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. Author manuscript; available in PMC: 2020 Jan 6.
Published in final edited form as: Drug Discov Today. 2017 Jun 13;22(10):1547–1556. doi: 10.1016/j.drudis.2017.05.011

Advances in antibody–drug conjugates: a new era of targeted cancer therapy

Samaresh Sau 1, Hashem O Alsaab 1,2, Sushil Kumar Kashaw 1,3, Katyayani Tatiparti 1, Arun K Iyer 1,4
PMCID: PMC6944323  NIHMSID: NIHMS884651  PMID: 28627385

Abstract

Antibody–drug conjugates (ADCs), a potent class of anticancer therapeutics, comprise a high-affinity antibody (Ab) and cytotoxic payload coupled via a suitable linker for selective tumor cell killing. In the initial phase of their development, two ADCs, Mylotarg®, and Adcetris® were approved by the US Food and Drug Administration (FDA) for treating hematological cancer, but the real breakthrough came with the discovery of the breast cancer-targeting ADC, Kadcyla®. With advances in bioengineering, linker chemistry, and potent cytotoxic payload, ADC technology has become a more powerful tool for targeted cancer therapy. In addition, ADCs with improved safety using humanized Abs with a unified ‘drug:antibody ratio’ (DAR) have been achieved. Concomitantly, there has been a significant increase in the number of clinical trials with anticancer ADCs with high translation potential.

Keywords: antibody drug conjugates, targeted cancer therapy, receptor mediated endocytosis, receptor overexpression, antibody bioengineering, advance linker, protein scaffold, Mylotarg, Adcetris, Kadcyla

Introduction

ADCs are an emerging class of targeted anticancer drug delivery agent that confer selective and sustained cytotoxic drug delivery to tumors [1]. An ADC can be divided into three main structural units (Figure 1a): the Ab; the cytotoxic agent; and the linker. Selecting a high-affinity Ab, stable linker, and potent cytotoxic payload enables the novel development of safe and efficient ADCs [2]. Many monoclonal Ab (mAb), such as avastin, rituximab, and cetuximab, are well known as standard treatments for solid tumors and hematological cancers [3]. By contrast, pristine chemodrugs, such as vinblastine, doxorubicin, and paclitaxel, have limited use for cancer treatment because of their nonspecific toxicity, thus narrowing the therapeutic window and increasing drug resistance [4,5]. Discovery of ADCs bridged the gap between the Ab and cytotoxic drug, creating highly specific anticancer agents with an improved therapeutic window [6]. The first FDA-approved ADC, Mylotarg®, a conjugate of the CD33 Ab and a calicheamicin payload, was developed for acute myelogenous leukemia (AML), although this was withdrawn from the market 10 years after its initial approval. Adcetris®, a conjugate of the CD30 Ab and monomethyl auristatin E, was approved for the treatment of lymphoma [2]. In 2013, Kadcyla® was commercialized for targeting HER-2-positive metastatic breast cancer, with emtansine (DM1) as the cytotoxic agent [1]. Although ADCs are designed against tumor-specific antigens, there are challenges associated with Ab immunogenicity, antigen expression, premature drug release, and low chemotherapeutic drug potency [7]. However, over time, advances in bioengineering have improved the safety profile of ADCs, particularly third-generation ADCs. In this review, we discuss current prospects for, and technical improvements in, ADCs towards developing safer and more efficacious personalized cancer medicine.

Figure 1.

Figure 1.

(a) Structural characteristics of earlier generation and advanced-generation antibody–drug conjugates (ADCs) [75]. (b) List of targetable tumor antigens: epidermal growth factor receptor (EFGF), platelet-derived growth factor receptor (PDGFR); tyrosine-protein kinase met (C-Met); epithelial cell adhesion molecule (EPCAM); carbonic anhydrase- IX (CA-9); tumor-associated calcium signal transducer 2 (TROP-2); vascular endothelial growth factors receptor-2 (VEGFR-2); prostate specific membrane antigen (PSMA); endothelin receptor-B (ET-B); matrix metallopeptidase-9 (MMP-9); and fibroblast activated protein (FAP) [1].

