DK166626B1 - ANTITUMORAL GLYCOPROTEINS, WHETHER CARBOHYDRATE UNITS HAVE BEEN MODIFIED, AND METHOD OF PRODUCING THEREOF. - Google Patents

ANTITUMORAL GLYCOPROTEINS, WHETHER CARBOHYDRATE UNITS HAVE BEEN MODIFIED, AND METHOD OF PRODUCING THEREOF. Download PDF

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DK166626B1
DK166626B1 DK277985A DK277985A DK166626B1 DK 166626 B1 DK166626 B1 DK 166626B1 DK 277985 A DK277985 A DK 277985A DK 277985 A DK277985 A DK 277985A DK 166626 B1 DK166626 B1 DK 166626B1
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glycoprotein
aqueous solution
ricin
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Franz Jansen
Pierre Gros
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Sanofi Sa
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Abstract

1. Claims for the contracting States : BE, CH, DE, FR, GB, IT, LI, LU, NL, SE Antitumoral glycoprotein which inactives ribosomes, whose carbohydrate units are modified by oxidation with the periodate ion, said glycoprotein differing from the whole ricin. 1. Claims for contracting State : AT Process for the obtention of an antitumoral glycoprotein whose carbohydrate units are modified, said glycoprotein differing from the whole ricin, characterized in that it consists in submitting an unmodified antitumoral glycoprotein to an oxydation with periodate ions.

Description

i DK 166626 B1in DK 166626 B1

Den foreliggende opfindelse angår hidtil ukendte antitumorale glycoproteiner, hvis kulhydratenheder er blevet modificeret ved oxidation med periodationen, hvorhos giycoproteinerne er forskellige fra det hele ricin.The present invention relates to novel antitumoral glycoproteins, whose carbohydrate units have been modified by oxidation with the periodization, wherein the giycoproteins are different from the whole ricin.

55

Narmere betegnet angår den foreliggende opfindelse hidtil ukendte glycoproteiner, der inaktiverer ribosomer og har en forlænget virkning, hvilke glycoproteiner adskiller sig fra det hele ricin.More particularly, the present invention relates to novel glycoproteins which inactivate ribosomes and have an extended action, which glycoproteins differ from the entire ricin.

1010

Udtrykket "glycoprotein, der inaktiverer ribosomer", således som det benyttes i den foreliggende beskrivelse og også i kravene, betegner ethvert stof, som bærer saccharidenheder tilhørende klassen af makromolekyler, der inaktiverer ribosomer og 15 følgelig inhiberer proteinsyntese i eucaryote celler, tilligemed ethvert fragment af det nævnte stof, som er i besiddelse af den samme inaktiverende egenskab, idet nævnte glycoprotein, som inaktiverer ribosomer, kan være af naturlig eller biosyntetisk oprindelse, idet det hidrører fra en celle, hvis geno-20 type er blevet modificeret til dette formål, og nævnte glycoprotein, som inaktiverer ribosomer, kan også være modificeret i dets aminosyrers funktionelle grupper, så at det let kan kobles til et antistof.The term "glycoprotein inactivating ribosomes", as used in the present specification and also in the claims, means any substance which carries saccharide units belonging to the class of macromolecules that inactivate ribosomes and consequently inhibit protein synthesis in eucaryotic cells, as well as any fragment of said substance possessing the same inactivating property, said glycoprotein which inactivates ribosomes may be of natural or biosynthetic origin, originating from a cell whose genotype was modified for this purpose, and said glycoprotein which inactivates ribosomes may also be modified in the functional groups of its amino acids so that it can be readily coupled to an antibody.

25 I beskrivelsen vil udtrykket "glycoprotein, der inaktiverer ribosomer", blive betegnet med symbolerne GPIR.In the specification, the term "glycoprotein inactivating ribosomes" will be denoted by the symbols GPIR.

I beskrivelsen betegner udtrykket "periodat" I04_-ionen, der i litteraturen også omtales som "metaperiodat".In the specification, the term "periodate" refers to the IO4_ ion, which is also referred to in the literature as "metaperiodate".

3030

Glycoproteinerne, som inaktiverer ribosomer (GPIR), er særlig anvendelige som mellemprodukter til fremstilling af immuno-toksiner ved hjælp af kobling med antistoffer.The glycoproteins that inactivate ribosomes (GPIR) are particularly useful as intermediates for the preparation of immunotoxins by coupling with antibodies.

35 US-patentskrift nr. 4.340.535 og beskrivelsen til de franske patentansøgninger nr. 2.504.010 og nr. 2.516.794 beskriver fremstillingen af anticancerforbindelser, omtalt som konjuga- DK 166626 Bl 2 ter, der opnås ved via en kovalent binding at koble ricins A-kæde med antistoffer eller anti stoffragmenter, som er rettet mod antigener båret af den celle, der skal nedbrydes. Produkterne af denne type er blevet betegnet og betegnes i den fo-5 religgende beskrivelse med det generiske navn immunotoksiner.35 U.S. Patent No. 4,340,535 and the disclosure to French Patent Application Nos. 2,504,010 and No. 2,516,794 disclose the preparation of anticancer compounds, referred to as conjugates, obtained by coupling via a covalent bond. ricin A-chain with antibodies or anti-drug fragments targeting antigens carried by the cell to be degraded. The products of this type have been designated and are designated in the prior religious description by the generic name of immunotoxins.

Der kendes konjugater, som er analoge med de tidligere beskrevne immunotoksiner indeholdende ricins A-kæde, hvilke konjugater også er egnede som anticancermedikamenter og hidrører 10 fra koblingen via en kovalent binding af antistoffer eller antistoffragmenter med andre glycoproteiner, der inaktiverer ri-bosomer, såsom især geloninet, der ekstraheres fra Gelonium multiflorum (eur. J. Biochem., 1981, 116, 447-454; CancerConjugates are known which are analogous to the previously described immunotoxins containing ricin A chain, which are also useful as anticancer drugs and are derived from the coupling via a covalent linkage of antibodies or antibody fragments with other glycoproteins which inactivate ribosomes, such as in particular the gelonin extracted from Gelonium multiflorum (eur. J. Biochem. 1981, 116, 447-454; Cancer

Res., 1984, 44, 129-133), eller den inhibitor, der ekstraheres 15 fra Momordica charantia (MOM) (US-patentskrift nr.Res., 1984, 44, 129-133), or the inhibitor extracted from Momordica charantia (MOM) (U.S. Pat.

4.368.149).4,368,149).

Disse glycoproteiner, der inaktiverer ribosomer (GPIR), og som har egenskaber svarende til de egenskaber, som ricins A-kæde 20 har, er stoffer med en molekylvægt af størrelsesordenen 20.000 og 30.000 (Cancer Survey, 1982, 1, 489-520).These glycoproteins that inactivate ribosomes (GPIR) and have properties similar to those of ricin A chain 20 are substances of the order of 20,000 and 30,000 (Cancer Survey, 1982, 1, 489-520).

Det er også kendt, at disse immunotoksiners cytotaksiske virkning kan potentieres ved hjælp af en række forskellige hjælpe- 25 stoffer, såsom ammoniumsalte, forskellige aminer eller visse carboxyl ionophorer, såsom monensin og nigericin.It is also known that the cytotoxic effect of these immunotoxins can be potentiated by a variety of excipients such as ammonium salts, various amines or certain carboxyl ionophores such as monensin and nigericin.

Uanset om immunotoksiners terapeutiske virkninger aktiveres eller ikke aktiveres, kan de imidlertid kun manifestere sig i 30 fuldt omfang, for så vidt i mmunotoksi net er i stand til via sin antistofdel at blive lokaliseret in vivo i den aktive form på målcellerne, der skal nedbrydes (sine qua non betingelse for at bringe enhver immunotoksinvirking til udtryk). Immuno-toksinets evne til at blive lokaliseret på målet afhænger 35 først og fremmest af immunotoks i nets evne til at forblive i blodstrømmen og de ekstracel1ulare væsker i den aktive form i tilstrækkelig lang tid til at det når frem til dets målceller DK 166626 B1 3 og i tilstrækkelige store koncentrationer, således at optagelsesgraden af de tilsvarende antigeniske steder bliver høj.However, whether the therapeutic effects of immunotoxins are activated or not activated, they can only fully manifest to the extent that in the immunotoxin network it is able to localize through its antibody in vivo in the active form of the target cells to be degraded ( its qua non condition to express any immunotoxin effect). The ability of the immunotoxin to be located on the target depends primarily on the ability of the immunotoxin to stay in the bloodstream and the extracellular fluids in the active form for a sufficient time to reach its target cells DK 166626 B1 3 and in sufficiently large concentrations such that the uptake rate of the corresponding antigenic sites becomes high.

Talrige undersøgelser har gjort det muligt.at fastslå immuno-5 toksiners plasmaeliminationskinetik efter intravenøs injektion i forskellige dyremodeller. Det fremgår, at efter injektion falder plasmamængden af biologisk aktivt immunotoksin meget hurtigt og meget betydeligt. I et typisk tilfælde, der involverer kaniner, i en model, som anvender et immunotoksin, der 10 ved hjælp af en arm indeholdende en disulfidbro er syntetiseret ved kobling af ricins-A-kæde med et monoklonalt antistof rettet mod humane T-lymfocyters (antistof T101) antigen T65, viser det sig således, at 97% af det på tidspunktet 0 i blod-strømmen tilstedeværende immunotoksin efter injektion forsvin-15 der i løbet af 30 min., og at 99,0% forsvinder i løbet af 17 timer. Denne hurtige forsvinden af immunotoksi net trækker ganske tydeligt fra med hensyn til, at dets fuldstændige cyto-toksiske kapacitet bringes til udtryk, ved at immunotoksi net hindres i et udstrakt tidsrum at mætte en høj andel af mål-20 antigenerne, der bæres af de celler, som skal nedbrydes. Endvidere viser sammenligning mellem immunotoksinernes plasmaeliminationskinetik og de tilsvarende ikke-konjugerede antistoffers plasmaeliminationskinetik, at antistofferne derimod forbliver i plasmaet i en høj mængde i relativt lange tidsrum, 25 således som det er velkendt. Imidlertid findes der altid en vis tilbageværende mængde af ikke-konjugerede antistoffer, endog i de mest højrensede immunotoksinpræparater. Via virkningen af de forskellige eliminationshastigheder for immunotiksi-ner og antistoffer bliver de ikke-konjugerede antistoffer, som 30 til at begynde med foreligger i meget lille mindretal, gradvist et flertal efter nogle få timer, og disse antistoffer bliver derfor gradvis ved konkurrence kraftige antigonister for fikseringen af immunotoksinerne til deres mål.Numerous studies have made it possible to determine the plasma elimination kinetics of immunotoxins after intravenous injection in various animal models. It is seen that after injection, the plasma amount of biologically active immunotoxin decreases very rapidly and very significantly. In a typical case involving rabbits, in a model employing an immunotoxin synthesized by means of an arm containing a disulfide bridge by coupling castor A chain with a monoclonal antibody directed against human T lymphocytes (antibody T101) antigen T65, it is found that 97% of the immunotoxin present at injection time at time 0 disappears after 30 minutes and 99.0% disappears within 17 hours. This rapid disappearance of immunotoxic networks is quite obvious in terms of its complete cytotoxic capacity being expressed by inhibiting immunotoxic networks for a prolonged period of time to saturate a high proportion of the target antigens carried by the cells. , which must be broken down. Furthermore, comparison of the plasma elimination kinetics of the immunotoxins with the plasma elimination kinetics of the corresponding non-conjugated antibodies, on the contrary, remains in the plasma for a high amount for a relatively long period of time, as is well known. However, there is always some residual amount of non-conjugated antibodies, even in the most highly purified immunotoxin preparations. Through the effect of the different elimination rates of immunotics and antibodies, the non-conjugated antibodies, which initially are in very small minority, gradually become a majority after a few hours, and these antibodies therefore gradually become potent antagonists for competition. the fixation of the immunotoxins to their targets.

35 Fordelen ved at forøge det vedvarende indhold af immunotoksi-ner i den aktive form i plasmaet med henblik på at forøge såvel varigheden som graden af optagelse af mål antigenerne og DK 166626 B1 4 følgelig af at forøge immunotoksinernes terapeutiske virkninger er derfor indlysende.The advantage of increasing the sustained content of immunotoxins in the active form in the plasma in order to increase both the duration and the degree of uptake of the target antigens and consequently of increasing the therapeutic effects of the immunotoxins is therefore obvious.

Endvidere har lokaliseringsforsøg in vivo med immunotoksinet 5 indeholdende ricins A-kæde, hvilket immunotoksin er blevet radiomærket og derpå injiceret i dyr uden et specifikt mål, vist, at i løbet af de første få minutter efter injektion bliver konjugatet fortrinsvis lokaliseret i leveren. Det samme gælder for ricins A-kæde, som følger det samme mønster, når 10 den injiceres i den ikke-koblede form. Dette antyder stærkt, at immunotoksinet fikseres i leveren via den cytotoksiske sub- i enhed, som det indeholder. Det er kendt, at ricins A-kæde er et glycoprotein, hvis polyoside grupper især indbefatter man- l nosegrupper og N-acetylglucosamingrupper, idet nogle mannose-15 grupper foreligger i terminale positioner (Agri. Biol . Chem., 1978, 42, 501). Eksistensen i leveren af receptorer, der er i stand til at genkende glycoproteiner med disse terminale man-nosegrupper, er også blevet fastslået. Endvidere har det vist sig, at de glycoproteiner, der genkendes af disse receptorer -20 idet sidstnævnte i alt væsentligt er til stede på Kupffer-cellerne - hurtigt elimineres fra blodstrømmen ved fiksering til disse celler, som metaboli serer dem. Dette er særlig godt dokumenteret i tilfælde af β-glucuronidsase og også i tilfælde af ribonuclease B (Arch. Biochem. Biophys., 1978, 188, 418; 25 Advances in Enzymology, udgivet af A. Meister, New York, 1974;Furthermore, in vivo localization experiments with the immunotoxin 5 containing the ricin A chain, which immunotoxin has been radiolabelled and then injected into animals without a specific target, have shown that during the first few minutes after injection, the conjugate is preferably localized in the liver. The same is true for ricin A chain, which follows the same pattern when injected into the uncoupled form. This strongly suggests that the immunotoxin is fixed in the liver via the cytotoxic subunit that it contains. It is known that the A chain of ricin is a glycoprotein whose polyoside groups include, in particular, mannose groups and N-acetylglucosamine groups, with some mannose groups being in terminal positions (Agri. Biol. Chem., 1978, 42, 501 ). The existence of the liver of receptors capable of recognizing glycoproteins with these terminal mannose groups has also been established. Furthermore, it has been found that the glycoproteins recognized by these receptors -20, the latter being substantially present on the Kupffer cells - are rapidly eliminated from the bloodstream by fixation to these cells which metabolize them. This is particularly well documented in the case of β-glucuronidase and also in the case of ribonuclease B (Arch. Biochem. Biophys., 1978, 188, 418; Advances in Enzymology, published by A. Meister, New York, 1974;

Pediat. Res., 1977, u, 816).Pediat. Res., 1977, u, 816).

Taget som helhed viser disse data, at den hurtige eliminering af immunotoksiner indeholdende ricins A-kæde kan forklares ved 30 levercellernes og især Kupf fer-cel lernes erkendelse af manno-segrupperne i ricins A-kæde.Taken as a whole, these data indicate that the rapid elimination of immunotoxins containing the ricin A chain can be explained by the recognition of the manno groups in the ricin A chain by the liver cells and especially the Kupf cells.

Undersøgelserne af plasmaeliminationskinetik udført med andre GPIR'er, f.eks. gelonin eyjer MOM, efter intravenøs injektion 35 i dyret, har vist, at ligesom i tilfælde af ricins A-kæde formindskes plasmaets QPiR-mængde meget hurtigt og meget væsentligt efter injekti0n> i et typisk tilfælde, hvor kaniner an- DK 166626 B1 5 vendes, viser det sig efter injektion af gelonin, der er renset ved hjælp af den beskrevne metode (J. Biol. Chem., 1980, 255, 6947-6953), at 93¾ af det i blodstrømmen på tidspunktet 0 efter injektion tilstedeværende gelonin forsvinder i løbet 5 af 1 time, og at 99,99% forsvinder i løbet af 24 timer.The studies of plasma elimination kinetics performed with other GPIRs, e.g. gelonin eye MOM, after intravenous injection 35 in the animal, has shown that, just as in the case of ricin A chain, the QPiR amount of plasma decreases very rapidly and very significantly after injection> in a typical case where rabbits are used. , after injection of gelonin purified by the method described (J. Biol. Chem., 1980, 255, 6947-6953), it appears that 93¾ of the gelonin present in the blood stream at time 0 after injection disappears in within 5 of 1 hour and 99.99% disappear within 24 hours.

Det er kendt, at oxidation af osidiske strukturer inklusive de 1 glycoproteiner indeholdte med periodationer forårsager overskæring af carbonkæden, når to tilstødende carbonatomer bærer 10 primære eller sekundære hydroxyIgrupper. Hvis de to tilstødende hydroxylgrupper er sekundære, således som det generelt er tilfældet i de i GPIR'er tilstedeværende cykliske oser, frembringer oxidation to aldehydgrupper på de carbonatomer, mellem hvilke overskæringen har fundet sted.It is known that oxidation of oscidic structures including the 1 glycoproteins contained with periodisations causes cutting of the carbon chain when two adjacent carbon atoms carry 10 primary or secondary hydroxy groups. If the two adjacent hydroxyl groups are secondary, as is generally the case in the cyclic oxides present in GPIRs, oxidation produces two aldehyde groups on the carbon atoms between which the cutting occurred.

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Det har nu vist sig, at når kulhydratenhederne i et antitumoralt glycoprotein er modificeret ved hjælp af oxidation med periodationer, forbliver nævnte glycoproteins biologiske virkning i det væsentlige uændret.It has now been found that when the carbohydrate units of an antitumoral glycoprotein are modified by periodic oxidation, the biological effect of said glycoprotein remains essentially unchanged.

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Det har også vist sig, hvilket er absolut uventet, at hvis kulhydratenhederne i et glycoprotein, som inaktiverer riboso-mer, modificeres ved oxidation med periodationer, opnås et nyt glycoprotein, der inaktiverer ribosomer, idet det nævnte nye 25 glycoprotein har den dobbelte egenskab i form at bevare sine biologiske virkninger og meget langsomt at blive elimineret fra blodstrømmen in vivo.It has also been found, which is absolutely unexpected, that if the carbohydrate units of a glycoprotein which inactivates ribosomes are modified by oxidation with periodizations, a new glycoprotein inactivating ribosomes is obtained, said new glycoprotein having the dual property of form to preserve its biological effects and very slowly to be eliminated from blood flow in vivo.

En dybtgående biokemisk undersøgelse af oxiderede og naturlige 30 GPIR'er har gjort det muligt at vise, at oxidationen af GPIR’er med periodat udelukkende involverer GPIR'ernes osidiske del og ikke har nogen virkning på sekvensen af aminosyrer-ne, der udgør peptiddelen deraf.An in-depth biochemical study of oxidized and natural 30 GPIRs has shown that the oxidation of GPIRs with periodate involves only the osidic portion of the GPIRs and has no effect on the sequence of the amino acids constituting the peptide portion thereof. .

35 Disse hidtil ukendte glycoproteiner, som inaktiverer ribosomer og har en forlænget virkning, omtales i det følgende ved hjælp af symbolerne GPIR-LA.35 These novel glycoproteins, which inactivate ribosomes and have a prolonged action, are hereinafter referred to by the symbols GPIR-LA.

DK 166626 B1 6DK 166626 B1 6

Endelig har det vist sig, at når disse hidtil ukendte glycop-roteiner, som inaktiverer ribosomer og har en forlænget virkning, kobles med antistoffer, bevarer de opnåede konjugater de for immutoksiner kendte biologiske egenskaber og har langsom 5 plasmaeliminationskinetik.Finally, when these novel glycoproteins, which inactivate ribosomes and have a prolonged action, have been found to be coupled with antibodies, the conjugates obtained retain the biological properties known for immotoxins and have slow plasma elimination kinetics.

Det vil bemærkes, at periodatoxidation har været foreslået som kendt teknik til fremstilling af konjugater bestående af et antistof eller et antistoffragment samt af et antitumoralt 10 stof. I dette tilfælde benyttes de ved hjælp af periodatoxidation af det antitumorale stof opnåede aldehydgrupper til fremstilling af det krævede konjugat. Disse aldehydgrupper indføres derfor midlertidigt for at muliggøre konjugationen af antistof og antitumoralt stof. EP offentliggørelsesskrift nr.It will be noted that periodate oxidation has been proposed as a known technique for the preparation of conjugates consisting of an antibody or antibody fragment and of an antitumoral substance. In this case, the aldehyde groups obtained by periodate oxidation of the antitumoral substance are used to prepare the required conjugate. Therefore, these aldehyde groups are temporarily introduced to allow for the conjugation of antibody and anti-tumor substance. EP disclosure no.

