Antibody-Drug Conjugates: The New Frontier of Chemotherapy

Abstract

In recent years, antibody-drug conjugates (ADCs) have become promising antitumor agents to be used as one of the tools in personalized cancer medicine. ADCs are comprised of a drug with cytotoxic activity cross-linked to a monoclonal antibody, targeting antigens expressed at higher levels on tumor cells than on normal cells. By providing a selective targeting mechanism for cytotoxic drugs, ADCs improve the therapeutic index in clinical practice. In this review, the chemistry of ADC linker conjugation together with strategies adopted to improve antibody tolerability (by reducing antigenicity) are examined, with particular attention to ADCs approved by the regulatory agencies (the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA)) for treating cancer patients. Recent developments in engineering Immunoglobulin (Ig) genes and antibody humanization have greatly reduced some of the problems of the first generation of ADCs, beset by problems, such as random coupling of the payload and immunogenicity of the antibody. ADC development and clinical use is a fast, evolving area, and will likely prove an important modality for the treatment of cancer in the near future.

Full text

Int. J. Mol. Sci. 2020, 21, 5510; doi:10.3390/ijms21155510 www.mdpi.com/journal/ijms
Review
Antibody-Drug Conjugates: The New Frontier of
Chemotherapy
Sara Ponziani 1,†, Giulia Di Vittorio 2, †, Giuseppina Pitari 1, Anna Maria Cimini 1, Matteo Ardini 1,
Roberta Gentile 2, Stefano Iacobelli 2, Gianluca Sala 2,3, Emily Capone 3, David J. Flavell 4,
Rodolfo Ippoliti 1 and Francesco Giansanti 1,*
1 Department of Life, Health and Environmental Sciences, University of L’Aquila, I-67100 L’Aquila, Italy;
sara.ponziani@guest.univaq.it (S.P.); giuseppina.pitari@univaq.it (G.P.);
annamaria.cimini@univaq.it (A.M.C.); matteo.ardini@univaq.it (M.A.); rodolfo.ippoliti@univaq.it (R.I.)
2 MediaPharma SrL, I-66013 Chieti, Italy; g.divittorio@mediapharma.it (G.D.V.);
r.gentile@mediapharma.it (R.G.); s.iacobelli@mediapharma.it (S.I.); g.sala@unich.it (G.S.)
3 Department of Medical, Oral and Biotechnological Sciences, University of Chieti-Pescara,
I-66100 Chieti, Italy; caponemily@gmail.com
4 The Simon Flavell Leukaemia Research Laboratory, Southampton General Hospital,
Southampton SO16 6YD, UK; davidf@leukaemiabusters.org.uk
* Correspondence: francesco.giansanti@cc.univaq.it; Tel.: +39-0862433245; Fax: +39-0862423273
† These authors contributed equally to this work.
Received: 14 July 2020; Accepted: 30 July 2020; Published: 31 July 2020
Abstract: In recent years, antibody-drug conjugates (ADCs) have become promising antitumor
agents to be used as one of the tools in personalized cancer medicine. ADCs are comprised of a drug
with cytotoxic activity cross-linked to a monoclonal antibody, targeting antigens expressed at higher
levels on tumor cells than on normal cells. By providing a selective targeting mechanism for
cytotoxic drugs, ADCs improve the therapeutic index in clinical practice. In this review, the
chemistry of ADC linker conjugation together with strategies adopted to improve antibody
tolerability (by reducing antigenicity) are examined, with particular attention to ADCs approved by
the regulatory agencies (the U.S. Food and Drug Administration (FDA) and the European Medicines
Agency (EMA)) for treating cancer patients. Recent developments in engineering Immunoglobulin
(Ig) genes and antibody humanization have greatly reduced some of the problems of the first
generation of ADCs, beset by problems, such as random coupling of the payload and
immunogenicity of the antibody. ADC development and clinical use is a fast, evolving area, and
will likely prove an important modality for the treatment of cancer in the near future.
Keywords: Mabs; Antibody-Drug Conjugate; cancer therapy; drug targeting; payload; cross-linking
1. Introduction
The twentieth century has been characterized by basic and applied research leading to the
discovery and use of an increasing number of cytotoxic chemotherapeutic compounds with the
ability to rapidly kill dividing cancer cells in preference to non-dividing healthy cells. The well-
known drawback of chemotherapy is due to the fact that these drugs, in addition to damaging cancer
cells, also damage healthy tissues; thus, causing side effects, sometimes with serious consequences.
The challenge is, therefore, to search for drug delivery systems that achieve high cytotoxic
efficacy against cancer cells, but with limited systemic toxicity. Antibody-drug conjugates (ADCs)
offer the promise of achieving this objective and increase the therapeutic index significantly.
The approach to targeted chemotherapy comes from Paul Ehrlich’s concept of the “magic bullet”
formulated at the beginning of the twentieth century [1]. The principle of this concept, to avoid side
effects, drugs must be guided and released into the tumor sites through association with ligands that

Int. J. Mol. Sci. 2020, 21, 5510 2 of 28
are overexpressed or selectively expressed in the tumor. Ehrlich’s proposal has been translated into
practical applications for therapy due to the development of monoclonal antibodies in the mid-70s,
combining the selectivity of recognition to the power of chemotherapeutic drugs [2]. To become a
pharmacologically active drug, monoclonal antibodies can be linked to either a radioisotope (giving
rise to Antibody radioimmunoconjugates, RAC), to a highly potent cytotoxic drug (antibody-drug
conjugates, ADCs) or protein toxins (producing immunotoxins) [3,4].
The production of ADCs face several vital issues, such as the target cell selection, the nature of
antigen, structure and stability of the antibody, the linker chemistry, and finally the cytotoxic
payload.
One of the first problems encountered in the use of antibodies was the fact that murine
antibodies are foreign proteins recognized as non-self by the human immune system that responds
by producing human anti-mouse antibodies (HAMA). HAMAs can have toxic effects due to immune-
complex formation in the patient and, thus, prevent further administration. With the technology of
recombinant DNA, Phage display, and transgenic mice, it is now possible to create of completely
human antibodies that are not immunogenic and greatly ameliorate such toxicities.
Chemotherapeutic drugs include antimetabolites (methotrexate, 6-mercaptopurine, 5-
fluorouracile, cytarabine, gemcitabine, etc.), molecules interfering with microtubule polymerization
(vinca alkaloids, taxanes), and molecules inducing damages on DNA (anthracyclines, nitrogen
mustards). The most recent generation of chemotherapeutic molecules include both DNA
damaging/alkylating agents (i.e., duocarmycin from Medarex/Bristol Mayer Squibb, Syntarge,
calicheamicin from Wyeth/Pfizer, indolino-benzodiazepine from Immunogen), and molecules
interfering with microtubule structure (i.e., maytansinoids, from immunogen, auristatin derivatives
from Seattle Genetics). These compounds can kill cells with extremely high potency so that severe
side effects greatly limit the administrable dose as a free drug. These compounds are therefore
considered as ideal payload components of ADCs with high therapeutic index [5].
The conjugation strategy and chemistry chosen to represent a key factor for the success of ADCs,
the homogeneity of ADC molecules being one of the main challenges in ADC design [2]. In deciding
in which chemical conjugation process to use, it is necessary to develop a strategy that allows the
reaction of those residues placed on the surface of the antibody through a chemical reactive group
present on the linker. These strategies, depending on the type of residue (mainly amino groups of
lysines or sulfhydryl groups of cysteines) that can lead to the production of mixed species whose
Drug-Antibody Ratio (DARs) is variable. When the DAR is poorly controlled, this phenomenon can
reduce the efficacy of the ADCs and furthermore increase aggregation possibility, the overall rate of
clearance and release of the payload systemically at an early stage [6], although higher DAR values
are beneficial for the overall potency. To improve the technology, focusing on obtaining
homogeneous ADCs with a high therapeutic index, site-specific conjugation technologies have now
been developed [7].
2. Basic Characteristics of the Conjugate
An ADC is composed of three different components (Figure 1): a monoclonal antibody, the
payload, and the linker that joins the first two components. Different types of conjugation chemistry
exist: as in the most common, linkage is obtained through lysine (-amine-group, -NH2 in the
deprotonated form) or cysteine (sulfhydryl-group, -SH). However, other conjugation strategies may
also be pursued (see below). Whatever the conjugation strategy, it is vital that this does not affect the
integrity and functionality of the antibody.

Int. J. Mol. Sci. 2020, 21, 5510 3 of 28
Figure 1. Schematic representation of various types of antibody-drug conjugates (ADCs) and their
components.
2.1. Monoclonal Antibody
In the development of ADCs for cancer treatment, the choice of the antigen and, consequently,
selection of the appropriate antibody plays a key role.
The antibody is chosen based on the molecular target recognition, with the highest affinity and
selectivity for the target. Ideally, it should recognize an overexpressed target only at the tumor site
to avoid delivering the pharmacological load inappropriately to non-target sites. For example, the
(human epidermal growth factor receptor 2) (HER2) receptor is more than 100 times overexpressed
in tumor tissues in comparison to the equivalent normal non-cancerous tissue [8].
The antigen against which the antibody is directed on the cancer cell should be present in high
copy number (>105/cell.) [9]. So far, several antigens have been reported overexpressed in cancer
tissues that can be exploited as targets for ADCs [10]. The antigen must be recognized and bound by
the antibody with a reasonable affinty (Kd ≤ 10 nM) to ensure rapid uptake in the target cell [11].
In the first generation of ADCs, in many cases murine antibodies being recognized as foreign
proteins generated a strong immune response with the production of anti-human antibodies that
potentially reduced their therapeutic efficacy. This problem has been partially solved through the use
of genetic engineering in second-generation ADCs, utilizing a mouse-human chimeric antibody
format. The “humanized” chimeric antibody contains the mouse light and heavy chain variable
regions that are linked to human constant regions. The chimeric ADCs showed promising results in
cancer treatment but sometimes the problem of decreased efficiency and human anti-chimeric
response were still present.
To overcome this problem, many efforts have been made to design a humanized monoclonal
antibody, which contain only murine complementary determining regions (CDRs) regions combined
with the human variable region [8] or fully human antibodies [12].
Usually, the antibodies used to construct ADCs are of the IgG1 class (Immunoglobulin G
Subclass 1) (~150 kDa), but since antibodies in ADCs exploit the Fab region to recognize the antigen
present at the end of light chains, only this region is essential to the antibody to carry out its function
as a specific carrier. Therefore, in some cases, smaller antibody formats (i.e., antibody fragments that
maintain the binding affinity for the receptor) have been used to create ADCs. These fragments can

Int. J. Mol. Sci. 2020, 21, 5510 4 of 28
be obtained by IgG cleavage following papain digestion or recombinant production to produce Fabs
and scFvs [13].
Selected antibodies and their derived ADCs can be directed against antigens that may or may
not induce internalization through receptor-mediated endocytosis (RME), and by this criterion,
ADCs can be classified as internalizing or non-internalizing.
2.1.1. Internalizing ADCs
Internalizing ADCs exploit RME to be internalized by target cells. In this case, the antibody
performs a fundamental role as it favors the internalization of the target antigen receptor, which
represents a crucial step for most ADCs to be effective. Although, as in the case of the anti-HER3
antibody EV20, the binding to the receptor and the internalization of receptor/antibody complex can
alone induce cell death and inhibition of tumor growth [14–16].
Following internalization, the ADC can follow different endocytic routes that crucially may have
profound effects on their cytotoxic efficacy. Clathrin-mediated and caveolae-mediated endocytosis
(CME) in which the receptor mediates endocytosis and, alternatively, clathrin-caveolin-independent
endocytosis, where the receptor does not mediate endocytosis [17]. The most common route to reach
the cell cytoplasm, adopted by various ADCs, is CME, which is target antigen dependent. Molecules,
such as epsin, dynamin, adaptor protein 2 (AP2), and phosphatidylinositol (4,5) bis-phosphate (PIP2)
may increase accumulation of ADCs on the surface of cellular membrane [18] and assist the
internalization of the ADC into the endo-lysosomal vesicle compartment.
Early endosomes form just below the membrane surface and usually endo-lysosomal vesicles
containing ADCs progress to form late endosomes, whose lumens are acidic and may lead to the
dissociation of antibodies from their receptors thus playing a vital role in recycling of antigen back to
the membrane surface and subsequently lead to fusion of the late endosomal vesicle with lysosomes.
The resulting pH decrease may also result in degradation of the ADC due to the numerous proteolytic
enzymes present in the acidic lysosomal compartment with subsequent release of the drug payload.
[19] (Figure 2).
Release of the drug within endolysosomal vesicles then results in the passive transport of drug
payload into the cytosol where it can exert its pharmacological effect, killing the cancer cells via a
molecule specific mechanism [20,21].

