Antibody-drug conjugates (ADCs) are novel targeted chemotherapeutic agents that utilize the specificity of monoclonal antibodies (mAbs) to deliver potent cell-killing agents to cancer cells that express the target antigen, thereby taking advantage of the best characteristics of both components. The experience and/or lessons learned from the first generation of conjugates have guided the development of more effective antitumor agents. Along with the development of the mAbs and cytotoxins, the design of chemical linkers to covalently bind these building blocks is making rapid progress but remains challenging. Encouragingly, the recent successes in these domains show that the next generation of antibody-drug conjugates has come of age.

•    Antibody−drug conjugates
•    Targeted cancer therapy
•    Linkers
•    Site-specific conjugation

Wang Z, Guravaiah N, Ning C, He Y, Yao L, et al. (2015) Antibody-Drug Conjugates: The Forefront of Targeted Chemotherapy for Cancer Treatment. J Drug Des Res 2(3): 1016.

ADC: Antibody−Drug Conjugate; AML: Acute Myeloid Leukaemia; HL: Hodgkin’s Lymphoma; NHL:Non-Hodgkin’s Lymphoma; ALCL: Anaplasia Large Cell Lymphoma; DLBCL: Diffuse Large B Cell Lymphoma; RCC: Renal Cell Carcinoma; SCLC: Small Cell Lung Cancer; NSCLC: Non-Small Cell Lung Cancer; CLL: Chronic Lymphocytic Leukaemia; EGFR: Epidermal Growth Factor Receptor; GPNMB: Glycoprotein NMB; PSMA: ProstateSpecific Membrane Antigen; NA: No Available

For cancer treatment, it is highly desirable to selectively target malignant cells instead of healthy tissues. However, cancer treatment usually remains a double-edged sword in the sense that therapeutic agents should be as aggressive as possible to kill the tumor cells, but it is precisely this aggressiveness that often causes severe side effects. It is for this reason that some promising chemotherapeutics cannot be applied systemically [1]. The therapeutic effect of most anticancer drugs in clinical use is limited by their general toxicity to proliferating cells, including some normal cells. Despite the fact that chemists constantly strive toward developing novel cytotoxic agents with unique mechanisms of action, many of these compounds still lack tumor selectivity and have not been therapeutically useful [2]. One promising approach that has been advanced through a series of preclinical and clinical studies is to combine the pharmacological potency of “small” cytotoxic drugs (0.3 to 1 kDa) and the high specificity of monoclonal antibodies (150 kDa) for tumor-associated antigen targets [3,4]. In this therapeutic strategy, different classes of cytotoxic agents are covalently linked to tumor-targeted monoclonal antibodies (mAbs) forming corresponding antibody−drug conjugates (ADCs; Figure 1).

Figure 1: Key characteristics of antibody-drug conjugates.

Antibody-drug conjugates are ideal candidates for targeted prodrug therapy. These types of drugs are vital members of the next generation of antibody-based therapy by taking advantage of specific molecular differences between healthy and diseased tissues. ADCs can be viewed as sophisticated delivery systems for antitumor cytotoxic drugs. This class of product typically requires mAbs that are internalized inside cells via receptor-mediated endocytosis. The mAb delivers drugs selectively to tumors by binding to antigens that are highly expressed on cancer cells, followed by internalization and intracellular drug release, which then unleashes its cytotoxic activity [5].

Clay B. Siegall (Seattle Genetics president and CEO) notes that ADCs offer formidable theoretical advantages over conventional chemotherapy in that they attach specifically to tumor cells with special target receptors while not affecting healthy cells that do not have relevant receptors. This strategy means that the cytotoxic agents delivered by ADCs can be much more potent than systemic chemotherapeutic agents, which do not discriminate between cancer cells and healthy cells. As such, intense research focused on optimization to increase the therapeutic indices of ADCs has been the spotlight of target cancer therapy. A safe and effective ADC such as brentuximab vedotin (Adcetris) should have many properties: antibody specificity, avidity and affinity for the antigens expressed on the surface of a cancer cell; antibody internalization so the ADC gets inside cancer cells; cytotoxin potency so the cancer cell is killed or stops dividing; and linker stability so the antibody and the cytotoxin stay together in the bloodstream but are cleaved inside a tumor cell to activate the drug payload (Figure 1) [6]. Each of these properties is essential, and trying to optimize one invariably means making changes to technology that has been created.