15 74.279 og FR offentliggørelsesskrift nr. 2.312.259 kan nævnes i denne henseende, hvilke offentliggørelsesskrifter ikke omtaler de ifølge den foreliggende opfindelse benyttede glycopro-teiner.15 74,279 and FR Publication No. 2,312,259 can be mentioned in this regard, which disclosures do not disclose the glycoproteins used in the present invention.

20 Ifølge den foreliggende opfindelse deltager aldehydgrupperne, som er indført på giycoproteinet ved hjælp af periodatoxidation, derimod ikke i dannelsen af de ovenfor definerede immu-notoksi ner.In contrast, according to the present invention, the aldehyde groups introduced on the glycoprotein by periodate oxidation do not participate in the formation of the immunotoxins defined above.

25 Den foreliggende opfindelse angår derfor, som hidtil ukendte produkter, strukturelt modificerede antitumorale glycoprotei-ner, hvis kulhydratenheder er blevet modificeret ved oxidation med periodationen. Den foreliggende opfindelse angår nærmere betegnet glycoproteiner, der inaktiverer ribosomer, og hvis 30 kulhydratenheder er blevet modificeret ved oxidation med periodationen, og som har den samme virkning som og en længere halveringstid end det umodificerede glycoprotein.The present invention, therefore, as novel products, relates to structurally modified antitumoral glycoproteins whose carbohydrate units have been modified by oxidation with the periodization. More particularly, the present invention relates to glycoproteins which inactivate ribosomes and whose 30 carbohydrate units have been modified by oxidation with periodization and which have the same effect and longer half-life than the unmodified glycoprotein.

Opfindelsen angår fortrinsvis glycoproteiner, som inaktiverer 35 ribosomer og har en forlænget virkning, og som opnås ved behandling af et glycoprotein, der inaktiverer ribosomer, og hvis thiolgrupper eventuelt er beskyttet, med en vandig opløs- DK 166626 B1 7 m'ng af et alkalimetalperiodat i et tidsrum fra 0,2 til 24 timer ved en tempertur fra 0 til 15°C og i fravær af lys, om ønsket afblokering af thiol grupperne samt isolation af slutproduktet ved hjælp af kendte metoder.The invention preferably relates to glycoproteins which inactivate 35 ribosomes and have a prolonged action, and which are obtained by treatment of a glycoprotein which inactivates ribosomes and whose thiol groups are optionally protected, with an aqueous solution of an alkali metal periodate. for a period of 0.2 to 24 hours at a temperature of 0 to 15 ° C and in the absence of light, if desired, to unblock the thiol groups and isolate the final product by known methods.

55

Ethvert antitumoralt glycoprotein kan i sine kulhydratenheder modificeres ved omsætning med peri odat ionen ifølge de kendte metoder.Any antitumoral glycoprotein can be modified in its carbohydrate units by reaction with the peri-odat ion according to the known methods.

10 Glycoproteinerne, som inaktiverer ribosomer, og som anvendes som foretrukne udgangsmaterialer for oxidation med periodatio-ner ifølge den foreliggende opfi ndel se,er alle GPIR'er, såsom ricins A-kæde, hvilke GPIR'er selv kun er meget svagt cytotok-siske, fordi de ikke kan fikseres til celler, men som på den 15 anden side efter kobling med et antistof, der genkender bestemte celler, bliver meget cytotoksiske over for disse celler, når først antistoffet har genkendt sit mål.The glycoproteins which inactivate ribosomes and which are used as preferred starting materials for oxidation with periodates according to the present invention are all GPIRs, such as ricin A chain, which GPIRs themselves are only very weakly cytotoxic because they cannot be fixed to cells but, on the other hand, after coupling with an antibody that recognizes certain cells, becomes highly cytotoxic to these cells once the antibody has recognized its target.

Repræsentative udgangsforbindelser er ricins A-kæde, gelonin 20 og det fra Momordica charantia ekstraherede stof (MOM), således som det opnås ved ekstraktion.Representative starting compounds are the A-chain of ricin, gelonin 20, and the extracted substance (MOM) extracted from Momordica charantia as obtained by extraction.

Andre GPIR'er, som kan anvendes som udgangsmaterialer for oxidation med periodationer, er som følger: 25Other GPIRs which can be used as starting materials for oxidation with periodisations are as follows:

Dianthin 30 fra Dianthus carryophy11usDianthin 30 from Dianthus carryophy11us

Dianthin 32 fraDianthin 32 from

Agrostin A fra Agrostemma githagoAgrostin A from Agrostemma githago

Agrostin B fra " " 30 Agrostin C f ra ‘ '' " HCI fra Hura crepitansAgrostin B from "30 Agrostin C from RA" HCl from Hura crepitans

Asparagus officinalis- inhibitor fra Asparagus officinalis 35 De samme stoffer produceret biosyntetisk ved hjælp af celler, hvis genotype er blevet modificeret til dette formål, er også egnede forbindelser.Asparagus officinalis inhibitor from Asparagus officinalis 35 The same substances produced biosynthetically by cells whose genotype has been modified for this purpose are also suitable compounds.

DK 166626 B1 8DK 166626 B1 8

Fragmenter af ovennævnte GPIR'er, forudsat at de bevarer hele eller en del af evnen til at inaktivere ribosomer, hvilken evne karakteriserer den GPIR, hvoraf de er afledt, kan også anvendes som udgangsmaterialer.Fragments of the above GPIRs, provided they retain all or part of the ability to inactivate ribosomes, which characterize the GPIR from which they are derived, can also be used as starting materials.

5 A-kæden i naturligt ricin, i hvilken mindst en af thiolgrup-perne er beskyttet, er en foretrukken udgangsforbindelse.The natural ricin A chain in which at least one of the thiol groups is protected is a preferred starting compound.

Nylige undersøgelser har vist, at ricins A-kæde omfatter 2 be-10 standdele betegnet Al og A2, der især adskiller sig med hensyn til deres polysaccharidenheder. De forsøg, som har været udført med A-kædens 2 bestanddele, har gjort det muligt at vise, at periodatoxidation finder sted på en meget ensartet måde i Al- og A2-kæden og giver disse 2 bestanddele identiske egen-15 skaber med hensyn til forbedring af pharmacokinetikken.Recent studies have shown that the A chain of ricin comprises 2 constituents designated A1 and A2, which differ in particular in their polysaccharide units. The tests that have been carried out with the 2 components of the A chain have made it possible to show that periodate oxidation takes place in a very uniform manner in the Al and A2 chains and gives these 2 components identical properties with respect to improving pharmacokinetics.

Fremstillingen af ricins rene A-kæde er beskrevet i US-patent-skrift nr. 4.340.535. Gelonin og MOM er også tidligere blevet beskrevet.The preparation of pure A-chain ricin is described in U.S. Patent No. 4,340,535. Gelonin and MOM have also been previously described.

2020

Beskyttelse af thiolgrupperne i de som udgangsmaterialer benyttede GPIR'er er ønskelig, når nævnte thiolgrupper er de grupper, som skal anvendes til kobling med antistoffet.Protection of the thiol groups in the GPIRs used as starting materials is desirable when said thiol groups are the groups to be used for coupling with the antibody.

25 Hvis andre funktionelle grupper anvendes til koblingen, f.eks. tyrosiners phenoliske hydroxylgruppe eller GPIR's aminogrupper eller carboxylgrupper, kan beskyttelse udføres eller undlades.If other functional groups are used for the coupling, e.g. tyrosines phenolic hydroxyl group or GPIR amino groups or carboxyl groups, protection can be performed or omitted.

Blokering udføres ved omsætning med et middel, der er i stand 30 til at substituere SH-grupperne med en gruppe, der efterfølgende kan fjernes ved reduktion eller thiol/disulfid-udveksling, f.eks. 2,2'-di nitro-5,5'-dithiodibenzoesyre (DTNB) eller 3-(pyridin-2-yl-disulfanylJpropionsyre eller alternativt dipyridyl-2,2'-di sulfid eller dipyridy1-4,4'-disulfid. Ved 35 fravær af en sådan behandling kan de frie thiolgrupper forsvinde under oxidationsreaktionen, i hvilket tilfælde de ikke kan gendannes fuldstændigt. Overskuddet af blokeringsmiddel DK 166626 Bl 9 fjernes ved hjælp af dialyse eller enhver anden passende behandl i ng.Blocking is effected by reaction with an agent capable of substituting the SH groups with a group which can subsequently be removed by reduction or thiol / disulfide exchange, e.g. 2,2'-di nitro-5,5'-dithiodibenzoic acid (DTNB) or 3- (pyridin-2-yl-disulfanylpropionic acid or alternatively dipyridyl-2,2'-di sulfide or dipyridyl-4,4'-disulfide. In the absence of such treatment, the free thiol groups may disappear during the oxidation reaction, in which case they cannot be fully recovered. The excess of blocking agent DK 166626 B1 9 is removed by dialysis or any other appropriate treatment.

Periodatoxidationsreaktionen udføres ved en sur pH-værdi mel-5 lem 3 og 7, fortrinsvis mellem 5 og 6,5.The periodate oxidation reaction is carried out at an acidic pH of between 3 and 7, preferably between 5 and 6.5.

Periodatet anvendes i overskud. Nærmere betegnet er koncentrationen af alkalimetalperiodat større end koncentrationen af de vicinale dioler, der er i stand til at blive oxideret. Koncen-trationer på 10 til 50 mM med hensyn til natr i umper i odat til koncentrationer på 1 til 10 mg/ml cytotoksisk sub-enhed er passende. Behandlingen udføres ved en temperatur mellem 0 og 15°C, fortrinsvis mellem 1 og 5*C, og i mørke, og den varer mellem 0,2 og 24 timer.The period is used in surplus. More specifically, the concentration of alkali metal periodate is greater than the concentration of the vicinal diols capable of being oxidized. Concentrations of 10 to 50 mM with respect to natr in umper in odat to concentrations of 1 to 10 mg / ml cytotoxic subunit are appropriate. The treatment is carried out at a temperature between 0 and 15 ° C, preferably between 1 and 5 ° C, and in the dark, and lasts between 0.2 and 24 hours.

1515

Reaktionen standses ved tilsætning af et middel, der forbruger det tilbageværende periodat, f.eks. et overskud af ethylen- glycol, og biprodukterne fjernes ved dialyse eller ved hjælp af enhver anden passende behandling. Det ved reaktionens afs-20 lutning opnåede produkt isoleres ved hjælp af konventionel tekn i k.The reaction is stopped by the addition of an agent consuming the remaining periodate, e.g. an excess of ethylene glycol and the by-products are removed by dialysis or by any other appropriate treatment. The product obtained at the end of the reaction is isolated by conventional techniques in k.

Hvis udgangsmaterialets thiolgrupper er blevet blokeret, gennemføres afblokering ved hjælp af de kendte metoder, f.eks.If the starting material thiol groups have been blocked, unblocking is carried out by the known methods, e.g.

25 ved omsætning med et reduktionsmiddel, der kan frigøre den tidligere blokerede thiolgruppe, såsom 2-mercaptoethanol, hvilket fører til det nye glycoprotein, der inaktiverer ribo-somer og har en forlænget virkning og er parat til at blive benyttet til f.eks. kobling med et antistof til opnåelse af et 30 immunotoksin.25 by reaction with a reducing agent which can release the previously blocked thiol group, such as 2-mercaptoethanol, which leads to the new glycoprotein which inactivates ribosomes and has an extended effect and is ready to be used, e.g. coupling with an antibody to obtain an immunotoxin.

I tilfælde af ricins A-kæde har det resulterende nye molekyle (omtalt ved hjælp af symbolerne A-La) følgende hovedegenskaber : - en molekylvægt, der ikke afviger signifikant fra den naturlige A-kædes molekylvægt. Såvidt det er muligt at se ved hjælp 35 DK 166626 B1 10 af polyacrylamidgradientelektrophorese frembringer denne modifikationsproces polymerer af proteinet i en meget ringe mængde og danner ikke nogen nedbrydningsprodukter.In the case of the A chain of ricin, the resulting new molecule (referred to by the symbols A-La) has the following main characteristics: - a molecular weight which does not differ significantly from the molecular weight of the natural A chain. As far as it is possible to see by means of polyacrylamide gradient electrophoresis, this modification process produces polymers of the protein in a very small amount and does not form any degradation products.

5 - et antal frie thiolgrupper, der er større end 0,7 pr. mol.5 - a number of free thiol groups greater than 0.7 per moth.

- en immunoreaktivitet over for kanin-antistoffer, der inhibe-rer ricins A-kæde, hvilken immunoreaktivitet ikke kan skelnes fra den naturlige A-kædes immunoreaktivitet.an immunoreactivity to rabbit antibodies that inhibit ricin A chain, which immunoreactivity cannot be distinguished from the natural A chain immunoreactivity.

10 - en inhiberende virkning på proteinsyntesen i en acellular model, der er større end 50% af den inhiberende virkning, som forårsages af en lige så stor mængde naturlig A-kæde.10 - an inhibitory effect on protein synthesis in an acellular model greater than 50% of the inhibitory effect caused by an equal amount of natural A chain.

15 - efter en enkelt intravenøs administration til kaniner i en dosis på ca. 0,4 mg/kg legemsvægt er endvidere mængden i plasmaet af A-kæde (A-La) med forlænget virkning 23 timer efter injektion større end 10% af den mængde, der er til stede på tidspunktet nul (i modsætning til 0,015% for den naturlige 20 A-kæde på dette tidspunkt), dvs, en stigning i plasmamængden med en faktor, der er meget større end 500.15 - after a single intravenous administration to rabbits at a dose of ca. Furthermore, 0.4 mg / kg body weight is the amount in the extended-chain A-La (A-La) plasma 23 hours after injection greater than 10% of the amount present at time zero (as opposed to 0.015% for the natural 20 A chain at this time), i.e., an increase in plasma volume by a factor much greater than 500.

Tilsvarende i tilfælde af gelonin har det ved periodatoxida-tion opnåede molekyle følgende hovedegenskaber: 25 - en molekylvægt, der ikke er signifikant anderledes end molekylvægten af naturligt gelonin.Similarly, in the case of gelonin, the molecule obtained by periodate oxidation has the following main properties: 25 - a molecular weight not significantly different from the molecular weight of natural gelonin.

- en immunoreaktivitet over for anti-gelonin kanin-antistof- 30 fer, der ikke kan skelnes fra det naturlige gelonins immuno reakt i vi tet.an immunoreactivity to anti-gelonin rabbit antibodies indistinguishable from the natural gelonin immunoreact in the wild.

- efter en enkelt intravenøs administration til kaniner i en dosis på ca. 0,3 mg/kg legemsvægt er endvidere mængden i plas- 35 maet af det modificerede gelonin 24 timer efter injektion større end 3% af den mængde, der er til stede på tidspunktet nul (i modsætning til 0,01% for det naturlige gelonin på dette DK 166626 B1 11 tidspunkt), dvs. en stigning i plasmamængden med en faktor, der er større end 200.- after a single intravenous administration to rabbits at a dose of approx. Furthermore, 0.3 mg / kg body weight is the amount in the plasma of the modified gelonin 24 hours after injection greater than 3% of the amount present at time zero (as opposed to 0.01% for the natural gelonin at this time), ie. an increase in plasma volume by a factor greater than 200.

Fremstillingen af konjugaterne eller immunotoksinerne ud fra 5 de g 1ycoproteiner, der inaktiverer ribosomer og har en forlænget virkning, udføres ved hjælp af en hvilken som helst fremgangsmåde, der hensigtsmæssigt vælges fra intervallet af fremgangsmåder beskrevet i US-patentskrift nr. 4.340.535. Hvis den valgte cytotoksiske sub-enhed naturligt indeholder mindst 10 en thiol, som gør den egnet til kobling, vil denne gruppe fortrinsvis blive benyttet til omsætning med det antistof eller anti stoffragment, der bærer en aktiveret disulfidgruppe. Hvis den valgte cytotoksiske sub-enhed ikke naturligt har en thiol-gruppe, der gør den egnet til kobling, kan i det mindste en 15 funktionel gruppe, der bærer en fri thiol, fortrinsvis blive indført kunstigt i den nævnte sub-enhed ved hjælp af en hvilken som helst kendt fremgangsmåde, og koblingen kan fortsættes som oven for anført. Indføringen af den nævnte funktionelle gruppe kan finde sted enten før oxidationstrinnet med perioda-20 tioner, i hvilket tilfælde det vil være nødvendigt for thiol-gruppen at blive blokeret under oxidationstrinnet og derefter blive afblokeret efter dette trin, eller efter oxidationstrinnet.The preparation of the conjugates or immunotoxins from 5 g of cycoproteins that inactivate ribosomes and have an extended effect is carried out by any method conveniently selected from the range of methods described in U.S. Patent No. 4,340,535. If the selected cytotoxic subunit naturally contains at least 10 a thiol which makes it suitable for coupling, this group will preferably be used for reaction with the antibody or antibody fragment carrying an activated disulfide group. If the selected cytotoxic subunit does not naturally have a thiol group which makes it suitable for coupling, at least one functional group carrying a free thiol may preferably be introduced artificially into said subunit by means of any known method and the coupling can be continued as above. The introduction of said functional group can take place either before the oxidation step of period 20, in which case it will be necessary for the thiol group to be blocked during the oxidation step and then be blocked after this step, or after the oxidation step.

25 Dette fører til modificerede immunotoksiner, som har opnået en ny karakter med hensyn til deres pharmacokinetiske egenskaber. Nærmere betegnet har det ved passende modifikation af den cytotoksiske sub-enhed været muligt til immunotoksiners specifikke cytotoksiske egenskaber uden at genere disse at tilføje 30 en ny egenskab, der er lige så naturlig, nemlig evnen til at udvise langsom plasmaeliminationskinetik.This leads to modified immunotoxins which have acquired a new character in their pharmacokinetic properties. Specifically, by appropriate modification of the cytotoxic subunit, it has been possible for the specific cytotoxic properties of immunotoxins without bothering them to add a new property that is equally natural, namely the ability to exhibit slow plasma elimination kinetics.

3535

Opfindelsen illustreres nærmere i det følgende under henvis ning til tegningen, hvor DK 166626 B1 12 fig. 1 viser plasmaeliminationskurven som funktion af tiden for A-kæden af intravenøst injiceret naturligt ricin, 5 fig. 2 viser tidens indflydelse på plasmakoncentrationen af A-kæde ved behandling med natriumperiodat, fig. 3 viser tidens indflydelse på plasmakoncentrationen af A-kæde og methyleret A-kæde ved behandling med natriumperiodat, fig. 4 viser plasmaeliminationskurverne for intravenøst inji-10 ceret naturligt gelonin og oxideret gelonin som funktion af tiden, fig. 5 viser plasmaeliminationskurveme som funktion af tiden for det oxiderede og det ikke-oxiderede MOM efter intravenøs injektion, 15 fig. 6 viser plasmaeliminationskurverne som funktion af tiden for det oxiderede diantin og det ikke-oxiderede diantin, fig. 7 viser plasmaeliminationskurverne som funktion af tiden for intravenøst injiceret IT T101 og IT (A-la) T101, fig. 8 viser dosis/virkning-kurver, der illustrerer cytotoksi-20 citet af IT (A-la) T101 og (A-la) over for CEM-celler målt ved inhibering af proteinsyntese, og fig. 9 viser kurver, der illustrerer den cytotoksiske virkning af immunotoksinerne IT (A-la) T101 og IT T101 på CEM-celler, målt ved hjælp af den clonogene prøve.The invention is further illustrated in the following with reference to the drawing, in which DK 166626 B1 12 FIG. Figure 1 shows the plasma elimination curve as a function of time for the A chain of intravenously injected natural ricin; Figure 2 illustrates the influence of time on the A-chain plasma concentration upon treatment with sodium periodate; Figure 3 shows the influence of time on the plasma concentration of A chain and methylated A chain upon treatment with sodium periodate; Figure 4 shows the plasma elimination curves for intravenously injected natural gelonin and oxidized gelonin as a function of time; Figure 5 shows the plasma elimination curves as a function of the time of the oxidized and non-oxidized MOM after intravenous injection; Figure 6 shows the plasma elimination curves as a function of the time of the oxidized diantine and the non-oxidized diantine; Figure 7 shows the plasma elimination curves as a function of time for intravenously injected IT T101 and IT (A-1a) T101; Figure 8 shows dose / effect curves illustrating the cytotoxicity of IT (A-1a) T101 and (A-1a) against CEM cells measured by inhibition of protein synthesis, and Figs. Figure 9 shows curves illustrating the cytotoxic effect of the immunotoxins IT (A-1a) T101 and IT T101 on CEM cells, measured by the clonogenic sample.

