Monoclonal antibody–drug conjugates

Review

10.1517/13543776.15.9.1087 © 2005 Ashley Publications Ltd ISSN 1354-3776 1087

Ashley Publicationswww.ashley-pub.com

Monthly Focus: Biologicals, Immunologicals & Drug Delivery

Monoclonal antibody–drug conjugatesPhilip R HamannChemical and Screening Sciences, Wyeth Research, 401 N. Middletown Rd., Pearl River, NY 10965, USA

This review covers cytotoxic antibody–drug conjugates for use in oncology.The focus is on drug conjugates of current interest, such as those of the tax-anes, maytansines, CC-1065 and the duocarmycins, the calicheamicins andother enediynes, and the auristatins. A few classes of drug conjugates fromearlier work are also mentioned, such as those of the antifolates, vincaalkaloids, and the anthracyclines. Also covered are some more recent linkersystems that are useful for making antibody–drug conjugates. This reviewdoes not cover conjugates of plant toxins, other bioactive proteins,enzymes (i.e., antibody-directed enzyme prodrug therapy [ADEPT]), radio-isotopes (photodynamic therapy), or conjugates made with secondarycarriers for the cytotoxic agent, such as liposomes or polymers.

Keywords: antibody, anthracycline, antifolate, auristatin, calicheamicin, CC-1065, chemoimmunoconjugate, conjugate, duocarmycin, maytansine, oncology, taxane, trichothecene, vinca alkaloid

Expert Opin. Ther. Patents (2005) 15(9):1087-1103

1. Introduction

Since the availability of monoclonal antibodies, there have been manyapproaches tried with these proteins in the hope of achieving Paul Ehrlich’sdream of a magic bullet [1-4]. Antibodies alone have shown some success inextending the lives of cancer patients but leave room for improvement. Many dif-ferent agents have been conjugated to try to improve on their activity, includingtraditional anticancer agents, cytotoxic natural products, phytotoxins, radio-isotopes, bioactive proteins, enzymes that activate prodrugs of cytotoxic agents,photosensitisers etc. So far, the FDA and/or other regulatory agencies haveapproved several naked antibodies, two radioimmunoconjugates, and one chem-oimmunoconjugate (Table 1). There are many more in various stages of clinicaltrials, and some of these will be mentioned.

The drugs that have been conjugated to antibodies include traditional chemo-therapy agents, such as doxorubicin and vinblastine, as well as more potentlycytotoxic derivatives of natural products, such as calicheamicin, maytansine, andduocarmycin. It is well accepted that these agents must be internalised into thetarget cells and then released from the antibody in order to exert their cytotoxiceffects. As a result of the requirement for drug release, the method of linking thedrug to the antibody has varied widely, and the sophistication of the linkers hasgenerally increased with time. The major mechanisms used to allow for cleavageof the drug from the antibody include hydrolysis in the acidic pH of the lyso-somes (hydrazones, acetals, and cis-aconitate-like amides), peptide cleavage bylysosomal enzymes (the cathepsins and other intracellular enzymes), and thereduction of disulfides. Linker systems will be discussed mostly as they apply tospecific drug conjugates, although some key methodologies are covered in theirown section as well.

1. Introduction

2. Antifolates

3. Vinca alkaloids

4. Anthracyclines

5. Taxanes

6. Maytansines

7. Cyclopropaindoles

8. Enediynes

9. Auristatin/dolastatin

10. Miscellaneous

11. Notable linker technology

12. Expert opinion

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Monoclonal antibody–drug conjugates

1088 Expert Opin. Ther. Patents (2005) 15(9)

Table 1. Approved antibody-based treatments in oncology.

Generic name Trade name Indication Approach US approval

Bevacizumab Avastin Colon Antibody 2004

Tositumomab Bexxar NHL RIT 2003

Alemtuzumab Campath CLL Antibody 2001

Cetuximab Erbitux Colon Antibody 2004

Trastuzumab Herceptin Breast Antibody 1998

Gemtuzumab ozogamicin Mylotarg AML CIT 2000

Edrecolomab Panorex Colon Antibody (1994)*

Rituximab Rituxan NHL Antibody 1997

Ibritumomab tiuxetan Zevalin NHL RIT 2002

*Approved in Germany only AML: Acute myeloid leukaemia; CIT: Chemoimmunotherapy; CLL: Chronic lymphocytic leukaemia; NHL: Non-Hodgkin’s lymphoma; RIT: Radioimmunotherapy.

2. Antifolates

Methotrexate (cf. 1a) is a classic antifolate and has commonlybeen conjugated to antibodies. It is an inhibitor of dihydro-folate reductase and hence interferes with the 1-carbon trans-fer reactions of folic acid that are key to many biologicalpathways, most importantly for cellular reproduction throughthe synthesis of nucleotides.

There appears to be little interest in antibody conjugates ofmethotrexate or its slightly more potent desmethyl analogueaminopterin. Early conjugation work by Cytogen Corp. [101]

discussed the γ-hydrazide derivative (1a), among others,which can be used to make conjugates with their antibodyoxidation technology [102]. Lilly worked on related conjugatesof these hydrazides at about the same time [103]. Noteworthyin this work is the use of bifunctional linkers (1b) that forman amide with the antibody and also a hydrolytically-labilehydrazone with the cytotoxic agent. The IC50 value in tissueculture was roughly 20 ng/ml of antifolate, and activity wasseen in a xenograft experiment at 2 mg/kg of antifolate.[Units in the area of antibody conjugates are problematicbecause of the various ways they are given. For in vitro experi-ments they can be given on a weight/ml or a molar basis. Forxenograft experiments they can be given as total dose, as perkg dose, or as per m2 dose. Most problematically, they canrefer to either the cytotoxic agent (with or without the linkerportion), the antibody, or the entire conjugate for either invitro or in vivo experiments. Unfortunately, the authors arenot always clear on the latter issue as well as the loading of thespecific batch of conjugate tested. Although this makes com-parisons difficult, this only adds to the ultimate difficulty ofhow to compare two conjugates that are made with differentcytotoxic agents, different linkers, and different antibodiesand that are tested by different methods on different cell lines.For these reasons the units used in this article are those used inthe references. It is felt that the potency relative to both anappropriate unconjugated drug control and, especially, a con-trol conjugate, are the best measures of success.] However, the

data on these conjugates, even though impressive at the time,are insufficient for complete evaluation. [Most antibody–drugconjugates are cytotoxic in vitro at some level and give variousdegrees of tumour growth inhibition in xenograft studies.However, in the author’s opinion, such data is best interpretedin comparison to a matched control conjugate with a non-targeting antibody.]

3. Vinca alkaloids

Vincristine and vinblastine are classic antitubulin agents.They bind to the growing end of microtubules, inhibitingtheir production. This results in a block in cell division and,ultimately, apoptosis. Desacetylvinblastine 2a was a commonvinca alkaloid to be derivatised for conjugation.

This class of conjugates also appears to be of little currentinterest. Lilly was active in the area of antibody conjugates ofthe vinca alkaloids. They discussed C-4 esters of aliphaticdiacids with the antibody attached to the second acid via anamide bond [104]. Similarly, Trouet [105] reported using C-4esters as the site of attachment to antibodies, using amino-diesters such as aspartate. In a different approach, Lilly hasdiscussed conjugates made with a hydrazide instead of an esterat C-3 [106]. That hydrazide was then attached to the lysineson the antibody via a bifunctional linker (e.g., 1b), such asthat used with methotrexate hydrazide. Such a conjugatemade with 4-acetylphenoxyacetic acid had an IC50 value ofbetween 50 and 100 ng/ml of vinca derivative, comparable tothe control drug tested, and conjugates with other bifunc-tional linkers were active in xenograft experiments. Thisincluded complete inhibition of tumour growth with freshlyimplanted tumour cells, but no data on a control conjugatefor either in vitro or in vivo experiments was given.

An interesting type of linker for antibody-drug conjugatesusing the same vinca hydrazide from Lilly’s earlier work wasalso reported [107]. Drug release in this case results from theaddition of a nucleophile to the methylene terminus of anα-methylene β-keto amide (2b, referred to as the BAMME

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Expert Opin. Ther. Patents (2005) 15(9) 1089

linker) followed by elimination of the vinca hydrazide(shown) or other amine or alcohol.

The Lilly desacetylvinblastine hydrazide conjugates havebeen profiled in the scientific literature. The conjugates com-pared were a carbohydrate-based conjugate made by themethod of Cytogen, a conjugate with the BAMME linker 2b,and a conjugate with linker 2c (the BAP linker). In general theconjugates were 3- to 25-fold less potent than the freehydrazide, with the lower potency correlating with higherlinker stability [5]. However, all the conjugates were active inxenograft experiments with survival as the end point. Someimprovement in survival was seen over that of a control conju-gate when the BAP linker (2c) was used. This construct wasthe most efficacious in a head-to-head xenograft comparison,perhaps because it shows the best compromise between stabil-ity in circulation and efficient release of the vinca hydrazide inthe lysosomes.

