Therapeutic antibodies for autoimmunity and inflammation

Antibodies have rapidly become a clinically important
drug class: more than 25 antibodies are approved for
humantherapyandmorethan240antibodiesarecurrently
in clinical development worldwide for a wide range of dis-
eases, including autoimmunity and inflammation (the
focus of this Review), cancer (the focus of the Review by
Weiner and colleagues in this issue,REF. 165), organ trans-
plantation, cardiovascular disease, infectious diseases and
ophthalmological diseases1
. The clinical success of anti-
bodies has led to a major commercial impact, with rapidly
growing annual sales that exceeded US$27 billion in 2007,
including 8 of the 20 top-selling biotechnology drugs2
.
This modern era of therapeutic antibodies originated
with the invention of hybridoma technology to gener-
ate mouse monoclonal antibodies in 1975 (REF. 3). Major
limitations of mouse antibodies as therapeutic agents
— immunogenicity, lack of effector functions and short
serum half-life — were subsequently identified and largely
overcome by the advent of antibody chimerization and,
later, humanization4
technologies in the mid-1980s.
Many antibody drugs, including those for autoimmunity
and inflammation (TABLE 1), are chimeric or humanized
versions of rodent antibodies.
Several approved antibody drugs and an increasing
proportion of antibodies entering clinical trials are of
human origin5
. These human antibodies are typically
derived from large phage display libraries expressing
human antibody fragments or transgenic mice engi-
neered with human immunoglobulin genes6
. Human
antibodies from transgenic mice are commonly devel-
oped as therapeutic agents without prior optimization6
.
By contrast, phage-derived antibodies may require
improvements in binding affinity for antigen or biological
potency that are routinely obtained by additional selec-
tion for desired phenotypes from phage display librar-
ies7
. Evolving technologies, including yeast, ribosome,
mRNA, mammalian and Escherichia coli display librar-
ies, as well as the direct cloning of human antibodies
from human blood or bone marrow-derived cells, might
also contribute to future therapeutic antibodies7
. Beyond
improved generation and optimization technologies, the
development of antibody drugs has benefited from better
choices and matching of target, antibody and patients8
, as
well as advances in the industrialization of recombinant
antibody production and purification processes9
.
Current therapeutic antibodies provide much expe-
rience to guide future antibody drug development —
strengths on which to build, limitations to overcome
and new opportunities to seize (BOX 1). The quest for bet-
ter therapeutic antibodies has gained great momentum
in recent years, motivated by a convergence of clinical,
scientific, technological and commercial factors10
. First,
there is a strong desire to improve on the clinical benefits
for patients that were achieved by first-generation thera-
peutic antibodies. Second, there is a growing understand-
ing of the mechanisms of action of antibody-based drugs
and, in some cases, their limitations, including mecha-
nisms of resistance. Third, technological advances are
available to optimize and overcome existing biophysical,
functional and immunogenic limitations of antibodies,
as well as to confer antibodies with new activities10–12
.
Fourth, the major commercial success of antibodies2
has
fuelled strong competition between companies that will
probably intensify as many approved and developing
*Department of Immunology,
Genentech, Inc.,1 DNA Way,
South San Francisco,
California 94080, USA.
‡
Department of Antibody
Engineering, Genentech, Inc.,
1 DNA Way,
South San Francisco,
California 94080, USA.
e-mails: acc@gene.com;
pjc@gene.com
doi:10.1038/nri2761
Effector functions
Fc-mediated antibody
properties that are involved
in target cell destruction:
antibody-dependent
cell-mediated cytotoxicity
(ADCC), antibody-
dependent cellular
phagocytosis (ADCP) and
complement-dependent
cytotoxicity (CDC).
Half-life
The time taken for the plasma
concentration of a drug to fall to
half of its original value. Initial
half-life and terminal half-life
refer to the first (distribution)
and second (elimination)
phase for bi-exponential
pharmacokinetics, respectively.
Therapeutic antibodies for
autoimmunity and inflammation
Andrew C. Chan* and Paul J. Carter‡
Abstract | The development of therapeutic antibodies has evolved over the past decade into
a mainstay of therapeutic options for patients with autoimmune and inflammatory diseases.
Substantial advances in understanding the biology of human diseases have been made and
tremendous benefit to patients has been gained with the first generation of therapeutic
antibodies. The lessons learnt from these antibodies have provided the foundation for the
discovery and development of future therapeutic antibodies. Here we review how key
insights obtained from the development of therapeutic antibodies complemented by
newer antibody engineering technologies are delivering a second generation of
therapeutic antibodies with promise for greater clinical efficacy and safety.
REVIEWS
NATURE REviEWS | Immunology volUME 10 | MAy 2010 | 301
focus on THERAPEuTIc AnTIBoDIEs
© 20 Macmillan Publishers Limited. All rights reserved
10

Table 1 | Monoclonal antibodies and Fc fusion proteins for autoimmunity and inflammation*
generic name (trade
name; sponsoring
companies)
Format Targets Development
stages
Diseases Proposed mechanisms of
action
Natalizumab (Tysabri;
Biogen Idec/Elan)
Humanized IgG4 α4 subunit
of α4β1
and α4β7
integrins
Approved MS and Crohn’s disease Receptor binding and
antagonism; inhibits leukocyte
adhesion to their counter receptor
(or receptors)
Vedolizumab
(MLN2; Millennium
Pharmaceuticals/
Takeda)
Humanized IgG1 α4β7
integrin
Phase III UC and Crohn’s disease Receptor binding and
antagonism; inhibits leukocyte
adhesion to their counter receptor
(or receptors)
Belimumab
(Benlysta; Human
Genome Sciences/
GlaxoSmithKline)
Human
(phage-produced) IgG1
BAFF Phase III SLE Ligand binding and neutralization
Atacicept (TACI–Ig;
Merck/Serono)
TACI ECD–Fc (IgG1)
fusion protein, modified
Fc to eliminate effector
functions
BAFF and
APRIL
Phase II/III SLE Ligand binding and neutralization;
blocks activation of TACI
Alefacept (Amevive;
Astellas)
LFA3 ECD–Fc (IgG1)
fusion protein
CD2 Approved Plaque psoriasis Inhibits LFA3–CD2 interaction and
blocks lymphocyte activation
Phase III GVHD
Otelixizumab
(TRX4; Tolerx/
GlaxoSmithKline)
Chimeric light chain,
humanized heavy chain,
IgG1 aglycosyl Fc
CD3 Phase III T1D Modulates T cell function
Teplizumab (MGA031;
MacroGenics/Eli Lilly)
Humanized IgG1with
mutated Fc
CD3 Phase III T1D Modulates T cell function
Rituximab (Rituxan/
Mabthera; Genentech/
Roche/Biogen Idec)
Chimeric IgG1 CD20 Approved Non-Hodgkin’s
lymphoma, RA (in
patients with inadequate
responses to TNF
blockade) and CLL
Sensitizes cells to chemotherapy;
induces apoptosis, ADCC and
CDC
Ofatumumab
(Arzerra; Genmab/
GlaxoSmithKline)
Human
(mouse-produced) IgG1
CD20 Approved CLL CDC and ADCC
Phase III RA
Ocrelizumab (2H7;
Genentech/Roche/
Biogen Idec)
Humanized IgG1 CD20 Phase III RA and SLE ADCC and CDC
Epratuzumab (hLL2;
Immunomedics/UCB)
Humanized IgG1 CD22 Phase III SLE and non-Hodgkin’s
lymphoma
ADCC and downregulation of
B cell receptor
Alemtuzumab
(Campath/
MabCampath;
Genzyme/Bayer)
Humanized IgG1 CD52 Approved CLL ADCC
Phase III MS
Abatacept (Orencia;
Bristol-Myers Squibb)
CTLA4 ECD–Fc,
mutated IgG1 Fc
CD80 and
CD86
Approved RA and JIA Inhibits T cell activation by binding
to CD80 and CD86, thereby
blocking interaction with CD28
Phase III UC and Crohn’s disease
Phase II/III SLE
Eculizumab
(Soliris; Alexion
pharmaceuticals)
Humanized IgG2 and
IgG4
C5
complement
protein
Approved Paroxysmal nocturnal
haemoglobinuria
Binds C5, inhibiting its cleavage
to C5a and C5b and preventing
the generation of the terminal
membrane attack complex
C5b–C9
Omalizumab (Xolair;
Genentech/Roche/
Novartis)
Humanized IgG1 IgE Approved Moderate to severe
persistent allergic
asthma
Ligand binding and receptor
antagonism, reduces release of
allergic response mediators from
mast cells and basophils
Canakinumab (Ilaris;
Novartis)
Human (mice) IgG1 IL-1β Approved Cryopyrin-associated
periodic syndromes
antagonism
Phase III SystemicJIA,neonatal-
onsetmultisystem
inflammatorydisease
andacutegout
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Phage display
Technology for displaying a
protein, such as an antibody
fragment, on the surface of a
bacteriophage that contains
the gene (or genes) encoding
the displayed protein (or
proteins), thereby physically
linking the genotype and
phenotype.
