Tumour therapy

A three component therapeutic system that comprises (i) a first compound; (ii) a catalytic macromolecule which does not specifically bind to a tumour antigen, but which is capable, following administration to the vascular compartment of a mammal, of being taken up by a tumour in the mammal, and is capable of converting the first compound into a second compound; and (iii) an inhibitor which, following administration to the said vascular compartment, reduces the level of the said catalytic conversion activity in the vascular compartment.

EXAMPLE 1 A Therapeutic System Using Ala-methotrexate Carboxypeptidases A and G2 The widely used agent methotrexate is a folic acid antagonist and its action is to block the conversion of folic acid, a dietary factor, to its reduced form 5-methyltetrahydrofolic acid (folinic acid, citrovorum factor) by the enzyme dihydrofolate reductase. Folinic acid is used in one carbon transfer in the synthesis of DNA. In the absence of folinic acid, cell reproduction is blocked in S phase and cell death follows. Methotrexate is used in the treatment of a wide range of malignant diseases. It also causes cell death in normal cell renewal tissues via the mechanisms already outlined. The magnitude of its effects is largely a function of the duration of tissue exposure to the drug, the longer the duration the greater the toxic effect. Susceptibility to the action of methotrexate is thought to result from polyglutamation of the drug which, by delaying its breakdown within and its excretion from the cell, favours prolonged action. The effect of methotrexate can be by-passed by folinic acid, generally given in the form of 5-formyl tetrahydrofolic acid. Carefully timed and dose-controlled administration of methotrexate with folinic acid has been found advantageous over the use of methotrexate alone in the treatment of some cancers. Thus large doses of methotrexate are commonly followed after 12-24 hours by folinic acid ‘rescue’. Similarly, administration of low dose methotrexate on alternate days and folinic acid on each succeeding day has proved to be a successful and low toxicity treatment for many patients with some forms of trophoblastic tumour (Bagshawe et al (1989) Brit. J. Ob. Gynae 96, 795-802). It has been shown (Kuefner et al (1989) Biochemistry 28, 2288-2297) that when methotrexate is modified by introducing an alanine moiety via an amide linkage to the a carboxyl of methotrexate the resulting compound is effectively excluded from cells and the toxicity of the alanine form is 50-100 fold less than that of native methotrexate in target cells in vitro. The alanine moiety is cleaved by the action of the enzyme carboxypeptidase A leaving native methotrexate. Alanine methotrexate is not metabolised by the enzyme carboxypeptidase G2. Carboxypeptidase G2 degrades methotrexate and natural folates by cleavage of the glutamic acid moiety. In the present example, the catalytic macromolecule of the system of the present invention comprises a macromolecule such as polyethylene glycol, a dextran or an immunoglobulin conjugated to carboxypeptidase A. Of course, a carboxypeptidase other than carboxypeptidase A but with the same substrate specificity may be used in place of carboxypeptidase A. Carboxypeptidase A is available from bovine and bacterial sources (for example from Calbiochem, Nottingham, UK) and is also present in human pancreas. The enzyme hydrolyses oligopeptides from the C terminal end of polypeptide chains, or from other compounds containing conjugated amino acids with a free carboxyl group. Carboxypeptidase A has a preference for aromatic residues. It is normally formed from mammalian sources by trypsinization of a complex assembly of three subunits produced by the pancreas. The CPA is conjugated to PEG or dextran by conventional techniques, for example as in WO 90/13540. The “first compound” is alanine methotrexate (Kuefner et al 1989) which is the prodrug from which the active drug methotrexate (the “second compound”) is generated by the action of carboxypeptidase A. The “inhibitor” is carboxypeptidase G2 conjugated to a macromolecular structure such as dextran. The purpose of this macromolecule is to confine CPG2 activity to the vascular compartment. For example, CPG2 may be coupled to soluble dextrans (Lomodex 40, Lomodex 70, Dextraven 110 and Dextraven 150, all trade marks; Fisons, Loughborough, Leics., UK) according to the method of Melton et al (1987). A volume of dextran preparation containing 1 g of dextran in 0.9% NaCl was diluted to 100 ml with 0.9% NaCl and reacted with cyanogen bromide (CNBr; Sigma, Poole, Dorset, UK). CNBr (0.5 g) was used for activating the 40- and 70,000 dalton dextrans and 0.4 g for the higher molecular weight dextrans. This reduction was necessary