AU593211B2 - Antitumoral glycoproteins modified on their carbohydrate units - Google Patents

Antitumoral glycoproteins modified on their carbohydrate units Download PDF

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AU593211B2
AU593211B2 AU43646/85A AU4364685A AU593211B2 AU 593211 B2 AU593211 B2 AU 593211B2 AU 43646/85 A AU43646/85 A AU 43646/85A AU 4364685 A AU4364685 A AU 4364685A AU 593211 B2 AU593211 B2 AU 593211B2
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chain
glycoprotein
ricin
aqueous solution
periodate
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Pierre Gros
Franz Jansen
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Sanofi SA
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    • 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
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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

COMMONWEALTH OF
A
Patents Act It ~E TE S P EC
(ORIGINAL)
CO0 M P I I F I C AT IO0N Class Int. Class Application Number 34 Lodged: Comnplete Specification Lodged Accepted Published 8.,cdon 49.
and Ig &4vr~gftp Priorities: Art 20 June 1984 13 February 1985 Name of Applicant *",A&1ress of Applicant Actual Inventor v Address for Service
SANOFI
Avenue George V, 75008 Paris, France.
Franz. JANSiEN; Pierre GROS F.B. RICE CO., Patent Attorneys, 28A Montague Street, BALMAIN. 20'4,.
~1 Complete Specification for the invention entitled-, ANTITUMORAL GLYCOPROTEINS MODIFIED ON THEIR CAflBOIIYDRAXTE UNITS.
The following statement is a full description of this invention including the best method of performing it known to us -1I- 1 Antitumoral glycoproteins modified on their carbohydrate units.
The present invention relates to new antitumoral glycoproteins whose carbohydrate units are modified by oxidation with the periodate ion.
More particularly, but without implying a limitation, the present invention refers to new glycoproteins which inactivate ribosomes and have a prolonged action.
The term "glycoprotein which inactivates ribohe-re, P er Aeecy(eA co-,A.
somes", askused in the present description and also in the claims, denotes any sust'nce c a rrias whl-ewhich inactivate ribosomes and consequently in- Shibit protein synthesis in eucaryotic cells, as well as 6 15 any fragment of the said substance which possesses the same inactivating property, it being possible for the $,so said glycoprotein which inactivates ribosomes to be of natural or biosynthetic 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 func- "tional 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.
S In the description, the term "periodate" denotes 0j the 104 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 4 340 535 and French Patent Applicatlons no. 2 504 010 and 1c1. 2 516 794 describe the pre- 3~ k 5 paration of Pnticancer products called conjugates, 1 A 2 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 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 whic(h are also suitable as anticancer drugs and result frbm 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- 0 o 6 florum (Eur. J. Biochem., 1981, 116, 447-454; Cancer 15 Res., 1984, 44, 129-133) or the inhibitor ext icted from Momordica charantia (MOM) Patent 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 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 amines or certain carboxylic ionophores such as S monensin and nigericin.
However, the therapeutic effects of immuno- S, toxins, thether activated or not, can only manifest themselves fully inasmuch as the immunotoxin is 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 depends first and foremost on the ability of the immuno- 1 1 S 1, 3 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 occupation 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 model,,. It is apparent that, after injection, the plasma level of biologically active immunotoxin decreases 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 S. 1 5 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 minutes and 99.9% disappears in 17 hours. This rapid 20 disappearance of the immunotoxin quite obviously detracts from the expression of its complete cytotoxic 4 0* capacity by preventing the immunotoxin from saturating, for a prolonged period, a high proportion of the target antigens carried by the cells to be destryed. Moreover, comparison of the plasma elimination kinetics of the immunotoxins with those of the corresponding non- .e 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 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 fl fore 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.
o Furthermore, in vivo localization experiments on the immunotoxin containing the A chain of ricin, radiolabelled and then injected into animals without a o specific target, have chown that, in the first few m 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 **19 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-glucuronidase arnd also in the case of rib inuclease 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, o have shown that, as in the case of the A chain of 4 00 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 15 gelonine purified by the method described 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.
20 It is known that the oxidation of osidic structures, including those contained in glycoproteins, with periodate ions causes the scission of the carbon Sichain wherever two adjacent carbon atoms carry primary or secondary hydroxyls. If the two djacent hydroxyls 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.
S2It 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 aubstantially unchanged.
It has also been found, absolutely unexpectedly, that if the carbohydrate units of a glycoprotein which 7.
6 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 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 exclusively 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 o ribosomes and have a prolonged action are referred to 15 below 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 20 known for immunotoxins and have slow plasma elimination kinetics.
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 glycoproteins which inactivate ribosomes and have a prolonged action and which are obtained by treatment of a glycoprotein which inactivates ribosomes, the thiol groups of which are optionally protected, with an 4, -7aqueous solution of an alkali metal periodate, for a period of 0.2 to 24 hours, at a temperature of 0 to 0 C and in the absence of light, unblocking of the thiol groups, if appropriate, and isolation of the final product by known methods.
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 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, to which are in themselves only very slightly cytotoxic 9 9 because they cannot fix to cells, but which, on the 15 other hand, after coupling with an antibody recognizing particular cells, become highly cytotoxic towards these cells once the antibody has recognized its target.
Representative starting compounds are the A S. 20 chain of ricin, gelonine and the substance extracted from Momordica charantia (MOM), as obtained by ex- S* traction.
