IE851511L - Modified glycoproteins - Google Patents

Modified glycoproteins

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Publication number
IE851511L
IE851511L IE851511A IE151185A IE851511L IE 851511 L IE851511 L IE 851511L IE 851511 A IE851511 A IE 851511A IE 151185 A IE151185 A IE 151185A IE 851511 L IE851511 L IE 851511L
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IE
Ireland
Prior art keywords
chain
glycoprotein
ricin
aqueous solution
periodate
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IE851511A
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IE58514B1 (en
Inventor
Franz Jansen
Pierre Gros
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Sanofi Sa
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Priority claimed from FR8409703A external-priority patent/FR2566271B1/en
Priority claimed from FR8502067A external-priority patent/FR2577137B1/en
Application filed by Sanofi Sa filed Critical Sanofi Sa
Publication of IE851511L publication Critical patent/IE851511L/en
Publication of IE58514B1 publication Critical patent/IE58514B1/en

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    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
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    • C07—ORGANIC CHEMISTRY
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    • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D231/12—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6819—Plant toxins
    • A61K47/6825—Ribosomal inhibitory proteins, i.e. RIP-I or RIP-II, e.g. Pap, gelonin or dianthin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6819—Plant toxins
    • A61K47/6825—Ribosomal inhibitory proteins, i.e. RIP-I or RIP-II, e.g. Pap, gelonin or dianthin
    • A61K47/6827—Ricin A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/56—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, attached to ring carbon atoms
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
    • C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C07D249/08—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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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

The present invention relates to new antitumoral glycoproteins which inactivate ribosomes, whose carbohydrate units are modified by oxidation with the periodate ion, said glycoproteins differing from the whole ricin.
More particularly, the present invention refers to new glycoproteins which inactivate ribosomes and have a prolonged action, said glycoproteins differing from the whole ricin.
The term "glycoprotein which inactivates ribosomes", as used in the present description and also in the claims, denotes any substance which carries saccharide units belonging to the class of macromolecules which inactivate ribosomes and consequently inhibit protein synthesis in eucaryotic cells, as well as any fragment of the said substance which possesses the same inactivating property, it being possible for the said glycoprotein which inactivates ribosomes to be of natural or biosyn-thetic origin, being derived from a cell whose genotype has been modified for this purpose, and it also being possible for the said glycoprotein which inactivates ribosomes to be modified on the functional groups of its amino acids so that it can easily be coupled with an antibody.
In the description, the expression "glycoprotein which inactivates ribosomes" will be denoted by the symbols GPIR.
In the description, the term "periodate" denotes the IOj^- ion, which is also referred to in the literature as "metaperiodate".
The glycoproteins which inactivate ribosomes (GPIR) are especially useful as intermediates in the preparation of immunotoxins by coupling with antibodies.
U.S. Patent Specification No. 4 340 535 and French Patent Applications nos. 2 504 010 and no. 2 516 794 describe the preparation of anticancer products called conjugates, which are obtained by the coupling, by means of a covalent bond, of the A chain of ricin with antibodies or antibody fragments directed against antigens carried by the cell to be destroyed. The products of this type 5 have been designated, and are designated in the present Application, under the generic name of immunotoxins.
Conjugates analogous to the previously described immunotoxins containing the A chain of ricin are known which are also suitable as anticancer drugs and 10 result from the coupling, by means of a covalent bond, of antibodies or antibody fragments with other glycoproteins which inactivate ribosomes, such as, in particular, the gelonine extracted from Gelonium multi-florum (Eur. J. Biochem., 1981, 116, 447-454; Cancer 25 Res., 1984, 44, 129-133) or the inhibitor extracted frctn ftanordica charantia (MOM) (U.S. Patent Specification No. 4 368 149).
These glycoproteins which inactivate ribosomes (GPIR), and which have properties similar to those of the A chain of ricin, are substances with a molecular 20 weight of the order of magnitude of 20,000 and 30,000 (Cancer Survey, 1982, 1, 489-520).
It is also known that the cytotoxic activity of these immunotoxins can be potentiated by a variety of adjuvant substances such as ammonium salts, various 25 amines or certain carboxylic ionophores such as monensin and nigericin.
However, the therapeutic effects of immunotoxins, whether activated or not, can only manifest themselves fully in as much as the immunotoxin is 30 capable, through its antibody part, of becoming localized in vivo, in the active form, on the target cells to be destroyed (sine qua non condition for any expression of immunotoxin activity). The capacity of the immunotoxin to become localized on the target de-35 pends first and foremost on the ability of the immuno- toxin to remain in the bloodstream and the extracellular fluids, in the active form, for sufficient lengths of time for it to reach its target cells and in sufficiently large concentrations for the degree of occu-5 pation of the corresponding antigenic sites to be high.
Numerous studies have made it possible to establish the plasma elimination kinetics of immunotoxins after intravenous injection into different animal models. It is apparent that, after injection, the jLo plasma level of biologically active immunotoxin de creases very rapidly and very substantially. Thus, in a typical case involving rabbits, in a model using an immunotoxin synthesized by coupling, with the aid of an arm containing a disulfide bridge, the A chain of ricin with a monoclonal antibody directed against the antigen T65 of human T lymphocytes (antibody T101), it appears that 97% of the immunotoxin present in the bloodstream at time 0 after injection disappears in 30 minutes and 99.9% disappears in 17 hours. This rapid 2q disappearance of the immunotoxin quite obviously de tracts from the expression of its complete cytotoxic capacity by preventing the immunotoxin from saturating , for a prolonged period, a high proportion of the target antigens carried by the cells to be destroyed. More-25 over, comparison of the plasma elimination kinetics of the immunotoxins with those of the corresponding non-conjugated antibodies shows that, on the contrary, the antibodies remain in the plasma at a high level for relatively long periods, as is well known. Now, there 30 is always a certain residual level of non-conjugated antibodies, even in the most highly purified immunotoxin preparations. Through the effect of the differential elimination rates of immunotoxins and antibodies, the non-conjugated antibodies, which are initially in a very small minority, gradually become a majority after a few hours and these antibodies therefore gradually become, by competition, powerful antagonists for the fixation of the immunotoxins to their targets.
The advantage of increasing the plasma persistence of immunotoxins, in the active form, in order to increase both the duration and the degree of occupation of the target antigens, and consequently to improve the therapeutic effects of the immunotoxins, is therefore clearly apparent.
Furthermore, in vivo localization experiments on the immunotoxin containing the A chain of ricin, radiolabeled and then injected into animals without a specific target, have shown that, in the first few minutes after injection, the conjugate becomes localized preferentially in the liver. The same applies to the A chain of ricin, which follows the same pattern when it is injected in the uncoupled form. This strongly suggests that the immunotoxin fixes in the liver via the cytotoxic sub-unit which it contains. It is known that the A chain of ricin is a glycoprotein whose polyosidic groups include especially mannose residues and N'-acetylglucosamine residues, some mannose residues being in terminal positions (Agri. Biol.
Chem., 1978, 42, 501). The existence, in the liver, of receptors capable of recognizing glycoproteins having these terminal mannose residues has also been established. Moreover, it has been shown that the glycoproteins recognized by these receptors - the latter being present essentially 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 beta-glucuro-nidase 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, 11, 816).
Taken as a whole, these data show that the rapid elimination of immunotoxins containing the A chain of ricin can be explained by the recognition of the mannose residues of the A chain of ricin by the liver cells and in particular the Kupffer cells.
The studies of plasma elimination kinetics carried out on other GPIRs, for example gelonine or MOM, after intravenous injection into the animal, have shown that, as in the case of the A chain of ricin, the plasma level of GPIR decreases very rapidly and very substantially after injection. Thus, in a typical case involving rabbits, after the injection of gelonine purified by the method described (J. Biol. Chem., 1980, 255, 6947-6953), it appears that 93% of the gelonine present in the bloodstream at time 0 after injection disappears in 1 hour and 99.99% disappears in 24 hours.
It is known that the oxidation of osidic structures, including those contained in glycoproteins, with periodate ions causes the scission of the carbon chain wherever two adjacent carbon atoms carry primary or secondary hydroxyls. If the two adjacent hydro-xyls are secondary, as is generally the case in the cyclic oses present in GPIRs, oxidation produces two aldehyde groups on the carbons between which the scission has taken place.
It has now been found that when the carbohydrate units of an antitumoral glycoprotein are modified by oxidation with periodate ions, the biological activity of the said glycoprotein remains substantially unchanged.
It has also been found, absolutely unexpectedly, that if the carbohydrate units of a glycoprotein which inactivates ribosomes are modified by oxidation with periodate ions, a new glycoprotein which inactivates ribosomes is obtained, the said new glycoprotein having the dual property of retaining its biological activities 5 and of being eliminated very slowly from the bloodstream in vivo.
An in-depth biochemical study of oxidized and native GPIRs has made it possible to demonstrate that the oxidation of GPIRs with periodate involves exclu-10 sively the osidic part of the GPIRs and has no action on the sequence of the amino acids constituting their peptide part .