Binding affinity
For two interacting molecules
this is the ratio of their
association (ka
) and
dissociation (kd
) rate constants:
Kd
= kd
÷ka
.
drugs target the same antigen and/or diseases. Together,
these major factors of target biology modulated by anti-
body properties can greatly affect the degree of clinical
efficacy achieved and the ultimate successful adoption
and use of a drug.
The main functions of igG are determined by their
interaction with four classes of naturally occurring
binding partners: antigen, Fc receptors for igG (FcγRs),
complement and the neonatal FcR (FcRn) (FIG. 1). Highly
selective antigen binding is a signature and functionally
crucial property of antibodies that is mediated by their
variable domains. Several igG functions are dependent on
interaction of the Fc region with other proteins: FcγRs for
antibody-dependent cell-mediated cytotoxicity (ADCC)
and antibody-dependent cellular phagocytosis (ADCP),
complement components for complement-dependent
cytotoxicity (CDC) and FcRn for long serum persist-
ence. Here, we first discuss the diverse mechanisms by
which therapeutic antibodies modulate the functions of
their target antigens to affect their biological behaviours
and therapeutic profile. These include binding of soluble
cytokines and growth factors, receptor blockade, cellular
depletion through ADCC, ADCP, CDC and induction of
apoptosis, receptor modulation and/or receptor signal-
ling. Thereafter, we discuss the strategies that are being
applied to create the next generation of antibody drugs.
Mechanisms of therapeutic antibody action
Cytokine and growth factor blockade. The first thera-
peutic antibody for the treatment of inflammatory
diseases was infliximab (Remicade; Centocor/Merck), in
1998, for the treatment of Crohn’s disease. infliximab,
Table 1 (cont.) | Monoclonal antibodies and Fc fusion proteins for autoimmunity and inflammation*
generic name (trade
name; sponsoring
companies)
Format Targets Development
stages
Diseases Proposed mechanisms of action
Mepolizumab (Bosatria;
GlaxoSmithKline)
Humanized IgG1 IL-5 Phase III Hyper-eosinophilic
syndrome
antagonism
Reslizumab (SCH55700;
Ception Therapeutics)
Humanized IgG4 IL-5 Phase III Eosinophilic
oesophagitis
antagonism
Tocilizumab (Actemra/
RoActemra; Chugai/
Roche)
Humanized IgG1 IL-6R Approved RA Receptor binding and ligand
blockade
Phase III JIA
Ustekinumab (Stelara;
Centocor)
Human (mice) IgG1 IL-12 and
IL-23
Approved Plaque psoriasis Ligand binding and receptor
antagonism
Phase III Psoriatic arthritis
Phase II/III Crohn’s disease
Briakinumab (ABT-874;
Abbott)
Human
(phage-produced)
IgG1
IL-12 and
IL-23
Phase III Psoriasis and plaque
psoriasis
antagonism
Etanercept (Enbrel;
Amgen/Pfizer)
TNFR2 ECD–Fc (IgG1)
fusion protein
TNF Approved RA, JIA, psoriatic
arthritis, AS and plaque
psoriasis
Neutralizes TNF activity by binding
soluble and transmembrane TNF and
inhibiting binding to TNFRs
Infliximab (Remicade;
Centocor/Merck)
Chimeric IgG1 TNF Approved Crohn’s disease, RA,
psoriatic arthritis, UC,
AS and plaque psoriasis
soluble and transmembrane TNF
and inhibiting binding to TNFRs;
induction of activated T cell and
macrophage apoptosis
Adalimumab (Humira/
Trudexa; Abbott)
Human
(phage-produced)
IgG1
TNF Approved RA, JIA, psoriatic
arthritis, Crohn’s
disease, AS and plaque
psoriasis
soluble and transmembrane TNF
and inhibiting binding to TNFRs;
lyses TNF-expressing cells by CDC;
induction of activated T cell and
macrophage apoptosis
Certolizumab pegol
(Cimzia; UCB)
Humanized Fab, PEG
conjugate
TNF Approved Crohn’s disease and RA Neutralizes TNF activity by binding
soluble and transmembrane and
Golimumab (Simponi;
Centocor)
Human
(mouse-produced)
IgG1
TNF Approved RA, psoriatic arthritis
and AS
soluble and transmembrane TNF and
ADCC, antibody-dependent cellular cytotoxicity; APRIL, a proliferation-inducing ligand; AS, ankylosing spondylitis; BAFF, B cell activating factor; CDC, complement-
dependent cytotoxicity; CLL, chronic lymphocytic leukaemia; CTLA4, cytotoxic T lymphocyte antigen 4; ECD, extracellular domain; GVHD, graft-versus-host
disease; IL, interleukin; JIA, juvenile idiopathic arthritis; LFA3, lymphocyte function-associated antigen 3; MS, multiple sclerosis; PEG, polyethylene glycol;
R, receptor; RA, rheumatoid arthritis; TACI, transmembrane activator and calcium-modulating cyclophilin ligand interactor; SLE, systemic lupus erythematosus;
T1D, type 1 diabetes; TNF, tumour necrosis factor; UC, ulcerative colitis. *Data sources include drug prescribing information, www.ClinicalTrials.gov, REF.1, REF. 163
and company web sites. Antibodies and Fc fusion proteins that have reached at least Phase II or III clinical trials are included, many are in less advanced stages for
additional indications. The αL integrin-specific antibody efalizumab (Raptiva/Xanelim; Genentech/Roche/Merck–Serono) was voluntarily withdrawn from the
market by its manufacturers in April 2009 because of its association with an increased risk of progressive multifocal leukoencephalopathy, a rare and usually fatal
disease of the central nervous system.
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Neonatal FcR
(FcRn). An Fc receptor that is
structually related to MHC
class I molecules and protects
IgG from degradation, resulting
in long serum half-life.
Additionally, FcRn mediates
IgG transfer from a mother to
her fetus, thereby providing
passive immunity.
Immunoadhesin
A fusion protein that combines
the functional domain of
a binding protein, such as a
receptor or ligand, with an
immunoglobulin Fc domain.
Such Fc fusion proteins can
endow binding proteins with
antibody-like properties
including long serum half-life
and effector functions.
a chimeric antibody with mouse variable domains
and human constant domains, binds both soluble
and membrane-associated tumour necrosis factor
(TNF)13
. over the past 12 years, four additional TNF
antagonists have been approved by the US Food and
Drug Administration (FDA) for human use, each with
distinguishing features14
. These include etanercept
(Enbrel; Amgen/Pfizer), a TNF receptor ii (TNFRii)
immunoadhesin fused to the Fc domains of human igG1
that neutralizes both TNF and lymphotoxin-α (also
known as TNFβ); adalimumab (Humira/Trudexa;
Abbott), a fully human igG1κ antibody derived from
phage display technology that inhibits TNF; certolizu-
mab pegol (Cimzia; UCB), a humanized Fabʹ frag-
ment, chemically conjugated to polyethylene glycol
(PEGylated), that neutralizes both membrane-associated
and soluble human TNF; and golimumab (Simponi;
Centocor), a fully human igG1κ TNF-specific antibody
created using genetically engineered mice (FIG. 2). The
TNF antagonists are presently the most successful class
of biological drug for inflammatory diseases, with total
worldwide sales of $16.4 billion in 2008, and they are
approved not only for the treatment of Crohn’s disease
but also for the treatment of rheumatoid arthritis, juve-
nile rheumatoid arthritis, psoriatic arthritis, ulcerative
colitis, ankylosing spondylitis and plaque psoriasis (see
http://www.pipelinereview.com). These five TNF antag-
onists show the evolution of antibody technologies over
the past two decades, beginning with the initial chal-
lenges of producing industrial quantities of the chimeric
antibody infliximab and the immunoadhesin etanercept
followed by the introduction of humanization proto-
cols, phage display technologies, use of genetically
modified mice expressing human immunoglobulins
and application of PEGylation to increase the half-life
of antibody fragments.
Box 1 | Strengths, limitations and future opportunities of therapeutic antibodies
Strengths
• The availability of several well-established and broadly applicable methods for antibody generation and optimization.
• A growing repertoire of technologies to redesign antibodies with modified or new properties to enhance their
clinical potential; for example, pharmacokinetic properties such as terminal half-life can be tuned from minutes
up to several weeks10–12
.
• A high success rate compared to other drugs: 17% for humanized antibodies from the first human trial to regulatory
approval5
.
• Well-established and broadly applicable production technologies9
.
• Often, but not invariably, well tolerated by patients.
• Broad experience and expanding knowledge of antibody drug development facilitates the generation of future
therapeutic antibodies.
limitations
• Expensive, reflecting high production costs and commonly large doses9
, potentially limiting patient access or clinical
applications.
• Clinical applications currently limited to cell surface or extracellular targets.
• Cannot be orally administered.
• The large size, particularly of the IgG format (~150 kDa), may limit tissue penetration.
• There is limited penetration of the central nervous system by IgG owing to inefficient penetration of the blood–brain
barrier.
Future opportunities
• Greater efficacy.
• Higher affinity or potency.
• Enhanced effector functions — antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular
phagocytosis (ADCP), complement-dependent cytotoxicity (CDC).
