Macromolecular covalent conjugates, methods for preparing and pharmaceutical compositions containing them

Description

[0001] The present invention relates to macromolecular covalent conjugates, methods for their preparation and pharmaceutical compositions containing them. More specifically, the method for making said conjugates involves linking two macromolecules through a linking agent having two or more active groups of different reactivity.

[0002] It is known to bind together macromolecules through an intermediate linking agent having a number of reactive groups and the complexes formed thereby have wide application. For example, enzymes may be linked to water-insoluble carriers and thereby insolubilised; drugs may be linked to carrier proteins (e.g. specific antibodies) to enable them to be localised at the site where the drug is required; or carrier proteins may be linked to liposomes containing entrapped drugs and derived from phospholipids having a reactive end group, again permitting localisation of drugs at specific sites.

[0003] The French Patent 23 34 954 discloses a reagent for enzymatic immunologic determinations comprising an ester of maleimidobenzoic acid and N-hydroxysuccinimide, which can be used for linking macromolecules, i.e. enzymes and antigens.

[0004] A common method of linking macromolecules comprises the reaction, in a single step of the two macromolecules and the linking agent. In such a reaction dimers or polymers of the macromolecules used are formed as well as the desired adduct. This dimerisation or polymerisation is disadvantageous because:-

(i) the yield of the desired product is reduced;

(ii) macromolecules, which are expensive and/or only obtained with difficulty, remain unreacted or become dimerised or polymerised;

(iii) the by-products may be undesirable and difficult to remove. Also, perhaps more importantly, single step coupling can lead to the formation of intramolecular

bonds, thus inhibiting or destroying the biological activity of the macromolecule.

[0005] We have now found that these problems may be mitigated or overcome by preparing macromolecular covalent conjugates in a stepwise manner by first reacting one of the macromolecules with a first reactive site on the linking agent and then reacting the intermediate so formed with the second macromolecule at a second reactive site on the linking agent, as shown in the following sequence:



in which M¹ is a first macromolecule; M² is a second macromolecule; and A is a first reactive group and B is a second reactive group on a linking group L. Strictly speaking, the group L need not remain unchanged during the steps, because there is no need for the groups A and B to be completely lost; they (or one of them) may become incorporated in a modified form in L. Accordingly the steps may be expressed as:



in which M¹, M², A, B and L are as defined above and L' may be the same as L or a modified version of L (derived from A and L) and L" may be the same as L' or a modified version of L' (derived from L' and B). For brevity hereinafter the symbol L is used solely but should be considered to include the possibilities of L' and L" as the context requires.

[0006] In order that such a stepwise reaction may be effected it is necessary that the conditions under which the first reaction (between M¹ and the group A) occurs are such that the second group B will not react with molecule M¹. This is important in linking macromolecules of biological origin which contain a' multiplicity of functional groups.

[0007] The subject matter of the present application therefore covers

(A) a macromolecular covalent conjugate derived from the linking of two macromolecules through a linking agent characterised in that the conjugate comprises a first macromolecular moiety derived from a macromolecule containing one or more amino groups and a second macromolecular moiety derived from a macromolecule containing one or more amino, carboxyl -₇C-H groups, said moieties being joined by a link derived from a linking agent having two or more reactive groups through reaction of one of these with an amino group or groups of the first macromolecule and of the remaining reactive group or groups with a carboxyl, amino or 7C-H group or groups of the second macromolecule, the first reactive group of the linking agent being a carboxyl group derivative in which the carbonyl group is activated in respect to reaction with an amino group, an isocyanate, an isothiocyanate, an iminoether, an iminothioether or a halogen substituted aromatic ring in which a halogen atom is activated in respect of reaction with an amino group, and being capable of reacting with an amino group or groups of the first macromolecule under conditions where the remaining reactive group or groups which is or are each an aziridine ring, an aryl azido group or a halogen atom which is in an a-position relative to a carbonyl group or is separated by two carbon atoms from a heteroatom carrying a lone pair of electrons, is or are substantially unreactive;

(B) a method of linking two macromolecules through linking agent characterised by:

(a) reacting a first macromolecule M¹ containing one or more amino groups with a linking agent A-L-B in which A is a first reactive group which is a carboxyl group derivative in which the carbonyl group thereof is activated in respect of reaction with an amino group, an isocyanate, an isothiocyanate, an iminoether, an iminothioether, or a halogen substituted aromatic ring in which the halogen atom is activated in respect of reaction with an amino group, L is a linking group joining A and B, and B is a second reactive group or further reactive groups which is or are each an aziridine ring, an aryl azido group or a halogen atom which is in an α-position relative to a carbonyl group or is separated by two carbon atoms from a heteroatom carrying a lone pair of electrons, under reaction conditions where the group A is reactive with amino groups but B is or are substantially unreactive to give an intermediate M'-L'-B in which L' is the same as L or is derived from L and A;

(b) reacting the intermediate M^l-L'-B with a second macromolecule M² containing one or more amino, carboxyl or

C―H groups under reaction conditions where the group or groups B is or are reactive with such a group to give a complex M¹―L"―M² in which L" is the same as L' or is derived from L' and B; and

(c) isolating the macromolecular covalent conjugate thereby produced; and

(C) a pharmaceutical composition comprising a biologically active macromolecular covalent conjugate as defined above together with a pharmaceutically acceptable carrier.

[0008] Linking agents suitable for use in the present invention include those in which the first group, A, will undergo reaction at a temperature, pH or level of irradiation at which the second group, B, is not reactive.

[0009] Reactive groups used as group A (the more reactive group) in such linking agents are (a) activated carboxyl groups, for example those of formula -COX in which X is a halogen atom, e.g. chlorine or bromine, (to give acid halides); an azido group -N₃; the residue of an organic acid, e.g. CH3C02- or

(to give acid anhydrides) or the residue of an alcohol e.g.

(to give activated esters) which will react with amino functions of macromolecules to give amide links;

(b) isocyanate or isothiocyanate groups which react with amino groups, for example, as follows:-

(c) iminoethers (or iminothioethers) which react with amino groups, for example, thus (for iminoethers)

(d) compounds bearing activated halogen groups on aromatic rings, e.g.

[0010] Suitable reactive groups B are activated halogen groups i,e, halogen groups two carbon atoms removed from a heteroatom carrying a lone pair of electrons (electron releasing group) for example halogen groups in radicals such as -S-CH₂CH₂-Cl or

(mustards) (which are unreactive at the lower temperatures at which the aforementioned activated carboxyl groups may be made to react) or halogen atoms in an a-position relative to a carbonyl group e.g. in a-halocarboxylic acids; the heterocyclic aziridine ring

which is stable at neutral or alkaline pH but which undergoes ring opening at acid pH; and an arylazido group which becomes reactive on irradiation with light.