These new glycoproteins which inactivate ribosomes and have a prolonged action are referred to below 15 by the symbols GPIR-La.
Finally, it has been found that when these new glycoproteins which inactivate ribosomes and have a prolonged action are coupled with antibodies, the conjugates obtained retain the biological properties known 20 for immunotoxins and have slow plasma elimination kine tics .
It will be noted that periodate oxidation has been proposed in the prior art for producing conjugates constituted of an antibody or of an antibody fragment 25 and of an antitumoral substance. In this case, the aldehyde groups obtained by periodate oxidation of the antitumoral substance are used for producing the required conjugate. These aldehyde groups are therefore introduced temporarily to allow the conjugating of antibody and 30 antitumoral substance. Patents EP-71* 279 and FR 2 312 259 can be cited to this effect, which patents do not mention the glycoproteins used in the present application.
In the present application, on the contrary, the aldehyde groups introduced on the glycoprotein by 35 periodate oxidation are not involved in the formation of the above-defined immunotoxins. - 6a - The present invention therefore relates, "by way of new products, to structurally modified, antitumoral glycoproteins whose carbohydrate units are modified "by oxidation with the periodate ion.
The present invention relates more particularly to glycoproteins which inactivate ribosomes, whose carbohydrate units are modified by oxidation with the periodate ion and which have the same activity as and a longer half-life than the unmodified glycoprotein.
The invention preferentially relates to glyco proteins which inactivate ribosomes and have prolonged action and which are obtained by treatment of a glycoprotein which inactivates ribosomes, the thiol groups of which are optionally protected, with an aqueous 15 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, unblocking of the thiol groups, if appropriate, and isolation of the final product by known methods.
The present invention provides an antitumoral glycoprotein which inactives ribosomes, whose carbohydrate units are modified by oxidation with the periodate ion, said glycoprotein differing from the whole ricin.
The present invention further provides a glycoprotein which inactivates ribosomes, whose carbohydrate units are modified by oxidation with the periodate ion and which has substantially the same activity as and a longer half-life than the unmodified glycoprotein which inactivates ribosomes, said glycoprotein differing from the whole ricin.
The present invention also provides a glycoprotein which inactivates ribosomes and has a prolonged action, said glycoprotein differing from the whole ricin, wherein it is obtained by treatment of an aqueous solution of a glycoprotein which inactivates ribosomes, the thiol groups of which 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, unblocking of the thiol groups, if appropriate, and isolation of the final product by known methods.
The present invention also provides a process for the preparation of the antitumoral glycoproteins of the invention wherein the process comprises subjecting the unmodified antitumoral glycoprotein to oxidation with periodate ions.
The invention further provides a process for the preparation of a glycoprotein which inactivates ribosomes and has a prolonged action, said glycoprotein differing from whole ricin, wherein it comprises treating an aqueous solution of a glycoprotein which inactivates ribosomes, the thiol groups of which 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, unblocking of the thiol group, if appropriate, and isolation of the final product by known methods.
In - Any antitumoral glycoprotein can be modified on its carbohydrate units by reaction with the periodate ion in accordance with the known methods.
The glycoproteins which inactivate ribosomes 5 and which are used as preferred starting materials for oxidation with periodate ions, according to the invention, are all GPIRs, such as the A chain of ricin, which are in themselves only very slightly cytotoxic because they cannot fix to cells, but which, on the 10 other hand, after coupling with an antibody recog nizing particular cells, become highly cytotoxic towards these cells once the antibody has recognized its target.
Representative starting compounds are the A 15 chain of ricin, gelonine and the substance extracted from Momordica charantia (MOM), as obtained by extraction.
Other GPIRs which are useful as starting materials for oxidation with periodate ions are as 20 follows: Dianthin 30 from Dianthus caryophyllus Dianthin 32 from » it Agrostin A from Agrostemma githago Agrostin B from » « Agrostin C from » ti IICI from Ilura crepitans Asparagus officinalis from Asparagus inhibitor officinalis The same substances produced biosynthetically by cells whose genotype has been modified for this purpose are also suitable compounds.
Fragments of the above GPIRs, provided they retain all or part of the property of inactivating 5 ribosomes which characterizes the GPIR from which they are derived, can also be used as starting materials.
The A chain of native ricin in which at least one of the thiol groups is protected is a preferred starting compound.
Recent studies have demonstrated that the A chain of ricin comprises 2 constituents denoted by A1 and A2, which differ especially in their polysaccharide units. The experiments which have been carried out on the 2 constituents of the A chain have made 15 it possible to demonstrate that periodate oxidation takes place in a similar way on the A1 and A2 chains and gives these 2 constituents identical properties of improving the pharmacokinetics.
The preparation of the pure A chain of ricin 20 is described in U.S. Patent Specification No. 4 340 535.
Gelonine and MOM are also described in prior art.
Protection of the thiol groups of the starting GPIRs is desirable when the said thiol groups are those which are to be used for coupling with the antibody. 25 Xf other functional groups are used for the coupling, for example the phenolic hydroxyl of the tyrosines or amino groups or carboxyl groups of the GPIR, protection can be carried out or not.
Blocking is carried out by reaction with an 30 agent capable of substituting the SH groups with a radical which can subsequently be removed by reduction or thiol/disulfide exchange, for example 2,2'-dinitro-5,5'-dithiodibenzoic acid (DTNB) or 3-(pyridin-2-yl-disulfanyl)propionic acid or alternatively dipyridyl-35 2,2'-disulfide or dipyridy1-4,41-disulfide. In the absence of such a treatment, the free thiols may disappear during the oxidation reaction, in which case they cannot be totally regenerated. The excess blocking agent is removed by dialysis or any other appropriate 5 treatment.
The periodate oxidation reaction is carried out at an acid pH of between 3 and 7, preferably of between 5 and 6.5.
The periodate is used in excess; more particu-10 larly, the concentration of alkali metal periodate is greater than the concentration of the vicinal diols capable of being oxidized; concentrations of 10 to 50 m M in respect of sodium periodate for concentrations of 1 to 10 mg/ml of cytotoxic sub-unit are suitable. The 15 treatment, carried out at a temperature of between 0 and °C, preferably of between 1 and 5°C, and in the dark, takes between 0.2 and 24 hours.
The reaction is stopped by the addition of a reagent which consumes the remaining periodate, for 20 example an excess of ethylene glycol, and the by-pro ducts are removed by dialysis or by any other appropriate treatment. The product obtained at the end of the reaction is isolated by conventional techniques.
If the thiol groups of the starting material 25 have been blocked, unblocking is effected by the known methods, for example by reaction with a reducing agent capable of freeing the previously blocked thiol group, such as 2-mercaptoethanol, giving the new glycoprotein which inactivates ribosomes and has a prolonged action, 30 ready to be used for example for coupling with an anti body to give an immunotoxin.
In the case of the A chain of ricin, the resulting new molecule (referred to by the symbols A-La) possesses the following main properties: 35 - a molecular weight which is not significantly differ- ent from that of the native A chain. As far as it is possible to see by polyacrylamide gradient electrophoresis, this modification process only produces polymers of the protein in a very small quantity 5 and does not produce any degradation products. - a proportion of free thiol groups greater than 0.7 per mo 1. - an immunoreactivity towards rabbit antibodies inhibiting the A chain of ricin which is indistinguish- able from the immunoreactivity of the native A chain. - an inhibitory activity on the protein synthesis in an acellular model which is greater than 50% of that caused by an equal quantity of native A chain. - finally, after a single intravenous administration 15 to rabbits at a dose of about 0.4 mg/kg of body weight, the plasma level of the prolonged-action A chain (A-La) 23 hours after injection is greater than 10% of the level present at time zero (as against 0.015% for the native A chain at this time) i.e. an increase in the 20 plasma level by a factor very much greater than 500 .
Likewise in the case of gelonine, the molecule obtained by periodate oxidation possesses the following main properties: - a molecular weight which is not significantly differ-25 ent from that of the native gelonine. - an immunoreactivity towards anti-gelonine rabbit antibodies which is indistinguishable from that of the native gelonine. - finally, after a single intravenous administration to 30 rabbits at a dose of about 0.3 mg/kg of body weight, the plasma level of the modified gelonine 24 hours after injection is greater than 3% of the level present at time zero (as against 0.01% for the native gelonine at this time) i.e. an increase in the plasma level by a 35 factor greater than 200 .
The preparation of the conjugates or immunotoxins from the glycoproteins which inactivate ribosomes and have a prolonged action is carried out by any process suitably chosen from the range of processes 5 described in U.S. Patent Specification No. 4 340 535. If the chosen cytotoxic sub-unit naturally contains at least one thiol making it suitable for coupling, this group will preferably be used by reaction with the antibody or antibody fragment carrying an activated disulfide 10 group. If the chosen cytotoxic sub-unit does not naturally possess a thiol group making it suitable for coupling, at least one functional group carrying a free thiol can preferably be introduced artificially into the said sub-unit by any known process and the coupling can be continued as indicated above. The introduction of the said functional group can take place either before the oxidation step with periodate ions, in which case it will be necessary for the thiol radical to be blocked during the oxidation step and 20 then unblocked after this step, or after the oxidation step .