25 De efterfølgende eksempler giver en bedre forståelse af opfin delsen uden at begrænse dens rammer.The following examples provide a better understanding of the invention without limiting its scope.

DK 166626 B1 13DK 166626 B1 13

Eksempel 1;Example 1;

Oxidation af den methylerede A-kæde, hvori SH-grupperne er blokeret med N-ethylmaleimid.Oxidation of the methylated A chain in which the SH groups are blocked with N-ethylmaleimide.

5 I - Fremstilling af ricins korrekt funktional iserede A-kæde 1) Hexamethylering af A-kæden5 I - Preparation of ricin's properly functionalized A-chain 1) Hexamethylation of the A-chain

10 Methyleringsreaktion udføres ved 0°C under omrøring i 0,2 MMethylation reaction is carried out at 0 ° C with stirring in 0.2 M

boratpuffer med pH 10 ved hjælp af fremgangsmåden ifølge Means og Feeney (Biochemistry 7, 2192 (1968)). 20 mg findelt borhy-drid (indeholdende 9,5 mCi/mmol) sættes til 35 ml A-kæde (3 mg/ml), efterfulgt af 350 mikroliter 6% formaldehyd (tilsat i 15 fem 70 mikroliter portioner fordelt over et tidsrum på 30 min.).pH 10 borate buffer by the method of Means and Feeney (Biochemistry 7, 2192 (1968)). 20 mg of finely divided boron anhydride (containing 9.5 mCi / mmol) is added to 35 ml of A chain (3 mg / ml), followed by 350 microliters of 6% formaldehyde (added in 15 to 70 microliters of portions over a period of 30 mine.).

Overskuddet af reagens fjernes ved hjælp af kontinuerlig dialyse mod 125 mM phosphatpuffer med pH 7 (40 liter ved 300 20 ml/h). Efter dialyse centrifugeres proteinopløsningen. 36,5 ml hexamethyleret A-kæde indeholdende 2,6 mg/ml opsamles.The excess reagent is removed by continuous dialysis against 125 mM phosphate buffer of pH 7 (40 liters at 300 20 ml / h). After dialysis, the protein solution is centrifuged. 36.5 ml of hexamethylated A chain containing 2.6 mg / ml are collected.

2) Blokering med N-ethylmaleimid 25 Den hexamethylerede A-kædes naturlige SH blokeres ved hjælp af metoden beskrevet i Methods in Enzymology 11, 541 (1967). For at gøre det, inkuberes ricins ricins A-kæde, som blev opnået i det foregående trin, i 2 timer ved 30°C i nærværelse af 20 ækvivalenter N-ethylmaleimid pr. mol A-kæde. Overskuddet af 30 reagens fjernes ved kontinuerlig dialyse mod 125 mM phosphat-puffer med pH 7, som i løbet af 20 timer fornys med en hastighed på 500 ml/ti me. Efter i nddampn i ng opnås 13 ml af en opløsning af ricins A-kæde indeholdende 7 mg/ml og ikke længere med thiolgrupper, som kan bestemmes ved hjælp af Ellman's reagens.2) N-ethylmaleimide blockage The natural SH chain hexamethylated A chain is blocked by the method described in Methods in Enzymology 11, 541 (1967). To do so, ricin ricin A chain obtained in the previous step is incubated for 2 hours at 30 ° C in the presence of 20 equivalents of N-ethylmaleimide per ml. mol A chain. The excess reagent is removed by continuous dialysis against 125 mM phosphate buffer of pH 7, which is renewed at a rate of 500 ml / h in 20 hours. After evaporation, 13 ml of a solution of castor A chain containing 7 mg / ml is obtained and no longer with thiol groups which can be determined by Ellman's reagent.

35 Det således opnåede produkt kaldes derefter hexamethyleret A-kæde (NEM).The product thus obtained is then called hexamethylated A chain (NEM).

DK 166626 B1 14 3) Periodatoxidation 6 ml af opløsningen af hexamethy1eret A-kæde (NEM), der er opnået som oven for anført, behandles med NaI04 (12,8 mg) i 40 5 min. i mørke ved pH 4,5 og ved 0°C. Omsætningen standses ved tilsætning af 600 mikroliter ethylenglycol, og reaktionsmediet dialyseres kontinuerligt mod 0,1 M carbonatpuffer med pH 10 (20 timer ved 500 ml/h).3) Periodate oxidation 6 ml of the hexamethylated A-chain (NEM) solution obtained as above is treated with NalO 4 (12.8 mg) for 40 min. in the dark at pH 4.5 and at 0 ° C. The reaction is quenched by the addition of 600 microliters of ethylene glycol and the reaction medium is continuously dialyzed against 0.1 M carbonate buffer of pH 10 (20 hours at 500 ml / h).

10 II - Enzymatisk virkning af A-kæden med forlænget virkning, målt på en acellular model10 II - Enzymatic action of the extended chain A chain, measured on an acellular model

Den fundamentale biologiske egenskab ved ricins A-kæde består i at inhibere proteinsyntesen i celler ved nedbrydning af den 15 ribosomale sub-enhed 60S.The fundamental biological property of ricin A chain consists in inhibiting protein synthesis in cells by degradation of the 15 ribosomal subunit 60S.

In vitro forskriften indebærer anvendelse af passende supplerede subcellulare fraktioner af rottelever, der er i stand til at inkorporere l^C-phenylalanin i nærværelse af en kunstig 20 messenger RNA: polyuridylsyre.The in vitro regulation involves the use of suitably supplemented subcellular fractions of rat liver capable of incorporating 1C-phenylalanine in the presence of an artificial 20 messenger RNA: polyuridyl acid.

Den til fremstilling af de subcellulare fraktioner og måling af inkorporeringen af l4C-phenylalanin benyttede procedure er en tilpasning af metoden beskrevet i Biochemica Biophysica Ac-25 ta 1973, 312, 608-615, under anvendelse af både en mikrosomal-fraktion og en cytosolfraktion af rottehepatocyter. Prøven indeholdende A-kæden indføres i form af en passende fortyndet opløsning i en 50 mM Tris HCl-puffer med pH 7,6 indeholdende 0,2% 2-mercaptoethanol og 15 mikrogram/ml kvægserumalbumin.The procedure used to prepare the subcellular fractions and to measure the incorporation of 14C-phenylalanine is an adaptation of the method described in Biochemica Biophysica Acts 1973, 312, 608-615, using both a microsomal fraction and a cytosol fraction of rat hepatocytes. The sample containing the A chain is introduced in the form of a suitably diluted solution in a 50 mM Tris HCl buffer of pH 7.6 containing 0.2% 2-mercaptoethanol and 15 micrograms / ml bovine serum albumin.

3030

Optæl!ingsdataene anvendes til i forhold til et kontrolmedium uden inhibitor at beregne den procentiske inhibering af inkorporeringen af l4C-phenylalanin i proteinerne for hvert reaktionsmedium indeholdende ricins A-kæde.The count data is used to calculate, relative to a control medium without inhibitor, the percent inhibition of the incorporation of 14C-phenylalanine into the proteins for each reaction medium containing the A chain of ricin.

Den inhiberende virkning blev bestemt. En ICsQ-værdi på 2,7 x 10“1° mol/liter iagttages for den oxiderede A-kæde. ICsg-vær- 35 DK 166626 B1 15 dien for den til kontrol benyttede A-kæde i forsøget er 1,03 x 10“1° mol/liter. Modifikationen forårsager derfor ikke et aktivitetstab for A-kæden.The inhibitory effect was determined. An IC 50 value of 2.7 x 10 “1 ° mol / liter is observed for the oxidized A chain. The ICsg value for the A-chain used for control in the experiment is 1.03 x 10 10 1 ° mol / liter. Therefore, the modification does not cause a loss of activity for the A chain.

5 Eksempel 2:Example 2:

Dette eksempel viser den langsomme eliminering af ricins A-kæ-de, der er modificeret med natriumperi odat, efter intravenøs injektion i dyret.This example illustrates the slow elimination of ricin A chain modified with sodium peri odat after intravenous injection into the animal.

10 I - Modifikation af ricins A-kæde med natriumperiodat10 I - Modification of ricin A chain with sodium periodate

1) Blokering af den naturlige SH-gruppe med DTNB1) Blocking the natural SH group with DTNB

15 Ricins A-kæde blev fremstillet og renset på den i US-patent-skrift nr. 4.340.535 beskrevne måde. 20 ækvivalenter af en opløsning af 2,2 '-dinitro-5,5'-dithiodibenzoesyre (DTNB), dvs.15 Ricin's A chain was prepared and purified in the manner described in U.S. Patent No. 4,340,535. 20 equivalents of a solution of 2,2'-dinitro-5,5'-dithiodibenzoic acid (DTNB), i.e.

385 mikroliter af en 0,1 M opløsning af DTNB i en 125 mM phosphatpuffer med pH 7 (denne opløsning indstilles til pH 7 20 med natriumhydroxid), sættes til 10 ml af en opløsning af ricins A-kæde indeholdende 5,6 mg/ml (med 0,84 thiolgruppe pr. A-kæde) i PBS-puffer (en puffer, der er 20 mM med hensyn til phosphat og 150 mM med hensyn til NaCl, med pH-værdi 7). Inkubation foretages i 20 min. ved 20°C. Derpå dialyseres 25 opløsningen mod PBS puffer ved 4°C til opnåelse af 53 mg A-kæde, der er blokeret på thiol-gruppen, i form af en opløsning indeholdende 5 mg/ml.385 microliters of a 0.1 M solution of DTNB in a 125 mM phosphate buffer of pH 7 (this solution is adjusted to pH 7 20 with sodium hydroxide) is added to 10 ml of a solution of ricin A-chain containing 5.6 mg / ml (with 0.84 thiol group per A chain) in PBS buffer (a buffer that is 20 mM for phosphate and 150 mM for NaCl, with pH 7). Incubation is done for 20 min. at 20 ° C. Then, the solution is dialyzed against PBS buffer at 4 ° C to obtain 53 mg of A chain blocked on the thiol group in the form of a solution containing 5 mg / ml.

2) Periodatoxidation af den blokerede A-kæde 30 120 mikroliter af en 0,5 M opløsning af natriumperiodat i vand sættes til 6 ml af en opløsning indeholdende 5 mg/ml blokeret A-kæde, og pH 6 indstilles med 1 M eddikesyre. Inkubation foretages i 16 timer ved 4°C i mørke. Oxidationsreaktionen 35 standses ved tilsætning af 620 mikroliter af en 1 M vandig opløsning af ethylenglycol. Efter inkubation i 15 min. ved 20°C, dialyseres reaktionsmediet ved 4°C mod PBS-puffer. Pe- DK 166626 B1 16 riodatoxidation frembringer et svagt præcipitat af protein, som fjernes ved centrifugering ved 10.000 x g i 30 min. Dette fører til 24 mg oxideret, blokeret A-kæde i en koncentration på 3,4 mg/ml.2) Periodic oxidation of the blocked A chain 30 120 microliters of a 0.5 M solution of sodium periodate in water are added to 6 ml of a solution containing 5 mg / ml blocked A chain and pH 6 is adjusted with 1 M acetic acid. Incubation is done for 16 hours at 4 ° C in the dark. The oxidation reaction 35 is quenched by the addition of 620 microliters of a 1 M aqueous solution of ethylene glycol. After incubation for 15 min. at 20 ° C, the reaction medium is dialyzed at 4 ° C against PBS buffer. Pe- DK 166626 B1 16 riodate oxidation produces a weak precipitate of protein which is removed by centrifugation at 10,000 x g for 30 min. This results in 24 mg of oxidized, blocked A chain at a concentration of 3.4 mg / ml.

5 3) Afblokering af thi olgrupperne.5 3) Unblocking thi ol groups.

2-mercaptoethanol tilsættes som reduktionsmiddel til en slut-koncentration på 1% til 6 ml oxideret, blokeret A-kæde inde-10 holdende 3,4 mg/ml i PBS-puffer. Inkubation foretages i 1 time ved 20°C. Opløsningen dialyseres derpå mod PBS-puffer ved 4°C. Dette fører til 19 mg oxideret A-kæde i en koncentration på 2,8 mg/ml.2-mercaptoethanol is added as reducing agent to a final concentration of 1% to 6 ml of oxidized blocked A chain containing 3.4 mg / ml in PBS buffer. Incubation is done for 1 hour at 20 ° C. The solution is then dialyzed against PBS buffer at 4 ° C. This leads to 19 mg of oxidized A chain at a concentration of 2.8 mg / ml.

15 Ved anvendelse af DTMB-teknikken (Methods in Enzymology, 1972, 25, 457 (Academic Press)), fastslås det, at den opnåede modificerede A-kæde har 0,70 fri thiolgruppe pr. mol. Den modificerede A-kædes molekylvægt er 30.000±3.000, bestemt ved hjælp af polyacrylamidgradientelectrophorese i nærværelse af 20 natriumdodecylsulfat.Using the DTMB technique (Methods in Enzymology, 1972, 25, 457 (Academic Press)), it is determined that the modified A chain obtained has 0.70 free thiol group per moth. The modified A-chain molecular weight is 30,000 ± 3,000, determined by polyacrylamide gradient electrophoresis in the presence of 20 sodium dodecyl sulfate.

Det tidligere opnåede A-kæde-præparat, hvori polysacchariden-hederne er blevet oxideret, blev undersøgt for dets enzymatiske virkninger til inhibering af proteinsyntese og for dets 25 pharmacokinetiske egenskaber.The previously obtained A chain preparation in which the polysaccharide moieties have been oxidized was investigated for its enzymatic effects for inhibiting protein synthesis and for its pharmacokinetic properties.

II - Enzymatisk virkning af A-kæde med forlænget virkning, målt på en acellular model 30 Den inhiberende virkning blev bestemt ved hjælp af den i eksempel 1 beskrevne teknik. En IC5Q“Værdi på 3 x 10“1° mol/liter iagttoges for den oxiderede A-kæde. ICsg-værdien for kontrol A-kæden ved forsøget er 1,2 x 10~1° mol/liter. Modifikationen forårsager derfor ikke et aktivitetstab for A-kæden.II - Enzymatic activity of extended chain A chain, measured on an acellular model 30 The inhibitory effect was determined by the technique described in Example 1. An IC5Q “value of 3 x 10” 1 ° mol / liter was observed for the oxidized A chain. The ICsg value for the control A chain in the experiment is 1.2 x 10 ~ 1 ° mol / liter. Therefore, the modification does not cause a loss of activity for the A chain.

35 DK 166626 B1 17 III - Farmakokinet i ske egenskaber hos A-kæden med forlænget virkning (A-La) A-kæden administreres til kaniner ved hjælp af en enkelt in-5 jektion i en ørevene. Mængden af injiceret A-kæde svarer til 0,415 mg/kg. Blodprøver udtages med mellemrum på heparin. Plasmaerne analyseres ved hjælp af en radioimmunometrisk prøve, der nedenfor betegnes ved forkortelsen RIM-1.35 DK 166626 B1 17 III - The Pharmacokin in Sparkling Properties of the Extended-Effect A (La-A) Chain The A-chain is administered to rabbits by a single injection into the ear veins. The amount of injected A-chain corresponds to 0.415 mg / kg. Blood samples are taken at intervals on heparin. The plasmas are analyzed by means of a radioimmunometric sample, designated below by the abbreviation RIM-1.

10 Denne teknik udmærker sig ved at bestemme A-kæden uden at modificere den. Bestemmelsen udføres i mikrotitreringsplader (f.eks.: "NUNC-TSP screening system" fra Poly Labo Block10 This technique is distinguished by determining the A chain without modifying it. The assay is performed in microtiter titre plates (eg: "NUNC-TSP screening system" from Poly Labo Block

France), hvis lågbærer hyperabsorberende spidser, der rager ned i bundens hulrum. Disse spidser udgør de faste faser.France), whose lid carries hyperabsorbent tips protruding into the bottom cavity. These tips form the solid phases.

15 Fåre-antistoffer, der inhiberer ricins A-kæde (betegnet i det følgende ved forkortelsen Acl), renset ved hjælp af affinitetskromatografi, absorberes på de faste faser. Til dette formål fordeles 200 mikroliter af en opløsning af Acl indholdende 10 mikrogram/ml i PBS-puffer i hulrummene. Spidserne 20 bringes først i kontakt med opløsningen af Acl i 24 timer ved 4°C og derpå med kalvefosterserum i 3 timer ved 20°C med henblik på mætning af alle fikseringsstederne. Den mættede immu-noabsorbent bringes derpå i 3 timer ved 20°C i kontakt med plasmaprøverne, som skal bestemmes i forskellige fortyndinger, 25 eller med opløsninger af A-kæde med kendte koncentrationer med henblik på at fastslå kalibreringskurven. Efter vaskning med en PBS-puffer bringes immunoabsorbenten i 2 timer ved 20eC i kontakt med fåre-antistof ferne, der inhiberer ricins A-kæde, som er blevet renset ved hjælp af affinitetskromatografi og 30 radiomærket (nedenfor betegnet ved forkortelsen Ac2). Radiomærkningen af Ac2 foretages med jod-125 i nærværelse af chloramin T ved metoden ifølge Greenwood og Hunter (Biochem J., 1963, 89, 114). Den specifikke aktivitet af de radiomærkede Ac2-antistoffer er 5 til 10 mikrocurie/mikrogram.Sheep antibodies that inhibit ricin A chain (designated herein by the abbreviation Acl), purified by affinity chromatography, are absorbed on the solid phases. For this purpose, 200 microliters of a solution of Acl containing 10 micrograms / ml is distributed in PBS buffer in the cavities. The tips 20 are first contacted with the solution of Acl for 24 hours at 4 ° C and then with fetal calf serum for 3 hours at 20 ° C for saturation of all the fixation sites. The saturated immunoabsorbent is then contacted for 3 hours at 20 ° C with the plasma samples to be determined in various dilutions, or with A-chain solutions of known concentrations to determine the calibration curve. After washing with a PBS buffer, the immunoabsorbent is contacted for 2 hours at 20 ° C with the sheep antibodies which inhibit ricin A chain which has been purified by affinity chromatography and radiolabelled (hereinafter abbreviated as Ac2). The radiolabelling of Ac2 is done with iodine-125 in the presence of chloramine T by the method of Greenwood and Hunter (Biochem J., 1963, 89, 114). The specific activity of the radiolabelled Ac2 antibodies is 5 to 10 microcurie / micrograms.

35 106 cpm radiomærkede Ac2-antistoffer indføres som 200 mikroli ter i en PBS-puffer indeholdende 0,1% kvægseruma1 bumin. Efter vaskning i PBS-puffer aftages spidserne, og mængden af bundet DK 166626 B1 1835 106 cpm of radiolabelled Ac2 antibodies are introduced as 200 microliters in a PBS buffer containing 0.1% bovine serum bumin. After washing in PBS buffer, the peaks decrease and the amount of bound DK 166626 B1 18

Ac2 roåles ved tælling af radioaktiviteten. Koncentrationen af A-kæde i prøverne, der skal bestemmes, måles ved henvisning til kalibreringskurven, der er frembragt ved indføring af A-kæden i forskellige kendte koncentrationer. Når A-kæden med 5 forlænget virkning injiceres i dyret, anvendes den samme A-kæde med forlænget virkning til at frembringe den tilsvarende kalibreringskurve.Ac2 is quenched by counting the radioactivity. The concentration of the A chain in the samples to be determined is measured by reference to the calibration curve produced by introducing the A chain at various known concentrations. When the A-extended-action A chain is injected into the animal, the same extended-action A-chain is used to produce the corresponding calibration curve.

Værdierne for koncentrationen af A-kæde i blodplasmaet, der 10 måles ved hjælp af denne teknik, er reproducerbare og pålidelige. Tærskelpåvisningen er 1 nanogram/ml. En undersøgelse af reproducerbarheden inden for og mellem forsøg fører til variationskoefficienter på mindre end 10% for koncentrationsværdier i intervallet fra 1 til 200 nanogram/ml.The values of the A chain concentration in the blood plasma measured by this technique are reproducible and reliable. The threshold detection is 1 nanogram / ml. A study of reproducibility within and between experiments leads to coefficients of variation of less than 10% for concentration values ranging from 1 to 200 nanograms / ml.

