Bioconjugate

Chemistry MARCH/APRIL, 1990

Volume 1, Number 2 0 Copyright 1990 by the American Chemical Society

REVIEW The Linkage of Cytotoxic Drugs to Monoclonal Antibodies for the Treatment of Cancer Geoffrey A. Pietersz Research Centre for Cancer and Transplantation, Department of Pathology, The University of Melbourne, Parkville, Victoria 3052, Australia. Received August 7, 1989 The search for selective chemotherapeutic agents for the eradication of infectious agents and cancer is of utmost importance. Most chemotherapeutic agents inhibit a critical metabolic pathway which is required for the target organism to survive (1,2). In the case of bacterial infection, chemotherapeutic agents such as cephalosporins and antifolates interfere specifically with bacterial cell wall synthesis or bacterial dihydrofolate reductase (DHFR),l with little or no side effects to the host, while for some retroviruses, drugs acting on reverse transcriptase have been useful. A great challenge has been the search for a selective agent for the treatment of cancer where there are few differences between the biochemical pathways of cancer cells and normal cells ( 3 ) . Indeed, most antineoplastic drugs inhibit metabolic pathways shared by both cancer and normal cell-the major differentiation being in the abnormal growth cycle of the cancer cell. Thus, rapidly proliferating cells in the hair follicles, bone marrow, and gastrointestinal tract are damaged by the antineoplastic agents. Several approaches have been used to produce antineoplastic agents with less side effects, such as design of analogues by chemical modification of

* List of abbreviations: DHFR, dihydrofolate reductase; MoAb(s),monoclonal antibody(ies);MTX, methotrexate; CBL, chlorambucil; N-AcMEL, N-acetylmelphalan; Dm, daunomycin; Ad, adriamycin; CDI, carbodiimide; NHS, N-hydroxysuccinimide; Br-Dm, 14-bromodaunomycin;Br-Ida, 14-bromoidarubicin; CEA, carcinoembryonic antigen; HSA, human serum albumin; DTT, dithiothreitol; BSA, bovine serum albumin; SMPB, N-succinimidyl4-(p-maleimidophenyl)butyrate; SPDP, N-succinimidyl3-(2-pyridylthio)propionate; MBS, m-maleimidobenzoic acid N-hydroxysuccinimide ester; Drug-COOH,drug containing a carboxyl group; MoAb-NH,, amino group of monoclonal antibody; Drug-NH,, drug containing an amino group; Dm-NH,, daunomycin amino group; MEL, melphalan.

the parent drug ( 4 ) ,genetic engineering of antibiotic producing microorganisms (5), and production of prodrugs (6) which are activated in the target tissue. However, of the many hundreds of drugs tested for cancer (thousands a t the National Cancer Institute) few have reached clinical usefulness-mainly because of nonspecific toxicity; i.e. they have a poor therapeutic index (7). Furthermore, many of the cytotoxic drugs currently in use are of little value in the commonest cancers, i.e. lung, colon, breast, and melanoma. There is clearly a major requirement for specific cytotoxic therapy which selectively kills tumor cells but spares normal cells. Can monoclonal antibodies convey such specificity to otherwise broadly cytotoxic drugs, and are such drug-antibody immunoconjugates the cancer treatment of the future? With the discovery of tumor-associated antigens and their detection by monoclonal antibodies (MoAb), another approach for producing selective antineoplastic drugs could be used (8,9). By covalently linking antineoplastic agents to MoAb reactive with tumor-associated antigens, these drugs can be targeted to the tumors (10). This concept was first suggested by Paul Ehrlich in the early 1900s (11). These drug-MoAb conjugates or immunoconjugates bind the tumor cells and are internalized and degraded in the lysosomes to release the drug or drugpeptide fragments which then act on the target-a particular enzyme, DNA, or RNA (12, 13). A number of tumor-associated antigens have now been identified and MoAbs have been produced for these (8,9). Several drugs (IO),toxins (14),radionuclides (15),immunomodulators (16,17),and enzymes (18)have been attached to MoAbs, and their effects in vitro as well as in vivo in mice and humans have been studied. This review will concentrate on the various chemical methods utilized to produce drug-MoAb conjugates. No attempt will be made to describe the biological activity of the various conjugates.

