Journal of Pharmacological and Toxicological Methods 64 (2011) 207212

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Journal of Pharmacological and Toxicological Methods

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j p h a r m t ox

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

a r t i c l e

i n f o

a b s t r a c t
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 specicity of a monoclonal antibody (mAb) to the cytotoxicity of a smallmolecule drug and, therefore, are promising new therapies for cancer. ADCs are prodrugs that are inactive in circulation but exert their cytotoxicity upon binding to the target cancer cell. Earlier unsuccessful attempts to generate ADCs with therapeutic value have emphasized the important role each component plays in determining the efcacy and safety of the nal ADC. Scientic advances in engineering antibodies for maximum efcacy as anticancer agents, identication of highly cytotoxic molecules, and generation of linkers with increased stability in circulation have all contributed to the development of the many ADCs that are currently in clinical trials. This review discusses parameters that guide the selection of the components of an ADC to increase its therapeutic window, provides a brief look at ADCs currently in clinical trials, and discusses future challenges in this eld. 2011 Published by Elsevier Inc.

Article history: Received 7 January 2011 Accepted 28 July 2011 Keywords: Targeted therapy Cancer therapy Cytotoxin delivery Antibody toxin conjugates

Contents 1. 2. 3. 4. Introduction . . . . . . . . . . . . Cytotoxins . . . . . . . . . . . . Monoclonal antibodies . . . . . . . Linker strategies . . . . . . . . . . 4.1. Acid-labile hydrazone linkers 4.2. Disulde-based linkers . . . 4.3. Peptide-based linkers . . . . 4.4. Noncleavable linkers . . . . 5. Salient features of ADCs . . . . . . 6. ADCs in clinical trials . . . . . . . 7. Challenges and future directions . . Acknowledgments . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 208 209 209 209 209 209 210 210 211 211 211 212

1. Introduction Despite several years of intensive research into cancer therapies, the prognosis for the average cancer patient is bleak. According to American Cancer Society projections for 2010, approximately 1.5 million new cancers were expected to be diagnosed and more than 500,000 patients were expected to die of cancer, with N 90% of these cancers being solid tumors (ACS, 2010). Most anticancer drugs currently in use target rapidly dividing cells by disrupting steps in
Corresponding author at: Non-Clinical Development Sciences, 35, Landsdowne Street, Cambridge, MA, 02139, USA. E-mail address: kadambi@mpi.com (V.J. Kadambi). 1056-8719/$ see front matter 2011 Published by Elsevier Inc. doi:10.1016/j.vascn.2011.07.005

the cell cycle or by targeting the pathways that control normal cell growth and malignant transformation (Bollag et al., 1995; Chow et al., 1988; Downward, 2003; Herbst and Khuri, 2003). These drugs do not explicitly discriminate between cancer cells and normal cells. This nonspecicity is responsible for many of the toxicities associated with these drugs, which are often dosed at suboptimal levels. Coupled with the emergence of drug-resistant cancer cell populations, it is clear that there is an urgent need for therapies that are selective and efcacious, with minimal toxicities. Much of the ongoing research in this area is focused on nding a way to increase the specicity of the smallmolecule drugs for the cancer cells. Antibody therapies offer many advantages, the main one being the specicity of each antibody for its target antigen. Since their discovery in

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Fig. 1. Schematic of an ADC. The schematic for the structure of an ADC is shown along with examples of antibodies, linkers and drugs used to make some of the ADCs currently in clinical trials.

the early 1970s, mAbs have been investigated as therapeutic agents and have enjoyed considerable success in the last 2 decades, with over 20% of mAbs that enter clinical trials receiving regulatory approval compared to 11% for small-molecule therapies (Kohler & Milstein, 1975; Reichert, Rosensweig, Faden, & Dewitz, 2005). Antibody-based therapies have been the focus of much recent research, as evidenced by the growing list of such therapies approved in the last few years or currently in clinical trials (Reichert, 2010). Of these, a large percentage are against oncology targets because a number of antigens have been identied as being overexpressed in cancer cells compared to normal tissues (Panchal, 1998). However, mAbs also have their disadvantages, including poor cytotoxicity and poor penetration into tumors. Combining these two therapies cytotoxic drugs with mAbs would provide the best of both worlds, as it were the specicity of a mAb and the cytotoxicity of the small molecule. The concept behind ADCs is simple use the exquisite specicity of a mAb for its antigen to selectively deliver a highly toxic drug to the cancer cell, thus increasing 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 inside the tumor cell (Doronina et al., 2003; Sutherland et al., 2006). Conjugation of the drug to the antibody inactivates the drug so that it is not toxic while in circulation. The mAb then delivers its cytotoxic payload to the antigen-expressing cancer cell, where internalization of the ADC and cleavage of the linker occur, releasing the cytotoxin in its active form to kill the targeted cancer cells. ADCs offer a unique opportunity to combine the best features of both small-molecule drugs and antibodies to create a single therapy that is highly specic and cytotoxic. As such, they have been the subject of intense research focused on optimization to increase the therapeutic indices of ADCs. The principles that guide the design of drugs and mAbs for use in ADCs differ from those that guide design of stand-alone small-molecule drugs and mAb therapies.