1515

Resultaterne af disse forsøg er vist i form af kurver, hvori tiden, udtryk i timer, er afsat efter abscissen, og plasmakoncentrationen af det målte produkt, angivet i procent af den teoretiske plasmakoncentration på tidspunktet 0, er afsat ef-20 ter en logaritmisk skala på ordinaten. Denne værdi, der kaldes den "relative plasmakoncentration" (RPC), beregnes ved anvendelse af følgende udtryk: RPC = koncentration målt ved tidspunktet t x jqq 25 injiceret mængde/plasmavolumenThe results of these experiments are shown in the form of curves in which the time, expression in hours, is plotted after the abscissa, and the plasma concentration of the measured product, expressed as a percentage of the theoretical plasma concentration at time 0, is plotted according to a logarithmic scale. on the ordinance. This value, called the "relative plasma concentration" (RPC), is calculated using the following terms: RPC = concentration measured at time t x yqq 25 injected amount / plasma volume

Plasmavolumenet betragtes som værende lig med 36 ml/kg af dyrets legemsvægt.The plasma volume is considered to be equal to 36 ml / kg of animal body weight.

30 Fig. 1 viser plasmaeliminationkurven, som funktion af tid, for naturligt ricins A-kæde, der er injiceret intravenøst. Denne kurve (kurve 1) har to faser. I den første fase forsvinder produktet meget hurtigt fra blodstrømmen, eftersom 0,1% af den injicerede dosis forbliver i plasmaet 3 timer efter injektion.FIG. Figure 1 shows the plasma elimination curve, as a function of time, for natural ricin A chain injected intravenously. This curve (curve 1) has two stages. In the first phase, the product disappears from the bloodstream very quickly, as 0.1% of the injected dose remains in the plasma 3 hours after injection.

35 I den anden fase er formindskelsen langsommere.35 In the second phase, the decrease is slower.

Når A-kæden er blevet oxideret i sine polysaccharidenheder, modificeres eliminationsprofilen dybtgående. Den første elimi- DK 166626 B1 19 nationsfase - som er ansvarlig for størstedelen af produktets forsvinden - undertrykkes praktisk talt, hvilket fører til en betydelig stigning i plasmamængderne af A-kæde. 20 timer efter injektion er koncentrationen af den oxiderede A-kæde 600 gange 5 større end i tilfældet af den umodificerede A-kæde (kurve 2).Once the A chain has been oxidized in its polysaccharide units, the elimination profile is profoundly modified. The first elimination phase, which is responsible for most of the disappearance of the product, is virtually suppressed, leading to a significant increase in plasma levels of A chain. 20 hours after injection, the concentration of the oxidized A chain is 600 times 5 greater than in the case of the unmodified A chain (curve 2).

Eksempel 3Example 3

Dette eksempel viser virkningen af periodatoxidation på den 10 med NEM blokerede A-kædes farmakokinet i ske egenskaber.This example demonstrates the effect of periodate oxidation on the 10 NEM-blocked A-chain pharmacokinetic properties.

1) Modifikation af ricins A-kæde a) blokering af den naturlige SH med N-ethyImaleimid 15 40 ml af en vandig opløsning af ricins A-kæde indeholdende 8 mg/ml (dvs. 4,1 mikromol A-kæde) behandles med en vandig opløsning af 2-mercaptoethanol, så at slutkoncentrationen er 1%.1) Modification of ricin A chain a) Blocking the natural SH with N-ethyl imaleimide 40 ml of an aqueous solution of ricin A chain containing 8 mg / ml (ie 4.1 micromoles A chain) is treated with a aqueous solution of 2-mercaptoethanol so that the final concentration is 1%.

2020

Opløsningen lades henstå i 1 time og dialyseres derpå kontinuerligt mod 125 mM phosphatpuf fer ved pH 7, der fornys i løbet af 40 timer med en hastighed på 300 ml/time. Ved anvendelse af Ellman's metode blev 0,9 ækvivalent SH bestemt pr.The solution is allowed to stand for 1 hour and then dialyzed continuously against 125 mM phosphate buffer at pH 7, which is renewed over 40 hours at a rate of 300 ml / hour. Using Ellman's method, 0.9 equivalent SH was determined per

25 mol af ricins A-kæde.25 moles of castor A chain.

Denne SH-gruppe blokeres med N-ethylmaleimid ved hjælp af metoden beskrevet i Methods in Enzymology, 11, 541 (1967). For at gøre dette inkuberes ricins A-kæde, som blev opnået i det 30 foregående trin, i 2 timer ved 30°C i nærværelse af 20 ækvivalenter N-ethylmaleimid pr. mol A-kæde. Overskudet af reagens fjernes ved hjælp af kontinuerlig dialyse mod 125 mM phosphatpuf fer med pH 7, som fornyes i løbet af 20 timer med en hastighed på 500 ml/time. Dette fører til 35 ml af en opløsning 35 af ricins A-kæde indeholdende 7 mg/ml, og den indeholder ikke længere thiolgrupper, der kan bestemmes ved hjælp af Ellman's reagens. Det således opnåede produkt kaldes i det følgende A-kæde (NEM).This SH group is blocked with N-ethylmaleimide by the method described in Methods in Enzymology, 11, 541 (1967). To do this, the A chain of ricin obtained in the previous 30 step is incubated for 2 hours at 30 ° C in the presence of 20 equivalents of N-ethylmaleimide per ml. mol A chain. The excess reagent is removed by continuous dialysis against 125 mM phosphate buffer of pH 7, which is renewed over 20 hours at a rate of 500 ml / hour. This leads to 35 ml of a solution 35 of ricin A chain containing 7 mg / ml and it no longer contains thiol groups which can be determined by Ellman's reagent. The product thus obtained is hereinafter referred to as A-chain (NEM).

DK 166626 B1 20 b) Periodatoxidation af A-kæden (NEM)DK 166626 B1 20 b) Periodic oxidation of the A chain (NEM)

Periodatoxidation af A-kæden (NEM) udføres ved anvendelse af den i eksempel 2 angivne procedure.Periodic oxidation of the A chain (NEM) is carried out using the procedure set forth in Example 2.

5 2) Den oxiderede A-kædes (NEM) egenskaber a) Den oxiderede A-kædes (NEM) enzymatiske aktivitet 10 Den inhiberende virkning på proteinsyntesen blev bestemt ved anvendelse af proceduren beskrevet i eksempel 1. De enzymatiske egenskaber viser sig at blive bevaret med en lC5o~værdi på 4,3 x 10"10 mol/1 for den oxiderede A-kæde (NEM).2) Properties of the oxidized A-chain (NEM) a) Enzymatic activity of the oxidized A-chain (NEM) 10 The inhibitory effect on protein synthesis was determined using the procedure described in Example 1. The enzymatic properties are found to be conserved with an IC 50 value of 4.3 x 10 "10 mol / l for the oxidized A chain (NEM).

15 b) Den oxiderede A-kædes (NEM) farmakokinetiske egenskaberB) The pharmacokinetic properties of the oxidized A chain (NEM)

Den oxiderede eller ikke-oxiderede A-kæde (NEM) administreres til kaniner i en enkelt injektion i ørevenen. Den injicerede mængde A-kæde svarer til 0,100 mg/kg. Plasmaprøverne opsamlet 20 på tidspunktet 23 h analyseres under anvendelse af den i eksempel 2 beskrevne immunometriske prøve RIM-1. Resultaterne er vist i den følgende tabel: 25 Plasmakoncentration 23 timer efter in jektion: A-kæde (NEM) 0,01%The oxidized or non-oxidized A chain (NEM) is administered to rabbits in a single injection into the ear vein. The amount of A-chain injected corresponds to 0,100 mg / kg. The plasma samples collected 20 at time 23 hours are analyzed using the immunometric test RIM-1 described in Example 2. The results are shown in the following table: 25 Plasma concentration 23 hours after injection: A-chain (NEM) 0.01%

Oxideret A-kæde (NEM) 6% 30 _____ 23 timer efter injektion er koncentrationen af den oxiderede A-kæde (NEM) 800 x større end i tilfælde af den umodificerede A-kæde (NEM).Oxidized A-chain (NEM) 6% 30 _____ 23 hours after injection, the concentration of oxidized A-chain (NEM) is 800 x greater than in the case of the unmodified A-chain (NEM).

35 DK 166626 B1 2135 DK 166626 B1 21

Eksempel 4Example 4

Dette eksempel viser vigtigheden af varigheden af den oxidative behandling for den oxiderede A-kædes farmakokinetiske egen-5 skaber.This example demonstrates the importance of the duration of the oxidative treatment for the pharmacokinetic properties of the oxidized A chain.

6 præparater af oxideret A-kæde fremstilles under anvendelse af den i eksempel 2 angivne procedure, med undtagelse af varigheden af natriumperiodatbehandlingen. Behandlingstiderne er 10 som følger: nul (reaktion standset øjeblikkeligt med ethylen-glykol), 20 minutter, 40 minutter, 2,5 timer, 4 timer og 18 timer.Six oxidized A-chain preparations are prepared using the procedure of Example 2, except for the duration of the sodium periodate treatment. The treatment times are 10 as follows: zero (reaction stopped immediately with ethylene glycol), 20 minutes, 40 minutes, 2.5 hours, 4 hours and 18 hours.

Disse forskellige præparater injiceres i kaniner, og de rela-15 tive plasmakoncentrationer af A-kæden måles efter 24 timer ved hjælp af den samme procedure som den i eksempel 1 benyttede.These various preparations are injected into rabbits and the relative plasma concentrations of the A chain are measured after 24 hours by the same procedure as used in Example 1.

Resultaterne er vist i fig. 2. Disse resultater viser, at 1) stigningen i plasmamængden af A-kæden i virkeligheden skyldes 20 periodatoxidation, fordi plasmakoncentrationen af A-kæde, når reaktionen standses øjeblikkeligt, er identisk med den for den naturlige A-kæde opnåede, og 2) det er nødvendigt for denne reaktions varighed at være relativ lang med henblik på opnåelse af optimale virkninger.The results are shown in FIG. 2. These results show that 1) the increase in plasma amount of the A chain is in fact due to 20 periodate oxidation because the plasma concentration of A chain, when the reaction is stopped immediately, is identical to that of the natural A chain obtained, and 2) it is necessary for the duration of this reaction to be relatively long in order to obtain optimal effects.

2525

Eksempel 5Example 5

Dette eksempel viser vigtigheden af varigheden af den oxidative behandling på den med NEM blokerede methylerede A-kædes 30 farmakokinetiske egenskaber.This example demonstrates the importance of the duration of the oxidative treatment on the pharmacokinetic properties of the NEM blocked methylated A chain.

1) Fremstilling af ricins funktionaliserede A-kæde a) Blokering af A-kædens naturlige SH med N-ethylmaleimid 35 DK 166626 B1 22 A-kædens naturlige SH blokeres med N-ethylmaleimid ved hjælp af denne samme procedure som den i eksempel 1 beskrevne.1) Preparation of ricin's functionalized A chain a) Blocking the natural chain of the A chain with N-ethylmaleimide 35 Blocking the natural SH of the A chain with N-ethylmaleimide by this same procedure as that described in Example 1.

b) Methylering af A-kæden 5b) Methylation of the A chain 5

Methyleringsreaktionen udføres ved 0eC under omrøring i 0,2 M boratpuffer ved pH 10 ved hjælp af metoden ifølge Means og Feeney (Biochemistry 7, 2192, 1968). 38 mg findelt borhydrid (indeholdende 47 mCi/mmol) sættes til 65,5 ml A-kæde (NEM) (3 10 mg/ml) efterfulgt af 1 ml 6% formaldehyd, som tilsættes i fem 200 mikroliterportioner fordelt over et tidsrum på 30 minutter.The methylation reaction is carried out at 0 ° C with stirring in 0.2 M borate buffer at pH 10 by the method of Means and Feeney (Biochemistry 7, 2192, 1968). 38 mg of finely divided borohydride (containing 47 mCi / mmol) is added to 65.5 ml of A-chain (NEM) (3 10 mg / ml) followed by 1 ml of 6% formaldehyde which is added in five 200 microliters aliquots over a period of 30 minutes. minutes.

Overskudet af reagens fjernes ved hjælp af kontinuerlig dialy-15 se mod 125 mM phsophatpuffer med pH 7 (40 ml). Efter dialyse centrifugeres proteinopløsningen. 63 ml methyleret A-kæde indeholdende 3 mg/ml opsamles.The excess reagent is removed by continuous dialysis against 125 mM pH 7 phsophate buffer (40 ml). After dialysis, the protein solution is centrifuged. 63 ml of methylated A chain containing 3 mg / ml are collected.

C) Periodatoxidation 20C) Periodic oxidation 20

Seks præparater af methyleret A-kæde (NEM) oxideres under anvendelse af proceduren beskrevet i eksempel 1, med undtagelse af varigheden af natriumperiodatbehandli ngen. Behandlingstiderne er som følger: nul (reaktions standset øjeblikkeligt med 25 ethylenglykol), 10 minutter, 40 minutter, 2,5 time, 4 timer og 18 timer.Six methylated A-chain (NEM) preparations are oxidized using the procedure described in Example 1, except for the duration of the sodium periodate treatment. The treatment times are as follows: zero (reaction stopped immediately with 25 ethylene glycol), 10 minutes, 40 minutes, 2.5 hours, 4 hours and 18 hours.

Disse forskellig præparater injiceres i kaniner, og de relative plasmakoncentrationer af A-kæden måles efter 23 timer ved 30 hjælp af den samme procedure som den i eksempel 2 anførte.These various preparations are injected into rabbits and the relative plasma concentrations of the A chain are measured after 23 hours by the same procedure as in Example 2.

Resultaterne er vist i fig. 3, kurve 2. Disse resultater viser, at for A-kæden (kurve 1): 35 1. skyldes stigningen i plasmamængden af den methylerede A- kæde (NEM) i virkeligheden periodatoxidation, fordi plasmakoncentrationen af methyleret A-kæde (NEM), når reaktionen standses øjeblikkeligt, er identisk med den for A-kæden opnåede; og DK 166626 B1 23 2. er det nødvendigt, at varigheden af denne reaktion er relativt lang med henblik på opnåelse af optimale virkninger.The results are shown in FIG. 3, curve 2. These results show that for the A chain (curve 1): 35 1. the increase in the plasma amount of the methylated A chain (NEM) is actually due to periodate oxidation, because the plasma concentration of methylated A chain (NEM), when the reaction is stopped immediately is identical to that obtained for the A chain; and DK 166626 B1 23 2. it is necessary that the duration of this reaction be relatively long in order to obtain optimal effects.

Eksempel 6 5Example 6 5

Dette eksempel viser, at når oxidationsreaktionen udføres separat med de to indgående molekylære varianter i A-kæden (Alkæde og A2-kæde) frembringer oxidationsreaktionen virkninger på hver af de to isomerer, der er analoge med virkningerne 10 beskrevet i eksempel 2 for ricins A-kæde.This example shows that when the oxidation reaction is carried out separately with the two input molecular variants of the A chain (Al chain and A 2 chain) the oxidation reaction produces effects on each of the two isomers analogous to the effects described in Example 2 for ricins A chain.

1) Separation af Al-kæden og A2-kæden 28 ml A-kæde indeholdende 10,9 mg/ml) (309 mg) i 125 ml phosp-15 hatpuffer med pH 7,0 påføres på en søjle af 112 ml konkavalin A/sepharose, der er ækvilibreret i den samme puffer. Al-kæden opnås i den første top ved vaskning med den samme puffer. A2-kæden eluerers med 0,1 M boratpuffer med pH 6,0, som er 0,5 M med hensyn til NaCl og 0,1 M med hensyn til α-methyImanno-2 0 sid.1) Separation of the Al chain and the A2 chain 28 ml of A chain containing 10.9 mg / ml) (309 mg) in 125 ml phosp-15 pH buffer buffer pH 7.0 is applied to a column of 112 ml concavalin A / sepharose equilibrated in the same buffer. The Al chain is obtained in the first peak by washing with the same buffer. The A2 chain is eluted with 0.1 M borate buffer of pH 6.0, which is 0.5 M with respect to NaCl and 0.1 M with respect to α-methylmanno-20 p.

Dette fører til 184 mg Al-kæde og 103 mg A2-kæde.This leads to 184 mg of Al chain and 103 mg of A2 chain.

Al-kæden og A2-kæden koncentreres ved hjalp af ultrafiltrering 25 under nitrogentryk. A2-kæden dialyseres mod 125 mM phosphat-puffer med pH 7,0.The Al chain and A2 chain are concentrated by means of ultrafiltration 25 under nitrogen pressure. The A2 chain is dialyzed against 125 mM phosphate buffer of pH 7.0.

Analyse af A-kæden ved hjælp af akrylamidgelgradientelektrop-horese med SDS viser tilstedeværelse af 2 bånd med forskellig 30 intensitet svarende til molekylvægte på 30.000 og 33.000. Al-kæden svarer til båndet med kraftigere intensitet og med molekylvægt 30.000, og A2-kæden svarer til båndet med svag intensitet og med molekylvægt 33.000.Analysis of the A chain by acrylamide gel gradient electrophoresis with SDS shows the presence of 2 bands of different intensity, corresponding to molecular weights of 30,000 and 33,000. The Al chain corresponds to the band of stronger intensity and molecular weight 30,000, and the A2 chain corresponds to the band of low intensity and molecular weight 33,000.

35 DK 166626 B1 24 2) Modifikation af ricins A-kæde og A2-kæde med natriumperiodat35 DK 166626 B1 24 2) Modification of ricin's A chain and A2 chain with sodium periodate

Denne modi f i kation foretages som beskrevet i eksempel 2. Præ-5 paraterne af A-kæde, hvori polysaccharidenhederne er blevet oxideret, blev undersøgt for deres enzymatiske aktiviteter til inhibering af proteinsyntese og for deres pharmakokinetiske egenskaber.This modification is made as described in Example 2. The A-chain preparations in which the polysaccharide units have been oxidized were tested for their enzymatic activities to inhibit protein synthesis and for their pharmacokinetic properties.

10 3) Enzymatiske virkninger af Al- og A2-kæder med forlænget virkning, målt på en acellular model3) Enzymatic effects of Al and A2 extended-chain chains, measured on an acellular model

Den inhiberende aktivitet blev bestemt som beskrevet i eksempel 1. Den iagttagne IC5Q-værdi er lig med 2,1 x 10"10 mol/1 15 og 2,1 x 10"1° mol/1 for henholdsvis den oxiderede Al-kæde og den oxiderede A2-kæde. ICsg-værdierne for de naturlige Al- og A2-kæder, som er kontrolkæderne ved forsøget er henholdsvis 1,9 x 10“1° mol/1 og 1 x 10~1° mol/1. Modifikationen af de separate varianter af A-kæden forårsager derfor ikke et tab af 20 deres enzymatiske aktivitet.The inhibitory activity was determined as described in Example 1. The observed IC5Q value equals 2.1 x 10 10 10 mol / l 15 and 2.1 x 10 "1 ° mol / l for the oxidized Al chain and the oxidized A2 chain. The ICsg values for the natural Al and A2 chains which are the control chains in the experiment are 1.9 x 10 “1 ° mol / 1 and 1 x 10 ~1 ° mol / 1 respectively. Therefore, the modification of the separate variants of the A chain does not cause a loss of their enzymatic activity.

4) Pharmakokinetiske egenskaber hos Al- og A2-kæderne (Al-La, A2-La) med forlænget virkning.4) Pharmacokinetic properties of the Al and A2 chains (Al-La, A2-La) with extended action.

25 Al- eller Al-La- kæden eller A2- eller A2-La-kæden administreres til kaniner ved hjælp en enkelt injektion i en ørevene (415 mikrogram A-kæde/kg). PIasmaoprøverne opsamlet efter 20 timer analyseres ved hjælp af den immunometri ske prøve RIM-1 (se eksempel 2). Resultat er vist i nedenstående tabel. Værdi-30 erne for A- og A-La-kæderne er angivet til sammenligning.The Al or Al-La chain or A2 or A2-La chain is administered to rabbits by a single injection into an ear vein (415 micrograms A chain / kg). The PIASMA samples collected after 20 hours are analyzed by the immunometric sample RIM-1 (see Example 2). Results are shown in the table below. The values for the A and A-La chains are given for comparison.

35 DK 166626 B1 2535 DK 166626 B1 25

Relativ plasmakoncentration 20 timer efter injektion A-kæde 0,012% 5Relative plasma concentration 20 hours after injection A chain 0.012% 5

Oxideret A-kæde (A-La) 10%Oxidized A-chain (A-La) 10%

Al-kæde 0,02%Al chain 0.02%

Oxideret Al-kæde (Al-La) 10% 10 A2-kæde 0,04%Oxidized Al chain (Al-La) 10% 10 A2 chain 0.04%

Oksideret A2-kæde (A2-La) 14% 20 timer efter injektion er koncentrationerne af Al-La og A2-La henholdsvis 500 og 350 gange større end i tilfælde af Al 15 og A2.Oxidized A2 chain (A2-La) 14% 20 hours after injection, the concentrations of Al-La and A2-La are 500 and 350 times higher, respectively, than in the case of Al 15 and A2.