The ADC is exposed to different conditions on its journey from the blood vessels to the molecular target in the tumor tissue. The mode of action at the cellular or molecular level is complex, with each step bringing its own challenges. ADCs must behave like a naked antibody when circulating in the bloodstream. In particular, the linker must be stable in the plasma due to decomposition or decay would release the cytotoxin before it can be delivered to the target site, which will inflict damage to healthy tissue. It is also necessary that the conjugated mAb retains high immunoaffinity. Attaching the cytotoxic compound to the mAb must not disturb its binding specificity [7,8]. Moreover, a sufficient intracellular concentration of the drug must be achieved. This is challenging because antigen targets on cell surfaces are often present in limited numbers and the internalization process for antigen-antibody complexes is frequently inefficient. Once internalized, the ADC is delivered to lysosomes, although there are some exceptions, where effective drug release takes advantage of the catabolic environment found within these organelles. Following release from the lysosome, the drug either binds to its pharmacological target or leaves the cell via active or passive processes [9]. ADCs have demonstrated the intracellular accumulation of released drug in antigen-positive cells but no intracellular accumulation in antigen negative cells, highlighting the antigen specificity of drug delivery by ADCs (Figure 2).

Figure 2: Mechanisms of drug delivery mediated by ADCs.

There have also been some reports that poorly internalized antigens can successfully be targeted with ADCs by potentially utilizing drug release strategies that occur in the tumor microenvironment rather than inside cells. For example, sub endothelial modified ECM components may serve as a target for ADCs, which do not require internalization for their activity [10].

Here, we will briefly discusses the low-molecular-weight cytotoxic drugs used in ADCs, the selection of tumor-specific antigens as targets, linker technologies and emerging technologies that seek to further advance this exciting area of research.

Drug payload

In spite of the extraordinary mode of action and potency of many cytotoxins, their development as single agent therapies has not been pursued because of delayed toxicity which limits the therapeutic dose range for treatment [11]. However, natural cytotoxins are well suited for the role of ADC payload because they often possess the potency, mechanism of action, chemical tractability, and structural properties required for effectiveness [12].Tumor-specific antibodies have been conjugated to these cytotoxic drugs, including small molecules that alkylate DNA, disrupt microtubules or bind DNA [6,13].

On the basis of the knowledge gained from first-generation ADCs, the following parameters in the cytotoxic component of the ADC were deemed to be highly pivotal for optimal activity. (1) The cytotoxic component was less immunogenic than toxins (e.g., ricin) that may be required; [14] (2) High potency of the effector molecule in vitro toward tumor cell lines, with IC values in the range of 0.01–10 nM. The use of ADCs bearing more highly potent effectors will increase the probability of delivering a therapeutic dose to tumor cells that have low antigen expression or have poor processing; [12,13,15] (3) A suitable functional group for linkage to an antibody. If a functional group is not already present, the desired substituent should be introduced at a suitable site to retain the potency of the parent drug. The key requirement was that the nature and position of the substituent would not diminish the potency of the parent molecule; [13] (4) The drug component of ADC should also be stable during preparation or storage and during circulation in a patient; [14] (5) Solubility and membrane permeability [16].

Promising data have been reported for ADCs composed of highly potent drugs such as calicheamicins, doxorubicin, maytansinoids, auristatins, and camptothecin. With much effort, important advances have been made on exploiting new drug types used in ADCs. For instance, pyrrolobenzodiaze-pine (PBD) dimer, α-Amanitin, duocarmycin analogs,Tubulysin B, cryptophycin analogs and soon have been explored as potent payloads for antibody-drug conjugates (Figure 3) [17-19].

Figure 3: New drug classes for ADCs.

Tumor-associated antigen selection

The goal of targeted therapy with mAb-drug conjugates is to achieve high degrees of therapeutic activity while sparing normal tissues from chemotherapeutic damage. Toward this end, the choice of the appropriate target antigen for ADCs is a critical parameter that affects the efficacy, therapeutic window, and toxicity profile of ADCs [20]. Ideally, the target antigens should be abundant and accessible. They should be expressed homogeneously, consistently and exclusively on the surface of cancer cells [21].It is crucial that antigens should have high tumor cell selectivity to limit toxicity and off-target effects[4]. Data from clinical trials of bivatuzumab mertansine suggested that tumor selectivity of the antibody is even more important than antigen density[22].In addition, antigen secretions should be minimal, as secreted antigens can bind the antibody in the circulation and could prevent sufficient antibody from binding to the tumor [23].. These desirable properties of antigens can lead to greater ADC potency, but they are rarely achieved [24].