[0011] Linking agents which are preferably used in the method of the invention are: acyl derivatives of chlorambucil (4-[p-bis(2-chloroethyl)aminophenyl]butyric acid), e.g.

and of 4-[p-(2-chloroethyl)aminophenyl]butyric acid, e.g.

the acyl groups of which will undergo reaction with primary or secondary amino groups at less than 10°C, preferably less than 5°C, and the chloroethyl amino groups of which will react with amino or carboxyl groups at 25 to 30°C. At a pH of about 9 the chloroethylamino groups react preferentially with amino groups in the macromolecule M² and as the pH is lowered the amount of such reaction with amino groups decreases, i.e. the reaction with carboxyl groups becomes relatively more important. A further way of increasing the difference in reactivities between the A and B groups, when the B group is a halogen group two carbon atoms removed from a heteroatom carrying a lone pair of electrons, is to carry out the A-group reaction in the presence of a high concentration, e.g. greater than 1 molar, of halide, e.g. chloride, ions as this reduces further the reactivity of the B group. When the B- group is reacted later, the reaction should preferably be carried out in the presence of a lower concentration of the halide ions, the halide ion concentration being reduced between steps, e.g. by ultrafiltration. This halide ion concentration adjustment is particularly suitable for the present class of linking agents.

[0012] Other linking agents that may be used include acyl derivatives of 2,4-dinitro-5-aziridinyl-benzoic acid, for example the mixed anhydride:

in which the acid anhydride group will react at alkaline or neutral pH with primary or secondary amino groups on the macromolecule M¹ to give amide links, with the aziridine ring being opened at acid pH by carboxyl groups on the macromolecule M²; and
acyl derivatives of 5-azido-2,4-dinitrobenzoic acid, e.g.

in which the acid chloride group reacts with amino groups under conditions of low radiation intensity e.g. in a dark room, the azide group then reacting, after elimination of a molecule of nitrogen, by insertion into a C-H bond when irradiated with visible light of a suitable higher intensity.

[0013] The linking group L of the linking agent serves as a carrier for the two reactive groups A and B and may be any group which serves such a purpose, provided that the differential activity between the groups A and B (as herein defined) is retained. The linking agent portion L may advantageously contribute to the differential reactivity of groups A and B. The group L used will generally be dicated by the availability or ease of synthesis of the linking agent A-L-B and conveniently the two groups A and B are attached to an aromatic ring or rings, preferably a benzene ring.

[0014] It is essential that the linking agent contains only one of the more reactive groups A in order to prevent dimer or polymer formation in the first step of the reaction. It is further preferable that only one group B is present in the linking agent, although the use of linking agents having two or more of such groups is not precluded.

[0015] In conducting the stepwise reaction hereinbefore described it is preferred to isolate and purify (for example by chromatography) the product of the first step before the second linking reaction is performed, to remove unreacted linking agent and, if desired, any unreacted macromolecule or any byproduct, the presence of which is undesirable. It will in general also be necessary to purify the final product to remove any unreacted starting materials or undesirable side product.

[0016] It is also to be understood that, in common with macromolecular chemistry in general, the products obtained by the method of the invention will not consist of a single chemical entity but will comprise a series of conjugates falling within a molecular weight band, by which they may be characterised. The degree of variation will depend upon the number and variety of functional groups in the macromolecules M¹ and M².

[0017] It is normally preferred that the products obtained by the method of the present invention have a ratio of 1:1 with respect to the two macromolecules M¹ and M². In order that such a ratio may be obtained it is desirable, in the reaction between M¹ and A-L-B, that the intermediate covalent conjugate obtained is of the form M¹―(L―B)_n, where n is greater than 1, and thus the intermediate covalent conjugate itself will not be a single chemical entity but a series of conjugates comprising macromolecules having a varying number of linking groups attached thereto. It is advisable to produce intermediate conjugates of this type as in the second step not all of the groups B in the linking molecules will react with the second macromolecule M² because of side reactions, for example with the solvent or with other components in the reaction mixture.

[0018] Control over the ratio of M¹ to M² is further complicated by the fact that in the second step two or more of the intermediate conjugates represented by M'-L-B may react together, rather than with M², to give further intermediates of the type represented by M^1-L-M^1-L-B which may themselves react with either M² or a further molecule M^l-L-B.

[0019] In both steps reaction techniques known in the art for controlling undesirable side reactions, e.g. using an excess of one component or by adding a component to the reaction mixture continuously during the course of the reaction, may be employed to try to obtain the desired ratio.

[0020] Any macromolecules containing suitable functional groups may be linked by using linking agents containing the afore-mentioned groups, although the method is particularly applicable to macromolecules of biological origin, such as peptides e.g. proteins, especially antigens, antibodies, enzymes, lipoproteins, serum albumins, toxins and toxoids. In peptides, the macromolecule M¹ reacts via its amino groups and macromolecule M² reacts via its amino or carboxyl groups, or C-H bonds.

[0021] The method of the present invention is particularly useful for linking together a chemotherapeutic agent, e.g. diphtheria toxin, to an antibody, i.e. immunoglobulin, since the complex so produced is thereby rendered site specific and the chemotherapeutic agent may be concentrated at the required. point, thereby reducing the dose required and hence side-effects whilst at the same time improving the therapeutic effect of the therapeutic agent.

[0022] The covalent conjugates of the present invention, especially the biologically active ones, may be used for a variety of purposes. For example, anti-cancer agents of improved selectivity for treatment of the disease in a mammal, including man, may be obtained by linking a tumour-specific antibody to the original anti-cancer agent. Improved immunosuppressive agents for treatment or prophylaxis in a mammal, including man of for example, graft versus host (GVH) disease or transplant (graft) rejection by a host mammal and autoimmune diseases may be obtained, for example, by linking one or more toxins to an anti-lymphocytic globulin. Further, anti-parasitic agents for treatment or prophylaxis of diseases in a mammal, including man, caused by parasites may be obtained by linking an antibody for the parasite e.g. Trypanosoma cruzi, to a toxin, e.g. diphtheria toxin. These complexes may be used in the treatment of the diseases referred to.

[0023] The covalent conjugates of the present invention may be used as reagents in enzyme linked immunosorbent assay (E.L.i.S.A) reactions. For this use an antibody to the substance being detected is labelled by being linked to an enzyme suitable for catalysing a readily monitored reaction. The conjugates obtained by the present process generally have greater activity than complexes produced by earlier methods.

