Journal of Pharmacological and Toxicological Methods 64 (2011) 207–212

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Journal of Pharmacological and Toxicological Methods

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Review

Antibody drug conjugates — Trojan horses in the war on cancer

U. Iyer, V.J. Kadambi ⁎Non-Clinical Development Sciences, Millennium Pharmaceuticals, Inc, Cambridge, MA 02139, USA

⁎ Corresponding author at: Non-Clinical DevelopmeStreet, Cambridge, MA, 02139, USA.

E-mail address: kadambi@mpi.com (V.J. Kadambi).

1056-8719/$ – see front matter © 2011 Published by Edoi:10.1016/j.vascn.2011.07.005

a b s t r a c t

a r t i c l e i n f o

Article history:Received 7 January 2011Accepted 28 July 2011

Keywords:Targeted therapyCancer therapyCytotoxin deliveryAntibody toxin conjugates

Antibody drug conjugates (ADCs) consist of an antibody attached to a cytotoxic drug by means of a linker.ADCs provide a way to couple the specificity of a monoclonal antibody (mAb) to the cytotoxicity of a small-molecule drug and, therefore, are promising new therapies for cancer. ADCs are prodrugs that are inactive incirculation but exert their cytotoxicity upon binding to the target cancer cell. Earlier unsuccessful attempts togenerate ADCs with therapeutic value have emphasized the important role each component plays indetermining the efficacy and safety of the final ADC. Scientific advances in engineering antibodies formaximum efficacy as anticancer agents, identification of highly cytotoxic molecules, and generation of linkerswith increased stability in circulation have all contributed to the development of the many ADCs that arecurrently in clinical trials. This review discusses parameters that guide the selection of the components of anADC to increase its therapeutic window, provides a brief look at ADCs currently in clinical trials, and discussesfuture challenges in this field.

nt Sciences, 35, Landsdowne

lsevier Inc.

© 2011 Published by Elsevier Inc.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2072. Cytotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2083. Monoclonal antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2094. Linker strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

4.1. Acid-labile hydrazone linkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2094.2. Disulfide-based linkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2094.3. Peptide-based linkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2094.4. Noncleavable linkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210

5. Salient features of ADCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2106. ADCs in clinical trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2117. Challenges and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212

1. Introduction

Despite several years of intensive research into cancer therapies,the prognosis for the average cancer patient is bleak. According toAmerican Cancer Society projections for 2010, approximately 1.5 mil-lion new cancers were expected to be diagnosed and more than500,000 patients were expected to die of cancer, with N90% of thesecancers being solid tumors (ACS, 2010). Most anticancer drugscurrently in use target rapidly dividing cells by disrupting steps in

the cell cycle or by targeting the pathways that control normal cellgrowth andmalignant transformation (Bollag et al., 1995; Chow et al.,1988; Downward, 2003; Herbst and Khuri, 2003). These drugs do notexplicitly discriminate between cancer cells and normal cells. Thisnonspecificity is responsible for many of the toxicities associated withthese drugs, which are often dosed at suboptimal levels. Coupled withthe emergence of drug-resistant cancer cell populations, it is clear thatthere is an urgent need for therapies that are selective and efficacious,with minimal toxicities. Much of the ongoing research in this area isfocused on finding a way to increase the specificity of the small-molecule drugs for the cancer cells.

Antibody therapies offer many advantages, the main one being thespecificity of each antibody for its target antigen. Since their discovery in

Fig. 1. Schematic of an ADC. The schematic for the structure of anADC is shown alongwithexamples of antibodies, linkers and drugs used to make some of the ADCs currently inclinical trials.

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the early 1970s,mAbs have been investigated as therapeutic agents andhave enjoyed considerable success in the last 2decades,withover20%ofmAbs that enter clinical trials receiving regulatory approval comparedto 11% for small-molecule therapies (Kohler & Milstein, 1975; Reichert,Rosensweig, Faden, & Dewitz, 2005). Antibody-based therapies havebeen the focus ofmuch recent research, as evidenced by the growing listof such therapies approved in the last few years or currently in clinicaltrials (Reichert, 2010). Of these, a large percentage are against oncologytargets because a number of antigens have been identified as beingoverexpressed in cancer cells compared to normal tissues (Panchal,1998). However, mAbs also have their disadvantages, including poorcytotoxicity and poor penetration into tumors.

