using a chemoenzymatic approach -...

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Developing site - Specifically Modified ADCs:

Using a Chemoenzymatic Approach

A special article collection companion to the webinar broadcast 23rd March 2016.

Table of Contents

IntroductionDeveloping Site-Specifically Modified ADCs Using a Chemoenzymatic Approach

pages 1-2Antibody-drug conjugates: current status and future directionsDrug Discovery Today, Volume 19, Number 7, July 2014, pages 869-881Heidi L. Perez, Pina M. Cardarelli, Shrikant Deshpande, Sanjeev Gangwar, Gretchen M. Schroeder, Gregory D. Vite, Robert M. Borzilleri pages 3-15

An emerging playbook for antibody–drug conjugates: lessons from the laboratory and clinic suggest a strategy for improving efficacy and safetyCurrent Opinion in Chemical Biology, Volume 28, October 2015, Pages 174-180Penelope M Drake, David Rabuka pages 16-22

Exploring the effects of linker composition on site-specifically modified antibody-drug conjugatesEuropean Journal of Medicinal Chemistry, Volume 88, 17 December 2014, Pages 3-9Aaron E. Albers, Albert W. Garofalo, Penelope M. Drake, Romas Kudirka, Gregory W. de Hart, Robyn M. Barfield, Jeanne Baker, Stefanie Banas, David Rabuka pages 24-30

An FDA oncology analysis of antibody-drug conjugatesRegulatory Toxicology and Pharmacology, Volume 71, Issue 3, April 2015, Pages 444-452Haleh Saber, John K. Leighton pages 31-39

Introduction

There are relatively few occasions in science that define a generation. Of course, the elucidation of the 3 dimensional structure of DNA was one. For my generation, however, I would suggest that a similarly powerful one was the discovery of monoclonal antibodies by Cesar Milstein at Mill Hill in London. Tools became available for the unambiguous estimation and examination of proteins. It also became clear that monoclonal antibodies could represent a potential class of therapeutic agents. Initially, despite the promise, the translation of monoclonal antibodies into useful medicines was somewhat underwhelming. There were many reasons for the difficulty of translation, some would be related to the poor choice of target, pharmacoki-netic and pharmacodynamics issues and issues related to immunogenicity. More recently, advances in protein engineering have addressed and, to a great extent, solved these issues. The number of monoclonal antibody therapies on the market and in devel-opment bears testament to the concept of using such molecules as therapeutics and the ingenuity of biologists and protein engineers.

One of the hottest topics in monoclonal antibody-based therapeutics at the moment is that of antibody-drug conjugates (ADCs). This approach is particularly attractive to the oncology therapeutic area and relies upon tethering a cytotoxic drug (generally) to a monoclonal antibody that targets the drug to the tumour cell and, upon internalisation, releases its toxic payload to the cell. The advantage of the approach is that there is a highly-efficient delivery of a lethal level of drug to a tumour. Such concentrations may be impossible to achieve in the patient because of cost and toxicity of systemic administration. In this eBook we have included articles to highlight some of the most up-to-date developments in ADC technology.

The first article in this supplement is by Haleh Saber and John K. Leighton of the US Food and Drug Admin-istration, Center for Drug Evaluation and Research, Office of Hematology and Oncology Products, 10903 New Hampshire Ave, Silver Spring, MD 20903,United States, entitled “An FDA oncology analysis of anti-body-drug conjugates”. The article deals with approaches that have been adopted as preclinical surrogates for first-in-human dose estimation and as part of a preclinical safety package. Analysis of the various approaches has allowed the authors to establish appropriate rules for the estimation of appropriate first-in-human doses and dose escalation studies to aid with Phase 1 clinical trial design.

The second article, from Penelope M Drake and David Rabuka of Catalent Pharma Solutions, Emeryville, CA 94608, USA, entitled “An emerging playbook for antibody–drug conjugates: lessons from the laboratory and clinic suggest a strategy for improving efficacy and safety”. The authors highlight the prominent posi-tion occupied by ADCs in the oncology drug portfolios of Pharmaceutical companies. They suggest that, the field has become somewhat bifurcated in that most of the developing strategies for development rely upon existing technologies. Another, minority, approach is concerned with developments in conjugation chemistry and linker optimisation. Such an approach indicates that there may be some advantages of such approaches over the more conventional methods. The authors suggest that the success of two pioneer ADCs, KadcylaTM and AdcetrisTM, may be as a result of somewhat unique target antigens and development of new therapeutic agents may require the use of novel linker techniques.

In the third of our four articles, the authors present some concepts which, to some degree, support the conclusions of Drake and Rabuka in that they suggest that the most effective ADCs will require careful

1

antigen selection and conjugation optimisation. The article: “Antibody–drug conjugates: current status and future directions” by Heidi L. Perez, Pina M. Cardarelli, Shrikant Deshpande, Sanjeev Gangwar, Gretchen M. Schroeder, Gregory D. Vite and Robert M. Borzilleri all from Bristol Myers Squibb, is a tour de force of the history, current status, mode of action, optimisation and future directions for this exciting class of biophar-maceuticals.

Last, but by no means least, is the article from Aaron E. Albers, Albert W. Garofalo, Penelope M. Drake, Romas Kudirka, Gregory W. de Hart, Robyn M. Barfield, Jeanne Baker, Stefanie Banas, David Rabuka entitled “Exploring the effects of linker composition on site-specifically modified antibody-drug conjugates”. As the title suggests, this article deals with how modification of the antibody-drug linker may improve potency or reduce the propensity to interact with multidrug transporter proteins. Optimising these parameters will lead to a drug with a much improved profile of an ADC and can be used to engineer improved properties.

Steve Carney was born in Liverpool, England and studied Biochemistry at Liverpool University, obtaining a BSc.(Hons) and then read for a PhD on the Biochemistry and Pathology of Connective Tissue Diseases in Manchester University, in the Departments of Medical Biochemistry and Histopathology. On completion of his PhD he moved to the Kennedy Institute of Rheumatology, London, where he worked with Professor Helen Muir FRS and Professor Tim Hardingham, on the biochemistry of experimental Osteoarthritis. He joined Eli Lilly and Co. and held a number of positions in Biology R&D, initially in the Connective Tissue Department, but latterly in the Neuroscience Department. He left Lilly to take up his present position as Managing Editor, Drug Discovery Today, at Elsevier. Currently, he also holds an honorary lectureship in Drug Discovery at the University of Surrey, UK. He has authored over 50 articles in peer-reviewed journals, written several book chapters and has held a number of patents.

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Drug Discovery Today � Volume 19, Number 7 � July 2014 REVIEWS

Recent advances in the discovery and development of antibody–drug conjugateshave led to FDA approvals and a rich clinical pipeline of promising

new cancer therapies.

Antibody–drug conjugates: currentstatus and future directions

Heidi L. Perez1, Pina M. Cardarelli2, Shrikant Deshpande2,Sanjeev Gangwar2, Gretchen M. Schroeder1, Gregory D. Vite1

and Robert M. Borzilleri1

1Bristol-Myers Squibb Research & Development, Princeton, NJ 08543, USA2Bristol-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.

IntroductionThe 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. ([email protected])

1359-6446/06/$ - see front matter � 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.drudis.2013.11.004 www.drugdiscoverytoday.com 869 3

mailto:[email protected]

http://dx.doi.org/10.1016/j.drudis.2013.11.004

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REVIEWS Drug Discovery Today � Volume 19, Number 7 � July 2014

Historical perspectiveThe origin of ADCs can be traced back over a century to the

German physician and scientist Paul Ehrlich, who proposed the

concept of selectively delivering a cytotoxic drug to a tumor via a

targeting agent (Fig. 1) [7,8]. Ehrlich coined the term ‘magic bullet’

to describe his vision, similar to the descriptors ‘warhead’ or

‘payload’ commonly used for the drug component of current

ADCs. Nearly 50 years later, Ehrlich’s concept of targeted therapy

was first exemplified when methotrexate (MTX) was linked to an

antibody targeting leukemia cells [9]. Early research relied on

available targeting agents, such as polyclonal antibodies, to enable

preclinical efficacy studies in animal models with both noncova-

lent-linked ADCs and later covalently linked ADCs [10–12]. In

1975, the landmark development of mouse mAbs using hybri-

doma technology by Kohler and Milstein greatly advanced the

field of ADCs [13]. The first human clinical trial followed less than

a decade later, with the antimitotic vinca alkaloid vindesine as the

cytotoxic payload [14]. Further advances in antibody engineering

enabled the production of humanized mAbs with reduced immu-

nogenicity in humans compared with the murine mAbs used for

early ADCs [15].

First-generation ADCs typically used clinically approved drugs

with well-established mechanisms of action (MOAs), such as anti-

metabolites (MTX and 5-fluorouracil), DNA crosslinkers (mitomy-

cin) and antimicrotubule agents (vinblastine) [16]. In addition to

1913

19501910 19401930 19601920

1958 19

Paul Ehrlich describedthe concept of a‘magic bullet’ anddrug targeting (i.e. a‘heptophore’ candeliver a ‘toxophore’selectively to a tumor)

MTX linked to anantibody directed

toward leukemia cells

ADCs proposedimmunoradioactiv

agent disclose

Nonlin

tested

NH2H2N

CO2H

CO2H

N

N

O

HN

N

N

N

FIGURE 1

Antibody–drug conjugate (ADC) timeline. Abbreviations: mAbs, monoclonal antibo

870 www.drugdiscoverytoday.com

the immunogenicity issues observed with murine mAbs, these

early attempts were met with limited success for several reasons,

including low drug potency, high antigen expression on normal

cells and instability of the linker that attached the drug to the mAb

[17]. Lessons learned from these initial failures led to a new

generation of ADCs, several of which entered and later failed

human clinical trials. For example, doxorubicin conjugate 1

(BR96-DOX) was designed using a bifunctional linker, wherein

the drug was appended via a hydrazone, and a maleimide enabled

conjugation to the BR96 antibody via cysteine residues (Fig. 2)

[18]. Although curative efficacy was observed in human tumor

xenograft models, the relatively low potency of doxorubicin

necessitated high drug:antibody ratios (DARs, eight per antibody)

and high doses of the ADC to achieve preclinical activity. In

clinical trials, significant toxicity was observed due to nonspecific

cleavage of the relatively labile hydrazone linker and expression of

the antigen target in normal tissue [19].

Further advancements, including higher drug potency and care-

fully selected targets, ultimately led to the first ADC to gain US Food

and Drug Administration (FDA) approval in 2000 (Mylotarg1,

gemtuzumab ozogamicin, 2) [20,21]. Despite initially encouraging

clinical results, Mylotarg1 was withdrawn from the market a

decade later owing to a lack of improvement in overall survival.

In 2011, following an accelerated approval process, a second ADC

(Adcetris1, brentuximab vedotin, 3) gained marketing approval

2000199019801970 2010

67

1972

1975

1983

1991

;ed

covalentked ADCin animal

models

Production ofmAbs usinghybridoma-based technology

Covalent linkedADCs tested inanimal models

Clinical trialsw/ ADC

vindesine-αCEA

Immunogenicityof mouse mAbs aserious limitationin developmentof ADCs

ADC w/ highlypotent

cytotoxincalicheamicin

First FDAapproved ADC

(Mylotarg®)

Mylotarg®

clinical failuresdue to MDR

Adcetris®

approved

Mylotarg®

withdrawnfrom market

Curative efficacy inhuman tumor

xenograft models w/BR96-DOX ADC

Kadcyla®

approved

2013

2001 2011

1993

1988

HumanizedmAbs reported

Drug Discovery Today

dies; MDR, multidrug resistance; MTX, methotrexate.

4


OMe

O

O

OH

OH

NOH

O

O

OHNH2

OH

3Adcetris®

(anti-CD30 auristatin conjugate)

OO

HN

NNH

O

SS

OHO

H

OONHO O

OO

S

OH

O

I

OOMe

OMe

NMeO

OOHO

MeO

O

OH

HN

O

N

O

O

S

2Mylotarg®

(anti-CD33 calicheamicin conjugate)

NHCO2Me

O N

OHN

ON

O

OMe O

N

OMe

HN

O

OH

NH

OHN

NH

NH2O

ONH

O

N OO

S

1BR96-DOX

(anti-BR96 doxorubicin conjugate)

H

O

NH

O

MeO OH

MeO NCl O O

O

O

N

N

O

OO

HNO

S

4Kadcyla®

(anti-Her2 maytansine conjugate)


FIGURE 2

Structures of antibody–drug conjugates (ADCs).


from the FDA for the treatment of Hodgkin’s (HL) and anaplastic

large-cell lymphomas (ALCL) [22,23]. Most recently, Kadcyla1 (ado-

trastuzumab emtansine, T-DM1, 4), which combines the huma-

nized antibody trastuzumab with a potent antimicrotubule cyto-

toxic agent using a highly stable linker, was approved for the

treatment of patients with Her2-positive breast cancer [24,25]. With

nearly 30 additional ADCs currently in clinical development, the

potential of this new therapeutic class might finally be coming to

fruition [26].

ADC designAlthough simple in concept, the success of a given ADC depends

on careful optimization of each ADC building block: antibody,

drug and linker (Fig. 3) [27]. The chosen antibody should target a

well-characterized antigen with high expression at the tumor site

and low expression on normal tissue to maximize the efficacy of

the ADC while limiting toxicity. Bifunctional linkers with attach-

ment sites for both the antibody and drug are used to join the two

components. With respect to the mAb, existing linker attachment

strategies typically rely on the modification of solvent-accessible

cysteine or lysine residues on the antibody, resulting in hetero-

geneous ADC populations with variable DARs. Given that low drug

loading reduces potency and high drug loading can negatively

impact pharmacokinetics (PK), DARs can have a significant impact

on ADC efficacy. In addition, the linker must remain stable in

systemic circulation to minimize adverse effects, yet rapidly cleave

after the ADC finds its intended target antigen. Upon antigen

recognition and binding, the resulting ADC receptor complex is

internalized through receptor-mediated endocytosis [28]. Once

inside the cell, the drug is released through one of several mechan-

isms, such as hydrolysis or enzymatic cleavage of the linker or via

degradation of the antibody. Typically, the unconjugated drug

should demonstrate high potency, ideally in the picomolar range,

to enable efficient cell killing upon release from the ADC.

Target antigens and antibody selectionAlthough the basic premise that a successful ADC should target a

well-internalized antigen with low normal tissue expression and

high expression on tumors remains true, the field is evolving to

refine these parameters. For example, antigen expression on nor-

mal tissues can be tolerated if expression on vital organs is minimal

or absent. The FDA approval of Kadcyla1 for Her2-positive breast

cancer highlights this point since Her2/neu, a member of the

epidermal growth factor receptor (EGFR) family, is not only

expressed in breast tissue, but also in the skin, heart and on

epithelial cells in the gastrointestinal, respiratory, reproductive

and urinary tracts [29]. In addition, prostate-specific membrane

antigen (PSMA) is an ADC target expressed both on prostate cancer

cells as well as normal prostate and endothelial tissue [30]. Given

that most patients with prostate cancer undergo surgery to remove

their prostate, selective expression relative to normal prostate cells

might not be crucial in this setting. Furthermore, apical expression

of PSMA on the kidney and gastrointestinal tract might prevent

the ADC from accessing these tissues. Other possible exceptions

include hematological malignancies in which normal target tis-

sues are able to regenerate, supported by the case of rituximab

where depletion of normal B cells was not a major safety issue in

patients [31]. Accessibility of the ADC to the target antigen is also

www.drugdiscoverytoday.com 871 5


Attachment site Linker Cytotoxin

Antibody

- Targets a well-characterizedantigen with high tumorexpression and limited normal

tissue expression- Maintains binding, stability,

internalization, PK, etc. when conjugated to a cytotoxin- Minimal nonspecific binding

- Typically through nonspecificmodification of cysteine or

lysine residues on the antibody- Mixture of conjugates with

variable drug:antibody ratios- Site-selective conjugation

technologies can produce more homogeneous ADCs

- Cleavable or noncleavable- Stable in circulation- Selective intracellular

release of drug (e.g. viaenzymatic cleavage or

antibody degradation)

- Highly potent- Non-immunogenic- Amenable to modifications for linker attachment- Defined mechanism of action


FIGURE 3

Key components of an antibody–drug conjugate (ADC). Abbreviation: PK, pharmacokinetics.

TABLE 1

Target antigens for ADCs in preclinical and clinical development

Indication Targets

NHL CD19, CD20, CD21, CD22, CD37, CD70, CD72,

CD79a/b and CD180

HL CD30

AML CD33

MM CD56, CD74, CD138 and endothelin B receptor

Lung CD56, CD326, CRIPTO, FAP, mesothelin, GD2,

5T4 and alpha v beta6

CRC CD74, CD174, CD227 (MUC-1), CD326 (Epcam),

CRIPTO, FAP and ED-B

Pancreatic CD74, CD227 (MUC-1), nectin-4 (ASG-22ME)and alpha v beta6

Breast CD174, GPNMB, CRIPTO, nectin-4 (ASG-22ME)

and LIV1A

Ovarian MUC16 (CA125), TIM-1 (CDX-014) and mesothelin

Melanoma GD2, GPNMB, ED-B, PMEL 17 and endothelinB receptor

Prostate PSMA, STEAP-1 and TENB2

Renal CAIX and TIM-1 (CDX-014)

Mesothelioma Mesothelin

Abbreviations: AML, acute myeloid leukemia; MM, multiple myeloma; CRC, colorectal

cancer.

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an important consideration. In addition to high interstitial pres-

sure in the tumor, endothelial, stromal and epithelial barriers can

limit ADC uptake, resulting in only a small percentage of the

injected dose reaching the intended tumor target [32]. From a

biology perspective, the design of an effective ADC relies on

selection of an appropriate target antigen, taking into account

tumor expression levels, rates of antigen internalization and anti-

body Fc format.

Tumor types

Table 1 highlights the broad range of hematologic and solid tumor

indications targeted by ADCs currently in preclinical or clinical

development. Several of these tumor-associated antigens exhibit

remarkable specificity, such as CD30 for HL and MUC16 for

ovarian cancer. Other antigens, such as CD74, are expressed in

multiple tumor types.

Antigen expression

In general, optimal ADC targets are homogeneously and selec-

tively expressed at high density on the surface of tumor cells.

Homogenous tumor expression, although preferred, is likely not

an absolute requirement owing to the ability of some ADCs to

induce bystander killing. Under these circumstances, a membrane-

permeable free drug liberated after intracellular cleavage of the

linker can efflux from the cell and enter neighboring cells to

facilitate cell death [33]. Most advanced ADCs in the clinic target

hematological indications, in part due to the largely homogeneous

expression of antigen in liquid tumors, despite frequently low

receptor densities. Although the treatment of solid tumors with

heterogeneous antigen expression might benefit from bystander

killing, the potential to harm normal cells could contribute to

systemic toxicity.