S* Other GPIRs which are useful as starting materials for oxidation with periodate ions are as follows: Dianthin 30 from Dianthus Scaryophyllus S* Dianthin 32 from Agrostin A from Agrostemma githago Agrostin B from Agrostin C from HCI from Hura crepitans Asparagus officinalis from Asparagus inhibitor officinalis The same substances produced biosyrthetically -8by 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 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 a, chain of ricin comprises 2 constituents denoted by Al and A2, which differ especially in their polysaccharide units. The experiments which have been carried a out on the 2 constituents of the A chain have made o 15 it possible to demonstrate that periodate oxidation 'takes place in a similar way on the Al and A2 chains and gives these 2 constituents identical properties of improving the pharmacokinetics.
The preparation of the pure A chain of ricin S,'t 20 is described in U.S. Patent 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.
If other functional groups are used for the coupling, for example the phenolic hydroxyl of the tyrosin's or amino groups or carboxyl groups of the GPIR, protection can be carried out or not.
Blocking is carried out by reaction with an 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'-dinitroacid (DTNB) or 3-(pyridin-2-yldisulfanyl)propionic acid or alternatively dipyridyl- 2,2'-disulfide or dipyridyl-4,4'-disulfide. In the r i i:_il -~~Liiiiii) )_-i;-?iiiiY~i .I 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 treatment.
The periodate oxidation reaction is carried out at an acid pH of between 3 and 7, preferably of between and The periodate is used in excess; more particularly, the concentration of alkali metal periodate is greater than the concentration of the vicinal diols C capable of being oxidized; concentrations of 10 to mM in respect of sodium periodate for concentrations of 1 to 10 mg/ml of cytotoxic sub-unit are suitable. The a G 15 treatment, carried out at a temperature of between 0 and Oro# 15 0 C, preferably of between 1 and 5 0 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-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,, If the thiol groups of the starting mateial 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, ready to be used for example for coupling with an antibody 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! a molecular weight which is not significantly differ- 7/ 10 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 and does not produce any degradation products.
a proportion of fxee thiol groups greater than 0.7 per mol.
an immunoreactivity towards rabbit antibodies inhibiting the A chain of ricin which is indistinguishable 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 hiy an equal quantity of native A chain.
finally, after a single intravenous administration S. 15 to rabbits at a dose of about 0.4 mg/kg of body to weight, the plasma level of the prolonged-action A chain a) 23 hours after injection is greater than 10% of the level present at time zero (as agaiast 0.015% for the native A chain at this time) L.e. an increase in the i 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; .t a molecular weight which .s not significantly differ- *t 25 ent from that of the native gelonine.
an immunoreactivity towards .,nti-gelonine rabbit t 4 ,tr *antibodies which is indistinguishable from that of the native gelonine.
finally, after a single intravenous adr,,inistrati n to rabbits at a dose of about 0.3 mg/kg of body weight, the plasma level of the modified golonine 24 hours after injection is groater than 3% of the level present at tinme zero (as against 0.01% for the native gelonine at this time) i.e. an increase in the plasma level by a factor greater than 200
L_
r 11 44 44 rto arr* 444 04 44 a i4e 6444 .a iI 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 described in U.S. Patent 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 disulfid~ group. If the chosen cytotoxic sub-unit does net 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 15 mp-li c.ca- e rb, conat i nue as iicat ea-bo-ve--T introduction of the said functional group can ake place either before the oxidation step with eriodate ions, in which case it will be necessary or the thiol radical to be blocked during the oxidaton step .nd 20 then unblocked after this step, or a er the oxidation step.
This gives modified immu otoxins which have acquired a new character as re ards their pharmacokinetic properties. More pc ticularly, by appropriate modification of the cytot ic sub--unit, it has been possible to add to the pecific cytotoxicity properties of immunotoxins, with ut interferLng with them, a new property which is st as intrinsic, namely the capacity to show sloaplasma elimination kinetics.
30 The ex mples which follow provide a clearer understandin of the invention without limiting its scope.
Oxida on of the methylated A chain in which the SH re _toced i, h Is I 4 I r IIa coupling can be continued as indicated above. As hereinafter defined this process is referred to as "functionalizing". Functionalisation may be achieved by methylation, as illustrated by Example 1. The introduction of the said functional group can take place either before the oxidation step with peripdate ions, in which case it will be necessary for the thiol radical to be blocked du ing the oxidation step and 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 modification of the cytotoxic sub-unit, it has been possible to add to the specific cytotoxicity properties of 15 immunotoxins, without nterfering with them, a new property which is just as intrinsic, namely the capacity to, show slow down plasma elimination kinetics.
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 groups "i are blocked with N-ethylmaleimide.
ell X W I Am*" -o 12 I Preparation of the correctl' functionalized A chain of ricin 1) Hexarethylation of the A chain The methylation reaction is carried out at O'C, with stirring, ini 0.2 M borate buffer of p11 10, by the method of Means and Feehoy (Biochemistry 7, 2192 (1968)). 20 mug of tritiated borohydride (containing ruCi/rumol) are added to 35 ,rl of L A chain (3 mg/ml), followed 4y 350 microliters of 6% formaldehyde (added in five 70 microliter portions spread over a period of minutes).
The excess reagent is removed by continuous dialysis against 125 mOd phosphate buffer of pHi 7 (40 1 at 300 After dialysis, the protein solut:xon is centrifugell. 36,5 ml of hexamethyloted A chain containing 2.6 mg/mI are collected, 2) Blocking with N-ethylmaleimide The natural SB1 of the hexamethylated A cha~in is blocked by the method described in Methods in Enzymology 11, 541 (1967), To do this, the A chain of ricin obtairted in the previous step is incubated for 2 hours at 301C in the presence of 20 equivalents oft N-ethylnlaleimi4'e per Mo o chain. The excess reagent is removed by continuous dialysis against 125 25 mM, plhosphate buffer of- pf! 7, which is renewed for ?Q hours at a rato of 500 ml/hour.Atter conceatrgtion, there is obtained 13 ml of a solution of kA chain of ricin containing 7 tmg/m. and no longer po,, sessing, thio2l groups which can be determined by E LbMAN's reagent. The product thus obtained is subsequentlty called hexamethylated A chain (NEM).
