antibodies

Review
Factors Affecting the Pharmacology of
Antibody–Drug Conjugates
Andrew T. Lucas 1,2,3 , Lauren S. L. Price 1 , Allison N. Schorzman 1 , Mallory Storrie 2 ,
Joseph A. Piscitelli 2 , Juan Razo 2 and William C. Zamboni 1,2,3, *
1 Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy,
University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; andrew_lucas@unc.edu (A.T.L.);
lslprice@email.unc.edu (L.S.L.P.); aschorz@email.unc.edu (A.N.S.)
2 UNC Eshelman School of Pharmacy, Chapel Hill, NC 27599, USA; mastorrie@gmail.com (M.S.);
joseph_piscitelli@unc.edu (J.A.P.); juanrazo@email.unc.edu (J.R.)
3 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill,
NC 27599, USA
* Correspondence: zamboni@email.unc.edu; Tel.: +1-919-843-6665; Fax: +1-919-966-5863

Received: 14 December 2017; Accepted: 1 February 2018; Published: 7 February 2018

Abstract: Major advances in therapeutic proteins, including antibody–drug conjugates (ADCs),

have created revolutionary drug delivery systems in cancer over the past decade. While these
immunoconjugate agents provide several advantages compared to their small-molecule counterparts,
their clinical use is still in its infancy. The considerations in their development and clinical use
are complex, and consist of multiple components and variables that can affect the pharmacologic
characteristics. It is critical to understand the mechanisms employed by ADCs in navigating biological
barriers and how these factors affect their biodistribution, delivery to tumors, efficacy, and toxicity.
Thus, future studies are warranted to better understand the complex pharmacology and interaction
between ADC carriers and biological systems, such as the mononuclear phagocyte system (MPS) and
tumor microenvironment. This review provides an overview of factors that affect the pharmacologic
profiles of ADC therapies that are currently in clinical use and development.

Keywords: antibody–drug conjugates; mononuclear phagocyte system; pharmacokinetics;

pharmacology; therapeutic proteins

1. Introduction
The number of available carrier-based drug systems for the treatment of cancer and other
diseases has seen exponential growth in the past three decades. As of 2013, there are more than
1600 nanotechnology-based products in the market, and almost 250 nanomedicine agents on the
market or in clinical trials, with more emerging at a rapid pace [1]. In addition, within the past 20 years,
the use and approval of monoclonal antibodies (mAbs) has risen sharply, both in the clinic as well as
in development, to advance a revolution in immunotherapy. While early mAb therapies were plagued
with toxicities due to immunogenicity, modern genetic engineering has led to the human/humanized
antibodies that we use today [2]. Research into antibody–drug conjugates (ADCs), conjugating highly
potent cytotoxic agents to targeted mAbs, has become a very active area of drug development for
the treatment of cancer. There are currently ~50 ADCs in 125 clinical trials with ~35 different ADC
formulations in >50 phase I/II studies in patients with solid tumors (Table 1) [3].
Even though these ADCs have been used for nearly two decades, there is still much to learn
about the factors that affect the disposition of ADCs and antibody-based therapies. Understanding the
factors affecting pharmacokinetic (PK) and pharmacodynamic (PD) variability, the exposure–response
relationship, and evaluating potential methods to individualize therapy of ADCs are essential to

Antibodies 2018, 7, 10; doi:10.3390/antib7010010 www.mdpi.com/journal/antibodies

Antibodies 2018, 7, 10 2 of 28

increasing their efficacy and reducing the toxicity of these agents. Furthermore, determining preclinical
and clinical toxicity and safety remain major challenges, due to the different properties between
protein-based and small molecule drugs. The high PK variability is clinically important for mAbs, and
especially for ADCs, as these agents have a narrow therapeutic index. In addition, the combination
of ADCs with other mAbs and immune modulators has a high likelihood of causing drug–drug
interactions, as these agents all undergo clearance by the mononuclear phagocyte system (MPS).
Thus, the evaluation of biomarkers of the MPS function, Fc receptors, and mediators and drug
exposures in MPS cells, are critically important to optimizing the treatment of ADCs alone and in
combination with other agents. In this review, we will summarize the factors that have been shown to
affect the pharmacokinetics (PKs) and pharmacodynamics (PDs) of mAb and ADC therapies, and the
goals of future research to better understand and predict their disposition.

2. Formulation Considerations
The use of antibodies as therapeutic agents or targeted carriers is a popular technique in hematology
and oncology, as demonstrated by the rapidly growing list of approved antibody-based drugs.
However, the majority of these therapies still rely on co-administration with traditional chemotherapy
to achieve meaningful clinical responses, and many others have demonstrated lower than anticipated
clinical efficacy. Thus, a significant effort has been devoted to enhancing mAb therapies through
various modifications, such as ADCs. These immunoconjugates are designed to exploit the specificity
of monoclonal antibodies to deliver potent cytotoxic drugs to tumors while limiting off-target exposure.
The drugs conjugated to the antibody are highly potent (IC50 < 10−9 M), and thus, are unable to be
safely dosed systemically without a carrier. The four current FDA-approved ADCs are ado-trastuzumab
emtansine (Kadcyla® , Genentech Inc., South San Francisco, CA, USA), brentuximab vedotin (Adcetris® ,
Seattle Genetics, Inc., Bothell, WA, USA), gemtuzumab ozogamicin (Mylotarg® , Pfizer Inc., Philadelphia,
PA, USA), and inotuzumab ozogamicin (Besponsa® , Pfizer Inc., Philadelphia, PA, USA). Examples of
ADCs in clinical trials include mirvetuximab soravtansine (an anti-folate receptor alpha-maytansinoid
conjugate) and ABT-414 (an anti-EGFR-auristatin conjugate) [4]. The structure and pharmacology of
ADCs are fundamentally different compared to standard small molecule (SM) drugs. These fundamental
differences between ADC agents and SM drugs are essential in understanding the differences in PK
and PD disposition of these agents.

2.1. Monoclonal Antibody Selection

The most common native antibody found in circulation is IgG, and the most commonly used
IgG isotype for therapeutic use is IgG1 [5]. The strength of the various effector functions varies
depending on the isotype of IgG antibody selected [6,7]. For instance, induction of antibody-dependent
cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) are stronger with
IgG1 and IgG3 isotypes, compared to other isotypes. Whereas, IgG3 is more efficient at inducing tumor
cell lysis despite having a relatively shorter half-life compared to IgG1/2/4 [6,7]. IgG4 antibodies also
have the unique trait of being able to exchange one-half of themselves with another IgG4, meaning
they can form new hybrids in circulation [8]. Finally, IgG2 antibodies have been targeted as potentially
favorable for therapeutic use, due to their tendency to form covalent dimers. Dimerization aids in
antibody–antibody associations, and can enhance the affinity and internalization of the antibody [6,9].
The proper selection of an antibody is important, especially as part of an ADC, as the antibody can
retain native/physiologic functions including immune activation, such as ADCC and CDC, or signal
inhibition or modulation [3,5].
Antibodies 2018, 7, 10 3 of 28

Table 1. Antibody–drug conjugates approved and under investigation (Phase II or higher).

Generic Name Brand Name Manufacturer Phase/Studies Open Target Antigen Linker Payload Indications
Brentuximab vedotin Adcetris Seattle Genetics Approved CD30 Cleavable (protease) MMAE Hematological
Gemtuzumab ozogamicin Mylotarg Pfizer Approved CD33 Cleavable (acid labile) Calicheamicin Hematological
Inotuzumab ozogamicin Besponsa Pfizer Approved CD22 Cleavable (acid labile) Calicheamicin Hematological
Trastuzumab emtansine Kadcyla Genentech Approved HER2 Non-cleavable DM1 Solid
Generic Name Investigational Name Manufacturer Phase/Studies Open Target Antigen Linker Payload Indications
Mirvetuximab Soravtansine IMGN-853 ImmunoGen I, II, III FOLRI 1 Cleavable (disulfide) DM4 Solid
Polatuzumab vedotin DCDS-4501A Genentech I, II, III CD79b Cleavable (protease) MMAE Hematological
Rovalpituzumab tesirine SC0001-SCX Stemcentrx I, I/II, II, III DLL3 Cleavable (protease) SCX Solid
Sacituzumab govitecan IMMU-132 Immunomedics I/II, II, III TROP2 EGP1 Cleavable (acid labile) SN-38 Solid
- AGS-16C3F Agensys II AGS-16/ENPP3 Non-cleavable MMAF Solid
Denintuzumab mafodotin SGN-CD19a Seattle Genetics II CD19 Non-cleavable MMAF Hematological
PSMA ADC - Progenics II PSMA Cleavable (protease) MMAE Solid
Anetumab Ravtansine BAY 94-9343 Bayer Healthcare I, I/II, II Mesothelin Cleavable (disulfide) DM4 Solid
Depatuxizumab Mafodotin ABT-414 Abbvie I, II EGFR Non-cleavable MMAF Solid
Enfortumab Vedotin ASG-22CE Astellas Pharma I, II Nectin 4 Cleavable (protease) MMAE Solid
Glembatumumab vedotin CDX-011 Celldex I/II, II gpNMB Cleavable (protease) MMAE Solid
Labetuzumab govitecan IMMU-130 Immunomedics I, II CEACAM5 Cleavable (acid labile) SN-38 Solid
Tisotumab Vedotin HuMax-TF Genmab Seattle Genetics I/II, II Tissue Factor Cleavable (disulfide) MMAE Solid
- CDX-014 Celldex I/II TIM-1 Cleavable (disulfide) MMAE Solid
- CX-2009 Cytomx I/II CD166 Cleavable (protease) DM4 Solid
- DT2219ARL OXS-1550 GT Biopharma I/II CD19 & CD22 Cleavable (protease) Modified diphtheria toxin Hematological
- HuMax-AXL Genmab I/II AXL Cleavable (protease) MMAE Solid
Indatuximab ravtansine BT-062 Biotest I/II CD138 Cleavable (disulfide) DM4 Hematological
Pinatuzumab vedotin DCDT-2980S Genentech I/II CD22 Cleavable (protease) MMAE Hematological
Abbreviations: MMAE, monomethyl auristatin E; DM1, mertansine; DM4, ravtansine; SCX, tesirine; MMAF, monomethyl auristatin F.
Antibodies 2018, 7, 10 4 of 28

2.2. Target Antigen

The selection of a target antigen is important, as it allows the antibody to recognize a target
on a tumor. An ideal target antigen is highly expressed with limited heterogeneity across a tumor,
has low expression on normal tissue, minimal antigen shedding to prevent binding with mAb in
circulation, and is well internalized by receptor-mediated endocytosis [3,6]. Baselga et al. reported
in patients with lymphoma and prostate cancers that a minimum value of tumor-antigen density
is a prerequisite for mAb/ADC efficacy [3]. However, another study has shown that efficacy is
independent of antigen density. Perez et al. found evidence that target antigens with low expression
can be effective as well [5,6]. This highlights that a desirable cutoff value and minimum threshold
for desired antigen density is highly variable and depends on many factors, such as internalization
rate and binding affinity [3]. Several recent studies have focused on targeting parts of the tumor
microenvironment to help increase the delivery of these agents to the tumor, to provide a more robust
anti-tumor effect [10–14].
Generally, experimental evidence would suggest that a correlation exists between available
antigen expression and ADC efficacy. However, an in vitro study evaluating an anti-CD22-DM1 ADC
against 18 different non-Hodgkin lymphoma (NHL) cell lines showed that sensitivity to the cytotoxic
payload (i.e., DM1) was more predictive of response than antigen expression levels [15]. This suggests
that patient selection based only on antigen expression may not guarantee anti-tumor efficacy, and
additional tumor and non-tumor markers may be necessary to select patients for ADC therapy.

2.3. Cytotoxic Payload

Compared to “naked” mAbs, ADCs also contain SM drugs conjugated to the targeting mAb.
These cytotoxic payloads are often highly potent, and are targeted to enter the tumor cells by nature of
antibody-mediated endocytosis, eventually resulting in the intracellular release of the drug from the
mAb carrier [3]. The potency of these agents (in the nM or pM range) typically preclude their use as
traditional SM intravenous therapies, due to systemic toxicity [16].
The first generation of ADCs contained payloads that were already traditionally used for
chemotherapy, such as doxorubicin and methotrexate, and other microtubule inhibitors and
DNA-damaging drugs [3]. Studies proved that first generation ADCs had limited success, with
high toxicity, poor chemical properties, and only 1–2% of the drug reaching the target tumor [3,17].
The second generation of ADCs focused on stabilizing linkers and payloads that were 100- to
1000-fold more potent than those in the first generation [18]. Second generation ADCs are exemplified
by ado-trastuzumab emtansine (Kadcyla® , Genentech Inc., South San Francisco, CA, USA) and
brentuximab vedotin (Adcetris® , Seattle Genetics, Inc., Bothell, WA, USA), which have a more stable
linker between the mAb and drug, and are FDA-approved ADCs. For example, ado-trastuzumab
emtansine contains the linker maleimidomethyl cyclohexane-1-carboxylate (MCC), which allows
the cytotoxic drug to remain stable in circulation until it is released into the tumor, improving
the overall therapeutic index of the ADC (Kadcyla, Genentech Inc., San Francisco, CA, USA).
Despite improvements and regulatory success of second generation ADCs complex issues still remain
in the selection mAb, linker, and active drug.