to prevent precipitation of the 110,000 and 150,000 dalton dextrans. The reaction mixture was vigorously stirred at room temperature and maintained at pH 10.7±0.1 units in a pH-stat (Radiometer, Copenhagen, Denmark) by addition of 2 M NaOH. The CNBr was added as a finely divided powder in two equal portions at an interval of 20 min; the second portion was allowed to react until the pH of the reaction mixture was stable at 10.7; the pH was then adjusted to 9.0 and the mixture dialysed against running water for 2 hr at 4° C. The pH was brought back to 9.0 with 1 M NaOH and 1 ml enzyme solution (1265 U; 2.3 mg) in 0.1 M Tris-HCl buffer, pH 7.3, was added. The mixture was reacted overnight at 4° C. after which 0.25 g glycine was added to block excess reactive sites. The mixture was stirred for a further 30 min and then concentrated to a volume of 40 ml in a model 202 concentrator using a PM10 ultrafiltration membrane (Amicon, Stonehouse, UK). The mixture (40 ml) was then chromatographed on a 1.3 liter bed volume of Sephadex G150 in a 4.4×87 cm column (Pharmacia, Uppsala, Sweden) and eluted with 0.05 M potassium phosphate buffer, pH 7.0. Fractions (10 ml) were collected and assayed for enzyme activity; carbohydrate content was determined by the phenol-sulphuric acid method (M. Dubois et al (1956) Analyt. Chem. 28, 350) using dextran-70 as standard in the range 0-100 &mgr;g/ml (Sephadex is a trade mark). The peak fractions were pooled and concentrated to a volume of 10-12 ml as before. Enzyme activity and carbohydrate content were determined and protein content measured by the Coomassie blue method (M. M. Bradford (1976) Analyt. Biochem. 72, 248) using bovine serum albumin fraction V as standard in the range 0-100 &mgr;g/ml. The concentrated material was filter sterilised (Millipore “Millex GS”, 0.22 &mgr;m pore size) and stored at −20° C. Millex GS is a trade mark. The macromolecule-carboxypeptidase A conjugate, if comprising enzyme of bovine origin, would be immunogenic. Similarly, a carboxypeptidase G2 macromolecule conjugate would also be immunogenic since CPG2 is bacterial in origin. It may be desirable, therefore, to reduce their immunogenicity or to employ means to induce immunosuppression or immune tolerance. 
 EXAMPLE 2 Method of Use Administration of a component capable of overcoming the host response to foreign protein may be started 48 hours before administration of the catalytic macromolecule. Initial tests may be performed to exclude as far as possible abnormal reaction by the patient to any of the protein components. The CPA conjugate is given intravenously, preferably by slow infusion, typically over 2 hours. Maximal tumour concentration of the CPA conjugate is achieved several hours later but at this time there are still high levels of CPA activity in plasma. It is desirable to eliminate this activity as far as possible before administering the prodrug. This elimination process is achieved by administration of a component (such as an anti-CPA antibody, which may be galactosylated) capable of achieving accelerated clearance or inactivation of CPA from non-tumour sites. This component is administered intravenously over several hours, typically 6-24 hours, or until enzyme is no longer detectable in plasma and may be infused at low concentration throughout the period of pro-drug administration. During this time enzyme in extracellular fluid diffuses back into the plasma as the plasma level of the enzyme falls. Tests for enzyme activity from plasma are continued for a period typically 8-24 hours to ensure that plasma CPA activity is not detectable. More of said component which eliminates the CPA activity is given if necessary. Alternative methods of accelerated clearance have been described. The CPG conjugate is then administered either by a series of bolus injections or by slow infusion, and it is preferred that the prodrug is administered simultaneously. Prodrug may be given as a series of bolus injections or by continuous infusion. Administration of the prodrug and CPG conjugate will normally continue for about 4-7 days. At about 7-10 days after administration of the CPA conjugate, it is desirable to review enzyme activity at tumour sites. Administration of the component capable of overcoming the host response to foreign protein is continued, the prodrug is discontinued, and the CPG conjugate may be discontinued. The CPA conjugate is reinfuse as previously, followed by the component which eliminates the plasma CPA activity as previously. Similar procedures to those previously described are followed before recommencing the prodrug. The cycle may be repeated. Limiting factors will be toxicity attributable to alanine methotrexate or the development of host antibodies to any of the foreign proteins employed. 
