molecule 20 and cultured in selection medium to isolate successfully transformed cells by well known methods, see e.g. Hinnen et al., Proc. Natl. Acad. Sci. USA, 75: 1929 (1978); and Miyajima et al., Mol. Cell. Biol., 4: 407 (1984). Transformed cells are grown under conditions compatible with cell growth and expression of the recombinant DNA, and the expressed protein is harvested by well known methods.

g. Fremstilling af opløseligt huTFh ved ekspression af rekombinante huTFh-kodende sekvenser.g. Preparation of soluble huTFh by expression of recombinant huTFh coding sequences.

Ekspressionen af opløseligt huTFh ud fra rekombinante 30 DNA-molekyler kan opnås i forskellige ekspressionsmedier på lignende måde som beskrevet i eksempel 16 for pre-huTFh og huTFh. I det eksempel produceres et 1133 bp segment indeholdende pre-huTFh-strukturgenet og med klæbende EcoRI-ender i eksempel 16a og manipuleres derefter i eksempel 16b-f, hvor-35 ved der fås vektorer, som er i stand til at eksprimere enten pre-huTFh eller huTFh i tre eksempelvise ekspressionsmedier, 71 DK 176220 B1 E. coli, S. cerevisiae og CHO-celler. På lignende måde manipuleres 805 bp segmentet indeholdende et strukturgen for opløseligt pre-huTFh og med klæbende EcoRI-ender og fremstillet i eksempel 16a ved metoderne beskrevet i eksempel 16b-f 5 til fremstilling af ekspressionsvektorer, der er i stand til at eksprimere en opløselig form af enten pre-huTFh eller huTFh (dvs. pre-huTFh-TR eller huTfh-TR) i de samme ekspressionsmedier .The expression of soluble huTFh from recombinant DNA molecules can be obtained in various expression media in a similar manner as described in Example 16 for pre-huTFh and huTFh. In that example, a 1133 bp segment containing the pre-huTFh structural gene and with adhesive EcoRI ends of Example 16a is produced and then manipulated in Examples 16b-f, thereby obtaining vectors capable of expressing either huTFh or huTFh in three exemplary expression media, E. coli, S. cerevisiae and CHO cells. Similarly, the 805 bp segment containing a structural gene for soluble pre-huTFh and with adhesive Eco RI ends and manipulated in Example 16a is manipulated by the methods described in Examples 16b-f 5 to produce expression vectors capable of expressing a soluble form. of either pre-huTFh or huTFh (i.e., pre-huTFh-TR or huTfh-TR) in the same expression media.

17. Inhibering af koagulering med polypep-10 tiderne P24-35 og Ό159-16917. Inhibition of coagulation by polypeptides P24-35 and Ό159-169

Polypeptiderne p24-35 og pl59-169, hvis aminosyre-sekvenser er vist i tabel 7, syntetiseres som beskrevet i eksempel 11.The polypeptides p24-35 and p159-169, whose amino acid sequences are shown in Table 7, are synthesized as described in Example 11.

1515

Tabel VIITable VII

Betegnelse3·) Amino syre sekvensDesignation3 ·) Amino acid sequence

p24-35 H-EWEPKPVNQVYT-OHp24-35 H-EWEPKPVNQVYT-OH

P159-169 Η-1YTLYYWKS S S SGKKTAK-OHP159-169 Η-1YTLYYWKS S S SGKKTAK-OH

2 0 _ a) Laboratoriebetegnelsen for hvert polypeptid repræ senterer den inkluderede aminosyresekvens som vist i fig. 1.A) The laboratory designation for each polypeptide represents the included amino acid sequence as shown in FIG. First

Polypeptiderne p24-35 og pl59-169 undersøges derefter for deres evne til kompetitiv inhibering af huTF-initieret 25 koagulering som beskrevet i eksempel 12. Resultaterne af denne undersøgelse er vist i fig. 12 og viser, at p24-35 og pl59-169 er i stand til at inhibere huTF-initieret koagulering henholdsvis 45 og 25% ved anvendelse i koncentrationer på 10 μΜ. Det skal bemærkes, at ved denne undersøgelse er 30 baggrundsinhiberinger som for peptiderne vist ved en udfyldt cirkel i fig. 12 lavere end ved undersøgelsen vist i tabel 4. Som resultat heraf betragtes polypeptider, der giver mindst 20% inhibering af koagulering ved en koncentration på 10 μΜ ved denne undersøgelse, som huTF-bindingssted-poly-35 peptidanaloge.Polypeptides p24-35 and p159-169 are then tested for their ability to competitively inhibit huTF-initiated clotting as described in Example 12. The results of this study are shown in Figs. 12 and shows that p24-35 and p159-169 are capable of inhibiting huTF-initiated coagulation at 45 and 25%, respectively, when used at concentrations of 10 μΜ. It should be noted that in this study, 30 background inhibitions as for the peptides are shown by a filled circle in FIG. 12 lower than in the study shown in Table 4. As a result, polypeptides giving at least 20% inhibition of coagulation at a concentration of 10 μΜ in this study are considered as huTF binding site polypeptide analogs.

72 DK 176220 B172 DK 176220 B1

Polypeptiderne p24-35 og pl59-169 udgør således huTFh-polypeptid-bindingssted-analoge ifølge opfindelsen. Det skal også bemærkes, at resultaterne opnået med p24-35, på baggrund af de lignende resultater opnået med polypeptid 5 p25-49, viser, at et huTFh-faktor VH/VIIa-bindingssted kan dannes af aminosyresekvensen, som disse to polypeptider har fælles, dvs. resterne 30-35 som vist i fig. 1 (-VNQVYT-).Thus, polypeptides p24-35 and p159-169 constitute huTFh polypeptide binding site analogs of the invention. It should also be noted that the results obtained with p24-35, based on the similar results obtained with polypeptide 5 p25-49, show that a huTFh factor VH / VIIa binding site can be formed by the amino acid sequence shared by these two polypeptides. , ie residues 30-35 as shown in FIG. 1 (-VNQVYT-).

18. De kinetiske forhold for inhibering af koagulering med anti-huTF-antistoffer 10 Til bestemmelse af tidsrummet, i hvilket anti-huTF- antistoffer er i stand til at inhibere huTF-initieret koagulering, måles tidsforløbet for inhibering ved anvendelse af inhiberingsbestemmelsen beskrevet i eksempel 10.18. The Kinetic Conditions for Inhibition of Coagulation with Anti-huTF Antibodies 10 To determine the length of time during which anti-huTF antibodies are capable of inhibiting huTF-initiated coagulation, the time course of inhibition is measured using the inhibition assay described in Example 10th

Ca. 1 ng af MAPS-isoleret monoklonalt TF8-5G9-antistof 15 fremstillet som beskrevet i eksempel 7 blandes i 100 μΐ HBS/BS med ca. 1 ng relipideret huTF fremstillet som beskrevet i eksempel 4. De forskellige blandinger, der er dannet på denne måde, holdes ved 37°C i varierende tidsrum fra ca. 1 til ca. 60 minutter til immunologisk binding af 20 anti-huTF-antistofmolekylet til huTF og dannelse af et immu-noreaktionsprodukt. På tidspunkterne anført i fig. 13 undersøges hver blanding derefter for huTF-prokoagulerende aktivitet som beskrevet i eksempel 2, og den procentiske inhibering udtrykkes derefter som beskrevet i eksempel 10.Ca. 1 ng of MAPS-isolated monoclonal TF8-5G9 antibody prepared as described in Example 7 is mixed in 100 μΐ HBS / BS with ca. 1 ng of precipitated huTF prepared as described in Example 4. The various mixtures formed in this manner are maintained at 37 ° C for varying periods of time from ca. 1 to approx. 60 minutes for immunologically binding the 20 anti-huTF antibody molecule to huTF and forming an immunoreaction product. At the times indicated in FIG. 13, each mixture is then examined for huTF procoagulant activity as described in Example 2, and the percent inhibition is then expressed as described in Example 10.

25 Fig. 13 viser resultaterne af en sådan kinetisk må ling, der viser, at inhiberinger af huTF-initieret koagulering på over 65% forekommer på mindre end 10 minutter ved den ved denne bestemmelse anvendte koncentration af antistof og renset huTF. Det antages, at der fås en mere hurtig og 30 fuldstændig inhibering ved højere koncentrationer af anti-huTF-antistof.FIG. Figure 13 shows the results of such a kinetic measurement showing that inhibitions of huTF-initiated coagulation greater than 65% occur in less than 10 minutes at the antibody and purified huTF concentration used in this assay. It is believed that more rapid and complete inhibition is obtained at higher concentrations of anti-huTF antibody.

19. Dosis-reaktion for inhibering af huTF--initieret koagulering med anti-huTF--antistoffer 35 Evnen af anti-huTF-antistofferne ifølge den forelig gende opfindelse til at inhibere huTF-initieret koagulering 73 DK 176220 B1 over et område af antistofdoseringer bestemmes ved metoderne beskrevet i eksempel 10 med følgende modifikationer. 1 ng relipideret huTF fremstillet ifølge eksempel 4 blandes i 0,1 ml HBS/BSA med forskellige mængder af monoklonalt 5 TF8-5G9-antistof isoleret som beskrevet i eksempel 7. De således fremstillede blandinger bibeholdes til dannelse af immunoreaktionsprodukter og undersøges derefter for huTF-prokoagulerende aktivitet som beskrevet i eksempel 10.19. Dose response for inhibition of huTF - initiated coagulation with anti-huTF antibodies 35 The ability of the anti-huTF antibodies of the present invention to inhibit huTF-initiated coagulation is determined over a range of antibody doses. by the methods described in Example 10 with the following modifications. 1 ng of precipitated huTF prepared according to Example 4 is mixed in 0.1 ml of HBS / BSA with various amounts of monoclonal 5 TF8-5G9 antibody isolated as described in Example 7. The thus prepared mixtures are maintained to form immunoreaction products and then tested for huTF. procoagulant activity as described in Example 10.

Resultaterne af en sådan dosis-reaktions-bestemmelse 10 er vist i fig. 14 og viser, at inhiberingerne er det halve af maksimum ved ca. 1-5 ng anti-huTF pr. ml ved den i denne undersøgelse anvendte koncentration af huTF.The results of such dose-response assay 10 are shown in FIG. 14, showing that the inhibitions are half the maximum at ca. 1-5 ng of anti-huTF per ml at the concentration of huTF used in this study.