This gives modified immunotoxins which have acquired a new character as regards their pharmacokinetic properties. More particularly, by appropriate 25 modification of the cytotoxic sub-unit, it has been possible to add to the specific cytotoxicity properties of immunotoxins, without interfering with them, a new property which is just as intrinsic, namely the capacity to show slow plasma elimination kinetics. 30 The examples which follow provide a clearer understanding of the invention without limiting its scope.
Example 1: Oxidation of the methylated A chain in which the SH 35 groups are blocked with N-ethylmaleimide. 1 - Preparation of the correctly functional i. zed A chain of ricin 1) Hexamethylation of the A chain The methylation reaction is carried out at 0°C, 5 with stirring, in 0.2 M borate buffer of pH 10, by the method of Means and Feeney (Biochemistry 7, 2192 (1968)). 20 mg of tritiated borohydride (containing 9.5 mCi/mmol) are added to 35 ml of A chain (3 mg/ml), followed by 350 microliters of 6% formaldehyde (added 10 in five 70 microliter portions spread over a period of minutes).
The excess reagent is removed by continuous dialysis against 125 mM phosphate buffer of pH 7 (40 1 at 300 ml/h). After dialysis, the protein solution is 15 centrifuged. 36.5 ml of hexamethylated A chain con taining 2.6 mg/ml are collected. 2) Blocking with N-ethylmaleimide The natural SH of the hexamethylated A chain is blocked by the method described in Methods in 20 Enzymology 11, 541 (1967). To do this, the A chain of ricin obtained in the previous step is incubated for 2 hours at 30°C in the presence of 20 equivalents of N-ethylmaleimide per mol of A chain. The excess reagent is removed by continuous dialysis against 125 mM phosphate buffer of pH 7, which is renewed for 20 hours at a rate of 500 ml/hour.After concentration, there is obtained 13 ml of a solution of A chain of ricin containing 7 me/ml and no longer possessing thiol groups which can be determined by ELLMAN1s reagent. The product thus obtained 30 is subsequently called hexamethylated A chain (NEM). 3) Periodate oxidation 6 ml of the solution of hexamethylated A chain (NEM) obtained above are treated with NalO^ (12.8 mg) for 40 minutes in the dark, at pH 4.5 and at 0°C. The reaction is 35 stopped by the addition of 600 microliters of ethylene glycol and the reaction medium is dialyzed continuously against 0.1 M carbonate buffer of pH 10 (20 h at 500 ml/h).
II - Enzvmntic activity of the prolonged-action A chain, measured on an acellular model The fundamental biological property of the A chain of ricin is to inhibit protein synthesis in cells by degradation of the ribosomal sub-unit 60S.
The in vitro protocol involves the use of appropriately complemented, subcellular fractions of rat liver capable of incorporating ^C-phenylalanine in the presence of an artificial messenger RNA: poly-uridylic acid.
The procedure employed for preparing the subcellular fractions and measuring the incorporation of 14 C-phenylalanine is an adaptation of the method described in Biochemica Biophysica Acta 1973, 312, 608-615, using both a microsomal fraction and a cytosol fraction of the rat hepatocytes. The sample containing the A chain is introduced in the form of a solution appropriately diluted in a 50 mM Tris HC1 buffer of pH 7.6 containing 0.2% of 2-mercaptoethanol and 15 micrograms/ml of bovine serum albumin.
The count data are used to calculate, relative to a control medium without inhibitor, the percentage 14 inhibition of the incorporation of C-phenylalanine into the proteins for each reaction medium containing A chain of ricin.
The inhibitory activity was determined. An IC^q of 2.7 • 10 ^ mol/1 is observed for the oxidized A chain. The ICc.n of the control A chain in the ex- -10 periment is 1.03*10 niol/l; therefore, the modification does not cause a loss of activity of the A chain. Example 2: This example demonstrates the slow elimination of the A chain of ricin modified with sodium periodate, after intravenous injection into the animal. I - Modification of the A chain of ricin with sodium periodate 1) Blocking of the natural SH with DTNB The A chain of ricin was prepared and purified in the manner indicated in U S. Patent Specification No. 4 340 535. 20 equivalents of a solution of 2,2'-dinitro-5,51-dithio-dibenzoic acid (DTNB), i.e. 385 microliters of a 0.1 M solution of DTNB in a 125 mM phosphate buffer of pH 7 (this solution is brought to pH 7 with sodium hydroxide), are added to 10 ml of a solution of A chain of ricin containing 5.6 mg/ml (with 0.84 thiol group per A chain) in PBS buffer (a buffer 20 mM in respect of phosphate and 150 mM in respect of NaCl, of pH 7). Incubation is left to proceed for 20 minutes at 20°C. The solution is then dialyzed against PBS buffer at 4°C to give 53 mg of A chain blocked on the thiol group, as a solution containing 5 mg/ml. 2) Periodate oxidation of the blocked A chain 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 of blocked A chain, brought to pH 6 with 1 M acetic acid. Incubation is left to proceed for 16 hours at 4°C in the dark. The oxidation reaction is stopped by the addition of 620 microliters of a 1 M aqueous solution of ethylene glycol. After incubation for 15 minutes at 20°C, the reaction medium is dialyzed at 4°C against PBS buffer. The periodate oxidation produces a slight precipitate of protein, which is removed by centrifugation at 10,000 x g for 30 minutes.
This gives 24 mg of oxidized blocked A chain at a concentration of 3.4 mg/ml. 3) Unblocking of the thiol groups 2-Mercaptoethanol is added as a reducing agent, at a final concentration of 1%, to 6 ml of oxidized blocked A chain containing 3.4 mg/ml in PBS buffer. Incubation is left to proceed for 1 hour at 20°C. The solution is then dialyzed against PBS buffer at 4°C.
This gives 19 mg of oxidized A chain at a concentration of 2.8 mg/ml.
Using the DTNB technique (Methods in Enzymology, 1972, 25, 457 (Academic Press)), it is determined that the modified A chain obtained has 0.70 free thiol group 10 per mol. The molecular weight of the modified A chain is 30 , 000± 1 , 000 , rietcrini ncd by polyncrylnmi.do gradient electrophoresis in the presence of sodium dodecyl-sulfate.
The previously obtained preparation of A chain 15 in which the polysaccharide units have been oxidized was studied for its enzymatic activities in the inhibition of protein synthesis and for its pharmacokinetic properties.
II - Enzvmatic activity of the prolonged-action A chain, 20 measured on an acellular model The inhibitory activity was determined by the technique described in example 1. An of 3 x 10"10 mol/1 is observed for the oxidized A chain. The IC^q of the control A chain in the experiment is 1.2 x 10-10 25 mol/1; therefore, the modification does not cause a loss of activity of the A chain.
III - Pharnacokinetic properties of the prolonged-action A chain (A -La) The A chain is administered to rabbits by means 30 of a single injection into a vein in the ear. The quantity of A chain injected corresponds to 0.415 mg/kg. Blood samples are taken at intervals on heparin. The plasmas are analyzed with the aid of a radioimmunometric test designated below by the abbreviation RIM-1. 35 This technique has the advantage of determining the A chain without modifying it. This determination is carried out in microtitration plates (for example: "N'U.\'C-TSP screening system" from Poly Labo Block France), the lid of which carries hyperabsorbent spikes which dip into the cavities in the base. These spikes constitute the solid phases. Ewe antibodies inhibiting A chain of ricin (designated below 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 in PBS buffer are divided up into the cavities. The spikes are brought into contact firstly with the solution of Acl for 24 h at 4 ° C and then with fetal calf serum for 3 h at 20°C in order to saturate all the fixation sites. The saturated immunoabsorbent is then brought into contact for 3 h at 20°C with the plasma samples to be determined at different dilutions, or with solutions of A chain of known concentrations in order to establish the calibration curve. After washing with a PBS buffer, the immunoabsorbent is brought into contact for 2 h at 20°C with the ewe antibodies inhibiting A chain of ricin, which have been purified by affinity chromatography and radiolabeled (designated below by the abbreviation Ac2). The radiolabeling of the Ac2 is effected 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 radiolabeled Ac2 antibodies is 5 to 10 microcuries/microgram. 10 cpm of radiolabeled Ac2 antibodies are introduced as 200 microliters into a PBS buffer containing 0.1% of bovine serum albumin.
After washing in PBS buffer, the spikes are detached and the quantity of bound Ac2 is measured by counting the radioactivity. The concentration of A chain in the samples to be determined is measured by reference to the calibration curve established by introducing the A chain at different known concentrations. When prolonged-action A chain is injected into the animal, this same prolonged-action A chain is used to establish the corresponding calibration curve.
