Antibody-drug conjugates (ADCs) are a therapeutic modality that enables the targeted delivery of highly potent cytotoxic payloads to tumors. This chapter describes the components of the ADCs and discusses the medicinal chemistry principles that guide the design of this class of therapeutics. A description of main classes of drugs and the linkers used to attach to monoclonal antibodies with an emphasis on the design, historical development, and linking strategies is presented. Clinical use of the approved ADCs for the treatment of cancer is briefly described.
KeywordsADCs Cancer Cytotoxic payloads Design Linker Medicinal chemistry Monoclonal antibodies
Antibody–drug conjugates (ADCs) can be described as a sum of components that are subject to several processes that lead to the desired outcome of selective delivery of a drug to an entity targeted by the specificity of the antibody. While the concept upon which ADCs are built appears deceptively simple, the elements involved in putting this concept into practice and the interdependencies between these components underscore the complexity of this family of drugs. The components of an ADC are monoclonal antibody, a set of linking sites targeted with bioorthogonal conjugation chemistry, a tether that bridges the linking site to the “prodrug,” the drug releasing moiety (sometimes called a “trigger”) that acts as both a tether and a means for selective release of the active “drug” to the desired tissue compartment and the drug. A set of processes are required for achieving efficacy with an ADC: plasma stability is a desired characteristic for ADCs as all ADCs known to date are administered IV, binding to the antigen situated on the surface of the cell is followed by internalization of the ADC-antigen complex that carries the ADC through the endosomal-lysosomal pathway leading to protein degradation by the action of hydrolases and release of the active drug followed by cytoplasmic and nuclear delivery of the active drug that interacts with the intracellular target. In case of anti-cancer drugs the cell dies and releases the drug into the tumor environment and, depending on the cell membrane permeability of the drug, can affect neighboring cells that do not express the antigen at the levels needed for a pharmacological effect, a process called “bystander effect.” The aim of this account is to describe the design principles of the components of ADCs and capture how those design principles translate into therapeutic outcomes. So far most of the applications of this therapeutic modality have been in Oncology where a highly cytotoxic drug is delivered to tumor cells. In an oncology setting minimizing the innate toxicity of the drug to healthy tissues becomes a critical design criterion.
2 Components of the ADCs
2.1 The Monoclonal Antibody
Early clinical experience with mAbs used murine monoclonal antibodies. It was quickly recognized that murine molecules led to development of immune responses (were immunogenic) that resulted in rapid clearance of the drug and impaired efficacy. The continuous improvements in antibody engineering technologies led to gradual elimination of murine elements in therapeutic mAbs as follows: chimeric format where variable regions of both HC and LC were murine, humanized format that used mouse CDR regions and finally, the currently preferred format for ADCs, fully human antibodies. The half-lives of human mAbs are up to 3 weeks, significantly longer than the murine mAb that could only be detected in the blood for a few days.
2.2 Biorthogonal Conjugation Chemistry
Two factors have shaped the process of attachment a drug to an antibody and those were the requirement that conjugation be performed under aqueous conditions within a narrow range of pH and the constraints imposed by the reactivity of the natural amino acid residues. The amino group in lysine residues and the thiol group in cysteines are functional groups that were widely used for conjugation to a drug by reaction with electrophiles.
In addition to lysine and cysteine one additional native amino acid – glutamine – was used for conjugation. Conjugation to glutamine uses the enzymatic recognition of a specific amino-acid sequence that contains the amino acid in the primary structure of the antibody. This approach leads to conjugates at a specified site of the antibody thus allowing for modulation of properties of the resulting conjugate. Glutamine conjugation to antibodies utilizes a bacterial transglutaminase (TG) isolated from Streptoverticillium mobaraense . Unlike common transglutaminases that can catalyze the formation of amide bonds between the primary amine of a lysine and the amide group of any glutamine, the bacterial TG can catalyze the formation of an amine bond between any primary amine and the amide group of sequence specific glutamines . A positional scan of antibody constant domains was performed by engineering a glutamine tag (LLQG) into surface accessible regions of an IgG1 antibody and several sites were identified that showed good biophysical properties and a high degree of conjugation .