Int. J. Mol. Sci. 2020, 21, 5510 5 of 28
Figure 2. Schematic illustration of the mechanism of drug delivery and release mediated by ADCs.
2.1.2. Non-Internalizing ADCs
The main pharmacological action of ADCs constructed with non-internalizing antibodies, relies
on the cytotoxic payload exerting a bystander effect upon reaching the target tumor site. In this
instance, once the ADC reaches the tumor site, proteolytic enzymes, or the reducing conditions in the
tumor extracellular environment, act to liberate the drug payload, which facilitate the entry of drugs
into the cells, by diffusion, pinocytosis, or other mechanisms. Once the released drugs start kill cancer
cells, they release additional reducing agents or proteases, which in turn catalyze further release of
drugs (Figure 2). This type of conjugates may also allow a by-stander effect on non-target cancer cells
that are near the main target tumor mass, due to diffusion of the released drug into neighboring
tumor cells of the drug [22].
It has been reported that an ADC directed against the alternatively spliced extracellular domain
A of fibronectin induces a potent anticancer effect following the release of its payload after tumor cell
death in the extracellular milieu. This allows the diffusion of the cytotoxic drug also into neighboring
cells, and amplification of the process determined by a further release of reducing agents (e.g.,
cysteine, glutathione) [23].
2.2. Linkers
The linker component of the ADC, through which the covalent chemical bond between the drug
and the antibody is created, should be chosen rationally, based on the mechanism of action of the
antibody (whether internalizing or not) and limit potential chemical modifications to the drug in
order to avoid loss of cytotoxicity. One of the main aims for the effective systemic delivery of an ADC
is that the drug is released only at the target site; the linker, thus, must be stable enough in a biological
environment (i.e., blood circulation) to avoid unwanted release of the pharmacological molecule.

Int. J. Mol. Sci. 2020, 21, 5510 6 of 28
There are two types of linkers available: cleavable and non-cleavable (Figure 3). The former can
be used either in the design of either an internalizing or not internalizing ADC, because the release
of the payload is required to take place in either the extracellular tumor environment, or within the
lysosome or cytosol. This is possible because the extracellular environment of the tumor is highly
reducing due to the presence of glutathione, which allows the release of payloads linked to the
antibody via thiolic bonds. It also allows payload release via the degradation of peptide bonds in the
presence of proteases such as Cathepsin B, whose overexpression in cancer drives its normal
lysosomal localization towards extracellular secretion [24]. A cleavable linker, therefore, exploits
differential conditions of reducing power or enzymatic degradation that can be present either outside
or inside the target cell. Due to the chemical reactions needed to release the payload, the site of
conjugation on the antibody is crucial to induce both stability in the plasma and availability to
reduction or degradation on/into the target cell [25,26]. Non-cleavable linker-based ADC must,
however, be internalizing, because to release their cytotoxic payload, the antibody component needs
to be degraded by lysosomal or cytoplasmic proteases [27]. Furthermore, drugs linked to such linkers
usually cannot exert a by-stander effect because upon degradation of the antibody by cellular
proteases, they are released as fragments of antibody peptides that have a poor ability to permeate
the cells. This type of non-cleavable linker has a higher efficiency for the treatment of tumors that
express an antigen at high levels (to achieve a good clinical response and tumor regression, 99% of
targeted cancer cells must be eliminated) or for hematological tumors [28,29].

Int. J. Mol. Sci. 2020, 21, 5510 7 of 28
Figure 3. Available cleavable linkers in ADC (A) Disulfide linker, cleaved by reducing agents; (B)
cathepsin B responsive linker, cleaved by Cathepsin B; (C) glycosidase-sensitive linker, cleaved by
gluconidase; (D) hydrazone linker, cleaved by acidic environment.
2.2.1. Cleavable Linkers
Figure 3 above summarizes the most commonly used cleavable linkers that are described in
detail in the sections that follow.
Disulfide Linkers

Int. J. Mol. Sci. 2020, 21, 5510 8 of 28
This type of linker is glutathione-sensitive. The disulfides are stable at physiological pH, in the
systemic blood stream, but they are vulnerable to nucleophilic attack by thiols. Human serum
albumin (HSA) represents the main thiol in plasma, being its concentration as high as > 400 mM.
Notwithstanding this high concentration, HSA fails to break the disulfide bond of ADC because its
residue containing free thiol (Cys34) is found near a cleft in the molecule that is not significantly
exposed to the solvent [30]. Conversely, disulfide-linked drugs resist reductive cleavage in the
circulation because the glutathione (GSH) concentration in the blood (5 µmol/L) is lower than in the
cytoplasm (1–10 mmol/L) allowing GSH thiol groups to be very effective in the cell cytoplasm also
due to its well exposed position and its small size [31]. This difference in reductive potential between
plasma and cytosol allows for the selective release of the intracellular payload of the ADCs. In
addition, cancer cells cause oxidative stress that generates high GSH levels. Low glutathione levels
in healthy tissues therefore discriminate release of the payload, also allowing the selective release of
payload in close proximity to the tumor. ADCs with disulfide linkers are often associated with
maytansinoid payloads, which were originally developed by Immunogen in 1992 [32]. To increase
the stability of the bond, methyl groups may be added to surround disulfides in the linker structure
[33], such as in the case of N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP) containing a single
methyl, or N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB) containing two methyl groups.
Some ADC designs use a direct disulfide bond between the drug and the antibody. In this variety
of ADC, the release of the drug is completely dependent on a strongly reducing tumor
microenvironment [34]. Recently, ADCs with a direct disulfide bond between engineered cysteine
residues and the thiols of maytansinoids payloads have been investigated [30]. By protecting
disulfides reduction through antibody hindrance, these ADCs have good in vivo stability in mouse
plasma. The results demonstrate that the DM3 payload is more stable than the DM1, given that only
10% of disulfide bonds are cleaved in plasma, a property that confers increased in vivo therapeutic
activity in a murine model [35]. The structure of the whole antibody thus represents a protective
environment significantly reducing the reductive release of the payload in the blood stream, but this
in turn may limit the efficiency of release once at the tumor site. Other studies have shown that by
creating an ADC using a small immunoprotein (SIP) antibody (small immunoprotein, comprised of
an IgG, including variable regions from heavy and light chains linked through peptide plus
additional C3 or C4 heavy chain proteins; see also below) and comparing the results with an
analogous ADC constructed with intact IgG, the release of the drug by the ADC-SIP occurs faster.
This is probably due to a more stable interchain disulfide bond in the SIP. However, by analyzing the
stability of ADCs in mouse plasma, a half-life greater than 48 h with IgG and less than 3 h with SIP
was determined. An analysis of the in vivo efficacy of the above compounds showed that the ADC-
SIP experienced an accumulation and therefore a greater release than the IgG-ADC, despite there
being a global accumulation of ADC-IgG after 24 h that was greater in the tumor than that observed
for the ADC-SIP [23].
Cathepsin B-Sensitive Linker
The cysteine protease Cathepsin B is normally found inside late endosomes and lysosomal
compartments in mammals. It is also implicated in tumor progression, being overexpressed by many
cancers [36]. The carboxydipeptidase activity of Cathepsin B allows the splitting of a dipeptide linker
that can bind a payload to the terminal C. This enzyme has various substrate target peptide sequences
with Phe-Arg being the most common [36]. In addition, it also preferentially recognizes sequences
such as valine-citrulline (Val-Cit) and phenylalanine-lysine (Phe-Lys) where the protease breaks a
peptide bond on the C-terminal side of Val-Cit, Val-Ala, or Phe-Lys. Some studies have shown that a
high pH basic environment increases the cleavage capacity [3] and that the hydrophobic residues
Phe, Val, and Ala allow cleavage with cathepsin B that has the effect of increasing the stability in
plasma. Sometimes, however, the payload can be too bulky in which case the use of a spacer that is
stable and that does not alter the drugs chemistry, and functionality is necessary. One of the most
used conjugation reagents is para-aminobenzyl carbamate (PABC) (Figure 4), that possesses a self-
cleavage ability allowing it to release the unmodified payload [35]. For example, linkers containing

Int. J. Mol. Sci. 2020, 21, 5510 9 of 28
Phe-Lys-PABC and Val-Cit-PABC, used for ADC with monomethyl-auristatin E (MMAE) payload,
have a half-life in plasma for Phe-Lys-PABC of 12 h compared to 80 h for Val-Cit-PABC 80 h. This
shorter half-life indicates that the linker with Phe-Lys-PABC is probably non-specific with the danger
that it may exert off-target toxicity [37].
Figure 4. PABC, p-aminobenzyl carbamate, CAS#:918132-66-8.
To summarize, it has been shown that if these types of linker are coupled with
paminobenzyloxycarbonyl (PABC) they work more efficiently as cleavable linkers (i.e., Val-Ala-
PABC) for ADCs [38]. The PABC group acts as a spacer separating the toxic payload from Val-Cit
sequence so that the active site of cathepsin B can gain better access to the cleavage sequence, thus,
more effectively exploiting its protease activity, particularly if a large molecular sized payload is
used. PABC is furthermore a self-immolate linker that, upon Cathepsin B cleavage, can undergo
hydrolysis releasing the free drug to which it is attached (i.e., monomethyl-auristatin E (MMAE))
[39,40].
Hydrazone Linker
Hydrazone linkers or other similar molecules that are pH-dependent, have quite a stable
structure at neutral pH (i.e., in the bloodstream at pH 7.4) and are hydrolyzed when they reach an
acidic cellular compartment such as the lysosome (pH < 5) or late endosomes (pH 5.5–6.2). However,
the degradation of this linker is not confined to the lysosome, but may, on occasion, also occur
extracellularly. ADCs with a hydrazone linker hydrolyze only slowly under physiological conditions,
with the slow release of the toxic payload [41]. A study with an antibody directed against mucin,
conjugated via an acid-labile linker, showed good therapeutic effects in a preclinical pancreatic cancer
model [42] where the tumor microenvironment is significantly more acidic than in normal tissues,
due to the enhanced glycolysis taking place in the tumor with the consequent production of lactate
to a level sufficient to induce extracellular cleavage of the linker. In mouse models, the slow release
of the circulatory payload has produced promising results, but only in the presence of payloads with
moderate cytotoxic activity. Payloads with higher cytotoxic activity, now widely used for the
production of ADCs, demand the use linkers with higher stability to avoid the undesired release of
the payload and resultant non-specific systemic toxicity [37].
Glycosidase-Sensitive Linkers
Glycosidases comprise hydrolytic lysosomal enzymes, such as β-glucuronidases that degrade β-
glucuronic acid residues into polysaccharides. They are found in lysosomes and work under
hydrophilic environments. β-glucuronidases, like cathepsin B, are also secreted in the necrotic areas
of some tumors. They are also enzymatically active in the extracellular environment [43]. ADCs that
contain β-glucuronic acid can reach a DAR = 8 without causing aggregation and without reducing
the hydrophobicity of the ADC. Indeed, this type of linker greatly reduce plasma clearance of ADCs,
thus increasing their efficacy in vivo [44]. It is also established that the use of Poly (Ethylene Glycol)
PEG linkers increases the hydrophilicity of β-glucuronic acid and, thereby, increases the activity and
efficiency of the ADC [30].

