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METHODS IN MOLECULAR BIOLOGY

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 Antibody-Drug Conjugates

Methods and Protocols

Edited by

L. Nathan Tumey
Department of Pharmacy and Pharmaceutical Sciences, Binghamton University, Binghamton, NY, USA
Editor
L. Nathan Tumey
Department of Pharmacy
and Pharmaceutical Sciences
Binghamton University
Binghamton, NY, USA

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-4939-9928-6 ISBN 978-1-4939-9929-3 (eBook)
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Preface

The design, preparation, and bioanalysis of antibody drug conjugates (ADCs) have become
progressively more sophisticated over the past decade. Thanks to the persistence of a handful
of research groups, ADCs are now viewed as a proven therapeutic modality for the targeted
delivery of chemotherapeutic agents. From the outside, the technology is deceptively
simple. A targeting antibody delivers a covalently attached payload to a cell type of interest,
wherein the molecule is internalized and lysosomally processed and the payload is released.
However, a quick look “under the hood” reveals that the failure rate of ADC discovery
programs is not so different than small molecule discovery efforts and that there remain a
tremendous number of unknown variables when designing these therapeutic agents. As
such, the diversity of ADC technology has exploded over the past 10 years in trying and
addressing the shortcomings in first-generation ADCs. ADCs in clinical and preclinical
development now employ a dizzying array of technology that spans multiple types of
antibody backbones, over a dozen varieties of payload classes, various cleavable and
non-cleavable linker chemistries, and numerous conjugation technologies. For the bench-
top scientists charged with evaluating and advancing these research programs, the technical
hurdles can seem overwhelming. This book has been designed keeping these scientists in
mind. We have pulled together a team of authors who are lab-based scientists who have
performed many of the key conjugations, bioanalyses, and in vitro assays for the ADCs that
are now in advanced preclinical and clinical evaluation. The goal of this book is not to
provide an exhaustive list of procedures, but rather to highlight some of the key methodol-
ogies that are commonly employed across multiple ADC programs. It is our hope that this
book will help to lower the “activation barrier” that is often present when entering a new
discipline and will provide a “toolbox” of sorts for the next generation of aspiring ADC
scientists who will, no doubt, advance the field into new and exciting therapeutic arenas.

Binghamton, NY, USA L. Nathan Tumey

v
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 An Overview of the Current ADC Discovery Landscape . . . . . . . . . . . . . . . . . . . . . 1
L. Nathan Tumey
2 Pushing the Envelope: Advancement of ADCs Outside of Oncology . . . . . . . . . . 23
Michael J. McPherson and Adrian D. Hobson
3 Conjugations to Endogenous Cysteine Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Durgesh V. Nadkarni
4 Site-Specific Conjugation to Cys-Engineered THIOMAB™ Antibodies . . . . . . . 51
Pragya Adhikari, Neelie Zacharias, Rachana Ohri,
and Jack Sadowsky
5 Transglutaminase-Mediated Conjugations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Yasuaki Anami and Kyoji Tsuchikama
6 Click Chemistry Conjugations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Tak Ian Chio and Susan L. Bane
7 Utilizing Solid-Phase to Enable High-Throughput, Site-Specific
Conjugation and Dual-Labeled Antibody and Fab Conjugates . . . . . . . . . . . . . . . 99
Sujiet Puthenveetil
8 Bridged Cysteine Conjugations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Matthew Bird, Joao Nunes, and Mark Frigerio
9 Antibody Conjugations via Glycosyl Remodeling . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Hanna Toftevall, Helén Nyhlén, Fredrik Olsson,
and Jonathan Sjögren
10 ADC Analysis by Hydrophobic Interaction Chromatography. . . . . . . . . . . . . . . . . 147
Ryan Fleming
11 Two-Dimensional Liquid Chromatography Coupled to High-Resolution
Mass Spectrometry for the Analysis of ADCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Soraya Chapel, Florent Rouvière, Morgan Sarrut,
and Sabine Heinisch
12 Drug Loading and Distribution of ADCs After Reduction
or IdeS Digestion and Reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
Elsa Wagner-Rousset, Olivier Colas, Yannis-Nicolas François,
Sabine Heinisch, Davy Guillarme, Sarah Cianférani, and Alain Beck
13 Analysis of ADCs by Native Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
Oscar Hernandez-Alba, Anthony Ehkirch, Alain Beck,
and Sarah Cianférani
14 High-Resolution Characterization of ADCs by Orbitrap LCMS . . . . . . . . . . . . . . 213
Jintang He, Surinder Kaur, and Keyang Xu
15 Conjugation Site Analysis by MS/MS Protein Sequencing . . . . . . . . . . . . . . . . . . . 221
Linjie Han, Yanqun Zhao, and Qunying Zhang