[0024] The amount of therapeutic conjugates required for therapeutic effect will of course vary with the particular conjugate used and the disease being treated. In general a suitable dose for a mammal of a covalent conjugate of the invention will lie in the range of 10-⁶ to 10 molar equivalents of the amount of the particular chemotherapeutic agent in the conjugate which would be required in its unbound form for the therapeutic effect obtained.

[0025] The carrier used in a pharmaceutical formulation may be any that is acceptable, i.e. does not have any substantially deleterious effect on the recipient. The carrier used for a particular administration will depend upon the route used, which would usually be parenteral; for example a liquid, e.g. aqueous, carrier would be suitable for parenteral administration, e.g. intravenous injection. A suitable aqueous carrier is water for injections.

[0026] The formulations of the present invention in liquid media may be freeze-dried for convenient storage.

[0027] The following Examples illustrate the invention.

Example 1

[0028] The coupling of anti-lymphocytic globulin to diphtheria toxin using chlorambucil residue as a linking group

Solution (A): 61 mg butyl chloroformate made up to 5.4 ml in dry dioxan.

Solution (B): 0.115 ml triethylamine made up to 10 ml in dry tetrahydrofuran.

[0029] Chlorambucil (25 ml) was dissolved in solution (B) (1.0 ml) and to an aliquot (0.3 ml) of the solution obtained was added solution (A) (0.3 ml). The reaction mixture was then stirred in an ice bath for 30 minutes.

[0030] The reaction mixture was added to a solution of horse anti-human lymphocytic globulin (ALG; 50 mg) in a mixture of saline borate (SB) buffer (0.05 M borate buffer containing 1.7% sodium chloride, fungicidal and bacteriostatic agents; pH 8.9-9.1) (5 ml) and dioxan (1.4 ml) and stirred at 4°C for 90 minutes to form a conjugate.

[0031] This solution was applied (at 4°C) to a jacketed Sephadex SG 25 (registered Trade Mark) column (Pharmacia) and eluted with SB buffer at a flow rate of 40 ml/hour.

[0032] The protein band (detected on an LBK flowthrough Spectrophotometric cell at 281 nm) (20 ml) was collected and concentrated to 2.5 ml in an Amicon ultrafiltration cell. A sample (0.1 ml) of the concentrate was diluted for spectrophotometric estimation of the level of binding of chlorambucil to the ALG (all done in a cold room at 4°C).

[0033] The concentrated conjugate solution (1 ml) was added to diphtheria toxin (1.05 ml) (25 mg/ml + 0.05 ml of 1^125-radio-labelled toxin) and stirred at room temperature for 30 hours.

[0034] A precipitate formed; the suspension was centrifuged (at 2,000 G), and the supernatant applied to a Sephadex SG 150 S/F (registered Trade Mark) column (Kl 6/90) (Pharmacia) and eluted with SB buffer. Fractions were collected and estimated spectrophotometrically. Samples (0.1 ml) of the fractions were also estimated by dissolving in scintillation fluid and counting.

[0035] The high molecular weight fraction having M.wt. of 180,000 to 250,000 comprised a series of conjugates of ALG and diphtheria toxin conjugated through chlorambucil, there being an average of from 1.0 to 1.5 moles of diphtheria toxin conjugated, through a chlorambucil residue, to each mole of ALG. A second high molecular weight fraction (M. wt >250,000) was also obtained.

(i) Integrity of the antigen-binding capacity of the conjugate

[0036] This was tested by indirect immunofluorescence. Human lymphoid cell-line cells (CLA₄ cells) were incubated (30 minutes, 37°C) with ALG or the conjugate, ALG-chlorambucil-diphtheria toxin, before washing and incubating with fluorescent rabbit and anti-horse antibodies. The cells were then washed three times and mounted on slides and examined under the fluorescence microscope. The resulting fluorescence is a measure of the amount of ALG or conjugate that bound to the cells during the first incubation so that the minimum concentration of antibody needed to produce visible fluorescence can be determined. The fluorescent titre for ALG-chlorambucildiphtheria toxin (M.wt 180,000 to 250,000) 1:1 was 3.0 x 10-⁵ mM which is insignificantly different from the titre for ALG alone (2.6 x 10-⁵ mM). Thus the linkage of diphtheria toxin to ALG did not affect the antigen binding capacity of the antibody. However, the conjugate of M.wt_>250,000 did show a somewhat reduced binding capacity, with a titre of 6.6 x 10-⁵ mM.

(ii) The cytotoxic properties of conjugated diphtheria toxin

[0037] This was tested using guinea-pig macrophages which non-specifically bind and uptake foreign proteins. The macrophages were contacted for four days with toxin or conjugates; cell survival at the end of this period was determined from the ability of adhering cells to pinocytose neutral red dye which was subsequently extracted and the optical density measured at 540 nm.

[0038] ALG-chlorambucil-diphtheria toxin (M.wt 180,000 to 250,000) was as toxic to the macrophages as was diphtheria toxin alone, killing down to 10^-12M, or about 50 molecules per cell. Thus the coupling of diphtheria toxin to ALG using chlorambucil does not inactivate the toxic properties of the toxin. The conjugate of higher M.wt. (_>250,000) did however show a twenty-fold reduction in its capacity to kill macrophages compared to diphtheria toxin.

The specific cytotoxic action of ALG-chlorambucildiphtheria toxin on human lymphoid cells

[0039] Having established that we could link ALG to diphtheria toxin in such a manner that neither antigen-binding capacity of the antibody nor toxic properties of the toxin were overly affected by conjugation, the conjugates were tested against their target cell, CLA₄, in vitro.

[0040] CLA₄ cells (2 x 10⁵ cells per 200 1 culture) in Hanks balanced salt solution containing 10% foetal calf serum were exposed to diphtheria toxin for 1 hour before washing five times. Cell survival was measured at the end of the 24 hours incubation by the ability of the cells to incorporate ³H-leucine into protein.

[0041] It was found that diphtheria toxin was not very effective at killing CLA₄ cells compared with other sensitive cells such as guinea pig macrophages and human fibroplasts. On prolonged (24 hours) exposure diphtheria toxin would only kill CLA₄ cells at concentrations down to 10-⁹M, and on short exposure no cell death was apparent even at the maximum concentration used of 10^-8M. However, the ALG-chlorambucil-diphtheria toxin conjugate (M.W. 180,000 to 250,000) was very potent and killed CLA₄ cells at concentrations down to 10-"M for both 1 hour and 24-hour exposures. Thus by linking diphtheria toxin to ALG its cytotoxic effect on 1 hour exposure to CLA₄ cells was improved by a factor of about 1000. ALG alone did not kill CLA₄ cells, and did not modify the toxicity of diphtheria toxin when presented simultaneously to the cells.