Glycoengineering was used for generation of site specific conjugates by either metabolic engineering or post-translational remodeling of native glycan located at Asn-297 conserved position in the Fc domain of the antibody. The objective of glycoengineering is modification of the glycan in a manner that a functional group becomes available for biorthogonal conjugation. Metabolic engineering was achieved by substituting the fucose residue with a functionalized fucose residue that had a reactive handle for conjugation. A thiofucose residue was incorporated in the mAb during expression in CHO cells in medium that contained the modified fucose. The resulting antibody was used to generate conjugates where maleimide-bearing payloads were conjugated at the thiofucose site. The efficiency of the process did not exceed 70% thus limiting the utility of this particular conjugation technique . Early efforts around post translational remodeling of native glycans have used a chemical oxidation step of the carbohydrate  or enzymatic introduction of sialic acid  to generate aldehyde functional group for oxime ligation. Engineering the active site of glycosyltransferases has provided tools for introducing chemically modified sugar substrates . Native IgGs were converted to a homogenous G0f glycoform population by using β-1,4-galactosidase from Streptococcus pneumoniae. The resulting antibody was modified with a synthetic galactose bearing a chemical handle at the C2 position by using mutant β4Gal-T1-Y289L . A keto- or an azido-group can be introduced in a site specific manner to generate an antibody with four functional groups. Further refinement of this technique was presented recently where an endoglycosidase was used to trim the antibody glycan to the fucosylated Acetylglucosamine (GlcNAc). Transfer of N-azidoacetylgalactosamine (GalNaz) in presence of β4Gal-T1-Y289L led to efficient installation of two azide groups onto the antibody .
Recent advances in development of methodologies for the genetic incorporation of unnatural amino acids into proteins  opened the possibility of site-selective modification of antibodies . The side chains of unnatural amino acids provide novel functional groups for biorthogonal chemistry thus enabling generation of stable conjugates. The two main functional groups introduced using genetic incorporation of non-natural amino acids are the keto- and azido- that showed utility for conjugation by oxime ligation and the Huisgen alkyne-azide cycloaddition (“click”) reaction, respectively. The incorporation of p-acetylphenylalanine into trastuzumab generated an antibody with ~2 acetyl groups that were used for conjugation with alkoxyamine to generate the oxime at pH 4.5 with >95% efficiency . N6-((2-azidoethoxy)carbonyl)-l-lysine was efficiently incorporated into trastuzumab and generated antibodies with ~2 azide groups that were efficiently conjugated by both strain promoted alkyl azide cycloaddition (SPAAC) and Cu(I) alkyl azide cycloaddition (CuAAC) to generate homogenous ADCs .