Although homogenous tumor expression is preferred, it is likely not an absolute requirement owing to the cell-lethal ability of some ADCs to induce bystander killing [25,26] or by combining an ADC with another therapeutic modality, such as cytotoxic chemotherapy[27].Especially on solid tumors with heterogeneous antigen expression, the bystander effect is one of the major mechanisms of action, as demonstrated by HuC242- DM1 in vivo xenograft studies with a heterogeneously expressed antigen, Can Ag [28]. Certainly, antigen expression on normal tissue can lead to on-target toxicity with ADCs in patients, but inefficient ADC localization to antigen-positive normal tissue is presumed to lower the risk of on-target toxicity [24,29]. Interestingly, the expression of antigen in some normal tissues does not necessarily preclude the development of an ADC. Prostate-specific membrane antigen (PSMA) is, for example, expressed on normal prostate and on prostate cancer cells, but it still has been applied in several ADC programs [25,30].This is the case when the normal tissue is either non-essential or insensitive to the action of the drug [31].

Another major and critical property of ADC targets is their ability to be internalized, which can be an intrinsic feature of the antigen by itself or it can be induced by the binding of the antibody to its antigen.4 Indeed, ADC internalization is crucial to reduce the side effect associated with the extracellular delivery of the drug payload. HER2 (target of T–DM1), for example, is internalized fairly quickly, which is also important, and it is not down regulated. Thus, once bound to the ADC antibody, HER2 does not disappear from the cell but keeps being produced. This factor is of prime interest to ensure specific drug internalization in cancer cells. To date, HER2 has been chosen as the target for the currently most clinically advanced ADCs in HER2-positive patients who are refractory to trastuzumab [4,32,33].

Conjugation technologies

It was recognized early on that conjugation technology is a critical aspect in generating effective ADCs, and optimization strategies can vary with the drug payload, the linker selected, and the antibody used, as conjugation methods significantly affect the potency, selectivity and the pharmacokinetics of the resulting conjugate [34].Traditionally, the cytotoxic agent is attached to the monoclonal antibody by a chemosynthetic linker. The linker should be sufficiently stable while in circulation to allow delivery of the intact ADC to the tumor sites, but, conversely, it should be sufficiently labile to allow release of the cytotoxic agent from the ADC once inside the targeted tumor cells [35]. The development of stable linkers together with the identification of relevant biomarkers has been a key to the success of a series of ADCs approved or in clinical trials [36].

Cytotoxic drugs are most often linked to the antibody via lysine or cysteine residues on the antibody by chemical linkers. In the case of lysine attachment, the linkage of effector molecules to the antibody could theoretically occur through the ε-amino group of lysine residues present on an antibody. In the case of cysteine attachment, the effector can be linked to reactive cysteine residues, activated by the reduction of internal hinge region cysteine disulfide bonds of the antibody or cysteine disulfides in the light chains to linking the heavy chains of the antibody [37-40]. The common methods for conjugating mAbs include alkylation of reduced interchain disulfides, acylation of lysines, and alkylation of genetically engineered cysteines. Stability of the drug-linker in circulation is important because it determines the long circulating half-life of the ADCs. Linkers differ in terms of plasma stability and in the mechanism of release [41]. Based on these differences, there are currently four different classes of linkers used in the ADCs approved or in clinical trial that broadly fall under two categories—cleavable and noncleavable linkers[42]. Moreover, some novel linkers, such as traceless linkers, are being exploited and evaluated [10].

After ADC internalization into the target cell, an intracellular release mechanism should cleave the linker to release the active drug. For cleavable linkers, the payload is released when the linker is cleaved by lysosomal enzymes or by the chemical environment of the intracellular compartment. Cleavable linkers usually release free drug or a simple derivative by hydrolytic or proteolytic means [2,43,44]. For non-cleavable linkers, the ADC is catabolized by proteases. Once in the lysosome, the antibody is fully catabolized while the linker remains intact, resulting in a released species that consists of the payload-linker bound to the remaining conjugated amino acid residue [43,44].

