molecules

Review Tubulin Inhibitor-Based Antibody-Drug Conjugates for Cancer Therapy

Hao Chen †, Zongtao Lin † ID , Kinsie E. Arnst, Duane D. Miller ID and Wei Li *

Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, 881 Madison Avenue, Room 561, Memphis, TN 38163, USA; [email protected] (H.C.); [email protected] (Z.L.); [email protected] (K.E.A.); [email protected] (D.D.M.) * Correspondence: [email protected]; Tel.: +1-901-448-7532; Fax: +1-901-448-6828 † These authors contributed equally to this work.

Received: 12 July 2017; Accepted: 29 July 2017; Published: 1 August 2017

Abstract: Antibody-drug conjugates (ADCs) are a class of highly potent biopharmaceutical drugs generated by conjugating cytotoxic drugs with specific monoclonal antibodies through appropriate linkers. Specific antibodies used to guide potent warheads to tumor tissues can effectively reduce undesired side effects of the cytotoxic drugs. An in-depth understanding of antibodies, linkers, conjugation strategies, cytotoxic drugs, and their molecular targets has led to the successful development of several approved ADCs. These ADCs are powerful therapeutics for cancer treatment, enabling wider therapeutic windows, improved pharmacokinetic/pharmacodynamic properties, and enhanced efficacy. Since tubulin inhibitors are one of the most successful cytotoxic drugs in the ADC armamentarium, this review focuses on the progress in tubulin inhibitor-based ADCs, as well as lessons learned from the unsuccessful ADCs containing tubulin inhibitors. This review should be helpful to facilitate future development of new generations of tubulin inhibitor-based ADCs for cancer therapy.

Keywords: antibody-drug conjugates; cytotoxic payloads; linker; monoclonal antibody; site-specific conjugation; tubulin inhibitors

1. Introduction Cancer is one of the leading causes of death in the United States, accounting for about half a million deaths every year [1]. In the early days of cancer treatment, conventional chemotherapy dominated the field with an improvement in cancer-related mortality. Early chemotherapeutic agents included, but were not limited to, DNA-damaging agents (such as nitrogen mustard, anthracyclines, and cyclophosphamide), tubulin inhibitors (such as the vinca alkaloids vinblastine and vincristine, and taxane diterpenes like taxol), and antifolate agents (such as methotrexate, pemetrexed, proguanil, pyrimethamine, and trimethoprim). A significant proportion of cancer patients suffer from acquired resistance or relapse after long-term chemotherapy treatment. Moreover, these drugs usually have nonspecific toxicity which kills not only tumor cells, but also damages normal tissues and causes serious side effects that often limit their efficacy [2]. In contrast to chemotherapy, targeted therapy is considered a more efficacious and safer cancer treatment and has gained increasing interest in recent years. Targeted therapy blocks the rapidly dividing cancer cells by interfering with the specific targeted molecules needed for tumor growth [3]. The main categories of current targeted cancer therapy are small molecules (e.g., serine/threonine kinase inhibitors and tyrosine-kinase inhibitors), monoclonal antibodies (mAbs), and antibody-drug conjugates (ADCs). One of the most successful small-molecule targeted therapeutic agents is imatinib mesylate, a kinase inhibitor used for chronic myelogenous leukemia and gastrointestinal stromal

Molecules 2017, 22, 1281; doi:10.3390/molecules22081281 www.mdpi.com/journal/molecules Molecules 2017, 22, 1281 2 of 28 tumor [4]. Other examples include erlotinib and bortezomib for treating non-small cell lung cancer Molecules 2017, 22, 1281 2 of 27 (NSCLC) and multiple myeloma, respectively [5,6]. Another emerging targeted therapy is monoclonal antibody(NSCLC) (mAb) and therapy,multiplealso myeloma, known respectively as immunotherapy [5,6]. Another [7]. The emerging mAbs aretargeted capable therapy of binding is specificmonoclonal tumor-associated antibody (mAb) antigens therapy, and also inducing known cancer as immunotherapy cell death through [7]. The antibody-dependent mAbs are capable of cell cytotoxicity,binding specific complement-dependent tumor-associated cytotoxicity,antigens and or inducing interference cancer with cell signaling death through pathways; antibody- thus they offerdependent a tumor-targeted cell cytotoxicity, treatment complement-dependent approach [8,9]. Targeted cytotoxicity, mAb therapy or interference is used to treatwith manysignaling cancers includingpathways; non-Hodgkin’s thus they offer lymphoma, a tumor-targeted colorectal treatmen cancer,t approach head/neck [8,9]. cancer, Target anded mAb breast therapy cancer. is However, used small-moleculeto treat many or cancers mAb targetedincluding therapy non-Hodgkin’s alone often lymphoma, shows colorectal inadequate cancer, therapeutic head/neck activity cancer, due and to its lowbreast cytotoxicity cancer. and However, weak penetrationsmall-molecule into or solid mAb tumors. targeted An therapy improved alone approach often shows to overcome inadequate these therapeutic activity due to its low cytotoxicity and weak penetration into solid tumors. An improved limitations is to create ADCs. approach to overcome these limitations is to create ADCs. ADC is a complex molecule composed of a mAb conjugated with a highly potent cytotoxic drug ADC is a complex molecule composed of a mAb conjugated with a highly potent cytotoxic drug (or payload)(or payload) through through an appropriate an appropriate linker linker (Figure (Figure1)[ 10 1), 11[10,11].]. An ADCAn ADC is designed is designed as targeted as targeted therapy, whichtherapy, targets which and kills targets only and cancer kills cellsonly whilecancer sparing cells while healthy sparing cells. healthy The development cells. The development of ADCs faced of a numberADCs of faced challenges a number in theof challenges early 1970s. in the The early first 197 set0s. of The ADCs, first preparedset of ADCs, by prepared conjugating by conjugating murine KS1/4 antibodymurine with KS1/4 the antibody cytotoxic with drug the methotrexatecytotoxic drug ormeth theotrexate BR96 antibodyor the BR96 conjugated antibody conjugated with doxorubicin, with weredoxorubicin, less active than were their less active parent than drugs their [12 parent–14]. drug The failures [12–14]. of The early failure ADCs of resultedearly ADCs from resulted several from factors: (a) theseveral chimeric factors: or murine(a) the antibodieschimeric or used murine in ADCs antibodies could elicitused immunogenicin ADCs could responses elicit immunogenic and cause fast clearanceresponses in circulation; and cause fast (b) theclearance cytotoxic in circulation; drug was (b) insufficiently the cytotoxic potent, drug was and insufficiently further conjugation potent, and might leadfurther to decreased conjugation potency; might (c) lead inappropriate to decreased linkage potency; and (c) theinappropriate poor selection linkage of antigenand the poor could selection lead to the failureof antigen of an ADC. could lead to the failure of an ADC.

FigureFigure 1. The1. The general general components components of ofADC. ADC. SelectionSelection pr principlesinciples of of antibody, antibody, comparison comparison between between non-specificnon-specific and and specific specific conjugations, conjugations, and descriptionsand descriptions on how on thehow drug the isdrug released is released and its mechanismand its of action.mechanism of action.

Later ADCs achieved success after a better understanding of the basic requirements for their Later ADCs achieved success after a better understanding of the basic requirements for their design. First, the application of humanized and human antibodies has greatly reduced the problems design.caused First, by theimmunogenicity. application of Second humanized, internalizing and human antigens antibodies as tumo hasr targets greatly allowed reduced the thedesign problems of causedmore by functional immunogenicity. antitumor Second, antibodies. internalizing Third, un antigensderstanding as tumor the targetsmechanisms allowed of theaction design (protein of more functionaluptake) antitumor of ADCs in antibodies. mammalian Third, cells helped understanding design different the mechanisms types of linkers, of action which (protein facilitated uptake) the of ADCsrelease in mammalian of payload cellsinside helped the tumors. design Finally, different thetypes discovery of linkers, of new which cytotoxic facilitated compounds the release with of payloadpicomolar inside IC the50 tumors.values further Finally, propelled the discovery the success of new of cytotoxic ADCs [15]. compounds ADCs are with now picomolar powerful IC 50 valuestherapeutics further propelled enabling the wider success therapeutic of ADCs window [15]. ADCss, improved are now pharmacokinetic/pharmacodynamic powerful therapeutics enabling wider therapeutic(PK/PD) windows, profiles, and improved enhanced pharmacokinetic/pharmacodynamic efficacies. The first ADC, gemtuzumab (PK/PD) ozogamicin profiles, (Mylotarg and enhanced®,

Molecules 2017, 22, 1281 3 of 28 efficacies.Molecules The 2017, first 22, 1281 ADC, gemtuzumab ozogamicin (Mylotarg®, Pfizer/Wyeth-Ayerst Laboratories,3 of 27 New York, NY, USA), was approved by the US Food and Drug Administration (FDA) in 2000 (later Pfizer/Wyeth-Ayerst Laboratories, New York, NY, USA), was approved by the US Food and Drug withdrawn from the market in 2010 due to toxicity concerns and no sufficient clinical benefit) for Administration (FDA) in 2000 (later withdrawn from the market in 2010 due to toxicity concerns and treatingno sufficient acute myeloid clinical leukemia.benefit) for It treating was followed acute myeloid by recent leukemia. approvals It was of followed brentuximab by recent vedotin approvals (SGN-35, ® Adcetrisof brentuximab, Seattle Genetics, vedotin (SGN-35, Inc., Bothell, Adcetris WA,®, USA)Seattle in Genetics, 2011 and Inc., ado-trastuzumab Bothell, WA, USA) emtansine in 2011 (T-DM1,and ® Kadcylaado-trastuzumab, Genentech, emtansine South San (T-DM1, Francisco, Kadcyla CA,®, Genentech, USA) in 2013. South Encouraged San Francisco, by CA, the USA) success in 2013. of these ADCs,Encouraged many new by the ADCs success have of beenthese developed.ADCs, many Currently,new ADCs morehave been than developed. 55 ADCs Currently, are under more various stagesthan of clinical55 ADCs evaluation are under various [16]. Since stages tubulin of clinical inhibitors evaluation are one [16]. of Since the most tubulin successful inhibitors cytotoxic are one drugsof incorporatedthe most successful in ADCs, cytotoxic this review drugs focuses incorporated on the development in ADCs, this ofreview tubulin focuses inhibitor-based on the development ADCs. of tubulin inhibitor-based ADCs. 2. General Considerations for ADC 2. General Considerations for ADC 2.1. Drug Release Mechanism of ADC 2.1. Drug Release Mechanism of ADC ADCs are an important class of highly potent biopharmaceutical drugs designed as prodrugs for the treatmentADCs of are cancers. an important The drug class release of highly mechanism potent biopharmaceutical of ADCs consists drugs of several designed key steps.as prodrugs The ADC firstfor binds the totreatment the tumor-associated of cancers. The drug antigen release onthe mech targetanism cells. of ADCs The resultingconsists of antigen-ADC several key steps. complex The is internalizedADC first by binds receptor-mediated to the tumor-associated endocytosis, antigen and on subsequently the target cells. transferred The resulting to late antigen-ADC endosome and complex is internalized by receptor-mediated endocytosis, and subsequently transferred to late lysosome where the cytotoxic drug is released either by cleavage of the linker or degradation of the endosome and lysosome where the cytotoxic drug is released either by cleavage of the linker or antibody. Finally, the released payload affects the intracellular target leading to apoptosis and cell degradation of the antibody. Finally, the released payload affects the intracellular target leading to deathapoptosis (Figure 2and)[ 17 cell–19 death]. This (Figure process 2) [17–19]. requires This concerted process efforts requires and concerted depends efforts on specific and depends properties on of eachspecific ADC component, properties of released each ADC drug component, ideally also released has adrug bystander ideally also effect. has a bystander effect.

