Ref 5

British Journal of DOI:10.1111/j.1476-5381.2010.01183.
BJP Pharmacology
www.brjpharmacol.org
REVIEW bph_1183 1470..1484

Correspondence
Matthew A Sleeman,
MedImmune Ltd, Milstein
Developing the next Building, Granta Park, Cambridge

CB21 6GH, UK. E-mail:
sleemanm@medimmune.com
generation of monoclonal ----------------------------------------------------------------
Keywords
antibody; rheumatoid arthritis;
antibodies for the treatment cytokine; engineering; phage

display; transgenic mice; pattern
recognition; toll-like receptors;
of rheumatoid arthritis biologics

----------------------------------------------------------------
Received
22 July 2010
Jamie Campbell1, David Lowe2 and Matthew A Sleeman1
Revised
1 22 October 2010
Department of Respiratory, Inflammation and Autoimmunity, MedImmune Ltd, Cambridge, UK,
2
Accepted
and Lead Generation, MedImmune Ltd, Cambridge, UK 30 November 2010
Rheumatoid arthritis is one of the commonest autoimmune diseases affecting 0.8% of the population. Over the last decade
the treatment of this chronic disease has been revolutionized by the use of monoclonal antibodies and fusion proteins,
targeting molecules like tumour necrosis factor alpha. Nevertheless, approximately one-third of subjects fail to respond to
these therapies and therefore significant unmet medical need remains. Following a decade of use, clinical, government and
regulatory agency expectations have changed for new antibodies therapies entering this highly competitive area. In this
review, we discuss the current advances being made in antibody engineering and how they are being considered and used in
the development of the next generation of antibodies to meet future expectations of healthcare providers, physicians and
patients. Moreover, we discuss how pattern recognition receptors may provide new antibody tractable targets that may break
the cycle of autoimmunity in rheumatoid arthritis.
Abbreviations
ACPA, anti-citrullinated peptide antibodies; ADCC, antibody-dependent cellular cytotoxicity; AE, adverse event; CDR,
complementarity determining region; CH, constant region heavy chain; CH1, constant region heavy chain domain 1;
CH2, constant region heavy chain domain 2; CH3, constant region heavy chain domain 3; CIA, collagen induced
arthritis; CL, constant region light chain; DAS, disease activity score; Fab, fragment antigen binding region; Fc, fragment
crystallizable region; FcRn, neonatal Fc receptor; FDA, Food and Drug Administration; HLA, human leukocyte antigen;
Ig, immunoglobulin; IL, interleukin; LPS, lipopolysaccharide (endotoxin); NLR, NOD-like receptors; PAMP, pathogen-
associated molecular patterns; PEG, polyethylene glycol; PK, pharmacokinetics; PRR, pattern recognition receptor; RA,
rheumatoid arthritis; s.c., subcutaneous; scFv, single chain variable fragment; SCW, streptococcal cell wall; TACI,
transmembrane activator and calcium modulating and cyclophilin ligand interactor; TH17, T Helper 17 cells; TLR,
toll-like receptor; TNF, tumour necrosis factor; VH, variable heavy chain; Vk, kappa light chain; VL, variable light chain
History of monoclonal antibodies in 20 per 100 000 person-year and a prevalence of more than
500 per 100 000. In the UK, approximately 387 000 people
the treatment of rheumatoid arthritis have been diagnosed with RA accounting for 0.8% of the
adult population (Symmons, 2002). Likewise in the USA, RA
Rheumatoid arthritis (RA) is a chronic systemic autoimmune affects more than 2 million people. Whilst the exact patho-
disease characterized by inflammation of the synovial joints, genesis of RA remains unclear, significant strides have been
which can result in pain, swelling and joint damage. It is one made over the last 20 to 30 years in our understanding and
of the commonest autoimmune diseases with an incidence of treatment of this disease. To date it is not known what the
1470 British Journal of Pharmacology (2011) 162 1470–1484 © 2011 MedImmune Ltd
British Journal of Pharmacology © 2011 The British Pharmacological Society
Engineering novel biologics for RA
BJP
triggers are that break immune tolerance and drive autoim- anti-metabolites, alkylating agents and especially glucocorti-
munity; however, it is known that genetic association of coids were used in various combinations to manage pain and
human leukocyte antigen HLA-DR1 and HLA-DR4 infers a inflammation in this disease in an attempt to slow down
50% risk of individuals developing RA. Currently 50–80% of disease progression. Whilst these drugs provided a degree of
RA patients have the autoantibody rheumatoid factor, typi- efficacy they were often associated with significant side effects
cally IgM and IgA molecules that bind Fc fragments of IgG, adding to the burden of co-morbidities often affecting these
and/or anti-citrullinated peptide antibodies (ACPA). Research patients. As such, a clear need for targeted therapies aimed at
has shown that presence of ACPA is a good predictor of rapid the modification of the disease process with an acceptable
disease as defined by progressive joint destruction (van der safety profile in patients with RA was sought. Like the treat-
Helm-van Mil et al., 2005). Moreover, Huizinga et al. (2005) ment of most chronic and disabling diseases this is a not a new
have demonstrated a link between ACPA-positive patients concept, with the idea of a ‘magic bullet’ being proposed by
and a number HLADRB1 alleles and a common shared Paul Ehrlich over 100 years ago. Ehrlich originally postulated
epitope within these HLA alleles. This has led to the hypoth- the existence of specific receptors that bind to certain antigens
esis that citrullinated peptides, following post-translational and hypothesized that these receptors either associated with
modification of amino acids on certain proteins, possibly due cells or distributed in the blood in response to an antigen
to viral infections or local injury in healthy individuals, bind interaction (reviewed in Drews, 2004). From this idea he
to these shared epitopes on HLA alleles, resulting in the evolved the concept of specific drugs that go straight to their
breaking of tolerance and generation of autoantibodies (Hill intended target, hence the ‘magic bullet’. One of the key
et al., 2003). Obviously, this hypothesis does not fit within all breakthroughs in reducing this concept to practice came
RA, for example ACPA negative patients; however, it does much later through the pioneering work of Kohler and Mil-
begin to provide us with a model of how tolerance may stein (1975) in which they demonstrated for the first time that
be broken in the majority of subjects. Once established, B cell precursors could be immortalized through fusion with
however, a range of inflammatory, macrophages, neutrophils, mouse myeloma cells to generate stable monoclonal antibody
T/B cells and stromal cells, such as synovial fibroblasts, producing cells. The ability of administered therapeutic anti-
appear to be central to the mechanisms of joint inflammation sera to treat bacterial infections had been established in 1891
and disease progression. At the sites of joint inflammation a by Emil von Behring and Shibasaburo Kitasato, but it wasn’t
broad panel of mediators, such as tumour necrosis factor until this identification of a method for isolating monoclonal
alpha (TNFa), interleukin (IL)-6, granulocyte macrophage antibodies of a defined specificity that the potential of this
colony-stimulating factor, matrix metalloproteinases, vascu- approach as a drug option became a real possibility. Early
larendothelial growth factor, receptor activator of nuclear clinical studies in cancer paved the way with humanized
kappa-B ligand cc chemokine ligand 2 and cc chemokine antibodies like CAMPATH-1H (alemtuzumab) (Hale et al.,
ligand 5 (reviewed in Feldmann et al., 1996), are all elevated 1988) demonstrating that by targeting an overexpressed spe-
contributing to increased inflammation, angiogenesis (due to cific antigen, such as CD52, one could provide clinical benefit.
the hypoxic nature of the inflammation) and joint damage By the mid 1980s early 1990s basic research in RA had
thus highlighting the extremely complex nature of the begun to identify TNFa (Buchan et al., 1988; Brennan et al.,
disease. This persistent inflammation also results in systemic 1989; Feldmann et al., 1990; Deleuran et al., 1992; Thorbecke
disease, with elevated acute phase proteins and increased et al., 1992; Williams et al., 1992) as potentially one of the key
circulating levels of cytokines such as IL-6 which contributes targets contributing to disease in RA. In vitro studies using
to the overall manifestation and morbidity of this condition. human primary synovial membrane cultures from RA joints
Due to the chronic and debilitating nature of the disease identified that these cells spontaneously produced TNFa and
this condition is associated with a lower quality of life, dis- had high levels of IL-1 (Buchan et al., 1988; Brennan et al.,
ability, premature death and unemployment. Consequently, 1989; Deleuran et al., 1992). Furthermore, it was shown that
the human, economic and healthcare costs are considerable. IL-1 production was TNFa-dependent by blocking IL-1 release
For example the direct real life costs for treating patients with using an anti-TNFa antibody in synovial membrane cultures
RA in France range from €6450 to €19618 per person, with the (Brennan et al., 1989). Since IL-1 was thought to be of signifi-
overall cost to the French National Health Authority of €222 cant importance in inflammatory arthritis, blocking its pro-
million (Maravic, 2010). Approximately 84% of these costs duction using neutralizing anti-TNFa antibodies could be of
are attributable to biologic therapies such as anti-TNFs. More- clinical benefit. In vivo evidence which strengthened the
over, within the UK the indirect and direct costs of RA are hypothesis came from studies which investigated the effect of
estimated at between £3.8 and £4.75 billion per year (Pugner TNFa in a mouse model of RA, collagen induced arthritis
et al., 2000). It has also been shown that these indirect costs (CIA). Repeated administration of recombinant TNFa
increase with disease severity, from approximately €5000 for increased severity and incidence of disease relative to control
minimal disease activity up to €20 000 in patients with severe mice (Thorbecke et al., 1992). Conversely intraperitoneal
disease (Lajas et al., 2003; Kobelt et al., 2008). To put these administration of a hamster anti-mouse TNFa monoclonal
figures in context the total economic cost to society in Europe antibody (15 mg·kg-1) reduced paw swelling and clinical score
and the USA has been estimated to be €45.3 and €41.6 billion when administered prophylactically and also therapeutically
respectively (Lundkvist et al., 2008). (Thorbecke et al., 1992; Williams et al., 1992). This effect was
Prior to the use of monoclonal antibodies and fusion found to be dose-dependent as administration of the antibody
proteins in RA, a large number of small molecules ranging at 2.5 mg·kg-1 had little effect on paw swelling or proportion of
from broad spectrum anti-inflammatories, such as non- joints with severe lesions (Williams et al., 1992). In summary,
steroidal anti-inflammatories and analgesics to anti-malarials, these studies had shown that TNF blockade reduces IL-1
British Journal of Pharmacology (2011) 162 1470–1484 1471

