Anti-cancer drugs containing the chain has ricine associated with an antibody antimelanomist and proceeded for their preparation.

In SAL (Sssaadosaatésieasfssn° 76, 27838<âaSogtssSs®1s7®28

and adding the n® 78 2C - G5& the I 2 October 1§7s&_I - 1% àumnâzsmsü has €lssit preparing the DS - psalmiteanticascéresui said ceajugoéfobtcmo RBM csaplage by binding c; the X ' how to alestechaâa® has " 3s the Klein® isâEaeïTO AAs

5 - protein such lo and SQT ' Pam antibodies? the mth. in transistor outline teætffiiagleSmllnafsaçso&T-

d.•hr " ttna© ginbniepable of reecaasaifer © caleefe&veaeKt "has" given the mylar jatlgse® © has mS bearing cells " not in will

such as the cancerous female pigs! "". Ls * property■main ' conjugates is&Te-® back agent * ^ tetesLqae specific cell®

10 intended targets *

. - Vtttil&e&tiæa antibody directed æoffifcreanfciffaos the OD differentiation Never © alluleg cancer had already criminal cbtesds " SWT tæaæsÆutela conjugates having specificity against eXfeiGS © of targets. The characterizing * astig&âeeâm ^ ied 'IDI - cells "sEséseese * 19' and the making aryl ^ astteorpsmmmMkbètigLfltt" other these eatiffcaeapernetteat D.⁹ ©®aims§3rd of cenjûgué * prieestant year®■spécifioifeS increased "facing of celtaicspartsueès of these" afciafci.fj&born "

■'■. i-& © pæéscsfciavsstieft *•providing one object as' uéiâeaEQ&T-© âss products called ceajatsls ^ " bbaaas to ' from M the chain of £, to the film of £<gielE "and 20 of an antibody Btsa®© lesaalaati" L®-9.ï§32s. ' teiaisi?

ôa hears FARs '" © ajeçttle' mS © ssléealas CD® © RSEs ertâfiLeâelâos e 00

which; the cbaîêâ & âo the ' SieSae. am Malssa - eœssiéa is pure. " sweats -VBE1 **

'd like to * - disulfide the m -antiesxpsesMaJlcaaîsfessai " © APs&®<the m e © seeeaasîfcssasélectivsasat U.S. antiçtsne. the ASA astasiéæils&bone "e" eâreus @ 0"

2 $'•; 6' to Ew " fifcieE ' how to eiaîaai, dressed with KLsâas & êabeen 'dâssiteêsias wears•patents' antériemrefe GBP-a-g-® épaïôtlea D.^Istâesaps® sesodLcffiaus: © sates of aSlanesiee 'fcœaiKs ·ο been sœtlcssé® donation "its literature I&îesfcifi ^ e transformer 4"' may be rsportesr " has particular λ 9s @ esc&the rg&εf. - EIT WEST&esa Isacte? OC. T-science? From WO 2183 * 7,

38 Providing one realize the L @ e ^ conjueés, ùù .aft aégeasaireqdè the çesâ&taea coupling carry ekæa " has at hand tsam&m da and an X % £ ^ aatarelliEE ATs TPN © ' or. artifieielleæcsnt rendering em aind er " creating the 'Maii ^ ^ sraieulf&DB srcgfelrfs © *, the' A chain Mr Heine present © naturelleasne its single - atom. sulfur for the desired coupling. C^! i§T-that of the thiol function of the re sidu.de cysteine included in the confectioneries and which connection which this chain the K to the chain ' b-isas complete toxin.

The antibody aatiaêlaasss is " die if fonctionthiol free, nor other sulfur atoms likely Other used for coupling. The change-amount of intraduire-to-artifieieilmsat on. the immunoglobulin molecule year bone more atoms' sulfur ultêrieurassent engageable in the disulfide bond to be set up with more soléeules © U-chain-Hein®.

■; according to 1' inventica, the preparation of the conjugate is

•performed by bringing together the chained confectioneries jth licise carrier - its free shush group with the antibody dated IsqaelLgroupment the shush was. artificially introduced sœss ee @ ms. natgESîgae is activated in the form of a disulfide® moiety with m ist, © rgaalque ℮℮ην β · aable=sulfur.

. The preparation USA coajogsé can ' be © represented

- by the scheme:

. THE RA "SHUSH + Α℮ DEGREES E" E " K ===E * ITS>."E=•■>CA - + KSE

wherein .:. Its. designates the extinguish to erffective powerU&Meine

'. The Ag denotes I ' antibody

· ·.. .1 ' denotes the radical activator.