4. Anthracyclines

Anthracylines have been a common class of drugs for chemo-immunoconjugates. Of these, doxorubicin (3a) has often beenthe choice for conjugation to antibodies, perhaps because ofits profile of activity versus solid tumours. The anthracyclinesintercalate into DNA and interfere with its function. Thisleads to eventual cleavage of the DNA by topoisomerase II. Asecondary mechanism of action is the generation of DNA-damaging free radicals by electron transfer reactions of theanthraquinone.

In work from the early 1980s, Cytogen disclosed deriva-tives with nucleophiles attached to the C-3′ nitrogen that can

be used to make conjugates with their carbohydrate-oxidationtechnique [108]. Many of these derivatives have hydrazides, butother related nucleophiles are discussed, such as hydrazines,O-linked hydroxy amines, and semicarbazides. Also disclosedare some conjugates at C-13 (hydrazones) and C-14(thioethers and tertiary amines) that can also be attached tooxidised antibodies through nucleophiles such as hydrazides.Although the conjugates showed more activity in xenograftexperiments with established tumours than controlconjugates, the effects were modest.

Hoechst disclosed conjugates of cytorhodin S, an anthra-cycline with a more complex carbohydrate at C-9 and anadditional aminosugar at C-12 [109]. Attachment to the anti-body is made through modification of the extra sugar residuesat C-9 to introduce either an activated carboxylic acid or amaleimide. The selectivity of the cytotoxicity of the conjugatewas shown by blocking its activity with excess antibody andby comparison to a control conjugate, but the conjugate was5-fold or more less potent than the anthracycline itself. Pub-lished data indicated that these conjugates showed modestactivity in xenograft experiments, possibly due to the lack of arelease mechanism [6].

Hoechst has also published on conjugates of anthracyclinesattached via an ester bond at C-4′ and maleimides attached toantibodies containing thiols [110]. Although these conjugatesshowed good potency in vitro, they may not be stable enoughin circulation to have good activity in vivo [7].

Bristol-Myers Squibb (BMS) has played a considerablerole in anthracycline conjugates, largely because of theiranti-LewisY antibody–doxorubicin conjugate (BR96-DOX),which has been actively pursued in clinical trials [8,9]. [This

N

N N

NN

NH

NH2

NH2

O

CONHNH2

OHO

1a Methotrexate hydrazide

2a Desacetyl vinblastine

2b Lilly BAMME linker 2c Lilly BAP linker

NH

O

OH

OO

1b Lilly linker

NH

O

OH

ONH

O

NH

N

N

N

OH

OH

OH

O

H

O

O

O

O

R NH

Antibody

O O O

NH

NH

O

(C-3)-Desacetyl vinblastine

3 4

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intellectual property is now owned by Seattle Genetics [201].]Conjugates made via C-13 keto hydrazones are disclosed inwhich the linker also contains a thioether or disulfide bond[111,112]. BR96-DOX is made with γ-maleimidocaproylhy-drazide as the linker, and with the antibody partially reducedto generate thiols for conjugation. It showed significantactivity in xenograft experiments at 5 – 20 mg/kg of doxoru-bicin and was 8- to 25-fold selectively cytotoxic in vitro withan IC50 of 400 nM of doxorubicin, but it was not morepotent than doxorubicin itself. Insufficient activity was seenas a single agent [10,11]. Although data indicated superioractivity in combination [1,12], it has been dropped from clin-ical development [203]. BMS also disclosed similar conjugatesmade with semicarbazides instead of hydrazides [113].

A more potent class of anthracyclines, making them ofinterest for conjugates, are the cyano morpholino and relatedderivatives that have modified amines. Pharmacia & Upjohnhave reported on such derivatives (3b), where a pyran ringattached to the C-14 hydroxyl is the site of hydrolytic drugrelease [114]. Over a broad range of antibodies and derivatives,the conjugates were 2- to 10-fold more cytotoxic than controlconjugates and some activity was seen in vivo. Immunogenhas also reported on morpholino and piperizino anthra-cyclines that have linkers attached to the morpholine or piper-azine [115]. Finally, Immunomedics has a patent applicationdisclosing anthracycline conjugates made with the SMCCbifunctional linker (a hydrazide-maleimide) [116], and Bruns-wick has disclosed morpholino anthracycline conjugates made

with sulfonylhydrazide containing linkers such as 4-hydrazino-sulfonylbenzoic acid [117].

Earlier publications on these potent anthracyclines showedsome promise [13], and there appears to be continued interestin these conjugates [14]. Hydrazone conjugates made with theC-13 keto group of morpholinodoxorubicin and γ-maleimi-docaproylhydrazide had IC50 values of 30 – 200 nM ofanthracycline with selectivities of 10- to 330-fold.

5. Taxanes

The taxanes, such as paclitaxel (4a), are unique microtubuleagents in that they bind to intact tubules and stabilise them.The resultant microtubules are non-functional, which resultsin cellular arrest. Immunogen has disclosed conjugates ofpaclitaxel and its derivatives [118,119], specifically conjugateslinked at the C-10 alcohol via ether, ester, or carbamate bondsto the taxane and attached to the antibody through mono-sulfides (with the maleimide on the antibody) or disulfides.The C-3′ carbon has an alkyl or aryl group attached instead ofthe phenyl and the C-3′ substituent is restricted to benzamideor t-butyl carbamate (similar to taxotere). More potent deriva-tives with either one or two electron withdrawing groups onthe C-2 benzoyl group were also discussed.

Conjugates of the taxanes with the linker attached at C-7and with the C-10 alcohol derivatised as a carbamate are alsodisclosed by Immunogen [120]. Also discussed are conjugateslinked at the C-2′ alcohol [121]. In specific examples, the

4a Paclitaxel

3a Doxorubicin

3b Pharmacia and Upjohn conjugate

OOMorpholino daunorubicin (C14)NH-Antibody

O

4b Immunogen derivative

O OH

O

OAcO

O

O

O

O

OH

NHO

OH

O

O

O

OSS

O

O

O

OH

OH

OH

O

O

OHNH2

OH

O

3'4'

1314

12

9

O OH

O

OAcO

O

AcO

O

O

OH

NHO

OH

710

2'3'

2

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C-3′carbon has an alkyl or aryl group attached, the C-3′ sub-stituent is either benzamide or t-butyl carbamate, and the C-2benzoyl group has one electron withdrawing group. Immuno-gen has also disclosed conjugates of taxanes with thiol linkersattached at a variety of sites on the molecule [122-125].

Some of the derivatives used to make such conjugates arecompared in two publication [15,16]. Their analogues focuson increasing the potency of their derivatives, and all ofthem have a C-3′ butenyl group and a C-3′ t-butyl car-bamate. It appears that taxanes with linkers attached to theC-10 hydroxyl group retain potency better, although thedata are not completely consistent. The most potent func-tionalised derivative (4b) also has two methoxy groups atpositions 2 and 5 of the C-2 benzoyl group. It has an IC50

value against two cell lines in tissue culture of 0.03 nM,which decreases only slightly to 0.045 – 0.08 nM with a3-methyldithiopropanoyl linker attached [16]. Paclitaxel/docetaxel had IC50 values of ∼ 2 nM.

Anti-EGFR conjugates have been studied for a similarderivative, but missing the two methoxy groups. In vitro, anIC50 of 1.5 nM of taxane derivative was seen versus A431 cells[15]. Although this is ∼ a 10-fold loss of potency versus the cor-responding unconjugated derivative with the linker attached,the cytotoxicity was shown to be antigen dependent by com-parison to a control conjugate ( > 300-fold selectivity) and byblocking with unconjugated antibody. The results of the onexenograft experiment presented showed that prolonged inhi-bition of the growth of 100 mm3 A431 tumours can beobtained with doses of 10 mg/kg of conjugate given daily for5 days. Necropsy at day 75 showed no evidence of viabletumour cells. Although these in vivo results are promising, acontrol conjugate was not reported. Using their more potentderivatives will, hopefully, reduce the IC50 values and thexenograft doses by 10-fold.

One other disclosure on taxanes from Immunogendescribed conjugates made with polyethylene glycol (PEG)-containing thiol-acid linkers attached via an ester to the C-7or C-10 alcohols [126]. The potency of the PEG-containingderivatives was comparable to the corresponding derivativeswithout the PEG group in 5/6 cell lines, with an IC50 value in3 non-multi drug resistant (MDR) lines of ∼ 1 nM of taxanederivative and in three MDR-positive lines being about 20 nM[17]. This average 20-fold resistance was an improvement overthat of paclitaxel, which averaged 300-fold.

Conjugates of paclitaxel derivatised with a diacid that isattached to diamine and conjugated to antibodies with glutar-aldehyde are also discussed [127]. Although stability data werenot presented, these conjugate may not have optimumstability in circulation.

6. Maytansines

Maytansine (5) is an ansa-macrolide that inhibits tubulinpolymerisation similarly to the vinca alkaloids. However, dueto its potency and resultant high toxicity, it is not used

clinically. Conjugates of the maytansines have been pursuedby Immunogen [18,19]. One disclosure discussed conjugates ofthe maytansinoids conjugated via disulfide linkers betweenpositions C-3, C-14, C-15, or C-20 of the maytansine to anti-bodies, and the detailed examples are made with maytansineitself and linkers attached to C-3 by replacing the acetyl of theacetamide with 3-mercaptopropanoyl [128]. This derivative,referred to as DM1, has been commonly conjugated to anti-bodies via a disulfide with 4-mercaptopentanoic acid [129].