Traditionally, the drug is conjugated non-selectively to solvent-accessible cysteine or lysine residues in the antibody [45]. The procedure can generate a heterogeneous mixture of up to 106 species with different molar ratios of drug to antibody ranging between 0 and 9 for the loading of warhead molecules, linked at different sites, each with distinct in vivo pharmacokinetic, efficacy and safety profiles [46-48]. These factors made the optimization of the biological, physical, and pharmacological properties of an ADC challenging. On the one hand, coupling at many different sites will lead to a heterogeneous mixture of ADCs, the components of which likely have distinct properties [49].On the other hand, there is limited stoichiometric control because of the large number of disulfide bonds and lysine residues in antibody molecules, which leads to a typical distribution of zero to eight toxins per antibody [47,50]. To assess the impact of drug stoichiometry on therapeutic potential, the investigation of cAC10-MMAE conjugates (anti-CD30 ADCs) with varied drug/antibody ratios was carried out by Seattle Genetics, Inc [51].The experimental design involved coupling MMAE to the cysteines that comprise the interchain disulfides of cAC10, creating an antibody-drug conjugate population, which was purified using hydrophobic interaction chromatography to yield antibody-drug conjugates with two, four, and eight drugs per antibody (E2,E4,and E8). The potency, maximum-tolerated dose and pharmacokinetic profiles of the distinct antibodydrug conjugates were then compared. Although antibody-drug conjugate potency in vitro was directly dependent on drug loading (IC50 values E8

Although, it was discovered that conventional conjugation strategies was liable to yield heterogeneous conjugates with relatively narrow therapeutic indexes [40].The good news is that linker technology is also rapidly improving. To limit these potential liabilities associated with traditional conjugation methods, intense efforts are being devoted to the development of methods for site-specific drug conjugation that will enable the production of homogeneous ADCs with defined sites and stoichiometries of drug attachment and with improved performance and batch-to-batch reproducibility [34,40-41]. The available schemes to generate such homogeneous ADCs includes site-specific conjugation using cysteine/unnatural amino acids [52,53-55],metabolic engineering of antibody carbohydrates for site-specific conjugation [56,57-58], enzymatic site-specific conjugation [59,60-64] and site-specific conjugation via native disulfide bond bridging [65].

Representative ADCs at different stages of clinical development

With approximately 40 antibody-drug conjugates in clinical trials and two FDA-approved drugs, it is clear that ADCs have demonstrated remarkable pre-clinical and clinical efficacy and may at some point obviate the need for systemic chemotherapy [37]. At least 30 ADCs were entered in to clinical trials from different organization in the recent past (2011-2014), it is about 4 folds higher than the drugs entered into clinical trials the in the years of 2008-2010 [66]. Table 1 lists the ADCs that are currently approved and most ADCs in clinical development. Up-to-date information on the clinical research on this topic can be acquired via the Thomson Pharma database and clinical trials. gov (http://clinicaltrials.gov/). After its approval, annual sales of brentuximab vedotin (Figure3) in the United States were US$136 million (October 2011 to September 2012) in a relatively small patient population. Genentech’s ADC ado-trastuzumab emtansine (Figure 4) was approved by the FDA in February 2013 following the positive outcome of the Phase III EMILIA trial [67].

Figure 4: Structures of brentuximab vedotin and ado-trastuzumab emtansine.

Fuelled by these successes, drug discovery companies like Abbott, Pfizer, Genentech, Bayer, Agensys, and Millennium are ramping up development of the burgeoning ADC pipeline. Much of the initial search for uses of ADCs was performed in hematological cancers, and five of eight ADCs in Phase II and III trials have been designed to treat blood cancers. Partly, this imbalance was driven by an early view that ADCs would penetrate poorly into solid tumors. However, the success of trastuzumab-DM1 in metastatic breast cancer has done away with the standpoint that ADCs are only useful against blood cancers. Coincidentally, ImmunoGen is taking a different tack with its mid?stage small cell lung cancer candidate lorvotuzumab mertansine (IMGN901) [12]. In terms of solid tumors, the field is pursuing a broader suite of targets and indications, and making more use of companion diagnostics.