• Target multiple disease mediators from distinct signalling pathways or that have redundant roles in the same pathway
using bispecific antibodies.
• Greater safety.
• Reduced effector functions.
• More ready reversibility of antibody action; for example, shorter half-life.
• Greater selectivity for target.
• Avoid activation of the target.
• Improved delivery.
• Lower or less frequent dosing using antibodies that are more potent or have a longer serum half-life.
• Increased bioavailability.
• Ability to cross the blood–brain barrier to reach the central nervous system.
• More facile ocular delivery, including sustained release, ocular penetration and half-life.
• Oral delivery.
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Nature Reviews | Immunology
FcγR
FcRn
IgG binding
partner
Protein strategies for
modifying interactions
Potential impact of
modifying interaction
Antigen Mutate V domain sequences
using display libraries and/or
rationale design
Mutate Fc sequence using display
libraries and/or rationale design;
select IgG isotype
Mutate Fc sequence
using display libraries and/or
rationale design
Antibody fragment lacking Fc
Glycosylation strategies
for modifying FcγR and
complement interactions
Aglycosylation
Non-fucosylation
Bisecting N-acetylglucosamine
↑ or ↓ ADCC
↑ or ↓ ADCP
↑ or ↓ CDC
↑ ADCC
↑ ADCC
↑ or ↓ half-life
↓ Half-life, ↓ CDC,
↓ ADCC and ↓ ADCP
↓ ADCC, ↓ ADCP and ↓ CDC
Altered binding affinity
or specificity
VL
CL
VH
CH1
CH2
CH3
Fc
region
Fab
Bisecting
N-acetylglucosamine
N-acetylglucosamine
Galactose
Sialic acid
Fucose
Asn297
Mannose
Core
Variable
Glycosylation
Complement
Human anti-chimeric
antibodies
(HACAs). The immune system
can develop an antibody
response to chimeric
antibodies. Binding of HACAs
to chimeric antibodies forms
immune complexes that can
shorten the half-life of the
therapeutic antibodies and
compromise clinical
effectiveness. Additionally
these immune complexes can
deposit in organs such as skin
and kidney causing adverse
events, including rashes and
glomerulonephritis.
Human anti-human
antibodies
(HAHAs). The immune system
can also develop an immune
response to human therapeutic
antibodies. HAHAs tend to be
less common and less severe
than HACAs.
Evolution of these technologies partially solved one
of the historic obstacles of antibodies — immunogenic-
ity. Patients with Crohn’s disease treated with inflixi-
mab develop human anti-chimeric antibodies (HACAs)
in 8–61% of patients15,16
. These HACAs can bind to the
therapeutic antibody limiting its half-life and clinical
effectiveness, as well as causing infusion-related ana-
phylaxis in some patients15,17
. The incidence of HACA
development can be attenuated by co-administration
of immunosuppressive agents in patients with Crohn’s
disease or with rheumatoid arthritis15,18
.
The advent of humanization protocols and develop-
ment of human antibodies have minimized this immuno-
genicity. However, patients treated with humanized
or human antibodies still develop human anti-human
antibodies (HAHAs). Adalimumab, a phage-derived
human antibody, has an incidence of 12% neutralizing
HAHAs in patients with rheumatoid arthritis receiving
monotherapy that is decreased to ~1% in patients also
treated with the immunosuppressant methotrexate19
.
in a prospective observational cohort of 121 patients
with rheumatoid arthritis treated with adalimumab,
HAHAs were detected in 34% of non-responders
compared with 5% of responders20
. Similarly, 6.3% of
patients with early onset rheumatoid arthritis treated
with golimumab, a human antibody derived from mice
expressing human immunoglobulin genes, developed
neutralizing HAHAs with the incidence of HAHAs
higher in non-methotrexate-treated (13.5%) than
methotrexate-treated patients (1.9–3.7%)21
.
Figure 1 | Engineering Igg structure and function. IgGs are ~150 kDa tetramers comprising pairs of identical heavy
and light chains linked by disulphide bonds (yellow bars). The heavy chains contain a variable domain, VH
, and three
constant domains, CH
1, CH
2 and CH
3, whereas the light chains contain a variable domain, VL
, linked to a single constant
domain, CL
. A signature property of antibodies is highly selective antigen binding mediated by their variable domains.
Human IgG, particularly IgG1 and IgG3, bound to an antigen on a target cell surface can interact with Fc receptors for
IgG (FcγRs) on effector cells and may support the destruction of target cells by antibody-dependent cell-mediated
cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), whereas interaction with the complement
component C1q may support killing by complement-dependent cytotoxicity (CDC). IgG antibodies, like other circulating
proteins, are taken up by vascular endothelial cells and other cells by pinocytosis. Subsequently, IgG can interact with the
salvage receptor, neonatal FcR (FcRn), in a pH-dependent manner with binding occurring in endosomes at pH 6.0–6.5,
followed by recycling and release at the cell surface at pH 7.0–7.4. Fc interaction with FcRn is mainly responsible for the
long serum half-life of IgG108
. Engineering of IgG variable domain sequences provides the means to tailor their
antigen-binding affinity or specificity10
. Fc amino acid sequence modification allows modulation of effector functions
(ADCC, ADCP and CDC) and/or half-life10–12,71
. Effector functions can be also be tuned by modifying Fc glycosylation. For
example, mutating the conserved aspargine, Asn297, prevents glycosylation to generate aglycosylated antibodies lacking
effector functions. ADCC can be enhanced by increasing the bisecting N-acetylglucosamine in the Fc carbohydrate or by
eliminating fucose11
. This figure is modified from Nature Reviews Immunology REF. 10.
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IL-6
TNF
TNF
Ligand blockade
Receptor blockade
Receptor downregulation
Depletion
Signalling induction
Infliximab*
Adalimumab*
Golimumab
Certolizumab pegol
Canakinumab
Briakinumab
Ustekinumab
Omalizumab*
Tocilizumab
Efalizumab*
Natalizumab
Vedolizumab
Abatacept‡
Efalizumab*
Omalizumab*
Otelixizumab*
Teplizumab*
Epratuzumab*
Otelixizumab*
Teplizumab*
Muromonab*
GA101*
Infliximab*
Adalimumab*
Rituximab*
Rituximab*
Ofatumumab
Ocrelizumab
GA101*
Alemtuzumab
Muromonab*
Epratuzumab*
IL-6R Soluble
IL-6R
TNFRI
TNFRI
Therapeutic antibody
Fab fragment
gp130 gp130
CD20
TCR–CD3
complex
αLβ2 integrin
Immunoadhesin
Therapeutic
antibody
FcγR
MAC
Lysis
Complement
components
Effector
cell
Belimumab
Eculizumab
Mepolizumab
Reslizumab
Etanercept‡
Atacicept‡
Alefacept‡
Half-lives of each of the TNF-specific antibodies
also define dosing schedules and modes of delivery.
infliximab has the shortest half-life (~8–10 days) among
the TNF-specific antibodies and is administered intra-
venously every 4–8 weeks. By contrast, adalimumab and
golimumab, both with half-lives of ~2 weeks, are admin-
istered subcutaneously every 2–4 weeks, respectively.
Subcutaneous administration is more convenient for
patients, particularly those with chronic diseases requir-
ing long-term therapy, as intravenous administration
requires visits to physicians’ offices or infusion centres.
Finally, the different formats of TNF-specific anti-
bodies have also revealed some interesting aspects con-
cerning the therapeutic mechanism (or mechanisms)
of efficacy. A proposed mechanism by which inflixi-
mab, and not etanercept, was thought to be efficacious
in Crohn’s disease was the ability of infliximab to bind
membrane-associated TNF and induce apoptosis of acti-
vated T cells and macrophages22–24
. However, the thera-
peutic success of certolizumab pegol and the preliminary
clinical efficacy of golimumab in Crohn’s disease, agents
that do not induce apoptosis of activated T cells or mac-
rophages, suggest that other mechanisms probably con-
tribute to the efficacy of TNF-specific agents in Crohn’s
disease14,25–27
.
The lessons learnt from the development of TNF-
neutralizing therapeutic agents are rapidly being applied
to other classes of therapeutic antibodies. Canakinumab
(ilaris; Novartis), an interleukin-1β (il-1β)-specific anti-
body approved for the treatment of cryopyrin-associ-
ated periodic syndrome (CAPS), builds on the clinical
experience of anakinra (Kineret; Amgen/Biovitrum), a
recombinant non-glycosylated form of the human il-1
receptor antagonist (il-1Ra) approved for the treatment
of rheumatoid arthritis28,29
. The long half-life of cana-
kinumab (~26 days) allows subcutaneous dosing every
8 weeks for children with CAPS. By contrast, anakinra
has a half-life of only 4 to 6 hours, necessitating daily
subcutaneous injections for patients with rheumatoid
arthritis. Ustekinumab (Stelara; Centocor), a human
igG1κ antibody that binds the p40 subunits of il-12 and
il-23, which has been approved for the treatment of
moderate to severe plaque psoriasis, has a median half-
life of ~21.6 days30,31
. Administration of ustekinumab
to patients with psoriasis requires only quarterly sub-
cutaneous injections following an initial loading dose.
interestingly, more frequent dosing seems to be required
for patients with Crohn’s disease and may reflect differ-
ences in the biology of il-12 and il-23 in these two
immunologically and genetically related diseases32
.