Current experimental evidence generally suggests that tumor

antigen density (expression level) does not directly correlate

with ADC efficacy [34]. When patient samples are accessible,

the number of receptors per cell can be quantified using flow


cytometry, immunohistochemistry (IHC) or radiolabeled satura-

tion-binding studies to assess the relation between target expres-

sion and efficacy [35]. In non-Hodgkin’s lymphoma (NHL) cell

lines, high CD79b expression was found to be a prerequisite for in

vitro response to an anti-CD79b auristatin conjugate (RG-7596,

Roche-Genentech); however, a wide range of sensitivities were

observed, indicating that a minimal expression threshold exists

[36]. Likewise, melanoma cells lines with receptor densities vary-

6



ing from 20,000 to 280,000 binding sites per cell were sensitive to

an anti-p97-auristatin conjugate [37]. This threshold level varies

among different targets based on the unique factors of the anti-

gen, such as rate of internalization and binding affinity for the

ADC. For example, approximately 5,000–10,000 copies of CD33,

the antigen target for Mylotarg1, are expressed per cell [38]. As

with Mylotarg1, no significant correlation was observed between

the activity of a preclinical anti-CD33 pyrrolobenzodiazepine

conjugate (SG-CD33A, Seattle Genetics) and CD33 levels in a

panel of acute myeloid leukemia (AML) cell lines [39]. An anti-

PSMA auristatin conjugate (PSMA ADC, Progenics/Seattle Genet-

ics) demonstrated potent in vitro cytotoxicity versus cells expres-

sing >105 PSMA molecules per cell, with 104 receptors per cell

serving as a threshold level [40]. For some tumor antigens, how-

ever, a relatively proportional relation between efficacy and

receptor expression level has been observed. In the case of an

anti-endothelin B receptor (EDNBR) auristatin conjugate,

improved efficacy against human melanoma cells lines and xeno-

graft tumor models generally correlated with increasing EDNBR

expression (1,500–30,000 copies per cell) [41].

Antigen internalization

Ideally, once an ADC binds to a tumor-associated target, the ADC–

antigen complex is internalized in a rapid and efficient manner.

Although poorly understood, various factors are likely to influence

the rate of internalization, such as the epitope on the chosen target

antigen bound by the ADC, the affinity of the ADC–antigen

interaction and the intracellular trafficking pattern of the ADC

complex [42–44]. For example, anti-Her2 antibodies that bind

distinct epitopes on Her2 have been shown to impact downstream

trafficking and lysosomal accumulation differentially, despite

binding to the same cell surface receptor [45]. Several ADCs,

including Adcetris1, have been shown to internalize with rates

similar to or greater than the corresponding unconjugated anti-

bodies [46–48,37]. Certain antigens mediate exceptionally rapid

accumulation of ADCs inside cells. When bound to ligand-acti-

vated EGFR, Her2 monomer is internalized at a rate up to 100-fold

greater than carcinoembryonic antigen (CEA) [49,50]. Likewise,

the catabolic rate of antibodies targeting CD74 is approximately

100 times faster than other antibodies that are considered to

rapidly internalize, such as anti-CD19 and anti-CD22 [51]. The

preclinical data for milatuzumab-DOX (Immu-110), an anti-CD74

doxorubicin conjugate in early clinical trials, suggest this agent is

equipotent to ADCs comprising more potent drug payloads that

target slower internalizing antigens [52].

Alternative approaches have been explored in which antigen

internalization is not required for efficient cell killing. The extra-

domain B (ED-B) of fibronectin is a marker of angiogenesis

undetectable in healthy tissue, but highly expressed around

tumor blood vessels [53]. Anti-ED-B antibodies have been shown

to localize to the subendothelial extracellular matrix of tumor

vasculature. Conjugation of these antibodies with a photosen-

sitizer has led to agents that selectively disrupt tumor blood

vessels upon irradiation, resulting in curative efficacy in mouse

models [54].

Impact of format

The biological activity of an antibody can depend on the interac-

tion of its Fc portion with cells that express Fc receptors (FcRs).

Therefore, selection of the appropriate antibody format for an

ADC is an important consideration. Broad understanding of the

relation between antibody Fc format and ADC function is lacking

since species differences in immune systems complicate preclini-

cal studies. In one study, McDonagh et al. conjugated anti-CD70

antibody immunoglobin G (IgG) variants (IgG1, IgG2 and IgG4) to

an auristatin (ADC toxin monomethyl auristatin F; MMAF) to

determine the effect of format on ADC function [55]. In addition,

the Fc regions of IgG1 and IgG4 were mutated (IgG1v1 and

IgG4v3) to examine the influence of IgG receptor (FcgR) binding.

Although all the ADCs demonstrated potent in vitro cytotoxicity

and were well tolerated in mice, the engineered IgGv1-MMAF

conjugate displayed improved antitumor activity and increased

exposure, which correlated with a superior therapeutic index

compared to the parent IgG1 conjugate.

In the absence of definitive guidelines for selecting an optimal

antibody format, all human IgG isotypes, except for IgG3, are

currently used for ADCs in clinical trials. IgG1, the most com-

monly used format, can potentially engage secondary immune

functions, such as antibody-dependent cellular cytotoxicity

(ADCC) or complement-dependent cytotoxicity (CDC). These

inherent effector functions could prove beneficial by providing

additional antitumor activity, as in the case of Kadcyla1, which

was shown to activate ADCC in preclinical models [56]. Adcetris1,

however, demonstrated minimal ADCC and no detectable CDC

despite its IgG1 format [57]. The absence of effector functions is

potentially advantageous as binding of an ADC to effector cells

could reduce tumor localization, hinder internalization and lead

to off-target toxicity [55]. Unlike IgG1, IgG2 and IgG4 typically

lack Fc-mediated effector functions. Mylotarg1 and inotuzumab

ozogamicin (CMC-544) exhibited no ADCC or CDC in preclinical

studies, consistent with their IgG4 format [58]. Overall, the con-

tribution of IgG effector functions to the efficacy, selectivity and

toxicity of ADCs is not yet well understood.

In addition to effector functions, ADCs often retain other

biological properties associated with their parent mAbs, such

as immunogenicity potential. Limited therapeutic efficacy of

early ADCs comprising murine mAbs prompted the develop-

ment of chimeric and humanized antibodies, which minimize

human immune response. Conversion of murine mAbs to

human IgGs also results in longer retention in systemic circula-

tion due to recognition by the human neonatal Fc receptor

(FcRn) and a greater ability to elicit ADCC [59]. Technologies

for the generation of fully human mAbs include the use of either

phage display or transgenic mouse platforms, in which a mouse

strain is engineered to produce human rather than mouse anti-

bodies [60].

Linker technology and stabilityThe identity and stability of a linker that covalently tethers the

antibody to the cytotoxic drug is crucial to the success of an ADC.

Sufficient linker stability is necessary to enable the conjugate to

circulate in the bloodstream for an extended period of time before

reaching the tumor site without prematurely releasing the free

drug and potentially damaging normal tissue. Once the ADC is

internalized within the tumor, the linker should be labile enough

to efficiently release the active free drug. Linker stability also

influences overall toxicity, PK properties and the therapeutic index

of an ADC. The lack of adequate therapeutic index for earlier



TABLE 2

Examples of ADC drug linkers

Cleavable linkers Noncleavable linkers

Valine-Citrulline (protease sensitive)

CytotoxinN-Maleimidomethylcyclohexane-1-carboxylate (MCC)

Cytotoxin

Hydrazone (acid-sensitive)

Cytotoxin

Maleimidocaproyl

Cytotoxin

Disulfide (glutathione-sensitive)

Cytotoxin Mercaptoacetamidocaproyl

Cytotoxin

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ADCs, such as BR96-DOX and Mylotarg1, has been attributed to

poor linker stability (Fig. 1) [19,61].

The two main classes of ADC drug linkers currently being

explored take advantage of different mechanisms for release of

the drug payload from the antibody (Table 2). The first is a

cleavable linker strategy, with three different types of release

mechanism within this class.

(i) Lysosomal protease sensitive linkers. This strategy utilizes

lysosomal proteases, such as cathepsin B (catB), that

recognize and cleave a dipeptide bond to release the free

drug from the conjugate [62]. Many ADCs in the clinic use a

valine–citrulline dipeptide linker, which was designed to

display an optimal balance between plasma stability and

intracellular protease cleavage [63]. This linker strategy was

successfully utilized by Seattle Genetics/Millennium in the

case of Adcetris1 [64].

(ii) Acid sensitive linkers. This class of linkers takes advantage of

the low pH in the lysosomal compartment to trigger

hydrolysis of an acid labile group within the linker, such

as a hydrazone, and release the drug payload. In preclinical

studies, hydrazone linker-based conjugates have shown

stability (t1/2) ranges from 2 to 3 days in mouse and human

plasma, which may not be optimal for an ADC [65].

Hydrazone linkers were used in Mylotarg1 (anti-CD33

calicheamicin conjugate) and recently in inotuzumab

ozogamicin (anti-CD22 calicheamicin conjugate) [66,67].

The withdrawal of Mylotarg1 from the market was attributed

to toxicities related to hydrazone linker instability, which

resulted in increased fatalities in patients treated with

Mylotarg1 plus chemotherapy as opposed to chemotherapy

alone [65]. Similarly, inotuzumab ozogamicin was recently

withdrawn from a phase III clinical trial owing to a lack of

improvement in overall survival.


(iii) Glutathione sensitive linkers. This strategy exploits the

higher concentration of thiols, such as glutathione, inside

the cell relative to the bloodstream. Disulfide bonds within

the linker are relatively stable in circulation yet are reduced

by intracellular glutathione to release the free drug. To

further increase plasma stability, the disulfide bond can be

flanked with methyl groups that sterically hinder premature

cleavage in the bloodstream [68]. This class of linker has been

used in several clinical candidates, such as SAR3419 (anti-

CD19 maytansine conjugate), IMGN901 (anti-CD56 may-

tansine conjugate) and AVE9633 (anti-CD33 maytansine

conjugate) developed by ImmunoGen and its partners [67].

The second strategy is one that uses noncleavable linkers. This

approach depends on complete degradation of the antibody after

internalization of the ADC, resulting in release of the free drug

with the linker attached to an amino acid residue from the mAb. As

such, noncleavable linker strategies are best applied to payloads

that are capable of exerting their antitumor effect despite being

chemically modified. This type of strategy has been used success-

fully by Genentech/Immunogen with Kadcyla1 (trastuzumab-

MCC-DM1). The released modified payload (lysine-MCC-DM1)

demonstrated similar potency compared with DM1 alone,

although the charged lysine residue is likely to impair cell perme-

ability and hence abate the bystander killing observed with the

free drug [69]. One potential advantage of noncleavable linkers is

their greater stability in circulation compared with cleavable

linkers. However, no significant difference in terminal half-life

(t1/2) values was observed in the clinic between Kadcyla1 [24],

which contains a noncleavable linker, and Adcetris1, which

employs a cleavable linker [22].

Preclinically, linker strategies continue to evolve [70,71].

Additional tumor-associated proteases, such as legumain, have

been identified that release the ADC payload in nonlysosomal

8



compartments (i.e. the endosome) [72]. Other nonprotease

enzymes have recently been exploited for the selective cleavage

of b-glucuronidase and b-galactosidase sensitive linkers in the

lysosome [73,74]. Demonstrating expanded utility, these

approaches enable drug linkage via a phenol functional group

in addition to a more traditional basic amine residue.

Cytotoxic agentsPayload classes and MOAs

There are two main classes of ADC payloads undergoing clinical

evaluation. The first class comprises drugs that disrupt microtu-

bule assembly and play an important role in mitosis. This class

includes cytotoxics, such as dolastatin 10-based auristatin analogs

(3, Adcetris1) [64] and maytansinoids (4, Kadcyla1) [75]. The

second class of payloads consists of compounds that target DNA

structure and includes calicheamicin analogs, such as Mylotarg1

(2), that bind the minor groove of DNA causing DNA double-

strand cleavage [76]. Duocarmycin analogs (MDX-1203, 5) [77]

participate in a sequence-selective alkylation of adenine-N3 in the

minor groove of DNA to induce apoptotic cell death (Fig. 4).

One common feature among these cytotoxic agents is that they

demonstrate at least 100–1000-fold greater potency in in vitro

NH

NONN

OHN

HN

NH

HN

O

O

HN

NH2O

OO

5Duocarmycin analog

Cl

NHO

HN

ONHO

N

O

O

S

7α-Amanitin

NHO

HN

O

HN

O

NH

O

O

HN

HNO

HN

O

NH

H

OH2N

HOH

O

NH

SO OH

O

OO

O

S

O

FIGURE 4

Representative antibody–drug conjugate (ADC) payload structures.

proliferation assays against a broad range of tumor cell lines

compared with conventional chemotherapeutic agents, such as

paclitaxel and doxorubicin [78,79]. The high potency of these

alternative payloads is crucial since only an estimated 1–2% of the

administered ADC dose will ultimately reach the tumor site,

resulting in low intracellular drug concentrations [80]. Unlike

earlier ADCs that failed to make a meaningful impact in the clinic

owing to low drug potency and suboptimal delivery, newer, more

potent cytotoxic compounds are now the focus of preclinical

research. For example, pyrrolobenzodiazepine (PBD) dimers 6

covalently bind the minor groove of DNA, resulting in a lethal

interaction due to cross-linking of opposing strands of DNA [81].

a-Amanitin 7, a cyclic octapeptide found in several species of the

Amanita genus of mushrooms, strongly inhibits RNA polymerase

II, leading to inhibition of DNA transcription and cell death [82].

Tubulysins 8, similar to auristatins and maytansine, inhibit tubu-

lin polymerization to induce apoptosis [83–85].

Addressing drug resistance

In addition to potency, the sensitivity of cytotoxic agents to

multidrug resistance (MDR) mechanisms is a factor to consider

in selecting the optimal payload for an ADC. Cancer cells have the

ability to become resistant to multiple drugs via increased efflux of

NH

ON

O

O

S

O

OMe

N

N

O

H

MeO N

N

O

HO

OMe

N

HN

ON

O O

S

N

HN

O

CO2H

O

8Tubulysin analog

6PBD

ON

O




Cysteine specific

Unnatural aminoacid insertion

Enzyme-assistedligation

(a) (b)

Transglutaminase SortaseFormylglycine-generating enzyme(chemoenzymatic)

Lysine

Cysteine,S-S reduction


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).

Review

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EYNOTEREVIEW

the drug by either P-glycoprotein (Pgp) or other multidrug-resis-

tance proteins (e.g. MRP1 and MRP3) [86]. The sensitivity of

cytotoxic drugs to MDR mechanisms can be measured in vitro.

In the case of Mylotarg1, in vitro cytotoxicity assays in AML cell

lines indicated that Pgp expression altered the potency of the

calicheamicin payload and that drug potency could be restored

by adding known efflux transporter antagonists to inhibit Pgp and

MRP-1 proteins [87]. These results were relevant for patients with

AML as levels of Pgp expression in the clinic were found to

correlate directly with responders and nonresponders [88,89].

Another interesting example related to MDR mechanisms

involves AVE9633, which comprises an anti-CD33 antibody linked

through a disulfide bond to the maytansine analog DM4. In vitro

data clearly demonstrated that the cytotoxicity of AVE9633 and

the DM4 free drug were highly dependent on the expression level

of Pgp protein in myeloid cell lines [90]. As with the calicheamicin

payload of Mylotarg1, the potency of DM4 could be restored in

Pgp-overexpressing cell lines by adding known inhibitors of Pgp.

However, Pgp activity was not found to be a major mechanism of

resistance for the AVE9633 conjugate in cells from patients with

AML. Reasons for the lack of correlation are unclear; other

mechanisms such as microtubule alteration were proposed for

chemoresistance to AVE9633.

Conjugation strategiesFor most ADCs in clinical development, conjugation of the drug

payload to the antibody involves a controlled chemical reaction

with specific amino acid residues exposed on the surface of the

mAb. Given that this process results in a mixture of ADC species

with variable DARs and linkage sites, alternative conjugation

strategies aimed at minimizing heterogeneity have been devel-

oped. In the overall design of an ADC, selection of the appropriate


drug-conjugation strategy significantly impacts efficacy, PK and

tolerability. As such, careful consideration of the various conjuga-

tion technologies for ADC generation is warranted (Fig. 5).

Chemical conjugation

In one type of chemical conjugation, a reactive moiety pendant to

the drug–linker is covalently joined to the antibody via an amino

acid residue side chain, commonly the e-amine of lysine. As

demonstrated with Mylotarg1, direct conjugation of lysine resi-

dues on gemtuzumab was achieved using an N-hydroxysuccini-

mide (NHS) ester appended to the drug–linker to form stable amide

bonds [91]. A two-step process can also be used in which surface

lysines on the antibody are first modified to introduce a reactive

group, such as a maleimide, and then conjugated to the drug–

linker containing an appropriate reactive handle (e.g. a thiol) [92].

Such a strategy was utilized in the case of Kadcyla1. Alternatively,

controlled reduction of existing disulfide bonds can liberate free

cysteine residues on the antibody, which then react with a mal-

eimide attached to the drug–linker. This approach, used in the

preparation of Adcetris1, takes advantage of the reducible disul-

fide bonds of IgG antibodies in which controlled conditions

enable reduction of only interchain disulfide bonds while intra-

chain disulfides remain unaffected, thus minimizing major struc-

tural disruptions to the antibody [19].

The random conjugation processes described above produce

heterogeneous mixtures of conjugated species with variable

DARs. Adding to the complexity, the site of conjugation could

be different for each ADC species containing even only one drug.

When lysines are used for conjugation, heterogeneity in overall

charge can impact solubility, stability and PK [93]. Therefore, the

clinical success of an ADC produced by random conjugation

depends on robust manufacturing processes that provide

the ability to monitor, control and purify the heterogeneous

10



products. Several organizations have developed expertise in this

area to overcome the process development and manufacturing

challenges associated with ADC commercialization [94].

Site-specific conjugation

Despite the success of Adcetris1 and Kadcyla1, considerable

enthusiasm for the next generation of ADCs has focused on the

development of homogeneous products derived via site-specific

conjugation. Currently, three strategies are at the forefront: inser-

tion of cysteine residues in the antibody sequence by mutation or

insertion, insertion of an unnatural amino acid with a bio-ortho-

gonal reactive handle, and enzymatic conjugation.

