WO2014181085A1

WO2014181085A1 - Tumour therapy

Info

Publication number: WO2014181085A1
Application number: PCT/GB2014/051329
Authority: WO
Inventors: Kenneth Dawson Bagshawe
Original assignee: Kenneth Dawson Bagshawe
Priority date: 2013-05-09
Filing date: 2014-04-30
Publication date: 2014-11-13
Also published as: GB2527477A; GB201308363D0; GB201518731D0

Abstract

There is disclosed a therapeutic system comprising: (i) a prodrug; (ii) an enzyme that has an active site which is capable of converting the prodrug into a drug; (iii) a vascular-targeting agent; and (iv) an inhibitor which, following administration, reduces the level of enzyme activity. There is also disclosed a therapeutic system for the treatment of cancer employing the four components (i) - (iv) in which the four components are combined into two groups of components. The two groups of components may be formulated or modified such that one component is metabolically active before the other.

Description

Tumour Therapy

Field of the Invention

This invention relates to tumour therapy, particularly to

therapeutic systems comprising prodrugs and to the use of

systems in treating tumours .

Background of the Invention

Cancer remains a major cause of human morbidity and death. It often remains symptom free and undiagnosed until it is too disseminated for complete surgical excision or even for control by radiotherapy. Cancer is a disease of cell proliferation. Normal cell

proliferation is a strictly controlled activity that supports the well-being of an organism as a whole. In contrast, cell

proliferation in cancer cells is uncontrolled and is ultimately detrimental to the whole organism.

One of the features of most forms of cancer is the formation of tumours. The accumulation of cancer cells in a normal tissue is initially sustained by the blood supply to that tissue. As the mass enlarges, cells are pushed away from blood capillaries into areas that are poorly oxygenated. Chemical signals are sent out that result in existing vessels sprouting new vessels that supply nutrients and remove waste products from cells in the interior cell mass of the tumour.

The search for new treatments for cancer over the past 60 years has resulted in the development of a substantial number of small drugs that are focussed on the mechanism of cell division and can reach cancer cells in most parts of the body. They have proved able to eliminate a small group of cancers even when widely metastasised but their effect on many cancers is less complete and generally fails to do more than slow their progression. A major problem that has been encountered in the use of such chemotherapeutic agents that attack cancer cell division is that the dose of such agents that can be safely administered is limited by the harmful effect that such agents have on essential normal cell division. This imposes a dose limit known as the maximum tolerated dose. Another major obstacle is the drug resistance of cancer cells. Cancer cells are

genetically unstable, so that any sizeable number of them includes many variants. Anti-cancer agents kill the variants that are susceptible to their action but those variants not susceptible to their action repopulate . Since the 1980' s there have been numerous attempts to improve the specificity of cytotoxic drugs by conjugating them to monoclonal antibodies directed at antigens over expressed on tumours and known as tumour associated antigens (TAA's) . Antibody drug conjugates (ADC's) targets are generally expressed by some normal tissues and there remain no antigenic markers for some common cancers.

It was found that antibodies lost specificity for the target antigen if loaded with more than four drug molecules . Two recent

developments have given impetus to ADC s One was the development of agents such as auristatins and maytansine that are highly toxic to cells when administered alone but when conjugated to an antibody they have low toxicity. The other development has been the

conjugation of drugs to antibodies by cleavable linkages. The antibody drug conjugates are designed to be internalised by antigen expressing cells and for them to be processed by the cell's

lysosomes where catalytic cleavage of the linker releases the drug to kill the cell and probably neighbouring cells. Two such ADC's have been licensed, one for the treatment of Hodgkin's lymphoma and another for the treatment of HER2 expressing breast carcinomas. It is reported (Chemical and Engineering News 2014, 92, 3, 13- 21) that more than 30 ADC's are currently being developed. Linkage chemistry and the analytical methods to ensure homogeneity of ADC s are highly complex . If it were possible to generate cytotoxic drugs inside tumours and restrict their action to tumours, it should be possible to generate in tumours a much higher concentration of cell killing drug than can be delivered safely by conventional means .

Duran-Reynals , Am. J. Cancer 1939, 35, 98-107 reported that new blood vessels formed within several types of experimental tumour were more permeable to large molecules than blood vessels in other tissues. More recently, Mach et al . , Nature 1974, 248, 704-706 and Goldenberg et al . , New Eng. J. Med. 1974, 298, 1384-1388 reported that intravenously injected radiolabelled antisera, directed at cancer cell surface antigens, were retained in tumour extracellular fluid. It became clear that the vasculature of the inner cell mass of a tumour is permeable to macromolecules , whereas the peripheral cells of tumours are supplied by the normal vasculature of the tissue in which the tumour is growing and is impermeable to

macromolecules. The duality of tumour vasculature can be

distinguished as intratumoural and peripheral. The extracellular fluid is presumed to be continuous throughout the tumour. The fact that blood vessels of tumours are leaky to circulating

macromolecules has, for example, been reported by Dvorak et al . , Am J Pathol 1988, 133, 95-109. Consequently, macromolecules accumulate non-specifically in tumours.

The use of prodrugs is a potential approach for overcoming some of the limitations of chemotherapeutic agents. Prodrugs are compounds that need to be transformed into the active drug before they exhibit their pharmacological action. One method of transforming an anticancer prodrug into the active drug is enzyme catalysis and a number of enzymes and prodrug systems have been reported, Rooseboom, et al . , Pharmacological Reviews 2004, 56, 53-102.

WO 89/10140 describes a strategy to achieve local activation of prodrugs at a tumour and thus achieve the generation of a cytotoxic drug in tumours. It comprises a three component system in which (i) a first component comprises an antibody-enzyme conjugate, wherein the antibody fragment is capable of binding with a tumour-associated antigen, and the enzyme is capable of converting a prodrug into a cytotoxic drug; (ii) a second component is a prodrug which is capable of conversion to a cytotoxic drug; and (iii) a third component which is an antibody directed at the active site of the enzyme for clearing the enzyme from the blood. This general system, which is often referred to as "antibody-directed enzyme prodrug therapy" (ADEPT) . The ADEPT system has also been discussed in

Bagshawe, et al . , Br. J. Can. 1987, 56, 531 and in Bagshawe, et al . , Br. J. Can. 1988, 58, 700-703.

A small scale clinical trial with the three component ADEPT system resulted in several patients with terminal cancer having extended survival, Bagshawe, et al . , Tumour Targeting 1995, 1, 17-29.

Subsequent clinical trials that attempted to develop the ADEPT methodology used a simplified version that omitted the antibody used for clearing the enzyme from the blood. However, these attempts have had disappointing results, see Francis et al., Brit J. Cancer

2002, 87, 600-607 and Mayer et al., Clin Cancer Res. 2006, 12, 6509-

6516.

In addition, a limitation for approaches that use antibody

conjugates to bind to a tumour associated antigen is that such antibodies are not currently available for the majority of cancers.

WO 98/24478 describes a macromolecule prodrug therapy system in which the delivery of enzymes to tumour sites can be effected without the use of specific antibodies by taking advantage of the non-specific accumulation of macromolecules at tumour sites. WO 98/24478 discloses administering a three-component system that comprises (i) a prodrug; (ii) a macromolecule component that comprises a conjugate or fusion of (a) an enzyme that is capable of activating the prodrug and (b) a macromolecule such as a

polyethylene glycol; and (iii) an inhibitor that reduces the activity of the enzyme.

Although conventional cytotoxic agents, in maximum tolerated doses, can kill a high proportion of tumour cells, the genetic diversity of human cancer cell populations is such that cells resistant to their action repopulate the tumour. More recently the use of a vascular- disrupting agent in combination with a conventional cytotoxic agent has been proposed by, for example, Gerber et al . , Clin Cancer Res 2011, 17, 6888-6896. Vascular-disrupting agents take advantage of the differences between the blood vessels of normal tissues and those of tumour tissues to target and destroy existing tumour vasculature .

Zoratto, Onco Targets and Therapy 2012, 20, 199-211 discloses the use of an antiangiogenic agent in combination with conventional cytotoxic agents in the treatment of colorectal cancer.

Antiangiogenic agents inhibit key factors required for new blood vessel development.

Pedley et al., Cancer Res 1999, 59, 3998-4003 discloses the

combination of a vascular-disrupting agent with an antibody directed at a tumour associated antigen that is linked to an enzyme for activation of a prodrug. Increased retention of the enzyme in tumours and increased efficacy in a mouse model were reported.

However, this study did not incorporate measures to ensure that the enzyme activity in the blood was reduced when the prodrug was administered. Furthermore, it is well recognised that antigenic targets are heterogeneously distributed in epithelial cancers and binding enzyme to them can result in non-uniform enzyme

distribution. Moreover, such antigenic markers are available only for a limited range of cancers.

There remains a need for therapeutic systems that alleviate the problems in the prior art. In particular, there is a need for therapeutic systems that are applicable to a much wider range of cancers, indeed to most carcinomas and sarcomas characterised by tumour formation. There is also a need for therapeutic systems that are capable of delivering a much higher concentration of cytotoxic drugs to cancer sites than can be achieved with conventional therapy or with antibody-drug conjugates without increased recipient toxicity. Summary of the Invention

In a first aspect, the present invention provides a therapeutic system comprising:

(i) a prodrug;

(ii) an enzyme that has an active site which is capable of converting the prodrug into a drug;

(iii) a vascular-targeting agent; and

(iv) an inhibitor which, following administration, reduces the level of enzyme activity.

In a further aspect, there is provided a therapeutic system for use in the treatment of a tumour in a patient wherein the therapeutic system comprises:

(i) a prodrug;

(ii) an enzyme that has an active site which is capable of converting the prodrug into a drug; and

(iii) a vascular-targeting agent; and

Without wishing to be bound by theory, the present invention makes use of the tumour vasculature and the ability of macromolecules to accumulate non-specifically in tumours. Many enzymes come into the category of macromolecules and are large enough not to be excreted in urine.

To take advantage of an enzymes' potential to activate prodrugs molecules it is necessary to achieve a situation in which the enzyme is present in an adequate concentration in tumour extracellular fluid but absent from blood and other tissue.