ADC research programs have been increasing quickly in recent years because of the tremendous potential shown by preceding clinical trials. Despite a record of success, many hurdles remain. History has offered a warning to researchers. In 2000, the FDA approved an ADC called Mylotarg for patients with acute myeloid leukemia. However, in June 2010 the drug was withdrawn by its manufacturer (Pfizer) because follow-up studies showed that the drug did not extend survival and was associated with a high rate of fatal toxicity [68]. The main problems of the drug is its linker instability (half-life of the conjugate in blood stream is 48 hours which is short for an ADC), which indicate the importance of linker design and that tremendous efforts in the design and validation of ADCs with higher structural homogeneity should be made [50]. In addition, the characterization of the quantitative assays used to measure ADC concentrations in support of pharmacokinetic, efficacy, and safety studies is of increasing importance [38]. Thankfully, in the effort to extend the technology and identify and optimize key parameters that lead to highly active and well-tolerated ADCs, numerous drug types, linker strategies, antigen targets and antibody formats have been investigated. Owing to improved technology and appropriate targeting, the clinical application of ADCs is accelerating rapidly.

Despite complexities in designing ADCs, the promise of this therapeutic class has generated intense interest in recent years. With so many companies seeking access to ADC technology and strengthening cooperation, the field looks poised to takeoff. Evolving clinical data will continue to drive technological advancements in the field, and further insight into the optimal design characteristics of effective ADCs will be best gained from these clinical datas. Progress in site-specific conjugation approaches, optimization of linkers with balanced stability, identification of novel targets, application of new cytotoxic agents and monitoring ADCs by dual radiolabelling [69] may pave the way for greater insight into the contribution of these various factors to ADC efficacy, safety and PK properties. Much work is underway to incorporate unnatural amino acids into the antibodies, which will allow novel connection chemistry to impact the nature of the ADCs and to investigate novel targets against solid tumors, such as antigens expressed selectively on the tumor vasculature. In the future, both the development of new ADCs and the exploration of combination treatments involving ADCs and other antineoplastic agents will continue and grow. Of course, the question whether the ADC technology can be applied beyond oncology to broader indications, such as immune disease, should be expounded.

Table 1: Antibody-Drug Conjugates currently in approval or in clinical trials.

This work was supported by the National Natural Science Foundation of China (81172927) and Hisun Pharmaceutical, China.

1. Schrama D, Reisfeld RA, Becker JC. Antibody targeted drugs as cancer therapeutics. Nat Rev Drug Discov. 2006; 5: 147-159.

2. Chari RV. Targeted cancer therapy: conferring specificity to cytotoxic drugs. Acc Chem Res. 2008; 41: 98-107.

3. Zhao RY, Erickson HK, Leece BA, Reid EE, Goldmacher VS, Lambert JM, et al. Synthesis and biological evaluation of antibody conjugates of phosphate prodrugs of cytotoxic DNA alkylators for the targeted treatment of cancer. J Med Chem. 2012; 55: 766-782.

4. Beck A, Haeuw JF, Wurch T, Goetsch L, Bailly C, Corvaia N. The next generation of antibody-drug conjugates comes of age. Discov Med. 2010; 10: 329-339.

5. Kularatne SA, Deshmukh V, Ma J, Tardif V, Lim RK, Pugh HM, et al. A CXCR4-targeted site-specific antibody-drug conjugate. Angew Chem Int Ed Engl. 2014; 55:11863-11867.

6. Carlson B. Antibody-Drug Conjugates: Where the Action Is: ADCs - The New Frontier. Biotechnol Healthc. 2012; 9: 28-31.

7. Ducry L, Stump B. Antibody-drug conjugates: linking cytotoxic payloads to monoclonal antibodies. Bioconjug Chem. 2010; 21: 5-13.

8. Panowksi S, Bhakta S, Raab H, Polakis P, Junutula JR. Site-specific antibody drug conjugates for cancer therapy. MAbs. 2014; 6: 34-45.

9. Alley SC, Okeley NM, Senter PD. Antibody-drug conjugates: targeted drug delivery for cancer. Curr Opin Chem Biol. 2010; 14: 529-537.

10. Casi G, Neri D. Antibody-drug conjugates: basic concepts, examples and future perspectives. J Control Release. 2012; 161: 422-428.