2. Cytotoxins A number of different classes of cytotoxins are available for the treatment of cancer, including taxanes, vinca alkaloids, anthracyclines, and others. These drugs target rapidly proliferating cells by disrupting different aspects of cell proliferation, including DNA replication, repair, translation, and cell division (Bollag et al., 1995; Chow et al., 1988; Herbst and Khuri, 2003). Because cancer cells have a high rate of proliferation, they are more susceptible to the cytotoxic effects of these drugs. Cytotoxins are, therefore, not truly specic for cancer cells and will also affect any normal cells that have high

proliferation rates. As a result, these drugs are often associated with dose-limiting toxicities of the gastrointestinal tract and bone marrow (Markman, 2003). The elucidation of the molecular pathways involved in development of cancer has provided many additional targets for the design of new cytotoxic drugs (Downward, 2003; Okada and Mak, 2004). One way to increase the therapeutic efcacy of a cytotoxin is to increase its selectivity by conjugating the drug to a tumor-specic antibody resulting in an ADC. Early attempts to create ADCs by attaching clinically approved drugs to mAbs were met with limited success, and one factor was thought to be the insufcient potency of the conjugated drug (Chari et al., 1992; Hinman et al., 1993; Liu et al., 1996). The hypothesis is that the low accumulation of the antibody at disease sites leads to suboptimal concentrations of the cytotoxic drugs at these sites (Liu et al., 1996). Therefore, the use of highly cytotoxic agents should provide better efcacy because even small amounts of these drugs can have potent tumoricidal effects. This prompted research into the use of cytotoxins that were 100 to 1000 times more potent than conventional chemotherapeutic agents because the conjugation of the drug to the antibody inactivates the drug while 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. Microtubules are well-established targets for cancer therapies because they play a key role in mitosis (Jordan & Wilson, 1998). The maytansinoids and auristatins are classes of cytotoxins that disrupt microtubule dynamics by inhibiting microtubule assembly (Cassady, Chan, Floss, & Leistner, 2004; Wu & Senter, 2005). Calicheamicin, a naturally occurring antibiotic, and its derivatives bind to the minor groove of DNA 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 1970s generated a great deal of interest due to their high cytotoxicity (Cassady et al., 2004). They were shown to be 100- to 1000-fold more potent than other microtubule-targeting agents such as vincristine and vinblastine (Chari et al., 1992). Maytansinoids, which are derived from a natural product, were used in clinical trials in the 1970s but were abandoned due to their toxicity prole and low therapeutic index (Cassady et al., 2004). Their high cytotoxicity, however, makes them ideal candidates for conjugation to antibodies, as targeted delivery to the cancer cells should greatly increase their therapeutic window. Several ADCs with maytansinoids as the cytototoxic payload are 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, are shown to be 50- to 200-fold more potent than the vinca alkaloids and doxorubicin, respectively (Doronina et al., 2003). Dolastatin and its synthetic derivatives were used in several clinical trials but failed to show activity against various cancers (Kindler et al., 2005; Perez et al., 2005). Auristatin conjugates currently in clinical trials include brentuximab vedotin (monomethyl auristatin E conjugated to anti-CD30 antibody) and CR011-vcMMAE (monomethyl auristatin E conjugated to anti-glycoprotein NMB antibody). Currently two auristatins, monomethyl auristatin E and monomethyl auristatin F, are being investigated as part of an ADC. Calicheamicin is another highly potent cytotoxin that has demonstrated cell killing at concentrations far below those of other standard chemotherapeutics (Hinman et al., 1993; Lee et al., 1989). Mylotarg (gemtuzumab ozogamicin), the rst ADC to win FDA approval, uses a calicheamicin derivative as the cytotoxin. Another calicheamicin conjugate in clinical trials is inotuzumab ozogamicin, in which calicheamicin is conjugated to inotuzumab (Dijoseph et al., 2006). Other drugs that are being investigated as components of ADCs include doxorubicin (a topoisomerase II inhibitor), SN-38 (a topoisomerase I inhibitor), and derivatives of paclitaxel (a microtubule inhibitor) (Govindan & Goldenberg, 2010).

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Apart from high potency, other important characteristics of a suitable cytotoxin for ADCs are low molecular weight and immunogenicity, availability of reactive functional groups that can be used in conjugation, and sufcient stability and solubility in aqueous solutions (Singh & Erickson, 2009).

