CA2124217A1 - Inactivation of cytotoxic drugs - Google Patents

Abstract

A three component kit of parts for use in a method of destroying target cells in a host is provided. The first component comprises a target cell-specific portion and an enzymatically active portion capable of converting a cytotoxic pro-drug into a cytotoxic drug. The second component is a cytotoxic pro-drug convertible by said enzymatically active portion to the cytotoxic drug. The third component comprises a portion capable of at least partly restraining the component from leaving the vascular compartment of a host when said compound is administered to the vascular compartment, and an inactivating portion capable of converting the cytotoxic drug into a less toxic substance.

Description

wo93/13806 ~ ~ 2 ~ ~ ~ 7 PCltGB93/00040 INACTIVATION OF CYTOTOXIC DRUGS

This invention relates to cytotoxic drll~ therapy and, more specifically. to theinac1i~ration of cytotoxic drugs to limit their undesirable side effects.
~:
One of the main limitations of conventional cytotoxic therapeutic agents is their lack of discrimination between cancer cells and normal replicating cells v~hich are essential for normal tissue inte~rity and body function. The effects of cytotoxic agents on these normal tissues limi~s ~he dosa~e of cytotoxic therapy I() or the duration of its administration. Cvtotoxic druo therapy is therefo interrupted at frequen~ in~en~als to allow normal tissll~ recover~. S~
interruption also allows recovery of surviving cancer cells and mav be important in allowing the emergence of dru~ resistance.

15 The duration of drug free intervals in therapy usually exceeds the duration of cytotoxic agent administration. This applies to all forms of cytotoxic therapy but is particularly evident in the case of those agents that interfere with DNA
replication during S phase of the cell cycle.

20 A method for the treatment of various cancers known as Antibody-Directed Enzvme Pro-drug Therapy tADEPT) has been described (Ba~shawe 1987 Br.
J. Cancer 56, 531-2; 1989 60~ 275-281) and is undergoing early clinical trial in which an antibody, or antibody fra~ment. directed at a tumour-associated antigen expressed by at least some cells in the cancer target, is conjugated to 25 an enzyme and used to convey that enzyme to the cancer sites. The enzyme in the conjugate is matched by a subsequently administered prodrug which is a relatively non-toxic substance and is a substrate for the enzyme. the end-product of the reaction being an active cytotoxic drug which is able to diffuse through the tumour and reach cells that do not express the target antigen. In 30 such therapy it is desirable that the enzyme used should not normally be present 2~2~7 WO 93/13806 PCI`/GB93/00040 . `~

.

in si~nificant amounts in human body fluids and that the prodrug is only subjectto conversion to active drug by the targeted enzyme (W088/07378).

In the example of choriocarcinoma xenografts i`n nude mice~ cited in W0 88/07378, clearance of the antibody enzyme conju~ate is accelerated by the presence of relatively large amounts of the target antigen in the plasma which results in immunoconjugate formation. In this case~ the accelerated clearance did not prevent localisation of the antibody enzyme conjugate at tumour sites althou~h, in general. rapid clearance of the antibody enzyme conjuQate from plasma results in poor tumour localisation.

In another exemplification of the antibody enzyme pro-drug principle (Senter et al (1988) Proc. I~atl. Acad. Sci. USA 85. 4842-4846 the targeted enzvme~
alkaline phosphatase, is widely distributed in body tissues includin~ plasma so lS that pro-drug activation mus~ occur at all these sites in addition to that generated at tumour sites. The tumour generated incremenl of active dmo under such conditions can only be small.

The objective of an antibody-enzyme pro-drug approach is ~o confine the action of cytotoxic aQents to tumour sites. This would be advantageous in allowing a _reater concentration of drug to act at tumour sites without incurring greatertoxic effects on normal tissues. Also~ many cytotoxic drugs currently in use are carcinogenic and survivors from one cancer may succumb to a cancer induced by treatment of the former cancer through the effect of the drugs or radiation on normal tissues; restricting mutagenic drugs to cancer sites would reduce this risk of iatrogenic cancer.
.:
A method of treatment in which an active cytotoxic drug is generated from a less toxic pro-drug by catalytic action can only achieve the objective~ as defined, if the catalyst is confined to the target site(s). When an antibody or WO93/13806 ~ ~ 2 ~ ~17 pcr/Gsg3/ooo4o fragment thereof, or a conjugate comprising an antibody or fragment thereof and an enzyme, is injected into a host bearing a tumour which expresses on the cell surface an antigen corresponding to the antibody, the antibody or conjugatepreferentially localises at tumour sites. That is to say, the concentration of enzyme at tumour sites is likely to be highér than in other tissues after an interval of hours or days. However, most of the adminis~ered antibody or conjugate is retained in other tissues. including the blood to some extent.
Although a patient with terminal cancer may have several kilo~rams of tumour it rarely represents more than 10% of body weight and treatment is ~enerally 10 undertaken when the body burden of cancer ran~es frol-1 a few grams to aboul I kilo~ram. Thus the advanta~e of a hi~her concentration of the enzyme achieved at tumour sites by the antibody-enzyme conjugate is offset bv the much ~reater volume of normal tissues retaining a low concentration of the enzyme. The enzyme-pro-drug reaction takes place in the interstitial fluid 15 (extracellular space) which in a tumour of I kilogram is unlikely to exceed I-200 millilitres whereas the plasma volume of an average adlllt male is ? ~S litres and the total extracellular space about 1~ litres. A consequence of these considerations is that a method of treatment based on an antibodv-enzyn~,e conjugate~ and a subsequently administered pro-dm~ is still subjected to a dose 20 limiting effect through cytotoxic drug action on normal tissues. It has been shown in nude mice bearing human colon cancer xenografts that if a pro-dru~
is given when enzyme is still present in plasma in sionificant amount that the pro-drug is rapidly activated with fatal effects ~Bagshawe ( 1989) Brit. J.
Cancer. 60~ 275-281).
Since antibody-enzyme conjugates ~enerally reach their maximum concentration at tumour sites within 12-24 hours and since they may take several days lo clear from plasma and other body fluids, it has also been shown that it is advantageous to accelerate the clearance of antibody-enzyme conju~ate and to 30 inactivate the specific enzyme present in the blood. Several means by which ~''.

~ 1 2 !~ h ~
WO 93/13806 Pcr/G~93/00040 this may be achieved have been described (WO89/10140).

Clearing and/or inactivating the enzyme in plasma has a marked effect~
allowing a greater amount of pro-dru, to be administered. Considerable S clinical and experimental experience has been gained with the use of a galactosylated anti-enzyme an~ibody which rapidly inactivates enzyme in blood after tumour localisation of the enzyme has been achieved. The galactosylation of the anti-enzyme antibody results in its rapid clearance from the blood ~hrough take-up by galactose receptors on hepatocytes and is necessary to 10 prevent its escape from lhe ~ascular compartlnent onto ~he tumour where it would inactive the enzyme. The tall in plasma enzyme concentration with the use of such a method~ in the other methods described in WO 89/10140 causes antibody enzyme conjuDates not l)oulld to antigen in normal tissues to diffuse back into the plasma compartment. Tlle enzvme returning to blood can, for 15 instance~ be inactivated by a slow infusion of anti-enzyme antibody. Althoughthese accelerated clearance and inactivating methods make antibodv enzyme pro-drug therapy widelv applicable some active drug still enters the vascular compartment and reaches cell renewal tissues~ particularly the haemopoietic tissues~ and can be dose limitino. Where a tumour is relativel~ large a 20 si~nificant amount of active drug will enter the blood by direct diffusion. or via ~he Iymphatic chain~ in addition to anv acti~e dru~ formed by residual enzyme activity in normal tissues.

Using these methods the eneration of a cytotoxic agent can be restricted to 25 those sites where the specific enzyme is located and predominantly to cancer sites.

A cytotoxic dru2 generated anywhere in the body diffuses throug~h the extracellular fluid and is able to reach nearby cells which do not express the 30 marker antigen to which the antibody is directed~ It may also pass back into ~WOg3/13806 2 i ~ 1~ 217 PCI`/GB93/00040 the vascular compartment and then be conveyed to cell renewal tissues. If the tumour mass is large and if the drug has a half-life of more than a few seconds then effects on normal tissues may still prove dose-limiting.

5 It is therefore undesirable for active drug to be present in the biood.

Anti-tumour antibodies exist which show little or no binding to haemopoietic tissue, but since haemopoiesis occurs within capillaries. or in proximitv to fenestrated capillaries, haemopoietic cells are highly vulnerable to the action of 10 cytotoxic agents present in blood.

This blood-mediated effect can be limited by ensuring that the active drug has a short half-life (Ba~shawe 1987). A drug generated at any point in the bod~
can reach~ via the circulating blood~ any other tissue within 20-30 seconds 1~ Some valuable cytotoxic agents possess relatively long half-lives and in the absence of an additional mechanism they may be excluded from consideration.
Also, it may prove difficult to identify hi~hly active short half-life dru~s that can.be generated from non-toxic prodrugs. A further consideration is tha~a very short half-life may limit the concentration of ac~ive dru~ that can b~
20 achieved at sites within a tumour mass that are remote from sites of antigen expression and therefore of active drug generation.

The objective of the present invention is to inhibit, at least partially~ in theblood any active drug which is present in blood, whether it has been generated 25 in blood, or gains access to the blood~ from the tumour or normal tissue.

One aspect of the present invention provides a compound comprising a portion capable of at least partly restraining the compound from leaving the vascular compartment of a host when said compound is administered to the vascular 30 compar,tment and an inactivating portion capable of converting a cytotoxic agent wo93/13806 ~ 1. 7 PCl`/GB93/00040 i~ '~

into a less toxic substance.

