GB2137210A

GB2137210A - Immunoglobulin conjugates

Info

Publication number: GB2137210A
Application number: GB08407766A
Authority: GB
Inventors: George Joseph Cullinan; George Ferdinand Rowland; Robin George Simmonds
Original assignee: Lilly Industries Ltd
Current assignee: Lilly Industries Ltd
Priority date: 1983-03-30
Filing date: 1984-03-26
Publication date: 1984-10-03
Also published as: NZ207675A; EP0121388A3; HU191647B; SU1362404A3; DK167884A; DE3482444D1; IE840772L; EP0121388B1; PL144887B1; AU570963B2; BG42837A3; FI841260A; KR840008286A; PT78322B; PL246931A1; FI841260A0; ATE53496T1; EG16175A; GR79899B; PT78322A

Abstract

A conjugate comprising a vinca moiety convalently linked at the 4-position via a group of the formula -OCOXCO- where X represents a single chemical bond or an optionally substituted C<sub>1-10</sub> chain, to an immunoglobulin or an immunoglobulin fragment.The conjugates are useful in the treatment of cancer.

Description

1 GB 2 137 210 A 1

SPECiFICATION lmmun.oglobulin Conjugates

This invention relates to novel immunoglobulin conjugates with pharmaceutical properties and, in particular, cytostatic activity.

A group of compounds which are cytostatic and widely employed in the treatment of cancer are, the vinca (indole-dihydroindole) alkaloids and drugs derived from them, The che moth 6 tape'utic use of these drugs is sometimes limited.id its effectiveness by unwanted side effects on the phtient and efforts to minimise such effects have been the subject of much res ' earch. For example, Britih Patent 2 090 837 describes a conjugate of vindesine, one of the vinba alkaloid.drugs referred to above, with an immunoglobulin, in which the vindesine moiety is linked directly at the -position of the vindesine 10 molecule. When the immunoglobulin is an antibody the conjugate concentrates at the site of action with a reduction in toxic effect on other tissues and a potential reduction of undesi rable gide-effects in the patient.

The invention provides a novel conjugate comprising a vinca moiety covalently linked at the 4- position via a group of the formula -OCOXCOwhere X represents a single chemical bon i doran 15 optionally substituted C,-,, chain,-to an immunoglobulin or an immunoglobulin fragment. The novel conjugate is a derivative of a vinca, such as for example, a 4-acetoxy or 4-hydroxy antineoplastic dimeric indole-dihydroindole alkaloid and can have one or more such vinca moieties covalently attached to it.

The immunogiobulin or fragment is an antibody or afragment'of an antibody with antigen 20 recognising properties. The preferred immunoglobulin material is an antibody or a fragment of an antibody adapted for recognition of antigens on the surface of unwanted cells of the type occurring in the human body. However immunoglobulin materials of other kinds are also included within the scope of the invention since they may be of use in treatment of animals and in control and assay experiments.

The novel conjugates are useful, inter alia, in the treatment. of.cancers. They are potentially more 25 effective and have fewer side effects by virtue, of their ability to increase the concentration of the cytotoxic drug at the site of action. The linkage of the drug moiety via the "spacer" group ---OCOXCO-frequently enables a greater drug concentration on the immunoglobulin to be produced, and can thus increase the efficacy of the conjugate. The invention also includes conjugates for use in an indirect system in which they are employed to recognise an antibody specific to the cell surface 30 antigen.

More particularly, the conjugates of the invention can be represented by the following formula a# 51 g17- 1 3L 1 7MC 9 0 H A 1 (I) OCOXCOI9 R H 0 A1 2 GB 2 137 210 A in which 1. represents an immunoglobulin or an immunoglobulin fragment and X is as defined above; and in which R' is COOH, COOC,-, alkyl or CO- RI, where R' is NH,, NI-I-C,-, alky], M-CH,CH2C1, 1 -pyrrolidyl, 1 - piperidinyl or NH-CH2CH2YCH3 where Y is S or 0; R 2 is H, CH3 or CHO; and when h' and R' are taken singly, R' is H, and one of R3 and R' is ethyl and the other is H or OH; and when R' and R' are taken together with the carbons to which they are attached, they form an oxirane ring, and R 3 is ethyl. The im m unoglobulin, or fragment, can be modified by one or more of the vinca residues shown.