The values of the concentration of A chain in the blood plasma measured by this technique are reproducible and reliable. The detection threshold is 1 nanogram/ml. A study of the reproducibility within and between experiments gives coefficients of variation 10 of 1 ess than 10% for concentration values within the range from 1 to 200 nanograms/ml.
The results of these experiments are represented in the form of curves in which the time, expressed in hours, is plotted on the abscissa and the plasma con-15 centration of the product measured, recorded in per cent of the theoretical plasma concentration at time zero, is plotted on a logarithmic scale on the ordinate. This value, called the "relative plasma concentration" (RPC), is calculated using the following expression: 20 concentration measured at time t RPC = X 100 quantity injected/plasma volume The plasma volume is considered to be equal to 36 ml/kg of the animal's body weight.
Figure 1 shows the plasma elimination curve, as a function of time, for the A chain of native ricin injected intravenously. This curve (curve 1) has two phases: in the first phase, the product disappears very rapidly from the bloodstream since only 0.1% of the dose 2q administered remains in the plasma 3 hours after injec tion. In the second phase, the decrease is slower.
When the A chain has been oxidized on its polysaccharide units, the elimination profile is profoundly modified: the first elimination phase - which is res-35 ponsible for the disappearance of the majority of the product - is practically suppressed, which leads to a considerable increase in the plasma levels of A chain. Twenty hours after injection, the concentration of the oxidized A chain is 600 times greater than in the case 5 of the unmodified A chain (curve 2).
Example 3: This example demonstrates the effect of periodate oxidation on the pharmacokinetic properties of the A chain blocked with NEM. 10 1 ) - Modification of the A chain of ricin a) Blocking of the natural SH with N-ethylmaleimide 0 ml of an aqueous solution of A chain of ricin containing 8 mg/ml (i.e. 4.1 micromol of A chain) are treated with an aqueous solution of 2-mercaptoethanol 15 so that the final concentration is 1 per cent.
The solution is left to stand for one hour and then dialyzed continuously against 125 mM phosphate buffer of pH 7, which is renewed for 40 hours at a rate of 300 ml/hour. Using Ellman's method, 0.9 equivalent 20 of SH was determined per mol of A chain of ricin.
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 step is incubated for 2 hours at 30°C 25 in the presence of 20 equivalents of N-ethylmaleimide per mol of A chain. The excess reagent is removed by continuous dialysis against 125 mM phosphate buffer of pH 7, which is renewed for 20 hours at a rate of 500 ml/hour. This gives 35 ml of a solution of A chain of 30 ricin containing 7 mg/ml and no longer possessing thiol groups which can be determined by Ellman's reagent. The product thus obtained is subsequently called A chain (NEM). b) Periodate oxidation of the A chain (NEM) Periodate oxidation of the A chain (NEM) is carried out using the procedure indicated in example 2. 2) - Properties of the oxidized A chain (NEM) a) Enzymatic activity of the oxidized A chain (NEM) The inhibitory activity on the protein syn-5 thesis was determined using the procedure described in example 1. The enzymatic properties are found to be maintained with an IC^q of 4.3*10~ mol/1 for the oxidized A chain (NEM). b) Pharmacokinetic properties of the oxidized A chain 10 (NEM) The oxidized or unoxidized A chain (NEM) is administered to rabbits by a single injection into a vein in the ear. The quantity of A chain injected corresponds to 0.100 mg/kg. The plasma samples collect-15 ed at time 23 h are analyzed using the immunanetric test RIM-1 as described in example 2. The results are shown in the table below: Plasma concentration 23 h after injection: A chain (NEM) Oxidized A chain (NEM) 0.01% 8% Twenty-three hours after injection, the concentration of the oxidized A chain (NEM) is 800 times greater than in the case of the unmodified A chain (NEM). Example &: This example demonstrates the importance of the duration of the oxidative treatment on the pharmacokinetic properties of the oxidized A chain.
Six preparations of oxidized A chain are prepared using the procedure indicated in example 2, except for the duration of the sodium periodate treatment. The treatment times are as follows: zero (reaction stopped immediately with ethylene glycol), 20 minutes, ^0 minutes, 2.5 hours, U hours and 18 hours.
These various preparations are injected into rabbits and the relative plasma concentration of the A 5 chain is measured after 23 hours by the same procedure as in example 1.
The results are shown in figure 2, on which the periodate treatment time is plotted as hours on the abscissae and the RPC is plotted on a logarithmic scale on the ordinates. These results indicate that 1) the increase in the plasma level of the A chain is indeed due to periodate oxidation because, when the reaction is stopped immediately, the plasma concentration of A chain is identical to that obtained for the native A chain, and 2) it is necessary for the duration of this reaction to be relatively long in order to obtain optimum effects. Example 5 : This example demonstrates the importance of the duration of the oxidative treatment on the pharmaco-20 kinetic properties of the methylated A chain blocked with NEM. 1) - Preparation of the functionalized A chain of ricin a) Blocking of the natural SH of the A chain with N-ethylmaleimide The natural SH of the A chain is blocked with N-ethylmaleimide by the same procedure as that described in example 1. b) Methylation of the A chain The methylation reaction is carried out at 0°C, 30 vith stirring, in 0.2 M borate buffer of pH 10, by the method of Means and Feeney (Biochemistry 7, 2192, 1968). 38 mg of tritiated borohydride (containing U7 mCi/mmol) are added to 65.5 ml of A chain (NEM) (3 mg/ml), followed by 1 ml of 6% formaldehyde added in five 200 micro-35 liter portions spread over a period of 30 minutes.
The excess reagent is removed by discontinuous dialysis against 125 mM phospate buffer of pH 7 C*0 ml).
After dialysis, the protein solution is centrifuged. 63 ml of methylated A chain containing 3 mg/ml are collected . c) Periodate oxidation 5 Six preparations of methylated A chain (NEM) are oxidized using the procedure indicated in example 1, except for the duration of the sodium periodate treatment. The treatment times are as follows: zero (reaction stopped immediately with ethylene glycol), 10 10 minutes, 40 minutes, 2.5 hours, 4 hours and 18 hours.
These various preparations are injected into rabbits and the relative plasma concentration ot the A chain is measured after 23 hours by the same procedure as in example 2.
The results are shown in figure 3, curve 2.
These results indicate that, as for the A chain (curve 1): 1. the increase in the plasma level of the methylated A chain (N'EM) is indeed due to periodate oxidation because, when the reaction is stopped immediately, the plasma concentration of methylated A chain (NEM) is identical to that obtained for the A chain j 2. it is necessary for the duration of this reaction to 25 be relatively long in order to obtain optimum effects.
Example 6: This example demonstrates that, when carried out separately on the two constituent molecular variants of the A chain (A1 chain and A2 chain), the oxidation reaction 30 produces effects on each of the two isomers which are analogous to those described in example 2 for the A chain of ricin. 1) - Separation of the A1 and A2 chains 28 ml of A chain containing 10.9 mg/ml (309 mg) 35 in 125 mM phosphate buffer of pH 7.0 are deposited on a colunn of 112 ml of concanavalin A/sepharose, equilibrated in the same buffer. The A1 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 in respect of NaCl and 0.1 M in respect of alpha-methylmannoside.
Thiii gives 184 mg of Al chain and 103 mg of A2 chain.
The Al and A2 chains are concentrated by ultrafiltration under nitrogen pressure; the A2 chain is dialyzed against 125 mM phosphate buffer of pH 7.0.
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 of MW 30,000 and the A2 chain corresponds to the band of weak intensity and of MW 33,000. 2) - Modification of the AT and A2 chains of ricin with sodiu~ periodate This modification is effected as described in example 2. The preparations of A chain in which the polysaccharide units have been oxidized were studied for their enzymatic activities in the inhibition of protein synthesis and for their pharmacokinetic properties. 3) - Enzymatic activities of the prolonged-action Al and A2 chains, measured on an acellular model The inhibitory activity was determined as described in example 1. The ICcq observed is equal to 2.1*10 ^ mol/1 and 2.1*10 * mol/1 for the oxidized Al and A2 chains respectively. The values of the native Al and A2 chains, which are the controls in the experiment, are 1.9*10"^ mol/1 and 1*10"^ mol/1 respectively. Therefore, the modification of the separate variants of the A chain does not cause a loss of their enzymatic activity. 4 ) - Pharmacokinetic properties of the prolonged-action Al and A2 chains (Al-La, A2-La) The Al or Al-La chain or the A2 or A2-La chain is administered to rabbits by a single injection into a vein in the ear (415 micrograms of A chain/kg). The plasma samples collected after 20 hours are analyzed with the aid of the immunoaetric test RIM-1 (see 10 example 2). The results are shown in the table below. The values for the A and A-La chains are indicated by way of comparison.