Int. J. Mol. Sci. 2020, 21, 5510 10 of 28
Another type of hydrolytic lysosomal enzyme, the β-galactosidases that degrade β-galactoside,
are also overexpressed in some types of cancer [45]. An ADC based on trastuzumab linked to MMAE
using a β-galactoside linker was shown to be more potent than an equivalent ADC based on a Val-
Cit-PABC linker. This formulation of ADC-β-galactoside-DM1 has also been shown to be more
efficient in vivo for the treatment of HER2+ breast tumors than the approved trastuzumab emtansine
(T-DM1) [35].
2.2.2. Non-Cleavable Linkers
The most used non-cleavable linkers are alkylic and polymeric. For example, the MCC amine-
to-sulfhydryl bifunctional cross-linker contains a cyclohexane ring structure that through steric
hindrance protects the resulting thioether bond from hydrolysis [46]. The greatest advantage of non-
cleavable versus cleavable linkers is their improved plasma stability; that results in reduced off-target
toxicity in comparison to conjugates with cleavable linkers and thus provides greater stability and
tolerability [47,48]. It is noteworthy that non-cleavable ADCs often have less activity against tumors
due to the heterogeneity of target antigen expression where a bystander effect is an important
contributor to therapeutic efficacy [49]. As described earlier, non-cleavable linkers require mAb
degradation within the lysosome after ADC internalization to release the drug to the site of
pharmacological activity in the cytosol. If the payload is linked to a charged amino acid such as lysine)
with a Pi < 9.5, this will prevent escape of the drug by diffusion through the cell membrane and result
in higher levels of drug-accumulation in the tumor cell which as a consequence should overcome the
limitations of any bystander effect. In summary the major advantage of non-cleavable linkers is that
they minimize drug release into the circulation thus limiting non-specific toxicity whilst maintaining,
good in vivo stability [50].
Usually, non-cleavable linkers contain a thioether or maleimidocaproyl group. Examples of non-
cleavable linker-based ADCs containing monomethyl auristatin F (MMAF), an anti-mitotic drug,
where it was demonstrated that the drug is more potent if linked via a simple alkyl chain to the
antibody. Conjugation effected with a non-reducible thioether linker demonstrated very good
activity in both in vitro and in vivo [51].
2.3. Payloads
Currently, most ADCs are constructed with two main families of highly toxic compounds, acting
either on microtubule or DNA structure. Among the first group, auristatins and maytansines
payloads both act as tubulin inhibitors and have been widely used for construction of ADCs. Both
molecules are potently cytotoxic against rapidly dividing cancer cells and have reduced toxicity to
normal cells. Alternatively, calicheamicins and PBDs are DNA-damaging agents, inducing cell death
by apoptotic mechanisms in all cells including cancer stem cells (CSCs), and for this reason, they do
exert severe side effects. There is also a third category of drug that targets specific enzymes essential
for cell survival. In general, the payloads suitable for an ADC must have: (a) good solubility in
aqueous solutions allowing an easier conjugation to the antibody and ensuring enough solubility to
ADC under physiological conditions; (b) a significantly higher cytotoxic activity (half maximal
inhibitory concentration (IC50) ranging from 0.01 to 0.1 nM) in comparison to clinically standard
chemotherapeutic agents; (c) induce cancer cell death by apoptotic mechanisms; and (d) possess an
appropriate functional group to facilitate conjugation to the antibody.
The most widely used commercialized drugs for ADC formulation comprise microtubule-
targeting agents. The choice of tubulin inhibitors as payloads is appropriate since rapid cellular
proliferation is one of the major discriminating features between cancerous and normal cells and
antimitotic agents are in principle less toxic to the normal cells [52]. Vinca alkaloid, laulimalide,
taxane, maytansine, and colchicine have all defined binding sites on microtubules. These molecules
(Figure 5) can be grouped in two main categories depending on their mechanism of action: tubulin
polymerization promoters (microtubule stabilizers) and tubulin polymerization inhibitors
(microtubule destabilizers) [53]. In particular, microtubule stabilizers inhibit the formation of
microtubules acting on the β-subunit of α-β tubulin dimers determining unregulated microtubule

Int. J. Mol. Sci. 2020, 21, 5510 11 of 28
growth, as in case of Auristatin. In contrast, the mechanism of action of microtubule destabilizers is
to block the polymerization of tubulin dimers by inhibiting the formation of mature microtubules, as
is the case for maytansinoids (Figure 6).
Figure 5. Mechanism of action of tubulin inhibitors payloads: polymerization promoters (microtubule
stabilizers) and tubulin polymerization inhibitors (microtubule destabilizers). In the figure are two
exemplifying drugs acting on microtubule formation: auristatins alters the formation of microtubules
by binding on the β-subunit of α-β tubulin dimers; thus, producing uncontrolled growth of
microtubules. Maytansines, on the contrary, stop tubulin dimers formation impairing the production
of mature microtubules.
Figure 6. Classical microtubule-targeting agents: maytansinoids (Left) and auristatin families (Right).
Auristatin is a dolastatin synthetic analog. The original drug was isolated from Dolabella
auricularia (sea hare) as dolastatin peptides, which successfully improved its water solubility to give
auristatin [54]. Auristatins block tubulin assembly and induce cell cycle arrest in G2/M phase, causing
cells to undergo apoptosis.

Int. J. Mol. Sci. 2020, 21, 5510 12 of 28
To prevent lysosomal payload degradation and to enhance drug efficacy two innovative
auristatin derivatives (monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF))
have been developed by Seattle Genetics. These two compounds are synthetic drugs derived by
design from structure-activity relationship (SAR) analysis. These two new molecules are different
due to a phenylalanine present at the C-terminus of MMAF that allows this latter compound to be
more membrane impermeable. In contrast, MMAE can exit the cell and thus diffuse to nearby cells
killing them through bystander effects [53].
Maytansinoids are derivatives of natural cytotoxic agents named maytansines, a family of toxins
originally isolated from the cortex of Maytenus serrata possessing macrolide structure. Maytansine
and maytansinoids alter microtubule polymerization thus inhibit the maturation of microtubules by
binding to or in close proximity to the vinblastine-binding site on the β-subunit of tubulin. This
consequently induces cell death through mitotic arrest [54].
ADCs that containing maytansinoid, are unfortunately substrates for multidrug resistance
protein 1 (MDR1), a critical protein of the cell membrane that acts by actively pumping a wide variety
of xenobiotics out of cells. To prevent this problem a series of hydrophilic linkers have been used in
ADC chemistry. These linkers allow for an increased drug content (DAR) in ADCs s and subsequent
increases in the amount of drug delivered to each target cell. The increased polarity introduced by
such linkers allows the formation of maytansinoid metabolites that are poor substrates for efflux
pumps thus overcoming MDR [55].
Maytansines are difficult to conjugate because they do not have reactive chemical groups. To
overcome this problem, a series of derivatives containing SH groups have been created examples of
which are, DM1 and DM4 that are substituted by methyl disulfide at the maytansine C3 N-acyl-N-
methyl-L-alanyl ester side chain [56].
A third type of antimitotic payload includes tubulysins characterized by higher affinity of
binding to the vinca domain of tubulin if compared with vinblastine. These agents exert a rapid
disruption of the cytoskeleton and subsequent disassembly of the mitotic apparatus in proliferating
cancer cells. This results in a block at G2/M of the cell cycle and subsequent apoptotic cell death [57]
(Figure 7).
Figure 7. Other microtubule-targeting agents.
Tubulysins possess high degree of selective cytotoxicity against human cancer cells due to their
rapid rate of division. Furthermore, they may also be effective against cancer cells overexpressing the

Int. J. Mol. Sci. 2020, 21, 5510 13 of 28
P-glycoprotein or which possess mutations in tubulin gene. Tubulysins are comprised of a family
with 14 different isoforms characterized by conserved core structure made of an L-isoleucine (Ile), a
tubuvaline (Tuv), and an N-methylD-pipecolic acid (Mep) unit.
The first targeted drug (EC0305) based on tubulysin has been recently obtained by linking
Tubulysin B to folic acid conjugate. Now several tubulysin D-based ADCs are under study [53].
To complete the family of drugs that bind to microtubules the following compounds are also
worth mentioning:
Cryptophycins, a class of cytotoxins more potent than MMAE and DM1, isolated from Nostoc
cyanobacteria induce tubulin depolymerization binding to microtubules. Cryptophycin-1 is the main
component, acting on many solid tumors and additionally MDR cancer cells.
Hemiasterlin from marine sponges are naturally occurring tripeptides acting as potent inhibitors
of cell growth. They bind to the tubulin vinca-site thus disrupting normal microtubule dynamics and
consequently inhibiting tubulin polymerization. Taltobulin (HTI-286) is a fully synthetic analog of
hemiasterlin and has been shown to be to be active against a variety of MDR cancer cell lines [53].
Cemadotin (LU103793) is a more hydrophilic synthetic pentapeptide analogous of dolastatin 15,
possessing strong antiproliferative activity through inhibition of microtubule assembly and tubulin
polymerization by binding at a novel site on tubulin. Cemadotin has been shown to be an effective
payload for ADC construction [53].
Rhizoxin, a compound isolated from Rhizopus microspores (a fungus able to be infectious for
humans causing mycosis) that binds to tubulin and causes inhibition of microtubule assembly [58].
Discodermolide is so far the most efficient natural promoter of tubulin assembly considered to
be a very promising candidate for future ADC development [53].
There are furthermore other tubulin inhibitors that have been investigated for their possible use
in ADC construction, such as taccalonolide A or B, taccalonolide AF or AJ, colchicine, epothilone A
and B, taccalonolide AI-epoxide, CA-4, laulimalide, paclitaxel, and docetaxel, together with their
synthetic analogous [53,59].
The second category of payload used for ADC construction is comprised of DNA-damaging
drugs. This class of payload may be more effective than microtubule inhibitors with IC50 values in
the picomolar, as opposed to the nanomolar range for microtubule inhibitors. This would make ADCs
constructed with DNA damaging drug payloads more potent and therefore better suited for targeting
antigens that are expressed at low levels on tumors. Furthermore, DNA-damaging drugs are fully
capable of apoptotically killing non-dividing cells including cancer stem cells when used in
combination with drugs that inhibit DNA repair and furthermore are capable of killing target cells at
any point in the cell cycle [60].
There are at least four mechanisms of action exerted by DNA-damaging agents, which are as
follows: (a) DNA double-strand breakage, (b) DNA alkylation, (c) DNA intercalation, and (d) DNA
cross-linking. The most used DNA-damaging payloads are pyrrolobenzodiazepine, duocarmycins,
doxorubicin, and calicheamicins [61] (Figure 8).
Figure 8. The four main mechanisms of action of DNA-damaging agents: DNA double-strand
breakers, DNA alkylators, DNA intercalators, and DNA cross-linkers. DNA-damaging agents. These
drugs can act at any phase of tumor cell life cycle.

Int. J. Mol. Sci. 2020, 21, 5510 14 of 28
Pyrrolobenzodiazepines (PBDs) were originally isolated from Streptomyces sp and are natural
products, possessing antibiotic and antitumor properties. PBD molecules bind in the minor groove
of double- stranded DNAs to the C2-amino groups of guanine residues.
PBDs forms an adduct PBD/DNA in the minor groove of DNA, leading to decreased DNA repair
and interfering with transcription factors binding to DNA, as well as to some enzyme functions
including RNA polymerase and endonucleases.
Currently, additional to natural isolated monomeric forms of PBDs, synthetic PBD dimers are
available, which in addition to forming monoadducts are also capable of forming intrastrand or
interstrand DNA cross-links [62] (Figure 9).
Figure 9. Examples of DNA-damaging drug payloads.
Duocarmycins, metabolites originally isolated from Streptomyces sp are powerful cytotoxic
substances because their mechanism of action involves alkylation of the DNA minor groove to form
a stable adduct. Duocarmycins specifically bind to a sequence of five-base-pair rich in AT-rich where
the central pyrroloindole may be easily accommodated. This results in irreversible DNA modification
compromising its architecture that finally leads to DNA cleavage and apoptotic cell death. There are
also synthetic analogs of duocarmycins available, such as adozelesin, bizelesin, and carzelesin.
Duocarmycins have impressively high cell cycle-independent cytotoxicity against a variety of
proliferating cancer cells in vitro with IC50 values in the pM range [63].
The duocarmycin analogous DUBA (duocarmycin-hydroxybenzamide-azaindole), representing
the duocarmycin final active drug metabolite, has been used to produce different new-generation
ADCs that have been tested in vitro and in vivo to verify their therapeutic efficacy. An example is
represented by SYD983, an anti-HER2 ADC, exerting clear anti-tumor activity in a mouse xenograft
model (BT-474) and showing enough stability in human and macaque primate plasma [64].
The high toxicity of duocarmycins and their analogous makes them desirable candidates to
maximize ADC cell-killing activity and also suggests that they may be effective agents to overcome
multi drug resistant (MDR) tumor cells [65].
Calicheamicins (LL-E33288) are a class of antibiotics that were discovered in Texas following a
search for novel fermentation-derived antitumor antibiotics that led to Micromonospora echinospora.
These compounds are a class of enediyne-containing DNA-cleaving antitumor agent with a potency
4000–10,000 times greater than DNA intercalating drugs, such as Adriamycin and other similar.

