REVIEWS

Building better monoclonal

antibody-based therapeutics
George J. Weiner
Abstract | For 20 years, monoclonal antibodies (mAbs) have been a standard component of
cancer therapy, but there is still much room for improvement. Efforts continue to build
better cancer therapeutics based on mAbs. Anticancer mAbs function through various
mechanisms, including directly targeting the malignant cells, modifying the host response,
delivering cytotoxic moieties and retargeting cellular immunity towards the malignant cells.
Characteristics of mAbs that affect their efficacy include antigen specificity, overall structure,
affinity for the target antigen and how a mAb component is incorporated into a construct
that can trigger target cell death. This Review discusses the various approaches to using
mAb-based therapeutics to treat cancer and the strategies used to take advantage of the
unique potential of each approach, and provides examples of current mAb-based treatments.

Cluster of differentiation
numbers
Numbers assigned to cell
surface molecules on the basis
of immunophenotyping. They
are often used to identify
different monoclonal
antibodies that bind to the
same antigen.
Holden Comprehensive
Cancer Center, University of
Iowa Hospitals and Clinics,
200 Hawkins Drive,
5970Z‑JPP, Iowa City,
Iowa 52242, USA.
e-mail:
george-weiner@uiowa.edu
doi:10.1038/nrc3930
More than two centuries have passed since we began to
understand the remarkable characteristics and potential
of antibodies1. Indeed, polyclonal antisera have been used
in the treatment of certain infectious diseases for decades2.
Soon after the first description of monoclonal anti-
bodies (mAbs) in 1975 (REF. 3) (a discovery that led to
a Nobel Prize 10 years later 4), mAbs were recognized
as unique biological tools and quickly became invalu-
able in pathological diagnosis and basic laboratory
investigation. Their ability to bind to specific antigenic
epitopes allowed for the rapid assessment of the molecu-
lar pheno­type of blood cells and, subsequently, of other
tissues. Molecules that were identified by mAb binding
were given cluster of differentiation numbers5 that are still
used extensively in diagnosis. mAbs are now used widely
in immunohistochemistry, flow cytometry and related
technologies. Aside from the use of mAbs as tools in
diagnosis, at the time they were first described there was
equal excitement about their therapeutic potential based
on the ability to manufacture mAbs of defined specificity
and class in essentially unlimited amounts. Theoretically,
this would allow for highly specific targeting of cancer
cells on the basis of their molecular phenotype.
However, early clinical results exploring mAb-based
therapeutics were disappointing 6, and until only 20 years
ago, some experts considered cancer treatment with
antibody-­based therapy a failed hypothesis. The first
mAbs evaluated in the clinic as cancer treatments were
murine mAbs. Although there were intriguing hints that
mAb therapy could be successful7, problems associated
with administering murine mAbs to humans limited
their clinical utility. These problems included the develop­
ment of an immune response against the therapeutic
mAb itself, the rapid clearance of the mAb and the sub-
optimal ability of the murine mAb to interact with the
human immune system in a manner that led to immune
destruction of the cancer.
Fortunately, persistent investigators continued to
explore how mAbs could be used in cancer treatment.
They evaluated various strategies, including using
immunoglobulin G (IgG) to target cancer directly, alter
the host response to cancer, deliver cytotoxic substances
to cancer and retarget the cellular immune response
towards cancer (TABLE 1).
The current era of successful mAb therapy began
with the development of techniques that allowed genetic
modification of murine mAbs to produce chimeric
mouse–human mAbs that behave immunologically in
most ways like naturally occurring human IgG8,9. Such
mAbs are less likely to be recognized by the host immune
system as a foreign antigen, have half-lives similar to
those of natural human IgG (generally 2–4 weeks) and
interact well with the effector arm of the human immune
system. They can be administered on a schedule that is
practical for patients (in many cases weekly or monthly),
and they are present in the circulation of patients at
therapeutic levels for months at a time. They distribute
to both the intravascular and the extravascular com-
partments and are present within the tumour mass for
long periods of time where they interact with malignant
cells, stromal cells, benign lymphocytes, the extracellular
matrix and the vasculature.

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Table 1 | Monoclonal antibody-based therapeutics

mAb-based Structure Characteristics of target Example of major ongoing research
therapeutic antigen questions
Antitumour mAbs Unmodified IgG or IgG modified to Tumour-associated surface Are IgGs with enhanced affinity for Fc receptors
mediate enhanced ADCC antigen more clinically effective than unaltered IgG?
Angiogenesis Unmodified IgG Host molecules that control What is the best way to evaluate clinical
inhibition angiogenesis response in patients treated with angiogenesis
inhibitors?
T cell checkpoint IgG1 (blocks checkpoint and Molecules that limit the How should we combine checkpoint
blockade mediates ADCC) or IgG4 (blocks anticancer T cell response blockade mAbs with each other, with other
checkpoint without mediating immunotherapeutics and with other anticancer
extensive ADCC) agents?
Radioimmunotherapy Unmodified IgG or mAb fragment Tumour-associated antigen that How can the logistics of administering
is not shed or present in the successful radioimmunotherapeutic agents be
circulation simplified to enhance their clinical utility?
Antibody–drug IgG modified with cleavable linker Highly specific tumour-associated What is the best combination of linkers and
conjugate and drug antigen that can internalize when drugs with each mAb and target antigen?
bound by a mAb
Bispecific antibody Variable regions from cancer-specific Tumour-associated antigen that is Can effective bispecific constructs that have
mAbs linked to variable regions not commonly absent in antigen- modified kinetics (thereby avoiding the logistic
specific for activating receptors on loss-resistant cancer variants complexities of continuous infusion) be
T cells developed?
Chimeric antigen Gene therapy approach to modifying Highly tumour-specific antigen Can very promising preliminary results be
receptor T cell T cells by inserting DNA coding for that is not commonly absent in extended to solid tumours, or will toxicity
the mAb variable region fused to DNA antigen-loss-resistant cancer be associated even with low levels of target
coding for signalling peptides variants antigen expression by benign cells?
ADCC, antibody-dependent cellular cytotoxicity; IgG, immunoglobulin G; mAb, monoclonal antibody.