Receptor blockade and receptor modulation. in addi-
tion to ligand blockade, therapeutic antibodies can
also block ligand–receptor interactions by targeting the
receptor (FIG. 2). These include antibodies that target
the il-6 receptor (tocilizumab (Actemra/RoActemra;
Chugai/Roche)), αl integrin (also known as CD11a
and lFA1) (efalizumab (Raptiva/Xanelim; Genentech/
Roche/Merck–Serono)), the α4 subunit of α4β1 and
α4β7 integrins (natalizumab (Tysabri; Biogenidec/Elan))
and α4β7 integrin (vedolizumab (MlN2; Millennium
Figure 2 | mechanisms of action of therapeutic antibodies. Five non-overlapping
mechanisms of action are depicted. Examples of therapeutic antibodies are listed for
each mechanism of action depicted. Ligand blockade with full length IgG therapeutic
antibodies (for example, infliximab, adalimumab or golimumab), antibody fragments
(forexample, certolizumab pegol) or receptor immunoadhesins (for example, etanercept
and those indicated with ‡
) can prevent ligands from activating their cognate receptors.
Binding of ligands (for example, interleukin-6 (IL-6)) to receptors (for example, IL-6R) can
also be blocked by antibodies directed to their cognate receptors and inhibit receptor
activation or function. Binding of cell surface receptors by antibodies can also result in
their internalization and downregulation to limit cell surface receptors that can be
activated by the ligand. Note that binding of cell surface receptors by antibodies (for
example, αL integrin by efalizumab) or binding of a ligand (for example, free serum IgE
by omalizumab) can indirectly also result in downregulation of cell surface receptors
available for cellular activation. Binding of cell surface receptors can result in depletion
of antigen-bearing cells through complement-mediated lysis and opsonization, as well
as Fc receptor for IgG (FcγR)-mediated clearance. Therapeutic antibodies can also
induce active signals that alter cellular fates. Binding of the T cell receptor (TCR)–CD3
complex by teplizumab can induce TCR-mediated signals and alter T cell functions
and differentiation. MAC, membrane attack complex; TNF, tumour necrosis factor;
TNFRI; TNF receptor I. *Antibodies with several mechanisms of action.
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10

Pharmaceuticals/Takeda)). Targeting of receptors adds a
secondary level of mechanistic activity as a subset of these
therapeutic antibodies not only blocks ligand binding but
also downregulates the cell surface expression of the tar-
geted receptors. However, this antibody-induced inter-
nalization of the target also results in antigen-induced
clearance of the therapeutic antibody and decreases its
serum half-life. For example, efalizumab not only binds
αl integrin to block interactions between αl integrin
and intercellular adhesion molecule 1 (iCAM1) but also
downregulates αl integrin expression by T cells, a crucial
co-receptor for T cell activation, as well as the expression
of other T cell co-stimulatory molecules. Hence, efalizu-
mab interferes with both T cell homing by interrupting
αl integrin–iCAM1 interactions and T cell activation by
downregulating co-stimulatory molecule expression33
.
Downmodulation of cell surface receptor expression
can also be achieved indirectly through ligand target-
ing. omalizumab (Xolair; Genentech/Roche/Novartis),
which is approved for the treatment of moderate and
severe allergic asthma, binds and prevents the secreted
form of igE from occupying the high affinity receptor
for igE (FcεRi) on mast cells and basophils34
. Whereas
binding sequesters allergic igE from allergen-mediated
activation of mast cells and basophils, the unoccupied
surface FcεRi undergoes internalization and results in
a decrease in cell surface FcεRi expression on dendritic
cells, mast cells and basophils35–37
. Hence, binding of igE
by omalizumab probably operates through two distinct
mechanisms: sequestration of the allergic igE and indi-
rect downregulation of cell surface FcεRs on mast cells
and basophils38
. Receptor downregulation might serve
as the basis for the efficacy of omalizumab in the treat-
ment of chronic idiopathic urticaria and angioedema
(CiU)39–41
. Approximately 40% of patients with CiU
have autoantibodies that recognize and can crosslink
the FcεRi α-subunit, triggering mast cell and basophil
activation42,43
. Downregulation of cell surface FcεRs by
omalizumab in these patients may attenuate the ability of
these autoantibodies to activate mast cells and basophils
to provide clinical improvement.
A theoretical concern for targeting cell surface recep-
tors, as opposed to their corresponding soluble ligands,
is a greater potential for inducing an immunogenic
response. Therapeutic antibodies that induce antigen-
dependent internalization through the endocytic path-
way can theoretically be processed as foreign antigens
and presented on MHC class ii molecules to initiate a
CD4+
T cell-dependent humoral response. However,
although this is possible, we are not aware of any data
that support this target class differential in immuno-
genicity of therapeutic antibodies. ongoing develop-
ment of therapeutic antibodies that target dendritic cell
surface antigens (such as DEC205; also known as ly75)
may be informative in this regard.
Depleting and signalling antibodies. Another class of
therapeutic antibodies binds to cell surface antigens —
for example, CD20, CD22 and CD52 — and depletes
antigen-bearingcells(FIG. 2).Rituximab(Rituxan/Mabthera;
Genentech/Roche/Biogen idec) and alemtuzumab
(Campath/MabCampath; Genzyme/Bayer), approved
first for the treatment of cancer, were subsequently tested
in autoimmune and inflammatory conditions. Rituximab,
a chimeric CD20-specific antibody, was approved in
2005forthetreatmentofpatientswithrheumatoidarthritis
who had inadequate responses to TNF blockade44
; rituxi-
mab and alemtuzumab (a humanized CD52-specific anti-
body)haveshownclinicalactivityinPhaseiiclinicaltrials
of the relapsing–remitting form of multiple sclerosis45,46
.
ocrelizumab (2H7; Genentech/Roche/Biogen idec),
which is a humanized CD20-specific antibody, is being
studiedinPhaseiiiclinicaltrialsofrheumatoidarthritis47
,
and epratuzumab (hll2; immunomedics/UCB), which
is a humanized CD22-specific antibody, is being studied
in Phase iii clinical trials of systemic lupus erythemato-
sus48
. This class of therapeutic antibodies operates mainly
through FcγR-mediated clearance of antigen-bearing
cells; although CDC can also contribute to their thera-
peutic effect under certain circumstances49–52
. Patients
with non-Hodgkin’s lymphoma treated with rituximab
have better prognosis if they express the FcγRiiiA val158
encoding allele, which has high affinity for igG1, than
patients expressing the FcγRiiiA Phe158 encoding allele,
which has lower affinity for igG1(REFS 53,54). Similarly, in
a small study of patients with SlE treated with rituximab,
B cell depletion was superior in patients with FcγRiiiA
val158 than those with FcγRiiiA Phe158 (REF. 55). Hence,
FcγR-mediated functions have an important role for this
class of therapeutic antibody and offer an opportunity to
design more efficacious therapies.
in addition to effector functions, the nature of the
antigen also contributes to the depleting potential of a
therapeutic antibody. Although antibodies such as efal-
izumab and natalizumab bind to αl and α4 integrins
expressed by T cells, these antibodies do not deplete
T cells. Many antigen-related factors, including cell sur-
face density, probably contribute to the depleting poten-
tial of a therapeutic antibody. CD20 is highly expressed
(~90,000 molecules) on normal B cells, but its expres-
sion level is highly variable in B cell malignancies56,57
.
lower levels of CD20 expression can inhibit the ability of
CD20-specific antibodies to mediate CDC58
. in addition,
expression of complement regulatory proteins (such as
CD59, CD46 and CD55) on the target cell can com-
promise complement-mediated killing of target cells59
;
however, the expression of these complement regula-
tory proteins does not predict clinical outcome follow-
ing rituximab treatment in patients with non-Hodgkin’s
lymphoma60
. Finally, antibody-mediated internalization
can also decrease the number of accessible cell surface
antibody–antigen complexes to affect both CDC- and
ADCC-mediated depletion of target cells. These mecha-
nistic data provide clues for the generation of future ther-
apeutic antibodies with improved effector function.
Astheclearanceofcellsismediated,inpart,bymobili-
zation of cells from organs into the blood and clearance of
antibody-bound targets by myeloid-derived cells (such as
Kupffer cells in the liver), non-circulating antigen-bear-
ing cells saturated with the therapeutic antibody seem
to be more resistant to antibody-mediated clearance50
.
Biopsies of the synovium of patients with rheumatoid
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Bispecific antibodies
Antibodies capable of binding
to two different antigens or two
distinct epitiopes on the same
antigen are known as
bispecific.
arthritis following treatment with rituximab contain
residual CD20-bearing B cells61,62
. in FvB mice, marginal
zone B cells in the spleen are not completely depleted by
CD20-specific antibody therapy50
. However, these mar-
ginal zone B cells are not intrinsically resistant to CD20-
specific antibody killing as forced mobilization of these
cells into the blood with blockade of αl integrin and α4
integrin renders them sensitive to depletion mediated by
CD20-specific antibody. Hence, strategies to mobilize
cells from their microenvironments may enhance the
depleting potential of therapeutic antibodies.