3. Monoclonal antibodies Although mAbs were discovered as early as 1975, it is only in the last 2 decades that they have demonstrated success in the clinical setting, with Rituxan being the rst mAb approved in 1997 (Kohler & Milstein, 1975). Early attempts to use murine mAbs in the clinical setting were unsuccessful because these antibodies were highly immunogenic, were not sufciently cytotoxic because they could not elicit potent antitumor responses, and had very short half-lives of less than 20 h (Christiansen & Rajasekaran, 2004). The development of techniques to generate chimeric or humanized antibodies has helped to overcome these limitations by reducing the immunotoxicity and increasing the halflives of the mAbs. Most of the antibodies currently in clinical testing are mouse mAbs that have been humanized (Reichert et al., 2005). It is important to note that, unlike cytotoxins, antibodies by themselves do not kill the target cell. Instead, by attaching to the target on the surface of the cancer cell, they induce cell death by two possible mechanisms. Binding of the antibody to the antigen on the surface of the cancer cell can elicit an immune response that leads to antibodydependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (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 to the cell can trigger a signaling cascade within the cell itself that results in apoptosis (Selenko et al., 2002). The type of antibody used in anticancer drugs is usually IgG1 because antibodies of this class can trigger both ADCC and CDC (Gelderman et al., 2004). Efforts have been made to modify the antibodies to be more effective at inducing immune responses and to increase their cytotoxic potential (Carter, 2006). Improved techniques for generating antibodies against highly validated targets have given rise to blockbuster mAb drugs, such as rituximab (target-CD20), trastuzumab (target-HER2), and cetuximab (target-epidermal growth factor receptor), whose sales amount to billions of dollars in revenue (Scolnik, 2009). However, despite these advances, there are still limitations to mAb therapeutics, including poor penetration into tumors, with only a small percentage of the administered mAb localizing within the tumor, and slow extravasation of the drug from the blood supply (Christiansen & Rajasekaran, 2004; Wu & Senter, 2005). Additionally, antibodies are expensive to generate, which increases the costs of the marketed drugs (Farid, 2007; Scott, 2005). Monoclonal antibody therapies are often not curative and may need to be combined with chemotherapy (Weiner, Surana, & Wang, 2010). As a result, there has been tremendous interest in nding a way to increase the therapeutic effectiveness of the mAb through attachment to a toxin, drugs, or radionuclides. Apart from the favorable properties required for a mAb to act as a stand-alone therapy, some additional factors must be considered while selecting antibodies for ADCs. The antibody should have sites amenable to conjugation to the drug such that binding to the antigen is not compromised. To facilitate attachment of the drug to the antibody, IgGs have also been engineered to have unique sites for conjugation that allow for generation of more homogenous ADCs (Junutula et al., 2008). Although it is assumed that the cytotoxin will play the major role in cell killing, additional cytotoxicity resulting from complement-dependent cytotoxicity and activation of immune effector cells by the antibody may be desirable, as long as it does not cause toxicity. For example, in the case of 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-mediated cytotoxicity (Ryan et al., 2010). Selection of the target antigen is also an important criterion in terms of both safety and effectiveness of the ADC. One obvious criterion is that the antigen must be present at the surface of the cell. A high level of expression of the target antigen in cancer cells, with little or no expression in normal cells, is clearly another primary requisite. However, a clear correlation between levels of antigen expression and sensitivity to the therapeutic mAb has not been shown (Walter, Raden, Kamikura, Cooper, & Bernstein, 2005). This suggests that other factors may also be critical in determining the efcacy of the ADC against a target antigen. Efcient internalization of the antigen is an important factor to consider, along with antigen levels, because the mechanism of action of ADCs is dependent on internalization after binding of the ADC (Sutherland et al., 2006; Walter et al., 2005). The presence of a cytotoxic payload on the ADC means that it is important to pick a target that is not shed into the blood by cleavage of the antigen from the cancer cells (Manshouri et al., 2003; Van Der Velden et al., 2004). The binding afnity of the antibody for the antigen and the number of antigen molecules on the 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 been the generation of suitable linkers for conjugating the antibody and the drug (Kemshead & Hopkins, 1993). The linker should be extremely stable in circulation since release of the cytotoxic payload before reaching the target would lead to nonspecic cell killing and associated toxicities. However, upon reaching the target cells, the linker must also be able to efciently release the drug in its active form to allow the drug to effect cell killing. Several strategies have been employed to produce linkers that satisfy both of these criteria. The binding of the antibody to the antigen usually leads to internalization of the conjugate by receptor-mediated endocytosis (Walter et al., 2005). Most of the linkers take advantage of the fact that the conjugate enters the cell through the acidic lysosomal compartment that is also rich in proteolytic enzymes, while some use the highly reducing environment of the cell itself. There are currently four different classes of linkers in use that broadly fall under 2 categories: cleavable and noncleavable linkers. 4.1. Acid-labile hydrazone linkers These were the rst linkers to be used and interestingly, this type of linker is also used in gemtuzumab ozogamicin, the only ADC to receive FDA approval. Although stable at the neutral pH of the blood, they undergo hydrolysis in the acidic environment of the cellular compartments (Ducry & Stump, 2010). These linkers have been associated with non-specic release of the drug in clinical studies (Ducry & Stump, 2010; Senter, 2009). 4.2. Disulde-based linkers The disulde bonds in these linkers are selectively cleaved in the cytosol because of the high intracellular concentration of glutathione. Previous work with plant protein toxins has shown that the stability of the disulde linkers can be increased by steric hindrance (Thorpe et al., 1987). 4.3. Peptide-based linkers Peptide-based linkers, as the name suggests, link the drug to the antibody by means of a peptide bond (Ducry & Stump, 2010). Release of the drug from the mAb occurs specically due to the action of lysosomal proteases. Thus, unlike the chemically labile linkers discussed thus far, peptide linkers combine greater systemic stability with rapid enzymatic release of the drug in the target cell.

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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 then targets either the DNA or the microtubules to kill the cell.

