CA1248874A - Process for the preparation of antitumoral glycoproteins modified on their carbohydrate units - Google Patents
Process for the preparation of antitumoral glycoproteins modified on their carbohydrate unitsInfo
- Publication number
- CA1248874A CA1248874A CA000483940A CA483940A CA1248874A CA 1248874 A CA1248874 A CA 1248874A CA 000483940 A CA000483940 A CA 000483940A CA 483940 A CA483940 A CA 483940A CA 1248874 A CA1248874 A CA 1248874A
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- chain
- ricin
- aqueous solution
- glycoprotein
- periodate
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
- 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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- C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
- 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
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- 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
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- 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
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- 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
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- 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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- C—CHEMISTRY; METALLURGY
- 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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- C—CHEMISTRY; METALLURGY
- 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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- C—CHEMISTRY; METALLURGY
- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Peptides Or Proteins (AREA)
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Abstract
ABSTRACT OF THE DISCLOSURE
The present invention relates to the preparation of glycoproteins by oxidation of their carbohydrate units with periodate ions. The resulting novel glycoproteins are useful as drugs and for the preparation of prolonged-action immunotoxins.
The present invention relates to the preparation of glycoproteins by oxidation of their carbohydrate units with periodate ions. The resulting novel glycoproteins are useful as drugs and for the preparation of prolonged-action immunotoxins.
Description
Process Eor the preparation of_antitumoral glycoproteins modified on their carbohydrate units.
The present invention relates to new antitumoral glycoproteins whose carbohydrate u~its are modified S by oxidation with the periodate ion.
~ lore particularly, but without implying a limita-tion, the present invention r~fers to the new glyco-proteins which inactivate ribosomes and have a pro-longed action.
The term "glycoprotein which inactivates ribo-somes", as used in the present description and also in the claims, denotes any substance which carries saccharide units belonging to the class of macromole-cules which inactivate ribosomes and consequently in-hibit 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 biosynthetic origin, being derived from a cell whose genotype has been modified for this purpose, and it also being possible Eor 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 denoced by the symbols GPIR.
In the description, the term "periodate" denotes the I04 ion, which is also referred to in the litera-ture 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 Applica-tions no. 81/07596 and no. 81!~1836 describe the pre-paration of anticancer products called conjugates, .,v , :,. -, ,. :
:: ':
: ~- ' ,., , :
~ -:
37~
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 App]lcation, under the generic name of immunotoxins.
Conjugates analogous to the previously des-cribed immunotoxins containing the A chain oE ricin are ~nown which are also suitable as anticancer drugs and result from the coupling, by means of a covalent bond, of antibodies or antibody fragments with other glyco-proteins ~hich inactivate ribosomes, such as, in par-ticular, the gelonine extracted from Gelonium multi-florum (Eur. J. Biochem., 1981, 116, 447-454; Cancer Res., 1984, 44, 129-133) or the inhibitor extracted from Momordica charantia (MOM) (U.S. 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 monensin and nigericin.~ - ~
HoweYer, the therapeutlc effects of immuno-toxins, whether activated or not, can only manifest themselves fully inasmuch as the immunotoxin~is capable, through its antlbody part, o~ becoming localized in vivo, in the active form, on the~target cells to~be destroyed (si~ne qua non~c~ondit~ton for an~y expression of immunotoxin activity). The capacity of the immunotoxin to become localized on the targe~ de-pends first and foremost~on the ab~l1ty of~ the immuno-:, ~9~
-- 3 ~
toxin to remain in the bloodstream and the extracellu-lar fluids, in the active form, for sufficient lengths of time for it to reach its target cells and in suffi-ciently large concentrations for the degree of occu-pation of the corresponding antigenic sites to be high.
~ umerous studies have made it possible to es-tablish the plasma elimination kinetics of immunotoxins after intravenous injection into different animal models. It is apparent that, after injection, the plasma level of biologically active immunotoxin de-creases very rapidly and very substantiallyO 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 O after injection disappears in 30 minutes and 99.9% disappears in 17 hours. This rapid
The present invention relates to new antitumoral glycoproteins whose carbohydrate u~its are modified S by oxidation with the periodate ion.
~ lore particularly, but without implying a limita-tion, the present invention r~fers to the new glyco-proteins which inactivate ribosomes and have a pro-longed action.
The term "glycoprotein which inactivates ribo-somes", as used in the present description and also in the claims, denotes any substance which carries saccharide units belonging to the class of macromole-cules which inactivate ribosomes and consequently in-hibit 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 biosynthetic origin, being derived from a cell whose genotype has been modified for this purpose, and it also being possible Eor 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 denoced by the symbols GPIR.
In the description, the term "periodate" denotes the I04 ion, which is also referred to in the litera-ture 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 Applica-tions no. 81/07596 and no. 81!~1836 describe the pre-paration of anticancer products called conjugates, .,v , :,. -, ,. :
:: ':
: ~- ' ,., , :
~ -:
37~
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 App]lcation, under the generic name of immunotoxins.
Conjugates analogous to the previously des-cribed immunotoxins containing the A chain oE ricin are ~nown which are also suitable as anticancer drugs and result from the coupling, by means of a covalent bond, of antibodies or antibody fragments with other glyco-proteins ~hich inactivate ribosomes, such as, in par-ticular, the gelonine extracted from Gelonium multi-florum (Eur. J. Biochem., 1981, 116, 447-454; Cancer Res., 1984, 44, 129-133) or the inhibitor extracted from Momordica charantia (MOM) (U.S. 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 monensin and nigericin.~ - ~
HoweYer, the therapeutlc effects of immuno-toxins, whether activated or not, can only manifest themselves fully inasmuch as the immunotoxin~is capable, through its antlbody part, o~ becoming localized in vivo, in the active form, on the~target cells to~be destroyed (si~ne qua non~c~ondit~ton for an~y expression of immunotoxin activity). The capacity of the immunotoxin to become localized on the targe~ de-pends first and foremost~on the ab~l1ty of~ the immuno-:, ~9~
-- 3 ~
toxin to remain in the bloodstream and the extracellu-lar fluids, in the active form, for sufficient lengths of time for it to reach its target cells and in suffi-ciently large concentrations for the degree of occu-pation of the corresponding antigenic sites to be high.
~ umerous studies have made it possible to es-tablish the plasma elimination kinetics of immunotoxins after intravenous injection into different animal models. It is apparent that, after injection, the plasma level of biologically active immunotoxin de-creases very rapidly and very substantiallyO 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 O after injection disappears in 30 minutes and 99.9% disappears in 17 hours. This rapid
2~ 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. ~ore-~5 over, comparison of the plasma elimination kinetics ofthe immunotoxins with those of the corresponding non-conjugated antibodies sho~s 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~immuno-toxin preparations. Through the effect of the differ-ential elimination rates of immunotoxins and~anti-bodies, the non-conjugated antibodies, which are initially in a very small m1nority, gradually become a . :
- ~ ~
7~
majority after a few hours and these antibodies there-fore gradually become, by competition, powerf~1l an-tagonists for the fi~ation of the immunotoxins to their targets.
The advantage of increasing the plasma persis-tence of immuno~oxins, 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, radiolabelled and then injected into animals without a specific target, have shown that, in the first few lS minutes after injection, the conjugate becomes local-ized 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 est~ab~
lished. Moreover, it has been~shown that the glyco-proteins recognized by these~re~ceptors - the latter bein8 present es~sentially on the Kupffer cel~ls -are rapidly elimin~at:ed from the bl~oodstream~by;~fixation to these cells~, which~metabolize them. This is par~
ticular]y well documented in the case of beta-glucuro-nidase and also i~n the cas~e of; r1bonuclease B; (Arch.
Biochem. Biophys.,~1978, 188,~418, Adva~nces in;~
: ~ : : : : : :
- : ~
Enzymology, published by A. Meister, New York, 1974;
Pediat. Res., 1977, ll, 8l6).
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 ~lO~I, 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% dis-` appears 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~t~o aldehyde groups on the carbons between which the scission has taken place.
It has now been found that when the carbo-hydrate units of an antitumoral glycoprotein are mod-ified by oxidation with periodate ions, the biological activity of the sald glyc~oproteLn~remains~substantlally -unchanged.
~ It has also been found,~ absolutely unexpectedly, that lf the carbohydra~te units~of a glycoproteln which ::
.
' , ' `, -:
inactivates ribosomes are modified by oxidation with periodate ions, a new glycoprotein which inactivates libosomes is obtained, the said new glycoprotein having the dual property of retaining its biological activit-ies and of being eliminated very slowly from the blood-stream in vivo.
~ n in-depth biochemical study of oxidized and native GPIRs has made it possible to demonstrate that the oxidation of GPIRs with periodate involves ex-cluslvely the osidic part of the GPIRs and has noaction 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 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 con-jugates obtained retain the biological properties 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 re:Lates more particularly to glycoproteins which inactivate ribosomes, whose carbohydrate units are modified by oxidat1on~with the periodate ion and which have the same activity as a~nd a longer half-life than the unmodified glycoprotein.~
The invention preferential~ly relates~to glyco-proteins which inactivate~ribosomes~and have~a pro-longed action and which are obtained by~treatment of a glycoprot~e1n which~inac~tivates ribosomes, t~he~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 O to 15C and in the absence of light, unblocking of the thiol groups, if appropriate, and isolation oE the final product by known methods.
Any antitumoral olycoprotein 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, which are in themselves only very slightly cytotoxic because they cannot fix to cells, but which, on the lS other hand, after coupling with an antihody recog-nizing particular cells, become highly cytotoxic to-wards these cells once the antibody has recognized its target.
Representative starting compounds are the A
chain of ricin, gelonine and the substance extracted ~rom Momordica charantia (MOM), as obtained by ex-traction.
Other GPIRs which are usefu] as starting matcrials Eor oxidation with periodate ions are as 25 follo~ls:
Dianthin 30 from Dianthus caryophyllus Dianthin 32 from " "
~grostin A Erom Agrostemma githago 30 Agrostin B from " "
Agrostin C from HCI from Hura crepitans Asparagus officinalis from Asparagus inhibitor officinalis The same substances produced b;osynthetically ~2~7~
by cells whose genotype has been modified for this pur-pose 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 protec`ted is a preferred starting compound.
Recent studies have demonstrated that -the A
chain of ricin comprises 2 constituents denoted by Al and A2, ~hich differ especially in their polysaccha-ride units. The experiments which have been carried out on the 2 constituents of the A chain have made 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 oE:~ the pure A chaln of rlcin is described in U.S. Pa~tent 4 340 535. Gelonine and ~IOM are also described in p~rior art.
Protection of the~thiol~ groups of the starting~
GPIRs is desirable when the~sald;~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 hyd~roxyl of the tyrosines or amino groups~-~or;~carboxy~l groups of the ;
GPIRj protection can'be;carr~ied~out or not~
Blocklng~is car~ried~out~by reaction~wlth an agent capab~le of substituting the~SH~gr~oups with a ~rad~ical~-~which ca~n;~subs~equ~ently~be~remo~ved~by,~re;duction~
or~thiol/disu`lfide~exchang~e;~ for~exa~mple~2,2~ dini~tro~
5~,5'~-dit~hiod~b~enzolc:aci~d~(DTNB)~or~3-~(p;yrid~i~n:-2-~yl~
disulfanyl)pro~pl~onlc~aci~d o~r alter~atlvely~dl~pyridyl~
2,2'-disulfide~o~r~d;ipyridyl-~4~,~4~-d~lsulflde.~In the~
- ~. : . : : ~
absence of such a treatment, the free thiols may dis-appear during the oxidation reaction, in which case they cannot be totally regenerated. The excess bloc}cing agent is removed by dialysls or any other appropriate 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-larly, the concentration of alkali metal periodate isgreater than the concentration of the vicinal diols capable of being oxidized; concentrations of 10 to 50 mM in respect of sodium periodate for concentrations of 1 to 10 mg/ml of cytotoxic sub-unit are suitable. Thè
i5 treatment, carried out at a temperature of between 0 and 15C, preferably of between 1 and 5C, 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 t for ; 20 example an excess of ethylene glycol, and the by-pro-ducts are removed by dialysis or by any other appro-priate treatment. The product obtained at the end of the reaction is isolated by conventional techniques.
If the thiol groups of the starting material 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 anti-body to give an immunotoxin.
