Figure S1. Comparison of the rate of adduct formation of the indolinobenzodiazepine dimer 6 and the pyrrolobenzodiazepine dimer SJG-136 with dsDNA prepared from annealing of single strand DNA 3'-TATAGATCTATA-5'. Figure S2. A, In vitro potency of anti-EpCAM ADC 1a toward antigen (+) HCT-15 cells without or with addition of blocking antibody. Figure S3. A, Structures of IGNs used in the DNA adduct formation assay. B, Time-dependent reversal of IGN-DNA adducts isolated from EOL-1 cells that had been exposed to the IGN diimine 7 or the monoimine 8, after heating to 90{degree sign} C. C, The ability of IGN 7 (diimine) and 8 (monoimine) to crosslink DNA was tested in an acellular comet assay with nuclear DNA from human normal lymphocytes. Figure S4. In vitro potency of anti-FRα ADC 4a toward antigen (+) KB cells without or with addition of an excess (1 x 10-6 M) blocking antibody. Figure S5. Comparison of the in vitro potency of IGN 9 and its sulfonated version towards the human Burkitt cell line Namalwa.

Extent of DNA adduct formation in EOL-1 (AML cells) exposed to the IGN diimine 7 or the monoimine 8 after a 5 h or 20 h exposure to the compounds

Supplementary Table S1: Amounts of catabolites generated upon treatment of cancer cells with CX and SMCC ADCs.

Comparative in vitro potency of IGNs with a diimine (1), monoimine (3), and diamine (2) towards hematologic and solid tumor cell lines

Effect of substitution of an indolino moiety (6) for a pyrrolo group (SJG-136) on in vitro potency

Supplementary Methods (S1-S3). Supplementary Method S1: Synthesis of di-, tri-, tetra-glycyl, and valine-citrulline-glycine peptide linkers and catabolites. Supplementary Method S2: Test of in vitro cytotoxicity, cell-cycle inhibition, and bystander cytotoxic activity of ADCs. Supplementary Method S3: Catabolism of [3H]-labeled and non-radiolabeled CX and SMCC ADCs by cancer cells.

Supplemental Figure S1. Activity of anti-FRα clones in an indirect cytotoxicity assay. Supplemental Figure S2. The antigen-dependent cell-mediated cytotoxic activity of M9346A antibody against Igrov-1 cells. Supplemental Figure S3. Activity of the M9346A antibody against KB xenograft tumors. Supplemental Figure S4. The bystander cytotoxic activity of IMGN853. Supplemental Figure S5. In vitro cytotoxicity of IMGN853 against KB cells (short exposure). Supplemental Figure S6. In vitro cytotoxicity of IMGN853, M9346A-SMCCDM1 and non-targeting human IgG1-SMCC-DM1 against KB cells (continuous exposure). Supplemental Figure S7. Catabolites of IMGN853 formed in KB cells in vitro. Supplemental Figure S8. Comparison of 3[H]-M9346A antibody and 3[H]-IMGN853 conjugate processing. Figure S9. IMGN853 activity against KB xenograft tumors. Supplementary Tables. Supplementary Methods.

Supplementary Figures (S1-S4). Supplementary Figure S1: In vitro cytotoxic activities of tetraglycyl (Gly4), triglycyl (Gly3), diglycyl (Gly2), and valine-citrulline-glycine (VCG)-linked anti-EGFR ADCs and a triglycyl-linked non-binding antibody ADC in PC-9, Ca9-22, HSC-2, H1975, A-431, and OSC-19 cells (3-4 DAR ADCs). Supplementary Figure S2: In vitro cytotoxic activities of anti-EpCAM CX and SMCC ADCs toward a low EpCAM antigen-expressing cell line, RPMI 8226 (~50,000 EpCAM per cell). The CX ADCs tested included a conjugate with a typical payload number (3.9 maytansinoid molecules per antibody molecule; 3.9 DAR) and conjugates with high payload numbers (8 and 9.6 DAR). The SMCC ADC had a typical payload number (4.3 DAR). Supplementary Figure S3: Binding-competition ELISA of catabolites of CX ADC (DM-CX1 and DM-CX2) and SMCC ADC (lysine-SMCC-DM1): inhibition by catabolites toward binding of anti-maytansine antibody to immobilized BSA-maytansinoid conjugate. Supplementary Figure S4: Test of bystander cytotoxic activity of anti-EGFR-CX-DM1 or SPDB-DM4 conjugate in mixed culture of EGFR-positive (Ca9-22) and EGFR-negative (Ramos) cells.