3) Periodate oxidatioa 6 ml of the solution of hoxamethyinted A chain (NiEN) obtained above are tr., ad with NaIQ 4 (12.8 mug) for 40 minutes in the dark, at phl 4.5 and at Q'C, The reaction is stopped by te addition of 600 microliter,, of ethylene
V
F,
13 t a ercr tctr Elt( re bP P
IILC
$I rLt
L
glycol and the reaction medium is dialyzed continuously against 0.1 M carbonate buffer of pH 10 (20 h at 500 ml/h).
II Enzymatic 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 The in vitro protocol involves the use of appropriately complemented, subcellular fractions of rat 14 live:- capable of incorporating 1 C-phenylalanine in the presence of an artificial messenger RNA: polyuridylic acid.
The procedure employed for preparing the sub- 15 cellular 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 introdeed 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 micrograms/ml of bovine serum albumin.
The count data are used to calculate, relative 25 to a control medium without inhibitor, the percentage 14 inhibition of the incorporation of 1C-phenylalanine into the proteins for each reaction medium containing A chain of ricin.
The inhibitory activity was determined. An
IC
50 fa 2.7 0 1 0 mol/l is observed for the oxidized A chail. The IC 5 0 of the control A chain in the ex- -10 periment is 103'10 0 ol/l; therefore, the modification does not cause a loss of activity of the A chain.
Exapple .2: This example demonstrates the slow elimination t4 S 4 *r 9 4 4 4 crsr 14of 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 4 340 535. equivalents of a solution of 2,2'-di'itro-5,5'-dithiodibenzoic icid (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 15 phosphate and 150 mM in respect of NaC1, of pH 7).
SIncubation is left to proceed for 20 minutes at The solution is then dialyzed against PBS buffer at c\c\ 4 to give 53 mg of A chain blocked on the thiol group, as a solution containing 5 mg/ml.
20 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 I with 1 M acetic acid. Incubation is left to proceed 25 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 0 C, the reaction medium is dialyzed at 4 C against PBS buffet. The periodate oxidation 30 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,
.NVI
/J
1
W
15 at a final concentration of 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 0 C. The solution is then dialyzed against PBS buffer at 4 0
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, A57 (Academic Press)), it is determined that the modified A chain obtained has 0.70 free thiol group per mol. The molecular weight of the modified A chain is 30,000±3,000, determined by polyacrylamide gradient electrophoresis in the presence of sodium dodecylsulfate.
The previously obtained preparation of A chain in which the polysaccharide units have been oxidized awas studied for its enzymatic activities in the inhibition of protein synthesis and for its pharmacokinetic properties.
S* II Enzymatic 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 IC 5 0 of 3 10 mol/1 is observed for the oxidized A chain. The IC of the control A chain in the experiment is 1.2 10 25 mol/l; therefore, the modification does not cause a loss of activity of the A chain.
,9 III Pharmacokinetic properties of the prolonged-action A chain (A-La) The A chain is administered to rabbits by means P *b 30 of a single injection into a vein in the ear. The •quantity ofiA 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 radioinmunometric ,test designated below by the abbreviation RI4-1.
c 35 This technique has the advantage of determining 9 tetdsgae eo y h brvlinRM -16the A chain without modifying it. This determination is carried out in microtitration plates (for example: "NUNC-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.
15 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 200C 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- 25 125 in the presence of chloramine T by the method of Greenwood and Hunter (Biochem 1963, 89, 114); the specific activity of the radiolabeled Ac2 antibodies is 6to 10 microcuries/micrograit. 10 cpm of radiolabeled Ac2 antibodies are introduced as 200 microliters into a 30 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 30 17 at different known concentrations. When prolongedaction 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 Achain 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 e periments gives coefficients of variation of less 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 or the abscissa and the plasma concentration of the product measured, recorded in per cent of the theoretical plasma concentration at time zero, is S plotted on a logarithmic scale on the ordinate. This value, called the "relative plasma concentration" (RPC), is calculated using the following expression: concentration measured at time t SRPC 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 a phases: in the first phase, the product disappears very rapidly from the bloodstream since only 0.1% of the dose S, 30 administered remains in the plasma 3 hours after injection. 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 responsible for the disappearance of the majority of the E~ 18 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 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.
1) Modification of the A chain of ricin a) Blocking of the natural SH with N-ethylmaleimide ml of an aqueous solution of A' chain of ricin containing 8 mg/ml 4.1 micromol of A chain) are treated with an aqueous solution of 2-mercaptoethanol so that the final concentration is 1 per cent.
4 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 hoirs 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.
B 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 0
C
in the presence of 20 equivalents of N-ethylmaleimide per mol of A chain. The excess reagent is removed by Scontinuous dialysis against 125 mM phosphate buffer of p 7, which is renewed for 20 hours at a rate of 500 ml/hour. This gives 35 ml of a solution of Achain of 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 19 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 synthesis was determined using the procedure described in example 1. The enzymatic properties are found to be -10 maintained with an IC50 of 4.3'10 mol/l for the oxidized A chain (NEM), b) Pharmacokinetic properties of the oxidized A chain
(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 collected at time 23 h are analyzed using the immunanetric trest RIK-1 as described in example 2. The results are *shown in the table below: 4.*4 4. .9 4.
4 4 *h Sr 4 4 4.44
I:
20 Plasma concentration 23 h after injection: A chain (NEM) 0.01% Oxidized A chain 8%
(NEIM)
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 4: 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 A I I I 20 immediately with ethylene glycol), 20 minutes, 40 minutes, 2.5 hours, 4 hours and 18 hours.