J Campbell et al.
BJP
production in vitro in human primary synovial cultures and in clinical studies and RA registries clearly demonstrates that
mouse models of arthritis would reduce disease pathology. patients on TNF therapy are at a higher risk for bacterial,
Based on these observations Feldmann and Maini took fungal or viral infections (Hyrich et al., 2008; Leombruno
the step to evaluate infliximab (cA2), a chimaeric anti-TNF et al., 2009; Strangfeld et al., 2009). In addition to this the
antibody, in a small cohort of RA patients (Elliot et al., 1993). debate also continues that inhibition of TNF may also lead to
Following single infusions of infliximab at 20 mg·kg-1 they an increased chance of malignancy (Symmons and Silman,
were able to show in this small cohort of subjects, a signifi- 2004); however, the data to date is equivocal. Currently all
cant reduction in signs and symptoms of the disease. Larger approved biologics in RA (except abatacept which has an
double-blinded placebo-controlled clinical trials confirmed infection risk warning in the label), carry black box warnings
that infliximab could produce response rates in patients with highlighting serious infections and in some cases malignan-
RA superior to methotrexate alone and could have a signifi- cies as key risks factors. As these new therapies are recombi-
cant effect on disease progression (Maini et al., 1999). Con- nant proteins one of the key factors in drug effectiveness is
sequently, infliximab was the first monoclonal antibody to be dependent on whether the patient generates an immune
approved for the treatment of RA patients with an inadequate response and human anti-human antibodies to the protein.
response to methotrexate while maintaining a generally For example, it is reported that approximately 10% of
acceptable safety profile. This breakthrough in the treatment patients treated with infliximab were positive for autoanti-
of RA with targeted biologic therapies to TNF has become one bodies to infliximab. Furthermore, antibody-positive patients
of the largest biologics success stories, with a market now were more likely to have higher rates of drug clearance,
estimated at greater than $8 billion per annum globally and reduced efficacy and experience infusion reactions.
five anti-TNF molecules (infliximab, etanercept, adalimumab, Therefore, considerable opportunity still remains to iden-
golimumab and certolizumab) approved either in the USA or tify new, safer, less immunogenic and improved therapies to
globally. Moreover, the confidence gained from using anti- treat RA. In spite of this unmet medical need in RA there is
TNF therapies as a first line biologic has allowed rheumatolo- also a clear recognition by the pharmaceutical industry that
gists to evaluate and successfully gain approval for a number the expectations of governmental agencies (www.nice.org.uk,
of other antibody or fusion protein-based therapies such as CG79 Rheumatoid Arthritis: full guideline), regulatory
rituximab (B cell depletion therapy), abatacept (inhibition of authorities, pharmaceutical competition, clinicians and
co-stimulation) and tocilizumab (anti-IL-6 receptor therapy). patients, as a consequence of a decade of successful use of
In conjunction with standard of care with small molecules, anti-TNF therapy, and more recently B cell depletion, inhibi-
such as methotrexate and glucocorticoids, this has given tion of co-stimulation and IL-6 receptor pathway discussed
rheumatologists a greater degree of clinical options to later in this article, has considerably changed market expec-
improve treatment of their patients and attain levels of effi- tations for new antibody therapies in this disease. Therefore,
cacy not previously achieved. This is exemplified by the fact these various healthcare bodies are seeking new therapies
that more than 1 million patients have been treated with with greater efficacy, improved safety characteristics, reduced
anti-TNF therapies. In trials irrespective of method of TNF costs and improved dosing intervals. In this review we
inhibition, the combination of methotrexate with anti-TNF outline some of the novel approaches being considered to
routinely achieve significant efficacy with 62% and 69% of meet future expectations for the treatment of RA and describe
patients achieving a 50% improvement in their disease at 1 how these pressures have also paralleled an evolution in how
year (Klareskog et al., 2004; Breedveld et al., 2006). Further- monoclonal antibodies are engineered and developed.
more, radiographic progression was also effectively stopped
in the majority of RA patients treated in combination. For
example in the TEMPO study (Klareskog et al., 2004) the Antibody structure
mean change in radiographic progression, as defined by total
sharp score, at week 52 was -0.5 (-1.5 to 0.0, 95% confidence The basic unit of all antibodies is the immunoglobulin (Ig)
interval) in the combination-treated group when compared molecule, a disulphide bond-linked tetramer, comprising two
with 2.8 (1.08 to 4.51, 95% confidence interval) in the meth- identical large chains of approximately 50 kDa in size, called
otrexate alone group. heavy chains, and two identical smaller chains of approxi-
Whilst anti-TNF and additional biologics therapies have mately 25 kDa in size, called light chains (Figure 1). Each
clearly revolutionized the treatment of RA, and arguably the heavy chain is made up of four protein domains, a variable
use of biologics in the pharmacopeia, there is still a signifi- (VH) domain which is directly involved in antigen binding
cant need for new therapies that provide additional benefit, and thus is specific for a given antibody, along with three
since nearly one-third of patients receiving anti-TNF therapy constant (CH) domains which are common to a specific anti-
fail to respond to the initial treatment. Furthermore, many body class and are involved in antibody functions such as
patients discontinue TNF because of adverse events (AE) or cytotoxicity (MacLennan et al., 1970), complement fixation
the development of a secondary resistance and a gradual loss (reviewed in Raghavan and Bjorkman, 1996) and serum
of effectiveness to these agents (Finckh et al., 2006) due in maintenance (reviewed in Ghetie and Ward, 2000). Similarly,
part to immunogenicity. One of the most important AEs each light chain is made up of two domains, a variable (VL)
associated with the use of TNF therapy is the increased risk of domain which together with the VH domain comprises the
infection. Screening, by chest X-ray, skin testing or whole antigen binding pocket of an antibody and a single constant
blood assays, is standard practice to reduce the chance of domain (CL) which is also common to all antibodies of a
reactivation of latent mycobacterium tuberculosis, a known given class and which is bound to the N-terminal CH domain
risk (Dixon et al., 2010). Moreover, meta-analysis from (called CH1) via a disulphide bond formed between cysteines
1472 British Journal of Pharmacology (2011) 162 1470–1484