[0042] Furthermore, when diphtheria toxin was coupled to a non-immune immunoglobulin which had no ability to bind to CLA, cells the conjugate was 50 fold less toxic than diphtheria toxin alone so that the improved toxicity of ALG-chlorambucil-diphtheria toxin seems to be specific for the target cell. This is supported by the finding that ALG-chlorambucil-diphtheria toxin was about twenty-fold less efficient at killing human fibroblasts, a cell to which ALG does not bind, than was diphtheria toxin alone.

Example 2

[0043] The coupling of human y globulin (HyG) to bovine serum albumin (BSA) using the sodium salt of 2,4-dinitro-5-aziridinyl-benzoic acid as a precursor for the linking agent

Solution (A): 0.16 g butyl choroformate made up to 2.35 ml in dry tetrahydrofuran

Solution (B): 0.15 ml triethylamine made up to 2.50 ml in DMSO.

[0044] The sodium salt of 2,4-dinitro-5-aziridinyl benzoic acid (20 mg) was dissolved at room temperature in solution B (0.2 ml) and cooled in ice until just solid when solution A (0.2 ml) was added. The mixture was stirred at room temperature until just fluid and then in an ice-water bath for 20 minutes.

[0045] A solution of I¹²⁵-radiolabelled bovine serum albumin (BSA) (50 mg in 1.5 ml SB buffer) was then added rapidly while stirring, and the reaction mixture was left at room temperature for 90 minutes during which time the pH was monitored and N/10 NaOH solution added to keep the pH at 8.0.

[0046] At the end of the 90 minutes the whole reaction mixture was applied to a K16/40 Sephadex SG 25 (registered Trade Mark) (Pharmacia) column and eluted with SB buffer at a flow rate of 40 ml/hour.

[0047] The protein band (detected as described in Example 1) (in 13 ml of eluant) was collected and a sample was diluted for spectrophotometric determination of the level of binding of 2-4-dinitro-5- aziridinyl benzoic acid to the BSA.

[0048] The protein band (conjugate) was concentrated to 2.0 ml in an Amicon ultra-filtration cell and the concentrate so obtained was added to HyG (50 mg) in SB buffer (2 ml). The pH at this point was 9.1.

[0049] The reaction mixture was stirred rapidly while the pH was adjusted to 5.3 by slow addition of 1 N hydrochloric acid from a microsyringe. When the addition of hydrochloric acid was complete the reaction mixture was stirred in a water bath at 40°C and the reaction followed spectrophotometrically, small samples being taken every 5 hours, diluted and examined by UV spectroscopy. After 20 hours a shift of Amax from 336 nm to 356 nm indicated that the reaction was complete and the pH was adjusted to 9.1 by slow addition, to the rapidly stirred mixture, of 1N sodium hydroxide.

[0050] A precipitate formed; the suspension was centrifuged and a portion of the supernatant (2.0 ml) was applied to a K16/90 Sephadex SG 150 (registered Trade Mark) S/F column (Pharmacia) and eluted with SB buffer.

[0051] Fractions from the column were collected, samples (0.1 ml) of the fractions were taken, dissolved in scintillation fluid and counted.

[0052] The high molecular weight fraction (M.wt >150,000) comprised a series of conjugates of bovine serum albumin and human (HyG) having an average of 9 moles of 2,4-dinitro-5-aziridinyl benzoic acid bound to each mole of BSA and to which conjugates was bound 80% of the labelled BSA. It was estimated that the conjugate band contained 1 to 2 molecules of BSA for each molecule of HyG.

Example 3

[0053] The coupling of proteins to liposomes

Solution (A): 0.1 ml butyl chloroformate made up to 2.16 ml in dry tetrahydrofuran

Solution (B): 0.1 ml triethylamine made up to 2.0 ml in dry DMSO

[0054] The sodium salt of 2,4-dinitro-5-aziridinyl benzoic acid (30 mg) was added to solution (B) (0.3 ml) and stirred at 0°C until just solid when solution (A) (0.3 ml) was added and stirred in a jacketed vessel at below 4°C for 20 minutes. An aliquot (0.4 ml) of the thus obtained reaction mixture was added to a phospholipid (dipalmitoyl phosphatidyl ethanolamine) (30 mg) in washed and dried chloroform (4 ml). The temperature was raised to 50°C and the reaction was followed by a thin layer chromatography (TLC) on aluminium backed silica gel TLC plates using:-

(1) CHCI₃: MeOH 20:5, or

(2) CHCl₃: MeOH:H₂O 70:30:5

[0055] After 2 hours no unreacted phospholipid was detectable in these systems. The reaction mixture was allowed to cool and applied to a dry silica gel column which was washed with chloroform (200 ml) to elute 2 minor bands. The solvent was then changed to chloroform:methanol:water (70:30:5), and the product first being adsorbed on to the top of the column and then eluted as two major bands; when the second band started to elute the solvent was changed to pure ethanol and all the material was eluted from the column.

[0056] The conjugate band (the first major band eluted) was dried by evaporation under reduced pressure at less than 40°C, redissolved in chloroform (20 ml), filtered through a Millipore (registered Trade Mark) filter (0.22,um pore size) to remove silica fines and dried by rotary evaporation (reduced pressure and ambient temperature). A yellow liquid residue (presumed to be largely water) remained and was removed by successive addition and evaporation under reduced pressure of dry benzene.

[0057] The product travels as a single sopt (Rf. 0.6) in the above mentioned TLC systems.

[0058] Liposomes are prepared from egg lecithin (10 mg) and dipalmitoyl phosphatidyl ethanolamine (2 mg) (obtained as described above) by the following method. The lipids are mixed and dried as a thin film under reduced pressure on a rotary evaporator at ambient temperature and then suspended in sodium chloride (1 ml, 73 ml) and phosphate buffer (1 mM; pH 7.0); (at this pH the aziridine ring of the 2,4-dinitro-5-aziridinyl benzoic acid residue is poorly reactive).

[0059] A solution (2 ml; 15 mg/ml) of bovine serum albumin (BSA) in sodium chloride (73 mM) and acetate buffer (5 mM) is then added and the pH of the mixture is adjusted to 4.1 with aqueous hydrochloric acid. At this pH BSA binds electrostatically to the liposomes (the liposomes are negatively charged, the BSA is not positively charged), and the 2,4-dinitro-5-aziridinyl benzoic acid residue reacts with suitable residues on the liposome. After an 8 hour incubation the pH is adjusted to 7.0 with aqueous sodium hydroxide and the liposomes are separated from unbound BSA by gel cchromato- graphy on Sepharose 6B.