Criteria for a successful ADC

Selection of targeting antigen

The selection of the targeting antigen the first and most important determining factor for a successful ADC. The ideal characteristics of a useful antigen are: (i) higher-fold expression in the tumor than in the healthy tissue; for example, Adcetris® targets the CD30 antigen, whose expression on the surface of mature and immature myeloid cells is high (90%–100%) in all patients with AML [8], whereas Kadcyla® targets the HER2 receptor, whose expression is almost 100-fold higher in cancer cells than in healthy cells [1]; (ii) internalization of antigen via endocytosis in the presence of ligand and its recycling back to the plasma membrane [9]; and (iii) homogeneous antigen expression in the tumor microenvironment and low antigen abundance in circulation [10] (Figure 1b). Therefore, given that ADCs target tumor-associated antigens, there should be minimum expression of such antigens on healthy cells to reduce any adverse effects [11]. For example, prostate-specific membrane antigen (PSMA) is expressed on the surface of cancer cells, whereas, in the healthy prostate, it is found in the cytoplasm; therefore, noncancer cells are not affected by PSMA-targeting ADCs. Thus, the indium (In111)-labeled PSMA-targeting Ab, ProstaScint® can be clinically used for the early detection of prostate cancer [12]. It is suggested that 10 000 antigens per cell is the minimum number of antigens required to ensure the selective delivery of lethal cytotoxic drugs to cancer cells [2]. A major challenge to solid tumor therapy arises from antigen expression, which varies with tumor volume, heterogeneity, and treatment [13]. In addition to specific and sufficient expression, an optimal targeting antigen should also stimulate effective ADC internalization [14]. This internalization efficiency depends on the choice of Ab, epitopes of the antigen, and type of target (Figure 2). It has been reported that some targets frequently internalize regardless of ligand binding, whereas others reside permanently on the cell surface [2] (Figure 2). It was thought that the anticancer efficacy of ADCs relies on their internalization by cancer cells. However, recent work on spliced domain fibronectin-conjugated maytansinoid (SIP-F8-SS-DM1) showed that the internalization of antigen is not always necessary to achieve a therapeutic effect [15]. This is because this disulfide-linked ADC is reduced at the subendothelial extracellular matrix of solid tumors and increases the local concentration of cytotoxic payload near the tumor vasculature, enabling the drug to diffuse to the neoplastic mass. Abs of ADCs are occasionally capable of inducing antigen-mediated anticancer activity in addition to the cytotoxicity of the ADC payload [16]. This occurs when the antigen inhibits the downstream signaling of cancer cells when binding with the Ab.

Figure 2.

Figure 2.

Mechanistic pathways of antibody–drug conjugate (ADC) internalization and cancer cell killing [2].

Other challenges to targeting tumor antigens with ADCs include high interstitial tumor pressure, physical and kinetic barriers, and a bystander effect associated with the heterogeneous expression of antigen [7]. The bystander effect is a phenomenon of nonspecific systematic toxicity that arises because of the diffusion of the membrane-internalized cytotoxic payload from antigen-positive tumor cells to neighboring antigen-negative healthy cells [17].

Antibody selection

Important properties of Abs in ADCs include their antigen affinity, target specificity, good retention, minimal immunogenicity, low cross-reactivity, and along circulation in plasma. Typically, the binding affinity of the Ab component of ADCs is in the range 0.1–1 nM. Murine Abs were used in first-generation ADCs, which caused severe immunogenicity by producing human antimouse Abs in patients; however, bioengineering research resulted in the discovery of chimerized, humanized, and fully human Abs [6]. Today, most Abs used in humans are either human derived or humanized. Humanized Abs mainly derive from human sources except for the complementarity-determining regions (CDRs), which have murine origins. In humanized Abs, mouse CDRs are either grafted or resurfaced to the human Fv surface and constant regions (Figure 3a) [18]. Kadcyla® is an example of a CDR resurfacing humanized ADC. Most Abs in ADCs utilized in clinical trials are human immunoglobulin (Ig)-G isotypes, particularly IgG1. Occasionally, the unmodified Fc regions of Abs bind to Fc-gamma receptors (FcγR) of immune cells, such as macrophages and natural killer cells, and induce Ab-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), resulting in induction of antitumor immune responses [7]. The effects of ADCC and CDC are particularly prominent in human IgG1 isotype mAbs rather than in IgG4, and IgG2 mAbs; for example, trastuzumab, which is the drug in Kadcyla®, was found to initiate ADCC and CDC-associated tumor cell killing.

Figure 3.

Figure 3.

(a) Schematic representation of fully human (blue), mouse (green), chimeric (fused mouse-originating antigen-binging domain with human constant domain, green-blue) and humanized [fused mouse CDR domain with a human immunoglobulin (Ig)-G backbone] antibody. Fab and Fc are antibody subdomains and Fv is a variable domain. (b) Advances in ADC technologies and representative images of first-generation (i), second-generation (ii) and third-generation (iii) ADCs. Adapted, with permission, from [7] (bi,ii), [76] (biii), [14] (biv) and [66] (bv).