When an enzyme is injected into a tumour bearing host, only a relatively small proportion of the injected dose enters tumour extracellular fluid and the majority of the dose remains in the blood. It is therefore necessary to ensure the injected enzyme that remains in the blood is inactivated and/or cleared from the blood. Administration of an inhibitor is used to inactive and/or accelerate clearance of the enzyme remaining in the blood. Accelerated clearance of the enzyme from blood is achieved by use of an

inhibitor that comprises a moiety that ensures that it clears rapidly from the blood via recticulo endothelial or hepatic routes and is, thus prevented from entering tumours where it could

potentially inactivate the enzyme in the tumour extracellular fluid.

An example of such a moiety is a glycosyl moiety that ensures rapid uptake by hepatic sugar receptors. Thus, enzyme loss from the blood is accelerated as compared to tumour extracellular fluid. In this way a great differentiation in enzyme concentration between normal and tumour tissues can be attained.

Detailed Description of the Invention

Defini tions

The term "tumour" is to be understood as referring to an abnormal mass of tissue, the growth of which exceeds and is uncoordinated with that of the normal tissues, and persists in the same excessive manner after cessation of the stimuli which evoked the change. The tumour may be present in any anatomical site in a patient.

The term "derivative" refers to the replacement of one or more, preferably from one to three, hydrogen atoms with one or more substitutents and/or the use of a pharmaceutically acceptable salt. Such substituents include such groups as Cl-6 acyl, C1-C6 alkyl, Cl- 6 alkanoyl, C1-C6 alkoxy, C1-C6 alkylthio, hydroxy, nitro, halo, carboxy, carboxylic esters (with a C1-C6 ester group) , cyano, C1-C6 haloalkyl, C1-C6 haloalkoxy, C1-C6 alkoxyalkyl, C1-C6

haloalkoxyalkyl , amino, carboxamido, amino, mono(Cl-C6 alkyl) amino, di (C1-C6 alkyl) amino, C1-C6 alkylsulfonyl , C1-C6 alkylsulfonylamino, aryl, arylalkyl and the like.

The general chemical terms used in the formulae above have their usual meanings. For example, the term "alkyl" includes such groups as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, 3-pentyl, neopentyl, hexyl, and the like. The term "aryl" refers to an aromatic ring or heteroaromatic ring and includes such groups as furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, isoxazolyl, isothiazolyl , imidazolyl, pyrazolyl,

tetrazolyl, phenyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiadiazolyl , oxadiazolyl, naphthyl, indanyl, fluorenyl, quinolinyl, isoquinolinyl , benzodioxanyl , benzofuranyl , benzothienyl, and the like. In some embodiments the aryl group has 3 to 15 ring members.

The terms "acyl" and "alkanoyl" include such groups as formyl, acetyl, propanoyl, butanoyl, pentanoyl, and the like.

The term "halo" means fluoro, chloro, bromo, and iodo.

It is understood that the terms described herein may be combined in chemically relevant ways. For example, the term "arylalkyl" refers to an optionally substituted aromatic ring or heteroaromatic ring linked to an alkyl chain, including but not limited to benzyl, tolyl, 2-, 3-, and 4-picolinyl, pyrimidinylethyl , 2- (thien-2- yl) propyl, and the like. In one embodiment the alkyl chain is a Cl- C6 alkyl chain.

Pharmaceutically acceptable salts of the compounds of formula (I) comprise the acid addition and base salts thereof.

Suitable acid addition salts are formed from acids which form non- toxic salts. Examples include the acetate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide,

hydroiodide/iodide , isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate , naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinofoate salts. Suitable base salts are formed from bases which form non-toxic salts. Examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine , diolamine, glycine, lysine,

magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts.

The term "antibody" includes any substance produced by an immune system in response to an antigenic compound. Suitably, an

"antibody" refers to an intact immunoglobulin or an antigen binding portion thereof that competes with the intact antibody for specific binding to the antigen. Antibodies include IgG, the various subclasses of IgG, IgA, IgD, IgE, IgM the subcomponents of

antibodies known as Fabl and Fab2, single chain antibodies with multispecific binding sites. An "immunoglobulin" is a tetrameric molecule. In a naturally occurring immunoglobulin, each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25,000 Daltons) and one "heavy" chain (about 50,000-70,000 Daltons) . The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa or lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. See generally, Fundamental Immunology Ch . 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes) . The variable regions of each light/heavy chain pair form the antibody binding site such that an intact immunoglobulin has two binding sites.

"Antibody fragment" means antigen-binding fragments and analogues of an antibody, typically including at least a portion of the antigen binding or variable regions (e.g. one or more CDRs) of the parental antibody. An antibody fragment retains at least some of the binding specificity of the parental antibody. Typically, an antibody fragment retains at least 10% of the parental binding activity when that activity is expressed on a molar basis. Preferably, an antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the parental antibody's binding affinity for the target. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab' )2, and Fv fragments; diabodies; linear antibodies;

single-chain antibody molecules, e.g., sc-Fv, unibodies (technology from Genmab) ; nanobodies (technology from Domantis); domain

antibodies (technology from Ablynx) ; and multispecific antibodies formed from antibody fragments . Engineered antibody variants are reviewed in Holliger and Hudson (2005) Nat. Biotechnol. 23:1126- 1136.

Therapeutic System

The therapeutic system comprises: (i) a prodrug; (ii) an enzyme that has an active site which is capable of converting the prodrug into a drug; (iii) a vascular-targeting agent; and (iv) an inhibitor which, following administration, reduces the level of enzyme activity.

Prodrug

The term "prodrug" as used in this application refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form [see, e.g., D.E.V. Wilman, "Prodrugs In Cancer Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382 (615th Meeting, Belfast 1986) and V. J. Stella et al . , "Prodrugs: A Chemical Approach To Targeted Drug Delivery", Directed Drug

Delivery, R. Borchardt et al . (ed.), pp.247-267 (Humana Press

1985) ] . The larger the differential in toxicity between the parent drug and the prodrug, the greater the amount of prodrug that can be

administered for an acceptable level of toxicity. Drugs with large differentials in toxicity compared to the prodrugs (e.g. 6500:1) are known and, for example, include a series of prodrugs based on duocarmycin, a potent alkylating agent as disclosed by Tietze et al., Chem. Eur. J., 2011, 17, 1922-1929.

The prodrug may be any anti-cancer prodrug that is capable of being converted by an enzyme into an anti-cancer drug. The anti-cancer drug may be any existing anti-cancer drug such as an alkylating agent; an agent which intercalates in DNA; an agent which inhibits any key enzymes such as dihydrofolate reductase, thymidine

synthetase, ribonucleotide reductase, nucleoside kinases or

topoisomerase; or an agent which effects cell death by interacting with any other cellular constituent. An example of a topoisomerase inhibitor is Etoposide.

Preferably the prodrug (i) is a prodrug of a cytotoxic drug and is selected from the group consisting of alcohols, adenosine-containing prodrugs, amino acid-containing prodrugs, beta-lactam-containing prodrugs, cephalosporin-containing prodrugs, cytosine-containing prodrugs, glucouronide-containing prodrugs, glutamate-containing prodrugs, glycosylated prodrugs, nitro-containing prodrugs, peptide- containing prodrugs, phenoxyacetamide-containing prodrugs,

substituted-phenoxyacetamide-containing prodrugs, phenylacetamide- containing prodrugs, substituted-phenylacetamide-containing

prodrugs, phenylacetyl-containing prodrugs, phosphate-containing prodrugs, purine-containing prodrugs, riboside-containing prodrugs; saccharide-containing prodrugs and sulfate-containing prodrugs.

Preferably the prodrug is selected from alcohols, adenosine- containing prodrugs, amino acid-containing prodrugs, cephalosporin- containing prodrugs, cytosine-containing prodrugs, glucouronide- containing prodrugs, glutamate-containg prodrugs, glycosylated prodrugs, beta-lactam-containing prodrugs, nitro-containing

prodrugs, peptide-containing prodrugs, phenoxyacetamide-containing prodrugs, substituted-phenoxyacetamide-containing prodrugs,

pheny1acetamide-containing prodrugs , substituted-phenylacetamide- containing prodrugs, phenylacetyl-containing prodrugs, phosphate- containing prodrugs, purine-containing prodrugs, riboside-containing prodrugs; saccharide-containing prodrugs and sulfate-containing prodrugs wherein the drug is selected from the group consisting of acetoaldehyde, 9-amino-camptothecin; aminopterin; 5- (Azaridin-4- hydroxy-amino-2-nitro-benzamide; bleomycins; 4- [N, N-bis (2- iodoethyl ) amino ] -phenol ; carbonothionic difluoride; carminomycin; dactinomycin; daunomycin; 4-desacetyl-vinblastine-3-carboxylic acid hydrazide [DAVLBHYD] ; doxorubicin; duocarmycins (including CC-1065, duocarmycin A and duocarmycin SA) ; epirubicin; esperamicins ;

etoposide; 2-fluoroadenine ; 5-fluorouracil ; ganciclovir-triphophate; methotrexate; 6-methylpurine [MeP] ; methylselenol ; mitomycins

(including mitomycin C) ; mustard and derivatives thereof (including melphalan, p- [N, -bis (2-chloroethyl ) -amino] phenol (POM), azobenzene mustards, benzoic acid mustard, mustard, aniline mustard, phenol mustard, phenylenediamine mustard, isophosphoramide mustard, phosphoramide mustard, p-hydroxyaniline mustard and fluoroinated derivatives thereof) ; oxazolidinone; palytoxin; cis-platinum and cis-platinum analogues (including 4' -carboxyphthalato (1, 2- cyclohexane-diamine) platinum) ; tenyposide; taxol; and derivatives of these drugs .