. the R^;L⁸ antibody substituted with U®-atèafe-dA-activated sulfur. is obtained from the L - ' antibody-on parts Eêiaâj by substitution to have-the reagent lui°îala&carrier. a atsæg sulfur activated according to scheme S

The Ag " + 9.ï^B R°S^IS^{the O} 9.ï "=*)=e ac ° s ° e - 3 £degrees

wherein :. Ac stands '■the antibody

.. Yen vis a covalent fisetionpsrasttarst function has the reagent on the protein

. S denotes a group which can carry the substituents yen and■simuitanfeent " X-s °:

. 1' denotes the erase! activator.

Mfoaetiesrâèl group F. is a® function capable 'of s® bind dede.façon' a VEE any® covalent $êasputties functions not the side chains of the amino acids constituting the protein to substitute. Among them, the amine functions end lysyîe radicals contained in the protein are particularly indicated " without this case, there may particularly represent:

" carboxyl group which may bind to the aminëas functions of the protein in the presence of a coupling agent such as a carfesdtimideand in particular a water soluble derivative such as ethyl "! (d-iéthylamino-to-3 carbodimide propyD and 3,

- a carboxylic acid chloride that is capable of reacting directly with the amino functions for the acylating,

■ > AU ester of said "activated" such as an ester of para-a-j orthoou nitro° or dinitrobenzoic pfaényle or n-hydroxy ester ^. the succinimide which reacts directly with the amine functions for the acylating,

- the m internal anhydride of a dicarboxylic acid such as for example the anhydride soccinic which reacts spontaneously with the healthy functions create amide bonds,

u.S. group laiiàester

THE NH

where JE - is' a

, group-alkyl responsive ' with - 1®5 - amino groups the protein according .de.

the * reaction:

THE H?

IS.

TIG process -■"*

NR

' € - ir₂ The m ^ WWB - - C - R₂ WHEREIN R IS OH + ^

THE V ' ;.. -...

S denotes a -■*. array.' functional group capable of reacting with the m® ehiol free. In particular, X may designate a group pyï ' iàyî " 2 - M. ^ greatly accelerated the pyridyl optionally substituted with one-or

alkyio radicals, halogen, carboxylic. X may also indicated

T-•

a group phéayleâa preferably substituted with one or more groups

the nitro - or eartesyllcpes. X-AEP still represent a group such alcoxycarboayledeny Lg - - méthonyearbonyle group.

- The IQ 1 general appointed all residue group capable simaltenement - carry the I "th Ou&atâ&uaiîte Y and -8," - e - X * it should be selected so as to not have functions. that may interfere during subsequent reactions with the reagents used and the products. synthesized ".. in particular, - the group, group a may distributed® management data of TSA. " with n comprised eatre. 1 and 10, or even a group®

RG is - CH2 -. ". "..•'; /••

HM -••-•". '. -■

.•wherein. The R ^ desired, hydrogen or 'ICU' group. © has d-lltyi ©; the L ' L to 8 carbon atoms® and RG denotes m-substituent inert® against.

10 - - - of reagents used afterward '. such as a carbamate group

". The NH-a-a-c "gold. where R" denotes a group-âilïyle-right 'eu branched - it. 5 -, .;

0 /.•;

1 à -, 5 atoms of carbon.. and: - the notsmasesit ' greoape tertiarybutyl®. .. '•. .. '- - The' of the reaction. . compound. % °1 - 8 - b * x admission.

IVBE1 Immunoglobulin is performed. *: - stage micropump. homogeneous at once more;

15. often in. the: eaiu'eaiu' have-a-buffer solution. © forsqv VBE1 solubility of reagent-'l'exige, it is possible ;'d ' added saddles, reaction to .20% header-volume of one-solvent - water-miscible organic

- such as alcohol and -." in particular 1®tetaaol - tertiary.

- " The-reaction is performed-to-to-to-temperature&biante -

-20 during a•temple - varying - of some 24 hours. teprès confectioneries. After ' dock, a microdialysis eliminates' tees molded products of low mass * -.

the IREs; and in. particular. - the excess - dferéactifs.This preceded■- T-perm

. D.^{the R} introducing a. number, of groûpefiisats substituents by SOI®:

. protein included. between; 1 and 5 if■la.-protein is a iEsœsjnôgïofeuiine .25 of ' class g, ; between 1 and 15 to the Al. protein is sine Tm " ogio *^:

- buline. of class it

EDD•using, of - such - ceaposés, the eoupîegeaveé; ", the chain has louse is made by. comfortable its presence in aqueous solution of both protéides s at a temperature not exceeding 3' 0^I TheS•30 during a time variant'.-the - 'few'. hour® to one day. The solution. '. obtained is dialyzed " toto potir remove products of low molecular weight, then the 'conjugate, perhaps, purified' by various methods known. ;®.••

The next thing of esempletaisux include In * ^ 35æ veation without limiting its scope ". - -

• Example 1

Conjugate obtained from human antiraelanomist antibody (antibody directed against the P antigen 97) group substituted activated disulfide and the chain of ricin confectioneries.

a) ^ Anticorgsantimélanome_humain_ (anti_p_97).