Building on early studies that explored the introduction of

surface cysteines on recombinant antibodies [95], several cysteine

engineered antibodies have been produced and tested for use in

site-specific attachment of cytotoxic drugs to yield homogeneous

ADCs [96]. Junutula et al. reported a class of THIOMAB-drug

conjugates (TDCs) prepared by taking advantage of: (i) phage

display techniques to identify ideal sites for mutation and pro-

duce antibodies with minimal aggregation issues, and (ii) meth-

ods to reduce and re-oxidize the antibody under mild conditions

to present only thiols of mutated cysteines for conjugation

[92,97]. Compared with a conventional, randomly conjugated

ADC, the analogous TDC displayed minimal heterogeneity with

similar in vivo activity, improved PK and a superior therapeutic

index. Moreover, McDonagh et al. engineered antibodies in

which interchain cysteines were replaced with serines to reduce

the number of potential conjugation sites, yielding ADCs with

defined DARs (two or four drugs per antibody) and attachment

sites [98]. Broad application of this approach to future ADCs will

depend on further studies to evaluate the effect of these mutations

on the overall stability and biological function of the engineered

antibody.

Encouraged by studies with cysteine engineered antibodies,

several investigators reasoned that the site and stoichiometry of

conjugation could be controlled by inserting unnatural amino

acids with orthogonal reactivity relative to the 20 natural amino

acids. Axup et al. genetically engineered an orthogonal amber

suppressor tRNA/aminoacyl-tRNA synthetase pair to insert site-

specifically p-acetylphenylalanine (pAcPhe) in recombinantly

expressed antibodies [99]. As a test case, pAcPhe was introduced

at one of several positions in the constant region of trastuzumab

(anti-Her2). These mutants were then conjugated to an alkoxy-

amine auristatin derivative via formation of a stable oxime bond.

The resulting chemically homogeneous ADCs demonstrated

improved PK compared with nonspecifically conjugated ADCs

and were highly efficacious in a Her2-positive human tumor

xenograft model. In addition to pAcPhe, other unnatural amino

acids are being explored through the use of appropriate tRNA–

aminoacyl-tRNA synthetase pairs [100]. Recently, in vitro transcrip-

tion and translation processes have also been developed and

optimized to insert unnatural amino acids in antibodies for site-

specific conjugation [101].

In addition to inserting unnatural amino acids into mAb

sequences, chemoenzymatic approaches have been explored to

generate bio-orthogonal reactive groups for selective conjugation.

Bertozzi and co-workers utilized formylglycine-generating enzyme

(FGE), which recognizes a CXPXR sequence and converts a

cysteine residue to formylglycine to produce antibodies with

aldehyde tags [102,103]. The reactive aldehyde functionality

can then undergo conjugation to the drug–linker via oxime

chemistry or a Pictet–Spengler reaction [104].

Harnessing enzymatic post-translational modification processes

for site-specific labeling of proteins is a recently reviewed approach

for the preparation of homogenous ADCs [105]. Bacterial trans-

glutaminase (BTG) catalyzes the ligation of glutamine side chains

with the primary e-amine of lysine residues, resulting in a stable

isopeptide bond. Jegar et al. exploited BTG to load four chelates on

a deglycosylated antibody with an N297Q mutation in a site-

specific manner [106]. Recently, Strop et al. conducted BTG-

assisted conjugations by inserting LLQG sequences at different

sites on an antibody [107]. These studies clearly demonstrated that

the site of conjugation has a significant impact on the stability and

PK of the ADC. Another enzyme, sortase A (SrtA), catalyzes hydro-

lysis of the threonine–glycine bond in a LPXTG motif to form a

new peptide bond between the exposed C-terminus of threonine

and an N-terminal glycine motif [108].

Next-generation ADCsKey clinical assetsThe nearly 30 ADCs currently in clinical development have been

reviewed in detail elsewhere [109], and representative examples of

the most advanced agents are summarized in Table 3. In addition

to the FDA-approved ADCs discussed in preceding sections, several

compounds are in late-stage clinical testing for both hematological

and solid tumor indications. Despite the withdrawal of Mylotarg1

from the market in 2010, promising results from ongoing clinical

studies have shown that when combined with chemotherapy

Mylotarg1 increased overall survival in patients with newly diag-

nosed AML compared to those treated with chemotherapy alone

[110]. Inotuzumab ozogamicin, which uses the same calicheami-

cin payload and cleavable hydrazone linker found in Mylotarg1,

recently failed to demonstrate improved survival in a phase III

study for patients with refractory aggressive NHL (Pfizer Inc. press

release; May 20, 2013). No unexpected safety concerns were iden-

tified, however, and phase III studies continue for acute lympho-

blastic leukemia (ALL) patients.

The vast majority of remaining ADCs in clinical development

use either auristatin [monomethyl auristatin E (MMAE) or MMAF]

or maytansinoid (DM1 or DM4) payloads, both potent inhibitors

of tubulin polymerization. Several MMAE conjugates with clea-

vable linkers are currently under evaluation in phase II studies for

various indications based on the target antigen. In general, these

agents were well tolerated in phase I trials with toxicities consis-

tent with the known mechanism of action for the auristatins (e.g.

neutropenia or neuropathy) [22,111]. SAR3419, an anti-CD19

DM4 conjugate with a cleavable disulfide linker, demonstrated a

dose-limiting toxicity (DLT) of reversible severely blurred vision in

a phase I study for refractory B cell NHL, but was well tolerated on a

modified dosing schedule [112]. Recently advanced to phase II

studies for colorectal cancer (CRC), labetuzumab-SN-38 employs a

cathepsin B-cleavable dipeptide linker and SN-38, the active meta-

bolite of the clinically used anticancer agent irinotecan, as a

payload. Initial phase I data indicated that labetuzumab-SN-38

was generally safe and well tolerated at effective clinical doses

[113]. Lorvotuzumab mertansine utilizes a maytansinoid payload

(DM1) and a disulfide linker to target CD56. No serious DLTs or


http://press.pfizer.com/press-release/pfizer-discontinues-phase-3-study-inotuzumab-ozogamicin-relapsed-or-refractory-aggress

http://press.pfizer.com/press-release/pfizer-discontinues-phase-3-study-inotuzumab-ozogamicin-relapsed-or-refractory-aggress


TABLE 3

Representative ADCs undergoing clinical evaluationa

Agent Sponsor (licensee) Status Indication Antigen Cytotoxin Linker

AdcetrisW (brentuximab

vedotin, SGN-35)

Seattle Genetics

(Millennium)

Launched HL, ALCL CD30 MMAE Cleavable, Val-Cit

KadcylaW (ado-trastuzumab

emtansine, T-DM1)

Roche-Genentech

(ImmunoGen)

Launched Her2+ metastatic

breast cancer

HER2 DM1 Non-cleavable, thioether

MylotargW (gemtuzumabozogamicin)

Pfizer (UCB) Withdrawn AML CD33 Calicheamicin Cleavable, hydrazone(Ac-But acid)

Inotuzumab ozogamicin

(CMC-544)

Pfizer (UCB) Ph III ALL, NHL CD22 Calicheamicin Cleavable, hydrazone

(Ac-But acid)

RG-7596 Roche-Genentech Ph II DLBCL, NHL CD79b MMAE Cleavable, Val-Cit

Glembatumumabvedotin CDX-011)

Celldex (SeattleGenetics)

Ph II Advanced breastcancer, melanoma

GPNMB MMAE Cleavable, Val-Cit

PSMA-ADC Progenics (Seattle

Genetics)

Ph II HRPC PSMA MMAE Cleavable, Val-Cit

SAR3419 Sanofi (ImmunoGen) Ph II Hematologic tumors CD19 DM4 Cleavable, disulfide

Labetuzumab-SN-38(IMUU-130)

Immunomedics Ph II Metastatic CRC CEACAM5 SN-38 Cleavable, Phe-Lys

Lorvotuzumab mertansine

(IMGN901)

ImmunoGen Ph I/II MM, solid tumors CD56 DM1 Cleavable, disulfide

Milatuzumab-DOX

(IMMU-110)

Immunomedics Ph I/II MM CD74 Doxorubicin Cleavable, hydrazone

BT-062 Biotest AG(ImmunoGen)

Ph I MM CD138 DM4 Cleavable, disulfide

BAY-94-9343 Bayer Schering

(ImmunoGen)

Ph I Solid tumors Mesothelin DM4 Cleavable, disulfide

ASG-5ME Astellas (SeattleGenetics)

Ph I Solid tumors AGS-5 MMAE Cleavable, Val-Cit

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.

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EYNOTEREVIEW

drug-related adverse events were reported in early-phase multiple

myeloma (MM) studies [114].

Although the modest potency of doxorubicin payloads limited

the efficacy of early ADCs (BR96-DOX), milatuzumab-DOX targets

CD74, an antigen with unique internalization and surface re-

expression, and is currently in phase I/II trials based on encoura-

ging preclinical efficacy in hematopoietic cancer xenograft models

[52]. Select agents in phase I trials include ADCs containing DM1

or DM4 cytotoxic drugs under evaluation by ImmunoGen, and

several ADCs with MMAE or MMAF developed by Seattle Genetics,

each targeting a different antigen across a variety of tumor indica-

tions. Available data for these and other phase I agents generally

provide initial evidence of efficacy and tolerability. Similar to

SAR3419 (anti-CD19 DM4 conjugate), the DM4-based anti-

mesothelin conjugate BAY-94-9343 has also been reported to

induce Grade 2 and 4 ocular toxicity [115].

ADC PKADCs typically retain the PK properties of the antibody compo-

nent, as opposed to the appended drug, and thus exhibit relatively

low clearance and long half lives. Compared with the unconju-

gated antibody, ADCs can exhibit somewhat higher clearance due


to introduction of an additional metabolic pathway (i.e. cleavage

of the drug from the antibody). In addition, ADCs with higher

DARs tend to clear faster than those with lower DARs [116].

Variable DARs and attachment sites, a consequence of current

random conjugation methods, result in heterogeneous ADCs with

PK parameters that can vary substantially compared to the uncon-

jugated antibody [117]. Each ADC component, along with their

respective metabolites, can potentially impact efficacy, safety and

tolerability [118]. Both the type of linker used and the site of

conjugation can influence the extent to which the drug is prema-

turely released from the antibody. Deconjugation of the payload

from the antibody can result in ADCs with lower DARs, reduced

efficacy and potentially increased toxicity owing to release of a

highly potent cytotoxic drug in systemic circulation.

The PK parameters of Adcetris1 and Kadcyla1 were evaluated in

mouse, rat and monkey in preclinical toxicity studies. Overall, these

ADCs demonstrated similar PK properties, albeit with a few differ-

ences in mouse and monkey. The t1/2 of Adcetris1 in mouse, rat and

monkey was 14, 10 and 2 days, respectively. The rapid clearance of

Adcetris1 in monkeys as compared with mouse or rat was hypothe-

sized to result from nontherapeutic antibodies, target-mediated

disposition and other factors [119]. In the case of Kadcyla1, the

12



t1/2 in mouse, rat and monkey was 5.7, 8.3 and 11.6 days, respec-

tively [120,121]. In humans, the PK characteristics of these two

conjugates were similar, with a t1/2 ranging from 3.5 to 5 days

[22,24]. Of note, the t1/2 of an ADC is often significantly shorter

in humans compared with other species. The optimal t1/2 for an ADC

remains to be determined, but the clinical success of Adcetris1 and

Kadcyla1 indicate that the range of 3–4 days is appropriate.

The long t1/2 typical of ADCs and mAbs results from FcRn

recycling [122]. In this process, antigen-independent internaliza-

tion by endothelial cells is followed by FcRn binding and then

FcRn-mediated return to the bloodstream. FcRn recycling essen-

tially protects ADCs from catabolism; however, diversion of FcRn-

bound ADCs to the lysosome can increase the risk of off-target

toxicities. Although the factors that influence this process are

poorly understood, the drug, linker, antibody and antigen can

each affect FcRn-mediated ADC trafficking [123]. Another

mechanism of off-target toxicity involves soluble cell-surface man-

nose receptors (MRs), which interact with agalactosylated glycans

on the antibody Fc domain [124]. Cell-surface MRs can internalize,

effectively delivering the ADC to the endosome and lysosome

compartments where the potent cytotoxic drug is released. Impor-

tantly, locations of off-target ADC activities reportedly coincide

with cell-surface MR locations. The shedding of antigen from the

tumor cell surface into circulation may also increase the risk of

toxicity. Binding of an ADC to shed antigen can, in some cases,

lead to higher ADC clearance and impaired tumor localization as

well as immune complex formation and accumulation in the

kidney [125].

To determine the effect of linker stability on PK and efficacy, the

noncleavable thioether linker of Kadcyla1 was compared to the

cleavable disulfide linker of a T-SPP-DM1 conjugate [121]. The

nonreducible thioether-linked Kadcyla1 demonstrated superior

PK with greater plasma exposure (area under the curve) and

increased maytansinoid tumor concentration. The disulfide-

linked ADC demonstrated higher plasma clearance owing to the

presence of the metabolically labile linker. Despite the difference

in PK, both conjugates had similar in vivo efficacy. It was hypothe-

sized that the drug released from the disulfide-linked T-SPP-DM1

conjugate would benefit from the bystander killing effect, whereas

Kadcyla1 ultimately liberates a maytansinoid appended to a

charged lysine residue, which limits diffusion to neighboring

tumor cells. Taken together, these results illustrate how minor

structural changes can profoundly impact ADC PK and efficacy.

In addition to the type of linker used to join the drug and

antibody, the conjugation site on the antibody has been shown to

influence stability and, therefore, PK. A recent study examined the

stability of MMAE conjugated to Her2 via a maleimide at various

site-specifically engineered cysteines [126]. Highly solvent acces-

sible conjugation sites were found to be labile, undergoing mal-

eimide exchange with reactive thiols in the plasma, such as

glutathione, albumin or free cysteine. At less accessible sites,

the succinimide ring of the linker underwent hydrolysis, which

served to protect the linker from maleimide exchange and resulted

in enhanced stability and efficacy. In a separate study, the stability

of monomethyl auristatin D (MMAD) conjugated to an anti-M1S1

antibody was examined using BTG to introduce the drug payload

site specifically at either the heavy or light chain [107]. The

conjugation site was found to influence stability and PK, with

ADCs appended to the heavy chain demonstrating a higher rate of

drug loss in rats via proteolysis of the valine–citrulline linker.

Interestingly, these results were species specific since both con-

jugates demonstrated comparable stability in mice, which also

serves to highlight the potential pitfall of performing safety and

efficacy studies in different species.

Concluding remarks and future directionsDespite complexities in designing ADCs, the promise of this

therapeutic class has generated intense interest for decades. A

robust clinical pipeline and the recent FDA approvals of Adcetris1

and Kadcyla1 suggest that the potential benefit of ADCs may

finally be realized. Evolving clinical data will continue to drive

technological advancements in the field. Current methods for

preclinical lead selection typically rely on systematic in vitro

evaluation of a matrix of various mAbs, linkers and cytotoxic

payloads. Whether in vitro models are sufficient to predict response

remains to be seen; until further understanding of ADCs is rea-

lized, early in vivo studies might be crucial. Progress in site-specific

conjugation modalities, optimization of linkers with balanced

stability and identification of novel, potent cytotoxic agents

should pave the way for greater insight into the contribution of

these various factors to ADC efficacy, PK and safety. Challenges in

target tumor selection will be addressed as the roles of antigen

expression, heterogeneity and internalization rate are further

elucidated. Guiding principles for the selection of an ideal anti-

body Fc format are, as of yet, lacking and prompt validation of

current assumptions regarding antibody-dependent properties,

such as specificity and immune effector functions. Ongoing efforts

to address these issues will continue to broaden the impact of

ADCs as targeted therapeutics for the treatment of cancer and

potentially other diseases.

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An emerging playbook for antibody–drug conjugates:lessons from the laboratory and clinic suggest astrategy for improving efficacy and safetyPenelope M Drake and David Rabuka

Available online at www.sciencedirect.com

ScienceDirect

Antibody–drug conjugates (ADCs) have become de rigueur for

pharmaceutical oncology drug development pipelines. There

are more than 40 ADCs undergoing clinical trials and many more

in preclinical development. The field has rushed to follow the initial

successes of KadcylaTM and AdcetrisTM, and moved forward with

new targets without much pause for optimization. In some

respects, the ADC space has become divided into the clinical

realm — where the proven technologies continue to represent the

bulk of clinical candidates with a few exceptions — and the

research realm — where innovations in conjugation chemistry

and linker technologies have suggested that there is much room

for improvement in the conventional methods. Now, two and four

years after the approvals of KadcylaTM and AdcetrisTM,

respectively, consensus may at last be building that these two

drugs rely on rather unique target antigens that enable their

success. It is becoming increasingly clear that future target

antigens will require additional innovative approaches. Next-

generation ADCs have begun to move out of the lab and into the

clinic, where there is a pressing need for continued innovation to

overcome the twin challenges of safety and efficacy.

Address

Catalent Pharma Solutions, Emeryville, CA 94608, USA

Corresponding author: Rabuka, David ([email protected])

Current Opinion in Chemical Biology 2015, 28:174–180

This review comes from a themed issue on Synthetic biomolecules

Edited by Christian Hackenberger and Peng Chen

http://dx.doi.org/10.1016/j.cbpa.2015.08.005

1367-5931/# 2015 Elsevier Ltd. All rights reserved.

IntroductionAntibody–drug conjugates (ADCs) are a class of drugs

that have long been heralded as the answer to nonspecific

toxicity induced by systemically-dosed chemotherapies.

As their name implies, ADCs comprise a small molecule

cytotoxic drug conjugated through a linker to an antibody

that directs the ADC to tumor cells expressing the cog-

nate antigen (Figure 1a). Upon internalization by the cell,

the ADC is degraded and the cytotoxic payload is re-

leased and diffuses into the cytosol or nucleus where it


binds to its targets and induces cell death (Figure 1b). In

theory, the targeting feature should endow ADCs with an

impressive therapeutic index — the difference between

the therapeutic and the toxic dose — and thus should

enable effective tumor eradication with minimal side

effects. This promise is now being tested by more than

40 molecules currently in clinical trials [1]. Two ADCs,

KadcylaTM and AdcetrisTM, are approved by the FDA for

treatment of HER2+ metastatic breast cancer and Hodg-

kin lymphoma or anaplastic large-cell lymphoma, respec-

tively. A third ADC, MylotargTM, approved to treat acute

myeloid leukemia, was voluntarily pulled from the mar-

ket after failure of the required post-approval study [2]. As

of early 2015, the majority of ADCs in clinical trials are in

Phase I studies [3]. The most significant hurdle for the

field will be to demonstrate that the ADC approach can be

broadly applied to generate drugs that are safe at effective

dose levels. Although promising results have been

reported for isolated therapies — such as the recently

released Phase I results from ImmunoGen’s anti-folate

receptor alpha-DM4 conjugate for the treatment of ovari-

an cancer [4] — the overall picture has yet to emerge. For

a summary of the clinical ADC pipeline, we refer readers

to the excellent review by Wang and Watanabe [1]. What

we do know is that the current iteration of ADCs has

limitations, including the targeting efficiency. It is esti-

mated that a very low percentage of the injected conju-

gate reaches its tumor target [5]. The remaining ADC is

eliminated predominantly by the hepatobiliary system

[6], inducing toxic side effects in the process. By boosting

the percentage of dosed ADC that reaches the intended

target, efficacy and safety can be increased in parallel.