En lignende dosis-reaktion gennemføres ved anvendelse af lyserede humane celler som kilde til huTF.A similar dose response is performed using lysed human cells as a source of huTF.

15 Den humane fibroblast-cellelinie GM1381 (NIGMS HumanThe human fibroblast cell line GM1381 (NIGMS Human

Genetic Mutant Cell Repository) dyrkes i Dulbecco's modificerede Eagle's medium (DMEM, Gibco Laboratories, Grand Island, NY) suppleret med 2 mM glutamin, 5% kalvefosterserum og antibiotika ved 37°C og under en atmosfære af 7 volumen-20 procent carbondioxid i luft. GM1381-cellerne dyrkes og høstes, og en pellet af 30xl06 celler fremstilles ved centri fugering og fryses ved -70°C. Denne frosne pellet optøes hurtigt ved tilsætning af 9 ml 15 mM β-octylglucopyranosid (Sigma) i HN-puffer (25 mM Hepes, 140 mM natriumchlorid, 25 pH-værdi 7,0} og holdes ved 37°C i 10 minutter til lyse af cellerne, hvorefter der iblandes 18 ml HN til dannelse af et cellelysat.Genetic Mutant Cell Repository) is grown in Dulbecco's modified Eagle's medium (DMEM, Gibco Laboratories, Grand Island, NY) supplemented with 2 mM glutamine, 5% fetal calf serum and antibiotics at 37 ° C and under an atmosphere of 7 volume-20 percent carbon dioxide in air . The GM1381 cells are grown and harvested, and a pellet of 30x106 cells is prepared by centrifugation and frozen at -70 ° C. This frozen pellet is rapidly thawed by the addition of 9 ml of 15 mM β-octylglucopyranoside (Sigma) in HN buffer (25 mM Hepes, 140 mM sodium chloride, pH 7.0) and kept at 37 ° C for 10 minutes to light. of the cells, after which 18 ml of HN is mixed to form a cell lysate.

Monoklonalt antistof TF8-5G9 isoleret som beskrevet i eksempel 7 fortyndes med 0,01% BSA (Sigma, RIA-kvalitet) 30 til de forskellige doseringer anført i fig. 15. 25 μΐ af hver antistoffortynding blandes derefter med 225 μΐ af det ovenfor fremstillede cellelysat og holdes ved 37°C i 60 minutter for at gøre det muligt for antistoffet at immuno-reagere med huTF, som er til stede i cellelysatet, og danne 35 et immunoreaktionsprodukt. Derefter blandes 50 μΐ af en 25 mM calciumchloridopløsning med 50 μΐ af opløsningen indehol- 74 DK 176220 B1 dende immunoreaktionsproduktet, hvorefter der tilsættes 50 μΐ citratbehandlet humant plasma til initiering af koagulering, De således dannede blandinger holdes ved 3 7°C, og tidsrummet mellem tilsætning af plasma og dannelse af et 5 koagel måles. Den effektive huTF-koncentration og den pro-centiske inhibering beregnes som beskrevet i eksempel 10.Monoclonal antibody TF8-5G9 isolated as described in Example 7 is diluted with 0.01% BSA (Sigma, RIA grade) 30 to the various dosages listed in FIG. 15. 25 μΐ of each antibody dilution is then mixed with 225 μΐ of the above cell lysate and maintained at 37 ° C for 60 minutes to allow the antibody to immunoreact with huTF present in the cell lysate to form 35 an immunoreaction product. Then, 50 μΐ of a 25 mM calcium chloride solution is mixed with 50 μΐ of the solution containing the immunoreaction product, then 50 μΐ citrate-treated human plasma is added to initiate coagulation, the mixtures thus formed are kept at 37 ° C and the time interval between plasma addition and formation of a clot are measured. The effective huTF concentration and the percent inhibition are calculated as described in Example 10.

Resultaterne af en dosis-reaktions-inhiberingsbestem-melse ved anvendelse af lysater af humane GM1381-celler som kilde til huTF er vist i fig. 15. Disse resultater viser, 10 at TF8-5G9-anti-huTF-antistof giver det halve af maksimal inhibering af denne cellelysatkilde til huTF ved ca. 8-10 ng antistof pr. ml.The results of a dose-response inhibition assay using lysates of human GM1381 cells as a source of huTF are shown in FIG. 15. These results show that TF8-5G9 anti-huTF antibody provides half of the maximum inhibition of this cell lysate source to huTF at ca. 8-10 ng antibody per ml.

20. Krydsreaktivitet af monoklonale antistoffer med ikke-human vævsfaktor 15 Vævsfaktor isoleres fra enten hjernevæv (rotte, kanin, okse, hund, får, svin og bavian) eller vævskulturceller (nyreceller af afrikansk grøn abe (COS-celler) . Væv eller celler optøes, befries for membraner, hakkes, homogeniseres i 1 ml kold acetone pr. g væv og filtreres under vakuum 20 gennem Whatman nr. 1-papir. Det faste stof resuspenderes i acetone og filtreres yderligere fem gange, hvorefter det lufttørres natten over og opbevares ved -30°C. Acetone-pulverne, som udgør 16-19% af udgangsvægten (våd), pulveriseres med en morter og pistil, resuspenderes i en koncentration 2 5 på 5% (vægt/volumen) i TBS indeholdende 5 mmol/liter EDTA og blandes 1 time ved omgivelsestemperatur. Fast stof fraskilles ved centrifugering ved 10.000 g i 30 minutter ved 20°C, og TF-holdige membraner opsamles ved centrifugering af den ovenstående væske ved 100.000 g i 1 time. Pelleten 30 resuspenderes i TBS og opbevares ved -80°C.20. Cross-reactivity of monoclonal antibodies with non-human tissue factor 15 Tissue factor is isolated from either brain tissue (rat, rabbit, ox, dog, sheep, pig and baboon) or tissue culture cells (kidney cells of African green monkey (COS cells). Tissue or cells are thawed) , free from membranes, chopped, homogenized in 1 ml of cold acetone per gram of tissue and filtered under vacuum 20 through Whatman No. 1 paper, resuspended in acetone and filtered five more times, then air dried overnight and stored at room temperature. -30 ° C. The acetone powders, which constitute 16-19% of the starting weight (wet), are pulverized with a mortar and pestle, resuspended at a concentration of 5% (5% by weight / volume) in TBS containing 5 mmol / liter EDTA and mixed for 1 hour at ambient temperature. Solid is separated by centrifugation at 10,000 g for 30 minutes at 20 ° C, and TF-containing membranes are collected by centrifugation of the above liquid at 100,000 g for 1 hour. be stored at -80 ° C.

Antistof-inhibering af animalsk TF (rå vævsekstrakter indeholdende TF-aktivitet) bestemmes på følgende måde. Lige volumener af TF (1 mg/ml) og ovenstående hybridoma-væske (fortyndet 1:10 med TBS/BSA) inkuberes i 2 timer ved 37°C, 35 Tilbageværende TF-aktivitet måles ved tilsætning af 100 μΐ af inkuberingsblandingen til 50 μΐ human faktor VII-frit 75 DK 176220 B1 plasma og 50 μΐ 50 mM calciumchlorid. Efter 1 minut ved 37°C tilsættes 50 μΐ af en 1:10-fortynding af homolog-art-serum som kilde til faktor VII, og der foretages en dobbeltbestemmelse af tidsrummet til dannelse af koagel.Antibody inhibition of animal TF (crude tissue extracts containing TF activity) is determined as follows. Equal volumes of TF (1 mg / ml) and the above hybridoma liquid (diluted 1:10 with TBS / BSA) are incubated for 2 hours at 37 ° C, 35 Remaining TF activity is measured by adding 100 μΐ of the incubation mixture to 50 μΐ human factor VII free 75 DK 176220 B1 plasma and 50 μΐ 50 mM calcium chloride. After 1 minute at 37 ° C, 50 μΐ of a 1: 10 dilution of homologous species serum is added as a source of factor VII and a double determination of clot formation time is made.

5 18 af de 24 monoklonale antistoffer inhiberer den prokoagulerende aktivitet af bavianhjerne-TF eller nyrecelleekstrakter af afrikansk grøn abe (tabel 8) . Imidlertid udviser ingen af de monoklonale antistoffer krydsreaktivitet med TF fra rotte, kanin, okse, hund, får eller svin, dvs.5 18 of the 24 monoclonal antibodies inhibited the procoagulant activity of baboon brain TF or kidney cell extracts of African green monkey (Table 8). However, none of the monoclonal antibodies exhibit cross-reactivity with TF from rat, rabbit, ox, dog, sheep or pig, ie.

10 ingen inhiberer evnen af disse TF-præparater til at fremskynde recalcificeringstiden af humant faktor VII-frit plasma i nærværelse af en kilde til homolog faktor VII. Ingen af antistofferne inhiberer den prokoagulerende aktivitet af kanin-TF bestemt med normalt humant plasma.10 none inhibit the ability of these TF preparations to accelerate the recalcification time of human factor VII free plasma in the presence of a source of homologous factor VII. None of the antibodies inhibit the procoagulant activity of rabbit TF determined with normal human plasma.

