CA1188681A - Cytotoxic products formed by covalent bonding of a chain of ricin with an antibody and the process for their preparation and use - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Carcinostatic products are formed by coupling, by covalent bonding, the A chain of ricin with an antibody which is capable of selectively recognising a given antigen at the surface of cancerous cells.
Such products can be made by the formation of a disulphide bridge between the free thiol group in the chain of ricin and an immunoglobulin or immunoglobulin fragment into which a thiol group or an activated disulphide group has been introduced. The A chain of ricin and the immunoglobulin are carefully purified before use.

Description

DESC~IPTION
~'CYTOTOXIC PRODUCTS FORMED BY COVALENT BONDING O~ THE A
CHAIN OF RICIN WITH AN ANTIBODY AND TEIE PROCESS FOR

THEIR PREPARATION AND US~"
- _ For many years, the treatment of cancer has given rise to multifarious research. More particularly, a very large number of substances have been proposed for the chemotherapeutic treatment of cancer by the more or less selective destruction of the cancerous cells.
Although it has been possible to achieve certain results, the chemotherapy of cancer is restricted by the non-specific toxicity o the antineoplastic agents towards normal high-growth cells such as, for example, the stem cells from blood lines. As a result, the effectiveness of the treatment is inadequate to permit the removal of the last cancerous cells, which cells will subsequently cause new growths of cancer.
In order to reduce the toxicity of carcinostatic agents towards normal cells, various experiments have been carried out [see in particular: C.R. Académie des Sciences de Paris 246 1,626 (1958); British Medical ~ournal 3, 495 tl972); Science 169, 68 (1970) and Cancer Research 35, 1,182 (1975)]. The aim of these experi-ments was to couple molecules having a cytotoxic activity with antibodies directed against the cancerous cells, in order to fix them solely to the target cells.
However, in practice, these experiments have not achieved notable results, essentially because the cytotoxic sub-8~

stance carried by the antibody remained active during its trans~er in-to the organism and was able to :inhibit the growth o~ normal cells be~ore encountering -the cancerous cells .
~le present invention relates -to active sub~
stances ~r the treatment of cancer, which consist of a cytotoxic agen-t coupled to an immunoglobulin which is speci~ic for cancerous cells. These subs-tances exhibit the characteris-tic tha-t they remain inactive during their lo transfer and cnly become active after fixation to -the target cells and penetration in-to these cells. The immunoglobulin used can be an an-tibody which is specific for a given antigen or can also be a fr~gment of this molecule, which possesses the capaci-ty of specific recog-nition with respect -to ~he anti~en, such as, ~or example, the fragments which ar~ usually denoted by the name Fab~
F(ab)' or F(ab')~. As regards the cytotoxic substance, the A chain of ricin seems to constitute a very valuable substance.
In f~c-t~ it is known that ricin, which is a toxic lectin extracted from the seeds of Ricinus communis, con-sist~ o~ the association of two polypeptide chains by means o~ a disulphide brid~e. me A chain, or "e~fectomer", has an intense cytotoxic activity by virtue of the inhibition of protein synthesis in the oells of eukaryotesO Howev~r, this A chain does not possess the property of effectively penetrating into the cells to exert its biological acti~ity therein. Further more, the B chain o~ ricin, or "haptomer", possesses the property of recognising saccharide units at the surface of the cells, ~Jith which units it creates an association o~ high affinity. Natural phenomena then make it possible to cause the ricin molecule to penetrate into the contents of the cell, where the A chain exerts its toxic activity. The toxici-ty of ricin is devoid of any specificity with respect to a particular type of cell, insoar as virhually all animal cells carry saccharide determinants which are recognised by -the B chain.
m e aim o~ the present invention is to produce artificial hybrid molecul~s, denoted by the term "conju-gatesl', in which the A chain of ricin is associated by means of a suitable covalent bond, preferably of the disulphide type, not to the B chain bu-t to a protein ~tructure ~hich is capable of selectively recognising a given antigen at the surface of the cells carrying this antig~nJ hs indicated above, this protein structure 20 will be an immunoglobulin which is speci~ic for the desired antigen, or any fra~ment of this immunoglobulin which possesses the same speci~icity of recognition.
m e cho~ce o~ th~ A chain of ricin as the cyto-toxic constituent of the conju~ates mainly results from 5 the followin~ facts:
an extremely high level of cytotoxic activity when, and only when, the A chain has penetrated into the 8~

contents of the -targe-t cells, a cyto-to~icity mechanism based on the distur-bance of a fundamen-tally vital func-tion of the cell, namely its capacity .for prote.in synthesis, and a very low level of non-specific toxicity, com~
pared with -the level of specific toxicity, insofar as the A chain does not by itself possess the property of penetratin~ into -the cells without being bonded to another suitable molecule which permits ~ixation to the cell membranes In the c~se of ricin, this suitable molecule is the B chain, and, in the case of the inven-tion~ it is a speci.~ic immunoglobulin which is capable of recognising a spec.ific receptor at the surface of the target cells and of creating, with this receptor, an association which possesses a high associa-tion constant.
An attempt to produce conjugates between immuno-globulins and constituent peptide chains of ricin has been rep~rted ~Annals of the New Y~rk Academy of Sciènces 277, 690 ~1976) ~ . However, the specifici-ty obtained in 20. vitro in this experiment is extremely low for various reasons, namely:
~ he A chain of ricin.was not isolated from the B chain be~are con~ugation with the antibody, the antibodies used w~re n~t pure and contained large amounts of non-antibody proteins and o~ antibodies other than those which are specifically directed against 'the target antigen, and ~ 6 ~

the coupling of the toxin with the antibody was carried out using glutaraldehyde, this being a product which is capable of causing the dena-turation of the anti-body or of the toxin by the ~orma-tion of briclges, in a non-specific manner, inside a pep-tide chain or between the various peptide chains, and this amounts to random polymerisation.
The present invention makes it possible to over-come all these difficulties and tn obtain conjuga-tes which possess a pronounced specificity for the target cells and can be used for the -treatment of cancer.
ISOLATION AND PURIFICATION OF RICIN AND OF ITS A CHAIN
The seed of Ricinus communis contains the toxic lectin known by the name ricin. I-t also contains another~ less -toxic, lectin which is deno-ted by the name agglutinin because o~ its agglutinating properties with respect to cells and, in particular, with respect to red blood cells. This agglutinin consists of two sub-units, each of which results, in the same way as ricin, from the association of two glycoprotein chains by means of a disulphide bridge.
~ inally, -the seed of Ricinus communis contains other proteins of diverse natures and also a large amount of lipids which are the constituents of castor ` 25 oil.
me extraction o~ ricin starts by grinding the seeds of Ricinus communis to produce a paste from which -the oil must be removed by means of a solvent for the lipids, for example by repeated extrac-tions with ethyl e-ther. After drying, the powder obtained is extracted cold by s-tirring with a solution of sodium chloride in a slightly acid mcdium~ preferably at a temperature which does not exceed 4C. ~fter separating off the sediments, the ex-tract is dialysed for a long -time, firstly against water and then against a buffer of low ionic strength ~TRIS-HCl, 10 mM, pH - 7.7). A slight precipitation occurs during dialysis, the precipitate is separated off by fil-tration or centrifuga-tion.
The extract thus obtained con-tains all the soluble proteins of -the Ricinus communis seed, namely ricin, agglu-tinin and various o-ther proteins. This solu-tion can be frozen a-t -20C1 at which temperature it keeps for several weeks.
'mepreparation ofpurericin fromthe crudeextract has already been described in the literature. In general, it involves chromatographic techniques, namely ion ex-change chromatography, chromatography on a molecular sieveor also affinity chromatography. Mos-t frequently, these various methods are combined with one another, giving rise to long and difficult techniques which cannot easily be applied to give large amounts of ricin. According to `25 the invention, it is possible to obtain pure ricin by means of a single affinity chromatography operation which malces it possible to separate the ricin successively from R ~ o~ ~n ~ ~ ~

the foreign proteins and then ~rorn the agglutinin. To do this, the crude extract is deposited on a column of Sepharose 4B~aIIagargel in the formof sphericalparticles, at aconcentr~t:ion of 4%, marlcetedby the Pharmacia Company) and then eluted in a sequential manner. Using a TRIS-HCl buffer~50 mM, pH - 7.7, the proteins in the seed which are not lectins are eluted, and then, using a galac-tose soluticn having a concentration of between 0.28 and o.56 ~M, the pure ricin is obtained. Finally, using a 0.1 M galactose solution, the agglutinin is obtained.
Total separationofthe constituents from one anotheris ach-ieved in a single chromatography operation i~ the volume of Sepharose ~B used is such that the totalamountofpro~
te~ introdu~edintothecolumn does not exceed the capacity of the latter.
After this step, ~ncentration by ultrafiltration makes it possible easily to obtain a solution of pure ricin, containing from 5 to 10 mg/ml of product in a buffer of low ionic strength. m e solution also con-tains a small amount of galactose(about 0.4 millimol/litre).
Frozenat ~20C,this solution cankeep ~or several weeks.
m e two constituent chains of ricin can be separ-ated after selectivesplittering of the single disulphide bridge which joins them. This splittering is carried `25 out by mean~ of a reducing agent, such as 2-mercapto-ethanol or dithiothreitol, of which the concentration ln the reaction medium must be at least 2 %.

D ~

~ 8~

The separation of -the ~wo chalns, by methods using ion exchange on various -types of support, has already been described, which methods are essentially based on the di:~ferences in -the isoelectric points o~ the 5 two chains. According to the invention, a process for separating the two chains A and B of ricin has been develope~ which utilises not only -the di~ference in the isoelec-tric points o~ -the two chains, but also their dif~erent affinities towards the polysaccharide chroma~
10 tographic supports containing galactose derivatives.
The use of such supports also exhibits the advantage that they retain any possible -traces of undivided ricin which could remain in the mixture.
In practice, the reducing agent (for exarllple 15 2-mercaptoethanol)is added, at ambient temperature, to the solution of ricin, obtained above, in the buffer of low ionic strength, until a concentra-tion of 2.5 % volume/
volume is reached, and the solution is then deposited on a column,consisting of DEAE CL Sepharose 6B (a gel mar-20 keted by the Pharmacia Company and used for ion exchangechromatography; it is prepared ~rom Sepharose, or agar gel, by crosslinking wi-th 2,3~dibromopropanol and remov-ing the sulphate groups by alkaline hydrolysis and then introducing diethylaminoethyl groups; concentration 6 %
25 ~n the gel), in the same buf~er containing the reducing agent. The two chains bind the co~umn by means of bonds which are ionic with respect to the DEAE

~18~

groups, and the B chain also becomes fixed to the Sepharose matrix by vir-tue o~ affinity.
The A chain is eluted by increasing the ionic strength and the pH, still in -the presence of the redu-cing agent so as -to prevent any recombination o~ the two chains with one ano-ther (elution buffer: O.l M TRIS~HCl, pH = 8.4, which is O.l M in respec-t of NaCl and contains

2.5 ~0 of 2-mercap-toe-thanol) Under these conditions, the B chain remains totally fixed; I-t can be eluted lO by the same buffer which is 0.2 M in respect of sodium chloride and O.l M in respect of galactose.
A varian-t of the process for separating the A and B chains consists in using, for chromatography, a s~pport on which the ion exchange and molecular sieving phenomena l5 occur simultaneously. Thus, using QAE Sephadex~A.50 (a strong basic ion exchanger obtained by fixing quater-nary ammoni~m groups to Sephadex, or dextran gel9 by means of ether bonds, and marketed by the Ph~rmac ia Company), the pure A chain can be eluted with -the TRIS-HCl ~uffer, 20 lO0 mM, pH = 8.4, containing 005 ~0 of 2-mercaptoethanol, whilst the B chain is eluted with the same buffer which, in addition, is 75 mM in respect of sodium chloride.
In either case, the choice o~ the chromatographic support is very important and various other supports 25 tested have not made it possible to achieve a good separ- .
ation o~ the A chain.
The A chai~ ob-tained by one or other of these 7~

processes was shown to be pu.re with respect to the vari-ous analytical criter.ia and do~s not require further purification. However, in order to effect its ~ubse-quent coupling with antibodies, it is necessary to have available fairly concentrated solutions which are free, in particular, from the reducing agent. In order to do this, the solution of A chain in TRIS, obtained above, is dialysed against a 10 mM phosphate buffer, pH = 6.5, and this simultaneously removes the TRIS, the sodium chloride, the 2-mercaptoethanol and the traces of galac-tose.