In the case of the A chain of ricin, the~result-ing new molecule (reEerred to by the symbols A~-La) possesses the following main properties: ~
- a mole~colar weight~which 15 not signlf1cant1y~d1ffer-, -:
:
-~;~4~
ent from that of the native A chain. As far as it ispossible to see by polyacrylamide gradient electro-phoresis, this modification process only produces polymers of the protein in a very small quantity and does not produce any degradation products.
- a proportion of free thiol groups greater than 0.7 per mol.
- an immunoreactivity to~ards rabbit antibodies in-hibiting the A chain of ricin ~hich is indistinguish-able from the immunoreactivity of the native A chain.- an inhibitory activity on the protein synthesis in an acellular model which is grea~er than 50% of that caused by an equal quantity of native A chain.
- finally, after a single intravenous administration to ra-bbits 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 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-ent~from that of the native gelonine.- an immunoreactivity towards anti-gelonine rabbit antibodies which is indistinguishable from that of~the native gelonine.
- finaIly, after a single intravenous administration to rabbits at a dose~of about 0.3 mg/kg of body weight, the plasma Ievel o~ the modified gelonine 24~hollrs after injection is greater than 3% of the level~present at time zero (as against 0.01%~for~ the nat:ive~gelon~in~e at this time) i.e. an increase in~ th~e~plasma leve~l by a factor greater ~than 200 .
:: ~
:: ::~ ::
"' The preparation of the conjugates or immuno-toxins from the glycoproteins which inactivate ribo-somes and have a prolonged action is carried out by any process suitably chosen from the range of processes descril)e~ in IJ.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 disulfide group. If the chosen cy,totoxic 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 then unblocked after this step, or after the oxidation step.
This gives modified immunotoxins which have acquired a new character as regards their pharmaco-kinetic properties. ~lore particularly, by appropriate 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 capa-city to show slow plasma elimination kinetics.
The examples which follow;provid,e a c~learer understanding of the~invention without l~imiting its scoye.
Exam~
Oxidation of the methylat~ed A chain in whïch the SH
groups are blocked w~ith N-ethy~lma~leimide.
:
~'~-: -, . .' ~
~ - .:, ` ' ~ .:. ' ' :
I - Preparation of the correctly functionalized A chain of ricin 1) Hexamethylation of the A chain The methylation reaction is carried out at 0C, with stirring, :in 0.2 M borate buffer of pH 10, by the method of Means and Feeney (Bi.ochemistry 7, 2192 (1968)). 20 mg of tritiated borohydride (containing 9.5 mCi/mmol) are add.ed to 35 ml of A chain (3 mg/ml), followed by 350 microliters of 6% formaldehyde (added in five 70 microliter portions spread over a period of 30 minutes).
The excess reagent is removed by continuous dialysis against 125 m~l phosphate buffer of pH 7 (40 l at 300 ml/h). After dialysis, the protein solution is centrifuged. 36.5 ml of hexamethylated A chain con-taining 2.6 mg/ml are collected.
2) Blocking with N-ethylmaleimide The natural Sl-l of the hexamethylated A chain is blocked by the method described in Methods in En~ymology 11, S~l (1967). To do this, the A chain of ricin obtained i.n the previous step is incubate(l for 2 hours at 30C ln the presence of 20 equivalents of N-ethylmal.eimide per mo:L of A chain. The excess re-agent is removed by continuous d:ialysis against 125 m~ phosphate buffer of pH 7, which is renewed for 20 hours at a rate of 500 ml/hour.After concentration, there is obtained l3 ml of a solution of ~ chain of ricin containin~ 7 m /ml ~n~
no longer possessing thiol groups which can be deter-- mined by ELLMAN's reagent. The product thus obtained is subsequently called hexamethylated li chain (NE~I).
- ~ ~
7~
majority after a few hours and these antibodies there-fore gradually become, by competition, powerf~1l an-tagonists for the fi~ation of the immunotoxins to their targets.
The advantage of increasing the plasma persis-tence of immuno~oxins, 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, radiolabelled and then injected into animals without a specific target, have shown that, in the first few lS minutes after injection, the conjugate becomes local-ized 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 est~ab~
lished. Moreover, it has been~shown that the glyco-proteins recognized by these~re~ceptors - the latter bein8 present es~sentially on the Kupffer cel~ls -are rapidly elimin~at:ed from the bl~oodstream~by;~fixation to these cells~, which~metabolize them. This is par~
ticular]y well documented in the case of beta-glucuro-nidase and also i~n the cas~e of; r1bonuclease B; (Arch.
Biochem. Biophys.,~1978, 188,~418, Adva~nces in;~
: ~ : : : : : :
- : ~
Enzymology, published by A. Meister, New York, 1974;
Pediat. Res., 1977, ll, 8l6).
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 ~lO~I, 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% dis-` appears 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~t~o aldehyde groups on the carbons between which the scission has taken place.
It has now been found that when the carbo-hydrate units of an antitumoral glycoprotein are mod-ified by oxidation with periodate ions, the biological activity of the sald glyc~oproteLn~remains~substantlally -unchanged.
~ It has also been found,~ absolutely unexpectedly, that lf the carbohydra~te units~of a glycoproteln which ::
.
' , ' `, -:
inactivates ribosomes are modified by oxidation with periodate ions, a new glycoprotein which inactivates libosomes is obtained, the said new glycoprotein having the dual property of retaining its biological activit-ies and of being eliminated very slowly from the blood-stream in vivo.
~ n in-depth biochemical study of oxidized and native GPIRs has made it possible to demonstrate that the oxidation of GPIRs with periodate involves ex-cluslvely the osidic part of the GPIRs and has noaction 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 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 con-jugates obtained retain the biological properties 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 re:Lates more particularly to glycoproteins which inactivate ribosomes, whose carbohydrate units are modified by oxidat1on~with the periodate ion and which have the same activity as a~nd a longer half-life than the unmodified glycoprotein.~
The invention preferential~ly relates~to glyco-proteins which inactivate~ribosomes~and have~a pro-longed action and which are obtained by~treatment of a glycoprot~e1n which~inac~tivates ribosomes, t~he~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 O to 15C and in the absence of light, unblocking of the thiol groups, if appropriate, and isolation oE the final product by known methods.
Any antitumoral olycoprotein 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, which are in themselves only very slightly cytotoxic because they cannot fix to cells, but which, on the lS other hand, after coupling with an antihody recog-nizing particular cells, become highly cytotoxic to-wards these cells once the antibody has recognized its target.
Representative starting compounds are the A
chain of ricin, gelonine and the substance extracted ~rom Momordica charantia (MOM), as obtained by ex-traction.
Other GPIRs which are usefu] as starting matcrials Eor oxidation with periodate ions are as 25 follo~ls:
Dianthin 30 from Dianthus caryophyllus Dianthin 32 from " "
~grostin A Erom Agrostemma githago 30 Agrostin B from " "
Agrostin C from HCI from Hura crepitans Asparagus officinalis from Asparagus inhibitor officinalis The same substances produced b;osynthetically ~2~7~
by cells whose genotype has been modified for this pur-pose 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 protec`ted is a preferred starting compound.
Recent studies have demonstrated that -the A
chain of ricin comprises 2 constituents denoted by Al and A2, ~hich differ especially in their polysaccha-ride units. The experiments which have been carried out on the 2 constituents of the A chain have made 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 oE:~ the pure A chaln of rlcin is described in U.S. Pa~tent 4 340 535. Gelonine and ~IOM are also described in p~rior art.
Protection of the~thiol~ groups of the starting~
GPIRs is desirable when the~sald;~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 hyd~roxyl of the tyrosines or amino groups~-~or;~carboxy~l groups of the ;
GPIRj protection can'be;carr~ied~out or not~
Blocklng~is car~ried~out~by reaction~wlth an agent capab~le of substituting the~SH~gr~oups with a ~rad~ical~-~which ca~n;~subs~equ~ently~be~remo~ved~by,~re;duction~
or~thiol/disu`lfide~exchang~e;~ for~exa~mple~2,2~ dini~tro~
5~,5'~-dit~hiod~b~enzolc:aci~d~(DTNB)~or~3-~(p;yrid~i~n:-2-~yl~
disulfanyl)pro~pl~onlc~aci~d o~r alter~atlvely~dl~pyridyl~
2,2'-disulfide~o~r~d;ipyridyl-~4~,~4~-d~lsulflde.~In the~
- ~. : . : : ~
absence of such a treatment, the free thiols may dis-appear during the oxidation reaction, in which case they cannot be totally regenerated. The excess bloc}cing agent is removed by dialysls or any other appropriate 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-larly, the concentration of alkali metal periodate isgreater than the concentration of the vicinal diols capable of being oxidized; concentrations of 10 to 50 mM in respect of sodium periodate for concentrations of 1 to 10 mg/ml of cytotoxic sub-unit are suitable. Thè
i5 treatment, carried out at a temperature of between 0 and 15C, preferably of between 1 and 5C, 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 t for ; 20 example an excess of ethylene glycol, and the by-pro-ducts are removed by dialysis or by any other appro-priate treatment. The product obtained at the end of the reaction is isolated by conventional techniques.
If the thiol groups of the starting material 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 anti-body to give an immunotoxin.
In the case of the A chain of ricin, the~result-ing new molecule (reEerred to by the symbols A~-La) possesses the following main properties: ~
- a mole~colar weight~which 15 not signlf1cant1y~d1ffer-, -:
:
-~;~4~
ent from that of the native A chain. As far as it ispossible to see by polyacrylamide gradient electro-phoresis, this modification process only produces polymers of the protein in a very small quantity and does not produce any degradation products.
- a proportion of free thiol groups greater than 0.7 per mol.
- an immunoreactivity to~ards rabbit antibodies in-hibiting the A chain of ricin ~hich is indistinguish-able from the immunoreactivity of the native A chain.- an inhibitory activity on the protein synthesis in an acellular model which is grea~er than 50% of that caused by an equal quantity of native A chain.
- finally, after a single intravenous administration to ra-bbits 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 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-ent~from that of the native gelonine.- an immunoreactivity towards anti-gelonine rabbit antibodies which is indistinguishable from that of~the native gelonine.
- finaIly, after a single intravenous administration to rabbits at a dose~of about 0.3 mg/kg of body weight, the plasma Ievel o~ the modified gelonine 24~hollrs after injection is greater than 3% of the level~present at time zero (as against 0.01%~for~ the nat:ive~gelon~in~e at this time) i.e. an increase in~ th~e~plasma leve~l by a factor greater ~than 200 .
:: ~
:: ::~ ::
"' The preparation of the conjugates or immuno-toxins from the glycoproteins which inactivate ribo-somes and have a prolonged action is carried out by any process suitably chosen from the range of processes descril)e~ in IJ.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 disulfide group. If the chosen cy,totoxic 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 then unblocked after this step, or after the oxidation step.
This gives modified immunotoxins which have acquired a new character as regards their pharmaco-kinetic properties. ~lore particularly, by appropriate 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 capa-city to show slow plasma elimination kinetics.
The examples which follow;provid,e a c~learer understanding of the~invention without l~imiting its scoye.
Exam~
Oxidation of the methylat~ed A chain in whïch the SH
groups are blocked w~ith N-ethy~lma~leimide.
:
~'~-: -, . .' ~
~ - .:, ` ' ~ .:. ' ' :
I - Preparation of the correctly functionalized A chain of ricin 1) Hexamethylation of the A chain The methylation reaction is carried out at 0C, with stirring, :in 0.2 M borate buffer of pH 10, by the method of Means and Feeney (Bi.ochemistry 7, 2192 (1968)). 20 mg of tritiated borohydride (containing 9.5 mCi/mmol) are add.ed to 35 ml of A chain (3 mg/ml), followed by 350 microliters of 6% formaldehyde (added in five 70 microliter portions spread over a period of 30 minutes).
The excess reagent is removed by continuous dialysis against 125 m~l phosphate buffer of pH 7 (40 l at 300 ml/h). After dialysis, the protein solution is centrifuged. 36.5 ml of hexamethylated A chain con-taining 2.6 mg/ml are collected.