4.4. Noncleavable linkers This class of thioether-containing linkers is noncleavable and release of the drug is postulated to occur through intracellular proteolytic degradation (Erickson et al., 2006; Lewis Phillips et al., 2008). One advantage of these linkers is their greater stability in circulation compared to the cleavable linkers; this can potentially improve the therapeutic index of the drug because it may be better tolerated (Lewis Phillips et al., 2008; Singh & Erickson, 2009; Polson et al., 2010). An ADC with trastuzumab linked to the maytansinoid, DM1, through a thioether linker has been shown to be better tolerated with a more favorable pharmacokinetic and safety prole compared to the disulde-linked ADC (Lewis Phillips et al., 2008). A disadvantage may be that poor internalization of the ADC may have a more negative impact on an ADC with a noncleavable linker compared to one with a cleavable linker because, in the latter, release of the drug by cleavage of the linker within the intracellular space may allow the 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 the ADC to the target antigen. As we have seen, careful selection of the different components of the ADC is critical to successful design of ADCs that could be used as cancer therapeutics. Apart from optimizing the component parts, efforts to build better ADCs have led to much research into all the characteristics of the ADC that may affect its therapeutic index, including conjugation of the antibody to the linker and the drug, drugantibody ratios, and the mechanism by which ADCs effect cell killing. The assembly of the ADC from the component parts is also an important step in determining the therapeutic potential of the ADC. Conjugation of the antibody to the drug should not alter the integrity of the antibody, the binding of the antibody to the antigen, or the biological activity of the drug upon reaching the target cell. The pharmacodynamic properties of the ADC must resemble that of the mAb while in circulation. Conjugation of the antibody to the drug occurs at cysteines or lysines on the antibody; the products of such conjugations are heterogeneous, with variations in the number of molecules of drug per antibody and in the sites of attachment (Junutula et al., 2008). To address this variation, efforts have been made to engineer reactive cysteine residues at specic sites in antibodies to allow drugs to be conjugated with dened stoichiometry. The drug-to-antibody ratio, or the number of drug molecules loaded per antibody, is also an important variable. Changing this ratio can affect the properties of the ADC. Increasing the number of drug molecules can potentially lead to higher concentrations of the drug at the target sites.

However, greatly modifying the antibody molecule can lead to loss of afnity for the target, aggregation and precipitation of the antibody due to lowered solubility, and faster clearance of the ADC (Firestone et al., 1996; Vater & Goldmacher, 2010). Currently, most ADCs have between 2 and 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, and 8 molecules of the cytotoxin MMAE on the anti-CD30 mAb, cAC10 (Hamblett et al., 2004). Drug loading did not affect binding of the ADC to the target in this case. The in vitro activity directly correlated with the number of drug molecules. However, the in vivo activity of the ADCs with 4 and 8 drug molecules per antibody was found to be equivalent. This was explained by the nding that clearance of the ADC was dependent on drug loading and that exposure is inversely correlated to the drug loading. Decreasing the number of drug molecules from 8 to 4 increased the therapeutic index of the ADC by 2 fold. This suggests that optimizing the drug to antibody ratio to maintain favorable pharmacokinetics while maximizing drug payload may be a helpful tool in creating better ADCs. Another feature unique to ADCs that can be manipulated to increase the potency of these therapeutics is the bystander killing effect. Some ADCs have been observed to effect killing of bystander antigen-negative cells present in the vicinity of the antigen-positive tumor cells. Studies to elucidate the mechanism of bystander cell killing by ADCs have indicated that metabolic products formed during intracellular processing of the ADCs may play a role (Doronina et al., 2003; Erickson et al., 2006; Kovtun et al., 2006). Neutral cytotoxic metabolites generated by metabolism of the ADCs in the antigenpositive cells can be released into the medium and can kill adjacent antigen-negative cells. Charged metabolites, however, may be prevented from diffusing across the membrane into the medium and cannot effect bystander killing. For example, maytansinoids linked to the antibody through disulde or thioether linkers undergo lysosomal degradation to give lysine adducts of the maytansinoid agent attached to the linker (Erickson et al., 2006). However, only the disuldelinked metabolites were further processed to give a neutral lipophilic metabolite that was more potent in in vitro cytotoxicity assays than the lysine adducts. The release of this lipophilic metabolite after degradation of the ADC in antigen-positive cells leads to killing of proximal antigen-negative cells. Bystander killing has also been observed as a result of diffusion of the free drug from the target cell after release of the drug from the ADC (Okeley et al., 2010). Manipulating the bystander killing effect through judicious use of linkers may be a valuable tool in targeting solid tumors with heterogeneous expression of the antigen.