vii
viii Contents

16 Conjugation Site Analysis of Lysine-Conjugated ADCs. . . . . . . . . . . . . . . . . . . . . . 235

Hua Sang, Ning Wan, Gaoyuan Lu, Yang Tian, Guangji Wang,
and Hui Ye
17 Characterization of ADCs by Capillary Electrophoresis . . . . . . . . . . . . . . . . . . . . . . 251
Wenjing Ning and Yanqun Zhao
18 Characterization of the Primary Structure of Cysteine-Linked
Antibody-Drug Conjugates Using Capillary Electrophoresis with
Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
Josiane Saadé, Rabah Gahoual, Alain Beck, Emmanuelle Leize-Wagner,
and Yannis-Nicolas François
19 Purification of ADCs by Hydrophobic Interaction Chromatography . . . . . . . . . . 273
Calvin L. Becker, Robert J. Duffy, Jorge Gandarilla,
and Steven M. Richter
20 Detection and Removal of Small Molecule and Endotoxin
Contaminants in ADC Preparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
Jeffrey Casavant, Anokha S. Ratnayake, Sujiet Puthenveetil,
and L. Nathan Tumey
21 Physical Stability Studies of Antibody-Drug Conjugates (ADCs)
Under Stressed Conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
Yilma T. Adem
22 Biophysical Methods for Characterization of Antibody-Drug Conjugates . . . . . . 313
Vamsi Krishna Mudhivarthi and Jianxin Guo
23 Determination of ADC Cytotoxicity in Immortalized Human Cell Lines . . . . . . 329
Shengjia Wu and Dhaval K. Shah
24 LC/MS Methods for Studying Lysosomal ADC Catabolism . . . . . . . . . . . . . . . . . 341
Andrew J. Bessire and Chakrapani Subramanyam
25 Assessing ADC Plasma Stability by LC-MS Methods . . . . . . . . . . . . . . . . . . . . . . . . 353
Cong Wei
26 Determination of ADC Concentration by Ligand-Binding Assays. . . . . . . . . . . . . 361
Hsuan-Ping Chang and Dhaval K. Shah