2.4. Linkers
To attach a cytotoxic payload to a mAb, the use of a chemical linker is required. Linkers are
comprised of functional groups, such as disulfides, hydrazones, and thioethers, to connect the mAb and
SM drug, and control the release, distribution, and delivery of the cytotoxic agent to the target cell [6,19].
If a drug in released from the mAb carrier prior to delivery to the tumor, it can cause severe off-target
toxicities by killing healthy cells. Because these linkers play an essential role in determining the PK
and therapeutic index of an ADC, they should ideally be stable enough to allow the ADC to release
the drug efficiently, but only at the targeted cells. Hydrophilic polyethylene glycol (PEG) linkers, used
Antibodies 2018, 7, 10 5 of 28

as Antibodies
a single 2018,
chain7, xor
FORarrangement,
PEER REVIEW are particularly attractive as a conjugation linker to improve 5 ofADC
28
solubility or reduce ADC aggregation [6,20]. Typically, linkers used in ADC formulations are classified
as improve
being either ADC “cleavable”
solubilityoror “non-cleavable”. Cleavable linkers
reduce ADC aggregation [6,20].respond to physiological
Typically, linkers usedstimuli,
in ADC such
as formulations are classified
low pH or proteolytic as beingtoeither
cleavage, “cleavable”
release or “non-cleavable”.
the cytotoxic drug from its ADC Cleavable linkers
carrier [21].respond
In theory,
to physiological
these stimuli, in
linkers are catalyzed such as lowcells,
a tumor pH ordue proteolytic cleavage,
to the presence, or to release the
increased cytotoxic
presence, drugcatalyst,
of their from
its ADC carrier [21]. In theory, these linkers are catalyzed in a tumor cells,
to allow for selective release of the cytotoxic agent [5,21]. An example of a cleavable linker is seen due to the presence, or in
increased presence,
brentuximab vedotin, of their the
where catalyst, to allow auristatin
monomethyl for selective release ofcytotoxic
E (MMAE) the cytotoxic agentis[5,21].
payload releasedAnby
example of a cleavable linker is seen in brentuximab vedotin, where the monomethyl
protease activity (Adcetris, Settle Genetics, Seattle, WA, USA). On the other hand, non-cleavable linkers auristatin E
(MMAE) cytotoxic payload is released by protease activity (Adcetris, Settle
rely on lysosomal degradation to release the cytotoxic payload after an ADC has been internalized by Genetics, Seattle, WA,
theUSA). On the other hand, non-cleavable linkers rely on lysosomal degradation to release the cytotoxic
cell [21]. In the lysosomal compartment, the process of intracellular proteolytic degradation reduces
payload after an ADC has been internalized by the cell [21]. In the lysosomal compartment, the
the ADC to the level of amino acids. For this type of linker to be effective, the cytotoxic agent must
process of intracellular proteolytic degradation reduces the ADC to the level of amino acids. For
maintain its activity, despite still being attached to part of the linker [22]. ADCs with non-cleavable
this type of linker to be effective, the cytotoxic agent must maintain its activity, despite still being
linkers are considered to have improved therapeutic index because of their greater plasma stability,
attached to part of the linker [22]. ADCs with non-cleavable linkers are considered to have
although new designs of cleavable linkers have significantly improved stability [23].
improved therapeutic index because of their greater plasma stability, although new designs of
It is clear
cleavable that have
linkers the disposition
significantly ofimproved
a mAb isstability
dependent [23].upon several factors (Figure 1), and not
just theIttherapeutic
is clear that theentity conjugated
disposition to it, is
of a mAb independent
the case of an ADC.
upon severalWhile
factorsmAbs
(Figuretrigger
1), andan effector
not just
function on their own, ADCs act more like prodrugs, and may or may not exert
the therapeutic entity conjugated to it, in the case of an ADC. While mAbs trigger an effector function an anti-cancer effect
until the SM drug is released from the mAb carrier. Thus, the pharmacology
on their own, ADCs act more like prodrugs, and may or may not exert an anti-cancer effect until the of mAbs and ADCs
areSMcomplex. As a result,
drug is released fromanalytical and PKThus,
the mAb carrier. studies must be performed
the pharmacology of mAbs to and
assess
ADCsthe are
disposition
complex. of
notAsjust conjugated
a result, analytical and released
and PK studiesforms
must ofbe
ADCs, but also
performed the effects
to assess of modifications
the disposition of not juston the mAbs
conjugated
after
andadministration.
released forms of ADCs, but also the effects of modifications on the mAbs after administration.

Figure
Figure 1. 1. Considerationsininthe
Considerations thedesign
designand
and development
development of
of antibody–drug
antibody–drugconjugates
conjugatesthat affect
that thethe
affect
pharmacokinetics and pharmacodynamics of the agents. PK, pharmacokinetic; PD,
pharmacokinetics and pharmacodynamics of the agents. PK, pharmacokinetic; PD, pharmacodynamic;
pharmacodynamic; DAR, drug–antibody ratios.
DAR, drug–antibody ratios.

3. Pharmacokinetic Considerations
3. Pharmacokinetic Considerations
The need to perform detailed PK studies of both conjugated and released forms of drug after
The need to of
administration perform
an ADC detailed PKtostudies
is critical of both conjugated
fully understand the PK and andPDreleased forms
disposition of of drugAn
ADCs. after
administration of an ADC is critical to fully understand the PK and PD
example of this need was demonstrated by gemtuzumab ozogamicin (Mylotarg ® , Pfizer Inc., disposition of ADCs.
AnPhiladelphia,
example of PA,this USA),
need the
wasfirst
demonstrated bythegemtuzumab ® Pfizer Inc.,
ADC to reach market after ozogamicin
gaining FDA (Mylotarg
approval in, 2000. It is
Philadelphia, PA, USA), the first ADC to reach the market after gaining FDA approval
made up of an extremely potent anti-tumor antibiotic, calicheamicin, coupled to an anti-CD33 IgG in 2000. It is
made
via aup of an extremely
pH-sensitive potent linker.
hydrolysable anti-tumor
Early antibiotic,
trials showedcalicheamicin, coupledrates
promising remission to anin anti-CD33
the high-riskIgG
viapopulation
a pH-sensitive hydrolysable
of older patients linker.
with Early trialsacute
relapsed showed promising
myeloid remission
leukemia (AML) rates in the
[24]. high-risk
However,
confirmatory
population trials
of older indicated
patients witha higher
relapsedincidence of earlyleukemia
acute myeloid fatality in patients
(AML) [24].receiving
However, gemtuzumab
confirmatory
ozogamicin, and it was voluntarily withdrawn from the market in 2010 [24]. In vitro
trials indicated a higher incidence of early fatality in patients receiving gemtuzumab ozogamicin, studies showed
that the linker was poorly thermostable, with rapid and extensive release of calicheamicin from the
Antibodies 2018, 7, 10 6 of 28

and it was voluntarily withdrawn from the market in 2010 [24]. In vitro studies showed that the
linker was poorly thermostable, with rapid and extensive release of calicheamicin from the antibody,
which resulted in toxicity to CD33-negative MOLT-16 cells [25]. This high systemic exposure of the
released active drug was implicated in the significant toxicities associated with administration of
gemtuzumab ozogamicin in patients [26]. Continued research using fractionated dosing regimens
established improved survival in patients with newly diagnosed AML [27,28]. Positive outcomes of
three investigator-initiated trials supported the FDA approval and reintroduction of gemtuzumab
ozogamicin with new dosing recommendations in September 2017 [29].

3.1. Pharmacokinetic Disposition

Absorption. Well known physiological barriers limit oral administration of protein-based drugs
and require that most of these agents be administered parenterally to reach systemic circulation [30].
As a result, mAb agents are typically administered either intravenously (iv) or subcutaneously
(sc). For oncology indications, iv infusion is the most frequent route of administration for ADCs.
By definition, these drugs have 100% bioavailability. By contrast, there are multiple FDA-approved
and commonly used mAbs indicated for inflammatory diseases that are administered via sc injection,
where bioavailability ranges from ~50–80% following sc administration (Humira, AbbVie Inc., Chicago,
IL, USA; Cimzia, UCB Inc., Smyrna, GA, USA; Simponi, Janssen Biotech Inc., Horsham, PA, USA;
Xolair, Genentech Inc., San Francisco, CA, USA) [31]. However, sc administration is highly improbable
for ADCs, due to the potential reactions to cytotoxic payloads and off-target toxicities mediated by
immune cells in the skin, which may cause local deposits of cytotoxic material.
Distribution. Due to their size and polarity, the distribution of ADCs is generally restricted to the
vascular and interstitial space [32]. Convective transport from blood vessels into tissues is slow and
reliant upon pressure gradients [32]. The local structure of both the blood vessel, including fenestration
size and membrane thickness, and the surrounding tissue, alters the rate of transport. For example,
the tight junctions of blood vessels in the brain effectively limit antibody penetration, resulting in
very low distribution of mAbs in brain tissue [32]. By contrast, tumors tend to have leaky vasculature
with large pore sizes, allowing increased convective transport of macromolecules into tumors [32].
These are similar barriers that have limited the tumor delivery of other carrier mediated agents, such
as nanoparticles and polymer conjugates [33,34]. However, these tumor barriers may be less of an
issue for ADCs, as they are smaller (~10 nm) than nanoparticles (~50–100 nm) [35].
Despite this advantage, numerous other factors within tumors may restrict or inhibit the
distribution of ADCs. For antibodies with high target affinity, the availability of target antigens
near blood vessels may restrict further distribution away from blood vessels due to rapid, tight antigen
binding. This phenomenon is known as the “binding site barrier”. Lee and Tannock investigated the
distribution of cetuximab and trastuzumab in mouse models of human epidermoid carcinoma (A431)
and breast adenocarcinoma (MDA-MB-231) [36]. They found that antibody distribution was dependent
on both time and dose administered, and that regions of hypoxia had poor antibody binding. At early
time points after dosing, antibody distribution in tumor was heterogeneous and concentrated near
blood vessels. Over time, and with increasing dose, the distribution became more homogenous, with
the exception of hypoxic tumor regions. Poor binding in hypoxic regions may have been due to low
antigen expression and/or decreased local blood flow [36]. Other tumor factors also play a role in the
local distribution of ADCs. Characterizing the interactions between tumor factors and ADCs may help
to improve the tumor delivery and efficacy of these agents.
ADCs are subject to the same distributive processes and barriers as mAbs, but additionally bear a
cytotoxic payload that can further affect their distribution. Both binding affinity and internalization
efficiency of the antibody component may be altered by the conjugated entities. These differences
may impact distribution hindrance, due to the binding-site barrier described above. In addition,
the conjugated SM drug has its own unique distribution profile once cleaved from the antibody.
Hydrophobic drugs may be able to cross membranes and distribute beyond the target cell while
Antibodies 2018, 7, 10 7 of 28

hydrophilic drugs are generally limited to the antigen-expressing cell that internalized the ADC.
Small molecule drugs that have increased permeability and distribution on their own can take
advantage of the “bystander effect” and may be beneficial in tumors with heterogeneous target
Antibodies 2018, 7, x FOR PEER REVIEW 7 of 28
expression. The protein binding characteristics of the released drug (e.g., % non-protein bound drug)
also influence The
expression. the exposure of active
protein binding drug [37,38].
characteristics of Breij et al. demonstrated
the released complete tumor
drug (e.g., % non-protein boundregression
drug)
in patient derived
also influence thexenograft
exposuremodels
of activeofdrug
solid[37,38].
tumorsBreij
following treatment with
et al. demonstrated an anti-tissue
complete factor mAb
tumor regression
conjugated
in patientto MMAE,
derived despite models
xenograft target antigen
of solidexpression in just treatment
tumors following 25–50% ofwith tumor an cells [39]. The
anti-tissue high
factor
response was believed
mAb conjugated to be despite
to MMAE, due to target
MMAE to cause
antigen a bystander
expression in just effect
25–50%[39]. Achieving
of tumor theThe
cells [39]. ideal
combination
high response of antibody
was believedand conjugated
to be due todrugMMAE characteristics is therefore
to cause a bystander important
effect to the efficacy
[39]. Achieving the ideal and
safety of ADCs, and is likely a unique balance for each target or disease.
combination of antibody and conjugated drug characteristics is therefore important to the efficacy
andMetabolism
safety of ADCs, and andElimination. The metabolism
is likely a unique balance for and each elimination of mAbs differs significantly
target or disease.
Metabolism
from traditional SMand Elimination.
drugs. The metabolism
SMs typically undergo renal and elimination
eliminationof ormAbs
phasediffers
I and IIsignificantly
metabolism,
from traditional
resulting in metabolitesSM drugs. SMs typically
with altered undergo
polarity, molecularrenalweight,
elimination or phasethat
and activity I and
mayII metabolism,
be more easily
resulting
excreted from in metabolites with altered polarity,
the body. Antibody-based molecular
therapeutics are weight,
cleared andvia aactivity
complex that may be more
combination ofeasily
specific
andexcreted from mechanisms
non-specific the body. Antibody-based therapeutics
(Figure 2). Degradation of are
ADCs cleared
occursvianonspecifically
a complex combination of
via proteolysis
specific and non-specific mechanisms (Figure 2). Degradation of ADCs occurs
in a variety of tissues, including the skin, muscle, and liver, due to macrophage uptake [40]. These cells nonspecifically via
mayproteolysis in a variety through
take up antibodies of tissues, including the
non-specific skin, muscle,
pinocytosis andand liver, due
degrade the to macrophage
engulfed uptakevia
antibodies
[40]. These cells may take up antibodies through non-specific pinocytosis and degrade the engulfed
lysosomal proteolysis.
antibodies via lysosomal proteolysis.
ADCs may also be cleared by several specific mechanisms. For antibodies with cellular targets,
ADCs may also be cleared by several specific mechanisms. For antibodies with cellular targets,
target-mediated clearance occurs when the antibody binds to the target cell and is internalized and
target-mediated clearance occurs when the antibody binds to the target cell and is internalized and
degraded. This clearance pathway is saturable, and may result in non-linear PKs, particularly with
degraded. This clearance pathway is saturable, and may result in non-linear PKs, particularly with
low doses and/or high expression of the target. Antibodies also bind to Fc-gamma receptors (FcγR)
low doses and/or high expression of the target. Antibodies also bind to Fc-gamma receptors (FcγR)
expressed
expressed ononcells
cellsofofthe
themononuclear phagocytesystem
mononuclear phagocyte system (MPS).
(MPS). Interaction
Interaction withwith FcγRs
FcγRs also also
leadsleads
to
to internalization
internalizationand andcatabolism
catabolism of mAbs. This pathway may be particularly
of mAbs. This pathway may be particularly important for ADCs with important for ADCs
with circulating
circulating or or secreted
secreted protein
protein targets,
targets, or or those
those that
that form
form larger
larger immune
immune complexes,
complexes, as as larger
larger
complexes tend to have a rapid and high binding
complexes tend to have a rapid and high binding to FcγRs [41]. to FcγRs [41].