In one aspect, the prodrug is selected from alcohol (e.g. ethanol) , etoposide phosphate; mitomycin phosphate; p- [N, iV-bis (2- chloroethyl ) amino ] -phenyl phosphate; N- (4-phosphonooxy) - phenylacetyl ) -doxorubincin; methotrexate-alanine, 4- ( [2- chloroethyl] [2-mesyloxyethyl] amino) -benzoyl-L-glutamic acid; 4-N,N- bis- (2-iodoethyl) aminophenoxycarbonyl L -glutamic acid; 1- ( 3 - Furanyl) -4-hydroxy-l-pentanone; N-3-bis (2-chloroethyl) -1,3,2- oxazaphosphinan-2 -amide-2 -oxide; (RS) -N, N-bis (2-chloroethyl ) -1 , 3 , 2- oxazaphosphinan-2-amine 2-oxide; 5-fluorocytosine; cyanophenylmethyl-beta-D-glucopyranosiduronic acid; p-hydroxyaniline mustard glucuronide; epirubicin glucuronide; glucuronidated Nor- nitrogen mustard; glucuronidated 9-amino-camptothecin; glucuronide mustard; nitrogen mustard-cephalosporin p-phenylene diamine; 7- (Phenylacetamido) -cephalosporin mustard; 7- (4- carboxybutanamido) cephalosporin mustard; cephalosporin-doxorubicin; cephalothin doxorubicin; vinblastine-cephalosporin;

vincaleukoblastin-23-oic acid, 04-deacetyl-, 2- ( ( (2-carboxy-8-oxo-7- ( ( 2 -thienylacetyl) amino) -5-thia-l-azabicyclo (4.2.0)oct-2-en-3- yl) methoxy) carbonyl) hydrazide, S-oxide, (3 (5S, 6R, 7R) ) ; cephalosporin mustard; cephalosporin- 4 ' -carboxyphthalato ( 1 , 2-cyclo- hexenediamine) platinum; PROTAX; cephalosporin mitomycin C;

cephalosporin carbamate derivative of melphalan; selenomethionine; trifluoromethionine ; 5- (Azaridin-l-yl) -2, 4-dinitrobenzamide ;

doxorubicin-lV-p-hydroxyphenoxy-acetamide ; melphalan-N-p- hydroxyphenoxy-acetamide ; N- (4 ' -hydroxyphenylacetyl ) palytoxin; N- (phenylacetyl ) doxorubicin; N- (phenylacetyl ) melphalan; doxorubicin-N phenoxyacetyl ; fludarabine; 6-methylpurine 2 ' -deoxyriboside ; 2- amino-9- { [ ( 1 , 3-dihydroxy-propan-2-yl ) oxy] methyl } - 6 , 9-dihydro-3if- purin-6-one; and derivatives thereof.

Preferably, the prodrug is water soluble.

Preferably, the prodrug is stable at room temperature and/or blood temperature .

Preferably, the prodrug is water soluble and is stable at room temperature and/or blood temperature.

Enzyme

Suitably the enzyme is any enzyme that is capable of converting a prodrug into a drug. A range of enzymes have been reported for converting prodrugs into their more active, cytotoxic drug forms. As used herein, the term "enzyme" includes any fragment of an enzyme that is capable of converting a prodrug into the cytotoxic drug. Preferably the enzyme has at least one epitope remote from the active site of the enzyme. The "active site" of the enzyme is the site at which the catalytic reaction of the enzyme occurs. Preferably the enzyme is of non-human origin. Such enzymes comprise enzymes that are not found in the human body. Such enzymes may comprise human enzymes that have one or more amino acid

substitutions so as to activate a prodrug that is not activated by the corresponding native enzymes. Examples of such enzymes are described in Afshar, et al . , Mol Cancer Ther 2009, 8, 185-193 and

Harding, et al . , Mol Cancer Ther 2005, 4, 1791-1800. Such minimally mutated human enzymes often have the advantage that they can be administered to a human without acting as an antigen. However, when minimally mutated human enzymes are used, the prodrug must be tailored to be a substrate for the mutated enzyme but not for the native enzyme.

Preferably the enzyme is selected from carbohydrate-cleaving enzymes, glycosyltranferases , hydrolases, lyases, nucleases, oxidoreductases , phosphorylases , phosphotransferases, proteases, DL- racemases and mixtures thereof.

More preferably, the enzyme is selected from amidases, deaminases, exopeptidases , endopeptidases , glycosidases , kinases, lactamases, lipases, lyases that cleave carbon-sulfur bonds, nucleases,

monooxygenases, oxidases, phosphatases, phosphorylases, reductases, sulfatases and mixtures thereof.

More preferably the enzyme is selected from alcohol dehydrogenase, alkaline phosphatases, aminopeptidases , arylacyl amidases,

arylsulfatases , azoreductases , carboxypeptidases , cyctochrome P450, cytosine deaminases, DT-diaphorases , beta-galactosidases , alpha- glactosidases , glucose oxidases, beta-glucosidases , beta- glucuronidases , beta-lactamases , lactoperoxidases , alpha- mannosidases , methionine gamma-lyases , ribonucleasess,

nitroreductases, pencillin amidases, purine-nucleoside phosphorylases , thrombolysins , thymidine kinases, urokinases, xanthine oxidases and mixtures thereof.

More preferably the enzyme is selected from alcohol dehydrogenase, alkaline phosphatase, carboxypeptidase, cyctochrome P450, cytosine deaminase, beta-galactosidase, beta-glucosidase, beta-glucuronidase, beta-lactamase , methionine gamma-lyase, nitroreductase, pencillin amidase, purine-nucleoside phosphorylase , thymidine kinase and mixtures thereof.

More preferably the enzyme is selected from alchol dehydrogenase (EC 1.1.1.1), alkaline phosphatase (EC 3.1.3.1), carboxypeptidase A (EC 3.4.17.1), carboxypeptidase G (EC3.4.19.9) , cyctochrome P450 (EC 1.14.14.1), cytosine deaminase (EC 3.5.4.1), beta-galactosidase (EC 3.2.1.23), beta-glucosidase (EC 3.2.1.21), beta-glucuronidase (EC 3.2.1.31), beta-lactamase (EC 3.5.2.6), methionine gamma-lyase (EC 4.4.1.11), nitroreductase (EC 1.1.284), pencillin amidase (EC

3.5.1.11), purine-nucleoside phosphorylase (EC 2.4.2.1), thymidine kinase (EC 2.7.1.21) and mixtures thereof.

Suitably the carboxypeptidase may be selected from carboxypeptidase A, B, G, Gl and G2.

Suitably the penicillin amidase may be selected from penicillin-V- amidase and penicillin-G-amidase .

In one aspect, the enzyme is an enzyme which does not specifically bind to a tumour antigen but is capable, following administration of being taken up by a tumour.

Preferably the enzyme is of a molecular size larger than the renal threshold for excretion. Preferably, the enzyme is larger than 60,000 Daltons, preferably larger than 70,000 Daltons. Following administration, the enzyme is capable of being taken up into a tumour. In one aspect the enzyme is a naked enzyme, that is the enzyme is not fused or conjugated to any carrier or targeting agent.

In a preferred aspect, the enzyme (ii) is fused or conjugated to a macromolecule. Hence, in such an aspect, this component contains (a) an enzyme and (b) a macromolecule.

Preferably, the macromolecule is a macromolecule which does not specifically bind to a tumour antigen, but which is capable, following administration, of being taken up by a tumour. Hence, for example, it does not consist of an antibody or part thereof which binds specifically to a tumour-associated antigen. It may

nevertheless comprise non-specific immunoglobulins.

Preferably, the macromolecule used m the invention is hydrophilic and is characterised by being soluble in body fluids and in

conventional fluids for parenteral administration. Suitably, the macromolecule is biodegradable so that systemic accumulation durin repeated administration is avoided. Clearly, however, it must not degraded so fast as to fail to accumulate at the tumour site.

The macromolecule may be conjugated to one or more enzyme molecules by simple chemical methods, using bi-functional agents which do not degrade the attached enzyme. Preferably, the starting macromolecule confers reduced immunogenicity on an immunogenic enzyme to which it is conjugated. acromolecules that are available as sub-units and are not

biodegradable may be 1inked by biodegradable linking units so that the non-biodegradable components are filtered through the kidneys and excreted in the urine .

Alternatively, it is preferred if the polymer used to make the macromolecule is not biodegradable then the molecular weight of any non-biodegradable portion of the conjugate should be less than the renal threshold so that after degradation of the biodegradable portion the residual non-biodegradable portion is excreted through the kidneys .

Whereas some macromolecules are not known to be internalised by cells others, such as N- (2-hydroxypropyl) methylacrylamide, are internalised through more than one mechanism (Duncan et al . , STP Pharma Sciences, 1996, 6, 237-263) .

Conveniently, the macromolecule may be any of a polyethylene glycol; a dextran; a polyamino acid, such as or may be polyaspartic acid or poly-L-lysine; or a non-tumour-specific protein such as an

immunoglobulin, an albumin, a transferrin; an hydroxypropyl

methylacrylamide; a copolymer of styrene and maleic anhydride; a polyvinyl pyrrolidone; or a polyethyleneimine .

Preferably, the macromolecule is a polyethylene glycol; a dextran; a polyamino acid; a non-tumour specific protein such as an

immunoglobulin, an albumin; or hydroxypropyl methylacrylamide.

Preferably, the macromolecule is polyethylene glycol (PEG) .

Derivatisation of proteins with polyethylene glycol has been demonstrated numerous times to increase their blood circulation lifetimes as well as decrease their antigenicity and immunogenicity . Use of PEG is discussed in Caliceti and Verovese, Advanced Drug Delivery Reviews, 2003, 55, 1261-1277.

Enzymes that are fused or conjugated to macromolecules such as PEG have been found to accumulate in tumours partly because they have prolonged half lives in blood.

Preferably, when fused or conjugated with a macromolecule the enzyme has a total molecular weight that exceeds that of the renal

threshold for urinary excretion. Preferably, when fused or

conjugated with a macromolecule the enzyme component has a total molecular weight of at least 60,000 Daltons, preferably at least 70,000 Daltons, as this helps the blood concentration to be sufficient to provide an effective blood: tumour concentration gradient. A molecular weight of up to at least 800 000 is generally suitable, for example up to 160,000. In one aspect, the total molecular weight of the enzyme component is at least 500,000

Daltons . The total molecular weight includes the molecular weight of the enzyme and the molecular weight of the macromolecule, if present, that is fused or conjugated thereto. Such a macromolecule is preferably one which is not readily captured by the

reticuloendothelial system. The molecular weights given exclude any water of hydration.

When a macromolecule is present, preferably the macromolecule has a molecular weight of at least 1000, more preferably, the

macromolecule has a molecular weight of at least 2000.