11. Hamann PR, Hinman LM, Beyer CF, Greenberger LM, Lin C, Lindh D, et al. An anti-MUC1 antibody-calicheamicin conjugate for treatment of solid tumors. Choice of linker and overcoming drug resistance. Bioconjug Chem. 2005; 16: 346-353.

12. Koehn FE. Natural Products and Cancer Drug Discovery; Teicher, B. A. Eds; 2013.

13. Gromek SM, Balunas MJ. Natural products as exquisitely potent cytotoxic payloads for antibody- drug conjugates. Curr Top Med Chem. 2015; 14: 2822-2834.

14. Alley SC, Benjamin DR, Jeffrey SC, Okeley NM, Meyer DL, Sanderson RJ, et al. Contribution of linker stability to the activities of anticancer immunoconjugates. Bioconjug Chem. 2008; 19: 759-765.

15. Kozak KR. Protein Chemistry; Phillips. Eds.; Springer, 2013; 3.

16. Kingston DG, Snyder JP. The quest for a simple bioactive analog of paclitaxel as a potential anticancer agent. Acc Chem Res. 2014; 47: 2682-2691.

17. Jeffrey SC, Burke PJ, Lyon RP, Meyer DW, Sussman D, Anderson M, et al. A potent anti-CD70 antibody-drug conjugate combining a dimeric pyrrolobenzodiazepine drug with site-specific conjugation technology. Bioconjug Chem. 2013; 24: 1256-1263.

18. Coccia MA, Terrett JA, King DJ, Pan C, Cardarelli J, Henning, AK. US Patent. 2010.

19. Low PS. Kularatane SA. US Patent. 2010.

20. Mathur R, Weiner GJ. Picking the optimal target for antibody-drug conjugates. Am Soc Clin Oncol Educ Book. 2013.

21. Damelin M, Zhong W, Myers J, Sapra P. Evolving Strategies for Target Selection for Antibody-Drug Conjugates. Pharm Res. 2015.

22. Riechelmann H, Sauter A, Golze W, Hanft G, Schroen C, Hoermann K, et al. Phase I trial with the CD44v6-targeting immunoconjugate bivatuzumab mertansine in head and neck squamous cell carcinoma. Oral Oncol. 2008; 44: 823-829.

23. Scott AM, Wolchok JD, Old LJ. Antibody therapy of cancer. Nat Rev Cancer. 2012; 12: 278-287.

24. Carter PJ, Senter PD. Antibody-drug conjugates for cancer therapy. Cancer J. 2008; 14: 154-169.

25. Perez HL, Cardarelli PM, Deshpande S, Gangwar S, Schroeder GM, Vite GD, et al. Antibody-drug conjugates: current status and future directions. Drug Discov Today. 2014; 19: 869-881.

26. Kovtun YV, Audette CA, Ye Y, Xie H, Ruberti MF, Phinney SJ, et al. Antibody-drug conjugates designed to eradicate tumors with homogeneous and heterogeneous expression of the target antigen. Cancer Res. 2006; 66: 3214-3221.

27. Clavio M, Vignolo L, Albarello A, Varaldo R, Pierri I, Catania G,et al. Adding low-dose gemtuzumab ozogamicin to fludarabine, Ara-C and idarubicin (MY-FLAI) may improve disease-free and overall survival in elderly patients with non-M3 acute myeloid leukaemia: results of a prospective, pilot, multi-centre trial and comparison with a historical cohort of patients. Br. J. Haematol. 2007,138:186-195.

28. Liu C, Tadayoni BM, Bourret LA, Mattocks KM, Derr SM, Widdison WC, et al. Eradication of large colon tumor xenografts by targeted delivery of maytansinoids. Proc Natl Acad Sci U S A. 1996; 93: 8618-8623.

29. Wolff BG, Bolton J, Baum R, Chetanneau A, Pecking A, Serafini AN, et al. Radioimmunoscintigraphy of recurrent, metastatic, or occult colorectal cancer with technetium Tc 99m 88BV59H21-2V67-66 (HumaSPECT-Tc), a totally human monoclonal antibody. Patient management benefit from a phase III multicenter study. Dis Colon Rectum. 1998; 41: 953-962.