In addition to monospecific Abs, bispecific Abs have been developed to enhance therapeutic functionality. These are used for targeting two antigens of tumor cells and tumor-associated immune cells [19]. In 2014, the FDA approved blinatumomab, a bispecific T cell engager (BiTE) for the treatment of Philadelphia chromosome-negative acute lymphoblastic leukemia (ALL) [20]. Few reports are available regarding bispecific ADC and most are still in preclinical investigations. One such bispecific ADC is bsHER2xCD63his-ADC, which targets two antigens of cancer cells, HER2 and CD63 (a protein that is responsible for communicating between the cell membrane and intracellular compartments) [20].

Linker selection

The linker has a key role in ADC outcomes because its characteristics substantially impact the therapeutic index, efficacy, and pharmacokinetics of the ADC [6,21]. The stable linkers in ADCs can maintain the Ab concentration in the blood circulation and do not release the cytotoxic drug before reaching the target, resulting in minimum off-target effects. However, the linker should be labile enough to rapidly release the cytotoxic drug once the ADC is internalized to the tumor cells [10]. Another critical consideration is how many drug molecules should be loaded onto the Ab: the so-called ‘DAR’. Therefore, optimization of DAR is necessary because attaching too few drug molecules will lead to lower efficacy, whereas attaching too many will alter the pharmacokinetics and further increase instability and toxicity [7]. The FDA has approved ADCs with proven activity that are manufactured by nonspecific conjugation to lysine residues of Abs; however, these generated an undesirable heterogeneous mixture of ADCs, with high DAR values. Therefore, research efforts are directed to the design of homogeneous ADCs with a high number of drug molecules stably linked to the Ab. The aim of most common ADCs is to attain a DAR value close to 4 [10,22].

Cleavable linker

The first type of cleavable linker can be classified into three types: hydrazone, disulfide, and peptide linkers. Each corresponds to a different tumor-specific intracellular condition: low pH sensitive, glutathione sensitive, and protease sensitive, respectively [23]. Acid-sensitive hydrazone linkers, which take advantage of the low pH (4~6) within the endosomes and lysosomes of cancer cells, undergo acid hydrolysis and release the cytotoxic payload [24,25].

Disulfide linkers are more stable in the circulation, and exploit higher concentrations of glutathione within cancer cells. Glutathione concentration is higher in cancer cells, because it is associated with cell survival and tumor growth, and is also elevated during cell stress conditions, such as hypoxia [2628].

Another form of cleavable linker is a (lysosomal protease-sensitive) peptide, which is more stable than the other forms of cleavable linker discussed above. It also enables improved control of drug release by attaching to the cytotoxic drug with mAbs [29]. Peptide-based linkers are optimized to release their toxic payload upon cleavage by distinct intracellular enzymes (proteases). For example, cathepsin B (a tumor-specific protease) recognizes and cleaves a specific dipeptide bond inside the tumor cells [30,31]. FDA-approved Adcetris® contains a cathepsin B-sensitive dipeptide linkage (valine-citrulline).

Another protease-sensitive linker, β-glucuronide, undergoes degradation and hydrolysis by β-glucuronidase, which is a lysosomal enzyme overexpressed in many cancers and utilized for selective payload release [32]. Burke et al. showed that the PEGylated self-immolative linker, β-glucuronide-conjugated monomethyl auristatin E (MMAE), had a DAR value of 8, and enhanced pharmacokinetic stability and potency in xenograft models compared with nonPEGylated linkers [33]. However, more research is required for the clinical improvement of glucuronic acid linker-based ADCs.

Noncleavable linkers

The other class of ADC linker comprises noncleavable thioether or maleimidocaproyl (mc), which both depend on the lysosomal enzymatic degradation of ADC to release the cytotoxic payload after internalizing to cancer cells [34,35]. For example, Kadcyla® was successfully designed to utilize a noncleavable mc-linker that links maytansinoid toxin to the HER2 Ab [36,37]. As a result of the stable linker, Kadcyla® is stable in serum for more than 3 days [38].

The advance design has extended the emergence of innovative linkers for toxic payload conjugation at the linker site instead of the Ab site. For example, SpaceLink Technology, developed by Syntarga, is a highly flexible linker-based ADC [39]. This linker can reversibly attach to the toxic payload and Ab. As a result, this allows the selection and optimization of the payload and linker–payload combination to generate ADCs with maximal therapeutic potency [39]. An excellent example of this promising technology is anti-HER2-duocarmycin conjugates, which exhibit in vivo antitumor efficacy with reduced off-target toxicity [39]. Thus, researchers are currently investigating several other innovative linkers for the safe and efficacious delivery of ADC to treat chronic diseases.