Combining these two therapies – cytotoxic drugs with mAbs –

would provide the best of both worlds, as it were— the specificity of amAb and the cytotoxicity of the small molecule. The concept behindADCs is simple — use the exquisite specificity of a mAb for its antigento selectively deliver a highly toxic drug to the cancer cell, thusincreasing the therapeutic window of both the drug and the antibody.Fig. 1 shows a general schematic of the structure of an ADC.

ADCs are prodrugs that require release of the cytotoxic drug insidethe tumor cell (Doronina et al., 2003; Sutherland et al., 2006).Conjugation of the drug to the antibody inactivates the drug so that itis not toxic while in circulation. The mAb then delivers its cytotoxicpayload to the antigen-expressing cancer cell, where internalizationof the ADC and cleavage of the linker occur, releasing the cytotoxin inits active form to kill the targeted cancer cells.

ADCs offer a unique opportunity to combine the best features ofboth small-molecule drugs and antibodies to create a single therapythat is highly specific and cytotoxic. As such, they have been thesubject of intense research focused on optimization to increase thetherapeutic indices of ADCs. The principles that guide the design ofdrugs and mAbs for use in ADCs differ from those that guide design ofstand-alone small-molecule drugs and mAb therapies.

2. Cytotoxins

A number of different classes of cytotoxins are available for thetreatment of cancer, including taxanes, vinca alkaloids, anthracy-clines, and others. These drugs target rapidly proliferating cells bydisrupting different aspects of cell proliferation, including DNAreplication, repair, translation, and cell division (Bollag et al., 1995;Chow et al., 1988; Herbst and Khuri, 2003). Because cancer cells havea high rate of proliferation, they are more susceptible to the cytotoxiceffects of these drugs. Cytotoxins are, therefore, not truly specific forcancer cells and will also affect any normal cells that have high

proliferation rates. As a result, these drugs are often associated withdose-limiting toxicities of the gastrointestinal tract and bone marrow(Markman, 2003). The elucidation of the molecular pathwaysinvolved in development of cancer has provided many additionaltargets for the design of new cytotoxic drugs (Downward, 2003;Okada and Mak, 2004).

One way to increase the therapeutic efficacy of a cytotoxin is toincrease its selectivity by conjugating the drug to a tumor-specificantibody resulting in anADC. Early attempts to create ADCs by attachingclinically approved drugs to mAbs were met with limited success, andone factor was thought to be the insufficient potency of the conjugateddrug (Chari et al., 1992; Hinman et al., 1993; Liu et al., 1996). Thehypothesis is that the low accumulation of the antibody at disease sitesleads to suboptimal concentrations of the cytotoxic drugs at these sites(Liu et al., 1996). Therefore, the use of highly cytotoxic agents shouldprovide better efficacy because even small amounts of these drugs canhave potent tumoricidal effects. This prompted research into the use ofcytotoxins that were 100 to 1000 times more potent than conventionalchemotherapeutic agents because the conjugation of the drug to theantibody inactivates the drugwhile it is in circulation (Chari et al., 1992;Hinman et al., 1993; Liu et al., 1996).

Cytotoxins currently being used in ADCs fall into two categories:those that target microtubules and those that target DNA. Microtu-bules are well-established targets for cancer therapies because theyplay a key role in mitosis (Jordan &Wilson, 1998). The maytansinoidsand auristatins are classes of cytotoxins that disrupt microtubuledynamics by inhibiting microtubule assembly (Cassady, Chan, Floss, &Leistner, 2004; Wu & Senter, 2005). Calicheamicin, a naturallyoccurring antibiotic, and its derivatives bind to the minor groove ofDNA and induce cell death by producing DNA strand breaks (Lee et al.,1989; Nicolaou & Smith, 1992).

The discovery of maytansine and its derivatives in the early 1970sgenerated a great deal of interest due to their high cytotoxicity(Cassady et al., 2004). They were shown to be 100- to 1000-fold morepotent than other microtubule-targeting agents such as vincristineand vinblastine (Chari et al., 1992). Maytansinoids, which are derivedfrom a natural product, were used in clinical trials in the 1970s butwere abandoned due to their toxicity profile and low therapeuticindex (Cassady et al., 2004). Their high cytotoxicity, however, makesthem ideal candidates for conjugation to antibodies, as targeteddelivery to the cancer cells should greatly increase their therapeuticwindow. Several ADCs with maytansinoids as the cytototoxic payloadare currently in clinical trials based on promising nonclinical results,including trastuzumab linked to DM1 (Lewis Phillips et al., 2008).