 EXAMPLE 3 Use of Quinazoline Antifolates A similar system to that disclosed in Examples 1 and 2 can be used with at least some members of a series quinazoline antifolates which have been described (Jones et al (1986) J. Med. Chem. 29, 468-472; Jodrell et al (1991) Brit. J. Cancer 64, 833-8; Harrap et al (1989) Advances in enzyme regulation 29, 161; Jackman et al (1991) Advances Enzyme Regulat. 31, 13; Jodrell et al (1990) Proc. Am. Assoc. Cancer Res. 31, 341. These agents differ from natural folates and methotrexate with respect to substitution for instance at the N 10 position and in the benzoyl ring but like natural folates and methotrexate have a terminal glutamate moiety linked to the benzoyl ring. Therefore at least some of the drugs in this series are inactivated by a peptide substitution such as an alanine in the &agr; position of the glutamate, and that such alanine- or other peptide-substituted derivatives are synthesized using the methodology described by Kuefner et al loc. cit. or variations thereof known in the art. Similarly, such quinazoline antifolates are deglutamated, and therefore inactivated, by carboxypeptidase G2 or a similar enzyme. These compounds are of particular interest because they act by inhibition of thymidylate synthetase. The chemical application of such drugs may be greatly extended by being administered in pro-drug form, converted to the active compound by carboxypeptidase A and the active drug in plasma degraded by carboxypeptide G2. 
 EXAMPLE 4 Production of Antibodies Discriminating Between Active Drug and Pro-drug In order to raise an antibody that would recognise the active drug (II) but not the pro-drug (I), a compound was synthesised which represented the region of greatest divergence between the two molecules (I) and (II). This region corresponds to the acid portion of the benzoic acid mustard drug (II). Since benzoic acid itself is not large enough to be immunogenic, it was considered that the most effective method of raising antibodies specific for the acid region would be to inoculate animals with a benzoic acid analogue that had been previously conjugated to keyhole limpet haemocyanin (KLH). A compound was synthesised that consisted of an L-lysine amino acid linked through an amide bond to aminobenzoic acid (VII). Then (VII) was conjugated to KLH by conventional methods using the &egr;-NH 2 groups on the lysine portion of the molecule, to produce the specific immunogen (VIII), all as described in WO 93/13806. 
 EXAMPLE 5 Production of a Monoclonal Antibody Reactive Against Carboxypeptidase G2 A monoclonal antibody raised against carboxypeptidase G2 is used for clearance and inactivation of residual enzyme activity at non-tumour sites. The monoclonal antibody was made in the following way. Balb/C mice (6-8 weeks old) were immunised with 50 &mgr;g CPG2 i.p. in incomplete Freund's adjuvant followed by two injections of CPG2 in complete Freund's adjuvant (50 &mgr;g CPG2 each, i.p.) at monthly intervals and with two daily injections (50 &mgr;g and 100 &mgr;g in PBS, i.v.) 2 days before fusion. Immune spleen cells were fused with non-immnunoglobulin secreting SP2/0 myeloma cells according to the hybridoma procedures of Köhler and Milstein (1975). The presence of anti-CPG2 antibodies was detected by a solid-phase indirect radioimimunoassay. A 1 &mgr;g ml −1 solution of CPG2 in 0.05 M phosphate buffer was placed in polyvinyl microtitre plates (100 ng, per well), allowed to dry, fixed with methanol and washed with PBS buffer containing a 0.05% Tween and 0.1% bovine serum albumim. Supernatant or purified antibody samples were diluted in PBS and incubated in the CPG2 coated microtitre plates (100 &mgr;l per well) at 37° C. for 4 h and then for −1 h with 125 I-labelled rabbit anti-mouse IgG. The wells were washed three times with PBS-Tween buffer between each stage and after final washing individual wells were cut and counted in a gamma counter. 