These challenges are beginning to be addressed on a

number of fronts (Figure 2).

Current ADC technologies and associatedchallengesDue to poor targeting efficiency, an ADC’s cytotoxic

payload must be sufficiently potent to induce target cell

death at low doses; subnanomolar activity is desired.

Only a handful of cytotoxic payloads are represented

on ADCs in the clinic, including the microtubule-bind-

ing auristatins and maytansinoids, and the DNA-target-

ing natural products pyrrolobenzodiazepine (PBD) and

calicheamicin [3,7]. Amanatin, an RNA polymerase II

inhibitor, is being evaluated in preclinical studies [8��].The linker joining the payload to the antibody may be

either cleavable — designed to be stable in the circulation

www.sciencedirect.com

16

http://crossmark.crossref.org/dialog/?doi=10.1016/j.cbpa.2015.08.005&domain=pdf


http://dx.doi.org/10.1016/j.cbpa.2015.08.005

http://www.sciencedirect.com/science/journal/13675931

An emerging playbook for antibody–drug conjugates Drake and Rabuka 175

Figure 1

(a)

(b)

Current Opinion in Chemical Biology

An idealized ADC acts as a ‘magic bullet’ directing the cytotoxic

payload to tumors. (a) The ADC binds to cognate antigen on the

tumor cell surface and is internalized. (b) The ADC traffics through the

endosomes to the lysosome, where it is degraded into component

pieces. The cytotoxic payload (star) diffuses through cell membranes

to reach its targets in the cytoplasm or nucleus.

but readily degraded after internalization by the target

cell — or noncleavable — designed to persist through an-

tibody degradation in the lysosome [9,10]. The current

generation of ADCs, which constitute nearly all of the

Figure 2

Conjugate structural features Effects on

Conjugationchemistry

Linkerchemistry

Cytotoxicpayload

Antibodyselectivityfor tumor

Tunable ADC structural characte

106

105

104

103

ng/m

L

Total ATotal A

0 100

0.0001 0.01 1 Drug (n

100

80

60

40

20

0

viab

ility

(%

)

ADC structural features that directly impact function can be compared throu

configuration for safety and efficacy. (left panel) Tunable features include lin

modifications that limit target-mediated or FcgR-mediated drug disposition

impact critical pharmacokinetic (PK) and pharmacodynamic (PD) attributes

PD largely governs treatment outcomes such as safety and efficacy. The ho

thereby offer patients excellent anti-tumor efficacy with minimal side effects


compounds in the clinic, are characterized by heteroge-

neous payload conjugation to lysine or cysteine residues.

The random nature of both conjugation approaches yields a

mixture of ADC species with drug-to-antibody ratios

(DARs) ranging from 0 to 8. The desired average DAR

is usually �3.5; this mixture includes some unconjugated

(and thus, inactive) antibody and some highly conjugated

antibody. The more highly-conjugated species appear to

be cleared faster by the liver [11,12�], likely due to their

increased hydrophobicity; in turn, this faster clearance

reduces efficacy and probably increases toxicity. Accord-

ingly, the heterogeneous mixtures found in conventional

ADCs include both inactive and more toxic isoforms, both

of which reduce the therapeutic index of the resulting

construct.

Clinical studies indicate that the drug-linker,not the target antigen, is driving toxicityA fascinating recent report by the FDA analyzed the

relationship between nonclinical study results and Phase

I study outcomes by looking at data from investigational

new drug applications filed for 20 ADCs from December

2012 to August 2013 [13��]. The authors found that ADCs

bearing the same drug-linker but with distinct target

antigens showed similar toxicity results, including the

PK/PD Treatment outcomes

Safety and efficacycharacteristics

ristics change functional readouts

Minimumefficacious dose

Therapeuticindex

Toxic dose

Dru

g do

se

ntibodyDC

200 300 400 500Hours post-dose

100 10000

M)

Dru

g do

se

Current Opinion in Chemical Biology

gh structure-activity relationship mapping to determine the optimal

ker and conjugation chemistries, cytotoxic payload selection, and

in non-tumor tissues. (middle panel) The structural features can directly

such as ADC distribution, half-life, and potency. (right panel) The PK/

pe for ADCs is that they will have a wide therapeutic index and

.


17

176 Synthetic biomolecules

affected organs and the maximum tolerated dose (MTD)

levels. The target antigen, even when the epitope was

present (e.g., in non-human primate studies) had no effect

on either the target organs affected or on the MTD. In fact,

the FDA concluded that the main driver of toxicity was the

drug-linker component. These findings strongly support

the observations that only a low percentage of an adminis-

tered antibody finds its way to the tumor (or indeed to any

target-expressing tissues), and that toxicity is primarily

driven by non-target mediated disposition. The vast major-

ity (�80%) of the injected dose is eliminated within the first

7 days through excretion in the bile and feces [6] — leading

to predictable and dose-limiting toxicities. Finding ways to

mitigate this non-specific elimination will be key to im-

proving the safety and efficacy of next-generation ADCs.

The relationship between linker chemistry,ADC stability, and conjugate efficacyIt may seem that improving the in vivo stability of the

conjugate would increase the proportion of payload that

reaches its target — and thus, improve efficacy. This

trend generally holds true, but there are a number of

factors that enter the equation [3,9,14]. First, there are

target-dependent requirements for linker-payloads,

which are likely related to cell surface expression levels

as well as internalization and recycling kinetics. Polson

et al. performed a systematic study comparing the

effects of a cleavable (SPP-DM1) and a noncleavable

(SMCC-DM1) linker carrying the maytansinoid pay-

load, DM1 [15]. Antibodies against a panel of seven

potential non-Hodgkin lymphoma antigens were con-

jugated to these linkers and tested for in vivo efficacy.

All of the ADCs with cleavable linkers showed efficacy,

while ADCs with noncleavable linkers were active

against only two of the seven target antigens. In addition

to revealing a relationship between target antigen, link-

er design, and efficacy, these data also highlight the

general importance of target antigen selection during

ADC development, a topic worthy of its own review

article [16��,17,18].

Second, there is a positive correlation between ADC

exposure and efficacy, such that ADCs with longer circu-

lating half-lives tend to show better efficacy if other

parameters (e.g., target antigen and cytotoxic payload)

are held equal. ADC half-lives are determined by at least

two components: linker stability and total antibody half-

life [19]. Conjugated antibodies often display shorter half-

lives as compared to their unconjugated counterparts

[12�,20,21]; evidence suggests that hydrophobicity is

the major driver of this increased clearance [11,22�]. In

cases where the total antibody half-life is relatively short,

Alley et al. showed that improving linker stability only

minimally improves the overall ADC exposure, and thus

has little effect on efficacy [23]. However, when the total

antibody half-life is relatively long, improving linker

stability can lead to better efficacy. Examples of this


effect came from both Pfizer and Seattle Genetics, who

took different approaches to efficiently hydrolyze malei-

mide-containing linkers for improved ADC stability; both

groups found that the new technologies led to longer

circulating ADC half-lives, longer overall exposure to the

conjugate, and improved efficacy against xenograft mod-

els [19,24].

Third, linker design can impact both active and passive

cell permeability of the ADC’s active metabolite. With

respect to active permeability, linker-payload suscepti-

bility to efflux by multidrug transporters (e.g., P-glyco-

protein/MDR1) relates to efficacy against multidrug

resistant cells. Namely, if other variables are held con-

stant, then linker-cytotoxins that are targets for MDR1-

mediated efflux have worse efficacy relative to those that

are not. Linker design can be exploited to reduce binding

of the cytotoxic payload to MDR1, and thus rescue ADC

efficacy in multidrug resistant cells. In general, linkers

that impart hydrophilicity, charge, or steric bulk show

reduced MDR1 binding, as the transporter prefers hydro-

phobic, weakly cationic, planar substrates [9,25,26]. With

respect to passive cell permeability, in vivo potency

against solid tumors is governed partially by the bystander

killing effect, where non-targeted (perhaps target anti-

gen-negative) cells are killed by cytotoxic payloads that

cross plasma membranes. Kellogg et al. observed that a

linker with intermediate plasma stability gave the best invivo efficacy results, in spite of having lower overall

exposure as compared to a noncleavable ADC bearing

the same payload [27]. The authors postulated that by-

stander killing, a trait that the noncleavable linker lacked,

contributed additional potency in vivo. Notably, the most

labile linker, which also could mediate bystander killing,

was cleared rapidly from the circulation and showed

relatively poor efficacy. Therefore, the linker that pro-

vided a balance of in vivo potency and exposure yielded

the best overall effect.

Finally, a number of groups have noted that the in vivostability of a given linker can vary depending upon the

model species [28–30], often with worse stability in

rodents and improved stability in primates. This likely

reflects increased enzymatic (e.g., protease or esterase)

activity in rodent plasma, leading to linker degradation.

The disparity in stability among species can make it

challenging to develop meaningful efficacy studies, which

are usually performed in immunocompromised rodent

models. In summary, there is not one best way to build

a linker for efficacy; rather the linker should be consid-

ered a tunable ADC feature that can be optimized for a

given target and payload.

The connection between linker stability, ADCpharmacokinetics, and conjugate tolerabilityThe relationship between ADC stability and safety is

similarly nuanced. Toxicity can be mediated both by


18


deconjugated cytotoxic payloads and by intact ADCs

that — through off-target effects — damage healthy

cells. The majority of deconjugated small molecules

are metabolically inactivated in the liver and cleared

through the biliary/fecal route. ADCs that are over-

conjugated with a hydrophobic payload can also be

cleared by the liver. Conventional, heterogeneous con-

jugates suffer from this issue, which can be alleviated by

using site-specific conjugation (discussed below) or by

increasing the hydrophilicity of the linker-payload

[11,22�]. A less often discussed avenue for toxicity is

through ADC interactions with Fcg receptors (FcgRs)

that are expressed on a variety of hematopoietic cells. A

recent study by Uppal et al. assessed the role of FcgIIRa

as a potential mechanism for thrombocytopenia follow-

ing treatment with T-DM1 (KadcylaTM) [31��]. The

authors found that the ADC inhibited a crucial step in

platelet development by exhibiting an FcgIIRa-depen-

dent cytotoxic effect against differentiating megakar-

yocytes. The data are particularly compelling because

thrombocytopenia is the dose-limiting toxicity associ-

ated with the clinical use of KadcylaTM. The recent

FDA analysis concluded that antibody isotype had no

impact on an ADC’s MTD or safety profile, suggesting

that immune-mediated effector functions (e.g., anti-

body-dependent cell-mediated cytotoxicity) were not

a major factor. However, the study did not rule out

FcgR-dependent uptake into hematopoietic cells as a

toxicity mechanism. Interestingly, overly conjugated/

hydrophobic ADCs may be at particular risk for this

clearance route, as low-affinity FcgRs (which bind to all

IgG isotypes) can only bind to aggregated or opsonized

antibody [32].

In animal models, if ADC hydrophobicity is held con-

stant, then ADCs with more stable linkers tend to show

improved tolerability. For example, in both the previ-

ously discussed linker studies [15,24], the more stable

linkers were less toxic when administered to rodents.

Interestingly, in humans the picture is more complex;

clinical data from a number of maytansinoid-based

ADCs made with different linkers indicates that toler-

ability is inversely related to stability [9]. Namely, the

most labile linker (the disulfide-based SPP-DM1)

has an MTD of 6.0–8.0 mg/kg every 3 wk. By contrast,

the intermediately labile and most stable linkers (the

hindered disulfide-based SPDB-DM4 and the nonclea-

vable SMCC-DM1, respectively) have MTDs of 3.5–7.0 and 3.6 mg/kg every 3 wk. The dose-limiting toxi-

cities are different for each of these linker-payload

combinations; the most labile linker is limited by liver

toxicity, while the most stable is limited by thrombo-

cytopenia. These observations bolster the conclusion

that a variety of factors contribute to toxicity, including

FcgR-mediated disposition to the hematopoietic com-

partment and deconjugated payload detoxification by

the liver.


Site-specifically conjugated ADCs can widenthe therapeutic windowA number of studies have shown that reducing the

heterogeneity of an ADC through the use of site-specific

conjugation approaches can simultaneously improve effi-

cacy, pharmacokinetics, and safety [33,34]. Furthermore,

the analytics are greatly simplified, leading to easier

quality control and batch-to-batch consistency. For all

of these reasons, the field is steadily moving towards site-

specific conjugation as a critical feature of the next-

generation ADC. Three site-specific ADCs are already

in the clinic, including two from Seattle Genetics bearing

PBD payloads [35,36], and one from Rinat-Pfizer carrying

a microtubule inhibitor [37]. A variety of approaches have

been developed to enable site-specific conjugation [10],

including the introduction of engineered cysteine resi-

dues (Genentech/Roche, Seattle Genetics, Medimmune)

[38�], the incorporation of non-natural amino acids

(Ambrx, Sutro) [39,40], and chemoenzymatic approaches

such as the SMARTagTM method (Catalent), which uses

an enzyme to install a bioorthogonal chemical group for

subsequent conjugation [41,42], and the transglutaminase

method (Rinat-Pfizer) [28], which uses an enzyme to

perform the ligation between payload and protein. The

relative merits of these approaches will be sorted out in

the coming months through in vivo efficacy and safety

outcomes, and through manufacturing feasibility studies.

It is likely that several, if not all, of them will eventually

be tested in humans.

In a satisfying example of convergence, the research

groups studying site-specifically conjugated ADCs made

using different approaches are coming to similar conclu-

sions. For example, while a higher DAR (more cytotoxic

payload per antibody) generally leads to increased poten-

cy in vitro, in vivo the best efficacy can sometimes be

obtained by using a lower DAR species, particularly when

comparing a site-specifically conjugated lower DAR to a

heterogeneously-conjugated higher DAR construct

[41,43,44]. This outcome also leads to an increased ther-

apeutic index and improved safety, as the same or better

efficacy can be achieved with less cytotoxic payload

[41,43,44]. Furthermore, conjugation site does not affect

ADC potency in vitro, but greatly impacts efficacy in vivo[28,41,45]. Evidence suggests that these observations are

likely explained by the improved pharmacokinetics of

site-specific relative to conventional conjugates, and the

fact that some site placements yield longer ADC half-

lives than others [46]. The latter point highlights a

particular strength of site placement: the ability to per-

form structure activity relationship (SAR) studies, where

the effect of payload placement on biophysical (e.g.,

aggregation) and functional parameters (e.g., pharmaco-

kinetics and efficacy) can be determined. While a number

of groups have demonstrated that site placement matters,

consistent rules for payload placement have yet to be

developed. It is likely that optimal site placement may

Current Opinion in Chemical Biology 2015, 28:174–180 19


vary according to the particular conjugation chemistries

and linker-payloads employed.

A multipronged strategy may be needed tominimize ADC targeting of healthy tissuesWhile the approaches outlined to improve ADC stability

and efficacy can improve the therapeutic index, at a

certain point this treatment window will be limited by

the tumor specificity of the target antigen. A recent

bioinformatics study surveyed healthy tissue mRNA

expression of target antigens for 27 ADCs that are

currently in the clinic [16��]. Most target antigens

showed poor differential expression between tumors

and normal tissues and were also widely expressed

across organs. This study highlights the fact that addi-

tional innovations are needed in order to improve ADC

tumor targeting. Target antigen selection is becoming

more sophisticated [7], and is beginning to acknowledge

the roles played by biological function, absolute — as

Figure 3

(4) Antigen-specifictumor targeting

(3) Site-specificconjugation

(2) Payloadand DAR

(1) Linker/conjugation chemistryCurrent Opinion in Chemical Biology

Design considerations for building better ADCs. (1) The choice of

conjugation and linker chemistry will govern many of the biophysical and

functional characteristics of the resulting ADC. Linker design can alter

stability, membrane permeability, and resistance to P-glycoprotein-

mediated efflux — all of which can impact efficacy. Furthermore, both

the linker and the conjugation chemistry can affect the overall

hydrophobicity of the construct, which directly relates to in vivo stability,

PK, and tolerability. (2) The choice of cytotoxic payload and the DAR

affect both efficacy and safety. The latter is at least partially dependent

on the conjugation and linker chemistry for success, that is, higher DAR

ADCs tend to need more solubilizing linkers to prolong PK and improve

efficacy. (3) The use of site-specific conjugation facilitates analytics and

offers the ability to perform structure-activity relationship mapping to

identify optimal ADC configurations for the best efficacy and safety. (4)

Modifications, perhaps to the CDR or Fc regions, should be introduced

to improve the specificity of ADC targeting to the tumor in order to

reduce off-target toxicity in healthy tissues. Whereas points 1–3 have

been explored to some extent by the community and a consensus

opinion has begun to form around how to approach these

considerations (as indicated by the solid arrows), point 4 represents a

new direction for the field and relatively little has been done as yet in

this vein (as indicated by the dashed arrow). It will remain to be seen

how this next challenge is best addressed.


well as relative — expression levels, internalization pa-

rameters, and biomarker availability [16��,17]; judicious

choices may lead to more specific drug delivery. Another

approach is to reduce the visibility of healthy tissue anti-

gens to ADCs by either predosing with unconjugated

antibody to saturate binding sites [47], or by using anti-

bodies with masked CDRs that are unmasked by tumor-

specific proteases to allow localized target binding [48].

ConclusionsThe emerging strategies for improving ADC performance

focus on tunable ADC features that affect ADC stability,

potency, and targeting efficiency — which together de-

termine the efficacy and safety profiles (Figure 3). These

in turn define the therapeutic index and govern whether

an efficacious dose can be safely administered. Increasing

the therapeutic index has been the motivation driving

ADC development; we are beginning to understand the

rules of the game and can start moving the ball down the

field towards that goal.

AcknowledgementsThe authors would like to thank Patrick Holder for fruitful discussions andRaghuveer Parthasarathy for Figure 1.