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
Contributors

YILMA T. ADEM  Pharmaceutical Development Department, Genentech (A Member of the

Roche Group), South San Francisco, CA, USA
PRAGYA ADHIKARI  Department of Protein Chemistry, Genentech, Inc., South San Francisco,
CA, USA
YASUAKI ANAMI  Texas Therapeutics Institute, The Brown Foundation Institute of Molecular
Medicine, The University of Texas Health Science Center at Houston, Houston, TX, USA
SUSAN L. BANE  Department of Chemistry, Binghamton University, State University of New
York, Binghamton, NY, USA
ALAIN BECK  Pierre Fabre Laboratories, IRPF—Centre d’Immunologie Pierre-Fabre
(CIPF), Saint-Julien-en-Genevois, France
CALVIN L. BECKER  Abbvie Inc., North Chicago, IL, USA
ANDREW J. BESSIRE  Medicine Design, Pfizer Worldwide R&D, Groton, CT, USA
MATTHEW BIRD  Abzena Ltd., Cambridge, UK
JEFFREY CASAVANT  Pfizer, Inc., Groton, CT, USA
HSUAN-PING CHANG  Department of Pharmaceutical Sciences, School of Pharmacy and
Pharmaceutical Sciences, The State University of New York, University at Buffalo, Buffalo,
NY, USA
SORAYA CHAPEL  Institut des Sciences Analytiques, CNRS, UMR 5280, Université de Lyon,
Villeurbanne, France
TAK IAN CHIO  Department of Chemistry, Binghamton University, State University of New
York, Binghamton, NY, USA
SARAH CIANFÉRANI  Laboratoire de Spectrométrie de Masse Bio-Organique (LSMBO),
IPHC, UMR 7178, Université de Strasbourg, CNRS, Strasbourg, France
OLIVIER COLAS  Pierre Fabre Laboratories, IRPF—Centre d’Immunologie Pierre-Fabre
(CIPF), Saint-Julien-en-Genevois, France
ROBERT J. DUFFY  Abbvie Inc., Redwood City, CA, USA
ANTHONY EHKIRCH  Laboratoire de Spectrométrie de Masse Bio-Organique (LSMBO),
IPHC, UMR 7178, Université de Strasbourg, CNRS, Strasbourg, France
RYAN FLEMING  Antibody Discovery and Protein Engineering, Biologic Therapeutics,
AstraZeneca, Gaithersburg, MD, USA
YANNIS-NICOLAS FRANÇOIS  Laboratoire de Spectrométrie de Masse des Interactions et des
Systèmes (LSMIS), UMR 7140 (Unistra-CNRS), Université de Strasbourg, Strasbourg,
France
MARK FRIGERIO  Abzena Ltd., Cambridge, UK
RABAH GAHOUAL  Laboratoire Vecteurs Pour l’Imagerie Moléculaire et le Ciblage
Thérapeutique (VICT), Faculté de Pharmacie, Université Paris Descartes, Paris, France
JORGE GANDARILLA  Abbvie Inc., North Chicago, IL, USA
DAVY GUILLARME  School of Pharmaceutical Sciences, University of Geneva, Geneva,
Switzerland; University of Lausanne, Lausanne, Switzerland
JIANXIN GUO  Pharmaceutical R&D, Pfizer, Inc., Chesterfield, MO, USA
LINJIE HAN  Process Analytical Chemistry, AbbVie Inc., North Chicago, IL, USA
JINTANG HE  Genentech Inc., South San Francisco, CA, USA