The method used to retain an inactivating agent in the vascular compartment will be determined in part by the nature of the inactivating agent which in turn5 may be determined by the nature of thè active dru~.

The retaining component must be biologically- compatible with all the functions of blood and the body's organs and should be biodegradable although degradation may take place slowly. It is a requirement that entry of the 10 retainin~ component into tumour extracellular should be minimàl It follows from the latter requir~ment that the inactivating portion should not be releasedin a free state from the retaining portion unless the free inactivating portion is rapidly removed from the blood thus limiting its access to tumour extracellular space.
The restraining portion may be a red blood corpuscle. Suitably~ the compound may be contained on or within red blood corpuscles by utilising techniques known in the art for causing molecules to enter into red blood corpuscles. T,he inactivating portion may be attached to the ervthrocyte membrane or mav be 20 made to enter the ervthrocyte and be retained therein.

A method of introducing desired agents into mammalian red blood cells without unacceptable loss of cell contents has been described ~US 4~31276 incolporated herein by reference). This method comprises (a) suspending and incubating the 25 cells in a solution containing a compound which readily diffuses into and outof the cells~ the concentration of the compound being sufficient to cause diffusion into the cells so that the contents of the cells become hvpertonic; ~b) rapidly creating a trans-membrane osmotic gradient by diluting the solution containing the hypertonic cells with an isotonic aqueous medium in the presence 30 of at least one agent to be introduced to cause diffusion of water into the cells ~ i 2~1217 wo 93/13806 Pcr/Gss3/00040 with consequent swelling and increase in permeability of the outer membranes;
and (c) maintaining the increase in permeability of the membranes for a time sufficient only to permit transport of the agent into the cells and diffusion ofsaid compound out of the cells. This method is especially useful with the S "osmotic pulse" mechanism of US4478824 for loading the cells.

US4652449 (incorporated herein by reference) describes a further method for encapsulation of a biologically-active substance in mammalian ervthrocytes which comprises (a) continuously feeding an aqueous suspension of ervthrocytes 10 into the main companment of a dialysis unit. the second compartment of which contains an aqueous solution which is hypotonic with respect to th~ er~throc~te suspension so as to cause Iysis of the erythrocvtes and (b) causin~ the erythrocyte Iysate ~o be in contact or contacted with the biolo~icall~ active compound(s) and then resealing the erythrocyte membranes bv increasing the - 15 oncotic and/or osmotic pressure of the Iysate.

Alternatively~ the restraining portion may be a liposome~ or ~ hi~h molecular weight ( ~ 25~00() Daltons, preferably at ieast 40.000 Daltons) pol~ mer such asdextram or a protein such as macroglobulin.
Liposomes with prolon~ed circulation times are also potential carriers t^or the inactivating portion. Liposomes with prolonged circulation times have been constructed in various ways. These include the use of gan~lioside GM 1 or a mixture of sphingomyelin~ egg phosphatidylcholine and cholesterol~ or other 25 relatively rigid carrier lipids. For each type of liposome, optimisation by sizin~
is also desirable to optimise prolonged intravascular residence. Gabizon and Papahadjopoulos (1988) Proc. Natl. Acad. Sci. USA 8~ 6949-695~) found a mean particle diameter of 100 nm to be optimal. However~ liposomes with a prolonged circulation time are suitable only if ~hey show no significant uptake 30 in tumours.
~.

2~ 2i~17 WO93/13806 pcr/Gs93/ooo4o Dextrans are polysaccharides consisting of cx-D-glucose units joined predominantly by 1-6 linkages. Partially hydrolysed and fractionated dextran has been widely used as a blood plasma expander. They are cleared by dextranases which are present in the liver, spleen, kidney, intestinal mucosa 5 and colon (Rosenfeld e~ al (1959) Biokhimia [Engl~ 24, 965-970: Ammon - (1963) En~ymologia 25, 245-251; Serry & Hehre (1956) J. Bacteriol. 71, 373-380). Dextrans are widely available with mean molecular weights of 40 kD
(Gentran 40. Rheomacrodex), 70-75 kD (Gentran 70 Macrodex) and 110 I;D.
They are potentially antigenic and some people have pre-existing antibodies~ but10 reactions to dextrans are reported to be no higher than to other widelv used pharmaceutical agents and are mild in character ~Goodman and Gilman. The Pharmacological Basis of T/lerapeurics. 8th Edition~ Pergamon Press. New York, Oxford, 1990).

15 Other polymeric drug carriers have been develope~ from carbohydrates~
peptides and lipids. Some of these may be suitable alternative restraining portions for the inactivating portion provided they fulfil the criteria defined here.

20 The restrainin~ portion may advanta~eously have a negative charge and mav have low lipophilicity.

Suitably., the restraining portion is biodegradable.

25 By biodegradable" we mean that the retaining portion is de~raded in the bodvof the patient, and therefore has a relatively short half-life, for example lessthan 72 hours, preferably less than 48 hours.

A second aspect of the present invention provides a method of inac~ivating a 30 cytotoxic agent in the vascular compartment of a host comprising administering I WO 93/1380Si ~ ~il r~ 2 11 7 Pcr/GB93/00040 to the host said compound.

Thus, it is preferred that the active drug is an alkylating a~ent and the methodof intravascular inactivation is by reaction with reduced glutathione, a reaction 5 which is catalysed by glutathione-S transferase. The non-protein thiol glutathione is regarded as the main endogenous reducing agent. Both glutathione and ~lutathione-S-transferase are present in normal erythrocvtes.
and it has been shown that ~lutathione levels in erythrocvtes correlate with responses to conventional chemotherapeutic agents in patients with breast 10 cancer, hi h le~els being associated with poor response (Herberos et al (1992) Tlle LallC~l 339. 1074-6). It has also been shown that the ~lutathione content of erythrocvtes can be substantiallv increased (3- to 4-fold) by a procedure of hypertonic dialysis and isotonic resealing (Fazi et al (1991) Biotech atld App.
Biochemis~r~ 14. 60-68).
1~
lnactivation of al~vlating agents in the blood is therefore possible using ervthrocvtes overloaded with glutathione. For this to be effective~ the active drug is required to be more readily inactivated than the pro-drug from which it is derived.
It is further preferred ~hat antibodies which discriminate between an active druo and its pro-drug are administered. Since a pro-druo~ usually~ possesses a cleavable moiety which is not present in the active drug, this provides a structural difference between the pro-drug and dru~ such tha. the said 2~ antibodies can bind onlv to the dru~. -Monoclonal antibodies have the advantage that they can be humanized thereb~limiting their immunogenicity. Such an antibody needs to be retained within the vascular compartment since it would otherwise inactivate active drug at 30 tumour sites. Restriction to the vascular compartment may be achieved by ~12~217 ~` `
WO 93/13806 Pcr/Gss3/ooo4o~ '~

galactosylating the antibody which confers a short half-life through uptake by hepatocyte galactose receptors (Sharma et al ~1990) Brit~ J. Cancer 61~ 6S9-662). The more galactose moieties on the antibody the shorter the half-life The short plasma half-life limits tissue penetration but to be effective would 5 require continuous intravenous infusion throughout the period of active dru~
generation.

Alternatively, antibody may be conjugated to a slowly biodeoradable polysaccharide such as a dextran.
::
lt is still further preferred ~hat an enzyme which degrades the activ~ druc~. orrenders it unable to cross cell membranes. and which has no effect~ or much less effect, on the pro-drug is administered. In certain circums~ances it is advantageous to use an inactivating enzyme compared to an inactivating 15 antibody since one enzyme molecule may inactivate a large number of active drug molecules whereas an antibody would act stoichiometrically (ie one antibody is required to inactivate one active dru~ molecùle~. A non-human enzyme has the disadvantage of beino immunogenic and therefore requi~es some form of irnmunolo~ical control but human enzymes includino 20 glucuronidases. kinases, sulphatases and other drug-inactivating enzymes may be used depending on the active dru~ substrate. Humanized catalytic antibodies would be advantageous.

An enzyme may be incorporated into erythrocytes by the osmotic technique 2~ described herein for the incorporation of ~lutathione, or incorporated in liposomes with prolonged circulation time. Alternatively~ an enzyme may be retained within the vascular compartment by conjugation to one of the many types of slowly biodegradable polymeric drug carrier (Krinick & Kope~ek (1991) in Targeted drug deliver~, pp 105-179 (Ed. R.L. Juliano)~ Springer 30 Verlag New York).

~:12217 PCT/GB ~ 3 / 0 0 0 40 13 D~C~ BER 199 A third aspect of the present invention provides a three companent kit of parts comprising a first component comprising a target cell-specific portion and an enzymatically active portion capable of converting a pro-drug into a cytotoxic drug; a second component that is a pro-drug convertible by said enzymatically S active portion to the cytotoxic drug; and a third component comprising a portion capable of at least partly restraining the said third component from leaving the vascular compartment of a host when said third component is .,~
administered to the vascular compartment, and an inactivating portion capable of converting the cytotoxic drug into a less toxic substance.
''`
In this way, the action of the cytotoxic agent may be sustained at tumour sites for significantly longer periods than is possible by conventional therapy, such that the clinical usefulness of the drug is substantially increased. In accordance -~
with the method of the present invention, a cytotoxic agent is generated at - 15 target cell sites, whereas any drug which reaches the vascular compartment is `;
at least partially destroyed or inactivated, thereby allowing prolonged action of the cytotoxic agent.

The entity which is recognised by the target cell-specific portior~ of the first20 component of the kit of parts may be any suitable entity which is expressed by tumour cells, virally-infected cells, pathogenic microorganisms, cells introduced as part of gene therapy or normal cells of the body which one wishes to destroy ` -for a particular reason. The entity must be present or accessible to the targeting portion in significantly greater concentrations in or on cells which are 25 to be destroyed than in any normal tissues of the host that cannot be functionally replaced by other therapeutic means.