Conjugates of DM1 that are in clinical trials or appear tobe of clinical interest include huC242-DM1 (cantuzumabmertansine, anti-CanAg) [20,21,22], BIWI1 (bivatuzumab mer-tansine, anti-CD44v6) [23], huN901-DM1 (BB-10901, anti-CD56) [24], MLN2704 (anti-PSMA) [25], and trastuzumabDM1 (anti-HER2) [130]. The data in these publications indi-cate that control conjugates do not in general show significantactivity in xenograft experiments, whereas the targeted conju-gates show pronounced effects. The most advanced of theseconjugates is cantuzumab mertansine. In preclinical evaluationversus colon cancer, this conjugate gave an IC50 of 0.03 nMwith ∼ 3 logs of selectivity versus a control cell line [20].In vivo, the conjugate cured Colo 205 xenografts and gavegood responses in both LoVo and HT-29 xenografts at0.3 mg/kg of maytansine given five times. In phase oneclinical trials there were some minor responses [21].

A recent publication from Immunogen discloses conjugatesmade from similar maytansine thiols, which differ in that theyhave one or two flanking substituents that would stabilise thedisulfide in a conjugate [131]. Examples are DM3 and DM4,which have either one or two flanking methyl groups, respec-tively. Although these thiols can be used to make conjugateswith hindered disulfides, they also report conjugates witheither maleimide or iodoacetamide-modified antibodies. BothDM3 and DM4 appear to give superior conjugates, withDM4 being somewhat more efficacious [26].

The IC50 value of one DM4 conjugate in cell culture was0.01 – 0.5 nM, reflecting the inherent sensitivity of the celllines to the maytansines. When conjugated to the anti-lymphoma (CD19) antibody huB4, it gave a 6 – 7 week delayin the growth of Ramos tumours at a dose of 10 ug/kg andapparent tumour ablation at both 200 and 400 µg/kg with noapparent toxicity as assessed by body weight measurements.However, insufficient data were presented to completely assessselectivity either in vitro or in vivo.

A recent publication disclosed maytansine conjugateswith linkers at C-9. This publication will be discussed inSection 11 [132].

7. Cyclopropaindoles

CC-1065 and the duocarmycins are the best know mem-bers of these highly elaborated di- and tri-indoles. Theybind to the minor grove of DNA and alkylate N3 of ade-nine. Several analogues of CC-1065 were examined in clin-ical trials by Upjohn, most notably adozelesin (6a), but were

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found to be too toxic. This class of cytotoxic agents has twoforms (cf. 6a and 6b) that readily interconvert depending onconditions. The two forms of a particular derivative havehad identical biological properties whenever they have beencompared.

Upjohn has disclosed derivatives that can be used to makeconjugates [133]. They report compounds containing themethylindole of adozelesin, but with the methyl group beingreplaced by alkyl, phenyl, or hydrogen. They disclose severalprodrugs at the phenol of the open form, including esters, car-bonates, carbamates, their thio-analogues, sulphonates, andmonosaccharides, including some containing solubilisingPEG, TRIS, and sulfate groups. They also disclose a variety ofDNA-binding units (ring systems B and C) and a wide varietyof possible linkers, including thiols, activated carboxylic acids,peptides, carbonyls, and hydrazides and related nucleophiles.More detailed examples include thiols and hydrazidesattached to the C ring system as linkers, as well as hydrazidesand acids attached to the prodrug of the phenol. Indoles andbenzofurans are included as DNA-binding units.

Immunogen was also working on these types of conjugatesduring the same timeframe as Upjohn. Immunogen disclosedconjugates made with the CBI (cyclopropabenzindole) alkyla-tion unit that has a benzo ring (6c) instead of the methyl-pyrrolo ring in CC-1065 and that was first made by Boger atScripps [27]. Their earliest disclosure is of derivatives attachedto antibodies via a linker containing a disulfide, and attachedto the C ring system of the DNA-binding portion [134]. The Band C units can be indole or benzofuran. Conjugates likewise

made with the CBI alkylation unit but with a wide variety ofDNA-binding units, such as single ring system B units andunits that contain an additional dihydropyrrolo ring that ispresent in CC-1065, are separately discussed [135]. Thedetailed examples in these two disclosures have disulfide link-ers attached to C-6 of the terminal ring system of the DNA-binding portion, either B or C, and have the methylpyrroloring of adozelesin.

Immunogen has published data on conjugates in the CBIseries [28]. Their DC1 derivative (6c) consists of the CBIDNA alkylation unit and two indoles in the DNA-bindingportion, the second of which is substituted by a 3-mercapto-propionamide at the 6-position. The IC50 values versus tar-get cells were ∼ 0.07 ng/ml of DC1 with selectivity of 2 – 3logs, as shown with either control cell lines or by blockingwith unconjugated antibody. Activity in a challenging dis-seminated lymphoma model was seen at doses as low as0.4 mg/kg of drug with a 2.7-fold increase in the duration ofsurvival or a 50% increase over that of a control conjugate.It is likely that these results may be improved by using morestable disulfides [26].

ImmunoGen has continued this work by trying to over-come the inherent insolubility of this class of drugs, such as bymaking phosphate prodrugs of the phenol [136]. This sameprodrug approach has also been used to try to overcome possi-ble stability issues associated with the DNA-alkylation unititself [137]. Prodrugs that have solublising units, such as piper-azine-containing carbamates, and linkers containing PEGunits are additionally reported [138].

6a Adozelesin (open form)

6b Du-86 (closed form) 6c Immunogen derivative

5 Maytansine

NH

O

N

Cl

OH

O

O

OO

O

O

O

N

O

3

15 14

20

9NH

OH

N

NHO

NH

OO

Cl

A

B

C

OH

N

NHO

NH

NHO

ClNH

SSR

O

A

B

C

NH

N

O

NHO

O

O

OOO

A

B

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Published data on such derivatives appeared in a poster pres-entation [29]. Immunogen has successfully added PEG units tothe linker arm to increase solubility, although a loss in activitywas seen with more than 4 ethylene glycol units. They showedthe synthesis of solublising carbamate prodrugs of the phenol,but presented no biological data. Lastly, they mentioned thephosphate prodrug of the phenol. It greatly increased watersolubility and was activated by various phosphatases. Conju-gates made with the phosphate prodrug had potency compara-ble to that seen previously without the phosphate.

Panorama Research has discussed antibody conjugates ofCBI derivatives with a single DNA-binding unit [139]. Otherconjugate work has been published by the Kyowa HakkoKogyo Co. [30]. They took Du-86 (6b) and extended the mid-dle of the three methoxy groups to an aminoethoxy group. Tothis they attached a valine, then alanine, and then a 600 DaPEG diacid. This was then conjugated to the antisilyl Lewisa

antibody KM231. Unfortunately, the conjugate had an IC50

value versus target cells of only 1 µM of Du-86 derivative.Medarex has three patent applications that disclose duocar-

mycin conjugates [140-142]. [Coulter Pharmaceuticals was soldto Medarex by Corixa in 2002 [202].] They all deal with conju-gates in which the DNA-alkylation unit has a pyrrolo ringwith alkyl or H in the 2-position of that ring and H, alkyl,ester, or amide in the 3-position. Detailed examples are withH and methyl at the 2-position and methyl ester at the3-position. The DNA-binding portions include one or twounits that are indole, benzofuran, and benzothiophene. Theydiscuss conjugates with disulfide, hydrazone, and peptidelinkers as the source of drug release from the antibody, withthe linkers being attached either to the DNA-binding portionor through a prodrug of the DNA-alkylation portion.

One of these patent applications discloses free phenols andtheir prodrugs such as carbamate, carbonate, phosphate, andester [140]. Prodrugs are discussed, including a peptide linkerattached to an antibody, and peptide linkers are also shownattached to the DNA-binding portion, each with examples ofPEG spacers. Another patent application discusses carbamateprodrugs with hydrazone linkers attached to the DNA-bind-ing portion [141]. It also discusses conjugates linked through apeptide attached to the phenol as a prodrug. The last patentalso discloses carbamate prodrugs, but with a disulfide groupin the linker instead of a peptide. It also discloses peptidelinkers attached to the DNA-binding portion [142]. Thesepatent applications contain a range of detailed examples. Interms of the linkers attached through prodrugs, there areexamples of peptides, hydrazones, disulfides, and also adisulfide with a hydrazone. Many of these linkers contain anN-alkyl carbamate as the prodrug, although there are alsocarbonates. Linkers attached to the DNA-binding portioninclude peptides, disulfides with or without a hydrazone,hydrazones with or without a PEG spacer, and simple amidebonds. These are attached at the C-5, C-6, C-7, or the indolenitrogen. Unfortunately, there is no biological data in thesepatent applications.