Some of the well known vinea alkaloids from which the above conjugates are derived have, for example, the vinca moiety of formula (1) above in which the 4-position is variously substituted as, for example, vinblastine in which R' is COOCH3, R 2 is methyl, R' is hydroxyl, R 4 is ethyl, R' is hydrogen and the substituent at the 4-position is acetoxy; vindesine in which R' is CONH2, R 2 is methyl, R 3 is 10 hydroxyl, R 4 is ethyl, R' is hydrogen and the substituent at the 4- position is -OH; vincristine in which R' is COOCH3, R' is formyi, R' is hydroxyl, R' is ethyl, R5 is hydrogen, and the substituent at the 4position is acetoxy; leurosidine in which R' is COOCH3, R' is methyl, R' is ethyl, R 4 is hydroxyl, R' is hydrogen and the substituent at the 4- position is acetoxy; VLB -A(4'-deoxyvinblastine) in which R' is COOCH3, R 2 is methyl, R' and R' are hydrogen, R 4 is ethyl and the substituent at the 4-position is acetoxy; VLB "B" (4'-deoxyleurosid!ne) in which R' is COOCI-l., R' is methyl, R 3 is ethyl, R4 is hydrogen, R' is hydrogen and the substituent at the 4-position is acetoxy; leurosine in which R' is COOCH3, R' is methyl, R' is ethyl, R4 and R' taken together form an a-epoxide (oxirane) ring and the substituent at the 4- position is acetoxy; and leuroformine (N-formyieurosine) in which R' is COOCH3, R' is formyi, R' is ethyl, R 4 and R' taken together form an a-epoxide ring and the substituent in the 4-position is acetoxy. 20 Literature references to the parent alkaloids of the 4-desacetyl derivatives are as follows:

leurosine (U.S. Patent No. 3,370,057), VLB (U.S. Patent No. 3,097,137), leurosidine (vinrosidine) and leurocristine (vincristine) (both U.S. Patent No. 3,205,220), desmethyl VLB (U.S. Patent No.

3,354,163), vindesine and other 3-carboxamides (U.S. Patent No. 4,203, 898), vinblastinoic acid, vincristinoic acid, etc. (U.S. Patent No. 4,012,390), 4'-epivincrist!ne (U.S. Patent No. 4,143,041) leuroformine, formylleurosine (U.S. Patent No. 4,279,816), and deoxy W13 'W' and "B" [Tetrahedron Letters, 783 (1958)1. Other vinca alkaloids are disclosed in US Patents 4, 166,810 and RE 30,560.

The group X represents a bond or an optionally substituted C,-,, chain and is ultimately derived from the corresponding dicarboxylic acid HOOCXCOOH. The group is preferably C,-, straight chain alkylene, C,-, branched alkylene, C2-,alkenylene, C3-,aWynylene, C3- 6CYCloa 1 kyle ne, phenylene, 30 hydroxy-substituted C,_,alkylene or a direct bond.

Groups illustrative of X include methylene, ethylene, propylene, butylene, vinyl, propenylene, butenylene, butynylene, ethynylene, hydroxyethylene, 1,2- dihydroxyethylene, 1,2-dimethyl ethylene, 1,2,3,4-tetrahydroxybutylene, 3,4-di methyl butylene, 1,4-cyclohexylene, 1,4-phenylene, 1,2-phenylene and the like. Preferably X is C,_,alkylene, Cl-4alkenylene, C2-,aWynylene, C3-6CYCloa [kyle ne or 35 phenylene and is especially Cl-,alkylene.