Transmembrane signalling
The process by which an
extracellular signal, mediated
by a natural ligand or an
alternative agent such as a
monoclonal antibody, binds to
a membrane receptor and
generates an intracellular
signal that can affect a broad
range of cellular functions,
including cell growth, cell
differentiation and cell death.
Complement-mediated
cytotoxicity
(CMC). Also known as
complement-­dependent
cytotoxicity. Cell death
resulting from the activation of
the complement cascade by a
monoclonal antibody that
leads to the formation of a
membrane attack complex on
the surface of the cell.
Antibody-dependent
cellular cytotoxicity
(ADCC). Lysis of a target cell by
an immune effector cell (such
as a natural killer cell,
monocyte, macrophage or
granulocyte) induced by the
recognition of an antibody
bound to the surface of the
target cell.
After many years of research, a variety of mAb-based
approaches to cancer therapy are being used with con-
siderable success, and progress using mAb therapeutics
that are based on various different strategies (FIG. 1) is
continuing at a remarkable pace. Oncologists now see
mAb-based cancer therapies as a vital component of
state‑of‑the-art cancer care, and even better mAb-based
treatments are on the horizon.
This Review provides an overview of recent pro-
gress in the development and application of mAb-based
approaches to cancer therapy, including a description of
mAbs and mAb-based constructs that directly target the
cancer, alter the host response to the cancer, deliver cyto-
toxic moieties to the cancer or redirect T cells towards
the cancer.
Targeting the cancer cell
mAbs have been used to target a wide variety of antigens
expressed on the surface of cancer cells10. Characteristics
that make antigens attractive as targets for mAb therapy
include the density and consistency of expression of the
target molecule by malignant cells, limited expression of
the target molecule on physiologically vital benign cells,
lack of high levels of soluble target and limited tendency
of antigen-negative tumour variants to emerge. Desirable
characteristics of target tumour antigens vary depending
on the mAb construct being considered, the nature of the
malignancy (for example, haematological versus solid
tumours) and the mechanism of action of such constructs.
In vitro and animal model data indicate that some
mAbs that target antigens on the surface of malignant
cells are able to induce apoptosis by direct transmem-
brane signalling11. There is also evidence that mAbs kill
target cells by complement-mediated cytotoxicity (CMC)12
and by inducing antibody-dependent cellular cytotoxicity
(ADCC)13. Determining which of these mechanisms
(FIG. 2) is most important for a given mAb in a given
clinical scenario remains a challenge.
mAb-mediated cell signalling. Some mAbs can induce the
death of malignant cells in vitro, and presumably in vivo, in
the absence of immune effector mechanisms. The strength
of this effect varies considerably depending on the mAb,
the target antigen and the target cell14,15. Assessment of
whether a given mAb can mediate signalling-­induced
death in the target cell in  vitro sometimes requires
crosslinking with secondary antibodies, such as those
specific to human IgG16. Secondary crosslinking results
in enhanced aggregation of receptors that can lead to a
more robust signal. This is not necessarily an artificial way
of replicating the crosslinking and signalling observed
in vivo, in which crosslinking of mAbs bound to malig-
nant cells probably occurs to a certain extent through the
binding of the constant region of IgG to Fc receptors (FcRs)
expressed by a variety of cells in the tumour microenvi-
ronment. It is not clear whether artificial crosslinking of
mAbs speeds up and strengthens the physiological effect
of mAb signalling so that it can be measured in a quick
laboratory assay, or by contrast, whether it provides such
a strong signal that non-physiological changes are induced
that may not be clinically relevant.
In vitro measurement of signalling needs to be inter-
preted with caution as it varies greatly from the clinical
environment17. In vitro studies usually use cells that have
been selected to grow rapidly and consistently outside the
body — that is, independent of their normal environment.