A recent publication indicates an interest in combininganalogues of the CBI core with the peptide linker system ofSeattle Genetics [31] (Section 11). Conjugates were madeeither with an amino-CBI carbamate (phenol replaced by thecorresponding aniline) or with a benzyl ether of an aza-CBIphenol (6d). These conjugates are relatively potent in tissueculture with selectivity versus a control conjugate of greaterthan 10-fold.

8. Enediynes

Enediynes are among the most cytotoxic of all classes of natu-ral products. Most of them are prodrugs of one type oranother, and commonly require reduction or nucleophilicaddition to activate or ‘trigger’ them. When bound in theminor groove of DNA, the resultant diradical abstracts hydro-gen atoms from the ribose residues on the backbone of theDNA, which leads to double-strand cleavage and, in manybut not all cases, apoptosis [32]. Antibody conjugates havebeen made both from derivatives of calicheamicin (7a), as wellas from neocarzinostatin (7c).

Earlier patents disclosed conjugates that were made frommethyltrithio-containing enediynes such as the calicheamicinsand esperamicins [143]. [American Cyanamid was acquired byAmerican Home Products in 1995, and they subsequentlychanged their name to Wyeth.] Using the natural products asstarting materials, a disulfide-containing linker was attachedby reacting the naturally-occurring trisulfide with a thiol. Awide range of linking chemistries were reported, although thespecific examples were made with either amide bonds to thelysines of the antibody, or through hydrazone bond formationwith oxidised carbohydrates on the antibody.

One conjugate related to those disclosed targets the tumourantigen PEM, a MUC1 variant, and has progressed toPhase II clinical trials [33]. Referred to as CMB-401, it con-tained only the disulfide as the source of drug release, whichwas found to be better for activity against MDR-resistant celllines [34]. The IC50 value was 15 ng/ml of calicheamicin for a1 h exposure to conjugate and 0.04 ng/ml for a 72 hour con-tinuous exposure, whilst in vivo, good activity was seenagainst established xenografts at doses of 50 – 400 µg/dose ofcalicheamicin for three doses [35]. This conjugate was selectiveboth in vitro and in vivo versus a control conjugate.

More recently, an additional linking strategy for the cali-cheamicins that involves incorporating an acyl hydrazonegroup as an additional release point in addition to thedisulfide already present and then attaching this linker tothe lysines of the antibody has been discussed [144]. The onlyFDA-approved chemoimmunoconjugate, gemtuzumab ozo-gamicin (Mylotarg®, 7b), which is approved for use in acutemyelogenous leukaemia is one of the examples [36,37].In vitro, Mylotarg was more potent and selective than theearlier amide and carbohydrate conjugates versus HL-60promyelocytic leukaemia cells [38,39]. It showed an IC50 valuein cell culture against HL-60 cells of 0.00046 ng/ml of

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calicheamicin with 78,000-fold selectivity vs. control cells.Mylotarg was shown to be curative of HL-60 xenografts inmice over a wide dose range.

Two related calicheamicin conjugates that differ in thetargeting antibody have been reported. CMC-544 (inotuzu-mab ozogamicin) targets the CD22 lymphoid antigen,makes use of the G544 antibody, and in some ways is toB cell lymphomas what Mylotarg is to leukaemia [40]. It hadan IC50 value of 0.01 – 0.5 nM against a panel of lymphoma

cell lines and was 20-fold more potent than a control conju-gate made from a non-binding antibody. It produced long-term, tumour-free survivors in various xenograft models andwas active in a disseminated model of B cell lymphoma. Theother conjugate targets the LewisY antigen and uses the anti-body designated Hu3S193 [41]. This conjugate was moreefficacious in several different solid tumour xenograft mod-els (gastric, colon, and prostate) than a control conjugate,producing cures in the gastric model.

N

O

NHO

O

O

O

N

Cl

NH

O

NH

NH

ONH

N

O

O

NH2

4

OO

O5

O

OOH

O

O

SO

O

NH

H

OHO

H

SSS

O

NH

O

O

ONH

I

O

O

O

OHOH

OH

O

O

7a γ-Calicheamicin

6d Seattle Genetics derivative

7b Mylotarg

Anti-CD33 antibody hP67.6

NH

ONH

O

NH

O

N

O

OOH

O

O

SO

O

H

OHO

H

O

NO

O

ONH

I

O

O

O

OHOH

OH

O

S

S

O

O

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A synthetic derivative named calicheamicin theta has alsobeen studied as an antibody conjugate. In this case, thethiol involved in triggering the calicheamicin is blocked as athioester and attachment is made through a disulfideattached to the aminosugar [42]. This material was made bya long total synthesis, limiting the amount of material thatis available [43]. Several conjugates of this agent have beenreported with potencies in vitro of as low as 0.1 pM of cali-cheamicin theta and with some reported activity in animalmodels [44-47].

Neocarzinostatin (7c) is an enediyne that is stabilised by anapoprotein, and conjugates have been made by attaching thatprotein to an antibody. Numerous publications discuss theconjugate of neocarzinostatin with the antiadenocarcinomaantibody A7 using traditional SPDP-based crosslinking strate-gies [145]. This conjugate was active in a xenograft model ofpancreatic cancer when injected either intravenously or intra-tumourally [48], and the same conjugate showed activityagainst gastric cell lines [49]. In vitro, the conjugate was10-fold more cytotoxic against antigen positive MKN45 cellsthan antigen negative MKN1 control cells with an IC50 valueof 70 ng/ml in neocarzinostatin equivalents. Tumour noduleformation was inhibited by 90% in a disseminated intraperi-toneal model. However, the effect was not significantly differ-ent than that seen with neocarzinostatin alone, although thediminished toxicity data indicated a wider therapeuticwindow for the conjugate.

Four other antibodies have been conjugated to neocarzi-nostatin [50]. These were prepared using thiol-maleimidecoupling to give a monosulfide linkage instead of thedisulfides produced by SPDP-based couplings. The antibod-ies used were 791T/36 (anti-gp72 targeting osteosarcomaand colorectal carcinoma), BW 431/26 (CEA, targetingcolon and stomach cancer), SWA11 (anti-SCLC-Ag clusterw4/gp45, targeting small-cell lung cancer), and OV-TL3(anti-OA3/p85, targeting ovarian carcinoma). The in vitroselectivity of the first three conjugates relative to antigen-negative cells was low, whereas the final one was ∼ 8-foldselective over background. The potency of these conjugatesranged 16 – 5100 ng/ml.

Conjugates of neocarzinostatin and the anticolon antibodyA7 have been in clinical trials. Patients given between 15 and90 mg of whole antibody conjugate showed no adverseeffects, and three out of eight patients with liver metastasessecondary to colon cancer showed a decrease in tumour sizeand three experienced pain relief, but this treatment had noeffect on other metastases [51]. However, most of these patientsmounted a human antimouse (HAMA) response against themurine antibody itself.

9. Auristatin/dolastatin

Dolastatin 10 is a potent tubulin inhibitor, similar in itsmechanism of action to maytansine and the vinca alkaloids,although it appears to occupy a somewhat different binding

site. Auristatin E differs from dolastatin 10 in the substitutionpattern of the phenethyl amine.

Seattle Genetics has a recently published patent applica-tion that discloses conjugates of auristatin E (AE, 8a) deriva-tives [146]. Modified pentapeptides are disclosed in which thethird residue is a γ-amino acid containing a β-methoxygroup, the fourth residue is the same as in auristatin E, andthe fifth residue is a derivatised phenethyl amine. Linkerexamples include a wide variety of hydrazones attacheddirectly or indirectly to the phenethyl amine residue.Attachment to the antibody is largely through maleimides,although α-bromo acetamides and reactive ester conjuga-tions are also exemplified. The most potent conjugatesexemplified have IC50 values in the low nanomolar range.Selectivity was modest in many cases, but over two logs ofselectivity was seen in one case versus a control cell line for aconjugate of auristatin E with 4-acetylbenzoic acid as thelinker esterified to the hydroxy group of the phenethylamine. The acetylbenzoate was attached via a hydrazone tothe antibody via 6-maleimidohexanoyl hydrazide.

A related auristatin E conjugate linked through the hydroxygroup on the phenethyl amine via 5-benzoylvaleric acid(AEBV) to either the anti-LewisY antibody BR96 or the anti-CD30 antibody cAC10 has been compared with a differentclass of auristatin E conjugates [52]. This class of conjugates ismade with auristatin E that is missing a terminal methylgroup from the dimethyl amine (MMAE) and is linkedthrough that amine via a cathepsin-labile dipeptide (Val-Citor Phe-Lys) and a ‘self-immolative linker’ (see Section 11)containing a para-amino benzyl carbamate (8b). This peptide-linked conjugate was shown to be more stable in plasma andto have a better in vitro profile (IC50 values of 5 – 90 ng/ml,selectivity of 50 to > 500-fold). The maximum tolerated dosesin mice paralleled the in vitro potency of the two derivativesbeing delivered, and both conjugate types made with BR96were capable of curing L2987 lung tumours in xenograftexperiments at doses where the control conjugates had noactivity. Even though the in vitro data indicated that the pep-tide-linked conjugates might be superior, insufficient in vivodata were presented to tell which conjugate type had thelargest therapeutic index.

