Antibody-drug conjugates: current status and future directions
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Drug Discovery Today Volume 00, Number 00 December 2013 REVIEWS
Recent advances in the discovery and development of antibody–drug conjugates
have led to FDA approvals and a rich clinical pipeline of promising
new cancer therapies.
Reviews KEYNOTE REVIEW
Antibody–drug conjugates: current
status and future directions
Heidi L. Perez1, Pina M. Cardarelli2, Shrikant Deshpande2,
Sanjeev Gangwar2, Gretchen M. Schroeder1, Gregory D. Vite1
and Robert M. Borzilleri1
1
Bristol-Myers Squibb Research & Development, Princeton, NJ 08543, USA
2
Bristol-Myers Squibb Research & Development, Redwood City, CA 94063, USA
Antibody–drug conjugates (ADCs) aim to take advantage of the specificity of
monoclonal antibodies (mAbs) to deliver potent cytotoxic drugs selectively
to antigen-expressing tumor cells. Despite the simple concept, various
parameters must be considered when designing optimal ADCs, such as
selection of the appropriate antigen target and conjugation method. Each
component of the ADC (the antibody, linker and drug) must also be
optimized to fully realize the goal of a targeted therapy with improved
efficacy and tolerability. Advancements over the past several decades have
led to a new generation of ADCs comprising non-immunogenic mAbs,
linkers with balanced stability and highly potent cytotoxic agents.
Although challenges remain, recent clinical success has generated intense
interest in this therapeutic class.
Introduction
The past decade has seen significant advances in new cancer treatments through the development
of highly selective small molecules that target a specific genetic abnormality responsible for the
disease [1,2]. Although this approach has seen great success in application to malignancies with a
single, well-defined oncolytic driver, resistance is commonly observed in more complex cancer
settings [3,4]. Traditional cytotoxic agents are another approach to treating cancer; however,
unlike target-specific approaches, they suffer from adverse effects stemming from nonspecific
killing of both healthy and cancer cells. A strategy that combines the powerful cell-killing ability
of potent cytotoxic agents with target specificity would represent a potentially new paradigm in
cancer treatment. ADCs are such an approach, wherein the antibody component provides
specificity for a tumor target antigen and the drug confers the cytotoxicity. Here, we present
key considerations for the development of effective ADCs and discuss recent progress in ADC
technology for application to the next wave of cancer therapeutics. Advances in other modalities
of antibody-mediated targeting, such as immunotoxins, immunoliposomes and radionuclide
conjugates, have been extensively reviewed elsewhere [5,6].
Corresponding author:. Borzilleri, R.M. (robert.borzilleri@bms.com)
1359-6446/06/$ - see front matter ß 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.drudis.2013.11.004 www.drugdiscoverytoday.com 1
Please cite this article in press as: H.L.. Perez, et al., Antibody–drug conjugates: current status and future directions, Drug Discov Today (2013), http://dx.doi.org/10.1016/
j.drudis.2013.11.004DRUDIS-1295; No of Pages 13
REVIEWS Drug Discovery Today Volume 00, Number 00 December 2013
Historical perspective the immunogenicity issues observed with murine mAbs, these
The origin of ADCs can be traced back over a century to the early attempts were met with limited success for several reasons,
German physician and scientist Paul Ehrlich, who proposed the including low drug potency, high antigen expression on normal
concept of selectively delivering a cytotoxic drug to a tumor via a cells and instability of the linker that attached the drug to the mAb
targeting agent (Fig. 1) [7,8]. Ehrlich coined the term ‘magic bullet’ [17]. Lessons learned from these initial failures led to a new
to describe his vision, similar to the descriptors ‘warhead’ or generation of ADCs, several of which entered and later failed
‘payload’ commonly used for the drug component of current human clinical trials. For example, doxorubicin conjugate 1
Reviews KEYNOTE REVIEW
ADCs. Nearly 50 years later, Ehrlich’s concept of targeted therapy (BR96-DOX) was designed using a bifunctional linker, wherein
was first exemplified when methotrexate (MTX) was linked to an the drug was appended via a hydrazone, and a maleimide enabled
antibody targeting leukemia cells [9]. Early research relied on conjugation to the BR96 antibody via cysteine residues (Fig. 2)
available targeting agents, such as polyclonal antibodies, to enable [18]. Although curative efficacy was observed in human tumor
preclinical efficacy studies in animal models with both noncova- xenograft models, the relatively low potency of doxorubicin
lent-linked ADCs and later covalently linked ADCs [10–12]. In necessitated high drug:antibody ratios (DARs, eight per antibody)
1975, the landmark development of mouse mAbs using hybri- and high doses of the ADC to achieve preclinical activity. In
doma technology by Kohler and Milstein greatly advanced the clinical trials, significant toxicity was observed due to nonspecific
field of ADCs [13]. The first human clinical trial followed less than cleavage of the relatively labile hydrazone linker and expression of
a decade later, with the antimitotic vinca alkaloid vindesine as the the antigen target in normal tissue [19].
cytotoxic payload [14]. Further advances in antibody engineering Further advancements, including higher drug potency and care-
enabled the production of humanized mAbs with reduced immu- fully selected targets, ultimately led to the first ADC to gain US Food
nogenicity in humans compared with the murine mAbs used for and Drug Administration (FDA) approval in 2000 (Mylotarg1,
early ADCs [15]. gemtuzumab ozogamicin, 2) [20,21]. Despite initially encouraging
First-generation ADCs typically used clinically approved drugs clinical results, Mylotarg1 was withdrawn from the market a
with well-established mechanisms of action (MOAs), such as anti- decade later owing to a lack of improvement in overall survival.