These various preparations are injected into rabbits and the relative plasma concentration of the A chain is measured after 23 hours by the same procedure as in example 1.
The results are shown in figure 2. 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 This example demonstrates the importance of the duration of the oxidative treatment on the pharmacokinetic properties of the methylated A chain blocked with NEM.
20 1) Preparation of the functionalized A chain of ricin a) Blocking of the natural SH of the A chain with Nethylmaleimide The natural SH of the A chain is blocked with N-ethylmaleimide by the same procedure as that described 25 in example 1.
b) Methylation of the A chain The methylation reaction is carried out at 0 0
C,
with stirring, in 0.2 M borate buffer of pH 10, by the method of Means and Feeney (Biochemistry 7, 2192, 1968).
30 38 mg of tritiated borohydride (containing 47 mCi/mmol) are added to 65.5 mi of A chain (NEM) (3 mg/ml), followed by 1 ml of 6% formaldehyde added in five 200 microliter portions spread over a period of 30 minutes.
The excess reagent is removed by discontinuous dialysis against 125 mM phosphate buffer of pH 7 (40 ml).
r r z~li ae
I~
21 9+ 4 9 94 *r 9 After dialysis, the protein solution is centrifuged.
F( ml of methylated A chain containing 3 mg/ml are -llected.
c) Periodate oxidation Six preparations of methylated A chain (NEM) are oxidized usin-g 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 minutes, 40 minutes, 2.5 hours, 4 hours and 18 hours.
These various preparations are injected into rabbits and the relative plasma concentration of 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 (NEM) is indeed due to periodate oxidation 20 because, when the reaction is stopped immediately, the plasma concentration of methylated A chain (NEM) is identical to that obtained for the A chain and 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 (Al 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 Al and A2 chains 28 ml of4 A chain containing 10.9 mg/ml (309 mg) 35 in 125 mM phosphate buffer of pH 7.0 are deposited on a 9* 9 94* i 9 *4 *4 *4 9
S
99994' 9I
.,I
nl 22 column of 112 ml of concanavalin 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 which is 0.5 M in respect of NaCI and 0.1 M in respect of alpha-methylmannoside.
This 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 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 a 30,000 and the A2 chain corresponds to the band of weak S. intensity and of MW 33,000.
2) Modification of the Al and A2 chains of ricin with 20 sodium 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.
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 IC observed is equal to 2.1*10 10 mol/i and 2.1.10 10 mol/ for the oxidized Al and A2 chains respectively. The IC 5 0 values of the native Al and A2 chains, which are the controls in the experiment, are 1.9*10 10 mol/1 and 1'0-1 0 mol/l respectively. Therefore, the modification of the i 6 1Plki~
I,
23 separate variants of the A chain does not cause a loss of their enzymatic activity.
4) Pharmacolcinetic 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 micrograns of A chain/kg). The plasma samples collected after 20 hours are analyzed with the aid of the immunometric test RIM-1 (see example 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 hours after injection A chain 0.012% Oxidized A chain (A-La) Al chain 0.02% Oxidized Al chain (Al-La) A2 chain 0.04% Oxidized A2 chain (A2-La) 14% Twenty hours after injection, the concentrations of Al-La and A2-La are respectively 500 and 350 times greater than in the case of At and A2, Example 7: This example describes the biochemical characteristics of the A chain and its variants, the Al chain and A2 chain, in the native form and in the oxidized form.
The A chains u.ed in these studios are prepared as described in examples 2 and 6.
I Carbohydrate compositions The carbohydrate compositions of these proteins are determined by gas chromatographic analyses using 24 qA 4 4, a 4 *4*4 4q 4* '4 4 4 *4~4 w 4, *4 Clamp's method (in Glycoproteins: their composition, structure and function (edited by A. Gottschalk), volume 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 5.58 or Al 4,54 or A2 6.24 or Oxidized A 2.27 or Al 2.07 or A2 3.33 or 15 Molar composition (On the basis of a molecular weighL of 30,625, the results are given with an average precision of or residue per molecule) Chains 20 Monosaccharides 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 5.2 1.43 1.29 2.26 Fucose 1.37 1.41 1.52 0 0 0 25 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 44 4 44 44 4 44 44 4 4 4444 4. 44 4* 4 g~ 4 4* ~44~ 4 3.17, 2.11 and 2.94 mannose residues, thp 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-acetylglucosam 4 ne residues are only slightly degraded.
II N-Terminal sequence The sequence of the N-terminal amitio acids of the A chain and its Al and A2 variants, in the native form and the oxidized form, was established with a protein sequencer by the procedures known to those skilled in the art. The results obtained are collated in the table below.
*4 I 4 444' #444 4 #4,4 6 #4 4 4 44.4 44 4* P4 1 o 4 4,44 4 4,44 *4 4 4 4 44 44 4 4 44 4 44 4#e, #4 4 44 44 #6 4 4~ 4 4 4 l~ 15 Chains Sequence of the 9 N-terminal amino acids Native A Ile-Phe-Pro-Lys-Gln--Tyr-Pro-Ile-Ile Al £le-Phe-I'ro-Lys-Gln-Tyr-Pro-Ile-Ile A2 Ile-Phe-Pro-t'ys-Gni-yr-Pro-lie-Ile Oi dized A Ile -Phe-P1ro-Lys--Gln-Tyr-Pro-Ile-ll Al Ile-Phe-Pro-Lys-Gln-Tyr-Piro-Ile,-Tle A2 Ile-Phe-Pro-Lys.-Gln-Tyr-.PrQ-Ile=le( It is found that the sequences of the 9 N-torminal amino acids ofC thp native and oxidized A, Al ind A2 chains are strictly identical to one ano-hexr, which demonstrates that the oxidative treatment L.~ave8 the protein chain intact. It is also found that the sequwnce 25 of the 9 N-terminal amino acids of the A chains is strictly identical to that previously describcd yV Funatsu, for the A chain of ricin (Agric 4 fliol. Chamn..