1043-1802/90/2901-0089$02.50/0 0 1990 American Chemical Society

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Table I. Commonly Used Chemical Transformations of Functional Groups of Drugs functional group reagent new functional group refs OH succinic OCOCH,CH,COOH 23, 24 anhydride CONHNH, 25, 26 COOR, hydrazine active ester 27, 28 COOH CDI/NHS CH(OH)CH(OH) periodate aldehyde 29 aldehyde 30 CH(OH)CH(NHJ periodate SMPB maleimide 31 NH, iodoacetic acid COCH,I 31 active ester succinic NHCOCH,CH,COOH 3 2 , 3 3 anhydride COCH, bromine COCH,Br 34,35 EARLY STUDIES ON IMMUNOCONJUGATES

The chemical methods used to couple cytotoxic drugs to MoAbs are similar to those used to couple small organic molecules to proteins such as bovine serum albumin for antibody production for immunoassay (19). In these cases vigorous reaction conditions could be used because no biological activity of the bovine serum albumin or hapten needed to be preserved. For immunoconjugate preparation mild conditions that do not denature the MoAb and interfere with the activity are essential. Some of the early work on immunoconjugate preparation was that of Mathe and colleagues in the mid-l950s, where methotrexate (MTX) was coupled to polyclonal rabbit antibodies raised against mouse leukaemia by diazotization of the MTX pterin amino group (20). Subsequently, in 1974 Davies’ (21) group examined the conjugation of chlorambucil (CBL)to polyclonal antibodies and made the remarkable observation that noncovalently linked CBL was active in vitro and in vivo as a covalently linked drug! This aspect of drug-antibody, interaction should always be borne in mind-i.e. that drugs can associate noncovalently with antibodies and give the appearance of high activity and of many molecules of drug being bound to antibody in vitro. However, in vivo, such linkages are unstable, with the drugs transferring to other proteins and having potential toxic side effects. The main point, however, is that MoAbs carrying noncovalently bound drug may appear to be active in vitro but will not be in vivo, so attention has to be paid to the removal of nonconjugated drug. In these early studies the exact nature of the bonds between drug and antibody was not clear and sufficient quality control on conjugates was not apparent. In more recent studies sophisticated methods have been used to prepare drug-monoclonal antibody conjugates. Coupling agents such as glutaraldelyde and carbodiimides are still being used although with caution due to the difficulty of reproducing these couplings in other laboratories. STRUCTURAL CONSIDERATIONS AND FUNCTIONAL GROUPS OF CYTOTOXIC DRUGS

Most cytotoxic agents are natural products or are produced by chemical synthesis, and from the biological activity of many analogues that have been produced, functional groups important for drug activity can be identified ( 2 2 ) . However, such drugs will not always have appropriate functional groups for coupling to MoAbs, and the groups available may need to be transformed into more suitable functional groups by chemical modification (Table I). For example, hydroxy groups may be easily transformed to carboxyl groups by succinylation or carboxylic ester groups transformed to hydrazides with hydrazine. However, modification of the groups essential for biological activity on the drug before or during

Pietersz

Table 11. Chemical Modification of Functional Groups of MoAb group NH,

ss carbohydrate

reagent SPDP MBS iminothiolane iodoacetic acid active ester DTT periodate

new grow activated disulfide maleimide SH COCH,I

ref

SH CHO

41 42

37 38 39 40

the coupling reaction will yield inactive conjugates, unless the drug is released when the immunoconjugate is degraded by the lysosomal enzymes. Several linkage strategies have been developed so that the drug is released after internalization and they will be discussed later. The release of the free drug may not always be necessary. Immunoconjugates of MTX made by reacting the y-carboxyl active ester with MoAb retain the ability to inhibit DHFR, and it is therefore likely that any MTX-peptide fragments resulting from lysosomal digestion will also inhibit DHFR (12, 36). Furthermore, CBL (28) and N-acetylmelphalan (N-AcMEL) (27)both retain alkylating activity while conjugated to MoAb and may not need release of free drug. Therefore, the absolute necessity for the drug to be released may depend on the mechanism of action of the drug. If steric constraints do not effect the binding of drug to the target, then drug-peptide fragments released by lysosomal degradation will be active. FUNCTIONAL GROUPS ON ANTIBODY MOLECULES