U. Iyer, V.J. Kadambi / Journal of Pharmacological and Toxicological Methods 64 (2011) 207212 Table 1 List of Antibody Drug Conjugates in Clinical Trials. ADC Gemtuzumab ozogamicin Inotuzumab ozogamicin (CMC-544) Trastuzumab-DM1 Target antigen CD33 (Siglec-3) CD22 (Siglec-2) HER2 (ErbB2) Linker, cytotoxic compound Hydrazone, calicheamicin Hydrazone, calicheamicin Thioether, maytansinoid Hindered disulde, maytansinoid Hindered disulde, maytansinoid Hindered disulde, maytansinoid Dipeptide, auristatin Hindered disulde, maytansinoid Dipeptide, auristatin Hindered disulde, maytansinoid Hindered disulde, maytansinoid Hydrazone, doxorubicin Dipeptide, auristatin Antibody hP67/6, humanized IgG4 G5/44, humanized IgG4 Trastuzumab, humanized IgG1 huN901, humanized IgG1 Tumor type AML B-cell lymphomas Metastatic breast cancer Developer Pzer (Wyeth) Pzer (Wyeth) Genentech/HoffmannLa Roche Immunogen Immunogen Immunogen Seattle Genetics/Millennium Biogen Idec CuraGen sano-aventis Biotest Immunomedics Status Withdrawn

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IMGN901 (huN901-DM1, CD56 (NCAM) BB10901) IMGN242 (huC242-DM4) CanAg IMGN388 Brentuximab vedotin (SGN-35) BIIB015 CRO11-vcMMAE SAR3419 BT062 hLL1-DOX SGN-75 Integrin CD30 Cripto Glycoprotein NMB CD19 Undisclosed CD74 CD70

Solid tumors, multiple myeloma huC242, humanized IgG1 Solid tumors Anti-integrin, IgG1 Anti-CD30 Anti-cripto IgG1 Anti-CRO11 Anti-CD19 IgG1 Undisclosed Milatuzumab Anti-CD70 Solid tumors Lymphomas Anti-cripto + solid tumors Breast cancer, melanoma B-cell Non-Hodgkin's lymphoma Multiple myeloma Multiple myeloma

Phase I and phase I/II, phase III combination Phase I, II, and phase III Phase I and phase I/II Phase I Phase I Phases I, II, and III Phase I Phase I/II, and phase II Phase I Phase I and phase I/II Phase I/II Phase I

Renal cell carcinoma, Seattle Genetics Non-Hodgkin's lymphoma

Further studies into the pharmaceutical properties of ADCs are critical to improving our understanding of ADCs and for providing additional guidance in designing better ADCs. 6. ADCs in clinical trials One of the rst ADCs to enter clinical trials was the drug doxorubicin linked via a hydrazone linker to a chimeric mAb against the LeY tetrasaccharide antigen on human carcinomas (Mosure, Henderson, Klunk, & Knipe, 1997; Saleh et al., 2000). The conjugate had 8 molecules of doxorubicin bound per antibody molecule and retained its afnity for the antigen (Firestone et al., 1996). In nonclinical studies, the ADC showed excellent activity at well-tolerated doses, with specic tumor cures in some cancer xenograft models (Firestone et al., 1996; Sjogren et al., 1997). The concentration of doxorubicin within the tumor was also shown to be higher in animals treated with the ADC than in animals treated with the maximum tolerated doses of unconjugated doxorubicin (Mosure et al., 1997). Despite such promising data, the drug failed to show clinical efcacy (Saleh et al., 2000; Tolcher et al., 1999). Several factors may have contributed to this failure, including low drug potency, instability of the linker, and gastrointestinal toxicity caused by expression of the target antigen in the gut (Tolcher et al., 1999). The current generation of ADCs has beneted greatly from the experience gained from earlier trials. As discussed, current ADC design uses more potent cytotoxins coupled to optimized mAbs via more stable linkers. Table 1 lists the ADCs currently in clinical development. Apart from the ADCs in clinical trials, a great deal of research is currently ongoing on many other ADCs. 7. Challenges and future directions As evidenced by the recent withdrawal of Mylotarg, the only ADC to have been approved, there are still many challenges involved with developing safe and efcacious ADC therapies. However, the data available from clinical trials of Mylotarg and other ADCs can be used to guide better design of ADCs. It has been demonstrated that Mylotarg could be internalized through nonspecic endocytosis not mediated through the target antigen (CD33); this nonspecicity may have contributed to some of the toxic effects seen with gemtuzumab ozogamicin (Jedema

et al., 2004). Additionally, high levels of CD33 in the peripheral blood consumed a large part of the dosed gemtuzumab ozogamicin and reduced target cell killing by this ADC (Van Der Velden et al., 2004). Increasing the dose could have caused complications resulting from a corresponding increase in toxicities. Data from clinical trials of bivatuzumab mertansine (a mertansinelinked CD44v6-targeting ADC) suggest that tumor selectivity of the antibody is more important than antigen density (Riechelmann et al., 2008). This ADC was withdrawn from clinical trials after the death of a patient due to skin toxicity. CD44v6, which is abundantly and consistently expressed in carcinomas, is also expressed on normal skin keratinocytes, and the toxicity was considered to be a result of CD44v6-mediated uptake of mertansine into skin keratinocytes. These failed ADCs provide valuable insight into what features of the ADC are most relevant for increasing the therapeutic index of future ADCs. Drug resistance resulting from the action of P-glycoprotein and efux pumps is a serious limitation of cancer therapies. Although it was hoped that ADCs would be able to circumvent this problem, the activity of many ADCs is poor in cells that express these efux pumps (Kovtun et al., 2010; Matsui et al., 2002; Takeshita et al., 2009). Strategies to develop ADCs that can circumvent resistance mechanisms have become a priority in ADC research (Kovtun et al., 2010). Although the concept behind the ADCs is relatively simple and has been known for a long time, the execution has proved challenging. The wealth of knowledge garnered over the past few years should be utilized. By careful selection of antigen targets, cytotoxic compounds, and linkers, it should be possible to minimize toxicities of conventional chemotherapy while maximizing the selectivity and potency of the ADC. Thus, ADCs represent an exciting new frontier in the treatment of cancer, and further research on overcoming limitations can have huge payoffs in terms of improving patient prognosis and quality of life.