The entity which is recognised will often be an antigen. Tumour-specific`
antigens, when they are expressed on the cell membrane or secreted into 30 tumour extra-cellular fluid, lend themselves to the role of targets for antibodies.

WO93/13806 2 ~. 2 4~ ~ 7 PCI'~GB93/00040 t~

The term "tumour" is to be understood as referring to all forms of neoplastic cell growth, including tumours of the lung, liver, skin, pancreas, colon~
prostate, uterus or breast~ The host is preferably a mammal, most preferably a human, but could in principle be any vertebrate.
S
The antigen-specific portion may be an entire antibody (usually, for convenience and specificity. a monoclonal antibody), a part or parts thereof (for example an Fab fra~ment or F(ab')~), a single chain antibody fragment or a synthetic antibody or part thereof. Th~ antibody component may be human or 10 humanised or may be a catalytic antibody. A conjugate comprising only part of an antibody may be advantageous by virtue of better tumour penetration and may be less likely to underPo non-specific binding due to the Fc part. Suitable monoclonal antibodies to selected antigens may be prepared by known techniques~ for example those disclosed in "Monoclonal Antibodies: A manual 1~ of techniques", H. Zola (CRC Press 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and Applications`', J.G.R. Hurrell (CRC Press. 1982).
All references mentioned in this specification are incorporated herein by reference. Bispecific antibodies may be prepared bv cell fusion. by reassociation of monovalent fra~ments or by chemical cross-linking of whole 20 antibodies. with one part of the resulting bispecific antibody being directed to the cell-specific antigen and the other to the enzyme. The bispecific antibody can be administered bound to the enzyme or it can be administered first, followed by the enzyme. Methods for preparing bispecific alltibodies are disclosed in Corvalen et al (1987) Cancer Immunol. Immunother. 24~ 1?7-132 25 and 133-137 and 138-143 and Gillsland et al (1988) Proc. Natl. Acad. Sci. USA 85, 7719-7723.

The variable heavy (VH) and variable light (VL) domains of the antibody are involved in antigen recognition~ a fact ~Irst recognised by early protease 30 digestion experiments. Further confirmation was found by "humanisation" of wo 93/13806 2 i 2 l~ 21 7 PCI/GB93/00040 rodent antibodies. Variable domains of rodent origin may be fused to constant domains of human origin such that the resultant antibody retains the anti~enic specificity of the rodent parènted antibody (Morrison et al (1984) Proc. Na Acad. Sci. USA 81, 6851-6~55).
S ''.
That antigenic specificity is conferred by variable domains and is independent of the constant domains is known from experiments involving the bacterial expression of antibody fragments, all con~aining one or more variable domains.
These molecules include Fab-like molecules (Better et al (1988) Scie/7ce 240.
1041); F~ molecules (Skerra et al (1988) Science 240~ 1038): sin~le-chain F~!
(ScFv) molecules where the VH and VL partner domains are lin~ed ~ia a flexible oligopeptide (Bird et al (1988) Science 242~ 423: Huston et al (1988) Proc. Natl. Acad. Sci. USA 85. S879) and single domain antibodies (dAbS?
comprising isolated V domains (Ward et al (1989) Natllre 341. ~44). A
general review of the techniques involved in the synthesis of antibodv fra~mentswhich retain their specific binding sites is to be found in Winter & ~lilstein (1991) Nature 349~ 293^299.

Bv `'ScFv molecules we mean molecules wherein the VH a-~d VL partner domains are linked via a flexible oligopeptide.

The advantages of using antibody fragments~ rather than whole antibodies. are several-fold. The smaller size of the fragments may lead to improved pharmacological properties. such as better penetration of solid tissue. Effectorfunctions of whole antibodies, such as complement binding~ are removed. Fab.
Fv~ ScFv and dAb antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of the said fragments.

Whole antibodies and F(ab')~ fragments are bivalent'~ Bv "bivalent" we 2~2~ 7 WO 93~13806 PCI'/GB93/00040 ' mean that the said antibodies and F(ab') fragments have two antigen combinin~
sites. In contrast, Fab, Fv, ScFv and dAb fragments are monovalent~ having only one antigen combining sites. Fragmentation of intact immunoglobulins to produce F(ab')~ fra~ments is disclosed by Harwood et ~1(1985) E~r. J. Cancer Clin. Oncol. 21, 1515-1522.

IgG class antibodies are preferred. `

Alternatively~ the entity which is recognised may or may not be antigenic but 10 can be recognised and selectively bound to in some other way. For example it may be a characteristic cell surface receptor such as the receptor for melanocvte-stimulating hormone (MSH) which is expressed in high numbers in melanoma cells. The cell-specific portion may then be a compound or part thereof which specifically binds to the entity in a non-immune sense. for -15 example as a substrate or analogue thereof for a cell-surface enzvme or as a messenger.

The virus-directed enzyme-pro-drug therapy (YDEPT) approach has been disclosed for the selective killing of neoplastic cells using the transcriptional 20 differences between normal and neoplastic cells to selectively drive expression of enzymes capable of convertin~ a pro-drug into a cvtotoxic dru~ (Huber et al (1991) Proc. Natl. Acad. Sci. USA 88, 8039-8043).

A difference in transcription between cells may be associated with tissue-2~ specific promoters, or may be due to changes in activator or repressor molecules in the neoplastic state. Thus in one example~ liver-associated albumin transcriptional regulatory sequences may be useful to drive ~he expression of inhibitor-inactivating enzymes in the treatment of patients with hepatocellular carcinoma. More transcriptional differences between normal and 30 neoplastic cells are being discovered all the time, and it is believed that many wO 93/13806 2 1 ~ ~ 2 17 pcr/Gss3/ooo4o of these differences may be exploited in the methods of the present inven~ion, Recombinant, replication-defective retroviruses which are suitable for deliverin,~
the genetic constructs (ie promoler pl'us gene encodin,~ inhibitor-inàctivatin~
5 enzyme) to target cells have been disclosed (Huber et a/ (1991) Proc. Natl Acad. Sci. USA 88. 8039-8043).

Thus, in one embodiment. the first component of the kit may be a system for deliverin~g a suitable ,~enetic construct for VDEPT.
Considerable ~ork has already been carried out on antibodies and fra~ments thereof to tumour-associated anti~ens and antibodies or antibody fra~ments directed at carcinoembryonic anti~en (CEA) and antibodies or their fragments directed at human chorionic gonadotrophin (hCG) can be conju,~ated to 15 carboxypeptidase G2 and the resulting conju~ate retains both antigen binding and catalytic function. Followin~ intravenous injection of these conju~ates theylocalise selectively in tumours expressing CEA or hCG respectively. Other antibodies are known to localise in tumours expressing the correspondinP
antigen. Such tumours mav be primary and metastatic colorectal sancer (CEA
20 and choriocarcinoma (hCG) in human patients or other forms of cancer.
Although such antibodv-enzyme conju~ates may also localise in some normal tissues expressing the respective anti,~ens. anti,~en expression is more diffusein normal tissues. Such antibody-enzyme conjugates may be bound to cell membranes via their respective anti~ens or trapped by anti~en in interstitial 25 space.

Examples of cell-specific anti~ens are ,_iven in Table 1, WO93/13806 Z 1 2 ~ 7 pcr/Gss3/ooo4o Table I

1. Tumour Associated Antigens ~; Antigen Antibodv Existin~ Uses Carcino-embryonic {C46 (Amersham) Imaging & Therapy of colon Antigen ~85A12 (Unipath~ /rectum tumours.

Placental Alkaline H17E2 (ICRF~ ]maQinQ & Therap~ of Phosphatase Travers & Bodmer) testicular and ovarian cancers.

Pan Carcinoma NR-LU-10 (NeoRx Imaging ~ Therap~ of Corporation) various carcinomas incl. small cell lunQ cancer.

Polymorphic HMFGI (Taylor- Ima~inQ & Therapv of Epithelial Mucin Papadimitriou. ovarian cancer~ pleural (Human milk fat ICRF) effusions.
globule) ,B-human Chorionic W 14 Targeting of enzyme (CPG2) Gonadotropin to human ~;eno~raft choriocarcinoma in nude mice (Searle et al (1981) Br.J. Cancer 44 137-144).

A carbohydrate on L6 (IgG2a)1 Targetin~ of alkaline Human Carcinomas phosphatase (Senter et al (1988) Proc. NaIl. Aca~. Sci.

~o 93/13806 ~ 1 2 ~ 2 1 7 PCI`/GB93/00040 ~ ~:

USA 85, 4842-4846 CD20 Anti~en on IF5 (IgG2a~2 Targeting of alkaline B Lymphoma (normal phosphatase (Senter et al and neoplastic) (1988) Proc. NaII. Acad. Sci. -85. 4~42-4846 Hellstrom et al (1986) Cancer Res. 46. 3917-3923 Clar~;e et al (1985) Proc. Natl. Acad. Sci. 82. 1766-1770 ' ~:
Other an~igens include alphafoetoprotein. Ca-125 and prostate specific anti~en.

2. Immune (~ell Antigens Pan T Lymphocyte OKT-3 (Ortho) As anti-rejection therapy ~
Surface Anti~en (CD3) for kidney transplants. .

B-lvmphocyte RFB4 (Janossv. lmmunotoxin therapy of 1 Surface AntiPen Roval Free cell Ivmphoma (CD22) Hospital) .

Pan T Iymphocyte H6S (Bodmer, Immunotoxin treatment of Surface Anligen Knowles ICRF, Acute Graft versus Host (CD5) Licensed to Xoma Disease, Rheumatoid Corp., USA) Arthritis.