The solution thus obtained is deposited on a column of carboxymethylcellulose, and the A chain is then eluted by simultaneously increasing the concentrati.on and the pH of the buffer from 10 mM, pH = 6.5~ to 125 mM, pR = 7~0, the buEfer beiny 1 mM in respect of EDTA. A
fairly concentrated solution (about 5 mg/ml), whlch is ready for the coupling reactions, is thus obtained.

The diluted solution thus obtained can also be concentra-20 ted and purified by subjecting said solution to a con-cen~ration step on C~ M. Sepharose ~ support, combining the ion exchange and affinity effects, and a solution presenting simultaneously a high concentration and a high leval o~E pureness, can be obtained. Furthermore, such a solution causes the ~ chain to deposit by cooling in a crystallised form.

Tha A chain thus prepared (in the state of concentrated solution or in the state of crystals) i9 free of non-~L8~

.speclific toxicity towards cellular sytems or animal bo~dies, and therefore can be used for producing conjuga-tes, the speclficity of which is strictly provided by the anti.body.

According to the invention, the preparation of the antibodies is carried out so as to give pure anti-bodies which are free from molecules without antibody actiYity, in order to impart the most complete specifi-city of action to the conjugates prepared subsequently,this being a characteristic which is not observed in the preparations described in the literature.

Immunisation is carried out repetitively for \

_ 12 -several mon-ths, in accordance with a conventional process, in order to achieveahyper-immunisation of the anlmals.
Several litres of i~munoserum are thus collected arld this can be s-tored a-t -20C before subjecting it to the puri-5 fication opera-tions.
The purification is effected by immunoadsorption using a Sepharose 4B gel,ac-tivated by cyanogen bromide, to which an an-tigen corresponding to the specific anti-body to be purified has been fixed. Af-ter $eparating 10 off the liquid phase9 intense washing makes it possible to remove all -the proteins which àre not fixed to the gel, and the antibodies fixed to the gel are then liberated with a suitable eluent. mis method can be applied to various antibodies of different specificities. Accord-15 ing to the invention, the population of antibodies in theserum can thus be fractionated so as to select the anti-bodies which have the highest affinity for the antigen.
With an excess of antibodies, relative to the amount of antigen fixed to the gel, the gel preferentially retains 20 the antibodies which have the highest affinity.
In practice, the process is carried out with an excess of antibodies which is such that only a fraction of the antibodies in the imm~e serum is fixed to the column, whilst the remainder are removed by washing. Under 25 these conditions, the average af~inity o~ the antibodies which are removed by washing is lower than that of the antibodies which are fixed to the column and subsequently eluted. .A popula-tion of an-tibodies is thus obtained which has a more homogeneous a:Efinity than the population present in the s-tarting immune serum.
If it :is desired to worlc with large amounts of antibodies, which requires columns of large dimensions, the amount of il~lune~erum to be used on a given gel, con-taining the antigen, is de-termined beforehand by carrying out a series of microimmunoadsorptions with increasing amounts of antiserum. De-termination of the antibodies lo in the eliminated li~uid phase by the radio.immunological .method malces it possible to de-termine the ratio of anti-gen/antibody which allows the undesirable fraction of the antibody initially introduced to escape into this phase.
PREPARATION OF THE A CHAIN OF RICINl~NTIBODY CpNJUGATES
The object of this part of the invention isto associate, by means of a covalent bond of the disulphide type, on the one hand, an immunoglobulin which is specific for a given antigen, or any fragment of this molecule which possesses -the capacity of specific recognition with respect.to the antigen, with, on -the other hand, the A
chain of ricin. l~e choice of a disulphide bond between the A chain and the immunoglobulin is based on the following arguments:
this type of bond is the type which exists in the natural ricin molecule, and it can be expected to be parti-cularly suitable for presenting the A chain in a conform-ation which facilitates its penetration into the cell, 8~
]4 _ whilst at bes-t retaining its fundamental biological pro-perty of inhibi-ting protein synthesis, this type of bond is biochemically labile/ which provides the A chain, coupled in this way, with the pos-sibility of bein~ liberated, from its carrier protein, inthe conten-ts of the cell, the A chain of ricin possesses a-single cys-teine residue in its s-tructure and hence only one SH grou~
capable of crea-ting a disulphide hond. Consequently, the conjugates formed by involving this SH group in a disulphide bridge will be chemically well defined and will in no way modify the structure of the A chain, thus ensuring the in-tegral reten-tion o~ its biological acti-vity, and there are efficient methods which make it pos-sible to produce such a disulphide bond under condi-tlons which are sufficiently mild to ensure the integrity o~
the biological properties of the protein constituents of the conjugates formed.
In order to produce such conjugates, the proteins to be coupled must each carry at least one sulphur atom which is naturally capable, or is artificially rendered capable, of creating the desired disulphide bond, whether these sulphur atoms already exist in the proteins or have ` 25 been chemically introduced into these proteins. As indicated above, the A chain of ricin naturally possesses only one sulphur atom permitting -the desired coupling.

15 ~

This is the sulphur atom in the thiol group of the single cystei~ residueincorporatedinthe A chain. As regards the .
immunoglobulin or its fragments, several cases mus-t be considered:
S 1) In the case of an entire immunoglobulin, neither a free thiol group norother sulphuratoms capableofbeingused for the couplin~ exist naturally in these pr!oteins. It will therefore be necessary, in this case, to introduce one or more sulphur atoms into th~ immunoglobulin mole-cule artificially so that:the biological proper-ties of the immunoglobulin are no-t profoundly impaired, and this sulphur atom, or these sulphur atoms, can subsequently be involved in -the disulphide bond to be established with one or more molecules of the A chain of ricin.
2) In the case of a Fab fragment, -the situation is abso-lutely identical to that described above.

3) If a fragment o~ the Fab' type is employed, it is 20 possible to use the sulphur atom present in the free . thiol group to carry out the coupling to the A chain.
However, it is also possible to use the artificial introduction of one or more sulphur atoms; in this case, it is necessary to block the free thiol group in a stable manner beforehand, for example by alkylation.

4) Finally, if it is desired to couple a F(ab')2 frag-ment of immunoglobulin, it is necessary, as in the case 16 _ of the whole immunoglobul.in, to introduce one or more sulphur~containing groups into F(ab~)2 artificially.
In all the cases in which one or more sulphur-containing radicals are introduced into the immunoglobulin or its fragments, it is necessary to avoid any substi-tu-tion in -the si-te for recognition of the an-tlgen or in its immediate environment, which substitu-tion could disturb the recognition proper-ties of -the antibody. In order to exclude this risk~ the site for recogni-tion of the anti-gen can be blocked -teMporarily, during the substi-tution reaction, by treating the an-tibody beforehand with the specific antigen, or with another antigen which possesses an adequate cross-reaction, or with a sui-table hapten.
The operation for temporary protection can be carried out:
either in the liquid phase, if the antibody-antigen (or hapten) complex is soluble in -the reaction medium, or in -the heterogeneous phase, if -this complex is spontaneously insoluble or also if it has been deliber-ately ren~ered insoluble by means o~ a suitable procedure, in particular by fixing -the antigen (or hapten) to an insoluble support so that the modified support thus obtained possesses an adequate affinity for the antibody.
` 25 After the substitution step, it will be necessary to unblock the site for recognition of the antigen) on the antibody, by means of a suitable procedure for removing _ 17 ~

the antigen, in order to regenerate the capacity of the antibody for specific recognition.
To produce the disulphide bridge bet~een the two proteins, i-t is not possible -to bring the two cons-titu-ents o~ the con;jugate, each carrying a SH yroup, intocontact with one another and to carry out an oxidation.
In fact, under -these condi-tions, the coupling reaction is an equilibrium reac-tion which is very difficul-t to drive to completion. Furthermore, the desired reaction is accompanied by the formation of polymers o~ each of the two constituents, which would result in a very low yield of the desirecl produc-t and the presence o~ impurities which are very difficul-t to remove.
According to the inven-tion, the conjugate is pre-pared by bringing one of the proteins, carrying its free~H group, into con-tact with the o-ther protein, in which theSH grbup is activated by conversion into a mixed di-sulphide with a suitab]e sulphur-containing organic radi-cal. ~he preparation o~ -the conjugate can `oe represen-ted by *he equation:
Pl SH ~ P2 S S X Pl-S-S-P2 ~ XSH
in which Pl and P2 represent the two proteins to be cou~ed and X denotes the activator radical. It is immediately apparent from this equation that, in each case9 the coup-` 25 ling reaction can be carried out in accordance with twovariants, depending on whether Pl represents the imm~no-globulin or its fragment and P2 reprbsents the A chain o~

~ l8 _ ricin, or vice versa.
~ r ~Oi^h ~1 represents_the an-t-body__r_a ~ra~
and P2 ~
To activate the free SH in the A chain of ricin,

5 the solution of A chain, prepared as indlcated above, is used, and it is subjected to an exchange reaction:
QSH ~ XSSX ~ A-S-S-X +XSH (1) in which ASH represents the A chain of ricin and X rep~
resents ~he activator radical O
In particular, X can deno-te a pyrid~2- or -4-yl group which is optionally substituted by one or more alkyl orhalogen radicals orcarboxylic acid groups, or X can also represent a phenyl nucleus which is optionally sub-stituted by one or more nitro or carboxylic acid groups.
15 Reaction (1) is an equilibrium reaction but the equili~
brium can easily be displaced towards the right by using a large molar excess of the reagen-t XSSX, which is gener-ally inexpensive and readily accessible. It is poss~ble to monitor the course of reaction (1) by ultraviolet or 20 visible spectrophotometry because the compound XSH which ; is formed shows an intense absorption in this region.
When the reaction has reached the desired degree o~ com-pletion, the excess of the reagent X-S-S-X, and also the reaction product X-SH, are removed by dialysis or filtra-25 tion on a molecular sieve in gel form. Finally9 a puresolution of the compound A-S-S-X in the chosen buffer i~
obtained. If necessaryJ this solution can be kept Bl _ 19 for several weeks af-ter freezing.
The immunoglobulin substituted by a SH group is also prepared. To do this, -the solution of immuno globulin obtained above is used ei-ther as such or after blocking its site for the recognition of the antigen, with the corresponding hapten, followed by removal of the excess hapten~ By reacting S-acetylmercaptosuccinic anhydride with this protein, it is possible to fix one or more S-ace-tylmercaptosuccinyl groups, per molecule of protein~by means of its free amino groups, and then to liberate the thiol groups by the action of hydroxylamine, as has been described [Archives of Biochemistry and Biophysics ll9, 4l-49 (1967)]. Dialysis makes it pos-sible to remoYe the excess reagents and also the reaction products of low molecular weight.
All these operations are carried out in a phos-phate buf~er at pH = 7.0 and at temperatures which do not exceed ambient temperature. The hapten which may have been used as a temporary blocking agent is removed from the solution finally obtained. If it proves necessary, this solution can be concentrated, for example by ultra-filtration. The coupling between the two reagents thus prepared is effected by simple contact in aqueoùs solutlon, at ambient temperature, for a time varying from a few hours to one day, in accordance with the ~uation:

~ 20 ~

Prot NH--C0-C'H-SII + A-S-S-X ~ Pro-t NHC0 CH-S-S-A t XSH

100(~ ~oo(~3 (4) The course o~ the reac-tion is followed by spectrophotometric determination of -the compound XSH
formed. The latter is removed by clialysis and a solution of-theexpec~d conjuga-te is ob-tained which must be further purified. In fact, i-t is essential, in particular, -to remove the molecules of A-S-S-X which have not reacted and which, if -they were present in the conjugate, could give rise to a no~-selective toxicity.
The purification can be effected by various known methods such as fractional precipita-tion with the aid of water~miscible or~anic solvents or of salts, gel perme-ation chromatography, or also affinity chromatography on lS a column ormed by an insoluble support on which the anti-gen (or the hapten) is fixed, against which antigen the antibody employed in the preparation of the conjugate is directed.
m ese purifica-tion methods can be applied directly to the dialysed solution originating from the coupling step. However, better results are obtained, and9 inl particular, the subsequent forma-tion of polymers of the conjugate is avoided, by the prior blocking o~ the SH
groups ~Jhi~h remain ~ree, ~it.h a reagent such as N-ethyl~
25 maleimide, ~2~ _ P represents the ntibo~y ~ n this case, the products required ~or the coup-l.ing are -the A chain of ricin and the immunoglobulin (or its fragment), which is substi-tuted by a group carrying one or more activated sulphur atoms. The A chain of ricin is used as obtained by the purification procedure described. The immunoglobulin substituted by an activated sulphur atom is prepared from the immunoglobulin itself by substitution with the aid of a reagent which itself carries an activated sulphur atom, in accordance with the equa-tion:
Prot ~ Y-R-S-S-X ~Prot-R-SS-X
in which Prot denotes the immunoglobulin, Y represents a group permitting the covalent fixation of the reagent to the protein, R denotes a group which can simultaneously carry the substituents Y and -S-SX, and X denotes the activator radical.
A reaction of this type has already been used for coupling two proteins (identical or different) by means o~
a disulphide bridge, but the application of this principle to the coupling of an immunoglobulin with the A chain o~
ricin is new.
The functional group Y is a group which is capable of bonding in a co~alent manner with any one of the groups carried by the side chains of the constituent aminoacids of the protein to be substitu-ted. Amongst these latter _ 22 _ groups, the terminal amino groups of lysyl radicals, which are present in the protein~ are particularly indicated.
In this case, Y can represent, in particular:
a carboxylic acid group which can bond to the amino groups of the protein in the presence of a coupling agent such as a carbodiimide and, in particular, a water-soluble deriva-tive such as l-ethyl-3-(3-diethylaminopropyl)-carbodiimide, a carboxylic acid chloride which is capable of 10 reac~ing directly with the amino groups in order to acyl-ate them, ~ a so-called "activated" ester, such as an ortho-or para-, nitro- or dinitro-phenyl ester or also a N-hydroxysuccinimide ester, which reacts directly with the 1S amino groups in order to acylate them, an internal anhydride o~ a dicarboxylic acid, such as, for example, succinic anhydride, which reacts spon-taneously with the amino groups in order to create amide bonds, or ~NH
an iminoester group -C \ ~in which Rl i~ an alkyl ~roup which reacts with the amino groups of the protein in accordance with the equation:
H

R10 ~ 2 ot N}~-C-R2 + RlOH

X denotes a ~unctional group which is capable of reacting 25 with a free thiol radical.
.: .
i In particular~ X can denote a pyrid-2 yl or pyrid-~-yl group which is op-tionally substituted by one or more alkyl, halogen or carboxylic acid radicals. X
can also denote a phenyl group which is preferably sub-5 stituted by one or more nitro or carbo~ylic acid groups.X can also represent an alkoxycarbonyl group such as the methoxycarbonyl group.
The radical R denotes any radical which is capable of simultaneously carrying the subs-tituents Y and S-S-X.
lo The radical chosen must not con-tain groups which are capable of interfering, in -the course of the subsequent r~actions, with the reagents used and the products syn-thesised. In particular~ the group R can be a group -(CH2)n, in which n is between 2 and 10, or also a group R3-~-Cll-in which RL denotes hydrogen or an alkyl group having from1 to 8 carbon atoms, and R3 denotes a substituent which is inert towards the reagents subse~uently used, such as an amide group -NH-C-OR5, in which R5 denotes a linear or O

20 branched alkyl group having from 1 to 5 carbon atoms, in particular the tert.-butyl group.
e reaction of the compound Y-R-S-S-X with the i~munoglobulin is carried out in ~he homogeneous liquid phase, most frequently in water or a buffer solution.

_2~ _ When required by the solubility of the reagents, it is possible to add, to -the reaction medium, up to 20 ~ by volume of a water-miscible organic solvent such as an alcohol, in par-tic-llar ter-tiary bu-tanol.
The reaction is carried out a-t ambient temperature for a time which varies from a few hours to 24 hoursJ
m ereafter, dialysis makes it possible to remove the pro-ducts of low molecular weight and, in particular, the excess reagents. lhis process makes it possible to introduce a number of substituent groups of between 1 and 5 per molecule of protein.
Using such compounds, the coupling with the A
chain of ricin is carried out by bringirlg the two proteins into contact with one another in aqueous solution,at a temperature which does not exceed 30C, for a time which ~aries from a few hours to one day. me reaction takes place in accordance with the equation:
Prot-~-S-S-X ~ ASH ~ Prot-R-S-S-A ~ XSH
in whi~h Prot-R-S-S-X represents the substituted immuno-globulin (or its fragment), activated on the sulphur atom,a~d ASH represents the A chain of ricin. The solution obtained is dialysed in order to remove the products of low molecular weight, and the conjugate can then be puri-fied by various known ~ethods, as indicated in the ~irst 2S process for the preparation of the conjugates.
m e characteristics of the present invention will be understood more clearly in the light of the following 8~
_ 25 -examples ~hich illus-trate the inven-tion without limiting the scope thereof.
In -the examples which follow, TPE will denote the 00125 M phosphate buffer, pH - 7.0~ which is 1 mM in 5 respect of the dlsodiurn salt of ethylenediaminetetra-acetic acid.
Rxample 1: Preparation of the ricin a) Extraction:
1 kg of whole unshelled seeds of Ricinus communis 10 is ground in a knife-type chopper to give a paste, and this paste is tri-turated carefully wi-th 2 li-tres o~ ethyl ether cooled to 4C. After removal of the ether, the lipid-removing operation is repeated a further two times with 2 litres of ether each time. After the last 15 removal of ether, the residue is dried in air and provides 400 g of fat~free powder. This powder is suspended in 3,400 ml of a 0.14 M solution of sodium chloride, coo~ed to 4C 9 and the pH of the suspension is brought to a value o~ 4 by adding acetic acid. m e suspension, cooled in 20 an ice bath, is subjected to a homogenisation operation involving rapid agitation in the bowl of a knife-type mill for 30 seconds, followed by a 30 minute rest period with cooling in an ice bath. This homogenisation/rest cycle is repea-ted 8 -times.
After completion o~ the extraction, the suspension is centrifuged and provides about 3~400 ml of a clear solution. The latter is transferred into a dialysis tube having a flat width of 10 cm, and is dialysed ~irs-tly against dis-tilled water (which is con-tinuously renewed) for 48 hours and then for 24 hours against the TRIS-HCl buffer, :L0 mM, pH = 7.7, which is con-tinuously renewed at a ra-te of 1 litre/hour. During the dialysis, a precipitate forms which is removed by filtration on a glass frit. 3,400 ml of a crude solution containing 12 mg/ml of proteins are thus collected. m is solution can be stored at -20C.
b~ Puriflcatlon A column having an internal diameter of 100 mm is filled with 5 li-tres of Sepharose 4B (Pharmacia) and equilibrated with the TRIS-HCl buf~er ~ 10 mM, pH = 7.7, cooled to 4C.
1,700 ml, that is to say half of the crude solu-tion obtained above, is deposited at the top of this I
column at a rate of 300 ml/hour. The column is then washed with 3 litres of TRIS-HCl buffer; 10 mM, pH = 7.7, whilst maintaining the same flow rate, and the filtrate, 20 containing the proteins which have not been fixed -to -the column by virtue of affinity~ is removed.
Elution is then carried out, still at the same flow rate9 with 4 litres of the same buffer containing 00050 g (0.28 millimol) of galacto~e per litre, and this 25 fraction is collected. Elution is then carried out, still with a flow rate of 300 ml/hour, with 4 litres of the same buffer containing 0.100 g (0.56 millimol) of _ 27 ~

galac-tose per litre. l~is fraction is combined with the preceding frac-tion to provide ~ litres of a solu-tion containing about 300 mcg of ricin per~ml.
In order to be able to re-use -the column for a subsequent operation, -the latter is eluted ~ith l litre of t~le q~RIs-Hcl buffer, pH = 7 7, which con-tains 18 g (0.1 mol) of galactose per litre, to provide a fraction which essentially contains agglutinin and is remo~ed~
After washing with a further l litre of -the same buffer l~ and then with several litres of the pure TRIS-~Cl buf~er, pH = 7.7, the column is again ready for use.
An identical operation is carried out on the other half of the crude solution obtained in paragraph a.
The elu-ted fraction is combined with the preceding frac-tion to produce 16 litres of a solution of ricin in the TRIS-HCl bu~fer, 10 mM, pH = 7.7, which con-tains an aver-age of 0.075 g of galactose per litre.
This solution is concentrated on an ultrafiltra-tion membrane, the porosity of which permits the passage of globular molecules of molecular weight ~ 30,000, in a cell of diameter 150 mm. 762 ml of a solution o~ ricin in the orlginal buffer, containing 6.7 mg/ml, is finally obtained. This solu-tion can be stored at -20C.
The ricin thus obtained possesses the following characteristics Molecular weig~t: 66,000 + 59000, determined by electro-phoresis in the presence of sodium dodecyl-sulphate in - 2~ -accordance with Journal of Biological Chemistry 244, 4,~06-4,~12 (1969), : 7.1, determined by 'free electrophoresis [Laboratory techniques in bio,chemistry and molecular biology, Volume 5, part II, page 501, Pub-l.ishers: T.S. WORK and E. WORK (North Hollànd)].
All the conventional analytic me-thods applied to the ricin thus obtained make it possible to allow the conclusion that -the product is pure.
Exam le 2- Pre aration of -the A chain of ricin P _ . P . _ __ 3.75 ml of 2-mercap,toethanol are added to 150 ml of the solution of ricin,obtained in Example l and con-taining about l g of ricin, and the solution is left for 2 hours at 20C.
This solution is then dcposi-ted on the top of a column of internal diameter 50 mm, filled wi-th 1,800 ml of DEAE CL Sepharose 6B (Pharmacia) and equilibra-ted'with the TRIS-HCl buffer, 0.1 M, pH = 8.4, which contains 2.5 % ~volume/volume) of 2-mercaptoethanol.
The column is washed ~ith 300 ml of the TRIS~HC1 bufer 0.1 M, pH = 8.4, which is O.l M in respect of sodium chloride and contains 2.5 % (volume/volume) of 2-mercaptoethanol. A single fraction is collected after the beginning of the loading operation (about 450 ml) and contains the A chain, identified by electrophoresis, at a concentration of about 0.89 mg/ml.
This fraction is carefully dialysed~in a dialysis 3~

tube having a flat width of 10 mm, against 10 mM phos-pha-te buf:Eer, pH 6.5, This solution is deposited on a column of internal diameter 25 mm , which contains 300 ml of carboxytnethylcellulose CM 52, marketed by Whatman~ and is equilibrated with the 10 mM phosphate buffer, pH = 6.5. Under these conditions of pH and ionic strength, the A chain becomes fixed and it is then eluted with TPE. 67 ml of a solution of ~ chain, con-taining 5.72 mg/ml, are thus collected.

The A chain possesses the following characteris-t iC8, determined in the same ways as those mentioned in the case of ri.cin (Example lj :

Molecular weiqht : 30,000 + 3,000.
isoelectric polnt : 7.5.

15 Furthermore, in accordance with th0 D'rNB tech.nique rMe-thods in Enzymology 2_, 457 (1972)tAcademic Press~ , 0.96 equivalent of SH was determined per mol of A chain, the latter having an estimated molecular weight of 30,000.
The column of DEAE CL Sepharose 6B used for the separa-tion can be regenerated, for re-use, by washing with the TRIS-HCl buffer,0~1 M,pH = 8.4, which is 0.1 M in res-pect of sodium chlori-3e and 0.1 M in respect of galactose and elutes the B chain fixed to the column. The column must then be re-equilibrated wi-th the TRIS-HCl buffer, 0.1 M, pH = 8.~, which contains 2.~% of 2-mercap-to-ethanol, for the purpose of a further preparation of the A chain.