Eksempel 7;Example 7;

Dette eksempel beskriver de biokemiske egenskaber hos A-kæden 20 og dens varianter, Al-kæden og A2-kæden, i den naturlige form og i den oxidererede form.This example describes the biochemical properties of the A chain 20 and its variants, the Al chain and the A 2 chain, in the natural form and in the oxidized form.

De til disse undersøgelser benyttede A-kæder fremstilles som beskrevet i eksemplerne 2 og 6.The A chains used for these studies are prepared as described in Examples 2 and 6.

25 I - Kul hydratsammensatn i nger25 I - Cool hydrate composition in batches

Disse proteiners kulhydratsammensætninger bestemmes ved hjælp af gaskromatografiske analyser under anvendelse af Clamp's me- 30 tode (in Glycoproteins: their composition, structure and function (udgivet af A. Gottschalk), bind 5A, side 300-321, Elsevier Publishing Co., Amsterdam, London, New York).The carbohydrate compositions of these proteins are determined by gas chromatographic analysis using Clamp's method (in Glycoproteins: their composition, structure and function (published by A. Gottschalk), Vol. 5A, pages 300-321, Elsevier Publishing Co., Amsterdam, London, New York).

De opnåede resultater er samlet i de to nedenstående tabeller.The results obtained are summarized in the two tables below.

35 DK 166626 B1 2635 DK 166626 B1 26

Procentetisk sammensætning Kæder Totale kulhydrater, % 5Percentage composition Chains Total carbohydrates,% 5

Naturligt A 5,58 + eller - 0,5Natural A 5.58 + or - 0.5

Al 4,54 + eller - 0,5 A2 6,24 + eller - 0,5 2q Oxideret A 2,27+eller-0,5Al 4.54 + or - 0.5 A2 6.24 + or - 0.5 2q Oxidized A 2.27 + or-0.5

Al 2,07 + eller - 0,5 A2 3,33 + eller - 0,5 15 Molær sammensætning (På basis af en molekylvægt på 30.625, anføres resultaterne med gennemsnitlig nøjagtighed på + eller - 0.5 gruppe pr. molekyle) 20 kæderAl 2.07 + or - 0.5 A2 3.33 + or - 0.5 Molecular Composition (Based on a molecular weight of 30,625, the results are given with average accuracy of + or - 0.5 group per molecule) 20 chains

Monosaccharidermonosaccharides

Naturlige Oxideret 2 5 A Al A2 A Al A2 N-Acetylglucosamin 1,89 1,48 2,15 1,74 1,50 2,37Natural Oxidized 2.5 A Al A2 A Al A2 N-Acetylglucosamine 1.89 1.48 2.15 1.74 1.50 2.37

Mannose 4,6 3,40 5,2 1,43 1,29 2,26 30 Fucose 1,37. 1,41 1,52 0 0 0Mannose 4.6 3.40 5.2 1.43 1.29 2.26 Fucose 1.37. 1.41 1.52 0 0 0

Xylose_11.6 1,481 1,671 0,36 0,48 0.62Xylose_11.6 1.481 1.671 0.36 0.48 0.62

Resultaterne dokumenterer, at periodat oxidation har nedbrudt en del af A-kædens sukkerarter. Pr. molekyle A-kæde er der en 3 5 gennemsnitlig formindskelse på 3,17, 2,11 og 2,94 mannose grupper, fucosegrupperne er fuldstændigt forsvundet, og der er en gennemsnitlig formindskelse på 1,24,1 og 1,05 xylosegrup- DK 166626 B1 27 per for henholdsvis A-, Al- og A2-kæderne. N-acetylglucosamin-grupperne er kun svagt nedbrudte.The results show that periodic oxidation has broken down part of the A-chain sugars. Pr. molecule A chain there is a mean decrease of 3.17, 2.11 and 2.94 mannose groups, the fucose groups have completely disappeared and there is an average decrease of 1.24.1 and 1.05 xylose groups. 166626 B1 27 per for the A, A1 and A2 chains respectively. The N-acetylglucosamine groups are only slightly degraded.

II - N-Terminal sekvens 5II - N-Terminal Sequence 5

Sekvensen af de N-terminale aminosyrer i A-k®den og dens Alog A2-varianter i den naturlige form og i den oxiderede form blev konstateret med en protein-sequencer ved hjælp af de procedurer, der kendes af fagmanden på området. De opnåede resul-10 tater er samlet i nedenstående tabel.The sequence of the N-terminal amino acids of the A-kd and its Alog A2 variants in the natural form and in the oxidized form was ascertained with a protein sequencer by the procedures known to those skilled in the art. The results obtained are summarized in the table below.

Kæder Sekvens af de 9 N-terminale aminosyrerChains Sequence of the 9 N-terminal amino acids

Naturligt A Ile-Phe-Pro-Lys-Gln-Tyr-Pro-Ile-Ile 15Natural A Ile-Phe-Pro-Lys-Gln-Tyr-Pro-Ile-Ile 15

Al Ile-Phe-Pro-Lys-Gln-Tyr-Pro-Ile-Ile A2 Ile-Phe-Pro-Lys-Gln-Tyr-Pro-Ile-IleAl Ile-Phe-Pro-Lys-Gln-Tyr-Pro-Ile-Ile A2 Ile-Phe-Pro-Lys-Gln-Tyr-Pro-Ile-Ile

Oxideret A Ile-Phe-Pro-Lys-Gln-Tyr-Pro-Ile-Ile 20Oxidized A Ile-Phe-Pro-Lys-Gln-Tyr-Pro-Ile-Ile 20

Al Ile-Phe-Pro-Lys-Gln-Tyr-Pro-Ile-Ile A2 Ile-Phe-Pro-Lys-Gln-Tyr-Pro-Ile-IleAl Ile-Phe-Pro-Lys-Gln-Tyr-Pro-Ile-Ile A2 Ile-Phe-Pro-Lys-Gln-Tyr-Pro-Ile-Ile

Det viser sig, at sekvensen af de 9 N-terminale aminoasyrer i 25 de naturlige og oxiderede A-, Al- og A2-kæder er fuldstændig identiske med hinanden, hvilket viser, at den oxidative behandling efterlader proteinkæden intakt. Det viser sig også, at sekvensen af A-kædernes 9 N-terminale aminosyrer er fuldstændig identisk med den tidligere af Funatsu beskrevne sek-30 vens for ricins A-kæde (Agric. Biol. Chem., (1979), 43, 2221).It turns out that the sequence of the 9 N-terminal amino acids in the natural and oxidized A, Al and A2 chains are completely identical to each other, which shows that the oxidative treatment leaves the protein chain intact. It is also found that the sequence of the A-chain N-terminal amino acids of the A chains is completely identical to the sequence previously described by Funatsu for the A chain of ricin (Agric. Biol. Chem., (1979), 43, 2221). .

III - Affinitet på concanavalin A/sepharose A-kæden, den med DTNB blokerede A-kæde [A(DTNB)J og kæderne 35 A(DTNB)-La, Al(DTNB), Al (DTNB)-La ,A2(DTNB) og A2(DTN8)-La afprøves for deres kapacitet til at fiksere til concanavalin A/sepharose. 1 ml af en opløsning af A-kæde indeholdende ca. 1 DK 166626 B1 28 mg/ml påføres på en søjle af 1 ml concanavali η A. Kromatografi efterfølges af måling af den optiske tæthed ved 280 nm. Efter vaskning med 125 mM phosphatbuffer med pH 7,0, indtil den første top, som ikke er tilbageholdt af concanavalin A, er vendt 5 tilbage til grundlinien, vaskes søjlen med 0,1 M boratbuffer med pH 6,0, som er 0,5 M med hensyn til NaCl og 0.1 M med hensyn til alfa-methylmannosid. Resultaterne, udtrykt som en procentdel af den optiske tæthed ved 280 nm, er sammenfattet i nedenstående tabel.III - Affinity on concanavalin A / Sepharose A chain, DTNB blocked A chain [A (DTNB) J and chains 35 A (DTNB) -La, Al (DTNB), Al (DTNB) -La, A2 (DTNB) ) and A2 (DTN8) -La are tested for their ability to fix to concanavalin A / Sepharose. 1 ml of a A-chain solution containing approx. 28 mg / ml is applied to a column of 1 ml concanavali η A. Chromatography is followed by measurement of the optical density at 280 nm. After washing with 125 mM phosphate buffer of pH 7.0 until the first peak not retained by concanavalin A is returned to baseline, the column is washed with 0.1 M borate buffer of pH 6.0 which is 0. 5 M for NaCl and 0.1 M for alpha-methylmannoside. The results, expressed as a percentage of the optical density at 280 nm, are summarized in the table below.

10 kæder % ikke tilbageholdt % elueret ved hjælp af Ialt __af con A__alpha-methvlmannosid__% A 53 27 80 15----10 chains% not retained% eluted using Total __ of con A__alpha methyl mannoside __% A 53 27 80 15 ----

Al(DTNB) 66 11 77Al (DTNB) 66 11 77

Al(DTNB)-La 78 5 83 A2(DTNB) 80 6 86 A2(DTNB)-La 73 0.4 73.4 20____ A2(DTNB) 25 70 95 A2(DTNB)-La 64 9 73 25 Det er kendt, at concanavalin A har en affinitet til glycop-roteiner med terminale mannoser. Det viser sig, at A-kæden, som indeholder sådanne grupper, kan binde.til con A. Dette er særlig tydeligt i tilfælde A2-kæden, som er den mere rigt med mannose substituerede isomer. Det viser sig også, at den oxi- 30 dative behandling nedbryder denne affinitet, som hænger sammen med nedbrydningen af sukkergrupperne ved en behandling af denne type.Al (DTNB) -La 78 5 83 A2 (DTNB) 80 6 86 A2 (DTNB) -La 73 0.4 73.4 20____ A2 (DTNB) 25 70 95 A2 (DTNB) -La 64 9 73 25 It is known that concanavalin A has an affinity for glycop-proteins with terminal mannoses. It turns out that the A chain containing such groups can bind to con A. This is particularly evident in the case of the A2 chain, which is the more abundant with mannose substituted isomers. It is also found that the oxidative treatment degrades this affinity, which is related to the degradation of the sugar moieties by a treatment of this type.

IV - Bestemmelse af E 1 promille.IV - Determination of E 1 per milliliter.

Absorptionskoefficienten ved 280 nm ((El promille) er den optiske tæthed ved 280 nm af en opløsning indeholdende 1 mg/ml, 35 DK 166626 B1 29 hvori proteinkoncentrationen bestemmes ved hjælp af FOLlN-prø-ven med et standardinterval af kvægserumalbumin.The absorption coefficient at 280 nm ((E1 promille) is the optical density at 280 nm of a solution containing 1 mg / ml, wherein the protein concentration is determined by the FOL1N sample with a standard range of bovine serum albumin.

Resultaterne er sammenfattet i følgende tabel: 5 A kæde 0,65 A(DTNB) kæde 1,12 10 A(DTNB)-La kæde 1,04The results are summarized in the following table: 5 A chain 0.65 A (DTNB) chain 1.12 10 A (DTNB) -La chain 1.04

Al(DTNB) kæde 1,07Al (DTNB) chain 1.07

Al(DTNB)-La kæde 1,02 15 A2(DTNB) kæde 0,95 A2(DTNB) -La kæde 0,95Al (DTNB) -La chain 1.02 A2 (DTNB) chain 0.95 A2 (DTNB) -La chain 0.95

Blokering af kædens thiolgruppe med DTNB frembringer en betydelig stigning i absorptionen ved 280 nm, som følge af indfø-20 ringen af nitrobenzoylgruppen.Blocking of the thiol group of the chain with DTNB produces a significant increase in absorption at 280 nm due to the introduction of the nitrobenzoyl group.

Efter oxidation iagtages ingen signifikant ændring af absorptionen ved 280 nm, hvilket viser, at oxidation ikke har påvirket aminosyrer, der er ansvarlige for absorptionen ved 280 25 nm* V - Isoelektrisk fokuseringAfter oxidation, no significant change in absorption is observed at 280 nm, which shows that oxidation has not affected amino acids responsible for absorption at 280 25 nm * V - Isoelectric focusing

Analyse af A-kæden ved hjælp af isoelektrisk fokusering frem-30 bringer et sæt bånd med isoelektriske punkter (pi), som ligger mellem 7.5 og 8.0 og er identiske for A-, Al- og A2-kæderne.Analysis of the A chain by isoelectric focusing produces a set of bands with isoelectric points (pi) that are between 7.5 and 8.0 and are identical for the A, Al and A2 chains.

Blokering af A-kædenå cystein med DTNB forårsager en udvidelse af båndene mod det sure område. Frigørelse af cysteinet med 35 mercaptoethanol fører disse bånd tilbage til den naturlige A-kædes placering.Blocking of the A-chain cysteine with DTNB causes an expansion of the bands toward the acidic region. Releasing the cysteine with 35 mercaptoethanol returns these bands to the location of the natural A chain.

OISLAND

DK 166626 B1 30DK 166626 B1 30

En sammenligning mellem de i soelektri ske punkter får de naturlige og oxiderede A-, Al- og Al-kæder viser, at ved fravær af et blokerende middel overføres alle de bånd, der er karakteristiske for A-kæden, med 0,5 pH-enhed mod sure pH-værdier.A comparison of the solar electric points with the natural and oxidized A, Al and Al chains shows that in the absence of a blocking agent, all the bands characteristic of the A chain are transferred with 0.5 pH. unit against acidic pH values.

5 Denne overførsel finder sted uden overlapning mellem de naturlige og oxiderede A-kæders pH-områder, hvilket synes at angive, at alle A-kædemolekylerne påvirkes af oxidation.5 This transfer takes place without overlap between the pH ranges of the natural and oxidized A-chains, which seems to indicate that all A-chain molecules are affected by oxidation.

Eksempel 8 10Example 8 10

Dette eksempel viser 1) den hurtige eliminering af naturligt gelonin, og 2) den langsomme eliminering af gelonin, der er modificeret med natriumperi odat, efter intravenøs injektion i dyret.This example shows 1) the rapid elimination of natural gelonin, and 2) the slow elimination of gelonin modified with sodium periodate after intravenous injection into the animal.

15 I - Modifikation af gelonin med natriumperi odatI - Modification of gelonin with sodium periodate

Geloninet blev fremstillet og renset udfra Gelonium multiflo-rum ved hjælp af den metode, som er blevet beskrevet (J. Biol.The gelonin was prepared and purified from Gelonium multiflo space by the method described (J. Biol.

20 Chem. (1980) 255, 6947-6953). Oxidationsreaktionen udføres under de samme betingelser som de, der i eksempel 2 er beskrevet for ricins A-kæde, med undtagelse af, at det trin, i hvilket thiolgrupperne blokeres med DTNB, udelades.Chem. (1980) 255, 6947-6953). The oxidation reaction is carried out under the same conditions as those described in Example 2 for the A chain of ricin, except that the step in which the thiol groups are blocked with DTNB is omitted.

25 Eftersom koblingen af gelonin med antistoffet generelt ikke foretages under anvendelse af gel oni nets naturlige thiol grupper, vil thiolgrupperne i virkeligheden blive indført kunstigt efter oxidationstrinnet ved hjælp af den teknik, der er beskrevet i Cancer Res., 1984, 44, 129-133. 21 mikroliter af en 30 o,5 M opløsning af natriumperiodat i vand sættes til 1 ml af en opløsning indeholdende 3 mg/ml gelonin i PBS-puffer, der er indstillet til pH 6 med 1 M eddikesyre. Inkubation foretages i 16 timer ved 4°C i mørke. Reaktionen standses ved tilsætning af 105 mikroliter af en 1M vandig opløsning af ethylenglycol.Since the coupling of gelonin with the antibody is generally not made using the natural thiol groups of the gel onion, the thiol groups will in fact be artificially introduced after the oxidation step by the technique described in Cancer Res., 1984, 44, 129-133. . 21 microliters of a 30.5 ml solution of sodium periodate in water is added to 1 ml of a solution containing 3 mg / ml gelonin in PBS buffer adjusted to pH 6 with 1 M acetic acid. Incubation is done for 16 hours at 4 ° C in the dark. The reaction is quenched by the addition of 105 microliters of a 1M aqueous solution of ethylene glycol.

35 Efter inkubation i 15 min ved 20°C, dialyseres reaktionsmediet ved 4eC mod PBS-puffer. Efter centrifugering ved 10.000 x g i 30 minutter, fører dette til 2,9 mg oxideret gelonin i en koncentration på 2,5 mg/ml.After incubation for 15 min at 20 ° C, the reaction medium is dialyzed at 4 ° C against PBS buffer. After centrifugation at 10,000 x g for 30 minutes, this results in 2.9 mg of oxidized gelonin at a concentration of 2.5 mg / ml.

DK 166626 B1 31DK 166626 B1 31

Ligesom ricins A-kæde består gelonins fundamentale egenskab i at inhibere proteinsyntese i eucaryote celler ved nedbrydning af den ribosomale sub-enhed 60 S (Biochem. J. (1982) 207, 505-509). Også i tilfælde af gelonin forårsager modifikationen 5 som følge af periodatoxidation ikke et aktivitetstab.Like the A chain of ricin, the fundamental property of gelonin consists in inhibiting protein synthesis in eucaryotic cells by degradation of the ribosomal subunit 60 S (Biochem. J. (1982) 207, 505-509). Also in the case of gelonin, the modification 5 due to periodate oxidation does not cause an activity loss.

II - Farmakokinetiske egenskaber hos gelonin med forlænget virkning 10 Naturligt gelonin eller gelonin, der er modificeret ved hjælp af de ovenfor forklarede procedurer, administreres til kaniner ved hjælp en enkelt injektion i en ørevene. Den injicerede ge-loninmængde er mellem 0,3 og 0,4 mg/kg. Blodprøver udtages med mellemrum på heparin. Plasmaerne analyseres ved hjælp af en 15 radioimmunometri sk prøve, der nedenfor betegnes ved forkortelsen RIM-2.II - Pharmacokinetic Properties of Extended-Effect Gelonin 10 Natural gelonin or gelonin modified by the procedures described above is administered to rabbits by a single injection into the ear veins. The amount of gelonin injected is between 0.3 and 0.4 mg / kg. Blood samples are taken at intervals on heparin. The plasmas are analyzed by means of a radioimmunometric sample, which is referred to below as the abbreviation RIM-2.

Denne prøve foretages ved hjælp af den samme teknik som blev benyttet til prøven RIH-1, med den undtagelse, at opløsningen 20 Acl her er en opløsning af anti-gelonin kanin-antistoffer, der er renset ved hjælp af affinitetskromatografi, idet Ac2-anti-stofferne er de samme radiomærkede antistoffer. Radiomærkningsproceduren er identisk med den procedure, der er beskrevet for metoden RIM-1. Koncentrationen af naturligt gelonin eller 25 modificeret gelonin i prøverne, der skal bestemmes, måles ved reference til en kalibreringskurve, der er fastlagt ved at indføre naturligt eller modificeret gelonin i forskellige kendte koncentrationer. Prøven RIM-2 har de samme pålidelig-heds- og reproducerbarhedskarakteristika, som er beskrevet for 30 metoden RIM-1. Resultaterne af disse forsøg er anført på samme måde som for ricins A-kæde i eksempel 2.This test is carried out by the same technique used for the RIH-1 sample, except that the solution 20 Acl here is a solution of anti-gelonin rabbit antibodies purified by affinity chromatography, -the substances are the same radiolabelled antibodies. The radiolabeling procedure is identical to the procedure described for the RIM-1 method. The concentration of natural gelonin or modified gelonin in the samples to be determined is measured by reference to a calibration curve determined by introducing natural or modified gelonin at various known concentrations. The RIM-2 sample has the same reliability and reproducibility characteristics described for the RIM-1 method. The results of these experiments are presented in the same manner as for the ricin A chain in Example 2.

Figur 4 viser plasmaeliminationskurverne som funktion af tid for naturligt gelonin og oxideret gelonin, der er i injiceret 35 intravenøst. Det naturlige gelonin forsvinder ligesom naturligt ricins A-kæde meget hurtigt fra blodstrømmen, eftersom 99,99% af det i blodstrømmen tilstedeværende gelonin forsvin- DK 166626 B1 32 der i løbet af 24 timer (kurve 1). Når geloninet er blevet oxideret i sine polysaccharidenheder, modificeres eliminationsprofilen dybtgående. 24 timer efter injektion er koncentrationen af det oxiderede gelonin 300 gange større end koncen-5 trationen af det naturlige gelonin (kurve 2).Figure 4 shows the plasma elimination curves as a function of time for natural gelonin and oxidized gelonin injected intravenously. Like natural ricin A chain, natural gelonin disappears from the bloodstream very quickly, as 99.99% of the gelonin present in the blood stream disappears within 24 hours (curve 1). Once the gelonin has been oxidized in its polysaccharide units, the elimination profile is profoundly modified. 24 hours after injection, the concentration of the oxidized gelonin is 300 times greater than the concentration of the natural gelonin (curve 2).