Figure 2. General mechanism of action of ADCs. The antibody of an ADC binds to tumor associated Figure 2. General mechanism of action of ADCs. The antibody of an ADC binds to tumor associated antigen. Upon binding, the ADC-antigen complex is internalized into the cancer cell. During the antigen. Upon binding, the ADC-antigen complex is internalized into the cancer cell. During the degradation of the complex, the released payload exerts its effects on the intracellular target leading degradationto apoptosis. of the complex, the released payload exerts its effects on the intracellular target leading to apoptosis. 2.2. Target and Antibody Selection for ADC 2.2. Target and Antibody Selection for ADC First, the selection of tumor-associated targets is critical for the success of ADCs as clinical anticancerFirst, the therapeutics. selection of Ideally, tumor-associated the target antige targetsns should is critical be expressed for the successabundantly of ADCsand uniformly as clinical anticanceron the therapeutics.surface of tumor Ideally, cells the and target be absent antigens in healthy should tissues be expressed and organs. abundantly However, and such uniformly ideal on the surface of tumor cells and be absent in healthy tissues and organs. However, such ideal antigens are Molecules 2017, 22, 1281 4 of 28 extremely rare, because an unrecognized antigen would trigger the immune response and lead to quick clearance [20]. In many cases, the optimal targets are expressed substantially higher in tumor cells than in normal cells. Such a difference can be identified by genomic, transcriptomic (microarray), and/or proteomic techniques [21]. It is worth noting that ADCs are efficacious over a wide range of antigen expression levels. For example, T-DM1 is an approved anti-human epidermal growth factor receptor 2 (HER2) ADC for metastatic breast cancer (MBC) and targets very high copy numbers of HER2 (in some cases, >3 million copies per cell) [22]. On the other hand, the ADC gemtuzumab ozogamicin targeting relatively low expression levels of CD33 (5000–10,000 receptors per cell) has shown potent activity in preclinical and clinical trials [23]. In addition, an optimal target must be localized on the tumor cell surface, can be recognized by its antibody, and has the ability of internalization upon ADC binding [24]. In addition to the selection of antigen, an optimal antibody is also important and has a significant impact on the therapeutic index, PK/PD profiles, and overall anticancer efficacy of an ADC. Antibodies can be classified into different types: IgA, IgG, IgD, IgE and IgM according to their biological properties and functional locations [25]. Based on the targets, currently used antibodies fall into the following categories, but are not limited to antibodies targeting leukocyte differentiation antigens (CD20, CD22, CD30, CD33, etc.), tyrosine kinase receptors (EGFR, EphA2, EphB2), glycoproteins (gpNMB, MUC16, PSMA, FAP, mesothelin, and TAG-72), sodium phosphate transporter (NaPi2b), oncoproteins (Cripto, ED-B, TMEFF2, CAIX), and hormone receptor (αv integrins) [26,27]. A suitable mAb in an ADC must retain high specificity and binding affinity to the target antigen on tumor cells, efficient receptor-mediated internalization, low immunogenicity, long circulation time, and antitumor activity against target cells after conjugating with the cytotoxic molecule through an appropriate linker [11,28]. First, the high affinity of a mAb to the target antigen is the most desirable characteristic, since it ensures good tumor localization and efficient internalization of ADCs into target cells. However, high affinity of the mAb may lead to fast binding to perivasculature with subsequent quick internalization of ADCs into the cells they first encounter, thus limiting further diffusion into the tumor [11]. Accordingly, the balance between penetration and retention ability of an antibody should be considered for a desired binding affinity [29]. Second, the molecular size is another important consideration. A full-length antibody having a long circulation half-life leading to a good chance of reaching the tumor site is thus favorable for an ADC. A long half-life, on the other hand, might be a risk for the degradation of an ADC and lead to nonspecific toxicity [11,29,30]. Preclinical studies have shown that smaller scaffold proteins or antibody fragments (single-chain variable fragments or Fab fragment) may achieve greater tumor penetration than might their parent antibodies [30,31]. Rapid clearance leading to a short half-life will still deter the clinical success of ADCs. Therefore, fine balances between molecular size and affinity and between tumor retention and penetration of an antibody will be critical for the success of an ADC [15,32].

2.3. Conjugation Strategy of Payload The chemistry involving the conjugation strategy of payload is another important consideration for designing and assessing ADCs with regard to their PK/PD profile, binding affinity, toxicity, and stability. The early approach for preparing ADCs involves random conjugation of a small-molecule drug to either amines on lysine residues or sulfhydryl groups on cysteine residues of the antibody. The conjugation forms a heterogeneous mixture with each subpopulation displaying different PK/PD and efficacy properties. In contrast, controlled site-specific conjugation has the potential to overcome the heterogeneity limitations and widen the therapeutic window of ADCs [33,34]. Initial controls of the heterogeneity involved selective reduction of the four intermolecular disulfide bonds within an IgG. However, such trials caused the drug-antibody ratio (DAR) distribution of ADCs to vary from 0 to 8. This diversity of ADCs not only resulted in great challenges for subsequent purification but also influenced the circulation half-life, efficacy, and even increased the potential of off-target toxicity. Moreover, the maleimide-based ADCs, wherein the payloads are attached via thioethers, are Molecules 2017, 22, 1281 5 of 28 prone to lose the payload through thiol exchange in the blood, though this limitation can be overcome by using cysteine bridging dibromomaleimides, bis-sulfone, and dibromopyridazinediones [35–37]. AnotherMolecules modification, 2017, 22, 1281 using cysteine bridging bifunctional groups is to reduce the maximum number5 of 27 Molecules 2017, 22, 1281 5 of 27 of cytotoxic drugs attached to a mAb from eight to four. 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(acid-labile while The release A ADC thatlinker linkers, the with mustcytotoxic protease non-cleavable cleavable be stableagent cleavable linkerenoughupon linker linkers,internalization involves in requiressystemic and hydrolysis,disulfide lysosomalcirculationof the linkers)ADC enzymatic degradationand within and be non-cleavable ablecancer reaction, to of cellsrapidly the [50].antibodylinkers or and reduction for (Table(Tablelinkers 1) [51]. 1) (acid-labile [51]. 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Summary the agent, toADC selectively of while commonlyis exposed that release [50]. usedwith the linkerscleavable cytotoxic for linker the drug development involvesbased on hydrolysis, ofthe ADCs. physiological enzymatic TableTable 1. 1.Summary Summary of of commonly commonly usedused linkers linkers for for the the development development of ADCs. of ADCs. reaction,environment or Tablereduction toTable which 1. 1.Summary Summarytheto ADCselectively ofisof exposed commonlycommonly release [50]. used used the linkers linkers cytotoxic for for the the developmentdrug development based of onADCs. of the ADCs. physiological AbbreviationTable 1. Summary Chemical of commonly Structure used linkers for the development of ADCs. Linker Type AbbreviationenvironmentAbbreviation to which the ADC Chemical Chemical is exposed Structure [50]. Linker Linker Type Type Abbreviation Table 1. Summary Chemical of commonly Structure used linkers for the development of Linker ADCs. Type AbbreviationAbbreviation Chemical Chemical StructureStructure Linker Linker Type Type MHHAbbreviationMHH Table 1. Summary Chemical of commonly Structure used linkers for the developmentChemicalChemical Linker labile of labileADCs. Type(acid (acid labile) labile) linker linker MHHMHH ChemicalChemical labile labile (acid (acid labile) labile) linker linker MHHMHH ChemicalChemical labile labile (acid (acidlabile) labile) linker linker Abbreviation Chemical Structure Linker Type MHH Chemical labile (acid labile) linker DSDM Disulfide-containing reducible linker DSDMDSDMDSDMMHH Disulfide-containingChemicalDisulfide-containingDisulfide-containing labile (acid reducible labile) reduciblereducible linker linker linkerlinker DSDMDSDM Disulfide-containingDisulfide-containing reducible reducible linker linker DSDM Disulfide-containing reducible linker

Sulfo-SPDB Disulfide-containing reducible linker Sulfo-SPDBDSDM Disulfide-containing reducible linker Sulfo-SPDBSulfo-SPDBSulfo-SPDB Disulfide-containingDisulfide-containingDisulfide-containing reducible reduciblereducible linker linker linker Sulfo-SPDBSulfo-SPDB Disulfide-containingDisulfide-containing reducible reducible linker linker

Sulfo-SPDB Disulfide-containing reducible linker MC-VC-PABC Enzymatically cleavable linker MC-VC-PABC Enzymatically cleavable linker MC-VC-PABC Enzymatically cleavable linker MC-VC-PABCMC-VC-PABCMC-VC-PABC EnzymaticallyEnzymaticallyEnzymatically cleavable cleavablecleavable linker linkerlinker MC-VC-PABC Enzymatically cleavable linker