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 REVIEWS

a Tumour-specific IgG b Angiogenesis inhibition c Checkpoint blockade the mAb results from the mAb interrupting the inter­
Tumour action between an activating ligand and the receptor,
cell
T cell
from inhibiting the dimerization of the receptor, or from
Receptor
having a direct effect on receptor signalling 19 (FIG. 2b).
Tumour PDL1 This complexity has been most extensively studied with
antigen VEGFR
the ERBB tyrosine kinase family of receptors20 and their
VEGF CTLA4 PD1
Complement PDL1- various ligands, and with mAbs such as trastuzumab
Effector cell specific and pertuzumab that recognize various receptor family
Ipilimumab Nivolumab members21. Individual receptors in this family can have
IgG multiple ligands, mAbs can alter dimerization proper-
ties and a mAb can have different signalling properties
d Radioimmunotherapy g CAR T cells depending on whether it is targeting a homodimeric or a
heterodimeric receptor 22. Understanding this complexity
is not just an academic exercise, as it can have a major
impact on the development and clinical testing of novel
Immunoconjugates Antigen-based therapeutics, including mAb combinations23. Resistance
retargeting of
cellular immunity to small molecules that inhibit signalling pathways can
develop with the emergence of cells that activate alterna-
tive or compensatory signalling pathways24. Similar pro-
e Antibody–drug f Bispecific cesses can lead to the emergence of resistance to mAbs
conjugate antibody
therapy therapy that mediate their therapeutic effects by signalling 25. As
CD3 with small molecules, the use of combination therapy is
one strategy to overcome this mechanism of resistance.
Figure 1 | Monoclonal antibody-based cancer therapeutic strategies.  Successful
monoclonal antibody (mAb) therapeutics have been based on a number of strategies.
Nature Reviews | Cancer Complement. The first studies demonstrating the very
Immunoglobulin G (IgG) molecules that bind to target cancer cells (part a) can mediate potent ability of complement fixation to enhance antibody-
antibody-dependent cellular cytotoxicity (ADCC) by immune effector cells, induce
mediated cytolysis were carried out more than a cen-
complement-­mediated cytotoxicity (CMC) or result in the direct signalling-induced
death of cancer cells (for example, herceptin and rituximab). IgG mAbs can also be used
tury ago. They involved the evaluation of the effect of
to inhibit angiogenesis (part b) (for example, bevacizumab) or to block inhibitory signals cobra venom on the lysis of red cells and bacteria26, and
(part c), thereby resulting in a stronger antitumour T cell response (for example, took place when our understanding of both antibodies
ipilimumab and nivolumab). Radioimmunoconjugates (part d) (for example, 131I and complement was obviously fairly limited. We have
tositumomab and ibritumomab tiuxetan) deliver radioisotopes to the cancer cells, since learned an amazing amount about complement in
whereas antibody–drug conjugates (part e) (for example, brentuximab vedotin and general and about how complement can have an impact
trastuzumab emtansine) deliver highly potent toxic drugs to the cancer cells. mAb on the efficacy of mAbs in particular12. We now know
variable regions are also used to retarget immune effector cells towards cancer cells that some (but not all) mAbs can mediate CMC27. The
through the use of bispecific mAbs that recognize cancer cells with one arm and ability of a given mAb to fix complement and to induce
activating antigens on immune effector cells with the other arm (part f) (for example,
CMC is partly dependent on antigen concentration, the
blinatumomab) or through a gene therapy approach in which DNA for a mAb variable
region fused to signalling peptides is transferred to T cells, thereby rendering them
orientation of the antigen in the membrane, and whether
chimeric antigen receptor (CAR) T cells (part g) specific for the tumour. CD3, T cell the antigen is present on the surface as a monomer or a
surface glycoprotein CD3 ε-chain; CTLA4, cytotoxic T lymphocyte-associated antigen 4; polymer. CMC can also depend on the mAb isotype and
PD1, programmed cell death protein 1; PDL1, PD1 ligand; VEGF, vascular endothelial the characteristics of the target cell, including whether
growth factor; VEGFR, VEGF receptor. the cell expresses complement-neutralizing molecules.
Even mAbs that are the same isotype and that target
the same antigen can vary in their ability to fix comple-
This can affect cell sensitivity to a wide variety of sig- ment 28. Type I human IgG1 CD20-specific mAbs (for
nals, and studying primary cells obtained directly from example, rituximab) crosslink CD20 tetramers and also
patients can avoid this problem. However, the evaluation fix complement. By contrast, type II IgG1 CD20‑specific
of primary cells requires extensive previous manipulation, mAbs (such as obinutuzumab) do not crosslink tetram-
Fc receptors including mincing, filtering and washing. This obliterates ers or fix complement well29. Antibody engineering that
(FcRs). A family of protein
receptors specific for an
the normal architecture of the microenvironment, alters alters either the constant region protein sequence or the
epitope on the constant region cell growth properties and often leads to the activation glycosylation of the mAb, gives developers control over
of an antibody. When FcRs on of apoptotic signalling pathways independently of addi- a number of characteristics, including the ability to fix
immune effector cells come tional therapy. In vitro signalling assays are usually carried complement 30,31 (FIG. 3).
into contact with an
out over a timescale of minutes to hours, whereas clinical Many studies exploring CMC use serum as a source
antibody-coated target cell,
this can result in immune responses to mAbs are measured over weeks and months. of complement, which highlights the importance of
effector cell activation (FcRs Despite these limitations, the detection of signalling- CMC in the circulation. Indeed, CMC seems to contri­
with immunoreceptor induced apoptosis has become a common approach to the bute most to the therapeutic effect of mAb in haemato-
tyrosine-based activation in vitro analysis of potential signalling effects of mAbs18. logical malignancies, in which target cells are exposed to
motifs (ITAMs)) or inhibition
(FcRs with immunoreceptor
Even when a mAb is known to alter signalling proper- complement in the circulation32. However, complement
tyrosine-based inhibitory ties and is effective clinically, it is difficult to use in vitro binding to target cells does not necessarily result in the
motifs (ITIMs)). assays to determine whether the therapeutic effect of lysis of the target cell. When a CD20‑specific mAb binds