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European Journal of Medicinal Chemistry 88 (2014) 3e9

Contents lists avai

European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Original article

Exploring the effects of linker composition on site-specificallymodified antibodyedrug conjugates

Aaron E. Albers, Albert W. Garofalo, Penelope M. Drake, Romas Kudirka,Gregory W. de Hart, Robyn M. Barfield, Jeanne Baker, Stefanie Banas, David Rabuka*

Redwood Bioscience, 5703 Hollis Street, Emeryville, CA 94608, USA

a r t i c l e i n f o

Article history:Received 5 June 2014Received in revised form21 August 2014Accepted 22 August 2014Available online 23 August 2014

Keywords:Antibodyedrug conjugateNoncleavable linkerAldehyde tag

* Corresponding author.E-mail address: [email protected]

http://dx.doi.org/10.1016/j.ejmech.2014.08.0620223-5234/© 2014 Elsevier Masson SAS. All rights re

a b s t r a c t

In the context of antibodyedrug conjugates (ADCs), noncleavable linkers provide a means to delivercytotoxic small molecules to cell targets while reducing systemic toxicity caused by nontargeted releaseof the free drug. Additionally, noncleavable linkers afford an opportunity to change the chemicalproperties of the small molecule to improve potency or diminish affinity for multidrug transporters,thereby improving efficacy. We employed the aldehyde tag coupled with the hydrazino-iso-Pictet-Spengler (HIPS) ligation to generate a panel of site-specifically conjugated ADCs that varied only in thenoncleavable linker portion. The ADC panel comprised antibodies carrying a maytansine payload ligatedthrough one of five different linkers. Both the linker-maytansine constructs alone and the resulting ADCpanel were characterized in a variety of in vitro and in vivo assays measuring biophysical and functionalproperties. We observed that slight differences in linker design affected these parameters in disparateways, and noted that efficacy could be improved by selecting for particular attributes. These studies serveas a starting point for the exploration of more potent noncleavable linker systems.

© 2014 Elsevier Masson SAS. All rights reserved.

1. Introduction

Antibodyedrug conjugates (ADCs) promise to alter the land-scape of anti-cancer therapeutics by targeting highly cytotoxic drugmolecules directly to cancer cells. The success of currentlyapproved ADCs has inspired a spate of research and developmentefforts in the area; dozens of new ADCs are in pre-clinical or clinicaltrials [1]. ADCs comprise a monoclonal antibody, a cytotoxicpayload, and a linker that joins them together [2]. The monoclonalantibody targets the payload to cells expressing the antigen on theirsurface, and the cytotoxic payload kills the cells upon internaliza-tion of the ADC. The linker is literally the central component of anADC; it contains the reactive group that governs the conjugationchemistry, and serves as a chemical spacer that physically connectsthe drug payload to the antibody. As such, the linker is also themostversatile aspect of the ADC. It can be modified in any number ofways to influence various drug/linker characteristics (e.g., solubil-ity) [3,4] and ADC properties (e.g., potency, pharmacokinetics,therapeutic index, and efficacy in multidrug resistant cells) [5e11].

(D. Rabuka).

served.

There are essentially two broad classes of ADC linkers; thosethat are chemically labile or enzymatically-cleavable, and thosethat are chemically stable or noncleavable [12]. Labile/cleavablelinkers are designed to keep the ADC intact when in circulation butrelease the drug payload upon internalization by the target cell.Some cytotoxic payloadsdfor example, MMAEdrequire a cleavablelinker, as they do not tolerate substitutions [13,14]. By contrast,other cytotoxic payloadsdfor example, maytansinedcan accom-modate substitutions while maintaining potency [15]. Such drugsare good substrates for the development of noncleavable linkers. Bydesign, noncleavable linkers do not contain chemical functional-ities that are readily susceptible to intracellular degradation.Therefore, after an internalized ADC is trafficked to the lysosome,the antibody moiety is proteolytically degraded into amino acidswhile the cytotoxic drug remains attached via the linker to anamino acid residue [16]. The retention of the linker as part of theactive metabolite allows for the modulation of the overall proper-ties of the metabolite (e.g., by altering hydrophobicity, length, andcharge) in order to improve potency.

We previously reported a novel site-specific ligation chemistrythat takes advantage of an aldehyde-tagged protein [17]. Thealdehyde tag is a straightforward means of site-specifically func-tionalizing proteins for chemical modification. The genetically-encoded tag consists of a pentapeptide sequence (CXPXR) that is

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A.E. Albers et al. / European Journal of Medicinal Chemistry 88 (2014) 3e94

specifically recognized by formylglycine-generating enzyme (FGE)[18-20]. During protein expression in cells, the cysteine residue inthe sequence is recognized by FGE and oxidized co-translationallyto formylglycine. The resulting aldehyde affords a bioorthogonalchemical handle for ligation (Fig. 1). Linkers terminating in a 2-((1,2-dimethylhydrazinyl)methyl)-1H-indole react with the alde-hyde by way of a hydrazino-iso-Pictet-Spengler (HIPS) reaction toform an azacarboline, resulting in a stable CeC bond joining theantibody and payload.

The aldehyde tag platform allows for site-specific conjugationthat yields a highly homogenous product. Accordingly, this tech-nology is well-suited for performing structure activity relationshipstudies in the context of an intact ADC. Here, we isolated linkercomposition as a single variable for optimization while the otherADC componentsdantibody backbone, cytotoxic payload, conju-gation site, drug-to-antibody ratio, and conjugation chemis-trydwere held constant. By characterizing a panel of five drug/linkers and their corresponding conjugates, we explored the impactof small changes in linker design on ADC potency and stability.

2. Materials and methods

2.1. Linker synthesis

Synthetic routes and analytical data are provided in theSupplemental materials.

2.2. Microtubule polymerization assay

We used the Tubulin Polymerization Assay Kit (Cytoskeleton)according to the manufacturer's instructions for the fluorescence-based test. All test articles were used at 3 mM.

2.3. Direct ELISA antigen binding

Maxisorp 96-well plates (Nunc) were coated overnight at 4 �Cwith 1 mg/mL of human HER2-His (Sino Biological) in PBS. The platewas blocked with ELISA blocker blocking buffer (ThermoFisher),and then the aHER2wild-type antibody and ADCswere plated in an8-step series of 2-fold dilutions starting at 100 ng/mL. The platewasincubated, shaking, at room temperature for 2 h. After washing inPBS 0.1% Tween-20, bound analyte was detected with a donkeyanti-human Fc-g-specific horseradish peroxidase (HRP)-conju-gated secondary antibody. Signals were visualized with Ultra TMB(Pierce) and quenched with 2 N H2SO4. Absorbance at 450 nmwasdetermined using a Molecular Devices SpectraMax M5 plate readerand the data were analyzed using GraphPad Prism.

Fig. 1. The aldehyde tag coupled with HIPS ligation yields site-specifically modified aenzyme (FGE) recognition sequence (CXPXR) is site-specifically inserted into the backboneThe aldehyde of formylglycine can then be reacted with nucleophiles to form a stable CeC

2.4. Bioconjugation, purification, and HPLC analytics

Humanized anti-HER2 IgG antibodies (15 mg/mL) bearing thealdehyde tag (LCTPSR) at the C-terminus of the heavy chain wereconjugated to maytansine-containing drug linkers (8 mol equiva-lents drug:antibody) for 72 h at 37 �C in 50 mM sodium citrate,50mMNaCl pH 5.5 containing 0.85% DMA and 0.085% Triton X-100.Free drug was removed using tangential flow filtration. Unconju-gated antibody was removed using preparative-scale hydrophobicinteraction chromatography (HIC; GE Healthcare 17-5195-01) withmobile phase A: 1.0 M ammonium sulfate, 25 mM sodium phos-phate pH 7.0, and mobile phase B: 25% isopropanol, 18.75 mM so-dium phosphate pH 7.0. An isocratic gradient of 33% B was used toelute unconjugated material, followed by a linear gradient of41e95% B to elute mono- and diconjugated species. To determinethe DAR of the final product, ADCs were examined by analytical HIC(Tosoh #14947) with mobile phase A: 1.5 M ammonium sulfate,25 mM sodium phosphate pH 7.0, and mobile phase B: 25% iso-propanol, 18.75 mM sodium phosphate pH 7.0. To determine ag-gregation, samples were analyzed using analytical size exclusionchromatography (SEC; Tosoh #08541) with a mobile phase of300 mM NaCl, 25 mM sodium phosphate pH 6.8.

2.5. In vitro cytotoxicity

The HER2-positive breast carcinoma cell line, NCI-N87, wasobtained from ATCC and maintained in RPMI-1640 medium (Cell-gro) supplemented with 10% fetal bovine serum (Invitrogen) andGlutamax (Invitrogen). 24 h prior to plating, cells were passaged toensure log-phase growth. On the day of plating, 5000 cells/wellwere seeded onto 96-well plates in 90 mL normal growth mediumsupplemented with 10 IU penicillin and 10 mg/mL streptomycin(Cellgro). Cells were treated at various concentrations with 10 mL ofdiluted analytes, and the plates were incubated at 37 �C in an at-mosphere of 5% CO2. After 6 d, 100 mL/well of CellTiter-Glo reagent(Promega) was added, and luminescence was measured using aMolecular Devices SpectraMax M5 plate reader. GraphPad Prismsoftware was used for data analysis, including IC50 calculations.

2.6. In vitro stability

ADCs were spiked into rat plasma at ~1 pmol (payload)/mL. Thesamples were aliquoted and stored at �80 �C until use. Aliquotswere placed at 37 �C under 5% CO2 for the indicated times and thenwere analyzed by ELISA to assess the anti-maytansine and anti-Fabsignals. A freshly thawed aliquot was used as a reference startingvalue for conjugation. All analytes were measured together on oneplate to enable comparisons across time points. First, analytes were

ntibodies. Using standard molecular biology techniques, a formylglycine-generatingof the antibody. FGE co-translationally oxidizes the cysteine residue to formylglycine.bond.

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A.E. Albers et al. / European Journal of Medicinal Chemistry 88 (2014) 3e9 5

diluted in blocking buffer to 20 ng/mL (within the linear range ofthe assay). Then, analytes were captured on plates coated with ananti-human Fab-specific antibody. Next, the payload was detectedwith an anti-maytansine antibody followed by an HRP-conjugatedsecondary; the total antibody was detected with a directly conju-gated anti-human Fc-specific antibody. Bound secondary antibodywas visualized with TMB substrate. The colorimetric reaction wasstopped with H2SO4, and the absorbance at 450 nm was deter-mined using a Molecular Devices SpectraMaxM5 plate reader. Dataanalysis was performed in Excel. Each sample was analyzed inquadruplicate, and the average values were used. The ratio of anti-maytansine signal to anti-Fab signal was used as a measure ofantibody conjugation.

Fig. 2. Inclusion of amino acid residues resulted in highly soluble maytansine-linker constructs with varied chemical composition. Five different maytansine-conjugated linkers were synthesized (as shown in Scheme 1) and characterized,both as free drugs and after conjugation to an a-HER2 antibody.

2.7. Xenograft studies

The animal studies were approved by Charles River LaboratoriesInstitutional Animal Care and Use Committee (IACUC). Female C.B-17 SCIDmicewere inoculated subcutaneously with 1�107 NCI-N87tumor cells in 50% Matrigel. When the tumors reached an averageof 112 mm3, the animals were given a single 5 mg/kg dose of ADC,trastuzumab antibody (untagged), or vehicle alone. The animalswere monitored twice weekly for body weight and tumor size.Tumor volume was calculated using the formula:

Tumor volume�mm3

�¼ w2 � l

2

where w ¼ tumor width and l ¼ tumor length.Tumor doubling times were obtained by averaging the tumor

growth rate curves from four groups of mice. Then, log10 cell killwas estimated using the formula:

log10 cell kill ¼ treated group TTE� control group TTE3:32� tumor doubling time

Treatment over control (T/C) ratios were determined by dividingthe tumor volume of the treatment group by the tumor volume ofthe control group at a designated time point.

3. Results and discussion

3.1. Linker design and synthesis

To examine the effect of linker composition, we tested a varietyof maytansine-linkers that contained functional groups anticipatedto aid in solubility, which improves bioconjugation yields [4].Initially, we used PEGn spacers (with n ¼ 2, 4, or 6), but found thatthe PEG group alone was not sufficiently hydrophilic to overcomethe very hydrophobic contributions from the maytansine and HIPScomponents. The conjugation efficiencies observed with linkerscontaining PEGn spacers alone were poor, e.g., 40% yield with aPEG6-maytansine linker conjugated to a C-terminally-tagged anti-body.We found that a simpleway to incorporate hydrophilicity wasby using amino acid residues as linker components (Fig. 2). In turn,this change resulted in a significant improvement in conjugationefficiency, e.g., 90% yield with a glutamic acid PEG2-maytansinelinker conjugated to a C-terminally-tagged antibody. Here, wetested the effect of using different amino acids as solubilizingagents by evaluating glutamic acid (Linkers 1, 4, and 5), asparagine(Linker 2), and phosphotyrosine (Linker 3). The latter was meant tofunction as a pro-drug, where the phosphorylated form would besoluble, but not membrane permeable. Once inside a cell, the linkerwas intended to be a substrate for phosphorylases, the action ofwhich would yield a more hydrophobic and membrane-permeable

active metabolite. We also incorporated a spacer element into thelinkersdeither PEG2 or n-propyldto improve conjugation effi-ciency and mitigate ADC aggregation. Finally, taking advantage ofthe hydrazino-iso-Pictet-Spengler (HIPS) chemistry, the linkersterminated in either a reactive 2-((1,2-dimethylhydrazinyl)methyl)indole (1, 2, 3, and 5) or 2-((1,2-dimethylhydrazinyl)methyl)pyrrolo[2,3-b]pyridine (4). The latter varied from the former by a singlenitrogen atom (Fig. 2), making it slightly more hydrophilic. Bothreactive groups enabled HIPS ligation of the linker-maytansine toaldehyde-tagged antibodies for ADC production.

A representative synthesis of the linkers is shown in Scheme 1.In the example, a pegylated, protected amino acid, 6, is coupled topentafluorophenyl ester, 7. The product, 8, is then coupled to N-deacetylmaytansine, 9, using HATU followed by hydrolysis of thetert-butyl ester and removal of the Fmoc-protecting group withpiperidine to give the final desired product, 1.

3.2. Linker composition did not alter the payload's ability to inhibitmicrotubule polymerization

As a first step, once the drug/linkers were in hand, we per-formed an in vitro microtubule polymerization assay to confirmthat the incorporated structural variations and elaborations tomaytansine did not impair the drug's ability to inhibit microtubulepolymerization (Fig. 3). As anticipated, due to the known toleranceof maytansine to substitutions at the N-acyl position [21], the panelof drug/linkers resulted in microtubule polymerization inhibitionsimilar to unmodified maytansine. A small spread of values wasnoted, but all werewithin 32% of maytansine itself. As shown in thenext section, these small differences did not appear to impact theIC50 of the drug/linkers when formulated as an ADC.

3.3. Bioconjugation and in vitro assessment of the ADC panel

Conjugation of the drug/linkers to a C-terminally aldehydetagged a-HER2 antibody was carried out by treating the antibody at37 �C with 8e10 equivalents of linker-maytansine in 50 mM so-dium citrate, 50 mMNaCl pH 5.5 containing 0.85% DMA and 0.085%Triton X-100, and the progress of the reaction was tracked byanalytical hydrophobic interaction chromatography (HIC). Uponcompletion, the excess payload was removed by tangential flow

26

Scheme 1. Representative linker synthesis.

0

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0 5 10 15 20 25 30 35 40 45 50 55 60

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resc

ence

(Arb

itrar

yU

nits

)

Minutes

Buffer

Maytansine

Paclitaxel

Vinblastine

Linker 1

Linker 2

Linker 3

Linker 4

Linker 5

Fig. 3. Linker selection does not hinder maytansine inhibition of microtubulepolymerization. The drug/linkers were tested in a microtubule polymerization assay.Free maytansine was included as a positive control. Paclitaxel and vinblastine wereincluded as a promoter and inhibitor, respectively, of microtubule polymerization. Thebuffer control indicates the polymerization rate of untreated microtubules. Relative tofree maytansine, the drug/linkers inhibited polymerization by the following amounts:Linker 1, 68%; Linker 2, 92%; Linker 3, 79%; Linker 4, 105%; Linker 5, 73%.


filtration, and the unconjugated antibody was removed by pre-parative HIC. These reactions were high yielding, with >90%conjugation efficiency (Table S1). After purification, the ADCscontained an average drug-to-antibody ratio (DAR) of 1.6 asdetermined by hydrophobic interaction chromatography(Figure S2). The drug distribution (ratio of DAR 1e2) was verysimilar among the ADCs made with different linkers (Table S2). Thepreparations were �95% monomeric as assessed by size-exclusionchromatography (data not shown).

With the ADCs in hand, we first askedwhether conjugationwiththe drug/linkers had altered the antibody affinity for the HER2antigen. To test this, we performed a direct-binding ELISA assayusing plates coated with human HER2-His, and compared the EC50of thewild-type (untagged and unconjugated) a-HER2 to the valuesobtained for the panel of a-HER2 ADCs (Fig. 4). Only minimal dif-ferences in affinity were noted, with most of the ADCs appearing tobind with slightly higher affinity than the wild-type antibody.

Next, we tested the in vitro cytotoxicity of the ADCs against theHER2-overexpressing gastric cell line, NCI-N87 (Fig. 5A). As acomparator, we also tested the cytotoxic activity of the corre-sponding free drug/linkers (Fig. 5B). Cell cultures were exposed to

varying concentrations of the analytes for 6 days, and then cellviability was measured by using a CellTiter-Glo assay, whichquantifies ATP levels. All ADCs exhibited picomolar activity, withIC50 values similar to or better than that observed after treatmentwith free maytansine. By contrast to the ADCs, the free drug/linkerswere overall less potent, generally showing IC50 values that were1000- to 2000-fold higher than the corresponding ADCs. Linkers 1and 4, which shared a glutamic acid-PEG2 scaffold, both had freedrug/linker IC50 values above 1 mM, ~10,000-fold higher than theADC versions of those compounds. In addition to the IC50 values, wenoted that the Linker 3 ADC, in spite of its measured picomolaractivity, failed to kill more than 70% of the cells, even at the highestdoses (Fig. 5A). The free version of Linker 3 did not suffer from thissame cytotoxic plateau, reducing cell viability by >93% at thehighest dose (Fig. 5B). We observed the same trends with the un-conjugated and conjugated versions of Linker 3 on a differentantibody and against a different cell line, suggesting that theplateau effect of this linker is translatable across platforms.Although the cytotoxic plateau is commonly observed in thesetypes of assays, the underlying mechanisms involved and the bio-logical significance of the effect is not clear.

As a final in vitro characterization of the ADC panel, we exam-ined the stability of the HIPS-conjugates in plasma for 14 days at37 �C. The assay consisted of an ELISA-basedmethod that comparedthe ratio of anti-payload to anti-Fc signals. As a group, the conju-gates exhibited a high degree of stability, with �85% payloadremaining after 7 days and �74% payload remaining after 14 days(Table 1). The glutamic acid-PEG2-containing scaffolds were themost stable over 14 days, both demonstrating more than 80%retention of payload. The most labile linker, Linker 2, only differedfrom the most stable linker by about 10% over 14 days.