A wide variety of methods are known in the art for fusing or conjugating proteins (such as enzymes) to macromolecules (such as PEGs) and a number of suitable methods are discussed in W098/24478 and in the references cited therein.

In a preferred aspect, the enzyme (ii) is fused or conjugated to a protected saccharide. Hence, in such an aspect, this component contains (a) an enzyme and (b) a protected saccharide. The protected saccharide is protected with a protecting group.

Suitable protecting groups are well-known in the art and, for example, may be found in Protecting Groups in Organic Synthesis, Theodora W. Greene and Peter G. . Wuts, published by John Wiley & Sons Inc. Preferably, the protected saccharide is protected with a phosphate protecting group.

Preferably, the protected saccharide comprises a mono-saccharide . Preferably, the protected saccharide comprises a saccharide selected from glucose and mannose.

In a further aspect, the enzyme (ii) is fused or conjugated to a macromolecule and a protected saccharide. Hence, in such an aspect, this component contains (a) an enzyme and (b) a macromolecule and (iii) a protected saccharide.

In a further aspect, the enzyme (ii) is administered in the form of a complex with an antibody or an antibody fragment that links to the enzyme at an epitope remote from the active site of the enzyme.

Such an antibody or antibody fragment is different from the agent (iii) of the therapeutic system and forms a complex by binding with a different epitope on the enzyme than that targeted by the agent (iii) . Suitably the antibody or antibody fragment may be a bivalent antibody which has binding sites for two different epitopes on the enzyme. Suitably the antibody may be bivalent or a multivalent. Suitably the antibody or antibody fragment is monoclonal and humanized. Suitably the antibody or antibody fragment is selected from IgG, IgA, IgD, IgE, IgM, Fabl and Fab2. Suitably the antibody or antibody fragment is an IgG antibody.

Prodrug and enzyme systems The prodrug and enzyme system may be any of those previously proposed. Suitable examples of prodrug and enzyme systems are included in the following table 1.

Table 1

Enzyme Prodrug Drug Ref .

Alkaline Etoposide phosphate Etoposide (1) phosphatase Mitomycin phosphate Mitomycin (2) p- [Ν,Ν-bis (2-chloroethyl) amino] - p- [N,N-bis (2- (3) phenyl phosphate (POMP) chloroethyl) - amino ] henol

(POM)

N- (4-phosphonooxy) - doxorubincin (4) phenylacetyl ) -doxorubincin

Carboxypeptidase Methotrexate-amino acids Methotrexate (5)

(e.g. Methotrexate-alanine) 4- ( [2-chloroethyl] [2- Benzoic acid (6) mesyloxyethyl ] amino) benzoyl-L- mustard glutamic acid (C DA)

4-N,N-bis (2-iodoethyl) amino- 4- [N, N-bis (2- (31) phenoxycarbonyl L -glutamic acid iodoethyl) mino] - [ZD2767P] phenol

Cytochrome P450 1- (3-Furanyl) -4-hydroxy-l- A furan epoxide (7) pentanone

[4-iodomeanol]

N-3-bis (2-chloroethyl) -1,3,2- Isophosphoramide (8) oxazaphosphinan-2-amide-2- mustard oxide

[Ifosfamide]

(RS) -N, N-bis (2-chloroethyl) - phosphoramide (8) 1,3, 2-oxazaphosphinan-2-amine mustard 2-oxide

[Cyclophosphamide]

Cytocine 5-fluorocytosine 5-fluorouracil (9) deaminase

Beta-glucosidase Cyanophenylmethyl-beta-D- (10) glucopyranosiduronic acid

Beta- p-hydroxyaniline mustard p-hydroxyaniline (10) glucuronidase glucuronide mustard

Epirubicin glucuronide Epirubicin (10)

Glucuronidated iVor-nitrogen Oxazolidinone (11) mustard

Glucuronidated 9-amino- 9-amino- (12) camptothecin camptothecin

Glucuronide mustard mustard (13)

Beta-lactamase Nitrogen mustard-cephalosporin phenylene diamine (10) p-phenylene diamine mustard

7- (Phenylacetamido) - phenylene diamine (14) cephalosporin mustard

mustard

[CM]

7- (4- phenylene diamine (15) carboxybutanamido) cephalosporin

mustard

[CCM]

doxorubicin derivatives; doxorubicin (16) including cephalosporin- doxorubicin [C-Dox] cephalothin doxorubicin doxorubicin (17) [PRODox]

Vinblastine derivative- vinvlastine (10) cephalosporin

Vincaleukoblastin-23-oic acid, 4-desacetyl- (18)

04-deacetyl-, 2- ( ( (2-carboxy-8- vinblastine-3- oxo-7- ( (2-thienylacetyl) amino) - carboxylic acid

5-thia-l-azabicyclo(4.2.0)oct-2- hydrazide

en-3-yl ) methoxy) carbonyl ) - [DAVLBHYD]

hydrazide, S-oxide, (3 (5S, 6R, R) )

[LY 266070]

Cephalosporin mustard mustard (10) cephalosporin-4 ' - 4' -carboxy(19) carboxyphthalato (1, 2-cyclo- phthalato (1,2- hexenediamine) platinum cyclohexane- [cephalosporin-DACCP, ] diamine) platinum

Taxol derivative synthesized taxol (20) by substituting the C-3'

position of cephalothin with 2'- (gamma-aminobutyryl ) taxol

[PROTAX]

cephalosporin mitomycin C mitomycin C (21) a cephalosporin carbamate 4- [bis (chloro- (22) derivative of melphalan

ethyl ) amino ] - [C-Mel]

phenylalanine

[melphalan]

Methionine Selenomethionine methyl selenol (23) gamma-lyase Trifluoromethionine Carbonothionic (24) difluoride

Nitroreductase 5- (Azaridin-l-yl-) -2,4- 5- (Azaridin-4- (25) dinitrobenz amide hydroxy-amino-2- [CB 1954] nitro-benzamide

Pencillin doxorubicin-iV-p-hydroxyphenoxy- doxorubicin (26) acetamide

amidase

[DPO]

melphalan-iV-p-hydroxyphenoxy- 4- [bis (chloro- (26) acetamide

ethyl ) amino ] - [MEIPO]

phenylalanine

[melphalan]

N- (4 ' - palytoxin (27) hydroxyphenylacetyl) palytoxin

[NHPAP] N- (phenylacetyl ) doxorubicin doxorubicin (4)

N- (phenylacetyl ) melphalan 4- [bis (chloro- (4) ethyl ) amino ] - phenylalanine

[melphalan]

Doxorubicin-N phenoxyacetyl doxorubicin (10)

Purine- Fludarabine 2-fluoroadenine (28) nucleoside 6-methylpurine 2'- 6-methylpurine (29) phosphorylase deoxyriboside [MeP]

[ ePdR]

Thymidine kinase 2-amino-9-{ [ ( 1 , 3-dihydroxy- Ganciclovir- (30) propan-2-yl) oxy] methyl } -6, 9- triphophate

dihydro-3/i-purin- 6-one

[Ganciclovir]

Other known prodrug and enzyme systems include aminopeptidases for 2-alpha-aminocyl MTC prodrugs; thrombolysin for thrombin prodrugs; aryl sulphatases for sulphated prodrugs; beta-glucuronidase for beta-glucuronomide anthracyclines ; alpha-galactosidase for

amygdalin; beta-galactosidase for beta-galactose anthracycline ;

azoreductase for azobenzene mustards and DT-diaphorase for 5- (Azaridin-l-yl-) -2, 4-dinitrobenzamide; glucose oxidase for glucose; and xanthine oxidase for xanthine.

Drug

Examples of cytotoxic drugs that can be derivatized into a prodrug form for use in this invention include, but are not limited to, drugs selected from the group consisting of acetoaldehyde, 9-amino- camptothecin; aminopterin; 5- (Azaridin-4-hydroxy-amino-2-nitro- benzamide; bleomycins; 4- [N, N-bis (2-iodoethyl) amino] -phenol;

carbonothionic difluoride; carminomycin; dactinomycin; daunomycin;

4-desacetyl-vinblastine-3-carboxylic acid hydrazide [DAVLBHYD] ;

doxorubicin; epirubicin; esperamicins ; etoposide; 2-fluoroadenine;

5-fluorouracil ; ganciclovir-triphophate; methotrexate; 6- methylpurine [MeP] ; methylselenol ; mitomycins (including mitomycin C) ; mustard and derivatives thereof (including melphalan, p-[N,N- bis (2-chloroethyl) -amino] phenol (POM), azobenzene mustards, benzoic acid mustard, mustard, aniline mustard, phenol mustard,

phenylenediamine mustard, Isophosphoramide mustard, phosphoramide mustard, p-hydroxyaniline mustard and fluoroinated derivatives thereof) ; oxazolidinone; palytoxin; cis-platinum and cis-platinum analogues (including 4' -carboxyphthalato (1, 2-cyclohexane-diamine) platinum); teniposide; taxol; and derivatives of these drugs.

The generated drug can escape into the blood and so access normal cell renewal systems. Hence, the generated drug should have a short half-life to minimize the risks of escaping from tumours where it is generated into the blood and so able to cause damage to normal cells. This can ensure that the cytotoxicity of the drug is largely confined to the tumour. In one aspect, the drug has a half-life of 10 minutes or less. Preferably, the drug has a half-life of 5 minutes or less; 2 minutes or less; 1 minute or less. More

preferably, the drug has a half-life of 1 minute or less.

The generated drug is concentration dependent over a wide range of concentrations. Suitable generated drugs that are concentration dependent over a wide range of concentrations are, for example, alkylating agents.

Vascular-targeted agent

The term "vascular-targeted agent" as used in this application refers to agents that target the tumour vascular supply to inhibit new vessel development and/or to destroy existing tumour

vasculature. These vascular-targeted agents comprise antiangiogenic agents and vascular-disrupting agents.

Antiangiogenic agents inhibit key factors required for new vessel development. In contrast, Vascular-disrupting agents take advantage of the differences between the blood vessels of normal tissues and those of tumour tissues to target and destroy existing tumour vasculature. In general, vascular-disrupting agents effect the abnormal blood vessels that supply the interior of tumour, the result of which is to produce excessive necrosis within a tumour. In one aspect, the vascular-targeted agent is an antiangiogenic agent .