 EXAMPLE 6 Reduction of Residual Enzyme Activity at Non-tumour Sites It is desirable to inactivate the enzymatic portion of the enzyme-macromolecule conjugate at non-tumour sites, but not at the tumour. One method of achieving this effect is to administer to the patient being treated using the compounds of the invention antibodies raised against the enzyme portion which have been conjugated with galactose residues. A monoclonal antibody directed at CPG2 inactivates the enzyme. To prevent the antibody inactivating the enzymes at tumour sites, additional galactose residues are conjugated to it so that it can still inactivate enzyme in plasma when it is given by intravenous route but the inactivating antibody is rapidly removed from the plasma by galactose receptors on hepatocytes. The galactosylated anti-CPG2 monoclonal antibody is given to eliminate enzymic activity in plasma and then to give an amount of the non-galactosylated anti-CPG2 monoclonal antibody to inactivate residual enzyme activity in other non-tumour tissues. The monoclonal antibody is galactosylated using the following protocol. A stock solution of the activated derivative was made up as follows. Cyanomethyl 2,3,4,6-tetra-O-acetyl-1-thio-&bgr;-D-galactopyranoside (Sigma C-4141) &lsqb;400 mg&rsqb; in anhydrous methanol (10 ml) was treated with 5.4 mg of sodium methoxide in 1 ml of anhydrous methanol at room temperature for 48 hours. The mixture was kept in a 25 ml Quickfit conical flask fitted with a slightly greased stopper. A stock solution of monoclonal antibody (1.3 mg/ml) is prepared in 0.25 M sodium borate buffer, pH 8.5. Aliquots of the required amount of activated galactosyl derivative (80, 40, 20, 10 &mgr;l) are dispensed into 3 ml glass ampoules and evaporated to a glassy residue in a stream of nitrogen. A solution of the antibody (200 &mgr;g) is added mixed until the residue is dissolved. After 2 hours at room temperature, the solution is dialysed against three changes of PBS. The preparations are scaled up by taking multiples of the volumes mentioned above. 
 EXAMPLE 7 Cytotoxic Therapy with Trimetrexate and Folinic Acid Rescue The present invention may provide a means to allow more continuous anti-folate action at tumour sites whilst protecting normal tissues from anti-folate effects, as in WO 93/13805. The first step is to initiate immunosuppressive or immune tolerance inducing agents and this will normally occur not less than 2 days before the compound of the present invention is administered. If the catalytic macromolecule is non-immunogenic this step is omitted. A macromolecule-CPG2 conjugate is given by intravenous or other appropriate route. After several hours has been allowed for the conjugate to localise at tumour sites an antibody is given. This may be directed at the active site on the enzyme in which case additional galactose residues are attached to the antibody to ensure its rapid clearance via hepatocyte galactose receptors. Alternative mechanisms for rapid clearance of the conjugate from non-tumour sites have been described above. When enzyme levels have fallen to very low or undetectable levels in plasma, trimetrexate is given by bolus or by continuous infusions with the aim of maintaining a constant plasma concentration. Concurrently with the trimetrexate, folinic acid is given at a dose level sufficient to protect the normal tissues from trimetrexate toxicity. Folinic acid reaching tumour sites where CPG2 is located is deglutamated, as is folic acid, and rendered inactive before it can enter cells and protect them from trimetrexate. In this way normal tissues may be protected by folinic acid from the action of trimetrexate whereas the protective molecule is rapidly degraded at tumour sites. Other antifolate drugs which, like trimetrexate are not degraded by CPG2, may be substituted for trimetrexate. 