MC-VC-PABCSMCC Non-cleavableEnzymatically bifunctional cleavable linker linker SMCC Non-cleavable bifunctional linker SMCCSMCC Non-cleavableNon-cleavable bifunctional bifunctional linker linker SMCCSMCC Non-cleavableNon-cleavable bifunctionalbifunctional linkerlinker SMCC Non-cleavable bifunctional linker Mal-PEG-NHS Non-cleavable spacer linker Mal-PEG-NHSSMCC Non-cleavableNon-cleavable bifunctional spacer linker linker Mal-PEG-NHSMal-PEG-NHS Non-cleavableNon-cleavable spacer spacer linker linker Mal-PEG-NHS Non-cleavable spacer linker Mal-PEG-NHSMal-PEG-NHS Non-cleavableNon-cleavable spacer spacer linker linker Mal-PEG-NHS Non-cleavable spacer linker GBC Enzyme-labile ß-Glucuronide linker GBC Enzyme-labile ß-Glucuronide linker GBCGBC Enzyme-labileEnzyme-labile ß-Glucuronide ß-Glucuronide linker linker

GBC Enzyme-labile ß-Glucuronide linker GBCGBCGBC Enzyme-labileEnzyme-labileEnzyme-labile ß-Glucuronide ß-Glucuronideß-Glucuronide linker linker linker Three major types of cleavable linkers are often used in ADCs: acid-cleavable linkers, protease- ThreeThree major major types types of of cleavable cleavable linkerslinkers are often ususeded in in ADCs: ADCs: acid-cleavable acid-cleavable linkers, linkers, protease- protease- cleavableThree linkers, major andtypes disulfide of cleavable linkers. linkers Acid-cleav are oftenable us linkersed in ADCs: such as acid-cleavable hydrazine linkers linkers, are protease-designed cleavablecleavable linkers, linkers, and and disulfide disulfide linkers. linkers. Acid-cleavAcid-cleavableable linkers linkers such such as as hydr hydrazineazine linkers linkers are are designed designed tocleavable be stable linkers, at a andneutral disulfide pH of linkers. circulation Acid-cleav but canable be linkers hydrolyzed such as hydrin lysosomesazine linkers with are a designedlow pH to tobe be stable stable at at a aneutral neutral pH pH ofof circulationcirculation but cancan bebe hydrolyzedhydrolyzed in in lysosomes lysosomes with with a lowa low pH pH toThree be stable major at types a neutral of cleavable pH of circulation linkers are but often can beus edhydrolyzed in ADCs: in acid-cleavable lysosomes with linkers, a low protease-pH ThreeThree major major types types of of cleavable cleavable linkers are are often often us useded in inADCs: ADCs: acid-cleavable acid-cleavable linkers, linkers, protease- protease- cleavable linkers, and disulfide linkers. Acid-cleavable linkers such as hydrazine linkers are designed cleavablecleavable linkers, linkers, and and disulfide disulfide linkers. linkers. Acid-cleavableable linkers linkers such such as hydr as hydrazineazine linkers linkers are designed are designed to beto stablebe stable at ata neutrala neutral pH pH of of circulationcirculation butbut cancan be be hydrolyzed hydrolyzed in lysosomesin lysosomes with witha low a pHlow pH to be stable at a neutral pH of circulation but can be hydrolyzed in lysosomes with a low pH

Molecules 2017, 22, 1281 6 of 28

Three major types of cleavable linkers are often used in ADCs: acid-cleavable linkers, protease-cleavable linkers, and disulfide linkers. Acid-cleavable linkers such as hydrazine linkers are designed to be stable at a neutral pH of circulation but can be hydrolyzed in lysosomes with a low pH environment. Mylotarg, for example, was generated by conjugating calicheamicin with mAb against the CD33 antigen through an acid-cleavable hydrazine linker [52]. Protease-cleavable linkers are also used to keep ADCs intact in systemic circulation and allow easy release of the cytotoxic drugs from ADCs by lysosomal enzymes within cancer cells. For example, valine-citrulline (Val-Cit) and phenylalanine-lysine (Phe-Lys), which confer excellent stability and PK/PD profile to ADCs, have been used in many ADCs for preclinical and clinical evaluations. In addition, the ADCs with disulfide linkers take advantage of reduced glutathione with high intracellular concentrations to release free drug inside the cell. ADCs with reducible disulfide linkers generate uncharged metabolites that can diffuse into neighboring cells and elicit bystander killing, which is essential for killing heterogeneous tumors [51,53]. ADCs with cleavable linkers have broader efficacy and faster rates of activating and releasing cytotoxic drugs for most cell lines. In contrast, ADCs with non-cleavable linkers can possibly provide increased plasma stability, greater therapeutic window, and reduced off-target toxicity. To reduce aggregation and improve the solubility of some ADCs, hydrophilic linkers such as β-glucuronide linker, Sulfo-SPDB, and Mal-PEG4-NHS were investigated [54,55]. In short, each type of linker has advantages and disadvantages, and each can be modified to achieve a fine balance between target efficacies and undesired toxicities [54].

2.5. Payload Selection for ADC The cytotoxic moiety (or payload) is the most important element of an ADC, and it should meet several requirements. First, the payload concentration in the target cells is often quite low, usually due to poor tumor localization and insufficient internalization. Therefore, a cytotoxic molecule with subnanomolar potency is required to exert optimal efficacy. In addition, the payload molecule should contain a functional group for conjugation or be capable of being chemically modified to generate an appropriate site whereby the original payload can be released from the ADC in tumor cells. Finally, the payload should be stable and soluble under physiological conditions. The payloads currently used for ADCs fall into the following four categories: tubulin inhibitor (e.g., maytansine analogs and auristatin analogs), DNA damaging agent (e.g., calicheamicin and pyrrolobenzodiazepine analogs), topoisomerase I inhibitor (e.g., SN-38), and RNA polymerase II inhibitor (e.g., α-amanitin).

3. Tubulin Inhibitors as Payloads of ADCs Microtubules, a dynamic assembly of α- and β-tubulin heterodimers (Figure3) [ 56,57] are involved in many cellular processes such as cell structure maintenance, cell division, and intracellular transport. Disruption of microtubules induces cell cycle arrest in the G2/M phase, which makes microtubules an attractive target for drug discovery. To date, microtubules have five known binding sites: vinca alkaloid binding site, taxane binding site, colchicine binding site, maytansine binding site, and the laulimalide binding site. Microtubule/tubulin inhibitors can be classified into two major categories according to their mechanisms of action: agents promoting tubulin polymerization and stabilizing microtubule structures (e.g., paclitaxel, epothilones, discodermolide and taccalonolides), and agents inhibiting tubulin polymerization and destabilizing microtubule structures (such as maytansinoids, auristatins, vinblastine and vincristine) (Figure4)[ 58,59]. The majority of the clinically used tubulin inhibitors are natural products and their synthetic derivatives, such as taxanes (e.g., paclitaxel) and vinca alkaloids (e.g., vinblastine) [60]. These earlier tubulin inhibitors, however, have intrinsic limitations including sensitivity to ATP-binding cassette transporter-mediated drug resistance and low cytotoxicity. These limitations resulted in studies leading to the discovery of new generations of tubulin inhibitors with greater potency. Maytansine was one of the first compounds with picomolar IC50 values and higher potency (100- to 1000-fold) than, for example, doxorubicin and paclitaxel. Molecules 2017, 22, 1281 7 of 28 Molecules 2017,, 22,, 12811281 7 of 27

Figure 3. Structure, polymerization and depolymerization of microtubules. Figure 3. Structure,Structure, polymerizationpolymerization andand dedepolymerization of microtubules.

Figure 4. ClassificationClassification ofof tubulintubulin stabilizersstabilizers anand destabilizers arresting cell cycle at G2/M phase. Figure 4. Classification of tubulin stabilizers and destabilizers arresting cell cycle at G2/M phase.

Molecules 2017, 22, 1281 8 of 28

Molecules 2017, 22, 1281 8 of 27 However, in many cases, the poor therapeutic window and lack of tumor specificity led to failure of potentHowever, tubulin in inhibitors many cases, alone the poor as an therapeutic anticancer wind agent.ow and ADCs lack of as tumor targeted specificity therapy led mightto failure be a promisingof potent approach tubulin inhibitors to address alone the limitationsas an antica ofncer single-agent agent. ADCs therapy. as targeted Currently, therapy tubulin might inhibitors be a arepromising one of the approach most validated to address payloads the limitations in ADCs of single-agent and have beentherapy. extensively Currently, investigated. tubulin inhibitors There areare two one approved of the most ADCs validated on the payloads market in today: ADCs T-DM1and have and been SGN-35, extensively which investigated. use tubulin There inhibitors are maytansine-derivativetwo approved ADCs DM1on the (tubulin market inhibitor today: T- targetingDM1 and maytansine SGN-35, which site) and use dolastatin-derivative tubulin inhibitors maytansine-derivative DM1 (tubulin inhibitor targeting maytansine site) and dolastatin-derivative MMAE (tubulin inhibitor targeting vinca alkaloid site) as warheads, respectively. Tubulin inhibitors MMAE (tubulin inhibitor targeting vinca alkaloid site) as warheads, respectively. Tubulin inhibitors as ADC payloads have achieved some commercial successes. However, these ADCs are still far from as ADC payloads have achieved some commercial successes. However, these ADCs are still far from the concept of ideal targeted therapy. Payloads which are active against drug-resistant tumors and the concept of ideal targeted therapy. Payloads which are active against drug-resistant tumors and have higher potency and better solubility, stability, tolerability, and therapeutic indices than current have higher potency and better solubility, stability, tolerability, and therapeutic indices than current payloads,payloads, will will be be beneficial beneficial for for the the success success ofof thethe nextnext generation of of ADCs ADCs [10]. [10 ].More More recently, recently, some some potentpotent tubulin tubulin inhibitors inhibitors suitable suitable forfor payloadspayloads of ADCs were were discovered discovered and and advanced advanced to toclinical clinical phasephase evaluation. evaluation. These These new new potential potential tubulin/microtubule tubulin/microtubule agents agents for for ADC ADC payloads payloads may may overcome overcome thethe current current limitations limitations of of anticancer anticancer therapies. therapies. ThisThis section highlights highlights both both representative representative tubulin tubulin inhibitorsinhibitors (Figure (Figure5) 5) that that have have already already been been used used inin ADCsADCs andand novel tubulin tubulin inhibitors inhibitors that that are are too too toxictoxic to to be be used used as as a singlea single agent agent but but are are promising promising candidatescandidates for for ADC ADC payloads. payloads.