~R~-o ,~ ~ r~

-30~

Concentratio _ of the A chain of ricin The A chain obtain&d in diluted solution is deposited on a chromatography column of internal dia-meter 16 ~m containing lO ml oE Sepharose ~ carboxy-methyl equilibrated with the same buffer. Under theseconditions, -the A chain becomes fixed, it is then elu-ted with the TPE buffer. The recovery is quantitative.

A deposit of 150 mg of A chai.n permits frac-tions -to be eluted at the average concentration of lO mg/ml of A chain, representing a total of 90% of the quantity deposited on the column.

The resulting concentrated solution is very pure.

Obtaininq A cha_n crystals A solution of A chain with a concentration equal to 10 mg/ml is left at 4C. After a few days, crystals develop, whose anal.ysis, after re-dissolution, shows that they possess the same physico-chemical (mole-cular weight, isoelectric point) and biological proper-ties (inhibition of the protein synthesis) as the A chain, from which they are issued.

_ 31 -Example 3: Pre~ara-t ~ f the A chain of ricin 1.75 ml of 2-mercap-toe-thanol are added to 10 ml of a solu-tion of ricin, obtained in accordance with the -technique of ~xample l and con-taining 4 mg/ml of ricin, 5 and -the solutiorl is left for 2 hours at 20C.
mis solution is deposited on a column containing 800 ml o~ QAE Sephadex A50 (Pharmacia) and equilibrated with th~ ~E~IS-~HCl buffer, 100 m~, pH = 8.4, which contains 0.5 % (volume/volume) of 2-mercaptoethanol. Elu-tion is lO carried out wi-th the same buffer.
From-the time Q~ de~osition, a170 ml fraction,contain-ing 0 9 mg/ml of A chain, is collected and then treated as indicated in Example 2, starting from the dialysis opera-tion. The A chain thus obtained possesses the same 15 charac-teristics as -those obtained in Example 2. In order to regenerate the column, the fixed B chain can be eluted with the TRIS-HCl bùffe~., 100 mM, p~I - 8.4, which is 0.15 M in respect of added sodium chloride, and the column can then be washed with -the TRIS-HCl buffer, 10 mM, 20 p~l = 7.7.
xample 4: Preparation ~
The term anti-DNP antibodies denotes antibodies which are specifically directed against the ~,4-dinitro-phenyl radical.
25 a) Immunisation of the animals:
____________~______________ The desired immunogen is produced by reacting 2,4-dinitrobenzenesulphonate ~ith bovine ~-globulin in i8~

- 32 ~

accordance with a conventional technique, leading to the fixation of 52 2,4~dinitrophenyl groups per molecule of protein (DNP52-BGG) By immunisation with this product, a fraction of the antibodies formed will be specifically directed against the hapten 2,4-dinitrophenyl.

Three male goats are each immunised by injection with 5 mg of DNp52-sGG in an emulsion obtained from 2 volumes of physiological salt solution, buffered to pH = 7.4,per volume of Freund's complete adjuvant The 10 first immunisation is followed by 7 booster injections spread out over one year. During the same period, 12 1 litre samples o blood are taken from the veins of each animal. The serum is prepared and kept at -20 C. Prior to the purification step, 17 samples are combined and in the pool thus obtained, the complement is destroyed by heating. Centrifugation is then carried out for 30 minu-tes at 20,000 x g in order to remove the denaturated pro-teins and the aggregates, and, finally, paper filtratiDn is carried out in order to retain the suspended fats.
20 The serum thus prepared is ready for the purification step b) Purification:
_ _ _ _ _ _ _ _ _ _ _ _ The purpose of this step is the specific adsorp-tion of the anti-D~P antibodies from the serum onto a 25 protein carrying the hapten 2,4-dinitrophenyl and fixed to a solid support. The free or non-specifically bound substances are subsequently removed and the pure anti-bodies are then eluted.

_ 33 _ The immunoadsorption gel is formed by coupling 1l630 mg of human serum albumin, carrying 35 dinitro-phenyl groups (DNP35-HSA), and 100 g (dry weight) of 5 Sepharose ~B in accordance with the conventional tech-nique. Spectrophotomctric determination at 280 nm and 360 nm of the wash ~aters after coupling makes it possible to establish a 95 % ef~iciency for the fixation of DNP35-~ISA. Two washing cycles are carried out: ~irs-tly at 10 pH = 3.0 (0.8 M glycine hydrochloride buffer whichislM in respect ofsodium chloride)and thenatpH =10.0 (0 1 M borate buffer which is 1 M in respect of sodium chloride). The gel is then equilibrated in the working bu~fer, the latter being a 0.1 M phosphate bu~fer, pH = 7.0, which contains 15 0.01 C/o (weight/volume) of sodium azide.
Adsorption of the antibodies:
Be~ore carrying out the adsorption onto the gel prepared above, it is necessary to determine the volume ratio of serum/gel to be used, so that only those anti-20 DNP antibodies possessing the strongest affinity are fixed to the support. In order to achieve this result, it was desided to fix only 50 % of the overall antibody activity o~ the serum to the column, leaving the other 50 % in the supernatant liquid.
For a given serum, the determination of the amount of gel to be used is carried out by working on small amounts under analytical conditions. 125 ~1 aliquots ~ 34 _ of the immunoadsorbent gel are brought into contact withvolumes of SerUDI ol 0.5 ml to 8 ml, increasing in a geo-metric progression wi-th a factor of 2. Af-ter incubation~
the superna-tant is de-termined with respect to its anti-DNP antibody activi-ty by a conventional radioimmunolo-gical method [Handbook of Experimen~al Immunology, Volume 1, chapter 15, pages 1-18, Publisher D M. ~IR, second Edition, 1973], using e-N-(2,4-dinitrophenyl)-L lysine -tri-tiated on the phenyl nucleus in the 3- and 5-positions.
Thus, for each experimen-t, it is possible to express the antibody activity which has remained inthe supernatant by a percen-tage (A) of the activity of the starting immu~ àerum before treatment with the gel, and to plot the corresponding curve A (%) versus f (volume ratio of serum/gel) This curve shows the amount of serum required, with 125 ~1 of gel, for 50 % of the anti-body activity to remain in the supernatant liquid. In the experiment described, this amount is 4 ml or also 32 ml of serum per ml of immunoadsorption gel.
With this ratio determined/ 6,150 ml of immun~
serum are stirred, in a lO litre container, with 193 ml of immunoadsorbent gel for 1 hour at ambient temperature and then overnight a-t 4C.
After centrifugation (l,OOOx g, lo min~thegelis separatedoffand the solid depositisreffuspended ~l~olume ofsupernatantliquidO Thissuspension is transferred onto a B~
, .

chromatography column (diame-ter 26m~ length 400mm)which is equipped with a cooling jacket at 4C, a device for recording the optical densities at 280 nm and a frac-tion collector. The column is then washed with 8 litres of 5 0.1 M phosphate buffer9 pH = 7.0, which contains 0.01 %
of sodium azide. Washing is carried out for 40 hours at 4C and -then for 24 hours at ambient tempera-ture.
~hen the washing is complete, the op-tical densi-ty at 280 nm is extremely low (OD C~o.04).
10 Elution of the antibodies:
l~e elution of the an-ti-DNP antibodies fixed to the column is effected wi-th a large excess of a solution of the hapten 2,4-dinitrophenol.
A`0.2 M solution of 2,4-dinitrophenol is used in 15 the same phosphate buffer used above, the acidity of which has been neutralised to pH = 7.0 by adding sodium hydroxide. The solution thus obtained must be kept in the absence of light. For -the elution, 2.8 litres o~
this solution are used and are passed through the column 20 at a rate of 125 ml/hour. In order to remove the 2,4-dinitrophenol from the eluted solution, the latter is immediatelyp~ssedthrough asecondcolumn which contains 300ml of Dowex 1 x 10 resin (an ion exchange resin consisting o~ a styrene/divinylbenzene polymer carrying quaternary 25 ammonium radicals)an1isequilibrated in the phosphate buffer used for washing.
The antibodies are eluted in a single peak o~

~ 36 proteins, Iollowed by a tail. On the one hand, the resul-ting fractions of optical density ~ 0.9 (fraction Bl, 700 ml), and, on the other hand, the other frac-tions (fraction B2, 2,000 ml), are combined. The concentra-tion 5 of protein i.n each of the fractions is determined in accordance with ~ournal of Immunologlcal Methods ~, lOl-ll9 (1971).
Fraction Bl contains 15.4 g, -that is to say 22 g/
litre,of antibodies and fraction B2 contains about l g, lO tha-t is to say 0.5 g/litr~ of antibodies. m e fractions are kept at -20C :in the O.l M phosphate buffer, pH = 7.0, used for washing, which contains O.Ol % of sodium azide.
The various physicochemical methods used, in particular agar gel thin layer electrophoresis, immuno-15 electrophor~sis and passage through a column of Sephadex G 200 (a dextran gel in the form of beads, obtained by crosslinking dextran fract.ions with epichlorohydrin a~d used for gel filtration~allow the conclusion that the antibodies ob.-tained are pure and, in particular, that 20 detectable amounts of albumin (less than l/500 of the antibodies) and of immunoglobulins M are absent in the preparations obtained.
e immunological activity of the purified anti-bodies was determined by the method of NAHM [Journal of ~25 Immunology ll9, 1,301 (1977)] and compared with that o~
the starting immun .serum and that of the antibodies pre-sent in the supernatant from immunoadsorption (frac~on Al).

~ ~ 37 -___ _ _ .
Mean association Affinity dispersion constant index _. _ ~ _ .
Irnmunoserum2 0 107mol~l O.50 Fraction Al5 . 105mol 1 0.22 Fraction Bl 1 1 0.75 These results indieate tha-t the purified an-ti-bodies ~frac-tion Bl) possess a higher Mean affinity than the s-tarting immune serum and, of course, than fraction Al, and a greater homogeneity of affinity tllan the immune ser~m.
Example 5: Preparation of the conju~ate obtained from anti-DNP antibodies containin,~ mercapto-succinyl ~roups and from the activated A_chain of riein a3 Pre~aration of_the aetivat_d A chain of ricin 15 ml of a solution of A chain in the phosphate buffer (EXample 2),eontaining 1.4 mg/ml of A chain, are mixed with 8 ml of a 1.425 mM solution of 2~2'-bipyridyl-disulphide in the same buffer. The solution is left tostand at ambient temperature and in the absence of light for 1 hour 30 minutes. A solution of A chain aetivated on the thiol group, containing 0.9 mg of protein per ml, is'thus obtained. After reaction, spectrophotometric determination at 343 nm indicates that the product formed eontains 0.9 activated group ~ -s-s- permoleculeofAchain.