Figure 2. Differences in the metabolism and elimination of small molecules drugs compared to
Figure 2. Differences in the metabolism and elimination of small molecules drugs compared to
antibodies and antibody–drug conjugates (ADCs).
antibodies and antibody–drug conjugates (ADCs).

Rather
Ratherthanthanattempting
attemptingtotoovercome
overcome this
this clearance mechanism,
mechanism, Kasturirangan
Kasturiranganetetal.al.recently
recently
published a report taking advantage of these phagocytic pathways to both neutralize
published a report taking advantage of these phagocytic pathways to both neutralize and clear IL-6 and clear IL-6
with a bispecific
with a bispecific antibody
antibody[42].
[42].Complexes
Complexes of a single
single antibody
antibodyandandsingle
singlecirculating
circulating target–antigen
target–antigen
complex,
complex, suchsuch as occurs
as occurs for for
IL-6,IL-6,
can can
evade evade clearance
clearance due due to weak
to weak FcγRFcγR interactions,
interactions, and extend
and extend antigen
antigen half-life via the FcRn recycling pathway. To counteract this effect, a bispecific
half-life via the FcRn recycling pathway. To counteract this effect, a bispecific antibody construct was antibody
construct
generated wastwo
from generated from two
mAbs targeting mAbs epitopes
different targetingofdifferent epitopes
IL-6, by joining ofsingle
the IL-6, chain
by joining the fragment
variable single
chain
(scFv) of variable fragment
one antibody with (scFv) of one antibody
the C terminus withantibody.
of the other the C terminus of the facilitates
This structure other antibody. This of
generation
structure facilitates generation of large branched immune complexes in the presence of IL-6 in vivo.
C57BL/6 mice dosed with IL-6 and either individual mAb displayed extended circulation of the mAb-
IL-6 complexes. By contrast, mice dosed with IL-6 and the bispecific antibody displayed rapid
clearance of the complexes from circulation with FcγR-dependent accumulation in the liver, a
Antibodies 2018, 7, 10 8 of 28

large branched immune complexes in the presence of IL-6 in vivo. C57BL/6 mice dosed with IL-6 and
either individual mAb displayed extended circulation of the mAb-IL-6 complexes. By contrast, mice dosed
with IL-62018,
Antibodies and7,the bispecific antibody displayed rapid clearance of the complexes from circulation 88with
Antibodies2018,
Antibodies
Antibodies 2018,7,
2018,
x FOR
FORPEER
7,7,xxxFOR
FOR PEERREVIEW
PEER
PEER REVIEW
REVIEW
REVIEW
of
of28
of
88of 28
28
28
FcγR-dependent accumulation in the liver, a primary MPS organ [42]. This novel approach demonstrates
primary
the
primary
primary MPS
potential
MPSfor
MPS organ
organ
organ [42].
[42]. This
improved
[42]. drug
This
This novel
novel
novel approach
design based demonstrates
approach
approach on
demonstrates
demonstrates the
the potential
an understanding
the potential
potential for
for improved
of antibody
for improved
improved drug
drug design
pharmacology
drug and
design
design
primary MPS organ [42]. This novel approach demonstrates the potential for improved drug design
based
basedon
based onan
interactions
on an understanding
anwith the MPS. of
understanding
understanding ofantibody
of antibodypharmacology
antibody pharmacologyand
pharmacology andinteractions
and interactionswith
interactions withthe
with theMPS.
the MPS.
MPS.
based on an understanding of antibody pharmacology and interactions with the MPS.
3.2.
3.2. Mononuclear Phagocyte System
3.2.Mononuclear
3.2.
3.2. MononuclearPhagocyte
Mononuclear
Mononuclear PhagocyteSystem
Phagocyte
Phagocyte System
System
System
As
As we strive to better understand the PKs and PDs of antibody-based drugs, itit has become
As we
As
As we strive
we
we strive to
strive
strive to better
to
to better understand
better
better understand the
understand
understand the PKs
the
the PKs and
PKs
PKs and PDs
and
and PDs of
PDs
PDs of antibody-based
of
of antibody-based drugs,
antibody-based
antibody-based drugs, it
drugs,
drugs,
has
has become
has
itit has become
become
become
apparent
apparent
apparent that
that
that similarities
similarities
similarities exist between
exist
exist these
between
between agents
these
these and
agents
agents nanoparticles
and
and (NPs;
nanoparticles
nanoparticles e.g., liposomes,
(NPs;
(NPs; e.g.,
e.g., polymeric
liposomes,
liposomes,
apparent that similarities exist between these agents and nanoparticles
apparent that similarities exist between these agents and nanoparticles (NPs; e.g., liposomes, (NPs; e.g., liposomes,
NPs, micelles,
polymeric conjugates) (Table 2). Factors affecting the PKs and PDs of NPs areofalso atarework for
polymeric NPs,
polymeric
polymeric NPs, micelles,
NPs,
NPs, micelles, conjugates)
micelles,
micelles, conjugates) (Table
conjugates)
conjugates) (Table 2).
(Table
(Table 2). Factors
2).
2). Factors affecting
Factors
Factors affecting the
affecting
affecting the PKs
the
the PKs and
PKs
PKs and PDs
and
and PDs of
PDs
PDs of NPs
of NPs are
NPs
NPs are also
are also at
also
also at
at
at
mAbs,
work
work and
for
for these
mAbs,
mAbs, mechanisms
and
and these
these are
mechanisms
mechanisms ultimately
are
are driven
ultimately
ultimately by the
driven
driven MPS.
by
by The
the
the MPS
MPS.
MPS. The
Theplays
MPS
MPS a significant
plays
plays a
a role
significant
significantin
work for mAbs, and these mechanisms are ultimately driven by the
work for mAbs, and these mechanisms are ultimately driven by the MPS. The MPS plays a significant MPS. The MPS plays a significant
the
role distribution, clearanceclearance
(CL), and(CL),activation of mAb and NP drugs. There is also highisvariability
role in
role
role in the
in
in the distribution,
the
the distribution, clearance
distribution,
distribution, clearance (CL),
clearance (CL), and
(CL), and activation
and
and activation of
activation
activation of mAb
of
of mAb and
mAb
mAb and NP
and
and NP drugs.
NP
NP drugs. There
drugs.
drugs. There is
There
There is also
is also high
also
also high
high
high
in the function
variability
variability in
in and
the
the phenotype
function
function and
and of the
phenotype
phenotype MPS. of
ofInthe
theaddition,
MPS.
MPS. In
In there is
addition,
addition, a bi-directional
there
there is
is a interaction
bi-directional between
interaction
variabilityin
variability inthe
the function
functionand and phenotype
phenotype of of the
the MPS.
MPS. InInaddition,
addition, there isaaabi-directional
there is bi-directional interaction
bi-directionalinteraction
interaction
the MPS and
between the NPs, and
MPS and NPs,
potentially
and mAbs, where
potentially mAbs,thewhere
physical the characteristics
physical of the carrier
characteristics of thealters the
between
between the
the MPS
MPS and
and NPs,
NPs, and
and potentially
potentially mAbs,
mAbs, where
where the
the
between the MPS and NPs, and potentially mAbs, where the physical characteristics of the physical
physical characteristics
characteristics of
of the carrier
the carrier
carrier
carrier
function
alters of the MPS [43].
altersthe
alters
alters thefunction
the
the functionof
function
function of
ofofthe
theMPS
the
the MPS [43].
MPS
MPS [43].
[43].
[43].
Table
Table 2. Summary of pharmacokinetic (PK) and pharmacodynamic (PD) similarities for nanoparticles
Table2.
Table
Table 2.Summary
2.
2. Summaryof
Summary
Summary ofpharmacokinetic
of
of pharmacokinetic(PK)
pharmacokinetic
pharmacokinetic (PK)and
(PK)
(PK) andpharmacodynamic
and
and pharmacodynamic(PD)
pharmacodynamic
pharmacodynamic (PD)similarities
(PD)
(PD) similaritiesfor
similarities
similarities fornanoparticles
for
for nanoparticles
nanoparticles
nanoparticles
(NPs)
(NPs) and
and antibody–drug
antibody–drug conjugates
conjugates (ADCs)
(ADCs) associated
associated with
with the
the mononuclear
mononuclear phagocyte
phagocyte system
system
(NPs)
(NPs)
(NPs) and
and
and antibody–drug
antibody–drug
antibody–drug conjugates
conjugates
conjugates (ADCs)
(ADCs)
(ADCs) associated
associated
associated with
with
with the
the
the mononuclear
mononuclear
mononuclear phagocyte
phagocyte
phagocyte system
system
system
(MPS)
(MPS) clearance.
clearance.
(MPS)
(MPS) clearance.
clearance.
(MPS) clearance.
Characteristic
Characteristic
Characteristic
Characteristic Cause
Cause
Cause
Cause Example
Example
Example
Example
Characteristic Cause Example
MPS
MPScells
MPS cellsare
cells areinvolved
are involvedin
involved inthe
in the
the
MPS cells are involved in the
High
Highdelivery
High delivery
delivery and
and
and distribution
distribution
distribution distribution
MPS cells
distribution
distribution and
are
and
and capture
involved
capture
capture in of
the
of
of
111 High delivery and
High distribution
delivery and distribution and capture of
distribution andincapture of these
1 1 to
to
to MPS
MPS
MPS organs
organs
organs
distribution to MPS organs these
these
these agents
agents
agents in
in liver
liver
liver and
and
and
to MPS organs these agents
agents in and
in liver liverspleen.
and
spleen.
spleen.
spleen. Liver
Liver
Liver Spleen
Spleen
Spleen
spleen. Liver Spleen
Faster
Faster clearance
clearance
FasterFaster
clearance is
isassociated
is associated
associated
Faster clearance is
clearance associated
is associated MPS
MPS
MPS is
isable
is able to
to
ableto recognize
recognize
torecognize
recognize and
and
and
with
with agents
agents
with agents
agents that
that
that have
have
have aaagreater
greater
greater MPS
MPS is
is able
able to recognizeandand
take
222 with
2
withthat have
agents that a greater
have a take
take
take
upup
up
up these
these
these
these “non-self”
“non-self”
“non-self”
“non-self” agents
agents
agents
agents to a
2 number
number
number of
of
greaterligands
ligands
number
of ligands
ligands linked
linked to
to
of ligands
linked to take up these “non-self” agents
number of linked to to
toaaaagreater
to greater
greater extent.
greaterextent.
extent.
extent.
the
linked
the
the carrier.
to the carrier.
carrier.
carrier. to greater extent.
the carrier.