2.3 The Linking Sites
Conjugation to antibodies is governed by two parameters: the position of the amino acid in the protein sequence that carries the conjugated moiety and the number of amino acid residues that are functionalized during the conjugation process known as the drug-to-antibody ratio (DAR). Both these parameters impact the in vivo behavior of ADCs. Stochastic conjugation to Lys or native cysteines generates a heterogeneous mixture of molecular species where the sites of conjugation are random and the drug-to-antibody ratio is defined as an average [33, 34]. Each of the molecular species of such a mixture would display a distinct behavior in vivo as a consequence of different pharmacokinetics . Discovery of solvent exposed engineered Cys IgGs was driven by the need to introduce a thiol-labelling site that did not adversely affect the structure or function of the antibody . Following expression in mammalian cells mutant cysteines were capped with either a Cys or glutathione. Efforts to find an uncapping reagent that did not affect the native disulfide bonds were partially successful when alkylation with iodoacetate of a serine to cysteine mutant at the position 442 in the Fc domain was attempted . These early discoveries set the stage for the development of antibodies containing engineered reactive cysteine residues at specific sites in antibodies that allow for drugs to be conjugated with defined stoichiometry without disruption of interchain disulfide bonds (termed THIOMABS) [38, 39]. These ADCs were produced by global reduction of blocked cysteine residues and interchain disulfides, subsequent oxidation in the presence of CuSO4 or dehydroascorbic acid to regenerate the interchain disulfide bonds, and then conjugation of the reactive cysteine thiol to maleimide reagents. This method generated site specifically modified ADCs with a homogenous distribution consisting of 92.1% species with two drugs/antibody. Importantly, the THIOMAB conjugate displayed a larger therapeutic index, as it was tolerated at much higher doses in animals and displayed better in vivo activity. The higher therapeutic index correlated with a higher in vivo stability of the THIOMAB when compared to equivalent stochastic ADCs. The authors hypothesized that engineered cysteines may be in relatively “protected” sites that resist proteolytic attack in circulation and they observed that the accessibility of a cysteine residue varies depending on the position of the mutated amino acid in the antibody protein sequence . In a subsequent study the THIOMAB team explored whether the conjugation site could modulate the stability of a cysteine-maleimide adducts. The Genentech team prepared three THIOMABs of trastuzumab in which the mutant cysteine residues were positioned at sites that differed by their local structural environment. One site was chosen based on high solvent accessibility, while the other two were relatively buried sites but one was located in a positively charged environment while the other was in a relatively neutral environment. The in vitro potency of the three ADCs was comparable for all sites but the serum stability showed significant differences in the stability of the ADCs: the solvent-accessible conjugate underwent rapid thiol exchange with serum albumin, the conjugate at the positively charged site showed succinimide hydrolysis, resulting in improved stability, and the conjugate at the neutral site exhibited intermediate behavior between the other two ADCs. Serum stability results correlated well with in vivo efficacy, pharmacokinetic properties, and toxicity. That study demonstrated for the first time that the structural and chemical dynamics of the conjugation site can be exploited to design optimal protein conjugates for therapeutic applications . A team at Seattle Genetics that were exploring conjugation of a highly hydrophobic drug pyrrolobenzodiazepine (PBD, see below) was faced with the challenge of finding a solution to the issues associated with stochastic conjugation of the maleimide-bearing PBD that led to formation of high percentage of aggregate. Scanning various conjugation sites with solvent accessible Cys mutants led to the discovery of position S239C on the HC where they conjugated the hydrophobic payload with only 1.6% aggregate. Thus they concluded that the recombinant construct provided superior ADCs compared to hinge disulfide conjugates in terms of ADC uniformity and aggregation levels .
2.4 The Drug
The choice of effective cytotoxic drugs for ADCs is governed by the relatively small number of antigen molecules on the cancer cell surface to which the antigen can bind (~104–106 receptors/cell) and by the efficiency of the internalization of cell-surface bound antigen–antibody complex and intracellular processing to release the active drug. Provided the intracellular delivery and release are efficient, the number of cytotoxic drug delivered to the individual cell by the ADC needs to be well above the number of cytotoxic agent molecules required to kill a cell. Thus cytotoxic drugs with potency in the pM range are needed. The early ADCs used drugs that were cancer chemotherapeutics as is the case with vinblastine analogs or doxorubicin. Following conjugation to an antibody both these drugs showed diminished potency as ADCs. This observation was attributed to the different modes of cellular uptake of the conjugated vs. non-conjugated drug. For cell membrane permeable drugs, the free diffusion can lead to high concentration of the drug inside the cell dependent on the dose of the administered drug. In addition to normal tissue target-mediated uptake that can be minimized by judicious choice of tumor-specific target, non-specific uptake by pinocytosis and plasma degradation of the ADC in circulation means that only ~1% of the administered ADC reaches the desired tumor tissue . From a medicinal chemistry perspective, the hydrophobicity of the drug and its impact upon aqueous solubility could present a significant challenge. Also medicinal chemists need to develop structure activity relationships that reveal sites on the cytotoxic drug that tolerate a linker and possess the required reactivity for attachment to an antibody as well as enable the release of the drug inside the cancer cell. All cytotoxic drugs that have been reported as ADC therapeutics are derived from naturally occurring molecules and have very large molecular surfaces that appear to be a common feature for all highly potent cytotoxic molecules. Consequently, the structural complexity of the cytotoxic drugs for ADCs presents a major challenge for development of such molecules. The intracellular targets of cytotoxic drugs are quite limited and two main mechanisms have been targeted successfully with ADCs, i.e. tubulin and DNA.