Relative plasma concentration 20 hours after injection A chain 0.012% Oxidized A chain (A-La) % A 1 chain 0.02% Oxidized Al chain (Al-La) % A 2 chain 0.04% Oxidized A2 chain (A2-La) 14% Twenty hours after injection, the concentrations 25 of Al-La and A2-La are respectively 500 and 350 times greater than in the case of Al and A2.
Example 7 : This example describes the biochemical characteristics of the A chain and its variants,the Al chain and 30 A2 chain, in the native form and in the oxidized form.
The A chains used in these studies are prepared as described in examples 2 and 6.
I - Carbohydrate compositions The carbohydrate compositions of these proteins 35 are determined by gas chromatographic analyses using Clamp's method (in Glycoproteins: their composition, structure and function (edited by A. Gottschalk), volume 5 A, p 300-321, Elsevier Publishing Co., Amsterdam, London, New York).
The results obtained are collated in the two tables below.
Percentage composition Chains Total carbohydrates, % Native A Al A2 Oxidized A Al A 2 .58 + or - 0.5 4.54 + or - 0.5 6.24 + or - 0.5 2.27 + or - 0.5 2.07 + or - 0.5 3.33 + or - 0.5 1 5 Molar composition (On the basis of a molecular weight of 30,625, the results are given with an average precision of + or -0.5 residue per molecule) Monosaccharides Chains Native Oxidized 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 .2 1.43 1.29 2.26 Fucose 1.37 1.41 1 .52 0 0 0 Xylose 1.6 1.48 1.67 0.36 0.48 0.62 These results prove that periodate oxidation has destroyed part of the sugars of the A chain. Per molecule of A chain, there is an average decrease of 3.17, 2.11 and 2.94 mannose residues, the fucose residues have completely disappeared and there is an average decrease of 1.24, 1 and 1.05 xylose residues for the A, Al and A2 chains respectively. The N-acetvlglucosamine 5 residues are only slightly degraded.
II - N-Terminal sequence The sequence of the N-terminal amino acids of the A chain and its Al and A2 variants, in the native form and in the oxidized form, was established with a protein 10 sequencer by the procedures known to those skilled in the art. The results obtained are collated in the table below.
Chains Sequence of the 9 N-terminal amino acids Native A Al A2 Ile-Phe-Pro-Lys-Gln-Tvr-Pro-Ile-Ile Ile-Phe-Pro-Lys-Gln-Tyr-Pro-Ile-Ile Ile-Phe-Pro-Lys-Gln-Tyr-Pro-Ile-Ile Oxidized A Al A 2 Ile-Phe-Pro-Lys-Gln-Tyr-Pro-Ile-Ile Ile-Phe-Pro-Lys-Gln-Tyr-Pro-Ile-Ile Ile-Phe-Pro-Lys-Gln-Tyr-Pro-Ile-Ile It is found that the sequences of the 9 N-terminal amino acids of the native and oxidized A, Al and A2 chains are strictly identical to one another, which demonstrates that the oxidative treatment leaves the protein chain intact. It is also found that the sequence 25 of the 9 N-terminal amino acids of the A chains is strictly identical to that previously described by Funatsu for the A chain of ricin (Agric. Biol. Chem., (1979), 43, 2221).
Ill - Affinity on concanavalin A/sepharose 30 The A chain, the A chain blocked with DTNB [A(DTNB)] and the A(DTNB)-La, Al(DTNB), Al(DTNB)-La, A2(DTNB) and A2(DTNB)-La chains are tested by their capacity to fix to concanavalin A/sepharose. 1 ml of a solution of A chain containing about 1 mg/ml is deposited on a column of 1 ml of concanavalin A. Chromatography is followed by measurement of the optical density at 280 nm. After washing with 125 mM phosphate 5 buffer of pH 7.0 until the first peak which is not retained by the concanavalin A has returned to the base line, the column is washed with 0.1 M borate buffer of pH 6.0, which is 0.5 M in respect of NaCl and 0.1 M in respect of alpha-methylmannoside. The 15 results, expressed as a percentage of the optical den sity at 2 SO p. in are summarized in the table below.
Chains % not retained % eluted by alpha- Total by the con A methylmannoside (%) A 53 27 80 A(DTNB) 66 11 77 A( DTNB )-La 78 83 A 1 ( DTN'B) 80 6 86 A 1 ( DTN B )-La 73 0.4 73.4 A 2(DTNB) 70 95 A 2 (DTNB )-La 64 9 73 It is known that concanavalin A has an affinity for glycoproteins with terminal mannoses. It is found that the A chain which contains such residues can bind to con A. This is particularly clear in the case of the A2 chain, which is the isomer more richly substituted with mannose. It is also found that the oxidative treatment destroys this affinity, which is coherent with the destruction of the sugar residues by a treatment of this type .
IV - Determination of the E l%o The absorption coefficient at 280 nm (E \%J) is the optical density at 280 nm of a solution containing 1 mg/ml, in which the protein concentration is determined by the FOLIN test with a standard range of bovine serum albumin.
The results are summarized in the table which follows: A chain 0.65 A(DTNB) chain A(DTNB)-La chain 1.12 1 .04 A 1(DTN B) chain A1(DTNB)-La chain 1 . 07 1.02 A2(DTNB) chain A 2(DTN B)-La chain 0.95 0.95 Blocking of the thiol group of the chain with DTNB produces a substantial increase in the absorption at 280 nm, due to the introduction of the nitrobenzoyl group.
After oxidation, no significant variation in the absorption at 280 nm is observed, demonstrating that oxidation has not affected amino acids responsible for the absorption at 280 nm.
V - Isoelectric focusing Analysis of the A chain by isoelectric focusing produces a set of bands with isoelectric points (pi) which are between 7.5 and 8.0 and are identical for the A, Al and A2 chains.
Blocking of the cysteine of the A chain with DTNB causes a widening of the bands towards the acid region; freeing of the cysteine with mercaptoethanol brings these bands back to the location of the native A chain.
Thus, a comparison of the isoelectric points of the native and oxidized A, Al and A2 chains shows that, in the absence of a blocking agent, all the bands characteristic of the A chain are transferred by 0.5 pH unit towards acid pH values. This transfer takes place with-5 out overlapping of the pH regions of the native and oxidized A chains, which seems to indicate that all the A chain molecules are affected by oxidation.
Example 8: This example demonstrates 1) the rapid elimin-10 ation of native gelonine, and 2) the slow elimination of gelonine modified with sodium periodate, after intravenous injection into the animal.
I - Modification of gelonine with sodium periodate The gelonine was prepared and purified from 15 Gelonium multiflorum by the method which has been des cribed (J. Biol. Chem. (1980) 255, 6947-6953). The oxidation reaction is carried out under the same conditions as those described for the A chain of ricin in example 2, except that the step in which the thiols are blocked with DTNB is omitted.
In fact, as the coupling of gelonine with the antibody is not generally performed using natural thiol groups of the gelonine, the thiol groups will be introduced artificially, after the oxidation .step, by the technique described in Cancer Res., 1984, 44, 129-133. 21 microliters of a 0.5 M solution of sodium periodate in water are added to 1 ml of a solution containing 3 mg/ml of gelonine in PBS buffer, brought to pH 6 with 1 M acetic acid. Incubation is left to proceed for 16 hours at 4°C in the dark. The reaction is stopped by the addition of 105 microliters of a 1 M aqueous solution of ethylene glycol. After incubation for 15 minutes 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 gives 2.9 mg of oxidized gelonine at a concentration of 2.5 mg/ml.
Like the A chain of ricin, the fundamental property of gelonine is to inhibit protein synthesis in eucaryotic cells by degradation of the ribosomal sub-5 unit 60 S (Biochem. J. (1982) 207, 505-509). In the case of gelonine too, the modification due to periodate oxidation does not cause a loss of activity. II - Pharmacokinetic properties of prolonged-action genonine N'ative gelonine or gelonine modified by the procedures explained above is administered to rabbits by a single injection into a vein in the ear. The quantity of gelonine injected is between 0.3 and 0.4 mg/kg. 31ood samples are taken at intervals on heparin. The 15 plasmas are analyzed with the aid of a radioimmunometric test designated below by the abbreviation RIM-2.
This test is performed by the same technique as used for the test RIM-1, except that the solution Acl here is a solution of anti-gelonine rabbit antibodies 20 purified by affinity chromatography, the Ac2 antibodies being the same antibodies radiolabeled. The radio-labeling procedure is identical to that described for the technique RIM-1. The concentration of native gelonine or modified gelonine in the samples to be 25 determined is measured by reference to a calibration curve established by introducing native or modified gelonine at different known concentrations. The test RIM-2 has the same reliability and reproducibility characteristics as described for the technique RIM-1. 30 The results of these experiments are represented in the same way as for the A chain of ricin in example 2 .
Figure 4 shows the plasma elimination curves, as a function of time, for native gelonine and oxidized gelonine, injected intravenously. The native gelonine, 35 like the A chain of native ricin, disappears very rapidly from the bloodstream since 99.99% of the gelonine present in the bloodstream disappears in 24 hours (curve 1).