metabolites (MTX and 5-fluorouracil), DNA crosslinkers (mitomy- In 2011, following an accelerated approval process, a second ADC
cin) and antimicrotubule agents (vinblastine) [16]. In addition to (Adcetris1, brentuximab vedotin, 3) gained marketing approval
First FDA
approved ADC
(Mylotarg®)
Curative efficacy in Kadcyla®
human tumor approved
xenograft models w/
H2N N NH2
BR96-DOX ADC
N
N
N Mylotarg®
N
withdrawn
Noncovalent Clinical trials ADC w/ highly
H from market
N CO2H linked ADC w/ ADC potent
O tested in animal vindesine- cytotoxin
CO2H models αCEA calicheamicin
1913 1958 1967 1975 1988 1991 2001 2011
1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010
1972 1983 1993 2013
Paul Ehrlich described MTX linked to an
the concept of a antibody directed
‘magic bullet’ and toward leukemia cells Production of Immunogenicity Adcetris®
drug targeting (i.e. a mAbs using of mouse mAbs a approved
‘heptophore’ can ADCs proposed; hybridoma- serious limitation
deliver a ‘toxophore’ immunoradioactive based technology in development
selectively to a tumor) agent disclosed of ADCs
Mylotarg®
clinical failures
Covalent linked Humanized
due to MDR
ADCs tested in mAbs reported
animal models
Drug Discovery Today
FIGURE 1
Antibody–drug conjugate (ADC) timeline. Abbreviations: mAbs, monoclonal antibodies; MDR, multidrug resistance; MTX, methotrexate.
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Drug Discovery Today Volume 00, Number 00 December 2013 REVIEWS
O
S
N HN
O
O
H O O HO
O N NHCO2Me
O OH N N S
O N S H
OH O H H
I O O O
OH S O N
HO
Reviews KEYNOTE REVIEW
O
OMe O OH O O OMe OH
OMe O N O
O O
HO
NH2 MeO
OH MeO
OH
1 2
BR96-DOX Mylotarg®
(anti-BR96 doxorubicin conjugate) (anti-CD33 calicheamicin conjugate)
O O OH
H H
N N N
O O O N N
H
N O OMe O OMe O N S O
N N
H H N HN
O O
O O O O
Cl O
NH MeO N
N O
O O O NH2
3 O 4
S
Adcetris® N O Kadcyla®
(anti-CD30 auristatin conjugate) H (anti-Her2 maytansine conjugate)
MeO OH
Drug Discovery Today
FIGURE 2
Structures of antibody–drug conjugates (ADCs).
from the FDA for the treatment of Hodgkin’s (HL) and anaplastic inside the cell, the drug is released through one of several mechan-
large-cell lymphomas (ALCL) [22,23]. Most recently, Kadcyla1 (ado- isms, such as hydrolysis or enzymatic cleavage of the linker or via
trastuzumab emtansine, T-DM1, 4), which combines the huma- degradation of the antibody. Typically, the unconjugated drug
nized antibody trastuzumab with a potent antimicrotubule cyto- should demonstrate high potency, ideally in the picomolar range,
toxic agent using a highly stable linker, was approved for the to enable efficient cell killing upon release from the ADC.
treatment of patients with Her2-positive breast cancer [24,25]. With
nearly 30 additional ADCs currently in clinical development, the Target antigens and antibody selection
potential of this new therapeutic class might finally be coming to Although the basic premise that a successful ADC should target a
fruition [26]. well-internalized antigen with low normal tissue expression and
high expression on tumors remains true, the field is evolving to
ADC design refine these parameters. For example, antigen expression on nor-
Although simple in concept, the success of a given ADC depends mal tissues can be tolerated if expression on vital organs is minimal
on careful optimization of each ADC building block: antibody, or absent. The FDA approval of Kadcyla1 for Her2-positive breast
drug and linker (Fig. 3) [27]. The chosen antibody should target a cancer highlights this point since Her2/neu, a member of the
well-characterized antigen with high expression at the tumor site epidermal growth factor receptor (EGFR) family, is not only
and low expression on normal tissue to maximize the efficacy of expressed in breast tissue, but also in the skin, heart and on
the ADC while limiting toxicity. Bifunctional linkers with attach- epithelial cells in the gastrointestinal, respiratory, reproductive
ment sites for both the antibody and drug are used to join the two and urinary tracts [29]. In addition, prostate-specific membrane
components. With respect to the mAb, existing linker attachment antigen (PSMA) is an ADC target expressed both on prostate cancer
strategies typically rely on the modification of solvent-accessible cells as well as normal prostate and endothelial tissue [30]. Given
cysteine or lysine residues on the antibody, resulting in hetero- that most patients with prostate cancer undergo surgery to remove
geneous ADC populations with variable DARs. Given that low drug their prostate, selective expression relative to normal prostate cells
loading reduces potency and high drug loading can negatively might not be crucial in this setting. Furthermore, apical expression
impact pharmacokinetics (PK), DARs can have a significant impact of PSMA on the kidney and gastrointestinal tract might prevent
on ADC efficacy. In addition, the linker must remain stable in the ADC from accessing these tissues. Other possible exceptions
systemic circulation to minimize adverse effects, yet rapidly cleave include hematological malignancies in which normal target tis-
after the ADC finds its intended target antigen. Upon antigen sues are able to regenerate, supported by the case of rituximab
recognition and binding, the resulting ADC receptor complex is where depletion of normal B cells was not a major safety issue in
internalized through receptor-mediated endocytosis [28]. Once patients [31]. Accessibility of the ADC to the target antigen is also
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Attachment site Linker Cytotoxin
Antibody
- Targets a well-characterized - Typically through nonspecific - Cleavable or noncleavable - Highly potent
antigen with high tumor modification of cysteine or - Stable in circulation - Non-immunogenic
expression and limited normal lysine residues on the antibody - Selective intracellular - Amenable to
tissue expression - Mixture of conjugates with release of drug (e.g. via modifications for
- Maintains binding, stability, variable drug:antibody ratios enzymatic cleavage or linker attachment
internalization, PK, etc. - Site-selective conjugation antibody degradation) - Defined mechanism
when conjugated to a cytotoxin technologies can produce more of action
- Minimal nonspecific binding homogeneous ADCs
Drug Discovery Today
FIGURE 3
Key components of an antibody–drug conjugate (ADC). Abbreviation: PK, pharmacokinetics.