This antibody was obtained using the method described in Proceedings of the national Academy science 1980, 77 (4), 2183-7.

It is the ultimate purification by dialysis against PBS (10 mm phosphate, 140 mm sodium chloride,

in pH: 7.4).

b) çhaîne_a_de_la_riçine

The ricin A was prepared and purified as well as it has been indicated in the previous requests Ta deman (Patent no. 78, 27838 and adding no. 79, 24655).

c) Anticorgs_an|ime1anomist humain_açtivé.

To 0.5 ml of a solution of 20 mg/ml of acid (pyridyi-to-2 disulfanyl) in tert-butanol -3 propionic, added 0.2 ml of a solution of 60 mg/ml. of éthyl L (diaéthylamino-to-3-propyl) carbodiimide and leaves -3 3 minutes at room temperature.

* ^ 30 jil.de added into the resulting solution to

1.66 ml of a solution of antibody to 2.4 mg/antimelanomist ml in the buffer. A PBS. NC leaves which incubation ' proceed for 20 hours.

f. èo °C. "?.>

, Then the solution is dialyzed in continuously during

3 days against 21 liters of PBS at 4^has C. and 4 mg of activated antibody has a concentration of 2.3 mg/ml..

, By assaying spectrophotooetric 343 nm to pyridine thionthion.e-a 2 released by exchange with reduced glutathione, it appears that having obtained a antibody bearing 2.6 ITAC groups' vateurs per mole of antibody.

d) conjugate.¹

With 1.3 ml of a solution of activated antibody in PBS (concentration of 2.3 mg/ml., either 3 mg of activated antibody), added 0.52 ml of a solution of ricin A chain in the same buffer (5.8 mg/ml of concentration of) ^ and incubated at 25®C for 20 hours ".

0a chromatography fetch the reaction mixture.

' gel column Sêphadex 100 grams. Mainly in each fraction, is held® the concentration in antibody by spgetrophetometry confectioneries 280 the TM &K. that the chain has by its inhibitory potency of the aeeliuiaireprotéosynthèsê measured on a system. Bone joins the identical moieties containing the conjugate and this provides about 1®g of the conjugate to the concentration of 0.2 RAGs/ai.

Analytical determinations performed allows⁴ * or emit show that the solution contains 50^gjnl/teakwood biolo chain has " giquèment activates, either about 1.4 seconds @ Is of chain has per mole of £I-d-® A.A=body.

-•a study conducted by eytofluoroaétrie Ea another show that the antibody antimilaneas. used, 1¹ antibody•corresponding activated and the conjugate of this. antibody with the chain. to of ricin have fluorescence histograms for very wising■affirm that - the antibody, has' has undergone no altering important during 'activating' réactionsd of and coupling to which it has been subjected and in particular that it remains capable, within the same conjugate, recognize the P antigen 97 against which it is directed.

. The conjugate, as ' 1." disclosure, obtained was a-étudiéétudié.en■précéderMsnt what concerned ' biological properties and plug specially its coaction anticancéireuse.

1) - Inhibition of nrôtéosynthêse

•the property. the chained■fundamental biological L ricin is inhibiting the keynote cell by altering the ribosomal subunit 60 sec.'

, - Was used here the m © odële CE llulaire. PK leg

measuring the effect substances studied on 1¹¹ ion is incorporated this

14

The c-lectin in EA-culturing cancer cells.

⁴ Cells used belong to the m-cell lined from oligomers® * 1477 uq■human melanoma which carries Hepatitis F.§?. these cells, after trypsiaation, are préineubéeshoisresrsoins at 24 to allow rêexpression surface antigens are optional "* lemant altered. After adding the ' substance to be examined, year the SFF. a catalyst © the immune. At the end of incubation, is carried out - iII assured rate incorporation.de ^ C leucine per the treated cells.

This measurement is performed by a technique

adapted the technique described in antitumor efficacy

14

1974, 249 (11), 3557 - 62 using the tracer C. leucine-for determining the level of keynote. The determination of incorporated radioactivity is performed here on the whole cells isolated by filtration,

From these determinations, one can draw the

dose/effect curves plotted by having the concentration of

14

materials investigated and ordered the incorporation of c-leucine is expressed as a percentage of the incorporation control cells in the absence of the substance to be analyzed.

This allows for each substance of interest which is the concentration which inhibits 50% of the incorporation of ^ c leucine or "inhibitory concentration 50" (IC 50).