WO 93/13806 2 1~ 4 2 17 PCI/GB93/00040~ ~

3. Infectious Agent-Related Anti~ens Mumps virus- An~i-mumps Antibody conjugated to related polyclonal Diphtheria toxin for antibody treatment of mumps.

Hepatitis B Anti HBs AP Immunotoxin a~ainst SurFace Anti~en Hepatoma.

It is likelv that the enzvmatically acti~e portion ot the first component will be active in isolation from the cell-specific portion but it is necessary only for it to be enzymatically active when (a) it is in combination with the cell-specific portion and (b) the compound is attached to or adjacent target cells.

lS It may not be necessary to use a conventional enzvme. Antibodies with catalytic capacity have been developed (Tramontano e~ al Science 234~ 1566-1570) and are known as 'abzymes'. These have the potential advantaoe of bein~ able to be humanized to reduce their immuno~enicitv.

The two portions of the first component of the kit of parts of the invention ma~be linked to~ether bv any of the convenlional ways of cross-linl;ino polypeptides, such as those generally described in O'Sullivan el al (1979!
- Anal. Biochem. 100~ 10û-108. For example~ the antibody p.ortion may be enriched with thiol groups and the enzyme portion reacted with a bifunctional agent capable of reacting with those thiol groups, for example the N-hydroxysuccinimide ester of iodoacetic acid (NHIA) or N-succinimidyl^3-(2-pyridyldithio)propionate (SPDP). Amide and thioether bonds~ for example achieved with m-maleimidobenzoyl-N-hydroxysuccinimide ester, are generally more stable i~1 ViYo than disulphide bonds.
-~

WO 93/13806 2 i~ 2 ' 2 ~ 7 PCI`/GB93/00040 It may not be necessary for the whole enzyme to be present in the first component (or the third component, if the third component metabolises the drug) of the kit of parts but, of course, the catalytic portion must be present 5 Alternatively, said first component may be produced as a fusion compound by recombinant DNA techniques whereby a length of DNA comprises respective regions encoding the two portions of the compound of the invention either adjacent one another or separated by a region encodin~ a linker peptide which does not destroy the desired properties of the compound. Conceivably. the two 10 portions of the compound may overlap whollv or partl~

The DNA is then expressed in a suitable host to produce a polypeptide comprising the compound of the invention. Thus. the DNA encodin~ the polypeptide constituting the compound of the invention may be used in 1~ accordance with known techniques. appropriately modified in view of the teachings contained herein~ to construct an expression vector, which is then used to transform an appropriate host cell for the expression and production of the polypeptide ofthe invention. Such techniques include those disclosed in~JS
Patent Nos. 4.440.859 issued 3 April 1984 to Rutter et al. 4.530~901 issued ~3 20 July 1985 to Weissman, 4,582,800 issued 15 April 1986 to Crowl. 4.677.063 issued 30 June 1987 tv Mark et al. 4.678.751 issued 7 Julv 1987 to Goeddel.

4~704,362 issued 3 November 1987 to Itakura et al . 4.710.463 issued December 1987 to Murray, 4,757.006 issued 12 Julv 1988 to Toole. Jr. et al~
4~766,075 issued 23 August 1988 to Goeddel et al and 4,810.648 issued 7 25 March 1989 to Stalker, all of which are incorporated herein by reference.

The DNA encoding the polypeptide constituting the compound of the invention may be joined to a wide variety of other DNA sequences for introduction into an appropriate host. The companion DNA will depend upon the nature of the 30 host, the manner of the introduction of the DNA into the host, and whether wo 93/13806 ~ ~L 2 4 2 1 7 pcr/GB93Jooo4o episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector~ such as a plasmid, in proper orientation and correct reading frame for expression. If necessary, 5 the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by the desired host. althoughsuch controls are generally available in the expression vector. The vector is then introduced into the host throu~h standard techniques. Generally. not all of the hosts will be transformed by the vector. Therefore~ it will be necessary l 0 to select for transformed host cells. One selection t~chnique involves incorporatin~ into the expression vector a DNA sequence~ with an~ necessary control elements. that codes for a selectable trait in the transformed cell. such as antibiotic resistance. Alternatively, the ~ene for such selectable trait can be on another vector, w hich is used to co-transform the desired host cell.
Host cells that have been transformed by the recombinant DNA of the invention are then cultured for a sufficient time and under appropriate conditions 1cnown to those skilled in the art in vie~ of the teachin~s disclosed herein to permit the expression of the polypeptide. which can then be recovered. --Many expression svstems are known. includin~ bacteria (for example E. coliand Bacillus subtilis), yeasts (for example 5`accharom!~ces cerevisiae)~
filamentous fungi (for example Aspergillus), plant cells, animal cells and insect cells.
The vectors include a procaryotic replicon~ such as the ColE 1 ori, for propagation in a procaryote, even if the vector is to be used for expression in other, non-procaryotic. cell types. The vectors can also include an appropriate promoter such as a procaryotic promoter capable of directin~ the expression 30 (transcription and translation) of the genes in a bacterial host cell. such as E.

wo93/13806 2 1 2 ~12 ~ 7 pcl/Gs93/ooo4o coli. transformed therewith.

A promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter S sequences compatible with exemplary bacterial hosts are typically pro~ided in plasmid vectors containin~ convenient restriction sites for insertion of a DNA
se~ment of the present invention.

Typical procaryotic vector plasmids are pUC18. pUCl9. pBR~ and pBR3~9 available from Biorad Laboratories (Richmond. CA. USA) and pTI~9A and pKK223-3 available from Pharmacia. Piscatawav~ NJ. USA.`

A typical mammalian cell vector plasmid is pSVL available t^rom Pharmacia.
Piscataway, NJ USA. This vector uses the SV40 late promoter to dn~e expression of cloned ~enes, the hi~hest level of expression bein~ found in T
antigen-producin~ cells. such as COS-l cells.

An example of an inducible mammalian expression veclor is pl~SG~ ~also available from Pharmacia. This vector uses the ~lucocort~coid-ind~lcible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of the cloned ~ene.

Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Strata~ene Cloning Systems~ La Jolla CA 92037 USA. Plasmids pRS403, pRS404. pRS405 and pRS406 are Yeast Inte~ratin~
plasmids (YIps) and incorporate the yeast selectable markers ~2is3, trpl, lel~2 and ura3. Plasmids pRS413-416 are Yeast Centromere plasmids (~'Cps) A variety of methods have been developed to operativelv link DNA to vectors 30 via complementary cohesive termini. For instance~ complementar~r 6 2 ~ 2 ~ 217 Pcr/Gss3/0oo4o ~ ;~

homopolymer tracts can be added to the DNA se~ment to be inserted to the vector DNA. The vector and DNA segment are then joined by hydro~en bonding between the complementary homopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors. The Dl~A segment. generated by endonuclease restriction digestion as described earlier~ is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase 1~ enz,vmes that 10 remove protrudin~. 3'-sin~le-stranded termini with their 3`-5`-exonucleol~ticactivities~ and fill in recessed 3'-ends with their polymerizinP activities.

The combination of these activities therefore generates blunt-ended DNA
segments. The blunt-ended segments are then incubated with a lar~e molar 15 excess of linker molecules in the presence of an enzyme that is able to catalvze the ligation of blunt-ended DNA molecules~ such as bacteriopha~e T4 DNA
ligase. Thus~ the products of the reaction are DNA se~ments carrvin~
polymeric linker sequences at their ends, These DNA se~ments are then cleaved with the appropriate restriction enzvme and li~ated to ~n expression 20 vector that has been cleaved with an enzyrne that produces termini compatiblewith those of the DNA segment. ;

Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International 25 Biotechnologies Inc, New Haven, CN, USA.

A desirable way to modify the DNA encodin~ the polypeptide of the invention is to use the polymerase chain reaction as disclosed- by Saiki et al (1988) Science 239. 487-491.

2i212-~
wo 93/13806 pcr/G~93/ooo4o In this method the DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers which themselves become incorporated into the amplified DNA. The said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression 5 vectors using methods known in the art.

Exemplary ~nera of veast contemplated to be useful in lhe practice of the present invention are Pic~lia, Saccharom~ces, Klu~veromvces, Canclida, Torulopsis, Hansenuln, Schi70saccharom~ces, Citeromvces. Pach! sole~l, 10 Debarom~ces. Metsc~lu~ ;o--!in, Rhodosporidium. Leucospondium. Botr~oasclls, Sporidiobolus, Endo~n! copsis. and the like. Preferred ~enera are those selectedfrom the group consistin~ of Pichia, Saccharomvces, Kluvverom!~ces. Yarrowia and Hansenula. Examples of Saccharomvces are Sacc~larom!!ces cere-i~isiae~
Saccharomy~ces italicus and Saccharomvces ro~xii. Examples of Klu! verom! ces 15 are Kluvveromyces frQgilis and Kl~vverom!~ces lactis. Examples of Hansenula are Hansenl~la pol!~morp~7a, Hansenula anomala and Hansenula c~psulala.
Yarrowia lipoh~tica is an example of a suitable Yarrowia species.

Methods for the transformation of S. cerevisiae are taught ~enerall~ in EP '~1 20 744, EP 258 067 and WO 90/01063, all of which are incorporated herein bv reference.

Suitable promoters for S. cerevisiae include those associated with the PG~'l gene, GALI or GAl,10 genes. CYCI, PH05, TRPI~ AD~ ADH~ the ~enes 25 for glyceraldehyde-3-phosphate dehydrogenase. hexokinase~ pyruvate decarboxylase, phosphofructokinase. triose phosphate isomerase~
phosphoglucose isomerase~ glucokinase. ~-mating factor pheromone~ a-mating factor pheromone~ the PRBI promoter, the G~J72 promoter~ and hybrid promoters involving hybrids of parts of 5' regulatory re~ions with parts of 5' 30 regulatory regions of other promoters or with upstream activation sites (eg the WO g3/138n6 2. 12 ~ 2 17 Pcr/Gss3/00040 ~ ~

promoter of EP-A-258 067).