2) Blocking with N-ethylmaleimide The natural Sl-l of the hexamethylated A chain is blocked by the method described in Methods in En~ymology 11, S~l (1967). To do this, the A chain of ricin obtained i.n the previous step is incubate(l for 2 hours at 30C ln the presence of 20 equivalents of N-ethylmal.eimide per mo:L of A chain. The excess re-agent is removed by continuous d:ialysis against 125 m~ phosphate buffer of pH 7, which is renewed for 20 hours at a rate of 500 ml/hour.After concentration, there is obtained l3 ml of a solution of ~ chain of ricin containin~ 7 m /ml ~n~
no longer possessing thiol groups which can be deter-- mined by ELLMAN's reagent. The product thus obtained is subsequently called hexamethylated li chain (NE~I).
3) Periodate oxidation 6 ml of the solution of hexamethylated A chain (NEM) obtained above are treated with NaI04 (12.8 mg) for 40 minutes in the dark, at pH 4.5 and at 0C. The reaction is stopped by the addition of 600 microliters of ethylene glycol and the reaction medlurn is dialyzed contlnuously against 0.1 M carbonate buffer of pH 10 (20 h at 500 ml/h)-II - Enzymatic activity of the prolon~ed-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 appro-priately complemented, subce]lular fractions of ratliver capable of incorporating 14C-phenylalanine in the presence of an artificial messenger RNA: poly-uridylic acid.
The procedure employed for preparing the sub-cellular fractions and measuring the incorporation of C-phenylalanine is an adaptation of the method des-cribed in Biochemica Biophysica Acta 1973, 312, 608-615, using both a microsomal fraction and a cytosol fraction of the rat hepatocytes. The sample contain-ing the A chain is introduced in the form of a solutionappropriately diluted in a 50 mM Tris HCl 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 inhibition of the incorporation o 1 C-phenylala~nine into the proteins for each reaction medium containing A chain of ricin.
The inhibitory activlty was determined. An IC50 of 2.7 ~10 l~mol/l i9 observed~for the~oxldized A chain~ The IC50 of thç control A chain in~the~ex-periment is 1.03-10 l mol/l; therefore,~the modifica-tion does not cause a los~s of a~ct1v~ity of the A~ch~in.
E~ample 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 rlcin ~lith sodium periodate l) 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. 20 equivalents oE a solution of 2,2'-dinitro-5,5'-dithio-dibenzoic acid (~TNB), i.e. 385 microliters of a 0.1 ~I solution of DT~B in a 125 mM phosphate buffer of pH
7 (this solution is brought to pH 7 with sodium hydro-xide), 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 20C.
The solution is then dialyzed against PBS buffer at 4C 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 ~ater are added to 6 ml of a solution con-taining 5 mg/ml of blocked A chain, brought to pH 6 ~ith l M acetic acid. Incubation is left to proceed for 16 hours at 4C 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 20C, the reaction medium is dialyzed at 4C against PBS buffer. The periodate oxidation produces a slight precipitate of protein, which is re-moved by centrifugation at lO,000 x g for 30 minutes.
This gives 24 mg of oxidized blocked A chain at a con~
centration of 3.4 mg/ml.
3~ Unblocking of the thiol groups 2-Mercaptoethanol is added as a reducing agent, ' : :
. ` ` ' , ~ ~ ` ~ :
:
~ - , .
, ~ ~
, 7~
at a final concentration of 1%, to 6 ml of oxidized blocked A chain containing 3.4 mg/ml in PBS buffer.
Inc~lbation is left to proceed for 1 hour at 20C. The solution is then dialyzed against PBS buffer at 4C.
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 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 dodecyl-sulfate.
The previously obtained preparation of A chain in which the polysaccharide units have been oxidized ~as studied for its enzymatic activities in the in-hibition of protein synthesis and for its pharmaco-kinetic properties.
II - Enzymatic activit~ of the prolonoed-action A chain, measured on an acellular modeI
The inhibitory activity ~as determined by the technique described in example 1. An IC50 of 3 10 ]
mol/l is observed for the oxidized A chain. The IC50 of the control A chain in the experiment isl~2 10 l moi/l; therefore, the modification-does not cause a loss of acti~ity of the A chain.
III - Pharmacokinetic~properties of the prolonged-action A chain (A-La) The A chain is administered to rabbits by means 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~analyz;ed w1th the ald of a radioimmunometric test designa;ted bel~ow~b~y~ the~abbreviation RIM-1. ~ ~
This technique~has the advantage of d~etermining ; :, ~:
. :
, :
..
~: ' 7~
the A chain wi-thout modifying it. This deter~ination is carried out in microtitration plates (for example:
"NUNC-TSP screening s'ystem" from Poly Labo Block France), the lid of ~hich 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 AcI
for 24 h at 4C and'then with fetal calf serum for 3 h at 20C in order to saturate all-the fixation sites.
The saturated immunoabsorbent is then brought into con-tact for 3 h at 20C with the plasma samples to be de-termined at different dilutions, or with solutions of A
chain o'f 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 20C
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 ofGreenwood and Hunter (Biochem J., 1963, 89, 114); the specific activity of the radiolabeled A'c2 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 determinéd is measured by reference to the calibration curve established by introducing the A chai'n .
.
~ ' ~
. '. : ' . ' ' 7~
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 repro-ducible and reliable. The detection threshold is 1 nanogram/ml. A study of the reproducibility within and bet~een experiments 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 on the abscissa and the plasma con-centration of the product measured, recorded in pe~ centof 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:
concentration measured at time t x 100 RPC =
quantity injectedlplasma volume The plasma volume is considered to be equal to 36 ml/kg of the animal's body weight.
'25 Figure 1 shows the plasma elimination curve, as a function of time, for the A chain of native ricin in-jected intravenously. This curve (curve l) has two phases: in the first phase~, the product d1sappears very~
rapidly from the blood'stream since only 0.1% of~the dose 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 i~ts poly-saccharide units,~the eliminati~on profile is profo'undly modlfled: the first-eliminaéion phase -;which~is res~
ponsible for the disappear~ance;~of~the majority of the;
: : : ::
., . :
: ~ , :
7~
product - is practically suppressed, which leads to a considerable increase in the plasma leve]s 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).
E~ample 3:
This example demonstrates the effect of periodate o~idation 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 40 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 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 oE 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, ll, 541 (1967). To do this, the A chain of ricin obtained in the previous step is incubated for 2 hours at 30C
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 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 (NE~I).
b) Periodate oxidation of~the A chain (NEM) Periodate oxidation of the A chaln (NE~I) is~
, :
~:
' ~' ~ ' ' :
:
7~
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-thesis was determined using the procedure described inexample l. The enzymatic properties are found to be maintained ~ith an IC50 of 4.3-10 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 collect-ed at time 23 h are analyzed using the immunometric test RI~-1 as described in example 2. The results are shown in the table below:
I
Plasma concentration 23 h after injection:
_ A chain (NEM) 0.01%
20 Oxidized A chain ~ 8%
(NEM) Twenty~three hours after injection, the con-centration 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 pharmaco-kinetic properties of the oxidized A chain.
Six preparations of oxidized A chain are pre-pared using the procedure lndicated in example~2, exceptfor the duration of the sodium periodate treatment. The treatment times are as Eollows: zero (reaction stopped .
. ~ :
.
immediately with ethylene glycol), 20 minutes, 40 min-utes, 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 5:
This example demonstrates the importance of the duration of the oxidative treatment on the pharmaco-kinetic properties-of the methylated A chain blocked with NE~I.
1) - Preparation of the functionalized A chain of ricin a) Blocking oE 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 t`hat described ~5 in example l.
b) Methylation of the A chain The methylation reaction is carried out at 0C, with stirring, in 0.2 M borate buffer of pH 10, by the method of Means and Feeney (Biochemistry~7~j 2192, 1968).
38 mg of tritiated borohydride (containing 47 mCi/mmol) are added to 65.5 ml of A chain (N~EM) (3 mg/ml),;~follo~-ed by l ml of 6% formaldehyde~added in~five~200 mlcro-liter portions spread over a period~of 30 minutes~
The excess reagent is r~emoved by discontinuous 35 dialysis agains;t 125 m~ phosphate buffer of~pil 7~(40 ml).
~: :
. . ~ :
- ' ' ' .. ~ .
7~
After dialysis, the protein solution is centrifuged.
63 ml of methylated A chain containing 3 mg/ml are collected.
- c) Periodate oxidation Six preparations of methylated A chain (NEM) are oxidized using the procedure indicated in example l,except for the duration of the sodium periodate treatment. The treatment times are as follows: ~ero (reaction stopped immediately with ethylene glycol), lO 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 ) :
l. the increase in the plasma level of the methylated A chain (NEM) 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 ;
and 2. it is necessary for the duration of this reaction to 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 produces effects on each oE the two isomers which~are analogous to those described in example 2 for the chain of ricin.
l) - Separation of the Al and A2 chains 28 ml of; A cha~in~containing lO.~mg/ml~(309 mgl in 125 mM phosphate~buf~fer of pU~7.0~are deposited o~n~a c ~ : :
. .
, column of 112 ml of concanavalin A/sepharose, equili-brated in the same bufrer. 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 NaC] and 0.1 ~ in res?ect of alpha-methylmannoside.
This gives 184 mg of Al chain and 103 mg of A2 chain.
The Al and A2 chains are concentrated by ultra-filtration under nitrogen pressure; the A2 chain isdialyzed against 125 m~l phosphate buffer of p~l 7Ø
Analysis of the A chain by acrylamide gel gradient electrophoresis with SDS shows the presence of 2 bands of different intensity, corresponding to molec-ular weights of 30,000 and 33,000. The Al chain corres-ponds to the band of stronger intensity and of MW
30,000 and the A2 chain corresponds to the band of weak intensity and of ~IW 33,000.
2) - ~lodification o the Al and A2 chains of ricin with 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 or their enzymatic activities in the inhibition of protein synthesis and for their pharmacokinetic prop-erties.
3) -Enzymatic activities of the prolonged-action Al and A2 chains, measured on an acellular model The inhibitory activity was determined as des-cribed in`example 1. The IC 0 observed is equal to2.1-10 10 mol/l and 2.1.10 1~ mol/l for the~oxidized Al and A2 chains respectively. The IC50 values of the native Al and A2 chains, which a~e the controls in the experiment, are 1.9-10 10 mol/l and 1 10-~1 mol/l respectively. Thereeore, the modlf1cation of the :
:
;,, '~
separate ~ariants of the A chain does not cause a loss of their enzymatic activity.
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 appro-priately complemented, subce]lular fractions of ratliver capable of incorporating 14C-phenylalanine in the presence of an artificial messenger RNA: poly-uridylic acid.
The procedure employed for preparing the sub-cellular fractions and measuring the incorporation of C-phenylalanine is an adaptation of the method des-cribed in Biochemica Biophysica Acta 1973, 312, 608-615, using both a microsomal fraction and a cytosol fraction of the rat hepatocytes. The sample contain-ing the A chain is introduced in the form of a solutionappropriately diluted in a 50 mM Tris HCl 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 inhibition of the incorporation o 1 C-phenylala~nine into the proteins for each reaction medium containing A chain of ricin.
The inhibitory activlty was determined. An IC50 of 2.7 ~10 l~mol/l i9 observed~for the~oxldized A chain~ The IC50 of thç control A chain in~the~ex-periment is 1.03-10 l mol/l; therefore,~the modifica-tion does not cause a los~s of a~ct1v~ity of the A~ch~in.
E~ample 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 rlcin ~lith sodium periodate l) 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. 20 equivalents oE a solution of 2,2'-dinitro-5,5'-dithio-dibenzoic acid (~TNB), i.e. 385 microliters of a 0.1 ~I solution of DT~B in a 125 mM phosphate buffer of pH
7 (this solution is brought to pH 7 with sodium hydro-xide), 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 20C.
The solution is then dialyzed against PBS buffer at 4C 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 ~ater are added to 6 ml of a solution con-taining 5 mg/ml of blocked A chain, brought to pH 6 ~ith l M acetic acid. Incubation is left to proceed for 16 hours at 4C 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 20C, the reaction medium is dialyzed at 4C against PBS buffer. The periodate oxidation produces a slight precipitate of protein, which is re-moved by centrifugation at lO,000 x g for 30 minutes.
This gives 24 mg of oxidized blocked A chain at a con~
centration of 3.4 mg/ml.
3~ Unblocking of the thiol groups 2-Mercaptoethanol is added as a reducing agent, ' : :
. ` ` ' , ~ ~ ` ~ :
:
~ - , .