Cytotoxic payloads for ADCs

The effectiveness of ADCs depends on the internalization of the ADC, followed by the release of active cytotoxic molecules inside the cytoplasm of tumor cells. For tumors with insufficient expression of targeting antigens, the potency of the payload should be sufficient to kill the cancer cells, even at low doses [40]. Given that ADCs currently require intravenous administration, it is crucial that the cytotoxic payload exhibits extended stability in the circulation. Apart from these physicochemical features, the chemical structure of cytotoxin should also allow conjugation to the linker while maintaining the internalization property of the Ab and promoting its antitumor effects [41]. The drug payload in the ADC should demonstrate a high potency therapeutic index because it is projected that only approximately 2% of the injected ADC dose will reach the tumor site, resulting in low intracellular drug delivery [2]. A drug that is suitable for use in an ADC should be potent enough to kill aggressively dividing cancer cells but not lethal to healthy cells. In fact, drugs used in clinical trials are limited to six to eight compounds; the majority of these payloads originate from natural product sources, and are categorized as follows [41]: (i) Microtubule inhibitors (DM1, DM4, MMAF, and MMAE); DNA synthesis inhibitors [calicheamicin, doxorubicin, duocarmycin, and pyrrolobenzodiazepines (PBD)]; and (iii) topoisomerase inhibitors [SN-38 (quinoline alkaloids)].

Consequently, an ideal payload for an ADC should have an in vitro subnanomolar IC50 (half maximal inhibitory concentration) value against tumor cell lines and a suitable functional group with adequate solubility in aqueous solutions for chemical conjugation with the Ab and for improving the solubility of the resulting ADC [42,43]. Derivatives of auristatin, maytansinoid, calicheamicin, duocarmycin, PBD, and amanitin are currently being utilized as cytotoxic payloads, and are described below [44].

Auristatin

Auristatin is a dolastatin 10-based auristatin analog [45]. Clinical trials of Dolastatin 10 did not progress because of its nonspecific toxicity. However, its synthetic derivatives MMAE and MMAF are currently used as cytotoxic payloads in ADCs. MMAE and MMAF function as mitotic inhibitors. The first and only FDA-approved auristatin-based ADC is Adcetris®, which is used for lymphoma and systemic anaplastic large cell lymphoma (sALCL) [40]. However, more than ten auristatin-based ADCs are currently in clinical trials [44] (Table 1).

Table 1.