 EXAMPLE 8 Cytotoxic Therapy with Thymidylate Synthetase Inhibitors and Thymidine Rescue The same principle may be applied to other anti-metabolite therapies. For instance, considerable effort has been directed in recent years to the development of agents that block the key enzyme thymidylate synthetase such as CB3717 and ICI D1694 (Jodrell et al 1991, BJC 64, 833-8). Such agents are likely to suffer the same limitations as conventional cytotoxic agents in that they also block thymidylate synthetase in normal cells. Their action is reversible by thymidine. Thymidine administration may therefore be used to protect normal tissues from thymidylate synthetase inhibitors whilst the protective effect of the thymidine and endogenous thymidine may be limited at tumour sites by a thymidine degrading enzyme, such as dihydrothymine dehydrogenase, or thymidine kinase which would inhibit entry of thymidine into the tumour cells. The thymidine inhibitor enzyme or agent is delivered by the non-specific macromolecule, in accordance with the invention. Dihydrothymine dehydrogenase can be obtained from normal human lymphocytes (Shiotani & Weber 1989 Cancer Res. 49, 1090-1094) and therefore has the advantage that a macromolecule-enzyme conjugate of low immunogenicity may be produced by its conjugation to a non-immunogenic macromolecule. 
 EXAMPLE 9 Tumour Take Up of PEG-enzyme Conjugate and of Non-tumour Specific IgG-enzyme Conjugate In LS174T human colon cancers xenografted in nude mice, 1.81% of administered dose of a 125 I-polyethylene glycol-carboxypeptidase G2 was found per gram of tumour at 24 h and 1.2% was still present at 72 h (see FIG. 1 ). Native 125 I-CPG2 gave comparable values of 0.35% and 0.09%. Comparison of tissue distribution of 125 I anti-CEA monoclonal antibody conjugated to carboxypeptidase G2 in LS174T (CEA positive) and CC3 (CEA negative) xenografts gave the following results. At 24 h after intravenous injection, LS174T contained 1.4%, whereas CC3 contained 0.9% of the injected dose per gram of tumour. At 48 h, LS174T had 1.2%, whereas CC3 had 0.7% and, at 7 days, LS174T had 0.35%, whereas CC3 had 0.3%. Thus, the amount of conjugate retained in the non-CEA producing tumour was more than half that retained in the CEA producing tumour (S. K. Sharma, Thesis for PhD examination, London University, 1991). It has further been shown (S. K. Sharma thesis), over a wide range of dosages of an antibody enzyme conjugate (20-300 &mgr;g per mouse), that the percentage of injected dose per gram of tumour remained constant within experimental limits (1.57-1.77%). Thus, the less efficient delivery of enzyme to tumour by non-specific macromolecule, compared with a tumour specific antibody, can be compensated by an increase in administered dosage of about 2-fold. A further investigation of the distribution of CPG2 conjugated to PEG in nude mice bearing the human colon carcinoma LS174T was undertaken. Materials and Methods Methoxypolyethylene glycol-p-nitrophenol carbonate of mol. wt. 5000 was obtained from Sigma. Methoxypolyethylene glycol-p-nitrophenol carbonate of mol. wt. 2000 was synthesised following the method described (Veronese, F. M., Largajolli, R., Boccu, E., Benassi, C. A. and Schiavon, O. (1985) Surface modification of proteins—Activation of monomethoxy-polyethylene glycols by phenyl chloroformates and modification of ribonuclease and superoxide dismutase. App. Biochem. Biotech. 11, 141-152). 