Acknowledgments The authors would like to thank Ganesh Iyer for critical review of the manuscript and help with gures and tables. The authors would also like to thank Julie Roy and Kathy Reckendorf for valuable assistance in editing and formatting this manuscript.

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U. Iyer, V.J. Kadambi / Journal of Pharmacological and Toxicological Methods 64 (2011) 207212 Manshouri, T., Do, K. -A., Wang, X., Giles, F. J., O'brien, S. M., Saffer, H., et al. (2003). Circulating CD20 is detectable in the plasma of patients with chronic lymphocytic leukemia and is of prognostic signicance. Blood, 101, 25072513. Markman, M. (2003). Managing taxane toxicities. Supportive Care in Cancer, 11, 144147. Matsui, H. T. A., Naito, K., Shinjo, K., Shigeno, K., Maekawa, M., Yamakawa, Y., et al. (2002). Reduced effect of gemtuzumab ozogamicin (CMA-676) on P-glycoprotein and/or CD34-positive leukemia cells and its restoration by multidrug resistance modiers. Leukemia, 16, 813819. McDonagh, C. F., Turcott, E., Westendorf, L., Webster, J. B., Alley, S. C., & Kim, K. (2006). Engineered antibody-drug conjugates with dened sites and stoichiometries of drug attachment. Protein Engineering Design & Selection, 19, 299307. Mosure, K. W., Henderson, A. J., Klunk, L. J., & Knipe, J. O. (1997). Disposition of conjugatebound and free doxorubicin in tumor-bearing mice following administration of a BR96doxorubicin immunoconjugate (BMS 182248). Cancer Chemotherapy and Pharmacology, 40, 251258. Nicolaou, K. C., & Smith, A. L. (1992). Molecular design, chemical synthesis, and biological action of enediynes. Accounts of Chemical Research, 25, 497503. Okada, H., & Mak, T. W. (2004). Pathways of apoptotic and non-apoptotic death in tumour cells. Nature Reviews. Cancer, 4, 592603. Okeley, N. M., Miyamoto, J. B., Zhang, X., Sanderson, R. J., Benjamin, D. R., Sievers, E. L., et al. (2010). Intracellular activation of SGN-35, a potent anti-CD30 antibody-drug conjugate. Clinical Cancer Research, 16, 888897. Panchal, R. G. (1998). Novel therapeutic strategies to selectively kill cancer cells. Biochemical Pharmacology, 55, 247252. Perez, E. A., Hillman, David W., Fishkin, Paul A., Krook, James E., Tan, Winston W., Kuriakose, Phillip A., et al. (2005). Phase II trial of dolastatin-10 in patients with advanced breast cancer. Investigational New Drugs, 23, 257261. Polson, A. G., Williams, M., Gray, A. M., Fuji, R. N., Poon, K. A., & McBride, J. (2010). AntiCD22-MCC-DM1: An antibody-drug conjugate with a stable linker for the treatment of non-Hodgkin's lymphoma. Leukemia, 24, 15661573. Reichert, J. M. (2010). Antibody-based therapeutics to watch in 2011. MAbs, 3, 7698. Reichert, J. M., Rosensweig, C. J., Faden, L. B., & Dewitz, M. C. (2005). Monoclonal antibody successes in the clinic. Nature Biotechonology, 23, 10731078. Riechelmann, H., Sauter, A., Golze, W., Hanft, G., Schroen, C., Hoermann, K., et al. (2008). Phase I trial with the CD44v6-targeting immunoconjugate bivatuzumab mertansine in head and neck squamous cell carcinoma. Oral Oncology, 44, 823829. Ryan, M. C., Kostner, H., Gordon, K. A., Duniho, S., Sutherland, M. K., Yu, C., et al. (2010). Targeting pancreatic and ovarian carcinomas using the auristatin-based anti-CD70 antibody-drug conjugate SGN-75. British Journal of Cancer, 103, 676684. Saleh, M. N., Sugarman, S., Murray, J., Ostroff, J. B., Healey, D., Jones, D., et al. (2000). Phase I