an important consideration. In addition to high interstitial pres- cytometry, immunohistochemistry (IHC) or radiolabeled satura-
sure in the tumor, endothelial, stromal and epithelial barriers can tion-binding studies to assess the relation between target expres-
limit ADC uptake, resulting in only a small percentage of the sion and efficacy [35]. In non-Hodgkin’s lymphoma (NHL) cell
injected dose reaching the intended tumor target [32]. From a lines, high CD79b expression was found to be a prerequisite for in
biology perspective, the design of an effective ADC relies on vitro response to an anti-CD79b auristatin conjugate (RG-7596,
selection of an appropriate target antigen, taking into account Roche-Genentech); however, a wide range of sensitivities were
tumor expression levels, rates of antigen internalization and anti- observed, indicating that a minimal expression threshold exists
body Fc format. [36]. Likewise, melanoma cells lines with receptor densities vary-
Tumor types
Table 1 highlights the broad range of hematologic and solid tumor TABLE 1
indications targeted by ADCs currently in preclinical or clinical Target antigens for ADCs in preclinical and clinical development
development. Several of these tumor-associated antigens exhibit Indication Targets
remarkable specificity, such as CD30 for HL and MUC16 for NHL CD19, CD20, CD21, CD22, CD37, CD70, CD72,
ovarian cancer. Other antigens, such as CD74, are expressed in CD79a/b and CD180
multiple tumor types. HL CD30
Antigen expression AML CD33
In general, optimal ADC targets are homogeneously and selec-
MM CD56, CD74, CD138 and endothelin B receptor
tively expressed at high density on the surface of tumor cells.
Lung CD56, CD326, CRIPTO, FAP, mesothelin, GD2,
Homogenous tumor expression, although preferred, is likely not
5T4 and alpha v beta6
an absolute requirement owing to the ability of some ADCs to
CRC CD74, CD174, CD227 (MUC-1), CD326 (Epcam),
induce bystander killing. Under these circumstances, a membrane-
CRIPTO, FAP and ED-B
permeable free drug liberated after intracellular cleavage of the
Pancreatic CD74, CD227 (MUC-1), nectin-4 (ASG-22ME)
linker can efflux from the cell and enter neighboring cells to
and alpha v beta6
facilitate cell death [33]. Most advanced ADCs in the clinic target
Breast CD174, GPNMB, CRIPTO, nectin-4 (ASG-22ME)
hematological indications, in part due to the largely homogeneous
and LIV1A
expression of antigen in liquid tumors, despite frequently low
Ovarian MUC16 (CA125), TIM-1 (CDX-014) and mesothelin
receptor densities. Although the treatment of solid tumors with
Melanoma GD2, GPNMB, ED-B, PMEL 17 and endothelin
heterogeneous antigen expression might benefit from bystander
B receptor
killing, the potential to harm normal cells could contribute to
Prostate PSMA, STEAP-1 and TENB2
systemic toxicity.
Current experimental evidence generally suggests that tumor Renal CAIX and TIM-1 (CDX-014)
antigen density (expression level) does not directly correlate Mesothelioma Mesothelin
with ADC efficacy [34]. When patient samples are accessible, Abbreviations: AML, acute myeloid leukemia; MM, multiple myeloma; CRC, colorectal
the number of receptors per cell can be quantified using flow cancer.
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Drug Discovery Today Volume 00, Number 00 December 2013 REVIEWS
ing from 20,000 to 280,000 binding sites per cell were sensitive to ADC is an important consideration. Broad understanding of the
an anti-p97-auristatin conjugate [37]. This threshold level varies relation between antibody Fc format and ADC function is lacking
among different targets based on the unique factors of the anti- since species differences in immune systems complicate preclini-
gen, such as rate of internalization and binding affinity for the cal studies. In one study, McDonagh et al. conjugated anti-CD70
ADC. For example, approximately 5,000–10,000 copies of CD33, antibody immunoglobin G (IgG) variants (IgG1, IgG2 and IgG4) to
the antigen target for Mylotarg1, are expressed per cell [38]. As an auristatin (ADC toxin monomethyl auristatin F; MMAF) to
with Mylotarg1, no significant correlation was observed between determine the effect of format on ADC function [55]. In addition,
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the activity of a preclinical anti-CD33 pyrrolobenzodiazepine the Fc regions of IgG1 and IgG4 were mutated (IgG1v1 and
conjugate (SG-CD33A, Seattle Genetics) and CD33 levels in a IgG4v3) to examine the influence of IgG receptor (FcgR) binding.
panel of acute myeloid leukemia (AML) cell lines [39]. An anti- Although all the ADCs demonstrated potent in vitro cytotoxicity
PSMA auristatin conjugate (PSMA ADC, Progenics/Seattle Genet- and were well tolerated in mice, the engineered IgGv1-MMAF
ics) demonstrated potent in vitro cytotoxicity versus cells expres- conjugate displayed improved antitumor activity and increased
sing >105 PSMA molecules per cell, with 104 receptors per cell exposure, which correlated with a superior therapeutic index
serving as a threshold level [40]. For some tumor antigens, how- compared to the parent IgG1 conjugate.
ever, a relatively proportional relation between efficacy and In the absence of definitive guidelines for selecting an optimal
receptor expression level has been observed. In the case of an antibody format, all human IgG isotypes, except for IgG3, are
anti-endothelin B receptor (EDNBR) auristatin conjugate, currently used for ADCs in clinical trials. IgG1, the most com-
improved efficacy against human melanoma cells lines and xeno- monly used format, can potentially engage secondary immune
graft tumor models generally correlated with increasing EDNBR functions, such as antibody-dependent cellular cytotoxicity
expression (1,500–30,000 copies per cell) [41]. (ADCC) or complement-dependent cytotoxicity (CDC). These
Antigen internalization inherent effector functions could prove beneficial by providing
Ideally, once an ADC binds to a tumor-associated target, the ADC– additional antitumor activity, as in the case of Kadcyla1, which
antigen complex is internalized in a rapid and efficient manner. was shown to activate ADCC in preclinical models [56]. Adcetris1,
Although poorly understood, various factors are likely to influence however, demonstrated minimal ADCC and no detectable CDC
the rate of internalization, such as the epitope on the chosen target despite its IgG1 format [57]. The absence of effector functions is
antigen bound by the ADC, the affinity of the ADC–antigen potentially advantageous as binding of an ADC to effector cells
interaction and the intracellular trafficking pattern of the ADC could reduce tumor localization, hinder internalization and lead
complex [42–44]. For example, anti-Her2 antibodies that bind to off-target toxicity [55]. Unlike IgG1, IgG2 and IgG4 typically
distinct epitopes on Her2 have been shown to impact downstream lack Fc-mediated effector functions. Mylotarg1 and inotuzumab
trafficking and lysosomal accumulation differentially, despite ozogamicin (CMC-544) exhibited no ADCC or CDC in preclinical
binding to the same cell surface receptor [45]. Several ADCs, studies, consistent with their IgG4 format [58]. Overall, the con-
including Adcetris1, have been shown to internalize with rates tribution of IgG effector functions to the efficacy, selectivity and
similar to or greater than the corresponding unconjugated anti- toxicity of ADCs is not yet well understood.