Examples 4-13

[0060] General method for coupling protein-1 to protein-2 using chlorambucil residue as linking group

Solution (A): 0.1 ml butyl chloroformate made up to 9.5 ml with dry dioxan

Solution (B): 0.1 ml triethylamine made up to 8.7 ml with dry tetrahydrofuran

[0061] Chlorambucil (7.5 mg) was dissolved in 0.3 ml solution (B) and to the stirred, cooled (2°C) solution was added 0.3 ml solution (A). After 30 minutes a pre-cooled solution of protein-1 (5 ml 10 mg protein-1 per ml in borate:NaCI (0.05 M:1.7%, pH 9) and dioxan (1.4 ml) was added quickly and stirring continued at 2°C for 1 t to 2 hours. The solution was then passed through a jacketed (2°C) column of Sephadex SG 25 and eluted with the same borate-saline buffer.

[0062] The protein containing eluate (20 ml)-detected with an LKB flow-through spectrophotometric cell at 280 nm-was run into a solution of protein-2 (1 ml 25 mg protein-2 per ml in the same borate-saline buffer). A small quantity of radio-iodine labelled (1¹²⁵) protein-2 was present to assist quantitation of the results.

[0063] The combined solutions were concentrated in an Amicon ultra-filtration cell down to 2 ml and then allowed to stand at 26°C for 24 hours. After centrifugation to remove any insoluble material the supernatant was passed down a column of Sephadex SG 200 superfine and eluted with the borate-saline buffer of pH 9.

[0064] Fractions were collected and the molecular weights were calculated from the elution volumes. The protein-2 content of the fractions was estimated from the gamma-radiation counts and the protein-1 content from the U.V. absorbance at 280 nm after deducting the contribution from protein-2.

[0065] Besides fractions containing unconjugated protein-1 and protein-2, fractions of M.wt 180,000 to 250,000 and >250,000 were obtained. The fraction of M.wt 180,000 to 250,000 consisted of conjugates of protein-1 and protein-2, there being an average of 1 to 1.5 molecules of protein-2 coupled to each molecule of protein-1.

[0066] For protein-1 it is possible to use normal horse and normal bovine immunoglobulin, horse antimouse thymocytic globulin, horse anti-human lymphocytic globulin and F(ab)₂ fragments (obtained by pepsin digestion) of normal horse immunoglobulin and of horse anti-human lymphocytic globulin. For protein-2 it is possible to use bovine serum albumin, diphtheria toxin and abrin.

[0067] When F(ab)₂ fragments are used as protein-1 it is preferable to use Sephadex SG 150 superfine for the final chromatographic separation and in this case the fraction of M.wt 130,000 to 200,000 contained predominantly a 1:1 protein-1 :protein-2conjugate.

[0068] The specific couplings set out in the Table have been carried out.

Examples 14-26

[0069] A portion of each of the covalent conjugates formed in Examples 1 to 13 is dispersed into separate aliquots of water for injections so as to form respective solutions suitable for injection.

Claims

1. A macromolecular covalent conjugate derived from the linking of two macromolecules through a linking agent characterised in that the conjugate comprises a first macromolecular moiety derived from a macromolecule containing one or more amino groups and a second macromolecular moiety derived from a macromolecule containing one or more amino, carboxyl or -7C-H groups, said moieties being joined by a link derived from a linking agent having two or more reactive groups through reaction of one of these with an amino group or groups of the first macromolecule and of the remaining reactive group or groups with a carboxyl, amino or 7C-H group or groups of the second macromolecule, the first reactive group of the linking agent being a carboxyl group derivative in which the carbonyl group is activated in respect to reaction with an amino group, an isocyanate, an isothiocyanate, an iminoether, an iminothioether or a halogen substituted aromatic ring in which a halogen atom is activated in respect of reaction with an amino group, and being capable of reacting with an amino group or groups of the first macromolecule under conditions where the remaining reactive group or groups which is or are each an aziridine ring, an aryl azido group or a halogen atom which is in an α-position relative to a carbonyl group or is separated by two carbon atoms from a heteroatom carrying a lone pair of electrons, is or are substantially unreactive.

2. A conjugate according to claim 1, in which the first and second macromolecular moieties are each derived from a peptide macromolecule.

3. A conjugate according to claim 2, in which the first and second macromolecular moieties are each derived from an antigen, antibody, enzyme, lipoprotein, serum albumin, toxin or toxoid.

4. A conjugate according to claim 1, 2 or 3, in which said remaining reactive group or groups of the linking agent is or are each an aziridine ring or a halogen atom which is in an a-position relative to a carbonyl group or which is separated by two carbon atoms from a heteroatom carrying a lone pair of electrons.

5. A conjugate according to any one of the preceding claims, in which the linking agent is an acyl derivative of chlorambucil, 4-[p-(2-chloroethyl)amino-phenyl]butyric acid or 2,4-dinitro-5-aziridinyl- benzoic acid which is formed at the carboxyl group thereof and in which the carbonyl group is activated in respect of reaction with an amino group.

6. A method of linking two macromolecules through a linking agent characterised by:

(a) reacting a first macromolecule M¹ containing one or more amino groups with a linking agent A-L-B in which A is a first reactive group which is a carboxyl group derivative in which the carbonyl group thereof is activated in respect of reaction with an amino group, an isocyanate, an isothiocyanate, an iminoether, an iminothioether, or a halogen substituted aromatic ring in which the halogen atom is activated in respect of reaction with an amino group, L is a linking group joining A and B, and B is a second reactive group or further reactive groups which is or are each an aziridine ring, an aryl azido group or a halogen atom which is in an a-position relative to a carbonyl group or is separated by two carbon atoms from a heteroatom carrying a lone pair of electrons, under reaction conditions where the group A is reactive with amino groups but B is or are substantially unreactive to give an intermediate M'-L'-B in which L' is the same as L or is derived from L and A;

(b) reacting the intermediate M'-L'-B with a second macromolecule M² containing one or more amino, carboxyl or 7C-H groups under reaction conditions where the group or groups B is or are reactive with such a group to give a complex M^1-L"-M² in which L" is the same as L' or is derived from L' and B; and

(c) isolating the macromolecular covalent conjugate thereby produced.

7. A method according to claim 6, in which the reaction conditions are adjusted in temperature, pH or radiation applied so as to cause the group or groups B to react under the new conditions with macromolecule M², although it or they had remained substantially unreacted with macromolecule M¹ under the previous conditions.

8. A method according to claim 6 or 7, in which the group or groups A is or are each a carboxyl group derivative being a halide, anhydride, azide or ester in which the esterifying group activates the carbonyl group in respect of reaction with an amino group.