, ~ ~
, 7~
at a final concentration of 1%, to 6 ml of oxidized blocked A chain containing 3.4 mg/ml in PBS buffer.
Inc~lbation is left to proceed for 1 hour at 20C. The solution is then dialyzed against PBS buffer at 4C.
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 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 dodecyl-sulfate.
The previously obtained preparation of A chain in which the polysaccharide units have been oxidized ~as studied for its enzymatic activities in the in-hibition of protein synthesis and for its pharmaco-kinetic properties.
II - Enzymatic activit~ of the prolonoed-action A chain, measured on an acellular modeI
The inhibitory activity ~as determined by the technique described in example 1. An IC50 of 3 10 ]
mol/l is observed for the oxidized A chain. The IC50 of the control A chain in the experiment isl~2 10 l moi/l; therefore, the modification-does not cause a loss of acti~ity of the A chain.
III - Pharmacokinetic~properties of the prolonged-action A chain (A-La) The A chain is administered to rabbits by means 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~analyz;ed w1th the ald of a radioimmunometric test designa;ted bel~ow~b~y~ the~abbreviation RIM-1. ~ ~
This technique~has the advantage of d~etermining ; :, ~:
. :
, :
..
~: ' 7~
the A chain wi-thout modifying it. This deter~ination is carried out in microtitration plates (for example:
"NUNC-TSP screening s'ystem" from Poly Labo Block France), the lid of ~hich 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 AcI
for 24 h at 4C and'then with fetal calf serum for 3 h at 20C in order to saturate all-the fixation sites.
The saturated immunoabsorbent is then brought into con-tact for 3 h at 20C with the plasma samples to be de-termined at different dilutions, or with solutions of A
chain o'f 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 20C
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 ofGreenwood and Hunter (Biochem J., 1963, 89, 114); the specific activity of the radiolabeled A'c2 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 determinéd is measured by reference to the calibration curve established by introducing the A chai'n .
.
~ ' ~
. '. : ' . ' ' 7~
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 repro-ducible and reliable. The detection threshold is 1 nanogram/ml. A study of the reproducibility within and bet~een experiments 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 on the abscissa and the plasma con-centration of the product measured, recorded in pe~ centof 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:
concentration measured at time t x 100 RPC =
quantity injectedlplasma volume The plasma volume is considered to be equal to 36 ml/kg of the animal's body weight.
'25 Figure 1 shows the plasma elimination curve, as a function of time, for the A chain of native ricin in-jected intravenously. This curve (curve l) has two phases: in the first phase~, the product d1sappears very~
rapidly from the blood'stream since only 0.1% of~the dose 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 i~ts poly-saccharide units,~the eliminati~on profile is profo'undly modlfled: the first-eliminaéion phase -;which~is res~
ponsible for the disappear~ance;~of~the majority of the;
: : : ::
., . :
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7~
product - is practically suppressed, which leads to a considerable increase in the plasma leve]s 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).
E~ample 3:
This example demonstrates the effect of periodate o~idation 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 40 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 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 oE 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, ll, 541 (1967). To do this, the A chain of ricin obtained in the previous step is incubated for 2 hours at 30C
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 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 (NE~I).
b) Periodate oxidation of~the A chain (NEM) Periodate oxidation of the A chaln (NE~I) is~
, :
~:
' ~' ~ ' ' :
:
7~
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-thesis was determined using the procedure described inexample l. The enzymatic properties are found to be maintained ~ith an IC50 of 4.3-10 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 collect-ed at time 23 h are analyzed using the immunometric test RI~-1 as described in example 2. The results are shown in the table below:
I
Plasma concentration 23 h after injection:
_ A chain (NEM) 0.01%
20 Oxidized A chain ~ 8%
(NEM) Twenty~three hours after injection, the con-centration 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 pharmaco-kinetic properties of the oxidized A chain.
Six preparations of oxidized A chain are pre-pared using the procedure lndicated in example~2, exceptfor the duration of the sodium periodate treatment. The treatment times are as Eollows: zero (reaction stopped .
. ~ :
.
immediately with ethylene glycol), 20 minutes, 40 min-utes, 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 5:
This example demonstrates the importance of the duration of the oxidative treatment on the pharmaco-kinetic properties-of the methylated A chain blocked with NE~I.
1) - Preparation of the functionalized A chain of ricin a) Blocking oE 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 t`hat described ~5 in example l.
b) Methylation of the A chain The methylation reaction is carried out at 0C, with stirring, in 0.2 M borate buffer of pH 10, by the method of Means and Feeney (Biochemistry~7~j 2192, 1968).
38 mg of tritiated borohydride (containing 47 mCi/mmol) are added to 65.5 ml of A chain (N~EM) (3 mg/ml),;~follo~-ed by l ml of 6% formaldehyde~added in~five~200 mlcro-liter portions spread over a period~of 30 minutes~
The excess reagent is r~emoved by discontinuous 35 dialysis agains;t 125 m~ phosphate buffer of~pil 7~(40 ml).
~: :
. . ~ :
- ' ' ' .. ~ .
7~
After dialysis, the protein solution is centrifuged.
63 ml of methylated A chain containing 3 mg/ml are collected.
- c) Periodate oxidation Six preparations of methylated A chain (NEM) are oxidized using the procedure indicated in example l,except for the duration of the sodium periodate treatment. The treatment times are as follows: ~ero (reaction stopped immediately with ethylene glycol), lO 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 ) :
l. the increase in the plasma level of the methylated A chain (NEM) 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 ;
and 2. it is necessary for the duration of this reaction to 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 produces effects on each oE the two isomers which~are analogous to those described in example 2 for the chain of ricin.
l) - Separation of the Al and A2 chains 28 ml of; A cha~in~containing lO.~mg/ml~(309 mgl in 125 mM phosphate~buf~fer of pU~7.0~are deposited o~n~a c ~ : :
. .
, column of 112 ml of concanavalin A/sepharose, equili-brated in the same bufrer. 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 NaC] and 0.1 ~ in res?ect of alpha-methylmannoside.
This gives 184 mg of Al chain and 103 mg of A2 chain.
The Al and A2 chains are concentrated by ultra-filtration under nitrogen pressure; the A2 chain isdialyzed against 125 m~l phosphate buffer of p~l 7Ø
Analysis of the A chain by acrylamide gel gradient electrophoresis with SDS shows the presence of 2 bands of different intensity, corresponding to molec-ular weights of 30,000 and 33,000. The Al chain corres-ponds to the band of stronger intensity and of MW
30,000 and the A2 chain corresponds to the band of weak intensity and of ~IW 33,000.
2) - ~lodification o the Al and A2 chains of ricin with 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 or their enzymatic activities in the inhibition of protein synthesis and for their pharmacokinetic prop-erties.
3) -Enzymatic activities of the prolonged-action Al and A2 chains, measured on an acellular model The inhibitory activity was determined as des-cribed in`example 1. The IC 0 observed is equal to2.1-10 10 mol/l and 2.1.10 1~ mol/l for the~oxidized Al and A2 chains respectively. The IC50 values of the native Al and A2 chains, which a~e the controls in the experiment, are 1.9-10 10 mol/l and 1 10-~1 mol/l respectively. Thereeore, the modlf1cation of the :
:
;,, '~
separate ~ariants 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 immunometric test RIM-l (see e~ample 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%
15 Oxidized A chain (A-La) 10%
Al chain 0.02%
Oxidized Al chain (Al-La) 10%
A2 chain 0.04%
Oxidiæed 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 Al and A2.
Example 7:
This example describes the biochemical character-istics 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 used in these studies are prepared as described in examples 2~and 6.
I - Carbohvdrate compo~iti;ons The carbohydrate~compositions of these~proteins are determined by~gas chromatographic analyses us1ng :
'' ~' ~ ' :, , ' ~ .
Clamp's method (in Glycoproteins: their composition, structure and function (edited by A. Gottschalk), volume
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%
15 Oxidized A chain (A-La) 10%
Al chain 0.02%
Oxidized Al chain (Al-La) 10%
A2 chain 0.04%
Oxidiæed 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 Al and A2.
Example 7:
This example describes the biochemical character-istics 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 used in these studies are prepared as described in examples 2~and 6.
I - Carbohvdrate compo~iti;ons The carbohydrate~compositions of these~proteins are determined by~gas chromatographic analyses us1ng :
'' ~' ~ ' :, , ' ~ .
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 Yorlc).
The results obtained are collated in the two tables below.
Percenta~e composition Chains Total carbohydrates, %
Native A 5.58 + or - 0.5 10Al 4.54 + or - O.S
A2 6.24 + or - 0.5 Oxidiæed A 2.27 + or - 0.5 Al 2.07 + or - 0.5 A2 3.33 + or - 0.5 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) _ Chains . . I
20MonosaccharidesNative Oxidized _ A Al A2 A Al A2 N-Acetylglucosamine 1.89 1.48 2.15 1.74 1.50 2.37 ~lannose 4.6 3.40 5.2 I.43 1.29 2.26 Fucose 1.37 1.41~ 1.52 O O
Xylose 1 6 1.48 1.67 0.~6 0.48 0.6 : ~ ~
These results prove that~periodate~oxidation has destroyed part of the~sugars o-E the~A chai~n. Per molecule of A chaln9~there is~an~a~vqrage decrease~of ::
~ ~ : . ' :
:::: : : :
. ~
,''"~
:
3.17, 2.11 and 2.9~ 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-acetylglucosamine residues are only slightly degraded.
II - N-Terminal sequence The sequence of the N-terminal amino acids oE
the A chain and its Al and A2variants, in the native form and in the o~idized 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.
Chains Sequenc of the 9 N-terminal amino acids Native A Ile-Phe-Pro-Lys-Gln-Tyr-Pro-Ile-Ile A1 Ile-Phe-Pro-Lys-Gln-Tyr-Pro-Ile-Ile A2 Ile-Phe-Pro-Lys-Gln-Tyr-Pro-Ile-Ile . .
O~idized A Ile-Phe-Pro-Lys-Gln-Tyr-Pro-Ile-Ile Al Ile-Phe-Pro-Lys-Gln-Tyr-Pro-Ile-Ile A2 Ile-Phe-Pro-L~Js-Cln-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 of the 9 N-terminal amino~ acids~ of the A chains~is strictly identical to that previously desc~ribed by Funatsu for the A chain of ricin (Agr~ic. Biol. Chem., (1979), 43, 2221).
III - Affinity on concanavalin A/sepharose ~
- 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 flx to concanav~a11n~ A~/sepharose. 1~ml~oE
:
. . . :
: ~: .
. ~ ~
: . : ~ . :
,, -: , ~ " ~ :
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 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 ith 0.1 ~I borate buffer of pH 6.0, which is 0.5 ~l in respect of NaCl and 0.1 ~l in respect of àlpha-methylmannoside. The results, expressed as a percentage of the optical den-sity at 280 nm are summari~ed in the table below.
.
Chains % not retained % eluted by alpha- Total by the con A methylmannoside (%) _ A(DTNB) 66 11 77 A(DTNB)-La78 5 83 _ Al(DTNB) 80 6 86 Al(DTNB~La 73 0.4 73.4' A2(DTNB) 25 70 95 A2(DTNB~La 64 9 73 _ _ It is known that concanavalin A has an affinity for glycoproteins with terminal mannoses. It is foun(l 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.
I~- Determination of _he E l~o The absorption coefficient ~t 280 Dm (E 1~is :
- : .,. ' , ~, , : ~ .
' ' :
g~
the optical density at 280 nm of a solution containing 1 mg/ml, in which the protein concentration is deter-mined by the FOLIN test with a standard range of bovine serum albumin.
The results are summarized in the table which follows:
.
A chain 0.6~
A(DTNB) chain 1.12 A(DTNB)-La chain 1.04 .
Al(DTNB) chain 1.07 Al(DTNB)-La chain 1.02 .
A2(DTNB) chain 0.95 A2(DTNB)-La chain 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 introductlon of the nitrobenzoyl group.
After oxidation, no significant variation in t`he absorption at 280 nm is observed, demonstrating that oxidation has not affected amlno acids responsible for the absorption at 280 nm.
Y - Isoelectric focusing Analysis of the A chain by isoel~ectric~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 thé~ cysteine~-with mercaptoethanol brings these bands ba~ck~to the location~o~f thé natlve A chain.
Thus, a compari~son o;f~the isoelec~tric points of : :
: .