The transcription termination signal is preferably the 3 flanking sequence of a eukaryotic gene which contains proper signals for transcription ~ermination 5 and polyadenylation; Suitable 3 flanking sequences may tor example be those of the gene naturally linked ~o ~he expression con~rol sequence used. ie may correspond to the promoter. Alternatively they may be differen~ in which case the termination si~nal of the S. cerevisiae AHDI ~ene is preferred 10 The second componen~ of the kit is a pro-drug which is relatively non-to~ic.
which is a substrate for the enzyme in the first componen~ of ~he ~;it and ~hicl~
is converted to a cytotoxic substance. The cytotoxic subs~ance may be any existing anti cancer drug such as an alkyla~ing agent~ an agent which interca-lates in DNA~ inhibits any key enzymes such as dihydrofolate reductase.
15 thymidine synthetase ribonucleotide reductase. nucleoside kinases or topoisomerase or effects cell death by interacting with any other cellular ;
constituent. Etoposide is~an example of a topoisomerase inhibitor. ~-It is evident that the inactivating portion of the compound of the invention 20 which forms the third component of the kit of parts must be chosen to suit the active drug that has been generated. It is also necessarv for most of the inactivation of the cytotoxic drug to be confined to the vascular compartment so as to avoid undesirable inactivation of the active d~ug at tar~et sites.

25 It will also be appreciated that the inac~ivating portion of the compound does not inactivate the prodrug which mày also be present in the blood~ in such a way that its conversion into the cytotoxic drug is prevented. lt is desirable tohave prodrug in the blood so that it can reach tumour sites where it iS
converted to the cytotoxic dru~. In accordance with preferred embodiments of 30 the present invention a means is therefore provided by which an active dru~

WO 93/13806 ~. 1 2 ~ 2 ~ 7 pcr/GB93/ooo4o generated from a prodrug at particular sites in the body can be inactivated in the blood of a patient without comparable inactivation of any prodru~ which is present in the blood.

The inactivating portion may be an enzymatically active portiôn~ capable of convertin~ the active dru~ into a less toxic substance. It may not be necessar~
for the whole enzyme to be present but~ of course, the catalytic portion must be present. In the case of methotrexate as the active drug, for example. the inactivating portion may be the enzvme carboxypeptidase G2 or another folate-10 deglutamating enzvme. which de~rades methotrexate therebv inactivatin~ it.

The bacterial enzymes carboxvpeptidase G I and G~ (CPG 1 and CPG2) de~radefolates including methotrexate by cleava~e of the ~erminal glutamic acid. The actions of the two enzymes are thought to be the same. The following description of preferred aspects of the invention refers to CPG2 but is equally applicable to CPGI and to any otlIer enzymes actin~ on the same substrates.
and to abzymes actin~ on the same substrates.

The isolation. purification and some of the properties of carboxypeptidase G2 20 from Pse~domonas sp. strain RS-16 have been disclosed by Sherwood et al (1984) Eur. J. Biochem. 14~ 447-453. The clonin~ of the ~ene encodin~ the said carboxypeptidase G2, its nucleotide sequence and its expression in E. coli have been disclosed by Minton et al (1984) Cene 31~ 31-38 and Minton et al (1983) J. Bacteriol. 1~6, 1222-1227. CP2(~2 is available from the Division of 25 Biotechnology, Centre for Applied Microbiological Research, Porton Down~
Salisbury. UK. Carboxypeptidase Gl (CPGI) is disclosed by Chabner el al ( 197~) Cancer Res. 3~. 21 14-21 19.

The inactivating portion may alternatively be a chemical moiety which reacts 30 spontaneously with the active cytotoxic dru~ or does so more effectivelv in the 2 i 7~ 17 pcrlGss3/ooo4o , 6 presence of an appropriate catalyst. For example. in the case of an alkylating agent as the drug, the inactivating portion may comprise a thiol-containin~
substance and inactivation is catalysed by ~lutathione transferase carried in oron macromolecular entities.

The inactivating portion may alternatively~ for example~ be an an~ibody or part thereof which binds to the active dru~ but not to the corresponding prodrug or does so only to a more limited extent. or which is catal~ticallv active towards the pro-dru~ thereby inactivatin2 it. but not active towards the dru~.
.
The production of antibodies to 1Ow molecular wei~ht colnpounds such as cytotoxic drugs may be facilitated bv the well l;nown technique of haptenisation, in which the low molecular wei2ht n~olecllle is conjuj~ated to a ;~
highly immunogenic protein~ s~lch as keyhole limpet haemocyanin or other 15 carrier molecule.

In situations where prodrujP is converted to active drug bv catalytic cleava~e of a moiety of the prodrug~ the production of an antibodv capable of discrimi~at-ing between prodru~ and active drug is favoured since~ in the prodrug~ the non-20 active moiety can sterically inhibit binding of an anti-dru~ an~ibodv to the thus hidden drug portion of the prodrug~

The inactivating portion may be conjugated to the restraining portion accordin~
to methods of linkage known in the art~ thereby to retain the compound of the 25 invention in the vascular compartment~

A further component may achieve accelerated clearance of antibody-enzyme conjugate and/or inactivation of the specific enzyme from non-tumour sites.
Several means by which this can be achieved have been described (PCT Patent Application WO 89/10140 & Baj~shawe~ 1989). For example~ in one method~

wo93/13806 ~12 ~ 7 Pcr/Gs93/00040 an antibody directed at the enzyme is employed. To prevent such an antibodv from combining with and possibly inactivating enzyme at tumour sites additional galactose residues are added which ensure that this second antibody and antibody-enzyme conjugate are quickly cleared from the blood bv bindin~
5 to galactose receptor rich hepatocytes.

The antibody used for clearing or inactivating the antibodv-enzvme conju~ate can be directed towards the antigen binding site on the antitumour antibody or the active site of the enzyme~ or any other site on the antibodv-enzvme 10 conjugate. Such antibodies may have additional galactose resid-les or other su~ars added to accelerate clearance or may be desialvlated. ~ialactosylation of the antibody results in its rapid clearance from the blood throu~h take-up bvgalactose receptors on hepatocytes. Alternatively~ or additon~lly~ the antibody-enzyme conjugate is galactosylated~ and given after the hepatic ~alactose 15 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 comple~; localises at tumour sites but followin~g cessation of galactose receptor blockade. the 20 galactosylated antibody-enzyme is quickly cleared via the available galactose receptors.

A component may also be required to avoid the constraints which could be imposed by the host immune response to foreign proteins. The nature of this 25 component may change with the stage of development of methods of immuno-logical control. Methods which can be used to overcome the host response to foreign protein are known in the art. Techniques for reducing the immuno~enicity of foreign proteins, applicable to antibody-enzvme conju~ates~
is that of conjugation to forms of polvethylene glycol (Wilkinson et al (19~7) 30 J. Immunol. 139~ 326-331).

?~1~24217 wo 93/13806 pcr/Gs93/ooo4o Alternatively, or additionally, the problem of immunogenicity may be overcome by administering immunosuppressors or immune tolerance inducing a~ents~
Cyclosporin and FKS06 are widely used drugs to achieve immunosuppression in tissue trans~lantation. Cyclosporin has been shown to delay host antibody 5 respone to foreign protein (Lederman el a/ (1988~ Br. J. Cancer 58, S62-566 and 654-657). Tolerance to foreign proteins when the host encounters the foreign problem for the first time after receiving an antibody directed at the CD4 epitope on lymphocytes has been disclosed (Waldman et al (1988~ pp 16-30 in Progress i~l A//erg!~ (Shizata & Woksman. Eds~ New Yorl;). Further 10 means to achieve lhis have been described elsewhere and mav chan~e as improvements occur in control of host antibodv responses to foreic~n anti~ens.
Catalytic antibodies (abzvmes) may be 'humanized' to reduce or remove their immunogenicity .

15 A fourth aspect of the present invention provides a method of destrovinQ tar~et cells in a host. the method comprising administration to the host of the variouscomponents described above.

The eomponents of the invention are administered in any suitabl~way~ usually 20 parenterally, for example intravenously, intraperitoneally or intravesically, in standard sterile, non-pyroQenic formulations of diluents and carriers.

When an antibody directed at a tumour associated antigen or an antibody-enzyme conjugate is injected into an appropriate tumour bearing host, only a 25 small fraction of the antibody or conju~ate localises at the tumour site and most of it remains in blood and other normal tissues for several days. Thus, although tumour concentration of the enzyme will be higher than in normal tissues, the volume of normal tissues is much greater. Thus~ to minimize the amount of enzyme residual in normal tissues and blood it may be desirable to 30 use the methods of the presenl invention in conjunction with the methods wo 93/13806 2 ~ ~ ~ ,?1 7 pcr/Gss3/ooo4o disclosed in WO 89/10140; Ba~shawe (1989) Brit. J. Cancer 60~ 75-281: and Sharma et al (I990) Brit. J. Cancer 61~ 659-662 for inactivating and clearin~
excess antibody-enzyme conjugate from the blood.

5 In attempting to achieve eradication of cancers it may not be possible to a-oid suppression of haemopoietic function (myelosuppression) althou~h for a uiven effect on a tumour target it is expected to be much less with the svst~m described herein. Similarly. for a ~iven de~ree of mvelosuppression a much greater tumour effect is expected. Growth factors or ~rowth inhibitory factors 10 actin~ on haemopoietic tissues ma~ sherefore be usef~ mplo~ed in combination with the svstem described herein.