~3~8~

The solution is purified by continuou~ di.alysis for 18 hours, a-t a tempera-ture of ~ C, against TPE. The pure solution is l~ept at -20C.
b) Preparation of the antl-DNP antlbody containln~
merca~-tosucclr~ r_u~s 0.3l ml of an aqueous solution of 2,4-dini-tro-phenol, con-taining 0.57 mg/ml, is added -to 3.3 ml of a solution of anti DNP antibodies (Example 4), containing 25.8 mg/ml of pure an~tibodies 9 &n~ the mixture is stirred for 10 minutes at ambient -temperat~re. This solution is added quantitatively to 3.06 mg of S-acetylmercapto-succinic a~lydride [Archives of Biochemistry and Bio-physics 96, 605-612 (1962)] and the mixture is s-tirred for 2 hours at ambient tempera-ture.
0 47 ml of a 0.1 M aqueous solution of hydroxyl-amine, pH = 8.0, is added to this solu-tion, and the mixture is left for 1 hour 30 minu-tes at ambien-t tempera-ture. m e prepara-tion is dialysed against 4 times 3 litres of TPE for 89 hours at 5C and the dialysed solution is then centrifuged at 27,000 x g for 10 mins at 4C. The supernatant is passed through a column of Dowex 1 x 8 resin of 200 to 400 mesh particle size, in the phosphate form, in order to remove the-2,L~-dinitro-phenol which is fixed to the site for recognition o~ the antigen ~ Elution is carried out with TPE at ambient temperature and -the solution is then concentrated by ~ 39 _ ultrafiltration. Finallyg 4.55 ml o~ a solution con-taining 17.0 mg/ml o:E an-tibodies con-taining mercapto-succinyl groups are ob-tained.
In accordance with the method described in Archives of Biochemistry and Biophysics 119~41-49 (1967), it is poss:Lble -to determine tha-t -the subs-tituted anti-body molecule carries 4 mercaptosuccinyl groups per mole-cule o~ antibody.
c) Con~u~ate 12.4 ml of the solution of activated A chain of ricin (prepared in accordance with the -technique of paragraph a), con-taining 1 mg/ml, tha-t is to say 0.413 ~mol, are mixed with 6.5 ml of a solution of an-ti-DNP
antibodies carrying 4 mercap-tosuccinyl groups (prepared in accordance with the method of paragraph b), containing 15 mg/ml, that is to say 0.658 ~mol~ The mix-ture is left to stand for 20 hours at ambient temperature and in the absence of light.
Spectrophotometric determination at 343 nm o~
the pyridine-2-thione liberated in -the reaction medium lndicates that 0.251 ~mol of A chain has been coupled to 0.658 ~mol of antibody. The preparation is dialysed against 3 times 5 litres of TPE for 75 hours at 5C and the resulting solution (17 ml) is filtered on a sterilis-ing membrane ~0.45~m) and kept at -20C. Purification is then carried out by filtration on Sephadex G 200 gel. Fbr this purpos~ 7 ml o~ the solution obtained above are centri-fuged at 27,000 x g f or 10 mins and the supernatant liquid is -treated with 0.6 ml of a solution of N-ethyl-maleimide, containing o.645 mg/ml in TPE, ~or 30 minu-tes, whils-t standing at ambient tempera-~re. The solution is ~iltered by the ascending technique on a column (diame-ter 26 mm) containing 473 ml of Sephadex G 200 gel The product is elutecl with TPE at a rate of 16 ml/hour and the effluent is collected in 2 ml fractions.
The concentra-tion of the ~ntibody in each fraction 10 is measured by spectrophotometry at 280 nm and the concen-tration of the A chain is measured by means of lts capa-city for inhibiting a~ellular protein synthesis. The existence o.f 2 elution peaks, con-taining both the anti-body and the A chain, is thus demons-trated and the frac-lS tions corresponding to each of these peaks are combined to give solutions a and b.
The conjugate, hav ng a mean molecular weight which is greater than or equal to 8009000~ corresponds to the firs-t peak (solution a). This preparation corres-20 ponds to a conjugate originating from antibodies whichhave been polymerised by means of intermolecular disul-phide bridges formed during the isolation of the anti-body containing mercaptosuccinyl groups.
me second peak (solution b) consists of proteins, 25 having a mean molecular weigh-t of 190,000, corresponding to conjugates in which the antibodyhasnot beenpolymerised.
The analytical determinations carried out make i-t _ ~ 1 possible to show -that solution a) con-tairls 423 ~g/ml of antibody and 20 ~g/ml of A chain, tha-t is to say an average of about 0.2 mol of A chain per mol of antibody.
Likewise,solution b) contains 171 ~g /ml of an-tibody and 13 ~g/ml of A chain, tha-t is to say an average of about 0.4 mol o~ A chain per mol of antibody.
Example_6. Pre~aration of the con~ e obtained filom anti-DN an sulphur atoms and from the A chain of ricin a) ~ rid 2-yldisul~hanyl)-~ro~ionlc acid This produc-t is ob-tained in two steps from 2,2'-dipyridyl disulphide without purifica-tion of the lnter-mediate.

~ ~ C12 ~ ~s~ 2-CooH ~i~
N~ S~S ~h -~ N SC1 il ~-S-C~-CH2-C-OH

4 g of 2,2'-dipyridyl disulphide are dissolved in ~g ml of methylene chloride, and chlorine is bubbled into the solution for about 30 minutes. A precipitate forms during the reaction. The mixture is evaporated lS to dryness under high vacuum and the dry residue is used for the following operation~
2. 4 g oi` 2-pyridine sulfenyl chloride are dissolved ln 40 ml of methylene chloride, and a solution of 1.75 g of 3-mercaptopropionic acid in 10 ml o~ methylene chloride 20 is added slowly, whilst stirring and cooling with an ice _ 42 _ bath. When the additlon is complete, the mix-ture is stirred at ambient -temperature for 15 hours. 50 ml of water are added and the pH is brought to 4.5 by adding 1 N sodium hydroxide solution~ m e organic phase is separated off and dried over sodium sulphate and the solvent is evaporated off to dryness. ~1 oily produc-t remains and this crystallises ~.8 g). It is purified by dissolution in 16.8 ml of a 0.5 M aqueous solution of sodium bicarbonate. Activated charcoal is added, the lo solution is filtered and theexpected product is then pre-cipitated by adding concentrated acetic acid (0.5 ml) so as to bring the pH -to between 3 and 3.5. m e precipi-tate is filtered off and dried under high vacuum and a solid (1.3 g~, melting point ~capillary method): 62-64 C~
is obtained.
b) Pre~ar__i_n_of activ_ted anti-DNP antibodies In order to alkylate any trace of a thiol group which could exist in the product used, 13 ml of a solution of anti-DNP antibodies, containing 23.6 mg/ml in a 0.1 M phosphate buffer, p~ = 7.0, are mixed with 1 ml of a solution of N-ethylmaleimide, containing 1.29 mg/ml in TPEo The mixture is stirred for 5 hours at ambient temperature and the solution is then dialysed continuously at 5C agains-t TPE for 15 hours at a rate of 500 ml/hour.
The contents of the dialysis bag (12 ml contain-ing 21.4 mg/ml) are mixed with 24 ml of water containing 19.9 mg of 1-ethyl-3-(3-dimethylaminopropyl)-carbodi-8~
-~3 _ imide, 55.7 ~g of 3-(pyri~l-2-yldisulphanyl)-propionic acid and 14 mg of l-hydroxybe~zo~triazole. The mixture is stirred for 16 hours at ambient temperature and -then centrifuged at 27,000 x cj fo~ lo minutes O The solu-tion is 5 dlalysed continuously at 5C agains-t TPE ~or 23 hours at a ra-te of 500 ml/hour. 34 ml of a solution con-taining 7.2 mg of protein per ml are -thus obtained. By spectro-photometric determina-tion at 343 nm of the pyridine-2-thione libera-ted by exchange wi-th -the reduced gluta-thione, lo it is found that an antibody carrying four activator groups per molecule of antibody has been obtained.
c) Con~ugate 16 ml of a solution of activated antibodies, ccn-taining 5.9 mg/ml and obtained as above, are mixed with 15 20 ml of a solution of A chain of ricin, containing 2.8 mg/ml in TPEo The mix-ture is lef-t to stand for 24 hours at ambient temperature and in the absence o~ light. It is centrifuged at 27,000 x g at 4 C for 10 minutes.
Determination of the pyridine-2-thione liberated 20 during the reaction indica-tes that 1.123 ~mols of A chain have been coupled to 0.638 ~mol of the antibody used, that is to say an average of 1.76 equivalents of A chain pe~ mol of antibody.
The solution is dialysed con-tinuously at 5C
25 against TPE at a rate of 720 ml/hour ~or 21 hoursO m e contents of the dialysis bag (34 ml) are treated with 1 ml of a solution of N-ethylmaleimide, containing 1205 mg/ml, _ 4~_ for 5 hours a-t ambient -tempera-ture, and are then kept at _20C .
This solution of conjugate is fractionated on a column of Sephadex G 200gelunder-the conditions described in Exarnple 5.
S 3 solutions are -thus obtained:
solution c) represen-ts the conjugate of mean molecular weight 300,000, solution d) represents the conjugate of molecular weight 190,000, and solution e) represents the conjugate of molecular weight 160,000, The composition of the conjugate in the ~arious solutions obtained is described in the following table:

! -- I ~g of A chain coupled Mols of A chain to the antibody per ml _ _ .
.of solution Mols of antibody - _ ~ _ solution c 13 0.9 solution d 48 1.0 20 solution e 3 0 2 .
_ _- _ . :

-- ~5 -Example 7 : Preparation of the conjuqate obtained from _ti-DNP a_tibodies substituted by an acti-_ated disulfide qroup and from the A ch_ln of .r cln.

a) 6-l3-(2-pyridyl disu~han~l)propionyl amlno)~ hexa-noic acid.
_ _ _ _ _ _ _ _ _ This acid is obtained in two steps from 3-(2-pyridyl-disulphanyl) propionic acid without puri-iication of the intermediate.

~ S - S - CH2CH2C - OH

' ~'\

~ ~ 2N ( 2~ 5 N S--S--CH 2 CH 2 C--O--N~ ` ~

~ 5 5 CH2CH2C--NH--CH2CH2CH2cH2cH2--C--OH

0.~30 g of 3-(2-pyridyl dlsulphanyl) propionic acid obtained as described in example 6 a) i9 dissolved in 3 ml of pyridin. The solution is cooled in an ice bath. 0.230 g oE N-hydroxysuccinimida in powder form and then 0.453 g of carbodiimid dicyclohexyl also in powder form are added to this solution. The mixture is stirred ~or 20 hours at 4 C. The reaction medium is filtered in order to remove the precipitate of dicyclo-hexylurea formed, and the precipitate is washed with py-10 ridin. The washing sGlution is evaporated to drynessunder high vacuum. The residue obtained is diluted in ethyl acetate. A new precipitate of dicyclohexylurea is formed; it is removed by filtering. The ethyl acetate i~ evaporated to dryness under high vacuum and subsequen-15 tly, the washing operation with ethyl acetate is repeatedtwice. 0.610 9 of the expected es-ter is thus obtained.

The product obtained is totally dissolved in 5 ml of tetrahydrofuran. Then 0.288 g of 6-amino caproic acid i9 added in solution wi-th 7.5 ml of water. Triethylamine 20 is added in stoic~iometric quantity with respect to the

6-amino caproic acid (0.222g) in order to maintain the pH
at 7Ø The reaction is further carried o~t at 4 C
under stirring for 20 hours. The reaction medium is evaporated to dryness under high vacuum. The residue is 25 diluted in 10 ml of water. The medium is acidiied up to pH 4.5 ~y adding acetic acid. A precipitate is for-med which is recovered by decantation and then solub ~ized in ethyl acetate. The solution is dried by magnesium sulphate, then filtered and evaporated to dryness under 30 high vacuum.

0.600 g of the product to be puri.fied is obtai~
ned. The purification is effected by division chroma-tography on a silica column, under the conditions defined by analysing the product in chromatography on thin layers in ~etone-chloroform-acetic acid mixture (15-80-5).

0.~10 g of the product obtained is deposited on a column of internal diameter 2 cm containing 160 ml of silica gel (No. 60, 60-230 mesh MERCK) equilibrated with the chloroform. The elution is effected in disconti-10 nuous gradient of methanol in the chloroform~ The pureproduct is eluted Wit~l the mixtuxe containing 2% of methanol. The solvents are evaporated to dryness under high vacuum.

The product obtained, which is light yellow and of pasty consistence, is characterized by its spectrum of nuclear magnetic resonance.

b) Preparation of activated anti-DNP antibodies ____________________________________________ To 9 ml of the solution of anti-D~P antibodies at a concentration of 11.4 mg/ml in the TPE buffer are 20 added 9 ml of a water/t.butanol 2/1 (v/v) mixture contai-ning 5.3 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodi-imide, and 13.7 mg of 6-~3-(2-pyridyl disulphanyl~ propio-lamino) hexanoic acid. The operations are then effec-ted as described in example 6.

The determination of the activated antibodies indicates an average substitution rate of 0.6 activator groups per mol of antibody.

~8E~
-~8-The solution of an-tibodies thus obtained is concentrat~d by ultrafiltration up to 11.1 mg/ml~

c) Con~ugate 48.8 mg of modified antibodies are reacted with 35.6 mg of A chain of ricin at 7.9 mg/ml in TPE.
The preparation is c~rried out under the conditions a]-ready described in example 6. The average coupling rate obtained is 0.5 A chain per mol of antihody. The purification of the conjugate by filtration is carried out as described in examp~e 6.