bodies [46–48,37]. Certain antigens mediate exceptionally rapid In addition to effector functions, ADCs often retain other
accumulation of ADCs inside cells. When bound to ligand-acti- biological properties associated with their parent mAbs, such
vated EGFR, Her2 monomer is internalized at a rate up to 100-fold as immunogenicity potential. Limited therapeutic efficacy of
greater than carcinoembryonic antigen (CEA) [49,50]. Likewise, early ADCs comprising murine mAbs prompted the develop-
the catabolic rate of antibodies targeting CD74 is approximately ment of chimeric and humanized antibodies, which minimize
100 times faster than other antibodies that are considered to human immune response. Conversion of murine mAbs to
rapidly internalize, such as anti-CD19 and anti-CD22 [51]. The human IgGs also results in longer retention in systemic circula-
preclinical data for milatuzumab-DOX (Immu-110), an anti-CD74 tion due to recognition by the human neonatal Fc receptor
doxorubicin conjugate in early clinical trials, suggest this agent is (FcRn) and a greater ability to elicit ADCC [59]. Technologies
equipotent to ADCs comprising more potent drug payloads that for the generation of fully human mAbs include the use of either
target slower internalizing antigens [52]. phage display or transgenic mouse platforms, in which a mouse
Alternative approaches have been explored in which antigen strain is engineered to produce human rather than mouse anti-
internalization is not required for efficient cell killing. The extra- bodies [60].
domain B (ED-B) of fibronectin is a marker of angiogenesis
undetectable in healthy tissue, but highly expressed around Linker technology and stability
tumor blood vessels [53]. Anti-ED-B antibodies have been shown The identity and stability of a linker that covalently tethers the
to localize to the subendothelial extracellular matrix of tumor antibody to the cytotoxic drug is crucial to the success of an ADC.
vasculature. Conjugation of these antibodies with a photosen- Sufficient linker stability is necessary to enable the conjugate to
sitizer has led to agents that selectively disrupt tumor blood circulate in the bloodstream for an extended period of time before
vessels upon irradiation, resulting in curative efficacy in mouse reaching the tumor site without prematurely releasing the free
models [54]. drug and potentially damaging normal tissue. Once the ADC is
Impact of format internalized within the tumor, the linker should be labile enough
The biological activity of an antibody can depend on the interac- to efficiently release the active free drug. Linker stability also
tion of its Fc portion with cells that express Fc receptors (FcRs). influences overall toxicity, PK properties and the therapeutic index
Therefore, selection of the appropriate antibody format for an of an ADC. The lack of adequate therapeutic index for earlier
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TABLE 2
Examples of ADC drug linkers
Cleavable linkers Noncleavable linkers
Cytotoxin
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Cytotoxin
N-Maleimidomethylcyclohexane-1-carboxylate (MCC)
Valine-Citrulline (protease sensitive)
Cytotoxin Cytotoxin
Hydrazone (acid-sensitive) Maleimidocaproyl
Cytotoxin
Cytotoxin Mercaptoacetamidocaproyl
Disulfide (glutathione-sensitive)
ADCs, such as BR96-DOX and Mylotarg1, has been attributed to (iii) Glutathione sensitive linkers. This strategy exploits the
poor linker stability (Fig. 1) [19,61]. higher concentration of thiols, such as glutathione, inside
The two main classes of ADC drug linkers currently being the cell relative to the bloodstream. Disulfide bonds within
explored take advantage of different mechanisms for release of the linker are relatively stable in circulation yet are reduced
the drug payload from the antibody (Table 2). The first is a by intracellular glutathione to release the free drug. To
cleavable linker strategy, with three different types of release further increase plasma stability, the disulfide bond can be
mechanism within this class. flanked with methyl groups that sterically hinder premature
(i) Lysosomal protease sensitive linkers. This strategy utilizes cleavage in the bloodstream [68]. This class of linker has been
lysosomal proteases, such as cathepsin B (catB), that used in several clinical candidates, such as SAR3419 (anti-
recognize and cleave a dipeptide bond to release the free CD19 maytansine conjugate), IMGN901 (anti-CD56 may-
drug from the conjugate [62]. Many ADCs in the clinic use a tansine conjugate) and AVE9633 (anti-CD33 maytansine
valine–citrulline dipeptide linker, which was designed to conjugate) developed by ImmunoGen and its partners [67].
display an optimal balance between plasma stability and The second strategy is one that uses noncleavable linkers. This
intracellular protease cleavage [63]. This linker strategy was approach depends on complete degradation of the antibody after
successfully utilized by Seattle Genetics/Millennium in the internalization of the ADC, resulting in release of the free drug
case of Adcetris1 [64]. with the linker attached to an amino acid residue from the mAb. As
(ii) Acid sensitive linkers. This class of linkers takes advantage of such, noncleavable linker strategies are best applied to payloads
the low pH in the lysosomal compartment to trigger that are capable of exerting their antitumor effect despite being
hydrolysis of an acid labile group within the linker, such chemically modified. This type of strategy has been used success-
as a hydrazone, and release the drug payload. In preclinical fully by Genentech/Immunogen with Kadcyla1 (trastuzumab-
studies, hydrazone linker-based conjugates have shown MCC-DM1). The released modified payload (lysine-MCC-DM1)
stability (t1/2) ranges from 2 to 3 days in mouse and human demonstrated similar potency compared with DM1 alone,
plasma, which may not be optimal for an ADC [65]. although the charged lysine residue is likely to impair cell perme-
Hydrazone linkers were used in Mylotarg1 (anti-CD33 ability and hence abate the bystander killing observed with the
calicheamicin conjugate) and recently in inotuzumab free drug [69]. One potential advantage of noncleavable linkers is
ozogamicin (anti-CD22 calicheamicin conjugate) [66,67]. their greater stability in circulation compared with cleavable
The withdrawal of Mylotarg1 from the market was attributed linkers. However, no significant difference in terminal half-life
to toxicities related to hydrazone linker instability, which (t1/2) values was observed in the clinic between Kadcyla1 [24],
resulted in increased fatalities in patients treated with which contains a noncleavable linker, and Adcetris1, which
Mylotarg1 plus chemotherapy as opposed to chemotherapy employs a cleavable linker [22].