Therapeutic antibodies that bind cell surface anti-
gens can also transmit intracellular signals required for
clinical efficacy and/or contribute to adverse events63
.
The mouse CD3-specific antibody muromonab-CD3
(oKT3; Janssen-Cilag), the first FDA-approved thera-
peutic antibody for the treatment of acute allograft
rejection in renal transplantation64
, crosslinks the T cell
receptor (TCR)–CD3 complex to induce transient
cytokine release, resulting in an acute and severe flu-like
syndrome and T cell depletion64
. Muromonab-CD3 has
an additional limitation in that ~50% of patients develop
neutralizing antibodies that diminish its therapeutic
effect65
. The beneficial activities of muromonab-CD3 are
mediated by its variable domains, whereas the undesir-
able mitogenic activity — stimulation of T cell prolif-
eration and cytokine production — requires interaction
between the muromonab-CD3 Fc region and FcγRs66
.
A second generation of CD3-specific antibodies
(teplizumab (MGA031; MacroGenics/Eli lilly) and
otelixizumab (TRX4; Tolerx/GlaxoSmithKline)) that are
under evaluation for the treatment of type 1 diabetes are
humanized antibodies with mutations that impair inter-
action with FcγRs (FIG. 2). These non-mitogenic antibod-
ies do not induce the same degree of T cell activation
and adverse events as observed with muromonab-CD3.
However, both these antibodies still activate T cells,
althoughtheirmechanisticeffectsarecomplex.First,these
antibodies seem to downregulate the level of cell surface
TCR–CD3 complexes. Although the TCR–CD3 complex
is continually internalized and recycled, internalization
of even unbound TCR–CD3 increases ~10-fold in the
presence of otelixizumab without change in the exocytic
rate67
. Second, in vitro crosslinking of T cells with teplizu-
mab induces TCR-mediated increases in free cytoplasmic
Ca2+
andanalteredpatternofcytokineproductionfavour-
ing il-10 rather than interferon-γ expression68
. Patients
treated with teplizumab developed increased numbers of
il-10-producing CD4+
T cells and an even greater expan-
sion of CD8+
CD25+
CTlA4+
FoXP3+
regulatory T cells69
.
Hence, this class of antibody seems to modulate T cell
function by altering T cells from an activating phenotype
to potentially a tolerizing or regulatory phenotype.
Next-generation therapeutic antibodies
Strategies for new antibody generation can be broadly
classified as modification of existing antibody properties
or the endowment of antibodies with new capabilities
(FIG. 1). Here we discuss examples of these two distinct
categories — modulation of Fc-mediated functions and
the generation of bispecific antibodies (FIG. 3) for dual
disease mediator blockade — that seem particularly
promising for the generation of antibody drugs for
autoimmunity and inflammation. We focus on igG as the
format most widely adopted for therapeutic antibodies.
Antibody fragments are also discussed as they are widely
used in engineering antibody properties, they provide
the building blocks to construct many new formats
(including bispecific antibodies) and they are becoming
important therapeutic agents in their own right70
.
Optimization of Fc-mediated antibody functions.
Modification of Fc-mediated activities is emerging as
one of the most promising ways to further increase the
clinical potential of antibodies10–12
. Modulation of effec-
tor functions (ADCC, ADCP and CDC) and pharmaco-
kinetic half-life are reviewed here in the context of
antibody development for autoimmunity and inflam-
mation. Engineering igG–binding partner interactions
is routinely accomplished through amino acid sequence
alterations in the antibody identified by selection from
display libraries of antibody fragments and/or structure-
guided design. Tailoring Fc glycosylation offers a pow-
erful additional method to modify some igG functions,
particularly enhancement of ADCC11
. Fc binding to
anotherligand,namelyproteinA,isubiquitouslyexploited
for chromatographic purification of antibodies, including
for therapeutic use9
.
Effector function minimization. For some antibody
therapies, antigen binding may be sufficient for achiev-
ing efficacy, and effector functions may be unnecessary
and a potential source of adverse events in patients.
Moreover, monovalent target binding may be necessary
to avoid unwanted activation of the target. Several dif-
ferent strategies are available to generate antibodies that
have minimal or no effector functions and, if need be,
that are also non-activating71
. indeed many antibody
drugs are designed with minimal effector functions, as
exemplified below with antibodies that have reached
Phase iii clinical trials or beyond in autoimmunity and
inflammatory diseases (TABLE 1).
As discussed previously, the clinical potential of
CD3-specific antibodies has been greatly improved by
Fc engineering to attenuate FcγR interactions and over-
come mitogenic activity and the development of a flu-like
syndrome, in conjunction with humanization to reduce
immunogenicity. indeed, two such humanized CD3-
specific antibodies — teplizumab and otelixizumab —
have advanced to Phase iii clinical trials (TABLE 1). These
CD3-specific antibodies use different means to mini-
mize FcγR binding: igG1 isotype with leu234Ala and
leu235Alamutationsforteplizumab72,73
andaglycosylated
igG1 with an Asn297Ala mutation for otelixizumab74
.
The igG4 isotype has also been selected for a few
therapeutic antibodies for which effector functions are
not desired75
, including natalizumab and reslizumab
(SCH55700; Ception Therapeutics) (TABLE 1). igG4
molecules are prone to exchange Fabʹ arms in vivo to
become functionally monovalent76
. However, this seem-
ingly undesirable attribute of igG4 for antibody drugs
can be readily prevented by mutation of the hinge
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a Antigen-binding building blocks
b Bispecific antibody fragments
c IgG-based bispecific antibodies
VL
VL
Fv scFv Diabody
Diabody scDiabody–Fc scDiabody–CH3
scDiabody
IgG–scFv scFv–IgG
VH VH
VL
VH
VL
VL
VH
VH
VL
VH
VL
VH
VL
VH
VL
VH
Antigen 1
Two variable domain
binding sites
Single variable domain
binding sites
Antigen 2
Tandem
scFv F(ab′)2
Hybrid hybridoma
Two-in-one IgG
(Fab′scFv)2
Fab–scFv
Tandem
scFv–Fc
scFv–Fc
knobs-into-holes
Knobs-into-holes with
common light chain
scFv–Fc–scFv
Dual V domain IgG
VL
CL
VH
CH1
VL
VH
IgG–V V–IgG
Figure 3 | modular Igg architecture supports numerous
bispecific antibody formats. a|Fvfragments,comprisinga
VL
andVH
domainpair,arethemostcommonantigen-binding
buildingblockinIgG.Fvfragmentsareroutinelyengineered
intoasingle-chain(sc)formatusingashortpeptidelinker(~15
residues)toconnectvariabledomainsineitherVH
–VL
orVL
–VH
topology156,157
.ConstructionofscFvfragmentswithshorter
linkers(typically1–10residues)allowsinter-chain,butnot
intra-chain,domainpairingtoformadimer—thediabody158
.
Singlevariabledomainantigen-bindingsitesarefoundinsome
IgGmoleculesfromcamelidsandcertainsharkspecies,andthis
formatisnowbeingusedforconstructingtherapeutics159
.
b|Antigen-bindingbuildingblocks,particularlyscFvand
diabodies,havebeenextensivelypermutatedwithother
antibodydomainsandproteindomains(notshown)tocreate
anincreasingrepertoireofalternativebispecificantibody
fragmentformats.Fcregionsareincludedincasesinwhich
specificeffectorfunctionsand/orlongserumhalf-lifeare
desired.Bycontrast,CH
3domainshavebeenusedfor
dimerizationincasesinwhichFc-mediatedfunctionsarenot
needed. c|Hybridhybridomatechnologiesprovidedthefirst
directroutetobispecificIgGbyco-expressionoftwodifferent
IgGspecificities160
,albeitcommonlywithlowyieldandpurity
as,beyondthedesiredbispecificIgG,co-expressionoftwo
differentantibodyheavyandlightchainscangiverisetoupto
nineunwantedantibodymolecules(notshown)161
.Efficient
constructionofbispecifichumanIgGwasachievedusing
phage-derivedantibodieswithacommonlightchain,to
circumventlightchainmispairing,andknobs-into-holes
engineering,todirecttheassemblyoftwodifferentheavy
chains162
.Phagedisplaytechnologyhasendoweda
monospecificantibodywiththeabilitytobindasecond
antigenwhilemaintaininghighaffinityforthefirstantigen—
‘two-in-one’IgG141
.Thebindingsitesarepartiallyoverlapping
sotheycanbindonlyoneantigenatatime.Severalbispecific
antibodyformatsarebothbispecificandbivalentforeach
antigen.Forexample,thedualvariabledomainIgGformatwas
createdbyappendingVL
andVH
domainswithsimilardomains
ofasecondantigen-bindingspecificity147,148
.Bispecific
antibodieshavealsobeengeneratedbyappendingeitherthe
aminoorcarboxylterminusofanIgGheavyorlightchainwitha
scFvorsinglevariable(V)domainofasecondbindingspecificity.