Antibody–drug conjugates (ADCs) combine the specificity of monoclonal antibodies with the potency of highly cytotoxic agents, potentially reducing the severity of side effects by preferentially targeting their payload to the tumour site. ADCs are being increasingly used in combination with other agents, including as first-line cancer therapies. As the technology to produce these complex therapeutics has matured, many more ADCs have been approved or are in late-phase clinical trials. The diversification of antigenic targets as well as bioactive payloads is rapidly broadening the scope of tumour indications for ADCs. Moreover, novel vector protein formats as well as warheads targeting the tumour microenvironment are expected to improve the intratumour distribution or activation of ADCs, and consequently their anticancer activity for difficult-to-treat tumour types. However, toxicity remains a key issue in the development of these agents, and better understanding and management of ADC-related toxicities will be essential for further optimization. This Review provides a broad overview of the recent advances and challenges in ADC development for cancer treatment.

In the fight against cancer, antibody–drug conjugates (ADCs) form a versatile class of chemotherapeutic agents by combining the exquisite binding affinity and selectivity of a monoclonal antibody with the high cell-killing potential of a cytotoxic payload. Although the concept was originally postulated more than 60 years ago, only in the past ten years have ADCs started to firmly establish themselves in the field of targeted cancer therapy, with currently ten approved drugs and many more (>80) in the clinical pipeline. This chapter is an introduction to antibody–drug conjugates in the broad sense, starting with a historical perspective of the development of ADC technologies, to be followed by an overview of marketed and clinically most advanced ADCs. Next, the essential considerations to design and develop a successful ADC are separately highlighted: the target; the antibody and the (cytotoxic) payload;a quick overview of conjugation chemistries to attach the payload to the antibody;and of course, the linker, the essential part in the middle.

Antibody-drug conjugates are an emerging class of cancer therapeutics constructed from monoclonal antibodies conjugated with small molecule effectors. First-generation molecules of this class often employed heterogeneous conjugation chemistry, but many site-specifically conjugated ADCs have been described recently. Here, we undertake a systematic comparison of ADCs made with the same antibody and the same macrocyclic maytansinoid effector but conjugated either heterogeneously at lysine residues or site-specifically at cysteine residues. Characterization of these ADCs in vitro reveals generally similar properties, including a similar catabolite profile, a key element in making a meaningful comparison of conjugation chemistries. In a mouse model of cervical cancer, the lysine-conjugated ADC affords greater efficacy on a molar payload basis. Rather than making general conclusions about ADCs conjugated by a particular chemistry, we interpret these results as highlighting the complexity of ADCs and the interplay between payload class, linker chemistry, target antigen, and other variables that determine efficacy in a given setting.

Creating antibody–drug conjugates (ADCs) by attaching cytotoxic effector molecules to antibodies that can selectively bind to targets on the surface of cancer cells is an approach for the selective delivery of cytotoxic agents to cancer cells while minimizing toxicity toward normal cells that lack the cell-surface target of the antibody. The successful application of this concept should yield ADCs that have a wider therapeutic index than that of small-molecule cytotoxic compounds (i.e. “classical” chemotherapy). Since the approvals of brentuximab vedotin in 2011 and ado-trastuzumab emtansine in 2013, two ADCs that contain potent tubulin-binding agents as their payloads, there has been an explosion of research in the field, with more than 65 ADC compounds in clinical evaluation at the end of 2017. In the last few years, medicinal chemists have generated a wide variety of cytotoxic compounds that kill cells by a variety of mechanisms, and that could serve as payloads for ADCs. The purpose of this chapter is to review the factors important for the design of ADCs, factors that medicinal chemists need to take into account when creating payloads and their linkers and when designing the payload-release mechanisms, all of which can influence the therapeutic effectiveness of ADCs in treating patients with cancer.