FigureFigure 5. 5.The The chemical chemical structures structuresof of tubulintubulin inhibitors and their their derivatives. derivatives. Microtubule Microtubule destabilizers: destabilizers: maytansinemaytansine and and its its derivatives, derivatives, dolastatin-10dolastatin-10 andand itsits derivatives,derivatives, cryptopycin-1, cryptopycin-1, cryptopycin-52, cryptopycin-52, tubulysinstubulysins D, D, hemiasterlin hemiasterlin and and its its derivative derivative (HTI-286),(HTI-286), colchicine and and CA4. CA4. Microtubule Microtubule stabilizers: stabilizers: paclitaxel,paclitaxel, discodermolide, discodermolide, taccalonolidestaccalonolides AA and B and their their derivatives derivatives (taccalonolide (taccalonolide AF AF and and taccalonolidetaccalonolide AJ), AJ), and and taccalonolide taccalonolide AI-epoxide,AI-epoxide, laulimalide, laulimalide, and and epothilones epothilones A and A and B. B.

Molecules 2017, 22, 1281 9 of 28 Molecules 2017, 22, 1281 9 of 27

3.1. Maytansinoids as ADC Payloads Maytansinoids are are anti-mitotic anti-mitotic tubulin tubulin inhibitors inhibitors derived derived from from maytansine maytansine (1) ( 1(Figure) (Figure 5),5), a abenzoansamacrolide benzoansamacrolide that that was was initially initially isolated isolated from from an an alcoholic alcoholic extract extract in in the the bark bark of the African shrubs Maytenus serrata and Maytenus buchananii in 1972 [[61].61]. Maytansine and maytansinoids bind to the maytansine site, resulting in the suppression of microtubule dynamics and causes cell cycle arrest in the G 2/M/M phase. phase. Maytansine Maytansine showed showed potent potent anticancer anticancer activity activity in in the the picomolar picomolar range against most of the tested cell lines, and it was 100- to 1000-times more potent than were clinically used anticancer drugs in vitro [[62].62]. Maytansine and its analogs were extensively evaluated in Phase I and Phase II 2 clinical trials, with a dose limitation of 1–2 mg/m2. .The The dose-limiting dose-limiting toxicities toxicities include include neurotoxicity, neurotoxicity, gastrointestinal toxicity,toxicity, weakness, weakness, nausea, nausea, vomiting, vomiting, and and diarrhea. diarrhea. The The poor poor therapeutic therapeutic window window and lackand lack of tumor of tumor specificity specificity led toled the to the ultimate ultimate termination termination of the of the investigations investigations on on maytansine maytansine alone alone as anas an anticancer anticancer agent agent [63 ].[63]. Due Due to the to the structural structural complexity complexity including including numerous numerous stereocenters, stereocenters, the total the synthesistotal synthesis of maytansine of maytansine and its and analogues its analogues is cost prohibitive is cost prohibitive even if it wereeven achievable if it were [achievable64]. Maytansine [64]. isMaytansine naturally generatedis naturally from generated ansamitocins from whichansami weretocins obtained which were from fermentingobtained from microorganism fermenting Actinosynnemamicroorganism pretiosumActinosynnema. Through pretiosum a semi-synthesis. Through a semi-synthesis strategy, a series strate of maytansinegy, a series analogsof maytansine (DM1, DM3analogs and (DM1, DM4) DM3 bearing and DM4) disulfide bearing or thiol disulfide groups, or whichthiol groups, allow covalentwhich allow linkage covalent with linkage mAbs, werewith obtainedmAbs, were in two obtained steps. The in precursor,two steps. ansamitocin The precursor, P-3 ( 2ansamitocin), was reduced P-3 by ( lithium2), was trimethoxyaluminumreduced by lithium hydridetrimethoxyaluminum to give maytansinol hydride (3 ).to It give was furthermaytansinol esterified (3). inIt was the presence further esterified of dicyclohexylcarbodiimide in the presence of anddicyclohexylcarbodiimide a Lewis acid (zinc chloride), and a toLewis give acid maytansinoid (zinc chloride), disulfides, to give which maytansinoid were subsequently disulfides, reduced which bywere dithiothreitol subsequently (DTT) reduced to yield by dithiothreitol DM1, DM3 and (DTT) DM4 to (Scheme yield DM1,1)[65 DM3,66]. and DM4 (Scheme 1) [65,66].

Scheme 1. Semi-synthesis of DM1, DM3 and DM4 from ansamitocin P-3.

The structure-activity relationship (SAR) studies on maytansine showed that the C4–C5 epoxide The structure-activity relationship (SAR) studies on maytansine showed that the C4–C5 epoxide moiety, the carbinolamide at C9, and double bonds at C11 and C13 are all required for optimal moiety, the carbinolamide at C9, and double bonds at C11 and C13 are all required for optimal activity. activity. The presence of the ester side chain (N-acyl-N-methyl-L-alanyl) at C3 is essential for activity The presence of the ester side chain (N-acyl-N-methyl-L-alanyl) at C3 is essential for activity and and their corresponding L-epimers is about 100-fold more potent than the unnatural N-methyl-D- their corresponding L-epimers is about 100-fold more potent than the unnatural N-methyl-D-alanyl alanyl moiety (4). The side chain, however, can be modified to afford a series of maytansinoids moiety (4). The side chain, however, can be modified to afford a series of maytansinoids bearing bearing disulfide or thiol groups that do not result in the loss of activity [66,67]. disulfide or thiol groups that do not result in the loss of activity [66,67]. Maytansinoids with potent cytotoxic activity are attractive candidates for ADC payloads. These Maytansinoids with potent cytotoxic activity are attractive candidates for ADC payloads. maytansinoids may be linked to antibodies via a non-cleavable linker or a cleavable linker. The T- These maytansinoids may be linked to antibodies via a non-cleavable linker or a cleavable linker. DM1 developed by Roche was prepared by reacting the antibody with succinimidyl 4-(N- The T-DM1 developed by Roche was prepared by reacting the antibody with succinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxylate (SMCC), followed by reaction of the modified antibody with the thiol-containing maytansinoid, which demonstrates increased plasma stability, greater therapeutic window, and reduced off-target toxicity compared to that with cleavable linkers (Scheme 2A)

Molecules 2017, 22, 1281 10 of 28 maleimidomethyl)cyclohexane-1-carboxylate (SMCC), followed by reaction of the modified antibody with the thiol-containing maytansinoid, which demonstrates increased plasma stability, greater therapeutic window, and reduced off-target toxicity compared to that with cleavable linkers Molecules 2017, 22, 1281 10 of 27 (Scheme2A) [ 68]. It was approved by the FDA to treat metastatic breast cancer, which was previously treated[68]. It with was ado-trastuzumab approved by the FDA and to a taxane.treat metastatic The maytansinoid breast cancer, ADC which with was reducible-linkage previously treated cleavage, with however,ado-trastuzumab is operated and by a taxane. different The mechanisms maytansinoid in ADC dissimilar with reducible-linkage physiological environmentscleavage, however, (i.e., is the persistentoperated higher by different concentration mechanisms of glutathione in dissimilar and physiological disulfide isomerase environmen enzymests (i.e., in the tumor persistent tissues higher vs. the intracellularconcentration compartment). of glutathione Representative and disulfide maytansinoidisomerase enzymes ADCs in based tumor on disulfidetissues vs. linker the intracellular are prepared throughcompartment). lysine residues Representative from maytan the antibodysinoid ADCs that canbased react on disulfide with N link-succinimidyl-4-(2-pyridyldithio)er are prepared through lysine butanoateresidues (SPDB)from the linker antibody to givethat can a modified react with antibody N-succinimidyl-4-(2-pyridyldithio)butanoate that contains a reactive disulfide. A(SPDB) thiol-bearing linker maytansinoidto give a modified is then antibody added that by contains replacing a reactive 2-thiopyridine disulfide. A thiol-bearing to give the ma maytansinoidytansinoid is then conjugates added (Schemeby replacing2B) [69 2-thiopyridine,70]. to give the maytansinoid conjugates (Scheme 2B) [69,70].

SchemeScheme 2. 2.(A (A) conjugation) conjugation of of DM1 DM1 toto antibodiesantibodies by maleimide maleimide linker linker to to produce produce antibody-SMCC-DM1 antibody-SMCC-DM1 ADCs.ADCs. SMCC: SMCC:N N-succinimidyl-4-(-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate;-maleimidomethyl)cyclohexane-1-carboxylate; (B) conjugation (B) conjugation of ofmaytansinoids maytansinoids (May: (May: DM1, DM1, DM3 DM3and DM4) and to DM4) antibodies to antibodies by disulfide by linker disulfide to produce linker antibody– to produce antibody–SPDB–MaySPDB–May ADCs. SPDB: ADCs. N-succinimidyl-4-(2-pyridyldithio)butanoate. SPDB: N-succinimidyl-4-(2-pyridyldithio)butanoate.