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10

sequence (Cys-Pro-Ser-Cys to Cys-Pro-Pro-Cys) or the
CH
3 domain76
. igG4 can bind to some FcγRs, includ-
ing FcγRi77,78
— an interaction that can be weakened by
point mutations to abolish ADCC activity79
.
Antibodies lacking effector functions can also be
created with hybrid isotype igG molecules that mimic
the desired attributes of their component isotypes. For
example, the complement component C5-specific anti-
body eculizumab (Soliris; Alexion pharmaceuticals)
(TABLE 1) uses a hybrid igG2–igG4 constant region that
includes the CH
1 domain and hinge region of a human
igG2 fused to the CH
2 and CH
3 domains of a human
igG4. Eculizumab lacks the ability to bind FcγRs and
activate complement, which are common attributes for
human igG2 and igG4, respectively80
.
Strategies to eliminate effector functions are readily
extended to Fc fusion proteins. For example, the cyto-
toxic T lymphocyte antigen 4 (CTlA4)–Fc fusion pro-
tein abatacept (orencia; Bristol-Myers Squibb) has five
mutations compared with human igG1: lys215Gln,
Cys220Ser, Cys226Ser, Cys229Ser and Pro238Ser81
(Eu
numbering82
). Multiple functional consequences of
these mutations include improved protein production,
elimination of inter-chain disulphide bonds and O-linked
glycosylation by virtue of the mutated hinge (Cys-Pro-
Ser-Cys to Ser-Pro-Pro-Cys), inefficient binding to FcγRs
and lack of ADCC and CDC activity12,81
.
A seemingly small risk in making Fc mutations to
ablate effector functions is that they might lead to del-
eterious structural perturbations. introducing the triple
mutation lys234Phe:lys235Glu:Pro331Ser (‘FES’) into
an igG1 molecule caused a substantial decrease in bind-
ing to FcγRs and the complement component C1q164
.
The structure of FES Fc is similar to other human Fc
structures164
. Thus the FES mutations designed to ablate
effector functions probably do so without causing undue
structural perturbation of the Fc domain.
Many different engineered antibody fragments have
been developed including several that have entered clini-
cal development70
. Many antibody fragments lack the igG
Fcregionanditsassociatedproperties—effectorfunctions
and extended half-life. As discussed previously, certolizu-
mab pegol, a humanized TNF-specific Fabʹ fragment, is
PEGylated to extend its pharmacokinetic half-life and is
the first FDA-approved PEGylated antibody fragment for
clinical use (TABLE 1). Whereas Fab fragments have a ter-
minal half-life of ~20 minutes, certolizumab pegol has a
half-life of ~14 days in patients, similar to its parent igG83
.
Enhancing effector functions. For some antibodies,
potent effector functions, as described previously for
rituximab, may be required for optimal clinical activ-
ity. Antibodies with efficient effector functions can be
identified directly by screening panels of antibodies84
,
engineering to improve the activities of existing antibod-
ies10–12
or a combination of both of these approaches.
Potential benefits from enhancing effector functions
include greater therapeutic efficacy, lower or less fre-
quent dosing and decreased cost of goods. Potential risks
from enhanced effector functions include more frequent
or severe adverse events in patients.
ADCC activity can be enhanced by engineering the
Fc protein sequence or tailoring the Fc carbohydrates
to strengthen binding to FcγRs, most importantly
FcγRiiiA11,12
. Detailed mutational analysis of igG1 iden-
tified the functionally important Fc residues for bind-
ing to the different FcγRs (FcγRi, FcγRiiA, FcγRiiB
and FcγRiiiA) and FcRn, as well as igG1 mutants with
increased binding to FcγRiiiA and increased ADCC
activity85
.ADCC-enhancingmutantshavealsobeeniden-
tified from yeast display libraries86
. Computational design
based on the structures of Fc–FcγR complexes together
with functional screening has identified several mutants
with up to ~100-fold higher affinity binding for FcγRiiiA.
one such mutant, Ser239Asp:Glu330leu:ile332Glu, gave
a ~100-fold increase in ADCC potency, with greater
maximal target cell killing in some cases87
. Fc protein
engineering to enhance ADCC is broadly applicable as
shown with variants of several therapeutically relevant
antibodies: trastuzumab (Herceptin; Genentech/Roche),
cetuximab (Erbitux; imClone Systems), rituximab and
alemtuzumab87
. ADCC-enhancing antibodies can kill
cells expressing lower levels of antigen, as shown with
trastuzumab variants87
— a potential benefit for improved
efficacy, albeit with unknown risk of killing non-target
cells expressing low levels of antigen.
ADCP activity can also be enhanced through Fc pro-
tein engineering. For example, a Gly236Ala mutation
increases igG1 affinity for FcγRiiA 70-fold, and it has a
15-fold improvement in the selectivity of binding to this
activating receptor over the related inhibitory receptor
FcγRiiB88
. The Gly236Ala mutation enhances phago-
cytosis of antibody-coated target cells by macrophages.
ADCPandADCCweresimultaneouslyenhancedbycom-
bining Gly236Ala and Ser239Asp:ile332Glu mutations
that enhance ADCP and ADCC, respectively88
.
Aglycosylated igG molecules bind poorly to FcγRs
and do not support ADCC. By contrast, bacterial dis-
play selection was used to identify a double mutant,
Glu382val:Met428ile, that endows aglycosylated igG1
with the ability to bind FcγRi with high (nanomolar)
affinity and support ADCC89
. Moreover, the binding
was remarkably selective for FcγRi with no detectable
binding to the other FcγRs. The Glu382val:Met428ile
mutations did not affect FcRn binding in vitro or serum
persistence in vivo. Thus, aglycosylated igG can be engi-
neered with selectivity profiles for FcγRs that are distinct
from those of glycosylated igG89
.
Asecondwidelyapplicableandcomparablysuccessful
method for increasing the igG ADCC activity is through
tailoring of Fc glycosylation11,90
. Antibody production in
a Chinese hamster ovary (CHo) cell line transfected to
overexpress N-acetylglucosaminyltransferaseiii resulted
in antibodies linked to carbohydrate with a bisect-
ing N-acetylglucosamine and more potent ADCC91
.
Decreasing the fucose content of the carbohydrates in
the Fc region can also enhance ADCC92,93
. Production
of antibodies devoid of fucose is readily achieved using
CHo cells that lack expression of fucosyltransferase 8
(REF. 94). Total elimination of fucosylated antibodies is
important to avoid competition with, and reduction
of, the activity of the more potent non-fucosylated
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Exposure
In the pharmacokinetic sense,
the area under the curve for a
plot of drug concentration
versus time.
antibodies95
. other host organisms are used to pro-
duce non-fucosylated antibodies, including extensively
engineered strains of Pichia pastoris96
.
Most licensed therapeutic antibodies are exten-
sively fucosylated11
, and their in vitro ADCC activity is
decreased by the presence of serum, presumably because
of competition for FcγRiiiA with high concentrations of
irrelevant igG. By contrast, non-fucosylated variants
of rituximab and trastuzumab have increased affinity
for FcγRiiiA and ~100-fold more potent ADCC activity
that is minimally affected by serum igG95
. The increases
in ADCC potency achieved from Fc mutations87
and
non-fucosylation95
seem to be similar as judged by
corresponding variants of trastuzumab and rituximab.
The binding site on human igG1 for complement
component C1q has been identified by mutational analy-
sis97
.Thisledtothegenerationofarituximabvariantwith
improved complement binding97
and ~twofold greater
CDC activity, albeit with slightly impaired ADCC activ-
ity98
. Enhancement of antibody CDC activity has also
been achieved using hinge-region mutations99
. increased
CDC activity has recently been achieved by creating a
hybrid isotype igG in which some igG1 residues were
replaced with corresponding igG3 sequences100
. A few
additional igG1 residues were required to restore effi-
cient binding to protein A to allow purification100
. An
antibody enhanced in both CDC and ADCC activities
was created by combining Fc protein mutations with
non-fucosylation100
.
The efficacy and safety of antibodies with enhanced
effector functions are challenging to assess preclinically
because of species differences between the immune sys-
tems of mice and humans. CD20-specific antibodies with
high ADCC activity have been evaluated in cynomolgus
macaques using B cell depletion as a surrogate for effi-
cacy87,101
.Severalantibodieswithpotenteffectorfunctions
have progressed into clinical development. For example,
human CD20-specific antibodies were obtained from
transgenic mice with potent CDC activity even against
tumour cell lines expressing high levels of complement
regulatory proteins, CD55 and CD59, that are resistant
to CDC by rituximab84
. one of these human CD20-
specific antibodies, ofatumumab (Arzerra; Genmab/
GlaxoSmithKline), is now licensed for the treatment
of chronic lymphocytic leukaemia and is under clinical
evaluation for rheumatoid arthritis, multiple sclerosis and
other cancers (TABLE 1). Another CD20-specific antibody,
ocrelizumab (2H7; Genentech/Roche/Biogen idec), has
in vitro ADCC and CDC activities that are higher and
lower than rituximab, respectively102
. ocrelizumab has
advanced to Phase iii clinical trials for rheumatoid
arthritis and systemic lupus erythematosus (TABLE 1).