Monoclonal antibodies have a range of reactive groups that can be utilized for coupling to drugs. Some of these are involved in preserving the tertiary structure and binding to antigens. Extensive covalent modification of MoAb by drug may lead to loss of solubility and antibody activity. The extent of possible modification of MoAb by drug will depend on the particular MoAb. Therefore, once a suitable coupling procedure has been designed, it is necessary to measure the antibody activity and protein recovery after modification of the particular MoAb with different molar ratios of drug to MoAb, resulting in a different number of bound-drug residues. Reactive groups may be introduced onto MoAb by (a) use of heterobifunctional cross-linkers, (b) reaction with sodium periodate to create aldehyde groups by carbohydrate oxidation, or (c) reaction with dithiotreitol to reduce inherent disulfides to expose free sulfhydryl groups (Table 11). SELECTED METHODS FOR COUPLING DRUGS T O MOABS

Direct Linkage of Drug to MoAb. Glutaraldehyde. Glutaraldehyde is used to cross-link two amino groups. The exact mechanism of this reaction is complex, but there is evidence to show that an initial Michael addition of one amino group is involved followed by Schiff base formation with the other amino group and aldehyde (Scheme I) (43). The major problem associated with the use of this reagent is polymerization. Several approaches are available to reduce such unwanted side reactions by conducting the reactions in two stagesfirst mix glutaraldehyde with one reactant, remove the excess glutaraldehyde, and then react with the second component. Despite these obvious disadvantages, adriamycin (Ad) (30) and daunomycin (Dm) ( 4 4 ) have been conjugated to MoAbs with the use of glutaraldehyde. The Schiff base formed between the aldehyde and amino group may be stabilized with sodium borohydride or sodium cyanoborohydride.

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Bioconjugate Chem., Vol. 1, No. 2, 1990 91

Scheme I.' Mechanism of Coupling of Drug (Drug-NH,) to Monoclonal Antibody (MoAb-NH,) with Glutaraldehyde

fi

fi

Drug-NHP + H--C-(CH,),

PH3

-C-H

-4

Drug-NH

=CH-(CH,),

-CH=NH--MoAb

MoAb-NHP

pH 7-8

t CH-N-Drug MoAb-NH CHO CH I -CH--(CH,),-CCH=C-(CH,),--CH-SI

I

I CH-C-H

II v

Under acidic conditions, Schiff bases are formed between antibody amino groups and aldehyde groups; at neutral or slightly basic pH a more complex reaction predominates. Scheme 11.' Coupling of Monoclonal Antibody Scheme III.* Coupling of Drugs to MoAbs via a (MoAb-NH,) to Active Ester Derivative of Drug (Drug-COOH)

-orO&ox

Dextran Intermediary

[email protected] &ug.NH.N

CDI 0

n

DrugNH-NHZ

\A

MoAb-NH, ""7-0

-01

I

Drug-C-NH-MoAb

The N-hydroxysuccinimide active ester reacts selectively

with the basic amino groups of MoAb.

Carbodiimide (CDI). This condensing agent reacts with amino groups and carboxyl groups to form an amide bond, and as with glutaraldehyde, polymerization occurs during this reaction (45). Attempts have been made to reduce the polymerization by preforming the activated derivative and then reacting with the MoAb (46). A more attractive method of coupling carboxyl-containing drugs to MoAb is the use of active ester. CDIs have been used for coupling adriamycin (30),daunomycin (30),and methotrexate (36, 47) to MoAbs. Active Ester. Esters formed between N-hydroxysuccinimide (NHS) and carboxylic acids react rapidly with amino groups and very slowly with water or hydroxy groups and has been the method of choice of coupling carboxylic acid containing drugs to MoAbs. Reaction of the active ester with an amino group gives rise to a stable amide linkage (Scheme 11). Methotrexate (36, 48), chlorambucil (28), and N-acetylmelphalan (27) have been coupled by using the active-ester method. Methotrexate and aminopterin can be selectively activated a t the ycarboxyl group with equimolar amounts of NHS and CDI (36, 48, 49). Recent work by Endo et al. suggested the possible reaction of MTX active esters with hydroxy groups available on MoAb (50). The resulting ester linkages can be hydrolyzed with hydroxylamine. Hydroxylamine treatment of these conjugates results in greater selective cytotoxicity between antibody-reactive and nonreactive cells. Compounds with primary or secondary hydroxyl groups can also be coupled to MoAb by the active-ester method, by first reacting with succinic anhydride to form the hemisuccinate, followed by reaction with NHS and CDI. The solubility of the active esters may be increased by using the water-soluble N-hydroxysulfosuccinimideinstead of NHS.