30. Wang X, Ma D, Olson WC, Heston WD. In vitro and in vivo responses of advanced prostate tumors to PSMA ADC, an auristatin-conjugated antibody to prostate-specific membrane antigen. Mol Cancer Ther. 2011; 10: 1728-1739.

31. Beck A, Lambert J, Sun M, Lin K. Fourth World Antibody-Drug Conjugate Summit: February 29-March 1, 2012, Frankfurt, Germany. MAbs. 2012; 4: 637-647.

32. Hughes B. Antibody-drug conjugates for cancer: poised to deliver? Nat Rev Drug Discov. 2010; 9: 665-667.

33. Wong DJ, Hurvitz SA. Recent advances in the development of antiHER2 antibodies and antibody-drug conjugates. Ann Transl Med. 2014; 2: 122.

34. Carter PJ, Senter PD. Antibody-drug conjugates for cancer therapy. Cancer J. 2008; 14: 154-169.

35. Zhao RY, Wilhelm SD, Audette C, Jones G, Leece BA, Lazar AC, et al. Synthesis and evaluation of hydrophilic linkers for antibody-maytansinoid conjugates. J Med Chem. 2011; 54: 3606-3623.

36. Burris HA. Developments in the use of antibody-drug conjugates. Am Soc Clin Oncol Educ Book. 2013.

37. Flygare JA, Pillow TH, Aristoff P. Antibody-drug conjugates for the treatment of cancer. Chem Biol Drug Des. 2013; 81: 113-121.

38. Kozak KR, Tsai SP, Fourie-O’Donohue A, Dela Cruz Chuh J, Roth L, Cook R, et al. Total antibody quantification for MMAE-conjugated antibodydrug conjugates: impact of assay format and reagents. Bioconjug Chem. 2013; 24: 772-779.

39. Alley SC, Benjamin DR, Jeffrey SC, Okeley NM, Meyer DL, Sanderson RJ, et al. Contribution of linker stability to the activities of anticancer immunoconjugates. Bioconjug Chem. 2008; 19: 759-765.

40. Garcia-Echeverria C. Developing second generation antibody-drug conjugates: the quest for new technologies. J Med Chem. 2014; 57: 7888-7889.

41. Bernardes GJ, Casi G, Trüssel S, Hartmann I, Schwager K, Scheuermann J, et al. A traceless vascular-targeting antibody-drug conjugate for cancer therapy. Angew Chem Int Ed Engl. 2012; 51: 941-944.

42. Iyer U, Kadambi VJ. Antibody drug conjugates - Trojan horses in the war on cancer. J Pharmacol Toxicol Methods. 2011; 64: 207-212.

43. Chari RV, Miller ML, Widdison WC. Antibody-drug conjugates: an emerging concept in cancer therapy. Angew Chem Int Ed Engl. 2014; 53: 3796-3827.

44. Doronina SO, Mendelsohn BA, Bovee TD, Cerveny CG, Alley SC, Meyer DL, et al. Enhanced activity of monomethylauristatin F through monoclonal antibody delivery: effects of linker technology on efficacy and toxicity. Bioconjug Chem. 2006; 17: 114-124.

45. Wang L, Amphlett G, Blättler WA, Lambert JM, Zhang W. Structural characterization of the maytansinoid-monoclonal antibody immunoconjugate, huN901-DM1, by mass spectrometry. Protein Sci. 2005; 14: 2436-2446.

46. Axup JY, Bajjuri KM, Ritland M, Hutchins BM, Kim CH, Kazane SA, et al. Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc Natl Acad Sci U S A. 2012; 109: 16101-16106.

47. Hamblett KJ, Senter PD, Chace DF, Sun MM, Lenox J, Cerveny CG, et al. Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin Cancer Res. 2004; 10: 7063-7070.

48. Kline T, Steiner AR, Penta K, Sato AK, Hallam TJ, Yin G. Methods to Make Homogenous Antibody Drug Conjugates. Pharm Res. 2014.

49. Shen BQ, Xu K, Liu L, Raab H, Bhakta S, Kenrick M, et al. Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates. Nat Biotechnol. 2012; 30: 184-189.

50. Junutula JR, Raab H, Clark S, Bhakta S, Leipold DD, Weir S, et al. Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat Biotechnol. 2008; 26: 925-932.