In one aspect, the vascular-targeted agent is a vascular-disrupting agent .

Preferably, the vascular-targeting agent is a vascular-disrupting agent . Where the vascular-targeting agent is an antiangiogenic agent it would form a combination therapy with the enzyme activated prodrug system. However, where the vascular-targeting agent is a vascular- disrupting agent the vascular-disrupting agent would directly contribute to the retention of the enzyme within the tumour

extracellular fluid.

Preferably the vascular-targeted agent includes but is not limited to Angiostatin, antiangiogenic antithrombin III, Angiozyme, ABT-627, anti-VEGF (vascular endothelial growth factor) antibodies (e.g.

Bevacizumab [Avastin] , DC101 and LL4), anti-aminophospholipid antibodies (e.g. bavituximab) , anti-Flk-1 antibodies, anti-Flt-1 antibodies and peptides, AVE 8062 [Ombrabulin] , Bay 12-9566,

Benefin, BMS-275291 , cartilage-derived inhibitor (CDI), CAI, CD59 complement fragment, CEP-7055, Col 3, Cilengitide, Combretastatin A- 4, Combretastatin A-4 phosphate, Combretastatin A-41 phosphate

(Oxi4503) , DMXAA [vadimezan, 5 , 6-dimethylxanthenone-4-acetic acid], Endostatin (collagenXVIII fragment), Denibulin HC1 ( N-029),

Experian (ADH-1), Fibronectin fragment, Gro-beta, Halofuginone , Heparinases, Heparin hexasaccharide fragment, HMV833, Human

chorionicgonadotropin (hCG) , 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors (e.g. statins, including rosuvastatin

[CRESTOR] , lovastatin [Mevacor] , atorvastatin [Lipitor] , pravastatin [Pravachol] , fluvastatin [Lescol] , pitavastatin [Livalo] , and simvastatin [Zocor] ) , I -862, Interferonalpha/beta/gamma,

Interferon inducible protein (IP-10), Interleukin-12 , Kringle 5

(plasminogen fragment) , Metalloproteinase inhibitors (TIMPs) , Matrix metalloproteinase inhibitors (e.g. membrane type 1 matrix metalloproteinase, batimastat, marimastat, prinostat and metastat) , 2- ethoxyestradiol, MMI 270 (CGS 27023A) , MoAblMC-lCl 1 , Neovastat, NM-3, Panzem, PI-88, Placental ribonuclease inhibitor, Plasminogen activator inhibitor, Platelet factor-4 (PF4), Prinomastat,

Prolactinl6kD fragment, Proliferin-related protein (PRP) , PTK 787/ZK 222594 [1- [4-chloroanilino] -4- [4-pyridylmethyl] phthalazine

succinate] , Retinoids, Solimastat, , sorafenib, sunitinib,

sunitinib maleate, Squalamine, SS 3304, SU 5416, SU6668, SU11248, Tetrahydrocortisol-S , Tetrathiomolybdate , Thalidomide,

Thiabendazole, Thrombospondin-l (TSP-1), TNP-470, Transforming growth factor-beta (TGF-b) , TZT-1026 (a dolastin analog), TZT-1027 [Soblidotin] , Vasculostatin, Vasostatin (calreticulin fragment) , VEGF-gelonin, ZD6126 [Phosphoric acid mono- (5-acetylamino-9, 10, 11- trimethoxy- 6 , 7-dihydro-5H-dibenzo [a, c] cyclohepten-3-yl ) ester] , ZD 6474 [Vandetanib] , vitaxin, Farnesyl transferase inhibitors (FTI), Biphosphonates , pharmaceutically acceptable salts thereof and mixtures thereof.

Preferably the vascular-targeted agent is selected from Bevacizumab [Avastin] (32), DC101, LL4 (33), AVE 8062 [Ombrabulin] (34), bavituximab (35) , Combretastatin A-4 (36) , Combretastatin A-4 phosphate, Combretastatin A-41 phosphate (Oxi4503) , DMXAA

[vadimezan] (37, 38), 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors (35) (e.g. statins, including rosuvastatin [CRESTOR] , lovastatin [Mevacor] , atorvastatin [Lipitor] , pravastatin

[Pravachol] , fluvastatin [Lescol] , pitavastatin [Livalo] , and simvastatin [Zocor] ) , Metalloproteinase inhibitors (TIMPs) , Matrix metalloproteinase inhibitors (39, 40) (e.g. membrane type 1 matrix metalloproteinase, batimastat, marimastat, prinostat and metastat) , Thiabendazole (41), ZD6126 (31) [Phosphoric acid mono- (5-acetylamino- 9,10, ll-trimethoxy-6, 7-dihydro-5if-dibenzo [a, c] cyclohepten-3-yl ) ester] , pharmaceutically acceptable salts thereof and mixtures thereof . In a further aspect, the vascular-targeted agent is selected from inhibitors of vascular endothelial growth factor (VEGF) ,

angiopoietin inhibitors, anti-aminophospholipid antibodies, anti- Flk-1 antibodies, anti-Flt-1 antibodies and peptides, cartilage- derived inhibitors, 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors, integrin blockers, vascular endothelial-cadherin inhibitors, tubulin inhibitors, cytokine inducers, Metalloproteinase inhibitors (TIMPs) , Matrix metalloproteinase inhibitors, Placental ribonuclease inhibitor, Plasminogen activator inhibitor, Farnesyl transferase inhibitors (FTI), pharmaceutically acceptable salts thereof and mixtures thereof. Where the vascular-targeted agent is an antibody or antibody fragments, preferably the antibody or antibody fragments are monoclonal and humanized.

Inhibitor

The therapeutic system comprises (iv) an inhibitor which, following administration, reduces the level of enzyme activity.

Preferably, the inhibitor comprises a moiety that ensures that it rapidly clears from the blood moiety.

The rapid clearance of the inhibitor from the blood ensures that negligible amounts of the inhibitor reach the tumour. Hence, whilst the inhibitor reacts with any enzyme it meets in the blood resulting in the rapid clearance of the enzyme from the blood, the inhibitor does not reach the tumour and so does not clear or inactivate enzyme in the tumour.

Suitably, the inhibitor is an enzyme-inactivating small compound.

Preferably, the inhibitor is an antibody, an antibody fragment, or an antibody mimetic (such as a DARPin) . DARPins (Designed Ankyrin Repeat Proteins) utilize the binding abilities of non-antibody polypeptides . They are derived from ankyrin proteins and their unique modular architecture features repeating structural units forming a stable protein domain with large modular target-binding surfaces. Additional information regarding repeating proteins such as DARPins can be found in US Patent Application Publication No. 2004/0132028 and International Patent Application Publication No. WO 02/20565. More preferably, the inhibitor is an antibody. Preferably, the inhibitor antibody is derivatised with a

polysaccharide. Suitable polysaccharides comprise galactose, mannose or other monosaccharides units. Preferably, the inhibitor antibody is derivatised with a polysaccharide comprising galactose units .

The inhibitor inhibits the activity of, or reduces the amount of, the enzyme in the vascular compartment, which results in reduction of enzyme in normal tissues thereby increasing the differential between the amount of or effect of enzyme in the tumour and in the rest of the body. Inactivation of the catalytic site of the enzyme and/or clearance of the enzyme may be achieved as in WO 89/10140.

Preferably, an antibody which binds at or near the catalytic site of the enzyme and thereby blocks it is used as the inhibitor. Such inhibitor antibodies (preferably monoclonal and humanized) may be raised or expressed by what are by now conventional techniques.

Accelerated clearance of the enzyme can be achieved by using an inhibitor antibody which has additional galactose, mannose or other saccharides added to accelerate clearance or may be desialylated . For example, galactosylation of the inhibitor antibody results in its rapid clearance from the blood through take-up by galactose receptors on hepatocytes . Alternatively, or additionally, the antibody-enzyme conjugate is galactosylated, and given after the hepatic galactose receptors have been blocked by asialo-bovine submaxillary gland mucoprotein or antibody directed at hepatic galactose receptor or other molecule with high affinity for

galactose receptor. The blocking substance is maintained in plasma for a period of up to 24 hours so that the antibody-enzyme complex localises at tumour sites but following cessation of galactose receptor blockade, the galactosylated antibody-enzyme is quickly cleared from blood via the available galactose receptors. The inhibitor antibody can be directed to any part of the catalytic macromolecule, since its function is to clear it, not (or at least not only) to block the catalytic action. Hence, in one aspect the inhibitor antibody is a non-inactivating antibody. Alternatively, small molecule enzyme inhibitors may be used; for example, these are described in WO 97/20580. For any given enzyme, the preferred small molecule inhibitors are those indicated as being preferred for that enzyme in WO 97/20580.

Preferably, the enzyme-inactivating small compound is a compound which inhibits the conversion of the first compound into the second compound to a useful extent. The extent of inhibition is preferably > 5%, more preferably > 10%, still more preferably > 50% and most preferably > 90%. Preferably, the inhibitor binds to the active site of the enzyme or to a site which influences the catalytic activity of the enzyme.

In further preference, the said inhibitor binds to the active site of the enzyme and in still further preference the said molecule is not exposed on the surface of the enzyme.

Preferably, the inhibitor is a relatively small molecule and it is further preferred if the molecule has a relative molecular mass of less than 10000.

It is also particularly preferred if the Ki of said enzyme, with respect to the inhibitor is < 100 microM, preferably < 1 micro , more preferably < InM and still more preferably substantially zero.

WO 97/20580 and Sharma et al . Cancer 1994, 73, 1114-1119 describe suitable inhibitors of CPG2.

Preferably, when the enzyme is Carboxypeptidase G2 the inhibitor is selected from antibodies with enzyme activation and clearance characteristics similar to Sb43 and Sb43 gal. Suitable other inhibitors for use with other enzymes (as listed) include: a) Carboxypeptidase A (Haenseler et al (1992) Biochemistry 31, 214-220: Hydrolyses terminal peptide linkage adjacent to free carboxyl group. Wide specificity, maximally active with aromatic side group (Figure 6. Possible inhibitors for this enzyme are also given, Figure 7.) b) Glucuronidase (Mitaku et al (1994) Ann. Oncol. 5 (Suppl. 5), 76: Sugar lactones are known to be inhibitors of this enzyme, such as D-saccharic acid-1 , -lactone for ss-glucuronidase . c) , -Lactamase (Svensson et al (1993) Bioconj . Chem. 3, 176-181: Clavulanic acid is a known inhibitor of this enzyme. Other

structures are also known sulbactam, thienamycin and imipenem.