AMWDDOTUN

1. D~IQCOOHsd Carbodiimide or Adive mlw ol drug 2. Perkdale oxidized

MoAborMoAbd

-w

aaent

(Drup),~-(Dextran).z-(MoAb).l

a Polyaldehyde dextran made by periodate oxidation of dextran may be sequentially reacted with drug and MoAb (path B), reacted with a diamine to introduce amino groups and then sequentially reacted with an activated drug and antibody (path C), or reacted with a drug hydrazide derivative (path A). Hydrazide. Hydrazides can be readily prepared from carboxylic acids or esters with hydrazine and the hydrazides thus formed react readily with aldehydes under acidic conditions to form hydrazones (Scheme 111, path A). Immunoglobulins have branched-chain carbohydrate at the hinge region and oxidation of this carbohydrate with periodate results in the production of aldehyde groups which can be reacted with a hydrazide derivative of the drug. Vinblastine (25) and methotrexate (26) have both been coupled to MoAb by using this procedure. The advantage of this method is the production of more immunoreactive conjugates than the active-ester method, due to site specific modification of MoAb. Halocarbonyl. a-Halocarbonyl compounds are reactive with sulfhydryl, amino, and carboxyl groups; for exam-

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ple, 14-Bromodaunomycin (Br-Dm) (32,34)and 14-bromoidarubicin (Br-Ida) (35) have been coupled to a variety of proteins. In the study where Br-Ida was used, the drug was linked via carboxylic groups and amino groups (35). The idarubicir. immunoconjugates had excellent in vivo activity although Br-Dm coupled via sulfhydryl groups resulted in inactive immunoconjugates (32). The N-iodoacetyl derivative of Ad, when coupled to sulfhydryl groups of MoAb, resulted in conjugates with similar activity to the N-iodoacetyl adriamycin derivative in vitro (31). In a recent study by Umemoto et al., a peptide derivative of MTX was coupled to MoAb by reaction of an iodoacetyl derivative (51). Miscellaneous. A number of other coupling procedures either unique to the particular drug or less widely used are available. Trenimon, an aziridinylquinone alkylating agent, was conjugated to a sulfhydryl-containing MoAb with retention of alkylating activity (52). Platinum salts have also been coupled to MoAb by simple mixing, even though the exact mechanism of coupling is not clear (53). The isocyanate derivative of chlorambucil has also been used to couple CBL to an anti-CEA MoAb ( 5 4 ) . Linkage of Drug to MoAb via an Intermediary. To increase the number of drug molecules that can be carried by the MoAb, inert intermediaries have been used. The drug is reacted with the intermediary and the resulting complex is coupled to the MoAb. Several intermediaries have been used, including modified dextrans, polyamino acids, and human serum albumin. Dextran. Dextran is a bacterial polysaccharide made up of a-D-glucopyranosyl units, and these dextrans can be derivatized in a variety of ways to introduce reactive groups and thereby be coupled to antibody (55). Polyaldehyde dextran, made by periodate oxidation, has been used as an intermediary for coupling cytosine arabinoside (29),bleomycin (56), daunomycin (57), adriamycin (58),and chlorin e6 (59) to MoAbs (Scheme 111, path B and C). Amino dextran synthesized from polyaldehyde dextran has been used to couple methotrexate to MoAb (60), resulting in an immunoconjugate with 23 residues of MTX per MoAb (Scheme 111, path C). Polyamino Acids. The property of poly(L-aminoacids) such as poly(L-glutamic acid) and poly(L-aspartic acid) to be digested by enzymes has been utilized by using it as an intermediary for carrying large amounts of drug. These polyamino acids have been used extensively in drug delivery as a means of increasing the therapeutic index of drugs (61). Two approaches have been used to couple drugs to MoAbs via a polyamino acid carrier. Firstly, polyamino acid may be derivatized with a cross-linking agent at the a amino group followed by derivatization with drug and then linking to MoAb (Scheme IV) (62). An alternative procedure is to synthesize the poly(Lamino acid) by polymerization of a suitably functionalized monomer. The latter method was used by Kat0 et al. to introduce a thiol group onto polyglutamic acid for coupling to MoAb in the final step (63). Human Serum Albumin. Human serum albumin (HSA) has also been used as a intermediary carrier for coupling methotrexate (64, 65) and mitomycin C (66) to monoclonal antibodies. In the approach by Garnett et al. (631,MTX was coupled to HSA with CDI and then reduced with DTT to expose the free thiol group on HSA and reacted with iodoacetylated MoAb. With this method, 38 residues of MTX were coupled to MoAb, and the resulting immunoconjugate was more active than free MTX. A serious problem in with the use of intermediaries is