3.4. In vivo efficacy of the ADC panel

To test the in vivo efficacy of the ADC panel, we assessed theconjugates using an NCI-N87 xenograft model in SCID mice. Com-pounds were administered as a single 5 mg/kg dose at the onset ofthe study. All ADCs were well-tolerated with no animal showing>10% weight loss up to 40 days post-treatment (Figure S1). Tumorgrowth was arrested, and some tumors were reduced in size aftertreatment with the a-HER2 ADCs (Fig. 6A), but not after treatmentwith the isotype control ADC (conjugated using Linker 1). Eventu-ally, tumors began to regrow in all animals, sooner in some groupsthan others, depending on the ADC used for treatment. By 60e70days post-dose, there were clear differences in mean tumor vol-umes among groups treated with an a-HER2 ADC; specifically, the

27

0.1 1 10 1000

1

2

3

[Ab ng/ml]

Abs

orba

nce

(A.U

.)Wild-type αHER2Linker 1Linker 2Linker 3Linker 4Linker 5

Fig. 4. a-HER2 ADCs conjugated with the panel of drug/linkers maintain theiraffinity for HER2 antigen. A direct-binding ELISA assay, using immobilized humanHER2-His protein as the capture reagent and an anti-human Fc-g-specific HRP-conjugated antibody as the detection reagent, was used to monitor the affinity of a-HER2 conjugates relative to the wild-type antibody. Standard curves of each analytewere generated and the EC50 value of each curve was calculated using GraphPad Prism.The calculated EC50 values (nM) were: Wild-type antibody, 8.72; Linker 1, 6.20; Linker2, 10.80; Linker 3, 7.10; Linker 4, 5.95; Linker 5, 7.23.

Table 1ADCs made with different linkers show similar stability in plasma at 37 �C.

ADC % Conjugate remaining after 7days

% Conjugate remaining after 14days

aHER2-Linker 1

93 81

aHER2-Linker 2

85 74

aHER2-Linker 3

93 77

aHER2-Linker 4

97 83

aHER2-Linker 5

95 77


mean tumor volumes ranged from 249 to 487 mm3 at day 60(Fig. 6A). In order to investigate this effect, we looked at thelog10 cell kill for tumors dosed with the various treatments(Table 2). The results indicated that treatment with ADCs conju-gated to Linkers 1 and 4 killed more tumor cells as compared totreatment with the other ADCs. Notably, these two linkers repre-sented the absolute minimum amount of chemical diversity, bothcontained the glutamic acid-PEG2 scaffold and differed from eachother by only a single nitrogen group in the azacarboline that formsduring ligation. This increased potency translated into a survivaladvantage for animals treated with ADCs conjugated to Linkers 1 or4 (Fig. 6B).

The efficacy of Linker 5 was reduced as compared to Linkers 1and 4, with which it shared the glutamic acid moiety. The results ofthis series of linkers suggest that, in this context, inclusion of the n-propyl spacer reduced efficacy as compared to the PEG2 spacer. Theother two linkers, which incorporated different amino acids on thePEG2 scaffold, had varying efficacy. Linker 2 showed an interme-diate log10 cell kill value (reflecting total cells killed throughout thecourse of the study), but was the best performer in the first 10 days

Fig. 5. Small changes in linker composition do not influence the in vitro cytotoxicity ofcytotoxicity in a 6 day assay. Free maytansine (black line) was included as a positive controcontrol to indicate specificity. (A) ADC IC50 values (reflecting the antibody concentrations exLinker 1, 170 pM; Linker 2, 160 pM; Linker 3, 110 pM; Linker 4, 96 pM; Linker 5, 120 pM;measured as follows: free maytansine, 405 pM; Linker 1, 1.58 mM; Linker 2, 342.5 nM; Link

of the study, reducing tumor volume more than any other treat-ment (Fig. 6A). Linker 3 had the poorest in vivo efficacy (p < 0.007,by the log-rank, ManteleCox, test). It is interesting to considerwhether the incomplete in vitro killing of NCI-N87 target cells byADCs conjugated to this linker is related todor perhaps predictiveofdits reduced in vivo efficacy as compared to the other ADCs.

Next, we selected two linkers from the initial panel to take into amultidose efficacy study. We chose Linker 1 on the merits of itsoverall potency, as measured by tumor growth, log10 cell kill, andsurvival. We chose Linker 2 because it showed the fastest initialtumor reduction, and we reasoned that perhaps this quick responsewould translate into increased efficacy in a multidose setting. Themultidose study employed NCI-N87 tumors in SCID mice. Animalswere dosed (10mg/kg) once aweek for four weeks. The experimentemployed two armsdwith dosing beginning when tumors reachedaverage volumes of either 180 or 400 mm3. a-HER2 ADCs madewith both Linkers 1 and 2 were highly active against the smallertumors (Fig. 7A), and resulted in very similar levels of tumor con-trol. By contrast, against the larger tumors, the a-HER2 ADC madewith Linker 2 showed superior efficacy, resulting in a greater levelof tumor inhibition as compared to the ADC made with Linker 1(Fig. 7B). Specifically, the treated/control tumor volumes at day 42were 0.39 and 0.26 for Linkers 1 and 2, respectively.

In conclusion, we developed a panel of C-terminally-conjugateda-HER2 ADCs bearing highly similar linkers, and observed thatrelatively minor structural changes led to dramatic differences inpotency both in vitro and in vivo against the NCI-N87 tumor model.Other biophysical parameters were less impacted. Specifically, weobserved only minor effects of linker architecture on inhibition ofmicrotubule polymerization (at the free drug/linker level), andantibody affinity (at the ADC level). With respect to in vitro cyto-toxicity, as a group, the ADCs were highly efficacious and yieldedvery similar IC50 values, with less than a 2-fold difference

aHER2 ADCs. NCI-N87 cells, which overexpress HER2, were used as targets for in vitrol, and an isotype control ADC (gray line) conjugated to Linker 1 was used as a negativecept in the case of the free drug) were measured as follows: free maytansine, 250 pM;isotype control ADC, could not be determined. (B) Free drug/linker IC50 values wereer 3, 125.8 nM; Linker 4, ~1 mM; Linker 5, 274.9 nM.

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urvi

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Linker 1

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Isotype ADC, Linker 1

Fig. 6. Linker composition affects the in vivo efficacy of aldehyde-tagged a-HER2 ADCs in an NCI-N87 tumor model. CB.17 SCID mice (8/group) were implanted subcutaneouslywith NCI-N87 cells. When the tumors reached ~113 mm3, the animals were given a single 5 mg/kg dose of an a-HER2 conjugated to Linkers 1-5 or of an isotype control antibodyconjugated to Linker 1. (A) Tumor growth was monitored twice weekly. (B) The differences in efficacy among the ADCs tested were reflected in survival curves. Animals wereeuthanized when tumors reached 800 mm3 or on day 112 of the study, whichever occurred first.

Table 2In vivo log10 cell kill of NCI-N87 tumor cells achieved by a single 5 mg/kg ADCdose.

a-HER2 ADC linker composition Log10 cell kill

Linker 1 1.24Linker 2 0.82Linker 3 0.65Linker 4 1.22Linker 5 0.92


encompassing the entire panel. However, one ADCdconjugatedwith Linker 3dexhibited a striking viability plateau in vitro, with32% viable cells remaining at the highest doses. By contrast to theADCs, the potency of the free drug/linkers varied morewidely, witha 12-fold difference encompassing the range of IC50 values. Inter-estingly, the rank order potency of the ADC did not directly corre-late with that of the free drug/linker. Furthermore, the

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Fig. 7. Multidose xenograft studies reveal differences in efficacy against larger tumors bsubcutaneously with NCI-N87 cells. Tumors were allowed to grow to either ~180 or 400 mmonce a week for four weeks with 10 mg/kg of an a-HER2 ADC conjugated to Linkers 1 or 2. Aeuthanized when tumors reached 800 mm3.

“completeness” of the cell cytotoxicity was not always the samebetween the corresponding ADC and free drug/linker analytes. Forexample, treatment with free Linker 3 abrogated all but 7% of theviable cells. With respect to in vivo cytotoxicity, ADCsmadewith allof the linkers inhibited growth of the HER2-overexpressing NCI-N87 xenograft to some extent. However, the log10 cell kill valuesachieved by the ADCs varied by up to 2-fold, and the median sur-vival time among the groups differed by 17 days, indicating thatlinker structure affected efficacy. Furthermore, we observed a dif-ference in the kinetics of tumor response to ADCs made withdistinct linkers, e.g., Linker 2 vs. Linker 1, whereby Linker 2 wasmore efficacious in the short term (1 wk), but the response wasshort lived. We were able to capitalize on this difference in a followup multidose xenograft study, in which the ADC with faster cyto-toxic kinetics showed superior efficacy against larger tumors.Therefore, we demonstrated that the sensitivity of our system tolinker design affords an opportunity to engineer next-generationADCs with optimized characteristics for improved efficacy.

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etween ADCs made with Linkers 1 and 2. CB.17 SCID mice (8/group) were implanted3 (Panels A and B, respectively) and then treatment was initiated. Animals were dosedrrows indicate dosing days. Tumor growth was monitored twice weekly. Animals were

29


Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ejmech.2014.08.062.

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Regulatory Toxicology and Pharmacology 71 (2015) 444–452

Contents lists available at ScienceDirect

Regulatory Toxicology and Pharmacology

journal homepage: www.elsevier .com/locate /yr tph

An FDA oncology analysis of antibody-drug conjugates q

http://dx.doi.org/10.1016/j.yrtph.2015.01.0140273-2300/Published by Elsevier Inc.

Abbreviations: AD, acceptable dose; ADC, antibody-drug conjugate; ADCC,antibody-dependent cellular cytotoxicity; FIH, first-in-human; HNSTD, highestnon-severely toxic dose; ICH, International Conference on Harmonization; LC/MS,liquid chromatography/mass spectrometry; mc, maleimidocaproyl linker; mAb,monoclonal antibody; MTD, maximum tolerated dose; NHP, non-human primate;NOAEL, no observed adverse effect level; PD, pharmacodynamics; PK, pharmacoki-netics; RP2D, recommended Phase 2 dose; SM, small molecule; STD10, severelytoxic dose in 10% of the animals; vc, valine–citrulline linker.

q Disclaimer: This article reflects the views of the authors and should not beconstrued to represent FDA’s views or policies.⇑ Corresponding author.

E-mail address: [email protected] (J.K. Leighton).

Haleh Saber, John K. Leighton ⇑US Food and Drug Administration, Center for Drug Evaluation and Research, Office of Hematology and Oncology Products, 10903 New Hampshire Ave, Silver Spring, MD 20903,United States

a r t i c l e i n f o

Article history:Received 16 January 2015Available online 7 February 2015

Keywords:Antibody-drug conjugatesFirst-in-human start dose selectionOncology drug development

a b s t r a c t

Antibody-drug conjugates (ADCs) are complex molecules composed of monoclonal antibodies conjugatedto potent cytotoxic agents through chemical linkers. Because of this complexity, sponsors have used dif-ferent approaches for the design of nonclinical studies to support the safety evaluation of ADCs and first-in-human (FIH) dose selection. We analyzed this data with the goal of describing the relationshipbetween nonclinical study results and Phase 1 study outcomes. We summarized the following data frominvestigational new drug applications (INDs) for ADCs: plasma stability, animal study designs and toxi-cities, and algorithms used for FIH dose selection. Our review found that selecting a FIH dose that is1/6th the highest non-severely toxic dose (HNSTD) in cynomolgus monkeys or 1/10th the STD10 inrodents scaled according to body surface area (BSA) generally resulted in the acceptable balance of safetyand efficient dose-escalation in a Phase 1 trial. Other approaches may also be acceptable, e.g. 1/10th theHNSTD in monkeys using BSA or 1/10th the NOAEL in monkeys or rodents using body weight for scaling.While the animal data for the vc-MMAE platform yielded variable range of HNSTDs in cynomolgus mon-keys, MTDs were in a narrow range in patients, suggesting that for ADCs sharing the same small moleculedrug, linker and drug:antibody ratio, prior clinical data can inform the design of a Phase 1 clinical trial.

Published by Elsevier Inc.

1. Introduction

Antibody-drug conjugates (ADCs) are a unique class of drugsconsisting of a monoclonal antibody (mAb) conjugated to a cyto-toxic drug (herein referred to as small molecules; SM) through achemical linker. Most ADCs in development are intended to treatcancer in patients with serious and life-threatening disease. Theantibody component generally directs the SM to a specific epitopeon the cancer cell where the ADC is internalized and releases thecytotoxic SM. The proposed advantage of developing an ADC con-jugate is to direct high concentrations of the SM to the tumor(via tumor-specific or overexpressed antigens) and thereby reduce

systemic exposure to the free SM and associated off-target adverseeffects (Sievers and Senter, 2013; Cancer Drug Discovery andDevelopment; Antibody-Drug Conjugates and Immunotoxins,2013; Roberts et al., 2013). Interest in developing ADCs for thetreatment of solid tumors and hematologic malignancies hasgrown in recent years with the introduction of new technologies,the validation of more tumor targets for anti-cancer therapies,and the approval of two ADCs. The recent FDA approval of twoADCs (brentuximab vedotin and ado-trastuzumab emtansine)and submission of completed Phase 1 study results for investiga-tional new drug applications (INDs) for ADCs, has provided enoughdata for FDA to conduct a preliminary summary analysis of non-clinical development programs for ADCs. FDA reviewed the non-clinical safety data submitted to support 20 separate INDapplications for ADCs with an emphasis on FIH dose selection, plas-ma stability, toxicities in animals, and toxicology study designs.

In the current generation of ADCs, the SMs used are genotoxicagents that target rapidly dividing cells (e.g., crypt cells and bonemarrow) in general toxicology studies by directly interacting withthe DNA or components of DNA synthesis or cell division. The SMsselected for use in ADCs are typically potent and poorly toleratedwhen used as free agents. The SM is covalently attached to theantibody via a linker intended to provide stability in plasma. The

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H. Saber, J.K. Leighton / Regulatory Toxicology and Pharmacology 71 (2015) 444–452 445

linker generally provides enough stability to allow the ADC toremain intact while in circulation and is labile enough to allowthe release of the small molecule drug after internalization insidethe cell. For protease-resistant noncleavable linkers, the SM isreleased after internalization of the ADC and proteolytic degrada-tion of the antibody moiety. The first generation of linkers includedthe acid-cleavable linker used in Mylotarg (ADC half-life of 1–2 days), but improvements in linker technologies have producedmore stable ADCs with half-lives of approximately one week(Ducry and Stump, 2010). Based on the INDs examined, linkersused were disulfide-based linkers, peptidic linkers such as the pro-tease cleavable valine–citrulline (vc) linker, and protease-resistantlinkers such as the maleimidocaproyl (mc) linker. The SMs con-tained in ADCs used in our analysis were mostly MMAE, MMAF,DM1, and DM4; other small molecules were also included, e.g.MED2460 (a DNA alkylating agent) and calicheamicin.

Estimating a FIH dose that is reasonably safe in preliminary clin-ical studies is an essential goal of a nonclinical development pro-gram. Ideally, the proposed FIH dose for anticancer therapiesshould avoid unacceptable toxicities while minimizing the time ittakes to deliver sufficient exposure to have the intended pharmaco-logical activity. Approaches used to estimate the FIH dose for smallmolecules or biological products may include applying a safety fac-tor to doses identified as tolerable in animal toxicity studies. The ani-mal dose can be converted to the human equivalent dose by scalingaccording to body surface area (BSA), as is typically done for smallmolecules, or according to body weight (BW) as done for some largemolecules, or less frequently by application of a hybrid PK/PD model(FDA, 2005; Haddish-Berhane et al., 2013).

Currently, there is no published guidance regarding method-ology for FIH dose selection for ADCs. Sponsors of IND applicationshave proposed various algorithms such as traditional approachesused for small molecules in oncology, approaches used for biologi-cal products in oncology or non-oncology settings, and scalingeither to BW or BSA. In our review, we reanalyzed animal toxicol-ogy data for 20 ADCs (2 approved and 18 in development) andcompared different methods for estimating the FIH dose basedon either: 1/10th STD10 (using BW or BSA for animal-to-humanconversions), 1/6th the HNSTD (using BW or BSA for conversion),1/10th HNSTD (using BW or BSA for conversion) and 1/10th NOAEL(using BW and BSA for conversion).

2. Methods

2.1. Data collection methodology

The FDA archival database was queried December 2012 throughAugust 2013 to identify INDs for inclusion in the analysis. Anti-body-drug conjugates selected were those that fell within thescope of ICH S9, i.e., drugs to treat advanced malignancies. Theintention was to select INDs that had completed Phase 1 clinicalinvestigations; although as discussed later for a few ADCs, thesponsors decided to inactivate the IND. The nonclinical programsfor these INDs were examined for the toxicology design, toxicityprofile seen in animals, determination of the FIH dose, and plasmastability. The toxicology studies previously reviewed by the FDAwere peer reviewed by one author (HS) and the following informa-tion was collected for each IND, when available: NOAEL, STD10,HNSTD, the isoform and target epitope of the antibody moiety,the type of linker, the structure and mechanism of action of thesmall molecule moiety, the human maximum tolerated dose(MTD) or recommended Phase 2 dose (RP2D). The time to reachthe human MTD/RP2D was identified as the time from the IND sub-mission to the date that the MTD/RP2D was reported. When anMTD or a RP2D was not identified, a human acceptable dose (AD)

was identified (see section below for the definition of AD). FIH doseapproaches were evaluated and the correlation between HNSTDsand human MTD, RP2D, or AD was assessed. Two ADCs in this ana-lysis have been approved; ado-trastuzumab emtansine and bren-tuximab vedotin. None of the ADCs examined had modificationson their antibody for reduced or increased antibody-dependentcellular cytotoxicity activity (ADCC), or site-specific linker attach-ment (e.g. by introducing non-conventional amino acids). Anti-body-drug conjugates that contain bacterial or plant-derivedtoxins and ADCs with radiolabeled SMs were excluded from thisanalysis.

Out of the 20 INDs examined, 16 were analyzed for FIH doseapproaches using data generated in cynomolgus monkeys withthe ADC. Four INDs were excluded due to the following reasons:IND inactivated before an MTD could be established (3 INDs), thetoxicology study design did not support the frequency of adminis-tration in patients (1 IND). For the latter IND, single doses of theADC were administered to monkeys; however, patients receivedtwice weekly doses of the ADC; the first dose in patients was abovethe MTD and doses were subsequently reduced.

Of the 20 INDs examined, 14 were analyzed for FIH doseapproaches using data generated in rodents with the ADC. Six INDswere excluded due to reasons described above or because the ADCwas not used in the rodents (either that a rodent study was notconducted or it was conducted with the small molecule only).