~L~J7~
the native and oxidized A, A1 and A2 chains shows that, in the ahsence oE a blocking agent, all the bands charac-teristic oE the A chain are transferred by 0.5 pH unit towards acid pH values. This transfer takes place with-out overlapping of the pH regions of the native andoxidized 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-1`0 ation of native gelonine, and 2) the slow elimination ofgelonine modified with sodium periodate, after intra-~enous 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 con-ditions 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 intro-duced 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 pll 6 with ;
1 M acetic acid. Incubation is~left ~:o proceed for 16 hours at 4C in the dark.~ The reaction is~stopped by the addition of 105 micraliters of a 1 M a~queous~sol-ution of ethylene glycol. ~After incubation for 15~min-utes at 20C, the reaction medium is dialyzed at 4C
against PBS bufer. A~fter centr~1fu~gation at IO,OOQ x g for 30 minutes, this givcs 2.9~mg oE~o~idi~zed gelonine : ~ I
- : ~
at a concentration of 2.5 mg/ml.
Like the i~ chain of ricin, the fundamental prop-erty of gelonine is to inhibit protein synthesis in eucaryotic cells by degradation of the ribosomal sub-,unit ~0 S (Biochem. J. (1982) 207, 505-509). In th,e case of gelonine too, the modification due to periodate' oxidation does not cause a loss o:E activity.
II - Pharmacokinetic properties of prolon~ed-action oenonine ~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.
Blood samples are taken at intervals on heparin. The plasmas are analyzed with the aid of a radio;mmunometric test designated below by the abbreviation RIM-2.
This test is performed by the same technique as used for the test RI~-l, except that the solution ~cl here is a solution of anti-gelonine rabbit antibodies purified by aEfinity c~hromatography, the ~c2 antibodies beillg the same antibodies radiolabeled. The radio-labeling procedure is ide~ntical to that described forthe technique RIM-l. The concentration of native gelonine or modified gelonine in the samples~to be 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 reliabi~lit~y and reproducibility~ :
- characteristics as described fo:r the technique~RIM
The results of these'experlments arc represente~d~in the same way~as for~the A chain, of rici.n in example 2.:
Figure 4 shows~:the~plasma elimination curves, .
. as a :function of time,~for native~gelonine and~oxidized gelonine, injected intra:venously.: The native~ge~lonine, like the A chain of~native~ricin, d~sappears very:ràpldly -:
' 7~
from the bloods-tream 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-?4 hours after injection, the concentration of theoxidized gelonine is 300 times greater than that of the native gelonine (curve 2).
Thus, as for the A chain of rlcin, 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 e~ample 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.
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 (Biochem1cal Journal (1980), 186, 443-452). The pharmacokinetic properties of native or modified MOM were established using radioactive GPIR
MOM~ The MOM is radiolabel~ed on the tyrosines~with radioactive iodine-125 in the-presence of chloramine T.
10 microliters of a solution of radioactive iodine-125 containing lOO ~Ci/ml, and 30 microliters o~ a solution~
of chloramine T containing 2.5 mgiml in water, are added to 100 microl1ters of~a~;solut1on~cont~ain~ing 1~m~g~/ml~of MOM in PBS buff~er. The~reaction~is left to~proceed for one minute at~ambient;te;mperature. The reaction is st~opped by the addltion;of~40~0 micro~liters of a solution o~ sodium metabisulf1te~¢ontainlng~0~.5 mg/ml.~The re--.
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action medium is chromatographed by gel Eiltration 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 ~he same conditions as described for the A chain of ricin in example 2, except that the step in which the thiols are ~0 blocked with DT~B is omitted and the concentration of protein is 125 times smaller (40 ~g/ml). 20 micro-liters 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 MO~I, brought to pH ~ with 1 M acetic acid. Incubation is left to proceed for 16 h at 4C 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 20C, the reaction medium is dialyzed at 4C 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/m].
The new molecule thus obtained has a molecular weight which is not significantly different from that ~5 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 polymersin a very small quantity and does not produce any degradation product.
2) - Pharmacokinetic properties of prolon~ed-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 3.5S
micrograms/kg, Blood samples aFe taken at intervals on * - Trademark .~` ..
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,, .. ., ~ -heparin. The plasmas (200 microliters) are incubated with trichloroacetic acid (TCA) (200 microliters at a concentration of 25%) for 30 minutes at 4C. After centrifugation, the radioactivity contained in the sedi-ment which can be precipitated by the acid is deter-mined. This method of analysis makes it possible to measure the plasma level of the intact ~IOM molecules, and any low-molecular degradation products whlch cannot be precipitated by TCA are not taken into account.
The results of these experiments are represented as the percentage of the initial radioactivity remain-in8 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 of 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 intra-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 1). When the~IOM has been oxidized on its sugar residues, the elimin-ation rate is reduced (c~rve 2): 8 hours after injection,level of oxidized MOM is 60 times grea~ter 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 e~limin-:
:
, ation of the MOM.
Example 10:
This example demonstrates:
1. the rapid elimination of GPIR Dianthin ex-tracted from Dianthus caryophyllus,and 2. the slow elimination of GPIR Dianthin mod-ified 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 radio-active Dianthin. The io~ination 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-ent from that of the native Dianthin 30.2) - Pharmacokinetic properties of prolon~ed-action Dianthin 30 The oxidized or unoxidized radiolabeled Dianthin is administered to rabbits by a single injection into a 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 MOM, disappears very rapidly from the bloodstream since 99.9% of the quantity i;nitially present disappears in 2 hours. On the other hand, when the Dianthin 30 has been oxidized on the carbohydrate residues, the~elimin-ation kinetics are slowed down considerably:~ 2 hours after injection, the Dlanthl~n level is 80 times greater , , .. .
than that of the unoxidized Dianthin. The level of o.Yidized 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 recog-nition process responsible for the rapid elimination of the Dianthin.
Example ll:
Conjugate obtained by the reaction of an antibody inhib iting human T cells (an antibody directed against the anti~en T65), substituted by activated disulfide groups, ~ith the oxidized A chain of ricin.
a) Antibody inhibiting human T cells (or antibody T101) This antibody was obtained by the method des-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) Rctivated antibodies inhibiting human T cells 20 microliters of a solution containing 60.3 mg/ml of 1-ethyl-3-dimethylaminopropyl-3-carbodiimide are added to 100 microliters of a solution containing ~0 mg/ml of 3-(pyridin-2-yldisulfanyl)propionic acid in tert.-butanol, and the mixture is left for 3 minutes at ambient ~emperature. 68 microliters of the solution thus obtained are added to 2 ml of an antibody solution containing 8.9 mg/ml in PBS buffer. The mixture~is stirred ~or 15 minutes at 30C and~then diaIyzed against PBS buffer at 4C. ~fter dialysis, the protein solution is centrifuged to give 15 mg of~activated antibody at~
a concèntration 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 act:ivated mixed~disulfide groups~per mol of antibody. ~
III) Pre~aration of the lmmuno~toxin~containln~_~roloneed-. .
action A chain of ricin 2.46 Ml of modified A chain conta:ining 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 20C. The solution is cen-trifuged and then purified by filtration on a Sephadex G100 column, the optical density of the efflue~t being measured at 280 nm. Combination of the fractions con-taining both the antibody and the A chain gives 15 mlof 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-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) TlOl, 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,~ wh`ich are abbreviated to IT (A-La)T101.
I - Procedure ~ ~
The conjugate prepared b~y the procedure explained in example-llis administered~to~ rabbits by a singlc in-jection into a vein in the ear.~ The quantity injected corresponds to 0`.415~mgtkg, expresse~d as~A~chain.
Blood samples~are take~n a~t intervals;on heparin.~ ~The~
plasmas are analyzed with~the~ aid~of a~r~adioimmunometric test with two sitesJ ~wh1ch lS ab~brevi~ated below to RIM_3.
This t~st is ~c~r-i.e~ out bV Ihe~ ~ame~ te~hn~que :: : : ` , : ., ~ :
, as that used for the test RIM-l, except that the solution Ac2 here is a so]ution of goat antibodies in-hibiting mouse IgG, purified by affinity chromatography and radiolabeled as described for the technique RIM-l.
The concentration of modified immunoto~in in the samples to be determined is measured by reference to a calibra-tion curve esta~lished by introducing the modified immunotoxin at different known concentrations. The test RI~l-3 has the same reliability and reproducibility characteristics as described for the technique R~ -1.
By ~ay 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 T101, substituted by activated disulfide groups, with the native A chain of ri~in. The preparation and the cytotoxic properties of this conjugate have been described in French Patent Application no.2 5l6 794 The results of these experiments are represented in the same way as for the uncoupled A chain of ricin in ex-ample 2II - Results Figure 7 shows the plasma elimination curves, as a function of time, for IT T101 and IT (A-La) T101, in-jected intravenously. Twenty-four hours after injection, the concentr&tion of active immunotoxin is 140 times greater Eor IT (A-La) TlOl than for IT T101. 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) T101 towards the target cells.
The fundamental bioIogical property of the A
chain of ricin is to inhibit protein synthesls in cells : ~ :
'h ' , by degradation of the ribosomal sub-unit 60S. The technique uses a cell model in ~hich the efEect of the substances studied on the incorporation o~ 14C_ leucine 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 anti-gen T65. The cells are incubated in the presence of the substance to be studied, and then, when incubation has ended, the degree of incorporation of 14C-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 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 wholecells isolated by filtration.
On the basis of these determinations, it is possiblè 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-tion of 14C-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-hibition of the incorporation of C-leucine, or "50%
inhibitory concentration" (IC50).
Figure 8 shows the curves obtained in the same experiment ~ith IT (A-La) T101 and the uncoupled ox-idized A~chain in the presence of 10 mM ammoniumchloride 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 1 M) which is aboilt 80,000 times greater than that of the uncoupled ox-idi~ed A chain, measur:d under the same conditions.
, Example 14:
This example demonstrates the comparative cyto-toxic efficacy oE IT (A-La) T101 and IT T101 towards CEM target cells, measured in a clonogenic test.
Immunotoxins are dedic~ted 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 poss-ible by optimum culture conditions in a gelled medium, applied to the CEM human lymphoid line.
I - Technique for measurin~ cytotoxicity_by the inh _ition of colony formation The medium used for cloning is the medium RPMI
1640 to which 1 mmol/l of sodium alpha-ketoglutarate, 1 mmol/l 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) is prepared in this medium, placed as a thin layer in small Petri dishes and solidified at +4C. The cells are mixed with a second, 0.275% agar solution kept at 37C, which is then deposited on the-first layer and solidified. These concentrations of agar were chosen after a preliminary study aimed at simultaneously opti-- mizing the cloning efficiency,~ the size of the colonies and the consistency oE the medium. After 15 days in the incubator, the colonies are counted using an auto-matic colony counter ("ARTEK", DYNATECH, U.S.A.). To determine the cloning efficiency and thus the exactnumber of cel~ls surviving the immunotoxin trèatment, it is essential to establish a calibration line~showing the number of cells i-noculated as a E~anc~tion~of the number of colonies formed. ~We~hav~e proved that~the cloning ~
efficiency indicated by this calibrat~ion-line is~prac- -: ~ :
`:
.
~L2 tically unaffected by the presence of a high proportion of dead cells, which is the situation naturally en-countered when the cells are treated with the immuno-toxin.
The immunotoxin treatrnent is carried out by in-cubating the cells in the exponential growth phase and at a concentration of 106/ml with the immunotoxin IT
(.~-La) TlOl or IT T101 at different concentrations, in a total volume of 1 ml of the medium RP~II 1640 contain-ing 10% of inactivated fetal calf serum and 10 mmol/l of ammonium chloride. The incubation takes place at 37C under an atmosphere containing 570 of carbon dioxide and ~ith horizontal shaking of the test-tubes (2500 rpm ~ith a "GIRATORY*G-2" shaker, NEW-BRUNSWICK). The cells are then washed and different dilutions are prepared, before mixing ~ith the agar solution, so that the number of ceIls surviving can be measured in the zone of maxi-mum sensitivity given by the calibration line. The results are expressed as the absolute number of cells ~0 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 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) T~101 and IT
T101 on the CEM cells~, in the presence of lO m~l ~ :
ammonium chIoridet as~a function of the immun~otoxin concentration (expressed as the~molarity of A~chain).