76 DK 176220 B176 DK 176220 B1

(Ti LD(Ten LD

tutu

Cn HCn H

c -η λ;c -η λ;

Eh mEh m

U i—I PQ CQfflCQPQCQ ffl M 0 ffl ffl Pdffl ffl C3 CQ ffl MU i — I PQ CQfflCQPQCQ ffl M 0 ffl ffl Pdffl ffl C3 CQ ffl M

-η e !!! s sssss sssss ss'is ssss! I-η e !!! s sssss sssss ss'is ssss! IN

A -HA -H

G C! Η Γΰ 4-1 rd — t+ * * * U h o (N η ω o π ^ co σ\ h m ^ (N co co > ^ io h cn <n h o o £ η 1/1 co co co h σι co co h Γ" æ co r- o σ\ r- -+ lt> r- r-- co co r-- cn -H > μ 03 Λ ~ H rn £ c! cn H niG C! Η Γΰ 4-1 rd - t + * * * U ho (N η ω o π ^ co σ \ hm ^ (N co co> ^ io h cn <nhoo £ η 1/1 co co h σι co co h Γ "æ co r- o σ \ r- - + lt> r- r-- co co r-- cn -H> µ 03 Λ ~ H rn £ c! cn H ni

O VD Oh ON 1/1 Lil h CO h C— [—^ VO LD LO r-· t~~ O Γ- LO 00 O OO VD Oh ON 1/1 Lil h CO h C— [- ^ VO LD LO r- · t ~~ O Γ- LO 00 O O

c.v> t*c| σ\ ση σι σι σι σι σι σι σι σι οι οι οι οι σ\ σι σο σ\ σι σι σι σι οι ίΝ 4-> οο Οc.v> t * c | σ \ ση σι σι σι σι σι σι σι οι οι οι οι σι σι σι σι σι οι ίΝ 4-> οο Ο

i—ι pc! + + + + + + + + + + + + + + + + + + + + + + + + Ii — ι PC! + + + + + + + + + + + + + + + + + + + + + + + + I

hl ffl Shl ffl S

tftf

CQ GCQ G

3 U3 U

H 0 4-3 10 0 £ (¾ +lii + + + + + + + + + + + + + + + + + + + + + i 4-3 O ι—ί D3 + + + + + + + + + + + + + + + + + + + + + + + + iH 0 4-3 10 0 £ (¾ + lii + + + + + + + + + + + + + + + + + + + + + i 4-3 O ι — ί D3 ++ + + + + + + + + + + + + + + + + + + + + + + + i

4J4J

OISLAND

QQ

. cn r- ro m in co cn ro in cn ^ ro σι ro ^ 1/1 «31 lo cn σι γν co cn ui ch. cn r- ro m in co cn ro in cn ^ ro σι ro ^ 1/1 «31 lo cn σι γν co cn ui ch

Η g ^ CO 1/1 ΡΊ Γ' CO LO CN ro M σι ^ H + Ol Cj) l/) (N ΙΟ N) CN CN ¢1 CO CNΗ g ^ CO 1/1 ΡΊ CO 'CO LO CN ro M σι ^ H + Ol Cj) l /) (N ΙΟ N) CN CN ¢ 1 CO CN

< Cu m in ^ «Η co in ι/i in ^ σ o oo i_n ooooorH r cn r- »p lo σι H ø u? co σι in ro co co co ^ γ-" lo P r^· ^ co co Ch coomoo^ h<Cu m in ^ «Η co in ι / i in ^ σ o oo i_n ooooorH r cn r-» p lo σι H ø u? co σι in ro co co co ^ γ- „lo P r ^ · ^ co co Ch coomoo ^ h

pei — CN CN CN CN (N CN CN CN CN (N CN (N H CN <N Η Γθ ΓΟ (N CNpei - CN CN CN CN CN (N CN CN CN CN (N CN (N H CN <N Η Γθ ΓΟ (N CN

g ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ +J i—li—I H r-H rHrHrHrHi—I rHrHi—I ri rH t—I i—l i—I ri i—1 <—I i—I rN i—\ i—i \—| o u o u o ooooo oooou ooooo oouuo o co tntTicn tn tn tn tn tn tn t n cn t n cn t n cn t n t n t n t n tn tn tn iji tn cng ^^^^^^^^^ + J i — li — IH rH rHrHrHrHi — I rHrHi — I ri rH t — I i — li — I ri i — 1 <—I i — I rN i— \ i —I \ - | o u o u o ooooo oooou ooooo oouuo o co tntTicn tn tn tn tn tn tn tn cn tn cn tn cn tn tn tn tn tn tn iji tn cn

Η MHH Η HHHHH MHHHH HHRHH HHHHH HΗ MHH Η HHHHH MHHHH HHRHH HHHHH H

CN OCN O

Η H O CN HΗ H O CN H

σ O h in r- æ σ-ι -+ m h + r- n1 N1 οι h co tj O H fc p w pq CO O EnQOPQO DQOUHH M U CP o o o in m η tH hhhhcn cn i/i in p p u> p r- co σ σ ον η h o g il) ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι h ¢5 oo co co σι σ\ cn on ch ch σι o\ σι o\ σι σι σι σι σι οι σι σι σι οι σι 0 tuPtiEti Ρ4 [ΪιΡ^ΡιηΡμΡμ pMl+F^tMF^ Ρν Ε+ Ε^ Ρη Ρ< cd 2 HHEh H HE-iÉ^HEh E-iHHHE-i Η ^ Η Η Η h h h Eh h Cu 77 DK 176220 B1 *·^ Medmindre andet er anført, er alle resultater opnået ved anvendelse af ovenstående væsker fra hybridoma-vævskultur ved en 1:10-fortynding. Radioimmunobestemmelses-resultaterne, udtrykt i tællinger pr. minut (cpm), under 5 anvendelse af 125I-TF mærket ved anvendelse af lactoperoxi-dase .σ O h in r- æ σ-ι - + mh + r- n1 N1 οι h co tj OH fc pw pq CO O EnQOPQO DQOUHH MU CP ooo in m η tH hhhhcn cn i / i in ppu> p r- co σ σ ον η hog il) ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι h ¢ 5 oo co co σι σ \ cn on ch ch σι o \ σι o \ σι σι σι σι οι σι σι σι οι σι 0 tuPtiEti Ρ4 [ΪιΡ ^ ΡιηΡμΡμ pMl + F ^ tMF ^ Ρν Ε + Ε ^ Ρη Ρ <cd 2 HHEh H HE-iÉ ^ HEh E-iHHHE-i Η ^ Ηh Cu 77 DK 176220 B1 * · ^ Unless otherwise stated, all results were obtained using the above liquids from hybridoma tissue culture at a 1:10 dilution. Radioimmunoassay results, expressed in counts per per minute (cpm), using the 125 I-TF labeled using lactoperoxidase.

2) Western blots gennemføres ved anvendelse af enten reduceret (R) eller ikke-reduceret (NR) TF.2) Western blots are performed using either reduced (R) or non-reduced (NR) TF.

3) Inhibering af koagulering af humant plasma frem-10 kaldt med renset humant hjerne-TF.3) Inhibition of coagulation of human plasma induced by purified human brain TF.

Inhibering af specifik 125I-faktor Vll/VIIa-binding til J82-celler. I nogle tilfælde (vist ved en stjerne) in-hiberer ovenstående væsker fra kultur i en fortynding på 1:10 ikke væsentligt faktor Vll/VIIa-binding, og der er 15 anført resultater for renset IgG i en koncentration på 10 /-ig/ml.Inhibition of specific 125I factor VII / VIIa binding to J82 cells. In some cases (shown by an asterisk), the above culture fluids at a 1:10 dilution do not significantly inhibit factor Vll / VIIa binding, and 15 results have been reported for purified IgG at a concentration of 10 ml.

5) Inhibering af koagulering af humant plasma fremkaldt af rå bavian-hjerneekstrakt (B) eller lyserede COS-celler (M) . Der er anført et bogstav for en art, hvis det 2 0 monoklonale antistof (MoAb) inhiberer den prokoagulerende aktivitet 60% eller mere.5) Inhibition of human plasma coagulation induced by crude baboon brain extract (B) or lysed COS cells (M). A letter for a species is indicated if the monoclonal antibody (MoAb) inhibits the procoagulant activity 60% or more.

Inhiberingen af prokoagulerende aktivitet, der udtrykkes af forskellige humane celler og væv, undersøges 25 mere detaljeret ved anvendelse af det monoklonale antistof TF8-5G9. TF8-5G9 neutraliserer funktionen af renset relipi-deret humant TF med mere end 90% ved IgG-koncentrationer på a 1 ^g/ml (fig. 16) . Evnen af dette monoklonale antistof til at inhibere den prokoagulerende aktivitet af humane 30 cellelysater og rå vævsekstrakter påvises også (tabel 9) . TF8-5G9 ved en IgG-koncentration på 10 ^g/ml inhiberer kvantitativt a 80% af den prokoagulerende aktivitet af rå hjerneekstrakt og placenta-acetone-pulver og af lyserede humane fibroblaster, blærecarcinomceller og endotoxinstimulerede 35 perifere mononucleære blodlegemer.The inhibition of procoagulant activity expressed by various human cells and tissues is examined in more detail using the monoclonal antibody TF8-5G9. TF8-5G9 neutralizes the function of purified relapsed human TF by more than 90% at IgG concentrations of α 1 µg / ml (Fig. 16). The ability of this monoclonal antibody to inhibit the procoagulant activity of human cell lysates and crude tissue extracts is also demonstrated (Table 9). TF8-5G9 at an IgG concentration of 10 µg / ml quantitatively inhibits α 80% of the procoagulant activity of crude brain extract and placental acetone powder and of lysed human fibroblasts, bladder carcinoma cells and endotoxin-stimulated peripheral blood mononuclear cells.

78 DK 176220 B178 DK 176220 B1

TABEL IXTABLE IX

Inhibering af koagulerende aktivitet af forskellige celler og væv med monoklonalt antistof TF8-5G9 5 TF-Aktivitet (% Inhibering)1^Inhibition of Coagulant Activity of Various Cells and Tissues with Monoclonal Antibody TF8-5G9 TF Activity (% Inhibition) 1

Intet anti -No anti -

Kilde til TF-aktivitet2> stof PAblOO TF8-5G9 10 renset human hjerne-TF 1569 1520 (3%) 245 (84%) rå hjerneekstrakt 2059 2059 (0%) 411 (80%) rå placentaekstrakt 1287 1344 (0%) 159 (88%) GM1381-fibroblaster (lyserede) 990 966 (2%) 143 (86%) 15 humane monocyter (lyserede) 2893 2745 (5%) 176 (94%) J82 blærecarcinoma- celler (lyserede) 882 902 (0%) 93 (89%) kanin-thromboplastin 2108 2108 (0%) 2157 (0%) 2 0 _Source of TF activity2> substance PAblOO TF8-5G9 10 purified human brain TF 1569 1520 (3%) 245 (84%) crude brain extract 2059 2059 (0%) 411 (80%) crude placental extract 1287 1344 (0%) 159 (88%) GM1381 fibroblasts (lysed) 990 966 (2%) 143 (86%) 15 human monocytes (lysed) 2893 2745 (5%) 176 (94%) J82 bladder carcinoma cells (lysed) 882 902 (0% 93 (89%) rabbit thromboplastin 2108 2108 (0%) 2157 (0%) 20

Renset humant hjerne-TF rekonstitueres i lipid-vesikler før undersøgelsen.Purified human brain TF is reconstituted in lipid vesicles prior to study.