9. A method according to claim 6, 7 or 8, in which the group or groups B is or are each an aziridine ring or a halogen atom which is separated by two carbon atoms from a heteroatom carrying a lone pair of electrons.

10. A method according to claim 8 in which the linking agent is a halide, anhydride, azide or ester derivative of chlorambucil, 4-[p-(2-chloroethyl)amino-phenyl]butyric acid, 2,4-dinitro-5-aziridinyl- benzoic acid, or 5-azido-2,4-dinitrobenzoic acid.

11. A method according to claim 10, in which the activated carbonyl group of the derivative of chlorambucil or of 4-[p-(2-chloroethyl)aminophenyl]butyric acid reacts with an amino group or groups in macromolecule M¹ at less than 10°C and the chloroethyl group or groups reacts or react with a carboxyl or amino group or groups in macromolecule M² at 25 to 30°C.

12. A method according to claim 10 or 11, in which the carbonyl group of the derivative of chlorambucil or of 4-[p-(2-chloroethyl)aminophenyl]butyric acid reacts, in the presence of a first concentration of halide ions, with an amino group or groups in macromolecule M¹ and the chloroethyl group reacts, in the presence of a second concentration of halide ions which is less than the first, with a carboxyl or an amino group or groups in macromolecule M².

13. A method according to any one of claims 6 to 12, in which the intermediate conjugate formed by the reaction of M¹ and A-L-B contains a number of linking group residues, -L-B, which is greater than 1.

14. A method according to any one of claims 6 to 13, in which the linking agent molecule contains only one group B.

15. A method according to any one of claims 6 to 14, in which the intermediate M^1-L'-B is isolated and purified before step (b).

16. A method according to any one of claims 6 to 15, in which M¹ and M² are each a peptide macromolecule.

17. A method according to claim 16, in which M¹ and M² are each an antigen, antibody, enzyme, lipoprotein, serum albumin, toxin or toxoid.

18. A method according to claim 17, in which either (a) one of the macromolecules is a diphteria toxin and the other is an antibody, or (b) one of the macromolecules is an antilymphocytic globin and the other is a toxin.

19. A pharmaceutical composition which comprises a biologically active conjugate according to any one of claims 1 to 5 or made by a process according to any of claims 6 to 18, together with a pharmaceutically acceptable carrier.

Revendications

1. Conjugué macromoléculaire covalent dérivé de la liaison de deux macromolécules au moyen d'un agent de couplage, caractérisé en ce que le conjugué comprend un premier élément macromoléculaire dérivé d'une macromolécule contenant un ou plusieurs groupements amino et un second élément macromoléculaire dérivé d'une macromolécule contenant un ou plusieurs groupements amino, carboxyle ou ₇C-H, lesdits éléments étant reliés par une liaison dérivée d'un agent de couplage ayant deux groupements réactifs ou davantage, par réaction de l'un de ceux-ci avec un groupement amino ou des groupements amino de la première macromolécule et du groupement ou des groupements réactif(s) restant(s) avec un groupement ou des groupements carboxyle, amino ou

C―H de la seconde macromolécule, le premier groupement réactif de l'agent de couplage étant un groupement dérivé du carboxyle dans lequel le groupement carbonyle est activé en ce qui concerne la réaction avec un groupement amino, un isocyanate, un isothiocyanate, un iminoéther, un iminothioéther ou un cycle aromatic halogéno-substitué dans lequel un atome d'halogène est activé par rapport à la réaction avec un groupement amino, et étant capable de réagir avec un groupement ou des groupements amino de la première macromolécule dans des conditions où le gropement ou les groupements réactif(s) restant(s) qui est ou qui sont chacun un cycle aziridine, un groupement aryle azido ou un atome d'halogène qui est en position a par rapport à un groupement carbonyle ou qui est séparé par deux atomes de carbone d'un hétéroatome portant une seule paire d'électrons, est ou sont sensiblement non réactif(s).

2. Un conjugué selon la revendication 1, dans lequel les premier et second éléments macromoléculaires sont chacun dérivés d'une macromolécule de peptide.

3. Un conjugué selon la revendication 2, dans lequel les premier et second éléments macromoléculaires sont chacun dérivés d'une antigène, d'un anticorps, d'une enzyme, d'une lipoprotéine, d'une sérum albumine, d'une toxine ou d'un toxoïde.

4. Un conjugué selon la revendication 1, 2 ou 3, dans lequel ledit groupement ou lesdits groupements réactif(s) restant(s) de l'agent de couplage est ou sont chacun un cycle aziridine ou un atome d'halogène qui est en position a par rapport à un groupement carbonyle ou qui est séparé par deux atomes de carbone d'un hétéroatome portant une seule paire d'électrons.

5. Un conjugué selon l'une quelconque des revendications précédentes, dans lequel l'agent de couplage est un dérivé acylé de chlorambucile, d'acide 4-[p-(2-chloroéthyl)amino-phényl] butyrique ou d'acide 2,4-dinitro-5-aziridinylbenzoïque qui est formé sur le groupe carboxylique de ceux-ci et dans lequel le groupement carbonyle est activé en ce qui concerne la réaction avec un groupement amino.

6. Une méthode de couplage de deux macromolécules à l'aide d'un agent de couplage, caractérisée par le fait que:

(a) l'on fait réagir une première macromolécule M¹ contenant un ou plusieurs groupements amino avec un agent de couplage A-L-B dans lequel A est un premier groupement réactif qui est un dérivé de groupement carboxylique dans lequel le groupement carbonyle est activé en ce qui concerne la réaction avec un groupement amino, un isocyanate, un isothiocyanate, un iminoéther, un iminothioéther, ou un cycle aromatique halogéno-substitué dans lequel l'atome d'halogène est activé en ce qui concerne la réaction avec un groupement amino, L est un groupement de liaison joignant A et B, et B représente un second groupement réactif ou d'autres groupements réactifs qui est ou qui sont chacun un cycle aziridine, un groupement aryl azido ou un atome d'halogène qui est en position a par rapport à un groupement carbonyle ou qui est séparé par deux atomes de carbone d'un hétéroatome portant une seule paire d'électrons, dan des conditions, réactionnelles où le groupement A est réactif vis-à-vis des groupements amino mais où B est (ou sont) sensiblement nonréactif(s), pour donner un composé intermédiaire M^1-L'-B dans lequel L' est identique à L ou est dérivé de L et A;

(b) l'on fait réagir l'intermédiaire M^1-L'-B avec une seconde macromolécule M² contenant un ou plusieurs groupements amino, carboxyle ou 7C-H dans des conditions réactionnelles où le groupement ou les groupements B est ou sont réactif(s) avec un groupement capable de donner un complexe M'-L"-M² dans laquel L" est identique à L' ou est dérivé de L' et B; et

(c) l'on isole le conjugué covalent macromoléculaire ainsi produit.