Similarly, auristatins, which are synthetic analogs of dolastatin, areshown to be 50- to 200-fold more potent than the vinca alkaloids anddoxorubicin, respectively (Doronina et al., 2003). Dolastatin and itssynthetic derivatives were used in several clinical trials but failed toshow activity against various cancers (Kindler et al., 2005; Perez et al.,2005). Auristatin conjugates currently in clinical trials include brentux-imab vedotin (monomethyl auristatin E conjugated to anti-CD30antibody) and CR011-vcMMAE (monomethyl auristatin E conjugatedto anti-glycoprotein NMB antibody). Currently two auristatins, mono-methyl auristatin E andmonomethyl auristatin F, are being investigatedas part of an ADC.

Calicheamicin is another highly potent cytotoxin that has demon-strated cell killing at concentrations far below those of other standardchemotherapeutics (Hinman et al., 1993; Lee et al., 1989). Mylotarg®(gemtuzumab ozogamicin), the first ADC to win FDA approval, uses acalicheamicin derivative as the cytotoxin. Another calicheamicinconjugate in clinical trials is inotuzumab ozogamicin, in whichcalicheamicin is conjugated to inotuzumab (Dijoseph et al., 2006).

Other drugs that are being investigated as components of ADCsinclude doxorubicin (a topoisomerase II inhibitor), SN-38 (a topo-isomerase I inhibitor), and derivatives of paclitaxel (a microtubuleinhibitor) (Govindan & Goldenberg, 2010).

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Apart from high potency, other important characteristics of asuitable cytotoxin for ADCs are low molecular weight and immuno-genicity, availability of reactive functional groups that can be used inconjugation, and sufficient stability and solubility in aqueoussolutions (Singh & Erickson, 2009).

3. Monoclonal antibodies

AlthoughmAbswere discovered as early as 1975, it is only in the last2 decades that they have demonstrated success in the clinical setting,with Rituxan® being the firstmAb approved in 1997 (Kohler &Milstein,1975). Early attempts to use murine mAbs in the clinical setting wereunsuccessful because these antibodies were highly immunogenic, werenot sufficiently cytotoxic because they could not elicit potent antitumorresponses, and had very short half-lives of less than20 h (Christiansen&Rajasekaran, 2004). The development of techniques to generatechimeric or humanized antibodies has helped to overcome theselimitations by reducing the immunotoxicity and increasing the half-lives of themAbs. Most of the antibodies currently in clinical testing aremouse mAbs that have been humanized (Reichert et al., 2005).

It is important to note that, unlike cytotoxins, antibodies bythemselves do not kill the target cell. Instead, by attaching to the targeton the surface of the cancer cell, they induce cell death by two possiblemechanisms. Binding of the antibody to the antigenon the surfaceof thecancer cell can elicit an immune response that leads to antibody-dependent cellular cytotoxicity (ADCC) and complement-dependentcytotoxicity (CDC), which in turn lead to cell lysis (Gelderman,Tomlinson, Ross, & Gorter, 2004; Van Meerten, Van Rijn, Hol,Hagenbeek, & Ebeling, 2006). Alternatively, binding of the antibody tothe cell can trigger a signaling cascadewithin the cell itself that results inapoptosis (Selenko et al., 2002).

The type of antibodyused in anticancer drugs is usually IgG1becauseantibodies of this class can trigger bothADCCandCDC (Geldermanet al.,2004). Efforts have been made to modify the antibodies to be moreeffective at inducing immune responses and to increase their cytotoxicpotential (Carter, 2006).