Ligesom for ricins A-kæde dokumenterer disse resultater således, at periodatoxidation har modificeret de sukkerarter, der er involveret i den genkendelsesproces, der ansvarlig for eli-1° minationen af gelonin, til punktet for hindring af denne genkendelse .As with the A chain of ricin, these results document that periodate oxidation has modified the sugars involved in the recognition process responsible for the elimination of gelonin to the point of obstruction of this recognition.

Eksempel 9: 15 Dette eksempel viser: 1. den hurtige eliminering af GPIR MOM, der erextraheret fra Momordica karantia, og 20 2. den langsomme eliminering af GPIR MOM, der er modificeret med natriumperiodat, efter intravenøs injektion i dyret.Example 9: 15 This example shows: 1. the rapid elimination of GPIR MOM that is extracted from Momordica quarantine, and 2. the slow elimination of GPIR MOM modified with sodium periodate after intravenous injection into the animal.

1) - Modifikation af GPIR MOM med natriumperiodat 25 GPIR MOM blev fremstillet og renset udfra Momordica karantia-frøs frøhvide ved hjælp af den metode, som er blevet beskrevet (Biochemical Journal (1980), 186, 443-452). Naturligt eller modificeret MOMs farmakokinetiske egenskaber blev fastslået 30 ved anvendelse af radioaktivt GPIR MOM. MOM radiomærkes på ty-rosinerne med radioaktivt jod-125 i nærværelse af chloramin T.1) - Modification of GPIR MOM with Sodium Periodate GPIR MOM was prepared and purified from Momordica quarantine seed seed white by the method described (Biochemical Journal (1980), 186, 443-452). The pharmacokinetic properties of natural or modified MOM were determined using radioactive GPIR MOM. MOM is radiolabeled on the tyrosine with radioactive iodine-125 in the presence of chloramine T.

10 mikroliter af en opløsning af radioaktivt jod-125 indeholdende 100 pCi/ml og 30 mikroliter af en opløsning af chloramin T indeholdende 2,5 mg/ml i vand sættes til 1O0 mikroliter af 35 en opløsning indeholdende 1 mg/ml MOM i PBS-puffer. Reaktionen lades forløbe i et minut ved omgivelsernes temperatur. Reaktionen standses ved tilsætning af 400 mikroliter af en opløs- DK 166626 B1 33 ning af natriummetabisulfit indeholdende 0,5 mg/ml. Reaktionsmediet kromatograferes ved hjælp af gelfi 1 trering på en søjle af "sephadex" G25 med PBS-puffer indeholdende 0,1% gelatine med henblik på at skille det radiomærkede protein fra det 5 uomsatte jod. Efter centrifugering ved 10.000 x g i 30 minutter fører dette til 80 mikrogram radiomærket Μ0Μ i en koncentration på 0.04 mg/ml.10 microliters of a solution of radioactive iodine-125 containing 100 µCi / ml and 30 microliters of a solution of chloramine T containing 2.5 mg / ml in water are added to 100 microliters of a solution containing 1 mg / ml MOM in PBS. buffer. The reaction is allowed to proceed for one minute at ambient temperature. The reaction is quenched by the addition of 400 microliters of a solution of sodium metabisulphite containing 0.5 mg / ml. The reaction medium is chromatographed by gel filtration on a column of "sephadex" G25 with PBS buffer containing 0.1% gelatin to separate the radiolabeled protein from the unreacted iodine. After centrifugation at 10,000 x g for 30 minutes, this leads to 80 micrograms of radiolabelled Μ0Μ at a concentration of 0.04 mg / ml.

Oxidationsreaktionen udføres under de samme betingelser som 10 beskrevet for ricins A-kæde i eksempel 2, med den undtagelse, at det trin, hvori thiolgrupperne blokeres med OTNB, udelades, og at koncentrationen af protein er 125 gange mindre (40 pg/ml }. 20 mikroliter af en 0,5 M opløsning af natriumperi odat i vand sættes til 1 ml af en opløsning indeholdende 0.04 mg/ml 15 radiomærket MOM, der er bragt til pH 6 med 1 M eddikesyre. Inkubation lades forløbe i 16 timer ved 4°C i mørke. Reaktionen standses ved tilsætning 100 mikroliter af en 1 M vandig opløsning af ethylenglycol. Efter inkubation i 15 minutter ved 20°C dialyseres ved 4°C mod PBS-puffer. Efter centrifugering ved 20 10.000 x g i 30 minutter fører dette til 32 mikrogram oxideret MOM i en koncentration på 0.021 mg/ml.The oxidation reaction is carried out under the same conditions as described for ricin A chain in Example 2, except that the step in which the thiol groups are blocked by OTNB is omitted and the concentration of protein is 125 times less (40 pg / ml}. 20 microliters of a 0.5 M solution of sodium periodate in water are added to 1 ml of a solution containing 0.04 mg / ml of 15 radiolabelled MOM brought to pH 6 with 1 M acetic acid and incubated for 16 hours at 4 ° The reaction is quenched by the addition of 100 microliters of a 1 M aqueous solution of ethylene glycol, after incubation for 15 minutes at 20 ° C, dialyzed at 4 ° C against PBS buffer, after centrifugation at 20,000 xg for 30 minutes to give 32 micrograms of oxidized MOM at a concentration of 0.021 mg / ml.

Det således opnåede nye molekyle har en molekylvægt, der ikke er signifikant forskellig fra det naturlige MOMs molekylvægt.The new molecule thus obtained has a molecular weight not significantly different from the molecular weight of the natural MOM.

25 Såvidt det er muligt at se ved hjælp af polyacrylamidgra-dientelektrophorese efter fremkaldelse med coomassie-blåt eller radioautograf i, frembringer modifikationsprocessen kun proteinpolymerer i en meget lille mængde og danner ikke noget nedbrydningsprodukt.As far as it is possible to see by polyacrylamide gradient electrophoresis after induction with coomassie blue or radio autograph, the modification process produces only protein polymers in a very small amount and does not form any degradation product.

30 2) - Farmakokinetiske egenskaber hos MOM med forlænget v i rkni nq30 2) - Pharmacokinetic properties of MOM with extended v i rkni nq

Det radiomærkede MOM, uanset om det er oxideret eller ikke er 35 oxideret ved hjælp af de ovenfor forklarede procedurer, administreres til kaniner ved hjælp af en injektion i en ørevene. Mængden af injiceret MOM er mellem 3,50 og 3,55 mikrogram/kg.The radiolabelled MOM, whether oxidized or not oxidized by the procedures described above, is administered to rabbits by injection into the ear veins. The amount of injected MOM is between 3.50 and 3.55 micrograms / kg.

DK 166626 B1 34DK 166626 B1 34

Blodprøver udtages med mellemrum på heparin. Plasmaerne (200 mikroliter) inkuberes med trichloreddikesyre (TCA) (200 mi-kroliter i en koncentration på 25%) i 30 minutter ved 4°C. Efter centrifugering, kan radioaktiviten indeholdt i det sedi-5 ment, som kan udfældes ved hjælp af syren, bestemmes. Denne analysemetode gør det muligt at måle plasmamængden af de intakte MOM-moleky1 er og hvilke som helst lavmolekylære nedbrydningsprodukter, som ikke kan udfældes ved hjælp af TCA, tages ikke i betragtning.Blood samples are taken at intervals on heparin. The plasma (200 microliters) is incubated with trichloroacetic acid (TCA) (200 microliters at a concentration of 25%) for 30 minutes at 4 ° C. After centrifugation, the radioactivity contained in the sediment which can be precipitated by the acid can be determined. This assay allows the plasma amount of the intact MOM molecules to be measured and any low molecular degradation products that cannot be precipitated by TCA are not taken into account.

1010

Resultaterne af disse eksperimenter er angivet som procenten af den oprindelige radioaktivitet, der forbliver i blodstrømmen som funktion af tiden. Denne værdi, som kaldes "procentdelen af den oprindelige plasmaradioaktivitet" (% IPR), bere-15 gnes under anvendelse af følgende udtryk: r x PV x 100 % I.P.R. = _The results of these experiments are given as the percentage of initial radioactivity remaining in the blood stream as a function of time. This value, which is called "the percentage of initial plasma radioactivity" (% IPR), is calculated using the following terms: r x PV x 100% I.P.R. = _

0.2 x R0.2 x R

20 hvor: r = radioaktivitet målt på tidspunktet t i 0,2 ml plasma , R = total injiceret radioaktivitet, 25 PV = plasmavolumen (betragtes som værende lig med 36 ml/kg af dyrets legemsvægt).Where: r = radioactivity measured at time t in 0.2 ml of plasma, R = total injected radioactivity, 25 PV = plasma volume (considered to be equal to 36 ml / kg of animal body weight).

Plasmaeliminationskurverne som funktion af tiden for det oxiderede eller ikke-oxiderede Μ0Μ efter intravenøs injektion er 30 vist i fig. 5. Μ0Μ forsvinder ligesom naturligt ricins A-kæde meget hurtigt fra blodstrømmen, eftersom 99.9% af det i blodstrømmen tilstedeværende MOM forsvinder i løbet af 8 timer (kurve 1). Når Η0Μ er blevet oxideret i sine sukkergrupper, formindskes elimineringshastigheden (kurve 2): 8 timer efter injektion er mængden af oxideret MOM 60 gange større end mængden af det ikke-oxiderede MOM. Disse resultater dokumenterer, at periodatoxidation har modificeret de sukkerarter, som 35 DK 166626 B1 35 indgår i den genkendelsesproces, der er ansvarlig for den hurtige eliminering af MOM.The plasma elimination curves as a function of the time of the oxidized or non-oxidized Μ0Μ after intravenous injection are shown in FIG. 5. Μ0Μ, like natural ricin's A chain, disappears from the bloodstream very quickly, as 99.9% of the MOM present in the bloodstream disappears within 8 hours (curve 1). When Η0Μ has been oxidized in its sugar groups, the elimination rate decreases (curve 2): 8 hours after injection, the amount of oxidized MOM is 60 times greater than the amount of the non-oxidized MOM. These results document that periodate oxidation has modified the sugars that are involved in the recognition process responsible for the rapid elimination of MOM.

Eksempel 10: 5Example 10: 5

Dette eksempel viser: 1. den hurtige eliminering af GPIR-Dianthin, der er extraheret fra Dianthus cariofyllus, og 10 2. den langsomme eliminering af GPIR-Dianthin, der er modificeret med natriumperiodat, efter intravenøs injektion i øret.This example shows: 1. the rapid elimination of GPIR-Dianthin extracted from Dianthus cariophyllus, and 2. the slow elimination of GPIR-Dianthin modified with sodium periodate after intravenous injection into the ear.

1) - Modifikation af Dianthin 30 med natriumperiodat 151) - Modification of Dianthin 30 with sodium periodate 15

Dianthin 30 blev fremstillet og renset ud fra Dianthus caryop-hyllusblade ved hjælp af den metode, som blevet beskrevet (Biochemical Journal (1981 ), 195, 339-4-05). Oxideret eller ikke -oxideret Dianthin 30‘s farmakokinetiske egenskaber blev 20 fastslået ved anvendelse af radioakt ivt Dianthin. Joder i ngs-og oxidationsreaktioner udføres under de samme betingelser som beskrevet for MOM i eksempel 9.Dianthin 30 was prepared and purified from Dianthus caryop shelf leaves by the method described (Biochemical Journal (1981), 195, 339-4-05). The pharmacokinetic properties of oxidized or non-oxidized Dianthin 30 were determined using radioactive Dianthin. Iodine in NgS and oxidation reactions are performed under the same conditions as described for MOM in Example 9.

Det således opnåede nye oxiderede Dianthinmolekyle har en 25 molekylvægt, der ikke er signifikant anderledes end molekylvægten af det naturlige Dianthin 30.The new oxidized Dianthin molecule thus obtained has a molecular weight which is not significantly different from the molecular weight of the natural Dianthin 30.

2) - Farmakokinetlske egenskaber hos Dianthin 30 med forlænget vi rkninq 302) - Pharmacokinetic properties of Dianthin 30 with prolonged drug 30

Det oxiderede eller ikke-oxiderede radiomærkede Dianthin administreres til kaniner ved hjælp en enkelt injektion i en ørevene. Plasmamængden af Dianthin måles ved hjælp af den samme procedure som beskrevet for MOM i eksempel 9. Fig. 6 viser 35 plasmaeliminationskurverne som funktion af tid for det oxiderede Dianthin (kurve 2) eller ikke-oxiderede Dianthin (kurve 1). Dianthin 30 forsvinder ligesom MOM meget hurtigt fra blod- DK 166626 B1 36 strømmen, eftersom 99,9¾ af den oprindeligt tilstedeværende mængde forsvinder i løbet af 2 timer. Når Dianthin 30 er ble-vete oxideret i kulhydratgrupperne, bliver eliminationskinetikken på den anden side gjort betydelig langsommere. To timer 5 efter injektion er dianthinmængden 80 gange større end mængden af ikke-oxideret Dianthin. Mængden af oxideret Dianthin forbliver høj udover 24 timer (3% af den oprindelige værdi efter 24 timer) .The oxidized or non-oxidized radiolabelled Dianthin is administered to rabbits by a single injection into the ear veins. The plasma amount of Dianthin is measured by the same procedure as described for MOM in Example 9. FIG. Figure 6 shows the plasma elimination curves as a function of time for the oxidized Dianthin (curve 2) or non-oxidized Dianthin (curve 1). Like MOM, Dianthin 30 disappears from the blood stream very quickly as 99.9¾ of the initially present volume disappears within 2 hours. On the other hand, when Dianthin 30 is oxidized in the carbohydrate groups, elimination kinetics are made significantly slower. Two hours 5 after injection, the amount of Dianthin is 80 times greater than the amount of non-oxidized Dianthin. The amount of Dianthin oxidized remains high beyond 24 hours (3% of the original value after 24 hours).

10 Atter her viser disse resultater, at periodatoxidation har modificeret de sukkerarter, der er involveret i den genkendelsesproces, som er ansvarlig for den hurtige eliminering af de Di anthi net.10 Again, these results show that periodate oxidation has modified the sugars involved in the recognition process responsible for the rapid elimination of the Di anthi networks.

15 Eksempel 11:Example 11:

Konjugat opnået ved omsætning af et antistof, som inhiberer humane T-celler (et antistof rettet mod antigenet T65), og som er substitueret med aktiverede disulfidgrupper, med ricins 20 oxiderede A-kæde.Conjugate obtained by reacting an antibody which inhibits human T cells (an antibody directed against the antigen T65) and which is substituted with activated disulfide groups, with the oxidized A chain of ricin.

a) Antistof, der inhiberer humane T-celler (eller antistof T101) 25 Dette antistof opnåedes ved hjælp af fremgangsmåden beskrevet i Journal of Immunology, 1980, 125(2), 725-737.a) Antibody Inhibiting Human T Cells (or Antibody T101) This antibody was obtained by the method described in Journal of Immunology, 1980, 125 (2), 725-737.

b) Ricins oxiderede A-kæde: Ricins A-kæde blev fremstillet på den i eksempel 2 angivne måde.b) Ricin's oxidized A-chain: Ricin's A-chain was prepared in the manner given in Example 2.

30 II) Aktiverede antistoffer der,inhiberer humane T- celler 20 mikroliter af en opløsning indeholdende 60.3 mg/ml 1-ethyl-3-dimethylaminopropyl-3-carbodiimid sættes til 100 mikroliter 35 af en opløsning indeholdende 20 mg/ml 3-(pyridin-2-yldi sy1fa-nyl) propionsyre i tert.-butanol, og blandingen lades henstå i 3 minutter ved omgivelsernes temperatur. 68 mikroliter af den DK 166626 B1 37 således opnåede opløsning sættes til 2 ml af en antistofopløs-ning indeholdende 8,9 mg/ml i PBS-puffer. Blandingen omrøres i 15 minutter ved 30eC og dialyseres derpå mod PBS-puffer ved 4°C. Efter dialyse centrifugeres proteinopløsningen til opnå-5 else af 15 mg aktiveret antistof i en koncentration på 7,9 mg/ml. Ved hjælp spektrofotometrisk analyse ved 343 nm af den ved udveksling med 2-mercatoethanol frigjorte pyridin-2-thion viser det sig, at det opnåede antistof bærer 3,8 aktiverede, blandede di sulfidgrupper pr. mol antistof.II) Activated antibodies that inhibit human T cells 20 microliters of a solution containing 60.3 mg / ml of 1-ethyl-3-dimethylaminopropyl-3-carbodiimide to 100 microliters of a solution containing 20 mg / ml of 3- (pyridine) 2-yldi-syphenyl-propionic acid in tert-butanol and the mixture is allowed to stand for 3 minutes at ambient temperature. 68 microliters of the solution thus obtained is added to 2 ml of an antibody solution containing 8.9 mg / ml in PBS buffer. The mixture is stirred for 15 minutes at 30 ° C and then dialyzed against PBS buffer at 4 ° C. After dialysis, the protein solution is centrifuged to give 15 mg of activated antibody at a concentration of 7.9 mg / ml. By spectrophotometric analysis at 343 nm of the pyridine-2-thione released by exchange with 2-mercatoethanol, it is found that the antibody obtained carries 3.8 activated mixed di sulfide groups per ml. mole of antibody.

10 III) Fremstilling af immunotoksinet indeholdende ricinets A-kæde med forlænget virkning 2,46 ml modificeret A-kæde indeholdende 2,87 mg/ml sættes til 15 1,5 ml af opløsningen af den som ovenfor anført opnåede opløs ning af aktiveret antistof (koncentration: 7,9 mg/ml, d.v.s.III) Preparation of the immuno-toxin containing the extended-action castor A chain 2.46 ml of modified A-chain containing 2.87 mg / ml is added to 1.5 ml of the solution of the activated antibody solution obtained as above ( concentration: 7.9 mg / ml, ie

11,8 mg aktiverede antistoffer) og blandingen inkuberes i 20 timer ved 20eC. Opløsningen centrifugeres og renses derpå ved filtrering på en "Sephadex" GlOO-søjle, idet afgangsstrømmens 20 optiske tæthed måles ved 280 nm. Kombination af fraktionerne indeholdende både antistoffet og A-kæden resulterer i 15 ml immunotoxinopløsning indeholdende 0,7 mg/ml, d.v.s. 10,5 mg. Denne opløsning indeholder 0.14 mg oxideret A-kæde koblet med antistoffet pr. ml.11.8 mg of activated antibodies) and the mixture is incubated for 20 hours at 20 ° C. The solution is centrifuged and then purified by filtration on a "Sephadex" GlOO column, measuring the optical density of the exit stream 20 at 280 nm. Combination of the fractions containing both the antibody and the A chain results in 15 ml of immunotoxin solution containing 0.7 mg / ml, i.e. 10.5 mg. This solution contains 0.14 mg of oxidized A chain coupled with the antibody per ml. ml.

2525

Den gennemsnitlige koblingsgrad i dette præparat er derfor 1,2 mol oxideret A-kæde pr. mol antistof.The average coupling rate in this preparation is therefore 1.2 moles of oxidized A chain per mole of antibody.

Immunotoxinet indeholdende ricins oxiderede A-kæde, IT (A-la) 30 T101, opnået som ovenfor anført, blev undersøgt for dets far- makokinetiske egenskaber og dets specifikke cytotoxiske egenskaber overfor målcellerne.The immunotoxin containing the oxidized A chain of ricin, IT (A-1a) T101, obtained as above, was investigated for its pharmacokinetic properties and its specific cytotoxic properties against the target cells.

Eksempel 12:Example 12:

Dette eksempel viser erhvervelsen af egenskaber til langsom plasmaelimination hos immunotoxinerne indeholdende ricins 35 DK 166626 B1 38 A-kæde med forlænget virkning, der forkortes til IT (A-La)TlOl.This example demonstrates the acquisition of slow plasma elimination properties of the immuno-toxins containing ricin's extended-chain A chain abbreviated to IT (A-La) T10.