Example 8 : Preparation of the conjuqate obtained from the F(ab)'2 fraqment of the anti-DNP anti-body and from the A chain of ricin.

a) Preparation of Fragment F(ab)' of the anti-DNP antibody.
________.______________________~_________________________ The solution of antibodies (example 4) is dyali-sed against the 120 mM acetate buffer, pH = 4.7. To 45 ml of this solution containing 500 mg of antibodies are added 3 ml of a solution of pepsine at a concentra-tion of 10 mg/ml in the same buffer (E.C. 3.4.4.1. pork pepsin, twice crystallised, a~ 3200 u/mg - SIGMA).
Incubation is maintained for 20 hours at 37 C. The progress of the hydrolysis is checked by electrophoresis in the presence of sodium dodecyl sulphate [(J. Biol.
Chem. 244, 4406-4412, (1969j~ by the disappearance of the strip containing 150~000 dalton and corresponding to the non-hydrolysed antibody.

B~
~.9 The hydrolysis is ~opped by adjusting the pH
of the reaction medium to 7.0 with the aid of a solution oE 1 N sodium, then the solution is centriEugated and the insoluble removed. The centrifugation supernatant is deposited on a column of internal diameter 50 mm con-taining 1800 ml o~ Sephadex G 150 ~ equilibrated with the 100 m~ phosphate buffer, pH - 7Ø

The elu-tion is effected with the same buffer :
the first peak ob-tained is collected in two fractions:
10 the first one (200 ml) contains 70 mg of Flab)'2 frag-ment of the antibody, and the second (100 ml) contains 70 mg of a mixture of an F(ab)'2 fragment with a conta-minating fragment of 50,000 dalton molecular weight.
The second peak contains protein fragments with an ave-15 rage molecular weight of 10,~000 dalton. It is removed.

The fragment F(ab)'2 obtained pure in the firstfraction of the first peak is concentrated by ultrafil-tration (procedure described in example 1) up to a con-centration of 16 mg/ml and stored at -20 C.

20 The ~ragment F(ab)'2 thus obtained shows the following characteristics :

Molecular weiqht : 95,000 dalton (determined after ca-libration of the Sephadex G 150 gel) 5 Isoelectric point : between pH 7.7 and 8.8 (obtained by isoelectrofocalisation on LKB plate).

b) Preparation of the activated fragment F(ab)' _________ _~_~ 2 To 15 ml of the F(ab)'2 solution at a concentra-tion of 15.6 mg/ml are added 15~3 ml o a mixture of water/t.butanol 2.1 (v/v) containing 17.3 mg of l-ethyl 3-(3-dimethylaminopropyl) carbocliimide and 50.4 mg of 3-(2-pyridyl disulphanyl)propionic acid. The mixture i9 incubated at 30 C for 20 hours. The solution i5 then dia]ysed continuously against the TPE buffer at 4 C for 23 hours at a rate of 500 ml~hour. Spectrophotometric 10 determination at 343 nm of the pyridine 2-thione libera-ted by exchange with the reduced glutathione indicates that the activation of fragment F(ab)'2 has lead to an average substitution rate oE 1.5 activator groups per mol of F(ab)'~.

The solution of activated F(ab)'2 thus obtained is concentrated by ultrafiltration up to lO mg/ml.

c) Con~ugate To 13.8 ml of the solution containing 10 mg/ml 20 of activated F(ab)'2 obtained as described above are added 13.8 ml of solution of A chain of ricin at 8 mg/ml in the TPE buffer.

The mixture is incubated at ambient temperature for 24 hours in a nitrogen atmosphere and in the absence 25 of light. ~The solution is then centrifugated at 27000 x g, at 4C for 10 minutes and the supernatant i9 recovered.
Determination of the pYridi~ 2-thione liberated during the reaction indicates that the average coupli~ rate is 1.2 equivalents of A chain per mol of F(ab)'2~

The solution i5 deposited on a column with internal diameter 50 mm containing 1730 ml of Sephade~
G 200 ~ Elution is carried out with the TPE buffer~
The fractions are collected under a volume of 8.4 ml and the an~ysis of the ractions is carried out as described in examples 5 and 6. The conjugation product comes out into a peak.

The fractions constituting the peak of the con-jugate are grouped into three solutions, respectively as follows :

. solution E : front part of the peak . solution g : central part of the peak . solution h : tail of the peak The analysis of solution g by determination of its inhibitory activity of the protein synthesis (test 1 and the determination of the proteins give the following resultsa yg of A chain coupled Equivalents of .
to F(ab)' per ml of A chain per mol solu~ion of F(ab)'~
_ Solution g 148 1.4 d) Concentration of the conjugate ________._______.___~________ In a first step, the concentration i5 caxr.ied out by ultr~filtration : 255 ml of solution g of the conjugate (at 1~ ~g/ml of A chain) are thus concentra-ted to 381 ~g/ml. The solution is then diluted 10 timesin distilled water so as to lower the ionic strength of the medium~

The solution obtained ~00 ml at 38 yg/ml of A chain) is deposited on a column of internal diameter 9 mm containing 5.7 ml of C.M. Sepharose ~ equilibrated with the 10 mM phosphate buffer, pH = 6.5 mixed with disodium salt of 1 mM ethylene diaminotetracic acid.

The depo~iting is carried out at a rate of 80 ml~hour. Under these conditions, the total conju-lS gate becomes fixed on th~ column. When deposi-ting is completed, 4.5 ml of the same buffer are left free, and elution of the conjugate is effected with the TPE
buf~er at the same rate.

The conjugate comes out into a single peak.
The tail of the peak containing little proteins is ~emo-ved. The remaining fractions are grouped and the con-centration in A chain is determined.

Determination indicates that the concentration operation on C.M. Sepharose ~ has allowed the concentra-tion of A chain of the conjugate to be brought to : 3.6 mg/ml. The operation is quantitative and the frac-tions constituting the solution at 3.6 mg/ml is overall 76% of the quantity of the conjugate deposited on the column.
xamE~ reparati n of the conjuqate obtain d Erom anti-_h~ 1.2 Iq~ antlbody and from the A cha1n of_ricin.

a) Preparation of the anti-Thy 1.2 IgM antibody __~_________________________________________ The pure IgM is prepared from the ascites liquid obtained from OLAC LTD ~Thy 1.2 F7 D5 monoclonal IgM
cytoxic antibody).

To 50 ml of ascites liquid containing the anti-body are added 15 g of powdered ammonium sulphate.
After dissolution, the mixture is incubated for one hour at ~ C and the whole is then centrifugated for 20 minu-tes at 17000 x g.

The supernatant after centrifugation is removed.
The deposit is dissolved in 20 ml of 100 mM phosphate buffer, pH = 7.0, then the solution is dialysed continuous-ly against 10 litres of the same buffer. The solution ob-tained is deposited on a column of internal diameter 50 mm containing 1800 ml o Sepharose 6B ~ equilibrated with the 100 mM phosphate buffer, pH = 7Ø Elution is car-ried out with the same bufer : the irst peak obtained corresponds to IgM aggregates. The second peak obtai-ned is collected in two fractions : the first ~raction (from the front part to the top of the peak) is consti-tuted by pure IgM; the second fraction contains a mix--5~-ture of IgM with a con-taminating protein of molecular weight estimated at about 660,000 dal-ton. This second fraction i5 ayain treated with ammonium sulpha-te and the precipitate obtained is centrifugated and re-dissol-ved in the ]00 mM phosphate buffer, p~l = 7Ø The solu-tion obtained is deposited on the Sepharose 6B ~ column, suitably washed after the first operation. Elution by the 100 mM phosphate buffer, pH = 7.0 gives rise, as in the first operation, to the production of two peaks.
The second peak obtained contains pure IgM. Each of both filtration operations on Sepharose 6B ~ has thus permitted a ~raction containing pure IgM to be obtained.
These two fractions are brought together and the whole is treated with ammonium sulphate at a final concen~ra-tion of 300 mg/ml, at 4 C. The preparation is centri-fugated for ~0 minutes at 17000 x g, then the deposit is diluted in the 100 mM phosphate buffer, pH = 7.4 and mixed with 400 mM sodium chloride.

All these operations make it possible to prepare 20 8 ml of a 901u-tion containing 6.4 mg/ml of pure IgM.
Analysis by electrophoresis in the presence of sodium dodecyl sulphate has been used for characterizing the fraction : the pure IgM thus obtained shows after analy-s~s two very similar bands of equal intensity corxespon-ding to molecular weights approximating 900,000 dalton.

b) preparation of the activated anti-Thy 1.2 antibody __________________________________________________ To 7.6 ml of the solution of I~M at a concentra-tion of 7.0 mg/ml are added 0.46 ml of a mixture of water/

t.butanol ]/5 (v/~) containing 2.9 mg of 1-3(3-dimethyl-amino prop~1) carbocliimide e-thyl and 7.7 mg of 3~~2-pyri-d~l disulphc~nyl)propi~onic acid. The mixture is incuba-ted ~or 20 hours at 30 C. The solution is then dialy-sed continuously against the 100 mM phosphate buffer,pH = 7.4, mi~ed with 400 mM sodium chloride and disodic salt o:E 1 mM ethylendiaminotetracetic acid. The dialy-i~ is continued for 23 hours at 4 C at the rate of 500 ml/hour~ The solution is subsequently centrifuga-10 ted at 27000 x g at 4 C for 10 minutes and the superna-tant is recovered. Spectrophotometric determination at 343 nm of the pyridine 2-thione liberated by exchanye with the reduced glu-tathione shows that the activati.on o the an-tibody has resulted in the average substitution rate of 13 activator groups per mol of IgM.

c) Conjugate _ _ _ _ _ _ _ _ _ The solution of A chain obtained as described in Example 2 is dialysed continuously against the 100 mM
phosphate buffer, pH = 7.4, mixed with 400 mM sodium chlo-20 ride and disodic salt of 1 mM ethylenediaminotetraceticacid. The dialysis is conducted at a rate of 500 ml~hour for 20 hours at 4~ C.

To 6.8 ml of the solution of activated IgM obtai-ned as described above are added 3.9 ml of dialysed A
chain, at a concentration of 7.4 mg/ml. The mixture is incubated for 24 hours at ambient temperature in an nitro-gen atmosphere and in the absence of light~ The deter-mination of the pyridine 2-thione liberated during the 6~:~

reaction indicates indicates that the average coupling xate is 9.7 equivalents of A chain per mol of IgM.

The solution is deposited on a column of internal diameter 50 mm containing 1727 ml of Sephaxose 6B ~ equi-librated with the conjugation buffe.r. Elution is conductedwith the same buffer. The fractions are collected under a volume of 3.7 ml and the analysis of -the fractions is carried out as described in examples 5 and 6 ~ The conjugation product comes out into two peaks~
10 The first corresponds to the conjugate with an average molecular wellght hiyher than that of the IgM~ The second peak corresponds to the conjugate with an a~erage mole-cular weight approximately equivalent to that of the IgM.
The central part of this peak (solution i) is retained.
15 The composition of the conjugate of solution i corresponds to the following charactexistics:

._ _ ~g of A chain coupled Equivalents of to IgM per mol A chain per mol . of solution of IgM
. - _ Solution i 23.7 3.2 The conjugates according to the invention and also the compounds used in the preparation of the said conjugates, were studied with respect to their biological properties, and very particularly, their carcinostatic action. An account of the principle of the various tests used is given below.