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REVIEWS

a Immune-mediated effects of tumour-specific IgG FcRs that then signal through ITAMs and induce effec-
tor cell activation13. Other cells express immuno­receptor
tyrosine-based inhibitory motifs (ITIMs), which can
NK cell Complement inhibit ADCC39. mAb-coated target cells can induce
ADCC
ITAM the production and release of cytokines by immune
CMC
effector cells that express FcRs40. These cytokines can
Tumour cell then activate other immune effector cells in the tumour
Tumour Lysis
antigen microenvironment 41. Thus, immune cell activation via
MAC
FcRs can contribute to direct ADCC, as well as to the
production of cytokines that contribute to the control
FcR of tumour growth in other ways.
Opsonization
Granulocyte Monocyte Interacting mechanisms of mAb-induced cytotoxicity.
There is growing evidence that there are extensive
b Direct effects of tumour-specific IgG
interactions — both synergistic and antagonistic
Inhibit receptor — between various mechanisms of action that can
dimerization
affect the antitumour effects of a single type of mAb.
Block ligand A mAb may be developed with one mechanism in
Induce apoptotic mind, but other mechanisms may also be important 42.
signalling Complement fixation has very complex effects43. On
the one hand, the CD20‑specific mAbs rituximab and
ofatumumab, and the CD52‑specific mAb alemtu-
Figure 2 | Mechanisms of action of monoclonal antibodies that target cancer
cells.  Monoclonal antibodies (mAbs) that bind directly to cancer cells can mediate zumab, rapidly kill target cells in vitro via CMC; on the
Nature Reviews
their antitumour effects through various mechanisms. These mechanisms | Cancer
are routinely other hand, complement fixation can block the inter-
identified in vitro but their relative contributions to clinical responses to mAb therapy action between mAbs and activating FcRs on NK cells,
is difficult to determine. a | mAbs can mediate antibody-dependent cellular and reduce ADCC as a consequence44. Thus, in some
cytotoxicity (ADCC) by immune effector cells that express immunoreceptor circumstances, complement fixation may induce CMC
tyrosine-based activation motifs (ITAMs), such as natural killer (NK) cells, monocytes, and enhance the response to mAbs, but in others it
macrophages and granulocytes. Fixation of complement can trigger the opsonization could inhibit ADCC, thereby blocking the response to
of the target cell and thereby enhance phagocytosis and lysis by monocytes and mAbs. Signalling that results from a mAb binding to a
granulocytes. Complement-mediated cytotoxicity (CMC) can directly result in target receptor on a cancer cell can alter the sensitivity of that
cell death through the development of a membrane attack complex (MAC). b | mAbs
target cell to ADCC11. mAb-induced cancer cell lysis
can also have direct effects on target cells by blocking the binding of an activating
ligand that is responsible for the survival of the cancer cell, inhibiting the dimerization can, at least in preclinical models, lead to enhanced
of a receptor, thereby blocking an activation signal or inducing an apoptotic signal by uptake of the target antigen and subsequent cross-
crosslinking a receptor. Such crosslinking of the receptor can be enhanced when a presentation by cells — such as dendritic cells —
mAb is bound to Fc receptor-expressing cells. IgG, immunoglobulin G. thereby leading to an enhanced T cell response 45.
Malignant B cells that express FcRs without activat-
ing motifs (that is, FcRs without ITAMs) may actu-
to a circulating chronic lymphocytic leukaemia (CLL) ally positively contribute to the antitumour effect of
cell and fixes complement, the entire antigen–mAb– a mAb. These FcRs can allow for autocrine crosslink-
complement complex may be sheared off the surface of ing of B cell-specific mAbs that bind both to target
the leukaemic cell as it circulates through the liver and antigens, through the variable region, and to the
spleen in a process known as ‘shaving’, or trogocytosis, FcRs expressed by the same cell or by neighbouring
which is mediated by FcR-expressing cells including malignant cells46.
Complement fixation
monocytes33. This results in circulating malignant cells An antigen that can be effectively targeted by a
Initiation of the complement
cascade by an antibody bound that temporarily lack the target antigen and therefore cancer-­specific mAb needs to be found on the surface
to an antigen. It can lead to evade mAbs specific for that antigen. The relative con- of the cancer cell in concentrations high enough to
complement-mediated centration of various complement components in the trigger one or more of the effector mechanisms out-
cytotoxicity of the target cell, extravascular fluid is not well understood and may be lined above once the mAb has bound to the surface.
as well as other complex
effects mediated by the
inadequate to mediate CMC34. It is generally accepted However, the antigen should not be expressed to a
activation of various that CMC has a limited role in the efficacy of mAbs that similar degree by a vital population of benign cells47 or
complement components. recognize target antigens on malignancies outside the found in high concentrations as a soluble antigen in the
vascular compartment: that is, solid tumours. circulation48. Efforts to identify mAbs that recognize
mAb isotype
novel tumour-associated antigens continue, although
The subtype of a monoclonal
antibody (mAb) based on the ADCC. mAbs can induce ADCC by binding to FcRs, it could be argued that most of the highly promising
amino acid sequence of the which are expressed by a variety of immune effector antigens that can be effectively targeted by unmodified
constant region. Isotypes cells, including natural killer (NK) cells, granulocytes, IgG have already been identified. Ongoing efforts to
(IgG1, IgG2, IgG3 and IgG4) monocytes and macrophages 35–38 (FIG.  2) . Some of enhance the efficacy of IgGs that directly target can-
vary in their ability to mediate
antibody-dependent cellular
these effector cells express FcRs with immunoreceptor cer cells involve the identification of mAbs with unique
cytotoxicity and complement- tyrosine-based activation motifs (ITAMs). To trigger signalling properties — such as HER2 (also known
mediated cytotoxicity. ADCC, mAbs bound to the target cell interact with as ERBB2) mAbs that vary in their ability to interfere