The system described herein may be used in conjunction ~vith other forms of therapy. These include conventional cvtotoxic agents. and use of multiple 15 enzyme delivery to inactivate more than one metabolite.

Similarlyt an enzyme delivered to tumour sites by an antibodv mav function both to activate a pro-drug and to inactivate a metabolite which protects nornlal tissues. Carboxypeptidase G2 as disclosed herein inactivates ~olinic acid at 20 tumour sites to leave the tumour cells unprotec~ed against trimetrexate. The same tumour located enzyme can activate a ben~oic acid pro-dru~ to form a cytotoxic mustard (as disclosed by Ba~shawe (1989) Brit. J. Cancer 60 275-281).

25 The compounds and methods of the present invention will be discussed in th.
following Examples and Figures with specific references to cytotoxic therap~
utilising the a~gent methotrexate which is inactivated by the enzym~
carboxypeptidase G2. The agent aminopterin is another powerful c~totoxic a~ent which is inactivated by the enzvme carbo~l;vpeptidase G2.

~ 12 17 WO 93/13806 PCI ~GB93/00040 3~) ,.
Figure 1 shows the structures of methotrexate and folinic acid.

Figure 2 shows the structures of (1) a pro-drug and (II) the cytotoxic drug (a benzoic acid mustard) produced after cleavage of (I) with carboxypeptidase G~
S ~:-Fi~ure 3 shows a scheme for conjugating aminobenzoic acid to kevhole limpet haemocyanin.

Figure 4 is a diagrammatic representation of the invention wherein the first 10 component is a target-cell specific antibodv coupled to carboxvpeptidase A
(CPA); the second component is alanine-methotrexate: and the third component is a restraining portion coupled to carboxypeptidase G2 (CPG2).

The invention relates to a general principle and mav be applied to other 15 cytotoxic agents.

Example 1: A therapeutic svstem usin~ Ala-methotrexate carbox~p~ptidases A and G2 20 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. ln the absence of folinic acid~ cell reproduction is blocked 25 in S phase and cell death follows. Methotrexate is used in the treatment of awide range of malignant diseases. It also causes cell death in normal cell renewal tissues via the mechanisms alreadv outlined. The magnitllde of i~s 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 30 methotrexate is thought to result from polyglutamation of the drug which~ bv .. .. , . .. _ .. ... . .. , .... ... . ~ . .. . . .. - ~. . . . . .

2 ~2~21'~ ~
wo 93/13806 pcrtGss3/ooo4o 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 advanta~eous over the use of methotrexate alone in the treatment of some cancers. Thus large doses of me~hotrexate are commonly followed after 12-24 hours by folinic acid 'rescue'. Similarly, administration of low dose methotrexate on al~ernate davs and folinic acid on each succeedinc~ day has proved to be a successflll and low toxicitv treatment for many patients with 10 some forms of trophoblastic ~ul-lollr (Baoshawe et ~l (19~9) Brit. J. Ob. G~1ae 96. 795-802).

1t has been shown (Kuefner et al ( 1989) Biochemistr 28~ ~28~-2297! that when methotrexate is modified bv introducing an alanine moietv via an amide linkage 1~ to the c~ carboxyl of methotrexate the resultin~ compound is effectively excluded from cells and the toxicity of the alanine form is 50-100 fold less than that of native methotrexate in tar~et cells i~1 ~itro. The alanine moiety is cleaved by the action of the enzyme carboxypeptidase A leavino natlve methotrexate. Alanine methotrexate is not metabolised bv- the enzvme 20 carboxypeptidase G2. Carboxypeptidase G2 deorades methotrexate and natural folates by cleava~e of the ~lu1amic acid moiety.

In the present example the first component of the system of the present invention comprises an antibody or antibody fragment conjugated to 25 carboxypeptidase A, directed at the tumour associated anti~en~
carcinoembryonic anti~en.

Of course, a carboxypeptidase other than carboxypeptidase A but with the same substrate specificity may be used in place of carboxvpeptidase A.
3~

WO93/13806 ~ 1 2 !~ 2 17 pcr/Gs93/ooo4o Carboxypeptidase A is available from bovine and bacterial sources (for example from Calbiochem, Nottingham, IJK) 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 5 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.

A further component may achieve accelera~ed clearance or inacti~ation of 10 carboxypeptidase A from non-tumollr sites.

The second component is alanine methotrexate (Kuefner et ~11 1989) which is the prodrug from which the active drug methotrexate is generated by the action of carboxypeptidase A.
The third component is carboxypeptidase G2 conju~ated to a macromolecular structure such as dextran. The purpose of the macromolecule is to confine CPG2 activity to the vascular compartment.

20 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 prepartion containing I g of dextran in 0.9~ NaCI was diluted to 100 ml with 0.9% NaCI and reacted with cyanogen bromide (CNBr:
25 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 hi~her molecular weight dextrans.
This reduction was necessary to pre~ent 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 l 0.1 units in a pH-stat (Radiometer.
30 Copenhagen, Denmark) by addition of 2 M NaOH. The CNBr was added as ~WO93/13806 ~ J~ g 7 pcr/Gss3/ooo4o 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 lo 9.0 and the mixture dialysed againstrunning water for 2 hr at 4C. The pH was brought back to 9.0 with I M
NaOH and 1 ml enzyme solution (1265 U; ~.3 mg) in 0.1 M Tris-HCI 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 ~o a volume of 40 ml in a model 202 concentrator usinP a P1~110 ultrafiltration membrane (Amicon Stonehouse. UK). The mixture (40 ml) was then chromato~raphed on a 1.3 litre bed ~olume ot` Sephadex Gl~0 in a 1.~ x ~7 cm column (Pharmacia Uppsala~ Sweden) and eluted with 0.0:~ ~I potassium phosphate buffer~ pH 7.0 Fractions ( 10 ml) were collected and assayed for enzyme activity; carbohydrate content was determined by the phenol-sulphllric acid method (M. Dubois et al ~ 1956) Analvt. C~lem. 28. 350) usin~ dextran-70 as standard in the ran~e 0-100 ~!ml (Sephadex is a trade mar~;).

The peak fractions were pooled and concentrated to a volume of 10-12 ml.as before. Enzyme activitv and carbohydrate content were determined and protein 20 content measured by the Coomassie blue method (M.M. Bradford (1976 Ana/~,t. Biocftem. 72. 248) usin~ bovine serum albumin fraction V as standard in the range 0-100 ~g/ml. The concentrated material was filter sterilised (Millipore "Millex G~"~ 0.22 ~m pore size) and stored at -20C. Millex GS
is a trade mar~.
The antibody-carboxypeptidase A conju~ate~ if comprisin~ antibody of murine ori~in and enzyme of bovine ori~in. ~ould be immunogenic. Similarl~ a carboxypeptidase G2 macromolecule conju~ate would also be immunoPenic since CPG2 is bacterial in origin. It may be desirable~ therefore~ to reduce 30 their immuno enicity or to employ means to induce in1munosuppression or ~1?421 ~ :`
wo 93/13806 pcr/Gs93/ooo4 immune tolerance.

Example 2: Method of use S Administration of a component capable of overcoming the host r~sponse to foreign protein may be started 48 hours before administration of Component 1.
Initial tests may be performed to exclude as far as possible abnormal reaction by the patient to any of the protein components. The antibod~-CPA conju~ate is given intravenously, preferably by slow infusiom tvpically over ? hours.
lO Maximal tumour concentration of the antibody-CPA conjll~ate is achie~ed several hours later but at this time there are still hi~h levels oi` CPA ~ctivity in plasma. It is desirable to eliminate this activi[y as far as possible before administering the prodrug. This elimination process is achieved by administra-tion of a component capable of achieving accelerated clearance or inactivation l5 of CPA from non-tumour sites. This componer~t is administered intravenously over several hours~ typically 6-2~ hours~ or until enzyll1e is no longer detectable in plasma and may be infused at low concentrati~n throu~hcut the period of pro-dru~ administration. ~uring this time enzvme in extracellular fluid diffuses back into the plasma as the plasma level of the enzvme falls.
20 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.

25 Administering component 3 is then commenced either bv a series of bolus injections or by slow infusion. ~ "

It is preferred that components 2 and 3 are started simultaneously (ie the pro-drug and the inactivatin~ compound).

r WO 93/13806 ~ 5~ iv 1 I PCI/GB93/00040 Pro-drug may be given as a series of bolus injections or by continuous infusion Administration of components 2 and 3 will normally continue for about 4-7 days but specific embodiments of Component 1 may ensure that sufficient enzyme actively persists at tumour sites for somewhat longer periods.
s At about 7-10 days after administration of component it is desirable to review enzyme activity at tumour sites. Administration of the component capable of overcoming the hos~ response to foreign protein is continued component 2 is discontinued. component 3 mav be discontinued.
~O ' .
Component I is reinfused as previousl~ followed by the component which eliminates the plasma CPA activitv as previously. Similar procedures to those previously described are followed before recommencing component ~.

1~ The cycle may be repeated. Limiting factors will be toxicity attributable to alanine methotrexate or the development of host an2ibodies to anv of the foreignproteins employed.