ix
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x Contributors

SABINE HEINISCH  Institut des Sciences Analytiques, CNRS, UMR 5280, Université de Lyon,
Villeurbanne, France
OSCAR HERNANDEZ-ALBA  Laboratoire de Spectrométrie de Masse Bio-Organique (LSMBO),
IPHC, UMR 7178, Université de Strasbourg, CNRS, Strasbourg, France
ADRIAN D. HOBSON  Abbvie Global Biologics, AbbVie Bioresearch Center, Worcester, MA,
USA
SURINDER KAUR  Genentech Inc., South San Francisco, CA, USA
EMMANUELLE LEIZE-WAGNER  Laboratoire de Spectrométrie de Masse des Interactions et des
Systèmes (LSMIS), UMR 7140 (Unistra-CNRS), Université de Strasbourg, Strasbourg,
France
GAOYUAN LU  School of Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu,
People’s Republic of China
MICHAEL J. MCPHERSON  Abbvie Global Biologics, AbbVie Bioresearch Center, Worcester,
MA, USA
VAMSI KRISHNA MUDHIVARTHI  Pharmaceutical R&D, Pfizer, Inc., Chesterfield, MO, USA
DURGESH V. NADKARNI  Pfizer Inc., Chesterfield, MO, USA
WENJING NING  Process Analytical Chemistry, AbbVie Inc., North Chicago, IL, USA
JOAO NUNES  Abzena Ltd., Cambridge, UK
HELÉN NYHLÉN  Genovis AB, Lund, Sweden
RACHANA OHRI  Department of Protein Chemistry, Genentech, Inc., South San Francisco,
CA, USA
FREDRIK OLSSON  Genovis AB, Lund, Sweden
SUJIET PUTHENVEETIL  AbbVie Bioresearch Center, R&D, Worcester, MA, USA; Pfizer, Inc.,
Groton, CT, USA
ANOKHA S. RATNAYAKE  Pfizer, Inc., Groton, CT, USA
STEVEN M. RICHTER  Abbvie Inc., North Chicago, IL, USA
FLORENT ROUVIÈRE  Institut des Sciences Analytiques, Université de Lyon, Villeurbanne,
France
JOSIANE SAADÉ  Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes
(LSMIS), UMR 7140 (Unistra-CNRS), Université de Strasbourg, Strasbourg, France
JACK SADOWSKY  Department of Protein Chemistry, Genentech, Inc., South San Francisco,
CA, USA
HUA SANG  Department of Pharmacy, Affiliated Hospital of Nantong University, Nantong,
Jiangsu, People’s Republic of China; Key Laboratory of Drug Metabolism and
Pharmacokinetics, State Key Laboratory of Natural Medicines, China Pharmaceutical
University, Nanjing, Jiangsu, People’s Republic of China
MORGAN SARRUT  Institut des Sciences Analytiques, Université de Lyon, Villeurbanne,
France
DHAVAL K. SHAH  Department of Pharmaceutical Sciences, School of Pharmacy and
Pharmaceutical Sciences, The State University of New York, University at Buffalo, Buffalo,
NY, USA
JONATHAN SJÖGREN  Genovis AB, Lund, Sweden
CHAKRAPANI SUBRAMANYAM  Medicine Design, Pfizer Worldwide R&D, Groton, CT, USA
YANG TIAN  School of Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu,
People’s Republic of China
HANNA TOFTEVALL  Genovis AB, Lund, Sweden
 Contributors xi

KYOJI TSUCHIKAMA  Texas Therapeutics Institute, The Brown Foundation Institute of

Molecular Medicine, The University of Texas Health Science Center at Houston, Houston,
TX, USA
L. NATHAN TUMEY  Department of Pharmacy and Pharmaceutical Sciences, Binghamton
University, Binghamton, NY, USA; Pfizer Inc., Groton, CT, USA
ELSA WAGNER-ROUSSET  Pierre Fabre Laboratories, IRPF—Centre d’Immunologie Pierre-
Fabre (CIPF), Saint-Julien-en-Genevois, France
NING WAN  Key Laboratory of Drug Metabolism and Pharmacokinetics, State Key
Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, Jiangsu,
People’s Republic of China
GUANGJI WANG  Key Laboratory of Drug Metabolism and Pharmacokinetics, State Key
Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, Jiangsu,
People’s Republic of China
CONG WEI  Drug Metabolism and Pharmacokinetics, Biogen Inc., Cambridge, MA, USA
SHENGJIA WU  Department of Pharmaceutical Sciences, School of Pharmacy and
Pharmaceutical Sciences, The State University of New York, University at Buffalo, Buffalo,
NY, USA
KEYANG XU  Genentech Inc., South San Francisco, CA, USA
HUI YE  Key Laboratory of Drug Metabolism and Pharmacokinetics, State Key Laboratory of
Natural Medicines, China Pharmaceutical University, Nanjing, Jiangsu, People’s
Republic of China
NEELIE ZACHARIAS  Department of Protein Chemistry, Genentech, Inc., South San Francisco,
CA, USA
QUNYING ZHANG  Process Analytical Chemistry, AbbVie Inc., North Chicago, IL, USA
YANQUN ZHAO  Process Analytical Chemistry, AbbVie Inc., North Chicago, IL, USA
 Chapter 1

An Overview of the Current ADC Discovery Landscape

L. Nathan Tumey

Abstract
The prototypical ADC mechanism involving antigen-mediated uptake and lysosomal release is both
elegantly simple and scientifically compelling. However, recent clinical-stage failures have prompted a
reevaluation of this delivery paradigm and have resulted in an array of new technologies that have the
potential to improve the safety and efficacy of up and coming programs. These innovations can generally be
categorized into seven areas that will be elaborated on in this chapter: (1) Exploiting new payload
mechanisms; (2) Increasing the drug–antibody ratio (DAR); (3) Increasing the antibody penetration;
(4) Overcoming ADC resistance mechanisms; (5) Increasing the efficiency of ADC uptake and processing;
(6) Mitigating off-target payload exposure; and (7) Employment of noncytotoxic payloads. It is our belief
that these seven areas capture the current “landscape” of innovations that are taking place in the design of
next-generation ADCs. Together, these advancements are reshaping the ADC field and providing a path
forward in the face of the recent clinical setbacks.