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 REVIEWS

Glycosylation with receptor heterodimerization49 — or the modifi- T cell receptor-like mAbs. Many tumour-associated
The post-translational cation of the IgG constant region to enhance its abil- antigens are not expressed on the surface of the cancer
attachment of a carbohydrate ity to interact with the human immune system (FIG. 3). cell. This has led to the generation of mAbs that bind to
moiety to a protein. The Fc Approaches to enhancing this interaction include peptides expressed by human leukocyte antigen (HLA)
region of immunoglobulin G
includes carbohydrate
changing the amino acid sequence50 or the glycosylation molecules55. These mAbs recognize peptides, such as
moieties. Altering the pattern of the IgG in a way that enhances interaction PR1 and WT1, that are derived from intracellular onco-
enzymes responsible for with FcRs on effector cells51. mAbs with such modifi- proteins56,57. Such mAbs are HLA-restricted (that is, they
glycosylation in a cell line that cations can have enhanced in vitro efficacy and seem only bind to antigen expressed by cells from patients
produces a monoclonal
to be safe in clinical trials52. One glycomodified mAb, with a given HLA genotype) and are in early develop-
antibody (mAb) can alter the
glycosylation of the mAb,
obinutuzumab, was recently approved by the US Food ment; however, they represent a novel approach to
thereby altering its ability to and Drug Administration (FDA) for the treatment of broadening the potential targets for mAb-based therapy.
activate immune effector CLL53. Obinutuzumab is unique in other ways, such
cells. as the manner by which it crosslinks its target antigen Altering the host response
CD20, and the actual therapeutic contribution of chang- Inhibiting angiogenesis. In the early 1970s, Folkman
ing IgG glycosylation remains to be determined. There and others hypothesized that tumours require new
have also been studies exploring mAbs that are based blood vessels to grow, and that the growth of such ves-
on other antibody isotypes, such as IgA54. These agents sels is stimulated by pro-angiogenic factors produced
have some intriguing properties but are not currently in by the cancer 58. A number of substances that stimulate
wide clinical development. intratumoural blood vessel growth were subsequently
identified, including vascular endothelial growth factor
(VEGF). mAbs (including bevacizumab) were produced
Heavy chain to inhibit angiogenesis by interfering with the ability
Light chain of VEGF to stimulate new intratumoural blood vessel
Variable region growth (FIG. 1). This, in turn, has an antitumour effect
Carbohydrate Constant region
by depriving a growing tumour of nutrients and oxy-
gen provided by the new blood vessels59. As such angio-
g Crosslink regions genesis inhibitors do not directly target the cancer and
a Glycomodified mAb from two mAbs
are generally assumed to inhibit cancer growth rather
than induce cancer cell death, they are most often used
in combination with cytotoxic agents60. An important
advantage of this approach is that it is not dependent
b Alter amino f Insert DNA for mAb on expression of a specific target antigen by the tumour
acids in variable region fused
constant to signalling peptide cell, but is instead based on the role of VEGF in neo­
region into T cell to induce angiogenesis, which is vital for the growth of tumours
expression of CAR in general. Bevacizumab has been shown to be effective,
c Use different e Link drug
human mAb d Link isotope to mAb with at least in some clinical trials and scenarios, in various
isotype to mAb with cleavable linker cancers, including colorectal, lung, breast, renal, brain
(e.g. IgG4) stable linker
and ovarian cancer61.
Figure 3 | Modifying monoclonal antibody structure.  Monoclonal antibody (mAb) The unique mechanism of action of angiogenesis
structure can be modified on the basis of the desired mechanism of action. inhibition creates a particular challenge in evaluating
Nature Reviews | Cancer
Immunoglobulin G1 (IgG1) is the most effective naturally occurring human IgG isotype efficacy 62. A standard approach to assess the response
at mediating antibody-dependent cellular cytotoxicity (ADCC). Glycomodified to most anticancer agents involves radiological determi-
afucosylated mAbs (part a) (such as obinutuzumab) demonstrate enhanced binding to nation of tumour shrinkage. It has been proposed that
IgG Fc receptors (FcγRs) and enhanced ADCC. Afucosylated mAbs are produced using angiogenesis inhibitors may temporarily shrink the size
cell lines that lack the enzymes that are responsible for fucosylation. Modifying the of a cancer by affecting vascularity, without having a
amino acid sequence of mAb Fc (part b), as was done to produce ocaratuzumab50, can
significant impact on the growth of the malignant cells.
also result in enhanced binding to FcγRs and enhanced ADCC. For mechanisms of action
in which ADCC is not desirable, IgG4 is a more appropriate isotype, as IgG4 mAbs do not Thus, although it remains controversial, some experts
mediate ADCC to the same degree as IgG1 (part c). Nivolumab, an IgG4 mAb that blocks claim that the evaluation of efficacy of angiogenesis
programmed cell death protein 1 (PD1) on T cells, is one such example. Producing radio- inhibitors should be based on overall survival rather
immunoconjugates involves linking the radioisotope to the mAb. A stable linker is most than on radiographical determination of tumour shrink-
desirable (part d) to limit the leakage of the free radioactive isotope. Conversely, optimal age63. Trials with survival as an end point are more chal-
antibody–drug conjugates (ADCs) use a cleavable linker (part e). To avoid nonspecific lenging, and robust debate continues over the clinical
toxicity, it is desirable for drugs used in ADCs to be cytotoxic once inside the target cell value of angiogenesis inhibition in various cancer types.
but non-toxic when bound to the mAb in the circulation. Linkers that are pH-sensitive or
enzymatically cleaved are now a standard component of ADCs. Chimeric antigen T cell checkpoint blockade. Years of basic immunology
receptor (CAR) T cells get their specificity from mAb variable regions but are a form of
research exploring the exquisite control of T cell
gene, not protein, therapy. They are produced by inserting DNA coding for the mAb
variable region fused to DNA coding for signalling peptides into T cells (part f). Bispecific immunity have demonstrated that co‑regulatory
antibodies require the removal of a functional constant region so that they do not ligand–receptor pairs have a central role in the balance
nonspecifically crosslink activating receptors and activate T cells (part g). The lack of a between activation and inhibition of antigen-specific
constant region on such constructs results in a short half-life, thus requiring continuous (and tumour-specific) T cells64. Such precise control is
infusion to achieve the desired exposure. central to the ability of the immune system to respond