(1979), 43, 2221).
III Affinity on concanavalin /ahoe The A chain., the A chaint blocked with WL'IP.
I:A(DTNB)] and the A(DT1,8l)-La, A1(tITNI3)t Ai(t0TNB)-LaO A2(b'rN4B) and A2(DTNB)-La chains are tested by their capacity to fix to concanavalin A/sepharoso. I ml of -4 1 1.
-~26a solution of A chain containing about 1 mg/mi is depositedI on a column of 1 ml of concanavalin A.
Chromatography iLs 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 which is not retained by the concanavalin A has returned to the base line, the column is washed w.,ith 0.1 M borate buffer of pH 6.0, which is 0.5 M in respect of NaCi and 0.1 M in respect of alpha-me thylma nno side. The resul ts. ceXressed as a percentage o-l che optical density at 280 nm are suumari7"-d in the table below.
Ch ai ns 7. not retained eluted by alpha- Total by the con A methy.rnannoside
M%
I a ma 4* 4 04.i4 44*.
4 4*44 44 p 4 4449 4* I 41 I 4 44t 9 4 9.4 4 4 44 4 9.9 It 4 15 rA 53_ 1 27 A(DNB6t6 11 77 A(DTNB)-L~k 78 5 83 Al1(DTNB) 80 6 86 Al (DT4B )-La 73 0.4 73.4 A2(MTR) 25 70 W2DTNBI)-La 64 9 73 It is known that concanavalin A has an affinity foc glycoproteins with terminial iiiannosos. 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 isomor more richly substituted with vinnose. It is also found that the oxidative treatment destroys this affinity, which is coherent with the destruction of the sugar residues by a treatment ofthis type.
IV -Determination of the B /6 Theoabsorption coefficient qt 280 nm (E U.c) is 27 -Ithe optical density at 280 nm of a solution containing 1 1 mg/mI, i:,i which the protein concentration is deter- Umined by the FOLIN test with a standard range of bovine serum albumin.
The results are summarizc~l in the table which f ollows A chain 0.65 A(DTNB) chain 1.12 A(flTNB).-Lai chain 1 .04 A1(DTNB) chain 1.07 Al(DTNB)-La chain 1.02 *1 p 88~48 *448 8 89*8 4 4.
88 8848 84 9* 88 4 8 8848 *884
A
4.
84 4 848 4 9 p.
88 8 84 8 84 9 4444 81 t~ 4 4 I ~r- AZ(EiTNB) cha-,n 0.95 A2(L'TNB)-La cha'!n 0.95 Blocking of the thiol group of the chain with 15 DTNB produces a substantial increase in the absorption at 280 nm, due to the introduction of the nitrobeazoyl group> After. oxidation, no significant variation in the absorption at 280 nrn is observed, demonstrating 20 that oxidotion has not affected amipo acids responsible for the absorption at 280 am.
V Isoelectric focusing Analysis of the A chain by iioele, tric focusing produces a set of bands with isoelectric points Qp) 25 which are between 7.5 and 8.0 and are identical foxr the A, Al and N2 chains.
Blocking of the cystei~ia of the A chain wtith DTNB causes a widening of the banels 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 Isoelp.,tric points of 28 the native and oxidized A, Al and A2 chains shows that, in the absence of a blocking agent, al. the bands characteristic of the A chain are transferred by 0.5 pH unit towards acid pH values. This transfer takes place without 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 elimination 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 Gelonium multiflorum by the method which has been described Biol. Chem. (1980) 255, 6947-6953). The "P91 oxidation reaction is carried out under the same con- S, ditions as those described for the A chain of ricin in 9o si example 2, except that the step in which the thiols are 20 blocked with DTNB is omitted.
.,4 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 25 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 solutiotn containing 3 amg/ml of gelonine in PBS buffer, brought to pH 6 with 1 M acetic acid. Incubation is left to proceed for 16 30 hours at 4°C in the dark. The reaction is stopped by the addition of 105 microliters of a 1 M aqueous sol- SUttion of ethylene glycol. After incubation for 15 minutes at 20°C, the reaction medium is dialyzed at 4°C against PBS buffer. After cei,trifugation at 10,000 x g for 30 minutes, this gives 2.9 mg of oxidized gelonine Ci J 1 K -29at 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 subunit 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 Native 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.
Blood samples are taken at intervals on heparin. The plasmas are analyzed with the aid of a radioimmunometric test 9, designated below by the abbreviation RIM-2.
This 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 9 20 purified by affinity chromatography, the Ac2 antibodies *being the same antibodies radiolabeled. The radiolabeling procedure is identical to that described for the technique RIM-1. The concentration of native S.r gelc ine or modified gelonine in the samples to be 25 determined is measured by reference to a calibration *a curve established by introducing native or modified gelonine at different known concentrations. The test t 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 4 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, like the A chain of native ricin, disappears very rapidly A I 30 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: 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 involved in the recognition process responsible for the elimination of the gelonine, to the point of preventing this recognition.
Example 9: 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, ,t after intravenous injection into the animal.
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.
l 10 microliters of a solution of radioactive iodine-125 containing 100 pCi/ml, and 30 microliters of a solution 30 of chloramine T containing 2.5 mg/ml in water, are added t to 100 microliters of a solution containing 1 mg/ml of e c 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 of sodium metabisulfite containing 0.5 mg/ml. The re- F 1 ;1 31 4.i .4 9, .9 91 4 9.