Other MMAE conjugates with the Val-Cit linker have alsobeen discussed. One is an anti-CD20 conjugate with rituxi-mab [53]. This conjugate has an IC50 of 40 ng/ml with at least3 logs of selectivity on Ramos cells, although the potency isonly ∼ 1000 ng/ml on two other cell lines. In vivo, an ∼ 30day delay in tumour growth was seen in a Ramos xenograftexperiment, with no activity being seen at the same dose withrituximab or a control conjugate. Activity was also seen in adisseminated model. An anti-CD30 conjugate of antibodycAC10 has also been discussed. IC50 values of 0.5 – 300 ng/mlwere seen on a range of target cell lines, with IC50 valuesagainst non-target cell lines being 3000 – 9000 ng/ml [54].Good activity was seen in a Karpas disseminated model at3 mg/kg with no activity seen with a control conjugate. This

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conjugate also had an in vivo half-life of 144 h in mice and230 h in cynomolgus monkeys [55].

A publication on anti-TMEFF2 conjugates has alsoappeared. TMEFF2 is an antigen that is expressed on normalprostate as well as prostate tumours [56]. The conjugate ofantibody Pr1 had an IC50 of ∼ 6 ng/mL of drug with 100-foldselectivity with respect to a control conjugate. It showed verygood activity against both LNCaP and CWR22 prostatetumour xenografts at 0.22 mg/kg of AE. There is also a publi-cation of an anti-EphB2 conjugate [57]. EphB2 is a solidtumour marker associated with progression. The conjugatehad an IC50 value of ∼ 6 ng/ml of AE, and showed activity intwo xenograft experiments.

One further conjugate type has been presented by SeattleGenetics [58]. This conjugate is similar to the MMAE con-struct but is made from a derivative (MMAF) containing a‘C-terminated phenylalanine... carboxylic acid’ that preventscell penetration of the free drug. This results in the free drugbeing 10- to 200-fold less potent than MMAE, even thoughthe conjugates are 10-fold more potent.

10. Miscellaneous

NeoRx has disclosed antibody conjugates of trichothecenes,such as verrucarin A (9) [147]. However, the minimal data pre-sented indicated that verrucarin lost much of its potency

when conjugated by their methods. Memorial-SloanKettering has disclosed conjugates of enediyne quinoneimines (10b) [148,149]. These are analogues of dynemicin (10a),which is an enediyne that is activated by quinone reduction.There has also been a recent publication discussing a conju-gate of the topoisomerase I inhibitor camptothecin (11) madewith a Val-Cit self-immolative linker [59]. Conjugates of thehsp90-binding agent geldanamycin (12) have been made atthe NCI [60], and these are exemplified in a recently publishedpatent application [150].

11. Notable linker technology

Although the focus of this review is on the drugs that havebeen conjugated, it is sometimes difficult to separate the drugitself from the linking technology. There are also linker sys-tems that have novel release mechanisms or other propertiesthat make them worth noting. These will be covered in thissection.

An historically important conjugation procedure is theCytogen method that relies on the conjugation of hydrazidesand other nucleophiles to the aldehydes generated by oxida-tion of the carbohydrates that naturally occur on antibodies[102]. Although still used occasionally, the exact structure ofthe hydrazones is difficult to determine and the kinetics ofdrug hydrolysis is difficult to control. Hydrazone-containing

7c Neocarzinostatin chromophore

NNH

N

O

O

O

N

O

NH

OH

OO

8a Auristatin E

8b Seattle Genetic's MC-Val-Cit-PABC-MMAE derivative

NNH

N

O

O

O

N

O

NH

OH

OOO O

NH

O

NH

O

NH

NH

N

OO

O

5

O

NH2

OO

O

O

O O

OH OO

OOH

OH

NH

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conjugates are now commonly made with introduced carbo-nyl groups that give the desired drug-release properties [61,103].

BMS has disclosed conjugates made with a linker that has adisulfide at one end, an alkyl chain in the middle, and ahydrazine derivative at the other end [151]. The hydrazinederivatives include semicarbazides, thiosemicarbazides, carba-zates, substituted ureas of carbohydrazides, and aryl hydra-zines with appended amides. Also exemplified are similarconjugates with a maleimide on the antibody for thiol attach-ment. The anthracyclines are used to exemplify this linkertechnology.

Linkers containing functional groups other than hydra-zones have the potential to be cleaved in the acidic milieu ofthe lysosomes. A patent application from Immunomedics dis-closes conjugates made from thiol-reactive linkers that con-tain a site other than a hydrazone that is cleavableintracellularly, such as esters, amides, and acetals/ketals [152].Camptothecin is used as the cytotoxic agent to illustrate theselinkers. Ketals made from a 5- to 7-membered ring ketoneand that has one of the oxygens attached to the cytotoxicagent, and the other to a linker for antibody attachment, havebeen previously disclosed [153]. The anthracyclines are againused in the examples.

Another class of pH-sensitive linkers that were used rela-tively early in the conjugates field are the cis-aconitates, whichhave a carboxylic acid juxtaposed to an amide bond. The car-boxylic acid accelerates amide hydrolysis in the acidic lyso-somes. There is a more recent patent publication thatdiscloses linkers that achieve that same type of hydrolysis rateacceleration with several other types of structures [132]. Someof the examples also include disulfides. The maytansinoidsconjugated with linkers attached at C-9 are used to illustratethese linkers.

Another potential release method for drug conjugates is theenzymatic hydrolysis of peptides by the lysosomal enzymes.Such conjugates have been of interest for some time [62].However, it is often difficult to attach a peptide to a cytotoxicagent in such a way that it is still a substrate for the desiredenzyme and the cytotoxic agent is released without anattached amino acid. One general solution has been disclosedby BMS [154]. This involves attaching the peptide via an amidebond to para-aminobenzyl alcohol and then making a

carbamate or carbonate between the benzyl alcohol and thecytotoxic agent (13). Cleavage of the peptide then leads to thecollapse, or self-immolation, of the aminobenzyl carbamate orcarbonate. The cytotoxic agents exemplified with this strategyincluded anthracyclines, taxanes, and mitomycin C. Com-plete details of their initial work has been published [63]. Theyhave recently published widely on conjugates made with thisstrategy and the auristatins (See Section 9).

A related patent application has been published that dis-closes a similar strategy, except that a phenol is released by col-lapse of the linker instead of the carbamate [64,155]. In anothervariation, disulfide reduction is used to initiate the collapse ofa para-mercaptobenzyl carbamate or carbonate [156].

Many of the cytotoxic agents conjugated to antibodies havelittle, if any, solubility in water, and that can limit drug loadingon the conjugate due to aggregation of the conjugate. Oneapproach to overcoming this is to add solublising groups to thelinker, and that has been the subject of several disclosures.Conjugates made with a linker consisting of PEG and a dipep-tide have been disclosed [157]. Examples have a PEG di-acid,thiol-acid, or maleimide-acid attached to the antibody, adipeptide spacer, and an amide bond to the amine of ananthracycline or a duocarmycin analogue. Another example isby Immunogen, who disclosed conjugates that are made with aPEG-containing linker disulfide bonded to a cytotoxic agentand amide bonded to an antibody [158]. Other approaches thatincorporate PEG groups appear to be beneficial in overcomingaggregation and limits in drug loading [17,30,31,65]. Overcomingthe tendency of conjugates to aggregate and the resultant limi-tations in loading is an active area of research, and it will beinteresting to see the variety of solutions that are forthcoming.

12. Expert opinion

There are three major parts to every chemoimmunoconjugate:the antibody, the drug, and the linker between them. All threeare crucial to the success of a conjugate. The antibody, a com-plete discussion of which is beyond the scope of this review,should target sufficient internalising antigen to lead to cyto-toxicity of the tumour cells only. The cytotoxic agent shouldbe potent enough that the amount of antibody that is inter-nalised delivers a lethal dose of the cytotoxic agent to the

O

O

O

O

O

O

O

O

OH

H

9 Verrucarin A

NHOH

OH O

O

OH

O

O

OH

O

N

O

R

O

R

R

RR

R

10a Dynemicin 10b Enediyne quinone imine

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tumour. The linker should be stable in circulation with ahalf-life significantly greater than that of the antibody, but itshould be cleaved to release the free cytotoxic agent at a rea-sonable rate intracellularly. If one of these components fails,the conjugate will likely fail. For example, if a tumour is sensi-tive to a certain cytotoxic agent and the antibody targets itwell, the conjugate may fail if the cytotoxic agent is notreleased properly, or if the linker and cytotoxic agent aredesigned with appropriate characteristics, the conjugate mayperform poorly because of a lack of internalisation.