2.4.1 Antimitotic Drugs
Disruption of microtubule dynamics impairs the ability of mitotic spindles to assemble and alter the architecture of the cytoskeleton, causing cell death . Due to their mechanism of action, antimitotic agents are particularly cytotoxic to cancer cells that divide faster than non-cancerous cells. However, normal tissues that contain rapidly dividing cells such as cells lining the digestive tract, hair follicles, and bone marrow can also be killed, causing undesired toxicity.
2.4.2 DNA Targeting Drugs
2.4.4 RNA Polymerase Inhibitors
SN-38 and the Camptothecins
2.5 The Tether
2.5.1 Non-cleavable Tethers
A non-cleavable tether does not contain a tumor or cancer cell-specific release unit, called a “trigger.” For a medicinal chemist, the use of a non-cleavable tether poses the challenge of finding a linking site on the cytotoxic drug where attachment of the tether will not impact cytotoxicity. Additionally, following cellular catabolism, the non-cleavable tether ADCs generates a species that contains a residual charged amino acid residue. In vitro assessment of the cytotoxicity of such species is not always trivial due to the potential low cell membrane permeability thus making the evaluation of linked drugs with non-cleavable tethers challenging. The particular nature of non-cleavable tethers has two apparently conflicting consequences: on one hand the stable linkage translates to higher plasma stability and reduced toxicity as shown by higher Maximum Tolerated Dose (MTD) in vivo. On the other hand, in tumors with heterogeneous expression of the antigen, the efficacy can be negatively affected by the reduced cell membrane permeability of the active species, leading to reduced ability of the resulting drug to kill neighboring cells.
MCC-DM-1 contains the thiosuccinimide moiety, which was shown to lead to drug loss following exchange with thiol-containing plasma protein, i.e. albumin. Two truly non-cleavable linkers that lacked the reversible thioether succinimide connection between the drug and antibody were shown to possess superior efficacy and stability relative to MCC-DM1. These two linkers are May-mc 23 and May-MPA 24 and the corresponding lysosomal released drugs are shown in Fig. 22 .
2.5.2 Cleavable Tethers
Cleavable tethers contain a spacer and a structural feature designed to release the drug inside the cell called a “trigger.” The spacer is usually a short carbon chain for most ADCs but for some is a series of polyethylene glycol units, which are designed to reduce the logP of the linked drug during the conjugation process and minimize the impact of drug lipophilicity upon the pharmacokinetics of the ADC. The trigger is a tool that exploits changes that occur in vesicles along the endosomal, lysosomal and cytoplasmic pathway following receptor-mediated endocytosis . The changes that have been exploited by trigger designs are: (1) gradual drop in pH from physiological range at the cell surface of 7.2–7.4 to 5.5–6 within the endosome and to ~5.0 in lysosome; (2) the activation of protein digesting enzymes at the lower pH of the lysosome; and (3) the increase in concentrations of reducing co-factors such as glutathione and cysteine and activation of enzymes that can reduce disulfide bonds .