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 o'it under the same 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 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 radiolabeled MOM, brought to pH 6 with 1 M acetic 15 acid. Incubation is left to proceed for 16 h at 4 0 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 0 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.
30 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 94 99 9r 9
Q
49* 4.
'I I 32 heparin. The plasmas (200 microliters) are incubated with trichloroacetic acid (TCA) (200 microliters at a concentration of 25%) for 30 minutes at 4 0 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 percentage 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: S* r x PV x 100 I.P.R. S0.2 x R S. where: r radioactivity measured at time t in 0.2 ml of plasma, R total radioactivity injected, PV plasma volume (considered to be equal to 36 ml/kg of the animal's body weight).
SThe plasma elimination curves, as a function of time, for the oxidized or unoxidized MOM after intra- 25 venous 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 When the MOM has been oxidized on its sugar residues, the elimination rate is reduced (curve 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- I S- 33 ation of the MOM.
Example This example demonstrates: 1. the rapid elimination of GPIR Dianthin extracted from Dianthus caryophyllus,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 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 radioactive Dianthin. The iodination and oxidation reactions are carried out under the same conditions as described t for MOM in example 9.
The new oxidized Dianthin molecule thus obtained *41 has a molecular weight which is not significantly differt 20 ent from that of the native Dianthin *9 2) Pharmacokinetic properties of prolonged-action Dianthin 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 measured by the same procedure as described for OM 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 Dianthin 30, like 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 kint:tics are slowed down considerably: 2 hours after injection, the Dianthin level is 80 times greater 34than 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 oxidation has modified the sugars involved in the recognition process responsible for the rapid elimination of the Dianthin.
Example 11: 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 A chain of ricin.
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) Oxidized A chain of ricin: The A chain of ricin was :tr prepared in the manner indicated in example 2.
SII) Activated antibodies inhibiting human T cells S 20 microliters of a solution containing 60.3 mg/ml of l-ethyl-3-dimethylaminopropyl-3-carbodiimide are added to 100 microliters of a solution containing mg/ml of 3-(pyridin yldisulfanyl)propionic acid in tert.-butanol, and the mixture is left for 3 minutes at ambient temperature. 68 microliters o'f 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 I stirred for 15 minutes at 300C and then dialyzed against A 10 PBS buffer at 4 0 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 prolongedi!,I i.
-r i i t *r I t 4tI~
I
t r i action A chain of ricin 2.46 ml of modified A chain containing 2.87 mg/ ml are added to 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 0 C. The solution is centrifuged 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 containing 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 specific cytotoxicity properties towards the target cells.
Example 12: This example demonstrates the acquisition of the property of slow plasma elimination of the immunotoxins containing prolonged-action A chain of ricin, which are abbreviated to IT (A-La)TIOI.
I Procedure The conjugate prepaied by the procedure explained in example 1 is administered to rabbits by a. single in- 30 jection into a vein in the ear. The quantity injected corresponds to 0.415 mg/kg, expressed as A chain.
Blood samples are taken at intervals on heparin. The plasmas are analyzed with the aid of a radioimmunnetric test with two sites, which is abbreviated below to RIM-3.
This test is carried out by the same technique II *1
I
c II7 I
I
1
I
36 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 affin.ity 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 RPI-3 has the same reliability and reproducibility 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 T101, which is obtained by the reaction of the same antibody T101, substituted by activated disulfide groups, with the native A chain of ricin. The preparation and Ot the cytotoxic properties of this conjugate have been described in French Patent Application no.
2 516 794 *44 o; 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 T101 and IT (A-La) T101, in- I jected intravenously. Twenty-four hours after injection, to 25 the concentration of active immurotoxin is 140 times greater for IT (A-La) T101 than for IT T101. This fact S demonstrates that the new pharmacokinetic properties *of the oxidized A chain are retained after coupling with an antibody.
t, 30 Example 13: This example demonstrates the retention of the S" 'specific cytotoxicity properties of IT (A-La) T101 towards the target cells.
The fundamental biological property of the A chain of ricin is to inhibit protein synthesis in cells 4 37 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 1Cleucine into cancerous cells in culture is measured.
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 1 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 radioactivity incorporated is determined here on the whole 10" cells isolated by filtration.
41 On the basis of these determinations, it is possible to draw the dose/effect curves, plotting, on t the abscissa, the molar concentration of A chain in the 20 substances studied, and, on the ordinate, the iicorpora- ~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 dctormine, for each sub- 25 stance studied, the concentration which causes a 50% in- 14 hibition of the incorporation of C-luucine, or inhi itory concentration" >Figure 8 shows the curves obtained in the same !A experiment with IT (A-La) TO11 and the uncoupled ox- Sr 30 idized 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) T101 has a very strong cytotoxic activity (IC50 5.5'10 2 M) which is about 80,000 times greater than that of the uncoupled oxidized A chain, measured under the same conditions.
ri.Li~Y;1 i l:l;-i 38 Example 14: This example demonstrates the comparative cytotoxic efficacy of IT (A-La) T101 and IT T101 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 up among several million dead cells. This is made possible by optimum culture conditions in a gelled medium, applied to the CEI human lyiiphoid line.