7. Une méthode selon la revendication 6, dans laquelle les conditions de réaction sont réglées en température, pH ou rayonnement appliqué de façon à provoquer la réaction du groupement ou des groupements B dans les nouvelles conditions avec la macromolécule M², alors que B était ou étaient resté(s) sensiblement non réactif(s) avec la macromolécule M¹ dans les conditions précédentes.

8. Une méthode selon la revendication 6 ou 7, dans laquelle le groupement ou les groupements A est ou sont chacun un dérivé du groupement carboxylique qui est un halogénure, un anhydride, un azide ou un ester dans lequel le groupement estérifiant active le groupement carbonyle en ce qui concerne la réaction avec un groupement amino.

9. Une méthode selon la revendication 6, 7 ou 8, dans laquelle le groupement ou les groupements B est ou sont chacun un cycle aziridine ou un atome d'halogène qui est séparé par deux atomes de carbone d'un hétéroatome portant une seule paire d'électrons.

10. Une méthode selon la revendication 8 dans laquelle l'agent de couplage est un halogénure, un anhydride, un azide ou un ester dérivé de chlorambucil, d'acide 4-[p-(2-chloroéthyl)amino-phényl] butyrique, d'acide 2,4-dinitro-5-aziridinylbenzoïque, ou d'acide 5-azido-2,4-dinitrobenzoïque.

11. Une méthode selon la revendication 10, dans laquelle le groupement carbonyle activé du dérivé de chlorambucile ou d'acide 4-[p-(2-chloroéthyl) amino-phényl] butyrique réagit avec un groupement amino ou des groupements amino dans la macromolécule M¹ à moins de 10°C et le groupement ou les groupements chloroéthyle réagit ou réagissent avec un groupement ou des groupements carboxyle ou amino dans la macromolécule M² à 25 à 30°C.

12. Une méthode selon la revendication 10 ou 11, dans laquelle le groupement carbonyle du dérivé de chlorambucile ou d'acide 4-[p-(2-chloroéthyl)amino-phényl] butyrique réagit, en présence d'une première concentration en ions halogénure, avec un groupement ou des groupements amino dans la macromolécule M¹, et le groupement chloroéthyle réagit, en présence d'une seconde concentration en ions halogénure qui est inférieure à la première, avec un groupement ou des groupements carboxyle ou amino dans la macromolécule M².

13. Une méthode selon l'une quelconque des revendications 6 à 12, dans laquelle le conjugué intermédiaire formé par la réaction de M¹ et de A-L-B contient un nombre de restes de groupements de liaison, -L-B, qui est supérieur à 1.

14. Une méthode selon l'une quelconque des revendications 6 à 13, dans laquelle la molécule d'agent de liaison contient seulement un groupement B.

15. Une méthode selon l'une quelconque des revendications 6 à 14, dans laquelle l'intermédiaire M'-L'-B est isolé et purifié avant l'étape (b).

16. Une méthode selon l'une quelconque des revendications 6 à 15, dans laquelle M¹ et M² sont chacun une macromolécule de peptide.

17. Une méthode selon la revendication 16, dans laquelle M¹ et M² sont chacun un antigène, un anticorps, une enzyme, une lipoprotéine, une sérum albumine, une toxime ou un toxoide.

18. Une méthode selon la revendication 17, dans laquelle soit (a) l'une des macromolécules est une toxine diphtérique et l'autre est un anticorps, soit (b) l'une des macromolécules est une globine antilymphocytaire et l'autre est une toxine.

19. Une composition pharmaceutique qui comprend un conjugué biologiquement actif qui est défini selon l'une quelconque des revendications 1 à 5 ou qui est préparé selon un procédé selon l'une quelconque des revendications 6 à 18, en combinaison avec un support pharmaceutique acceptable.

Ansprüche

1. Makromolekulares covalentes Kinjugat, hergestellt durch Verbinden von zwei Makromolekülen durch ein verbindendes Agens, dadurch gekennzeichnet, daß das Konjugat umfaßt einen ersten makromolekularen Anteil, der abgeleitet ist von einem Makromolekül, das eine oder mehr Aminogruppen enthält, und einen zweiten makromolekularen Anteil, der abgeleitet ist von einem Makromolekül, das eine oder mehr Amino-, Carboxyl- oder >C­C-Gruppen enthält, wobei diese Anteile durch eine Brückenbindung miteinander verbunden sind, die abgeleitet ist von einem verbindenden Agens mit zwei oder mehr reaktiven Gruppen durch Umsetzung einer derselben mit einer Aminogruppe oder -gruppen des ersten Makromoleküls und der restlichen reaktiven Gruppe oder Gruppen mit einer Carboxyl-, Amino- oder jC-H-Gruppe oder -Gruppen des zweiten Makromoleküls, wobei es sich bei der ersten reaktiven Gruppe des verbindenden Agens handelt um ein Carboxylgruppenderivat, in dem die Carbonylgruppe aktiviert ist in bezug auf die Reaktion mit einer Aminogruppe, ein Isocyanat, ein Isothiocyanat, einen Imonoäther, einen Iminothioäther oder einen halogensubstituierten aromatischen Ring, in dem ein Halogenatom aktiviert ist in bezug auf die Reaktion mit einer Aminogruppe, und mit einer Aminogruppe oder-gruppen des ersten makromoleküls unter Bedingungen reagieren kann, unter denen die restliche reaktive Gruppe oder Gruppen, die ist oder jeweils sind ein Aziridinring, eine Arylazidogruppe oder ein Halogen-atom, das sich in einer a-Position, bezogen auf eine Carbonylgruppe, befindet oder durch zwei Kohlenstoffatome von einem ein freies Elektronenpaar tragenden Heteroatom getrennt ist, im wesentlichen nicht-reaktiv ist oder sind.

2. Konjugat nach Anspruch 1, worin der erste und der zweite makromolekulare Anteil jeweils von einem Peptid-Makromolekül abgeleitet sind.

3. Konjugat nach Anspruch 2, worin der erste und der zweite makromolekulare Anteil jeweils von einem Antigen, Antikörper, Enzym, Lipoprotein, Serumalbumin, Toxin oder Toxoid abgeleitet sind.

4. Konjugat nach Anspruch 1, 2 oder 3, worin die restliche reaktive Gruppe oder Gruppen des verbindenden Agens ist oder jeweils sind ein Aziridinring oder ein Halogenatom, das sich in einer a-Position, bezogen auf eine Carbonylgruppe, befindet oder das durch zwei Kohlenstoffatome von einem ein freies Elektronenpaar tragenden Heteroatom getrennt ist.