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Opsonization
The process by which a target
is marked for phagocytosis or
for destruction by phagocytes.
This term is often used to
describe phagocytosis of
microbial pathogens. In the
case of monoclonal antibody
therapy of cancer, the target is
a cancer cell.
Checkpoint blockade
Inhibitory pathways limit T cell
activation in order to maintain
self-tolerance and prevent
autoimmunity. Checkpoint
blockade involves blocking
these inhibitory pathways, and
thereby allowing for to a more
robust T cell response.
Breakthrough therapy
designation
US Food and Drug
Administration (FDA)
designation if a new drug is
intended to treat a serious or
life-threatening disease and
preliminary clinical evidence
suggests that it provides a
substantial improvement over
existing therapies.
366 | JUNE 2015 | VOLUME 15 www.nature.com/reviews/cancer
to infection and to avoid the autoimmunity that could
result from an uncontrolled T cell response. Over the
past decade, mAbs that interfere with the inhibitory
signals responsible for limiting T cell activation have
been developed. These mAbs, called checkpoint blockade
mAbs, can maintain the T cell activation phenotype and
enhance T cell-mediated lysis (FIG. 1). In the case of can-
cer, such mAbs can induce a more robust and sustained
antitumour T cell response64,65.
Cytotoxic T lymphocyte-associated antigen 4
(CTLA4; also known as CD152) is a receptor expressed
by activated T cells that results in the downregula-
tion of the T cell response. Physiologically, CTLA4
and other negative regulators of T cell activation have
a role in blunting the T cell response. Such blunting
of the T cell response is important for avoiding auto­
immunity but is undesirable when it comes to the gen-
eration and maintenance of an effective antitumour
T cell response66. Blocking this negative signal would
be expected to enhance and maintain such a response,
and this has proven to be the case. Ipilimumab, a mAb
that blocks CTLA4, has been approved by the FDA and
has had a major impact on the treatment of patients
with melanoma67. Clinical responses to ipilimumab
can be profound and long-lasting. Ongoing studies in
various cancers are exploring regimens that incorporate
ipilimumab, both alone and in combination with other
agents, particularly when there is reason to think that
such therapy could be inducing a T cell response, such
as in the case of vaccines or agents that induce antigen
release68. The toxicity of checkpoint blockade mAbs
is also unique and, as would be expected, includes
autoimmunity 69.
Several mAbs that interfere with a different T cell
regulatory pathway, the programmed cell death pro-
tein 1 (PD1) receptor–PD1 ligand 1 (PDL1) axis, are
also generating considerable excitement. These include
mAbs that bind to the ligand and others that bind to the
receptor 70,71. Both approaches have been very promis-
ing in early clinical trials, and we are sure to learn much
more about the relative value of these approaches in the
years ahead. Indeed, the FDA has recently approved two
PD1‑specific mAbs (pembrolizumab and nivolumab) for
the treatment of melanoma. A mAb against PDL1 has
been granted breakthrough therapy designation by the FDA
on the basis of its promise in early phase clinical trials70.
Whereas CTLA4 regulates de novo immune responses,
the PD1–PDL1 axis has a greater influence on ongoing
T cell immune responses. Thus the concept of check-
point blockade is similar, but the efficacy and toxicity
of mAbs that target these two pathways are likely to be
different. For instance, a PD1‑specific mAb has been
found to be effective in patients with melanoma refrac-
tory to CTLA4‑specific therapy 72. Combination trials
exploring the inhibition of CTLA4 and PD1 together are
promising 73, although blocking both pathways does raise
concerns about enhanced autoimmunity.
The PD1–PDL1 axis seems to be particularly impor-
tant in classic Hodgkin lymphoma in which Reed–
Sternberg cells have recently been shown to express
PDL1 and PDL2, which allows them to turn off T cells.
© 2015 Macmillan Publishers Limited. All rights reserved
This helps to explain the hallmark of Hodgkin lym-
phoma that has intrigued haematologists for decades:
how malignant Reed–Sternberg cells are able to survive
despite being surrounded by benign immune cell infil-
trates. An early phase clinical trial of single-agent PD1
blockade in relapsed or refractory Hodgkin lymphoma
demonstrated very positive results74.
Delivering cytotoxic moieties
Military analogies have been a part of immunology since
the days of Paul Ehrlich and his description of anti­bodies
as “magic bullets” (REF. 1). In more recent years, the
phrase ‘smart bombs’ has been used to describe mAbs
that deliver cytotoxic moieties to cancer cells. The types
of payload that have been successfully delivered by mAb-
based smart bombs are quite varied and include radio­
active molecules, cytotoxic small molecules and cellular
components of the immune system (FIG. 3).
Radioimmunoconjugates. Treatment of thyroid can-
cer with 131I was the first highly effective targeted
cancer treatment and was successful because of the
specificity of elemental iodine for the thyroid. 131I is a
β-particle emitter that also emits a modest amount
of γ-radiation and so can be used in both diagnosis
(imaging) and treatment. This success led investiga-
tors to explore approaches to delivering radioisotopes
directly to target molecules expressed by other cancers
using various carrier molecules, including antibodies.
Attempts to use such radioimmunoconjugates to treat
cancer began with polyclonal antibodies before mAb
technology was available. Order and colleagues75 pro-
duced polyclonal antisera by immunizing various species
(including rabbits, pigs, monkeys and cows) with human
ferritin. Antisera from these animals were labelled with
131
I and administered to patients with tumours that
were known to express high levels of ferritin receptor 75.
The results, particularly the images demonstrating evi-
dence for some targeting of the agent to the tumour,
were intriguing, but the use of polyclonal antisera from
various species demonstrated little clinical efficacy and
posed many problems. These included significant con-
sistency and quality-control issues, and the development
of host immune responses against the foreign proteins
(similar to classic serum sickness)76.
mAb technology solved some of these problems and
resulted in a renewed interest in radioimmunoconju-
gates and radioimmunotherapy. However, challenges
have persisted and have limited the clinical utility of
radioimmunotherapy. Radioisotopes continually decay
and cause nonspecific radiation damage to normal tis-
sue when the agent is in the circulation or is taken up
nonspecifically by normal tissues77. Bone marrow is
particularly radiosensitive and is therefore affected by
circulating radioimmunoconjugate. The kidney and the
liver receive high doses of radiation because of their role
in clearing the radioimmunoconjugate and free radio-
isotope, and only a small fraction (typically 0.01–0.001%
per gram of tumour) of the injected radiation dose ends
up in the tumour, even for the most specific agents77.
Optimizing the very narrow therapeutic window of