~57 _ 1) Inhibition of ero-tein synthesis __ __ The fundamental biological proper-ty of the A
chain of ricin is to inhibit microsomal proteln synthesis by damaginG the ribosomal 60S sub--uni t .
5 a) Acellu].ar rnodel (Test 1) l~e in vitro procedure corresponding to a non cellular protein synthesis model uses subcellular frac-tions of rat liver, which are sui-tably complemented and capable of incorporating C-phenylalanine in the presence of an ar-ti~icial messenger RNA, namely polyuridylic acid.
The method of opera-tion employed for preparing the subcellular fractions and for measuring the incorpor-ation of 14C~phenylalanine is an adaptation of the method described in Biochemica Biophysica Acta ~, 608~615 (1973), employing both a microsomal frac-tion and a cytosol fraction of rat hepatocytes. The sample containing the A chain is introduced in the form of a suitably dilute;l solution in a 50 mM TRIS-HCl buffer9 pH = 7.6, containing 0.2 ~0 of 2-mercaptoethanol and 15 ~gtml of bovine serum albumin.
From the counting data, the percentage inhibition of the incorporation of 14C~phenylalanine in the proteins, for each reaction medium containing the A chain of ricin, is calculated relative to a control medi~ without inhi~
25 bitor.
Fromtheseresults it ispossibletocalculate that 186~
_58 concentration ofthe Achain inthe reaction medium which inhibits -the .incorporation of radioactivi-ty into -the proteins by 50 %~ Thi.s 50 % inhibitory concentration (IC 50) makes it possible to charac-terise any preparation 5 conta.ining the A chain.
I~e same procedure also makes it possible to deter~ine -the A chain presen-t in a sample by comparing the percen-tage inhibition, shown by this sample, with a standard curve obtained under the same conditions using 10 solutions having a }cnown concentration of pure A chain.
b) Cellular rnodel (Tes-t 2) ______~________ This test measures the effect of the substances studied on the incorporation of 14C-leucine into cancerous cells in a culture.
The cells used are HeLa cells which originate ~rom a biopsy of human cervical adenocarcinoma and are kept in a continuous line by means of monolayer culture.
These cells are incubated in the presence of pre-parations of the substances to be studied and are then 20 subJected, after incubation, to a measurement of the.ir degree of incorporation of 14C-leucine. mis measure-ment is carried out in accordance with a technique adap-ted from the technique described in Journal of Biological Chemistry 249 (11), 3,557-3,562 (1974), using the tracer 2S 14C-leucine for determining the degree of protein syn-thesis. Determination of the incorporated radioactivity is effected in this case on the whole cells isolated by i 6~.
_ 59 -filtration.
From these determin~tions,it is possible to plo-t the dose/ef~ec-t curves showing, on the abscissa, the concentra-tion of the ~ubstances stuclied, and, on the ordinate, the incorpora-tion o:f 14C-leucine~ expressed as a percentage o~ the .incorporation by -the control cells in the absence of -the substance to be studied.
The concen-tration which inhibits the incorpora-tion of 14C-leucine by 50 %, or the "50 % inhibitory concentration" (IC 50), can thus be determined for each substance studied. It was shown that the measurement of incorporation of 14C-leucine in the whole cells resul-ted in the determination of IC 50 values which are identi-cal to those ob-tained by the conventional method of protein synthesis measuremen-t.
If it is desired to study conjuga-tes prepared with the anti-DNP antibodies, the HeLa cells must be able to be recognised by -these antibodies and must there~ore carry hap-ten units on their surface. The HeLa cells are 20 hence converted into target cells by labelling with a suitable hapten. In practice, it was decided to use the 294,6-trinitrophenyl group, or TNP, as -the hapten.
The fixation of the TNP groups to the HeLa cells is effected by the action of 2,4,6-trinitrobenzenesulphonate ~5 (TNBS) in accordance wi-th an adaptation of the techniques described in Biochimica Biophysica Acta 2~, 79-90 (1972) and Journal of Immunology 111, 930-937 (1973)o The modification oE the method essentially con-sists in choosing the reaction parame-ters, namely a temperature of 40 C and stopping the reaction with an excess of lysine after a reaction time of 15 seconds.

This labelling was retained for various rea~ons namely :

the ]ow toxicity of TNBS to the ~IeLa cells, the high level of cross-reaction between DNP
and TNP, and ]o comparable levels of incorporation of 4C-1PU-cine between labelled cells and unlabelled cells.

The concentration of TNBS in the reaction medium ~10 mg~ml) causes the fixation, to each cell, of 7.10 hapten units accessible to the antibodies. The associa-tion constant between anti-DNP antibodies and hapten units i9 1.2. 107.

For the conjugate with anti-Thy 1.2 specifi-city, the cells used belong to the WEHI-7 line (lymphoma of Balb/C mice) obtained from SALK I~STITUTE - SAN DIEG0 (California). These cells are not subjected to any label-ling 9 tep because they naturally carry the Thy 1.2 anti-gen on their surface. Incubation with the conjugate and then measurement of the degree of protein synthesis are e~fected as described above.

2) ~ qlu-tination The hemmagglutination test (~ournal Of Biolo-gival Chemistry 249, 803 (197~) is intended for compa-ring the capacity of the various preparations studied to become fixed to the membrane of ~luman blood cells of group 0, Rhesus negative.

The test can be :

either direct (test 3), by bringing the subs-tance to be studied into contact with red hlood cells, which leads, depending on the dose of substance, to a negative or positive response, ........ ............. ......... .. .... . . ... .
\

6~
_ 62 _ or indirect (test 4). In thi.s case 9 a:Eter the direc-t test, the detection of the substance which may be fixed to the red bloocl . cells is sensi.-tised by adding, to the red blood cells separated from -the reaction medium by decantation, an im~u~ serum containing anti-bodies which are capable of recognising the substance studied, and the inheren-t hemagglutinant actlvity of which has been removed by adsorption onto red blood cells of the same group~

1) ~ (Tes-t 5) The study of -the acute -toxicity is carried out by -the intraperitoneal or intravenous injec-tion of the . substance to be studied, brough-t into isotonic solution, to batches of animals. The animals are observed for

7 days and the mortality is noted for each dose studied m e 50 % lethal dose (LD 50) is then determined.
2) fect of the conjugates on the development of can-cerous cells .~_ The model chosen consists in studying -the effect of the substances to.be tested on the development, in the form of solid tumours, of cançerous cells injected into the animal.
The experimental method consists in injecting 25 HeLa cells, carrying the hapten TNP, into "nude" mice (Bomlholtgaard congeneric nude/nude mice), known for ~ot rejecting allogenic or xenogenic grafts, and in treating

8~
_ 63 _ these mice by simultaneous or subsequent injection with the compound to be 5 tudied.
Various tes-ts were carried ou-t:
a) Test 6: Prc-incuba-tion of the HeLa-TNP cells wi-th the _ __ ____ 5 conjuga-te, followed by subcutaneous injection into the animal.
The size of -the tumours which may have developed is then determined by measuring 2 orthogonal diameters ofthe-tumourin accordancewiththe techniquedescribed in 10 Annales d'IrNmunologie ~Institut Pasteur) 124 C, 56~-572 (1973~.
b) Tes-t 7: Intraperitoneal injection of the HeLa~TNP
_ _ _ _ __ cells, ~ollowed by the injection, also in-to the intra-peritoneal cavity, of the compound to be st~ldied.
At the end of -the experiment) the size of the . tumours is determined, after sacrificing the animals by measuring the weight of the tumours which have been collected, The various in vitro and in vivo tests thus des-20 cribed were used ~or studying the properties of the conjugates and also of the substances used ~or the prepar-a~ion of the conjugates. .
RICIN AND A CHAIN
~ _ .. _ .
The ricin and the A chain were subjected to the 25 various in vitro tests and also to the in vivo toxici-ty test. Furthermore, the A chain purified by concentration step on C.M. Sep~arose (indicated by (A) chain in the table) has been subjected to the same tests The results obtain~d are given in Table I.

These results indicate that ricin possesses a strong capacity for inhibiting protein synthesis, both in an acellular system and in a cel~ular system. In an .acellular medium, the A chain also strongly inhi-bits protein synthesis but, on the other hand, it can-not exert its effect in a cellul~r medium when it is separated from the B chain.

The same phenomenon is observed in the hemagglutination tests and the acute toxicity test, in which tests ricin proves to be much more active than the A chain.

These results furthermore indicate that the concentration step on C.M. Sepharose makesit possible to again reduce the non-specific toxicity of the A chain - by substantially increasing the pureness of the prepara-tion. The extra pure A chain thus produced is free of non-specific toxicity ~ith respect to a cellular system.
This is clearly shown by the activity ratio between ricin and the ~A) chain in this test, which is higher than 10000.

Exhaustive absorption tests of the A chain were conducted using successively excesses of HeLa cells and red blood cells whic}~ have been brought into contact with the A chain, and by measuring the cellular toxi-city of the A chain in the absorption supernatant (test 2) :

. The cellular toxicity of the solution of A chain not concentrated on C.M. Sepharose is lowered by absorp-tion and thus brought to the toxicity level of the solution of A chain which is concentrated on C.M. Sepharose and not absorbed.

. On the contrary, the toxicity of the pre-paration concentration on C.M. Sepharose is no longer lowered by the absorption tests, which confirms the botal lack of affinity of the (A) chain thus prepa-red towards the plasma membranes of the cells, and consequently the extreme degree of pureness obtained.

... . . .. ... .. .... . .. .. ..... ... ..... , .. . .. , .. . _ ~ ~

\

8~
_ 66 -ANTI-DNP ANTIBODIES
In the test for -the inhibition of protein syn -thesis iIl a cellular system, the anti-DNP antibodies do not produce any effec-t up to the highest concentration tested~ namely 10 ~.
Furthermore, in the acute toxicity test, no indica-tion of toxicity was recorded up to the highest dose tested, namely 1~6 mg of ant~body per animal.
CONJUGATES
The purpose of a first series of experiments is to show the integrity of the properties of the A chain, on the one hand, and of the antibody, on the other hand 9 in the conjuga-tes prepared.
First of all, by means of radioimmunological test (Handbook of Experimental Immunology, Volume 1, chapter 15, pages 1 to 18, Publisher D.M. WEIR, 2nd edition, 1973), it was possible -to establish that the anti DNP antibody activity of the conjugate was equivalent to the activity 20 of the antibody by itself.
Furthermore, as regards the A chain of the conau~
gate, the inhibitory activity with respect to a-cellular protein synthesis was determined, on the one hand, on a sample of con~ugate (Example 4, solution a), and, on the 25 other hand, on another identical cample~ incubated for 30 minutes with a 0.2 % strength solution of 2-mercapto-ethanol in order to spli~ the disulphide bridge.

An IC 50 of 59.4 ng/ml of A chain was obtalned in -the 1st case and an IC 50 of 9.2 ng/ml of A chain was obtained in the second case, this being an activity ratio of 1 to 6.6.
This shows that the main par-t o:E the inhibitory activity with respect to protein synthesis only appears after the conjugate has been treated with 2-mercap-to-ethanol in order to break the disulphide bond created.
The fact that an inhibitory activity is detectablewithout treatment with 2-mercaptoethanol does not contradict this conclusion insofar as the medium used for determination contains thiols which are partly involved in the liberation of the A chain coupled to the antibody.
In conclusion, the conjugates obtained according to the invention retain the properties of each of their constituents.
The purpose of another series of tests is to determine the therapeutic effectiveness of the con~ugates.
a) Effect on the cellular model ITest 2~
Figures 1 and 2 give the curves showing the per-centage inhibition of incorporation of 14C-leucine by the TNP-labelled HeLa cells, as a function of the dose of conjugate used, expressed in concentrations (nM) of A
chain. The conjugate used in these figures is the conjugate of anti-DNP antibodies and of the A chain such as described in Examples 5 and 6. By way of comparison, the action of the conjugate on unlabelled HeLa cells is also shown (broken lines).

These curves were obtained, for figures 1 and 2 respectively, with ~he conjugate of Example 5 (solu-tion b) and with the conjugate of Example 6 (solution e).
These curves show that the 50% inhibitory effect with respect to cellular protein synthesis of the HeLa-TNP
cells is obtained for concentrations (IC 50 = ll x lO ~M
in the case of solution b and 7.5 x 10 M in the case of solution e) which are very much lower than those required to obtain the same effect with unlabelled HeLa cells (IC 50 = 130 x lO M in the case of solution b and lO00 x M in the case of solutinn e).

Furthermore, if bovine serum albumin - DNP
(l mg/ml) is added to the incubation medium, the inhibi-tory effect of the conjugate is entirely cancelled.