I - Procedure 5I - Procedure 5

Konjugatet fremstillet ved den i eksempel 11 forklarede procedure administreres til kaniner ved hjælp af en enkelt injektion i en ørevene. Den injicerede mængde svarer til 0,415 mg/kg, udtrykt som A-kæde. Blodprøver udtages med mellemrum på 10 heparin. Plasmaerne analyseres ved hjælp af en radioimmunome-trisk prøve med to steder, der nedenfor er forkortet til RlM-3.The conjugate prepared by the procedure explained in Example 11 is administered to rabbits by a single injection into the ear veins. The amount injected corresponds to 0.415 mg / kg, expressed as A chain. Blood samples are taken at intervals of 10 heparin. The plasmas are analyzed by a two-site radioimmunometric assay, abbreviated below to RlM-3.

Denne prøve udføres ved hjælp af den samme teknik, som blev 15 benyttet til prøven RIM-1, med den undtagelse, at opløsningen Ac2 her er en opløsning af gedantistoffer, der inhiberer mus IgG, renset ved hjælp af affinitetskromatografi og radiomærket som beskrevet for metoden RIM-1.This test is performed by the same technique used for the RIM-1 sample, except that the solution Ac2 here is a solution of goat antibodies that inhibit mouse IgG, purified by affinity chromatography and radiolabelled as described for the method. RIM-1.

20 Koncentrationen af modificeret immunotoxin i de til bestemmelse beregnede prøver måles ved reference til en kalibreringskurve, der er frembragt ved at indføre det modificerede immu-notoxin i forskellige koncentrationer. Prøven RIM-3 har de samme pål i deligheds-reproducerbarhedsegenskaber som beskrevet 25 for metoden RIM-1.The concentration of modified immunotoxin in the assays to be determined is measured by reference to a calibration curve generated by introducing the modified immunotoxin at various concentrations. The RIM-3 sample has the same piles in partition reproducibility properties as described for the RIM-1 method.

Til sammenligning udføres en kontrolundersøgelse under de samme betingelser med konjugatet omtalt som IT T101, der opnås ved omsætning af det samme antistof T101, substitueret med ak-30 tiverede disulfidgrupper, med ricins naturlige A-kæde. Dette konjugats fremstilling og cytotoksiske egenskaber er beskrevet i fransk fremlæggelsesskrift nr. 2 516 794. Resultaterne af disse forsøg er angivet på samme måde som for ricins ukoblede A-kæde i eksempel 1.For comparison, a control study is conducted under the same conditions with the conjugate referred to as IT T101, obtained by reaction of the same antibody T101, substituted with activated disulfide groups, with the natural A chain of ricin. The preparation and cytotoxic properties of this conjugate are described in French Patent Specification No. 2,516,794. The results of these experiments are given in the same manner as for the unlinked A chain of ricin in Example 1.

35 DK 166626 B1 39 II - Resul tater35 DK 166626 B1 39 II - Result tater

Fig. 7 viser plasmaeliminationskurverne som funktion af tiden for IT T101 og IT (A-La) T101, der er injiceret intravenøst.FIG. Figure 7 shows the plasma elimination curves as a function of time for IT T101 and IT (A-La) T101 injected intravenously.

5 Fireogtyve timer efter injektion er koncentrationen af aktivt immunotoxin 140 gange større for IT (A-La) T101 end for IT T101. Dette faktum viser, at den oxiderede A-kædes nye farma-kokinetiske egenskaber bibeholdes efter kobling med et antistof.Twenty-four hours after injection, the concentration of active immunotoxin is 140 times greater for IT (A-La) T101 than for IT T101. This fact shows that the new pharmacokinetic properties of the oxidized A chain are retained after coupling with an antibody.

1010

Eksempel 13;Example 13;

Dette eksempel viser bevarelsen af de specifikke cytotoksiske egenskaber hos IT (A-La) T101 overfor målcellerne.This example demonstrates the conservation of the specific cytotoxic properties of IT (A-La) T101 against the target cells.

1515

Ricins A-kædes fundamentale biologiske egenskab består i at inhibere proteinsyntesen i celler ved nedbrydning af den ribo-somale sub-enhed 60S. Metoden benytter en cellemodel, i hvilken virkningen af de undersøgte stoffer på inkorporeringen af 20 l^C-leucin i cancerceller under dyrkning måles.The fundamental biological property of ricin A chain consists in inhibiting protein synthesis in cells by degradation of the ribosomal subunit 60S. The method uses a cell model in which the effect of the investigated substances on the incorporation of 20 µC leucine into cancer cells during culture is measured.

De benyttede celler tilhører CEM-cellelinien hidrørende fra en human T-leukæmi, der bærer antigenet T65. Cellerne inkuberes i nærværelse af det stof, der skal undersøges, og efter afslut-25 tet inkubation måles inkorporeringsgraden af l^C-leucin i de på denne måde behandlede celler.The cells used belong to the CEM cell line derived from a human T-leukemia carrying the T65 antigen. The cells are incubated in the presence of the substance to be examined, and upon completion of incubation, the degree of incorporation of 1 ° C leucine into the cells treated in this way is measured.

Denne måling foretages ved hjælp af en tilpasset metode i forhold den i Journal of Biological Chemistry 1974, 249(11), 30 3557-3562, beskrevne ved anvendelse af sporstoffet cl^-leucin til bestemmelse af proteinsyntesegraden. Den inkorporerede radioaktivitet bestemmes her på hele celler, som er isoleret ved hjælp af filtrering.This measurement is made by a customized method relative to that described in Journal of Biological Chemistry 1974, 249 (11), 3557-3562, using the trace element C1 -leucine to determine the degree of protein synthesis. Here, the incorporated radioactivity is determined on whole cells isolated by filtration.

35 p§ basis af disse bestemmelser er det muligt at optegne dosis/ virkning-kurverne, idet man efter abscissen afsætter den molære koncentration af A-kæde i stofferne, der skal undersøges, DK 166626 B1 40 og efter ordinaten afsætter inkorporeringen af cl^·— 1 eucin udtrykt som en procentdel af inkorporeringen i kontrol cel 1 er under fravær af noget stof, der påvirker proteinsyntese.On the basis of these provisions, it is possible to record the dose / effect curves, after the abscissa, the molar concentration of A-chain in the substances to be investigated is deposited, and after the ordinance the incorporation of Cl - 1 eucin expressed as a percentage of the incorporation into control cell 1 is in the absence of any substance affecting protein synthesis.

5 Det er således muligt for hvert stof, der skal undersøges, at bestemme den koncentration, som forårsager en 50% inhibering af inkorporeringen af Cl4-leucin, eller "50% inhiberende koncentration" (IC50)· 10 Fig. 8 viser de kurver, der opnås ved det samme forsøg med IT (A-La) T101 og den ukoblede oxiderede A-kæde i nærværelse af 10 mM ammoni umchlorid i inkubationsmediet. Det kan af denne figur ses, at IT (A-La) T101 har en meget kraftig cytotoxisk virkning (IC50 = 5.5 · 10~12M), hvilket er ca. 80.000 gange 15 mere end virkningen af den ukoblede oxiderede A-kæde, målt under de samme betingelser.Thus, it is possible for each substance to be tested to determine the concentration which causes a 50% inhibition of the incorporation of Cl 4 leucine or "50% inhibitory concentration" (IC 50). Figure 8 shows the curves obtained by the same experiment with IT (A-La) T101 and the uncoupled oxidized A chain in the presence of 10 mM ammonium chloride in the incubation medium. It can be seen from this figure that IT (A-La) T101 has a very potent cytotoxic effect (IC50 = 5.5 · 10 ~ 12M), which is approx. 80,000 times 15 more than the effect of the uncoupled oxidized A chain, measured under the same conditions.

Eksempel 14 20 Dette eksempel viser den forholdsmæssige cytotoxiske effektivitet af IT (A-La) T101 og IT T101 overfor CEM-målcel lerne, målt ved en clonogen prøve.Example 14 20 This example shows the relative cytotoxic efficacy of IT (A-La) T101 and IT T101 against the CEM target cells, as measured by a clonogenic sample.

Immunotoxiner er forudbestemt til udslettelse af hver eneste 25 af målcellerne. Denne opførsel kan kun bedømmes med en meget følsom metode; prøver til inhibering af kolonidannelse fremby-der denne mulighed, fordi en enkelt overlevende celle kan afsløres blandt flere millioner døde celler. Dette muliggøres ved hjælp af optimale dyrkningsbetingelser i et geleret me-30 dium, der anvendes til den humane CEM lymfoide linie.Immunotoxins are predetermined to annihilate every 25 of the target cells. This behavior can only be judged by a very sensitive method; colony inhibition samples offer this possibility because a single surviving cell can be revealed among millions of dead cells. This is made possible by optimum culture conditions in a gelled medium used for the human CEM lymphoid line.

I- Metode til måling af cytotoxititet ved inhibering af kolonidannelse 35 Det til cloning benyttede medium er mediet RPMI 1640, hvortil 1 mmol/1 natrium-alfa-ketog1utarat, 1 mmol/1 natriumoxalace- tat, 5% inaktiveret kalvefosterserum og 10% i nakt i veret heste- DK 166626 B1 41 serum sættes. En første 0f3 % agaropløsning (Agarose type VII, SIGMA laboratories) fremstilles i dette medium, placeres som et tyndt lag i små Petriskåle og størknes ved + 4°C. Cellerne blandes med en anden ved 37°C opbevaret 0,275% agaropløsning, 5 som derpå påføres på det første lag og størknes. Disse koncentrationer af agar blev valgt efter en forudgående undersøgelse roed henblik på samtidig optimering af kloningseffektiviteten, koloniernes størrrelse og mediets konsistens.I Method for Measuring Cytotoxicity by Inhibition of Colony Formation 35 The medium used for cloning is the medium RPMI 1640 to which 1 mmol / 1 sodium alpha-ketoglutarate, 1 mmol / 1 sodium oxalacetate, 5% inactivated fetal serum, and 10% in the nude in the verte-horse serum. An initial 0f3% agar solution (Agarose type VII, SIGMA laboratories) is prepared in this medium, placed as a thin layer in small Petri dishes and solidified at + 4 ° C. The cells are mixed with another stored at 37 ° C 0.275% agar solution, which is then applied to the first layer and solidified. These concentrations of agar were selected after a previous study aimed at simultaneously optimizing cloning efficiency, size of colonies and media consistency.

10 Efter 15 dage i inkubatoren tælles kolonierne under anvendelse af en automatisk kolonitæller ("ARTEK", DYNATECH, U.S.A.). Til bestemmelse af kloningseffektiviten og således det nøjagtige antal celler, der overlever immunotoxinbehandlingen, er det vigtigt at tilvejebringe en kalibreringslinje, der viser an-15 tallet af podede celler som funktion af antallet af dannede kolonier. Det er blevet godtgjort, at kloningseffektiviteten angivet ved hjælp af denne ka1 i brer ings1 inje er praktisk taget upåvirket af tilstedeværelsen af en stor mængde døde celler, hvilket er den situation, der naturligt kommer til at forelig-20 ge, når cellerne behandles med immunotoxinet.After 15 days in the incubator, the colonies are counted using an automatic colony counter ("ARTEK", DYNATECH, U.S.A.). In order to determine the cloning efficacy and thus the exact number of cells surviving the immunotoxin treatment, it is important to provide a calibration line showing the number of seeded cells as a function of the number of colonies formed. It has been demonstrated that the cloning efficiency indicated by this cell in the broad range is virtually unaffected by the presence of a large number of dead cells, which is the situation that naturally occurs when the cells are treated with the immunotoxin. .

Immunotoxinbehandl i ngen udføres ved inkubation af cellerne i den eksponentielle vækstfase og i en koncentration på 10®/ml med immunotoxinet IT (A-La) T101 eller IT T101 i forskellige 25 koncentrationer i en samlet mængde på 1 ml af mediet RPMI 1640 indeholdende 10% inaktiveret kalvefosterserum og 10 mmol/liter ammoniumchlorid. Inkubationen finder sted ved 37°C under en atmosfære indeholdende 5% carbondioxid og med horisontal rystning af prøverørene (2500 omdrejninger pr. min. med en "GIRA-30 TORY G- 2" ryster, NEW-BRUNSWICK). Cellerne vaskes derpå, og forskellige fortyndinger fremstilles før blanding med agaropløsningen, så at antallet af overlevende celler kan måles i zonen med maximal følsomhed angivet ved hjælp af kalibreringslinjen. Resultaterne udtrykkes som det absolutte antal overle-35 vende celler, ekstrapoleret fra kloningseffektiviteten, under DK 166626 B1 42 anvendelse af følgende relation: absolut antal overlevende celler: C x dImmunotoxin treatment is performed by incubating the cells in the exponential growth phase and at a concentration of 10® / ml with the immunotoxin IT (A-La) T101 or IT T101 at various concentrations in a total amount of 1 ml of the medium RPMI 1640 containing 10 % inactivated fetal calf serum and 10 mmol / liter ammonium chloride. The incubation takes place at 37 ° C under an atmosphere containing 5% carbon dioxide and with horizontal shaking of the test tubes (2500 rpm with a "GIRA-30 TORY G-2" shaker, NEW-BRUNSWICK). The cells are then washed and various dilutions are prepared before mixing with the agar solution so that the number of surviving cells can be measured in the zone with maximum sensitivity indicated by the calibration line. The results are expressed as the absolute number of surviving cells, extrapolated from the cloning efficiency, using the following relation: absolute number of surviving cells: C x d

EE

5 hvor C er antallet af kloner pr. Petriskål, d er fortyndingsfaktoren for det undersøgte cellepræparat, og E er kloningseffektiviteten angivet ved ka1ibrerings1 injens hældning. Hvert punkt svarer til gennemsnittet af tre prøver.5 where C is the number of clones per Petri dish, d is the dilution factor for the cell preparation examined, and E is the cloning efficiency indicated by the slope of calibration1 injection. Each point corresponds to the average of three samples.

10 II - Resultater10 II - Results

Fig. 9 viser kurverne for den cytotoksiske virkning af immuno-toxinerne IT (A-La) T101 og IT T101 på CEM-cellerne i nærvæ-relse af 10 mM ammoniumchlorid, som funktion af immunotoxin-koncentrationen (udtrykt som molariteten af A-kæde). Det fremgår, at effektiviterne af disse to produkter er af samme størrelsesorden. Den resulterende formindskelse af antallet af celler er ekstremt stor i begge tilfælde, eftersom mængden af 20 tilbageværende overlevende celler er af størrelsen 0,001% af begyndelsesværdien ved koncentrationer så lave som 10“H M. Denne virkning er overordentlig specifik, eftersom det ved disse koncentrationer blev godtgjort, at den ukoblede A-kæde eller et uspecifikt immunotoxin ikke har nogen virkning på 25 disse celler.FIG. Figure 9 shows the curves for the cytotoxic effect of the immunotoxins IT (A-La) T101 and IT T101 on the CEM cells in the presence of 10 mM ammonium chloride as a function of the immunotoxin concentration (expressed as the A chain molarity). It appears that the efficiencies of these two products are of the same magnitude. The resulting decrease in the number of cells is extremely large in both cases, since the amount of 20 remaining surviving cells is 0.001% of the initial value at concentrations as low as 10 ° H M. This effect is exceedingly specific, as at these concentrations demonstrated that the uncoupled A chain or unspecific immunotoxin has no effect on these cells.

Dette eksempel viser, at IT (A-La) T101 har specifikke cyto-toxicitetsegenskaber, der er praktisk taget identiske med konventionelt IT TIOl's cytotoxicitetsegenskaber.This example shows that IT (A-La) T101 has specific cytotoxicity properties that are virtually identical to conventional IT TIO1 cytotoxicity properties.

3030

Eksempel 15Example 15

Konjugat opnået ved omsætning af et antistof, som inhiberer humane T-celler (et antistof rettet mod antigenet T65), og 35 som er substitueret med aktiverede disulfidgrupper, med ricins oxiderede og funktionaliserede A-kæde (NEM), idet koblingen finder sted mellem de aktiverede di sulfidgrupper.og A-kædens funktional iserede sukkergrupper.Conjugate obtained by reacting an antibody that inhibits human T cells (an antibody directed against antigen T65) and substituted with activated disulfide groups, with ricin's oxidized and functionalized A chain (NEM), the coupling taking place between the activated di sulfide groups. and the A-chain functionalized sugar groups.

DK 166626 B1 43 1) - Fremstilling af immunotoksinet a) Fremstilling af den funktionaliserede A-kæde 5 A-kæden blokeres med N-ethylmaleimid på dens SH-gruppe og oxideres derpå i 18 timer ved hjælp af den i eksempel 3 beskrevne procedure.DK 166626 B1 43 1) - Preparation of the immunotoxin a) Preparation of the functionalized A chain The 5 A chain is blocked with N-ethylmaleimide on its SH group and then oxidized for 18 hours by the procedure described in Example 3.

Kobling med cystamin 10Coupling with cystamine 10

Efter dialyse mod carbonatpuffer med pH 9,5 inkuberes 5,2 ml af en proteinopløsning indeholdende 4.65 mg/ml sammen med 18 mg cystaminhydrochlorid i 2 timer ved 25°C. Denne inkubation efterfølges af reduktion med natriumborhydrid (200 ekvivale-15 nter pr mol A-kæde, d.v.s. 156 mikroliter af en opløsning indeholdende 17,6 mg i 1 ml 0,1 N NaOH) i 2 timer ved 25°C.After dialysis against carbonate buffer with pH 9.5, 5.2 ml of a protein solution containing 4.65 mg / ml is incubated with 18 mg of cystamine hydrochloride for 2 hours at 25 ° C. This incubation is followed by reduction with sodium borohydride (200 equivalents per mole of A chain, i.e., 156 microliters of a solution containing 17.6 mg in 1 ml of 0.1 N NaOH) for 2 hours at 25 ° C.

Overskuddet af reagens fjernes ved hjælp af kontinuerlig dialyse mod 125 mM phosphatpuffer med pH 7. Den fixerede cystamins 20 disulfidbro reduceres derpå med 2-mercaptoethanol til en slut-koncentration på 5 procent i 1 time ved 30° C, idet dette efterfølges af yderligere kontinuerlig dialyse mod 125 mM phosphatpuffer med pH 7 (20 liter ved 300 ml/time). Efter dialyse og centrifugering bestemtes 0,25 SH pr mol A-kæde ved 25 Ellman's metode.The excess reagent is removed by continuous dialysis against 125 mM phosphate buffer of pH 7. The disulfide bridge of the fixed cystamine 20 is then reduced with 2-mercaptoethanol to a final concentration of 5 percent for 1 hour at 30 ° C, followed by further continuous dialysis against 125 mM phosphate buffer of pH 7 (20 liters at 300 ml / h). After dialysis and centrifugation, 0.25 SH per mole of A chain was determined by 25 Ellman's method.

b) Fremstilling af antistoffet (se eksempel 11) c) Koblingsreaktion 30 1,5 ml af opløsningen af ricins modificerede A-kæde (d.v.s.b) Preparation of the antibody (see Example 11) c) Coupling reaction 30 1.5 ml of the solution of the modified A chain of ricin (i.e.

0,058 mikromol) sættes til 211 mikroliter af opløsningen af det som ovenfor anført opnåede aktiverede antistof (d.v.s. 0,006 mikromol). Blandingen lades reagere i 18 timer ved 30eC. Reaktionsmediet dialyseres derpå mod PBS-puffer (10 mM med 35 hensyn til phosphat og 140 mM med hensyn til natriumchlorid, pH 7,4). Efter centrifugering og undersøgelse ved hjælp af polyacrylamidgradientelektroforese viser det sig, at det DK 166626 Bl 44 opnåede immunotoxin har en gennemsnitlig koblingsgrad for det- t, te præprarat på 0,8 A-kæde (NEM) pr. mol antistof.0.058 micromol) is added to 211 microliters of the solution of the activated antibody obtained as indicated above (i.e. 0.006 micromol). The mixture is allowed to react for 18 hours at 30 ° C. The reaction medium is then dialyzed against PBS buffer (10 mM with 35 phosphate and 140 mM with sodium chloride, pH 7.4). After centrifugation and examination by polyacrylamide gradient electrophoresis, it turns out that the immunotoxin obtained by DK 166626 B144 has an average coupling rate for this tea preparation of 0.8 A chain (NEM) per mole of antibody.