Key words ADC, Resistance, Toxicity, Conjugation, DAR, Tumor penetration, Payload

1 Introduction

Drug discovery is not for the faint of heart. As anyone who has
spent time in the field knows, promising therapeutic agents in late-
stage trials can essentially disappear at a moment’s notice after a
disastrous clinical report; and drugs that seemed destined for
obscurity can suddenly become major blockbusters. For good or
for bad, the field of antibody–drug conjugates (ADCs) has proven
to be a microcosm of the broader drug-discovery engine. The past
10 years of ADC discovery has seen incredible scientific innovation
coupled with massive clinical failures and unexpected successes. The
goal of this chapter is to highlight some of the changes in the field
in recent years and to offer a “snapshot” of the most promising
innovations that are currently being explored.
Typical ADCs consist of three key components: An antibody
targeting vehicle, a cytotoxic drug, and a covalent linker that con-
nects the system together. Upon binding to the receptor on the
target cell, the ADC is internalized via a clathrin-mediated

L. Nathan Tumey (ed.), Antibody-Drug Conjugates: Methods and Protocols, Methods in Molecular Biology, vol. 2078,
https://doi.org/10.1007/978-1-4939-9929-3_1, © Springer Science+Business Media, LLC, part of Springer Nature 2020

1
2 L. Nathan Tumey

Fig. 1 Prototypical ADC uptake mechanism

endocytosis and trafficked to the lysosome. Lysosomal proteolysis

cleaves the covalent linkage between the antibody and payload, thus
allowing the payload to escape the lysosome and bind to its intra-
cellular target (Fig. 1). The antibody is designed to target antigens
that are uniquely expressed on the tissue of interest, typically cancer
cells. Great effort is typically employed to ensure that the selected
antigen has minimal expression in normal tissue, especially in highly
perfused tissue. The linker is carefully selected to be rapidly cleaved
in the lysosome while having minimal cleavage during its extended
time in circulation (often 2 weeks or more) [1]. Linker design is
also critically important in determining propensity for aggregation
and the rate of premature linker cleavage in plasma [1, 2]. The ADC
payload must be exquisitely potent due to the limited number of
receptor interactions that are possible at a given time. Note that
unlike passive diffusion, the rate of drug entry into the cell is easily
saturated once all receptors have been engaged with an antibody
partner. Thus, it is typically believed that only low-nM intracellular
concentrations of active drug can be realized using ADC technol-
ogy. Once the payload is released from the antibody (depending on
its physical properties) it has the potential to freely diffuse through
the cell membrane and affect nearby non-antigen expressing cells
thus inducing the so-called bystander activity.
The above description represents the idealized scenario and
deliberately neglects to mention many of the key “unknowns” in
the field where innovation is most ripe. For the sake of organizing
our discussion, we will categorize recent technological innovations
into seven areas: (1) Exploiting new payload mechanisms;
 An Overview of the Current ADC Discovery Landscape 3

(2) Increasing the drug–antibody ratio (DAR); (3) Increasing the

antibody penetration; (4) Overcoming ADC resistance mechan-
isms; (5) Increasing the efficiency of ADC uptake and processing;
(6) Mitigating off-target payload exposure; and (7) Employment of
noncytotoxic payloads. These areas of innovations are by no means
exhaustive and certainly we will not address every technologically
interesting advancement that has been reported in recent years.
However, it is our belief that these seven areas capture the current
“landscape” of innovations that are taking place in the design of
next-generation ADCs.