Immunoglobulins specific to antigens on the surface of cells to be killed, and techniques for their production from the serum of immunised animals or by culturing hybridomas secreting monocional products, are well known. The preferred type of antibody for use in the invention is an immunoglobulin of the lgC class. Some representative immunoglobulins areas follows, monoor polyclonal antibodies 40 to (i) human or animal tumour associated antigens (ii) human B- and T-cell antigens (iii) human]a antigens (iv) vital, fungal and bacterial antigens (v) cells involved in human inflammatory or allergic reactions mentioned:

0) - 50 (ii) (iii) 0v) (v) (vi) (vii) (viii) Of the preferred antibodies to human or animal tumour associated antigens there may be 19 from goats or sheep immunised with carcinoembryonic antigen lg from rabbit antiacute lymphoblastic leukemia serum lg from various primate antisera raised against acute lymphoblastic leukemia, acute myieoblastic leukemia, chronic lymphoblastic leukemia and chronic granulocytic leukemia ]g from goats or sheep immunised with lung carcinoma material monocional lg from mouse hydribomas secreting anti-human colorectal carcinoma antibodies monocional lg from mouse hydridomas secreting anti-human rnelanoma antibodies monoclonal lg from mouse hybridomas secreting antibodies reacting with human leukemia cells monoclonal]g from mouse hybridomas secreting antibodies reacting with human neuroblastoma cells (ix) monoclonal lg from mouse hybridomas secreting antibodies reacting with human breast cancer antigens (x) monoclonal lg from mouse hybridomas secreting antibodies reacting with human ovarian carcinoma cells 3 GB 2 137 210 A 3 (xi) monoclonal lg from mouse hybridomas secreting antibodies reacting with human osteosarcoma cells (xii) monoclonal lg from mouse hybridomas secreting antibodies to lung carcinoma.

As indicated above, the conjugate can also be made with immunoglobulin fragments, referred to as Fab, FaW or Rab% or lgM monomer derived from an antibody by, for example, proteolytic enzyme digestion. Such materials and methods of preparation are well known and it may be mentioned that preferred proteolytic enzymes are pepsin and papain.

Preferred conjugates of the invention are those of formula (1) above in which R' is COOMe or CONH2, R 2 is methyl or formyl, R3 is hydroxyl, R' is ethyl and Rr' is hydrogen, notably those derived from desacetylvinblastine and vindesine. Preferably the hydrophilic group X takes the value Cl-, alkylene 10 and lg is preferably a monoclonal antibody to human or animal tumour antigen.

The conjugates of the invention can be prepared by reacting an immunoglobulin or an immunoglobulin fragment with a hemi-acid derivative comprising a vinca moiety having a group of the formula -OCOXCOZ attached at the 4-position, where X represents a bond or an optionally substituted C,-,, chain and Z is a leaving group.

More particularly the process of the invention comprises reacting an immunoglobulin or immunoglobulin fragment with a compound of the formula is-# 4? 40 H 1 0 1 A a A A A (II) CH3 CHni 0COXICICIZ R R in which X, W, R', R 3, R 4 and R 5 have the values given for these radicals in formula (1) above and Z is a leaving group.

In the above formula (11) Z is a leaving group which can be any of the well known groups employed in peptide chemistry to establish a covalent amide link (-CONI-1- ) between the conjugate and a free amino group on the immunoglobulin molecule or fragment. Such groups are well known in the art and are discussed for example, in Peptide Synthesis by M. Bodanszky, Y. S. Klausner and M. A.

Ondetti, Second Edition (1976) John Wiley & Sons, notably pages 85 to 136. It may be mentioned 25 that Z can be an azide (-N,) group, a halogen atom for example bromine and especially chlorine, an acyloxy group of the formula WC0.0 where R' is an aliphatic or aromatic residue such as for example Cl-, alky], an alkoxy group preferably Cl-, alkoxy or an aryloxy group, a methanesulphonyloxy, tosyloxy or benzenesulphonyloxy group, an imidazolyl radical or the residue of an N-acyl hydroxyla mine derivative, for example where Z is succinimidoxy, phthalimidoxy or benzotriazolyloxy. Preferred 30 examples are those compounds in which Z is the residue of an N- acylhydroxyla mine for instance the N hydroxysuccinimide esters prepared by use of 1 -cyclohexyl-3-(2- morpholinoethyi)-carbodiimide metho-p-toluenesulphonate or 1,3-dicyclohexyi-carbodlimide, or via a mixed anhydride such as that 4 GB 2 137 210 A obtained by using isobutyl chloroformate. When Z is an imidazoly] radical it can be prepared by use of carboxyl di-imidazole and when Z is R'CO.0, where R 'S Cl-3alkyl especially ethyl and isobutyl, it can be prepared by use of 1 -ethoxycarbonyi-2-ethoxy-1,2-dihydroquinoline and isobutyl chloroformate, respectively.