Improved techniques for generating antibodies against highlyvalidated targets have given rise to blockbuster mAb drugs, such asrituximab (target-CD20), trastuzumab (target-HER2), and cetuximab(target-epidermal growth factor receptor), whose sales amount tobillions of dollars in revenue (Scolnik, 2009). However, despite theseadvances, there are still limitations to mAb therapeutics, includingpoor penetration into tumors, with only a small percentage of theadministered mAb localizing within the tumor, and slow extravasa-tion of the drug from the blood supply (Christiansen & Rajasekaran,2004; Wu & Senter, 2005). Additionally, antibodies are expensive togenerate, which increases the costs of the marketed drugs (Farid,2007; Scott, 2005). Monoclonal antibody therapies are often notcurative and may need to be combined with chemotherapy (Weiner,Surana, & Wang, 2010). As a result, there has been tremendousinterest in finding a way to increase the therapeutic effectiveness ofthe mAb through attachment to a toxin, drugs, or radionuclides.

Apart from the favorable properties required for a mAb to act as astand-alone therapy, some additional factors must be considered whileselecting antibodies for ADCs. The antibody should have sites amenableto conjugation to the drug such that binding to the antigen is notcompromised. To facilitate attachment of the drug to the antibody, IgGshave also been engineered to have unique sites for conjugation thatallow for generation of more homogenous ADCs (Junutula et al., 2008).Although it is assumed that the cytotoxin will play themajor role in cellkilling, additional cytotoxicity resulting from complement-dependentcytotoxicity and activation of immune effector cells by the antibodymaybedesirable, as longas it does not cause toxicity. For example, in the caseof SGN-75 (monomethyl auristatin F linked to an anti-CD70 antibody,h1F6), it has been demonstrated that the antibody backbone retains its

ability to engage effector cells and induce complement-mediatedcytotoxicity (Ryan et al., 2010).

Selection of the target antigen is also an important criterion in termsof both safety and effectiveness of the ADC. One obvious criterion is thatthe antigen must be present at the surface of the cell. A high level ofexpression of the target antigen in cancer cells, with little or noexpression innormal cells, is clearly another primary requisite. However,a clear correlationbetween levels of antigenexpression andsensitivity tothe therapeutic mAb has not been shown (Walter, Raden, Kamikura,Cooper, & Bernstein, 2005). This suggests that other factors may also becritical in determining the efficacy of the ADC against a target antigen.

Efficient internalization of the antigen is an important factor toconsider, along with antigen levels, because the mechanism of action ofADCs is dependent on internalization after binding of the ADC(Sutherland et al., 2006;Walter et al., 2005). The presence of a cytotoxicpayload on the ADCmeans that it is important to pick a target that is notshed into the blood by cleavage of the antigen from the cancer cells(Manshouri et al., 2003; VanDerVelden et al., 2004). Thebinding affinityof the antibody for the antigen and the number of antigen molecules onthe cell surface might also affect the potency of the ADC (Carter, 2006).

4. Linker strategies

One of the biggest challenges in the development of ADCs has beenthe generation of suitable linkers for conjugating the antibody and thedrug (Kemshead & Hopkins, 1993). The linker should be extremelystable in circulation since release of the cytotoxic payload beforereaching the target would lead to nonspecific cell killing andassociated toxicities. However, upon reaching the target cells, thelinkermust also be able to efficiently release the drug in its active formto allow the drug to effect cell killing. Several strategies have beenemployed to produce linkers that satisfy both of these criteria.

The binding of the antibody to the antigen usually leads tointernalization of the conjugate by receptor-mediated endocytosis(Walter et al., 2005). Most of the linkers take advantage of the factthat the conjugate enters the cell through the acidic lysosomalcompartment that is also rich in proteolytic enzymes, while some usethe highly reducing environment of the cell itself. There are currentlyfour different classes of linkers in use that broadly fall under 2categories: cleavable and noncleavable linkers.

4.1. Acid-labile hydrazone linkers

These were the first linkers to be used and interestingly, this typeof linker is also used in gemtuzumab ozogamicin, the only ADC toreceive FDA approval. Although stable at the neutral pH of the blood,they undergo hydrolysis in the acidic environment of the cellularcompartments (Ducry & Stump, 2010). These linkers have beenassociated with non-specific release of the drug in clinical studies(Ducry & Stump, 2010; Senter, 2009).

4.2. Disulfide-based linkers

The disulfide bonds in these linkers are selectively cleaved in thecytosol because of the high intracellular concentration of glutathione.Previousworkwithplantprotein toxinshas shownthat the stabilityof thedisulfide linkers canbe increasedby steric hindrance (Thorpeet al., 1987).