Because of the variations in these three components as wellas the manner in which the conjugates are tested, it is difficult,if not impossible, to make fair comparisons between the vari-ous conjugates that have been reported. However, some com-ments about the three components may be useful. Themajority of human or humanised antibodies have reasonablehalf-lifes in circulation. Chimeric antibodies carry the risk ofinducing an immune response and murine antibodies rou-tinely cause an immune response in patients. The half-life ofan antibody can be affected by the conjugation of the cytotoxicagent [66], making assessment of the half-life of the conjugatein comparison to the native antibody important. The targetantigen should be expressed uniformly and in high copynumber on the cells and internalise the conjugate efficiently.[Antigen heterogeneity, both in terms of copy number andantigen negative tumour cells, is a real problem for many anti-gens. By-stander effects are possible, but are beyond the scopeof this review.] The amount that needs to be internalised willdepend on the potency of the cytotoxic agent, but it is theabsolute amount internalised that is important, which makesdrug loading important. And, of course, the antigen shouldnot be present in significant numbers on normal cells where itcan be bound by the conjugate.

The cytotoxic agent is more difficult to assess. Although theanthracyclines, specifically doxorubicin, continue to be popularagents for new studies [65,67], they may not have sufficientpotency in many cases. The more potent anthracyclines, such asmorpholinodoxorubicin, seem to make reasonable conjugates,

but do not appear to be of significant current interest. Some ofthe potent cytotoxic agents that are of current interest are thecalicheamicins, the CC-1065/duocarmycin analogues, themaytansines, the potent taxanes, and, most recently, theauristatins. All of these exhibit subnanomolar potency in tissueculture, with the calicheamicins as a family appearing to be themost potent and the taxanes appearing to be the least potent.Research into improving the potency of the taxanes with theintent of making conjugates appears to be continuing [68].

There is published data that the most potent member of aclass of cytotoxic agents does not always make the conjugatewith the widest therapeutic window [69]. Consistent with this,it has been shown that reducing the drug loading on one con-jugate, which decreases the potency of the conjugate as awhole, actually increases the effectiveness of that conjugate[66]. The reasons for these examples are not clear, althoughmultiple possibilities exist. One interesting possibility is that itmay not be possible to get favourable pharmacokinetic prop-erties with the extremely low doses of conjugate used withthese most potent conjugates.

It is, of course, the potency of the conjugate itself that isimportant. The cytotoxic agent that is being considered maynot penetrate cells well on its own, leading to a lack of potencyin tissue culture, whilst the conjugate of that particular agentmay be very potent if internalisation and drug release occurs[58]. This might be a favourable circumstance if the linker hassome instability in circulation and the released cytotoxic agentcan be cleared without end organ toxicity. However, some ofthe antigen-independent toxicity of conjugates in vivo willalways be due to non-specific uptake of the intact conjugate.

Aside from having the desired potency, there is the issue ofthe stability of the cytotoxic agent in circulation. For thosecytotoxic agents where data are available, this does not seemto be a problem, although it might be speculated that the tri-chothecene conjugates with a cyclic ester bond might be sus-ceptible to some esterases, leading to their loss in potency[147]. Antibodies are commonly taken up into lysosomes,where the drug must also be stable, but there is no published

O

NH

Peptide-antibody

Drug-X

O

O

S

S-Linker-antibody

Drug-X

O

O

NH

Peptide-antibody

Drug

13 Self-immolative linkers

11 Camptothecin

N O

N

O

O

OH OO

NH

O

H2NCO2

OH

O

O

O

12 Geldanamycin

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data on the lysosomal stability of these drugs. Indeed, when aconjugate of a new cytotoxic agent appears to be well designedin all other aspects, but its conjugate lacks potency, lysosomalstability might be worth examining.

Of the five classes of agents mentioned, two are DNAactive agents (calicheamicin and CC-1065/duocarmycin)and three are antimitotics that act on tubulin (maytansine,the taxanes, and auristatin). Whether agents active againstone of these targets have an advantage over the others forconjugates is difficult to say. A more important considera-tion, perhaps, is the possible induction of resistance mecha-nisms. There is data to suggest that both MDR and defectsin apoptosis are resistance mechanisms for Mylotarg [70,71].The taxanes are also susceptible to MDR, although the morepotent derivatives less so, but the CC-1065/duocarmycinstend to be much less susceptible to this resistance mecha-nism [72]. However, DNA-alkylators as a class suffer fromtheir own resistance mechanisms, as do antimitotics and allother classes of cytotoxic agents. Resistance considerationsmay help dictate to some extent what combination therapyis tried with these conjugates in the future.

The structure, potency, and even resistance mechanismsof a particular cytotoxic agent is not completely separatefrom the linker used to attach it to the antibody. With cali-cheamicin, using disulfide reduction as the only mechanismof drug release led to more activity against MDR-resistantxenografts [34], although it is possible that this has as muchto do with the structure of the released drug than anythingto do with the release mechanism per se. With all cytotoxicagents, a linker must be attached in a position that does notalter the potency undesirably. That has been possible for thecommon cytotoxic agents.

Hydrazones, disulfides, and peptides are three commonmethods of drug release from conjugates. These rely on acid

hydrolysis in the lysosomes, reduction in an unknowncellular compartment, and enzymatic hydrolysis in the lyso-somes, respectively. Hydrazones have the disadvantage ofbeing cleaved slowly whenever they are in solution. This isstructure dependent and can, therefore, be controlled [144].Disulfides can also be reduced in circulation, where the glu-tathione concentration is ∼ 1% of intracellular levels. Again,this can be controlled by the structure of the disulfide, withthe more stable disulfides having increased serum stability[26,69]. Peptide linkers may offer the promise of the highestselectivity between serum and intracellular cleavage becausethe reaction is enzymatic [55]. However, there are alsoenzymes in serum that may cleave these linkers at an appre-ciable rate making complete serum stability an ideal thatmay never be reached. Most importantly, the answer towhether a few per cent of linker cleavage per day in circula-tion adds to toxicity or detracts from efficacy has not beendemonstrated definitively.

In the final analysis, conjugates of the calicheamicins, theCC-1065/duocarmycin analogues, the maytansines, thepotent taxanes, and the auristatins are all worth following asthey progress through the clinic and as future constructs aredisclosed. When assessing them, one needs to consider theantibody and the linking chemistries that are used, as well asthe specific model of cancer for which they are tested. The rel-ative merits of these conjugates and their potential for successwill become clearer only after considerably more direct com-parisons are published and the results of more clinical trialsare reported.

Acknowledgement

I would like to thank J Upeslacis for his help in discussing andediting this manuscript.

BibliographyPapers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers.

1. ROSS JS, GRAY K, SCHENKEIN D: Antibody-based therapeutics in oncology. Expert Rev. Anticancer Ther. (2003) 3:107-121.

• Covers antibody therapeutics in clinical trials and contains an extensive table.

2. MEYER DL, SENTER PD: Recent advances in antibody drug conjugates for cancer therapy. Annu. Rep. Med. Chem. (2003) 38:229-236.

• Orientated towards chemoimmunoconjugate and organised by linker type.

3. MILENIC DE: Monoclonal antibody-based therapy strategies: providing options for the cancer patient. Curr. Pharm. Rev. (2002) 8:1749-1764.

• Comprehensive review of all antibody uses in oncology.

4. GARNETT MC: Targeted drug conjugates: principles and progress. Adv. Drug Deliv. Rev. (2001) 171-216.

•• Although not as recent, a very comprehensive review of chemoimmunoconjugates.

5. APPELGREN LD, BAILEY DL, BRIGGS SL et al.: Chemoimmunoconjugate development for ovarian carcinoma therapy: preclinical studies with vinca alkaloid-monoclonal antibody constructs. Bioconjug. Chem. (1993) 4:121-126.

6. IWAHASHI T, TONE Y: USUI J et al.: Selective killing of carcinoembryonic-antigen (CEA)-producing cells in vitro by the immunoconjugate cytorhodin-S and CEA-reactive cytorhodin-S antibody CA208. Cancer Immunol. Immunother. (1989) 30:239-46.

7. HERMENTIN P, DOENGES R, GRONSKI P et al.: Attachment of rhodosaminylanthracyclinone-type anthracyclines to the hinge-region of monoclonal antibodies. Bioconjug. Chem. (1990) 1:100-107.

8. TRAIL PA, WILLNER D, LASCH SJ et al.: Cure of xenografted human carcinomas by BR96-doxorubicin immunoconjugates. Science (1993) 261:212-215.

• Seminal paper on BR96-DOX conjugate.

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9. SMITH S: Technology evaluation: SGN-15, Seattle Genetics, Inc. Curr. Opin. Mol. Ther. (2001) 3:295-302.

10. SALEH MN SUGARMAN S, MURRAY J et al.: Phase I trial of the anti-Lewis Y drug immunoconjugate BR96-doxorubicin in patients with Lewis Y-expressing epithelial tumors. J. Clin. Oncol. (2000) 18:2282-2292.

11. TOLCHER AW, SUGARMAN S, GELMAN KA et al.: Randomized Phase II study of BR96-doxorubicin conjugate in patients with metastatic breast cancer. J. Clin. Oncol. (1999) 17:478-484.

12. TRAIL PA, WILLNER D, BIANCHI AB et al.: Enhanced antitumor activity of paclitaxel in combination with the anticarcinoma immunoconjugate BR96-doxorubicin. Clin. Cancer Res. (1999) 5:3632-3638.