Int. J. Mol. Sci. 2020, 21, 5510 15 of 28
The mechanism of action of calicheamicins after cell entry and nuclear diffusion is due to drug
targeting and binding to the minor groove of DNA, causing double-strand breaks that induce
apoptotic cell death [66].
Calicheamicins are extremely powerful drugs acting at sub-pM concentrations but also
unfortunately exert significant non-specific toxicity, damaging the DNA of all cells. Their high
toxicity means that they cannot be used directly as a single therapeutic agent in cancer treatment.
Their inherent characteristics (i.e., high cytotoxicity, relatively small molecular size, mechanism of
action) have however made calicheamicins useful payloads for the construction of ADCs [54].
Camptothecin (CPT) is a natural compound isolated from Camptotheca acuminata and is an
inhibitor of the nuclear enzyme topoisomerase I. CPT molecules inhibit both DNA and RNA
synthesis in mammalian cells, and have demonstrated to be strongly cytotoxic against a wide range
of experimental tumors. Unfortunately, several clinical trials have shown considerable toxicity
problems in patients due to their low solubility and resultant adverse side effects. To circumvent
these limitations, camptothecin analogs topotecan (TPT) and irinotecan (camptothecin-11, CPT-11)
that show improved water solubility have been approved by the FDA. These molecules were tested
in clinical practice, and demonstrated significant antitumor activity and reduced toxicity [67].
SN-38 and DX-8951f are two additional CPT-analogs that have been used as ADC payloads. SN-
38, an active CPT-11 metabolite that exploits inhibition of DNA topoisomerase to exert its anticancer
activity [68].
In addition to all of the above-mentioned payloads, other molecules available also act as DNA-
damaging agents for incorporation into newly emerging ADCs. Among these compounds, particular
mention should be given to iSGD-1882 (DNA minor groove cross-linker derived from PBD dimers),
centanamycin (binds to DNA and alkylates or intercalates into DNA), PNU-159682 (an anthracycline
metabolite) [69], and uncialamycin (an enediyne natural product isolated from Streptomyces uncialis)
[70], all active on different cancer cell lines, and finally indolinobenzodiazepine dimers (IGNs) bind
to the DNA minor groove leading to DNA cross-linking [71].
Alternative Payloads.
In addition to all the payloads discussed above, other molecules are available whose cytotoxicity
is based on different mechanisms of action that include the direct induction of apoptosis, spliceosome,
and RNA polymerase inhibition.
Bcl-2 family members, including Bcl-xL, are overexpressed in cancer and the BH3- binding
domain on Bcl-xL has been targeted. Examples of such targeting agents comprise two anti-EGFR-Bcl-
xL ADCs both of which possessed reasonable anti-tumor activity [72].
The spliceosome is an attractive target in cancer therapy, and thailanstatins have been shown to
inhibit RNA splicing by the binding to different spliceosome subunits [61]. Thailanstatin A in fact
was demonstrated to bind to the SF3b subunit of the spliceosome blocking RNA splicing and was
used in the generation of an ADC (anti-Her2-thailanstatin). The Spliceostatins are potent spliceosome
inhibitors of natural origin with interesting and potentially useful anticancer activities [61].
The final class of promising payloads are the transcription inhibitors targeting RNA polymerase
II. Example of these compounds are the amatoxins, macrocyclic peptides produced by mushrooms
of the genus Amanita, that are powerful and selective inhibitors of RNA polymerase II, thus resulting
in the inhibition of protein synthesis [73].
β-amanitin has been covalently coupled to a MUC1-targeting mAb and this ADC has proven to
be specifically cytotoxic against the human breast carcinoma cell lineT47D [74].
α-amanitin was efficiently targeted to cancer cells through an anti-HER2 mAb, with an IC50 value
in the pM range. Moreover, α-amanitin has also been covalently linked to an EpCAM-targeting mAb,
showing effective antiproliferative activity both in vitro and in vivo. An anti-PSMA-α-amanitin ADC
has been recently observed to have in vivo antitumor activity when coupled using a stable and
cleavable linker [56].
Amatoxins are highly water soluble, a property that facilitates the conjugation process and
reduces ADC aggregation. Their low molecular weight, after release, allows for rapid kidney

Int. J. Mol. Sci. 2020, 21, 5510 16 of 28
excretion in the urine. Amatoxins are also highly active against MDR cancer cells because they
represent poor substrates for MDR mechanistic processes [71].
It should also be mentioned that payloads for conjugation to antibody can also include
proteinaceous enzymes from plants (e.g., saporin, ricin A chain) [4,20] or bacterial toxins (PE,
Pseudomonas exotoxin, DT, Diphtheria toxin) which induce cell death by irreversibly inhibiting
protein synthesis catalytically [75,76]. Although this latter class of toxin molecule when conjugated
to an antibody is commonly known as an immunotoxin, it is not considered a small molecule drug.
The enzymatic nature of proteinaceous toxins as a payload represents added value since a single
molecule may be sufficient to fatally intoxicate an individual cell. A variety of different linkers and
payloads has been investigated over the years and because these are totally protein constructs, fully
recombinant toxins are possible making this a promising production strategy [4].
The Figure 10 below summarizes all the payload categories discussed above in section 2.3.
Figure 10. Summary diagram of the different classes of cytotoxic molecules used in ADC construction.
3. Conjugation Strategies
Most ADCs exploit the presence of lysine and cysteine residues within the polypeptide structure
of the antibody as the point of conjugation. The average IgG1 molecule for example, possesses
approximately 90 lysine residues, but only 30 of these are accessible for conjugation, so theoretically
the number of covalently coupled payloads could range from 1 to 30. Amide or amidine bond
formation on the side chain of lysine is the most common reaction to effect covalent cross-linking of
the antibody to the payload through exploitation of the reactive groups of linkers (i.e., N-
hydroxysuccinimide esters, NHS; imidoesters) [77]. Figure 11 shows the main reactions used in the
cross-linking procedures.

Int. J. Mol. Sci. 2020, 21, 5510 17 of 28
Figure 11. Main reactions used in the cross-linking procedures. (A) Lysine amide coupling, (B)
Maleimide Alkylation, and (C) thiol-reactive conjugation.
The lysine-amide coupling conjugation is not site-specific and requires a pre-conjugation
derivatization of the antibody and/or the payload in order for conjugation to proceed, very often
using thiolic or citrulline-valine as linkers [77]. Alternatively, conjugation via cysteines requires that
a partial reduction of the antibody is undertaken or a thiol-containing reagent (e.g., Trout’s reagent)
is used to introduce additional-SH groups available for the conjugation. This may cause
destabilization of the whole IgG molecule and introduce structural heterogeneity into the final
product. IgG1 has four disulfide bridges, two that link the heavy to the light chains, and two in the
hinge region, which bond together the two half-heavy chains of the whole antibody [78]. As one of
the fundamental points of conjugation chemistry is the precise control of the drug Antibody Ratio
(DAR), a recently used strategy is to achieve site-specific coupling of the payload by engineering the
structure of the antibody. For example, the substitution of three cysteines in the hinge region with
three serines yields an IgG molecule that fully retains its interactions between heavy and light chains
[79]. Thus, through such modification of the cysteine residues, this leaves only two reactive cysteines,
one on each chain, to yield an ADC product containing up to two molecules of drug per antibody. By
refining the conjugation conditions, it is possible to obtain an extremely homogeneous product with
the presence of the only conjugate with exactly two molecules of drug per antibody molecule (DAR
2) [23,34,79].

Int. J. Mol. Sci. 2020, 21, 5510 18 of 28
4. Site-Specific Enzymatic Conjugation
It is possible to use enzymatic methods to perform a site-specific controlled conjugation. This
requires enzymes that react with the antibody and can induce a site- or amino acid sequence-specific
modification. The most used enzymes are: sortase, transglutaminase, galactosyltransferase, and
syaliltransferase. Sortase A from Staphylococcus aureus binds the LPXTG sequence and breaks the
bond between glycine and threonine linking an oligoglycine (oligo-G) that can be used to bind the
desired payload. A transglutaminase derived from Streptomyces mobaraensis catalyzes
transpeptidation and recognizes an LLQG motif that has been inserted into a genetically engineered
antibody, giving rise to a convenient site-specific ADC conjugation site. An application of a
transglutaminase conjugation method gave rise to improvements in DAR for ADCs constructed with
a branched linker that allowed for the loading of multiple payload molecules. Anami and coworkers
developed an innovative conjugation method utilizing a branched linker on an anti-HER2
monoclonal antibody with MTGase, without a reduction in antibody binding affinity leading to the
production of a homogeneous ADC molecular population with a remarkable increased DAR (up to
8) using monomethyl auristatin F as the payload [80].
The linkers used contain a lysine scaffold to generate a branch point and PEG spacers to increase
ADC mobility. For MTGase-mediated antibody-linker conjugation, the presence of a primary amine
is necessary as well as the presence of two reactive azide groups to link with the payloads [46]. Using
MTGase this azide-linker can be bound to the glutamine residue Q295 in the IgG heavy chain. To
generate an ADCs with DAR 2 the chosen payloads can be bound through azide-alkyne cyclization
using a linear or branched linker to generate a DAR 4 ADC. This improved ADC showed increased
in vitro cytotoxicity against HER2-expressing breast cancer cell lines compared to ADCs produced by
more traditional methods [46].
An N-Glycan engineering strategy takes advantage of conserved Asn297 (N297) within the Fc
domain in all IgG classes. In order to create a reactive aldehyde group on the N-glycan terminus it is
possible to employ either β-1,4-galactosyltransferase (GalT) or α-2,6-sialyltransferase (SialT) enzymes
to achieve this. The aldehyde groups enzymatically created are then used to conjugate amino-oxy-
functionalized payloads [80]. Recently Bruins and coworkers used a mushroom tyrosinase to oxidize
the exposed tyrosine residues on antibody to generate a 1,2-quinone, which can then be subjected to
a nucleophilic reaction with thiols or amines from the side chains of amino acids such as cysteine,
lysine, histidine, or any thus modified payload [81]. A further new recent strategy to improve ADC
stability is site-specific conjugations using antibody engineered to incorporate non-natural amino
acids (nnAA). The introduction of nnAA with orthogonal reactive functional groups (aldehyde,
ketone, azido, or alkynyl tag) increases the homogeneity of ADCs and enables novel bioorthogonal
chemistry that utilizes reactive groups that are different from the classical thiol or amine reactions.
The most used nnAA or strategies are: seleno-cysteine, p-azidomethyl-L-phenylalanine (pAMF) p-
acetyl phenylalanine (p-AcPhe), FGE (formylglycine generating enzyme) azide or alkynyl nnAA or
glycan. To improve ADC stability, Transtuzumab was engineered to introduce p-AcPhe that could
react through the carbonyl group (ketone) with a drug containing an alkoxy-amine to produce an
oxime [82]. To achieve this, engineered new cell lines or cell free protein expression systems (OCFS:
Open Cell Free Synthesis) were generated that possess the transcriptional machinery capable of
inserting the a nnAA exactly where desired. In this system, the most important element needed for
nnAA incorporation is a aminoacyl tRNA synthetase (aaRS) that charges a specific tRNA with the
nnAA [83,84].
5. Approved ADCs and Future Perspectives
The ADC gemtuzumab ozogamicin, also known with the commercial name of Mylotarg®
produced by Pfizer Inc., was the first ADC approved twenty years ago by the U.S. Food and Drug
Administration (FDA). Mylotarg® was used to target the CD33 (Cluster of differentiation 33, sialic
acid binding Ig-like lectin 3 (Siglec3)).