Compounds of formula (11) are prepared by reacting the corresponding free acid in which Z in the 5 above formula (11) is OH, with the appropriate activating molecule, in an inert solvent such as for example dimethylformamide and preferably at a temperature of from -200C to 750C. The free acids (Z is OH) are prepared according to the following methods:

First the appropriate 4-desacetyl indole-dihydroindole is prepared by the procedure of, for example, Hargrove (United States Patent 3,392,173).

The 4-desacetyl compound is then acylated with a carboxylic acid anhydride of the formula in which X has the values given above. Alternatively, an acylating group of the formula Z1COXCOZ2 in which Z1 is an activating group and Z2 is a carboxy protecting group, can be used, and the carboxy protecting group removed to yield the compound of formula (11) in which Z is OH. Such compounds are 15 then converted to the activated compound by reaction with the appropriate activating molecule.

When an indole-dihydroindole is to be reacted with succinic anhydride or the like to prepare a compound of formula (11) wherein R' is COOH, the C3 ester group as well as the C-4 ester group must be hydrolyzed initially to yield, for example from VLB, a 4-desacetyl vinblastinoic acid-see United States Patent 4,012,390. Next, the C-3 carboxyl group must be protected with a carboxy protecting group as defined above. This C-2 carboxy protected derivative having a free hydroxyl at C-4 is then reacted as above with an anhydride. The resulting compound can then be manipulated chemically to yield compounds according to (11) in which Z is an activating moiety provided reaction conditions are neutral or basic, thus avoiding removal of the C-3 carboxy protecting group. After the desired terminal group, Z, is in place, the carboxy protecting group at C-3 can be removed to yield compounds according to (11) in which R' is COOK When X in the compouri.d to be prepared is hydroxy dihydroxy or tetrahydroxy C'1-4alkylene; i.e., a linking group derived from malic, tartaric acid or saccharic acid, it is necessary in preparing the compounds to protect the hydroxy or hydroxyls with a protecting group such as a pyranyl group. A trialkylsily] group such as a trimethylsilyl group can also be used. In the instance of a vicinal dihydroxy 30 compound such as tartaric acid, an acetal; i.e., an isopropylidene or cyclohyeoxylidene derivative, can be used.

When X is a direct bond (the linking group is formed from oxalic acid), oxalylchloride cannot be used since the hemioxalate may cyclize with the 3-hydroxy]. However, an oxalate half ester or an oxalic acid derivative of the formula Cf-CO-CO-V can be used and the ester hydrolyzed or the carboxy 35 protecting group removed during the simultaneous conversion to an activated moiety, R-CO-CO Z1.

Reaction of immunoglobulin or immunoglobulin fragment with compound of formula (11) is preferably carried out in an aqueous med.i.um and at a temperature of from 51C to 251C, for example at room temperature, and at a pH of 7.5 to 9.5, preferably 8.0 to 9.0. The process results in the attachment by covalent linkage of one or more vinca residues at the free amino groups of the immunoglobulin molecule, for example, amino groups derived from lysine residues. The number of residues attached will depend on the concentration of the reactants and the duration of the reaction but the average number is usually for examplefrom 1 or 3 to 14 or 20.

For example in carrying out the reaction, a solution of the compourid of formula (11) in a suitable 45 solvent such as dimethylformamide is slowly added dropwise to 'a buffered solution of immunoglobulin in for example 0.34 M borate buffer at pH 8.6.The conjugate is isolated by gel filtration and stored in saturated ammonium sulphate solution being-readily brought back into solution by dialysis with a buffer solution for example a phosphate buffered saline pH 7.4, or alternatively it can be stored in a refrigerator at 40C or frozen at for example -200C.

Evaluation of the conjugate can be carried out using well known techniques such as affinity chromotography. The efficacy of the conjugate can be estimated by counting the number of viable cells after treatment of a suspension of tumour cells with the conjugate, or from measurements of the uptake of tritiated uridine. Protein and drug concentrations are determined by measuring optical densities of conjugate solutions at two wavelengths, for example 270 and 280 rim, and relating the values obtained to those for the free drug and unconjugated immunoglobulin at the same two wavelengths.