List of ADCs in clinical trialsa,b

Clinical trial stage ADC name Antibody/Linker Cytotoxic drugs Cancer type (s) Antigen targeted
Phase I ASG-22ME Hu IgG1/valine-citrulline (V.C) MMAE Urothelial and other malignant solid tumors Nectin-4
SAR566658 Hz IgG1/DS6 disulfide Maytansinoid Malignant neoplasm, triple-negative breast cancer CA6
BAY 94–9343 H IgG1/disulfide, HuIgG1/SPDB Maytansinoid, (DM4) Mesothelin-positive solid tumors Mesothelin
IMGN388 IgG1/disulfide Maytansinoid Solid tumors Integrin αv
BIIB015 IgG1/disulfide Maytansinoid Anti-Cripto, refractory solid tumors Cripto-1
SGN-75 Anti D70/dipeptide Auristatin Renal cell carcinoma CD70
AGS-22M6E Anti-Nectin fully human IgG/dipeptide Auristatin Malignant solid tumors Nectin4
IMGN529 K7153A humanized IgG1 thioether Maytansinoid B cell malignancies CD37
AMG595 Anti-EGFRvIII fully human IgG1/thioether Maytansinoid Glioblastoma EGFRvIII
RG7593/DCDT2980S Hz IgG1/valine-citrulline MMAE NHL CD22
SAR566658 Hu IgG1/SPDB Maytansine (DM4) Solid tumors CA6
Labestuzumab-SN-38 HzIgG1/phenylalanine-lysine SN38 Colorectal cancer CD66e/CEACAM5
AGS-16C3F Hu IgG2/maleimidocaproyl MMAF Renal cell carcinoma ENPP3
SGN-CD33A Hz PDB dimer AML CD33
SGN-CD19A Hz IgG1/maleimidocaproyl MMAF B cell lymphoma CD19
SGN-LIV1A Hz IgG1/V.C MMAE Metastatic breast cancer LIV-1
RG7596 Hz IgG1/V.C MMAE NHL CD79b
ASG-5ME Hu IgG2/V.C MMAE Pancreatic, gastric, an prostate cancers SLC44A4
BAY 79–4620 Hu IgG1/V.C MMAE Advanced solid tumors CA-IX
AGS-67E Hu IgG2/V.C MMAE Lymphoid malignancies, leukemia CD37
AMG-172 Hu IgG1/MCC Maytansine (DM1) Clear cell renal cell carcinoma, renal cell adenocarcinoma and carcinoma CD27L
AGS15E Hu IgG2/[cleavable] MMAE Metastatic urothelial cancer SLITRK6
GSK2857916 Hz IgG1/maleimidocaproyl MMAF Multiple myeloma BCMA
IMGN289 SMCC Maytansine (DM1) EGFR-positive solid tumors EGFR
AMG 595 SMCC Maytansine DM1 Advanced malignant glioma, anaplastic astrocytomas, glioblastoma multiforme EGFRvIII
IMGN242 (huC242-DM4) Humanized IgG1/huC242 disulfide Maytansinoid Solid tumors CanAg
Phase I/II Immunomedics (IMMU)-110 (hLL1-DOX) Milatuzumab hydrazone Doxorubicin Multiple myeloma CD74
Lorvotuzumab mertansine (IMGN901) Humanized IgG1/huC242 disulfide Maytansinoid Multiple myeloma, solid tumors CD56
IMMU-132 Hz IgG1/CL2A CPT-11 SN38 Colorectal cancer, gastric adenocarcinoma, esophageal cancer, hepatocellular carcinoma TROP-2
Milatuzumab doxorubicin Hz IgG1/hydrazone Doxorubicin Multiple myeloma CD74
HuMax®-TF Hu IgG1/V.C MMAE Solid tumors Tissue Factor
Phase II SAR3419 (huB4-DM4) huB4/humanized IgG1 disulfide, Hz IgG1/SPDB DM4 B cell NHL CD19
BT062 ChIgG4/SPDB, anti-CD138 chimeric IgG4 disulfide DM4 Multiple myeloma CD138, Syndecan1
Glembatumumab vedotin (CDX-011) Hu IgG2/valine-citrulline, anti-CR011 dipeptide MMAE Breast cancer melanoma, squamous cell carcinoma of lung gpNMB
Anti-PSMA ADC Hu IgG1/V.C MMAE Prostate cancer PSMA
MLN0264 Hu IgG1/V.C MMAE Gastrointestinal malignancies Guanylyl cyclase C
Lorvotuzumab mertansine Hz IgG1/SPP DM1 Solid tumors CD56
PSMAADC Anti-PSMA fully human IgG1/dipeptide Auristatin Metastatic, hormone-refractory prostate cancer PSMA
Phase III Inotuzumab ozogamicin (CMC 544) Humanized IgG4/G5/44 hydrazone Calicheamicin B- cell lymphomas, NHL CD22
FDA Approved Gemtuzumab ozogamicin (mylotarg®), Now terminated Hu IgG4/hydrazone calicheamicin AML CD33
Trastuzumab-emtansine (Kadcyla®) HzIgG1 trastuzumab/thioether DM1 Metastatic breast cancer HER2
Brentuximab vedotin (Adcetris®) Ch IgG1/V.C MMAE Hodgkin’s lymphoma CD30
Terminated LOP628 HzIgG1/(noncleavable) Maytansine AML/C-Kit-positive solid tumors cKit
b

Abbreviations: Ch, chimeric antibody; Hu, fully human; Hz, humanized.

Maytansionids

Maytansinoids (DMs) are thiol derivatives of maytansines and their function is similar to that of Vinca alkaloids. Maytansines bind to the ‘plus’ end of the growing microtubule and block the polymerization of tubulin dimers, thus preventing the formation of mature microtubules [44,46]. However, maytansine derivative (DM1 or DM4)-conjugated ADCs can selectively deliver the payload in cancer cells, thus improving the therapeutic index. Some maytansinoid-based ADCs are currently in clinical trials for solid and hematological cancers [7]. Among these, trastuzumab-DM1 (Kadcyla®), a maytansinoid-linked HER-2-targeting ADC, was approved by the FDA for use against HER-2-positive metastatic breast cancer.