The potent antibiotics in this family possess beta-lactamase inhibitory properties and thus run into thousands of derivatives.

It will be appreciated that salts of the inhibitor molecules may be used in the practice of the invention.

Treatment

The therapeutic system described herein is for use in the treatment of a tumour in a patient. The patient may be a human or a mammal such as domestic animals (e.g. a dog, a cat, etc.) . The therapeutic system described herein is not specifically designed for the treatment of leukaemias, isolated cancer cells or

micrometastases (smaller than 1-2 mm in diameter) . Conventional chemotherapy is generally effective in treating isolated cancer cells and micrometastase .

In a further aspect, the treatment comprises the sequential

administration of:

(a) the enzyme;

(b) the vascular-targeting agent;

(c) the inhibitor which, following administration, reduces the level of enzyme activity; and

(d) the prodrug. The above system and components (a) - (d) may have the further features as described above with regard to the individual

components .

The tumour may be present in any anatomical site in the patient. Preferably, the therapeutic system described herein is for use in the treatment of a tumour originating in the appendix, bladder, blood cells, bone, brain, breast, colon, liver, lung, lymph glands, naso pharyngeal, pancreas, prostate, rectum, skin, stomach, uterus, and of unknown primary origin. Tumours of unknown origin are not uncommon and may result from a histological diagnosis based on an accessible metastasis. The repeated use in patients of enzymes and/or antibodies of non- human origin is likely to require the use of de-immunised molecules, humanised molecules, immunosuppressive measures or tolerisation strategies . A particular issue may be immunogenicity arising from administration of an enzyme of non-human origin is that it may result in a host B cell antibody response which results in the production of antiserum. This consists of a multiplicity of antibodies directed at a

multiplicity of epitopes on the enzyme. In the early phase of this immune response Ig antibodies (MW 900,000 Daltons) are usually highly represented. Amongst the other classes of antibodies the main components of a matured response are IgG class 1 and 2

antibodies (MW typically around 150,000 Daltons) . Fab2 fragments that lack the Fc component typically have a MW around 100,000

Daltons and Fab fragments around 50,000 Daltons.

Ways of addressing this issue have been reported in relation to the bacterial enzyme beta-lactamase . Two groups have reported de- immunised beta-lactamase by replacing non-human amino acid sequences with human sequences, see Afshar, et al . , Mol Cancer Ther, 2009, 8, 185-193, and Harding et al., Mol Cancer Ther, 2005, 4, 1791-1800. Osipovitch et al . , Prot. Eng Design & Sel . , 2012, 25, 612-623 has reported an algorithm for the de-immunisation of foreign proteins in general and has applied it to beta-lactamase resulting in various alternative humanized forms of beta-lactamase. EP1501540 discloses reducing T-cell epitopes on GPG2.

In a clinical trial of ADEPT using CPG2, cyclosporine was

administered to some patients with the objective of delaying the immune response. Use of cyclosporine in this trial delayed the immune response from approximately 14 to approximately 21 days following exposure of the patient to CPG2. However, cyclosporine was also found to increase the toxicity of the treatment.

More recently, Nowak et al . , Cancer Res, 2002, 62, 2353-2358, reported that a subject's antibody response to soluble immunogens was delayed and/or prevented by co-administration with gemcitabine, an antimetabolite drug that has been licensed for the treatment of several cancers . An alternative strategy that has the potential to deliver multiple cycles of therapy is to switch to using an alternative enzyme- prodrug system when an antibody response is observed for a first enzyme-prodrug system. Such a strategy has been used in the treatment of childhood acute lymphoblastic leukaemia with the enzyme asparaginase derived from alternative sources.

A further aspect of the present invention provides a method of destroying target cells in a host, the method comprising

administration to the host of the various components described above .

The components of the invention are administered in any suitable way, usually parenterally, for example intravenously,

intraperitoneally or intravesically, in standard sterile, non- pyrogenic formulations of diluents and carriers. The blood

concentration of the treatment compounds and indices of renal, hepatic and haemopoietic function should be measured at suitable intervals. Typically, after administration of pegylated enzyme, vascualar disruptive agent and enzyme inactivating agent, the concentration of the enzyme in the tumour is greater than its concentration in the blood.

Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents

(e.g., pH of from about 3 to about 9) . For some applications, however, the components may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water. The preparation of parenteral formulations under sterile conditions

(e.g., by lyophilization) may be readily accomplished using standard pharmaceutical techniques.

The solubility of compounds which are used in the preparation of parenteral solutions may be increased through appropriate

formulation techniques, such as the incorporation of solubility- enhancing agents . Formulations for parenteral administration may be formulated to be immediate or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release. Thus, the components may be formulated as a suspension, a solid, a semi-solid, or a thixotropic liquid for administration as an implanted depot providing modified release of the active compound. Examples of such formulations include drug-coated stents and semisolids and suspensions comprising drug-loaded poly (DL-lactic-co-glycolic) acid (PGLA) microspheres.

Suitably the therapeutic system is formulated so that the four components (i) - (iv) are provided as two constituent parts. Hence, the four components are formulated into two groups of components.

In one aspect, the therapeutic system is formulated to comprise:

(a) a first constituent part comprising two components selected from components (i) , (ii), (iii) and (iv); and

(b) a second constituent part comprising the remaining two components not present in the first constituent part (a) . In a second aspect, the therapeutic system is formulated to comprise :

(1) a first constituent part comprising one component selected from components (i) , (ii), (iii) and (iv) ; and

(2) a second constituent part comprising the remaining three components not present in the first constituent part (1) .

Suitably each constituent part is formulated or modified such that one component is metabolically active before any other component present in that constituent part.

Suitably each constituent part is formulated as a biodegradable matrix. Suitable biodegradable matrices may be selected from biodegradable polymers [e.g. poly (lactic-co-glycolic acid) PLGA] , natural compounds (e.g. collagen; Lee, et al., Int J Pharm, 2001, 221, pages 1-22) and liposomes (Trift, et al . , Exp Biol Med, 2001, 226, pages 559-564; Park, et al . , J Control Rel, 2001, 74, pages 95-113) .

The biodegradable matrices may be a solid, gel or a liquid.

Solid dosage forms such as biodegradable microspheres consisting of PLGA have been used as an injectable depot system for drugs, peptides and proteins (Cleland, et al . , Adv. Drug Del Rev, 1997,

28, pages 71-84; Yang, et al . , J Control Rel, 2001, 75, pages 115- 128; and Cleland, et al . , J Control Rel, 2001, 72, pages 13-24). Such systems require a suspension vehicle to allow injection of the microspheres.

Injectable gels generally comprise a solvent to dissolve the matrix and the therapeutic agent (Gutowska, et al . , Anal Rec, 2001, 263, pages 342-349) . More suitably, the biodegradable matrices is selected from biodegradable nanospheres and liposomes. Such systems modify the release of the entrapped components as they circulate through the body or accumulate at a target site. For example, the accumulation of liposomes containing a therapeutic agent such as doxorubicin occurs in solid tumours (Gabizon, Cancer Res, 1992, 52, pages 891-896) .

More suitably, the biodegradable matrices is a liposome. Most suitably, the biodegradable matrices is a liposome comprising polyethylene glycol PEG lipids, Liposomes comprising PEG lipids are protected from serum protein binding and phagocytic

recognition and this protection increases the circulating half- life as compared to conventional liposomes .

Suitably a constituent part is modified by adding a physiological cleavable group, such as a phosphate, to one component so that component is metabolically active before any other component present in the constituent part. Examples of chemically modifying pharmaceutical agents by adding various physiologically cleavable groups are disclosed in W0199302519 , US8,592,427 and

WO2000075164.

An enzyme or enzyme-macromolecule may be administered by slow intravenous infusion in a dosage sufficient to locate an optimal concentration of enzyme at tumour sites. After allowing time

(typically 24h) for the enzyme to localise at tumour sites, the vascular-targeting agent may be administered by slow intravenous diffusion. After allowing time for the vascular targeting agent to act and trap enzyme within the tumour the inhibitor which reduces the level of enzyme activity may be administered by slow intravenous diffusion. This inhibitor may be a monoclonal antibody directed at the enzyme. The inhibitor accelerates the clearance of the enzyme from the blood. In this way a great differentiation in enzyme concentration between normal and tumour tissues can be attained. When enzyme activity is no longer detectable in blood samples, a prodrug which is a substrate for the enzyme is given by repeated bolus injections or by infusion and continued until the enzyme concentration in the tumour falls below an effective value. It may not be possible to avoid drug generated in tumours from getting into blood and suppressing haemopoietic function

(myelosuppression) although for a given effect on a tumour target it is expected to be much less with the system described herein. It is feasible that an agent can be developed that would inactivate or reduce the activity of drug in the blood. Such an agent would not only protect normal cell renewal tissues but would also allow for the use of drugs with longer half-lives. Brief Description of the Drawings

Embodiments of the present invention will now be described further, with reference to the accompanying drawings, in which: Figure 1 shows in vitro tumour cell survival of LS174T cells, a human colon carcinoma;

Figure 2 shows carboxypeptidase G2 clearance and tumour retention in LS174T colon cancer xenografted nude mice;

Figure 3 shows clearance and tumour retention of polyethylene glycol-carboxypeptidase G2 in LS174T colon cancer xenografted nude mice;

Figure 4 shows the relative tumour volume (RTV) for Enzyme-Prodrug therapy compared to untreated control in LS174T colon cancer xenografted nude mice;

Figure 5 shows the RTV for Enzyme-Prodrug therapy compared to untreated control in MKN-45 gastric cancer xenografted nude mice; Figure 6 shows the RTV for Enzyme-Prodrug therapy compared to untreated control in CAPAN-1 pancreatic cancer xenografted nude mice; and

Figure 7 shows the RTV for enzyme-prodrug therapy compared to untreated control in SW1222 colon cancer xenografted nude mice.