trial of the anti-Lewis Y drug immunoconjugate BR96-doxorubicin in patients with Lewis Y Expressing epithelial tumors. Journal of Clinical Oncology, 18, 22822292. Scolnik, P. A. (2009). mAbs: A business perspective. MAbs, 2, 179184. Scott, C. T. (2005). The problem with potency. Nature Biotechonology, 23, 10371039. Selenko, N., Majdic, O., Jger, U., Sillaber, C., Stckl, J., & Knapp, W. (2002). Crosspriming of cytotoxic t cells promoted by apoptosis-inducing tumor cell reactive antibodies. Journal of Clinical Immunology, 22, 124130. Senter, P. D. (2009). Potent antibody drug conjugates for cancer therapy. Current Opinion in Chemical Biology, 13, 235244. Singh, R., & Erickson, H. K. (2009). Antibodycytotoxic agent conjugates: preparation and characterization. Methods in Molecular Biology, 525. (pp. 123) : SpringerLink. Sjogren, H. O., Isaksson, M., Willner, D., Hellstrom, I., Hellstram, K. E., & Trail, P. A. (1997). Antitumor activity of carcinoma-reactive BR96-doxorubicin conjugate against human carcinomas in athymic mice and rats and syngeneic rat carcinomas in immunocompetent rats. Cancer Research, 57, 45304536. Sutherland, M. S., Sanderson, R. J., Gordon, K. A., Andreyka, J., Cerveny, C. G., Yu, C., et al. (2006). Lysosomal trafcking and cysteine protease metabolism confer targetspecic cytotoxicity by peptide-linked anti-CD30-auristatin conjugates. Journal of Biological Chemistry, 281, 1054010547. Takeshita, A., Shinjo, K., Yamakage, N., Ono, T., Hirano, I., Matsui, H., et al. (2009). CMC544 (inotuzumab ozogamicin) shows less effect on multidrug resistant cells: analyses in cell lines and cells from patients with B-cell chronic lymphocytic leukaemia and lymphoma. British Journal of Haematology, 146, 3443. Thorpe, P. E., Wallace, P. M., Knowles, P. P., Relf, M. G., Brown, A. N. F., Watson, et al. (1987). New coupling agents for the synthesis of immunotoxins containing a hindered disulde bond with improved stability in vivo. Cancer Research, 47, 59245931. Tolcher, A. W., Sugarman, S., Gelmon, K. A., Cohen, R., Saleh, M., Isaacs, C., et al. (1999). Randomized phase II study of BR96-doxorubicin conjugate in patients with metastatic breast cancer. Journal of Clinical Oncology, 17, 478. Van Der Velden, V. H. J., Boeckx, N., Jedema, I., Te Marvelde, J. G., Hoogeveen, P. G., Boogaerts, M., et al. (2004). High CD33-antigen loads in peripheral blood limit the efcacy of gemtuzumab ozogamicin (Mylotarg[reg]) treatment in acute myeloid leukemia patients. Leukemia, 18, 983988. Van Meerten, T., Van Rijn, R. S., Hol, S., Hagenbeek, A., & Ebeling, S. B. (2006). Complement-induced cell death by rituximab depends on CD20 expression level and acts complementary to antibody-dependent cellular cytotoxicity. Clinical Cancer Research, 12, 40274035. Vater, C. A., & Goldmacher, V. S. (2010). Antibody-cytotoxic compound conjugates for oncology. Macromolecular Anticancer Therapeutics (pp. 331369). Humana Press. Walter, R. B., Raden, B. W., Kamikura, D. M., Cooper, J. A., & Bernstein, I. D. (2005). Inuence of CD33 expression levels and ITIM-dependent internalization on gemtuzumab ozogamicin-induced cytotoxicity. Blood, 105, 12951302. Weiner, L. M., Surana, R., & Wang, S. (2010). Monoclonal antibodies: Versatile platforms for cancer immunotherapy. Nature Reviews Immunology, 10, 317327. Wu, A. M., & Senter, P. D. (2005). Arming antibodies: Prospects and challenges for immunoconjugates. Nature Biotechonology, 23, 11371146.