alone [65]. Similarly, inotuzumab ozogamicin was recently Preclinically, linker strategies continue to evolve [70,71].
withdrawn from a phase III clinical trial owing to a lack of Additional tumor-associated proteases, such as legumain, have
improvement in overall survival. been identified that release the ADC payload in nonlysosomal
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Drug Discovery Today Volume 00, Number 00 December 2013 REVIEWS
compartments (i.e. the endosome) [72]. Other nonprotease proliferation assays against a broad range of tumor cell lines
enzymes have recently been exploited for the selective cleavage compared with conventional chemotherapeutic agents, such as
of b-glucuronidase and b-galactosidase sensitive linkers in the paclitaxel and doxorubicin [78,79]. The high potency of these
lysosome [73,74]. Demonstrating expanded utility, these alternative payloads is crucial since only an estimated 1–2% of the
approaches enable drug linkage via a phenol functional group administered ADC dose will ultimately reach the tumor site,
in addition to a more traditional basic amine residue. resulting in low intracellular drug concentrations [80]. Unlike
earlier ADCs that failed to make a meaningful impact in the clinic
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Cytotoxic agents owing to low drug potency and suboptimal delivery, newer, more
Payload classes and MOAs potent cytotoxic compounds are now the focus of preclinical
There are two main classes of ADC payloads undergoing clinical research. For example, pyrrolobenzodiazepine (PBD) dimers 6
evaluation. The first class comprises drugs that disrupt microtu- covalently bind the minor groove of DNA, resulting in a lethal
bule assembly and play an important role in mitosis. This class interaction due to cross-linking of opposing strands of DNA [81].
includes cytotoxics, such as dolastatin 10-based auristatin analogs a-Amanitin 7, a cyclic octapeptide found in several species of the
(3, Adcetris1) [64] and maytansinoids (4, Kadcyla1) [75]. The Amanita genus of mushrooms, strongly inhibits RNA polymerase
second class of payloads consists of compounds that target DNA II, leading to inhibition of DNA transcription and cell death [82].
structure and includes calicheamicin analogs, such as Mylotarg1 Tubulysins 8, similar to auristatins and maytansine, inhibit tubu-
(2), that bind the minor groove of DNA causing DNA double- lin polymerization to induce apoptosis [83–85].
strand cleavage [76]. Duocarmycin analogs (MDX-1203, 5) [77] Addressing drug resistance
participate in a sequence-selective alkylation of adenine-N3 in the In addition to potency, the sensitivity of cytotoxic agents to
minor groove of DNA to induce apoptotic cell death (Fig. 4). multidrug resistance (MDR) mechanisms is a factor to consider
One common feature among these cytotoxic agents is that they in selecting the optimal payload for an ADC. Cancer cells have the
demonstrate at least 100–1000-fold greater potency in in vitro ability to become resistant to multiple drugs via increased efflux of
O NH2
HN
O S
Cl O O
H H
O N N N
N N
H H H
N N O O
N O
N O
O N
H
5
Duocarmycin analog N
H N O O H
O
NH O N N
OMe MeO
O
S O O OMe
HN
N N
O H
O
O
O O 6
HO O
H H PBD
N
HN N O O
H
O
O HN S O O
HO H
N O OH O N N O
O S N H N N
H O
HN N O S HN
O O
HN N O
H2N H
O O CO2H
7 8
α-Amanitin Tubulysin analog
Drug Discovery Today
FIGURE 4
Representative antibody–drug conjugate (ADC) payload structures.
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(a) (b)
Cysteine specific
Lysine
Reviews KEYNOTE REVIEW
Unnatural amino
acid insertion
Cysteine,
S-S reduction
Enzyme-assisted
ligation
Formylglycine- Transglutaminase Sortase
generating enzyme
(chemoenzymatic)
Drug Discovery Today
FIGURE 5
Random and site-specific conjugation strategies. Antibody–drug conjugate (ADC) products of random conjugation comprise chemically heterogeneous species
(a), whereas site-specific conjugation methods produce fairly homogeneous product profiles (b).
the drug by either P-glycoprotein (Pgp) or other multidrug-resis- drug-conjugation strategy significantly impacts efficacy, PK and
tance proteins (e.g. MRP1 and MRP3) [86]. The sensitivity of tolerability. As such, careful consideration of the various conjuga-
cytotoxic drugs to MDR mechanisms can be measured in vitro. tion technologies for ADC generation is warranted (Fig. 5).
In the case of Mylotarg1, in vitro cytotoxicity assays in AML cell Chemical conjugation
lines indicated that Pgp expression altered the potency of the In one type of chemical conjugation, a reactive moiety pendant to
calicheamicin payload and that drug potency could be restored the drug–linker is covalently joined to the antibody via an amino
by adding known efflux transporter antagonists to inhibit Pgp and acid residue side chain, commonly the e-amine of lysine. As
MRP-1 proteins [87]. These results were relevant for patients with demonstrated with Mylotarg1, direct conjugation of lysine resi-
AML as levels of Pgp expression in the clinic were found to dues on gemtuzumab was achieved using an N-hydroxysuccini-
correlate directly with responders and nonresponders [88,89]. mide (NHS) ester appended to the drug–linker to form stable amide
Another interesting example related to MDR mechanisms bonds [91]. A two-step process can also be used in which surface
involves AVE9633, which comprises an anti-CD33 antibody linked lysines on the antibody are first modified to introduce a reactive
through a disulfide bond to the maytansine analog DM4. In vitro group, such as a maleimide, and then conjugated to the drug–
data clearly demonstrated that the cytotoxicity of AVE9633 and linker containing an appropriate reactive handle (e.g. a thiol) [92].