GB 2 137 210 A 5 The novel conjugates of the invention are useful in the treatment of cancers and as such are preferably prepared for use in formulations suitable for injection. Thus the invention includes a pharmaceutical formulation, for example an injectable preparation, comprising a conjugate of the invention together with a pharmaceutically-acceptable carrier or diluent such as are well known in the art. It is preferably in unit dosage form each dosage containing for example from 0.0 1 to 2 mg of the active ingredient (in terms of the vinca drug moiety).

The novel conjugates are effective over a wide dosage range and for example for the treatment of adult humans dosages per week will normally fall within the range of 1 to 10 mg/kg (vinca drug moiety) m ore usually in the range of from 3 to 9 mg/kg. However it will be understood that the amount 10. of compound actually administered will be determined by a physician in the light of the relevant 10 circumstances, including the condition to be treated and the chosen route of administration.

The invention is illustrated by the following Preparations and Examples.

Preparation 1 4-Suceinoyi-Desacetylvinblastine Two g of 4-desacetylvinblastine were dissolved in pyridine to which solution were added 2 g of 15 succinic anhydride. The reaction mixture was stirred at ambient temperature for 5 hours. (Temperatures in the range 0-501C may be used for this reaction.) The volatile constituents were removed by evaporation in vacuo and the residue taken up in CH2C12. The CH2C12 layer was washed with 5V6 aqueous sodium bicarbonate, and then with water. The organic layer was dried and the solvent removed therefrom in vacuo to give 4-succinoyi- desacetylvinblastine.

The above procedure was used to prepare the following 4-succinoylvindesine 4-succinoyi-desacetylvincristine 4-succinoyl-4'epideoxydesacetylvinblastine A similar procedure was used to prepare 4-glutaroyl desacetylvinblastine using glutaric anhydride 25 in place of succinic anhydride.

In any of the above acylations, any incidental acylation of the 3-OH group can be reversed by treatment with wet silica gel, according to the procedure of Hargrove, United States Patent 3,392,173.

Alternatively, the compounds can be purified from any 3-acyl derivative or other by-products of the reaction by chromatography, conveniently over silica gel-with an ethyl acetate/methanol solvent 30 mixture as the eluant.

Preparation 2 Activated 4-Succinoy]-Desacetylvinblastine One gram of 4-suceinoyi-vindesine was mixed with 380 mg of Nmethyl morph oil ne in 20 mi of methylene dichloride, and 390 mg of isobutyl chloroformate were added. The reaction mixture was 35 stirred at about 01C. under a nitrogen atmosphere for about 45 minutes. 795 mg of N hydroxysuccinimide were added and the reaction mixture heated at reflux temperature under N, with stirring for about 45 minutes. The reaction mixture was cooled and the cooled mixture washed with deionized water and then dried immediately with Na,SO, The drying agent was separated by filtration and the filtrate evaporated to dryness in vacuo.

EXAMPLE 1 Vindesine-Succinoyl-Rabbit Anti-Mouse 19G Conjugate To a solution of 4-succinoyl-vindesine (25 mg) in dry dimethylformamide (DMF) (0.4 mi) was added with stirring 0.3 mi of an 11.4 mg/ml solution of N-hydroxysuccinimide in dry DIVIF, followed by 0.3 mi of a 41.7 mg/ml solution of 1 -cyclohexyl-3(2-morpholinoethyl)- carbodiimide metho-ptoluenesulphonate in dry DIVIF. The mixture was kept at room temperature in the dark for 48 hours giving a 25 mg/mi solution of 4-succinoyi-vindesine Whydroxysuccinimide ester.

pi of the above solution was added with rapid stirring to 0.9 m] of a 10. 7 mg/mi solution of rabbit anti-mouse 19G in 0.34M borate buffer pH 8.6. The mixture was stirred at room temperature for 5.5 hours and the product isolated by gel filtration on a 1 x27 em (21 mi) column of Bio-Gel P-6 equilibrated with phosphate buffered saline. The excluded peak was collected (4.9 ml) and assayed for protein and vindesine by spectroscopy at 270 and 280 nm. The conjugate so prepared contained 7.9 moles vindesine per mole of lg.