BJP
VL
Fv
CL
VH
CH1
CH2
CH3
Murine Human
Chimaeric Humanized
Figure 1
Evolution of therapeutic antibody development. The original monoclonal antibodies such as Orthoclone OKT3 were murine proteins. Later
developments include chimaeric antibodies such as infliximab, which is made up of a murine Fab genetically fused to a human Fc region as well
as humanized antibodies, whereby murine complementarity determining regions (CDRs) are grafted onto human IgG frameworks. More recently
human antibodies, such as adalimumab have been developed using phage display or transgenic methods.
in these two domains. This symmetrical modular structure of cytes, it was quickly shown that the new monoclonal
Ig molecules has been central to the key developments in antibodies could provide a degree of clinical benefit (Hale
antibody engineering that have revolutionized the field over et al., 1988). Initial studies proved challenging due to the
the last 25 years, and that has been the driving force for the onset of human anti-mouse neutralizing antibodies due to
recent expansion in the development and commercialization the recognition of the non-self murine protein. If one could
of antibody therapeutics. overcome this immunogenic response it was hypothesized
that the use of monoclonal antibodies might have broader
utility in autoimmunity (Herzog et al., 1987; Horneff et al.,
Engineering monoclonal antibodies 1991a,b; Wendling et al., 1993).
By using the newly emerging field of molecular biology
Following Kohler and Milstein’s invention of the hybri- to re-engineer mouse antibodies with functionally equiva-
doma method for immortalizing immunized B lympho- lent human amino acids, initially by replacing the murine