The concept of exploiting the specific binding properties of monoclonal antibodies as a mechanism for selective delivery of cytotoxic agents to tumor cells is an attractive solution to the challenge of increasing the therapeutic index of cell-killing agents for treating cancer. All three parts of an antibody-drug conjugate (ADC)-the antibody, the cytotoxic payload, and the linker chemistry that joins them together-as well as the biologic properties of the cell-surface target antigen are important in designing an effective anticancer agent. The approval of brentuximab vedotin in 2011 for treating relapsed Hodgkin's lymphoma and systemic anaplastic large cell lymphoma, and the approval of ado-trastuzumab emtansine in 2013 for treating HER2-positive metastatic breast cancer, have sparked vigorous research in the field, with >65 ADCs currently in clinical evaluation. This review highlights the ADCs that are approved for marketing, in pivotal clinical trials, or in at least phase II clinical development for treating both hematologic malignancies and solid tumors.

Attaching a cytotoxic "payload" to an antibody to form an antibody-drug conjugate (ADC) provides a mechanism for selective delivery of the cytotoxic agent to cancer cells via the specific binding of the antibody to cancer-selective cell surface molecules. The first ADC to receive marketing authorization was gemtuzumab ozogamicin, which comprises an anti-CD33 antibody conjugated to a highly potent DNA-targeting antibiotic, calicheamicin, approved in 2000 for treating acute myeloid leukemia. It was withdrawn from the US market in 2010 following an unsuccessful confirmatory trial. The development of two classes of highly potent microtubule-disrupting agents, maytansinoids and auristatins, as payloads for ADCs resulted in approval of brentuximab vedotin in 2011 for treating Hodgkin lymphoma and anaplastic large cell lymphoma, and approval of ado-trastuzumab emtansine in 2013 for treating HER2-positive breast cancer. Their success stimulated much research into the ADC approach, with >60 ADCs currently in clinical evaluation, mostly targeting solid tumors. Five ADCs have advanced into pivotal clinical trials for treating various solid tumors-platinum-resistant ovarian cancer, mesothelioma, triple-negative breast cancer, glioblastoma, and small cell lung cancer. The level of target expression is a key parameter in predicting the likelihood of patient benefit for all these ADCs, as well as for the approved compound, ado-trastuzumab emtansine. The development of a patient selection strategy linked to target expression on the tumor is thus critically important for identifying the population appropriate for receiving treatment.

Antibody-drug conjugates (ADCs) are being actively pursued as a treatment option for cancer following the regulatory approval of brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine (Kadcyla). ADCs consist of a cytotoxic agent conjugated to a targeting antibody through a linker. The two approved ADCs (and most ADCs now in the clinic that use a microtubule disrupting agent as the payload) are heterogeneous conjugates with an average drug-to-antibody ratio (DAR) of 3-4 (potentially ranging from 0 to 8 for individual species). Ado-trastuzumab emtansine employs DM1, a semisynthetic cytotoxic payload of the maytansinoid class, which is conjugated via lysine residues of the antibody to an average DAR of 3.5. To understand the effect of DAR on the preclinical properties of ADCs using maytansinoid cytotoxic agents, we prepared a series of conjugates with a cleavable linker (M9346A-sulfo-SPDB-DM4 targeting folate receptor α (FRα)) or an uncleavable linker (J2898A-SMCC-DM1 targeting the epidermal growth factor receptor (EGFR)) with varying DAR and evaluated their biochemical characteristics, in vivo stability, efficacy, and tolerability. For both formats, a series of ADCs with DARs ranging from low (average of ∼2 and range of 0-4) to very high (average of 10 and range of 7-14) were prepared in good yield with high monomer content and low levels of free cytotoxic agent. The in vitro potency consistently increased with increasing DAR at a constant antibody concentration. We then characterized the in vivo disposition of these ADCs. Pharmacokinetic analysis showed that conjugates with an average DAR below ∼6 had comparable clearance rates, but for those with an average DAR of ∼9-10, rapid clearance was observed. Biodistribution studies in mice showed that these 9-10 DAR ADCs rapidly accumulate in the liver, with maximum localization for this organ at 24-28% percentage injected dose per gram (%ID/g) compared with 7-10% for lower-DAR conjugates (all at 2-6 h post-injection). Our preclinical findings on tolerability and efficacy suggest that maytansinoid conjugates with DAR ranging from 2 to 6 have a better therapeutic index than conjugates with very high DAR (∼9-10). These very high DAR ADCs suffer from decreased efficacy, likely due to faster clearance. These results support the use of DAR 3-4 for maytansinoid ADCs but suggest that the exploration of lower or higher DAR may be warranted depending on the biology of the target antigen.