CA4P (180mg/kg per mouse) was substituted for DMXAA in this study.

The invention will now be described in more detail with reference to the following Examples wherein: Examples

Abbreviations

BIP = bis-iodo phenol prodrug

CA4P = Combretastatin 4 phosphate

CPG2 = carboxypeptidase G2

Da = Daltons

DMSO = dimethyl sulfoxide

DMXAA = 5,5- Dimethyl xanthenone-4-acetic acid (Sigma)

ID =injected dose

PEG = polyethylene glycol, m.wt 2300 Da (Sigma)

PEG-CPG2 = pegylated CPG2 (estimated 20 PEGs per molecule of CPG2) SB43 gal = an in-house murine monoclonal antibody that inactivates CPG2 and is galactosylated (it was prepared by conventional methods known in the art such as those described in Sharma, S.K., et al . , Br J Cancer, 1990, 61: p. 659-62)

uM= micro mole

One unit of CPG2 is defined as the amount of enzyme required to hydrolyse 1 umol of methotrexate per minute per ml of reaction mixture at 37° C.

CPG2 PEGylation method Carboxypeptidase G2 was dissolved to 2mg/ml in phosphate-buffered saline (PBS) pH 7.5. The PEGylation strategy was by direct

modification of surface lysines on CPG2 using an N-hydroxy

succinamide (NHS) based PEG-linker. The PEGylation reagent used was m-dPEG®4g, a 49-unit PEG polymer with a reactive NHS-ester terminal for lysine conjugation. This was dissolved in dry DMSO to give a lOOm stock. After optimization of conditions, PEGylation was carried out at a 20-fold molar excess (assuming a monomeric

molecular weight for CPG2=41695.4 and PEG molecular weight=2315.70) where 0.1ml of 2mg/ml CPG2 was reacted with 0.96 microlitre at room temperature for 30 mins. Unreacted PEG was removed by exhaustive dialysis or desalt column in PBS pH 7.5 buffer. PEGylation was confirmed by running samples on a 15% SDS polyacrylamide gel and staining with Instant Blue Commassie reagent. PEGylation can be seen as a polysisperse shift in molecular weight between 50-72kDa. This corresponds to up to 10 PEG chains attached per CPG2 monomer, with the mean number of PEG chains being 8-9 per CPG2 monomer, 16-18 PEG chains per functional CPG2 homodimer.

Reference

Hermanson, G.T. (2008) . Bioconjugate Techniques Chapter 3 'The reactions of bioconj ugation' . Academic Press.

Example 1 : Method of use

Initial tests may be performed to exclude as far as possible abnormal reaction by the patient to any of the protein components. The enzyme is given intravenously, preferably by slow infusion, typically over 2 hours. Maximal tumour concentration of the enzyme is achieved several hours later but at this time there are still high levels of enzyme activity in plasma. Whilst the concentration of enzyme in blood and tumour is high (typically within 24 hours of giving the enzyme) administration of the anti-vascular agent is given by short intravenous infusion, at a dosage based on published clinical trials using the same anti-vascular agent. After allowing a suitable interval for the anti-vascular agent to shut down the intra-tumour blood vessels, typically 6-24 hours, intravenous infusion of the enzyme inhibitor begins to reduce the enzyme concentration in blood. This begins with an infusion of a low concentration of inhibitor to avoid a high concentration of immune complex formation in blood. After the concentration of enzyme in blood has been reduced, infusion of a higher concentration of inhibitor is continued until the enzyme is no longer detectable in blood. If there is evidence of leak back of enzyme from tumours into blood the infusion with low concentration inhibitor can be continued throughout prodrug administration. Prodrug administration by a series of bolus injections, or by intravenous infusion, commences as soon as enzyme is no longer detectable in blood and may continue as long as there is evidence of a sufficient concentration of enzyme in tumour. The anti-vascular agent may be repeated after 4 or 7 days . Following administration (typically 48-72 hours) of an

immunogenic protein an agent that suppresses an immune response to the antigen may be given.

The cycle may be repeated. Limiting factors will be toxicity attributable to prodrug/drug or the development of host antibodies to any of the foreign proteins employed.

Example 2

Mouse Matrigel™ Pug Angiogenesis Assays

To confirm the effects of a composition on angiogenesis, a mouse Matrigel™ plug angiogenesis assay can be used. Various growth factors (e.g., IGF-1, bFGF or VEGF) (250 ng) and Heparin (0.0025 units per/mL) are mixed with growth factor reduced Matrigel™ as previously described (Montesano, et al . , J. Cell Biol. 1983,

97:1648-1652; Stefansson, et al . , J. Biol. Chem. 2000, 276:8135- 8141) . Compositions described herein or control antibodies can be included in the Matrigel™ preparations utilizing one or more dosage groups of animals. In control experiments, Matrigel™ is prepared in the absence of growth factors. Mice are injected subcutaneously with 0.5 mL of the Matrigel™ preparation and allowed to incubate for one week. Following the incubation period, the mice are sacrificed and the polymerized Matrigel™ plugs surgically removed. Angiogenesis within the Matrigel™ plugs is quantified by two established methods, including immunohistochemical analysis and hemoglobin content

(Furstenberger , et al . , Lancet. 2002, 3:298-302; Volpert, et al . , Cancer Cell 2002, 2(6) :473-83; and Su, et al . , Cancer Res. 2003, 63:3585-3592) . For immunohistochemical analysis, the Matrigel™ plugs are embedded in OCT, snap frozen and 4 micro metre sections

prepared. Frozen sections are fixed in methanol/acetone (1:1) . Frozen sections are stained with polyclonal antibody directed to CD31. Angiogenesis is guantified by microvascular density counts within 20 high powered (200. times.) microscopic fields.

Haemoglobin content can be guantified as described previously (Schnaper, et al . , J. Cell Physiol. 1993, 256:235-246; Montesano, e al., J. Cell Biol. 1983, 97:1648-1652; Stefansson, et al . , J. Biol. Chem. 2000, 276:8135-8141; and Gigli, et al . , J. Immunol. 1986, 100:1154-1164) . The atrigel™ implants are snap frozen on dry ice and lyophilized overnight. The dried implants are re-suspended in 0.4 mL of 1.0% saponin (Calbiochem) for one hour, and disrupted by vigorous pipetting. The preparations are centrifuged at 14,000 x g for 15 minutes to remove any particulates. The concentration of haemoglobin in the supernatant is then determined directly by measuring the absorbency at 405 nm and compared to a standard concentration of purified haemoglobin.

Example 3 : In vivo Studies in Immunodeficient Athymic Nude Mice Bearing Human Colon Carcinoma

In these studies enzyme administration was by intravenous route and other agents were given by intra-peritoneal route. Repeated intravenous injections in mice are difficult so that repeated injections are given via intra-peritoneal route. In human patients such drugs are most conveniently administered by controlled intravenous infusion.

Studies were carried out, in vivo, in immunodeficient athymic nude mice bearing human tumour xenografts . Mice were maintained and humanely killed in conformity with national Home Office regulations

Human cancer xenografts growing in mice have murine vasculature. DMXAA was chosen as the vascular-targeting agent because it causes disruption of the internal vasculature of human xenografts growing in mice and because of cost considerations. However, other targeting agents may be more effective in humans and some current clinical trials use combretastatin A4 Phosphate as a vascular disruptive agent.

The tumours were established by injecting specified types of human cancer cells subcutaneously in one flank. Each mouse was injected with 5 xlO⁶ cultured cancer cells to establish the xenografts. Cell lines were maintained in standard culture conditions.

At the time of administration of the first agent, the tumours were established and about 0.5-0.7cm diameter. Values relate to 3 mice per group for clearance and retention studies and 5-8 mice per group for therapy studies unless otherwise stated.

The studies illustrating the action of the agents employed the cancer cell line known as LS174T, a moderate to poorly

differentiated human colon carcinoma that has proved resistant eradication by several cancer drug protocols.

The dosages used in the therapy studies are based on those published in other applications and the time interval between agents was largely based on convenience. No attempt has been made to optimise dosages or time intervals between the agents used.

The pegylated carboxypeptidase G2 used in the present

exemplification studies was found, in retrospect, to have

deteriorated during the course of the studies, so that the amount of enzyme delivered to tumours, during the DMXAA studies, was less than expected. The estimated loss was 50% by the start of the therapy experiments and more than 75% by their completion. The results on tumour growth obtained during the course of the studies are

therefore less than might be expected with optimal reagents.

Study of the Effect of Administering Varying Concentrations of CPG2 in Combination with a Constant Concentration of Prodrug upon Survival of LS174T Cells A study was carried out to measure the survival of LS174T cells at 72 hours after 1 hour exposure to 0.1 uM of prodrug BIP and various concentrations of CPG2. The results are shown in Figure 1 where the concentration of CPG2 varies from the left hand column, culture medium control (CPG2 concentration of 0), through to the right hand column which shows the effect of CPG2 1 unit/ml. The height of each column indicates the % cell viability compared to medium only control at the concentrations of enzyme shown. % Cell viability of treated samples relative to the untreated control cells was calculated using the formula:

100 x [ (OD of treated sample) / (OD of untreated sample) ] . The control untreated cell viability taken as 100%.

From Figure 1 it can be seen that 60% of cells survived and, hence, that 40% of cells were killed by 1 hour exposure to a concentration of CPG2 at 1 unit/ml in combination with 0. luM BIP prodrug. Figure 1 also shows that 30% of cells were killed by concentration of CPG2 as low as 0.06 enzyme units/ml in combination with 0. luM prodrug for one hour exposure.

Study Comparing the Effect of Administering CPG2 and PEG-CPG2 upon the Clearance of Enzyme from Blood and Enzyme Retention in the Tumour

Figure 2 shows the results of a study of the tumour and blood concentrations of unmodified CPG2 in LS174T xenografts following intravenous injection of 65 enzyme units of native CPG2 at time 0.

The enzyme (CPG2) concentration in blood and tumour was measured by an indirect high-performance liquid chromatography assay using methotrexate as a substrate (Sharma et al 2005, Clin Cancer Res.

11 : pages 814-825) . The results of a study of the tumour and blood concentrations of CPG2 in mice bearing LS174T xenografts following intravenous injection of 25 enzyme units of PEG-CPG2 is shown in Figure 3.