The in vivo stability of the maytansinoid ADCs with two disulfide-linkages (huC242-DM1 and The in vivo stability of the maytansinoid ADCs with two disulfide-linkages (huC242-DM1 huC242-DM4) and a thioether-linked conjugate (huC242-SMCC-DM1) was proved through the andlatter’s huC242-DM4) exceptional and half-life a thioether-linked of 134 h in mice, conjugatea half-life superior (huC242-SMCC-DM1) to that of huC242–DM4 was proved and huC242– through theDM1 latter’s (102 exceptionalh and 47 h, respectively) half-life of 134[70,71]. h in The mice, in vitro a half-life cytotoxicity superior studies to that showed of huC242–DM4 that all of these and huC242–DM1conjugates were (102 highly h and potent 47 h, respectively)with IC50 values [70 ranging,71]. The fromin vitro3.5 tocytotoxicity 15 pM. Erikson studies and co-workers showed that all[70,72] of these evaluated conjugates the weremetabolism, highly and potent indicated with ICth50at valuesthe sole ranging metabolite from of 3.5the tothioether-linked 15 pM. Erikson andhuC242-SMCC-DM1 co-workers [70,72 was] evaluated lysine-SMCC-DM1 the metabolism, (Scheme and 3A), indicatedwhile the conjugates that the sole of huC242-DM1 metabolite ofand the thioether-linkedhuC242-DM4 with huC242-SMCC-DM1 disulfide linkers produced was lysine-SMCC-DM1 DM1 and S-methyl-DM1 (Scheme (35A),) (Scheme while 3B). the conjugatesLikewise, ofthe huC242-DM1 disulfide-linked and huC242-DM4DM4 conjugates with generated disulfide DM4 linkers and S produced-methyl-DM4 DM1 (6) and (SchemeS-methyl-DM1 3C). These (5) (Schemeuncharged3B). Likewise,metabolites, the S disulfide-linked-methyl-DM1 and DM4 S-methyl-DM4, conjugates are generated able to ef DM4flux andfromS the-methyl-DM4 cell, diffuse (6) (Schemeinto bystander3C). These cells, uncharged and induce metabolites, cell death [53,70S-methyl-DM1,72–75]. This may and explainS-methyl-DM4, why ADCs are with able cleavable to efflux fromlinkers the cell,have diffuse a broader into efficacy bystander for cells,most andcell lines. induce Many cell deathmaytansinoid [53,70,72 ADCs–75]. Thisare substrates may explain of the why ADCsmultidrug with cleavable transporter linkers MDR1 have (also a broader known efficacyas P-glycop forrotein most cellor P-gp), lines. Manyso several maytansinoid novel hydrophilic ADCs are substrateslinkers (Sulfo-SPDB of the multidrug and Mal-PEG4-NHS) transporter MDR1 were (also developed known asto P-glycoproteinevade MDR1-mediated or P-gp), drug so several resistance novel hydrophilic(Scheme 4A) linkers [76,77]. (Sulfo-SPDB and Mal-PEG4-NHS) were developed to evade MDR1-mediated drug resistance (Scheme4A) [76,77].

Molecules 2017, 22, 1281 11 of 28 Molecules 2017, 22, 1281 11 of 27 Molecules 2017, 22, 1281 11 of 27

SchemeScheme 3. 3.(A ()A antibody-SMCC-DM1) antibody-SMCC-DM1 generatesgenerates metabolitemetabolite Lys-SMCC-DM1. Lys-SMCC-DM1. SMCC: SMCC: N-succinimidyl-N-succinimidyl- Scheme 3. (A) antibody-SMCC-DM1 generates metabolite Lys-SMCC-DM1. SMCC: N-succinimidyl- 4-(4-(N-maleimidomethyl)cyclohexane-1-carboxylate;N-maleimidomethyl)cyclohexane-1-carboxylate; ( (BB) )metabolism metabolism of of antibody-SPDB-DM4. antibody-SPDB-DM4. SPDB: SPDB: 4-(N-maleimidomethyl)cyclohexane-1-carboxylate; (B) metabolism of antibody-SPDB-DM4. SPDB: N N-succinimidyl-4-(2-pyridyldithio)butanoate; (CC) metabolism of antibody-SPDB-DM4. -succinimidyl-4-(2-pyridyldithio)butanoate;N-succinimidyl-4-(2-pyridyldithio)butanoate; ((C) metabolism of of antibody-SPDB-DM4. antibody-SPDB-DM4.

Scheme 4. (A) the chemical structure of mAb-PEG-Mal-DM1 and mAb-sulfo-SPDB-DM4. PEG4: Scheme 4. (A) the chemical structure of mAb-PEG-Mal-DM1 and mAb-sulfo-SPDB-DM4. PEG4: Schemehydrophilic 4. (A tetraethylene) the chemical glycol, structure sulfo-PDB: of mAb-PEG-Mal-DM1 sulfo N-succinimidyl-4-(2-pyridyldithio)butanoate; and mAb-sulfo-SPDB-DM4. PEG4:(B) hydrophilic tetraethylene glycol, sulfo-PDB: sulfo N-succinimidyl-4-(2-pyridyldithio)butanoate; (B) hydrophilicthe tubulin tetraethylene inhibitor glycol,MMAF-based sulfo-PDB: ADCs; sulfo (CN)-succinimidyl-4-(2-pyridyldithio)butanoate; the hydrazone functionalities, β-glucuronide (B) the the tubulin inhibitor MMAF-based ADCs; (C) the hydrazone functionalities, β-glucuronide tubulincontaining inhibitor linkers, MMAF-based quaternary ammonium ADCs; (C) thelinker hydrazones or rebridged functionalities, ado-trastuzumab-basedβ-glucuronide ADCs containing using containing linkers, quaternary ammonium linkers or rebridged ado-trastuzumab-based ADCs using linkers,MMAE quaternary or MMAF ammoniumas payload. linkers or rebridged ado-trastuzumab-based ADCs using MMAE or MMAE or MMAF as payload. MMAF as payload.

Molecules 2017, 22, 1281 12 of 28

3.2. Auristatins as ADC Payloads

TheMolecules auristatins 2017, 22, 1281 are derived from the natural product dolastatin-10 (7) (Figure5), which12 is of isolated 27 from the sea hare Dolabella auricularia found in the Indian Ocean and the coastal waters of Japan in an 3.2. Auristatins as ADC Payloads extremely low abundance [78]. Dolastatin-10 and its analogs inhibit tubulin-dependent GTP binding and blockThe the auristatins binding of are vinca derived alkaloids from tothe tubulin natural in product a noncompetitive dolastatin-10 manner (7) (Figure [78,79 5),]. Dolastatin-10which is has demonstratedisolated from the a wide sea hare spectrum Dolabella of anticancerauricularia found activity in the against Indian a multitudeOcean andof the lymphomas, coastal waters leukemia, of Japan in an extremely low abundance [78]. Dolastatin-10 and its analogs inhibit tubulin-dependent and solid tumors with initial studies demonstrating IC50 values in the subnanomolar range. However, GTP binding and block the binding of vinca alkaloids to tubulin in a noncompetitive manner [78,79]. dolastatin-10 failed in Phase I and Phase II trials for non-small cell lung, melanoma, colorectal, ovarian, Dolastatin-10 has demonstrated a wide spectrum of anticancer activity against a multitude of prostate, breast, and pancreatobiliary tumors as a single agent due to its high cytotoxic activity and lymphomas, leukemia, and solid tumors with initial studies demonstrating IC50 values in the narrowsubnanomolar therapeutic range. window However, [80,81 dola]. statin-10 failed in Phase I and Phase II trials for non-small cell Tolung, achieve melanoma, appropriate colorectal, cytotoxic ovarian, prostate, ADC payloads, breast, and monomethyl pancreatobiliary auristatin-E tumors as a (MMAE,single agent8) and monomethyldue to its auristatin-Fhigh cytotoxic (MMAF, activity 9and) were narrow designed therapeutic based window on the [80,81]. structure of auristatin. Both MMAE and MMAFTo achieve showed appropriate no degradation cytotoxic in plasma,ADC payloads, in human monomethyl liver lysosomal auristatin-E environment, (MMAE, 8) orand by the actionmonomethyl of proteases. auristatin-F As free (MMAF, toxins, the 9) were cytotoxicities designed based of MMAE on the structure and MMAF of auristatin. are less Both potent MMAE than that of dolastatinand MMAF 10 showed in lymphoma no degradation cells in vitroin plasma,. The in ADC human conjugating liver lysosomal MMAE environment, or MMAF or with by the cAC10, however,action showed of proteases. similar As free cytotoxicity toxins, the against cytotoxici CD30ties +oflymphoma MMAE and cellsMMAF with are dolastatin-10less potent than [15 that,82 –84]. of dolastatin 10 in lymphoma cells in vitro. The ADC conjugating MMAE or MMAF with cAC10, In addition, MMAE and MMAF are fully synthetic drugs, which are relatively easy to modify when however, showed similar cytotoxicity against CD30+ lymphoma cells with dolastatin-10 [15,82–84]. optimizing their physical properties and characteristics. Likewise, MMAE and MMAF are unique linear In addition, MMAE and MMAF are fully synthetic drugs, which are relatively easy to modify when pentapeptidesoptimizing containing their physical unnatural properties amino and acidscharacteristics. with slight Likewise, modifications MMAE inand the MMAFN- or Care-position, unique and retainlinear their pentapeptides potency in the containing form of the unnatural ADC payloads. amino acidsIn vitrowith studiesslight modifications concluded that in the MMAF N- orwas C- less cytotoxicposition, than and was retain MMAE, their because potency thein the charged form of carboxylic the ADC payloads. acid group In hindersvitro studies its ability concluded to diffuse that into the cellsMMAF [71, 83was]. Differentless cytotoxic linking than strategieswas MMAE, including because acid-labilethe charged or carboxylic proteolytically acid group cleavable hinders linkers its as well asability non-cleavable to diffuse linkersinto the have cells been [71,83]. evaluated Different in auristatinlinking strategies ADCs. Theincluding proteolytically acid-labile cleavable or linkersproteolytically are introduced cleavable to keep linkers ADCs as stablewell as in non-cl systemiceavable circulation linkers have and been allow evaluated their easy in auristatin release of the cytotoxicADCs. drugs The byproteolytically specific intracellular cleavable linkers proteases are suchintroduced as cathepsin to keep B ADCs [85]. Thestable ADC in systemic SGN-35 was circulation and allow their easy release of the cytotoxic drugs by specific intracellular proteases such prepared by conjugating MMAE to a CD30-specific mAb via a protease-cleavable dipeptide linker, as cathepsin B [85]. The ADC SGN-35 was prepared by conjugating MMAE to a CD30-specific mAb valine–citrullinevia a protease-cleavable (val-cit) as dipeptide stated above linker, [17 valine–c,86–89].itrulline The val-cit (val-cit) peptide as stated can above be hydrolyzed [17,86–89]. The through lysosomalval-cit proteases, peptide can then be thehydrolyzed paraaminobenzyl through lysosomal (PAB)-MMAE proteases, subsequently then the paraaminobenzyl undergoes self-immolation (PAB)- to releaseMMAE the subsequently original MMAE undergoes (Scheme self-immolation5)[ 85]. This to is release one of the the original ADCs MMAE on the market(Scheme approved5) [85]. This by the FDAis in one August of the 2011.ADCs on the market approved by the FDA in August 2011.

SchemeScheme 5. Metabolism 5. Metabolism and and self-immolation self-immolation of mc-Val-Cit-PABC-MMAE. mc: mc: maleimidocaproyl. maleimidocaproyl. Val: Val: valine.valine. Cit: Cit: citrulline. citrulline. MMAE: MMAE: monomethylauristatin monomethylauristatin E. E.