Lutter et al, "The Use of a New Series of Cleavable Protein-Crosslinkers on the Escherichia Colli Ribosome' FEBS Letters, vol. 48, No. 2, Nov. 1974 pp. 288-292. Lambert et al, "The Subunit Interface of the Escherichia Coli Ribosome” J. Mol. Biol. (1981) 149, pp. 451-476. Wang et al, "The Behavior of Cleavable Crosslinking Reagents Based on the Disulfide Group', Israel Journal of Chemistry, vol. 12, Nos. 1-2, 1974 pp.375-389. Carlsson et al, "Protein Thiolation and Reversible Protein-Protein Conjugation' Biochem J. (1978) 173, pp. 723-737. Primary Examiner-John Kight Assistant Examiner-Nathan M. Nutter

AbstractA triglycyl peptide linker (CX) was designed for use in antibody–drug conjugates (ADC), aiming to provide efficient release and lysosomal efflux of cytotoxic catabolites within targeted cancer cells. ADCs comprising anti-epithelial cell adhesion molecule (anti-EpCAM) and anti-EGFR antibodies with maytansinoid payloads were prepared using CX or a noncleavable SMCC linker (CX and SMCC ADCs). The in vitro cytotoxic activities of CX and SMCC ADCs were similar for several cancer cell lines; however, the CX ADC was more active (5–100-fold lower IC50) than the SMCC ADC in other cell lines, including a multidrug-resistant line. Both CX and SMCC ADCs showed comparable MTDs and pharmacokinetics in CD-1 mice. In Calu-3 tumor xenografts, antitumor efficacy was observed with the anti-EpCAM CX ADC at a 5-fold lower dose than the corresponding SMCC ADC in vivo. Similarly, the anti-EGFR CX ADC showed improved antitumor activity over the respective SMCC conjugate in HSC-2 and H1975 tumor models; however, both exhibited similar activity against FaDu xenografts. Mechanistically, in contrast with the charged lysine-linked catabolite of SMCC ADC, a significant fraction of the carboxylic acid catabolite of CX ADC could be uncharged in the acidic lysosomes, and thus diffuse out readily into the cytosol. Upon release from tumor cells, CX catabolites are charged at extracellular pH and do not penetrate and kill neighboring cells, similar to the SMCC catabolite. Overall, these data suggest that CX represents a promising linker option for the development of ADCs with improved therapeutic properties. Mol Cancer Ther; 15(6); 1311–20. ©2016 AACR.