the DM4 free drug were highly dependent on the expression level Such a strategy was utilized in the case of Kadcyla1. Alternatively,
of Pgp protein in myeloid cell lines [90]. As with the calicheamicin controlled reduction of existing disulfide bonds can liberate free
payload of Mylotarg1, the potency of DM4 could be restored in cysteine residues on the antibody, which then react with a mal-
Pgp-overexpressing cell lines by adding known inhibitors of Pgp. eimide attached to the drug–linker. This approach, used in the
However, Pgp activity was not found to be a major mechanism of preparation of Adcetris1, takes advantage of the reducible disul-
resistance for the AVE9633 conjugate in cells from patients with fide bonds of IgG antibodies in which controlled conditions
AML. Reasons for the lack of correlation are unclear; other enable reduction of only interchain disulfide bonds while intra-
mechanisms such as microtubule alteration were proposed for chain disulfides remain unaffected, thus minimizing major struc-
chemoresistance to AVE9633. tural disruptions to the antibody [19].
The random conjugation processes described above produce
Conjugation strategies heterogeneous mixtures of conjugated species with variable
For most ADCs in clinical development, conjugation of the drug DARs. Adding to the complexity, the site of conjugation could
payload to the antibody involves a controlled chemical reaction be different for each ADC species containing even only one drug.
with specific amino acid residues exposed on the surface of the When lysines are used for conjugation, heterogeneity in overall
mAb. Given that this process results in a mixture of ADC species charge can impact solubility, stability and PK [93]. Therefore, the
with variable DARs and linkage sites, alternative conjugation clinical success of an ADC produced by random conjugation
strategies aimed at minimizing heterogeneity have been devel- depends on robust manufacturing processes that provide
oped. In the overall design of an ADC, selection of the appropriate the ability to monitor, control and purify the heterogeneous
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Drug Discovery Today Volume 00, Number 00 December 2013 REVIEWS
products. Several organizations have developed expertise in this aldehyde tags [102,103]. The reactive aldehyde functionality
area to overcome the process development and manufacturing can then undergo conjugation to the drug–linker via oxime
challenges associated with ADC commercialization [94]. chemistry or a Pictet–Spengler reaction [104].
Site-specific conjugation Harnessing enzymatic post-translational modification processes
Despite the success of Adcetris1 and Kadcyla1, considerable for site-specific labeling of proteins is a recently reviewed approach
enthusiasm for the next generation of ADCs has focused on the for the preparation of homogenous ADCs [105]. Bacterial trans-
development of homogeneous products derived via site-specific glutaminase (BTG) catalyzes the ligation of glutamine side chains
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conjugation. Currently, three strategies are at the forefront: inser- with the primary e-amine of lysine residues, resulting in a stable
tion of cysteine residues in the antibody sequence by mutation or isopeptide bond. Jegar et al. exploited BTG to load four chelates on
insertion, insertion of an unnatural amino acid with a bio-ortho- a deglycosylated antibody with an N297Q mutation in a site-
gonal reactive handle, and enzymatic conjugation. specific manner [106]. Recently, Strop et al. conducted BTG-
Building on early studies that explored the introduction of assisted conjugations by inserting LLQG sequences at different
surface cysteines on recombinant antibodies [95], several cysteine sites on an antibody [107]. These studies clearly demonstrated that
engineered antibodies have been produced and tested for use in the site of conjugation has a significant impact on the stability and
site-specific attachment of cytotoxic drugs to yield homogeneous PK of the ADC. Another enzyme, sortase A (SrtA), catalyzes hydro-
ADCs [96]. Junutula et al. reported a class of THIOMAB-drug lysis of the threonine–glycine bond in a LPXTG motif to form a
conjugates (TDCs) prepared by taking advantage of: (i) phage new peptide bond between the exposed C-terminus of threonine
display techniques to identify ideal sites for mutation and pro- and an N-terminal glycine motif [108].
duce antibodies with minimal aggregation issues, and (ii) meth-
ods to reduce and re-oxidize the antibody under mild conditions Next-generation ADCs
to present only thiols of mutated cysteines for conjugation Key clinical assets
[92,97]. Compared with a conventional, randomly conjugated The nearly 30 ADCs currently in clinical development have been
ADC, the analogous TDC displayed minimal heterogeneity with reviewed in detail elsewhere [109], and representative examples of
similar in vivo activity, improved PK and a superior therapeutic the most advanced agents are summarized in Table 3. In addition
index. Moreover, McDonagh et al. engineered antibodies in to the FDA-approved ADCs discussed in preceding sections, several
which interchain cysteines were replaced with serines to reduce compounds are in late-stage clinical testing for both hematological
the number of potential conjugation sites, yielding ADCs with and solid tumor indications. Despite the withdrawal of Mylotarg1
defined DARs (two or four drugs per antibody) and attachment from the market in 2010, promising results from ongoing clinical
sites [98]. Broad application of this approach to future ADCs will studies have shown that when combined with chemotherapy
depend on further studies to evaluate the effect of these mutations Mylotarg1 increased overall survival in patients with newly diag-
on the overall stability and biological function of the engineered nosed AML compared to those treated with chemotherapy alone
antibody. [110]. Inotuzumab ozogamicin, which uses the same calicheami-
Encouraged by studies with cysteine engineered antibodies, cin payload and cleavable hydrazone linker found in Mylotarg1,
several investigators reasoned that the site and stoichiometry of recently failed to demonstrate improved survival in a phase III
conjugation could be controlled by inserting unnatural amino study for patients with refractory aggressive NHL (Pfizer Inc. press
acids with orthogonal reactivity relative to the 20 natural amino release; May 20, 2013). No unexpected safety concerns were iden-
acids. Axup et al. genetically engineered an orthogonal amber tified, however, and phase III studies continue for acute lympho-
suppressor tRNA/aminoacyl-tRNA synthetase pair to insert site- blastic leukemia (ALL) patients.