Molecules 2017, 22, 1281 13 of 28

ADCs have also been designed to conjugate to cAC10 with mal-caproyl-vc-PAB MMAF and mal-caproyl-MMAF to give a protease-cleavable linkage and a non-cleavable linkage, respectively (Scheme4B) [ 90,91]. Initial studies showed that the maximum tolerated dose (MTD) with non-cleavable linkage is higher than that with cleavable linkage. However, in some cases, the cytotoxicity of MMAF ADCs with cleavable linkage is more potent than that with non-cleavable linkage. The acid-labile cleavable linkers, such as hydrazone functionalities, β-glucuronide containing linkers, or quaternary ammonium linkers have also been used for several auristatin ADCs for PK profile evaluation (Scheme4C) [ 75,92,93]. To date, these compounds have not advanced to clinical trials. Most recently, a novel technology using cysteine rebridged ado-trastuzumab-MMAE to produce a homogeneous and stable conjugate with a DAR of 4 was investigated, and the conjugate was found to be superior in efficacy to T-DM1, as evaluated in a low HER2 expressing JIMT-1 xenograft model (Scheme4C) [94].

3.3. Cryptophycins as ADC Payloads The cryptophycins are a family of 16-membered cyclic depsipeptides, first isolated from terrestrial blue-green algae in 1990 [95]. They are potent tubulin-binding antimitotic agents. The most abundant component, cryptophycin-1 (10) (Figure5), has demonstrated excellent activity against a broad spectrum of solid tumors with IC50 values in the picomolar range [96]. Significantly, it was not an effective substrate for the P-gp in multiple-drug resistant (MDR) cancer cell lines [97]. However, the intrinsically low bioavailability of cryptophycin-1 caused by its instability and solubility rendered it inefficacious in vivo. Chemists continued to explore and modify the scaffold, leading to the synthesis of hundreds of analogs [98]. The SAR can be summarized as follows: for unit A, the β-configuration of benzylic epoxide is essential and more potent than are the α-configured epoxides. The opening of the epoxide in the case of chlorohydrins also showed potent cytotoxicity. A series of para-substituted phenyls, such as hydroxyl, amino, carboxyl, ester, and amide derivatives, were designed, synthesized, and were proven to be more potent than ortho- or meta-substituted analogs. More importantly, the nature of the para-substitution can be modified as a conjugating site for the preparation of cryptophycin ADCs. Sanofi filed a patent based on the analogs described above, and they demonstrated effective antitumor activity with potentially improved physical, chemical, and biological profiles [99–103]. For unit B, the retention of D-tyrosine and benzene ring bearing para-methoxy or ortho-hydroxy is necessary for the activity. Studies showed that the B segment bearing the 5-fluorine substituted benzene ring lost bioactivity [99,104]. The C fragment structure-activity studies have focused on the C6 position, and it has been determined that the introduction of a bulky substituent such as a benzyl or isopropyl group resulted in a considerable reduction of bioactivity, while geminal methyl groups or a cyclopropyl group in the C6 position enhanced anticancer activity. In addition, it lost most activity if the C fragment was transformed into an alpha-amino acid, and the 16-membered ring property is essential for bioactivity, though the replacement of the ester bond between the C and D units with an amide derivative did not influence cytotoxicity. Nahrwold and co-workers reported that the replacement of an amide bond between units B and C by a triazole ring via Cu(I)-mediated “click” cyclization led to a reported IC50 value of 3.2 nM against the MDR tumor cell line KB-V1. Norbert Sewald’s group introduced the side chain at C6 containing an allyl and azide, and the resulted analogs were highly active in the cytotoxicity assay using the human cervix carcinoma cell line KB-3-1 and its MDR subclone KB-V1 [100,105–107]. The unit D side chain shows only slight variations relative to activity. Norbert Sewald’s group first introduced a side chain containing an ester bond or free carboxylic acid group. The functionalization of unit D precursors still retained good activity and provided a new site for biological coupling [108–110]. Cryptophycin-52 (Cr-52, LY355703, 11) was designed by Eli Lilly, and its reported potency is 40- to 400-fold greater than that of paclitaxel or vinca alkaloids, making it a promising clinical candidate [111–114]. The mechanism of action involves suppressing microtubule dynamics and arresting cells in the G2/M phase. However, the co-crystallization data between Cr-52 and tubulin has not yet been Molecules 2017, 22, 1281 14 of 28 solved, and the details of the binding site remain elusive. The Cr-52 structure is an analogue to cryptophycin-1, containing two hydroxy acids (units A and D) and two amino acids (units B and C). Due toMolecules the steric 2017, 22 hindrance, 1281 of the geminal dimethyl group, it is more resistant against ester hydrolysis14 of 27 than arecryptophycin-1, cryptophycin-1 containing derivatives two hydroxy [103]. acids After (units numerous A and D) preclinical and two amino studies, acids Eli(units Lilly B and advanced C). Cr-52Due to clinicalto the steric trials, hindrance with aims of the togeminal define dimethyl the MTD, group, recommended it is more resistan dose,t against pattern ester of hydrolysis toxicity, and 2 pharmacokineticthan are cryptophycin-1 profile. Two derivatives Phase I [103]. clinical After trials numerous were performed,preclinical studies, and a Eli dose Lilly of advanced 1.5 mg/m Cr- was recommended52 to clinical for Phasetrials, with II evaluation aims to define on a day the 1MTD, and 8recommended schedule every dose, 21 dayspattern [111 of]. toxicity, and Despitepharmacokinetic these findings, profile. theTwo treatment Phase I clinical effect fortrials NSCLC were performed, was not achieved and a dose as expected.of 1.5 mg/m Twenty-six2 was patientsrecommended were enrolled, for Phase of whom II evaluation 25 were evaluableon a day 1 and for toxicity8 schedule and every response. 21 days There [111]. were no responders when the weeklyDespite dosethese wasfindings, lowered the treatment from 1.5 mg/meffect fo2 rto NSCLC 1.125 mg/mwas not2. achieved Median survivalas expected. was Twenty- 4.1 months, six patients were enrolled, of whom 25 were evaluable for toxicity and response. There were no and toxicity was predominantly neurologic in the form of peripheral neuropathy and constipation [113]. responders when the weekly dose was lowered from 1.5 mg/m2 to 1.125 mg/m2. Median survival was Another study was performed to determine the activity of LY355703 and to characterize its toxicity 4.1 months, and toxicity was predominantly neurologic in the form of peripheral neuropathy and profileconstipation in patients [113]. with Another platinum-resistant study was performed advanced to ovarian determine cancer, the activity but only of modestLY355703 activity and to was observedcharacterize in these its patients toxicity [profile114]. in patients with platinum-resistant advanced ovarian cancer, but only Cr-52modest failed activity in was Phase observed II clinical in these studies patients due [114]. to the lack of efficacy and its high toxicity at the chosen doses.Cr-52 Although failed in Phase the cryptophycins II clinical studies were due unable to the lack to advance of efficacy to stand-aloneand its high toxicity agents, at desirable the characteristicschosen doses. including Although high the potency, cryptophycins relative were hydrophilicity, unable to advance lack of P-gpto stand-alone susceptibility, agents, and desirable a common resistancecharacteristics mechanism including make ithigh an attractivepotency, relative ADC payload.hydrophilicity, Additionally, lack of P-gp the parasusceptibility,-substituted and phenyl a can becommon modified resistance for the mechanism attachment make of an it an antibody attractive without ADC payload. losing activity.Additionally, Sanofi the designedpara-substituted a series of phenyl can be modified for the attachment of an antibody without losing activity. Sanofi designed a ADCs targeting EphA2 receptors by using hu2H11 antibodies coupling Cr-52. They were evaluated series of ADCs targeting EphA2 receptors by using hu2H11 antibodies coupling Cr-52. They were in MDA-MB-231 cell lines, and showed high bio-activity in early evaluation (Scheme6A). However, evaluated in MDA-MB-231 cell lines, and showed high bio-activity in early evaluation (Scheme 6A). furtherHowever, documented further antitumor documented activity antitumor of Cr-52 activity ADCs of Cr-52 has ADCs not been has reported.not been reported. Vishal A. Vishal Verma A. and coworkersVerma used and cryptophycinscoworkers used as cryptophycins potent payloads as potent for ADCs payloads through for ADCs either through a cleavable either or a non-cleavablecleavable linkeror and non-cleavable achieved satisfactorylinker and achievedin vitro satisfactoryactivity [115 in]. vitro Further activity investigation [115]. Further may investigation qualify Cr-52 may as an acceptablequalify candidate Cr-52 as an of acceptable ADCs for candidate clinical evaluation.of ADCs for clinical evaluation.

SchemeScheme 6. (A 6.) the(A) tubulinthe tubulin inhibitor inhibitor cryptophycins-based cryptophycins-based ADCs; ADCs; (B (B) the) the tubulin tubulin inhibitor inhibitor tubulysins- tubulysins- based ADCs containing quaternary ammonium linkers; (C) the tubulin inhibitor taxoid-based ADCs. based ADCs containing quaternary ammonium linkers; (C) the tubulin inhibitor taxoid-based ADCs. 3.4. Tubulysins as ADC Payloads 3.4. Tubulysins as ADC Payloads Tubulysins are cytotoxic natural products, originally isolated from myxobacterial cultures [116]. TubulysinsStructurally, are tubulysins cytotoxic are natural tetrapeptides, products, composed originally of D-methylpipecolate isolated from myxobacterial (D-Mep), L-isoleucine cultures (L- [116]. Structurally,Ile), L-tubuvaline tubulysins (L-Tuv), are tetrapeptides,and L-tubuphenylalanine composed (L-Tup) of D-methylpipecolate residues. Among them, (D-Mep), tubulysinL-isoleucine D (12) (L-Ile),(FigureL-tubuvaline 5) possesses (L-Tuv), the most and L potent-tubuphenylalanine activity with IC (L50-Tup) values residues. between Among0.01 and them, 10 nM, tubulysin exceeding D (12) (Figureother5) possesses tubulin modifiers the most including potent activity epothilones, with vinbla IC 50 valuesstine, and between paclitaxel 0.01 (Taxol) and 10 by nM, 20- to exceeding 1000-fold. other tubulinThe modifiers mechanism including of action epothilones,of tubulysins resembles vinblastine, peptide and paclitaxelantimitotics (Taxol) dolastatin-10, by 20- phomopsin to 1000-fold. A, The mechanismand hemiasterlin, of action ofinducing tubulysins the depletion resembles of cell peptide microtubules, antimitotics arresting dolastatin-10, cells in the G phomopsin2/M phase, and A, and