References
A. C. S. (2010). Cancer facts & gures 2010. American Cancer Society. Bollag, D. M., McQueney, P. A., Zhu, J., Hensens, O., Koupal, L., Liesch, J., et al. (1995). Epothilones, a new class of microtubule-stabilizing agents with a taxol-like mechanism of action. Cancer Research, 55, 23252333. Carter, P. J. (2006). Potent antibody therapeutics by design. Nature Reviews Immunology, 6, 343357. Cassady, J. M., Chan, K. K., Floss, H. G., & Leistner, E. (2004). Recent developments in the maytansinoid antitumor agents. Chemical and Pharmaceutical Bulletin, 52, 126. Chari, R. V. J., Martell, B. A., Gross, J. L., Cook, S. B., Shah, S. A., Blattler, W. A., et al. (1992). Immunoconjugates containing novel maytansinoids: promising anticancer drugs. Cancer Research, 52, 127131. Chow, K. C., Macdonald, T. L., & Ross, W. E. (1988). DNA binding by epipodophyllotoxins and N-acyl anthracyclines: Implications for mechanism of topoisomerase II inhibition. Molecular Pharmacology, 34, 467473. Christiansen, J., & Rajasekaran, A. K. (2004). Biological impediments to monoclonal antibody-based cancer immunotherapy. Molecular Cancer Therapeutics, 3, 14931501. Dijoseph, J. F., Dougher, M. M., Kalyandrug, L. B., Armellino, D. C., Boghaert, E. R., Hamann, P. R., et al. (2006). Antitumor efcacy of a combination of CMC-544 (inotuzumab ozogamicin), a CD22-targeted cytotoxic immunoconjugate of calicheamicin, and rituximab against Non-Hodgkin's B-Cell lymphoma. Clinical Cancer Research, 12, 242249. Doronina, S. O., Toki, B. E., Torgov, M. Y., Mendelsohn, B. A., Cerveny, C. G., Chace, D. F., et al. (2003). Development of potent monoclonal antibody auristatin conjugates for cancer therapy. Nature Biotechonology, 21, 778784. Downward, J. (2003). Targeting RAS signalling pathways in cancer therapy. Nature Reviews Cancer, 3, 1122. Ducry, L., & Stump, B. (2010). Antibody-drug conjugates: Linking cytotoxic payloads to monoclonal antibodies. Bioconjugate Chemistry, 21, 513. Erickson, H. K., Park, P. U., Widdison, W. C., Kovtun, Y. V., Garrett, L. M., Hoffman, K., et al. (2006). Antibody-maytansinoid conjugates are activated in targeted cancer cells by lysosomal degradation and linker-dependent intracellular processing. Cancer Research, 66, 44264433. Farid, S. S. (2007). Process economics of industrial monoclonal antibody manufacture. Journal of Chromatography B, 848, 818. Firestone, R. A., Willner, D., Hofstead, S. J., King, H. D., Kaneko, T., Braslawsky, G. R., et al. (1996). Synthesis and antitumor activity of the immunoconjugate BR96-Dox. Journal of Controlled Release, 39, 251259. Gelderman, K. A., Tomlinson, S., Ross, G. D., & Gorter, A. (2004). Complement function in mAb-mediated cancer immunotherapy. Trends in Immunology, 25, 158164. Govindan, S. V., & Goldenberg, D. M. (2010). Immunoconjugate anticancer therapeutics. Macromolecular Anticancer Therapeutics (pp. 371391). Humana Press. Hamblett, K. J., Senter, P. D., Chace, D. F., Sun, M. M. C., Lenox, J., Cerveny, C. G., et al. (2004). Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clinical Cancer Research, 10, 70637070. Herbst, R. S., & Khuri, F. R. (2003). Mode of action of docetaxel a basis for combination with novel anticancer agents. Cancer Treatment Reviews, 29, 407415. Hinman, L. M., Hamann, P. R., Wallace, R., Menendez, A. T., Durr, F. E., & Upeslacis, J. (1993). Preparation and characterization of monoclonal antibody conjugates of the calicheamicins: A novel and potent family of antitumor antibiotics. Cancer Research, 53, 33363342. Jedema, I., Barge, R. M. Y., Van Der Velden, V. H. J., Nijmeijer, B. A., Van Dongen, J. J. M., Willemze, R., et al. (2004). Internalization and cell cycle-dependent killing of leukemic cells by gemtuzumab ozogamicin: rationale for efcacy in CD33-negative malignancies with endocytic capacity. Leukemia, 18, 316325. Jordan, M. A., & Wilson, L. (1998). Microtubules and actin laments: dynamic targets for cancer chemotherapy. Current Opinion in Cell Biology, 10, 123130. Junutula, J. R., Raab, H., Clark, S., Bhakta, S., Leipold, D. D., Weir, S., et al. (2008). Sitespecic conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nature Biotechonology, 26, 925932. Kemshead, J. T., & Hopkins, K. (1993). Uses and limitations of monoclonal antibodies (MoAbs) in the treatment of malignant disease: A review. Journal of the Royal Society of Medicine, 86, 219224. Kindler, H. L., Tothy, Peter K., Wolff, Robert, Mccormack, Richard A., Abbruzzese, James L., Mani, Sridhar, et al. (2005). Phase II trials of dolastatin-10 in advanced pancreaticobiliary cancers. Investigational New Drugs, 23, 489493. Kohler, G., & Milstein, C. (1975). Continuous cultures of fused cells secreting antibody of predened specicity. Nature, 256, 495497. Kovtun, Y. V., Audette, C. A., Mayo, M. F., Jones, G. E., Doherty, H., Maloney, E. K., et al. (2010). Antibody-maytansinoid conjugates designed to bypass multidrug resistance. Cancer Research, 70, 25282537. Kovtun, Y. V., Audette, C. A., Ye, Y., Xie, H., Ruberti, M. F., & Phinney, S. J. (2006). Antibody-drug conjugates designed to eradicate tumors with homogeneous and heterogeneous expression of the target antigen. Cancer Research, 66, 32143221. Lee, M. D., Manning, J. K., Williams, D. R., Kuck, N. A., Testa, R. T., & Borders, D. B. (1989). Calicheamicins, a novel family of antitumor antibiotics. Journal of Antibiotics, 42, 10701087. Lewis Phillips, G. D., Li, G., Dugger, D. L., Crocker, L. M., Parsons, K. L., Mai, E., et al. (2008). Targeting HER2-Positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate. Cancer Research, 68, 92809290. Liu, C., Tadayoni, B. M., Bourret, L. A., Mattocks, K. M., Derr, S. M., Widdison, W. C., et al. (1996). Eradication of large colon tumor xenografts by targeted delivery of maytansinoids. Proceedings of the National Academy of Sciences of the United States of America, 93, 86188623.