At least eight effector function-enhanced antibodies
have progressed into clinical development. For example,
GA-101 (Roche/GlycArt) is a humanized type ii CD20-
specific antibody that is glyco-engineered (bisected
and non-fucosylated) to enhance ADCC and is in
Phase ii clinical trials for non-Hodgkin’s lymphoma103
.
other clinical stage glyco-engineered antibodies include
a non-fucosylated humanized il-5R-specific antibody
MEDi-563 (BioWa/Medimmune). A single intravenous
dose of MEDi-563 of only 30 μg/kg — much lower
than for most antibody drugs — was well tolerated
and induced robust and reversible blood eosinopenia
in all six mildly asthmatic patients treated11,104
. ADCC-
improved antibodies containing Fc mutations have also
entered clinical development, including the humanized
CD30-specific antibody Xmab2513 (Xencor) that is in
a Phase i clinical trial for Hodgkin’s and anaplastic large
cell lymphomas.
A distinct mode of depleting CD20-specific antibod-
ies has emerged for the treatment of B cell malignancies
that has implications potentially for future depleting
therapies in autoimmune and inflammatory diseases.
CD20-specific antibodies can be defined as either type i
(exemplified by rituximab) or type ii (such as tositu-
momab (Bexxar; Corixa/GlaxoSmithKline))105
. Type ii,
but not type i, CD20-specific antibodies induce an actin-
dependenthomotypicadhesionandaggregationofCD20-
bearing cells106
. Through an unknown mechanism, type ii
antibodies trigger lysosome destabilization and release
of lysosomal contents to mediate a caspase- and B cell
lymphoma 2 (BCl-2)-independent form of cell death.
Should these mechanisms prove effective in human
malignancies, they offer a distinct therapeutic option to
deplete all CD20-bearing cellular niches.
Pharmacokinetic half-life extension. The terminal
half-life for chimeric, humanized and human antibody
drugs in the circulation varies widely (~3–27 days)107
.
Engineering antibodies to increase their serum half-
life and exposure offers the potential benefits of greater
efficacy, lower or less frequent dosing, lower cost and
enhanced localization to the target.
The interaction between the Fc region of igG and the
recycling receptor FcRn has a key role in igG homeostasis
and is largely responsible for the long half-life of igG108
:
~21 days in humans for igG1 (REF. 109). igG, like other
circulating proteins, is taken up by vascular endothelial
cells and other cells by pinocytosis. Subsequently,igG can
interact with the FcRn, in a pH-dependent manner with
binding occurring in endosomes at pH 6.0–6.5, followed
by recycling and release at the cell surface at pH 7.0–7.4.
Fc engineering to modulate the FcRn interaction
and extend igG half-life is discussed briefly here and in
greater depth elsewhere10,12
. An important early milestone
was the identification of Fc mutations from phage display
libraries that strengthen Fc binding to FcRn and prolong
the half-life of mouse igG in mice110
. Subsequently, Fc
mutations (Thr250Gln:Met428leu; ‘Ql’) were identified
that increase the binding affinity of a human igG2 for
human FcRn at pH 6.0 but not at pH 7.4 (REF. 111). These
Ql mutations confer a similar pH-dependent increase in
binding of a human igG2 to rhesus FcRn and a ~twofold
increase in serum half-life in rhesus macaques111
. The Ql
mutations had a similar effect in extending the half-life
of a human igG1 in rhesus macaques112
. The Ql muta-
tions also increased the binding affinity of a TNF-specific
igG1 to FcRn from cynomolgus macaques, albeit without
extending half-life for unknown reasons113
. Alternative
mutations have been identified that extend the half-life
of antibodies in non-human primates. For example, the
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10

triple Fc mutation, Met252Tyr:Ser254Thr:Thr256Glu
(‘yTE’) increases the half-life of a humanized respira-
tory syncytial virus (RSv)-specific igG1 in cynomolgus
macaques by three–fourfold114
. The X-ray crystallo-
graphic structure of the yTE Fc is similar to other Fc
structures, strongly suggesting that the yTE mutations
do not significantly perturb the Fc structure115
.
Fc mutations that extend pharmacokinetic half-
life can also enhance in vivo efficacy, which has been
recently shown for the first time116
. The double muta-
tion Met428leu:Asn434Ser (‘lS’) engineered into the
vascular endothelial growth factor (vEGF)-specific
antibody bevacizumab (Avastin; Genentech) extended
serum half-life by ~threefold in cynomolgus mon-
keys and by ~fourfold in engineered mice expressing
human, but not mouse, FcRn116
. Moreover, the lS muta-
tions increased the antitumour activity of bevacizumab
in a xenograft study in mice. in addition, a humanized
version of the chimeric epidermal growth factor receptor
(EGFR)-specific antibody cetuximab engineered with the
lS mutations out-performed cetuximab in vivo, extended
half-lifeincynomolgusmonkeys(~threefold)andinengi-
neered mice (~fivefold), and had superior antitumour
activity in mice.
Studies in primates with engineered antibodies (see
above) support the feasibility of extending the plasma
half-life of therapeutic antibodies by tailoring the interac-
tionofFcwithFcRn.indeed,thehumanizedRSv-specific
igG1 with yTE mutations (MEDi-557) is currently in
Phase i clinical trials to explore this possibility. The half-
life-extending yTE mutations have also been successfully
combined with mutations that enhance ADCC as judged
by potent in vitro activity in cytotoxicity assays114
.
Species differences in FcRn–Fc interactions between
mice and humans add much complexity to the preclini-
cal analysis of antibodies engineered for extended half-
life117
. Transgenic mice expressing human FcRn may
offer a partial solution to this challenge118
. An engineered
igG that binds to FcRn at both neutral and slightly acidic
pH is more rapidly cleared than wild-type igG119
. Thus,
maintaining the strict pH dependence of Fc binding to
FcRn is apparently crucial for supporting the long half-
life of igG. An igG engineered to bind FcRn with higher
affinity and reduced pH dependence potently inhibits
the interaction of FcRn with endogenous igG and rapidly
lowers the concentration of circulating igG in mice120
.
These FcRn-blocking antibodies known as ‘Abdegs’ may
have therapeutic potential for reducing pathogenic igG
levels in antibody-mediated autoimmune diseases120
.
Pharmacokinetic half-life reduction. For some therapeu-
tic antibodies, it may be advantageous to decrease their
terminal half-life to decrease exposure, improve safety
or, in the case of some imaging applications, improve
the target/non-target localization ratios. A large reduc-
tion in antibody half-life — from weeks to hours or
less — is readily achieved by using small antibody frag-
ments susceptible to renal elimination and that lack
a functional Fc region to preclude recycling by FcRn.
indeed, two unmodified Fab fragments are licensed for
human therapy: the integrin-specific Fab abciximab
(ReoPro; Eli lilly) for the prevention of platelet-mediated
clot in coronary angioplasty and the vEGF-specific Fab
ranibizumab (lucentis; Genentech) for the treatment of
neovascular (wet) age-related macular degeneration.
it may be desirable to tune the pharmacokinetic
properties of some antibody drugs to achieve half-lives
that are intermediate between small antibody fragments
and igG. This can be accomplished by Fc mutations that
attenuate the interaction with FcRn and decrease anti-
body half-life, as shown by pharmacokinetic optimiza-
tion of single-chain (sc)Fv–Fc antibody fragments for
tumour imaging121
. Antibody fragments with a wide
range of terminal half-lives — from a few hours to days
— can be generated through site-specific PEGylation122
.
Several PEGylated proteins are licensed for human
therapy123
including one PEGylated antibody fragment,
certolizumab pegol, that has igG-like pharmacokinetic
behaviour. The pharmacokinetic properties of PEGylated
proteins can be tuned by varying the size and extent of
branching of the PEG molecules, sites of attachment and
number of PEG molecules attached per protein123
.
Several alternative strategies have been developed to
extend the half-lives of antibody fragments and other
proteins including genetic fusion to albumin124
, igG Fc125
or an unstructured recombinant polypeptide (XTEN)126
,
engineering them with the ability to bind long-circulating
proteins such as albumin127
or igG128
, or by creating new
glycosylation sites129
. Fc mutations that impair interaction
with FcRn can decrease antibody half-life as exemplified
by pharmacokinetic optimization of scFv–Fc antibody
fragments for tumour imaging121
.
Optimization of antigen-binding domains
Engineering of antibody variable domains to increase
the binding affinity for antigen or tune the binding spe-
cificity has been extensively reviewed elsewhere10
and is
not discussed further here. Two major strategies gaining
momentum for endowing antibodies with new activities
are antibody–drug conjugates130
and bispecific antibod-
ies131,132
, which are mainly used for targeting cytotoxic
drugs and effector cells, respectively, to kill tumour cells.
Bispecific antibodies have additional potential applica-
tions including dual blockade of disease mediators133
,
as discussed here.