13. MUELLER BM, REISFELD RA, SILVEIRA MH, DUNCAN JD, WRASIDLO WA: Pre-clinical therapy of human melanoma with morpholino-doxorubicin conjugated to a monoclonal antibody directed against an integrin on melanoma cells. Antibody Immunoconjug. Radiopharm. (1991) 4:99-106.

14. KING HD, STAAB AJ, PHAM-KAPLITA K et al.: BR96 conjugates of highly potent anthracyclines. Bioorg. Med. Chem. Lett. (2003) 13:2119-2122.

15. OJIMA I, GENG X, WU X et al.: Tumor-specific novel taxoid-monoclonal antibody conjugates. J. Med. Chem. (2002) 45:5620-5623.

16. MILLER LM, ROLLER E, WU X et al.: Synthesis of potent taxoids for tumor-specific delivery using monoclonal antibodies. Bioorg. Med. Chem. Lett. (2004) 14:4079-4082.

17. MILLER LM, ROLLER EE, ZHAO RY et al.: Synthesis of taxoids with improved cytotoxicity and solubility for use in tumor-specific delivery. J. Med. Chem. (2004) 47:4802-4805.

18. CHARI RVJ, MARTELL BA, GROSS JL et al.: Immunoconjugates containing novel maytansinoids: Promising anticancer drugs. Cancer Res. (1992) 52:127-131.

19. LIU C, CHARI RVJ: The development of antibody delivery systems to target cancer with highly potent maytansinoids. Expert Opin. Investig. Drugs (1997) 6:169-172.

20. LIU C, TADAYONI BM, BOURRET LA et al.: Eradication of large colon tumor xenografts by targeted delivery of maytansinoids Proc. Natl. Acad. Sci. USA (1996) 93:8618-8623.

21. HELFT PR, SCHILSKY RL, HOKE FJ et al.: A Phase I study of cantuzumab mertansine administered as a single intravenous infusion once weekly in patients with advanced solid tumors. Clin. Cancer Res. (2004) 10:4363-4368.

22. SMITH SV: Technology evaluation: cantuzumab mertansine, Immunogen. Curr. Opin. Mol. Ther. (2004) 6:666-674.

• Good overall review of cantuzumab mertansine.

23. LIEBISCH P: EPPINGER S, SCHÖPFLIN C: CD44v4, a target for novel antibody treatment approaches is frequently expressed in multiple myeloma and associated with deletion of chromosome arm 13q. Haematologica (2005) 90:489-493.

24. TASSONE P, GOZZINI A, GOLDMACHER V et al.: In vitro and in vivo activity of the maytansinoid immunoconjugate huN901-N2′-deacetyl-N2′-(3-mercapto-1-oxopropyl)-maytansine against CD56+ multiple myeloma cells. Cancer Res. (2004) 64:4629-4636.

25. HENRY MD, WEN S, SILVA MD, CHANDRA S, MILTON M, WORLAND PJ: A prostate-specific membrane antigen-targeted monoclonal antibody-chemotherapeutic conjugate designed for the treatment of prostate cancer. Cancer Res. (2004) 64:7995-8001.

26. WILHELM SD, WIDISON WC, WHITEMAN KR et al.: Tumor-specific delivery of novel maytansinoids: Synthesis and biological evaluation. 229th ACS National Meeting, San Diego, CA (2005) MEDI-442.

• Improved maytansine conjugates. Need to wait for full publication.

27. BOGER DL, YUN W: CBI-TMI: synthesis and evaluation of a key analog of the duocarmycins. Validation of a direct relationship between chemical solvolytic stability and cytotoxic potency and confirmation of the structural features responsible for the distinguishing behavior of enantiomeric pairs of agents. J. Am. Chem. Soc. (1994) 116:7996-8006.

28. CHARI RVJ, JACKEL KA, BOURRET LA et al.: Enhancement of the selectivity and antitumor efficacy of a CC-1065 analog through immunoconjugate formation. Cancer Res. (1995) 55:4079-4084.

29. ZHAO RY, CHARI R, CAVANAGH E et al.: New water soluble CC-1065 analog prodrugs: design, synthesis and evaluation. 224th ACS National Meeting, Boston, MA (2002) MEDI-147.

30. SUZAWA T, NAGAMURA S, SAITO H, OHTA S, HANAI N, YAMASAKI M: Synthesis of a novel duocarmycin derivative DU-257 and its application to immunoconjugate using poly(ethylene glycol)-dipeptidyl linker capable of tumor specific activation. Bioorg. Med. Chem. (2000) 8:2175-2184.

31. JEFFREY SC, TORGOV MY, ANDREYKA JB et al.: Design, synthesis, and in vitro evaluation of dipeptide-based antibody minor groove binder conjugates. J. Med. Chem. (2005) 48:1344-1358.

32. CORTAZZO M, SCHOR NF: Potentiation of enediyne-induced apoptosis and differentiation by Bcl-2. Cancer Res. (1996) 56:1199.

33. CHAN SY, GORDON AY, COLEMAN RY et al.: A Phase II study of the cytotoxic immunoconjugate CMB-401 (hCTM01-calicheamicin) in patients with platinum-sensitive recurrent epithelial ovarian carcinoma. Cancer Immunol. Immunother. (2003) 52:243-248.

34. HAMANN PR, HINMAN LM, BEYER CF et al.: An anti-MUC1 antibody-calicheamicin conjugate for treatment of solid tumors, choice of linker and overcoming drug resistance. Bioconjug. Chem. (2005) 16:346-353.

•• Demonstrates that the best linker may not be universal but depends on target antigen/cell type.

35. HAMANN PR, HINMAN LM, BEYER CF: A calicheamicin conjugate with a fully humanized anti-MUC1 antibody shows potent antitumor effects in breast and ovarian tumor xenografts. Bioconjug. Chem.16:354-360 (2005).

36. BROSS PR, BEITZ J, CHEN G et al.: Approval summary: gemtuzumab ozogamicin in relapsed acute myeloid leukemia. Clin. Cancer Res. (2001) 7:1490-1496.

• See also [73].

37. DEANGELO DJ, LIU D, STONE R: Preliminary report of a Phase II study of gemtuzumab ozogamicin in combination with cytarabine and daunorubicin in patients < 60 years of age with de novo acute myeloid leukemia. Proc. Am. Soc. Clin. Oncol. (2003) 22:a2325.

• Meeting abstract on one of the continuing studies with this conjugate, which indicates an 84% CR rate.

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38. HAMANN PR, HINMAN LM, BEYER CF et al.: An anti-CD33 antibody-calicheamicin conjugate for treatment of acute myeloid leukemia. Choice of linker. Bioconjug. Chem. (2002) 13:40-46.

39. HAMANN PR, HINMAN LM, HOLLANDER I et al., Gemtuzumab ozogamicin, a potent and selective anti-CD33 antibody-calicheamicin conjugate for treatment of acute myeloid leukemia. Bioconjug. Chem. (2002) 13:47-58.

•• Describes preclinical characterisation of Mylotarg and how the linker was designed.

40. DIJOSEPH JF, ARMELLINO DC, BOGHAERT ER et al.: Antibody-targeted chemotherapy with CMC-544: a CD22-targeted immunoconjugate of calicheamicin for the treatment of B-lymphoid malignancies. Blood (2004) 103:1807-1814.

41. BOGHAERT ER, SRIDHARAN L, ARMELLINO DC et al., Antibody-targeted chemotherapy with the calicheamicin conjugate hu3S193-N-acetyl gamma calicheamicin dimethyl hydrazide targets LewisY and eliminates LewisY-positive human carcinoma cells and xenografts. Clin. Cancer Res. (2004) 10:4538-4549.

42. LODE HN, REISFELD RA, HANDGRETINGER R, NICOLAOU KC, GAEDICKE G, WRASIDLO W: Targeted therapy with a novel enediyne antibiotic calicheamicin θI

1 effectively suppresses growth and dissemination of liver metastases in a syngeneic model of murine neuroblastoma. Cancer Res. (1998) 58:2925-2928.

43. NICOLAOU KC: The battle for calicheamicin γ1I. Angew. Chem., Int. Ed. Engl. (1993) 32:1377-85.

44. BURCHARDT CA, UTTENREUTHER FMM, FISCHER P, GAEDICKE G, WRASIDLO W: Cytotoxic effects of new immunoconjugates in different neuroblastoma cell-lines. J. Cancer Res. Clin. Oncol. (1998) 124:137.

45. BERNT KM, LODE HN, HAGEMEIER C, GAEDICKE G, WRASIDLO W: CD19 targeted calicheamicin theta is effective against high risk ALL. Blood (2000) 96:467a.

46. KNOLL K, WRASIDLO W, SCHERBERICH JE, GAEDICKE G, FISCHER P: Targeted therapy of experimental renal cell carcinoma with a novel conjugate of monoclonal antibody 138H11 and calicheamicin θI

1. Cancer Res. (2000) 60:6089-6094.

47. SCHMIDT CS, WRASIDLO W, SCHERBERICH JE, GAEDICKE G, FISCHER P: Chemoimmunoconjugates with the monoclonal antibody 138H11 for targeting the cytotoxic prodrug calicheamicin θ to renal cell carcinomas. Tumor Target. (1999) 4:271-277.