Example 3: Use of quinazoline antifo!ates --A similar system to that disclosed in Examples I and 2 can be used with atleast some members of a series quinazoline antifolates which have been described (Jones et al (1986~ ~. Med. Chem. 29~ 468-472; Jodrell et ~1(1991) Brit. J. Cancer 64, 833-8; Harrap et al (1989) Advances in en~me regulatio~l 25 29 161; Jackman et al (199l) Advances En~yme 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 Nl 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 30 some of the drugs in this series are inactivated bv a peptide substitu~ion such ~ ~ 2 ~ 7 WO 93/t3806 PC~/GB93/00040 as an alanine in the ~ position of the ~lutamate. and that such alanine- or other peptide-substituted derivatives are synthesized using the methodology described by Kuefner et al loc. ci~. or variations thereof known in the art. Similarly, such quinazoline antifolates are de~lutamated, and therefore inactivated, by 5 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-dru~ form~ converted to the active compound by carboxypeptidase A and the active dmg in plasma degraded by carboxypeptide G2.
Example 4: Production of ~ntibodies discrimin~tino bet~veen ~ctive dru~
and pro~

In order to raise an antibody that would recognise the active drug (II) but not 15 ~the pro-drug (1), a compound was synthesised which represented the region ofgrèatest divergence between the two molecules (I) and (~I? (Figure 3). This region corresponds to the acid portion of the benzoic acid mustard dru~
Since benzoic acid itself is not lar~e enou~h to be immunogenic, it w~s considered that the most effective method of raising antibodies specific for the20 acid region would be to inoculate animais 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 li-nked throu~h an amide bond to aminobenzoic acid (VII). Then (VII) was conjugated to KLH by conventional methods usin~ the ~-NH~ groups on the Iysine portion 25 of the molecule, to produce the specific immuno~en (VIII).

1. Preparation of (lV) The N~.N~-di-tert-Benzyloxycarbonyl-L-lysine ~IV) was liberated from its 30 dicyclohexylammonium salt (Ill). Briefly, (III) ~4 mmol) was suspended in 2:12 ~2~ ~
WO 93/13806 Pcr/Gs93/ooo4o ethyl acetate (100 ml), then washed with cold citric acid (10%). The or~anic layer was separated, dried over sodium sulphate and evaporated to drvness to (IV), a white gum. Yield 100%. NMR (Me~SO-~6) ~1.4 (bd.s. 2 H)~ 1.6 (m.
2H), 2.9 (d, 2H)~ 3.8 (m, IH), 6.7 (m~ lH). 7.0 (d~ IH) S
2. Preparation of (V~) Compound (IV) was coupled to 4-arninobenzoyl tert-butvl ester (V ) b~ a modification of a peptide couplin~ literature method (( 19~91 ./ ~1e~1. Che~l. 31 10 163). To a stirred solution of (IV) (2 mmol) and l~-meth~ orpholine (~
mmol) in THF (2 ml). cooled to -20C. was add~d isobllt~l chloro~ormate (2 mmol~. ARer 10 minutes. a suspension of (V) (2 mlllol) in THF (' ml) containing N-methvlmorpholine (2 mmol) ~as added . The stirrin~ ~ as continued for lO minutes at -20C~ and then the mixt~lre allowed to warm to 15 room temperature. The N-methylmorpholine hydrochloride was filtered off and the filtrate evaporated to dryness. The resultin~ crude~ ixtur~ ~as twice chromatographed on silica ~el to give the novel pllre product as an oran<le oil (Vl~. Yield 4%. NMR (Me~S0-~6) o 1.36 (s~ 9H). 1.3~ (s~ 13H)~ Is.
9H)~ 1.61 (m~ 2H). 2.90 (d. 2H). 4 02 (m. IH). 6.73 (m~ lH). 7.00 (d. IH!.
20 7.71 (d, 2H), 7.85 (d, 2H). 10.22 (s~ IH) Mass Spectrllm Ill,''Z 5'1 (M).

3. Preparation of (VI~) The deprotection of compound (Vl) (0.1 mmol) was effected by suspension in 2S TFA (2 mL). After 40 minutes, the solution was evaporated to drvness to ~ive the novel product (VII) as a white solid. Yield 100%. NMR (Me~S0-~6) o 1.40 (m, 2H), l.~S (m~ 2H)~ 1.82 (m, 2H)~ 2.76 (m~ 2H)~ 3.97 (m~ lH)~ ?.6~
~bd.s, 2H), 7~73 ~d, 2H)~ 7.97 (d. 2H), 8.27 (bd.s. 2H). 10.7~ (s. IH~ Mass Spectrum (FAB) m/z 265 ([M + H~]) '~2 ~217 wo 93/U806 pcr/Gss3/ooo4o 4. Preparation of (VIII) The coupling of compound (VII) to keyhole limpet haemocyanin (KLH~ was effected by a modification of the literature method of Hancock and Evan ((1992) Methods in Molecular Biolog~ 10, 23). Briefly, (VII) was added to KLH (equivalent weight for weight) and the mixture adjusted with sodium bicarbonate and glutaraldehyde to a final concentration of O.IM and 0.05~
respectively. The mixture was stirred for 24 hours before the addition of ~Iycine ethyl ester to a final concentration of 0. lM . The reaction mixture ~ as left 30 minutes before the addition of cold acetone (36 ml). After a further 30 minutes. the precipitated conjugate was centrifuged the sllpernatant remo~ed and the pellet air-dried to yield a pink solid. The conjugate was stored at -20C prior to inoculation.

5. Preparation of (VIII) for inoculation The conjugate was suspended in saline (0.9%) to a final concentration of 1 mg/ml. The suspension was then emulsified in Freund's adjuvant (Comp1ete or Incomplete~. prior to inoculation.
Polyclonal and monoclonal antibodies were raised to the conjugate usinP
methods well known in the art.

In the case of the monoclonal antibodies the supernatant from clones of 2~ hybridoma cells grown under conventional conditions was tested for binding to pro-drug and its cytotoxic derivative. Most clones reacted with both pro-drug and active drug. The supernatant from two clones bound only to active druP.

~ W093/t3806 2 ~ 2 ~ 217 PCr/GB93/00040 3 9 . .
Example 5: Use with ADEPT

The ADEPT concept uses an antibody-enzyme conjugate to generale a cytotoxic drug from an inactive precursor at tumour sites. The presen~ invention is used S in conjuction with ADEPT. Active drug is generated at the tumour site usin~
lhe ADEPT antibody-enzyme conjugate. Anv ac~ive drug entering the blood compartment is cleared from the blood usin~ the inactivating agent c~upled to the restraining portion.

10 In the case of ADEPT treatment. nude mice bearin~ human choriocarcinoma (CC3) tumours rec~ived '9 unils of CPG~ ~onjuoated to anti-HCG (~'1 1 Fab~ -as disclosed in WO 88/07378)~ and after ~ or 48 hours recei\,ed pro-drug (41 ~M/kg). The amollnt of inacti~atin~ agent is adjllsted to ~ e an optimal protective effect.
': ~
Example 6: Prepar~tion of a monoclonal ~ntibod~ reacti- e a~ainst ~: -c~rcinoembrvonic anti~en Purified CEA was pr~pared from metastases of colonic tumour. Radio-20 iodination to a specific activity of 6 ~LCi~ was carried out by the iodogen method. Dilution buffer was prepared as a 0.15M sodium phosphate buffer~
pH 7.4, containing 0.1% bovine serum albumin. The studies at low ionic strength were carried out in 0.02M Tris-HCI buffer at pH 7.4.

25 Immunisation schedule: Monoclonal antibody A~B~ was raised against purified. heat-treated CEA using the following procedure. One milli~ram of purified CEA was heated at 85C for ~ mill in 0.05 ~1 phosphate buffer (pH

7) at a concentration of I mg ml~~. After mixing with I ml of 107Q aqueous potassium aluminium sulphate (alum). the pH was adjusted with constant 30 stirring to 6.5-7 by dropwise addition of NaOH solution. After stirrin~ at room 2i~7 WO 93113806 PCl-/GB93/000411 temperature for 30 min the resultin~ precipitate was washed three times in saline. It was then mixed with 10' formalised Borde~ella pertussis (kindlv supplied by Wellcome Research Laboratories). Three different immunisation schedules were used.
s Spleen cells from the immunised mice were then fused with either SP2/0-A~T
14 or P3-NS/I-Ag 4-1 myeloma cells (Flow Laboratories~ Ul~) and the hybridomas producing anti-CEA cloned by sin~le cell transfer.

10 Example 7: Prel~aration of F~ab')~ fra~ments of A~B~ .

The monoclonal anti-CEA (A5B~) used in this study has been described previously and chosen for its low cross-reactivity with NCA and its stability onimmunopurification and radiolabellin~. F(ab')~ fra~ments were prepared by the method Lamoyi and Nisonoff (1983) J. Immunol. Methods ;6~ 235-2~. After separation of the digest mixture on Sephacryl S-200. the fractions were analysed by SDS-PAGE using a 7.5% ~el~ The fraction containino the F(ab'!~
was concentrated and dialysed a~ainst 0.15M phosphate buffer. pH 7. B~th intact A~B7 and the fragment were shown to be immunological1y active and relatively homogeneous by electroblotting of the SDS ~el onto nictrocellulose paper and overlayin~ with ~5I-labelled CEA. Intact AcB~ and its F(ab')~
fragment were radiolabelled by the chloramine T method to specific activities of 5.6 and 5.2 ~Ci/,ug respectively. Both labelled preparations were shown to retain immunological activity by solid-phase radioimmunoassay using CEA
coupled to amino-cellulose (Ro~ers et a/ (1983) El~r. J. Callcer Cli/n 0~7Co/.
19, 629-639). An excess of 60% activity was retained in each case.

`~ WO 93/13806 2 i 2 ~ 2 ~ 7 PCr/Gs93tOo040 Example B: Production of a monoclonal antibod reactive a~ainst carbox~peptidase A

A monoclonal antibody raised a~ainst carboxypeptidase is used for makin~
bispecific antibody (see next Example) and for clearance and inactivation of residual enzyme activity at non-tumour sites.