2) - Immunotoxinet IT (A(NEM)-La-cysteamin) TIOl's egenskaber 52) - Immunotoxin IT (A (NEM) -La-cysteamine) TIOl Properties 5

Specifik cytotoksisk aktivitetSpecific cytotoxic activity

Det viser sig, at dette immunotoxin, fremstillet ved den ovenfor forklarede procedure, har en meget kraftig cytotoxisk 1° virkning på CEM-målcellerne (IC50 = l,2xlO“H M, fastslået ved fremgangsmåden beskrevet i eksempel 13) b) PlasmaelimineringIt appears that this immunotoxin produced by the procedure described above has a very potent cytotoxic 1 ° effect on the CEM target cells (IC 50 = 1.2 x 10 9 H determined by the procedure described in Example 13) b) Plasma elimination

Immunotoxinet administreres til kaniner ved hjælp af en enkelt injektion i en ørevene (50 mikrogram A-kæde/kg). Plasmaprøver, 15 er opsamlet efter 22 timer, analyseres ved hjælp af immun- oprøven RIA-3 (eksempel 12). Resultaterne er vist i nedenstående tabel. Værdierne for IT T101 er angivet til sammenligning.The immunotoxin is administered to rabbits by a single injection into the ear veins (50 micrograms A chain / kg). Plasma samples, 15 collected after 22 hours, are analyzed by immunoassay RIA-3 (Example 12). The results are shown in the table below. The values for IT T101 are given for comparison.

2020

Relativ plasmakoncentration 22 timer efter injektion IT (A(NEM)-La-cysteamin)T101 2,4% 25 it T101 0,08%Relative plasma concentration 22 hours after injection IT (A (NEM) -La-cysteamine) T101 2.4% 25 it T101 0.08%

Toogtyve timer efter injektion er koncentrationen af IT indeholdende modificeret A-kæde 30 gange større end i tilfælde af IT T101.Twenty-two hours after injection, the concentration of IT containing modified A chain is 30 times greater than in the case of IT T101.

3030

Eksempel 16;Example 16;

Konjugat opnået ved omsætning af et antistof, der inhiberer humane T-celler (et antistof rettet mod antigenet T65), og som 3 5 er substitueret med aktiverede disulfidgrupper, med ricins me-thylerede, oxiderede og funktionaliserede A-kæde (NEM), idet koblingen finder sted mellem de aktiverede disulfidgrupper og A-kædens modificerede sukkergrupper.Conjugate obtained by reaction of an antibody that inhibits human T cells (an antibody directed against the antigen T65) and which is substituted with activated disulfide groups, with the methylated, oxidized and functionalized A chain (NEM) of ricin, with the coupling takes place between the activated disulfide groups and the modified A-chain modified sugar groups.

DK 166626 B1 45 1) - Fremstilling af immunotoxinet a) Fremstilling af den funktionaliserede A-kæde 5 A-kæden blokeres med N-ethylmaleimid på sin SH-gruppe og me-thyleres og oxideres derpå i 18 timer ved hjælp af den i eksempel 5 beskrevne metode.DK 166626 B1 45 1) - Preparation of the immunotoxin a) Preparation of the functionalized A chain 5 The A chain is blocked with N-ethylmaleimide on its SH group and then methylated and oxidized for 18 hours by means of the example 5 method described.

Kobling med cystamin 10Coupling with cystamine 10

Efter dialyse mod 0,1 M carbonatpuffer med pH 9,5 inkuberes 18,5 ml af en proteinopløsning indeholdende 2,5 mg/ml sammen med 35,6 mg cystaminhydrochlorid i 2 timer ved 25°C. Denne inkubation efterfølges af reduktion med natriumborhydrid (200 15 ækvivalenter pr. mol A-kæde, dvs. 395 mikroliter af en opløsning indeholdende 17,6 mg i 1 ml 0,1 N NaOH) i 2 timer ved 25°C.After dialysis against 0.1 M carbonate buffer of pH 9.5, 18.5 ml of a protein solution containing 2.5 mg / ml is incubated with 35.6 mg of cystamine hydrochloride for 2 hours at 25 ° C. This incubation is followed by reduction with sodium borohydride (200 equivalents per mole of A chain, i.e. 395 microliters of a solution containing 17.6 mg in 1 ml of 0.1 N NaOH) for 2 hours at 25 ° C.

Reagensoverskuddet fjernes ved kontinuerlig dialyse mod 125 mM 20 phosphatpuffer med pH 7. Den fikserede cystamins disulfidbro reduceres derpå med 2-mercaptoethanol til en slutkoncentration på 5% i 1 time ved 30eC, idet dette efterfølges af yderligere kontinuerlig dialyse mod 125 mM phosphatpuffer med pH 7 (20 1 ved 300 ml/time). Efter dialyse og centrifugering, bestemtes 25 0,32 SH pr. mol A-kæde ved hjælp af Ellman's metode.The excess reagent is removed by continuous dialysis against 125 mM 20 phosphate buffer at pH 7. The disulfide bridge of the fixed cystamine is then reduced with 2-mercaptoethanol to a final concentration of 5% for 1 hour at 30 ° C, followed by further continuous dialysis against 125 mM phosphate buffer with pH 7 (20 L at 300 ml / h). After dialysis and centrifugation, 0.32 SH mole A chain using Ellman's method.

b) Fremstilling af det modificerede antistof En opløsning indeholdende 2,12 mg N-succinimidyl-3-pyridin-2-yldithiopropionat i ethylalkohgol 30 sættes til 23,5 ml af en opløsning af antistof T101 indeholdende 4,4 mg/ml (dvs. 0,68 mikromol). Denne blanding omrøres i 30 min. ved 25°C og dialyseres derpå mod 125 mM phosphatpuf-fer med pH 7. Efter dialyse, centrifugeres proteinopløsningen til opnåelse af 23,5 ml af en opløsning indeholdende 4,2 mg 35 modificeret antistof pr. ml.b) Preparation of the Modified Antibody A solution containing 2.12 mg of N-succinimidyl-3-pyridin-2-yldithiopropionate in ethyl alcohol 30 is added to 23.5 ml of a solution of antibody T101 containing 4.4 mg / ml (i.e. 0.68 micromoles). This mixture is stirred for 30 min. at 25 ° C and then dialyzed against 125 mM phosphate buffer of pH 7. After dialysis, the protein solution is centrifuged to obtain 23.5 ml of a solution containing 4.2 mg of 35 modified antibody per ml. ml.

Ved hjælp af spektrophotometrisk analyse ved 343 nm af den ved udveksling med 2-mercaptoethanol frigjorte pyridin-2-thion vi DK 166626 B1 46 ser det sig, at det opnåede antistof bærer 3,2 aktiverede, blandede disulfidgrupper pr. mol antistof.By means of spectrophotometric analysis at 343 nm of the pyridine-2-thione released by exchange with 2-mercaptoethanol, it appears that the antibody obtained carries 3.2 activated mixed disulfide groups per ml. mole of antibody.

c) koblingsreaktion 5 7,3 ml af opløsningen af ricins modificerede A-kæde (dvs.c) Coupling Reaction 5 7.3 ml of the solution of modified ricin A chain (i.e.

0,275 mikromol) sættes til 781 mikroliter af den som oven for anført opnåede opløsning af aktiveret antistof (dvs. 0,022 mikromol). Blandingen lades reagere i 18 timer ved 30eC. Reaktionsmediet dialyseres derpå mod PBS-puffer (10 mM med hensyn 10 til phosphat, 120 mM med hensyn til natriumchlorid, pH 7,4).0.275 micromol) is added to 781 microliters of the activated antibody solution obtained above (i.e., 0.022 micromol). The mixture is allowed to react for 18 hours at 30 ° C. The reaction medium is then dialyzed against PBS buffer (10 mM for 10 for phosphate, 120 mM for sodium chloride, pH 7.4).

Efter centrifugering og undersøgelse ved hjælp af polyacryla-midgradientelectrophorese viser det sig, at det opnåede immu-notoksin har en gennemsnitlig koblingsgrad for dette præparat 15 på 0,8 oxideret, methyleret A-kæde (NEM) pr. mol antistof.After centrifugation and examination by polyacryla mid-gradient electrophoresis, it is found that the obtained immunotoxin has an average coupling rate of this preparation of 0.8 oxidized methylated A chain (NEM) per mole of antibody.

Immunotoksfnet indeholdende ricins methylerede, oxiderede A-kæde (NEM), opnået som oven for anført, blev undersøgt for dets pharmakokinetiske egenskaber og dets specifikke cytotok-20 s icitetsegenskaber over for målcellerne.The immunotoxin containing methylated ricin oxidized A chain (NEM) obtained as above was investigated for its pharmacokinetic properties and its specific cytotoxicity properties against the target cells.

2) Egenskaber hos immunotoksinet IT (methyleret A(NEM)-la-cysteamin) T101 25 a) Specifik cytotoksicitetsvirkning2) Properties of the immunotoxin IT (methylated A (NEM) -la cysteamine) T101 25 a) Specific cytotoxicity effect

Det viser sig, at dette immunotoksin, fremstillet ved den oven for forklarede procedure, har en meget kraftig cytotoksisk virkning på CEM-målcel lerne (IC50 = 7 x fastslået ved den i eksempel 13 beskrevne metode).It appears that this immunotoxin produced by the above-described procedure has a very potent cytotoxic effect on the CEM target cells (IC 50 = 7 x determined by the method described in Example 13).

30 b) PlasmaelimineringB) Plasma elimination

Immunotoksinet administreres til kaniner ved hjælp af en enkelt injektion i en ørevene (81 mikrogram-A-kæde/kg). Plasmaprøverne opsamles efter 24 timer og analyseres ved hjælp 35 af immunprøven RIA-3 (eksempel 12). Resultaterne er vist i nedenstående tabel.The immunotoxin is administered to rabbits by a single injection into the ear veins (81 micrograms A chain / kg). The plasma samples are collected after 24 hours and analyzed by 35 of the RIA-3 immunoassay (Example 12). The results are shown in the table below.

DK 166626 B1 47 Værdierne for IT T101 er angivet til sammenligning.DK 166626 B1 47 The values for IT T101 are given for comparison.

Relativ p1asmakoncentrat i _ on efter 22 timer 5 IT (methyleret A(NEM)-La-cysteamin) 1,4% T101 IT T101 0,08% 10 __ 22 timer efter injektion er koncentrationen af IT indeholdende modificeret A-kæde 17,5 gange større end i tilfælde af IT T101.Relative Plasma Concentrate in _on after 22 hours 5 IT (methylated A (NEM) -La-cysteamine) 1.4% T101 IT T101 0.08% 10__ 22 hours after injection, the concentration of IT containing modified A chain is 17.5 times greater than in the case of IT T101.

1515

Eksempel 17Example 17

Toksicitet af i mus injiceret A-kæde med forlænget virkning.Toxicity of prolonged effect A-injected A-chain.

2Q Det var vigtigt at undersøge den samlede toksikologiske virkning af den oxiderede A-kæde på hele dyret (idet immunotoksi-nernes toksicitet er af samme størrelsesorden som toksiciteten af A-kæden i lige molære doser). Dette blev foretaget ved bestemmelse af den 50% dødelige dosis af den oxiderede A-kæde, 25 administreret intravenøst til Charles River, France CDl-mus ved sammenligning med den 50% dødelige dosis af den naturlige A-kæde.2Q It was important to investigate the overall toxicological effect of the oxidized A chain on the whole animal (since the toxicity of the immunotoxins is of the same order of magnitude as the toxicity of the A chain in equal molar doses). This was done by determining the 50% lethal dose of the oxidized A chain, administered intravenously to Charles River, France CD1 mice by comparison with the 50% lethal dose of the natural A chain.

De fundne værdier er vist i den følgende tabel.The values found are shown in the following table.

30 ld50 (mikrogram/mus) 35 Naturlig A-kæde 55030 ld50 (micrograms / mouse) 35 Natural A-chain 550

Oxideret A-kæde 800Oxidized A-chain 800

Claims (13)

1. Antitumoralt glycoprotein, som inaktiverer ribosomer (GPIR), kendetegnet ved, at dets kulhydratenheder er modificeret ved oxidation med periodationen, hvorhos gly-coproteinet er forskelligt fra det hele ricin. DK 166626 B11. Antitumoral glycoprotein which inactivates ribosomes (GPIR), characterized in that its carbohydrate units are modified by oxidation with the periodation, wherein the glycoprotein is different from the whole ricin. DK 166626 B1 2. Glycoprotein ifølge krav 1, kendetegnet ved, at det har i det væsentlige den samme virkning som og en længere halveringstid end det umodificerede glycoprotein, som i nakt i -verer ribosomer. 5Glycoprotein according to claim 1, characterized in that it has substantially the same effect as and a longer half-life than the unmodified glycoprotein which, in the nude, produces ribosomes. 5 3. Glycoprotein ifølge krav 2, kendetegnet ved, at det er opnået ved behandling af en vandig opløsning af et glycoprotein, som inaktiverer ribosomer, og hvis thiolgrupper eventuelt er beskyttede, med en vandig opløsning af et alkali- 10 metalperiodat i et tidsrum fra 0,2 til 24 timer, ved en temperatur fra 0 til 10°C og i fravær af lys, at thiolgrupperne om ønsket afblokeres, og at slutproduktet isoleres ved hjælp af kendte metoder. 15 4.. Glycoprotein ifølge krav 3, kendetegnet ved, at den vandige opløsning af glycoprotein er en vandig opløsning af ricins A-kæde.Glycoprotein according to claim 2, characterized in that it is obtained by treating an aqueous solution of a glycoprotein which inactivates ribosomes and whose thiol groups are optionally protected, with an aqueous solution of an alkali metal period for a period of 0 , For 2 to 24 hours, at a temperature of 0 to 10 ° C and in the absence of light, if desired, the thiol groups are unblocked and the final product isolated by known methods. Glycoprotein according to claim 3, characterized in that the aqueous solution of glycoprotein is an aqueous solution of the A chain of ricin. 5. Glycoprotein ifølge krav 3, kendetegnet ved, at 20 ricins A-kæde er funktionaliseret, f.eks. ved methylering.Glycoprotein according to claim 3, characterized in that 20 ricins A chain is functionalized, e.g. by methylation. 6. Glycoprotein ifølge krav 3, kendetegnet ved, at den vandige opløsning af glycoprotein er en vandig opløsning af gelonin. 25Glycoprotein according to claim 3, characterized in that the aqueous solution of glycoprotein is an aqueous solution of gelonin. 25 7. Glycoprotein ifølge krav 3, kendetegnet ved, at den vandige opløsning af glycoprotein er en vandig opløsning af GPIR MOM.Glycoprotein according to claim 3, characterized in that the aqueous solution of glycoprotein is an aqueous solution of GPIR MOM. 8. Glycoprotein ifølge krav 3, kendetegnet ved, at den vandige opløsning af glycoprotein er en vandig opløsning af GPIR Dianthin 30. 1 Glycoprotein ifølge krav 3, kendetegnet ved, at 35 den vandige opløsning af glycoprotein er en vandig opløsning af Dianthin 32, Agrostin A, Agrostin B, Agrostin C, HCI eller Asparagus officinalis inhibitor. DK 166626 B1Glycoprotein according to claim 3, characterized in that the aqueous solution of glycoprotein is an aqueous solution of GPIR Dianthin 30. A glycoprotein according to claim 3, characterized in that the aqueous solution of glycoprotein is an aqueous solution of Dianthin 32, Agrostin. A, Agrostin B, Agrostin C, HCl or Asparagus officinalis inhibitor. DK 166626 B1 10. Glycoprotein ifølge krav 4, kendetegnet ved, at det er opnået enten udfra en A-kæde i ricin, som er naturlig ricins A-kæde eller et fragment af naturlig ricins A-kæde, eller udfra en A-kæde i ricin eller et fragment deraf fremstil- 5 let biosyntetisk ved hjælp af en celle, hvis genotype er blevet passende modificeret.Glycoprotein according to claim 4, characterized in that it is obtained either from an A-chain in ricin, which is A-chain of natural ricin or a fragment of A-chain of natural ricin, or from an A-chain in ricin or a fragments thereof prepared biosynthetically by a cell whose genotype has been suitably modified. 11. Glycoprotein ifølge krav 4 eller 9, kendetegnet ved, at det er opnået ved behandling af en vandig opløsning af 10 ricins A-kæde, hvori mindst en af thiolgrupperne er beskyttet ved omsætning med 2,2'-di nitro-5,5'-dithiodibenzoat, med en vandig opløsning af natriumperiodat i et tidsrum fra 0,2 til 24 timer ved en temperatur på ca 4°C og i fravær af lys, behandling af blandingen med 2-mercaptoethanol samt isolation af 15 det resulterende produkt ved hjælp af kendte metoder.Glycoprotein according to claim 4 or 9, characterized in that it is obtained by treating an aqueous solution of 10 ricins A chain, wherein at least one of the thiol groups is protected by reaction with 2,2'-di nitro-5.5. -dithiodibenzoate, with an aqueous solution of sodium periodate for a period of from 0.2 to 24 hours at a temperature of about 4 ° C and in the absence of light, treatment of the mixture with 2-mercaptoethanol and isolation of the resulting product by means of of known methods. 12. Glycoprotein ifølge krav 6, kendetegnet ved, at det er opnået ved behandling af en vandig opløsning af gelonin med en vandig opløsning af natriumperiodat, i et tidsrum fra 20 0,2 til 24 timer, ved en temperatur på ca 4eC og i fravær lys, behandling af blandingen med 2-mercaptoethanol og isolation af det resulterende produkt ved hjælp af kendte metoder.Glycoprotein according to claim 6, characterized in that it is obtained by treating an aqueous solution of gelonin with an aqueous solution of sodium periodate, for a period of from 0.2 to 24 hours, at a temperature of about 4 ° C and in the absence. light, treating the mixture with 2-mercaptoethanol and isolating the resulting product by known methods. 13. Fremgangsmåde til fremstilling af et antitumoralt glycop-25 rotein ifølge krav 1, kendetegnet ved, at det umo- dificerede, antitumorale glycoprotein underkastes oxidation med periodationer.A method of preparing an antitumoral glycoprotein according to claim 1, characterized in that the unmodified antitumoral glycoprotein is subjected to oxidation with periodic periods. 14. Fremgangsmåde til fremstilling af et glycoprotein ifølge 30 krav 2, kendetegnet ved, at en vandig opløsning af et glycoprotein, som i nakt i verer ri bosomer, og hvis thiolgrupper eventuelt er beskyttet, behandles med en vandig opløsning af et alkal imetalper iodat i et tidsrum fra 0,2 til 24 timer ved en temperatur fra 0 til 15°C og i fravær af lys, at thiol-35 gruppen eventuelt afblokeres, og at slutproduktet isoleres ved hjælp af kendte metoder. DK 166626 B1Process for the preparation of a glycoprotein according to claim 2, characterized in that an aqueous solution of a glycoprotein, which is in the nude in bosoms and whose thiol groups are optionally protected, is treated with an aqueous solution of an alkaline metal metal iodate in a time range of 0.2 to 24 hours at a temperature of 0 to 15 ° C and in the absence of light, optionally unblocking the thiol-35 and isolating the final product by known methods. DK 166626 B1 15. Fremgangsmåde ifølge krav 14, kendetegnet ved, at det benyttede udgangsmateriale er enten en A-kæde i ricin, som er naturlig ricins A-kæde eller fragment af naturlig ri-cins A-kæde, eller en A-kæde i ricin eller et fragment deraf, 5 der er produceret biosyntetisk ved hjælp af en celle, hvis genotype er blevet passende modificeret. 10 15 20 25 30 35Process according to claim 14, characterized in that the starting material used is either an A-chain in ricin, which is A-chain of natural ricin or fragment of natural ricin's A-chain, or an A-chain in ricin or a fragments thereof, 5 which have been biosynthetically produced by a cell whose genotype has been appropriately modified. 10 15 20 25 30 35
DK277985A 1984-06-20 1985-06-19 ANTITUMORAL GLYCOPROTEINS, WHETHER CARBOHYDRATE UNITS HAVE BEEN MODIFIED, AND METHOD OF PRODUCING THEREOF. DK166626B1 (en)

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FR8409703A FR2566271B1 (en) 1984-06-20 1984-06-20 NOVEL CYTOTOXIC CONJUGATES FOR USE IN THERAPEUTICS AND PROCESS FOR OBTAINING SAME
FR8502067A FR2577137B1 (en) 1985-02-13 1985-02-13 ANTI-TUMOR GLYCOPROTEINS, MODIFIED ON THEIR CARBOHYDRATES
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US6610299B1 (en) 1989-10-19 2003-08-26 Aventis Pharma Deutschland Gmbh Glycosyl-etoposide prodrugs, a process for preparation thereof and the use thereof in combination with functionalized tumor-specific enzyme conjugates
US7241595B2 (en) 1989-10-20 2007-07-10 Sanofi-Aventis Pharma Deutschland Gmbh Glycosyl-etoposide prodrugs, a process for preparation thereof and the use thereof in combination with functionalized tumor-specific enzyme conjugates
US6475486B1 (en) 1990-10-18 2002-11-05 Aventis Pharma Deutschland Gmbh Glycosyl-etoposide prodrugs, a process for preparation thereof and the use thereof in combination with functionalized tumor-specific enzyme conjugates
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