J Campbell et al.
BJP
Ig constant domains with human equivalents (Figure 1), the mouse and human VH and VL genes (Taylor et al., 1992; 1994),
overall immunogenicity of the molecule could be reduced ultimately culminating in the generation of mice with four
(Knight et al., 1995). This led to a generation of so-called germ line modifications, two targeted disruptions of endog-
‘chimaeric antibodies’ (Boulianne et al., 1984; Morrison enous mouse heavy and k light chain genes and the intro-
et al., 1984; Neuberger et al., 1985) with a number of these duction of human heavy and k light chain transgenes (Green
types of antibodies, such as infliximab and rituximab et al., 1994; Lonberg et al., 1994). Therefore, by combining
gaining successful approval in RA in 1999 and 2006 respec- early work by Kohler and Milstein (1975) with this latest
tively. Nevertheless, immunogenicity due to human anti- discovery one would be able to generate fully human VH Vk
chimaeric antibodies, hypersensitivity and consequently IgGs as drug molecules in the mouse. As with phage display,
infusion-associated reactions, as described in their black box these discoveries provided the basis for commercialization,
warning label, and in some cases a resultant lack of efficacy leading to the formation of the companies Abgenix and
remained common issues with these antibodies. A key Medarex. To date four new fully human monoclonal antibod-
breakthrough to surmount these effects was the develop- ies derived from this technology have been approved by the
ment of complementarity determining region (CDR) loop FDA, panitumab (Abgenix/Amgen), for the treatment of
grafting by Winter and colleagues (Jones et al., 1986). metastatic colorectal cancer (Jakobovits et al., 2007), goli-
This led to the generation of new so-called ‘humanized’ mumab (Medarex/Centocor) as a new subcutaneous anti-TNF
monoclonal antibodies with minimal murine amino acids therapy approved for the treatment of RA (Smolen et al.,
(Figure 1) in their sequences (Jones et al., 1986; Verhoeyen 2009), psoriatic arthritis and ankylosing spondylitis, ofatu-
and Riechmann, 1988). Currently the only humanized anti- mumab an additional fully human CD20 antibody for the
body approved for the treatment of RA is tocilizumab, an treatment of chronic lymphocytic leukaemia (Lemery et al.,
anti-IL-6R antibody (Nishimoto and Kishimoto, 2008), 2010) and ustekinumab for plaque psoriasis (Tan et al., 2010).
although a number of these formats have been approved in Both phage display and transgenic mice are now well vali-
oncology (trastuzumab, bevacizumab), asthma (omali- dated and robust technologies for the generation of high
zumab) and multiple sclerosis (natalizumab). potency human antibodies and consequently most of the
The generation of recombinant antibodies of fully human growing number of antibodies entering clinical trials are now
origin was realized with the development of two alternative completely human in origin (Reichert et al., 2005).
technologies; phage display of human antibody fragments
(McCafferty et al., 1990), and transgenic mice expressing
human Ig genes (Green et al., 1994; Lonberg et al., 1994). Antibody fragments as therapies
One key limitation of monoclonal antibody therapies (inflix-
imab, adalimumab, rituximab and golimumab) for RA is their
Phage display of antibody fragments costly manufacture compared with small molecule-based
pharmaceuticals, due partly to their production via mamma-
Using filamentous bacteriophage displaying an antibody frag-
lian cell fermentation. There is therefore much activity
ment comprising the VH and VL domains linked together with
towards moving to cheaper production methods, such as
a flexible amino acid linker sequence (known as a single
bacterial expression of antibodies. The most advanced
chain variable fragment or scFv) as a fusion with one of its
product based on these new antibody formats is certolizumab
coat proteins, McCafferty et al. (1990) demonstrated that spe-
pegol (Figure 2), which has been approved for the treatment
cific phage could be recovered on the basis of antigen binding
for RA and Crohn’s disease in the USA (certolizumab has not
in vitro. Using this approach large libraries of human VH and
been approved for the treatment of Crohn’s disease by the
VL gene sequences were prepared from peripheral blood lym-
European Medicines Agency). By utilizing only the Fab’ frag-
phocytes from naive non-immunized donors. Using such
ment of a humanized antibody to TNFa, this molecule can be
libraries, large panels of scFv could be rapidly enriched for
expressed in Escherichia coli obviating the need for mamma-
antigen binding and then screened for functional activity, in
lian cell-based expression. Expression of full Ig molecules is
an analogous fashion to small molecule chemical libraries.
more difficult in bacterial systems, due to the oxidizing envi-
Using this approach Cambridge Antibody Technology (now
ronment of the bacterial periplasm, which is not readily con-
MedImmune Ltd) and Abbott Laboratories developed the first
ducive to the formation of the multiple cysteine bonds that
completely human monoclonal antibody, adalimumab, to
order the tertiary structure of the molecule. Additionally,
gain approval by the Food and Drug Administration (FDA)
post-translational modifications, notably glycosylation, are
in 2003 for the treatment of patients with moderate to
not readily achieved in bacterial host systems.
severe RA.
Certolizumab, being a recombinant Fab’ fragment, is
claimed to exhibit high expression by E. coli fermentation.
The lack of an Fc region (Ig CH2 and CH3 domains), which
Transgenic mice maintains serum antibody levels via interaction with the
neonatal Fc receptor (FcRn) (Roopenian and Akilesh, 2007),
In parallel with this development other groups employed the results in Fab fragments having serum half-lives of less than
use of transgenic mice capable of producing fully human Igs, 24 h (Weir et al., 2002). Therefore, in order to maintain an
this concept first being hypothesized as early as the mid economical and practical dosing regime, certolizumab
1980s (Alt et al., 1985). Over the next decade a range of needed to be modified to extend the serum half-life. To that
transgenic mice were developed containing mixtures of end a 40 kDa polyethylene glycol (PEG) moiety was coupled