4.3. Peptide-based linkers

Peptide-based linkers, as the name suggests, link the drug to theantibody by means of a peptide bond (Ducry & Stump, 2010). Releaseof the drug from the mAb occurs specifically due to the action oflysosomal proteases. Thus, unlike the chemically labile linkersdiscussed thus far, peptide linkers combine greater systemic stabilitywith rapid enzymatic release of the drug in the target cell.

Fig. 2.Mechanism of Action of ADCs. The ADC is internalized by receptor-mediated endocytosis and cleavage of the linker occurs within the cell to release the cytotoxin, which thentargets either the DNA or the microtubules to kill the cell.

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4.4. Noncleavable linkers

This class of thioether-containing linkers is noncleavable and releaseofthe drug is postulated to occur through intracellular proteolyticdegradation (Erickson et al., 2006; Lewis Phillips et al., 2008). Oneadvantage of these linkers is their greater stability in circulation comparedto the cleavable linkers; this canpotentially improve the therapeutic indexof the drug because it may be better tolerated (Lewis Phillips et al., 2008;Singh & Erickson, 2009; Polson et al., 2010). An ADC with trastuzumablinked to the maytansinoid, DM1, through a thioether linker has beenshown to be better tolerated with a more favorable pharmacokinetic andsafety profile compared to the disulfide-linked ADC (Lewis Phillips et al.,2008). A disadvantage may be that poor internalization of the ADC mayhave a more negative impact on an ADC with a noncleavable linkercompared to one with a cleavable linker because, in the latter, release ofthe drug by cleavage of the linkerwithin the intracellular spacemay allowthe drug to permeate the cell (Ducry & Stump, 2010).

5. Salient features of ADCs

Fig. 2 shows the sequence of events that occur upon binding of theADC to the target antigen.

As we have seen, careful selection of the different components ofthe ADC is critical to successful design of ADCs that could be used ascancer therapeutics. Apart from optimizing the component parts,efforts to build better ADCs have led to much research into all thecharacteristics of the ADC that may affect its therapeutic index,including conjugation of the antibody to the linker and the drug, drug-antibody ratios, and the mechanism by which ADCs effect cell killing.

The assembly of the ADC from the component parts is also animportant step in determining the therapeutic potential of the ADC.Conjugation of the antibody to the drug should not alter the integrity ofthe antibody, the binding of the antibody to the antigen, or the biologicalactivity of the drug upon reaching the target cell. The pharmacodynamicproperties of the ADCmust resemble that of themAbwhile in circulation.Conjugation of the antibody to the drug occurs at cysteines or lysines onthe antibody; the products of such conjugations are heterogeneous, withvariations in thenumberofmoleculesofdrugperantibodyand in the sitesof attachment (Junutula et al., 2008). To address this variation, effortshave beenmade to engineer reactive cysteine residues at specific sites inantibodies to allow drugs to be conjugated with defined stoichiometry.

The drug-to-antibody ratio, or the number of drugmolecules loadedper antibody, is also an important variable. Changing this ratio can affectthe properties of the ADC. Increasing the number of drugmolecules canpotentially lead to higher concentrations of the drug at the target sites.

However, greatly modifying the antibody molecule can lead to loss ofaffinity for the target, aggregation and precipitation of the antibody dueto lowered solubility, and faster clearance of the ADC (Firestone et al.,1996;Vater&Goldmacher, 2010). Currently,mostADCshavebetween2and 4 drug molecules per antibody molecule (Hamblett et al., 2004;McDonagh et al., 2006).

Hamblett et al. have studied the effects of loading 2, 4, and8 molecules of the cytotoxin MMAE on the anti-CD30 mAb, cAC10(Hamblett et al., 2004). Drug loading did not affect binding of the ADC tothe target in this case. The in vitro activity directly correlated with thenumberof drugmolecules.However, the in vivo activityof theADCswith4 and 8 drug molecules per antibody was found to be equivalent. Thiswas explained by the finding that clearance of the ADC was dependenton drug loading and that exposure is inversely correlated to the drugloading. Decreasing the number of drugmolecules from 8 to 4 increasedthe therapeutic index of the ADC by 2 fold. This suggests that optimizingthe drug to antibody ratio tomaintain favorable pharmacokineticswhilemaximizing drug payload may be a helpful tool in creating better ADCs.