25 2) I de to søjler til højre er der anført tilbage værende TF-aktivitet i millienheder målt efter behandling med det anførte rensede IgG og som gennemsnittet af to bestemmelser. Prøverne inkuberes med 10 μg/ml IgG i 30 minutter ved 37°C før måling af den tilbageværende TF-aktivitet.25 2) In the two columns to the right, residual TF activity in milliseconds is measured after treatment with the purified IgG indicated and as the average of two determinations. The samples are incubated with 10 μg / ml IgG for 30 minutes at 37 ° C before measuring residual TF activity.

30 Værdierne i parentes er den procentiske inhibering. I hvert enkelt tilfælde er dette den relative værdi i forhold til aktiviteten af den samme prøve, der ikke er behandlet med antistof.30 The values in parentheses are the percent inhibition. In each case, this is the relative value of the activity of the same antibody-treated sample.

21. Faktor Vll-bindingsundersøgelser 35 Da binding af faktor Vll/VIIa til TF er nødvendig til samling af det funktionelle TF:Vll/Vlla-prokoagulerende kompleks, 79 DK 176220 B1 undersøges evnen af de monoklonale antistoffer anført i tabel VIII til at neutralisere TF-aktivitet via blokering af bindingen af faktor Vll/VIIa til TF.21. Factor VII binding studies 35 As binding of factor VII / VIIa to TF is required for assembly of the functional TF: VII / VIIa procoagulant complex, the ability of the monoclonal antibodies listed in Table VIII to neutralize TF is investigated. activity via blocking the binding of factor Vll / VIIa to TF.

Human vævsfaktor-medieret binding af faktor VII til 5 overfladen af J82-blærecarcinom-celler er blevet godt karakteriseret, jf. Fair et al., J. Biol. Chem., 262:11692 (1987). Følgelig undersøges virkningerne af de monoklonale antistoffer på samlingen af celleoverflade-huTF:VIl/VIIa-komplekset ved forinkubering af J82-celler med antistof og derefter 10 mængdebest emme Ise af den specifikke binding af 12^1-faktor VII/VIIla.Human tissue factor-mediated binding of factor VII to the surface of J82 bladder carcinoma cells has been well characterized, cf. Fair et al., J. Biol. Chem., 262: 11692 (1987). Accordingly, the effects of the monoclonal antibodies on the assembly of the cell surface huTF: VI1 / VIIa complex are investigated by preincubating J82 cells with antibody and then 10 quantitatively determining the specific binding of 12 µl factor VII / VIIa.

J82-celler dyrkes indtil sammenflydning i 12-brønds kulturplader som beskrevet af Fair et al., J. Biol. Chem. 262:11692 (1987) , vaskes med puffer A (137 mM natriumchlorid, 15 4 mM kaliumchlorid, 11 mmol/liter glucose, 5 mM natriumazid, 10 mM ΗΕΡΕΞ, pH-værdi 7,45) og inkuberes i 2 timer ved 37°C med 0,7 ml puffer A indeholdende renset monoklonalt antistof IgG eller en 1:10-fortynding af ovenstående væske fra hybri-domakultur. Calciumchlorid og 125I-faktor Vll/VIIa tilsættes 20 til slutkoncentrationer på henholdsvis 5 mM og 1 nM og inkuberes med celler i yderligere 2 timer ved 37°C. Cellemonolag vaskes derefter 5 gange med kold puffer B (140 mM natriumchlorid, 0,5% ΒΞΑ, 5 mM tris-HCl, pH-værdi 7,45), lyseres i 1 ml 0,2 M natriumhydroxid, 1% SDS, 10 mM EDTA, 25 og lysatet tælles i en r-tæller. Den specifikke binding bestemmes ved fratrækning af ikke-specifikt bundet radioaktivitet (12^1-faktor Vll/VIIa knyttet til celler i nærværelse af et 10 0 ganges molært overskud af umærket faktor VII/Vila) .J82 cells are grown until confluent in 12-well culture plates as described by Fair et al., J. Biol. Chem. 262: 11692 (1987), washed with Buffer A (137 mM sodium chloride, 4 4 mM potassium chloride, 11 mmol / liter glucose, 5 mM sodium azide, 10 mM ΗΕΡΕΞ, pH 7.45) and incubated for 2 hours at 37 ° C with 0.7 ml of buffer A containing purified monoclonal antibody IgG or a 1:10 dilution of the above liquid from hybridoma culture. Calcium chloride and 125 I factor VII / VIIa are added 20 to final concentrations of 5 mM and 1 nM, respectively, and incubated with cells for an additional 2 hours at 37 ° C. Cell monolayer is then washed 5 times with cold buffer B (140 mM sodium chloride, 0.5% ΒΞΑ, 5 mM tris-HCl, pH 7.45), lysed in 1 ml of 0.2 M sodium hydroxide, 1% SDS, 10 mM EDTA, 25 and the lysate are counted in an r counter. The specific binding is determined by subtracting non-specifically bound radioactivity (12 µl factor VII / VIIa attached to cells in the presence of a 10 0 fold molar excess of unlabeled factor VII / Vila).

Den procentiske inhibering af specifik binding bestemmes 30 for J82-celler behandlet med monoklonale antistoffer i forhold til kontrolceller behandlet med 9 dele puffer A og 1 del kulturmedium.The percent inhibition of specific binding is determined for J82 cells treated with monoclonal antibodies relative to control cells treated with 9 parts buffer A and 1 part culture medium.

Når faktor Vll/VIIa bindes til TF, bliver det ikke normalt inkorporeret, men for at eliminere muligheden for 35 TF-inkorporering fremkaldt af antistofbinding forgiftes J82-cellerne metabolisk med 5 mM natriumazid. Der opnås 80 DK 176220 B1 lignende resultater, hvadenten cellerne behandles med azid eller ej.When factor VII / VIIa binds to TF, it is not normally incorporated, but to eliminate the possibility of TF incorporation induced by antibody binding, the J82 cells are metabolically poisoned with 5 mM sodium azide. 80 DK 176220 B1 similar results are obtained, whether or not the cells are treated with azide.

Resultaterne af denne undersøgelse er anført i tabel 8 ovenfor. Alle 23 monoklonale antistoffer, der inhiberer 5 TF-aktivitet, blokerer også faktor Vll/VIIa-binding. Som forventet bevirker det monoklonale antistof, der ikke inhiberer TF-aktivitet, TF9-10H10, ikke blokering af faktor VII-binding.The results of this study are listed in Table 8 above. All 23 monoclonal antibodies that inhibit TF activity also block factor VII / VIIa binding. As expected, the monoclonal antibody that does not inhibit TF activity, TF9-10H10, does not block factor VII binding.

22. Inhering af faktor Xa-dannelse 10 af J82-celler.22. Inhibition of Factor Xa Formation 10 of J82 Cells.

Hastighederne for faktor Xa-dannelse af huTF:Vll/VIIa-komplekset på J82-celler bestemmes ved en dobbeltbestemmelse under anvendelse af flerbrønds-kulturpladebestemmelsen beskrevet af Fair et al., J. Biol . Chem. , 262:11692 (1987) 15 med følgende modifikationer. Celler dyrkes i 12-brønds-plader og forinkuberes i 2 timer ved 37°C med forskellige koncentrationer af renset IgG-fraktion af monoklonale antistoffer før bestemmelsens påbegyndelse, som beskrevet ovenfor for faktor Vll/VIIa-binding til J82-celler. Der anvendes en 20 enkelt koncentration af faktor Vll/VIIa (1 mM) ved bestemmelsen. Med intervaller på 5, 10 og 15 minutter efter tilsætning af faktor X til en slutkoncentration på 50 /zg/ml fjernes 50 μΐ ovenstående væske og sættes til 550 μΐ 50 mM Tris-HCl, 225 mM natriumchlorid, 50 mM EDTA (pH-værdi 8,2).The rates of factor Xa formation of huTF: V11 / VIIa complex on J82 cells are determined by a double determination using the multi-well culture plate assay described by Fair et al., J. Biol. Chem. , 262: 11692 (1987) with the following modifications. Cells are grown in 12-well plates and preincubated for 2 hours at 37 ° C with various concentrations of purified IgG fraction of monoclonal antibodies prior to assay, as described above for factor VII / VIIa binding to J82 cells. A single concentration of factor VIII / VIIa (1 mM) is used in the determination. At intervals of 5, 10 and 15 minutes after addition of factor X to a final concentration of 50 µg / ml, 50 μΐ of the above liquid is removed and added to 550 μΐ 50 mM Tris-HCl, 225 mM sodium chloride, 50 mM EDTA (pH value) 8.2).

25 Efter tilsætning af chromogent faktor Xa-substrat (50 μΐ 3,4 mM "S-2222", Helena Labs, Beaumont, TX), bestemmes faktor Xa-aktiviteten ved måling af hastigheden for forøgelse af absorptionen ved 405 nm i et Beckman DU-30 spektrofotometer med kinetisk analysemodul. Baggrundshydrolyse af "S-2222" 30 med den ovenstående væske fra J82-celler inkuberet i fraværelse af faktor Vll/VIIa trækkes fra hver bestemmelse.After addition of chromogenic factor Xa substrate (50 μΐ 3.4 mM "S-2222", Helena Labs, Beaumont, TX), factor Xa activity is determined by measuring the rate of increase of absorption at 405 nm in a Beckman DU -30 spectrophotometer with kinetic analysis module. Background hydrolysis of "S-2222" 30 with the above liquid from J82 cells incubated in the absence of factor V11 / VIIa is subtracted from each assay.

Den procentiske inhibering ved antistofbehandlingen beregnes i forhold til celler, der ikke er forinkuberet med antistof.The percent inhibition of antibody treatment is calculated relative to cells not preincubated with antibody.