15 Figure 3 shows the results obtained with the conjugate formed from the A chain of ricin and the F(ab)'2 fragment of anti-DNP IgG. The data are the same as those of figures 1 and 2. The results are given for :

(I) ricin Ic 50 (in A chain) S.5. lO M.
20 (II) tha A chain IC 50 9. lO M.
- (III) the conjugate on HeLa IC 50 cannot be deter-mined ~lO M.

Figure 4 shows the inhibition of the incorpora-tion o C leucine into WEHI-7 cells by using the conju-25 gate formed from the A chain and the anti-Thy 1.2 IgM.
The data are the same as thosè of figures l and 2. The 6~

results ars given for :
(I) the ricin IC 50 (in A chain) 2.4. 10 M
(I~) the A chain IC 50 8.1 10 M
(III) the conjugate IIC 50 (in A chain) 5.2 10- M

Finally, it should be noted that the conju~ate formed from the A chain of ricin and the anti-DNP IgG
by using 6-~3-(2-pyridyl disulphanyl)propionylamino~
hexanoic acid as the activator of IgG, has a toxicity and a specificity identical to those of the conjugate prepared in Example 6.

It can be concluded that the conjugates prepa-red according to the invention have a specific action.
By administering a suitably chosen dose of the conjugate, it is possible to act only on the cells which carry the antigen corresponding to the antibody used.

The formation of a disulphide bridge between the A chain and an antibody specific of an antigen, which is naturally carried by cancerous cells, makeS
it possible to destroy the cancerous cells with a very high efficiency and an important specificity of action.

b) In vivo tests _ ._ _ _ _ _ _ _ _ _ _ _ _ A. T_st~conducted on the eonl~qate of A_chain and of anti-DNP antibody (solutions c_and d, Exa~ple 6) 1) Te_t 6 described above was used with ~eLa-TNP cells, at a concentration of 10 cells per ml, incu-bated Eor 90 minutes at 37~ C with the conjugate (solu-tion c), at a concentration of 10 M e~pressed in terms of A chain.

After centrifugation (700 x g, 10 minutes), the deposit is re-suspended in PBS (a buffered isotonic solution without calcium or magnesium, J. Exp. Med. 99 167 (1954)) at a concentration of 107 cells per ml.
The animals (1~ weeks old) receive 0.1 ml of this sus-pension, that is to say lo cells, by subcutaneous admi-nistration.

A cuntrol batch receives cells not incubatedwith the conjugate, The animals are observed dailyand/this is accom-panied by measurement of the surface area of the tumours.
Any tumour of which the surface area is greater than or equal to 10 mm (sensitivity limit of the test) is considered to have taken.

Figure 5 represents the cumulative percentage of developed tumours having a surface area~ 10 mm , as a function of time, after having injected, into the mice, either ~eLa cells (curve in solid line) or T~P-labelled Hela cells (curve in broken lines) or HeLa cells label-led with TNB and pre-incubated with the conjugate (curve in broken lines and dotted lines). The effectiveness of the conjugate, which strongly reduces the development of tumours, can be established from the figure.

Observa-tion up to the 50th day shows the deve-lopment of tumours in 80% of the cases for HeLa cells which have not been treated with the conjugate, and in 70% of the cases Eor the HeLa-TNP cells which have not been treated with the conjugate.

This percentage is only 20% in the case of HeLa-TMP cells which have been pre-incubated with the conju-gate.

rhe difference is statiscally significant at the 5% level.

2) Te~t 7 is used by the intraperitoneal injec-tion,into the animals (10 weeks old), of 2 .10 HeLa TNP cells suspended in 0.1 ml of a culture medium.

0.1 ml of conjugate, containing 36 Jug of A chain (solution d concentrated 7.5 times by ultrafiltration) is then injected.

A control batch receives the same injection of HeLa cells but does not receive any treatment with the conjugate.

On the 25~h day after the start of the experi-ment, the animals are sacrificed and the tumours are weighed.

Figure 6 shows the decimal logarithm of the weight of the tumours in mg for each of the animals 36~

of the two batches, the control animals being on tha left and the treated animals being on the right. The threshold of detection is 5 mg, this corresponding to a decimal loyarithm of 0.7.

From this figure, it can be established that, in the case o-E the control batch, tumour development occured in 1~ cases out of 15 (14 points above the threshold of detection and only 1 point below). Con-versely, in the case of the treated animals, tumour development only occured in 1 case out of 15 (only 1 point above the threshold of detection).

B. Tes-ts conducted on the conjuqate of the A chain and of the anti-Thy 1.2 antibody (solution i, Example 9).

The cancerous cells used are of the WEHI 22. 1 line (obtained from SALK INSTITUTE, SAN DIEGO, California) and originate from a lymphoma of Balb/C mice. They are injected into Balb/C o~a/ined from BOMEOLTGAARD (Denmark) and are kept from their birth and for the duration of the test in a ~one free of specific pathogenic organisms (SPF).

In the test, the WEEI 22.1 cells kept in conti-nuous culture are collected on the third ~ay after trans-plant of the culture, washed by centrifugation and re-suspended in cn isotonic phosphate buffer (PBS) at a con-centration of 12.106 cells/ml.

0.2 ml of the suspension is injected by intra-peritoneal administration into mices previously placed ~ 73 -at random in their cages and carrying an individual mar-ki~g on their fee-t.

In mice not treated with the conjugate, the in-traperitoneal injection of WEHI ~2.1 cells is followed by the evolution of these cells into tumours dispersed in the peritoneum with formation of an ascites. This evolution of the cells is accompanied with an increase in the body weight. The ascites is detected by obser-ving an increase in the abdominal volume.

One hour after injection ~jf the cells, the ani-mals treated with the conjugate receive 1 ml of solution of the conjugate (solution i, 23.7 mg/ml of A chain) by intraperitoneal administration, previously dialysed ex-tensively against the PBS buffer, and then sterilized hy filtration. The batches of control animals receive 1 ml of PBS buffer containing no conjugate.

On the 7th day after inoculation of the cells, the treated animals receive a second injection of the conjugate in the same conditions, and the control animals a second iniection of PBS buffer.

The results of the test are seen in figures 7 and 8. Figure 7 describes the chronology of the forma-tion of ascites in the treated and in the non-treated animals. Figure 8 describes the evolution of the body weight in treated and in non-treated animals. The evolution of the weight is expressed in terms of weight increase ~grams) with respect to the weight of the same animals 3 days before inoculation of the cells.

Figure 7 (abcissa representing the number of days after inocuLation of the WEHI 22.1 cells, and the ordlnate representing the percenta~e of animals showing an ascites) shows the existence of an inhibition of the formation of ascites in animals treated with the conju-gate. This inhibition is proved by the ascites foxming at different times (curve in continuous line represen-ting the treated animals, and that in dotted line re-presenting the non~treated animals).

Figure 8 (abcissa representing the number of days after inoculation of the WEHI 22.1 cells and the ordinate the average increase (grams) in the body weight), also shows an inhibition of the increase in the body weight in animals treated with the conjugate (continuous line) compared with the control animals (dotted line).

These observations are confirmed by the statis-tical analysis of the results :

. the inhibition by the conjugate of the forma-tion of ascites is significant at the 1% threshold of reliance (test X2).

. The inhibition by the conjugate of the weight increase is significant at the 5% threshold of reliance (test of comparison o~ the gradients of linear adjustments).

The in vivo test makes it possible to demons-trate the anti~cancerous activit~ of the conjugate formed from the A chain of rlcin and the anti-Thy 1.2 IgM.
Such activity is shown hy the inhibition of the develop-ment of WE~II 22.1 cancerous cells in~ected into Balb/Cmice.

The conjugates prepared according to the inven-tion exhibit a specificity of action which is such that it is possible to envisage their use in human therapy for the treatment of cancer. They are prepared for use by injection and can be used either by ~hemselves or in association with another cancer treatment.

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Claims (16)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for the preparation of a cytotoxic product having a covalent disulphide bond between a cytotoxic compound and an antibody, the process comprising forming a disulphlde bridge between the A chain of ricin and the antibody, the antibody being an immunoglobulin or an immuno-globulin fragment and being specific for a given antigen carried by the cells which are to be destroyed.

2. The process of claim 1 wherein the antibody is an anti-DNP antibody.

3. The process of claim 1 wherein the antibody is the F(ab)'2 fragment of the anti-DNP antibody.

4. The process of claim 1 wherein the antibody is the anti-Thy 1.2 IgM.

5. The process of claim 1, wherein a compound of the formula P1-SH is reacted with a compound of the formula P2-S-S-X in accordance with the equation: P1SH + P2-S-S-X ? P1-S-S-P2 + XSH, in which when P1 is the radical of the A chain of ricin to which the free thiol group of this molecule is fixed, P2 is an immunoglobulin or an immunoglobulin fragment, or conversely when P1 is an immunoglobulin or an immunoglobulin fragment, P2 is the radical of the A chain of ricin, and X is an organic activator radical.

6. The process of claim 5, wherein P1 is an immuno-globulin or an immunoglobulin fragment into which a free thiol group is to be introduced, the recognition site of the said immunoglobulin or immunoglobulin fragment is first blocked by prior treatment with the antigen, or with another antigen exhibiting a cross-reaction, or with a hapten, the immunoglobulin or immunoglobulin fragment, protected in this way, then being reacted with .delta. -acetyl-mercaptosuccinic anhydride, the thiol groups are liberated by the action of hydroxylamine, and the recognition site is finally unblocked by removing the antigen or hapten.

7. The process of claim 5, wherein P2SH represents the A chain of ricin, and the latter is converted into the disulphide by the exchange reaction:
P2SH + X - S - S - X ? P2 - S - S - X + XSH

in which X represents an activator radical selected from the pyrid-2-yl or pyrid-4-yl group which is optionally substituted by one or more alkyl, halogen or carboxylic acid groups, or phenyl which is optionally substituted by one or more nitro or carboxylic acid groups, and the reaction is carried out with a large molar excess of the reagent XSSX, which is removed at the end of the reaction by dialysis or filtration on a molecular sieve in gel form.

8. The process of claim 6 or 7, wherein all the reactions are carried out at pH 7.0 in a phosphate buffer and at ambient temperature.

9. The process of claim 5, wherein P1SH represents the A chain of ricin and P2 represents the immunoglobulin or a fragment thereof, and P2 is converted into the activated disulphide in accordance with the equation:
P2 + Y - R - S - S - X ? P - R - S - S - X
in which Y represents a functional group which is capable of covalently bonding to any one of the functional groups carried by the side chains of the constituent aminoacids of the immunoglobulin, R represents a group -(CH2)n-, n being an integer from 2 to 10, or a group R3-CH-CH-R4 , R4 denoting a hydrogen atom or an alkyl group of 1 to 8 C atoms, and R3 denoting a substituent which is inert towards the reactants subsequently used, the reaction being carried out in the homogeneous liquid phase and at ambient temperature.

10. The process of claim 5, wherein after all the reactions are complete, the conjugate obtained is purified, in order to remove the unreacted compound P2-S-S-X therefrom.

11. The process of claim 10 wherein the unreacted compound is removed by fractional precipitation with a water-miscible organic solvent.

12. The process of claim 10 wherein the unreacted compound is removed by chromatography.

13. A cytotoxic product, consisting in compounds called "conjugates", wherein the A chain of ricin is coupled by a disulphide bridge to a protein structure which is an antibody, an immunoglobulin, or an immunoglobulin fragment, the protein structure being capable of selectively recognising an antigen presented at the surface of the carrier cells, whenever prepared by the process claims in claim 1, or by an obvious chemical equivalent thereof.

15. A product as claimed in claim 13, wherein the antibody is an anti-DNP antibody, whenever prepared by the process of claim 2, or by an obvious chemical equivalent thereof.

15. A product as claimed in claim 13, wherein the antibody is the F(ab)'2 fragment of the anti-DNP
antibody, whenever prepared by the process of claim 3, or by an obvious chemical equivalent thereof.

16. A product as claimed in claim 13, wherein the antibody is the anti-Thy 1.2 IgM, whenever prepared by the process of claim 4, or by an obvious chemical equivalent thereof.