It is apparent that the efficacies oL these two products are of the same order of magnitude. The * - Trademark :: ; ::
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resulting reduction in the number of cells is extremely large in both cases since, for concentrations as loi~ as 10 11 ~l, 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 immunoto~in 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.
Example 15:
Conjugate obtained by the reaction of an antibody in-hibiting human T eells (an antibody directed against the antigen T65), substituted by activated disulfide groups, with the oxidized and functionalized A chain (NE~I) 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 functionalized A chain The A chain is blocked with N-ethylmaleimide on its SH group and then oxidized for 18 hours by the pro-cedure described in example 3.
CouPlino with cystamine 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 25C. This incubation is followed by reduc-tion with sodium borohydride (200 equivalents per~ mol of A chain, i.e. 156 microliters of a solutlon contain-ing 17.6 mg in 1 ml of 0.1 N NaOH) for 2 hours at 25C.
The excess reagent is removed by continuous dialysis a~ainst 125 m~l phosphate bufEer of pll 7. The disulfide bridge of the fixed cystamine is then reduced with 2-mercaptoethanol at a final con~centration of 5 : ' ' !
'' ~- ' `
' .
per cent for 1 hour at 30C, this being followed by further continuous dialysis against 125 mM phosphate buffer of pH 7 (20 l 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 reactibn -1.5 ml of the solution of modified A'chain ofricin (i.e. 0.058 micromol) are added to 211 micro-liters of the solution of activated antibody obtainedabove (i.e. 0.006 micromol~. The mixture is left to react for 18 hours at 30C. The reaction medium is the-n dialyzed against P'BS buffer (10 mM in respect of phosphate and 140 m~l in respect of sodium chloride, pH 7.4). After 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) - Propertie$ of the immunotoxin IT (A(NEM)-La-cysteamine) T101 ~ -Specific cytotoxicity activity It is found that this immunotoxin, prepared by the procedure explained above, has a very strong cyto-toxic activity on the CEM target cells (IC50 = 1.2~10 M, established by the method described in example 13).
b) Plasma elimination ` ~ -The immunotoxin lS administered to rab~bits by a single in~jection into a,veln in~the ear (50 m1~crograms - of A chain/kg). The plasma samples~collected after 22 hours are analyzed with the aid of the immunoassay RIA-3 (example 12). The results are sho~n in the table~
below. The values~for IT T101 are 1ndicated~by way of comparison.
~ 42 -_ ._ Relative plasma concentration 22 hours after injection IT (A(NEM)-La-cysteamine)T101 2.4%
IT Tl01 0.08%
Twenty-two hours after injection, the concentra-tion of IT containing modified A chain is 30 times greater than in the case oE IT T101.
Example 16:
Conjugate obtained by the reaction of an antibody in-hibiting human T cells (an antibody directed against the anti8en 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 resi-dues of the A chain.
l) - Preparation of the immunotoxin - a) Preparation of the functlonalizèd 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 c~stamine After dialysis against 0.1 ~I carbonate buffer o pH 9.5, 18.5 ml of a protein solution~containing 2.5 mg/ml are incubated with;35.6 mg of cystamine hydro-chloride for 2 hours at 25C. This incubation is followed by reduction with sodium borohy~dride (200 ~
equivalents per mol of A chain, i.e. 39S microIiters of a solution containing 17.6 mg in 1 ml of 0.1 N~NaOH) for 2 hours at 25C.
The excess rèagen~t is removed by continuous dialysis against 125~mM phosph-ate buffer of pH 7. The disulEide bridge oL the fix~e~d 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 m~ phosphate buffer of pH 7 (201 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 ~3.5 ml of a solution of antibody TlOlcontaining 4.4 m~/ml (i.e. 0.68 micromol). The mixture is stirred for 30 minutes at 25C 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 ~ith 2-mercapto-ethanol, it is found that the antibody obtained carries 3.2 activated mixed disulfide groups per mol of anti-body.
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 oE the solution of activated antibody obtained above (i.e. 0.022 micromol). The mixture is left to react for 18 hours at 30C. 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 coup-ling for this preparation of 0.8 oxldized methylated A
chain (NEM) per mol of antibody. ~
The immunotoxin containin~ methylated oxi~d~ized A chain (NE~) of ricin, obtained as indicated above, ~as studied for its;pharmacokinet~lc~properties and its 5 specific cytotoxicity properties~toi~ards the target cells.
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2) - Properties of the immunotoxin IT (methylated A(NEM)-la-cystcamine) T10l a) Specific cytotoxicity activity It is found that this immunotoxin, prepared by the procedure explained above, has a very strong cyto-toxic activity on the CEM target cells (IC50 = 7-10 12 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 T101 are indicated by way of comparison.
Relative plasma concentration after 22 hours IT (methylated A(NEM)-La-cysteamine) 1.4%
Tl01 IT TlOl 0.08%
. . . _ _ Twenty-two~hours after injection, the concentra-tion of IT containing modified A chain is 17.5 times 20` greater than in the case of IT T101.
Examplè 17:
Toxicity of the prolon~ed-action A chain injected into mice It was important to check the overall toxico-logical impact of the oxidized A chain on the wholeanimal (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 intra-venously to Charles River Fran~ce CDl mice, by comparison :~,; ; :
- , ' ' . -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 in-crease in the plasma level of the A chain when thelatter has been modified by oxidation, the toxicity of - the product is not only not increased but, on the con-trary, 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 anti-body used to prepare the immunotoxin. The optimum ad-ministration conditions and the treatment~time willhave to be determined in each case according to the subject and the nature of the disease to be treated.
In more general terms, antitumoral glyco-proteins 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 - 30 an antitumoral glycoprotèin whose carbohydrate units are modified by oxidation with the~periodate ion 1 S
brought in-to a form suitable f~or~administration by injection and preferably intravenous administration.
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.
The results obtained are collated in the two tables below.
Percenta~e composition Chains Total carbohydrates, %
Native A 5.58 + or - 0.5 10Al 4.54 + or - O.S
A2 6.24 + or - 0.5 Oxidiæed A 2.27 + or - 0.5 Al 2.07 + or - 0.5 A2 3.33 + or - 0.5 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) _ Chains . . I
20MonosaccharidesNative Oxidized _ A Al A2 A Al A2 N-Acetylglucosamine 1.89 1.48 2.15 1.74 1.50 2.37 ~lannose 4.6 3.40 5.2 I.43 1.29 2.26 Fucose 1.37 1.41~ 1.52 O O
Xylose 1 6 1.48 1.67 0.~6 0.48 0.6 : ~ ~
These results prove that~periodate~oxidation has destroyed part of the~sugars o-E the~A chai~n. Per molecule of A chaln9~there is~an~a~vqrage decrease~of ::
~ ~ : . ' :
:::: : : :
. ~
,''"~
:
3.17, 2.11 and 2.9~ 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-acetylglucosamine residues are only slightly degraded.
II - N-Terminal sequence The sequence of the N-terminal amino acids oE
the A chain and its Al and A2variants, in the native form and in the o~idized 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.
Chains Sequenc of the 9 N-terminal amino acids Native A Ile-Phe-Pro-Lys-Gln-Tyr-Pro-Ile-Ile A1 Ile-Phe-Pro-Lys-Gln-Tyr-Pro-Ile-Ile A2 Ile-Phe-Pro-Lys-Gln-Tyr-Pro-Ile-Ile . .
O~idized A Ile-Phe-Pro-Lys-Gln-Tyr-Pro-Ile-Ile Al Ile-Phe-Pro-Lys-Gln-Tyr-Pro-Ile-Ile A2 Ile-Phe-Pro-L~Js-Cln-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 of the 9 N-terminal amino~ acids~ of the A chains~is strictly identical to that previously desc~ribed by Funatsu for the A chain of ricin (Agr~ic. Biol. Chem., (1979), 43, 2221).
III - Affinity on concanavalin A/sepharose ~
- 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 flx to concanav~a11n~ A~/sepharose. 1~ml~oE
:
. . . :
: ~: .
. ~ ~
: . : ~ . :
,, -: , ~ " ~ :
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 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 ith 0.1 ~I borate buffer of pH 6.0, which is 0.5 ~l in respect of NaCl and 0.1 ~l in respect of àlpha-methylmannoside. The results, expressed as a percentage of the optical den-sity at 280 nm are summari~ed in the table below.
.
Chains % not retained % eluted by alpha- Total by the con A methylmannoside (%) _ A(DTNB) 66 11 77 A(DTNB)-La78 5 83 _ Al(DTNB) 80 6 86 Al(DTNB~La 73 0.4 73.4' A2(DTNB) 25 70 95 A2(DTNB~La 64 9 73 _ _ It is known that concanavalin A has an affinity for glycoproteins with terminal mannoses. It is foun(l 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.
I~- Determination of _he E l~o The absorption coefficient ~t 280 Dm (E 1~is :
- : .,. ' , ~, , : ~ .
' ' :
g~
the optical density at 280 nm of a solution containing 1 mg/ml, in which the protein concentration is deter-mined by the FOLIN test with a standard range of bovine serum albumin.
The results are summarized in the table which follows:
.
A chain 0.6~
A(DTNB) chain 1.12 A(DTNB)-La chain 1.04 .
Al(DTNB) chain 1.07 Al(DTNB)-La chain 1.02 .
A2(DTNB) chain 0.95 A2(DTNB)-La chain 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 introductlon of the nitrobenzoyl group.
After oxidation, no significant variation in t`he absorption at 280 nm is observed, demonstrating that oxidation has not affected amlno acids responsible for the absorption at 280 nm.
Y - Isoelectric focusing Analysis of the A chain by isoel~ectric~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 thé~ cysteine~-with mercaptoethanol brings these bands ba~ck~to the location~o~f thé natlve A chain.
Thus, a compari~son o;f~the isoelec~tric points of : :
: .
~L~J7~
the native and oxidized A, A1 and A2 chains shows that, in the ahsence oE a blocking agent, all the bands charac-teristic oE the A chain are transferred by 0.5 pH unit towards acid pH values. This transfer takes place with-out overlapping of the pH regions of the native andoxidized 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-1`0 ation of native gelonine, and 2) the slow elimination ofgelonine modified with sodium periodate, after intra-~enous 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 con-ditions 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 intro-duced 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 pll 6 with ;
1 M acetic acid. Incubation is~left ~:o proceed for 16 hours at 4C in the dark.~ The reaction is~stopped by the addition of 105 micraliters of a 1 M a~queous~sol-ution of ethylene glycol. ~After incubation for 15~min-utes at 20C, the reaction medium is dialyzed at 4C
against PBS bufer. A~fter centr~1fu~gation at IO,OOQ x g for 30 minutes, this givcs 2.9~mg oE~o~idi~zed gelonine : ~ I
- : ~
at a concentration of 2.5 mg/ml.
Like the i~ chain of ricin, the fundamental prop-erty of gelonine is to inhibit protein synthesis in eucaryotic cells by degradation of the ribosomal sub-,unit ~0 S (Biochem. J. (1982) 207, 505-509). In th,e case of gelonine too, the modification due to periodate' oxidation does not cause a loss o:E activity.
II - Pharmacokinetic properties of prolon~ed-action oenonine ~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.
Blood samples are taken at intervals on heparin. The plasmas are analyzed with the aid of a radio;mmunometric test designated below by the abbreviation RIM-2.
This test is performed by the same technique as used for the test RI~-l, except that the solution ~cl here is a solution of anti-gelonine rabbit antibodies purified by aEfinity c~hromatography, the ~c2 antibodies beillg the same antibodies radiolabeled. The radio-labeling procedure is ide~ntical to that described forthe technique RIM-l. The concentration of native gelonine or modified gelonine in the samples~to be 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 reliabi~lit~y and reproducibility~ :
- characteristics as described fo:r the technique~RIM
The results of these'experlments arc represente~d~in the same way~as for~the A chain, of rici.n in example 2.:
Figure 4 shows~:the~plasma elimination curves, .
. as a :function of time,~for native~gelonine and~oxidized gelonine, injected intra:venously.: The native~ge~lonine, like the A chain of~native~ricin, d~sappears very:ràpldly -:
' 7~
from the bloods-tream 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-?4 hours after injection, the concentration of theoxidized gelonine is 300 times greater than that of the native gelonine (curve 2).
Thus, as for the A chain of rlcin, 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 e~ample 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.