4-&lsqb;N,N-bis(2-chloroethyl)amino&rsqb;-phenoxy-carbonyl-L-glutamic acid (phenol mustard prodrug) was synthesised according to the published procedure and its structure is shown in the figure (Dowell, R., Springer, C. J., Davies, D. H., Hadley, E. M., Burke, P. J., Boyle, F. T., Melton, R. G., Connors, T. A., Blakey, D. C. and Mauger, A. B. (1996). New mustard prodrugs for antibody-directed enzyme prodrug therapy (ADEPT): Alternative to the amide link. J. Med. Chem. 39, 1100-1105). (This prodrug is described in WO 94/02450.) 1 Either 516 mg of methoxypolyethylene glycol p-nitrophenyl carbonate of mwt 5000 or 206.4 mg of methoxypolyethylene glycol p-nitrophenyl carbonate of mwt 2000 was added to 38.13 mg of CPG2 in 6.7 ml of 0.2 M sodium phosphate buffer pH 7.2 (Veronese, F. M., Largajolli, R., Boccu, E., Benassi, C. A. and Schiavon, O. (1985) Surface modification of proteins—Activation of monomethoxy-polyethylene glycols by phenyl chloroformates and modification of ribonuclease and superoxide dismutase. App. Biochem. Biotech. 11, 141-152). After 1 hr at room temperature unreacted PEG was removed in an ultrafiltration cell (Amicon) using an XM-50 membrane and 0.1 M sodium phosphate buffer, pH 7.2 as the dialysing solution. Polyethylene glycol modified CPG2 was separated from unmodified protein by FPLC gel filtration using a superose S/12 HR column (Pharmacia) equilibrated with 0.1 M sodium phosphate buffer pH 7.2 and eluted with the same buffer. Animal Studies The following figures (FIGS. 2 to 6 ) illustrate the biodistribution of carboxypeptidase G2 conjugated to PEG in nude mice bearing the human colon carcinoma LS174T. The mice were injected intravenously with the specified amount of enzyme (measured as enzyme units) using 4 mice per time point. Mice were killed at 24 h, 48 h, and 72 h after injection, in some cases at 96 h and 120 h. CPG2 activity in plasma was measured by spectrophotometric assay using methotrexate as the substrate. CPG2 activity in tumour and other tissues was measured by HPLC using methotrexate as the substrate. Unconjugated CPG2 clears rapidly from blood tumour and normal tissues. The biodistribution of native CPG2 and PEG-CPG2 at 72 hours is illustrated in FIGS. 7 and 8 . FIG. 2 ( a ). CPG2 conjugated to PEG 5000 mwt, average 32 PEG molecules per molecule of CPG2, 20 &mgr;g per mouse. Figure shows percentage of injected dose per gram in tissues. FIG. 2 ( b ). The corresponding tumour to normal tissue ratios. FIG. 3 ( a ). CPG2 conjugated to PEG 2000 mwt, average 27-PEG molecules per molecule of CPG2, 25 enzyme units per mouse. FIG. 3 ( b ). The corresponding tumour to organ ratios. FIG. 4 ( a ). Biodistribution of CPG2 activity following use of galactosylated SB43 (clearing antibody). Mice were given CPG2 conjugated to PEG 2000 mwt, average 22 mols PEG per molecule of CPG2, at time 0. Galactosylated monoclonal antibody SB43 was given 19, 20 and 21 hrs later by IP route. The mice were killed at 24 h post CPG2. FIG. 4 ( b ). Tumour to organ ratios with above protocol. FIG. 5 . CPG2 activity in LS174T tumours comparing 20 units of CPG2 conjugated to PEG 5000 or to monoclonal antibody A5B7-(Fab′) 2 conjugated to CPG2. FIG. 6 . Nude mice bearing LS174T tumours were given 25 enzyme units of CPG2-PEG (22 PEG molecules, PEG 5000) followed at 20 hr, 23 h, 26 h by galactosylated SB43. At 48 h post CPG2-PEG they received 25 mg/kg of a phenol prodrug intraperiotoneally, repeated to a total of 3 doses at 1 hr intervals. In relation to FIG. 4 ( a ) and 4 ( b ) these show good tumour to normal tissue concentration and ratios following use of the clearing antibody. However, it is not possible to administer the clearing antibody as efficiently in the mouse as in the human patient. At the therapeutic level in the LS174T tumour model I have shown significant growth delay compared to untreated controls when PEG 5000 at 22 PEGs per CPG2 are used