Inhiberingskurverne for behandling af J82-celler med 35 de monoklonale antistoffer TF9-2C4 og TF9-5B7 viser, at hastigheden for faktor Xa-dannelse inhiberes af lignende 81 DK 176220 B1 antistofkoncentrationer som dem, der inhiberer faktor VII-binding (fig. 17). Det ikke-inhiberende (ikke-neutralise-rende) monoklonale antistof TF9-10H10 har ringe eller ingen effekt på prokoagulerende aktivitet, faktor VII/VIla-binding 5 eller hastigheden for faktor Xa-dannelse ved IgG-koncentrationer op til 10 μ%/τη1, og det samme gælder det monoklonale antistof PAblOO, der tjener som kontrol (ikke vist).The inhibition curves for the treatment of J82 cells with the monoclonal antibodies TF9-2C4 and TF9-5B7 show that the rate of factor Xa formation is inhibited by similar antibody concentrations as those which inhibit factor VII binding (Fig. 17). . The non-inhibitory (neutralizing) monoclonal antibody TF9-10H10 has little or no effect on procoagulant activity, factor VII / VIa binding 5, or the rate of factor Xa formation at IgG concentrations up to 10 μ% / τη1. , and the same applies to the PAblOO monoclonal antibody which serves as a control (not shown).

23. Inhibering af faktor X-aktivering på J82-celler ved kompetitiv binding af 10 huTFh-polypeptider til faktor VII/Vila23. Inhibition of Factor X Activation on J82 Cells by Competitive Binding of 10 huTFh Polypeptides to Factor VII / Vila

Som det er velkendt, er cellulær aktivering af koa-gulerings-protease-kaskaderne knyttet til en heterogen gruppe af sygdomme, der betegnes konsumptive thrombohæmorrhagiske 15 sygdomme. Koagulations-protease-kaskaden initieres mest almindeligt på celleoverflader ved høj affinitetsbinding af faktor VII/VIIa til dens membranreceptor og essentielle co-faktor, vævsfaktor (TF) . Det bimolekylære prokoagulerende kompleks af TF og faktor VII/VIIa (TF:VII/VIIa), aktiverer 20 faktor X og IX ved begrænset proteolyse, der tilsidst fører til thrombindannelse og fibrinafsætning. Udover rollen af TF ved hæmostase er initiering af koagulations-protease-kaskaden af TF blevet impliceret i dissemineret intravaskulær koagulation og i thrombogenese, jf. Niemetz et al., Blood, 25 42:47 (1973) og Bevilacqua et al., J. Exp. Med., 160:618 (1984) . TF er et vigtigt effektormolekyle, der eksprimeres på overfladen af monocyter og endotheliale celler som reaktion på inflammatoriske mediatorer og ved cellulære immunreaktioner .As is well known, cellular activation of the coagulation protease cascades is associated with a heterogeneous group of diseases known as consumptive thrombohemorrhagic diseases. The coagulation protease cascade is most commonly initiated on cell surfaces by high affinity binding of factor VII / VIIa to its membrane receptor and essential co-factor, tissue factor (TF). The bimolecular procoagulant complex of TF and factor VII / VIIa (TF: VII / VIIa) activates 20 factors X and IX by limited proteolysis, eventually leading to thrombin formation and fibrin deposition. In addition to the role of TF in hemostasis, initiation of the coagulation protease cascade of TF has been implicated in disseminated intravascular coagulation and in thrombogenesis, cf. Niemetz et al., Blood, 25:42 (1973) and Bevilacqua et al., J. Exp. Med., 160: 618 (1984). TF is an important effector molecule that is expressed on the surface of monocytes and endothelial cells in response to inflammatory mediators and in cellular immune responses.

30 Evnen af huTFh-polypeptiderne ifølge den foreliggende opfindelse til at binde faktor VII/VIIa og derved inhibere dannelsen af et TF:Vll/VIla-kompleks, som er i stand til at aktivere faktor X, er undersøgt.The ability of the huTFh polypeptides of the present invention to bind factor VII / VIIa and thereby inhibit the formation of a TF: VIII / VIa complex capable of activating factor X has been investigated.

50 μΐ af en opløsning indholdende en huTF-polypep-35 tidanalog i en koncentration på 100 μΜ i TBS sættes til hver brønd af en 96-brønds fladbundet polystyren-bestemmel- 82 DK 176220 B1 sesplade. Derefter blandes der i hver brønd 25 μΐ af en opløsning indeholdende faktor VII/VIIa isoleret som beskrevet i eksempel 3 i en koncentration på 1 nm i TBS, og der iblandes yderligere 2 5 μΐ 2 0 mM calciumchlorid i TBS, og den 5 fremkomne blanding holdes ved stuetemperatur i 30 minutter.50 µl of a solution containing a huTF polypeptide analog at a concentration of 100 µΜ in TBS is added to each well of a 96 well flat bottom polystyrene assay plate. Then, in each well, 25 μΐ of a solution containing factor VII / VIIa isolated as described in Example 3 was mixed at a concentration of 1 nm in TBS, and an additional 25 μΐ 20 mM calcium chloride in TBS was added and the resulting mixture kept at room temperature for 30 minutes.

Humane J82-blærecellecarcinom-celler (ATCC HTB 1) dyrkes som beskrevet af Fair et al., J. Biol. Chem., 262:11692-11698 .Human J82 bladder cell carcinoma cells (ATCC HTB 1) are grown as described by Fair et al., J. Biol. Chem., 262: 11692-11698.

5 x 104 J82-celler suspenderes i 50 μΐ TBS og blandes 10 i hver brønd af polystyren-bestemmelsespladen efter den ovennævnte bibeholdelsesperiode. Umiddelbart derefter iblandes 25 μΐ faktor X, isoleret som beskrevet af Fair et al., J, Biol. Chem., 262:11692-11698 (1987) i en koncentration på 100 nM i TBS og 50 μΐ af Xa-chromogent substrat "S-2222" 15 (1 mg/ml i TBS), og den fremkomne blanding holdes i 2 minut ter ved stuetemperatur til dannelse af en opløsning indeholdende chromogent reaktionsprodukt.5 x 10 4 J82 cells are suspended in 50 μΐ TBS and mixed 10 in each well of the polystyrene assay plate after the aforementioned retention period. Immediately thereafter, 25 μΐ of factor X, isolated as described by Fair et al., J, Biol, is added. Chem., 262: 11692-11698 (1987) at a concentration of 100 nM in TBS and 50 μΐ of Xa chromogenic substrate "S-2222" 15 (1 mg / ml in TBS) and the resulting mixture was maintained for 2 minutes. at room temperature to form a solution containing chromogenic reaction product.

Den dannede mængde chromogent produkt bestemmes ved måling af den optiske tæthed (OD) ved 405 nm, idet der an-20 vendes et V-max 96-brønds spektrofotometer (Molecular Devices, Mountain View, California). Kontroller med PBS i stedet for polypeptid eller uden tilsat faktor VII gennemføres også til bestemmelse af de maksimalt og minimalt mulige 0D-værdier. Resultaterne af disse målte inhiberinger er vist i 25 tabel X.The amount of chromogenic product formed is determined by measuring the optical density (OD) at 405 nm using a V-max 96-well spectrophotometer (Molecular Devices, Mountain View, California). Controls with PBS instead of polypeptide or without added factor VII are also performed to determine the maximum and minimum possible 0D values. The results of these measured inhibitions are shown in Table X.

30 35 83 DK 176220 B130 35 83 DK 176220 B1

Tabel XTable X

Inhibering af X-aktiverina på J82-celler ved anvendelse af huTF-polvpeptider 5 huTFh-polypeptider Optisk tæthed1) PBS 0,960 ± 0,083 ingen faktor VIl/VTIa 0,005 + 0,001 pi-18 1,007 ± 0,087 10 pl-30 1,098 ± 0,028 pli-28 0,687 ± 0,071 p24-25 0,477 ± 0,017 p26-49 0,437 ± 0,020 p40-71 0,814 ± 0,053 15 p72-104 0,781 ± 0,047 p94-123 0,818 ± 0,055 pl21-155 0,889 ± 0,067 pl44-159 0,507 ± 0,053 pl4 6 -16 7 0,004 ± 0,001 20 pl57-169 0,389 ± 0,035 pl61-190 0,600 ± 0,023 pi90-209 0,625 ± 0,031 p204-22 6 0,715 ± 0,042 p244-263 0,619 ± 0,047 25 _ D Inhibering af faktor X-aktivering (Xa-dannelse) betragtes som signifikant, hvis den optiske tæthed (OD) er ca. 0,500 eller mindre.Inhibition of X-activin on J82 cells using huTF polypeptides 5 huTFh polypeptides Optical density1) PBS 0.960 ± 0.083 no factor VI1 / VTIa 0.005 + 0.001 pi-18 1.007 ± 0.087 10 pI-30 1.098 ± 0.028 pI-28 0.677 ± 0.071 p24-25 0.477 ± 0.017 p26-49 0.437 ± 0.020 p40-71 0.814 ± 0.053 p72-104 0.781 ± 0.047 p94-123 0.818 ± 0.055 pl21-155 0.889 ± 0.067 pl44-159 0.507 ± 0.053 pl4 6-16 7 0.004 ± 0.001 20 pl57-169 0.389 ± 0.035 pl61-190 0.600 ± 0.023 pi90-209 0.625 ± 0.031 p204-22 6 0.715 ± 0.042 p244-263 0.619 ± 0.047 25 D Inhibition of Factor X Activation (Xa Formation) is considered significant if the optical density (OD) is approx. 0.500 or less.

30 Resultaterne af denne undersøgelse viser, at huTFh- polypeptiderne p24-25, p26-49, pl44-159, pl46-167 og pl57-169 binder til faktor VII/VIIa og inhiberer dens evne til at danne et TF: Vll/VIIa-kompleks, der kan aktivere faktor X. Disse resultater viser, at en huTFh-bindingssted-polypep-35 tidanalog ifølge den foreliggende opfindelse kan anvendes til at inhibere koagulering.The results of this study show that the huTFh polypeptides p24-25, p26-49, pl44-159, pl46-167 and pl57-169 bind to factor VII / VIIa and inhibit its ability to form a TF: Vll / VIIa complex, which can activate factor X. These results show that a huTFh binding site polypeptide analog of the present invention can be used to inhibit coagulation.

84 DK 176220 B1 2 4 . In vivo-inhibering af koagulering med monoklonale Anti-huTFh-antistoffer84 DK 176220 B1 2 4. In vivo inhibition of coagulation with monoclonal Anti-huTFh antibodies

Sepsis på grund af gramnegative bakterier involverer ofte en choktilstand, der tilsidst kan føre til døden. For-5 styrrelser af det hæmostatiske system er tæt knyttet til udviklingen af en chok-tilstand. Taylor et al., J. Clin. Invest., 79:918-825 (1987) har vist, at exogent tilsat ak tiveret protein C, et naturligt forekommende antikoagulerende enzym, forhindrer koagulopatisk reaktion og dødelige effek-10 ter af "koncentrationer af E. coli hos bavianer.Sepsis due to gram-negative bacteria often involves a state of shock that can eventually lead to death. Disorders of the hemostatic system are closely related to the development of a shock condition. Taylor et al., J. Clin. Invest., 79: 918-825 (1987) have shown that exogenously added activated protein C, a naturally occurring anticoagulant enzyme, prevents coagulopathic response and lethal effects of E. coli concentrations in baboons.