48. OTSUJI E, YAMAGUCHI T, TSURUTA H et al.: The effect of intravenous and intra-tumoral chemotherapy using a monoclonal antibody–drug conjugate in a xenograft model of pancreatic cancer. Eur. J. Surg. Oncol. (1995) 21:61-65.

49. OKAMOTO K, YAMAGUCHI T, OTISUJI E et al.: Targeted chemotherapy in mice with peritoneally disseminated gastric cancer using monoclonal antibody-drug conjugate. Cancer Lett. (1998) 122:231-236.

50. MAIBUCHER A, SCHONLAU F, GOTTSCHALK U, KOHNLEIN W, GARNETT: Neocarzinostatin immunoconjugates: In vitro evaluation of therapeutic potential. Pharm. Sci. Commun. (1994) 4:253-261.

51. TAKAHASHI T, YAMAGUCHI T, KITAMURA K et al.: Clinical application of monoclonal antibody-drug conjugates for immunotargeting chemotherapy of colorectal carcinoma. Cancer (1988) 61:881-888.

52. DORONINA S, MENDELSOHN B, TOKI B et al.: Development of potent monoclonal antibody auristatin conjugates for cancer therapy. Nat. Biotechnol. (2003) 21:778-784.

53. LAW C-L, CERVENY CG, GORDON KA et al.: Efficient elimination of B-lineage lymphomas by anti-CD20 auristatin conjugates. Clin. Cancer Res. (2004) 10:7842-7851.

54. FRANCISCO JA, CERVENY CG, MAYER DL et al.: cAC10-vcMMAE, an anti-CD30-monomethyl auristatin E conjugate with potent and selective antitumor activity. Blood (2003) 102:1458-1465.

55. SANDERSON RJ, HERING MA, JAMES SF et al.: In vivo drug-linker stability of an anti-CD30 dipeptide-linked auristatin immunoconjugate. Clin. Cancer Res. 11:843-852.

56. ARAF DEH, BHASKAR V, IBSEN E et al.: Preclinical validation of antiTMEFF2-auristatin E-conjugated antibodies in the treatment of prostate cancer. Mol. Cancer Ther. (2004) 3:921-931.

57. MAO W, LUIS E, ROSS S et al.: EphB2 as a therapeutic antibody drug target for the treatment of colorectal cancer. Cancer Res. (2004) 64:781-788.

58. DORONINA SE, TOKI BE, TORGOV MY et al.: Immunoconjugates comprised of drugs with impaired cellular permeability: A new approach to targeted therapy. 228th ACS National Meeting, Philadelphia, PA (2004) MEDI-010.

• An interesting approach. Need to wait for full publication.

59. WALKER MA, DUBOWCHIK GM, HOFSTEAD SJ, TRAIL PA, FIRESTONE RA: Synthesis of an immunoconjugate of camptothecin. Bioorg. Med. Chem. Let. (2002) 12:217-219.

60. MANDLER R, KOBAYASHI H, HINSON ER, BRECHBEIL MW, WALDMAN TA: Herceptin-geldanamycin immunoconjugates: pharmacokinetics, biodistribution, and enhanced antitumor activity. Cancer Res. (2004) 64:1460-1467.

61. HAMANN PR, HINMAN LM, HOLLANDER I et al.: A potent and selective anti-CD33 antibody–calicheamicin conjugate for treatment of acute myeloid leukemia. Bioconjug. Chem. (2002) 13:7-58.

62. ABOUD-PIRAK E, SERGENT T, OTTE-SLACHMUYLDER C, ABARCA J, TROUET A, SCHNERIDER YJ et al.: Binding and endocytosis of a monoclonal antibody to a high molecular weight human milk fat globule membrane-associated antigen by cultured MCF-7 breast carcinoma cells. Cancer Res. (1988) 48:3188-3196.

63. DUBOWCHIK GM, FIRESTONE RA, PADILLA L et al.: Cathepsin B-labile linkers for lysosomal release of doxorubicin from internalizing immunoconjugates: Model studies of enzymatic drug release and antigen-specific in vitro anticancer activity. Bioconjug. Chem. (2002) 13:855-869.

64. TOKI BE, CERVENY CG, WAHL AF, SENTER PD: Protease-mediated fragmentation of p-amidobenzyl ethers: a new strategy for the activation of anticancer prodrugs. J. Org. Chem. (2002) 67:1866-1872.

65. SUZAWA T, NAGAMURA S, SAITO H: Enhanced tumor cell selectivity of adriamycin-monoclonal antibody conjugates via a poly(ethyleneglycol)-based cleavable linker. J. Control Release (2002) 79:229-249.

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66. HAMBLETT KJ, SENTER PD, CHACE DF et al.: Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin. Cancer Res. (2004) 10:7063-7070.

•• Shows some of the considerations that need to be addressed with the degree of drug loading.

67. DUBOWCHIK GM, FIRESTONE RA, PADILLA L: Cathepsin B-labile dipeptide linkers for lysosomal release of doxorubicin from internalizing immunoconjugates: model studies of enzymatic drug release and antigen-specific in vitro anticancer activity. Bioconjug. Chem. (2002) 13:855-869.

• Describes the work behind the auristatin peptide linkers.

68. BALOGLU E, MILLER ML, ROLLER EE et al.: Synthesis and biological evaluation of novel taxoids designed for targeted delivery to tumors. Bioorg. Med. Chem. Lett. (2004) 14:5885-5888.

69. HINMAN LM, HAMANN R, MENENDEZ AT, WALLACE R, DURR FE, UPESLACIS J: Preparation and characterization of monoclonal antibody conjugates of the calicheamicins: a novel and potent family of antitumor antibiotics. Cancer Res. (1993) 53:3336-3342.

70. MATSUI H, TAKESHITA A, NAITO K. et al.: Reduced effect of gemtuzumab ozogamicin (CMA-676) on P-glycoprotein and/or CD34-positive leukemia cells and its restoration by multidrug resistance modifiers. Leukemia (2002) 16:813-819.

71. AMICO D, BARBUI AM, ERBA E, RAMBALDI A, INTRONA M, GOLAY J: Differential response of human acute myeloid leukemia cells to gemtuzumab ozogamicin in vitro: Role of Chk1 and Chk2 phosphorylation and caspase 3. Blood (2003) 101:4589-4597.

72. SAVARAJ N, WU CJ, LAMPIDIS TJ, TAPIERO H, CHUA L, FEUN LG: In vitro cytotoxicity of U-73975 in multidrug resistant and cisplatin resistant cells. Cell. Pharmacol. (1994) 1:103-106.

73. BROSS PR, BEITZ J, CHEN G et al.: Approval summary: gemtuzumab ozogamicin in relapsed acute myeloid leukemia (correction to previous reference). Clin. Cancer Res. (2002) 8:300.

• See also [36].

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108. CYTOGEN CORP.: US4950738 (1990).

109. HOECHST JAPAN LTD: US5028698 (1991).

110. HOECHST, AK: EP0298385 (1988).

111. BRISTOL-MYERS SQUIBB CO.: US5122368 (1992).


113. BRISTOL-MYERS: US5137877 (1992).

114. PHARMACIA & UPJOHN: US5776458 (1998).

115. IMMUNOGEN, INC.: US6630579 (2003).

116. IMMUNOMEDICS, INC.: US20040202666 (2004).

117. BRUNSWICK CORP.: EP0476408 (1992).

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128. IMMUNOGEN, INC.: US5208020 (1993).

129. IMMUNOGEN, INC.: US6441163 (2002).

130. GENENTECH: US2002035736 (2002).

131. IMMUNOGEN, INC.: US20040235840 (2004).

132. SANTI D, MYLES DC, METCALF B, HUTCHINSON R, ASHLEY G: US20030109682 (2003).

133. PHARMACIA & UPJOHN CO.: US5739350 (1998).

134. IMMUNOGEN, INC.: US5475092 (1995).

135. IMMUNOGEN, INC.: US5846545 (1998).

136. IMMUNOGEN, INC.: US6756397 (2004).

137. IMMUNOGEN, INC.: US2003199519 (2003).

138. IMMUNOGEN, INC.: US20040109867 (2004).

139. PANORAMA RES.: US5843937 (1998).

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144. AMERICAN CYANAMID: US5773001 (1998).

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150. UNITED STATES DEPT OF HEALTH & HUMAN SERVICES: US2004204395 (2004).


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Websites

201. http://www.seattlegenetics.com/about/alliances.htmSeattle Genetics.

202. http://www.medarex.com/cgi-local/item.pl/20020523-555512Medarex, press release archive.

203. http://www.seattlegenetics.com/news/index.htm Seattle Genetics, press release (7/6/2005).

AffiliationPhilip R HamannChemical and Screening Sciences, Wyeth Research, 401 N. Middletown Rd., Pearl River, NY 10965, USATel: +1 (845) 602 3423; Fax: +1 (845) 602 5561;E-mail: [email protected]

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Documents

Monoclonal antibody–drug conjugates