When the gelonine has been oxidized on its polysaccharide units, the elimination profile is profoundly modified: 5 24 hours after injection, the concentration of the oxidized gelonine is 300 times greater than that of the native gelonine (curve 2).
Thus, as for the A chain of ricin, these results prove that periodate oxidation has modified the sugars 10 involved in the recognition process responsible for the elimination of the gelonine, to the point of preventing this recognition.
Example ^: This example demonstrates: 1. the rapid elimination of GPIR MOM extracted from Momordica charantia, and 2. the slow elimination of GPIR MOM modified with sodium periodate, after intravenous injection into the animal. 20 1) _ Modification of GPIR MOM with sodium periodate The GPIR MOM was prepared and purified from the endosperm of Momordica charantia seeds by the method which has been described (Biochemical Journal (1980), 186, 443-452). The pharmacokinetic properties of native 25 or modified MOM were established using radioactive GPIR MOM. The MOM is radiolabeled on the tyrosines with radioactive iodine-125 in the presence of chloramine T. 10 microliters of a solution of radioactive iodine-125 containing 100 jiCi/ml, and 30 microliters of a solution 30 of chloramine T containing 2.5 mg/ml in water, are added to 100 microliters of a solution containing 1 mg/ml of MOM in PBS buffer. The reaction is left to proceed for one minute at ambient temperature. The reaction is stopped by the addition of 400 microliters of a solution 35 of sodium metabisulfite containing 0.5 mg/ml. The re action medium is chromatographed by gel filtration on a column of Sephadex G25 with PBS buffer containing 0.1% of gelatine in order to separate the radiolabeled protein from the unreacted iodine. After centrifugation at 10,000 x g for 30 minutes, this gives 80 micrograms of radiolabeled MOM at a concentration of 0.04 mg/ml.
The oxidation reaction is carried out under the sarce conditions as described for the A chain of ricin in example 2, except that the step in which the thiols are blocked with DTNB is omitted and the concentration of protein is 125 times smaller (40 /m 1). 20 microliters of a 0.5 M solution of sodium periodate in water are added to i ml of a solution containing 0.04 mg/ml of radiolabeled MOM, brought to pH 6 with 1 M acetic acid. Incuoation is left to proceed for 16 h at 4°C in the dark. The reaction is stopped by the addition of 100 microliters of a 1 M aqueous solution of ethylene glycol. After incubation for 15 minutes 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 gives 32 micrograms of oxidized MOM at a concentration of 0.021 mg/ml.
The new molecule thus obtained has a molecular weight which is not significantly different from that of the native MOM. As far as it is possible to see by polyacrylamide gradient electrophoresis after development with coomassie blue or radioautography, the modification process only produces protein polymers in a very small quantity and does not produce any degradation product. 2) - Pharmacokinetic properties of prolonged-action MOM The radiolabeled MOM, whether or not oxidized by the procedures explained above, is administered to rabbits by a single injection in a vein in the ear. The quantity of MOM injected is between 3.50 and 3.55 micrograms/kg. Blood samples are taken at intervals on heparin. The plasmas (200 microliters) are 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 is determined. This method of analysis makes it possible to measure the plasma level of the intact MOM molecules, and any low-molecular degradation products which cannot be precipitated by TCA are not taken into account.
The results of these experiments are represented as the jjercL-ii tugo of the initial radioactivity remaining in the bloodstream as a function of time. This value, which is called the "percentage of the initial plasma radioactivity" (% IPR), is calculated using the following expression: r x PV x 100 % I.P.R. = 0.2 x R where: r = radioactivity measured at time t in 0.2 ml o r plasma , R = total radioactivity injected, PV = plasma volume (considered to be equal to 36 ml/kg of the animal's body weight).
The plasma elimination curves, as a function of time, for the oxidized or unoxidized MOM after intravenous injection are shown in figure 5. The MOM, like the A chain of native ricin, disappears very rapidly from the bloodstream since 99.9% of the MOM present in the bloodstream disappears in 8 hours (curve 1). When the MOM has been oxidized on its sugar residues, the elimination rate is reduced (curve 2): 8 hours after injection, level of oxidized MOM is 60 times greater than that of the unoxidized MOM. These results prove that periodate oxidation has modified the sugars involved in the recognition process responsible for the rapid elimin- ation of the MOM.
Example 10: This example demonstrates: 1. the rapid elimination of GPIR Dianthin ex-5 tracted from Dianthus caryophy11 us,and 2. the slow elimination of GPIR Dianthin modified with sodium periodate, after intravenous injection into the animal. 1 ) - Modification of Dianthin 30 with sodium periodate 10 The Dianthin 30 was prepared and purified from the leaves of Dianthus caryophyllus by the method which has been described (Biochemical Journal (1981), 195, 339-405). The pharmacokinetic properties of oxidized or unoxidized Dianthin 30 were established using radio-15 active Dianthin. The iodination and oxidation reactions are carried out under the same conditions as described for MOM in example 9.
The new oxidized Dianthin molecule thus obtained has a molecular weight which is not significantly differ-20 ent fro.r. that of the native Dianthin 30. 2) - P h n r m >'i c o kinetic properties of prolonged-action Dianthin 30 The oxidized or unoxidized radiolabeled Dianthin is administered to rabbits by a single injection into a 25 vein in the ear. The plasma level of Dianthin is meas ured by the same procedure as described for MOM in example 9. Figure 6 shows the plasma elimination curves, as a function of time, for the oxidized Dianthin (curve 2) or unoxidized Dianthin (curve 1). Dianthin 30, like 30 MOM, disappears very rapidly from the bloodstream since 99.9% of the quantity initially present disappears in 2 hours. On the other hand, when the Dianthin 30 has been oxidized on the carbohydrate residues, the elimination kinetics are slowed down considerably: 2 hours after injection, the Dianthin level is 80 times greater than that of the unoxidized Dianthin. The level of oxidized Dianthin remains high beyond 24 hours (3% of the initial value at 24 hours).
Here again, these results prove that periodate 5 oxidation has modified the sugars involved in the recog nition process responsible for the rapid elimination of the Dianthin.
PL x a m p 1 e 11 : Conjugate obtained by the reaction of an antibody inhib-10 iting human T cells (an antibody directed against the antigen T65), substituted by activated disulfide groups, with the oxidized A chain of ricin. a) Antibody inhibiting human T cells (or antibody T101) This antibody was obtained by the method des-15 cribed in Journal of Immunology, 1980, 125(2), 725-737. b) Oxidized A chain of ricin: The A chain of ricin was prepared in the manner indicated in example 2.
II) Activated antibodies inhibiting human T cells microliters of a solution containing 60.3 20 n; g / m 1 of l-ethyl-3-dimethylaminopropyl-3-carbodiimide are added to 100 microliters of a solution containing 20 mg/ml of 3-(pyridin -2- yldisulfanyl)propionic acid in tert.-butanol, and the mixture is left for 3 minutes at ambient temperature. 68 microliters of the solution 25 thus obtained are 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 30 a concentration of 7.9 mg/ml. By spectrophotometric analysis at 343 nm of the pyridine-2-thione released by exchange with 2-mercaptoethanol, it is found that the antibody obtained carries 3.8 activated mixed disulfide groups per mol of antibody.
III) Preparation of the immunotoxin containing prolonged- action A chain of ricin 2.46 ml of modified A chain containing 2.87 mg/ mi are added Lo 1.5 ml of the solution of activated antibody obtained above (concentration: 7.9 mg/ml, i.e. 11.8 mg of activated antibodies) and the mixture is incubated for 20 hours at 20°C. The solution is cen-trifuged and then purified by filtration on a Sephadex G100 column, the optical density of the effluent being measured at 280 nm. Combination of the fractions con-10 taining both the antibody and the A chain gives 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.
The average degree of coupling in this prepara-15 tion is therefore 1.2 mol of oxidized A chain per mol of antibody .
The immunotoxin containing oxidized A chain of ricin, IT (A-la) T101, obtained as indicated above, was studied for its pharmacokinetic properties and its 20 specific cytotoxicity properties towards the target cells.
Example 1 2 : This example demonstrates the acquisition of the property of slow plasma elimination of the immunotoxins 2 R containing prolonged-action A chain of ricin, which are abbreviated to IT (A-La)TlOl.
I - Procedure The conjugate prepared by the procedure explained in example 11 is administered to rabbits by a single in-^ jection into a vein in the ear. The quantity injected corresponds to 0.415 mg/kg, expressed as A chain.
Blood samples ore taken at intervals on heparin. The plasmas are analyzed with the aid of a radioimmuncrnetric test with two sites, which is abbreviated below to RIM-3.
This test is carried out by the same technique as that used for the test RIM-1, except that the solution Ac2 here is a solution of goat antibodies inhibiting mouse IgG, purified by affinity chromatography and radiolabeled as described for the technique RIM-1.