Figure 3 shows prolonged retention of a much lower dose of pegylate CPG2 enzyme activity in blood compared with that of the native enzyme (Figure 2) . It also shows much higher uptake and prolonged retention of pegylated enzyme in tumour compared with the native enzyme. Whereas the unmodified enzyme has a half-life in blood of approximately 2 hours (Figure 2) that of PEG-CPG2 has a half-life i blood of approximately 2 days (Figure 3) .

Study Comparing the Effect on Tumour Retention of Enzyme in Mice of Administering PEG-CPG2 with or without the addition of DMXAA

A study was carried out comparing the effect that the addition of DMXAA had on the retention of enzyme in tumour in mice bearing LS174T xenografts given PEG-CPG2. The tumour and blood

concentrations of enzyme in mice bearing LS174T xenografts were measured following intravenous injection of 20 enzyme units of PEG- CP at time zero in a sample group of mice and in a control group of mice. In the sample group of mice this was followed 24 hours later by intraperitoneal injection of 25 mg/ kg of DMXAA.

The results for the control group of mice injected with PEG-CPG2 alone are given in Table 2.

Table 2

The results for the sample group of mice injected with PEG-CPG2 DMXAA are given in Table 3 Table 3

This shows the effect of DMXAA on retention of enzyme in tumour o at various time points post PEG-CPG2 injection. Compared with the control group of mice administered PEG-CPG2 alone, mice in the sample group that were also administered with DMXAA resulted in 80% greater enzyme retention in tumour at 72 hours after PEG-CP2 inj ection .

Study Comparing the Effect on Retention of Enzyme in Tumour in Mice of Administering PEG-CPG2 and DMXAA with or without the Addition of SB43 gal

A study was carried out comparing the effect on retention of enzyme in mice of administering PEG-CPG2 and DMXAA with or without the further addition of SB43 gal. Mice bearing LS174T xenografts were intravenously injected with 20 enzyme units of PEG-CPG2 at time 0, and this was followed by intraperitoneal injection of 25 mg/Kg of DMXAA 24 hours post PEG-CPG2 administration. In a sample group of the mice this was followed by three injections of SB43 gal (50ug per mouse per intraperitoneal injection) after a further 16 hours. A control group of mice did not undergo the injections with SB43 gal. The tumour and blood concentrations of CPG2 in LS174T xenografts were measured and the results of this study are summarised in Table Table 4

The tumour to blood ratio (at 72 hours) of the mean enzyme

concentrations were calculated for both the control group, where

SB43 gal was absent, and for the sample group, where SB43 gal was administered, and are provided in Table 5.

Table 5

These results show the removal of enzyme activity from blood by addition of SB43 gal. The removal of enzyme activity from normal tissues is not shown but closely follows blood levels. The level of enzyme activity in the tumour also falls but is sustained at an effective level for several days. As can be seen, the ratio of the enzyme that is present in the tumour as compared to that in the blood is two orders of magnitude greater when SB43 gal is used.

Example : In vivo Studies of Therapy of Human Carcinomas

Xenografted into Immunodeficient Athymic Nude Mice In vivo Study of Therapy of Human Colon Carcinomas Xenografted into Immunodeficient Athymic Nude Mice Protocol

A study was carried out of the therapy of five LS174T tumours in xenografted nude mice. The mice received intravenous injection of PEG-CPG2 (20 enzyme units) at time 0, followed 24 hours later by intraperitoneal injection of DMXAA (25 mg/Kg), followed at 48 hours by SB43-gal (50ug per mouse per intraperitoneal injection) . BIP was administered intraperitoneal at 70 mg/Kg in three doses at hourly intervals, starting 24 hours after the first injection of SB43 gal.

Figure 4 shows the results of the growth (measured as the relative tumour volume, RTV) of matched tumours that received no treatment, and mice receiving PEG-CPG2, DMXAA, SB43gal, and BIP on day 4.

These results show that a single cycle of therapy effected growth delay of 20 days.

Tumour measurements were carried out in three dimensions

(length, width and height) and volume calculated as: (length x width x height) /2 (see Sharma et al, 2005, Clin Cancer Res. 11: pages 814-825) .

Tumour volume is shown relative to the tumour volume (RTV) at the time of initiation of the therapy.

In vivo Study of Therapy of Human Gastric Carcinomas Xenografted into Immunodeficient Athymic Nude Mice

The above protocol was used except that the mice were xenografted with a gastric carcinoma (MKN-45) rather than with LS174T. Figure 5 shows the results of the growth (measured as the relative tumour volume, RTV) of matched tumours that received no treatment, and mice receiving PEG-CPG2, DMXAA, SB43gal, and BIP on day 4. The treated tumours showed considerable growth delay from a single cycle of therapy in comparison with the untreated control tumours. In vivo Study of Therapy of Human Pancreatic Carcinomas Xenografted into Immunodeficient Athymic Nude Mice

The above protocol was used except that the mice were xenografted with a pancreatic carcinoma (CAPAN-1) rather than with LS174T.

Figure 6 shows the results of the growth (measured as the relative tumour volume, RTV) of matched tumours that received no treatment, and mice receiving PEG-CPG2, D XAA, SB43gal, and BIP on day 4. The treated tumours showed considerable growth delay from a single cycle of therapy in comparison with the untreated control tumours.

In vivo Study of Therapy of Human Colon Carcinomas Xenografted into Immunodeficient Athymic Nude Mice with the Substitution of

Compretastatin-A 4P (CA4P) for DMXAA

The above protocol was used, including using mice xenografted with SW1222 tumours, but with the substitution of CA4P (180 mg/kg) for DMXAA (Figure 7) . Figure 7 shows the results of the growth (measured as a relative tumour volume RTV) of matched tumours that received no treatment, and mice receiving PEG-CPG2, CA4P, SB43gal and BIP on day 4.

The treated tumours showed growth delay from a single cycle of therapy in comparison with untreated control tumours.

Conclusions

Although alkylating agents are generally thought to be ineffective against colorectal, pancreatic and gastric carcinomas when given by conventional route and dosage in the clinic, the above experiments indicate efficacy when used in the enzyme activated prodrug system described herein.

Furthermore, it should be noted that the treatment protocol used has not been optimised in terms of drug concentration, nor in terms of timing for the LS174T tumours, nor for any of the other tumour models used here. Is should also be noted that the therapy used in the mice did not result in significant weight loss nor was there any other evidence of serious toxicity.

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41 Che et al . , Plos Biology 2012, 10, 1-13. All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variation in the described system and method of the invention will be apparent to those skilled in the art without departing from the spirit and scope of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the following claims .

Claims

1. A therapeutic system comprising:

(i) a prodrug;

(iii) a vascular-targeting agent; and

2. A therapeutic system according to claim 1, wherein the prodrug (i) is a prodrug of a cytotoxic drug and is selected from the group consisting of alcohols, adenosine-containing prodrugs, amino acid- containing prodrugs, beta-lactam-containing prodrugs, cephalosporin- containing prodrugs, cytocine-containing prodrugs, glucouronide- containing prodrugs, glutamate-containing prodrugs, glycosylated prodrugs, nitro-containing prodrugs, peptide-containing prodrugs, phenoxyacetamide-containing prodrugs , substituted-phenoxyacetamide- containing prodrugs, phenylacetamide-containing prodrugs,

substituted-phenylacetamide-containing prodrugs, phenylacetyl- containing prodrugs, phosphate-containing prodrugs, purine- containing prodrugs, riboside-containing prodrugs; saccharide- containing prodrugs and sulfate-containing prodrugs.

3. A therapeutic system according to claim 1 or 2, wherein the enzyme is selected from carbohydrate-cleaving enzyme molecules, glycosyltranferases , hydrolases, lyases, oxidoreductases ,

phosphorylases , phosphotransferases, proteases, DL-racemases and mixtures thereof.

4. A therapeutic system according to any one of the preceding claims, wherein the enzyme is selected from alkaline phosphatases, aminopeptidases , arylacyl amidases, arylsulfatases , azoreductases , carboxypeptidases , cyctochrome P450, cytocine deaminases, DT- diaphorases , beta-galactosidases , alpha-glactosidases , glucose oxidases, beta-glucosidases , beta-glucuronidases , beta-lactamases , lactoperoxidases , alpha-mannosidases , methionine gamma-lyases , nitroreductases, pencillin amidases, purine-nucleoside

phosphorylases , thrombolysins , thymidine kinases, urokinases, xanthine oxidases and mixtures thereof.

5. A therapeutic system according to claim 4, wherein the

carboxypeptidase is selected from carboxypeptidase A,

carboxypeptidase B, carboxypeptidase G, carboxypeptidase Gl and carboxypeptidase G2.

6 . A therapeutic system according to any one of the preceding claims, wherein the vascular-targeting agent is an antiangiogenic agent .

7. A therapeutic system according to any one of claims 1-5, wherein the vascular-targeting agent is a vascular-disrupting agent.

8. A therapeutic system according to any one of the preceding claims, wherein the inhibitor is an enzyme-inactivating small compound .

9 . A therapeutic system according to any one of the preceding claims, wherein the inhibitor is an antibody.

10. A therapeutic system according to claim 9 , wherein the

inhibitor antibody is derivatised with a polysaccharide.

11. A therapeutic system according to any of claims 1 to 10, wherein the enzyme (ii) is fused or conjugated to a macromolecule .

12. A therapeutic system according to claim 11, wherein the macromolecule is selected from a polyethylene glycol; a dextran; a polyamino acid; a non-tumour-specific protein; an hydroxypropyl methylacrylamide; a copolymer of styrene and maleic anhydride; a polyvinyl pyrrolidone; or a polyethyleneimine .

13. A therapeutic system according to any of claims 1 to 12, wherein the enzyme (ii) is fused or conjugated to a protected saccharide .

14. A therapeutic system for use in the treatment of a tumour in a patient wherein the therapeutic system comprises:

(i) a prodrug;

(iii) a vascular-targeting agent; and

15. A therapeutic system for use in the treatment of a tumour in a patient according to claim 14, wherein the treatment comprises the sequential administration of:

(a) the enzyme;

(b) the vascular-targeting agent;

(d) the prodrug.