with the and CPG2 clearance antibody SB43 and the phenol prodrug. I have investigated PEG (mol wt 5000) in the range of 13-32 PEG molecules per enzyme molecule (CPG2, mol wt 83,000); the higher the molecular weight the higher and more prolonged is the plasma concentration of enzyme achieved. The higher the plasma concentration the higher tumour concentration in the tumour model LS174T. Since one objective is to get as much enzyme into the tumour as possible and prolong the period for which an effective concentration of enzyme is sustained in the tumour the conjugate giving the highest concentration of enzyme in blood is preferred. However, the choice of conjugate also has to take account of the need to clear enzyme from blood after the desired concentration of enzyme has been achieved in the tumour(s) and before prodrug is given. A conjugate which is difficult to clear from blood would be disadvantageous so that compromise between these two requirements may be necessary. The downside is that the high and persistent plasma concentration of PEG (5000) 32-CPG2 indicates that more clearing antibody should be used to lower the concentration of active CPG2 in the plasma. In comparing PEG 5000 with PEG 2000, my studies show that tumour concentrations of enzyme are similar but plasma levels with PEG 2000 are lower. Thus, PEG with a molecular weight of 2000 in the range 20 to 40 PEG molecules per molecule of enzyme is preferred, giving a total molecular weight in the range of around 130-160 kDal, but associated water of hydration gives a molecular size equivalent to molecules of much greater weight. 
 EXAMPLE 10 PEG-NQO2 Conjugation Data NQO2 is a much smaller enzyme (mol. wt. 24000) than CPG2 (mol. wt. 80000). In order to ensure that NQO2 is not readily excreted by the kidneys after intravenous administration and thus fail to localise in tumours, it is desirable to ensure that the molecular weight of the conjugate is >70000. This was achieved by derivitisation and conjugation to polyethylene glycol mol. wt. 5000 (Peg 5000). Attachment of 14 molecules or more of Peg 5000 to each molecule of NQO2 resulted in a conjugate of estimated molecular weight of 90000. 2 TABLE 1 Coupling Moiety (boxed) and general METHOD structure of adduct PEG-Cyanuric chloride 2 PEG-succinimidyl active ester (succinimidyl succinate) 3 PEG-succinate mixed anhydride 4 PEG- phenylchloroformates 5 PEG- carbonyldiimidazole 6 PEG-succinimide carbonate 7 Poly (PEG-MA) anhydride 8 PEG-maleimide 9 PEG-acetaldehyde no coupling moiety present or ethylene oxide unit CH 3 (—O—CH 2 —CH 2 ) n —O—CH 2 —CH 2 —NH-protein PEG-sulphonic no coupling moiety present halogenide CH 3 (—O—CH 2 —CH 2 ) n —NH-protein PEG-phenyl glyoxal 10 METHOD Bond Co-product reference PEG-Cyanuric HCl (1,2,3) chloride PEG-succinimidyl amide bond N-hydroxy- (4,5,6) active ester bond succinimide ester (succinimidyl succinate) PEG-succinate mixed amide bond ester plus (7) anhydride ester bond ester-modified protein PEG- carbamate substituted (8) phenylchloroformates phenol PEG- carbamate imidazole (9,10) carbonyldiimidazole PEG-succinimide carbamate (11) carbonate Poly (PEG-MA) amide bond (12) anhydride PEG-maleimide amide bond oxidised (13,14) cyano- borohydride PEG-acetaldehyde secondary amine sulphonic acid (15) PEG-sulphonic secondary amine (16) halogenide PEG-phenyl glyoxal secondary amine (17) 3 TABLE 2 I25 I-PEG-CPG2 distribution in LS174T xenografts 24 hrs % ID/g tissue 1.61 ± 0.3 0.63 ± 0.07 0.98 ± 0.15 0.58 ± 0.09 0.36 ± 0.06 0.14 ± 0.038 1.81 ± 0.47 72 hrs 0.38 ± 0.08 0.26 ± 0.04 0.38 ± 0.06 0.14 ± 0.025 0.10 ± 0.02 0.03 ± 0.01 1.2 ± 0.21 
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