Another feature unique to ADCs that can be manipulated toincrease the potency of these therapeutics is the bystander killingeffect. Some ADCs have been observed to effect killing of bystanderantigen-negative cells present in the vicinity of the antigen-positivetumor cells. Studies to elucidate the mechanism of bystander cellkilling by ADCs have indicated that metabolic products formed duringintracellular processing of the ADCs may play a role (Doronina et al.,2003; Erickson et al., 2006; Kovtun et al., 2006). Neutral cytotoxicmetabolites generated by metabolism of the ADCs in the antigen-positive cells can be released into the medium and can kill adjacentantigen-negative cells. Charged metabolites, however, may be pre-vented from diffusing across the membrane into the medium andcannot effect bystander killing. For example, maytansinoids linked tothe antibody through disulfide or thioether linkers undergo lysosomaldegradation to give lysine adducts of themaytansinoid agent attachedto the linker (Erickson et al., 2006). However, only the disulfide-linked metabolites were further processed to give a neutral lipophilicmetabolite that was more potent in in vitro cytotoxicity assays thanthe lysine adducts. The release of this lipophilic metabolite afterdegradation of the ADC in antigen-positive cells leads to killing ofproximal antigen-negative cells.

Bystander killing has also been observed as a result of diffusion ofthe free drug from the target cell after release of the drug from theADC (Okeley et al., 2010).

Manipulating the bystander killing effect through judicious useof linkers may be a valuable tool in targeting solid tumors withheterogeneous expression of the antigen.

Table 1List of Antibody Drug Conjugates in Clinical Trials.

ADC Target antigen Linker, cytotoxiccompound

Antibody Tumor type Developer Status

Gemtuzumab ozogamicin CD33(Siglec-3)

Hydrazone,calicheamicin

hP67/6, humanizedIgG4

AML Pfizer (Wyeth) Withdrawn

Inotuzumab ozogamicin(CMC-544)

CD22(Siglec-2)

Hydrazone,calicheamicin

G5/44, humanizedIgG4

B-cell lymphomas Pfizer (Wyeth) Phase I and phase I/II,phase III combination

Trastuzumab-DM1 HER2 (ErbB2) Thioether,maytansinoid

Trastuzumab, humanizedIgG1

Metastatic breast cancer Genentech/Hoffmann-La Roche

Phase I, II, and phaseIII

IMGN901 (huN901-DM1,BB10901)

CD56 (NCAM) Hindered disulfide,maytansinoid

huN901, humanized IgG1 Solid tumors, multiplemyeloma

Immunogen Phase I and phase I/II

IMGN242 (huC242-DM4) CanAg Hindered disulfide,maytansinoid

huC242, humanized IgG1 Solid tumors Immunogen Phase I

IMGN388 Integrin Hindered disulfide,maytansinoid

Anti-integrin, IgG1 Solid tumors Immunogen Phase I

Brentuximab vedotin(SGN-35)

CD30 Dipeptide, auristatin Anti-CD30 Lymphomas SeattleGenetics/Millennium

Phases I, II, and III

BIIB015 Cripto Hindered disulfide,maytansinoid

Anti-cripto IgG1 Anti-cripto+solid tumors Biogen Idec Phase I

CRO11-vcMMAE GlycoproteinNMB

Dipeptide, auristatin Anti-CRO11 Breast cancer, melanoma CuraGen Phase I/II, and phase II

SAR3419 CD19 Hindered disulfide,maytansinoid

Anti-CD19 IgG1 B-cell Non-Hodgkin'slymphoma

sanofi-aventis Phase I

BT062 Undisclosed Hindered disulfide,maytansinoid

Undisclosed Multiple myeloma Biotest Phase I and phase I/II

hLL1-DOX CD74 Hydrazone,doxorubicin

Milatuzumab Multiple myeloma Immunomedics Phase I/II

SGN-75 CD70 Dipeptide, auristatin Anti-CD70 Renal cell carcinoma,Non-Hodgkin's lymphoma

Seattle Genetics Phase I

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Further studies into the pharmaceutical properties of ADCs arecritical to improving our understanding of ADCs and for providingadditional guidance in designing better ADCs.