3.1 Antigen Binding and Internalization
At a cellular level, multiple factors contribute to efficient targeting of the cancer cells and subsequent tumor destruction: the antigen copy number expressed on the surface of the cancer cell (as mentioned above), the affinity of ADC for the antigen, the rate and efficiency of internalization of the ADC-antigen complex, the rate of recycling of the complex back to the cell surface and the efficiency of trafficking to the appropriate compartment for drug release and ultimately achieving the interaction with the intracellular target leading to cell death all play critical roles in the success of targeted cancer therapy. Most of these characteristics are antibody target dependent and judicious target selection will have a significant impact on the clinical success of the ADC . However, the efficiency of internalization of the ADC could be induced by conjugation of the drug to the non-internalizing mAbs. Several reports showed that upon binding of unconjugated mAb to the antigen, the antibody/antigen complex remains on the surface while conjugation to drugs such as auristatins L49 mAb  or anti-CD-20  led to molecules that efficiently internalize once bound to cell surface antigen. Whether this applies to any drug conjugated to non-internalizing mAbs still remains to be demonstrated.
3.2 ADC Intracellular Processing
Trafficking and intracellular fate of ADCs following internalization has been the object of several detailed studies [118, 122, 123]. Anti-CD30 conjugates bearing the protease cleavable tether mc-VC-MMAE and the non-cleavable mc-MMAF were generated and were shown to display comparable binding and internalization rates with both ADCs showing efficient and antigen-dependent cancer cell killing. Immunofluorescence microscopy showed that the ADC localized to lysosomes following 16 h incubation in L540cy cells. When inhibitors of trafficking (ammonium chloride) were used prior to addition of ADCs to the cells the total intracellular level of ADC was significantly diminished while flow cytometry and fluorescence microscopy showed accumulation of ADC at the surface of the cell. Inhibitors of cathepsin B-mediate proteolysis significantly enhanced the intracellular levels of ADCs. The lysosomal metabolism of the ADC caused the released of the drug was probed by using inhibitors of cysteine proteases that showed subdued cytotoxicity when incubated with the cancer cell pretreated with inhibitor. Interestingly, when the inhibitors were added to the cells hours after the ADC treatment there was minimal effect upon cytotoxicity .
4 Clinical Experience
4.1 Hematological Cancers
4.1.1 Gemtuzumab Ozogamicin
Gemtuzumab ozogamicin (Mylotarg) was approved by the FDA in 2000 and was the first ADC to reach the market. It is a humanized IgG4 mAb directed against CD33, a surface antigen present in 85–90% of acute myeloid leukemia (AML), conjugated by random Lys conjugation to N-Ac-γ1I dimethyhydrazide (DMH) (Fig. 11) with an average of DAR4. It was prescribed as a monotherapy in patients over the age of 60 with AML who were not candidates for cytotoxic chemotherapy. Mylotarg was withdrawn in 2010 after a phase III study showed no clinical benefit and a higher risk of fatal adverse events .
4.1.2 Brentuximab Vedotin
Brentuximab vedotin (Adcetris) is composed of a mouse IgG1 anti-CD30 mAb conjugated to mc-VC-MMAE via maleimides to native cysteine residues with an average DAR of 4. Adcetris binds to the cell surface CD30 with high affinity (3 nM) and has high potency against CD30+ Hodgkin lymphoma and ALCL tumor cells in vitro (IC50 values of 3–50 pM) . The clinical MTD of Adcetris was 1.8 mg/kg every 3 weeks. At this dose objective responses were obtained, including complete responses (4 of 12 patients) and partial responses (2 of 12 patients) in relapsed CD30-positive lymphomas  in a first Phase I study. In a second Phase I trial of brentuximab vedotin carried out to test its effects, when administered weekly the dosing range was 0.4–1.4 mg/kg, the MTD was 1.2 mg/kg and the overall response rate (ORR) was 59%, with 34% complete responses . Adcetris was approved for the treatment of patients with relapsed or refractory CD30+ Hodgkin lymphoma following autologous stem cell transplant (ASCT) or patients not eligible for ASCT who have failed at least two other chemotherapy treatments. Adcetris has also been approved for patients with anaplastic large cell lymphoma (ALCL) as a second line following a phase II study where 86% of patients showed an overall response rate and 54% complete responses . The most common adverse reactions were peripheral sensory neuropathy, neutropenia, fatigue, nausea, and thrombocytopenia.