Calicheamicin

Calicheamicin, a potent DNA-targeting agent, is used as a toxic payload for ADCs [47]. Calicheamicin recognizes the minor groove of the TCCTAGGA sequence of DNA and inhibits DNA replication [44]. Mylotarg®, a calicheamicin γ1- anti-CD33 conjugate, has an acid-cleavable hydrazone linker. Mylotrag® was withdrawn from market 10 years after its initial approval, because of adverse effects such as premature release of calicheamicin, low conjugation efficiency of drug to Ab, exchange of Fab arm with serum Ab, and unwanted binding of ADC to CD33-positive liver cells [1]. CMC-544, a calicheamicin-linked CD22 (IgG4)-targeting ADC has been pursued in multiple clinical trials for different hematological cancers, such as ALL and non-Hodgkin’s lymphoma (NHL) [48].

Duocarmycin

Duocarmycin, a potent antitumor antibiotic, has an IC50 of 40–100 pM [49]. It targets the minor groove of DNA, where it alkylates adenine bases. Synthetic analogs of duocarmycin, such as CC1065, is are to be a potent payload for ADCs; currently, BMS-936561 (MDX-1203) is in a Phase I clinical trial [50]. CC1065-conjugated ADCs are efficacious against multidrug resistant (MDR) cell lines (DLD-1 and HCT-15) and showed promising in vivo antitumor effect at lower dose [44].

Amatoxin

Amatoxin is a cyclic peptide that binds RNA polymerase II, leading to the inhibition of DNA transcription and ultimately programmed cell death [51]. ChiHEA125-Ama is anti-EpCAM linked to α-amanitin ADC [52].

Other ADC payloads

Some miscellaneous cytotoxic payloads, such as derivatives of PBDs and centanamycin (indole carboxamide), are also considered as potent payloads for ADC development. These molecules bind to double strands of DNA and either alkylate or intercalate the DNA, thereby inhibiting DNA replication. The PBD-containing ADCs (SGN-CD33A and SGN-CD70A) are currently in Phase I clinical trials [44,53] (Table 1). PBDs are naturally produced and intercalate specific regions of DNA within the tumor cell [44]. The blocking of cancer cell division potentially inhibits tumor growth without distorting its DNA helix structure, thereby avoiding emergent drug resistance [54].

Doxorubicin

Doxorubicin binds to DNA by intercalation and inhibits DNA synthesis [55]. For example, milatuzumab-conjugated doxorubicin ADC (IMMU-110) has undergone Phase I/II clinical trials for CD74-positive relapsed multiple myelomas [56].

The primary challenge to developing a new cytotoxic drug is that it should have a high therapeutic index value; therefore, a low dose of drug would be enough to kill the tumor cells, resulting in the reduction of adverse effects.

Advances in ADC technology

Since the breakthrough discovery of the first FDA-approved ADC, Mylotrag®, anticancer therapy research has diversified, resulting in the development of several novel, safer ADCs. Herein, we describe advances in the development of ADCs towards targeted anticancer drug delivery.

First-generation ADCs

The primary focus in the design of first-generation ADCs was faster body clearance to reduce adverse effects. To achieve this, ADCs were made with murine-derived Ab backbones; however, in humans, these generated immunogenic human antimouse Abs, which accelerated the clearance of ADCs by the immune system [44]. The choice of antigen in first-generation ADCs was not specific to tumor cells, resulting in adverse effects. The linkers of ADCs were also not stable enough in the circulation and the toxic payload used was less potent (micromolar IC50 range). For example, BR96-Dox, an anti-Lewis Y-targeting BR96-mAb conjugated with doxorubicin, was terminated in Phase II clinical trials against Lewis Y-expressing epithelial tumors [57]. Similarly, the FDA-approved CD33-targeting Mylotrag® was also withdrawn from market. Both ADCs showed acute adverse effects and morbidity in patients, attributed to acid-labile weak hydrazone linkers and nonspecific antigen expression in healthy cells [58].