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 (Biochem1cal Journal (1980), 186, 443-452). The pharmacokinetic properties of native or modified MOM were established using radioactive GPIR
MOM~ The MOM is radiolabel~ed on the tyrosines~with radioactive iodine-125 in the-presence of chloramine T.
10 microliters of a solution of radioactive iodine-125 containing lOO ~Ci/ml, and 30 microliters o~ a solution~
of chloramine T containing 2.5 mgiml in water, are added to 100 microl1ters of~a~;solut1on~cont~ain~ing 1~m~g~/ml~of MOM in PBS buff~er. The~reaction~is left to~proceed for one minute at~ambient;te;mperature. The reaction is st~opped by the addltion;of~40~0 micro~liters of a solution o~ sodium metabisulf1te~¢ontainlng~0~.5 mg/ml.~The re--.
, "
... , ": . ; , -~: .. :., .,, ,. :
7~
action medium is chromatographed by gel Eiltration 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 ~he same conditions as described for the A chain of ricin in example 2, except that the step in which the thiols are ~0 blocked with DT~B is omitted and the concentration of protein is 125 times smaller (40 ~g/ml). 20 micro-liters 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 MO~I, brought to pH ~ with 1 M acetic acid. Incubation is left to proceed for 16 h at 4C 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 20C, the reaction medium is dialyzed at 4C 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/m].
The new molecule thus obtained has a molecular weight which is not significantly different from that ~5 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 polymersin a very small quantity and does not produce any degradation product.
2) - Pharmacokinetic properties of prolon~ed-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 3.5S
micrograms/kg, Blood samples aFe taken at intervals on * - Trademark .~` ..
.~ - .
.,:
- : , ~, "
,, .. ., ~ -heparin. The plasmas (200 microliters) are incubated with trichloroacetic acid (TCA) (200 microliters at a concentration of 25%) for 30 minutes at 4C. After centrifugation, the radioactivity contained in the sedi-ment which can be precipitated by the acid is deter-mined. This method of analysis makes it possible to measure the plasma level of the intact ~IOM molecules, and any low-molecular degradation products whlch cannot be precipitated by TCA are not taken into account.
The results of these experiments are represented as the percentage of the initial radioactivity remain-in8 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 of 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 intra-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 1). When the~IOM has been oxidized on its sugar residues, the elimin-ation rate is reduced (c~rve 2): 8 hours after injection,level of oxidized MOM is 60 times grea~ter 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 e~limin-:
:
, ation of the MOM.
Example 10:
This example demonstrates:
1. the rapid elimination of GPIR Dianthin ex-tracted from Dianthus caryophyllus,and 2. the slow elimination of GPIR Dianthin mod-ified 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 radio-active Dianthin. The io~ination 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-ent from that of the native Dianthin 30.2) - Pharmacokinetic properties of prolon~ed-action Dianthin 30 The oxidized or unoxidized radiolabeled Dianthin is administered to rabbits by a single injection into a 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 MOM, disappears very rapidly from the bloodstream since 99.9% of the quantity i;nitially present disappears in 2 hours. On the other hand, when the Dianthin 30 has been oxidized on the carbohydrate residues, the~elimin-ation kinetics are slowed down considerably:~ 2 hours after injection, the Dlanthl~n level is 80 times greater , , .. .
than that of the unoxidized Dianthin. The level of o.Yidized 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 recog-nition process responsible for the rapid elimination of the Dianthin.
Example ll:
Conjugate obtained by the reaction of an antibody inhib iting human T cells (an antibody directed against the anti~en T65), substituted by activated disulfide groups, ~ith the oxidized A chain of ricin.
a) Antibody inhibiting human T cells (or antibody T101) This antibody was obtained by the method des-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) Rctivated antibodies inhibiting human T cells 20 microliters of a solution containing 60.3 mg/ml of 1-ethyl-3-dimethylaminopropyl-3-carbodiimide are added to 100 microliters of a solution containing ~0 mg/ml of 3-(pyridin-2-yldisulfanyl)propionic acid in tert.-butanol, and the mixture is left for 3 minutes at ambient ~emperature. 68 microliters of the solution thus obtained are added to 2 ml of an antibody solution containing 8.9 mg/ml in PBS buffer. The mixture~is stirred ~or 15 minutes at 30C and~then diaIyzed against PBS buffer at 4C. ~fter dialysis, the protein solution is centrifuged to give 15 mg of~activated antibody at~
a concèntration 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 act:ivated mixed~disulfide groups~per mol of antibody. ~
III) Pre~aration of the lmmuno~toxin~containln~_~roloneed-. .
action A chain of ricin 2.46 Ml of modified A chain conta:ining 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 20C. The solution is cen-trifuged and then purified by filtration on a Sephadex G100 column, the optical density of the efflue~t being measured at 280 nm. Combination of the fractions con-taining both the antibody and the A chain gives 15 mlof 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-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) TlOl, 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,~ wh`ich are abbreviated to IT (A-La)T101.
I - Procedure ~ ~
The conjugate prepared b~y the procedure explained in example-llis administered~to~ rabbits by a singlc in-jection into a vein in the ear.~ The quantity injected corresponds to 0`.415~mgtkg, expresse~d as~A~chain.
Blood samples~are take~n a~t intervals;on heparin.~ ~The~
plasmas are analyzed with~the~ aid~of a~r~adioimmunometric test with two sitesJ ~wh1ch lS ab~brevi~ated below to RIM_3.
This t~st is ~c~r-i.e~ out bV Ihe~ ~ame~ te~hn~que :: : : ` , : ., ~ :
, as that used for the test RIM-l, except that the solution Ac2 here is a so]ution of goat antibodies in-hibiting mouse IgG, purified by affinity chromatography and radiolabeled as described for the technique RIM-l.
The concentration of modified immunoto~in in the samples to be determined is measured by reference to a calibra-tion curve esta~lished by introducing the modified immunotoxin at different known concentrations. The test RI~l-3 has the same reliability and reproducibility characteristics as described for the technique R~ -1.
By ~ay 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 T101, substituted by activated disulfide groups, with the native A chain of ri~in. The preparation and the cytotoxic properties of this conjugate have been described in French Patent Application no.2 5l6 794 The results of these experiments are represented in the same way as for the uncoupled A chain of ricin in ex-ample 2II - Results Figure 7 shows the plasma elimination curves, as a function of time, for IT T101 and IT (A-La) T101, in-jected intravenously. Twenty-four hours after injection, the concentr&tion of active immunotoxin is 140 times greater Eor IT (A-La) TlOl than for IT T101. 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) T101 towards the target cells.
The fundamental bioIogical property of the A
chain of ricin is to inhibit protein synthesls in cells : ~ :
'h ' , by degradation of the ribosomal sub-unit 60S. The technique uses a cell model in ~hich the efEect of the substances studied on the incorporation o~ 14C_ leucine 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 anti-gen T65. The cells are incubated in the presence of the substance to be studied, and then, when incubation has ended, the degree of incorporation of 14C-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 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 wholecells isolated by filtration.
On the basis of these determinations, it is possiblè 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-tion of 14C-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-hibition of the incorporation of C-leucine, or "50%
inhibitory concentration" (IC50).
Figure 8 shows the curves obtained in the same experiment ~ith IT (A-La) T101 and the uncoupled ox-idized A~chain in the presence of 10 mM ammoniumchloride 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 1 M) which is aboilt 80,000 times greater than that of the uncoupled ox-idi~ed A chain, measur:d under the same conditions.
, Example 14:
This example demonstrates the comparative cyto-toxic efficacy oE IT (A-La) T101 and IT T101 towards CEM target cells, measured in a clonogenic test.
Immunotoxins are dedic~ted 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 poss-ible by optimum culture conditions in a gelled medium, applied to the CEM human lymphoid line.
I - Technique for measurin~ cytotoxicity_by the inh _ition of colony formation The medium used for cloning is the medium RPMI
1640 to which 1 mmol/l of sodium alpha-ketoglutarate, 1 mmol/l 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) is prepared in this medium, placed as a thin layer in small Petri dishes and solidified at +4C. The cells are mixed with a second, 0.275% agar solution kept at 37C, which is then deposited on the-first layer and solidified. These concentrations of agar were chosen after a preliminary study aimed at simultaneously opti-- mizing the cloning efficiency,~ the size of the colonies and the consistency oE the medium. After 15 days in the incubator, the colonies are counted using an auto-matic colony counter ("ARTEK", DYNATECH, U.S.A.). To determine the cloning efficiency and thus the exactnumber of cel~ls surviving the immunotoxin trèatment, it is essential to establish a calibration line~showing the number of cells i-noculated as a E~anc~tion~of the number of colonies formed. ~We~hav~e proved that~the cloning ~
efficiency indicated by this calibrat~ion-line is~prac- -: ~ :
`:
.
~L2 tically unaffected by the presence of a high proportion of dead cells, which is the situation naturally en-countered when the cells are treated with the immuno-toxin.
The immunotoxin treatrnent is carried out by in-cubating the cells in the exponential growth phase and at a concentration of 106/ml with the immunotoxin IT
(.~-La) TlOl or IT T101 at different concentrations, in a total volume of 1 ml of the medium RP~II 1640 contain-ing 10% of inactivated fetal calf serum and 10 mmol/l of ammonium chloride. The incubation takes place at 37C under an atmosphere containing 570 of carbon dioxide and ~ith horizontal shaking of the test-tubes (2500 rpm ~ith a "GIRATORY*G-2" shaker, NEW-BRUNSWICK). The cells are then washed and different dilutions are prepared, before mixing ~ith the agar solution, so that the number of ceIls surviving can be measured in the zone of maxi-mum sensitivity given by the calibration line. The results are expressed as the absolute number of cells ~0 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 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) T~101 and IT
T101 on the CEM cells~, in the presence of lO m~l ~ :
ammonium chIoridet as~a function of the immun~otoxin concentration (expressed as the~molarity of A~chain).
It is apparent that the efficacies oL these two products are of the same order of magnitude. The * - Trademark :: ; ::
:: ~
, ~ : ~ . :
, ., , - ., ' .';
.
::
resulting reduction in the number of cells is extremely large in both cases since, for concentrations as loi~ as 10 11 ~l, 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 immunoto~in 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.
Example 15:
Conjugate obtained by the reaction of an antibody in-hibiting human T eells (an antibody directed against the antigen T65), substituted by activated disulfide groups, with the oxidized and functionalized A chain (NE~I) 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 functionalized A chain The A chain is blocked with N-ethylmaleimide on its SH group and then oxidized for 18 hours by the pro-cedure described in example 3.
CouPlino with cystamine 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 25C. This incubation is followed by reduc-tion with sodium borohydride (200 equivalents per~ mol of A chain, i.e. 156 microliters of a solutlon contain-ing 17.6 mg in 1 ml of 0.1 N NaOH) for 2 hours at 25C.
The excess reagent is removed by continuous dialysis a~ainst 125 m~l phosphate bufEer of pll 7. The disulfide bridge of the fixed cystamine is then reduced with 2-mercaptoethanol at a final con~centration of 5 : ' ' !
'' ~- ' `
' .
per cent for 1 hour at 30C, this being followed by further continuous dialysis against 125 mM phosphate buffer of pH 7 (20 l 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 reactibn -1.5 ml of the solution of modified A'chain ofricin (i.e. 0.058 micromol) are added to 211 micro-liters of the solution of activated antibody obtainedabove (i.e. 0.006 micromol~. The mixture is left to react for 18 hours at 30C. The reaction medium is the-n dialyzed against P'BS buffer (10 mM in respect of phosphate and 140 m~l in respect of sodium chloride, pH 7.4). After 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) - Propertie$ of the immunotoxin IT (A(NEM)-La-cysteamine) T101 ~ -Specific cytotoxicity activity It is found that this immunotoxin, prepared by the procedure explained above, has a very strong cyto-toxic activity on the CEM target cells (IC50 = 1.2~10 M, established by the method described in example 13).
b) Plasma elimination ` ~ -The immunotoxin lS administered to rab~bits by a single in~jection into a,veln in~the ear (50 m1~crograms - of A chain/kg). The plasma samples~collected after 22 hours are analyzed with the aid of the immunoassay RIA-3 (example 12). The results are sho~n in the table~
below. The values~for IT T101 are 1ndicated~by way of comparison.