High
Highinterpatient
High interpatient PK
PKand
PK
High interpatient
interpatient PKPD
and
and PD
and
PD MPS
MPS
MPS function
function
MPS function
function is
isishighly
is highlyvariable
highly
highly variable
variable
variable
333 3High interpatient PK and PD MPS function is highly variable
3 variability
PD variability
variability
variability in
in
in patients
in patients
patients
patients
variability in patients

MPS
MPS
MPS uptake of
uptake
uptake ofofparticles
of particleshas
particles has a
has
Non-linear/saturable
Non-linear/saturable clearance
Non-linear/saturable
clearance
MPS
MPS uptake
uptake of particles
particles has
has aaa
4Non-linear/saturable clearance
444 Non-linear/saturable clearance maximum
maximum
maximum capacity
capacity
capacity
maximum capacity that
that
that
capacity that cancan
can
that can be
be
can be
be
4 clearance
at
at
at high
high
high
at high
doses.
doses.
doses.
doses. maximum be saturated
at high doses. saturated
saturated
saturated
saturated

Body Body weight,

weight, body body MPS function is altered in
Body
Body
Body weight,
weight,
composition, body
weight,bodybodyhabitus
body MPS
MPS
MPS
MPS function
function
function
function is
is altered
is altered
altered
is altered in
in
in
in patients
555 composition,
5composition, body
composition, bodyhabitus
body
are covariateshabitus
habitus are
are
are
related patients
patients
patients with
with
with
with large large
large
bodylarge
mass body
body
body mass
mass
mass
and mass
weight
5 composition, body habitus are patients with large body
covariates
covariates related
related
to
covariates related to
to
related to clearance.
clearance.
clearance.
to clearance.
clearance. and
andweight
and weight
weight
covariates and weight
Metastatic Tumors Liver

MPS
MPS
MPS function
function
function is
isisincreased
is increased
increased in
in
in
Metastatic
MetastaticTumors
Tumors
Metastatic Tumors
Liver
Liver
Liver

MPS function
MPS functionis increased
increased in in
Tumor
Tumor
Tumor burden
burden
burden
Tumor is
is
is
burden a
a
a covariate
covariate
covariate
is a covariate patients
patients
patients
patients &&&
& animals
animals
animals
animals with
with
withwith large
large
large
large tumor
666
6 6Tumor burden is a covariate
related to
patients & animals with large
related
related
related toclearance
to
to clearance
related to clearance
clearance
clearance
tumor
tumor
tumor
tumor
burden,
burden,
burden, especially
especially
especially
burden,
burden, when tumors
especially
especially
when
when
when
when are
tumors
tumors are
are
tumors are present
present in
present
are present
present inthein
in the
liver.
the
in the liver.
liver.
the liver.
liver.
tumors

NPs
NPs and
NPs and mAbs
and mAbs both
mAbs both demonstrate
both demonstrate aaaa high
demonstrate high delivery
high delivery and
delivery and distribution
and distribution to
distribution to the
to the MPS
the MPS organs,
MPS organs, which
organs, which
which
NPs and mAbs both demonstrate high delivery and distribution to the MPS organs, which
include
include
include liver,
liver,
liver,spleen,
spleen,
spleen, and
and
and lung.
lung.
lung.NPs
NPs
NPs are
are
are well
well
well known
known
known to
to
to accumulate
accumulate
accumulate and
and
and be
be
be cleared
cleared
cleared through
through
through these
these
these MPS-
MPS-
MPS-
include liver, spleen, and lung. NPs are well known to accumulate and be cleared through these MPS-
associated
associated
associated organs
organs
organs [44–46].
[44–46].
[44–46]. Likewise,
Likewise,
Likewise, high
high
high relative
relative
relative distribution
distribution
distribution of
of
of trastuzumab
trastuzumab
trastuzumab to
to
tothe
the
the liver,
liver,
liver,spleen,
spleen,
spleen, and
and
and
associated organs [44–46]. Likewise, high relative distribution of trastuzumab to the liver, spleen, and
lungs
lungs has
has
lungshas been
been
hasbeen observed
observed
beenobserved
observedin in
in mouse
mouse
inmouse models
models
mousemodels
modelsof of
of cancer
cancer
ofcancer [47,48].
[47,48].
cancer [47,48]. The
The
[47,48].The
TheCL CL
CL
CLofof
of NPs
NPs
ofNPs
NPsandand
and mAbs
mAbs
and mAbs
mAbsis is
is attributed
attributedto
attributed
isattributed to
to
lungs to
their
their
their recognition
recognition
recognition and
and
and uptake
uptake
uptake by
by
by the
the
the MPS
MPS
MPS in
in
in these
these
these organs.
organs.
organs. Additionally,
Additionally,
Additionally, high
high
high interpatient
interpatient
interpatient variability
variability
variability
their recognition and uptake by the MPS in these organs. Additionally, high interpatient variability
is
is associated
is associated with
associated with NP
with NP pharmacology.
NP pharmacology. Schell
pharmacology. Schell et
Schell et al.
et al. reported
al. reported aaaa meta-analysis
reported meta-analysis that
meta-analysis that compared
that compared the
compared the
the
is associated with NP pharmacology. Schell et al. reported meta-analysis that compared the
Antibodies 2018, 7, 10 9 of 28

NPs and mAbs both demonstrate a high delivery and distribution to the MPS organs, which
include liver, spleen, and lung. NPs are well known to accumulate and be cleared through these
MPS-associated organs [44–46]. Likewise, high relative distribution of trastuzumab to the liver, spleen,
and lungs has been observed in mouse models of cancer [47,48]. The CL of NPs and mAbs is attributed
to their recognition and uptake by the MPS in these organs. Additionally, high interpatient variability is
associated with NP pharmacology. Schell et al. reported a meta-analysis that compared the variability
of patients receiving a liposomal therapy with patients receiving the SM counterpart. The results
showed a significant increase in PK variability (i.e., plasma area under the curve (AUC)) for liposomes
(66%) compared with SM (31%) in patients receiving similar doses [49]. In comparison, the interpatient
variability in CL among patients receiving trastuzumab has been reported at 43% (95% CI: 0.213 to
0.238) [50]. Lastly, the CL of NPs is non-linear, likely due to saturation of the MPS [51]. Such saturable
CL has also been observed for mAbs as well [52].
Physical characteristics of the drug and of the patient also affect the PKs of NPs and mAbs.
NPs demonstrate an accelerated CL when there are a greater number of ligands linked to the carrier,
as is the case with actively targeted NPs when compared to non-active NPs. Gabizon et al. have
shown that liposomes formulated with folic acid on their surface were cleared faster than the liposome
without the folic acid ligand [53]. This is paralleled for ADCs like ado-trastuzumab emtansine, where
trastuzumab is conjugated to emtansine, and clears more rapidly (3-fold) in mice and humans than
trastuzumab alone [54–56]. This is attributed to the ability of the MPS to recognize and take up these
more hydrophobic, non-self-agents. Furthermore, a patient’s body habitus has been shown to affect
the pharmacology of both NP and mAb drugs. Population PK studies of trastuzumab in patients with
HER2+ metastatic breast cancer have shown that body weight is a covariate of CL where increased
body weigh correlates with increased CL [50,57]. This is consistent with the altered MPS function
for patients with a larger body mass and weight, which have higher MPS function and higher CL of
NPs [43]. Finally, tumor burden is also a covariate related to CL of NPs, and increased CL is observed
with the number of metastatic sites [58,59]. This same effect is observed with mAb drugs, exemplified
in the population PK analysis of trastuzumab, that showed baseline tumor burden was a significant
covariate for CL and volume of distribution [50]. MPS function and NP CL are higher in tumor bearing
mice compared to non-tumor bearing mice, and in patients with higher tumor burden and certain
tumors, especially when tumors are present in liver [58–60].
While the MPS may play a role, the importance of this pathway is incompletely understood, and
probably differs for different antibodies depending on their antigen specificity and isotype. With the
heavy genetic manipulation of mAbs being optimized for higher affinities to targets, improved
specificity, and reduced CL rates, an unwanted consequence appears to be an increased incidence
of patients demonstrating unexpected or highly variable PK profiles [61,62]. In several studies,
common factors known to affect mAbs (target binding, FcRn binding, whole blood stability, anti-drug
antibodies (ADA)) were evaluated and shown to not account for the variability in the PKs of mAbs
and ADCs [63–65]. These results suggest that the variable PK of these agents in certain patients may
be due to an increase in low affinity receptor binding, an increased off-target binding and/or inter-
and intrapatient variability in MPS function [66].
Two ADCs that preferentially target antigens on tumor cells, that have demonstrated
clinical efficacy with manageable safety profiles, are brentuximab vedotin and ado-trastuzumab
emtansine [4,67,68]. Both of these agents utilize tubulin-binding agents as their cytotoxic payload,
but ado-trastuzumab emtansine uses a non-cleavable linker, while brentuximab vedotin uses a
cleavable linker. Both agents incorporate an average DAR of 3.5–4 (though the products are actually
heterogeneous with DAR ranging from 0 to 8) [69]. Ado-trastuzumab emtansine has also been shown
to have non-specific distribution to highly perfused organs without demonstrating accumulation,
similar to the unconjugated “naked” mAb trastuzumab [70]. Brentuximab vedotin demonstrated
similar PK results as ado-trastuzumab emtansine above (i.e., ADC and naked mAb tissue distribution
profiles), though the ADC seems to present slight accumulation in the liver [71,72]. This data would
Antibodies 2018, 7, 10 10 of 28

suggest that certain formulation characteristics of brentuximab vedotin may make it more recognizable
to the host’s immune system and MPS compared to the naked mAb, resulting in hepatic accumulation
due to MPS-based clearance. However, it is unknown if this hepatic accumulation is the result of ADC
linker type (i.e., cleavable and/or not as stable), leading to MPS-related accumulation or that released
drug quickly
Antibodies accumulates
2018, 7, in the liver.
x FOR PEER REVIEW 10 of 28
ADCs also demonstrate high interpatient variability and distribution patterns that make
understanding the dose–response
between ado-trastuzumab emtansinerelationship
dose anddifficult. The PK of
PK parameters inado-trastuzumab
plasma is presented emtansine, total
in Figure 3.
mAb,
ThereandwasDM1high were evaluated
interpatient PK in combination
variability with paclitaxel in
in ado-trastuzumab a phase Ib/IIa
emtansine AUC and trialCmax
in patients with
in plasma,
HER2-positive
with increasing metastatic breast
variability (i.e.,cancer
CV%)administered iv every threethe
as patients approached weeks
MTD [73].
(3.6The relationship
mg/kg). between
Another ADC,
ado-trastuzumab
anetumab ravtansine emtansine
(BAYdose and PKisparameters
94–9343), in plasma is anti-mesothelin
a novel fully-human presented in Figure 3. There was high
immunoglobulin G1
interpatient PK variability
antibody conjugated in ado-trastuzumab
to the maytansinoid tubulinemtansine AUC DM4.
inhibitor and Cmax in plasma,
Anetumab with increasing
ravtansine AUC in
variability (i.e., CV%) assimilar
plasma demonstrates patients highapproached the variability,
interpatient MTD (3.6 mg/kg).
which wasAnother
foundADC, anetumab
to not ravtansine
be associated with
(BAY 94-9343), is a novel fully-human anti-mesothelin immunoglobulin G1 antibody
anti-drug antibody (ADA) titers [74]. In addition, there was an overlap of anetumab ravtansine conjugated to the
maytansinoid
plasma exposures tubulin inhibitor
at doses DM4.
of 5.5, 6.5Anetumab
(MTD), and ravtansine AUC
7.5 mg/kg. Theinhigh
plasma
PK demonstrates
variability of similar high
these ADCs
interpatient variability,
may be associated withwhich was found
variability in theto not be
MPS, associated
especially with anti-drug
considering a lackantibody (ADA) titers
of a relationship [74].
between
In
PKaddition,
variability there
and was
ADAan overlap
titers. of anetumab ravtansine plasma exposures at doses of 5.5, 6.5 (MTD),
and 7.5 mg/kg. The high PK variability of these ADCs may be associated with variability in the MPS,
especially considering a lack of a relationship between PK variability and ADA titers.

Figure 3.
Figure 3. Relationship
Relationshipbetween
between ado-trastuzumab
ado-trastuzumab emtansine
emtansine dose andand
dose AUCAUCor Cmax in plasma.
or Cmax Mean
in plasma.
± standard
Mean deviation
± standard (SD) of(SD)
deviation patients for each
of patients fortreatment group group
each treatment are represented by the by
are represented black
thebar. There
black bar.
was high
There was interpatient pharmacokinetic
high interpatient (PK) variability
pharmacokinetic in ado-trastuzumab
(PK) variability emtansine,
in ado-trastuzumab especially
emtansine, when
especially
approaching
when important
approaching PK doses
important (i.e., maximum
PK doses tolerable
(i.e., maximum dose).dose).
tolerable The high PK variability
The high of ado-
PK variability of
trastuzumab emtansine may be associated with variability in the mononuclear phagocyte
ado-trastuzumab emtansine may be associated with variability in the mononuclear phagocyte system system (MPS).
CV%, coefficient
(MPS). of variation.
CV%, coefficient of variation.

4. Physical
4. Physical Characteristics
Characteristics of
of ADCs
ADCs

4.1. Size
4.1. Size
Therapeutic proteins
Therapeutic proteins come in aa range
range ofof sizes,
sizes, and
and differences
differences can
can affect
affect the
the PK
PKdisposition
disposition inin
variousways.
various ways. Examples
Examples such
suchas
as full
full IgG
IgG mAbs,
mAbs, FabFab fragments
fragments (Fabs),
(Fabs), serum
serumalbumin,
albumin, andand streptavidin
streptavidin
are just
are just aafew
fewthat
thathave
havebeen
beenexamined.
examined. These These proteins
proteins were
were injected
injected into
into SCID
SCID mice
mice bearing
bearing HeLa
HeLa
tumor xenografts,
tumor xenografts, and
and blood
blood concentrations
concentrations were measuredmeasured over the course
course of 77 days
days [75].
[75]. Full
Full IgGs
IgGs
had the
had the slowest
slowest clearances
clearances compared
compared to to streptavidin
streptavidin and and body
body surface
surface area
area (BSA),
(BSA), and
and Fabs
Fabs had
had the
the
highest clearance, which was directly related to its molecular weight [75]. While Fab fragments are
one-third the size of full IgG antibodies (~50–66 kDa vs. ~150 kDa, respectively), the clearance is 10-
fold greater (1.1 mL/h vs. 0.1 mL/h) though the Vd is similar between all sizes (~1.5 mL) [75]. The
clearance value for Fabs is greater because these molecules are smaller than IgGs, and as such, they
are also cleared in more traditional pathways (i.e., hepatic excretion and renal elimination) than via
Antibodies 2018, 7, 10 11 of 28

highest clearance, which was directly related to its molecular weight [75]. While Fab fragments are
one-third the size of full IgG antibodies (~50–66 kDa vs. ~150 kDa, respectively), the clearance is
10-fold greater (1.1 mL/h vs. 0.1 mL/h) though the Vd is similar between all sizes (~1.5 mL) [75].
The clearance value for Fabs is greater because these molecules are smaller than IgGs, and as such,
they are also cleared in more traditional pathways (i.e., hepatic excretion and renal elimination) than
via cellular interaction and endocytosis, such as interaction with the MPS or proteolytic degradation.
In terms of pharmacological benefit, larger proteins (full IgG mAbs or ADCs) could increase the
amount of time an agent remains in the body, leading to prolonged dosing intervals and increased
time of exposures, which leads to prolonged therapeutic effect.
Conjugating SM drugs to antibodies has been shown to be safe and effective, with regards to
patient care, though few examples exist of smaller proteins (such as Fabs) as carriers. The Chinese
Food & Drug Administration (CFDA) previously approved Metuximab-I131 , a murine IgG1 anti-CD147
F(ab)2 conjugated to iodine-131, for the treatment of liver cancers. Metuximab-I131 had a longer half-life
when compared to the naked Fab fragment (1.96 h vs. 0.6 h, respectively) [76]. Similarly, naptumomab
estafenatox (an anti-5T3 Fab conjugated to a staphylococcal enterotoxin) has a terminal half-life of
1.38 h, demonstrating similar PK to that of Metuximab-I131 [77]. Based on these data, conjugated Fabs
have a lower clearance and longer terminal half-life values compared to “naked” Fabs. This may be
due to a reduction in the ability or rate of traditional clearance pathways, or the impact of additional
clearance pathways due to the increased size of the agent based on the size of the conjugated drug.
In addition, PK of Fab-ADCs may not be affected by concomitant chemotherapy compared to full mAbs,
as naptumomab estafenatox administered in combination with docetaxel resulted in a non-significant
change in half-life [77].