2 Exploiting New Payload Mechanisms

A full discussion of the breadth of cytotoxic payloads employed in

ADCs is beyond the scope of this chapter and has been reviewed
elsewhere [3]. However, it is worth noting that many of these new
classes of payloads are mechanistically related to the present cohort
of clinically validated payloads. For example, the clinical success of
auristatin and maytansinoid payloads (tubulin-binding agents) has
prompted enormous efforts to identify alternative structural classes
of molecules that exploit this mechanism. This has resulted in the
advancement of tubulysin [4–6], eribulin [7], cryptophycin [8],
and cemadotin [9] ADCs, in addition to new structural variants
of auristatin [10–12] and maytansinoid payloads [13, 14]. Likewise,
the clinical success of calicheamycin ADCs has inspired a new
generation of DNA-damaging payloads including uncialamycin
[15], pyrrolo[2,1-c] [1, 4] benzodiazepines (PBDs) [16–18], and
duocarmycins [19, 20]. While these payloads continue to advance
through preclinical and clinical development, recent high-profile
failures of multiple ultra-potent DNA damaging ADCs [21, 22]
have prompted considerable interest in alternative anti-proliferative
mechanisms.
Building on the success of tubulin-binding agents, a team from
Beyer reported a series of ADCs that block the activity of kinesin
spindle protein (KSP), an ATP-dependent microtubule motor that
is involved in the separation of centrosomes during mitosis
[23]. Interestingly, in spite of the presence of an obvious amine
handle for linker conjugation, these payloads were initially attached
by a noncleavable linker to a maleimide conjugation handle.
(ADC1) Conjugation via endogenous cysteine residues resulted
in DAR3-4 ADCs that exhibited sub-nM potency against antigen
expressing cell lines. While not explicitly stated, a design theme for
this series of ADCs seems to be the delivery of an impermeable
payload that minimizes bystander activity while maximizing intra-
cellular payload concentration. As such, a cleavable ADC (ADC2)
was designed to release a highly polar zwiterionic lysine-adduct, as
shown in Fig. 2. Indeed, the released species for these ADCs were
4 L. Nathan Tumey

H2 N O

HN

O O
H H
HO2C N N
N N S
F H
N O O
O

O N O H O H
N O N NH ADC2
F N N
H S N
HO O N O
NH2 NH2
O F
O
O O
ADC1
HO
O
F O H O H
N H
N
NH
O HN
H O O
O O O N N N O
O OH
H H N S O N
O O H H O
N HO H
H N
O N N
ADC3 ADC4 O H H
O
O NH2

F O NH2
O O
H
O N N
H O N N S
O
N N N O
H N N O
N N S H

ADC5 O O ADC6

H2N O

HN

SO3H O O
H H
N CO2H N N
N N (CH2)5 N
H S
N O O O
O N O O
HN
N O
N S
ADC7

Fig. 2 Recently disclosed payload classes for use in ADCs

shown to be minimally effective in a cytotoxicity assay in spite of

their impressive efficacy as conjugates. Interestingly, both ADCs
appear to be designed to release an amino-acid linked payload that
is perhaps a substrate of an amino acid transporter that has been
speculated to be an important mechanism of lysosomal escape [24].
In 2016, a team from Pfizer reported generating ADCs that
deliver a potent RNA splicing inhibitor known as thialanstatin A
(also known as spliceostatin) [25]. Unlike auristatins and maytan-
sinoids, these compounds do not have a readily available amine or
thiol handle that is suitable for conjugation. Instead, a carboxylic
acid on the payload was directly attached to lysine residues of the
antibody by a “linker-less” NHS-ester-mediated conjugation.
(ADC3) Similar to early-generation auristatin ADCs, the loading
was found to significantly impact the efficacy. The optimal efficacy
was observed with an ADC with a DAR of 3.2. Both high-loaded
(DAR 4.2) and low-loaded (DAR 2.2) ADCs were less efficacious.
Interestingly, a later report showed that the hinge-cysteine conju-
gated thialanstatin A resulted in quite weak in vivo efficacy. Thus, in
 An Overview of the Current ADC Discovery Landscape 5