Int. J. Mol. Sci. 2020, 21, 5510 19 of 28
myeloid associated leukocyte differentiation antigen expressed by myeloid leukemia cells
(CD33+ AML). Currently, Mylotarg® is indicated for the treatment of patients diagnosed since at least
two years with recurrent or refractory CD33+ AML [85].
The Mylotarg® ADC was produced using a random conjugation technique with an amide bond
interposed between the cleavable linker, hydrazone acetyl butyrate with the antibody attached to the
calicheamicin payload via a lysine sidechain on the antibody [86]. The history of its approval has been
complicated due to unexpected toxicities, in particular veno occlusive disease (VOD) in the liver in a
significant proportion of patients. Myelotarg was initially approved by the FDA in the USA in 2000
but then voluntarily withdrawn from the market in 2011 following toxicity-related deaths and a lack
of good clinical evidence showing its therapeutic benefits. Subsequently however, lower dose studies
have demonstrated its safety and have clearly shown it to be of clinical benefit in a subset of AML
patients [87].
In 2017, Myelotarg was once again approved by the FDA [88] and immediately following this
approval another calicheamicin-based ADC using the same linker technology (linker-antibody bond
and cytotoxin, bystander effect) inotuzumab ozogamicin (also known as Besponsa®) directed against
the B-cell restricted differentiation antigen CD22 [89]. Besponsa®, was approved for use in the EU for
the treatment of acute lymphoblastic leukemia currently under orphan drug status [90,91].
The second US, EU, and Japan approved ADC was brentuximab vedotin (Seattle Genetics, Inc.
and Takeda Pharmaceutical Company Ltd.). The commercial name for this ADC is ADCETRIS®
(Seattle Genetics Inc., n.d.) and is indicated for the treatment of Hodgkin’s lymphoma targeting the
Reed-Sternberg cell-associated antigen, CD30. This ADC was constructed using a protease-cleavable
mc-VC-PABC linker and Monomethyl auristatin E (MMAE) as the cytotoxic drug payload [92]. The
chemistry of linking method to provide a bystander effect is achieved through a dithiol bond via to a
cysteine residue on the antibody. Adcetris® (brentuximab vedotin) has been approved by FDA in
2011 [93].
The final and most recent approved ADC at the time of writing is trastuzumab-emtansine (Roche
Registration GmbH) sold under the commercial name Kadcyla®. The Trastuzumab (commercial name
Herceptin) is a monoclonal antibody used as a naked antibody to treat HER2+ breast cancer by
targeting the antigen HER-2 (Human Epidermal growth factor Receptor) and triggering host-
mediated antibody dependent cellular cytotoxicity (ADCC) while simultaneously downregulating
EGFR-mediated growth signaling thereby inhibiting tumor growth [94]. The ADC Kadcyla® uses a
maytansinoid derivative as the anti-neoplastic drug payload (DM-1) and a non-cleavable SMCC
(amide antibody-linker) as linker. This ADC shows reduced bystander effect, strongest activity
compared to Herceptin in certain conditions [86] and has been approved in the US, EU and Japan
since 2013 [95–97].
Over the past two years, the FDA approved two new ADCs: Polivy® (Polatuzumab vedotin) and
Lumoxiti® (Moxetumomab pasudotox). The Polivy® is a humanized monoclonal antibody, directed
against CD79B (an antigen expressed by Large B-Cell lymphoma). Polivy is the first commercial
therapeutic ADC produced using a site-specific covalent bond conjugated to the synthetic
monomethyl auristatin E (MMAE) via engineered cysteines (THIOMABs) and using a protease-
cleavable peptide linker to increase the plasma stability [98].
The Lumoxiti® is the first approved recombinant ADC. It is an innovative linkerless ADC is
produced by genetic fusion between the Fv fragment of an anti-CD22 monoclonal with the 38 kDa
fragment (PE38) of Pseudomonas exotoxin A [99].
We can underline that all the above-described approved ADCs (except the unique recombinant
linkerless ADC Lumoxiti®) were developed using conventional random conjugation methods [100].
Table 1 reports shows all the approved and marketed ADCs.
Table 1. ADCs currently approved for clinical use.
Name Antigen
Target
Type of Cancer
Target
Linker Type Status

Int. J. Mol. Sci. 2020, 21, 5510 20 of 28
Mylotarg®
(Gemtuzumab
ozogamicin)
CD33 Myeloid leukemia B-
cell lymphoma
Cleavable linker
(hydrazone acetyl
butyrate)
marketed
Besponsa®
(Inotuzumab
ozogamicin)
CD22 Lymphoblastic B
leukemia
Cleavable linker
(hydrazone acetyl
butyrate)
marketed
Adcetris®
(Brentuximab vedotin)
CD-30 Hodgkin’s lymphoma Protease-cleavable mc-
VC PABC
marketed
Kadcyla®
(Trastuzumab
emtansine)
HER-2 HER2+ Breast cancer Non cleavable thioether
linker
marketed
Polivy®
(Polatuzumab vedotin)
CD79B Large B Cell
lymphoma
Protease-cleavable marketed
Lumoxiti®
(Moxetumomab
pasudotox)
CD22 Refractory hairy cell
leukemia
Recombinant covalently
fused (linkerless)
marketed
6. Future Perspectives
The approved ADCs are mostly indicated for the treatment of hematological malignancies and,
with a few exceptions, their clinical activity has largely failed for solid tumors. The reasons for these
failures may be attributed to the large molecular size of the ADC molecule that shows poor
penetration into the tumor mass, thus resulting in poor in vivo efficacy [11]. For this reason, other
forms of reduced sized antibodies such as single chain fragments of variable regions (scFv), i.e., v
regions joined by a linker peptide, have been investigated, Also in the form of heterodimers of IgG
and IgE, a small divalent immunoprotein (SIP, 75 kDa) or “minibody”, a homodimer stabilized by a
disulfide bond to its C-terminal [13]. The most explored antibody derivative variants are the dsFv
and scFv. They are made of Vh and VL domains linked through an interchain disulfide bond (dsFv)
genetically engineered and linked covalently with a hydrophilic linker to form an scFv. Due to their
modular nature, they can undergo multimerization into homo and hetero oligomers (diabody,
triabody, tetrabody) strengthening antigen binding affinity and diversifying the different
functionalities. The sdAbs (single domain antibodies) are smaller than scFvs, comprising 15-KDa Vh,
Vl, or Vhh domains, also termed nanobodies, and containing the antigen domain in the terminal region
of the hinge. Similarly, to scFv, these nanobodies can form homodimers increasing the binding
affinity for the target antigen or formed into heterodimers with bispecific properties. Bispecific
antibodies can interact simultaneously with two antigens on the same target cell, a property that
potentially allows for an increase in the therapeutic window while decreasing the non-specific effects
on non-target cells [101]. SIP antibodies have high affinity to their antigen and their turnover occurs
in the liver. The technology for producing SIP antibodies was developed by Neri et al. [79] against
fibronectin and other vascular antigens. These antigens, common in tumors, are stable and accessible.
In addition, SIP have two C-terminal cysteines that allows a disulfide bridge with various payloads
[102]. All these small fragments of antibodies as Fab, diabody and scFv, penetrate more rapidly into
solid tumors but have a reduced serum half-life and undergo rapid renal elimination. This means
that they are often eliminated before adequate absorption takes place at the tumor site.
Depending on the tumor under treatment, it is necessary to adequately choose and modify the
Fc portion on the antibody to have the best possible response, especially to take advantage of the
effect of the ADCC combined with other mechanisms of cell killing exerted via direct antibody-
mediated cell signaling [2].
In addition to the above-mentioned ADCs, there are also other constructs and strategies to attack
cancer cells that involve the conjugation of toxins or chemotherapeutic drugs to ligands or proteins

Int. J. Mol. Sci. 2020, 21, 5510 21 of 28
that are overexpressed on the target cell. The most used ligands as carriers can be proteins or peptides.
Another strategy is to use peptide-drug-conjugates that are made up of small, synthetic peptides
[103–108]. These molecules appear to have an even faster penetration and elimination than the small
antibody fragments we have described [102].
Nanomedicine is one of the formulation-based technologies to increase bioavailability of drugs.
Nanotechnology can provide new treatment options for tumors due to the great potential for selective
targeting and controlled drug release. Increasingly more attention is being paid to antibodies and
their fragments as targeting ligands able to bind specific receptors that are overexpressed on tumor
cells [109] for the delivery of nanoparticles.
Non-targeted nanoparticles such as liposomal-based preparations [110] polymeric [111] and
metallic nanoparticles [112,113] are readily available for the conjugation with antibodies and drugs,
potentially opening the possibility to develop theragnostic (therapeutics and diagnostics) agents.
These formulations can reduce the toxicity profiles of the payloads and improve the therapeutic
widow. One example is Doxil1, which has been on the market for 20 years as a liposomal preparation
of doxorubicin, and is now being improved by PEGylation [114].
Antibody conjugate nanoparticles (ACNPs) are formed from a combination of ADC and
nanotechnologies. ACNPs similarly to ADCs use antibodies to specifically target cancer cells for the
delivery of encapsulated drugs.
Many ACNPs have been tested in clinical trials, but to date none has yet reached phase III trials
[115].
In recent years, great progress has been made in developing effective nanoparticle-based drug
targeting using conjugated antibodies. In addition, the use of antibody fragments combined with
advances in molecular design are overcoming some of the problems associated with the large
molecular size of unmodified antibodies [109].
With the adoption of strategies that improve the ability of ACNP to reach the tumor site to
facilitate active targeting together with additional studies that are still needed to define and refine
conjugation technology, size, shape and surface charge of nanoparticles will likely lead in the future
to useful outcomes for these targeting reagents.
7. Conclusions
More than 80 ADCs are currently under investigation and are in various stages of clinical
development for cancer treatment [116]. Current evidence indicates that the field of ADCs is a very
promising one, even though in past years they have faced a number of clinical failures. Recent
advances in technology now provide all of the necessary elements required for the facile production
of humanized monoclonal antibodies, site-specific conjugation protocols, various potent cytotoxic
payloads with different mechanisms of action, adaptable linker technologies, together with advanced
analytic techniques [117]. With the availability of the new technologies and biomarker selection
strategies, ADCs are set to represent an important contribution to the future of immuno-oncology.
Author Contributions: Conceptualization, F.G. and R.I.; Methodology, G.S. and S.I.; Software, S.P.; Validation,
F.G., R.I.; Resources, S.P., R.G. and G.D.V.; Data Curation, S.P., G.P., E.C., M.A., R.G.; Writing-Original Draft
Preparation, S.P., G.D.V., F.G., D.J.F and R.I.; Writing-Review & Editing, A.M.C., G.S., S.I., D.J.F. and R.I.;
Supervision, F.G., D.J.F. and R.I.; Project Administration, R.I., and S.I.; Funding Acquisition, R.I., G.S. and S.I.
All authors have read and agreed to the published version of the manuscript.
Funding: F.G., A.M.C., R.I. are supported by University of L’Aquila, Fondi RIA di Ateneo. E.C. is recipient of an
AIRC fellowship. G.S. is supported by AIRC (IG: 18467). S.P. program is funded by the Italian Ministry of
Instruction, University, and Research under the national project PON ricerca e innovazione 2014–2020. M.A.
supported by MIUR-Ministero dell'Istruzione, dell'Università e della Ricerca (Ministry of Education, University
and Research) under national project PONRI 2014–2020 (N AIM1887574, CUP: E18H19000350007) co-funded by
European Union through FSE and FESR.
Conflicts of Interest: G.S. and S.I. are shareholders of Mediapharma s.r.l. The other authors have no potential
conflict of interest to report.