4.2. Drug–Antibody Ratio (DAR)

The drug–antibody ratio (DAR), or number of drug molecules conjugated to a single ADC, is
critical in determining efficacy. Optimal DAR to achieve the greatest clinical efficacy has yet to be
determined, and may be highly dependent on other ADC variables. In addition, DAR varies within
a single product, and controlling this heterogeneity is complicated, due to the number of lysine
residues exposed on the antibody surface, such as for ado-trastuzumab emtansine, and the cysteine
residues used in creating interchain disulfide bonds, such as for brentuximab vedotin [3]. With too
few molecules attached, the ADC may not provide an adequate cytotoxic response or anti-tumor
efficacy [3]. On the other hand, too many drug molecules conjugated to an ADC can become unstable,
be rapidly recognized by the immune system, produce altered PK and PD properties, and increased
toxicity [78].
It has been shown previously that a higher DAR leads to greater in vitro potency, though the
increased DAR also affects the pharmacological properties of the agent [78,79]. For instance, the
conjugation of MMAE in the creation of ADCs, or doxorubicin in high DAR ADCs, resulted in
increased aggregation, both believed to be due to their added hydrophobicity [78,80,81]. To offset the
hydrophobic detriments of cytotoxic payloads, hydrophilic-based polymer linkers are being evaluated.
Preliminary studies of these hydrophilic linkers show that higher DARs (up to a DAR of 20) increase
potency, but also maintain the desired biological/pharmacological properties in vivo [79,82].
There have been extensive studies on the effects of DAR on the properties of ADCs. One group
prepared a series of maytansinoid-coupled ADCs with a DAR range from 2 to 14, using both cleavable
and non-cleavable linkers, to evaluate the preclinical and clinical effects of DAR, including in vivo
stability, efficacy, and tolerability [79,83]. Their results showed that agents with a high DAR (average 10,
range 7–14) using both linker formats had faster distribution and clearance rates and decreased
efficacy in vivo (Figure 4) [79,83]. On the other hand, ADCs with a DAR less than 6 displayed similar
clearances, superior in vivo efficacy, and were well tolerated [79,83]. Hambelet et al. demonstrated
similar conclusions in a study evaluating MMAE conjugated to a CD30 antibody using a MC–vc–PAB
linker [78]. When DARs were manipulated to increase the cytotoxic payload with either 2, 4, or 8 drugs
Antibodies 2018, 7, 10 12 of 28

per antibody, the clearance increased [78]. These results may be related to the difference in the rate of
uptake of these agents by the MPS and other non-target cells.
Additionally, the site of conjugation can play a role in augmenting the pharmacology of an
ADC. Strop et al. created an ADC with an average DAR ~1.7, but directed conjugation using a novel
enzymatic conjugation to precise points on the antibody backbone to create homogenous solutions [84].
Their study reported how the site of conjugation affected linker stability in a species-dependent
manner, where conjugation on the light chain appears to be cleaved more quickly compared to heavy
chain conjugation in mice, but stable in rats [84]. Additionally, heavy chain conjugation demonstrated
Antibodies 2018, 7, x FOR PEER REVIEW 12 of 28
dramatically different PK and faster rate of drug loss, compared to either light chain conjugated ADCs
or naked
the role of mAbs in rats [84].
the linker, linkerThe uses of and
position, suchcytotoxic
technologies can allow
payload for increased
to optimize studies of the and
the pharmacology role
of the linker,
improve linker position,
the therapeutic indexand cytotoxic
of these payload to optimize the pharmacology and improve the
agents.
therapeutic index of these agents.

Figure 4.
Figure 4. Pharmacokinetic studies evaluating
Pharmacokinetic studies evaluating antibody–drug conjugate (ADC)
antibody–drug conjugate (ADC) concentrations using
concentrations using
radiolabeled ADCs. Clearance of M9346A–sulfo-SPDB–[3H]DM4 (A) and
radiolabeled ADCs. Clearance of M9346A–sulfo-SPDB–[3H]DM4 (A) and J2898A–SMCC–[3H]DM1 J2898A–SMCC–[3H]DM1
(B) conjugates
(B) from plasma
conjugates from plasma ofof CD-1
CD-1 mice,
mice, that
that were
were injected
injected iv
iv as
as aa single
single 10
10 mg/kg dose, were
mg/kg dose, were
measured by
measured by counting
counting the
the radioactivity
radioactivity in
in plasma
plasma arising
arising from
from the
the tritium
tritium label
label on
onthe
themaytansinoid.
maytansinoid.
These findings suggest that maytansinoid conjugates, regardless of linker type, with drug–antibody
These findings suggest that maytansinoid conjugates, regardless of linker type, with drug–antibody
ratios (DAR)
ratios (DAR) ranging
ranging from
from 22 to
to 6,
6, have
have aa better
better therapeutic
therapeutic index
index than
than conjugates with very
conjugates with very higher
higher
DAR (>9). Adapted with permission from [79]. Copyright 2017, American Chemical
DAR (>9). Adapted with permission from [79]. Copyright 2017, American Chemical Society. Society.

4.3. Surface
4.3. Surface Modifications
Modifications
Modifying formulation
Modifying formulation and
and structures
structures ofof drug
drug carriers
carriers is
is aa common
common practice
practice as as aa method
method to to alter
alter
the way
the way the the body
body handles
handles anan agent.
agent. The
The most
most common
common way way to
to modify
modify these
these agents
agents isis by
by glycosylation
glycosylation
and PEGylation. These modifications can be made on the antibody or the linker,
and PEGylation. These modifications can be made on the antibody or the linker, and may reduce and may reduce the
the
rate of
rate of clearance.
clearance.
Glycosylation. The
Glycosylation. Thepost-translational modification
post-translational of proteins
modification through the
of proteins additionthe
through of carbohydrates
addition of
(i.e., glycans) to amino acid side chains is a process known as glycosylation.
carbohydrates (i.e., glycans) to amino acid side chains is a process known as glycosylation. This natural modification
This
has beenmodification
natural associated with
hasthe production
been associatedof therapeutic proteins inofeukaryotic
with the production therapeutic cellproteins
lines andiniseukaryotic
dependent
on several
cell lines andfactors, includingon
is dependent the cell line
several and culturing
factors, includingconditions
the cell line[85].
andHowever,
culturingboth the amount
conditions [85].
and location of glycosylation dramatically affects the disposition of these
However, both the amount and location of glycosylation dramatically affects the dispositionproteins, such as regulating
of these
receptor binding
proteins, such asand Fc effector
regulating functions
receptor [86]. and
binding In addition, N-linked
Fc effector glycosylation
functions has beenN-linked
[86]. In addition, used for
glycosylation has been used for promoting the systemic residence time, especially for smaller
proteins like diabodies [87]. While conflicting studies have shown that glycosylation of the Fc region
may affect clearance, it is generally acknowledged that high mannose-5 glycan forms may be cleared
more rapidly compared to other glycoforms [88,89]. Ado-trastuzumab emtansine was
glycoengineered to include terminal sialic acids [90]. This allows for payload addition without
Antibodies 2018, 7, 10 13 of 28

promoting the systemic residence time, especially for smaller proteins like diabodies [87]. While conflicting
studies have shown that glycosylation of the Fc region may affect clearance, it is generally acknowledged
that high mannose-5 glycan forms may be cleared more rapidly compared to other glycoforms [88,89].
Ado-trastuzumab emtansine was glycoengineered to include terminal sialic acids [90]. This allows for
payload addition without remodeling the entire antibody to avoid potential interference with drug
loading. However, the analysis of heterogeneous populations (due to different levels of glycosylation and
glycoforms) makes thorough analysis of different drug formulations difficult to compare.
PEGylation. PEGylation, the addition of non-immunogenic PEG polymer, is another suitable
option for modifying antibodies to overcome disadvantages of certain biologics. In general, PEGylation
provides improved agent solubility, decreased immunogenicity, and prolonged residence time in the
body [91–93]. This occurs as PEG protects against enzymatic degradation, slows filtration by the
kidneys, and slow recognition by the MPS [94]. However, PEGylation can also influence binding
affinity/potency to its target, due to steric limitations, thus, individual conjugation sites and control of
conjugation should be evaluated. The main use of PEGylation on ADCs is as a linker to improve the
solubility and limit aggregation. In a study by Burke et al, drug-like PEG side chains were examined
to determine PK parameters [95]. The group used a dose of 3 mg/kg MMAE in Sprague-Dawley
rats, and monitored the amount of antibody over time. The length of the PEG-chain was varied
from 2 to 24 PEG-block polymers, and each ADC was conjugated with 8 MMAE drug molecules [95].
After iv administration in the rats, the increased length of the PEG-chain resulted in lower clearance [95].
This relationship held true for PEG2 and PEG4 , but from PEG8 –PEG24 there were only minor differences
between the PK parameters [95]. Using PEG on Fab fragments also increased the circulation time [93].

4.4. Charge and pH Engineering

The charge of a protein is a critical variable in determining its electrostatic/non-specific
interactions with other materials found within circulation and tissues. A fundamental property
of proteins is their isoelectric point (pI), or the pH at which the antibody carries no net electrical charge,
and traditionally falls in a range between 8 and 9 [96]. However, due to current manufacturing and
isolation techniques, ADCs will be heterogeneous in relation to the surface charge and pI properties
within the entire protein pool. Shifts in the isoelectric point of even one pI unit can produce measurable
changes in tissue distribution and kinetics [97,98]. Cationization (where the pI is raised/more basic)
of antibodies have been associated with increased blood clearance and higher tissue accumulation,
tending to adhere to more anionic sites on the cell surface [96]. One study assessed the effect of
increasing the pI of the antibody by 2 units (from pI 7 to 9) and found a 28-fold increase in plasma
clearance [99]. However, minor changes to the pI (typically < 1.0 unit) did not appear to affect
mAb function or PK, suggesting that minor changes in formulation pI may not warrant additional
PK considerations [96,100]. Cationization of the antibody drug has also been used to encourage
extravasation, antigen binding, and receptor-mediated endocytosis of antibodies into the target
cells due to electrostatic interactions (positively charged antibody and the negatively charged cell
membrane), resulting in non-specific membrane flow [96,101,102]. Therefore, it is important to isolate
and characterize charge variants, and evaluate the PK disposition of specific isolates, as variability in
PK could be due to a heterogenous pool of agents.

5. Host-Associated Factors and Disease Status

5.1. Sex and Body Habitus

Historically, it is controversial if sex and/or body weight play a role in the disposition of mAb
agents, though examples do exist. A population analysis of 671 patients enrolled in the five phase I to III
studies of ado-trastuzumab emtansine found that serum albumin, serum AST, tumor burden, and body
weight all demonstrated significant effects on ado-trastuzumab emtansine PK [103]. Of these factors,
body weight demonstrated the greatest effect, where patients with higher baseline weight had larger
Antibodies 2018, 7, 10 14 of 28

effects on both CL and Vc [103,104]. These data also proved to be the most sensitive in affecting the
steady-state AUC and Cmax concentrations of drug [103]. Similar effects were detected for brentxumiab
vedotin in a population PK analysis of 314 patients evaluated in five trials. In this evaluation, body
weight and BSA affected clearance and volume of distribution [105]. In both cases, patients with larger
body habitus had a higher CL and lower exposure of the agents. Both of these studies support the
decision to utilize weight-based dosing strategies for these ADCs, though variability still exists despite
weight-based dose normalization. The higher CL in patients with larger body habitus and the inability
of weight-based dosing to fully account for the differences in PKs are consistent with higher MPS
Antibodies 2018, 7, x FOR PEER REVIEW 14 of 28
function in overweight and obese patients [43].