The monoclonal antibodv was made in the following wav. Balb/C mice (6-8 weeks old) were immunised with ~0 ~1~ CPA i.p. in incomplete Freund's 10 adjuvant followed bv two injec(ions of CPA in complete Freund's adjuvant (50 ~L CPA eacll. i.p.) at monthlv inter~als and with two dail~ injeclions (50 ,u~
and 100 ,uo in PBS~ i.v.) 2 days before fusion. Immune spleen cells were fused with non-immunoglobulin secretin~ SP2!0 myeloma cells accordino to the hybridoma procedures of Kohler and Milstein (1975~.
The presence of anti-CPA antibodies was detected by a solid-phase indirect radioimmunoassay. A 1 ,~ ml-~ solution of CPA in 0.0~ M phosphate buffer was placed in polyvinyl microtitre plates (100 ng per well)~ allowed to drv~
fixed with methanol and washed with PBS buffer containin! a Q.055~ Tween ~0 and 0.1% bovine serum aibumin. Supernatant or purified antibody samples were diluted in PBS and incubated in the CPA coated microtitre plates (100 ~1 per well) at 37C for 4 h and then for 1 h with ~-5l-labelled rabbit anti-mouse lgG. The wells were washed three times with PBS-Tween buffer between each sta~e and after final washing individual wells were cut and counted in a gamma 25 counter.

Example 9: l~ispecif~lc antibod- reacti~e aoainst CPA and CEA

The hybridoma producing AsB7, a monoclonal antibody reactive a~ainst CEA
30 has been disclosed by Harwood et al (1986) Brit. J. Cancer 54 7~-82~ and a 2 ~

method of generating a hybridoma~ a monoclonal antibody reactive against CPA
is disclosed in Example 8.

The fusion protocol allows any two antibody-producing hybridomas to be fused S and has been diclosed previously (Clark & Waldmann (1987) J. Nall. Cancer Insl. 79, 1393-1401). Briefly, ~ x 106 to 3.5 x 107 cells of one parental hybridoma that have been previously renderedhypoxanthine/aminopterinl-thymidine (HAT) sensitive by selection for a hypoxanthine phosphoribosyltransferase-negative varian~ were fused at I 1:1 or 10:1 ratio.
10 using 1 ml of a ~0% (wt/vol) solution of polvettlvlene glvcol. with the second parental hybridoma cells that had been pretrea~ed witll a letllal dose of 10 mM
iodoacetamide. Excess polyethylene glycol was washed out and the cells were plated at concentrations from 8 x 105 per ml to x 105 per ml into ~4-well plates in bicarbonate-buffered Iscove's modified Dulbecco`s medium tlMDM) 15 supplemented with 5% (vol/vol) fetal calf serum. After 24 hr in culture~
hybrid hybridomas were selected for in medi~ l containin~ HAT.

Example 10: Reduction of residual enzvme acti-itv at non-tumour sitçs -20 It is desirable to inactivate the enzymatic portion of the enzvme-antibody conjugate at non-tumour sites. but not a~ the tumour. One method of achievin~
this effect is to administer to the patient being treated using the compounds ofthe invention antibodies raised against the enzyme portion which have been conjugated with galactose residues.
A monoclonal antibody directed a~ CPA inactivates the enzyme. To prevent the antibody inactivatin~ the enzymes at tumour sites additional ~alaclose residues are conjugated to it so that it can still inactivate enzyme in plasma when it isgiven by intravenous route but the inactivating antibody is rapidly removed 30 from the plasma and ~alac~ose receptors on hepa~ocytes.

`~Wo 93/13806 ~ ~ 2 `~121 ~ pcr~Gsg3/ooo4û

~3 The galactosylated anti-CPA monoclonal antibody is given to eliminate enzymic activity in plasma and then to give an amount of the non-~alactosylated an~i-CPA monoclonal antibody to inactiva~e residual enzvme activity in other non-tissues.
The monoclonal antibody is galactosylated usin~ the following protocol: A
stock solution of the activated derivative was made up as follows:
Cyanomethyl 2,3,4,6-tetra-O-acetyl-l-thio-,B-D-~,alactopyranoside (Si~ma C-4141) [400 mg] in anhydrous methanol (10 ml) ~vas treated with 5.4 mc~ of 10 sodium methoxide in 1 ml of anhydrous methanol at room t~mperature for 4~
hours. The mixture was kept in a ~:~ Inl Quic};fit conical flask fitted with a slightly greased stopper.

A stock solution of monoclonal antibody (1.3 m~/ml) is prepared in 0.25 M
15 sodium borate buffer, pH 8.5. Aliquots of the required amount of activated galactosyl derivative (80~ 40~ 20. 10 ~1) are dispensed into ~ ml ~lass ampoulesand evaporated to a glassy residue in a stream of nitro~en. A solution of ihè
antibody (200 ,ug) is added mixed until the residue is dissolved. Aft~r 2 hours at room temperature. the solution is dialysed a~ainst thr~e chan~es of PBS.
The preparations are scaled up by taking multiples of the volumes mentioned above.

212~121'~
wo 93/13806 pcr/Gs93/ooo4~ ~;

14 ., REFERENCES

I . Bagshawe (1987) Br. J. Cancer 56, 531 -2; (1989) 60~ 275-281.

2. M.M. Bradford (1976) Anal~. Biochem. 72, 248.

3. Corvalan e~ al (1987) Cancer Immunoh Immunother. 24 127-132~ I 33-137 and 1~8-143.

-1. M. Dubois el al (1956) Alln/~t. CIIen1. 28 350.

5. J.G.R.- H~lrrell (CRC Press 1982) "Monoclonal Hybridoma Anti-bodies: Techniques and Applications'.

6. Kuefner e~ al (1989) Biochemistr~ 28~ 2288-2297.

7. O'Sullivan et al (1979) Ana/. Bioc~lem. 100~ 100-108.

8. H. Zola (CRC Press 1988) "1~1onoclonal Antibodies: A manual of techniques-.

9. Meltonetnl(1987)Bioc11em. Pllarm. 36(1), 105-112.

10. Melton et al (1987) Biochem. Pharm. 36(1), 113- 121.

11. Kohler & l~Iilstein (1975) Nature 256~ 495.

Claims (25)

1. A compound comprising a portion capable of at least partly restraining the compound from leaving the vascular compartment of a host when said compound is administered to the vascular compartment and an inactivating portion capable of converting a cytotoxic agent into a less toxic substance.

2. A compound according to Claim 1 wherein the portion capable of restraining the compound from leaving the vascular compartment of a host is a macromolecular portion.

3. A compound according to Claim 2 wherein the macron.olecular portion is a macroglobulin, liposome, dextran or other high molecular weight polymer.

4. A compound according to Claim 1 wherein the portion capable of restraining the compound from leaving the vascular compartment of a host is a red blood corpuscle.

5. A compound according to any one of the preceding claims wherein the inactivating portion comprises an enzymatically active portion.

6. A compound according to Claim 5 wherein the inactivating portion comprises at least the catalytic portion of carboxypeptidase G2 which is capable of degrading the cytotoxic agents methotrexate and aminopterin to less toxic substances.

7. A compound according to Claim 5 wherein the inactivating portion comprises at least the catalytic portion of glutathione-S-transferase PCT/GB?3/00040 which is capable of inactivating cytotoxic alkylating agents to less toxic substances.

8. A compound according to any one of Claims 1 to 4 wherein the inactivating portion comprises an antibody or part thereof which binds to the cytotoxic agent.

9. A compound according to any one of Claims 1 to 4 wherein the inactivating portion comprises a thiol which binds to the cytotoxic agent.

10. A compound according to Claim 9 wherein the thiol is glutathione or an analogue thereof.

11. A method of at least partially destroying a cytotoxic agent in a vascular compartment of a host comprising administering to the host a com-pound according to any one of Claims 1 to 10.

12. A three component kit of parts comprising a first component compris-ing a target cell-specific portion and an enzymatically active portion capable of converting a cytotoxic pro-drug into a cytotoxic drug; a second component that is a cytotoxic pro-drug convertible by said enzymatically active portion to the cytotoxic drug; and a third component comprising a portion capable of at least partly restraining the said third component from leaving the vascular compartment of a host when said third component is administered to the vascular compartment, and an inactivating portion capable of converting the cytotoxic drug into a less toxic substance.

13. A kit of parts according to Claim 12 further comprising a means of removing or inactivating the enzymatically active portion of said first component at non-target cell sites.

14. A kit of parts according to either one of Claims 12 and 13 wherein the target cell-specific portion comprises an antibody or part thereof.

15. A kit of parts according to any one of Claims 12 to 14 wherein the target cell-specific portion recognises and selectively binds to a tumour cell.

16. A kit of parts according to any one of Claims 12 to 15 wherein the enzymatically active portion of the first component comprises at least the catalytic portion of carboxypeptidase A

17. A kit of parts according to Claim 16 wherein the drug is methotrexate or aminopterin or a quinazoline antifolate.

18. A kit of parts according to Claim 17 wherein the inactivating portion of the third component is at least the catalytic portion of carboxypeptidase G2.

19. A kit of parts according to any one of Claims 12 to 15 wherein the drug is an alkylating agent

20. A kit of parts according to Claim 19 wherein the inactivating portion is a thiol.

21. A kit of parts according to Claim 19 wherein the inactivating portion is at least the catalytic portion of glutathione-S-transferase.

22. A kit of parts according to any one of Claims 12 to 18 wherein the restraining portion of the third component is a macroglobulin, liposome, high molecular weight polymer or red blood corpuscle.

23. A kit of parts according to any one of Claims 12 to 22 for use in a method of destroying target cells in a host involving administration to the host of the various components.

24. A method of destroying target cells in a host. the method comprising administering to the host (i) a first component as defined in any one of Claims 12 to 15, (ii) a second component as defined in any one or Claims 12, 13, 17 and 19 and (iii) a third component as defined in any one of Claims 12, 13, 18 and 20 to 22.

25. A method according to Claim 24 wherein each component is administered by an intravenous route.