EXAM P LE 2 Vindesine-Succinoyl-Mouse-MonocionaI Anti-Osteogenic Carcinoma Conjugate 300 AI of a 28 mg/mi solution of 4-succinoyl-vindesine Whydroxysuccinimide ester in dry DIVIF, prepared in a similar manner to that described in Example 1, was added with rapid stirring to 1.5 mi of a 19.8 mg/mi solution of mouse-monoclonal anti-osteogenic sarcoma in 0. 34M borate buffer pH 8.6.

The mixture was stirred at room temperature for 4 hours, then clarified by centrifugation and the product isolated by gel filtration of the supernatant on a 1.5x26.5 em (46.8 mi) column of Bio-Gel P-6, 60 equilibrated with phosphate buffered saline. The excluded peak was collected (5.52 mi) and assayed 6 GB 2 137 210 A 6 for vindesine and protein by spectroscopy at 270 and 280 nm. The conjugate so prepared contained 8.7 moles vindesine per mole of lg.

EXAMPLE 3

Vindesine-Succinoyi-Mouse-MonocionaI Anti-Melanoma Antigen Conjugate 200 A] of a 20 mg/mi solution of 4-suceinoyi-vindesine Whydroxysuccinimide ester in dry DIVIF, prepared in a similar manner to that described in Example 1, was added with rapid stirring to 1.0 m] of a 21.2 mg/mi solution of mouse-monoclonal anti-melanoma antigen in 0.34M borate buffer pH 8.6.

The mixture was stirred at room temperature for 4 hours and the product isolated by gel filtration on a 1.5x26 em (45.9 mi) column of Bio-Gel P-6 equilibrated with phosphate buffered saline. The excluded peak was collected (12.31 m]) and assayed for vindesine and protein by spectroscopy at 270 and 280 10 nm. The conjugate so prepared contained 9.1 moles vindesine per mole of 19.

EXAMPLE 4 Vi ndesi n e-S u cei noyi-M ouse-Mo noel ona I-Anti-Ca rci noem bryon!c-Antig en Conjugate 1,0 mi of a 22 mg/mi solution of 4-suceinoyi-vindesine Whydroxysuccinimide ester in dry DIVIF, prepared in a similar manner to that described in Example 1, was added with rapid stirring to 7.0 mi of is a 21.4 mg/m] solution of mouse-monoclonal anti-carcinoembryonic antigen in 0.34M borate buffer pH 8.6. The reaction was stirred at room temperature for 4 hours, then diluted with 3.5 mi of 0.7M sodium chloride/0.05M phosphate buffer pH 7.4 and the product isolated by gel filtration on a 3.2x24.4 em (196 mi) column of Bio-Gel P-6 equilibrated with phosphate buffered saline. The excluded peak was collected (28.24 mi) and assayed for vindesine and protein by spectroscopy at 270 and 280 nm. The 20 conjugate so prepared contained 5.8 moles vindesine per mole of lg.

EXAMPLE 5

Desacetylvinblastine-Succinoyi-Rabbit Anti-Mouse 19 Conjugate To a solution of 4-succinoyi-desacetylvinblastine (18 mg) in dry DIVIF (0. 4 mi) was added with stirring 0.3 mi of a 7.97 mg/mi solution of Whydroxysuccinimide in dry DIVIF, followed by 0.3 m] of a 25 14.4 mg/m] solution of 1,3-dicyclohexylcarbodiimide in dry DIVIF The mixture was kept at room temperature in the dark for 19 hours giving an 18 mg/m] solution of 4- succinoyi-desacetylvinblastine Whydroxysuccinimide ester.

AI of the above solution was added with rapid stirring to 1.8 m] of an 8. 02 mg/mi solution of rabbit anti-mouse lg in 0.34M borate buffer pH 8.6. The mixture was stirred at room temperature for 30 3.5 hours, then clarified by centrifugation and the product isolated from the supernatant by gel filtration on a 1.5x25 em (44 mO column of Bio-Gel P-6 equilibrated with phosphate buffered saline.