radioimmunotherapy requires careful choosing of the
mAb, the radioisotope and the chemistry that links
the two. Experience over the past 25 years has taught
us that radioimmunoconjugates directed towards solid
tumours can be useful as diagnostic agents, but that they
have limited therapeutic impact owing to radioresistance
of the tumours78. A radioimmunoconjugate consisting of
a radioiodinated mAb that targets necrotic tumour cells
has been approved for clinical use in China79. Ongoing
studies are evaluating radioimmunotherapy combined
with chemotherapy.
Lymphoma is uniquely suited for radioimmuno­
therapy because of the availability of highly specific
target antigens and its relative radiosensitivity. Two
radio­immunoconjugates, ibritumomab tiuxetan80 and
tositumomab81, have been approved by the FDA as
therapeutic agents for the treatment of lymphoma. They
are both based on CD20‑specific mAbs but use differ-
ent isotopes (90Y for ibritumomab tiuxetan and 131I for
tositumomab). The logistics of administration, need
for dosimetry and pharmacokinetics of these two agents
are different, but their clinical efficacies and toxic effects are
similar. Despite the clear clinical efficacy and limited
toxicity of lymphoma radioimmunotherapy, clinical
use of this modality has been surprisingly limited. This
seems to be due to the complex logistics of providing
radioimmunotherapy (it requires experienced nuclear-
medicine physicians), and the emergence of a number
of other new therapies for lymphoma that are less com-
plex to deliver. Recent and ongoing studies are explor-
ing additional clinical questions related to lymphoma
radioimmunotherapy, including the timing of therapy
(at diagnosis versus at relapse), which lymphoma sub-
types should be treated (given that it is most effec-
tive for follicular lymphoma) and how therapy should
be combined with other treatments, including bone
marrow transplantation82 and novel agents83.
Ongoing studies are also exploring radioimmuno-
conjugates that contain novel radioisotopes, including
α-emitters84. The advantage of α-emitters is that the
ionizing radiation is delivered very locally (within just
a few cell diameters), which limits radiation exposure
to cells that express the target antigen and spares benign
neighbouring cells. This highly specific delivery of
radiation allows the treatment of malignancies such as
leukaemia that are not geographically isolated but that
are highly radiosensitive. The radiochemistry of work-
ing with these isotopes is challenging because of their
very short half-life and because the production of iso-
topes with promising properties, such as 211At, requires
a cyclotron85. However, not all α-particle-emitting iso-
topes are short-lived. Preliminary preclinical and clinical
results using α-particles are intriguing 86 and studies are
ongoing.
Antibody–drug conjugates. In the 1970s and 1980s, a
number of investigators began developing and testing
immunotoxins composed of mAbs linked to a variety of
very potent protein toxins, such as ricin, pseudomonas
exotoxin and diphtheria toxin. For an immunotoxin
to be effective, it needs to bind to a surface antigen on
NATURE REVIEWS | CANCER VOLUME 15 | JUNE 2015 | 367
© 2015 Macmillan Publishers Limited. All rights reserved
REVIEWS
a cancer cell, enter the cell by endocytosis and traffic
within the cell to the site where it can kill the cell. The
toxins need to be modified by removing the moieties
responsible for nonspecific binding to normal cells, so
that cellular targeting and uptake is controlled by mAb
specificity. Initial studies of immunotoxins were carried
out using protein conjugation techniques, such as the
use of heterobifunctional reagents to link the mAb to
the toxin. Subsequent efforts involved the development
of recombinant immunotoxins. These agents were found
to be highly potent, but the immunogenicity and non-
specific toxicity of protein toxins posed major problems
and limited further development 87.
Many of the important lessons learned from the study
of immunotoxins were applied to the development and
testing of antibody–drug conjugates (ADCs), a field that
is showing great promise88. ADCs combine the cytotoxic
potential of drugs with the specifıcity of mAbs and theor­
etically overcome the limitations of both nonspecifıc
cytotoxic drugs and specifıc but often ineffective mAbs.
When choosing the target antigen and the mAb for ideal
ADCs, desirable characteristics are different from those
used for unlabelled mAbs89. Tumour antigens for ADCs
can be expressed at lower concentrations compared with
unlabelled mAbs because the delivery of a highly toxic
drug by a small number of ADC molecules can result
in the death of the target cancer cell. Depending on the
drug used, only a few molecules delivered intracellularly
may be capable of killing a target cell. Internalization
properties are also important. For unlabelled mAbs, it is
helpful for the mAb–antigen complex to remain on the
surface of the cell so that it can be recognized by the host
immune system. For ADCs, internalization needs to take
place so that the drug can be released inside the cancer
cell and mediate its toxic effect.
ADCs use small molecules instead of protein tox-
ins and thereby reduce immunogenicity. Drugs used
as components of ADCs include potent drugs such as
calicheamicin, which binds to the minor groove in DNA
and causes strand scission90; monomethyl auristatin E,
which blocks polymerization of tubulin91; maitansine,
which inhibits the assembly of microtubules92; and,
most recently, pyrrolobenzodiazepines, which crosslink
DNA93. These are extremely potent drugs; indeed, some
were evaluated as stand-alone chemotherapy agents and
rejected because of their toxicity at very low doses90,94.
The linker that connects the mAb to the drug is vital.
It needs to attach the drug to the mAb in a manner that
does not alter the specificity of the mAb, to render the
drug non-toxic while bound to the mAb, to remain
stable in the circulation and to release the drug in the
appropriate intracellular compartment when the ADC
is internalized, so that the drug can kill the target cell95.
Linker motifs include disulfides, hydrazones, peptides
and thioethers. Some are cleavable, whereas others are
not, based on the properties of the mAb and the desired
distribution of the cytotoxic agent within the cancer cell.
As with radioimmunoconjugates, ADCs seem to be
particularly effective in lymphoma, but they are also
showing promise in solid tumours. Brentuximab vedo-
tin (for lymphoma)96,97 and ado-trastuzumab emtansine
REVIEWS
Bispecific antibody
(Also known as bifunctional
antibody). An engineered
monoclonal antibody (mAb)
that is composed of fragments
of two different mAbs that bind
to two different antigens. In
cancer therapy, it typically
binds to an activating antigen
on an immune effector cell with
one arm and to a tumour-
associated antigen on a cancer
cell with the other arm, thereby
retargeting the immune
effector cell towards the target
cancer cell.
Chimeric antigen receptor
(CAR). An engineered receptor
that grafts an alternative
specificity onto an immune
effector cell, most often a
T cell. Most of the current
constructs for engineered
receptors include a single-chain
monoclonal antibody variable
region that recognizes the
target cell and is linked to
activating transmembrane
domains that activate the T cell
when it comes into contact
with a target cell.
368 | JUNE 2015 | VOLUME 15 www.nature.com/reviews/cancer
(for breast cancer)98 were the first ADCs to be approved
by the FDA. The field is rapidly expanding with more
than 30 ADCs currently in development and in clinical
trials99, and many have shown encouraging preliminary
results100,101. The ADC field is currently in its infancy,
and major advances based on the design of new ADCs,
and a better understanding of how to use them, are sure
to come.
An approach that combines targeting of the cancer
cell directly with altering the tumour microenviron-
ment is the use of immunocytokines that can mediate
their antitumour effect through both the direct effect
of the mAb and the ability to deliver a cytokine, such as
interleukin (IL‑2) or granulocyte–macrophage colony-
stimulating factor (GM‑CSF), to the tumour. Most notable
in this area of research have been immunocytokines based
on ganglioside-­specific GD2, with clinical development
mostly centred on the treatment of neuroblastoma102,103.
These agents have shown remarkable efficacy in this
rare childhood malignancy. A modified version of this
immuno­cytokine, designed to mediate reduced comple-
ment fixation that is thought to decrease its neurotoxicity,
has also shown promise in an early phase clinical trial104.
Retargeting T cells
Tumour immunology has taught us that T cell immu-
nity is key to the immune rejection of many cancers.
The initial step of cell-mediated cytolysis involves the
formation of conjugates between T cells and target
cells. The appropriate engagement of receptors and
co‑­receptors on T cells is followed by the triggering of
cytotoxic responses that result in the death of the cancer
cell. Two broad approaches to combining the specificity
of mAbs with the power of T cell immune responses
have been explored with the hope of enhancing immune
rejection of cancer (FIG. 3). Both of these approaches are
designed to retarget large numbers of T cells towards the
tumour by bypassing the need for the T cell receptor to
recognize target antigens as processed and expressed by
target cell major histocompatibility complexes (MHCs).
Bispecific antibodies. The first approach involves the
creation of bispecific antibody-like molecules that have one
arm that binds to the target cell and one arm that binds
to activating receptors on cytotoxic cells, such as T cells
or NK cells. Most bispecific antibodies involve constructs
that bind to an antigen on a cancer cell with one arm
and to T cell surface glycoprotein CD3 ε-chain (CD3) on
T cells with the other arm. This, as with many of the strat-
egies based on using mAbs to treat cancer, is not a new
concept, but it has benefited from a steady improvement
in technology and lessons learned from earlier preclinical
and clinical studies105. For example, early studies demon-
strated that bispecific antibodies with intact Fc activate
T cells nonspecifically and result in unacceptable toxicity.
Smaller bispecific molecules lacking Fc have short half-
lives and need to be given by continuous infusion, but
these molecules result in less nonspecific immune activa-
tion than do intact bispecific mAbs. Recent positive clini-
cal trials with the bispecific mAb blinatumomab led to
its approval by the FDA. Additional bispecific antibodies
© 2015 Macmillan Publishers Limited. All rights reserved
are under development, including some with different
structures that have longer half-lives and that may not
require continuous infusion106,107.
Chimeric antigen receptor T cells. Another example
of how persistence has paid off in mAb-based cancer
immuno­therapy is demonstrated by the decades of
research that preceded the current high level of excitement
surrounding chimeric antigen receptor (CAR) T cells108.
CAR T cells are genetically modified to respond to target
cells expressing a given antigen, and consist of a mAb vari-
able region linked to a T cell-activating motif. A number
of investigators worked for many years to refine various
antigen-specific constructs and approaches to transferring
those constructs into primary T cells109. Recent clinical
trials based on some of the newer constructs show that
CAR T cells can lead to robust therapeutic responses110,111.
They also result in significant, but increasingly manage-
able, toxicity owing to cytokine storm that occurs as a
consequence of massive activation and proliferation of
CAR T cells; activation and proliferation are induced
when CAR T cells come into contact with a large tumour
burden. CAR T cells are unique among mAb-based treat-
ment strategies in that the antitumour effect can expand
over time without additional therapy as the CAR T cells
divide and the tumour specificity of the CAR T cells is
passed on to daughter cells.
Indeed, long-lived CAR T memory cells have been
observed in a number of patients 112. This memory
response represents both an advantage and a challenge.
On the one hand, it probably contributes to the very grati-
fying long-term clinical responses that are seen in some
patients treated with CAR T cells. On the other hand, such
long-term immune memory can result in long-term, per-
haps even permanent, depletion of any cells that express
the target antigen. In the case of B cell malignancies, long-
term loss of benign B cells that express CD19 may be an
acceptable toxicity. However, most tumour antigens are
not as specific as CD19, and long-term toxicity to vital
normal tissues can result if such tissues express even low
levels of the target antigen recognized by CAR T cells. This
may limit the number and types of cancers that can be
treated with this approach.
Bispecific antibodies and CAR T cells each have advan-
tages and disadvantages. Bispecific antibodies are ‘off-the-
shelf ’ reagents and can be stopped if autoimmune toxic
effects are observed, but they are challenging to admin-
ister. CAR T cells need to be produced individually for
each patient, but they expand in response to tumour and
can result in long-term antitumour immune responses.
Both approaches can result in the rapid activation of large
numbers of T cells interacting with large numbers of cells
expressing the target antigen113. The resulting massive
release of cytokines can result in a clinical cytokine storm
with potentially devastating physiological effects, includ-
ing cardiovascular collapse114. Cytokine storms can be
limited by stopping infusion for bispecific antibodies or by
using mAbs against certain cytokines such as IL-6‑specific
mAbs for CAR T cells. Accumulating experience with
CAR T cells is leading to the development of protocols
to prevent or treat this challenging complication114.
 REVIEWS