Molecules 2017, 22, 1281 15 of 28

hemiasterlin, inducing the depletion of cell microtubules, arresting cells in the G2/M phase, and finally triggering cell apoptosis. Interestingly, microtubule depolymerization by tubulysins could not be prevented by preincubation with epothilone B and paclitaxel. In competition experiments, tubulysins interfered with vinblastine binding to tubulin in a non-competitive manner. In addition, tubulysins are less effective substrates of P-gp in MDR cancer cell lines [117]. Based on computer-aided drug design and biological electronic principles, a series of tubulysin derivatives with good biological activity have been synthesized. The clinical pharmacological data of these compounds have not yet been reported. Tubulysins are highly toxic tubulin-targeting agents with a narrow therapeutic window, making them interesting for target cancer therapy. Folate, ADCs, and polymeric tubulysin-peptide targeting nanoparticles enable tubulysins to achieve wider therapeutic indexes and improve their aqueous solubility [118–121]. Li et al. developed a biparatopic HER2-targeting ADC using tubulysin as payload, which showed superior antitumor activity over T-DM1 in tumor models representing various patient subpopulations [120]. The recently developed tubulysin ADCs with a novel quaternary ammonium linker have great stability profiles and potent anticancer activity based on in vitro and in vivo evaluation (Scheme6B) [ 93]. Other than this, tubulysin ADCs are quite different from MMAE/MMAF and DM1/DM4 ADCs. The specific structural requirements of the tubulysin ADCs for optimal activity are still ambiguous at this stage. Further research on tubulysins as the ADC payloads may provide additional options for anticancer treatment. SAR studies on tubulysins are shown in Figure5: (a) the amine group of the Mep residue at the N-terminus is essential for activity; (b) the (R)-configuration of the O-acetate is preferred, and it influences tubulin assembly; (c) N,O-acetal groups can be eliminated without affecting cellular activity, and introducing a hydrophobic group improves bioactivity; and (d) the carboxylic acid group and the 3R configuration in the fragment D are essential for the activity [122,123].

3.5. Hemiasterlins as ADC Payloads Hemiasterlin (13, Figure5), a natural product, is a member of a small family of cytotoxic tri-peptides that were initially isolated from marine sponges [124]. Hemiasterlin and its synthetic analog HTI-286 (14) bind to the vinca binding site on the tubulin, inhibit the polymerization, and trigger mitotic arrest and subsequent apoptosis [125]. Studies demonstrated that hemiasterlin and its analogs stabilize the binding of colchicines to tubulin. They also inhibit the binding of vinblastine and dolastatin-10 to tubulin in noncompetitive and competitive manners, suggesting that hemiasterlin and dolastatin toxins share common microtubule protein binding sites, which is consistent with recently published structural biologic data [126,127]. Total synthesis of hemiasterlin and its analogs has been accomplished, and the SAR studies showed that the olefin at C2 and C3, the L-isopropyl and the L-methyl amino group at C4, and the geminal β,β-dimethyl group are all essential for activity. The N-methylindole could be replaced by phenyl and methyl groups, while still retaining potent antimitotic activity. HTI-286 and its analogue with a para-methoxyphenyl group are even more potent than hemiasterlin [128]. HTI-286 is less sensitive to P-gp protein than the current antimicrotubule agents such as paclitaxel, docetaxel, vinorelbine, and vinblastine. Phase I clinical trials in cancer patients dosed once every 21 days showed no objective responses. Side effects of HTI-286 include neutropenia, hair loss, and pain [129–131], leading to ultimate termination of phase II trials [130]. However, the preclinical evaluation of ADC micelle systems using hemiasterlins as payloads showed potent cytotoxicity against a broad range of tumor cells, reduced toxicity, and excellent therapeutic window. Hemiasterlin ADC is advanced as a potential clinical candidate for cancer therapy [132].

3.6. Paclitaxel and Docetaxel as ADC Payloads Paclitaxel (Taxol, 15, Figure5), an amitotic inhibitor that binds and stabilizes microtubules, was first separated from the bark of the rare Pacific yew, later characterized by Wani et al. [133]. Paclitaxel Molecules 2017, 22, 1281 16 of 28 and docetaxel (16) represent the taxane family of drugs, which showed remarkable efficacy against advanced solid tumors such as ovarian and breast cancer. In 1991, Holmes et al. reported the first clinical trial of paclitaxel to treat MBC with 250 mg/m2 over 24 h, and a 57% overall response was observed [134]. Despite these positive therapeutic features, paclitaxel seriously suffered from the lack of tumor specificity, MDR, poor aqueous solubility and dose-limiting toxicities including alopecia, nausea and vomiting, joint and muscle pain, peripheral neuropathy, and bone marrow suppression. To attenuate these side effects, many research groups have proposed that chemical coupling of mAbs with paclitaxel or its analogs would allow target delivery to tumor cells with increased efficacy and reduced side effects [135–137]. The first paclitaxel ADC with a cleavable ester linker was prepared by linking the paclitaxel C20 hydroxyl group to glutaric anhydride to give 20-glutarylpaclitaxel, whose carboxylic acid group then formed a peptide bond with the antibody. The resulted ADC showed better cytotoxic activity in vitro than paclitaxel or paclitaxel plus mAb, however, it showed only limited efficacy in vivo. In addition, Ojima et al. developed an EGFR-targeting ADC using a second-generation taxoid (C-10 methyl-disulfanylpropanoyl taxoid) as payload. This ADC showed remarkable target-specific antitumor activity in vivo (Scheme6C) [ 136]. However, it remains elusive whether the effects are truly due to immunologically specific drug delivery or not. In short, the paclitaxel-based ADCs did not advance into clinical trials since paclitaxel and its analogs display insufficient cytotoxicity in many cell lines. It has thus been suggested that they are not suitable for ADCs. The development of next generation taxoids with IC50 of 10–100 pM will be the research direction using paclitaxel analogs as payloads. The structure-activity relationship studies of paclitaxel showed that C-3’ with an alkyl or alkenyl group, C3’-N with a tert-butoxycarbonyl group, and acylation at C-10 are important to improve its activity against drug-resistant cancer cells. In addition, the benzoate at the C-2 can be replaced by alkenyl and alkyl groups, which provide nonaromatic second-generation toxoids [138].

3.7. Other Tubulin Inhibitors as Potential ADC Payloads Other tubulin inhibitors (Figure5) investigated include taccalonolide A or B ( 17 or 18), taccalonolide AF or AJ (19 or 20), taccalonolide AI-epoxide (21), discodermolide (22), epothilone A(23) and B (24), laulimalide (25), colchicine (26), CA-4 (27), and their respective synthetic derivatives. Although these compounds were not successful while being used alone as anticancer agents, they had the potential to be used as ADC payloads. Discodermolide remains the most potent natural promoter of tubulin assembly, is effective against paclitaxel-resistant cell lines, and also has synergistic effects with paclitaxel [139,140]. The epoxidation of taccalonolide yielded its analogs with strong cytotoxicity and unique interaction mechanisms (specifically, covalent bonding) to tubulin; thus, these are promising candidates for developing a new generation of irreversible tubulin inhibitor-based ADC drugs [141–143]. Epothilone, laulimalide, colchicine, and CA-4 are easy to be chemically modified, and they are not P-gp substrates and are active against P-gp related drug-resistance. As a result of being introduced to better conjugation groups (sites), these inhibitors could possibly be used as ADC payloads [144–148]. Future studies on tubulin inhibitor-based ADC payloads may help elucidate the drug resistance mechanisms and thus allow the design of drugs that are active against drug-resistant tumor cells but not P-gp substrates. In addition, all tubulin inhibitors so far target β-tubulin; discovering compounds with different tubulin inhibition mechanisms (such as α-tubulin inhibition) will open alternative avenues and provide a new class of payloads with different tolerability and therapeutic index from currently available ADCs [149]. Another direction may take advantage of hybrid payloads targeting different binding sites on tubulin simultaneously to overcome the intrinsic limitations of drug resistance and to improve the activity of payload drugs synergistically. The tubulin inhibitor-based ADCs in preclinical and/or clinical trials and their corresponding status, therapeutic indications, targets, mAbs, linkers, and payloads are summarized in Table2. Data were generated from the Thompson Pharma Partnering database and clinical trials. Molecules 2017, 22, 1281 17 of 28

Table 2. Summary of the preclinical and clinically tested ADCs employing the maytansinoid, dolastatin, cryptophycin hemiasterlin, tubulysin and taxol as payloads.

ADC Status Therapeutic Area Target mAb Linker Payload Trastuzumab emtansine (T-DM1) Approved Breast cancer HER2 Trastuzumab SMCC DM1 a Hematologic-blood cancer, sarcoma, neuroblastoma, Lorvotuzumab mertansine (IMGN901) Phase II CD56 huN901 SPP DM1 SCLC, MM IMGN529 Phase II NHL CD37 K7153A SMCC DM1 IMGN289 Phase I Squamous Cell carcinoma, NSCLC, solid tumors EGFR J2898A SMCC DM1 AMG 172 Phase I RCC CD27L Anti-CD27L MCC DM1 AMG 595 Phase I Glioma, AA EFGRvIII Anti-EGFRvIII SMCC DM1 MLN-2704 Discontinued Prostate cancer PSMA MLN-591 SPP DM1 Cantuzumab mertansine (HuC242-DM1) Discontinued NSCLC, pancreatic cancer, colorectal cancer, solid tumors CanAg huC242 SPP DM1 Mirvetuximab soravtansine (IMGN853) Phase II Solid tumors, NSCLC, ovarian cancer Folate receptor 1 M9346A sSPDB DM4 a Anetumab ravtansine (BAY94-9343) Phase II NSCLC, Mesothelioma, solid tumors Mesothelin Anti-mesothelin SPDB DM4 Coltuximab ravtansine (SAR3419) Phase II LBCL CD19 huB4 SPDB DM4 BT-062 Phase I/II MM CD138 nBT062 SPDB DM4 SAR-428926 Phase I Solid tumors LAMP-1 853K3 SPDB DM4 SAR-566658 Phase I Solid tumors CA6 huDS6 SPDB DM4