I Technique for measuring cytotoxicity by the inhibition of colony formation The medium used for cloning is the medium RPHI
S
t t 1640 to which I mmol/l of sodium alpha-ketoglutarate,
I
1 mmol/1 of sodium oxaloacetate, 5% of inactivated fetal calf er'im and 10% of inactivated horse serum are added. A first, 0.3% agar luution (Agarose type VII, SIGMA laboratories) 20 is prepared in this medium, placed as a thin layer in small Petri dishes and solidified at +4 0 C. The cells are mixed with o second, 0.275% agar solution kept at 37"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, To determine the cloning efficiency and thus the exact number of cells surviving the immunotoxin treatment, it c 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 efficiency 4 rtdicated by this calibration line is prac-
I;
.1 a w- 39 tically unaffected by the presence of a high proportion of dead cells, which is the situation naturally encountered when the cells are treated with the immupotoxin.
The immunoto.xin treatment is carried out by incubating the cells in the exponential growth phase and at a concentration of 10 /ml with the immunotoxin IT (A-La) TI01 or IT T101 at different concentrations, in a total volume of 1 ,ni of the medium RPMI 1640 containing 10% of inactivated fetal calf serum and 10 mmol/l of ammonium chloride. The incubatiin 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 "GIRATORY G-2" shaker, NEW-BRUNSWICK). The cells are then washed and different dilutions are prepared, before mixing with the agar solution, so that the number "tt of cells surviving can be measured in the zone of maxim.um sensitivity given by the calibration line. The V' results are expressed as the absolute aumber of cells 4, 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 the dilution factor of the cell preparation examined 25 and E is the cloning efficiency established from the slope of the calibrat'on line. Each point corresponds Kto the average of three tests.
II Results o Figure 9 shows the curves of the cytotoxic activity of the immunotoxins IT (A-La) TI01 and IT T10I on the CEN cells, in the presence of 10 mM ammonium chloride, as a function of the immunotoxin concentration (expressed as the molarity o:f A chain).
It is apparent that the efficacies of these two products are of the same order of magnitude. The 40 resulting reduction in the number of cells is extremely large in both cases since, for concentrations as low as 0 1 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 p ved that the uncoupled A chain or a non-specific immunotoxin has no effect on these cells.
This example demonstrates that IT (A-La) T101 has specific cytotoxicity properties which are virtually identical to those of conventional IT T101.
SExample Conjugate obtained by the reacti-n of an antibody inhibiting human T cells (an antibody directed against the antigen T65), substituted by activated disulfide groups, with the oxidized and functionalized A chain (NEM) of ricin, the coupling taking place between the activated disulfide groups and the functionalized sugar residues i of the A chain.
S1) Preparation of the immunotoxin 4 20 a) Preparation of the functionalized A chain The A chain is blocked with N-ethylmaleimide on its SH group and then oxidized for 18 hours by the procedure des,cribed in example 3.
4 Coupling with cystamine 25 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 hydiochloride for I 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 0
C.
The excess reagent is removd 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 41 per cent for 1 hour at 30 0 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, 0.25 SH per mol of A chain was determined by Ellman's method.
b) Preparation of the antibody (see example 11) c) Coupling reaction ml of the solution of modified A chain of ricin 0.058 micromol) are added 'o 211 microliters of the solution of activated antibody obtained above 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, pH After centrifugation and examination by polyacrylamide gradient electrophoresis, it is found that the immunotoxin obtained has an average degree of coupling for chis b Preparation of 0.8 A chain (NE) per mol of antibody.
2) Poperties of the immunotoxin IT (A(NEM)-Iacysteamine) T101 Spercific cytotoxicity activity It is foundl that this immunotoxin, prepared by the procedure explained above, has a very strong cyto- Stoxic activity on he CEM target cells (IC 50 d1.210 M, established by the method described in example 13).
1 b) Plasma elimination ent eThe immunotoxin is administered to rabbits by a single injection into avein in the ear (50 micrograms Sof A chain/kg). The plasma samples collected after 22 hours are analyzed wi t he aid of the immunoassay RIA- 1- tt 3 (example 12). The restlts are shown in the table below. The values IT TIOI are indicated by way of comparison.
r ,71211 42 Relative plasma concentration 22 hours after injection IT (A(NEM)-La-cysteamine)T101 2.4% IT T101 0.08% Twenty-two hours after injection, the concentration of IT containing modified A chain is 30 times greater than in the case. o IT T101.
Example 16: Conjugate obtained by the reaction of an antibody inhibiting human T cells (an antibody directed against the antiger. 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.
15 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 Counlin with cvstamine 4* 4* After dialysis against 0.1 M carbonate buffer of pH 9.5, 18.5 ml of a protein solution containing 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 botohydride (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 1,25 mM phosphate buffer of pH 7. The disulfide bridge of the fixed cystamine is then reduced with 2-mercaptoethanol at a final concentration of i-i 43per cent for 1 hour at 30 0 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-yldithiopropionate in ethyl alcohol is added to 23.5 ml of a solution of antibody T101 containing 4.4 mg/ml 0.68 micromol). The mixture is stirred for 30 minutes at 25°C and then dialyzed against 125 mM phosphate buffer of pH 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-mercaptoethanol, 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 0.275 micromol) are added to 781 microliters of the solution of activated antibody obtained above 0.022 micromol). The mixture is left to react for 18 hours at 30 0 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 polyacrylamide 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.
S 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.
S-
44 2) Properties of the immunotoxin IT (methylated A(NEM)la-cysteamine) TIO a) Specific cytotoxicity activity It is found thaL this immunotoxin, prepared by the procedure explained above, has a very strong cytotoxic activity on the CEM target cells (IC 7-10 12
M
50 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 TI01 are indicated by way of comparison.
Relative plasma concentration after 22 hours 4i *44.~
I
I
44~t.
4. 4~ 4, 1 a 4u 4 4~44 44 1 4 44 4 4 44 4 64 4* 4 4444
I
I
IT (methylated A(NEM)-La-cysteamine) 1.4%
TIO
IT TIOI 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 TI01.