5. Kunjugat nach einem der vorhergehenden Ansprüche, worin das verbindende Agens ein Acylderivat von Chlorambucil, 4-[p-(2-Chloroethyl)amino-phenyl]buttersäure oder 2,4-Dinitro-5- aziridinylbenzoesäure ist, das an ihrer Carboxylgruppe gebildet wird und in dem die Carbonylgruppe in bezug auf die Reaktion mit einer Aminogruppe aktiviert ist.

6. Verfahren zum Verbinden von zwei Makromolekülen durch ein verbindendes Agens, gekennzeichnet durch die:

a) Umsetzung eines ersten Makromoleküls M¹, das eine oder mehr Aminogruppen enthält, mit einem verbindenden Agens A-L-B, worin A eine erste reaktive Gruppe ist, bei der es sich handelt um ein Carboxylgruppenderivat, in dem die Carbonylgruppe desselben aktiviert ist in bezug auf die Reaktion mit einer Aminogruppe, ein Isocyanat, ein Isothiocyanat, einen Iminoäther, einen Iminothioäther oder einen halogensubstituierten aromatischen Ring, in dem das Halogenatom in bezug auf die Reaktion mit einer Aminogruppe aktiviert ist, L eine A und B verbindende Gruppe ist und B eine zweite reaktive Gruppe oder weitere reaktive Gruppen ist, die ist oder jeweils sind ein Aziridinring, eine Arylazidogruppe oder ein Halogenatom, das sich in einer a-Position, bezogen auf eine Carbonylgruppe, befindet oder durch zwei Kohlenstoffatome von einem ein freies Elektronenpaar tragenden Heteroatom getrennt ist, unter solchen Reaktionsbedingungen, unter denen die Gruppe A mit Aminogruppen reaktiv ist, jedoch die Gruppe oder Gruppen B im wesentlichen nicht-reaktiv ist oder sind, unter Bildung eines Zwischenprodukts M¹―L'―B, worin L' die gleiche Bedeutung wie L hat oder von L und A abgeleitet ist;

b) Umsetzung des Zwischenprodukts M^1-L'-B mit einem zweiten Makromolekül M², das eine oder mehr Amino-, Carboxyl- oder 7C-H-Gruppen enthält, unter solchen Reaktionsbedingungen, unter denen die Gruppe oder Gruppen B mit einer solchen Gruppe reaktiv ist oder sind unter Bildung eines Komplexes M'-L"-M², worin L" die gleiche Bedeutung wie L' hat oder von L' und B abgeleitet ist; und

c) Isolierung des dabei gebildeten makromolekularen kovalenten Konjugats.

7. Verfahren nach Anspruch 6, worin die Reaktionsbedingungen in bezug auf Temperatur, pH-Wert oder Bestrahlung so eingestellt werden, daß die Gruppe oder Gruppen B unter den neuen Bedingungen mit dem Makromolekül M² reagieren, obgleich sie unter den vorherigen bedingungen mit dem Makromolekül M¹ in wesentlichen nicht-umgesetzt verblieben war oder waren.

8. Verfahren nach Anspruch 6 oder 7, worin die Gruppe oder Gruppen A ist oder jeweils sind ein Carboxylgruppenderivat, bei dem es sich um ein Halogenid, Anhydrid, Azid oder Ester handelt, worin die veresternde Gruppe die Carbonylgruppe in bezug auf die Reaktion mit einer Aminogruppe aktiviert.

9. Verfahren nach Anspruch 6, 7 oder 8, worin die Gruppe oder Gruppen B ist oder jeweils sind ein Aziridinring oder ein Halogenatom, das durch zwei Kohlenstoffatome von einem ein freies Elektronenpaar tragenden Heteroatom getrennt ist.

10. Verfahren nach Anspruch 8, worin das verbindende Agens ein Halogenid, Anhydrid, Azid oder Esterderivat von Chlorambucil, 4-[p-(2-Chlorethyl)amino-phenyl]buttersäure, 2,4-Dinitro-5-aziridinyl- benzoesäure oder 5-Azido-2,4-dinitrobenzoesäure ist.

11. Verfahren nach Anspruch 10, worin die aktivierte Carbonylgruppe des Derivats von Chlorambucil oder von 4-[-(2-Chloroethyl)aminophenyl]buttersäure mit einer Aminogruppe oder -gruppen in dem Makromolekül M¹ bei weniger als 10°C reagiert und die Chlorethylgruppe oder -gruppen mit einer Carboxyl- oder Aminogruppe oder -gruppen in dem Makromolekül M^2-bei 25 bis 30°C reagiert oder reagieren.

12. Verfahren nach Anspruch 10 oder 11, worin die Carbonylgruppe des Derivats von Chlorambucil oder 4-[p-(2-Chlorethyl)aminophenyl]buttersäure in Gegenpwart einer ersten Konzentration von Halogenidionen mit einer Aminogruppe oder -gruppen in dem Kakromolekül M¹ reagiert und die Chlorethylgruppe in Gegenwart einer zweiten Konzentration Halogenidionen, die geringer ist als die erste, mit einer Carboxyl- oder einer Aminogruppe oder -gruppen in dem Makromolekül M² reagiert.

13. Verfahren nach einem der Ansprüche 6 bis 12, worin das durch die Reaktion von M¹ und A-L-B gebildete Zwischenprodukt-Konjugat eine Anzahl von Verbindungsgruppen-Resten-L-B enthält, die größer als 1 ist.

14. Verfahren nach einem der Ansprüche 6 bis 13, worin das Molekül des verbindenden Agens nur eine Gruppe B enthält.

15. Verfahren nach einem der Ansprüche 6 bis 14, worin das Zwischenprodukt M^l-L'-B vor der Stufe (b) isoliert und gereinigt wird.

16. Verfahren nach einem der Ansprüche 6 bis 15, worin M¹ und M² jeweils ein Peptid-Makromolekül sind.

17. Verfahren nach Anspruch 16, worin M¹ und M² jeweils ein Antigen, Antikörper, Enzym, Lipoprotein, Serumalbumin, Toxin oder Toxoid sind.

18. Verfahren nach Anspruch 17, worin entweder (a) eines der Makromoleküle ein Diphtherie-Toxin und das andere ein Antikörper oder (b) eines der Makromoleküle ein Antilymphozyten-Globin und das andere ein Toxin sind.

19. Pharmazeutische Zusammensetzung, die umfaßt ein biologisch aktives Konjugat nach einem der Ansprüche 1 bis 5 oder hergestellt nach einem Verfahren gemäß einem der Ansprüche 6 bis 18, zusammen mit einem pharmazeutisch akzeptablen Träger.