order to retain the lysine-based conjugation method, a site-specific

method that employed multivalent peptidic linkers was reported
[26]. In short, a small lysine-containing peptide was allowed to
react with the NHS-activated thialanstatin and subsequently
“clicked” into place using a site-specific azide handle. The resulting
homogeneous DAR4 thialanstatin conjugates exhibited efficacy in a
Her2 model at doses as low as 1.5 mg/kg. Interestingly, while prior
reports of site-specific conjugation methods typically focus on sta-
bility and metabolism issues [27], this report shows that site of
conjugation can play a significant role in in vitro cytotoxicity.
Presumably this is due to site-dependent lysosomal catabolism,
similar to the mechanism reported by Bessire [28].
Continuing with the theme of blocking RNA processing, a
number of reports have emerged describing inhibitors of RNA
polymerase, the key enzyme responsible for the formation of
mRNA. The centrality of this mechanism for all biological processes
suggests that RNA polymerase inhibitors will have toxicity across
both proliferating and quiescent cells. The most well-studied of the
RNA polymerase inhibitors are toxins derived from the Amanita
genus of mushrooms, known collectively as amatoxins. An early
amatoxin linked to an anti-EpCAM antibody exhibited efficacy in a
pancreatic cancer line following a 2 mg/kg dose, but exhibited
severe toxicity at doses of 6 mg/kg and higher [29]. Building on
this work, a number of patents have now been filed on amanatin-
based ADCs, such as the one shown in Fig. 2 [30–32] (ADC4).
While amanatin ADCs (like their small-molecule payloads) have
been known to exhibit severe toxicity, a serendipitous observation
reported in 2015 may render a path forward for this class of
molecules. A well-known tumor suppressor gene known as TP53
is frequently found to be deleted in human tumors, thus leading to
uncontrolled cell growth. A key subunit of RNA polymerase,
known as POLR2A, is a genetic neighbor of TP53, and is fre-
quently downregulated in hemizygous TP53 deletions, thus ren-
dering the cells hypersensitive to RNA polymerase inhibitors
[33]. Thus, amatoxin-ADCs may be particularly useful in TP53-
downregulated tumors.
In a move away from mitotic and transcriptional inhibitors,
teams from Novartis and Seattle Genetics simultaneously disclosed
ADCs designed to block cellular metabolism by inhibiting the
formation of nicotinamide adenine dinucleotide (NAD+)
[34, 35]. The ADCs, shown in Fig. 2 (ADC4 and ADC5), deliver
potent inhibitors of nicotinamide phosphoribosyltransferase
(NAMPT), a critical enzyme involved in the biosynthesis of
NAD+. Unlike some of the aforementioned mechanisms, inhibitors
of NAMPT were demonstrated to be cytotoxic irrespective of the
mitotic state of the cells. Perhaps most importantly, a null-targeted
version of ADC5 was shown to be well tolerated in rats at doses of
up to 100 mg/kg—well above the dose that is typically tolerated by
6 L. Nathan Tumey

DNA-damaging and tubulin-binding ADCs. Efficacy was reported

in a variety of tumor xenograft models at doses of 10–20 mg/kg.
Rather than directly blocking an essential metabolic pathway, a
recent report demonstrates an interesting approach to sensitize cells
to apoptosis by inhibiting an anti-apoptotic protein known as
Bcl-xL. While the specific details have not yet been published, the
team from AbbVie has reported a series of ADCs, exemplified by
ADC7, which enhance the activity of docetaxel at doses of
3–10 mg/kg [36, 37]. Importantly, these ADCs do not appear to
result in thrombocytopenia, a common side effect of Bcl-xL inhibi-
tors that have been evaluated in the clinic. Given the immense
progress in our understanding of cell signaling in recent years,
agents that induce apoptosis and autophagy are certain to become
more widely explored as ADC payloads.