5.2.
5.2. Chemical
2018, Modulators
Chemical
Antibodies 7, x FOR PEERof
Modulators Immunity
Immunity in
ofREVIEW in Blood
Blood 14 of 28

While traditional population

While traditional population PK PK studies
studies have
have routinely
routinely used
used patient
patient covariates
covariates to to evaluate
evaluate PK PK
5.2. Chemical Modulators of Immunity in Blood
differences
differences across patient populations, various blood chemistry factors know to regulate immunity
across patient populations, various blood chemistry factors know to regulate immunity
and tissue
Whileeffectors
and tissue have
traditional
effectors have also been
population
also beenPK evaluated.
studies have
evaluated. As
As the MPS
MPS appears
routinely
the to
to play
used patient
appears play aa significant
covariates
significant role
role in
to evaluate in the
PK
the
disposition
differences of mAb
across agents,
patient sex hormones
populations, and
various chemokines
blood that
chemistry regulate
factors
disposition of mAb agents, sex hormones and chemokines that regulate the MPS may also affect the
know MPS to may also
regulate affect
immunity the
PK
andPK
the disposition
tissue
dispositionof these
effectors have
of agents
these similarly
alsoagents
been evaluated.
similarly[106–108].
As the Several
[106–108]. studies
MPSSeveral
appears tohave
studiesplayhaveademonstrated
significant role
demonstrated that sex
inthat
the
hormones
disposition have
of in
mAb vitro
agents, regulatory
sex hormoneseffectsandon lymphocyte
chemokines and
that macrophage
regulate
sex hormones have in vitro regulatory effects on lymphocyte and macrophage functions, including the MPS functions,
may also including
affect the
macrophage
PK disposition
macrophage FcɣR
Fc Rofexpression,
these agents
expression, which
which can
can lead
similarly to
to increased
lead[106–108].
increased clearance
Several of
of IgG-coated
studies
clearance materials
materials [109–115].
have demonstrated
IgG-coated that sex
[109–115].
Certain
hormones
Certain hormones, such
have insuch
hormones, as regulatory
vitroas calcitriol, the most on
effects
calcitriol, the most potent form of
lymphocyte
potent form of vitamin
vitamin D3,
D3, also
and macrophage also appear to
to be
functions,
appear be important
including
important
mediators
macrophage of FcɣRs
FcɣR and function,
expression, and
which have
can previously
lead to been
increased shown
clearance toofregulate
IgG-coated
mediators of Fc Rs and function, and have previously been shown to regulate key immune cytokines key immune
materials cytokines
[109–115].
in
in mononuclear
Certain hormones,
mononuclear phagocytes
phagocytes (IL-1, IL-6,
such as calcitriol,
(IL-1, IL-6,theTNFa)
mostand
TNFa) and T lymphocytes
potent
T (IL-2,D3,
form of vitamin
lymphocytes (IL-2, INFg) [116–121].
appear to These
also[116–121].
INFg) be
These effects
important
effects
may
may be
be more
mediators of prominent
more FcɣRs in
in tissues,
and function,
prominent where
tissues,and haveactivated
where previously
activated macrophages
been shown
macrophages can
canto be affected
regulate
be affected keyby locally
locally produced
byimmune cytokines
produced
calcitriol
calcitriol and cytokines,
in mononuclear
and cytokines,
phagocytesreaching pharmaceutically-relevant
(IL-1, pharmaceutically-relevant
reaching IL-6, TNFa) and T lymphocytes concentrations
(IL-2, INFg)and
concentrations and influencing
[116–121]. cellular
Thesecellular
influencing effects
immune
may be more
immune responses.
prominent in tissues, where activated macrophages can be affected by locally produced
responses.
calcitriol and cytokines, reaching pharmaceutically-relevant concentrations and influencing cellular
5.3. Renal responses.
immune or Hepatic Impairment
According to the FDA, the assessment of mAb agents in special populations, populations, such such asas renal or
5.3. Renal or Hepatic Impairment
hepatic impairment,
impairment, is not necessary [122,123]. This is because renal and hepatic impairment is not
likelyAccording
to alter thetoPK thedisposition because the of
FDA, the assessment excretion
mAb agentsby theinkidneys
special and liver are not
populations, such as renal or
significantly
involved in the clearance
hepatic impairment, is not and disposition
necessary of mAbs
[122,123].
disposition of mAbs agents.
becauseIn
This isagents. addition,
Inrenal case
case reports
and hepatic
addition, in patients
impairment is not
undergoing hemodialysis
likely to alter the PK disposition
hemodialysis becausethat
have reported therenal impairment
excretion did not affect
by the kidneys the PKs
and liver are of
PKs not
of bevacizumab,
significantly
bevacizumab,
cetuximab,
involved inrituximab,
the clearance and disposition
or trastuzumab Whileagents.
of mAbs
[124–126]. most agents may notcase
In addition, be affected,
reports thein kidneys
patients
may play a role
undergoing in the elimination
hemodialysis of agents
have reported thatcapable of passing glomerular
renal impairment did not affectfiltration
the PKs(~<60 kDa)
kDa) [127].
of bevacizumab,
(~<60 [127].
These agentsrituximab,
cetuximab, that pass or thetrastuzumab
cutoff typically experience
[124–126]. While a gradual decrease
most agents may in
nottheir clearance,
be affected, theand thus
kidneys
increased
may play aexposure,
role in the with
elimination
increasing of agents capable of passing
renal impairment glomerular
[127]. However, filtration
Czock et al.(~<60 kDa)3-fold
reported [127].
reduction
These agents
reduction inCL
in CL ofofpass
that lowmolecular
low molecular
the weight
cutoff weight
typically peptides
andand
experience
peptides proteins
a gradual
proteins (<50
kDa)kDa)
decrease
(<50 in patients
ininpatients
their with with
clearance, severe
and
severe thus
renal
increased
renal
failure exposure,
failure
or end or
stage withdisease
endrenal
stage increasing
renal diseaserenal
(ESRD) impairment
(ESRD)
[128]. Future [127]. are
[128].studies
Future However,
studies
needed Czock
are toneeded et al.
determineto reported
determine 3-fold
the effects the
for
reduction
effects
smaller for in CL of
smaller
antibody low molecular
antibody
fragments fragments weight
(e.g., Fab, scFv,peptides
(e.g., Fab,
adAb)scFv, and
and proteins
adAb)
with (<50conjugation
and with
conjugation kDa)
to form in patients
to form
ADCs, with
as severe
ADCs,
cytotoxicas
cytotoxic
payloads payloads
renal failure or end
provide provide
additional additional
stage renal disease
mechanisms mechanisms
(ESRD) of alteration.
[128].
of alteration. Future studies are needed to determine the
effects for smaller antibody fragments (e.g., Fab, scFv, adAb) and with conjugation to form ADCs, as
5.4.
5.4. Neonatal
Neonatal
cytotoxic Fc Receptor
Fc
payloadsReceptor
provide(FcRn)
(FcRn)
additional mechanisms of alteration.
The role
role ofofFcRn
FcRnininsupporting
supportingthe the prolongation
prolongation of of
serum
serum IgGIgGhalf-life in circulation
half-life has been
in circulation well
has been
5.4. Neonatal Fc
characterized Receptor
[129]. Its (FcRn) was discovered in the 1960s as a transporter of maternal antibodies
existence
well characterized [129]. Its existence was discovered in the 1960s as a transporter of maternal
to a fetus,
antibodies
The rolethus its original
to aoffetus,
FcRn thus itsname of the
original
in supporting name
the neonatal Fc receptor
of the neonatal
prolongation (FcRn)
Fc receptor
of serum [130].
(FcRn)
IgG half-life This receptor
in[130]. ishas
widely
This receptor
circulation beenis
expressed
widely within
expressed within
well characterized cells of various
[129].cells
Its of organs
various was
existence throughout
organs the
throughout
discovered body
in the[131].
the body FcRn
1960s[131]. does
as a FcRn not bind to its
does notofbind
transporter ligand
to its
maternal
(i.e.,
ligand “Fc
antibodies region”
(i.e., “Fc
to or CH2–CH3
region”
a fetus, or its
thus interface)
CH2–CH3
original name at physiological
interface)
of theat neonatal pHFc (pH
physiological pH= 7.0–7.5),
receptor (pH but
= 7.0–7.5),
(FcRn) only
[130]. butinonly
This the acidic
in the
receptor is
environment
acidic
widelyenvironment
expressedof anwithin
endocytic
of ancells vacuole
endocytic (pH
vacuole
of various < 6.5)
(pH
organs when
< 6.5)exposed
throughout when histidine
the exposed
body residues
histidine
[131]. FcRn does on FcRn
residues
not bind become
on FcRn
to its
protonated
become and
“Fcincrease
protonated
ligand (i.e., and their
region” affinity
increase
or CH2–CH3 theirtoaffinity
the Fc region
interface)to at of
Fc IgGs
thephysiological
region[132]. It is(pH
of IgGs
pH generally
[132]. It isbelieved
generally
= 7.0–7.5), that
but only serum
believed
in the
IgGs
that
acidic are
serum first internalized
IgGs
environment are first via various vacuole
of aninternalized
endocytic pathways
via various(pH(nonspecific
pathways
< 6.5) when endocytosis
(nonspecific or fluid-phase
endocytosis
exposed histidine or pinocytosis)
residuesfluid-phase
on FcRn
become protonated
pinocytosis) and increaseendosome,
into an intracellular their affinity to the
before Fc can
they region
bindoftoIgGs
FcRn[132].
to beItrecycled
is generally believed
and expelled
thatofserum
out IgGs are and
the endosome first cell
internalized
[131]. via various pathways (nonspecific endocytosis or fluid-phase
As mentioned
pinocytosis) into anpreviously,
intracellular FcRn contributes
endosome, to the
before theyoverall PKto
can bind profile
FcRn ofto mAb-based therapeutics
be recycled and expelled
in
outseveral potential and
of the endosome ways.cellFcRn
[131]. is widely expressed throughout the body, including within
parenchymal and hematopoietic
As mentioned previously, FcRn cellscontributes
in fat, kidneys,
to theliver,
overallmuscle, skin,ofspleen,
PK profile and placenta,
mAb-based but
therapeutics
may have differing
in several potential functions
ways. FcRn based is on the tissue.
widely Chenthroughout
expressed et al. examined the effect
the body, of FcRn
including on
within
 Antibodies 2018, 7, 10 15 of 28