The results obtained with the conjugate prepared in the previous example (ITHM) are represented by Figure 1. On the character, are also represented the curves obtained respectively with the ricin (R.), the chain has ri NICE (ARs) and a conjugate ricin A chain radical containing anti - dinitrophenyl (NFB)

(SD 10), conjugate which does not have any affinity for the assayed cells '.

It is appreciated that the conjugate on this fig.

" 9 studied (ITHM) has a strong cytotoxic activity (IC 50 * 5x10 meters), about 400 times greater than that of 1 ¾ ^ ricin A chain.

, Furthermore, the DNP antibody conjugate (SD 10) has not effect on the m cell 1477 up to the highest concentration

tested (10"⁶ M). However, this same conjugate in DS - 10 appears cytoenzymology

-9

toxic with an IC 50 10 m to close when contacted with the same m cell 1477 previously made artificially oxidoreductase " teuses radical DPN. The latter two findings demonstrate the ' special character of the cytotoxic activity of the conjugate ITHM, * 2) - Inhibition of colony formation. *

The cultured cells melanoma m in 1477 are peeled from their support by the Versene solution (PBS containing L * ethylenediaminetetraacetic acid) or by trypsirisation. These cells are seeded ^ ^ 2x10 proportion of cells per plate of 5 cm diameter containing the following culture medium:

'*

" I-a 640 uSffietnc EMI medium)."&àâitiàmâ thru " me from glut&' Z- u-&, cIO&%~

- . bonate sodium 2 g/i, 15% of séruafessai inactivated calf

-, ' (Seromad) and antibiotics (penicillins, - b-amphoterieittê. and streptomycin).

24 Hours tackle, the - eêiiiaiessêat processed ATs?®®varied® concentrations® of the conjugate to be studied.

as the 4th confectioneries eeaparsiseasfèsss d series " experiments is carried out with îslieise,th" BS INSERM_O " i ^® thence Chains 'has 4®.'. ricin, 'd ' other parê, finally with a T @ the m conjugate ' PAA specific for this cell line (conjugate of efea&AEs to of ss.ieia® and in Asti<SU=

the anti-DNP - body - (SD 10)).. ■ ■

Tackles again 24 hours, the culture medium.

is removed and replaced by fresh medium 1®IEA ©, free 4th cytotoxic substance "_

'._; 10 th 15 j of mobile homes - more gsrd_has the ls® ATEs @ ' 4®eeicéies that have developed. is déteminé tackle " © leratic® with only solution of crystal violet using ', d'un. 6 №ρ℮℮κ ¾ nntcsatic ' of œloaiea.

(Artek system 880)...

This, œëtbpdô. allows. the detection 4^I the® amount also-fault, as the natural © 10 cells. © a viable, by: I5 is®&e e and that. this ee.p® be verified. in using. 4 e- f®attêls cultures. 11 a been equal "'also proven' that the forming die eoterlgè © ac-to-strictly ptopôr" - tionnesy the K the. concentration-to-'a-'initiale of eolfelesy - ^ P minus in the 10 to 10 ^ ^ limitede bs cells " '. '•

'.' */ -. The results " obtained sæfi presented. in Figure. - 2, upon which © '* carried-ordered © 6 ©-to-eoordotœéas logarithmic, the number of colony -' by moth © 6 in abscissa the concentration of the ". product. Experiments have been carried out to the Rieine (R.) - s r efeSae β of the lice ' (ARs)- ls GSE-to-âuâu.it stored characteristics marital. salojt 1' disclosure (ïflF). '

t-®' conjugate Xï® a.m. has high activity&yj-PALs

. - the ' last melanoma cell is tee © th aces.<; concentrating confectioneries svirea ^ '=9

•2 îflO m in conjugate. This concentration is well knowncesparèMe "* gm

à .than, the lice LC 10 sec. ii) © lornqcedaaSaeSieiæ 4s to fear the

the II must reach 10 "® of £ii to obtain the seated dffsfc.

May also be noted that the conjugate the DS 10 did

8•

any activity up to 2 x 10 m the rained high concentration to which it has been tested.

Inferences from this experiment confirm fully those of the inhibition experiment of keynote.

The conjugates prepared according to the invention may therefore a high specificity of action through to human melanoma cell lines. They can therefore be used in human therapy in the treatment of melanomas and possibly other conditions, cancerous or not, that would be responsive to the antibody used.

These conjugates are conditioned for use by injection. They may be used either alone or in combination with another treatment of cancerous condition concerned and; in particular, associated with other immunosuppressant drugs to delay and thence the patient's natural immune response against the foreign protein to its organism that is the conjugate.

To remove all of the cancer cells, the treatment should be carried out with a sufficient dose of codjugué and treatment time will have to be determined in each case depending on the subject and the nature of the disorder to be treated...