6. ADCs in clinical trials

One of the first ADCs to enter clinical trials was the drug doxorubicinlinked via a hydrazone linker to a chimeric mAb against the LeYtetrasaccharide antigen on human carcinomas (Mosure, Henderson,Klunk,&Knipe, 1997; Salehet al., 2000). The conjugatehad8molecules ofdoxorubicin bound per antibodymolecule and retained its affinity for theantigen (Firestone et al., 1996). In nonclinical studies, the ADC showedexcellent activity at well-tolerated doses, with specific tumor cures insomecancerxenograftmodels (Firestoneet al., 1996; Sjogrenet al., 1997).The concentration of doxorubicinwithin the tumorwas also shown to behigher in animals treated with the ADC than in animals treated with themaximum tolerated doses of unconjugated doxorubicin (Mosure et al.,1997). Despite such promising data, the drug failed to show clinicalefficacy (Saleh et al., 2000; Tolcher et al., 1999). Several factors may havecontributed to this failure, including low drug potency, instability of thelinker, and gastrointestinal toxicity caused by expression of the targetantigen in the gut (Tolcher et al., 1999).

The current generation of ADCs has benefited greatly from theexperience gained from earlier trials. As discussed, current ADC designuses more potent cytotoxins coupled to optimized mAbs via morestable linkers. Table 1 lists the ADCs currently in clinical development.

Apart from the ADCs in clinical trials, a great deal of research iscurrently ongoing on many other ADCs.

7. Challenges and future directions

As evidenced by the recent withdrawal of Mylotarg, the only ADC tohave been approved, there are still many challenges involved withdeveloping safe and efficacious ADC therapies. However, the dataavailable from clinical trials of Mylotarg and other ADCs can be used toguidebetter designofADCs. It hasbeendemonstrated thatMylotarg couldbe internalized through nonspecific endocytosis not mediated throughthe target antigen (CD33); this nonspecificity may have contributed tosome of the toxic effects seen with gemtuzumab ozogamicin (Jedema

et al., 2004). Additionally, high levels of CD33 in the peripheral bloodconsumeda large part of thedosedgemtuzumabozogamicin and reducedtarget cell killing by this ADC (Van Der Velden et al., 2004). Increasing thedose could have caused complications resulting from a correspondingincrease in toxicities.

Data from clinical trials of bivatuzumabmertansine (amertansine-linked CD44v6-targeting ADC) suggest that tumor selectivity of theantibody is more important than antigen density (Riechelmann et al.,2008). This ADCwas withdrawn from clinical trials after the death of apatient due to skin toxicity. CD44v6, which is abundantly andconsistently expressed in carcinomas, is also expressed on normalskin keratinocytes, and the toxicity was considered to be a result ofCD44v6-mediated uptake of mertansine into skin keratinocytes.

These failed ADCs provide valuable insight into what features of theADCaremost relevant for increasing the therapeutic indexof futureADCs.

Drug resistance resulting from the action of P-glycoprotein and effluxpumps is a serious limitation of cancer therapies. Although it was hopedthat ADCswould be able to circumvent this problem, the activity ofmanyADCs is poor in cells that express these efflux pumps (Kovtun et al., 2010;Matsui et al., 2002; Takeshita et al., 2009). Strategies to developADCs thatcan circumvent resistance mechanisms have become a priority in ADCresearch (Kovtun et al., 2010).

Although the concept behind the ADCs is relatively simple and hasbeen known for a long time, the execution has proved challenging. Thewealth of knowledge garnered over the past few years should beutilized. By careful selection of antigen targets, cytotoxic compounds,and linkers, it should be possible to minimize toxicities of conventionalchemotherapywhilemaximizing the selectivity andpotencyof theADC.Thus, ADCs represent an exciting new frontier in the treatment ofcancer, and further research on overcoming limitations can have hugepayoffs in terms of improving patient prognosis and quality of life.

Acknowledgments

The authorswould like to thankGanesh Iyer for critical reviewof themanuscript andhelpwithfigures and tables. The authorswould also liketo thank Julie Roy and Kathy Reckendorf for valuable assistance inediting and formatting this manuscript.

212 U. Iyer, V.J. Kadambi / Journal of Pharmacological and Toxicological Methods 64 (2011) 207–212

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