The excluded peak was collected (6.47 mi) and assayed for desacetylvinblastine and protein by spectroscopy at 270 and 280 nm. The conjugate so prepared contained 9.0 moles desacetylvinblastine per mole of lg.

EXAMPLE 6

Desacetylvincristine-Succinoy]-Mouse-MonocionaI Anti-Melanoma Antigen Conjugate To a solution of 4-suceinoyi-desacetylvincristine (10 mg) in dry DMF (0.1 m]) was added with stirring 0.2 mi of a 6.5 mg/mi solution of Whydroxysuccinimide in dry DIVIF, followed by 0.2 mi of a 24.0 mg/mi solution of 1 -cyclohexyi-3-(2-morpholinoethyi)-carbodiimide metho-p-toluenesuiphonate 40 in dry DMF. The mixture was kept at room temperature in the dark for 65 hours giving a 20 mg/mi solution of 4-succinoyi-desacetylvincristine Whydroxysuccinimide ester.

p] of the above solution was added with rapid stirring to 1.0 mi of a 22. 7 mg/mi solution of mouse-monoclonal anti-melanoma antigen in 0.34M borate buffer pH 8.6. The mixture was stirred at room temperature for 4 hours and the product isolated by gel filtration on a 3.2x24.5 em (197 m]) 45 column of Bio-Gel P-6 equilibrated with phosphate buffered saline. The excluded peak was collected (14.16 mO and assayed for protein and desacetylvineristine by spectroscopy at 270 and 280 nm. The conjugate so prepared contained 14.3 moles of -desacetylvincristine per mole of lg.

EXAMPLE 7

Desacetylvinblastine-Succinoy]-Mouse Monoclonal Anti-Lung Adenocarcinoma Conjugate 350 AI of 14.7 mg/mi solution of 4-suceinoyi-desacetylvinblastine Whydroxysuccinimide ester, in DMF was added with rapid stirring to 2.0 m] of a 20.0 mg/mi solution of mouse monoclonal anti-lung small cell carcinoma antibody in 0.34 M borate buffer pH 8.6. After stirring at room temperature for 4 hours the reaction mixture was adjusted to pH 7.4 using 1 N HCI and clarified by centrifugation. The product was isolated by gel filtration on a 2.0x22.0 em (67.0 m]) column of Bio-Gel ' P-6 equilibrated 55 with phosphate buffered saline. The excluded peak was collected (9.7 ml) and assayed for desacetylvinblastine and protein by spectrometry at 270 and 280 nm. The conjugate so prepared contained 7.5 moles desacetylvinblastine per mole of]g.

In a similar manner were prepared using the appropriate isolated Whydroxysuccinimide esters:

vindesine-succinoyl-mouse monoclonal anti-carcinoembryonic antigen conjugate, 7 GB 2 137 210 A 7 conjugate.

vindesinesuccinoyi-mouse monoclonal anti-lung adenocarcinoma conjugate, desacetylvinblastine-glutaroyi-mouse monoclonal ahti-carcinoembryonic antigen conjugate, 4'-epideoxy desacetylvinblastine-suceinoyi-mouse monoclonal anti-carcinoembryonic antigen EXAMPLE 8

A preparation of cells growing in culture was dispensed into microtitre tubes at a level of 105 cells per tube. After concentrating the cells by centrifugation, 0.2 mi aliquots of various concentrations of conjugate preparations were added. The cells were resuspended, incubated at 37 'C for 30 minutes, then centrifuged and the supernatant removed. The cells were then suspended in tissue culture medium and dispensed into microtitre culture trays at a level of 101 cells per well. After six days in 10 culture the number of cells present in each well was determined and compared with a cell preparation treated with phosphate buffered saline alone.