Second-generation ADCs

The limitations and failures of first-generation ADCs were eliminated in second-generation ADCs. The premature release of drugs because of the unstable hydrazone linker in Mylotrag® has been avoided in second-generation FDA-approved ADCs, by using different linkers, such as the valine-citrulline (cathepsin cleavable) linker in Adcetris® and a thioester (noncleavable) linker in Kadcyla ®. The cytotoxic payloads used in second-generation ADCs are also more potent than in first-generation ADCs. For example, tubulin-targeting agents, such as MMAE used in Adcetris® is approximately 100–1000-fold stronger than DNA-intercalating doxorubicin of BR96-Dox [59]. The IC50 of MMAE is approximately 1 nM in different human cancer cell lines, whereas doxorubicin IC50 is in the 1–6 μM range [60]. Despite the improvement in cytotoxic payloads and the introduction of stable linkers, second-generation ADCs have significant limitations in terms of their heterogeneous DAR, resulting from stochastic coupling strategies between the Ab and drug [61]. Typically, chemical conjugation between the drug and Ab occurs via the lysine or cysteine residue of the mAb, which generates DAR (range 0–8) with an average value of 3–4. Therefore, heterogeneous ADCs can contain a mixture of unconjugated, partially conjugated, and overconjugated Abs and, therefore, there will be competition between unconjugated Abs and drug-conjugated species for antigen binding that can diminish the activity of the ADC. By contrast, overconjugation of the drug to the Ab can result in Ab aggregation, a decrease in stability, incremental increases in nonspecific toxicity, and a reduction in the half-life of ADCs in the circulation [22]. Overall, heterogeneous ADCs have a limited therapeutic index and tumor penetration abilities, which are associated with the induction of drug resistant in the tumor microenvironment.

Third-generation ADCs

The aforementioned concerns regarding the heterogeneous DARs of second-generation ADCs have been addressed in third-generation ADCs. Site-specific conjugation has been introduced to produce homogenous ADCs with well-characterized DARs and desired cytotoxicities [62]. The site-specific conjugation of the drug to Ab provides a single-isomer ADC with a uniform DAR value. Such ADCs can be made using bioengineered Abs containing site-specific amino acids, such as cysteine, glycan, or peptide tags [63]. For example, precise site-specific conjugation of MMAE to human IgG was developed by replacing the Ala114 amino acid of the CH1 domain of the IgG Ab with cysteine to create a selectively engineered Ab, called THIOMAB [64]. The site-specific coupling of drug with THIOMAB creates THIOMAB drug conjugates (TDCs) that have homogeneous DARs, low hydrophobicity, and better safety profile for patients [62]. Alternative approaches to site-specific drug conjugation include: (i) a thio-bridge approach that links drugs to the interchain disulfide bond of Abs (four per mAb) [14]; (ii) introduction of unnatural amino acids, such as p-acetylphenylalanine, or noncanonical amino acids, such as phenylselenocysteine, using a encoded gene of the amber stop codon (TAG), along with corresponding orthogonal tRNA/aminoacyl-tRNA synthetase [65]. Recently, a novel chemoenzymatic approach, SMARTag™, was used for the site-selective modification of Abs that generated uniform DAR in ADCs. In this method, the formylglycine-generating enzyme (FGE) recognition sequence was inserted at a specific location in the Ab backbone using molecular biology techniques. In eukaryotic cell FGE, an endogenous enzyme of eukaryotic cells catalyzes the conversion of the cystine to a formylglycine residue (fGly) [66]. The aldehyde group of fGly is involved in the site-specific reaction of drugs, such as PBDs, auristatin, and duocarmycin. SMARTag™-based ADCs, such as CD22–4AP, are a proprietary technology of Catalent’s pharma solution and preclinical studies of CD22–4AP showed it to have a promising therapeutic index with minimum adverse effects and to avoid the development of drug resistance. Similarly, other enzymatic strategies, such as SMAC-TAG™ and TG-ADC™, have been developed for site-specific ADC preparation [67] (Figure 3b). Efforts are continuing to develop more efficient cytotoxic payloads in third-generation ADCs so that a smaller amount of drug is needed to achieve the desired therapeutic effect and to eliminate drug-resistant tumor cells. The development of PBDs, derivatives of tricyclic antibiotics, has attracted interest over other DNA-alkylating agents. PBDs are potent compounds, some of which have subpicomolar IC50 and do not show cross-resistance with other chemotherapeutics, such as cisplatin [68]. Four PBD-containing ADCs are currently in clinical trials: SC16LD6.5 is in Phase II trials for small cell lung carcinoma; SGN-CD33A is in Phase II trials for AML; SGN-CD70A is used to treat patients with CD70-positive cancer, and ADCT-301 is in Phase I trials for CD25 lymphoma [69].