specifically p-acetylphenylalanine (pAcPhe) in recombinantly The vast majority of remaining ADCs in clinical development
expressed antibodies [99]. As a test case, pAcPhe was introduced use either auristatin [monomethyl auristatin E (MMAE) or MMAF]
at one of several positions in the constant region of trastuzumab or maytansinoid (DM1 or DM4) payloads, both potent inhibitors
(anti-Her2). These mutants were then conjugated to an alkoxy- of tubulin polymerization. Several MMAE conjugates with clea-
amine auristatin derivative via formation of a stable oxime bond. vable linkers are currently under evaluation in phase II studies for
The resulting chemically homogeneous ADCs demonstrated various indications based on the target antigen. In general, these
improved PK compared with nonspecifically conjugated ADCs agents were well tolerated in phase I trials with toxicities consis-
and were highly efficacious in a Her2-positive human tumor tent with the known mechanism of action for the auristatins (e.g.
xenograft model. In addition to pAcPhe, other unnatural amino neutropenia or neuropathy) [22,111]. SAR3419, an anti-CD19
acids are being explored through the use of appropriate tRNA– DM4 conjugate with a cleavable disulfide linker, demonstrated a
aminoacyl-tRNA synthetase pairs [100]. Recently, in vitro transcrip- dose-limiting toxicity (DLT) of reversible severely blurred vision in
tion and translation processes have also been developed and a phase I study for refractory B cell NHL, but was well tolerated on a
optimized to insert unnatural amino acids in antibodies for site- modified dosing schedule [112]. Recently advanced to phase II
specific conjugation [101]. studies for colorectal cancer (CRC), labetuzumab-SN-38 employs a
In addition to inserting unnatural amino acids into mAb cathepsin B-cleavable dipeptide linker and SN-38, the active meta-
sequences, chemoenzymatic approaches have been explored to bolite of the clinically used anticancer agent irinotecan, as a
generate bio-orthogonal reactive groups for selective conjugation. payload. Initial phase I data indicated that labetuzumab-SN-38
Bertozzi and co-workers utilized formylglycine-generating enzyme was generally safe and well tolerated at effective clinical doses
(FGE), which recognizes a CXPXR sequence and converts a [113]. Lorvotuzumab mertansine utilizes a maytansinoid payload
cysteine residue to formylglycine to produce antibodies with (DM1) and a disulfide linker to target CD56. No serious DLTs or
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TABLE 3
Representative ADCs undergoing clinical evaluationa
Agent Sponsor (licensee) Status Indication Antigen Cytotoxin Linker
W
Adcetris (brentuximab Seattle Genetics Launched HL, ALCL CD30 MMAE Cleavable, Val-Cit
vedotin, SGN-35) (Millennium)
KadcylaW (ado-trastuzumab Roche-Genentech Launched Her2+ metastatic HER2 DM1 Non-cleavable, thioether
emtansine, T-DM1) (ImmunoGen) breast cancer
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MylotargW (gemtuzumab Pfizer (UCB) Withdrawn AML CD33 Calicheamicin Cleavable, hydrazone
ozogamicin) (Ac-But acid)
Inotuzumab ozogamicin Pfizer (UCB) Ph III ALL, NHL CD22 Calicheamicin Cleavable, hydrazone
(CMC-544) (Ac-But acid)
RG-7596 Roche-Genentech Ph II DLBCL, NHL CD79b MMAE Cleavable, Val-Cit
Glembatumumab Celldex (Seattle Ph II Advanced breast GPNMB MMAE Cleavable, Val-Cit
vedotin CDX-011) Genetics) cancer, melanoma
PSMA-ADC Progenics (Seattle Ph II HRPC PSMA MMAE Cleavable, Val-Cit
Genetics)
SAR3419 Sanofi (ImmunoGen) Ph II Hematologic tumors CD19 DM4 Cleavable, disulfide
Labetuzumab-SN-38 Immunomedics Ph II Metastatic CRC CEACAM5 SN-38 Cleavable, Phe-Lys
(IMUU-130)
Lorvotuzumab mertansine ImmunoGen Ph I/II MM, solid tumors CD56 DM1 Cleavable, disulfide
(IMGN901)
Milatuzumab-DOX Immunomedics Ph I/II MM CD74 Doxorubicin Cleavable, hydrazone
(IMMU-110)
BT-062 Biotest AG Ph I MM CD138 DM4 Cleavable, disulfide
(ImmunoGen)
BAY-94-9343 Bayer Schering Ph I Solid tumors Mesothelin DM4 Cleavable, disulfide
(ImmunoGen)
ASG-5ME Astellas (Seattle Ph I Solid tumors AGS-5 MMAE Cleavable, Val-Cit
Genetics)
SGN-75 Seattle Genetics Ph I NHL, RCC CD70 MMAF Non-cleavable, MC
IMGN529 ImmunoGen Ph I Hematologic tumors CD37 DM1 Non-cleavable, thioether
SAR-566658 Sanofi (ImmunoGen) Ph I Solid tumors DS6 DM4 Cleavable, disulfide
a
Abbreviations: CEACAM5, carcinoembryonic antigen cell adhesion molecule 5; HRPC: hormone refractory prostate cancer; MC: maleimidocaproyl; RCC: renal cell carcinoma; SN-38, 7-
ethyl-10-hydroxycamptothecin.
drug-related adverse events were reported in early-phase multiple to introduction of an additional metabolic pathway (i.e. cleavage
myeloma (MM) studies [114]. of the drug from the antibody). In addition, ADCs with higher
Although the modest potency of doxorubicin payloads limited DARs tend to clear faster than those with lower DARs [116].
the efficacy of early ADCs (BR96-DOX), milatuzumab-DOX targets Variable DARs and attachment sites, a consequence of current
CD74, an antigen with unique internalization and surface re- random conjugation methods, result in heterogeneous ADCs with
expression, and is currently in phase I/II trials based on encoura- PK parameters that can vary substantially compared to the uncon-
ging preclinical efficacy in hematopoietic cancer xenograft models jugated antibody [117]. Each ADC component, along with their
[52]. Select agents in phase I trials include ADCs containing DM1 respective metabolites, can potentially impact efficacy, safety and
or DM4 cytotoxic drugs under evaluation by ImmunoGen, and tolerability [118]. Both the type of linker used and the site of
several ADCs with MMAE or MMAF developed by Seattle Genetics, conjugation can influence the extent to which the drug is prema-
each targeting a different antigen across a variety of tumor indica- turely released from the antibody. Deconjugation of the payload
tions. Available data for these and other phase I agents generally from the antibody can result in ADCs with lower DARs, reduced
provide initial evidence of efficacy and tolerability. Similar to efficacy and potentially increased toxicity owing to release of a
SAR3419 (anti-CD19 DM4 conjugate), the DM4-based anti- highly potent cytotoxic drug in systemic circulation.