In one experiment, human melanoma cells were treated with either vindesine-succinoyl-mousemonoclonal anti-melanoma antigen conjugate (Example 3) or desacetylvincristine-succinoyi-mousemonoclonal antimelanoma antigen conjugate (Example 6) and the effects on cell growth determined, 15 with the following results:

Cell Growth as Conjugated Drug Percentage of Phosphate Concentration, Buffered Saline Treated Conjugated Drug AgImi Control Cells 20 Vindesine 50 16.0 5.0 61.8 0.5 84.2 Desacetylvincristine 50 21.5 5.0 74.5 25 0.5 100.0 In another experiment, human colo-rectal carcinoma cells were treated with vindesine-succinoyl mouse-monoclonal anti-carcinoembryonic antigen conjugate (Example 4), with the following results:

Cell Growth as % of Phosphate Conjugated Drug Buffered Saline Treated 30 Concentration, Ag/mi Control Cells 3.2 39.9 89.9 104.8 EXAMPLE 9

In order to test the in vivo efficacy of the conjugates as anti-tumour agents, groups of athymic mice were implanted subcutaneously with cell suspensions of a human colo- rectal carcinoma and subsequently treated with intra-peritoneal injections twice weekly for five weeks. Mice were injected with either phosphate-buffered saline, mouse monoclonal antibody (anti- carcinoembryonic antigen) or 40 a vindesine conjugate of this antibody prepared as described in Example 4. The dose of antibody injected each time per mouse was between 3.3 and 3.7 mg. The dose of vindesine in the conjugate injected each time per mouse was between 119 and 128 Ag.

61 Days after tumour implantation, the groups receiving PBS or antibody alone were killed, and the tumours excised and weighed. Mice receivingvindesine-antibody conjugates were killed 90 days 45 after tumour implantation. The mean tumour weights are shown below:

8 GB 2 137 210 A 8 Treatment Group No. of Mice Mean Tumour Weight (mg) S.D.

PBS Antibody alone 11 Vindesine-antibody conjugate 8 1793 1609 527 477 22+19

Claims

1. A conjugate comprising a vinca moiety covalently linked at the 4position via a group of the formula -OCOXCO- where X represents a single chemical bond or an optionally substituted Cl_,, 10 chain, to an immunoglobulin oran immunoglobulin fragment.

2. A conjugate according to claim 1 represented as follows c 4 loll.43, 1.,5 174' s\ 7 CH3 OCOXCOIg N0H R1 (I) in which]g represents an immunoglobulin or an immunoglobulin fragment and X is as defined in claim 1; and in which R' is COOH, CO0C1-,alkyl or CO- R', where R' is NH, NH-C,-3alky], NI-I-CH2CH,Cl, 1 -pyrrolidyl, 1 -piperidinyl or NI-i-CH,Cl-1,YCH, where Y is S or 0; R 2 is H, CH3 or CHO; and when R' and R' are taken singly, R' is H, and one of R' and R' is ethyl and the other is H or OH; and when R' and R' are taken together with the carbons to which they are attached, they form an oxirane ring, and R' is ethyl.

3. A conjugate according to either of claims 1 and 2 in which the group X'S C1-4 straight chain alkylene, C,-, branched alkylene, C2-4 alkylene, C3-4 alkynylene, C3-1 cycloalkylene, phenylene, 20 hydroxy-substituted C,-, alkylene or a direct bond.

4. A conjugate according to claim 3, in which X is C,-.4 alkylene.

5. A conjugate according to any of the preceding claims in which the immunoglobulin or immunoglobulin fragment is derived from an antibody to human or animal tumour associated antigens.

6. A conjugate according to claim 5 which is derived from a monoclonal antibody.

7. A conjugate according to any of claims 2 to 6 in which R' is COOMe or CONH2, R' is methyl or formyl, R' is hydroxy, R' is ethyl, R 5 is hydrogen, X is C1-4 alkylene and lg is a monoclonal antibody to human or animal antigen.

8. A conjugate according to any of the preceding claims which comprises 3 to 14 vinca residues.

9 GB 2 137 210 A 9. A pharmaceutical formulation comprising a conjugate according to any of claims 1 to 8.

10. A process for producing a conjugate according to claim 1 which comprises reacting an immunoglobulin or immunoglobulin fragment with a hemi-acid derivative comprising a vinca moiety having a group of the formula -OCOXCOZ attached at the 4-position, where X represents a bond or 5 an optionally substituted C,-,, chain and Z is a leaving group.

Printed in the United Kingdom for Her Majesty's Stationery Office, Demand No. 8818935, 10/1984. Contractor's Code No. 6378. Published by the Patent Office, 25 Southampton Buildings, London, WC2A lAY, from which copies may be obtained.