Evnen af et antikoagulerende monoklonalt antistof ifølge opfindelsen til at inhibere koagulering in vivo undersøges ved anvendelse af bavianmodellen for septisk chok beskrevet af Taylor et al., se ovenfor. Bavianer med en 15 vægt på 7-8 får lov at faste natten over før undersøgelsen og immobiliseres om morgenen før forsøget med ketamin (14 mg/kg, intramuskulært) . Natriumpentobarbital indgives derefter i hjernevenen via et percutant kateter til opretholdelse af et let niveau af kirurgisk anæstesi (2 mg/kg 20 ca. hvert 45. minut). En femoral vene frilægges aseptisk og kateteriseres i det ene bagben til udtagelse af blodprøver.The ability of an anticoagulant monoclonal antibody of the invention to inhibit coagulation in vivo is tested using the baboon model of septic shock described by Taylor et al., Supra. Babies with a weight of 7-8 weight are allowed to fast overnight before the study and are immobilized in the morning before the ketamine trial (14 mg / kg, intramuscularly). Sodium pentobarbital is then administered into the brain vein via a percutaneous catheter to maintain a slight level of surgical anesthesia (2 mg / kg 20 approximately every 45 minutes). A femoral vein is aseptically released and catheterized in one hind leg for blood sampling.

Det percutane kateter anvendes til infusion af E. coli og andre midler, herunder det monoklonale antistof TF9-5B7, som har vist sig at krydsreagere med bavian-TF ifølge eksem-25 pel 20. Efter en ækvilibreringsperiode på 30 minutter modtager dyrene i løbet af ca. 10 minutter en infusion af enten 500 μg/kg eller 150 Mg/kg af det monoklonale antistof TF9-5B7 (MAPS - isoleret som i eksempel 7 og derefter dialyseret mod sterilt normalt saltvand til en koncentration på 30 0,58 Mg/ml) eller 500 Mg/kg af et irrelevant monoklonalt antistof.The percutaneous catheter is used for infusion of E. coli and other agents, including the monoclonal antibody TF9-5B7, which has been shown to cross-react with baboon TF according to Example 20. After an equilibration period of 30 minutes, the animals receive during ca. 10 minutes an infusion of either 500 µg / kg or 150 Mg / kg of the monoclonal antibody TF9-5B7 (MAPS - isolated as in Example 7 and then dialyzed against sterile normal saline to a concentration of 0.58 Mg / ml) or 500 Mg / kg of an irrelevant monoclonal antibody.

Efter indgivelse af monoklonalt antistof og en ækvi-libreringsperiode på 30 minutter modtager hvert dyr en LD-^q dosis af E. coli (ca. 101^ organismer, en mængde, der frem-35 kalder død på grund af septisk chok ca. 8-16 timer efter infusionen). E. coli indgives ved infusion over en periode 85 DK 176220 B1 på 2 timer. Resultaterne af denne undersøgelse er vist i tabel 11.After administration of monoclonal antibody and an equilibration period of 30 minutes, each animal receives an LD 50 dose of E. coli (about 101 µg organisms, an amount that causes death due to septic shock about 8 -16 hours after the infusion). E. coli is administered by infusion over a period of 2 hours. The results of this study are shown in Table 11.

Tabel 11 5 In vivo-standsning af letalitet af septisk chok hos bavianerTable 11 5 In vivo arrest of septic shock lethality in baboons

Dosis, Hæmo- E. coliDose, Hemo E. coli

Gruppe MoAb ug/kg stase1^ Infuseret Død 10 I. Kontrol TF9-5B7 500 Normal Nej Nej II. Kontrol HB2^ 500 Normal Ja Ja III. Undersøgelse TF9-5B7 500 Normal Ja Nej TF9-5B7 150 Normal Ja Nej 15 ___Group MoAb ug / kg stasis1 ^ Infused Death 10 I. Control TF9-5B7 500 Normal No No II. Control HB2 ^ 500 Normal Yes Yes III. Study TF9-5B7 500 Normal Yes No TF9-5B7 150 Normal Yes No 15 ___

Forskellige hæmostatiske parametre, herunder blodtryk, aktivering af koagulering og fibrinnedbrydningsproduk-ter, bestemmes efter indgivelse af monoklonalt antistof (MoAb), men før infusion af E. coli.Various hemostatic parameters, including blood pressure, activation of coagulation and fibrin degradation products, are determined after administration of monoclonal antibody (MoAb) but before infusion of E. coli.

20 2) HB er et monoklonalt antistof af samme klasse og underklasse som TF9-5B7, men immunoreagerer med et irrelevant antigen.2) HB is a monoclonal antibody of the same class and subclass as TF9-5B7, but immunoreact with an irrelevant antigen.

Som det fremgår af tabel 11, overlever bavianer, der 25 modtager det monoklonale antistof TF9-5B7, udsættelse for en LDlOO-dosis af E. coli. Både dosen på 150 /ig/kg og dosen på 500 /ig/kg af det monoklonale antistof beskytter. Desuden dæmpes den kraftige hypotension, koagulationskaskadeaktivering og nedbrydning af fibrin, der er knyttet til koagulo-30 pati, tydeligt hos dyrene, der modtager det monoklonale antistof TF9-5B7.As shown in Table 11, baboons receiving the monoclonal antibody TF9-5B7 survive exposure to an LD100 dose of E. coli. Both the dose of 150 µg / kg and the dose of 500 µg / kg of the monoclonal antibody protect. In addition, the severe hypotension, coagulation cascade activation and degradation of fibrin associated with coagulopathy are clearly attenuated in the animals receiving the monoclonal antibody TF9-5B7.

35 86 DK 176220 B1 25. Karakterisering af den lette kæde af 58 kDa huTF-heterodimeren som hæmoglobin-g-kæde35 86 DK 176220 B1 25. Characterization of the 58 kDa light chain huTF heterodimer as a hemoglobin g chain

Immunoaffinitetsisoleret huTF karakteriseres yder-5 ligere ved Western blot-analyse til identificering af komponenterne af 58 kDa huTF-heterodimeren, nemlig 47 kDa og 12,5 kDa proteinerne beskrevet i eksempel 4.Immunoaffinity-isolated huTF is further characterized by Western blot analysis to identify the components of the 58 kDa huTF heterodimer, namely the 47 kDa and 12.5 kDa proteins described in Example 4.

Western blot -analyse, gennemføret som beskrevet i eksempel 6c, gennemføres ved anvendelse af immunoaffinitets-10 isoleret huTF fremstillet som beskrevet i eksempel 9, renset humant hæmoglobin eller molekylvægtsstandarder som prøverne, der underkastes elektroforese. Hvor det er anført, inkluderes 50 mM dithiothreitol i prøvepufferen til reduktion af disul f idbindingerne . Western blots immunoreageres som anført 15 ved anvendelse af ikke-immun-kanin-IgG, kanin-anti-huTF-IgG fremstillet ved anvendelse af velkendte metoder eller kanin-anti-humant hæmoglobin-IgG fra Dako (Santa Barbara, California) . De første to IgG-præparater MAPS-II-isoleres som beskrevet i eksempel 7.Western blot analysis, performed as described in Example 6c, is performed using immunoaffinity-10 isolated huTF prepared as described in Example 9, purified human hemoglobin, or molecular weight standards as the samples subjected to electrophoresis. Where indicated, 50 mM dithiothreitol is included in the sample buffer to reduce the disulphide bonds. Western blots are immunoreacted as indicated using non-immune rabbit IgG, rabbit anti-huTF IgG prepared by well known methods, or rabbit anti-human hemoglobin IgG from Dako (Santa Barbara, California). The first two IgG MAPS-II preparations are isolated as described in Example 7.

20 Resultaterne af den ovennævnte Western blot-analyse er vist i fig. 18. Anti-huTF-IgG immunoreagerer kun med 47 kD-båndet af reduceret huTF og ikke med 12,5 kDa-båndet (panel A, bane 3), medens det samme IgG immunoreagerer med både 58 og 47 kDa-formerne af ikke-reduceret huTF (panel 25 A, bane 4) . Disse resultater stemmer overens med en identifikation af huTF som 47 kDa-komponenten af 58 kDa-hetero-dimeren. Anti-hæmoglobin-IgG immunoreagerer på Western blots kun med 58 kDa-båndet i den ikke reducerede huTF-prøve og ikke med 47 kDa-monomeren (panel B, bane 4). Imidlertid 30 immunoreagerer anti-hæmoglobin-IgG med 12,5 kDa-båndet i den reducerede huTF-prøve (panel B, bane 3) og immunoreagerer med 12,5 kDa renset humant hæmoglobin-protein (panel B, bane 2) . Der er ingen reaktivitet med et ikke-immun-kanin-IgG.The results of the aforementioned Western blot analysis are shown in Figs. 18. Anti-huTF IgG immunoreacts only with the 47 kD band of reduced huTF and not with the 12.5 kDa band (panel A, lane 3), while the same IgG immunoreacts with both the 58 and 47 kDa forms of reduced huTF (panel 25A, lane 4). These results are consistent with an identification of huTF as the 47 kDa component of the 58 kDa hetero-dimer. Anti-hemoglobin IgG immunoreact on Western blots only with the 58 kDa band in the non-reduced huTF sample and not with the 47 kDa monomer (panel B, lane 4). However, anti-hemoglobin IgG immunoreact with the 12.5 kDa band in the reduced huTF sample (panel B, lane 3) and immunoreact with 12.5 kDa purified human hemoglobin protein (panel B, lane 2). There is no reactivity with a non-immune rabbit IgG.

35 87 DK 176220 B135 87 DK 176220 B1

De ovenfor anførte resultater støtter den konklusion, at 58 kDa formen af ikke-reduceret huTF består af 4 7 kDa huTF-protein, som er disulfid-bundet til hæmoglobin.The above results support the conclusion that the 58 kDa form of unreduced huTF consists of 47 kDa huTF protein disulfide-bound to hemoglobin.