BJP
Murine mAb
HTNF40 CDR’s
VL
Human
VH Addition of
IgG1Fab 40 kDa PEG
via free cysteine
CL CH1
Free Cys
Certolizumab
pegol
Figure 2
Molecular structure of certolizumab pegol. CDR, complementarity determining region; PEG, polyethylene glycol.
to the Fab’ fragment via a free (unbonded) cysteine left from Affinity maturation
the hinge region of the original Ig sequence, resulting in a
molecule with significantly extended serum persistence (Weir A fundamental principal of in vitro protein engineering plat-
et al., 2002). forms, such as phage, yeast or ribosome display is the ability
Therefore, over the last decade of treatment of RA we have to generate large numbers of variants containing mutations
already seen how commercial competition, innovation and in controllable positions in the protein sequence, coupled
clinical requirements have applied evolutionary pressure on with the ability to select for those variants with a defined
research and development strategies for monoclonal anti- characteristic, such as affinity for a given target (reviewed in
body therapies for the treatment of RA. As a consequence of Carter, 2006; Dufner et al., 2006). A wide range of mutagen-
this, we have a broad range of biologics and antibodies esis strategies are available, ranging from random approaches,
approved for the treatment of RA (Table 1). such as error-prone PCR (Hawkins et al., 1992) or DNA
shuffling (Stemmer, 1994) to more targeted approaches,
whereby one or more of the CDR loops is mutated (recent
Targeting the next generation of examples including Steidl et al., 2008; Gerhardt et al., 2009).
therapies in rheumatoid arthritis Variants with improved binding kinetics can be isolated by
tailoring the selection conditions such that they are prefer-
Whilst significant unmet medical need remains within RA, entially enriched, for example by lowering the concentration
there is also a realization by governments, healthcare provid- of the antigen (Hawkins et al., 1992; Schier et al., 1996), or
ers, regulators and patients that new therapies must not only increasing the time of incubation of with antigen (e.g. Boder
tackle the signs and symptoms of the disease, but they must et al., 2000). Using such approaches has led to many
also be easily delivered and cost-effective. Moreover, they must examples of in vitro matured antibodies with affinities beyond
clearly demonstrate additional benefit over existing therapy to those naturally found in the immune response, with several
justify their use. As a consequence of their plasticity, antibod- examples exhibiting equilibrium dissociation constants in
ies make ideal molecules to ‘tailor’ these drugs to very specific the femtomolar range (Boder et al., 2000; Steidl et al., 2008).
requirements, that is, protein therapeutics by design.
As discussed, we have already witnessed the second gen-
eration of anti-TNF therapy, such as golimumab (Centocor), a Antibody pharmacokinetics
subcutaneously delivered anti-TNF to overcome the short
comings of the companies original chimaeric iv formulated As noted above, in vitro affinity maturation strategies have
product (infliximab; Centocor). The ability of companies to resulted in antibodies with extremely strong affinities for
drive new antibody therapies with improved bioavailability their given antigen, but to understand whether this increase
has been realized by a robust understanding of human IgG will provide clinical benefit, biotech and pharmaceutical
pharmacokinetics (PK) (Ternant and Paintaud, 2005) and our companies increasingly use theoretical models of antibody
ability to increase the strength of the antibody antigen inter- PK (Agoram, 2009). The strength that can be attributed to
action through mutation of the variable regions and selection these models is largely based on the predictable nature of
for higher affinity variants, in a process akin to affinity matu- antibody PK. The half-life of human IgG is typically between
ration in vivo. 14 and 21 days primarily due to the antibodies pH-dependent

BJP
Table 1
J Campbell et al.
Currently approved antibody and antibody based therapies for the treatment of subjects with rheumatoid arthritis
Date of approval
Generic Target Route of for rheumatoid
name antigen Antibody format administration/frequency Company arthritis
Infliximab TNFa Chimaeric IgG1 i.v. infusion/3 mg·kg-1 every 8 weeks Centocor Ortho Biotech Inc. 1 Apr 1999†
Etanercept* TNFa Soluble fusion protein of p75 extra s.c. dose/50 mg weekly Amgen/Pfizer (formerly 2 Nov 1998
cellular domain + IgG1 Fc domain Immunex/Wyeth)

Adalimumab TNFa Human IgG1 s.c. dose/40 mg eow Abbott Laboratories 31 Dec 2002
Golimumab TNFa Human IgG1 s.c. dose/50 mg monthly Centocor Ortho Biotech Inc. 29 Apr 2009
Certolizumab TNFa Pegylated Human Fab’2 s.c. dose/400 mg weeks 0, 2 and 4 followed by 200 mg UCB Inc. 18 Nov 2009‡
pegol eow
Rituximab CD20 Chimaeric IgG1 i.v. infusion/2 ¥ 1000 mg 2 weeks apart. Subsequent Genentech Inc./Roche/Biogen Idec 28 Feb 2006§
courses every 24 weeks or based on clinical evaluation
Tocilizumab IL-6R Humanized IgG1 i.v. infusion/4 mg·kg-1 monthly or up to 8 mg·kg-1 Genentech Inc./Roche 8 Jan 2010
monthly based on clinical response
Abatacept* CD80 & Soluble fusion protein of CTLA-4 + i.v. infusion/500 mg (<60 kg), 750 mg (60 to 100 kg), Bristol Myers Squibb/Biogen Idec 23 Dec 2005
CD86 IgG1 Fc domain 1000 mg (>100 kg) dosed at weeks 0, 2 and 4 and
then monthly thereafter
*Both molecules are technically not monoclonal antibodies; however, as they include the Fc domain of IgG1 and have been approved in the treatment of rheumatoid arthritis these
molecules have been included.
†
Infliximab was first approved by the FDA on 28 August 1998 for the treatment of moderate to severely active Crohn’s disease and the treatment of fistulizing Crohn’s disease.
‡
Certolizumab pegol was first approved by the FDA on 22 April 2008 for reducing the signs and symptoms of Crohn’s disease and maintaining clinical response in adult patients with
moderately to severely active disease.
§
Rituximab was first approved by the FDA on 26 November 1997 for the treatment of patients with relapsed or refractory low-grade or follicular, B cell non-Hodgkin lymphoma.
BJP
Serum, pH7.4
Cell
Membrane
Uptake
Release
Endothelial
Cell
Endosome
pH=6
IgG binds FcRn
Recycling of bound IgG
Unbound IgG = Antibody