Int. J. Mol. Sci. 2020, 21, 5510 22 of 28

Int. J. Mol. Sci. 2020, 21, 5510 23 of 28
References
1. Strebhardt, K.; Ullrich, A. Paul Ehrlich’s magic bullet concept: 100 years of progress. Nat. Rev. Cancer 2008,
8, 473–480.
2. Hoffmann, R.M.; Coumbe, B.G.T.; Josephs, D.H.; Mele, S.; Ilieva, K.M.; Cheung, A.; Tutt, A.N.; Spicer, J.F.;
Thurston, D.E.; Crescioli, S.; et al. Antibody structure and engineering considerations for the design and
function of Antibody Drug Conjugates (ADCs). OncoImmunology 2018, 7, e1395127,
doi:10.1080/2162402X.2017.139512.
3. Sochaj, A.M.; Świderska, K.W.; Otlewski, J. Current methods for the synthesis of homogeneous antibody-
drug conjugates. Biotechnol. Adv. 2015, 33, 775–784.
4. Giansanti, F.; Flavell, D.J.; Angelucci, F.; Fabbrini, M.S.; Ippoliti, R. Strategies to Improve the Clinical Utility
of Saporin-Based Targeted Toxins. Toxins (Basel) 2018, 10, 82, doi:10.3390/toxins10020082.
5. Panowski, S.; Bhakta, S.; Raab, H.; Polakis, P.; Junutula, J.R. Site-specific antibody drug conjugates for
cancer theraphy. mAbs 2014, 6, 34–45.
6. Singh, S.K.; Luisi, D.L.; Pak, R.H. Antibody-Drug Conjugates: Design, Formulation and Physicochemical
Stability. Pharm. Res. 2015, 32, 3541–3571, doi: 10.1007/s11095-015-1704-4.
7. Zhou, Q. Site-specific conjugation for ADC and beyond. Biomedicines 2017, 5, 64,
doi:10.3390/biomedicines5040064.
8. Khongorzul, P.; Ling, C.J.; Khan, F.U.; Ihsan, A.U.; Zhang, J. Antibody-Drug Conjugates: A Comprehensive
Review. Mol. Cancer Res. 2020, 18, 3–19, doi:10.1158/1541-7786.MCR-19-0582.
9. Chari, R.V.J.; Miller, M.L.; Widdison, W.C. Antibody-drug conjugates: An emerging concept in cancer
therapy. Angew. Chem. Int. Ed. Engl. 2014, 53, 3796–3827.
10. Weidle, U.H.; Maisel, D.; Klostermann, S.; Schiller, C.; Weiss, E.H. Intracellular proteins displayed on the
surface of tumor cells as targets for therapeutic intervention with antibody-related agents. Cancer Genom.
Proteom. 2011, 8, 49–63.
11. Gauzy-Lazo, L.; Sassoon, I.; Brun, M.P. Advances in Antibody-Drug Conjugate Design: Current Clinical
Landscape and Future Innovations. Slas Discov. 2020, 20, 2472555220912955, doi:10.1177/2472555220912955.
12. Brüggemann, M.; Osborn, M.J.; Ma, B.; Hayre, J.; Avis, S.; Lundstrom, B.; Buelow, R. Human antibody
production in transgenic animals. Arch. Immunol. Exp. (Warsz) 2015, 63, 101–108.
13. Aguiar, S.; Dias, J.; Manuel, A.M.; Russo, R.; Gois, P.M.P.; da Silva, F.A.; Goncalves, J. Chimeric Small
Antibody Fragments as Strategy to Deliver Therapeutic Payloads. Adv. Protein Chem. Struct. Biol. 2018, 143–
182, doi:10.1016/bs.apcsb.2018.03.002.
14. Sala, G.; Rapposelli, I.G.; Ghasemi, R.; Piccolo, E.; Traini, S.; Capone, E.; Rossi, C.; Pelliccia, A.; Di Risio, A.;
D’Egidio, M.; et al. EV20, a NovelAnti-ErbB-3 Humanized Antibody, Promotes ErbB-3 Down-Regulation
and Inhibits Tumor Growth In Vivo. Transl. Oncol. 2013, 6, 676–684.
15. Prasetyanti, P.R.; Capone, E.; Barcaroli, D.; D’Agostino, D.; Volpe, S.; Benfante, A.; van Hooff, S.; Iacobelli,
V.; Rossi, C.; Iacobelli, S.; et al. ErbB-3 activation by NRG-1β sustains growth and promotes vemurafenib
resistance in BRAF-V600E colon cancer stem cells (CSCs). Oncotarget 2015, 6, 16902–16911.
16. Ghasemi, R.; Rapposelli, I.G.; Capone, E.; Rossi, C.; Lattanzio, R.; Piantelli, M.; Sala, G.; Iacobelli, S. Dual
targeting of ErbB-2/ErbB-3 results in enhanced antitumor activity in preclinical models of pancreatic
cancer. Oncogenesis. 2014;3(8):e117. doi:10.1038/oncsis.2014.31
17. Conner, S.D.; Schmid, S.L. Regulated portals of entry into the cell. Nature 2003, 422, 37–44.
18. Kalim, M.; Chen, J.; Wang, S.; Lin, C.; Ullah, S.; Liang, K.; Ding, Q.; Chen, S.; Zhan, J. Intracellular trafficking
of new anticancer therapeutics: Antibody-drug conjugates. Drug Des. Devel. 2017, 11, 2265–2276,
doi:10.2147/DDDT.S135571.
19. Rusten, T.E.; Vaccari, T.; Stenmark, H. Shaping development with ESCRTs. Nat. Cell Biol. 2011, 14, 38–45.
20. Capone, E.; Giansanti, F.; Ponziani, S.; Lamolinara, A.; Iezzi, M.; Cimini, A.; Angelucci, F.; Sorda, R.;
Laurenzi, V.; Natali, P.G.; et al. EV20-Sap, a novel anti-HER-3 antibody-drug conjugate, displays promising
antitumor activity in melanoma. Oncotarget 2017, 8, 95412–95424, doi:10.18632/oncotarget.20728.
21. Capone, E.; Lamolinara, A.; D’Agostino, D.; Rossi, C.; De Laurenzi, V.; Iezzi, M.; Sala, G.; Iacobelli, S. EV20-
mediated delivery of cytotoxic auristatin MMAF exhibits potent therapeutic efficacy in cutaneous
melanoma. J. Control Release 2018, 277, 48–56.
22. Staudacher, A.H.; Brown, M.P. Antibody drug conjugates and bystander killing: Isantigen-dependent
internalisation required? Br. J. Cancer 2017, 117, 1736–1742, doi:10.1038/bjc.2017.367.

Int. J. Mol. Sci. 2020, 21, 5510 24 of 28
23. Dal Corso, A.; Gebleux, R.; Murer, P.; Soltermann, A.; Neri, V. A non-internalizing antibody-drug conjugate
based on an anthracycline payload displays potent therapeutic activity in Vivo. J. Control. Release 2017, 264,
211–218.
24. Mohamed, M.M.; Sloane, B.F. Cysteine cathepsins: Multifunctional enzymes in cancer. Nat. Rev. Cancer
2006, 6, 764–775.
25. Lewis Phillips, G.D.; Li, G.; Dugger, D.L.; Crocker, L.M.; Parsons, K.L.; Mai, E.; Blättler, W.A.; Lambert,
J.M.; Chari, R.V.; Lutz, R.J.; et al. Targeting HER2-positive breastcancer with trastuzumab-DM1, an
antibody-cytotoxic drug conjugate. Cancer Res. 2008, 68, 9280–9290, doi:10.1158/0008-5472.CAN-08-1776.
26. Dorywalska, M.; Strop, P.; Melton-Witt, J.A.A.; Hasa-Moreno, A.; Farias, S.E.; Galindo Casas, M.; Delaria,
K.; Lui, V.; Poulsen, K.; Loo, C.; et al. Effect of attachment site on stability of cleavable antibody drug
conjugates. Bioconjug. Chem. 2015, 26, 650–659, doi:10.1021/bc5005747.
27. Lu, J.; Jiang, F.; Lu, A.; Zhang, G. Linkers Having a Crucial Role in Antibody-Drug Conjugates. Int. J. Mol.
Sci. 2016, 17, 561, doi:10.3390/ijms17040561.
28. Chari, R.V. Targeted cancer therapy: conferring specificity to cytotoxic drugs. Acc Chem Res. 2008; 41, 98–
107, doi: 10.1021/ar700108g.
29. Dorywalska, M.; Strop. P.; Melton-Witt, J.A.; Hasa-Moreno, A.; Farias, S.E.; Galindo Casas, M.; Delaria, K.;
Lui, V.; Poulsen, K.; Sutton, J.; et al. Site-Dependent Degradation of a Non-Cleavable Auristatin-Based
Linker-Payload in Rodent Plasma and Its Effect on ADC Efficacy. PLoS ONE 2015, 10, e0132282,
doi:10.1371/journal.pone.0132282.
30. Pillow, T.H.; Sadowsky, J.D.; Zhang, D.; Yu, S.F.; Del Rosario, G.; Xu, K.; He, J.; Bhakta, S.; Ohri, R.; Kozak,
K.R.; et al. Decoupling stability and release in disulfide bonds with antibody-small molecule conjugates.
Chem. Sci. 2017, 8, 366–370, doi:10.1039/c6sc01831a.
31. Wu, B.; Zhang, G.; Shuang, S.; Choi, M.M. Biosensors for determination of glucose with glucose oxidase
immobilized on an eggshell membrane. Talanta 2004, 64, 546–553, doi:10.1016/j.talanta.2004.03.050.
32. Chari, R.V.; Martell, B.A.; Gross, J.L.; Cook, S.B.; Shah, S.A.; Blättler, W.A.; McKenzie, S.J.; Goldmacher,
V.S. Immunoconjugates containing novel maytansinoids: Promisinganticancer drugs. Cancer Res. 1992, 52,
127–131.
33. Saito, G.; Swanson, J.A.; Lee, K.D. Drug delivery strategy utilizing conjugation viareversible disulfide
linkages: Role and site of cellular reducing activities. Adv. Drug Deliv. Rev. 2003, 55, 199–215.
34. Giansanti, F.; Capone, E.; Ponziani, S.; Piccolo, E.; Gentile, R.; Lamolinara, A.; Di Campli, A.; Sallese, M.;
Iacobelli, V.; Cimini, A.; et al. Secreted Gal-3BP is a novel promising target for non-internalizing Antibody-
Drug Conjugates. J. Control. Release 2018, 294, 176–184, doi:10.1016/j.jconrel.2018.12.018.
35. Bargh, J.; Isidro-Llobet, A.; Parker, J.; Spring, D. Cleavable linkers in antibody–drug conjugates. Chem. Soc.
Rev. 2019, 48, 4361–4374, doi:10.1039/C8CS00676H.
36. Dubowchik, G.M.; Mosure, K.; Knipe, J.O.; Firestone, R.A. Cathepsin B-sensitive dipeptide prodrugs. 2.
Models of anticancer drugs paclitaxel (Taxol), mitomycin C and doxorubicin. Bioorganic. Med. Chem. Lett.
1998, 8, 3347–3352, doi:10.1016/s0960-894x(98)00610-6.
37. Doronina, S.O.; Toki, B.E.; Torgov, M.Y.; Mendelsohn, B.A.; Cerveny, C.G.; Chace, D.F.; DeBlanc, R.L.;
Gearing, R.P.; Bovee, T.D.; Siegall, C.B.; et al. Development of potent monoclonal antibody auristatin
conjugates for cancer therapy. Nat. Biotechnol. 2003, 21, 778–784, doi:10.1038/nbt832.
38. Jain, N.; Smith, S.W.; Ghone, S.; Tomczuk, B. Current ADC Linker Chemistry. Pharm. Res. 2015, 32, 3526–
3540, doi:10.1007/s11095-015-1657-7.
39. Dubowchik, G.M.; Firestone, R.A.; Padilla, L.; Willner, D.; Hofstead, S.J.; Mosure, K.; Knipe, J.O.; Lasch,
S.J.; Trail, P.A. Cathepsin B-labile dipeptide linkers for lysosomal release of doxorubicin from internalizing
immunoconjugates: Model studies of enzymatic drug release and antigen-specific in vitro anticancer
activity. Bioconjug. Chem. 2002, 13, 855–869.
40. Caculitan, N.G.; Dela, C.; Chuh, J.; Ma, Y.; Zhang, D.; Kozak, K.R.; Liu, Y.; Pillow, T.H.; Sadowsky, J.;
Cheung, T.K.; et al. Cathepsin B Is Dispensable for Cellular Processing of Cathepsin B-Cleavable Antibody-
Drug Conjugates. Cancer Res. 2017, 77, 7027–7037, doi:10.1158/0008-5472.CAN-17-2391
41. Laguzza, B.C.; Nichols, C.L.; Briggs, S.L.; Cullinan, G.J.; Johnson, D.A.; Starling, J.J.; Baker, A.L.; Bumol,
T.F.; Corvalan, J.R. New antitumor monoclonal antibody-vinca conjugates LY203725 and related
compounds: Design, preparation, and representative in vivo activity. J. Med. Chem. 1989, 32, 548–555.