into an intracellular endosome, before they can bind to FcRn to be recycled and expelled out of the
endosome and cell [131].
As mentioned previously, FcRn contributes to the overall PK profile of mAb-based therapeutics in
several potential ways. FcRn is widely expressed throughout the body, including within parenchymal
and hematopoietic cells in fat, kidneys, liver, muscle, skin, spleen, and placenta, but may have differing
functions based on the tissue. Chen et al. examined the effect of FcRn on biodistribution of an
Fc-containing IgG1 in wild type (WT) and FcRn-knockout (KO) mice [133]. The fat, muscle, and skin
of KO mice had a decreased tissue/blood exposure ratio versus WT mice, while the kidney, liver, and
Antibodies
spleen had 2018, an7, increased
x FOR PEERratio. REVIEW The differing effects on tissue/blood exposure ratios of FcRn knockout 15 of 28
may be explained by differing functions of FcRn in each tissue. For example, in liver and spleen, FcRn
circulation,
may primarily while
serveintomuscle transport and skin, FcRn
antibodies out of may tissue be backresponsibleinto systemic for delivering
circulation,antibodies while in muscle from
circulation to the tissues [133].
and skin, FcRn may be responsible for delivering antibodies from circulation to the tissues [133].
Several
Several discussions
discussions have have been been published
published on on how how optimization
optimization of of thethe interaction
interaction with with FcRn FcRn may may
alter intracellular transport, thus leading to longer
alter intracellular transport, thus leading to longer half-lives (and reduced clearance) of mAb-based half-lives (and reduced clearance) of mAb-based
drugs. Modulation of
drugs. Modulation of the
theFcRn–IgG
FcRn–IgGinteraction, interaction,totoimprove improvePK PK parameters,
parameters, has has been been a strategy
a strategy used used by
by several investigators to either extend (improving
several investigators to either extend (improving efficacy and reducing dosing frequency) or shorten efficacy and reducing dosing frequency) or
shorten (for diagnostic evaluation or control for
(for diagnostic evaluation or control for known toxicities) the half-life of an agent. The correlation known toxicities) the half-life of an agent. The
correlation
between FcRn between binding FcRn binding
affinity and affinity
systemic andhalf-life
systemichas half-life has been investigated
been investigated extensively extensively
for a number for a
number
of mAbsof[66,134–138].
mAbs [66,134–138]. Several Severalreviewsreviews have also have also discussed
discussed how mutations how mutations in the in Fc the region Fc region
affect
affect the FcRn–IgG interaction, and are directly related
the FcRn–IgG interaction, and are directly related to observed differences in half-life [136,139–141]. to observed differences in half-life [136,139–
141].
Five Fivemutationsmutations of note of notehave havebeen been previously
previously reviewed,
reviewed, and andhave havebeen beendemonstrated
demonstrated to to extend
extend
serum
serum IgG half-life [142,143].
IgG half-life [142,143]. However, However, other other studies studies contradict contradict these these reports,
reports, raising raising questions questions on on
the
the interaction between FcRn and in vivo clearance [138,144,145]. Cross-species differences in
interaction between FcRn and in vivo clearance [138,144,145]. Cross-species differences in thethe
FcRn–IgG
FcRn–IgG interaction
interaction have have been been evaluated
evaluated to to determine
determine the the relevance
relevance in in the
the use use of ofpreclinical
preclinical models, models,
especially mice. Because of a stronger affinity
especially mice. Because of a stronger affinity of human IgG antibodies for murine FcRn of human IgG antibodies for murine FcRn (~15-fold
(~15-fold
greater
greater than thanhuman human FcRn),
Antibodies
FcRn), 2018,
human human
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of theFcRn FDA, isthe
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[122,123].
prolongation hemodialysis
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is special have
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Fc Receptor
hepaticprovide(FcRn)
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is mechanisms
PK
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necessary of ofbecause
therapeutic
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renal cetuximab,
[122,123]. failure the
This or5.4. proteins
end Neonatal
rituximab,
excretion
is becausestage [168].
by Fc
or Receptor
the
renal trastuzumab
disease
kidneys
and (FcRn)
hepatic [124–126
(ESRD)
and liver
impai [1 a
Pertuzumab (Perjeta ® ) is awell novel
characterized [129]. Its existence was discovered in the 1960s as a transporte
humanized recombinant mAb directed against HER2 withInand
According to theThe FDA, the likely
assessment to alter
involved of the
mAb inPK disposition
agents
the in
clearance special because
and 5.4.
effects Neonatal
may
the
populations,
disposition for play
excretion Fc
smaller a
such
ofReceptor
role antibody
by
as
mAbs in the the
renal (FcRn)
kidneys
agents. fragments
elimination
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of
liver
addition, agents Fab,
are not
case scF
cap
role of FcRn antibodies
in supporting to a fetus, thus its original
the prolongation name of
of serum IgG thehalf-life
neonatalThe role inFc receptor
circulation
of FcRn in (FcRn)
has supporting
been [130]. Tt
a distinct
hepatic mechanism
impairment, 5.4.isNeonatal
not of action
Fc Receptor
necessary from
involved[122,123]. trastuzumab.
(FcRn)
undergoing
in theThis hemodialysis
clearance
is To and
because evaluate
renal have
disposition
and potential
cytotoxic
reportedThese
hepatic of DDIs
payloads
agents
that
mAbs renal
impairment associated
provide
that
agents. pass
impairment is Innot with
additional
the cutoff
did
addition, not mechanisms
typically
caseaffect expe
the
reports P
well characterized widely [129]. Its expressed
existence within
was cells discoveredof various inThe organs
the role
1960s of as
well FcRn
throughout in supporting
characterized
a transporter the body[129]. of the
[131].maternal prolongati
FcRn
Its does
existenc
co-administration
likely to alter theantibodies of other agents
PK disposition with
undergoing
because the pertuzumab,
cetuximab, hemodialysis
excretion rituximab,
by phase
thehave or Ireported
kidneys studies
trastuzumab and evaluating
increased
that
liver [124–126].
renalare PK
impairment
not ofWhile
exposure, pertuzumab
significantly most
with
did notagents
increasing
affect maythe not
renal
PKs be
imp
of ab
b
The to rolea fetus,
of FcRn ligand
thus in its(i.e.,
supporting “Fc region”
original name or the
of
the prolongation CH2–CH3neonatal well ofinterface)
Fc receptor
serum
characterized IgG at half-life
physiological
antibodies
(FcRn)[129].[130]. to
inIts pH (pH
acirculation
fetus,
This
existence thus
receptor =was
hasits7.0–7.5),
original
is
beendiscov
given at
involved in the
thesame dose, and
clearance alonedisposition
and in may
cetuximab, combination
rituximab,
ofplay mAbs a role withor
in
agents. trastuzumab
trastuzumab
the elimination
In addition, 5.4. and
[124–126].
of docetaxel,
Neonatal
reduction
agents
case Fc
While
reports capable in
Receptor
in CL patients
most
in of (FcRn)
low
agents
passing
patients with
molecular
may not
glomerular weight
be affected, pep
filtratio
widely expressed within
well characterized acidic
[129]. environment
cells variousof
Itsofexistence an endocytic
organs
was throughout
discovered vacuole
antibodies inthe the body (pH
to1960sawidely< 6.5)
fetus,
[131]. as expressed
FcRn when
athus its
does
transporter exposed
original
within
not of bind histidine
name
cells to its
maternal the
of vario resi n
metastatic
undergoing breast cancer,
hemodialysis have were may
reportedcompared
playThese
that a role[169,170].
renalagentsin the that
impairment Thepass
elimination mean
the
did not serum
cutoff
of agents
affect Cmin
typically
renal
thecapable
PKs for of pertuzumab
experience
failure of or
passing
bevacizumab, end a gradual
stage after
glomerular renal decrease disease
filtration in their
(ESRD
(~<6
ligand (i.e., “Fc
antibodies to aregion” become
fetus, thus protonated
or CH2–CH3
its originalinterface) and increase
name of the their
at physiological
neonatal
widely affinity
The Fc pHtoligand
receptor
role
expressed the
of (pHFcRn Fc
(FcRn)
within region
=(i.e.,7.0–7.5),
in cells “Fc
[130].ofregion”
supporting IgGs
ofbutThis only
various [132].
orin
receptor
the It
CH2–CH3
the
prolongati
organs isisgene
thr
administration, alone or in combination with trastuzumab and docetaxel, effects were
for 35.0
smaller µg/mL antibody and fragments (e.g.,
cetuximab, rituximab,
acidic
widely or trastuzumab
environment
expressed These
that
within agents
of[124–126].
an cellsthat
increased
serum endocytic ofWhile
IgGs pass
exposure,
are
variousvacuoletheorgans
most
first cutoff
agents
with
internalized typically
<may
increasing
(pHthroughout 6.5)ligand
well not
via
when experience
be
renalaffected,
various
exposed
(i.e.,
the
characterized impairment
“Fc
body a[131].
pathways
acidic gradual
the
histidine
region” kidneys
environment
[129]. FcRn decrease
or[127].
(nonspecific
residues
CH2–CH3
Its does
existence of in
However,
not an
on their Czock
endocytosis
endocytic
FcRn
interface)
bind was to its Fa
clearan
discov atet
63.6 µg/mL, respectively. The ~2-fold reduction
reduction in
in clearance
CL ofincreasing
low and increase in
cytotoxic exposure payloads of pertuzumab
provide additional mechan
may play a role in become
ligand (i.e., “Fc increased
the elimination
protonated of agents
and
pinocytosis)
region” exposure,
orcapable
increase CH2–CH3 their
into ofan with
passing
affinity
intracellular
interface) to molecular
glomerular
the Fc renal
acidic
atendosome, region
physiological
antibodies weight
impairment
filtration
environment
of IgGs
before
to peptides
(~<60
pHbecome [127].
kDa)
[132].
they
a fetus, (pH ofcan and
an [127].
=protonated
thusIt is
bind
7.0–7.5), proteins
However,
endocytic
itsgenerally
to but and
FcRn
original (<50
Czockvacuole
onlybelieved
increase
to be
name kDa)
inetrecycle
of al.
(pH
the inrep
thei
the pn<
suggests
These agentsthat that combining
pass the mAbtypically
cutoff agentsrenal
reduction resultsin
experienceCL in of
failure a DDI low
a
or that
gradual
end alters
molecular
stage decrease the
weight
renal PKs in and
peptides
their
disease PDs
clearance,
(ESRD) ofand mAbs proteins
and
[128]. orthus ADCs.
Future (<50 kDa)
studies inare patients
neede
that serum
acidic IgGs areout
environment first
ofofan internalized
theendocytic
endosome via andvarious
vacuole cell(pH pathways
[131]. < become
6.5) when
widely (nonspecific exposed
protonated
expressed that withinserum
endocytosis
histidine
and IgGs residues
increase
cells are
of or first
fluid-phase
their
various internalized
onorgans
affinity FcRn tothr th
increased Rilotumumab
exposure, is aincreasing
with fully human renal renal monoclonal
effects
failure impairmentfor
or end antibody
smaller [127].
stage antibody against
However,
renal hepatocyte
fragments 5.4. (e.g.,
Neonatal growth
Fab, FcFuture
scFv,factor
Receptor adAb) (HGF)(FcRn)
and with conjugatio
become
pinocytosis) protonated
into an and Asincrease
intracellular mentioned their
endosome, previously,
affinitybefore todisease
FcRn
the
they that
Fc Czock
can
ligand (ESRD)
serum
contributes
region
bind etof
(i.e., al.
to [128].
IgGs
“Fc to
IgGs
FcRnreported
are
the
pinocytosis)
region” be 3-fold
first
tooverall
[132]. studies
Itinternalized
recycled
or is
intoPK
CH2–CH3 are
profile
generally
an and needed
via
intracellularof
expelled various
mAb-base
believed
interface) to
endo de
at p
and the only
reduction in CLout known
of low ligand
molecular for the
weight
effects METcytotoxic
for receptor
peptides
smaller payloads[171].
and
antibody The
provide
proteins PK
fragments (<50of rilotumumab
additional
kDa)
(e.g., in
Fab,mechanisms
patients
scFv, has been
with
adAb) of evaluated
alteration.
severe
and with conjugation to for
thatof serum
the endosome IgGs are inand several
first cell potentialvia
internalized
[131]. ways.
various FcRn is widely
pathways
acidic
pinocytosis) Theexpressed
(nonspecific
environment intoout
rolean ofofintracellular
the
ofFcRn throughout
endocytosis
endosome
an endocytic
in supporting orand the
fluid-phase
endosome, cell
vacuole body,
the [131].(pH
before
prolo inc
in failure
renal severalor trials
end now,
stage inrenal
combinationcytotoxic
disease with
(ESRD) other
payloads [128].traditional
provide
Futureadditional anti-cancer
studies therapies
mechanisms
are needed toand of other targeted
alteration.
determine the[131].
As mentioned
pinocytosis) into parenchymal
previously,
an intracellular FcRn and hematopoietic
contributes
endosome, beforeto the cells
theyout
overall
become in
of
canwell fat,
the PK
bind kidneys,
endosome
profile
protonated to
characterizedFcRn ofliver,
As andand
to mAb-based
mentioned muscle,
cell
increase
be[129]. recycled skin,
Itspreviously,
therapeutics
their
andaffinity
existence spleen,
expelled FcRn
was toand dc
th
agents
effects [172–175].
for smaller A recent
antibody review of the Fab,
5.4. preclinical
Neonatal Fcand Receptorclinical (FcRn) data, to date, oftorilotumumab states
inout of the fragments
several potential
endosome may (e.g.,
ways.
and have cellFcRn scFv,
differing
[131]. is adAb) functions
widely and with
expressed based conjugation
that onAs the
throughout
serum mentioned
antibodies IgGs inform
tissue. tothe aChen
several
are ADCs,
body,
first
previously,
fetus, et as
potential
thus al. its examined
including
internalized FcRn ways. viawithin
contributes
original the effe
FcRn
various
name isp
to
of
that DDIs
cytotoxic do notprovide
payloads exist when co-administered
additional 5.4. Neonatal
mechanisms Fcwith ofother
Receptor therapeutic proteins [171]. However, reference
(FcRn)
alteration.
parenchymal As mentioned biodistribution
and hematopoietic
previously, The FcRn roleof
cells aninFc-containing
fat, kidneys,
contributes
of FcRn to the
in supporting IgG1
liver,
in several
overall
pinocytosis) in
widelythe wild
muscle, PK type
profile
into
prolongation
expressed an(WT)
parenchymal
potentialskin, spleen,
ofways. andserum
mAb-based
intracellularof
within and
andFcRn-knockout
FcRn
cells hematopoietic
placenta,
is various
endosome,
IgG
of widely
therapeutics
half-life but (KO) in cm
cells
expr
before
organ
PK studies evaluating monotherapy with panitumumab showed an AUC of 34,100 ± 9260 h·ug/mL
may have differing
in several potential fat, muscle,
functions
Theways.
well and
based
FcRn
rolecharacterized
of FcRn skin
ison inofthe
widelyKO
supporting
[129]. mice
tissue. Itshad
expressed parenchymal
thea of
Chen
out
existence decreased
et
theal.
throughout
ligand
prolongation endosome
was (i.e.,and
examined
may “Fcofhematopoietic
tissue/blood
have
the
discovered and
serum the
body,
region” cell inexposure
differing
effect
IgG cells
including
[131].
orthe offunctions
CH2–CH3
half-life
1960s FcRninin
ratio fat,
versus
within
as on kidne
a based
interfac
circulati
trans W
5.4.and
Neonatal 227 ± 36.7
CmaxFcofReceptor (FcRn) µg/mL, while dual therapy of panitumumab and rilotumumab at the same
biodistribution
parenchymal of and anthe
well kidney,
Fc-containing
hematopoietic
antibodies
characterized liver, IgG1
cells
to and inspleen
in
a fetus,
[129]. wild
fat,
Itsthus had
type
kidneys,
existence an
its (WT)may increased
liver,
originalwas and
have
acidic
As name ratio.
FcRn-knockout
muscle, differing
environment
mentioned
discovered theThe
biodistribution
of skin, differing
spleen,
functions
previously,
neonatal
in the (KO)
of an
1960sof
Fc mice
andan effects
based Fc-containing
[133].
placenta,
endocytic
FcRn
receptor
as on on
acontributes The
the tissue/b
(FcRn)
transporter but
vacuole tissu Ig(
to
[13
dose results in an AUC of 61,400 ± 1620 h·µg/mL and Cmax of 346 ± 88.6 µg/mL [171]. This is
The role of fat, may
FcRn muscle,
inhave and
supporting skin
differing ratios
offunctions
antibodies
the KO ofmice
FcRn
to aexpressed
prolongation
widely knockout
had
based
fetus, aof decreased
on
thus serum may
theoriginal
its
within be
IgGcells biodistribution
explained
tissue/blood
tissue. in Chen
name
half-life
of several
become
various of
inby thediffering
exposure
et al.
circulation
organs fat,ofmuscle,
examined
potential
protonated
neonatal an
ratio Fc-containing
functions
has
throughout versus
ways.
Fc and
the
andreceptor
been of
skin
WT
effect
FcRn FcRn
increase
the IgG1
mice,
of ofKO
is
(FcRn)
body inFcRn
widely
their in
each
while
mice
[131]. wild
[130]. tissue
on had
affinity typ
expr
FcRn Thi
an approximately 80% increase in AUC exposure and 52% increase in Cmax [171]. This study also
well characterized the kidney,Itsliver,
biodistribution
[129]. and
of in
anliver
widely
existence spleen
ligand
was and
Fc-containing
expressed had spleen,
(i.e.,
discovered an IgG1
“Fc
within FcRn
increased inthe
region”
incells may
wild of primarily
ratio.
or type
various
1960s The (WT)
parenchymal
fat,
CH2–CH3
as serve
differing
amuscle,
that
organs and serum
transporter to
and
interface) transport
effects
the
FcRn-knockout
and
throughout skin
IgGskidney,
ofaton
hematopoietic
ofare antibodies
tissue/blood
KO liver,
first
physiological
the
maternal (KO)
mice
body andmice
had out
cells
internalized
[131]. spleen
pH of
exposure
a[133].
in(pH
FcRn tissue
fat,
decreased had
viaThe
=kidne
does back
anti
vario
7.0–7 n
demonstrated altered PK of rilotumumab when combined with other mAbs. The mean plasma AUC
antibodies to a fetus,ratios of FcRn
fat, thus
muscle, knockout
and
its original skinname
ligand ofmay KOofbe
acidic
(i.e., mice
“Fc explained
had a decreased
environment
the region”
neonatal by
or Fcof differing
an endocytic
CH2–CH3
receptor the
tissue/blood
may(FcRn)kidney,
functions have vacuole
pinocytosis)
interface) [130]. liver,
ofdiffering
atFcRn
exposure ratios
(pH
This and
in
into
physiological of<each
ratio spleen
FcRn
functions
receptoran6.5) tissue.
versus when
intracellular
pH
is had
knockout WTFor
based anmice,
exposed
(pH increased
example,
may
on
=endosome, be
while
the
7.0–7.5), rati
expla
tissu
histidine be
bu
of rilotumumab monotherapy on cycle 5 and rilotumumab at 10 mg/kg plus 10 mg/kg bevacizumab
widely expressed intheliver
within and
kidney,cells spleen,
liver, FcRn
acidic
of various may primarily
and environment
spleen
become
organs had
protonated
throughout serve
anofincreased
an the tobody
endocytic
and transport
ratio.
increase The
vacuole
[131]. antibodies
out
their differing
biodistribution
ratios of of
FcRn FcRn
(pH thein
affinity
does outliver
effects
of toan
<knockout
endosome
6.5)
not of and
tissue
when
the
bind on
Fc-containing
Fc may
tospleen,
and back
tissue/blood
region
its be
exposed cell into
FcRn IgG1
explained
of[131]. systemic
may
exposure
histidine
IgGs in by
[132]. primari
wild Ittyp
differ
residu is
were 55,900 µg/mL·h and 82,700 µg/mL·h, respectively—an increase in serum AUC of 47% after the
ligand (i.e., “Fc region” ratios of orFcRnCH2–CH3 knockout
become thatmay serum
protonated
interface) beatexplained
IgGsandare
physiological byfirst
increase differing in
fat,
internalized
pH their (pH liver
functions
muscle,
affinity and
= 7.0–7.5),via tospleen,
As of
and
variousFcRn
mentioned
the but skin
Fc FcRn
onlyinofeach
pathways
region KO may
previously,
in ofthe primarily
tissue.
mice
IgGs had For
(nonspecificFcRn
[132]. serve
a example,
decreased to tra
isendocy
Itcontribut
genera ti
addition of bevacizumab [171,172]. Further studies are needed to determine if these sizeable increases
acidic environment in liver
of anand spleen,
endocytic FcRn
thatvacuole
serum may IgGs
pinocytosis) (pH primarily
are
< 6.5)
into serve
firstwhen
an to transport
internalized
exposed
intracellular thevia in antibodies
kidney,
various
histidine
endosome, several liver,
pathways
residues out and
potential
before of on
they tissue
spleenFcRn ways.
(nonspecific
can back
had
bindFcRn into
an systemic
increased
toendocytosis
FcRn is to berati
widely or
rec
in AUC are clinically relevant.
become protonated and increase their out ofinto
affinity
pinocytosis) thethe
to endosome
anFc regionand
intracellular cell
of IgGs ratios
[131].
endosome, [132]. parenchymal
of FcRn
Itbefore
is generally knockout
they and
can hematopoietic
maytobeFcRn
believed
bind explained tocells by
be recycled indiffer
fat, ka
However, in comparison to SM drugs, there are far fewer evaluations of how therapeutic
that serum IgGs are first internalized out of via the various
endosome pathways
As mentioned and cell [131]. inFcRn
(nonspecific
previously, livermay
endocytosis and have
contributes spleen, differing
or FcRn
fluid-phase
to may
the overall functions primarily based
PK profile serve on tothe
of mAb tra
antibodies may affect the other ADC PK and/or PD. Data with the use of an ADC in combination
pinocytosis) into an intracellular endosome, Asinmentioned
several
before they potential
previously,
can bind ways.FcRn
to FcRn FcRn tobiodistribution
contributes is recycled
be widely to theexpressed ofexpelled
overall
and an PK Fc-containing
throughout
profile of mAb-based IgG1
the in body wil
out of the endosome and cell [131]. in several parenchymalpotential and ways. hematopoietic
FcRn is widely cells inexpressed
fat, muscle, fat, kidneys,and skin liver,
throughout of KOmuscle, micethe had skin,
body, spleen
a decreasinclu
As mentioned previously, FcRn parenchymal may have
contributes andto the hematopoietic
differing
overallfunctions PK profile cells basedinofthefat, kidney,
kidneys,
on
mAb-based the tissue.liver,
liver,and
therapeutics muscle,
Chen spleen et skin,
al.had an increased
spleen,
examined and thep
in several potential ways. FcRnmay biodistribution
have
is widely differing
expressed of throughout
an Fc-containing
functions basedthe on ratiosIgG1tissue.
the
body, inincluding
of wildChen
FcRn type
knockout (WT)
et
within al. may and
examined FcRn-knockout
be explained the effect by (K d
parenchymal and hematopoietic biodistribution cells infat, fat,muscle,
kidneys,of an and Fc-containing
liver,
skinmuscle,of KO mice IgG1
skin,had inwild
in
spleen,liver
a decreased and spleen,
type (WT)
placenta, and
tissue/blood FcRn but may
FcRn-knockout primarily
exposure (KO)
ratio serve mit
vers
may have differing functions based onthe
fat, muscle, thekidney,
and
tissue. skin liver,
of KO
Chen and etmicespleen
al. had examinedhad an the
a decreased increased tissue/blood
effect ratio.
of FcRn The exposure
on differing ratio effects versus on WT tiss
Antibodies 2018, 7, 10 17 of 28