~ 42 -_ ._ Relative plasma concentration 22 hours after injection IT (A(NEM)-La-cysteamine)T101 2.4%
IT Tl01 0.08%
Twenty-two hours after injection, the concentra-tion of IT containing modified A chain is 30 times greater than in the case oE IT T101.
Example 16:
Conjugate obtained by the reaction of an antibody in-hibiting human T cells (an antibody directed against the anti8en 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 resi-dues of the A chain.
l) - Preparation of the immunotoxin - a) Preparation of the functlonalizèd 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 c~stamine After dialysis against 0.1 ~I carbonate buffer o pH 9.5, 18.5 ml of a protein solution~containing 2.5 mg/ml are incubated with;35.6 mg of cystamine hydro-chloride for 2 hours at 25C. This incubation is followed by reduction with sodium borohy~dride (200 ~
equivalents per mol of A chain, i.e. 39S microIiters of a solution containing 17.6 mg in 1 ml of 0.1 N~NaOH) for 2 hours at 25C.
The excess rèagen~t is removed by continuous dialysis against 125~mM phosph-ate buffer of pH 7. The disulEide bridge oL the fix~e~d 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 m~ phosphate buffer of pH 7 (201 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 ~3.5 ml of a solution of antibody TlOlcontaining 4.4 m~/ml (i.e. 0.68 micromol). The mixture is stirred for 30 minutes at 25C 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 ~ith 2-mercapto-ethanol, it is found that the antibody obtained carries 3.2 activated mixed disulfide groups per mol of anti-body.
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 oE the solution of activated antibody obtained above (i.e. 0.022 micromol). The mixture is left to react for 18 hours at 30C. 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 coup-ling for this preparation of 0.8 oxldized methylated A
chain (NEM) per mol of antibody. ~
The immunotoxin containin~ methylated oxi~d~ized A chain (NE~) of ricin, obtained as indicated above, ~as studied for its;pharmacokinet~lc~properties and its 5 specific cytotoxicity properties~toi~ards the target cells.
:
:: ~
. '' ` ~
~:, , ' :
:~ ~
2) - Properties of the immunotoxin IT (methylated A(NEM)-la-cystcamine) T10l a) Specific cytotoxicity activity It is found that this immunotoxin, prepared by the procedure explained above, has a very strong cyto-toxic activity on the CEM target cells (IC50 = 7-10 12 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 T101 are indicated by way of comparison.
Relative plasma concentration after 22 hours IT (methylated A(NEM)-La-cysteamine) 1.4%
Tl01 IT TlOl 0.08%
. . . _ _ Twenty-two~hours after injection, the concentra-tion of IT containing modified A chain is 17.5 times 20` greater than in the case of IT T101.
Examplè 17:
Toxicity of the prolon~ed-action A chain injected into mice It was important to check the overall toxico-logical impact of the oxidized A chain on the wholeanimal (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 intra-venously to Charles River Fran~ce CDl mice, by comparison :~,; ; :
- , ' ' . -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 in-crease in the plasma level of the A chain when thelatter has been modified by oxidation, the toxicity of - the product is not only not increased but, on the con-trary, 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 anti-body used to prepare the immunotoxin. The optimum ad-ministration conditions and the treatment~time willhave to be determined in each case according to the subject and the nature of the disease to be treated.
In more general terms, antitumoral glyco-proteins 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 - 30 an antitumoral glycoprotèin whose carbohydrate units are modified by oxidation with the~periodate ion 1 S
brought in-to a form suitable f~or~administration by injection and preferably intravenous administration.
`` ` ' ' , ~ ~ : ` :
' ::
.
Claims (16)
1. Glycoprotein having an antitumoral action, whose carbohydrate units are modified by oxidation with the periodate ion.
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.
3. Glycoprotein which inactivates ribosomes and has a prolonged action and which is obtained by treatment of an aqueous solution of a glycoprotein which inactiv-ates 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 temp-erature of 0 to 15°C and in the absence of light, un-blocking of the thiol groups, if appropriate, and isolation of the final product by known methods.
4. Glycoprotein according to claim 3, wherein the aqueous solution is an aqueous solution of A chain of ricin.
5. Glycoprotein according to claim 3, wherein the A
chain of ricin is functionalized.
chain of ricin is functionalized.
6. Glycoprotein according to claim 5, wherein the A chain of ricin is functionalized by methylation.
7. Glycoprotein according to claim 3, wherein the aqueous solution is an aqueous solution of gelonine.
8. Glycoprotein according to claim 3, wherein the aqueous solution is an aqueous solution of GPIR MOM.
9. Glycoprotein according to claim 3, wherein the aqueous solution is an aqueous solution of GPIR Dianthin 30.
10. Glycoprotein according to claim 3, wherein the aqueous solution is an aqueous solution of a glycoprotein selected from the group consisting of Dianthin 32, Agrostin A, Agrostin B, Agrostin C, HCI or Asparagus officinalis inhibitor.
11. Glycoprotein according to claim 4 which 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 produced biosynthetically by a cell whose genotype has been appro-priately modified.
12. Glycoprotein according to claim 11, which 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'-dithio-dibenzoate, with an aqueous solution of sodium periodate, for a period of 0.2 to 24 hours, at a temperature of about 4°C and in the absence of light, treatment of the mixture with 2-mercaptoethanol and isolation of the resulting pro-duct by known methods.
13. Glycoprotein according to claim 7, which is obtained by treatment of an aqueous solution of gelonine with an aqueous solution of sodium periodate, for a period of 0.2 to 24 hours, at a temperature of about 4°C and in the absence of light, treatment of the mixture with 2-mercaptoethanol and isolation of the resulting product by known methods.
14. A process for the preparation of an antitumoral glyco-protein as claimed in claim 1, which comprises subject-ing the unmodified antitumoral glycoprotein to oxidation with periodate ions.
15. A process for the preparation of a glycoprotein which inactivates ribosomes and has a prolonged action, which comprises treating an aqueous solution of a glyco-protein which inactivates ribosomes, the thiol groups of which are optionally protected, with an aqueous solu-tion 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.
16 . The process as claimed in claim 15, 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.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR8409703A FR2566271B1 (en) | 1984-06-20 | 1984-06-20 | NOVEL CYTOTOXIC CONJUGATES FOR USE IN THERAPEUTICS AND PROCESS FOR OBTAINING SAME |
| FR8409703 | 1984-06-20 | ||
| FR8502067 | 1985-02-13 | ||
| FR8502067A FR2577137B1 (en) | 1985-02-13 | 1985-02-13 | ANTI-TUMOR GLYCOPROTEINS, MODIFIED ON THEIR CARBOHYDRATES |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1248874A true CA1248874A (en) | 1989-01-17 |
Family
ID=26224020
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000483940A Expired CA1248874A (en) | 1984-06-20 | 1985-06-13 | Process for the preparation of antitumoral glycoproteins modified on their carbohydrate units |
Country Status (14)
| Country | Link |
|---|---|
| EP (1) | EP0172045B1 (en) |
| JP (1) | JPS6112628A (en) |
| KR (1) | KR930000058B1 (en) |
| AT (1) | ATE40700T1 (en) |
| AU (1) | AU593211B2 (en) |
| CA (1) | CA1248874A (en) |
| DE (1) | DE3568167D1 (en) |
| DK (1) | DK166626B1 (en) |
| ES (1) | ES8605681A1 (en) |
| GR (1) | GR851497B (en) |
| IE (1) | IE58514B1 (en) |
| IL (1) | IL75484A0 (en) |
| NZ (1) | NZ212439A (en) |
| PT (1) | PT80662B (en) |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2577135B1 (en) * | 1985-02-13 | 1989-12-15 | Sanofi Sa | LONG-TERM ACTION IMMUNOTOXINS COMPRISING A GLYCOPEPTITIDE CONSTITUENT MODIFYING RIBOSOMES MODIFIED ON ITS POLYSACCHARIDE PATTERNS |
| US4911912A (en) * | 1985-12-20 | 1990-03-27 | Sanofi | Ribosome-inactivating glycoproteins, modified by oxidation of their osidic units and reduction, and in vivo prolonged-action immunotoxins containing such a glycoprotein |
| 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 |
| FR2601679B1 (en) * | 1986-07-15 | 1990-05-25 | Sanofi Sa | IMMUNOTOXINS, PREPARATION METHOD AND PHARMACEUTICAL COMPOSITIONS CONTAINING SAME |
| DE4106389A1 (en) * | 1991-02-28 | 1992-09-03 | Behringwerke Ag | FUSION PROTEINS FOR PRODRUG ACTIVATION, THEIR PRODUCTION AND USE |
| US6610299B1 (en) | 1989-10-19 | 2003-08-26 | Aventis Pharma Deutschland Gmbh | Glycosyl-etoposide prodrugs, a process for preparation thereof and the use thereof in combination with functionalized tumor-specific enzyme conjugates |
| 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 |
| US5250532A (en) * | 1991-04-11 | 1993-10-05 | Dowelanco | 3,4,N-trisubstituted-4,5-dihydro-1H-pyrazole-1-carboxamides and their use as insecticides |
| GB9808485D0 (en) * | 1998-04-21 | 1998-06-17 | Univ Cambridge Tech | Improvements relating to immunotherapy |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IL47372A (en) * | 1975-05-27 | 1979-10-31 | Yeda Res & Dev | Fab'dimers bound to daunomycin or adriamycin,their preparation and pharmaceutical compositions containing same |
| FR2437213A1 (en) * | 1978-09-28 | 1980-04-25 | Cm Ind | CYTOTOXIC PRODUCTS FORMED BY COVALENT BINDING OF THE CHAIN TO RICIN WITH AN ANTIBODY AND THEIR PREPARATION METHOD |
| JPS5616418A (en) * | 1979-07-20 | 1981-02-17 | Teijin Ltd | Antitumor protein complex and its preparation |
| JPS5843926A (en) * | 1981-09-08 | 1983-03-14 | Suntory Ltd | Selective carcinostatic agent |
-
1985
- 1985-06-09 PT PT80662A patent/PT80662B/en not_active IP Right Cessation
- 1985-06-11 IL IL75484A patent/IL75484A0/en not_active IP Right Cessation
- 1985-06-13 CA CA000483940A patent/CA1248874A/en not_active Expired
- 1985-06-13 AU AU43646/85A patent/AU593211B2/en not_active Ceased
- 1985-06-17 NZ NZ212439A patent/NZ212439A/en unknown
- 1985-06-18 IE IE151185A patent/IE58514B1/en not_active IP Right Cessation
- 1985-06-18 EP EP85401201A patent/EP0172045B1/en not_active Expired
- 1985-06-18 DE DE8585401201T patent/DE3568167D1/en not_active Expired
- 1985-06-18 AT AT85401201T patent/ATE40700T1/en not_active IP Right Cessation
- 1985-06-19 GR GR851497A patent/GR851497B/el unknown
- 1985-06-19 DK DK277985A patent/DK166626B1/en not_active IP Right Cessation
- 1985-06-19 KR KR1019850004354A patent/KR930000058B1/en not_active IP Right Cessation
- 1985-06-19 ES ES544337A patent/ES8605681A1/en not_active Expired
- 1985-06-20 JP JP60135217A patent/JPS6112628A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| KR930000058B1 (en) | 1993-01-06 |
| GR851497B (en) | 1985-11-25 |
| DE3568167D1 (en) | 1989-03-16 |
| AU593211B2 (en) | 1990-02-08 |
| IL75484A0 (en) | 1985-10-31 |
| KR860000315A (en) | 1986-01-28 |
| AU4364685A (en) | 1986-01-02 |
| ATE40700T1 (en) | 1989-02-15 |
| EP0172045B1 (en) | 1989-02-08 |
| JPH0582400B2 (en) | 1993-11-18 |
| EP0172045A1 (en) | 1986-02-19 |
| PT80662A (en) | 1985-07-01 |
| NZ212439A (en) | 1989-01-27 |
| ES8605681A1 (en) | 1986-04-01 |
| IE851511L (en) | 1985-12-20 |
| ES544337A0 (en) | 1986-04-01 |
| IE58514B1 (en) | 1993-10-06 |
| JPS6112628A (en) | 1986-01-21 |
| PT80662B (en) | 1986-12-09 |
| DK166626B1 (en) | 1993-06-21 |
| DK277985D0 (en) | 1985-06-19 |
| DK277985A (en) | 1985-12-21 |
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