Pietersz

Scheme IV." Linkage of Daunomycin to MoAb via a Poly(L-glutamic acid) Intermediary COOH

COOH

I

I

Poly(L-glutamicacid) is activated at the amino terminal with

SPDP, and the drug containing an amino group is coupled to poly(L-glutamic acid) with CDI. The reduced polymer is then reacted with an antibody substituted with a maleimide group. Scheme V.a Linkage of Drug (Drug-NH,) to Monoclonal Antibody (MoAb-NH,) via a cis-Aconityl Linkage + 0

Drug-NH,

H

m

~

o

a

~

~

~

0

a The basic amino group of the drug reacts with cis-aconitic anhydride to give a dicarboxylic acid, which can be selectively coupled by the carboxyl group to MoAb. Under acidic conditions the drug is liberated from the MoAb. the possibility of obtaining conjugates with more than one carrier molecule per antibody molecule, which results in decreased yields. Lysosome-Sensitive Linkers. The passage of antigen antibody complexes to the lysosome by endocytosis has led to the design of several linkages that are cleaved by either lysosomal enzymes or lower pH (4.5-5) to release free drug. Early work by Trouet et al. (67) demonstrated that the peptides LeuAlaLeu and AlaLeuAlaLeu when used as linkers between drug and BSA released the free drug when treated with lysosomal hydrolases. Several other linkers have been synthesized that have similar properties-e.g. GlyPheLeuGly (51). MTX has been coupled to MoAb with this linker. The use of cis-aconitic anhydride to link drugs to proteins was demonstrated by Shen et al. (68), where the cis-aconityl derivative of daunomycin was linked to poly(L-lysine). Exposure of these conjugates to pH 4.5-5 results in the release of drug (Scheme V). Immunoconjugates of daunomycin (69) and adriamycin (70) made by using this procedure have shown antitumor effects in vitro and in vivo. Several other linkers have been synthesized recently that may be utilized for coupling drugs to MoAbs (71). Prodrug Approach. Immunoconjugates, once admin-

o

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Biocon/ugafeChem., Vol. 1, No. 2, 1990 93

Review

istered to animals, will circulate and eventually be removed from the circulation, presumably by the reticuloendothelial system. If the drug is not detoxified a t this stage, drug toxicity will be seen. MTX immunoconjugates are more toxic than free MTX in animals, especially if given daily ( 2 6 ) , presumably due to rapid clearance of MTX by the kidney and accumulation of immunoconjugate in the reticuloendothelial system. Alternatively, the drug may dissociate from the MoAb and harm nontumor tissue. With these in mind, the use of nontoxic prodrugs for immunoconjugation is an attractive concept. These prodrugs may be drugs with charged groups to prevent free diffusion across the cell membrane, defective by modification of groups involved in active transport, or requiring lysosomal cleavage for activation. This is an area that has not been explored even though cis-acotinyl derivatives and peptide derivatives of drugs may be considered as prodrugs. Melphalan was converted to Nacetylmelphalan by acetylation of the amino group, and this modification resulted in a 100-fold decrease in cytotoxicity due to defective transport (27). Conjugation of N-AcMEL to MoAb resulted in a conjugate 3-fold less toxic than melphalan. N -AcMEL conjugates were not toxic to mice at a dose of 16 mg/kg. However, NAcMEL and MEL had an LD, of 115and 6 mg/kg, respectively. Another method of utilizing prodrugs for targeting is their use in conjunction with enzyme-MoAb conjugates. Enzymes such as carboxypeptidase (72)or alkaline phosphatase (73) can be coupled to monoclonal antibodies and targeted to tumors and nontoxic prodrugs may be administered (64,65). The prodrugs are transformed to the drug at the vicinity of the tumor and diffuse into the tumor. For enzyme targeting of this type, MoAbs to noninternalizing antigens are necessary. Careful consideration has to be given in choosing enzymes such that prodrugs are not activated in normal tissue by endogenous enzyme; therefore alkaline phosphatase is not an ideal choice. Another approach worth considering is the linking of photoactivatable moieties such as chlorin e6 (59)and hematoporphyrin (74)to MoAbs. These agents are inactive until activated (cf. prodrugs) by light of a particular wavelength to emit singlet oxygen. Since singlet oxygen is a diffusable product, the conjugates do not need to be internalized and are capable of killing bystander cells lacking tumor antigen. Moreover, the light activation can also be directed to the tumor and thereby activate the photodrug locally, allowing MoAbs with slight cross-reactivity with healthy tissues to be used. In Vitro Testing. Coupling of drug to MoAbs may lead to partial or complete loss of antibody activity and/ or drug activity. Therefore both activities of the immunoconjugate should be carefully monitored at each step. Several approaches can be used to test for antibody activity and a review of this aspect is found in Pietersz et al. (10). The drug activity may be tested by using a cytotoxicity assay measuring the inhibition of DNA, RNA, or protein synthesis (10). The colorimetric tetrazolium assay (MTT) which utilized a yellow, water-soluble dye which is transformed to a blue formazon by live cells can also be used. For certain drugs such as alkylating agents and dihydrofolate reductase inhibitors, the functional integrity of the bound drug can be determined by measuring the drugs ability to alkylate 4-(4-nitrobenzyl)pyridine (28) or inhibit DHFR, respectively (36). The selectivity of immunoconjugates may be tested by comparing the inhibition of growth of antibody-reactive and nonreactive cell lines. However, lack of selectivity does not necessarily imply unsuitable conjugates (35). This point could be illustrated with reference to idarubicin-antibody con-