with other antibodies have only recently begun to be evaluated for efficacy and synergistic effects
in preclinical models. Thus, while data is still lacking with ADCs, our growing knowledge of DDIs
after administration of multiple antibodies would suggest this is an important factor to evaluate in
preclinical models or formulation selection.

7. The Next Generation of ADCs

Several hurdles remain for antibodies and ADC to overcome, such as low delivery efficiency,
heterogeneity of target antigen expression, and target expression on normal tissues. While the success
of maytansinoid and auristatin ADCs is notable, further research is being performed with even more
potent cytotoxic compounds (EC50 in the pM range) and novel mechanisms of action, as a means to
increase the therapeutic window. Of note, pyrrolobenzodiazepine (PBD) dimers and duocarmycin
analogs have demonstrated cytotoxicity at 10-fold lower concentrations than those of maytansinoid and
auristatin ADCs [176,177]. An example in development is SGN-CD70A, a PBD-conjugated anti-CD70
ADC for the treatment of renal cell carcinoma and NHL, which showed anti-tumor activity in preclinical
mouse models when dosed at 0.1–0.3 mg/kg of ADC [178]. On the other hand, a HER2 conjugated
duocarmycin analog (vc–seco–DUBA) has shown tumor growth inhibition in patient derived xenografts,
despite low HER2 expression, after a single dose of 1 mg/kg [179]. There has been skepticism about the
utility of these drugs due to their short half-life (~1 h) after release, but this characteristic may ultimately
be of benefit as potential systemic toxicities would be expected to be minimal [180].
There is a considerable emphasis being placed on the optimization and understanding of DAR
effects. Of note, substantial effort is currently focused on determining how to direct the site(s) of
conjugation and create more homogenous products with a narrow DAR range. THIOMAB was the first
approved that used this particular strategy, utilizing strategic cysteine residues [4]. Others have begun
to add sequence tags, such as SMARTag and TG-ADC, to direct enzymatic drug conjugation [181–183].
Beyond even the number of conjugation sites on a particular carrier, optimal conjugation site selection
must also be considered, as site accessibility and linker stability were inversely correlated with
intermediate accessible sites in demonstrating increase efficacy and safety [184]. While advancements
are creating even higher homogenous DARs, it is still unknown if this leads to an improved therapeutic
window while also increasing efficacy/safety.
Instead of altering the cytotoxic payload, others have altered the antibody carrier to affect function,
such as using smaller antibody fragments (e.g., scFv, minibodies, sdAb, diabodies). While this may
result in increased drug delivery into the tumor microenvironment and provide greater clinical effects,
these ADCs will also result in both faster clearance and larger volumes of distribution, changing how
we administer these biologic agents from other antibodies and ADCs. Additional studies will provide
insight if more frequent dosing of these smaller antibody agent can provide similar or improved
clinical efficacy while being more tolerable due to their more rapid clearance.

8. Conclusions
The addition of ADCs within the field of medicine has led to great advances in our fight with
treating hematologic and solid tumor malignancies, but significant challenges still remain to be solved.
However, there are still several more innovative and novel formulations under development and in
clinical trials, as immunotherapies begin to take focus. In addition, the use of predictive biomarkers or
screening tools other than time-intensive IHC methods will be essential, to ensure effective treatments
are targeted towards patients who will gain the most benefit.
While progress has been made in gaining knowledge on the PKs and PDs of mAbs and ADCs,
increased understanding of the mechanistic aspects of their disposition and how this relates to efficacy
and safety are needed. There are many properties that make ADCs unique compared to an active SM
drug. However, antibody-based therapies may also have new toxicities associated with enhanced
distribution to specific organs and toxicities associated with the components of the carrier. It has been
shown that physical properties, the MPS, host-associated factors, and pharmacologic-associated factors
Antibodies 2018, 7, 10 18 of 28

all contribute in varying degrees to altering the PKs and PDs, in preclinical models and in patients.
Thus, the pharmacology of ADCs is highly complex. Thus, several challenges still exist to optimize
ADC therapies before the field can make further improvements, including DAR coupling strategies,
the limited tumor penetration of these agents, and the development of resistance.
There is still much to learn about the clinical applications of biologic-based therapies, but the
success of early agents, such as Kadcyla and Adcetris, have emboldened further research into improved
treatments and optimizing outcomes based on personal phenotypes. Areas of research that can aid in
our understanding of how these agents are handled and how we may predict their actions in patients,
include pharmacogenomics, cellular function (probing the MPS), more sensitive and accurate analytical
PK methods for determining drug and carrier components, and coupling of phenotypic probes with
relevant PK and PD models.

Acknowledgments: Work performed by authors is partially supported by NIH/NCI (1 U54 CA151652-01)

Carolina Center of Cancer Nanotechnology Excellence and the UNC University Cancer Research Fund. The content
is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
The authors have no other relevant affiliations or financial involvement with any organization or entity with a
financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart
from those disclosed.
Author Contributions: Andrew T. Lucas and William C. Zamboni conceived the topic; Lauren S. L. Price
and Allison N. Schorzman contributed Section 3; Mallory Storrie contributed to the writing of Section 2;
Joseph A. Piscitelli contributed to the writing of Sections 4.1 & 4.3; Juan Razo contributed to the writing of
Section 4.4; Andrew T. Lucas wrote remaining sections of the manuscript and drew/requested permissions for
the figures. Andrew T. Lucas, William C. Zamboni, Allison N. Schorzman, and Lauren S. L. Price helped and
corrected text and figures.
Conflicts of Interest: William C. Zamboni holds equity in and licensed patent on a MPS probe. All other authors
have no conflicts of interest to disclose.

Abbreviations
ADA anti-drug antibodies
ADC Antibody–drug conjugate
ADCC antibody-dependent cell-mediated cytotoxicity
AML acute myeloid leukemia
BSA body surface area
CDC complement-dependent cytotoxicity
CEA carcinoembryonic antigen
CFDA Chinese Food & Drug Administration
CL clearance
DAR drug–antibody ratio
DDI drug–drug interaction
DLT dose limiting toxicity
ESRD end stage renal disease
Fabs Fab fragments
FcγR Fc-gamma receptors
FcRn neonatal Fc receptor
GEMMs genetically engineered mouse models
HGF hepatocyte growth factor
iv intravenously
KO knockout
mAb monoclonal antibody
MCC maleimidomethyl cyclohexane-1-carboxylate
MMAE monomethyl auristatin E
MPS mononuclear phagocyte system
NHP non-human primate
NP nanoparticle
OS overall survival
Antibodies 2018, 7, 10 19 of 28

PBD pyrrolobenzodiazepine
PD pharmacodynamic
PEG polyethylene glycol
PFS progression-free survival
pI isoelectric point
PK pharmacokinetic
sc subcutaneously
scFv single chain variable fragment
SM small molecule
WT wild type

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