Example 17: Toxicity of the prolonged-action A chain injected into mice It was important to check the overall toxicological 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 lethal dose of the oxidized A chain, administered intravenously to Charles River France CDI mice, by comparison 45 with that of the native A chain.
The values found are indicated in the table which follows.
(micrograms/mouse) Native A chain 550 Oxidized A chain 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 15 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 ,t 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 (14)

1. A glycoprotein having a cytotoxic action which inactivates ribosomes as hereinbefore defined, whose carbohydrate units are modified by oxidation with the periodate ion and which has substantially the same .u ivity as and a longer half-life than the unmodified glycuprotein which inactivates ribosomes.
2. Glycoprotein which inactivates ribosomes as hereinbefore defined and has a prolonged action and which 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 0 C and in the absence of light, unblocking of the thiol groups, if appropriate, and isolation of the final product by known methods.
3. Glycoprotein according to claim 2, wherein the aqueous solution of a glycoprotein is an aqueous solution S/ of the A chain of ricin.
4. Glycoprotein according to claim 2, wherein the A chain of ricin is functionalized as hereinbefore defined.
Glycoprotein according to claim 4, wherein the A •S chain of ricin is functionalized as hereinbefore defined by methylation.
6. Glycoprotein according to claim 2, wherein the 0" ;aqueous solution of a glycoprotein is an aqueous solution of gelonine.
7. Glycoprotein according to claim 2, wherein the .I aqueous solution of a glycoprotein is an aqueous solution of GPIR MOM.
8. Glycoprotein according to claim 2, wherein the aqueous solution of a glycoprotein is an aqueous solution of GPIR Dianthin k O 1 47
9. Glycoprotein according to claim 2, wherein the aqueous solution is an aqueous solution of a glycoprotein selected from the group consisting of Diathin 32, Agrostin A, Agrostin B, Agrostin C, HCI or Asparagus officinalis inhibitor.
Glycoprotein according to claim 3 which is dcrived either from the A chain of native ricin or a fragment thereof, or from the A chain of ricin or a fragment thereof produced biosynthetically by a cell whose genotype has been modified to produce at least the A chain of ricin.
11. Glycoprotein according to claim 10, which is obtained by treatment of an aqueous solution of the A chain of ricin, at least one of the thiol groups of which is protected by reaction with a 2,2'-dinitro-5,5'-dithio- dibenzoate, with an aqueous solution of sodium periodate, for a period of 0.2 to 24 hours, at a temperature of about 4 0 C and in the absence of light, treatment of the mixture with 21mercaptoethanol. and isolation of the resulting product by known methods.
12. Glycoprotein according to claim 6, which is obtained by treatment of an aqueous solt~lion of gelonine with an aqueous solution of sodium periodate, for a period of 0.2 to 24 hours, at a temperature of about 4 0 C and in the absence of light, treatment of the mixture with t 2-mercaptoethanol and isolation of the resulting product by known methods.
13. A process for the preparation of an antitumoral m, glycoprotein as claimed in claim 2, which comprises subjecting the unmodified antitumoral glycoprotein to oxidation with periodate ions. 'C 48
14. A process for the preparation of a glycoprotein which inactivates ribosomes as hereinbefore defined and has a prolonged action, which 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 0 C and in the absence of light, unblocking of the thiol group, if appropriate, and isolation of the final product by known methods. The process as claimed in claim 14, wherein the glycoprotein 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 V t^ fragment thereof produced biosynthetically by a cell whose genotype has been appropriately modified. S*; DATED this 25 day of September 1989 SANOFI Patent Attorneys for the Applicant: RICE CO. 4 e 9 1X
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IL80973A (en) * 1985-12-20 1992-08-18 Sanofi Sa Modified ribosome-inactivating glycoproteins,their preparation,immunotoxins containing them and pharmaceutical compositions containing such immunotoxins
FR2602682B1 (en) * 1986-08-12 1988-12-02 Sanofi Sa LONG-DURING IN VIVO IMMUNOTOXINS COMPRISING A RIBOSOME INHIBITING GLYCOPROTEIN MODIFIED BY OXIDATION OF OSID PATTERNS AND FORMATION OF A SCHIFF BASE
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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
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
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Publication number Priority date Publication date Assignee Title
US4340535A (en) * 1978-09-28 1982-07-20 C M Industries Cytotoxic products formed by covalent bonding of the A chain of ricin with an antibody and the process for their preparation and use
EP0023401A2 (en) * 1979-07-20 1981-02-04 Teijin Limited Antitumor protein hybrid and process for the preparation thereof
EP0074279A2 (en) * 1981-09-08 1983-03-16 Suntory Limited Selective anti-tumour agents

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GR851497B (en) 1985-11-25
KR930000058B1 (en) 1993-01-06
DK277985D0 (en) 1985-06-19
DK277985A (en) 1985-12-21
JPH0582400B2 (en) 1993-11-18
DE3568167D1 (en) 1989-03-16
EP0172045A1 (en) 1986-02-19
EP0172045B1 (en) 1989-02-08
NZ212439A (en) 1989-01-27
CA1248874A (en) 1989-01-17
AU4364685A (en) 1986-01-02
IE851511L (en) 1985-12-20
JPS6112628A (en) 1986-01-21
ES544337A0 (en) 1986-04-01
ES8605681A1 (en) 1986-04-01
PT80662A (en) 1985-07-01
IL75484A0 (en) 1985-10-31
DK166626B1 (en) 1993-06-21
KR860000315A (en) 1986-01-28
PT80662B (en) 1986-12-09
IE58514B1 (en) 1993-10-06
ATE40700T1 (en) 1989-02-15

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