Int. J. Mol. Sci. 2020, 21, 5510 25 of 28
42. Govindan, S.V.; Cardillo, T.M.; Sharkey, R.M.; Tat, F.; Gold, D.V.; Goldenberg, D.M. Milatuzumab-SN-38
conjugates for the treatment of CD74+ cancers. Mol. Cancer 2013, 12, 968–978, doi:10.1158/1535-7163.MCT-
12-1170.
43. Tranoy-Opalinski, I.; Legigan, T.; Barat, R.; Clarhaut, J.; Thomas, M.; Renoux, B.; Papot, S. β-Glucuronidase-
responsive prodrugs for selective cancer chemotherapy: An update. Eur. J. Med. Chem. 2014, 74, 302–313,
doi:10.1016/j.ejmech.2013.12.045.
44. Lyon, R.P.; Bovee, T.D.; Doronina, S.O.; Burke, P.J.; Hunter, J.H.; Neff-LaFord, H.D.; Jonas, M.; Anderson,
M.E.; Setter, J.R.; Senter, P.D. Reducing hydrophobicity of homogeneous antibody-drug conjugates
improves pharmacokinetics and therapeutic index. Nat. Biotechnol. 2015, 33, 733–735, doi:10.1038/nbt.3212.
45. Kolodych, S.; Michel, C.; Delacroix, S.; Koniev, O.; Ehkirch, A.; Eberova, J.; Cianférani, S.; Renoux, B.;
Krezel, W.; Poinot, P.; et al. Development and evaluation of β-galactosidase-sensitive antibody-drug
conjugates. Eur. J. Med. Chem. 2017, 142, 376–382, doi:10.1016/j.ejmech.2017.08.008.
46. Lambert, J.M.; Chari, R.V. Ado-trastuzumab Emtansine (T-DM1): An antibody-drug conjugate (ADC) for
HER2-positive breast cancer. J. Med. Chem. 2014, 57, 6949–6964.
47. Kovtun, Y.V.; Audette, C.A.; Ye, Y.; Xie, H.; Ruberti, M.F.; Phinney, S.J.; Leece, B.A.; Chittenden, T.; Blättler,
W.A.; Goldmacher, V.S. Antibody-drug conjugates designed toeradicate tumors with homogeneous and
heterogeneous expression of the targetantigen. Cancer Res. 2006, 66, 3214–3221
48. Oflazoglu, E.; Stone, I.J.; Gordon, K.; Wood, C.G.; Repasky, E.A.; Grewal, I.S.; Law, C.L.; Gerber, H.P. Potent
anticarcinoma activity of the humanized anti-CD70 antibody h1F6 conjugated to the tubulin inhibitor
auristatin via an uncleavable linker. Clin. Cancer Res. 2008, 14, 6171–6180.
49. Polson, A.G.; Calemine-Fenaux, J.; Chan, P.; Chang, W.; Christensen, E.; Clark, S.; de Sauvage, F.J.; Eaton,
D.; Elkins, K.; Elliott, J.M.; et al. Antibody-drug conjugates for the treatment of non-Hodgkin’s lymphoma:
Target and linker-drug selection. Cancer Res. 2009, 69, 2358–2364.
50. Sau, S.; Alsaab; H.O.; Kashaw; S.K.; Tatiparti, K.; Iyer, A.K. Advances in antibody-drugconjugates: A new
era of targeted cancer therapy. Drug Discov. Today 2017, 22, 1547–1556, doi:10.1016/j.drudis.2017.05.011.
51. Doronina, S.O.; Mendelsohn, B.A.; Bovee, T.D.; Cerveny, C.G.; Alley, S.C.; Meyer, D.L.; Oflazoglu, E.; Toki,
B.E.; Sanderson, R.J.; Zabinski, R.F.; 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.
52. Lencer, W.I.; Blumberg, R.S. A passionate kiss, then run: Exocytosis and recycling of IgG by FcRn. Trends
Cell Biol. 2005, 15, 5–9.
53. Chen, H.; Lin, Z.; Arnst, K.E.; Miller, D.D.; Li, W. Tubulin inhibitor-based antibody-drug conjugates for
cancer therapy. Molecules 2017, 22, 1281.
54. Anderl, J.; Faulstich, H.; Hechler, T.; Kulke, M. Antibody–Drug Conjugate Payloads. Methods Mol. Biol.
2013, 1045, 51–70, doi:10.1007/978-1-62703-541-5_4.
55. Zakacs, G.; Paterson, J.K.; Ludwig, J.A.; Booth-Genthe, C.; Gottesman, M.M. Targeting multidrug resistance
in cancer. Nat. Rev. Drug Discov. 2006, 5, 219–234.
56. Leung, D.; Wurst, J.M.; Liu, T.; Martinez, R.M.; Datta-Mannan, A.; Feng, Y. Antibody Conjugates-Recent
Advances and Future Innovations. Antibodies (Basel). 2020, 9, 2, doi: 10.3390/antib9010002.
57. Kaur, G.; Hollingshead, M.; Holbeck, S.; Schauer-Vukasinovic, V.; Camalier, R.F.; Domling, A.; Agarwal, S.
Biological evaluation of tubulysin A: A potential anticancer and antiangiogenic natural product. Biochem.
J. 2006, 396, 235–242.
58. Prota, A.E.; Bargsten, K.; Diaz, J.F.; Marsh, M.; Cuevas, C.; Liniger, M.; Steinmetz, M.O. A new tubulin-
binding site and pharmacophore for microtubule destabilizing anticancer drugs. Proc. Natl. Acad. Sci. USA
2014, 111, 13817–13821.
59. Dumontet, C.; Jordan, M.A. Microtubule-binding agents: A dynamic field of cancer therapeutics. Nat. Rev.
Drug Discov. 2010, 99, 790–803, doi:10.1038/nrd3253.
60. Kastenhuber, E.R.; Lowe, S.W. Putting p53 in Context. Cell 2017, 170, 1062–1078,
doi:10.1016/j.cell.2017.08.028.
61. Yaghoubi, S.; Karimi, M.H.; Lotfinia, M.; Gharibi, T.; Mahi-Birjand, M.; Kavi, E.; Hosseini, F.; Sineh Sepehr,
K.; Khatami, M.; Bagheri, N.; et al. Potential drugs used in the antibody-drug conjugate (ADC) architecture
for cancer therapy. J. Cell. Physiol. 2020, 235, 31–64, doi:10.1002/jcp.28967.
62. Antonow, D.; Thurston, D.E. Synthesis of DNA-interactive pyrrolo[2 ,1-c][1,4]benzodiazepines (PBDs).
Chem. Rev. 2011, 111, 2815–2864.

Int. J. Mol. Sci. 2020, 21, 5510 26 of 28
63. Tietze, L.F.; Schmuck, K. Prodrugs for targeted tumor therapies: Recent developments in ADEPT, GDEPT
and PMT. Curr. Pharm. Des. 2011, 17, 3527–3547.
64. Dokter, W.; Ubink, R.; van der Lee, M.; van der Vleuten, M.; van Achterberg, T.; Jacobs, D.; Loosveld, E.;
van den Dobbelsteen, D.; Egging, D.; Mattaar, E.; et al. Preclinical profile of theHER2-targeting ADC
SYD983/SYD985: Introduction of a new duocarmycin-basedlinker-drug platform. Mol. Cancer 2014, 13,
2618–2629, doi:10.1158/1535-7163.MCT-14-0040-T.
65. Rinnerthaler, G.; Gampenrieder, S.P.; Greil, R. HER2 Directed Antibody-Drug-Conjugates beyond T-DM1
in Breast Cancer. Int. J. Mol. Sci. 2019, 20, 1115, doi:10.3390/ijms20051115.
66. Gebleux, R.; Casi, G. Antibody-drug conjugates: Current status and future perspectives. Pharm. Ther. 2016,
167, 48–59.
67. Adams, D.J.; Dewhirst, M.W.; Flowers, J.L.; Gamcsik, M.P.; Colvin, O.M.; Manikumar, G.; Wani, M.C.; Wall,
M.E. Camptothecin analogues with enhanced antitumor activity at acidic pH. Cancer Chemother. Pharm.
2000, 46, 263–271.
68. Starodub, A.N.; Ocean, A.J.; Shah, M.A.; Guarino, M.J.; Picozzi, V.J.; Vahdat, L.T.; Thomas, S.S.; Govindan,
S.V.; Maliakal, P.P.; Wegener, W.A. First-in-human trial of a novel anti-trop-2 antibody-SN-38 conjugate,
sacituzumab govitecan, for the treatment of diverse metastatic solid tumors. Clin. Cancer Res. 2015, 21, 3870–
3878.
69. Yu, Q.; Ding, J. Precision cancer medicine: Where to target? Acta Pharmacol. Sin. 2015, 36, 1161–1162,
doi:10.1038/aps.2015.93.
70. Chowdari, N.S.; Pan, C.; Rao, C.; Langley, D.R.; Sivaprakasam, P.; Sufi, B.; Derwin, D.; Wang, Y.; Kwok, E.;
Passmore, D.; et al. Uncialamycin as a novel payload for antibody drug conjugate (ADC) based targeted
cancer therapy. Bioorganic. Med. Chem. Lett. 2019, 29, 466–470, doi:10.1016/j.bmcl.2018.12.021.
71. Kim, E.G.; Kim, K.M. Strategies and advancement in antibody- drug conjugate optimization for targeted
cancer therapeutics. Biomol. Ther. (Seoul) 2015, 23, 493–509.
72. Hennessy, E.J. Selective inhibitors of Bcl-2 and Bcl-xL: Balancing antitumor activity with on-target toxicity.
Bioorganic. Med. Chem. Lett. 2016, 26, 2105–2114.
73. Hallen, H.E.; Luo, H.; Scott-Craig, J.S.; Walton, J.D. Gene family encoding the major toxins of lethal Amanita
mushrooms. Proc. Natl. Acad. Sci. USA 2007, 104, 19097–19101.
74. Danielczyk, A.; Stahn, R.; Faulstich, D.; Löffler, A.; Märten, A.; Karsten, U.; Goletz, S. PankoMab: A potent
new generation anti-tumour MUC1 antibody. Cancer Immunol. Immunother. CII 2006, 55, 1337–1347,
doi:10.1007/s00262-006-0135-9.
75. Kaplan, G.; Mazor, R.; Lee, F.; Jang, Y.; Leshem, Y.; Pastan, I. Improving the In Vivo Efficacy of an Anti-Tac
(CD25) Immunotoxin by Pseudomonas Exotoxin A Domain II Engineering. Mol. Cancer Ther. 2018, 17, 1486–
1493, doi:10.1158/1535-7163.MCT-17-1041.
76. Kaplan, G.; Lee, F.; Onda, M.; Kolyvas, E.; Bhardwaj, G.; Baker, D.; Pastan, I. Protection of the Furin
Cleavage Site in Low-Toxicity Immunotoxins Based on Pseudomonas Exotoxin A. Toxins 2016, 8, 217,
doi:10.3390/toxins8080217.
77. Tsuchikama, K.; An, Z. Antibody-drug conjugates: Recent advances in conjugation and linker chemistries.
Protein Cell 2018, 9, 33–46, doi:10.1007/s13238-016-0323-0.
78. Liu, H.; May, K. Disulfide bond structures of IgG molecules: Structural variations, chemical modifications
and possible impacts to stability and biological function. mABs 2012, 4, 17–23, doi:10.4161/mabs.4.1.18347.
79. Gébleux, R.; Wulhfard, S.; Casi. G.; Neri, D. Antibody Format and Drug Release RateDetermine the
Therapeutic Activity of Noninternalizing Antibody-Drug Conjugates. Mol. Cancer Ther. 2015, 14, 2606–2612,
doi:10.1158/1535-7163.MCT-15-0480.
80. Anami, Y.; Xiong, W.; Gui, X.; Deng, M.; Zhang, C.C.; Zhang, N.; An, Z.; Tsuchikama, K. Enzymatic
conjugation using branched linkers for constructing homogeneous antibody-drug conjugates with high
potency. Org. Biomol. Chem. 2017, 15, 5635–5642, doi:10.1039/c7ob01027c.
81. Bruins, J.J.; Westphal, A.H.; Albada, B.; Wagner, K.; Bartels, L.; Spits, H.; van Berkel, W.J.H.; van Delft, F.L.
Inducible, Site-Specific Protein Labeling by Tyrosine Oxidation-Strain-Promoted (4 + 2) Cycloaddition.
Bioconjug. Chem. 2017, 28, 1189–1193, doi:10.1021/acs.bioconjchem.7b00046.
82. Axup, J.Y.; Bajjuri, K.M.; Ritland, M.; Hutchins, B.M.; Kim, C.H.; Kazane, S.A. Synthesis of site-specific
antibody-drug conjugates using unnatural amino acids. Proc. Natl. Acad. Sci. USA 2012, 109, 16101–16106.
83. Tian, F.; Lu, Y.; Manibusan, A.; Sellers, A.; Tran, H.; Sun, Y.; Phuong, T.; Barnett, R.; Hehli, B. A general
approach to site-specific antibody drug conjugates. Proc. Natl. Acad. Sci. USA 2014, 111, 1766–1771.