degraded in
lysosome
= FcRn receptor
Figure 3
Theoretical model of neonatal Fc receptor (FcRn)-based IgG recycling.
interaction with the FcRn and its molecular weight studies are underway (clinical trial.gov, NCT00578682) in
(~150 kDa) preventing rapid clearance through the renal fil- healthy volunteers for an anti-respiratory syncytial virus anti-
tration. Free IgG, that is, not bound to antigen, in the circu- body with extended half-life. Nevertheless, preclinical studies
lation is passively taken up by endothelial cells through have been reported with an anti-IL-6 antibody subcutane-
endocytosis and compartmentalized within cells in endo- ously delivered antibody with extended half-life (Moisan
somes. As the pH drops from pH 7.4 to pH 6 within the et al., 2009) as a potential therapy for the treatment of
endosome the free IgGs bind in a pH-dependent manner to inflammation, autoimmune diseases and cancer.
FcRn on the endosomal membrane protecting those antibod-
ies from degradation through lytic vesicles. As these endo-
somes are recycled back to the cell’s plasma membrane the Bispecifics
pH then is restored to neutral, its affinity for FcRn reduces
and the antibody is released back into circulation (Figure 3). The key strength of monoclonal antibodies is their high
Within RA the utility of infrequent dosing for subcutane- specificity for their target molecule. However, for complex
ous formulations has been adopted by clinicians and patients diseases like RA it is perhaps unlikely that any one single
to reduce the number of injections with s.c. dosing gradually cytokine or molecule is responsible for driving the pathology.
moving from weekly dosing (etanercept), to once every 2 Therefore, to potentially increase the efficacy of new thera-
weeks (adalimumab) and most recently, with golimumab, to pies in RA with antibodies, one may need to consider
monthly dosing. Therefore, there is clear evidence of a need targeting multiple mechanisms. Due to the high cost of
for reduced dosing intervals. One way this is being considered manufacture of these agents, dosing two independent anti-
is through pioneering work by Dall’Acqua et al. (2006). They bodies is not currently a viable option. To overcome this
hypothesized that by increasing an antibody’s binding affin- hurdle, many groups have begun to consider ways in which
ity to FcRn then theoretically the serum half-life should be more than one pathway can be targeted at the same time with
extended. By mutating the Fc domain of human IgG1 they a single antibody by generating molecules that are bi- or
identified three residues M252Y/S254T/T256E that increased multispecific (Wu et al., 2007; Dimasi et al., 2009). To date,
the pH-dependent affinity of Fc 10-fold from 2249 ⫾ 53 nM only one bispecific molecule has been approved for therapeu-
to 210 ⫾ 56 nM for FcRn and consequently extended the tic use, catumaxomab, a bispecific chimaeric rat/mouse anti-
antibody’s half-life (T1/2) three- to fourfold in cynomolgus body targeting EpCAM and CD3, which has been approved
monkeys from 5.7 ⫾ 1.4 days to 21.2 ⫾ 9.1 days in non- for treatment of malignant ascites in Europe (Fresenius
human primates. Likewise, more recently additional residues Biotech/TRION Pharma). However, approaches such as this
have been identified (Yeung et al., 2009; Zalevsky et al., 2010) are showing extremely promising preclinical findings in
that can also extend half-life. If these in vivo studies allometri- many different disease indications.
cally scale to man, then it is possible that we could soon see Whilst targeting multiple pathways seems like an obvious
anti-cytokine therapies with once quarterly dosing intervals. next step, identifying the most appropriate partnership is
As yet no data has been reported in humans although clinical challenging as it assumes that both the pharmacodynamics