The concentration of modified immunotoxin in the samples to be determined is measured by reference to a calibration curve established by introducing the modified immunotoxin at different known concentrations. The test RIM-3 has the same reliability and reproducibility 10 characteristics as described for the technique RIM-1.
By way of comparison, a control study is carried out under the same conditions with the conjugate called IT TlOl, which is obtained by the reaction of the same antibody TlOl, substituted by activated disulfide groups, 15 with the native A chain of ricin. The preparation and the cytotoxic properties of this conjugate have been described in French Patent Application no.2 516 794 The results of these experiments are represented in the same way as for the uncoupled A chain of ricin in ex-20 ample 2.
II - Results Figure 7 shows the plasma elimination curves, as a function of time, for IT TlOl and IT (A-La) TlOl, injected intravenously. Twenty-four hours after injection, 25 the concentration of active immunotoxin is 140 times greater for IT (A-La) TlOl than for IT TlOl. This fact demonstrates that the new pharmacokinetic properties of the oxidized A chain are retained after coupling with an antibody.
Example 13: This example demonstrates the retention of the specific cytotoxicity properties of IT (A-La) TlOl towards the target cells.
The fundamental biological property of the A 35 chain of ricin is to inhibit protein synthesis in cells by degradation of the ribosomal sub-unit 60S. The technique uses a cell model in which the effect of the 14 substances studied on the incorporation of C-leucine into cancerous cells in culture is measured. 5 The cells used belong to the CEM cell line derived from a human T leukemia which carries the antigen T65. The cells are incubated in the presence of the substance to be studied, and then, when incubation 14 has ended, the degree of incorporation of C-leucine by the cells treated in this way is measured.
This measurement is made by a technique adapted from the one described in Journal of Biological Chemistry 14 1974, 249(11), 3557-3562, using the tracer C-leucine to determine the degree of protein synthesis. The radio- activity incorporated is determined here on the whole cells isolated by filtration.
On the basis of these determinations, it is possible to draw the dose/effect curves, plotting, on the abscissa, the molar concentration of A chain in the substances studied, and, on the ordinate, the incorpora- 14 tion of C-leucine expressed as a percentage of the incorporation by control cells in the absence of any substance affecting protein synthesis.
It is thus possible to determine, for each sub- stance studied, the concentration which causes a 50% in- 14 hibition of the incorporation of C-lcucine, or "50% inhibitory concentration" (IC^q).
Figure 8 shows the curves obtained in the same experiment with IT (A-La) TlOl and the uncoupled oxidized A chain in the presence of 10 mM ammonium chloride in the incubation medium. It can be seen on this figure that the IT (A-La) TlOl has a very strong -1 2 cytotoxic activity (IC^q = 5.5*10 M) which is about 80,000 times greater than that of the uncoupled ox-35 idized A chain, measured under the same conditions.
Exannle 14; This example demonstrates the comparative cytotoxic efficacy of IT (A-La) TlOl and IT TlOl towards CEM target cells, measured in a clonogenic test.
Immunotoxins are dedicated to the eradication of every single one of the target cells. This performance can only be evaluated with a highly sensitive technique; tests for the inhibition of colony formation offer this possibility because a single surviving cell can be shown 10 up among several million dead cells. This is made possible by optimum culture conditions in a gelled medium, applied to the CEM human lymphoid line.
I - Technique for measuring cytotoxicity bv the inhibition of colony formation 15 The medium used for cloning is the medium RPMI 1640 to which 1 mmol/1 of sodium alpha-ketoglutarate, 1 mmol/1 of sodium oxaloacetate, 5% of inactivated fetal calf serum and 10% of inactivated horse serum are added. A first, 0.3% agar solution (Agarose type VII, SIGMA laboratories) 20 is prepared in this medium, placed as a thin layer in small Petri dishes and solidified at +4°C. The cells are mixed with a second, 0.275% agar solution kept at 3 7 J C , which is then deposited on the first layer and solidified. These concentrations of agar were chosen 25 after a preliminary study aimed at simultaneously optimizing the cloning efficiency, the size of the colonies and the consistency of the medium. After 15 days in the incubator, the colonies are counted using an automatic colony counter ( "ARTEK"*, DYNATECH , U.S.A.). To 30 determine the cloning efficiency and thus the exact number of cells surviving the immunotoxin treatment, it is essential to establish a calibration line showing the number of cells inoculated as a function of the number of colonies formed. We have proved that the cloning 35 efficiency indicated by this calibration line is prac * Trade Mark tically unaffected by the presence of a high proportion of dead cell:;, which is the situation naturally encountered when the cells are treated with the immunotoxin.
The immunotoxin treatment is carried out by in cubating the cells in the exponential growth phase and at a concentration of 10^/ml with the immunotoxin IT (A-La) TlOl or IT TlOl at different concentrations, in a total volume of 1 ml of the medium RPMI 1640 contain-10 ing 10% of inactivated fetal calf serum and 10 mmol/1 of ammonium chloride. The incubation takes place at 37°C under an atmosphere containing 5 % of carbon dioxide and with horizontal shaking of the test-tubes (2500 rpm with a "GIRAT0"Y G-2"*shaker, NEW-BRCN'SWICK) . The cells 15 are then washed and different dilutions are prepared, before mixing with the agar solution, so that the number of cells surviving can be measured in the zone of maximum sensitivity given by the calibration line. The results are expressed as the absolute number of cells 20 surviving, extrapolated from the cloning efficiency, using the following relationship: absolute number of cells surviving: C x d E where C is the number of clones per Petri dish, d is 25 the dilution factor of the cell preparation examined and E is the cloning efficiency established from the slope of the calibration line. Each point corresponds to the average of three tests.
II - Results Figure 9 shows the curves of the cytotoxic activity of the immunotoxins IT (A-La) TlOl and IT TlOl on the CEM cells, in the presence of 10 mM ammonium chloride, as a function of the immunotoxin concentration (expressed as the molarity of A chain). 35 It is apparent that the efficacies of these two products are of the same order of magnitude. The * Trade Mark - AO - resulting reduction in the number of cells is extremely large in both cases since, for concentrations as low as 10 M, the proportion of residual cells surviving is of the order of 0.001% of the initial value. This effect is highly specific since, at these concentrations, it was proved that the uncoupled A chain or a non-specific immunotoxin has no effect on these cells.
This example demonstrates that IT (A-La) TlOl has specific cytotoxicity properties which are virtually identical to those of conventional IT TlOl.
Example 15: Conjugate obtained by the reaction of an antibody inhibiting human T cells (an antibody directed against the antigen T65), substituted by activated disulfide groups, with the oxidized and functiona1ized A chain (NEM) of ricin, the coupling taking place between the activated disulfide groups and the functionalized sugar residues of the A chain. 1 ) - Preparation of the immunotoxin a) Preparation of the ftinctionnlizcd A chain The A chain is blocked with N-ethylmaleimide on its SH group and then oxidized for 18 hours by the procedure described in example 3 .
Coupling with cvstamine After dialysis against carbonate buffer of pH 9.5, 5.2 ml of a protein solution containing 4.65 mg/ml are 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 mol 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.
The excess reagent is removed by continuous dialysis against 125 mM phosphate buffer of pH 7. The disulfide bridge of the fixed cystamine is then reduced with 2-mercaptoethanol at a final concentration of 5 - Al - per cent for 1 hour at 30°C, this being followed by further continuous dialysis against 125 xM phosphate buffer of pH 7 (20 1 at 300 ml/hour). After dialysis :ind contrifugation, 0.25 SH per mol of A chain was 5 determined by Ellman's method. b) Preparation of the antibody (see example 11) c) Coupling reaction 1.5 ml of the solution of modified A chain of ricin (i.e. 0.058 micromol) are added to 211 micro-10 liters of the solution of activated antibody obtained above (i.e. 0.006 micromol). The mixture is left to react for 18 hours at 30°C. The reaction medium is then dialyzed against PBS buffer (10 mM in respect of phosphate and 140 mM in respect of sodium chloride, pll 7.A). After 15 centrifugation and examination by polyacrylamide grad ient electrophoresis, it is found that the immunotoxin obtained has an average degree of coupling for this preparation of 0.8 A chain (NEM) per mol of antibody. 2) - Properties of the immunotoxin IT (A(N'EM)-La-20 cvsteamine) TlOl Specific cytotoxicity activity It is found that this immunotoxin, prepared by the procedure explained above, has a very strong cytotoxic activity on the CEM target cells (IC-q = 1.2*10~^ 25 M, established by the method described in example 13). b) Plasma elimination The immunotoxin is administered to rabbits by a single injection into a vein in the ear (50 micrograms of A chain/kg). The plasma samples collected after 22 30 hours are analyzed with the aid of the immunoassay RIA- 3 (example 12). The results are shown in the table below. The values for IT TlOl are indicated by way of comparison.
Relative plasma concentration 22 hours after injection IT ( A( NEM)-La-cysteamine) T101 IT T101 2.4% 0.08% Twenty-two hours after injection, the concentration of IT containing modified A chain is 30 times greater than in the case of IT TlOl.