Det antages derfor nu, at let-kæde-komponenten på 5 12,5 kDa af 58 kDa-heterodimeren beskrevet i eksempel 4 er α-kæden af hæmoglobin, og at dens tilknytning til 47 kDa-huTF-proteinet er et kunstprodukt af huTFh-isoleringsproceduren .Therefore, it is now assumed that the light chain component of 5 12.5 kDa of the 58 kDa heterodimer described in Example 4 is the α chain of hemoglobin and that its association with the 47 kDa huTF protein is an art product of huTFh. the insulation procedure.

10 Sammenfatning og diskussion af resultaterne i eksempel 1-2510 Summary and discussion of the results in Examples 1-25

Der er beskrevet et bibliotek af 24 monoklonale antistoffer mod human hjerne-TF, opnået ud fra to forskellige cellefusioner. Immunspecificiteten af hvert monoklonalt 15 antistof er karakteriseret ved dot-blot, Western blot og radioimmunobestemmelse. De fleste monoklonale antistoffer reagerer med humant TF under alle tre betingelser og med både nativt og denatureret TF. Et af de monoklonale antistoffer, TF8-5G9, er blevet anvendt med held til rutinemæssig 20 oprensning af TF-proteinet. Det adsorberer kvantitativt TF-aktivitet fra vævsekstrakter og giver konsekvent udbytter af renset humant TF i mg-mængder.A library of 24 monoclonal antibodies against human brain TF obtained from two different cell fusions has been described. The immune specificity of each monoclonal antibody is characterized by dot blot, Western blot and radioimmunoassay. Most monoclonal antibodies react with human TF under all three conditions and with both native and denatured TF. One of the monoclonal antibodies, TF8-5G9, has been used successfully for routine purification of the TF protein. It adsorbs quantitatively TF activity from tissue extracts and provides consistent yields of purified human TF in mg amounts.

Alle undtagen et af de monoklonale antistoffer neutraliserer stærkt den funktionelle aktivitet af renset human 25 hjerne-TF. Selvom adskillige monoklonale antistoffer viser sig at krydsreagere med TF fra bavian og abe, inhiberer ingen af antistofferne koagulering af faktor VII-frit humant plasma initieret af thromboplastin fra rotte, kanin, okse, hund, får eller svin i nærværelse af homolog faktor VII.All but one of the monoclonal antibodies strongly neutralize the functional activity of purified human brain TF. Although several monoclonal antibodies appear to cross-react with baboon and monkey TF, none of the antibodies inhibit coagulation of factor VII free human plasma initiated by rat, rabbit, bovine, dog, sheep or pig thromboplastin in the presence of homologous factor VII.

30 Endvidere inhiberes initieringen af koagulering af normalt humant plasma med kanin-hjerne-thromboplastin ikke af nogle af disse monoklonale antistoffer, hvilket støtter konklusionen, at inhiberingen af human TF-prokoagulerende aktivitet ikke skyldes interferens af antistofferne med opløselige 35 plasmakoagulationsproteiner, herunder faktor Vll/VIIa.Furthermore, the initiation of coagulation of normal human plasma by rabbit brain thromboplastin is not inhibited by any of these monoclonal antibodies, which supports the conclusion that the inhibition of human TF procoagulant activity is not due to interference of the antibodies with soluble plasma coagulation proteins, including factor V / II. VIIa.

88 DK 176220 B188 DK 176220 B1

Den enkleste basis for inhiberingen af TF-prokoagu-lerende aktivitet med anti-TF-antistoffer er blokering af faktor Vll/VIIa-binding. Som forventet ophæver alle 23 anti-koagulerende (neutraliserende) monoklonale antistoffer den 5 specifikke binding af faktor Vll/VIIa til J82-celler, hvilket stemmer overens med den primære receptorfunktion af TF.The simplest basis for the inhibition of TF procoagulant activity with anti-TF antibodies is the blocking of factor VII / VIIa binding. As expected, all 23 anti-coagulant (neutralizing) monoclonal antibodies abrogate the 5 specific binding of factor Vll / VIIa to J82 cells, which is consistent with the primary receptor function of TF.

Dette underbygges yderligere ved dosisbestemmelse af udvalgte rensede monoklonale antistoffer, hvorved halvmaksimal in-hibering af faktor VII-binding og halvmaksimal inhibering 10 af hastigheden for faktor Xa-dannelse forekommer ved lignende IgG-koncentrationer.This is further substantiated by dose determination of selected purified monoclonal antibodies, whereby half-maximal inhibition of factor VII binding and half-maximal inhibition of the rate of factor Xa formation occur at similar IgG concentrations.

Et monoklonalt antistof mod humant TF er for nyligt blevet beskrevet af Carson et al., Blood, 70:490 (1987) at inhibere TF-aktivitet, tilsyneladende ved at inferere med 15 faktor Vll/VIIa-binding, selvom dette ikke er undersøgt direkte. At 23 ud af 24 af de her beskrevne monoklonale antistoffer neutraliserer TF-aktivitet stærkt, er bemærkelsesværdigt. Det har været erfaringen med monoklonale antistoffer mod forskellige humane koaguleringsproteiner, at 20 kun en mindre andel neutraliserer funktionel aktivitet. Det er usandsynligt, at hybridomaerne alle er søsterkloner på grund af forskellene i deres reaktiviteter, herunder krydsreaktiviteter med primat-TF. Desuden viser igangværende epitop-kortlægningsundersøgelser, at mindst tre særskilte, 25 ikke-konkurrerende antistofbindingssteder genkendes af dette panel af monoklonale antistoffer. Det er derfor usandsynligt, at den store andel af neutraliserende monoklonale antistoffer mod TF er konsekvensen af få immunodominante epitoper, som også har fælles funktion. TF-aminosyresekvensen forudsiges 30 da også ved metoden ifølge Hopp et al.. Mol. Immunol., 20:483 (1983) at indeholde flere antigene determinanter.A human TF monoclonal antibody has recently been described by Carson et al., Blood, 70: 490 (1987) to inhibit TF activity, apparently by inferring with 15 factor VII / VIIa binding, although not directly investigated. . That 23 out of 24 of the monoclonal antibodies described here strongly neutralize TF activity is remarkable. It has been the experience of monoclonal antibodies against various human coagulation proteins that only a minor proportion neutralizes functional activity. The hybridomas are unlikely to be all sister clones due to the differences in their reactivities, including cross-reactivities with primate TF. In addition, ongoing epitope mapping studies show that at least three distinct, 25 non-competitive antibody binding sites are recognized by this panel of monoclonal antibodies. Therefore, the high proportion of neutralizing monoclonal antibodies against TF is unlikely to be the consequence of few immunodominant epitopes, which also have common function. The TF amino acid sequence is then also predicted by the method of Hopp et al., Mol. Immunol., 20: 483 (1983) to contain several antigenic determinants.

Den lille størrelse af TF kan delvis forklare, hvorfor så mange monoklonale anti-TF-antistoffer blokerer faktor Vll/VIIa-binding. TF forudsiges ud fra cDNA-kloning at have 3 5 et ekstracellulært domæne på 2 5 kDa, ekslusive glycosylering. Derfor kan antistof- og faktor Vll/VIIa-molekyler udvise 89 DK 176220 B1 sterisk hindring ved binding til det meget mindre ekstracel-lulære domæne af TF. Hypotesen med sterisk hindring stemmer overens med observationen af, at concanavalin A inhiberer TF-aktivitet (Pitlick. J. Clin. Invest., 55:175 (1975)}, da 5 kulhydratgrupperne på TF sandsynligvis ikke er nødvendige for funktionen (Nakamura, Throm. Hemost., 58:135 (1987)).The small size of TF may partially explain why so many monoclonal anti-TF antibodies block factor VII / VIIa binding. TF is predicted from cDNA cloning to have an extracellular domain of 2.5 kDa, excluding glycosylation. Therefore, antibody and factor VII / VIIa molecules may exhibit steric hindrance upon binding to the much smaller extracellular domain of TF. The steric hindrance hypothesis is consistent with the observation that concanavalin A inhibits TF activity (Pitlick. J. Clin. Invest., 55: 175 (1975)}, since the 5 carbohydrate groups on TF are probably not required for function (Nakamura, Throm , Hemost., 58: 135 (1987)).

Det er blevet overvejet, at den faktor VII-afhængige prokoagulerende aktivitet, der eksprimeres af forskellige celler og væv, kunne skyldes mere end en molekyleart af TF-10 lignende proteiner med lignende funktion. Imidlertid inhiberer det monoklonale antistof TF8-5G9 kvantitativt den prokoagulerende aktivitet af rå hjerne- og placentaekstrakter og af lyserede fibroblaster, blærecarcinomceller og perifere mononucleære blodlegemer. Selvom disse resultater ikke er 15 udtømmende, støtter de konklusionen, at cellulære prokoagulerende aktiviteter, som i øjeblikket tilskrives TF, er antigent relaterede, hvis de ikke er identiske. Dette stemmer overens med resultatet, at der formentlig er et enkelt gen for TF.It has been considered that the factor VII-dependent procoagulant activity expressed by different cells and tissues could be due to more than one molecular species of TF-10 like proteins with similar function. However, the monoclonal antibody TF8-5G9 quantitatively inhibits the procoagulant activity of crude brain and placental extracts and of lysed fibroblasts, bladder carcinoma cells and peripheral mononuclear blood cells. Although these findings are not exhaustive, they support the conclusion that cellular procoagulant activities currently attributed to TF are antigenically related, if not identical. This is consistent with the result that there is probably a single gene for TF.

20 Det er fornyligt blevet påvist, at de letale effekter af septisk chok kan forhindres hos bavianer ved infusion af aktiveret protein C, et antikoagulerende protein, der virker på mellemstadier i koagulerings-protease-kaskaden.It has recently been demonstrated that the lethal effects of septic shock can be prevented in baboons by infusion of activated protein C, an anticoagulant protein acting at intermediate stages in the coagulation-protease cascade.