Plasma stability data were reviewed for 8 INDs; several INDsdid not contain in vitro plasma stability.

All 20 INDs were examined for: toxicities in animals, pharmaco-logic relevance of the animal species, and design of toxicologystudies. Of the 20 INDs, 16 were used to obtain the time betweenthe IND submission and reaching an MTD/RP2D/AD.

2.2. Selection of STD10, HNSTD and NOAEL

The IND-enabling studies in rodents and in monkeys were peer-reviewed by one author (HS) to define or confirm the STD10

(rodents), the HNSTD (monkeys) or the NOAEL in both species. Inevery application reviewed, monkeys were the non-rodent specieschosen for toxicity testing. If a dose exceeded the STD10 (e.g. morethan 10% mortality), the next lower dose was defined as the STD10.Similarly, if a dose in the toxicology study in monkeys resulted inunacceptable toxicities, a dose below it was defined as the HNSTD.Estimation of an STD10 or the HNSTD based on modeling as report-ed in some INDs was not done. Eight potential FIH doses were cal-culated from the pivotal toxicology studies using six approaches:1/6th HNSTD (BW), 1/6th HNSTD (BSA), 1/10th HNSTD (BW), 1/10th HNSTD (BSA), 1/10th STD10 (BW), 1/10th STD10 (BSA), 1/10th NOAEL (BW), or 1/10th NOAEL (BSA).

2.3. FIH-dose selection and its proximity to the human MTD

The human MTD or RP2D was obtained from the sponsor’s sub-mission or publicly available information when such informationcould not be obtained from the FDA archive. The date when theMTD/RP2D was first reported and the source of information wererecorded. If the human MTD/RP2D was not reached or the informa-tion was not available, the safety database in FDA’s archival systemwas searched to obtain an ‘‘acceptable dose’’ (AD). An AD is onethat is not clearly identified as being the MTD, but was the nextlowest dose clinically tested. This could be a dose that was expand-ed to further evaluate adverse effects, e.g. a dose above it presentedwith dose-limiting toxicities (DLTs) and hence the sponsor reducedthe dose and expanded the number of patients at the lower dose toevaluate toxicities.

To provide consistent comparison across INDs when assessingthe various approaches to setting the FIH dose, it was necessary

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446 H. Saber, J.K. Leighton / Regulatory Toxicology and Pharmacology 71 (2015) 444–452

to normalize the number of dose escalations required to reach theMTD/RP2D/AD by using an assumption that all escalations were100% (i.e., dose-doubling). The point of this exercise was to exam-ine various approaches to setting a start dose relative to the humanMTD/RP2D/AD, not to assess appropriate escalation schemes. TheFIH dose approach was considered acceptable if it resulted in adose that was below the MTD/RP2D/AD and took at least one dosedoubling to reach the human MTD. Of note, ‘‘1 dose doubling’’being acceptable indicates that the first two dose cohorts did notshow DLTs. The FIH dose algorithm was considered unsafe if thedose obtained was at or above the human MTD/RP2D/AD or if adose doubling of the FIH dose would be above the human MTD/RP2D/AD. While acceptable, the FIH dose may be too low if 4 ormore artificial dose doublings are required to reach the humanMTD; this is based on the observation that in Phase 1 trials of ADCs,except for the first dose escalation, actual escalations were usuallyless than 100% and 4 artificial escalations in our analysis maytranslate into more than 7 actual escalations in a Phase 1 trial.See an example of how the number of escalations to reach thehuman MTD/RP2D/AD was estimated using toxicity data from ratsand monkeys for ADCs #1 and 2 (Table 1).

3. Results

3.1. Design of IND-enabling GLP toxicology studies

For the INDs examined, general toxicology studies were con-ducted in 2 species, a rodent and a non-rodent. The rodent wasmouse or rat; the non-rodent was cynomolgus monkey. The study

Table 1Examples of possible FIH start dose calculations and estimated escalations to reach a hum

GLP toxicology study FIH dos

BSA con

ADC#1MTD = 3 mg/kg (Q3 W dosing)

Cynomolgus monkeyADC, given IV, Weekly � 4, at 1, 3,6 mg/kg

1/6th HThree d2.5 mg/

HNSTD: 6 mg/kg (72 mg/m2) 1/10thFour dohuman

NOAEL: 3 mg/kg (36 mg/m2) 1/10thFour dohuman

RatADC given IV, Weekly � 4, at 3, 10,30 mg/kgSTD10: 30 mg/kg (180 mg/m2)

1/10thTwo dohuman

NOAEL: 3 mg/kg (18 mg/m2) 1/10thkg)Five dohuman

ADC#2RP2D dose = 3.6 mg/kg (Q3 Wdosing)

Cynomolgus monkeyADC, given IV; single dose at 3, 10,and 30 mg/kg

1/6th HOne do

HNSTD = 30 mg/kg (360 mg/m2) 1/10thOne do

NOAEL = 3 mg/kg (36 mg/m2) 1/10thFive do

Cynomolgus monkeyADC, given IV; every 3 weeks � 4 at0, 3, 10, and 30 mg/kg

1/6th HTwo do1/10th

HNSTD = 10 mg/kg (120 mg/m2) Three dNOAEL: not identified 1/10 NRatADC, IV, at 6, 20, 60 mg/kg

1/10th

STD10 = 20 mg/kg (120 mg/m2) Three d

NOAEL: not identified 1/10th

Q3W: every 3 weeks.Note: Each dose escalation is assumed to be 100% increase from the previous dose.

designs used varied greatly; however, a consistent feature was thatall sponsors used 3 (or more) dose levels of the ADC in the pivotalstudy conducted in cynomolgus monkeys. The ADC was not alwaysused in the rodent. Some sponsors also studied the effects of thesmall molecule alone, the linker-small molecule, or the free anti-body. These studies were conducted as separate studies or armswere added into the study conducted with the ADC.

3.2. Toxicities in animals

If the ADC did not bind to the animal target, toxicities in rodentand cynomolgus monkey in the IND-enabling studies wereobserved mainly in the hematopoietic system, liver, and reproduc-tive organs for all ADCs examined and also in the skin and kidneysfor some ADCs (Table 2). Toxicities to the hematopoietic system,liver, and reproductive organs were directly related to the SM.Toxicities to the kidneys observed for some ADCs may be a directeffect or may have been secondary to an immunogenic response.For the ADCs examined, the antibody did not bind to the targetin rodents but many bound to the intended target in cynomolgusmonkey. When the target was minimally expressed in the monkey(e.g. target was a tumor antigen), toxicities were comparable to theINDs where no binding was reported. When the target was highlyexpressed, DLTs were mainly due to the toxicities in thehematopoietic system and liver toxicity did not occur in the IND-enabling toxicology studies, with the only exception being ado-trastuzumab emtansine. While ado-trastuzumab emtansine recog-nizes the target epitope in the monkey and the target is highlyexpressed in this species, hepatotoxicity was evident in single dose

an MTD.

e calculation

version BW conversion

NSTD: 12 mg/m2 (0.3 mg/kg) 1/6th HNSTD: 1 mg/kgose escalations will result inkg

One dose escalation will result in a humandose of 2 mg/kg

HNSTD: 7.2 mg/m2 (0.19 mg/kg) 1/10th HNSTD = 0.6 mg/kgse escalations will result in adose of 3 mg/kg

Two dose escalation will result in a humandose of 2.4 mg/kg

NOAEL: 3.6 mg/m2 (0.1 mg/kg) 1/10th NOAEL: 0.3 mg/kgse escalations will result in adose of 1.6 mg/kg

Three dose escalations will result in a humandose of 2.4 mg/kg

STD10 = 18 mg/m2 (0.5 mg/kg)se escalations will result in adose of 2 mg/kg

1/10th STD10 = 3 mg/kgThis dose is at the human MTD. No doseescalation could be done.

NOAEL = 1.8 mg/m2 (0.05 mg/ 1/10th NOAEL = 0.3 mg/kg

se escalations will result in adose of 1.6 mg/kg

Three does escalations will result in a humandose of 2.4 mg/kg

NSTD: 60 mg/m2 (1.6 mg/kg) 1/6th HNSTD: 5 mg/kgse escalation to 3.2 mg/kg The dose is above the RP2D

HNSTD: 36 mg/m2 (1 mg/kg) 1/10th HNSTD: 3 mg/kgse escalation to 2 mg/kg Dose escalation will result in a dose above the

RP2DNOAEL: 3.6 mg/m2 (0.1 mg/kg) 1/10th NOAEL: 0.3 mg/kgse escalations to 3.2 mg/kg Three dose escalations to 2.4 mg/kgNSTD: 20 mg/m2 (0.5 mg/kg) 1/6 HNSTD: 1.6 mg/kgse escalations to 2 mg/kg One dose escalation to 3.2 mg/kgHNSTD: 12 mg/m2 (0.3 mg/kg) 1/10 HNSTD: 1 mg/kgose escalations to 2.4 mg/kg One dose escalation to 2 mg/kg

OAEL: cannot be done 1/10 NOAEL: cannot be doneSTD10 = 12 mg/m2 (0.3 mg/kg) 1/10th STD10 = 2 mg/kg

ose escalations to 2.5 mg/kg Dose escalation will result in a dose that isabove 3.6 mg/kg human dose

NOAEL: Could not be done 1/10th NOAEL = could not be done

33

Table 2Examples of INDs with prominent organ toxicities observed in nonclinical studies. Each row represents a separate ADC.

Target binding in cynomolgusmonkeys/Linker-SM

Rodents Cynomolgus monkeys

Yes1/mc-MMAF

Hematopoietic system, kidney, testes, skin, lung, liver, uterus Hematopoietic system, kidney, testes, skin, lung, liver, uterus

Yes1/Peptide-based-MED2460

Hematopoietic system, liver, male and female reproductive organs Hematopoietic system, liver, kidney, male and femalereproductive organs

NI2/vc-MMAE

Hematopoietic system, liver, kidney, skin, male reproductive organs Hematopoietic system

No/disulfide-DM4

Hematopoietic system, liver, GI tract, kidney, male and female reproorgans, nerve cells, skin

Hematopoietic system, liver, GI tract, kidney, male and femalereproductive organs, nerve cells, skin

Yes/vc-MMAE

Hematopoietic system, liver, male reproductive organs Hematopoietic system

Yes/vc-MMAE


Yes/vc-MMAE


Yes3/Disulfide-DM4

Hematopoietic system, GI tract liver, eye, skin, neuropathy, male andfemale reproductive organs

Hematopoietic system, GI tract liver, neuropathy, eye, skin

No/Disulfide-DM4

Hematopoietic system, GI tract, liver, male and female reproductiveorgans, skin (lesions)

Hematopoietic system, GI tract, liver, male and femalereproductive organs., skin (lesions)

No/Acid-cleavable-calicheamicin

Hematopoietic system, liver, GI tract, male and female reproductiveorgans, kidney and peripheral neuropathy

Hematopoietic system, liver, GI tract, male and femalereproductive organs

Yes/vc-MMAE

Hematopoietic system, liver, male reproductive organs Hematopoietic system, male reproductive organs

Yesmc-DM1

Hematopoietic system, liver, kidney, male reproductive organs Hematopoietic system, liver, kidney, neuropathy

1 Target is minimally expressed in cynomolgus monkeys.2 NI: no information.3 Minimal binding/mainly surface epithelium cells.


toxicity studies. Neurotoxicity was observed for several ADCswhere the SM was a microtubule inhibitor, as is expected for thisclass of cytotoxic agents.

3.3. Plasma stability

It is assumed that the higher the stability of the ADCs in plasma,the less early release of the small molecule and potentially the lessoff-target toxicities. Plasma stabilities were therefore examined tosee whether lower plasma stabilities resulted in lower STD10,HNSTD, or human MTDs. For many INDs, in vitro plasma stabilitystudies were not conducted, for some of the INDs this was possiblydue to the development programs preceding the publication of theICH S9 guidance. In vitro plasma stability was measured by incuba-tion of the ADC in human or animal plasma, usually for 96 h. Sta-bility was measured by LC/MS/MS or by an ELISA assay. The lownumber of INDs (n = 8) with plasma stability data and differentapproaches used for determination of plasma stability made it dif-ficult to draw a conclusion on the role of plasma stability on toxi-city (animal STD10, HNSTD and human MTD).

Generally, lower plasma stability was reported when the intactADC was measured (e.g. ELISA was used or LC/MS/MS was usedafter capturing the intact ADC through ELISA to measure the con-jugated SMs) and higher stability reported when the amount offree/unbound small molecule was measured (through LC/MS/MSalone). For methods used to detect ADC and SM see Cancer DrugDiscovery and Development, Antibody-Drug Conjugates andImmunotoxins, 2013.

For an ADC utilizing the vc-MMAE platform, the human plasmastability indicated that the amount of intact ADC was approximate-ly 70% using ELISA to detect the conjugated antibody (intact ADC);�60% using LC/MS/MS to detect the conjugated MMAE post-ELISA(intact ADC), and 99% using LC/MS/MS alone to detect the freeMMAE (Table 3). For this ADC, similar findings were observed inplasma from most species. While the two methods for measuringthe intact ADC versus the free MMAE agreed that at least someof the small molecule will be released in plasma, the percent ofADC remaining intact were different.

3.4. Initiation of the IND to the RP2D/MTD

Based on the INDs examined, the median time to reach theMTD/RP2D/AD was 2 years (range < 1 –5 years). However, someINDs had unknown dates of initial patient treatment after IND sub-mission. Some INDs exceeded the MTD and the slow de-escalationcontributed to the length of time required to reach the MTD. Insome cases, a slow escalation scheme appeared to result in alengthy Phase 1 trial. There are many other variables that couldaffect the length of time required to identify the MTD/R2D, includ-ing slow patient accrual, unanticipated safety signals, manufactur-ing problems, and the FIH dose selected. For one of the INDs, thetiming between the submission of the IND and reporting of theDLTs was 9 months. For this IND, the sponsor used 1/20th STD10

(BSA approach) to select the FIH dose and an accelerated dose esca-lation clinical design. However, the MTD was exceeded and treat-ment-related deaths occurred under this IND, indicating a steepthreshold for toxicity of this drug and the need for careful titrationdesigns above certain dose levels. Many of the INDs used a 3 + 3design that is commonly proposed for dose escalations in Phase1 clinical trials (Simon et al., 1997).

3.5. Predicting human MTDs

For a given small molecule, the human MTD appears to remainin a relatively tight range regardless of the antibody target or iso-type, if the linker, the ratio of small molecule-to-antibody, and thefrequency of administration remain the same. For example, forADCs with vc-MMAE given once every 3 weeks with a SM:Ab ratioof approximately 4, the human MTD ranged from 1.8 mg/kg to2.4 mg/kg (Table 4). The human MTD was independent of the anti-body isotype used (IgG1 or IgG2), indicating a limited role for anti-body-mediated effector functions (e.g., ADCC) in causing toxicities.In addition the MTD was independent of the antibody target.Though the number or examples is small (n = 3), DM4-containingADCs with disulfide linkers had human MTDs of 150–168 mg/m2

(4.1–4.5 mg/kg), when given once every 3 weeks or with a compa-rable schedule (e.g. Days 1 and 8, every 28 days for one of the

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Table 3Stability data from one ADC program using vc-MMAE platform: Concentration of ADC after 96 h incubation at 37 �C reported as a percentage relative to time zero.

Species source of plasma Intact ADC measured Intact ADC calculated based on free MMAE

ELISA LC/MS/MS1 Free MMAE measured by LC/MS/MS Intact ADC calculated

Human 71.8 60.1 0.3 99.7Monkey 89.1 55.1 0.2 99.8Rat 73.3 56.6 1.6 98.4Mouse 69.3 64.3 25 75Vehicle 90.5 97.9 0.1 99.9

1 Antibodies (ADC and unconjugated mAb) were isolated by ELISA, MMAE was enzymatically separated from the ADC and the MMAE measured by LC/MS/MS. The MMAEreleased represents those that remained conjugated to the ADC.


INDs). Too few examples were included in our analysis for othersmall molecules (n = 1 or 2) to determine if a narrow MTD orRP2D range holds true for their linker-SM subgroups.

Our analysis suggests that prior human clinical experience forADCs utilizing vc-MMAE given every 3 weeks with SM:Ab ratio of4 may inform the starting dose and escalation plan for subsequentADCs utilizing this platform. The importance of using prior clinicaldata (when available) from other ADCs using the same linker andSM cannot be overstated as human toxicities seen with ADCs canbe severe. For ADCs using the vc-MMAE platform, severe motorand sensory neuropathy as well as bone marrow toxicity, includingtreatment-related deaths, have been seen at doses above 1.8 mg/kggiven on an every 3 week cycle.

For the MMAE and DM4 containing ADCs, the safety profile inanimals was also independent of the antibody isotype or the target

Fig. 1. Data from 8 INDs; for one of the INDs two sets of data are presented.

Fig. 2. Human MTD/RP2D/AD fall within narrow range based on linker-small molecule mX-axis represents an IND.

of the antibody but the HNSTDs did not appear to have a similarlytight range as the human MTDs (Tables 4 and 5). This may be dueto variable frequency of administration used in animals, the largegap between the doses, and lack of pre-specified definition of DLTsfor toxicology studies. There were some consistencies noted how-ever: for vc-MMAE ADCs administered once every 3 weeks for 4doses, the HNSTD ranged from 5 to 6 mg/kg; for single dose studiesusing disulfide linker-DM4 ADCs, the HNSTDs were 10–15 mg/kg.A graphical view of HNSTD (monkeys) and MTD (humans) rangesis presented in Figs. 1 and 2, respectively.

The gap between the doses (low dose versus mid dose versushigh dose) may be too wide in animals to accurately determinethe HNSTD. If there is mortality or severe toxicities at a dose level,the dose below it is defined as the HNSTD, even if this lower doseshows no toxicity. This may occur when there is a large gapbetween the two dose levels. Thus, the HNSTD is the highestnon-severely toxic dose ‘‘tested’’ in that study. Finally, the defini-tion of HNSTD is subjective. There are no set criteria to defineseverity of toxicities in animals, for instance in terms of percentchanges in clinical pathology parameters. In patients, clinical andlaboratory adverse events are monitored utilizing CTCAE criteriaand pre-specified dose-limiting toxicities are defined. The dose isthen adjusted until a true MTD is achieved.

3.6. FIH-dose approach and the number of escalations to reach thehuman MTD

When using the BSA approach for animal-to-human dose con-version, 1/6th the HNSTD and 1/10th HNSTD in cynomolgus mon-keys or 1/10th the STD10 in rodents resulted in a median of 2.5–3dose doublings to reach the human MTD/RP2D/AD (see Table 6).

oiety. Linkers: vc for MMAEs; disulfide for DM4; mc for MMAF. Each number on the

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Table 4Nonclinical HNSTD and human MTD for vc-MMAE-containing ADCs utilizing an every 3 week administration schedule.