IMGN388 Discontinued Solid tumors Integrin αv Anti-integrin αv SPDB DM4 BIIB015 Discontinued Solid tumors Crypto Anti-cripto SPDB DM4 AVE-9633 Discontinued AML CD33 huMy9-6 Undisclosed DM4 Cantuzumab ravtansine (huC242-DM4) Discontinued Gastric cancer, pancreatic cancer CanAg huC242 SPDB DM4 Adcetris (SGN-35) Approved HL, ALCL CD30 cAC10 (SGN-30) VC MMAE b Glembatumumabvedotin (CDX-011) Phase II Breast cancer, melanoma, osteosarcoma, NSCLC GPNMB CR-011 VC MMAE Pinatuzumab vedotin (DCDT2980S) Phase II NHL, DLBCL CD22 Anti-CD22 VC MMAE Polatuzumab vedotin (DCDS4501A) Phase II NHL, DLBCL, CLL CD79b Anti-CD79b VC MMAE Lifastuzumab vedotin (DNIB0600A) Phase II Ovarian cancer, NSCLC NaPi2b Anti-NaPi2b VC MMAE PSMA ADC Phase II Prostate cancer PSMA Anti-PSMA VC MMAE DMOT-4039A Phase I Pancreatic cancer, ovarian cancer MSLN MMOT-0530A VC MMAE AGS-22M6E (ASG-22CE) Phase I Genitourinary cancer, solid tumors Nectin-4 Anti-Nectin-4 VC MMAE SGN-LIV1A Phase I Breast cancer LIV1 Anti-LIV1 VC MMAE HuMax-TF-ADC Phase I Solid tumors tissue factor TF-011 VC MMAE AGS-15E Phase I urothelial SLITRK6 AGS15 VC MMAE DLYE-5953A Phase I breast cancer, NSCLC, solid tumors LY6E Anti-Ly6E VC MMAE DEDN-6526A Phase I Melanoma EDNRB Anti-EDNRB VC MMAE Molecules 2017, 22, 1281 18 of 28

Table 2. Cont.

ADC Status Therapeutic Area Target mAb Linker Payload AGS-67E Phase I Hematologic-blood cancer, AML CD37 Anti-CD37 VC MMAE MLN-0264 Discontinued Pancreatic cancer, gastric cancer GCC 5F9 VC MMAE ASG-5ME Discontinued Pancreas cancer, Prostate cancer, gastric cancer AGS-5 Anti-AGS-5 VC MMAE Bay 79-4620 Discontinued Solid tumors CA9 3ee9 VC MMAE Sofituzumab vedotin (RG-7458) Discontinued Ovarian cancer, pancreatic cancer MUC16 Anti-MUC16 VC MMAE Vandortuzumab vedotin (DSTP-3086S) Discontinued Prostate cancer STEAP1 Anti-STEAP1 VC MMAE Denintuzumab mafodotin Phase II DLBL, follicular lymphoma CD19 Anti-CD19 MC MMAF b (SGN-CD19A) AGS-16M8F Phase II RCC ENPP3 Anti-AGS-16 MC MMAF Glioblastoma multiforme, NSCLC, brain cancer, solid Depatuxizumab mafodotin (ABT-414) Phase II EGFRvIII ABT-806 MC MMAF tumors ARX-788 Phase I Breast cancer Her2 Anti-Her2 PEG4 MMAF J6M0-mcMMAF (GSK-2857916) Phase I MM BCMA J6M0 MC MMAF Vorsetuzumab mafodotin (SGN-75) Discontinued NHL, RCC CD70 HIF6 MC MMAF PF-06263507 Discontinued Solid tumors 5T4 Anti-5T4 MC MMAF MEDI-547 Discontinued Ovarian cancer, solid tumors EphA2 1C1 MC MMAF Hu2H11-SPDB-Ex1 Preclinical Breast cancer, colon cancer, EphA2 hu2H11 SPDB Cryptophycin analogue c Hu2H11-SPDB-Ex2 Preclinical Breast cancer, colon cancer, EphA2 hu2H11 SPDB Cryptophycin analogue Hu2H11-SPDB-Ex5 Preclinical Breast cancer, colon cancer, EphA2 hu2H11 SPDB Cryptophycin analogue Hu2H11-SPDB-Ex6 Preclinical Breast cancer, colon cancer, EphA2 hu2H11 SPDB Cryptophycin analogue Hu2H11-Ex17 Preclinical Breast cancer, colon cancer, EphA2 hu2H11 Undisclosed Cryptophycin analogue Hu2H11-Ex18 Preclinical Breast cancer, colon cancer, EphA2 hu2H11 Undisclosed Cryptophycin analogue Hu2H11-Ex19 Preclinical Breast cancer, colon cancer, EphA2 hu2H11 Undisclosed Cryptophycin analogue VDC-886 Preclinical Prostate cancer, breast cancer, NHL pl-CS DBL1-ID2a Undisclosed KT886 d PEG-poly(glutamic NC-6201 Preclinical Breast cancer, pancreatic cancer, colon cancer EGFR NCAB001 E7974 d acid benzyl ester) MEDI-4276 (AZ13599185-Trastuzumab) Phase I Breast cancer, gastric cancer HER2 39S MC AZ13599185 e 131I-TUB-OH-Trastuzumab Preclinical Breast cancer HER2 Trastuzumab - TUB-OH e 131I-TUB-OMOM-Trastuzumab Preclinical Breast cancer HER2 Trastuzumab - TUB-OMOM e DX-109 Preclinical Breast cancer HER2 anti-HER2 Thioether Tub-001 e Quaternary αCD30–glucQ-Tub Preclinical HL, ALCL CD30 AC10 Tubulysin M e Ammonium Hemisuccinamide- α-CEA-680-PTX Preclinical Pancreatic cancer CEA Anti-CEA DyLight™ 680 − Paclitaxel f 4 × PEG Note: payload based on a maytansinoid; b dolastatin; c cryptophycin; d hemiasterlin; e tubulysin and f taxol. The reasons for discontinued ADCs as follows: MLN-2704: sufficient therapeutic window was not achievable; Cantuzumab mertansine (HuC242-DM1): unforeseen or unacceptable toxicities; IMGN388: strategic reasons; AVE-9633: absence of evidence of clinical activity; Cantuzumab ravtansine (huC242-DM4): ocular toxicities; MLN-0264: lack of efficacy; ASG-5ME: strategic reasons; Bay 79-4620: safety reasons; some others: unknown. Molecules 2017, 22, 1281 19 of 28

4. Conclusions and Final Remarks Encouraged by the in-depth understanding of ADCs for targeted therapy in oncology and the approvals of SGN-35 and T-DM1 by the FDA, ADCs are rapidly moving from empiricism to rational design. Nevertheless, ADCs are far from fulfilling the concept of a “magic bullet” specifically delivering cytotoxic drugs to tumor cells. Additionally, toxicity of ADCs to normal cells/tissues caused by undesired payload release needs to be considered. Studies have indicated that the delivery efficacy of ADCs into the target tumor cells was far less than 1% [150]. The limitations of ADCs must be addressed by future studies to propel this concept forward into relevant clinical regimens. Successful development of ADCs requires vast understanding of each ADC component, connecting each part with sufficient efficiency, expanding the therapeutic index, and improving the PK/PD profile. In addition, a deeper understanding of antigen targets, mechanisms of internalization, roles of novel linkers, executions of appropriate antibodies, and properties of the payload (such as cytotoxicity, specificity, solubility, and stability) are all essential criteria for the overall efficacy of ADCs. Advancement and implementation of site-specific conjugation technologies must be investigated and used to improve the safety and efficiency of ADCs as well. In short, the convergence of a thorough understanding of ADC-related technologies will be the driving force behind the design and development of novel ADCs, which will be promising tools for targeted cancer therapy in the future.

Acknowledgments: This work is partially supported by NIH/NCI grants R01CA148706 to WL and DDM. Zongtao Lin would like to thank the Alma and Hal Reagan Fellowship from the University of Tennessee Health Science Center. We thank Richard Redfearn at UTHSC for editorial assistance. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH/NCI. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

ADCs Antibody-drug conjugates ALCL Anaplastic large-cell lymphoma AML Acute myelogenous leukemia BCMA B-cell maturation antigen CA9 Carbonic anhydrase IX CEA Carcinoembryonic antigen CLL Chronic lymphocytic leukemia DAR Drug-to-antibody ratio DLBCL Diffuse large B cell lymphoma DTT Dithiothreitol EDNRB Endothelin receptor type B EGFR Epidermal growth factor receptor ENPP3 Ectonucleotide pyrophosphatase/phosphodiesterase family member 3 EphA2 Ephrin type-A receptor 2 EphB2 Ephrin type-B receptor 2 FDA Food and Drug Administration GCC Guanyl cyclase C GPNMB Transmembrane glycoprotein NMB GTP Guanosine triphosphate HER2 Human epidermal growth factor receptor 2 HL Hodgkin’s lymphoma IgG Immunoglobulin G LBCL Large B-cell lymphoma LY6E Lymphocyte antigen 6 complex mAb Monoclonal antibody MBC Metastatic breast cancer MC Maleimidocaproyl Molecules 2017, 22, 1281 20 of 28

MCC 4-(N-Maleimidomethyl)cyclohexane-1-carboxylate MDR Multiple-drug resistant MDR1 Multidrug resistance protein 1 MM Multiple myeloma MMAE Monomethyl auristatin E MMAF Monomethyl auristatin F MTD Maximum tolerated dose MUC16 Mucin 16 NHL Non-Hodgkin’s lymphoma NSCLC Non-small cell lung cancer PEG Polyethylene glycol PK/PD Pharmacokinetic/pharmacodynamics PSMA Prostate specific membrane antigen RCC Renal cell carcinoma SAR Structure-activity relationship SCLC Small-cell lung cancer SLITRK6 SLIT and NTRK-like protein 6 SMCC Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate SPDB N-Succinimidyl-4-(2-pyridyldithio)butanoate SPP N-Succinimidyl 4-(2-pyridyldithio) pentanoate sSPDB Sulfo-SPDB STEAP1 Six transmembrane epithelial Antigen of prostate 1 TAG-72 Tumor-associated glycoprotein-72 vc Valine-citrulline

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Sample Availability: Samples of the compounds are not available from the authors.

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