51. Hamblett KJ, Senter PD, Chace DF, Sun MM, Lenox J, Cerveny CG, et al. Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin Cancer Res. 2004; 10: 7063-7070.

52. McDonagh CF, Turcott E, Westendorf L, Webster JB, Alley SC, Kim K, et al. Engineered antibody-drug conjugates with defined sites and stoichiometries of drug attachment. Protein Eng Des Sel. 2006; 19: 299-307.

53. Junutula JR, Flagella KM, Graham RA, Parsons KL, Ha E, Raab H, et al. Engineered thio-trastuzumab-DM1 conjugate with an improved therapeutic index to target human epidermal growth factor receptor 2-positive breast cancer. Clin Cancer Res. 2010; 16: 4769-4778.

54. Wada R, Erickson HK, Lewis Phillips GD, Provenzano CA, Leipold DD, Mai E, et al. Mechanistic pharmacokinetic/pharmacodynamic modeling of in vivo tumor uptake, catabolism, and tumor response of trastuzumab maytansinoid conjugates. Cancer Chemother Pharmacol. 2014; 74: 969-980.

55. Cao H, Yamamoto K, Yang LX, Weber R. Brentuximab vedotin: first-line agent for advanced Hodgkin lymphoma. Anticancer Res. 2013; 33: 3879-3885.

56. O’Shannessy DJ, Dobersen MJ, Quarles RH. A novel procedure for labeling immunoglobulins by conjugation to oligosaccharide moieties. Immunol Lett. 1984; 8: 273-277.

57. Laguzza BC, Nichols CL, Briggs SL, Cullinan GJ, Johnson DA, Starling JJ, et al. New antitumor monoclonal antibody-vinca conjugates LY203725 and related compounds: design, preparation, and representative in vivo activity. J Med Chem. 1989; 32: 548-555.

58. Abraham R, Moller D, Gabel D, Senter P, Hellström I, Hellström KE. The influence of periodate oxidation on monoclonal antibody avidity and immunoreactivity. J Immunol Methods. 1991; 144: 77-86.

59. Madej MP, Coia G, Williams CC, Caine JM, Pearce LA, Attwood R, et al. Engineering of an anti-epidermal growth factor receptor antibody to single chain format and labeling by Sortase A-mediated protein ligation. Biotechnol Bioeng. 2012; 109: 1461-1470.

60. Agarwal P, Kudirka R, Albers AE, Barfield RM, De Hart GW, Drake PM, et al. Hydrazino-Pictet-Spengler ligation as a biocompatible method for the generation of stable protein conjugates. Bioconjug Chem. 2013; 24: 846-851.

61. Carrico IS. Chemoselective modification of proteins: hitting the target. Chem Soc Rev. 2008; 37: 1423-1431.

62. Kudirka R, Barfield RM, McFarland J, Albers AE, De Hart GW, Drake PM, et al. Generating site-specifically modified proteins via a versatile and stable nucleophilic carbon ligation. Chem Biol. 2015; 22: 293-298.

63. Yokoyama K, Nio N, Kikuchi Y. Properties and applications of microbial transglutaminase. Appl Microbiol Biotechnol. 2004; 64: 447-454.

64. Spolaore B, Raboni S, Ramos Molina A, Satwekar A, Damiano N, Fontana A. Local unfolding is required for the site-specific protein modification by transglutaminase. Biochemistry. 2012; 51: 8679-8689.

65. Schumacher FF, Nunes JP, Maruani A, Chudasama V, Smith ME, Chester KA, et al. Next generation maleimides enable the controlled assembly of antibody-drug conjugates via native disulfide bond bridging. Org Biomol Chem. 2014; 12: 7261-7269.

66. Mullard A. Maturing antibody-drug conjugate pipeline hits 30. Nat Rev Drug Discov. 2013; 12: 329-332.

67. Zolot RS, Basu S, Million RP. Antibody-drug conjugates. Nat Rev Drug Discov. 2013; 12: 259-260.

68. Ornes S. Antibody-drug conjugates. Proc Natl Acad Sci USA. 2013; 110: 13695.

69. Cohen R, Vugts DJ, Visser GW, Stigter-van Walsum M, Bolijn M, Spiga M, et al. Development of novel ADCs: conjugation of tubulysin analogues to trastuzumab monitored by dual radiolabeling. Cancer Res. 2014; 74: 5700-5710.