Ever since cancer patients were first treated with cytotoxic agents with the goal of eradicating the tumor tissue, oncologists have looked to widen the therapeutic window for these agents. The goal of combination chemotherapy, pioneered by Emil “Tom” Frei and others [1], was to increase antitumor efficacy of cytotoxic drug therapy, without substantially increasing overall toxicity to the patient, by using agents with nonoverlapping dose-limiting toxicities. However, such modalities have proven only partially effective at the maximum achievable doses, limited by the severe side effects of the cytotoxic agents used. Attaching cytotoxic effector molecules to an antibody to form an antibody–drug conjugate (ADC) provides a mechanism for the selective delivery of the cytotoxic payload to cancer cells via the specific binding of the antibody moiety to cancer-selective cell surface molecules. This simple concept was thought to be a particularly attractive solution to the challenge of finding a way to increase the therapeutic window of the cytotoxic agent (Figure 1.1). Furthermore, conjugation of a small molecular weight cytotoxic agent to a large hydrophilic antibody protein is expected to restrict penetration of the cytotoxic compound across cellular membranes of antigen-negative normal cells, providing an additional mechanism by which the therapeutic index of the small molecule cytotoxin is widened, beyond that of targeted delivery. Thus, from the perspective of a medicinal chemist, an ADC is a prodrug that can only be activated within tumor cells and is excluded from normal cells by virtue of conjugation to a protein. In addition, giving the in vivo distribution properties of an antibody to the small molecular weight cytotoxic agent has the potential to reduce its systemic toxicity. Typical Antibody–Drug Conjugates

Antibody-drug conjugates (ADCs) have become a widely investigated modality for cancer therapy, in part due to the clinical findings with ado-trastuzumab emtansine (Kadcyla). Ado-trastuzumab emtansine utilizes the Ab-SMCC-DM1 format, in which the thiol-functionalized maytansinoid cytotoxic agent, DM1, is linked to the antibody (Ab) via the maleimide moiety of the heterobifunctional SMCC linker. The pharmacokinetic (PK) data for ado-trastuzumab emtansine point to a faster clearance for the ADC than for total antibody. Cytotoxic agent release in plasma has been reported with nonmaytansinoid, cysteine-linked ADCs via thiol-maleimide exchange, for example, brentuximab vedotin. For Ab-SMCC-DM1 ADCs, however, the main catabolite reported is lysine-SMCC-DM1, the expected product of intracellular antibody proteolysis. To understand these observations better, we conducted a series of studies to examine the stability of the thiol-maleimide linkage, utilizing the EGFR-targeting conjugate, J2898A-SMCC-DM1, and comparing it with a control ADC made with a noncleavable linker that lacked a thiol-maleimide adduct (J2898A-(CH2)3-DM). We employed radiolabeled ADCs to directly measure both the antibody and the ADC components in plasma. The PK properties of the conjugated antibody moiety of the two conjugates, J2898A-SMCC-DM1 and J2898A-(CH2)3-DM (each with an average of 3.0 to 3.4 maytansinoid molecules per antibody), appear to be similar to that of the unconjugated antibody. Clearance values of the intact conjugates were slightly faster than those of the Ab components. Furthermore, J2898A-SMCC-DM1 clears slightly faster than J2898A-(CH2)3-DM, suggesting that there is a fraction of maytansinoid loss from the SMCC-DM1 ADC, possibly through a thiol-maleimide dependent mechanism. Experiments on ex vivo stability confirm that some loss of maytansinoid from Ab-SMCC-DM1 conjugates can occur via thiol elimination, but at a slower rate than the corresponding rate of loss reported for thiol-maleimide links formed at thiols derived by reduction of endogenous cysteine residues in antibodies, consistent with expected differences in thiol-maleimide stability related to thiol pKa. These findings inform the design strategy for future ADCs.

The clinical success of Adcetris® (brentuximab vedotin) and Kadcyla® (ado-trastuzumab emtansine) has sparked clinical development of novel ADCs. These powerful anti-cancer agents are designed to allow specific targeting of highly potent cytotoxic agents to tumor cells while sparing healthy tissues. Despite the use of tumor-specific antibodies, the emerging clinical data with ADCs indicates that adverse effects frequently occur before ADCs have reached their optimal therapeutic dose, resulting in a relatively narrow therapeutic window. This review summarizes the therapeutic window of ADCs currently in clinical development, along with some strategies that may help to widen the window.