J Campbell et al.
BJP
and PK are identical for each arm of the antibody, something for both non-Hodgkin lymphoma and RA. However, as rit-
that is not necessarily the case. Moreover, early attempts at uximab depletes greater than 90% of peripheral blood B
using two biologics anti-inflammatories in RA, for example cells in RA, it is not clear how improvements in ADCC will
anakinra + etanercept, resulted in a significant increase in ultimately lead to significant improvements in the clinical
incidental infections that was greater than standard of care response. Alternative approaches to improve efficacy in RA
(Genovese et al., 2004). Thus targeting multiple pathways by B cell depletion have targeted different B cell antigens
may also increase side effects and in some way begin to such as CD19 (CLB-CD19, MEDI-551) and CD22 (Epratu-
resemble the side effect profiles typically associated with the zumab) which are more broadly expressed in the B cell
development of new small molecule compounds. lineage. Recently, an afucosylated CD19 antibody (MEDI-
551) with enhanced effector function has started a Phase I
clinical trial for the treatment of systemic sclerosis (clinical-
Increased effector function trials.gov, NCT00946699), and therefore it will be interest-
ing to determine whether targeting CD19 demonstrates any
Whilst anti-TNF therapies have revolutionized the treatment significant differences in efficacy or safety over therapies
of RA, one criticism of these agents is that they do not such as rituximab.
directly address the key question of autoimmunity and only
target a downstream effector of inflammation, rather than
the core cause of the disease. Professor J. Edwards hypoth- Autoimmunity and pattern recognition
esized that to tackle autoimmunity one must first address the
source of the autoantibodies which stimulate the perpetua- As discussed previously, targeting TNF was and still remains,
tion of an inflammatory response, and therefore target those a major breakthrough in the treatment of RA. However, over
self-perpetuating B cells responsible for the production of time it has become apparent that many individuals do not
these autoantibodies (Edwards and Cambridge, 1998). Using respond to these therapies or respond in such a minor way to
rituximab (a B cell depleting therapy then licensed for the provide only a modicum of clinically measurable benefit that
treatment of non-Hodgkin lymphoma) in patients with RA, does not translate into a meaningful improvement in their
this group demonstrated profound and lasting responses for condition. Consequently, new therapies continue to be tri-
at least 6 months after a single treatment cycle of two 1 g alled in RA. New potential approaches currently in develop-
infusions in five patients. This result was later confirmed in a ment include targeting the macrophages and granulocyte
randomized Phase IIa study involving 161 patients with macrophage colony-stimulating factor pathway (MedIm-
active RA where rituximab provided significant clinical mune Ltd, Nycomed, Morphosys), IL-17A pathway (Ely Lilly,
benefit and that this response was associated with a signifi- Novartis, Amgen) and additional anti-B cell therapies, anti-B
cant decrease in autoantibodies such as rheumatoid factor lymphocyte-stimulating factor (HGS/GSK), anti-CD22 (UCB),
and anti-cyclic citrullinated peptide (Edwards and Cam- TACI-Ig (Zymogenetics/Serono) and anti-CD20 approaches
bridge, 2001; Leandro et al., 2002; Edwards et al., 2004). The (GSK/Genmab/trubion). Many of these therapies are designed
most interesting observation was that the response was sus- to specifically address the signs and symptoms of the disease,
tained for 7–33 months after a single treatment cycle and that but it has also been shown that treating patients early enough
the failure to respond or disease relapse was often associated in their disease progression can result in attainable clinical
with insufficient depletion of autoantibody producing B cells remission within RA (Quinn et al., 2005). Therefore, over the
or return of circulating B cells, respectively, thus clearly estab- next decade a greater emphasis is likely to be placed on
lishing a link between the B cell and disease pathology (Cam- therapies that also have the potential to induce remission
bridge et al., 2006; Cohen et al., 2006; Edwards et al., 2006; (Smolen et al., 2010).
Leandro et al., 2006; Popa et al., 2007). Consequently, the The theory that an unresolved bacterial or viral infection
chimaeric antibody rituximab was approved for the treat- provides the initial trigger for RA has been hypothesized for
ment of RA in 2006. This approach demonstrated that by many years (Gibson, 1928; Cecil and Nicholls, 1931). As our
identifying a specific target antigen one could remove a spe- understanding of the immune response has advanced this
cific subset of cells and provide clinical benefit. Thus a further hypothesis may now be revised in the context of pattern
two CD20 B cell depleting antibodies are currently in clinical recognition receptors (PRRs). PRRs such as toll-like receptors
development (ofatumumab and TRU-015). (TLRs) and NOD-like receptors (NLR) are expressed on a
With the success of rituximab a number of approaches variety of cells of both myeloid and non-myeloid lineage. For
are being considered to enhance effector function to either the purposes of this review we will concentrate on TLRs as
improve efficacy and/or increase the duration of pharmaco- NLRs are intracellularly expressed and are therefore not yet
dynamic activity, resulting in infrequent dosing regimens. an antibody tractable target class. Since their identification in
Engineering the Fc domain of monoclonal antibodies has 1997 (Medzhitov et al., 1997), TLRs have become integral to
resulted in a number of antibody variants with enhanced our understanding of innate (Janeway and Medzhitov, 2002)
ADCC (antibody-dependent cellular cytotoxicity) activity and to some extent adaptive immune responses (Iwasaki and
when compared to wild-type antibodies. This is typically Medzhitov, 2004; Reynolds et al., 2010). This family of PRRs
achieved by engineering amino acids in the Fc domain (of which there are now 10 human members) recognize
(Lazar et al., 2006) or by modifying the carbohydrates at the various microbial pathogen-associated molecular patterns
glycosylation site Asn297 in the Fc domain (Shinkawa et al., (PAMPs) (Medzhitov and Janeway, 1997, O’Neill et al., 2009).
2003). Thus a number of second generation CD20 B cell However, the scope of TLRs are not restricted to detecting
depleting antibodies are currently in clinical development microbes such as bacteria, fungi and viruses as numerous