2.1 Increasing the Based on the classical ADC mechanism outlined in Fig. 1, it is clear
Drug–Antibody Ratio that the intracellular drug concentration will be related to the
(DAR) number of receptors that are able to engage with an ADC. Low
numbers of receptors will naturally result in low cellular uptake. In
order to compensate for this, a classical approach has been to
increase the drug–antibody ratio (DAR) in order to maximize the
number of drugs that are delivered per binding event. This can
naturally lead to examples of the so-called super-stoichiometric
activity, wherein a modest increase in drug loading can lead to a
dramatic increase in biological activity. One example of this can be
seen in the 2011 report from Zhao in which an increase in the
loading of a maytansinoid conjugate by 2.5-fold led to an increase
in the in vitro ADC activity by approximately 50-fold [38]. An even
more dramatic example can be seen in the aforementioned thailan-
statin ADCs in which increasing the loading of an anti-Her2 con-
jugate from ~1.6 to 4.5 increased the in vitro cytotoxicity against a
moderate-expresser (MDA-MB-361-DYT2) by approximately
2500-fold. These examples illustrate the benefits of increasing the
drug loading [25].
However, based on the seminal work of Hamblett [39], it has
long been known that increasing the drug loading often has an
adverse effect on pharmacokinetic exposure. For this reason, the
vast majority of clinical-stage ADCs has employed ADCs with DAR
values of ~2-4. However, the past 5 years has seen a dramatic shift in
this paradigm due to advances in our understanding about how
biophysical properties of ADCs impact their PK. Hydrophobicity
has been found to be a key driver of these findings. A fascinating
example of this was reported in 2015 by Lyon. In this study, the
authors incorporated a glycolytic-release chemistry and a PEG
chain within the linker (ADC8) in order to “mask” the hydropho-
bicity of the MMAE payload (Fig. 3) [10]. In doing so, they were
able to develop a DAR8 ADC that had equivalent pharmacokinetics
to the naked antibody. This has now become a common strategy
 An Overview of the Current ADC Discovery Landscape 7

O O O O O
O
m n q
OH OH OH O OH O

O O

O O S
O NH O N O
24 H H
O MMAE NH O N
O N

O O
H O O
N
O N S ADC9
HN O
O N N O O O H
H H N
O O HO
HO
OH
O
HO OH O
ADC8
N O
OH H
H N

N
HO

Fig. 3 Strategies for increasing the DAR of ADCs by maximizing the polarity of the linker (ADC8) and by
incorporating polymeric linkers (ADC9)

and DAR8 intra-chain cysteine ADCs are now routinely used

provided that the linker-payload is sufficiently polar to prevent
accelerated clearance [35, 40, 41]. Additionally, a variety of chemi-
cal strategies have been used to incorporate polar functional groups
into the linker with the explicit goal of decreasing the hydropho-
bicity of the ADC. This includes the incorporation of phosphates
[42, 43], sulfates [38], tertiary amines [44], and quaternary
amines [6].
While it is now commonly known that polar functionality in the
linker can “mask” the hydrophobicity of a payload, it is less com-
monly known that the site of conjugation can have a dramatic
impact on ADC hydrophobicity. Several recent studies have
recently demonstrated that carefully chosen sites of conjugation
can allow for the attachment of very hydrophobic payloads with
minimal impact on hydrophobicity, as measured by HIC [27, 45,
46]. Using these sites, conjugates with DAR as high as eight has
been achieved with a minimal impact on PK [46]. It should be
noted that these sites have the potential to be useful locations for
the attachment of dendrimeric or polymeric linkers, as described
below.
Using the polar linker technology described above, a maximal
DAR of 8 can be achieved using endogenous cysteine residues.
However, an alternative strategy has been developed in which a
dendrimeric or polymeric linker is used in order to increase the
loading far beyond eight. Interestingly, this strategy was first
reported back in 1991—long before the current cohort of clinical
ADCs had been developed [47]. More recently, a team from Mer-
sana has developed a poly-1-hydroxylmethylethylene hyrdoxy-
methylformal (PHF) linker that can be tuned for various levels of
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