4.2 Solid Tumors
4.2.1 Ado-Trastuzumab Emtansine
Ado-trastuzumab Emtansine (T-DM1, Kadcyla)  is composed of trastuzumab, an anti-Her2 humanized IgG1 antibody random conjugated via Lys to non-cleavable SMCC-DM1 with an average DAR of 3.5. In vitro potency of trastuzumab-MCC-DM1 in a panel of human breast cancer cell lines expressing Her2 was IC50 4–15 ng/mL . The clinical MTD was 3.6 mg/kg every 3 weeks with a t½ of 3.5 days . In early 2013, T-DM1 was approved as a new therapy for patients with HER2-positive, late-stage (metastatic) breast cancer. Currently, T-DM1 is the only ADC approved for treating metastatic breast cancer that overexpresses the HER2 antigen [131, 132].
5 Future Directions and Perspective
Therapeutic ADCs for cancer treatment are experiencing encouraging success with the approval of Adcetris and Kadcyla and promising results with several clinical candidates in advanced clinical trials [133, 134]. The recent advances in linker development led to a breakthrough in the ability to achieve good clinical efficacy with limited toxicity. The two parameters that define the therapeutic index of ADCs are Minimum Effective dose (MED) and MTD and while ADCs present certain advantages over chemotherapy with regard to systemic toxicity still most current ADCs are dosed at or close to the maximum tolerated dose. The most encouraging aspect of ADC development is that targeted therapy, with very few examples, has delivered on its promise of showing efficacy against tumors defined by a particular target. The main challenge for next generation of ADCs is to improve their tolerability. While on-target toxicity can be addressed by a careful choice of target antigen, the off-target toxicity can be addressed with medicinal chemistry. Highly potent drugs have contributed to the success of the current generation of ADCs but all have shown significant dose-limiting toxicities . Generation of homogenous ADCs is a reality with the establishment of site-specific conjugation technology used by ADCs currently in the clinic. This technology, along with the advances in control of the hydrophobicity of linked drugs should lead to significant improvement in pharmacokinetics to a point where the half-lives of the ADCs should closely resemble the ones for the parent mAbs. The small fraction of current ADCs reaching the tumor  presents an excellent opportunity for improving the therapeutic index as just a small improvement in efficient targeting could have a great impact on both the MED and MTD. The non-specific uptake of ADC via target-independent internalization, i.e. pinocytosis, was recognized as an area of intervention . Exploiting the genetic differences between tumor and normal cells is in its infancy and medicinal chemists and cell biologists are faced with a great opportunity to harness these yet unknown devices that will enhance the ability to kill cancer cells with future ADCs. Uncovering tumor-specific mechanisms of release will require development of new triggers or combination of triggers within the same tether or linked drug. The suggested dependence of lysosomal escape upon the structure of the linked drug gave us a glimpse into the types of opportunities available to ADC medicinal chemists . From a structural perspective, the interaction of the linked drugs with the antibody is far from being understood. Developing an understanding of these interactions could provide design principles for medicinal chemists when building linked drugs for mAbs. Such an advance could be achieved by making use of molecular modelling or, ideally, by obtaining crystal structures of ADCs.
The efficacy of ADCs is dependent on whether the intracellularly released active species is a substrate of multidrug resistance (MDR) proteins. Most of the ADCs in clinical trials use a small set of cytotoxic warheads: auristatin and maytansinoids. Resistance, innate or acquired, to these drugs could lead to loss of clinical efficacy . It was shown that the chemical structure of the tether can enable the active species to evade efflux mechanisms. Medicinal chemists will always be challenged to discover new chemical warheads that are not efflux substrates, while still displaying the required potency for ADCs. The interaction of medicinal chemists with protein engineers, oncologists, cell biologists, and clinicians will result in better ADCs. These interactions present excellent opportunities to demonstrate that medicinal chemistry is a central science that can provide innovative solutions to the problems currently faced by this fascinating field of scientific endeavor.
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