Int. J. Mol. Sci. 2020, 21, 5510 27 of 28
84. Zimmerman, E.S.; Heibeck, T.H.; Gill, A.; Li, X.; Murray, C.J.; Madlansacay, M.R.; Tran, C.; Uter, N.T.; Yin,
G.; Rivers, P.J.; et al. Production of site-specific antibody-drug conjugates using optimized non-natural
amino acids in a cell-free expression system. Bioconjug. Chem. 2014, 25, 351–361, doi:10.1021/bc400490z.
85. Norsworthy, K.J.; Ko, C.W.; Lee, J.E.; Liu, J.; John, C.S.; Przepiorka, D.; Farrell, A.T.; Pazdur, R. FDA
Approval Summary: Mylotarg for Treatment of Patients with Relapsed orRefractory CD33-Positive Acute
Myeloid Leukemia. Oncologist 2018, 23, 1103–1108, doi:10.1634/theoncologist.2017-0604.
86. Ricart, A.D. Antibody-drug conjugates of calicheamicin derivative: Gemtuzumab ozogamicin and
inotuzumab ozogamicin. Clin. Cancer Res. 2011, 17, 6417–6427, doi:10.1158/1078-0432.CCR-11-0486.
87. Tanimoto, T.; Tsubokura, M.; Mori, J.; Pietrek, M.; Ono, S.; Kami, M. Differences in drugapproval processes
of 3 regulatory agencies: A case study of gemtuzumabozogamicin. Invest. New Drugs 2013, 31, 473–478,
doi:10.1007/s10637-012-9877-8.
88. FDA (2017a) FDA Approves Mylotarg for treatment of acute myeloid leukemia [WWW]. Available online:
https://www.fda.gov/newsevents/newsroom/pressannouncements/ucm574507.htm. (accessed on 1
September 2017).
89. FDA (2017b) FDA Approves New Treatment for Adults with Relapsed or Refractory Acute Lymphoblastic
Leukemia [WWW]. Available online:
https://www.fda.gov/newsevents/newsroom/pressannouncements/ucm572131.htm. (accessed on 17
August 2017).
90. EMA (2017) Besponsa. Inotuzumab ozogamicin [WWW]. Available online:
http://www.ema.europa.eu/ema/index.jsp?curl=pages/medicines/human/medicines/004119/human_med_
002109.jsp&mid=WC0b01ac058001d124. (accessed on 28 June 2017).
91. Lamb, Y.N. Inotuzumab Ozogamicin: First Global Approval. Drugs 2017, 77, 1603–1610, doi:10.1007/s40265-
017-0802-5.
92. Moek, K.L.; de Groot, D.J.A.; de Vries, E.G.E.; Fehrmann, R.S.N. The antibody-drug conjugate target
landscape across a broad range of tumour types. Ann. Oncol. 2017, 28, 3083–3091,
doi:10.1093/annonc/mdx541.
93. Dan, N.; Setua, S.; Kashyap, V.K.; Khan, S.; Jaggi, M.; Yallapu, M.M.; Chauhan, S.C. Antibody-Drug
Conjugates for Cancer Therapy: Chemistry to Clinical Implications. Pharmaceuticals (Basel) 2018, 11, 32,
doi:10.3390/ph11020032.
94. EMA (2018b) Herceptin. Trastuzumab [WWW]. Available online:
http://www.ema.europa.eu/ema/index.jsp?curl=pages/medicines/human/medicines/000278/human_med_
000818.jsp&mid=WC0b01ac058001d124 (accessed on 14 May 2018).
95. EMA (2018c) Kadcyla. Trastuzumab emtansine [WWW]. Available online:
http://www.ema.europa.eu/ema/index.jsp?curl=pages/medicines/human/medicines/002389/human_med_
001712.jsp&mid=WC0b01ac058001d124 (accessed on 14 May 2018).
96. FDA (2013) Drug Approval Package. Kadcyla (ado-trastuzumab emtansine) injection [WWW]. Available
online: https://www.accessdata.fda.gov/drugsatfda_docs/nda/2013/125427Orig1s000TOC.cfm (accessed
on 18 May 2018).
97. PMDA (2013b) Trastuzumab emtansine. Review report [WWW]. Available online:
http://www.pmda.go.jp/files/000153735.pdf (accessed on 14 May 2018).
98. Deeks, E.D. Polatuzumab Vedotin: First Global Approval. Drugs 2019, 79, 1467–1475, doi:10.1007/s40265-
019-01175-0.
99. Dhillon, S. Moxetumomab Pasudotox: First Global Approval. Drugs 2018, 78, 1763–1767,
doi:10.1007/s40265-018-1000-9.
100. Yoder, N.C.; Bai, C.; Tavares, D.; Widdison, W.C.; Whiteman, K.R.; Wilhelm, A.; Wilhelm, S.D.; McShea,
M.A.; Maloney, E.K.; Ab, O.; et al. A Case Study Comparing Heterogeneous Lysine- and Site-Specific
Cysteine- Conjugated Maytansinoid Antibody-Drug Conjugates (ADCs) Illustrates the Benefits of Lysine
Conjugation. Mol. Pharm. 2019, 16, 3926–3937, doi:10.1021/acs.molpharmaceut.9b00529.
101. Goulet, D.R.; Atkins, W.M. Considerations for the Design of Antibody-Based Therapeutics. J. Pharm. Sci.
2020, 109, 74–103.
102. Deonarain, M.P. Miniaturised ‘antibody’-drug conjugates for solid tumours? Drug Discov. Today Technol.
2018, 30, 47–53, doi:10.1016/j.ddtec.2018.09.006.

Int. J. Mol. Sci. 2020, 21, 5510 28 of 28
103. Cimini, A.; Mei, S.; Benedetti, E.; Laurenti, G.; Koutris, I.; Cinque, B.; Cifone, M.G.; Galzio, R.; Pitari, G.; Di
Leandro, L.; et al. Distinct cellular responses induced by saporin and a transferrin-saporinconjugate in two
different human glioblastoma cell lines. J. Cell Physiol. 2012, 227, 939–951, doi:10.1002/jcp.22805.
104. Della Cristina, P.; Castagna, M.; Lombardi, A.; Barison, E.; Tagliabue, G.; Ceriotti, A.; Koutris, I.; Di
Leandro, L.; Giansanti, F.; Vago, R.; et al. Systematic comparison of single-chain Fvantibody-fusion toxin
constructs containing Pseudomonas Exotoxin A or saporinproduced in different microbial expression
systems. Microb. Cell Fact. 2015, 14, 19, doi:10.1186/s12934-015-0202-z.
105. Giansanti, F.; Di Leandro, L.; Koutris, I.; Pitari, G.; Fabbrini, M.S.; Lombardi, A.; Flavell, D.J.; Flavell, S.U.;
Gianni, S.; Ippoliti, R. Engineering a switchable toxin: Thepotential use of PDZ domains in the expression,
targeting and activation ofmodified saporin variants. Protein Eng. Des. Sel. 2010, 23, 61–68,
doi:10.1093/protein/gzp070.
106. Giansanti, F.; Sabatini, D.; Pennacchio, M.R.; Scotti, S.; Angelucci, F.; Dhez, A.C.; Antonosante, A.; Cimini,
A.; Giordano, A.; Ippoliti, R. PDZ Domain in the Engineeringand Production of a Saporin Chimeric Toxin
as a Tool for targeting Cancer Cells. J. Cell Biochem. 2015, 116, 1256–1266, doi:10.1002/jcb.25080.
107. Provenzano, E.A.; Posteri, R.; Giansanti, F.; Angelucci, F.; Flavell, S.U.; Flavell, D.J.; Fabbrini, M.S.; Porro,
D.; Ippoliti, R.; Ceriotti, A.; et al. Optimization of construct design and fermentation strategy for the
production ofbioactive ATF-SAP, a saporin based anti-tumoral uPAR-targeted chimera. Microbcell Fact.
2016, 15, 194.
108. Dhez, A.C.; Benedetti, E.; Antonosante, A.; Panella, G.; Ranieri, B.; Florio, T.M.; Cristiano, L.; Angelucci, F.;
Giansanti, F.; Di Leandro, L.; et al. Targeted therapy of human glioblastoma via delivery of a toxinthrough
a peptide directed to cell surface nucleolin. J. Cell Physiol. 2018, 233, 4091–4105.
109. Marques, A.C.; Costa, P.J.; Velho, S.; Amaral, M.H. Functionalizing nanoparticles with cancer-targeting
antibodies: A comparison of strategies. J. Control. Release 2020, 320, 180–200.
110. El Maghraby, G.M.; Arafa, M.F. Liposomes for enhanced cellular uptake of anticancer agents. Curr. Drug
Deliv. 2020, doi:10.2174/1567201817666200708113131.
111. Sun, H.; Erdman, W.; Yuan, Y.; Mohamed, M.A.; Xie, R.; Gong, S.; Cheng, C. Crosslinked polymer
nanocapsules for therapeutic, diagnostic, and theranostic applications. Wiley Interdiscip. Rev. Nanomed.
Nanobiotechnol. 2020, e1653, doi:10.1002/wnan.1653.
112. Jindal, M.; Nagpal, M.; Singh, M.; Aggarwal, G.; Dhingra, G.A. Gold Nanoparticles- Boon in Cancer
Theranostics. Curr. Pharm. Des. 2020, doi:10.2174/1381612826666200701151403.
113. Ardini, M.; Huang, J.; Sánchez, C.S.; Mousavi, M.Z.; Caprettini, V.; Maccaferri, N.; Melle, G.; Bruno, G.;
Pasquale, L.; Garoli, D.; et al. Live Intracellular Biorthogonal Imaging by Surface Enhanced Raman
Spectroscopy using Alkyne-Silver Nanoparticles Clusters. Sci. Rep. 2018, 8, 1265.
114. Wang, H.; Zheng, M.; Gao, J.; Wang, J.; Zhang, Q.; Fawcett, J.P.; He, Y.; Gu, J. Uptake and release profiles
of PEGylated liposomal doxorubicin nanoparticles: A comprehensive picture based on separate
determination of encapsulated and total drug concentrations in tissues of tumor-bearing mice. Talanta 2020,
208, 120358, doi:10.1016/j.talanta.2019.120358.
115. Johnston, M.C.; Scott, C.J. Antibody conjugated nanoparticles as a novel form of antibody drug conjugate
chemotherapy. Drug Discov. Today Technol. 2018, 30, 63–69, doi:10.1016/j.ddtec.2018.10.003.
116. Coats, S.; Williams, M.; Kebble, B.; Dixit, R.; Tseng, L.; Yao, N.S.; Tice, D.A.; Soria, J.C. Antibody-Drug
Conjugates: Future Directions in Clinical and Translational Strategies to Improve the Therapeutic Index.
Clin. Cancer Res. 2019, 12, doi:10.1158/1078-0432.CCR-19-0272.
117. Drake, P.M.; Rabuka, D. Recent Developments in ADC Technology: Preclinical Studies Signal Future
Clinical Trends. Bio. Drugs 2017, 31, 521–531, doi:10.1007/s40259-017-0254-1.
© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).