Bispecific antibodies. The concept of bispecific antibod-
ies was first suggested nearly 50 years ago134
, and it is
finally coming of age for the generation of therapeutic
agents131,132
. Historically, a major obstacle to developing
bispecific antibodies as therapeutic agents has been the
difficulty in designing these complex molecules with
favourable drug-like properties and producing them in
sufficient quantity and quality to support drug develop-
ment.Bispecificantibodiesaspotentialtherapeuticagents
have undergone a renaissance in recent years, facilitated
by the advent of numerous alternative formats and
improved methods for their more efficient generation,
optimization and production132,133,135
(FIG. 3). Moreover,
greater understanding of disease pathogenesis is leading
to better choices of target antigen and antibody pairs.
Here we propose ideal attributes of bispecific antibodies
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Valency
For antibodies, including
bispecific antibodies, the
number of binding sites
for each cognate antigen.
for drug development based on experience with antibod-
ies and other biological drugs. Additionally, advances in
selected technologies for generating bispecific antibodies
for therapeutic applications are presented (FIG. 3).
The most successful clinical application of bispecific
antibodies to date has been in oncology by redirect-
ing cytotoxic T cells to kill tumour cells. This concept
was originally proposed in 1985 (REF. 136) and has been
explored in the context of several different bispecific anti-
body formats. The most successful format identified to
date for this application is a tandem scFv (FIG. 3) known
as a ‘BiTE’ (bispecific T cell engager). Blinatumomab
(MT103; Micromet/Medimmune), a BiTE specific for
CD19 and CD3, has been used to treat patients with non-
Hodgkin’s lymphoma137
. Complete and partial responses
were observed with blinatumomab administered at doses
as low as 0.15 μg per m2
per day137
, that is, doses at least
10,000 times lower than those typically used for thera-
peutic igG1 antibodies for targeting tumours. BiTEs are
rapidly cleared, necessitating administration by a port-
able mini-pump; however a possible benefit is fine dos-
ing control. Adverse events with blinatumomab include
neurologicalsymptomsthatareseeminglyreversible.This
early clinical success with BiTEs has encouraged further
pursuit of this format, including targeting of many differ-
ent tumour antigens131
. Several additional applications of
bispecificantibodieshavebeeninvestigated133,135
,including
blockade of several disease mediators as discussed here.
Designing bispecific antibodies as therapeutic agents.
An ideal bispecific antibody format for therapeutic appli-
cations would be broadly applicable to different combi-
nations of target antigens without requiring extensive
customizationforindividualantibodypairs.Routinehigh-
level expression (gram per litre) of bispecific antibody will
probably be needed to support manufacturing for clini-
cal development, preferably in well established and widely
available host production systems such as CHo cells or
E. coli. Bispecific antibodies should preserve the antigen-
binding affinity and beneficial biological activities of their
component monospecific antibodies. Bispecific antibody
design preferably facilitates purification to homogene-
ity and recovery in high yield. Simultaneous binding of
both antigens to the bispecific antibody is desirable and
may be essential depending on the application. Selection
of the bispecific antibody format to match the valency of
each component antibody to the biology of the target
antigens may be necessary, and is readily achievable from
the many alternative formats (FIG. 3). Bispecific antibodies
need favourable physicochemical properties such as high
solubility and stability plus low propensity to aggregate,
as suggested by successful biological drugs. Bispecific
antibodies have pharmacokinetic properties that are suit-
able for their intended clinical application — long serum
half-life in many cases.
Dual blockade of disease mediators with bispecific
antibodies. The pathogenesis of many human diseases
involves several mediators that function in distinct sig-
nalling pathways or that have redundant roles in the
same pathway. Simultaneous blockade of several different
disease mediators may lead to greater therapeutic effi-
cacy and/or benefit more patients than targeting indi-
vidual disease mediators133
. This dual targeting concept
was initially explored in oncology with bispecific anti-
bodies that bind vEGFR1 and vEGFR2 (REF. 138), EGFR
and insulin-like growth factor receptor 1 (iGFR1)139,140
.
More recently the human epidermal growth factor
receptor 2 (HER2)-specific antibody trastuzumab was
engineered using phage display technology to create a
‘two-in-one’ antibody (FIG. 3c) that can bind either HER2
or vEGF with high affinity; it was shown to inhibit both
HER2- and vEGF-mediated cell proliferation in vitro
and tumour progression in mouse models141
.
Although this combinatorial strategy is currently
being pursued in many cancers, combinatorial therapy
has been limited, to date, by significant safety issues when
used in inflammatory diseases. Combination of il-1β
and TNF blockade by anakinra and etanercept, respec-
tively, as well as dual targeting of TNF (by etanercept)
and CD80 and CD86 (by abatacept (orencia; Bristol-
Myers Squibb)) did not result in any significant additive
or synergistic clinical effects, but rather in a substantial
increase in infectious complications142,143
. Nonetheless,
dual targeting of more appropriate pathways may pro-
vide greater efficacy without significantly increased
toxicities. in turn, bispecific antibodies seem preferable
in these cases, as opposed to developing two unapproved
therapeutic antibodies, for reasons that include lower
costs, simpler clinical drug development schemes and
more traditional regulatory pathways.
The pro-inflammatory cytokines il-1α and il-1β
have key and seemingly redundant roles in the patho-
genesis of various diseases144
. indeed, blockade of both
il-1α and il-1β in a mouse model of collagen-induced
arthritis using a combination of monospecific neutraliz-
ing antibodies was more efficacious than either individual
antibody145,146
. These antibody combination studies pro-
vide a strong rationale for developing therapeutic agents
thatneutralizeboth il-1αandil-1β,ideallywithoutinter-
fering with endogenous il-1Ra147,148
. identifying a single
antibody that binds to both il-1α and il-1β has proved
elusive, as these cytokines share only ~20% identity. By
contrast, a new bispecific tetravalent igG-like molecule
known as dual variable domain igG (DvD-igG; FIG. 3c)
was generated that neutralizes both mouse il-1α and
il-1β and has similar in vivo potency to a combination of
the two monospecific igGs from which it was derived148
.
ADvD-igGbindinghumanil-1αandil-1βwasthencre-
ated, allowing clinical evaluation147
. A further DvD-igG,
comprising il-12-specific and il-18-specific antibodies,
suggests that this format may be broadly applicable148
.
DvD-igG can match or at least approach the bind-
ing affinity of the parent antibodies, although some
optimization may be required; for example, antibody
selection, order of antigen-binding variable domains
and design of linkers connecting variable domains147,148
.
DvD-igG can be generated with many attributes condu-
cive to drug development, including robust expression
in mammalian cells and facile purification by protein A
chromatography, as well as favourable physicochemical
and pharmacokinetic properties147,148
.
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10

Functional blockade of additional cytokine pairs has
been achieved with several different bispecific antibody
formats. T helper 17 (TH
17) cells may be associated with
the pathogenesis of some allergic diseases such as allergic
contact dermatitis, atopic dermatitis and asthma149
. Dual
targeting of il-23, a TH
17 cell growth and survival factor,
and il-17A, a pro-inflammatory cytokine produced by
TH
17 cells, may be more effective than blockade of either
il-23 or il-17A alone in inflammatory diseases involv-
ing TH
17 cells150
. Selection from phage display libraries
was used to generate and optimize stable and high affin-
ity scFv neutralizing either il-17A or il-23. Bispecific
antibodies binding to both il-17A and il-23 were then
generated by incorporating these monospecific scFv
into a several previously described bispecific antibody
formats150
: tandem scFv–Fc151
, scFv–Fc–scFv152
and igG–
scFv153
(FIG. 3). Bispecific antibodies were identified that
potently neutralize both il-17A and il-23 with good
stability and pharmacokinetic properties. These findings
support the design principle of constructing bispecific
antibodies from stable monomeric building blocks154
.
Concluding remarks
Much has been learnt from the clinical experiences of
the first generation of therapeutic antibodies. We have
reviewed here some of the insights derived from the
first decade of therapeutic antibodies in autoimmunity
and inflammation as well as a subset of the technologi-
cal advances that will enhance the capabilities of thera-
peutic antibodies. The experiences from this second
generation of therapeutic antibodies will undoubtedly
inform our next generation of therapeutic antibodies.
Although not discussed in this Review, there is much to
be learnt about how antibody delivery, as well as other
factors, can alter the distribution of antibody in the body.
These include oral delivery of antibodies, development
of sustained release platforms that can be used in the eye
to avoid frequent intravitreous injections and improved
access of therapeutic antibodies into the brain. Although
rituximab and alemtuzumab have shown preliminary
evidence of efficacy in the relapsing–remitting form of
multiple sclerosis, steady state cerebrospinal fluid levels
of rituximab in relapsing–remitting multiple sclerosis are
low (<0.25 to <0.1% of serum levels)155
. A better under-
standing of the factors that regulate the dynamics of
antibody circulation between the systemic and central
nervous system compartments may allow the design of
therapeutic antibodies with improved access to cerebral
spinal fluid and application of therapeutic antibodies in a
wide range of neurological diseases. We are merely at the
dawn of antibody-based therapy and have much to look
forward to as we begin this exciting journey.
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