BJP
endogenous ‘ligands’ have been proposed (Bauer et al., 2009; nificantly reduced in TLR4-/- and, but to a lesser extent,
McCormack et al., 2009; Drexler and Foxwell, 2010). There- TLR2-/- mice. In contrast, TNF-induced inflammation was
fore, in the context of PRRs the infection theory can be reduced to a lesser extent with TLR4-/- mice (Abdollahi-
revised thus: microbial ligands acting through PRRs induce Roodsaz et al., 2009). Therefore, it was not blockade of the
the release of inflammatory mediators, for example TNFa/ initial pro-inflammatory stimulus (i.e. IL-1) that reduced
IL-1b which cause cellular and tissue damage. This resultant inflammation but blockade of endogenously released TLR
damage induces the release or up-regulation of inflammatory ligands in response to IL-1. This is supported by the observa-
proteins some of which contain damage-associated molecular tion that TLR4-/- mice are protected in the IL-1Ra-/- spon-
patterns or ‘Danger signals’ (Matzinger, 1994) which may taneous arthritis model (Abdollahi-Roodsaz et al., 2008b).
then drive the autoimmune pathogenesis of RA by acting Interestingly, TLR2-/- mice in this model appeared to exac-
back through PRRs (van der Heijden et al., 2000; McCormack erbate in this system clearly highlighting that work is needed
et al., 2009). Therefore, targeting TLRs with neutralizing anti- to fully understand the role of these pathways in RA. In
bodies may break this inflammatory loop and could be a addition, it was also hypothesized that pattern recognition
therapeutic approach in the treatment RA. may also be involved in driving T Helper 17 (TH17) cell and
Expression of TLR2 and 4 have been confirmed in RA IL-17 release in this system, which is a known clinical target in
synovium (Radstake et al., 2004; Ospelt et al., 2008), RA RA currently under clinical investigation. This hypothesis has
peripheral blood monocytes (Iwahashi et al., 2004) and cells in part been recently substantiated in that a TH17 mouse
from RA synovial fluid (Seibl et al., 2003; Huang et al., 2007). autoimmune experimental autoimmune encephalomyelitis
Furthermore, in vitro stimulation of RA synovial cells demon- model has been shown to be TLR2-dependent (Reynolds et al.,
strates that these cells are hyper-responsive to both TLR2 and 2010). Antibody intervention studies in models of arthritis
TLR4 microbial ligands [peptidoglycan and lipopolysaccha- have not as yet been reported; however, the effects of a TLR4
ride (LPS) respectively], when compared to normal cells antagonist (B. quintana) has been evaluated in both the CIA
(Huang et al., 2007). Further evidence for the involvement of model and the IL-1Ra-/- arthritis model (Abdollahi-Roodsaz
TLRs in RA is the inhibition of spontaneous TNF production et al., 2007). It is believed that LPS from this pathogen acts as
from human synovial membrane cultures by overexpression a TLR4 antagonist by competing with other TLR4 agonists and
of dominant negative versions of TLR2/4 adaptor molecule preventing signalling through the TLR4/MD2 complex,
MyD88 and Mal (Sacre et al., 2007). Moreover, endogenous although this has as yet not been formally demonstrated. In
TLR4 ligand came from studies investigating IL-1b and TNF- these models B. quintana attentuated arthritis in both models
induced joint inflammation (Abdollahi-Roodsaz et al., 2009). whether dosed prophylactically or therapeutically and
Washouts from murine patellas stimulated with IL-1b reduced IL-1b levels in the CIA arthritic joint. This type of
induced murine TLR4 complex-dependent IL8 production approach is currently being evaluated in the clinic with a PhIII
from transfected human embryonic kidney cells. Further- study in severe sepsis (Clinicaltrials.gov NCT00334828) (Kim
more, this response was inhibited by the TLR4 antagonist et al., 2007; Park et al., 2009; Tidswell et al., 2010) but as yet
Bartonella quintana. has not been trialled in autoimmune disease.
Numerous in vivo studies have strengthened the hypoth- These above data suggest the possible involvement of the
esis of the role of pattern recognition in arthritis. Zymozan (a role of pattern recognition in driving the pathogenesis of RA.
known TLR2 ligand) induced arthritis was significantly It is of note that microbial ligands have been detected in RA
reduced in TLR2-/- mice (Frasnelli et al., 2005). In a strepto- synovial joints (van der Heijden et al., 2000) although they
coccal cell wall (SCW) induced arthritis model TLR2-/- mice have also been detected in non-arthritic joints (Schumacher
did not develop joint swelling or inhibition of cartilage syn- et al., 1999). Whatever the trigger, be it microbial PAMPs or
thesis whereas TLR4-/- mice developed joint inflammation inflammatory cytokines, the search is on to identify an endog-
similar to wild-type mice (Joosten et al., 2003). Subsequent enous ‘ligand’ which drives RA. Numerous ‘proposed’ TLR
studies which looked at both early and late time points in ligands have also been detected and there is some compelling
SCW-induced arthritis reproduced this data for the acute evidence for their involvement in the inflammation observed
phase (days 1–2) of the disease, with a clear protective effect in in RA. A by no means exhaustive list would include heat shock
TLR2-/- mice (Abdollahi-Roodsaz et al., 2008a). However, the proteins, fibronectin, hyaluronan, high-mobility group
chronic phase (day 28) joint inflammation was significantly protein-B1 (reviewed in Miyake, 2007; O’Neill, 2008; Bauer
reduced in TLR4-/- and not TLR2-/- mice. The authors sug- et al., 2009; Huang and Pope, 2009; Andersson and Harris,
gested that the shift in dependence from TLR2 to TLR4 may 2010) and tenascin C (Midwood et al., 2009; Erridge, 2010;
correlate with the shift from innate to adaptive immune Goh et al., 2010). The issue with all of these studies is the
response. This type of studies may provide an insight into the ability to convincingly show that the observed effect of the
initial events that trigger RA; however, it would be virtually ligand is not due to LPS contamination (Tsan and Gao, 2007).
impossible to treat this initial event from a pharmaceutical Despite the strong rationale for TLRs in RA, there have
perspective because it is unknown which pathogens might been no studies investigating the inhibitory effects of anti-
trigger this event or identify which proportion of subjects that bodies in this disease or in vivo models of RA. Neutralizing
would in turn be predisposed to develop RA. However, evi- anti-mouse TLR2 (Meng et al., 2004) and TLR4 (Akashi et al.,
dence that endogenous ligands signalling through PRRs 2000) are commercially available. Moreover, potent anti-
maybe be involved in established disease can be gained from mouse (Daubeuf et al., 2007) and anti-human TLR4 MAbs
models using adenoviral expression of IL-1 and TNF locally in (Dunn-Siegrist et al., 2007) developed by NovImmune are
joints to induce joint inflammation and damage. In this currently in pre-clinical studies and it would be interesting to
system it was shown that IL-1-driven inflammation was sig- speculate how these would perform in the various models of

J Campbell et al.
BJP
induced and spontaneous arthritis. Targeting a receptor Acknowledgements
involved in bacterial sensing would of course also have safety
implications in terms of host defence and these would need We would like to thank Ian K Anderson and Tristan J.
to be monitored accordingly. Interestingly, a small clinical RA Vaughan for their support and critical review of this manu-
study involving 23 subjects with active disease [disease activ- script. This work was supported by MedImmune Ltd.
ity score (DAS) 28 > 3.2 at baseline] treated with chaperonin
10 (XToll™), a downstream inhibitor of TLR signalling, sug-
gested a significant improvement in disease activity, as deter-
mined by DAS28, from baseline to day 84 (Vanags et al., 2006) Conflict of interest
irrespective of dose. However, as this was not a placebo-
controlled study, further studies are warranted to determine JC, DL and MS are all employees of MedImmune Ltd (a
the relative significance of this change in DAS. Nevertheless, wholly owned subsidiary of AstraZeneca). MedImmune cur-
these support the notion that inhibition of TLR signalling rently has a number of monoclonal antibodies in preclinical
may provide clinical benefit in these subjects. and clinical development for the treatment of RA (CAM-
Whether antibody inhibitors of TLRs will drive subjects 3001), systemic lupus erythematosus (MEDI-545) and sys-
into remission is another question. Pattern recognition is the temic sclerosis (MEDI-551).
first event involved in mounting an inflammatory response
to a pathogen; moreover, once established, the resulting cel-
lular damage and endogenous ligands may continue to signal
through these self same receptors perpetuating the inflamma-
tion. In RA it has been hypothesized that this process is References
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British Journal of DOI:10.1111/j.1476-5381.2010.01183.

REVIEW bph_1183 1470..1484

Developing the next Building, Granta Park, Cambridge

generation of monoclonal ----------------------------------------------------------------

antibodies for the treatment cytokine; engineering; phage

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British Journal of Pharmacology (2011) 162 1470–1484 1479

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