De i den foreliggende beskrivelse omtalte undersøgelser 25 viser, at monoklonale antistoffer, der inhiberer TF-aktivitet, er høj specifikke in vivo-antikoagulerende midler, idet de ved at blokere initieringen af koagulerings-protease-kaskaden forhindrer det forbrug af plasmakoaguleringsfaktorer, der normalt er knyttet til patologisk aktivering af 30 intravaskulær koagulering.The studies disclosed in the present disclosure indicate that monoclonal antibodies that inhibit TF activity are highly specific in vivo anticoagulants, by blocking the initiation of the clotting protease cascade which prevents the consumption of plasma coagulation factors that are normally associated with pathological activation of 30 intravascular coagulation.

58 kDa-formen af huTF beskrevet i eksempel 4 er påvist at være en disulfid-bundet heterodimer af 47 kDa-TF-proteinet og et ca. 12,5 kDa polypeptid, der nu er identificeret im-munokemisk og ved delvis aminosyresekvensbestemmelse som a~ 35 kæden af hæmoglobin. Tidligere antagelser om, at 58 kDa-båndet kunne udgøre en naturligt forekommende heterodimer 90 DK 176220 B1 form af cellulært TF, er formentlig ukorrekt, idet det er sandsynligt, at 58 kDa-heterodimeren dannes under isoleringen .The 58 kDa form of huTF described in Example 4 has been shown to be a disulfide-linked heterodimer of the 47 kDa TF protein and a ca. 12.5 kDa polypeptide, now identified immunochemically and by partial amino acid sequencing as the α ~ 35 chain of hemoglobin. Previous assumptions that the 58 kDa band could constitute a naturally occurring heterodimer in cellular TF form are probably incorrect, since the 58 kDa heterodimer is likely to form during the isolation.

Qf-Kæden af hæmoglobin har en enkelt cysteingruppe, 5 og TF forudsiges ud fra cDNA at have en enkelt cysteingruppe i det cytoplasmiske domæne. TF har også fire cysteingrupper i det ekstracellulære domæne, men mindst to må 'være involveret i disul fidbinding mellem kæder, da TF-funktionen går tabt efter reduktion. Den enkelte cysteingruppe i det cyto-10 plasmiske domæne af TF bibeholdes formentlig, ligesom cyste-iner i de fleste cytosoliske proteiner, i den reducerede tilstand. Denne (eller mindre sandsynligt en anden cysteingruppe af TF) kan være let tilgængelig for blandet disulfid-dannelse efter cellelyse, og det foreslås, at oxidation 15 under isoleringsproceduren medfører dannelse af en disulfid-binding mellem cysteinresten af det cytoplasmiske domæne af TF og hæmoglobin. En støtte for denne konklusion er observationen af, at heterodimer-dannelsen tilsyneladende er tidsafhængig, idet minimering af tidsrummet mellem detergent-20 ekstraktion af TF fra hjerne-acetone-pulver og binding til immunoaffinitetsmatrixen formindsker mængden af dannet hete-rodimert TF. Den formodede 96 kDa TF-dimer kan også dannes ved en lignende mekanisme under isoleringen.The Qf chain of hemoglobin has a single cysteine group, 5 and TF is predicted from cDNA to have a single cysteine group in the cytoplasmic domain. TF also has four cysteine groups in the extracellular domain, but at least two must be involved in disul fid bonding between chains as TF function is lost upon reduction. The single cysteine group in the cyto-10 plasmic domain of TF is presumably, like the cysteines in most cytosolic proteins, in the reduced state. This (or less likely another cysteine group of TF) may be readily available for mixed disulfide formation after cell lysis, and it is suggested that oxidation 15 during the isolation procedure results in the formation of a disulfide bond between the cysteine residue of the cytoplasmic domain of TF and hemoglobin. Supporting this conclusion is the observation that heterodimer formation appears to be time dependent, minimizing the time between detergent extraction of TF from brain acetone powder and binding to the immunoaffinity matrix decreases the amount of heterodermated TF formed. The putative 96 kDa TF dimer can also be formed by a similar mechanism during the isolation.

En anti-hæmoglobin-antistofsøjle binder specifikt 58 25 kDa-heterodimeren, men fjerner ikke kvantitativt alle stofferne med højere molekylvægt, der observeres i de immunoaf finitetsrensede TF-præparater. Spormængder af andre mindre bånd med molekylvægte over 47 kDa observeres at reagere med anti-TF-antistoffer. Disse stoffer i mindre mængder, omfat-30 tende en del af 58 kDa-båndet, kan være blandede disulfider dannet mellem TF og andre uidentificerede proteiner.Specifically, an anti-hemoglobin antibody column binds the 58 kDa heterodimer, but does not quantitatively remove all of the higher molecular weight substances observed in the immunoaffinity-purified TF preparations. Trace amounts of other smaller bands with molecular weights above 47 kDa are observed to react with anti-TF antibodies. These minor amounts, comprising a portion of the 58 kDa band, may be mixed disulfides formed between TF and other unidentified proteins.

Claims (10)

91 DK 176220 B1 PATENTKRAV.91 DK 176220 B1 PATENT REQUIREMENT. 1. Monoklonalt antistof, som binder human vævsfaktorprotein, til anvendelse ved forebyggelse af sepsis.1. Monoclonal antibody which binds human tissue factor protein for use in the prevention of sepsis. 2. Monoklonalt antistof, som binder human vævsfaktor-5 protein, til anvendelse ved behandling af thrombohæmorrha- giske lidelser.2. Monoclonal antibody which binds human tissue factor protein for use in the treatment of thrombohemorrhagic disorders. 3. Monoklonalt antistof, som binder human vævsfaktorprotein, til anvendelse ved inhibering af koagulation.3. Monoclonal antibody which binds human tissue factor protein for use in coagulation inhibition. 4. Monoklonalt antistof ifølge ethvert af kravene 1- 10 3, hvor det monoklonale antistof yderligere reagerer med en human vævsfaktor-bindingssted-polypeptidanalog.The monoclonal antibody according to any one of claims 1- 10, wherein the monoclonal antibody further reacts with a human tissue factor binding site polypeptide analog. 5. Monoklonalt antistof ifølge krav 4, hvor det monoklonale antistof reagerer med en sekvens valgt blandt følgende :The monoclonal antibody of claim 4, wherein the monoclonal antibody reacts with a sequence selected from the following: 15 EPKPVNQVYTVQISTKSGDWKSKC, VFGKDLIYTLYYWKSS SSGKKT, SSSGKKTAKTNTNEFLIDVDKGENYCFSV, SGTTNTVAAYNLTWKSTNFKTILEWEPKPV, TKSGDWKSKCFYTTDTECDLTDEIVKDVKQTY,15 EPKPVNQVYTVQISTKSGDWKSKC, VFGKDLIYTLYYWKSS SSGKKT, SSSGKKTAKTNTNEFLIDVDKGENYCFSV, SGTTNTVAAYNLTWKSTNFKTILEWEPKPVTTDKTYDVTKDVK 20 KSGDWKSKC, ECDLTDEIVKDVKQTY, LARVFSYPAGNVESTGSAGEPLYENSPEFTPYLC, YENSPEFTPYLEMNIGQPTIQSFEQVGTKV, ogKSGDWKSKC, ECDLTDEIVKDVKQTY, LARVFSYPAGNVESTGSAGEPLYENSPEFTPYLC, YENSPEFTPYLEMNIGQPTIQSFEQVGTKV, and 25 QAVIPSRTVNRKSTDSPVEC; og i det væsentlige ikke immunoreagerer med et polypeptid med formlen vist i fig. 1 fra position 204 til position 226.25 QAVIPSRTVNRKSTDSPVEC; and substantially do not immunoreact with a polypeptide of the formula shown in FIG. 1 from position 204 to position 226. 6. Anvendelse af et monoklonalt antistof, som binder human vævsfaktor-protein, ved fremstilling af et medikament 30 til behandling af sepsis.Use of a monoclonal antibody that binds human tissue factor protein in the manufacture of a medicament 30 for the treatment of sepsis. 7. Anvendelse af et monoklonalt antistof, som binder human vævsfaktor-protein, ved fremstilling af et medikament til behandling af thrombohæmorrhagiske lidelser.Use of a monoclonal antibody that binds human tissue factor protein in the manufacture of a medicament for the treatment of thrombohemorrhagic disorders. 8. Anvendelse af et monoklonalt antistof, som binder 35 human vævsfaktor-protein, ved fremstilling af et medikament til inhibering af koagulation. 92 DK 176220 B1Use of a monoclonal antibody that binds 35 human tissue factor protein in the preparation of a coagulation inhibiting drug. 92 DK 176220 B1 9. Anvendelse ifølge ethvert af kravene 6 til 8, hvor det monoklonale antistof er som anført i krav 4 eller 5 . DK 176220 B1 -30 -20Use according to any one of claims 6 to 8, wherein the monoclonal antibody is as claimed in claims 4 or 5. DK 176220 B1 -30 -20 -10 ME TPAVJPRVPRP ETAVARTLLL GWVFAQVAGA 10 20 30 40 SGxTNTVAAY NLTWKSTNFK TILEWEPKPV NQVYTVQIST 50 60 70 80 KSGDWKSKCF YTTDTECDLT DEIVKDVKQT YLARVFSYPA 90 100 110 120 GNVESTGSAG EPLYENSPEF TPYLETNLGQ PTIQSFEQVG 130 140 150 160 TKVNVTVEDE RTLVRRNNTF LSLRDVFGKD LIYTLYYWKS 170 180 190 200 SSSGKKTAKT NTNEFLIDVD KGENYCFSVQ AVIPSRTVNR 210 220 230 240 KSTDSPVECM GQEKGEFREI FY IIGAWFV VIILVIILAI 250 260 SLHKCRKAGV GQSWKENSPL NVS FIG. I-10 ME TPAVJPRVPRP ETAVARTLLL GWVFAQVAGA 10 20 30 40 SGxTNTVAAY NLTWKSTNFK TILEWEPKPV NQVYTVQIST 50 60 70 80 KSGDWKSKCF YTTDTECDLT DEIVKDVKQT YLARVFSYPA 90100110120 GNVESTGSAG EPLYENSPEF TPYLETNLGQ PTIQSFEQVG 130140150160 TKVNVTVEDE RTLVRRNNTF LSLRDVFGKD LIYTLYYWKS 170180190200 SSSGKKTAKT NTNEFLIDVD KGENYCFSVQ AVIPSRTVNR 210220230240 KSTDSPVECM GQEKGEGEFREI FOR IIGAWFV VIILVIILAI 250 260 SLHKCRKAGV GQSWKENSPL NVS FIG. IN