jugates, where specific conjugates are 5-10 times more active on the target cell line than the nonspecific conjugates. However, in vivo nonspecific conjugates do not show any antitumor effects, and no toxic effects due to the conjugates are apparent. The exact mechanism by which nonspecific conjugates demonstrate cytotoxicity in vitro is not known. These problems emphasize the need for more appropriate cytotoxicity and specificity assays that mimic the in vivo behavior of immunoconjugates. Suitability of immunoconjugates as specific antitumor agents will only be apparent from efficacy in preclinical studies using animal models (10). Despite certain drawbacks such as the inability to predict effects due to crossreactivity of MoAb with normal human tissues, animal models can be extremely useful for the characterization of immunoconjugates. Ideally, preclinical testing should include tests for stability in vitro and in vivo, biodistribution studies using radiolabeled drug, and doseresponses studies to compare antitumor efficacy and toxicity. CONCLUSION

This review has concentrated primarily on the chemical methods and strategies utilized for immunoconjugation of drugs. The techniques for coupling cytotoxic drugs to MoAbs have become increasingly more sophisticated, with the design of novel linkers and protecting groups which confer unique properties, such as stability in serum, with sensitivity to lysosomal or hydrolytic enzymes. The majority of drugs used in immunoconjugates have been limited to those commonly used in the clinic for the treatment of cancer and are not necessarily the most optimal for conjugation to antibodies. The conjugation of very cytotoxic, lower molecular weight compounds previously discarded due to their toxicity should now be investigated for immunoconjugation, as binding to antibody increases specificity and reduces the toxicity of such drugs. Such potent immunoconjugates may result in better antitumor efficacy by counteracting the low uptake of drug to the tumor. The access of immunoconjugates to the tumor is still a major problem and is a serious limitation for the treatment of larger tumors, but the production of small, single chain antibody molecules by genetic engineering techniques may overcome this. Because of the difficulty in delivery of large amounts of antibody to large tumours, the final place of immunoconjugate in the treatment of cancer may be in conjunction with other modes of therapy for treating minimum, residual disease in the adjuvant setting. However, this remains to be proven. LITERATURE CITED (1) (1975) Antibiotics (J. W. Corcoran and F. E. Hahn, Eds.) Vol. 111, Springer-Verlag; Berlin. (2) (1981) Molecular Actions and Targets for Cancer Chemotherapeutic Agents (A. C. Sartorelli, J. S. Lazo, and J. R. Bertino, Eds.) Academic Press, New York. (3) Weber, G. (1983) Biochemical strategy of cancer cells and the design of chemotherapy: G. H. A. Clowes Memorial Lecture. Cancer Res. 43, 3466-3492. (4) (1980) Anticancer Agents Based on Natural Product Models (J.M. Cassady and J. D. Douros, Eds.) Vol. 16, Medicinal Chemistry, Academic Press, New York. (5) Hutchinson, C. R., Borell, C. W., Olten, S. L., StutzmanEngwall, K. J., and Wang, Y. (1989) Drug discovery and development through the genetic engineering of antibioticproducing microorganisms. J. Med. Chem. 32,929-937. (6) Carl, P. L., Chakravarty, P. K., Katzenellenbogen, J. A., and Weber, M. J. (1980) Protease-activated "prodrugs" for cancer chemotherapy. Proc. Natl. Acad. Sei. U.S.A. 77,22242228.

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