SM: Ab Antibody isotype Animal dosing schedule HNSTD (mg/kg) Human dosing schedule Human MTD/RP2D/AD

3.2–4.8 Human IgG1 Every other week � 3 9 Q3W 2.3 mg/kg (only 1 patient received this dose)�3.5 Human IgG2 Q3 W x 4 6 Q3W *1.8 mg/kg�3.5 Humanized IgG1 Single dose 3 Q3W 2.4 mg/kg�3.5 Humanized IgG1 Q3W � 4 5 Q3W 2.4 mg/kg�4 Chimeric IgG1 Q3W � 11 3 Q3W 1.8 mg/kg�4.5 Human IgG2 Weekly � 4 1 Q3W 1.88 mg/kg�4 Human IgG1 Q3W � 4 5 Q3W 2.3 mg/kg�3.7 Humanized IgG1 Q3W � 4 5 Q3W 2.4 mg/kg

* The MTD is also dependent on the frequency of administration. When the frequency of dosing was increased to weekly administration, the MTD dropped to 1.2 mg/kg forthis ADC.

Table 5Monkey HNSTD and human MTD for disulfide bond-DM4-containing ADCs.

SM:Ab Antibody isotype Animal dosing schedule HNSTD (mg/kg) Human dosing schedule Human MTD/RP2D/AD

3.5 Humanized IgG1 Single dose 10 Days 1/8 every month 150 mg/m2 (4.1 mg/kg)3.9 Humanized IgG1 Single dose 10 Q3W *160 mg/m2 (4.3 mg/kg)3.5 Humanized IgG1 Single dose 15 Q3W 168 mg/m2 (4.5 mg/kg)

* The MTD is also dependent on the frequency of administration. A weekly administration reduced the MTD to 55 mg/m2.

Table 6Median number of escalations and the range for approaches that could be potentially used to select the FIH dose.

*Approach Species Median number for dose doublings to reach human MTD/RP2D/AD Range for dose doubling to reach human MTD/RP2D/AD

1/6th HNSTD (BSA) Monkey 2.5 1–51/10th HNSTD (BSA) Monkey 3 1–51/10th STD10 (BSA) Rodent 3 2–41/10th NOAEL (BSA) Monkey 5 4–71/10th NOAEL (BSA) Rodent 6 5–71/10th NOAEL (BW) Monkey 4 2–51/10th NOAEL (BW) Rodent 3 3–5

* One ADC containing the first generation of linker was excluded. This linker is no longer used in ADCs.


For one IND, 1/6th HNSTD (BSA approach) was below but close tothe human MTD; this ADC, however, contained the first generationof linker which is unstable and no longer used in any ADCs. ThisIND was excluded from this analysis as the linker is no longer uti-lized. When using the BW approach for conversions, 1/6th theHNSTD (6 INDs) or 1/10th the HNSTD (2 INDs) in monkeys, or 1/10th the STD10 in rodents (10 INDs) resulted in a FIH dose thatwas above the MTD or too close to the human MTD such that adose doubling could not be done. One-tenth the NOAEL incynomolgus monkeys or rodents using the BW approach resultedin a median dose doublings of 3–4 to reach the human MTD/RP2D/AD. One-tenth the NOAEL using the BSA approach resultedin a median of 5–6 dose doublings (range of 4–7) to reach thehuman MTD/RP2D/AD.

For one IND, the sponsor selected the FIH dose of the ADC basedon the doses of the free antibody previously administered topatients under a separate IND. The free antibody was given at dos-es up to 8 mg/kg to patients with acceptable toxicities; however,the dose of ADC which contained that same antibody resulted intwo DLTs at 4 mg/kg and the ADC dose was hence de-escalated.This further re-emphasizes that the conjugated small moleculedrives the human toxicity making the free antibody less informa-tive for FIH dose decisions. Therefore, it is recommended that theFIH dose of the ADC not be based on the doses of the free antibody.

4. Conclusions and discussion

Over the years the improvement in linker technologies hasresulted in ADCs with improved stability. More stable productstogether with the understanding of new targets in sub-populationsand two recent ADC approvals have encouraged sponsors to

develop more ADCs. Our review has identified that there is littleconsistency in the design of toxicology studies and approachestaken to select the FIH dose. Based on accumulating data fromnon-clinical and clinical development, we have generated severalconsiderations for future ADC development.

4.1. Toxicology studies to support FIH trials

A common feature of nonclinical programs to support clinicaltrials was to conduct toxicology studies in the non-human primate(NHP) with 3 (or more) doses of the ADC. While this is done withthe clinical candidate, one sponsor conducted the toxicology studyin the NHP using arms of both the clinical candidate and thecynomolgus surrogate. A toxicology study with the clinical candi-date is considered sufficient at this time as results indicate thatdose limiting toxicities are related to the small molecule, indepen-dent of target binding. Conducting the study with a surrogate mayalso need additional characterization of the surrogate (e.g. epitopebinding, activity and potency, and PK) to find the relevance of theresults and how to use the data for FIH dose selection. The toxicol-ogy study in the NHP with the clinical candidate has providedsufficient information to set the human starting dose and to gainknowledge on organ toxicities for clinical monitoring. For INDsexamined, when the ADC was used in the rodent, the study couldalso be used to select the FIH dose and to define the toxicity profileof the product. In general, the studies conducted in rodents variedgreatly across INDs examined. For several ADCs, the sponsors con-ducted a study with the free small molecule in a rodent species.The advantage of the study with the free small molecule is thatthe sponsor can cross-reference to that IND for another submissionin which the same small molecule is linked to a different antibody,

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which simplifies the design of a nonclinical study and also reducesanimal use. Investigating the toxicities associated with the freelinker may not be necessary; toxicology studies conducted withthe ADC is expected to identify potential toxicities associated withthe linker. Moreover, toxicities were comparable and related to thesmall molecule when both arms of the SM and linker-SM wereincluded in the toxicology studies, indicating that linker-relatedtoxicities may be minimal compared to toxicities related to thesmall molecule.

In clinical trials, ADCs were usually dosed weekly, every otherweek, or every 3 weeks. At times, the sponsors conducted a morefrequent dosing in animals (e.g. weekly) if they had uncertaintiesregarding the clinical schedule and to potentially dose patientsmore frequently, hence to support multiple schedules in patients.

In line with ICH S9, some sponsors conducted single dose toxi-cology studies to support every-3-week dosing in patients. Inregard to FIH dose selection, single dose animal toxicology studiessupported a dosing schedule of once every 3 weeks for ADCs whenthe BSA approach was used for scaling (based on 5 single dosestudies in rodents and 5 single dose studies in cynomolgus mon-keys). Since the number of INDs with single-dose administrationis small, more data needs to be collected to ensure that single dosestudies support every-3-week schedule of administration inpatients. For one of the INDs where an old generation acid-cleav-able linker was used, 1/6th HNSTD based on BSA resulted in aFIH dose that was too close to the human MTD. However, this link-er is cleaved by esterases, resulting in a relatively unstable ADCwith a short half-life; this linker is no longer used.

4.2. Selection of FIH dose based on animal data

For small molecules, a common approach, and one discussed inICH S9, has been to use 1/10th the severely toxic dose to 10% of theanimals (STD10), identified in rodents (DeGeorge et al., 1998).When the non-rodent is used to set the clinical start dose, thenthe dose chosen is generally 1/6th the highest non-severely toxicdose (HNSTD). The approach used to select the FIH dose for smallmolecules in oncology is acceptable for ADCs. The approach of 1/10th NOAEL using BW for conversion, (used for certain biologicalproducts) is also acceptable. In addition, 1/10th HNSTD using BSAfor scaling is acceptable as it produced results that were compara-ble to 1/6th HNSTD (BSA approach) and 1/10th STD10 (BSAapproach). One-sixth the HNSTD, 1/10th the HNSTD and 1/10thSTD10 using BW for conversion may not be safe, as this may resultin a FIH dose that is at or above the human MTD, or slightly belowbut too close to the human MTD. One-tenth the NOAEL using theBSA for conversion may be too conservative, resulting in a medianof 5–6 (range of 4–7) dose doublings to reach the human MTD.Most Phase 1 FIH studies utilize a Fibonacci-type algorithm fordose escalation. While the first one or two escalations may utilize100% escalations, additional dose increments are typically less than100%. Therefore, when 1/10th the NOAEL BSA approach is used toset the FIH dose, the number of escalations to reach the humanMTD may be more than 7 steps. Hence this tradeoff in safety (lowerstarting dose) may be offset by a sub-therapeutic dose in the firstfew cohorts as well as an increased time to reach the MTD. Becausethe SM mediates the toxicities of ADCs, the FIH dose should not bebased on doses of the free antibody studied in animals or previous-ly tested in humans.

In a recent review, Ponce (2011) assessed the data of LeTourneau et al. (2010) and concluded that by using the 1/10thSTD10 approach the frequency of unacceptable toxicity in Phase 1trials was low. In the analysis of Le Tourneau, of 81 molecularlytargeted agents studied, the median number of dose levels to reachan MTD or maximum administered dose (MAD) was 5 (range of 1–14 dose levels). For large molecules when no pharmacologically-

relevant animal model is available, or for biologicals with immuneagonist properties, the start dose is usually conservative, e.g.,defined by using an approach based on minimally anticipated bio-logical level (MABEL). For biopharmaceuticals with a pharmaco-logically relevant animal species, when the product is not animmune agonist and drug-related toxicities have been clearlydetected in animals, 1/10th of NOAEL has been a commonly usedapproach to select the FIH dose. Regardless of which method isused, all available scientific data (including pharmacokinetics andpharmacodynamics) are also used to justify the proposed humandose.

4.3. Toxicities observed in patients treated with FDA approved ADCs

There have been three ADCs approved for use in the UnitedStates. The first approved (in 2000), Mylotarg (gemtuzumabozogamicin), is composed of a recombinant humanized IgG4 anti-body to CD33 conjugated to calicheamicin using an acid-cleavablelinker. Calicheamicin binds to DNA creating double-stranded DNAbreaks and CD33 is an antigen expressed primarily on cells of mye-loid lineage. Mylotarg was approved under the acceleratedapproval pathway in 2000 for treatment of acute myeloid leukemia(AML) (Bross et al., 2001). The application was subsequently with-drawn in 2010 because the post-marketing study required foraccelerated approval failed to verify the clinical efficacy of Mylo-targ, particularly in the context of severe toxicities that becamemore apparent in the post-marketing period (Przepiorka et al.,2013).

Upon internalization of the ADC, the calicheamicin is expectedto be released by hydrolysis of the hydrazone in lysosomes of theCD33-positive target cells. However, esterases and carbonyl reduc-tase are also reported to be important in the metabolism/hy-drolysis of gemtuzumab ozogamicin. This may result in a higherthan expected release of the small molecule, resulting in the smallmolecule-related toxicities. The rather short half-life of Mylotarg(1–2 days) compared to ADCs with newer generation linkers fur-ther supports a lower stability compared to contemporary ADCplatforms (Ducry and Stump, 2010). Toxicities seen in human sub-jects receiving Mylotarg are thought to be related to calicheamicinincluding severe and fatal liver toxicity, hepatic veno-occlusive dis-ease, and pulmonary toxicity. Post-marketing data also revealedcases of severe hypersensitivity reactions, including fatalities, indi-cating that the antibody component may also contribute totoxicity.

Adcetris (brentuximab vedotin) is a CD30-directed ADC utiliz-ing a chimeric IgG1 antibody attached to the microtubule inhibitor,monomethyl aurastatin E (MMAE) via a protease cleavable valine–citrulline (vc) linker. Adcetris was granted accelerated approved in2011 for treatment of Hodgkin lymphoma (HL) and systemicanaplastic large cell lymphoma (ALCL) based on high rates of dur-able objective responses in single arm trials (de Claro et al., 2012).Adverse events for this ADC related to MMAE, include neutropeniaand potentially severe cumulative peripheral sensory and motorneuropathy. During post-marketing surveillance, Adcetris-relatedhepatotoxicity was identified as a new safety issue and the FDAlabel was updated to include hepatotoxicity in the warnings andprecautions section. (FDA, 2014).

Kadcyla (ado-trastuzumab emtansine) is an ADC targeting Her2(Poon et al., 2013; Amiri-Kordestani et al., 2014). The antibody moi-ety is a humanized anti-HER2 IgG1, trastuzumab, which is covalent-ly linked to the microtubule inhibitor DM1 (a maytansine derivative)via a protease-resistant, the thioether linker MCC (4-[N-maleimido-methyl] cyclohexane-1-carboxylate). Kadcyla was approved in 2013for treatment of HER2-positive, metastatic breast cancer. The boxedwarning of Kacyla also contains information on hepatotoxicity. Theelimination half-life in humans is 3.5–4 days. Protease-resistance

37


(aka non-degradable) linkers are expected to increase the half-life ofthe ADC; however, this did not hold for Kadcyla which shows compa-rable or slightly reduced half-life compared to Adcetris.

Overall, there are common toxicities in humans many of whichare related to the small molecule moiety, e.g. myelosuppressionand related effects (neutropenia, sepsis, and hemorrhage), hepato-toxicity, and neurotoxicity. Infusion-related toxicities reported inpatients may be related to the protein component and the largesize of these molecules. While the ADC platforms are designed todeliver these potent cytotoxic agents to the tumor, it is clear thatsystemic toxicities consistent with the small molecule are a risk.The cumulative nature of some adverse events such as neuro-toxicity further complicate optimal dose selection for those treat-ments intended to be administered chronically until diseaseprogression.

4.4. Toxicity profile and MTDs

Based on our review of IND-enabling GLP toxicology studies,when there was no binding to the epitope, toxicities occuredmainly in the liver, hematopoietic system, and reproductiveorgans for all INDs examined, and in the skin (lesions) or inthe kidney for a few INDs. While not common, additional toxici-ties occasionally occurred, e.g. ocular toxicity (Table 2). Whenthe ADC binds to its epitope, as was the case for 5 ADCs testedin monkeys, DLTs observed in monkeys were mainly related totoxicities in the hematopoietic system. The only exceptionappears to be Kadcyla, where liver toxicity was prominent aftera single dose administration in the monkey, even though it doesbind to the monkey Her-2. While this finding is not clearlyunderstood, possible reasons for this exception may include theADC not readily accessing the target and hence high amountsof circulating ADC are taken up by the liver, or the ADC beingrelatively unstable (or a fraction of the ADC being less stable)resulting in some early release of DM1.

For the approved ADCs, many of the toxicities reported, espe-cially those considered dose limiting, are related to the small mole-cule moiety and include bone marrow suppression, hepatotoxicityand neuropathy. As toxicities are related mainly to the small mole-cule and the release of the small molecule is dependent on the link-er used for conjugation, for ADCs using the same linker and smallmolecule, prior human data can be informative. For instance, thedose-finding trials for vc-MMAE containing ADCs have demon-strated similarities with respect to dose-related toxicities. Basedon data from 8 ADC development programs utilizing vc-MMAEplatforms, the recommended Phase 2 human doses are between1.8 mg/kg and 2.4 mg/kg when the ADC is given every 3 weeks.Doses above 2.4 mg/kg have been poorly tolerated, including casesof severe bone marrow toxicity, septic deaths and severe motorneuropathy and doses below 1.0 mg/kg have reported very fewdose-limiting toxicities. With regard to ADC development utilizingvc-MMAE platforms with a SM:Ab ratio of approximately 4, dosesabove 2.4 mg/kg should be explored with extreme caution. Fur-thermore, the peripheral neuropathy seen with MMAE is frequent-ly cumulative; necessitating careful dose modification rules forneuropathy.

4.5. Factors defining the human MTD

Factors that could affect the MTD include the chemistry of thelinker, dose schedule, the small molecule to antibody ratio, andthe small molecule itself. Our review suggests that the antibodyitself does not appear to have a large effect on the MTD. Withrespect to linker, the first generation linkers (acid-cleavable hydra-zone) produced ADCs with a short half-life (e.g. 1–2 days) and lowstability. A linker may increase or decrease the small molecule-re-

lated toxicities by changing the stability of the ADC and result inincreased or reduced release of the small molecule in the plasma.Ducry and Stump (2010) examined the effect of linkers on the plas-ma half-life and concluded that a hydrazone linker (as was used inMylotarg) results in a shorter half-life when compared to two otherlinkers, phenylalanine-lysine and valine–citrulline. While plasmastability data were examined with an to attempt to identify anyrelationship between linker stability and the human (or animal)MTD, no conclusion could be made based on limited data availableand different experimental methods used. It was noted that whenthe intact ADC was measured, plasma stability was reported to beless than when the free small molecule was measured. Further dia-log may be needed with industry regarding the value of varioustechniques employed and to harmonize the approach used formeasuring plasma stability.

Based on human and animal toxicity data, the small moleculeappears to dictate the dose limiting adverse events seen, and there-fore the MTD. The higher the SM: Ab mean ratio, the lower theMTD is anticipated to be. For the INDs examined the ratio wasapproximately 4. Other potential factors may include prior treat-ments; e.g. for ADC containing doxorubicin as the small molecule,prior treatment with doxorubicin may reduce the threshold forcardiac toxicity.

In summary, despite great interest in ADC development, thereremains little uniformity regarding best practices for designinganimal toxicity studies, calculating a FIH starting dose or Phase 1clinical dose escalation strategies. Our review finds that for calcu-lation of human starting dose, 1/6th the HNSTD in cynomolgusmonkeys or 1/10th the STD10 in rodents using the BSA approachfor animal-to-human conversion generally resulted in an accept-able balance of safety and efficiency in dose escalation. Otheracceptable approaches are 1/10th HNSTD in cynomolgus monkeysusing the BSA scaling and 1/10th NOAEL in rodents or monkeysusing the BW scaling. FIH dose selection algorithms that were con-sidered unsafe consist of: 1/6th HNSTD, 1/10th HNSTD, and 1/10thSTD using BW for animal-to-human dose conversions. While theanimal data for the vc-MMAE platform yielded a variable rangeof HNSTDs in cynomolgus monkeys, the human MTD were report-ed with a tighter range and there was a steep dose-dependentthreshold for toxicities in patients. Overall, for all the INDs includ-ed in our analysis, there was a good correlation between HNSTDand human MTD. For ADCs sharing the small molecule drug, thesame linker, and the same SM:Ab ratio, available prior clinical datacan inform the design of a safe yet efficient Phase 1 clinical trialdesign. As the database available for nonclinical and clinical stud-ies using novel ADCs grows, additional analysis will be useful toinform ADC drug development program design andimplementation.

Acknowledgments

The authors would like to thank Paul Kluetz and Anthony Mucciof the Food and Drug Administration for their constructivecomments.

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