Clinical trials of ADCs

The FDA withdrawal of mylotarg® (10 years after its initial approval) was a lesson learned for redesigning ADC components. Researchers have made significant progress in improving conjugation technology, Ab bioengineering, linker chemistry, and the identification of potent drugs has resulted in more successful clinical outcomes for ADCs (Table 1). This led to the FDA approval of Adcetris® and Kadcyla®, and additional clinical trials, such as 50 active trials for solid tumors and 20 active clinical trials for hematological cancers [44,70]. Pharma Source’s trend reports (www.pharmsource.com/trend/adc-market-opportunity-for-cmos) on ADCs reported that more than 15 clinical trials were pursued in 2015, six to eight trials were completed in 2016, and that this trend will continue in 2017. The promising ADC, inotuzumab ozogamicin (CMC 544), was recently withdrawn for the treatment of relapsed NHL. However, CMC-544 is in active Phase III trials for ALL, and was recognized as an orphan drug by the FDA. Some Phase I trials show some success in the use of drugs from the maytansinoid or auristatin classes of molecules for various cancers. Furthermore, the most commonly used Ab is IgG1, although there are trials on going with IgG2 and IgG4. Despite the strong clinical outcome, the cost of ADC development remains a major limitation. For example, the cost of treatment with the mAb trastuzumab is approximately US$50 000 per year, and the price almost doubles for the trastuzumab-DM1 conjugate Kadcyla®. The development of ADCs is currently directed at decreasing the costs of production by using cheaper nonanimal recombinant mAb technologies and repurposing existing drugs [71]. Therefore, based on the success rate of these clinical trials, it is evident that there is much scope for the further study of ADCs.

Concluding remarks: challenges and hope for ADCs

Extensive research is on going to improve all the components of ADCs that can enhance their targetability and therapeutic efficacy against tumors. A better understanding of ADC-targeting strategies can speed up the FDA approval rate of ADCs and drastically increase the number of clinical trials, especially in solid tumors. However, the failure of some ADCs, such as Mylotrag®, IMGN242-for gastric cancer in Phase II trials, and SGN15-for ovarian cancer in Phase II trials, is mostly attributed to payload-related toxicity and low therapeutic index [44]. These limitations are being addressed in various ways, such as the development of homogenous ADCs with narrowed DARs, using bispecific Ab with enzyme-activated linkers, and high potent payloads [20,69]. Another challenge to the clinical success of ADCs is the evaluation of viable biomarkers, which will ensure the effectiveness of ADCs for selective cancer targeting. Immunohistology (IHC) is a widely used method of determining antigen expression and requires tissue biopsies, which sometimes limits its use. Recently, other efforts, such as the use of circulating tumor cells and imaging techniques, avoid the limitations of IHC-based methods and are being used to determine the patient population likely to respond to ADC therapy [72]. Another approach to increasing the binding affinity of ADCs is to use protein scaffolds, which have well-defined 3D polypeptide frameworks and their binding affinity to antigens is in the nM to pM range. Clinically utilized protein scaffolds are categorized into two classes: non-Ig scaffolds, such as the Kunitz domain scaffold-based DX88 and affibody scaffold-based ABY-220; and Ab-derived scaffolds, such as the BiTE scaffold-based blinatumomab and nanobody scaffold-based Alx-0141 [73]. These scaffolds appear promising in the development of higher-affinity ADCs. A major hurdle to ADCs targeting solid tumors is the tumor stroma, which is a thick, connective, functionally supportive framework layer comprising tumor and nontumor cells. This tumor stroma has been identified as crucial factor in promoting tumor growth, angiogenesis, metastasis, and immune suppression. Recently, emphasis has shifted to inhibiting fibroblast activation protein (FAP)-expressing tumor stromal cells [74]. FAP-targeting mAbs are still in development and only a few have been utilized in clinical trials. Therefore, it is hoped that, in the near future, tumor stroma-targeting ADC could open a new therapeutic window for the treatment of solid tumors.

highlights.

  • ADC represents a new class of targeted anticancer therapeutics

  • Elucidates selection criteria for tumor specific antigen and ADC development

  • Smart linkers and fixed DAR value for reducing side effects of ADCs are covered

  • Current clinical trial trends and market prediction of ADCs are summarized

Acknowledgments

K.T. would like to acknowledge an AGRADE scholarship from the Wayne State University Graduate School to pursue MSc studies in the Iyer Lab, Department of Pharmaceutical Sciences, Wayne State University. A.K.I. acknowledges US National Institutes of Health/National Cancer Institute (NIH/NCI) grant R21CA179652 and Wayne State University start-up funding for research support.

Footnotes

Teaser: Discovery of antibody–drug conjugates (ADCs) bridged together by smart linkers created a powerful entity that can deliver highly efficacious anticancer agents targeted to tumor cells and tissues with an improved therapeutic window. Advancement of protein engineering, linker chemistry, and new cytotoxic payloads herald ADCs as safe and effective anticancer therapeutics for personalized medicine.

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