Example 16: Conjugate obtained by the reaction of an antibody inhibiting human T cells (an antibody directed against the antigen T65), substituted by activated disulfide groups, with the methylated, oxidized and functionalized A chain (NEM) of ricin, the coupling taking place between the activated disulfide groups and the modified sugar residues of the A chain. 1) - Preparation of the immunotoxin a) Preparation of the functionalized A chain The A chain is blocked with N-ethylmaleimide on its SH group and then methylated and oxidized for 18 hours by the method described in example 5.
Coupling with cystamine After dialysis against 0.1 M carbonate buffer of pH 9.5, 18.5 ml of a protein solution containing 2.5 mg/ml are 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 mol 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.
The excess reagent is removed by continuous dialysis against 125 mM phosphate buffer of pH 7. The disulfide bridge of the fixed cystamine is then reduced with 2-mercaptoethanol at a final concentration of 5 per cent for 1 hour at 30°C, this being followed by further continuous dialysis against 125 mM phosphate buffer of pH 7 (20 1 at 300 ml/hour). After dialysis and centrifugation, 0.32 SH per mol of A chain was determined by Ellman's method. b) Preparation of the modified antibody A solution containing 2.12 mg of N-succinimidyl 3-pyridin-2-vldithiopropionate in ethyl alcohol is added to 23.5 ml of a solution of antibody TlOl containing 4.4 mg/ml (i.e. 0.68 micromol). The mixture is stirred for 30 minutes at 25°C and then dialyzed against 125 mM phosphate buffer of pll 7. After dialysis, the protein solution is centrifuged to give 23.5 ml of a solution containing 4.2 mg of modified antibody per ml.
By spectrophotometric analysis at 343 nm of the pyridine-2-thione released by exchange with 2-mercapto-ethanol, it is found that the antibody obtained carries 3.2 activated mixed disulfide groups per mol of antibody. c) Coupling reaction 7.3 ml of the solution of modified A chain of ricin (i.e. 0.275 micromol) are added to 781 microliters of the solution of activated antibody obtained above (i.e. 0.022 micromol). The mixture is left to react for 18 hours at 30°C. The reaction medium is then dialyzed against PBS buffer (10 mM in respect of phosphate, 120 mM in respect of sodium chloride, pH 7.4).
After centrifugation and examination by poly-acrylamide gradient electrophoresis, it is found that the immunotoxin obtained has an average degree of coupling for this preparation of 0.8 oxidized methylated A chain (NEM) per mol of antibody.
The immunotoxin containing methylated oxidized A chain (NEM) of ricin, obtained as indicated above, was studied for its pharmacokinetic properties and its specific cytotoxicity properties towards the target cells. _ 44 - 2) - Properties of the immunotoxin IT (methylated A(NEM)-1 a-cvsteamine) TlOl a) Specific cytotoxicity activity It is found that this immunotoxin, prepared by the procedure explained above, has a very strong cyto- -1 2 toxic activity on the CEM target cells (IC^q = 7*10 M established by the method described in example 13). b) Plasma elimination The immunotoxin is administered to rabbits by a single injection into a vein in the ear (81 micrograms of A chain/kg). The plasma samples collected after 24 hours are analyzed with the aid of the immunoassay RIA-3 (example 12). The results are shown in the table below. The values for IT TlOl are indicated by way of comparison Relative plasma concentration after 22 hours IT (methylated A(NEM)-La-cysteamine) TlOl IT TlOl 1.4% 0.08% Twenty-two hours after injection, the concentration of IT containing modified A chain is 17.5 times greater than in the case of IT TlOl.
Example 17: Toxicity of the prolonged-action A chain injected into mice It was important to check the overall toxico-logical impact of the oxidized A chain on the whole animal (the toxicity of the immunotoxins being of the same order of magnitude as that of the A chain at 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 that of the native A chain.
The values found are indicated in the table which follows.
LD50 (micrograms/mouse) Native A chain Oxidized A chain 550 800 These results show that the toxicity of the oxidized A chain is lower than that of the native A chain. This means that, despite a considerable increase in the plasma level of the A chain when the latter has been modified by oxidation, the toxicity of the product is not only not increased but, on the contrary, substantially reduced.
The immunotoxins containing modified cytotoxic sub-units can therefore be used as drugs in human therapy. These modified immunotoxins can be used for the treatment of cancerous or non-cancerous diseases where the target cells would be recognized by the antibody used to prepare the immunotoxin. The optimum administration conditions and the treatment time will have to be determined in each case according to the subject and the nature of the disease to be treated.
In more general terms, antitumoral glycoproteins whose carbohydrate units are modified by oxidation with the periodate ion, and which have a longer half-life than the corresponding unmodified antitumoral glycoproteins, are useful as drugs.
Therefore, according to a further feature, the present invention relates to antitumoral drugs in which an antitumoral glycoprotein whose carbohydrate units are modified by oxidation with the periodate ion is brought into a form suitable for administration by injection and preferably intravenous administration.

Claims (18)

1. Antitumoral glycoprotein which inactives ribosomes, whose carbohydrate units are modified by oxidation with the periodate ion, said glycoprotein differing from the whole ricin.
2. Glycoprotein which inactivates ribosomes, whose carbohydrate units are modified by oxidation with the periodate ion and which has substantially the same activity as and a longer half-life than the unmodified glycoprotein which inactivates ribosomes, said glycoprotein differing from the whole ricin.
3. Glycoprotein which inactivates ribosomes and has a prolonged action, said glycoprotein differing from the whole ricin, wherein it is obtained by treatment of an aqueous solution of a glycoprotein which inactivates ribosomes, the thiol groups of which are optionally protected, with an aqueous solution of an alkali metal periodate, for a period of 0.2 to 2k hours, at a temperature of 0 to 15°C and in the absence of light, unblocking of the thiol groups, if appropriate, and isolation of the final product by known methods. H.
4. Glycoprotein according to claim 3, wherein the aqueous solution of glycoprotein is an aqueous solution of A chain of ricin.
5. - Glycoprotein according to claim U, wherein the A chain of ricin is functionalized, for example by methylation.
6. Glycoprotein according to claim 3, wherein the aqueous solution of glycoprotein is an aqueous solution of gelonine.
7. - Glycoprotein according to claim 3, wherein the aqueous solution of glycoprotein is an aqueous solution of GPIR MOM.
8. Glycoprotein according to claim 3, wherein the aqueous solution of glycoprotein is an aqueous - 47 - solution of GPIR Dianthin 30.
9. Glycoprotein according to claim 3, wherein the aqueous solution of glycoprotein is an aqueous solution of Dianthin 32, Agrostin A, Agrostin B, Agrostin C, HCI or Asparagus officinalis inhibitor.
10. Glycoprotein according to claim U , wherein it is obtained either from an A chain of ricin which is the A chain of native ricin or a fragment of A chain of native ricin, or from an A chain of ricin or a fragment thereof produeed biosynthetically by a cell whose genotype has been appropriately modified.
11. Glycoprotein according to one of claims U or 9, wherein it is Obtained by treatment of an aqueous solution of A chain of ricin, at least one of the thiol groups of which is protected by reaction with 2,2'-dinitro-5,5'-dithiodibenzoate, with an aqueous solution of sodium periodate, for a period of 0.2 to 24 hours, at a temperature of about U°C and in the absence of light, treatment of the mixture with 2-mercaptoethanol and isolation of the resulting product by known methods.
12. Glycoprotein according to claim 6, wherein it is obtained by treatment of an aqueous solution of gelonine with an aqueous solution of sodium periodate, for a period of 0.2 to 2h hours, at a temperature of about 1+°C and in the absence of light, and isolation of the resulting product by known methods.
13. A process for the preparation of an antitumoral glycoprotein according to claim 1, wherein it comprises subjecting the unmodified antitumoral glycoprotein to oxidation with periodate ions. lU .
14. A process for the preparation of a glycoprotein which inactivates ribosomes and has a prolonged action, said glycoprotein differing from whole ricin , wherein it comprises treating an aqueous solution of a _ 48 _ glycoprotein which inactivates ribosomes, the thiol groups of which are optionally protected, with an aqueous solution of an alkali metal periodate, for a period of 0.2 to 2k hours, at a temperature of 0 to 15°C and in the absence of light, unblocking of the thiol group, if appropriate, and isolation of the final product by known methods.
15. Process according to claim lH , wherein the starting material used is either an A chain of ricin which is the A chain of native ricin or a fragment of A chain of native ricin, or an A chain of ricin or a fragment thereof produced biosynthetically by a cell whose genotype has been appropriately modified.
16. A process according to claim 13 or claim 14, substantially as described in the Examples.
17. A glycoprotein whenever prepared by a process according to any of claims 13 to 16.
18. A glycoprotein according to any of claims 1 to 3, substantially as herein described. MACLACHLAN & DONALDSON Applicants' Agents 4 7 Merrion Square DUBLIN 2.
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