Additional studies are needed to determine the relative
efficacy, toxicity and value of these two exciting new
strategies for using mAbs to treat cancer.
Conclusion
Cancer mAb therapy has come a long way since the days
when unmodified murine mAbs were first explored as
anticancer agents. A wide variety of mAb-based strate-
gies have proven to be useful in treating patients with
cancer, including: the use of unlabelled IgG that binds
directly to cancer cells, mAbs that alter the active host
response to the cancer, immunoconjugates that deliver
cytotoxic moieties to the cancer and constructs that use
the specificity of mAbs to retarget cellular immunity
towards the cancer cell. The demonstration of clinical
efficacy for each of these approaches was made possible
by the dedication and persistence of investigators who
would not give up on a good idea.
Indeed, investment in mAb-based therapy of can-
cer from both the public and the private sectors and
the speed of progress have never been greater. Recent
advances and positive clinical results in checkpoint
blockade, ADCs and retargeting T  cells via CAR
T cells and bispecific antibodies are particularly excit-
ing. We are in a period of unprecedented progress
in mAb-based therapy with the rapid development
of new therapeutic agents and constructs, enhanced
understanding of their biological effects, and grow-
ing clinical experience based on both clinical trials
and community use of FDA-approved drugs. With
dedicated attention to basic, translational and clinical
research geared towards additional progress, we will
certainly be able to build even better anticancer mAbs
in the years ahead. Equally important will be studies
evaluating how best to use these agents, alone and in
combination, to better serve our patients.

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Acknowledgements
The author would like to acknowledge support from the
US National Institutes of Health grants P30 CA86862 and
P50 CA97274.
Competing interests statement
The author declares no competing interests.