mesothelin conjugate BAY-94-9343 has also been reported to The PK parameters of Adcetris1 and Kadcyla1 were evaluated in
induce Grade 2 and 4 ocular toxicity [115]. mouse, rat and monkey in preclinical toxicity studies. Overall, these
ADCs demonstrated similar PK properties, albeit with a few differ-
ADC PK ences in mouse and monkey. The t1/2 of Adcetris1 in mouse, rat and
ADCs typically retain the PK properties of the antibody compo- monkey was 14, 10 and 2 days, respectively. The rapid clearance of
nent, as opposed to the appended drug, and thus exhibit relatively Adcetris1 in monkeys as compared with mouse or rat was hypothe-
low clearance and long half lives. Compared with the unconju- sized to result from nontherapeutic antibodies, target-mediated
gated antibody, ADCs can exhibit somewhat higher clearance due disposition and other factors [119]. In the case of Kadcyla1, the
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t1/2 in mouse, rat and monkey was 5.7, 8.3 and 11.6 days, respec- influence stability and, therefore, PK. A recent study examined the
tively [120,121]. In humans, the PK characteristics of these two stability of MMAE conjugated to Her2 via a maleimide at various
conjugates were similar, with a t1/2 ranging from 3.5 to 5 days site-specifically engineered cysteines [126]. Highly solvent acces-
[22,24]. Of note, the t1/2 of an ADC is often significantly shorter sible conjugation sites were found to be labile, undergoing mal-
in humans compared with other species. The optimal t1/2 for an ADC eimide exchange with reactive thiols in the plasma, such as
remains to be determined, but the clinical success of Adcetris1 and glutathione, albumin or free cysteine. At less accessible sites,
Kadcyla1 indicate that the range of 3–4 days is appropriate. the succinimide ring of the linker underwent hydrolysis, which
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The long t1/2 typical of ADCs and mAbs results from FcRn served to protect the linker from maleimide exchange and resulted
recycling [122]. In this process, antigen-independent internaliza- in enhanced stability and efficacy. In a separate study, the stability
tion by endothelial cells is followed by FcRn binding and then of monomethyl auristatin D (MMAD) conjugated to an anti-M1S1
FcRn-mediated return to the bloodstream. FcRn recycling essen- antibody was examined using BTG to introduce the drug payload
tially protects ADCs from catabolism; however, diversion of FcRn- site specifically at either the heavy or light chain [107]. The
bound ADCs to the lysosome can increase the risk of off-target conjugation site was found to influence stability and PK, with
toxicities. Although the factors that influence this process are ADCs appended to the heavy chain demonstrating a higher rate of
poorly understood, the drug, linker, antibody and antigen can drug loss in rats via proteolysis of the valine–citrulline linker.
each affect FcRn-mediated ADC trafficking [123]. Another Interestingly, these results were species specific since both con-
mechanism of off-target toxicity involves soluble cell-surface man- jugates demonstrated comparable stability in mice, which also
nose receptors (MRs), which interact with agalactosylated glycans serves to highlight the potential pitfall of performing safety and
on the antibody Fc domain [124]. Cell-surface MRs can internalize, efficacy studies in different species.
effectively delivering the ADC to the endosome and lysosome
compartments where the potent cytotoxic drug is released. Impor- Concluding remarks and future directions
tantly, locations of off-target ADC activities reportedly coincide Despite complexities in designing ADCs, the promise of this
with cell-surface MR locations. The shedding of antigen from the therapeutic class has generated intense interest for decades. A
tumor cell surface into circulation may also increase the risk of robust clinical pipeline and the recent FDA approvals of Adcetris1
toxicity. Binding of an ADC to shed antigen can, in some cases, and Kadcyla1 suggest that the potential benefit of ADCs may
lead to higher ADC clearance and impaired tumor localization as finally be realized. Evolving clinical data will continue to drive
well as immune complex formation and accumulation in the technological advancements in the field. Current methods for
kidney [125]. preclinical lead selection typically rely on systematic in vitro
To determine the effect of linker stability on PK and efficacy, the evaluation of a matrix of various mAbs, linkers and cytotoxic
noncleavable thioether linker of Kadcyla1 was compared to the payloads. Whether in vitro models are sufficient to predict response
cleavable disulfide linker of a T-SPP-DM1 conjugate [121]. The remains to be seen; until further understanding of ADCs is rea-
nonreducible thioether-linked Kadcyla1 demonstrated superior lized, early in vivo studies might be crucial. Progress in site-specific
PK with greater plasma exposure (area under the curve) and conjugation modalities, optimization of linkers with balanced
increased maytansinoid tumor concentration. The disulfide- stability and identification of novel, potent cytotoxic agents
linked ADC demonstrated higher plasma clearance owing to the should pave the way for greater insight into the contribution of
presence of the metabolically labile linker. Despite the difference these various factors to ADC efficacy, PK and safety. Challenges in
in PK, both conjugates had similar in vivo efficacy. It was hypothe- target tumor selection will be addressed as the roles of antigen
sized that the drug released from the disulfide-linked T-SPP-DM1 expression, heterogeneity and internalization rate are further
conjugate would benefit from the bystander killing effect, whereas elucidated. Guiding principles for the selection of an ideal anti-
Kadcyla1 ultimately liberates a maytansinoid appended to a body Fc format are, as of yet, lacking and prompt validation of
charged lysine residue, which limits diffusion to neighboring current assumptions regarding antibody-dependent properties,
tumor cells. Taken together, these results illustrate how minor such as specificity and immune effector functions. Ongoing efforts
structural changes can profoundly impact ADC PK and efficacy. to address these issues will continue to broaden the impact of
In addition to the type of linker used to join the drug and ADCs as targeted therapeutics for the treatment of cancer and
antibody, the conjugation site on the antibody has been shown to potentially other diseases.
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12 www.drugdiscoverytoday.com
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